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A structural investigation of the capsular antigens of two E. coli strains K26 and K49 Beynon, Linda M. 1988

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A STRUCTURAL INVESTIGATION OF THE CAPSULAR ANTIGENS OF TWO E. c o l l STRAINS K26 AND KZf9 by LINDA M. BEYNON B.A. (Hons), Open University, United Kingdom, 1981 M.Sc, University of B r i t i s h Columbia, Canada, 1985 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (DEPARTMENT OF CHEMISTRY) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA JULY 1988 ©LINDA M. BEYNON In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of CM £ ro i ^ T R y The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/81) i i ABSTRACT Diseases caused by encapsulated bacteria such as E. c o l i are among the most prevalent i n the world. The polysaccharide capsule (K antigen) i s an important factor i n the virulence and pathogenicity of E. c o l i bacteria. Serological c l a s s i f i c a t i o n of these bacteria i s also based mainly on the immunologically dominant capsular polysaccha-r i d e , due to i t s location at the b a c t e r i a l c e l l surface. In order to understand the role played by the K antigens i n b a c t e r i a l infections, and the chemical basis of sero-l o g i c a l d i f f e r e n t i a t i o n , the systematic structural i n v e s t i -gation of a l l the capsular polysaccharides of E. c o l i (74 serotypes) i s underway i n this laboratory and others. Presented i n this thesis are the structures of the K a n t i -gens of E. c o l i K26 and K49 serotypes. K26 capsular polysaccharide was studied using techni-ques such as methylation analysis, 8-elimination, ;Smith degradation and p a r t i a l hydrolysis. The oligosaccharides produced by the p a r t i a l acid hydrolysis were analysed by g.c.-c.i.-m.s. To aid inthe characterization of these oligo-saccharides, a ' l i b r a r y ' of r e l a t i v e retention times and c . i . mass spectra of authentic standards ( d i - , t r i - , and tetra-saccharides) was prepared. The results from these analyses, together with data from n.m.r. spectroscopy of the native polysaccharide and derived oligosaccharides, allowed the following structure to be assigned to E. c o l i i i i K26 p o l y s a c c h a r i d e . _3_0<_L-Rhaj- (1 -3) -^-D-Galp- (1 -3) -^-D-Gl cA p- (1 -3) -o(-L-Rha£- (1 -3) -oC-L-Rhap- (1 • 1 o<-L - Rhap CH 3 \ J O O H E. c o l i K 4 9 c a p s u l a r a n t i g e n c o n t a i n s two amino a c i d s , s e r i n e and t h r e o n i n e , a m i d i c a l l y l i n k e d t o the c a r b o x y l group of g l u c u r o n i c a c i d . Techniques used i n the s t r u c t u r a l e l u c i d a t i o n were raethylation a n a l y s i s , a c e t o l y s i s , amino a c i d a n a l y s i s , HF h y d r o l y s i s , p a r t i a l a c i d h y d r o l y s i s and Smith d e g r a d a t i o n . The o l i g o s a c c h a r i d e s generated by the three l a t t e r methods v/ere a n a l y s e d by g.c.-c.i.-m.s. and n.m.r. sp e c t r o s c o p y . A b a c t e r i o p h a g e - a s s o c i a t e d enzyme degradation of the K / + 9 a n t i g e n y i e l d e d a product (P1) which c o n s i s t e d of a s i n g l e r e p e a t i n g u n i t (see below). R e s u l t s from the a n a l y s e s of P1 and the c h e m i c a l l y produced o l i g o s a c c h a r i d e s were i n agreement w i t h the f o l l o w i n g assignment f o r the s t r u c t u r e of the E. c o l i Kif9 c a p s u l a r p o l y s a c c h a r i d e . Thr(Ser) -Zf-^-D-GlcAjj-C 1 - 6 )-^-D-Galj2-( 1 - 6 )-£-D-Glctp-( 1 - 3 ) -^-D-GalNAcj?-( 1-i v TABLE OF CONTENTS Page ABSTRACT • . i i TABLE OF CONTENTS i v LIST OF APPENDICES i x LIST OF TABLES x LIST OF FIGURES x i i i LIST OF SCHEMES . • • • x v i i LIST OF ABBREVIATIONS x v i i i IMMUNOLOGY GLOSSARY xix ACKNOWLEDGEMENTS . . . . . x x i i i I. INTRODUCTION * 1.1 B a c t e r i a l P o l y s a c c h a r i d e s - types and l o c a t i o n s 3 1.2 Serology, Chemistry and B i o s y n t h e s i s o f E. c o l i Capsular P o l y s a c c h a r i d e s . k 1.3 Immunology of B a c t e r i a l P o l y s a c c h a r i d e s . 8 1.3.1 S t r u c t u r e and A n t i g e n i c i t y of B a c t e r i a l P o l y s a c c h a r i d e s 9 V 1 .3.2. C r o s s - r e a c t i o n s of B a c t e r i a l Poly-s a c c h a r i d e s 10 1.4 The Role of Capsular P o l y s a c c h a r i d e s i n the V i r u l e n c e and P a t h o g e n i c i t y of E. c o l i B a c t e r i a 11 1.5 B a c t e r i a l Capsular P o l y s a c c h a r i d e Vaccines 13 I I PROCEDURES FOR THE STRUCTURAL ANALYSIS OF BACTERIAL CAPSULAR POLYSACCHARIDES 1 6 I I . 1 I s o l a t i o n and P u r i f i c a t i o n 1? II.2 Chemical A n a l y s i s 19 11.2.1 Monosaccharide Composition . . . . . 20 11.2.2 Ring S i z e and Linkage P o s i t i o n . . . . 22 11.2.3 Anomeric C o n f i g u r a t i o n 25 11.2.4 C o n f i g u r a t i o n of Sugars (D or L) . . . . 27 11.2.5 L o c a t i o n of Non-carbohydrate S u b s t i t u e n t s . . . . . . 27 11.2.6 Determination of the Monosaccharide Sequence by S p e c i f i c P o l y s a c c h a r i d e Degradations . 29 11.2.6.1 P a r t i a l A c i d H y d r o l y s i s 30 11.2.6.2 fl-Elimination 31 v i 11.2.6.3 Periodate Oxidation and Smith Hydrolysis 32 11.2.6.4 Lithium/Ethylenediamine Degradation . . 33 11.2.6.5 Anhydrous Hydrogen Fluoride Hydrolysis . 36 11.2.6.6 Reductive Cleavage . 37 11.3 Enzymatic Analysis of Capsular Antigens . . . . . 39 11.3.1 Exo Enzymes 39 11.3.2 Endo Enzymes 41 11.3.2.1 Nature and Mode of Penetration of E. c o l i Capsule Bacteriophage . . . . 41 11.3.2.2 Isolation and Selection of Bacteriophage 42 11.3.2.3 Enzymology of Endoglycanases 46 11.3.2.4 Applications 47 11.3.2.5 Conclusions 48 11.4 Instrumental Analysis 49 11.4.1 Separation Techniques 49 II.4.1.1 Gas Liquid Chromatography . . . . . . . 50 11.4.2 Mass Spectrometry 54 v i i I I . 4.3 Nuclear Magnetic Resonance (n.m.r.) Spectroscopy 67 BIBLIOGRAPHY 74 I I I STRUCTURAL STUDIES ON E. c o l i K26 CAPSULAR POLYSACCHARIDE USING G.C-C.I-M.S. CAPILLARY GAS CHROMATOGRAPHY REFERENCE DATA 82 I I I . 1 A b s t r a c t 83 111.2 I n t r o d u c t i o n 83 111.3 R e s u l t s and D i s c u s s i o n 83 111.4 Conclusion 101 111.5 Experimental 104 IV STRUCTURAL INVESTIGATION OF THE CAPSULAR POLY-SACCHARIDE FROM E. c o l i 09 : K26 : H" 109 TV.1 A b s t r a c t 110 IV.2 I n t r o d u c t i o n 110 IV.3 R e s u l t s and D i s c u s s i o n 111 TV.4 Conclusion 137 IV.5 Experimental 137 V BACTERIOPHAGE-ASSOCIATED ENZYMATIC DEGRADATION OF E. c o l i K49 CAPSULAR POLYSACCHARIDE 145 v i i i V.1 A b s t r a c t 146 V.2 I n t r o d u c t i o n 146 V.3 R e s u l t s and Di s c u s s i o n 147 V .4 Conclusion 154 V. 5 Experimental 155 VI STRUCTURAL INVESTIGATION OF AN AMINO ACID CONTAINING CAPSULAR POLYSACCHARIDE FROM E. c o l i SEROTYPE 08 : K49 = H21 159 V I . 1 A b s t r a c t 160 VI.2 I n t r o d u c t i o n 160 VI.3 R e s u l t s and Discussion 161 VI.4 Conclusion 190 VI. 5 Experimental 190 BIBLIOGRAPHY 197 i x LIST OF APPENDICES Appendix Page I 1H- and 1^C-N.m.r. s p e c t r a 200 X LIST OF TABLES Table Page I. 1 Polysaccharides of Bacteria, Frequently found i n Human Flora, that Cross-react with the Polysaccharide Capsules of Human Pathogenic Bacteria 15 II . 1 Description of Cap i l l a r y Columns used to Analyse Oligosaccharides 53 II I . 1 Relative Retention Times of Methylated Disaccharides on DB 17, DB 225, and DB 5 Capill a r y Columns 87 111.2 Relative Retention Times of Methylated Trisaccharides on DB 17 Capillary Column 89 111.3 Relative Retention Times of Methylated T r i -and Tetra-saccharides on DB 1 and DB 5 Cap i l l a r y Columns 92 111 .4 Relative Retention Times and Ions obtained on G.c-c.i.-m.s. of Methylated Disaccha-rides obtained from E. c o l j K26 Capsular Polysaccharide . . . . 97 IV.1 N.m.r. Data for E. c o l i K26 Capsular Poly-saccharide and Derived Oligosaccharides . . . 1 13 IV.2 Methylation Data for E. c o l i K26 Polysaccha-ride and Derived Products 1 1 9 x i IV.3 R e l a t i v e Retention Times and Ions obtained on G.c.-c.i.-m.s. of Methylated O l i g o s a c c h a r i d e s obtained from E. c o l i K26 P o l y s a c c h a r i d e . . . . 124 IV.Zf Reducing End Determination of F r a c t i o n s C and E from E. c o l i K26 Capsular P o l y s a c c h a r i d e . . . . 131 IV. 5 A n a l y s i s of the Product of a L i t h i u m Ethylene-diamine Degradation of E. c o l i K26 Capsular Pol y s a c c h a r i d e u s i n g G.c.-c.i.-m.s. . . . . . . . 135 V. 1 1H-n.m.r. Data f o r the Bacteriophage Degradation Product ( 1) and E. c o l i K49 Capsular Poly-saccharide 149 V .2 Sugar A n a l y s i s of K49 P o l y s a c c h a r i d e and the Bacteriophage Degradation Product (1) . . . . 152 V . 3 M e t h y l a t i o n Data f o r the Bacteriophage Degradation Products 1 & 2 152 V. Zf Reducing End Determination of the Bacteriophage Degradation Products 1 & 2 153 V I . 1 M e t h y l a t i o n Data f o r K49 P o l y s a c c h a r i d e and Derived O l i g o s a c c h a r i d e s 1 64 VI.2 C . i . Mass S p e c t r a l Data f o r the Reductive Cleavage of K49 P o l y s a c c h a r i d e 167 VI.3 Methanolysis Data f o r E. c o l i K49 Capsular P o l y s a c c h a r i d e • 1 6 7 x i i VI.4 R e l a t i v e Retention Times and Ions obtained on G.c.-c.i.-m.s. of Methylated D i s a c c h a r i d e s obtained from E t c o l i K49 Capsular Poly-sac c h a r i d e • 170 VI.5 L . d . i . - f . t . - i . c . r . P o s i t i v e Ion Spectrum of O l i g o s a c c h a r i d e 3a 176 VI.6 Reducing End Determination of 3b 177 VI.7 N.m.r. Data f o r E. c o l i K49 P o l y s a c c h a r i d e and Derived O l i g o s a c c h a r i d e s 180 x i i i LIST OF FIGURES F i g u r e Page 1.1 E l e c t r o n micrograph of t h i n s e c t i o n of an encapsulated E. c o l i organism 5 1.2 Schematic diagram of the E. c o l i c e l l envelope • • • 5 11.1 Reduction of c a r b o x y l i c a c i d i n aqueous s o l u t i o n 26 11.2 P e r i o d a t e o x i d a t i o n and Smith degradation of an h y p o t h e t i c a l p o l y s a c c h a r i d e 34 II.3- B a s i c morphological types of bacteriophage with types of n u c l e i c a c i d 43 11.4 Diagram of the Coliphage T2 v i r i o n 43 11.5 V i r a l p e n e t r a t i o n of the b a c t e r i a l c a psular p o l y s a c c h a r i d e kk 11.6 Fragmentation pathways of some a l d i t o l a c e t a t e s and p a r t i a l l y methylated a l d i t o l a c e t a t e s . • 57 11.7 Fragment ions formed by homolytic cleavage of g l y c o s i d i c bonds of a non-reducing d i s a c c h a r i d e 62 11.8 Fragmentation pathways of p o l y s a c c h a r i d e s u s i n g f.a.b-m.s 6 5 x i v III . 1 Fragment i o n s of methylated m a l t o t r i o s e 89 III.2 a Fragment ions of methylated melezitose 91 III.2b G . c . - c . i . mass spectrum of methylated melezitose . 91 III.3 a Gas Chromatogram of a methylated t e t r a s a c c h a r i d e u s i n g a DE ' 1 c a p i l l a r y column 93 III.3b Gas Chromatogram of a methylated t e t r a s a c c h a r i d e u s i n g a DB 5 c a p i l l a r y column 93 III.k Reference chart f o r the p r e l i m i n a r y i d e n t i f i c a t i o n of o l i g o s a c c h a r i d e s from t h e i r r e l a t i v e r e t e n t i o n times 93 III.5 a Fragment i o n s of methylated 3-.Q-o<-L-rhamnopyranosyl -L-rhamnose 99 III.5b G . c . - c . i . mass spectrum of methylated 3-Q.-v<-L-rhamnopyranosyl-L-rhamnose . . . . . . . . . . . 99 III.6 a Fragment i o n s of methylated 3-P.-K-L-rhamno-pyranosyl-D-galactose 100 III.6b G . c . - c . i . mass spectrum of methylated 3-.Q-K.-L-rhamnopyranosyl-D-galactose . . . . . . . . . . . 100 III.7 a Fragment i o n s of reduced and methylated 3-0.-K-L-rhamnopyranosyl-D-galactose 102 III.7b G . c . - c . i . mass spectrum of reduced and methylated 3-0-oC-L-rhamnopyranosyl-D-galactose 102 III.8 a Fragment i o n s of methylated 3-0_-p-D-glucopyran-uronosyl-L-rhamnose • ^ 03 XV I I I . 8b G . c . - c . i . mass spectrum of 3-.Q-j3-D-glucopyran-uronosyl-L-rhamnose 103 IV. 1 M e t h y l a t i o n a n a l y s i s of E. c o l j K26 p o l y -s a c c h a r i d e . . 120 IV.2a Oas Chromatogram of methylated F r a c t i o n C usi n g DB 1 c a p i l l a r y column 123 IV.2b G . c . - c . i . mass spectrum of methylated C1 . . . 123 IV.3a Gas Chromatogram of methylated F r a c t i o n D •u s i n g DB 5 c a p i l l a r y column 12? IV.3b G . c - c . i . mass spectrum of methylated component D1 127 IV.3c G . c . - c . i . mass spectrum of component D2 . . . 128 IV.Zfa Gas Chromatogram of methylated F r a c t i o n E u s i n g DB 5 c a p i l l a r y : c o l u m n 129 IV.^b G . c . - c . i . mass spectrum of methylated component E1 129 IV.5 F.a.b. mass spectrum of p e r a c e t y l a t e d G . . . . 133 VI.1 Fragment i o n s of methylated l a and methylated, reduced l a 170 VI.2 S t r u c t u r e of fragment i o n i n d i c a t i n g presence of a ( 1 - 6 ) - l i n k e d a l d o b i o u r o n i c a c i d 171 VI.3 Fragment i o n s of methylated 2 172 x v i VI .4 C i . mass s p e c t r a of the three components o f the a l d o b i o u r o n i c a c i d p resent i n o l i g o s a c c h a r i d e 3a . • 174 V I . 5 S t r u c t u r e s of 3 a and 3b ob ta ined by HF h y d r o l y s i s of K49 p o l y s a c c h a r i d e . . . . . 177 V I . 6 Fragment i o n s of the two components of the" methy la ted Smith Product (4) 179 x v i i LIST OF SCHEMES Scheme Page 11.1 Reductive cleavage of pyranosides and furanosides 26 11.2 Ammonia c i . - i n d u c e d g l y c o e i d i c cleavage of b u t y l o t - D-glucopyranoside 62 x v i i i LIST OF ABBREVIATIONS Ac - a c e t y l CTAB = cetyltrimethylammonium bromide c.i.-m.s. = chemical ionization-mass spectrometry DMSO = d i m e t h y l s u l f o x i d e e. i.-m.s. = e l e c t r o n impact-mass spectrometry f . a.b. = f a s t atom bombardment Gal = g a l a c t o s e Galp = galactppyranose GalNAc = 2-acetamido-2-deoxygalactose(galactosamine) Glc = glucose G l c l = glucofiiranose g. c.-m.s. = gas chromatography-mass spectrometry GlcA = g l u c u r o n i c a c i d Hex = hexose h. p . l . c . = high pressure l i q u i d chromatography i . r . = i n f r a - r e d l . d . i . - f # t , - i . c . r . = l a s e r d e s o r p t i o n i o n i z a t i o n f o u r i e r transform i o n c y c l o t r o n resonance Me = methyl mol. wt. = molecular weight n.m.r. J=: .nuclear magnetic resonance Pyr = py r u v i c a c i d a c e t a l s = seconds; min. = minutes; h = . hours Ser = Se r i n e Thr = Threonine TFA = t r i f l u o r o a c e t i c a c i d f6 fc bacteriophage = phage p . f . u . =- plaque forming u n i t s x i x IMMUNOLOGY GLOSSARY ALTERNATIVE PATHWAY (of complement a c t i v a t i o n ) A pathway f o r the a c t i v a t i o n of complement, which does not depend upon the b i n d i n g of antibody by antigen (as the c l a s s i c a l pathway does) but upon the presence of p a r t i c u l a r constant c h a r a c t e r i s t i c s of other macromolecules found, f o r example, i n the membranes of c e r t a i n b a c t e r i a . ANAMNESTIC RESPONSE The immunological response to antigen subsequent to the response evoked by a primary encounter with that a n t i g e n . ANTIBODY Serum p r o t e i n s of the gamma-globulin type s y n t h e s i z e d as a r e s u l t of the exposure of an i n d i v i d u a l to a n t i g e n . They combine s p e c i f i c a l l y with that a n t i g e n and are c l a s s i f i e d as IgG, IgM, IgA, IgE, or IgD on the b a s i s of the nature of t h e i r heavy chains (see immunoglobulin). ANTIBODY SPECIFICITY The s p e c i f i c i t y of antibody f o r a p a r t i c u l a r a n t i g e n i c determinant. ANTIGEN A m a t e r i a l which induces the production of s p e c i f i c a n t i b o d i e s or r e a c t i o n s of c e l l - m e d i a t e d immunity a g a i n s t i t s e l f . Whether a m a t e r i a l a c t s as an antigen i n a p a r t i c u l a r i n d i v i d u a l i s a f e a t u r e of the i n d i v i d u a l as w e l l as of the m a t e r i a l . ANTIGENIC DETERMINANT Part of an antigen molecule, bound by s p e c i f i c antibody, a g a i n s t which s p e c i f i c immune r e a c t i o n s are d i r e c t e d . ANTISERUM A serum c o n t a i n i n g a n t i b o d i e s a g a i n s t a p a r t i c u l a r a n t i g e n . B-CELL A p u t a t i v e antibody-forming c e l l . The name was o r i g i n a l l y an a b b r e v i a t i o n f o r 'bursa-derived c e l l ' , however XX the d e s i g n a t i o n i s now used f o r c e l l s of s i m i l a r p o t e n t i a l i n s p e c i e s without a w e l l - d e f i n e d bursa. CLASSICAL PATHWAY (of complement a c t i v a t i o n ) A sequence of complement componentstriggered by a n t i b o d i e s which have bound s p e c i f i c a n t i g e n . CLONAL SELECTION Antigen s e l e c t s c e l l s of s u i t a b l e (comple-mentary) s p e c i f i c i t y v i a a n t i b o d y - l i k e r e c e p t o r s on the c e l l s u r f a c e and thereby s t i m u l a t e s the c e l l s to p r o l i f e r a t e and form clones of c e l l s a c t i v e a g a i n s t the a n t i g e n . COMPLEMENT A group of serum p r o t e i n s which work together i n a h i g h l y organized f a s h i o n to give r i s e to a number of d i s t i n c t consequences, predominant among which are: l y s i s of f o r e i g n c e l l s and enhancement of phagocytosis. HEAVY CHAINS The l a r g e r of the two s o r t s of polypeptide chains which make up a l l antibody molecules. I t i s the heavy chain t h a t determines the c l a s s of an antibody. The heavy chain types are found as f o l l o w s : ^ r-chain i n IgG, /tc-chain i n IgM, c<-chain i n IgA, g-chain i n IgE and b-chaih i n IgD. IMMUNOGEN A m a t e r i a l which can induce an immune response i n an i n d i v i d u a l . IMMUNOGLOBULINS Serum p r o t e i n s composed of l i g h t and heavy c h a i n s . A n t i b o d i e s are immunoglobulins and are c l a s s i f i e d as f o l l o w s : IgG T h i s present i n serum i n by f a r the highest concentra-t i o n of a l l antibody c l a s s e s . I t has y-heavy chains and four s u b c l a s s e s , and i s the only c l a s s that passes through the human p l a c e n t a . IgM The antibody c l a s s with the highest molecular weight ( c o n t a i n i n g ten l i g h t and ten heavy c h a i n s ) . When bound to ant i g e n i t i s p a r t i c u l a r l y e f f i c i e n t i n complement a c t i v a t i o n v i a the c l a s s i c a l pathway. IgM contains >-heavy chains and xx i an a d d i t i o n a l peptide to s t a b i l i z e i t s l a r g e polymeric form. ( J c h a i n ) . IgA The c l a s s of antibody found i n mucous s e c r e t i o n s and s e r v i n g as a f i r s t l i n e of defence a g a i n s t disease agents. In a d d i t i o n to the normal l i g h t chains and c l a s s - d e t e r m i n i n g °l-heavy chain IgA has a s p e c i a l peptide which f a c i l i t a t e s i t s s e c r e t i o n and another polypeptide ( J chain) which i s i n v o l v e d i n i t s p o l y m e r i z a t i o n to the dimeric form ( f o u r l i g h t and four heavy c h a i n s ) . Two sub c l a s s e s have been documented. IgE Although present i n serum i n only very s m a l l amounts, IgE exerts a pronounced e f f e c t by b i n d i n g mast c e l l s . Has €-heavy c h a i n . IgD The c l a s s of antibody found i n the lowest f r e e concentra-t i o n i n serum, although i t i s o f t e n found a s s o c i a t e d with c e l l s . I t has £-heavy chains. LIGHT CHAIN The smal l e r of the two types of polypeptide chains which make up a l l antibody molecules, the other types being c a l l e d heavy c h a i n . There are two b a s i c types of l i g h t chain: Aand x . LYMPHOCYTE The b a s i c c e l l of adaptive immunity. Morphologi-c a l l y , i t i s a simple c e l l with very l i t t l e cytoplasm. Two w e l l documented and d i s t i n c t types are the B - c e l l and T - c e l l . MACROPHAGE A c e l l a c t i v e i n the phagocytosis of f o r e i g n m a t e r i a l and i n promoting antibody-forming responses. MAST CELL A c e l l which contains prominent granules of the mediators of anaphylaxis (immediate-type h y p e r s e n s i t i v i t y ) which i t can r e l e a s e i n t o the e x t r a c e l l u l a r space. MEMORY CELLS Lymphocytes produced by a previous encounter with a n t i g e n . They are s p e c i f i c f o r that antigen and t r i g g e r e d by i t s subsequent p r e s e n t a t i o n . OPSONIZATION The attachment of c e r t a i n a n t i b o d i e s to antigen, x x i i which f a c i l i t a t e s i t s subsequent attachment to and phagocyto-s i s by c e r t a i n c e l l s . PHAGOCYTOSIS The a c t i v e engulfment of p a r t i c l e s by c e l l s such as macrophages and n e u t r o p h i l s . T-CELLS (thymus-derived c e l l s ) Lymphocytes which are respon-s i b l e f o r c e l l - m e d i a t e d immunity and helper/suppressor f u n c t i o n s i n antibody formation. x x i i i A CKNOWLEDGEMENTS During my time working under the supervision of Dr. Dutton, he has been u n f a i l i n g l y kind, generous with his time and ready to give advice when i t was required. Without his help and encouragement my time at the University of B.C. would have been much less pleasant. Grateful thanks are also due to Zamas Lam for a l l his help, especially for the preparation of the figures for thi s thesis. Thanks are also due to the st a f f of the m.s. service, i n p a r ticular G. K. Eigendorf, the mechanical and e l e c t r i c a l / electronic shops, and to the s t a f f of the n.m.r. services. As a part of Dr. Dutton's group, I have had the opportunity to meet and work with v i s i t i n g professors, post-doctoral fellows and graduate students from various countries. Their friendship, help and advice has been invaluble to me during my years i n Vancouver. Although I cannot mention a l l their names here, I would l i k e to extend my thanks to Dr. H. Parolis and Dr. L. A. S. Parolis for their assistance during the f i r s t months and the l a s t months of my time working for Dr. Dutton. F i n a l l y I am grateful for the f i n a n c i a l assistance given by the chemistry department i n the form of a Teaching Assistantship. 1 CHAPTER I INTRODUCTION 2 I. INTRODUCTION Although carbohydrate-containing macromolecules are ubi q u i t o u s i n l i v i n g organisms and i n c l u d e a) g l y c o p r o t e i n s , proteoglycans and peptidoglycans; b) g l y c o l i p i d s and l i p o -p o l y s a c c h a r i d e s ; c) t e i c h o i c a c i d s and d) n u c l e i c a c i d s , not a l l of these molecules are c l a s s i f i e d as p o l y s a c c h a r i d e s . True p o l y s a c c h a r i d e s may be def i n e d as those carbohydrate poly-mers which c o n t a i n p e r i o d i c a l l y r e p e a t i n g u n i t s l i n k e d pre-dominantly, though not n e c e s s a r i l y e n t i r e l y , by O - g l y c o s i d i c bonds. Using t h i s d e f i n i t i o n the category of p o l y s a c c h a r i d e s i n c l u d e s such macromolecules as l i p o p o l y s a c c h a r i d e s and proteoglycans, but excludes n u c l e i c a c i d s and g l y c o p r o t e i n s -the carbohydrate chain of which may c o n s i s t of up to 2 0 sugar r e s i d u e s , but no r e p e a t i n g s t r u c t u r e s are u s u a l l y observed. P o l y s a c c h a r i d e s have a wide v a r i e t y of f u n c t i o n s i n nature, and may act as storage m a t e r i a l s , or as s t r u c t u r a l components. Starc h and glycogen are examples of pol y s a c c h a r i d e food r e s e r v e s while c e l l u l o s e , a f i b r o u s p o l y s a c c h a r i d e , and carrageenan, a gel-forming p o l y s a c c h a r i d e , are examples of the two c l a s s e s of s t r u c t u r a l p o l y s a c c h a r i d e s . T h i r d l y , p o l y s a c c h a r i d e s may f u n c t i o n as p r o t e c t i v e m a t e r i a l s . The exudate gums from p l a n t s , which s e a l o f f i n j u r i e s to prevent i n f e c t i o n by microbes, are one example of a p r o t e c t i v e p o l y -s a c c h a r i d e , but the h i g h l y s p e c i f i c a n t i g e n i c andiimmunogenic e x t r a c e l l u l a r p o l y s a c c h a r i d e s from microorganisms are perhaps the predominant example of t h i s genre. 3 P o l y s a c c h a r i d e s , both p l a n t and m i c r o b i a l , are becoming i n c r e a s i n g l y important i n the commercial world. The value of most p o l y s a c c h a r i d e s r e s i d e s i n t h e i r a b i l i t y to a l t e r the b a s i c p r o p e r t i e s of water (e.g. t h i c k e n i n g and g e l l i n g ) . They are used e x t e n s i v e l y i n the food i n d u s t r y as e m u l s i f i e r s and s t a b i l i z e r s , and p l a y an important r o l e i n c o n t r o l l i n g the te x t u r e , f l a v o u r , c o l o u r and appearance of foods. P o l y -s a c c h a r i d e s a l s o perform as t h i c k e n i n g and s i z i n g agents i n the paper, p a i n t and t e x t i l e i n d u s t r i e s and are added to adhesives to c o n t r o l the v i s c o s i t y of the product. However the major use of p o l y s a c c h a r i d e s i s i n the o i l i n d u s t r y where they are employed as d r i l l i n g f l u i d s and i n o i l rec o v e r y enhancement. O i l f i e l d a p p l i c a t i o n s would appear to be the area i n which the l a r g e s t p o t e n t i a l market f o r novel p o l y -s a c c h a r i d e s (e.g. m i c r o b i a l p o l y s a c c h a r i d e s ) e x i s t s . 1.1 B a c t e r i a l P o l y s a c c h a r i d e s - types and l o c a t i o n Many b a c t e r i a , both gram-positive and gram-negative produce e x t r a c e l l u l a r p o l y s a c c h a r i d e s . As these p o l y -s a c c h a r i d e s are at the sur f a c e of the b a c t e r i a they are the p r i n c i p a l p a r t i c i p a n t s i n most of the i n t e r a c t i o n s between the b a c t e r i a and the host c e l l immune system. They may be present as a d i s c r e t e capsule (K antigen) surrounding the b a c t e r i a l c e l l ( F i g . 1.1), or as a loose slime unattached to the c e l l u l a r s u r f a c e . The presence of a capsule f r e q u e n t l y " denotes a pathogenic bacterium, while a mutation which produces an acapsular s t r a i n renders the bacterium non-pathogenic. Gram-negative b a c t e r i a d i f f e r from gram-positive b a c t e r i a i n that they have an outer membrane surrounding the peptidoglycan l a y e r that comprises the c e l l w a l l (Fig.1 . 2 ) . The main component of the outer membrane of gram-negative b a c t e r i a i s l i p o p o l y s a c c h a r i d e . Where the l i p o p o l y s a c c h a r i d e has a h y d r o p h i l i c , high molecular weight p o l y s a c c h a r i d e c h a i n (0 antigen) t h i s i s the main anti g e n of the b a c t e r i a and i s equ i v a l e n t i n f u n c t i o n to the exopolysaccharide. Gram-p o s i t i v e b a c t e r i a have a d d i t i o n a l components w i t h i n the p e p t i -doglycan l a y e r - one a r e l a t i v e l y simple p o l y s a c c h a r i d e and the other a t e i c h o i c a c i d . 1.2 Serology, Chemistry and B i o s y n t h e s i s of E. c o l i Capsular P o l y s a c c h a r i d e s E. c o l i i s a gram-negative, m o t i l e , non-sporing b a c i l l u s , m o r p h o l o g i c a l l y i d e n t i c a l with other members of the f a m i l y E n t e r o b a c t e r i a c e a e . E. c o l i s t r a i n s , a c q u i r e d by i n g e s t i o n d u r i n g the f i r s t few days of l i f e , are predominant among the ae r o b i c commensal f l o r a present i n the gut of humans and animals. A h e a l t h y person w i l l have more than one E. c o l i s t r a i n present, but v a r i a t i o n s occur over a p e r i o d of time -some s t r a i n s p e r s i s t f o r r e l a t i v e l y long time i n t e r v a l s while others are t r a n s i e n t . Outside' the gut E. c o l l , are r e s p o n s i b l e f o r u r i n a r y t r a c t i n f e c t i o n s and some s t r a i n s with e n t e r o t o x i c c h a r a c t e r i s t i c s a l s o cause g a s t r o e n t e r i t i s e s p e c i a l l y i n i n f a n t s . F i g . 1.1 Electron micrograph of thin section of an encapsulated E. c o l i organism. The capsule was s t a b i l i z e d with monoclonal antibody to K antigen before sample was embedded and sectioned. polysaccharide capeul. llpopolysaccharide (0 .ntlg.n) outar a.abrana perlplaaalc apaca p . p t l d o g l y c . n Inner aeabrane VJ1 c y t o p l a s m Schematic diagram of the E. c o l i c e l l envelope, p, membrane protein; l p , lipoprotein; pe, perlplasmic enzyme; ps, permease; pm, inner membrane protein 6 E. c o l i b a c t e r i a l s t r a i n s or serotypes are c l a s s i f i e d a c c o r d i n g to the nature of t h e i r c e l l s u r f a c e a n t i g e n s . These comprise the afore-mentioned somatic 0 antigen (164 t y p e s ) , the capsular K antigen (7h t y p e s ) , and the p r o t e i n -aceous f l a g e l l a r H antigen (56 t y p e s ) ^ . Capsular (K) antigens of E. c o l i are a c i d i c p o l y s a c c h a r i d e s and on the b a s i s of chemical, p h y s i c a l , and m i c r o b i o l o g i c a l c h a r a c t e r i s t i c s can be d i v i d e d i n t o two groups . Group I c o n s i s t s of high molecu-l a r weight p o l y s a c c h a r i d e s with a low charge density/low e l e c t r o p h o r e t i c m o b i l i t y . Hexuronic a c i d s normally form t h e i r a c i d i c component, they are expressed at a l l growth temperatures and are h e a t - s t a b l e at pH 5-6. These high molecular weight antigens ( f o r m e r l y termed A antigens) form d i s c r e t e capsules and are co-expressed only with the 08 and 09 a n t i g e n s . The b a s i s of t h i s r e g u l a t o r y r e s t r i c t i o n i s unknown. The K antigens of group II have a higher charge d e n s i t y / e l e c t r o p h o r e t i c m o b i l i t y , are not expressed at low growth temperature and are t h e r m o l a b i l e at pH 5-6. They are co-expressed with many 0 antigens and t h e i r a c i d i c components are the more unusual sugars such as 2-keto-3-deoxy-D-manno-o c t u l s o n i c a c i d (KDO). C l a s s i f i c a t i o n s of t h i s type have t h e i r l i m i t a t i o n s however, s t r u c t u r a l and s e r o l o g i c a l s t u d i e s ^ have shown that the same p o l y s a c c h a r i d e may e i t h e r occur as a group I a n t i g e n , or as an a c i d i c l i p o p o l y s a c c h a r i d e , although such occurrences are r a r e . Both group I and group II K antigens have the same ba s i c s t r u c t u r a l p a t t e r n . They c o n s i s t of r e p e a t i n g u n i t s 7 of up to s i x monosaccharide r e s i d u e s , they may be l i n e a r or branched and o f t e n have non-carbohydrate s u b s t i t u e n t s (e.g. _0-acetyl, pyruvate and phosphate). When present i n a n t i g e n i c p o l y s a c c h a r i d e s pyruvate a c e t a l s are the determinant group i . e . the major s i t e of antibody s p e c i f i c i t y (see s e c t i o n 1.2). Amino a c i d s have a l s o been i d e n t i f i e d as s u b s t i t u e n t s on a few c a p s u l a r p o l y s a c c h a r i d e s . L - rThreonine and L - s e r i n e were i d e n t i f i e d as being a m i d i c a l l y l i n k e d to E. c o l i K54 c a p s u l a r p o l y s a c c h a r i d e and the K/fO antigen a l s o has L-threonine as a s u b s t i t u e n t ^ B i o s y n t h e s i s of the c a p s u l a r p o l y s a c c h a r i d e i s f a s t and l i k e l i p o p o l y s a c c h a r i d e s y n t h e s i s i n v o l v e s l i p i d - l i k e saccha-r i d e i n t e r m e d i a t e s . In E. c o l i . s y n t h e s i s of new capsule occurs at a l i m i t e d number of s i t e s on the cytoplasmic mem-brane and there i s evidence that t r a n s p o r t of the completed p o l y s a c c h a r i d e from i t s s i t e of s y n t h e s i s to the c e l l e x t e r i o r i n v o l v e s the same s i t e s , where cytoplasmic and outer membranes are i n c l o s e c o n j u n c t i o n , as does the t r a n s p o r t of l i p o p o l y s a c c h a r i d e ^ . How the e x t r a c e l l u l a r p o l y s a c c h a r i d e remains a s s o c i a t e d with the b a c t e r i a l c e l l s u r f a c e i s as yet unknown. However, the recent i d e n t i f i c a t i o n of a l i p i d a t tached to the r e d u c i n g end of a i ~fraction of the p o l y -s a c c h a r i d e p r e p a r a t i o n s from E. c o l i strains-^ suggests that c a p s u l a r p o l y s a c c h a r i d e s a s s o c i a t e with the b a c t e r i a l c e l l outer membrane through hydrophobic i n t e r a c t i o n s . 8 1.3 Immunology of B a c t e r i a l P o l y s a c c h a r i d e s The immune system c o n s i s t s of a complex network of c e l l s (lymphocytes and macrophages) and molecules ( a n t i b o d i e s and complement) which c i r c u l a t e throughout the "bloodstream and lymphatic system, s e a r c h i n g out and d e s t r o y i n g or i n a c t i v a t -i n g any f o r e i g n i n v a d e r s . The i n t e r a c t i o n of three main types of c e l l s produces an immune response; the T c e l l s which, when a c t i v a t e d by an antigen, d i f f e r e n t i a t e i n t o v a r i o u s subsets e.g. k i l l e r c e l l s and he l p e r or suppressor c e l l s , B c e l l s which , i n co-operation -with a T lymphocyte and a macrophage, are r e s p o n s i b l e f o r the production of antibody, and the macrophage - a l a r g e mononuclear, phagocytic c e l l . The major r o l e of the macrophage appears to be one of T p r e s e n t i n g ' the antig e n to the lymphocytes. I t a l s o has su r f a c e r e c e p t o r s f o r immunoglobulins and f o r the t h i r d component of complement (C3) • The complement cascade can be a c t i v a t e d i n two ways; the c l a s s i c a l pathway i s i n i t i a t e d by an antibody-antigen complex, to which complement binds, the a l t e r n a t e pathway does not r e q u i r e the b i n d i n g of antigen to antibody and i s i n i t i a t e d d i r e c t l y at the C3 step by pol y -s a c c h a r i d e s such as zymosan and l i p o p o l y s a c c h a r i d e s . Thus, the a l t e r n a t i v e pathway of complement provides a f i r s t defense a g a i n s t an i n v a d i n g microorganism before a n t i b o d i e s have been produced. The end r e s u l t of both pathways i s comple-ment-mediated phagacytosis by the macrophage. When a c t i v a t e d by a T - c e l l , p r e v i o u s l y primed by 9 antigen, B c e l l s normally undergo c l o n a l expansion to produce antibody-producing c e l l s and memory c e l l s which are a v a i l a b l e f o r response when subsequently presented with the same a n t i g e n . However, some high molecular weight molecules with r e p e a t i n g sequences of a n t i g e n i c determinants e.g. Q p o l y s a c c h a r i d e s are T - c e l l independent". No memory c e l l s are produced by B - c e l l s when the antigen i n v o l v e d i s T - c e l l independent. Thus, when immunized with a T - c e l l independent a n t i g e n , an immunologically naive subject, w i l l not r a i s e a secondary response on re-exposure to the same a n t i g e n . T h i s , of course, has important consequences when c o n s i d e r i n g the use of a p o l y s a c c h a r i d e v a c c i n e f o r young c h i l d r e n (see S e c t i o n 1.5). 1.3.1 S t r u c t u r e and A n t i g e n i c i t y of B a c t e r i a l P o l y s a c c h a r i d e s Molecular weight i s an important c r i t e r i o n i n determin-i n g the a n t i g e n i c i t y of a molecule. Dextrans with an average molecular weight of 90,000 are immunogenic while those of average molecular weight 50,000 or lower are not. A n t i -bodies are r a i s e d a g a i n s t only s m a l l p o r t i o n s of the p o l y -s a c c h a r i d e molecule c o n s i s t i n g of between two and four r e s i d u e s i . e . the a n t i g e n i c determinant. The c l a s s i c a l i n t e r p r e t a t i o n by Kabat^ of h i s f i n d i n g that f o r l i n e a r p o l y -s a c c h a r i d e s a n t i b o d i e s bind p r e f e r e n t i a l l y to a t e r m i n a l non-red u c i n g sugar has r e c e n t l y been challanged by Glaudemans 1 0. In the l i g h t of h i s f i n d i n g s he r a t i o n a l i z e d t h a t an a n t i -10 body w i l l have one s u b s i t e with higher r e l a t i v e g l y c o s y l a f f i n i t y than any other s u b s i t e , but that i t s l o c a t i o n i s not n e c e s s a r i l y p e r i p h e r a l . In branched p o l y s a c c h a r i d e s the immunodominant sugars are u s u a l l y those t h a t form the branches ' , and as pointed out p r e v i o u s l y (see S e c t i o n 1.2) where non-carbohydrate s u b s t i t u e n t s are present e.g. pyruvate 1 3 a c e t a l s , they have been shown to be immunodominant -'. 