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ADVANCES IN MACROMOLECULAR CARBOHYDRATE RESEARCH

Volume 7

1 997

This Page Intentionally Left Blank

ADVANCES IN MACROMOLECULAR CARBOHYDRATE RESEARCH Editor:

ROBERT J. STURGEON Department of Biological Sciences Heriot- Watt University Edinburgh, Scotland

VOLUME 1

1997

@) jAI PRESS INC. Greenwich, Connecticut

London, England

Copyright © 1997 by JAI PRESSINC 55 Old Post Road, No. 2 Greenwich, Connecticut 06836 JAI PRESSLTD. 38 Tavistock Street Covent garden London WC2E 7PB England All rights reserved. No part of this publication may be reproduced, stored on a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, filming, recording, or otherwise, without prior permission in writing from the publisher. ISBN: 1-55938-323-2 Manufactured in the United States of America

CONTENTS

LIST OF CONTRIBUTORS PREFACE

Robert J. Sturgeon

vii

ix

ARABINANS AND ARABINAN DEGRADING ENZYMES

G. Beldman, H.A. Schols, S.M. Pitson, M.J.F. Searle-van Leeuwen, and A.G.J. Voragen

STRUCTURAL ELUCIDATION OF THE N-LINKED OLIGOSACCHARIDES OF GLYCOPROTEINS USING HIGH pH ANION-EXCHANGE CHROMATOGRAPHY

Kevin D. Smith, Elizabeth F. Hounsell, John M. McGuire, Moira A. Elliot, and Heather G. Elliot

NEOGLYCOCONJUGATES AS ARTIFICIAL ANTIGENS: CHEMICAL ASPECTS

Nikolay K. Kochetkov and Anatoly Ya. Chernyak

65

93

GLYCOSYLATION PATTERNS IN MUCUS GLYCOPROTEINS

Amalia Slomiany, Chinnaswamy Kasinathan, and Bronislaw Slomiany

INDEX

177 213

This Page Intentionally Left Blank

LIST OF CONTRIBUTORS

C. Beldman

Department of Food Science Wageningen Agricultural University Wageningen, The Netherlands

Anatoly Ya. Chernyak

N.D. Zelinsky Institute of Organic Chemistry Russian Academy of Sciences Moscow, Russia

Heather G. Elliot

Department of Pharmaceutical Sciences University of Strathclyde Glasgow, Scotland

Moira A. Elliot

Department of Pharmaceutical Sciences University of Strathclyde Glasgow, Scotland

Elizabeth F. Hounsell

Department of Biochemistry and Molecular Biology University College London London, England

Chinnaswamy

Research Center University of Medicine and Dentistry of New Jersey Newark, New Jersey

Kasinathan

Nikoiay K. Kochetkov

N.D. Zelinsky Institute of Organic Chemistry Russian Academy of Sciences Moscow, Russia

John M.

McGuire

Department of Pharmaceutical Sciences University of Strathclyde Glasgow, Scotland

LIST OF CONTRIBUTORS

SM. Pitson

Department of Food Science Wageningen Agricultural University Wageningen, The Netherlands

HA. Schols

Department of Food Science Wageningen Agricultural University Wageningen, The Netherlands

M.j.F. Searle-van Leeuwen

Department of Food Science Wageningen Agricultural University Wageningen, The Netherlands

Amalia

Research Center

Slomiany

University of Medicine and Dentistry of New Jersey Newark, New Jersey Bronislaw

Slomiany

Research Center University of Medicine and Dentistry of New Jersey Newark, New Jersey

Kevin D. Smith

Department of Pharmaceutical Sciences University of Strathclyde Glasgow, Scotland

A.G.j. Voragen

Department of Food Science Wageningen Agricultural University Wageningen, The Netherlands

PREFACE

The decision to introduce a new series, Advances in Macromolecular Carbohydrate Research, was taken as a result of the remarkable advances which have been made in recent years in the understanding of the chemistry, biochemistry, and biology of carbohydrates. By far the most significant advances have been made in knowledge of the structure and function of carbohydrates in the macromolecular state, whether polysaccharides, glycoproteins, or glycolipids. New terms created to describe such work—^for example Glycobiology, Glycotechnology, and Glycoengineering—now form part of the everyday vocabulary of those interested in and/or working in the field of carbohydrates. Many of the advances achieved arise from the design and implementation of new analytical techniques, such as nuclear magnetic resonance spectroscopy and mass spectrometry, which allow the study of minute amounts of material. This new knowledge has already been applied, for example, in the synthesis of carbohydrate therapeutics, in the engineering of proteins such as carbohydrate hydrolases, for the conversion and modification of polysaccharides, and in the use of recombinant DNA technology to produce glycoproteins of importance in health and disease.

X

PREFACE

In compiling this volume, authors were invited to submit chapters which are not necessarily exhaustive reviews, but which address those areas of the subject in which the authors believe the most important advances are being made. The chapters therefore reflect the interests and views of the individual authors and do not attempt to provide exhaustive citations of early literature. This volume contains four chapters. In "Arabinans and Arabinan Degrading Enzymes", Beldman et al. review an area which is becoming more and more important to plant biochemists, food technologists, and nutritionists. The chapter deals with the isolation of arabinans together with their structural and physical characteristics. Current understanding of the mode of action of arabinan degrading enzymes is of importance in food technology. "Structural Elucidation of the A^-Linked Oligosaccharides of Glycoproteins" is covered by Smith et al. The authors demonstrate the value of high pH anion-exchange chromatography in the study of the heterogeneity of 7V-glycosylated structures in glycoproteins. Functional studies have demonstrated that, in addition to other properties, the oligosaccharide chains of glycoproteins are important determinants of the overall biological activity of these macromolecules. It was established a number of years ago that many disease-creating microorganisms contain polysaccharides or glycoconjugates on their outer cell surfaces, and that these molecules can be used to prepare vaccines for the protection of an animal species against those diseases. Kochetkov and Chemyak discuss the chemical aspects of the "Production of Neoglyconjugates as Artificial Antigens". The increasing use of neoglycoconjugates has led to a rapid development of new synthetic approaches to their production. Mucus glycoproteins (mucins), unlike the more commonly studied animal glycoproteins which bear mainly //-linked oligosaccharides contain almost exclusively 0-glycosidically-linked carbohydrate. Slomiany et al., in reviewing the gylycosylation patterns, concentrate on addressing the area of research relating to apomucin synthesis and co-translational modifications. It is clear that research initiatives will continue to influence advances in carbohydrate research for many years to come. The chapters presented here of necessity focus on a relatively small window of what is a vast subject, and provide valuable insight into a number of aspects

Preface

xi

of a rapidly developing field that will undoubtedly continue to increase in importance in the future. Robert J. Sturgeon Editor

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ARABINANS AND ARABINAN DEGRADING ENZYMES

G. Beldman, H.A. Schols, S.M. Pitson, MJ.F. Searle-van Leeuwen, and A.G.J. Voragen

Note: This chapter was originally accepted for publication in 1992. An Appendix at the end of the chapter contains updated material from 1992-1997. I. Introduction II. Substrates A. Arabinans in General B. Arabinoxylans C. Arabinogalactans D. Isolation of Arabinans E. Pretreatment of Raw Material F. Extraction G. Purification

Advances in Macromolecular Carbohydrate Research Volume 1, pages 1-64. Copyright © 1997 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-323-2

1

2 3 3 5 5 8 8 9 9

:

BELDMANETAL.

H. Characterization of Isolated Arabinans 11 III. Enzymes 20 A. Classification of Arabinanases 20 B. Cooperative Effects during Enzymic Degradation of Arabinose Containing Polysaccharides . . . 33 C. Purification and Assay Methods for Arabinanases . . 36 IV. Concluding Remarks 40 V. Appendix: Review of the Literature 1992-1997 41 A. Substrates 41 B. Enzymes 42 Acknowledgments 55 References 56

I. INTRODUCTION Polysaccharides containing L-arabinose residues are important in many plant tissues. They occur as either homoglycans, generally associated with pectins or, in a number of plant tissues, in a genuine, natural form, or as heteroglycans such as arabinoxylans, arabinogalactans, and arabinogalactan proteins [1-7]. Here we have reviewed arabinans and arabinan degrading enzymes. This subject is becoming more and more important to plant biochemists, food technologists, and nutritionists. Plant biochemists are interested in the biosynthesis of arabinans, their functions as a cell wall constituent in cell wall cohesion, cell growth, metabolic exchanges between plant tissues and soil, and in germination [8-11]. Arabinans have also attracted the attention of food technologists because of their role in fruit and vegetable processing and their occurrence in by-products of some processed plant crops [12—19]. Recently, nutritional aspects of arabinans as component of dietary fiber and their possible physiological activities are mentioned [20-22]. Medical research shows that some arabinans exhibit biological activities in humans [23-25]. The food industry is also interested in arabinan degrading enzymes where their application in fruit and vegetable processing are explored in upgrading of arabinan-rich by-products and in bioconversion of plant biomass [12,14,15,18,19,26-28].

Arabinans and Arabinan Degrading Enzymes

3

Arabinan degrading enzymes also have become essential analytical tools in the elucidation of the fine structure of arabinans [19,29,30]. In this review we have collected information on the occurrence of arabinans and arabinan degrading enzymes. The isolation of the arabinans and their structural and physical characteristics are considered. Also, the isolation, purification, and characterization of enzymes able to degrade these arabinans are covered. To this end, the mode of action of arabinan degrading enzymes is related to their substrate specificity. The possible role of arabinans and the arabinan degrading enzymes in food technology are discussed. Earlier reviews related to this subject are from Whitaker [31] and Kaji [32]. IL SUBSTRATES A. Arabinans in General Arabinans, mainly composed of a-L-arabinofuranosyl residues, are generally arranged in (l->5)-linked chains with varying n u m b e r s of r e s i d u e s s u b s t i t u t e d with other a-Larabinofuranosides at the 0 2 and/or 0 3 position. In some sources, they have been isolated by nondegrading extraction as homopolysaccharides. Other arabinans contain small amounts of additional sugar residues of uncertain significance. Another class of arabinans, containing other constituent sugars, including galacturonic acid, and isolated under degrading conditions, probably originates from the residual stubs of pectins [1-6]. A schematic structure of arabinan is shown in Figure 1. Studies on pectins from many sources clearly reveal that pectin is not a homopolysaccharide, but is constituted of a backbone of (axialaxial) a-(l~>4)-linked D-galacturonopyranosyl units interrupted at intervals by the insertion of a-(l->2)-linked L-rhamnopyranosyl residues in adjacent or alternate positions [1,6,31]. Other constituent sugars are attached in side chains to this rhamnogalacturonan backbone. They comprise D-galactose, L-arabinose, and D-xylose—^the more common ones—^and D-glucose, D-mannose, L-fucose, and D-glucuronic acid which are found

BELDMANETAL

HOCH.

Figure 1. Schematic structure of a highly branched arabinan.

less frequently. Most of these sugars occur in short side chains, one of three units long, glycosidically linked to 04 and 03 of L-rhamnopyranose or 02 or 03 of some of the galacturonosyl residues. The main sugars, D-galactose and L-arabinose, are present in more complex chains with structures similar to genuine arabinans and arabinogalactans and with chain lengths which can be considerable [2-6]. Pectins with arabinans attached have been described for apples [19,20,22,33-35], sugar beets [36-40], suspension-cultured sycamore cell walls [41], rape-seed [42], apricots [43,44], tomatoes [45], carrots [46,47], cabbage [21,48], cell walls of mung bean hypocotyls [8], horsebean roots [10], onions [49,50], pears [12], and angelica [51]. By degrading extracted pectins specifically in the galacturonan backbone by Pelimination [52—54] or enzymatically with endo-^oXygalacturonase, e«^o-pectin lyase or e/i^fo-pectate lyase [35,55— 61], oligogalacturonic acid fragments, and rhamnogalacturonan fractions of higher molecular mass in which virtually all of the neutral sugars were concentrated could be isolated. From these results it was concluded that there is an intramolecular distribution in which the neutral sugars are concentrated in blocks of more highly substituted rhamnogalacturonan regions ("hairy"), separated by unsubstituted regions ("smooth") containing almost exclusively D-galactopyranosyluronic acid residues [62].

Arabinans and Arabinan Degrading Enzymes

5

Rhamnogalacturonans rich in neutral sugars were also isolated from other cell walls using pectolytic enzymes [6,49,63-68] and this can be seen as a confirmation of this concept. B. Arabinoxylans

Arabinose also occurs in considerable amounts in plant cell walls as a constituent of arabinoxylans and arabinogalactans. Arabinoxylans have a homopolymeric backbone of (l->4)linked p-D-xylopyranosyl residues substituted with (1-^2)linked and/or (l->3)-linked a-L-arabinofuranosyl residues as in the endosperm of annual plant cereals and together with (1-^2)linked a-D-glucuronic acid residues and/or its 4-0-methyl ether in soft woods, cereals, brans, hulls, corn cobs, straw, etc. In addition, the xylopyranosyl residues may be acetylated on 02 and/or 03 [2,4,5,69]. Arabinoxylans often contain ferulic and j!?-coumaric acids which are bound to C5 of the arabinofuranosyl side groups. Through oxidative dimerization of these ferulic acid residues arabinoxylan chains can be cross-linked and rendered insoluble [70,71]. C. Arabinogalactans

Arabinogalactans have been grouped by Clarke et al. [7] into three main structural types: the arabino-4-galactans (type I), the arabino-3,6-galactans (type II), and polysaccharides with arabinogalactan side chains like pectic substances and gum exudates (type III). Arabino-4-galactans have a linear chain of (l-^4)-linked P-D-galactopyranosyl residues with 20 to 40% aL-arabinofuranosyl residues (1^5)-linked in side chains connected in general to 03 of D-galactopyranosyl residues [2,6]. Arabinogalactan type II is a highly branched polysaccharide with ramified chains of P-D-galactopyranosyl residues joined by (l->3)- and (l->6)-linkages mainly in the exterior chains. Most of the side chains are terminated with L-arabinofuranosyl residues and to some extent L-arabinopyranosyl residues occur [2,4,5,6].

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8

BELDMANETAL

D. Isolation of Arabinans

The many methods of isolation of arabinans are described in the literature. Table 1 shows the most important sources from which arabinans have been obtained. This table also gives the literature references. Where the references given in the table do not cover aspects of pretreatment, extraction, purification, and characterization, appropriate references are given in the text. E. Pretreatment of Raw Material

Various fractional extraction procedures have been developed. For biologically active plant material these procedures usually start with grinding of the material in warm ethanol or methanol. Subsequently the material is washed with methanol, ethanol, or acetone to inactivate endogenous enzymes instantaneously, to remove alcohol soluble solids, as well as to exchange water [9,24,16,52,72]. For other raw materials, procedures start with clean-up steps to remove interfering compounds with an ethanol—benzene mixture [11,73,74] sometimes preceded [11] or followed [73] by treatment with 80% ethanol for delignification. Oleaginous materials are first defatted with boiling light petroleum [52] or a chloroform—petroleum mixture [72]. Of vegetable tissues the alcohol insoluble residues (AIR) were prepared first which were then ball-milled in an aqueous medium. In this stage, arabinan containing polysaccharides already solubilize from cabbage AIR [48]. After ball-milling, the ground material was deproteinated enzymically (pronase) in an appropriate buffer. Lipids and pigments are normally removed by washing with a mixture of phenol, acetic acid, and water. The bulk of starch can be removed by treatment with aqueous 9% dimethyl sulphoxide [21,22] or with amyloglucosidase [75]. In this latter case arabinans were extracted simultaneously. Siddiqui and Wood [17] started from the hot water insoluble residue of rapeseed which, prior to extraction of the arabinans, was further pretreated with a 2% EDTA solution.

Arabinans and Arabinan Degrading Enzymes

9

F. Extraction

After a clean-up treatment, most of the powdered materials were extracted with water. Hot water (80-100 °C) was used for the extraction of sawdust of white Willow and maritime pinewood, the inner bark of Rosa glauca stems, mung bean hypocotyl cells [8], soybean cotyledon meal, roots of horse beans, rapeseed [16], and cabbage AIR. Water at ambient temperature was used for the extraction of mustard seed and the roots of marshmallow. Parsnip was extracted with 0.2 M acetate buffer (pH 4.5) and rapeseed with 0.5% ammonium oxalate. Mustard seed embryos were extracted with a 2% EDTA solution (pH 7.5, 90 °C) followed by hot water. For cowpea, 10% trichloroacetic acid at 4 °C was used for extraction which was reported to be nondegradative [76]. Azuki beans were extracted with 5% (w/w) sodium hydroxide and aspen bark with 0.5%) (w/w) ammonium oxalate followed by 10%) (w/w) sodium carbonate and 1%) (w/w) sodium hydroxide. Hot (100 °C) solutions of calcium hydroxide or 0.5 M sodium hydroxide containing sodium borohydride were used for the extraction of sugar beet pulp [5,40,77]. Pectic arabinans are usually obtained by sequential extraction with (hot) water, ammonium oxalate, dilute HCl, and alkali solutions (apples, carrots, onions, potatoes, cabbage; for references see Table 3). G. Purification

For most extracts (dialyzed, concentrated) purification starts with graded fractionation with aqueous ethanol mixtures up to 80% ethanol. Extracts of Rosa glauca stems [11] and cabbage cell wall material [21] have been treated first with copper acetate to precipitate polyuronides. The aqueous extract of soybean cotyledon meal was partitionated with a mixture of chloroform and amyl alcohol, and the azuki bean extract was destarched and deproteinated prior to ethanol fractionation. In general the arabinans remain solubilized in the supernatant fraction (parsnip, grape juice, rapeseed, azuki beans, horsebean roots, aspen bark, white willow sawdust, mustard seed embryo). Marshmal-

10

BELDMANETAL

low and maritime pine arabinan were recovered from the precipitate. In the extracts of cowpea and mustard seed meal, the arabinans were precipitated with acetone. From extracts of maritime pine sawdust, Rosa glauca stems and soybean cotyledon meal "pure" arabinan fractions were obtained by reprecipitation with 70-90% ethanol in water solutions as well as with acetone. Arabinan extracts from grape juice and maritime pine sawdust were first decolorized by treatment with polyamide and active carbon respectively. Many isolates of arabinans (from cowpea, Vicia faba roots, azuki beans, white willow bark and aspen, rapeseed cotyledon meal, mustard seed embryo's [9], cabbage [21,48], apple [20], onion [77] and sugar beet [29,30] cell wall material, and marshmallow roots) were further purified by DEAE-ion-exchange chromatography. These anion-exchange columns were used with various buffer systems such as carbonate, phosphate, formate, or acetate. The neutral arabinans are eluted with water or with the starting (low molarity) buffer. Hirst et al. [9] and Siddiqui and Wood [17] used the anion exchanger in the borate form to separate arabinans from a neutral galactose-rich polysaccharide. Since borate is able to form complexes with arabinans, which bind strongly to anion-exchange columns, high concentrations of borate buffer are necessary to elute the complexes from the columns. The pure arabinans can be recovered after dialysis and freeze drying. Borate was removed as volatile methylborate after adding methanol. The final purification step in the isolation of arabinans from apple [20] and mustard seed embryo's [9] was obtained by precipitation with cetyl trimethyl ammonium hydroxide. Arabinans from aspen bark [78], sugar beet [29], marshmallow roots [23] and rapeseed [17] were purified by size-exclusion chromatography. Jones and Tanaka [40] purified their sugar beet arabinan in acetylated form. Churms et al. [14] and Voragen et al. [19] isolated arabinan fractions from apple juice concentrates by filtration and centrifugation, with further purification by cold water washing and reprecipitation. Pectic fragments which are covalently attached are removed from pectic arabinans by a treatment with hot alkali. Under these conditions the galacturonan backbone is degraded and arabinans linked to stubs of the

Arabinans and Arabinan Degrading Enzymes

11

pectic backbone remain [13,83]. Backinowsky et al. [79] describe and stereospecific chemical synthesis of an 1,5-a-L-arabinan via polycondensation of 3-0-benzoyl-l,2-0-[(l-e«rfocyano) ethylidene]-5-0-trityl-P-L-arabinofuranose. H. Characterization of Isolated Arabinans Methods of Analysis

The most important characteristics of isolated and purified arabinans are their arabinose content, optical rotation, molecular mass, and glycosidic linkage composition which can be revealed by either periodate consumption or methylation analysis. Structural information can also be obtained by nuclear magnetic resonance (NMR) spectroscopy. The methods used and collected data from literature are summarized in Table 1. Various techniques have been used to characterize isolated arabinans. The purity of the preparations can be deduced from their sugar composition. This is determined generally by gas chromatography of the alditol acetates of trimethylsilyl derivatives of the sugar moieties released after acid hydrolysis. Another characteristic of arabinans, which often has been determined, is optical rotation ([a]^). Mostly measurements have been made in aqueous systems; however temperatures of measurement varied. Values ranging between —88° to —178° have been reported. For the synthetic arabinan, an [a]2o of—130° was measured. The high negative values and the fact that the specific rotation increased to reach values of +65°-+100° upon acid hydrolysis, were seen as evidence that the arabinose residues are in the furanose form and have the a-configuration. In the last decade, this unreliable method has been used less frequently and conformational information is now derived mostly from NMR spectroscopy. Molecular weights have been determined by size exclusion chromatography using Sephadex G50 or G75 columns with dextran standards for calibration. Reported molecular mass values are in the range of 5 to 22 kDa. The linear arabinan isolated from hazy apple juice concentrate had a molecular mass of 10 kDa; for the synthetic linear arabinan a value

12

BELDMANETAL

between 2 and 3 kDa was measured [14,79]. Initially, information on the glycosidic linkage composition was obtained by periodate oxidation and by Smith degradation. More recently methylation analysis has been used. Values for periodate consumption in the range of 0.66-0.77 mole per arabinose residue have been reported for branched arabinans and a value of 1.07 is reported by Tagawa and Kaji [29,86] for the linear (l->5)L-arabinan which they obtained by digestion of a branched arabinan with an arabinofuranosidase. Both values are in good agreement with the theoretical values which can be calculated for both structures. Smith degradation resulted in the formation of mainly glycerol and L-arabinose [16,11,73,76]. Methylation Analysis, The relative proportions of methyl ethers of arabinitol acetates obtained from methylation analysis of isolated, pure arabinans are summarized in Table 2 and for pectin-associated arabinans in Table 3. The variety of partially methylated arabinitol acetates which were identified point to highly branched polysaccharides. Analysis of methylated Smith degradation products indicated that (1^5)-linked a-L-arabinofuranosyl residues form the backbone of arabinans [13,16,20,80]. These studies also showed that arabinans have both isolated and adjacent branching residues in the backbone and that there are no substantial proportions of linear sequences [13,16,20,80], Evidence for the presence of an (1^5)-a-L-arabinan backbone can also be derived from the observation that branched arabinans can be enzymatically linearized to (l->5)-a-L-arabinans [19,29,30,89]. Such linear arabinans, which are poorly soluble in cold water, have been isolated from apple and pear juice concentrates [12,14,15,84]. Aspinall and Fanous [20] examined the methylated reaction products derived from Smith degradation of apple arabinan directly by GLS/MS and found arabinofiiranosylglycerol and two isomers of arabinobiosylglycerols for which they presume that one has a (1 ->5) intersugar linkage and the other a (1 -^3)-linkage. Branching at 03 and double branching at 02 and 03 of (l-»5)-linked a-L-arabinofiiranosyl residues is common in most arabinan preparations; they are either absent, or only present to a small extent in the linearized preparations [14,29,67]. They also occur in the purified arabinans from sugar beet, peanut, and in the pectic arabinan fractions

Table 2. Methylation Analysis of Pure Arabinans from Various Sourcesa Source

A

W

Maritime Pine (Pinus sp.; sawdust) Aspen (Populus sp.) White Willow (Salk sp.) Marsh Mallow (Althaea oficinalis) Rosa glauca I I1A IIB Parsnip (Pastinaca sativa) Horse bean I ( Kcia faba) Horse bean I1 (Kciafaba) Cow pea (Kgna sinensis) Azuki bean I (Phaseolus radiatus L.) Azuki bean I1 (Phaseolus radiatus L.) Soybean (Glycine m a ) Peanut (Arachishypogaea) Rapeseed (Brassica napus) Rapeseed (Brassica napus)

1,sT(pyr) Linked

1,2Linked

1.3Linked

1,2,3Linked

1.2.5Linked

2,44

-

0,28

-

0,13

0,45

0,09 0,06 0,03

1,26 0,59 0,82

-

-

-

0,11 0,03

-

0,21 0,14 0,21

1 1 I 2 1 1 1 1

0,04

-

0,20 0,18 0,13

-

0,17 0,08 0,lO

-

-

-

-

-

-

-

-

-

-

-

-

-

0,9 0,47 0,46 1,25 4,1 3,7 2,61 0,49

-

-

24

1

-

0,59

-

52 83 16 17

1 1 1 1

-

0,77 1 1 0,64

-

Re$

Tgur)

74

1

0,04

78 73 23

1

11 11 11 75 10 10 76 24

1

1

-

-

-

-

-

1,3,5- 1,2,3,5Linked Linked

T/B

DB(%)

0,11

1,30

18

0,37 0,22 0,21

0,31 0,34 0,34

0,91 1,02 0,94

37 42 42

-

1,8 0,85 0,14 0,43

0,4 0,31 0,35 0,43 0,37 0,51 0,34 0,lO

0,25 0,19 0,20 0,13 0,15 tr 0,24 0,21

0,97 1,30 1,18 1,45 0,40 0,73 1,04 1,05

36 35 38 25 33 22 22 43

-

-

0.47

0,15

0,23

0,93

44

-

-

0,15 1 0 tr

0,36

0,28

-

-

0,88 0,64

0,25 0,18

0,93 1 0,72 1

42 33 44 41

-

-

-

(continued)

Table 2. Continued Source

Ref:

Tgur)

Rapeseed (Brassica napta) Mustard seed (Brassica sp.) Mustard seed (Brassica sp.) Mustard seed (Brassica sp.) Mustard seed (Brassica sp.) Lemon peel Apple (Malta sp.) Apple UFR Apple UFR ara Apple haze Apple haze Grapes (Mtis sp.) Cabbage (Brassica oleracea capitata) Sugar beet (Beta sp.) Sugar beet linear Sugar beet Sugar beet linear Sugar beet

42 52 9 72 13 85 20 67 67 67 14 18 21

1 1 1 1 1 1 1 1 1 1 1 1 1

30 30 29 29 89

1 1 1 1 1

1.5-

1,2-

1.3-

1,2,3-

1,2,5-

1,3,5-

1,2,3,5-

Linked

Linked

Linked

Linked

Linked

Linked

Linked

Nore: 'T = terminally linked arabinosyl residue; 1.5 = linked arabinosyl residue (etc.); DB = degree of branching (number of branching points as percentage of total arabinosyl residues); TIB = ratio terminally linked residues: branching points.

