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Use of 2D N.M.R. and bacteriophages in structural studies of some E. coli antigens Kuma-Mintah, Agyeman 1989

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USE OF 2D N.M.R. AND BACTERIOPHAGES IN STRUCTURAL STUDIES OF SOME E. COLI ANTIGENS BY AGYEMAN KUMA-MINTAH B.Sc. (Hons), University of Science and Technology, Ghana, 1982 M.Sc, University of British Columbia, Canada, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in DEPARTMENT OF CHEMISTRY WE ACCEPT THIS THESIS AS CONFORMING TO THE REQUIRED STANDARD THE UNIVERSITY OF BRITISH COLUMBIA May, 1989 ® Agyeman Kuma-Mintah In p resen t i ng this thesis in partial fu l f i lment of the requ i remen ts for an a d v a n c e d d e g r e e at the Univers i ty of Brit ish C o l u m b i a , I agree that t he Library shal l m a k e it f reely avai lable fo r re fe rence and s tudy. I fur ther agree that pe rm iss i on fo r ex tens ive c o p y i n g of this thesis fo r scho lar ly p u r p o s e s may b e g ran ted by the h e a d of m y depa r tmen t o r by his o r her representa t ives . It is u n d e r s t o o d that c o p y i n g o r pub l i ca t i on of this thesis for f inancial ga in shal l no t b e a l l o w e d w i t h o u t m y wr i t ten p e r m i s s i o n . D e p a r t m e n t of T h e Univers i ty of Brit ish C o l u m b i a V a n c o u v e r , C a n a d a D E - 6 (2/88) i i ABSTRACT The capsular polysaccharide of Escherichia c o l i K31 has been found by methylation analysis and n.m.r. spectroscopy to be based on the hexasaccharide shown. The sequence of the repeating unit was deduced from the combined results of /3-elimination, lithium ethylenediamine degradation, and hydrogen fluoride and selective hydrolyses. The nature of the anomeric linkages, established by chromic acid oxidation, was confirmed by coupled ^C-n.m.r. spectroscopy. Two dimensional n.m.r. studies on a low molecular weight polymer obtained by bacteriophage depolymerization confirmed the structure given below. •2) -a-D-Glcp- (l->3) -0-D-Galp- (l-»3) -cr-D-GlcpA- (1-^ 2) -ct-L-Rhap- (l-*2) -a-L-Rhap- (1 4 1 a-L-Rhap-The importance of bacteriophage-borne enzymes in the generation of lower polymers very suitable for homonuclear (^ H) two dimensional n.m.r. studies i s demonstrated. E. c o l i K33 has been shown to cross react with Klebsiella K58. Chemical and two-dimensional n.m.r. studies gave the K antigen of E.  c o l i K33 as a tetrasaccharide repeating unit i i i OAc |2 - —3)-a-D-Glc(l-4)-0-D-GlcA(l->3)-a-L-Fuc(l--3 2 4 \ / C / \ HOOC CH3 1 a-D-Gal and this structure is quite identical to that of Klebsiella K58. The use of homonuclear two-dimensional ^H-spin correlated n.m.r. experiment as a very convenient method for the location of acetate i n the repeating unit of a polysaccharide i s illustrated using E. c o l i K33 polysaccharide as an example. The identification of a phosphate ester in the repeating unit of E.  c o l i K46 using n.m.r. spectroscopy is reported. Detailed n.m.r. study on this antigen is well documented in this communication. The f i r s t occurrence of a 3,6-dideoxyamino sugar residue i n an E. c o l i K antigen (i.e. E. c o l i K45 K antigen) and the finger print of i t s proton chemical shifts is also shown. i v TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS iv LIST OF APPENDICES v i i i LIST OF TABLES ix LIST OF FIGURES x i LIST OF ABBREVIATIONS xv ACKNOWLEDGEMENTS x v i i I. INTRODUCTION 1 1.1 Immunological importance of bacterial exopolysaccharides 2 1.2 Chemistry and serology of E. c o l i capsular polysaccharide 7 1.3 Bacteriophages 10 1.4 Nuclear magnetic resonance spectroscopy 14 II. METHODOLOGY 21 II.1 Pulse Fourier transform nuclear magnetic resonance spectroscopy (F.T.-n.m.r.) 21 II.1.1 The F.T.-n.m.r. experiment 23 - II.1.2 ^C-N.m.r. spectroscopy via spin echo experiments (attached proton test) 25 V 11.1.3 Basic theory of two dimensional n.m.r. spectroscopy 27 11.1.4 Two dimensional spin correlated n.m.r. spectroscopy 29 II.1.4a Homoscaler-correlated 2D n.m.r. spectroscopy 29 II.1.4b Heteronuclear correlated 2D n.m.r. spectroscopy 31 11.1.5 Sequence analysis using ^H-n.m.r. methods 32 11.2 Chemical methods 33 11.2.1 Isolation and purification 34 11.2.2 Sugar analysis 36 II.2.2a Total hydrolysis 36 II.2.2b Characterization and quantification of sugars 37 II.2.2c Determination of the configuration (D or L) of sugars 38 11.2.3 Position of linkage 39 II.2.3a Methylation analysis 39 II.2.3b Characterization and quantitation of methylated sugars 41 11.2.4 Sugar sequence 42 II.2.4a Periodate oxidation and Smith degradation 44 II.2.4b Uronic acid degradation (/3-elimination) 45 II.2.4c Partial hydrolysis 47 II.2.4d Lithium ethylenediamine degradation 48 11.3 Mass spectrometry 49 11.3.1 Electron impact-mass spectrometry 50 11.3.2 Chemical ionization (CI) mass spectrometry 52 v i 11.3.3 Fast atom bombardment - mass spectrometry (f.a.b.-m.s.) 53 11.3.4 Laser desorption Fourier transform ion cyclotron resonance (L.d.i.-F.t.-i.c.r) mass spectrometry 55 III. STRUCTURAL STUDIES OF E. COLI K31 CAPSULAR POLYSACCHARIDE BY CHEMICAL METHODS 57 I I I . l The structure of Escherichia c o l i K31 antigen 57 111.1.1 Introduction 57 111.1.2 Results and discussion 57 111.1.3 Experimental 63 IV. STRUCTURAL STUDIES ON FOUR E. COLI CAPSULAR POLYSACCHARIDES USING MODERN N.M.R. TECHNIQUES 75 IV.1 Introduction 75 IV.2 -^H chemical shift assignment of the sugar residue in E. c o l i K44 capsular polysaccharide 76 IV.2.1 Introduction 76 IV.2.2 Results and discussion 76 IV.2.3 Experimental 84 IV.3 Sequencing and location of acetate in E. c o l l K33 capsular polysaccharide 86 IV.3.1 Introduction 86 IV.3.2 Results and discussion 86 IV.3.3 Experimental 102 v i i IV.4 Sequencing of a hexasaccharide repeating unit (E. c o l i K31 polysaccharide) by homonuclear 2D n.m.r. spectroscopy 104 IV.4.1 Introduction 104 IV.4.2 Results and discussion 104 IV.4.3 Experimental 113 V. CONCLUDING REMARKS 115 VI. BIBLIOGRAPHY 117 APPENDIX I 128 APPENDIX II 168 APPENDIX III 177 APPENDIX IV 192 v i i i LIST OF APPENDICES Appendix Page I 1-H chemical shift assignment of 3,6-dideoxy-amino sugar residue i n E. c o l i capsular polysaccharide and structural studies on E. c o l i K46 polysaccharide 128 II Bruker 2D f i l e s employed in this study 168 III H^, 1 3C and 2D n.m.r. spectra 177 IV Polysaccharide antigens of Escherichia c o l i 192 ix LIST OF TABLES Methylation analyses of E. c o l i K31 polysaccharide and derived products N.m.r. data for derived products of E. c o l i K31 capsular polysaccharide Determination of anomeric configuration ( 1 3C, XH coupled) N.m.r. data for the native E. c o l i K44 polysaccharide ^-N.m.r. data for E. c o l i K44 native polysaccharide •^3C-N.m.r. data of K33 polysaccharide and derived products l3C-^-H Coupled n.m.r. experimental data ^-N.m.r. data for E. c o l i K33 deacetylated polysaccharide •^H-N.m.r. data 2D (COSY n.m.r. experiment) for E. c o l i K33 polysaccharide N.O.e. data for E. c o l i K33 polysaccharide •^H-N.m.r. data for E. c o l i K33 deacetylated polysaccharide (on AM400 Bruker n.m.r. spectrometer and experiment performed at 300°K) N.O.e. data for E. c o l i K33 deacetylated polysaccharide (on AM400 Bruker n.m.r. spectrometer and experiment performed at 300 °K) •^H-N.m.r. data for E. c o l i K31 polysaccharide N.O.e. data for E. c o l i K31 polysaccharide X VII.1 Methylation analysis of E. c o l i K46 polysaccharide 130 VII.2 l-H-N.m.r. data for E. c o l i K46 polysaccharide 137 VII.3 N.O.e. data for E. c o l i K46 polysaccharide 139 VII.4 13C-N.m.r. chemical shifts and 3 1P- 1 3C coupling constants and E. c o l i K46 native polysaccharide 143 VII.5 1 3C- 1H Coupled n.m.r. experimental data 145 VII.6 XH Chemical shift data of E. c o l i K46 dephosphorylated product 146 VII.7 123C-N.m.r. data of K46 dephosphorylated polysaccharide 147 VII.8 -^H-N.m.r. data for E. c o l i K46 bacteriophage polysaccharide (Px) 152 VII.9 N.O.e. data of lower molecular weight polymer (P x) 152 VII.10 ^-N.m.r. data for E. c o l i K45 native polysaccharide 164 LIST OF FIGURES Diagrammatic representation of the c e l l surface of Gram-positive and Gram-nagative bacteria Diagrammatic representation of the c e l l surface envelope of Gram-negative bacteria Basic morphological types of bacteriophage with the types of nucleic acid A diagrammatic i l l u s t r a t i o n of a bacteriophage A schematic diagram i l l u s t r a t i n g the steps in the infection of a bacterium by a bacteriophage Different regions in the n.m.r. (^•H and 1 3C) spectra of polysaccharides Illustration of an F.T.-n.m.r. experiment Time sequence of modem pulse experiments Illustration of a basic two dimensional n.m.r. experiment Mass spectra of 1,3,4,5-tetra-O-acetyl-2,6-di-O-methylglucitol and 1,2,5,6-tetra-O-acetyl-3,4-di-0-methylglucitol Fragmentation pathways of some part i a l l y methylated a l d i t o l acetates C.i.-mass spectrum and fragmentation pattern of methylated aldobiouronic acid (GlcA — Rha) C.i.-mass spectrum and fragmentation pattern of methylated HF product A2 x i i III. 3 C.i.-mass spectrum and fragmentation pattern of methylated HF product A3 62 IV. 1 Homonuclear ^H-spin correlated (COSY) n.m.r. spectrum of the native polysaccharide (K44) 80 IV.2 One step ^H-spin coherence transfer (C0SYHGR1) n.m.r. spectrum of native polysaccharide (K44) 81 IV. 3 Two step ^-H-spin coherence transfer (C0SYHGR2) n.m.r. spectrum of native polysaccharide (K44) 82 IV.4 Heteronuclear (^3C-^H) correlated n.m.r. spectrum of the native polysaccharide (K44) 83 IV.5 ^3C-N.m.r. spectra of deacetylated polysaccharide (K33) 88 IV.6 Homonuclear ^H-spin correlated (COSY) n.m.r. spectrum of native polysaccharide (K33) 91 IV.7 Homonuclear ^H-spin correlated (COSY) n.m.r. spectrum of deacetylated polysaccharide (K33) 92 IV.8 One step relay H^ spin coherence transfer (C0SYHGR1) spectrum of deacetylated polysaccharide (K33) 93 IV.9 Two step relayed H^ spin coherence transfer (C0SYHGR2) spectrum of deacetylated polysaccharide (K33) 94 IV.10 Homonuclear dipolar correlated 2D n.m.r. (NOESYHG) spectrum of deacetylated polysaccharide (K33) at 338°K 97 IV.11 Homonuclear dipolar correlated 2D n.m.r. (NOESYHG) spectrum of deacetylated polysaccharide (K33) at 338°K 101 IV.12 Homonuclear ^H-spin correlated (COSYHG) spectrum of a lower molecular weight polymer (Pn) derived from K31 native polysaccharide 108 IV.13 One step relayed H^ spin coherence transfer (COSYR1HG) spectrum of a lower molecular weight polymer (Pn) derived from K31 native polysaccharide 109 x i i i IV.14 Two step relay spin coherence transfer (C0SYR1HG) spectrum of a lower molecular weight polymer (Pn) derived from K31 native polysaccharide 110 IV.15 Homonuclear dipolar correlated 2D n.m.r. (NOESYHG) spectrum of a lower molecular weight polymer (Pn) derived from K31 native polysaccharide 111 IV.16 ^-N.m.r. spectrum of Pn (K31) 112 VII. 1 ^3C-N.m.r. (% decoupled) spectrum of native polysaccharide (K46) 132 VII.2 l3C-N.m.r. (*H decoupled) spectrum of dephosphorylated product (K46) 133 VII.3 13C-N.m.r. ATP spectrum of dephosphorylated product (K46) 134 VII.4 31P-N.m.r. ( XH coupled) of native polysaccharide (K46) 135 VII.5 Homonuclear ^H-spin correlated (COSY) n.m.r. spectrum of native polysaccharide (K46) 138 VII.6 Two step relay H^ spin coherence transfer (C0SYHGR2) spectrum of native polysaccharide (K46) 140 VII.7 Homonuclear dipolar correlated 2D n.m.r. (NOESYHG) spectrum of native polysaccharide (K46) 141 VII.8 Heteronuclear (^H-^3C) correlated spectrum of native polysaccharide (K46) 142 VII.9 Homonuclear ^-H-spin correlated (COSY) n.m.r. spectrum of dephosphorylated product (K46) 148 VII.10 One step relay H^ spin coherence transfer (COSYRCT) spectrum of dephosphorylated product (K46) 149 VII.11 Two step relay H^ spin coherence transfer (C0SYRCT2) spectrum of dephosphorylated product (K46) 150 xiv Heteronuclear (i-JC-1H) correlated n.m.r. spectrum of dephosphorylated product (K46) Homonuclear ^H-spin correlated (COSY) n.m.r. spectrum of a lower molecular weight polymer (Pjj) derived from K46 native polysaccharide One step relayed spin coherence transfer (C0SYR1HG) n.m.r. spectrum of a lower molecular weight polymer (P^) derived from K46 native polysaccharide Two step relayed spin coherence transfer (C0SYHGR2) n.m.r. spectrum of a lower molecular weight polymer (P^) derived from K46 native polysaccharide Homonuclear dipolar correlated 2D n.m.r. (NOESY) spectrum of a lower molecular weight polymer (P^) derived from K46 native polysaccharide Homonuclear H^ spin correlated n.m.r. (COSY) spectrum of native polysaccharide (K45) X V LIST OF ABBREVIATIONS Glc — glucose; Glcp — glucopyranose Gal — galactose; Galf — galactopyranose Rha — rhamnose GlcA - glucuronic acid GlcNAc — 2-acetamido-2-deoxyglucose GalNAc - 2-acetamido-2-deoxygalactose PMAA - part i a l l y methylated a l d i t o l acetate TFA = trifluoroacetic acid DMSO - dimethylsulfoxide Pyr - pyruvic acid acetal Ac — acetyl Me — methyl mol. wt. — molecular weight s — seconds; min - minutes; h - hours; d — days i . r . - infra-red n.m.r. - nuclear magnetic resonance g.l.c. - gas-liquid chromatography HPLC - high pressure li q u i d chromatography m.s. - mass spectrometry g.l.c.-m.s. — gas liquid chromatography - mass spectrometry I.e.-m.s. — liq u i d chromatography - mass spectrometry e.i.-m.s. — electron impact - mass spectrometry c.i.-m.s. - chemical ionization - mass spectrometry xvi fast atom bombardment - mass spectrometry bacteriophage plaque forming units room temperature atomic mass unit homonuclear spin correlated n.m.r. spectroscopy Relay COSY — one step relayed spin coherence transfer Two step relay COSY - two step relayed spin coherence transfer NOESY - homonuclear dipolar correlated 2D n.m.r. n.O.e. - nuclear Overhauser effect HETCOR - 1H- 1 3C heteronuclear chemical s h i f t correlated spectroscopy F.t. - Fourier transform f.a.b.-m.s. p.f.u. r. t. a.m.u. COSY xv i i ACKNOWLEDGEMENTS I would like to express my sincere gratitude to Professor G.G.S. Dutton for his guidance and interest throughout the course of this thesis. I am thankful to Dr. H. Parolis, Dr. S.N. Ng and Dr. P. Ph i l l i p s for their encouragement and helpful discussions. My grateful thanks to Dr. S.O. Chan, Liane Darge and Marietta Austria for their assistance. My special thanks to Rani Theeparajah for typing this thesis. - 1 -I. INTRODUCTION Carbohydrates are polyhydroxyaldehydes, ketones (usually in the acetal or hemiacetal forms) or substances which may be hydrolyzed by dilute acid to these compounds. Carbohydrate-containing macromolecules are of widespread occurrence in most l i v i n g organisms.^ In nature, carbohydrates are often linked to other molecules such as proteins and li p i d s to form: (i) glycoproteins, proteoglycans and peptidoglycans, ( i i ) glycolipids and lipopolysaccharides, ( i i i ) teichoic acids, and (iv) nucleic acids. Polysaccharides are carbohydrate polymers but may not be confined solely to O-glycosidically linked carbohydrates. Polysaccharides are of widespread occurrence i n nature and have significant commercial and biological importance. They have uses in numerous industries^ ranging from foods, pharmaceuticals, textiles, papers, paints, cosmetics, and tertiary o i l recovery to the treatment of environmental pollutants. The u t i l i t y of polysaccharides in these industries is due to their gelling, thickening, emulsifying, binding, coating, and film forming properties. The biological importance of poly-saccharides is threefold: architectural, nutritional and as specific agents. Their importance is shown in many facets of human l i f e where they are components of antigens, enzymes, nucleic acids, and c e l l surface glycoconjugates.3»^ Polysaccharides are important in the plant world, i n which cellulose i s the principal structural component and starch serves as the main energy bank. In insects and Crustacea, chitin provides skeletal support. Carbohydrates also serve as a renewable - 2 -source of carbon, for energy storage and as a readily manipulated natural source of ch i r a l i t y . 1.1 Immunological importance of bacterial exopolysaccharides The role of polysaccharides in immunology dates from 1917 with the report by Dochez and Avery-* of a "specific soluble substance" secreted by pneumococci during growth. These "specific soluble substances" were shown to be polysaccharides and in fact this was the f i r s t time that any material other than protein had been shown to be antigenic. The protective effect exhibited by antibodies raised to pneumococcal polysaccharides against pneumococcal infections provided proof of the importance of these substances in immunology.^•^ It was demonstrated that a relatively small portion of the polysaccharide is the major site of antibody specificity. The presence of the same determinant group in several polysaccharides was shown to be responsible for their serologi-cal cross-reactivity; that i s , the capacity of a polysaccharide from one species of bacterium to precipitate the polysaccharide-specific anti-bodies of another. Heidelberger has illustrated the use of cross-reactions in immunological analysis of polysaccharides.^ In his study he was able to predict the presence of structural features before they were determined. The use of polysaccharides as antigens and immunogens has contributed greatly to the classification and identification of bacteria, to a better understanding of the immune response, to the definition of the active site in antigen-antibody interactions and to - 3 -the detection and prevention of human disease caused by invasive organisms. More recently nuclear magnetic resonance spectroscopy has been applied to study the conformation of antigens during their interaction with antibodies. This chapter i s not intended to be exhaustive but to provide a general overview of structural studies of polysaccharides. Exopolysaccharide i s a generic name for a l l forms of bacterial polysaccharides found outside the c e l l wall. Most bacteria, either Gram-positive or Gram-negative produce extracellular polysaccharide^ (see Fig. I.1.1). Structure elucidation has revealed that most bacte-r i a l polysaccharides are composed of oligosaccharide repeating u n i t s . ^ Relatively l i t t l e is known about the function of microbial exopolysac-charides in contrast to the extensive data on their primary chemical structures. It has been proposed^ that they may be involved i n one or a l l of the following: (i) energy storage or reserve ( i i ) virulence, protection against phagocytosis ( i i i ) protection against desiccation and predators (iv) adhesion, and (v) as a general barrier Exopolysaccharides are the outermost mediators between the organism and i t s environment, the f i r s t portal of entry and the last barrier to excretion. The bacterial surface thus contains components which play an important role i n recognition processes.12 The c e n envelope (compris-ing the outer membrane and capsule) contains antigens which induce the formation of antibodies i n man and animals, and which react serologi-- 4 -p»ptn Cytoplasmic mombrtnt GRAM POSITIVE GRAM NEGATIVE Fig. I.I.I: Diagrammatic representation of the c e l l surface of Gram-positive and Gram-negative bacteria Capsular polysaccharide (K antigen) LieopotrMcchpnd* cftxnt ( 0 antigen) Phospholipid OUTER MEMBRANE PERIPLASMS SPACE ffOO kpOp'Otvin Sound lipoprotein CYTOPLASMIC MEMBRANE Fig. I . l . I I : Diagrammatic representation of the c e l l surface envelope of Gram-negative bacteria - 5 -cally with these antibodies.^- 3 The immune system includes a l l the structures and processes that provide a defense against potential pathogens. These defenses can be grouped into non-specific and specific categories. Non-specific defense mechanisms include barriers to penetration of the body and internal defense: (a) Phagocytic cells engulf invading pathogens (b) Interferons are polypeptides secreted by cells infected with viruses that help protect other cells from v i r a l infections Specific immune responses are directed against specific antibodies and are a function of lymphocytes. B lymphocytes secrete antibodies and provide humoral immunity. K i l l e r T lymphocytes, acting through the secretion of lymphokines, provide cell-mediated immunity (i.e. they come into contact or close proximity to secrete lymphokines in order to k i l l victim c e l l s ) . The structure of antibodies allows them to combine with antigens in a specific fashion and thereby activate other elements of the immune system to protect against pathogens. The aim of the immunochemical analysis of polysaccharide antigens which combines serological and chemical studies is to define antigenic determinants (or immunodominant sugars) within the polysaccharide as chemical expression of i t s immuno-logical character. These immunodominant residues, which form the major contributors to serological specificity, can be a monosaccharide linked in a specific manner, an oligosaccharide or even non-carbohydrate in nature, e.g. acetal-linked pyruvic acid, or 0 and N-acetyl groups.^ In acidic capsular polysaccharides such as those of Klebsiella. E. c o l i . - 6 -and Pneumococcus. the charged constituents are often part of the anti-genic determinants. 1 3 In branched polysaccharides, the immunodominant residues may be located i n the side chain. Bacterial polysaccharides have the same antigenic determinants expressed many times over due to their repetitive structure. Molecular weight and conformation are also c r i t i c a l parameters in the antigenicity of a polysaccharide. The sero-group-specific polysaccharides of Neisseria meningitis (at molecular weights of 50,000-130,000) are good immunogens in man, but the response is much weaker with preparations that have a molecular weight of 30,000. 1 5• 1 6 The accumulated evidence 1 5" 1 8 suggests that 45,000 ± 5,000 is the molecular weight above which polysaccharides are immunogenic and below which their immunogenicity f a l l s off rapidly. Immunization using vaccines, has been u t i l i z e d by man for a long time, for disease prevention. Vaccines derived from k i l l e d bacteria (or virus) preparations, live "attenuated" bacteria and toxoid are common and s t i l l effective. The f i r s t use of bacterial polysaccharides as a vaccine to combat pneumococcal infection was tested on U.S. Army recruits during World war II. The post World war II era saw renewed interest In prevention of diseases by immunization, due to widespread and multiple antibiotic resistance. The serious problem in developing polysaccharide vaccines is the i n a b i l i t y of these antigens (vaccines) to develop protective levels of serum antibodies in infants and young children (i.e. they give rise to only IgM antibodies).^0»21 This difference in immune response is thought to be due to the T-cell Independence of polysaccharide antigens, the participation of T cells being essential for the induction of IgG antibodies and memory c e l l s . 2 1 - 7 -There have been several attempts to overcome this problem by conjugating polysaccharides to an antigenic protein in order to form a T-cell-dependent antigen.23-25 ^h e potential use of microbial polysaccharides in cancer research has been reported by whistler et al.^6 The structural studies of bacterial polysaccharides are very important i n the light of bacterial infections, the production of protective vaccines and their potential as noncytotoxic antitumour drugs. To further the understanding of the chemical basis for serologi-cal differentiation, a knowledge of the primary structures of bacterial polysaccharides is required. It also forms the basis for the under-standing of their three dimensional structures in the so l i d state and in solution and for an appreciation of the ways in which polysaccharides are biosynthesized and degraded. With these and latent potential applications of bacterial polysaccharides i n mind, the task of elucidat-ing the detailed chemical structure of seventy-four E. c o l i capsular polysaccharides is currently being undertaken in this and other labora-tories. However, in this thesis, the emphasis is on the use of bacte-riophage born enzymes and two dimensional nuclear magnetic resonance spectroscopy as a probe for investigating the structures of capsular polysaccharides, from E. c o l i serotypes K31, K33, K44 and K46. 1.2 Chemistry and serology of E. c o l i capsular polysaccharides The organism Escherichia c o l i belongs to the family Enterobacteriaceae whose normal habitat is the intestinal tract of man and animals.^ - 8 -E. c o l i . f i r s t isolated from faeces by Escherich In 1885, i s often found in human urinary tract infections and is associated with severe infan-t i l e diarrhea. 2 8 Enterobacteriaceae are Gram-negative bacteria and cla s s i f i c a t i o n of the family (Enterobacteriaceae) by Edwards and Ewing2^ (shown i n Table 1.1) has been updated by Kauffmari.3^ An interest i n Escherichia c o l i in recent years from both human and veterinary medicine has been followed by an interest in the surface structure of these bacteria because of their special role in pathophysiological processes, their usefulness in epidemiological studies and their importance for the normal immunological status of the host. Within the bacterial species of E. c o l l there are many serotypes, each of which produces different extracellular polysaccharides. The serotyping scheme i s based on the c e l l surface antigens (see Fig. I . l . I I ) . These comprise the capsular or K antigens (74 polysaccharide types), the somatic 0 antigens (164 lipopolysaccharide types) and the flagellar H antigens (56 proteinaceous types). 3^ The 0 antigen i s the 0-specific polysaccharide of the c e l l wall lipopolysaccharide. It is a thermostable surface antigen (the bacteria keep their immunogenic, agglutinating and agglutinin-binding capacity after boiling). The structure and known properties of some bacterial lipopolysaccharides have been reported. 1 3 • > 3 1 Most E. c o l i strains have a unique K antigen. The K antigens are capsular and envelope antigens and a l l are polysaccharides except for two that are proteins (K88 and K99). These polysaccharides are made up of oligosaccharide repeating units, varying in size from one to seven sugar residues. 