1 .3.2 C r o s s - r e a c t i o n s of B a c t e r i a l P o l y s a c c h a r i d e s A n t i b o d i e s r a i s e d by immunization c o n s i s t of hetero-geneous subpopulations each s p e c i f i c f o r a p a r t i c u l a r part of the a n t i g e n . Some of the a n t i b o d i e s r a i s e d a g a i n s t one p a r t i c u l a r antigen may a l s o r e a c t with another antigen i f the u second a n t i g e n has some s t r u c t u r a l f e a t u r e s i n common with the f i r s t . C r o s s - r e a c t i o n s are common i n b a c t e r i a l poly-s a c c h a r i d e s and canbe used to e s t a b l i s h the s t r u c t u r e of determinant groups. However, r e s u l t s may be misl e a d i n g as a p o s i t i v e r e a c t i o n i s good evidence f o r a common determinant» but l a c k of a c r o s s - r e a c t i o n does not n e c e s s a r i l y mean there are no common s t r u c t u r a l f e a t u r e s - r e a c t i o n may be prevented by s t e r i c hindrance. H e i d e l b e r g e r used t h i s approach to p r e d i c t some of the s t r u c t u r a l f e a t u r e s of E. c o l i K26 c a p s u l a r p o l y s a c c h a r i d e (see S e c t i o n ;IV .2). The c r o s s -r e a c t i v i t y of p o l y s a c c h a r i d e s may a l s o f i n d uses i n the development of v a c c i n e s (see S e c t i o n 1 . 5 ) . 11 I.h The Bole of Capsular P o l y s a c c h a r i d e s i n the V i r u l e n c e and P a t h o g e n i c i t y of E. c o l i B a c t e r i a B a c t e r i a l v i r u l e n c e may be regarded as the inva d i n g o r g a n i s m s ' a b i l i t y to evade the immune system while m u l t i -p l y i n g and e v e n t u a l l y k i l l i n g the host. In an organism such as E. c o l i . the ca p s u l a r p o l y s a c c h a r i d e i s an important v i r u l e n c e f a c t o r . Both the 0 and K antigens p r o t e c t the c o l i b a c t e r i a a g a i n s t the b a c t e r i c i d a l a c t i o n of phagocytes 14* and complement, although the capsule i s the more e f f e c t i v e . I n g e s t i o n by a macrophage u s u a l l y r e q u i r e s that the b a c t e r i a l c e l l be opsonized i . e . coated with antibody. In the absence of s p e c i f i c a n t i - c a p s u l a r a n t i b o d i e s the b a c t e r i a m u l t i p l y and acute i n f e c t i o n r e s u l t s . Noncapsulated b a c t e r i a are u s u a l l y phagocytized r a p i d l y as the u n d e r l y i n g components of the b a c t e r i a l c e l l w a l l have the a b i l i t y to d i r e c t l y a c t i v a t e the a l t e r n a t i v e pathway of complement without having p r e v i o u s l y bound a n t i b o d i e s . A l t e r n a t i v e l y , the presence of a capsule may a i d b a c t e r i a l s e l f - d e f e n s e by e x e r t i n g a camouflage effect-^. The b a c t e r i a are not recognized as f o r e i g n by the host's immune system and so s p e c i f i c host defense ( i . e . the formation of a n t i b o d i e s ) i s not i n i t i a t e d . E. c o l i KI c a p s u l a r a n t i g e n 1 ^ due to i t s s t r u c t u r a l s i m i l a r -i t y to g a n g l i o s i d e s of the host c e l l e x e r t s such an e f f e c t and E . c o l i KI b a c t e r i a are e s p e c i a l l y v i r u l e n t . Capsules are a l s o determinant -factors intthe? 12 pathogenicity of E. c o l i bacteria. B a c t e r i a l pathogenicity i s a complex phenomenon and includes many processes such as b a c t e r i a l adhesion, evasion of host defenses and production of toxins. However, i f E. c o l i bacteria are grouped together according to diseases they cause then some in t e r e s t -ing patterns emerge '. Bacteria with highly charged surface polysaccharides penetrate into or through the gut wall and cause ex t r a - i n t e s t i n a l diseases. E. c o l i bacteria with low molecular weight polysaccharides from thin capsules are t y p i c a l l y associated with urinary tract i n f e c t i o n s , bacteraemia and neonatal meningitis, while E. c o l i which penetrate into but not through the gut wall, causing dysen-try, are not encapsulated but have aci d i c 0 antigens. Enteropathogenic and enterotoxigenic strains have only neu-t r a l 0 antigens and no capsules. Thus, for a given patho-genic i n f e c t i o n i t appears that there i s a p a r t i c u l a r combination of b a c t e r i a l surface polysaccharides and the pathogenic properties of E. c o l i appear to be controlled by the differences i n charge. Furthermore, there i s evidence that the structural p r i n c i p l e s underlying pathogenic mechanisms are not r e s t r i c t e d to one b a c t e r i a l species but are more general. For example, meningitis i n young children i s caused by N. meningitidis and also E. c o l i K1. Both bacteria take the same invasive route and must penetrate several functional b a r r i e r s . The capsular antigens of both these pathogens are i d e n t i c a l 1 The mechanism of penetration and the role of the negatively 13 charged polysaccharide i n this process i s unknown but the role of the acid capsular polysaccharide appears to be of great importance. 1.5 B a c t e r i a l Capsular Polysaccharide Vaccines Diseases caused by encapsulated bacteria are among the most prevalent i n the world. The mortality rate from mening-i t i s and pneumonia i s s t i l l high despite therapy with a n t i -b a c t e r i a l drugs. : Furthermore, many strains of encap-sulated bacteria have become resistant to a n t i b i o t i c s . It was the increasing resistance of meningococci microorganisms to sulphonamides which provided the o r i g i n a l drive to develop vaccines from meningococcal capsular polysaccha-19 rides . There are now three b a c t e r i a l capsular poly-saccharide vaccines licensed i n the U.S.A., meningococcal, pneumococcal and H. influenzae type b. Other vaccines u t i l i z i n g polysaccharides from grounB Streptococcus Ia, l b , II and I I I , K l e b s i e l l a , Pseudomonas aeruginosa and E t cpLj are i n the various stages of c l i n i c a l studies and vaccine 1Q development . The u t i l i z a t i o n of capsular polysaccharides as vaccines has only been p a r t i a l l y successful due to the f a i l u r e of infants and young children (under 2 years), unlike adults, to develop protective lev e l s of serum antibodies. In young children polysaccharide vaccines give r i s e to only Ig M antibodies and there i s no anamnestic response, whereas 14 p r o t e i n v a c c i n e s induce both Ig M and Ig G a n t i b o d i e s and an immunological memory. T h i s d i f f e r e n c e i n immune response i s thought to be due to the T - c e l l independence of polysaccha^ r i d e antigens (see S e c t i o n 1 .3)• A s o l u t i o n , c u r r e n t l y being i n v e s t i g a t e d , i s the enhancement of the immunogenicity of the pure p o l y s a c c h a r i d e s by c o n v e r t i n g them to thymus-dependent a n t i g e n s . One way of a c h i e v i n g t h i s i s by conjuga-t i n g them to protexn c a r r i e r s I t has been observed: that most a d u l t animal sera c o n t a i n a n t i b o d i e s to p o l y s a c c h a r i d e antigens of v a r i o u s pathogenic b a c t e r i a even where there was no p o s s i b l e contact with these organisms. These p r o t e c t i v e a n t i b o d i e s appear to have been e l i c i t e d i n response to the presence of non-pathogenic b a c t e r i a , i n the i n t e s t i n a l and pharyngeal f l o r a , which c a r r y antigens with some s t r u c t u r a l f e a t u r e s i n common with those of pathogenic b a c t e r i a l a n t i g e n s . Scheerson and 22 Robbins fed non-pathogenic E. c o l j K100 b a c t e r i a to a d u l t v o l u n t e e r s and found that a n t i b o d i e s s p e c i f i c f o r H.  i n f l u e n z a e type b p o l y s a c c h a r i d e were induced. Thus, d e l i b e r a t e i n t e s t i n a l c o l o n i z a t i o n by c r o s s - r e a c t i v e s t r a i n s c ould r e s u l t i n the b i o s y n t h e s i s of a n t i - c a p s u l a r polysaccha-r i d e antigens and provide an a l t e r n a t i v e to v a c c i n a t i o n . Table 1.1 l i s t s some of the normal human f l o r a which could be r e s p o n s i b l e f o r n a t u r a l immunity to groups A, B, C N. m e n i n g i t i s and type b H. i n f l u e n z a e . TABLE 1.1 Polysaccharides of Bacteria, Frequently Found in Human Flora, That Cross-react with the Polysaccharide Capsules of Human Pathogenic Bacteria Pathogen Cross-reacting organism Structure Neisseria meningitidis Group A Group B Group C Haemophilus influenzae Type b B. pumili6 S. fecalis E. c o l i KI E. c o l i K92 E. c o l i K 100 S. aureus B. pumilis B. subt i l i s L. plantarum 2-acetamido-2-deoxymannosyl phosphate residues •+8)-D-NeupAc(2* and i t s 0Ac + variant -»8)-D-NeupAc( 2—9)-D-Neu^Ac( 2-r -v3)-D-Ribf (1 ~2M>-ribitol(5-0-P-» OH teichoic acids containing r i b i t o l phosphate 16 CHAPTER I I PROCEDURES FOR THE STRUCTURAL ANALYSIS OF BACTERIAL CAPSULAR POLYSACCHARIDES (CPS) 17 I I . PROCEDURES FOR THE STRUCTURAL ANALYSIS OF BACTERIAL CAPSULAR POLYSACCHARIDES (CPS) I I . 1 I s o l a t i o n and P u r i f i c a t i o n The main o b j e c t i v e when i s o l a t i n g , a polysaccharide i s to o b t a i n a high y i e l d of chemically pure homogeneous m a t e r i a l . High molecular-weight capsular polysaccharides are u s u a l l y i s o l a t e d from b a c t e r i a grown on a s o l i d medium, whereas low molecular-weight polysaccharides are i s o l a t e d p r e f e r e n t i a l l y from l i q u i d b a c t e r i a l c u l t u r e s . A f t e r h a r v e s t i n g , the b a c t e r i a are k i l l e d by the a d d i t i o n of phenol s o l u t i o n and the b a c t e r i a l c e l l s are removed by u l t r a -c e n t r i f u g a t i o n . The a c i d i c capsular polysaccharide may then be s e l e c t i v e l y p r e c i p i t a t e d from the supernatant by the a d d i t i o n of c a t i o n i c detergents such as c e t y l t r i m e t h y l -ammonium "bromide (CTAB) s o l u t i o n . The quaternary ammonium s a l t complexes with the c a r b o x y l groups of the a c i d i c cap-s u l a r polysaccharide r e s u l t i n g i n t h e i r c o - p r e c i p i t a t i o n . A f t e r removal, by low speed c e n t r i f u g a t i o n , the polysaccha-ride-CTAB complex i s d i s s o c i a t e d i n sodiumlchloride s o l u t i o n and the polysaccharide r e p r e c i p i t a t e d , as i t s sodium s a l t , i n t o e t h a n o l . Unambiguous proof f o r the homogeneity of the polysaccha-r i d e cannot be obtained, the most that may be e s t a b l i s h e d i s a l a c k of heterogeneity which can be demonstrated by: 1) Gel f i l t r a t i o n or molecular s i e v e chromatography, 18 where s e p a r a t i o n i s based on d i f f e r e n c e s i n molecular s i z e . I f the column i s c a l i b r a t e d then the r e l a t i v e molecular weight may a l s o be obtained?^. 2) E l e c t r o p h o r e s i s d i s t i n g u i s h e s molecules on the b a s i s of i o n i z a b l e groups but i s l e s s r e a d i l y adapted f o r p r e p a r a t i v e purposes. 3) Ion exchange chromatography i s used to f r a c t i o n a t e b a c t e r i a l p o l y s a c c h a r i d e s c o n t a i n i n g c a r b o x y l i c a c i d s . The c r o s s - l i n k e d g e l s c a r r y i n g an a p p r o p r i a t e f u n c t i o n a l group e.g. d i e t h y l a m i n o e t h y l ( D E A E ) - c e l l u l o s e 2 Z f are u s u a l l y u t i l i z e d because of t h e i r high c a p a c i t i e s , but have the disadvantage of undergoing l a r g e a l t e r a t i o n s i n volumes with changes i n i o n i c s t r e n g t h of the e l u a n t . 4-0 A f f i n i t y chromatography i s based on the a b i l i t y of some p r o t e i n s or l e c t i n s to r e v e r s i b l y b i n d , non-covalently, carbo-hydrate polymers. These p r o t e i n s or l e c t i n s are s p e c i f i c f o r i n d i v i d u a l sugars with a c e r t a i n g l y c o s i d i c c o n f i g u r a t i o n . Concanavalin A, from the jack bean, i s one such example which, when bound to an i n e r t support such as Sephadex, has been used to f r a c t i o n a t e ] i - g l y c o s i d i c g l y c o p e p t i d e s 2 ^ . Although t h i s technique i s p o t e n t i a l l y very u s e f u l , the f r a c t i o n a t i o n of p o l y s a c c h a r i d e s by t h i s method has not been u t i l i z e d o f t e n to date. Constancy of chemical composition and p h y s i c a l p r o p e r t i e s a r e the most important c r i t e r i a f o r absence of h e t e r o g e n e i t y i n a p o l y s a c c h a r i d e and may be e s t a b l i s h e d by one of the f o l l o w i n g : h y d r o l y s i s (sugar composition); n u c l e a r magne-19 t i c resonance spectroscopy ( f u n c t i o n a l group d e t e r m i n a t i o n or a n a l y s i s f o r a c e r t a i n type of sugar e.g. deoxy sugar); p h y s i c a l p r o p e r t i e s ( o p t i c a l r o t a t i o n , v i s c o s i t y ) . I f f u r t h e r p u r i f i c a t i o n i s necessary the p o l y s a c c h a r i d e may be t r e a t e d with s p e c i f i c enzymes such as protease or RNase. Contaminating RNA may a l s o be removed by p r e c i p i t a t i o n of a CTAB-RNA complex p r i o r to the formation o f the CTAB-26 p o l y s a c c h a r i d e p r e c i p i t a t e . II.2 Chemical A n a l y s i s Once a pure, non-heterogeneous b a c t e r i a l c a p s u l a r p o l y s a c c h a r i d e has been obtained then the s t r u c t u r a l and c o m p o s i t i o n a l a n a l y s i s i s begun. P o l y s a c c h a r i d e s are p o l y -d i s p e r s e , so any i n f o r m a t i o n obtained on mol e c u l a r s i z e or composition w i l l o n l y g i v e the average molecular weight or r e l a t i v e p r o p o r t i o n s of the sugar c o n s t i t u e n t s . B a c t e r i a l c a p s u l a r p o l y s a c c h a r i d e s c o n s i s t of r e g u l a r r e p e a t i n g u n i t s of up to seven monosaccharide u n i t s and any s t r u c t u r a l d e t e r m i n a t i o n must answer the f o l l o w i n g q u e s t i o n s : -1) How many and what type o f monosaccharides are present i n the r e p e a t i n g u n i t ? 2) What are the r i n g s i z e s o f the sugars and where are t h e i r l i n k a g e p o s i t i o n s ? 3) What are the c o n f i g u a t i o n s of the anomeric l i n k a g e s ? 4) Are the sugars i n t h e i r D- or L- c o n f i g u r a t i o n s ? 5) Do the r e p e a t i n g u n i t s c a r r y any non-carbohydrate sub-20 s t i t u e n t s e.g. amino acids or pyruvate? 6) F i n a l l y the most d i f f i c u l t problem to solve, what i s the sequence of the sugars i n the repeating unit? There are some excellent texts and reviews on the procedures and techniques used to f i n d the answers to these P7 PR questions '* . In the following section techniques which have been used i n the course of t h i s work e.g. reductive cleavage 2^, HF h y d r o l y s i s ^ 0 ' ^ 1 and lithium/ethylenediamine deg^adation^ 0 ,^ 2 w i l l be disc ussed, along with the more routine procedures such as acid hydrolysis. II.2.1 Monosaccharide Composition Depolymerization of a polysaccharide i s normally the f i r s t step i n determining i t s q u a l i t a t i v e and quantitative monosaccharide composition. A l l monosaccharides are degraded to some extent by a c i d hydrolysis, so a balance must be achieved between maximum depolymerization and minimum destruction of the sugars. It may be desirable i n some z cases to use d i f f e r e n t hydrolytic conditions for the analysis of d i f f e r e n t sugar residues i n the same polysaccharide. D u t t o n ^ has reviewed the disadvantages and advantages of various acids commonly used for hydrolysis. Incomplete hydrolysis i s usually associated with the presence of a uronic a c i d residue or an amino sugar. Advantage of t h i s difference i n l a b i l i t y 6f the g l y c o s i d i c bonds i s taken when determination of the sequence of sugars i n the polymer 21 n e c e s s i t a t e s the g e n e r a t i o n of o l i g o s a c c h a r i d e s (see S e c t i o n I I . 2 . 6 ) . To o b t a i n complete d e p o l y m e r i z a t i o n , when a u r o n i c a c i d i s present as a p o l y s a c c h a r i d e component, r e d u c t i o n to the c o r r e s p o n d i n g hexose i s u s u a l l y e f f e c t e d . T h i s i s norm-a l l y a c h i eved by s u c c e s s i v e treatments of the p o l y s a c c h a r i d e with a w a t e r - s o l u b l e c a r b o d i i m i d e and sodium b o r o h y d r i d e ^ ^ ( F i g . I I . 1 ) . An a l t e r n a t i v e method i n v o l v e s treatment of the p o l y s a c c h a r i d e with methanolic hydrogen c h l o r i d e to c l e a v e most of the g l y c o s i d i c bonds. Methyl g l y c o s i d e s and methyl e s t e r s are formed s i m u l t a n e o u s l y and r e d u c t i o n of the e s t e r s to t h e i r c orresponding a l c o h o l s p r i o r to h y d r o l y s i s ensures 35 t h a t a l l the sugar r e s i d u e s are r e l e a s e d ^ . 2-Amino-2-deoxyhexoses are the amino sugars most common-l y found as p o l y s a c c h a r i d e c o n s t i t u e n t s . They are normally present as N - a c e t y l d e r i v a t i v e s and, s i n c e a c i d h y d r o l y s i s r e s u l t s i n N - d e a c e t y l a t i o n , the r e s u l t i n g 2-amino-2-deoxy-g l y c o s i d e s are r e s i s t a n t to h y d r o l y s i s . Vigorous c o n d i t i o n s are then r e q u i r e d f o r complete d e p o l y m e r i z a t i o n of the p o l y -s a c c h a r i d e . An a l t e r n a t i v e procedure i s to N - d e a c e t y l a t e the amino sugar, and a t the same time c l e a v e a l l other g l y c o s i d i c l i n k a g e s , by h y d r o l y s i s of the p o l y s a c c h a r i d e . Subsequent treatment of the h y d r o l y s a t e with n i t r o u s a c i d w i l l deaminate the 2-amino-2-deoxysugar forming a 2,5-anhydrohexose r e s i d u e with concomitant cleavage of the remaining 2-amino- 2-deoxyglycosidic l i n k a g e s ^ . The use o f anhydrous hydrogen f l u o r i d e i n p o l y s a c c h a -r i d e s t r u c t u r a l a n a l y s i s i s becoming more p r e v a l e n t . By 22 m a n i p u l a t i n g the temperature a t which the r e a c t i o n occurs e i t h e r s e l e c t i v e bond cleavage (see S e c t i o n I I . 2 . 6 . 5 ) or complete d e p o l y m e r i z a t i o n may be a c h i e v e d . Treatment of a p o l y s a c c h a r i d e with anhydrous HF a t room temperature f o r 3 h i s n o r m a l l y s u f f i c i e n t to c l e a v e even r e s i s t a n t 2-amino-2 - d e o x y g l y c o s i d i c l i n k a g e s . . A n a l y s i s of the i n d i v i d u a l sugar mixtures i s i n the f i r s t i n s t a n c e achieved by paper chromatography (see S e c t i o n 4.1). For q u a n t i t a t i v e and q u a l i t a t i v e i n f o r m a t i o n , v o l a t i l e d e r i v a t i v e s are prepared and a n a l y s e d by g a s - l i q u i d chroma-tography (see S e c t i o n II.4.1.1). A n a l y s i s of some c l a s s e s of sugars may be performed s p e c t r o p h o t o m e t r i c a l l y . As many of these methods i n v o l v e treatment of the polysaccha-r i d e with a c i d , p r i o r h y d r o l y s i s i s unnecessary. H y d r o l y s i s i s accompanied by decomposition to give chromogens ( u s u a l l y f u r a n or p y r r o l e d e r i v a t i v e s ) which form c o l o u r e d compounds w i t h s p e c i f i c reagents-^ . I I . 2 . 2 Ring S i z e and Linkage P o s i t i o n Once the number and i d e n t i t i e s of the sugars i n the p o l y s a c c h a r i d e have been determined, the next s t e p i n a s t r u c t u r a l e l u c i d a t i o n i s the d e t e r m i n a t i o n of t h e i r l i n k a g e p o s i t i o n and r i n g s i z e . M e t h y l a t i o n a n a l y s i s i s s t i l l the method o f choice f o r o b t a i n i n g t h i s i n f o r m a t i o n . The b a s i s of t h i s technique i s the formation of a c i d -s t a b l e e t h e r groups where p r e v i o u s l y f r e e sugar h y d r o x y l 23 groups existed. Hydrolysis of the methylated polysaccha-ride y i e l d s p a r t i a l l y methylated sugars with free hydroxyl groups only where there were formerly inter-sugar linkages. The presence of a methoxyl group at either C-if or C-5 defines the sugar as either a furanose or pyranose. Separa-tion and characterization of the v o l a t i l e derivatives of the methylated sugars are achieved by g.c.-m.s. The p a r t i a l l y or f u l l y methylated sugars may be i d e n t i f i e d on the basis of t h e i r mass spectra (see Section I I . 4 « 2 ) . A successful methylation depends on completely i o n i z -ing the free hydroxyl groups to give alkoxides which undergo a nucleophilic reaction with the a l k y l a t i n g agent, usually methyl iodide. The Hakomori methylation-^, where the poly-saccharide dissolved i n dimethyl-.sulphoxide ( D M S O ) i s f i r s t treated with the base sodium methylsulphinylmethanide, i s s t i l l the most commonly employed method. During a Hakomori methylation any uronic acid residues present w i l l be e s t e r i -f i e d . Complete depolymerization of the polysaccharide may then be achieved by reduction, using lithium aluminium hydride, of the uronic ester to i t s corresponding hexose. Completeness of methylation of the polysaccharide may be ascertained by the absence of O-H stretching vibrations i n the IR spectrum. In the event of an incomplete methylation a second Hakomori methylation cannot be carried out on a uronic acid-containing polysaccharide - uronic esters being very susceptible to base-catalyzed p-elimination (see Section I I . 2 . 6 . 2 ) . In such cases, the o r i g i n a l Purdie methylation^ 0 24 i n which methyl i o d i d e i s both the s o l v e n t and a l k y l a t i n g agent, with s i l v e r oxide as base, i s g e n e r a l l y the method s e l e c t e d . More r e c e n t l y 0H~, H~ and "OtBu have been used as the b a s i c reagents i n m e t h y l a t i o n r e a c t i o n s ^ E x c e l l e n t y i e l d s were obtained, and fewer i m p u r i t i e s were observed i n the gas chromatograms, with these r e a g e n t s . A l k a l i - l a b i l e carbohydrates do not withstand the s t r o n g n u c l e o p h i l i c c o n d i t i o n s of the Hakomori m e t h y l a t i o n ^ . For such p o l y s a c c h a r i d e s , treatment with methyl t r i f l u o r o m e t h a n e -sulphonate i n t r i m e t h y l phosphate i s recommended^^. How-ever, experiences i n t h i s l a b o r a t o r y and o t h e r s ^ . ' ^ i n d i c a t e d t h a t t h i s method, while s u i t a b l e f o r the methy-l a t i o n of lower molecular weight o l i g o s a c c h a r i d e s , was u n s u c c e s s f u l i n the m e t h y l a t i o n of p o l y s a c c h a r i d e s , probably due to t h e i r low s o l u b i l i t y i n t r i m e t h y l phosphate. There are two s i g n i f i c a n t disadvantages a s s o c i a t e d with m e t h y l a t i o n a n a l y s i s : 1) where n e i t h e r the 0-4 or 0-5 p o s i t i o n are methoxylated then m e t h y l a t i o n a n a l y s i s does not p r o v i d e i n f o r m a t i o n on r i n g s i z e or l i n k a g e p o s i t i o n e.g. 2,3,6-tri-O-methyl-D-galactose could r e s u l t from e i t h e r a 4 - 0 - s u b s t i t u t e d D-galactopyranose or a 5-P_-substituted D - g a l a c t o f u r a n o s e , and 2) the method i s time consuming. To overcome these problems G r a y ^ has developed a new method f o r c a r r y i n g out m e t h y l a t i o n a n a l y s i s . The main f e a t u r e of t h i s method i s the r e g i o s p e c i f i c r e d u c t i v e cleavage of the g l y c o s i d i c carbon-oxygen bond u s i n g t r i e t h y l s i l a n e as the r e d u c i n g agent, i n the presence of e i t h e r t r i m e t h y l s i l y l -25 t r i f l u o r o n e t h a n e sulphonate (TMSOTf) or boron t r i f l u o r i d e e t h e r a t e (BF^Et20). Pyranosides are converted to 1,5-a n h y d r o a l d i t o l d e r i v a t i v e s (1) and f u r a n o s i d e s are converted to 1 , 4 - a n h y d r o a l d i t o l d e r i v a t i v e s ( 2 ) , thus the l i n k a g e p o s i t i o n and r i n g s i z e are e s t a b l i s h e d s i m u l t a n e o u s l y (see Scheme I I . 1 ) . Furthermore, the a n h y d r o a l d i t o l s are v o l a -t i l e and i t i s unnecessary to c a r r y out a f u r t h e r sequence of r e a c t i o n s to convert the products to d e r i v a t i v e s s u i t a b l e f o r g.c.-m.s a n a l y s i s . Using the r e d u c t i v e cleavage method t h e r e f o r e , both disadvantages of the c o n v e n t i o n a l m e t h y l a t i o n a n a l y s i s are overcome. A t h i r d advantage of the r e d u c t i v e cleavage method i s , t h a t by c a r e f u l s e l e c t i o n of an appro-p r i a t e c a t a l y s t , i t i s p o s s i b l e to achieve p a r t i a l or t o t a l r e d u c t i v e cleavage. T h i s s e l e c t i v i t y may be a p p l i e d to sequence s t u d i e s of a p o l y s a c c h a r i d e (see Section'II.2.6.6) II.2.3 Anomeric C o n f i g u r a t i o n The monosaccharide r e s i d u e s i n a p o l y s a c c h a r i d e may be l i n k e d e i t h e r e q u a t o r i a l l y ( p-linked) or a x i a l l y («*-link-ed) . D e t e r m i n a t i o n . of the c o n f i g u r a t i o n of the g l y c o -s i d i c l i n k a g e s may be achieved by one of the f o l l o w i n g methods; 1) chromium t r i o x i d e o x i d a t i o n - where the ^ - l i n k e d pyrano-s i d i c sugars of a p e r - a c e t y l a t e d p o l y s a c c h a r i d e are degraded p r e f e r e n t i a l l y t o the <*-linked s u g a r s ^ , 2) measurement of o p t i c a l rotation**®, 26 CH2OMe ( i ) reductive j O cleavage OMe ( i i ) Ac jav / \ o \ 0 M e / r AcoNl / OMe OMe as above CH2OMe AcO — OMe Scheme II.1 Reductive cleavage of pyranosides and furanosldes RCOOH -r RCOO'H H+ 0H R" NHR' N0BH4 pH 5-7 RCH20H NoBH4 [R?H! + 0=6 + H L J NHR' F i g . II.1 Reduction of.carboxylic acid i n aqueous solu t i o n . E.D.C. = l-ethyl-3-(-dimethylaminopropyl)-carbodiimide; C.M.C. = 1-cyclohexyl-3~(2-morpholinoethyl)carbodiimide metho-j>-toluene sulphonate* 27 3) measurement of the c o u p l i n g c o n s t a n t s and the chemical s h i f t s of sugars from both the 1^C- and 1H-n.m.r. s p e c t r a (see S e c t i o n II./+.3) of the p o l y s a c c h a r i d e . k) use of s p e c i f i c enzymes e.g. exoglycanases, to p r e f e r e n t -i a l l y degrade a sugar with a c e r t a i n anomeric c o n f i g u r a t i o n e.g. yS-D-glucosidase (see S e c t i o n II.3.1). 11.2.4 C o n f i g u r a t i o n of Sugars (D or L) The c o n f i g u r a t i o n of the monosaccharide r e s i d u e s of a p o l y s a c c h a r i d e may a l s o be determined by a v a r i e t y of d i f f e r e n t t e c h n i q u e s . These i n c l u d e ; the measurement of the o p t i c a l r o t a t i o n of i s o l a t e d s u g a r s ^ , formation of the a l d i t o l a c e t a t e s or p a r t i a l l y methylated a l d i t o l a c e t a t e s of i s o l a t e d sugars and measurement of t h e i r c i r c u l a r d i c h r o i s m ^ 0 r the use of s p e c i f i c oxidases e.g. D-g-lucose-o x i d a s e ^ 0 . However, the method of ch o i c e i n the work presented here was the formation o f diastereomers by the r e a c t i o n o f the i s o l a t e d monosaccharides with a c h i r a l a l c o h o l ^ 1 a n d s e p a r a t i o n of the g l y c o s i d e s by g.c. 11.2.5 L o c a t i o n of Non-carbohydrate S u b s t i t u e n t s E. c o l i c a p s u l a r p o l y s a c c h a r i d e s s u b s t i t u t e d with non-carbohydrate groups occur n a t u r a l l y . O-Acetyl and pyruvate s u b s t i t u e n t s are the most common and t h e i r presence and q u a n t i t a t i o n per r e p e a t i n g u n i t can be deduced from the 28 H-n.m.r. spectrum of the n a t i v e p o l y s a c c h a r i d e (see S e c t i o n I I . 4 . 3 ) . Removal of a pyruvate s u b s t i t u e n t i s r e l a t i v e l y easy, o f t e n a u t o h y d r o l y s i s of an a c i d i c p o l y s a c c h a r i d e i s s u f f i c i e n t . Comparison of the g.c.-m.s. data from the m e t h y l a t i o n a n a l y s e s of the p y r u v y l a t e d and d e p y r u v y l a t e d p o l y s a c c h a r i d e w i l l r e v e a l the l o c a t i o n of the pyruvate. For example, the appearance of 2,3,4-tri-£)-methylrhamnose and the concomitant disappearance of 2-^-methylrhamnose a f t e r d e p y r u v y l a t i o n , l o c a t e d the pyruvate s u b s t i t u e n t of E. c o l i K26 c a p s u l a r p o l y s a c c h a r i d e on p o s i t i o n s 0-3 and 0-4 of a t e r m i n a l rhamnose u n i t . Determination of the a b s o l u t e c o n f i g u r a t i o n of the pyruvate s u b s t i t u e n t may a l s o 53 be accomplished with the a i d of n.m.r. sp e c t r o s c o p y data^ . O-Acetyl s u b s t i t u e n t s are b a s e - l a b i l e and cannot be l o c a t e d u s i n g the Hakomori m e t h y l a t i o n . Prehm m e t h y l a t i o n ^ of a s u i t a b l y s u b s t i t u t e d o l i g o s a c c h a r i d e , or replacement of the 0 - a c e t y l groups with the s t a b l e 0-methyl group, u s i n g methyl v i n y l e t h e r as a p r o t e c t i v e r e a g e n t ^ are two commonly used techniques f o r the p r e c i s e l o c a t i o n of any 0 - a c e t y l s u b s t i t u e n t s . E. c o l i c a p s u l a r p o l y s a c c h a r i d e s s u b s t i t u t e d with amino a c i d s a r e more unusual, but r e c e n t l y the s t r u c t u r e s of two U 5 such p o l y s a c c h a r i d e s have been elucidated^"». In the f i r s t i n s t a n c e , the presence of amino a c i d s may be determined by h y d r o l y s i s of the p o l y s a c c h a r i d e and a n a l y s i s of the h y d r o l y s a t e by paper chromatography. Both amino a c i d s and amino sugars are d i s t i n g u i s h e d by the c o l o u r r e a c t i o n they 29 g i v e w i t h n i n h y d r i n a f t e r h e a t i n g ^ . The i d e n t i t i e s of the amino a c i d s may be confirmed u s i n g an automatic amino, a c i d analyser"^.* a f t e r v i g o r o u s h y d r o l y s i s of the p o l y s a c c h a r i d e to d e s t r o y the sugar r e s i d u e s . I I . 2 . 6 Determination of the Monosaccharide Sequence by S p e c i f i c P o l y s a c c h a r i d e Degradations The g e n e r a t i o n of s m a l l fragments or o l i g o s a c c h a -r i d e s i s the f i r s t s t e p i n e l u c i d a t i n g the sequence of the sugar r e s i d u e s i n a p o l y s a c c h a r i d e . A wide v a r i e t y of chemical techniques are a v a i l a b l e f o r the g e n e r a t i o n of o l i g o s a c c h a r i d e s , some of the more important methods are d i s c u s s e d i n t h i s s e c t i o n . Each technique w i l l y i e l d a d i f f e r e n t o l i g o s a c c h a r i d e , and by c a r e f u l s e l e c t i o n of a p p r o p r i a t e degradation techniques, the a c q u i s t i o n of two d i f f e r e n t but complementary o l i g o s a c c h a r i d e s may be s u f f i -c i e n t to g i v e the complete s t r u c t u r a l sequence. In a d d i t i o n the n.m.r. data from these o l i g o s a c c h a r i d e s w i l l a l l o w the assignment of the anomeric s i g n a l s and thus p r o v i d e c o n f i r m -a t i o n o f anomeric c o n f i g u r a t i o n s . Bacteriophage-mediated enzyme cleavage of a p o l y s a c c h a r i d e i s an a l t e r n a t i v e to chemical degradation and i s d i s c u s s e d i n more d e t a i l i n S e c t i o n II.3.2 30 II.2.6.1 P a r t i a l A c i d H y d r o l y s i s " 1 ' The method most wi d e l y used to determine sequence i s graded h y d r o l y s i s with a c i d , f o l l o w e d by s e p a r a t i o n and i d e n t i f i c a t i o n of the r e s u l t a n t o l i g o s a c c h a r i d e s . The main drawback of t h i s method i s the need f o r r e l a t i v e l y l a r g e amounts of m a t e r i a l , as fragments formed d u r i n g the h y d r o l y -s i s may be degraded s t i l l f u r t h e r as the r e a c t i o n proceeds; the y i e l d of the r e s u l t a n t o l i g o s a c c h a r i d e s i s u s u a l l y low. Furthermore, the r a t e s of h y d r o l y s i s of the v a r i o u s g l y c o -s i d i c l i n k a g e s may be only m a r g i n a l l y d i f f e r e n t and complex mixtures of o l i g o s a c c h a r i d e s are o b t a i n e d . There are how-ever, some g l y c o s i d i c l i n k a g e s which are r e l a t i v e l y r e s i s t a n t to a c i d h y d r o l y s i s and p o l y s a c c h a r i d e s which c o n t a i n such l i n k a g e s may fragment with a h i g h degree of s e l e c t i v i t y . G l y c o s i d u r o n i c a c i d s are much more r e s i s t a n t to a c i d h y d r o l y s i s than t h e i r c o r r e s p o n d i n g n e u t r a l g l y c o s i d e s . Graded a c i d h y d r o l y s i s of g l y c u r o n i c a c i d - c o n t a i n i n g p o l y -s a c c h a r i d e s w i l l u s u a l l y y i e l d the a c i d i c d i s a c c h a r i d e ( a l d o b i o u r o n i c a c i d ) and o f t e n h i g h e r a c i d i c o l i g o s a c c h a -r i d e s . 2-Amino-2-deoxyglycosides are a l s o r e s i s t a n t to a c i d h y d r o l y s i s . The i n d u c t i v e e f f e c t of the a l r e a d y protonated 2-amino group reduces the l i k e l i h o o d of f u r t h e r p r o t o n a t i o n o f the g l y c o s i d i c oxygen. Conversely, some g l y c o s i d i c l i n k a g e s are more s u s c e p t i b l e to a c i d h y d r o l y s i s . Weak g l y c o s i d i c l i n k a g e s are a s s o c i a t e d w i t h furanose and deoxy sugar r e s i d u e s . 6-Deoxyhexoses are 31 common i n b a c t e r i a l p o l y s a c c h a r i d e s but t h e i r g l y c o s i d i c l i n k a g e s are o n l y h y d r o l y s e d a t f i v e times the r a t e of the c o r r e s p o n d i n g hexoses. However, 3,6-dideoxyhexopyranosides 57 which occur i n some b a c t e r i a l p o l y s a c c h a r i d e s ^ are hydro-l y s e d more r e a d i l y . A c e t o l y s i s ^ i s complementary to a c i d h y d r o l y s i s as the r e l a t i v e r a t e s of cleavage of the g l y c o s i d e s i n the two r e a c t i o n s are sometimes r e v e r s e d . Whereas (1-6)-linkages are r e l a t i v e l y s t a b l e to a c i d h y d r o l y s i s , they are p r e f e r e n t -i a l l y s p l i t d u r i n g a c e t o l y s i s . Neuraminic a c i d ( s i a l i c a c i d ) l i n k a g e s which are very l a b i l e to a c i d h y d r o l y s i s and e a s i l y decomposed may remain i n t a c t d u r i n g a c e t o l y s i s and thus a l l o w the i s o l a t i o n of s i a l i c a c i d o l i g o m e r s ^ . II.2.6.2 / ^ - E l i m i n a t i o n 6 0 ' 6 1 G l y c o s i d i c l i n k a g e s are g e n e r a l l y s t a b l e to a l k a -l i n e c o n d i t i o n s . However, when a s u i t a b l e e l e c t r o n - w i t h -drawing group i s p r e s e n t , base c a t a l y s e d y i - e l i m i n a t i o n s may occur, w i t h concomitant cleavage of the g l y c o s i d i c l i n k a g e s . E a s e - c a t a l y s e d degradations from hexuronic a c i d s take p l a c e when the u r o n i c a c i d i s a) e s t e r i f i e d and b) s u b s t i t u t e d a t the 0-4 p o s i t i o n . During the y s - e l i m i n a t i o n the 0-4 s u b s t i -t u e n t s are e l i m i n a t e d with the formation of hex -4-enopyrano-s i d u r o n a t e r e s i d u e s . While g l y c o s i d u r o n i c a c i d l i n k a g e s are a c i d - r e s i s t a n t , the hex - 4-enopyranosiduronic a c i d l i n k a g e s are v e r y a c i d - l a b i l e and may be e a s i l y c l e a v e d 32 under m i l d c o n d i t i o n s . The " a g l y c o n i c " h y d r o x y l group which was f o r m a l l y s u b s t i t u t e d by the hexuronic a c i d i s exposed, but the other g l y c o s i d i c l i n k a g e s remain i n t a c t . L i n d b e r g and c o w o r k e r s 6 0 ' 6 1 developed the r e a c t i o n sequence most wid e l y used. The p o l y s a c c h a r i d e i s methylated a c c o r d i n g to the Hakomori procedure and re-treatment of the f u l l y methy-l a t e d p o l y s a c c h a r i d e with f r e s h base g i v e s s u b s t a n t i a l d e g r a d a t i o n . Although ya-elimination i s a u s e f u l method f o r determining the sugar and the p o s i t i o n to which the hexuronic a c i d i s a t t a c h e d , i t s u f f e r s from two disadvantages which to date have l i m i t e d i t s u s e f u l n e s s ; 1) s t r u c t u r a l i n f o r m a t i o n i s l o s t when f u r t h e r degradation of the r e d u c i n g sugars occurs and 2) there are few s a t i s f a c t o r y methods f o r separa-t i n g mixtures of p a r t i a l l y methylated o l i g o s a c c h a r i d e s (see S e c t i o n III.2). R e c e n t l y , however, g.c.