Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Structural studies of some bacterial capsular polysaccharides from the family Enterobacteriaceae:Klebsiella… Vee San Lim, Andrew 1986

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1986_A1 L54_8.pdf [ 8.65MB ]
Metadata
JSON: 831-1.0060450.json
JSON-LD: 831-1.0060450-ld.json
RDF/XML (Pretty): 831-1.0060450-rdf.xml
RDF/JSON: 831-1.0060450-rdf.json
Turtle: 831-1.0060450-turtle.txt
N-Triples: 831-1.0060450-rdf-ntriples.txt
Original Record: 831-1.0060450-source.json
Full Text
831-1.0060450-fulltext.txt
Citation
831-1.0060450.ris

Full Text

STRUCTURAL STUDIES OF SOME BACTERIAL CAPSULAR POLYSACCHARIDES FROM THE FAMILY E n t e r o b a c t e r l a c e a e : K l e b s l e l l a SEROTYPES K79 AND K35 AND E s c h e r i c h i a c o l i SEROTYPE K44 by ANDREW VEE SAN LIM B.Sc. (Hons.) U n i v e r s i t y o f Wales, I n s t i t u t e o f S c i e n c e and Technology, U n i t e d Kingdom, 1980. M . S c , U n i v e r s i t y o f B r i t i s h Columbia, Canada, 1983. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (DEPARTMENT OF CHEMISTRY) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA SEPTEMBER 1986 © ANDREW VEE SAN LIM 1986 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of 0\&MV3qgN The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 D e d i c a t e d w i t h l o v e and g r a t i t u d e t o my p a r e n t s MARY SIEW LAN AND MICHAEL TEN CHYE LIM f o r t h e i r l o v e and encouragement i i ABSTRACT K l e b s i e l l a and E s c h e r i c h i a c o l i are among the most f r e q u e n t l y found p a t h o g e n i c E n t e r o b a c t e r i a . S e r o l o g i c a l c l a s s i f i c a t i o n o f t hese two E n t e r o b a c t e r i a i s based m a i n l y on the i m m u n o l o g i c a l l y dominant c a p s u l a r p o l y s a c c h a r i d e s (K a n t i g e n s ) , due t o t h e i r l o c a t i o n a t the outermost s u r f a c e o f the b a c t e r i a l c e l l . I n o r d e r t o u n d e r s t a n d the c h e m i c a l b a s i s o f s e r o l o g i c a l d i f f e r e n t i a t i o n and the immunochemistry o f these a n t i g e n s , the s y s t e m a t i c s t r u c t u r a l i n v e s t i g a t i o n o f a l l the c a p s u l a r p o l y s a c c h a r i d e s o f K l e b s i e l l a (77 s e r o t y p e s ) and E. c o l i (74 s e r o t y p e s ) i s c u r r e n t l y b e i n g u n d e r t a k e n i n t h i s and o t h e r l a b o r a t o r i e s . To date, the s t r u c t u r e o f the c a p s u l a r p o l y s a c c h a r i d e s o f a p p r o x i m a t e l y s e v e n t y K l e b s i e l l a s e r o t y p e s and t h i r t y E. c o l i s e r o t y p e s have been e l u c i d a t e d . These p o l y s a c c h a r i d e s were found to be unique i n each c a s e . As a c o n t r i b u t i o n t o the immunology o f 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 , the s t r u c t u r e s o f the K a n t i g e n s from K l e b s i e l l a K79 and K35, and E.  c o l i K44 were d e t e r m i n e d and a r e p r e s e n t e d i n t h i s t h e s i s . The t e c h n i q u e s o f sugar a n a l y s i s , m e t h y l a t i o n , chromic a c i d o x i d a -t i o n , d e a m i n a t i o n , b a s e - c a t a l y z e d u r o n i c a c i d d e g r a d a t i o n , Smith degra-d a t i o n and p a r t i a l h y d r o l y s i s were 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 o f t h e s e p o l y s a c c h a r i d e s . Methods such as g a s - l i q u i d chromatography, gas chromatography-mass s p e c t r o m e t r y , g e l p e r m e a t i o n , ion-exchange and paper-chromatography were used to i s o l a t e and c h a r a c t e r i z e 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 d e g r a d a t i v e p r o c e d u r e s . N.m.r. s p e c t r o s c o p y (^H and l ^ C ) was w i d e l y used i n the c h a r a c t e r i z a t i o n o f the p o l y s a c c h a r i d e s and i i i o f d e r i v e d p o l y - and o l i g o - s a c c h a r i d e s . I n a few i n s t a n c e s , n.m.r. s p e c t r o s c o p y and mass s p e c t r o m e t r y were used t o d e l i n e a t e the sequence o f the su g a r s i n the s t r u c t u r e o f the p o l y - and o l i g o - s a c c h a r i d e s . The sum o f these experiments e s t a b l i s h e d the f o l l o w i n g s t r u c t u r e s . 3)-0-D-Ga!-(l—3)-P-r>GlcA-(J-»2)-Q-L-Rha-(l-»3)-o-L-Rha-(l-»3)-a-L-Rha-(l— 4 T l o-D-GIc 6 t 1 o-D-GIc K l e b s i e l l a K79 c a p s u l a r p o l y s a c c h a r i d e -»3)-a-D-Galp-(l-.3)-a-D-Manp-(l-*3)-a-D-Manp-(1-»3)-/3-r>Glc/>-(l-. 6 4 2 \ / t C 1 / \ /3-D-GlcAp H,C CO :H K l e b s i e l l a K35 c a p s u l a r p o l y s a c c h a r i d e 4)-0-D-GlcAp- (1-3) -a-L-Rhap- (l-»4)-a-D-GlcpNAc- (l-*6)0-D-GalpNAc- ( 1 -E, c o l i K4A c a p s u l a r p o l y s a c c h a r i d e i v The s t r u c t u r e f o r the K l e b s i e l l a K79 c a p s u l a r p o l y s a c c h a r i d e r e p r e s e n t s the s e c o n d example o f a "5+2" p a t t e r n i n the 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 s e r i e s . The "4+1" s t r u c t u r a l p a t t e r n o f K l e b s i e l l a K35 c a p s u l a r p o l y s a c c h a r i d e , w i t h t h e h e x u r o n i c a c i d i n the s i d e c h a i n , i s s i m i l a r t o b u t y e t d i f f e r e n t from those o f K l e b s i e l l a K9 and K59. 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 o f E. c o l i K44 r e p r e s e n t s the f i r s t i n s t a n c e o f E. c o l i K a n t i g e n s ( o f group A) to c o n t a i n two d i f f e r e n t 2-acetamido-2-deoxyhexoses. F o r a l m o s t e v e r y s t r a i n o f b a c t e r i a , t h e r e e x i s t s a b a c t e r i o p h a g e ( b a c t e r i a l v i r u s , d enoted by <f>) t h a t i n f e c t s i t and r e p l i c a t e s w i t h i n the b a c t e r i a l c e l l . A s s o c i a t e d w i t h these b a c t e r i o p h a g e s a r e enzymes ( e n d o g l y c a n a s e s ) t h a t s p e c i f i c a l l y c l e a v e the c a p s u l a r p o l y s a c c h a r i d e s o f the h o s t b a c t e r i a t o o l i g o s a c c h a r i d e s c o m p r i s i n g one o r more r e p e a t -i n g u n i t s o f the p o l y s a c c h a r i d e . These o l i g o s a c c h a r i d e s when c o u p l e d to c a r r i e r p r o t e i n s have been u s e d i n the p r o d u c t i o n o f p r o t e c t i v e v a c c i n e s . I n our l a b o r a t o r y , however, these b a c t e r i o p h a g e - b o r n e endo-g l y c a n a s e s a r e b e i n g u s e d t o g e n e r a t e o l i g o s a c c h a r i d e s i n the s t r u c t u r a l e l u c i d a t i o n o f 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 . The b a c t e r i o p h a g e s f o r K l e b s i e l l a K79 and E. c o l i K44 were i s o l a t e d from sewage and p r o p a g a t e d on t h e i r h o s t s t r a i n s . The b a c t e r i o p h a g e - i n d u c e d depolymer-i z a t i o n s o f the c a p s u l a r p o l y s a c c h a r i d e s o f K l e b s i e l l a K79 and E. c o l i K44 a r e r e p o r t e d i n t h i s t h e s i s . The o l i g o s a c c h a r i d e s i s o l a t e d were c h a r a c t e r i z e d u s i n g the t e c h n i q u e s d e s c r i b e d . The sum o f t h e s e exper-iments demonstrated t h a t the e n d o g l y c a n a s e a s s o c i a t e d w i t h K l e b s i e l l a <£79 had y S - g a l a c t o s i d a s e a c t i v i t y . The endoglycanase from E. c o l i <£44 V e x h i b i t e d ^ - N - a c e t y l - g a l a c t o s a m i n i d a s e a c t i v i t y which i s n o v e l , i n t h a t i t i s the f i r s t r e p o r t e d a c t i o n o f t h i s n a t u r e i n the b a c t e r i o p h a g e s i s o l a t e d f o r the s p e c i e s E. c o l i . v i TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS v i LIST OF APPENDICES x i LIST OF TABLES x i i LIST OF FIGURES x i v LIST OF SCHEMES x v i LIST OF ABBREVIATIONS x v i i ACKNOWLEDGEMENTS x i x I. INTRODUCTION 1 1.1 Immunological importance o f b a c t e r i a l e x o p o l y s a c c h a r i d e s 3 1.2 C h e m i s t r y and s e r o l o g y o f 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 8 1.3 C h e m i s t r y and s e r o l o g y o f 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 10 I I . METHODOLOGY OF STRUCTURAL ANALYSIS OF BACTERIAL CAPSULAR POLYSACCHARIDES 12 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 14 v i i I I . 2 A n a l y s i s o f monosaccharides 15 II.2.1 A c i d h y d r o l y s i s 16 I I . 2.2 C h a r a c t e r i z a t i o n and q u a n t i t a t i o n o f sugars 18 I I . 2.3 D e t e r m i n a t i o n o f the c o n f i g u r a t i o n (D o r L) o f sugars 20 II.2.4 D e t e r m i n a t i o n o f anomeric c o n f i g u r a t i o n . . 21 I I . 3 P o s i t i o n o f l i n k a g e - 22 11.3.1 M e t h y l a t i o n a n a l y s i s 22 11.3.2 A n a l y s i s o f n o n - c a r b o h y d r a t e c o n s t i t u e n t s . . 26 I I . 4 G e n e r a t i o n o f o l i g o s a c c h a r i d e s 27 II.4.1 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 29 I I . 5 I n s t r u m e n t a t i o n 31 11.5.1 N u c l e a r magnetic resonance s p e c t r o s c o p y . . 31 11.5.2 Mass s p e c t r o m e t r y 39 I I I . GENERAL EXPERIMENTAL PROCEDURES 47 I I I . l P r e p a r a t i o n and i s o l a t i o n o f 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 48 111.1.1 Media and g e n e r a l m i c r o b i a l c o n d i t i o n s . . 48 111.1.2 I s o l a t i o n o f 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 49 111.1.3 I s o l a t i o n o f 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 51 I I I . 2 N.m.r. s p e c t r o s c o p y 51 v i i i I I I . 3 Sugar a n a l y s i s 52 111.3.1 M e t h a n o l y s i s 52 111.3.2 F o r m a t i o n o f a l d i t o l a c e t a t e s 53 111.4 M e t h y l a t i o n a n a l y s i s 54 111.4.1 Hakomori m e t h y l a t i o n 54 111.4.2 R e d u c t i o n o f u r o n i c e s t e r 55 111.4.3 F o r m a t i o n o f p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s 55 111.5 Paper chromatography 56 111.6 G a s - l i q u i d chromatography and mass s p e c t r o m e t r y . . 57 I I I . 7 G e l and ion-exchange chromatography 59 111.8 O p t i c a l r o t a t i o n , c i r c u l a r d i c h r o i s m and i n f r a r e d s p e c t r o s c o p y 60 111.9 P r o p a g a t i o n o f b a c t e r i o p h a g e 61 111.9.1 P r e p a r a t i o n o f b a c t e r i a l lawn 61 111.9.2 B a c t e r i o p h a g e p l a q u e assay 61 I I I . 9.3 Tube l y s i s 62 111.9.4 Sm a l l f l a s k l y s i s 62 111.9.5 Wash-bottle l y s i s 63 I V . STRUCTURAL INVESTIGATION OF K l e b s i e l l a AND E. c o l i CAPSULAR POLYSACCHARIDES 64 IV.1 S t r u c t u r e o f the c a p s u l a r p o l y s a c c h a r i d e o f K l e b s i e l l a s e r o t y p e K79 65 i x IV.1.1 A b s t r a c t 65 IV.1.2 I n t r o d u c t i o n 65 IV.1.3 R e s u l t s and d i s c u s s i o n 66 IV.1.4 C o n c l u s i o n 78 IV.1.5 E x p e r i m e n t a l 79 IV.2 S t r u c t u r e o f the c a p s u l a r p o l y s a c c h a r i d e o f K l e b s i e l l a s e r o t y p e K35 87 IV.2.1 A b s t r a c t 87 IV.2.2 I n t r o d u c t i o n 87 IV.2.3 R e s u l t s and d i s c u s s i o n 88 IV.2.4 C o n c l u s i o n 101 IV.2.5 E x p e r i m e n t a l 102 IV. 3 E. c o l i s e r o t y p e K44: an 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 c o n t a i n i n g two 2-acetamido-2-deoxyhexoses 108 IV.3.1 A b s t r a c t 108 IV.3.2 I n t r o d u c t i o n 108 IV.3.3 R e s u l t s and d i s c u s s i o n . 109 IV.3.4 C o n c l u s i o n 123 IV. 3.5 E x p e r i m e n t a l 123 V. BACTERIOPHAGE-INDUCED DEPOLYMERIZATION OF BACTERIAL CAPSULAR POLYSACCHARIDES 132 V. l Nature and mode o f p r o p a g a t i o n o f b a c t e r i o p h a g e . . 133 V. l . l E n doglycanases a s s o c i a t e d w i t h b a c t e r i o p h a g e s 137 V.2 B a c t e r i o p h a g e - i n d u c e d d e p o l y m e r i z a t i o n o f K l e b s i e l l a K79 c a p s u l a r p o l y s s a c c h a r i d e 140 X V.2.1 I n t r o d u c t i o n 140 V.2.2 R e s u l t s and d i s c u s s i o n 140 V.2.3 C o n c l u s i o n 147 V.2.4 E x p e r i m e n t a l 148 V.3 B a c t e r i o p h a g e - i n d u c e d d e p o l y m e r i z a t i o n o f E. c o l i K44 c a p s u l a r p o l y s a c c h a r i d e 155 V.3.1 I n t r o d u c t i o n 155 V.3.2 R e s u l t s and d i s c u s s i o n 155 V.3.3 C o n c l u s i o n 163 V.3.4 E x p e r i m e n t a l 163 VI . CONCLUDING REMARKS 167 V I I . BIBLIOGRAPHY 171 x i LIST OF APPENDICES Appendix Page I Q u a l i t a t i v e a n a l y s i s and chemotyping o f 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 183 I I G e l - p e r m e a t i o n chromatograms 185 I I I *H and 1 3C-N.m.r. s p e c t r a 188 IV C h e m i c a l i o n i z a t i o n mass s p e c t r a 227 x i i LIST OF TABLES T a b l e Page I I . 4.I Some o f the commonly used d e g r a d a t i o n t e c h n i q u e s i n p o l y s a c c h a r i d e c h e m i s t r y 28 I I I . l . I Growth media f o r the K l e b s i e l l a b a c t e r i a 48 I I I . 1. I I Growth media f o r the E. c o l i b a c t e r i a 49 IV. 1.I Sugar a n a l y s i s o f K l e b s i e l l a K79 and d e r i v e d p r o d u c t s 67 I V . l . I I N.m.r. d a t a f o r K l e b s i e l l a K79 c a p s u l a r 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 . . . . 69 I V . 1 . I l l M e t h y l a t i o n a n a l y s e s o f K l e b s i e l l a K79 p o l y s a c c h a r i d e and d e r i v e d p r o d u c t s 72 IV.2.I Sugar a n a l y s i s o f K l e b s i e l l a K35 p o l y s a c c h a r i d e and d e r i v e d p r o d u c t s 89 I V . 2 . I I N.m.r. d a t a f o r K l e b s i e l l a K35 c a p s u l a r 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 . . . 90 I V . 2 . I l l M e t h y l a t i o n a n a l y s e s o f K l e b s i e l l a K35 p o l y s a c c h a r i d e and d e r i v e d p r o d u c t s 94 IV.3.I Sugar a n a l y s i s o f E. c o l i K44 p o l y s a c c h a r i d e and d e r i v e d p r o d u c t s 110 I V . 3 . I I N.m.r. d a t a f o r E. c o l i K44 p o l y s a c c h a r i d e and d e r i v e d p r o d u c t s 113 x i i i IV. 3 . I l l M e t h y l a t i o n a n a l y s e s o f E. c o l i K44 p o l y s a c c h a r i d e and d e r i v e d p r o d u c t s 117 V. 2.I N.m.r. d a t a f o r K79<jJD and the K l e b s i e l l a K79 p o l y s a c c h a r i d e 144 V. 2 . I I M e t h y l a t i o n a n a l y s e s o f K79<£D 146 V.3.I N.m.r. d a t a f o r PI and the n a t i v e E. c o l i K44 p o l y s a c c h a r i d e 158 V.3. I I M e t h y l a t i o n a n a l y s e s o f PI and P1R 161 x i v LIST OF FIGURES F i g u r e Page I . I . I Diagrammatic r e p r e s e n t a t i o n o f the c e l l s u r f a c e o f G r a m - p o s i t i v e and Gram-negative b a c t e r i a . . . 4 I. l . I I Diagrammatic r e p r e s e n t a t i o n o f the c e l l s u r f a c e e n v e l o p e o f Gram-negative b a c t e r i a . : 4 I I . 5.1 D i f f e r e n t r e g i o n s i n the n.m.r. s p e c t r a o f p o l y s a c c h a r i d e s 34 IV.2.I Diagrammatic r e p r e s e n t a t i o n o f the ^H-n.m.r. s p e c t r a o f K l e b s i e l l a K35 p o l y s a c c h a r i d e and d e r i v e d p o l y s a c c h a r i d e s 98 I V . 2 . I I Digrammatic r e p r e s e n t a t i o n o f the ^ C-n.m.r. s p e c t r a o f K l e b s i e l l a K35 p o l y s a c c h a r i d e and d e r i v e d p o l y s a c c h a r i d e s 98 I V . 2 . I l l (a) ^H-N.m.r. spectrum o f p a r t i a l l y d e p y r u v y l a t e d K35 p o l y s a c c h a r i d e ; (b) Smith d e g r a d a t i o n p r o d u c t SD; (c) -^C-N.m.r. spectrum o f p a r t i a l l y d e p y r u v y l a t e d K35 p o l y s a c c h a r i d e 100 IV. 3.I 1H- and 1 3C-N.m.r. s p e c t r a o f E. c o l i K44 c a p s u l a r p o l y s a c c h a r i d e I l l V. I . I B a s i c m o r p h o l o g i c a l types o f b a c t e r i o p h a g e s w i t h the types o f n u c l e i c a c i d 134 V . l . I I A diagrammatic i l l u s t r a t i o n o f a b a c t e r i o p h a g e 134 xv V . l . I I I A s c h e m a t i c diagram i l l u s t r a t i n g the s t e p s i n the i n f e c t i o n o f a b a c t e r i u m by a b a c t e r i o p h a g e 136 V . l . I V B a c t e r i a l lawn w i t h h a l o e d p l a q u e s 137 V.2.I C o r r e l a t i o n between b a c t e r i a l count and the o p t i c a l d e n s i t y f o r K l e b s i e l l a K79 b a c t e r i a . . . 151 V.3.I ^H-N.m.r. s p e c t r a showing the anomeric r e g i o n o f (a) the crude p r o d u c t , (b) contaminant 2, (c) PI, and (d) E. c o l i K44 c a p s u l a r p o l y s a c c h a r i d e 157 xv i L IST OF SCHEMES Scheme Page II.1.1 Deamination sequence o f some amino s u g a r s . . . . 19 I I . 5 . I F r a g m e n t a t i o n pathways o f some p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s 41 II.5 . I I Some mass s p e c t r a l f r a g m e n t a t i o n pathways f o r p e r m e t h y l a t e d g l y c o p y r a n o s i d e s . . . 44 IV.1.I M e t h y l a t i o n a n a l y s i s o f the K l e b s i e l l a K79 c a p s u l a r p o l y s a c c h a r i d e 73 I V . l . I I P e r i o d a t e o x i d a t i o n - Smith d e g r a d a t i o n o f K l e b s i e l l a K79 c a p s u l a r p o l y s a c c h a r i d e 75 IV.2.I M e t h y l a t i o n a n a l y s i s o f the n a t i v e and d e p y r u v y l a t e d K l e b s i e l l a K35 c a p s u l a r p o l y s a c c h a r i d e 95 IV.3.I M e t h y l a t i o n a n a l y s i s o f the c a r b o d i i m i d e r e d u c e d E. c o l i K44 c a p s u l a r p o l y s a c c h a r i d e . . . 118 IV. 3.II P e r i o d a t e o x i d a t i o n - Smith d e g r a d a t i o n o f the E. c o l i K44 c a p s u l a r p o l y s a c c h a r i d e . . . . 112 V. 3.I Mass fragments o b s e r v e d i n the c h e m i c a l i o n i z a t i o n mass spectrum o f m e t h y l a t e d PI . . . . 162 x v i i LIST OF ABBREVIATIONS G l c - g l u c o s e ; G l c p - g l u c o p y r a n o s e G a l - g a l a c t o s e ; G a l f - g a l a c t o f u r a n o s e Man - mannose G l c A — g l u c u r o n i c a c i d GlcNAc - 2-acetamido-2-deoxyglucose GalNAc — 2-acetamido-2-deoxygalactose Gro - g l y c e r o l PMAA - p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e TFA - t r i f l u o r o a c e t i c a c i d DMSO - d i m e t h y l s u l f o x i d e Pyr - p y r u v i c a c i d a c e t a l Ac - a c e t y l Me - me t h y l mol. wt. - m o l e c u l a r weight s - seconds; min. - minutes; h - h o u r s ; d - days i . r . - i n f r a - r e d n.m.r. — n u c l e a r magnetic resonance g . l . c . - g a s - l i q u i d chromatography HPLC - h i g h p r e s s u r e l i q u i d chromatography m.s. — mass s p e c t r o m e t r y g.l.c.-m.s.« gas l i q u i d chromatography - mass s p e c t r o m e t r y I.e.-m.s.- l i q u i d chromatography - mass s p e c t r o m e t r y e.i.-m.s.- e l e c t r o n impact - mass s p e c t r o m e t r y c.i.-m.s.— c h e m i c a l i o n i z a t i o n - mass s p e c t r o m e t r y x v i i i f a s t atom bombardment b a c t e r i o p h a g e p l a q u e f o r m i n g u n i t s room temperature atomic mass u n i t x i x ACKNOWLEDGEMENTS I t has been a r e w a r d i n g e x p e r i e n c e to work under the s u p e r v i s i o n o f P r o f e s s o r Dutton. I have b e n e f i t t e d g r e a t l y from h i s d i r e c t i o n , i n s i g h t and g e n e r o s i t y d u r i n g the time spent i n h i s l a b o r a t o r y . F o r a l l these, I e x t e n d my g r a t e f u l thanks t o him. My i n t e r a c t i o n s w i t h the many v i s i t i n g p r o f e s s o r s , p o s t d o c t o r a l f e l l o w s and graduate s t u d e n t s from v a r i o u s c o u n t r i e s have n o t o n l y been s t i m u l a t i n g i n t e l l e c t u a l l y , b u t a l s o c u l t u r a l l y . To a l l these p e o p l e , I g r a t e f u l l y acknowledge t h e i r f r i e n d s h i p , c o o p e r a t i o n and a s s i s t a n c e . Thanks a r e a l s o due to the s t a f f o f the M.S. S e r v i c e , the Mechani-c a l and E l e c t r i c a l / E l e c t r o n i c Shops, and e s p e c i a l l y Dr. S.O. Chan and the f r i e n d l y s t a f f o f the N.m.r. S e r v i c e . I s h o u l d a l s o l i k e t o thank R a n i T h e e p a r a j a h f o r the c a r e f u l t y p i n g o f t h i s t h e s i s . The f i n a n c i a l a s s i s t a n c e g i v e n by the Ch e m i s t r y Department i n the form o f a T e a c h i n g A s s i s t a n t s h i p f o r the d u r a t i o n o f the time sp e n t h e r e i s a l s o g r a t e f u l l y acknowledged. My s t a y i n Vancouver has been made most memorable by the g o o d w i l l shown towards me by a group o f V a n c o u v e r i t e s whose names a r e too numer-ous to be mentioned h e r e . To these p e o p l e , I g r a t e f u l l y acknowledge t h e i r f r i e n d s h i p and may they be b l e s s e d and rewarded f o r t h e i r k i n d -ness . F i n a l l y , and most i m p o r t a n t l y , the work p r e s e n t e d i n t h i s t h e s i s would have been a l o t h a r d e r w i t h o u t the l o v e , u n d e r s t a n d i n g and encouragement o f my w i f e , Mei Yeng. I am g r a t e f u l and g r e a t l y i n d e b t e d XX or h e r p e r s e v e r a n c e and s a c r i f i c e d u r i n g the d u r a t i o n o f our 'student e a r s ' a t U.B.C. 1 CHAPTER I INTRODUCTION 2 I. INTRODUCTION The term ' g l y c a n ' i s t h e s y s t e m a t i c g e n e r i c name g i v e n t o p o l y -s a c c h a r i d e s i n w h i c h a l a r g e number o f g l y c o s e ( m o n o s a c c h a r i d e ) r e s i d u e s a r e j o i n e d by O - g l y c o s i d i c l i n k a g e s . P o l y s a c c h a r i d e s a r e t h e r e f o r e c o n d e n s a t i o n p o l y m e r s i n w h i c h each i n t e r s ugar g l y c o s i d i c l i n k a g e i s formed from the g l y c o s y l m o i e t y o f a h e m i a c e t a l and a h y d r o x y l group o f a n o t h e r s u g a r u n i t a c t i n g as an a c c e p t o r m o l e c u l e o r a g l y c o n e . I n N a t u r e , c a r b o h y d r a t e s a r e o f t e n l i n k e d t o o t h e r m o l e c u l e s such as p r o t e i n s and l i p i d s t o form: ( i ) g l y c o p r o t e i n s , p r o t e o g l y c a n s and p e p t i d o g l y c a n s , ( i i ) 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 , ( i i i ) t e i c h o i c a c i d s , and ( i v ) n u c l e i c a c i d s . P o l y s a c c h a r i d e s a r e o f w i d e s p r e a d o c c u r r e n c e and t h e i r r o l e , b o t h c o m m e r c i a l l y and b i o l o g i c a l l y , i s o f s i g n i f i c a n t i m p o r t a n c e . They have uses i n numerous i n d u s t r i e s ^ r a n g i n g from f o o d s , p h a r m a c e u t i c a l s , t e x t i l e s , p a p e r s , p a i n t s , c o s m e t i c s , and t e r t i a r y o i l r e c o v e r y t o the t r e a t m e n t o f e n v i r o n m e n t a l p o l l u t a n t s . The t h i c k e n i n g and g e l l i n g p r o p e r t i e s o f the p o l y s a c c h a r i d e s a r e the main f e a t u r e s c o n t r i b u t i n g t o t h e i r u s e f u l n e s s , w h i l e t h e i r e m u s i f y i n g , b i n d i n g , c o a t i n g and f i l m f o r m i n g a b i l i t i e s p l a y a s e c o n d a r y r o l e . P o l y s a c c h a r i d e s s e r v e t h r e e p u r p o s e s i n l i v i n g s y s t e m s : a r c h i t e c t u -r a l , n u t r i t i o n a l , and as s p e c i f i c a g e n t s . T h e i r i m p o r t a n c e i s e x h i -b i t e d i n many f a c e t s o f human l i f e where t h e y a r e components o f a n t i -b o d i e s , enzymes, n u c l e i c a c i d s , and c e l l s u r f a c e g l y c o c o n j u g a t e s 2 . P o l y s a c c h a r i d e s a l s o p l a y a v i t a l r o l e i n the p l a n t w o r l d , i n whi c h c e l l u l o s e i s the p r i n c i p a l s t r u c t u r a l component and s t a r c h s e r v e s as a - 3 -main energy store. I n Crustacea and i n s e c t s , c h i t i n p r o v i d e s s k e l e t a l s u p p o r t . C a r b o h y d r a t e s a r e a l s o o f i n t e r e s t as a renewable so u r c e of carbon, f o r energy s t o r a g e , and as a r e a d i l y m a n i p u l a t e d n a t u r a l source o f c h i r a l i t y . 1.1 Immunological importance o f b a c t e r i a l e x o p o l y s a c c h a r i d e s . S e v e r a l s p e c i e s o f Gram-negative b a c t e r i a and a few s p e c i e s of G r a m - p o s i t i v e b a c t e r i a p o s s e s s c a p s u l e s which s u r r o u n d the c e l l s i n the form o f a t h i c k o u t e r l a y e r c o n s i s t i n g o f p o l y s a c c h a r i d e ^ (see F i g . I . I . I ) . The term e x o p o l y s a c c h a r i d e i s a g e n e r i c name f o r a l l forms 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 found o u t s i d e the c e l l w a l l . Comparative s t r u c t u r a l a n a l y s i s has r e v e a l e d t h a t most b a c t e r i a l p o l y s a c c h a r i d e s have the same g e n e r a l s t r u c t u r a l f e a t u r e namely, they a r e composed of o l i g o s a c c h a r i d e r e p e a t i n g u n i t s ^ . R e l a t i v e l y l i t t l e i s known about the f u n c t i o n o f m i c r o b i a l e x o p o l y s a c c h a r i d e s i n c o n t r a s t to the w e a l t h of i n f o r m a t i o n about t h e i r p r i m a r y c h e m i c a l s t r u c t u r e s . I t has been suggested'' t h a t they may be i n v o l v e d i n one or a l l o f the f o l l o w i n g : ( i ) energy s t o r a g e o r r e s e r v e ( i i ) v i r u l e n c e , p r o t e c t i o n a g a i n s t p h a g o c y t o s i s ( i i i ) p r o t e c t i o n a g a i n s t d e s i c c a t i o n and p r e d a t o r s ( i v ) a d h e s i o n , and (v) as g e n e r a l b a r r i e r s . The l o c a t i o n o f 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 a t the outermost - 4 -Pepti i Capsule Outer Membrane P«plidoglycan Cytoplatmic membrane GRAM POSITIVE GRAM NEGATIVE F i g . I . I . I : Diagrammatic r e p r e s e n t a t i o n o f the c e l l s u r f a c e o f G r a m - p o s i t i v e and Gram-negative b a c t e r i a . Divalent ions (* ) ttatxlitmf the outer membrane Mmor outer membrane . protein WW] Dili ! Po»m C a p s u l a r p o l y s a c c h a r i d e (K a n t i g e n ) Lipooolytaccnaride eha.ni ( 0 a n t i g e n ) Phospholipid O U T E R M E M B R A N E I P E R I P L A S T I C S P A C E Free lipoprotein Bovnd lipoprotein C Y T O P L A S M I C M E M B R A N E ftBptrdofiycan F i g . I . l . I I : Diagrammatic r e p r e s e n t a t i o n o f the c e l l s u r f a c e envelope o f Gram-negative b a c t e r i a . ( r e a d a p t e d from r e f . 5 ) 5 s u r f a c e o f the c e l l g i v e s them i m p o r t a n c e . They a r e t h e o u termost m e d i a t o r s between the o r g a n i s m and i t s e n v i r o n m e n t , the f i r s t p o r t a l o f e n t r y and the l a s t b a r r i e r t o e x c r e t i o n . F u r t h e r m o r e , the c e l l e n v e l o p e ( c o m p r i s i n g t h e o u t e r membrane and c a p s u l e ) c o n t a i n s a n t i g e n s w h i c h i n d u c e t h e f o r m a t i o n o f a n t i b o d i e s i n man and a n i m a l s , and w h i c h r e a c t Q s e r o l o g i c a l l y w i t h t h e s e a n t i b o d i e s 0 . I t a l s o c o n t a i n s r e c e p t o r s f o r b a c t e r i o p h a g e s . P o l y s a c c h a r i d e s o f the c a p s u l e s a r e w e l l s u i t e d t o be a g e n t s o f s p e c i f i c i t y and d i v e r s i t y because t h e y a r e c a p a b l e o f g r e a t e r d i v e r s i t y p e r u n i t s t r u c t u r e t h a n o t h e r t y p e s o f m a c r o m o l e c u l e s . The b a c t e r i a l s u r f a c e thus c o n t a i n s components w h i c h p l a y an i m p o r t a n t r o l e - . Q m r e c o g n i t i o n p r o c e s s e s . The i m m u n o l o g i c a l d e f e n c e system o f h i g h e r o r g a n i s m s i s b a s e d on i t s c a p a c i t y t o r e c o g n i z e s t r u c t u r e s as a l i e n s o r as p a r t o f i t s e l f . I f t h e a l i e n s t r u c t u r e i s c a p a b l e o f i n d u c i n g the f o r m a t i o n o f a n t i b o d i e s , i t i s c a l l e d an a n t i g e n . I n d e a l i n g w i t h a n t i g e n i c b a c t e r i a l e x o p o l y -s a c c h a r i d e s , t h e i r immunogenic ( i . e . t h e i r c a p a c i t y t o i n d u c e the f o r m a t i o n o f a n t i b o d i e s ) and t h e i r a n t i g e n i c ( i . e . t h e i r r e a c t i v i t y w i t h a n t i b o d i e s ) a s p e c t s have t o be c o n s i d e r e d . R e c e n t l y , e x t e n s i v e r e v i e w s on the immunology o f 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 have been o i n p u b l i s h e d 0 ' 1 - . The b a s i s o f t h e s e i m m u n o l o g i c a l phenomena i s the r e c o g n i t i o n o f p a r t i a l s t r u c t u r e s o f the p o l y s a c c h a r i d e a n t i g e n s by a n t i b o d y m o l e c u l e s o r immune c e l l r e c e p t o r s . Hence the aim of the immunochemical a n a l y s i s o f p o l y s a c c h a r i d e a n t i g e n s , w h i c h combines s e r o l o g i c a l and c h e m i c a l s t u d i e s , i s t o d e f i n e a n t i g e n i c d e t e r m i n a n t s ( o r immunodominant s u g a r s ) w i t h i n the p o l y s a c c h a r i d e as c h e m i c a l e x p r e s -s i o n o f i t s i m m u n o l o g i c a l c h a r a c t e r . These a n t i g e n i c d e t e r m i n a n t s , - 6 -which form the major c o n t r i b u t o r s t o s e r o l o g i c a l s p e c i f i c i t y , can be a monosaccharide l i n k e d i n a s p e c i f i c manner, an o l i g o s a c c h a r i d e o r even n o n - c a r b o h y d r a t e i n n a t u r e e.g. a c e t a l - l i n k e d p y r u v i c a c i d , 0- and N - a c e t y l s ^ . I n 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 s such as those of K l e b s i e l l a . E. c o l i and Pneumococcus. the cha r g e d c o n s t i t u e n t s a r e o f t e n o p a r t o f the a n t i g e n i c d e t e r m i n a n t s 0 . I n br a n c h e d 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 i n the s i d e c h a i n s . Due to t h e i r r e p e t i t i v e s t r u c t u r 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 have the same a n t i g e n i c d e t e r m i n a n t s e x p r e s s e d many times over. The same a n t i g e n i c d e t e r m i n a n t however, may be p r e s e n t i n s e v e r a l p o l y s a c c h a r i d e s . As a consequence, i t i s r e c o g n i z e d by i t s homologous a n t i b o d i e s i n d i f f e r e n t p o l y s a c c h a r i d e s , no m a t t e r i n what organisms t h e s e d e t e r m i n a n t s are produced. These p o l y s a c c h a r i d e s t h a t a r e immuno-l o g i c a l l y r e l a t e d are s a i d t o c r o s s - r e a c t s e r o l o g i c a l l y . H e i d e l b e r g e r and coworkers-'-2"-'--' have used these c r o s s - r e a c t i o n s f o r the immunological d e t e r m i n a t i o n o f the s t r u c t u r e s o f many p o l y s a c c h a r i d e s . Furthermore, a n t i s e r a o f known s p e c i f i c i t y are o f g r e a t importance i n the s e r o l o g i c a l c l a s s i f i c a t i o n and r a p i d d i a g n o s i s o f b a c t e r i a , e s p e c i a l l y i n an e p i -demic. C r o s s - r e a c t i o n s were a l s o found between man and b a c t e r i a , i n v o l v i n g the c a r b o h y d r a t e m o i e t y o f b l o o d group s u b s t a n c e s and t i s s u e a n t i g e n s o f the h e a r t , k i d n e y , and more g e n e r a l l y , o f thr. HL-A a n t i g e n s ( t h e major h i s t o c o m p a t i b i l i t y a n t i g e n s o f man). As a means o f d i s e a s e p r e v e n t i o n , immunization u s i n g v a c c i n e s has been u t i l i z e d by man f o r a l o n g time. V a c c i n e s d e r i v e d from k i l l e d b a c t e r i a ( o r v i r u s ) p r e p a r a t i o n s , l i v e ' a t t e n u a t e d ' b a c t e r i a , and t o x o i d s a r e common and s t i l l e f f e c t i v e . The f i r s t use o f a b a c t e r i a l - 7 -p o l y s a c c h a r i d e as a v a c c i n e t o combat pneumococcal i n f e c t i o n s d u r i n g W orld War I I was r e p o r t e d by H e i d e l b e r g e r ^ - 0 . The sequence and mode o f l i n k a g e o f the sugar r e s i d u e i n the p o l y s a c c h a r i d e a r e r e c o g n i z e d by the immune system o f the body and the a n t i b o d i e s formed i n r e s p o n s e r e a c t n o t o n l y w i t h the p o l y s a c c h a r i d e a n t i g e n s , b u t a l s o w i t h the whole b a c t e r i a i n subsequent i n f e c t i o n s . S i n c e t h e n many e x o p o l y s a c c h a r i d e s o f b a c t e r i a , sometimes j u s t the h a p t e n i c o l i g o s a c c h a r i d e c o u p l e d t o a p r o t e i n c a r r i e r , have been used i n the p r o d u c t i o n o f v a c c i n e s - ^ . M i c r o b i a l p o l y s a c c h a r i d e s have a l s o shown p o t e n t i a l i n the a r e a o f c a n c e r r e s e a r c h ^ . E x t e n s i v e s t u d i e s have been r e p o r t e d on n o n c y t o t o x i c and h o s t - m e d i a t e d a n t i t u m o r p o l y s a c c h a r i d e s from v a r i o u s s o u r c e s amongst them 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 ^ - 0 • ^ . The s t r u c t u r a l s t u d i e s o f 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 r e v e r y i m p o r t a n t i n the l i g h t o f b a c t e r i a l i n f e c t i o n s , the p r o d u c t i o n o f p r o t e c t i v e v a c c i n e s , and t h e i r p o t e n t i a l as a n o n c y t o t o x i c , a n t i t u m o r drug. F urthermore, the knowledge o f the p r i m a r y o r c o v a l e n t s t r u c t u r e o f b a c t e r i a l p o l y s a c c h a r i d e s e x p l a i n s the b a s i s f o r t h e i r s e r o l o g i c a l c l a s s i f i c a t i o n s . I t a l s o forms the b a s i s f o r an u n d e r s t a n d i n g o f t h e i r t h r e e d i m e n s i o n a l s t r u c t u r e s i n the s o l i d s t a t e and i n s o l u t i o n , and f o r an a p p r e c i a t i o n o f the ways i n which p o l y s a c c h a r i d e s a r e b i o s y n t h e s i z e d and degraded. W i t h these and the l a t e n t p o t e n t i a l o f b a c t e r i a l p o l y -s a c c h a r i d e s i n mind, the fundamental t a s k o f e l u c i d a t i n g the d e t a i l e d c h e m i c a l s t r u c t u r e o f s e v e n t y - s e v e n K l e b s i e l l a and s e v e n t y - f o u r 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 i s c u r r e n t l y b e i n g u n d e r t a k e n i n t h i s and o t h e r l a b o r a t o r i e s around the w o r l d . I n t h i s t h e s i s , the d e t e r m i n a t i o n o f the c h e m i c a l s t r u c t u r e o f the K - a n t i g e n o f K l e b s i e l l a s e r o t y p e s K79 and K35, 8 K35, and E. c o l i s e r o t y p e K44 a r e p r e s e n t e d i n S e c t i o n IV. The b a c t e r i o p h a g e - i n d u c e d d e p o l y m e r i z a t i o n o f t h e c a p s u l a r p o l y s a c c h a r i d e s o f K l e b s i e l l a K79 and E. c o l i K44 a r e a l s o i n c l u d e d i n S e c t i o n V. 1.2 C h e m i s t r y and s e r o l o g y o f 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 genus K l e b s i e l l a c o m p r i s e s G r a m - n e g a t i v e , n o n - m o t i l e b a c t e r i a o f t h e f a m i l y E n t e r o b a c t e r i a c e a e and the t r i b e K l e b s i e l l e a e 2 ^ • 2 ^ . The common h a b i t a t o f t h e s e m i c r o - o r g a n i s m s a r e the upper r e s p i r a t o r y , i n t e s t i n a l , and g e n i t o - u r i n a r y t r a c t s o f h e a l t h y c a r r i e r s . A l t h o u g h most s t r a i n s a r e n o t t o x i c , the p a t h o g e n i c ones however cause pneumonia 9 9 and m e n i n g i t i s ^ . I s o l a n t s a r e i d e n t i f i e d and c l a s s i f i e d by b i o c h e m i c a l r e a c t i o n s i n t o t h r e e s p e c i e s namely, K. pneumoniae. K . r h i n o s c h l e r o m a t i s 91 and K. ozaenae" 1 1. These b a c t e r i a c a n be f u r t h e r s u b d i v i d e d i n t o s e r o t y p e s a c c o r d i n g t o t h e i r s u r f a c e a n t i g e n s 2 3 ' 2 ^ . These are the c a p s u l a r (German; K a p s e l ) o r K a n t i g e n s , o f w h i c h t h e r e a r e s e v e n t y -s e v e n r e c o g n i z e d s e r o t y p e s ; and the s o m a t i c (German; ohne Hauch) o r 0 a n t i g e n s c o n s i s t i n g o f l i p o p o l y s a c c h a r i d e s , o f w h i c h t h e r e a r e n i n e s e r o t y p e s (see F i g . I . l . I I ) . U n l i k e most E. c o l i b a c t e r i a , the K l e b s i e l l a b a c t e r i a do n o t p o s s e s s f l a g e l l a o r H a n t i g e n s as t h e y a r e n o n - m o t i l e . S i n c e most K l e b s i e l l a b a c t e r i a a r e h e a v i l y e n c a p s u l a t e d w i t h t h e h e a t s t a b l e c a p s u l a r p o l y s a c c h a r i d e , t h e 0 a n t i g e n i s c o m p l e t e l y s h i e l d e d . C o n s e q u e n t l y , s e r o l o g i c a l c l a s s i f i c a t i o n i s b a s e d s o l e l y on the i m m u n o l o g i c a l l y more s i g n i f i c a n t c a p s u l a r K a n t i g e n e.g. K l e b s i e l l a s e r o t y p e s K35 and K79. The numbers o f t h e s e K a n t i g e n s have no s i g n i f i -9 cance w h a t s o e v e r , t h e y were d e s i g n a t e d as new s e r o t y p e s were d i s c o v e r e d . 9 S 9 ft Nimraich" 1 J 1 ^ D has r e p o r t e d t h e q u a l i t a t i v e c o m p o s i t i o n o f most o f th e K l e b s i e l l a K a n t i g e n s , and c a t e g o r i z e d them i n t o c h e m o t y p e s 2 ^ (see A p p e n d i x I ) . To d a t e , a p p r o x i m a t e l y s e v e n t y o f t h e s e K l e b s i e l l a p o l y s a c c h a r i d e s have been s t u d i e d and t h i r t y - t w o d i f f e r e n t s t r u c t u r a l p a t t e r n s ( l i n e a r , b r a n c h e d and c o m b - l i k e ) have emerged from the s t r u c -t u r e s p r o p o s e d . The d i v e r s i t y o f s t r u c t u r e s p r e s e n t e d by the K a n t i g e n s , w h i c h have t h e same q u a l i t a t i v e c o m p o s i t i o n , j u s t i f i e s t h e i r s e r o l o g i c a l d i f f e r e n t i a t i o n . I n t h e i r r e v i e w on b a c t e r i a l p o l y s a c c h a -r i d e s , Kenne and L i n d b e r g " have r e p o r t e d the s t r u c t u r e s o f some 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 b u t a more u p d a t e d l i s t o f s i x t y -s e v e n s t r u c t u r e s has been c o m p i l e d by K a r u n a r a t n e 2 o \ S e r o l o g i c a l c r o s s r e a c t i o n s i n v o l v i n g a number o f them have been r e p o r t e d - * - 3 • 2 ^ > 3 ^ . L i k e most b a c t e r i a l p o l y s a c c h a r i d e s , t h e K l e b s i e l l a p o l y s a c c h a r i d e s a r e composed o f o l i g o s a c c h a r i d e r e p e a t i n g u n i t s , w h i c h c o m p r i s e t h r e e t o seven s u g a r r e s i d u e s , and exceed m o l e c u l a r w e i g h t s o f a m i l l i o n . The p o l y s a c c h a r i d e s a r e a c i d i c i n n a t u r e , due t o the p r e s e n c e o f u r o n i c a c i d s ( D - g a l a c t u r o n i c and D - g l u c u r o n i c a c i d s ) o r a k e t o a c i d . Commonly o c c u r r i n g hexose s u g a r s i n K l e b s i e l l a p o l y s a c c h a r i d e s a r e D - g a l a c t o s e , D - g l u c o s e , D-mannose, L-rhamnose and L - f u c o s e . These hexoses n o r m a l l y o c c u r i n the p y r a n o s e c o n f i g u r a t i o n , b u t g a l a c t o s e i n the f u r a n o s e c o n f i g u r a t i o n has been r e p o r t e d i n K123-*-, K 1 4 3 2 and K 4 1 3 3 . I n a d d i t i o n , n o n - c a r b o h y d r a t e s u b s t i t u e n t s such as a c e t y l and 1 - c a r b o x y e t h y l i d e n e ( a c e t a l - l i n k e d p y r u v i c a c i d ) may be p r e s e n t . I n c e r t a i n K l e b s i e l l a p o l y s a c c h a r i d e s , t h e s e n o n - c a r b o h y d r a t e s u b s t i t u e n t s have been r e p o r t e d t o be i m p o r t a n t immunodeterminant g r o u p s ^ ' 10 -1.3 C h e m i s t r y and s e r o l o g y o f E s c h e r i c h i a 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 . E s c h e r i c h i a c o l i . which b e l o n g s to the f a m i l y E n t e r o b a c t e r i o c e a e and the genus E s c h e r i c h i a . i s a u b i q u i t o u s b a c t e r i u m . I t i s commonly found i n the i n t e s t i n a l t r a c t o f mammals and i s a Gram-negative, non-s p o r i n g and, u s u a l l y , p e r i t r i c h o u s and f i m b r i a t e b a c i l l u s 3 - ' . Non-p a t h o g e n i c s t r a i n s have formed the work-horses o f b i o c h e m i s t s and g e n e t i c i s t s f o r e l u c i d a t i n g many g e n e r a l b i o l o g i c a l phenomena. A l t h o u g h the m a j o r i t y o f E. c o l i s t r a i n s are ha r m l e s s , some s e r o t y p e s o f E. c o l i cause s e v e r e d i s e a s e s i n man and a n i m a l s . These i n c l u d e a n t e r i t i s , u r e t h r i t i s , s e p t i c a e m i a , m e n i n g i t i s and o t h e r s y n d r o m e s 3 ^ • 3 ? . W i t h i n the b a c t e r i a l s p e c i e s o f E. c o l i . t h e r e a r e many s e r o t y p e s . The s e r o t y p i n g scheme i s based on the c e l l s u r f a c e a n t i g e n s (see F i g . I . l . I I ) . These comprise the c a p s u l a r or K a n t i g e n s (74 p o l y s a c c h a r i d e t y p e s ) , the somat i c 0 a n t i g e n s (164 l i p o p o l y s a c c h a r i d e t y p e s ) , and the f l a g e l l a r H a n t i g e n s (56 p r o t e i n a c e o u s t y p e s ) 3 - \ The c a p s u l a r p o l y -s a c c h a r i d e s (K a n t i g e n s ) have been f u r t h e r s u b d i v i d e d by Kauffmann 3^ i n t o t h r e e groups (A, B, and L) on the b a s i s o f t h e i r 0 a n t i s e r u m a g g l u t i n a b i l i t y and h e a t s t a b i l i t y . Hence the E. c o l i s t r a i n t h a t was i n v e s t i g a t e d i n t h i s t h e s i s can be s e r o t y p e d as E. c o l i 08:K44(A):H-. In r e c e n t y e a r s w i t h more o f the c h e m i c a l s t r u c t u r e s o f E. c o l i K a n t i g e n s r e p o r t e d , $ r s k o v and c o w o r k e r s 3 9 s u g g e s t e d t h a t t h e i r nomencla-t u r e be r e s t r i c t e d to a c i d i c p o l y s a c c h a r i d e K a n t i g e n s and p r o t e i n K a n t i g e n s . To date, o n l y two p r o t e i n K a n t i g e n s have been r e p o r t e d i . e . K 8 8 4 0 and K 9 9 3 9 . 