Table 3. Methylation Analysis of Heterogeneous Pectic Arabinans 1,5A

VI

Source

Apple (Malus sp.) UFR Cold buffer Hot buffer H5 HI0 Carrot (Daucus carota) KA2

KB4 PS 1 C1 11,

Ref:

1.2-

1,3-

1.2.3-

1.2.5-

1.3.5- 1,2,3,5-

Tcur) T(pyr) Linked Linked Linked Linked Linked Linked Linked Ara (%)

67 49 82 20 34 34

1 1 1 1 1 1

-

0,04

-

1,88 0,34 0,77 0,78 0,66 1,32

22 22 46 59

1 1 1 1

-

0,73 1,08 0,23 2,45

-

-

0,32 0,18 0,17

-

0,2 0,29 0,35 0,44

-

-

0,44 0,24 0,50 0,56 0,46 0,63

-

-

-

-

0,51 0,l

0,25 0,12

-

0,43 0,33

-

-

-

-

-

-

0,46 0,55

1,49 0,39

-

-

tr 0,20

-

0,12 0,02 0,11

55

T/B

DII ("4)

22

1,04 1,19 0,76 0,69

19 41 45 52

17 11

0,76 0,78

39 37

0,26 0,49

30 19

1,02 0,65

40 49

0,05 0,18

12 32

10 1,78

4 14

(continued)

Table 3. (Continued) 1.5-

1.2.3-

Onion (Alliurn sepa)

49

1

0,23

-

-

I 1

-

0,Ol 8

-

50 50

-

-

-

-

1,73

-

-

-

65 65

1 1

-

-

-

-

-

-

-

60 60 60

1 1 1

0,32 0,71 1,93

0,03 0,16

-

-

0,02 0,11 0,lO

48 48 81 81

1 1 1 1

0,88 0,72 1,05 0,9

-

0,11

-

-

-

-

-

-

-

-

-

-

K3B

Q\

1.3-

Re$

%A

A

1,2-

Source

Potato (Solanurn tuberosurn) I 111 Grape (Atis vinifera L.) WSP HP AIRPEL Cabbage (Brassica oleracea capitata) AIR CWU Hot H,O Oxal.

1,2,5-

1.3.5- 1,2,3,5-

T f i r ) T(pyr) Linked Linked Linked Linked Linked Linked Linked Ara (%)

0,15 -

-

-

-

-

8,64 0,07

-

-

T/B

DB (%)

Arabinans and Arabinan Degrading Enzymes

17

of onions and potatoes. No double branching was found in the grape arabinan. Branching at 02 only is also fairly common, although to a lower extent than branching at 03. Arabinans from beans, particularly horse beans, show a high level of branching at 02. In arabinans isolated from most other sources, however, 02 substitution is virtually absent (rapeseed, mustard seed, grape, cabbage, and parsnip). This is also true for pectic arabinan fractions obtained from apples, carrot, grape berries, cabbage, onion, and potato. Several investigators indicated the presence of longer side chains containing some contiguous (l->3)-linked arabinofuranosyl residues [11,20,30,34,46,59,60,67, 73,74,80], some of which may be branched [30]. In arabinans from sugar beet [90], apple, grape, and cabbage (l->2)-linked arabinofuranosyl residues were encountered. The only report of the presence of di-substituted arabinofiiranosyl residues at 02 and 03 in their sugar beet arabinan is by Weinstein and Albersheim [30]. Terminal arabinopyranosyl residues were found in maritime pine, aspen, white willow, marshmallow, Rosa glauca, mustard seed, sugar beet, onion and potato. NMR Analysis, A more sophisticated method to study the molecular structure of arabinans is the use of NMR spectroscopy, either in the ^H- or ^^C-mode [8,10,23,35,77,79]. ^^C NMR is particularly usefiil as the anomeric signals of a-L-arabinofiiranose residues have a very characteristic chemical shift (106—109 ppm). This enables unambiguous assignment of ^^C-NMR signals of arabinans in the anomeric region. However, the NMR data mentioned in the literature vary as a result of differences in the temperature of recording the spectra and the use of various reference systems. The most complete ^^C-NMR data of differently linked arabinofiiranose residues are listed by Capek et al. [23]. In this study the assignments are given for a-L-arabinofiiranosyl residues, variously linked by (l->5), (l->3), and (l->2) bonds at 30 °C using DMSO (39.45 ppm relative to TMS) as internal standard. The ^^C-NMR spectrum is shown in Figure 2 and the corresponding data are presented in Table 4. In most studies, a good correlation is obtained between the results of methylation analysis and NMR. Arabinans present in the side chains of pectins can also be studied by NMR without destruction of the intact polysaccharide but quantitative conclusions should be treated with caution since

18

BELDMANETAL

' ' ' I '

Figure 2. The ^^C-NMR spectrum of the L-arabinan (3% in D2O) (reprinted with permission from ref. 23).

signal intensities may be affected by differences in segmental motion between the arabinan side chains and the pectic backbone [77] or by other unidentified factors. In general, NMR can be used as a quick and effective method to study the structure of water-soluble arabinans. Other Characteristics Churms et al. [14] characterized their microcrystalline linear L-arabinan also by X-ray crystallographic analysis and deduced Table 4. ^^C NMR Data for the L-Arabinan from the Roots of Althaea officinalis L. [23] Chemical Shifts (ppm) Sugar Residues Terminal 1,2,3,5-linked 1,2,5-linked 1,3,5-linked 1,5-linked

C-I 108.7 107.6 107.6 108.15 108.15

Note: ^ e s e signals could not be assigned.

C-2

C-3

C-4

82.5 86.1 88.2 81.4 82.2

77.8 83.4 76.4 84.3 77.8

85.2 82.5 83.4 82.5 83.4

C-5 62.3 — 67.35' 68.00 67.65

Arabinans and Arabinan Degrading Enzyfnes

19

from the diffractogram that its molecular structure was ordered and repetitive. By electron microscopy they also showed that the aggregates which formed upon cooling of a heated solution of linear arabinan were ellipsoidal with major and minor axes of 1700 and 840 nm, respectively. Tagawa and Kaji [29] determined intrinsic viscosities of 19.5 and 23.7 g cm~^ and densities of 1.593 and 1.524 for branched and linear beet arabinans, respectively. Churms et al. [14] also reported a hydrolysis rate constant of A: = 154 X 10"^ s~^ for the (l-^5)-arabinofuranosyl linkages in the solid apple haze arabinan in trifluoroacetic acid solution of pH 2.0 and 100 °C. In our laboratory we estimated the energy of activation {E, kcal/mole) for hydrolysis of branched and linear arabinans in 0.185 molar potassium malate buffers (to mimic apple juice) by measuring formation of reducing groups. For linear arabinans we calculated E values of 28.3, 23.1, and 27.9 kcal/mole at pH's of 2.0, 3.0, and 3.5, respectively; for highly branched sugarbeet arabinan these values were 27.9, 24.1, and 25.2 kcal/mole, respectively. These results indicate that there is not much difference in the susceptibility of the various arabinofuranosylic linkages in arabinans to acid degradation at enhanced temperatures. Guillon et al. [39] found that the terminal arabinopyranosyl residues of the arabinan side chains in sugar beet pectin carried about 20 to 30% of the feruloyl groups present in sugar beet pectins. Peroxidase and persulfate ions can catalyze the oxidative coupling of pectin molecules and give gelling properties to sugar beet pectin [37]. Structural Features

All arabinans reviewed here, which originate from various plant sources, show essentially the same general structural features. Structural differences are apparent in degree of branching; sites of substitution at 02, 0 3 or 02, and 0 3 ; the presence of sequences of (l->3)-linked residues; and the presence of pyranose residues. The degree of branching can be defined as terminal arabinosyl units, expressed as percentage of total

20

BELDMANETAL

arabinosyl units or as sites of branching relative to total arabinosyl units. When measured by methylation analysis, the disparity between values can be ascribed to undermethylation resulting in overestimation of branching points, to preferential loss of the volatile, highly methylated terminal arabinosyl residues [75], or to the presence of arabinans of oligomeric nature. The ratio between terminal arabinosyl units and number of branching sites is therefore a good indication for the efficiency of the methylation analysis of polymeric arabinans. A ratio <1 may indicate undermethylation and/or underestimation of terminal arabinosyl residues. A ratio >1 can be explained by linkage of terminal arabinofuranosyl residues to polymers other than arabinan and by the presence of oligomeric arabinans. Evaluation of the calculated ratios indicates that for about 10% of the purified arabinan samples (Table 2) and for 40% of the pectin associated arabinans (Table 3) the ratio was >1.1; for about 40% of the purified samples and 20% of the pectin-associated arabinans the ratio was <0.9. Degrees of branching varied between 23 and 45 for purified arabinans with a ratio of ca. 1, and between 23 and 43 for the corresponding pectic arabinans. From this evaluation it appears that there are no fundamental differences between purified and pectic arabinans. The ratio between terminal arabinosyl units and number of branching sites is more often found to be >1 in pectic arabinans than in purified arabinans which can be easily explained by the presence of other arabinosyl-carrying polysaccharides. In pectic arabinans branching at 0 2 tends to occur less frequently, while (l-^2)-linked arabinofuranosyl residues are encountered more frequently.

III. ENZYMES A. Classification of Arabinanases

As is the case for most polysaccharide degrading enzymes, arabinanases can also be classified on the basis of their mode of action, i.e. endo- or exo-acting. Enzymes releasing L-arabinose from polysaccharides such as beet arabinan, arabinoxylans.

Arabinans and Arabinan Degrading Enzymes

21

and arabinogalactans, or from arabino-oligosaccharides and synthetic substrates such as /7-nitrophenyl-a-L-arabinofuranoside, are named arabinosidases or arabinofuranosidases. These enzymes act by an ejco-manner and are systematically classified as a-L-arabinofuranoside arabinofiiranohydrolases (EC 3.2.1.55). Arabinanases degrading polysaccharides with a (l-^5)-a-Larabinan chain by an endo-fsishion are called endoA^S-a-Larabinanases. The systematic name is 1-^5-a-L-arabinan l->5-a-L-arabinanohydrolase (EC 3.2.1.99), according to the enzyme nomenclature [87]. In a review from 1984, Kaji subdivided the arabinofuranosidases according to the source from which they were isolated [32]. He discriminates Aspergillus niger types and Streptomyces purpurascenses types of a-L-arabinofuranosidases. The first type releases side-chain L-arabinosyl residues of branched Larabinan, L-arabinoxylan, and L-arabinogalactan, and is also active towards synthetic substrates, arabino-oligosaccharides, and linear (l-^5)-a-L-arabinan. The latter enzyme acts only on low molecular weight substrates such as arabino-oligosaccharides and/7-nitrophenyl-a-L-arabinofuranoside and is inactive towards polymers. In the course of our research on polysaccharide degrading enzymes, we encountered arabinose releasing enzymes for which we could not use this classification [88] or for which this division would at least give rise to confusion since both types of enzymes mentioned above were found in A. niger [19,89]. We therefore propose to classify the arabinanases on the basis of their mode of action and substrate specificity. The following classes can be distinguished: • a-L-arabinofuranosidase, not active towards polymers (EC 3.2.1.55) • a-L-arabinofuranosidase, active towards polymers • a-L-arabinofiiranohydrolase, specific for arabinoxylans • ejco-a-L-arabinanase, not active on j[?-nitrophenyl-a-L-arabinofiiranoside • p-L-arabinopyranosidase • endo' 1 ^5-a-L-arabinanase (EC 3.2.1.99)

22

BELDMANETAL

a-L'Arabinofuranosidase, Not Active Towards Polymers (Arafur A)

Screening of several actinomycetes by Komae et al. [90] resulted in the discovery of an a-L-arabinofuranosidase from Streptomyces purpurascens which was able to hydrolyze/>-nitrophenyl-a-L-arabinofuranoside and arabino-oligosaccharides. The enzyme was inactive towards arabinosyl linkages present in polysaccharides. Considering this substrate specificity, the enzyme is a true a-L-arabinofuranosidase. The native form of this enzyme is an octamer composed of eight subunits with a molecular weight of 62,000. An integral purification study of arabinanases from A. niger by Rombouts et al. [89] showed the existence of a similar type of arabinofuranosidase (Arafur A). This enzyme appeared to have a molecular weight of 128,000. But this was later reassessed as 83,000 [138]. In Table 5 a comparison is made between some properties of the enzymes from A. niger and S. purpurascens. Earlier investigations on A. niger arabinanases failed to identify arabinofuranosidase A, probably because a heat treatment was introduced to facilitate the isolation of another type of arabinofuranosidase [91,92]. This latter enzyme was shown to be more heat-stable then arabinofuranosidase A [89]. Enzyme stability studies indicated the existence of two types of arabinofuranosidase in^. niger [31,93]. One form appeared to be stable at pH 4 and the other one at pH 7. However, in the original paper it was stated that this was caused by two forms of the same enzyme, being another type than arabinofuranosidase A. The existence of arafur A as a product of Bacillus subtilis FII was also reported by Weinstein and Albersheim [30]. a-L'Arabmofuranosidase^ Active towards Polymers (Arafur B)

Purified a-L-arabinofuranosidases are commonly measured using /?-nitrophenyl-a-L-arabinofuranoside as substrate. The majority of isolated arabinanases appeared also to be active towards (l->3)- and (l->5)-linked arabinosyl residues which are present in beet arabinan. The nomenclature of Rombouts et al. [89] will

Arabinans and Arabinan Degrading Enzymes

23

Table 5. Some Properties of a-L-Arabinofuranosidase A from A. niger and S. purpurascens M^(Da) Optimum pH K^ (mMol/L) ^cat(^in~')

pl Reference

A. niger

S. purpurascens

83,000 4.1 0.6' 15.7x10^' 6-^.5 89,138

62,000 6.5 0.082^ 5.5x10^*^ 3.9 90

Notes: "pH 5.0; 30 °C; /7-nitrophenyl-a-L-arabinofuranoside. V H 6.5; 30 °C; p-nitrophenyl-a-L-arabinofuranoside.

be used, referring to this type of enzyme as arabinofuranosidase B (Arafur B). Properties of the enzyme has been reviewed by Kaji [32] and Whitaker [31]. Its appearance in nature is extensive, including fungi, bacteria, actinomycetes, yeasts, protozoa, and plants. However, in order to compare the different arabinofuranosidases B properly, discussion will be restricted to those enzymes which have been purified and characterized in more detail. Arabinofuranosidases type B have been isolated from the microbial sources: A, niger, Cortitium rolfsi, Rhodotorula flava, Streptomyces massasporeus, Streptomyces sp. 17-1, Bacillus subtilis, Ruminococcus albus, Dichomitus squalens, and Trichoderma reesei. The enzyme has also been purified from plant sources including: Scopolia japonica, Lupinus luteus, and Daucus carota (carrots). Table 6 summarizes some properties of arabinofuranosidase B. Molecular weights of arafur B are generally below 100,000. The native form of the enzyme from R, albus is probably a tetramer with a molecular weight of 305,000-310,000 [101]. Most of the purified enzymes are optimally active at a pH between 3.7 and 6.0. An optimum pH for bacterial arabinofuranosidases appears to be more in the neutral region. C. rolfsii and R. flava produce an enzyme which is active at extreme acidic conditions. K^ values of all purified arabinofuranosidases are of the same order of magnitude. Abberative values have been obtained when phenyl-a-L-arabino-

24

BELDMANETAL

furanoside was used as substrate instead of j!?-nitrophenyl- aL-arabinofuranoside. The two—^probably different—strains of ^. niger produce arabinofuranosidases with different properties, especially with respect to molecular weights and isoelectric points (Table 6). The best characterized enzyme has been isolated from^. niger. From several lines of evidence, it has been proven that this enzyme hydrolyzes a-(l~>3)- as well as a-(l->5)-linkages in beet arabinan. The rate of hydrolysis of a-(1^3)-linkages of the side chains is much higher than of the a-(l-->5)-linkages in the back bone. This is mainly caused by the relatively higher concentration of accessible a-(l->3)-linked arabinosyl residues present in the side chains. However, the affinity, expressed as \/K^ (mol/L)~^ for both linkages is in the same order of magnitude [89]. The K^ for a branched arabinan isolated from apple juice and expressed as terminally linked arabinosyl residues appeared to be 3.7 X 10"^ (mol/L). After enzymic removal of the branches this value was 2.9 x 10"^ (mol/L) which indicates even a slightly higher affinity for the a-(l->5)-linkages. The ability to hydrolyze both linkages in beet arabinan is not only demonstrated for arafur B from A. niger, but is also observed for the enzymes from C. rolfsii, R. flava, S. massasporeus, Streptomyces sp. 17-1, B. subtilis, M, fructigena, D. squalens, and T. reesei, as well as from carrot cells. Recently, we made a detailed study of the linearization of sugar beet arabinan by arafur B. Sugar beet arabinan (0.2%) in 0.05 M sodium acetate buffer (pH 4.0) was incubated with arafur B. (60 U/50 ml) and the release of arabinose was followed by measuring the increase in reducing sugars after set time intervals. The residual polymeric arabinan remaining after dialysis and freeze-drying of the reaction mixture was analyzed by methylation studies. Part of the results are summarized in Figure 3, which shows the percentage of degradation and the relative proportions of the various glycosidic linkages in sugar beet arabinan plotted against incubation time with arafur B. These relative proportions are expressed in percentages of the total of glycosidic linkages present in the starting material. From this figure it can be concluded that the proportion of terminal

Table 6. Some Properties of a-L-Arabinofuranosidase B from Different Sources Source Aspergillus niger Aspergillus niger Corticium rolfsii RhodotorulaJava Streptomyces massasporeus Streptomyces sp. No 17-1 Bacillus subtilis Ruminococcus albus Monilinia fmctigena Dichomitus squalens Trichoderma reesei Scopolia japonica Lupinus luteus Daucus carota

M, (Da)

Optimum pH

PI

Kma (mMol/L)

kcoP

(min-')

Reference

26

BELDMANETAL

^

40

C

c

O -z

a c c

o

4 5 6 incubation time (h) Figure 3. Debranching of highly branched sugar beet arabinan by arabinofuranosidase B. Relative proportions of the various glycoside linkages are plotted against incubation time (a t-ara/; +1,5 araf; O 1,3-ara/; A 1,3,5-ara/; o 1,2,5-ara/; v 1,2,3,5-ara/).

arabinofuranosyl residues decreases rapidly and reflects the increase in reducing sugars. It also can be seen that the proportion of (l-^5)-linked a-L-arabinofuranosyl residues doubly branched at 02 and 03, declines while the proportions of residues singly branched, at either 02 or 03 remained almost constant. However, it must be realized that new single-substituted residues are formed by the debranching of the double-substituted residues. The proportion of a-(l->3)-linked arabinofuranosyl residues did not change. Based on these results it can be concluded that arafur B does not discriminate between single and double substitution nor between sites of substitution (02 or 03) of arabinofuranosyl residues in the backbone. a-(1^3)-Linkages in longer side chains appear to be more resistant towards enzymic degradation. Heteropolysaccharides, like arabinoxylan and arabinogalactan, have been used as substrates for arafur B. Several enzymes mentioned above are able to release arabinose at least partially

Arabinans and Arabinan Degrading Enzymes

27

from these substrates. Neukom et al. [109] and Tagawa and Kaji [29] have found an almost complete release of arabinosyl residues from wheat arabinoxylan using the A. niger enzyme, while arafur B from T. reesei liberates only 50% of arabinose groups from wheat straw arabinoxylan [105]. Andrewartha et al. [110] investigated an enzyme which was isolated from the commercial preparation, Pectinol 59-L. Only 18% of the arabinosyl residues of wheat arabinoxylan could be removed. The enzyme was called an arabinofuranosidase and cited as such in the literature [32], but its activity on/7-nitrophenyl-a-L-arabinofuranoside was not reported. Based on reaction rates, beet arabinan is a better substrate than wheat a r a b i n o x y l a n for a r a b i n o f u r a n o s i d a s e B [29,89,99,104]. Taking into account the proportion of arabinosidic branches in beet arabinan (33%) and in wheat arabinoxylan (30-50%), this means that the a-(l->3)-linkages in the former substrate are more easily hydrolyzed than the a-(l-^2)and a-(l->3)-linkages in the latter polysaccharide. a^'Arabinofuranohydrolase, Specific for Arabinoxylans Several enzymes capable of releasing arabinosyl residues, present as side chains in arabinoxylans, are described in the literature. Mainly, two groups can be distinguished: the arabinofuranosidase type B and a type of enzyme belonging to the group of nonspecific xylanases which can hydrolyze the xylan backbone as well as release the arabinosyl residues [111]. While investigating the enzymic degradation of arabinoxylans, Kormelink et al. [88] found a new enzyme in the culture filtrate of Aspergillus awamori which did not fit this classification. It was highly specific for arabinosidic linkages in arabinoxylans from oat spelts, wheat, or barley and therefore called l~>4-pD-arabinoxylan arabinofuranohydrolase (AXH). Some properties of this enzyme are listed in Table 7. Neither jc^-nitrophenyl-aL-arabinofuranoside, nor a whole range of synthetic substrates could be hydrolyzed. Neither branched nor linear arabinans were degraded by this enzyme. Also other heteropolysaccharides, containing arabinose, were resistant to hydrolysis.

28

BELDMANETAL

Table 7. Some Properties of 1 ->4-p-L-Arablno-D-xylan Arabinofuranohydrolase from Aspergillus awamori [88,120] i^(Da) pH-optimum Temp.-optimum (°C) Activity towards:^ /7-Nitrophenyl-a-L-arabinofuranoside Arabinoxylan from oat spelts Arabinoxylan from wheat Arabinan from sugar beet UFR from apple*' Linear arabinan Arabinogalactan from citrus Arabinogalactan from potato Arabinogalactan from coffee Arabinogalactan from larch wood

32,000 5.0 50 + + -

Notes: ^+ Release of arabinose; — no release of arabinose. \jltrafiltration retentate isolated according to Voragen et al. [19].

A comparison has been made for the activity of AXH and arabinofuranosidase B from A. niger on arabinoxylan from oat spelts. On short incubation time arafur B (at a higher protein concentration) could not release a measurable amount of arabinose from this substrate, while 1,4-P-D-arabinoxylan arabinofuranhydrolase readily hydrolyzed 43% of the arabinosidic linkages. Similar results have been obtained with wheat arabinoxylan. On this substrate the specific activities of AXH and arabinofuranosidase B were 22 U/mg and 0.9 U/mg, respectively. Kormelink et al. [88] suggest different specificities of these two types of enzymes for the different types of glycosidic linkages present at the xylan backbone as a-(l-^3)- and a(l~>2)-linked arabinosyl residues. exo-a-i-Arabinanases^ Not Active on p-Nitrophenyl-a-i-arabinofuranoside Using beet arabinan as a substrate Kaji and Shimokawa [112] isolated an arabinanase from Erwinia carotovora lAM 1024 with

Arabinans and Arabinan Degrading Enzymes

29

unusual properties. The enzyme produced arabinotriose from beet arabinan in an ^xo-fashion. It was not active towards aryl arabinosides and heteropolysaccharides containing arabinose as side chains. It was considered not to act on the (l->3)-arabinofuranosyl side chains of the branched beet arabinan and to be specific for a-(l-^5)-linked arabinofuranosyl residues. In contrast, a linear arabinan was not attacked, thus establishing that this enzyme is not an ^wt/o-arabinanase. It is probable that an exo-aXtdick occurs on side chains of beet arabinan, which most likely contain three arabinose residues. Small amounts of arabinobiose in the reaction product also suggest the ability of the enzyme to split off dimeric side chains. In recent work, Lahaye and Thibault [113] claim the existence of an exo-arabinanase as the product of ^. niger (var. aculeatus). The enzyme appeared to be a glycoprotein with properties as listed in Table 8. The major product from beet arabinan was arabinobiose, together with the formation of a small amount of trisaccharide. Considering these products, together with the low activity of the enzyme on carboxymethylarabinan, these investigators conclude that this arabinanase is an exo-acting enzyme. Table 8. Some Properties of exoArabinanase from Aspergillus niger (var. aculeatus) [113] M^ (Da) pH-optimum Temp.-optimum (°C) pl Activity (nKat/ml) towards: Reduced arabinan from sugar beet Arabinogalactan from potato Arabinogalactan from larch wood Polygalacturonic acid /?-Nitrophenyl-a-L-arabinofuranoside /7-Nitrophenyl-P-D-galactopyranoside Carboxymethylarabinan Gum arabic

67,000 4.0 60 2.85 28.8 0 0 0 1.2 0 2.0 0

30

BELDMANETAL

^'I'Arabinopyranosidase So far, only enzymes which are able to release arabinose of the a-L-furanose form, have been discussed. This chapter deals with another type of arabinanase, namely P-L-arabinopyranosidase. Commonly p-L-arabinopyranosides as well as a-D-galactopyranosides and a-D-fucopyranosides are good substrates for a-galactosidases because of the similarity of the glycosidic linkage and the orientation of hydrogen and hydroxyl groups at C2, C3, and C4. Dey [114] discovered an activity, not being an a-galactosidase, but yet able to hydrolyze P-L-arabinopyranosides. The enzyme was isolated from Cajanus indicus seeds, until now the only source for this enzyme mentioned in the literature. Characteristics of P-L-arabinopyranosidase were given in this first publication and supplemented in a next paper [115]. Some of the properties are compiled in Table 9. Probably a carboxyl group and a histidine imidazolium group are involved with the enzyme substrate complex and influence the activity [114]. There is a cooperative catalytic action of a proton donor, being the imidazolium ion, and a nucleophile in the form of a carboxyl group. Probably this is a two-step mechanism which includes the formation of a glycosyl—enzyme intermediate. The enzyme also catalyzes a ^ra«5-glycosylation Table 9. Some Properties of P-L-Arabinopyranosidase from Cajanus indicus Seeds [114,115] M^(Da) pH-opt pi ^.(M) Activity towards:^ />-Nitrophenyl-p-L-arabinopyranoside /7-Nitrophenyl-a-D-galactopyranoside p-Nitrophenyl-P-D-galactopyranoside p-Nitrophenyl-a-D-fucopyranoside Note: ^+ activity; - no activity.