3 2 Common monosaccharides that have been reported in E. c o l i polysaccharides are D-hexopyranoses - 9 -(galactose, glucose, and mannose); D-pyranosyluronic acids (galacturonic and glucuronic acids); L-deoxyhexoses (fucopyranose and rhamnopyranose); 3-deoxy-D-manno-octulosonic acid (KDO) and 5-acetamino-3,5-dideoxy-D-glycero-D-galacto-nonulopyranosonic acid (NeuNAc). Immunodominant non carbohydrate substituents that occur i n E. c o l i polysaccharides include N and O-acetyl, phosphate, and 1-carboxyethylidene (acetal-linked pyruvic acid) groups. In their review on bacterial polysaccharides Kenne and Lindbergh l i s t e d the proposed structures of E. c o l i K antigens known up u n t i l 1982. An updated l i s t of the proposed struc-tures for the E. c o l i K and 0 antigen has been compiled i n Appendix IV. The K antigens consist of three groups (A, B and L). By electrophoretic means two groups of K antigens can be differentiated; those with high electrophoretic mobility (L antigen) and those with very low electropho-retic mobility (A and B antigens). Inspection by electron microscopy revealed that the acidic polysaccharides with low molecular weight (high electrophoretic mobility) form thin, patchy capsules while those with high molecular weight (low electrophoretic mobility) form thick and copious capsules. It has been shown that E. c o l i strains with 0 and K antigens exhibiting the same immunoelectrophoretic pattern could cause the same disease. 3^ In four cases, the chemical structure of the capsular polysaccha-rides of E. c o l i and Klebsiella were found to be identical. They are E.  c o l i K30 and Klebsiella K20, 3 3 E. c o l i K32 and Klebsiella K55, E. c o l i K33 and Klebsiella K58 and E. c o l i K42 and Klebsiella K63. 3 3 Recently, the cross-reaction of the K and 0 antigens of E. c o l i with the antisera from othe micro-organisms were reported. 3^" 3& - 10 -1.3 Bacteriophages Exopolysaccharases from bacteria 3? and bacteriophage-associated endoglycanases 3 8"^ are two main sources of enzymes that hydrolyze bacterial capsular polysaccharides. The former are isolated in low yields. Bacteriophages may be obtained in high yield and hence be used to a great advantage in our and other laboratories for the structural elucidation of bacterial capsular polysaccharides. Bacteriophages are viruses that infect bacteria, multiply within them and eventually k i l l them.^1"^3 Bacteriophages are cl a s s i f i e d by Bradley into morphological groups^ (see Fig. 1.3.1). The types A-C contain two strands of DNA, while type D has one. These four types are unique to phages. Types E and F contain a strand of RNA and a strand of DNA respectively. In a study on phages active on the capsular polysac-charides of the genus Klebsiella Rieger-Hug and Stirm reported that thirty of these phages belong to the Bradley type C, twelve to type B and three to type A. E. c o l i viruses are however known to belong to a l l six Bradley morphological types and these coliphage have been studied.^ 3 Bacteriophages are specific regarding the species of bacteria they w i l l infect although some have a broad or less restricted host range. This spec i f i c i t y depends on the presence of a specific receptor site on the c e l l surface. Protease can be used to demonstrate the bacteriophage receptor site for E. c o l i strains.^ 5 The v i r a l infection of a host by bacteriophages i s usually characterized by four phases (see Fig. I. 3 . I l l ) . These are: (i) adsorption of the phage particle onto the susceptible host . 11 -2-DKA 2-DNA 2-DNA 1-DNA 1-RNA 1-DNA Fig. 1.3.1: Basic morphological types of bacteriophage with the types of nucleic acid Bead Fig. I.3.II: A diagrammatic i l l u s t r a t i o n of a bacteriophage - 12 -Fig. I . 3 . I l l : A schematic diagram i l l u s t r a t i n g the steps in the infection of a bacterium by a bacteriophage ( i i ) injection of the v i r a l DNA (or RNA) into the host ( i i i ) replication of the phage nucleic acid and phage protein at the expense of the metabolic process of the host (iv) phage maturation and release which results i n the lys i s of the host c e l l - 13 -The presence of a phage on Its host bacterial lawn can be detected by Its characteristic plaque morphology, i.e. the plaque proper (clear spot with lyzed cells) i s surrounded by a halo (translucent spot) i n which the bacterial growth is decapsulated. The halo formation is due to the production of excess free spikes, which have been shown to contain the capsule depolymerase, during the biosynthesis of progeny virus i n the host c e l l . These spikes diffuse from the plaque and catalyze the hydrolysis of the surrounding bacterial capsules.^ Bacteriophage-borne glycanases^ m a y be hydrolases^ 8 or lyases.^9,50 These enzymes (glycanases) which occur in bacteriophages are generally specific for one or a few substrates. Kinetic studies on the bacte-riophage -borne enzymes revealed that the activity i s inhibited by products and high substrate concentrations.^ Depolymerization of bacterial capsular polysaccharides by the use of phage-borne enzymes allows: (i) the generation of selectively cleaved oligosaccharides (in high yields) corresponding to one or more repeating units. 3^ ( i i ) acid or base labile non-carbohydrate 5 1 groups (e.g. 0-acetyl and acetal-linked pyruvic acid) to remain intact on the oligosaccha-ride repeating unit. This is d i f f i c u l t to achieve using chemical methods of degradation. Bacteriophage generated oligosaccharide may be subsequently used (i) for the verification of the structures of the original polysaccharides; ( i i ) as substrates for mass-spectrometry and X-ray diffraction studies; ( i i i ) in the study of conformation in s o l u t i o n ; 5 3 (iv) as a source of complex and novel oligosaccharides and (v) for coupling as haptens to - 14 -immunoglobins52 for immunological studies. In this study bacteriophage borne enzymes were employed in generat-ing low molecular weight polymers from E. c o l i capsular polysaccharides. This enables the preparation of concentrated solutions which have reasonable nuclear Overhauser effect (n.O.e.). Increasing sample concentration enhances the signal to noise ratio (i.e. sensitivity) during nuclear magnetic resonance spectroscopic studies. 1.4 Nuclear magnetic resonance spectroscopy The f i r s t n.m.r. experiments were carried o u t 5 ^ ' 5 5 in 1945, but useful chemical applications became possible only after the discovery of the chemical shift e f f e c t 5 ^ i n 1949. Today the subject has expanded so that i t is of equal importance with the old established branches of spectroscopy [e.g. vibrational (infrared) and electronic (ultraviolet)]. •^H, 1 3C, ^ F , 3 1 P and nuclei have a spin quantum number (I) of 1/2 and because of their nuclear magnetic moment precess in an applied magnetic f i e l d . The spin of a nucleus aligns i t s e l f with one of two possible orientations with respect to the magnetic f i e l d . The parallel spin [i.e. parallel to direction of external magnetic f i e l d (B Q)] is more stable (lower energy) than the anti-parallel configuration or align-ment. At resonance with an applied radio frequency f i e l d , the magnetic moment of the spins parallel to the magnetic f i e l d absorbs energy and f l i p s to the higher-energy antiparallel spin state. The amount of energy required to execute this f l i p is dependent on the magnetic f i e l d - 15 -strength and the environment (i.e. electronic shielding). The induced magnetic f i e l d due to circulation of an electronic charge cloud opposes the primary applied f i e l d (B G). Thus electrons w i l l shield nuclei from the influence of the f i e l d B Q. This shielding can be taken into account by using an effective f i e l d B at the nucleus, given by B = B Q (1-a). The dimensionless number a is a small fraction, usually l i s t e d in parts per million and i s known as the shielding constant. (i) Chemical shift The phenomenon of chemical shift arises because of shielding (screening) of the nuclei from the external magnetic f i e l d by the electrons, discussed i n the previous section. Since the shielding effect is caused by the electronic environment, values of a w i l l vary with the position of the nucleus i n the molecule. The resonance condition i s u — |7/2ir|BQ(l-a) where v and a are the Larmor (resonance) frequency and shielding constant respectively. The chemical shift (6) is the difference between the Larmor frequency of the nucleus and that of a reference compound (e.g. TMS). The chemical sh i f t i s expressed in parts per million of the applied f i e l d and is intimately related to several effects such as orientation, electronegativity, aromatic ring effect and anisotropic effect. The n.m.r. spectrum of a carbohydrate can be divided into three regions for a *H spectrum and four for a ^ 3C spectrum, i.e. the high f i e l d region, the ring region, the anomeric region and la s t l y (only for •LJC spectra), the carbonyl group region (see Fig. 1.4.1). • 16 -Anomeric region Ring region High f i e l d region 1 H —n.m.r. spectrum Carbonyl region Anomeric region Ring region High f i e l d region c - o r 17S c - i £ 0 0 0 1 1 0 C - l - p o BCOR — I — 1 C 0 13 HCOH CHjOH BCN f 3 £ H 3 C K 0 « 3 C O - C - 0 £ H - f 0 C—n m.r spectrum Fig. 1.4.1: Different regions i n the n.m.r. (XH- and 1 < 3 C) spectra of polysaccharides - 17 -In the high f i e l d region (between S 1.0-2.5 in the ^-n.m.r. spectrum and S 15-30 i n the 1 3C spectrum), the methyl resonances of acetal-linked pyruvic acid, N- and 0-acetyl groups and 6-deoxy-hexoses (e.g. L-rhamnose and L-fucose) can be detected. It has been shown that the stereochemistry (R or S) of the acetalic carbon of the pyruvic acid can be differentiated by the chemical shift of the methyl groups. 5 8 In the ring region, the ^H-n.m.r. spectrum, (fi 3.0-4.5) is not well resolved and therefore assignments are d i f f i c u l t . Using 2D n.m.r. experiments 5^-^ chemical s h i f t assignment in the ring region can be simplified. In contrast to ^H-n.m.r. spectra, the ring region (6 60-85) of 1 3C is more resolved due to a larger sweep width. O-Glycosylation and/or O-alkylation results in the carbon atom(s) involved being deshielded (by 7-11 ppm) so as to produce signals well separated from other ring carbons (6 80 ± 5). This is called the "a-effeet". However a carbon atom immediately adjacent to that carbon w i l l be slightly shielded (1-2 ppm), and this i s the "^-effect". These a and 0-shifts (which are different from a and /3-anomeric signals) have been used in the assignment of 1 3C signals of oligo- and polysaccharides and consequently delineating the structural sequence.^3 The anomeric region of -^ H-n.m.r. spectra i s S 4.5-5.8 and that for 1 3C is S 93-110. Contrary to ^H-n.m.r. the o anomeric carbons appear upfield from the /9-anomeric carbons due to a shielding effect. It has been found that increased shielding of a 1 3C nucleus is accompanied by a decrease in the shielding of the appended proton, i.e. 1 3C and H^ shift are affected inversely.^ 3 In the n.m.r. ( 1 3C and ^H) spectra of an oligo or polysaccharide, the number of anomeric signals and their correspond-- 18 -ing integrals indicate the number of sugar residues per repeating unit; furthermore the linkage configuration (a and 0) can be determined from the combined measurements of the chemical shift and coupling constant. Signals for some non-anomeric protons have been reported to occur in the anomeric r e g i o n ^ ' ^ 5 but this ambiguity can be resolved by 2D homonu-clear spin correlated n.m.r. experiments.5^.60 For 1 3C spectra, any signal in the extreme downfield region of S 170-180 indicates the presence of a carbonyl group which could be associated with N or 0-acyl group, pyruvic acid or uronic acid. ( i i ) The relative area or integral of individual signals The relative intensities of absorption signals for different nuclei are equal to the relative number of the nuclei producing the s i g n a l s . ^ The number of anomeric linkages, relative amounts of 6-deoxy sugars, 0-acetyl, N-acetyl and 1-carboxyethylidene substituents can be deter-mined by computing the integrals of their corresponding signals. For oligo or poly-saccharides this parameter permits a rapid quantitative analysis of the ratio of a and f) linkages. However, quantitation based on signal integration is often not reliable in the proton-decoupled 1 3C spectrum due to saturation and n.O.e. effects. Nevertheless, comparison of integrals of 1 3C nuclei with the same number of hydrogen atoms often yields accurate information about their relative amounts. - 19 -( i i i ) Coupling Spin-spin coupling is a scalar coupling which is administered through bonds. In the simplest cases (referred to as first-order spectra) these features occur as s p l i t t i n g of the resonance signals due to each coupling nucleus. If there are only two nuclei with non-zero spin in a molecule under consideration, having spins 1^ and I2, then i t is found that the resonance of spin 1^ is split-into 2I2 + 1 lines of equal intensity and that of spin I2 is similarly s p l i t into 21^  + 1 lines. From spin-spin coupling patterns structural information on nuclear environment may be obtained. The magnitude of the spin-spin coupling is given by a spin-spin coupling constant (usually simply referred to as a coupling constant and written J j ^ for interaction between spins j and k). The variation of the spectrometer operating frequency does not affect the magnitude of spectral s p l i t t i n g (i.e. coupling constant) as such coupling constants are always expressed in Hertz (Hz) and never in ppm. In a f i r s t order spectrum, the magnitude of the coupling constants can be measured directly from the. spectrum. Coupling constants can also be predicted using the Karplus^ qua-tion and these values are usually in good agreement with observed values. However, deviations occur when substituents are of different electronegativity.^® The Karplus equation gives an approximate relationship between the three-bond v i c i n a l coupling constant ( 3J) and the dihedral angle (<f>) between the protons. 8.5 cos 2^ - 0.28 0° < <(> < 90° 3 J (HI, H2) - or 9.5 cos 2^ - 0.28 90° < <f> < 180° - 20 -The values are maximum when the dihedral angle (4>) is 0° or 180°, and minimum when i t is 90°. Although the coupling constant is influenced by other parameters (such as electronegativity, angle strain and bond length), i t is useful for the assignment of trans-diaxial protons (<f> — 180°, ^-linked, 3 J i t 2 7-9 Hz) and equatorial-axial or equatorial-equa-t o r i a l protons (<f> — 60, a-linked, 3 J ^ 2 I " 3 Hz). These 3J-values can be applied to hexopyranoses such as D-galactose, D-glucose, D-glucuronic acid and 2-acetamido-2-deoxyhexopyranoses (e.g. GlcNAc and GalNAc). Due to the equatorial proton at C-2, L-rhamnose and D-mannose have a different set of 3 J values (i.e. a-anomer 3 J ^ 2 = 2 Hz, ^-anomer 3 J ^ 2 "* 1 Hz). The J constants of anomeric protons are thus useful in predict-ing the anomeric configuration (a and /?) and the overall conformation (pyranose/furanose and chair/boat forms) of the monosaccharides. Most of the ^ 3C spectra presented in this thesis were recorded in proton-decoupled mode. However, -^3C spectra may also be correlated with the proton spectra to provide information on ^3C--'-H coupling constants (^Cl.Hl) This is usually performed by the 'gated' decoupling or by the single frequency off resonance' decoupling technique. The ^"Jci HI value is useful in differentiating anomeric pairs in the pyranose form, since they differ by 10 Hz,^>^ e.g. ^JC1,H1 ^ o r a 0-rhamnoside is -160 Hz and ^"Jfjl HI ^ o r a n a-rhamnoside is -169 Hz. In the structural studies on the E. c o l i K31 polysaccharide the anomeric configuration of the six sugar residues in the repeating unit were confirmed by their ^JQI h1 v a l u e s • Recently Bock and Pedersen reported the determination of ^ c i HI v a l u e s of some carbohydrate samples through the measurements of 1 3 C s a t e l l i t e signals in ^ -n.m.r. spectra (500 MHz). 7 0 - 21 -II. METHODOLOGY E. c o l i capsular polysaccharides have complex and immensely diversified structural patterns 1^ the structural elucidation of which necessitates the use of different chemical methods as well as spectros-copic techniques. Since the structure of these polysaccharide immunog-ens i s intimately linked to their effectiveness as vaccines, the development of a technique for rapid structural determination would be invaluable. In this thesis, It has been demonstrated that bacteriophage borne enzymes and two dimensional nuclear magnetic resonance (2D-n.m.r.) spectroscopy are powerful tools in such an investigation. The methodol-ogy of the two dimensional n.m.r. techniques employed in this study is illustrated in this chapter. The structure of the E. c o l i polysaccha-rides determined by 2D-n.m.r. were f i r s t elucidated by chemical and mass spectrometric techniques. An overview of these techniques is also presented in this chapter. II.1 Pulse Fourier transform nuclear magnetic resonance spectroscopy (F.T.-n.m.r.) N.m.r. is quite insensitive compared to optical spectroscopy (e.g. UV or IR) because of the very small population excess i n the lower energy state. One way to improve the sensitivity is by signal averaging in which case the rate of scan of the spectral width is very important. In F.t.-n.m.r. the rate of scan of the spectral width far super-- 22 -cedes that of continuous wave (CW) n.m.r. hence more experiments can be accomplished using the F.t.-n.m.r. technique. The introduction of the F.t. method has not only enhanced the sensitivity of high resolution n.m.r. spectroscopy, i.e. allowing measurements to be made on less sensitive nuclei of the periodic table, but also has paved the way for the development of a large number of new experimental techniques. The use of programmable pulse transmitters and the separation of the experiment into preparation, evolution and detection have made new n.m.r. experiments possible. In particular the concept of two-dimensional (2D) spectroscopy has opened up new p o s s i b i l i t i e s Important for the analysis of complicated spectra and is able to provide informa-tion not otherwise accessible. In structural studies of carbohydrates, one dimensional (ID) n.m.r. is mostly used as a backup procedure to chemical methods. The ring proton region (-4.5 ppm to -3.5 ppm) of lD-n.m.r. spectra i s quite com-plicated but may be simplified using 2D-n.m.r. spectroscopy. 1 1 8-120,149 E. c o l i polysaccharides are immunogens and quick structural elucidation of these immunogens may be necessary. The use of 2D-n.m.r. spectroscopy is suitable for this purpose. The use of 2D-n.m.r. in the structural studies of the capsular polysaccharides from the E. c o l i serotypes K31, K33, and K46 is demonstrated In this study. An overview of the n.m.r. methods used i s presented in this chapter. Since a rigorous mathematical treatment i s complicated and does not necessarily improve the comprehensibility, the chapter attempts to give an i l l u s t r a t i v e presentation of these techniques within the framework of the Bloch vector model. - 23 -III.1.1 The FT-n.m.r. e x p e r i m e n t 1 5 0 ' 1 5 1 ' 1 5 2 The basic principle of the Fourier transform n.m.r. experiment is recalled i n this s e c t i o n . 1 5 0 ' 1 5 1 ' 1 5 2 For the nucleus of interest, the resonance signals of different Larmor frequency present in the spectral window chosen form the so-called macroscopic magnetization (M) of magnitude Mo, parallel to the external f i e l d B Q (Fig. II.l a ) . The strong radio frequency (RF) p u l s e 1 5 1 produced by a radio frequency c o i l on the X-axis carries M away from the Z-axis to the X, Y plane. The duration and the power of the RF pulse determine the direction of M after the pulse (with the power of the RF sources used in modern spectrometers this process requires 5-20 Ms)• If a so-called 90° x or w/2 pulse is applied, M points along the positive Y-axis (Fig. I l . l . b ) . The longitudinal or Z-magnetization is thus transformed into a trans-verse magnetization. Within the framework of the classical macroscopic description of experiments, not only i s the macroscopic magnetization M a sum of components but also each individual vector i s the vector sum of the various nuclear magnetic moments that have the same chemical environment and therefore precess with the same Larmor frequency. The Larmor frequencies of the various nuclear magnetic moments present vary and thus, the vector M splits into i t s components (Fig. II.Ic). The concept of the "rotating frame" 5 3 is very convenient when describing an F.t.-n.m.r. experiment. It uses a coordinate system K 1 that rotates in the same sense and with the same frequency vQ as the rotating f i e l d vector of the RF f i e l d . In the rotating frame, vectors that correspond 24 O) 1 b) i x y X c) d) s a) Macroscopic magnetization M in the laboratory frame; b) transverse magnetization Mx v after a 90° x pulse; the effect of pulses is described by the "right-hand rule", vhere the thumb gives the direction of the pulse and the . bent fingers the rotational sense of the magnetization vector; c) Larmor precession of the individual nuclear magnetic moments of different Larmor frequency in the laboratory frame; d) as c), however, i n the rotating frame K'(x',y',z) (taken from R. Benn and H. Gunter's a r t i c l e in Angew. Chem. Int. Ed. Engl., 22, 350-380, 1983) Fig. II.1: e) Free induction decay (FID) or time signal of a n.m.r. line. The damped sine vave i s characterized by the time constant T£* and the frequency v± T2* i s the effective transverse relaxation time that contains contributions from transverse relaxation and from f i e l d inhomogeneity (taken from R. Benn and H. Gunter's a r t i c l e i n Angew. Chem. Int. Ed. Engl., 22, 350-380, 1983) e) u - 25 -to signals with frequencies > uQ rotate clockwise, whereas those corresponding to signals with < vQ rotate anti-clockwise, a signal with i>i = uQ is static in the rotating frame (Fig. III.Id). It is helpful to remember that each vector is characterized by i t s Larmor frequency, by i t s orientation in the rotating frame and by i t s l i f e time. The Larmor frequency determines the position of the signal in the spectrum (i.e. chemical shift) whereas the orientation at the beginning of data accumulation determines the phase with respect to the rotating frame and hence signal phase. The magnetic vector rotating in the X-Y plane produces a voltage in the receiver c o i l that is detected as the n.m.r. signal. The loss of transverse magnetization is governed by transverse relaxation processes as well as f i e l d inhomogeneity. A plot of the resultant damped os c i l l a -tion with decay time is the free induced decay (FID) (Fig. II.Ic). The induced voltage is converted to numerical form by analogue-to-digital converters. Fourier transform of this time domain data yields the well known n.m.r. signal or spectrum. II.1.2 13C-n.m.r. spectroscopy via spin echo experiments. (Attached  proton test) The modulation of transverse magnetization through spin-spin coupl-i n g 1 ^ ' ^ - ^ can be used in a simple manner in 13C-n.m.r. spectroscopy via spin echo experiments with gated ^H-decoupling to distinguish between signals of quaternary C atoms and CH, CH2 and CH3 groups.^5.178,179 I n - 26 -i t s simplest form, known as SEFT, 1 / B' i / y i t yields singlets that d i f f e r by 180° i n their phase. The SEFT pulse sequence which relies on spin echo modulation through *J ( 1 3C, *H) coupling is shown. PW 1B0 180 *•#* •»****» ******* OBSERVE Dl # * D2 * # D2+D3 * * D3 ACQUISITION *•**«#*** ****** ********* ************ *********** ******************************** DECOUPLE * * ****** During the D2 + D3 period transverse magnetization i s modulated through spin-spin coupling to the proton. When D2 + D3 - ^-/j i t i s possible i n only one experiment to distinguish between signals of quaternary and methylene carbon atoms, on the one hand, and those of methine and methyl carbons on the other, since for both groups of resonances a phase difference of 180° exists. The signals of quaternary carbon atoms and those of CH2 groups have a positive and those of CH and CH3 groups a negative phase. In carbohydrate research the SEFT (attached proton test) experiment is used to differentiate a carbon atom at position 6 (i.e. CH2) from the other ring carbon atoms (i.e. CH) of a sugar residue. Thus from this experiment the investigator i s able to deduce the number of sugar residues i n the repeating unit of a polysaccharide that are non-deoxy at C-6. This i s well documented i n Chapter four of this thesis. - 27 -II.1.3 Basic theory of two dimensional n.m.r. spectroscopy The common feature of pulse F.t.-n.m.r. experiments is the time sequence preparation - evolution - detection shown in Fig. II.2. This time sequence also forms the basis of two-dimensional n.m.r. spectro-scopy (2D n . m . r . ) ; * 1 5 5 • w i t h the important difference, however, that the evolution time t^ within a sequence of pulse cycles is now a variable. In 2D-n.m.r. experiments the receiver signal is also dependent on the evolution period t^ because over n experiments each t^ is increased by a constant time increment At^. The receiver signal is thus a function of t^ and t2. Fourier transformation with respect to t£ i n i t i a l l y yields n conventional spectra whose data points on the time axis t^ define the modulation frequency, which can be determined by a second Fourier transformation (Fig. II.3). Two frequency variables F^ and F2 are obtained in 2D-n.m.r. experiments. The f i r s t Fourier transformation with respect to t£ (F2) yields the resonance frequency and the second Fourier transformation with respect to t^ the modulation frequency (i.e. coupling constant, chemical shift due to coherence transfer etc.). For the graphical display of 2D-n.m.r. the procedures currently used are stacked plot 1-^» 1 5 8 and contour p l o t . 