11 As w i t h the 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 a c i d i c p o l y s a c c h a r i d e K a n t i g e n s o f E. c o l i a r e composed o f r e p e a t i n g u n i t s , v a r y i n g i n s i z e from one t o s i x s u g a r residues 4-''. Common monosaccha-r i d e s t h a t have been r e p o r t e d i n E. c o l i p o l y s a c c h a r i d e s a r e D-pyrano-hexoses ( g a l a c t o s e , g l u c o s e and mannose); D - p y r a n o s y l u r o n i c a c i d s ( g a l a c t u r o n i c and g l u c u r o n i c a c i d s ) ; deoxyhexoses ( L - f u c o p y r a n o s e and L-rhamnopyranose); 3-deoxy-D-manno-octulosonic a c i d (KDO) and 5-acetami-d o - 3 , 5 - d i d e o x y - D - g l y c e r o - D - g a l a c t o - n o n u l o p y r a n o s o n i c a c i d (NeuNAc). S i m i l a r l y , as f o r K l e b s i e l l a p o l y s a c c h a r i d e s , immunodominant non-c a r b o h y d r a t e s u b s t i t u e n t s i n c l u d e N- and 0- a c e t y l s , p h o s p h a t e , and 1 - c a r b o x y e t h y l i d e n e ( a c e t a l - l i n k e d p y r u v i c a c i d ) . I n t h e i r r e v i e w on b a c t e r i a l p o l y s a c c h a r i d e s , Kenne and L i n d b e r g h l i s t e d t h e p r o p o s e d s t r u c t u r e s o f E. c o l i K a n t i g e n s known up u n t i l 1982. S i n c e t h e n a few more s t r u c t u r e s have been r e p o r t e d , e.g. K 9 4 2 , K 1 4 4 3 , K 1 2 4 4 , K 2 6 4 5 , K 2 8 4 6 , K 3 2 4 7 , K 3 3 4 8 , K 3 4 4 9 , and K44 ( t h i s t h e s i s ) . An u p d a t e d l i s t o f th e p r o p o s e d s t r u c t u r e s f o r t h e E. c o l i K and 0 a n t i g e n s has been c o m p i l e d by A l t m a n ^ ^ . K54 i s the f i r s t 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 t o have an amino a c i d i n i t s r e p e a t i n g unit-'-'-. I n f o u r c a s e s , the c h e m i c a l s t r u c t u r e s o f the c a p s u l a r p o l y s a c c h a r i d e s o f E. c o l i and K l e b s i e l l a were f o u n d t o be i d e n t i c a l . They a r e : E. c o l i K30 and K l e b s i e l l a K 2 0 5 2 , E. c o l i K32 and K l e b s i e l l a K55, E. c o l i K33 and K l e b s i e l l a K58, and E. c o l i K42 and K l e b s i e l l a K 6 3 5 2 . R e c e n t l y , the c r o s s - r e a c t i o n s o f the K and 0 a n t i g e n s o f E. c o l i w i t h the a n t i s e r a f r o m o t h e r m i c r o - o r g a n i s m s were r e p o r t e d - ' 3 " . - 12 CHAPTER I I METHODOLOGY OF STRUCTURAL ANALYSIS OF BACTERIAL CAPSULAR POLYSACCHARIDES I I METHODOLOGY OF STRUCTURAL ANALYSIS OF POLYSACCHARIDES. The c a p s u l a r p o l y s a c c h a r i d e s o f K l e b s i e l l a and E. c o l i a r e h e t e r o p o l y s a c c h a r i d e s made up o f r e p e a t i n g u n i t s 0 , i n c o n t r a s t t o most p l a n t p o l y s a c c h a r i d e s ( e . g . exudate gums, a l g a l p o l y s a c c h a r i d e s ) - ' 0 w h i c h l a c k a d e f i n i t e o r g a n i z e d s t r u c t u r e . W i t h p o l y s a c c h a r i d e s t h a t c o n s i s t o f r e p e a t i n g u n i t s , the s t r u c t u r a l s t u d y w i l l a l l o w a d e f i n i t e p r i m a r y c h e m i c a l s t r u c t u r e t o be e s t a b l i s h e d . F o r a c o m p lete c h a r a c t e r i z a t i o n o f t h e p o l y s a c c h a r i d e r e p e a t i n g u n i t , a s t r u c t u r a l s t u d y must e s t a b l i s h : ( i ) t h e q u a l i t a t i v e and q u a n t i t a t i v e m o n o s a c c h a r i d e c o m p o s i t i o n ( i i ) t h e c o n f i g u r a t i o n (D o r L) o f t h e r e s p e c t i v e m o n o s a c c h a r i d e s ( i i i ) t he l i n k a g e p o s i t i o n s o f the m o n o s a c c h a r i d e s i n the p olymer ( i v ) t h e anomeric n a t u r e (a or /3) o f t h e g l y c o s i d i c l i n k a g e s (v) t h e sequence o f the m o n o s a c c h a r i d e s i n the r e p e a t i n g u n i t , and ( v i ) t h e p r e s e n c e and l i n k a g e p o s i t i o n s o f n o n - c a r b o h y d r a t e c o n s t i -t u e n t s . From t h e e x t e n s i v e s t u d i e s c o n d u c t e d on b a c t e r i a l p o l y s a c c h a r i d e s 0 , the e x i s t e n c e o f complex and w i d e l y d i v e r s e p a t t e r n s o f p o l y s a c c h a r i d e r e p e a t i n g u n i t s has been r e v e a l e d . The e l u c i d a t i o n o f any one s t r u c t u r e o f s u c h c o m p l e x i t y n e c e s s i t a t e s t h e use o f a c o m b i n a t i o n o f d i f f e r e n t c h e m i c a l a n a l y s e s and methods, w h i c h u s u a l l y i n v o l v e d e g r a d a t i o n p r o c e -d u r e s t o a g r e a t e r o r l e s s e r e x t e n t . I n the f o l l o w i n g s e c t i o n s , some o f t h e known c h e m i c a l methods u s e d i n 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 p o l y s a c c h a r i d e s w i l l be b r i e f l y d e s c r i b e d . These p r o c e d u r e s w i l l n o t be d e s c r i b e d i n d e t a i l as r e l e v a n t i n f o r m a t i o n i s r e a d i l y a v a i l a b l e i n many t e x t s and r e v i e w s J ' > J O . A g e n e r a l o v e r v i e w o f the methodology and - 1 4 -r e c e n t s i g n i f i c a n t developments w i l l be p r e s e n t e d . I I . 1 I s o l a t i o n a n d p u r i f i c a t i o n o f 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 . The o v e r a l l o b j e c t i v e s i n i s o l a t i n g a p o l y s a c c h a r i d e , as w i t h any o t h e r type o f macromolecule, a r e to o b t a i n t h a t m a t e r i a l i n as h i g h a y i e l d and i n as c h e m i c a l l y pure and homogeneous a form as p o s s i b l e . The problems u s u a l l y e n c o u n t e r e d are t h o se o f : ( i ) i s o l a t i o n w i t h o u t degra-d a t i o n ; ( i i ) p u r i f i c a t i o n so t h a t the p o l y s a c c h a r i d e i s o b t a i n e d f r e e o f c o n t a m i n a t i n g m a t e r i a l s e.g. l i p i d s , p r o t e i n s , and n u c l e i c a c i d s ; and ( i i i ) f r a c t i o n a t i o n t o g i v e s u b s t a n c e s t h a t a r e c h e m i c a l l y and p h y s i -c a l l y homogeneous to w i t h i n a c c e p t a b l e l i m i t s . I n the c a s e o f c a p s u l a r p o l y s a c c h a r i d e s from b a c t e r i a , the l y o p h i l i c n a t u r e o f the p o l y s a c c h a -r i d e s enhances t h e i r i s o l a t i o n i n a homogeneous form. The b a c t e r i a l c e l l w a l l components a r e l e s s s o l u b l e i n aqueous media due to t h e i r c l o s e a s s o c i a t i o n w i t h the m a t r i x and o t h e r macromolecules. S e v e r a l p r o c e d u r e s have been used i n the i s o l a t i o n o f p o l y s a c c h a -r i d e s from p l a n t and m i c r o b i a l o r i g i n s such as: f r a c t i o n a l p r e c i p i t a -t i o n ; s e l e c t i v e p r e c i p i t a t i o n by complex f o r m a t i o n w i t h K +, C u 2 + , C a 2 + , B a 2 + and b o r a t e s a l t s , i n s o l u b l e complex f o r m a t i o n w i t h dyes such as C a l c o f l u o r White 2R and Congo Red; e l e c t r o p h o r e s i s ; g e l chromatography, i o n exchange chromatography on DEAE-Sephadex, and a f f i n i t y chromatogra-phy u s i n g C o n c a n a v a l i n A-^. I n the c a s e o f 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 s from K l e b s i e l l a and E. c o l i . i s o l a t i o n was p e r f o r m e d by a non s o l v e n t p r e c i p i t a t i o n o f the aqueous s o l u t i o n ( i . e . s u p e r n a t a n t - 15 -from the u l t r a c e n t r i f u g a t i o n o f the b a c t e r i a l s u s p e n s i o n ) w i t h an a c e t o n e - e t h a n o l m i x t u r e ^ , f o l l o w e d by a s e l e c t i v e p r e c i p i t a t i o n w i t h C e t a v l o n ( c e t y l t r i m e t h y l ammonium b r o m i d e ) ^ s o l u t i o n . The q u a t e r n a r y ammonium f u n c t i o n a l i t y o f C e t a v l o n complexes w i t h the c a r b o x y l groups o f the a c i d i c p o l y s a c c h a r i d e , 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 . ( F o r d e t a i l s , see S e c t i o n I I I . l ) . Any n e u t r a l p o l y s a c c h a r i d e s e.g. (0 a n t i g e n s from the l i p o p o l y s a c c h a r i d e s ) p r e s e n t w i l l remain i n s o l u t i o n and c a n be i s o l a t e d s e p a r a t e l y . The p u r i t y and homogeneity ( o r the absence o f h e t e r o g e n e i t y ) o f the p o l y s a c c h a r i d e i s o l a t e d a r e the n c h e c k e d by g e l chromatography (when t h e column i s c a l i b r a t e d , t h i s w i l l a l s o g i v e the r e l a t i v e m o l e c u l a r weight o f the p o l y s a c c h a r i d e ) ^ , u l t r a - c e n t r i f u g a t i o n * * 3 , e l e c t r o p h o r e s i s * * 4 > 65 _ O u c h t e r l o n y double d i f f u s i o n a s s a y ^ and n.m.r. s p e c t r o s c o p y . The c o n s t a n c y o f the c h e m i c a l c o m p o s i t i o n o f the p o l y s a c c h a r i d e can be e s t a b l i s h e d by one o r more o f the f o l l o w i n g p r o c e d u r e s : sugar and m e t h y l a t i o n a n a l y s e s , o p t i c a l r o t a t i o n , and s o l u t i o n p r o p e r t i e s such as v i s c o s i t y . I I . 2 A n a l y s i s o f monosaccharides The d e t e r m i n a t i o n o f p o l y s a c c h a r i d e c o m p o s i t i o n r e q u i r e s the i d e n t i f i c a t i o n and q u a n t i t a t i v e e s t i m a t i o n o f the sugar c o n s t i t u e n t s . In a d d i t i o n , a n a l y s i s i s n e c e s s a r y f o r n o n - c a r b o h y d r a t e s u b s t i t u e n t s such as N-and 0- a c e t y l s , s u l f a t e , phosphate, and a c e t a l - l i n k e d p y r u v i c a c i d . I n t h e i r r e view, A m i n o f f and c o w o r k e r s ^ 7 have l i s t e d methods f o r the a n a l y s i s o f t h e s e n o n - c a r b o h y d r a t e s u b s t i t u e n t s . C e r t a i n o f these - 16 -s u b s t i t u e n t s can be a n a l y z e d non d e s t r u c t i v e l y by i . r . and n.m.r. s p e c t r o s c o p y . The use o f n.m.r. s p e c t r o s c o p y i n t h e p r e l i m i n a r y a n a l y -s i s o f p o l y s a c c h a r i d e s i s e s p e c i a l l y i m p o r t a n t , s i n c e the e a r l y r e c o g n i -t i o n o f t h e number o f sugar components and u n u s u a l f e a t u r e s i n the r e p e a t i n g u n i t , w i l l i n d i c a t e the need f o r s u i t a b l e c h e m i c a l l y b a s e d a n a l y t i c a l p r o c e d u r e s . II.2.1 A c i d h y d r o l y s i s The f i r s t s t e p i n the s t r u c t u r a l s t u d y o f a p o l y s a c c h a r i d e i s the q u a l i t a t i v e and q u a n t i t a t i v e d e t e r m i n a t i o n s o f t h e m o n o s a c c h a r i d e s r e l e a s e d on t o t a l h y d r o l y s i s . The h y d r o l y t i c c o n d i t i o n s ( t y p e and c o n c e n t r a t i o n o f a c i d , t e m p e r a t u r e and d u r a t i o n ) u s e d a r e i m p o r t a n t f o r a q u a n t i t a t i v e r e l e a s e o f s u g a r s w i t h minimum d e g r a d a t i o n . Commonly us e d a c i d s a r e t r i f l u o r o a c e t i c , h y d r o c h l o r i c , s u l f u r i c and f o r m i c . I n h i s r e v i e w , D u t t o n D ^ l i s t e d the a dvantages and d i s a d v a n t a g e s o f d i f f e r e n t a c i d s and t h e h y d r o l y t i c c o n d i t i o n s used. More r e c e n t l y , the use o f h y d r o g e n f l u o r i d e f o r s e l e c t i v e and t o t a l h y d r o l y s i s o f b a c t e r i a l p o l y s a c c h a r i d e s has been reported°9-72 T r i f l u o r o a c e t i c a c i d and h y d r o -c h l o r i c a c i d were the a c i d s o f c h o i c e used i n t h e work p r e s e n t e d i n t h i s t h e s i s . M o r e o v er, t h e y a r e b o t h e a s i l y removed under r e d u c e d p r e s s u r e . 7 ^ Capon' J has r e p o r t e d t h a t t h e h y d r o l y t i c r a t e s o f d i f f e r e n t g l y c o s i d i c l i n k a g e s v a r y g r e a t l y . 2 - A m i n o - 2 - d e o x y g l y c o s i d i c and 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 a r e more r e s i s t a n t t o a c i d h y d r o l y s i s t h a n hexose and 6-deoxyhexose g l y c o s i d i c l i n k a g e s . I n the case o f u r o n i c - 17 -a c i d , the p r e s e n c e o f e l e c t r o n w i t h d r a w i n g c a r b o x y l group s t a b i l i z e s the u r o n o s y l l i n k a g e through the h e t e r o c y c l i c o x y g e n 7 4 . To h y d r o l y z e a c i d i c p o l y s a c c h a r i d e s a method d e v e l o p e d i n t h i s l a b o r a t o r y , i n v o l v e s the use o f m e t h a n o l y s i s 7 - ' . Treatment o f the a c i d i c p o l y s a c c h a r i d e w i t h m e t h a n o l i c hydrogen c h l o r i d e c l e a v e s most g l y c o s i d i c bonds t o form the methyl g l y c o s i d e s and a t the same time, the methyl e s t e r s o f the u r o n i c a c i d s . R e d u c t i o n o f the u r o n i c e s t e r s t o t h e i r c o r r e s p o n d i n g a l c o h o l and subsequent a c i d h y d r o l y s i s ensures complete r e l e a s e o f sugar r e s i -dues. A l t e r n a t i v e l y , the r e d u c t i o n o f u r o n i c a c i d t o hexose r e s i d u e s may be a c h i e v e d b e f o r e h y d r o l y s i s by s u c c e s s i v e t r e a t m e n t s o f the a c i d i c p o l y s a c c h a r i d e w i t h 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 boro-h y d r i d e 7 6 . Amino sugars 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 o c c u r most commonly as 2-acetamido-2-deoxyhexoses (e.g. GalNAc and Glc N A c ) . 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 f r e e amino group becomes p r o t o n a t e d i n a c i d i c media and the sugar i s the n h i g h l y r e s i s t a n t t o h y d r o l y s i s . A l t e r n a t i v e s to a c i d h y d r o l y s i s a r e methanoly-s i s 7 ^ and a c e t o l y s i s - h y d r o l y s i s 7 7 . More r e c e n t l y , the v e r s a t i l e h ydrogen f l u o r i d e h y d r o l y s i s was r e p o r t e d 7 * ^ • 7 2 to c l e a v e b o t h u r o n o s y l and 2 - a c e t a m i d o - 2 - d e o x y g l y c o s i d i c l i n k a g e s w i t h r e l a t i v e ease. Some members o f our r e s e a r c h group a r e c u r r e n t l y c o n d u c t i n g h y d r o l y t i c s t u d i e s on v a r i o u s p o l y s a c c h a r i d e s ( e s p e c i a l l y those c o n t a i n i n g amino s u g a r s ) u s i n g t h i s h y d r o f l u o r i n o l y s i s t e c h n i q u e . However, f o r the amino s u g a r - c o n t a i n i n g p o l y s a c c h a r i d e o f E. c o l i K44, t r e a t m e n t w i t h 2M h y d r o c h l o r i c a c i d a t 95° f o r 7 h g i v e s r e a s o n a b l e p r o p o r t i o n s o f n e u t r a l hexoses t o amino s u g a r s . 18 -An a l t e r n a t i v e approach t o the a n a l y s i s o f amino s u g a r s i s t o p e r f o r m b a s i c N - d e a c e t y l a t i o n b e f o r e h y d r o l y s i s , o r t o a l l o w N-deacety-l a t i o n t o t a k e p l a c e d u r i n g h y d r o l y s i s o f a l l b u t t h e 2-amino-2-deoxy-g l y c o s i d i c l i n k a g e s , and t h e n t o s u b m i t t h e amino s u g a r s t o n i t r o u s a c i d 7 ft deamination'°. 2-Amino-2-deoxyhexoses when t r e a t e d w i t h n i t r o u s a c i d undergo c y c l i c r e a r r a n g e m e n t s , e.g. 2-amino-2-deoxyglucose forms 2,5-anhydro-D-mannose, and 2 - a m i n o - 2 - d e o x y g a l a c t o s e forms 2,5-anhydro-D - t a l o s e (see Scheme I I . 1 . 1 ) . S e v e r a l methods have been r e p o r t e d f o r N - d e a c e t y l a t i o n o f N - a c e t y l a t e d hexosamines. The two commonly used methods a r e ( i ) h y d r a z i n o l y s i s ^ (anhydrous h y d r a z i n e w i t h c a t a l y t i c amount o f h y d r a z i n e s u l f a t e ) , and ( i i ) t r e a t m e n t w i t h sodium h y d r o x i d e I n aqueous d i m e t h y l s u l f o x i d e w i t h t h i o p h e n o l as oxygen s c a v e n g e r 0 ^ . II.2.2 C h a r a c t e r i z a t i o n and q u a n t i t a t i o n o f monosaccharides from t o t a l h y d r o l y s i s . The c l a s s i f i c a t i o n o f s u g a r s i n t o b r o a d c l a s s e s e.g. p e n t o s e s , d e o x yhexoses, h e x o s e s , u r o n i c a c i d s , amino s u g a r s and s i a l i c a c i d s , by c o l o r i m e t r i c a n a l y s e s 0 - 1 i s p o s s i b l e , b u t has l i m i t e d a p p l i c a t i o n f o r i n d i v i d u a l c h a r a c t e r i z a t i o n . The c h a r a c t e r i z a t i o n and q u a n t i t a t i o n o f s u g a r s r e l e a s e d upon h y d r o l y s i s o f a p o l y s a c c h a r i d e a r e n o r m a l l y p e r -Q O formed by g . l . c . . T h i s method r e q u i r e s t h e f o r m a t i o n o f d e r i v a t i v e s o f s u f f i c i e n t v o l a t i l i t y and adequate s t a b i l i t y . An e x t e n s i v e r e v i e w of t h i s t e c h n i q u e , the v o l a t i l e d e r i v a t i v e s and g . l . c . columns used, has OR ,0 ,OGIy NH2 OR *"^ )D' equatorial 2-amino OGly e \ RO/ • y_j/CH=0Gly RO glycoiidic cleavage 6 r • HO-Gly liberation of aglycone R'O OR' CH=0R epime • t C2 rizatlon I I ROH OR' CHO liberation of 3-O-glycoayl euEstituent 1^9 H 2 N OR equatorial 3-amino Rbn/0 ;'y RO OR Rb -» \ OHC OR' Rb-,/0 ROH liberation of 2-O-glycoayl auEatituent ROH liberation of 4-O-glyeoayl •uEatituent y r axial 4-amino CH 3 .-0 0-C . H 3 ^ 0 Ac H3C Ov NHAc Scheme II . 1 . 1 : Deaminatlon sequence o f some amino-sugars . - 20 -been p u b l i s h e d by D u t t c m ^ ' 8 3 . The two most w i d e l y used d e r i v a t i v e s are a l d i t o l a c e t a t e s 8 4 and a c e t y l a t e d a l d o n o n i t r i l e s 8 - ' ~ 8 7 . T h i s approach e l i m i n a t e s the problem o f m u l t i p l e d e r i v a t i v e f o r m a t i o n when d i f f e r e n t r i n g forms and/or d i f f e r e n t anomeric forms are g e n e r a t e d from r e d u c i n g s u g a r s or from e q u i l i b r i u m m i x t u r e s o f methyl g l y c o s i d e s . The a c y c l i c d e r i v a t i v e s a r e c h a r a c t e r i z e d by r e t e n t i o n times and, i f n e c e s s a r y , f o r sugars o f u n u s u a l s t r u c t u r a l f e a t u r e s , from mass s p e c t r a . K o n t r o h r and Q Q K o c s i s 0 0 have r e c e n t l y r e p o r t e d the s e p a r a t i o n o f e i g h t e p i m e r i c 2-aminoaldohexoses as amino h e x i t o l a c e t a t e s by g . l . c . Paper chromato-g r a p h y 8 9 , 9 ^ i s a l s o used to g i v e a q u a l i t a t i v e a n a l y s i s o f the sugars r e l e a s e d a f t e r a c i d h y d r o l y s i s . More r e c e n t l y , h i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h y 9 9 2 i s used, b o t h q u a l i t a t i v e l y and q u a n t i t a t i v e l y , on u n d e r i v a t i z e d s u g a r s . I I . 2 . 3 D e t e r m i n a t i o n o f the c o n f i g u r a t i o n (D o r L) o f the monosaccharides. Many sugars e.g. g l u c o s e and x y l o s e , have been found n a t u r a l l y i n o n l y the one e n a n t i o m e r i c D form. O t h e r s such as g a l a c t o s e , mannose, a r a b i n o s e , rhamnose and f u c o s e , have been found i n b o t h e n a n t i o m e r i c forms, a l t h o u g h n o t n e c e s s a r i l y i n the same p o l y s a c c h a r i d e . The D or L c o n f i g u r a t i o n o f the sugars can be d e t e r m i n e d u s i n g the f o l l o w i n g methods: ( i ) the use o f s p e c i f i c o x i d a s e s (e.g. D-glucose and D - g a l a c t o s e o x i d a s e s ) and e x o h y d r o l a s e s (e.g. / 3 - D - g l u c u r o n i d a s e ) 9 3 > 9 4 , - 21 -( i i ) t h e i s o l a t i o n o f the d i f f e r e n t s u g a r s and measurement o f t h e i r o p t i c a l r o t a t i o n 9 - * • , ( i i i ) the g . l . c . s e p a r a t i o n o f enantiomers, u s i n g a c h i r a l s t a t i o n a r y phase o r c o n v e r t i n g the enantiomers i n t o d i a s t e r e o m e r s u s i n g such c h i r a l agents as ( + ) - 2 - o c t a n o l 9 7 » 9 8 , and ( - ) - 2 - b u t a n o l 9 9 and s e p a r a t i o n on a n o n - c h i r a l phase, and ( i v ) the method u s e d i n our l a b o r a t o r y i s to c o n duct c i r c u l a r dichroism-'-OO measurements a t 213 nm on a l d i t o l a c e t a t e s , a c e t y -l a t e d a l d o n o n i t r i l e s o r p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s , where the a c e t o x y l group a c t s as a chromophore. I I . 2 . 4 D e t e r m i n a t i o n o f a n o m e r i c c o n f i g u r a t i o n . The monosaccharides p r e s e n t i n a p o l y s a c c h a r i d e can be g l y c o s i d i -c a l l y l i n k e d e i t h e r i n an a ( a x i a l l y o r i e n t e d ) o r /3 ( e q u a t o r i a l l y o r i e n t e d ) c o n f i g u r a t i o n . The assignment o f anomeric c o n f i g u r a t i o n to s p e c i f i c l i n k a g e s i n an o l i g o - o r p o l y - s a c c h a r i d e can be e s t a b l i s h e d by the f o l l o w i n g methods: ( i ) measurement o f the o p t i c a l r o t a t i o n 9 ^ • 9 ^ , ( i i ) the use o f s p e c i f i c enzymes ( e x o g l y c a n a s e ) ^ 1 e.g. a- and jS-D-g l u c u r o n i d a s e , a- and /3-D-galactosidase, ( i i i ) the use o f chromium t r i o x i d e o x i d a t i o n ^ 2 o n p e r a c e t y l a t e d c a r b o h y d r a t e samples, where o n l y the ) S - l i n k e d sugars are degraded i n p r e f e r e n c e t o the a - l i n k e d s u g a r s , and ( i v ) measurement o f the c o u p l i n g c o n s t a n t s and c h e m i c a l s h i f t s o f the - 22 -v a r i o u s sugars i n the n.m.r. s p e c t r a o f o l i g o - and p o l y -s a c c h a r i d e s (see S e c t i o n I I . 5 . 1 ) . I I . 3 D e t e r m i n a t i o n o f p o s i t i o n s o f l i n k a g e s o f monosaccharides and n o n - c a r b o h y d r a t e c o n s t i t u e n t s . A f t e r e s t a b l i s h i n g the n a t u r e and number o f 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 o f the p o l y s a c c h a r i d e , the subsequent s t e p i s to d e t e r -mine the p o s i t i o n s i n which the sugars are l i n k e d i n the c h a i n . In a d d i t i o n , the p o s i t i o n ( s ) o c c u p i e d by n o n - c a r b o h y d r a t e c o n s t i t u e n t s such as 0 - a c e t y l and a c e t a l - l i n k e d p y r u v i c a c i d , and the sugar t o which these s u b s t i t u e n t s a r e a t t a c h e d have to be e s t a b l i s h e d . I I . 3 . 1 M e t h y l a t i o n a n a l y s i s 1 0 3 M e t h y l a t i o n a n a l y s i s i s the o l d e s t , but s t i l l by f a r the most w i d e l y used p r o c e d u r e f o r l i n k a g e a n a l y s i s i n c a r b o h y d r a t e polymers. The method i s based on the complete e t h e r i f i c a t i o n ( m e t h y l a t i o n ) o f f r e e sugar h y d r o x y l groups i . e . those not i n v o l v e d i n r i n g f o r m a t i o n , i n t e r -sugar g l y c o s i d i c l i n k a g e s , or c a r r y i n g b a s e - s t a b l e s u b s t i t u e n t s (e.g. a c e t a l - l i n k e d p y r u v i c a c i d ) . The i n t e r g l y c o s i d i c bonds o f the permethy-l a t e d c a r b o h y d r a t e sample are s u b s e q u e n t l y h y d r o l y z e d (2 M t r i f l u o r o -a c e t i c a c i d a t 95° f o r 16 h f o r K l e b s i e l l a K79 and K35 p o l y s a c c h a r i d e s , and 2 M h y d r o c h l o r i c a c i d a t 95° f o r 7 h f o r E. c o l i K44 p o l y s a c c h a -- 23 -r i d e ) ; t h e r e l e a s e d s u g a r s a r e r e d u c e d and c o n v e r t e d t o p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s f o r g.l.c.-m.s. a n a l y s i s . The u n m e t h y l a t e d p o s i t i o n s o f t h e s u g a r s r e p r e s e n t s i t e s o f l i n k a g e , e x c e p t i n the c a s e s o f u r o n i c a c i d r e s i d u e s o r r e s i d u e s w i t h p y r u v i c a c i d a c e t a l a t t a c h e d . The n a t u r e o f u r o n i c a c i d s can be i d e n t i f i e d by c o m p a r i s o n o f 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 o f the a c i d i c p o l y s a c c h a r i d e w i t h t h o s e o f t h e r e d u c e d p o l y s a c c h a r i d e , o r the r e d u c e d p o l y s a c c h a r i d e w i t h the c o r r e s p o n d i n g r e d u c e d r e m e t h y l a t e d p r o d u c t . F o r a l l t h e s e s u g a r s the p r e s e n c e o f a m e t h o x y l group a t C-4 o r C-5 d e f i n e s t h e r i n g s i z e as p y r a n o s e o r f u r a n o s e , r e s p e c t i v e l y . However, c e r t a i n p a r t i a l l y methy-l a t e d s u g a r s do n o t c a r r y m e t h o x y l groups a t e i t h e r p o s i t i o n s , and h e r e m e t h y l a t i o n a n a l y s i s does n o t 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 t y p e . R e c e n t l y , Gray and cowo r k e r s 1 0 4 ' 1 0 5 have r e p o r t e d the r e d u c t i v e - c l e a v a g e method whereby the l i n k a g e p o s i t i o n ( s ) and r i n g form o f each m o n o s a c c h a r i d e r e s i d u e a r e 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 . T h i s method i n v o l v e s the i o n i c h y d r o g e n a t i o n o f a l l g l y c o s i d i c c a rbon-oxygen bonds i n the f u l l y m e t h y l a t e d p o l y s a c c h a r i d e w i t h t r i e t h y l s i l a n e c a t a -l y z e d by b o r o n t r i f l u o r i d e e t h e r a t e o r 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 -s u l f o n a t e . T h i s r e d u c t i v e c l e a v a g e a f f o r d s a s e r i e s o f p a r t i a l l y methy-l a t e d a n h y d r o a l d i t o l s , w h i c h a r e s u b s e q u e n t l y a c e t y l a t e d and a n a l y s e d by g.l.c.-m.s. To summarize, m e t h y l a t i o n a n a l y s i s 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 the f o l l o w i n g : ( i ) number and t y p e o f s u g a r s p e r r e p e a t i n g u n i t ; t h e s e d a t a w i l l be complementary t o the s u g a r a n a l y s i s d a t a ( i i ) r i n g s i z e o f the s u g a r r e s i d u e ( i i i ) l i n k a g e p o s i t i o n s 24 ( i v ) i d e n t i t y o f the t e r m i n a l u n i t s and b r a n c h i n g u n i t ( s ) (v) the p o s i t i o n s of b a s e - s t a b l e (e.g. p y r u v i c a c i d a c e t a l ) and base l a b i l e (e.g. O - a c e t y l ) s u b s t i t u e n t s , and ( v i ) the degree o f p o l y m e r i z a t i o n f o r an o l i g o s a c c h a r i d e a l d i t o l . S e v e r a l m e t h y l a t i o n p r o c e d u r e s 1 * ^ " 1 1 1 have been dev e l o p e d , but the method used t h r o u g h o u t i n the work p r e s e n t e d i n t h i s t h e s i s was the 11 o 1 -1 o Hakomori J-' L^ • • L J- J p r o c e d u r e . The p o l y s a c c h a r i d e , d i s s o l v e d i n d i m e t h y l s u l f o x i d e , i s f i r s t t r e a t e d w i t h sodium m e t h y l s u l f i n y l m e t h a n i d e (sodium d i m s y l ) and s u b s e q u e n t l y r e a c t e d w i t h methyl i o d i d e . The s t r o n g base, which i s the c o n j u g a t e base o f the s o l v e n t , ensures complete a l k o x i d e f o r m a t i o n . E f f i c i e n t m e t h y l a t i o n o f f r e e h y d r o x y l and acetamido ( o f amino s u g a r s ) groups i s u s u a l l y a c h i e v e d i n one s t e p , as i n d i c a t e d by the absence o f the h y d r o x y l s t r e t c h i n g v i b r a t i o n s i n i n f r a - r e d s p e c t r o -scopy. However i n cases o f i n c o m p l e t e m e t h y l a t i o n , a subsequent P u r d i e m e t h y l a t i o n 1 1 4 (Ag20 i n r e f l u x i n g methyl i o d i d e ) i s conducted. A second Hakomori m e t h y l a t i o n i s never conducted on an a c i d i c o l i g o - o r p o l y -s a c c h a r i d e as e x t e n s i v e d e g r a d a t i o n by ^ - e l i m i n a t i o n would take p l a c e on a d d i t i o n o f f r e s h base 1 1-'' 1 1*'. The c h a r a c t e r i z a t i o n o f p a r t i a l l y m e t h y l a t e d monosaccharides r e l e a s e d on t o t a l h y d r o l y s i s o f the p e r m e t h y l a t e d o l i g o - o r p o l y -s a c c h a r i d e i s c o n v e n i e n t l y performed by paper chromatography 8 9 , 9'-'. The p apers on development w i t h p _ - a n i s i d i n e and h e a t i n g , g i v e a 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 o f the m e t h y l a t e d sugars a c c o r d i n g to t h e i r m o b i l i t i e s and the d i f f e r e n t c o l o r s formed (see S e c t i o n IV.1.5). F u r t h e r q u a l i t a -t i v e and q u a n t i t a t i v e a n a l y s i s o f the sugars (as p a r t i a l l y m e t h y l a t e d - 25 -a l d i t o l a c e t a t e s ) i s p erformed u s i n g g . l . c . by comparing the r e t e n t i o n times on v a r i o u s columns w i t h known v a l u e s and co-chromatography w i t h a u t h e n t i c samples. I n c a s e s o f a m b i g u i t i e s , c o n f i r m a t i o n i s sought by g.l.c.-m.s. a n a l y s i s . E x t e n s i v e reviews on the a p p l i c a t i o n o f g . l . c . t o c a r b o h y d r a t e a n a l y s i s have been p u b l i s h e d 0 ^ » ^ 3 . The v o l a t i l e p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s (PMAAs) are w i d e l y u s ed because s i m p l e and w e l l - r e s o l v e d chromatograms a r e o b t a i n e d d u r i n g g . l . c . a n a l y s i s . The r e l a t i v e r e t e n t i o n times-'--'-^ (on packed and c a p i l l a r y g . l . c . columns) and mass s p e c t r a ^ - 0 * o f PMAAs o f many n e u t r a l monosaccharides have been p u b l i s h e d . The r e t e n t i o n times and mass s p e c t r a l d a t a f o r v a r i o u s m e t h y l a t e d d e r i v a t i v e s o f 2-amino-2-deoxyhexoses (e . g . GalNAc and GlcNAc) have a l s o been r e p o r t e d ^ • . Base l i n e s e p a r a t i o n s o f m i x t u r e s o f PMAAs a r e p o s s i b l e on packed columns made o f SP-2340 (75% c y a n o p r o p y l s i l i c o n e ) , SP-1000, ECNSS-M ( e t h y l e n e s u c c i n a t e - c y a n o e t h y l s i l i c o n e c o p o l y m e r ) , and OV-225 (25% p h e n y l , 25% c y a n o p r o p y l , m e t h y l s i l i c o n e ) , a l l o f which are p o l a r m a t e r i a l s . The use o f c a p i l l a r y columns i n the l a s t decade has g r e a t l y enhanced the v e r s a t i l i t y o f g . l . c . Fused s i l i c a columns are t h e r m a l l y s t a b l e a t h i g h temperatures, g i v e good r e s o l u t i o n and d e t e c t samples i n the microgram range. The s t a t i o n a r y l i q u i d phases u s e d i n t h i s s t u d y were DB-17 and SE-30. Lomax and C o n c h i e ^ ^ have r e p o r t e d a computer-a s s i s t e d i d e n t i f i c a t i o n system f o r PMAAs u s i n g more than s i x t y PMAAs s e p a r a t e d on a g l a s s c a p i l l a r y column o f SP-1000. U s i n g t h i s method, the y were a b l e t o i d e n t i f y a u t o m a t i c a l l y l a r g e numbers o f unknown peaks i n m e t h y l a t e d b i o l o g i c a l samples. - 26 -I I . 3 . 2 A n a l y s i s o f n o n - c a r b o h y d r a t e c o n s t i t u e n t s I n K l e b s i e l l a and 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 , t h e commonly fo u n d n o n - c a r b o h y d r a t e c o n s t i t u e n t s a r e a c e t a t e and a c e t a l l i n k e d p y r u v a t e groups. The N - a c y l group, u s u a l l y a s s o c i a t e d w i t h 2-acetami-do-2-deoxy hexoses e.g. GalNAc and GlcNAc, i s n o t c o n s i d e r e d to be a s u b s t i t u e n t b u t forms an i n t e g r a l p a r t o f these s u g a r s . Q u a n t i t a t i o n o f the O - a c e t y l group p e r r e p e a t i n g u n i t o f a p o l y s a c c h a r i d e c a n be meas-u r e d from i t s ^H-n.m.r. spectrum (from i n t e g r a t i o n o f p r o t o n s ) . The f o l l o w i n g methods can be u s e d f o r the p r e c i s e d e t e r m i n a t i o n o f the O - a c e t y l p o s i t i o n on the sugar moiety: ( i ) XH- and •L->C-n.m. r . s p e c t r o s c o p y ; and more r e c e n t l y w i t h the a p p l i c a t i o n o f 2D-n.m.r. t e c h n i q u e s 4 7 . ( i i ) t h e method o f de B e l d e r and N o r r m a n 1 2 1 . ( i i i ) Prehm m e t h y l a t i o n 1 ^ 7 , and ( i v ) p e r i o d a t e o x i d a t i o n which p r o v i d e s i n f o r m a t i o n on a c e t a t e c o n t a i n -i n g s u g a r s o n l y i f the a c e t a t e s u b s t i t u e n t and g l y c o s i d i c l i n k a g e s a r e s u i t a b l y l o c a t e d . The p r e s e n c e o f a c e t a l - l i n k e d p y r u v i c a c i d i s b e s t i d e n t i f i e d and q u a n t i f i e d by n.m.r. s p e c t r o s c o p y (see S e c t i o n I I . 5 . 1 ) . As a c o n f i r m a -t i o n , p y r u v i c a c i d can be i d e n t i f i e d , a f t e r i t s r e l e a s e from a p o l y s a c -c h a r i d e by m i l d h y d r o l y s i s , u s i n g 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e 1 2 2 _ o r by l a c t a t e d e h y d r o g e n a s e 1 2 3 . p o s i t i o n o f the p y r u v i c a c i d a c e t a l and the sugar r e s i d u e I n the polymer can be e s t a b l i s h e d by comparing the g.l.c.-m.s. d a t a o f the m e t h y l a t i o n a n a l y s e s o f the n a t i v e p o l y s a c c h a -- 27 -r i d e and t h e 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 . The appearance o f 2 , 4 , 6 - t r i - O - m e t h y l g a l a c t o s e and t h e c o n c o m i t a n t d i s a p p e a r a n c e o f 2 - O - m e t h y l g a l a c t o s e showed t h a t the a c e t a l i s l i n k e d a t 0-4 and 0-6 o f the g a l a c t o s y l r e s i d u e i n the K l e b s i e l l a K35 p o l y s a c c h a r i d e . N.m.r. s p e c t r o s c o p y has a l s o been i n v a l u a b l e i n d e t e r m i n i n g t h e c o n f i g u r a t i o n (R o r S) about t h e a c e t a l i c c a r b o n o f t h e p y r u v i c a c i d ^ 2 ^ . I I . 4 G e n e r a t i o n o f o l i g o s a c c h a r i d e fragments f o r the d e t e r m i n a t i o n o f monosaccharide sequence. The f r a g m e n t a t i o n o f a p o l y s a c c h a r i d e i n t o s m a l l e r o l i g o s a c c h a r i d e u n i t s by the use o f v a r i o u s d e g r a d a t i v e methods, f o l l o w e d by the 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 o f t h e s e o l i g o s a c c h a r i d e s i s t h e key t o e s t a b l i s h i n g t h e sequence o f ' s u g a r s i n the p o l y s a c c h a r i d e . I n a d d i t i o n , n.m.r. a n a l y s e s o f t h e s e o l i g o s a c c h a r i d e s a l l o w unambiguous s i g n a l a s s i g n m e n t s and p r o v i d e i n f o r m a t i o n on anomeric c o n f i g u r a t i o n s . There a r e many t y p e s o f s p e c i f i c d e g r a d a t i o n r e a c t i o n s on p o l y s a c c h a r i d e s w h i c h g i v e r i s e t o d i f f e r e n t y e t complementary d a t a . Summarized below i n t a b u l a r form i s a l i s t o f some o f the more commonly u s e d d e g r a d a t i o n t e c h n i q u e s i n p o l y s a c c h a r i d e c h e m i s t r y . The use o f b a c t e r i o p h a g e - b o r n e enzymes as a' means o f s e l e c t i v e l y c l e a v i n g b a c t e r i a l p o l y s a c c h a r i d e s w i l l be t r e a t e d i n more d e t a i l i n S e c t i o n V. As each b a c t e r i a l p o l y s a c -c h a r i d e i s u n i q u e i n i t s s t r u c t u r a l c h a r a c t e r i s t i c s , i t i s the t a s k o f the i n v e s t i g a t o r t o d e c i d e on the d e g r a d a t i v e p r o c e d u r e s t h a t w i l l b e s t s u i t t he p o l y s a c c h a r i d e under i n v e s t i g a t i o n . T a b l e I I . 4 . I : Some o f the c h e m i s t r y commonly used d e g r a d a t i o n t e c h n i q u e s i n p o l y s a c c h a r i d e Procedure Mode of Cleavage 1. Par t ia l acid hydro lya le 5 8 using mineral acids (TFA, HC1, HjSO^), tr i f luoroacetolyala or acetolys is , hydrofluorinolysls, nercaptolysls, methanolysis and autohydrolysls. 2. Smith d e g r a d a t i o n 1 2 5 - 1 2 6 (Periodate oxidation followed by select ive hydrolysis of acyc l ic acetals). - the di f ferent ratea of hydrolysis of g lycosidic linkages are dependent on the following factors, e.g. r ing s ize , configuration, conformation, posit ion of linkage, poslton of sugar and ag lycone 7 ' ' 7 * . - oxidative degradation of the C-C bond between v i c i n a l d lo l s . - except for suitably branched sugars, l->3 hexo-pyranose and l-»4 linked 2-acetanldo-2-deoxy hexose, the rest of the hexopyranoses are degraded. 3. Chromium tr loxlde o x i d a t i o n 1 0 2 - select ive oxidative degradation of equatorlal ly orientated (0-1Inked) peracetylated glycopyrano-sldes. 4. Nitrous acid desalination 78 select ive degradation of amino sugars through r ing contraction on deaalnatlon (see Schema I I . 1 . 1 ) . S. Base catalysed 0-allmlnatlona from hezuronate r e s i d u e s 1 1 5 , 1 1 ' select ive degradation of hexuronate reslduea leaving a l l other glycosidic linkages intact . 6. Base catalysed sulfone degrada-t i o n 1 2 7 7. Svensson degradat ion 1 2 8 conversion of a free primary hydroxyl group of a sugar residue to Its sulfone derivative followed by base catalyzed degradation. oxidation of a free hydroxyl group followed by base catalyzed degradation. 8. Hoffmann-Veermann and ralated degradations, and oxidative d e c a r b o x y l a t i o n 5 8 - 1 2 9 see references. 9. Degradation of hexuronlc acid using l ithium In ethylene d l amine1™ select ive degradation of hexuronlc acids. 10. Enzymatic methods (Endoglyca-n a s e ) 1 3 1 (I) bacteriophaee-borne enzy-B e s 1 3 2 . l 5 3 (II) enzymes of bacterial o r ig ins^' ' cleavage of the polysaccharide at spec i f i c glycosidic linkages, see section V. Information - provides a range of oligosaccharides, from dlsaccharlde to pentasaccharide - oligosaccharides with acid lab i le substituents e .g . acetate and pyruvic acid acetal , may be obtained depending on hydrolyt ic conditions. - provides complementary data for methylation analysis - ol igosaccharide s ize dependent on linkage sequence of augar residues. - oligosaccharides with suitably positioned substituents e .g . acetate and pyruvic acid aceta l , can be obtained. - provides Information on anomeric configuration of surviving sugars. - good means of generating oligosaccharides from predominantly a- l inked polysaccharides. • a l ta of attachment of amino augara. - augar sequences in the v i c i n i t y of the amino sugars. - a l tes of attachment of hexuronlc residues. - generation of oligosaccharide without the hexuronlc residue. - location of primary hydroxyl groups not Involved In g lycosid ic l inkages. - sequence* of sugar residues. - augar aequences in the v i c i n i t y of hexuronlc acid residues Including characterization of glycosyl substituents. - provides Information s imi lar to base-catalyzed ^-elimination reaction, except that reaction Is conducted on underlvatlzed polysaccharide. - generation of oligosaccharides comprising of one or more repeating uni ts . 29 I I . 4 . 1 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 o b t a i n e d f r o m d e g r a d a t i o n . D e g r a d a t i o n o f p o l y s a c c h a r i d e s , whether a c h i e v e d c h e m i c a l l y o r e n z y m a t i c a l l y , u s u a l l y g i v e s m i x t u r e s o f o l i g o m e r s , t o g e t h e r w i t h some u n r e a c t e d p o l y m e r i c m a t e r i a l and c o n t a m i n a n t s from s i d e r e a c t i o n s . To o b t a i n t h e d e s i r e d o l i g o s a c c h a r i d e s , the c h o i c e o f t h e s e p a r a t i o n p r o c e d u r e ( s ) i s dependent on t h e s i z e o f the p r o d u c t , d e r i v a t i z e d o r non d e r i v a t i z e d , b a s i c o r a c i d i c and l y o p h i l i c o r l y o p h o b i c . The s e p a r a t i o n and p u r i f i c a t i o n t e c h n i q u e s u s e d i n t h i s s t u d y were d i a l y s i s 1 3 ^ , paper c h r o m a t o g r a p h y 9 ^ , paper e l e c t r o p h o r e s i s 8 9 ' 1 3 * ' , u l t r a c e n t r i f u g a t i o n , i o n - e x c h a n g e ' c h r o m a t o g r a p h y 1 3 7 ' 1 3 8 , a d s o r p t i o n c h r o m a t o g r a p h y 1 3 9 , g e l p e r m e a t i o n chromatography*" 2.140 a n c i t h i n - l a y e r c h r o m a t o g r a p h y 1 4 1 . As the t h e o r y and a p p l i c a t i o n s o f t h e s e t e c h n i q u e s have been w e l l docu-mented, t h e y w i l l n o t be d i s c u s s e d h e r e . H i g h p r e s s u r e l i q u i d chromatography (HPLC) H i g h p r e s s u r e l i q u i d chromatography o r h i g h p e r f o r m a n c e l i q u i d c h romatography i s one o f the most u s e f u l s e p a r a t i o n t e c h n i q u e s a v a i l a b l e t o d a y 1 4 2 ' 1 4 3 . A l t h o u g h t h i s t e c h n i q u e was n o t u s e d i n t h i s s t u d y , a d i s c u s s i o n i s i n c l u d e d h e r e because o f i t s p o t e n t i a l i n the f u t u r e . HPLC has been recommended as a method f o r r o u t i n e c a r b o h y d r a t e a n a l y s i s because one i s a b l e t o a v o i d the d e r i v a t i z a t i o n s t e p r e q u i r e d by g . l . c . M o reover, i t has the advantage o f a h i g h r e s o l v i n g power and speed o f s e p a r a t i o n when compared t o o t h e r t e c h n i q u e s . I n c o n t r a s t t o g . l . c . - 30 -where s e l e c t i v i t y i s a c h i e v e d by columns w i t h a wide v a r i e t y o f s t a t i o n -a r y l i q u i d phases, o n l y a l i m i t e d number o f s u p p o r t s are a v a i l a b l e f o r HPLC e.g. alumina, s i l i c a , i o n exchangers, p o l a r bonded phases, and r e v e r s e d phase m a t e r i a l . To overcome t h i s problem, r e s o l u t i o n i s enhanced by v a r i a t i o n o f column temperature o r c o m p o s i t i o n o f the m o b i l e l i q u i d phase e.g. by use o f more than one s o l v e n t ; the a d d i t i o n o f methanol or a c e t o n i t r i l e to b u f f e r s o l u t i o n s ; v a r i a t i o n o f the pH and i o n i c s t r e n g t h o f b u f f e r s o l u t i o n s ; or by the a d d i t i o n s o f s m a l l amounts o f amines, a c i d s , b u f f e r s , e l e c t r o l y t e s or d e t e r g e n t s t o the mobile l i q u i d p h a s e s ^ 2 . HPLC as a s e p a r a t i o n t e c h n i q u e , c a n be used b o t h a n a l y t i c a l l y and p r e p a r a t i v e l y f o r the f o l l o w i n g : ( i ) e s t i m a t i o n o f r e l a t i v e m o l e c u l a r weight and homogeneity o f p o l y -s a c c h a r i d e s ( i i ) s u gar a n a l y s i s o f monosaccharides ( w i t h o u t d e r i v a t i z a t i o n ) from o l i g o - and p o l y - s a c c h a r i d e s ( i i i ) m o n i t o r the e x t e n t o f a p r e p a r a t i v e d e g r a d a t i o n r e a c t i o n e.g. a n a l y s i s o f the h y d r o l y z a t e s from p a r t i a l h y d r o l y s i s ( i v ) p r e p a r a t i v e s e p a r a t i o n o f n o n d e r i v a t i z e d or 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 from d e g r a d a t i o n r e a c t i o n s , and (v) s e p a r a t i o n o f c a r b o h y d r a t e m i x t u r e s f o l l o w e d by immediate mass s p e c t r a l a n a l y s i s , when the HPLC i n s t r u m e n t i s l i n k e d to a mass s p e c t r o m e t e r as i n I.e.