25,900 3-4.6 4.4 0.83 X 10"^ + -

Arab!nans and Arabinan Degrading Enzymes

31

reaction in which the anomeric conformation of the product is retained. One could speculate on the function of p-L-arabinopyranosidase in plant tissue. Maybe the enzyme is involved in the breakdown of highly ramified polysaccharides such as larch arabinogalactan. Arabinosyl residues are present in branches of this polysaccharide in both the a-L-furanose and the p-L-pyranose forms [4]. endo' 1-^5Hi'L'Arabinanases

The existence of an e«(io-arabinanase was first noticed in 1963 as a product of Clostridium felsineum (var. sikokianum) [116]. Since then, the enzyme has been purified from other bacterial sources, including Bacillus subtilis F-11 [30,117] and B. subtilis IFO 3022 [118]. An enzyme from this last source is able to disintegrate potato tissue in the absence of a pectinase, which is commonly necessary to macerate this type of tissue. An enzyme, with similar properties, has been isolated from B. subtilis IFO 3134 [119]. Considering its ability to release protopectin from beet pulp, this enzyme has been called "protopectinase-C". However, according to its properties one has to conclude that it is actually an e«(io-arabinanase with the same characteristics as the e«^o-arabinanases from the B. subtilis strains mentioned before. ewrfo-Arabinanase was also found in a fungal preparation from A. niger [89] in 1988. In previous investigations on arabinofuranosidase B from A. niger [91,92] the existence of this enzyme remained unnoticed because a heat treatment was introduced into the purification procedure. ^«Jo-Arabinanase as well as arafur A appeared to be relatively more sensitive to heat in activation than arafur B. Arabinanase from B. subtilis F-11 was active towards linear (l->5)-a-L-arabinan and branched beet arabinan, but inactive towards phenyl-a-L-arabinofuranoside, arabinoxylan, arabinogalactan, and gum arable. With respect to this substrate specificity, the other purified enJo-arabinanases resembled the B. subtilis F-11 enzyme. In general linear (l->5)-a-L-arabinan ap-

32

BELDMANETAL

peared to be a much better substrate than the branched beet arabinan. For instance, after prolonged incubation (120 h) Kaji found a hydrolysis limit of the first substrate of 23.3% while the branched polysaccharide was only hydrolyzed by 3.3% [117]. The activity of ^«Jo-arabinanase on arabinans declines during the course of the reaction as the (l-^5)-a-L-arabinofuranosyl sequences become shorter or more highly substituted [19]. Some properties of the purified e«Jo-arabinanases mentioned in the literature, including protopectinase-C, are summarized in Table 10. Yoshihara and Kaji [118] concluded that the enzyme from B. subtilis IFO 3022 is evidently the same as the endoA-^5-a-harabinanase from B. subtilis F-11. We already mentioned that this is also true for the e«Jo-arabinanase from B. subtilis IFO 3134. However, the fungal enzyme firom^. niger showed somewhat different properties, not only with respect to molecular weight, pH-optimum, and isoelectric point, but also with respect to the product formation. The bacterial e«Jo-arabinanases produce arabinose and arabinobiose as end-products, while arabiTable 10. Some Properties of Purified endo-Arabinanases B. subtilis

M^ (Da) pH-optimum pl Activity towards: Linear (l->5)-a-L-arabinan Beet arabinan /7-Nitrophenyl-a-LArabinofuranoside Products after prolonged incubation^ Reference

F-11

IFO 3022

IFO 3134

A. niger

32,000 6.0 9.3

33,000 6.0 7.9; 9.7'

30,000 6.0 9.0

35,000 5.0 4.5-5.5

+ + —

+ + —

+ + —

+ + —

A1+A2

A1+A2

A,+A2

A2+A3

30,117

118

119

19,89

Notes: ^Reference 32. ^Ai arabinose; A2 arabinobiose; A3 arabinotriose.

Arabinans and Arabinan Degrading Enzymes

33

nobiose and arabinotriose accumulate in the reaction mixture using the fungal enzyme. B. Cooperative Effects during Enzymic Degradation of Arabinose Containing Polysaccharides Arabinans

It has been shown that A. niger produces arabinofuranosidases A and B, as well as e«t/o-arabinanase. These enzymes can act in concert during the degradation of branched arabinans [19]. Combinations of these arabinanases were made according to Table 11 and their activities were expressed as a percentage of the sum of activities of single enzymes. ewrfo-Arabinanase shows a strong synergistic interaction with both arabinofuranosidases. The mechanism of both synergistic effects, however, is different. Arafur A, on its own, is unable to degrade the polymeric substrate. Its synergistic interaction is only expressed when endoarabinanase forms oligo-arabinosides, which are a good substrate for the furanosidase. The synergism between arafur B and e«t/o-arabinanase can be explained as follows: arafur B liberates linear l->5-a-L-arabinosyl sequences, which are better substrates for the endo-enzymt. In turn, the oligomeric products are new substrates for the furanosidase. As could be expected from their substrate specificities, there was no synergism between the two arabinofuranosidases. In the experiments where the enzyme is combined with itself, resulting Table 11. Relative Activities of Arabinanases Towards Branched Arabinan (UFR) Separately and in Combinations [19]^ Arabinofuranosidase Enzyme

A

B

endO'Arabinanase

Arabinofuranosidase A Arabinofuranosidase B e«t/o-Arabinanase

80 109 191

63 143

79

Note: *Activities of combinations are expressed in % of the sum of activities of single enzymes.

34

BELDMANETAL.

OH

"

""'

OH

L-arabi no-oligosaccharides

Figure 4. Mode of action of the arabinanases of A. niger in the breakdown of highly branched arabinan.

in a double dosage, the increase of activity is less than proportional. The authors ascribe this to the relatively high enzyme dosage and reaction time. The conclusion from these observations is that the arabinanase complex of ^. niger can completely break down branched L-arabinans to arabinose, according to a reaction scheme presented in Figure 4. Most likely this model is also valid for the degradation of such a branched substrate by the arabinanase complex of 5. subtilis, composed of a similar mixture of enzymes. Arabinoxylans

Kormelink et al. [120] studied the degradation of arabinoxylan by combinations of enzymes. Arabinose releasing enzymes, be-

Arabinans and Arabinan Degrading Enzymes

35

Table 12. Relative Activities of Arabinoxylan Degrading Enzymes Towards Wheat Arabinoxylan Separately and in Combinations [88]^ Enzyme AXH' Arabinofiiranosidase B ewflfo-Xylanase

AXH 109 103 133

Arafur B^

endo-Xylanase

88 142

100

Notes: ^Activities of combinations are expressed in % of the sum of activities of single enzymes. **! ,4-p-D-Arabinoxylan arabinofuranohydrolase. '^a-L-Arabinofuranosidase B.

ing a substrate-specific 1,4-p-D-arabinoxylan arabinolfuranohydrolase (AXH) from A. awamori [88] and an arabinofiiranosidase B fi'om A. niger, were combined with an ^«Jo-xylanase ft*om A. awamori and incubated with wheat arabinoxylan. The degradation of arabinoxylan was measured by a reducing sugar assay. Clear synergism was found for the combination of endoxylanase and an arabinose releasing enzyme (Table 12). This is valid for AXH, as well as for arafur B. Similar cooperative effects between arabinofuranosidase and e«
The activity of arabinofuranosidase B from A. niger towards potato arabinogalactan is only very limited [9,121]. De Vis et al. [121] showed that potato arabinogalactan degradation was

36

BELDMANETAL

Table 13. Relative Activities of Potato Arabinogalactan Degrading Enzymes Separately and in Combinations [121]^ endo- Galactanase Enzyme e«^o-Galactanase A e«(i(9-Galactanase D e«(io-Arabinanase

A

D

endo-A rabinanase

92 88 130

85 121

52

Note: ^Activities of combinations are expressed in % of the sum of activities of single enzymes.

not enhanced when this enzyme was combined with an endogalactanase. Kinetic experiments with e«Jo-arabinanase from A. niger had already shown that side chains of potato arabinogalactan are composed of linear (l-^5)-a-L-arabinans with only a few branches. This was confirmed by the cooperative action of an ^wrfo-galactanase and an ^«(io-arabinanase during the hydrolysis of this type of arabinogalactan. The degradation could considerably be accelerated when both enzymes acted together (Table 13). A more progressive decrease of the molecular weight of potato arabinogalactan by the action of endo-galactanase was obtained when arabinose-containing side chains were removed by e^zcfo-arabinanase [121]. C. Purification and Assay Methods for Arabinanases

Arabinanases are commonly extracellular enzymes and can be isolated from the culture filtrates of relevant microbial sources. For that purpose 'classical' techniques have been used, such as: precipitation, ultrafiltration, ion-exchange chromatography (on CM- as well as on DEAE-modified carriers), gel filtration, isoelectric focusing, and adsorption on hydroxylapatite. Methods to isolate arabinofuranosidase B from A. niger have been reviewed recently [92]. By omitting the heat treatment, generally used by these investigators, Rombouts et al. were able to purify the complete arabinan degrading enzyme complex [89]. In this procedure a cross-linked alginate column was used to selectively remove contaminating polygalacturonase activity. An

Arabinans and Arabinan Degrading Enzymes

37

affinity chromatography technique was developed by Waibel et al. [122] using cross-linked arabinan or arabinan linked to activated Sepharose 6B. It was found that only one of the two arabinofuranosidases present in the crude enzyme preparations which were used for these experiments could bind to the column. Most likely this is an arabinofuranosidase B, known to have a high affinity for the polysaccharide. Obviously the arabinofuranosidase which passed the column unbound must have been arabinofuranosidase A. Lahaye and Thibault [113] and Lahaye et al. [128] used affinity chromatography on divinylsulphonesubstituted Sepharose and chelating Sepharose (in the Cu^^form) to separate arabinanases and galactanases from A. niger (var. aculeatus). Chromatofocusing on Mono-P appears to be an efficient method to remove a persistent contamination of arabinofuranosidases from an e«^o-arabinanase from A. aculeatus (unpublished results). During fractionation of arabinanases a reliable, simple, and quick assay is needed to identify them. The most widely used assay for arabinosidases is based on the hydrolysis of /?-nitrophenyl-a-L-arabinoside. The hydrolysis product, p-nitrophenol, can be measured at 400 nm after adjustment of the pH to 9.0. Screening of fractions using this substrate alone is not sufficient because it is impossible to discriminate between arabinofuranosidase type A and B, as well as to identify an endoarabinanase. For that purpose arabinans with different degrees of branching can be used; these include (l-^5)-a-L-arabinan (linear), beet arabinan (heavily branched), and UFR from apple juice (moderately branched) [19]. After incubation the increase of reducing sugars is generally measured with the Nelson-Somogyi method [123]. Activity towards (l-^5)-a-L-arabinan indicates the presence of an ^«(io-arabinanase, but only if hydrolysis of the synthetic substrate is negligible. Fractions with activity towards beet arabinan may contain arafur B, while both enzymes mentioned here may be present if there is activity towards UFR [19,89]. In mixtures of arabinanases, e/i^fo-arabinanase can selectively be measured by inhibition of arabinofuranosidases by L-arabonic acid y-lactone [106,108]. Very convenient methods to measure

38

BELDMANETAL.

endo-arabinanase have been developed by McCleary using substituted linear (l~>5)-a-L-arabinans [124]. These substrates include carboxymethyl debranched arabinan, Procion Brilliant Red linear arabinan, and Remazol Brilliant Blue-or-Black-CM-linear arabinan. For practical applications we prefer the substrate dyed with Procion Red. After incubation with enzyme the unreacted substrate is precipitated in 75% ethanol and the dyed hydrolysis products are measured at 520 nm. Advanced methods to identify endo- or ejo-acting arabinanases have been introduced by the use of fast and high performance liquid chromatography (FPLC and HPLC. respectively). These methods have been used to develop a rapid screening procedure for arabinanases in crude mixtures of polysaccharidases, but are also applicable for xylanases [125]. Basically a rapid separation of the proteins by FPLC (Mono-Q column) is followed by identification of hydrolysis products by

5 -

D(?5

c

s JD c

1 ^^ Df?2

c o

GL

-

30 min.

1i

CLI

Q

2-

15 min.

A J

- ^5 min. ^

c)

A

1

JJi

J 1 ll

nn 1 UhiH....

i,

j L-Jl

LXi.i.XAAAAAAAA,^..^,.s^

1

10

10

20

30

40 5( retention time (min)

Figure 5. Analysis of an enc/o-arabinanase digest of a linear arabinan by HPLC (Carbopac PA 1; sodium acetate gradient; pulsed amperometric detection).

Arabinans and Arabinan Degrading Enzymes

39

^ "

15 MIN

^m

3 0 MIN

"

1 2 3 4 5 6 7 8 9

4 5 MIN

^

6 0 MIN

N—i

6 0 0 MIN

10 1 1 1 2 13 14 15 16

ARABINOSE UNITS Figure 6. Reaction products formed by endoarabinanase in the various stages of degradation of a linear arabinan.

sugar analysis on an Aminex HPX 87 P column [126], or determination of the molecular weight distribution by gel permeation chromatography on a series of TSK-columns [36]. Recently we applied ion-exchange chromatography on a HPLC system equipped with the Dionex CarboPac PAl column and a pulsed amperometric detection system for the separation and identification of arabinanases. It appeared to be a very versatile method to identify the mode of action of these enzymes. Beet linear (l->5)-a-L-arabinan was incubated with ewrfo-arabinanase from A. aculeatus and at several times samples were taken and analyzed by HPLC (Figure 5). The chromatographic conditions will be published elsewhere. Monomer and oligomers could be detected up to a degree of polymerization of 23. The relatively high peaks represent the linear oligosaccharides, whereas smaller peaks in between these peaks probably originate from branched oligomers. However, this has still to be proven by structural analysis such as NMR. The method described was used to study the intermediary reaction products of linear arabi-

40

BELDMANETAL.

nan in various stages of the breakdown (Figure 6). At an inital stage of reaction, relatively small amounts of low molecular weight products are present in the reaction mixture. As the reaction proceeds more and more small oligomers accumulate, with arabinotriose, -biose and -tetraose being the most important products. An accurate evaluation of peak area's in the region of DP 9-16 (Figure 6, insertion) showed us a 'valley point' at DP 13, especially for a reaction time of 45 and 60 minutes. Apparently the enzyme has a relatively higher affinity for oligomers of that size. We do not know whether this is caused by structural features of the enzyme (i.e. the number of sub-sites) or of the substrate (i.e. its 3D structure in solution). This phenomenon can only be investigated further with kinetic experiments using purified oligosaccharides.

IV. CONCLUDING REMARKS In this contribution, we have tried to bring together the present knowledge on arabinans and arabinan degrading enzymes. This knowledge is far from complete. In recent years particularly, food technologists have shown an urgent need for more information. This need arises from problems in fruit juice processing like fouling of membranes in ultrafiltration of fruit juices and haze formation of fruit juice concentrates, as well as interest by the food industry to find applications for arabinan-rich byproducts. Recently, McCleary et al. [127] succeeded in preparing an enzymically-debranched arabinan from sugar beets which can gel to a creamy paste and which has potential food applications. It is necessary to obtain more knowledge on the chemical structure of pectic arabinans and their enzymic modification and degradation, and to have more insight in the relation between chemical structure and physical behavior. This is also of importance to understand their role as a dietary fiber. For the degradation and tailored modification of arabinans, the active and most efficient enzymes have to be identified and their action patterns studied at a molecular level. This will boost the production of these enzymes and improve their properties by DNA technology.

Arabinans and Arabinan Degrading Enzymes

41

V. APPENDIX: REVIEW OF THE LITERATURE 1992-1997 The preceding sections of this review were prepared and accepted for publication in 1992. Since there has been considerable research into arabinans and arabinan degrading enzymes in the last few years, at our request, the editor allowed us to review the recently published findings which are summarized below. A. Substrates

Although the structural features of a number of arabinose-rich polysaccharides have been published in the last few years, a relatively small number of these studies give additional information to that already discussed in the previous sections. While not discussed further here, arabinan-rich pectic molecules have also been briefly investigated recently in relation to their role in the processing of fruits and vegetables [129] and their possible application in foods [130]. Recently, an arabinan fragment isolated from the water-soluble fraction of dehuUed rapeseed has been described in detail [131]. However, since this fraction still contained about 16% galactose and relatively high amounts of 1,2,5-linked arabinose, it would appear to resemble the highly branched arabinogalactan from rapeseed [132] rather than the free rapeseed arabinan described by the same researchers [16]. Cooper et al. [133] described in detail the physical properties of an enzymically-debranched arabinan from sugar beet, isolated according to the patent of McCleary [127]. It was demonstrated that these arabinans form gels that show the progressive structural breakdown and flow characteristics of fat-continuous spreads, rather than the abrupt fracture typically found for normal polysaccharide gels. Furthermore, the possible use of debranched arabinans as a fat-replacer was demonstrated in a number of products, including low fat spreads, ice cream, and chilled and frozen desserts [127,133]. This same debranched arabinan fraction motivated Chandrasekaran et al. [134] to study the molecular architecture using

42

BELDMANETAL.

X-ray diffraction and computer modeling methods in order to explain the functional properties of these polysaccharides. They concluded that the arabinan fraction was able to form microcrystallites and exists in at least three crystalline allomorphs differing in the conformation of the twofold helical structure. The conformational flexibility of the arabinofuranose ring was studied by molecular mechanics software by Cros et al. [135]. In a following study [136], the same authors used high resolution NMR and computerized molecular modeling to study the conformational behavior of arabinobiose. They also extrapolated their findings to regular polymeric arabinans generating arabinan chains displaying right- and left-handed chirality and a wide range of repeating units per turn of helix. Advanced NMR spectroscopy techniques on a 600-MHz apparatus was used by Eriksson et al. [131] to characterize the above mentioned arabinan fraction from rapeseed. Using HMQC, NOESY, and TOCSY experiments, they were able to assign most peaks in the rather complex ^H and ^^C NMR spectra of the highly branched arabinan. In conclusion, it can be still stated that freely occurring arabinans are not frequently extracted from plant materials, and it is considered that they are, in general, an integrated part of pectic molecules or arabinogalactans. Their role in the plant matrix, in plant-derived products, as well as food ingredients should be studied in more detail to enable a better understanding of their structure-function relationships. B. Enzymes Classification of Arabinanases

A considerable number of arabinanases from a variety of microbial and plant sources have been purified and characterized in the last few years (1992-1997). Most of these recently isolated enzymes appear to fit broadly into the classification system we have proposed based on their mode of action and substrate specificity. Some of these arabinanases are discussed below. Again, only enzymes that have been adequately purified and

Arabinans and Arabinan Degrading Enzymes

43

characterized well enough to allow their classification have been included in this appendix. a-i-Arabinofuranosidase A (Not Active Towards Polymers; Arafur A). Several recently isolated arabinofuranosidases appear to be of the arafur A type since they can hydrolyse aryl-a-L-arabinofuranosides, arabino-oligosaccharides, and arabinoxylan oligosaccharides, but have very little, or no activity against arabinofuranosyl linkages in polymeric substrates such as branched arabinan, arabinoxylan, or arabinogalactan. Some physicochemical properties of these arabinofuranosidases are given in Table 14. Some arafur A's are monomeric proteins with molecular weights of around 40-80 kDa, while the native form of the enzymes from B. polymyxa [139], B. stearothermophilus [141], S. lividans [144], and B. xylanolyticus [143] are much larger, being composed of several subunits. The Table 14. Some Properties of Arabinofuranosidase A's from Different Sources Source Aspergillus aculeatus Ara2 Aspergillus niger N400 Bacillus polymyxa Bacillus stearothermophilus LI Bacillus stearothermophilus T-6 Bacillus subtilis Bacteriodes xylanolyticus Streptomyces lividans Wheat Ara I

MW(Da)

Optimum pH pl

37,000

4.0-4.5

83,000 163,000' 110,000^

3.4 6.5 7.0

256,000' 5.5-6.0

137 3.3 4.7

0.68 1.19 0.22

2.74 X 10^ 3.49 X 10^ 1.21 X 10^

138 139 140

6.5

0.42

4.80 X 10^

141

0.5

9.46 X 10^

142 143

0.6

1.24x10^

7.0 61,000 366,000^^ 5.5-6.0 69,000' 49,000

6.0 4.5

^m

(mM) feat (mirf ) Reference

4.6

Notes: **Composed of two subunits of 65,000 Da and one subunit of 33,000 Da. ''Composed of two subunits of 52,500 and 57,500 Da. *^Composed of four subunits of 64,000 Da. '^Composed of six subunits of 61,000 Da. *Gelfiltrationresults in a MW estimate of 380,000 Da.

144 145

44

BELDMANETAL

characterized fungal arafiir A's have quite acidic pH optima (pH 3.0-4.1), while that of their bacterial counterparts generally appear at higher pH (5.5-7.0). AT^^ values for arafUr A's with;7-nitrophenyl-a-Larabinofiiranoside are similar and appear to be slightly lower than for most arafiir B 's examined, possibly indicating a general higher affinity for this substrate. Recent studies with the arafur A's fromv4. niger and B. subtilis, the two most extensively characterized enzymes of this type, have contributed important information regarding the substrate specificity of this enzyme class. During studies of the enzymic degradation of alkali-extractable wheat-flour arabinoxylan, Kormelink et al. [146] examined the action of the A. niger arafur A on isolated arabinoxylan-derived oligosaccharides. While this enzyme was not active against polymeric arabinoxylan, it readily cleaved all (l-^3)-a-linked arabinofuranosyl groups from singly substituted xylopyranosyl residues in arabinoxylan oligosaccharides, irrespective of whether the substituted xylopyranosyl residue was in a terminal or nonterminal position. Further recent work in our laboratory has also suggested that this enzyme has similar activity against (1^2)-a-linked arabinofuranosyl groups of singly substituted xylopyranosyl residues (K.M.J. Van Laere, unpublished work). In contrast, no hydrolysis of arabinofuranose from doubly substituted xylopyranosyl residues was observed [146]. Similar action patterns on small arabinoxylan oligosaccharides were also found for the B. subtilis arafur A [142]. Kaneko and Kusakabe [147] have shown recently, using chemically synthesized arabinobiose derivatives, that the B. subtilis arafur A effectively hydrolyzes all possible linkages occurring between two a-L-arabinofuranosyl residues (i.e., ( 1 ^ 2 ) - , ( 1 ^ 3 ) - and (l->5)-a-L-linkages). Perhaps surprisingly, due to it's less frequent occurrence in natural substrates, highest activity was shown against the (l->2)-a-L-arabinofuranosyl linkage, while the lowest hydrolysis rate was seen against the more commonly occurring (l->5)-a-L-linkage. Further examination of the action of this enzyme on a small branched arabino-oligosaccharide derivative (methyl 3,5-di-O-a-L-arabinofuranosyla-L-arabinofuranose) also suggested that the (1^3)-a-L-

Arabinans and Arabinan Degrading Enzymes

45

arabinofuranosyl linkage in this substrate was cleaved in preference to the (l-->5)-a-L-linkage [147]. a-i-Arabinofuranosidase B (Active Towards Polymers; Arafur B). The majority of new arabinofiiranosidases examined in the last few years appear to be of the arafur B type. Physicochemical properties of these enzymes, summarized in Table 15, appear similar to those of previously characterized arafur B's discussed earlier (Table 6). While all arafur B's, by definition, have activity against aryla-L-arabinofuranosides and a-L-arabinose containing oligo- and polysaccharides, some of the recently isolated enzymes appear to show some diversity in their substrate specificities. For example, most arafur B's hydrolyze branched arabinan and also have at least some activity against linear arabinan, arabinoxylan, and arabinogalactan, although the relative rate of hydrolysis of these substrates varies markedly between different enzymes. However, an arafur B recently isolated from soybean seedlings was reported to have no activity against arabinoxylan or arabinogalactan, even though it hydrolyzed both linear and branched arabinans [165]. A similar substrate specificity was also seen with an arafur B from C. acetobuylicum [155], while other Arafur B's from P. capsulatum [158] and B. fibrisolvens [154] were reported to show no activity against arabinogalactan, although branched arabinan and arabinoxylans were hydrolyzed rapidly. Two other arabinofuranosidases with more unusual substrate specificities were recently isolated from G. candidum [157]. Like typical arafur A's, these enzymes hydrolyzed pnitrophenyl-a-L-arabinofuranoside but had no activity against branched beet arabinan. However, irrespective of this, they are probably best considered as arafur B's due to their ability to hydrolyze arabinogalactan and arabinoglucuronoxylan. An arafur B recently isolated from A. awamori [148] is also unusual since it appears capable of releasing feruloyl and pcoumaroyl arabinose residues from arabinoxylans. An arafur B from T. emersonii may also have this ability [160], although other arafurs so far examined do not [154,158]. Although details of the action pattern of A. niger arafur B on branched arabinans have been known for some time (see

46

BELDMANETAL.