1 5 ^ The stacked plot i s , in most cases, aesthetically appealing, but often badly arranged and therefore d i f f i c u l t to analyze. The contour plot is a cross-section through the stacked plot parallel to the X,Y-plane at a chosen height. In this way one obtains a clearly arranged diagram of contour lines that is easy to analyze, i n particular for correlated spectra. - 28 -Preparation Evolution Fig. II.2: Time sequence of modern pulse experiments Fig. II.3: Two-dimensional n.m.r. spectroscopy through amplitude and phase modulation (a, b respectively). The figure shows the situation after the f i r s t Fourier transformation. The modulation of the signals results from a periodic perturbation of the spin system during the evolution period. Repeated Fourier transformation of the time signal S(tj_) yields the frequency F^ (taken from R. Benn and H. Gunter's a r t i c l e in Angew. Chem. Int. Ed. Engl. 22, 350-380, 1983). - 29 -II.1.4 Two dimensional spin correlated n.m.r. spectroscopy Maudsley and E r n s t ^ 0 were among the f i r s t to propose and realize two dimensional spin correlated n.m.r. experiments. Experimentally they require an additional time interval, the so-called mixing time, 1*' 1' 1 1 9 between evolution and detection periods. In correlated 2D-n.m.r. spectra both frequency axes (F^ and F2) contain chemical shifts. Two types of 2D correlated spectra can be distinguished: both dimensions may be coupled through coherent transfer of transverse magnetization (scalar correlation 5 9"*' 2•160) o r through incoherent transfer of magnetization (two-dimensional n.O.e. s p e c t r a , 1 1 9 two-dimensional chemical exchange spectra). II.1.4a Homoscalar-correlated 2D-n.m.r. spectroscopy Two-dimensional correlations for homonuclear spin systems (for example A = X = 1H) are known as COSY 5 9- 1 1 8 and SECSY60. Both frequency axes F^ and F2 contain the Larmor frequencies of the same nucleus, e.g. the 8 (1H) values. The COSY experiments done in this study involved using pre-saturation of solvent With two power levels (i.e. SI and S2). In these experiments a modified Bruker program1*"2 with a pulse sequence D1-D3-90°X-D0-D2-90°X-F1D HG(S1)-(S2) DO (see appendix II) was used. After the preparation period (i.e. DI and D3), a 90° x pulse produces transverse magnetization. The magnetization - 30 -vectors precess in the X,Y plane according to their Larmor frequencies and their spin-spin coupling constant J . For the case (J(A X) 1* ^ ) > the A magnetization through the scalar A,X- interaction in the x,y plane also depends on the Larmor frequencies I/JJ and v^. The 2D spectrum therefore contains the characteristic off-diagonal signals (cross peaks) with coordinates 6% a n d $x ^ A« which indicate a scalar spin-spin coupling between A and X. The coupling scheme of each monosaccharide can be obtained using proton-proton 2D chemical shift correlation spectroscopy (COSY)^ 3 based on the fact that v i c i n a l protons exhibit off-diagonal cross peaks in the 2D spectrum due to scalar coupling. The pattern of coupling for a sugar residue can be traced out using an unambiguous assignment (e.g. H-l) as the starting point. The overlap of proton resonances often leads to ambiguities in establishing connectivities in the COSY spectrum. These can be resolved using multiple-relay-COSY, in which correlations are transferred from nucleus to nucleus within a spin system. In this study (Chapter A) H-l of each sugar residue was identified by inspection, H-2 as a cross-peak in the COSY spectrum and the remaining protons were identified by a series of multiple-relay-COSY experiments. Another method for investigating homonuclear spin systems is spin echo correlated spectroscopy (SECSY),^0 in which the frequency differences (Fj^ axis) are plotted against chemical s h i f t (F2 axis). Proton assignment of spectra containing severe spectral overlaps have been made using the homonuclear Hartman-Hahn (HOHAHA) method^^ • 169 which provides high-resolution phase-sensitive spectra that display both direct and relayed connectivities. - 31 -II.1.4b Heteronuclear correlated 2D-n.m.r. spectroscopy In heteronuclear correlated 2D-n.m.r. (HETCOR) experiments 1 2 0 the Larmor frequencies of two different types of nuclei e.g. and 1 3C are related through scalar coupling. The fundamental concept of heteronuclear correlated (HETCOR) experiments has been documented by Benn and Gunter. 1 6 5 The HETCOR (*H and 1 3C) map consists of -^H chemical shifts along the axis and the 1 3C chemical shifts along the F2 axis. Various types of connectivity can be investigated using these shift-correlated experiments, e.g. one-bond couplings, long-range couplings or delayed correlation. The relative insensitivity of these experiments has been dramatically improved by the introduction of new ^-detected 1H- 1 3C correlation maps 1 6 4' 1 6 6- 1 6 7 suitable for f u l l spectral analysis of small quantities of oligosaccharides (e.g. 3.5 mg sample of a trisaccharide1*'*') and polysaccharides (e.g. 10 mg sample of Haemophilus  influenzae type capsular polysaccharide 1*" 7) . Homonuclear 2D-correlated experiments (e.g. COSY, multiple relay COSY) can provide most of the 1H assignments required for identification of the sugar residues. Once the % signals have been identified, the 1 3C assignments follow directly from 1H- 1 3C heteronuclear chemical shift correlation spectroscopy (HETCOR). Alternatively, i f the 13C-n.m.r. spectrum is assigned then the HETCOR experiment can be used to interpret the spectrum. •LH--LJC three-bond coupling ( Jfl-C) from the anomeric proton across the oxygen atom to the aglycone carbon skeleton could provide informa-tion on linkage s i t e . 1 * ' 8 ' * ' 3 ' 1 7 0 Recently the selective INEPT experiment - 32 -has been used to detect and measure intra- and inter-residue long-range ^H-l^C c o u p l i n g s . T h e sensitivity of these methods has been greatly improved by indirect observation of ^ 3C via *H detection.^®•1*>7 Coupling across the glycosidic bond may possibly be detected also from the heteronuclear analogue of the relay-COSY e x p e r i m e n t . j n this study, the investigator only had access to a spectrometer lacking reverse detection capability and this technique, therefore, could be not be employed. II.1.5 Sequence analysis using —H-n.m.r. methods Conformational analysis of oligosaccharides has shown that the exoanomeric effect operates such that the anomeric and aglyconic protons of glycosidic bonds are in virtual van der Waals contact.^3,174 This makes possible the use of nuclear Overhauser effect (n.O.e.) experiments to yield intra and inter-ring couplings, because the arrangement of the protons is suitable for dipolar coupling. This type of experiment can either be done in ID, usually as n.O.e. difference spectroscopy, or 2D (NOESY) 1 1 9. The contour map obtained for a NOESY experiment is analogous to the COSY plot, except that the cross-peaks are due to dipolar coupling. The identification of inter-ring coupling in the n.O.e. experiment 1 7^ may be obtained by comparing COSY and NOESY plots. The success of these n.O.e. experiments requires that the molecule being examined should experience a measurable amount of n.O.e. and that the pertinent signals are adequately resolved. Compounds of intermediate - 33 -molecular weight that experience a detectable amount of n.O.e. without losing spectral resolution are most amenable to these studies. In this investigation low molecular weight polymer generated by bacteriophage-borne glycanase degradation was employed in the NOESY experiments. The usual way of sequencing polysaccharides is by examining the oligosaccharide fragments derived from them. These fragments are more suitable to spectroscopic studies than their parent polymers, but experience small n.O.e. effects at high fields. The problems associated with n.O.e. investigations of small water-soluble molecules can be diminished by using spin-locked n.O.e. spectroscopy 1*^ or by derivatization (e.g..O-acetylation) 1 7-* and use of an organic solvent. In delayed COSY experiments, scalar coupling (^JHH) between the anomeric and aglyconic protons may provide sequencing information. 1 7 1 This approach is complicated by interference from five-bond coupling. II.2 Chemical methods The methodology of structural studies of polysaccharide using conventional chemical methods includes: i ) qualitative and quantitative estimation of the sugar constituents i i ) analysis for non-carbohydrate substituent groups (O-acetyl, N-acetyl, phosphate etc.) i i i ) determination of the linkage configuration iv) determination of the position of linkage v) determination of the sugar sequence - 34 -Some known analytical methods employed for these goals are described in the following sections. II.2.1 Isolation and p u r i f i c a t i o n 7 1 ' 7 5 A major task in polysaccharide chemistry is obtaining the material under investigation i n a pure form. In this study the polysaccharide of interest i s the K antigen. The isolation and purification process involves three stages: i) bacterial growth and harvest of crude polysaccharide components; i i ) the isolation of the polysaccharide such that i t is free from low molecular ' weight material and other high molecular weight material, and i i i ) Isolation of a single, monodispersed, polysaccharide species. The purity of the polysaccharide, the absence of heterogeneity, rather than the presence of homogeneity can be demonstrated by many independent techniques such as nuclear magnetic resonance spectroscopy, optical rotation measurements, e l e c t r o p h o r e s i s 7 1 , 7 2 and gel-permeation chromatography.73 Escherichia c o l i bacteria serotypes K31, K33, K44 and K46 were received as stab cultures from Dr. Ida Orskov (Copenhagen). These bacterial cultures were streaked on Mueller-Hinton agar plates at 37°C u n t i l large, Individual capsular colonies were obtained. E. c o l i K31 was grown by inoculating a Mueller-Hinton broth medium with single - 35 -colonies and incubating the resultant li q u i d culture at 37°C on Mueller-Hinton agar for four days. The lawn was scraped off and the capsular bacteria were k i l l e d by adding phenol solution and s t i r r i n g the mixture for five hours. The phenol in this mixture (polysaccharide component plus bacterial debris) was dialyzed out. The polysaccharide components were separated from the high molecular components (e.g. bacterial cells) by ultracentrifugation. Isolation from aqueous solution by the addition of a water-miscible solvent 7^ (e.g. ethanol, acetone) resulted in the precipitation of neutral and acidic polysaccharides. The resultant stringy precipitate was dissolved in water and treated with Cetavlon7-* (cetyltrimethylammonium bromide) solution, which selectively precipitated the acidic polysaccharide. The neutral polysaccharide present remained in solution and the precipitate (acidic polysaccharide) was separated from the supernatant by centrifu-gation. Dissolution of the precipitate in 4M sodium chloride, precipitation with ethanol and centrifugation were carried out twice. The f i n a l precipitate was dissolved in water, dialyzed for two days and freeze dried to y i e l d the purified K31 capsular polysaccharide. Isolation and purification of E. c o l i serotypes K33, K44 and K46 were carried out as described for E. c o l i K31. Chromatographic techniques such as gel-permeation and ion-exchange chromatography may be used to enhance the purity and homogeneity of capsular polysaccharides. Traces of low molecular weight contaminants in the capsular polysaccharides were removed by gel-permeation chromatography (Bio-Gel P2). - 36 -II.2.2 Sugar analysis II.2.2a Total h y d r o l y s i s 7 6 ' 8 1 The i n i t i a l step in the structural study of a polysaccharide is the quantitative acid hydrolysis of the polysaccharide into individual monosaccharides with minimum degradation. Dutton 7 6 reviewed the advantages and disadvantages in the use of different acids. Sulfuric acid, hydrochloric acid, formic acid and trifluoroacetic acid are the most commonly used acids. Trifluoroacetic acid is easily removed under diminished pressure and thus is being used increasingly instead of the mineral acids. The conditions of hydrolysis must be carefully chosen and controlled. To. attain the correct hydrolysis condition the hydroly-zate may be monitored by paper chromatography high performance liquid chromatography (h.p.l.c.) or gas liquid chromatography (g.I.e.). The quantitative hydrolysis of E. c o l i K31 polysaccharide into i t s individual monosaccharides with minimum degradation was attained using 2M trifluoroacetic acid for 20 hours. The hydrolytic rates of glycosidic linkages vary greatly. Uronosyl linkages in acidic polysaccharides are more resistant to acid hydrolysis because of the presence of electron acceptor carboxyl groups which stabilize the uronosyl linkages through the heterocyclic oxygen. One of the means of overcoming the resistance of the uronosyl linkage to acid hydrolysis, is the reduction of a l l carboxyl functionalities in the acidic polysaccahride by conversion into carbodiimide 7 7 derivatives followed by sodium borohydride reduction. An alternative method of - 37 -reduction developed in our laboratory 7^ to overcome the resistance of uronosyl linkages to acid hydrolysis, involves methanolysis of the glycosyl linkages with the simultaneous esterification of the carboxylic acid, which is then reduced to the corresponding alcohol. The resulting mixture of neutral glycosides is hydrolyzed with 2M trifluoroacetic acid, to ensure complete release of sugar residues. Methanolic hydrogen chloride has the advantage of being less destructive to deoxy sugars and s i a l i c acids.80,81 Karunaratne has discussed the hydrolytic conditions for the hydrolysis of polysaccharides containing amino sugars7® which w i l l therefore not be duplicated in this thesis. II.2.2b Characterization and quantification of sugars / b»° Z'^ J-The sugars released upon acid hydrolysis can be analyzed qualitatively using paper chromatography,®2'®3 high performance li q u i d chromatography,®^ paper electrophoresis,®5 and thin layer chromatogra-phy.®^ Colorimetric®7'®® analysis can be used in the classification of sugars into broad classes (hexoses, pentoses, uronic acids, deoxy or amino sugars and s i a l i c acid) but has limited applications for individ-ual characterization. High performance li q u i d chromatography (h.p.l.c.) can also be used for quantitative analysis of underivatized sugars. H.p.l.c. however has a limited number of stationary phases compared to gas-liquid chromatography. The monosaccharides, released upon acid hydrolysis of a polysaccha-ride, can be converted to volatile derivatives and analyzed using - 38 -gas-liquid chromatography ( g . l . c ) . G.l.c. offers reproducible quanti-fication and characterization of sugar residues of polysaccharides. An extensive review of this technique has been reported by Dutton. 7^' 8 9 Sugars can be analyzed as their v o l a t i l e trimethylsilyl (TMS) deriva-tives but the existence of anomeric forms of sugars at equilibrium yields a complicated chromatogram of multiple peaks. This problem was solved by converting the acyclic sugar alditols into the vo l a t i l e acetates, trifluoroacetates or trimethylsilyl ethers. A l d i t o l trifluoro acetates show parti a l de-esterification on the column and the TMS derivatives of the alditols give poor r e s o l u t i o n 9 0 on g.l.c. A l d i t o l acetates have good resolution and short retention times 9 1 and therefore, were used throughout this investigation. Sugars separated by g.l.c. are usually confirmed by g.l.c.-mass spectrometry. II.2.2c Determination of the configuration (D or L) of sugars^~* 3 In general, chromatographic separation methods and spectroscopic analyses do not distinguish between enantiomers. However enantiomers can be separated by g.l.c. using a chiral column or converting the enantiomers into diastereomers using chiral reagents (for example, (-)-2-butanol, (+)-2-octanol, or (+)-1-phenylethanethiol) and separation on a non-chiral phase. 9 2» 9 3» 9^ The D and L configuration of sugars can be determined by the isolation of the different monosaccharides and measurement of their optical rotation [a]n. Specific oxidases (e.g. D-glucose and - 39 -D-galactose oxidases) and enzymes can be used for the determination of D and L configuration of sugars. The method employed i n determining the D and L configuration of sugar i n this investigation was by circular dichroism^ 7 measurement at 213 run on a l d i t o l acetates, acetylated aldononitrile or pa r t i a l l y methylated a l d i t o l acetates, where the acetoxy group acts as a chromophore. II.2.3 Position of linkage II.2.3a Methvlation a n a l y s i s 9 6 This technique involves the complete etherification of the free hydroxyl groups of the sugar residues i n oligo- and polysaccha-r i d e s 9 6 , 9 7 which acts as a label in distinguishing the original unlinked positions from the linked position. Methylation analysis i s routinely employed in the structural characterization of complex carbohydrates as a means to establish (i) linkage positions; ( i i ) number and types of sugar per repeating unit; ( i i i ) identity of terminal unit(s), branching unit(s); and (iv) the position of base-stable substituents (e.g. pyruvate). In the early days, methyl ethers were formed by repeated reaction with dimethyl sulfate and sodium hydroxide.9® Treatment of a polysaccharide with silve r oxide in boiling methyl iodide according to Purdie and I r v i n e 9 9 gives a f u l l y methylated polysaccharide. Purdie's - 40 -method was considerably improved by Kuhn and colleagues 1 0 0 who used N,N-dimethylformamide as a solvent in conjunction with methyl iodide and silver oxide. A more convenient method for methylating polysaccharides was devised by Hakomori. 1 0 1 This involves the treatment of a polysac-charide with sodium methylsulfinyl methanide (dimsyl sodium) and methyl i o d i d e . 1 0 2 Most undermethylations, by the Hakomori method or not, are due to incomplete dissolution of the sample. The solubility of a polysaccharide in the appropriate organic solvent may be enhanced by careful de-ionization of the polysaccharide, (for example, using Amberlite IR-120 (H+) resin). In cases where the Hakomori methylation gives an "undermethylated" product, complete methylation can be achieved by using the Purdie method. A. second Hakomori methylation is never conducted on a methylated acidic oligo- or poly- saccharide as this w i l l result in /9-eliminatiori. Methyl trifluoromethane sulfonate is a milder base and, in the presence of 2,6-ditertiarybutylpyridine and trimethyl-phosphate as s o l v e n t , 1 0 3 effects methylation without cleavage of acyl groups. Polysaccharides containing uronic acids may be reduced with lithium aluminum hydride after the permethylation step. The methylated material is usually purified by dialysis, extraction and gel-permeation chromatography (Sephadex LH 20). The completeness of methylation can be verified by i . r . spectroscopy (absence of hydroxyl absorption at 3600 cm"1) or by analysis of the methoxyl content. The methylated polymer is usually hydrolyzed using 2M trifluoroacetic acid at 95°C for about 18 h. The pa r t i a l l y methylated monosaccharides released on hydrolysis are reduced to their alditols and acetylated to give vo l a t i l e p a r t i a l l y methylated a l d i t o l acetate derivatives for - 41 -g.l.c. and g.l.c.-m.s. analyses. Uremic acids may be identified by comparison of methylation analysis results of the acidic polysaccharide with those of the methylated-reduced polysaccharide. II.2.3b Characterization and quantitation of methylated  sugars 7 6 •89,104.10.5,44 Partially methylated monosaccharides released on total hydrolysis of the permethylated oligo- or polysaccharide can be characterized using paper chromatography.104 The methylated sugars are detected with p_-anisidine hydrochloride spray^ following by heating at 110°C for 5 min. These sugars are then characterized according to their relative mobilities (Rf values) and the different colours formed. Gas-liquid chromatography (g.l.c.) is the most widely used technique for quantitative and qualitative analysis of methylated sugars. A review of the applications of g.l.c. to carbohydrate analysis has been published by D u t t o n . 7 6 , 8 9 The methylated sugars were analyzed as their p a r t i a l l y methylated a l d i t o l acetates during this work. The identifica-tion and quantitation of these par t i a l l y methylated a l d i t o l acetates were made by consideration of the relative retention times and co-chromatography with authentic samples. For unambiguous identification of p a r t i a l l y methylated a l d i t o l acetates, g.l.c. results should be confirmed using g.l.c.-m.s. Studies done on the fragmentation of par t i a l l y methylated a l d i t o l acetates have been reported.105 The primary fragments are formed as a result of . 42 -fission of the C-C bond in the al d i t o l chain and this cleavage follows the preferential order indicated. CHOMe > CHOMe The intensity of the primary ion decreases with increasing molecular weight. Secondary ions can be obtained by loss of acetic acid (m/z 60), ketene (m/z 42), methanol (m/z 32), methyl acetate (m/z 74), methoxy-methyl acetate (m/z 104) or acetoxymethyl acetate (m/z 132). Fig. II. 1 illustrates the differences in the mass spectra of 1,3,4,5-tetra-O-acetyl-2,6-di-O-methyl-D-glucitol and 1,2,5,6-tetra-0-3,4-di-O-methyl-D-glucitol. II.2.4 Sugar sequence The elucidation of the sequence of sugars in a polysaccharide involves the isolation and characterization of oligosaccharide fragments. Lindberg and coworkers 1 0 6 have reviewed the various techniques employed in the specific degradation of polysaccharides. The degradation techniques employed in this investigation are discussed in this section. CHOMe CHOAc > CHOAc CHOAc - 43 -100 -i 8 0 -6 0 -4 0 -20 0 43 117 87 58 •° 74 I I I ' ? 3 IRI 1 8 5 ^ 231 305 I A . I ' M V ' I I ' F ! J'F,l"l.V.VCVirMl..f'-"'"f" 50 100 150 2 0 0 250 ^ T I1 I I I I I I I | t I I I I I I I I | I I I I I I I I I | 3 0 0 ' 3 5 0 (a) lOO-i 8 0 -6 0 -4 0 -2 0 1 0 129 43 8 7 A ! 111 j.! iA i \ i fib i ' t n ' ' I 1 1 5 0 • 100 113 189 159 i 11 i i i i £ I ^ T \ I r ? M 111 i | i i i i 11 i i i | i i i 111 111 | 150 2 0 0 2 5 0 (b) 3 0 0 3 5 0 4 0 0 Fig. II.1: Has* spectra of <a) l,3,4,5-tetra-p.-acetyl-2,6-dimethyl-g l u c l t o l . (b) l,2,5,6-tetra-0-acetyl-3,4-dlmethylglucitol - 44 -II.2.4a Periodate oxidation and Smith d e g r a d a t i o n 1 0 7 ' 1 1 2 Oxidative cleavage of the C-C bond of v i c i n a l diols by sodium metaperiodate is of importance in the structural determination of poly-saccharides 1 0 7 and i t s uses are two fold. Firs t , as an analytical technique using small amounts of material and secondly, as a preparative technique namely Smith degradation. 1 1 2 Oxidations are usually carried out In aqueous media with the water soluble metaperiodate Ion. Over-oxidation may be prevented by performing the reaction in the dark at about 4°C. 1 0 8 The periodate consumption can be monitored spectrophoto-m e t r i c a l l y 1 0 8 and the results i l l u s t r a t e the number of periodate sensitive sugars per repeating unit i n a polysaccharide. Except for terminal side-chains and 1-6 linked sugars, one mole of periodate is consumed for every oxidizable hexose in a repeating u n i t . 1 0 9 The "polyaldehyde" produced, on periodate oxidation of a polysaccharide, is usually reduced with sodium borohydride into the polyol. Cis glycols are observed to oxidize faster than trans and some trans glycol are resistant to oxidation i f fixed in an unfavourable conforma-t i o n . 1 1 0 Ebisu et a l . selectively oxidized the terminal /3-D-galacto-pyranosyl residues in the Pneumococcus S-14 polysaccharide leaving the l-*4 linked ^ -D-glucose units in the main chain i n t a c t . 1 1 1 In a similar exercise, the selective oxidation of the terminal D-glucose, and l-»4 linked galactose over l->2 linked glucuronic acid in E. c o l i K34 was achieved. 1 1 3 For analytical purpose, methylation analysis or sugar analysis is mostly performed on small quantities of the polyol. - 45 -Smith degradation is an important modification of periodate oxida-tion devised by Smith and coworkers. 1 1 2 It gives valuable information on the sequence of sugar residues in a polysaccharide. Smith degrada-tion" involves periodate oxidation followed by mild acid hydrolysis on large quantities of the resultant polyol. This mild acid hydrolysis of the polyol results in the cleavage of the acetal linkages leaving the glycosidic linkages intact. Smith degradation yields oligo- or polysac-charides and these oligo- or polysaccharides may be characterized by sugar or methylation analysis. II.2.4b Uronic acid degradation f f l - e l i m i n a t i o n t 1 0 6 ' 1 1 4 ' 1 1 5 ' 1 1 6 ' 1 1 7 Base catalyzed degradation can be employed to generate defined oligosaccharide fragments from an acidic p o l y s a c c h a r i d e . 1 0 6 • 1 1 4 • 1 1 5 The carboxylic functionality of the uronic acid i n an acidic polysaccharide is esterified on methylation. The esterified uronic acid residue is a strong electron-withdrawing group and thus enhances the acidity of the ring proton at C-5. When the methylated polysaccharide is treated with base (sodium methylsulfinyl methanide), the proton at C-5 is removed followed by a ^-elimination of the 4-0-substituent with the formation of hex-4-eno-pyranosiduronate residues. 1 1 6 The main steps of this degradation are outlined as follows: - 46 -Aspinall and Rosell have shown i n their experiments that, under conditions normally used for base degradations, complete loss of uronic acid residues occurs and that an acid hydrolysis is unnecessary. 1 1 7 The free hydroxyl group exposed after ^-elimination, can be labelled by alkylation with methyl iodide, ethyl iodide or trideuteromethyl iodide. The resultant alkylated oligosaccharide is analyzed by g.l.c.-m.s. of the p a r t i a l l y methylated a l d i t o l acetates. In the structure elucidation of E. c o l l K31, the site of attachment of the uronic acid unit was determined by comparing g.l.c.-m.s. results of the /9-elimination and that of the methylation analysis of the native polysaccharide. - 47 -II.2.4c Partial hydrolysis Partial hydrolysis followed by characterization of the product(s) is often u t i l i z e d i n structural studies of carbohydrates. The method is of particular value when a polymer contains a limited number of acid-labile glycosidic linkages, which may be cleaved without significant hydrolysis of the other glycosidic linkages. It is advisable, therefore, to perform some p i l o t experiments in order to determine optimal conditions for the par t i a l acid hydrolysis. Many factors seem to influence the rate of hydrolysis, including the ring size, configuration, conforma-tion, and polarity of the sugar as well as the size and polarity of the aglycon. 1 2 1 Capon has reviewed the rate constants for the acid-catalyzed hydrolysis of a large number of glycosides. 1 2 2 In general, furanosides are hydrolyzed more readily than pyranosides. Deoxyglyco-pyranosides are more acid labile than glycopyranosides and aminosugars. In this investigation selective hydrolysis of a side chain rhamnosyl linkage over an i n chain one was attained using 0.1M TFA for 25 min (see Chapter 4). Uronic acids are relatively resistant to hydrolysis compared to neutral sugars and graded hydrolysis of acidic polysaccha-rides leads to isolation of acidic disaccharides (aldobiouronic acids) and higher oligosaccharides. In the structural studies of E. c o l i K31 capsular polysaccharide the uronic acid degradation (/3-elimination) result was confirmed by isolation of an aldobiouronic acid (GlcA-—-Rha; see Chapter 3). Mort and co-workers 1 2 3 have reported that using anhydrous liquid hydrogen fluoride (HF), there was a large enough variation in the rates - 48 -of glycosidic bond cleavage of amino and neutral sugars at 0°C, that amino sugar linkages remained intact while those of neutral sugars were cleaved. It has also been illustrated that di f f e r e n t i a l cleavage of neutral and acidic sugars could be obtained using HF at below zero t e m p e r a t u r e s . K u o and Mort^S u s i n g extremely mild conditions (-40°) to cleave gellan gum, obtained preferential cleavage of a over fi linkages in HF and the resultant tetrasaccharide had i t s acetate and glycerate substituents intact. In the present study p a r t i a l hydrolysis using HF below -40°C was used in the structural investigation of E. c o l i serotypes K31 and K46 polysaccharides. II.2.4d Lithium ethvlenediamine degradation Mort and Bauer^G have shown that 3-linked glycosyluronic acid-containing polysaccharides can be cleaved using lithium metal dissolved in ethylenediamine. Subsequently i t was demonstrated by Albersheim et al.