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Structural analysis of the capsular polysaccharide of Escherichia coli K34 and studies of the glycanase… Kuma-Mintah, Agyeman 1985

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STRUCTURAL ANALYSIS OF THE CAPSULAR POLYSACCHARIDE OF ESCHERICHIA COLI K34 AND  STUDIES OF THE GLYCANASE ACTIVITY  OF SPECIFIC BACTERIOPHAGE ENZYMES  BY  AGYEMAN KUMA-MINTAH .Sc. (Hons.), U n i v e r s i t y o f S c i e n c e and Technology, Ghana, 1982  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  IN  THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF CHEMISTRY  WE ACCEPT THIS THESIS AS CONFORMING TO THE REQUIRED STANDARD  THE UNIVERSITY OF BRITISH COLUMBIA NOVEMBER, 1985  Agyeman Kuma-Mintah. 1985  In  presenting  degree freely  at  of  department publication  partial  fulfilment  University of  British  Columbia,  for  and  the  available  copying  this  this  by this  in  reference  thesis  or of  thesis  for  his thesis  study.  scholarly  or for  her  purposes  of  The University of British 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  DE-6(3/81)  ig  l t f  ,  Columbia  b^cewgiPiZ, i f gr  requirements that  agree  may  be  It  is  representatives.  financial gain  the  I agree  I further  permission.  Department  of  shall  not  that  the  permission  granted  an  advanced  Library shall  by  understood be  for  allowed  the  for  make  extensive  head  that without  it  of  copying my  my or  written  ii  ABSTRACT  To f u r t h e r the u n d e r s t a n d i n g differentiation, rides,  produced  the s t r u c t u r a l by  of  of  this  and  other  capsular  polysaccha-  (E. c o l i ) s t r a i n s has  laboratories.  The  structural  E. c o l i K34 c a p s u l a r p o l y s a c c h a r i d e and r e l a t e d  are r e p o r t e d i n t h i s  studies  communication.  N.m.r. s p e c t r o s c o p y capsular  investigation  the v a r i o u s E s c h e r i c h i a c o l i  been embarked upon i n elucidation  o f the chemical b a s i s f o r s e r o l o g i c a l  and  sugar  analysis  data  on  polysaccharide indicated that i t consists of a  E.  coli  pentasaccharide  r e p e a t i n g u n i t w i t h g l u c o s e , g a l a c t o s e and g l u c u r o n i c a c i d as i t s components.  The  nature  ^H-n.m.r. s p e c t r o s c o p y fully  acetylated  on  the  the  and confirmed  polysaccharide  periodate oxidation studies  of  Smith  capsular  by the r e s u l t s o f o x i d a t i o n o f the with  chromic and  polysaccharide  consist  - G l c A p ^ D - Galpi-^D-Galpi-  0 1 D-Galp  4  1  D-Glcp  0  Methylation,  acid  degradation  on the c a r b o x y l  unit,  3  acid.  uronic  and  polymer, o f E. c o l i K34 show the s t r u c t u r e t o  a  sugar  anomeric l i n k a g e s was e s t a b l i s h e d by  hydrolysis  0  K34  0  of  a  reduced  repeating  iii  All  the sugar r e s i d u e s  i n this capsular polysaccharide  were a s s i g n e d the  D - c o n f i g u r a t i o n from c i r c u l a r d i c h r o i s m measurements. tion  of  the  bacteriophage  a-glucose  was  confirmed  by  generated o l i g o s a c c h a r i d e w i t h  treatment  group 09 E. c o l i  the immunodominant either  be  occurrence  1 —> of  1  strains  sugar o f E.  E.  coli  K34  and  (K28, K31, K32 and K33), we suggest t h a t  2 l i n k e d g l u c u r o n i c a c i d o r the t e r m i n a l g l u c o s e .  The  —>  2  E. using  coli  coli  linked  In t h i s  K34  K34  capsular  glucuronic  acid  and  K31  capsular  E.  coli  K34  polysaccharide.  polysaccharides  bacteriophages.  were  Characteriza-  generated o l i g o s a c c h a r i d e s r e v e a l e d  E. c o l i K34 and K31 b a c t e r i o p h a g e - b o r n e glycanases respectively.  in  to be r e p o r t e d i n b a c t e r i a l  t h e i r corresponding  t i o n o f the b a c t e r i o p h a g e  glucosidase  K34  may  i s the f i r s t  depolymerized  E. c o l i  polysaccharide  polysaccharide study  of  configura-  a-D-glucosidase.  From the r e s u l t s o f the c r o s s - r e a c t i o n between other  The D  that  the  are g a l a c t o s i d a s e and  iv TABLE OF CONTENTS Page ABSTRACT  i i  TABLE OF CONTENTS  iv  LIST OF APPENDICES  vii  LIST OF TABLES  viii  LIST OF FIGURES  ix  LIST OF SCHEMES  X  ACKNOWLEDGEMENTS  xi  I.  INTRODUCTION  II.  