Table 15. Some Properties of Arabinofuranosidase B's from Different Sources Source

MW (Da)

Optimum pH

pi

Km (mM)

kcdX

(mirT^)

Reference

Aspergillus Ara I 37,000 3.0-3.5 137 aculeatus Aspergillus 4.6 3.6,3.2 1.39 64,000 148 awamori Aspergillus 4.0 3.3 0.679 6.46x10^ 65,000 149 nidulans A. niger 5-16 4.0 3.5 67,000 150 (intracellular) ^. «/gerN400 138 3.8 3.5 0.52 1.58x10' 67,000 A. niger 151 4.6 3.0 1.03 52,900' Aspergillus 152 5.0 3.9 34,300 sojae 153 Aspergillus 4.0 Ara I 39,000 7.5 terreus 4.0 Aral! 59,000 8.3 4.0 Ara III 59,000 8.5 154 0.7 3.38 X 10' 248,000'' 6.0-6.5 6.0 Butyrivibrio flbrisolvens 155 5-5.5 8.15 4.0 3.42x10' Clostridium ace94,000 tobutylcum 156 Clostridium 5.0 52,000' stercorarium 157 Ara I 80,000 4.00 0.63 1.68x10' Geotrichum 4.1 Aral! 69,000 3.85 0.83 2.00 X 10^ candidum 4.1 158 4.15 0.18 2.06 X 10^ Ara I 64,500 Penicillium 4.0 4.54 1.3 4.82 X lO'* capsulatum Ara II 62,700 4.0 7.34 1.3 1.26x10^ 159,160 55,000 Phanerochaete 2.5 chrysosporium 161 8.8 10 Ara I 38,000 4-7 Streptomyces 8.3 12.5 Ara II 60,000 diastaticus 162 3.5 0.16 6.3 X 10' 210,000^ 3.2 Talaromyces emersonii 163 0.1 92,000' Thermomono6.0 sporafusca 164 3.8 Daucus carota Ara i f 80,000 5.6 0.22 1.47x10^ 165 0.53 Soybean 4.8 87,000 166 2.2 68 Spinach 4.8 Ara II 68,000 4.2 Notes: "Gel filtration. ^Composed of eight subunits of 31,000 Da. ''Gel filtration results in a MW estimate of 195,000 Da. '^Composed of two subunits of 105,000 Da. ^Composed of two subunits of 46,000 Da. 'Ara I appears in Table 6.

Arabinans and Arabinan Degrading Enzymes

47

earlier [137]), recent examination of the hydrolysis of arabinoxylan and isolated arabinoxylan-derived oligosaccharides by this enzyme has provided new insights into its substrate specificity. Against arabinoxylan oligosaccharides this enzyme is quite specific, only removing arabinofuranosyl residues from singly substituted, nonreducing terminal xylopyranosyl residues of these substrates. No activity is shown against arabinofuranosyl residues of singly substituted nonterminal, or doubly substituted xylopyranosyl residues in these substrates [146]. This type of action pattern on arabinoxylan oligosaccharides would appear common for arafur B's, since similar results have been described for an intracellular A. niger arafur B [150] and an arafur B from B. fibrisolvens [154]. Furthermore, since this action is different from that of arafur A-type enzymes on these substrates (see earlier), Kaneko et al. [142,147,150] have proposed that this is one way of easily differentiating between these two enzyme classes. However, in an apparent contradiction, there are indications that A. niger arafur B primarily removes arabinofuranosyl residues from doubly substituted xylopyranosyl residues during action on polymeric arabinoxylan [146], although detailed studies have not been performed. Arabinoxylan Arabinofuranohydrolase (a-i-Arabinofuranosidase Specific for Arabinoxylans; AXIH), Enzymes with similar specificity for arabinofuranosyl linkages in arabinoxylans as the A. awamori AXH [88] have been recently isolated from Pseudomonas fluorescens [167], wheat [145], Bifidobacterium adolescentis [168], and T, reesei (R. Kavitha, unpublished work). Two further similar enzymes have been isolated from B. polymyxa, although in addition to hydrolyzing arabinoxylans, these enzymes also have very low, but detectable activity against aryl-a-L-arabinofuranosides, but no other arabinose containing substrates [169]. Physicochemical properties of some of these enzymes are given in Table 16. Our examination of the action patterns of A. awamori AXH on arabinoxylans and arabinoxylan-derived oligosaccharides [146,170] have revealed its high specificity for both (l->2)and (l->3)-a-L-arabinofuranosyl residues of singly substituted xylopyranosyl residues in these substrates. No activity was

48

BELDMANETAL

Table 16. Some Properties of (1 ->4)-p-D-Arabinoxylan Arabinofuranohydrolases from Different Sources Source Bacillus polymyxa Bifidobacterium adolescentis Pseudomonas fluorescens Wheat

AF64 AF53'

MW(Da)

Optimum pH

64,000 53,000 100,000

6.5 6.5 6.0

pl 8.7 9

Reference 169 168

59,000

167

40,000

145

Note: ^This protein probably arises from the proteolytic cleavage of ca. 100 amino acids from the C-terminus of AF64 [169].

shown by this enzyme toward arabinofuranosyl residues of doubly substituted xylopyranosyl residues in these substrates [146,170]. Our recent analysis of the B. adolescentis AXH [168] indicated that this enzyme had a different specificity, since it only hydrolyzed (l-^3)-a-L-arabinofuranosyl residues of doubly substituted xylopyranosyl residues of arabinoxylans and arabinoxylan oligosaccharides. No activity was shown against the (l-»2)-a-L-arabinofuranosyl residues of these doubly substituted xylopyranosyl residues, or against arabinofuranosyl residues of singly substituted xylopyranosyl residues of these substrates [168]. The discovery of such highly specific enzymes raises the possibility that other AXH's with different specificity may also exist. In light of this, we have proposed a nomenclature for these enzymes that more accurately indicates their individual specificities [168]. AXH's that remove arabinofuranosyl residues specifically from singly (mono) substituted xylopyranosyl residues are given a postfix m, while a d postfix indicates those AXH's acting specifically on arabinofuranosyl residues of doubly substituted xylopyranosyl residues. Furthermore, AXH's acting on only one type of a-L-arabinofuranosyl linkage (i.e., either ( 1 ^ 2 ) - or (l->3)-a-L-linkages) are given a number to indicate the linkage cleaved, while the number is omitted for enzymes acting on both linkage types. Thus, the A. awamori enzyme

Arabinans and Arabinan Degrading Enzymes

49

[88] is designated AXH-m, while the enzyme from B. adolescentis is designated AXH-(i3 [168]. endo- 1-^5-a-i-Arabinanase. e«(io-Arabinanases have been recently described from SQWQval Aspergillus species, namely^, aculeatus [137], A. nidulans [149], and a few further A. niger strains [138,171,172]. The physicochemical properties of these enzymes, summarized in Table 17, are comparable to those of the previously examined ewtio-arabinanases (see Table 10). The action patterns of all recently purified Aspergillus endoarabinanases examined appear similar to that of the A. niger enzyme previously discussed [19,89] since arabinobiose and arabinotriose are the major final hydrolysis products released from linear arabinan [137,151,171,173]. However, comparison of the initial (transient) hydrolysis products released from this substrate by the A. niger and A. aculeatus ewrfo-arabinanases revealed some distinct differences in their apparent action patterns [137]. Our further recent studies on the action of these enzymes against linear arabino-oligosaccharides have shown that these observed differences result, at least in part, from variations in the substrate binding regions of these two enzymes. Table 17. Some Properties of endo-0 -^5)-a-L-Arabinanases from Different Sources A. aculeatus A. nidulans MW (Da) Optimum pH pl Activity towards: Linear (l->5)-a-L-arabinan Beet arabinan p-Nitrophenyl a-Larabinofiiranoside Products after prolonged incubation* Reference

A. niger N400

A. niger

45,000 5.5

40,000 5.5 3.25

43,000 4.6 3.0

42,500 4.8 2.9

++ + —

++ + —

++ + —

++ + —

A2 + A3

Note: ^A2 arabinobiose; A3 arabinotriose.

137

A2+A3

149

138, 149

172,173

50

BELDMANETAL.

with the A. niger enzyme having five binding subsites and the A. aculeatus enzyme six [174]. The extent and rate of branched arabinan hydrolysis by all ewrfo-arabinanases appears affected by the degree of substitution on the arabinan backbone by (l->3)- or (l->2)-a-L-arabinofuranosyl residues [19,30,117,137,173]. ^^C-NMR studies of hydrolysis products released from branched arabinan by the A. niger ^«(io-arabinanase [175] indicated that the smallest branched oligosaccharide released was a tetrasaccharide with an (1^3)-a-L"arabinofuranosyl residue linked to the central residue of (l-^5)~a-L-arabinotriose [i.e., 3^-a-L-arabinofuranosyl (l-»5)-a-L-arabinotriose]. This suggested a requirement for this enzyme of at least one unsubstituted a-L-arabinofuranosyl residue on either side of the substituted residue before hydrolysis can occur. Comparison of the hydrolysis products formed by action of each of the Aspergillus e«(io-arabinanases on branched arabinans gave similar distribution of branched oligomeric products [137], possibly indicating that the two ^«rfo-arabinanases have a similar tolerance for branched sites, although further work is needed to confirm this. Multifunctional Enzymes with Arabinanase Activity. A variety of multifunctional enzymes have been described with apparent arabinanase activity. Enzymes with both endo-xyldinase and arabinofuranosidase activity, the so-called "arabinose-releasing endo-xylanases", have been proposed to exist for many years [111,176-178]. In arecentstudy [179], ew^o-xylanase 1 from Fibrobacter succinogenes was found to first remove arabinofiiranosyl residues from arabinoxylan before acting in an endo manner on the remaining debranched xylan backbone. Several other enzymes have been reported with similar action, although the contamination of these enzymes by an arabinofuranosidase, or more likely an AXH, has long been suspected. As Coughlan [178] suggested, the question regarding the true existence of these enzymes could finally be answered if cloned enzymes are shown to exhibit the same combined activities. Enzymes with both P-D-xylopyranosidase and a-L-arabinofuranosidase activity have also been reported. Again, the purity of such enzymes has been questioned, although in contrast to

Arabinans and Arabinan Degrading Enzymes

51

the "arabinose-releasing e«(io-xylanases", firm evidence to support the existence of these enzymes has been reported in the form of multifunctional cloned enzymes [180-182]. The properties of some of these enzymes with high arabinofuranosidase activity are shown in Table 18. From this data it can be seen that these enzymes generally appear to have much higher affinity for p-nitrophenyl-P-D-xylopyranoside than /?-nitrophenyl-a-Larabinofuranoside, and thus are often considered as P-D-xylosidases with the catalytic flexibility to also hydrolyze a-L-arabinofuranosides [159,160,176]. Inhibition studies [184] have suggested that the bifunctional activity of these enzymes may reside in a single catalytic site, a proposal that some workers [180,182,184] have rationalized on the basis of the structural similarity of P-D-xylopyranosyl and a-L-arabinofuranoysl residues around CI, C2 and C3, similar to that discussed earlier to account for arabinopyranosidase activity seen with some galactosidases [145,185].

Table 18. Some Properties of Bifunctional P-D-Xylosidase/a-L-Arabinofuranosidases from Different Sources Clostridium Thermoanaerobacter T. reesei ethanolyticus stercorarium MW (Da) Optimum pH pl pNPA' K^{mM) ^cat ("^in ')

Radish

Spinach (Ara I)

64,000 4.5 4.7

118,000 4.8 4.2

212,000' 7.0

170,000*^ 5.0-5.2

100,000 4.0 4.7

17.6 8.85 X 10^

4.6 9.10 X 10^

>2.5

9.7 1.2 4.67 X 10^ 73.2

2.5 3.13 X 10^ 182

0.038 1.56 xlO"^ 181

0.08

0.95 3.56 X 10^ 184

pNPX^ ^m ^cat ("^in ' )

References

Notes: ^Composed of four subunits of 53,000 Da. ''Composed of two subunits of 85,000 Da. *^;7-Nitrophenyl-a-L-arabinofuranoside. p-Nitrophenyl-p-D-xylopyranoside.

183

166

52

BELDMANETAL

Classification ofArabinanases Based on Amino Acid Sequence Similarity, Traditionally, glycosyl hydrolases have been classified on the basis of substrate specificity and action patterns [186]. However, generally this classification gives little information regarding the structure and catalytic mechanisms of these enzymes, nor was it intended to. Therefore, to compliment this traditional classification and better reflect the structural features and evolutionary development of these enzymes, a classification system for glycosyl hydrolases based on amino acid sequence similarity has been recently devised [187—189]. Currently 60 glycosyl hydrolase families are recognized, although more families will undoubtedly arise as the number of known enzyme sequences increase. Members of those families to which enzymes with arabinanase activity have been classified are given in Table 19. This classification system has already proved useful in predicting the catalytic mechanisms of arabinanases [202], and is sure to become invaluable in identifying key residues and predicting active site structures when more detailed information is obtained on a few members of these families [203]. Catalytic Mechanisms ofArabinanases

Enzymic hydrolysis of glycosidic linkages can occur via two major mechanisms which result in either net retention, or inversion of anomeric configuration [204]. Both hydrolytic mechanisms involve general acid catalysis and require two critical residues: a proton donor and a nucleophile/base. Inverting enzymes operate by a single displacement reaction [205], involving protonation of the glycosidic oxygen, followed by nucleophilic attack on the anomeric carbon by water. With retaining enzymes a double displacement mechanism operates [205], with the hydrolysis proceeding through either a covalent glycosyl-enzyme intermediate, or an oxocarbonium ion intermediate stabilized electrostatically by enzyme carboxylate(s) [204]. It is now recognized that enzymes with similar substrate specificities and action patterns do not necessarily catalyze hydrolysis with the same stereochemical outcome. However, members of a given glycosyl hydrolase family do exhibit the

Arabinans and Arabinan Degrading Enzymes

53

Table 19. Classification of Arabinanases Based on Amino Acid Sequence Similarity [187-189] Accession Numbers Glycosyl Hydrolase Family 43

Source Aspergillus niger Bacillus polymyxa Bacillus pumilus Bacillus subtilis Bacillus subtilis Bacillus sp. K-17 Bacteriodes ovatus

Enzyme Endo-arabinanase {abnA) Xylanase D^ {xynD) p-Xyl {xynB) Endo-arabinanase {abnA) ORF (J3A) p-Xyl p-Xyl/Arafur {xsa) p-Xyl/Arafur ixylB) Arafur

Butyrivibrio flbrisolvens Butyrivibrio flbrisolvens Clostridium p-Xyf cellulolyticum Clostridium p-Xyl/Arafur stercorarium ixylA) Prevotella ruminicola p-Xyl {xynB) 51

54

Ax2if\xr A(abfA) Aspergillus niger Streptomyces lividans Arafur {abfA) Bacteriodes ovatus Arafur I (asdl) Bacteriodes ovatus Arafur II (asdll) Bacillus Arafur'^ stearothermophilus Bacillus subtilis Arafur (abfA) Arafur (asd) Bacillus subtilis

SWISS- EMBL/ PROT Genbank References P42256 L23430

190

P45796 X57094

191

P07129 X05793 Z75208

192 193

P42293 D31856 P49943 U04957

194 195 196

P45982 M55537

180

U55187 197 P48790 D13268

182

P48791 Z49241

198

P42254 L29005 P53627 U04630 U15178 U15179

199 144 141

Z75208 Z75208

193 193

Arafur B (abJB) P42255 X74777 Aspergillus niger Synechocystis sp. Arafur (abfB) D64004 Trichoderma koningii Arafur/Xyl {xyll) P48792 U38661 Trichoderma reesei Arafur (abfl) Z69252

200

Non-classified^ Pseudomonas fluorescens

AXH (xynC)

P23031

201 167

Notes: ^Corresponding genes are shown in parentheses. ^This enzyme was originally considered to be an e« Jo-xylanase [191], however, recent flirther analysis has indicated that is probably an AXH-type enzyme [169]. '^Classification based on partial (N-terminal) amino acid sequence. No similar sequences currently known to allow classification.

54

BELDMANETAL

same stereoselectivity [206,207], which is not surprising since they share a common fold and active-site topology [208—210]. Therefore, mechanistic data for glycosyl hydrolases can best be interpreted in terms of membership in these glycosyl hydrolase families. Glycosyl hydrolase family 43 contains enzymes catalyzing hydrolysis with inversion of anomeric configuration, as determined for the A. niger endo-amhinanasQ [202] and P-xylosidases from B. pumilus [211] and C cellulolyticum [197]. In contrast, members of glycosyl hydrolase families 51 and 54 act with retention of anomeric configuration, determined by following the stereochemistry of hydrolysis of the A. niger arafur A and arafur B, respectively [202]. The stereochemistry of hydrolysis of several other unsequenced arabinanases not currently classified into glycosyl hydrolase families are also known. All a-L-arabinofuranosidases examined to date act with retention of anomeric configuration, including those from A. aculeatus, A. awamori, Humicola insolens, P. capsulatum, B. subtilis [202], and also Monilinia fructigena [212] for which detailed mechanistic data have been determined [see ref 204]. As discussed earlier, the C. indicus P-L-arabinopyranosidase also acts with retention of configuration [115]. Like its A. niger counterpart, the A. aculeatus endo-arabinanase catalyzes hydrolysis with inversion of anomeric configuration [202]. However, the 5 . subtilis IFO 3134 ewJo-arabinanase (protopectinase-C) has apparent glycosyl transferase activity [213,214], which would suggest that this enzyme may act with retention of configuration [204], although a detailed examination has not been performed. New Substrates and Assays for Arabinanases

The last few years have seen considerable advancement in the understanding of arabinanase action and specifity, with a major reason for this being the recent availability of pure, welldefined arabinose-containing oligosaccharides for use as substrates for these enzymes. For example, purified (l->5)-a-L-

Arabinans and Arabinan Degrading Enzymes

55

arabino-oligosaccharides (up to arabinooctaose) are now commercially available [124], as are a number of aryl-a-L-arabinofuranosides [215]. Several recent studies have reported the isolation and structural characterization of a variety of arabinoxyIan-derived oligosaccharides [216-218], many of which have been used in combination with HPLC and NMR analysis to provide valuable information on arabinanase specificity [146,147,168]. Other useful substrates for arabinanase characterization have been recently synthesized, including arabinobiose derivatives with different linkages (i.e., methyl 0-a-L-arabinofuranosyl-(l-^2)-a-L-arabinofuranoside, methyl 0-a-L-arabinofuranosyl-(l->3)-a-L-arabinofuranoside, and methyl 0-a-L-arabinofuranosyl-(l->5)-a-L-arabinofuranoside) [219], and a branched arabinotriose derivative (methyl 3,5-di(9-arabinofuranosyl-a-L-arabinofuranoside) [220]. Finally, some recently developed arabinanase assays are worth mentioning. A specific endo-arabinansiSQ assay kit is now commercially available [124], employing a dyed and cross-linked substrate (Azurine-cross-linked linear arabinan). This substrate is reported to be very specific for e«Jo-arabinanase and can be used to detect activity in solution in the presence of large excesses of other pectic enzymes, including arabinofuranosidases, or to localize activity in electrophoresis gels [124]. Similarly, Yoshida et al. [221] have developed a sensitive method for detecting arabinofuranosidase activity in isoelectric focused gels based on the use of a colorimetric substrate, 6-bromo-2naphthyl-a-L-arabinofuranoside. Dunkel and Amado [222] have also recently developed a rapid, semiquantitative cujy-plate (diffusion) assay useful for detecting ew^io-arabinanase activity in column chromatography fractions based on the use of Procion Brilliant Red arabinan (discussed earlier) as substrate.

ACKNOWLEDGMENTS We would like to thank Dr. Gerhard A. de Ruiter for critically reading the manuscript, particularly the section dealing with NMR analysis, and Mrs. Helga Belling and Mrs. Gerda van Laar-Engelen for typing the manuscript. We would also like to thank Dr. Bemard Henrissat for kindly providing

56

BELDMANETAL

information relating to the classification of arabinanases by amino acid sequence similarity.

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STRUCTURAL ELUCIDATION OF THE N-LINKED OLIGOSACCHARIDES OF GLYCOPROTEINS USING HIGH pH ANION-EXCHANGE CHROMATOGRAPHY

Kevin D. Smith, Elizabeth F. Hounseil, John M. McGuire, Moira A. Elliott, and Heather G. Elliott

I. Introduction II. The Occurrence of A^-Glycosylation Sites

Advances in Macromolecular Carbohydrate Research Volume 1, pages 65-91. Copyright © 1997 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-323-2

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III. IV. V. VI. VII.

The Structure of A^-Glycosylation The Glycoform Concept The Chromatography of A^-Linked Chains High pH Anion-Exchange Chromatography The Diversity of HPAEC A. Monosaccharide Composition B. Sialic Acids C. Oligosaccharides VIII. Applications of HPAEC References

68 71 73 75 76 76 78 80 87 89

I. INTRODUCTION Protein glycosylation is a major modification which occurs during and after translation and is characterized by extensive structural heterogeneity. Functional studies have concluded that the oligosaccharide chains of glycoproteins are important determinants of overall biological activity through influencing both physiochemical (solubility, folding, conformation, stability, and protease resistance) and intermolecular (circulating half-life, immunogenicity, intracellular trafficking) properties [1]. Thus, the knowledge of the oligosaccharide structures of a glycoprotein, in addition to protein primary structure, is a prerequisite for the study of structure—function relationships. Oligosaccharide chains are ordered structures composed of various monomer units called monosaccharides (commonly Lfucose, D-mannose, D-galactose, A^-acetyl-D-galactosamine and A^-acetyl-D-glucosamine) and charged molecules (sialic acids) in a specific sequence. The latter are normally present as A^acetylneuraminic acid (Neu5Ac) and its partially acetylated forms, such as 4-0-acetyl-7V^-acetylneuraminic acid, which in turn may be substituted with lactyl, methyl, or phosphate groups [2], or as A^-glycolylneuraminic acid (NeuSGc) and its 0-acetylated derivatives. Oligosaccharide chains consist of the aforementioned monosaccharide residues linked by glycosidic bonds, from the CI hydroxyl group (C2 in the case of the sialic acids), in either a or p anomeric configuration, to any other hydroxyl group of the adjacent residue. A typical oligosaccharide chain

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contains from 2 to 20 residues arranged in a branched structure and covalently linked to the protein through the reducing end of one of the chain residues. Thus, the seven common monosaccharides found in the oligosaccharide chains of the mammalian cell surface may be assembled into many different structures by means of different linkages, by chain branching, and by the inclusion of substituents such as sulfate, phosphate, and acetyl groups. The oligosaccharide to protein linkages of glycoproteins occur either through the formation of an amide bond with asparagine (A^-glycosylation) or through a hydroxyl side chain of serine or threonine (0-glycosylation). Both A^- and O-glycosylation can occur on many different types of protein but the former predominate on plasma glycoproteins and the latter on glycoproteins of epithelial origin. This article will concentrate on the heterogeneity of A^-glycosylated structures between glycoproteins and review the use of high pH anion exchange chromatography (HPAEC) with pulsed electrochemical detection to detect these differences.

II. THE OCCURRENCE OF N-GLYCOSYLATION SITES The amino acid sequence of a glycoprotein can be used to predict the possible locations of A^-glycosylation sites: the presence of the consensus sequence asparagine-X-serine/threonine (Asn-XSer/Thr), where X is any amino acid except proline, normally indicates an A^-glycosylation site [3]. However, examples of A^glycosylation at asparagines outwith a consensus sequence exist in egg phosvitin [4], human von Willebrand factor [5] and the heavy chain of bovine protein C [6]. Earlier research [7] concluded that only one-third of sites possessing the consensus tripeptide were A/^-glycosylated. Komfeld and Kornfeld [8] provided an explanation stating the requirement for effective A^glycosylation of proteins as being "a sufficient pool of completely assembled and glycosylated lipid-linked oligosaccharide donor, an adequate activity of oligosaccharyltransferase, and a properly oriented and accessible Asn-X-Ser/Thr sequence in the acceptor". The critical point concerns the attainment of

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a favorable conformation for glycosylation to occur which has been predicted as being a p-turn [9,10] or loop structure. These are generally located at the surface of proteins, thus being accessible to glycosyltransferases. Additionally the p-turn or loop represents a spatial arrangement favoring the formation of a hydrogen bond between the amide group of asparagine and the oxygen from the hydroxyl group of serine or threonine in the consensus signal. This is viewed as critical [11] to TV-linked glycosylation, perhaps due to the mechanism of the biosyntheses enzymatic reactions. Proline cannot equal X in the tripeptide sequence (in Asn-X-Ser/Thr) since it cannot achieve the conformation necessary for the occurrence of the aforementioned hydrogen bonding. Bause et al., [11] reported the formation of a disulfide bond near or around a consensus sequence limits the adoption of the required information. Yet and Wold [12] indicated that A^-linked oligosaccharide chains may interact with aromatic but not charged amino acid side chains within the three amino acid residues on either side of the glycosylated asparagine. This explains the observation that chymotryptic cleavages at the carboxy terminals of leucine, tyrosine, phenylalanine, tryptophan, and histidine residues, but not tryptic cleavages at the carboxy terminals of arginine or lysine residues, within this region are inhibited.