-m.s. has been used to separate and i d e n t i f y o l i g o s a c c h a r i d e s and o l i g o s a c c h a -r i d e a l d i t o l s c o n t a i n i n g up to seven sugars . In the work presented here o l i g o s a c c h a r i d e a l d i t o l s c o n t a i n i n g f o u r sugars were s u c c e s s f u l l y separated and i d e n t i f i e d by g . c . - c . i . m.s. (see S e c t i o n IV.3). II.2.6.3 P e r i o d a t e O x i d a t i o n and Smith H y d r o l y s i s 6 ^ " 6 ^ The p e r i o d a t e o x i d a t i o n of w a t e r - s o l u b l e carbo-h y d r a t e s i s an important a n a l y t i c a l technique. Sodium meta-p e r i o d a t e i s used to c l e a v e those sugars which c o n t a i n c i s g l y c o l s , the r e s u l t a n t "polyaldehyde" i s then reduced with 3 3 sodium borohydride to give a "polyalcohol".(Fig. I I . 2 ) . Smith degradation i s the controlled acid hydrolysis of the oxidized and reduced polysaccharide, i n which the modified sugars containing a c y l i c acetals are hydrolysed with d i l u t e acid at room temperature, leaving the remaining g l y c o s i d i c linkages i n t a c t . The r e s u l t of t h i s reaction sequence may be, depending on the r e l a t i v e positions of periodate-r e s i s t a n t sugars, oligosaccharides or low molecular weight products consisting of sugar residues g l y c o s i d i c a l l y linked to fragments such as g l y c e r o l or a t e t r i t o l (see F i g . I I . 2 ) . Characterization of the degradation products gives consider-able s t r u c t u r a l information and the Smith degradation i s frequently used i n s t r u c t u r a l polysaccharide chemistry. However, the success of the Smith degradation depends on ensuring a l l cleavable d i o l and t r i o l groups have been oxidized and that the hydrolysis step i s completely s e l e c t i v e . The periodate oxidation step may be monitered spectrophoto-66 m e t r i c a l l y , but i t i s le s s simple to ascertain that there i s complete cleavage of the a c y c l i c acetals but no detectable hydrolysis of the g l y c o s i d i c linkages. Dutton and G i b n e y ^ developed a method i n which the hydrolysis step was monitored by g.c. of the t r i m e t h y s i l y l derivatives of the products. I I . 2 . 6 . 4 Lithium/Ethylenediamine Degradation^ 0* 5 2 Lithium metal dissolved i n ethylenediamine was demonstrated to cleave 3 - l i n k e d glycosyluronic acid-contain-34 CH 2OH CH 2OH CH 2OH CH 2OH O . J O _ J O J O HO OH HO OH OH ( i ) NalO^ ( i i ) NaBH. OH 0.5M TFA, 16 h HO F i g . II.2 Periodate oxidation and Smith degradation of a hypothetical polysaccharide. 35 ing polysaccharides "by Mort and Bauer^ . Subsequently i t 32 was demonstrated by Albersheim and coworkers that t r e a t -ment of carbohydrates with lithium cleaves the glycosyluronic acid residues regardless of the i r positions of substitution by other glycosyl residues. Lithium dissolved i n an amine solvent i s a powerful SO reducing agent. The Birch reduction and.reduction of aldehydes, ketones and carboxylic acids, and conjugated a l k e n e s ^ " ^ 5 are a l l examples of the applications of t h i s reagent. Neutral glycosyl residues are stable i n t h i s reaction but any O-methyl substituents or pyruvate acetals w i l l be removed. In fact, Mort and Bauer 5 0 were attempting to remove an _0-methyl substituent from a polysaccharide when they discovered that lithium i n ethylenediamine degrades glycosyluronic acid residues. The lithium reaction i s s i m i l a r to a p-elimination i n that the polysaccharide i s cleaved at the glycosyluronic acid residues. Such s p e c i f i c cleavages at selected sugar residues make the subsequent tasks of separation and charac-t e r i z a t i o n of the fragment oligosaccharides far easier. The lithium degradation i s p a r t i c u l a r l y valuable because i t cleaves underivatized polysaccharides thus allowing the products to be used for both s t r u c t u r a l analysis and studies on t h e i r b i o l o g i c a l a c t i v i t y . 36 II.2.6.5 Anhydrous Hydrogen F l u o r i d e H y d r o l y s i s Since 1965 l i q u i d HF has been used f o r the f i n a l d e p r o t o n a t i o n s t e p i n peptide and p r o t e i n s y n t h e s i s . In 1977 Mort and Lamport 5* 7 found t h a t u s i n g HF, there was a l a r g e enough v a r i a t i o n i n the r a t e s of g l y c o s i d i c bond cleavage of amino and n e u t r a l sugars a t 0°, t h a t amino sugar l i n k a g e s remained i n t a c t while those of n e u t r a l sugars were broken. More r e c e n t l y M o r t ^ and Mort and B a u e r 5 0 d i s c o v e r e d that d i f f e r e n t i a l cleavage of n e u t r a l and a c i d i c sugars' c o u l d be obtained u s i n g HF a t below zero temperatures. Furthermore, under m i l d c o n d i t i o n s primary and secondary e s t e r s are s t a b l e i n l i q u i d HF but under harsher c o n d i t i o n s (e.g. s e v e r a l hours at room temperature) a c y l groups may migrate or be l o s t completely'' 7^. Kuo and M o r t ^ u s i n g extremely m i l d c o n d i t i o n s (-40°) to c l e a v e g e l l a n gum, found not o n l y was i t p o s s i b l e to o b t a i n p r e f e r e n t i a l c l e a v -age of ©cover p l i n k a g e s i n HF, but a t e t r a s a c c h a r i d e thus produced had the l a b i l e s u b s t i t u e n t s a c e t a t e and g l y c e r a t e s t i l l a t t a c h e d . P r e v i o u s s t r u c t u r a l s t u d i e s ' ^ undertaken on the p o l y s a c c h a r i d e had r e p o r t e d o n l y the presence of an a c e t y l group s i t u a t e d on approximately one i n three of the r e p e a t i n g t e t r a s a c c h a r i d e u n i t s . H e r e i n l i e s one of the main advantages of HF h y d r o l y s i s - i t i s p o s s i b l e to produce o l i g o s a c c h a r i d e s with a c i d or base l a b i l e s u b s t i t u e n t s s t i l l i n p l a c e , so t h e i r p o i n t of attachment to the p o l y s a c c h a r i d e may be determined. Bacteriophage-mediated enzymic cleavage 37 i s another depolymerization technique with the same capa-b i l i t y . Unfortunately t h i s technique, while producing the repeating unit of a polysaccharide i s tedious, time con-suming, yie l d s are often poor a n d v i t i s not always possible to i s o l a t e a bacteriophage capable of cleaving a given polysaccharide. II.2.6.6 Reductive Cleavage In addition to i t s advantages over the conventional methylation analysis, reductive cleavage possesses another important property, i . e . s e l e c t i v i t y . Total reductive cleavage i s achieved using trimethylsilyltrifluoromethane sulphonate (TMSOTf) as a c a t a l y s t , whereas selective reductive cleavage i s possible with boron t r i f l u o r i d e etherate ( E F ^ E t 2 0 ). For example the gl y c o s i d i c bond between 6-linked glycopyranosyl residues i s r e l a t i v e l y stable to reductive cleavage using B F ^ E t 2 0 . This was demonstrated by Gray and coworkers 2^ during studies on per-methylated pullulan - a l i n e a r polysaccharide consisting of a trisaccharide repeating unit containing one cx-l,6-linked and two c<-l,^-linked £-glucopyranosyl residues. With. TMSOTf as catalyst a l l linkages were cleaved, but with B F ^ E t 2 0 as c a t a l y s t , under the same reaction conditions, the products were the If-Q-acetyl-1 , 5-anhydro-D-glucitol ( l ) and the disaccharide anhydroalditol derivative ( 3 ) . B F ^ E t 2 0 was also i n e f f e c t i v e i n catalysing the reductive cleavage 38 of permethylated l a m i n a r i n ^ ? , a B-1 , 3 - l i n k e d D-glucan branched at some 0 - 6 p o s i t i o n s . R e i n h o l d and c o w o r k e r s ^ i n c o r p o r a t e d the advantages of the r e d u c t i v e cleavage method i n t o a sequencing s t r a t e g y f o r permethylated p o l y s a c c h a r i d e s . They a l t e r e d the p u b l i s h e d r e a c t i o n c o n d i t i o n s ^ to generated mixtures of o l i g o s a c c h a r i d e s e.g. p - c y c l o d e x t r i n was s u b j e c t e d to p a r t i a l r e d u c t i v e cleavage. On s e p a r a t i o n of the products by h . p . l . c , peaks were observed corresponding to the mono-mer through to the open-chain heptamer. C o n s i d e r a b l e v a r i a -t i o n s i n g l y c o s i d i c bond s t a b i l t y were a l s o observed by R e i n h o l d and coworkers''''7. For example o c d - ^ ) l i n k a g e s were almost completely c l e a v e d while ;5(l-2) l i n k a g e s remained e s s e n t i a l y i n t a c t . Such s e l e c t i v i t y i n r e d u c t i v e cleavage suggests t h a t the method w i l l become of i n c r e a s i n g importance i n s t r u c t u r a l a n a l y s i s . CH 2OMe MeO 39 I I . 3 Enzymatic A n a l y s i s of Capsular A ntigens C l a s s i c a l chemical techniques of s t r u c t u r a l a n a l y s i s , f o r example, p a r t i a l a c i d h y d r o l y s i s and Smith degradation, have been supplemented to an e v e r - i n c r e a s i n g degree by enzymatic methods, the two types of a n a l y s i s u s e f u l l y comple-menting each o t h e r . Enzymatic d e p o l y m e r i z a t i o n of carbo-hydrate polymers occurs i n e i t h e r an endo or exo f a s h i o n . Enzymes which degrade p o l y s a c c h a r i d e s i n an exo manner can be f u r t h e r s u b - d i v i d e d i n t o g l y c o s i d a s e s and exoglycanases. Eoth these enzymes r e l e a s e sugar r e s i d u e s from the non-r e d u c i n g end of t h e i r s u b s t r a t e s , and i f these c o n t a i n r e p e a t i n g u n i t s then r e l e a s e i s s e q u e n t i a l . II.3.1 Exo Enzymes Exoglycanases and g l y c o s i d a s e s have d i f f e r e n t sub-s t r a t e requirements, but they are most e a s i l y d i s t i n g u i s h e d by t h e i r r e l a t i v e h y d r o l y t i c r a t e s with o l i g o s a c c h a r i d e s of i n c r e a s i n g degree of p o l y m e r i z a t i o n (DP). For exoglycanases, Ymax i n c r e a s e s and Km decreases as DP i n c r e a s e s from f o u r to s i x . Although only the t e r m i n a l u n i t i s r e l e a s e d , f o r e f f e c t i v e h y d r o l y s i s the exoglycanase needs fo u r to s i x u n i t s f o r r e c o g n i t i o n and b i n d i n g . H y d r o l y s i s u s u a l l y occurs with i n v e r s i o n of c o n f i g u r a t i o n a t the C-1 hydroxyl 50 e.g. t h i s takes p l a c e with exo-1,3 -B-D-glucanase^ which has been i s o l a t e d from a v a r i e t y of microorganisms, p a r t i c u -40 l a r y yeasts. Glycosidases recognize the non-reducing terminal sugar, whose glycosi d i c bond i s cleaved. Km and Vmax do not vary s i g n i f i c a n t l y with increasing DP i n a homologous series of oligosaccharides. Configuration i s normally retained at the C-1 hydroxyl, and unlike exoglycanases, glycosidases generally are less s p e c i f i c i n the i r hydrolysis require-ments. Many glycosidases hydrolyse dimers linked (1-2), (1-3), (1-4), and (1-6), although the r e l a t i v e rates d i f f e r . However, a l l glycosidases are completely s p e c i f i c for the 79 anomeric configuration and one enantiomer . In s t r u c t u r a l analysis of b a c t e r i a l capsular polysaccharides, glycosidases are commonly used to remove the non-reducing terminal sugar from oligosaccharides obtained by conventional chemical degradation. The i d e n t i t y of the new terminal residue may then be established by methylation analysis. I f the oligo-saccharide consists of two or three sugars of the appropri-ate type and configuation, these may be removed sequentially using p a r t i a l enzyme hydrolysis to give important information on anomeric configurations and sugar sequence. This approach 8 0 was used by Amemura and coworkers , who successively re-moved two non-reducing ^-glucose residues from the side chain of an oligosaccharide obtained from the e x t r a c e l l u l a r polysaccharide of Rhizobium t r i f o l i i 4S. 41 II.3.2 Endo Enzymes In r e c e n t years a n o v e l technique has been developed i n our l a b o r a t o r y and o t h e r s , u t i l i z i n g b a c t e r i o p h a g e -borne endoglycanases to depolymerize c a p s u l a r p o l y s a c c h a -r i d e s . V i r u s - a s s o c i a t e d enzymes may be c l a s s i f i e d a c c o rd-i n g to the type of r e a c t i o n they c a t a l y z e and the genus of the b a c t e r i a l host: 1) The h y d r o l y a s e s - these are e i t h e r glycanases ( g l y c o -81 —83 s i d e h y d r o l a s e s ) d e p o l y m e r i z i n g e x o p o l y s a c c h a r i d e s ^ or the O - s p e c i f i c s i d e chains of c e l l w a l l l i p o p o l y s a c c h a r i d e s , or they are " d e a c e t y l a s e s " which remove a c e t y l s u b s t i t u e n t s 85 from the b a c t e r i a l s u r f a c e p o l y s a c c h a r i d e s . 2) The l y a s e s - these are l e s s common but a few have been o r an found f o r K l e b s i e l l a c a p s u l a r p o l y s a c c h a r i d e s The enzymes (carbon-oxygen l y a s e s ) produce o l i g o s a c c h a r i d e s with non-reducing t e r m i n a l unsaturated g l y c u r o n i c a c i d r e s i d u e s . II.3.2.1 Nature and Mode of P e n e t r a t i o n of E. c o l i Capsule Bacteriophage (K phages) A t the b e g i n n i n g of an i n f e c t i o n a bacteriophage (f6), capable of a t t a c k i n g an encapsulated bacterium, i s presented w i t h a g l y c o c a l i x surrounding the host c e l l . T h i s exopoly-s a c c h a r i d e capsule may be as much as ten times the diameter of the i n f e c t i n g v i r u s , thus such bacteriophage c a r r y exo-p o l y s a c c h a r i d e - d e g r a d i n g enzymes. Such v i r u s e s are not adsorbed to s i m i l a r s t r a i n s with s e r o l o g i c a l l y and c h e m i c a l l y 42 d i f f e r e n t c a p s u l e s . B a c t e r i o p h a g e s have been c l a s s i f i e d by B r a d l e y i n t o s i x m o r p h o l o g i c a l types (see F i g . I I . 3 ) . S t i r m and Freund-M o l b e r t ^ found most K phages belong to B r a d l e y group C c a r r y i n g " s p i k e s " d i r e c t l y l i n k e d to the head or o c c a s i o n -a l l y through a v i s i b l e base p l a t e (see F i g . I I . 4 ) . L a t e r s t u d i e s by S t i r m and coworkers^ 0'^ 1 demonstrated t h a t the glycanase enzymes o f t e n take the form of t a i l - s p i k e s on the b a c t e r i o p h a g e . Bayer and coworkers 7 have shown t h a t the v i r i o n p e n e t r a t e s the capsule by d e s t r u c t i o n of i t s p o l y s a c c h a r i d e r e c e p t o r s . I t forms a tunnel-shaped path i n i t s descent ( F i g . I I . 5 a and 5b) to the outer membrane, where i t may d i f f u s e l a t e r a l l y u n t i l i t i s p o s i t i o n e d over the adhesion s i t e s , a t which i n n e r and outer membranes are f u s e d . In the f i n a l s teps of the a d s o r p t i o n process the v i r u s comes i n t o c o n t a c t with the adhesion s i t e and i t s DNA ( o r RNA) i s r e l e a s e d i n t o the host c e l l . Once i n s i d e r e p l i c a t i o n of the phage n u c l e i c a c i d and phage p r o t e i n s takes p l a c e a t the expense of the metabolic processes of the host c e l l . The f i n a l s t e p i n the i n f e c t i o n process i s the packaging of v i r a l DNA i n t o the p r o t e i n coat of the v i r a l head, and r e l e a s e of the phage progeny by l y s i s of the host c e l l . I I . 3 - 2 . 2 I s o l a t i o n and S e l e c t i o n of Bacteriophage Bacteriophage are found i n the n a t u r a l h a b i t a t W5 B E 2-DNA 2-DNA 2-DNA 1-DNA 1-fiNA 1-DNA F i g . I I . 3 B a s i c m o r p h o l o g i c a l types of bacteriophage with the types o f n u c l e i c a c i d . of Wi tnclMtd ty • prot*tB F i g . I I . 4 Diagram of the Coliphage T2 V i r i o n F i g . II.5a At temperatures of 0 to k° the path of the v i r i o n i s preserv ed92. F i g . II.5b Phage K29 a f t e r penetration of the capsule at 37°. V i r u s p a r t i c l e s are found over the c e l l ' s outer membrane^2. 45 of t h e i r host bacteria i . e . i n the case of Enterobacteri-aceae. i n human of animal faeces or sewage. To i s o l a t e a s p e c i f i c bacteriophage large samples of sewage are mixed with concentrated nutrient broth and then with an a c t i v e l y growing culture of the host bacteria. A f t e r incubation a d i l u t i o n s e r i e s of the enrichment culture i s placed on b a c t e r i a l lawns of the host. I f an appropriate bacterio-phage i s present i t can be detected by i t s c h a r a c t e r i s t i c plaque morphology. The plaque proper of the virus (clear spot i n which the b a c t e r i a l c e l l s are lysed) may be surround-ed by a halo (translucent spot) i n which the bacteria have been decapsulated. The halo i s the r e s u l t of overproduction of free enzymatically active spikes i n addition to complete virus p a r t i c l e s . The /49 i s o l a t e d p reviously^ 5 to depoly-merize E. c o l i K49 capsular polysaccharide exhibited t h i s t y p i c a l plaque morphology (see Section V). Generally viruses are propagated i n l i q u i d culture. If o p t i c a l density measurements are used to follow c e l l density, the host cultures can be infected at the appropriate time to ensure l y s i s of the host c e l l before the medium i s depleted. Using t h i s method i t i s possible to obtain v i r a l suspensions with 1 0 ^ to 1 0 ^ plaque forming units (p.f.u.) per mL. The b a c t e r i a l debris i s removed by low speed centrifugation and the crude v i r a l suspension may be used d i r e c t l y for polysaccharide depolymerization , or the bacteriophage may be precipitated with ethylene g l y c o l , c o l l e c t e d by low speed centrifugation and p u r i f i e d by i s o -46 p y c n i c u l t r a c e n t r i f u g a t i o n through a C s C l d e n s i t y g r a d i e n t . Close to 1 0 1 ^ p . f . u . may be ob t a i n e d from 1 L of l y s a t e , s u f f i c i e n t to depolymerize l 'gm of c a p s u l a r p o l y s a c c h a r i d e . The phage suspensions can be s t o r e d a t 4 ° over c h l o r o f o r m f o r s e v e r a l months wi t h o u t l o s s of i n f e c t i v i t y or enzymatic a c t i v i t y . I I . 3 . 2 . 3 Enzymology of Endoglycanases O p Geyer and coworkers , i n an e x c e l l e n t r e v i e w on the c u r r e n t p o s i t i o n of p o l y s a c c h a r i d e d e g r a d a t i o n by b a c t e r i o p h a g e - a s s o c i a t e d enzymes, presented data on the enzymology of the b a c t e r i o p h a g e - c a t a l y s e d r e a c t i o n s . They concluded t h a t a l l the bacteriophage enzymes they had analysed so f a r showed no dependence on s p e c i f i c i o n s and r e q u i r e d an optimum pH of between 7 and 8 . K i n e t i c data o b t a i n e d w i t h K l e b s i e l l a g l y c a n a s e s showed t h a t M i c h a e l i s -Menten or Lineweaver-Burk p l o t s c o u l d be drawn, f o r the r e l a t i o n s h i p between s u b s t r a t e c o n c e n t r a t i o n and v e l o c i t y , w i t h complete bacteriophage as c a t a l y s t s . The r e a c t i o n v/as a l s o i n h i b i t e d by i t s p r o d u c t s . S u b s t r a t e s p e c i f i c i t y was s t u d i e d by Rieger-Hug and S t i r m ^ . A p p r o x i m a t e l y 5 0 glycanase cleavage s i t e s from K l e b s i e l l a c a p s u l a r p o l y s a c c h a r i d e s were i d e n t i f i e d and the r e s u l t s were summarized as f o l l o w s : 1 ) In most cases, the p o l y s a c c h a r i d e c h a i n s were c l e a v e d on e i t h e r s i d e of the n e g a t i v e charge ( g l y c u r o n i c a c i d or k7 pyruvate a c e t a l ) , but reducing glycuronic acids were not produced. 2) Often, the reducing end sugars were substituted at position 3» 3) In the majority of cases ^ - g l y c o s i d i c linkages were hydrolysed. 4) Where one polysaccharide was acted upon by several phage enzymes, more often than not, the same bond was cleaved by the d i f f e r e n t agents. II.3.2.4 Applications Much of the current i n t e r e s t i n capsular poly-saccharide-specific bacteriophages and the i r respective glycanases a r i s e s from th e i r usefulness or pote n t i a l use-fulness i n : 1) I d e n t i f i c a t i o n of capsular polysaccharide types when antisera are not available^**. 2) Degradation of capsular polysaccharide to produce ol i g o -saccharide repeating units with a c i d - or base-labile sub-sti t u e n t s i n t a c t , i n order to v e r i f y the structure of the 95 o r i g i n a l polysaccharide^ . 3) Production of substrates for n.m.r. analysis - better resolved spectra are obtained with oligosaccharides than 96 with polysaccharides • 4) Production of oligosaccharides for synthesis of protein-op coupled vaccines , and as p o t e n t i a l l y s p e c i f i c a n t i b a c t e r i a l 48 agents a g a i n s t p l a n t and animal pathogens • II.3.2.5 C o n c l u s i o n s Although there are some obvious advantages to be gained from the use of bacteriophage-enzyme degradation there are a l s o drawbacks a s s o c i a t e d with t h i s technique, e s p e c i a l l y when employed with E. c o l i c a p s u l a r p o l y s a c c h a -r i d e s . F or K l e b s i e l l a c a p s u l a r p o l y s a c c h a r i d e s , good y i e l d s of pure products were obtained with crude phage suspensions whereas, i n t h i s l a b o r a t o r y y i e l d s of products from E. c o l i c a p s u l a r p o l y s a c c h a r i d e s were found to be lower. Furthermore the products were contaminated w i t h an i m p u r i t y 7 " 5 s e p a r a t i o n of which proved d i f f i c u l t . T h i s problem was overcome by p u r i f i c a t i o n of the v i r a l oo suspension''-' but t h i s lengthens an a l r e a d y time consuming p r o c e s s . HF h y d r o l y s i s , though not q u i t e as s e l e c t i v e as phage degradation can achieve v e r y s i m i l a r r e s u l t s (see S e c t i o n II.2.6.5) i n a f r a c t i o n of the time. In the s t r u c t u r a l a n a l y s i s of a c a p s u l a r p o l y s a c c h a r i d e , a HF h y d r o l y s i s may y i e l d a product or products with l a b i l e sub s t i t u e n t s i n t a c t , thus a l l o w i n g the sugar sequence and p o s i t i o n of s u b s t i t u e n t s to be e s t a b l i s h e d , . e l i m i n a t i n g the n e c e s s i t y of und e r t a k i n g a l e n g t h y bacteriophage d e g r a d a t i o n . However, any degr a d a t i o n technique, whether chemical or enzymatic, w i l l r e s u l t i n a product or products which r e q u i r e s e p a r a t i o n , p u r i f i c a t i o n and c h a r a c t e r i z a t i o n . 49 Techniques which are commonly employed to achieve these ends are discussed i n the following section. II.4 Instrumental Analysis Throughout the s t r u c t u r a l elucidation of a capsular polysaccharide instrumental techniques are used to charac-te r i z e and/or separate the products of chemical degrada-tions. The choice of separation technique i s dependent on the size of the product, i t s a c i d i t y or b a s i c i t y , and whether i t i s derivatised or not. II.4.1 Separation Techniques The separation and p u r i f i c a t i o n techniques used i n t h i s study were d i a l y s i s , paper chromatography^ , gel 101 25 permeation chromatography ' J and gas l i q u i d chromato-g r a p h y 5 5 ' 1 0 2 . As the theory and applications of the f i r s t three techniques are well documented they w i l l not be d i s -cussed here. High pressure l i q u i d chromatography i s probably one of the most useful separation techniques but has not been u t i l i z e d i n t h i s work. There are however many valuable 105-105 books and a r t i c l e s on t h i s subject . G.c. i s d i s -cussed i n d e t a i l as a large proportion of the r e s u l t s presented i n t h i s thesis were obtained through analysis of oligosaccharides by g.c.-m.s. (see Sections I I I . 3 , IV.3 and V I . 3 ) . 50 I T . 4 . 1 . 1 Gas Liquid Chromatography 5 5'^ 0 2 The separation, quantitation and preliminary i d e n t i f i c a t i o n of the products of the various chemical or enzymatic degradations of polysaccharides, whether these are monosaccharides or oligosaccharides may be achieved by gas l i q u i d chromatography. Most carbohydrates are not s u f f i c i e n t -l y v o l a t i l e to be used for g.c. and therefore are converted to v o l a t i l e derivatives before analysis. In. a c l a s s i c a l paper published i n 1963 Sweeley and 1 06 coworkers revolutionized the separation of carbohydrate mixtures by demonstrating that the t r i m e t h y l s i l y l deriva-tive of carbohydrates are r e a d i l y formed and are v o l a t i l e . A disadvantage of t r i m e t h y l s i l y l a t i o n i s the appearance of multiple peaks i n the chromatogram due to the isomeric forms of a reducing sugar present at equilibrium i.e.<=><and anomers of the furanosides and pyranosides. This m u l t i p l i c i t y of peaks may be a useful diagnostic tool but may also unnecessarily complicate the chromatogram. To eliminate t h i s problem a monosaccharide may be converted to 1 06 i t s oxime or i t s a l d i t o l and then acetylated to form'a v o l a t i l e compound. Jones and colleagues separated a wide range of a l d i t o l acetates, but were not successful i n separa-ing D - g l u c i t o l and D - g a l a c t i t o l 1 ^ ' 1 °^. I t v/as not u n t i l the development of new l i q u i d phases that the separation of a l d i t o l acetates became the most widely used method for analysing carbohydrate mixtures. Another important advance 51 was made by L i n d b e r g and coworkers 7 who showed that many methylated compounds could a l s o be separated as t h e i r a l d i t o l a c e t a t e s and, u n l i k e many other methods used i n s t r u c t u r a l p o l y s a c c h a r i d e chemistry, m e t h y l a t i o n a n a l y s i s 110 p r o v i d e s q u a n t i t a t i v e i n f o r m a t i o n The s e p a r a t i o n of o l i g o s a c c h a r i d e s i s b a s i c l y the same as the s e p a r a t i o n of monosaccharides. O l i g o s a c c h a r i d e s have 106 been an a l y s e d as t h e i r t r i m e t h y l s i l y l d e r i v a t i v e s and a l s o as t h e i r t r i m e t h y l s i l y l o l i g o s a c c h a r i d e a l d i t o l s 1 1 1 . The lower v o l a t i l i t y of carbohydrate a c e t a t e s has prevented many o l i g o s a c c h a r i d e s from being separated i n t h i s form. O l i g o s a c c h a r i d e s are separated more s u c c e s s -f u l l y as t h e i r more v o l a t i l e methylated d e r i v a t i v e s e s p e c i -a l l y those of higher molecular weights (see S e c t i o n I I I ) . E a r l i e r a n a l y s i s of carbohydrate mixtures by g.c. used packed columns and with the advent of a new phase, E C N S S M (a n i t r i l e s i l i c o n e - p o l y e s t e r copolymer) s u c c e s s f u l separa-1 1 2 t i o n of a l d i t o l a c e t a t e s and methylated a l d i t o l a c e t a t e s 1 b e c a m e f e a s i b l e . The i n t r o d u c t i o n of the open t u b u l a r columns gave a dramatic i n c r e a s e i n s e p a r a t i o n over the c o n v e n t i o n a l packed columns. Open t u b u l a r columns are l e n g t h s of c a p i l l a r y t u b i n g which have been coated with a s t a t i o n a r y phase and d i f f e r from c o n v e n t i o n a l packed columns i n t h a t they have a hi g h e r s p e c i f i c gas p e r m e a b i l i t y and s m a l l amounts of s t a t i o n a r y phase i n a uniform f i l m ( 0 . 1 -0 . 4 / tm) which s i g n i f i c a n t l y c o n t r i b u t e s to column e f f i c i e n c y . Furthermore, the 'openness'of the column enables columns of 52 g r e a t e r l e n g t h s to be used,generating a l a r g e r number of t h e o r e t i c a l p l a t e s per column. The most noteworthy d i f f e r e n c e between packed and c a p i l l a r y columns i s the i n c r e a s e d s e n s i -t i v i t y w i t h open t u b u l a r columns, due i n p a r t to the over-a l l i n e r t n e s s p o s s i b l e with a g l a s s c a p i l l a r y system. The i n t r o d u c t i o n of the bonded phase fused s i l i c a columns r e p r e -sented a major breakthrough i n column t e c h n o l o g y 1 1 5 , now columns c o u l d be used up to temperatures of 3 5 0 ° , with none of the i n c r e a s e i n column a c t i v i t y with i n c r e a s i n g tempera-tu r e observed with some of the e a r l i e r s t a t i o n a r y phases. T h i s i s an important f a c t o r to be taken i n t o c o n s i d e r a t i o n when a n a l y s i n g l a r g e r m olecular weight o l i g o s a c c h a r i d e s which r e q u i r e h i g h e r temperatures f o r e l u t i o n from the column. In t h i s study bonded phase columns were used f o r analyses of carbohydrate m i x t u r e s . L i s t e d i n Table II.1 are the columns employed, the p o l a r i t y and composition of each column and the types of samples analysed by each column. In gas chromatography, the b a s i c process c o n s i s t s of a s o l u t e p a r t i t i o n i n g between two phases, the s t a t i o n a r y l i q u i d and the mobile gas phase. The e q u i l i b r i u m constant i s determined o n l y by the compound, the l i q u i d phase and the temperature and i s independent of column type. K D = kp k = p a r t i t i o n or c a p a c i t y r a t i o , p - phase r a t i o The t o t a l amount of time a s o l u t e spends on the column i s TABLE I I . 1 D e s c r i p t i o n of c a p i l l a r y columns used to analyse o l i g o -s a c c h a r i d e s . Column P o l a r i t y Composition Temperature Range O l i g o s a c c h a r i d e Analysed DB 1 DB 5 DB 17 DB 225 Non-polar 100% Dimethyl- - 6 0 ° to 325 / 3 5 0 ° p o l y s i l o x a n e Non-polar 95# Dimethyl-(5%) - 6 0 ° to 3 2 5 / 3 5 0 ° d i p h e n y l -p o l y s i l o x a n e Intermediate 100% Methyl-phenyl-poly-P o l a r s i l o x a n e 50% Cyanopropyl-methyl - (50%)-methylphenyl-p o l y s i l o x a n e 40° to 280 / 3 0 0 ° 40° to 2 2 0 / 2 4 0 ° T r i s a c c h a r i d e s T e t r a s a c c h a r i d e s D i s a c c h a r i d e s T r i s a c c h a r i d e s T e t r a s a c c h a r i d e s D i s a c c h a r i d e s T r i s a c c h a r i d e s D i s a c c h a r i d e s ?• J & W S c i e n t i f i c S t a t i o n a r y Phases; Durabond S e r i e s - c r o s s l i n k e d and bonded b See S e c t i o n I I I , Tables 1 , 2 , and 3 54 i t s r e t e n t i o n time. Since a l l s o l u t e s must spend the same amount of time i n the gas phase, k which i s a measure of how much time the compound spends i n the l i q u i d phase i s r e l a t e d to the r e t e n t i o n time. The phase r a t i o ^ i s the r a t i o between the gas volume i n the column and the volume of the s t a t i o n a r y phase. Open t u b u l a r columns are charac-t e r i z e d by r e l a t i v e l y l a r g e p v a l u e s and r e l a t i v e l y s m a l l k v a l u e s . Column temperature has a very marked e f f e c t on a n a l y s i s , k i s i n v e r s e l y p r o p o r t i o n a l to column tempera-t u r e , the lower the temperature the h i g h e r the c a p a c i t y r a t i o , i n d i c a t i n g t h a t the s o l u t e i s spending more time i n the l i q u i d phase, l e a d i n g to i n c r e a s e d r e t e n t i o n times. By c a r e f u l c h o i c e of a p p r o p r i a t e temperature programme, column type ( l e n g t h and p o l a r i t y ) , c a r r i e r gas (type and flow r a t e ) , i d e n t i f i c a t i o n of each compound i n a mixture i s p o s s i b l e on the b a s i s of r e t e n t i o n data. However, even wi t h the high r e s o l u t i o n p o s s i b l e today with c a p i l l a r y columns, o f t e n s e p a r a t i o n of d i f f e r e n t compounds i s not s u f f i c i e n t f o r unambiguous i d e n t i f i c a t i o n . Thus f o r complete c h a r a c t e r i z a t i o n of a compound, a combination of gas chroma-tography and mass spectrometry i s used. II.4 .2 Mass Spectrometry Mass spectrometry has t r a d i t i o n a l l y been an impor-t a n t t o o l f o r chemists, but w i t h the a v a i l a b i l i t y of combina-t i o n gas chromatography-mass spectrometry (g.c.-m.s.) 55 i n s t r u m e n t a t i o n t h i s technique has been s u c c e s s f u l l y used f o r i d e n t i f i c a t i o n of i n d i v i d u a l components i n complex mi x t u r e s . Carbohydrate chemists use g.c.-m.s. f o r r e s o l v -i n g and i d e n t i f y i n g monosaccharide mixtures generated d u r i n g l i n k a g e a n a l y s i s , e i t h e r as t h e i r as a l d i t o l a c e t a t e s or p a r t i a l l y methylated a l d i t o l a c e t a t e s . S e p a r a t i o n and c h a r a c t e r i z a t i o n of v o l a t i l e d e r i v a t i v e s of o l i g o s a c c h a r i d e s , 62 c o n s i s t i n g of up to seven sugars , are a l s o p o s s i b l e u s i n g g.c.-m.s. Mass spectrometry of monosaccharides.- The fragmentation of a l d i t o l a c e t a t e s i s simple and e a s i l y i n t e r p r e t e d . A l d i t o l a c e t a t e s do not g i v e a molecular i o n , but upon e l e c t r o n impact (M-CH3CO2) i s found i n low abundance. However, mass spectrometry does not d i f f e r e n t i a t e between the d i f f e r e n t s t e r e o i s o m e r s , hence the mass spectrum of D-glucitol„ hexa-a c e t a t e i s r e p r e s e n t a t i v e of a l l p e r a c e t y l a t e d h e x i t o l s (Fig.II. 6 a ) and r e l a t i v e r e t e n t i o n times are necessary f o r complete i d e n t i f i c a t i o n . The base peak i n the s p e c t r a of a l l a l d i t o l a c e t a t e s i s the a c e t y l i u m i o n m/z 43 ( C H ^ c J ^ O ) . Primary fragment i o n s are formed by the e l i m i n a t i o n of an acetoxy group or by cleavage of the a l d i t o l c h a i n . I n t r o -d u c t i o n of an acetamido f u n c t i o n enhances the r e l a t i v e abundance of i o n s formed by cleavage at t h i s g r o u p 1 1 1 1 ^ ( F i g . I I . 6 b ) . Secondary fragments are produced by the e l i m i n a t i o n of a c e t i c a c i d (m/z 60), ketene (m/z 42) , or a c e t i c anhydride (m/z 102). 56 Because of the w e l l - e s t a b l i s h e d behaviour of a l d i t o l a c e t a t e s upon e l e c t r o n impact, these d e r i v a t i v e s are w e l l s u i t e d f o r the i d e n t i f i c a t i o n of sugars. T e t r o s e s , pentoses, hexoses and heptoses g i v e c h a r a c t e r i s t i c s p e c t r a t h a t can be i n t e r p r e t e d 1 1 6 . E l e c t r o n - i m p a c t mass spectrometry (e.i.-m.s.) i s a l s o commonly used i n the a n a l y s i s of permethylated a l d i t o l a c e t a t e s . Once again a molecular i o n i s not observed, and the s m a l l d i f f e r e n c e s i n r e l a t i v e i n t e n s i t i e s of peaks f o r s t e r e o i s o m e r s with the same s u b s t i t u t i o n p a t t e r n , e.g. 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y l h e x i t o l s are i n s u f f i c i e n t f o r unambigu-ous i d e n t i f i c a t i o n . The main peaks i n the mass spectrum r e s u l t from fragment i o n s formed by primary f i s s o n between two adjacent carbons i n the c h a i n - f i s s i o n between two ad j a c e n t methoxylated groups being p r e f e r r e d over f i s s i o n between a methoxylated and an a c e t o x y l a t e d carbon, which i s i n t u r n p r e f e r r e d over f i s s i o n between two a c e t o x y l a t e d g r o u p s 1 1 * * ' 1 1 ^ . Secondary fragment i o n s are formed by s i n g l e or c o n s e c u t i v e l o s s of a c e t i c a c i d , ketene, methanol (m/z 32) or formaldehyde (m/z 30) . I t i s p o s s i b l e t h e r e f o r e , to determine the s u b s t i t u t i o n p a t t e r n by comparison of the mass spectrum of a p a r t i a l l y methylated a l d i t o l a c e t a t e with that of an a u t h e n t i c standard or by a n a l y s i n g i t a c c o r d i n g to the above p r i n c i p l e s ( F i g . I I . 6 c ) . When a methoxyl and an a c e t -amido group are a d j a c e n t , f i s s i o n between these two groups i s p r e f e r r e d ( F i g . II.6d). The s t r o n g e s t primary fragment i o n f o r a 2-amino-2-deoxyhexose i s at m/z 158 and the 57 CH~0Ac 1 2 f _ L . . XcOCH HCOAc HCOAc CH-.OAC _ 217 _ 189 - 1 6 1 (a) CH~0Ac 360 _ _ | _ \ HCNHAc AcOCH CH 20Ac (b ) CH 20Ac r-l i—t l 277 233 " ~ I - - 1 1 ? MeOCH RCOMe - - 7 i L 189 CH 20Ac (c) CH 20Ac HCNMeAc MeOCH HCOMe 202 HCOAc 1 CH 20Ac (d) F i g . I I . 