-m.s. 31 -I I . 5 I n s t r u m e n t a t i o n Some o f the a n a l y t i c a l t e c h n i q u e s used i n t h i s work e.g. o p t i c a l r o t a t i o n 1 4 4 , c i r c u l a r d i c h r o i s m 1 4 4 , i n f r a - r e d and u l t r a - v i o l e t s p e c t r o p h o t o m e t r y have been w e l l documented i n many t e x t s . The p o w e r f u l and v e r s a t i l e a n a l y t i c a l t e c h n i q u e s o f n u c l e a r magnetic resonance s p e c t r o s c o p y and mass s p e c t r o m e t r y , which were e x t e n s i v e l y used i n t h i s s tudy, w i l l be d i s c u s s e d more t h o r o u g h l y i n t h i s S e c t i o n . I I . 5.1 N u c l e a r magnetic resonance (n.m.r.) s p e c t r o s c o p y . 1 4 - J " 1 - 3 ^ Due to i t s n o n - d e s t r u c t i v e n a t u r e , n.m.r. s p e c t r o s c o p y i s w i d e l y used f o r the s t r u c t u r a l , c o n f i g u r a t i o n a l , and c o n f o r m a t i o n a l a n a l y s i s o f c a r b o h y d r a t e s (mono-, o l i g o - and p o l y - s a c c h a r i d e s ) and t h e i r d e r i v a -t i v e s . Advances i n i n s t r u m e n t a t i o n e.g. i n t r o d u c t i o n o f s u p e r c o n d u c t i n g s o l e n o i d s , F o u r i e r t r a n s f o r m a t i o n and s o p h i s t i c a t e d computers, have enhanced the v e r s a t i l i t y o f t h i s t e c h n i q u e and have a l l o w e d c o m p l i c a t e d n.m.r. experiments to be performed. N.m.r. experiments have, t r a d i t i o n -a l l y , been c o n d u c t e d on c a r b o h y d r a t e s o l u t i o n s . R e c e n t l y however, s o l i d s t a t e n.m.r. experiments u s i n g c r o s s p o l a r i z a t i o n - m a g i c a n g l e s p i n n i n g t e c h n i q u e s have been r e p o r t e d f o r p o l y s a c c h a r i d e s 1 - * 3 . The p r o t o n s p i n - l a t t i c e r e l a x a t i o n times (T^) o f some mono-, o l i g o - and p o l y -s a c c h a r i d e s have been r e p o r t e d by H a l l and P r e s t o n 1 ^ 4 ' 1 ^ ^ . They a l s o showed t h a t T^ v a l u e s may p r o v i d e a p o w e r f u l method f o r e s t a b l i s h i n g 32 c o n f o r m a t i o n s o f o l i g o s a c c h a r i d e s i n s o l u t i o n s . Bock and cowork-e r s 1 5 6 , 1 5 7 ftave r e p o r t e d s t u d i e s o f c a r b o h y d r a t e c o n f o r m a t i o n i n s o l u t i o n by t h e use o f xH-n.m.r. s p e c t r o s c o p y and p r o t o n n u c l e a r O v e r h a u s e r enhancement (n.O.e.) e f f e c t s i n c o m b i n a t i o n w i t h an e m p i r i c a l e v a l u a t i o n o f t h e p r e f e r r e d c o n f o r m a t i o n u s i n g the h a r d - s p h e r e s exo-anomeric e f f e c t (HSEA) c a l c u l a t i o n . W i t h the r e c e n t i n t r o d u c t i o n o f two d i m e n s i o n a l (2D) n.m.r. s p e c t r o s c o p i c t e c h n i q u e s 1 ^ 8 " i* > 3, i n f o r m a t i o n w h i c h was f o r m e r l y i n a c c e s s i b l e ( e . g . s i g n a l c o n n e c t i v i t i e s and J c o u p l i n g c o n s t a n t s ) c a n now be o b t a i n e d from t h e complex c a r b o h y d r a t e s p e c t r a . Many r e v i e w s on t h e a p p l i c a t i o n o f n.m.r. (1^145-147 a n ( j 13(-;148-152) s p e c t r o s c o p y t o c a r b o h y d r a t e a n a l y s i s a r e a v a i l a b l e , b u t the i n f o r m a t i o n p r e s e n t e d h e r e emphasizes t h e d i r e c t a p p l i c a t i o n o f t h i s t e c h n i q u e t o t h e s t r u c t u r a l e l u c i d a t i o n o f o l i g o - and p o l y - s a c c h a r i d e s . The f a c t t h a t 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 l t h o u g h o f m o l e c u l a r w e i g h t >10^, g i v e w e l l - r e s o l v e d and i n t e r p r e t a b l e s p e c t r a a t t e s t t o t h e i r w e l l - o r d e r e d r e g u l a r i t y and r e p e a t i n g u n i t s t r u c t u r e s . I n the s t r u c t u r a l s t u d i e s o f c a r b o h y d r a t e s , u s e f u l i n f o r m a t i o n c a n be o b t a i n e d from t h e f o l l o w i n g n.m.r. p a r a m e t e r s . ( i ) C h e m i c a l s h i f t The c h e m i c a l s h i f t o f a XH o r i J C n u c l e u s i n a m o l e c u l e i s dependent on i t s c h e m i c a l and magnetic e n v i r o n m e n t , and i s c l o s e l y r e l a t e d t o the s u b s t i t u t i o n a l , o r i e n t a t i o n a l and e l e c t r o n e g a t i v i t y e f f e c t s o f the n e i g h b o r i n g groups. The n.m.r. sp e c t r u m o f a ca r b o -h y d r a t e c a n be d i v i d e d i n t o t h r e e r e g i o n s f o r a XH sp e c t r u m and f o u r , - 33 -f o r a 1 3 C spectrum, i . e . the h i g h f i e l d r e g i o n , the r i n g r e g i o n , the anomeric r e g i o n , and l a s t l y ( o n l y f o r 1 3 C spectrum), the c a r b o n y l group r e g i o n (see F i g I I . 5 . 1 ) . I n the h i g h f i e l d r e g i o n (between 5 1.0-2.5 i n the xH-n.m.r. spectrum, and 15-30 p.p.m. i n the x 3 C s p ectrum), the m e t h y l re s o n a n c e s o f a c e t a l - l i n k e d p y r u v i c a c i d , a c e t a t e s (N- and 0 - a c y l g r o u p s ) , and 6-deoxyhexoses e.g. L-rhamnose and L - f u c o s e , can be d e t e c t e d . The r e l a t i v e p r o p o r t i o n s o f these c o n s t i t u e n t s i n the r e p e a t -i n g u n i t can be q u a n t i f i e d by measurement o f the s i g n a l i n t e g r a l s (see l a t e r ) i n the xH-n.m.r. spectrum. I t has been shown t h a t th e s t e r e o -c h e m i s t r y (R o r S) o f the a c e t a l i c c a r b o n o f the p y r u v i c a c i d can be d i f f e r e n t i a t e d by the c h e m i c a l s h i f t o f the m e t h y l g r o u p 1 2 4 n i F a b i o and c o w o r k e r s 1 ^ have r e p o r t e d t h a t on p a r t i a l d e p y r u v y l a t i o n o f the K l e b s i e l l a K46 p o l y s a c c h a r i d e (see a l s o K l e b s i e l l a K35 i n S e c t i o n I V . 2 ) , the i n d u c e d ' t w i n n i n g ' e f f e c t p r o v i d e s i n v a l u a b l e i n f o r m a t i o n on the sequence o f sugar r e s i d u e s i n the v i c i n i t y o f the p y r u v y l a t e d / d e p y r u v y -l a t e d s u g a r . A n o t h e r ' t w i n n i n g ' phenomenon i n 1 3C-n.m.r. s p e c t r a which i s i n d u c e d by the i n c o m p l e t e d e u t e r a t i o n o f the sugar h y d r o x y l groups, has been r e p o r t e d by R e u b e n 1 ^ t o p r o v i d e i n f o r m a t i o n on sugar sequence i n p o l y s a c c h a r i d e s . The p o s i t i o n ( s ) o f attachment to sugar r e s i d u e s o f n o n - c a r b o h y d r a t e c o n s t i t u e n t s e.g. O - a c e t y l and p y r u v i c a c i d a c e t a l may be d e t e r m i n e d by 2D-n.m.r. t e c h n i q u e s . The p r e s e n c e o f l e s s common and/or u n u s u a l sugars i s sometimes d e t e c t e d i n the h i g h f i e l d r e g i o n o f the spectrum (see F i g I I . 5 . 1 ) . I n the r i n g r e g i o n , the xH-n.m.r. spectrum, (5 3.0-4.5) i s n o t w e l l - r e s o l v e d and t h e r e f o r e assignments a r e d i f f i c u l t . H a l l 1 4 7 has p r o p o s e d measures to overcome t h i s 'hidden r e s o n a n c e s ' problem, and they - 3 4 -Anomeric r e g i o n R i n g r e g i o n H i g h f i e l d r e g i o n r l n | p r o t e s t (Via a % f oar ^ l.» l.» 1.4 t.l 2 >•• ••• ••» >•* | 1 H— n.m.r. spectrum i 0 «(....«.) C a r b o n y l r e g i o n Anomer i c r e g i o n R i n g r e g i o n H i g h f i e l d r e g i o n c=o 1 7 5 6 a 6 C - l - p a HCOR HCOH CH 2 OH HCN H I 1 i 1 1 r— 1 1 r - r n o i o o eo 70 13 C-n.m.r. spectrum C H 3 C H 3 C K 0 C H 3 C O-C-0 CH^to 1 1 h 6 0 5 0 3 0 V- 1 —H 2 0 0 p . p .in . F i g . I I . 5 . 1 : D i f f e r e n t r e g i o n s i n the n.m.r. ( 1 H - and 1 3 C ) s p e c t r a o f p o l y s a c c h a r i d e s . 35 -i n c l u d e d e r i v a t i z a t i o n o f c a r b o h y d r a t e samples, the use o f d i f f e r e n t s o l v e n t s , p aramagnetic s h i f t r e a g e n t s , double resonance t e c h n i q u e s and h i g h e r f i e l d s p e c t r o m e t e r s . More r e c e n t l y , 2D n.m.r. experim e n t s have been u s e d t o s i m p l i f y s p e c t r a o f o l i g o - and p o l y - s a c c h a r i d e s ( d e r i v a t -i z e d and u n d e r i v a t i z e d ) ; and from t h e i r J c o n n e c t i v i t i e s , sugar sequen-ce was e s t a b l i s h e d . I n c o n t r a s t t o xH-n.m.r. s p e c t r a , the r i n g r e g i o n (60-85 p.p.m.) o f x 3 C s p e c t r a i s w e l l r e s o l v e d . The s i g n a l due to the c a r b o n c a r r y i n g the N - a c y l group n o r m a l l y r e s o n a t e s between 50-55 p.p.m. The s i g n a l s due to carbons o f p r i m a r y a l c o h o l s a r e v e r y d i s t i n c -t i v e a t 60-65 p.p.m, which can be d i f f e r e n t i a t e d as l i n k e d o r n o n - l i n k e d by t h e i r c h e m i c a l s h i f t s ( n o n - l i n k e d , 60-62 p.p.m.; when l i n k e d , they are s h i f t e d 7-10 p.p.m. d o w n f i e l d ) . The s i g n a l s due t o the carbons o f seco n d a r y a l c o h o l s appeared a t 75 ± 5 p.p.m.; b u t on 0 - g l y c o s y l a t i o n and/or O - a l k y l a t i o n , the c a r b o n ( s ) i n v o l v e d i s s u f f i c i e n t l y d e s h i e l d e d (by 7-11 p.p.m.) as to produce a s i g n a l w e l l s e p a r a t e d from the o t h e r r i n g carbons (80 ± 5 p.p.m.). T h i s i s c a l l e d the ' a - e f f e c t ' . However carbons i m m e d i a t e l y a d j a c e n t t o t h a t c a r b o n w i l l be s l i g h t l y s h i e l d e d (1-2 p.p.m.), and t h i s i s the '/9 - e f f e c t ' . These a- and /9- s h i f t s (which are d i f f e r e n t from a- and /9- anomeric s i g n a l s ) have been used i n the 1 ^  assignment o f X J 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 , and c o n s e q u e n t l y d e l i n e a t i n g the s t r u c t u r a l s e q u e n c e 1 4 8 . In the anomeric r e g i o n o f the xH-n.m.r. s p e c t r a (6 4.5-5.8), a d i v i s i o n a t 6 5.0 was a c c e p t e d whereby s i g n a l s o f pyra n o s e s a p p e a r i n g u p f i e l d o f i t a r e a s s i g n e d t o f3-linkages ( a x i a l p r o t o n s ) and those s i g n a l s a p p e a r i n g d o w n f i e l d are a s s i g n e d to a - l i n k a g e s ( e q u a t o r i a l p r o t o n s ) . F o r the x 3 C spectrum (between 93-110 p.p.m.), a d i v i s i o n a t - 36 -101 p.p.m. was a c c e p t e d , b u t c o n t r a r y to xH-n.m.r., the a-anomeric carbons appear u p f i e l d o f the /J-anomeric carbons due to a s h i e l d i n g e f f e c t . I t has been found t h a t i n c r e a s e d s h i e l d i n g o f a 1 3 C n u c l e u s i s accompanied by a d e c r e a s e i n the s h i e l d i n g o f the appended p r o t o n , i . e . 1 3 C and XH s h i f t s are a f f e c t e d i n v e r s e l y 1 4 8 . I n the n.m.r. ( 1 3 C and 1H) spectrum o f an o l i g o - o r p o l y - s a c c h a r i d e , the number o f anomeric s i g n a l s and t h e i r c o r r e s p o n d i n g i n t e g r a l s i n d i c a t e the number o f sugar r e s i d u e s p e r r e p e a t i n g u n i t ; f u r t h e r m o r e the l i n k a g e c o n f i g u r a t i o n (a or can be d e t e r m i n e d from the combined measurements o f the c h e m i c a l s h i f t and c o u p l i n g c o n s t a n t . R e c e n t l y , some non-anomeric c a r b o h y d r a t e s i g n a l s have been r e p o r t e d t o appear i n the anomeric r e g i o n . Examples are the H-2 o f D-mannose 1^, H-5 o f D - g a l a c t u r o n i c 1 6 6 a c i d and the p o s i t i o n o f a c e t y l a t i o n ( 1 3 C and XH) o f b r a n c h e d sugar r e s i d u e s 4 7 . Hence, c a u t i o n i s r e q u i r e d when a s s i g n i n g the number o f sugar r e s i d u e s p e r r e p e a t i n g u n i t s o l e l y on the b a s i s o f the n.m.r. s p e c t r a . Other c o l l a b o r a t i v e e v i d e n c e from sugar and m e t h y l a t i o n a n a l y s e s i s n e c e s s a r y . The e f f e c t on the c o r r e s p o n d i n g r i n g p r o t o n , by m o n o a c e t y l a t i o n o f d i f f e r e n t p o s i t i o n s o f methyl a-L-rhamnoside i s c u r r e n t l y b e i n g i n v e s t i g a t e d i n t h i s l a b o r a t o r y . F o r -L-'C s p e c t r a , any s i g n a l i n the extreme downf i e l d r e g i o n o f 170-180 p.p.m. i n d i c a t e s the p r e s e n c e o f a C=0 group which c o u l d be a s s o c i a t e d w i t h an N- or 0 - a c y l group, p y r u v i c a c i d o r u r o n i c a c i d . For a c e t a t e s and p y r u v a t e s , the c h e m i c a l s h i f t o f the s i g n a l s i n t h i s c a r b o n y l group r e g i o n combined w i t h those i n the h i g h f i e l d r e g i o n w i l l a l l o w t h e i r i d e n t i t y and q u a n t i t y to be determined. 37 ( i i ) The r e l a t i v e a r e a o r i n t e g r a l o f i n d i v i d u a l s i g n a l s The number o f p r o t o n s r e s o n a t i n g a t each p a r t i c u l a r f r e q u e n c y can be d e t e r m i n e d from the a r e a o f the s i g n a l p roduced. C o n s e q u e n t l y , a q u a n t i t a t i v e e s t i m a t i o n o f the r a t i o o f a- to /3-linkages, number o f 6-deoxyhexoses, a c e t a t e s , p y r u v a t e s e t c . p r e s e n t i n the ^-H-n.m.r. spectrum can be performed. However, q u a n t i t a t i o n b a s e d on s i g n a l i n t e g r a t i o n i s n o t o f t e n r e l i a b l e i n the p r o t o n - d e c o u p l e d ^ 3 C spectrum due t o s a t u r a t i o n and n.O.e. e f f e c t s . N e v e r t h e l e s s , comparison o f i n t e g r a l s o f C c a r r y i n g the same number o f hydrogen atoms o f t e n y i e l d s a c c u r a t e i n f o r m a t i o n about t h e i r r e l a t i v e amounts. Co n s e q u e n t l y , by c o u n t i n g the number o f s i g n a l s i n the spectrum o f a p o l y s a c c h a r i d e , the number o f 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 c a n be determined, e.g. the t o t a l o f 28 c a r b o n s i g n a l s i n the spectrum o f E. c o l i K44 p o l y s a c c h a r i d e c o n f i r m e d t h a t i t comprised a 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 . F o r o l i g o s a c c h a r i d e s , the anomeric s i g n a l s (^ 3C and ^H) o f the r e d u c i n g end can be d i s t i n g u i s h e d from o t h e r anomeric s i g n a l s because they d i s p l a y the e f f e c t o f m u t a r o t a t i o n , showing two s e p a r a t e s i g n a l s f o r the a- and ^-anomers. The anomeric s i g n a l s o f the r e d u c i n g end can a l s o be d e t e r m i n e d by comparing the n.m.r. s p e c t r a o f the o l i g o s a c c h a -r i d e t o t h a t o f the o l i g o s a c c h a r i d e a l d i t o l . Furthermore, the degree o f p o l y m e r i z a t i o n o f an o l i g o s a c c h a r i d e can be d e t e r m i n e d by comparing the number o f anomeric s i g n a l s (•LJC and XH) to t h a t o f the r e d u c i n g end ( c o u n t e d as o n e ) . - 38 -( i i i ) C o u p l i n g c o n s t a n t (J v a l u e ) The r e l a t i o n s h i p betwen the t h r e e - b o n d v i c i n a l c o u p l i n g c o n s t a n t ( 3 J ) and the d i h e d r a l a n g l e (4>) between the p r o t o n s i s g i v e n a p p r o x i -m a t e l y by the K a r p l u s 1 * * ? e q u a t i o n : 3 J (H1.H2) ' 8.5 c o s 2 <t> - 0.28 0° < <f> < 90° ^ 9.5 c o s 2 <{> - 0.28 90° < <j> < 180 c The v a l u e s a r e maximum when <f> i s 0° o r 180°, and minimum when i t i s 90°. The c o u p l i n g c o n s t a n t s p r e d i c t e d by the use o f t h e s e e q u a t i o n s agree r e a s o n a b l y w e l l w i t h the o b s e r v e d v a l u e s . D e v i a t i o n s however, o c c u r when s u b s t i t u e n t s a r e o f d i f f e r e n t e l e c t r o n e g a t i v i t y e.g. p e r a c e t y l a t e d d e r i v a t i v e s 1 * * ^ A l t h o u g h the c o u p l i n g c o n s t a n t i s i n f l u e n c e d by o t h e r parameters ( s u c h as e l e c t r o n e g a t i v i t y , a n g l e s t r a i n and bond l e n g t h ) , i t i s u s e f u l f o r t h e assignment o f t r a n s - d i a x i a l p r o t o n s (<f> = 180°, ) 3 - l i n k e d , J\ 2 7 - 9 Hz), and e q u a t o r i a l - a x i a l o r e q u a t o r i a l - e q u a t o r i a l p r o t o n s (<j> - 60, a - l i n k e d , " ^ 1 2 1 - 3 H z ) • These 3 J - v a l u e s can be a p p l i e d t o hexopyranoses l i k e D - g a l a c t o s e , D-glucose, D - g l u c u r o n i c a c i d and 2-acetamido-2-deoxyhexopyranoses (e.g. GlcNAc and GalNAc). Due to the e q u a t o r i a l p r o t o n a t C-2, L-rhamnose and D-mannose have a d i f f e r e n t s e t o f 3 J v a l u e s , i . e . a-anomer 2 ^ 2 Hz, /3-anomer 3 J ^ 2 = 1 Hz. The J c o n s t a n t s o f anomeric p r o t o n s a r e thus u s e f u l i n e s t a b l i s h i n g the anomeric c o n f i g u r a t i o n (a o r /S) and the o v e r a l l c o n f o r m a t i o n ( p y r a -n o s e / f u r a n o s e and c h a i r / b o a t forms) o f the monosaccharides. Most o f the 1 3 C s p e c t r a p r e s e n t e d i n t h i s t h e s i s were r e c o r d e d i n 39 the p r o t o n - d e c o u p l e d mode. Such s p e c t r a g i v e simple and w e l l r e s o l v e d s i g n a l s which are e a s i l y i n t e r p r e t e d . However, s p e c t r a may a l s o be c o r r e l a t e d w i t h p r o t o n s p e c t r a to p r o v i d e i n f o r m a t i o n on -^3C--'-H c o u p l i n g c o n s t a n t s ( ^ c l H i ) • T h i s i s u s u a l l y performed by the 'gated' d e c o u p l i n g o r by the s i n g l e f r e q u e n c y o f f resonance d e c o u p l i n g t e c h -n i q u e . The ^JQI HI v a l u e i s u s e f u l i n d i f f e r e n t i a t i n g anomeric p a i r s i n the pyranose form, s i n c e they d i f f e r by - 10 H z ^ ' ' ^ " , e.g. ^JQI j j l ^ o r a /?-rhamnoside i s 160 Hz and ^ H ^ HI f ° r a n a-rhamnoside i s 169 Hz. In the s t r u c t u r a l s t u d i e s on the E. c o l i K44 p o l y s a c c h a r i d e , the anomeric c o n f i g u r a t i o n o f the f o u r 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 was c o n f i r m e d by t h e i r ^Jci HI v a l u e s . R e c e n t l y , Bock and Pedersen-'-'7! r e p o r t e d the d e t e r m i n a t i o n o f ^ c i HI v a l u e s o f some c a r b o h y d r a t e samples through the measurements o f s a t e l l i t e s i g n a l s i n ^H-n.m.r. s p e c t r a (500 MHz). II.5.2 Mass s p e c t r o m e t r y Mass s p e c t r o m e t r y i s an im p o r t a n t t e c h n i q u e i n c a r b o h y d r a t e s t r u c -t u r a l a n a l y s i s . I t i s based on the f r a g m e n t a t i o n o f o r g a n i c m o l e c u l e s under e l e c t r o n impact, and d i f f e r e n t i a t i o n o f the r e s u l t i n g p a r t i c l e s by use o f the mass-to-charge r a t i o . The v e r s a t i l i t y o f t h i s t e c h n i q u e i s g r e a t l y i n c r e a s e d when l i n k e d to a g a s - l i q u i d or l i q u i d chromatography, i . e . g.l.c.-m.s. and I.e.-m.s. Mass s p e c t r o m e t r y i n c a r b o h y d r a t e a n a l y s i s p r o v i d e s i n f o r m a t i o n on: ( i ) the s u b s t i t u t i o n p a t t e r n s i n p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e - 40 -(PMAA) d e r i v a t i v e s ( i i ) the p o s i t i o n o f the methyl and methylene groups i n deoxysugars, and the amino group i n amino deoxysugars ( i i i ) the sugar sequence i n o l i g o s a c c h a r i d e s , and ( i v ) the v a r i o u s forms of the monosaccharides e.g. between c y c l i c and a c y c l i c , pyranose and f u r a n o s e , pentose and hexose, and a l d o s e and k e t o s e . Mass s p e c t r o m e t r y , however, does not d i f f e r e n t i a t e between d i a -s t e r e o m e r i c compounds e.g. g l u c o s e , g a l a c t o s e and mannose. E x t e n s i v e r e v i e w s ^ 0 * > 172-175 o n t ^ e a p p l i c a t i o n s o f mass s p e c t r o m e t r y to carbo-h y d r a t e a n a l y s i s a r e a v a i l a b l e . E l e c t r o n impact - mass s p e c t r o m e t r y . The e l e c t r o n - i m p a c t t e c h n i q u e i s the method commonly used i n the a n a l y s i s o f PMAAs, p e r a c e t y l a t e d a l d i t o l s and p e r a c e t y l a t e d aldono-n i t r i l e s i n t h i s l a b o r a t o r y . The h i g h energy (70 eV) beam o f e l e c t r o n s l e a d s to e x t e n s i v e f r a g m e n t a t i o n o f the sugar d e r i v a t i v e s a l o n g s p e c i f i c pathways and u s u a l l y r e s u l t s i n a weak or no m o l e c u l a r i o n peak. P r i m a r y f r a g m e n t a t i o n s from PMAAs (see Scheme I I . 5.1) a r i s e by Q-cleavage w i t h , i n g e n e r a l , p r e f e r r e d f o r m a t i o n o f : ( i ) i o n s o f lower m o l e c u l a r weight ( i i ) i o n s from c l e a v a g e between two m e t h o x y l - b e a r i n g c a r b o n atoms, and ( i i i ) i o n s from c l e a v a g e between a m e t h o x y l - b e a r i n g and an a c e t o x y l -- 41 Primary fragmentation: CH,OAc J17 HCOMe HCOAc I HCOAc I CH, CH,OAc U7 HCOMe 7 ~ t 205 J61 _| HCOMe 1 6 1 MeOCH HCOAc CH,OMe 45 Secondary fragmentation: Me HC= I HCOMe I HCOAc I CH,OMe m/e 205 -AcOH HC=OMe COMe - II CH I CH,OMe m/e 145 CH,OAc HCOMe -HC=OMe © m/e 161 -AcOH CH, II ^ - COMe -HC=OMe m/e 101 -CH.O CH, II C II © I Me m/e 71 CH,OMe m/e 161 e HC=OMe I CH II HCOMe m/e 101 © HC=OMe I COAc -II CH, © „„ HC=OMe -CH.CO | — C=0 I CH, m/e 129 m/e 87 i * - 4 2 116 « 15B 145 « 5oiT" - 6 0 MeOCH - 6 0 202 >142 T I T T 1 m ' i i i» i i i I Scheme I I . 5 . I : F r a g m e n t a t i o n pathways o f some p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s . 42 b e a r i n g c a r b o n atom w i t h marked p r e f e r e n c e f o r the methoxyl-b e a r i n g s p e c i e s t o c a r r y the p o s i t i v e charge; b u t ( i v ) i o n s formed by s c i s s i o n between two a c e t o x y l - b e a r i n g c a r b o n atoms a r e g e n e r a l l y o f low abundance. There i s l i t t l e tendency f o r c h a i n s c i s s i o n to take p l a c e a d j a c e n t t o a deoxygenated c a r b o n atom b u t the pr e s e n c e o f such a u n i t i s u s u a l l y a p p a r e n t from the i n c r e a s e i n m/z v a l u e s by 14 a.m.u. Some t y p i c a l fragment i o n s a r e i l l u s t r a t e d i n Scheme I I . 5.1. P r i m a r y fragment i o n s undergo a s e r i e s o f subsequent e l i m i n a t i o n s t o g i v e s e c o n d a r y fragments, i n c l u d i n g l o s s e s by ^ - e l i m i n a t i o n o f a c e t i c a c i d (m/z 60), o r methanol (m/z 32); l o s s e s by a - e l i m i n a t i o n o f a c e t i c a c i d (m/z 60) but not o f methanol; and l o s s e s v i a c y c l i c t r a n s i t i o n s t a t e s o f formaldehyde methoxymethyl a c e t a t e , o r ac e t o x y m e t h y l a c e t a t e . The mass s p e c t r a o f PMAAs o f 2-amino-2-deoxyhexoses have a l s o been w e l l documented^•-^6>177^ c h a r a c t e r i s t i c base peak b e i n g t h a t o f m/z 116. The p r i m a r y f r a g m e n t a t i o n i s l a r g e l y governed by the acetamido group, and f i s s i o n i s almost e x c l u s i v e l y between the methylacetamido group and the a d j a c e n t methoxyl or a c e t o x y l group. The secondary fragments a r e s i m i l a r l y formed as i l l u s t r a t e d i n Scheme I I . 5.1. Chem i c a l i o n i z a t i o n - mass s p e c t r o m e t r y ( c . i . - m . s . ) Chemical i o n i z a t i o n - mass s p e c t r o m e t r y i s an a l t e r n a t i v e method o f a n a l y s i s which i s complementary t o e.i.-m.s. Due to i t s s o f t e r methods - 43 -o f i o n i z a t i o n (8-20 eV), the c.i.-mass s p e c t r a g i v e m o l e c u l a r or q u a s i -m o l e c u l a r i o n s and i n g e n e r a l g i v e much more abundant i o n s i n the high-mass range (500-2000 a.m.u.). but t h e i r s i m p l e r s p e c t r a p r o v i d e l e s s i n f o r m a t i o n on i n d i v i d u a l monosaccharide s t r u c t u r e . C.i.-m.s. i s p a r t i c u l a r l y u s e f u l f o r a n a l y z i n g 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 . The a b i l i t y to a n a l y z e i n t a c t o l i g o s a c c h a -r i d e s i s s e v e r e l y l i m i t e d by the thermal i n s t a b i l i t y and v e r y low v o l a t i l i t y o f those compounds. However, c o n v e r s i o n t o v o l a t i l e d e r i v a -t i v e s l i k e a c e t a t e s , methyl e t h e r s , and t r i m e t h y l s i l y l e t h e r s , p e r m i t s t h e i r a n a l y s i s by c.i.-m.s. The c.i.-m.s. a n a l y s i s on the o l i g o s a c c h a -r i d e s o b t a i n e d i n the s t r u c t u r a l a n a l y s i s o f K l e b s i e l l a K79 and E. c o l i K44 p o l y s a c c h a r i d e s was performed on t h e i r p e r m e t h y l a t e d d e r i v a t i v e s . W i t h the i n c r e a s i n g p o t e n t i a l o f HPLC f o r the 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 d e r i v a t i v e s and the a v a i l a b i l i t y o f I.e.-m.s., mass s p e c t r a l a n a l y s i s o f m i x t u r e s o f h i g h e r m o l e c u l a r weight c a r b o h y d r a t e s w i l l be extended. The mass s p e c t r o m e t r i c b e h a v i o r o f p e r m e t h y l a t e d o l i g o s a c c h a r i d e s has been e x t e n s i v e l y i n v e s t i g a t e d - ^ * * - 1 8 0 > a n d the f r a g m e n t a t i o n pathways p r o c e e d i n a manner s i m i l a r t o t h a t o f the monosaccharide d e r i v a t i v e s . Scheme I I . 5 . I I shows the f r a g m e n t a t i o n nomenclature f i r s t d e v i s e d by C h i z h o v and Kochetkov-'-^ 2 and l a t e r m o d i f i e d by K o v a c i k e t . a l . ^ ^ . The most e a s i l y r e c o g n i z a b l e fragments i n the c . i . mass s p e c t r a o f permethy-l a t e d o l i g o s a c c h a r i d e a l d i t o l s a r e those from n o n r e d u c i n g u n i t s , i . e . , the , i o n s : f o r p e r m e t h y l a t e d hexose (m/z 219); p e r m e t h y l a t e d deoxyhexose (m/z 189); p e r m e t h y l a t e d p e ntose (m/z 175); p e r m e t h y l a t e d h e x u r o n i c e s t e r (m/z 233); and p e r m e t h y l a t e d 0- and N-2-acetamido-- 44 -A series C H 2 O R „ R j O O R j B series C H j O R . *tO-( > - O R , R j O O R j C H . O R , , R, = Me R 4 0 C H , O R 6 R 3 0 O R 2 O R : C H , O R 6 C H = 0 R * 0 - S ^ O R , R 3 0 O R 2 R4O-S R j O C H j O R t R . O H J ~ J ^ O R , ' -OMe • C H O R 2 C H j O R . C H = 0 M e t V • / - O R , + R 4 0 -• C H O R 2 O R , O R , C H , O R „ + I! ' C H = 0 R.O=Y^ ^OR, R j O O R ; C H j O R , C H = 0 + M e O C H — O R , E series C H 2 O R e R 4 O - / V-OR, R 3 0 O R 2 R 4 Q — ^ — O R , + R „ 0 C H 2 R j O O R 3 Scheme I I . 5 . I I : Some mass s p e c t r a l f r a g m e n t a t i o n pathways f o r p e r m e t h y l a t e d g l y c o p y r a n o s i d e s . - 45 -2-deoxyhexose r e s i d u e s (m/z 260). Furthermore, i n f o r m a t i o n on the g e n e r a l n a t u r e o f unbranched i n t e r n a l u n i t s c a n be o b t a i n e d by d e t e c t i n g i n the m/z v a l u e s f o r fragment i o n s o f h i g h e r mass i n c r e m e n t s o f 204 (he x o s e ) , 174 (6-deoxyhexose), 160 ( p e n t o s e ) , 218 ( h e x u r o n i c a c i d ) , and 245 (2-acetamido-2-deoxyhexose) r e s p e c t i v e l y . These a s p e c t s o f mass s p e c t r o m e t r y , t h e r e f o r e , a l l o w c o n c l u s i o n s t o be drawn c o n c e r n i n g the sequences o f sugar u n i t s when these a r e o f d i f f e r e n t t y p e s . On the whole, v e r y l i t t l e i n f o r m a t i o n can be o b t a i n e d c o n c e r n i n g s t e r e o c h e m i s -t r y , e i t h e r o f i n d i v i d u a l r e s i d u e s o r o f the c o n f i g u r a t i o n o f g l y c o s i d i c l i n k a g e s . N e v e r t h e l e s s , some i n f o r m a t i o n c a n be o b t a i n e d on l i n k a g e t y p e s from an e x a m i n a t i o n o f f r a g m e n t a t i o n pathways. F a s t a t o m b o m b a r d m e n t - m a s s s p e c t r o m e t r y ( f . a . b . - m . s . ) . A n o t h e r mass s p e c t r o m e t r i c t e c h n i q u e g a i n i n g a wide a p p l i c a t i o n i n c a r b o h y d r a t e a n a l y s i s i s t h a t o f f . a . b . - m . s . 1 8 2 T y p i c a l l y , a sample i s d i s s o l v e d i n a s u i t a b l e s o l v e n t (e.g. water, g l y c e r o l , t h i o - g l y c e r o l , methanol o r c h l o r o f o r m ) and the sample i s i n t r o d u c e d as a m a t r i x onto the probe. The sample i s the n bombarded w i t h a r g o n o r xenon atoms p o s s e s s i n g 7-8 KV o f energy and the p o s i t i v e and or n e g a t i v e i o n s t h a t a r e r e l e a s e d a r e r e c o r d e d . The f r a g m e n t a t i o n i n f o r m a t i o n p r o v i d e d by t h i s t e c h n i q u e i s s i m i l a r t o t h a t o f c.i.-m.s. ex c e p t t h a t 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 ( o b t a i n e d from b a c t e r i o p h a g e - i n d u c e d o r c h e m i c a l l y - i n d u c e d d e g r a d a t i o n s ) can be used. A member o f our r e s e a r c h group i s c u r r e n t l y c o n d u c t i n g - 46 e x t e n s i v e f.a.b.-m.s. and c.i.-m.s. a n a l y s e s o f a range o f o l i g o s a c c h a -r i d e s , w i t h the o b j e c t i v e o f b u i l d i n g up a l i b r a r y o f mass s p e c t r a l d a t a which c o u l d be used l a t e r f o r d e l i n e a t i n g the s t r u c t u r a l sequence o f o l i g o s a c c h a r i d e s . 47 CHAPTER I I I GENERAL EXPERIMENTAL PROCEDURES 48 -GENERAL EXPERIMENTAL PROCEDURES I I I . l P r e p a r a t i o n and i s o l a t i o n o f 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 . I I I . 1 . 1 Media and g e n e r a l m i c r o b i a l c o n d i t i o n s The b a c t e r i a l growth media used and t h e i r c o m p o s i t i o n s were as f o l l o w s : T a b l e I I I . l . I : Growth media f o r the K l e b s i e l l a b a c t e r i a . I n g r e d i e n t s N u t r i e n t N u t r i e n t S u c r o s e - y e a s t e x t r a c t b r o t h agar agar medium D i f c o b a c t o peptone 5 g 5 g D i f c o b a c t o b e e f e x t r a c t 3 g 3 g 5 g NaCl 2 g 2 g 5 g H 20 ( d i s t i l l e d ) 1 L 1 L 2.5 L D i f c o agar 15 g 37.5 g s u c r o s e 75 g K H 2 P 0 4 2.5 g MgS0 4-7 H 20 0.625 £ CaS0 4 1.25 g - 49 -T a b l e I I I . l . I I : Growth media f o r the E. c o l i b a c t e r i a . I n g r e d i e n t M u e l l e r H i n t o n M u e l l e r H i n t o n b r o t h agar D i f c o Bacto M u e l l e r 21 g 21 g H i n t o n B r o t h NaCl 2 g 2 g H 20 ( d i s t i l l e d ) 1 L 1 L D i f c o agar 15 g S t e r i l i z a t i o n o f gl a s s w a r e and media was pe r f o r m e d i n an American S t e r i l i z e r model 57-CR f o r 20 min a t 121° and 15-20 p . s . i . M i c r o b i o l o g -i c a l a s e p t i c t e c h n i q u e (a t e c h n i q u e which keeps the working environment as f r e e from contaminants as p o s s i b l e ) was employed d u r i n g the h a n d l i n g o f m i c r o o r g a n i s m s . Agar p l a t e s were made by p o u r i n g the s t e r i l e l i q u i d agar (-45°) i n t o P e t r i d i s h e s (8.5 cm diameter) and a l l o w i n g them to s e t a t room temperature. A l o o p i n o c u l a t o r , s t e r i l i z e d by h e a t i n g u n t i l r e d - h o t (15s) and the n c o o l e d , was used f o r b a c t e r i a l c o l o n y t r a n s f e r . I I I . 1 . 2 I s o l a t i o n o f 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 2 ^ » ^ 4 . Stab c u l t u r e s o f K l e b s i e l l a s e r o t y p e s K79 and K35 were r e c e i v e d from Dr. Ida ^ r s k o v (WHO I n t e r n a t i o n a l E s c h e r i c h i a C e n t e r , Copenhagen). A c t i v e l y growing c u l t u r e s o f b a c t e r i a were p r o p a g a t e d from s i n g l e 50 -c o l o n i e s by r e - p l a t i n g s e v e r a l times on n u t r i e n t agar. P l a t e s were i n c u b a t e d o v e r n i g h t (37°) and then h e l d a t 7 ° . F o r l a r g e s c a l e p r o d u c t i o n o f c a p s u l a r p o l y s a c c h a r i d e s , i n o c u l a (100 mL, 4 h b r o t h c u l t u r e s grown from a s i n g l e c o l o n y ) were poured on to a s t e r i l e , s u c r o -s e - y e a s t e x t r a c t - a g a r medium (2.5 L i n a met a l t r a y 60 cm x 40 cm). The c u l t u r e s were i n c u b a t e d ( 3 d ) a t room temperature. The b a c t e r i a l lawn produced was d i s i n f e c t e d by s p r a y i n g w i t h 1% p h e n o l s o l u t i o n and then s c r a p e d o f f the agar s u r f a c e and added t o an e q u a l volume o f 2% phenol s o l u t i o n . A f t e r s t i r r i n g f o r -48 h a t 4 ° , u l t r a c e n t r i f u g a t i o n (4 h a t 15° on a Beckmann L3-50 u l t r a c e n t r i f u g e u s i n g r o t o r type 35 a t 31000 r.p.m. o r 80000 g) was used t o sediment the dead b a c t e r i a l c e l l s . The v i s c o u s s u p e r n a t a n t , was poured i n t o 4:1 e t h a n o l - a c e t o n e m i x t u r e to p r e c i p i t a t e the p o l y s a c c h a r i d e . The r e s u l t a n t s t r i n g y p r e c i p i t a t e was r e - d i s s o l v e d i n a minimal q u a n t i t y o f water and r e - p r e c i p i t a t e d w i t h 10% C e t a v l o n ( i . e . C H 3 ( C H 2 ) 1 5 N + ( C H 3 ) 3 B r " ) . The i n s o l u b l e C e t a v l o n - a c i d i c p o l y s a c c h a r i d e complex was i s o l a t e d by c e n t r i f u g a t i o n (20 min) and d i s s o l v e d i n NaCl s o l u t i o n (4 M). The p o l y s a c c h a r i d e , i n the s a l t s o l u t i o n , was p r e c i p i t a t e d w i t h 4:1 e t h a n o l - a c e t o n e m i x t u r e , r e d i s s o l v e d i n NaCl s o l u t i o n (4 M), and d i a l y z e d (mol. wt. c u t o f f 14,000) a g a i n s t r u n n i n g tap water (3 d ) . The c o n c e n t r a t e d r e t e n t a t e was u l t r a c e n t r i -f u g e d ( u s i n g p r e v i o u s l y d e s c r i b e d c o n d i t i o n s ) and the s u p e r n a t a n t l y o p h i l i z e d t o g i v e a white s t y r o f o a m - l i k e m a t e r i a l . 51 I I I . 1 . 3 I s o l a t i o n o f E s c h e r i c h i a 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 1 8 - 5 > x 8 b. A s t a b c u l t u r e o f E. c o l i 08:K44(A):H- was o b t a i n e d from Dr. Ida /0rskov (WHO I n t e r n a t i o n a l E s c h e r i c h i a Center, Copenhagen). M u e l l e r -H i n t o n b r o t h and agar were used to grow the b a c t e r i a . An a c t i v e l y -growing c u l t u r e o f E. c o l i K44 i n M u e l l e r - H i n t o n b r o t h (100 mL) was sp r e a d a c r o s s t r a y s o f M u e l l e r - H i n t o n agar (2.5 L / t r a y ) and i n c u b a t e d (3 d) a t room temperature. The s l i m e was c o l l e c t e d and p u r i f i e d a c c o r d i n g to the p r o c e d u r e o u t l i n e d f o r K l e b s i e l l a p o l y s a c c h a r i d e s . I I I . 2 N u c l e a r magnetic resonance ( i J c - i 8 / and -"-H-n.m.r . • L B B ) s p e c t r o s c o p y . N.m.r. s p e c t r a were r e c o r d e d on Bruker WH-400 and V a r i a n XL-300 i n s t r u m e n t s . XH-N.m.r. s p e c t r a were r e c o r d e d e i t h e r a t ambient or e l e v a t e d temperatures (90 ± 5°) and acetone (5 2.23) was used as an i n t e r n a l s t a n d a r d . Samples were deuterium-exchanged by d i s s o l v i n g i n D 20 (99.9%) and f r e e z e - d r i e d ( 3 x ) . Carbo h y d r a t e samples (10-20 mg) d i s s o l v e d i n D 20 were examined i n 5 mm diameter, n.m.r. tubes. JC-N.m.r. s p e c t r a were r e c o r d e d a t ambient temperature, and acetone was used as the i n t e r n a l s t a n d a r d (31.07 p.p.m.). The c h e m i c a l s h i f t s , f o r b o t h the XH- and JC-n.m.r. s p e c t r a , a r e g i v e n r e l a t i v e to t h a t of e x t e r n a l sodium 4 , 4 - d i m e t h y l - 4 - s i l a p e n t a n e s u l f o n a t e ( D . S . S . ) 1 8 9 . For C-n.m.r. experiments, samples (30-50 mg) were d i s s o l v e d i n the minimum o f D 20 and examined i n n.m.r. tubes o f 5 or 10 mm dia m e t e r . The t y p i c a l number o f scans which i s r e q u i r e d f o r a good s i g n a l - t o - n o i s e i- 5C-n.m.r. spectrum ranges between 70,000-100,000. I I I . 3 Sugar a n a l y s i s The q u a l i t a t i v e and q u a n t i t a t i v e d e t e r m i n a t i o n s o f the sugars p r e s e n t i n the p o l y - and o l i g o - s a c c h a r i d e samples were conducted by g . l . c . a n a l y s i s o f t h e i r d e r i v e d a l d i t o l a c e t a t e s . The procedure d e s c r i b e d h e r e i s not used f o r samples c o n t a i n i n g amino - s u g a r s . I I I . 3 . 1 M e t h a n o l y s i s 7 5 . For the d e t e c t i o n o f u r o n i c a c i d s i n c a r b o h y d r a t e s , samples were t r e a t e d i n the f o l l o w i n g way b e f o r e t o t a l h y d r o l y s i s and c o n v e r s i o n o f sugars t o t h e i r a l d i t o l a c e t a t e s . P o l y - and o l i g o - s a c c h a r i d e samples (3-12 mg) d r i e d i n vacuo were r e f l u x e d w i t h 3% HC1 i n methanol (1 mL a c e t y l c h l o r i d e i n 20 mL anhydrous methanol) on a steam-bath (-95°) f o r 8-16 h. A f t e r n e u t r a l i z a t i on o f the a c i d w i t h PbC0 3, the r e a c t i o n m i x t u r e was c e n t r i f u g e d . The r e s u l t a n t s u p e r n a t a n t was d e c a n t e d and c o n c e n t r a t e d to d r y n e s s . The r e s i d u e was d i s s o l v e d i n anhydrous metha-n o l (5 mL) and reduced w i t h NaBH 4 (20 mg) f o r 2 h a t ambient tempera-t u r e . The excess b o r o h y d r i d e was decomposed w i t h A m b e r l i t e IR 120 (H +) r e s i n which was s u b s e q u e n t l y removed by f i l t r a t i o n . The f i l t r a t e was e v a p o r a t e d to d r y n e s s and the r e s i d u a l b o r i c a c i d was removed by co-53 e v a p o r a t i o n w i t h CH3OH (3 x 5 mL). The samples thus o b t a i n e d were f u r t h e r t r e a t e d as d e s c r i b e d below. I I I . 3 . 2 F o r m a t i o n o f a l d i t o l a c e t a t e s 1 9 0 , 1 9 1 . H y d r o l y s i s . - Car b o h y d r a t e samples (3-12 mg) were h y d r o l y z e d u s i n g 2 M t r i f l u o r o a c e t i c a c i d (TFA) on a steam-bath (-95°). P o l y -s a c c h a r i d e samples were h y d r o l y z e d f o r -16 h and o l i g o s a c c h a r i d e s between 4-6 h. The TFA was removed by c o - e v a p o r a t i o n w i t h H2O ( 3 x 5 mL). The e x t e n t o f h y d r o l y s i s was checked by paper chromatography i n s o l v e n t s 1 and 3 (see S e c t i o n I I I . 5 ) . R e d u c t i o n . - The r e l e a s e d s u g a r s , d i s s s o l v e d i n H 20 (5-10 mL), were r e d u c e d w i t h NaBH^ (20 mg) a t ambient temperature (20 min). The excess NaBH4 was decomposed w i t h A m b e r l i t e IR 120 (H +) r e s i n and the s o l u t i o n f i l t e r e d . The f i l t r a t e was e v a p o r a t e d t o dry n e s s and the r e s i d u a l b o r i c a c i d removed by c o - e v a p o r a t i o n w i t h CH3OH ( 3 x 5 mL). A c e t y l a t i o n . - The a l d i t o l s , d r i e d i n vacuo (20 min), were a c e t y l a t e d u s i n g 1:1 a c e t i c a n h y d r i d e - p y r i d i n e (30 min, 9 5 ° ) . The exce s s A c 2 0 was c o n v e r t e d t o i t s e s t e r by ad d i n g e t h a n o l and removed by e v a p o r a t i o n . The p y r i d i n e was removed by c o - e v a p o r a t i o n w i t h H 20 ( 4 x 5 mL). The p e r a c e t y l a t e d a l d i t o l s were s u c c e s s i v e l y p u r i f i e d by e x t r a c -54 -t i o n u s i n g the f o l l o w i n g i m m i s c i b l e m i x t u r e s : CHC^-H^SO^ (10%), CHCl 3-NaHC03 ( s a t u r a t e d ) and CHCI3-H2O. The a l d i t o l a c e t a t e s were d i s s o l v e d i n CHCI3 and a n a l y z e d by g . l . c . on column A or E (see S e c t i o n I I I . 6 ) . I I I . 4 M e t h y l a t i o n a n a l y s i s . Sodium m e t h y l s u l f i n y l m e t h a n i d e (2M) was p r e p a r e d a c c o r d i n g t o the pr o c e d u r e d e s c r i b e d by S a n d f o r d and Conrad-^ 2>1°3_ T h i s i n v o l v e d h e a t i n g the anhydrous NaH (3 g ) , w i t h anhydrous d i m e t h y l s u l f o x i d e (DMSO, 30 mL) f o r 2 h a t 55° w i t h a c o n t i n u o u s N 2 purge. I I I . 4 . 1 Hakomori m e t h y l a t i o n i i Z • i i : > A c a r b o h y d r a t e sample (3-30 mg), d r i e d i n vacuo and i n the f r e e a c i d form ( o b t a i n e d by p a s s i n g the aqueous s o l u t i o n t h r o u g h a column of A m b e r l i t e IR 120 (H +) r e s i n ) , was d i s s o l v e d w i t h s t i r r i n g i n anhydrous DMSO (2-10 mL) under a N 2 atmosphere. Sodium m e t h y l s u l f i n y l m e t h a n i d e (2M, 1-5 mL) was added t o the s o l u t i o n v i a a s y r i n g e and s t i r r i n g was c o n t i n u e d f o r another 3 h a t ambient temperature. The r e a c t i o n m i x t u r e was c o o l e d (~4°) and CH3I (3-15 mL) was added. When the i n i t i a l l y murky r e a c t i o n m i x t u r e was t r a n s f o r m e d t o a c l e a r , p a l e - y e l l o w s o l u t i o n (-2 h ) , the ex c e s s CH3I was e v a p o r a t e d o f f . M e t h y l a t e d o l i g o s a c c h a r i d e s were r e c o v e r e d by e x t r a c t i o n u s i n g CH 2C1 2-H 20. M e t h y l a t e d p o l y s a c c h a -55 r i d e s were f i r s t d i a l y z e d (mol. wt. c u t o f f 13,500) a g a i n s t r u n n i n g tap water o v e r n i g h t and the p r o d u c t i n the r e t e n t a t e was e x t r a c t e d u s i n g CH2CI2• The m e t h y l a t e d sample was d e s a l t e d by p a s s i n g through a column (1 cm x 10 cm) o f Sephadex LH-20, u s i n g 1:1 C H C I 3 - C H 3 O H as e l u a n t . M e t h y l a t i o n was c o n s i d e r e d complete when the i n f r a - r e d ( i . r . ) spectrum o f the m e t h y l a t e d p r o d u c t was d e v o i d o f h y d r o x y l a b s o r p t i o n s ( a t 3640 cm" 1 and 3200-3500 cm" 1). 111.4.2 R e d u c t i o n of the u r o n i c e s t e r A sample o f p e r m e t h y l a t e d m a t e r i a l (3-30 mg) was r e d u c e d w i t h L 1 A I H 4 (30-50 mg) i n anhydrous oxolane ( i . e . t e t r a h y d r o f u r a n , 3-10 mL) a t ambient temperature o v e r n i g h t and f u r t h e r r e f l u x e d f o r 1 h a t 95°. The excess L i A l H ^ was d e s t r o y e d by the dropwise a d d i t i o n o f e t h a n o l and the p r e c i p i t a t e formed was d i s s o l v e d i n HCl ( 1 0 % ) . The m e t h y l a t e d , r e d u c e d p r o d u c t was r e c o v e r e d from the aqueous s o l u t i o n by C H C I 3 e x t r a c t i o n . I . r . s p e c t r o s c o p i c a n a l y s i s o f the sample ( d i s s o l v e d i n C C l ^ ) showed r e d u c t i o n to be complete when no c a r b o n y l a b s o r p t i o n peak (-1730 cm" 1) was d e t e c t e d and the c o n c o m i t a n t appearance o f h y d r o x y l a b s o r p t i o n s i g n a l s were observed. 