Carbohydrate Chemistry Volume 14 Part II Macromolecules

A Review of the Literature Published during 1980

By J. F. Kennedy

The Royal Society of Chemistry

Copyright © 1983 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-0-85186-132-6

Contents

1 Introduction, 1,
2 General Methods, 5,
3 Plant and Algal Polysaccharides, 21,
4 Microbial Polysaccharides, 51,
5 Glycoproteins, Glycopeptides, and Animal Polysaccharides, 92,
6 Enzymes, 230,
7 Glycolipids and Gangliosides, 345,
8 Chemical Synthesis and Modification of Oligosaccharides, Polysaccharides, Glycoproteins, Enzymes, and Glycolipids, 375,
Author Index, 508,

CHAPTER 1

Introduction

BY J. F. KENNEDY

The Introduction to the Part II Report was revised extensively in Volume 12. That Chapter, and the additions in Volume 13, covered in detail: ‘Scope and Coverage of the Report’; ‘Organization, Nomenclature, and Use of the Report’; ‘Significant Advances in Macromolecular Carbohydrate Chemistry’; and ‘Conclusions and Readership’, is classified ,as ‘essential reading’ for any new reader of ‘Carbohydrate Chemistry’ Part II Reports.

This present Chapter essentially builds upon the corresponding chapters of the two previous Volumes the former of which may be regarded as the ground state.

1 Scope and Coverage of the Report

This is largely unchanged from that of the two preceding Volumes; again the journal coverage list has been modified to include new titles. However, the current trend of economies in various University and other institutional libraries will undoubtedly lead to cutbacks in their current holdings of less well read journals. If a considerable amount of material becomes unavailable directly to Reporters, it may be that again on grounds of cost it will not be possible to sustain backup through the Inter Library Loans system.

The continuing inclusion of reports on principal carbohydrate (-containing) macromolecules in the more recently inaugurated Specialist Periodical Reports series entitled ‘Macromolecular Chemistry’, is neither a duplication of, nor a competitor to, ‘Carbohydrate Chemistry, Part II’. For the specialists, and the readers who want information on the broader field of both synthetic and neutral macromolecules, the similarities and differences between them, and an overview of the uses of all types of macromolecules including carbohydrates, proteins, and nucleic acids, a copy of each volume of ‘Macromolecular Chemistry’ is appropriate for their bookshelves. However, the relevance of that title is not confined to the Chapter on carbohydrates – since numerous chapters include items on the chemistry and interactions of carbohydrates in general polymer chemistry and bio-medical applications.

2 Organization, Nomenclature, and Use of the Report

Draft documents from the Nomenclature Committee of the International Union of Pure and Applied Chemistry (IUPAC) for various aspects of carbohydrate nomenclature are under consideration. One final document, entitled ‘Con-formational Nomenclature for Five- and Six-Membered Ring forms of Mono-saccharides and their Derivatives (Rec. 1980)’, has been published and definitive documents on abbreviated terminology of oligosaccharide chains, nomenclature of unsaturated monosaccharides, symbols for specifying the conformation of polysaccharide chains, nomenclature of branched-chain monosaccharides, and polysaccharide nomenclature can be expected to appear in the very near future.

In the area of nomenclature, there has been considerable discussion of late on the terminologies of the words phytohaemagglutinin, haemagglutinin, and lectin. Whereas the underlying etymology of some of these words is obvious, the relative utilities of the words has come into question. As new discoveries have been made, it has been necessary to adjust from the original belief that such types of molecules were only endogenous to plants. Various workers in the field have discussed nomenclature under the heading ‘What should be called a lectin?’ as follows.

‘During the past decade, interest in lectins (also referred to as agglutinins, phytohaemagglutinins, phytoagglutinins, and protectins) has increased enormously. The term lectin has frequently been used to describe substances that differ markedly from those classically considered lectins, that is plant seed carbohydrate-binding proteins. Nevertheless, the discovery of lectin-like substances in widely diverse sources (bacteria, fungi, fish-sera, snails, etc.) appears to warrant an expansion of the term, For the sake of clarity we would suggest the following definition of “lectin”.