III. THE STRUCTURE OF N-GLYCOSYLATION So far we have dealt with A^-glycosylation at the level presented in most biochemical textbooks; however this secondary modification is not as simplistic as it seems, The A/-linked oligosaccharide structures of the same glycoprotein are capable of exhibiting a high degree of subtle variation in structure. Individual chains can be heterogeneous in terms of the number of outer chain branches or antennae [normally mono- (1), bi- (2), tri- (3), or tetra- (4) antennary], monosaccharide composition and sequence, the intrachain linkages, and degree of sialylation. All A^-linked oligosaccharides have a common pentasaccharide

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core (Structure I) linked to asparagine, since they originate from a common precursor. a-Man-( 1 ->3)-|3-Man-( 1 -»4)-p-GlcN Ac-( 1 -^4)-p-GlcN Ac-( 1 ^ Asn 6

t

1 a-Man I The biosynthesis of A^-linked oligosaccharides is initiated by the transfer of a lipid-linked intermediate to the forming polypeptide in the endoplasmic reticulum (ER). Passage through the ER and the Golgi expose the immature chain to a variety of synthesizing and cleaving enzymes. This results in three distinct classes of TV-linked oligosaccharide chains which can be categorized according to their branch constituents: (a) mannose only (high mannose iV-glycans); (b) alternating 7V-acetylglucosamine and galactose residues terminated by sialic acid (a sialyllactosamine sequence), with the possibility of intrachain substitution with A^-acetylglucosamine or fucose or sialic acid (complex A^-glycans; Table 1); or (c) attributes of both high mannose and complex chains (hybrid A^-glycans). The heterogeneity of A^-linked oligosaccharide chains occurs in the monosaccharides linked to the a-(l->3)- and a-(l->6)linked mannose residues in the core region furthest from the asparagine. It is normally manifested as differences in the number of branches, the presence of peripheral sugars such as fucose, or the number of sialic acids, all of which affect the relative hydrophobicity and charge of the moiety. Complex and hybrid chains can exist as biantennary, triantennary, tetraantennary, and pentaantennary structures, and even in nonmammals, hexaantennary structures. The elongation of branch structures through substitutions of peripheral residues is most prominent in complex chains. In addition to those shown in Table 1, Kornfeld and Kornfeld [8] summarize the most common linkages as being a-Fuc-(l->2) or a-Gal-(l->3) or [a-Neu5Ac-(2->8)]„-aNeu5Ac-(2->3) [n = 8-12] to the terminal galactose residue on the lactosamine branch sequence; and a-Fuc-(1^3) or a-

70

SMITH ETAL

Table 1. A Generalized Structure for the N-Glycosidically Linked Chains of Glycoproteins a-Neu5 Ac-(2-^3/6).p-Gal-( 1 ->3/4)-p-GlcNAc-1 ^ 6 a-Neu5 Ac-(2-^3/6)-p-Gal-( 1 ^3/4)-p-GlcNAc-( 1 ^2)-a-Man-1

C

i ^ 6 p-Man-(1^4)-R 3 ^ a-Neu5 Ac-(2->3/6)-p-Gal-( 1 ^3/4)-p-GlcNAc-( 1 ^6)-a-Man-1

B

A

t a-Neu5 Ac-(2->3/6)-p-Gal-( 1 ^3/4)-p-GlcNAc-1 R = -p-GlcNAc-(l->4)-p-GlcNAc-(l^Asn A complex chain consisting of branches: (i) A and B is biantennary and capable of being de-, mono-, or bisialylated; (ii) A, B, and D is Type I triantennary and capable of being de-, mono-, bi-, or trisialylated; (iii) A, B, and C is Type II triantennary and capable of being de-, mono-, bi-, or trisialylated (iv) A, B, C, and D is tetraantennary and capable of being de-, mono-, bi-, tri-, or tetrasialylated

Neu5Ac-(2-^6) to the A^-acetylglucosamine on the lactosamine branch sequence. A well-known substitution in high mannose chains is the addition of one or more phosphate groups on the outer mannose residues (mannose-6-phosphate) which occurs in mammalian lysosomal hydrolases and allows binding to the mannose-6-phosphate receptor. Occasionally the sialyllactosamine branch structures of complex A^-linked glycoproteins are incompletely processed as illustrated by Montreuil [13] for ovotransferrin and ovomucoid and by Yamashita et al. [14] for ovalbumin. A large number of nonconforming A^-linked structures occur as a result of disease such as the absence of one A^-acetyglucosamine residue from the pentasaccharide core in

N-Linked Oligosaccharides

71

human myeloma IgM [15]; or agalactosylation (the absence of both sialic acid and galactose residues) on the outer antennae of IgG in diseases such as rheumatoid arthritis, Crohn's disease, and tuberculosis [16].

IV. THE GLYCOFORM CONCEPT A property common to A^-linked glycosylation is the presence of glycosylated variants (glycoforms) of a single protein which have identical amino acid sequences and thus identical glycosylation sites but different glycosylation patterns. This heterogeneity can be explained by the fact that different cell types have different complements of processing enzymes which act upon the precursors of oligosaccharide chains in different ways. These variations reflect both the source of the molecule (cell or tissue) and the particular physiological and biochemical conditions existing at the time of release [17]. In particular, the presence of disease may alter the structures of the oligosaccharide chains giving rise to novel glycoforms of the same glycoprotein. The glycosylation pattern of a disease specific glycoform can be completely different from the normal pattern, or variation may be confined to one particular site. Additionally the basis of heterogeneity could be the presence or absence of a single monosaccharide which would be the difference between a structure being antigenic or not (Table 2). It is not within the scope of this article to document every published instance of novel A^-linked structures in disease since many excellent articles are already in print [16-18]. We will confine our interest to bovine fetuin, a-1-acid glycoprotein (AGP), and epidermal growth factor receptor (EGFR), for which HPAEC analytical data will be presented later. Bovine fetuin is an a-globulin present in fetal calf serum at a high concentration which diminishes gradually during the first weeks of life. It is a single polypeptide which is 22% glycosylated, shared unevenly between three 0-linked [19,20] and three A^-linked sites [19,21]. The considerable heterogeneity of structure at each A^-linked glycosylation site [22] appears to be due to sialylated variants of three structures. The majority of

72

SMITH ETAL

Table 2. Common Oligosaccharide Antigens

A—B- —c

t f

D Antigen

A

B

E

c

Blood group a-GalNAc- |3-Gal-(l-> — A (l->3) — a-GalBlood group (3-Gal-(l-> B (1^3) — Blood group a-Fucp-Gal-(l-> H (l->2) — p-Gal-(l-^4) (3-GlcNAcLewis X

D

E

a-Fuc(l->2) a-Fuc(l->2) —

—

—

Sialyl Lewis X Lewis A

a-NeuAc- p-Gal-(l->4) p-GlcNAc(2^3) (1^ — p-Gal-(l-^3) p-GlcNAc-

—

Sialyl Lewis A

a-NeuAc- p-Gal-(l->3) p-GlcNAc(2^3) (1^

—

—

— — a-Fuc(1^3) a-Fuc(1^3) a-Fuc(l->4) a-Fuc-

the A^-linked chains are triantennary with P-Gal-(l->4)-PGlcNAc-( 1^2/4) lactosamine antennae [23] with a type I branching pattern (Table 1); i.e., one antenna on the a-Man(l->6) and two on the a-Man-(l-^3) linked residues. Other minor populations consist of biantennary chains with Gaipi-4GlcNAc (lactosamine) sequences and triantennary chains with p-Gal-(1^3)-p-GlcNAc-(l->2/4) (lactosamine) sequences. Sialylated variants of the biantennary and the two triantennary structures have resulted in the identification of approximately 23 oligosaccharide chains in bovine fetuin. This glycoprotein therefore provides an excellent standard for HPAEC. AGP (orosomucoid) is a major serum glycoprotein that is classified as one of the positive acute phase reactants since its plasma concentration becomes elevated two- to fivefold in some disease states [24]. Human AGP is a tightly folded, monomeric

N'Linked Oligosaccharides

73

polypeptide, glycosylated with five asparaginyl-linked complex oligosaccharide chains accounting for approximately 45% of the molecular weight, of which 11% is sialic acid [25]. In certain diseases, both the total concentration of the glycoprotein and the relative proportions of the glycoform variants are altered markedly [17,26]. In cancer and other acute phase inflammatory conditions there is an increase in the proportion of glycoforms with biantennary chains, while in pregnancy, liver damage, and chronic inflammatory disorders such as rheumatoid arthritis the proportion of the biantennary glycoforms is decreased [17,26]. Recently, it has been reported that the absolute amount of sialyl Lewis X (SLX) determinants (Table 2) substituted AGP molecules and the number of SLX per molecule were enhanced under various acute inflammatory conditions and in patients with rheumatoid arthritis [27]. Epidermal growth factor receptor (EGFR) is the membrane bound mediator of the effects of EOF. The mature cell surface receptor, molecular weight 175 kDa and 20% glycosylation [28], is overproduced by cancer cells such as the A431 human epithelial cell line. The latter system also produces a soluble 115kDa protein with a sequence corresponding to the extracellular domain of the membrane receptor [29]. EGFR is one of the few membrane receptors in which glycosylation is an essential requirement for the interaction with its ligand [30]. Within the glycosylated population of EGFR, possession of the terminal blood group A specific GalNAc residue correlates with a low affinity for EGF [31].

V. THE CHROMATOGRAPHY OF N-LINKED CHAINS A complete structural characterization of individual A^-linked chains, in terms of its oligosaccharide structures, requires the isolation of each oligosaccharide chain together with the determination of the composition and anomeric nature of the constituent monosaccharides and their sequence and linkage positions. This would normally require significant (microgram) quantities of each oligosaccharide which may not be practically feasible. The heterogeneity of the oligosaccharides of glyco-

74

SMITH ETAL

proteins requires that chromatography be an essential part of any structural elucidation procedure. Several chromatographic methods are used for the analysis of the released oligosaccharides [32-34], which together with specific enzyme digestion greatly extend the amount of structural information capable of being elucidated. Classically this has been carried out for A^-linked chains of glycopeptides by Bio-Gel P4 chromatography and stepwise exoglycosidase digestion [22,35-37]. The susceptibility of an oligosaccharide to purified exoglycosidases (specific for anomeric configuration and nonreducing terminal sugar residue) is indicated by changes in the GPC elution profile. This technique is very time-consuming, only separates according to size, and problems may arise with the purity and specificity of the glycosidases. However, the recent introduction of an automated version of this technique results [38] in the ability to sequence picomole amounts of oligosaccharide structures. HPLC modes such as gel permeation, metal (lithium, barium or potassium) loaded cation-exchange, normal (aminopropyl bonded silica) phase, and reverse (octadecylsilica) phase have all been utilized extensively. However the optimum resolution of oligosaccharides is difficult to achieve. The differing chromatographic behavior of oligosaccharides on reverse-phase and amino-bonded (or amino-modified) columns allows a combination of these two systems which can often separate oligosaccharides not resolved by one system alone [39,40]. Reverse phase chromatography has also proved useful for the separation of oligosaccharides, for example as the pyridylamino or benzoyl derivatives [41—43]. The utilization of a two-column HPLC technique provides a 2D map of oligosaccharide which can be interpreted analytically [44-47], particularly when pyridylamino derivatives are used [48—51]. The highly sensitive fluorescence detection of pyridylamino derivatives is counterbalanced by the loss of sialic acid from sialylated oligosaccharides; however the use of l-(p-methoxy)phenyl-3-methyl-5-pyrazolone (PMPMP) in labeling reactions has been shown not to cause desialylation [52]. Recently, a porous graphitized column support has been shown not only to exhibit similar properties to reverse phase

N-Linked Oligosaccharides

75

but also to separate closely related oligosaccharide isomers [53,54]. Anion exchange chromatography using alkali-susceptible silicabased columns can also be used for separation at acidic pH. For example, the separation of bi-, tri- and tetra-sialyl A^-linked oligosaccharides can be achieved on columns of AX-5 ion-exchange HPLC resin with an increasing potassium phosphate gradient [55]. Additional separations of isomeric oligosaccharides which differ only in the linkage position of the terminal Nacetylneuraminic acids, e.g., a-Neu5Ac-(2^3/6) residues linked to P-Gal-(l->4)-p-Glc [35], are achieved on these columns and amine-bonded phases such as aminopropylsilica when eluted in the presence of acetonitrile. Ion suppression amine absorption chromatography (ISAA-HPLC) introduced by Baenziger's group [39,40] also resolves oligosaccharide isomers which differ only in NeuAc linkage. ISAA-HPLG utilizes amine-bearing bonded phase columns from which oligosaccharides are eluted with glacial acetic acid/acetonitrile titrated to pH 5.5 with triethylamine. The technique overcomes the main disadvantage of normal ionexchange chromatography in that size as well as net charge contribute to the separation. Earlier work by Green and colleagues [58] demonstrated that the addition of borate to the eluent in the analysis of neutral sugars resulted in the formation of anionic oligosaccharide—borate complexes and subsequent separation by anion-exchange chromatography.

VI. HIGH pH ANION-EXCHANGE CHROMATOGRAPHY One of the most significant developments in the chromatogaphy of A/'-linked oligosaccharides has been the emergence of anionexchange separation using quaternary-ammonium-bonded pellicular resins (CarboPAc PA-1 or PA-100) at high pH elution. The use of strong alkali exploits the weakly acidic property of the hydroxyl groups of monosaccharides (and thus oligosaccharides) at pH>12. The resulting oxyanions can be chromatographed as anions, without pre-derivatization or the use of additives in the mobile phase, on the basis of molecular size, monosac-

76

SMITH ETAL

charide composition, and linkage of monosaccharide residues [59-61]. Excellent separations of the oxyanion derivatives of isomers and structurally closely related neutral and acidic oligosaccharides have been achieved [59-69]. The spherical pellicular resin contributes towards the high resolution of the technique by locating all the ionic groups on the surface resulting in the elimination of diffusion [60]. The technique is further enhanced by using pulsed amperometric detection (PAD), to give additional potential for 2D oligosaccharide mapping with sensitivity in the picomole range [70]. It utilizes the principle of triple amperometry to overcome the problem of electrode contamination by the oxidized product of carbohydrates associated with normal amperometry [71]. For many years the major disadvantage of HPAEC was the inability to further analyze eluted fractions due to the high salt content of the eluent. This problem has been largely overcome by the introduction of suppressor systems.

VII. THE DIVERSITY OF HPAEC HPAEC has the capacity to supplement and extend the existing techniques of structural elucidation of A^-linked oligosaccharides, particularly with regard to the rapid analysis of the monosaccharide, sialic acid, and oligosaccharide contents of small amounts of A^-linked glycoproteins without structural derivatization. A. Monosaccharide Composition

The ability to determine the monosaccharide composition for an individual glycoprotein is extremely important. Apart from verifying that the protein is glycosylated, it can indicate the type of glycosylation present and the identity and quantity of each monosaccharide present. The presence of A^-acetylgalactosamine usually indicates (9-glycosylation; while significant amounts of mannose are indicative of A^-glycosylation. The technique is useful as a mechanism to monitor the qualitative and quantitative changes in glycosylation which may occur during

N-Linked Oligosaccharides

77

disease, or to establish the consistency of the product after the recombinant production of a glycoprotein. The most cost-effective method for obtaining monosaccharides from intact oligosaccharide structures is by acid hydrolysis; for example incubation with 2 M trifluoroacetic acid for 4 h at 100 °C [71]. These conditions are optimal for the cleavage of all intrachain linkages in oligosaccharides and, with the exception of NeuSAc (which is destroyed), liberate the monosaccharide components in quantitative amounts [71]. Although direct analysis of the hydrolysate is possible, better profiles are obtained after ion-exchange chromatographic purification to remove the peptide and amino acid components of the digest. Monosaccharide compositional analysis using HPAEC is a rapid and economical method with the generation of profiles from one microgram of glycoprotein in less than 15 minutes [72]. The mobile phase of 160 mM sodium hydroxide ensures that the monosaccharides are present as their oxyanions and the order of elution is then correlated with their individual pK^ values. Additionally, since each of the hydroxyl groups in an individual monosaccharide has a slightly different pK^ value, the loss of a specific hydroxyl group, for instance in the formation of a glycosidic bond, alters the elution position of a monosaccharide by changing its pK^ value. Thus, a mixture of galactose, 2-deoxygalactose, and 6-deoxygalactose (fucose) can be separated to baseline resolution (Figure 1). The advantages of HPAEC for monosaccharide composition, namely its rapidity and reproducibility with no requirement for sample derivatization, has resulted in the examination of glycan chains derived from a wide variety of glycoproteins [73 and references therein]. The compositional profiles for standard monosaccharides and the recombinantly produced extracellular domain of EGFR are illustrated (Figure 2). In the standard profile the order of elution is largely correlated to the pK^ value although the positions of galactose and 7V-acetyl-D-glucosamine are interconvertable with concentration [60]. The amounts of A^-acetyl-D-glucosamine, mannose, and galactose in the oligosaccharide chains of EGFR are consistent with the presence of complex bi- or triantennary

78

SMITH ETAL

50 H

I/)

LL

>

E

c

Q UJ Q_

Figure 1. The HPAEC separation of (1) fucose, (2) 2-deoxygalactose, and (3) galactose by HPAEC. The chromatography was carried out as described in Smith et al. [75].

A^-linked structures (Table 3). The presence of fucose could signify the appearance of an antigenic determinant on the outer chain. Fucose (a-Fuc) and A'^-acetyl-D-galactosamine are present in amounts which suggest that one-third of the oligosaccharide structures could contain blood group A (terminal a-Fuc and aGalNAc) or H (terminal a-Fuc) antigens. B. Sialic Acids

The sialic acids are a family of nine carbon carboxylated sugars which confer a formal negative charge on certain classes— hybrid and complex—of A^-linked chains through terminating the branch antennae (see Table 1). This results in sialic acids, or any oligosaccharide chain containing them, binding more strongly to anion-exchange resins. Thus, in comparison to neutral and amino sugars, stronger elution conditions are required to elute the retained charged molecules from the column. This is achieved by the application of an increasing linear gradient of sodium acetate (50-250 mM sodium acetate over 30 min) in addition to the constant alkaline pH conferred by 100 mM

N-Linked Oligosaccharides

79

GalNAc

100-lA

>

I 50C

o

Q. tf) Q» OH

a

IXJ Q-

10

I

15

100-

> E

en a lU CL

50 H

Fuc

Gal GalNAcyv

Man

10

15

Figure 2. The HPAEC separation of (A) 50 ng each of fucose (Fuc), N-acetylgalactosamine (GalNAc), galactose (Gal), N-acetylglucosamine (GlcNAc) and mannose (Man); (B) the monosaccharides hydrolyzed from 2 jj,g of rEGFR. The chromatography was carried out as described in Smith et al. [75].

80

SMITH ETAL

Table 3. The Monosaccharide Composition of rEGFR as Determined by HPAEC (Figure 2) Monosaccharide

Amount per 2 [ig rEGFR

Fucose A^-Acetylgalactosamine Galactose A^-Acetylglucosamine Maimose

14.4 ng 12.1 ng 67.8 ng 236.6 ng 131.7 ng

Amount per mol rEGFR 4.8 mol 3.0 mol 20.7 mol 58.8 mol 40.2 mol

sodium hydroxide. The sialic acid content of a glycoprotein can be determined after hydrolysis either by enzyme (neuraminidase, 10 mU, 37 °C, 1 h) or acid (0.1% hydrochloric acid, 70 °C, 1 h). HPAEC is capable of separating the two major sialic acids, A^-glycolyl- and A/'-acetylneuraminic acid, by more than 10 minutes on the sodium acetate gradient despite only differing in structure by one extra hydroxyl group (Figure 3A). Unfortunately, this gradient is unable to separate the individual O-acetylated forms (reviewed in ref. 74) of each sialic acid. Aliquots (45 pmol) of the recombinant form of EGFR was desialylated using both enzyme digestion (Figure 3B) and acid hydrolysis (Figure 3C). The protein was found by resorcinol staining to contain the equivalent amount of sialic acid to 485 pmole of standard iV-acetylneuraminic acid, which is consistent with the presence of bi- or trisialylated chains on each of the 11 potential A^-glycosylation sites. However the HPAEC profiles show that the time of the released product did not correlate with the retention of the standards. This may represent a five-deaminated derivative of sialic acid (H rather than NHCOCH3 at C5 of Neu5Ac) resulting from the cell type used in the recombinant production. However this type of modification has not been previously reported. C.

Oligosaccharides

So far, HPAEC has been discussed in the context of determining the monosaccharide and sialic acid compositional analysis of glycoproteins and/or individual oligosaccharide chains.

NeuSGc

mm

100 n (/) > E

Si

c o

Q. V)

50H

Q UJ Q.

:L 10

20 min

30

40

Figures. HPAECprofilesof(A)N-acetylneuraminicacid (NeuSAc)and N-glycolylneuraminic acid (NeuSGc); (B) sialic acid from rEGFR released by neuraminidase; (C) sialic acid from rEGFR released by HCI hydrolysis. The chromatography was carried out as described in Smith etal. [75]. 81

82

SMITH ETAL.

However the diversity of the technique extends to the qualitative determination of the iV-linked oligosaccharides present on a glycoprotein as a whole or at a particular glycosylation site. It is particularly useful in the resolution of complex sialylated structures. The TV-linked structures are released enzymically using peptide: A^-glycosidase F [PNGase F, peptide-N'^-Cacetyl-P-glucosaminyl)asparagine amidase] which catalyses the hydrolysis of the A^,jV'-diacetylchitobiose bond adjacent to the asparagine residues of all A^-linked oligosaccharides [71]. The reaction mechanism differs from that of endo-glycosidasQS D, H, and F. These enzymes cleave the glycosidic linkage between the two GlcNAc residues. The released oligosaccharides, each bearing a reducing terminal p-GlcNAc residue, are resolved by HPAEC using an acetate gradient (50-200 mM IM sodium acetate in 40 min in the presence of 100 mM sodium hydroxide) similar to that used for sialic acids [72,75]. An HPAEC profile for the A^-linked oligosaccharides released from bovine fetuin by PNGase F gives a complex pattern (Figure 4). The initial separation is in terms of the number of sialic 100

> E g o

triantcmary 2|

50-

Q.

Q UJ £L

10

20

30 min

1^ AO

50

60

1 70

Figure 4. HPAEC profile of 200 pmol of the N-linked oligosaccharides released from bovine fetuin using PNGase F. The chromatography was carried out as described in Smith et al. [75].

N-L inked Oligosaccharides

83

acid residues per chain and thus the degree to which an individual structure is retained on the column. Thus fetuin can be shown to contain bisialylated, trisialylated, and tetrasialylated structures. The presence of fucosylation causes an oligosaccharide to elute earlier than its non-fucosylated counterpart [67,68,76]. Thereafter, separation occurs within each charge band on the basis of size (a bisialylated, biantennary chain will be retained to a lesser extent than a bisialylated, triantennary chain) and, for similarly sized structures, in terms of isomeric differences normally differing intrachain linkages. The involvement of one or more hydroxyl groups of a monosaccharide in glycosidic bonds to other monosaccharides will alter its ionization. Thus two oligosaccharides which have identical monosaccharide sequences but different glycosidic linkage positions can be separated. This effect can be illustrated by reference to Figure 4 and structures II-IV. Structures II and IV are separated due to differing linkages between the Gal and GlcNAc in the lactosamine branches [63], i.e. -P-Gal-(l->3)-p-GlcNAc-(l-^4) (structure IV) elutes after -P-Gal-(l-^4)-P-GlcNAc-(l->4) (structure II). A similar effect occurs with the linkages between sialic acids [65]; the greater the proportion of (2-^3) to (2->6) linkages the longer the elution time; structure II is a trisialylated chain which contains two a-Neu5Ac-(2->6) and one a-Neu5Ac-(2-^3) linkages while

a-Neu5Ac-(2->6)-p-Gal-(l-»4)-p-GlcNAc-(l->2)-a-Man-l

i 6 p-Man-(l->4)-R 3

t

a-Neu5Ac-(2->6)-p-Gal-(1^4)-P-GlcNAc-(l->6)-a-Man-l 4

t

a-Neu5Ac-(2->3)-P-Gal-(l-^4).p-GlcNAc-l II

84

SMITH ETAL.

a-Neu5Ac-(2->3)-P-Gal-(l->4)-P-GlcNAc-(l->2)-a-Man-l

i 6 P-Man-(l-»4)-R 3

t a-Neu5 Ac-(2-»6)-p-Gal-( 1 ■^4)-p-GlcNAc-( 1 -»6)-a-Man-1 4

t

a-Neu5 Ac-(2-^3)-P-Gal-( 1 -M)-p-GlcNAc-1 III a-Neu5Ac-(2->3)-p-Gal-(l-»4)-p-GlcNAc-(l-^2)-a-Man-l

i 6 P-Man-(l->4)-R 3

t

a-Neu5Ac-(2-^6)-p-Gal-(l->4)-P-GlcNAc-(1^6)-a-Man-l 4

t

a-Neu5Ac-(2->3)-p-Gal-(l-^3)-p-GlcNAc-l IV R = -P-GlcNAc-(1^4)-p-GlcNAc-(l-).Asn

Structure III contains the reverse. Reduction of the released iV-linked oligosaccharides by alkaline borohydride treatment [71] decreases the retention times in the profile (Figure 5) without any major decrease in response or resolution [77]. PNGase F treatment of rEGFR and HPAEC analysis of the released oligosaccharides (Figure 6) gave one major peak at approximately 35 minutes in the region corresponding to a relatively slow eluting biantennary chain, perhaps as result of trisialyla-

N-L inked Oligosaccharides

85 Reduced triantennary

Figure 5. HPAEC profile of 1 nmol of the N-linked oligosaccharides released from bovine fetuin and then reduced to alditols by alkaline borohydride treatment [71 ]. The chromatography was carried out on a Dionex-500 HPAEC system using the gradient of McGuire et al. [77].

100-1

Figure 6. HPAEC profile of the N-linked oligosaccharides released from 45 pmol of rEGFR The chromatography was carried out as described in Smith et al. [75].