6 Fragmentation pathways "of some a l d i t o l acetates and some p a r t i a l l y methylated a l d i t o l ace ta tes . 58 secondary fragment i o n at m/z 116, d e r i v e d from m/z 158 by 114,115 the l o s s of ketene . Reduction of the p a r t i a l l y methylated sugar with sodium b o r o d e u t e r i d e , w i l l r e s o l v e any a m b i g u i t i e s i n d e t e r m i n i n g which carbon atom i s C-1. Mass spectrometry of o l i g o s a c c h a r i d e s . - Mass spectrometry p r o v i d e s r a p i d i n f o r m a t i o n on the type and sequence of monosaccharides present i n an o l i g o s a c c h a r i d e . C o n v e n t i o n a l techniques e l e c t r o n impact ( e . i . ) and chemical i o n i z a t i o n ( c i . ) of samples i n t r o d u c e d by g . c or v i a d i r e c t probe i n l e t , r e q u i r e the o l i g o s a c c h a r i d e to be converted to a v o l a t i l e d e r i v a t i v e p r i o r to i o n i z a t i o n . However, those o l i g o s a c c h a r i d e s with more than twelve r e s i d u e s w i l l p robably have vapour pr e s s u r e s that are too low even i n a d e r i v a t i z e d form. S e v e r a l new methods, where d e s o r p t i o n of samples i n an i o n i z e d form d i r e c t l y from the condensed s t a t e takes p l a c e , have been employed to study u n d e r i v a t i z e d o l i g o s a c c h a r i d e s . Secondary i o n mass spectrometry ( s . i . m . s . ) , f a s t atom bombardment ( f . a . b . ) , plasma d e s o r p t i o n ( p . d . ) , f i e l d desorp-t i o n ( f . d . ) , electrohydrodynamic i o n i z a t i o n (e.h.m.s.) and thermal d e s o r p t i o n ( t . d . ) are s e v e r a l examples of the d e s o r p t i o n i o n i z a t i o n used to c h a r a c t e r i z e g l y c o c o n j u g a t e s and other b i o m o l e c u l e s of i n t e r m e d i a t e atomic weight. Only the techniques employed i n t h i s study w i l l be d i s c u s s e d here, however, f o r f u r t h e r r e f e r e n c e there are s e v e r a l r e c e n t reviews which d e s c r i b e i n d e t a i l methods of d e s o r p t i o n 59 i o n i z a t i o n and t h e i r a p p l i c a t i o n s . E l e c t r o n i o n i z a t i o n mass spectrometry.- Much of the s t r u c -t u r a l work i n v o l v i n g d i r e c t a n a l y s i s of o l i g o s a c c h a r i d e s has been c a r r i e d out u s i n g e.i.-m.s. of v o l a t i l e d e r i v a t i v e s . P e r m e t h y l a t i o n i s the p r e f e r r e d approach as methyl groups c o n t r i b u t e l e a s t to the molecular weight. The sequence of monosaccharide u n i t s of a permethylated o l i g o s a c c h a r i d e can be d i r e c t l y deduced from i t s e . i . mass spectrum. The most v a l u a b l e fragment i o n s (A - s e r i e s ) are formed by homolytic cleavage of g l y c o s i d i c bonds with the p o s i t i v e charge being r e t a i n e d on the r i n g oxygen. A fragment i o n at m/z 219 i n d i c a t e s t h a t the t e r m i n a l non-reducing sugar i s a hexose. O l i g o s a c c h a r i d e s with a t e r m i n a l deoxyhexose or N - a c e t y l -hexosamine have i n t e n s e i o n s a t m/z 189 and 2 6 0 r e s p e c t i v e l y . S i m i l a r l y , cleavage of an i n t e r n a l g l y c o s i d i c l i n k a g e w i l l g i v e m/z 423 f o r a t e r m i n a l non-reducing hexose d i s a c c h a r i d e ( F i g . I I . 7 ) . A second s e r i e s of i o n s r e s u l t i n g i n the homo-l y t i c cleavage on the other s i d e of the g l y c o s i d i c bond a l s o g i v e s sequence i n f o r m a t i o n . Permethylated a l d o b i o u r o n i c a c i d s f o l l o w fragmentation r o u t e s s i m i l a r to permethylated 121 n e u t r a l d i s a c c h a r i d e s . g i v i n g an i n t e n s e fragment i o n at m/z 201 r e s u l t i n g from the l o s s of methanol from the t e r m i n a l non-reducing hexuronic a c i d (m/z 233)• Very l i t t l e other i n f o r m a t i o n can be a c q u i r e d from the e . i . spectrum of per-methylated d e r i v a t i v e s . In some i n s t a n c e s the fragment i o n s may g i v e i n f o r m a t i o n on s p e c i f i c g l y c o s i d i c l i n k a g e s , these fragmentation processes have been d i s c u s s e d i n d e t a i l by 60 v a r i o u s a u t h o r s 1 2 2"" 1 2 Z f . Permethylated o l i g o s a c c h a r i d e s a l d i t o l s may be i n v e s t i -gated by combined g.c.-m.s. In c o n t r a s t to permethylated o l i g o s a c c h a r i d e s they g i v e s i n g l e peaks on g.c. and t h e i r mass s p e c t r a are s i m p l e r . The nature of the non-reducing sugar i s i d e n t i f i e d by A - s e r i e s fragments, and the l i n k a g e p o s i t i o n i d e n t i f i e d by i n s p e c t i o n of the fragments formed by cleavage of the a l d i t o l c h a i n (see mass spectrometry of mono-s a c c h a r i d e s ) . Sequence i n f o r m a t i o n obtained by e.i.-m.s. of permethylated o l i g o s a c c h a r i d e s has been used i n suppport of s t r u c t u r a l s t u d i e s on the c a p s u l a r p o l y s a c c h a r i d e s of K l e b s i e l l a K 9 1 2 5 and K i f 7 1 2 6 . Chemical i o n i z a t i o n mass spectrometry.- Chemical i o n i z a t i o n mass spectrometry g e n e r a l l y a f f o r d s h i g h e r i n t e n s i t i e s of molecular i o n s and fragment i o n s i n the h i g h mass range, as compared to e l e c t r o n impact mass spectrometry, due to i t s " s o f t e r " i o n i z a t i o n ( 8 - 2 0 eV) technique. Standard i n s t r u -mental procedures f o r c i . r e q u i r e the sample t o be i n t r o -duced i n t o the i o n chamber as a gas, t h e r e f o r e l i k e e . i . , c i . r e q u i r e s the formation of v o l a t i l e d e r i v a t i v e s t o a v o i d p y r o l y t i c decomposition d u r i n g v a p o r i z a t i o n . Most i n v e s t i g a t i o n s of the c i . mass s p e c t r a of h i g h e r molecular weight o l i g o s a c c h a r i d e s have used permethylated d e r i v a t i v e s ^ ' 1 2 ' ' 7 . The reagent gases employed are most f r e q u e n t l y methane, isobutane or ammonia which produce the co r r e s p o n d i n g r e a c t i n g s p e c i e s CH^ , C ^ H ^ , and NH^ . The 61 proton donating a b i l i t y of these gases decreases i n the above order and thus, fragment i o n s are abundant i n the methane and isobutane s p e c t r a of o l i g o s a c c h a r i d e s , whereas molecular weight r e l a t e d i o n s dominate the ammonia c . i . mass s p e c t r a . As i n e . i . , c.i.-m.s. y i e l d s fragment i o n s from cleavage a t e i t h e r s i d e of the g l y c o s i d i c bond. These cleavages are a l s o observed under f.a.b. i o n i z a t i o n , and n e u t r a l gas c o l l i s i o n s . R e i n h o l d and c o w o r k e r s 1 2 ^ i n t r o d u c e d butyl<X-D-gl u c o p y r a n o s i d e i n t o the mass spectrometer v i a gas chroma-tography, ammonia c . i . - i n d u c e d g l y c o s i d i c cleavage gave the pyranoxonium i o n and subsequent adduct formation with n e u t r a l ammonia gave ah even-mass i o n at m/z 236 (Scheme II.2). An a l t e r n a t i v e e x p l a n a t i o n f o r the formation of t h i s fragment i o n i s the d i r e c t a t t a c k of ammonia at C1 and e l i m i n a t i o n of the b u t y l group to g i v e an a l t e r n a t i v e i s o m e r i c s t r u c t u r e (Scheme II.2 pathway b ) . The use of as the reagent gas gave r e s u l t s which supported the c o v a l e n t n i t r o g e n 1 27 attachment '. R e i n h o l d and h i s c o l l e a g u e s concluded that s i n c e the g l y c o s i d i c cleavage r e p r e s e n t e d the only major fragmentation i n the c . i . mass spectrum of the b u t y l g l y c o -s i d e t h e i r r e s u l t s i n d i c a t e t h a t , under those experimental c o n d i t i o n s , ammonia c.i.-m.s. would be the most a p p p r o p r i a t e energy probe to o b t a i n a combination of molecular weight and sequence'inf ormation v/ith l a r g e r o l i g o s a c c h a r i d e s . T h i s c o n c l u s i o n had a l s o been reached independently by t h i s r e s e a r c h e r and was the m o t i v a t i o n f o r the work presented i n S e c t i o n I I I . Furthermore the use of combination g.c.-m.s. 62 Scheme II.2 Ammonia c i . - i n d u c e d g l y c o s i d i c cleavage of butyl <x-D-glucopyranoside MeO OMe F i g . II.7 Fragment ions formed by homolytic cleavage of g l y c o s i d i c bonds of a non-reducing disaccharide using g . c -X D . S . 63 g i v e s mass s p e c t r a uncontaminated with by-products from the m e t h y l a t i o n r e a c t i o n and thus are more e a s i l y i n t e r p r e t e d . F a s t atom bombardment mass spectrometry ( f . a . b . ) . - F.a.b.-m.s. l i k e c.i.-m.s. g i v e s molecular weight r e l a t e d i o n s and fragment i o n s which may be used i n sequence assignments. L i k e other d e s o r p t i o n i o n i z a t i o n techniques, i t has the added advantage t h a t i t may be used on n o n - d e r i v a t i z e d samples. However, u n d e r i v a t i z e d carbohydrates produce ambiguous f r a g -ment i o n s on f.a.b. a n a l y s i s , r e s u l t i n g from m u l t i p l e c l e a v -ages, and as a g e n e r a l r u l e unambiguous sequencing r e q u i r e s I pQ the study of d e r i v a t i v e s i The d e r i v a t i v e s normally used f o r f.a.b-m.s are the same as those used f o r e . i or c . i . -m.s., i . e . permethylated or p e r a c e t y l a t e d o l i g o s a c c h a r i d e s . A c e t y l a t i o n i s p r e f e r r e d as i t i s a " c l e a n e r " r e a c t i o n and s p e c t r a can be a c q u i r e d w i t h i n 30 min. of the s t a r t of the a c e t y l a t i o n . F.a.b.-m.s. i n v o l v e s the t r a n s f e r of k i n e t i c energy from a beam of h i g h l y e n e r g e t i c atoms such as argon or xenon, to a matrix such as g l y c e r o l , and then to the sample. T h i s technique i s c l o s e l y r e l a t e d to s.i.m.s. which uses a beam of i o n s r a t h e r than atoms but, i n c o n t r a s t to s.i.m.s. experiments, f.a.b.-mass s p e c t r a l a s t s e v e r a l minutes, and sometimes hours, i n s t e a d of seconds. Both p o s i t i v e and neg-a t i v e i o n s are produced and e i t h e r can be r e c o r d e d . P o s i -t i v e i o n s (M + H ) + or (M + c a t i o n ) + r e s u l t from p r o t o n a t i o n or c a t i o n i z a t i o n r e s p e c t i v e l y , while n e g a t i v e i o n s are pre-64 dominantly (M - H)"". The type of the pseudomolecular i o n i s determined by the composition of the matrix and the chemical nature of the sample. A l l f.a.b. s p e c t r a have abundant pseudomolecular i o n s f o r both the sample and matrix, but a l s o have a r e l a t i v e l y h igh l e v e l of chemical " n o i s e " r e s u l t i n g i n a s i g n a l a t every mass number up to the molecular i o n r e g i o n (see S e c t i o n IV.3) . Two other types of s i g n a l s are a l s o present - c l u s t e r i o n s which are m a t r i x d e r i v e d and fragment i o n s . Fragmentation pathways appear to be common to a l l c l a s s e s of p o l y s a c c h a r i d e s . D e l l , i n her review on f.a.b.-m.s. presented a scheme f o r each fragmentation pathway (see F i g . I I . 8 ) . The schemes are s p e c u l a t i v e however, as no r i g o r o u s s t u d i e s , u s i n g i s o t o p i c a l l y l a b e l l e d compounds, have been undertaken to confirm the cleavages and hydrogen s h i f t s . F.a.b.-m.s. i s a powerful technique f o r examining mix-t u r e s of carbohydrates but, u n l e s s the components have very d i f f e r e n t chemical s t r u c t u r e s , a l l w i l l g i ve the same mole-c u l a r i o n s and the r e l a t i v e abundances w i l l not n e c e s s a r i l y r e f l e c t the r e l a t i v e c o n c e n t r a t i o n s of the components. F.a.b.-m.s. i s the procedure of c h o i c e f o r c o n f i r m i n g the l o c a t i o n of O-acyl groups present i n an o l i g o s a c c h a r i d e . P e r a c e t y l a t i o n with t r i f l u o r o a c e t i c anhydride/CD^COOD ensures t h a t n a t u r a l 0 - a c e t y l groups w i l l be d i s t i n g u i s h e d from the i n t r o d u c e d a c e t y l f u n c t i o n s . The type and number of n a t i v e a c y l groups i s then determined by the d i f f e r e n c e between the Pathway A Description: glycosidic cleavage to form an oxonium ion; charge retained un nonreducing end; positive-ion mode only; often referred to as A,-type cleavage, because of similarity to one of the cleavages seen in electron impact-mass spectrometry. CH.OH -6s] CH.OH OH HO OH CH.OH O HO Pathway B Description: glycosidic cleavage with a hydrogen transfer; charge retained on reducing end; positive- and negative-ion modes; often referred to as 0-cleavage. ^ / w . + or - H 1 CH.OH HO OH Pathway C Description: glycosidic cleavage with a hydrogen transfer; charge retained on the nonreducing end; positive- and negative-ion modes. CH.OH CH.OH J-o J-°. HO OH H O H OH CH.OH f— c J V-OH + or - H HO OH Pathway D Description: ring cleavage; charge retained on reducing end; ions are 28 mass units heavier than those formed in Pathway B; positive- and negative-ion modes. CH.OH CH.OH rO J -O O ' V> X O HO OH HO OH CH,OH O / - q . A . HO OH + or - H Pathway E Description: ring cleavage; charge retained on nonreducing end; infrequent pathway in the positive-ion mode, but a major pathway in the negative mode, because a stable, enolate anion results from loss of the enolic hydrogen atom; ions are 42 mass units higher than those formed in Pathway C . CH.OH CH.OH OH HO OH ] CH.OH Fig. II.{ Fragmentation pathways of polysaccharides using f.a.b. m.s. ON 128 F i g . II.8 Fragmentation pathways of polysaccharides using f.a.b.-m.s. 66 c a l c u l a t e d m olecular weight and the a c t u a l molecular weight, obtained from the f.a.b.-m.s. The exact r e s i d u e , c a r r y i n g the a c y l s u b s t i t u e n t , may be determined by c a l c u l a t i o n of the mass of each sequence i o n . G.c.-m.s., e.i.-m.s., and c.i.-m.s. are today r e l a t i v e l y r o u t i n e a n a l y t i c a l procedures, but i n c r e a s i n g l y f.a.b.-m.s. i s being employed as a t o o l i n s t r u c t u r a l a n a l y s i s . At present c.i.-m.s. shows g r e a t e r s e n s i t i v i t y and b e t t e r o v e r a l l s i g n a l - t o - n o i s e , but improvements i n t h i s area are a n t i c i p a t e d i n f.a.b.-m.s. i n . t h e next few. y e a r s . 67 II.Zf.3 Nuclear Magnetic Resonance (n.m.r.) Spectroscopy N.m.r. spectroscopy has become an increasingly important to o l i n the characterization of polysaccharides and ol i g o -saccharides. I t i s non-destructive,i.e. the polymer has no need of modification or degradation and i s recovered i n t a c t . The most useful n.m.r parameters i n carbohydrate analysis are: chemical s h i f t s , integration, coupling con-stants, nuclear Overhauser enhancement (n.O.e.), and spin-l a t t i c e r e l a x t i o n times (T-]). Only those parameters which have been u t i l i z e d i n thi s study are discussed here. Information on the application of n.O.e. and s p i n - l a t t i c e relaxation times i n s t r u c t u r a l analysis i s avail a b l e i n some 1 pq i excellent reviews and a r t i c l e s published on these topics 7 ' -Chemical s h i f t s - The chemical s h i f t of a given nucleus represents the frequency at which i t resonates when placed i n an external magnetic f i e l d , but the actual magnetic f i e l d that a nucleus sees i s modified by the electron density surrounding i t . As chemical s h i f t s and coupling constants of sugars and t h e i r derivatives have been es s e n t i a l for s t r u c t u r a l analysis of poly-and oligo-saccharides, great emphasis has been placed on t h e i r assignments 1-^ 1' 1^ 2. A ' l i b r a r y ' of spectra for known compounds i s useful for r e f e r -ence when tackling a spectrum of an unknown carbohydrate, although each carbohydrate n.m.r. spectrum i s unique. The n.m.r spectrum of a polysaccharide can be divided 68 i n t o three ( H) or four ( ^ C) r e g i o n s . These are the high f i e l d r e g i o n , the r i n g proton/carbon r e g i o n , the anomeric r e g i o n and the c a r b o n y l r e g i o n ( 1 5 C ) . The methyl groups of pyruvate, a c e t a t e (N- and O-acyl groups), and 6-deoxyhexoses e.g. L-rhamnose resonate i n the high f i e l d r e g i o n - between 61.0 and 6*2.5 f o r the 1H-n.m.r. spectrum and 15-30 p.p.m. 1 X i n the vC-n.m.r. spectrum. Furthermore, the abs o l u t e s t e r e o c h e m i s t r y of the a c e t a l carbon of p y r u v i c a c i d can be assigne d from the chemical s h i f t of the methyl g r o u p 5 5 , and p a r t i a l d e p y r u v y l a t i o n of a p o l y s a c c h a r i d e induces a twinn-1 XX i n g e f f e c t which may provide u s e f u l s t r u c t u r a l i n f o r m a t i o n . The h i g h f i e l d r e g i o n may a l s o e x h i b i t s i g n a l s which i n d i c a t e 86 the presence of l e s s common sugars . The r i n g proton r e g i o n of the ^ H-n.m.r spectrum (63.0-54.0) i s l e s s w e l l r e s o l v e d and, t h e r e f o r e , r e l a t i v e l y l e s s use-f u l than the hig h f i e l d or anomeric r e g i o n . In c o n t r a s t , the r i n g r e g i o n of 1 5C-n.m.r. s p e c t r a i s b e t t e r r e s o l v e d (60-85 p.p.m.) and p r o v i d e s i n f o r m a t i o n on the l i n k a g e p o s i t i o n s of the sugar r e s i d u e s . The carbon of a primary a l c o h o l g i v e s a d i s t i n c t i v e s i g n a l between 60-62 p.p.m. i f n o n - l i n k e d . I f the sugar i s s u b s t i t u t e d a t the Q-6 p o s i t i o n then t h i s resonance i s s h i f t e d 7-10 p.p.m. do w n f i e l d . The presence of a carbon c a r r y i n g a N-acyl group e.g. an amino sugar can be determined by a s i g n a l i n the r e g i o n 50-55 p.p.m. A l s o i n t h i s r e g i o n o f the spectrum appear s i g n a l s from other com-ponents w i t h a C-N bond e.g. the cx-C of s e r i n e and a l a n i n e r e s o n a t e a t 59.7 and 53»7 p.p.m. r e s p e c t i v e l y . :S9 S i g n a l s due to secondary a l c o h o l s w i l l resonate a t 75-5 P.p.m. but on O - g l y c o s y l a t i o n and/or 0,-alkylation w i l l s h i f t d o w n f i e l d to 80^5 p.p.m. ( c<-effeet). Carbons ad j a c e n t to the s u b s t i t u t e d carbon are however s l i g h t l y s h i e l d e d ( 1 -2 p.p.m.,B - e f f e e t ) . These s h i f t s have been used to a s s i g n 1 5 C s i g n a l s o f o l i g o - and p o l y - s a c c h a r i d e s 1 5 0 . The anomeric r e g i o n of ^H-n.m.r. s p e c t r a (S4.3- 5.8) has been a r b i t a r i l y d i v i d e d a t 65.0 - s i g n a l s due to pyranose r e s i d u e s appearing downfield of i t are a s s i g n e d to cx-linkages ( e q u a t o r i a l p r o t o n s ) , and those appearing u p f i e l d are assigned to p-linkages ( a x i a l p r o t o n s ) . The d i v i s i o n f o r the anomeric r e g i o n of the 1 5 C spectrum (93-110 p.p.m.) i s a t 101 p.p.m., however, as decreased s h i e l d i n g of a proton i s accompanied by s h i e l d i n g of an a t t a c h e d 1 5 C nucleus, 1 5 C and ^ s h i f t s a re r e v e r s e d ! 5 0 . T h i s a r b i t a r y d i v i s i o n breaks down f o r sugars of the manno- c o n f i g u r a t i o n e.g. L-rhamnose where both (X and p sugars resonate, c l o s e to 65.0. F o r t u n a t e l y the measurement of —HI c o u P l i n S c onstants from the 1 5C-n.m.r. spectrum enables the anomeric c o n f i g u r a t i o n s to be a s s i g n e d (see l a t e r ) . As a g e n e r a l r u l e , the number of s i g n a l s i n the anomeric r e g i o n corresponds to the number of s i g n a l s i n the r e p e a t i n g u n i t of the p o l y s a c c h a r i d e , and t h e i r l i n k a g e s can be determined from combined measurements of chemical s h i f t s and c o u p l i n g c o n s t a n t s . R e c e n t l y , however, there have been cases of non-anomeric carbohydrate resonances appear-i n g i n the anomeric r e g i o n . Annison 1 5** r e p o r t e d t h a t H-2 of an a c e t y l a t e d rhamnose r e s i d u e was appearing a t S5.52 i n the • 70 1H-n.m.r. spectrum of E . c o l i K32 p o l y s a c c h a r i d e . Thus care must be taken when a s s i g n i n g the number of sugar r e s i d u e s i n the r e p e a t i n g u n i t of a p o l y s a c c h a r i d e from i t s n.m.r. spectrum, c o n f i r m a t o r y evidence must be obtained from chemical a n a l y s e s . Carbonyl groups resonate i n the extreme low f i e l d r e g i o n (170-180 p.p.m.) of the 1 5 C s p e c t r a . These resonances may a r i s e from a C=0 group of a u r o n i c a c i d , N- or 0 - a c y l group, p y r u v i c a c i d or amino a c i d . Changes i n pH w i l l a f f e c t the s p e c t r a of b a s i c and a c i d i c carbohydrates. Advantage of t h i s p r o p e r t y was taken when determining the p o s i t i o n of s u b s t i t u t i o n of a m i d i c a l l y - l i n k e d amino a c i d s i n E. c o l i KZf9 p o l y s a c c h a r i d e (see S e c t i o n VI.3). R e l a t i v e areas or i n t e g r a l s of i n d i v i d u a l s i g n a l s - The r e l a t i v e areas under each peak are d i r e c t l y r e l a t e d to the number of protons r e s o n a t i n g a t a p a r t i c u l a r frequency. Thus the number of sugar r e s i d u e s per r e p e a t i n g u n i t of a p o l y -s a c c h a r i d e can be estimated and the r e l a t i v e r a t i o s of c<-to j 5 - l i n k a g e s . E s t i m a t i o n s of the r a t i o s of deoxysugars and s u b s t i t u e n t s such as pyruvate per r e p e a t i n g u n i t may be o b t a i n e d by comparison of the i n t e g r a l of t h e i r methyl group 1 35 to t h a t of the anomeric protons . However, q u a n t i t a t i o n i s not r e l i a b l e from s i g n a l i n t e g r a t i o n i n a proton de-coupled 1 5 C spectrum as s a t u r a t i o n and n.O.e. e f f e c t s w i l l d i s t u r b s i g n a l i n t e n s i t i e s . N e v e r t h e l e s s , i t i s p o s s i b l e to compare i n t e g r a l s of 1 5 C s i g n a l s w i t h the same number of 71 a t t a c h e d hydrogens, and f o r some simple p o l y s a c c h a r i d e s the number of carbon s i g n a l s r e f l e c t s the number of sugar r e s i d u e s per r e p e a t i n g u n i t . The n.m.r s p e c t r a o f o l i g o s a c c h a r i d e s e x h i b i t two s i g n a l s f o r a r e d u c i n g sugar i . e . mutarotation r e s u l t s i n the presence of both the cx- and p-anomers. The degree of p o l y m e r i z a t i o n can be determined by comparison o f the number of anomeric s i g n a l s to those of the r e d u c i n g sugar. C o u p l i n g constants ( J value) - Coupling constants are the r e s u l t of s p i n - s p i n c o u p l i n g s between two or more n u c l e i p o s s e s s i n g magnetic moments. The magnitude of the s p l i t t i n g , measured i n Hz, i s the c o u p l i n g c o n s t a n t . The c o u p l i n g constant i s independent of the magnetic f i e l d but, as s p i n -s p i n i n t e r a c t i o n s are mediated through the bonding e l e c t r o n s , i t s magnitude i s dependent on the d i s t a n c e , the chemical bond type, the nature of the two n u c l e i and the bond angle between them. Three bond c o u p l i n g s are very i n f o r m a t i v e about the s p a c i a l r e l a t i o n s h i p between two n u c l e i . The 1 56 K a r p l u s - t y p e J angular dependence f o r three bond c o u p l i n g i n which t h e r e i s a minimum value a t 90° and maximum values at 0° and 180° i s w e l l e s t a b l i s h e d and a p p l i c a b l e to 1H- 1H, 1 5 C - 1 5 C and 1 H - 1 5 C three-bond c o u p l i n g s i n carbohydrates. In the p y r a n o s y l system the -^H ax'ial-H a x i a l (180°) and the 5 J H . e q u a t o r i a l - H . a x i a l or e q u a t o r i a l - e q u a t o r i a l (60°) have v a l u e s of approximately 7-8 Hz and 1-3 Hz r e p e c t i v e l y . J con s t a n t s o f anomeric protons t h e r e f o r e provide i n f o r m a t i o n 72 on the anomeric c o n f i g u a t i o n and r i n g s i z e v . 1 z s p e c t r a , although normally a c q u i r e d i n the decoupled mode, may a l s o be r e c o r d e d with "gated" c o u p l i n g or s i n g l e frequency o f f resonance d e c o u p l i n g to o b t a i n i n f o r m a t i o n on JC- H c o u p l i n g c o n s t a n t s . The -H1 c o u p l i n g shows a c l e a r dependence on the o r i e n t a t i o n of the s u b s t i t u e n t at C-l and t h e r e f o r e p r o v i d e s i n f o r m a t i o n on anomeric c o n f i g u r a -1 ^ 8 ^ 1 3 t i o n . An e q u a t o r i a l l y o r i e n t e d H-1 has a ^C1-H1 value of 169-171 Hz and an a x i a l l y o r i e n t e d H-1 has a somewhat lower J - v a l u e (158-162 Hz). In the l a s t few years a v a r i e t y of two-dimensional (2D) n.m.r. techniques has been used to e l u c i d a t e complex carbo-hydrate structures 139*140^ Homonuclear chemical s h i f t c o r r e l a t i o n (COSY) has a i d e d s i g n a l assignments i n 1H s p e c t r a , and ^ C assignments can be obtained from h e t e r o n u c l e a r chem-i c a l c o r r e l a t i o n experiments t h a t r e l a t e the proton spectrum to the carbon spectrum. In those cases i n which only p a r t i a l assignment of the proton spectrum can be made a two dimen-s i o n a l , h e t e r o n u c l e a r RELAY experiment can be used to i d e n t i f y a d j a c e n t , protonated ^ C n u c l e i . R e c e n t l y , a two-dimen-s i o n a l NOE experiment and COSY experiment were used to d e t e r -mine o l i g o s a c c h a r i d e i n t e r g l y c o s i d i c l i n k a g e s . Bundle and c o w o r k e r s 1 ^ used c o n v e n t i o n a l 1H- and ^C-n.m.r data t o -gether w i t h 2D n.m.r experiments to a s s i g n the s t r u c t u r e of the 0 a n t i g e n from S a l m o n e l l a landau. T h e i r c o n c l u s i o n s were however t h a t w i t h e l a b o r a t e n.m.r. experiments i t i s p o s s i b l e 73 to e s t a b l i s h the s t r u c t u r e " of complex p o l y s a c c h a r i d e s , but the most e f f e c t i v e approach was a combination of c l a s s i c a l methods wit h high r e s o l u t i o n n.m.r. a t hig h f i e l d s t r e n g t h . C o n v e n t i o n a l m e t h y l a t i o n a n a l y s i s i s s t i l l the most e f f e c t i v e means of e s t a b l i s h i n g l i n k a g e p o s i t i o n s and n.O.e. e x p e r i -ments may then be used on the p o l y s a c c h a r i d e or d e r i v e d o l i g o s a c c h a r i d e s to determine the sequence of sugar u n i t s . 74 BIBLIOGRAPHY 1. I. 0rskov, F. 0rskov, B. Jann and K. Jann, B a c t e r i o l . Rev., 41 U977) 667-710. 2. K. Jann and B. Jann, Prog. A l l e r g y , 33 (1983) 53-79. 3. K. Jann and B. Jann, Rev. I n f e c t . D i s . , 9, Su p p l . 5 (1987) S517-S526.and r e f . t h e r e i n . 4. P. Hofmann, B. Jann and K. Jann, Carbohydr. Res., 139 (1985) 261-271. 5. T. Dengler, B. Jann and K. Jann, Carbohydr. Res., 150 (1986) 233-240 6. M.E. Bayer and H. Thurow, J . B a c t e r i o l . , 130 (1977) 911-936. 7. D.T. Fearson and K.F. Austen, N. E n g l . J . Med., 303 (1980) No. 5, 259-263. 8. C T . Bi s h o p and H.J. Jenn i n g s , i n G.O. A s p i n a l l ( E d . ) , "The P o l y s a c c h a r i d e s " , V o l . 1, Academic P r e s s , New York, (1982) pp. 291-330. 9. E.A. Kabat, J . Immunol. 84 (1960) 8 2 - 8 5 . 10. C.P.J. Glaudemans, M o l e c u l a r Immun., 23, 8 (1986) 917-918. 11. C.E. B a l l o u , P.N. Lipk e and W.C. Raschke, J . B a c t e r i o l . , 117 (1974) 461-467. 12. W.C. Raschke and C.E. B a l l o u , B i o c h e m i s t r y , 11 (1972) 3807-3816. 13. W.F. Dudman and M. H e i d e l b e r g e r , S c i e n c e , 164 (1969) 954-955 14. W.C. Van D i j k , H.A. Verbrugh, M.E. van der T o l , R. P e t e r s and J . Verhoef, I n f e c t . Immun., 25, 2 (1979) 603-609. 15. A.S. Cross, K.S. Kim, D.G. Wright, J.C. Sado f f and P. Gemski. J . I n f . D i s . , 154 (1986) 497-503. 16. E . J . McGuire and S.B. B i n k l e y , B i o c h e m i s t r y , 3 (1964) 247-251. 17. K. Jann and B. Jann, Spec. P u b l . Soc. Gen. M i c r o b i o l . , V o l . 13, N o . ( V i r u l e n c e of E. c o l i ) . (1985) 157-176/ 75 18. A.K. Bhattacharjee, H.J. Jennings. C P . Kenny, A. Martin and.I.C.P. Smith, J . B i o l . Chem., 250 (1976) 1926-1932 19. Chi-Jen Lee, Molecular Immun., 24, No. 10 (1987) 1005-1019 and r e f . therein. 20. R. Schneerson, 0. Barrera, A. Sutton and J.B. Robbins, J . Exp. Med., 152 (1980) 361-376. 21. R. Schneerson, J.B. Robbins, J.C. Parke, C. B e l l , J . J . Schlesselman, A. Sutton, Z. Wang, 6. Schiffman, A. Karpas and J . Shiloach, Infect. Immun., 52 (1986) 519-528. 22. R. Schneerson and J.B. Robbins, New Engl. J . Med., 292 (1975) 1093-1095. 23. S.C. Churms, Adv. Carbohydr. Chem. Biochem., 25 (1970) 13-51. 24. H. Neukom and W. Kuendig, Methods Carbohydr. Chem., 5 (1965) 14-17. 25. S. Narasimhan, J.R. Wilson. E. Martin and H. Schachter, Can. J . Biochem., 57 (1979) 83-96. 26. K. Jann, Spec. Publ. Soc. Gen. Microbiol., V o l . 13 No.(Virulence of E. c o l i ) (1985) 375-379. 27. B. Lindberg, J . Lonngren, and S. Svensson, Adv. Carbohydr. Chem. Biochem., 31 (1975) 185-240. 28. G.O. A s p i n a l l , i n G.O. A s p i n a l l (Ed.), "The Polysaccha-r i d e s " , Vol.1, Academic Press, New York, (1982) 36-131. 29. G.R. Gray, Methods Enzymol., (1987) 138, No.(Complex Carbohydr., PT.E), 26-38 and r e f . therein. 30. A.J. Mort and W.D. Bauer, J . B i o l . Chem., 257 (1982) No.4, 1870-1875. 31. M.P. Sanger and D.T.A. Lamport, Anal. Biochem., 128 (1983) 66-70. 32. J.M. Lau, M. McNeil, A.G. D a r v i l l and P. Albersheim, Carbohydr. Res., 168 (1987) 219-243. 33. G.G.S. Dutton, Adv. Carbohydr. Chem. Biochem., 28 (1973) 11-160. 34. R.L. Taylor and H.E Conrad, Biochemistry, 11 (1972) 1383-1388). 35. G.G.S. Dutton and M.-T Yang, Can. J . Chem., 59 (1977) 179-192. 76" 36. M. W i l l i a m s , Adv. Carbohydr. Chem. Biochem., 3* (1975) 9-79 37. A . J . Mort and D.T.A. Lamport, A n a l . Biochem., 82 (1977) 289-309. 38. Z. Dis c h e , Methods Carbohydr. Chem., I (1962) 477-514. 39. S. - I . Hakomori, J . Biochem. (Tokyo), 55 (1964) 205-208. 40. T. Purdi e and J.C. I r v i n e , J . Chem. S o c , 83 (1903) 1021-1037. 41. I . Ciucanu and F. Kerek, Carbohydr. Res., 131 (1984) 209-217. 42. J . F i n n e . T. K r u s i u s and H. Rauvala, Carbohydr. Res., 80 (198O) 336-339. 43* D.M.W. Anderson, I.CM. Dea, P.A. Maggs and A.C. Munro, Carbohydr. Res., 5 (1967) 489-491 . 44. P. Prehm, Carbohydr. Res., 78 (1980) 372-374. 45. M. -S. Kuo and A . J . Mort, Carbohydr. Res., 145 (1986) 247-265. 46. H. P a r o l i s , p e r s o n a l communication. 47. S.J. Angyal and K. James, Aust. J . Chem., 23 (1970) 1209-1221. 48. C F . Snyder, H.L. Frush and H.S. I s b e l l , Methods Carbohydr. Chem., I (1962) 524-534. 49. G.M. B e b a u l t , J.M. B e r r y , Y. -M.Choyy G.G.S. Dutton, N. F u n n e l l , L.D. Hayward and A.M. Stephen, Can J . Biochem., 51 (1973) 324-326. 50. J . J . Marshal, Adv. Chem. Biochem., 30 (1974) 257-370. 51. K. Leonte i n , B. Li n d b e r g and J . Lonngren, Carbohydr. Res., 62 (1978) 359-362. 52. G.J. Gerwig, J.P. Kamerling and J.F.G. V l i e g e n t h a r t , Carbohydr. Res., 62 (1978) 349-357. Carbohydr. Res., 77 (1979) 1-7 53. P.J. Garegg, P.E. Jansson, B. Li n d b e r g , F. L i n d h , J . Lonngren, I . Kvarns.trom, and W. Nimmich, Carbohydr. Res., 78 (1980) 127-132. 54. A.N. de B e l d e r and B. Norrman, Carbohydr. Res., 8 (1968) 77 55. J.K.N. Jones, Methods Carbohydr. Chem., I (1962) 21-31 . 56. T. Devenyi and J . Gergely, ' Amino a c i d , Peptides and P r o t e i n s ' , E l s e v i e r P u b l i s h i n g Company, Inc., New York, (1974). 57. 0. L u d e r i t z , K. Jann and R. Wheat, i n 'Comprehensive Biochemistry', M. F l o r k i n (Ed.), E l s e v i e r , Amsterdam, 26A (1968) 105-228. 58. R.D. Guthrie and J.F. McCarthy, Adv. Carbohydr. Chem. Biochem., 22 (1967) 11-23. 59. B. Baynard and J . M o n t r e u i l , Carbohydr. Res. 24 (1972) 427-443. 60. B. L i n d b e r g , J . Lonngren,and J.L. Thompson, Carbohydr. Res., 28 (1973) 351-357 61. B. Lindberg and J . Lonngren, Methods Carbohydr. Chem., 7 (1976) 142-148. 62. H. K a r l s s o n , Ingemar C a r l s t e d t and G. Hansson, Febs. L e t t s . , 226 (1987) 23-27. 63. G.W. Hay, B.A. Lewis and F. Smith, Methods Carbohydr. Chem., 5 (1965) 357-361. 64. G.W. Hay, B.A. Lewis and F. Smith, Methods Carbohydr., 5 (1965)361-370. 65. J.M. B o b b i t t , Adv. Carbohydr. Chem., 11 (1956) 1-41. 66. G.O. A s p i n a l l and R.J. F e r r i e r , Chem. and Ind. (London), (1957) 1216. 67. G.G.S. Dutton and K.B. Gibney, Carbohydr* Res., 25 (1972) 99-105. 68. A.J. B i r c h , Q. Rev. Chem. S o c , 4 (1970) 69-93. 69. A.W. B u r g s t a h l e r , L.R. Worden and T.B. Lewis, J . Organ. Chem., 28 (1963) 2918-2919. 70. A.O. Bedenbaugh, J.H. Bedenbaugh, W.A.Bergin and J.D. Adkins, J . Am. Chem. S o c , 92 (1970) 5774-5775. 71. J.W. Huffman and J.T. Ch a r l e s , J . Am. Chem. Soc. 90 (1968) 6486-6492. 72. H. Adkins and R. G i l l e s p i e , Org. Syn. C o l l . , 3 (1955) 671-673. 73. H.A. Smith, B.J.L. Huff, W.J. Powers I I I and D Caine, J . Org. Chem., 32 (1967) 2851-2856. 78 74. A.J. Mort, Abstr. Pap. Am. Chem. Soc. Meet., 181 (1981) CARB-49. 75. J . Lenard, Chem. Rev., 69 (1969) 625-638. 76. P. -E. Jansson, B. Lindberg and P.A. Sanford, Carbohydr. Res., 124 (1983) 135-139. 77. V.N. Reinhold, E. Coles and S. A. Carr, J . Carbohydr. Chem. , 2 ( 1 ) (1983) 1-18. 78. D. Rolf and G.R. Gray, J . Am. Chem. S o c , 105 (1982) 3539-3541. 79. E.T. Reese, F.W. Parrish and Mettlinger, Carbohydr. Res., 18 (1971) 381-388. 80. A. Amemura, T. Harada, M. Abe and S. Higashi, Carbohydr. Res., 115 (1983) 165-174. 81. G.-G.S. Dutton, J.L. Di Fabio, D.M. Leek, E.H. M e r r i f i e l d , J.R. Nunn and A.M. Stephen, Carbohydr. Res., 97 (1981) 127-138. 82. H. Geyer, K. Himmelspach, B. Kwiatkowski. S. Schlecht and S. Stirm, Pure Appl. Chem., 55 (1983; 637-653, and re f . therein. 83. D. Rieger-Hug and S. Stirm, Virology, 113 (1981) 363-378. 84. A.A. Lindberg, i n I.W. Sutherland (Ed.), 'Surface Carbo-hydrates of the Prokaryotic C e l l ! , Academic Press, New York, (1977) PP 289-356. 85. B. Kwiatkowski, H. Beilharz and S. Stirm, J . Gen. V i r o l . , 29 (1975) 267-280. 86. L.A.S. P a r o l i s , Ph.D. Thesis, Rhodes University (1985). 87. N. Ravenscroft, A.M. Stephen and E. H. M e r r i f i e l d , S. A f r . J . S c i . , 81 (1985) 381-382. 88. D.E. Bradley, B a c t e r i d . Rev., 31 (1967) 230-314. 89. S. Stirm and E. Freund-Molbert, J . V i r o l . , 8 (1971), 330-342. 90. W. Bessler, F. Fehmel, E. Freund-Molbert, H. Knufermann and S. Stirm, J . V i r o l . , 15 (1975) 976-984. 91. W. Bessler, E. Freund-Molbert,H."Knufermann, C. Rudolph, H. Thurow and S. Stirm, V i r o l . 56 (1973) 134-151. 79 92..' M.E. Bayer, H. Thurow and M.H. Bayer, V i r o l . , 94 (1979) 95-118. 93. L.M. Beynon, M.Sc. T h e s i s , U n i v e r s i t y of B r i t i s h Columbia (1985). 94. S. Tomlinson and P.W. T a y l o r , J . V i r o l . , 55 (1985) 374-378. 95. G.Q .S . Dutton and D.N. Karanaratne, Carbohydr. Res., 138 (1985) 277-291. 96. G.G .S . Dutton, K.L. Mackie, A.V. Savage. D. Rieger-Hug and S. S t i r m , Carbohydr. Res., 84 (1980) 161-170. 97. H. W i l l i a m s Smith and M.B. Huggins , J • Gen. M i c r o b i o l . , 129 (1983) 2659-2675 98. A . V . S . Lim, Ph.D. U n i v e r s i t y of B r i t i s h Columbia (1986). 99. A. Kuma-Mintah, p e r s o n a l communication. 100. W.W. B i n k l e y , Methods Carbohydr. Chem., 5 (1965) 54-55. 101. R.L. W h i s t l e r and A.K.M. Anisuzzaman, Methods Carbohydr. Chem., 8 (1980) 45-53. 102. G.G.S. Dutton, Adv. Carbohydr. Chem..Biochem., 30 (1973) 9-110. 103. G.D. McGinnis and P. Fang, Methods Carbohydr. Chem., 8 (1980) 33-43. 104. M. McNe i l , A.G. D a r v i l l , P. Aman, L. -E. Franzen and P. Albe r s h e i m , Methods Enzymol., 83 (1982) 3-45. 105. L.R. Snyder and J . J . K i r k l a n d , ! ' I n t r o d u c t i o n to Modern L i q u i d Chromatography', John Wiley ( E d . ) , New York, (1974). 106. C C . Sweeley, R. B e n t l e y , M. Makita and W.W. W e l l s , J . Amer. Chem., S o c , 85 (1963) 2497. 107. S.W. Gunner, J.K.N. Jones and M.B. P e r r y , Chem. Ind. (London), (1961) 225. 108. S.W. Gunner, J.K.N. Jones and M.B. P e r r y , Can. J . Chem., 39 (1961) 1892. 109. H. B j o r n d a l , B. L i n d b e r g and S. Syensson, A c t a Chem. Scand., 21 (1967) 1801. 110. H.O. Bouveng and B. L i n d b e r g , Adv. Carbohydr. Chem. Biochem., 15 (1960) 53. 80 111. J . Karkkainen, Carbohydr. Res. 11 (1969) 247-256. 112. J.S. Sawardeker, J.H. Sloneker and A.R. Jeanes, Anal. Chem., 37 (1963;1602-1604. 113. R» Dandeaneau and E.H. Zerenner, J . H.R.C. & C C , 2 (1979) 351. 