‘A lectin (from the Latin legere: to choose) is a sugar-binding protein or glycoprotein of non-immune origin which agglutinates cells and/or precipitates glycoconjugates. Lectins bear at least two sugar-binding sites, agglutinate animal and plant cells (most commonly erythrocytes, unmodified or enzyme-treated) and/or precipitate polysaccharides, glycoproteins, and glycolipids. The specificity of a lectin is usually defined in terms of the monosaccharide(s) or simple oligosaccharides that inhibit lectin-induced agglutination (or precipitation or aggregation) reactions. Although first discovered in plants, lectins also have been found in many organisms from bacteria to mammals. A lectin may be soluble in biological fluids (e.g. concanavalin A) or membrane-bound (e.g. rabbit or rat liver lectins). There are several other types of sugar-binding proteins including sugar-specific enzymes (glycosidases, glycosyltransferases, glycosyl kinases, glycosylpermeases, glycosylepimerases, …), transport proteins, hormones (thyroid-stimulating hormone, follicle-stimulating hormone, …), and toxins (ricin, abrin, modeccin, …), etc.

‘Under some conditions, sugar-specific enzymes with multiple combining sites agglutinate cells (and/or precipitate glycoconjugates) and so act as a lectin. In spite of similarities to true lectins from the same source, toxins that bear only one sugar-binding site should not be called lectins since they do not agglutinate cells or precipitate glycoconjugates.’

In connection with use of Chapter 6 on Enzymes, readers are reminded of the existence of a special index suffixed to the Chapter. This index is invaluable since it enables readers to locate references to a particular enzyme speedily, and it also can be read as a listing of the enzymes which are principally related to carbohydrate hydrolysis, oxidation, and isomerization. As has been generally adopted in this Series, transferase enzymes are dealt with in conjunction with the macromolecular carbohydrate, the biosynthesis of which they contribute to, and therefore appear in the relevant Chapter rather than specifically and only in Chapter 6.

3 Significant Advances in Macromolecular Carbohydrate Chemistry

An increasing dislike by the legislative authorities for the use of polysaccharide (starch) derivatives in foodstuffs means that the development of derivatives for commercial applications must be confined to other uses. In particular, oxidized starches are coming to be questioned re their safety in foods such as bread, etc.

Mention should be made of the increasing use of 1H and 13C n.m.r. spectroscopy in structural studies on plant polysaccharides. Particular use has been made in studies on water-insoluble polymers where the polymer has been sohbilized by derivatization. Anthraquinone and its 2-sulphonic acid derivative continue to be of use in preventing the β-elimination reaction of polysaccharides in alkaline solutions.

The inability of certain tomato fruits to ripen is caused by the absence of a specific poly-D-galacturonanase isoenzyme.

The number of citations in the field of glycoproteins, proteoglycans, and animal polysaccharides has increased dramatically, and for the first time there are more than one thousand references cited in Chapter 5. Techniques for the preparation of monoclonal antibodies are now being widely applied, and numerous examples of the isolation and application of monoclonal antibodies to well-defined glycoproteins and polysaccharides are reported.

The minimum sequence of monosaccharides in heparin necessary for binding to antithrombin III has now been identified as eight. The location of sulphate ester groups in the octasaccharide is also important for the retention of biological activity.

The amount of attention being afforded the types and levels of glycoside hydrolase enzymes involved in the manifestation of clinical conditions and related subjects continues to increase, and the various enzymes responsible for the breakdown of a number of carbohydrate(-containing) molecules have now been identified. The various amylolytic enzymes also attract attention and there is considerable activity in the field of generation of new substrates particularly for fully automated systems. Undoubtedly, the number of identifiable poly-saccharide-degrading enzymes is steadily increasing, but the fuller understanding of their properties, action patterns, and mechanisms of action follows at a slower pace. The field of cellulases still provides plenty of scope for elucidation of their functional relationships.