86

SMITH ETAL

loo-

se-

1

1

10

20

1 30 min

1

40

1 50

1

60

1 70

Figure 7. HPAEC profile of the N-linked oligosaccharides released from rEGFR after fucosidase treatment. The chromatography was carried out as described in Smith et al. [75].

tion, or a fast eluting trisialylated chain. Treatment with a fucosidase which removes inner core a-Fuc-(l-^6) linked residues (Structure V), prior to PNGase F treatment, significantly increased the elution time of the peak (Figure 7) to a time compatible with a non-fucosylated triantennary structure.

a-Man-(l->3)-P-Man-(l->4)-p-GlcNAc-(l->4)-|3-GlcNAc-(l-^Asn 6 6

t

1 p-GlcNAc-( 1 ->6)-a-Man 4

t

1 p-GlcNAc

t

1 a-Fuc

N-Linked Oligosaccharides

87

Vni. APPLICATIONS OF HPAEC The ability to generate an oligosaccharide profile for a specific glycoprotein provides a sensitive diagnostic mechanism to monitor changes in glycosylation between the normal molecule and those generated under abnormal (disease) conditions or in a different system, e.g. recombinant production. As mentioned earlier, the glycosylation pattern at each of the five glycosylation sites of a-1-acid glycoprotein (AGP) is known to alter. In certain diseases abnormally glycosylated variants (glycoforms) which may be functionally significant, are expressed. We have compared the glycosylation patterns of AGP from patients with contrasting degrees of rheumatoid arthritis using high pH anion exchange chromatography (Figure 8) after deglycosylation of the glycoprotein (Table 4). The conclusion from a limited study is that the glycosylation pattern of AGP in rheumatoid arthritis can be directly correlated with the disease activity. Results indicate that a stepwise increase in the mean grade of disease activity (II->III->IV) is correlated with an increase in the biantennary portion of total AGP and a significant reduction in the content of tri- and tetraantennary chains. This type of pattern has previously only been reported in acute inflammatory conditions [79]; normally patients with chronic symptoms have dis-

Table 4. The Classification of the Patients Used to Study Alterations in the Glycosylation Patterns of AGP during Rheumatoid Arthritis (Figure 8)

Patient 1 2 3 Abbreviations: Hb ESR MGDA

Disease Duration (years)

ESR (mm/h)

Hb (g/dl)

MGDA [78]

16 14 2

20 50 106

12.1 13.1 10.1

II III IV

Hemoglobin level Erythrocyte sedimentation rate Mean grade of disease activity

88

SMITH ETAL

PATIENT 1

100 H

triantennary

> E

I

tetraantennary

Q UJ

a.

OH 20

25

30

n

35

40

\

1

1

45

50

55

45

50

55

45

50

55

100 H PATIENT 2

>

Q

ID

OH 15

20

1^

25

I

30

35

"1— 40

1 ^

I

PATIENT 3

100-

20

25

-T 30

r. 35 mm

40

Figure 8. HPAEC profile of the N-linked oligosaccharides released from AGP isolated from rheumatoid human plasma of patients with varying disease activity. The chromatography was carried out on a Dionex-300 HPAEC system using the gradient of Smith et al. [75].

N-L inked Oligosaccharides

89

played an increased expression of tri- and tetraantennary chains to the detriment of biantennary structures. REFERENCES [1] [2] [3] [4] [5]

Varki, A., Glycobiology, 3 (1993) 97-130. Schauer, R., Adv. Carbohydr. Chem. Biochem., 40 (1982) 131-234. Gavel, Y. and von Heijne, G., Prot. Eng., 3 (1990) 433-443. Marshall, R.D., Ann. Rev. Biochem., 41 (1971) 673-702. Titani, K., Kumar, S., Takio, K., Ericsson, L.H., Wade, R.D., Ashida, K., Walsh, K.A., Chopek, M.W., Sadier, J.E. and Fujikawa, K., Biochemistry, 25 (1986) 3171-3184. [6] Stenflo, J. and Femlund, J., J. Biol. Chem., 257 (1982) 12180-12190. [7] Kronquist, K.E. and Lennarz, W.J., J. Supramol. Struct, 8 (1978) 51-55. [8] Komfeld, R. and Komfeld, S., Ann. Rev. Biochem., 54 (1985) 631-664. [9] Marshall, R.D., In Smellie, R.M.S. and Beeley, J.G. (eds.). The Metabolism and Function of Glycoproteins, Biochemical Symposia No. 40, The Biochem. Soc, London (1974) 17-26. [10] Avanov, A. Ya., Mol. Biol., 25 (1991) 237-249. [11] Bause, E., Biochem. J., 209 (1983) 331-336. [12] Yet, M.G. and Wold, R, Arch. Biochem. Biophys., 278 (1990) 356-364. [13] Montreuil, J., Adv. Carbohdr. Chem. Biochem., 37 (1980) 157-223. [14] Yamashita, K., Tachibana, Y. and Kobata, A., J. Biol. Chem., 253 (1978) 3862-3869. [15] Miller, R, Immunochemistry, 9 (1972) 217-228. [ 16] Rademacher, T.W, Parekh, R.B. andDwek, R.A., Ann. Rev. Biochem 57 (1988) 785-838. [17] van Dijk, W., Turner, G.A. and Mackiewicz, A., Glycosylation and Disease, 1 (1994)5-14. [18] Lis, L. and Sharon, N., Eur. J. Biochem., 218 (1993) 1-27. [19] Begbie, R., Biochim. Biophys. Acta., 371 (1974) 549-567. [20] Spiro, R.G. and Bhoyroo, V.D., J. Biol. Chem., 249 (1974) 5704^5717. [21] Spiro, R.G., Adv. Protein Eng., 27 (1973) 349-367. [22] Takasaki, S. and Kobata, A., Biochemistry, 25 (1986) 5709-5715. [23] Baenziger, J.U. and Fiete, D., J. Biol. Chem., 254 (1979) 789-795. [24] Baumann, H. and Gauldie, J., Mol. Biol. Med., 7 (1990) 147-159. [25] Schmid, K., Kaufmann, H., Isemura, S., Bauer, R, Emura, J., Motoyama, Ishiguro M.T. andNanno, S., Biochemistry, 12 (1973) 2711-2722. [26] Turner, G.A., Clin. Chim. Acta., 208 (1992) 149-171. [27] De Graaf, T.W, Van der Stelt, M.E., Anbergen, M.G. and van Dijk, W, J. Exp. Med., 177(1993)657-666. [28] Mayes, E.L.V. and Waterfield, M.D., EMBO. J., 3 (1984) 531-537. [29] Soderquist, A.M., Stoscheck, C. and Carpenter, G., J. Cell. Physiol. 136 (1988) 447-454.

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[30] Slieker, L.J. and Lane, M.D., J. Biol. Chem., 260 (1985) 687-690. [31] Defize, L.H.K., Amdt-Javin, D.J., Jovin, T.M., Boonstra, J., Meisenhelder, J., Hunter, T., de Hey, H.T. and de Laat, S.W., J. Cell. Biol., 107 (1988) 939-949. [32] Honda, S., Anal. Biochem., 140 (1984) 1^7. [33] Hicks, K.S., Adv. Carbohydr. Chem. Biochem., 46 (1988) 17-72. [34] Hounsell, E.F., In Lim, C.K., (ed.), HPLC of Small Molecules, IRL Press, Oxford, (1986) 49-^8. [35] Yamashita, K., Mizouchi, T. and Kobata, A., Meth. EnzymoL, 83 (1982) 105-126. [36] Sutton, C.W., O'Neill, J.A. and Cottrell, J.S., Anal. Biochem., 218 (1994) 3^M6. [37] Pfeiffer, G., Schmidt, M., Strobe, K-H. and Geyer, R., Eur. J. Biochem., 186 (1989)273-286. [38] Edge, C.J., Rademacher, T.W., Wormald M.R., Parekh, R.B., Butters, T.D., Wing, D.R. andDwek, R.A., Proc. Natl. Acad. Sci. USA, 89 (1992) 6338-6342. [39] Hounsell, E.F., Jones, N.J. and Stoll, M.S., Biochem. Soc. Trans., 15 (1985) 1061-1064. [40] Hounsell, E.F., Lawson, A.M., Feeney, J., Gooi, H.C., Pickering, N.J., Stoll, M.S., Lui, S.C. and Feizi, T., Eur, J. Biochem., 148 (1985) 367-377. [41] Honda, S., Akao, E., Suzuki, S., Okuda, M., Kakehi, K. andNakamura, J., Anal. Biochem., 180 (1989) 351-357. [42] Daniel, RF., de Feudis, D.F., Lott, LT. and McCleu, R.H., Carbohydr. Res. 97 (1981)161-189. [43] Webb, J.W, Jiang, K., Gillece-Castro, B.L., Tarentino, A.L., Plummer, T.H., Byrd, J.C, Fisher, S.J. and Burlingame, A.L., Anal. Biochem., 163 (1988) 337-349. [44] Bendiak, B. and Gumming, D.A., Carbohydr. Res., 151 (1986) 89-103. [45] Arbatsky, N., Martynova, M.D., Zhelrova, A.O., Derevitskaya, V.A. and Kochetkov, N.K., Carbohydr. Res., 187 (1989) 165-171. [46] Bendiak, B., Harris-Brandts, M., Michnik, S.W., Carver, J.P. and Gumming, D.A., Biochemistry, 28 (1989) 6491-6499. [47] Kakehi, K. and Honda, S., J. Chromatog., 379 (1986) 27-55. [48] Tomiya, N., Awaya, J., Kurono, M., Endo, S., Arata, Y. and Takahashi, N., Anal. Biochem., (1988) 171, 73-90. [49] Oku, H., Hase, S. and Ikenaka, T., Anal. Biochem., 185 (1990) 331-334. [50] Hase, S. and Ikenaka, T., Anal. Biochem., 184 (1990) 135-138. [51] Tomiya, N., Lee, YC., Yoshida, T., Wada, Y, Awaya, J., Kurono, M. and Takahashi, N., Anal. Biochem., 193 (1991) 90-100. [52] Kakehi, K., Suzuki, S., Honda, S. and Lee, Y C , Anal. Biochem., 199 (1991) 256-268. [53] Davies, M., Smith, K.D., Harbin, A-M. and Hounsell, E.F., J. Chromatog., 609 (1992)125-131. [54] Davies, M., Smith, K.D., Harbin, A-M., Carruthers, R.A., Chai, W., Lawson and Hounsell, E.F., J. Chromatog., 646 (1993) 317-326. [55] Baenziger, J.U. and Notowicz, M., Anal. Biochem., 112 (1981) 357-361.

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[56] Green, E.D. and Baenziger, J.U., Anal. Biochem., 158 (1986) 42-^9. [57] Green, E.D., Adelt, G., Baenziger, J.U., Wilson, S. and van Halbeek, H., J. Biol. Chem., 263 (1988) 18253-18268. [58] Green, E.D., Baenziger, J.U. and Boime, I., J. Biol. Chem., 260 (1985) 1563115638. [59] Spellman, M.W., Anal. Chem., 62 (1990) 1714-1722. [60] Lee, Y.C., Anal. Biochem., 189 (1990) 151-162. [61] Hardy, M.R., Meth. Enzymol., 179 (1989) 76-82. [62] Hardy, M.R. and Townsend, R.R., Meth. Enzymol, 230 (1994) 208-225. [63] Hardy, M.R. and Townsend, R.R., Proc. Natl. Acad. Sci. USA, 85 (1988) 3289-3293. [64] Hardy, M.R. and Townsend, R.R., Carbohydr. Res., 188 (1989) 1-7. [65] Townsend, R.R., Hardy, M.R., Gumming, D.A., Carver, J.R and Bendiak, B., Anal. Biochem., 182 (1989) 1-8. [66] Townsend, R.R., Hardy, M.R. and Lee, Y.C., Meth. Enzymol., 179 (1989) 65-76. [67] Pfeiffer, G., Geyer, H., Kalsner, L, Wendorf, R and Geyer, H., Biomed. Chromatog., 4, (1990) 193-199. [68] Basa, L.J. and Spelhnan, M.W., J. Chromatog., 499 (1990) 205-220. [69] McGuire, J.M., Douglas, M. and Smith, K.D., Carbohydr. Res., 292 (1996) 1-9. [70] Hermentin, R, Witzel, R., Doenger, R., Bauer, R., Haupt, H., Patel, T, Parekh, R.B and Brazel, D., Anal. Biochem., 206 (1992) 419-429. [71] Davies, M.J., Smith, K.D. and Hounsell, E.F., Meth. Molec. Biol., 32 (1993) 129-141. [72] Smith, K.D., Davies, M.J., and Hounsell, E.R,: Meth. Molec. Biol. 32 (1993) 143-155. [73] Hardy, M.R. and Townsend, R.R., Glycobiology, 1 (1991) 139-147. [74] Varki, A., Glycobiology, 2 (1992) 25-40. [75] Smith, K.D., EUiott, M.A., Elliott, H.G., McLaugWin, CM., Wightman, P and Wood, G.C., J. Chromatog. Biomed. AppL, 661 (1994) 7-14. [76] Anumula, K.R. and Taylor, PB., Eur. J. Biochem., 195 (1989) 269-280. [77] McGuire, J.M., Elliott, M.A., Elliott, H.G. and Smith K.D., Carbohydr. Res., 270(1995)63-69. [78] Mallya, R.K. and Mace, B.E.W., Rheumatol, and Rehabil, 20 (1981) 14-17. [79] Raynes, J., Biomedicine, 36 (1982) 77-86.

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NEOGLYCOCONJUGATES AS ARTIFICIAL ANTIGENS: CHEMICAL ASPECTS

Nikolay K. Kochetkov and Anatoly Ya. Chernyak

I. Introduction 94 II. The Principles of Design and Architecture of Glycoconjugates 95 III. Glycoconjugates with Oligosaccharide as a Carbohydrate Moiety 98 A. Carbohydrate Carriers of Specificity: Their Preparation . . 98 B. Conjugation to a Polymer Carrier 106 C. Preparation of Oligosaccharide Derivatives Suitable for Conjugation 116 D. Discussion 130 E. Polymerization as a Method for Preparing Glycoconjugates with a Synthetic Polymer Carrier . . . . 132

Advances in Macromolecular Carbohydrate Research Volume 1, pages 93-175. Copyright © 1997 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-323-2

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IV. Neoglycoconjugates with Polysaccharide as a Carbohydrate Moiety A. Utilization of Natural Polysaccharides B. Utilization of Synthetic Polysaccharides V. Conclusion Acknowledgments Notes References

152 153 161 164 166 166 166

I. INTRODUCTION Novel aspects have appeared in the chemistry and biochemistry of carbohydrates in the last 10 to 20 years. It became clear that their role in the functioning of living cells is much wider and more important than was long believed. This is primarily because in many carbohydrate structures information is encoded which determines, in particular, cell-to-cell and cell-to-cytoplasm interactions [1,2]. This can be best exemplified by the well-known role of surface or capsular polysaccharides of microbial cells which determine their antigenic properties [3] or of group-specific animal and human glycoproteins, the carbohydrate chains of which are responsible for group specificity [4]. Studies on the essence and mechanisms of these finest biological events require, naturally, not only the knowledge of the structures of the carbohydrate-containing compounds involved but also of the very compounds under study in sufficient amounts and, even more critical, of an appropriate purity. If additionally it is taken into account that in many cases their concentration in nature is extremely low and they are the components of very intricate complexes or mixtures, it becomes evident that their isolation for subsequent experimental studies may require processing of a vast amount of a natural material and the application of skilled labor. In this connection semisynthetic compounds became of definite importance in the very beginning of studies on biological specificity of carbohydrate structures. They were prepared by

Neoglycoconjugates Synthesis

95

covalent attachment of a carbohydrate molecule under investigation, or its fragment, to a high molecular weight carrier, frequently a protein. This approach, which was initiated by the classical works of Landsteiner [5] on the synthesis of artificial antigens and further developed by Goebel et al. [6], is currently very popular. Compounds of this type with a carbohydrate fragment immobilized on a high molecular weight carrier are named neoglycoconjugates^ and constitute an independent and rapidly expanding class of polymers. Neoglycoconjugates, which in a broad sense involve both synthetic analogs and modified carbohydrates with complex substituents, in particular, have found not only biomedical and pharmacological applications [7] but also some technical uses. The latter were usually developed quite independently although some approaches that resulted were also used for the preparation of neoglycoconjugates of biological significance. This review deals mainly with the latter group which are employed as artificial antigens. Other studies not related directly to this topic will be briefly discussed. Methods for preparation of neoglycoconjugates, mostly neoglycoproteins, and the diversity of their uses have been covered in several excellent reviews published a decade ago [8-10]. The rapidly increasing use of compounds of this kind for the solution of many problems in life sciences has resulted in the equally rapid development of synthetic approaches to obtain them. Relevant novel data appearing in the last few years are essential for practical work in this field and are also reviewed here.

II. THE PRINCIPLES OF DESIGN AND ARCHITECTURE OF GLYCOCONjUGATES Generally, the problem of synthesis of glycoconjugates can be considered as covalent incorporation of carbohydrate fragments into a macromolecule. This was traditionally performed by conjugation of a carbohydrate moiety of a given specificity, e.g. a hapten in the synthesis of artificial antigens to an existing polymeric support (route A, Scheme 1). The support which is most

NIKOLAY K. KOCHETKOV and ANATOLY YA. CHERNYAK

96 SUGAR

SUGAR (carbohydrate monomer)

(polymer carrier )

( noncar bo hydrate monomer)

SUGAR

SUGAR

Scheme 1.

often used is a protein [8], although sometimes it is a synthetic polypeptide [11] or some other macromolecule. A novel alternative approach for the preparation of neoglycoconjugates consists of the copolymerization of carbohydrate fragments (haptens in the case of antigens) bearing functional groups suitable for polymerization with non-carbohydrate monomers (route B, Scheme 1) [12,13]. In this case the polymeric carrier is formed in the very process of synthesis of the neoglycoconjugate. When using neoglycoconjugates as models of the natural carbohydrate-containing biopolymers it should be kept in mind that quite often their overall design differs considerably from that of natural compounds and results in the alteration of biological properties. This should be taken into account when planning glycoconjugate synthesis. Thus, artificial antigens, which mimic bacterial ones, are usually built on to a central polymer chain to which oligosaccharides are laterally attached which, in turn, correspond to a

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97

repeating unit of a natural polysaccharide. This is essentially in contrast to the design of the natural prototypes: for instance, somatic antigens of a Gram-negative microbial cell surface (the so-called 0-antigens) contain long polysaccharide chains comprising 10 or more repeating units [14], while capsular antigens are polysaccharides of extremely high molecular mass also built of repeating oligosaccharide units [3]. These differences in design may result in disappearance from the neoglycoconjugate structure of certain elements typical of the natural prototype and the appearance of new ones. Thus, the fragment A-^C inherent to the prototype is modified when present as a component of a neoglycoconjugate shown in Scheme 2: the monosaccharide A is not linked to the residue C of the neighboring repeating unit but is coupled to a carrier to leave C unsubstituted. The relative proportion of these Cterminal residues can be much higher than in the natural structure. This, in turn, can alter to a certain extent the serological specificity of neoglycoconjugates, especially if it is taken into

RU (repeating unit)

RU

«~

spacer

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NIKOLAY K. KOCHETKOV and ANATOLY YA. CHERNYAK

account that monosaccharide residues at the nonreducing end of oligo- and polysaccharides are, as a rule, immunodominant [3]. It is thus of importance to identify the fragment of the carbohydrate polymer which is introduced into the neoglycoconjugate, with the artificial antigen in particular. Two fundamentally different types of artificial antigens should be distinguished: one contains oligosaccharide or in extreme cases even monosaccharide fragments, while the other less frequently comprises polysaccharide chains attached to a macromolecular support. Distinction between these two types of artificial antigens is associated not so much with different conjugation procedures (although this is also the case, cf. Section IV) but rather with the aims of their preparation. In fact, based on its general character of the principle of regularity, repetition for the majority of the natural polysaccharide antigens leads to the conclusion that the information on the antigenic specificity is encoded, for the most part, in the structure of one-two repeating oligosaccharide units. Therefore, artificial antigens, with short oligosaccharide fragments, simulate partial antigenic specificities and serve as an indispensable tool in studies of immunochemistry of natural carbohydrate-containing antigens. Conjugation of purified polysaccharide preparations to a polymeric carrier pursues another goal; namely the preparation of an immunogenic material which can be used for eliciting anti-polysaccharide antisera and serve as a model of polysaccharide vaccines for prophylaxis of infectious diseases. These two kinds of glycoconjugates differ considerably in the nature of the starting carbohydrates and in techniques of their immobilization on a high molecular weight carrier. Therefore their preparation will be discussed here separately. III. GLYCOCONJUGATES WITH OLIGOSACCHARIDE AS A CARBOHYDRATE MOIETY A. Carbohydrate Carriers of Specificity: Their Preparation

The preparation of an artificial carbohydrate antigen requires the oligosaccharide fragment to be present in a pure state and

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99

in a sufficient amount. Natural glycoconjugates themselves are sources for the oligosaccharides from which they can be isolated by means of chemical or enzymic degradation. However, the oligosaccharide fragments obtainable by these methods are few compared to those which exist in natural glycoconjugates since it is not always possible to split linkages specifically by chemical methods and substrate specificity of enzymes sets its limits for enzymatic degradation. Thus the series of oligosaccharides which may actually be prepared from the natural sources can be rather limited. Chemical Degradation

Solvolytic splitting of glycosidic linkages is the most widespread method for isolation of oligosaccharides from natural polysaccharides. Hydrolysis with mineral or organic acids [15] under controlled conditions quite often splits the most labile glycosidic bonds present in a given structure rather selectively. Thus, for instance, furanosidic linkages and glycosidic linkages of deoxy- and dideoxysugars can be split with sufficient selectivity. On the other hand, glycosidic linkages of hexuronic acids are more resistant to hydrolysis than those of neutral sugars. Thus, partial acid hydrolysis of the capsular polysaccharide of Streptococcus pneumoniae type 3 (or type 8) afforded cellobiouronic acid [16] which was used to prepare one of the first artificial antigens for modeling bacterial polysaccharides [17]: ^3)-P-D-GlcA-(l->4)-p-D-Glc-(1^3)-|3-D-GlcA-(l->4)-p.D-Glc-(l->

P-D-GlcA-(l->4)-D-Glc

The resistance of glycosidic linkages towards acetolysis can sometimes differ from that towards acid hydrolysis, as is the case with (l->6)-linkages [18]. Splitting of natural polysaccharides by this method gives rise to peracetylated fragments. A new and promising method for selective scission of glycosidic linkages is the solvolysis with anhydrous hydrogen fluoride (Scheme 3) [19]. Variation of conditions, mainly reaction

100

NIKOLAY K. KOCHETKOV and ANATOLY YA. CHERNYAK

"'»(A)

rVo„

R'OH

Scheme 3.

temperature, affects the specificity of splitting and produces different oligosaccharides from the same starting material. The regularities of the solvolysis with hydrogen fluoride have been reviewed [19]. According to the accepted mechanism (Scheme 3), reaction with hydrogen fluoride results in the formation of glycosyl fluorides. Their subsequent fate is determined by the mode of treatment of the reaction mixture. Addition of water (pathway A) gives reducing oligosaccharides, while treatment with methanol (pathway B) affords methyl glycosides. If treatment of glycosyl fluorides with other alcohols suitable for subsequent conjugation (e.g. allyl alcohol) also gives rise to glycosides, the solvolysis with hydrogen fluoride can become a valuable method for production of oligosaccharides which can be directly employed in designing neoantigens (see below. Section III.E). Oligosaccharides can also be released by other methods specific for the degradation of carbohydrate-containing biopolymers, e.g. Smith degradation [20]. The presence in the starting structure of a sequence of several monosaccharides which are not oxidized with periodate is the prerequisite for preparation of an oligosaccharide fragment (Scheme 4) [21].

Neoglycoconjagates

Synthesis

101

OOH

HO

OH

CH3

NHAc

CH2OH

NHAc

Scheme 4.

The elimination of oligosaccharide fragments, which are the components of glycoproteins, can also be effected by solvolytic methods. This usually results in a complex mixture of oligosaccharides due to heterogeneity of oligosaccharide chains in the starting glycoprotein and nonspecific degradation of carbohydrate chains. Whenever intact oligosaccharide fragments of glycoproteins are required, specific methods are employed. To split oligosaccharide chains of glycoproteins attached to serine or threonine residues through 0-glycosidic linkage, Pelimination is most often used. It is performed by the action

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NIKOLAY K. KOCHETKOV and ANATOLY YA. CHERNYAK

of an alkali in the presence of a reducing agent which blocks chain degradation from the reducing end. Conventionally, the reagent used is 1 M NaBH^ + 50 mM NaOH (50 °C, 16 h) [22]. Treatment with this reagent, however, results in release of not only 0-linked oligosaccharide chains but also 10-13% of A^-linked chains due to reductive splitting of A^-glycosylamide bonds. This side reaction can be suppressed if degradation is carried out in the presence of Cd^"^, resulting in the selective splitting of 0-linked carbohydrate chains [23]. Alkaline acetolysis may also be used for the same purpose [24]. Under conditions specified (1:1 acetic acid—acetic anhydride, saturated with sodium acetate, 80-85 °C, 100-200 h) Olinked chains are split selectively and in high yield (75-90%), with A^-linked chains being unaffected. This degradation method is based on P-elimination and the oligosaccharides liberated are simultaneously acetylated. A mild method for chemical splitting of iV-glycosylamide bonds in glycoproteins was recently developed. This involves treatment with lithium borohydride in 70% aqueous tert-hutyl alcohol followed by hydrolysis of glycosylamines produced with aqueous acetic acid (Scheme 5). This allows oligosaccharide chains of

-OH p NH-■OvNHCCHjCH .OH C-NH^HCO... RON—{ § \f NHAc

LIBHt, 45, 5 h

NH4HCO3

||p„3.,

30* 6-7 days pOH

Scheme 5.