METHODOLOGY OF STRUCTURAL STUDIES ON POLYSACCHARIDES  11  11.1  I s o l a t i o n and p u r i f i c a t i o n  12  11.2  Sugar analysis  14  11.2.1  Total hydrolysis  14  11.2.2  Characterization and quantitation of sugars  16  Determinationof the configuration (D or L) of sugars  17  11.2.3  11.3  1  P o s i t i o n of linkage  18  11.3.1  Methylation analysis  18  11.3.2  Characterization and quantitation of methylated sugars  22  V  11.4  11.5  III.  23  11.4.1  Periodate oxidation and Smith hydrolysis  23  11.4.2  Uronic acid degradation  26  11.4.3  Bacteriophage degradation  29  Determination of anomeric resonance  30  11.5.1  Nuclear magnetic resonance spectroscopy  30  11.5.1.1  ^H-n.m.r. spectroscopy  30  11.5.1.2  l^C-n.m.r. spectroscopy  35  11.5.2  Chromium trioxide oxidation  37  11.5.3  Other techniques  38  .RESULTS AND DISCUSSION  40  111.1  Composition and n.m.r. studies  41  111.2  Chromium trioxide oxidation  44  111.3  Methylation analysis  44  111.4  Periodate oxidation - Smith hydrolysis  46  111.5  Selective Smith degradation  49  111.6  Determination of the configuration (D or L) of the sugar  50  I s o l a t i o n of bacteriophages ((f>31 and <£34) and cross-reactions  51  Depolymerization with E. c o l i K31 bacteriophage (c£31)  54  Depolymerization with E. c o l i K34 bacteriophage (<£34)  56  111.7 111.8 111.9  IV.  Sugar sequence  EXPERIMENTAL  61  IV.1  62  General methods  vi  IV.2  I s o l a t i o n and p u r i f i c a t i o n o f E. c o l i capsular polysaccharide  66  IV.3  Sugar a n a l y s i s and c o m p o s i t i o n  67  IV.4  Chromium t r i o x i d e o x i d a t i o n  69  IV.5  Methylation analysis  69  IV.6  Uronic  71  IV.7  Carbodiimide  IV.8  P e r i o d a t e o x i d a t i o n and Smith h y d r o l y s i s o f  a c i d degradation r e d u c t i o n o f K34 p o l y s a c c h a r i d e  c a r b o x y l reduced  K34 p o l y s a c c h a r i d e  IV.9  S e l e c t i v e Smith d e g r a d a t i o n  IV.10  Determination of  71  72 74  o f the c o n f i g u r a t i o n (D o r L)  the sugars  75  IV.11  Serological cross-reactions  75  IV.12  Bacteriophage d e p o l y m e r i z a t i o n o f E. c o l i K31 capsular polysaccharide B a c t e r i o p h a g e d e p o l y m e r i z a t i o n o f E. c o l i K34 capsular polysaccharide  76  IV.13  V.  K34  BIBLIOGRAPHY  77 80  Vll  LIST OF APPENDICES  Appendix I  Page The known structures of the Escherichia c o l i  K antigens  87  II  Mass Spectra  96  III  ^-H and C-n.m.r. spectra 13  103  viii LIST OF TABLES  Table  Page  1.1  Familia Enterobacteriaceae  II.1  Three regions of a carbohydrate n.m.r. spectrum  33  111.1  Sugar analysis of K34 polysaccharide and derived products  42  N.m.r. data f o r E. c o l i K34 capsular polysaccharide and derived products  43  Methylation analysis of K34 polysaccharide and derived products  45  Configuration of sugar residues of E. c o l i K34 capsular polysaccharide  51  III. 5  Propagation of bacteriophage #31  '54  111.6  Determination of the reducing end of E. c o l i K31 oligosaccharide i s o l a t e d after bacteriophage degradation of E. c o l i K31 polysaccharide  55  111.7  Propagation of bacteriophage #34  56  111.8  Proton n.m.r. data (400 MHz) f o r the oligosaccharide generated i n bacteriophage depolymerization of E. c o l i K34 capsular polysaccharide  58  Determination of the reducing end of E. c o l i K34 oligosaccharide isolated after bacteriophage degradation of E. c o l i K34 polysaccharide  59  111.2 111.3 111.4  111.9  ix LIST OF FIGURES  Figure 1.1  Page Schematic representation of the Gram-positive and Gram-negative c e l l w a l l of b a c t e r i a  4  Basic morphological types of bacteriophages with the types of n u c l e i c a c i d  7  1.3  The mechanics of i n f e c t i o n by bacteriophage  9  1.4  The structures of capsular polysaccharides of K l e b s i e l l a K58 and E s c h e r i c h i a c o l i K33  10  11.1  Mass spectra of (a) 1,3,4,5-tetra-O-acetyl 2 , 6 - d i m e t h y l g a l a c t i t o l (b) 1,2,5,6-tetra-03,4-dimethylglucitol  24  11.2  Structure of K l e b s i e l l a K44 bacteriophage oligosaccharide  30  11.3  R e l a t i o n s h i p between d i h e d r a l angle (#) and coupling constants f o r a- and /?- hexoses  34  111.1  C a l i b r a t i o n graph of absorbance versus  47  111.2  Periodate consumption by K34 polysaccharide w i t h respect to time  47  111.3  Separation of the depolymerization products of E. c o l i K34 by gel-permeation chromatography (Bio-Gel P-2)  57  1.2  IO4VIO3"  X LIST OF SCHEMES  Scheme  II.1  Page  Methylation analysis polysaccharide  o f E. c o l i  K34 20  II.2  Fragmentation p a t t e r n s o f a l d i t o l  II.3  S e l e c t i v e Smith d e g r a d a t i o n polysaccharide  derivatives  o f E. c o l i  21  K34 27  xi  ACKNOWLEDGEMENTS  I would l i k e Dutton  to express my s i n c e r e g r a t i t u d e t o  f o r h i s guidance  and  interest  Professor  G.G.S.  throughout the c o u r s e o f t h i s  thesis. I am t h a n k f u l  to  my  colleagues  i n the  laboratory  f o r their  encouragement and h e l p f u l d i s c u s s i o n s . My  grateful  thanks  t o Dr. S.O. Chan and the s t a f f o f the n.m.r.  s e r v i c e s and Dr. G. E i g e n d o r f services for their  and the s t a f f  of  the mass  spectrometry  assistance.  My s p e c i a l thanks t o Rani Theeparajah f o r t y p i n g t h i s t h e s i s .  xii  DEDICATED TO  MY GRANDPARENTS  - 1-  CHAPTER I  INTRODUCTION  2 I.  