111.4.3 F o r m a t i o n o f p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s (PMAAs) ( i ) H y d r o l y s i s - A sample o f m e t h y l a t e d m a t e r i a l (3-20 mg) was 56 h y d r o l y z e d u s i n g 2M TFA a t 95°. M e t h y l a t e d p o l y s a c c h a r i d e s were h y d r o l y z e d f o r 16-20 h and m e t h y l a t e d o l i g o s a c c h a r i d e s 6-8 h. The TFA was removed by c o - e v a p o r a t i o n w i t h (3x) and the e x t e n t o f h y d r o l y s i s checked by paper chromatography i n s o l v e n t 4. ( i i ) R e d u c t i o n - The h y d r o l y z a t e was d i s s o l v e d i n (5-10 mL) and r e d u c e d w i t h NaBH^ (20-50 mg). The r e s t o f the workup i s as p r e v i o u s l y d e s c r i b e d (see S e c t i o n I I I . 3.2). ( i i i ) A c e t v l a t i o n - T h i s i s as p r e v i o u s l y d e s c r i b e d f o r a l d i t o l a c e t a t e s . The PMAAs, d i s s o l v e d i n CHCI3, were a n a l y z e d by g . l . c . on columns B, C o r E (see S e c t i o n I I I . 6 ) . A l l g . l . c . a n a l y s e s were checked f o r r e p r o d u c i b i l i t y and i d e n t i f i c a t i o n o f peaks ( u s i n g r e l a t i v e r e t e n -t i o n t i mes) were c o n f i r m e d by g.l.c.-m.s. I I I . 5 Paper c hromatography 0^ ,90 A n a l y t i c a l paper chromatography was pe r f o r m e d by the de s c e n d i n g method u s i n g Whatman No. 1 paper and the f o l l o w i n g s o l v e n t systems; (1) 18:3:1:4 e t h y l a c e t a t e - - a c e t i c a c i d - - f o r m i c a c i d - - w a t e r (2) 2:1:1 1 - b u t a n o l - - a c e t i c a c i d - - w a t e r ( f r e s h l y p r e p a r e d ) (3) 8:2:1 e t h y l a c e t a t e - - p y r i d i n e - - w a t e r , and (4) 4:1:5 1 - b u t a n o l - - e t h a n o l - - w a t e r (upper ph a s e ) . - 57 -D e s c e n d i n g p r e p a r a t i v e paper chromatography was p e r f o r m e d u s i n g Whatman No. 1 paper and s o l v e n t s 1 and 2. Paper s t r i p s , c o n t a i n i n g c a r b o h y d r a t e m a t e r i a l , were c u t and soaked i n water o v e r n i g h t . The aqueous s o l u t i o n s were f i l t e r e d , c o n c e n t r a t e d and f r e e z e - d r i e d . Chromatograms were d e v e l o p e d u s i n g the f o l l o w i n g t e c h n i q u e s : ( i ) a l k a l i n e s i l v e r n i t r a t e ( i i ) s p r a y i n g w i t h p . - a n i s i d i n e h y d r o c h l o r i d e i n aqueous 1- b u t a n o l , f o l l o w e d by h e a t i n g a t 110° f o r 5-10 min, and ( i i i ) n i n h y d r i n i n acetone f o l l o w e d by h e a t i n g a t 110° f o r 5 min. I I I . 6 G a s - l i q u i d c h r o m a t o g r a p h y ( g . 1. c . ) ° 4 » • L J - / a n d M a s s s p e c t r o m e t r y 1 1 8 » 1 7 2 - 1 7 5 A n a l y t i c a l g . l . c . s e p a r a t i o n s were p e r f o r m e d u s i n g t h e f o l l o w i n g i n s t r u m e n t s : ( i ) a H e w l e t t - P a c k a r d 5700 i n s t r u m e n t f i t t e d w i t h d u a l f l a m e - i o n i z a t i o n d e t e c t o r s . S t a i n l e s s - s t e e l columns (1.8 m x 3 mm) were used w i t h a N 2 c a r r i e r - g a s f l o w r a t e o f 20 mL/min; ( i i ) a H e w l e t t - P a c k a r d 5890A c a p i l l a r y gas chromatograph f i t t e d w i t h flame i o n i z a t i o n d e t e c t o r s . A f u s e d s i l i c a c a p i l l a r y column (15 m x 0.256 mm) was used w i t h a h e l i u m c a r r i e r - g a s f l o w r a t e o f 1.1 mL/min. The f o l l o w i n g p a c k i n g m a t e r i a l and programmes were used f o r the HP 5700 i n s t r u m e n t : 58 (A) 3% o f SP-2340 on S u p e l c o p o r t (100-120 mesh), programmed a t 195° f o r 4 min, and then a t 2°/min to 260° •(B) 3% o f OV-225 on Gas Chrom Q (100-120 mesh), i s o t h e r m a l a t 170°, or programmed a t 180° f o r 4 min, and th e n a t 2°/min t o 230°, and (C) 5% o f SP-1000 on Gas Chrom Q (100-120 mesh), i s o t h e r m a l a t 220°. P r e p a r a t i v e g . l . c . was performed on an F & M model 720 d u a l column i n s t r u m e n t f i t t e d w i t h t h e r m a l c o n d u c t i v i t y d e t e c t o r s . S t a i n l e s s - s t e e l columns (1.8 m x 6.3 mm) were used w i t h a h e l i u m c a r r i e r - g a s f l o w r a t e o f 60 mL/min. S e p a r a t i o n s were conducted i n (D) 3% o f SP-2340 on S u p e l c o p o r t (100-120 mesh), programmed from 190° a t 4°/min to 240° ( f o r a l d i t o l a c e t a t e s ) , o r from 160° a t 2°/min to 240° ( f o r PMAAs). On the HP 5890A i n s t r u m e n t , the f u s e d s i l i c a c a p i l l a r y column (E) was c o a t e d w i t h DB-17 (100% m e t h y l p h e n y l - p o l y s i l o x a n e ) and programmed a t e i t h e r ( i ) 180° f o r 2 min and th e n 5°/min t o 220° ( f o r p e r a c e t y l a t e d a l d i t o l s and o r a l d o n o n i t r i l e s ) , or ( i i ) 180° f o r 1 min and then 2°/min t o 250° ( f o r PMAAs). G.l.c.-m.s. a n a l y s e s were performed on a K r a t o s MS80RFA i n s t r u m e n t w i t h h e l i u m as the c a r r i e r - g a s a t a f l o w r a t e o f 25 mL/min. The c a p i l -l a r y columns used f o r the s e p a r a t i o n s were: (E) DB-17 (0.25 mm x 15 m) . (F) DB-225 (0.25 mm x 15 m), programmed a t 180° f o r 4 min,' then at 10°/min t o 250°; and (G) SE-30 (0.25 mm x 15 m), programmed a t 60° f o r 1 min, then at 10°/min to 250°. 59 -The mass s p e c t r a were r e c o r d e d a t 70 eV w i t h an i o n i z a t i o n c u r r e n t o f 100 fiA and an i o n sour c e a t 200°. Chemical i o n i z a t i o n , mass s p e c t r o m e t r i c ( c . i . - m . s . ) a n a l y s e s o f p e r m e t h y l a t e d o l i g o s a c c h a r i d e s were p e r f o r m e d on a Nermag R10-10 , quadrupole mass s p e c t r o m e t e r w i t h ammonia, i s o b u t a n e o r methane as the r e a g e n t gases. F a s t atom bombard-ment mass s p e c t r o m e t r y (f.a.b.-m.s.) was performed on an AEIMS9 i n s t r u -ment, f i t t e d w i t h a s a d d l e - f i e l d gun o p e r a t i n g a t 1 mA and between 7-8 kV. Xenon was us e d as the bombarding gas. I I I . 7 GelLW and i o n - e x c h a n g e c h r o m a t o g r a p h y . P r e p a r a t i v e g e l - p e r m e a t i o n chromatography was performed u s i n g columns (2.5 x 100 cm) o f B i o - G e l P-2 o r P-4 (both 400 mesh). The v o i d volume o f the column and the e f f i c i e n c y o f p a c k i n g were d e t e r m i n e d u s i n g B l u e D e x t r a n 2000 ( 0 . 2 % ) . The c o n c e n t r a t i o n o f the samples f r a c t i o n a t e d on the column ranged from 40-100 mg/mL. The e l u a n t used was 500:5:2 w a t e r - - p y r i d i n e - - a c e t i c a c i d a t a f l o w r a t e o f 10 mL/h. F r a c t i o n s (2.3 mL) were c o l l e c t e d , l y o p h i l i z e d , weighed and the e l u t i o n p r o f i l e p l o t t e d . S e l e c t e d samples were p i c k e d f o r p r e l i m i n a r y c h a r a c t e r i z a t i o n s by paper c h r o m a t o g r a p h i c and ^ H-n.m.r. s p e c t r o s c o p i c a n a l y s e s . P e r m e t h y l a t e d c a r b o h y d r a t e samples (3-50 mg) were d e s a l t e d by p a s s i n g through a column (1 cm x 10 cm) of Sephadex LH-20 w i t h 1:1 CH3OH--CHCI3 as the e l u a n t . Ion-exchange chromatography f o r the s e p a r a t i o n o f 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 was performed on a column (2 cm x 30 cm) o f 60 -Bio-Rad AG1-X2 (200-400 mesh, formate form). The n e u t r a l f r a c t i o n was e l u t e d w i t h water and the a c i d i c f r a c t i o n w i t h f o r m i c a c i d ( 1 0 % ) . D e c a t i o n i z a t i o n o f n e u t r a l and a c i d i c c a r b o h y d r a t e samples was e f f e c t e d by p a s s i n g the aqueous s o l u t i o n s through a column o f A m b e r l i t e IR 120 (H +) r e s i n . Aqueous n e u t r a l c a r b o h y d r a t e samples were d e - a n i o n i z e d by passage t h r o u g h a column o f A m b e r l i t e IR 45 (OH") r e s i n . I I I . 8 O p t i c a l r o t a t i o n 9 6 , c i r c u l a r d i c h r o i s m 1 0 0 and i n f r a - r e d s p e c t r o p h o t o m e t r y O p t i c a l r o t a t i o n s were measured on aqueous s o l u t i o n s a t 23-25° i n a 10 cm c e l l w i t h a P e r k i n - E l m e r model 141 p o l a r i m e t e r . C i r c u l a r d i c h r o i s m (c.d.) s p e c t r a were r e c o r d e d w i t h a J a s c o J-500 A a u t o m a t i c r e c o r d i n g s p e c t r o p o l a r i m e t e r f o r s o l u t i o n s i n a q u a r t z c e l l o f 0.3 mL c a p a c i t y and a p a t h l e n g t h o f 1 cm. Samples were d i s s o l v e d i n s p e c t r o s c o p i c - g r a d e a c e t o n i t r i l e and the s p e c t r a were r e c o r d e d i n the range o f 200-290 nm. The i n f r a - r e d ( i . r . ) s p e c t r a o f m e t h y l a t e d d e r i v a t i v e s , d i s s o l v e d i n s p e c t r o s c o p i c - g r a d e CC1^ } were r e c o r d e d on a P e r k i n - E l m e r model 457 s p e c t r o p h o t o m e t e r . 61 -I I I . 9 P r o p a g a t i o n o f b a c t e r i o p h a g e s ^ 0 0 . I I I . 9 . 1 P r e p a r a t i o n o f b a c t e r i a l lawn. The b a c t e r i a l lawn r e q u i r e d f o r b a c t e r i o p h a g e p l a q u e a s s a y was p r e p a r e d as f o l l o w s . An agar p l a t e ( n u t r i e n t agar f o r K l e b s i e l l a . M u e l l e r - H i n t o n agar f o r E. c o l i ) was d r i e d , u p s i d e down and l i d o f f , a t 37° i n an i n c u b a t o r (1 h ) . A c t i v e l y - g r o w i n g b a c t e r i a l c u l t u r e (2 mL) was p i p e t t e d on t o the agar s u r f a c e and a f t e r 15 min a t 37°, excess l i q u i d was d r a i n e d o f f . The p l a t e was then i n c u b a t e d a t 37°, f i r s t w i t h the l i d p a r t i a l l y on, then u p s i d e down f o r 1 h. I l l . 9 . 2 B a c t e r i o p h a g e p l a q u e a s s a y The d e t e r m i n a t i o n o f b a c t e r i o p h a g e c o n c e n t r a t i o n ( i . e . p l a q u e assay) was pe r f o r m e d as f o l l o w s . B a c t e r i o p h a g e s u s p e n s i o n s were s e r i -a l l y d i l u t e d (0.3 mL to 2.7 mL s t e r i l e b r o t h ) t o 1 0 " ^ . One s m a l l drop (20 nL o f each d i l u t i o n ) was added to the p r e p a r e d h o s t b a c t e r i a l lawn a t p o s i t i o n s near the p e r i m e t e r o f the p l a t e . A f t e r o v e r n i g h t i n c u b a -t i o n a t 37°, p l a q u e s ( c l e a r s p o t s ) sometimes s u r r o u n d e d by h a l o s were ob s e r v e d . A t the h i g h e s t d i l u t i o n , s i n g l e p l a q u e s marked the p r o l i f e r -a t i o n o f i n d i v i d u a l b a c t e r i o p h a g e p a r t i c l e s . The phage c o n c e n t r a t i o n of the o r i g i n a l p r e p a r a t i o n was c a l c u l a t e d u s i n g the e x p r e s s i o n below. 62 No. o f p l a q u e s x d i l u t i o n B a c t e r i o p h a g e t i t e r =  ( i n p l a q u e - f o r m i n g - u n i t s / m L ) d r o p s i z e ( m L ) 111.9.3 Tube l y s i s . An a c t i v e l y - g r o w i n g b a c t e r i a l c o l o n y was i n o c u l a t e d i n t o s t e r i l e b r o t h (5 mL) and i n c u b a t e d a t 37° w i t h s h a k i n g , u n t i l the b a c t e r i a l c u l t u r e t u r n e d t u r b i d (3-4 h ) . S t e r i l e b r o t h ( 5 x 4 mL) i n c u l t u r e t e s t - t u b e s was th e n i n o c u l a t e d w i t h the b a c t e r i a l c u l t u r e (0.5 mL) and f u r t h e r i n c u b a t e d . A t 30 min i n t e r v a l s , c o n s e c u t i v e tubes were i n o c u -l a t e d w i t h a b r o t h s o l u t i o n (0.5 mL) o f b a c t e r i o p h a g e . C o n t i n u e d i n c u b a t i o n r e s u l t e d i n the f i r s t few tubes c h a n g i n g from the t u r b i d s o l u t i o n t o a c l e a r s o l u t i o n due to b a c t e r i a l c e l l l y s i s . A f t e r the l a s t tube had c l e a r e d (-5 h a f t e r the f i r s t b a c t e r i o p h a g e i n o c u l a t i o n ), CHCI3 (1 mL) was added t o a l l the tubes and the m i x t u r e s were v o r t e x e d . Combinations o f tubes t h a t c l e a r e d , f o l l o w e d by c e n t r i f u g a t i o n (20 min) y i e l d e d c l e a r phage s o l u t i o n s which were pl a q u e a s s a y e d t o determine the b a c t e r i o p h a g e c o n c e n t r a t i o n s . 111.9.4 S m a l l f l a s k l y s i s . T h i s t e c h n i q u e i s s i m i l a r to the tube l y s i s e x c e p t t h a t l a r g e r volumes a r e used. S t e r i l e b r o t h (5 x 48 mL) i n Erlenmeyer f l a s k s (125 - 63 -mL) was i n o c u l a t e d w i t h a c t i v e l y - g r o w i n g b a c t e r i a l c u l t u r e (1 mL) and i n c u b a t e d a t 37°. A t 30 min i n t e r v a l s , a l i q u o t s (1 mL) o f b a c t e r i o p h a g e s o l u t i o n (from tube l y s i s o r o t h e r w i s e ) were added c o n s e c u t i v e l y t o the f l a s k s . C o n t i n u e d i n c u b a t i o n and work up was p e r f o r m e d as f o r tube l y s i s . I I I . 9 . 5 Wash b o t t l e l y s i s . Three w a s h - b o t t l e s , each c o n t a i n i n g s t e r i l e b r o t h (200 mL) were i n o c u l a t e d w i t h a c t i v e l y - g r o w i n g b a c t e r i a l c u l t u r e (10 mL). Growth was m a i n t a i n e d a t 37° w i t h a e r a t i o n , e f f l u e n t a i r b e i n g p a s s e d through aqueous p h e n o l t o s t e r i l i z e i t . A few drops o f Dow a n t i f o a m FG-10 e m u l s i o n were added t o each w a s h - b o t t l e . Absorbance was m o n i t o r e d on samples o f b a c t e r i a l c u l t u r e (3 mL) withdrawn a t i n t e r v a l s o f 20-30 min. When an o p t i c a l d e n s i t y v a l u e was r e a c h e d t h a t c o r r e s p o n d e d t o the a p p r o p r i a t e number o f c e l l s i n r e l a t i o n t o the q u a n t i t y o f phage t o be expended (see F i g . V . 2 . I ) , the b a c t e r i o p h a g e s u s p e n s i o n was added t o each o f t h e t h r e e w a s h - b o t t l e s . F o r optimum b a c t e r i o p h a g e p r o p a g a t i o n , e x p e r i e n c e i n d i c a t e d t h a t the b a c t e r i o p h a g e t o b a c t e r i a l c e l l r a t i o s h o u l d be a p p r o x i m a t e l y 3:1. When the o p t i c a l d e n s i t y o f the c u l t u r e had f a l l e n t o ne a r i t s b l a n k v a l u e (~ 3 h ) , i n d i c a t i n g complete b a c t e -r i a l l y s i s , work up and p l a q u e a s s a y f o l l o w e d the u s u a l p r o c e d u r e . - eu CHAPTER IV STRUCTURAL INVESTIGATION OF THE CAPSULAR POLYSACCHARIDES OF K l e b s i e l l a AND E s c h e r i c h i a c o l i 65 IV.1 STRUCTURE OF THE CAPSULAR POLYSACCHARIDE OF K l e b s i e l l a SEROTYPE K79 IV.1.1 A b s t r a c t The s t r u c t u r e o f the c a p s u l a r p o l y s a c c h a r i d e from K l e b s i e l l a K79 was d e t e r m i n e d by the t e c h n i q u e s o f m e t h y l a t i o n , p e r i o d a t e o x i d a t i o n , / 3 - e l i m i n a t i o n , chromic a c i d o x i d a t i o n and p a r t i a l h y d r o l y s i s . N.m.r. s p e c t r o s c o p y (^H and ^ 3C) was used e x t e n s i v e l y t o e s t a b l i s h the n a t u r e o f the anomeric l i n k a g e s o f the p o l y s a c c h a r i d e and o f d e r i v e d o l i g o s a c -c h a r i d e s o b t a i n e d through d e g r a d a t i v e p r o c e d u r e s . The p o l y s a c c h a r i d e was found t o have the h e p t a s a c c h a r i d e , "5+2" r e p e a t i n g u n i t : 3) -,9-D-Gal- (1-3) -/9-D-GlcA- (1-2) -a-L-Rha- (1-3) -a-L-Rha- (1-3) -a-L-Rha-(1-4 t 1 a-D-Glc 6 t 1 a-D-Glc IV.1.2 I n t r o d u c t i o n The b a c t e r i a l genus K l e b s i e l l a i s d i v i d e d i n t o 77 d i f f e r e n t s e r o t y p e s , which are d i s t i n g u i s h e d by the s t r u c t u r e o f t h e i r c a p s u l a r p o l y s a c c h a r i d e s . S t r u c t u r a l a n a l y s e s o f about 70 o f t h e s e c a p s u l a r 66 p o l y s a c c h a r i d e s have thus f a r been d e s c r i b e d . As p a r t o f our c o n t i n u i n g e f f o r t t o e x p l a i n the s e r o l o g y o f t h i s genus on a s t r u c t u r a l b a s i s , we now r e p o r t our r e s u l t s on K79. K l e b s i e l l a s e r o t y p e K79 i s one o f the f o u r s t r a i n s ( t h e o t h e r s a r e K18, K19 and K 4 1 ) 6 whose c a p s u l a r p o l y s a c c h a r i d e s a r e composed o f D - g a l a c t o s e , D - g l u c o s e , D - g l u c u r o n i c a c i d and L-rhamnose. Four o t h e r s t r a i n s (K12, K36, K55 and K70)° b e s i d e s h a v i n g the same su g a r composi-t i o n , have i n a d d i t i o n a c e t a t e and/or 1 - c a r b o x y e t h y l i d e n e s u b s t i t u e n t g r oups. R e c e n t l y , the s t r u c t u r e o f t h e c a p s u l a r p o l y s a c c h a r i d e from K l e b s i e l l a K50 was r e p o r t e d ^ 9 4 as t h e f i r s t example o f a "5 + 2" t y p e s t r u c t u r e w h e r e i n t h e b r a n c h p o i n t i s a D - g l u c o s y l u n i t . The p a t t e r n f o r K79 i s a l s o o f t h e "5 + 2" t y p e b u t i n t h i s c a s e , i t i s t h e u r o n i c a c i d r e s i d u e t h a t i s the b r a n c h p o i n t . The s t r u c t u r e i s , t h e r e f o r e , n o v e l i n t h i s s e r i e s . The r h a m n o t r i o s e u n i t i n t h i s p o l y s a c c h a r i d e i s a l s o n o t e w o r t h y as s i m i l a r p a r t i a l s t r u c t u r e s a r e t o be f o u n d i n K l e b s i e l l a K32 and K34 ( r e f . 6 ) . IV.1.3 R e s u l t s and d i s c u s s i o n C o m p o s i t i o n and n.m.r. s p e c t r a . - K l e b s i e l l a K79 b a c t e r i a were grown on an a g a r medium and t h e a c i d i c p o l y s a c c h a r i d e was p u r i f i e d by two p r e c i p i t a t i o n s w i t h c e t y l t r i m e t h y l a m m o n i u m b r o m i d e 1 8 4 . The p r o d u c t was m o n o d i s p e r s e by g e l - p e r m e a t i o n chromatography (M r = 3 x 1 0 6 ) and had [ a ] D +18°, w h i c h compares w e l l w i t h the v a l u e o f +19° c a l c u l a t e d by u s i n g Hudson's r u l e s o f i s o r o t a t i o n 9 - 5 . A n a l y s i s o f t h e a c i d i c p o l y -67 s a c c h a r i d e gave g a l a c t o s e , g l u c o s e and rhamnose i n the molar r a t i o s o f 1.20:2.00:2.00 ( T a b l e IV.1.1, column I ) . When the u r o n i c a c i d i n the p o l y s a c c h a r i d e was r e d u c e d 7 6 , the molar q u a n t i t i e s o f g l u c o s e and rhamnose i n c r e a s e d ( T a b l e IV.1.1, column I I ) . T h i s i n d i c a t e d t h a t the g l u c u r o n i c a c i d i s l i n k e d to a rhamnosyl u n i t , which was l a t e r c o n f i r m e d by a / 3 - e l i m i n a t i o n r e a c t i o n . The K79 p o l y s a c c h a r i d e i s thus comprised o f g a l a c t o s e , g l u c o s e , g l u c u r o n i c a c i d and rhamnose i n the r a t i o s o f 1:2:1:3. C i r c u l a r d i c h r o i s m measurements 1^"-* on the a l d i t o l a c e t a t e s demonstrated rhamnose to be o f the L - c o n f i g u r a t i o n , and the g l u c o s e and g l u c u r o n i c a c i d t o be o f the D c o n f i g u r a t i o n . G a l a c t o s e was shown to TABLE I V . 1 . I : Sugar a n a l y s i s o f K l e b s i e l l a K79 p o l y s a c c h a r i d e and d e r i v e d p r o d u c t s . S u g a r M o l a r r a t i o s 5 ' b (as a l d i t o l a c e t a t e ) I I I I I I IV V VI V I I G a l a c t o s e 1.20 1.00 1.00 1.00 - 0.26 1.00 G l u c o s e 2.00 2.77 0.93 0.95 1.00 1.00 0.97 G l y c e r o l - - + c 0.2 c -1 , 2 - P r o p a n e d i o l - - - c -Rhamnose 2.00 2.75 1.85 1.75 1.51 1.47 0.85 a U s i n g SP-2340 column programmed from 195° f o r 4 min, and t h e n a t 2°/min to 260°. ^ I, o r i g i n a l a c i d i c p o l y s a c c h a r i d e ; I I , c a r b o d i i m i d e -reduced p o l y s a c c h a r i d e ; I I I , p o l y o l from p e r i o d a t e o x i d a t i o n ; IV, t e t r a s a c c h a r i d e i s o l a t e d from Smith d e g r a d a t i o n ; V, chromium t r i o x i d e o x i d a t i o n o f o r i g i n a l p o l y s a c c h a r i d e ; VI, chromium t r i o x i d e , o x i d a t i o n o f c a r b o d i i m i d e - r e d u c e d p o l y s a c c h a r i d e ; V I I , c a r b o x y l - r e d u c e d t r i s a c -c h a r i d e 5, o b t a i n e d from p a r t i a l h y d r o l y s i s . c Q u a n t i t a t i o n i n a c c u r a t e due to h i g h v o l a t i l i t y . - 68 -have the D c o n f i g u r a t i o n by the c i r c u l a r d i c h r o i s m c u r v e o f the 2 , 4 , 6 - t r i - O - m e t h y l d e r i v a t i v e 1 0 0 . The ^H-n.m.r. spectrum o f the p o l y s a c c h a r i d e i n d i c a t e d the pr e s e n c e o f seven anomeric p r o t o n s ( T a b l e I V . I . I I ) . These c o r r e s p o n d to f o u r a, two /3, and one b o r d e r l i n e s i g n a l which was p r o v e d by chromic a c i d d e g r a d a t i o n 1 ^ t o be t h a t o f an a l i n k a g e . There a r e thus f i v e a and two /3 l i n k a g e s i n the r e p e a t i n g u n i t 1 ^ D . From the h i g h - f i e l d r e g i o n 1 ^ 0 o f the spectrum, i t was f u r t h e r deduced t h a t : ( i ) t h r e e 6-deoxy sugar u n i t s were p r e s e n t , and ( i i ) a c e t a t e and p y r u v a t e , which are common f e a t u r e s o f 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 absent. These r e s u l t s were s u b s t a n t i a t e d by the 1 3C-n.m.r. spectrum 1-* 0, which i n c l u d e d a s i g n a l a t 173 p.p.m., a t t r i b u t e d t o the C = 0 group o f 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 l l z • L l J i . - A n a l y s e s were c a r r i e d out on the p o l y s a c c h a r i d e ( i ) i n i t s o r i g i n a l form, ( i i ) a f t e r r e d u c t i o n o f the u r o n i c a c i d f o l l o w i n g m e t h y l a t i o n (see Scheme I V . 1 . I ) , and ( i i i ) a f t e r r e m e t h y l a t i o n o f p r o d u c t from ( i i ). The r e s u l t s , p r e s e n t e d i n T a b l e I V . 1 . I l l , columns I t o I I I c o n f i r m the concept o f a h e p t a s a c c h a r i d e r e p e a t i n g u n i t . They show t h a t the b r a n c h p o i n t i s a g l u c o s y l u r o n i c r e s i d u e l i n k e d a t C-3 and C-4, and t h a t one o f the g l u c o s e u n i t s i s a t e r m i n a l , n o n - r e d u c i n g r e s i d u e . P e r i o d a t e o x i d a t i o n 1 ^ . - C a r b o x y l reduced K79 p o l y s a c c h a r i d e ' ' 0 - 6 9 -T a b l e I V . l . I I : N . M . R . d a t a f o r K l e b s i e l l a K 7 9 c a p s u l a r p o l y s a c c h a r i d e s a n d d e r i v e d o l i g o s a c c h a r i d e s . Compound" 'H-N.m.r. data ** l,f Integral (p.p.m.) (Hi) (noofH) Auignmetu' "C-N.m.r. data (p.p.m.) Assignment 1 I 3 „ . 1 3 _ , I 3 _ . 1 2 . _ R h » R h a — - G i l . C l c _ Gro» a a 0 0 5.08 t 2 R h » 103.99 4.69 b 2 - R h » 103.02 R h * - R h a -1.31 > 6 0 CHjOf R h i 102.84 61 73 17.50 C-6 ofhcioKS C-6o( R h i R h » - ^ R h » - L 2 R h » J - ^ O i ] - O M e 5.40 0 5 (permeihylated in C D O , ) 5.10 5.02 4.80 1.31 1.25 2.0 1.0 0.5 - R h » R h » 3 G » l - L i G l c A-4 ^ R r « - O H 5.23 « 0.5 -i - R h a O H 104 01 Gat—.— p P o p 5 3 4 84 i 0 5 _ i _ R h . - — O H 97.29 — R h a - - - O H P P 4.65 8 1.0 0»V—— 93.38 -i - R h a O H 0 ° 4.50 I 1.0 _J_GlcA-—- 17.30 C - « o f R h a 1.31 t 3 C H . o f R h . - 7 0 -Compound >H Hmr daw (tarn) WD Integral -C-N.m t 4mu tppm.) Auignmmi' -LRnJL20llJ l^cAJ'Rn^ Rn._L • P .i P o « I i C l c 6 | 1° I I C k K79 Polysaccharide S.62 5.33 3.05 495 4 65 4 49 1.0 1.0 2.0 1.0 1.0 G l c -- R h » 3 . 4 , i l c A -179 60 173. on 104 79 103.81 103.57 102 77 C > 0 of acetone C - O o f CIcA 3.4 . a l -i l c A -- R h a -- R h » 1.32 C H , of Rha 101.69 99.37 61 86 17.55 Clc o C-6 of heio«*< C-6o( Rha 3 1 \j- . 1 3 - . 1 2 , — R h a Cal . G l c - g -• P , P I C l c 6 1 3 , I C l c Reduced K79 Polyucchiride 3.46 3.37 507 4.97 4.66 4 49 1.32 1.0 1.0 2 0 1.0 1.0 1.0 9.0 G k -3 -Rha 0 3.4 , 0 C H , of Rha 179.30 104.72 103.59 102.80 102 75 101.78 99.70 C - O o f acetone 3 r- , P -Rha G l c -61.83 17.50 C-6o( heicnts C-6 of Rha - 71 a For the o r i g i n of compounds 1 , 2 , and 5 , see text. b Chemical s h i f t r e l a t i v e to sodium-4,4-dimethyl-4-silapentane-l-sulfonate ( D S S ) 1 ^ . c j r e v . _ broad, unable to assign accurate coupling constant; s - s i n g l e t ; t - t r i p l e t ; m - m u l t i p l e t . d For example —2-Cal-g— r e f e r s to the anomeric proton of a 3-linked g a l a c t o s y l residue i n the ^-anomeric c o n f i g u r a t i o n . The absence of a numerical p r e f i x i n d i c a t e s a (terminal) nonreducing group. e Chemical s h i f t r e l a t i v e to Me^Si. * As f o r d, but for anomeric 1 3 C n u c l e i . See Appendix III f o r a l l n.m.r. spectra. 72 T a b l e I V . 1 . I l l : M e t h y l a t i o n a n a l y s e s o f K l e b s i e l l a K79 p o l y s a c c h a r i d e and d e r i v e d p r o d u c t s . M e t h y l a t e d s u g a r 3 M o l % b ' c (as a l d i t o l a c e t a t e s ) I d I I I I I IV V VI V I I V I I I IX 2,3,4-Rha - -3,4-Rha 16 15 2,4-Rha 34 28 2,4,6-Gal 16 14 2,3,4,6-Gal - -2,3,4,6-Glc 16 14 2,4,6-Glc - -2,3,4-Glc 16 14 2,4-Glc - -2,6-Glc - -2-Glc _ 15 20 19 25 17 15 - - . . . 30 29 50 30 40 55 42 14 25 28 18 20 5 -36 15 - - 18 - 21 -22 13 - - 5 - 15 34 14 25 - - - -2,3,4,6-Glc = l , 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 . V a l u e s a r e c o r r e c t e d by use o f the e f f e c t i v e , c a r b o n - r e s p o n s e f a c t o r s g i v e n by A l b e r s h e i m e t a l ^ u . U s i n g an OV-225 column programmed from 180° f o r 4 min, and then a t 2°/niin to 230°. I, o r i g i n a l a c i d i c p o l y s a c c h a r i d e ; I I , a f t e r r e d u c t i o n o f u r o n i c e s t e r ; I I I , p r o d u c t from r e m e t h y l a t i o n a f t e r r e d u c t i o n o f u r o n i c e s t e r ; IV, p r o d u c t from p e r i o d a t e o x i d a t i o n ; V, p r o d u c t from Smith d e g r a d a t i o n ; VI, p r o d u c t from f3-elimination and r e m e t h y l a t i o n ; V I I , p r o d u c t from p r e p a r a t i v e p - e l i m i n a t i o n ; V I I I , p r o d u c t from the chromic a c i d - d e g r a d e d reduced p o l y s a c c h a r i d e ; IX, m e t h y l a t e d and r e d u c e d t r i s a c c h a r i d e from p a r t i a l h y d r o l y s i s . - 7 3 -Klebsiella K79 POLYSACCHARIDE ( H +) 1. HAKOMORI METHYLATION a. SODIUM DIMSYL/DMSO b. CH,I S c h e m e I V . 1 . I : M e t h y l a t i o n a n a l y s i s o f t h e K l e b s i e l l a K 7 9 c a p s u l a r p o l y s a c c h a r i d e . - 74 -consumed f i v e moles o f p e r i o d a t e p e r r e p e a t i n g u n i t , which i s i n agree-ment w i t h t h e o r y . Sugar a n a l y s i s o f the p e r i o d a t e - o x i d i z e d p r o d u c t showed t h a t g a l a c t o s e , g l u c o s e and rhamnose s u r v i v e d i n the r a t i o s 1.00:0.93:1.85 ( T a b l e IV.1.I, column I I I ) . M e t h y l a t i o n a n a l y s i s o f t h i s same p r o d u c t r e v e a l e d t h a t the g l u c o s e u n i t which s u r v i v e d the degrada-t i o n i s a c t u a l l y the r e d u c e d g l u c u r o n i c a c i d r e s i d u e (see T a b l e I V . 1 . I l l , column I V ) . A p r e p a r a t i v e Smith d e g r a d a t i o n 1 ^ , 1 9 8 ^ c o n d u c t e d on the c a r b o x y l r e d u c e d K79 p o l y s a c c h a r i d e , y i e l d e d the o l i g o s a c c h a r i d e 1 (see Scheme I V . l . I I ) , which on a c i d h y d r o l y s i s gave g a l a c t o s e , g l u c o s e and rhamnose i n the r a t i o s o f 1.00:0.95:1.75 ( T a b l e I V . 1 . I , column I V ) . N.m.r. a n a l y s e s ( T a b l e I V . l . I I ) on 1 c o n f i r m e d i t to be a t e t r a s a c c h a -r i d e and i n d i c a t e d the rhamnosyl u n i t s to be a - l i n k e d 1 ^ ^ , whereas the g a l a c t o s e and g l u c o s e u n i t s a r e ^ - l i n k e d . From the m e t h y l a t i o n d a t a ( T a b l e I V . 1 . I l l , column V) a n o n - r e d u c i n g , t e r m i n a l rhamnosyl u n i t i s i n d i c a t e d ; the p r e s e n c e o f a 2 , 4 , 6 - t r i - O - m e t h y l g l u c o s e d e r i v a t i v e i n d i c a t e s t h a t the b r a n c h p o i n t r e s i d u e o f the K79 p o l y s a c c h a r i d e , g l u c u r o n i c a c i d , has a s i d e c h a i n l i n k e d t o i t a t C-4. C h e m i c a l -i o n i z a t i o n , mass s p e c t r o m e t r i c a n a l y s i s ^ 0 0 (see mass spectrum numbered 1 i n Appendix IV) o f the p e r m e t h y l a t e d 1 showed peaks a t m/z 771, 567, 363 and 189, among o t h e r s . The s t r u c t u r e o f 1 i s g i v e n below and the s o u r c e s o f some p e r t i n e n t fragments from m e t h y l a t e d 1 a r e i n d i c a t e d . I I I I I I I I a-L-Rha-(1-3)-a-L-Rha-(1-3)-0-D-Gal-(1-3)-0-D-Glc-(1-2)-glycerol I I I I t i l l m/z 189 363 567 771 1 - 7 5 -K l e b s i e l l a K 7 9 POLYSACCHARIDE CARBODIDUDI WEDUCTIOK COOH •-CBjOB COMPOUND 1 S c h e m e I V . l . H : P e r i o d a t e o x i d a t i o n - S m i t h d e g r a d a t i o n o f K l e b c a p s u l a r p o l y s a c c h a r i d e . / S - E l i m i n a t i o n ^ u . A b a s e - c a t a l y z e d u r o n i c a c i d d e g r a d a t i o n o f the p e r m e t h y l a t e d p o l y s a c c h a r i d e , f o l l o w e d by r e m e t h y l a t i o n , gave the r e s u l t shown i n T a b l e I V . 1 . I l l , column V I . The p r e s e n c e o f 2 , 3 , 4 - t r i - O - m e t h y l -rhamnose, i n p l a c e o f the p r e v i o u s l y o b s e r v e d 3,4-di-0-methylrhamnose, c o n f i r m e d t h a t the g l u c u r o n i c a c i d i s l i n k e d t o p o s i t i o n 2 o f a rhamnose u n i t . A d e c r e a s e o b s e r v e d i n the amount o f 2 , 3 , 4 - t r i - O - m e t h y l - g l u c o s e was a t t r i b u t e d to the d e g r a d a t i o n o f t h i s sugar on l i b e r a t i o n and exposure to the base 2*- 1 2. Hence, t h i s r e s u l t i n d i c a t e s t h a t the r e p e a t i n g u n i t o f K79 p o l y s a c c h a r i d e has an i s o m a l t o s e s i d e - c h a i n ; from the a n a l y s e s c o n d u c t e d on 1, t h i s s i d e c h a i n i s l i n k e d t o C-4 o f the g l u c u r o n i c a c i d r e s i d u e . A p r e p a r a t i v e , b a s e - c a t a l y z e d u r o n i c a c i d d e g r a d a t i o n c o n d u c t e d on the p e r m e t h y l a t e d p o l y s a c c h a r i d e , w i t h the i n t e n t i o n o f g e n e r a t i n g o l i g o s a c c h a r i d e s , f o l l o w e d by m e t h y l a t i o n , gave o n l y 2 as the o l i g o m e r i c p r o d u c t . H y d r o l y s i s o f 2, f o l l o w e d by c o n v e r s i o n o f the p r o d u c t s t o the p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s , i n d i c a t e d the p r e s e n c e o f a t e r m i n a l rhamnose, two 3 - l i n k e d rhamnoses, and a 3 - l i n k e d g a l a c t o s e (see T a b l e I V . 1 . I l l , column V I I ) . ^H-N.m.r. a n a l y s i s o f the p e r m e t h y l a t e d 2 r e v e a l e d the p r e s e n c e o f f o u r anomeric p r o t o n s ( T a b l e I V . l . I I ) . The s i g n a l s a t 6* 5.40 and S 4.80 can be a t t r i b u t e d t o a r e d u c i n g g a l a c t o s y l r e s i d u e . A n a l y s i s o f 2 by c h e m i c a l i o n i z a t i o n , mass s p e c t r o m e t r y 2 ^ ^ showed peaks a t m/z 741, 537, 363 and 189 among o t h e r s (see mass spec-trum numbered 2 i n Appendix I V ) . T h i s c o n f i r m e d the s t r u c t u r e o f 2 as shown. 77 a-L-Rha - (1—3) -a-L-Rha-(1-3)-a-L-Rha-(1-3)-a-D-Gal I I I I I I I I I m/z 189 363 537 741 2 ( p e r m e t h y l a t e d ) Attempts to i s o l a t e the s i d e - c h a i n i s o m a l t o s e r e s i d u e were not s u c c e s s -f u l . I t i s p o s s i b l e t h a t the p e r m e t h y l a t e d a - G l c - ( 1 - 6 ) - G l c ~ O H was degraded f u r t h e r under the b a s i c c o n d i t i o n s used, as has been n o t i c e d p r e v i o u s l y ^ O ^ ^ n t h e c a s e G f K l e b s i e l l a K26. Chromic a c i d o x i d a t i o n 1 ^ . . A n a l y s e s were c o n d u c t e d on ( i ) the p e r a c e t y l a t e d o r i g i n a l p o l y s a c c h a r i d e , and ( i i ) the p e r a c e t y l a t e d c a r b o d i i m i d e - r e d u c e d p o l y s a c c h a r i d e . The r e s u l t s , p r e s e n t e d i n T a b l e I V . 1 . I , columns V and VI show t h a t g a l a c t o s e and g l u c u r o n i c a c i d have been degraded and t h e r e f o r e are ft - l i n k e d . The r e s u l t from the m e t h y l a t i o n a n a l y s i s o f the p r o d u c t from ( i i ) ( T a b l e I V . 1 . I l l , column V I I I ) i s s i m i l a r to t h a t from the / 3 - e l i m i n a t i o n , e x c e p t t h a t the 3 - l i n k e d / J - g a l a c t o s y l r e s i d u e , which has been degraded, i s now d i m i n i s h e d . The p r e s e n c e o f o n l y one molar r a t i o o f 2 , 3 , 4 - t r i - O - m e t h y l -rhamnose p e r r e p e a t i n g u n i t shows t h a t the g a l a c t o s y l r e s i d u e must be l i n k e d to the g l u c u r o n i c a c i d . T h i s i s i n agreement w i t h the data p r e v i o u s l y o b t a i n e d . An attempt to g e n e r a t e s e l e c t i v e l y c l e a v e d o l i g o s a c c h a r i d e s by chromic a c i d o x i d a t i o n o f the p e r a c e t y l a t e d , c a r b o d i i m i d e - r e d u c e d 78 p o l y s a c c h a r i d e , gave two p r o d u c t s , 3 and 4, i s o l a t e d by paper chromatography a f t e r d e a c e t y l a t i o n o f the o x i d i z e d r e s i d u e . S i g n a l s a t 6 5.03 (1.5H), 4.92 (1H), and 1.31 (9H) i n the ^H-n.m.r. spectrum o f 3 i n d i c a t e d i t to be a r h a m n o t r i o s e (see spectrum numbered 4 i n Appendix I I I ) , b u t the spectrum o f 4 was n o t d e f i n i t i v e . Poor y i e l d s o f 3 and 4 p r e c l u d e d f u r t h e r a n a l y s e s . 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 o f the n a t i v e p o l y s a c c h a -r i d e , w i t h the i n t e n t i o n o f i s o l a t i n g the i s o m a l t o s e s i d e - c h a i n , f o l -lowed by s e p a r a t i o n on paper chromatography, gave f o u r compounds. Compound 5 ( E l a c t o s e 0 - 9 8 ) 1 S o f the most i n t e r e s t because i t p o s s e s s e s a m o b i l i t y comparable to t h a t o f i s o m a l t o s e . However, on the b a s i s o f i t s sugar a n a l y s i s ( T a b l e IV.1.I, column V I I ) , n . m . r . - s p e c t r a l d a t a ( T a b l e I V . l . I I ) , and m e t h y l a t i o n a n a l y s i s ( T a b l e I V . 1 . I l l , column I X ) , the s t r u c t u r e o f 5 i s deduced to be as f o l l o w s jfl-Gal- (1-3) -/3-GlcA- (1-2) -Rha 5 IV.1.4 C o n c l u s i o n The s t r u c t u r e o f the c a p s u l a r p o l y s a c c h a r i d e from K l e b s i e l l a K79 i s thus based on the h e p t a s a c c h a r i d e r e p e a t i n g - u n i t shown i n the A b s t r a c t , and i s c o n s i s t e n t w i t h the q u a l i t a t i v e a n a l y s i s r e p o r t e d by Nimmich ° (see Appendix I ) . S e v e r a l attempts to o b t a i n the s i n g l e r e p e a t i n g u n i t by means o f b a c t e r i o p h a g e - i n d u c e d d e p o l y m e r i z a t i o n o f the K79 p o l y s a c c h a -r i d e f a i l e d . A t e t r a m e r o f the h e p t a s a c c h a r i d e r e p e a t i n g u n i t was o b t a i n e d i n s t e a d (see S e c t i o n V.2). IV.1.5 E x p e r i m e n t a l G e n e r a l methods - The g e n e r a l e x p e r i m e n t a l p r o c e d u r e s a r e as d e s c r i b e d i n S e c t i o n I I I . P r e p a r a t i o n and p r o p e r t i e s o f K79 c a p s u l a r p o l y s a c c h a r i d e s - A c u l t u r e o f K l e b s i e l l a K79, o b t a i n e d from Dr. Ida J2h:skov, Copenhagen, was grown as p r e v i o u s l y d e s c r i b e d 1 ^ , and the p o l y s a c c h a r i d e was p u r i f i e d by two p r e c i p i t a t i o n s w i t h cetyltrimethylammonium bromide. The p u r i f i e d p o l y s a c c h a r i d e (0.95 g p e r l i t r e o f medium) was shown to be homogeneous by gel-chromatography (see Appendix I I ) I t s m o l e c u l a r mass was e s t i m a t e d as 3 x 10~ 6 d a l t o n s , and i t had [ a ] D +18° (c 0.91, w a t e r ) . N.m.r. s p e c t r o s c o p y (^C and ^H) was performed on the o r i g i n a l m a t e r i a l , 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 r e c o r d e d i n T a b l e I V . l . I I . A n a l y s i s o f K79 p o l y s a c c h a r i d e - A sample (10 mg) o f K79 80 -p o l y s a c c h a r i d e was h y d r o l y z e d w i t h 2M t r i f l u o r o a c e t i c a c i d f o r 18 h on a steam b a t h . A f t e r removal o f the a c i d by r e p e a t e d c o e v a p o r a t i o n w i t h water, paper chromatographic a n a l y s e s ( s o l v e n t s 1 and 3) o f the h y d r o l y -z a t e showed D - g a l a c t o s e , D-glucose, L-rhamnose and an a l d o b i o u r o n i c a c i d . The r e s u l t s o f the g . l . c . a n a l y s i s , on column A, o f the sugars as t h e i r a l d i t o l a c e t a t e s are shown i n T a b l e IV.1.I, column I. R e d u c t i o n of K79 p o l y s a c c h a r i d e - F o l l o w i n g the p u b l i s h e d p r o c e d u r e ^ , K79 p o l y s a c c h a r i d e (1.0 g) was r e d u c e d u s i n g 1 - c y c l o h e x y l -3 - ( 2 - m o r p h o l i n o e t h y l ) - c a r b o d i i m i d e metho-p_-toluenesulfonate (4 g) and aqueous sodium b o r o h y d r i d e (3M, 250 mL). The n e u t r a l p o l y s a c c h a r i d e (960 mg) o b t a i n e d a f t e r two t r e a t m e n t s had the c o m p o s i t i o n shown i n T a b l e I V . 1 . I , column I I . P r e p a r a t i v e g . l . c . (column D), f o l l o w e d by measurements o f the c i r c u l a r d i c h r o i s m s p e c t r a x U U , showed the g l u c i t o l h e x a a c e t a t e to be o f the D c o n f i g u r a t i o n , and the r h a m n i t o l p e n t a a c e t a t e to be o f the L c o n f i g u r a t i o n . M e t h y l a t i o n a n a l y s i s - A sample o f K79 p o l y s a c c h a r i d e i n the f r e e a c i d form (96 mg), o b t a i n e d by p a s s i n g the aqueous s o l u t i o n through a column o f A m b e r l i t e IR-120 (H +) r e s i n , was m e t h y l a t e d by the Hakomori 119 11^ p r o c e d u r e x , d ' • L i . The p r o d u c t , r e c o v e r e d a f t e r d i a l y s i s and p u r i f i e d by e x t r a c t i o n w i t h d i c h l o r o m e t h a n e and passage through a column o f Sephadex LH-20, showed complete m e t h y l a t i o n (no h y d r o x y l a b s o r p t i o n i n the i . r . spectrum). One p a r t (30 mg) was h y d r o l y z e d (2M TFA, 20 h, 95°) a f t e r 81 which paper chromatography i n s o l v e n t 4 r e v e a l e d f i v e components; 2, 3 , 4 , 6 - t e t r a - O - m e t h y l g l u c o s e (ExMG 1 • 0 u > pi- nk spot) ; 2,4- and 3,4-di-O-methylrhamnoses ( E T M G 0.85, g r e e n i s h brown s p o t ) ; 2 , 3 , 4 - t r i - O - m e t h y l -g l u c o s e (SxMG 0.83, b r o w n i s h p i n k s p o t ) ; 2 , 4 , 6 - t r i - O - m e t h y l g a l a c t o s e (SXMG O-^ 1' P i n ^ s p o t ) ; and a m e t h y l a t e d a l d o b i o u r o n i c a c i d ( E T M G 0.50, p i n k s p o t ) . A n a l y t i c a l f i g u r e s f o r the n e u t r a l sugars a r e p r e s e n t e d i n T a b l e I V . 1 . I l l , column I. A second p o r t i o n of the m e t h y l a t e d p o l y s a c -c h a r i d e (60 mg) was r e d u c e d w i t h l i t h i u m aluminum h y d r i d e i n r e f l u x i n g o x o l a n e . The p r o d u c t was d i v i d e d i n t o two e q u a l p a r t s , one o f which was h y d r o l y z e d and a n a l y z e d w i t h the r e s u l t s shown i n T a b l e I V . 1 . I l l , column I I . The paper chromatograms o f t h i s h y d r o l y z a t e u s i n g s o l v e n t 4 showed the d i s a p p e a r a n c e o f the s p o t a t E T M G 0.50 and the a d d i t i o n a l appearance of 2 - 0 - methylglucose ( E T M G 0-26, p i n k ) . The o t h e r h a l f o f the m e t h y l a t e d r e d u c e d p o l y s a c c h a r i d e was r e m e t h y l a t e d 1 ' ^ > a n c j h y d r o l y z e d whereupon paper chromatography showed the d i s a p p e a r a n c e o f the s p o t a t R.TMG 0.26 and the appearance o f 2 , 6 - d i - 0 - m e t h y l g l u c o s e ( E T M G 0.56). The g . l . c . r e s u l t s a r e g i v e n i n T a b l e I V . 1 . I l l , column I I I . G . l . c . - m . s . a n a l y s e s were co n d u c t e d w i t h columns E and F. 1 , 3 , 5 - T r i - 0 - a c e t y l - 2 , 4 , 6 -t r i - O - m e t h y l g a l a c t i t o l was i s o l a t e d by p r e p a r a t i v e g . l . c . (column D) and found to g i v e a p o s i t i v e c.d. c u r v e , i n d i c a t i n g t h a t the g a l a c t o s e had the D c o n f i g u r a t i o n 1 0 0 . P e r i o d a t e o x i d a t i o n ± y ' and Smith d e g r a d a t i o n - ^ 0 > i y ° o f n e u t r a l K79 p o l y s a c c h a r i d e - A sample o f the c a r b o d i i m i d e - r e d u c e d K79 p o l y s a c c h a -r i d e (20 mg) was d i s s o l v e d i n water (5 mL) and sodium m e t a p e r i o d a t e - 82 -s o l u t i o n (0.03M, 5 mL) was added. The r e a c t i o n was c o n d u c t e d a t room temperature and i n the dark. A l i q u o t s (0.1 mL) were withdrawn p e r i o d -i c a l l y , d i l u t e d 250 times w i t h water, and the a b s o r b a n c e s 1 9 7 o f the r e s u l t i n g s o l u t i o n s were measured i n a G i l f o r d s p e c t r o p h o t o m e t e r , model 240 a t 223 ran. The p e r i o d a t e consumption r e a c h e d a p l a t e a u a f t e r about 50 h and a p p r o x i m a t e l y 0.08 mmoles IO4" had been consumed, e q u i v a l e n t t o 5 moles o f IO4" p e r mole o f r e p e a t i n g u n i t . E t h y l e n e g l y c o l (0.2 mL) was added t o d e s t r o y the excess p e r i o d a t e , the p o l y a l d e h y d e was r e d u c e d w i t h sodium b o r o h y d r i d e , and the s o l u t i o n was n e u t r a l i z e d w i t h a c e t i c a c i d ( 5 0 % ) , d i a l y z e d and l y o p h i l i z e d t o y i e l d the p o l y o l (12 mg). Sugar and m e t h y l a t i o n a n a l y s e s o f the p o l y o l gave the r e s u l t s shown r e s p e c t i v e l y i n T a b l e I V . 1 . I , column I I I and T a b l e I V . 