NHAc

Neoglycoconjugates Synthesis

103

A^-glycoproteins to be obtained in yields of 60-80% [25]. A combination of these two methods [23,25] allows sequential and selective splitting of O- and then A^-linked carbohydrate chains of A^,0-glycoproteins to be performed [26]. Enzymic Degradation The advantage of enzymic hydrolysis is its regio- and stereospecificity, which allows splitting of one particular type of glycosidic linkage in a natural carbohydrate-containing biopolymer, albeit the very isolation of an enzyme, estimation of its substrate specificity, and optimization of the degradation procedure requires considerable preliminary work. An interesting and rather widespread access to oligosaccharides from natural sources is based on enzymic cleavage of bacterial polysaccharides by bacteriophages [27]. The tail region of a phage is a natural compartment for the glycosidases responsible for cleavage. If phage-induced hydrolysis is combined with simultaneous dialysis, a set of large fragments (di-, tri-, tetra-, and higher oligomers of a repeating unit) can be prepared [28]. These fragments are hardly accessible by partial solvolysis or specific chemical degradation. This hydrolytic procedure does not imply isolation of an enzyme in a pure state; it simply consists of incubation, at 37 °C, of abacterial lipopolysaccharide preparation with a purified inactivated bacteriophage in a dialysis bag. The oligosaccharides thus released are separated by gel-chromatography. However, practical application of this approach also requires serious preliminary work involving the search for an appropriate phage, specific to a given polysaccharide chain, and characterization of substrate specificity of glycanases of the phage. This can be exemplified by degradation of Salmonella 0-polysaccharides. Studies of 18 bacteriophages specific to LPS of serological groups A, B, and Dj revealed all of them to contain eM(io-a-rhamnosidases which catalyze splitting of the a-L-Rhap(1—>3)-D-Galp bond [29]. They differ from each other in subtle details of substrate specificity (Scheme 6). The presence of an a-glucose substituent at position 4 of a galactose residue pre-

104

NIKOLAY K. KOCHETKOV and ANATOLY YA. CHERNYAK

R 1

a-D-Glc 1

4

i

3 4 l-a-D-Man-(l-^)- a-L -Rha-(1 -»3)-a-D-Gal-(l^ ft phages P22, P27 ® phages 9N A, KB 1© R 1

a-D-Glc 1

i

i

6 3 -^ 2)-a-D-Man-( 1 ->4)-a-L-Rha-( 1 ->3)-a-D-Gal-( 1 -> i\

phages P22, P27 e phages 9NA, KB 1®

R = 3,6-dideoxy-a-hexosyl (D-arabino, D-xylo, D-ribo) Scheme 6,

vents hydrolysis of a rhamnosyl—galactose linkage under the action of phage 9NA and KBl glycanases, whereas phages P22 and P27 are active. Quite an opposite situation is observed if galactose bears an a-glucose substituent at position 6. The presence of a 3,6-dideoxyhexose residue attached at position 3 of mannose is the prerequisite for the phage P22 to manifest its rhamnosidase activity irrespective of the nature of the 3,6-dideoxyhexose. Thus, enzymic hydrolysis allows the isolation of oligosaccharide fragments containing 3,6-dideoxyhexoses, the latter being immunodominant sugars. These fragments cannot be achieved if conventional hydrolytic procedures are applied. This was taken into account when conditions for preparativescale degradation of Salmonella lipopolysaccharides had to be chosen—^the most frequently used was phage P22 [30]. Enzymatic hydrolysis has not apparently been applied to the preparative-scale isolation of oligosaccharide fragments of glycoproteins despite the existence of specific enzymes which en-

Neoglycoconjugates Synthesis

105

sure highly selective splitting of nondegraded carbohydrate chains. Flavobacterium meningosepticum is the source for peptide A^-glycosidase F which cleaves the 7V-glycosylamide bond between asparagine residues and carbohydrate chains [31]. OLinked carbohydrate chains can be split off by using a 0-glycan-peptide hydrolase (0-glycopeptide e«^o-D-galactosylN-acetyl-a-D-galactosaminyl hydrolase, EC 3.2.1.97) from Diplococcus pneumoniae [32a]. Substrate specificity of this enzyme is confined to asialo-oligosaccharide chains; moreover, it is only disaccharide p-D-Gal-(l-^3)-D-GalNAc that is the major product."^ Synthesis of Oligosaccharides

A versatile method for preparation of oligosaccharide fragments representing biological determinants is by chemical synthesis. In principle, this route allows access to any oligosaccharide, including structural analogs of the natural determinant fragments. Analogs of this kind are often very useful in elucidation of functionally important structural details of carbohydrate antigens [33,34]. Preparation of the first artificial carbohydrate antigens made use of available mono- and disaccharides [17,35]. However, the progress in this field necessitated the synthesis of more complex oligosaccharides and this was one of the most powerful stimuli for development of oligosaccharide synthesis. On the basis of better comprehension and by developing synthetic methods the solution to more and more complex problems is becoming possible. This can be exemplified by syntheses of oligosaccharides which are repeating units of microbial polysaccharides (cf. Table III in ref. 36) and carbohydrate fragments of several glycoproteins (ref. 37 and references cited therein; ref. 38). Today the number of reports on the synthesis of oligosaccharides is literally compared with an avalanche that has swept over scientific journals. A chemical synthesis of oligosaccharides is a more complicated problem than that of polypeptides or polynucleotides. It is practically devoid of general solutions, and the synthesis of any particular oligosaccharide is a rather specific challenge [39].

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NIKOLAY K. KOCHETKOV and ANATOLY YA. CHERNYAK

Nevertheless, synthesis of oligosaccharide structures comprising up to 10 monosaccharide residues is currently quite a realistic task [38,40]. The modern methodology of oligosaccharide synthesis is covered in several excellent reviews [39,41,42] and will not be dealt with here. What should be emphasized is that if synthetic oligosaccharides are to be used for the preparation of neoglycoconjugates, their synthesis should be designed and accomplished in such a way as to produce the target oligosaccharides directly in a form suitable for subsequent conjugation. As a rule, the synthetic oligosaccharides are obtained as O- or S-glycosides with a fixed configuration of a glycosidic bond and an aglycone with a reactive functionality. This circumstance may seriously affect the general strategy of the synthesis, especially in the case of complex structures because problems of the oligosaccharide synthesis, as such, are supplemented by those associated with the introduction and transformation of a more or less complex aglycone. B. Conjugation to a Polymer Carrier

Covalent attachment of carbohydrate determinants to a polymer carrier (conjugation) is the key stage in preparation of neoglycoconjugates. Several versions of conjugation have been developed, each requiring preliminary derivatization of an oligosaccharide. The generally accepted methods for conjugation will be discussed first followed by ways to prepare the corresponding oligosaccharide derivatives. Polymer Carriers

Most often neoglycoconjugates, especially artificial antigens, are prepared as neoglycoproteins [8-10] by attachment of a carbohydrate moiety to a protein. In the majority of cases, easily accessible serum proteins such as bovine, rabbit, or human albumin are employed as a protein carrier. These proteins, molecular weight of about 67,000-69,000, can be purified and obtained in a crystalline state. They are readily soluble in water.

Neoglycoconjugates Synthesis

10 7

and are convenient for conjugation. Hemocyanin (M^9,000,000) of different origins [43] or the hempseed protein, edestin (M^ 310,000) [44,45], are less commonly used. The latter exhibits higher immunogenicity than albumins but its utilization is hampered by its solubility only at high pH values. This creates difficulties both at the conjugation step and at isolation of the carbohydrate-protein conjugate. The use of other protein carriers is also documented. They include diphtheria toxin [46], bacterial protein, porin (Mj. 34,000-36,000), isolated from the outer membrane of Salmonella typhimurium [47], cytochrome c (Mj. 12,300), and some others. Of especial interest is the utilization of porin for conjugation with oligosaccharide fragments of 0-antigen from the same bacteria since it affords a neoglycoconjugate of enhanced protective effect. This is believed to be associated with the ability to elicit antibodies against two independent targets on a bacterial cell at the same time [47]. The use of synthetic polypeptides, such as poly-L-lysine (M^ 70,000) [48] or poly-(L-aspartic acid) [49] as the protein carriers are less common. Methods for Conjugation of Oligosaccharides with Protein Carriers As a rule, covalent attachment of oligosaccharide determinants to a protein carrier does not require any preliminary modification of the latter. Free 8-amino groups of L-lysine residues (e.g. in the case of BSA they amount to 57) or free carboxylic groups that are involved in coupling, by means of conventional organic reactions with carbohydrate residues, may result in the formation of a C-N bond. Formation of a N=N-X Bond (X = NH, C). Diazo coupling is historically the first conjugation method and was the most widespread for a long time. It dates back to the work of K. Landsteiner [5] and it is the modified procedure by Westphal and Feier [50] that is usually employed (Scheme 7). This procedure involves the introduction of the 4-aminophenyl moiety into the oligosaccharide; diazotization leads to an

108

NIKOLAY K. KOCHETKOV and ANATOLY YA. CHERNYAK

NaN02, HCl,

Sug-O^NH^

„ . , ; ^ / ^

Sug-0-<^N.N d

HO protein, NQHCO3, 0, 15 min

'CH2-profein

Sug-0 Y O V N-N-NH-prof ein

Scheme 7.

arenediazonium derivative which is immediately coupled to a protein. This method is characterized by a low specificity of bond formation. The highly reactive diazonium derivative interacts with a protein to produce at least two types of linkages, viz. azo-linkages with the residues of aromatic amino acids (e.g. tyrosine) and triazene linkages with free amino groups (e-amino group of lysine). Besides this nonspecificity of carbohydrate-toprotein bond formation, other drawbacks of the method involve the introduction of the immunogenic aromatic azo-group and instability of the diazonium salts under conjugation conditions. Formation ofC(=X)NH Bond (X = O, S, NH). The predominant part of conjugation methods with proteins is the involvement of the free amino groups of a protein molecule in the formation of a bond of the-C(=X)NH type (X = O, S, NH). It is primarily the amide bond (X = O) which can be formed through a carboxyl group present in a carbohydrate determinant and activated by carbodiimide, via mixed carboxyl anhydrides, or as acyl azide, with these being the three principal activation methods. Activation with carbodiimide is applied mainly to the oligosaccharides bearing a free COOH-group, e.g. to those of

Neoglycoconjugates Synthesis

109

which the reducing end is oxidized with hypoiodite into an aldonic acid residue [51]. An alternative version consists in conjugation of an oligosaccharide derivative having a free amino group, e.g. glycosylamine with carboxyl groups of a protein [52] or polypeptide [49]. Carboxyl activation is performed using water-soluble carbodiimide, e.g. l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), since conjugation is normally carried out in aqueous solution (Scheme 8). Among the drawbacks of this method are the necessity to use large excess (sometimes, up to 50-fold) of a carbohydrate component to achieve an acceptable degree of substitution in the conjugate produced, EDC-induced cross-linking of the carrier, and rather drastic reaction conditions (optimum pH is 4.75) which render some proteins insoluble, e.g. diphtheria toxin, or edestin [30]. The mixed anhydride method is based on the activation of the carboxyl group of the carbohydrate component (aldonic acid or glycoside with a COOH-containing aglycone) by treating it with isobutyl chloroformate in aqueous DMF in the presence of tri(«-butyl)amine (Scheme 9) [53,54]. Rather extreme conjugation conditions limit the use of this method, especially in the case of base-sensitive proteins, although the reaction itself is very efficient. With 3-4-fold excess of carbohydrate component to protein amino group, quite acceptable degrees of incorporation of carbohydrate determinants (8-15 mol per mol of BSA) [53] can be achieved. The acyl azide method used for conjugation of peptide haptens to protein carriers [55,56] was adapted for coupling 8-car-

Sug-COOH + R*N=C=NR

PH4-7 1 T HgN-proteln ; *- Sug-C-O-i-NHR -pjj-frg H *^ * h 25°, 2-6 0

►Sug-C-NH-proteln + RN ' HCONHR Scheme 8.

110

NIKOLAY K. KOCHETKOV and ANATOLY YA. CHERNYAK

Sug-COOH

tao-BuOCOCl,

J J , r—► Sug-C-0-C-OBu^ aq. BMP. BU3N.°0

►

H2N-proteln. 9 : ► Sug-C-NH-proteln + CO9 + iso-BuOH pH 10.5-12, 20^. 15-20 h "^ Scheme 9.

boxyoctyl glycosides to proteins (Scheme 10) [57]. In spite of preparation of acyl azides in a two-step procedure, this conjugation method is very efficient. At a 1:1 ratio of carbohydrate component to protein amino group the degree of substitution of lysine residues in BSA can exceed 50%. Nitrous acid, required to convert acyl hydrazide into acyl azide, is usually generated from tert-hutyl nitrite in DMF by treatment with 4M HCl in 1,4-dioxane with excess nitrous acid being decomposed by sulfamic acid [58], Alternatively, nitrosyl chloride can also be employed [55]. The conversion of hydrazide into azide can be simplified by using dinitrogen tetroxide as the nitrosating agent [59]. This modification gives excellent results in the synthesis of numerous neoglycoconjugates possess-

ing specificities of Shigella flexneri. Streptococcus A, and Salmonella polysaccharides [59]. Another improvement consists

Sug-0(CH2)8C00CH3

N%NI|'%0. [HONQ], 25^, ^^ ^ ^^ ^ i^ Sug-0(CHg)8C0NHNH2 45 mln ^

H2N-proteln, Y 'Sug-0(CHo)QGON« -z ► Sug-0(Cll5)pCNH-proteln ^ ^ ^ pH 9.0. 0^. 20 h ^8 ^ Scheme 10.

111

Neoglycoconjagates Synthesis

in replacing DMF with methyl sulfoxide [60] which is a better solvent for glycosides containing a polymethylene aglycone. By the use of isothiocyanates for preparation of conjugates, disadvantages inherent in diazo coupling are overcome to a considerable extent. Oligosaccharides with j!?-aminophenyl groups are used resulting in the formation of thiocarbamide bonds -NH(C=S)NH- (Scheme 11). The concept of the method arises from the chemistry of proteins because the activated derivative of a carbohydrate determinant is the protected phenyl isothiocyanate, i.e. Edman reagent [61]. Treatment of aminophenyl derivatives with thiophosgene affords isothiocyanates which react under conditions specified (Scheme 11) with free amino groups of proteins; interaction with -SH and -OH groups present in a protein molecule takes place only under more vigorous conditions [62]. The conjugation procedure is very efficient. With 10-fold excess of isothiocyanate to protein amino groups it is possible to modify up to 90% of amino groups of BSA [62]. Conditions originally developed [63] for the conversion of aminoderivatives into isothiocyanates (pH 2.0, 80% ethanol, 25 °C, 1.5 h) are rather harsh for oligosaccharides with acid-labile glycosidic bonds. Mild alkaline conditions (pH 8.0, 80% ethanol, 20 ^C, 2 h) are more acceptable [64], and by performing the reaction in a twophase system the hazards of using toxic thiophosgene are di-

-0-^^M<2

^

Sug.O-
H2N protein. pH 8-9, 4-25;

6-72 h

Sug-O-O^VNHCNH-protein

Scheme 11

112

NIKOLAY K. KOCHETKOV and ANATOLY YA. CHERNYAK

^-NH2 a q . NaHCO^, .'^ R-NHCS^Na^ 0.01 M NaQH^ p-dloxanef 2 0 " , ethanol 12 h

^'^^^^^

Scheme 12.

minished [65]. Unlike diazonium salts, isothiocyanates are rather stable and react more selectively. However, neoglycoconjugates obtainable by the procedure inevitably contain an immunogenic hydrophobic aromatic fragment. Nevertheless, the isothiocyanate method has found wide practical applications. It should be emphasized that there are no obstacles for its adaptation to conjugation of carbohydrates having an aliphatic amino group in the aglycone, as shown by Banoub et al. [66]. An alternative procedure for conversion of amines into isothiocyanates (Scheme 12) developed by Elbert and Cerny [67] can favor further development of the isothiocyanate method.

nOH /

KOH

°\

/"OH -•-

H(^Jl/

OH NH 0\NHCSCH3

H

?^"3

6%

H2N-C=NH

—;

50 17 days

j-OH NH / - Q NHCNH-protcin

H2N-piDtein, pH 8.5, 0* 48 h

NQOH.

-^-

.kJ OH

Scheme 13.

Neoglycoconjugates

Synthesis

113

Few examples are known of conjugation of carbohydrates with proteins through guanidine -NHC(=NH)NH- or amidineCH2C(=NH)NH- bonds. Guanidination (Scheme 13) consists of the interaction of a free mono- or oligosaccharide with iS-methylisothiourea under very harsh conditions to give moderate yields of l-(p-glycosyl)-2-methylisothiourea which then slowly reacts with free amino groups of a protein to form guanidines [68]. This reaction is not, however, specific since histidine residues in the protein can also be affected. Amidination reactions are more promising despite multistep procedures required to prepare the carbohydrate amidination reagents, 2-imino-2-methoxyethyl 1-thioglycosides (Scheme 14) [69]. The peracetylated glycosyl bromide is boiled in acetone with thiourea and the iS'-(per-0-acylglycosyl)isothiourea hydrobromide formed is converted in a one-pot procedure into 1-

^i >*~0.

S H2N-C-NH2

♦

c^N5C(NH2)2f Br" OAc

P

C

pOH

Vj^H2CN

K2CO3. NQHSO3

\

/

H2N-protcin, pH 7-10. 4-20* 2 h*^

001M MeONa,

J ~ 0 SCH2COCH3

30-3?: 24-48 h

rOH NH / — 0 . scH2CNH-protein C^_/

Scheme 14.

yn

114

NIKOLAY K. KOCHETKOV and ANATOLY YA. CHERNYAK

thiosugar and then, by S'-alkylation with chloroacetonitrile, into the S-cyanomethyl 1-thioglycoside. Removal of 0-acetyl groups and simultaneous conversion of the cyanomethyl group into imido ester is performed by treatment with sodium methoxide to produce 2-imino-2-methoxyethyl 1-thioglycosides which react readily with the free amino groups of proteins. Under alkaline conditions and with a 5- to 10-fold excess of the imido ester, about 50-60% of amino groups in BSA and other proteins are modified by carbohydrates. This method has found application mainly in the conjugation of mono- and disaccharides. Quantitation of the degree of amidination in this case can easily be accomplished because the thioglycoside linkage undergoes selective mercuric salt-catalyzed hydrolysis [70]. Formation of C-NH Bonds. Conjugation methods involving reductive amination result in C—NH bond formation (Scheme 15). The method is based on the shift of equilibrium between an aldehyde and the respective Schiff base, formed with the amino group of the protein, by means of selective reduction of the aldimine into secondary amine [71]. The aldehyde group may be specially introduced in an aglycone portion of the carbohydrate determinant [72,73]. On the other hand, the reducing oligosaccharide itself possesses an aldehyde group masked as a hemiacetal. In the latter case the concentration of the free aldehyde group in solution is low and therefore conjugation of unprotected oligosaccharides proceeds very slowly, often taking several days [71]. The use of sodium cyanoborohydride as a reducing agent is advantageous since, unlike sodium borohydride, it reduces selectively aldimines and not aldehyde groups. In order to achieve a reasonable extent of modification of proteins, large excesses of the aldehyde components are required that, in turn, result in the formation of tertiary as well as secondary amines [74]. Application of this conjugation procedure to reducing oUgosaccharides results in transformation of the cyclic structure of the monosaccharide at the reducing HgN-protein Sug~CH=0 ^=^

NaBHaCN

^ Sug-CH=N-proteln pH T~9^ Sug-CHgNH-proteln Scheme 15.

Neoglycoconjugates

Synthesis

115

terminus into acyclic 1-amino-1-deoxyalditol. With this unit, a new specificity arises in the neoglycoconjugate that, provided small molecular weight oligosaccharides are immobilized, lowers the merits of this method. Conjugation methods wherein the C-NH bond is produced include the stepwise substitution of chlorine atoms in the bifunctional reagent cyanuric chloride (2,4,6-trichloro-5-triazine) [75,76] with a carbohydrate component and a protein (Scheme 16). Nucleophilic substitution of one chlorine atom in cyanuric chloride with an amino derivative of an oligosaccharide essentially deactivates the remaining chlorine atoms so that no disubstitution takes place (Scheme 16). This first step is usually carried out at Q-5 °C. Increase in the reaction temperature to 45 "^C enables nucleophilic substitution of the second chlorine atom. Provided the protein amino group acts as a nucleophile at the second step, a neoglycoprotein is obtained with an asymmetric disubstituted chlorotriazine moiety at the junction site. Besides introducing this hydrophobic and possibly antigenic fragment, solubility problems may also be a disadvantage of this method. The use of 0-acetylated sugar derivatives may be necessary in the first step. Deacetylation of the conjugates proCi

R-NH2

♦

ci

IOJL

R « Sug. Sug(OAc)„

-^r-^

^

'lOj ^

CI Ur\H

H2N-proteln.

CI Sug-HN^ ^ N ^ N H --protein p

Scheme 16.

^"^* " * ° " *

116

NIKOLAY K. KOCHETKOV and ANATOLY YA. CHERNYAK

duced thereafter, may not completely overcome the solubility problem [54]. The use of cyanuric chloride in the preparation of glycoconjugates based on polysaccharides will be dealt with in Section IV.A. C. Preparation of Oligosaccharide Derivatives Suitable for Conjugation To effect conjugation of an oligosaccharide with a protein the former should possess a functional group which enables its successful condensation to amino groups of the protein by one of the aforementioned methods. The existence of such a group should be envisaged for a synthetic oligosaccharide. But provided an oligosaccharide is prepared by degradation of a natural product and produces a reducing species, this should be properly functionalized. Derivatization of Reducing Oligosaccharides The transformation of oligosaccharides into derivatives, which can be conjugated, does not require, as a rule, preliminary hydroxyl protection and subsequent deprotection. This is very advantageous, albeit the cost is the "loss" of a monosaccharide residue at the reducing end of the oligosaccharide. This is due to the fact that almost all the transformations, with the exception of formation of glycosylamines (cf. Scheme 5) [77,78], results in elimination of the natural cyclic form and configuration of the glycosidic linkage of this monosaccharide unit. The most frequent are the following transformations: 1. The reducing (terminal) monosaccharide residue is oxidized into an aldonic acid (Scheme 17) in order to activate the resulting carboxyl group with carbodiimide (Scheme 8) or via mixed anhydride (Scheme 9) prior to conjugation. 2. The monosaccharide unit is converted into 1-amino-1-deoxyalditol by reductive amination. If amino groups of a protein are involved in this slow transformation, i.e. the reductive amina-

Neoglycoconjugates Synthesis

11 7

IH

pOH \2. N Q O H

\

SugC^N

/ AH

)-0H

►

OH

SugO

V -

COOH 6H

pOH

J—OH

H2N—protein

SugO

^

yCONH-protein

Scheme 17,

tion is carried out in the presence of the protein, the product is directly a neoglycoprotein [74,79] (cf. Scheme 15). Alternatively, the reaction can be performed with 2-(4-aminophenyl)ethylamine (Scheme 18) [80]. The oligosaccharide is dissolved in the reagent [80] or in anhydrous dimethyl sulfoxide as a solvent. Anhydrous conditions in this case favor dehydration of a carbinolamine intermediate [81]. If reductive amination with sodium cyanoborohydride is performed in aqueous solution at pH 8.0 [82], it is the aromatic amino group that is involved in the reaction [83] so that another 1-amino-1-deoxyalditol is formed (cf Scheme 18):

CH2CH2NH2

The aromatic amino group, introduced by reductive amination, can be used effectively in the conjugation with a protein (Scheme 18) by means of the diazo coupling procedure [84] or the isothiocyanate method [82]. Reductive amination of oligosaccharides with 4-trifluoroacetamidoaniline (TFAN) (pH 6, 15-48 h) also affords

A A

03

-

SugO ~ l i = N C H 2 C H 2 e N ~

I

ugo~~~HC~CH2eN=-NH-profein

NaBHG

-

C H ~ N H C H ~ ( H ~ ~ N H ~ sugo

sugo

Scheme 18.

C H ~ N H C H ~ C H ~ ~ N H \ N rHi n- P ~ O ~ 5

Neoglycoconjugates Synthesis

119

r-OH /—OH

TFAN. NaBH^CN, <;iijn/\

/

pH 6,15-48 h

SugO

6-

CH2NH-^^NHC0CF3 OH

|-0H 25%

20:

NH(pH.

3h

/ O H

SugO

^ ,^^ ^ - ^ C- H 2 N H"H ^ ^^-NH2 )-h OH

Scheme 19.

aminoalditol derivatives [83]. The separation of mixtures of TFAN-derivatives of oligosaccharides by HPLC has been suggested as an advantage of this derivatization method. Treatment with aqueous ammonia generates free aromatic amino groups to be used for conjugation with a protein (Scheme 19). 3. Reducing monosaccharide residues can be used in heterocyclic system formation as it takes place, e.g. in the preparation of l-(3-nitrophenyl)flavazole derivatives (Scheme 20a) [85]. In order to obtain a neoglycoconjugate the aromatic nitro group is then reduced to an amino group and a diazo coupling is employed. When this approach was applied, for instance, to a tetrasaccharide prepared by partial acid hydrolysis of the 0-specific polysaccharide from Salmonella illinois, the reducing (terminal) rhamnose residue became a building block for the flavazole heterocycle so that the neoantigen obtained was practically devoid of the 0:3 serological specificity associated with this monosaccharide (Scheme 20b) [86]. Elimination of an intact monosaccharide residue from the reducing terminus of an oligosaccharide can be a disadvantage in using the above-mentioned methods for preparing the free oligosaccharides for conjugation. This has to be taken into account when artificial antigens of a predetermined specificity,

NIKOLAY K. KOCHETKOV and ANATOLY YA. CHERNYAK

120

a ^

+

NH2

n

CHOH ^ CHOH CHOH I I CHOH i

100. +

HN 4.5 h "2^^

NO

0:3

a Glc I

i3-Gal-{1——6) i3 Man-d—^4)-Rha

X

N =N--NH-edestin

B-Man

Scheme 20.

especially with short oligosaccharide determinants, are to be prepared. This is because modifications to the reducing end of the oligosaccharide may induce additional antigenic sites in a neoglycoconjugate molecule. Antibodies can be raised against these sites [87] as well as against the region of linkage of these fragments, especially aromatic ones, with a protein [88].