^27 that treatment of carbohydrates with lithium cleaves the glycosyluronic acid residues regardless of their positions of substitu-tion by other glycosyl residues. The lithium reaction is similar to a /9-elimInation In that the polysaccharide is cleaved at the glycosyl-uronic acid residues. The lithium degradation is particularly valuable because i t cleaves underivatized polysaccharides thus allowing the products to be used for both structural analysis and studies on their biological activity. This type of degradation was used in the structural elucidation of E. c o l i K31 capsular polysaccharide. - 49 -II.3 Mass spectrometry Mass spectrometry is mostly applied i n carbohydrate chemistry as a backup procedure to "wet chemical" methods. In the last two decades the growth i n chemical instrumentation has been phenomenal, largely due to the advances in the electronic industry. Mass spectrometry is playing an increasingly important role in carbohydrate research. 1 2 8 This technique is based on the fragmentation of ionized molecules and differentiation of the resulting particles by use of the mass-to-charge ratio. Mass spectrometry in carbohydrate analysis provides information on: i) the substitution patterns in pa r t i a l l y methylated a l d i t o l acetate (PMAA) derivatives i i ) the position of the methyl and methylene groups i n deoxy sugars and the amino group in amino deoxy sugars i i i ) the sugar sequence in oligosaccharides, and iv) the various forms of the monosaccharides, e.g. between cyclic and acyclic, pyranose and furanose, pentose and hexose and aldoses and ketose. Mass spectrometry, however, does not differentiate between diastereo-meric compounds, e.g. glucose, galactose and mannose. A l l mass spectrometers have four components: (a) the ion source, where the sample under Investigation i s ionized and transferred into the gas phase; (b) the analyzer, where the molecular or fragment ions are mass analyzed according to their mass to charge ratio; (c) the - 50 -detector, where the resolved ions are detected, amplified and their intensity measured; (d) the vacuum system, which provides a stable environment for the above mentioned processes. There are numerous methods for ion production and their relative importance is continuously changing with the advent of new techniques. The choice of ionization technique i s dictated by the nature of sample and information sought. The main objective is to produce as many ions as possible with a beam composition which accurately represents the structural features of the sample. II.3.1 Electron impact-mass spectrometry This is the most popular technique for generating ions i n the gas phase. An electron beam of 70 eV is the maximum for most ion efficiency curves and the excess energy acquired by the molecule causes i t to undergo single or multi-stage fragmentation. Hence, the molecular ions are often not observed in the case of carbohydrate mass spectrometric analysis. EIMS is usually performed on volatile, low mass samples like a l d i t o l acetates. Fragmentation patterns typical of pa r t i a l l y methylated a l d i t o l acetates are illustrated in Fig. II.5. Primary fragment ions undergo a series of subsequent eliminations, including ^-eliminations, to give secondary fragments, by loss of acetic acid (m/z 60) or methanol (m/z 32). Losses by a-elimination of acetic acid (m/z 60), but not of methanol, and losses via cyclic transition states of formaldehyde Prim*TY fragmentation: - 51 -CH.OAC , „ B C O M . BCOAc I BCOAc I C H , CK.OAC I , „ B C O M . ""~T." Ki it} _j BCOMe 1 8 1 I BCOAc _ CH,OMe 4 5 MrOCH Secondary fragmentation: B C — O M * BCOMe »AcOH | BCOAc CH,OMe mlt 10 & Me BC« COMe - L i CH,OMe mlt 14S CK,OAc BCOMe • B C » g M e mlt 161 •At OH - covst B C - O M * e mlt 101 • C K , 0 C H , r « « I Me • i / r 71 € B C - O M f BCOAc CH,OMe ml* 161 :<»SM* BC« I CH BCOM* mlt 101 COAc Z - < f « 0 ^ H , C H , mlt 129 100 i 80 60 40 74 43 20-« 0 -*•»* 87 II6 • 4 2 I l 6 — I 5 8 | 15^5205 » r 99 29 I42 1 5 8 - T _ 2 0 2 ; ^ 1 4 2 ,205 50 100 50 45 200 250 300 Fig. II.5: Fragmentation pathways of some p a r t i a l l y methylated a l d i t o l acetates - 52 -methoxymethyl acetate or acetomethyl acetate may also occur. The mass spectra of par t i a l l y methylated a l d i t o l acetates of 2-amino-2-deoxyhexoses have also been reported,129,130,131 t n e characteristic base peak being that of m/z 116. The primary fragmenta-tion i s dependent on the position of the acetamido group, and fi s s i o n is almost exclusively between the methylacetamido group and the adjacent methoxyl or acetoxyl group. The secondary fragments are similarly formed as illustrated in Scheme II.3.1. II.3.2 Chemical ionization (CI) mass spectrometry Chemical ionization was f i r s t introduced by Munson and Field in 1966.1^2 ionization is achieved by gas phase ion/molecule reactions. Thus the reagent gas, introduced into the ionization chamber in large excess, is ionized by energetic electrons. This is followed by ion/molecule reactions between the primary ions and the neutral reagent gas which produces the CI reagent ions. Therefore the CI reagent ions have much lower energy than the ionizing electrons. Consequently CI is a milder form of ionization, where frequently the molecular ion can be observed i n abundance. The extent of fragmentation is dependent on the reagent gas used. The most common reagent gases used i n carbohydrate research are methane and ammonia.I33 The CI ionization mode employed in this study was desorption chemical ionization (i.e. to desorb material from external probes which are positioned directly within the CI reagent ion plasma). - 53 -C.i.-m.s. i s particularly useful for analyzing derivatized oligo-saccharides and oligosaccharide alditols (i.e., C.i.-m.s. ionization is in gas phase so conversion to Volatile derivatives such as acetates, methyl esters etc. i s essential for C.i.-m.s. analysis). C.i.-m.s. was used i n the structural studies of the capsular polysaccharides for E.  c o l i serotypes K31 and K46. The mass spectrometry behaviour of permethylated oligosaccharides has been extensively investigated, 134-1135 a n ( j t ^ e fragmentation pathways proceed in a manner similar to those of the monosaccharide derivatives. Scheme II.5.II shows the fragmentation nomenclature f i r s t devised by Chizhov and Kochetkovl36 and later modified by Kovacik et al.^37 C.i.-m.s. provides relevant information on the sequences of sugar units when these are of different types. On the whole, very l i t t l e information can be obtained concerning the stereochemistry, either of individual residues or of glycosidic linkages. Nevertheless some information can be obtained on linkage types from an examination of the fragmentation pathways. II.3. 3 Fast atom bombardment - mass spectrometry (f.a.b.-m.s.) F.a.b.-m.s. is gaining a wide application In carbohydrate r e s e a r c h . T y p i c a l l y , a sample is dissolved in a suitable solvent (e.g. water, glycerol, thioglycerol, methanol or chloroform) and is introduced as a matrix onto the probe. The sample is then bombarded with argon or xenon atoms possessing 7-8 keV of energy and the positive - 54 -A Knes CH,OR RjO OR3 CH,OR, , 0 - ( ^OR, » KO-(% R.O OR, CH,OR. OR j B series CH,OR. l . O - ^ ^ - O R , R,0 OR, CH,OR. ^OOM^ O R , RjO OR; J tenes R40 CH,OR. CH=6*> OR, R,0 OR, . R.O R,0 OR, OR, CH,OR, • + I: CH=0 R4Q=s^  ^-ORi R,0 OR: CH.OR, R,0—(j^-X-OR, •CHOR, CH,OR. CH=Q MeC? R.O-•CHOR, CH,OR. CH=0 ,CH-OR, MeO: J . E tene* CH,OR. R , 0 - ^ VOR, R.O OR, R.O—( V-OR, + R.O CH, R.O OR, Scheme II.3.I: Some mass spectral fragmentation pathways for permethylated glycopyranosides - 55 -and/or negative ions are released and analyzed. The fragmentation information provided by f.a.b.-m.s. is similar to that of c.i.-m.s. The advantage of this technique is the a b i l i t y to analyze underivatized samples. Lam 1 3 9 obtained excellent sequencing information on some bacteriophage generated oligosaccharides using this technique. II.3.4 Laser desorption ionization Fourier transform ion cyclotron  resonance (L.d.i.-F.t.-i.c.r.) mass spectrometry Laser radiation is coherent, monochromatic, directional and intense. The high Intensity of a laser beam enables sample vaporization to be on a short time scale. After vaporization, c o l l i s i o n between neutral and primary ions In the plasma can lead to formation of secondary ions. The dominant process is cationization by a l k a l i i o n s . 1 ^ The most popular ionization techniques for oligosaccharide m.s. analysis are desorption chemical ionization and fast-atom bombardment (f.a.b.). These desorption techniques, give abundant sequence informa-tion, but provide l i t t l e or no information on the positions of linkage or the anomeric configuration at the linkage. Mass analyzed ion kinetic energy experiments have been used to confirm the anomeric configura-t i o n 1 ^ with some success and tandem-m.s. experiments (like c o l l i s i o n -activation dissociation) have been used to distinguish the position of l i n k a g e . j h e disadvantage of both methods is that the oligosaccha-ride, unless of very low molecular weight, must be derivatized prior to - 56 -analysis. Positive ion laser desorption .ionization Fourier transform ion cyclotron resonance spectroscopy (l.d.i.-f.t.-i.c.r.)1^3-146 j j a s been used for the mass-spectral analysis of glycosides. It has recently been reported that positive ion l . d . i . - f . t . - i . c . r . provides both sequence information and a tentative indication of some linkage posi-t i o n . 1 4 7 It has also been suggested that negative l . d . i . - f . t . - i . c . r . on underivatized oligosaccharides may provide potential information on the anomeric configuration of the individual monosaccharides. 1 4 8 - 57 -III. STRUCTURAL STUDIES OF E. COLI CAPSULAR POLYSACCHARIDES BY CHEMICAL METHODS In this section the chemical study on the K antigen of E. c o l i serotype K31 Is presented. Detailed n.m.r. study on this K antigen is reported i n the next chapter. I I I . l The structure of Escherichia c o l i K31 antigen 111.1.1 Introduction The capsular (K) antigen of Escherichia c o l i K31 has been designa-t e d 3 0 as heat stable (type A) and may, therefore, i n the absence of amino sugars, be expected to resemble those of Klebsiella. A partial structure of E. c o l i K31 has been proposed earlier by other workers 3 1 and we now report the complete structural elucidation of this antigen. 111.1.2 Results and discussion Composition. Analysis of the native polysaccharide before and after reduction 7 7 of the uronic acid gave galactose, glucose, and rhamnose in the ratios of 1.00:0.98:2.95 and 1.00:1.89:2.98, respect-ively. A composition of galactose, glucose, glucuronic acid, and rhamnose in the ratios of 1:1:1:3 was consistent with n.m.r. data - 58 -suggesting a hexasaccharide repeating unit. Methylation. Methylation 1 0 1 analyses without and with reduction of the uronic acid gave the results shown in Table I I I . l , columns I and II, from which i t may be deduced that a rhamnose residue occupies a terminal position and the glucuronic acid unit constitutes the branch point. Methylation analysis of the product (PH) obtained by selective partial hydrolysis indicates (Table I I I . l , column III) that the lateral rhamnose unit i s linked to the glucuronic acid at C-4. The glucuronic acid was shown by a ^-elimination experiment 1 1 5 to be linked to C-2 of a rhamnose unit (Table I I I . l , column IV). In order to confirm the identity of the sugar to which the glucuronic acid is linked a portion of the hydrolyzate used to determine the sugar composition of the native polysaccharide was methylated and analyzed directly by g.l.c. Subsequent to the elution of a mixture of permethylated monosaccharide methyl glycosides a single peak appeared which was shown by c.I.-m.s. to give a molecular ion + [M + NH4] - 456 consistent with the formulation of the aldobiouronic acid as GlcA — Rha (Fig. I I I . l ) . The potential of capillary columns to separate neutral and acidic materials has been noted p r e v i o u s l y 1 8 0 and avoids tedious separations on ion-exchange resins. On the basis of these methylation experiments the par t i a l structure -•3) -GlcA- (l-»2) -Rha- (1-4 Rha may be written. - 59 -Table I I I . l : Methylation analyses of E. c o l i K31 polysaccharide and derived products Methylated sugars 3 Mole % b (as a l d i t o l acetates) II III IV V VI VII 3,4-Rha 39. ,7 33. .9 43. ,6 26. ,8 - 41. ,54 27. .6 2,3,4-Rha 14. .0 16. .9 5. .1 31. .1 - 22. ,72 29. .3 2,4,6-Gal 20. .8 17. .3 16. .2 20. .4 50.9 - 21. .4 2-Glc - 13, .8 5, .2 - - 17, .54 -2,4-Glc - - 9 .0 - - - -3,4,6-Glc 18 .2 18, .1 21 .0 21 .8 - 19, .54 21, .7 2,3,4,6-Glc _ - _ - 49.1 - _ 2,3,4,6-Glc •= l,5-di-0-acetyl-2,3,4,6-tetra-0-methylglucitol etc. Values are corrected using the effective carbon response factors given by Sweet et a l . ; determined on a DB 17 column programmed for 1 min at 180" then to 250° at 2°/min. I, K31 polysaccharide; II, K31 polysaccharide, uronic ester reduced after methylation; III, product PR from selective pa r t i a l hydrolysis; IV, product from ^-elimination and remethylation; V, product A2 from HF hydrolysis; VI, product from chromium trioxide oxidation; VII, product F l from Li-ethylene-diamine degradation. - 60 -The configuration of the rhamnose was established as L and of the other sugars as D by comparison of the circular dichroism spectra of their methylated derivatives with standards.^ 5 COOr/e M e Q KOMe / I I I MeO | 223|249 R=Me, [M+NH44]=456 i n •i •< I0I 2 0 1 * n * I * ( l a T i M < O a u 7 5 u l 3 7 5 1 6 9 2 4 9 11111 I"I 11 \ i ,', <• 4 5 6 111111 * 1 1 1 0 0 2 0 0 1111 I ' I 1111 i \ 1 1 i ri 111 1111 1111 i 3 0 0 4 0 0 5 0 0 Fig. I I I . l : C.i.-mass spectrum and fragmentation pattern of methylated aldobiouronic acid (GlcA — Rha) Llthium-ethvlenediamine degradation. The polysaccharide was treated with lithium In ethylenediamine and the reducing oligosaccharide (Fl) present i n the mixture was isolated by gel-permeation and paper chromatography. N.m.r. spectroscopy (^ H and 1 3C) and methylation data (Table I I I . l , column VII) showed the product F l to be a tetrasaccharide - 61 -containing two deoxy sugar residues and the reducing end was determined by the method of Morrison 2 0 0 to be galactose which therefore in the polymer is linked to glucuronic acid at C-3.. The tetrasaccharide F l , i s , therefore, (Glc,Rha,Rha)-Gal and since two of the three rhamnose residues have already been located i t remains to establish whether the sequence in F l and, hence, in the native poly-saccharide is Rha-Rha-Glc-Gal or Rha-Glc-Rha-Gal. Partial hydrolysis of the native polysaccharide with hydrofluoric acid distinguished between these p o s s i b i l i t i e s . Hydrofluoric acid hydrolysis. From this hydrolysis two disaccha-rides (A2 and A3) were isolated, the latter in only small amount. Methylation analyses of A2 (Table I I I . l , column V) showed i t to be Glc-(l->3)Gal and c.i.-m.s. gave [M + NH4] - 472 consistent with A2 being a hexose-hexose disaccharide (Fig. III.2). Methylated A3 similarly gave + [M + NH4] - 412 (Fig. III.3) indicative of a disaccharide composed of two deoxyhexose units which, from the methylation results of the original polysaccharide, must be Rha-(l->2)-Rha. It therefore follows that the sequence in the main chain is Glc — Gal — GlcA — Rha — Rha N.m.r. spectroscopy. The proton spectrum of the native polysaccha-ride showed six signals in the anomeric region between 6 5.24 and S 4.82 (Table III.2 and Appendix III). Each signal integrated to approxi-mately 1 H but only the signal at S 4.82 showed a measurable coupling - 62 -CH2OMe CH2OMe O . J MeO/ 1 Ov 235 MeO R=Me [ M + N H 4 + ] = 4 7 2 » 4 4 0 4 7 2 5 0 0 6 0 0 7 0 0 Fig. III.2: C.i.-mass spectrum and fragmentation pattern of methylated HF product A2 MeO i 189*j 189 |205 1 4 9 2 0 5 I 8 9 | 3 9 4 1 7 8 X. • .iT. 4 1 2 1 0 0 2 0 0 3 0 0 4 0 0 R=Me, [M+NHU+]=412 Fig. III.3: C.i.-mass spectrum and fragmentation pattern of methylated HF product A3 - 63 -constant of 8 Hz, the others were broad due, presumably, to the visco-s i t y of the solution. In addition two signals at S 1.31 and S 1.28 integrating to approximately 9 H were observed (Table III.2 and Appendix III). The proton decoupled ^3C-spectrum likewise showed six signals in the anomeric range between 104.73 and 95.76 p.p.m. together with signals at 176.94, 17.57 and 17.45 p.p.m. indicative of uronic acid and 6-deoxy-sugar (Table III.2 and Appendix III). In a proton coupled spectrum the signal at 104.73 p.p.m. exhibited a coupling constant (JC,H) °^ 160.35 Hz whereas the others gave values between 168.52-172.88 Hz (Table III.3). Both the proton and the 1 3C spectra suggest that the K31 polysaccharide Is composed of a hexasaccharide repeating unit with a single ^-glycosidic linkage. This was confirmed by a chromium trioxide oxidation^^ when galactose was the only sugar completely degraded. Detailed n.m.r. studies on a lower molecular weight polymer (gener-ated by depolymerization of native polysaccharide with v i r a l endoglyca-nase) are illustrated in the next Chapter. These studies afforded the confirmation of the structure of E. c o l i K31 capsular antigen using modern pulse n.m.r. techniques. III.1.3 Experimental General methods. Solutions were concentrated on a rotary evaporator with bath temperatures not exceeding 40°C. Frozen solutions were obtained using a dry ice-acetone mixture and lyophilized on a - 64 -Table III.2: N.m.r. data for derived products of E. c o l l K31 capsular polysaccharide Coapound *H-n.».r. data data d" Ji 2 Integral Aaalgmtenta p.p.a.d Aaalgmenta c <6*> Native polysaccharide BhaJ-2£haL^lcl-2Gal-OH a a a 5.24 b 1 2-Rhaa- 176.94 00 of uronic 5.13 b 1 2-Clca- acid 5.11 b 0.9 2-Rhao- 104.73 3-Call-5.03 b 0.9 3,4-ClcAa- 101.80 Rhao-4.87 b 0.9 Rhao- 100.80 3.4-ClcAa-A.82 8 0.9 3-Cal0- 100.21 2-Rhaa-1.31 of Rha'a 96.47 2-Glca-1.2B CH3 95.76 2-Rhao-17.57/17.45 CH3 of Rha'a 5.25 b 1 2-Rhaa-5.13 3 1.2 2-Clca-4.99 <1 1.3 Khaa-4.95 b 0.2 3-Cala-4.65 8 0.8 3-Cal^-1.31 8 3 1.29 8 3 CH3 of Rha'a Chemical s h i f t downfield from sodium-4,4-dimethyl-4-silapentane-l-sulfonate (DSS) Key: b - broad, unable to assign accurate coupling constant For example 3-Gal/9- refers to anomeric proton of a 3-linked galactosyl residue in the ^ -anomeric configuration Chemical s h i f t In ppm downfield from DSS - 65 -Table III.3: Determination of anomeric configuration ( i JC, iH coupled) Chemical s h i f t Chemical shift Chemical shift Coupling Configura-decoupled nuclei downfield upfield constant tion (ppm) (ppm) (ppm) 104. ,73 105, ,794 103. ,586 165, ,60 101. ,80 102. .952 100, .647 172, .88 a 100. .80 101. .923 99, ,650 170, ,46 a 100. ,21 101, ,165 98. ,874 171. .82 a 96, .47 97, .592 95, .345 168, .52 a 95, .72 96 .847 94, .595 168 .90 a G.l.c.-m.s. analysis on methylated product from chromium trioxide oxidation indicated that the sugar residue with ^-configuration is galactose and the rest of the sugars have the a configuration. - 66 -Unitrap II freeze-dryer. Circular dichroism spectra (c.d.) were recorded on a Jasco J-500A automatic recording spectropolarimeter, with a quartz c e l l of 0.3 mL capacity and a path length of 0.1 cm. C.d. samples were prepared by dissolving the appropriate a l d i t o l acetate in spectroscopic grade acetonitrile. The c.d. spectra were recorded i n the range 210-240 nm. The infrared (i . r . ) spectra of methylated derivatives were recorded on a Perkin Elmer model 457 spectrophotometer. The solvent used for sample preparation was spectroscopic grade carbon tetrachloride. Analytical paper chromatography was performed by the descending method using Whatman No. 1 paper and the following systems: (1) 18:3:1:4 ethyl acetate-acetic acid-formic acid-water; (2) 8:2:1 ethyl acetate-pyridine-water; and, (3) upper phase of 4:1:5 1-butanol-ethanol-water. Preparative paper chromatography was carried out using Whatman No. 3 paper and solvent system 1. Chromatograms were either developed with alkaline silver n i t r a t e 1 9 4 or by heating at 100°C for 10 min after being sprayed with p-anisidine hydrochloride 4 4 i n aqueous 1-butanol. Sugars and oligosaccharides were detected by these methods. A Bio-Gel P-2 (400 mesh) column (2.5 x 100 cm) was used for preparative gel-permeation chromatography. The void volume of the column and the efficiency of packing were determined using blue dextran (0.2%). The concentration of the samples applied to the column ranged from 40-100 mg/mL. Eluant was d i s t i l l e d water at a flow rate of approximately 6 mL/h. Fractions were collected, freeze-dried, weighed and the elution profile was obtained. Sephadex LH-20 was used to purify large molecular weight carbohydrate material that is soluble in organic - 67 -solvent, e.g. permethylated oligo- and polysaccharides. A Hewlett-Packard 5890 instrument equipped with dual flame-ioniza-tion detectors was used for analytical g.l.c. separations. A Hewlett-Packard 3392A integrator was used to quantify the peak areas. Open tubular (capillary) columns were used with a helium carrier-gas flow rate of 48 mL/min. The columns used were: (A) fused s i l i c a capillary column (DB-17-15N); (B) fused s i l i c a capillary column (DB-225-15N). Preparative g.l.c. was carried out with F & M model 720 dual column instrument f i t t e d with thermal conductivity detectors. Stainless-steel columns (1.8 m x 6.3 mm) were used with carrier-gas helium flow-rate of 60 mL/min. This column was packed with 3% of SP-2340 on Supelcoport (100-200 mesh) and programmed from 175°C to 240°C at l°/min. G.l.c.-m.s. analyses were performed with a V.C. Micromass 12 instrument equipped with a Watson-Biemann separator. Spectra were recorded at 70 eV with an ionization current of 100 A and an ion source at 200°C. The columns used for the separation were (A) and (B). 13C-N.m.r. and ^H-n.m.r. spectra were recorded on Varian XL-300 and Bruker WH-400 instruments respectively. Acetone was used as an internal standard for both ^H-n.m.r. (2.23 p.p.m.) and 13C-n.m.r. (31.07 p.p.m.) spectroscopy. H^-N.m.r. spectra were recorded at elevated temperature and chemical s h i f t values are given relative to that of external sodium 4,4-dimethyl-4-silapentanesulfonate (taken as zero). H^-N.m.r. samples were prepared by dissolving i n D2O and lyophilizing three times from D2O solutions. These samples were dissolved i n D2O and submitted in 5 mm diameter n.m.r. tubes. 13C-N.m.r. spectra were recorded at ambient temperature. Samples were dissolved in the minimum of D2O and submitted - 68 -in n.m.r. tubes of diameter size 5 mm. In our laboratory, we generally proceed as follows for the isolation of E. c o l i bacteriophages. Large samples of sewage are mixed with concentrated Mueller-Hinton broth and then with an actively growing culture of the bacteria for which a virus shall be isolated. This mixture is then incubated (37°C) overnight. The bacteria in this mixture are k i l l e d by adding chloroform and shaking vigorously. The crude bacteriophage solution is separated from the bacterial debris by centrifugation. The bacterial lawn needed for bacteriophage assay i s prepared as follows. A Mueller-Hinton agar plate was dried, upside down at 37°C in an incubator for 2 hours. Actively growing bacterial culture (3 mL) was pipetted on to the agar surface and after 20 min at 37°C, excess li q u i d was drained off. The plate was then incubated at 37°C for 1 hour to produce the bacterial lawn. An assay for determining bacteriophage concentration i s described as follows: a 0.3 mL portion of bacteriophage suspension was diluted ten-fold by adding 2.7 ml of st e r i l e broth. A 0.3 mL portion of the resultant solution was further diluted ten-fold (in a similar manner) and the process repeated on subsequent solutions u n t i l a dilution range of l O' 1 - 10" 1 0 was obtained. One small drop of bacteriophage suspension was spotted on the bacterial lawn by means of a st e r i l e pipette drawn to a fine t i p . After overnight incubation at 37°C, the number of plaques observed for the highest dilution are counted. The counts of plaque-forming units (p.f.u.) per mL of undiluted phage suspension were calculated based on the volume of the bacteriophage - 69 -solution applied, the number of plaques and the dilution that gave those plaques. The methods employed in building up the concentration of bacterio-phage to a level sufficient for degrading the polysaccharide in question are tube l y s i s and flask l y s i s . (a) Tube l y s i s An actively growing culture of E. c o l i was obtained by successive replating on agar plates. A colony of this actively growing bacteria was picked up into s t e r i l e broth (5 mL) and incubated at 37°C u n t i l the bacterial culture became turbid (4 hours). Sterile broth ( 5 x 5 mL) in culture test-tubes was then inoculated with the bacterial culture (0.5 mL) and incubated at 37°C. After 30 minutes of incubation, bacterio-phage suspension was added to the test-tubes consecutively at 30 minutes interval. Continued incubation results in gradual clearing of the cloudy solution due to c e l l l y s i s . After the last tube had cleared (about 5 hours after the f i r s t addition of bacteriophage) a few drops of chloroform were added to each tube to prevent bacterial growth. The last two tubes were combined and the bacteriophage solution was separ-ated from the bacterial debris by centrifugation. (b) Flask lv s i s This technique is quite similar to the tube l y s i s except that large volumes of bacteriophage are produced. Aliquots (48 mL) of s t e r i l e - 70 -Mueller Hinton broth, in six Erlenmeyer flasks (125 ml), were each inoculated with 1 mL of actively growing culture. At 30 minute inter-vals, bacteriophage suspension (1 mL from the tube ly s i s or otherwise) was consecutively added to the flasks. The procedure was then continued as described for tube l y s i s . Isolation and purification of E. c o l i K31 capsular polysaccharide. The medium used for the growth of the bacteria was Mueller Hinton agar:beef extract (3.0 g), acid hydrolyzate of casein (17.5 g), starch (1.5 g), and agar (12.0 g) per l i t e r of water. St e r i l i z a t i o n of glassware and Mueller Hinton medium was done in an American S t e r i l i z e r model 57-CR for 15 minutes at 121° and 15-20 p.s.i. E. c o l i K31 culture was obtained from Dr. Ida |6rskov (WHO Interna-tional Escherichia Center, Copenhagen). Actively growing colonies of E.  c o l i K31 were propagated by replating several times on to Petri dishes (layered with s t e r i l e Mueller Hinton agar); a single colony being selected each time the bacteria were to be plated. Overnight growth of bacteria at 37"C was sufficient. Broth (100 mL) was inoculated with E. c o l i K31 bacteria and incubated for 4 hours. Actively growing E.  c o l i K31 bacteria were poured onto a ste r i l e , Mueller Hinton agar medium (in a metal tray 60 x 40 cm) and incubated for four days at 37°C. The K31 bacteria were scraped from the agar surface, diluted with 1% phenol solution and stirred at 4°C for 5 hours. The mixture of polysaccharide, bacterial c e l l s and other debris was ultracentrifuged (for 4 hours at 15°C"on a Beckman L3-50 ultracentrifuge using rotor 45 T i at 31000 r.p.m. or 80000 g) to separate the dead bacterial cells from the - 71 -polysaccharide solution. The viscous honey-coloured supernatant was precipitated with ethanol. The resultant stringy precipitate was dissolved i n water and treated with Cetavlon 1 9 3 (cetyltrimethylammonium bromide). The Cetavlon-polysaccharide complex was dissolved i n 4M NaCl solution, precipitated into ethanol, redissolved i n water and dialyzed against d i s t i l l e d water (two days). The polysaccharide was isolated as a styrofoam-like material by lyophilization. The isolated polysaccha-ride was further purified by gel permeation chromatography using a Bio-Gel P2 column (100 cm x 2.5 cm). Sugar analysis and composition. Hydrolysis of a sample (20 mg) of K31 polysaccharide with 2 M trifluoroacetic acid (TFA) for 20 h at 95°, removal of excess acid by coevaporation with water, followed by paper chromatography (solvent 1) showed glucose, galactose, rhamnose, and an aldobiouronic acid. The neutral sugars released were analyzed as a l d i t o l acetates by g.l.c. column DB 17, programmed from 180° to 220°C at 5°/min. A portion of this hydrolyzate was methylated 1 0 1 and subjected to g.l.c. using the program 145°, 1 min then 2°/min to 155°, 1 min, 3"/min to 220°, 1 min and then to 250°C at 3°/min. Under these conditions the permethylated monosaccharides were eluted between 1.9 and 4.9 min and the methylated aldobiouronic ester methyl glycoside at 16.0 min. When + this component was analyzed by c.i.