1 . I l l , column IV. A s o l u t i o n o f c a r b o d i i m i d e - r e d u c e d K79 p o l y s a c c h a r i d e (520 mg) i n water (200 mL) was mixed w i t h sodium m e t a p e r i o d a t e s o l u t i o n (60 mL, 0.5M) and k e p t i n the dark a t room temperature. A f t e r 3 d, the excess p e r i o d a t e was d e s t r o y e d by the a d d i t i o n o f e t h y l e n e g l y c o l (10 mL). A f t e r d i a l y s i s f o r 1 d, the l y o p h i l i z e d p o l y a l d e h y d e , r e d i s s o l v e d i n water, was r e d u c e d to the p o l y o l w i t h sodium b o r o h y d r i d e , and the s o l u t i o n was n e u t r a l i z e d w i t h a c e t i c a c i d ( 5 0 % ) , d i a l y z e d , and l y o p h i -l i z e d . A second t r e a t m e n t was conducted, and a f t e r s i m i l a r workup the p o l y o l was r e c o v e r e d ( y i e l d 400 mg). A sample o f the p o l y o l (360 mg) was s u b j e c t e d t o s e l e c t i v e h y r o l y -s i s w i t h 0.5M TFA f o r 24 h a t room temperature. Paper c h r o m a t o g r a p h i c a n a l y s i s o f the h y d r o l y z a t e i n s o l v e n t system 1 (41 h) gave a s p o t a t R.Glc 0.74. No monosaccharides were o b s e r v e d . S e p a r a t i o n by p r e p a r a t i v e paper chromatography u s i n g s o l v e n t 1 (2 d) gave a t e t r a s a c c h a r i d e , 1 - 83 -(120 mg). Sugar and m e t h y l a t i o n a n a l y s e s on 1 gave the r e s u l t s as shown r e s p e c t i v e l y i n T a b l e IV.1.I, column IV and T a b l e I V . 1 . I l l , column V. Fo r n.m.r. a n a l y s e s , see T a b l e I V . l . I I . A sample o f 1 (23 mg) was m e t h y l a t e d by the Hakomori p r o c e d u r e 1 1 2 , 1 1 3 and the p r o d u c t was a n a l y z e d by c h e m i c a l - i o n i z a t i o n (methane) m.s. 2^. The spectrum showed peaks a t m/z 189, 363, 567, 771 and 890 (see mass spectrum numbered 1 i n Appendix I V ) . U r o n i c a c i d d e g r a d a t i o n ^ " 1 - M e t h y l a t e d K79 p o l y s a c c h a r i d e (70 mg) d r i e d i n vacuo. was d i s s o l v e d i n a m i x t u r e o f 19:1 d i m e t h y l s u l f o x i d e - 2 , 2 - d i m e t h o x y p r o p a n e (20 mL) t o g e t h e r w i t h a t r a c e o f p.-to-l u e n e s u l f o n i c a c i d and s t i r r e d under n i t r o g e n . Sodium m e t h y l s u l f i n y l -methanide (2M, 6 mL) was added and the r e a c t i o n m i x t u r e was l e f t s t i r -r i n g f o r 18 h a t room temperature. M e t h y l i o d i d e was added t o the f r o z e n m i x t u r e and i t was s t i r r e d f o r a n o t h e r 7 h a t room temperature, t h e n the e x c e s s base was n e u t r a l i z e d by a d d i n g 50% a c e t i c a c i d . The m e t h y l a t e d , degraded p r o d u c t was r e c o v e r e d by p a r t i t i o n between d i c h l o -romethane and water (3 x 20 mL). A p o r t i o n o f t h i s p r o d u c t was hydro-o l y z e d (2M TFA, 6 h, 95.) and the sugars r e l e a s e d were a n a l y z e d as d e s c r i b e d p r e v i o u s l y f o r the m e t h y l a t i o n a n a l y s i s ( T a b l e I V . 1 . I l l , column V I ) . The f r a c t i o n a t i o n o f the r e s t o f the m e t h y l a t e d , degraded p r o d u c t on a Sephadex LH-20 column (1.2 x 50 cm), u s i n g 1:1 c h l o r o f o r m - - m e t h a n o l as e l u a n t , gave two main f r a c t i o n s . The f i r s t f r a c t i o n was deduced to be p o l y m e r i c as e v i d e n c e d by ^H-n.m.r., t . l . c . and sugar a n a l y s e s (as - 84 t h e i r p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s ) . The second f r a c t i o n was c o n s i d e r e d to be a m e t h y l a t e d t e t r a s a c c h a r i d e 2 on the b a s i s o f ^•H-n.m.r. ( T a b l e I V . l . I I ) and g . l . c . d a t a ( T a b l e I V . 1 . I l l , column V I I ) . C h e m i c a l i o n i z a t i o n , (methane) m.s.^ 0 0 o f 2 gave a spectrum showing peaks a t m/z 189, 363, 537, 741 and 758 (see mass spectrum numbered 2 i n Appendix I V ) . Chromic a c i d o x i d a t i o n 1 " - To a s o l u t i o n o f the o r i g i n a l K79 p o l y s a c c h a r i d e (15 mg) i n formamide (5 mL) were added a c e t i c a n h y d r i d e (4 mL) and p y r i d i n e (4 mL), and the m i x t u r e was s t i r r e d a t room tempera-t u r e f o r 36 h. F o l l o w i n g d i a l y s i s f o r 2 d, the r e t e n t a t e was f r e e z e -d r i e d , and the a c e t y l a t e d 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 a second a c e t y l a t i o n t r e a t m e n t . A f t e r s i m i l a r workup, the 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 was d i s s o l v e d i n g l a c i a l a c e t i c a c i d (3.5 mL) ; CrC>3 (100 mg) was added and the m i x t u r e was s t i r r e d f o r 20 h a t room temperature. The p r o d u c t , i s o l a t e d by p a r t i t i o n between c h l o r o f o r m and water, was ana-l y z e d as a l d i t o l a c e t a t e s (see T a b l e IV.1.I, column V ) . Some c a r b o d i i m i d e - r e d u c e d K79 p o l y s a c c h a r i d e (95 mg) was a c e t y l a t e d as d e s c r i b e d above. Complete a c e t y l a t i o n was checked by i . r . s p e c t r o -scopy (absence o f h y d r o x y l group between 3650 to 3000 cm" 1). A f t e r passage t h r o u g h a Sephadex LH-20 column (acetone as s o l v e n t ) , the 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 was d i s s o l v e d i n g l a c i a l a c e t i c a c i d (25 mL), Cr03 (280 mg) was added, and the m i x t u r e was s t i r r e d a t room temperature f o r 27 h. The p r o d u c t , i s o l a t e d by p a r t i t i o n between c h l o r o f o r m and water, was a n a l y z e d as f o l l o w s . (a) Sugar a n a l y s i s : A 85 p o r t i o n o f the p r o d u c t (10 mg) was h y d r o l y z e d (2M TFA), reduced w i t h sodium b o r o h y d r i d e and a c e t y l a t e d w i t h 1:1 a c e t i c a n h y d r i d e - p y r i d i n e (see S e c t i o n I I I . 3 . 2 f o r g e n e r a l e x p e r i m e n t a l c o n d i t i o n s ) . See T a b l e IV.1.I, column VI f o r g . l . c . a n a l y s i s . (b) M e t h y l a t i o n a n a l y s i s : A p o r t i o n o f the p r o d u c t (15 mg) was m e t h y l a t e d by the Hakomori p r o c e -dure-1--1-^' -L-LJ, h y d r o l y z e d (2M TFA), r e d u c e d w i t h sodium b o r o h y d r i d e and a c e t y l a t e d t o g i v e p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s (see S e c t i o n I I I . 4 f o r g e n e r a l e x p e r i m e n t a l c o n d i t i o n s ) . F o r the g . l . c . a n a l y s i s , see T a b l e I V . 1 . I l l , column V I I I . The remainder o f the chromic a c i d - o x i d i z e d p r o d u c t (35 mg) was d e a c e t y l a t e d by s t i r r i n g i n sodium h y d r o x i d e (0.5M) s o l u t i o n f o r 26 h a t room temperature, and then reduced w i t h sodium b o r o h y d r i d e . Paper chromatography o f the p r o d u c t , a f t e r passage t h r o u g h A m b e r l i t e IR-120 (H +) r e s i n and removal o f the b o r a t e by c o e v a p o r a t i o n w i t h methanol, showed two predominant s p o t s (Sgi c 1.17 and RQXC 0-24 o r E l a c t o s e 1-0). S e p a r a t i o n by p r e p a r a t i v e paper chromatography ( s o l v e n t 2) gave two p r o d u c t s , 3 (4 mg) and 4 (5 mg). The c h a r a c t e r i z a t i o n o f 3 as a rh a m n o t r i o s e by ^H-n.m.r. a n a l y s i s i s d e s c r i b e d i n the R e s u l t s s e c t i o n . 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 K79 p o l y s a c c h a r i d e (630 mg) i n 0.025M H2SO4 was h e a t e d f o r 1.5 h on a steam b a t h . A f t e r n e u t r a l i z i n g w i t h PbC03 and c e n t r i f u g a t i o n , the s u p e r n a t a n t was d i a l y z e d (mol. wt. c u t o f f 3400) 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 z a b l e m a t e r i a l (480 mg) 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 (0.5M TFA, 95°, 1.5 h ) , f o l l o w e d by d i a l y s i s . The d i a l y z a b l e m a t e r i a l - 86 -from the h y d r o l y s e s (620 mg) was p a s s e d through a column o f A m b e r l i t e IR 120 ( H + ) r e s i n , a f t e r which paper chromatography r e v e a l e d the p r e s e n c e o f monosaccharides ( g a l a c t o s e , g l u c o s e and rhamnose) and f o u r o l i g o s a c -c h a r i d e s . P r e p a r a t i v e paper chromatography ( s o l v e n t 2) o f the h y d r o l y -z a t e gave f o u r compounds. Compound 5 (12 mg, E l a c t o s e 0-98), which was i s o l a t e d pure, was o f the most i n t e r e s t because i t s m o b i l i t y i s compara-b l e to t h a t o f i s o m a l t o s e . However, 5 i s deduced to be an a c i d i c t r i s a c c h a r i d e on the b a s i s o f i t s n.m.r. ( T a b l e I V . l . I I ) , sugar a n a l y s i s on the c a r b o x y l - r e d u c e d 7 5 p r o d u c t ( T a b l e IV.1.I, column V I I ) , and m e t h y l a t i o n a n a l y s i s w i t h r e d u c t i o n o f the u r o n i c e s t e r ( T a b l e I V . 1 . I l l , column I X ) . ^H-N.m.r. a n a l y s e s o f the o t h e r t h r e e o l i g o s a c c h a r i d e s r e v e a l e d t h a t the samples were n o t homogeneous and hence were n o t f u r t h e r examined. - 87 -IV.2 STRUCTURAL INVESTIGATION OF THE CAPSULAR POLYSACCHARIDE OF K l e b s i e l l a SEROTYPE K35 IV.2.1 A b s t r a c t The s t r u c t u r e o f the c a p s u l a r p o l y s a c c h a r i d e (K a n t i g e n ) o f K l e b s i e l l a K35 has been e s t a b l i s h e d as h a v i n g the p e n t a s a c c h a r i d e r e p e a t i n g u n i t shown ("four p l u s one" t y p e ) . The s t r u c t u r a l i n v e s t i g a -t i o n u t i l i z e d the t e c h n i q u e s o f m e t h y l a t i o n , / ^ - e l i m i n a t i o n , Smith d e g r a d a t i o n and p a r t i a l h y d r o l y s i s . N.m.r. s p e c t r o s c o p y ( 1H and -^C) was u s e d e x t e n s i v e l y t o e s t a b l i s h the c o n f i g u r a t i o n s o f the anomeric l i n k a g e s and to d e l i n e a t e the sequence o f the sugars i n the s t r u c t u r e o f the p o l y s a c c h a r i d e . •3) - Q-D-Galp- (1—3) -a-D-Manp-(1-3)-a-D-Manp-(1-3)-£-D-Glcp-(1-6 4 2 \ / t C 1 / \ £-D-GlcAp_ H 3C C0 2H IV.2.2 I n t r o d u c t i o n K l e b s i e l l a s e r o t y p e K35 i s one o f 17 s t r a i n s whose c a p s u l a r p o l y s a c c h a r i d e s a r e composed o f D - g l u c u r o n i c a c i d , D - g a l a c t o s e , D-glu-cose and D-mannose^ 5>26 E i g h t o f these p o l y s a c c h a r i d e s have 1-carboxy-88 e t h y l i d e n e s u b s t i t u e n t s , and i n t h i s subgroup, the s t r u c t u r e s of K l e b s i e l l a K7, K13, K26, K30, K31, K46 and K69 have been e s t a b l i s h e d 6 . As p a r t o f our c o n t i n u i n g s t u d y to e x p l a i n the s e r o l o g y and immuno-c h e m i s t r y o f the c a p s u l a r p o l y s a c c h a r i d e s o f t h i s genus on a s t r u c t u r a l b a s i s , we now r e p o r t our r e s u l t s on K35, the e i g h t h member o f t h i s sub group. IV. 2 . 3 R e s u l t s and d i s c u s s i o n C o m p o s i t i o n and n.m.r. s p e c t r a - K l e b s i e l l a K35 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 p u r i f i e d by two p r e c i p i t a t i o n s w i t h c e t y l t r i m e t h y l a m m o n i u m b r o m i d e 1 8 4 . The p r o d u c t was monodisperse by g e l - p e r m e a t i o n chromatography (M r = 9 x 10 6, see e l u t i o n p r o f i l e i n Appendix I I ) and had +66°, which compares r e a s o n a b l y w e l l w i t h the v a l u e o f +59° c a l c u l a t e d by u s i n g Hudson's r u l e s o f i s o r o t a t i o n 9 ^ . The p r e s e n c e o f g a l a c t o s e , g l u c o s e , mannose and an a l d o b i o u r o n i c a c i d i n the a c i d h y d r o l y z a t e o f the p o l y s a c c h a r i d e was o b s e r v e d by paper chromatography. G . l . c . a n a l y s i s o f t h i s same h y d r o l y -z a t e gave the molar r a t i o o f g a l a c t o s e : g l u c o s e r m a n n o s e as 1:00:1.08:1.03 (see T a b l e IV.2.I, column I ) . When the u r o n i c a c i d was reduced, f o l l o w -i n g m e t h a n o l y s i s 7 ^ , the molar p r o p o r t i o n s o f g l u c o s e and mannose i n c r e a s e d (see T a b l e IV.2.I, column I I ) . T h i s i n d i c a t e d t h a t the g l u c u r o n i c a c i d i s l i n k e d to a mannosyl u n i t , which was l a t e r c o n f i r m e d by a / 3 - e l i m i n a t i o n r e a c t i o n ^ u x and the i s o l a t i o n o f an a l d o b i o u r o n i c a c i d . The i n c r e a s e i n g l u c o s e and mannose was l e s s t h a n expected, 89 -T a b l e I V . 2 . I : S u g a r A n a l y s i s o f K l e b s i e l l a K 3 5 P o l y s a c c h a r i d e a n d D e r i v e d P r o d u c t s S u g a r M o l a r r a t i o s 5 ' ( a s a l d i t o l a c e t a t e ) I 5 I I I I I I V V G a l a c t o s e 1 . 0 0 1 . 0 0 1 . 1 4 0 . 9 3 1 . 0 0 G l u c o s e 1 . 0 8 1 . 2 1 1 . 0 0 1 . 0 0 M a n n o s e 1 . 0 3 1 . 4 0 2 . 0 7 0 . 9 3 0 . 9 8 U s i n g a n S P - 2 3 4 0 c o l u m n p r o g r a m m e d f r o m 1 9 5 ° f o r 4 m i n , a n d t h e n a t 2 ° / m i n t o 2 6 0 ° . I , o r i g i n a l a c i d i c K 3 5 p o l y s a c c h a r i d e ; I I , c a r b o x y l - r e d u c e d p o l y s a c c h a r i d e ; I I I , S m i t h - d e g r a d e d p o l y s a c c h a r i d e ; I V , c a r b o x y l -r e d u c e d t r i s a c c h a r i d e ; V , n e u t r a l d i s a c c h a r i d e . presumably due to in c o m p l e t e r e d u c t i o n o f the u r o n i c a c i d . A composi-t i o n o f Gal-.Man: G l c : G l c A o f 1:2:1:1 was, however, c o r r o b o r a t e d b o t h by n.m.r. s p e c t r o s c o p y and by m e t h y l a t i o n a n a l y s i s . The xH-n.m.r. spectrum o f the p o l y s a c c h a r i d e i n d i c a t e d ( F i g . IV.2.Ia) the p r e s e n c e o f f i v e anomeric p r o t o n s , c o r r e s p o n d i n g t o t h r e e a- and two / ? - l i n k a g e s x 9 6 ; a l s o , one 1 - c a r b o x y e t h y l i d e n e a c e t a l 1 2 4 per r e p e a t i n g u n i t was d e t e c t e d . These r e s u l t s were f u r t h e r s u b s t a n t i a t e d by the x 3C-n.m.r. s p e c t r u m 1 - ^ (see F i g u r e I V . 2 . I I a and T a b l e I V . 2 . I I ) . C i r c u l a r d i c h r o i s m measure-m e n t s 1 ^ on the a l d i t o l a c e t a t e s showed g l u c u r o n i c a c i d , g l u c o s e and mannose to have the D - c o n f i g u r a t i o n . G a l a c t o s e and the b r a n c h - p o i n t - 90 -Table IV.2.II: N.m.r. data f o r K l e b s i e l l a K35 capsular polysaccharide and derived oligosaccharide. Compound' •H " C t* hi Integral Assignment* 9 Assignment Ip.pm.) (Hz) (no. ofH) and symbol" (ppm.) and symbol* C I c A - ^ M a n P 5.30 % 0.73 —Man— a 102.51 G l c A ? (A2) 4.95 i 0.27 ~ M i n J 94.55 - ? - M a n ^ G l c A - ^ M a o-Licic P o 4.54 8 1.00 G l c A j 92.90 —Man— a 5.35 > 1.00 —Man— a 102.44 G l c A ? (A3) 5.24 3 0.40 a 99.75 —Man— a 4 6 6 8 0.60 96.80 A G I C J 4.54 8 1.00 G l c A T 93.14 i c . c -a Gal-J——Man a 5.29 > 1.00 G a t -or <N:» 5.17 t 066 —Man— o 4.91 % 0.34 ~ M a r > J 3 . , 1 3 . , 1 3 . . 1 3 _ , I . „ Gal Man Man Glc—s- 5.38 6 4 ° " " * A H j C C O O H (SO) 5.25 5.15 4.76 1.59 1.00 1.00 1.00 1.00 3.00 6 4 " A H , C C O O H -^ Man— a -^ Man— a [DI 104.85 IE] 102.89 101.84 101.54 25.84 •iWan— a - i G a l — 6 4 ° A H , C C O O H a CHjOfpyr IA] |C] ID] IE) IF] (DP2) 5.37 5.15 4.74 4.54 2.00 1.00 1.00 1.00 — M a n — —Man— a GlcA-s-[D] IE] IB] 104.40 103.32 IC) 102.89 | A] 101.35 99.38 G k A j -'-Man— a 2 -2-Man— 21 " |A'J IB] IC] |D'] IE) - 91 Compound' 'H >>C (p.p.m.) (Hz) Integral Assignment* (no. ofH) and symbol1 (p.p.m.) Assignment and symbol* C-6 of hexosct 3 . , 1 3 u 1 3 . . 1 3 _ . 1 . , , — G a l Man Man Glc——• 5.37 6 4 ° " 2 " fi X » H j C C O O H 1 (33%) GIcA ( D P I ) 5.15 2.00 4.73 3 _ ,1 3 , , 1 3 . . 1 3 _ . Gal Man Man——Glc-6 4 ° • 2 -A * H j C C O O H 1 Glc A KJ5 polysaccharide ID.D') 104.89 1.00 1.00 i-Man— —Man— a |E] ICJ 104.54 103.50 103.12 |A.A<) 101.78 c - L l o . , 6 4 X H j C C O O H |A] 2 - G k - L 2 - G a l P G l c A -—Man— a 3, 6 4 " H 3 C C O O H | A ' l IB] IC] [D] 4.56 7 1.00 G k A - IB] 101.38 -2-Gal— a |D' 1.58 t 1.00 CHjOfpyr IF] 99.51 —Man— 2 | ° IE) 25.83 CHjOf pyr IH 5.36 b 2.00 •^Gal— ID) 173.73 C - O o f G I c A 6 4 ° A H j C C O O H —Man— » l * IE] 172.85 C - O o f pyr 5.14 2 1.00 -^-Man— a |C] 104.87 IA] 4.74 7 1.00 ±G\cj |A] 103.45 G l c A - IB] 4.57 7 1.00 G l c A - IB] 103.05 -^Man— o IC] 1.58 t 3.00 CHjOf pyr 1" 101.84 -5-Gal— ID] 6 4 ° HjC C O O H 99 43 —Man— (El 25.81 I C H j of pyr {K config) IF] 92 a For the o r i g i n of compounds A2, A3, N2, SD, DP2, and DPI, see text. b Chemical s h i f t downfield from sodium-4,4-dimethyl-4-silapentane-l-sulfonate (DSS). c Key: b - broad, unable to assign accurate coupling constant; s - s i n g l e t . d For example -Man—jr— r e f e r s to the anomeric proton of a 3-linked mannosyl residue i n the Q-anomeric c o n f i g u r a t i o n . The absence of a numerical p r e f i x i n d i c a t e s a (terminal) nonreducing group. e See Fig s . 1 and 3. f Chemical s h i f t i n p.p.m. downfield from DSS. 8 As f o r d, but f o r anomeric 1 3 C n u c l e i . See Fig s . 2 and 3. See Appendix 111 for a l l n.m.r. spectra. - 93 -mannose were shown to po s s e s s the D - c o n f i g u r a t i o n by the c i r c u l a r d i c h r o i s m c u r v e s o f t h e i r 2 , 4 , 6 - t r i - 0 - m e t h y l and 4,6-di-0-methyl d e r i v a t i v e s 1 0 0 r e s p e c t i v e l y . M e t h y l a t i o n a n a l y s e s /> 1 1' 3 - A n a l y s e s were conducted ( i ) on the o r i g i n a l p o l y s a c c h a r i d e , ( i i ) a f t e r r e d u c t i o n o f the u r o n i c e s t e r f o l l o w i n g m e t h y l a t i o n , ( i i i ) a f t e r r e m e t h y l a t i o n o f p r o d u c t ( i i ) , and ( i v ) on the 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 t h r e d u c t i o n o f the u r o n i c e s t e r f o l l o w i n g m e t h y l a t i o n . The r e s u l t s , p r e s e n t e d i n T a b l e I V . 2 . I l l , columns I to IV, c o n f i r m the concept o f a p e n t a s a c c h a r i d e r e p e a t i n g u n i t and show t h a t : ( i ) the b r a n c h p o i n t i s a mannosyl r e s i d u e l i n k e d a t 0-2 and 0-3, ( i i ) the g l u c u r o n i c a c i d i s a t e r m i n a l , n o n - r e d u c i n g group, and ( i i i ) the 1 - c a r b o x y e t h y l i d e n e a c e t a l i s l i n k e d a t 0-4 and 0-6 of the g a l a c t o s y l r e s i d u e (see Scheme I V . 2 . I ) . A / 3 - e l i m i n a t i o n exper-201 iment"'0-1- demonstrated t h a t the u r o n i c a c i d i s d i r e c t l y l i n k e d t o the mannose a t 0-2 (see T a b l e I V . 2 . I l l , column V ) . Smith d e g r a d a t i o n 1 ^ 1 3 ' i y ° - P e r i o d a t e o x i d a t i o n o f the n a t i v e p o l y s a c c h a r i d e was f o l l o w e d by r e d u c t i o n and s e l e c t i v e h y d r o l y s i s w i t h d i l u t e a c i d . The r e c o v e r e d m a t e r i a l SD was p o l y m e r i c , c o n f i r m i n g t h a t the u r o n i c a c i d i s l a t e r a l l y a t t a c h e d , and the n.m.r. s p e c t r a (see T a b l e IV. 2. I I ) showed the d i s a p p e a r a n c e o f a /3-signal a t 6 4.56 (J-j^ 2 1 Hz) 1 and a t 103.45 p.p.m. ( i J C ) . The f u l l r e t e n t i o n o f the p y r u v i c a c i d m e thyl s i g n a l a t 6 1.59 i n the ^H-n.m.r., i n d i c a t e s the s t a b i l i t y to - 94 -T a b l e I V . 2 . I l l : M e t h y l a t i o n A n a l y s e s o f K l e b s i e l l a K35 P o l y s a c c h a r i d e and D e r i v e d P r o d u c t s M e t h y l a t e d s u g a r 3 M o l e % b ' c (as a l d i t o l a c e t a t e ) I I I I I I IV V VI V I I V I I I IX 2,3,4,6-Gal 52 2,4,6-Gal 20 2-Gal 24 18 19 - 23 22 -2,3,4,6-Glc 21 2,4,6-Glc 26 21 21 20 25 27 - 33 -2,3,4-Glc 21 - 20 - - 44 34 -4,6-Man 24 20 19 20 -3,4,6-Man 56 33 2,4,6-Man 26 20 20 20 54 51 - - 48 a 2,3,4,6-Glc - 1.5-d i - O - a c e t y l - 2 , 3 , 4 , 6 - t e t r a - O - m e t h y l g l u c i t o l , e t c . k U s i n g an SP-1000 column a t 220° i s o t h e r m a l . V a l u e s a r e c o r r e c t e d by use o f the e f f e c t i v e , c a r b o n - r e s p o n s e f a c t o r s g i v e n by A l b e r s h e i m e t a l 2 0 3 . -c I, o r i g i n a l a c i d i c p o l y s a c c h a r i d e ; I I , u r o n i c e s t e r r e d u c e d a f t e r m e t h y l a t i o n ; I I I , r e m e t h y l a t i o n a f t e r r e d u c t i o n o f u r o n i c e s t e r ; IV, 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 t h u r o n i c e s t e r r e d u c e d a f t e r m e t h y l a t i o n ; V, p r o d u c t from / 9 - e l i m i n a t i o n and r e m e t h y l a t i o n ; VI, Smith-degraded p o l y s a c c h a r i d e ; V I I , m e t h y l a t e d and reduced d i s a c -c h a r i d e A2; V I I I , m e t h y l a t e d and r e d u c e d t r i s a c c h a r i d e A3; IX, n e u t r a l d i s a c c h a r i d e N2. a c i d o f the p y r u v i c a c i d a c e t a l l i n k e d t o p o s i t i o n s 4 and 6 of the g a l a c t o s e u n i t . T h i s ; i s i n agreement w i t h the d i f f i c u l t y e n c o untered d u r i n g d e p y r u v y l a t i o n (see E x p e r i m e n t a l ) . In c o n t r a s t , the a c y c l i c a c e t a l p r o d u c t o b t a i n e d by r e d u c t i o n f o l l o w i n g p e r i o d a t e o x i d a t i o n o f - 95 -K l e b s i e l l a K35 POLYSACCHARIDE 1. HAXOMOKI KtTHYUTlON a. SODIUM DWSTL/DHSO k. CHjI DtfTXlVTLATTE U S K U U C U U I 2. 2M TFA. 95 , 17h BO •VLTT K UVTHTH) «SJ raLTIACCBAaUDK 1 . NaBH, REDUCTION 2 . Ac.O/PTRIDINI J. C.L.CrK.S. WHERE X - OMe O CJ W S c h e m e I V . 2 . I : M e t h y l a t i o n a n a l y s i s o f t h e n a t i v e a n d d e p y r u v y l a t e d K l e b s i e l l a K35 c a p s u l a r p o l y s a c c h a r i d e . - 96 the p o l y s a c c h a r i d e was h i g h l y s u s c e p t i b l e t o h y d r o l y s i s w i t h d i l u t e a c i d . Sugar a n a l y s i s o f SD showed o n l y n e u t r a l sugars (see T a b l e I V . 2 . I , column I I I ) , whereas the m e t h y l a t i o n a n a l y s i s gave r e s u l t s i d e n t i c a l to those o b t a i n e d by / ^ - e l i m i n a t i o n (see T a b l e I V . 2 . I l l , column V I ) . P a r t i a l h y d r o l y s i s - P a r t i a l h y d r o 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 w i t h a c i d was f o l l o w e d by s e p a r a t i o n o f the a c i d i c and n e u t r a l f r a c t i o n s v i a ion-exchange chromatography. The n e u t r a l f r a c t i o n con-t a i n e d p r e d o m i n a n t l y monosaccharides and a d i s a c c h a r i d e N2 which was s e p a r a t e d by paper chromatography. S e p a r a t i o n o f the a c i d i c f r a c t i o n , by paper chromatography, y i e l d e d t h r e e o l i g o s a c c h a r i d e s A2, A3 and A4 (which was i n d i c a t e d to be a m i x t u r e o f o l i g o s a c c h a r i d e s ) . On the b a s i s o f t h e i r n.m.r. s p e c t r a l d a t a (see T a b l e I I ) and t h e i r m e t h y l a t i o n a n a l y s e s (see T a b l e I V . 2 . I l l , columns V I I to I X ) , the s t r u c t u r e s o f these compounds were shown to be as f o l l o w s : £-GlcA-(l-2)-Man A2 /3-GlcA- (1-2)-a-Man-(1-3)-Glc A3 a-Gal-(1-3)-Man N2 The sum o f these experiments e s t a b l i s h e s the s t r u c t u r e o f the K l e b s i e l l a K35 p o l y s a c c h a r i d e as 97 D C E A »3)-a-D-Galp-(1-3)-a-D-Manp-(1-3)-a-D-Manp-(1-3)-0-D-Glcp-(1-6 4 2 \ / t C 1 / \ /3-D-GlcAp. H 3C C0 2H B F which i s c o n s i s t e n t w i t h the n.m.r. s p e c t r a o b t a i n e d . The i n d i v i d u a l sugar r e s i d u e s i n the s t r u c t u r e above have been d e s i g n a t e d by the l e t t e r s A-F to f a c i l i t a t e d i s c u s s i o n o f these s p e c t r a i n the f o l l o w i n g s e c t i o n . N.m.r. stu d y on o r i g i n a l , d e p y r u v y l a t e d and Smith-degraded p o l y -s a c c h a r i d e s - The n u m e r i c a l n.m.r. d a t a f o r these p o l y s a c c h a r i d e s are assembled i n T a b l e I V . 2 . I I , and the d i f f e r e n t s p e c t r a a r e compared s c h e m a t i c a l l y i n F i g u r e s IV.2.I and I V . 2 . I I , where the h e i g h t o f each v e r t i c a l l i n e i s p r o p o r t i o n a l to the number o f p r o t o n s or c a r b o n atoms. From the c h e m i c a l s h i f t o f the methyl group o f the a c e t a l (F, F i g u r e s IV.2.Ia and I V . 2 . I I a ) , i t was p o s s i b l e to a s s i g n the R c o n f i g u r a t i o n to the a c e t a l c a r b o n atom. I t has been s h o w n 1 2 ^ t h a t the c h e m i c a l s h i f t s f o r the methyl groups o f the a c e t a l d i f f e r a c c o r d i n g t o whether these groups a r e a x i a l o r e q u a t o r i a l . The d i f f e r e n c e i s e s p e c i a l l y pronounced i n s p e c t r a , where the v a l u e s a r e -18 p.p.m. f o r the a x i a l methyl groups and -26 p.p.m. f o r the e q u a t o r i a l 1 2 ^ . P a r t i a l d e p y r u v y l a t i o n o f a p o l y - or o l i g o - s a c c h a r i d e may l e a d to the t w i n n i n g o f c e r t a i n s i g n a l s 1 6 ^ . M i l d h y d r o l y s i s o f K35 p o l y s a c c h a -- 98 --E I-(d)SD D E C D+E 540 500 4.50 (C) DP 2 C A B 540 500 4.50 (b)DPi V A 160 160 r~n i i I T r~r 5.40 5.00 _ 4.50 1.60 Figure I V . 2 . 1 : 'H-n.m.r. spectra (400 MHz) of (a) K35 polysaccharide, (b) partially depyruvylated (~66°c) K35 polysaccharide, (c) completely depyruvylated K35 polysaccharide, and (d) Smith-degraded poly-saccharide. The height of each vertical line is proportional to the proton integral. £ I -(d)SD A C DE - r — i — f — r ~ - i — i — i — i — r 105 100 (c) DP 2 A' BC D' E i i r T I I T i r 105 100 (b) DP, BC ^ E i i i r 105 i I r 100 (a) K35 A BC D E 105 1 — r n — i — i — too 6 (p.p.m.) -7A /A -TA 26 - 1 — 26 26 26 Figure I V . 2 . I I : 1JC-N.m.r. spectra (100.6 MHz) of (a) K35 polysaccharide, (b) panially depyruvylated (~66%) K35 polysaccharide, (c) completely depyruvylated K35 polysaccharide, and (d) Smith-degraded poly-saccharide. Due to the n.O.e. effect, peak quantitation is inaccurate. Peak heights for anomeric signals are derived from the corresponding 'H-spectrum. r i d e gave a p r o d u c t DPI whose i- 5C-n.m.r. spectrum showed p a i r s o f s i g n a l s A,A 1 and D ^ 1 f o r r e s i d u e s A and D r e s p e c t i v e l y . The r a t i o s o f the h e i g h t s o f A:A 1 and D i D 1 were s i m i l a r , and as h y d r o l y s i s o f the a c e t a l p r o g r e s s e d to c o m p l e t i o n ( p r o d u c t DP2) o n l y s i g n a l s o f A 1 and D 1 were obs e r v e d . T h i s i s i l l u s t r a t e d by comparison o f F i g u r e s I V . 2 . l i b and I V . 2 . l i e w h i l e the a c t u a l anomeric r e g i o n o f DPI i s d e p i c t e d i n F i g u r e I V . 2 . I I I c . T w i n n i n g was l e s s n o t i c e a b l e i n the p r o t o n spectrum but the s m a l l u n a s s i g n e d peak (Y i n F i g u r e I V . 2 . I l i a ) , which was l a r g e r i n the spectrum o f DPI, may be due to t h i s phenomenon, a l t h o u g h t h i s s i g n a l was absent i n the spectrum o f DP2. S i g n a l X i s u n a s s i g n e d , but i t i n t e g r a t e d f o r l e s s than one p r o t o n . I t may be n o t e d t h a t removal o f the a c e t a l group causes an u p f i e l d s h i f t o f the s i g n a l s A and D. T w i n n i n g o f the s i g n a l due to the a - D - g a l a c t o p y r a n o s y l r e s i d u e , which i s s u b s t i t u t e d by the p y r u v i c a c i d , i s to be e x p e c t e d . The e f f e c t on the anomeric s i g n a l o f the /?-D-glucopyranosyl r e s i d u e may be r a t i o n a l i z e d by i n s p e c t i o n o f a Framework m o l e c u l a r model which shows t h a t the c a r b o x y l f u n c t i o n o f the a c e t a l group on the g a l a c t o s e s h o u l d have a d e s h i e l d i n g e f f e c t on the anomeric c a r b o n o f i t s p o s t e r i o r n e i g h b o r ( 3 - G l c ) but m i n i m a l i n f l u e n c e on i t s a n t e r i o r n e i g h b o r (3-Man ). The assignment a o f s i g n a l A t o the /9-D-glucopyranosyl u n i t and o f s i g n a l B to the / 3 - 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 f o l l o w s from the d i s a p p e a r a n c e o f the l a t t e r s i g n a l , i n b o t h the and -^C-n.m.r. s p e c t r a , on Smith d e g r a d a t i o n o f the K35 p o l y s a c c h a r i d e (compare F i g u r e s IV.2.Ia and IV.2.Id; I V . 2 . I I a and I V . 2 . I I d ; and I V . 2 . I l i a and I V . 2 . I I I b ) . The s p e c t r a a l s o demonstrate t h a t the anomeric s i g n a l s o f the b r a n c h p o i n t D-mannose r e s i d u e (E) s h i f t e d on removal o f the u r o n i c a c i d s i d e c h a i n , - 100 -F i g u r e I V . 2 . I l l : (a) iH-N.m.r. spectrum (400 MHz) o f p a r t i a l l y d e p y r u v y l a t e d K35 p o l y s a c c h a r i d e , (b) Smith degrada-t i o n p r o d u c t SD; (c) x 3 C spectrum (100 MHz) o f p a r t i a l l y d e p y r u v y l a t e d K35 p o l y s a c c h a r i d e . - 101 -u p f i e l d i n the X H case and d o w n f i e l d i n 1 3 C c a s e . A r e f e r e e has s u g g e s t e d t h a t we s h o u l d c o n s i d e r a r e p e a t i n g u n i t o f t h e form -D-E-A-C- b u t t h i s would n o t a c c o u n t f o r t h e f o r m a t i o n o f o l i g o s a c c h a r i d e N2 (D-C). Furthermore, a m o l e c u l a r model shows t h a t the c a r b o x y l group o f the a c e t a l would now d e s h i e l d a-D-mannopyranosyl r e s i d u e ( C ) , y e t t h e s e s i g n a l s ( XH and i 3 C ) a r e c l e a r l y u n p e r t u r b e d by p r o g r e s s i v e h y d r o l y s i s o f the a c e t a l s u b s t i t u e n t . IV.2.4 Conelus i o n The p o l y s a c c h a r i d e from K l e b s i e l l a K35 has a "4 + 1" type p a t t e r n , s i m i l a r t o the p o l y s a c c h a r i d e s i s o l a t e d from K l e b s i e l l a K9 and K59 ( r e f . 6) w h e r e i n the immunodominant group, a t e r m i n a l , n o n r e d u c i n g D-glucu-r o n i c a c i d i s i n the s i d e - c h a i n . However, i n K35, t h e r e i s an a d d i -t i o n a l immunodominant f e a t u r e , namely a 1 - c a r b o x y e t h y l i d e n e s u b s t i t u -e n t 3 4 ( a c e t a l l i n k e d t o 0-4 and 0-6 o f a D - g a l a c t o s e r e s i d u e . I n t h e i r e x t e n s i v e s t u d y on the b a c t e r i o p h a g e - i n d u c e d d e p o l y m e r i -z a t i o n o f the c a p s u l a r p o l y s a c c h a r i d e o f 74 K l e b s i e l l a b a c t e r i a , Rieger-Hug and S t i r m 1 3 2 showed t h a t the e ndoglycanase a s s o c i a t e d w i t h the b a c t e r i o p h a g e (c635) f o r K l e b s i e l l a K35 has g l u c o s i d a s e a c t i v i t y . They a l s o o b s e r v e d t h a t most b a c t e r i o p h a g e endoglycanase g e n e r a l l y c l e a v e 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 on e i t h e r s i d e o f the sugar c a r r y i n g t h e n e g a t i v e c h a r g e ( s ) . W i t h the s t r u c t u r e o f K l e b s i e l l a K35 c a p s u l a r p o l y s a c c h a r i d e e l u c i d a t e d , i t i s now o b v i o u s t h a t the carbox-y l i c group o f the p y r u v i c a c i d on the g a l a c t o s y l u n i t (and n o t the 102 g l u c u r o n i c a c i d ) i s the dominant n e g a t i v e charge r e c o g n i z e d by <j>35. T h i s /3-glucosidase a c t i v i t y a s s o c i a t e d w i t h <j>35 i s i n agreement w i t h the pr o p o s e d s t r u c t u r e f o r the K35 p o l y s a c c h a r i d e . IV.2.5 E x p e r i m e n t a l G e n e r a l methods - The g e n e r a l e x p e r i m e n t a l p r o c e d u r e s are as d e s c r i b e d i n S e c t i o n I I I . P r e p a r a t i o n and p r o p e r t i e s o f K35 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 o f K l e b s i e l l a K35, o b t a i n e d from Dr. Ida ^ r s k o v , Copenhagen, was grown as p r e v i o u s l y d e s c r i b e d 1 * ^ , and the p o l y s a c c h a r i d e was p u r i f i e d by two p r e c i p i t a t i o n s w i t h C e t a v l o n . The p u r i f i e d p o l y s a c c h a r i d e (1.5 g from 7.5 L o f medium) had [q]q +66° (c 0.15, w a t e r ) . A n a l y s i s by g e l chromatography (9 cm x 100 cm column o f Sepharose 4B w i t h 5 mL/h of M NaCl e l u a n t , column c a l i b r a t e d w i t h d e x t r a n s ) showed t h a t the p u r i f i e d p o l y s a c c h a r i d e was monodisperse and had a r e l a t i v e m o l e c u l a r mass o f 9 x 10° d a l t o n s (see e l u t i o n p r o f i l e i n Appendix I I ) . N.m.r. s p e c t r o s c o p y was p e r f o r m e d on the o r i g i n a l K35 p o l y s a c c h a r i d e ; the p r i n c i p a l s i g n a l s and t h e i r assignments, f o r b o t h the ^H- and 1^C-n.m.r. s p e c t r a , are r e c o r d e d i n T a b l e I V . 2 . I I . H y d r o l y s i s o f the p o l y s a c c h a r i d e - H y d r o l y s i s o f a sample (11 mg) - 103 -o f K35 p o l y s a c c h a r i d e was e f f e c t e d w i t h 2M t r i f l u o r o a c e t i c a c i d (TFA) f o r 15 h a t 95°, t h e n the a c i d was removed by s u c c e s s i v e e v a p o r a t i o n s w i t h water. Paper chromatography ( s o l v e n t s 1 and 3), showed the p r e s e n c e o f g a l a c t o s e , g l u c o s e , mannose and an a l d o b i o u r o n i c a c i d (EGXC 0.47). A q u a n t i t a t i v e a n a l y s i s o f the n e u t r a l sugars as t h e i r a l d i t o l a c e t a t e s i s p r e s e n t e d i n T a b l e IV.2.I, column I . A p o r t i o n o f the K35 p o l y s a c c h a -r i d e (10 mg) was m e t h a n o l y z e d ^ o v e r n i g h t i n 3% HCI-CH3OH, and the p r o d u c t s were r e d u c e d w i t h sodium b o r o h y d r i d e , h y d r o l y z e d , and a n a l y z e d , w i t h the r e s u l t s shown i n T a b l e IV.2.I, column I I . A l d i t o l a c e t a t e s f o r c.d. m e a s u r e m e n t s 1 ^ w e r e o b t a i n e d by g . l . c , u s i n g column D. M e t h y l a t i o n a n a l y s i s - A sample o f K35 p o l y s a c c h a r i d e i n the f r e e a c i d form (30 mg) was m e t h y l a t e d by the Hakomori p r o c e d u r e 1 1 2 , 1 1 3 . The p r o d u c t r e c o v e r e d a f t e r d i a l y s i s and e x t r a c t i o n w i t h methylene c h l o r i d e , showed complete m e t h y l a t i o n (no h y d r o x y l a b s o r p t i o n i n the i . r . spec-trum) . One p a r t (11 mg) was h y d r o l y z e d w i t h 2M TFA a f t e r which paper chromatography i n s o l v e n t 4 t h e n r e v e a l e d f i v e components, namely 2,4,6-tri-0-methylglucose (ETMG 0.80), 2,4,6-tri-O-methylmannose (0.75), 4,6-di-0-methylmannose (0.50), 2-0-methylgalactose (0.18) and a r e l a t i v e l y f a i n t p i n k s p o t (0.09) which i s a t t r i b u t e d t o an a c i d com-pound. A n a l y t i c a l f i g u r e s f o r the n e u t r a l sugars a r e p r e s e n t e d i n T a b l e I V . 2 . I l l , column I. A second p o r t i o n (22 mg) o f the m e t h y l a t e d p o l y s a c -c h a r i d e was r e d u c e d w i t h l i t h i u m aluminum h y d r i d e i n r e f l u x i n g o x o l a n e . The p r o d u c t (19 mg) was d i v i d e d i n t o h a l v e s , one o f which was h y d r o l y z e d and a n a l y z e d , w i t h t h e r e s u l t s shown i n T a b l e I V . 2 . I l l , column I I . The - 104 -o t h e r h a l f was r e m e t h y l a t e d 1 - ^ , 1 1 3 ^ h y d r o l y z e d and c o n v e r t e d to p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e d e r i v a t i v e s . The g . l . c . r e s u l t i s g i v e n i n T a b l e I V . 2 . I l l , column I I I . A l l m e t h y l a t i o n a n a l y s e s were c o n f i r m e d by g.l.c.-m.s. D e p y r u v y l a t e d K35 p o l y s a c c h a r i d e - An aqueous s o l u t i o n o f K35 p o l y s a c c h a r i d e (70 mg, pH 2.75) was a u t o h y d r o l y z e d f o r 4 h on a steam-b a t h . D i a l y s i s (mol. wt. c u t o f f 3500) a g a i n s t d i s t i l l e d water, f o l l o w e d by l y o p h i l i z a t i o n o f the r e t e n t a t e y i e l d e d 55 mg o f p r o d u c t . ^H-N.m.r. a n a l y s i s o f t h i s p r o d u c t showed t h a t o n l y -30% of the a c e t a l - l i n k e d p y r u v i c a c i d had been removed. A f t e r f u r t h e r h y d r o l y s i s (0.02M TFA, 1 1/2 h, 95°) o f the p o l y s a c c h a r i d e and workup by a s i m i l a r p r o c e d u r e , n.m.r. a n a l y s e s (see DPI i n T a b l e I V . 2 . I I ) i n d i c a t e d t h a t -33% o f the p y r u v a t e s t i l l remained i n t a c t . A t h i r d a u t o h y d r o l y s i s (4 1/2 h, 95°) c o n d u c t e d on the p a r t i a l l y 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 , a c c o m p l i s h e d complete d e p y r u v y l a t i o n (see DP2 i n T a b l e I V . 2 . I I ) . A sample (10 mg) o f the d e p y r u v y l a t e d K35 p o l y s a c c h a r i d e was s u c c e s s i v e l y m e t h y l a t e d by the Hakomori p r o c e d u r e 1 1 " ^ , 113 ^ r e d u c e d w i t h l i t h i u m aluminum h y d r i d e i n r e f l u x i n g oxolane, h y d r o l y z e d and c o n v e r t e d to p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s . The g . l . c . r e s u l t i s g i v e n i n T a b l e I V . 2 . I l l , column IV. The d i s a p p e a r a n c e of 2-O-methylgalactose (see columns I - I I I ) and the appearance o f 2 , 4 , 6 - t r i - O - m e t h y l g a l a c t o s e shows t h a t p y r u v a t e i s l i n k e d as an a c e t a l to 0-4 and 0-6 o f the g a l a c t o s e r e s i d u e . The p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s f o r c.d. m e a s u r e m e n t s 1 0 0 were o b t a i n e d by g . l . c , u s i n g column D. - 105 -U r o n i c a c i d d e g r a d a t i o n 2 ^ 1 - M e t h y l a t e d K35 p o l y s a c c h a r i d e (11 mg), d r i e d i n vacuo was d i s s o l v e d i n 19:1 Me2SO-2,2-dimethoxypropane (8 mL) c o n t a i n i n g a t r a c e o f p . - t o l u e n e s u l f o n i c a c i d , 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 . Sodium m e t h y l s u l f i n y l m e t h a n i d e (2M, 3 mL) was added, and the m i x t u r e was s t i r r e d f o r 18 h a t room temperature. The m i x t u r e was f r o z e n , methyl i o d i d e was added, the m i x t u r e was melted, and s t i r r e d f o r 2 h a t room temperature, and the excess methyl i o d i d e was e v a p o r a t e d . The m e t h y l a t e d , degraded p r o d u c t was i s o l a t e d by p a r t i t i o n between c h l o r o f o r m and water (3 x 15 mL), the p r o d u c t was d r i e d , and h y d r o l y z e d w i t h 2M TFA, and the sugars r e l e a s e d were a n a l y z e d as d e s c r i b e d p r e v i o u s l y f o r the m e t h y l a t i o n a n a l y s i s (see T a b l e I V . 2 . I l l , column V ) . Smith d e g r a d a t i o n 1 2 6 - 1 9 8 - A s o l u t i o n o f K35 p o l y s a c c h a r i d e (53 mg) i n water (40 mL) was mixed w i t h NaI04 (0.5 g) and NaC104 (0.2 g ) , and k e p t s t i r r i n g i n the dark a t room temperature. A f t e r 60 h, e t h y l e n e g l y c o l (0.5 mL) was added, and the m i x t u r e was r e d u c e d w i t h NaBH^. The exc e s s o f NaBH^ was decomposed w i t h f o r m i c a c i d (50%) and the p r o d u c t was d i a l y z e d (mol. wt. c u t o f f 3500) a g a i n s t 0.25M TFA f o r 48 h. F u r t h e r d i a l y s i s a g a i n s t d i s t i l l e d water, and passage t h r o u g h a column o f A m b e r l i t e IR 120 (H +) r e s i n , f o l l o w e d by c o n c e n t r a t i o n and l y o p h i l i -z a t i o n gave the p o l y o l SD (30 mg). N.m.r. a n a l y s e s (see T a b l e IV.2.II) o f SD showed t h a t the t e r m i n a l g l u c u r o n i c a c i d had been degraded, and t h a t the p y r u v i c a c i d a c e t a l was u n a f f e c t e d by 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 used. R e s u l t s f o r the sugar and m e t h y l a t i o n a n a l y s e s are 106 g i v e n i n T a b l e IV.2.I, column I I I and T a b l e I I I , column VI r e s p e c t i v e l y . P a r t i a l h y d r o l y s i s - The K35 p o l y s a c c h a r i d e (450 mg) was h y d r o l y z e d w i t h 0.5M TFA f o r 2 h a t 95°. A f t e r removal o f the TFA by s u c c e s s i v e e v a p o r a t i o n s w i t h water, the h y d r o l y z a t e was d i a l y z e d (mol. wt. c u t o f f 3500) a g a i n s t d i s t i l l e d water. The l y o p h i l i z e d r e t e n t a t e (170 mg) was s u b j e c t e d t o f u r t h e r h y d r o l y s i s (0.5M TFA 1 h, 95°) and the same workup p r o c e d u r e f o l l o w e d . A s o l u t i o n o f the combined, l y o p h i l i z e d d i a l y z a t e (380 mg) was s e p a r a t e d on a column o f Bio-Rad AG1-X2 (formate) ion-exchange r e s i n , t o g i v e a n e u t r a l (150 mg) and an a c i d i c f r a c t i o n (200 mg). The n e u t r a l f r a c t i o n was e l u t e d w i t h water, and the a c i d i c f r a c t i o n w i t h f o r m i c a c i d ( 1 0 % ) . P r e p a r a t i v e paper c h r o m a t o g r a p h i c s e p a r a t i o n o f the a c i d i c f r a c t i o n , u s i n g s o l v e n t 2, gave an a l d o b i o u r -o n i c a c i d A2 (18 mg), an a l d o t r i o u r o n i c a c i d A3 (26 mg), a m i x t u r e A4 (12 mg) o f a l d o t e t r a o u r o n i c a c i d s (as a n a l y z e d by ^H.n.m.r. and m e t h y l a t i o n a n a l y s i s ) , and h i g h e r m o l e c u l a r weight o l i g o s a c c h a r i d e s (65 mg) which remained a t the o r i g i n . Paper chromatography o f the n e u t r a l f r a c t i o n showed t h a t i t comprised p r e d o m i n a n t l y monosaccharides ( g a l a c -t o s e , g l u c o s e and mannose), p l u s a n e u t r a l d i s a c c h a r i d e N2 (11 mg) which was s e p a r a t e d by p r e p a r a t i v e paper chromatography u s i n g s o l v e n t 2. Each o l i g o s a c c h a r i d e was s u b j e c t e d t o sugar a n a l y s i s and methyla-t i o n a n a l y s i s (see S e c t i o n s I I I . 3 and I I I . 4 ) . F o r sugar a n a l y s i s the a c i d i c o l i g o s a c c h a r i d e s were t r e a t e d w i t h 3% HC1 i n anhydrous methanol f o r 8 h on a steam b a t h , t h e n the methyl e s t e r groups i n the p r o d u c t s were r e d u c e d w i t h sodium b o r o h y d r i d e i n anhydrous methanol. T h i s was - 107 f o l l o w e d by h y d r o l y s i s w i t h 2M TFA, r e d u c t i o n to the a l d i t o l s , and a c e t y l a t i o n w i t h 1:1 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 s o f the g . l . c . a n a l y s e s (column A) ar e g i v e n i n T a b l e I V . 2 . I . The n e u t r a l d i s a c c h a r i d e N2 was i n i t i a l l y h y d r o l y z e d and a n a l y z e d s i m i l a r l y . M e t h y l a t i o n s were conducted by the method o f Hakomori 1-'- 2,113 ( t h e a c i d i c o l i g o s a c c h a r i d e s b e i n g reduced w i t h l i t h i u m aluminum h y d r i d e i n anhy-drous oxolane a f t e r m e t h y l a t i o n ) a f t e r which h y d r o l y s i s w i t h 2M TFA, sodium b o r o h y d r i d e r e d u c t i o n , and a c e t y l a t i o n gave 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 , which were a n a l y z e d by g . l . c . and g.l.c.-m.s. (see T a b l e I V . 2 . I I I ) . The n.m.r. d a t a f o r each o l i g o s a c c h a r i d e are g i v e n i n T a b l e I V . 2 . I I . 