114. H. Bjorndal, CG. H e l l e r q v i s t , B. Lindberg and S. Svensson, Angew. Chem. Int. Ed. Engl., 9 (1970) 610-620. 115. J . LSnngren and S. Svensson, Adv. Carbohydr. Chem. Biochem., 29 (1974) 41 -106. 116. P. -E. Jansson, L. Kenne, H. Liedgren, B. Lindberg and J . Lonngren, Chem. Comm. University Stockholm, 8 (1976). 117. V.N. Reinhold and S.A.Carr, Mass Spectrom. Rev., 2 (1983) 153-221. 118. G.W. Wood, Mass Spectrom. Rev., 1 (1982) 63-102. 119. K.L. Rinehart, Science, 218 (1982) 254-260. 120. K.L. Busch and R.G. Cooks, Science, 218 (1982) 247-254. 121. V. Kovacik, S. Bauer and J . Rosik and P. Kovac, Carbohydr. Res., 8 (1968) 282-290. 122. O.S. Chizhov and N.K. Kochetkov, Adv. Carbohydr. Chem. Biochem., 21 (1966) 39-93. 123. H. Rauvala, J.Finne, T. Krusius, J . Karkkainen, Adv. Carbohydr. Chem..Biochem., 38 (1981) 389-416. 124. E.G. De Jong, W. Heerma, G.Dijkstra, Biomed. Mass Spectrom. 7 (1980) \21~J>\ . 125. B. Lindberg, J . Lonngren, J.L. Thompson and W. Nimmich, Carbohydr. Res., 25 (1972) 49-57. 126. H. Bjorndal, B. Lindberg, J . Lonngren. K.G. Rosel l and W. Nimmich, Carbohydr. Res., 27 (1973) 373-378. 127. V.N. Reinhold, Methods Enzymol., (1987) 138, No.(Complex Carbohydr., PT.E.), 59-84 and r e f . therein. 128. A. D e l l , Adv. Carbohydr. Chem. Biochem., 45 (1987) 19-72 and r e f . therein. 129. C C Sweeley, H.A. Nunez, Ann. Rev. Biochem., 54 (1985) 765-801 . 81 130. P.A.J. Gorin, Adv. Carbohydr. Chem. Biochem., 38 (1981) 13.-1 Ok. 131. K. Bock, H. Thorgersen, Ann. Ren. NMR Spectrosc. 13 (1982) 1-57. 132. K. Bock, C. Pedersen, Adv. Carbohydr. Chem. Biochem., 41 (1983) 27-66. 133. J.L. Di Fabio, G.G.S. Dutton and H. P a r o l i s , Carbohydr. Res., 133 (1984) 125-133. 134. G. Annison and G.G.S. Dutton, Carbohydr. Res., 168 (1987) 89-102. 135. CM. Bebault, Y.M. Choy, G.G.S. Dutton, F. Funnell. A.M. Stephen and M.T. Yang, J . of B a c t e r i o l . , 113 (1973) 1345-1347. 136. M. Karplus, J . Am. Chem. S o c , 85 ( 1 9 6 3 ) 2870-2871 . 137. C. Altona, C.A.G. Haasnoot, Org. Magn. Reson., 13 (1980) 417-429. 138. K. Bock, I. Lundt and C. Pederson, Tetrahedron Lett., 13 (1973) 1037-1043. 139. A « Bax, W. Egan and P. Kovac, J . Carbohydr. Chem., 3 (1984) 593-611. 140. S.L. Patt, J . Carbohydr. Chem., 3 (1984) 493-511. 141. D.R. Bundle, M. Gerken and M.B. Perry, Can. J . Chem., 64 (1986). 235-264. 82 CHAPTER III STRUCTURAL STUDIES ON E. c o l i K26 CAPSULAR POLYSACCHARIDE USING G.C.-C.I.-M.S. CAPILLARY GAS CHROMATOGRAPHY REFERENCE DATA 83 III STRUCTURAL STUDIES ON E. c o l i K26 CAPSULAR POLYSACCHARIDE USING G.C.-C.I.-M.S. CAPILLARY GAS CHROMATOGRAPHY REFERENCE DATA 111.1 ABSTRACT A number of methylated oligosaccharides were analysed by combined c a p i l l a r y gas chromatography chemical i o n i z a -tion mass spectrometry. Relative retention times were obtain-ed for d i - t r i - and tetra-saccharide standards using DB 17, DB 225, DB 1 and DB 5 c a p i l l a r y columns. Glucose d i - and tri-saccharides d i f f e r i n g only in their position of l i n k -age were well resolved and separation of «- and p- anomers of 1,4- and 1,6- linked disaccharides was also achieved. Pseudo-molecular ions and prominent fragment ions, from chemical i o n i z a t i o n mass spectra, were used to characterize the oligosaccharides. This procedure was then applied to the characterization of a sample composed of a mixture of oligosaccharides obtained during the study of the struc-ture of E. c o l i K26 capsular antigen. 111.2 INTRODUCTION The successful analysis of oligosaccharides, produced by various chemical techniques from polysaccharides, i s to a large extent dependent on the purity and homogeneity of the sample. Separation and p u r i f i c a t i o n of oligosaccharide 84 mixtures by the usual methods, e.g. preparative paper chroma-tography and gel permeation chromatography, can be time consuming and, where complex mixtures are to be separated, may only be p a r t i a l l y successful. In addition, where the oligosaccharides are available i n only small amounts, or have s i m i l a r Rf values or molecular weights, separation i s not f e a s i b l e . Hence a method for simultaneously separating, purifying and characterizing mixtures of oligosaccharides i s desirable, but few such techniques are a v a i l a b l e . Fast atom bombardment mass spectrometry (f.a.b.-m.s.) has been used to analyse d i r e c t l y mixtures of oligosaccharides, how-ever the sample must be pure and contain oligosaccharides of d i f f e r e n t molecular weights for successful characterization. Furthermore the r e l a t i v e abundance of ions w i l l not necess-a r i l y r e f l e c t the r e l a t i v e amounts of the components 1. A d i f f e r e n t approach i s to couple permethylated oligosaccha-rides with a fluorophore at t h e i r reducing ends, separate by p h.p.l.c. and then sequence by d . c . i . mass spectrometry . The most r e a d i l y available and simplest method for simultaneously separating, p u r i f y i n g and characterizing a mixture of oligosaccharides i s g.c.-m.s. There are many examples of the analysis of permethylated oligosaccharide and oligosaccharide a l d i t o l s by g.c.-e.i.-m.s.^ -^. . The sequence of monosaccharide units can be deduced from the e . i . mass spectrum, and i n some cases the position of s p e c i f i c g l y c o s i d i c linkages can be i n f e r r e d . The use of g . c . - c . i . -m.s. affords higher i n t e n s i t i e s of the molecular ion and 8 5 l a r g e r fragment i o n s r e l a t i v e to g.c.-c.i.-m.s., and thus improves the a b i l i t y to d i s c r i m i n a t e between d i f f e r e n t o l i g o s a c c h a r i d e d e r i v a t i v e s . A r e c e n t review by Sweeley c and Nunez gave no r e f e r e n c e s f o r the a p p l i c a t i o n of t h i s combination of techniques to o l i g o s a c c h a r i d e s t r u c t u r a l c h a r a c t e r i z a t i o n , but s i n c e the work presented here was completed, at l e a s t one paper has been p u b l i s h e d i n which g.c.-c.i.-m.s. was used to a n a l y s e a c i d i c and n e u t r a l o l i g o -s a c c h a r i d e s o b t a i n e d from an anti-complementary a c i d i c h e t e r o g l y c a n . However, the a u t h o r s , p r i o r to a n a l y s i s by g.c.-c.i.-m.s., separated the a c i d i c and n e u t r a l f r a c t i o n s , and the a c i d i c methylated o l i g o s a c c h a r i d e a l d i t o l s were c a r b o x y l - r e d u c e d . In the work presented here, the p r i o r s e p a r a t i o n of a c i d i c and n e u t r a l f r a c t i o n s was not found to be e s s e n t i a l i n the c h a r a c t e r i z a t i o n of o l i g o s a c c h a r i d e mixtures by g.c.-c.i.-m.s. Thus, the a n a l y s i s of a r e l a t i v e -l y s m a l l amount of m a t e r i a l , c o n s i s t i n g of a mixture of t h r e e d i s a c c h a r i d e s , a l l o w e d the sequence of sugars i n the p o l y -meric backbone of E. c o l i c a p s u l a r p o l y s a c c h a r i d e to be determined. -The a n a l y s i s , by g.c.-c.i.-m.s., of the mixture was g r e a t l y f a c i l i t a t e d by having a v a i l a b l e , f o r comparison,the r e l a t i v e r e t e n t i o n times and mass s p e c t r a l data of a u t h e n t i c s t a n d a r d s . Although i t may have been p o s s i b l e to f i n d the r e l a t i v e r e t e n t i o n times of some o l i g o s a c c h a r i d e s by a s s i d u o u s -l y s e a r c h i n g the l i t e r a t u r e , s o f t e n the r e l e v a n t data i s not a c c e s s i b l e . U s e f u l i n f o r m a t i o n can be l o s t as authors may 86 not i n d i c a t e , i n e i t h e r the t i t l e or a b s t r a c t o f a paper, t h a t g.c.-m.s. has been used to i d e n t i f y o l i g o s a c c h a r i d e s . Furthermore i n work presented i n e a r l i e r p a p e r s 5 packed columns r a t h e r than c a p i l l a r y columns were employed and, as r e l a t i v e r e t e n t i o n times d i f f e r depending on the column used, such r e s u l t s are of l i m i t e d use. For these reasons p r i o r t o a n a l y s i s of the o l i g o s a c c h a r i d e mixture from E . c o l i K26 c a p s u l a r a n t i g e n a s e r i e s of d i - t r i - and t e t r a - s a c c h a r i d e s were methylated and analysed by g.c.-c.i.-m.s. u s i n g v a r i o u s c a p i l l a r y columns. I I I . 3 RESULTS AND DISCUSSION C a p i l l a r y gas chromatography chemical i o n i z a t i o n mass  spectrometry of permethylated d i s a c c h a r i d e s . - C a p i l l a r y gas chromatography a n a l y s i s of permethylated d i s a c c h a r i d e s show-ed t h a t u s i n g DB17 and DB 225 c a p i l l a r y columns, anomeric forms c o u l d be w e l l r e s o l v e d (Table I I I . l ) . In a d d i t i o n , d i s a c c h a r i d e s d i f f e r i n g i n o n l y t h e i r l i n k a g e p o s i t i o n s gave r e l a t i v e r e t e n t i o n times (R^) which allowed complete r e s o l u -t i o n . Where s a t i s f a c t o r y r e s o l u t i o n was not achieved with a DB 17 c a p i l l a r y column, as i n the case of l a m i n a r i b i o s e (5) and c e l l o b i o s e (2 ) , which have comparable r e l a t i v e r e t e n t i o n times on DB 17 (1.18, 1.35 and 1.18, 1.33 r e s p e c t i v e l y ) , adequate r e s o l u t i o n was achieved with a DB 225 c a p i l l a r y column (1 .23,1 .43 and 1 .30, 1.44 r e s p e c t i v e l y ) . Most d i -s a c c h a r i d e s gave two peaks on g.c. a n a l y s i s . These peaks 87 TABLE III.1 Relative Retention Times of Methylated Disaccharides on DB 1?, DB 225 and DB 5 Capillary Columns Oligosaccharide Trivial Name Relative retention times 6 DB 1?b DB 225b DB 5C 4-0-a-D-Glucopyranosyl-D-glucose 4-O-P-D-Glucopyranosyl-D-glucose 6-0-a-D-Glucopyranosyl-D-glucose 6-0-p-D-GlucopyranosyI-D-glucose 3- 0-p-D-Glucopyranosyl-D-glucose 2-O-P-D-Glucopyranosyl-L-rhamnose 4- 0-P-D-Glucopyranosyl-L-rharnnose P-D-Fructopyranosyl-a-D-glucopyranose 6-0-a-D-Glucopyranosyl-D-fructofuranose 6-0-a-D-Galactopyranosyl-D-glucose 4-0- P-D-Galactopyranosyl-L-rhamnose 4-O-o-D-Mannopyranosyl-L-rhamnose 4-O-P-D-Mannopyranosyl-L-rhamnose 4-O-a-L-RhamnopyranosyI-L-rhamnosc 2-Acetamido-2-deoxy-4-(O-a-L-rhamnopyranosyl)-D-glucosc 6-O-p-D-Glucopyranuronosyl-D-galactose (1) Maltose 1.32 1.42 1.39 1.51 1.61 1 .48 (2) Ccllobiose 1.18 1.30 1 .29 1.33 1.44 1.36 (3) Isomaltose 1.23 1.43 1.26 1.50 (4) Genriobiose 1.31 1.43 1.43 1.52 1.66 1.58 (5) Laminaribiosc 1.18 1.35 1.23 1.43 (6) 0.75 0.73 0.81 0.92 1 .02 0.89 (7) 0.91 0.90 0.93 (8) Sucrose 1.00 1.00 1.00 (9) Palatinose 1.32 1.39 1 .32 1.40 1.51 1.41 (10) Melibiose 1.34 1.43 1.35 1.54 1.68 1.50 (ID - 1.0? 1.10 0.97 (12) 0.95 0.99 0.87 1.07 1.16 0.99 (13) 1.10 1.16 1.05 1.23 1.53 1.13 (14) 0.76 0.82 0.74 (15) 2.38 3.82 2.17 2.41 (16) 1.76 2.15 1.84 1.90 1.98 2.07 2.38 2.54 2.61 1.91 a Relative to that of methylated sucrose: DB 17 - 5.98 min., DB 225 -. 4.92 min., DB 5 -n3.25 min. 0 Programmed at 210 for 1 min. then 4Vmin. to 24g . Programmed at 210°for 2 min. then 6 /min. to 290 . c 88 r e s u l t from the presence of both the o(- and ^-.anomers of the t e r m i n a l r e d u c i n g sugars. There were two main excep-t i o n s . Monosaccharides with a manno- c o n f i g u r a t i o n give predominantly one anomer and thus d i s a c c h a r i d e s with rhamnose as the t e r m i n a l r e d u c i n g sugar e i t h e r gave a s i n g l e peak (7,11, and l i f ) , or two peaks where one was r e l a -t i v e l y i n s i g n i f i c a n t (12 and 13). On the other hand, a 6-9 l i n k e d g a l a c t o s e , on being methylated by the Hakomori 7 procedure, w i l l g i v e both furanose and pyranose products. As a r e s u l t , 16 produced four peaks i n the chromatogram. C a p i l l a r y gas chromatography chemical i o n i z a t i o n mass spectrometry of permethylated t r i - and t e t r a - s a c p h a r j d e s . -High molecular weight methylated o l i g o s a c c h a r i d e s were a l s o separated and a n a l y s e d by g.c.-c.i.-m.s. u s i n g a DB 17 column. The non-reducing t r i s a c c h a r i d e s , r a f f i n o s e an,d mele-z i t o s e , e l u t e f a s t e r (Rt 3.61.and 3*10 r e s p e c t i v e l y ) than comparable r e d u c i n g o l i g o s a c c h a r i d e s such as m a l t o t r i o s e (R^ 4.21, 4*53)• M a l t o t r i o s e and i s o m a l t o t r i o s e were w e l l r e s o l v e d (Rt 3.95, 4.29 and 4.21, 4.50 r e s p e c t i v e l y ) con-f i r m i n g t h a t h i g h e r molecular weight o l i g o s a c c h a r i d e s d i f f e r -i n g s o l e l y i n t h e i r p o s i t i o n of l i n k a g e c o u l d be separated by g . c ( T a b l e III.2). C . i . mass s p e c t r a p r o v i d e d mole-c u l a r weight i n f o r m a t i o n and sequence i n f o r m a t i o n , e.g. m a l t o t r i o s e gave a pseudo-molecular i o n (M + NH^) + a t m/z 676 and a fragment a t m/z 423 ( F i g . III.1), whereas m e l e z i t o s e with the same pseudo-molecular i o n had a s t r o n g fragment i o n 89 TABLE III.2 R e l a t i v e R e t e n t i o n Times o f M e t h y l a t e d T r i s a c c h a r i d e s on DB17 C a p i l l a r y Column Structure Trivial Name Relative Retention Times I II 0-a-D-GlcE-(l-3)-0-B-D-Fruf-(2-l)-0-a-D-GlcE M e l e z i t o s e 3.10 1.0 6»-a-D-GalD-(l-6)-<9-a-D-GlcD-(l-2)-C>-B-D-Fruf Raffinose 3.61 1.28 [0-a-D-GlcjH 1 -4)-]-20-a-D-Glcp. Maltotriose 3.95 1.71 4.29 2.01 [0-a-D-GlcD-(l-6)-]- 20-a-D-GlcE Isomaltotriose 4.21 1.93 4.53 2.23 I — retention times relative to sucrose (5.35) and obtained with a programme of 210° for 1 min then 4°/min to 280° II — retention times relative to melizitose (3.48) and obtained with an isothermal programme at 290° F i g . I I I . 1 Fragment ions of methylated maltotriose 391—— 423 (M + N H 4 ) + = 676 X = OMe 90 at m/z 377 ( F i g . III.2 a and 2b) . T e t r a s a c c h a r i d e s , stachyose and m a l t o t e t r o s e , were a l s o a n a l y s e d by g.c.-c.i.-m.s. u s i n g a DB 17 c a p i l l a r y column ( i s o t h e r m a l 2 9 0 ° ) . With t h i s column, r e l a t i v e r e t e n t i o n times f o r the t e t r a s a c c h a r i d e s are s u b s t a n t i a l l y l o n g e r than those of the t r i s a c c h a r i d e s l e a d i n g to peak broaden-i n g . However, the mass s p e c t r a obtained e x h i b i t pseudo-molecular i o n s (M + NH^) + f o r both compounds at m/z 880 together with the expected fragment i o n s at m/z 219, 4-23 and 395. To overcome the problem of peak broadening the t r i - and t e t r a - s a c c h a r i d e s were analysed u s i n g DB 1 and DB 5 c a p i l l a r y columns (Table I I I . 3 ) . DB 1 i s a non-polar column and samples e l u t e d f a s t e r than from the s l i g h t l y more p o l a r DB 5» when u s i n g the same temperature programme. Thus, peak widths were narrow u s i n g DB,1 but some r e s o l u t i o n was l o s t ( F i g . I I I .3 a ) . R e s o l u t i o n was improved by u s i n g DB 5 but the longer r e l a t i v e r e t e n t i o n times obtained with t h i s column r e s u l t e d i n the h i g h e r molecular weight o l i g o -s a c c h a r i d e s g i v i n g chromatograms with broader peaks ( F i g . I I I . 3b) . However, the peak widths were s t i l l f a r l e s s than those o b t a i n e d with DB 17. Thus, DB 5 would appear to be the column of choice f o r a n a l y s i n g an unknown sample which may c o n t a i n both h i g h and low molecular weight o l i g o s a c c h a -r i d e s . Although s i m i l a r d i s a c c h a r i d e s are s l i g h t l y l e s s w e l l r e s o l v e d than with the more p o l a r columns DB 17 and DB 225 (Table I I I . 1 ) , adequate s e p a r a t i o n of lower molecular weight o l i g o s a c c h a r i d e s can be achieved with a programme t h a t 91 F i g . I I I . 2 a Fragment i o n s of methylated m e l e z i t o s e (M + N H 4 ) + = 676 X = OMe F i g . I I I . 2 b G . c . - c . i . mass spectrum of methylated m e l e z i t o s e 187 •3 ,uj),ll,|ll|,ltl) 4. J 4 236 377 , ; , . , 1 J . , I , . J , I J L W .0 100 ISO 140 ICO IM 200 230 340 3*0 MO 300 320 340 SCO 380 J < 676 i ' " ^ " " " . l o 4 4 . X " X ' soo ' " ^ o " " »io ' '"^0 " .oo ' «io ' ..o »«° W O TOO TABLE III.3 Relative Retention Times of Methylated T r i - and Tetra-6accharide8 on DB 1 and DB 5 Cap i l l a r y Columns Oligosaccharide T r i v i a l Name Relative Times Retention DB 1 a DB 5° 0-«-D-Glcp-( 1 -3)-£-p-D-Fruf-(2-1 )-Q-o(-D-Glcp Melezitose 4.81 3.34 0-«-D-Galp-(1-6)-Q-«-D-Glcp-(1-2)-0-p-D-Fruf Raffinose 5.53 3.73 [o-K-D-Glcp-C 1 -if)-J2-Q-«-D-Glcp Maltotriose 6.71 6.79 4.24 4.31 Jo-x-D-Glcp-O -6)-j^-0.-(X-D-Glcp Isomaltotriose 6.35 6.61 4.14 ' 4.28 O-oc-D-Galtv- (1 -6)-0-<X-D-Galp- (1 -6) -0-oc-D-Glct>-(1-27-B-D-Frucf Stachyose 10.60 7.22 Jo-<*-D - Glcp-( 1 -4)-J -0-o(-D-Glcp Maltotetrose; 11.97 12.12 8.89 9.21 Retention times r e l a t i v e to sucrose (1.54 min.) and obtained with a programme of 210 for 2 min., then 6 /min. to 290 . ° Retention times r e l a t i v e to sucrose (3«25 min.) and obtained with a programme of 210 for 2 min., then 6 /min. to 290 . 93 F i g . III.3 a Gas Chromatogram of a methylated tetrasaccharide using a DB 1 C a p i l l a r y Column co o u o col T3 •H & O O cs. CO crj +> CP EH - I F i g . III.3b Gas Chromatogram of a methylated tetrasaccharide using a DB 5 C a p i l l a r y Column <D TJ •H U o o as CO Ct) +> CD — EH 94 also allows higher molecular weight oligosaccharides to be eluted i n a reasonable space of time. These model experiments made i t possible to use the retention time obtained for an oligosaccharide (with a p a r t i -cular column and temperature programme) to make a preliminary assessment of the number and type of sugars i t contains ( F i g . I I I . 4 ) . The molecular ion peak from the c i . mass spectrum provides confirmation of the number of sugars and type of sugars i . e . a c i d i c sugars, deoxy sugars or amino sugars, present i n the oligosaccharide. I f the oligosaccha-ride consists s o l e l y of sugar residues with d i f f e r e n t molecular weights then the sequence of the sugar can be obtained. Complete characterization by c i . mass spectrometry i s not possible as no d i s t i n c t i o n i s possible between diastereomers such as glucose and galactose. The usefulness of t h i s technique was demonstrated when a minor f r a c t i o n (F), obtained by p a r t i a l hydrolysis of E. c o l i K26 polysaccharide, was shown to be a mixture of three oligosaccharides. Characterization of these three oligo-saccharides provided ;strong evidence for the structure of the K26 polymeric backbone. The problems which a r i s e during the characterization of a capsular polysaccharide are s p e c i f i c to the i n d i v i d u a l polysaccharide-- one may have an unusual substituent, an-other may contain a previously unknown sugar. While the K26 polysaccharide has no unusual substituents or sugars, i t s t r u c t u r a l determination proved more d i f f i c u l t than was F i g . III.k Reference Chart for the Preliminary Identification of Oligosaccharides from their Relative Retention times a I . I 1 u 10 Time (min.) 15 20 Deoxyhex-deoxyhex - : r HexA-Hex • Deoxyhex-Hex HexNAc-Hex Reducing Trieaccharide Hex-Hex Non-reducing trisaccharide a DB 17, 210° for 1 min. then Wmin. to 2^0° 96 f i r s t anticipated due to the high proportion of rhamnose residues i n i t s repeating u n i t . From previous work 1 0 i t was known that a l l the rhamnose units are ^ - l i n k e d and a l l except a terminal residue are 3-substituted. Furthermore, the polysaccharide contains a 3 ,4-linked glucuronic acid:: and a 3-linked galactose residue both ^ - l i n k e d . A s e l e c t i v e hydrolysis of the native polysaccharide had indicated that the side chain consists of a single rhamnose residue linked to position 4 of the glucuronic a c i d . Thus, the remaining three rhamnose and one galactose residues were assigned to the main chain. A mild p a r t i a l hydrolysis of the K26 poly-saccharide, followed by gel permeation chromatography, gave among others a f r a c t i o n F which was then either methylated, or reduced with sodium borohydride prior to methylation. G.c.-c.i.-m.s. revealed that the methylated f r a c t i o n F consisted of three disaccharides F1, F2, and F3 with r e l a t i v e retention times of 0.70, 1.06 and 1.26 respectively (Table III.4 ) . The mass spectrum of Fl had a pseudomolecular ion (M + NH/j.)+ at m/z 412 and fragment ions at m/z 380, 363, and 189 (Table I I I .4 , F i g . HI.5a and 5b) . Both the r e l a t i v e retention time and mass spectrum were consistent with those of a deoxyhexose-deoxyhexose disaccharide. The mass spectrum of F2 exhibited a pseudomolecular ion (M + NH^)4" at m/z 442 and fragment ions at m/z 410, 393, 219 and 189 (Table III.4 , F i g . III.6a and 6b) . The pseudomolecular ion and fragment ions indicated that the disaccharide was composed of a hexose TABLE III. 4 RELATIVE RETENTION TIMES AND IONS OBTAINED ON G.C.-C.I.-M.S. OF METHYLATED DISACCHARIDES OBTAINED FROM E. COU K26 CAPSULAR POLYSACCHARIDE Oligosaccharide fraction F1 Rha^  F1 ' RhaJ F 2 Rha-F 2 ' W»-P3 GlcA-P3» GlcA-:Rha •Rha-ol •Gal •Gal-ol •Rha •Rha-ol Relative retention time" (min) 0.70 0.58 1.06 0.84 1.21 0.94 Chemical ionization mass spectral ions [m/z] (Relative abundance) [M+NH41+ 412 (17) 442 (12) 456 (68) 472 (26) [M+H]H 411 (8) 441 (19) 455 (10) [M+NH4-MeOH]+ [M+H-McOH]+ 380 (18) 410 (10) 424 (12) 363 (35) 379 (51) 393 (38) 409 (10) 407 (8) 423 (20) • DB 17,210° for 1 min 4°/min 240°. Retention times relative to that of sucrose (5.98 min). b H and H* represent the non-reducing and reducing sugars respectively in samples. TABLE III.4 (continued) AOH2+ A + H,+b H + b 189 (100) 223 205 189 (78) (16) (100) 219 189 (93) (94) 253 235 189 (93) (72) (100) 189 233 (9) (30) 223 205 233 (69) (13) (30) [A-MeOH]+ [H'-MeOH]+ [H-MeOH]+ 157 (95) 173 157 (9) (45) 187 157 (68) (86) 203 157 (2) (60) 157 201 (40) (100) 173 201 (28) (100) 99 F i g . I l l . 5a Fragment ions of methylated 3-O-a-L-rhaninopyranosyl-L-rhamnose X X ) / 1 X | N / j j ! 157—— 189 331-*- 363 (M + NH4)+ = 412 X = OMe Fig. III.5b G.c.-c.i. mass spectrum of methylated 3-0-o> L-rhamnopyranosyl-L-rhamnose 363 380 412 11111 l'i 111 111 I 'M )111 ' | 11111111 q'ft 11 i*i 111111 ft* i 1111 ri 1111111 p 11111111| 11111 i'i i' 111' IU III U l Iff IU li* »•• * * • « i 4«l 189 - -i I I'l'I'ITl I'l'I'I'I'I'l'I'I'I'I'I'I'I'I'I'I'ITI'l I I'I'I'I'I'I | ITITI'I 111|TITI I 11 I I |'l I I I I'l'l'l l| I I I i 11 11 i | 111111111 11 U U III IH 141 1(1 III ti l III »•! I I I I I I I I'I I I I l'i I I I M II 100 F i g . I I I . 6a Fragment ions of methylated 3-O-a-L-rhamnopyranosyl-D-galactose 393 i ! X i ; 1 X i i i i 219 189 157 125 (M + NH 4 ) + = 442 X = OMe F i 'K . I I I . 6 b G . c . - c . i . mass spectrum of methylated 3-g-<*-L- rhamnopyranosy l -D-ga lac tose •E » J 393 MI|WlMl' l l l |Mll l l l ' l|'.'r ' f?l . l |" l l l . ' l l l | JM tit U l » • *•* n r f 410 442 <•• <M ••• 10. s »• M *> <r Jf 189 219 i |'i i I'I'I'I I'I i,'. 11111 i'r. i u i n I iff 1»» 101 and a deoxyhexose r e s i d u e . The r e d u c i n g sugar was i d e n t i f i e d when, on c o n v e r s i o n of t h i s d i s a c c h a r i d e to i t s a l d i t o l (F1 1) no fragment i o n a t m/z 219 was observed, i n s t e a d a new fragment i o n appeared a t m/z 2 3 5 - ( F i g . III.7 a and 7b) . T h e r e f o r e F2 was i d e n t i f i e d as a deoxyhexose-hexose d i s a c c h a -r i d e . The t h i r d component F3 gave a mass spectrum with a pseudomolecular i o n (M +NH/t)+ a t m/z 456 and two prominent fragment i o n s a t m/z 201 and 233 (Table I I I . 4 , F i g . 8 a and 8b). Thus, t h i s component was i d e n t i f i e d as the a l d o b i o u r o n i c a c i d which has been p r e v i o u s l y shown to be present i n E. c o l i K26 c a p s u l a r p o l y s a c c h a r i d e 1 1 . The r e s u l t s of t h i s g . c . - c . i . -m.s. a n a l y s i s t o g e t h e r with m e t h y l a t i o n data from the n a t i v e p o l y s a c c h a r i d e allowed the f o l l o w i n g s t r u c t u r e s to be ass i g n e d to the components of f r a c t i o n F. F1 3-0-cx-L-rhamnopyranosyl-L-rhamnose F2 3-0-«-L-rhamnopyranosyl-D-galactose F3 3-Q - r i- :D-glucopyranuronosyl-L-rhamnose I I I . 4 CONCLUSION Making the assumption t h a t the m i l d h y d r o l y t i c c o n d i t i o n s d i d not c l e a v e the a l d o b i o u r o n i c a c i d u n i t , a n a l y s i s by g . c -c.i.-m.s. of a mixture c o n t a i n i n g three o l i g o s a c c h a r i d e s allowed the assignment of the f o l l o w i n g s t r u c t u r e to the backbone of the K26 biopolymer. 102 F i g . I I I . 7a Fragment ions of reduced and methylated 3-0-oC-L-rhamnopyranosyl-D-galactose 157- 189 235 (M + H) + = 441 X = OMe F i g . III.7b G.c.-c.i. mass spectrum of reduced and methylated 3-2-OC-L-rhamnopyranosyl-D-galactose I 1 • 1 ' T l i t III irt tn • ttt ttt t n 103 F i e . I I I . 8 a Fragment ions of methylated 3-Q-p-D-glucopyranuronosyl-L-rhamnose 1 169-*— 2 0 1 - * - 233 357—- 407 (M + N H 4 ) + = 456 X = OMe F i e . I I I . 8 b G . c . - c . i . mass spectrum of methylated 3-Q-«j _D-gluc opyranuronosyl-L-rhamnose I f f © 9 f -0 • f _ a *o T f _ c C f Jc-«e t f _ a- 4 f — 4-1 I f _ c -•— t f _ C cn I f 456 Z •in I"" 375 I I I I I I—I I I i u i ? i • » « m • » « « • « » » 201 1*0 »• V c I " > - 11 «" M 7 1* c . a 111 169 233 I ' l ' " 1 ! ' l"1!"'1!1 l""!""!1 'I T"'l"' I •I 1'"'' ' I ' " 1 I '' | I I I I | I I I I I ' 7! IH | » IU IJt «»• « • 1M »»» 104 3 1 3 1 3 1 3 1 3 , 1 GlcA Rha Rha Rha Gal p a a a P This assignment was confirmed by subsequent analyses of other oligosaccharides produced during the p a r t i a l acid hydrolysis (see Section TV). The usefulness of g.c.-c.i.-m.s. analysis i s limited at present by the lack of r e l a t i v e retention times data, however as novel oligosaccharides are generated during the course of s t r u c t u r a l studies on polysaccharides t h i s drawback w i l l be overcome. III.5 EXPERIMENTAL Materials.- Maltose and sucrose were purchased from B.D.H. (Toronto, ON, Canada); cellobiose from Pfansteihl (Waukegan, IL 60085} U.S.A.); melezitose dehydrate and palatinose from A l d r i c h Chemical Co. (Milwaukee, WI 55233, U.S.A. ); maltotriose, isomaltotriose, isomaltose, malto-tetraose and gentiobiose from Sigma Chemical Co. (St. Louis, MO 63178, U.S.A.); ra f f i n o s e and melibiose from Eastman Organic Chemicals (Rochester 3, NY. U.S.A.); and laminari-biose from Koch-Light Laboratories L td. (Colnbrook, Bucks, U.K.). The s y n t h e t i c a l l y prepared disaccharides (6,7,11,12,13, and 14) were obtained from Dr. 6.M. Bebault, the amino 105 sugar d i s a c c h a r i d e (15) from A. Kuma-Mintah, and the a c i d i c d i s a c c h a r i d e (16) from Dr. A.M. Stephen. General methods.- D e i o n i z a t i o n s were performed on a column of Am b e r l i t e IR-120(H +) r e s i n (20x1.5 cm). S o l u t i o n s were conc e n t r a t e d under d i m i n i s h e d pressure a t 3 7 ° . A n a l y t i c a l paper chromatography was performed by the descending method, u s i n g Whatman No. 1 paper with the f o l l o w i n g s o l v e n t systems: (A) 18 : 3 : 1 k e t h y l a c e t a t e -a c e t i c a c i d - formic a c i d - water, (B) 8 : 2 : 1 e t h y l a c e t a t e - p y r i d i n e - water, (C) if : 1 : 5 1-butanol - a c e t i c a c i d - water. Chromatograms were developed u s i n g a l k a l i n e s i l v e r n i t r a t e . S e p a r a t i o n o f o l i g o s a c c h a r i d e s was achi e v e d u s i n g g e l permeation chromatography performed on a column of B i o - g e l P2 (95 x 3 cm) with a c i d i f i e d d i s t i l l e d water (1 drop HCOOH per l i t r e R^O) as the e l u a n t . O l i g o -s a c c h a r i d e f r a c t i o n s were l o c a t e d by the p h e n o l - s u l p h u r i c 12 a c i d method . M e t h y l a t i o n . - The m e t h y l a t i o n of samples (5 nig) was performed by the procedure of Hakomori^, as m o d i f i e d by 1 3 Sandford and Conrad -/. Reduc t i o n . - To a s o l u t i o n of the o l i g o s a c c h a r i d e excess sodium borohydride was added, and the mixture was s t i r r e d a t room temperature (3 h ) . The excess NaBH^ was removed by IR-120 (H +) r e s i n , the s o l u t i o n was f i l t e r e d and co-evapor-at e d w i t h methanol (3x) . P a r t i a l a c i d h y d r o l y s i s . - K26 p o l y s a c c h a r i d e (1.6 g) d i s s o l v e d i n H 2S0^ (200 mL, 0.025M) was heated on a steam 106 bath f o r 1.5 h. The s o l u t i o n was neu t r a l i z e d with lead carbonate and the p r e c i p i t a t e was removed by c e n t r i f u g a t i o n . The supernatant was dialysed against d i s t i l l e d water and the dial y s a t e was concentrated, deionized and analysed by paper chromatography (solvent systems A and B). Rhamnose appeared to be the only component of the d i a l y s a t e . The retentate was l y o p h i l i s e d and subjected to a second mild a c i d h ydrolysis (0.5M TFA, 1.5 h, 95°). The excess aci d was removed under diminished pressure and the f i n a l traces were n e u t r a l i z e d with sodium bicarbonate. The hydrolysate was de-salted on a column of Sephadex G10 (86 x 3 cm) and separated by gel permeation chromatography. On an a l y s i s by paper chromatography (solvent system C ) of the f r a c t i o n s obtained, a r e l a t i v e l y pure f r a c t i o n F was observed ( R Q & ^ O.49). G.c.-c.i.-m.s. data f o r the methylated f r a c t i o n F and reduced and methylated f r a c t i o n F are shown i n Table I I I . 4 . Car>illarv gas chromatography.- A n a l y t i c a l g.c. was performed using a Hewlett-Packard 5890A Gas chromatograph f i t t e d with dual flame i o n i z a t i o n detector and a 3392A recording i n t e g r a t o r . Methylated disaccharides were separated on three d i f f e r e n t 13 m x 0.25y*. ( i . d . ) fused s i l i c a c a p i l l a r y columns, DB 17, DB 225 and DB 5 ( J & W S c i e n t i f i c , Rancho Cardova, CA 95670, U.S.A.). The pro-grammes used were as follows. For both DB'. 17 and DB 225 i n i t i a l temperatures 210° for 1 min., then a rate of 4°/min. to bring the temperature to 240°. For DB 5» i n i t i a l tempera-107 t u r e 210° f o r 2 min., then a r a t e of 6°/min. to 290°. The r e l a t i v e r e t e n t i o n times are l i s t e d i n Table I I I . 1 . T r i -s a c c h a r i d e s were separated on DB 17, DB 5 and a l s o DB 1 (15 x 0.25y& ( i . u . ) ) . Two temperature programmes were used with the DB 17 column, a) as f o r d i s a c c h a r i d e s but i n c r e a s i n g the temperature to 280°, b) i s o t h e r m a l a t 290 ° . The r e l a t i v e r e t e n t i o n times are l i s t e d i n Table I I I . 2 . For DB 1 and BB 5 the i n i t i a l temperature 210° f o r 2 min., then a r a t e of 6°/min. to 290°. The same temperature programme was used f o r the s e p a r a t i o n o f t e t r a s a c c h a r i d e s on DB- 1 and DB 5. The r e t e n t i o n times r e l a t i v e to sucrose are given i n Tab l e I I I . 3 . G.c.-c.i.-m.s.- A n a l y s i s of o l i g o s a c c h a r i d e s was performed on a V a r i a n V i s t a 6000 s e r i e s Gas Chromatograph coupled d i r e c t l y to a D e l s i Nermag R10-10C quadrupole mass spectrometer, or a C a r l o Erba ^160 Gas Chromatograph coupled d i r e c t l y to a K r a t o s MS 80RFA double f o c u s i n g mass spectrometer. Both mass spectrometers are f i t t e d with a chemical i o n i z a t i o n source, and the peaks e l u t e d were i o n i z e d by ammonia as the chemical i o n i z a t i o n reagent gas, with a source pressure of 0.1 T o r r , i o n source temperature of 175°, and an e l e c t r o n v o l t a g e of 72 eV. ACKNOWLEDGMENT T h i s r e s e a r c h was supported by the N a t u r a l S c i e n c e s and E n g i n e e r i n g Research C o u n c i l o f Canada. The author would l i k e to thank Dr. I . 0rskov f o r p r o v i d i n g a c u l t u r e of 108 E. c o l i K 2 6 , and Z. Lam and C. M. Moxham f o r running the g.c-c.i.-m.s. Thanks a l s o the B. Lee f o r m e t h y l a t i o n of some of the d i s a c c h a r i d e standards. 109 CHAPTER IV STRUCTURAL INVESTIGATION OF THE CAPSULAR POLY-SACCHARIDE FROM E. c o l i 09 : K26 : H" n o IV STRUCTURAL INVESTIGATION OF THE CAPSULAR POLYSACCHARIDE FROM E. c o l i 09 : K26 : H" IV.1 ABSTRACT The s t r u c t u r e of E. c o l l 09a : K26 : H" has been i n v e s -t i g a t e d u s i n g m e t h y l a t i o n a n a l y s i s , p a r t i a l a c i d h y d r o l y s i s , Smith degradation and ^ - e l i m i n a t i o n . Nuclear magnetic reson-ance spectroscopy ( 1H a n d ^ C ) v/as used to c h a r a c t e r i z e the c o n f i g u r a t i o n o f the anomeric l i n k a g e s of the p o l y s a c c h a r i d e and o l i g o s a c c h a r i d e s . The p o l y s a c c h a r i d e was found to have a hexasaccharide, "5 + V" r e p e a t i n g u n i t , the t e r m i n a l rhamnose r e s i d u e being s u b s t i t u t e d with a 1 - c a r b o x y e t h y l i -dene group. -3-«<-L-Rhap-( 1 -3)-/-D-Qalp-( 1 -3)-^-D-01cAp-( 1 -3)-c<-L-Rhap-( 1 -3)-c<-L-Rhap-( 1 -<X-L - Rhap tOH IV.2 INTRODUCTION The complexity of the E. c o l i K26 c a p s u l a r polysaccha-111 r i d e r e p e a t i n g u n i t i s due i n pa r t to the high p r o p o r t i o n of L-rhamnosyl u n i t s , and i n p a r t to the nature of the l i n k -ages i n the polymeric backbone which are e x c l u s i v e l y (1-3) A high p r o p o r t i o n of the same sugar r e s i d u e , l i n k e d v i a the same p o s i t i o n , makes i t d i f f i c u l t to a c c u r a t e l y q u a n t i f y the t o t a l number of sugars i n the r e p e a t i n g u n i t of a polysaccha-r i d e , from n.m.r. data or m e t h y l a t i o n a n a l y s e s . A back-bone of 3 - l i n k e d sugars means that a p e r i o d a t e o x i d a t i o n followed by Smith degradation cannot be used to produce o l i g o s a c c h a r i d e s f o r sequence d e t e r m i n a t i o n . Furthermore, 3 - l i n k e d sugars are s u s c e p t i b l e to 'pe e l i n g * r e a c t i o n s on treatment with base 1**. Immunochemical c r o s s - r e a c t i o n s undertaken by H e i d e l -berger had p r e d i c t e d t h a t a rhamnose u n i t would occupy a non-reducing t e r m i n a l p o s i t i o n , that D - g l u c u r o n i c a c i d was p o s s i b l y a branch p o i n t and that a t l e a s t one 3 - l i n k e d rhamnose r e s i d u e was p r e s e n t 1 ^ . These p r e d i c t i o n s were s u b s t a n t i a t e d by the chemical evidence obtained. We now r e p o r t the r e s u l t s of the s t r u c t u r a l i n v e s t i g a t i o n of the c a p s u l a r a n t i g e n from E. c o l i 09a : K26 : H~. IV.3 RESULTS AND DISCUSSION Composition and n.m.r. s p e c t r a . - E. c o l i K26 b a c t e r i a were grown on an agar medium, and the a c i d i c p o l y s a c c h a -r i d e was i s o l a t e d and subsequently p u r i f i e d by p r e c i p i t a -t i o n with cetyltrimethylammonium bromide (CTAB). The pro-112 duct was mbnodisperse by gel-permeation chromatography (M y = 1 x 107), andy a f t e r depyruvylation had -13° (c 1.8, water). G.c. analysis of the a l d i t o l acetates, prepared from, the products of an acid hydrolysis of the polysaccharide, gave rhamnose, galactose and glucose i n the molar r a t i o s 1.0 : 1.0 : 0.2. Reduction of the carboxyl acid function, p r i o r to acid hydrolysis, increased the molar quantities of rhamnose and glucose. The repeating unit was thus deter-mined to be a hexasaccharide, consisting of rhamnose, galac-tose and glucuronic acid i n the r a t i o s of 4 : 1 : 1 • These re s u l t s were obtained during the course of preliminary s t r u c t u r a l analysis and a bacteriophage-enzyme degradation of K26 polysaccharide 1^. The concomitant increase i n the amount of the rhamnose residue with that of the glucose residue a f t e r reduction, suggested that the glucuronic acid i s linked to a rhamnosyl unit, t h i s was l a t e r confirmed by a ^ - e l i m i n a t i o n reaction. The r e l a t i v e configurations of the sugars were assigned by the formation of the (-)-2-octylglycosides and co-injec-tion with authentic standards 1^. A l l the rhamnose r e s i -dues were shown to be of the L-configuration, galactose and glucuronic acid of the D-configuration. The ^H-n.m.r. spectrum of the native polysaccharide indicated the presence of s i x anomeric protons (Table IV.1). These correspond to three cx(6 5 • 11)» one /5(6"4.74) and one borderline s i g n a l (64.91) of r e l a t i v e i n t e n s i t y two, which was shown by the use of a single frequency o f f resonance TABLE IV.1 N.m.r, Dat^ for E. c o l i K26 Capsular Polysaccharide and Derived Oligosaccharides Compound H-N.ra.r data 13 C-Nm.r. data '1,2 Integral Assignment0 P.p.m.d Assignment6 (p.p.m.) (Hz) (no. of H) (p.p.m.) ^Rha U G a l LioicA URha LiRha 1 -<x fi Jfl p <* * 1! Rha pyr K26 polysaccharide 5.10 4.91 4.74 1.59 1.35 8 b b B 3 2 1 2 12 { -3HhaV Rha1-M Q I C A L CH 3 (pyr) CH 3 (Rha) 104.16 103.59 103.11] 102.99j 102.04 62.21 23.61 17.61 J G a l l l ^ G l c A l -^Rha1-Rha 1-/ " -pyr C-6 (hex) CHj (pyr) C-6 (Rha) K26 polysaccharide (autohydrolysis) 5.06 -^Rha- 173.85 C=0 (GlcA) TABLE IV. 1 (continued) 4.87 4.71 1.31 K26 polysaccharide 5.08 (autohydrolyeie) 4.88 P« 7 4.85 4.80 1.32 ''Gal RhaL 3A GlcA-CH^  (Rha) 104.57 103.671 103.65J 103.04" 103.00^ 102.97 99.94 61.93 17.47 ?Gal! -4GICA' 3*4 GlcA 1 RhaW C-6 (hex) CHj (pyr) j R h a l ^ -iGalV Rha1-3A GlcA1-CHj (Rha) TABLE IV.1 (continued) K 2 6 polysaccharide 5*08 s (selective hydrolysis) 4.81 8 4 . 