-m.s. a value of [M. + NH4] - 456 was obtained. A portion (49.5 mg) of K31 polysaccharide (H + form) was dissolved in water (30 mL), l-cyclo-hexyl-3-(2-morpholinoethyl)-carbodiimide metho-p_-- 72 -toluenesulfonate (CMC, 423 mg) was added and reduction was achieved by the addition of aqueous sodium borohydride (3M, ca 100 mL) with continu-ous adjustment of the pH to 7. The product (61 mg), isolated by dialyses and lyophilization, was analyzed as a l d i t o l acetates following hydrolysis with 2 M TFA. Methylation analysis. The capsular polysaccharide (30 mg, H + form) was methylated by the method of Hakomori, 1 0 1 dialyzed, partitioned between dichloromethane and water and purified on Sephadex LH 20 to give a product which showed no absorption at 3625 cm"1. Analytical results on this material are given i n Table I I I . l , column I and on a portion which was refluxed overnight with lithium aluminum hydride In oxolane in Table I I I . l , column II. Uronic acid degradation. A sample (25 mg) of methylated K31 polysaccharide was dried and then, with a trace of p_-toluenesulfonic acid, was dissolved in 19:1 dimethyl sulfoxide--2,2-dimethoxypropane (12 mL) and the flask was sealed under nitrogen. Dimethylsulfinyl anion (5 mL) was added and allowed to react for 18 h at room temperature. Methyl iodide (3 mL) was added to the cooled reaction mixture and s t i r r i n g was continued for 7 hours. The methylated degraded product was isolated by partition between chloroform and water and was then purified by gel permeation chromatography on Sephadex LH-20. The degraded product was hydrolyzed with 2 M TFA for 8 h at 95° and analyzed by g.l.c. (Table I I I . l , column IV). - 73 -Selective partial hydrolysis. Polysaccharide (60 mg) was hydrolyzed with 0.1 M TFA for 25 min at 95°C. After dialysis the retentate (PH) was methylated 1 0 1 followed by reduction of the carboxyl function with lithium aluminum hydride. The analytical results are presented in Table I I I . l , column IV. Reaction with lithium in ethylenediamine. Dry K31 polysaccharide (150 mg) was suspended in dry ethylenediamine (21 mL) and six pieces of lithium wire (3 mm x 3 mm, hexane washed) were added. The intense blue color obtained was maintained for 1 h by the addition of smaller pieces of lithium. The reaction was terminated by the addition of dry methanol (4 mL) with the flask cooled in ice water. The excess ethylenediamine and methanol were removed in vacuo over sulfuric acid and sodium hydroxide. Glacial acetic acid (3 mL) was added to the residue, with external cooling to decompose lithium methoxide followed by the addition of an equal volume of water. The resultant product was purified by ion-exchange chromatography (Bio-Rad AG 50X8 resin) and gel permeation chromatography (Bio Gel P2). A pure oligosaccharide (Fl) was isolated by preparative paper chromatography. Methylation data on F l is reported in Table I I I . l , column VII. The proton n.m.r. spectrum showed signals at 6 5.25 (b, 1H), 5.13 (3 Hz, 1.2H), 4.99 (b, 1.3H), 4.95 (b, 0.2H), and 4.65 (8 Hz, 0.8H). Hydrofluoric acid hydrolysis. The native polysaccharide was hydrolyzed by HF at -40°C for 15 mins and the products A2 (~8 mg) and A3 (-2 mg) were separated by paper chromatography. Methylation data for A2 74 are given i n Table I I I . l , column V. G.1.c.-c.i.-m.s. of methylated A2 + + gave [M + NH4] - 472 and methylated A3 gave [M + NH/J - 412 (Fig. III.3) Chromium trioxide oxidation. Acetylated polysaccharide (25 mg) was dissolved i n acetic acid and treated with chromium trioxide (100 mg) for 2 h at 50°C. The results of methylation analysis of the product are shown in Table I I I . l , column VI. Bacteriophage depolymerization. The phage was isolated from Vancouver sewage and propagated by tube and flask l y s i s to a concentra-tion of 1.2 x 10^ p.f.u. mL"l. Bacteriophage was added to an aqueous solution of 150 mg of K31 polysaccharide. Depolymerization was carried out for 5 h at 37"C after which the solution was heated to 85°C for 5 min and lyophilized. The crude product was deionized by passage through a column of Amberlite IR 120 (IT*") and the eluate was concentrated and added to a column of Bio-Gel P2 (400 mesh) which was eluted with water at 20 mL h'^. The low molecular weight polymer (Pn) was collected between 133-150 mL to give 80 mg. IV. STRUCTURAL STUDIES ON THREE E. c o l i CAPSULAR POLYSACCHARIDES USING MODERN N.M.R. TECHNIQUES IV.1 Introduction Until recently n.m.r. was used as a back-up technique in carbohydrate research. With the introduction of modern computers pulse n.m.r. techniques have been employed extensively in structural elucidation of carbohydrates. The advantages of using principally n.m.r. techniques in structural studies of carbohydrates are that the method: (i) is non-destructive ( i i ) requires smaller samples than chemical methods ( i i i ) is fast. Capsular polysaccharides are immunogenic and the use of fast tech-niques, such as modern n.m.r. methods, in their structural elucidation may be essential. As an on-going process in this and other laboratories we wish to build a good data base and hence establish n.m.r. as a reliable routine technique for structural studies of polysaccharides. In this investigation, mostly low molecular weight polymer, generated by v i r a l glycan depolymerization of natural capsular polysaccharide, was used for n.m.r. studies. The advantage of using these lower molecular weight polymers is that they give good resolution as well as n.O.e.'s. - 76 -IV.2 Chemical shift assignment of the sugar residues i n E. c o l i K.44 capsular polysaccharide IV.2.1 Introduction The structure of the capsular polysaccharide from Escherichia c o l i 08:K44(A):H- (K44 antigen) has been est a b l i s h e d 1 9 6 using the techniques of methylation /J-elimination, deamination, Smith hydrolysis and n.m.r. spectroscopy. 4)£-D-GlcAp- (l-O) -a-L-Rhap- (l-»4) -a-D-GlcpNAc- (l-*6) -/5-D-GalpNAc- (l-> This capsule is unique because i t has two amino-sugar residues linked to each other as well as uronic acid i n i t s repeating unit. This is a model study to assign the chemical shifts of the sugar residues in the repeating unit of the K44 polysaccharide. IV.2.2 Results and Discussion The ^-n.m.r. spectrum of the native polysaccharide (Table IV.1) showed four anomeric proton signals:19® an a-linkage at 6 4.95 (J i ( 2 3 Hz), two ^-linkages at 6 4.72 (Jj.,2 8 Hz, 1H) and S 4.60 (J i f 2 8 Hz, 1H) and a borderline signal at 6 4.89 (b, 1H). In the ^ -n.m.r. spectrum of the native polysaccharide, methyl protons of rhamnose appeared at S 1.34 (b, '3H) and two signals attributed to methyl protons of the N-acyl groups were observed at 6 2.09 (s, 3H) and 6 2.06 (s, 3H), respectively. Table IV 1: N.M.R. data for PI and the native E. colt K44 polysaccharide. Compound* H^-N.m.r. data 13C-N.m. r. 1 J l , 2 (Hz) Integral proton Asslgnnent P.p.m. Assignment 4clcAl-3RhJL5cicNAcl-ficalMAcl-P a a P 4.96 4.B9 3 b 1.0 1.0 ^ClcNAc^ a ^Rhal-a 173.80 1 175.00 J 1 C-0 of ClcNAc \ and | CalNAc K44 polyssaccharlde (Na*) 4.70 4.61 2.09 8 8 8 1.0 1.0 3.0 * C1CA1-P ficalNAci-P CH} of N-Ac (ClcNAc) 172.30 104.77 102.89 C-0 (ClcA) 4C1CAI-P ^CalNAci-P 2.06 S 3.0 CH3 of N-Ac (CalNAc) 101.46 J-Rhal-a 1.33 5 3.0 CH} (Rha) 98.90 ScicNAcl-a 61.08 54.79 52.90 C-6 (ClcNAc) C-2 (ClcNAc) C-2 (CalNAc) 23.21 N-S-CHj (CalNAc) 22.67 N-C-CHj (ClcNAc) 17.52 C-6 (Rha) - 78 -The 13C-n.m.r. spectrum of the native polysaccharide showed (Table IV.1) a total of twenty eight carbon signals and these, coupled with the . presence of only four anomeric carbon s i g n a l s , 1 9 7 confirmed a tetra-saccharide repeating unit for the K44 polysaccharide. The three C«=0 signals i n the downfield region of the spectrum (between 170-180 ppm) agree with the presence of two acetamido and one carboxyl group associated with the polysaccharide. The signals at 54.84 ppm and 53.08 ppm can be attributed to C-2 of glucos- and galactos-amine. In the upfield region of the spectrum, three signals corresponding to the methyl carbon of the two N-acyl groups and the rhamnose residue were observed. The anomeric configuration of the sugar residues in the repeating units were established 1 9 6 by a proton-coupled 13C-n.m.r. experiment on the native polysaccharide. !H chemical shift assignments: The sugar residues were a r b i t r a r i l y labelled A to D in the order of decreasing chemical s h i f t of their H-l resonances. H-l and H-2 resonances of a l l the sugar residues were established by a COSY n.m.r. experiment (Fig. IV.1 and Table IV.2). H-3 and H-4 of residue C were established by one and two step relay experi-ments (Figs. IV.2, IV.3 and Table IV.2). Complete assignment of 1H resonances of the rhamnosyl residue was achieved by taking F2 slides in the Fi dimension and walking through the 2D COSY spectrum. H-3, H-4 and H-5 of residue D were established by walking through a 2D COSY spectrum (Fig. IV.1). H-6 and H-6' of residue A were established from a heteronuclear correlated experiment. This assignment made possible for the assignment of H-5, H-4 and H-3 of residue A. - 79 -Table IV.2: *H N.m.r. data for E. c o l i K44 native polysaccharide Symbol Sugar residue H-l H-2 H-3 H-4 H-5 H-6/H-6' A -4)GlcNAcl-» 4.94 3.96 3.71 4.17 3.90 3.87/3.74 B -3)a-Rha(l- 4.89 4.29 3.75 3.54 3.46 1.33 C -+4)/5-GlcA(l- 4.71 3.46 3.64 3.82 D -6)/9-GalNAc(l- 4.58 3.95 3.78 3.63 3.47 Based on the composition and methylation r e s u l t s i y b for the native polysaccharide and on comparison of ^ H-n.m.r. data for the native poly-saccharide with those of monosaccharide methyl glycosides, residue A was identified as the 4-linked a-glucosamine, residue B as the 3-linked a-rhamnose, C as the ^ -glucuronic acid and D as the 6-linked /9-galactos-amine. - 80 -Fig. IV.1: Homonuclear -Ml-spin correlated (COSY) n.m.r. spectrum of native polysaccharide (K44). - 81 -6 6 o e e D1 • s* o D2 B Q i c a CJ C2 " P P M -1 .0 -1.5 -2.0 -2.5 -3.0 -3.5 -4.0 -4.5 -5.0 -5.5 ' I' l ' ' l 1 i ' ' i 1 i 1 1 i ' i 1 • \ 1 i • ' | ' | ' ' | 1 1 • ' i • i • • i • i 1 • i • i 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 PPM Fig. IV.2: One step ^ - s p i n coherence transfer (COSY HGR1) n.m.r. spectrum of native polysaccharide (K4A). - 82 -0 e •ii Dl£f" D2 D3 C4 C3 C2 BS3 SS3 & B1 * ' P P M -2.8 -3.0 -3.2 -3.4 -3.6 -3.8 -4.0 -4.2 -4.4 -4.6 -4.8 -5 .0 1 ' I ' I 1 I 1 I ' I ' I ' | ' | • 1 i | i i i 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 PPM Fig. IV.3: Two step ^-H-spin coherence transfer (COSY HGR2) n.m.r. spectrum of native polysaccharide (K44). - 83 -•H, . 1.6 . 2.0 . 2.B . 8.0 a.B . 4.0 . 4.8 B.O I i I • I ' i I • I . I i I . I • I— 110 100 BO 80 70 60 SO 40 90 20 Fig. IV.4: Heteronuclear ( 1 3C- 1H) correlated n.m.r. spectrum of native polysaccharide (K44) r - 84 -IV.2.3 Experimental L General methods: Spectra were recorded for samples in 5 mm diameter tubes at 400 MHz using a Bruker WH400 spectrometer equipped with an Aspect 3000 computer. Homonuclear two-dimensional spectroscopy was performed using Bruker D15B871 software. A l l 2D experiments were per-formed with suppression of HOD resonance. A l l homonuclear experiments were performed with quadrature detection in the F l dimension and a total of 256 t^ increments of 96 or 112 scans each were recorded with minimum delay between pulses of 1.2 s and 1024 real data points in t2- A l l 2D time domain data sets were symmetrized after Fourier transformation. Symmetrized 2D spectra were compared with the unsymmetrized one to check for any artifacts introduced during symmetrization. COSY: Native polysaccharide (K44) was lyophilized twice from deuterium oxide and dissolved at a concentration of 410 mg mL"'1. The deuterium exchange was done at 338°K to enhance polysaccharide solubility. This sample was used for the 2D n.m.r. experiments which were conducted at 338°K. The ID n.m.r. (^ H) experiment was done at 368°K and 1 3C n.m.r. at 300°K. The pulse sequence used in the COSY experiment i s given i n section II.4.1 (appendix II). The acquisition parameters are given below: Sweep width i n F l dimension (SW1) - 1000 Hz Fixed delay to enhance effects from small J(D2) - 0.000003 Relaxation delay (Dl) - 1.3 is Delay for evolution of shut and coupling (DO) - 0.000003 (with increments of 0.5/SW1 for 256 times) Pre-saturation with power SI - 40L - 85 -Power S2 for evolution - 50L Additional delay for switching (D3) - 0.002 s 90° excitation pulse (PI) - 19.5 us Mixing pulse (145°)/90° - 9.8 /*s/19.5/is Number of scans per experiment (NS) - . 96 Number of experiments (NE) - 256 Relay COSY (1 step and 2 step): The pulse sequence for the relay COSY experiments is given in Appendix II. The acquisition parameters for the one step relay COSY n.m.r. experiment are as follows: Sweep width in F l dimension (SW1) = 1000 Hz Relaxation delay (Dl) = 1.2 s Pre-saturation with power SI - ' 30 L Delay for switching (D3) - 0.000003 Power S2 for evolution •= 45 L Delay for evolution of shifts (DO) = 0.000003 (with increments of 0.5/SW1 for 256 times) Second coherence period (D2) - 0.035, etc. 90° pulse creating XY-magnetization = 14.3 /xs 180° pulse for refocus chemical shifts - 28.6 ps Number of experiments (NE) = 256 Number of scans per experiment (NS) = 96 The acquisition parameters for the two step relay COSY n.m.r. experiment are illustrated below. Sweep width in F l dimension (SW1) - 1000 Hz Relaxation delay (Dl) - 1.5 s Pre-saturation with power SI - 30 L Delay for switching (D5) - 0.002 Power S2 for evolution - 63 L Delay for evolution of shifts (DO) - 0.000003 (with increments of 0.5/SW1 for 256 times) Second coherence period (D2) - 0.035 Third coherence period (D3) - 0.035 90° pulse creating XY-magnetization (P1-PH2) - 14.3 ps 90° pulse applied to complete 1st coherence transfer (P1-PH2) - 14.3 ps 180° pulse to refocus chemical shifts (P2-PH2) - 28.6 ps 90° pulse applied to complete 2nd coherence transfer (P1-PH2) - 14.3 ps 90° pulse for third coherence transfer (P1-P4) - 14.3 ps Number of experiments (NE) - 256 Number of scans per experiment (NS) — 96 - 86 -IV.4 Sequencing and location of acetate In E. c o l i K33 capsular polysaccharide using N.M.R. IV.4.1 Introduction The capsular (K) antigen of Escherichia c o l i K33 has been designated as heat stable (type A) and may, therefore, in the absence of amino sugars, be expected to resemble those of Klebsiella. E. c o l i K33 has been shown to cross react with Klebsiella K58. In collaboration with Dr. B.A. Lewis, structural studies using conventional chemical techniques gave a partial structure of K33 polysaccharide -3)a-D-Glc(l-4)0-D-GlcA(l->?)-a-L-Fuc- (1-\3 2/ ? C / \ HOOC CH3 1 a-D-Gal In this section of the thesis, the use of 2D n.m.r. spectroscopy as a convenient method for the location of acetate in the polysaccharide is demonstrated. The use of 2D n.m.r. for sequencing the sugar residues in the repeating unit of the polysaccharide is also reported. IV.4.2 Results and discussion 13C-N.m.r.: 13C-n.m.r. data of the native polysaccharide are given in Table IV.3. The 1 3C broad band decoupled n.m.r. spectrum (75 MHz) of the deacetylated polysaccharide (Fig. IV.5 and Table IV.3) showed - 87 -Table IV.3: 13 C N.m.r. data of K33 polysaccharide and derived product Product Chemical s h i f t (ppm) Assignments Native polysaccharide 174.74 174.70 174.07 104.60 101.40 101.30 100.30 99.50 97.73 62.64 61.14 23.58 21.27 16.07 CO of ketal pyruvic acid CO of acetyl group CO of GlcA C-l of /3-GlcA C-6 of /5-Gal C-6 of a-Glc CH3 of acetyl group CH3 of pyruvic acid ketal CH3 of Fuc Deacetylated polysaccharide 174.70 173.14 104.48 101.23 100.11 99.98 62.46a 60.89a 22.99 16.10 CO pyruvic acid ketal CO of GlcA —GlcA-a — G a l - a —Fuc-a — G l c - o C-6 of Gal C-6 of Glc CH3 of pyruvic acid ketal CH3 of Fuc a These assignments have been confirmed by the attached proton test (ATP) experiment - 68 -cn cn O «o m TO r- in *- mo o o 00 *- °i O f\i CM CM CM I I H » « t l te t i m i CM cn o / / i 1''i' ' ' ' i i ' ' ' ' i ' ' ' ' i ' ' ' • i " ' ' i ' ' ' ' i ' ' • ' i 1111111'IIi 1111111 200 180 160 140 120 100 80 60 40 20 PPM 200 . 1 8 0 160 140 120 100 80 60 40 20 0 PPM Fig. IV.5: 1 3 C H.a.r. spectra of deacetylated polysaccharide (K33) (a) Broad band decoupled (b) Attached proton test experiment (ATP) * Incomplete deacetylatlon - 89 -four major signals for anomeric carbons at 104.476, 101.225, 100.107 and 99.976 ppm. Among other 1 3C signals, are 1 3C signals at 22.987 and 16.099 ppm corresponding to CH3 of pyruvate and fucose residues respectively. The C-6 methylene carbons were clearly differentiated from methine ring carbons in the 1 3C attached proton test (APT) n.m.r. spectrum (Fig. IV.5). The 1 3C signals at 174.70 and 173.135 ppm correspond to carbonyl carbons of pyruvate and glucuronic acid residues respectively. A proton-coupled 13C-n.m.r. spectrum of the deacetylated, depyruvy-lated polysaccharide (see spectrum in Appendix III) showed signals at 104.46 ppm ( 1 J C l , H l 162.7125 Hz), 101.214 ppm ( 1 H C 1 H 1 174.1875 Hz), 100.103 ppm ( ^ c i . H l 171 1425 Hz) and 99.476 ( ^ c i . H l 171.7575 Hz) (Table IV.4). The ^ J c i HI values reported for the a and /9 glycosidic linkages of 6-deoxy and hexopyranoses are -169 and —160 Hz respec-t i v e l y . 6 8 , 6 9 Hence there are one p and three a linkages in the repeating unit of the K33 polysaccharide. 2D N.m.r. experiments (see later) afforded the assignment of the glucuronic acid anomeric linkage as 0 and the rest of the sugar residues in the repeating unit as a. H^ Chemical shift assignments (deacetylated polysaccharide). The sugar residues were ar b i t r a r i l y labelled by their H-l resonances. H-l, H-2, H-3 resonances of a l l the sugar residues were established by COSY (Fig. IV.7) and one relay COSY (Fig. IV.8) n.m.r. experiments. The H-4 resonance of the glucuronic acid residue was assigned from the two step relay COSY spectrum (Fig. IV.9). The H-5 resonance (6 4.0096) was located based on the diagonal cross peak for H-4 (6" 3.9162) in the COSY - 90 -Table IV.4: ^C-l-H Coupled n.m.r. experiment data Chemical shift Chemical shift Chemical shift -^C-H Anomeric ^H-decoupled ^H-coupled difference coupling configu-resonance) ppm between down- constant ration and sugar downfield upfield f i e l d & upfield ^JcH residue resonance resonance resonance 104.46 105.2246 103.0548 2.17 162.71 (Glc A) 101.21 101.9939 99.6715 2.32 174.19 (Gal) 100.10 100.8915 98.6096 2.28 171.14 (Fuc) 99.48 100.3368 98.0467 2.29 171.76 (Glc) spectrum. The H-5 signal for the fucose was assigned by the additional window provided by the methyl resonances. Based on the composition, methylation results and comparison of the ^H-n.m.r. data of the deacetylated polysaccharide with those for monosaccharide methyl glycosides, residue D was identified as 4-linked /9-glucuronic acid, residue B as branched point fucose, residue A as 3-linked a-glucose, and residue C as terminal galactose. - 91 -'A |Glc(H1) Glc(H2) • - O Glc(H3), ~ i 1 1 r~ 5.5 5.0 4.5 4.0 ^PPM -1.0 -1.5 -2.0 -2.5 - 3:0 -3.5 -4.0 -4.5 -5.0 -5 .5 i 1 r -3.5 3.0 2.5 PPM i 1 r 2.0 1.5 1.0 Fig. IV.6: Homonuclear *H-spin correlated (COST) n.m.r. spectrum of 'native' polysaccharide (K.33) - 92 -PPM -1.0 -1.5 -2.0 -2.5 -3 .0 -3.5 -4.0 -4.5 -5.0 -5.5 i 1 1 1 1 1 1 1 i r-5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 PPM Fig. IV.7: Homonuclear ^ - s p l n correlated (COSY) n.m.r. spectrum of deacetylated polysaccharide (K33) - 93 -PPM I .1.0 -1.5 -2.0 -2.5 -3.0 -3 .5 -4.0 -4.5 -5.0 -5.5 —I 1 1 1 1 1 1 I I i 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 PPM Fig. IV.8: One step relay XH spin coherence transfer (C0SYHGR1) spectrum of deacetylated polysaccharide (K33) - 94 -Fig. IV.9: Two step relayed XH spin coherence transfer (C0SYHGR2) spectrum of deacetylated polysaccharide (K33) - 95 -Location of acetate. The glucose residue (A) shows a diagonal cross peak between H-l (S - 5.3780) and H-2 (6 - 5.1482) in the COSY spectrum (Fig. IV.6 and Table IV.6) of the native polysaccharide. This diagonal cross peak is however absent i n the COSY spectrum (Fig. IV.7 and Table IV.5) of the deacetylated polysaccharide. This is an i l l u s t r a t i o n that the acetyl group is located on position 2 of the glucose residue. The down f i e l d s h i f t of a ring proton to the anomeric region (6 6.00 to 4.5) due to acetylation is known.65 Sequencing. The sequence of the sugar residues i n the polysaccha-ride back bone was obtained from a NOESY experiment 1 8 5 (Fig. VI.10 and Table IV.7). Interresidue n.O.e. contacts established that A was linked to D and that D was linked to B, thus giving the following partial sequence. OAc 4-2 (B) -3)-a-Glc(l-4) -/3-GlcA(l-»3) -a-Fuc(l-> (A) (D) 4 t - 96 -Table IV.5: lH-N.m.r. data for E. c o l i K33 deacetylated polysaccharide Symbol Sugar HI H2 H3 H4 H5 H6/H6' Residue A Glc 5. .42 3. .76 3. .76 3. .56 3. .63 B Fuc 5. .28 4. .06 4. .12 - 4. .42 1.32/1,30 C Gal 5. .27 3. .81 3. .95 - -D GlcA 4, .63 3. .49 3. .82 3. .92 4. .01 Table IV.6: ^-N.m.r. data 2D (COSY n.m.r. experiment) for E. c o l i K33 polysaccharide Symbol Sugar Residue HI H2 H3 H4 A Glc 5.38 5.15 . 4.27 4.19 B Fuc 5.28 4.06 5.25 3.78 5.16 3.72 D 4.56 3.47 - 97 -Fig. IV.10: Homonuclear dipolar correlated 2D n.m.r. (NOESYHG) spectrum of deacetylated polysaccharide (K33) at 338°K - 98 -Table IV.7: N.O.e. data for E. c o l l K33 polysaccharide Sugar Residue Symbol Interresidue Intraresidue contact contact •3)-a-Glc-(l- A 3.92, H-4 (D) 3.76, 3.56 •3)-a-Fuc(l- B 3.76 a 4.06 4 I -l)-a-Gal C - 3.81 -4)-£-GlcA(l- D 4.12, H-3 (B) 4.01 suspect H2 and H3 coinciding hence this n.O.e. contact could not be assigned poor resolution; the linkage between Gal and Fuc was established by ^-elimination experiment - 99 -The COSY, relay COSY and NOESY n.m.r. experiments were repeated on another n.m.r. spectrometer ( AM 400 MHz Bruker n.m.r. spectrometer) for the following reasons. (a) to check the va l i d i t y of n.O.e. contacts (b) to obtain additional sequencing information. The sequencing information (Table IV.8, Table IV.9 and Fig. IV.11) is identical to what was previously obtained. Chemical s t u d i e s 1 9 5 conducted on this polysaccharide suggest the structure, -•3)-a-D-Glc(i-4)0-D-GlcA(l-*?)Fuc(l-* 3 2 ? \ / C / \ HOOC CH3 1 'a-D-Gal Based on 2D n.m.r. and chemical data we report the complete structure of E. c o l i K33 K antigen as: OAc I 2 -»3)-a-D-Glc(l-*4)-0-D-GlcA(l-»3)-a-L-Fuc(l-3 2 4 \ / C / \ HOOC CH3 1 a-D-Gal The K antigens of E. c o l i K33 and Klebsiella K58 are similar and these two strains have been shown to cross react. - 100 -Table IV.8: 1H-N.m.r. data for E. c o l l K33 deacetylated polysaccharide (on AM400 Bruker n.m.r. spectrometer and experiment performed at 300°K) Symbol Sugar HI H2 H3 H4 H5 Residue A Glc 5. .40 3. .72 3. .72 3. .52 3.64 B Fuc 5. .28 4. .05 4. .12 3. .93 -C Gal 5. .23 3, .82 3. .93 - -D GlcA 4. .58 3, .46 3. .79 3. .88 _ Table IV.9: N.O.E. data for E. c o l i K33 deacetylated polysaccharide (on AM400 Bruker n.m.r. spectrometer and experiment performed at 300°K) Sugar Residue Symbol Interresidue Interresidue contact contact -3)-QG1C(1- A 3.88 H-4 (D) 3.72 -3)-a-Fuc(l-> B 3.72a 4.01 4 t ->l)-a-Gal C - D 3.81, 3.75 -4)-/9-GlcA(l- D 4.12 H-3 (B) 3.96 suspect H2 and H3 coinciding and this n.O.e. contact could not be assigned poor resolution - 101 -Fig. IV.11: Homonuclear dipolar correlated 2D n.m.r. (NOESYHG) spectrum of deacetylated polysaccharide(K33) at 300°K - 102 -IV.3.3 Experimental General methods. The general experimental procedures are as described in Section III.1.3. Isolation and purification of E. c o l i K33 capsular polysaccharide. E. c o l i K33 culture was obtained from Dr. Ida jfcskov, WHO International Escherichia Centre, Copenhagen). Actively growing colonies of E. c o l i K33 were propagated and grown as described in Section III.1.3. Isola-tion and purification of polysaccharide were done as previously described (Section III.1.3). Peacetvlation. 150 mg of K33 native polysaccharide was dissolved in 0.1 M NaOH solution and heated at 80°C for 4 h. The resultant product was dialyzed (mol. wt. cut off 3500) against d i s t i l l e d water, followed by lyophilization of the retentate. The retentate was purified by gel permeation chromatography (Bio-Gel P2, column size 92 cm x 2.6 cm). Depyruwlation. 70 mg of deacetylated polysaccharide was dissolved in 0.1% acetic acid and heated at 80"C for 20 h. The resultant product was dialyzed and the retentate was lyophillzed. The resultant product was purified on a Bio Gel P2 column (92 cm x 2.6 cm). N.m.r. studies. General methods are as discussed in Section IV.2.3. COSY Experiment on native polysaccharide. Native polysaccharide was - 103 -lyophilized twice from deuterium oxide and dissolved at 20 mg mL"1. The experiment was performed at 338°K. The acquisition parameters were identical to those of the COSY experiment in Section IV.2.4 apart from using 32 transients per experiment. 