It has been established that Krabbes disease is caused by an accumulation of D-galactosylsphingosine (psychosine). The glycolipid reaches toxic levels and kills oligodendroglial cells.

The first report has been made of the presence of L-fucose residues in the more complex glycolipid fraction of human brain tissue.

Considerable interest continues to be taken in the synthesis of the repeating units of bacterial polysaccharides. The behaviour of cycloamylose inclusion complexes, particularly those of drugs, has been the subject of a number of studies. Polysaccharides have been used extensively as support matrices for the preparation of affinity chromatography media and of immobilized enzymes, Numerous immobilized derivatives of cells have been prepared and applied to problems in both research and industry.

Special Issues of Carbohydrate Research have continued in the pattern of being published in honour of persons with international reputations in carbohydrate chemistry and their achievements. Professor Stephen J. Angyal and his work on, inter alia, chromium trioxide oxidation of glycosides and the use of the reaction in structural studies on polysaccharides, and the complexation of sugar with alkaline-earth and rare-earth ions and the use of this complexation to facilitate the synthesis of rare methyl glycosides as well as in the separation of mixtures of methyl glycosides, have been so honoured.

Published versions of the Tate and Lyle Carbohydrate Chemistry Award Lecture entitled ‘Transition-metal oxide chelates of carbohydrate-directed macromolecules’, and ‘From carbohydrates to enzyme analogues’, have appeared.

4 Conclusions and Readership

As already indicated, ‘Carbohydrate Chemistry’ enjoys a wide readership and utility. However, it is the experience of Senior Reporters and Reporters that well-established reputable groups overseas are not readily aware of the Series. Whereas the Royal Society of Chemistry has increased its publicity and sales, programme all readers are encouraged to support the Series by advertising it as widely as possible in the interests of economy and survival.

CHAPTER 2

General Methods

BY R. J. STURGEON

1 Gas-Liquid Chromatography

The use of g.l.c. for the measurement of free sugars and sugar alcohols in biological fluids has been reviewed. A chiral stationary phase, N-propionyl-L-valyl-t-butylamide polysiloxane, has been used for the separation of monosaccharides as their alditol acetates by capillary column g.l.c.

The alditol acetates of the common neutral sugars and three hexa-acetates, and the peracetates of 3-O-methyl- and 4-O-methyl-D-glucitol can be resolved in short analysis times. The determination by g.l.c. of neutral sugars as aldono-nitrile acetates, and of amino-sugars as O-methyl oxime acetates, in glycoproteins has been reported. An improved method is also described for converting neutral sugars to oximes, which can be either converted to the trimethylsilyl derivatives or, upon acetylation, derivatized to aldononitrile acetates.

Levels of D-mannose in human serum have been measured using the aldono-nitrile acetate derivatives. The method has been used for measuring D-mannose in patients as an indicator of the presence of invasive candidiasis. Dissolved carbohydrates in natural water have been estimated by g.l.c. of the derived alditol acetates.

The use of peracetylated aldononitrile derivatives of aldoses and peracetylated ketose oxime derivatives of ketoses for g.l.c.-m,s. studies has been reported in an accurate discriminatory method for the identification and quantitation of aldoses, ketoses, and small oligosaccharides. These derivatives may also be used in the separation and identification of products arising from methylation-fragmentation structural analysis.

D-Fructose has been determined as the O-methyl oxime trimethylsilyl derivative by g.l.c. using glass capillary columns. The D-fructose derivative is readily separated from D-glucose, D-galactose, L-rhamnose, and L-arabinose.