7 1 8 1 . 3 2 5-6 K 2 6 polysaccharide 5.08 s (carbodiimide 4 . 9 7 s reduced) 8 4 . 7 4 8 1 . 3 2 s JGalL 1 0 4 . 4 3 1 0 3 . 8 5 103.81 -IG ICA 1— 1 0 3 . 0 6 2Qal' CH.j (Rha) 1 0 3 . 0 1 1 0 2 . 9 3 1 0 1 . 4 4 9 9 . 6 5 61.86 2 3 . 0 0 1 7 . 4 9 2V+GlcAV J?GlcA1— ^Rha 1-fit RbV-II pyr RhaV C-6 (hex) CH3 (pyr) CH 3 (Rha) -Rha^ Rha^ J a i l 3,4, Glci CH 3 (Rha) TABLE IV. 1 (continued) K26 polysaccharide 5.08 6 3 (Smith degraded) 4.81 8 1 4.75 7 0.7 4.63 0.3 1.32 6 9 Oligosaccharide A 5.10 8 0.6 QlcAlJRha P 4.85 8 o.k 4.78 ) 4.77J 7-8 1 Fraction D 5.10 1-2 0.7 GlcA1—?Rha1—^Rha f> * 5.08 8 0.5 4.88 S 0.3 4.751 4.70J 7-8 1.5 1.32 6 jRha1--2Gal -^GlcA-CHj (Rha) -^ RharOH -^ Pha^ OH GlcAV P -^ Rha'-OH 104.81 -^Rha1— 104.03 jRha1--OH 102.94 P , 94.78 GlcA1-P 94.20 CHj (Rha) 17.63 CH, (Rha) GlcA^-—Rha1—OH -5Rha1j0H a Chemical shift relative to internal acetone assigned at 62.23 downfield from external sodium 4,4-dimethyl-4-8ilapentane-1-sulphonate ( D . S . S . ) . B Key: b = broad, unable to assign accurate coupling constant; s = singlet. °E.g. -^ GlcA1- refers to the anomeric proton of a i^-linked glucuronosyl residue in the B-anomeric configuration. The absence of a numeral prefix indicates a non-reducing terminal group. Chemical shift relative to internal acetone assigned at 31.07 e 1 ^  p.p.m. downfield from external D . S . S . As for c, but for anomeric JZ nuclei. All n.m.r. spectra are given in Appendix I. 117 decoupled experiment (SFORD) to r e s u l t from one <x and one /S l i n k a g e . In the high f i e l d r e g i o n of the spectrum, the two s i g n a l s observed were a s s i g n e d to the methyl groups of f o u r rhamnose u n i t s (81.35) and a 1-carboxyethylidene (61.59) which was present on two out of three r e p e a t i n g u n i t s . D e p y r u v y l a t i o n of the n a t i v e p o l y s a c c h a r i d e r e s u l t e d i n the l o s s of the s i g n a l a t $1.59. °n a d j u s t -ment of the pH of the p o l y s a c c h a r i d e s o l u t i o n from pH 3 to pH7, s i g n i f i c a n t changes occurred i n the anomeric r e g i o n . The resonance at 64«91 r e s o l v e d i n t o two s i g n a l s (84.88, ^ 8 Hz, 64.85) and the resonance at 64.74 s h i f t e d d ownfield to 64.80. Complete assignment of s i g n a l s i n the anomeric r e g i o n was accomplished on the b a s i s of the s p e c t r a of the p o l y s a c c h a r i d e and d e r i v e d o l i g o s a c c h a r i d e s (Table IV.1). The -n.m.r. spectrum of the n a t i v e p o l y s a c c h a r i d e showed a s t r o n g s i g n a l a t 17.61 p.p.m corresponding"to the C-6 atoms of the rhamnose r e s i d u e s and a s i g n a l a t 23.61 p.p.m. which was a s s i g n e d to the methyl group of a pyru-vate s u b s t i t u e n t . The resonance at 62.21 p.p.m. was a s s i g n e d to a primary a l c o h o l i c group i n d i c a t i n g t h a t the g a l a c t o s e i s not l i n k e d through 0-6. In the anomeric r e g i o n f i v e s i g n a l s a t IO4.I6, 103.59, 103.11, 102.99 (double) and 102.04 p.p.m. c o u l d be seen, one of them of double i n t e n s i t y . Removal of the pyruvate s u b s t i t u e n t r e s u l t e d i n the s h i f t of the resonance at 102.04 p.p.m. u p f i e l d to 99*94 p.p.m., and removal of a rhamnose u n i t by s e l e c t i v e h y d r o l y s i s gave a polymer whose 1^C spectrum 118 l a c k e d a s i g n a l a t t h i s resonance. There were however s m a l l r e s i d u a l resonances a t 101.44 p.p.m. and 99.65 p.p.m. Thus the s i g n a l a t 102.0i+ p.p.m. i n the n a t i v e p o l y s a c c h a r i d e was as s i g n e d to a t e r m i n a l rhamnose sugar with a pyruvate a c e t a l s u b s t i t u e n t . The resonance at 103.59 p.p.m. i n the n a t i v e p o l y s a c c h a r i d e was as s i g n e d to the branched g l u c -u r o n i c a c i d . T h i s assignment was made as the s i g n a l a t 103.8 p.p.m., i n the 1-^C-n.m.r. spectrum of the s e l e c t i v e l y h y d r o l y s e d p o l y s a c c h a r i d e , was twinned. T h i s twinning would r e s u l t from some g l u c u r o n i c a c i d r e s i d u e s s t i l l r e t a i n i n g a t e r m i n a l rhamnose r e s i d u e . M e t h y l a t i o n a n a l y s i s ^ * A n a l y s e s of the a) methylated n a t i v e p o l y s a c c h a r i d e , b) depyru v y l a t e d , methylated p o l y -s a c c h a r i d e , c) dep y r u v y l a t e d , methylated, reduced and r e -methylated p o l y s a c c h a r i d e , gave the r e s u l t s shown i n Table TV.2, columns I to I I I . The i n c r e a s e i n the 2,3 J'+-tri-.Q-methylrhamnose, a f t e r the removal of the pyruvate s u b s t i -tuent, a l o n g with the l o s s of the 2-^-methylrhamnose d e r i v a t i v e l o c a t e d the a c e t a l group on a t e r m i n a l rhamnose r e s i d u e . .'The r e s u l t s are c o n s i s t e n t with the presence of a hexasaccharide r e p e a t i n g u n i t , with g l u c u r o n i c a c i d as the branch p o i n t , l i n k e d at C-3 and C-4 ( F i g . I V . 1 ) . ^ - e l i m i n a t i o n ^ A b a s e - c a t a l y z e d u r o n i c a c i d degradation of the permethylated p o l y s a c c h a r i d e , f o l l o w e d by remethyla-t i o n , , gave the r e s u l t s shown i n Table IV.2, column IV. The TABLE TV.2 Methylation Data for E. c o l i K26 Polysaccharide and Derived Products Methylated sugar a Mole R a t i o ' (as a l d i t o l acetates) I II III IV V VI VII VIII 1.2.4.5- «ha 5.9 2,3,4-Rha 6.4 9.4 12.5 36.1 4.6 3.7 2,4-Rha 57.5 29.1 36.7 72.2 22.2 45.7 2?.2 7.1 2.3.4.6- Gal 3. 7 3 # 7 2-Rha 14.9 2,3,4-Glc 20.1 2,4,6-Gal 27.3 15.1 12.8 21.1 8.6 18.0 14.0 2,6-Glc 8.3 2,4-Glc 3 i 9 5.3 2-Glc 4 . 6 a 2,3,4,6-Gal = 1,5-di-0-acetyl-2,3,4,6-tetra-Q-methylgalactitol, etc. Values are corrected by use of the e f f e c t i v e , carbon-response . factors given by Albersheim and coworkers 50. c I, native polysaccharide; I I , depyruvylated polysaccharide; I I I , depyruvylated, uronic ester reduced, reraethylated polysaccharide; IV, product from p-elimination; V, product from selective hydrolysis (1)-uronic ester reduced; VI, product from selective hydrolysis(2) : VII, Smith degradation product; VIII, reduced oligosaccharide B, afer reduction of uronic ester. .120 E. c o l i K26 CPS (H +) CH 2 OMe COOMe Hakomori methylation ( i ) Sodium dimsyl/DMSO ( i i ) CH 3I MeO) ° . ) ° . k MeO A ° . K MeO / ° . K MeO / °.0 OMe OMe N OMe > OMe > 0 M e MeO / " ' C H 3 MeO OMe CH 2 OMe MeO / ° MeO / 0 C H , V o H Ym V e N OMe ( i ) LiAlH^ ( i i ) Remethylation ( i i i ) 2M TFA, I* h OH CH2OMe (OH y OH MeO A 0 I / C H 3 • OH MeO OMe \ | / H O \ | / OMe 0 M e ( i ) NaBH. OH OMe MeO-AcO-• OAc -OMe • OMe AcO-MeO-C H , OAc -OMe ( i i ) Ac 20/pyridine ( i i i ) G.c.-m.s. •OAc -OMe AcO-• OAc • OMe •OAc MeO-•OAc AcO — • OMe C H , • OAc -OMe •OAc F i g . IV.1 Methylation analysis of E. c q l i K26 polysaccharide. 121 i n c r e a s e i n the amount of r e l a t i v e moles of 2 , 3 , 4 - t r i - 0 -methylrhamnose i n d i c a t e d that g l u c u r o n i c a c i d i s l i n k e d to p o s i t i o n 3 of a rhamnose u n i t . T h i s a l d o b i o u r o n i c a c i d had been i s o l a t e d and c h a r a c t e r i z e d p r e v i o u s l y by L e e k 1 1 . S e l e c t i v e h y d r o l y s i s . - M i l d a c i d h y d r o l y s i s of the n a t i v e p o l y s a c c h a r i d e gave, a f t e r d i a l y s i s , a polymer from which approximately 30% of the t e r m i n a l rhamnose r e s i d u e had been removed. The presence of 2,4-di-0.-methylglucose and 2-0-methylglucose among the products of the methylated reduced polymer i n d i c a t e d t h a t the t e r m i n a l rhamnose i s l i n k e d to p o s i t i o n k of the g l u c u r o n i c a c i d (Table IV.2, column V ) . T h i s experiment was c a r r i e d out d u r i n g p r e v i o u s work but i s i n c l u d e d here f o r completeness 1^. A second s e l e c t i v e h y d r o l y s i s almost completely removed the t e r m i n a l rhamnose and, as no other t e r m i n a l sugar was observed the s i d e c h a i n was deduced to c o n s i s t of a s i n g l e rhamnose r e s i -due (Table IV.2, column V I ) . 1 ft P e r i o d a t e O x i d a t i o n and Smith degradation .- Depyruvylated K26 p o l y s a c c h a r i d e was s u b j e c t e d to Smith deg r a d a t i o n . A polymeric product was obtained c o n f i r m i n g that the back-bone of the p o l y s a c c h a r i d e c o n s i s t s s o l e l y of 3 - l i n k e d r e s i d u e s (Table IV.2, column V I I ) . P a r t i a l h y d r o l y s i s . - A c i d h y d r o l y s i s of the s e l e c t i v e l y h y d r o l y s e d p o l y s a c c h a r i d e y i e l d e d f o u r o l i g o s a c c h a r i d e s , 122 a f t e r s e p a r a t i o n by paper chromatography. Only two, A and B were present i n s u f f i c i e n t q u a n t i t i e s and of s u f f i c i e n t p u r i t y t o a l l o w a n a l y s i s by ^-n.m.r spectroscopy (Table IV.1) and m e t h y l a t i o n . O l i g o s a c c h a r i d e A was deduced to be the a l d o b i o u r o n i c a c i d by comparison of i t s 1H-n.m.r. spectrum with t h a t of a p r e v i o u s l y i s o l a t e d s a m p l e 1 1 . From the the sugar a n a l y s i s of the reduced, methylated, r e -duced o l i g o s a c c h a r i d e (Table IV.2, column V I I I ) the f o l l o w -i n g s t r u c t u r e was deduced f o r B. £-D-GlcA-( 1 _3)-£x-L-Rha-( 1 -3) -L-Rha A second p a r t i a l h y d r o l y s i s was undertaken to t r y to generate an o l i g o s a c c h a r i d e which would al l o w the sequence of the two remaining sugars to be determined Four f r a c -t i o n s C,D,E,F were o b t a i n e d , a f t e r s e p a r a t i o n of the pro-ducts on a B i o - g e l P2 column. The permethylated o l i g o -s a c c h a r i d e f r a c t i o n were analysed by g.c.-c.i.-m.s. (Table IV.3). F r a c t i o n C had a main component (C1) with a r e l a -t i v e r e t e n t i o n time of 5.80 min. ( F i g . 2 a ) . The mass spectrum e x h i b i t e d a pseudomolecular i o n (M + NH^) + a t m/z 820, and fragment i o n s a t m/z 233, 201 and 205 ( F i g . 2b). These i o n s were c o n s i s t e n t w i t h those of an a l d o t e t r a o -u r o n i c a c i d with the f o l l o w i n g is true ture: P-D-GlcA-(1 -3) - o(-L-Rha- (1-3) -c<-L-Rha- (1 -3) -L-Rha - o l 123 2a CI o t> c rt c as > •H -P cd rH CD tt CD O c as T3 c .a CD > •H +> rt r H CD tt 1 too 150 2b 200 350 ' ' I ' ' 650 J3 .» 250 300 ~r 350 4oe _L_ 400 450 500 730 ' ' 1 ' ' 850 sei - P L " I " S50 F i g . IV.2a Gas chromatogram of methylated F r a c t i o n C u s i n g DB 1 c a p i l l a r y column. F i g . IV.2b C i . mass spectrum of methylated Component C1 TABLE IV . 3 R e l a t i v e Retention Times and Ions obtained on G.c-c.i.-m.s. of Methylated Oligosaccharide A l d i t o l s obtained from E. c o l i K26 P o l y s a c c h a r i d e . Oligosaccharide F r a c t i o n R e l a t i v e Retention Time a (area#) Chemical i o n i z a t i o n mass (Re l a t i v e abundance) s p e c t r a l ions (m/z) (M + NH,) + (M + H) + A0H 2 + A + b C1 G l c A ^ R h a ^ R h a l ^ R h a - o l 5.80 (34) 820 (18) 223 (25) 205 (9) D1 G l c A ^ R h a ^ R h a - o l 3.34 (14) 646 (18) 223 (10) 205 (2) D2 Gal 1-^GlcA 1-=^Rha-ol /3 fi 3.54 (4) 676 (39) 223 (4) 205 (7) E1 Rhal^Rha 1-=^Gal-ol 3.02 (33) 632 615 (13) (4) 253 (80) 235 (13) a DB 5» 210° f o r 2 min. then 6°/min. to 290°. Retention times r e l a t i v e to that of sucrose (3.24 min.). b A and H represent the reducing and non-reducing sugars r e s p e c t i v e l y . TABLE IV.3 (continued) H + b (A-Me0H)+ (H-MeOH)+ Other fragment ions 233 (9) 173 (33) 201 (100) 424 233 (13) 201 (100) 407, 424 (11) (20) 219 (7) 187 (23) 437, 405 (1) (1) 189 (61) 157 (20) 380. 363, (42) (32) 205 (52) 126 F r a c t i o n C a l s o contained other h i g h e r molecular weight components. U n f o r t u n a t e l y t h e i r c.i.-mass s p e c t r a d i d not e x h i b i t pseudomolecular i o n s or high molecular weight fragment i o n s t h e r e f o r e t h e i r s t r u c t u r e s c o u l d not be-deduced. F r a c t i o n D had a main component (D1) with a r e l a t i v e r e t e n t i o n time of 3.34 min. ( F i g . 3a ) . The pseudomole-c u l a r i o n (M + NH^) + a t m/z 646 and fragment i o n s a t m/z 407, 233 and 201 (Table IV.3 , F i g . 3b) i n d i c a t e d t h a t D1 was the a l d o t r i o u r o n i c a c i d which had been i s o l a t e d d u r i n g the p r e v i o u s p a r t i a l h y d r o l y s i s . A second component D2 was a l s o p r e s e n t , with a r e l a t i v e r e t e n t i o n time of 3.34 min. ( F i g . 3a) . A s t r o n g pseudo-mol e c u l a r i o n (M + NH^) + a t m/z 676 ( F i g . 3c ) . and fragment i o n s of 437, 405, 205 and 219 were i n agreement with the f o l l o w i n g s t r u c t u r e f o r D2: £-D-Gal-(1-3)-£-D-GlcA-(1-3)-L-Rha-ol A t h i r d minor component, had a r e l a t i v e r e t e n t i o n time of 3»90 min. and a s t r o n g pseudomolecular i o n a t m/z 660 and fragment i o n s a t m/z 219 and 187. T h i s component was thus deduced t o be D2 i n which the t e r m i n a l r e d u c i n g rhamnose r e s i d u e had not been reduced to i t s a l d i t o l . F r a c t i o n E had a main component (E1) wi t h a r e l a t i v e r e t e n t i o n time of 3.02 min. ( F i g . 4a) . The pseudomolecular i o n (M + NH,) + a t m/z 632 and fragment i o n s a t ra/z 363, 253 127 D1 •D2 900 t o o 6 9 0 7 0 0 7 3 0 90O B50 900 930 F i g . IV.3a Gas chromatogram of methylated Fraction D using D B 5 c a p i l l a r y column F i g . IV.3b G.c.-c.i. mass spectrum of methylated Component DI 128 129 4b 4J too 4>H 262 j 23: , t 'I 239 300 331 347 350 487^  ^ 3j0 325 400 450 Q) -<D > • H --P 05 • CD 374 5SS " 1 r-r-p 630 661 712 740 I ' ''Pi'I • ••' ' V 770 831 eoo 914 343 " I'' ) 950 998 100 F i g . IV.4a Gas chromatogram of methylated F r a c t i o n E u s i n g DB 5 c a p i l l a r y column. F i g . IV.4b G . c . - c . i . mass spectrum of methylated Component El 130 235 and 189 ( F i g . 4b) suggested the f o l l o w i n g s t r u c t u r e f o r E1: oc-L-Rha- (1 -3) - tX-L-Rha- (1-3) -D-Gal-ol A second component of f r a c t i o n E had a r e l a t i v e r e t e n -t i o n time of 3.34 min. and a pseudomolecular i o n a t m/z 646. T h i s component was again i d e n t i f i e d as the a l d o t r i o -u r o n i c a c i d . F r a c t i o n F was a l s o analysed by g.c-c.i.-m.s. of the permethylated sample. F o r the r e s u l t s of the a n a l y s i s see S e c t i o n I I I . Reduction of f r a c t i o n s C and E, f o l l o w e d by h y d r o l y s i s and c o n v e r s i o n of the monosaccharides to t h e i r p e r a c e t y l a t e d a l d i t o l n i t r i l e s , confirmed the i d e n t i t i e s o f the r e d u c i n g sugars p r e s e n t i n each f r a c t i o n (Table IV.4). M e t h y l a t i o n a n a l y s i s c a r r i e d out on f r a c t i o n s C and E confirmed t h a t the two f r a c t i o n s comprised of mixtures of o l i g o s a c c h a -r i d e s ( r e s u l t s not shown). Lithiu m / E t h y l e n e d i a m l n e d e g r a d a t i o n . - Treatment of p o l y -s a c c h a r i d e s c o n t a i n i n g u r o n i c a c i d s w i t h l i t h i u m e t h y l e n e -diamine was found t o c l e a v e the polymer where a u r o n i c a c i d was p r e s e n t 1 ^ . U n l i k e the product of a ^ - e l i m i n a t i o n r e a c t i o n the o l i g o s a c c h a r i d e i s ob t a i n e d i n an u n d e r i v a t i s e d form and i s t h e r e f o r e amenable to f u r t h e r chemical or enzymatic d e g r a d a t i o n . The removal of one or more sugars 131 TABLE IV.4 Reducing End Determination of F r a c t i o n s . C and E from E. c o l i K26 Capsular P o l y s a c c h a r i d e A c e t y l a t e d F r a c t i o n F r a c t i o n d e r i v a t i v e C E of (mole (mole r a t i o ) r a t i o ) R h a m n o n i t r i l e 2.3 2.1 Rhamnitol 1.0 1.0 G a l a c t o n i t r i l e 0.8 1.0 G a l a c t i t o l 0.3 0.4 132 from the non-reducing end of an o l i g o s a c c h a r i d e , by e i t h e r a p e r i o d a t e o x i d a t i o n or enzymatic h y d r o l y s i s are two techniques commonly used to determine the sequence of monsaccharide u n i t s i n such an o l i g o s a c c h a r i d e (see S e c t i o n I I . 2 amd 11*3 r e s p e c t i v e l y ) . T h e r e f o r e , i t was decided to apply the l i t h i u m ethylenedamine degradation to the K26 e x t r a c e l l u l a r p o l y s a c c h a r i d e , i n order to gener-ate a n e u t r a l t e t r a s a c c h a r i d e . A Smith degradation would then be used to remove the non-reducing t e r m i n a l sugar. C h a r a c t e r i z a t i o n of the remaining t r i s a c c h a r i d e a l d i t o l would confirm the sequence of sugars i n the K26 p o l y s a c c h a -r i d e backbone. K26 p o l y s a c c h a r i d e was suspended i n ethylenediamine and r e a c t e d with l i t h i u m f o r 1 h. A f t e r s e p a r a t i o n of the pro-duct G by g e l permeation chromatography, a p o r t i o n was submitted f o r fast-atom-bombardment mass spectrometry. The f.a.b. mass spectrum (Fig.IV.5) of p e r a c e t y l a t e d G e x h i b i t e d A-type i o n s f o r the sequence:-• j • • Deoxyhex—j— Deoxyhex—•—Deoxyhex — i — H e x —!—X 273 J 503 J ll>y 1021 j An unexplained s i g n a l a t m/z 1327, p o s s i b l y an (M + H) i o n , w i t h weaker s i g n a l s a t m/z 1344 and 1349 b e i n g (M +NH^) + and (M + N a ) + r e s p e c t i v e l y , suggested the presence of a 20 c h e m i c a l l y degraded product a t t a c h e d to the hexose r e s i d u e • A n a l y s i s of |he permethylated o l i g o s a c c h a r i d e a l d i t o l 133 F i g . iv. 5 F.a.b. mass spectrum of peracetylated G. 134 by g.c.-c.i.-m.s. i n d i c a t e d t h a t the sample c o n s i s t e d predominantly of a rhamnose t r i s a c c h a r i d e a l d i t o l and s m a l l e r amounts of a rhamnose d i s a c c h a r i d e a l d i t o l and a non-reduced rhamnose-rhamnose d i s a c c h a r i d e (Table I V . 5 ) . No h i g h e r molecular weight o l i g o s a c c h a r i d e was observed. These r e s u l t s i n d i c a t e d t h a t f u r t h e r degradation of the p o l y s a c c h a r i d e had take p l a c e on treatment with l i t h i u m / ethylenediamine. T h i s phenomenon i s w e l l known and i n v o l v e s a base c a t a l y s e d ^ - e l i m i n a t i o n ( u s u a l l y i n aqueous s o l u t i o n ) t o give i n i t i a l l y 3-deoxyhex-2-enopyranose. T h i s e l i m i n a t i o n occurs most e a s i l y when there i s a 3-0 sub-s t i t u e n t to p r o v i d e a good l e a v i n g group. Since ^ - e l i m i n -a t i o n exposed the next sugar i n the o l i g o s a c c h a r i d e as a r e d u c i n g u n i t , d egradation may proceed stepwise a l o n g the c h a i n i n a process known as "peeling"!**. K26 polysaccha-r i d e with i t s backbone of 3 - l i n k e d sugars was i d e a l l y s e t up f o r t h i s process to occur. The f.a.b. mass spectrum supported t h i s c o n c l u s i o n as i t e x h i b i t e d (M + H ) + i o n s at m/z 607 and 837 c o n f i r m i n g the presence of rhamnodiol and r h a m n o t r i o l o l i g o m e r s . The spectrum d i d however e x h i b i t a fragment i o n a t m/z 1021 which was i n agreement with the presence o f a h i g h e r molecular weight o l i g o s a c c h a -r i d e , c o n s i s t i n g of three deoxyhexose u n i t s and a hexose u n i t . T h e r e f o r e these r e s u l t s were c o n f i r m a t i o n of the sequence of sugars i n the main c h a i n of the K26 p o l y s a c c h a -r i d e a l t h o u g h unambiguous c h a r a c t e r i z a t i o n of G was not. TABLE IV.5 Analysis of the Product of a Lithium Ethylenediamine Degradation of E . c o l i K26 Capsular Polysaccharide using G.c.-c.i.-m.s. Oligosaccharide Relative Retention Time a (area#) Chemical i o n i z a t i o n mass (Relative abundance) spectral ions (m/z) (M+NH^)+ (M+NH. . -MeOHV (M+H -MeOH) A0H+ Rha 1—^Rha-ol 0.56 ( 3 ) 428 ( 3 ) 223 (100) Rha^Rha «< 0.69 ( 3 ) 412 (11) 380 (25) 363 (20) Rhal-^Rha 1—^Rha-ol 2.39 ( 9 ) 602 (10) 223 (25) a DB 1 7 , ' 2 1 0 ° for 1 min. then 4°/min. to 290° TABLE IV.5 (continued) A + H + (A-MeOH)+ 206 189 173 (26) (50) ( D 189 (34) 205 189 173 (83) (70) (6) (H-MeOH)+ Other fragment ions 157 (6) 157 (9) 157 (25) 363 (50) 137 a c h i e v e d . IV. Zf CONCLUSION The s t r u c t u r e of K26 c a p s u l a r p o l y s a c c h a r i d e i s as shown i n the a b s t r a c t . The p o s t u l a t e d s t r u c t u r e i s i n agreement with the c r o s s - r e a c t i o n r e s u l t s p r o v i d e d by 1 5 H e i d e l b e r g e r ' and the s t r u c t u r e s of the d e r i v e d o l i g o s a c c h a -r i d e s as determined by g.c-c.i.-m.s. IV.5 EXPERIMENTAL General methods.- The g e n e r a l procedures and i n s t r u -mentation were the same as those d e s c r i b e d i n S e c t i o n I I I . 5 , w i t h these a d d i t i o n s . The i n f r a r e d ( i . r . ) s p e c t r a o f methyla-ted samples ( d i s s o l v e d i n CCl^) were recorded on a P e r k i n -Elmer model 457 spectrophotometer. A c e t y l a t i o n of a l d i t o l s was performed with a c e t i c a n h y d r i d e - p y r i d i n e (1:1) f o r 30 min. a t 100°. A n a l y t i c a l paper chromatography and p r e p a r a t i v e paper chromatography, f o r s e p a r a t i o n of o l i g o s a c c h a r i d e s , were performed u s i n g s o l v e n t s A or B. O l i g o s a c c h a r i d e f r a c t i o n s a f t e r g e l permeation chromatography, were l o c a t e d e i t h e r by 12 the p h e n o l - s u l p h u r i c a c i d method or by measurement of t h e i r r e f r a c t i v e i n d e x . A l d i t o l a c e t a t e s were analysed by g.c. u s i n g a DB 17 column and a programme of 180° f o r 2 min., and then 5 ° / m i n » t o 220°. P a r t i a l l y methylated a l d i t o l a c e t a t e s were analysed 138 u s i n g the same column and a programme of 180 f o r 1 min., and then 2°/min. to 250°. Methylated o l i g o s a c c h a r i d e s were ana l y s e d u s i n g a DB 5 c a p i l l a r y column and a programme of 210° f o r 2 min, then the temperature was r a i s e d to 290° a t a r a t e of 6°/min. R e t e n t i o n times r e l a t i v e t o sucrose are l i s t e d i n Table IV.3. Higher molecular weight o l i g o -s a c c h a r i d e s were l o c a t e d u s i n g a DB 1 column and a programme of 2Zf0° f o r 1 min., then 7°/min. to 290°. 1H-N.m.r. and 1^C-n.m.r. s p e c t r a were r e s p e c t i v e l y r e c o r d e d w i t h a Bruker WH-400 instrument a t a temperature o f 95° and a V a r i a n XL-300 instrument a t ambient temperature. Acetone was used as an i n t e r n a l standard (62,23 f o r 1H-n.m.r. and .31.07 P.p.ra. f o r ^C-n.m.r. s p e c t r o s c o p y ) , and a l l v a l u e s measured a g a i n s t e x t e r n a l sodium 4,4-dimethyl-if-s i l a p e n t a n e - 1 - s u l p h o n a t e . Samples (10-15 mg) were deu-terium exchanged by f r e e z e - d r y i n g s o l u t i o n s i n T>^) ( t h r e e c y c l e s ) , and the r e s i d u e was d i s s o l v e d i n D 20 f o r t r a n s f e r to 5 nun diameter tubes. P r e p a r a t i o n and p r o p e r t i e s of K26 c a p s u l a r p o l y s a c c h a r i d e . - A c u l t u r e of E s c h e r i c h i a c o l i K26 b a c t e r i a , obtained from Dr. I . 0rskov (Copenhagen), was grown on Mu e l l e r - H i n t o n agar ( c o n t a i n i n g 0.5$ (w/v) of NaCl) f o r 6 days a t 37°. The a c i d i c c a p s u l a r p o l y s a c c h a r i d e was i s o l a t e d and p u r i -f i e d by CTAB p r e c i p i t a t i o n as d e s c r i b e d p r e v i o u s l y 2 1 . The p u r i f i e d p o l y s a c c h a r i d e was shown to be homogeneous by g e l chromatography on Sepharose 4B and i t s molecular mass was estimated as 1 x 10^ d a l t o n s . The n a t i v e and depyru-139 v y l a t e d p o l y s a c c h a r i d e s were examined by H- and 13c-n.m.r and the p r i n c i p a l s i g n a l s and t h e i r assignments are reco r d e d i n Table IV.1. M e t h y l a t i o n a n a l y s i s . - A sample of K26 p o l y s a c c h a r i d e (40 mg) was converted to i t s f r e e a c i d form and methylated by the Hakomori p r o c e d u r e ^ ' 1 3 , The product showed no a b s o r p t i o n f o r h y d r o x y l groups i n i t s i . r . spectrum. The methylated p o l y s a c c h a r i d e was h y d r o l y s e d with 2M TFA (18 h, 95°) and the r e s u l t i n g p a r t i a l l y methylated sugars were ana l y s e d as t h e i r a l d i t o l a c e t a t e s d e r i v a t i v e s by g.c.-m.s ( Table IV.2, column I ) . A second sample (17 mg) of po l y s a c c h a -r i d e was d e p y r u v y l a t e d , by passage of an aqueous s o l u t i o n through a column of I.R. 120 (H*) r e s i n ( x 4 ) , and sub-sequent l y methylated. A p a r t of the product was h y d r o l y s e d and a n a l y s e d as bef o r e (Table IV,2, column I I ) . The remainder (10mg) was reduced with l i t h i u m aluminium hy-d r i d e i n oxolane (18 h, R.T.), remethylated and h y d r o l y s e d (2M TFA, 4 h, 95°)/ The g.c. r e s u l t s f o r the p a r t i a l l y methylated a l d i t o l a c e t a t e s a re given i n Table IV.2, column I I I . ^ - e l i m i n a t i o n 1 P e r m e t h y l a t e d K26 p o l y s a c c h a r i d e (30 mg) was d i s s o l v e d i n dimethyl s u l p h o x i d e and 2 , 3 - d i -methoxypropane (19 ' 1, 20 mL) and a t r a c e of p- t o l u e n e -s u l p h o n i c a c i d was added. Sodium m e t h y l s u l f i n y l m e t h y l i d e (2M, 10 mL) was added and the s o l u t i o n was s t i r r e d , under n i t r o g e n , o v e r n i g h t a t room temperature. Methyl i o d i d e (3 ML) was added to the c o o l e d r e a c t i o n mixture and the 140. mixture was s t i r r e d f o r a f u r t h e r 3 h. The methylated, u r o n i c a c i d degraded product was recovered by p a r t i t i o n between chloroform and water, and f u r t h e r p u r i f i e d by pass-age through a column of Sephadex LH 20. The product was hyd r o l y s e d (2M TFA, 4 h, 95°) and analysed as i t s p a r t i a l l y methylated a l d i t o l a c e t a t e s by g.c.-m.s. (Table IV.2, column I V ) . S e l e c t i v e h y d r o l y s i s . - K26 p o l y s a c c h a r i d e (580 mg) was d i s s o l v e d i n TFA (0.1M) and heated f o r 15 min. on a steam bath. The excess a c i d was removed by repeated co-evapora-t i o n with water, and the r e s i d u e was d i s s o l v e d i n H 20 and d i a l y s e d o v e r n i g h t . The n o n d i a l y s a b l e m a t e r i a l (230 mg) was l y o p h i l i z e d and a p o r t i o n (5 mg) was methylated, reduced and h y d r o l y s e d . G.c-m.s. a n a l y s i s of the p a r t i a l l y methy-l a t e d a l d i t o l a c e t a t e s showed t h a t 50& of a t e r m i n a l rham-nose u n i t had been removed (Table IV.2, column V ) . A" second s e l e c t i v e h y d r o l y s i s on K26 p o l y s a c c h a r i d e (80 mg) u s i n g TFA (10 mL, 0.1M, 30 min., 95°), a f t e r s e p a r a t i o n of the product on B i o - G e l P2, gave a product which had a t l e a s t 80$ of the t e r m i n a l rhamnose removed. T h i s r e s u l t and the presence of a s m a l l s i g n a l a t 23.00 p.p.m. allowed the assignment o f a r e s i d u a l s i g n a l a t 101.44 p.p.m. to rhamnose wi t h i t s pyruvate s u b s t i t u e n t s t i l l a t t a c h e d , and the s i g n a l a t 99.65 p.p.m. was a s s i g n e d to the t e r m i n a l rhamnose without a pyruvate s u b s t i t u e n t , i n the spectrum of the s e l e c t i v e l y h y d r o l y s e d product . 1 8 P e r i o d a t e O x i d a t i o n and Smith deg r a d a t i o n A sample of 141 depyruvylated K26 p o l y s a c c h a r i d e (15 mg) was d i s s o l v e d i n sodium metaperiodate s o l u t i o n (0.1M, 10 mL). The s o l u t i o n was kept i n the dark a t room temperature. A f t e r 72 h, ethylene g l y c o l (0.5 mL) was added and the polyaldehyde was reduced with sodium bor o h y d r i d e . The excess NaBH^ was n e u t r a l i s e d with a c e t i c a c i d (50%) and the product was d i a l y s e d a g a i n s t tap water. The r e s u l t a n t p o l y o l was l y o -p h i l i z e d and subsequently h y d r o l y s e d with 0.5M TFA (16 h, 25°). The product was d e s a l t e d by passage through a Sepha-dex G10 column and a p o r t i o n (5 mg) was methylated and hy d r o l y s e d (2M TFA, 18 h, 100°). The p a r t i a l l y methylated sugars were ana l y s e d as t h e i r a l d i t o l a c e t a t e s by g.c.-m.s. (Table IV.2, column V I I ) . P a r t i a l h y d r o l y s i s . - A s o l u t i o n o f the s e l e c t i v e l y h y d r o l y -sed polymer i n M TFA was heated f o r 1 h (95°). - A f t e r removal o f the excess TFA by co - e v a p o r a t i o n with water, the product was d i a l y s e d (mol. wt. c u t - o f f 3,500) a g a i n s t d i s -t i l l e d water. The n o n - d i a l y s a b l e m a t e r i a l was l y o p h i l i z e d and s u b j e c t e d to a second h y d r o l y s i s (M TFA, 1 h, 95°), f o l l o w e d by d i a l y s i s . The d i a l y s a t e s were combined, l y o -p h i l i z e d and the o l i g o s a c c h a r i d e s were subsequently separa-ted by p r e p a r a t i v e paper chromatography ( s o l v e n t A ) . Two a c i d i c o l i g o s a c c h a r i d e s (A and B) were obtained of s u f f i c i e n t p u r i t y and y i e l d to m e r i t f u r t h e r a n a l y s i s . On the b a s i s of i t s ^ H-n.m.r. spectrum,, A (5 mg) was deduced to be the a l d o b i o u r o n i c a c i d 1 1 and was not analysed f u r t h e r . An aqueous s o l u t i o n of B (5 mg) was reduced w i t h excess NaBH^ 142 (3 h, R.T.) and the reduced o l i g o s a c c h a r i d e was methylated reduced and analysed as bef o r e (Table IV.2, column V I I I ) . The procedure used f o r the second p a r t i a l h y d r o l y s i s has been d e s c r i b e d i n S e c t i o n I I I . 5 . A f t e r s e p a r a t i o n of the h y d r o l y s a t e on B i o - G e l P2, f o u r f r a c t i o n s were obtained (C, D, E and F ) . Each f r a c t i o n was analysed by n.m.r. spect r o s c o p y and g.c.-c.i.-m.s. of i t s reduced, methylated d e r i v a t i v e ( Table IV.3) . P e r a c e t y l a t e d a l d o n i t r i l e s . - A sample o f f r a c t i o n s C and E (2 mg) were reduced with sodium borohydride (3 h, R.T.), the r e d u c i n g sugar converted to i t s a l d i t o l . The excess borohydride was removed by the a d d i t i o n of I.R. 120 H* r e s i n , the s o l u t i o n was f i l t e r e d and co-evaporated to dryness w i t h methanol (3x). The reduced samples were h y d r o l y s e d (2M HC1, 18 h, 100°) and evaporated to dryness. The a l d o s e s were r e a c t e d under anhydrous c o n d i t i o n s , w i t h 5$ hydroxylamine HCl i n p y r i d i n e (0.2 mL/mg of a l d o s e ) , f o r 15 min. a t 9 5 ° , to produce the oximes. A f t e r c o o l i n g , a c e t i c anhydride (0.2 mL/mg of aldose) was added and heat-i n g continued f o r a f u r t h e r 30 min. to dehydrate the oxime to the n i t r i l e and to a c e t y l a t e the f r e e h y d r o x y l groups. G.c. a n a l y s i s gave the r e s u l t s shown i n Table IV.4. L i t h i u m / Ethylenediamine d e g r a d a t i o n . - K26 po l y s a c c h a -r i d e (130 mg) was d r i e d and suspended i n ethylenediamine (7 mL), with s t i r r i n g , f o r h. Three s m a l l p i e c e s of l i t h i u m wire ( 3 x 3 mm), washed i n hexane, were added at 15 min. i n t e r v a l s . The r e a c t i o n began almost immediately and a deep bl u e c o l o u r was maintained f o r 1 h. The r e a c t i o n 143 was terminated by the a d d i t i o n of 1 - 2 mL anhydrous methanol, the r e a c t i o n mixture being c o o l e d i n i c e water. The excess ethylenediamine and methanol were removed under vacuum, over H 2S0^ and NaOH, f o r 3 h. One or two drops c o n c e n t r a t e d CH^COOH were added to the r e s i d u e , with c o o l -i n g , to d e s t r o y the l i t h i u m methoxide, and the product then d i s s o l v e d i n H^O (4 mL). The mixture was d e s a l t e d on a Sephadex 610 column, the f r a c t i o n s c o n t a i n i n g sugar ( d e t e r -12 mined by the p h e n o l - s u l p h u r i c a c i d t e s t ) were pooled and separated on a B i o - G e l P2 column. The a c e t y l a t e d o l i g o s a c c h a r i d e G was analysed by f.a.b.-m.s and methyla-t i o n of G, f o l l o w e d by a n a l y s i s by g.c.-c.i.-m.s. gave the r e s u l t s shown i n T a b l e TV. 5 Determination of a b s o l u t e c o n f i g u r a t i o n . - A s o l u t i o n of c arbodiimide-reduced K26 p o l y s a c c h a r i d e (16.0 mg) i n 2M TFA was h y d r o l y s e d o v e r n i g h t (95°)• The monosaccharides were separated by p r e p a r a t i v e chromatography ( s o l v e n t B ) . Each sugar (2 mg) was r e f l u x e d i n ( - ) - 2 - o c t a n o l p l u s one drop TTFA (95°, 18 h ) . The products were co n c e n t r a t e d to dryness under vacumn (50°) and a c e t y l a t e d ( 1 : 1 a c e t i c a c i d — p y r i d i n e , i h, 95°)• The a c e t y l a t e d o c t y l g l y c o -s i d e s were ana l y s e d by g.c.-m.s. (DB 17 a t 180° f o r 2 min. then 5°/min. to 220°) and the a b s o l u t e c o n f i g u r a t i o n s of the sugars determined by c o - i n j e c t i o n with a u t h e n t i c s t a n d a r d s . ACKNOWLEDGEMENT This research was supported by the Natural Sciences and Engineering Research Council of Canada. The author would l i k e to thank S.C. Churms for providing the M r for K26 polysaccharide and Z. Lara and C. M. Moxham for running the g.c.-c.i.-m.s. 145 CHAPTER V BACTERIOPHAGE-ASSOCIATED ENZYMATIC DEGRADATION OF E. c o l i K 4 9 CAPSULAR POLYSACCHARIDE 146 V BACTERIOPHAGE-ASSOCIATED ENZYMATIC DEGRADATION OF E. c o l i K^q CAPSULAR POLYSACCHARIDE V.1 ABSTRACT A bacteriophage-associated enzyme degradation was undertaken on the native polysaccharide of E. coli, K49. 1H-N.m.r. spectroscopy, reducing end determination and methylation analysis were used to characterize the pro-duct 1. Using t h i s information i t was possible to deduce two possible a l t e r n a t i v e s for the saccharide sequence i n the tetrasaccharide repeating unit of E. c o l j K49 capsular polysaccharide. The correct assignment for 1 was made aft e r the i s o l a t i o n of a aldobiouronic acid, from K 4 9 polysaccharide, by p a r t i a l hydrolysis.(see Section V I . 3 ) . 1 GlcA 1 ^Gal 5 ^Glc 1 5GalNAc — OH V . 2 INTRODUCTION Preliminary s t r u c t u r a l studies of E. c o l i K49 poly-saccharide had suggested i t contains a tetrasaccharide repeating unit comprising glucose, galactose, 2-acetamido-2-deoxygalactose and glucuronic a c i d . The polysaccharide was also found to have two amino acids, threonine and serine, present as substituents. In the normal course of events, the bacteriophage-associated endoglycanase i s expected to 147 cleave the polysaccharide so as to produce a non-reducing terminal glucuronic a c i d . Furthermore, the reducing end 22 sugars formed are usually substituted at position 3 . In previous cases where amino acids are attached to E. c o l i capsular polysaccharides 2-^ » 2 Z f, they have been found to be amidically-linked to the glucuronic acid residue. This was l a t e r found to be true for K49 polysaccharide (see Section VI.3). It was informative to discover that the s u b s t i t u t i o n of the glucuronic acid residues i n no way altered the expected s i t e of cleavage - the endoglycanase produced by tfk9 exhibited £-N-acetyl-galactosaminidase a c t i v i t y . The f i r s t reported example of a bacteriophage-associated enzyme of t h i s nature, i s o l a t e d from E„ c o l i 25 phages, was reported by Lim . V.3 RESULTS AND DISCUSSION E. c o l i f6k9i i s o l a t e d previously from sev/age 1 0, exhibited small plaques with large halos.when spotted on a lawn of i t s host bacteria. Halos are usually attributed to over production of endoglycanase and are a sign of a strong phage. The phage was propagated on i t s host s t r a i n E. c o l i K49, using Mueller-Hinton broth as a medium. Propagation was car r i e d out u n t i l a t o t a l of 0.4 x 10 1^ phage p a r t i c l e s was achieved. 