108 -IV.3 E. c o l l SEROTYPE K44: AN ACIDIC CAPSULAR POLYSACCHARIDE CONTAINING TWO 2-ACETAMID0-2-DE0XYHEX0SES. IV.3.1 A b s t r a c t The s t r u c t u r e o f the c a p s u l a r p o l y s a c c h a r i d e from E s c h e r i c h i a c o l i 08:K44(A):H- (K44 a n t i g e n ) has been e s t a b l i s h e d u s i n g the t e c h n i q u e s o f m e t h y l a t i o n , / ? - e l i m i n a t i o n , d e a m i n a t i o n and Smith d e g r a d a t i o n . N.m.r. s p e c t r o s c o p y (±JC and XH) was used e x t e n s i v e l y t o e s t a b l i s h the n a t u r e o f the anomeric l i n k a g e s o f the p o l y s a c c h a r i d e and o f o l i g o s a c c h a r i d e s d e r i v e d t h r o u g h d e g r a d a t i v e p r o c e d u r e s . The K a n t i g e n comprises r e p e a t -i n g u n i t s o f the l i n e a r t e t r a s a c c h a r i d e shown. T h i s a c i d i c p o l y s a c c h a -r i d e r e p r e s e n t s the f i r s t i n s t a n c e , i n t h i s s e r i e s o f E. c o l i K a n t i g e n s ( o f group A) t o c o n t a i n two d i f f e r e n t 2-acetamido-2-deoxyhexoses. 4) -/3-D-GlcAp- (1-3) -a-L-Rhap- (1-4) -a-D-GlcpNAc- (l-6)/3-D-GalpNAc- ( 1 -IV.3.2 I n t r o d u c t i o n The b a c t e r i a o f the s p e c i e s E s c h e r i c h i a c o l i . a r e c l a s s i f i e d on the b a s i s o f 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 c a p s u l a r or K a n t i g e n s (74 p o l y s a c c h a r i d e t y p e s ) , the somatic 0 a n t i g e n s (164 l i p o -p o l y s a c c h a r i d e t y p e s ) , and the f l a g e l l a r H a n t i g e n s (56 p r o t e i n a c e o u s t y p e s ) . The c a p s u l a r p o l y s a c c h a r i d e s (K a n t i g e n s ) can be f u r t h e r s u b d i v i d e d i n t o t h r e e groups (A, B, and L) on the b a s i s o f t h e i r 0 - 109 -a n t i s e r u m a g g l u t i n a b i l i t y and h e a t s t a b i l i t y 3 8 . To d a t e , t h e c h e m i c a l s t r u c t u r e s o f a p p r o x i m a t e l y 27 o f these i m m u n o l o g i c a l l y s i g n i f i c a n t K a n t i g e n s have been r e p o r t e d . As p a r t o f our c o n t i n u i n g program t o c o r r e l a t e p r i m a r y c h e m i c a l s t r u c t u r e w i t h s e r o l o g i c a l and immunological a c t i v i t i e s o f t h i s s p e c i e s , we now r e p o r t our r e s u l t s on the E. c o l i K44 c a p s u l a r p o l y s a c c h a r i d e . I V . 3 . 3 R e s u l t s and D i s c u s s i o n C o m p o s i t i o n and n.m.r. s p e c t r a - The p o l y s a c c h a r i d e from E. c o l i was i s o l a t e d and p u r i f i e d a c c o r d i n g t o the p r o c e d u r e p r e v i o u s l y d e s c r i -b e d 4 6 . The a c i d i c p o l y s a c c h a r i d e o b t a i n e d from two c e t y l t r i m e t h y l a m m o -nium bromide p r e c i p i t a t i o n s had [ a ] j j 2 ^ + 5 0 . 4 ° . D e t e r m i n a t i o n o f the n e u t r a l and amino sugars as a l d i t o l a c e t a t e s r e v e a l e d the p r e s e n c e o f rhamnose, glucosamine and g a l a c t o s a m i n e i n the p o l y s a c c h a r i d e (see T a b l e IV.3.I, column I ) . The f o r m a t i o n o f e q u i m o l a r p r o p o r t i o n s o f 2,5-anhydromannitol and 2 , 5 - a n h y d r o t a l i t o l on d e a m i n a t i o n o f some h y d r o l y z a t e s c o n f i r m e d the p r e s e n c e o f glucosamine and g a l a c t o -samine i n the p o l y s a c c h a r i d e (see T a b l e I V . 3 . I , column I I I ) . Both the c a r b o x y l - r e d u c e d methyl g l y c o s i d e s ^ - * , and the c a r b o d i i m i d e - r e d u c e d p o l y s a c c h a r i d e ^ 6 on a n a l y s i s as a l d i t o l a c e t a t e s showed the p r e s e n c e o f g l u c o s e (0.95 and 0.68 moles r e s p e c t i v e l y , see T a b l e I V . 3 . I , columns I I and I V ) . Thus E. c o l i K44 c o n s i s t s o f a 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 o f e q u i m o l a r p r o p o r t i o n s o f g a l a c t o s a m i n e , glucosamine, g l u c u r o n i c a c i d 110 -TABLE IV . 3 . I : Sugar a n a l y s i s o f E. c o l i K44 p o l y s a c c h a r i d e and d e r i v e d p r o d u c t s . S u g a r 5 M o l a r r a t i o s 5 ' b (as a l d i t o l a c e t a t e ) I I I I I I IV V VI V I I Rhamnose 1.00 1.00 Glucosamine 0.61 0.40 Ga l a c t o s a m i n e 0.48 0.28 Gl u c o s e 0.95 2,5-Anhydromannitol 2 , 5 - A n h y d r o t a l i t o l G l y c e r o l E r y t h r i t o l 1.00 1.00 1.00 1.00 1.00 0.76 0.65 0.47 0.35 0.90 0.68 0.77 0.54 0.42 0.15£ 0.19£ 0.67 5 U s i n g DB-17 c a p i l l a r y column programmed from 180° f o r 2 min, and then a t 5°/min t o 220°. ^ I, n a t i v e a c i d i c p o l y s a c c h a r i d e u s i n g 2 M HC1 a t 95° f o r 7 h; I I , methanolyzed c a r b o x y l - r e d u c e d p o l y s a c c h a r i d e ; I I I , d e a m i n a t i o n o f p r o d u c t s from I I ; IV, c a r b o d i i m i d e - r e d u c e d p o l y s a c c h a -r i d e ; V, methanolyzed c a r b o x y l - r e d u c e d compound 1; V I , p o l y o l from p e r i o d a t e o x i d a t i o n ; V I I , d i s a c c h a r i d e from Smith d e g r a d a t i o n . 2. Quan-t i t a t i o n i s i n a c c u r a t e due to i t s h i g h v o l a t i l i t y . and rhamnose. 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 o f the p o l y s a c c h a r i d e was more c l e a r l y demonstrated i n the n.m.r. s p e c t r a (see F i g . I V . 3 . I ) . The •^H-n.m.r. spectrum o f the n a t i v e p o l y s a c c h a r i d e showed f o u r anomeric p r o t o n s i g n a l s 1 9 6 : an a - l i n k a g e a t 8 4.95 ( J ^ 2 3 Hz, 1H), two ^ - l i n k a g e s a t 5 4.72 ( J 1 2 8 Hz, 1H) , and 6 4.60 (J]_ 2 8 H z > 1 H ) > 5 n d 5 F i g u r e I V . 3 . I : . LU- and 1 3C-N.m.r. s p e c t r a o f E. c o l i K44 c a p s u l a r p o l y s a c c h a r i d e . 112 b o r d e r l i n e s i g n a l a t 5 4.89 (b, 1H). A f t e r passage through a column o f c a t i o n - e x c h a n g e r e s i n , the p o l y s a c c h a r i d e (now i n i t s a c i d i c form) showed a d r a m a t i c d o w n f i e l d s h i f t o f one o f the anomeric s i g n a l s i n the •'-H-n.m.r. spectrum i . e . from 6 4.70 to 6 4.80. T h i s s i g n a l i s a t t r i b u t e d t o the g l u c u r o n o s y l r e s i d u e and the J v a l u e showed t h a t i t has a /?-anomeric c o n f i g u r a t i o n . The d o w n f i e l d s h i f t o f the g l u c u r o n o s y l anomeric p r o t o n i s p r o b a b l y due t o the i n d u c e d d e s h i e l d i n g e f f e c t on p r o t o n a t i o n o f the c a r b o x y l group. I n the •'-H-n.m.r. spectrum o f the n a t i v e p o l y s a c c h a r i d e , methyl p r o t o n s o f rhamnose appeared a t 5 1.34 (b, 3H), and two s i g n a l s a t t r i b u t e d t o methyl p r o t o n s o f the N - a c y l groups were o b s e r v e d a t 6 2.09 ( s , 3H), and 5 2.06 ( s , 3H), r e s p e c t i v e l y . The •'-^C-n.m.r. spectrum o f the n a t i v e p o l y s a c c h a r i d e (see F i g . IV.3.I) showed a t o t a l o f twenty e i g h t c a r b o n s i g n a l s and t h i s , c o u p l e d w i t h the p r e s e n c e o f o n l y f o u r anomeric carbon r e s o n a n c e s - ' ^ , c o n f i r m e d a 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 f o r the K44 p o l y s a c c h a r i d e . The t h r e e C = 0 s i g n a l s i n the d o w n f i e l d r e g i o n o f the spectrum (between 170-180 p.p.m.) agree w i t h the p r e s e n c e o f two acetamido and one c a r b o x y l group a s s o c i a t e d w i t h the p o l y s a c c h a r i d e . The s i g n a l s a t 54.84 p.p.m. and 53.08 p.p.m. can be a t t r i b u t e d to C-2 o f g l u c o s - and g a l a c t o s - amine. In the u p f i e l d r e g i o n o f the spectrum, t h r e e s i g n a l s c o r r e s p o n d i n g to the methyl carbons o f the two N - a c y l groups and the rhamnose r e s i d u e were ob s e r v e d . A p r o t o n - c o u p l e d -^C-n.m.r. spectrum o f the n a t i v e p o l y s a c c h a r i d e (see spectrum numbered 30 i n Appendix I I I ) showed s i g n a l s a t 104.2 p.p.m. ( 1 J c i , H l 1 6 0 - 9 H z > . 102.9 p.p.m. ( 1 J c i i H l 1 5 8 - 9 H z ) . 101.3 p.p.m. ( l j C l , H l 1 6 7 H z ) > a n d 9 8 - 9 P-P- m- ( l j C l , H l 1 7 1 H z ) • T h e l j C l , H l v a l u e s T a b l e I V . 3 . I I : N . M . R . d a t a f o r E . c o l l K 4 4 p o l y s a c c h a r i d e a n d d e r i v e d p r o d u c t s . C o m p o u n d * ^ - N . r a . r . d a t a « C • N . m . , r . d a t a 6 ° J l , 2 C ( H z ) I n t e g r a l p r o t o n A s s i g n m e n t * 3 P . p . , m . e A s s i g n m e n t * G l c A ^ — 3 - R h a ~ 0 H P 4 . 6 8 b 1 . . 0 G l c A i — P 5 . 1 7 b 0 . . 9 ^ R h a OH a 1 1 . 3 4 b 3 . 0 C H 3 ( R h a ) R h a U l G l c N A c L - i c r o S a a 4 . 9 6 b 1 . 0 ^ G l c N A c — a 1 0 1 . . 6 0 R h a ^ -a 4 . 9 0 b 1 . 0 R h a i -Q 9 9 . 0 5 - ^ C l c N A c i -a 2 2 . 0 9 b 3 . 0 C H 3 o f N - A c 7 7 . 6 2 C - 4 ( G l c N A c ) 1 . 3 4 b 3 . 0 C H 3 o f R h a 7 3 . 4 9 C - l ( G r o ) 6 2 . 3 2 C - 3 ( G r o ) 6 1 . 1 C - 6 ( G l c N A c ) 5 4 . 8 5 C - 2 ( G l c N A c ) ft 2 2 . 7 3 N - C - C H 3 1 7 . 4 8 C - 6 ( R h a ) C o m p o u n d 3 A H - N . m . r . d a t a 1 3 C - N . m . r . d a t a * b J l , 2 C ( H z ) I n t e g r a l p r o t o n A s s i g n m e n t 1 * P . p . m . e A s s i g n m e n t * ^ G l c A i - ^ R h a i - ^ l c N A c i - ^ G a l N A c i - 4 9 6 3 1 0 ^ G l c N A c i - 1 7 5 . 8 0 P a a p a 4 8 9 b 1 0 1 7 5 0 0 K44 p o l y s a c c h a r i d e (Na ) a 4 7 0 8 1 0 ^ G l c A1 -P 1 7 2 3 0 4 6 1 8 1 0 ^ G a l N A c i — P 1 0 4 7 7 2 0 9 s 3 0 C H 3 o f N - A c ( G l c N A c ) 1 0 2 8 9 2 0 6 s 3 0 C H 3 o f N - A c ( G a l N A c ) 1 0 1 . 4 6 1 3 3 5 3 0 C H 3 ( R h a ) 9 8 . 9 0 8 2 . 9 8 8 0 . 9 3 7 7 . 7 4 6 1 . 0 8 5 4 . 7 9 5 2 . 9 0 2 2 . 6 7 1 7 . 5 2 N - a c e t y l • o f G l c N A c a n d G a l N A c C - 0 C - 0 C - 0 ( G l c A ) ^ G l c A 1 -P S c a l N A c i -P 3 R h a ! -a ^ G l c N A c 1 -a C - 4 ( G l c A ) C - 3 ( R h a ) C - 4 ( G l c N A c ) C - 6 ( G l c N A c ) C - 2 ( G l c N A c ) C - 2 ( G a l N A c ) 2 3 . 2 1 N - C - C H 3 ( G a l N A c ) N - E - C H 3 ( G l c N A c ) C - 6 ( R h a ) a F o r t h e o r i g i n o f c o m p o u n d 1 a n d 2 , s e e t e x t . D C h e m i c a l s h i f t r e l a t i v e t o i n t e r n a l a c e t o n e a s s i g n e a t S 2 . 2 3 d o w n f i e l d f r o m e x t e r n a l s o d i u m 4 , 4 - d i m e t h y l - 4 - s i l a p e n t a n e - l - s u l f o n a t e ( D . S . S . ) . c K e y : b -b r o a d , u n a b l e t o a s s i g n a c c u r a t e c o u p l i n g c o n s t a n t ; s - s i n g l e t . & F o r e x a m p l e 3 . R n a - r e f e r s t o t h e a n o m e r i c p r o t o n o f a 3 - l i n k e d r h a m n o s y l r e s i d u e i n t h e a - a n o m e r i c c o n f i g u r a t i o n . T h e a b s e n c e o f a n u m e r i c a l p r e f i x i n d i c a t e s a ( t e r m i n a l ) n o n r e d u c i n g g r o u p . £ C h e m i c a l s h i f t r e l a t i v e t o i n t e r n a l a c e t o n e a s s i g n e d a t 3 1 . 0 7 p . p . m . d o w n f i e l d f r o m e x t e r n a l D . S . S . £ A s f o r d , b u t f o r a n o m e r i c n u c l e i . 6 G r o - g l y c e r o l . A l l n . m . r . s p e c t r a a r e g i v e n i n A p p e n d i x I I I - 116 -r e p o r t e d f o r the a and (3 g l y c o s i d i c l i n k a g e s o f 6-deoxyhexo- and hexo-pyranoses a r e -169 and -160 Hz r e s p e c t i v e l y 1 6 8 > 1 6 9 . Hence t h e r e a r e two a and two /? l i n k a g e s i n the r e p e a t i n g u n i t o f the K44 p o l y s a c c h a r i d e . A chromium t r i o x i d e o x i d a t i o n 1 9 - ^ c o n d u c t e d on the p e r a c e t y l a t e d c a r b o d i i m i d e - r e d u c e d p o l y s a c c h a r i d e showed, a f t e r p a s s i n g the a c i d h y d r o l y z a t e t h r o u g h a column o f c a t i o n - e x c h a n g e r e s i n ( t o remove the b a s i c 2-amino-2-deoxysugars), rhamnose as the o n l y n e u t r a l sugar. Chromium t r i o x i d e i n a c e t i c a c i d has been shown 1^ 2 to s e l e c t i v e l y degrade p e r a c e t y l a t e d p y r a n o s e - s u g a r s i n the /3- anomeric c o n f i g u r a t i o n . Hence t h i s r e s u l t i n d i c a t e s the p r e s e n c e o f an a-rhamnosyl r e s i d u e and a /3-glucuronosyl r e s i d u e i n the K44 p o l y s a c c h a r i d e . C i r c u l a r d i c h r o i s m m e a s u r e m e n t s 1 ^ on the a l d i t o l a c e t a t e s showed rhamnose, to have the L - c o n f i g u r a t i o n , whereas g l u c u r o n i c a c i d , 2 - a c e t a m i d o - 2 - d e o x y g a l a c t o s e and 2-acetamido-2-deoxyglucose have the D - c o n f i g u r a t i o n . M e t h y l a t i o n a n a l y s i s - E. c o l i K44 p o l y s a c c h a r i d e was m e t h y l a t e d a c c o r d i n g t o the Hakomori p r o c e d u r e 1 1 2 > l l 3 . H y d r o l y s i s o f the methy-l a t e d p o l y s a c c h a r i d e was c a r r i e d out u s i n g two d i f f e r e n t a c i d c o n d i t i o n s (see T a b l e I V . 3 . I l l , columns I and I I ) . The d e r i v a t i v e s d e t e c t e d by g . l . c . and i d e n t i f i e d by g.l.c.-m.s. were 2,4-di-0-methylrhamnose, 2-deoxy-2-N-methylacetamido-3,6-di-O-methylglucose ( h e r e a f t e r r e f e r r e d to as 2 , 3 , 6 - t r i - O - m e t h y l g l u c o s a m i n e ) and 2-deoxy-2-N-methylacetamido-3 , 4 - d i - 0 - m e t h y l g a l a c t o s e ( h e r e a f t e r r e f e r r e d t o as 2 , 3 , 4 - t r i - 0 - m e t h y l -g a l a c t o s a m i n e ) . M e t h y l a t i o n a n a l y s i s o f the c a r b o x y l - r e d u c e d ' ' 6 117 TABLE I V . 3 . I l l : M e t h y l a t i o n a n a l y s e s o f E. c o l i K44 p o l y s a c c h a r i d e and d e r i v e d p r o d u c t s . M e t h y l a t e d s u g a r M o l e % b (as a l d i t o l a c e t a t e ) I c I I I I I IV V VI V I I 2,4-Rha 7.4 60.6 35.8 56.4 43.2 2,3,4-Rha 48.5 65.3 2,3,4-Glc 43.5 2,3,6-Glc 24.3 3,6-GlcN(Me)Ac 52.9 23.9 27.2 45.6 26.7 34.6 3,4-GalN(Me)Ac 39.6 15.7 12.5 5.8 1,4-Ery 27.0 1-Gro 4 . 1 d a 2,4-Rha = l , 3 , 5 - t r i - 0 - a c e t y l - 2 , 4 - d i - 0 - m e t h y l r h a m n i t o l ; 3,6-GlcN(Me)Ac = 1 , 4 , 5 - t r i - 0 - a c e t y l - 2 - d e o x y - 2 - ( N - m e t h y l ) a c e t a m i d o - 3 , 6 - O - m e t h y l g l u c i t o l ; E r y = E r y t h r i t o l and Gro = G l y c e r o l . ^ U s i n g DB-17 c a p i l l a r y column programmed from 180° f o r 1 min, and then a t 2°/min t o 250°. c I, o r i g i n a l a c i d i c p o l y s a c c h a r i d e a c e t o l y z e d w i t h 0.9 M H2SO4 i n 95% AcOH at 80° f o r 15 h; I I , o r i g i n a l a c i d i c p o l y s a c c h a r i d e h y d r o l y z e d u s i n g 2 M HC1 a t 95° f o r 5 h; I I I , c a r b o d i i m i d e - r e d u c e d p o l y s a c c h a r i d e ; IV, m e t h y l a t e d and red u c e d 1; V, p r o d u c t from / 3 - e l i m i n a t i o n and rem e t h y l a -t i o n ; V I, p o l y o l from p e r i o d a t e o x i d a t i o n ; V I I , d i s a c c h a r i d e from Smith d e g r a d a t i o n . d Due to h i g h v o l a t i l i t y , q u a n t i t a t i o n was i n a c c u -r a t e . p o l y s a c c h a r i d e showed, i n a d d i t i o n , the pr e s e n c e o f 2 , 3 , 6 - t r i - 0 - m e t h y l -g l u c o s e (see T a b l e I V . 3 . I l l , column I I I ) . These r e s u l t s showed t h a t the glucosamine and g l u c u r o n i c a c i d are 4 - l i n k e d , w h i l e the rhamnose i s 3 - l i n k e d and the g a l a c t o s a m i n e i s 6 - l i n k e d (see Scheme I V . 3 . I ) . There COOH - 1 1 8 -t . c o l l I** POLYSACCHARIDE B U c CARBODIIKIDE REDUCTIOK COOK - CHjOH HAKOWORI METHYLATION •.SODIUM D1MSYL h. CH I •NMe INMe 2M ECI 95*7h «,x ra,m C}" w BOC^™ "O" X BO X BNHe BNH* WHERE X - OMe • OAc ' OHt ' OAc J ' ON . OAc • OAc 2 . AtjO/miDIKE 3. C.L.CrM.S. Ac OAc OAc OHe M.O-HeO-. OAC . OAC S c h e m e I V . 3 . I : M e t h y l a t i o n a n a l y s i s o f t h e c a r b o d i i m i d e r e d u c e d E . c o l i K44 c a p s u l a r p o l y s a c c h a r i d e . - 119 -were no b r a n c h p o i n t s o r t e r m i n a l s u g a r s , which i m p l i e d 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 . D e amination o f the p o l y s a c c h a r i d e - The p o l y s a c c h a r i d e was N - d e a c e t y l a t e d w i t h DMSO/H20 and NaOH 8 0, and the removal o f the N - a c e t y l s u b s t i t u e n t was v e r i f i e d by xH-n.m.r. s p e c t r o s c o p y . The r e s u l t i n g polymer was deaminated w i t h NaNC^/HOAc and the p r o d u c t s e p a r a t e d by g e l - p e r m e a t i o n chromatography. Two f r a c t i o n s were o b t a i n e d , the slower-moving f r a c t i o n ( e l u t e d i n the r e g i o n c o r r e s p o n d i n g t o monosaccharides) f a i l e d t o g i v e p o s i t i v e r e s u l t s due to the low y i e l d . Sugar and methy-l a t i o n a n a l y s e s (see T a b l e s IV.3.I and I V . 3 . I l l ) c o n d u c t e d on the second f r a c t i o n 1, showed t h a t i t c o n t a i n e d the a l d o b i o u r o n i c a c i d GlcAL^-Rha-OH. The pres e n c e o f 2 , 3 , 4 - t r i - O - m e t h y l g l u c o s e , on methyla-t i o n f o l l o w e d by r e d u c t i o n o f the u r o n a t e e s t e r , showed t h a t the g l u c u -r o n i c a c i d e x i s t s i n the p y r a n o i d c o n f i g u r a t i o n . I n the ^H-n.m.r. spectrum (see T a b l e I V . 3 . I I ) the s i g n a l a t 8 4.68 (broad, 1.0 H) i s a t t r i b u t e d t o the /3-glucuronosyl r e s i d u e , whereas the s i g n a l a t 8 5.17 (broad, 0.9 H) i s a t t r i b u t e d to the rhamnosyl r e d u c i n g end. B a s e - c a t a l y z e d / S - e l i m i n a t i o n 2 ^ ! - The p e r m e t h y l a t e d n a t i v e p o l y s a c c h a r i d e was t r e a t e d w i t h sodium m e t h y l s u l f i n y l m e t h a n i d e f o r 18 h, and the p r o d u c t was m e t h y l a t e d . G . l . c . a n a l y s i s (see T a b l e I V . 3 . I l l , column V) o f the sugars as p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s (PMAAs) showed 2 , 3 , 6 - t r i - 0 - m e t h y l g l u c o s a m i n e and s m a l l q u a n t i t i e s o f 2 , 3 , 4 - t r i -120 O - methylgalactosamine. The low amount o f the l a t t e r i s p r o b a b l y due to i t b e i n g the r e d u c i n g sugar (on l i b e r a t i o n o f the g l u c u r o n i c a c i d d u r i n g @ - e l i m i n a t i o n ) u n d e r g o i n g d e g r a d a t i o n i n the b a s i c medium. T h i s same o b s e r v a t i o n was n o t e d f o r K l e b s i e l l a K26^02 and K 7 9 ^ u 5 ( t h i s t h e s i s ) p o l y s a c c h a r i d e s . I n b o t h c a s e s , the r e d u c i n g sugars o b t a i n e d , a f t e r a b a s e - c a t a l y z e d / 9 - e l i m i n a t i o n , were 6 - l i n k e d . The 2,4-di-0-methyl-rhamnose from the m e t h y l a t i o n o f the n a t i v e p o l y s a c c h a r i d e (see T a b l e I V . 3 . I l l , columns I and I I ) was r e p l a c e d by 2,3,4-tri-O-methylrhamnose, thus i n d i c a t i n g t h a t the g l u c u r o n i c a c i d was l i n k e d to 0-3 o f the rhamnose r e s i d u e . These r e s u l t s p e r m i t the assignment o f the t e n t a t i v e p a r t i a l sequence f o r the K44 r e p e a t i n g u n i t : . . . — G a l N A c l - ^ G l c A i — 2 - R h a — . . . Smith d e g r a d a t i o n - P e r i o d a t e o x i d a t i o n y / o f the c a r b o d i i m i d e -r e d u c e d p o l y s a c c h a r i d e was f o l l o w e d by r e d u c t i o n and the p o l y o l was i s o l a t e d a f t e r d i a l y s i s a g a i n s t d i s t i l l e d water. The •'•H-n.m.r. spectrum o f the p o l y o l showed two predominant anomeric s i g n a l s a t 5 4.96 (1.0 H) and 5 4.89 (1.0 H), w i t h g r e a t l y d i m i n i s h e d s i g n a l s a t 6 4.72 (0.2 H), and 5 4.60 (0.1 H). Sugar (see T a b l e IV.3.I, column VI) and m e t h y l a t i o n (see T a b l e I V . 3 . I l l , column VI) a n a l y s e s c o nducted on the p o l y o l showed t h a t the 3 - l i n k e d rhamnose and 4 - l i n k e d 2-acetamido-2-deoxyglucose s u r v i v e d the p e r i o d a t e o x i d a t i o n ; whereas the 6 - l i n k e d 2-acetami-d o - 2 - d e o x y g a l a c t o s e and 4 - l i n k e d g l u c o s e (reduced from g l u c u r o n i c a c i d ) 121 -were degraded (see Scheme I V . 3 . I I ) . T h i s r e s u l t i s i n agreement w i t h the e v i d e n c e from the m e t h y l a t i o n a n a l y s e s o f the c a r b o x y l - r e d u c e d K44 p o l y s a c c h a r i d e (see T a b l e I V . 3 . I I I ) . To ensure complete d e g r a d a t i o n by p e r i o d a t e , a second p e r i o d a t e o x i d a t i o n was c o n d u c t e d on the p o l y o l , and worked up i n a s i m i l a r manner. The p o l y o l was the n s u b j e c t e d t o a Smith d e g r a d a t i o n 1 ^ , whereby the a c y c l i c a c e t a l s were s e l e c t i v e l y c l e a v e d i n p r e f e r e n c e to g l y c o s i d i c ( o r c y c l i c a c e t a l ) l i n k a g e s . D i a l y s i s o f the Smith-degraded p r o d u c t f o l l o w e d by s e p a r a t i o n u s i n g g e l - p e r m e a t i o n chromatography gave compound 2. Sugar (see T a b l e IV.3.I, column V I I ) and m e t h y l a t i o n (see T a b l e I V . 3 . I l l , column V I I ) a n a l y s e s o f 2 showed t h a t i t comp r i s e d the d i s a c c h a r i d e R h a l - ^ G 1 C N A C 1 - ! G l y c e r o 1 a a The p r e s e n c e o f g l y c e r o l , the p r o d u c t o f the Smith degraded 6 - l i n k e d GalNAc r e s i d u e , c o n f i r m e d t h a t the amino-sugars a r e a d j a c e n t to each o t h e r i n the n a t i v e p o l y s a c c h a r i d e . The assignments f o r the anomeric s i g n a l s o f 2 a r e p r e s e n t e d i n T a b l e V I . 3 . I I . These assignments were made by comparison w i t h the n.m.r. d a t a o f compound 3, which was o b t a i n e d as f o l l o w s . A p e r i o d a t e o x i d a t i o n on compound 2 f o l l o w e d by r e d u c t i o n gave compound 3. The ^H-n.m.r. spectrum o f 3 showed o n l y one anomeric s i g n a l a t S 4.97 ( J ^ 2 ^ Hz, 1H), and a methyl resonance s i g n a l a t S 2.06 ( s , - 1 2 2 -E . c o l l K 4 4 P O L Y S A C C H A R I D E CARB0DIIM1DE REDUCTION COOH CBjOH 1. 2 MOLES 10^" 2. NaBH. P O L Y O L 1. 0.5H TFA, «8h 2, NaBH, GLYCEROL COMPOUND 2 . 3 . 1 1 : P e r i o d a t e o x i d a t i o n - S m i t h d e g r a d a t i o n o f t h e E K 4 4 c a p s u l a r p o l y s a c c h a r i d e . - 123 3H), which i s a t t r i b u t e d to the N - a c e t y l o f the 2-acetamido-2-deoxy-g l u c o s e r e s i d u e (see T a b l e I V . 3 . I I ) . The d e g r a d a t i o n o f the t e r m i n a l rhamnosyl r e s i d u e , as i n d i c a t e d by the absence o f the s i g n a l s a t 6 4.90 and 5 1.34, agrees w i t h the m e t h y l a t i o n d a t a o f compound 2. A Smith d e g r a d a t i o n conducted on compound 3 f o l l o w e d by a m e t h y l a t i o n a n a l y s i s (see T a b l e I V . 3 . I l l , column V I I I ) r e v e a l e d the p r e s e n c e o f 2,3,4,6-t e t r a - O - m e t h y l g l u c o s a m i n e . IV.3.4 C o n c l u s i o n These experiments demonstrate t h a t the s t r u c t u r e o f the c a p s u l a r p o l y s a c c h a r i d e from E. c o l i K44 i s based on the 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 . S e p a r a t e e v i d e n c e f o r t h i s c h e m i c a l s t r u c t u r e was o b t a i n e d from the b a c t e r i o p h a g e - i n d u c e d degrada-t i o n s t u d i e s on the K44 p o l y s a c c h a r i d e (see S e c t i o n V . 3 ) . IV.3.5 E x p e r i m e n t a l G e n e r a l methods - See S e c t i o n I I I . P r e p a r a t i o n and p r o p e r t i e s o f E. c o l i K44 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 o f E. c o l i K44, o b t a i n e d from Dr. Ida ^ r s k o v (WHO I n t e r n a -t i o n a l E s c h e r i c h i a Center, Copenhagen) was p r o p a g a t e d on M u e l l e r - H i n t o n b r o t h and agar. The K44 p o l y s a c c h a r i d e was p r e p a r e d ^ 0 as d e s c r i b e d i n 124 -S e c t i o n I I I . 1 . 3 . The p u r i f i e d p o l y s a c c h a r i d e (-0.3 g per l i t r e o f medium) was i s o l a t e d by two p r e c i p i t a t i o n s w i t h c e t y l t r i m e t h y l a m m o n i u m bromide and had [a]n +50.4°. N.m.r. ( x 3 C and XH) s p e c t r o s c o p y was per f o r m e d on the n a t i v e p o l y s a c c h a r i d e i n the sodium s a l t and the f r e e a c i d forms, 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 a r e r e c o r d e d i n T a b l e I V . 3 . I I . A p r o t o n - c o u p l e d ± 3C-n.m.r. experiment, u s i n g the s i n g l e f r e q u e n c y o f f - r e s o n a n c e d e c o u p l i n g (SFORD) t e c h n i q u e , was per-formed on the n a t i v e K44 p o l y s a c c h a r i d e t o e s t a b l i s h the n a t u r e o f the anomeric c o n f i g u r a t i o n s (see spectrum numbered 30 i n Appendix I I I ) . Sugar a n a l y s e s o f the p o l y s a c c h a r i d e - A sample (7 mg) o f K44 p o l y s a c c h a r i d e was h y d r o l y z e d w i t h 2M HC1 f o r 7 h on a steam b a t h (-95°). A f t e r removal o f the a c i d by c o e v a p o r a t i o n w i t h water ( 4 x 5 mL), paper c h r o m a t o g r a p h i c a n a l y s e s o f the h y d r o l y z a t e i n s o l v e n t s 1 and 3 (see S e c t i o n I I I . 5 ) showed the pr e s e n c e o f rhamnose, glucosamine, g a l a c t o s a m i n e and a slow-moving component ( p r o b a b l y the a l d o b i o u r o n i c a c i d ) . Sodium b o r o h y d r i d e r e d u c t i o n (30 min, r . t . ) o f the h y d r o l y z a t e i n aqueous medium was f o l l o w e d by a c i d i f i c a t i o n w i t h 50% a c e t i c a c i d and e v a p o r a t i o n t o d r y n e s s . The r e s i d u a l b o r i c and a c e t i c a c i d s were co-e v a p o r a t e d w i t h methanol ( 4 x 5 mL). A c e t y l a t i o n o f the d r i e d r e s i d u e u s i n g 1:1 p y r i d i n e - A c 2 0 (30 min, 90°) was f o l l o w e d by the u s u a l work up pr o c e d u r e f o r a l d i t o l a c e t a t e s (see S e c t i o n I I I . 3 . 2 ) . The m i x t u r e o f a l d i t o l a c e t a t e s was d i s s o l v e d i n CHCI3 and a n a l y z e d by g . l . c . u s i n g the DB-17 c a p i l l a r y column and programmed a t 180° f o r 2 min, and then 5°/min to 220° (see T a b l e IV.3.I, column I ) . - 125 -A sample (11 mg) o f the K44 p o l y s a c c h a r i d e was methanolyzed 7-' w i t h 3% HC1 i n methanol (16 h, 95°) and the a c i d was n e u t r a l i z e d w i t h PDCO3. The r e s u l t a n t w h i t e PbCl2 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 (20 min). R e d u c t i o n w i t h NaBH 4 i n anhydrous methanol (3 h, r . t . ) was f o l l o w e d by a d d i t i o n o f 50% a c e t i c a c i d and the d r i e d p r o d u c t was c o - e v a p o r a t e d w i t h methanol ( 3 x 5 mL). The r e s i d u e o b t a i n e d was h y d r o l y z e d w i t h 2M HCl (6 h, 95°) and a p o r t i o n o f the h y d r o l y z a t e was redu c e d (aqueous NaBH^, 30 min, r . t . ) and c o n v e r t e d i n t o a l d i t o l a c e t a t e s f o l l o w i n g the p r o c e d u r e p r e v i o u s l y d e s c r i b e d . F o r g . l . c . d a t a , see T a b l e I V . 3 . I , column I I . The o t h e r p o r t i o n was d e a m i n a t e d ^ 0 ^ (1 h, r . t . ) w i t h 33% a c e t i c a c i d (1 mL) and 5% aqueous NaN0 2 (1 mL). T h i s was f o l l o w e d by d i l u t i o n w i t h water (5 mL) and the s o l u t i o n was f r e e z e d r i e d . The p r o d u c t d i s s o l v e d i n water, was red u c e d w i t h NaBD 4 (2 h, r . t . ) and worked up as p r e v i o u s l y d e s c r i b e d . A c e t y l a t i o n u s i n g 1:1 p y r i d i n e - A c 2 0 (30 min, 95°) f o l l o w e d by g . l . c . a n a l y s i s gave the r e s u l t s i n T a b l e I V . 3 . I I I . C a r b o d i i m i d e - r e d u c t i o n o f K44 p o l y s a c c h a r i d e - F o l l o w i n g the p u b l i s h e d p r o c e d u r e 7 q , K44 p o l y s a c c h a r i d e (250 mg) was red u c e d u s i n g 1 - c y c l o h e x y l - 3 - ( 2 - m o r p h o l i n o e t h y l ) - c a r b o d i i m i d e m e t h o - p - t o l u e n e s u l f o n a t e (1 g) and aqueous NaBH 4 (3 M, 150 mL). N e u t r a l p o l y s a c c h a r i d e was o b t a i n e d a f t e r two t r e a t m e n t s . A sample (10 mg) o f t h i s n e u t r a l p o l y -s a c c h a r i d e was h y d r o l y z e d (2 M, HCl, 7 h, 95°) and c o n v e r t e d to i t s a l d i t o l a c e t a t e s u s i n g the p r o c e d u r e s d e s c r i b e d . The sugar compositon i s as shown i n T a b l e IV.3.I, column IV and the m e t h y l a t i o n d a t a i n T a b l e - 126 -I V . 3 . I l l , column I I I . F o r the n.m.r. ( XH and C) s p e c t r a , see Appendix I I I . P r e p a r a t i v e g . l . c . (column D) o f the a l d i t o l a c e t a t e s f o l l o w e d by measurements o f the c i r c u l a r d i c h r o i s m s p e c t r a 1 0 0 , showed r h a m n i t o l p e n t a a c e t a t e t o have the L c o n f i g u r a t i o n ; whereas g l u c i t o l h e x a a c e t a t e , 2 - a c e t a m i d o - 2 - d e o x y g l u c i t o l p e n t a a c e t a t e and 2-acetamido-2-deoxy-g a l a c t i t o l p e n t a a c e t a t e a r e o f the D c o n f i g u r a t i o n . These c.d. s p e c t r a were compared t o those o f the c o r r e s p o n d i n g a u t h e n t i c a l d i t o l a c e t a t e s . Chromic a c i d o x i d a t i o n 1 " - To a s o l u t i o n o f the c a r b o d i i m i d e -r e d u c e d K44 p o l y s a c c h a r i d e (15 mg) i n formamide (5 mL) were added A C 2 O (4 mL) and p y r i d i n e (4 mL), and the m i x t u r e was s t i r r e d a t room tempera-t u r e f o r 2 d. F o l l o w i n g d i a l y s i s (mol. wt. c u t o f f 13,500) a g a i n s t r u n n i n g t ap water, the r e t e n t a t e was f r e e z e - d r i e d , and the a c e t y l a t e d p o l y s a c c h a r i d e was s u b j e c t e d t o a second a c e t y l a t i o n . A f t e r s i m i l a r workup, the 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 (checked by i . r . s p e c t r o s c o p y f o r absence o f h y d r o x y l absorbance) was d i s s o l v e d i n g l a c i a l AcOH ( 3 . 5 mL), Cr03 (100 mg) was added, and the m i x t u r e was s t i r r e d f o r 24 h a t room temperature. The p r o d u c t , i s o l a t e d by p a r t i t i o n u s i n g CHCI3 - H 2 O , was d e s a l t e d on a column o f Sephadex LH-20 u s i n g acetone as e l u a n t . The degraded p e r a c e t y l a t e d p r o d u c t was h y d r o l y z e d (2 M, HC1, 7 h, 95°) and re d u c e d w i t h NaBH 4 (30 min, r . t . ) . Treatment o f the r e a c t i o n m i x t u r e w i t h A m b e r l i t e IR 120 (H +) r e s i n , b e s i d e s d e s t r o y i n g the excess NaBH 4 a l s o removes the b a s i c amino suga r s , f o l l o w e d by f i l t r a t i o n gave o n l y n e u t r a l s u g a r s . These were then a c e t y l a t e d w i t h 1:1 p y r i d i n e - - A C 2 O and - 127 -a n a l y z e d by c a p i l l a r y g . l . c . where r h a m n i t o l p e n t a a c e t a t e was the o n l y sugar ob s e r v e d . M e t h y l a t i o n a n a l y s i s - A sample o f the n a t i v e K44 p o l y s a c c h a r i d e (40 mg) i n the f r e e a c i d form ( o b t a i n e d by p a s s i n g an aqueous s o l u t i o n o f the p o l y s a c c h a r i d e through a column o f A m b e r l i t e IR 120 H + r e s i n ) was m e t h y l a t e d by the Hakomori p r o c e d u r e 1 1 2 ' 1 1 3 . T h i s i n v o l v e d d i s s o l v i n g the p o l y s a c c h a r i d e i n dry DMSO (7 mL) and treat m e n t w i t h sodium methyl s u l f i n y l m e t h a n i d e (2 M, 4 mL) f o r 2 h under a N 2 atmosphere. M e t h y l i o d i d e (10 mL) was added to the c o o l e d m i x t u r e and s t i r r e d u n t i l a c l e a r p a l e - y e l l o w s o l u t i o n was o b t a i n e d (-2 h ) . The p r o d u c t , r e c o v e r e d a f t e r d i a l y s i s (mol. wt. c u t o f f 13,500) a g a i n s t r u n n i n g tap water, was e x t r a c t e d u s i n g CH2CI2 and p a s s e d through a column o f Sephadex LH-20 ( u s i n g 1:1 MeOH-CHCl3 as e l u a n t ) . An i . r . s p e c t r o p h o t o m e t r i c a n a l y s i s o f the p u r i f i e d p r o d u c t (37 mg) showed complete m e t h y l a t i o n i . e . no h y d r o x y l a b s o r p t i o n . The same p r o c e d u r e was per f o r m e d f o r a sample (10 mg) o f the c a r b o d i i m i d e - r e d u c e d p o l y s a c c h a r i d e . One p a r t (12 mg) was h y d r o l y z e d u s i n g 2 M HC1 f o r 7 h a t 95°. The o t h e r p a r t (15 mg) was a c e t o l y z e d 7 7 u s i n g 0.9 M H2SO4 i n 95% AcOH a t 80° f o r 15 h, f o l l o w e d by a d d i t i o n o f H2O (2 mL) and f u r t h e r h e a t e d (2 h ) . The p e r m e t h y l a t e d c a r b o d i i m i d e - r e d u c e d p o l y s a c c h a r i d e was h y d r o l y z e d u s i n g 2 M HC1 f o r 7 h a t 95°. The e x t e n t o f h y d r o l y s i s i n each case was m o n i t o r e d by paper chromatography i n s o l v e n t 4. The c o n v e r s i o n o f the suga r s i n t o p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s (PMAAs) was a c h i e v e d by the same p r o c e d u r e d e s c r i b e d f o r unmethylated s u g a r s . F o r g . l . c . - 128 -d a t a , see T a b l e I V . 3 . I l l , columns I - I I I . For c o n f i r m a t i o n , g.c.-m.s. a n a l y s e s were conducted. D e a m i n a t i o n o f K44 p o l y s a c c h a r i d e - N - D e a c e t y l a t i o n o f the p o l y s a c c h a r i d e was performed 8'-' by d i s s o l v i n g w i t h s t i r r i n g a sample (18 mg) i n a m i x t u r e o f H 20 (0.5 mL), DMSO (2.5 mL), NaOH (200 mg), and a drop o f t h i o p h e n o l . The r e a c t i o n m i x t u r e was k e p t a t 80° f o r 15 h. The e x c e s s base was n e u t r a l i z e d w i t h 2 M HC1 and the s o l u t i o n was d i a l y z e d (mol. wt. c u t o f f 13,500) a g a i n s t r u n n i n g tap water o v e r n i g h t . The r e t e n t a t e was c o n c e n t r a t e d , c e n t r i f u g e d and the s u p e r n a t a n t was f r e e z e -d r i e d . The removal o f the N - a c e t y l s u b s t i t u e n t was v e r i f i e d by xH-n.m.r. s p e c t r o s c o p y . The r e s u l t i n g polymer, d i s s o l v e d i n H 20 (0.5 mL) , was deaminated 2*- 1 4 by s t i r r i n g a t room temperature f o r 1 h w i t h AcOH (33%, 1 mL) and NaN0 2 (5%, 1 mL). Water (3 mL) was added and the r e a c t i o n m i x t u r e f r e e z e -d r i e d . The p r o d u c t , d i s s o l v e d i n H 20 (2 mL), was r e d u c e d w i t h NaBD^ (50 mg, 2 h ) . A d d i t i o n o f A m b e r l i t e IR 120 (H +) r e s i n d e s t r o y e d the excess NaBD^ and d e - c a t i o n i z e d the r e a c t i o n m i x t u r e . The f i l t r a t e o b t a i n e d on f i l t r a t i o n was c o - e v a p o r a t e d w i t h MeOH ( 4 x ) . The p r o d u c t was f r a c t i o n -a t e d on a column (2.5 cm x 25 cm) o f B i o - G e l P-2 u s i n g 5:2:500 p y r i d i n e -a c e t i c a c i d - w a t e r as e l u a n t . The e l u t i o n p r o f i l e o b t a i n e d i n d i c a t e d the p r e s e n c e o f two compounds. C h a r a c t e r i z a t i o n by sugar and m e t h y l a t i o n a n a l y s e s on b o t h f r a c t i o n s was performed f o l l o w i n g the procedure d e s c r i b e d i n S e c t i o n I I I . 3 . 1 and I I I . 4 . Due to i t s low y i e l d , the slower-moving component f a i l e d to g i v e p o s i t i v e r e s u l t s . For the sugar, 129 -n.m.r. and m e t h y l a t i o n a n a l y s e s o f the o t h e r f r a c t i o n , see T a b l e s IV.3.I, I V . 3 . I I and I V . 3 . I I I . B a s e - c a t a l y z e d / ^ - e l i m i n a t i o n 2 ^ - A d r i e d sample (20 mg) o f m e t h y l a t e d K44 p o l y s a c c h a r i d e was d i s s o l v e d i n 19:1 DMSO-2,2-dimethoxy-propane (10 mL) c o n t a i n i n g a t r a c e o f p _ - t o l u e n e s u l f o n i c a c i d . Sodium m e t h y l s u l f i n y l m e t h a n i d e (2 M, 5 mL) was added and the m i x t u r e was s t i r r e d a t room temperature f o r 18 h. The degraded polymer was remethy-l a t e d by a d d i n g CH3I (10 mL) to the c o o l e d s o l u t i o n and f u r t h e r s t i r r e d (1 h ) . The m e t h y l a t e d degraded p r o d u c t was i s o l a t e d by e x t r a c t i o n u s i n g CH2CI2-H2O and f u r t h e r p u r i f i e d by passage through a column o f Sephadex LH-20 u s i n g 1:1 MeOH-CHCl3 as e l u a n t . The p r o d u c t was h y d r o l y z e d (2 M HC1, 7 h, 95°) and the sugars r e l e a s e d were a n a l y z e d as p r e v i o u s l y d e s c r i b e d f o r the m e t h y l a t i o n a n a l y s i s (see T a b l e I V . 3 . I l l , column V ) . P e r i o d a t e o x i d a t i o n 1 9 7 and Smith d e g r a d a t i o n 1 2 6 o f c a r b o d i i m i d e -r e d u c e d K44 p o l y s a c c h a r i d e - A sample (190 mg) o f the c a r b o d i i m i d e -r e d u c e d p o l y s a c c h a r i d e was d i s s o l v e d i n H2O (120 mL) and NalO^ (1.42 g) was added. The r e a c t i o n was con d u c t e d a t room temperature and i n the dark (4 d ) . E t h y l e n e g l y c o l (0.1 mL) was added t o d e s t r o y the excess p e r i o d a t e (2 h) and the r e a c t i o n m i x t u r e was d i a l y z e d (mol. wt. c u t o f f 13,500) a g a i n s t d i s t i l l e d water. The c o n c e n t r a t e d r e t e n t a t e was reduced w i t h aqueous NaBH^ (1 h, r . t . ) and excess NaBH4 was d e s t r o y e d w i t h 50% AcOH. D i a l y s i s (mol. wt. c u t o f f 13,500) o f the r e a c t i o n m i x t u r e - 130 -a g a i n s t d i s t i l l e d H 20 f o l l o w e d by l y o p h i l i z a t i o n o f the r e t e n t a t e y i e l d e d the p o l y o l (127 mg). The c o m p o s i t i o n o f the p o l y o l i s shown i n T a b l e s IV.3.I and I V . 3 . I I I . See T a b l e I V . 3 . I I f o r the n.m.r. d a t a . A s e c o n d p e r i o d a t e o x i d a t i o n was r e p e a t e d on the p o l y o l and worked up f o l l o w i n g the p r o c e d u r e d e s c r i b e d . Smith d e g r a d a t i o n o f t h e p o l y o l u s i n g 0.5 M TFA (2 d, r . t . ) was f o l l o w e d by d i a l y s i s (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 H2O. The d i a l y z a t e , a f t e r r e p e a t e d e v a p o r a t i o n to d r y n e s s w i t h water (3x) t o remove the TFA, was d e i o n i z e d by passage t h r o u g h s e p a r a t e columns o f A m b e r l i t e IR 120 ( H + ) r e s i n and A m b e r l i t e IR 45 (OH") r e s i n . S e p a r a t i o n o f the Smith-degraded p r o d u c t u s i n g a B i o - G e l P-2 column and water as the e l u a n t gave compound 2 (77 mg). Sugar (see T a b l e IV.3.I, column V I I ) , n.m.r. (see T a b l e I V . 3 . I I ) , and m e t h y l a t i o n (see T a b l e I V . 3 . I l l , column V I I ) a n a l y s e s showed t h a t compound 2 i s a n e u t r a l d i s a c c h a r i d e g l y c o s i d e . Compound 2 (27 mg) was s u b j e c t e d to a p e r i o d a t e o x i d a t i o n (400 mg, NalO^ i n 25 mL H 20) f o r 1 d a t room temperature and i n the dark. A f t e r d e s t r o y i n g the e x c e s s NaI04 w i t h e t h y l e n e g l y c o l (0.1 mL), the r e a c t i o n m i x t u r e was c o n c e n t r a t e d to -10 mL and r e d u c e d w i t h NaBH4 (30 min, r . t . ) . A f t e r t r e a t m e n t w i t h 50% AcOH and r e p e a t e d c o - e v a p o r a t i o n w i t h MeOH ( 4 x 5 mL), the aqueous s o l u t i o n was d e i o n i z e d to g i v e compound 3. The ^H-n.m.r. spectrum o f 3 showed one anomeric s i g n a l a t S 4.97 ( J ^ 2 3 Hz, 1H), and i n the h i g h f i e l d r e g i o n , a m e t h y l resonance s i g n a l a t 5 2.06 ( s , 3H). Smith d e g r a d a t i o n o f compound 3 u s i n g 0.5 M TFA o v e r n i g h t a t room temperature was f o l l o w e d by r e p e a t e d e v a p o r a t i o n w i t h H 20 ( 4 x 5 mL). A m e t h y l a t i o n a n a l y s i s c o n d u c t e d on t h i s r e s i d u e o n l y showed the 131 p r e s e n c e o f 2,3,4,6-tetra-O-methylglucosamine. IV.3.6 Acknowledgement Some o f the i n i t i a l work conducted on the E. c o l i K44 c a p s u l a r p o l y s a c c h a r i d e has been r e p o r t e d by D.N. K a r u n a r a t n e ^ 8 and was l a t e r r e p e a t e d by m y s e l f i n the c o u r s e o f t h i s s t r u c t u r a l s t u d y . - 132 -CHAPTER V BACTERIOPHAGE-INDUCED DEPOLYMERIZATION OF BACTERIAL CAPSULAR POLYSACCHARIDES - 1 3 3 -O V . B A C T E R I O P H A G E - I N D U C E D D E P O L Y M E R I Z A T I O N OF 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 . T h e r e a r e t w o m a i n s o u r c e s o f e n z y m e s t h a t h y d r o l y z e 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 , n a m e l y e x o p o l y s a c c h a r a s e s f r o m b a c t e r i a 1 3 4 a n d b a c t e r i o p h a g e - a s s o c i a t e d e n d o g l y c a n a s e s 1 3 1 " 1 3 3 . T h e f o r m e r a r e i s o l a t e d i n l o w y i e l d s , w h e r e a s i t i s t h e l a t t e r t h a t h a v e b e e n u s e d t o a g r e a t a d v a n t a g e i n o u r a n d o t h e r l a b o r a t o r i e s f o r t h e s t r u c t u r a l e l u c i d a t i o n o f 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 . V . l N a t u r e a n d m o d e o f p r o p a g a t i o n o f b a c t e r i o p h a g e B a c t e r i o p h a g e s o r ' p h a g e s ' a r e v i r u s e s t h a t i n f e c t b a c t e r i a , m u l t i p l y i n g w i t h i n t h e h o s t c e l l a n d e v e n t u a l l y k i l l i n g i t . 2 0 6 - 2 0 8 M a n y d i f f e r e n t s t r a i n s o f p h a g e s h a v e b e e n i s o l a t e d a n d c h a r a c t e r i z e d s i n c e T w o r t f i r s t r e p o r t e d t h e d i s c o v e r y o f a b a c t e r i o p h a g e i n 1 9 1 5 . P h a g e s a r e t h e m o s t e x t e n s i v e l y s t u d i e d g r o u p o f v i r u s e s t o d a y , d e s p i t e b e i n g t h e l a s t m a j o r g r o u p t o b e r e c o g n i z e d . T h i s I s l a r g e l y d u e t o t h e t e c h n i c a l l y e a s i e r p r o p a g a t i o n a n d m a n i p u l a t i o n o f p h a g e s c o m p a r e d t o o t h e r t y p e s o f v i r u s e s . M o r e o v e r , t h e y a r e r e l a t i v e l y e a s y t o i s o l a t e f r o m a n y b a c t e r i a l e n v i r o n m e n t e . g . g a s t r o i n t e s t i n a l t r a c t a n d s e w -1 3 3 S t r u c t u r a l l y , p h a g e s a r e m o r e c o m p l e x t h a n o t h e r v i r u s t y p e s . B r a d l e y 2 * ^ h a s c a t e g o r i z e d t h e s e p h a g e s i n t o s i x m o r p h o l o g i c a l t y p e s , A - F ( s e e F i g . V . l . I . ) . T h e t y p e s A - C c o n t a i n t w o s t r a n d s o f D N A , w h i l e - 134 -2 - D N A 2 - D N A 2 - D N A 1 - D N A 1 - R N A 1 - D N A F i g u r e V . I . I : B a s i c m o r p h o l o g i c a l t y p e s o f b a c t e r i o p h a g e s w i t h the t y p e s o f n u c l e i c a c i d . Head F i g u r e V . l . I I : A diagrammatic i l l u s t r a t i o n o f a b a c t e r i o p h a g e . 