2D Experiment on deacetylated polysaccharide. K33 native polysaccha-ride (100 mg) was dissolved in 0.1 M NaOH and heated at 80°C for 6 h. Dialysis (mol. wt. cut off 3500) against d i s t i l l e d water was followed by lyophilization of the retentate which was further purified by gel permeation chromatography. The resultant product was lyophilized twice from deuterium oxide and dissolved at a concentration of 30 mg mL"1. This sample was used for a l l 2D n.m.r. and 13C-n.m.r. experiments for deacetylated polysaccharide. The acquisition parameters for the COSY and relay COSY experiments were identical to those given in Section IV.2.4. A number of NOESY experiments were performed and the best results were attained using a mixing time (D9) of 300 ms with no random variation (i.e. V9 •= 0). The other NOESY parameters are: Sweep width in F l dimension (SW1) = 920 Hz Relaxation delay (Dl) «= 1.2 s Pre-saturat ion with power SI — 40 L Delay for evolution of shifts (DO) - 0.0002 (with increments of 0.5/SW1 for 256 times) 90° excitation pulse (PI) - 16ps Mixing pulse 90° (P2) - 16 ps Detection pulse 90° (P3) - 16 ps Number of scans per experiment (NS) — 112 Number of experiments (NE) - 256 - 104 -IV.4 Sequencing of a hexasaccharide repeating unit (E. c o l i K31 polysaccharide) by homonuclear 2D n.m.r. spectroscopy IV.4.1 Introduction The capsular antigen of Escherichia c o l i K31 has been designated 3 0 as heat stable (type A) and may, therefore, i n the absence of amino sugars be expected to resemble those of Klebsiella. A part i a l structure of E. c o l i K31 has been proposed earlier by other workers. 3^ Structural studies on this polysaccharide using conventional chemical methods suggested the repeating unit given below. 2) -a-D-Glcp- (1-+3) -0-Galp- (l->3) -a-D-GlcpA- (l-»2) -a-L-Rhap- (l-*2) -a-L-Rhap-(1-4 A a-L-Rhap-This structure was confirmed by 2D n.m.r. studies on lower molecular weight polymer (Pn) generated by bacteriophage glycanase degradation of the native polysaccharide. ' IV.4.2 Results A polysaccharide (Pn) of significantly lower molecular weight and viscosity than the native polysaccharide which was prepared by depoly-merization with a v i r a l endoglycanase was used i n a l l the 2D experi-ments . - 105 -The sugar residues were ar b i t r a r i l y labelled A to F in order of decreasing chemical shift of their H-l resonances. The results of COSY 1 6 3 (Fig. IV.12 and Table IV.10) afforded the assignments of H-l and H-2 resonances of a l l the sugar residues i n the repeating unit. Further assignments were made using COSY data after interpreting the data for the relay COSY experiments. One and two step relay COSY spectra (Figs. IV.13, IV.14 and Table IV.10) afforded the assignment of H-3 and H-4 resonances of most of the sugar residues i n the repeating unit. The assignment of the ^H signals for the a-6-deoxy residues A, C, and E were greatly f a c i l i t a t e d by the additional window provided by the methyl resonances. Thus following the cross-peaks from H-l of A, C and E the respective H-2, H-3 and H-4 resonances were established while from the H-6 resonances, the H-5 resonances and their respective connectivities to H-4 were established (Fig. IV.12). The assignment of the resonances for the glucuronic acid residues was likewise f a c i l i t a t e d by the easily recognized H-5 doublet ( 3J 4.0 Hz) at 6 4.3538 (Figs. IV.12 and IV.16). Only the resonances of F could not be completely assigned. Based on the composition and methylation results for the native polysaccharide and on the comparison of the ^H-n.m.r. data for Pn with those for monosaccharide methyl glycosides, residue E was identified as the terminal a-rhamnose, residues A and C as 2-linked a-rhamnoses, B as the 2-linked a-glucose residue, D as the 3,4-linkaged a-glucuronic acid and F as the 3-linked ^-galactose residue. The sequence of the sugar residues i n the repeating unit was con-firmed by data from a NOESY experiment 1 8 5 (Table IV.11). Interresidue n.O.e. con t a c t s 1 8 6 established that A was linked to B, D to C, and E and - 106 -Table IV.10: 1H-N.m.r. data for E. c o l i K31 polysaccharide Symbol Residue H-l H-•2 H--3 H-•4 H-•5 H-•6 H-•6' A 2-Rhaa- 5.25a «D 4. .11 3. .86 3. .50 3. .71 1. 32 B 2-Glca- 5.13 (3) 3. 69 3. ,96 3. ,50 3. ,95 3. 78 3. 86 C 2-Rhaa- 5.10 «D 4. .10 3, .89 3, .56 3. ,75 1. 32 D 3,4-GlcAa- 5.03 (3) 3. .86 4, .13 3, .77 4. .35 -E Rhaa- 4.84 (<D 3. ,96 3. .85 3. .42 4, ,42 1. 29 F 3-Gal0- 4.82 3. ,66 3, .74 4. .09 - -Determined at 400 MHz, measured from internal acetone at S 2.23. Spectra were recorded at 343°K. The sample used was the low M.W. polymer Pn, see text for details. - 107 -Table IV.11: N.O.E. data for E. c o l i K31 polysaccharide Sugar Residue Symbol Inter-residue Inter-residue contact 3 contacts 2-Rhaa- A 3. 69, H-2 (B) 4. ,11 2-Glca- B 3. • 74, H-3 (F) 3. .69 2-Rhaa- C - 4. .10 3,4-GlcAa- D 4, .10, H-2 (C) 3. .86 Rhaa- E 3, .79, H-4 (D) 3. .96 3-Gal/3- F 4. .13, H-3 (D) 3. ,66b See footnote a, Table II. May be due to scalar correlation effect. - 108 -i 1 1 1 n 1 1 1 r 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 PPM Fig. IV.12: 2D H-H spin-correlated (COSYHG45) spectrum of Pn - 109 -Fig. IV. 13: One step relayed spin coherence transfer (C0SYR1HG) spectrum of Pn - 110 -Fig. IV.14: Two step relayed *H spin coherence transfer (C0SYHGR2) spectrum of Pn J - I l l -Fig. -IV.15: Homonuclear dipolar correlated 2D-n.m.r. (NOESY) spectrum of Pn - 112 -PPM Pig. XV. 16: -^H-n.m.r. spectrum of Pn * indicating B5 of GlcA - 113 -F were linked to D, thus giving the sequence shown. Interestingly, n.O.e.s were observed between A-l and C-l, and C-l and D-l. In both cases, H-l of the glycosyl residue i s located between the proton at the bridge (H-2) and the equatorial neighbouring proton (H-l) of the glycosyl-linked sugar residue. This observation is common for a-D-mannose-type residues linked by a glycosyl group at 0-2.187-192 The proposed structure differs i n several respects from the partial one published previously. 3! The main difference is the absence in the latter of a lateral rhamnose unit although a cross-reaction with Pneumococus suggested i t s presence. 31 It is known, however, that lateral rhamnose residues are sometimes fortuitiously eliminated during purification steps. B F D C A -»2) -a-D-Glcp- (l-»3) -p,-D-Galp(l->3) -a-D-GlcpA- (l->2) -a-L-Rhap- (l-*2) -a-L-Rhap- (1-4 1 a-L-Rhap-E IV.4.3 Experimental General methods are as discussed in Section IV.2.4. COSY. Phage degraded polymer Pn was lyophilized twice from deute-rium oxide and dissolved at a concentration of 25 mg mL"l. This sample was used for a l l the 2D n.m.r. experiments which were conducted at 343°K. The pulse sequence used in the COSY experiment is given in - 114 -Section II.4.1 (Appendix II). The acquisition parameters were identical to those of the COSY experiment in Section IV.2.4 apart from using 112 transients per experiment. Relay COSY (one and two step). The pulse sequence for the relay COSY experiments i s given i n Appendix II. The acquisition parameters for the one step and two step relay COSY n.m.r. experiment were identical to those of the relay COSY experiments given i n Section IV.2.4. NOESY. Several NOESY experiments were performed and the best results were attained using a mixing time (D9) 0.25s with no random variation (i.e. V9 - 0). The other NOESY parameters were identical with those given i n Section IV.2.3. - 115 -CONCLUDING REMARKS Knowledge of the primary structure of bacterial polysaccharides besides explaining the serology of the different bacterial strains on a molecular level, , has important biological, medical and commercial significance. E. c o l i capsular polysaccharides lik e other bacterial polysaccharides consist of regular repeating units. N.m.r. has or i g i -nally been used as a supportive technique to chemical methods in structural studies of carbohydrates. The goal i n this study is to obtain complete structures of E. c o l i antigens using only n.m.r. methods and hence to establish this as a reliable routine process in this laboratory. During this investigation: i . the complete structure of E. c o l i K31 antigen was established using chemical methods. i i . the complete sequence and linkage of the sugar residues in the hexasaccharide repeating unit of E. c o l i K31 capsular polysac-charide was established by homonuclear 2D n.m.r. i i i . the use of 2D n.m.r. as a convenient method for locating the position of acetate in the repeating unit of capsular polysac-charides i s also demonstrated. iv. the complete sequence and linkage of the sugar residues in the repeating unit of E. c o l i K46 polysaccharide was established by 2D n.m.r. (the use of n.m.r. methods for the identification and location of the position of phosphate diester in the repeating - 116 -unit of polysaccharides is illustrated using E. c o l i K46 polysaccharide as an example). While major advances in the application of n.m.r. spectroscopy to structural studies of carbohydrate biopolymers have been made in recent years, the a b i l i t y to identify sugar residues in the repeating unit of complex bacterial polysaccharides using only n.m.r. data has always been a d i f f i c u l t task for carbohydrate n.m.r. spectroscopists. It is hoped that efforts in using only n.m.r. spectroscopy in the complete structure elucidation of bacterial polysaccharides w i l l be achieved in the next few years. - 117 -BIBLIOGRAPHY 1. G.O. Aspinall i n G.O. Aspinall (Ed . ) i "The Polysaccharides", Vol. 1, Academic Press, New York, (1982), p. 1. 2. P.A. Sandford and J. Baird in G.O. Aspinall (Ed.), "The Polysac-charides", Vol. 2, Academic Press, New York, (1983), pp. 411-490. 3. N. Sharon i n "Complex Carbohydrates: Their Chemistry, Biosynthesis and Functions", Addison-Vesley Publishing Company, Massachusetts, (1975). 4. J.F. Kennedy and C.A. White in "Bioactive Carbohydrates: In Chemis-try, Biochemistry and Biology", E l l i s Horwood Limited, England, (1983). 5. A.R. Dochez and O.T. Avery, J. Exp. Med., 26, 477-493, (1917). 6. CM. MacLeod, R.G. Hodges, M. Heidelberger, and W.C. Bernhard, J. Exp. Med., 82, 445-465, (1945). 7. M. Heidelberger, CM. MacLeod, J.J. Kaiser and B. Robinson, J. Exp. Med., 83, 303-320, (1946). 8. M. Heidelberger, Res. Immunochem. Immunobiol., 3, 1-40, (1978). 9. S.M. Hammond, P.A. Lambert, and A.N. Rycroft, i n "The Bacterial Cell Surface", Kapitan Szabo Publishers, Washington, D.C, (1984). 10. L. Kenne and B.Lindberg, in G.O. Aspinall (Ed.), "The Polysaccha-rides", Vol. 2, Academic Press, New York, (1983), pp. 287-363. 11. W.F. Dudman in I.W. Sutherland (Ed.), "Surface Carbohydrates of the Prokaryotic C e l l " , Academic Press, New York, (1977), pp. 287-363. 12. K. Jann and B. Jann, in I.W. Sutherland (Ed.) "Surface Carbohydrates of the Prokaryotic Cell", Academic Press, New York, (1977), pp. 247-287. 13. C.T. Bishop and H.J. Jennings, in G.O. Aspinall (Ed.), "The Poly-saccharides", Vol. 1, Academic Press, New York, (1982), pp. 291-330. 14. K. Jann and 0. Westphal, i n M. Sela (Ed.), "The Antigens", Vol. I l l , Academic Press, New York, (1975), pp. 1-125. 15. B.L. Brandt, M.S. Artenstein, and C.D. Smith. Infect. Immun., 8, 590-596, (1973). - 118 -16. E.C. Gotschlich, M. Rey, R. Trian, and K.J. Sparks, J. Clin. Invest., 51, 89-96, (1972). 17. I.A. Rudbach, J. Immunol., 106, 993-1001, (1971). 18. E. Metu, H.Y. Whang, and H. Mayer, in E.H. Kass and S.M. Wolff (Eds.), "Bacterial Lipopolysaccharides" pp. 48-51, Univ. of Chicago Press, Chicago, I l l i n o i s , (1973). 19. M. Heidelberger, Trends Biochem. Sc. 7, 261-263, (1982). 20. R. Gold, M.L. Lepow, I. Goldschneider, T.L. Draper, and E.C. Gotschlich, J. Clin. Invest., 66, 1536-1547, (1975). 21. R. Gold, M.L. Lepow, I. Goldschneider, and E.C. Gotschlich, J. Infect. Dis., 136, 531-535, (1977). 22. H. Bradley-Mullen, Immunology 40, 521-527, (1980). 23. W.E. Paul, D.H. Katz, and B. Benacerraf, J. Immunol., 107, 685-688, (1971). 24. E.C. Beuvery, F. Miedema, R.W. van Delft, and J. Nagel, "Seminars in Infectious Disease, Bacterial Vaccines", Vol. 4, (Ed.) by J.B. Robbins, J.C. H i l l , and J.C. Sadoff. Thieme-Stratton Inc., New York, (1982). 25. R. Schneerson, 0. Barrera, A. Sutton, and J.B. Robbins, J. Exp. Med., 152, 361-376, (1980). 26. R.L. Whistler, A.A. Bushway, P.P. Singh, W. Nakahara, and R. Tokuzen, Adv. Carbohydr. Chem. Biochem., 32, 235-275, (1976). 27. E.M. Cooke, "Escherichia c o l i and Man", Churchill Livingstone, London, (1974). 28. F. Kauffmann, J. Immunol., 57, 71, (1949). 29. P.R. Edwards and W.H. Ewing, "Identification of Enterobacteriaceae" 3rd Edition, Burgess Publishing Company, Minneapolis, (1972). 30. F. Kauffmann, "The Bacteriology of Enterobacteriaceae". 3rd Edition, Burgess Publishing Company, Minneapolis, (1972). 31. I. ^ rskov, F. J^rskov, B. Jann, and K. Jarm, Bacteriol. Rev., 41, 667-710, (1977). 32. K. Jann and B. Jann, Prog. Allergy 33, 53-79, (1983). - 119 -33. G.G.S. Dutton in V. Crescenzi, I.CM. Dea, and S.S. Stivala (Eds.), "New Developments in Industrial Polysaccharides", Gordon and Breach Amsterdam, pp. 7-26, (1985). 34. M. Heidelberger, K. Jann, and B. Jann, J. Exp. Med., 162, 1350-1358, (1985). 35. C. Adlam, J.M. Knights, A. Mugridge, J.C. Lindon, J.M. Williams, and J.E. Beesley, J. Gen. Microbiol., 131, 1963-1972, (1985). 36. H. Peters, M. Juers, B. Jann, K.N. TImis, and D. Bitter-Suermann, Infect. Immunol., 50, 459-466, (1985). 37. I.W. Sutherland, in I.W. Sutherland (Ed.), "Surface Carbohydrates of the Prokaryotic C e l l " , Academic Press, New York, pp. 209-245, (1977). 38. N.K. Matheson and B.V. McCleary, in G.O. Aspinall (Ed.), "The Polysaccharides", Vol. 3, Academic Press, New York, pp. 1-105, (1985). 39. D. Rieger-Hug and S. Stirm, Virology, 113, 363-378, (1981). 40. H. Geyer, K. Himmelspack, N. Kwiatkowski, S. Schlecht, and S. Stirm, Pure Appl. Chem., 55, 637-653, (1983). 41. M. Adams, "Bacteriophages", Interscience Publishers Inc., New York, (1959) . 42. I. Douglas, "Bacteriophages", Chapman and Hall, London, (1975). 43. C.K. Mathews, "Bacteriophage Biochemistry", Van Nostrand Reinhold Co., New York, (1971). 44. D.E. Bradley, Bacteriol. Rev., 31, 230-314, (1967). 45. R. Morona, J. Tommassen, and U. Henning, Eur. J. Biochem., 150(1), 161-169, (1985). 46. W. Bessler, E. Freund-Molbert, H. Knufermann, C. Rudolph, H. Thurow, and S. Stirm, Virology, 56, 134-151, (1973). 47. M.H. Adams and D.H. Park, Virology, 2, 719-736, (1956). 48. H. Beilharz, B. Kwiatkowski, and S. Stirm, Acta Biochem. Polon., 25, 207-219, (1978} 49. I.W. Davidson, C.J. Lawson, and I.W. Sutherland, J. Gen. Micro-b i o l . , 98, 233-239, (1977). - 120 -50. H. Niemann, A. Birch-Andersen, E. Kjems, B. Mansa, and S. Stirm, Acta Pathol. Scand. Sect., B84, 145-153, (1976). 51. G.G.S. Dutton and D.N. Karunaratne, Carbohydr. Res., 138, 277-291, (1985). 52. C.P.J. Glaudemans, Adv. Carbohydr. Chem. Biochem., 31, 313-346, (1975) . 53. K. Bock, Pure and Appl. Chem., 55, 605-622, (1983). 54. E.M. Purcell, H.C. Torrey, and R.V. Pound, Phys. Rev., 69, 37, (1946). 55. F. Block, W.W. Hansen, and M.E. Packard, Phys. Rev., 69, 127(L), 680(A), (1946). 56. W.G. Proctor and F.C. Yu, Phys. Rev., 77, 717, (1950). 57. W.G. Dickinson, Phys. Rev., 77, 736, (1950). 58. P.J. Garegg, P.E. Jansson, B. Lindberg, F. Lindh, J. Lonngren, I. Kvarnstrom, and W. Nimmich, Carbohydr. Res., 78, 127-132, (1980). 59. W.P. Aue, E. Bartholdi, and R.R. Ernst, J. Chem. Phys., 64, 2229, (1976) . 60. K. Nagayama, A. Kumar, K. Wuthrich, R.R. Ernst, J. Magn. Res., 40, 321, (1980). 61. G. Wagner, J. Magn. Res., 55, 151, (1983). 62. A. Bax and G. Drobny', J. Magn. Res., 61, 306, (1985). 63. P.A.J. Gorin, Adv. Carbohydr. Chem. Biochem., 38, 13-104, (1981). 64. B. Matsuhiro, A.B. Zanlungo, and G.G.S. Dutton, Carbohydr. Res., 11-18, (1981). 65. G. Annison, G.G.S. Dutton, and E. Altman, Carbohydr. Res., 168, 89-102, (1987). 66. G. Kotowyez and R.U. Lemieux, Chem. Rev., 73, 669-698, (1973). 67. M. Karplus, J. Chem. Phys., 30, 11-15, (1959). 68. K. Bock and C. Pedersen, J. Chem. Soc. Perkin Trans., 2, 293-297, (1974) . 69. K. Bock and C. Pedersen, Acta Chem. Scand., Ser. B., 29, 258-264, (1975) . - 121 -70. K. Bock and C. Pedersen, Carbohydr. Res., 145, 135-140, (1985). 71. D.H. Northcote, Methods Carbohydr. Chem., 5, 49-53, (1965). 72. M. Breen, H.G. Weinstein, L.J. Black, M.S. Borcherding, and R.A. Sitting, Methods Carbohydr. Chem., 7, 101-115, (1966). 73. S.C. Churms, Adv. Carbohydr. Chem. Biochem., 25, 13-51, (1970). 74. R.L. whistler and J.L. Sannella, Methods Carbohydr. Chem., 5, 34-36, (1965), 75. J.E. Scott, Chem. Ind. (London), 168-169, (1955). 76. G.G.S. Dutton, Adv. Carbohydr. Chem. Biochem., 28, 11-160, (1973). 77. H.E. Conrad and R.L. Taylor, Biochemistry 11, 1383-1388, (1972). 78. D.N. Karunaratne, Ph.D. Thesis, University of British Columbia, April, (1985). 79. G.G.S. Dutton and M.T. Yang, Can. J. Chem., 51, 1826-1832, (1973). 80. J.F. Codington, K.B. Linsley, and C. Silber, Methods Carbohydr. Chem., 7, 226-232, (1976). 81. S. Inoue, G. Matsumura, and Y. Inoue, Anal. Biochem., 125, 118-124, (1982) . 82. K. Macek, i n E. Heftmann (Ed.), "Chromatography", 2nd Edn., Reinhold Publishing Corporation, New York, (1967), pp. 139-164. 83. I. Smith and J.G. Feinberg, "Paper and Thin Layer Chromatography and Electrophoresis", 2nd Edn., Longman, London, (1972). 84. A. Pryde and M.T. Gilbert, "Application of High Performance Liquid Chromatography", Chapman and Hall Ltd., London, (1979). 85. H. Weigel, Adv. Carbohydr. Chem., 18, 61-97, (1963). 86. R.E. Wing and J.N. BeMiller, Methods Carbohydr. Chem., 6, 42-59, (1972). 87. Z. Dische, Methods Carbohydr. Chem., 1, 477-514, (1962). 88. D. Aminoff, W.W. Binkley, R. Scaffer, and R.W. Mowry, in W. Pigman and D. Horton (Eds.), "The Carbohydrates", 2nd Edn., Vol. 2B, Academic Press, New York, (1970), pp. 739-807. 89. G.G.S. Dutton, Adv. Carbohydr. Biochem., 30, 9-110, (1974). - 122 -90. J.S. Sawardeker, J.H. Sloneker, and A.R. Jeanes, Anal. Chem., 37, 1602-1604, (1965). 91. S.W. Gunner, J.K.N. Jones, and M.B. Perry, Can. J. Chem., 39, 1892-1899, (1961). 92. G.J. Gerwig, J.P. Kamerling, and J.F.G. Vliegenthart, Carbohydr. Res., 62, 349-357, (1978). 93. K. Leontein, B. Lindberg, and J. Lonngren, Carbohydr. Res., 62, 359-362, (1978). 94. M.R. L i t t l e , Carbohydr. Res., 105, 1-8, (1982). 95. G.M. Bebault, J.M. Berry, Y.M. Choy, G.G.S. Dutton, N. Funnell, L.D. Hayward, and A.M. Stephen, Can. J. Chem., 51, 324-326, (1973). 96. B. Lindberg, Methods Enzymol., 28, 178-195, (1972). 97. P.E. Jarmsson, L. Kenne, H. Liedgreen, B. Lindberg, and J. Lonngren, Chem. Commun. Univ. Stockholm, 8, (1976). 98. W.N. Haworth, J. Chem. Soc, 107, 8-16, (1915). 99. T. Purdie and J.C. Irvine, J. Chem. Soc, 83, 1021-1037, (1903). 100. R. Kuhn, H. Trischmann, and J. Low, Angew. Chem., 67, 32, (1955). 101. S.I. Hakomori. J. Biochem. (Tokyo), 55, 205-208, (1964). 102. P.A. Sandford and H.E. Conrad, Biochemistry, 5, 1508-1516, (1966). 103. P. Prehm, Carbohydr. Res., 78, 372-374, (1980). 104. E.L. Hirst, L. Hough, and J.K.N. Jones, J. Chem. Soc, 928-933, (1949). 105. H. Bjbrndal, B. Lindberg, and S. Svensson, Carbohydr. Res., 5, (1967). 106. B. Lindberg, J. Lonngren, and S. Svensson, Adv. Carbohydr. Chem. Biochem., 31, 125-240, (1975). 107. H.O. Bouveng and B. Lindberg, Adv. Carbohydr. Chem., 15, 53-89, (1960). 108. G.W. Hay, B.A. Lewis, and F. Smith, Methods Carbohydr. Chem., 5, 357-361, (1965). - 123 -109. G.W. Hay, B.A. Lewis, and F. Smith, Methods Carbohydr. Chem., 5, 377-380, (1965). 110. J.M. Bobbit, Adv. Carbohydr. Chem., 11, 1-41, (1956). 111. S. Ebisu, J. Lonngren, and I.J. Goldstein, Carbohydr. Res., 58, 187-191, (1977). 112. I.J. Goldstein, G.W. Hay, B.A. Lewis, and F. Smith, Methods Carbohydr. Chem., 5, 361-370, (1965). 113. G.G.S. Dutton and A. Kuma-Mintah, Carbohydr. Res., 169, 213-220, (1987). 114. B. Lindberg and J. Lonngren, Methods Carbohydr. Chem., 7, 142-148, (1976) . 115. B. Lindberg, J. Lonngren, and J.L. Thompson, Carbohydr. Res., 28, 351-357, (1973). 116. J. Kiss, Adv. Carbohydr. Chem. Biochem., 29, 229-303, (1974). 117. G.O. Aspinall and K.G. Rosell, Carbohydr. Res., 57, C22-C23, (1977) . 118. A. Bax and R. Freeman, J. Magn. Res., 44, 542, (1981). 119. G. Bodenbausen, H. Kogler, and R.R. Ernst, J. Magn. Res., 58, 370, (1984). 120. A. Bax and G. Morris, J. Magn. Res., 42, 501, (1981). 121. J.N. BeMiller, Adv. Carbohydr. Chem. Biochem., 22, 25-108, (1967). 122. B. Capon, Chem. Rev., 69, 407, (1969). 123. A.J. Mort and D.T.A. Lamport, Anal. Biochem., 82, 289-309, (1977). 124. A.J. Mort, Abstr. Am. Chem. Soc. Meet., 181, CARB-49, (1981). 125. M.S. Kuo and A.J. Mort, Carbohydr. Res., 145, 247-265, (1986). 126. A.J. Mort and W.D. Bauer, J. Biol. Chem., 257, No. 4, 1870-1875, (1982). 127. J.M. Lau, M. McNeil, A.G. Darvill, and P. Albersheim, Carbohydr. Res., 168, 219-243, (1987). 128. V.N. Reinhold and S.A. Car, Mass Spectrom. Rev., 2, 153, (1983). - 124 -129. K. Stellner, H. Saito, and S.I. Hakomori, Arch. Biochem. Biophys., 155, 464-472, (1973). 130. H. Bjorndal, C.G. Hellerquist, B. Lindberg, and S. Svensson, Angew. Chem. Internat. Edn., 9, 610-619, (1976). 131. A.K. Bhattacharjee and H.J. Jennings, Carbohydr. Res., 51, (1976). 132. B. Munson, Anal. Chem., 43, 28A, (1971). 133. K.I. Harada, S. Ito, N. Takade, and T. Suzuki, A. Biomed. Mass Spectrom., 10, 5, (1983). 134. J. Karkkainen, Carbohydr. Res., 14, 27-33, (1970). 135. J. Karkkainen Carbohydr. Res., 17, 11-18, (1971). 136. N.K. Kochetkov and O.S. Chizhov, Adv. Carbohydr. Chem. Biochem., 21, 39-93, (1966). 137. V. Kovacik, S. Bauer, J. Rosik and P. Kovac, Carbohydr. Res., 8, 282-294, (1968). 138. A. Dell, H.R. Morris, H. Egge, H. Van Nicolai, and G. Strecker, Carbohydr. Res., 115, 41-52, (1983). 139. Z. Lam, M.Sc. Thesis, University of British Columbia, (1987). 140. F.W. Rollgen and H.R.Z. Schulten, Naturforch., 30a, 1683, (1975). 141. G. Puzo, J. C. Prome, and J.I. Fournie, Carbohydr. Res. , 140, 131-134, (1985). 142. E.G. de Jong, W. Heerman, and G. Dijkatra, Biochem. Mass Spec-trom., 7, 127-131, (1980). 143. D.A. McCrery, E.B. Ledrord Jr., and M.L. Gross, Anal. Chem., 54, 1435-1437, (1982). 144. R.B. Cody, J. Kinsinger, S. Ghjaderi, J.L. Amster, F. McLafferty, and F.W. Brown, Anal. Chim. Acta, 178, 43-66, (1985). 145. M.L. Coates and C.L. Wilkins, Anal. Chem., 59, 197-200, (1987). 146. C.L. Wilkins, D.A. Weil, C.L.P. Yang, and CF. Ijamies, Anal. Chem., 57, 520-524, (1985). 147. Z. Lam, M.B. Comisarow, G.G.S. Dutton, D.A. Weil, and A. Bjarnason, .Rapid Commun. Mass Spectrom., 1, 83-86, (1987). - 125 -148. Z. Lam, M.B. Comisarow, G.G.S. Dutton, D.A. Weil, and A. Bjarnason, Carbohydr. Res., 180, C1-C7, (1988). 149. J. Dabrowski, P. Hanfland, H. Egge, and U. Dabrowsky, Arch. Biochem. Biophys., 210, 405-411, (1981). 150. T.C. Farrar, E.D. Becker, Pulse and Fourier-Transform NMR, Academic Press, New York, (1971). 151. D. Shaw, Fourier Transform NMR Spectroscopy, Elsevier, Amsterdam, (1976). 152. H. Gunter, NMR Spectroscopy, Wiley, Chichester, (1980). 153. H.C. Torrey, Phys. Rev., 76, 1059, (1949). 154. R. Freeman and G.A. Morris, Bull. Magn. Reson., 1, 5, (1979). 155. G.A. Morris, i n A.G. Marshall (Ed.) Fourier Hadamard and Hulbert Transformations in Chemistry, Plenum, New York, (1982). 156. A. Bax, Two-dimensional Nuclear Magnetic Resonance in Liquids, D. Reidel, Dordrecht, (1982). 157. L. Muller, A. Kumar, and R.R. Ernst, J. Chem. Phys., 63, 5490, (1975). 158. G. Bodenhausen, R. Freeman, R. Niedermeyer, D.L. Turner, J. Magn. Reson., 26, 133, (1977). 159. R.K. Hester, J.L. Ackerman, B.L. Neff, and J.S. Waugh, Phys. Rev. Lett., 36, 1081, (1967). 160. A.A. Maudsley and R.R. Ernst, Chem. Phys. Lett., 50, 368, (1977). 161. K. Nagayama, P. Bachmann, K. Wuthrich, and R.R. Ernst, J. Magn. Reson., 31, 113, (1978). 162. CO. Chan, Personal communication, Chemistry Department, Univer-sity of Bri t i s h Columbia, Vancouver, B.C., Canada. 163. A. Bax, R. Freeman, and G.A. Morris, J. Magn. Res., 42, 164-168, (1981). 164. M.F. Summers, L.G. M a r z i l l i , and A. Bax, J. Am. Chem. Soc, 108, 4284-4294, (1986), (and references therein). 165. R. Benn and H. Gunter, Angew. Chem. Int. Ed. Engl., 22, 350-380, (1983). 166. L. Lemer and A. Bax, Carbohydr. Res., 166, 35-46, (1987). - 126 -167. R.A. Byrd, W. Egan, M.F. Summers, and A. Bax. Carbohydr. Res., 166, 47-58, (1987). 168. A.S. Perlin, in "MTP Int. Rev. Sci: Org. Chem. Ser. Two", Vol. 7, Carbohydrates 1-34, in G.O. Aspinall (Ed.), Butterworth, London, (1976) (and references therein). 169. A. Bax and D.G. Davis, J. Magn. Reson,, 65, 355-360 (1985). 170. M.L. Hayes, A.S. Serianni, and R. Barker, Carbohydr. Res., 100, 87-101, (1982). 171. A. Bax, W. Egan, and P. Kovac, J. Carbohydr. Chem., 3, 593-611, (1984). 172. G.A. Morris, Magn. Reson. in Chem., 24, 371-403, (1986). 173. H. Thogersen, R.U. Lemieux, K. Bock, and B. Meyer, Can. J. Chem., 60, 44-57, (1982). 174. D. Bundle, M. Gerken, and H.B. Perry, Can. J. Chem., 64, 255-264, (1986). 175. M.A. Bernstein and L.D. Hall, J. Am. Chem. Soc, 104, 5553-5555, (1982). 176. E.L. Hahn and D.E. Maxwell, Phys. Rev., 88, 1070, (1952). 177. S. Stempfle and E.G. Hoffman, Z. Naturforsch., 25A, 200, (1970). 178. D.W. Brown, T.T. Nakashima, and D.L. Rabenstein, J. Magn. Reson., 45, 302, (1981). 179. D.L. Rabenstein and T.T. Nakashima, Anal. Chem., 51, 1465S, (1979) . 180. L.M. Beynon and G.G.S. Dutton, Carbohydr. Res., 179, 419-423, (1988). 181. I.A. Morrison, J. Chromatogr., 108, 361-364, (1975). 182. G.A. Pearson, J. Magn. Reson. 64, 487-500, (1985). 183. D.R. Bundle, I.CP. Smith, and H.R. Jennings. J. Biol. Chem., 249, 7, 2275-2281 (1974). 184. J. Hoffman and B. Lindberg, Methods Carbohydr. Chem., 8, 117-122, (1980) . 185. J. Dabrowski, P. Hanfland, H. Egge, and N. Dabrowski, Arch, Biochem. Biophys., 210, (1981). - 127 -186. A. Kumar, R.R.Ernst, and K. Wuethrich, Biochem. Biophys. Res. Commun., 95, 1-6, (1980). 187. A.A. Grey, S. Narashimhan, J.R. Brisson, H. Schachter, and J.P. Carver, Can. J. Biochem., 60, 1123-1131, (1982). 188. J.R. Brisson and J.P. Carver, J. Biol. Chem.-, 258, 1431-1434, (1983) . 189. S.W. Homans, R.A. Dwek, D.L. Fernandes, and T.W. Rademacher, FEBS Lett., 164, 231-235 (1983). 190. S.N. Bhattacharyya, W.S. Lynn, J. Dabrowski, K. Trauner, and W.E. Hull, Arch. Biochem. Biophys., 231, 72-85, (1984). 191. H. Paulsen, T. Peters, V. Sinwell, R. Lebuhn, and B. Meyer, Justus Liebegs Ann. Chem., 489-509, (1985). 192. J. Dabrowski and M. Hauck, Carbohydr. Res., 180, 163-174, (1988). 193. J.E. Scott. Chem. Ind. (London), 168-169, (1955). 194. V. Smirnyagin, C.T. Bishop, and F.P. Coper, Can. J. Chem., 43, 3109, (1965). 195. B.A. Lewis, Personal communication. Chemistry Department, Univer-sity of British Columbia, 196. G.G.S. Dutton, D.N. Karunaratne, and A.V.S. Lim, Carbohydr. Res., 183, 111-122, (1988). 197. K. Bock, and C. Pedersen, Adv. Carbohydr. Chem. Biochem., 41, 27-66, (1983). 