26 The method of Dutton and colleagues , s l i g h t l y modi-f i e d , was employed i n the depolymerization of K49 polysaccha-148 r i d e . Products from p r e v i o u s phage degradations of E. c o l i c a p s u l a r p o l y s a c c h a r i d e s , undertaken i n t h i s l a b o r a -10 25 27 t o r y » -7» were contaminated by an i m p u r i t y which proved d i f f i c u l t to remove. Tb minimize the p r o b a b i l i t y of the product from the j6 d e g r a d a t i o n of K49 p o l y s a c c h a r i d e being contaminated, the degr a d a t i o n was c a r r i e d out i n a d i a l y s i s bag. Furthermore, there i s evidence that glycanase a c t i v i -t y i s i n h i b i t e d by the r e l e a s e of i t s p r o d u c t s 2 ^ . There-f o r e , i t was hoped t h a t the y i e l d of the product would be i n c r e a s e d under such r e a c t i o n c o n d i t i o n s . Niemann and coworkers had a l s o demonstrated that f o r the depolymeriza-t i o n of K l e b s i e l l a K13 p o l y s a c c h a r i d e by /rf13> r e a c t i o n v e l o c i t y decreased a t s u b s t r a t e c o n c e n t r a t i o n s of above 28 ImM . Thus, the phage-polysaccharide mixture was made up to a c o n c e n t r a t i o n of*0.5mM, incubated f o r two days at 37° w h i l e d i a l y s i n g a g a i n s t d i s t i l l e d water. The cloudy d i a l y s a t e s were pooled, U l t r a c e n t r i f u g e d , exchanged wit h I.R. 120 H + r e s i n .and c o n c e n t r a t e d . S e p a r a t i o n of the product, on a B i o - G e l P4 column, gave two products, one which e l u t e d from the column i n the v o i d volume 1 and a second which e l u t e d l a t e r 2. 1H-N.m.r. s p e c t r o s c o p y ; - The n.m.r. data f o r the higher m o l e c u l a r weight product 1 and the b a s e - t r e a t e d K49 p o l y s a c c h a r i d e a re reco r d e d i n Table V.1. The presence of a s i g n a l a t low f i e l d (S5.50) which c o u l d be assign e d to the tX-anomer of a r e d u c i n g sugar confirmed t h a t the TABLE V.1 ^H-n.m.r. Data f o r the Bacteriophage Degradation Product ( 1 ) and E. c o l i K49 Capsular Polysaccharide. Compound H-n.m.r. data 6 a (p.p.m.) J1,2 (Hz) Integral Assignment -^GlcA V ^ G a l - r ^ G l c U^GalNA c L K49 polysaccharide (base treated) G l c A 1 — ^ G a l ^ G l c 1-^GalNA c f P f P1 4.62 4.60 4.58 4.53 4.41 2.04 1.25 7-8 7-8 7-8 s 8 7-8 7-8 7-8 1 1 3 3 0.4 0.8 1 3 3 Thr(Ser) ^GlcA 1-i a i e V ' 3 P 1 — GalNAc — i G . i ' r / CH3 (acetaraido) CH3 (threonine) -^GalNAc 1«0H ' GlcA 1-- S i c 1 / — GalNAc ^ OH CH3 (acetamido) CH3 (threonine) -P-aChemical s h i f t r e l a t i v e to i n t e r n a l acetone assigned at 6"2.23 downfield from external sodium 4,4-dimethyl-4-silapentane-1-sulphonate (D.S.S.) b Key: b = broad, unable to assign accurate coupling constant; s = s i n g l e t . c E.g. J±GlcA~ r e f e r s to the anomeric proton of a ^ - l i n k e d glucurono8yl residue i n the p-anomeric configuration. The absence of a numeral p r e f i x i n d i c a t e s a non-reducing terminal group. 150 sample although a p p a r e n t l y of high molecular weight was an o l i g o s a c c h a r i d e . The r e l a t i v e i n t e n s i t y of the s i g n a l i n comparison to t h a t of an i n cha i n anomeric proton (40 : 80)suggested t h a t the sample was a s i n g l e r e p e a t i n g u n i t i . e . P1 of the p o l y s a c c h a r i d e . Reduction of the o l i g o s a c c h a r i d e with NaBH^ r e s u l t e d i n the l o s s of the s i g n a l a t S5.50 and the apparent i n c r e a s e i n i n t e n s i t y of the s i g n a l at §4.58. However, r e s o l u t i o n o f the spectrum a f t e r r e d u c t i o n was poor and i t was decided that i t would not be p o s s i b l e to o b t a i n a good spectrum. The ^-n.m.r spectrum of the lower molecular weight f r a c t i o n 2 showed two main s i g n a l s i n the anomeric r e g i o n (55.36, S"5«10 b r o a d ) . These same •contaminant' anomeric s i g n a l s were a l s o observed i n the 1H-n.m.r. spectrum of the products from the phage-depolymerization of the c a p s u l a r p o l y s a c c h a r i d e s of E. c o l i K 2 6 1 0 , K 3 1 2 7 and K 4 4 2 5 . Sugar a n a l y s e s . - The o l i g o s a c c h a r i d e 1 was h y d r o l y s e d , u s i n g i n c r e a s i n g l y v i g o r o u s c o n d i t i o n s , but the r a t i o s of the a l d i t o l a c e t a t e s produced d i f f e r e d from those of the n a t i v e p o l y s a c c h a r i d e (see Table V . 2 ) . The r e l a t i v e l y high amount of glu c o s e was a t t r i b u t e d to some of the contaminant remaining a f t e r s e p a r a t i o n on B i o - G e l P 4 . A n a l y s i s of the h y d r o l y s a t e by paper chromatography gave the same r e s u l t s as a n a l y s i s o f the n a t i v e p o l y s a c c h a r i d e . Both amino a c i d s , t h r e o n i n e and s e r i n e , were i d e n t i f i e d from t h e i r R f v a l u e s . 151 Reducing; end d e t e r m i n a t i o n . - The t e r m i n a l r e d u c i n g sugar of 1 was converted to i t s a l d i t o l u s i n g sodium borohydride and the o l i g o s a c c h a r i d e was h y d r o l y s e d . The a l d o s e s r e l e a s e d were converted to t h e i r p e r a c e t y l a t e d a l d o n i t r i l e s . No evidence f o r the presence of a r e d u c i n g end sugar, as i t s a l d i t o l a c e t a t e , was observed on a n a l y s i s of the sample by g.c.-m.s. The presence of g a l a c t o n i t r i l e and g l u c o -n i t r i l e i n a 1 : 1 r a t i o d i d , however, show that n e i t h e r of those r e s i d u e s formed the r e d u c i n g end of the o l i g o s a c c h a -r i d e (Table V . 4 ) . M e t h y l a t i o n a n a l y s i s . - A n a l y s i s was conducted on the reduced o l i g o s a c c h a r i d e a l d i t o l 1• Because r e d u c t i o n of the c a r b o x y l f u n c t i o n of the methylated n a t i v e p o l y s a c c h a -r i d e had been u n s u c c e s s f u l ( l a t e r shown to be a consequence o f the u r o n i c a c i d b e i n g a m i d i c a l l y s u b s t i t u t e d with amino a c i d s , see S e c t i o n VI.3) the methylated o l i g o s a c c h a r i d e was not reduced p r i o r to h y d r o l y s i s . On a n a l y s i s of the p a r t i a l l y methylated a l d i t o l a c e t a t e s by g.c.-m.s., n e i t h e r a r e d u c i n g end sugar nor a non-reducing end r e s i d u e were observed. The presence of 2,3,4-tri-£-methylglucose and 2,3»4-tri-0-methylgalactose i n a 1 : 1 r a t i o d i d however i n d i c a t e t h a t glucose and g a l a c t o s e form the middle r e s i d u e s of the t e t r a s a c c h a r i d e (Table V . 3 ) . A n a l y s i s by g.c.-c.i.-m.s. of the methylated contami-nant 2 i d e n t i f i e d i t as a t r i s a c c h a r i d e composed of three hexose r e s i d u e s . The mass spectrum e x h i b i t e d a (M + NH^) + pseudomolecular i o n peak a t m/z 676 and fragment i o n s at 152 TABLE V.2 Sugar A n a l y s i s of K49 Polysaccharide and Bacteriophage Degradation Product ( 1) Sugar Mole % (as a l d i t o l acetate) I II III Glucose 2 .0 1.5 2.7 Galactose 1 . 9 1.5 1.0 Galactosamine 1 .1 1.3 O.if I, polysaccharide hydrolysed with i+M HC1 (k h, 9 5 ° ) . I I , polysaccharide hydrolysed with HF (3 h, R.T). I I I , PI hydrolysed with 4M HC1 (3 h, 9 5 ° ) . TABLE V .3 Methylation Data f o r the Bacteriophage Degradation Products 1 & 2. Methylated Mole r a t i o b -S ugar a (as a l d i t o l acetate) 2,3,4,6-Glc 36.1 2,3 ,4 ,6-Galc 9.2 2,3,4-Glc 1.0 22.5 2,3,4-Gal 0.98 8.6 a 2 ,3,4,6-Glc = 1 ,5-di-£-acetyl-2,3,4,6-tetra-O- m e t h y l g l u c i t o l , e t c . b Values are c o r r e c t e d by use of the e f f e c t i v e carbon-response f a c t o r s given by Albersheim and coworkers5°. The presence of two non-reducing terminal Sugars suggests that the sample i s a mixture of two o l i g o s a c c h a r i d e s . 153 TABLE V.if Reducing End Determination of the Bacteriophage Enzyme Degradation Products 1 & 2 A c e t y l a t e d Mole Ratio• D e r i v a t i v e of ^ G l u c o n i t r i l e G a l a c t o n i t r i l e G l u c i t o l 1 .0 1.0 1 .5 0.7 0.2 154 m/z i+23, 219 and 187. 1 6 1 6 2 G l c - ^ - G l c — G l c ~ 0 H H y d r o l y s i s of the methylated 2 gave the r e s u l t s l i s t e d i n Table V.3 The s t r u c t u r e of 2 was determined to be as shown above. The r e d u c i n g end r e s i d u e was confirmed by formation of the p e r a c e t y l a t e d a l d o n i t r i l e s from the reduced, h y d r o l y s e d o l i g o s a c c h a r i d e (Table V . 4 ) . V.4 CONCLUSION The data obtained from the a n a l y s i s of 1 allowed some c o n c l u s i o n s to be drawn about the s t r u c t u r e of the t e t r a -s a c c h a r i d e r e p e a t i n g u n i t of the n a t i v e K49» I t was c l e a r t h a t the glucose and g a l a c t o s e r e s i d u e s were l i n k e d together. As i t i s known t h a t a phage-associated endoglycanase normally 22 does not produce a t e r m i n a l r e d u c i n g u r o n i c r e s i d u e , then there are two p o s s i b l e a l t e r n a t i v e s t r u c t u r a l sequences f o r 1, and t h e r e f o r e f o r the r e p e a t i n g u n i t of the n a t i v e poly-^ s a c c h a r i d e : 1a GlcA G a l G l c GalNAc ~ OH 1b GlcA G l c G a l G a l N A c — O H When l a t e r work r e s u l t e d i n the i s o l a t i o n o f the 155 a l d o b i o u r o n i c a c i d (see S e c t i o n VI.3) then 1b was e l i m i n -a t e d . The assignment of the sugar sequence was confirmed when a second d i s a c c h a r i d e was i s o l a t e d . Although a n a l y s i s of t h i s d i s a c c h a r i d e by g.c.-c.i.-m.s. gave r e t e n t i o n times and a pseudomolecular i o n compatible with a hexose-hexosamine d i s a c c h a r i d e : , h y d r o l y s i s produced on l y 2»3»4,6-tetra-Q.-methylglucose. Other o l i g o s a c c h a r i d e s obtained from the n a t i v e p o l y s a c c h a r i d e c o n t a i n i n g the galactosamine r e s i d u e , but not a t the r e d u c i n g t e r m i n a l , presented no problem on m e t h y l a t i o n a n a l y s i s and a 3-l i n k e d galactosamine was observed (see S e c t i o n V I . 3 ) . Thus, the absence of galactosamine on m e t h y l a t i o n a n a l y s i s appears to be a s s o c i a t e d with i t s presence a t the r e d u c i n g end of an o l i g o s a c c h a r i d e . T h i s o b s e r v a t i o n s u b s t a n t i a t e s the assignment of galactosamine as the r e d u c i n g t e r m i n a l r e s i d u e of 1. V.5 EXPERIMENTAL Gener a l methods.- Instrumentation and g e n e r a l procedures were b a s i c l y the same as those d e s c r i b e d f o r E. c o l i K26. However, a B i o - G e l P4 column was used to s e p a r a t e the phage product and n i n h y d r i n was employed f o r de v e l o p i n g chroma-tograms^^.when i t was necessary to i d e n t i f y amino a c i d s . Propagation o f f& 9 . - Phage $9, i s o l a t e d p r e v i o u s l y 1 0 , was propagated on i t s host bacterium E t c o l j KZf9 u s i n g M u e l l e r -156 Hinton b r o t h as a medium. S u c c e s s i v e propagations u s i n g the technique of f l a s k l y s i s were conducted as f o l l o w s : An o v e r n i g h t b a c t e r i a l c u l t u r e (2 mL) was added to M u e l l e r - H i n t o n b r o t h (100 mL x 6) and the c u l t u r e shaken a t 3 7 ° A f t e r 1 h, 2 mL of phage suspension i n b r o t h (cone. 2 . 4 x 1 0 1 0 p.f.u./mL) was added to the cloudy b a c t e r i a l c u l t u r e and the mixture was shaken u n t i l i t c l e a r e d (1 h ) . The b a c t e r i a l d e b r i s was removed by low speed c e n t r i f u g a t i o n and c h l o r o f o r m was added to the 6 suspension to prevent f u r t h e r b a c t e r i a l growth. A s e r i a l d i l u t i o n of the phage suspension was prepared and u s i n g a c a l i b r a t e d Pasteur p i p e t t e , drawn to a f i n e p o i n t , a drop of each d i l u t i o n was a p p l i e d to a lawn of the host bacterium. The p.f.u./mL were c a l c u l a t e d from the number of phage plaques formed, a f t e r i n c u b a t i o n of the lawn o v e r n i g h t . Three propagations were necessary to o b t a i n a s u f f i -c i e n t q u a n t i t y of phage. The 6 suspensions were pooled ( 2 . 4 L, cone. 1.1 x l O 1 ^ p . f . u . ) , c o n c e n t r a t e d (1 L ) , d i a l y s e d a g a i n s t d i s t i l l e d water (48 h) and r e c o n c e n t r a t e d to 500 mL (cone. 0 .4 x l O 1 ^ p.f.u.) D e p o l v m e r i z a t i o n o f E. c o l i K4Q- p o l y s a c c h a r i d e and i s o l a t i o n of the product.- The phage suspension was added to the p o l y s a c c h a r i d e (200mg) to g i v e a f i n a l c o n c e n t r a t i o n of-~0.5mM (pH 6.9). The r e a c t i o n mixture was t r a n s f e r r e d to a d i a l y s i s tube, a few drops of c h l o r o f o r m were added and d e p o l y m e r i z a t i o n was c a r r i e d out f o r 2 d a t 3 7 ° . 157 D i a l y s i s was c a r r i e d out a g a i n s t d i s t i l l e d water ( 3 x 1 L ) . The d i a l y s a t e s were subsequently combined, u l t r a -c e n t r i f u g e d , exchanged with I.R. 120 H + r e s i n ( 2 x ) , c o n c e n t r a t e d and d e s a l t e d on a SephadexGIO column ( y i e l d 30 mg). T h i s product was then separated i n t o two f r a c t i o n s (1 and 2) on a B i o - G e l Plf column. The y i e l d s of the o l i g o -s a c c h a r i d e s a f t e r s e p a r a t i o n were (1) 7 mg, (2) 8 mg. The products were analysed by 1H-n.m.r. spec t r o s c o p y . The data i s presented i n Table V.1. Sugar a n a l y s i s . - A sample of 1 (1 mg) was h y d r o l y s e d u s i n g ifM HC1 f o r k h a t 100°. A f t e r removal of the excess a c i d , under d i m i n i s h e d p r e s s u r e , the h y d r o l y s a t e was analysed by paper chromatography, u s i n g s o l v e n t s A and B. Glucose, g a l a c t o s e , galactosamine and threonine and s e r i n e were a l l observed t o be present i n the h y d r o l y s a t e . How-ever, on a n a l y s i s of the h y d r o l y s e d sugars as t h e i r a l d i t o l a c e t a t e s , by g.c.-m.s., a r e l a t i v e l y h i g h amount of glucose and low amounts of galactosamine, i n comparison to the n a t i v e p o l y s a c c h a r i d e , were observed. V a r y i n g the h y d r o l y t i c c o n d i t i o n s d i d not r e s u l t i n any v a r i a t i o n s i n the r e l a t i v e amounts of the sugars (Table V . 2 ) . Reducing end d e t e r m i n a t i o n . - Samples of 1 and 2 (5 mg) were reduced w i t h sodium bo r o h y d r i d e , A p o r t i o n of each (1 mg) was h y d r o l y s e d (/+M TFA, 1h 125°) and converted to t h e i r p e r a c e t y l a t e d a l d o n i t r i l e s u s i n g the method d e s c r i b e d 158 i n S e c t i o n I V . 5 . M e t h y l a t i o n a n a l y s i s . - The remaining o l i g o s a c c h a r i d e a l d i t o l (1) and non-reduced (2) were methylated a c c o r d i n g to the Hakomori procedure^. The presence of excess base was checked a f t e r 1 h by removal of an a l i q u o t from each r e a c t i o n mixture and i t s . a d d i t i o n to a few g r a i n s of t r i -phenylmethane. The development of a deep r e d c o l o u r confirmed the presence of excess base. The methylated products were p u r i f i e d by e x t r a c t i o n with CH 2 C1 2/H 2 0 . Methylated 2 was anal y s e d by g.c.-c.i.-m.s. p r i o r to h y d r o l y s i s . Each sample was h y d r o l y s e d with 2M TFA f o r 6 h a t 9 5 ° . The r e s u l t s of these m e t h y l a t i o n a n a l y s e s are g i v e n i n Table V . 3 . ACKNOWLEDGEMENT T h i s r e s e a r c h was supported by the N a t u r a l S c i e n c e s and E n g i n e e r i n g Research C o u n c i l of Canada. The author would l i k e to thank Dr. I . 0rskov f o r p r o v i d i n g a c u l t u r e of E. c o l i KZf9. 159 CHAPTER VI STRUCTURAL INVESTIGATION OF AN AMINO ACID CONTAINING CAPSULAR POLYSACCHARIDE FROM E. c o l i SEROTYPE 08 : Ki+9 : H21 1 6 0 V I . STRUCTURAL INVESTIGATION OF AN AMINO ACID CONTAINING CAPSULAR POLYSACCHARIDE FROM E. c o l i SEROTYPE 08 : K 4 9 : H21 VI.1 ABSTRACT The E. c o l i K49 c a p s u l a r a n t i g e n and the o l i g o s a c c h a -r i d e s d e r i v e d from i t , by p a r t i a l a c i d h y d r o l y s i s , HF h y d r o l y s i s and Smith d e g r a d a t i o n , were s t u d i e d u s i n g 1H-and ^c-n.m.r. spectroscopy, g.c.-c.i.-m.s. and me t h y l a t i o n a n a l y s i s . The Ki+9 p o l y s a c c h a r i d e . c o n s i s t s of -*4)-J?-D-GlcA-( 1 -»6)-P-D-Gal-( 1 ->6)-£-D-Glc-( 1 -»3) -£-D-GalNAc-( 1 -> r e p e a t i n g u n i t s . The g l u c u r o n i c a c i d r e s i d u e s are s u b s t i -t u t e d , i n the molar r a t i o s of 3 : V , with L - t h r e o n i n e and s e r i n e a m i d i c a l l y l i n k e d to the c a r b o x y l groups. The K49 p o l y s a c c h a r i d e has a r e l a t i v e molecular weight of 420 000. VI.2 INTRODUCTION P y r u v y l a t i o n , or Q - a c e t y l a t i o n , i s common i n m i c r o b i a l p o l y s a c c h a r i d e s , but s u b s t i t u t i o n with amino a c i d s i s r e l a t i v e l y r a r e . Amino a c i d s are found i n t e i c h o i c - a c i d s where they are e s t e r - l i n k e d ^ . N - A c e t y l a l a n i n e has been encountered a m i d i c a l l y l i n k e d to the c e l l w a l l p o l y s a c c h a -r i d e s o f E. c o l i OITZf^1 a n d S. a u r e u s ^ 2 . S e r i n e i s amidi-c a l l y l i n k e d through i t s amino group to the S h i g e l l a b o v d i i 161 type 8 a n t i g e r r ^ and the l i p o p o l y s a c c h a r i d e of Proteus  m i r a b i l i s s t r a i n 1959^ c o n t a i n s a m i d i c a l l y l i n k e d l y s i n e . The c a p s u l a r p o l y s a c c h a r i d e from Haemophilus i n f l u e n z a type d c o n t a i n s t h r e e amino a c i d s , a l a n i n e , s e r i n e and thre o n i n e l i n k e d to C-6 of the D-mannosyluronic r e s i d u e s as a m i d e s ^ T h e r e are two pr e v i o u s examples of E. c o l i c a p s u l a r p o l y s a c c h a r i d e s with amino a c i d s u b s t i t u e n t s . The K5k p o l y s a c c h a r i d e c o n s i s t s of - » 3 ) - p- D- QlcA-( l *3)-oc-L-Rha-(1* r e p e a t i n g u n i t s , of which 85^ of the g l u c u r o n i c a c i d r e s i d u e s are s u b s t i t u t e d i n the r a t i o s of 9 : 1 w i t h L - t h r e o n i n e and L - s e r i n e , a m i d i c a l l y l i n k e d to the c a r b o x y l g r o u p 2 ^ . The KZfO p o l y s a c c h a r i d e c o n s i s t s of ->4)-p-D-GlcA-( 1 ->if)i<X-D-GlcNAc-( 1 -»6)-«.-D-GlcNAc-( 1 -» r e p e a t -i n g u n i t s , a l l g l u c u r o n i c a c i d r e s i d u e s are s u b s t i t u t e d a m i d i c a l l y with L - s e r i n e 2 ^ , That the s e r i n e s u b s t i t u e n t was the immunodominant group was i n d i c a t e d by the f a c t t h a t E. c o l i K 5 p o l y s a c c h a r i d e d i d not r e a c t with an a n t i -KZfO antiserum. The K 5 p o l y s a c c h a r i d e has the same r e p e a t i n g u n i t as the KZfO p o l y s a c c h a r i d e but l a c k s the s e r i n e s u b s t i -t u e n t . The s t r u c t u r e of the c a p s u l a r K49 a n t i g e n of E. c o l l i 08 : K/f9 : H21 , which i s an amino a c i d - c o n t a i n i n g p o l y -s a c c h a r i d e , i s now r e p o r t e d . V I . 3 RESULTS AND DISCUSSION I s o l a t i o n and c h a r a c t e r i z a t i o n . - E. c o l i K49 b a c t e r i a were 162 grown on s o l i d medium a t room temperature. The a c i d i c p o l y s a c c h a r i d e was i s o l a t e d by a sequence of p r e c i p i t a -t i o n with cetyltrimethylammonium bromide (CTAB )37, e x t r a c -t i o n with aqueous sodium c h l o r i d e and p r e c i p i t a t i o n with with e t h a n o l . The product was monodisperse by g e l -permeation chromatography (M p = ^ 20 000). Treatment of the n a t i v e p o l y s a c c h a r i d e with d i l u t e base (0.1M NaOH) r e s u l t e d i n a decrease i n the r e l a t i v e m olecular weight (Mr = 80 000). G.c. a n a l y s i s of the a l d i t o l a c e t a t e s , prepared from the products o f an a c i d h y d r o l y s i s of the n a t i v e p o l y s a c c h a r i d e , gave g l u c o s e , g a l a c t o s e and g a l a c t o s -amine i n the molar r a t i o s 2.0 : 1 ,9 ' l . l . H y d r o l y s i s at room temperature f o r 3 h with anhydrous HF gave gl u c o s e , g a l a c t o s e and galactosamine i n the molar r a t i o s 1 .5 •' 1 «5 1 .3 (see Table V . 2 ) . The a b s o l u t e c o n f i g u r a t i o n s of glucose and g a l a c t o s e were determined as D by g.c. of t h e i r (-)-2-o c t y l e s t e r d e r i v a t i v e s . Amino a c i d a n a l y s i s .revealed t h a t the p o l y s a c c h a r i d e a l s o c o ntained s e r i n e and threonine r e s i d u e s i n the molar p r o p o r t i o n s of 0.1 and 0.J+ r e s p e c t t i v e l y , r e l a t i v e t o galactosamine, however, 1H-n.m.r. data showed t h a t t h r e o n i n e was present i n an approximately -1 : 1 r a t i o w i t h galactosamine (see below). The a b s o l u t e c o n f i g u r a t i o n s of th r e o n i n e and galactosamine were assig n e d as L and D r e s p e c t i v e l y by the formation of t h e i r (+)-2-b u t y l e s t e r d e r i v a t i v e s . The a b s o l u t e c o n f i g u r a t i o n o f s e r i n e c o u l d not be e s t a b l i s h e d although three attempts were made to prepare the (+)-2-butyl e s t e r d e r i v a t i v e . 163 Nature of the amino a c i d s u b s t i t u t i o n . - The amino a c i d s could not be removed from the p o l y s a c c h a r i d e by m i l d t r e a t -ment with d i l u t e base (0.1M NaOH, 2 h, R.T.), i n d i c a t i n g t h a t they were a m i d i c a l l y bound, which was i n agreement wit h n.m.r. data (see below). Degradation of the p o l y -s a c c h a r i d e o c c u r r e d when more d r a s t i c c o n d i t i o n s were used (1M NaOH, 6 h, 50°, under n i t r o g e n ) . M e t h a n o l y s i s and r e d u c t i o n of the n a t i v e K49 p o l y s a c c h a r i d e had p r e v i o u s l y i n d i c a t e d t h a t a g l u c u r o n i c a c i d r e s i d u e was a l s o present i n the r e p e a t i n g u n i t 1 0 . However, treatment of the p o l y -38 s a c c h a r i d e with carbodiimide-sodium borohydride d i d not i n c r e a s e the r e l a t i v e amount of glucose on h y d r o l y s i s of the reduced p o l y s a c c h a r i d e , i n d i c a t i n g t h a t the amino a c i d s were amide-linked to the g l u c u r o n i c a c i d . M e t h y l a t i o n a n a l y s i s . - The K49 p o l y s a c c h a r i d e was methy-l a t e d a c c o r d i n g to the Hakomori^ method and then h y d r o l y s e d . The p a r t i a l l y methylated sugars were converted to t h e i r a l d i t o l a c e t a t e s and ana l y s e d by g.c.-m.s. The r e s u l t s , shown i n Tab l e VI.1, column I , i n d i c a t e d t h a t the K49 p o l y s a c c h a r i d e c o n t a i n e d a 6 - l i n k e d glucose and a 6-l i n k e d g a l a c t o s e r e s i d u e . A 3 - l i n k e d 2-amino-2-deoxy-hexose r e s i d u e was a l s o present but had been i n c o m p l e t e l y h y d r o l y s e d . Reduction, with l i t h i u m aluminium h y d r i d e , of the e s t e r f u n c t i o n of the methylated p o l y s a c c h a r i d e was attempted, but agai n was u n s u c c e s s f u l . T h i s was a f u r t h e r i n d i c a t i o n t h a t t h e r e was an amide l i n k a g e between TABLE V I . 1 M e t h y l a t i o n Data f o r K49 P o l y s a c c h a r i d e and Derived O l i g o s a c c h a r i d e s Methylated s u g a r a M o l b , c  (as a l d i t o l a cetate) 1 2 3 4 1,2,3,4,5-Gal 0.84 2,3,4,6-Glc 2.73 4.35 2,3,4-Glc 5.33 4.90 2,3,4-Gal 4.09- 4.15 2.82 2,4,6-GalNAc 2.09 1.88 2.61 a 2,3,4,6-Glc = 1 , 5 - d i - 0 - a c e t y l - 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y l g l u c i t o l , e t c . b Values are c o r r e c t e d by use of the e f f e c t i v e , carbon-response f a c t o r s given by Albersheira and coworkers^ 0. c 1, methylated p o l y s a c c h a r i d e ; 2, methylated, u r o n i c e s t e r reduced 1a; 3, methylated 3a; 4, reduced, methylated 3b. 165 the g l u c u r o n i c a c i d and the amino a c i d s . Dengler and co-w o r k e r s 2 ^ found t h a t they faced the same problem d u r i n g the s t r u c t u r a l e l u c i d a t i o n of E. c o l i K40 p o l y s a c c h a r i d e . Reductive c l e a v a g e . - Methylated K49 p o l y s a c c h a r i d e was r e d u c t i v e l y c l e a v e d , u s i n g t r i m e t h y l s i l y l t r i f l u o r o m e t h a n e -sulphonate as the c a t a l y s t , and the procedure of Langenhove 39 and R e i n h o l d . The products were a c e t y l a t e d , e x t r a c t e d w i t h 1M NaHCO^/dichloromethane and the aqueous l a y e r was evaporated. A f t e r r e - a c e t y l a t i o n of the aqueous f r a c t i o n , both f r a c t i o n s were ana l y s e d by g.c.-c.i.-m.s. (Table V I . 2 ) . The r e d u c t i v e l y c l e a v e d products were i d e n t i f i e d on the b a s i s of t h e i r mass s p e c t r a and by comparison of t h e i r r e t e n t i o n times with those of a u t h e n t i c standards. I t was hoped t h a t t h i s procedure would a l l o w the d i r e c t i d e n t i f i c a -t i o n of the u r o n i c a c i d present i n the K49 p o l y s a c c h a r i d e as the n e c e s s i t y f o r the r e d u c t i o n of the c a r b o x y l i c a c i d f u n c t i o n i s e l i m i n a t e d . No h i g h e r molecular weight products, r e s u l t i n g from a u r o n i c a c i d r e s i d u e a m i d i c a l l y s u b s t i t u t e d with s e r i n e or threonine, were present i n the gas chromato-gram. The expected anhydro d e r i v a t i v e s f o r the glucose (5) g a l a c t o s e (6) and galactosamine (3) were i d e n t i f i e d by g.c.-m.s. Two peaks were observed f o r the amino sugar d e r i v a t i v e - an i n t r a m o l e c u l a r r e a c t i o n occurs r e s u l t i n g i n the formation of 4 from 3^°« F o r t u i t o u s l y , a s m a l l amount of methyl 2-fi-acetyl-3,6-anhydro-4»5-di-iQ-methyl-L-gulonate (1) was observed i n the chromatogram (Table VI.2). 166 COOMe AcO-H ACQ OMe OMe methyl 2-0-acetyl-3,6-anhydro -4,5-di-Q-methylr-L-gulonate 1 methyl 3-Q-acetyl-2,6-anhydro -4,5-di-0»L-methyl-L-gulonate 2 CH 2OMe MeO I °^ OAc 1 amino -D-galactopyranose ^OMe MeO' OMe 6-0-acetyl-1 ,5-anhydro-2,3,4 £-§-*cet£!f! ,5" a n? y dJ?:^ , 3 , Z f t r i - O - m e t h y l - D - g l u c i t o l t r i - Q - m e t h y l - D - g a l a c t i t o l * 5 6 167 TABLE VI. 2 C. l . Mass Spectral Data for the Reductive Cleavage of K49 Polysaccharide Compound Kole$ a (M + H) + 1 0.4 263 3 0.9 348 4 8.0 289 5 6.6 249 6 5.1 249 Ratios of compounds 3 and 4 cannot be directly compared to those of co-pounds 1 , 5 and 6 as the quantities rere obtained fro* t"-o different fractions i . e . aqueous and organic <see text). TABLE VI.3 Methanolyele Data for E. c o l i K49 Capsular Polysaccharide P a r t i a l l y methylated methylglycoside Mass Spectral Data Main Ions leading to Characterization (m/z) C . l . E . l . a CH20Me 296 247 (MeoJ °v (M +NH 4) + J, 75>K 271 MeONI ( D1 177 OMe COOMc (oMe \~OMc AcONJ / OMe 8 310 J, 75) K 2 71 (M+NH^)* B] 204 K 2 71<10 CH2OMe 320 142 ™>}-\ ( h + h ) + «:i 5 7 / V _ n w . H1 1 5 7 V-OMe ^ n 6 ( a b 8 e n t ) JJI-CH3 COCH3 to the scheme proposed by Zolotarev 168 Vodonik and G r a y H had demonstrated t h a t 1 i s the product obt a i n e d , when a p o l y s a c c h a r i d e c o n t a i n i n g a 4 - l i n k e d D - g l u c o p y r a n o s y l u r o n i c a c i d r e s i d u e i s r e d u c t i v e l y c l e a v e d , r a t h e r than the expected product 2. Thus, r e d u c t i v e cleavage does not e s t a b l i s h whether the u r o n i c a c i d r e s i d u e i s a i+-linked pyranoside or a 5 - l i n k e d f u r a n o s i d e . M e t h a n o l y s i s .- To v e r i f y the presence of a u r o n i c a c i d and to determine i t s p o s i t i o n of s u b s t i t u t i o n , a methylated K49 p o l y s a c c h a r i d e sample was methanolysed and a c e t y l a t e d . The p a r t i a l l y methylated methyl e s t e r methyl g l y c o s i d e of a u r o n i c a c i d may be d i r e c t l y analysed by g.c-m.s., and the p o s i t i o n of l i n k a g e determined from the fragment i o n s present i n the e . i . mass spectrum** 2. Fragmentation path-ways f o r permethylated u r o n i c a c i d r e s i d u e s are analogous to those of permethylated hexopyranosides, the e x c e p t i o n being t h a t fragment i o n s c o n t a i n i n g C-5 and C-i+ (with a p p r o p r i a t e s u b s t i t u t i o n s ) a re absent. T h e i r low s t a b i l i t y and d i s i n t e g r a t i o n to i o n s i n the lower mass range of the spectrum i s presumably due to the presence of the e l e c t r o n -a c c e p t i n g methoxycarbonyl g r o u p ^ . Fragment i o n s c o n t a i n i n g C-6 w i l l , of course, be s h i f t e d by 1J+ mass u n i t s i n compari-son to those of a permethylated hexopyranoside. The c . i . mass s p e c t r a of the products from the methylated, methanolysed, a c e t y l a t e d K49 p o l y s a c c h a r i d e gave pseudo-molecular i o n s which confirmed the presence of two methyl mono-£)-acetyl-tri-£>-methylglycosides, a methyl mono-O-acetyl-169 di-O-methylglycuronate and a methyl mono-p.-acetyl-2-deoxy-di-0-methyl-N(methylacetamido)-glycoside (see Table V I . 3 ) . From the corresponding e . i . mass s p e c t r a the p o s i t i o n of the 0 - a c e t y l s u b s t i t u e n t of each r e s i d u e c o u l d be determined f o l l o w i n g the scheme proposed by Fournet and c o w o r k e r s ^ . The data confirmed the m e t h y l a t i o n a n a l y s i s r e s u l t s and furthermore, the presence of methyl (methyl 4 - f i - a c e t y l - 2 , 3 -d i - 0 - m e t h y l g l y c o p y r a n o s i d e ) u r o n a t e (8) f i n a l l y p r ovided c o n c l u s i v e evidence f o r the presence of a u r o n i c a c i d r e s i d u e , which was ^ - s u b s t i t u t e d , i n K49 p o l y s a c c h a r i d e (see Table V I . 3 ) . P a r t i a l a c i d h y d r o l y s i s . - When the K49 p o l y s a c c h a r i d e was hy d r o l y s e d v/ith 1M HC1 a t 95° f o r 1 h, p r e p a r a t i v e paper chromatography of the n e u t r a l i s e d h y d r o l y s a t e r e v e a l e d t h r e e a c i d o l i g o s a c c h a r i d e s l a ( RQ a^ 0.30), 1b ( R Q a l 0.40) and 1c ( R g a i 0.13). A f t e r m e t h y l a t i o n , g.c.-c.i.-m.s. of l a r e v e a l e d the presence of fragment i o n s a t m/z 233» 219 and 201. Although the pseudomolecular i o n was not observed, the fragment i o n s were i n agreement with 1a being an a l d o -b i o u r o n i c a c i d (Table V I . 4 , P i g . V I . 1 ) . On c a r b o x y l r e d u c t i o n of the methylated o l i g o s a c c h a r i d e , the fragment i o n a t m/z 233 disappeared and a s t r o n g fragment i o n at m/z 205 appeared. A pseudomolecular i o n (M + NH^) + a t m/z 458 was a l s o observed ( T a b l e VI.4) s u g g e s t i n g t h a t 1a was composed of a hexuronic a c i d and a hexose r e s i d u e . The presence of a fragment i o n a t m/z 367 i n d i c a t e d t h a t 170 TABLE Vl.k Relative Retention Times and Ions obtained on G.c.-c.i.-m.s. of Methylated Disaccharides obtained from E. c o l l K*f9 Capsular Polysaccharide Compound Relative Retention time 8 (min) Chemical i o n i z a t i o n mass s p e c t r a l ions (m/z) (Relative abundance) (M+NHi,)+ (M+H)* (M+H- . H'0H2 MeOH) H,+b H+b ( H'-MeOH) + (H-MeOH)H G l c A ^ Q a l 1.76 la 1.90 1.98 2.07 219 (32) 233 (11) 187 (10) 201 (50) Glc'-r^Gal r i»58 237 (7) (15) 219 (19) 20S (56) 187 . (5) 173 (15) GlcL'-GalNAc 2 2.57 2.65 3.03 1*96 k6U 278 (6) (20) (10) 260 (15) s ? 228 (90) 187 (27) a Relative to to 2V3°. that of sucrose (5.98 min.) using DB 17 programmed at 210° for 1 min. then tf°/min. H' and H represent the reducing and non-reducing sugars respectively. i g . VI.1 Fragment ions of methylated 1a and methylated, reduced 1a 171 the a l d o b i o u r o n i c a c i d 1a contained a (1-*6) l i n k a g e ^ . ( s e e F i g . VI.2) S — O C H 3 ^ r—rO^ \) V — O C H 3 ^ S — O- C H 2 - CH= O — C I T 5 Y^OCH 3 X ° C H 3 •CH-OCH3 m / z 3 6 7 s = u r o n i c a c i d r e s i d u e F i g . VI.2 S t r u c t u r e of fragment i o n i n d i c a t i n g presence of a (1-6) l i n k e d a l d o b i o u r o n i c a c i d . Comparison of the r e l a t i v e r e t e n t i o n times of the methy-l a t e d d i s a c c h a r i d e l a with those of an a u t h e n t i c standard, i d e n t i f i e d the a l d o b i o u r o n i c a c i d as 6-£i-?-D-glucopyran-ur o n o s y l - D - g a l a c t o s e (Table V I .4). C o n f i r m a t i o n of t h i s assignment was obtained when h y d r o l y s i s of methylated l a gave 2 , 3 » 4-tri-0-methylglucose.and 2 , 3>4-tri - J 2-methylgalactose. The amount of 2 , 3»4-tri-p_-methylglucose i n c r e a s e d r e l a t i v e to t h a t of 2 , 3 , 4 - t r i - C j - r a e t h y l g a l a c t o s e on h y d r o l y s i s of methylated, reduced l a (Table VI.1, column I I ) . Me t h y l a t i o n a n a l y s i s of l b and 1c i n d i c a t e d t h a t these o l i g o s a c c h a r i d e s were a l s o the a l d o b i o u r o n i c a c i d , the d i f f e r e n t ^Q a^ va l u e s were assumed to be due to the presence of the amino a c i d s u b s t i t u e n t s . The assumption was born out 172 by the n.m.r. data (see below). Hydrogen flu o r i d e hydrolysis.-Treatment of the K49 poly-saccharide with anhydrous HF, at 0 ° for 1 h, gave one main product 2, which eluted from a Biogel P2 column i n the d i -saccharide region. On analysis, by paper chromatography, i t was found to be a neutral oligosaccharide with R G a l 0.39. The methylated oligosaccharide 2 was analysed by g . c . - c . i . -m.s. The r e l a t i v e l y long retention times (see Table VI.4) indicated that i t was probably a disaccharide containing an amino sugar and a hexose residue. This was confirmed by the presence of a pseudomolecular ion (M + H) + at m/z 496, along with fragment ions at m/z 260 and 219 (Table VI.4, F i g . V I . 3 ) . MeO OMe NCH 3 COCH 3 187*—219 2 6 0 — » 228 (M + H ) T = 496—»464 F i g . VI.3 Fragment ions of methylated 2. 173 H y d r o l y s i s of methylated 2, f o l l o w e d by the formation of the p a r t i a l l y methylated a l d i t o l a c e t a t e s , r e v e a l e d , on a n a l y s i s by g . c , the presence of 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y l -g l u c o s e . No peak with the c o r r e c t r e t e n t i o n time f o r 2-deoxy-2-N-methylacetamido-4>6-di-Cj-methylgalactose was observed. Two other HF h y d r o l y s e s were a l s o undertaken on K49 p o l y s a c c h a r i d e , a t 0° f o r 15 min., both i n the presence and absence of methanol. The presence of methanol induces the formation of a methyl g l y c o s i d e a t the r e d u c i n g t e r m i n a l of any o l i g o s a c c h a r i d e s formed, and minimises s i d e r e a c t i o n s of the sugar f l u o r i d e s on removal of the HF by e v a p o r a t i o n . One main product 3a was obta i n e d , i n good y i e l d ( 2 1 $ ) , on s e p a r a t i o n by g e l permeation chromatography of the HF hydro-l y s a t e obtained i n the presence of methanol. M e t h y l a t i o n a n a l y s i s of 3a gave the r e s u l t s shown i n Table V I . 1 , column I I I . T h i s data together with the n.m.r. spectroscopy data suggested t h a t 3a was a t e t r a s a c c h a r i d e with a non-reducing t e r m i n a l glucose r e s i d u e . A n a l y s i s of the methylated sample by g.c.-c.i.-m.s., u s i n g DB 1 or DB 5 c a p i l l a r y columns was u n s u c c e s s f u l i n t h a t the t e t r a s a c c h a r i d e was not observed. However, s m a l l amounts of the a l d o b i o u r o n i c a c i d s t i l l sub-s t i t u t e d w i t h i t s amino a c i d s were observed to be present i n the sample. The a l d o b i o u r o n i c a c i d gave three main components on g.c.-c.i.-m.s -. a n a l y s i s , with s m a l l pseudo-molecular i o n s ('M + H ) + a t m/z 598(A), 584(B), and 566(C) Component A a l s o had ansexeeptionaltystrong-fragment i o n WO-Jfi fi COOMe ^H-CHOM. eN CH, OMe COOMe 9H-CHjDMt MeN « » » nt CO OMe J I6 -H9 ^ 1 4< * * T 100 i w I 330 «00 430 300 Jaijiliiilit<^A,rJ 246 2 | « ,.I..f??i.,,<y?^ r. 100 ISO 200 M O 300 MO Me COC ' 4»CH N CHj CO MeO . OMe OMe J98-U0 114 343 4« 4 0 4 4 6 4 1 ' 1 130 200 230 300 400 « 0 " I " 300 F l K . Vl.lf C . i . mass spectra of the three components (A, B, and C) of the al d o b i o u r o n i c a c i d present i n olig o s a c c h a r i d e 3a 175 at m/z 362 and o t h e r s a t m/z 330 and 303> showing t h a t t h r e o -nine was a m i d i c a l l y l i n k e d to the g l u c u r o n i c a c i d r e s i d u e ( F i g . V I . 