INTRODUCTION  Carbohydrates are polyhydroxyaldehydes acetal  or  hemiacetal  forms  or  d i l u t e a c i d to these compounds. are o f widespread occurrence are carbohydrate cally  linked  The  is  o f t e n found  severe  and  Edwards and An  Polysaccharides  not be c o n f i n e d s o l e l y to O - g l y c o s i d i -  P o l y s a c c h a r i d e s are o f g r e a t  whose  coli  belongs  first  to  i n human u r i n a r y t r a c t i n f e c t i o n s and  in  of  diarrhea^.  Escherichia  these  the  family  i s o l a t e d from f a e c e s by E s c h e r i c h i n  Enterobacteriaceae of  coli  bacteria  pathophysiological process,  are  1.1)  has been updated by  an  because  (Enterobacteriaceae)  Kauffmann^.  their  in  the  special  coli  their usefulness i n epidemiological  are  e x t r a c e l l u l a r polysaccharides. cell  for  1.1.  These  a  Gram-negative Simplified  pictures  Gram-negative and Gram-positive polysaccharides,  together  bacteria  with  of  surface role  t h e i r importance f o r the normal immunological s t a t u s o f the Escherichia  by  from both human and  interest of  1885,  Gram-negative  family  i n recent years  man  i s associated with  the  v e t e r i n a r y medicine has been f o l l o w e d by  and  commercial^  normal h a b i t a t i s the i n t e s t i n a l t r a c t o f  Ewing^ (shown i n Table  structure  by  i n most l i v i n g organisms^.  classification  interest  be h y d r o l y z e d  macromolecules  Escherichia  E. c o l i  infantile  bacteria  the  importance.  Enterobacteriaceae animals^.  which may  substances  polymers but may  organism  and  usually in  Carbohydrate-containing  carbohydrates.  and b i o l o g i c a l ^  or ketones,  which the  in  studies host. produce bacterial  b a c t e r i a are shown i n F i g . the  flagella  H  antigen  3 TABLE  1.1  Genera  Tribes  A.  B.  C.  Eschericheae  Klebsielleae  Proteae  constitute bacteria. coli  the 0,  principal  H and  bacteria  immunogens  K antigens  (Fig.  1.1)  and  i.  Escherichia  ii.  Shigella  iii.  Salmonella  iv.  Citrobacter  i.  Klebsiella  ii.  Enterobacter  iii.  Hafnia  iv.  Serratia  i.  Proteus  ii.  Morganella  iii.  Rettgerella  iv.  Providencia  and  a r e l o c a t e d on  antigens the  cell  of  the surface  coli  E. of  E.  could stimulate production of antibodies  A  - C o p u l a ' Polyiotchorid*  Copult  .Xapfjlo' Prottm  r\jptidoglyeon With TtKhoK Acid Polymers  M t n o r Of C t l l  with iviOkis fntmbron*  proteins, tnzrmts one  Ctll Woll  Cytoplasmic Membrane  The envelope of the Gram-positive c e l l wall  The envelope of the Gram-negative c e l l wall  Fig.  1.1  Schematic representation of the Gram-positive and the Gram-negative c e l l wall of bacteria. J.E. McCartney,  From T.J. Mackie and  "Medical Microbiology", V o l . 1: "Microbial  Infections", 13th edn., C h u r c h i l l Livingstone, Edinburgh, 1978.  - 5 (immunoglobulins antigens).  As  produced surface  by  immune  system  which  interact  with  components o f the b a c t e r i a , these a n t i g e n s are  implicated  i n the complex h o s t v e r s u s micro-organism i n t e r a c t i o n and are  not  responsible  only  against  the  f o r the  invading  encapsulated  bacteria  bacteria.  s t i m u l a t i o n o f the human immune system but  also  f o r the  0 and K a n t i g e n s  virulence  o f the  were shown t o be c a r b o h y d r a t e  i n n a t u r e by Toenniessen^>^ i n 1914. The  0 antigen  i s the 0 - s p e c i f i c p o l y s a c c h a r i d e  lipopolysaccharide.  I t i s a thermostable s u r f a c e  keep t h e i r immunogenic, a g g l u t i n a t i n g after  boiling.)  The  structure  and  and  of  the  antigen  cell  (the b a c t e r i a  agglutinin-binding  known  wall  properties  capacity  of bacterial  l i p o 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 - ^ >11>12 The  H antigen  of motile  E. c o l i  Most capsular two  a r e heat l a b i l e p r o t e i n s c o n t a i n e d  E. c o l i  s t r a i n s have a unique K a n t i g e n .  and envelope a n t i g e n s  of oligosaccharide  repeating  basis  antigens,  their structures  groups  of  of  three K  electrophoretic  the  weight  flagella  antigens  need (A,  can  mobility  revealed  units.  The K a n t i g e n s a r e  polysaccharides  For a c l e a r  to  be  B and L ) .  be (L  (A and B  except  These p o l y s a c c h a r i d e s  elucidated.  for  a r e made up  understanding  a n t i g e n i c s p e c i f i c i t y o f the E.  groups  electrophoretic mobility microscopy  and a l l a r e  (K88 and K99).  chemical  of  the  bacteria.  that are proteins  consist  in  o f the  coli  The  capsular  K  antigens  By e l e c t r o p h o r e t i c means two  differentiated: antigen) antigens).  