198. J.F.G. Vliegenthart, L. Dorland, and H. Van Halbeek, Adv. Carbo-hydr. Chem. Biochem., 41, 209-374, (1983). 199. H.J. Jennings and I.CP. Smith, Methods Enzymol. , 50C, 39-50, (1978). 200. I.A. Morrison, J. Chromatogr., 108, 361-364, (1975). 201. J.H. Bradbury and G.A. Jenkins, Carbohydr. Res., 126, 125-156, (1984) . 202. J.H. Banoub and D.H. Shaw, Can. J. Chem., 59, 877-879, (1981). - 128 -APPENDIX I H Chemical Shift Assignments of 3,6-dideoxy Amino Sugar Residue in E. c o l l K45 Capsular Polysaccharide and Structural Studies on E. c o l i K46 K Antigen - 129 -STRUCTURAL STUDIES ON E. COLI K46 POLYSACCHARIDE Introduction The occurrence of amino-sugars in the capsular polysaccharides of E.  c o l i i s known. Most of the reported K antigens of E. c o l i are acidic. In this investigation structural studies on a K antigen containing an amino sugar and a phosphate ester are reported. Results and Discussion Composition. Analysis of the native polysaccharide before and after reduction 7 7 both gave glycerol, rhamnose, glucose, galactose and glucosamine in the ratio 0.5:0.7:1.08:1.08:1. N.m.r. studies (see later) suggest the presence of phosphate ester and a three carbon frag-ment in addition to four sugar residues i n the repeating unit of the K46 polysaccharide, thus suggesting that this polysaccharide consists of a pentasaccharide repeating unit with a composition of rhamnose, glucose, galactose, glucosamine, glycerol in a ratio of 1:1:1:1:1. Methylation. Methylation^Ol analyses gave the results shown in Table VII.I from which i t may be deduced that a glucose residue occupies a terminal position and a galactose unit constitutes the branch point. - 130 -Table VII.1: Methylation analysis of E. c o l l polysaccharide Sugar Residue Methylated sugar a (as a l d i t o l acetates) Mole % b -*3)-Rha-(l-* Glc(l-+ •2)-Gal(l-3 2,4-Rha 2,3,4,6-Glc 4,6-Gal 27.28 30.10 21.77 >3)-GlcNAc(l-» 4,6-GlcNAc 20.10 2,3,4,6-Glc - l,5-di-0-acetyl-2,3,4,6-tetra-0-methylglucitol etc. Values are corrected using the effective carbon response factors given by Sweet et a l . ; determined on a DB-17 column programmed for 1 min. at 180°C then 250°C at 2°/min. Dephosphorylation. 60 mg of dephosphorylated product was obtained by treating 100 mg of native polysaccharide with 48% aqueous hydrogen fluoride (for 72 h at 4°C) and purifying by gel permeation chromato-graphy (Bio Gel P2). 5 mg of dephosphorylated product was treated with anhydrous hydrogen fluoride at 20°C for 6 h. The resultant product was converted to a l d i t o l acetates by sodium borodeuteride reduction and acetylation. G.l.c. results indicate that the dephosphorylated product contains - 131 -glycerol, rhamnose, glucose, galactose and glucosamine i n ratios of 1:1:1:1:1. Detailed n.m.r. studies were performed on the dephosphorylated product (see later). The dephosphorylated product i s awaiting FAB-MS analysis. N.m.r. studies. -^H-N.m.r. (400 MHz, 300°K) spectrum of native polysaccharide showed four signals i n the anomeric region at 6 4.97 (s, 1H), 4.79 (d, 1H, Jm - -8 Hz), 4.75 (d, 1H, - -8 Hz), and 4.66 (d, 1 H , Jm 8 Hz), also signals at 2.05 (s, 3H, CH3 of NAc) and -1.30 (d, 3H, Jjm - -7.5 CH3 of rhamnose). Consistent with the results, the 1 3C-n.m.r. (75 MHz, 300°K) spectrum showed characteristic signals at 103.730, 102.864, 101.553 and 99.706 ppm corresponding to four anomeric carbons. The 13C-n.m.r. spectrum of the native CPS contains 28 carbon resonances (Fig. VII.1) but the heteronuclear correlated spectrum (Fig. VII.8) indicated 29 cross-peaks (see later) (i.e. two carbon resonances coincided). This observation suggests the presence of a three carbon fragment (glycerol) i n the repeating unit i n addition to four sugar residues. Due to splittings i n four- of the 1 3C s i g n a l s 1 8 3 ' 1 9 9 (Fig. VII.1 i.e. 1 3 C broad band ^ decoupled spectrum), the presence of an element with spin - 1/2 apart from *H and 1 3C in the repeating unit of the native polysaccharide was suspected. 31P-N.m.r. (122 MHz) (see Fig. VII.4) indicated the presence of a phosphorus ester i n the repeating unit of the native polysaccharide. 2D N.m.r. studies (see later) suggested that the phosphorus ester i s linked at C-3 to a galactose residue. - 132 -CO O 1*3 I m T CM O tri to to CM 1 200 • i " " i " 1 180 160 140 120 100 80 60 40 CM 20 0 PPM •>- O) CM d co to I I u o CO I I 1 CM CO O) CD cd co CO CO I •M ) m o coco in in co co I' I oo CM K> CO I m CO CO oo I „ ^ s CO I I , o CO CO in m l 80 • | i i i i | i n n | i i i i | i n i | • •• I • • • 11 . , • 11 . 7 0 «!•••• I 1 "I '"I 60 PPM Fig. VII.1: l3C-K.m.r. (*H decoupled) spectrum of K46 native polysaccha ride (I) f u l l spectrum ( i i ) ring region ( 9 0 - 6 0 ppm) - 133 -q I •-NO if) ro 00 O O O 01 V s * 7 CM 200 180 160 140 120 100 80 i 60 40 To" PPM co »- o> CM oo oo e n ' : oo co co_ r ^ N 1 as 1 0 1 0 . CM * cq co q > L J J J 11 FvJ:,^ ~80 •ifj f O CM CD I •r-' O CO CO I I m in I • • A « « Mr .V •.' J • i 70 i 60 PPM Fig. VII.2: 13C-K.«i.r. (}u decoupled) spectrum of K46 dephosphorylated product, ( i ) f u l l spectrum (11) ring region. 134 To 100 80 TO" 20 CN OO I d oo I CO cn co. n < - o o co co »— to' co" co iri Is* r>» «o CM •* oo m T O 70 co co co * - T-r - CO CO ^ 0> CM «— T- O CO CO CO CO CO I II I 60 PPM CO to in I PPM Fig. VII.3: 13C-H.B.r.-AFT spectrum of K46 dephosphorylated product (1) f u l l spectrum; ( i i ) ring region - 135 -i i | i i i i | i i : i ; i i i i j i i i i | n i i | M i i i i i i i | i i ; i | i i i i | i : i i | h ^ V I 1.0 C S 0.6 .^4- 0 2 C O —0 2 —0.4—0.6 —0.8 -PPM Fig. VII.4: J1P-n.m.r. ( AH coupled) of native polysaccharide (K46) - 136 -The sugar residues were arbi t r a r i l y labelled A to D in order of decreasing chemical shift of their H-l resonances. The results of COSY 1 6 3 (Fig. VII.5 and Table VII.2) afforded the assignment of H-l and H-2 resonances of most of the sugar residues in the repeating unit. Further assignments were made using COSY results after interpreting the data for the heteronuclear shift correlated 2D n.m.r. experiment. One and two step r e l a y 6 2 spectra (Fig. VII.6 and Table VII.2) afforded the assignments of H-3 and H-4 resonances of most of the sugar residues in the repeating unit. The entire J system of the residue B was relayed in the two step relayed COSY experiment (Fig. VII.6). This may be due to long T l and T2 since B is the lateral residue. However, this was not further investigated because of machine time. The assignment of the H^ signals for the a-6-deoxy residue A was greatly f a c i l i t a t e d by the additional window provided by the methyl resonance. The heteronuclear shift correlated 2D n.m.r. experiment using the CHORTLE technique 1 8 2 w a s then performed and permitted the unambiguous assignments of most carbons in the repeating unit (Fig. VII.8 and Table VII.4). The correlation of the methylene carbons with the corresponding proton signal via the CHORTLE experiment led to assignment of some of the resonances of the ^H-n.m.r. spectrum (Table VII.2). C-2 of the aminohexosyl residue has a characteristic resonance at about 55 ppm.2^1 H-2 of the residue C correlated with carbon resonance at 55.340 ppm in the heteronuclear correlated spectrum (Fig. VII.8, Table VII.2 and Table VII.4). Thus C was identified as glucosamine. H-2 resonance (3.247 ppm) of residue B (Table VII.2) is typical of - 137 -Table VII.2: -^H-n.m.r. data for E. c o l l K46 polysaccharide Sugar • Symbol Residue H-l H-l H-2 H-3 H-4 H-5 H-6 H-61 A -»3)a-Rha(l- -4.97 - 4.28 3.88 3.49 3.91 1.30 B £-Glc(l- 4.79 - 3.25 3.50 3.38 3.43 3.74* 3.91* C -3)-0-GlcNAc(l- 4.75 - 3.89 3.92 -3.59 3.49 3.78* 3.77* D -*2)-0-Gal(l-» 4.66 E glycerol 4.05* 3.93* 3.74 / 3 - g l u c o s e . B a s e d on this evidence and methylation results (see later) residue B was identified as terminal /?-glucose. Methyl residue resonance at 1.298 ppm showed 1H spin correlation (i.e. C O S Y spectrum Fig. V I I . 5 and Table V I I . 2 ) to H-5 of residue A. Based on this evi-dence, the fact that H - l resonance i s a singlet and methylation data suggests that residue A is 3-linked a-rhamnose. Residue D was then assigned as 2,3-linked ^-galactose residue and E as a glycerol. The anomeric configurations of the sugar residues in the repeating unit were established by a 1 3 C-1H coupled n.m.r. experiment (Table V I I . 5 and spectrum in Appendix I I I ) . - 138 -r 0 mo A1 i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—r 5.1 4.9 4.7 4.5 4.3 4.1 3.9 3.7 3.5 3.3 3.1 PPM Fig. 'VII.5: Homonuclear •LH-spin correlated (COSY) n.m.r. spectrum of native polysaccharide (K46) - 139 -Table VII.3: N.O.e. data for E. c o l i K46 polysaccharide Symbol Sugar Interresidue Intraresidue a Residue n.O.e. n.O.e. -3)-a-Rha-(l- 3.94(H2ofD) 4.22 B -£-Glc-(l- 3.92 3.25, -3.50, 3,38 -3)-/3-Glc-NAc-(l- 3.87 (H3 of A) -3.50 D -2)-0-Gal-(l- 3.93 4.31, 3.90 a could be scaler correlated effect - 140 -- 141 -- 142 -VII.8: Heteronuclear (•LH-J-SC) correlated spectrum of native polysaccharide (K46) - 143 -Table VII.4: 13C-N.m.r. chemical shifts* and 3 1 P - 1 3 c coupling constants (in parentheses) for E. c o l i K46 native polysaccharide Symbol Sugar C-l C-2 C-3 C-4 C-5 C-6 Residue A -3)-a-Rha(l- 99.71 70.81 80.97 71.80 80.97 17.57 B 0-Glc(l- 101.55 74.54 76.81 70.47 76.81 61.78 C -3)-/9-GlcNAc(l- 102.86 55.34 82.14 68.92 75.90 61.65 D -2)-0-Gal(l- 103.73 -77.52 -78.55 68.68 75.63 61.33 3 (7.95 Hz) (4.4 Hz) t E Glycerol 65.83 76.23 60.62 (3.7 Hz) (5.25 Hz) In ppm from Internal acetone (1% 31.070 ppm) - 144 -The sequence of the sugar residues in the repeating unit was obtained by data from a NOESY experiment 1 8 9 (Table VII.3 and Fig. VII.7). Inter-residue n.O.e. contacts 1 8^ established that A was linked to D, C to A, B to C and D to E, thus giving the sequence below. 0 n CH20-P-0-I 0. -*3)-0-Galp(l-K>-CH 2 J CH20H 1 /9-Glcp- (1-+3) -0-GlcNAcp(l->3) -a-Rhap-Methylation analysis data indicate that the galactose residue is linked at C-2 and C-3. The sp l i t t i n g of 1 3C resonances 1 9 9' 2 0 1 of C-2 and C-3 of the galactose residue gave an indication that the phosphate diester residue i s scalar coupled to these carbons. Sugar analysis and 13C-n.m.r. data on the dephosphorylated product (Pn) indicate that the Pn is a pentasaccharide repeating unit. The ^ -n.m.r. spectrum of Pp does not show anomerization of the galactose residue. These evidences and NOESY results (i.e. galactose i s linked at C-2 to the rhamnose residue) suggests that the phosphate diester i s linked to C-3 of the galactose residue and C-l of the glycerol. The H-l and H-2 resonances of most of the sugar residues in the repeating unit of the dephosphorylated product (Pn) are obtained from COSY data (Fig. VII.9 and Table VII.6). The H-3 and H-4 resonances of most of the sugar residues in Pn were established from one and two step - 145 -Table VII.5: ^C-^H Coupled n.m.r. experimental data Chemical shift (^H-decoupled resonance) ppm and sugar residue Chemical shift •^H-coupled downfield resonance upfield resonance Chemical shift difference between down-f i e l d & upfield resonances «C-H coupling constant ^CH 103.73 (0-Gal) 104.799 102.629 -2.17 162.75 102.86 (/3-GlcNAc) 103.903 101.707 -2.20 164.70 101.55 (/3-Glc) 102.629 100.438 2.19 164.31 99.71 (a-Rha) 100.776 98.512 2.26 169.80 - 146 -Table VII.6: chemical shift data of K46-dephosphorylated polysaccharide Sugar Symbol Residue H-l H-2 H-3 H-4 H-5 H6/H6 •3)a-Rha(l- -4.97 4.22 3.88 3.51 3.86 1.29 B /5-Glc(l- 4.81 3.30 3.51 3.42 0)-/9-GlcNAc(l- 4.80 3.82 3.93 3.60 D -2)-£-Gal(l- 4.60 3.79 3.86 3.92 E Glycerol - 147 -Table VII.7: 13C-N.m.r. chemical s h i f t s 3 for E. c o l l dephosphorylated product Symbol Sugar C-l C-2 C-3 C-4 C-5 C-6 Residue A -3)-a-Rha(l- 99.77 70.92 74.31 71.75 69.81 17.40 B 0-Glc(l- 101.82 74.66 76.63 76.01 C -3)-/9-GlcNAc(l- 103.37 55.61 69.62 69.04 D -2 ) - 0-Gal(l- 103.51 78.55 80.99 82.41 3 t E Glycerol . . . . In ppm from internal acetone (1% 31.070 ppm) - 148 -52 4.8 4.4 i 1 1 r 4.0 3.6 32 2.8 PPM i ~i r 2.4 2.0 1.6 12 Fig. VII.9: Homonuclear ^H-spin correlated (COSY) n.m.r. spectrum of dephosphorylated product (K46) - 149 -PPM -12 -1.6 - IB -24 -2.8 e e -32 -3.6 -4.0 CI Bf A l * • Dl D3D2 . c 3C2b2B2 A2 A3 -4.4 -4.8 -52 ~ i 1 1 1 1 1 1 1 1 1 r ~ 52 4.8 4.4 4.0 3.6 32 2.8 24 L8 1.6 12 PPM Fig. VII.10: One step relay 1H spin coherence transfer (COSYRCT) spectrum of dephosphorylated product (K46) - X3U -6 0 ci Bl A l ? . 03 C4 • •>. *>«SOO 0 C3C2B3B4B2 A2- A3 A4 i 0 i i 1 1 1 1 1 1 1 1 r -52 48 44 4.0 3.6 32 28 2.4 2.0 1.6 12 PPM PPM -1.2 -1.6 -2.0 -2.4 -28 -3.2 -3.6 -4.0 -4.4 -48 -52 Fig. VII.11: Tvo step relay 1H spin coherence transfer (COSYRCT) spectrum of dephosphorylated product (K46) - 151 -relay COSY experiments (Figs. VII.10, VII.11 and Table VII.6). The extra window provided by the methyl group resonance afforded the assignment of the chemical shift of resonance of residue A. Residue A was assigned as 3-linked a rhamnose, B as later a l /?-glucose, C as 3-linked glucosamine, D as 2,3-linked /?-galactose and E as three carbon fragment (Table VII.6). This assignment is based on previous results (Table VII.2). A heteronuclear correlated n.m.r. experiment on Pn afforded the assignment of carbon resonances, C-l to C-4 of most of sugar residues. Pn i s awaiting FAB mass spectrometric analysis. A polysaccharide (Px), of significantly lower molecular weight and viscosity than the native polysaccharide was prepared by depolymeriza-tion with a v i r a l endoglycanase and was used for 2D n.m.r. studies. The use of Px enabled the preparation of more concentrated samples than was used for the previous 2D n.m.r. study. In fact this 2D n.m.r. study was an attempt to reproduce the n.O.e. contacts in Table VII.3 using a lower molecular weight polymer. The sugar residues were ar b i t r a r i l y labelled A' and D' i n order of decreasing chemical shift of their H-l resonances. The results of COSY 1 6 3 (Fig. VII.13 and Table VII.8) gave the assignments of H-l and H-2 resonances of a l l the sugar residues i n the repeating unit. One and two step relay COSY62 spectra (Figs. VII.14, VII.15 and Table VII.8) afforded the assignments of H-3 and H-4 resonances of the rhamnosyl, glucosyl and 2-acetamido-2-deoxyglucosyl residues. The assignment of H-5 of the rhamnosyl residue was achieved by the additional window provided by methyl resonances. Partial sequencing of the sugar residues in the repeating unit was - 152 -Table VII.8: H^-N.m.r. data for E. c o l i K46 bacteriophage polysaccha-ride (Px) Sugar residue H-l H-2 H-3 H-4 H-5 H-6 H6] A' (Rha) 4.96 4.20 3.86 3.50 3.85 1.33£ B' (Glc-NHAc) 4.82 3.82 3.95 3.52 C (0-Glc) 4.78 3.28 3.53 3.42 D' (jS-Gal) 4.64 3.91 3.93 3.81 a chemical shift values of CH3 of rhamnosyl residue Table VII.9: N.O.e. data of lower molecular weight polymer (Px) Symbol Sugar residue Interresidue n.O.e. Intraresidue n.O.e. A' -*3)-a-Rha-(l-» 4.20 B' -*3)-p>-GlcNAc-(l- 3.87 (H3 of A) C' /3-Glc-(l- 3.28 -2,3)-Gal-(l-* 3.95 scalar effect - 153 -Fig.-VII.12: Heteronuclear ("C-^ -H) correlated n.m.r. spectrum of K46 dephosphorylated product - 154 -Fig. VII.13: Homonuclear ^-H-spin correlated (COSY) n.m.r. spectrum of a lover molecular veight polymer (Px) derived from K46 native polysaccharide - 155 -5.4 5.0 4.6 4.2 3.6 3.4 3.0 2.6 2.2 1.8 1.4 PPM Fig. VII.14: One step relayed XH spin coherence transfer (C0SYR1HG) n.m.r. spectrum of a lover molecular velght polymer (Pz) derived from K46 native polysaccharide - 156 -PPM Fig.- VII.15: Two step relayed XH spin coherence transfer (C0SYHGR2) n.m.r. spectrum of a lover molecular weight polymer (Pz) derived from K46 native polysaccharide - 157 -7] PPM -.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 .5 PPM Fig. VII.16: Homonuclear dipolar correlated 2D n.m.r. (NOESY) spectrum of a lover molecular weight polymer (Px) derived from K46 native polysaccharide - 158 -) obtained by data from a NOESY experiment 1 8 5 (Table VII.9 and Fig. VII.16). Interresidue n.O.e. con t a c t s 1 8 6 established that B' was linked to A' thus giving the sequence shown. (B)'. - (A)' G l c N A c ^ Rha Chemical studies and n.m.r. studies on the native polysaccharide and bacteriophage product (Px) afforded the total sequence of the sugar residues i n the repeating unit of the polysaccharide as shown below. 0 II CH20-P-0-I I I °--•3)-0-Galp(l-M3-CH 2 CH20H 0-Glcp- (l-»3) -0-GlcNAcp(l-»3) -o-Rhap-Experimental General methods. The general experimental procedures are as described i n Section III.1.3. Isolation and purification of E. c o l i K46 capsular polysaccharide. E. c o l i K46 culture was obtained from Dr. Ida j^rskov, WHO International Escherichia Centre, Copenhagen). Actively growing colonies of E. c o l i - 159 -K46 were propagated by replating several times onto Petri dishes (layered with s t e r i l e Mueller Hinton agar), a single colony being selected each time the bacteria were to be plated. This culture was grown as described i n Section III.1.3. The K46 bacteria were scraped from the agar surface, diluted with 1% phenol solution and stirred at 4°C for 6 h. The mixture of polysaccharide bacterial c e l l s and other debris was ultracentrifuged (for 4 h at 15°C on a Beckman L3-50 ultracentrifuge using rotor 45 T i at 31,000 r.p.m. or 80,000 g) to separate the polysaccharide from the dead bacterial c e l l s . The viscous honey-colored supernatant was precipitated with ethanol. The resultant stringy precipitate was dissolved in water. The resultant solution was treated with a few drops of Cetavlon (cetyltrimethylammonium bromide) and was centrifuged. The supernatant was further treated with Cetavlon and kept at 4°C overnight. This fractional precipitation was adapted because the f i r s t precipitate was impure. The Cetavlon-polysaccharide complex was dissolved i n 4M NaCl solution, precipitated into ethanol, redissolved i n water and dialyzed against d i s t i l l e d water (for two days). The polysaccharide was isolated as a styrofoam-like material by lyophilization. The isolated polysaccharide was further purified by gel permeation chromatography using Sephadex S-400 column (100 cm x 2.5 cm). Sugar analysis and composition. 10 mg of K46 polysaccharide was treated with anhydrous hydrogen fluoride for 6 h at 25°C, then the acid was removed by evaporation and co - d i s t i l l e d with 5% acetic acid. The neutral sugars were analyzed as a l d i t o l acetates by g.l.c.-m.s. A portion of K46 polysaccharide (10 mg) was methanolyzed overnight in 3% - 160 -HCI-CH3OH, and the resultant product was reduced with sodium borohy-dride, hydrolyzed and analyzed by g.l.c.-m.s. as a l d i t o l acetates. Methylation analysis. The native polysaccharide (20 mg) was methylated by the method of Hakomori.^l dialyzed, partitioned between dichloromethane and water and purified on a column of Sephadex LH20 to give a product which showed no absorption at 3625 cm"1. The methylated polymer was converted to a l d i t o l acetates and analyzed by g.l.c.-m.s. Determination of reducing end.^OO A sample (7 mg) of compound 2 (i.e. lower molecular weight compound) was converted to i t s a l d i t o l by NaBH4 reduction. This a l d i t o l was hydrolyzed using 4M TFA for 0.5 h at 122°C. After removing the excess TFA, the anhydrous residue was treated with 5% hydroxylamine hydrochloride i n pyridine (1 mL) and heated at 95°C for 20 min. Anhydrous acetic anhydride (1 mL) was added to the cooled reaction mixture and further heated (at 95°C for 25 min). Removal of excess acetic anhydride and pyridine was attained by the addition of ethanol and repeated c o d i s t i l l a t i o n with water. Pure a l d i t o l acetate and acetylated aldononitriles were obtained by water/ chloroform extraction and evaporation of the chloroform phase. The a l d i t o l acetate and acetylated aldononitriles were analyzed by g.l.c.-m.s. on a DB-17 column programmed at 180°C, 5°C per minute to 220°C. Dephosphorvlatlon of native polysaccharide. Native polysaccharide (100 mg) was dissolved i n 48% aqueous hydrogen fluoride and stirred at - 161 -4°C for 72 h. The reaction mixture was then neutralized using 50% ammonium hydroxide solution. The reaction mixture was desalted on Sephadex G10. The isolation of dephosphorylated polysaccharide was by gel permeation chromatography on Bio-Gel P2 column (100 cm by 2.5 cm). This resultant product (60 mg) was analyzed by 1 3C, ^H, 3 1 P n.m.r. and 2D n.m.r. 5 mg of dephosphorylated product was dried under vacuum for 18 h and treated with anhydrous hydrogen fluoride for 6 h. The excess hydrogen fluoride was removed by evaporation and c o - d i s t i l l a t i o n with 5% acetic acid aqueous solution. The resultant product was reduced by sodium borodeuteride and analyzed by g.l.c.-m.s. as a l d i t o l acetates. Bacteriophage depolymerization. The phage was isolated from Vancouver sewage and propagated by tube and flask l y s i s to a concentra-tion of 1.3 x mL'1. Depolymerization and isolation of low molecular weight polymer (Px) were conducted as described In Section III.1.3. 140 mg of K46 polysaccharide was used i n depolymerization and 60 mg of Px was isolated. N.m.r. studies General methods for 2D homonuclear experiments are as discussed i n Section IV.2.4. The native polysaccharide was lyophilized twice from deuterium oxide and dissolved at a concentration of 30 mg mL'1. 13C-N.m.r. spectra were obtained using samples i n 5 mm diameter tubes at 300°K on a Varian XL 300 spectrometer operating at 75 MHz i n the pulsed Fourier transformed - 162 -mode with complete proton decoupling. 1 3C- 3 1P Coupling constants were obtained from the expanded ring region (90 ppm to 60 ppm) of the 13C-n.m.r. spectrum at 75 MHz. A heteronuclear 1 3C- 1H shift correlated experiment was done on a Bruker AM 400 spectrometer using the CHORTLE technique (carbon-hydrogen correlations from one-dimensional polariza-tion-transfer spectra by least-square a n a l y s i s ) . e x p e r i m e n t was performed at 300"K on a sample of a native CPS in deuterium oxide (300 mg mL"1). 128 experiments were performed using 1072 transients per experiment. The acquisition parameters of the COSY and relay COSY (i.e. one and two step) experiments were identical to that given in Section IV.2.4. These experiments were performed on the AM 400 Bruker spectrometer. The pulse sequence for the NOESY experiments is given in Appendix II. Several NOESY experiments were performed and the best results were obtained using a mixing time (D9) of 0.255s with no random variation (V9=0). The other details are: Sweep width in F l dimension (SW1) - 920 Hz Relaxation delay (Dl) - 1.2 s Pre-saturation with power SI - 40 L Delay for evolution of shifts (DO) •=• 0.0002 (with increments of 0.5/SW1 for 256 times) 90° excitation pulse (PI) - 16 ps Mixing pulse 90° (P2) - 16 ps Detection pulse 90° (P3) - 16 ps Number of scans per experiment (NS) - 112 Number of experiments (NE) - 256 31P-N.m.r. experiments were performed on a Varian XL300 at 122 MHz in the pulse Fourier transform mode with no proton decoupling. Phage degraded polymer Px was lyophilized twice from deuterium oxide and dissolved at a concentration of 35 mg mL"1. This sample was used - 163 -for the 2D n.m.r. experiments a l l of which were conducted at 343° K. The acquisition parameters of the COSY experiment were identical to those given in Section IV.2.3 except for using 112 transients per experiment. Apart from the second coherence period (D2) and third coherence period (D3), the acquisition parameters for relay COSY experiments were identical to those given in Section IV.2.3. The second coherence period (D3) was varied in a number of experiments to attain the best relayed spin coherence transfer. Several NOESY experiments were performed and the best results were attained using a mixing time (D9) of 0.26 s with no random variation (i.e. V9 - 0). The other NOESY parameters were identical with those given in this section. Chemical shift assignment of 3.6-dideoxvamino sugar residue in E.  co l i K45 capsular polysaccharide Introduction The repeating unit of the K antigen of E. c o l i K45 includes a 3,6-dideoxyamino sugar residue but the complete structure has not been established. Further work w i l l be done to establish the structure. The chemical shift assignment of the 3,6-dideoxyamino sugar residue is reported. Results and discussion The repeating unit of E. c o l i K45 polysaccharide contains a 3,6-di-- 164 -deoxy amino sugar and the mass spectrum of the a l d i t o l a c e t a t e 2 0 2 of this amino sugar is shown below. !I4 230 100 200 258 300 400 500 Table VII.10: •LH N.m.r. data for E. c o l i K45 native polysaccharide Symbol Sugar residue H-l H-2 H-3 H-4 H-5 H-6/H-6' 4.94 4.04 3.84 3.63 3.51 4.62 3.88 3.56 4.30 3.83 3,6-dideoxy 4.54 3.62 3.57 3.81 3.95 1.26 amino sugar 4.48 3.53 3.92 3.74 3.66 - 165 -The H-l, H - 2 , H - 3 , and H-4 resonances of a l l the sugar residues i n the repeating unit were assigned from the COSY n.m.r. experiment data (Fig. V . l and Table V . l ) . Complete assignment of the proton resonances of residue C was afforded by the additional window provided by C H 3 resonance. Residue C was assigned as 3,6-dideoxyamino sugar. An attempt to isolate a pure form of the 3,6-dideoxyamino sugar for K45 polysaccharide by paper chromatography was unsuccessful. It was anticipated that a 2 D - C 0 S Y phase-sensitive double quantum f i l t e r n.m.r. experimental result on the pure form of the 3,6-dideoxyamino sugar could have provided complete structure of this amino sugar. The isolation of a l d i t o l acetates of the 3,6-dideoxyamino sugar and i t s analysis by X-ray crystallographic w i l l be pursued. Although the complete structure of the 3,6-dideoxyamino sugar is unknown, this study provides the finger print of the proton chemical shifts of this amino sugar. Experimental General methods for 2 D homonuclear experiments are as discussed in Section IV.2.4. The acquisition parameters of the COSY experiment were Identical to those given i n Section IV.2.4 except for using 48 transients per experiment. C 0 S Y-45 (45° mixing pulse P 2 - 9.8 /is) and COS 90 (90° mixing pulse P 2 - 19.5 us) experiments were performed. COSY-45 provided better results (see Table V . l and Fig. V . l ) . - 166 -Native polysaccharide (K45) was lyophilized twice from deuterium oxide and dissolved at 25 mg mL"1. COSY experiment (Table V . l and Fig. V.l) were performed at 338°K. - 167 -PPM PPM Fig. VII.17: Homonuclear ^ - s p i n correlated (COSY) n.m.r. spectrum of native polysaccharide (KA5) - 168 -APPENDIX II Bruker 2D Files Employed in this Study - 169 -; COSY.AU ; HOMONUCLEAR SHIFT-CORRELATED 2-D NMR (JEENER) ; W.P.AUE, E.BARTHOLDI, R.R.ERN5T, J.CHEM.RHYS. 64, 2229 U976) ? K.NA6AYAMA ET AL, J.MAGN.RES. 40, 321 (1980) ; DI — 90 - DO - 90 OF: 45 - FID ; SYMMETRIC MATRIX WITH SHIFTS AND COUPLINGS IN Fl , F2 ; OFF-DIAGONAL PEAKS CORRELATE SPINS WHICH SHARE A f SCALAR COUFLING J. .1 2E 2 DI |RELAXATION 3 PI PHI |90 DEG EXCITATION PULSE A DO (EVOLUTION OF SHIFTS AND COUPLINGS 5 P2 PH2 {MIXING PULSE, 90 OR 45 DEG £ 60=2 jACQUIRE FID 7 WR *tl | STORE FID 8 IF #1 ;INCREMENT FILE NUMBER 9 IN=1 ;INCREMENT DO AND LOOP FOR NEXT EXPER. 