135 type D has one. These f o u r types a r e unique t o phages. Types E and F c o n t a i n a s t r a n d o f RNA and a s t r a n d o f DNA r e s p e c t i v e l y . A diagram-m a t i c i l l u s t r a t i o n o f a phage i s shown i n F i g . V . l . I I . I n an e x t e n s i v e s t u d y on phages a c t i v e 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 o f the genus K l e b s i e l l a . Rieger-Hug and S t i r m 1 - ^ 2 r e p o r t e d t h a t t h i r t y o f these phages b e l o n g t o the B r a d l e y type C, twelve o f type B and t h r e e o f type A. E . c o l i v i r u s e s a r e , however, known to b e l o n g t o a l l s i x B r a d l e y m o r p h o l o g i c a l t y p e s , and these c o l i p h a g e s have been e x t e n s i v e l y s t u -d i e d 2 1 0 . When phages i n f e c t and m u l t i p l y w i t h i n a s u s c e p t i b l e h o s t , they l y s e the c e l l by p r o d u c i n g an enzyme (lysozyme) t h a t a t t a c k s the murein o f the c e l l w a l l , weakening i t so t h a t i t b u r s t s and l i b e r a t e s the progeny phages w i t h i n . The l y t i c c y c l e o f a v i r u l e n t phage i n v o l v e s the f o l l o w i n g s t a g e s 2 0 8 (see F i g . V . l . I I I . ) : ( i ) a d s o r p t i o n o f the phage onto the s u s c e p t i b l e h o s t by r e c o g n i t i o n o f s p e c i f i c r e c e p t o r s which can be f l a g e l l a , p i l i , c a p s u l e s , l i p o - p o l y s a c c h a r i d e s , t e i c h o i c a c i d - p e p t i o d o g l y c a n complexes and s u r f a c e p r o t e i n s . An e x t e n s i v e s t u d y o f t h i s p h a g e - r e c e p t o r i n t e r a c t i o n has been r e v i e w e d by L i n d b e r g 2 1 1 ( i i ) i n j e c t i o n o f v i r a l DNA ( o r RNA) i n t o the h o s t c e l l ( i i i ) r e p l i c a t i o n o f the phage n u c l e i c a c i d and phage p r o t e i n a t the expense o f the m e t a b o l i c p r o c e s s e s o f the h o s t , and ( i v ) phage m a t u r a t i o n and r e l e a s e which r e s u l t s i n the death o f the h o s t c e l l . 136 F i g u r e V . l . I l l : A schematic diagram i l l u s t r a t i n g t h e s t e p s i n the I n f e c t i o n o f a b a c t e r i u m by a b a c t e r i o p h a g e . The p r e s e n c e o f a phage on i t s h o s t b a c t e r i a l lawn can be d e t e c t e d by i t s c h a r a c t e r i s t i c plaque morphology i . e . the p l a q u e p r o p e r ( c l e a r s p o t w i t h l y s e d c e l l s ) i s surrounded by a h a l o ( t r a n s l u c e n t s p ot) i n which the b a c t e r i a l growth i s d e c a p s u l a t e d (see F i g . V . 2 . I V ) . Plaque s i z e and morphology are c h a r a c t e r i s t i c o f a g i v e n phage type and thus - 137 -a r e o f t e n u s e f u l i n i s o l a t i o n and d i s t i n g u i s h i n g d i f f e r e n t phages. The h a l o f o r m a t i o n i s due to the p r o d u c t i o n o f excess f r e e s p i k e s , which have been shown to c o n t a i n the c a p s u l e depolymerase, d u r i n g the b i o -s y n t h e s i s o f progeny v i r u s i n the h o s t c e l l . These s p i k e s d i f f u s e from the p l a q u e and c a t a l y z e the h y d r o l y s i s o f the s u r r o u n d i n g b a c t e r i a l c a p s u l e s ' 1 . V . l . l E n d o g l y c a n a s e s a s s o c i a t e d w i t h b a c t e r i o p h a g e S t r u c t u r a l s t u d i e s c onducted on 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 have r e v e a l e d polymers b u i l t up o f r e p e a t i n g u n i t s , and e x h i b i t i n g many d i f f e r e n t s t r u c t u r a l p a t t e r n s ^ . F o r most o f these c a p s u l a r p o l y s a c c h a -- 138 -r i d e s , t h e r e e x i s t s one ( v e r y r a r e l y more) s p e c i f i c phage c o n t a i n i n g an enzyme which i s c a p a b l e o f d e g r a d i n g a p a r t i c u l a r c a p s u l a r p o l y s a c c h a -r i d e i n t o o l i g o s a c c h a r i d e s c o r r e s p o n d i n g to one o r more r e p e a t i n g T O O u n i t s x . These phage-borne enzymes are c a t e g o r i z e d a c c o r d i n g to the type o f r e a c t i o n c a t a l y z e d , and the genus o f the b a c t e r i a l h o s t . The commonly found enzymes are the endoglycanases ( g l y c o s i d e h y d r o l a s e s ) , but r e c e n t l y some l y a s e s have been r e p o r t e d e.g. K l e b s i e l l a b a c t e r i o -phages <£5 2 13, ^ 6 4 2 1 ^ and <£14^ 2. These l y a s e s ( o r e l i m i n a s e s ) a c t on e x o p o l y s a c c h a r i d e s to produce o l i g o s a c c h a r i d e s w i t h n o n - r e d u c i n g , t e r m i n a l u n s a t u r a t e d g l y c u r o n i c a c i d s . These types o f v i r a l p e n e t r a s e s have been s t u d i e d u s i n g phages f o r E. c o l i . K l e b s i e l l a . S a l m o n e l l a . S h i g e l l a , P r o t e u s , Pseudomonas, Rhizobium and S t r e p t o c o c c u s 1 ^ 3, R e c e n t l y , Kwiatkowski and c o w o r k e r s 2 1 5 r e p o r t e d on the endo-N-a c e t y l n e u r a m i n i d a s e a c t i v i t y o f the b a c t e r i o p h a g e s i s o l a t e d f o r E. c o l i K235 and K92. I n an e x c e l l e n t r e v i e w on the d e g r a d a t i o n o f b a c t e r i a l s u r f a c e c a r b o h y d r a t e s by v i r u s - a s s o c i a t e d enzymes, Geyer e t a l 1 - ^ p r e s e n t e d the c u r r e n t knowledge on the f o l l o w i n g a s p e c t s o f phages: i s o l a t i o n and p u r i f i c a t i o n , e l e c t r o n m i c r o s c o p y and enzymology. In a c o m p a r a t i v e s t u d y o f h o s t c a p s u l e depolymerases a s s o c i a t e d w i t h f i f t y -f i v e K l e b s i e l l a phages, Rieger-Hug and S t i r m 1 ^ 2 r e p o r t e d the f o l l o w i n g o b s e r v a t i o n s : ( i ) i n most c a s e s c l e a v a g e o c c u r s on e i t h e r s i d e o f the sugar u n i t c a r r y i n g the n e g a t i v e c h a r g e ( s ) , but r e d u c i n g g l y c u r o n i c a c i d s a r e n o t produced; ( i i ) most o f t e n , the r e d u c i n g end sugar formed i s s u b s t i t u t e d a t C-3; ( i i i ) i n the m a j o r i t y o f c a s e s , / 9 - g l y c o s i d i c l i n k a g e s a r e h y d r o l y z e d ; - 1 3 9 -( i v ) i n m o s t p o l y s a c c h a r i d e s w h i c h a r e a c t e d u p o n b y s e v e r a l p h a g e e n z y m e s , t h e s a m e g l y c o s i d i c b o n d s a r e c l e a v e d b y t h e d i f f e r e n t a g e n t s . D e p o l y m e r i z a t i o n o f 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 b y t h e u s e o f p h a g e - b o r n e e n z y m e s a l l o w s : ( i ) t h e g e n e r a t i o n o f s e l e c t i v e l y c l e a v e d o l i g o s a c c h a r i d e s ( i n h i g h y i e l d s ) c o r r e s p o n d i n g t o o n e o r m o r e r e p e a t i n g u n i t s 1 3 2 ; ( i i ) a c i d - o r b a s e - l a b i l e n o n - c a r b o h y d r a t e s u b s t i t u e n t s 2 1 ^ > 2 1 7 ( e . g . O - a c e t y l , a n d a c e t a l - l i n k e d p y r u v i c a c i d ) t o r e m a i n i n t a c t o n t h e o l i g o s a c c h a r i d e r e p e a t i n g u n i t . T h i s i s d i f f i c u l t t o a c h i e v e u s i n g c h e m i c a l m e t h o d s o f d e g r a d a t i o n . T h e s e o l i g o s a c c h a r i d e s g e n e r a t e d b y p h a g e a c t i o n m a y s u b s e q u e n t l y b e u s e d : ( I ) f o r t h e v e r i f i c a t i o n o f t h e s t r u c t u r e s o f t h e o r i g i n a l p o l y s a c c h a r i d e s ; ( i i ) a s s u b s t r a t e s f o r n . m . r . , m a s s - s p e c t r o m e t r i c a n d x - r a y d i f f r a c t i o n s t u d i e s ; ( i i i ) i n t h e s t u d y o f c o n f o r m a t i o n s i n s o l u t i o n 1 ^ 6 . ( i v ) a s a s o u r c e o f c o m p l e x a n d n o v e l o l i g o s a c c h a r i d e s , a n d ( v ) f o r c o u p l i n g a s h a p t e n s t o i m m u n o g l o b u l i n s 2 1 8 f o r i m m u n o l o g i c a l s t u d i e s . P h a g e s a r e o f t e n d e s i g n a t e d b y t h e G r e e k l e t t e r <f>, f o l l o w e d b y t h e n u m b e r o f t h e s e r o t y p e t h a t a c t s a s t h e h o s t s t r a i n e . g . <f>79 i s t h e p h a g e t h a t i s p r o p a g a t e d o n , a n d d e p o l y m e r i z e s t h e c a p s u l a r p o l y s a c c h a -r i d e o f K l e b s i e l l a K 7 9 . I n t h e f o l l o w i n g s e c t i o n , d e p o l y m e r i z a t i o n s u s i n g (f>79 a n d # 4 4 , o n t h e c a p s u l a r p o l y s a c c h a r i d e s o f K l e b s i e l l a K 7 9 a n d E . c o l i K 4 4 r e s p e c t i v e l y , a r e p r e s e n t e d . 140 -V.2 BACTERIOPHAGE-INDUCED DEPOLYMERIZATION OF THE CAPSULAR POLYSACCHA-RIDE OF K l e b s i e l l a K79 V.2.1 I n t r o d u c t i o n The purpose o f u s i n g <f>19 to degrade the c a p s u l a r p o l y s a c c h a r i d e o f K l e b s i e l l a K79 was to gen e r a t e o l i g o s a c c h a r i d e s r e p r e s e n t i n g s u b u n i t s o f the p o l y s a c c h a r i d e . The h e p t a s a c c h a r i d e r e p e a t i n g u n i t o f the K79 p o l y s a c c h a r i d e , which was not o b t a i n e d u s i n g c h e m i c a l d e g r a d a t i v e methods (see S e c t i o n I V . l . ) , makes i t an i n t e r e s t i n g s u b s t r a t e f o r s e l e c t i v 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 - b o r n e e n d o g l y c a n a s e . These 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 phage d e p o l y m e r i z a t i o n o f the K79 p o l y s a c c h a r i d e , were th e n to be used as model compounds f o r n.m.r. and m a s s - s p e c t r a l s t u d i e s . Moreover, d e t e r m i n a t i o n o f the r e d u c i n g end o f th e s e o l i g o s a c c h a r i d e s h e l p s t o c h a r a c t e r i z e the g l y c o s i d a s e a c t i v i t y o f the enzyme a s s o c i a t e d w i t h $79. T h i s knowledge c o u l d be u s e f u l i n the f u t u r e . V.2. 2 R e s u l t s and D i c u s s s i o n I s o l a t i o n o f $79 - $79 was i s o l a t e d from sewage u s i n g the s e r o l o g i -c a l t e s t - s t r a i n f o r K l e b s i e l l a K79 a n t i g e n as a p r o s p e c t i v e h o s t , and s e l e c t i n g f o r pla q u e morphology. When i t was o r i g i n a l l y i s o l a t e d , $79 gave r e a s o n a b l e s i z e d h a l o e d p l a q u e s (3mm diam e t e r ) on the K79 b a c t e r i a l - 1 4 1 -l a w n . H o w e v e r w i t h p r o l o n g e d $ 7 9 p r o p a g a t i o n , t h e p l a q u e s d e c r e a s e d i n s i z e t o ' p i n h o l e s ' ( - 1 m m d i a m e t e r ) a n d e v e n t u a l l y l o s t t h e i r h a l o s a l t o g e t h e r . C r o s s - r e a c t i v i t y o f K l e b s i e l l a K 7 9 w i t h o t h e r K l e b s i e l l a p h a g e s -I n a n e x t e n s i v e s t u d y e m p l o y i n g s e v e n t y - f o u r s e r o l o g i c a l l y d i f f e r e n t K l e b s i e l l a s t r a i n s , R i e g e r - H u g a n d S t i r m 1 3 2 t e s t e d t h e h o s t r a n g e o f f i f t y - f i v e K l e b s i e l l a p h a g e s . T h e y r e p o r t e d t h a t t h e v i r a l d e p o l y m e r -a s e s w e r e f o u n d t o b e v e r y s p e c i f i c i . e . 3 3 c r o s s - r e a c t i n g w i t h n o n e , 1 8 w i t h o n e , 2 w i t h t w o a n d 1 e a c h w i t h t h r e e o r f o u r h e t e r o l o g o u s p o l y s a c -c h a r i d e s . H o w e v e r n o p h a g e - c r o s s r e a c t i o n o n K l e b s i e l l a K 7 9 w a s r e p o r t e d . A s t h e e n z y m e a c t i v i t y a s s o c i a t e d w i t h t h e i s o l a t e d $ 7 9 h a d w e a k -e n e d ( i n d i c a t e d b y i t s ' p i n h o l e ' p l a q u e s ) w i t h p r o l o n g e d p r o p a g a t i o n , a n a l t e r n a t i v e p h a g e w h i c h i s a c t i v e o n K 7 9 b a c t e r i a w a s s c r e e n e d f r o m t h e s t o c k o f K l e b s i e l l a p h a g e s t h a t w e h a v e i n o u r c o l l e c t i o n . T h u s b a c t e r i a l l a w n s o f K l e b s i e l l a K 7 9 w e r e t e s t e d f o r e n z y m i c c r o s s -r e a c t i o n s u s i n g s u s p e n s i o n s o f t h e f o l l o w i n g K l e b s i e l l a p h a g e s : $ 1 , $ 5 , $ 1 7 , $ 2 1 , $ 4 4 , $ 4 6 a n d $ 7 0 . T h e c r i t e r i a f o r t h e c h o i c e o f t h e s e p h a g e s w e r e : ( i ) t h e p h a g e e x h i b i t s s t r o n g e n z y m i c a c t i v i t y o n i t s h o s t , a n d ( i i ) t h e c a p s u l e o f t h e h o s t b a c t e r i u m h a s s t r u c t u r a l s i m i l a r i t i e s w i t h t h a t o f K 7 9 . H o w e v e r , n o p l a q u e w a s o b s e r v e d f o r a l l o f t h e p h a g e s t e s t e d . T h i s r e s u l t r e i n f o r c e s t h e o b s e r v a t i o n b y R i e g e r - H u g a n d S t i r m 1 3 2 c o n c e r n i n g t h e h i g h s p e c i f i c i t y o f m o s t b a c t e r i o p h a g e s . - 1 4 2 -P r o p a g a t i o n o f # 7 9 - # 7 9 w a s p r o p a g a t e d a c c o r d i n g t o t h e s t a n d a r d p r o c e d u r e s o f v i r o l o g y 2 0 6 . A t o t a l o f 1 . 2 5 x 1 0 1 3 p . f . u . o f # 7 9 w a s e v e n t u a l l y o b t a i n e d a f t e r e n c o u n t e r i n g a l o t o f d i f f i c u l t i e s p r o p a g a t i n g t h e p h a g e t o a h i g h c o n c e n t r a t i o n . T h e p r o b l e m w a s a t t r i b u t e d t o t h e p o o r e n z y m e a c t i v i t y a s s o c i a t e d w i t h # 7 9 . I n # 7 9 p l a q u e a s s a y , p i n h o l e - s i z e d p l a q u e s w i t h o u t h a l o s w e r e o b s e r v e d , a n d i t w a s o n t h e b a s i s o f t h e s e ' p i n h o l e s ' t h a t # 7 9 w a s e n u m e r a t e d . A l t m a n a n d c o w o r k e r s h a v e r e p o r t e d 2 1 9 e n c o u n t e r i n g d i f f i c u l t i e s i n t h e p r o p a g a t i o n o f E . c o l i # 2 8 w h i c h i n c i d e n t a l l y a l s o g a v e p i n h o l e - s i z e d p l a q u e s . D e p o l y m e r i z a t i o n o f K 7 9 p o l y s a c c h a r i d e a n d i s o l a t i o n o f p r o d u c t S t i r m a n d c o w o r k e r s f i r s t r e p o r t e d 2 1 2 t h e u s e o f 1 0 1 3 p . f . u . o f # 1 1 t o d e g r a d e l g o f K l e b s i e l l a K l l c a p s u l a r p o l y s a c c h a r i d e r e s u l t i n g i n g o o d y i e l d s o f o l i g o s a c c h a r i d e s . E v e r s i n c e t h e n , t h e u s e o f 1 0 1 3 p . f . u . o f p h a g e t o d e g r a d e 1 g o f h o s t p o l y s a c c h a r i d e h a s b e e n g e n e r a l l y p r a c -t i c e d . H o w e v e r , d u e t o t h e w e a k e n z y m e a c t i v i t y a s s o c i a t e d w i t h # 7 9 a n e x c e s s o f # 7 9 w a s u s e d i n t h e d e p o l y m e r i z a t i o n o f t h e K 7 9 p o l y s a c c h a -r i d e . T h e d e p o l y m e r i z a t i o n o f t h e K 7 9 p o l y s a c c h a r i d e w a s c o n d u c t e d u s i n g t h e m e t h o d o f D u t t o n a n d c o w o r k e r s 2 2 0 . T h e p h a g e - p o l y s a c c h a r i d e m i x t u r e w a s i n c u b a t e d f o r 3 d a y s , c h l o r o f o r m b e i n g a d d e d t o p r e v e n t b a c t e r i a l g r o w t h . T h e m i x t u r e w a s c o n c e n t r a t e d a n d t h e n d i a l y z e d . T h e d i a l y z a t e w a s t r e a t e d w i t h c a t i o n - e x c h a n g e r e s i n , c o n c e n t r a t e d a n d t h e r e s i d u e f r a c t i o n a t e d b y g e l - p e r m e a t i o n c h r o m a t o g r a p h y ( s e e A p p e n d i x I I ) t o g i v e a s i n g l e p r o d u c t K 7 9 # D . - 1 4 3 -C h e m i c a l a n a l y s i s o f K 7 9 $ D - S u g a r a n a l y s i s c o n d u c t e d o n K 7 9 $ D g a v e r h a m n o s e , g l u c o s e a n d g a l a c t o s e i n t h e m o l a r r a t i o s o f 2 . 5 : 1 . 6 : 1 . 0 . T h e g l u c u r o n o s y l r e s i d u e w a s n o t d e t e c t e d i n t h e g . l . c . a s t h e c a r b o x y l g r o u p w a s n o t r e d u c e d . T h i s r e s u l t i s s i m i l a r t o t h a t o b t a i n e d f o r t h e K 7 9 p o l y s a c c h a r i d e ( s e e T a b l e I V . 1 . I ) . I n o r d e r t o d e t e r m i n e t h e n a t u r e o f t h e r e d u c i n g e n d a n d t h e d e g r e e o f d e p o l y m e r i z a t i o n , t h e m e t h o d d e v i s e d b y M o r r i s o n 2 2 1 w a s u s e d . A s a m p l e o f K 7 9 $ D w a s r e d u c e d t o i t s a l d i t o l , a n d t h e s u g a r s r e l e a s e d u p o n h y d r o l y s i s w e r e c o n v e r t e d t o a l d i t o l a n d a l d o n o n i t r i l e a c e t a t e s . T h e r e s u l t f r o m t h e g . l . c . a n a l y s i s s h o w e d t h e p r e s e n c e o f p e r a c e t y l a t e d a l d o n o n i t r i l e s o f r h a m n o s e , g l u c o s e a n d g a l a c t o s e , a n d t h e a l d i t o l a c e t a t e o f g a l a c t o s e , t h u s i n d i c a t i n g t h a t g a l a c t o s e i s t h e r e d u c i n g e n d . T h e m o l a r p r o p o r t i o n o f t h e g a l a c t o s e r e d u c i n g e n d ( d e t e c t e d a s g a l a c t i t o l h e x a a c e t a t e ) t o t h e i n - c h a i n g a l a c t o s y l r e s i d u e ( d e t e c t e d a s a l d o n o n i t r i l e a c e t a t e ) i s 0 . 3 5 : 1 . 0 0 . T h i s i n d i c a t e s t h a t K 7 9 $ D c o n t a i n e d a t o t a l o f 2 8 m o n o s a c c h a r i d e s i . e . a t e t r a m e r o f t h e h e p t a -s a c c h a r i d e r e p e a t i n g u n i t . M e t h y l a t i o n o f K 7 9 $ D a n d K 7 9 $ D a l d i t o l , f o l l o w e d b y r e d u c t i o n o f t h e u r o n i c e s t e r a n d c o n v e r s i o n t o p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s r e v e a l e d t h e c o m p o u n d s l i s t e d i n T a b l e V . 2 . I I . T h e s e r e s u l t s a r e s i m i l a r t o t h o s e o f t h e m e t h y l a t e d a n d c a r b o x y l - r e d u c e d n a t i v e K 7 9 p o l y s a c c h a r i d e ( s e e T a b l e I V . 1 . I I I ) . T h e p r e s e n c e o f 1 , 2 , 4 , 5 , 6 - p e n t a -O - m e t h y l g a l a c t i t o l , i n t h e m e t h y l a t i o n a n a l y s i s o f K 7 9 $ D a l d i t o l , c o n f i r m e d g a l a c t o s e a s t h e t e r m i n a l r e d u c i n g e n d . M o r e o v e r , t h e 0 . 1 2 m o l e % d e c r e a s e i n t h e 2 , 4 , 6 - t r i - O - m e t h y l g a l a c t o s e , c o m p a r e d t o t h e c o r r e s p o n d i n g d a t a i n T a b l e V . 2 . I I , c o l u m n I I , i s a l s o i n d i c a t i v e o f t h e Table V.2.I: N.M.R. data f o r K794P and the native K79 polysaccharide. Compound* 1H-N.n.r. data l3C-N.m r. 6 b J l , 2 C Integral Assignment 0 P.p.m e Assignment* (Hz) proton i R h a l u J c a l l - ^ C l c A i - i R h a l - l R h a l - 5.62 s 1.0 £<31c 173.00 C-0 of GlcA o P 4 P a a a a 5.33 s 1.0 Glc 104.79 3-Cal 1 a P Glc 5.05 s 2.0 3-Rha 103.81 UiGlcA 6 a P a 4.95 s 1.0 2Rha a 103.57 iRha a I > Glc 4.65 8 1.0 2c*l 102.77 2.Rha P a 4.49 8 1.0 i ^ C l c A 101.69 ^ C l c P a K79 polysaccharide 1.32 s 9 CH 3 of Rha 99.37 Glc a 61.86] 2cal [c-6 -l- P 61.83J Glc a 17.55 C-6 of ^ Rha a 17.48 C-6 of ^ Rha C o m p o u n d 3 ^ - N . m . r . d a t a 1 3 C - N . m . : r . 6 b J l . 2 C (Hz) I n t e g r a l p r o t o n A s s i g n m e n t 0 P . p . m e A s s i g n m e n t * K 7 9 # D 5 . 5 4 s 1 . . 0 ^ G l c a 1 0 4 . 8 0 3cal P 5 . 2 9 s 1 , . 0 G l c a 1 0 3 . 7 9 3 - ^ G l c A P 5 . 0 8 s 2 . 0 3-Rha a 1 0 3 . 5 3 2Rha a 4 . 9 3 s 1 . 0 2-Rha a 1 0 2 . 9 4 2 R h a a 4 . 7 5 8 1 . 0 3 - G a l P 4 . 4 9 8 1 . 0 3 - ^ G l c A P 1 0 1 . 7 3 £ G 1 C — oc 1 . 3 3 b 9 . 0 C H 3 o f R h a 9 9 . 6 5 G l c — — aC 1 7 . 5 3 C H 3 o f R h a a F o r t h e o r i g i n o f K 7 9 # D , s e e t e x t . b C h e m i c a l s h i f t r e l a t i v e t o i n t e r n a l a c e t o n e a s s i g n e d a t 6 2 . 2 3 d o w n f i e l d f r o m e x t e r n a l s o d i u m 4 , 4 - d i m e t h y l - 4 - s i l a p e n t a n e - l - s u l f o n a t e ( D . S . S . ) . c K e y : b - b r o a d , u n a b l e t o a s s i g n a c c u r a t e c o u p l i n g c o n s t a n t . - F o r e x a m p l e - R h a A j - r e f e r s t o t h e a n o m e r i c p r o t o n o f a 3 - l i n k e d r h a m n o s y l r e s i d u e i n t h e a - a n o m e r i c c o n f i g u r a t i o n . T h e a b s e n c e o f a n u m e r i c a l p r e f i x i n d i c a t e s a ( t e r m i n a l ) n o n r e d u c i n g g r o u p . - C h e m i c a l s h i f t r e l a t i v e t o i n t e r n a l a c e t o n e a s s i g n e d a t 3 1 . 0 7 p . p . m . d o w n f i e l d f r o m e x t e r n a l D . S . S . - A s f o r d , b u t f o r a n o m e r i c 1 3 C n u c l e i . A l l n . m . r . s p e c t r a a r e g i v e n i n A p p e n d i x l I I . - 146 -Ta b l e V . 2 . I I : M e t h y l a t i o n a n a l y s e s o f K 7 9$D M e t h y l a t e d s u g a r 3 Mole % b (as a l d i t o l a c e t a t e s ) I c I I I I I 3,4-Rha 15 14.7 14.3 2,4-Rha 28 25.1 28.0 2,4,6-Gal 14 15.3 13.3 1,2,4,5,6-Gal 1.7 d 2,3,4,6-Glc 14 16.7 14.9 2,3,4-Glc 14 15.3 15.1 2-Glc 15 12.8 12.5 a 2,4-Rha - l , 3 , 5 - t r i - 0 - a c e t y l - 2 , 4 - d i - 0 - m e t h y l r h a m n i t o l ; b U s i n g DB-17 c a p i l l a r y column programmed from 180° f o r 1 min, t h e n 2°/min t o 250°. c I, o r i g i n a l a c i d i c K79 p o l y s a c c h a r i d e , m e t h y l a t e d and reduced; I I , m e t h y l a t e d and r e d u c e d K79$D; I I I , m e t h y l a t e d and r e d u c e d K79$D a l d i t o l . d Due t o i t s h i g h v o l a t i l i t y , q u a n t i t a t i o n was i n a c c u r a t e . t e t r a m e r i c r e p e a t i n g - u n i t n a t u r e o f K79$D. The n.m.r. (^H and 1 3 C ) s p e c t r a o f K79$D showed seven anomeric s i g n a l s , which c l o s e l y resemble those o f the K79 p o l y s a c c h a r i d e ( s e e T a b l e V . 2 . I ) . On r e d u c t i o n o f K79$D to i t s o l i g o s a c c h a r i d e a l d i t o l , no d i s t i n c t changes were o b s e r v e d i n the ^H-n.m.r. spectrum. Due to the 147 -h i g h degree o f p o l y m e r i z a t i o n o f K79$D, no d i s a p p e a r a n c e o f the r e d u c i n g g a l a c t o s e s i g n a l s w a s o b s e r v e d on r e d u c t i o n . S i m i l a r l y f o r the same re a s o n , a f a s t atom bombardment-mass s p e c t r o m e t r i c a n a l y s i s o f K79$D and p e r m e t h y l a t e d K79$D a l d i t o l f a i l e d t o g i v e c o n c l u s i v e e v i d e n c e . V.2.3 C o n c l u s i o n The sum o f these experiments i n d i c a t e K79$D to have the f o l l o w i n g s t r u c t u r e . G l c A J — ^ R h a ^ R h a i - ^ R h a l 4 P  a a a 1 G l c 6 a 1 G l c ^ 9 ^ G a l i - l G l c A ^ R h a i - ^ R h a i - ^ R h a i - ^ G a l - O H P a a a P 4 a 1 G l c 6 a 1 G l c The endoglycanase a s s o c i a t e d w i t h $79 has y 9 - g a l a c t o s i d a s e a c t i v i t y . I n t e r e s t i n g l y , the p o s i t i o n o f c l e a v a g e i n d u c e d by $79 agre e s w i t h the o b s e r v a t i o n r e p o r t e d by Rieger-Hug and S t i r m 1 3 2 i _ e _ c l e a v a g e o c c u r s on e i t h e r s i d e o f the sugar u n i t c a r r y i n g the n e g a t i v e charge, b u t r e d u c i n g g l y c u r o n i c a c i d s a r e not produced. A n o t h e r phage a c t i v e on the K l e b s i e l l a K79 b a c t e r i a was l a t e r 148 -i s o l a t e d from Grahamstown (S. A f r i c a ) sewage by Dr. H. P a r o l i s . Attempts a t p r o p a g a t i o n by P. G e h r s 2 2 2 r e v e a l e d the pr e s e n c e o f two types o f pl a q u e on the K79 b a c t e r i a l lawn i . e . s m a l l ' p i n h o l e ' p l a q u e s and l a r g e h a l o e d p l a q u e . T h e i r r e l a t i o n s h i p i s s t i l l t o be i n v e s t i g a t e d . Other i n v e s t i g a t o r s have a l s o r e p o r t e d b a c t e r i o p h a g e s which gave r i s e t o s m a l l p l a q u e s , and on phage d e g r a d a t i o n o f t h e i r r e s p e c t i v e h o s t p o l y s a c c h a -r i d e r e s u l t s i n o l i g o s a c c h a r i d e s o f s i z e s r a n g i n g from two to f i v e r e p e a t i n g u n i t s ^ . V . 2 . 4 E x p e r i m e n t a l G e n e r a l methods - See S e c t i o n I I I . I s o l a t i o n o f K l e b s i e l l a #79 - A sample o f Vancouver sewage (900mL), t e n f o l d n u t r i e n t b r o t h (lOOmL), and an a c t i v e l y - g r o w i n g K l e b s i e l l a K79 c u l t u r e (30 mL n u t r i e n t b r o t h ) were combined and i n c u b a t e d w i t h s h a k i n g a t 37° f o r 1.5d. A sample (50 mL) o f t h i s e n richment c u l t u r e was t r a n s f e r r e d i n t o a f l a s k , CHCI3 (lOmL) was added and t h o r o u g h l y mixed by s h a k i n g v i g o r o u s l y (20 min.). A f t e r c e n t r i f u g a -t i o n (20 min.), the s u p e r n a t a n t was p i p e t t e d i n t o a s t e r i l e f l a s k . A few drops o f CHCI3 were added t o p r e v e n t b a c t e r i a l growth. T h i s s o l u t i o n was the n s c r e e n e d f o r #79 by the 'plaque a s s a y t e c h n i q u e ' (see S e c t i o n I I I . 9 . 2 ) u s i n g K l e b s i e l l a K79 as the h o s t . #79 was s e l e c t e d on the b a s i s o f i t s p l a q u e morphology-'- 3 2 • 2 1 2 , i n d i c a t e d by the presence o f - 1 4 9 -a p l a q u e w i t h a h a l o ( s e e F i g . V . l . I V ) . P u r i f i c a t i o n o f t h e $ 7 9 w a s c a r r i e d o u t b y f o u r c o n s e c u t i v e s i n g l e p l a q u e i s o l a t i o n s . C r o s s - r e a c t i v i t y o f K l e b s i e l l a K 7 9 w i t h o t h e r K l e b s i e l l a p h a g e s -T h e s c r e e n i n g f o r c r o s s r e a c t i v i t y b e t w e e n t h e K 7 9 b a c t e r i a a n d o t h e r p h a g e s w a s c o n d u c t e d b y t h e ' p l a q u e a s s a y t e c h n i q u e ' . T h e f o l l o w i n g K l e b s i e l l a p h a g e s w e r e u s e d f o r t h e s c r e e n i n g : $ 1 , $ 5 , $ 1 7 , $ 2 1 , $ 4 4 , $ 4 6 a n d $ 7 0 . B a c t e r i a l l a w n s o f t h e K 7 9 b a c t e r i a w e r e p r e p a r e d ( s e e S e c t i o n I I I . 9 . 1 ) . T o t h e d r i e d s u r f a c e o f t h e b a c t e r i a l l a w n w a s p l a c e d a d r o p l e t o f t h e v a r i o u s p h a g e s u s p e n s i o n s ( u n d i l u t e d a n d a t 1 0 " 1 d i l u -t i o n ) . A f t e r o v e r n i g h t i n c u b a t i o n , t h e b a c t e r i a l l a w n s w e r e c h e c k e d f o r p l a q u e s . N o p l a q u e s w e r e o b s e r v e d f o r a l l t h e p h a g e s s c r e e n e d . K l e b s i e l l a K 7 9 b a c t e r i a l g r o w t h c u r v e - T h e r e l a t i o n s h i p o f c e l l c o u n t p e r m L t o o p t i c a l d e n s i t y r e a d a t 5 4 5 nn I n a d e n s i t o m e t e r w a s d e t e r m i n e d i n t h e a b s o r b a n c e r a n g e 0 . 0 3 - 0 . 0 9 a s f o l l o w s . A f l a s k c o n t a i n i n g n u t r i e n t b r o t h ( l O O m L ) w a s i n o c u l a t e d w i t h a c o l o n y o f K 7 9 b a c t e r i a a n d i n c u b a t e d a t 3 7 ° w i t h a e r a t i o n . A f e w d r o p s o f D o w a n t i - f o a m F G - 1 0 e m u l s i o n w e r e a d d e d t o p r e v e n t e x c e s s i v e f o a m i n g . A l i q u o t s ( 3 m L ) w e r e r e m o v e d a t 3 0 m i n . i n t e r v a l s a n d s e r i a l l y d i l u t e d ( 1 0 " 5 t o 1 0 " 8 ) w i t h n u t r i e n t b r o t h . A n a l i q u o t ( 0 . 3 m L ) f r o m e a c h o f t h e d i l u t e d s o l u t i o n w a s i n c u b a t e d a t 3 7 ° o n n u t r i e n t a g a r o v e r n i g h t . I n d i v i d u a l c o l o n i e s o f b a c t e r i a w e r e c o u n t e d a n d t h e r e l a t i o n s h i p - 1 5 0 -b e t w e e n a b s o r b a n c e a n d b a c t e r i a l c o u n t w a s p l o t t e d ( s e e F i g . V . 2 . I . ) . P r o p a g a t i o n o f # 7 9 - # 7 9 w a s p r o p a g a t e d , u s i n g n u t r i e n t b r o t h a s m e d i u m , a c c o r d i n g t o t h e s t a n d a r d p r o c e d u r e s o f v i r o l o g y 2 0 6 . T h i s i n v o l v e d s u c c e s s i v e p r o p a g a t i o n s u s i n g i n c r e a s i n g a m o u n t s o f K 7 9 b a c t e -r i a l c u l t u r e a n d # 7 9 i . e . t u b e l y s i s , f l a s k l y s i s a n d w a s h - b o t t l e l y s i s ( s e e S e c t i o n I I I . 9 f o r d e t a i l s ) . T h e o p t i m a l c o n c e n t r a t i o n o f b a c t e r i a f o r w h i c h t h e m a x i m a l p r o p a g a t i o n o f # 7 9 w o u l d b e o b t a i n e d w a s e s t i m a t e d f r o m t h e g r o w t h c u r v e o f K l e b s i e l l a K 7 9 ( s e e F i g . V . 2 . I . ) . A r a t i o o f t h r e e p h a g e p a r t i c l e s p e r c o l o n y w a s u s e d . A t o t a l o f 1 . 2 5 x 1 0 1 3 p . f . u . o f # 7 9 w a s e v e n t u a l l y a c h i e v e d . D e p o l y m e r i z a t i o n o f K 7 9 p o l y s a c c h a r i d e a n d i s o l a t i o n o f p r o d u c t T h e d e p o l y m e r i z a t i o n w a s c a r r i e d o u t u s i n g t h e m e t h o d o f D u t t o n a n d c o w o r k e r s 2 2 0 . A n u t r i e n t b r o t h s o l u t i o n o f # 7 9 w a s c o n c e n t r a t e d ( - 4 0 0 m L ) a n d d i a l y z e d ( m o l . w t . c u t o f f 1 3 , 5 0 0 ) a g a i n s t d i s t i l l e d w a t e r ( p H ~ 7 . 0 ) f o r 2 d . T h e l i g h t b r o w n s o l u t i o n w a s c o n c e n t r a t e d ( - 5 0 0 m L ) a n d a p l a q u e a s s a y w a s c o n d u c t e d o n i t . A t o t a l o f 1 . 2 5 x 1 0 1 3 p . f . u . o f # 7 9 r e m a i n e d . T h i s # 7 9 s o l u t i o n w a s a d d e d t o t h e K 7 9 p o l y s a c c h a r i d e s o l u t i o n ( 6 4 0 m g K 7 9 p o l y s a c c h a r i d e i n 3 5 0 m L d i s t i l l e d w a t e r ) a n d i n c u b a t e d a t 3 7 ° . C h l o r o f o r m ( 3 0 m L ) w a s a d d e d t o p r e v e n t b a c t e r i a l g r o w t h . A l e s s v i s c o u s m i x t u r e w a s o b s e r v e d a f t e r 3 d , i n d i c a t i n g s o m e d e p o l y m e r i z a t i o n o f p o l y s a c c h a r i d e h a d o c c u r r e d . T h e m i x t u r e w a s c o n c e n t r a t e d a n d d i a l y z e d ( m o l . w t . c u t o f f 3 , 5 0 0 ) a g a i n s t d i s t i l l e d 151 -Absorbance (545 nm) F i g u r e V . 2 . I : C o r r e l a t i o n b e t w e e n b a c t e r i a l c o u n t a n d t h e a b s o r b a n c e f o r K l e b s i e l l a K 7 9 b a c t e r i a . - 1 5 2 -w a t e r ( 7 x ) . A M o l i s c h 8 1 • 2 2 3 t e s t c o n d u c t e d o n a c o n c e n t r a t e d s a m p l e o f t h e d i a l y z a t e s h o w e d a p o s i t i v e r e a c t i o n . T h e c o m b i n e d d i a l y z a t e s w e r e c a t i o n - e x c h a n g e d u s i n g a c o l u m n o f A m b e r l i t e I R 1 2 0 ( H + ) r e s i n . T h e c o n c e n t r a t e d e l u a n t w a s f r a c t i o n a t e d o n a c o l u m n ( 2 . 5 c m x 1 0 0 c m ) o f B i o - G e l P - 2 ( 4 0 0 m e s h , v o i d v o l u m e 1 3 0 m L ) u s i n g 5 0 0 : 5 : 2 w a t e r - p y r i d i n e -a c e t i c a c i d a s s o l v e n t . T h e c h r o m a t o g r a m s h o w e d a s i n g l e b r o a d p e a k a w a y f r o m t h e v o i d v o l u m e o f t h e c o l u m n ( s e e A p p e n d i x I I ) . P a p e r c h r o m a t o g r a p h i c ( i n s o l v e n t 1 ) a n d n . m . r . a n a l y s e s o f s e l e c t e d s a m p l e s s h o w e d c l o s e s i m i l a r i t i e s b e t w e e n t h e m . H e n c e t h e s e s a m p l e s w e r e c o m b i n e d a n d l a b e l l e d K 7 9 $ D ( 2 4 7 m g ) . S u g a r a n a l y s i s - A s a m p l e ( l O m g ) o f K 7 9 $ D w a s h y d r o l y z e d u s i n g 2M T F A f o r 1 4 h a t 9 5 ° . A f t e r r e m o v a l o f t h e e x c e s s a c i d b y r e p e a t e d c o e v a p o r a t i o n w i t h w a t e r ( 3 x ) , t h e h y d r o l y z a t e w a s c h r o m a t o g r a p h e d o n p a p e r i n s o l v e n t 1 . T h e r e l e a s e d s u g a r s w e r e r e d u c e d w i t h a q u e o u s N a B H ^ ( r . t . , 2 0 m i n . ) a n d a c e t y l a t e d u s i n g 1 : 1 p y r i d i n e - A C 2 0 ( a t 9 5 ° f o r 2 0 m i n . ) . T h e w o r k u p p r o c e d u r e f o r b o t h r e a c t i o n s w a s a s p r e v i o u s l y d e s c r i b e d ( s e e S e c t i o n I I I . 3 . 2 . ) . G . l . c . a n a l y s i s o f t h e a c e t y l a t e d a l d i t o l s u s i n g D B - 1 7 c a p i l l a r y c o l u m n o n p r o g r a m ( i ) s h o w e d r h a m n o s e , g l u c o s e a n d g a l a c t o s e i n t h e m o l a r p r o p o r t i o n s 2 . 5 : 1 . 6 : 1 . 0 . R e d u c t i o n t o K 7 9 $ D a l d i t o l - A s a m p l e o f K 7 9 $ D ( 8 0 m g ) w a s r e d u c e d w i t h a q u e o u s N a B H ^ a t r . t . f o r 4 h . T h e e x c e s s N a B H 4 w a s d e c o m p o s e d b y a d d i n g A m b e r l i t e I R 1 2 0 ( H + ) r e s i n , a n d t h e r e s u l t a n t - 153 -b o r i c a c i d was removed by c o e v a p o r a t i o n w i t h MeOH ( 4 x ) . A 1H-n.m.r. a n a l y s i s on K79eiD a l d i t o l d i d not show any s i g n i f i c a n t changes. D e t e r m i n a t i o n o f r e d u c i n g end and degree o f p o l y m e r i z a t i o n 2 2 - ' - - A sample o f K79#D a l d i t o l (10 mg) was h y d r o l y z e d u s i n g 2M TFA f o r 14 h a t 95°. A f t e r removing the excess TFA, the anhydrous r e s i d u e was t r e a t e d w i t h 5% h y d r o x y l a m i n e h y d r o c h l o r i d e i n p y r i d i n e (5 mL) and h e a t e d a t 95° f o r 20 min. Anhydrous A C 2 O (5 mL) was added t o the c o o l e d r e a c t i o n m i x t u r e and f u r t h e r h e a t e d ( a t 95° f o r 25 min). Removal o f excess A C 2 O , p y r i d i n e and hy d r o x y l a m i n e h y d r o c h l o r i d e f o l l o w e d the p r o c e d u r e as p r e v i o u s l y d e s c r i b e d i n S e c t i o n I I I . 3.2. G . l . c . a n a l y s i s on DB-17 c a p i l l a r y column u s i n g program ( i ) r e v e a l e d the a c e t y l a t e d aldono-n i t r i l e s o f rhamnose, g l u c o s e , g a l a c t o s e , and g a l a c t i t o l h e x a a c e t a t e i n the molar r a t i o s o f 2.90:1.00:0.35 r e s p e c t i v e l y . M e t h y l a t i o n a n a l y s i s - M e t h y l a t i o n o f K79#D (10 mg) and K79#D a l d i t o l (10 mg) was e f f e c t e d u s i n g the Hakomori procedure 1-'- 2 > 1 1 3. The p r o d u c t s , a f t e r C H 2 C l 2 e x t r a c t i o n and passage t h r o u g h a column o f Sephadex LH-20 r e s i n , were shown t o be f u l l y m e t h y l a t e d by i . r . s p e c t r o -scopy. The m e t h y l a t e d sample was red u c e d u s i n g L i A l H ^ i n r e f l u x i n g o x o l a n e f o r 1.5 h and worked up as p r e v i o u s l y d e s c r i b e d . H y d r o l y s i s of the reduced, m e t h y l a t e d sample u s i n g 2M TFA was co n d u c t e d f o r 7 h a t 95°, and the e x t e n t o f h y d r o l y s i s was m o n i t o r e d by paper chromatography i n s o l v e n t 4. The r e l e a s e d sugars were re d u c e d w i t h aqueous NaBH^ - 154 -( r . t . , 20 min) and a c e t y l a t e d u s i n g 1:1 p y r i d i n e - A c 2 0 (95° , 20 min). Fo r g . l . c . d a t a , see T a b l e V . 2 . I I . Mass s p e c t r o m e t r i c a n a l y s e s - F a s t atom bombardment mass s p e c t r o m e t r i c a n a l y s e s ( f o r e x p e r i m e n t a l d e t a i l s , see S e c t i o n I I I . 6) o f K79#D and p e r m e t h y l a t e d K79#D a l d i t o l gave poor mass s p e c t r a . Due to the l i m i t e d range o f the d e t e c t o r (-2000 a.m.u.) compared t o the h i g h m o l e c u l a r masses o f K79#D and p e r m e t h y l a t e d K79#D a l d i t o l , the d e s i r e d m o l e c u l a r peaks and t h e i r fragments were not d e t e c t e d . A c h e m i c a l i o n i z a t i o n mass s p e c t r o m e t r i c a n a l y s i s c onducted on the p e r m e t h y l a t e d K79#D a l d i t o l a l s o f a i l e d to g i v e c o n c l u s i v e e v i d e n c e . - 155 -V . 3 B A C T E R I O P H A G E - I N D U C E D D E P O L Y M E R I Z A T I O N O F T H E C A P S U L A R P O L Y S A C C H A R I D E O F E . c o l i K 4 4 . V . 3 . 1 I n t r o d u c t i o n T h e s t r u c t u r a l s t u d y o n E . c o l i K44 c a p s u l a r p o l y s a c c h a r i d e h a s e s t a b l i s h e d 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 c o m p r i s i n g r h a m n o s e , g l u c u r o n i c a c i d , 2 - a c e t a m i d o - 2 - d e o x y g l u c o s e a n d 2 - a c e t a m i d o - 2 - d e o x y -g a l a c t o s e . T h e p r e s e n c e o f t w o a m i n o s u g a r s i n t h e r e p e a t i n g u n i t o f t h e p o l y s a c c h a r i d e m a k e s i t a n o v e l s u b s t r a t e f o r b a c t e r i o p h a g e - i n d u c e d d e p o l y m e r i z a t i o n . I t w o u l d b e i n t e r e s t i n g t o f i n d o u t i f t h e a m i n o s u g a r s h a d a n y I n f l u e n c e o n t h e g l y c o s i d a s e a c t i v i t y o f t h e e n d o g l y c a -n a s e a s s o c i a t e d w i t h t h e E . c o l i $44. M o r e o v e r , t h e p r e s e n c e o f t h r e e d i f f e r e n t t y p e s o f m o n o s a c c h a r i d e s ( i . e . o n t h e b a s i s o f t h e i r m o l e c u l a r w e i g h t s ) i n t h e r e p e a t i n g u n i t m a k e s t h e o l i g o s a c c h a r i d e s , g e n e r a t e d f r o m t h e p h a g e d e p o l y m e r i z a t i o n o f t h e p o l y s a c c h a r i d e , i d e a l m o d e l s u b s t r a t e s f o r c h a r a c t e r i z a t i o n b y n . m . r . a n d m a s s s p e c t r o m e t r y . V . 3 . 2 R e s u l t s a n d D i s c u s s i o n D e p o l y m e r i z a t i o n o f K 4 4 p o l y s a c c h a r i d e a n d i s o l a t i o n o f p r o d u c t s E . c o l i $44 w a s p r o p a g a t e d o n i t s h o s t s t r a i n E . c o l i K44 u s i n g M u e l l e r - H i n t o n b r o t h a s m e d i u m . P r o p a g a t i o n w a s c o n d u c t e d f o l l o w i n g t h e s t a n d a r d p r o c e d u r e s o f v i r o l o g y 2 0 ^ . L i k e K l e b s i e l l a $79, $44 e x h i b i t e d 156 s m a l l p l a q u e s (1 mm d i a m e t e r ) w i t h h a l o s . These c o u l d be a t t r i b u t e d to t h e i r l i m i t e d endoglycanase p r o d u c t i o n o r weak enzymic a c t i v i t y . N o n e t h e l e s s , p r o p a g a t i o n o f #44 was c o n t i n u e d u n t i l a t o t a l o f -1.0 x 1 0 1 3 p . f . u . was o b t a i n e d . The method o f Dutton and c o w o r k e r s 2 2 0 , was used i n the depolymer-i z a t i o n o f the E. c o l i K44 p o l y s a c c h a r i d e . The p h a g e - p o l y s a c c h a r i d e m i x t u r e was i n c u b a t e d f o r two days and the d e p o l y m e r i z e d p r o d u c t s were s e p a r a t e d from the m i x t u r e by d i a l y s i s . The d i a l y z a t e , a f t e r t reatment w i t h c a t i o n - e x c h a n g e r e s i n , gave a p o s i t i v e r e s u l t w i t h the M o l i s c h 81 92"^ r e a g e n t 0 1 • ^ J . Paper chromatographic a n a l y s i s o f t h i s crude p r o d u c t showed the p r e s e n c e o f some contaminants and an o l i g o s a c c h a r i d e ( S l a c t o s e 0.33). The 1H-n.m.r. spectrum o f t h i s crude p r o d u c t i s d e p i c t e d i n F i g . V.3.1. S e p a r a t i o n o f t h i s p r o d u c t by p r e p a r a t i v e paper chromatography gave two f r a c t i o n s , PI (the s i n g l e r e p e a t i n g u n i t o f the K44 p o l y s a c c h a r i d e ) and a f a s t e r - m o v i n g component 2, which was l a t e r shown to be a contami-nant from the d e p o l y m e r i z a t i o n m i x t u r e . A n a l y s i s o f the u n d e r i v a t i z e d PI by fast-atom-bombardment mass s p e c t r o m e t r y gave i n c o n c l u s i v e r e s u l t s due to the poor spectrum o b t a i n e d . Comparative •LH-n.m.r. stu d y - The n.m.r. (-"-H and i J C ) d a t a o f P i are p r e s e n t e d i n T a b l e V.3.1. A comparative a n a l y s i s o f the anomeric r e g i o n o f the ^H-n.m.r. spectrum o f the crude p r o d u c t , PI, 2 and the E.  c o l i K44 p o l y s a c c h a r i d e i s shown i n F i g . V.3.1. In F i g . V.3.1, the anomeric s i g n a l s marked w i t h an X i n d i c a t e the contaminant from the (a) 1 5 7 (b) 5 . 0 p.p.m. 5 . 3 6 4 . 6 4 x J 4 . 9 8 x (c) 570 p. p.m. (d) p .p .m. F i g u r e V . 3 . I : . ITQ " P.p.m. ^ H - n . m . r . s p e c t r a s h o w i n g t h e a n o m e r i c r e g i o n o f ( a ) t h e c r u d e p r o d u c t , ( b ) c o n t a m i n a n t 2 , ( c ) P I , a n d ( d ) c o l i K 4 4 c a p s u l a r p o l y s a c c h a r i d e . Table IV.3.1: N.M.R. data f o r PL and the native E. c o l l K44 polysaccharide. Compound8 ^H-N.m.r. data 13C-N.m.r. «° J l . 2 C (Hz) Integral proton Assignment 4* P.p.m.6 Assignment' ^GlcAl-JRhaJLJkJlcNAcl-iCalNAcl-P a a P K44 polyssaccharlde (Na +) 4.96 3 1.0 4.89 b 1.0 4.70 8 1.0 4.61 8 1.0 2.09 s 3.0 2.06 s 3.0 1.33 5 3.0 ^GlcNAcl-<k ^RhaJ-a ^GICAI-P ^GalNAc*-P CH 3 of N-Ac (GlcNAc) CH 3 of N-Ac (GalNAc) CH 3 (Rha) 175.80 175.00 172.30 104.77 102.89 101.46 98.90 61.08 54.79 52.90 ) 0 0 of GlcNAc and GalNAc C-0 (GlcA) 4cicAl-P ^GalNAc^-P 3-Rhal-a ^GlcNAci-a C-6 (GlcNAc) C-2 (GlcNAc) C-2 (GalNAc) 0 II 23.21 N-C-CH3 (GalNAc) 0 22.67 N-C-CH3 (GlcNAc) 17.52 C-6 (Rha) PI 5.36 4.96 4.89 4.70 4.60 2.09 2.06 1.34 b b X X ^GlcNAc— a 3-Rha— a G l c A — P G a l N A c — P CH 3 of N-Ac (GlcNAc) CH 3 of N-Ac (GalNAc) CH 3 (Rha) 175.87 I C-0 of N-Ac [ of GlcNAc and 175.81 J GalNAc 104.75 G l c A — P 103.00 X 102.27 *GalNAc— P 101.60 i R h a — a 100.46 X 98.94 ^GlcNAc— a 61.26 C-6 (GlcNAc) 54.80 C-2 (GlcNAc) 52.95 C-2 (GalNAc) 0 23.30 N-C-CH3 (GalNAc) g 22.64 N-C-CH3 (GlcNAc) 17.55 C-6 Rha a For the o r i g i n of PI, see text. D 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 5 2.23 downfield from external sodium 4 l4-dimethyl-4-silapentane-l-sulfonate ( D . S . S . ) . c Key: b - broad, unable to assign accurate coupling constant; s - s i n g l e t . - For example 3-Rha- r e f e r s to the anomeric proton of a 3 - l i n k e d rhamnosyl residue In the er-anomeric configuration. The absence of a numerical p r e f i x indicates a (terminal) nonreducing group. X r e f e r s to the s i g n a l a t t r i b u t e d to the contaminant from the depolymerization mixture. £ 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 3 1 . 0 7 p.p.m. downfield from external D . S . S . spectra are given i n Appendix I I I . •£ As for d, but for anomeric * 3C n u c l e i . A l l n.m.r. - 160 -d e p o l y m e r i z a t i o n m i x t u r e , a s c l e a r l y e v i d e n c e d b y F i g . V . 3 . 1 ( b ) . U n f o r t u n a t e l y , t h i s c o n t a m i n a n t c o u l d n o t b e t o t a l l y r e m o v e d f r o m P I , a s s h o w n i n F i g . V . 3 . 1 ( c ) . T h e s e s a m e ' c o n t a m i n a n t ' a n o m e r i c s i g n a l s i . e . a t 5 5 . 3 6 a n d S 4 . 