and  those those  with  Inspection  t h a t the a c i d i c p o l y s a c c h a r i d e s  with  by  very  high low  electron  w i t h low m o l e c u l a r  ( h i g h e l e c t r o p h o r e t i c m o b i l i t y ) form t h i n p a t c h y  capsules  while  - 6those  with  high  molecular  weight  t h i c k and copious c a p s u l e s .  (low electrophoretic mobility)  I t h a s b e e n shown t h a t E. c o l i  0 a n d K a n t i g e n s e x h i b i t i n g t h e same i m m u n o e l e c t r o p h o r e t i c cause  form  strains  with  pattern could  t h e same disease-'--'. B a c t e r i a a r e r e c o g n i z e d b y t h e immune s y s t e m  antibody-antigen system  interactions.  The  of  a  host  a n t i b o d i e s produced  o f man o r a n i m a l s a g a i n s t a p a r t i c u l a r  E.  coli  through  b y t h e immune  strain  may  be  dependent on t h e a n t i g e n i c s p e c i f i c i t y  of the K antigen of the s t r a i n i n  question.  relatively  I t i s known  polysaccharide is  is  that  the major s i t e  termed the determinant  several  monosaccharide  specificity. sugar.  only  small  A determinant  residues,  one o f w h i c h  of  and t h i s  g r o u p may  consist  a  part of  c o n t r i b u t e s most t o t h e  r e s i d u e i s termed  non-carbohydrate  portion  of antibody s p e c i f i c i t y  groupie.  This monosaccharide  Certain  a  the  immunodominant  groups such as p y r u v a t e  and 0 - a c e t y l  19  may f u n c t i o n a s a n t i g e n i c d e t e r m i n a n t s - 1  antigen  (polysaccharide)  antigenic  w i t h i n them.  will  site  infect  the  bacteriophage of  a  host  These a r e :  cell  although  some  regarding  and  multiply  into morphological  groups^^  the  have a b r o a d  depends on t h e p r e s e n c e  surface.  receptor site by  are c l a s s i f i e d  Most a r e s p e c i f i c  This s p e c i f i c i t y on  the  f o r the i d e n t i f i c a t i o n o f the  (40 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  Bacteriophages  ( s e e F i g . 1.2).  range.  Structure elucidation of  determinant.  Bacteriophages  they  i s necessary  .  Protease  f o r E. c o l i  bacteriophages  species  of  bacteria  or less restricted of a specific  c a n be u s e d strains-*--^.  receptor  t o demonstrate The v i r a l  onto  the  infection  i s u s u a l l y c h a r a c t e r i z e d by four  ( i ) a d s o r p t i o n o f t h e phage p a r t i c l e  host  phases.  the s u s c e p t i b l e  7  2-DNA  F i g . 1.2  2-DNA  2-DNA  1-DNA  1-RNA  1-DNA  Basic morphological types of bacteriophages with the type n u c l e i c acid (from r e f . 100).  - 8host,  ( i i ) i n j e c t i o n o f the v i r a l  replication of  of  DNA  results  RNA)  into  i n the l y s i s  o f the h o s t c e l l  (see F i g . 1.3).  (glycanases), one  which  or  a  few  bacteriophage-borne  enzymes  products  substrate  and  enzymes  high  occur  in  Bacterio-  Kinetic  These  are g e n e r a l l y  studies  on  these  r e v e a l e d t h a t the a c t i v i t y i s i n h i b i t e d by concentrations^-^.  Bacteriophage-borne  ( g l y c a n a s e s ) a r e capable o f d e p o l y m e r i z i n g p o l y s a c c h a r i d e s i n t o  o l i g o s a c c h a r i d e repeating u n i t s without immunologically  significant  O-acetyl  Bacteriophage-depolymerization in  bacteriophages,  substrates.  release  lyases-*-^, 104  1  specific for  host, ( i i i )  ( i v ) phage m a t u r a t i o n and  phage -borne glycanases-'-O-'- may be h y d r o l a s e s - ^ or enzymes  the  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  the m e t a b o l i c p r o c e s s o f the h o s t ,  which  (or  structural  studies  the and  removal  of  the  possibly  p y r u v i c a c e t a l groups-^»20  and r e l a t e d s t u d i e s are an important  on carbohydrate  polymers.  tool  Dutton and coworkers  are c u r r e n t l y i n v e s t i g a t i n g the p o s s i b i l i t y o f e l u c i d a t i n g the s t r u c t u r e of  E.  coli  capsular  polysaccharides using bacteriophage  2D-n.m.r. s p e c t r o s c o p y and F.A.B. mass Stirm different  and  Rieger-Hug  Klebsiella  Klebsiella  strains,  spectrometry.  employing tested  bacteriophages^.  degradation,  seventy-four the  These  host  range  Klebsiella  g l y c a n a s e s were found to be v e r y s p e c i f i c  serologically of f i f t y - f i v e  virus-associated  (33 c r o s s - r e a c t i n g w i t h  R e c e n t l y Beynon r e p o r t e d a study o f the c r o s s - a b s o r p t i o n o f c o l i b a c t e r i o p h a g e s w i t h a number o f E. c o l i react  when  Heidelberger the presence  their has  immunodominant  groups  strains^-^. are  similar  none).  certain  Antigens in  E.  cross  nature.  used c r o s s - r e a c t i o n s e x t e n s i v e l y i n the p r e d i c t i o n o f  o f some  structural  features  before  they  were  verified  - 9 -  Fig.  1.3  The Mechanics of Infection by Bacteriophage A.  Free phage.  B.  Phage attaches to c e l l wall with f i b r e s , base plate in close contact with outer layers of c e l l wall.  