10 EXIT PH1=A0 AO AO AO Al Al Al Al ;PHASE PROGRAMS CANCEL AXIAL A2 A2 A2 A2 A3 A3 A3 A3 ; PEAKS (SCANS 1-2), SELECT N-TYF'E ;PEAKS (SCANS 3-4), SUPPRESS F2 PH2=A0 A2 Al A3 Al A3 A2 AO {QUAD IMAGES (SCANS 5-S:> , AND CAN Al A3 A2 AO A2 AO A3 Al {ARTEFACTS FROM PI (SCANS 9-l£>. ;PROGRAM REQUESTS FILENAME WITH .SER EXTENSION ; N E DEFINES NUMBER Or FIDS = TD1 ;USE OP, NS = 4,8, OR 16 (COMPLETE PHASE CYCLE> ;PS = 2 OR 4 ;RD=PW=0 ;D1 = 1-5+-T1 ;P1 = 90 DEG ;F2 = 90 DEG FOR MAX. SENSITIVITY { = 45 DEG FOR MINIMAL DIAGONAL (GOOD FOR TIGHT AB SYSTEMS> ; AND 'TILTED' CORREL. PEAKS (SIGNS Or COUPLINGS). {DO = 3E-6 INITIAL DELAY {IN = 0.5/SW1 «= 2*DW {NDO = 1 { I2D = 1, SWl=SW/2 {CHOOSE SW AND SI SD THAT HZ/PT = CA. 2-£ HZ {TYPICALLY USE TD * SI, NO ZERO-FILLING IN F2 { NE « SI/4, ZERO-FILL IN Fl {MATRIX CAN BE SYMMETRIZED ABOUT DIAGONAL - 170 -•LIST C0SYH6.AU FILE: COSYHS .AU f COSYHG.AU 1 HOMONUCLEAR SHIFT-CORRELATED 2-D NMR (JEENER) » USING PRE-SATURATION Or SOLVENT WITH TWO POWER LEVELS. i ALSO ALLOWS FOR FIXED DELAY AS IN COSYLR.AU « » HG<S1)-<S2> — D 0 I Dl - D3 -90 - DO -D2-90 OR 45-D2 - FID ; SYMMETRIC MATRIX WITH SHIFTS AND COUPLINGS IN F l , F2 ; OrF-DIAGONAL PEAKS CORRELATE SPINS WHICH SHARE A • SCALAR COUPLING J. 1 ZE Dl HG SI ;RELAXATION, PRE-SATURATION WITH POWER SI D3 S2 ;SWITCH TO MIN. POWER. S2 FOR EVOLUTION 3 PI PHI ISO DEG EXCITATION PULSE 4 DO ;EVOLUTION OF SHIFTS AND COUPLINGS D2 ;FIXED DELAY TO ENHANCE EFFECTS FROM SMALL J 5 P2 PH2 I MIXING PULSE, 90 OR 4 5 DEG £ u ^ 60=2 DO ;ACQUIRE FID WITH DEC. GATED OFF 7 WR *1 jSTORE FID B IF #1 1 INCREMENT FILE NUMBER 9 IN=1 I INCREMENT DO AND LOOP FOR NEXT EXPER. 10 EXIT PHI-AO AO AO AO Al Al Al Al A2 A2 A2 A2 A3 A3 A3 A3 PH2-A0 A2 Al A3 Al A3 A2 AO Al A3 A2 AO A2 AO A3 Al ;PROGRAM REQUESTS FILENAME - 171 -FILE: COSYRCT .AU ; CDSYRCT.AU | COSY WITH 1-STEP RELAYED COHERENCE TRANSFER (MAGNITUDE MODE) ;^ G.WAGNER, JMR 55, 151 <B3). j^ A.BAX fc G.DROBNY, JMR £1,20£ <B5) 5 D1-90-D0-90-D2-1B0-D2-90-FID ; CORRELATION CROSS-PEAKS CAN BE OBTAINED FROM SPINS A AND X ; IN AN AMX SYSTEM WHEN J(AX) IS TOO SMALL. 1 ZE 2 Dl ;RELAXATION DELAY 3 PI PHI j90 DEG PULSE CREATES XY—MAGN. 4 DO . ;EVOLUTION OF SHIFTS 5 PI PH2 |COMPLETE FIRST COHERENCE TRANSFER, E.G. ; SPIN A TO M DEPENDS ON SIN(FT*J(AM:>*DCO £ D2 ;SECOND COHERENCE PERIOD 7 P2 PH2 ;REFOCUS CHEMICAL SHIFTS B D2 9 Fl F'H3 | COMPLETE SECOND TRANSFER (EG. M TO X > ; EFFICIENCY DEPENDS ON ; SIN (PI *2D2*J (AM.) )*SIN (FI*2D2* J < MX ) 10 G0=2 PH4 ;AC0U1RE FID 11 WR *1 ;STORE FID IN .SEP FILE 12 IF 01 13 1N=1 ;LDOP FOR NEXT EXPERIMENT 14 EXIT PH1=A0 AO AO AO AO AO AO AO ;SCANS 1-2 SUPPRESS AXIAL PEAKS Al Al Al Al Al Al Al Al ;SCANS 3-4 FOR Fl QUAD (N-TYPE) A2 A2 A2 A 2 A2 A2 A2 A2 A3 A3 A3 A3 A3 A3 A3 A3 PH2-A0 AO Al Al A2 A2 A3 A3 ;SCANS 5-8 SUPPRESS NOESY PEAKS Al Al A2 A2 A3 A3 AO AO ;FURTHER CYCLING FOR F2 QUAD <0P A2 A2 A3 A3 AO AO XA1 Al A3 A3 AO AO Al Al A2 A2-PH3=A0 A2 Al A3 AO A2 Al A3 Al A3 A 2 AO Al A3 A2 AO A2 AO A3 Al A2 AO A3 Al A3 Al AO A2 A3 Al AO A2 PH4=R0 RO R2 R2 RO RO R2 R2 RI RI R3 R3 RI RI R3 RS R2 R2 RO RO R2 R2 RO RO R3 R3 RI RI R3 R3 RI RI ;D2 « CA. 0.5/<JCAM)+J(MX)> WHEN COUPLINGS DO NOT DIFFER BY I MORE THAN FACTOR 2 ; • CA. 0.5/< l.£«J(MAX> ) OR AT MOST 0.5/< 1.3*J<MAX> > j TO COVER A WIDER RANGE OF J. I NULLING CONDITIONS OF D2=0.5/J SHOULD BE AVOIDED |NS«B«N ;P1=90, P2=1B0 I OTHERWISE PARAMETERS AS FOR COSY. ;SEE ALSO •REC0SY2.AU' - 172 -FILE: C0SYR1HG.AU C0SYR1HG.AU COSY WITH 1-STER RELAYED COHERENCE TRANSFER (MAGNITUDE MODE) WITH 2 LEVELS SOLVENT SIGNAL SUPPRESSION BY PRESATURAT10N G.WAGNER, JMR 55, 151 (B3). A.BAX G.DROBNY, JMR £1,306 <B5) HG(S1)-(S2) DO DI - D3-90-D0-90-D2-180-D2-90-FID CORRELATION CROSS-PEAKS CAN BE OBTAINED FROM SPINS A AND X IN AN AMX SYSTEM WHEN J(AX) IS TOO SMALL. 1 ZE 2 DI HG D3 S2 3 PI PHI 4 DO 5 PI PH; 6 D2 7 P2 PH: & D2 9 PI PH: SI 10 G0=2 11 WF: * 12 IF # 13 IN=1 14 EXIT DC FH4 {RELAXATION DELAY, PRE-SAT WITH POWER SI {SWITCH TO MIN. POWER S2 FOR EVOLUTION {90 DEG PULSE CREATES XY-MAGN. {EVOLUTION OF SHIFTS {COMPLETE FIRST COHERENCE TRANSFER, E.G. { SPIN A TO M DEPENDS ON 51N(PI *J(AM)*D0) {SECOND COHERENCE PERIDD {REFOCUS CHEMICAL SHIFTS COMPLETE SECOND TRANSFER (EG. M TO X) EFFICIENCY DEPEND5 ON SIN(PI«2D2* J(AM))* SIN(PI* 2D2* J(M X) ACQUIRE FID WITH DEC. GATED OFF STORE FID IN .SEP FILE {LOOP FOR NEXT EXPERIMENT F-H1=A0 AO AO AO AO AO AO AO Al Al Al Al Al Al Al Al A2 A2 A 2 A 2 A2 A2 A 2 A2 A3 A3 A3 A3 A3 A3 A3 A3 PH2=A0 AO Al Al A 2 A2 A3 A3 Al Al A 2 A 2 A3 A3 AO AO A2 A2 A3 A3 AO AO Al Al A3 A3 AO AO Al Al A2 A2 PH3=A0 A2 Al A3 AO A2 Al A3 Al A3 A2 AO Al A3 A2 AO A2 AO A3 Al A2 AO A3 Al A3 Al AO A2 A3 Al AO A2 PH4=R0 RO R2 R2 RO RO R2 R2 Rl Rl R3 R3 Rl Rl R3 R3 R2 R2 RO RO R2 R2 RO RO R3 R3 Rl Rl R3 R3 Rl Rl ;SCANS 1-2 SUPPRESS AXIAL PEAKS ;SCANS 3-4 FOR Fl QUAD (N—TYPE) ;SCANS 5-B SUPPRESS NOESY PEAKS {FURTHER CYCLING FOR F2 QUAD (DP) D3 *= 2 MSEC TO SWITCH DEC POWER SI «= 30L, S2 = 45L D2 = CA. 0.5/(J(AM)+J(MX)> WHEN COUPLINGS DO NOT DIFFER BY MORE THAN FACTOR 2 = CA. 0.5/( 1.6*JCMAX) ) OR AT MOST 0.5/( 1.3*J(MAX) ) TO COVER A WIDER RANGE OF J. NULLING CONDITIONS OF D2=0.5/J SHOULD BE AVOIDED NS=B*N PI=90, P2=1B0 OTHERWISE PARAMETERS AS FOR COSY. - 173 -FILE: C0SYRCT2.AU ; C0SYRCT2.AU ; COSY WITH 2-BTEP RELAYED COHERENCE TRANSFER (MAGNITUDE MODE > j yG.WAGNER, JMR 55, 151 CB3>. ; -A.BAX V G.DROBNY, JMR 61,306 (B5> 5 90-D0-90-D2-1B0-D2-90-D3-1B0-D3-90-FID ; CORRELATION CROSS-PEAKS CAN BE OBTAINED FDR SPIN A j FROM SPINS M,Q,X IN AN AMQX SPIN SYSTEM. 1 ZE 2 Dl 3 PI PHI 4 DO 5 PI PH2 6 D2 . 7 P2 PH2 B D2 9 PI PH3 10 D3 11 P2 PH2 12 D3 13 PI PH4 14 60=2 PH5 15 WR #1 16 IF #1 17 IN=1 IB EXIT {RELAXATION DELAY 90 DEG PULSE CREATES XY-MAGN. EVOLUTION OF SHIFTS COMPLETE FIRST COHERENCE TRANSFER, E.G. SPIN A TO M DEPENDS ON SIN(PI«J(AM>*DCU SECOND COHERENCE PERIOD REF0CU5 CHEMICAL SHIFTS ;COMPLETE SECOND TRANSFER (EG. M TO 0) ;THIRD TRANSFER FROM 0 TO X ;ACQUIRE FID ;STORE FID IN .SER FILE ;LOOP FOR NEXT EXPERIMENT PH1=A0 PH2=A0 AO AO AO Al Al Al Al A2 A2 A2 A2 A3 A3 A3 A3 PH3=A0 AO A2 A2 Al Al A3 A3 PH4=A0 A2 AO A2 Al A3 Al A3 PH5=R0 RO RO RO R2 R2 R2.R2 JSC JSC JSC ANS 1-2 SUPPRESS AXIAL PEAKS ANS 3-4 FOR Fl OUAD (N-TYPE) ANS 5-e SUPPRESS NOESY PEAKS ;FOR F2 OUAD PHASE CYCLING (OP, jCYCLOPS) ALL PHASES MUST BE jINCREMENTED IN 30 DEG STEPS. jFOR LINEAR SPIN SYSTEM AMQX, THE TRANSFER FUNCTION IS j SIN(PI*J(AM>*2D2)SIN(PI*J(MQ)*2D2)* ; SIN(PI*J(MQ)»2D3)SIN(PI*J(QX)*2D3) ;SET D2 « CA. 0.5/(1.6*J), WHERE J «= LARGER OF J(AM), J(MD) jSET D3 « . . . . .. J(MQ), J(QX) ;NS=16*N jPl=90, P2=1B0 : OTHERWISE PARAMETERS AS FOR COSY. ;SEE ALSO REC0SY3.AU - 174 -FILE: CDBYR2HG.AU ; C0SYR2HG.AU ; COSY WITH 2-STEP' RELAYED COHERENCE TRANSFER (MAGNITUDE MODE) ; AND 2-LEVEL RR'ESATURAT I ON OF SOLVENT SIGNAL ; MODIFICATION MADE BY S. ORSON CHAN, DEFT OF CHEM, UBC ^ G.WAGNER, JMR 55, 151 (83). -J A.BAX «< G.DROBNY, JMR 61,306 <85) j HGCS1)-(S2) DO { DI - D5-90-D0-90-D2-1B0-D2-90-D3-1B0-D3-90-FID ; CORRELATION CROSS-PEAKS CAN BE OBTAINED FOR SPIN A ; FROM SPINS M,Q,X IN AN AMQX SPIN SYSTEM. 1 ZE 2 DI HG SI D5 S2 3 PI PHI 4 DO 5 PI PH2 6 D2 7 P2 PH2 8 D2 9 PI PH3 10 D3 11 F2 PH2 12 D3 13 PI PH4 14 G0=2 FHl 15 WR *1 16 IF #1 17 IN=1 IB EXIT RELAXATION DELAY, PRESATURATION WITH POWER SI SWITCH TO MIN. POWER S2 FOR EVOLUTION 90 DEG PULSE CREATES XY-MAGN. EVOLUTION OF SHIFTS COMPLETE FIRST COHERENCE TRANSFER, E.G. SPIN A TO M DEPENDS ON SIN(PI *J(AM)•DO) SECOND COHERENCE PERIOD REFOCUS CHEMICAL SHIFTS COMPLETE SECOND TRANSFER (EG. M TO 0) ;THIRD TRANSFER FROM Q TD X DO {ACQUIRE FID WITH DEC. GATED OFF {STORE FID IN .SER FILE ;LOOP FOR NEXT EXPERIMENT PH1=A0 PH2=A0 AO AO AO Al Al Al Al A^L A+1. AA±1 A » J Aj Atii A3 PH3=A0 AO A2 A2 Al Al A3 A3 PH4=A0 A2 AO A2 Al A3 Al A3 PH5=R0 RO RO RO R2 R2 R2 R2 {SCANS 1-2 SUPPRESS AXIAL PEAKS {SCANS 3-4 FOR Fl QUAD (N-TYPE) {SCANS 5-B SUPPRESS NOESY PEAKS {FOR F2 QUAD PHASE CYCLING (QP, {CYCLOPS) ALL PHASES MUST BE {INCREMENTED IN 90 DEG STEPS. FOR LINEAR SPIN SYSTEM AMQX, THE TRANSFER FUNCTION IS SIN(PI#J(AM)#2D2)SIN(PI*J(MQ)«2D2)* SIN(PI*J(MQ)*2D3>SIN(PI*J(QX)*2D3) SET D2 = CA. 0.5/(1.6*J), WHERE J » LARGER OF J(AM), J(MQ) SET D3 = " " " J(MQ), J(QX) D1=1-5*T1 D5=2 MSEC TO SWITCH DEC. POWER NS=16*N Pl=90, P2=1B0 OTHERWISE PARAMETERS AS FOR COSY. SEE ALSO REC0SY3.AU - 175 -FILE: NOESYHG .AU NDEEYHG.AU HOMONUCLEAR DIPDLAR-CORF:ELATED 2-D NMR (MAGNITUDE MODE) WITH FRE-SATURATION OF SOLVENT. DIPOLAR COUPLING MAY EE DUE TO NDE DR CHEMICAL EXCHANGE. Dl - 90 - DO - 90(OR 45) - D9 - 90(OR 45) - FID SYMMETRIC MATRIX WITH SHIFTS AND COUPLINGS IN Fl, F2 OFF-DIAGONAL PEAKS CORRELATE SPINS WHICH SHARE A DIPOLAR COUPLING. SCALAR COUPLING CORRELATIONS ARE STRONGLY REDUCED BY RANDOM VARIATION DF THE MIXING TIME D9. 2E Dl H6 S3 PI PHI DO P2 D9 P3 PH2 6 7 PH3 B GD=2 PH4 DO S WR #1 10 IF 4*1 11 IN=1 12 EXIT RELAXATION WITH PRE-SATURATION 90 DEG EXCITATION PULSE EVOLUTION OF SHIFTS AND COUPLINGS MIXING PULSE, 90 (OR 45) DEG MIXING TIME FOR Z—MAGN. EXCHANGE DETECTION PULSE, 90 (OR 45) DEG ACQUIRE FID WITH DEC. GATED OFF STORE FID INCREMENT FILE NUMBER INCREMENT DO AND LOOP FOR NEXT EXPER, PH1=A0 PH2=A0 A2 Al A3 PH3=A0 AO Al Al A2 A2 A3 A3 Al Al A2 A2 A3 A3 AO AO PH4=R0 R2 R2 RO R2 RO RO R2 RI R3 R3 RI R3 RI RI R3 ;SCANS 1-2 SUPPRESS AXIAL PEAKS ;SCANS 2-4 GIVE Fl QUAD (N-TYPE) ;SCANS 5-B SUPPRESS DBL. QUANTUM ;SCANS 9-16 FOR DP PROGRAM REQUESTS FILENAME WITH .SER EXTENSION NE DEFINES NUMBER OF FIDS = TD1 NS = 4, B OR 16 (COMPLETE PHASE CYCLE) DS = 2 OR 4 F:D=PW=0 Dl = 1-5*T1 S3 = DEC. POWER FOR PRE-SATURATIDN, SHDULD BE AS LOW AS POSSIBLE TO AVOID BLOCH-SIEGERT EFFECTS (30-40L). PI = 90 DEG, P2 AND P3 «= NORMALLY 90 DEG BUT CAN BE 45 DEG TO GIVE REPRESENTATION LIKE COSY-45. DO « 3E-6 INITIAL DELAY IN = 0.5/SW1 = 2*DW NDO = 1 I2D • 1, SWl=SW/2 D9 = MIXING TIME = CA. Tl FOR SMALL MOLECULES (EXTF:EME NARROWING LIMIT) OR CA. 50-200 MSEC FOR LARGE MOLECULES WITH CROSS—RELAXATION (SPIN-DIFFUSION). V9: D9 WILL BE VARIED RANDOMLY BY MAX. V9 V. OF ITS VALUE TO SUPPRESS ZEFlD-QUANTUM J-CROSS PEAKS (COSY); CHOOSE V9 SO THAT D9 IS VARIED BY CA. +/- 20 MSEC TO SUPPRESS J-CROSS PEAKS BETWEEN SPINS WHOSE SHIFTS DIFFER BY >50 HZ. TYPICALLY USE TD = SI, NO ZERO-FILLING IN F2 NE « SI/4, ZERO-FILL IN Fl MATRIX CAN BE SYMMETRIZED ABOUT DIAGONAL - 176 -FILE: XHCORR . AUR XHCORR.AUR HETEF.'ONUC. SHIFT-CORRELATED 2-D NMR (CRD DECOUPLING) USING POLARIZATION TRANSFER FROM 1H TO X VIA J(XH). A.BAX S< G.MORRIS, J. MAGN. RES. 42, 501 (Bl) 1H: DO - 90 - DO - - DO - D3 - 90 BB X: DI -1B0- 90 - D4 - FID F2 DOMAIN: BB DEC. X-NUCLEUS SPECTRUM Fl DOMAIN: X-NUCLEUS DECOUPLED 1H SPECTRUM WITH J(HH) J(XH) MUST BE > 1/T2 ZE DI DO SI P1:D PHI DO P4 PH4 DO D3 B P1:D PH2 9 D4 S2 10 60=2 PH; 11 D4 DO 12 WR «1 13 IF #1 14 IN=1 15 EXIT ;1H RELAXATION, SET DEC. FDR PULSING ;90 DEG 1H PULSE ?EVOLUTION OF 1H SHIFTS AND COUPLINGS - ;180 DEG X PULSE TO DECOUPLE X FROM 1H 5 FURTHER EVOLUTION ;WAIT FOR OPTIMUM POLARIZATION OF X-H ; 1H DOUBLET P3 PH3 ;90 DEG 1H PULSE COMPLETES POLAR. ; TRANSFER, 90 DEG X PULSE CREATES I DETECTABLE X,Y-MAGN. :WAIT FOR ANTI-PHASE X-NUCLEUS MULTIPLETS TO REPHASE CPD ;ACQUIRE BB DEC. X-NUCLEUS FID, 1H SHIFTS AND J(HH). GATE DEC. OFF STORE FID INCREMENT FILE NUMBER INCREMENT DO, LOOP FOR NEXT EXPER. MODULATED PHI =B0 PH2 =B0 B2 Bl B3 PH3 =A0 AO AO AO AO AO AO AO Al Al Al Al Al Al Al Al A2 A2 A2 A2 A2 A2 A2 A2 A3 A3 A3 A3 A3 A3 A3 A3 PH4 =A0 AO AO AO A2 A2 A2 A2 PH5 =R0 R2 Rl R3 RO R2 Rl R3 Rl R3 R2 RO Rl R3 R2 RO R2 RO R3 Rl R2 RO R3 Rl R3 Rl RO R2 R3 Rl RO R2 {NS=4*N 5PROGRAM REQUESTS FID FILENAME WITH .SER EXTENSION fNE DEFINES THE NUMBER OF EXPERIMENTS «=TD1 FOR 1H DI •= 1-5*T1 FOR 1H 51 « OH, MAX. POWER FOR PULSING 52 «= NORMAL POWER FOR CPD DECOUPLING DO = 3E-6 INITIAL DELAY PI «= 90 DEG 1H PULSE P3,P4 = 90,180 X PULSE D3 = 0.5/J(XH) FOR MAX. POLARIZATION TRANSFER D4 = 0.25/J(XH) TO OBSERVE ALL MULTIPLICITIES = 0.5/J(XH) TO OBSERVE XH DOUBLET MULTIP. ONLY RD=PW=0 - 1 7 7 -APPENDIX III N.m.r. Spectra Spectrum 3 O-VL coupled spectrum)n.m.r. (75 MHz at 300°K) spectrum of E. c o l l K46 native polysaccharide (M O - 181 -PPM 1 - 182 -Spectrum 5 2 D 2 step relay COST n.m.r. spectrum (C0SYRCT2) spectrum of native polysaccharide (300°K) —I 1 1 1 1 1 ' f ' 5.0 4.0 3.0 2.0 1.0 PPM Spectrum 6 HETCOR n.m.r. partial spectrum of E. c o l l K46 native polysaccharide 1 1 1 1 1 1 1 1 1 I I t i l l ! 84.0 80.0 76.0 72.0 68.0 64.0 60.0 56.0 PPM 3.2 3.4 3.6 3.8 4.0 4.2 4.4 PPM CO u> Spectrum 7 Heteronuclear correlated (13C-1H) n.m.r. partial spectrum of K46 dephosphorylated product l I 85.0 80.0 75.0 70.0 65.0 PPM 60.0 55.0 50.0 45.0 Spectrum 8 ^-n.m.r. spectrum (400 MHz 368°K) of E. coll K33 natlve-polysaccharlde Spectrum 9 1 3C (^-coupled) n.m.r. (75 MHz at 300°K) of E. coli K33 deacetylated, depyruvylated polysaccharide. Spectrum 10 ^-n.m.r. spectrum (400 MHz, 368°K) of E. c o l l K33 deacetylated polysaccharide 00 i 1—|—I 1 1—I 1 1 — I — I — I — | 1 — I — i 1 [ — i 1 — t — i — | — ( — i 1 — i — | 1 >.—i 1—|—> ! r — i • • • r 1—| • • ! 1 1—i 1 1 r ~ ; 5. 'i ' j . 0 a. 5 a. 0 3.5 • 3. 0 2.5 2.0 1 . [J 1.0 .5 PPB Spectrum 11 1 3C (J-H-decoupled) n.m.r. (75 MHz, 300°K) spectrum of K31 native polysaccharide Co CO Spectrum 12 " C (^-coupled) n.m.r. (75 MHz, 300"K) spectrum of K31 native polysaccharide Spectrum 13 "C decoupled) n.m.r. (75 lithium degraded product (Fl) MHz, 300°K) of K31 polysaccharide - 192 -APPENDIX IV POLYSACCHARIDE ANTIGENS OF ESCHERICHIA COLI -193 -STRUCTURES OF E- c e l l K-ANTICENS Kl -8)-O-H«UB5AC-(2-K2«. -0-t.O-*)-a-D-C«la-(l-2)-Clyc«rol-(l- ),n—(O-P-0-5)-«-fi-C«U-(l-2)-Clye«rol-(l- ) n-K2«b,K62 I 2/3 I "2/3 I OH I OAc OAc K2, (K2a), nenacatylatad K62 (K2ab), *e«tylatad K3 -2) -a-L-Bhas- (1-3) -o-l,-Rh*E- (l-»3) -a-i-Rhas- d-3 I 3 " ? 2 2 S S S — 6-fi-acetyl-4-d«oxy-2-btxulo«ontc acid K4 -4)-/J-D-C1C8A-(1-3)-^-D-C1CENAC-(1-3 t l K5 -4)-0-£-G1CBA-U-4)-O-£-C1CRNAC-(1-K6 -3)-0-E-Ribl-(W)-0-Kdofi-(2-" 2 T i 0-E-RlbX -2)-^-g-Rlbf-(l-2)-^-£-Ribl-(l-7)-o-Kd02-(2-X7 (K56) -J)-^-D-IUnBNAcA-(l-^i)-^-C-ClCE-(l-L K8 -3)-o-J-ClcEHAc-(l-3)-^-J[-Clc8A-(l-3)-^-£-CalfiNAc-(l-.2)-^-£-C«lE-(l-I OAc K9 -3)-f-£-6als-(l-3)-^-JJ-CalBKAc-(i-4)-o-J-CalB-(l-A)-o-»«u85Ae-(2-OAc K12 (K82> -.3)-o-L-Rhas-(l-2)-o-L-Rhaa-(l-5)-^-Kdofi-(2-7/8 I - 194 -K13 -.3)-0-£-RibX-(l-7)-0-Kdofi-(2- K13, 0-ae«tyl en 4 of Rdo (K20.K23) K20. O-acetyl on 5 of Rib K23, nonacatylatcd K14 ~6)-0-£-CalBNAc-(l-5)-0-Rdos-(2-OAc (-60%), OProplonyl (-10*) X15 -*4)-A-£-Clc£NAc-U-5).l-Xdo£-(2-0 RIB, K22 -2)-0-B-Rlbf-(l-2)-fi-Ubitol-(5-O-P-O- K22. aonaeatylatad J OH OAc OAc (-30*) 8 K 1 9 -3)-P-E-RlbI-(l-4)-^-KdoB-(2-K 2 6 -3)-o-^-Rh«B-(l-3)-^ -g-Calfi-(l-3)-^-£-Clc8A-(l-3)-o-^-Rh«B-(l-3)-o-i-Rh«B-4 — t H02C 3 1 X o-i-Rhaa H3C 4 K 2 7 -4) -O - J - C I C E - (1-4)-O-B>-C1CBA- (1-3) -o-J,-Fucs- (1-t 1 e-^-Cals K 2 8 -3)-o-D-Clcs-(1-4)-J-D-CICBA-(1-4)-O-L-FUCB-(1-* 2/3 t •: 1 OAc (-70%) J-fi-Cals K29 •»2)-*-g-IUitt-(l-3)-l-£-Clcs-(l-3)-l-£-Cle£A-(l-3)-o-£-CalB- (1-4 T H02C 4 1 X *-B-ClcB-(l-2)-B-B-ManB H3C 6 " R30 -2)-o-D-Kans-(l-3)-^-fi-CalB-(l-3 T I l-&-ClcfiA-(l-3)-o-fi-GalB - 195 -•2)-o-B-Clca-(l-3)-^-£.C«lB-(l-3)-^-B-6XeEA-(X-.2)-^-i-Rh«S-(l-2)-#-l-Rh«£-(l-~ 4 t 1 a-L-Khafi OAe I 2 -4)-o-L-Rh«8-(1-3)-J-D-Clcs-(1-I 1 i-£-ClcfiA-(1-3)-«-£-C«lfi •3)-o-D-Clcs-(1-*)-*-B-Clc»A-(1-4)-o-L-Pucfi-(1-V T B 3 C C0 2H 1 contains fi-acetyl group* •2) -^•C_-C1CEA- (1-4).0-£-Cal£- (1-3)-fi-g-Gals- (1-1 o-g-CleB-(l-4).^.£-C«lE •3)-0-D-C»l8-(l-3)-o-fi-C«lBA-(l-2)-o-E-JUns-(l-""4 -T i o-B-tUnc -3)-0-g-Clefi-(1-3)-o-£-C«lfi-(1-4 t 1 o-D-Calj 6 4 X o) HjC C0 2H -6)-o-g-ClcE-(l-4)-^-{-ClcBA-(l-2)-o-J-KanE-(l-3)-^-£-ClcE-(l-t 1 a-£-Cal£ -4).^ .J.ClcEA-(l-4)-o-5-ClcBNAe-(l-€)-o-£-ClcBNAe-(l-J-Str (aaide) -3)-o-g-CalB-(l-3)-o-J-CalsA-(l-3)-o-l<-rucs-(l--4)-^-£-ClcBA-(l-3)-o-l<-RhaB-(l-4)-o-5l-CleBNAc.(l-6)-^-£-CalENAc-(l-- 196 -fi-ClcfiA-(1-6)-a-D-Cals-(1-6)-«-fi-Clcs-(1-3)-«-C-ClcsNAc•(1-" t 1-Thr (73%). £-S«r (25%) (aide) 0 I -3) ^ .fi-ClesNAc-(l-O-P-0-I OH OAc OAc <-»0%) i I -3)-o-D.C»l8-(l-0-r-0-t OH 2 OAc, OProp (-10%) -3). »-B-c«U- (1-6)-fi-J-ClcjA- (1-L •3)-^-fi-ClcsA-(l-3)-«-L-Rh*s-(1-6 S H»Thr (90%), J-Ser (10%)] (-85%. aside) OAc (40%) I 2 -^ •)-^ -S-ClcBA-(l-4).«-fi-ClcfiA-(l-3).«-C-IUn£-(l-" 4 6 V H3C C02H •3)-^-E-IUbI-{l-2)-^-B-BJ.bi-(l-«)-*-Kdoi-(2-2 OAc (-65%) BhaT »2/4)-ClcA-(l-2/6)-IUn-(l-3)-IUn-(l-3)-Clci!Ac-(l-K«n-(l-3).|Un-(l-3)-CleNAc-(l-t 2/6 1 1 In* 1 ClcA - 197 -K87 -^)-^-2-CleaA-(l-3)-l,-PucaNAe-(l-3)-ClcsNAc-(l-6)-C«lB-(l-f ' 1 OAc? 0-Clcj ? I OAc? K92 -8)-a-K«ua5Ac-(2->9)-o-»«UE5Ac-<2-K93 -3) - J -D-C«U- ( 1 - 4 ) - J - D - C I C B A - ( 1 -5 6 I I OAc OAc K95 -3).^-|-Rlbl-(l-8)-rdoI-(2-randonly 0-«cctylated 0 II K 1 0 0 -3)-^-5-RlbI-(l-2)-g-Ribitol-(5->0-P-0-OH - 198 -ftZTTJUEHCES TOR K-AKTICEKS XI t.J. HeCuira and S.S. Blnkley, Bloehenlstrv. 3 (1966) 247-251. X2.X62 K. Jann end M.A. Schaldt. FTM5 meroblol. Lett.. 7 (1980) 79-81. B. riaehar. H.A. Schaldt. B. Jam. and X. Jann, aioehealstrv. 21 (1982) 1279-1204. K3 T. Dangler, X. •laailapach. B. Jam. and X. J a n . Carbohydr. Res.. 178 (1988) 191-201. K4 K. Jans and B. Jam, RUT. J. »<peh«n . In press. K5 W.F. Vans. M.A. Schmidt. B. Jann. and K. Jam, far. 3. Hochen . 116 (1981) 359-364. K6 a) P. Messner and F.K. linger. Blochea aloohys. Res Caaaun.. 96 (1980) 1003-1010. b) H.J. Jennings, X.-C Koaall, and B.C. John*on, Carbohydr. Res.. 105 (1982) 45-56. K7 F.-P. Tsui, B.A. Boyklns. and V. Egan. Cerbehydr. Res.. 102 (1982) (K56) 263-271. KS L.A.S. Parolls and H. ParolIs, Abstracts. 14th Tntsrastlonsl Cerbohv-drate Svamosiug. Stockholm. 1988 p. 145. K9 C C S . Button. M. Parolla, and L.A.S. Parolls, Cerbehvdr. Res.. 170 (1987) 193-206. K12.K82 M.A. Schaldt and K. Jam, TTf.S Microbiol Lett. - 14 (1982) 69-74. X13(K20.K23) *.F. Varm and K. Jann. Infect . 25 (1979) 85-92. V.F. Varm, T. Sodarstroa, V. Egan, F.-P. Tsui, B. Schneerson, I. 0rskov, and F. Vrekov, lnf»rt Temun. . 39 (1983) 623-629. X14 t. Jam. P. Hofaarm, and K. Jam. Cerbohydr Res 120 (1983) 131-141. K1S H. Tarn, Unpublished results. X18.X22 H.-L. Bodriguex. B. Jam. and X. Jam. Carbohydr. Res.. 173 (1988) 943-253. K19 B. Jam, X. Ahrens, T. Dangler, and X. Jam. Carbohydr. Res.. 177 (1988) 273-277. K26 L.N. tteynon and C C S . Button. Cerbehvdr »e« i/y u»M) «iy-«*j. L.M. Bavnon, PhD. Thetis. Pniversltv of British Cdugble V . M . u v . r £UAdA. 1988. K27 X. Jam, B. Jam, K.F. Schneider, F. Brskov, and I. 0rakov, Eur. J j>eh««.. S (1968) 456-465. A.K. CheJtrabort?. Mecroaol. Chea.. 183 (1982) 2881-2887. K28 E. Altaan and C C S . Button. Carbohydr Res.. 138 (1985) 293-303. - 199 -K29 Y.-K. Choy, T. M w l , I. Prank, and S. Stlra. J- Vlrel.. 16 (1975) 581-590. K30 D. Hungerer, X. Jann, 1. Jann, P. «Jrsko-», and I. eftskcv. Xur. 3. Ufifihiau. 2 (1967) 115-126. 4.X. Chakraberty, H. PrteboUn. and S. Stirs. J- Bacterid. 141 (1980) 971-972. X31 C C S . Dutton, 4. Xum*-Uintah, and S. Kg. Abstracts. 14th International Carbohydrate Svnmoslua. StoeVhola. 1988 p. 86. X32 C Annlcon. C C S . Dutton. and I. Altaan. Carbohydr. Res. . 168 (1987) 89-102. R33 B.A. Levis, Unpublished raaulta. X34 C C S . Button and A. Xum*-Uintah. C.rhohvdr. Res . 169 (1987) 213-220. K36 H. Parolia. L.A.S. Parolla, and S.H.R. Stanley. Carbohydr. Res. . 175 (1988) 77-83. K37 A.N. Anderson, H. Parolis, and L.A.S. Parolla. Carhnhydr R«« 163 (1987) 81-90. K39 H. Parolla. L.A.S. Parolla, and R.D. Vanttr. Carbohydr. Res.. In press. X40 I. Dangler. B. Jann, and X. Jann. Carbohydr. Res.. 150 (1986) 233-240. K42 H. Nlaaann, AX. Chakraborty, H. mabolln. and S. Stirs, J Baeta-r l o l . . 133 (1978) 390-391. K44 C C S . Dutton, D.R. Xamnaratna, and A.V.S. Ll». Carbohydr. t i i . . 183 (1988) 111-122. K49 L.H. Baynon. Ph.D. Thesis. University of British ColimMa. Vancouver. £40*41, 1988. XS1 B. Jann. T. Dangler, and X. Jam. TP45 meroblol. Lart 29 (19B5) 257-261. K52 P. Bofaann, B. Jam. and X. Jam. Eur. J. Bloeh.i. 147 (1985) 601-609. K53, K93 4. lax. H.T. Suanara. V. Xgan. I. Culrgls. 1. Schneerson, J.B. Bobbins, P. afrakov. J. eft-star, and S.P. Vaan. Crt.ol.vdr ».. 173 (1988) 33-64. K54 P. b a m , B. Jam. and X. Jam, Carbohydr •«. . 1J» (1985) 261-271. K53 4.1. 4adaraon and H. Parolla. Abstracts, lath Tr,t.matlonal Carboy. drate Svsmoslun. Stockholm. 1988 ». SI. K74 R. Ahrena, B. Jam. X. Jam, and H. Brada. Carbohydr. B.« 179 (1988) 223-231. K85 X. Jam, B. Jam. F. Vrskov. and I. fJrskov. Blaehea T. . 346 (1966) 368-385. X87 L. Tareaay. B. Jam. and X. Jam, Eur. J. Bloehen. 23 (1971) 505-514. - 200 -M.R. Lifely. J.C. Lindon, J.H. Hlllleas, and C. Moreno. Cerbohvdr ESJU. 143 (1985) 191-205. V. Egan. T.-Y. Liu, D. Dorov, J.S. Cohan, J.D. Bobbins, E.C. Cot-achlleh, and J.B. Bobbins. Biochemistry. 16 (1977) 3687-3692. T. Bcngler. 8. Jann, and K. Jam, Cerbohvdr. Res.. 1*2 (1985) 269-276. F.-P. Tsui, V. Egan, M.F. Suaaera. B.A. Byrd. B. Schnearaon, and J.B. Bobbin*. Cerbohvdr. Res 173 (1988) 65-74. . s STRUCTURES OF O-AKTICENS OF F. eoll ^)-^-fi-Clcs^e-(l-3)-a-l-Rhaa-(l-2)-a-l-RhaE-(l-3)-^-L-Rhajj-(l-~ 2 t 1 e-g-Fucj3HAc -3)-«-fi-ClcsNAe-(l-2)-a-L-Rhaa-(l-«)-o-B-CleB-(l-3)-o-L-FueEKAe-(l-3 " ~ t 1 a-S-Clcs -•3) -«-B-Kans- (1-*) -«-D-Kann.- (1-3) -e-D-ClesKAe-(1-4) -e-B-CaloHAe- (1-* 2 t 1 0-g-ClcH -3)-o-B-ClcBNAc-(l-3)-^-D-0jilB4NAe-(l-2)-o-D-ManE-(l-4)-^-fi-Cal8-(l-s. 3 t 1 o-^ -Rhas B-QuloNAc - 4-acetaaldo-4,6-dideoxy-B-glucopvTanose o-g-Kan£3Me- (l-J-3) -0-g-Kann.- (1-2) -a-g-Hann.- (1-2)-a-g-Mans- (1-rh fi-Man3Ma - 3-fl-nethyl-g-aannose n - -10 -3)-a-g-h^na-(l-3)-a-fi>-Mar«-(l-2)-a-g-h^nB-(l-2)-a-g-M*na-(l-2)-B-g-ManB--3)-a-g-Kana-(l-3)-a-g-KanB-(l-2)-a-g-Manfi-(l-2)-a-g-Mans-(l-- 201 --3) - 0-fi-Clea.NAe- (1-3) -*-L-Ih*.B- <l-3) -o-L-Ehag-(1-3) •o-D-Calfi- (1-2 t 1 •-g-Fue^HAcyl Acyl - Acotyl (60%) er £-3-hydrojrybutyxyl (40%) -2)-o-Eh«a-(l-*)-o-C*lE-(l-6)-«-ClcB-(l-3).ei-CleBNAc-(l-3 t 1 0-ClcoNAc -2) •'•B-B.lbX- (1-4) -o-B-Cals- (1--4)-o-fi-C»lB-(l-2)-«-C-»Mbi-(l-~ 3 1 1 O - £ - C»1B o-L-RKtB " 1 4 3 -3) -O - ^ - F U C B N A C- (1-3)-4-g-ClcsNAc - (1-4) -o-J.-Clc8- (1-t 1 ^-B-Cles -3)-ofi-C»lj-(l-3)-^-fi-C«lBNAc-(l-6)-^-C-ClcBRAc-(l-1 o-Colfi-(1-2)-^-fi-C*la Col — 3,6-dld«oxy-l-sjlfi-h«xoi« -3) .^-fi-ClesNAc- (l-4)-a-fi-IUnfi- (1-4) .o-fi-JUnfi- (1-" 3 "2/3 t I 1 OAC ••s S - 3-fl-((RJ-l-c*rboxy«thyl]-L^rti*amopyr«no«« -3)-^-g-ClcsNAc-(l-2)-o-a-Rh«B-(l-2)-o-4-Rh*B-(l-.2)-o-£-C«lB-(l--3)-0-fi-ClefiNAe.(1-3)-o-B-C«ls-(1-4)-o-i-Rhafi-(1-~ ~ 4 t 1 l-g-Mans - 202 --3)-0-J-Clcs!Uc-(l-4)-J-J.-Clc8KAc-U-^ l)-«.l-PueB- (1-2) - ^ - J - C»1B- (1-3)-«-J-C*1BNAC-(1-3) . ^ - J -C*laNAe-(l-t 1 ••£>CmlB ••Cel£ 1 i •3) - J-£-GlcBNAe- (1-4) -a-£-Clca- (1-4) -«-g-C«lB- (1-t ••Col£ Col — 3.<-dldoo«V'L-xvlo-h«»o«« -3)-o-E-ClcfiNAe-(1-4)-«-fi-QulB3N-(1-3)-J-fi-Ribi-(1-4)-*-fi-C»lB-(1-j •-•c«tyl-l-S«r (aaldo) C>I1B3N — 3-«»lno-3,<-dld«OTy-^-fi-|,lueo7yT«no»» -3) - J-fc-ClsNAc- (1-3) -o-£-C«ls- (l-«) - J-J-CalX- (1-T I *-S-(l-6)-o-£-Clca S - 4-fi-[(E)-l-e»rboxy»thyl]-J-jlucopyT«noie -4)-^-D-CICBNAC-(1-3)-0-D-CleaHAc-(1-3)- J-1,-Rh«8-(1-Y « HjC C02H -3)-O-J.-C*1BNAC-(l-2)-a-£-P«rBNAc-(l-3)-«-fc.Fuca- (l-4)-0-£-Clcs- (1-P»r — 4-aalno-4,6-dld«osy>o-g-BaBnopyr«no*e - 203 -u m o t c E s FOR O-AFTICDIS 02 P.-E. J I U I O S , H. Lemhola. >. Lindberg, 0. Llndqulst, end I.B. tvanaen, c«rhBh»dr 1 . . 161 (19(7) 273-279. 04 P.-E. Janeson, B. Lindberg, M. Ogunl««l, t.B. Branson, and C. Vrangiell, C.rhohydr Res . 1)4 (19S4) 283-291. 06 P.-I. Jena>on, B. Lindberg, J. Lonngren, C. Ortage, and S.B. flranaon. Cer-BfltlYdr. nBi. 131 (1984) 277-283. 07 V.L. L ' T O V. A.S. Shaahkov. B.A. Daltriev, B.K. Kochetkov. B. Jam. and X. Jam, Carbohydr. P..... 126 (1984 ) 249-259. 08 P.-E. Janaaon, J. Lonngren, C. Vldaala, X. Leonteln, X. Sltttangren, S.B. Svanaon, C. Vrangaall, A. Ball, and P.K. Ti l l a r , Cerbohvdr «»« 145 (1985) 59-66. X. Baaka and X. Jam, Eur. J. Bloehea. 91 (1972) 320-328. 09 P. Praha, B. Jam, and X. Jam, Eur. J Bloehea . 67 (1976) 53-56. 09a L.A.S. Parolls, H. Parolls, and C C S . Button, Csrhohvdr Res . 155 (1986) 272-276. 010 L. Kama, B. Lindberg, C. Lugowakl, and S.B. Ivans on, Cerbohvdr. Re« 151 (1986) 349-358. OlBac D.S. Cupta, B. Jam. and X. Jam, T»f..-r T—,m AS (1984) 203-209. 020 V.N. Vasll'ev and X.T. lakhartrva, Bloors Khla. 2 (1976) 199-206. 020ae V.R. Vasll'ev. X.T. Zakharova. and A.S. Shaahkov, Bloory. Khla. . 8 (1982) 120-125. 025 L. Kama, B. Lindberg, J.K. Madden, B.A. Lindberg. and P. Caaskl, Jr., Csr-hohvdr 122 (1983) 249-256. 055 B. Lindberg, F. Llndh, J. Lftmgren, A.A. Lindberg, and S .B. Breneoa, Csrbo-» " 97 (1981) 103-112. 058 B.A. Baitrier, T.A. Xnirol. l.K. Kaehetkcre, B. Jam, and X. Jam, Eur- J • BlBfihjpi. 79 (1977) 111-115. - 2 0 4 -069 C. Krblng. L. C O B * , B. TfarffciTg. 6. steuaann, and B . •iamlea. Q u H a h j A l ^ . Bee.. 96 (19T7) 371-376. 07S C. Krblng, L. Km*, ft. Lindberg. and S. Haaaaxetroa. Cerbohvdr t.« 60 (197S) 400-403. 071 Jenaaon, B . Undbarg, C. Vldaala. and K. La on tain. earbahvdr t « « . . 163 (1987) 17-92. 066 It. Andaraaon, a. Caxlta, K. Laocteia, V. Undqulst and K. tlattangran. Abetreet* 14th tntemetltmal Carbohvdrete Svemoelua. StocVhola. 1988 p. 127. 0111 K. Eklind, P.J. Caragg, L. Kann*. A. A. Undbarg, and B . Undbarg, Abitraete 9th International Carbohydrate Svapoilun. London. 1978 p. 493. 0114 L. Kann* and B. Undbarg. In The Polyaaccharidaf. Vol. 2. Ed. CO. aaplnall, (1983) 287-363. 0124 B.A. Daltrlev. V.L. L'vov, B.K. Kochetkov, B . Jann, and X. Jann, Eur. J. Bloehea. 64 (1976) 491-498. 0169 A. Adeysye, t.'t. Janaeon, B . Lindberg. B . Aba**, and S.B. Svanson, Cerbo-TUVflr. Kci,. 176 (1988) 231-236. 0157 K.B. Parry. L. Mac La an, and B.V. Crlfflth, Can J Bloehea Cell aiol.. 64 (1986) 21-28. PUBLICATIONS 1. G.G.S. Dutton and A. Kuma-Mintah, "Structural Studies of E. c o l i serotype K3V, Carbohydrate Research, 169 (1987), 213-220. 2. G.G.S. Dutton, A. Kuma-Mintah and H. P a r o l i s , "The structure of E. c o l i K31 antigen", Carbohydrate Research (in press). 3. G.G.S. Dutton, A. Kuma-Mintah, B.A. Lewis and S.N. Ng, "The structure of E. c o l i K33 antigen", manuscript to be submitted for p u b l i c a t i o n . A. G.G.S. Dutton, A. Kuma-Mintah, S.N. Ng and H. P a r o l i s , "The location of phosphate diester and sequencing of the sugar residues i n E. c o l i KA6 antigen by 2D NMR and FAB-M.S.", manuscript to be submitted for publica t i o n . PAPERS S t r u c t u r a l S t u d i e s on E . C o l i s e r o t y p e s K34 and K46. ( X H I t h I n t e r n a t i o n a l C a r b o h y d r a t e Symposium, August 1987). S t r u c t u r a l S t u d i e s on t h e C a p s u l a r P o l y s a c c h a r i d e of E . C o l i s e r o t y p e K31. ( X l V t h I n t e r n a t i o n a l C a r b o h y d r a t e Symposium, August 1988, Stockholm, Sweden). Mass s p e c t r o m e t r y and n u c l e a r magnetic resonance s p e c t r o s c o p y on E . c o l i K46. B a c t e r i o p h a g e g e n e r a t e d o l i g o s a c c h a r i d e . ( C o n j o i n t M e e t i n g on I n f e c t i o u s D i s e a s e s , November 1988, C a l -gary, Canada). 

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