4 ) . Component B was found to be the s e r i n e analogue of A ( F i g . V I . 4 ) . The e . i . mass spectrum of C r e v e a l e d t h a t t h i s component was an a r t e f a c t , d e r i v e d from A by the l o s s of methanol ( F i g . V I . 4 ) . T h i s a r t e f a c t was a l s o observed by 23 Hofmann and c o l l e a g u e s ^ d u r i n g the a n a l y s e s , by e.i.-m.s., of three components a c q u i r e d a f t e r s e p a r a t i o n , by Sephadex LH-20, of the methylated a l d o b i o u r o n i c a c i d d e r i v e d from E. c o l i K54 p o l y s a c c h a r i d e . L a s e r d e s o r p t i o n i o n i z a t i o n f o u r i e r transform i o n c y c l o t r o n resonance spectroscopy ( L . d . i . - f . t . - i . c . r . ) has been shown to g i v e m olecular or pseudomolecular i o n s from u n d e r i v a t i z e d o r g a n i c molecules, and u l t r a h i g h mass r e s o l u -t i o n . The technique a l s o shows promise f o r complete sequenc-i n g and f o r d e t e r m i n a t i o n of some l i n k a g e p o s i t i o n s f o r the 45 u n d e r i v a t i z e d , l i n e a r o l i g o s a c c h a r i d e . The pseudo-molecular i o n s and some fragment i o n s from the l . d . i . - f . t . -i . c . r . p o s i t i v e i o n spectrum of 3a are shown i n Table VI.5. These r e s u l t s are i n agreement with 3a having the s t r u c t u r e g i v e n i n F i g . V I . 5 . A second HF h y d r o l y s i s , c a r r i e d out i n the absence of methanol, was quenched with a n e u t r a l i s i n g suspension (CaCO^/ CHgC^/dry i c e ) . A g a i n . 5a s i n g l e o l i g o s a c c h a r i d e 3b was o b t a i n e d (12$ y i e l d ) , a f t e r s e p a r a t i o n of the products by g e l permeation chromatography. A n a l y s i s showed t h a t a g a l a c t o s e r e s i d u e formed the r e d u c i n g t e r m i n a l of 3b (Table TABLE VI.5 L . d . i . - f . t . - i . c . r . P o s i t i v e Ion Spectrum of Ol i g o s a c c h a r i d e 3a obtained from K49 P o l y s a c c h a r i d e by HF H y d r o l y s i s a Observed C a l c u l a t e d R e l a t i v e Maes Proposed Mass MaBS I n t e n s i t y E r r o r Structure^ 3 arau amu % ppm 890 .9755 8 9 1 . 3 0 6 5 4 . 0 -371 8 8 9 . 8 7 3 2 889 .2699 9 . 4 678 875.4570 875 .2542 5 . 7 232 859.6471 859 .2803 4 . 4 427 8 2 9 . 6 2 0 2 829.2488 11.1 448 815 .2331 8 1 5 . 5 3 9 6 4 . 8 376 813 .5631 813.2748 1 5 . 8 354 7 1 1 . 5 6 8 5 711 .2431 5.1 458 7 1 3 . 5 6 9 3 7 1 3 . 2 0 1 4 4 . 5 516 7 2 7 . 6 1 3 6 727 .2171 4 . 4 545 679.4385 679.2169 4 . 3 326 6 5 1 . 3 6 9 5 6 5 1 . 2 2 2 0 7 . 9 226 524 .3131 5 2 4 . 1 3 7 6 7.8 334 5 0 8 . 3 1 2 7 5 0 8 . 1 6 3 7 7 . 7 293 4 2 2 . 2 0 1 3 422.1059 1 7 . 0 226 406.2118 4 0 6 . 1 3 2 0 15.3 197 404 .1631 404 .0954 3 9 . 4 168 3 8 8 . 1 9 5 3 388 .1214 100.0 190 3 6 6 . 2 0 5 7 3 6 6 . 1 3 9 5 7 . 5 181 a This data waB provided by Z. Lam b Thr= threonine, Ser = s e r i n e , Hex = hexose, hexose. • Loss of ketene from a c t y l (Thr,HexA,2Hex,HexNAc,20Me)+Na + +H2O (Thr,HexA ,2Hex,HexNAc ,20Me)+K + (Ser,HexA ,2Hex,HexNA c,20Me)+K + (Ser,HexA,2Hex,HexNA c,20Me)+Na+ *(Thr,HexA,2Hex,HexNAc ,20Me)+K +-H 2 0 »(Ser,HexA,2Hex,HexNAc,20Me)+K +-H 20 *(Thr,HexA,2Hex,HexNA c,20Me)+Na +-H20 (Thr,HexA,Hex,HexNAc,20Me)+Na + (Ser,HexA,Hex,HexNAc,20Me)+K + (Thr,HexA,Hex,HexNAc ,20Me)+K + (Ser,HexA,Hex,HexNAc,20Me)+Na +-H 20 *(Thr,HexA,Hex,HexNAc,20Me)+Na +-H 20 (Thr,Hex,HexA,20Me)+K + (Thr,Hex,HexA,20Me)+Na + (Hex,HexNAc)+K + (Hex,HexNAc)+Na + (Hex,HexNAc)+K +-H 2 0 (Hex,HexNAc)+Na +-H 20 (Hex,HexNAc)+H +-H pO HexA = hexuronic a c i d , HexNAc = 2-amino -2-deoxy-177 TABLE VI.6 -Reducing End Determination of O l i g o s a c c h a r i d e 3b A c e t y l a t e d Mole R a t i o D e r i v a t i v e of G l u c o n i t r i l e i f .5 G a l a c t o n i t r i l e 1.9 G a l a c t i t o l 3.1 COOMe [—CH(CH3)OR NH OH NH OH OH COCH 3 3a R = CH 3 3b R = H F i g . V I . 5 S t r u c t u r e s of 3a and 3b obtained by HF hydrolyses from K49 p o l y s a c c h a r i d e . 178 VI.6) and m e t h y l a t i o n a n a l y s i s of reduced 3b confirmed that t h i s o l i g o s a c c h a r i d e a l s o had a glucose r e s i d u e as the non-reducing t e r m i n a l (Table VI.1 , column I V ) . Thus, m e t h y l a t i o n a n a l y s i s of 3b confirmed t h a t K49 p o l y s a c c h a r i d e c o n t a i n e d the d i s a c c h a r i d e : G a l 1 — ^ G l c P e r i o d a t e o x i d a t i o n and Smith d e g r a d a t i o n . - O x i d a t i o n of K49 p o l y s a c c h a r i d e , f o l l o w e d by borohydride r e d u c t i o n and m i l d 1 Pi a c i d h y d r o l y s i s (Smith degradation ), r e s u l t e d i n the com-p l e t e l o s s of the glucose and g a l a c t o s e r e s i d u e s . A number of products were observed on s e p a r a t i o n of the h y d r o l y s a t e on B i o - g e l P2. The main product 4> on h y d r o l y s i s , and a n a l y s i s by paper chromatography, was found to c o n t a i n galactosamine, s e r i n e and t h r e o n i n e . On a n a l y s i s , by g.c.-c.i.-m.s., of methylated 4 two components were found to be present, with r e t e n t i o n times r e l a t i v e to t h a t of sucrose of Z+.22 min. and 4*27 min. No molecular i o n s were seen, but the fragmentation p a t t e r n s , of each component, shown i n F i g . VI.6a and 6b i n d i c a t e d t h a t 2-acetamido-2-deoxygalactb-pyranosyl-(1-»4)-glucuronopyranose a c i d forms p a r t of the s t r u c t u r e of E. c o l i K49 p o l y s a c c h a r i d e . N.m.r. spe c t r o s c o p y a) K49 p o l y s a c c h a r i d e . - The 1 3C-n.m.r. data f o r K49 p o l y -s a c c h a r i d e i s g i v e n i n T a b l e V I . ? . The presence of threonine was i n d i c a t e d by the s i g n a l s a t 20.50, 59.90 and 68.49 p.p.m. 179 (a) MeO NCH 3 C O C H 3 228-260 COOMe J^CH(R)OMe 507 OMe CH2OMe 290 (R = CH3) 406-374-342 MeO 228-260 258-226 F i g . VI.6 Fragment ions of the two components of the methylated Smith product ( 4 ) , TABLE VI.7 N.m.r. Data for E, c o l i K49 Polysaccharide and Derived Oligosaccharides Compound H-n.m.r. data 13 C-n.m.r. data * "1,2 (p.p.m.) (Hz) Integral Assignment P.p.m. Assignment >GlcA 1 - ^ 6 a l ^ G l c 1 - i G a l N A c -f* r P P K49 polysaccharide 4.63 4.61 4.58 4.43 2.05 1.25 7 s See below 1 3 3 171.29 174.85 176.33 105.87 105.05 104.67] 104.60/ 101.24 81.78 68.49 62.58 59.90 56.73 52.70 23-90 20.50 Thr C=0 (GlcA) C=0 (Thr) C=0 (N-acetamido) •io . iL, J a i c £ ,Thr(Ser) itdlcA 1-p -^GalNAc1— C-3 (GalNAc) C-3 (Thr) C-6 (GalNAc) C-2 (Thr) C-2 (Ser) C-2 (GalNAc) CH^ (N-acetamido) CH-j (Thr) TABLE VI.7 (continued) Kif9 polysaccharide 4.62 ' (base-treated) 4.60 7-8 4.58, 4.53 7-8 4.41 7-8 2.04 8 1.25 7-8 GlcAl_§Gal 5.28 s,:; P 7-8 1a 4.59 4.50 7-8 1.20 6 Thr GicA1_JGal P lb 5.28 b 4.61 b 1.25 5 Ser) 1 1 3 0.33 1 0.66 0.30 0.33 1.30 .1 -^GalNAc --«Gal'T * CH3 (acetamido) (threonine) •^Gal^OH -^Gal^-OH GlcAL CH3 (Thr) -^ Gal-^ OH r Thr(Ser) l 1 GlcA-^ c 1 f -^GalVOH P CH3 (Thr) 170.40 175.37 105.17 104.45 103.93 103.88 171.37 103.63 97.27 93.19 19.98 Thr C=0 (GlcA) C=0 (N-acetamido) ^al±-i G l c ^ Thr(Ser) IcA 100.34 —GalNAc jj-81.27 C-3 (GalNAc) 61.96 C-6 (GalNAc) 52.02 C-2 (GalNAc) 23.26 CH3 (N-acetamido) 20.09 CH3 (Threonine) Thr C=0 (GlcA) Thr(Ser) GlcA—_ fi 1 " -2Gali-0H fi 1 P —Gal^—OH CH3 ( Thr) TABLE VI.7 (continued) Glc1-^GalNAc 2 4.75 4.57 -4.52 2.05 1-2 8 8 b GlcV^OalNAcL^GlcA1—^Gal-OMe 4.84 p fin P 3a (pH 3 - 4 ) P 4.61 4.58 4.56 4.53 3.42 J 7-8 0.64 0.20 1 3 ^GalNAc1-OH -^GalNAc-OH P Glc' 175.581 175.28/ 105.121 104.92J 95.92 91.95 81.141 CRj 81.141 (N-acetamido) 78.04J 61.981 61.75J 61.22 53.791 49.75/ C=0 (N-acetamido) 23 22 Glc^-—GalNA q'-OH - P ^GalNAc-OH OC C-3 (GalNac) C-6 (GalNAc) C-6 (Glc) C-2 (GalNAc) !oo} m 3 ( N- a c etamido) 0.8 3.4 -^ Gal^ OMe Glc-P -^GalNAc— P —Gal—OMe 3 OCH^  175.81 170.26 105.08 104.65 104.23 (Thr,. C=0 N-acetamido) Thr C=0 (GlcA) GlcL-6 ^ ^Gal^OMe P TABLE VI.7 (continued) 2.02 1.23 Glc 1—^GalNAc 1—^GlcAl_§GalLoH 5.27 3b 4.59 4.58 4.56 4.54' 2.02 1.23 (N-acetamido) CH3 (Thr) 103.81 100.31 100.28 61 .90 61 .23 56.08 51.98 23.23 19.99 P —GalNAc^--^Gal^Me C-6 (GalNAc) C-6 (Glc) 0CH3 C-2 (GalNAc) CH3 (N-acetamido) CH3 (Thr) -Gal^- OH 'hr IcA 1 GlcV -Gal^-OH -^GalNAc— P CH, (N-acetamido) CH3 (Thr) 175.61 170.13 104.97 103.53 100.14 97.12 93.05 C=0 (N-acetamido) C=0 (GlcA) Glc1_. hr IcA P ^GalNAc 1_ -^GaljOH J?Gal1— OH TABLE VI.7 (continued) 81.05 61.86 61.18 51.95 23.25 20.12 C-3 (GalNAc) C-6 (GalNAc) C-6 (Glc) C-2 (GalNAc) CHj (N-acetamido) CH^ (Thr) Smith Degradation Product 4 4.67 7-8 4.47 b " 4-43 b , 4.40 b 2.08 s 1.26 6 GalNAc unassigned 3 CH, (N-acetamido) 3 CH3 (Thr) 175.80 C=0 (N-acetamido) 174.47 C=0 (Thr) 101.81 GalNAc y 61.86 C-6 (GalNAc) 53.50 C-2 (GalNAc) 23.17 CH3 (N-acetamido) 19.86 CH3 (Thr) Chemical s h i f t r e l a t i v e to i n t e r n a l acetone assigned at 62.23 downfield from external sodium 4»4-dimethyl-4-silapentane-1 sulphonate (D.S.S.). b Key: b = broad, unable to assign accurate coupling constant; s = s i n g l e t . c E.g. -^GlcA^- r e f e r s to the anomeric proton of a L - l i n k e d glucuronosyl residue i n the B-anomeric configuration. The absence of a numeral p r e f i x i n d i c a t e s * j a non-reducing terminal group. Chemical s h i f t r e l a t i v e to i n t e r n a l acetone assigned at 31.07 e 1 ^ p.p.m. downfield from external D.S.S. As for c, but for anomeric JZ n u c l e i . A l l n.m.r. spectra are given i n Appendix I. 185 The s i g n a l at 52.70 p.p.m. was ass i g n e d to the C-2 of galactosamine, and the resonance a t 23.90 p.p.m. assign e d to a N - a c e t y l methyl group, i n d i c a t i n g t h a t the amino sugar i s N - a c e t y l a t e d . A l e s s i n t e n s e s i g n a l at 56.73 p.p.m. was a s s i g n e d to the C-2 of s e r i n e . The 62.58 p.p.m. s i g n a l corresponds to the C-6 of the amino sugar, the presence of a s i n g l e f r e e -CrLjOH group i s i n agreement with the chemical a n a l y s i s . The resonance f o r a r i n g carbon a t 81 .78 p.p.m. was ass i g n e d to C-3 of the 3 - l i n k e d g a l a c t o s -amine r e s i d u e . The anomeric r e g i o n of the spectrum e x h i b i t e d f o u r s i g n a l s , downfield of 101 p.p.m., a t 101.24, 104 . 6 7 , 105.05 and 105 .87 p.p.m. i n d i c a t i n g a l l the sugars were f 1 13 l i n k e d . T h i s assignment was confirmed by the H - 1 — VC-1 s c a l a r c o u p l i n g c o n s t a n t s i . e . ^ c i - H l between 161 Hz and 164 Hz f o r a l l resonances. The s i g n a l a t 104.6? p.p.m. had a shoulder a t 104.60 p.p.m. and t h e r e f o r e was t e n t a t i v e l y a s s i g n e d to the g l u c u r o n i c a c i d r e s i d u e which was e i t h e r s u b s t i t u t e d with t h r e o n i n e or s e r i n e . The low f i e l d r e g i o n of the spectrum showed three s t r o n g carbonyl-carbon resonances a t 171.29, 174 .85 and 176.33 pip.m. These s i g n a l s were a s s i g n e d to the C=0 f u n c t i o n s of the g l u c u r o n i c a c i d r e s i d u e w i t h t h r e o n i n e , the thr e o n i n e s u b s t i t u e n t and the N-acetamido group r e s p e c t i v e l y . Two s m a l l e r resonances a t 171.04 and 174 .60 p.p.m. were a t t r i b u t e d , r e s p e c t i v e l y , t o the C=0 of g l u c u r o n i c a c i d s u b s t i t u t e d with s e r i n e and to the s e r i n e r e s i d u e i t s e l f . Treatment o f the n a t i v e p o l y s a c c h a r i d e with d i l u t e base 1 8 6 r e s u l t e d i n the l o s s of the carbonyl resonance at 174.85 p.p.m. and the l o s s of the two s i g n a l s assigned to the C-2 atoms of threonine and serine (59.90 and 56.73 p.p.m r e s p e c t i v e l y ) . These r e s u l t s suggest that the amino a c i d s were e s t e r - l i n k e d . However, the s i g n a l at 20.09 p.p.m. assigned to CH^ of threonine remained. Furthermore, chemical a n a l y s i s of the base-treated polysaccharide con-firmed that the aminoaacids had not been removed. G.c-c.i.-m.s. data from methylated 3a was a l s o i n agreement with the amino a c i d s being a m i d i c a l l y - l i n k e d . A p o s s i b l e explanation f o r t h i s apparent c o n t r a d i c t i o n i s that there are both e s t e r - l i n k e d and amide-linked amino a c i d s u b s t i t u -ents on the K49 polysaccharide. The assignments of the resonances for threonine, s e r i n e and the amino a c i d s u b s t i t u t e d glucuronic a c i d were made on the b a s i s of chemical s h i f t comparison with model compounds^ . The 1H-n.m.r. spectrum of the native polysaccharide (Table VI.7) a l s o e x h i b i t e d s i g n a l s f o r the methyl protons of threonine (51 .25) and the N-acetamido group ( £ 2 . 0 5 ) . These resonances, i n each batch of K49 polysaccharide produced, were always present i n a 1 : 1 r a t i o i n d i c a t i n g that the threonine substituent was present on every repeating u n i t , contrary to the amino a c i d a n a l y s i s r e s u l t s which suggested threonine was present on l e s s than h a l f . The spectrum of the base-treated polysaccharide was b e t t e r resolved and a coupling constant of 7-8 Hz was observed f o r the reson-ance at 51 . 25 . The r e s o l u t i o n of the anomeric 187 region was also improved and signals for three overlapping doublets ( J 1 2 7-8 Hz, r e l a t i v e i n t e n s i t y two) and two doublets (J^ 2 7 - 8 H z ^ a t H - 5 3 a n d 64,41, w©re observed. The glucuronic acid was assumed to produce two signals i n the anomeric region, r e s u l t i n g from i t s substitution with either serine or threonine. Thus, both the H- and VC-n.m.r. data were i n good agreement with the suggested t e t r a -saccharide repeating u n i t . b) The aldobiouronic acid ( 1 ) . - The tabulated 1H- and ^ C -n.m.r. data for the aldobiouronic acid 1, along with the data for the other oligosaccharides, i s o l a t e d from K 4 9 polysaccharidesare presented i n Table VI.7• The anomeric region of the spectrum of 1b exhibited signals at 103.63, 97-27 and 93.19 p.p.m. which corresponds well with the signals obtained for an authentic standard of GlcAL^Gal (see Spectrum no .20) . In addition, the spectrum had a resonance at 19.98 p.p.m. and two extra resonances i n the r i n g proton region which indicated the presence of threonine. The 1H-n.m.r. spectra of 1b also exhibited a signal for the methyl group of threonine (61 . 21 , J 1 2 6-7). The anomeric region had resonances at S4.61 and55*28. In comparison the spectrum of l a which did not carry an amino aci d substituent exhibited three anomeric resonances at55.28, S4.59 and £ 4 . 5 0 . In the ^ -spectrum of the authentic standard the signa l at 64.51 was twinned, i n d i c a t i n g t h i s s i g n a l resulted from the glucuronic acid residue attached to either the «<- or p-anomer of the galactose residue. I t 188 was t h e r e f o r e concluded t h a t s u b s t i t u t i o n of the g l u c u r o n i c a c i d by an amino a c i d r e s u l t e d i n a s h i f t downfield of i t s resonance from 84.50 i n 1a to 64.61 i n 1b. c) The amino sugar d i s a c c h a r i d e ( 2 ) . - The 1 3 C spectrum very c l e a r l y supported the chemical evidence of N - a c e t y l g a l a c t o s -amine forming the r e d u c i n g end of the d i s a c c h a r i d e 2. A t e r m i n a l r e d u c i n g sugar w i l l not only g i v e two s i g n a l s f o r C-1 , r e s u l t i n g from the presence of the and 8-anomers, but the resonances of the other C :atoms may a l s o be twinned. Each of the resonances which c o u l d be u n e q u i v o c a l l y a s s i g n e d to an N - a c e t y l a t e d amino sugar e x h i b i t e d t h i s c h a r a c t e r i s t i c twinning e.g. the CH-^  resonance gave two s i g n a l s at 25.11 and 22.88 p.p.m. and the C-2 resonance e x h i b i t e d an even more pronounce twinning, w i t h s i g n a l s a t 53.79 and 49 .45 p.p.m. Furthermore, two resonances a t low f i e l d which c o u l d be a s s i g n e d to the C-3 of a 3 - l i n k e d amino sugar were p r e s -ent (81.14 and 78 . 0 4 ) . Some of the s i g n a l s f o r C atoms of a sugar l i n k e d to the r e d u c i n g sugar may a l s o show twinnings. Fo r 2, the resonances due to C-1, C-2 and C-3 of the g l u c o s y l r e s i d u e were a l s o twinned, but with s m a l l e r d i f f e r e n c e s i n chemical s h i f t s . The above assignments were made on the b a s i s of chemical s h i f t comparisons with model compounds^'** 7. The ^H-spectrum of 2 contained some extraneous s i g n a l s which were not a s s i g n e d . These may have r e s u l t e d from the incomplete replacement o f f l u o r i d e i o n s with OH i o n s a t C-1 of the r e d u c i n g sugar. In the anomeric r e g i o n resonances a t 85.26 and 64.75 were a s s i g n e d to the oc- and p-anomers 189 of the r e d u c i n g galactosamine r e s i d u e and the s i g n a l at &4-57 was as s i g n e d to the non-reducing glucose r e s i d u e . The resonance a t 52.05» assi g n e d to the methyl group of the N-acetamido s u b s t i t u e n t , showed the twinning t y p i c a l of a resonance a s s i g n e d to a r e d u c i n g end sugar. d) O l i g o s a c c h a r i d e s 3a and "5b.- The ^ C s p e c t r a were ob t a i n e d , a t both pH 3-4 and pH 7, f o r 3a (see S p e c t r a 26 and 27, T a b l e V I . 7 ) • A s h i f t was observed f o r the c a r b o n y l -carbon resonance of thre o n i n e from 177.01 to 175.81 p.p.m. on l o w e r i n g the s o l u t i o n pH, while the carbonyl-carbon resonance o f g l u c u r o n i c a c i d showed no a p p r e c i a b l e s h i f t . T h i s demonstrates the c o v a l e n t attachment of t h i s amino a c i d to C-6 of the g l u c u r o n i c a c i d . The n.m.r. data presented (Table VI.7) i s i n agreement with the chemical and mass s p e c t r a l a n a l y s e s of 3a and 3b, i . e . they are tetramers w i t h a glucose r e s i d u e a t t h e i r non-reducing t e r -m i n a l s . The presence of a methyl g l y c o s i d e a t the r e d u c i n g end of 3b i s i n d i c a t e d by a s h i f t downfield of the carbon resonances o f the <x- and ^-anomers of the g a l a c t o s e r e s i d u e from 93.05 and 97.12 p.p.m. i n 3b to 100.31 and 104.65 p.p.m. i n 3 a . The l a t t e r chemical s h i f t s b e i n g t y p i c a l o f the <X-and p-forms of a methyl g a l a c t o s i d e residue^®. A l s o s i g n i f i c a n t i s the presence o f a s i g n a l a t 56.08 p.p.m. i n the 1^C spectrum of 3a, demonstrating the presence of a methyl g l y c o s i d e or the e s t e r i f i c a t i o n of the -C00H groups of the amino a c i d s . e) The Smith degraded product ( 4 ) . - The ^ C spectrum of 4 190 was c o n s i s t e n t w i t h the presence of a s i n g l e i n t a c t mono-sa c c h a r i d e (101.81 p.p.m.) which was i d e n t i f i e d as N-a c e t y l g a l a c t o s a m i n e (23.17, 53.50 and 175.80 p.p.m.). The presence of t h r e o n i n e was i n d i c a t e d by s i g n a l s a t 19 .86 and 17Zf.Zf7 p.p.m. The ^-n.m.r. spectrum had s i g n a l s a t 51.26 and 6"2.08 which were as s i g n e d r e s p e c t i v e l y to the CH^ groups of the threonine and N-acetamido s u b s t i t u e n t s (see Table V I . 7 ) . VI.k CONCLUSION The K49 p o l y s a c c h a r i d e c o n t a i n s two amino a c i d s , s e r i n e and t h r e o n i n e , as s u b s t i t u e n t s competing f o r one s u b s t i t u t i o n s i t e . A s i m i l a r s i t u a t i o n i s found i n E. c o l i K54 p o l y s a c c h a r i d e 2 ^ . The p o l y s a c c h a r i d e of E. c o l i KZf9 has a s t r u c t u r e based on a l i n e a r t e t r a s a c c h a r i d e r e p e a t i n g u n i t as shown i n the a b s t r a c t . VI.5 EXPERIMENTAL General methods.- Instrumentation and g e n e r a l pro-cedures were b a s i c l y the same as those d e s c r i b e d f o r E. c o l i K26 and the bacteriophage-borne enzyme degr a d a t i o n of K49 c a p s u l a r p o l y s a c c h a r i d e . B a c t e r i a and c u l t i v a t i o n . - E. c o l i 08 : K49 : H21 was o b t a i n e d from Dr. I . 0rskov (Copenhagen) and grown on M u e l l e r 191 Hinton agar c o n t a i n i n g 0.5% (w/v) NaCl. I s o l a t i o n and p u r i f i c a t i o n of the KLQ p o l y s a c c h a r i d e . -A f t e r h a r v e s t i n g , the b a c t e r i a l c e l l s were suspended i n 2% phenol and s t i r r e d (R.T., 18 h ) . The b a c t e r i a l c e l l s were removed by u l t r a c e n t r i f u g a t i o n and the supernatant was f r e e z e -d r i e d . The a c i d i c c a p s u l a r p o l y s a c c h a r i d e was obtained by f r a c t i o n a l p r e c i p i t a t i o n w i t h cetyltrimethylammonium bromide as d e s c r i b e d by J a n n ^ . The procedure was m o d i f i e d as f o l l o w s ; i n s t e a d o f the a d d i t i o n o f water to the super-natant remaining a f t e r the removal of the CTAB-RNA p r e c i p i -t a t e, i n o r d e r to o b t a i n a second p r e c i p i t a t e of CTAB-poly-s a c c h a r i d e , the supernatant was conc e n t r a t e d and p r e c i p i t a t e d w i t h e t h a n o l (3 v o l . ) . The p r e c i p i t a t e was then d i s s o l v e d i n water (0.1-1% w/v), and p u r i f i e d by r e p r e c i p i t a t i o n as i t s CTAB s a l t (3 mg CTAB/mg p o l y s a c c h a r i d e ) 2 1 . On g e l permeation chromatography the product was shown to be mono-d i s p e r s e (M r = 420 000). A n a l y t i c a l procedures.- 2-Acetamido-2-deoxygalactose, t h r e o n i n e and s e r i n e were determined ( a f t e r h y d r o l y s i s -6N HC1, 110°, 24 h) wit h an automatic amino a c i d a n a l y s e r . To determine the a b s o l u t e c o n f i g u r a t i o n o f th r e o n i n e and galactosamine, K49 p o l y s a c c h a r i d e (50 mg) was hy d r o l y s e d (4M TFA, 1 h, 125°) and the h y d r o l y s a t e separated by paper chromatography. The r e s i d u e s were converted to t h e i r (+)-2-b u t y l e s t e r d e r i v a t i v e s by the a d d i t i o n of ( + )-2-butanol 192 (1 mL) and t r i f l u o r o a c e t i c a c i d (1 drop) and h e a t i n g to 100 f o r 18 h. F o r the galactosamine r e s i d u e , p r i o r re-N-a c e t y l a t i o n ( A c 2 0 (1 mL), k h, R.T.), was necessary. Excess (+)-2-butanol was removed under a stream of N 2 and the products were a c e t y l a t e d ( A c 2 0 / p y r i d i n e , 30 min., 95°) and a n a l y s e d by g.c.-m.s. (DB 17 - 160° f o r 2 min. 2°/min. to 220°) i n comparison with a u t h e n t i c s t a n d a r d s . The a b s o l u t e c o n f i g u r a t i o n of glucose and g a l a c t o s e was a s s i g n e d by the formation of t h e i r ( - ) - 2 - o c t y l e s t e r d e r i v a t i v e s as d e s c r i b e d i n S e c t i o n IV.3. For a n a l y s i s of c o n s t i t u e n t sugars, samples of n a t i v e p o l y s a c c h a r i d e (2-5 mg) were e i t h e r heated to 100° f o r k h with i+M HCL, or to 125° with ifM TFA f o r 1 h, or h y d r o l y s e d with anhydrous HF f o r 3 h at room temperature. The h y d r o l y -s i s products were converted to t h e i r a l d i t o l a c e t a t e s and a n a l y s e d by g.c.-m.s. (see"Table V.2). M e t h y l a t i o n a n a l y s i s . - K49 p o l y s a c c h a r i d e ( H + form, 7 mg) was methylated a c c o r d i n g to the Hakomori procedure^. A p o r t i o n was h y d r o l y s e d (2M HC1, 6 h, 100°), reduced with sodium borohydride, a c e t y l a t e d and analysed by g.c. and g.c.-m.s. (Table VI.1, column'I). Reductive c l e a v a g e . - T r i m e t h y l s i l y l t r i f l u o r o m e t h a n e -sulphonate (100->uL), t r i e t h y l s i l a n e (100/*L) and d i c h l o r o -methane (250 /*L) were combined a t 0 ° . T h i s r e d u c i n g agent (100 /«.L) was added to a d r i e d r e s i d u e of permethylated K49 193 p o l y s a c c h a r i d e (2 rag). The s o l u t i o n was heated f o r 1 h a t 45° i n a temperature b l o c k . To terminate the r e d u c t i v e cleavage and to d e r i v a t i z e a l l l i b e r a t e d h y d r o x y l groups, a c e t i c anhydride (50 /*!>) was added and the s o l u t i o n was warmed to if0° f o r 15 min. A f t e r the a d d i t i o n o f d i c h l o r o -methane (0.5 mL), the product was e x t r a c t e d with 1M NaHCO^ (3 x 1 mL). The aqueous f r a c t i o n was evaporated under d i m i n i s h e d p r e s s u r e , a c e t y l a t e d ( A c 2 0 / p y r i d i n e , 30 min., 95°)» and p u r i f i e d by e x t r a c t i o n with d i c h o l o r o -methane from an aqueous s o l u t i o n by the a d d i t i o n of water (1 mL) and dichloromethane (5 mL). Both the aqueous f r a c t i o n and the o r g a n i c f r a c t i o n were analysed by g.c. and g.c.-c.i.-m.s., u s i n g a DB 17 c a p i l l a r y column and ammonia as the reagent gas with a temperature programme of 110° f o r 1 min., then i n c r e a s i n g to 220° a t a r a t e of 6°/ min. (Table V I . 2 ) . M e t h a n o l v s i s . - Methylated K49 p o l y s a c c h a r i d e (3 mg) was r e f l u x e d with 3^ methanolic HC1 (1 mL, 2/f h, 8 0 ° ) . The s o l u t i o n was n e u t r a l i s e d w i t h s i l v e r carbonate, f i l t e r e d , e v a p o r t a t e d t o dryness under a stream of gas, and a c e t y l a t e d . The p a r t i a l l y methylated methyl g l y c o s i d e s were a n a l y s e d by g.c.-c.i.-m.s. and g.c.-e.i.-m.s. The r e s u l t s a re gi v e n i n Table V I . 3 . P a r t i a l a c i d h y d r o l y s i s . - A s o l u t i o n of Klf9 p o l y s a c c h a -r i d e (365 mg) i n HC1 (1M, 100 mL) was r e f l u x e d f o r 1 h, 194 n e u t r a l i s e d with l e a d carbonate and c e n t r i f u g e d to remove the p r e c i p i t a t e . The h y d r o l y s a t e was d i a l y s e d a g a i n s t d i s -t i l l e d water and the d i a l y s a t e was c o n c e n t r a t e d . A f t e r d e i o n i z a t i o n w i t h IR-120 (H +) r e s i n , the o l i g o s a c c h a r i d e s were sep a r a t e d by paper chromatography. Three a c i d i c o l i g o s a c c h a r i d e s were obtained 1a, 1b, and 1c ( ^ G a i 0.30, 0.40 and 0.13, y i e l d s 6 mg, 6 mg, and 8 mg r e s p e c t i v e l y ) . M e t h y l a t i o n and m e t h y l a t i o n and r e d u c t i o n of 1a, followed by a n a l y s i s by g.c.-c.i.-m.s. gave the r e s u l t s shown i n Table VI.4 Hydrogen f l u o r i d e h y d r o l y s i s . - K49 p o l y s a c c h a r i d e (100 mg) was t r e a t e d with anhydrous HF (3-5 mL) a t - O 0 f o r 1 h. The r e a c t i o n was quenched by pouring the mixture i n t o a s l u r r y of dry. i c e / c a l c i u m carbonate/ dichloromethane 1^. The s o l v e n t was removed under a stream of N 2, the sample d i s s o l v e d i n water and the p r e c i p i t a t e removed by c e n t r i -f u g a t i o n . A f t e r s e p a r a t i o n of the products on a B i o - g e l P2 column, a n e u t r a l o l i g o s a c c h a r i d e 2 was o b t a i n e d ( B G a l 0.39, y i e l d 5 mg). A f t e r m e t h y l a t i o n 2 was f i r s t a nalysed by g.c.-c.i.-m.s. ( T a b l e VI.4) and then h y d r o l y s e d (2M HC1, 6 h, 100°), converted to permethylated a l d i t o l a c e t a t e s and a g a i n a n a l y s e d by g.c.-m.s. K49 p o l y s a c c h a r i d e (125 mg) was d r i e d o v e r n i g h t iunder vacuum) and r e a c t e d w i t h anhydrous HF a t 0° f o r 15 min i n the presence of 0.5 mL anhydrous m e t h a n o l ^ . The HF was removed by vacuum a s p i r a t i o n through a sodium hydroxide 195 t r a p , and the l a s t t r a c e s removed under a stream of gas. The product was d e s a l t e d on a Sephadex G10 column and separated on B i o - g e l P2. The main product 3a ( 2 1 % y i e l d ) was methylated, h y d r o l y s e d (2M HC1, 6 h, 95°), reduced with sodium borohydride and a c e t y l a t e d . The mix-t u r e was analysed by g.c.-m.s (Table V I . 1 , column I I I ) . A sample (1 mg) was submitted to N i c o l e t A n a l y t i c a l Instrument f o r a n a l y s i s by l a s e r d e s o r p t i o n i o n i z a t i o n f o u r i e r transform i o n c y c l o t r o n resonance spectroscopy ( l . d . i . - f . t . - i . c . r . ) . The r e s u l t s are shown i n Table VI.5. A second sample of K49 p o l y s a c c h a r i d e (100 mg) was sub-m i t t e d to a HF h y d r o l y s i s u s i n g the same c o n d i t i o n s as above, but without the a d d i t i o n of methanol. Instead of removing the HF by vacuum a s p i r a t i o n the sample was n e u t r a l i s e d , c e n t r i f u g e d and separated i n the same manner as 2. A sample (7 mg) of the main product 3b ( y i e l d 12%) was reduced with NaBH^ (3 h, R.T.), n e u t r a l i s e d with 10% a c e t i c a c i d i n methanol and coevaporated with methanol ( 3 x ) , under a stream of n i t r o g e n . The o l i g o s a c c h a r i d e a l d i t o l (1 mg) was h y d r o l y s e d (ifM TFA, 1 h, 125°) and the a l d o s e s r e l e a s e d converted to t h e i r p e r a c e t y l a t e d a l d o n i t r i l e d e r i v a t i v e s u s i n g the method d e s c r i b e d i n S e c t i o n IV.5. The o l i g o s a c c h a r i d e a l d i t o l (5 mg) was methylated, h y d r o l y s e d (2M HC1, 6 h, 100°), reduced w i t h NaBH^ and a c e t y l a t e d w i t h a c e t i c a n h y d r i d e / p y r i d i n e . The r e s u l t i n g p a r t i a l l y methylated a l d i t o l a c e t a t e s were ana l y s e d by g.c. and g.c.-m.s. (see Table V I , column I V ) . 196 P e r i o d a t e o x i d a t i o n and Smith d e g r a d a t i o n . - The K49 p o l y s a c c h a r i d e (90 mg) was o x i d i s e d i n 0.1M NalO^ (10 mL, 72 h, R.T.), and the product was reduced with NaBH^ (3 h, R.T.). Excess reagents were removed by d i a l y s i s and the product was l y o p h i l i s e d . S e l e c t i v e h y d r o l y s i s was c a r r i e d out u s i n g 0.5M TFA f o r 24 h. Excess a c i d was removed i n vacuo and the products were separated on B i o - g e l P2. Paper chromatography, u s i n g s o l v e n t A, of the products obtained v i a the h y d r o l y s i s of the Smith product 4, i n d i c a -t e d i t contained galactosamine, s e r i n e and t h r e o n i n e . A f t e r m e t h y l a t i o n , 4 was anal y s e d by g.c.-c.i.-m.s. (see F i g . V I . 6 ) . Each o l i g o s a c c h a r i d e ( 1 a , l b , 1c, 2, 3 a , 3b and 4) p r i o r to any chemical a n a l y s i s was s t u d i e d by both 1H-and ^C-n.m.r sp e c t r o s c o p y . The r e s u l t s are t a b u l a t e d i n Table VI.7 and the s p e c t r a are d i s p l a y e d i n Appendix 1. .ACKNOWLEDGEMENT T h i s r e s e a r c h was supported by the N a t u r a l S c i e n c e s and En g i n e e r i n g Research C o u n c i l o f Canada. The author would l i k e to thank Dr. L.A.S. P a r o l i s f o r p r o v i d i n g the M r of K49 p o l y s a c c h a r i d e , Dr. I . T a y l o r f o r the amino a c i d a n a l y s i s r e s u l t s and Z. Lam f o r running the g . c . - c . i . - m . s . 197 BIBLIOGRAPHY 1. A. D e l l , Adv. Carbohydr. Chem. Biochem., 45 (1987) 19-72. 2. V.N. R e i n h o l d , E. Coles and S.A. Car r , J . Carbohydr. Chem. 2 (1983) 1-18. 3. J . Karkkainen, Carbohydr. Res., 17 (1971) 11-18. 4. M.W. Spellman, M. McNeil, A.G. D a r v i l l , P. Albersheim and A. D e l l , Carbohydr. Res., 122 (1983) 131-133. 5. I . Mononen, Carbohydr. Res.,104 (1982) 1-9. 6. C'.-C. Sweeley and H.A. Nunez, Ann. Rev. Biochem., 54 (1985) 765-801 and r e f . t h e r e i n . 7. E.G. De Jong. W. Heerma and G. D i j k s t r a , Adv. Mass Spectrom., 8 (1979) 1314-1320. 8. H. Yamada. H. Yiyohara and Y. Otsuka, Carbohydr. Res., 170 (1987) 181-191. 9. S. - I . Hakomori, J . Biochem. (Tokyo), 55 (1964) 205-208. 10. L.M. Beynon, M . S c , U n i v e r s i t y o f B r i t i s h Columbia ( 1 9 8 5 ) . 11. D. Leek, M.Sc., U n i v e r s i t y of B r i t i s h Columbia (1982). 12. M. Dubois, K.A. G i l l e s , J.K. Hamilton, P.A. Rebers and F. Smith, A n a l Chem., 28 (1956) 350. 13. P.A. Sanford and H.E. Conrad, B i o c h e m i s t r y , 5 (1966) 1508-1517. 14. G.O. A s p i n a l l , i n G.O A s p i n a l l ( E d), "The Polysaccha-rides"*, V o l . 1, Academic P r e s s , New York, (1982) 36-131. 15. H e i d e l b e r g e r , p e r s o n a l communication. 16. K. L e o n t e i n , B. L i n d b e r g and J . Lonngren, Carbohydr. Res., 62 (1978) 359-362. 17. B. L i n d b e r g , J . Lonngren and J.L.'Thompson, Carbohydr. Res., 28 (1973) 351-357.• 18. I . J . G o l d s t e i n , G.W. Hay, B.A. Lewis and F. Smith, Methods Carbohydr. Chem., 5 (1965) 361-370. 19. A . J . Mort and W.D. Bauer, J . B i o l . Chem., 257 (1982) 1870-1875. 198 20. A. D e l l , personal communication. 21. K. Okutani and G.G.S. Dutton, Carbohydr. Res., 86 (1980) 259-271. 22. D. Rieger-Hug and S. Stirm, Virology, 113 (1981) 363-378. 23. P. Hofmann, B. Jann and K. Jann, Carbohydr. Res., 139 (1985) 261-271 . 24. T. Dengler, B. Jann and K. Jann, Carbohydr. Res., 150 (1986) 233-240. 25. A.V.S. Lim, Ph.D., University of B r i t i s h Columbia, (1986)• 26. G.G.S. Dutton, J.L. Di Fabio, D.M. Leek, E.H. M e r r i f i e l d , J.R. Nunn and A.M. Stephen, Carbohydr. Res., 97 (1981) 127-138. 27. A. Kuma-Mintah, M.Sc., University of B r i t i s h Columbia (1985). 28. H. Niemann, H. Beilharz and S. Stirm, Carbohydr. Res., 60 (1978) 353-366. 29. J.K.N. Jones, Methods Carbohydr. Chem., I (1962) 21-31. 30. K.W. Knox and A.J. Wicken, B a c t e r i o l . Rev., 37 (1973) 215-257. 31. V.L. L'Vov, N.V.Tochtamysheva, A.S. Shaskov, B.A"; Dmitriev . and K. Capek, Carbohydr. Res.,;112 (1983) 233-239. 32. S. Hanessian and T. Haskell, J . B i o l . Chem., 329 (1964) 2758-2764. 33. V.L. L'vov, N.V.. Tochtamysheva, A.S. Shaskov, B.A. Dmitriev and N.K. Kochetkov, Bioorg. Khim., 9 (1983) 60-73. 34. W. Gromska and H. Mayer, Eur. J . Biochem., 62 (1976) 391-399. 35. P. Branefors-Helander, L, Kenne, B. Lindberg, K;Peterson and P. Unger, Carbohydr. Res., 97 (1981) 285-291. 36. F.P. Tsui, R. Schneerson, R.A. Boykins, A.B. Karpas and W. Egan, Carbohydr. Res., 97 (1981) 293-306. 37. K. Jann, i n M. Sussman (Ed.),*The Virulence of Escherichia  coli*. Academic Press, London, (1985) 375-379. 38. R.L.Taylor and H.E. Conrad, Biochemistry, 11 (1972) 1383-1388. 199 39. A. Van Langenhove and V.N. Re i n h o l d , Carbohydr. Res. 143 (1985) 1-20. 40. J.A. Bennek, M.J. R i c e and G.R. Gray, Carbohydr. Res., 157 (1986) 125-137. 41. S.A. Vddnik and G. R. Gray, Carbohydr. Res., 175 (1988) 93-102. 42. B. Fournet, G. S t r e c k e r , Y. Leroy and J . M o n t r e u i l , A n a l . Biochem., 116 (1981) 489-502. 43. K. Kovac'ik, S. Bauer, J . Rosik and P. Kovac, Carbohydr. Res., 8 (1968) 282-290. 44. B.M. Z o l o t a r e v , A. Ya. Ott and O.S. Chizhov, Adv. Mass Spectrom. 7B (1978) 1371-1375. 45. Z . Lam, M.B. Comisarow, G.G.S. Dutton, D.A.Weil and A. Bja r a n s o n , Rap. Commun. Mass Spectrom., 1 (5) (1987) 83-86. 46. K. Bock, C. Pedersen and H. Pedersen, Adv. Carbohydr. Chem. Biochem., 42 (1984) 193-225. 47. K. Izumi, Carbohydr. Res., 170 (1987) 19-25. 48. K. Bock and H. Thjzfgersen, Ann. Rep. NMR S p e c t r o s c . 13 (1982) 1-57. 49. M.P. Sanger and D.T.A. Lamport, A n a l . Biochem., 128 (1983) 66-70. 50. P; Al b e r s h e i m , R.H. Shapiro and D.P. Sweet, Carbohydr. Res., 5 (1975) 199-225. 200 APPENDIX I N.M.R. SPECTRA Spectrum No.k E . c o l l K26 p o l y s a c c h a r i d e - autohydrolysed 100 80 60 40 20 P P M Spectrum No. 5 E. c o l i polysaccharide (depyruvylated) pH 7 1H-n.m.r. 400 MHz E«. c o l i polysaccharide ( s e l e c t i v e l y hydrolysed -r L02 E . c o l i polysaccharide (carbodiimide reduced) ^-n.m.r. 400 MHz 08 _ Spectrum No, 10 Spectrum No.11 E. c o l i K26 - F r a c t i o n D GlcALiRha 1-^Rha 1 P H-n.ra.r. .75 MHz O co o \ -4-* rvj O co o rvj I I I I I ' I ) I I I I I I I I' I I I I I I I I I i 2 0 1 0 0 8 0 Spectrum No. 12 o 4) c o +> CD CJ T1 i i i i | i i i 6 0 i i i i | i i i i I i i 4 0 2 0 P P M E . c o l i K/f9 Bacteriophage degradation product 1 GlcA 1—^Gall-^Glc 1-^GalNA c P P 'H-n.m.r. 400 MHz 95 Spectrum No.13 2.23 acetone ^ ^ ^ ^ r ro Spectrum No.14 E» c o l l K49 n a t i v e p o l y s a c c h a r i d e 1 3 C - n.m.r. 75 MHz in o LPs O • • rH V £ > f-rH rH t>-C\J * J - | H o o rH rH 180 H&6 140 ^ o ,100 Spectrum No.15 •Pt, .coli Kk9 polysaccharide (base-treated) ^ - n . m . r . 400 MHz 95° Spectrum N o .18 022 E. c o l i KZf9 O l i g o s a c c h a r i d e 3a (pH 3-4) G l c L _ 3 GalNAcl-^GlcAl-§Gal~ OMe 1 3C-n.m.r. 75 MHz co • O - VD <- OJ • O [>-J . ,11 i n >,nliL*li i i ,1.1 I J . I . U I , 1. 0 0 • o o O O 9% o 1— • • o o 1— o \ \ 1 6 0 140 I 2 0 1 00 Spectrum No.26 E . a c o l i K49 Oligosaccharide 3b Glc^GalNA C!-^GlcAl-§Gal P 13 C-n.m.r 75 MHz I 74 I 7 ; . 1 1 I 1 1 1 j . 1 i . 1 , I : . j i . . . 170 16a O D W CN -31 O I A * • I A CN f A « - i O • ! <- O o O r A ' I ' 1 ' ' I ' 1 4 0 1 2 0 I " iOC Spectrum No.29 j I | I I i I | I ! I I I I I I I J I I I I | I I ' 1 J > ' 6 0 4 0 2 0 

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