C.  Sheath contracts and central core i s pushed through the c e l l wall and DNA transfer begins.  D.  Transfer of DNA completed. Phage head i s now early events of phage growth cycle begins.  empty and  (From T.J. Mackie and J.E. McCartney, "Medical Microbiology", Vol. 1, "Microbial Infections", 13th edn., C h u r c h i l l Livingstone, Edinburgh, 1978).  - 10 chemically^ ~ .  Dutton e t a l . observed t h a t E. c o l i K33 c r o s s  with  K58 s i n c e 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 a r e i d e n t i c a l  Klebsiella  in structure  play  ( F i g . 1.4)21.  It  i s c l e a r t h a t b a c t e r i a l a n t i g e n s o f t h e type  a  r o l e i n pathogenic p r o c e s s e s .  c a l l s f o r chemical embarked  upon.  polysaccharide antigenic  analysis In  (K  this  antigen)  of bacterial  antigens,  which  has  above roles been  study, the s t r u c t u r e o f E s c h e r i c h i a c o l i K34 was  elucidated  determinant was deduced.  respective  discussed  The u n d e r s t a n d i n g o f these  and  bacteriophage  the nature  o f the  I n the course o f t h i s t h e s i s two 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 (K31 and K34) were their  reacted  (#31 and  #34)  depolymerized  and  the  with  degradation  products c h a r a c t e r i z e d .  — > 3 ( -a-D-Glcp.- ( 1 — > 4 ) -/3-D-GlcpA- ( 1 — > ) -a-L-Fucp- ( l - > 3 2 \ / C OAc a-D-Galp_ / \ Me COOH  K l e b s i e l l a K58  >3(-a-D-Glcp.- (1—>4) -0-D-GlcpA-(1—>) -o-L-Fucp- ( l - > 3  2  +0Ac  \ / C Me  / \ COOH  Q-D-Galp.  E. c o l i K33 Fig.  1.4  The structures of capsular polysaccharides of K l e b s i e l l a K58 and Escherichia c o l i K33.  - 11 -  CHAPTER I I  METHODOLOGY OF STRUCTURAL ANALYSIS OF POLYSACCHARIDES  12  II.  METHODOLOGY OF STRUCTURAL ANALYSIS OF POLYSACCHARIDES  Bacterial  polysaccharides  structural patterns^ complexity different  .  and t h e i r chemical  E.  have complex and immensely  coli  capsular  structural  methods as w e l l  diversified  polysaccharides  have  such  elucidation  necessitates the use o f  as  involving  methods  the use o f  enzymes. The  methodology o f s t r u c t u r a l  (i)  qualitative  (ii)  analysis acetyl,  studies ofpolysaccharides  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 ;  f o r non-carbohydrate phosphate  s u b s t i t u e n t groups  determination  o f the linkage configuration;  (iv)  determination  o f the p o s i t i o n o f linkage;  (v)  determination  o f the sugar  known  ( O - a c e t y l , N-  etc.);  (iii)  Some  includes  analytical  sequence.  methods  employed  f o r these  goals  are  described i n the following sections.  II.1  ISOLATION AND  PURIFICATION " 23  27  A major task i n 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 interest  i s the K  i n a pure form. antigen.  The  chemistry I nthis  i s obtaining the material  study  the polysaccharide o f  i s o l a t i o n and p u r i f i c a t i o n  process  13 -  involves three (i)  stages:  b a c t e r i a growth and h a r v e s t o f crude p o l y s a c c h a r i d e  (ii)  the i s o l a t i o n o f the p o l y s a c c h a r i d e such t h a t i t i s f r e e from molecular material,  (iii)  components;  weight  material  and  other  high  molecular  weight  and  i s o l a t i o n o f a s i n g l e , monodispersed, p o l y s a c c h a r i d e s p e c i e s . p u r i t y o f the p o l y s a c c h a r i d e , the absence o f h e t e r o g e n e i t y , than  the  presence  independent  of  homogeneity  techniques  spectroscopy, and  such  as  can nuclear  chromatography'  1  individual  on  capsular  inoculating  the  f o r f o u r days. from  the  agar  s o l u t i o n and mixture  colonies  Mueller-Hinton  incubating  magnetic  agar  medium  were  received  at  37°C  until  E. c o l i K34  with  single  was  large, grown by  colonies  and  r e s u l t a n t l i q u i d c u l t u r e a t 37°C on M u e l l e r - H i n t o n The  lawn o f c a p s u l a r b a c t e r i a was  surface.  stirring  The  K34  the mixture  (polysaccharide  h a r v e s t e d by  f o r f i v e hours. plus  The  agar  scraping  b a c t e r i a were k i l l e d by adding  component  as  These b a c t e r i a l c u l t u r e  plates  were o b t a i n e d .  broth  resonance  electrophoresis^>24  and K34  Ida Orskov (Copenhagen).  Mueller-Hinton  rather  .  E s c h e r i c h i a c o l i b a c t e r i a s e r o t y p e K31 stab c u l t u r e s from Dr.  The  be demonstrated by many  o p t i c a l r o t a t i o n measurements,  gel-permeation  were s t r e a k e d  low  phenol  in  b a c t e r i a d e b r i s ) was  phenol this  dialyzed  out. The molecular  polysaccharide components  components  (e.g.  bacterial  I s o l a t i o n from aqueous s o l u t i o n by  the  were  separated  cells)  by  addition  from  the  high  ultracentrifugation. of  a  water-miscible  - 14 9  solvent^-" neutral  ft  (e.g. and  was d i s s o l v e d ammonium  ethanol,  i n water  polysaccharide.  