Neoglycoconjugates Synthesis

121

Thus, the conversion of reducing oligosaccharides into glycosylamines with retention of the cyclic form of a monosaccharide residue at the reducing end seems promising. This transformation can be accomplished on treatment with ammonium hydrogen carbonate to give P-glycosylamines in a yield of 50-80% (Scheme 5) [77,78]. However, in this case the glycosidic oxygen is replaced by nitrogen. Synthesis of Oligosaccharides as Glycosides with an Aglycone Spacer

Synthetic oligosaccharides are used for conjugation, as a rule, in the form of O- and iS-glycosides with an aglycone carrying a reactive, functional group. This serves to form a linkage with a protein and is at some distance from the glycosidic linkage being separated by an aromatic ring or aliphatic chain with or without heteroatoms. The nature and length, in particular, of this spacer part of a molecule determines the distance between the carbohydrate fragment and the protein chain of a carrier, essentially influencing the properties of the neoglycoconjugate prepared. The functionalized glycosides are obtained via corresponding glycosylating agents, the most usual of which are: glycosyl halides; 1,2-0-orthoesters, and oxazolines [39]; 1,2O-cyanoethylidene derivatives [89]; 1-O-trichloroacetimidates [41]; and thioglycosides [42]. No special problem arises when a functionalized aglycone is introduced into short oligosaccharide chains. On the contrary, conversion of higher oligosaccharides into glycosyl donors, e.g. glycosyl halides, can become somewhat troublesome [90]. Therefore, in the case of higher oligosaccharides, it is advisable to introduce the aglycone spacer at early stages of synthesis of glycosides. A"blockwise" strategy of synthesis is practicable, where an oligosaccharide chain with the preformed aglycone spacer or its precursor (prespacer) is elongated with the use of mono- or disaccharide synthons. On the other hand, the thioglycoside-based strategy [42] facilitates introduction of an aglycone spacer at final stages of the oligosaccharide synthesis [91].

NIKOLAY K. KOCHETKOV and ANATOLY YA. CHERNYAK

122

Table 1 lists typical aglycone spacers of the synthetic oligosaccharide determinants which are used for conjugation with protein or polypeptide carriers. The aglycone spacers are used in introducing one of the following principal functions: amine, carboxyl (or alkoxycarbonyl), or aldehyde into molecules, offering the advantage of chemical potentialities which will be used in the covalent bond formation during conjugation. The relative diversity of conjugation procedures reflects the search for optimal, versatile methods for coupling, under mild conditions, carbohydrate Table 1. Aglycone Spacers Used in Conjunction with Protein Carriers Aglycone Spacer

Conjugation Procedure

-o-f^,

References 17, 50, 62, 64,92

•NH2

Diazo-coupling, carbodiimidefacilitated amidation, isothiocyanate method

—OCHs-nf

)—^NH2

-0(CH2)2-Y

V"NH2

Diazo coupling, isothiocyanate method

0(CH2)2S—f^

y ^NH2

Isothiocyanate method

96

Cyannric chloride method Carbodiimide-faciUtated amidation Carbodiimide-faciUtated amidation Mixed anhydride method

54 97

-OCH2CH2NH2 -S(CH2)2NHCO(CH2)6NH2 -OCH2COOH -0(CH2)8COOH -O(CH2)8C00Me -O(CH2)2S(CH2)2C00Me

! [

-0CH2CH=0 ] -S(CH2)„CH=0 -S(CH2)„CONHCH2CH=0 I («=1,5) J -SCH2C(=NH)0Me

Acyl azide method

Reductive amination Amidination

93—95

98 54 57-59 60, 96, 99, 100 101 102 73, 74, 103, 104 69, 105

Neoglycoconjugates Synthesis

12 3

determinants of a given type to a protein carrier. It is necessary to ensure a sufficient degree of incorporation of the determinants with the desirable physicochemical and biological properties into a neoglycoconjugate. It is for this purpose that the aglycone spacers are used possessing the predetermined characteristics such as optimum size, appropriate conformational flexibility, a minimal antigenicity, and reduced possibilities for nonspecific hydrophobic interactions. In spite of the fact that aromatic fragments possess their own considerable antigenicity [5,11], hydrophobic aromatic spacers are still frequently employed, preferably in combination with the isothiocyanate coupling procedure [64,83,92,95,96]. The role of a functional group, which ensures linking to a protein, usually involves the 4-aminophenyl group. This may be attached as the 0-glycoside to the carbohydrate moiety either directly or through a short aliphatic chain. A conformationally more flexible, nine-carbon long, 8-carboxyoctyl aglycone was introduced by Lemieux et al. [57,58]. This makes the distance from the carbohydrate moiety to the carrier equal to 140 nm. Coupling via this spacer can be achieved in either of two ways: the carboxyl-containing aglycone is linked to a protein following carbodiimide [54] or mixed anhydride activation, or the aglycone with a methoxycarbonyl group is attached following conversion into acyl azide [58]. The octamethylene spacer is insignificantly antigenic but it tends to bind nonspecifically to antibodies due to hydrophobicity as shown by inhibition of immunoprecipitation studies [58,106]. The polymethylene spacers are thought to possess the pronounced tendency to coil in aqueous solutions, thus lessening their effective lengths [107]. A spacer of the same length but of lower hydrophobicity was employed in the synthesis of the oligosaccharide determinant of the T-antigen (Thomsen-Friedenreich antigen) [108]. It contained a hydrophilic amide group in the middle of a chain and a promising approach of a stepwise chain elongation was used (Scheme 21). First, a 2-(benzylideneamino)ethyl group aglycone was introduced which, following hydrolysis of a labile C = N

124

NIKOLAY K. KOCHETKOV and ANATOLY YA. CHERNYAK rOAc ^

r-OAc

)*

^ HaH2CH2N.CH^

S—f N3

—

^

^-^

\

^-Y0CH2CH2N
rOAc 6 0 % AcOH

L

eo\ 15 min

\

H00C(CH2)4C00CH3

S-/^^^^2CH2NH2

^ ^

DCC

CH2CH2NHCO(CH2)4COOCH3

Scheme 21.

bond in a Schiff base and condensation with monomethyl adipate, was converted into a peptide-like spacer. Two novel aglycone spacers of dioxa-type were used in the synthesis of a repeating unit of the Streptococcus pneumoniae Type XIV capsular polysaccharide, viz. 7-methoxycarbonyl-3,6-

i-CAc /~OvO(CH2)20(CH2)20CH2COOCH3

HO(CH2)20(CH2)20CH2COOCH3, /

Hg(CN)2, DCM

^

7

pOAc

/ NHAc

u

NHAc

|-OAc rOA — 0 0(CH2)20(CH2)20{CH2l2N3 3. J/~^y

H0(CH2)20(CH2)20(CH2)2N3, AgOTf,

TMM. OCM NHAc

Scheme 22.

Scheme 23.

126

NIKOLAY K. KOCHETKOV and ANATOLY YA. CHERNYAK

dioxaheptyl- and 8-azido-3,6-dioxaoctyl-ones (Scheme 22) [109]. Catalytic reduction of the azido group in the latter aglycone to the amino group enables its coupling to macromolecular carriers. Other spacers of the same type are shown in Scheme 23 [110]. Reaction of acylglycosyl bromides with 3—5 equivalents of dior triethylene glycol under Helferich conditions affords monoglycosides. Tosylation of the free hydroxyl group followed by nucleophilic substitution of the tosyloxy group allows the introduction of an azido functionality into the aglycone [110]. The principle of stepwise elongation of a spacer avoids the presence of a long-chain aglycone at all the stages of the synthesis and technical difficulties associated therewith. What is even more important is that using this approach the size and functionalization of the spacer may be varied for the same carbohydrate determinant. Among the examples, besides those already cited, are chain elongation of (co-aminoalkyl)aminoethyl spacer (Scheme 24) [97], of aminopropyl spacer (Scheme 25) [111], as well as some transformations of the allyl aglycone (Scheme 26) [112,113].

. NH

2 0 ; 30 min

(^

P

- 0 SCH2CH2NH2 / OAc

Ac O^CH2CH2NHCO(CH2)5NHCOCF3

,

pOH OH"

/"OvSCH2CH2NHCO(CH2)5NH2 SH

Scheme 24.

Neoglycoconjugates

127

Synthesis

QAc

Ox

+

^ '

H0(CH2l3NHC0CF3

0(CH2)3NHC0CF3

NHAc

^",

-OH pun

— \7

Amberlyst Ar26 (OH"),

/-0^(CH2)3NH2

C VoCOICHjl^COOEf

aq. MeOH

NHAc

pOH 0(CH2)3NHC0{CH2)4C00Et

NHAc

Scheme 25.

-OH

p: s'°> C

yOCH2CH=CH2

i

r

OH

-^

i

OH pOH

HSCH2CH2NH2

OH 0

0. CH2CH»0

Ph3P=CHC00Mc

.ho CH2CH=CHC00Me

S'^_/0(CH2)3S(CH2)2NH2 OH OH

H2 -

Pd/C

j—0. {CH2)3COOMe

Scheme 26,

HOCHLW2b, ?SOH,

Mpq-td~teno

uUIc

,u2h

u ~ c w u ~ B~t4

4-------1---

0 % ~

M M , Sfi-Lutidine

OAc

hapter,s-inhibitot~

YN-pmiin

neooniqens

Scheme 27.

O h

Neoglycoconjugates Synthesis

129

The "pre-spacer" strategy as a systematic principle was realized by Magnusson and co-workers (Scheme 27) [114]. The 2-bromoethyl aglycone, which was used as a pre-spacer, could easily be introduced by conventional glycosylation methods using glycosyl halides or oxazolines [96]. This aglycone could also be introduced into the natural reducing mono- and oligosaccharides in the form of per-0-acetates prepared beforehand [115,116]. This conversion does not affect the cyclic form of a monosaccharide residue at the reducing terminus of an oligosaccharide, unlike transformations of reducing oligosaccharides mentioned above. Nucleophilic substitution of bromine by functionalized thiols completes the conversion of the pre-spacer into a spacer [117]. It is at this step that the length and the nature of a spacer can be varied. Glycosides prepared in this manner are further converted into neoantigens by using suitable conjugation methods, viz. via acyl azides or isothiocyanates [60,96,99,100]. In the case of long-chain thiols, neoglycolipids have also been prepared [99,117]. The use of 3-bromo-2-(bromomethyl)propan-l-ol (dibromoisobutyl alcohol) instead of 2bromoethanol in the glycosylation reaction opens the way for the preparation of double-chain bis(sulfide)neoglycolipids resembling natural glycosphingolipids or glycoglycerolipids (Scheme 28) [118].

PAC

uo

OAc

r

Br

BFo, ether

OAc . . OAc

<^^

130

NIKOLAY K. KOCHETKOV and ANATOLY YA. CHERNYAK

If bromine in 2-bromoethyl glycosides is reduced catalytically, the ethyl glycosides obtained can be used as inhibitors in immunochemical studies [96,99,117]. It is worthwhile noting that the 2-bromoethyl aglycone can be introduced at early steps of the oligosaccharide synthesis because it is compatible with the majority of standard organic reactions used in carbohydrate chemistry. The 2-bromoethyl group remains intact under hydrogenolysis conditions if acetic acid is used as the reaction solvent [96]. D.

Discussion

In conclusion, the evaluation of different methods for preparation of glycoconjugates can be made. The classical procedure of Landsteiner—Goebel, which is based on diazo coupling, is now mainly of historical interest. Guanidination and amidination methods are rather complicated and relatively less efficient, therefore they are applicable to a limited set of accessible monoand disaccharides. The most popular methods currently used for coupling oligosaccharides with protein carriers to obtain neoglycoconjugates are based on isothiocyanate and acyl azide procedures. The former was applied to aminoaryl glycosides [62,64] and to reducing oligosaccharides following their reductive amination with 2-(4-aminophenyl)ethylamine [82]. It is by this approach that a Swedish group has prepared artificial 0-antigens with specificity for Salmonellae [29,119]. The acyl azide coupling method is applied to glycosides with a (carboxy)polymethylene aglycone spacer and thereby Lemieux et al. prepared neoglycoconjugates which mimic group-specific glycoproteins and glycolipids [120,121]. In designing neoantigens, besides the purely chemical aspects, are the essentially important immunobiological aspects. The procedure chosen, including the conjugation method, should give rise to a neoglycoconjugate which functions effectively as an artificial antigen. It would be of interest, in this context, to compare immunobiological properties of neoglycoconjugates with the same carbohydrate portion and polymeric carrier prepared using differing conjugation methods. Conclusions can

Neoglycoconjugates Synthesis

131

Table 2. Comparison of Antibody Titers in Rabbits Immunized with Various S. typhimurium Octaprotein Conjugates''^ ELISA Endpoint Titers x KT^ of Antibodies Against:

S. typhimurium Immunogen

S. typhimurium SH 4809 LPS (0:4,5,12)

Carrier Protein

76,240 240 87,500 37,500 1,800

7,640 <1.0 <1.0 25,000 9,300

Octa-ITC-BSA(DS~ll) Octa-EDC-BSACDS-T) Heat-killed cells Octa-ITC-DTCDS-lS) Octa-ITC-PO(DS-15) Notes:

^Octa = a-D-Gal/7( 1 ->2)-a-D-Man/7( 1 ^4)-a-L-Rhap-( 1 -^3) -a-D-Ga\p-{ 1 ->2)-a-D-Marv?( 1 ->4)-L-Rha^ 3 3

t

1 a-Abep

t

1 a-Abcp

''Reproduced with permission from ref. 119.

only be made by direct comparison of neoantigens in one series of immunobiological assays. Unfortunately, studies of this kind are extremely rare, although one such report deals with the immunogenicity of neoantigens differing only in the mode of conjugation [119] (Table 2). Cleavage of the 0-specific polysaccharide of S. typhimurium by a bacteriophage P22-associated ^«(io-rhamnosidase [30] afforded a dimer of the repeating unit of the polysaccharide. This oligosaccharide was coupled to bovine serum albumin (BSA) either by the isothiocyanate (ITC) method [following reductive amination with 2-(4-aminophenyl)ethylamine] or by the carbodiimide (EDC) procedure (following oxidation of the monosaccharide residue at the reducing terminus into aldonic acid). In its ability to raise anti0-specific antibodies, the thiocarbamide-linked conjugate (octa-ITC-BSA) is much the same as whole heat-killed S, typhimurium bacteria, whereas that prepared by the EDC-procedure (octa-EDC-BSA) is about 300 times less efficient. For several conjugates of the same octasaccharide with different protein carriers [BSA, diphtheria toxin (DT), porin (PO)], which were prepared using the same procedure, titers of anti0-specific antibodies vary in an unpredictable manner (Table

132

NIKOLAY K. KOCHETKOV and ANATOLY YA. CHERNYAK

2). Each of the conjugates elicited antibodies against the carrier, the titers of which amounting to 50-70% of those obtainable upon immunization with the respective unconjugated carrier protein [119]. E. Polymerization as a Method for Preparing Glycoconjugates with a Synthetic Polymer Carrier

Another general approach in preparation of neoglycoconjugates consists of conversion of a carbohydrate fragment into a macromolecular state via polymerization (Scheme 29) [12,13,122,123]. In this case an oligosaccharide derivative with a group suitable for polymerization (e.g. double bond) undergoes copolymerization with a second non-carbohydrate monomer. The macromolecular carrier is thereby the result of copolymerization of a carbohydrate and a non-carbohydrate monomer, the latter forming a linear polymeric skeleton with built-in residues bearing pendant carbohydrate fragments. The number of oligosaccharide determinants in a neoglycoconjugate and their distribution along the linear chain of the macromolecular carrier can be controlled by means of a non-carbohydrate monomer.

SugO

rf

-CH-CH2 CH2=CH-X

X X X

0^4

Scheme 29.

Neoglycoconjugates Synthesis

133

Neoglycoconjugates of this type, which are linear polymers modified with carbohydrates (as branches), can be referred to as "pseudopolysaccharides." The interest in their synthesis (mainly on the basis of available mono- and disaccharides) arose initially in connection with the problem of elaboration of polymeric drugs [124] and attempts to carry out solid-phase glycoside synthesis [125]. Design of Pseudopolysaccharides

Polymerization and copolymerization are used to prepare polymers of this type, as well as modification of a preformed polymeric carriers that can be exemplified by nucleophilic ring-opening in a protected 6-(9-epoxypropyl-galactose with polyvinyl alcohol [126] followed by deprotection or formation of hydrazones of malto-oligosaccharides upon reaction with polyacrylic hydrazide [127]. Polymerization is carried out with ester or ether derivatives of carbohydrates (monosaccharides, as a rule) containing a double bond [128-133]. If a substituent occupies any but the anomeric position, the product of polymerization is a polymer carrying reducing sugars (Scheme 30) [128]. Polymerization of 1-0- or 1-A^-substituted derivatives of mono- and oligosaccharides affords polymeric conjugates with carbohydrate-to-carrier bonds which are closer to those in natural glycoconjugates [134—136] (cf. Scheme 31) [135]. On the other hand, direct addition polymerization of mono- and disaccharides with diisocyanates gives pseudopolysaccharides of another type wherein carbohydrate residues are incorporated in the main chain of a synthetic polymer (Scheme 32) [137]. The a p p l i c a t i o n of m e t h o d s for the p r e p a r a t i o n of pseudopolysaccharides to informational oligosaccharides gives rise to synthetic polymers which are of potential biological interest. Thus, for instance, radical polymerization of A^-(4-vinylbenzyl)aldonamides derived from malto-oligosaccharides gives water-soluble polystyrene polymers with oligosaccharide substituents (Scheme 33) [138-139]. The polymers obtained with maltose (n = 2) or maltotriose (« = 3) moieties in each repeating

R =

( R=

-C0C=CH2

-CH2CH«CH2»

-CH=CH2 M 2 8 1 .

-CO(CH2)ioOCOC=CH2

1130)

Scheme 30. pOBz Nia2-6H20.

BzoW

20: 2 days

MeONQ

Scheme 31. 134

135

Neoglycoconjugates Synthesis

0-C«N-(CH2l4-N-C«0

0CNH(CH2)4NH?0

Scheme 32.

KIO. 40*

40 min

HO

H2NCH2-^^CH-CH2 70.

KjSjOg.

—

2-13 h

^

60. 10 16 h

CH2-CH—}j

Scheme 33.

CH3C0NMe2. ► 17; 24 h

136

NIKOLAY K. KOCHETKOV and ANATOLY YA. CHERNYAK

"—0,0CH2m=CH2 +

(NH4)2S208. TEMED. CH2=CHCONH2 -

20:

2h

CONH9 .CH2CHHCH2CH)jr-+n

HO-

CH2

unit are recognized by concanavalin A, with precipitation being more rapid in the latter case [138]. Specific lectin-precipitating properties are also exhibited by water-soluble O-glycosylpolyacrylamide polymers prepared by copolymerization of allyl glycosides of monosaccharides (o-Man, D-Gal, L-FUC, D-GalNAc) with acrylamide (Scheme 34) [136]. Artificial Antigens Prepared by Copolymerization

Studies described earlier for the preparation of carbohydratecontaining polymers have served as a basis for development of a new approach to artificial antigens, which is being elaborated in the authors' laboratory [12,123]. Antigens of a novel type, with serological specificity of Salmonella 0-antigens [12,13,122], did not contain protein but in certain cases elicited a humoral antibody response in immunized experimental animals [140]. It is shown thereby that it is not necessarily a protein carrier that is a prerequisite for immunogenicity [5,11]. It is with this approach that neoglycoconjugates of a copolymer type

Neoglycoconjugates

Synthesis

13 7

and also with specificity of group-specific glycoproteins have been obtained [141]. Starting Monomers and Copolymerization. Oligosaccharide determinants for preparation of neoglycoconjugates were initially synthesized as allyl glycosides [142,143] or allyloxyalkyl glycosides (Scheme 35) [141]. In many cases it is in the form of allyl glycosides that oligosaccharides are prepared (cf. ref 144 and refs. cited therein) and this is an advantageous aspect of these monomers. The merits of the allyl group include its serving as a good protection for the anomeric hydroxyl group, its acting as a TLC tracer in the course of oligosaccharide synthesis, and its removal under mild conditions [145]. In addition, it undergoes facile terminalfiinctionalization(Scheme 26) that allows the conversion of the same carbohydrate fragment into neoglycoconjugates of various types, including conjugates with protein carriers (Scheme 36) [146]. Polymerization of oligosaccharide monomers as allyl or allyloxyalkyl glycosides should result, in principle, in polymers with carbohydrate branching in each monomer unit (cf. Scheme 33). Such a dense arrangement of carbohydrate chains in a polymer can become a negative factor because efficient functioning of a multivalent neoglycoconjugate as an antigen may require quite

lOlCHjlnly

J—On /70>X)(CH2l,0CH2CH-CH2 SugO

— / NHAc

n -

3,4

SugoA.^ I X - OH. NHAc; y.Q. y=:1.

Scheme 35.

n = 3.4 )

138

NIKOLAY K. KOCHETKOV and ANATOLY YA. CHERNYAK

CH 2=CHC0NH 2 R0CH2CH=CH2

^1^^ ►-[-(CH2CH)2.-CH2CH-]j^ CHo 'Z

1. 02 . MeoS

iR

H2N-BSA ( o r TT) R0CH2CH=0

NaBH3ClJ, pH T-Of

R0CH2CH2NH-(BSA)

37°. 3 days R = a-Neu5-Ac Scheme 36. definite epitope density [147]. The use of copolymerization of a carbohydrate monomer with a non-carbohydrate monomer allows control over broad limits of the incorporation of the determinant groups in a final neoantigen, as the non-carbohydrate monomer acrylamide which is used, easily undergoes radical polymerization induced by various initiators. Polymerization of acrylamide and its copolymerization with hydrophilic monomers give rise to water-soluble linear polymers of rather high molecular mass. Polyacrylamide is practically neutral as a polymeric carrier. Cationic charges are zero and anionic ones, due to partial hydrolysis of carboxamide groups, are negligible. The polymers are stable over a broad pH range, resistant to microbial degradation, and nontoxic in contrast to acrylamide itself. Radical copolymerization of allyl- and allyloxyalkyl glycosides with acrylamide proceeds under very mild conditions, that is in aqueous solution in the presence of 0.1% (w/v) ammonium persulfate and 0 . 1 - 0 . 2 % (v/v) N,N,N\N'-tQtr2imethylethylenediamine (TEMED) for 2-12 h at 20 ^C. The carbohydrate moiety of the oligosaccharide monomers is not affected as judged from ^^C NMR spectroscopy [142,143]. These oligosaccharide-polyacrylamide conjugates can be isolated by gel-filtration or by precipitation with methanol from an aqueous solution. They are very water-soluble and exhibit M^ 100,000 or greater, as shown from analytical ultracentrifugation and

Neoglycoconjagates Synthesis

139

ultrafiltration data [142]. Quantitation of carbohydrates in the neoantigens can be achieved by carbohydrate analysis or by means of nondestructive methods, viz. by ^^C NMR spectroscopy where the relative intensity of signals for the carbohydrate moiety and the carrier is considered, or by optical rotation measurements because it is only the carbohydrate moiety of the polymeric glycoconjugate that possesses optical activity [142]. Copolymerization of acrylamide with allyl glycosides (or, more generally, alkenyl glycosides or alkenyl 1-thioglycosides [148]) suffers from a disadvantage associated with different reactivities of double bonds under the polymerization conditions: it is lower for the allyl group. Therefore, to introduce into the polymer the desired amount of carbohydrate determinants it is necessary to use an excess of the allyl glycoside and this excess has to be determined experimentally. This drawback can be overcome if aglycones of the glycosides used bear an acrylamide fragment which is close in reactivity to that of acrylamide itself. Aglycones of this type were first employed to incorporate carbohydrate ligands into polyacrylamide gels, the latter being used in cell adhesion studies [149] and for affinity chromatography of lectins [150]. Different variants of aglycones with acrylamide moieties, which were used in syntheses of polymeric neoantigens, are shown in Schemes 37 [151-153], 38 [111], 39 [154], and 40 [112,155—157]. Preparation of these aglycones involves acryloylation of co-aminoalkyl or aminoaryl glycosides, sometimes in combination with a stepwise elongation of the allyl aglycone. Thereby carbohydrate determinants with an allyl prespacer can be transformed into glycosides with an acrylamidecontaining aglycone which is more efficient as regards to polymerization (Scheme 40) [155-157]. Thioglycoside derivatives of acrylamide can be obtained by monoaddition of 1-thiosugar to the double bond of co,o)'-alkylene-bisacrylamides (Scheme 41) [150,158]. However, linear polymers prepared by copolymerization of thioglycosides with acrylamide so far have been used for the noncovalent incorporation into a cross-linked polyacrylamide network with formation of a gel for affinity chromatography [150].

140

NIKOLAY K. KOCHETKOV and ANATOLY YA. CHERNYAK

pOH /~^^0(CH2)nNHX 00(CH2)nNH2 OH

X = COCF3. C00CH2Ph n = 2, 6 CH2-CHCOCI. Amberly$tA-26(HC0^

pOH /

\0(CH2)2N3

\

T""™'

Scheme 37.

The aforementioned methods for preparation of carbohydrate ligands to be converted into polymeric conjugates have been applied to synthetic oligosaccharides (Schemes 37-41). The recently suggested methods for introducing an olefmic group at

,

0

HO(CH2)3NHCOCF3

V~0^(CH2)3NHCOCF3

N ^ f 'CH3 H,

Amberlyst A-26 (OH')

"NHAC

pOH 0(CH2)3NH2

|CH2«CHCO)20.

/-^(CH2)3NHC0CH=CH2

MeOM NHAc

Scheme 38.

Neoglycoconjugates

Synthesis

141

pOH

pOH

-NO2

H2 - PtO

^°^"^0"-NH9

OH

pOH CH2=CHC0Cl. K2CO3

/ ~ ~ 0 o-^KNHCOCH=CH2 \

*"

^ OH

Scheme 39.

the reducing end of an oligosaccharide [78,159] open perspectives for preparation of neoantigens of this type from oligosaccharides isolated from natural sources. The first procedure (Scheme 42) is advantageous for derivatization of relatively long oligosaccharides as it is connected with the transformation of a reducing monosaccharide residue into a 1-amino-1-deoxyalditol derivar-OH

r-OH

/~"O^H2CH