Hexitols occurring in biological fluids have been analysed as their per-O-acetyl derivatives by g.l.c.-m.s. with selected ion-monitoring. The method has been developed and applied to the analysis of D-galactitol and other polyols in amniotic fluid as an adjunct to enzyme studies on amniotic fluid cells in the antenatal diagnosis of galactosaemia and measurement of galactokinase activity.

The thermal degradation of mono- and oligo-saccharides occurring in culture fluids as a result of microbial growth has been studied by g.l.c.-m.s. Aldopentoses degrade to furfural, whereas aldohexoses yield acetic acid, furfural, methyl furfural, levulinic acid, and hydroxymethylfurfural. Oligo-saccharides give degradation products that reflect the structures of the component monosaccharides.

The carbohydrates and organic acids of uremic serum have been monitored using g.l.c.-chemical ionization m.s. of the O-TMS-ethers.

2 Column and Ion-exchange Chromatography

Practical aspects of gel-permeation chromatography for separation of carbohydrates have been reviewed. Molecular sieving and non-ionic adsorption in polysaccharide gels have been discussed in the XII Hopkins Memorial Lecture.

The applications of a commercially available liquid chromatography analysec to provide a fully automated qualitative and quantitative analysis of neutral mono- and oligo-saccharides with on-line computer calculation of the data have been described. Oligosaccharides (d.p. 2–15) may be routinely separated, with a degree of separation superior to that which is achieved using gel-filtration techniques under gravity or with peristaltic pump-aided flow.

Gels synthesized by cross-linking guar gum with epichlorhydrin in aqueous propan-2-ol have been described. The flow properties of the gels are satisfactory only at moderate pressures. The degree of cross-linking was controlled and one of the products was used in the separation of proteins (mol. wt. 5 x 103-7 x 104). The problems associated with the column packings used in the characterization of dextran by aqueous gel-permeation chromatography have been investigated. Fractionating range, efficiency, short analysis time, and long-term stability are the major factors taken into account, but none of the solumn packings investigated is considered to be ideal.

Liquid chromatographic separations of poly-, oligo-, and mono-saccharides have been achieved.” Polysaccharides and oligosaccharides are separated by gel-permeation chromatography, whereas monosaccharides are resolved on macro-porous anion-exchange resins.

A new detection technique for the location of non-reducing oligosaccharides has been reported. These sugars are hydrolysed by passing the eluant from liquid chromatographic columns through a small reaction column packed with a strongly acidic cation-exchanger. The reducing saccharides which are released may then be detected after reaction with 4-hydroxybenzoic acid hydrazide.

Oligosaccharides and their degradation products have been separated on Bio-Gel P-4. Differences of one D-glucose equivalent could be distinguished for oligomers at least as large as 15 D-glucose equivalents. The method has been applied to the analysis of glycoprotein-derived oligosaccharides. Although various charged species such as phosphorylated or sialylated oligosaccharides show aberrant mobilities when eluted with water, their proper mobilities are obtained by removal of the charged function from the oligosaccharide or by using high salt concentrations.

The anomalous behaviour of some sugars on gel chromatography has been studied. The separation of hexoses from 6-deoxyhexoses by gel filtration on Bio-Gel P-2 is not related solely to molecular size.

Six methyl ethers of methyl α-L-rhamnopyranoside have been isolated by liquid chromatographic separation on silica gel of the products of partial methylation of methyl α-L-rhamnopyranoside.

Uronic acids and their oligomers derived from pectic substances have been separated and determined using a commercial liquid chromatograph. Mono-saccharides normally found in glycoprotein hydrolysates have been chromatographed as free sugars and as tritium-labelled alditols on anion-exchange resins. The presence of amino-acids in the hydrolysates does not interfere with the analysis.

Details of a fully automated ion-exchange chromatographic analysis of neutral monosaccharides and oligosaccharides have been published.

(Continues…)Excerpted from Carbohydrate Chemistry Volume 14 Part II Macromolecules by J. F. Kennedy. Copyright © 1983 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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