9 8 w e r e a l s o o b s e r v e d i n t h e ^ H - n . m . r . s p e c t r u m o f t h e p r o d u c t s f r o m t h e p h a g e - d e p o l y m e r i z a t i o n o f t h e c a p s u l a r p o l y s a c c h a r i d e s o f E . c o l i K 2 6 4 5 , K 3 1 4 9 a n d K 3 4 4 9 . T h i s ' c o n t a m i n a n t ' a p p e a r s t o b e p e c u l i a r o n l y f o r t h e p r o d u c t s i s o l a t e d f r o m t h e p h a g e - d e p o l y m e r i z a t i o n u s i n g E . c o l i p h a g e s b u t n o t w i t h K l e b s i e l l a p h a g e s . M e t h y l a t i o n a n a l y s e s - A n a l y s e s w e r e c o n d u c t e d o n P I a n d i t s c o r r e s p o n d i n g o l i g o s a c c h a r i d e a l d i t o l P 1 R , w h i c h w a s o b t a i n e d b y a s o d i u m b o r o h y d r i d e r e d u c t i o n o f P I . T h e r e s u l t s p r e s e n t e d i n T a b l e V . 3 . I I a r e i n a g r e e m e n t w i t h t h e m e t h y l a t i o n d a t a o f t h e c a r b o d i i m i d e -r e d u c e d 7 6 E . c o l i K 4 4 c a p s u l a r p o l y s a c c h a r i d e . T h e p r e s e n c e o f 2 , 3 , 4 -t r i - O - m e t h y l g l u c o s e a n d 2 - N - m e t h y l a c e t a m i d o - l , 3 , 4 , 5 - t e t r a - 0 - m e t h y l -g a l a c t i t o l i n t h e a n a l y s i s o f t h e m e t h y l a t e d a n d r e d u c e d P 1 R i n d i c a t e : ( i ) g l u c u r o n i c a c i d a s t h e t e r m i n a l , n o n - r e d u c i n g e n d , a n d ( i i ) t h e 6 - l i n k e d N - a c e t y l - g a l a c t o s a m i n e a s t h e t e r m i n a l r e d u c i n g s u g a r . T h i s e v i d e n c e w a s f u r t h e r s u b s t a n t i a t e d b y t h e c h e m i c a l i o n i z a t i o n m a s s s p e c t r u m o f t h e p e r m e t h y l a t e d P I . C . i . - m . s . a n a l y s i s o f p e r m e t h y l a t e d P I - M o s t o f t h e f r a g m e n t s d e t e c t e d i n t h e c . i . - m . s . o f p e r m e t h y l a t e d P I i n d i c a t e d a s e q u e n t i a l d e g r a d a t i o n o f t h e o l i g o s a c c h a r i d e p r o g r e s s i n g f r o m t h e g l u c u r o n i c a c i d - 161 -T a b l e V . 3 . I I : M e t h y l a t i o n a n a l y s e s o f PI and P1R M e t h y l a t e d s u g a r a (as a l d i t o l a c e t a t e ) Mole % b I I I I I 2,4-Rha 2,3,4-Glc 3,6-GlcN(Me)Ac 3,4-GalN(Me)Ac l,3,4,5-GalN(Me)Ac 35.8 24.3 27.2 12.5 46 31 22 48 35 10 a 2,4-Rha = l , 3 , 5 - t r i - 0 - a c e t y l - 2 , 4 - d i - 0 - m e t h y l r h a m n i t o l ; 3,6-GlcN(Me)Ac 1 , 4 , 5 - t r i - O - a c e t y l - 2 - d e o x y - 2 -(N-methyl)acetamido-3,6-di-0-methyl-DB-17 c a p i l l a r y column programmed from 180' f o r 1 g l u c i t o l . u U s i n g min, t h e n 2° to 250°. c I, C a r b o d i i m i d e - r e d u c e d " 3 E. c o l i K44 c a p s u l a r p o l y s a c c h a r i d e ; I I , m e t h y l a t e d and h y d r o l y z e d PI; I I I , m e t h y l a t e d and r e d u c e d P1R. d e g r a d a t i o n i n d u c e d on p r o l o n g e d a c i d h y d r o l y s i s . end o f the m o l e c u l e (see Scheme V.3.1 and mass spectrum numbered 3 i n Appendix I V ) . T h i s i s e x e m p l i f i e d by the fragments a t m/z 928, 679, 505, and 260 which can o n l y be e x p l a i n e d by a c l e a v a g e from the g l u c u -r o n i c a c i d end. The i o n a t m/z 505 i n d i c a t e s t h a t the two amino sugars are a d j a c e n t to each o t h e r whereas the i o n s a t m/z 434 and 189 are due to the l o s s o f the N-methylacetamido group from the amino s u g a r s . T h i s mass s p e c t r a l e v i d e n c e i s another p r o o f o f the s t r u c t u r a l sequence of the r e p e a t i n g u n i t i n the E. c o l i K44 c a p s u l a r p o l y s a c c h a r i d e . - 162 -O - M e m / z 434 WHERE X = OMe S c h e m e V . 3 . 1 : M a s s f r a g m e n t s o b s e r v e d I n t h e c h e m i c a l i o n i z a t i o n m a s s s p e c t r u m o f m e t h y l a t e d P I . 163 V.3.3 C o n c l u s i o n The sum o f these experiments i n d i c a t e d PI t o have the f o l l o w i n g s t r u c t u r e . G1cAi-^Rha^-^G1cNAci-^GalNAc-OH P a a ^ |44 The endo g l y c a n a s e a s s o c i a t e d w i t h E. c o l i #44 was demonstrated to e x h i b i t / 9 - N - a c e t y l - g a l a c t o s a m i n i d a s e a c t i v i t y . T h i s i s the f i r s t r e p o r t e d example o f t h i s n a t u r e from the b a c t e r i o p h a g e s i s o l a t e d from the s p e c i e s E. c o l i . U n f o r t u n a t e l y , the problem w i t h the 'contaminant' was not f u l l y r e s o l v e d . To overcome t h i s problem, i t i s s u g g e s t e d t h a t f u t u r e propa-g a t i o n and d e p o l y m e r i z a t i o n u s i n g E. c o l i phages s h o u l d be co n d u c t e d i n a s y n t h e t i c medium such as P-medium. Moreover, the use o f p u r i f i e d 919 phage p a r t i c l e s ^ - 1 ' ' i n the d e p o l y m e r i z a t i o n r e a c t i o n s h o u l d a l s o be c o n s i d e r e d . V.3.4 E x p e r i m e n t a l G e n e r a l methods - See S e c t i o n I I I . I s o l a t i o n and p r o p a g a t i o n o f #44 #44 was i s o l a t e d from - 164 -Grahamstown (S. A f r i c a ) sewage by P r o f . Dutton u s i n g the pr o c e d u r e as p r e v i o u s l y d e s c r i b e d (see S e c t i o n V.2.4). I t was p r o p a g a t e d on i t s h o s t s t r a i n E. c o l i K44 u s i n g M u e l l e r - H i n t o n b r o t h as medium. S u c c e s s i v e p r o p a g a t i o n s u s i n g the t e c h n i q u e o f tube l y s i s and f l a s k l y s i s (see S e c t i o n I I I . 9 ) were conducted. A t o t a l o f -1.0 x 1 0 1 3 p . f . u . o f $44 was e v e n t u a l l y o b t a i n e d . D e p o l y m e r i z a t i o n o f E. c o l i K44 p o l y s a c c h a r i d e and i s o l a t i o n o f p r o d u c t s - D e p o l y m e r i z a t i o n o f the K44 p o l y s a c c h a r i d e was c a r r i e d out u s i n g the method o f Dut t o n and c o w o r k e r s 2 2 0 . The M u e l l e r - H i n t o n s u s p e n s i o n o f $44 was c o n c e n t r a t e d (-400 mL) and d i a l y z e d (mol. wt. c u t o f f 13,500) a g a i n s t v o l a t i l e b u f f e r (7.7 g CH 3C0 2NH 4 and 4.3 g ( N H 4 ) 2 CO3 i n 1 L H 20, pH = 7.0). The r e t e n t a t e was c o n c e n t r a t e d to g i v e a l i g h t brown s o l u t i o n (-400 mL). A phage as s a y c o n d u c t e d on t h i s $44 s u s p e n s i o n gave a t o t a l count o f 8.0 x l O 1 2 - 1.0 x 10^ p . f . u . P u r i f i e d E. c o l i K44 c a p s u l a r p o l y s a c c h a r i d e (380 mg) d i s s o l v e d i n v o l a t i l e b u f f e r (50 mL) was mixed w i t h the s o l u t i o n (400 mL) o f $44 and i n c u b a t e d a t 37". Some c h l o r o f o r m was added t o p r e v e n t b a c t e r i a l growth. A f t e r two days, the s i g n i f i c a n t l y l e s s v i s c o u s phage-p o l y s a c c h a r i d e m i x t u r e was d i a l y z 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 d i a l y z a t e was c o n c e n t r a t e d and pa s s e d through a ca t i o n - e x c h a n g e column o f A m b e r l i t e IR 120 (H +) r e s i n . A M o l i s c h t e s t ° i , i i J c o n d u c t e d on a c o n c e n t r a t e d sample o f the e l u a n t gave a p o s i t i v e r e s u l t , thus i n d i c a t i n g the pres e n c e o f s u g a r s . The l y o p h i -l i z e d p r o d u c t was a brownish s t y r o f o a m - l i k e m a t e r i a l (120 mg). The - 165 -•'-H-n.m.r. spectrum o f t h i s crude p r o d u c t i s d e p i c t e d i n F i g . V . 3 . 1 . Paper c h r o m a t o g r a p h i c a n a l y s i s o f t h i s crude p r o d u c t showed t h r e e f a s t moving components ( m o s t l y contaminants) and a s p o t t h a t c o u l d be the d e s i r e d o l i g o s a c c h a r i d e ( E l a c t o s e 0-33). S e p a r a t i o n o f t h i s crude p r o d u c t by p r e p a r a t i v e paper chromatogra-phy i n s o l v e n t 2 gave two f r a c t i o n s , PI (90 mg) and a f a s t e r - m o v i n g component 2. The n.m.r. ( 1H and 1 3 C ) d a t a o f PI i s p r e s e n t e d i n T a b l e V.3.1. A comparison between the •'-H-n.m.r. spectrum o f PI, 2, and the E.  c o l i K44 p o l y s a c c h a r i d e showed the p r e s e n c e o f r e s i d u a l contaminant i n PI. (see F i g . V . 3 . I ) . No c o n c l u s i v e e v i d e n c e c o u l d be o b t a i n e d from the f.a.b.-mass spectrum o f the u n d e r i v a t i z e d PI; the absence o f the molecu-l a r i o n peak, c o u p l e d w i t h the e x t e n s i v e f r a g m e n t a t i o n and n o i s y back-ground makes the mass spectrum d i f f i c u l t to i n t e r p r e t . M e t h y l a t i o n a n a l y s i s o f PI - A sample (15 mg) o f PI was methy-l a t e d by the Hakomori p r o c e d u r e 1 1 2 • 1 1 3 . The m e t h y l a t e d p r o d u c t was r e c o v e r e d by e x t r a c t i o n w i t h d i c h l o r o m e t h a n e and f u r t h e r p u r i f i e d by passage t h r o u g h a column o f Sephadex LH-20. An i . r . s p e c t r o s c o p i c a n a l y s i s o f t h i s sample showed complete m e t h y l a t i o n . The 1H-n.m.r. o f t h i s sample ( d i s s o l v e d i n CDCI3) showed peak s i g n a l s , which made i t d i f f i c u l t t o judge i f the r e s i d u a l 'contaminant' had been removed d u r i n g the p u r i f i c a t i o n o f the m e t h y l a t e d PI. A p o r t i o n o f the m e t h y l a t e d PI was a n a l y z e d by c h e m i c a l i o n i z a t i o n mass s p e c t r o m e t r y u s i n g methane as the r e a g e n t gas (see Scheme V.3.I and Appendix I V ) . H y d r o l y s i s o f the r e m a i n i n g p o r t i o n u s i n g 2M HCl f o r 7 h was f o l l o w e d by sodium boro-166 h y d r i d e r e d u c t i o n o f the sugars r e l e a s e d , and a c e t y l a t i o n t o t h e i r p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s . The p r o c e d u r e s f o r t h i s sequence o f r e a c t i o n s were d e s c r i b e d p r e v i o u s l y (see S e c t i o n IV.3.4). The g . l . c . d a t a a r e p r e s e n t e d i n T a b l e V . 3 . I I , column I I . (NaBH 4) R e d u c t i o n o f PI t o P1R - A p o r t i o n o f PI (70 mg) was t r e a t e d w i t h aqueous sodium b o r o h y d r i d e and s t i r r e d a t ambient tempera-t u r e f o r 4 h. The excess b o r o h y d r i d e was d e s t r o y e d by a d d i n g a c e t i c a c i d ( 5 0 % ) . The b o r i c a c i d formed was removed by c o - e v a p o r a t i n g w i t h methanol ( 4 x ) . A second sodium b o r o h y d r i d e r e d u c t i o n was r e p e a t e d f o l l o w e d by s i m i l a r workup p r o c e d u r e . An aqueous s o l u t i o n o f the r e s i d u e was t h e n poured i n t o a m i x t u r e o f e t h a n o l - a c e t o n e ( 4 : 1 ) . The p r e c i p i t a t e was sedimented by c e n t r i f u g a t i o n , r e d i s s o l v e d i n water and f r e e z e - d r i e d t o g i v e a f l u f f y w h i t e p r o d u c t , P1R (40 mg). A sample (15 mg) o f P1R was m e t h y l a t e d by the Hakomori p r o c e -119 11^ dure- 1- 1^ • J - x J and worked up f o l l o w i n g the u s u a l p r o c e d u r e f o r m e t h y l a t e d o l i g o s a c c h a r i d e s . The p e r m e t h y l a t e d sample was r e d u c e d w i t h L i A l H ^ i n r e f l u x i n g t e t r a h y d r o f u r a n o v e r n i g h t , and the excess L i A l H ^ was d e s t r o y e d by the dropwise a d d i t i o n o f e t h a n o l . The Al(0H)3 p r e c i p i t a t e formed was d i s s o l v e d ( w i t h s t i r r i n g ) i n 10% HC1, and the m e t h y l a t e d sample recov-e r e d by e x t r a c t i o n s w i t h CHCI3 (3x). The m e t h y l a t e d , r e d u c e d ( c o n f i r m e d by i . r . s p e c t r o s c o p y ) sample was h y d r o l y z e d u s i n g 2M HCl a t 95° f o r 16 h, and the sugars r e l e a s e d were reduced w i t h aqueous NaBH4 f o l l o w e d by a c e t y l a t i o n t o t h e i r p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s . The g . l . c . d a t a a r e shown i n T a b l e V . 3 . I I , column I I I . - 167 -CHAPTER VI CONCLUDING REMARKS 168 CONCLUDING REMARKS The i n f o r m a t i o n d e r i v e d from s t r u c t u r a l s t u d i e s o f b a c t e r i a l p o l y s a c c h a r i d e s , b e s i d e s e x p l a i n i n g the s e r o l o g y o f the d i f f e r e n t b a c t e r i a l s t r a i n s on a m o l e c u l a r l e v e l , has i m p o r t a n t b i o l o g i c a l , m e d i c a l and commercial s i g n i f i c a n c e . T h i s i n c l u d e s the p r o d u c t i o n o f p r o t e c t i v e p o l y s a c c h a r i d e v a c c i n e s 1 0 and n o n c y t o t o x i c a n t i - t u m o r d r u g s 1 7 , and uses i n many i n d u s t r i a l and domes t i c a p p l i c a t i o n s 1 . The use o f b a c t e r i a l p o l y s a c c h a r i d e s i n these f i e l d s , a p a r t from xanthan gums 1, i s s t i l l a t the e a r l y s t a g e o f development. The number o f b a c t e r i a l p o l y s a c c h a r i d e s f o r which the p r i m a r y c h e m i c a l s t r u c t u r e i s known i s i n c r e a s i n g v e r y r a p i d l y . The r e g u l a r r e p e a t i n g s t r u c t u r e o f b a c t e r i a l p o l y s a c c h a r i d e s makes s t r u c t u r a l a n a l y s i s l e s s c o m p l i c a t e d , when compared to c a r b o h y d r a t e polymers o f lower o r g a n i z a t i o n , e.g. p l a n t p o l y s a c c h a r i d e s . Moreover, the time and e f f o r t r e q u i r e d f o r s t r u c t u r a l e l u c i d a t i o n have been g r e a t l y r e d u c e d i n r e c e n t y e a r s . These advances can be a t t r i b u t e d t o the use o f s p e c i f i c b a c t e r i o p h a g e - b o r n e e ndoglycanases and h i g h l y s e l e c t i v e c h e m i c a l degra-d a t i o n t e c h n i q u e s , i n c o n j u n c t i o n w i t h more s o p h i s t i c a t e d and computer-i z e d c h r o m a t o g r a p h i c , n.m.r. s p e c t r o s c o p i c and mass s p e c t r o m e t r i c methods. Other m u t u a l l y i n t e r a c t i n g f a c e t s o f t h e i r c h e m i s t r y have to be s t u d i e d b e f o r 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 can be u t i l i z e d t o t h e i r maximum c a p a c i t y . These i n v o l v e s t u d y i n g the r e l a t i o n s h i p between p r i m a r y s t r u c t u r e and ( i ) c o n f o r m a t i o n ( i n s o l i d s t a t e and s o l u t i o n ) , - 169 ( i i ) p h y s i c a l p r o p e r t i e s , ( i i i ) b i o l o g i c a l a c t i v i t i e s , ( i v ) b i o s y n t h e -s i s , (v) i n t e r a c t i o n s w i t h p r o t e i n s . I n f a c t , s t u d i e s i n many o f these a r e a s have a l r e a d y begun i n the l a s t d e c a d e 1 " ^ . 170 CHAPTER V I I BIBLIOGRAPHY 171 -V I I . BIBLIOGRAPHY 1. P.A. S a n d f o r d and J . B a i r d , 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 . 2, Academic P r e s s , New York (1983), pp. 411-490. 2. N. Sharon i n "Complex C a r b o h y d r a t e s : T h e i r C h e m i s t r y , B i o s y n t h e s i s and F u n c t i o n s " , Addison-Wesley P u b l i s h i n g Company, M a s s a c h u s e t t s , (1975). 3. J . F . Kennedy i n " P r o t e o g l y c a n s - B i o l o g i c a l and Ch e m i c a l A s p e c t s i n Human L i f e " , E l s e v i e r S c i e n t i f i c P u b l i s h i n g Company, Amsterdam, (1979) . 4. J . F . Kennedy and C A . White i n " B i o a c t i v e C a r b o h y d r a t e s : In Chemis-t r y , B i o c h e m i s t r y and B i o l o g y " , E l l i s Horwood L i m i t e d , England, (1983) . 5. S.M. Hammond, P.A. Lambert, and A.N. R y c r o f t , i n "The B a c t e r i a l C e l l S u r f a c e " , K a p i t a n Szabo P u b l i s h e r s , Washington D.C., (1984). 6. L. Kenne and B. L i n d b e r g , 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 . 2, Academic P r e s s , New York, (1983) pp. 287-363. 7. W.F. Dudman, i n I. S u t h e r l a n d ( E d . ) , " S u r f a c e C a r b o h y d r a t e s o f the P r o k a r y o t i c C e l l " , Academic P r e s s , New York, (1977) pp. 357-414. 8. C.T. B i s h o p and H.J. J e n n 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. K. J a n n and B. Jann, i n I. S u t h e r l a n d ( E d . ) , " S u r f a c e C a r b o h y d r a t e s o f the P r o k a r y o t i c C e l l " , Academic P r e s s , New York, (1977) pp. 247-287. 10. H.J. J e n n i n g s , Adv. Carbohydr. Chem. Biochem., 44 (1983) 155-208. 11. K. Jann and 0. Westphal, i n M. S e l a ( E d . ) , "The A n t i g e n s " , V o l . I l l , Academic P r e s s , New York, (1975) pp. 1-125. 12. M. H e i d e l b e r g e r and G.G.S. Dutton, J . Immunol., I l l (1973) 857-859 . 13. M. H e i d e l b e r g e r and W. Nimmich, Immunochemistry, 13 (1976) 67-80. 14. J.Y. Lew and M. H e i d e l b e r g e r , Carbohydr. Res., 52 (1976) 255-258. 15. M. H e i d e l b e r g e r , Res. Immunochem. Immunobiol., 3 (1973) 1-40. - 172 16. M. H e i d e l b e r g e r , Trends Biochem. Sc., 7 (1982) 261-263. 17. R.L. W h i s t l e r , A.A. Bushway, P.P. Singh, W. Nakahara, and R. Tokuzen, Adv. Carbohydr. Chem. Biochem., 32 (1976) 235-275. 18. J.R. Crook, W.K. Ot t o , a n d R . S . Jones, Proc. Soc. Exp. B i o l . Med., 109 (1962) 552-556. 19. K. Miyamoto, R. K o s h i u r a , T. Hasegawa, and N. Kato, Jpn. J . Phar-macol. , 36 (1984) 51-57. 20. I . $ r s k o v , i n Bergey's Manual o f D e t e r m i n a t i v e B a c t e r i o l o g y , 8th edn., (1974) pp. 321-324. 21. P.R. Edwards and W.H. Ewing, " I d e n t i f i c a t i o n o f E n t e r o b a c t e r i a c -eae", Burgess P u b l i s h i n g Company, M i n n e a p o l i s , (1972). 22. H.R. Morgan, i n R. Dubos, and J.G. H i r s c h ( E d s . ) , " B a c t e r i a l and M y c o t i c I n f e c t i o n s o f Man", J.B. L i p p i n c o t t Company, P h i l a d e l p h i a , (1965) pp. 610-648. 23. I . #rskov, A c t a P a t h o l . M i c r o b i o l . Scand., 38 (1956) 375-384. 24. I. ,0rskov, and M.A. F i f e - A s b u r y , I n t e r n a t . J . S y s t e m a t i c Bacte-r i d . , 27 (1977) 386-387. 25. W. Nimmich, Z. Med. M i k r o b i o l . Immunol., 154 (1968) 117-131. 26. W. Nimmich, A c t a B i o l . Med. Ger., 26 (1971) 397-403. 27. W. Nimmich, Z. A l l g . M i k r o b i o l . , 19 (1979) 343-347. 28. D.N. Ka r u n a r a t n e , Ph.D. T h e s i s , U n i v e r s i t y o f B r i t i s h Columbia, (1985). 29. M. H e i d e l b e r g e r , W. Nimmich, J . E r i k s e n , G.G.S. Dutton, S. S t i r m , and C.T. Fang, A c t a Path. M i c r o b i o l . Scand. S e c t . C , 83 (1975) 397-405. 30. M. H e i d e l b e r g e r , W. Nimmich, J . E r i k s e n , and S. S t i r m , A c t a Path. M i c r o b i o l . Scand. S e c t . B, 86 (1978) 313-320. 31. G.G.S. Dut t o n and A.V. Savage, Carbohydr. Res., 83 (1980) 351-362. 32. G.G.S. Dutton, H. P a r o l i s , and L.A.S. P a r o l i s , Carbohydr. Res., 140 (1985) 263-275. 33. J.-P. J o s e l e a u , M. Lapeyre, M. Vignon, and G.G.S. Dutton, Carbo-h y d r . Res., 67 (1978) 197-212. 173 -34. M. H e i d e l b e r g e r , W.F. Dudman, and W. Nimmich, J . Immunol., 104 (1970) 1321-1328. 35. F. Kauffmann, "The B a c t e r i o l o g y o f E n t e r o b a c t e r i a c e a e " , 3 r d edn., Burgess P u b l i s h i n g Company, M i n n e a p o l i s , (1972). 36. E.M. Cooke, " E s c h e r i c h i a c o l i and Man", C h u r c h i l l L i v i n g s t o n e , London, (1974). 37. M. Sussman ( E d . ) , "The V i r u l e n c e o f E s c h e r i c h i a c o l i " , Academic P r e s s , London, (1985). 38. F. Kauffmann, A c t a Path. M i c r o b i o l . Scand., 20 (1943) 21-44. 39. I. /Zhrskov, F. 0 r s k o v , B. Jann, and K. Jann, B a c t e r i o l . Rev., 41 (1977) 667-710. 40. I. jZhrskov, A. B i r c h - A n d e r s o n , J.P. Duguid, J . Stenderup, and F. ^ r s k o v , I n f e c t . Immun., 47 (1985) 191-200. 41. K. Jann and B. Jann, Prog. A l l e r g y , 33 (1983) 53-79. 42. L.A.S. P a r o l i s , Ph.D. T h e s i s , Rhodes U n i v e r s i t y , (1985). 43. B. Jann, P. Hofmann, and K. Jann, Carbohydr. Res., 120 (1983) 131-141. 44. M.A. Schmidt and K. Jann, Eur. J . Biochem., 131 (1983) 509-517. 45. L.M. Beynon, M.Sc. T h e s i s , U n i v e r s i t y o f B r i t i s h Columbia, (1985). 46. E. Altman and G.G.S. Dutton, Carbohydr. Res., 138 (1985) 293-303. 47. G. An n i s o n , G.G.S. Dutton, and E. Altman, Carbohydr. Res., i n p r e s s . 48. G.G.S. Dut t o n and B.A. Lewis, Carbohydr. Res., i n p r e s s . 49. A. Kuma-Mintah, M.Sc. T h e s i s , U n i v e r s i t y o f B r i t i s h Columbia, (1985) . 50. E. Altman, Ph.D. T h e s i s , U n i v e r s i t y o f B r i t i s h Columbia, (1984). 51. P. Hofmann, B. Jann, and K. Jann, Carbohydr. Res., 139 (1985) 261-271. 52. G.G.S. Dutton, i n V. C r e s c e n z i , I.CM. Dea, and S.S. S t i v a l a ( E d s . ) , "New Developments i n I n d u s t r i a l P o l y s a c c h a r i d e s " , Gordon and Breach Sc. P u b l i s h e r s , Amsterdam, (1985) pp. 7-26. - 174 -53. M. H e i d e l b e r g e r , K. Jann, and B. Jann, J . Exp. Med., 162 (1985) 1350-1358. 54. C. A d l am, J.M. K n i g h t s , A. Mugridge, J.C. L i n d o n , J.M. W i l l i a m s , and J . E . B e e s l e y , J . Gen. M i c r o b i o l . 131 (1985) 1963-1972. 55. H. P e t e r s , M. J u e r s , B. Jann, K.N. Timmis, and D. B i t t e r - S u e r m a n n , I n f e c t . Immun., 50 (1985) 459-466. 56. 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 . 2, Academic P r e s s , New York, (1983) . 57. B. L i n d b e r g , J . Lbnngren, and S. Svensson, Adv. Carbohydr. Chem. Biochem., 31 (1975) 185-240. 58. 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 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. 36-131. 59. 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 P o l y s a c c h a r i d e s " , V o l . 2, Academic P r e s s , New York, (1983) pp. 19-34. 60. R.L. W h i s t l e r and J . L . S a n n e l l a , Methods Carbohydr. Chem., 5 (1965) 34-36. 61. J . E . S c o t t , Methods Carbohydr. Chem., 5 (1965) 38-44. 62. S.C. Churms, Adv. Carbohydr. Chem. Biochem., 25 (1970) 13-51. 63. J.H. Pazur and L.S. F o r s b e r g , Methods Carbohydr. Chem., 8 (1980) 211-217. 64. D.N. N o r t h c o t e , Methods Carbohydr. Chem., 5 (1965) 49-53. 65. M. Breen, H.G. W e i n s t e i n , L . J . B l a c k , M.S. B o r c h e r d i n g and R.A. S i t t i g , Methods Carbohydr. Chem., 7 (1966) 101-115. 66. 0. O u c h t e r l o n y , Prog. A l l e r g y , 6 (1962) 30-154. 67. D. Aminoff, W.W. B i n k l e y , R. S c h a e f f e r , and R.W. Mowery, i n W. Pigman and D. H o r t o n ( E d s . ) , "The C a r b o h y d r a t e s " , 2nd edn., Aca-demic P r e s s , New York (1970) pp. 739-807. 68. G.G.S. Dutton, Adv. Carbohydr. Chem. Biochem., 28 (1973) 11-160. 69. A . J . Mort and D.T.A. Lamport, A n a l y t i c a l Biochem., 82 (1977) 289-309. 70. A . J . Mort, J.-P. U t i l l e , G. T o r r i , and A.S. P e r l i n , Carbohydr. Res., 121 (1983) 221-232. 71. A . J . Mort, Carbohydr. Res., 122 (1983) 315-321. 175 -72. M.P. Sanger and D.T.A. Lamport, A n a l y t i c a l Biochem., 128 (1983) 66-70. 73. B. Capon, Chem. Rev., 69 (1969) 407-498. 74. J.N. B e M i l l e r , Adv. Carbohydr. Chem. Biochem., 22 (1967) 25-108. 75. G.G.S. D u t t o n andM.-T. Yang, Can. J . Chem., 59 (1977) 179-192. 76. H.E. T a y l o r and R.L. Conrad, B i o c h e m i s t r y , (1973) 1383-1388. 77. K. S t e l l n e r , H. S a i t o , and S.-I. Hakomori, A r c h . Biochem. Bi o p h y s . , 155 (1973) 464-472. 78. J.M. W i l l i a m s , Adv. Carbohydr. Chem. Biochem., 31 (1975) 9-79. 79. Z. Yosizawa, T. Sato, and K. Schmid, Biochim. B i o p h y s . A c t a , 121 (1966) 417-420. 80. L. Kenne and B. L i n d b e r g , Methods Carbohydr. Chem., 8 (1980) 295-296. 81. Z. D i s c h e , Methods Carbohydr. Chem., 1 (1962) 477-514. 82. V.N. R e i n h o l d , Methods Enzymol., 25 (1972) 244-249. 83. G.G.S. Dutton, Adv. Carbohydr. Chem. Biochem., 30 (1974) 9-110. 84. J.H. S l o n e k e r , Methods Carbohydr. Chem., 6 (1972) 20-24. 85. B.A. D m i t r i e v , L.V. Backinowsky, O.S. Chizhov, B.M. Z o l o t a r e v , and N.K. Kochetkov, Carbohydr. Res., 19 (1971) 432-435. 86. R.H. Furneaux, Carbohydr. Res., 113 (1983) 241-255. 87. C.C. Chen andG.D. Mc G i n n i s , Carbohydr. Res., 90 (1981) 127-130. 88. T. K o n t r o h r and B. K o c s i s , J . Chromatogr., 291 (1984) 119-125. 89. S.C. Churms, i n G. Zweig, and J . Sherma ( E d s . ) , "C.R.C. Handbook o f Chromatography", C.R.C. P r e s s I n c . , (1982). 90. E. Heftman ( E d . ) , "Chromatography", 2nd edn., R e i n h o l d P u b l i s h i n g C o r p o r a t i o n , New York (1967). 91. G.D. McG i n n i s and P. Fang, Methods Carbohydr. Chem., 8 (1980) 33-43. 92. E.F. H o u n s e l l , J.M. R i d e o u t , N.J. P i c k e r i n g , and C.K. Lim, J . L i q . Chromatogr., 7 (1984) 661-674. 176 93. J.H. Pazur, Methods Enzymol., 9 (1966) 82-87. 94. J . J . M a r s h a l l , Adv. Carbohydr. Chem. Biochem., 30 (1974) 276-370. 95. C.S. Hudson, J . Am. Chem. S o c , 31 (1909) 66-86. 96. A.L. Stone, Methods Carbohydr. Chem., 7 (1976) 120-138. 97. K. L e o n t e i n , B. L i n d b e r g , and J . LSnngren, Carbohydr. Res., 62 (1978) 359-362. 98. P.A. G o r i n , M. I a c o m i n i , M.A.L. F e i j o , S.M.W. Za n i n , and L. Hogge, Ar g . B i o l . Techno1., 28 (1985) 387-398. 99. G.J. Gerwig, J.P. K a m e r l i n g and J.F.G. V l i e g e n t h a r t : ( i ) Carbohydr. Res., 62 (1978) 349-357. ( i i ) Carbohydr. Res., 77 (1979) 1-7. 100. G.M. B e b a u l t , J.M. B e r r y , Y.-M. Choy, G.G.S. Dutton, N. F u n n e l l , L.D. Hayward, and A.M. Stephen, Can. J . Chem., 51 (1973) 324-326. 101. Y.T. L i and S.C. L i , Methods Carbohydr. Chem., 7 (1976) 221-225. 102. S.J. A n g y a l and K. James, A u s t . J . Chem., 23 (1970) 1209-1215. 103. H. Rauvala, J . F i h n e , T. K r u s i u s , J . K a r k k a i n e n , and J . J a r n e f e l t , Adv. Carbohydr. Chem. Biochem., 38 (1980) 389-416. 104. D. R o l f and G.R. Gray, J . Am. Chem. S o c , 104 (1982) 3539-3541. 105. J.U. Bowie and G.R. Gray, Carbohydr. Res., 129 (1984) 87-97. 106. J . Arnap, L. Kenne, B. L i n d b e r g , and J . Lonngren, Carbohydr. Res., 44 (1975) C5-C7. 107. P. Prehm, Carbohydr. Res., 78 (1980) 372-374. 108. J . F i n n e , T. K r u s i u s , and H. Rauvala, Carbohydr. Res., 80 (1980) 336-339. 109. L.R. P h i l l i p s and B.A. F r a s e r , Carbohydr. Res., 90 (1981) 149-152. 110. T. N a r u i , K. T a k a h a s h i , M. Kob a y a s h i , and S. S h i b a t a , Carbohydr. Res., 103 (1982) 293-295. 111. P.J. H a r r i s , R.J. Henny, A.B. Blakeney, and B.A. Stone, Carbohydr. Res., 127 (1984) 59-73. 112. S.-I. Hakomori, J . Biochem. ( t o k u o ) , 55 (1964) 205-208. 113. H.E. Conrad, Methods Carbohydr. Chem., 6 (1972) 361-364. - 177 114. T. P u r d i e and J.C. I r v i n e , J . Chem. S o c , 83 (1903) 1021-1037. 115. J . K i s s , Adv. Carbohydr. Chem. Biochem., 29 (1974) 229-303. 116. B. L i n d b e r g and J . Lonngren, Methods Carbohydr. Chem., 7 (1976) 142-148. 117. H.G. Jones, Methods Carbohydr. Chem., 6 (1972) 25-41. 118. P.-E. Jan s s o n , L. Kenne, H. L i e d g r e n , B. L i n d b e r g , and J . Lonngren, Chem. Commun., U n i v e r s i t y Stockholm, 8 (1976). 119. C.G. Wong, S.-S.J. Sung, and C.C. Sweeley, Methods Carbohydr. Chem., 8 (1980) 55-66. 120. J.A. Lomax and J . Conchie, J . Chromatogr., 236 (1982) 385-394. 121. A.N. de B e l d e r and B. Norrman, Carbohydr. Res., 8 (1968) 1-6. 122. J.H. S l o n e k e r and D.G. Or e n t a s , Nature, 194 (1962) 478-479. 123. A.G. H a d j i v a s s i l i o u and S.V. R i e d e r , C l i n . Chim. A c t a , 19 (1968) 357-361. 124. P.J. Garegg, P.E. Jan s s o n , B. L i n d b e r g , F. L i n d h , J . Lonngren, I. Kvarnstrom, and W. Nimmich, Carbohydr. Res., 78 (1980) 127-132. 125. R.D. G u t h r i e , Adv. Carbohydr. Chem., 16 (1961) 105-157. 126. I . J . G o l d s t e i n , G.W. Hay, B.A. Lewis, and F. Smith, Methods Carbo-h y d r . Chem., 5 (1965) 361-370. 127. H. B j o r n d a l and B. Wagstrom, A c t a Chem. Scand., 23 (1969) 3313-3320. 128. S. Svensson and L. Kenne, Methods Carbohydr. Chem., 8 (1980) 67-71. 129. O.S. C h i z h o v and N.K. Kochetkov, Methods Carbohydr. Chem., 8 (1980) 123-125. 130. A . J . Mort and W.D. Bauer, J . B i o l . Chem., 257 (1982) 1870-1875. 131. N.K. Matheson and B.V. McCle a r y , 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 . 3, Academic P r e s s , New York, (1985) pp. 1-105. 132. D. Rieger-Hug and S. S t i r m , V i r o l o g y , 113 (1981) 363-378. 133. H. Geyer, K. Himmelspach, N. Kwiatkowski, S. S c h l e c h t , and S. S t i r m , Pure. A p p l . Chem., 55 (1983) 637-653. 178 -134. I.W. S u t h e r l a n d , i n I.W. S u t h e r l a n d ( E d . ) , " S u r f a c e C a r b o h y d r a t e s o f the P r o k a r y o t i c C e l l " , Academic P r e s s , New York, (1977) pp. 209-245. 135. W.W. B i n k l e y , Methods Carbohydr. Chem., 5 (1965) 54-55. 136. H. W e i g e l , Adv. Carbohydr. Chem., 18 (1963) 61-97. 137. J . F . Kennedy and J . E . Fox, Methods Carbohydr. Chem., 8 (1980) 3-12. 138. 0. Samuelson, Methods Carbohydr. Chem., 6 (1972) 65-75. 139. A. Thompson, Methods Carbohydr. Chem., 1 (1962) 36-42. 140. R.L. W h i s t l e r and A.K.M. Anisuzzaman, Methods Carbohydr. Chem., 8 (1980) 45-53. 141. R.E. Wing and J.N. B e M i l l e r , Methods Carbohydr. Chem., 6 (1972) 42-64. 142. A. Pryde and M.T. G i l b e r t , " A p p l i c a t i o n s o f Hi g h Performance L i q u i d Chromatography", Chapman and H a l l L t d . , London, (1979). 143. M. M c N e i l , A.G. D a r v i l l , P. Aman, L.-E. Franz e n , and P. A l b e r s h e i m , Methods Enzymol., 83 (1982) 3-45. 144. R.J. F e r r i e r , i n W. Pigman and D. H o r t o n ( E d s . ) , "The Carbohy-d r a t e s " , 2nd edn., V o l . IB, Academic P r e s s , New York (1980) pp. 1354-1375. 145. L.D. H a l l , Adv. Carbohydr. Chem. Biochem., 19 (1964) 51-93. 146. B. Coxon, Adv. Carbohydr. Chem. Biochem., 27 (1972) 7-83. 147. L.D. H a l l , Adv. Carbohydr. Chem. Biochem., 29 (1974) 11-40. 148. P.A.J. G o r i n , Adv. Carbohydr. Chem. Biochem., 38 (1981) 13-104. 149. K. Bock and H. Th^gersen, Annual R e p o r t s on NMR S p e c t r o s c o p y , 13 (1982) 2-55. 150. K. Bock and C. Pedersen, Adv. Carbohydr. Chem. Biochem., 41 (1983) 27-66. 151. K. D i l l , E. Berman, and A.A. P a v i a , Adv. Carbohydr. Chem. Biochem., 43 (1985) 2-49. 152. J.H. Bradbury and G.A. J e n k i n s , Carbohydr. Res., 126 (1984) 125-156. 179 -153. P.E. P f e r r e r , J . Carbohydr. Chem., 3 (1984) 613-639. 154. C M . P r e s t o n and L.D. H a l l , Carbohydr. Res., 37 (1974) 267-282. 155. L.D. H a l l and C M . P r e s t o n , Carbohydr. Res., 49 (1976) 3-11. 156. K. Bock, Pure and A p p l . Chem., 55 (1983) 605-622. 157. K. Bock, M. M e l d a l , D.R. Bundle, T. I v e r s e n , B.M. P i n t o , P.J. Gar-egg, I. Kvarnstrom, T. Norberg, A.A. L i n d b e r g , and S.B. Svensson, Carbohydr. Res., 130 (1984) 35-53. 158. S.L. P a t t , J . Carbohydr. Chem., 3 (1984) 493-514. 159. A. Bax, W. Egan, and P. Kovac, J . Carbohydr. Chem., 3 (1984) 593-611. 160. F. Michon, J.B. B r i s s o n , R. Roy, F.E. Ashton, and H.J. J e n n i n g s , B i o c h e m i s t r y , 24 (1985) 5592-5598. 161. J . Dabrowski, H. Egge, and U. Dabrowski, Carbohydr. Res., 114 (1983) 1-9. 162. F. Cavagna, H. Deger, and J . P u i s , Carbohydr. Res., 129 (1984) 1-8. 163. D.R. Bundle, M. Gerken, andM.B. P e r r y , Can. J . Chem., 64 (1986) 255-264. 164. J . - L . D i F a b i o , G.G.S. Dutton, and H. P a r o l i s , Carbohydr. Res., 133 (1984) 125-133. 165. J . Reuben, J . Am. Chem. S o c , 107 (1985) 1747-1755. 166. B. M a t s u h i r o , A.B. Zanlungo, and G.G.S. Dutton, Carbohydr. Res., 97 (1981) 11-18. 167. M. K a r p l u s , J . Chem. Phys., 30 (1959) 11-15. 168. G. Kotowycz and R.U. Lemieux, Chem. Rev., 73 (1973) 669-698. 169. K. Bock and C. Pedersen, J . Chem. Soc. P e r k i n T r a n s . , 2 (1974) 293-297. 170. K. Bock and C. Pedersen, A c t a Chem. Scand., Ser. B, 29 (1975) 258-264. 171. K. Bock and C. Pedersen, Carbohydr. Res., 145 (1985) 135-140. 172. N.K. Kochetkov and O.S. Chizhov, Adv. Carbohydr. Chem. Biochem., 21 (1966) 39-93. 180 173. J . Lonngren and S. Svensson, Adv. Carbohydr. Chem. Biochem., 29 (1974) 41-106. 174. G.R. W a l l e y and O.C. Dermer ( E d s . ) , " B i o c h e m i c a l A p p l i c a t i o n s o f Mass Sp e c t r o m e t r y " , W i l e y - I n t e r s c i e n c e , New York, (1980). 175. D.C. De Jongh, i n W. Pigman and D. Hor t o n ( E d s . ) , "The Carbo-h y d r a t e s " , 2nd edn., V o l . IB, Academic P r e s s , , New York, (1981), pp. 1327-1353. 176. H. B j o r n d a l , C.G. H e l l e r q u i s t , B. L i n d b e r g , and S. Svensson, Angew. Chem. I n t e r n a t . Edn., 9 (1976) 610-619. 177. A.K. B h a t t a c h a r j e e and H.J. J e n n i n g s , Carbohydr. Res., 51 (1976) 253-261. 178. J . K a r k k a i n e n , Carbohydr. Res., 14 (1970) 27-33. 179. J . K a r k k a i n e n , Carbohydr. Res., 17 (1971) 1-10. 180. J . K a r k k a i n e n , Carbohydr. Res., 17 (1971) 11-18. 181. V. K o v a c i k , S. Bauer, J . R o s i k , and P. Kovac, Carbohydr. Res., 8 (1968) 282-294. 182. A. D e l l , H.R. M o r r i s , H. Egge, H. Van N i c o l a i , and G. S t r e c k e r , Carbohydr. Res., 115 (1983) 41-52. 183. H.R. M o r r i s , A. D e l l , M. P a n i c o , and R.A. McDowell, A n a l . Chem. Symp. Ser., 24 (1985) 363-377. 184. K. O k u t a n i and G.G.S. Dutton, Carbohydr. Res., 86 (1980) 259-271. 185. 0. Westphal and K. Jann, Methods Carbohydr. Chem., 5 (1965) 83-91. 186. K. Jann, i n M. Sussman ( E d . ) , "The V i r u l e n c e o f E s c h e r i c h i a c o l i " , Academic P r e s s , London (1985) pp. 375-379. 187. B. Coxon, Methods Carbohydr. Chem., 6 (1972) 513-539. 188. H.J. J e n n i n g s and I . C P . Smith, Methods Carbohydr. Chem., 8 (1980) 97-105. 189. G.V.D. T i e r s and R.I. Coon, J . Org. Chem., 26 (1961) 2097-2098. 190. L.A. T o r e l l o , A . J . Y a t e s , and D.K. Thompson, J . Chromatogr., 202 (1980) 195-209. 191. A.B. Blakeney, P.J. H a r r i s , R.J. Henry, and B.A. Stone, Carbo-h y d r . Res., 113 (1983) 291-299. - 181 -192. H.E. Conrad, Methods Carbohydr. Chem., 6 (1972) 361-364. 193. H. Ra u v a l a , Carbohydr. Res., 72 (1979) 257-260. 194. E. Altman and G.G.S. Dutton, Carbohydr. Res., 118 (1983) 183-194. 195. J . Hoffman and B. L i n d b e r g , Methods Carbohydr. Chem., 8 (1980) 117-122. 196. J.F.G. V l i e g e n t h a r t , L. D o r l a n d , and H. Van Halbeek, Adv. Carbo-h y d r . Chem. Biochem., 41 (1983) 209-374. 197. G.O. A s p i n a l l and R.J. F e r r i e r , Chem. Ind. (London), (1957), 1216. 198. G.G.S. D u t t o n and K.B. Gibney, Carbohydr. Res., 25 (1972) 99-105. 199. G.G.S. Dutton, E.H. M e r r i f i e l d , C. L a f f i t e , F. P r a t v e i l - S o s a , and R. Wylde, Org. Magn. Reson., 20 (1983) 154-158. 200. O.S. Chizhov, V . I . Kadentsev, A.A. Solov'yov, P.F. Levonowich, and R.C. Dougherty, J . Org. Chem., 41 (1976) 3425-3428. 201. B. L i n d b e r g , J . Lonngren, and J . L . Thompson, Carbohydr. Res., 28 (1973) 351-357. 202. J . D i F a b i o and G.G.S. Dutton, Carbohydr. Res., 92 (1981) 287-298. 203. P. A l b e r s h e i m , R.H. S h a p i r o , and D.P. Sweet, Carbohydr. Res., 5 (1975) 199-216; 217-225. 204. J.A. C i f o n e l l i , Methods Carbohydr. Chem., 7 (1976) 139-141. 205. G.G.S. D u t t o n and A.V.S. Lim, Carbohydr. Res., 144 (1985) 263-276. 206. M. Adams, " B a c t e r i o p h a g e s " , I n t e r s c i e n c e P u b l i s h e r s I n c . , New York, (1959). 207. J . Douglas, " B a c t e r i o p h a g e s " , Chapman and H a l l , London, (1975). 208. C.K. Matthews, " B a c t e r i o p h a g e B i o c h e m i s t r y " , Van N o s t r a n d R e i n h o l d Co., New York, (1971). 209. D.E. B r a d l e y , B a c t e r i o l . Rev., 31 (1967) 230-314. 210. S. S t i r m and E. Freund-Mdlbert, J . V i r o l . , 8 (1971) 330-342. 211. A.A. L i n d b e r g , i n I.W. S u t h e r l a n d ( E d . ) , " S u r f a c e C a r b o h y d r a t e s o f the P r o k a r y o t i c C e l l " , Academic P r e s s , New York, (1979) pp. 289-356. 182 212. W. B e s s l e r , E. Freund-Molbert, H. Knufermann, C. Rudolph, H. Thurow, and S. S t i r m , V i r o l o g y , 56 (1973) 134-151. 213. J.E.G. Van Dam, H. Van Halbeek, J.P. K a m e r l i n g , J.F.G. V l i e g e n t h a r t , H. Snippe, M. J a n s z e , and J.M.N. W i l l e r s , Carbohydr. Res., 142 (1985) 338-343. 214. N. R a v e n s c r o f t , 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. 215. B. Kwiatkowoki, B. Boschek, H. T h i e l e , and S. S t i r m , J . V i r o l . , 45 (1983) 367-374. 216. A.V.S. Lim, M.Sc. T h e s i s , U n i v e r s i t y o f B r i t i s h Columbia, (1983). 217. G.G.S. D u t t o n and D.N. Ka r u n a r a t n e , Carbohydr. Res., 138 (1985) 277-291. 218. C.P.J. Glaudemans, Adv. Carbohydr. Chem. Biochem., 31 (1975) 313-346. 219. E. Altman, G.G.S. Dutton, and A.M. Stephen, S. A f r . J . S c i . , 82 (1986) 45-46. 220. G.G.S. Dutton, J . L . D i F a b i o , 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. 221. I.A. M o r r i s o n , J . Chromatogr., 108 (1975) 361-364. 222. P. Gehrs, B.Sc. T h e s i s , U n i v e r s i t y o f B r i t i s h Columbia, (1986). 223. H. M o l i s c h , Monatoch. Chem., 7 (1886) 108. 183 APPENDIX I K l e b s i e l l a CAPSULAR POLYSACCHARIDES (K1-K83). QUALITATIVE ANALYSES AND CHEMOTYPE GROUPING - 184 -Klebsiella capsular polysaccharides (K1-K83). Qualitative analysis and chemotype grouping G l c A G a l G l c 8 P , 1 1 P , 15, 25, 2 7 p , 51 G l c A G a l M a n 20, 2 1 P , 2 9 P 4 2 p 43, 66, 7 4 p G l c A G a l R h a 9, 9 * , 47, 52, 81, 83 G l c A G l c M a n 2, 4, 5 P 24 G l c A G l c R h a 17, 23, 44, 45, 71 G l c A G l c F u c 1, 54 G l c A G a l G l c M a D 7 P 1 0 , 1 3 P 2 6 p 28, 3 0 p 3 1 p 3 3 P 4 6 p 50, 59, 60, 61 , 62 , 6 9 p G l c A G a l G l c F u c 16, 5 8 p G l c A G a l G l c R h a 1 2 P 18, 19, 3 6 P 41, 5 5 P 7 0 P 79 G l c A G a l M a n R h a 40, 53, 8 0 p G l c A G l c M a n F u c 6 p G l c A G l c M a n R h a 6 4 p 6 5 p G l c A G a l G l c M a n F u c 6 8 p G l c A G a l G l c M a n R h a 1 4 P , 67 G a l A G a l M a n 3 P , 49, 57 G a l A G l c R h a 34, 48 G a l A G a l F u c 63 P y r A G l c R h a 72 P y r A G a l R h a 32 P y r A G a l G l c R h a 56 K e t o A G a l G l c 22, 37, 38 G l c A g l u c u r o n i c a c i d G a l galactose G a l A g a l a c t u r o n i c a c i d M a n mannose P y r A p y r u v i c a c i d R h a rhamnose K e t o A rare u r o n i c a c i d F u c fucose G l c glucose p y r u v i c a c i d present i n a d d i t i o n - 185 -APPENDIX I I GEL CHROMATOGRAMS I ' ' ' • I ! 1 i ! Hi1 j^'^Wtw (Lfl • fa i ! 10 ^00 i ^ 2,0 y> 40 5" 10 3e 3o lto DO 5"0 E l u t i o n p r o f i l e o f t h e K l e b s i e l l a K79 a n d K35 c a p s u l a r p o l y s a c c h a r i d e s s e p a r a t e d b y g e l c h r o m a t o g r a p h y Elution profile of the .eparation of K79$D by gel permeation chromato-graphy (using Bio Gel P2) / x Number of te s t - t u b e s (2.33mL/test.- tube) - 188 -APPENDIX I I I N.M.R. SPECTRA K l e b s i e l l a K79, Compound 1 Rh a i - ^ R h a ^ G a l i - ^ G l c i - ^ G l y c e r o 1 1 3C-n.m.r. 100 MHz, amb. temp. 103.02 103.99 Spectrum No. 2 17.50 73 Acetone 31.07 K l e b s i e l l a K 7 9 , C o m p o u n d 2 R h a i - ^ R h a i - ^ R h a i - ^ G a l - C M e ° a a ( p e r m e t h y l a t e d 1H-n.m.r. 400 M H z , a m b . temp. K l e b s i e l l a K79, Compound 5 Spectrum No. 6 G a l ^ G l c A i - ^ R h a P P C-n.m.r. 100 MHz, amb. temp 104.01 lAcetone 31.07 K l e b s i e l l a K79 p o l y s a c c h a r i d e 3 R h a l ^ a l l ^ G l c A l _ 2 R h a l _ 3 R h a l _ P 4 a 1 G l c 6 a 1 G l c P a a H n.m.r. 400 MHz, 95° 5.05 5.62 5.33 4.65 4.49 4.95 5 Spectrum No. 7 1.32 K l e b s i e l l a K79 p o l y s a c c h a r i d e l R h a l ^ G a l l ^ l c A l _ 2 R h a l _ ^ R h a l _ a 1 G l c 6 a 1 G l c 1 J C n.m.r. 100 MHz, amb. temp. 103.57 102.77 Spectrum No. 8 Acetone 31.07 17.55 61.86 ^}iw'nrwiimrfr^n ^mf' r I V ' lr | l I I " 1 I K l e b s i e l l a K 7 9 polysaccharide, reduced 3 R h a L _ 3 G a l I _ 3 G L C -ORhal-^Rhal-7 Spectrum No. 9 102.80 103.59 104.72 Uidii i fLirt i i i i l iW 102.75 101.78 99.70 Spectrum No. 10 K l e b s i e l l a K35, Compound A2 GlcAi-2-Man P 1 3 C n.m.r. 100 MHz, amb. temp. 102.51 104.55 92.92 Spectrum No. 12 Acetone 31 .07 K l e b s i e l l a K35, Compound A3 GlcAi-^-Mani-^Glc P a 1 3 C n.m.r. 100 MHz, amb. temp. Spectrum No. 14 102.44 61 .55 Acetone 31 . 102.89 101.54 101.84 Spectrum No. 17 K l e b s i e l l a K35, Compound SD 3 G a l i - 3 M a n l - 3 M a n i - 2 c i c i -Y a o a p H 3C COOH 13„ C n.m.r. 100 MHz, amb. temp. Acetone 31,07 5 ' 3 7 5.15 T Spectrum No. 19 K l e b s i e l l a K35, Compound DP2 3 G a l l ^ M a n l _ J M a n l _ ^ G 1 c l _ a a 2 a P P 1 G l c A 13 C n.m.r. 100 MHz, amb. temp. Acetone K l e b s i e l l a K35, Compound DPI % a l i - ^ M a n ^ J M a n i - ^ G 1 c i -P H 3C COOH 1 GlcA 13 C n.m.r. 100 MHz, amb. temp. Spectrum No. 21 A c e t o n e 31.07 K l e b s i e l l a K35 p o l y s a c c h a r i d e i G a l i - ^ M a n ^ M a n ^ G l c ^ a a 2  a P P H 3C COOH 1 GlcA 13 C n.m.r. 100 MHz, amb. temp. 103.05 103.45 172.85 173.73 Spectrum No. 23 Acetone 31.07 E. c o l i K44, Compound 2 R h a ^ L G 1 C N A C 1 _ L G l y c e r o 1 a a 1 3 C n.m.r. 100 MHz, amb. temp. Spectrum No. 26 Acetone 31.07 22. E. c o l i K 4 4 , p o l y s a c c h a r i d e (Na ^ G l c A ^ R h a i - ^ G l c N A c i - ^ G a l N A c l P a a l-H n.m.r. 400 MHz, 95° Spectrum No. 29 E- c o l i K44 p o l y s a c c h a r i d e ^ G l c A i - l R h a i - A c i c N A c i - i G a l N A c l -P a a p 1 3 C n.m.r. 100 MHz, amb. temp. 104.79 80.93 101.46 98.91 82.98 17.52 E. c o l i K44 p o l y s a c c h a r i d e (Na +) —G1 c A i - ^ R h a i - ^ G l c N A c i — ^ - G a l N A c — P a a p 1 3 C - 1 H c o u p l e d n.m.r. (SFORD expt.) 100 MHz, amb. temp. Spectrum No. 30 E. c o l i K44 p o l y s a c c h a r i d e (reduced) ^ G l c l - l R h a i - ^ - G l c N A c i - ^ G a l N A c ^ P a a p 1 3 C n.m.r. 100 MHz, amb. temp. Spectrum No. 32 .87 58,20 Acetone 31 .07 O K l e b s i e l l a K79, Compound K79$D 1H-n.m.r. 400 MHz, 95" 10D Spectrum No. 33 1.33 Acetone 2.23 2 K l e b s i e l l a K79, Compound K79#D 1 3C-n.m.r. 100 MHz, amb. temp. 102.94 Spectrum No. 34 17.53 Acetone 31.07 E. c o l i K44, crude p r o d u c t % n.m.r. 400 MHz, 95° Spectrum No. 35 E. c o l i K44, Compound PI G l c A i - ^ R h a i - ^ G l c N A c i - ^ G a l N A c /3 a a 1 H n.m.r. 400 MHz, 95° T Spectrum No. 37 E. c o l l K44, Compound p i G l c A l ^ R h a i - ^ l c N A c i - 6 - G a l N A c P Spectrum No. 38 5C n.m.r. 100 MHz, amb. temp. 1 7 5 . 0 0 12C - 227 -APPENDIX IV C.I. - MASS SPECTRA K l e b s i e l l a K79, Compound 1 (permethylated) CH^ as reagent gas Mass spectrum No. 1 K l e b s i e l l a K79, Compound 2 (permethylated) CH 4 as reagent gas Mass spectrum No. 2 i i 81 *5| 11 '7 >43 eo l oo i 2 0 i i y i o j fTTTf TTTT(TTTTT^TT I I I o-L-Rh«-(l-3)-o-L-Rh«.(l-.3)-o-L-Rh«-(l-3)-o-D-C«l I I I I I I I t I 189 363 537 7&1 329 2m 140 K-O 180 200 220 240 2G0 280 3O0 320 340 360 360 400 420 440 X50 460 480 W) 5.T0 940 560 •}>#} 60O 6iO 640 G60 680 TOO 720 740 760 780 800 t o 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0060450/manifest

Comment

Related Items