and  treated  solution, The  neutral  and the p r e c i p i t a t e  sodium  The  like  final  as  paper  be  used  capsular polysaccharides. in  both  E.  coli  (cetyltrimethyl-  2 7  present,  the a c i d i c  remained  of  the  precipitate  K34  was  dissolved  in  in  Chromatographic  gel-permeation to  E.  were  K34 was  techniques  ion-exchange  enhance the p u r i t y and homogeneity o f  T r a c e s o f low m o l e c u l a r and  and  in  water,  I s o l a t i o n and p u r i f i c a t i o n o f E, c o l i K31  d e s c r i b e d f o r E. c o l i K34.  may  of  precipitate  d r i e d to y i e l d the p u r i f i e d E. c o l i  chromatography,  chromatography  Cetavlon  Dissolution  precipitate  f o r two days and f r e e z e  out  stringy  p r e c i p i t a t i o n w i t h e t h a n o l and c e n t r i f u g a t i o n  capsular polysaccharide. carried  with  precipitation  ( a c i d i c p o l y s a c c h a r i d e ) was s e p a r a t e d from  4M  c a r r i e d out t w i c e .  the  The r e s u l t a n t  polysaccharide  s u p e r n a t a n t by c e n t r i f u g a t i o n . chloride,  in  which s e l e c t i v e l y p r e c i p i t a t e d  the  dialyzed  results  a c i d i c polysaccharides.  bromide)  solution  acetone)  weight  contaminants  c o l i K31 c a p s u l a r p o l y s a c c h a r i d e s were  removed by g e l - p e r m e a t i o n chromatography ( B i o - G e l P2).  II.2  SUGAR ANALYSIS  II.2.1  Total  The the  hydrolysis  initial  quantitative  monosaccharides  2 8  "  3 3  step i n the s t r u c t u r a l study o f acid hydrolysis with  minimum  a  polysaccharide  o f the p o l y s a c c h a r i d e i n t o  degradation.  9p  Dutton  z o  is  individual  reviewed  the  -  advantages  and  disadvantages i n the use  acid, hydrochloric most  mineral and  p r e s s u r e and  acids.  controlled.  hydrolyzate  The In  may  quantitative individual 2M  a c i d , formic  commonly used a c i d s .  diminished  linkages  a c i d and  trifluoroacetic  attaining  acid  the  of h y d r o l y s i s must be  c a r e f u l l y chosen  The  of  E.  correct  the  monitored by paper chromatography or h . p . l . c .  hydrolysis  the  of  the  monosaccharides  coli  K34  hydrolysis  polysaccharide  w i t h minimum d e g r a d a t i o n was  into  its  attained  using  hours.  h y d r o l y t i c rates of g l y c o s i d i c linkages vary g r e a t l y . Uronosyl i n a c i d i c polysaccharides  stabilizes  the  of  uronosyl  are more r e s i s t a n t to a c i d h y d r o l y s i s  e l e c t r o n ' acceptor  linkages  acid hydrolysis,  i s the  reduction  carboxyl  groups  the  of a l l carboxyl  uronosyl  by d e r i v a t i z a t i o n i n t o c a r b o d i i m i d e ^  followed  by  borohydride  reduction  developed i n our  uronosyl  linkages  to  reduction.  laboratory ^ 3  acid  An  a l t e r n a t i v e method of  to overcome  hydrolysis,  involves  the  resistance  of  m e t h a n o l y s i s of  the  simultaneous e s t e r i f i c a t i o n o f the  a c i d , which i s then reduced to the  corresponding a l c o h o l .  mixture of n e u t r a l glycosides  hydrolyzed  is  with  to ensure complete r e l e a s e o f sugar r e s i d u e s . the  acids  advantage o f b e i n g l e s s d e s t r u c t i v e  3 2 , 3 3  .  Karunaratne has  discussed  to  derivatives  2  g l y c o s y l l i n k a g e s w i t h the  c h l o r i d e has  linkage  One  f u n c t i o n a l i t i e s i n the  a c i d i c polysaccharide sodium  which  through the h e t e r o c y c l i c oxygen.  o f the means o f overcoming the r e s i s t a n c e o f  sialic  are  condition  because o f the presence  acid,  Sulphuric  thus i s b e i n g used i n c r e a s i n g l y i n s t e a d conditions  be  of d i f f e r e n t a c i d s .  T r i f l u o r o a c e t i c a c i d i s e a s i l y removed under  t r i f l u o r o a c e t i c a c i d f o r 20 The  15  2M  The  carboxylic resulting  trifluoroacetic  M e t h a n o l i c hydrogen to deoxy sugars  the h y d r o l y t i c  and  conditions  - 16 f o r the h y d r o l y s i s o f  polysaccharides  would t h e r e f o r e not be  duplicated i n this thesis.  I I . 2. 2  C h a r a c t e r i z a t i o n and  The  sugars  containing  q u a n t i f i c a t i o n of  released  upon  acid  amino  sugars^ '- ~ 0  hydrolysis  q u a l i t a t i v e l y u s i n g paper c h r o m a t o g r a p h y ^ ' ^ , h i g h chromatography^, graphy-^. of  paper  electrophoresis-^,  C o l o r i m e t r i c - ^ •^®  sugars  individual  sialic  can  be  analyzed  thin  liquid  l a y e r chromato-  (hexoses, pentoses, u r o n i c  a c i d s , deoxy or  acid)  however  can  5  performance  but  High  has  limited  performance  has  a  applications  liquid  a l s o be used f o r q u a n t i t a t i v e a n a l y s i s  H.p.l.c.  4  and  classification  chracterization.  (h.p.l.c.) sugars.  and  and  54  Of)  a n a l y s i s can be used i n the  sugars i n t o broad c l a s s e s  amino  sugars  for  chromatography  of  underivatized  l i m i t e d number o f s t a t i o n a r y phases  compared to g a s - l i q u i d chromatography. The  monosaccharides,  polysaccharide,  can  be  released  upon  rides.  An  characterization  extensive  Dutton^• (TMS)  and  .  review o f  Sugars  can  d e r i v a t i v e s but  equilibrium problem was volatile  be  this  acetates,  of  the e x i s t e n c e  converting  G.l.c.  of  offers  sugar r e s i d u e s  technique  analyzed  y i e l d s a complicated s o l v e d by  hydrolysis  c o n v e r t e d to v o l a t i l e d e r i v a t i v e s and  u s i n g g a s - l i q u i d chromatography ( g . l . c ) . quantification  acid  has  anomeric  forms  analyzed  reproducible of  been  as t h e i r v o l a t i l e  polysacchareported  sugar  by  trimethylsilyl of  sugars  chromatograph o f m u l t i p l e peaks. the a c y c l i c  of  alditols  t r i f l u o r o a c e t a t e s or t r i m e t h y l s i l y l e t h e r s .  into  at This the  Alditol  - 17 -  t r i f l u o r o a c e t a t e s show p a r t i a l d e - e s t e r i f i c a t i o n on the column and TMS  derivatives  of  the  alditols  give  poor  A l d i t o l a c e t a t e s have good r e s o l u t i o n and s h o r t t h e r e f o r e , were used throughout  resolution^ retention  this investigation.  spectrometry.  II.2.3  (D o r L) o f  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  In g e n e r a l , chromatographic analyses  do  not  s e p a r a t i o n methods  d i s t i n g u i s h between enantiomers.  can be s e p a r a t e d by g . l . c . u s i n g  a  enantiomers  using  into  (-)-2-butanol,  diastereomers  D  chiral  and  3  s e p a r a t e d by  sugars 44-47  and  spectroscopic  However or  enantiomers  converting  reagents  the  ( f o r example,  and  L  •^ .  configuration  i s o l a t i o n o f the d i f f e r e n t optical  column  g.l.c.  ( + ) - 2 - o c t a n o l , o r ( + ) - 1 - p h e n y l e t h a n e t h i o l ) and s e p a r a t i o n  on a n o n - c h i r a l p h a s e ^ • ^ The  chiral  on  times^  Sugars  g . l . c . are u s u a l l y c o n f i r m e d by g.l.c.-mass  2  the  rotation  of  sugars can be determined by  monosaccharides  [ a ] .  Specific  n  and  measurement  oxidases  (e.g.  of  the  their  D-glucose  and  D - g a l a c t o s e o x i d a s e s ) and enzymes can be used f o r the d e t e r m i n a t i o n o f D and L c o n f i g u r a t i o n o f sugars. The sugar 213  method  i n this nm  on  methylated chromophore.  employed  i n d e t e r m i n i n g the D and L c o n f i g u r a t i o n o f  i n v e s t i g a t i o n was alditol  alditol  acetates, acetates,  by c i r c u l a r d i c h r o i s n A acetylated where  the  7  measurement  aldononitrile acetoxy  group  at  or p a r t i a l l y acts  as  a  -. 18 II. 3  POSITION OF LINKAGE  II.3.1  Methylation a n a l y s i s " 4 8  This  technique  involves  h y d r o x y l groups o f the rides  4 8  sugar  5 5  the complete e t h e r i f i c a t i o n o f the f r e e residues  in  oligo-  and  poly-  saccha-  ' ^ which a c t s as a l a b e l i n d i s t i n g u i s h i n g the o r i g i n a l  unlinked  4  p o s i t i o n from the l i n k e d p o s i t i o n . employed  Methylation  analysis  is  routinely  i n the s t r u c t u r a l c h a r a c t e r i z a t i o n o f complex c a r b o h y d r a t e s  a means to e s t a b l i s h  ( i ) linkage p o s i t i o n s ;  sugar p e r r e p e a t i n g u n i t ; unit(s);  and  (iv)  the  (iii)  ( i i ) number and  types  i d e n t i t y of terminal u n i t ( s ) ,  position  of  base-stable  as of  branching  substituents  (e.g.  pyruvate). In with  the e a r l y days, methyl e t h e r s were formed by r e p e a t e d r e a c t i o n  dimethyl  sulfate  polysaccharide methylated  with  and  sodium  s i l v e r oxide  hydroxide ^.  Treatment  5  i n b o i l i n g methyliodide  p o l y s a c c h a r i d e a c c o r d i n g to P u r d i e  and  gives a  Irvine ^. 5  of fully  Purdie's  S9  method  was  considerably  improved  by  formamide as a s o l v e n t i n c o n j u n c t i o n oxide.  A  more  convenient  d e v i s e d by H a k o m o r i ^ . 5  with  sodium  iodide due  5 4  to  .  careful  with  dissolution  methyl  methanide be of  (dimsyl  of  used N,N-dimethyliodide  and  silver  the  of a  the  sample.  sodium)  polysaccharide,  The may (for  was  polysaccharide and  methyl  i t the Hakomori method or not,  i n the a p p r o p r i a t e o r g a n i c s o l v e n t  de-ionization  who  T h i s i n v o l v e s the treatment  Most u n d e r m e t h y l a t i o n s ,  polysaccharide  JZ  method f o r m e t h y l a t i n g p o l y s a c c h a r i d e s  methylsulfinyl  incomplete  Kuhn -  are  s o l u b i l i t y of a be  enhanced  example,  by  using  - 19 -  Amberlite  IR-120 (H )  resin).  +  g i v e s an "undermethylated"  product,  by u s i n g the P u r d i e method. conducted result  on a m e t h y l a t e d  i n /3-elimination  In cases where the  A  solvent-'-'.  second  Hakomori  acidic oligo-  methylation  (see S e c t i o n I I . 4 . 2 ) .  Methyl  tion  absorption  content.  The  trifluoroacetic  3600  methylated acid  cm"-'-)  polymer  or  is  by  to  give  dialysis,  analysis  usually  a t 95°C f o r about 18 h.  volatile  partially  The  with  extraccomplete-  (absence  partially  using  alditol  acetate  Uronic  i d e n t i f i e d by comparison o f m e t h y l a t i o n a n a l y s i s  be  r e s u l t s o f the a c i d i c reduced  polysaccharide.  polysaccharide  with  those  of  Scheme  and  and  may  (see  2M  methylated  to t h e i r a l d i t o l s  methylated  of  o f the methoxyl  hydrolyzed  The  analyses  as  derivatives for g.l.c. acids  g.l.c.-m.s.  groups  be reduced  i . r . spectroscopy  monosaccharides r e l e a s e d on h y d r o l y s i s are reduced acetylated  of a c y l  chromatography (Sephadex LH 20).  at  will  step.  m a t e r i a l i s u s u a l l y p u r i f i e d by  ness of m e t h y l a t i o n can be v e r i f i e d by hydroxyl  never  trimethylphosphate  P o l y s a c c h a r i d e s c o n t a i n i n g u r o n i c a c i d s may  and g e l - p e r m e a t i o n  is  trifluoromethane  cleavage  o f 2 , 6 - d i t e r t i a r y b u t y l p y r i d i n e and  methylated  achieved  or p o l y - s a c c h a r i d e as t h i s  l i t h i u m aluminum h y d r i d e a f t e r the p e r m e t h y l a t i o n The  methylation  complete m e t h y l a t i o n can be  i s a m i l d e r base and e f f e c t s m e t h y l a t i o n without i n the presence  Hakomori  the  I I . 1).  methylated-  20 f  <rQ}l  K34  r  polysaccharide  O*e  _  Ox.  I - OH  <5 .0*  1.  3,4-OMe .-Glucose  4.  2,4,6-OMe -Galactose  2.  2,6-_OMe -Galactose  5.  2,3,4,6-OMe^-Glucose  3.  2,3,6-OMe -Galactose  2  3  3  1)  11)  MsIH  (  -E_0/s>rTldll.t  OAC  -OAC  -OAc  -OAc  OAc  -OKf  "OMe  "OH*  AcO • OHe — O A c  Scheme II.1  -OUt  AcO • -OAc  3Ac  -Ont  *—One  - OAc  -0!_  Methylation analysis of E. c o l i K3A polysaccharide  21 Primary fragmentation:  QJ t  117 161  2  HCCMe + MeOCH + HCCMe  205 161  I  HCQAc 45  CH CMe 2  Secondary fragmentation:  ©  0  HC=CMe  HC=0Me  I  HCCMe  -AcOH  I  HCOAc  I  9  CCMe II  CH  I  2  HCCMe  CH. -AcOH  CH aie  m/z 205  l V z 145  I  CH_  2  CCMe B>CMe  H&gfe  CH CMe 2  CH_0Ac  —>  2  rtVz 101  m/ 161 z  -AcOH HC-OMe HCOAc  ©  CH CMe 2  rr/z 161  -CH CO 2  HC=CMe  I c=o I  CH Scheme I I . 2  3  rr/z 87 Fragmentation patterns of a l d i t o l d e r i v a t i v e s  II© H C  V  Q / H  Me ir/z 71  - 22 II.3.2  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 m e t h y l a t e d sugars  2 8  >  4 1  >  5 6  >  5 7  P a r t i a l l y m e t h y l a t e d monosaccharides of  the p e r m e t h y l a t e d  oligo-  u s i n g paper c h r o m a t o g r a p h y ^ . _>-anisidine min.  hydrochloride  Gas-liquid technique sugars.  The m e t h y l a t e d sugars a r e d e t e c t e d  spray^^  chromatography  f o r quantitative  f o l l o w e d by h e a t i n g a t liO°C f o r 5  (g.l.c.)  according  i s the most  and q u a l i t a t i v e  A review o f the a p p l i c a t i o n s o f g . l . c . 2 8  partially  analysis  to  widely of  their  analysis  are analyzed  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 d u r i n g t h i s work. partially  used  methylated  to carbohydrate  The m e t h y l a t e d sugars  4  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 o n o f these acetates  with  (Rf v a l u e s ) and the d i f f e r e n t c o l o u r s formed.  has been p u b l i s h e d by D u t t o n - ! . their  hydrolysis  o r p o l y - s a c c h a r i d e can be c h a r a c t e r i z e d  These sugars a r e then t e n t a t i v e l y i d e n t i f i e d  relative mobilities  as  r e l e a s e d on t o t a l  methylated  The  alditol  a r e made by c o n s i d e r a t i o n o f the r e l a t i v e r e t e n t i o n times and  co-chromatography w i t h a u t h e n t i c samples. For unambiguous i d e n t i f i c a t i o n  of partially  methylated  a c e t a t e s , g . l . c . r e s u l t s s h o u l d be c o n f i r m e d u s i n g g.l.c.-m.s.  alditol Studies  done on the f r a g m e n t 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 been  reported . 5 7  The p r i m a r y  fragments  a r e formed  f i s s i o n o f t h e C-C bond i n the a l d i t o l c h a i n and t h i s the p r e f e r e n t i a l o r d e r shown below.  have  as a r e s u l t o f  cleavage  follows  23 -  CHOMe  CHOAc  CHOMe  > CHOMe  The  intensity  weight.  the p r i m a r y  i o n decreases w i t h i n c r e a s i n g m o l e c u l a r  Secondary i o n s c a n be o b t a i n e d by l o s s o f a c e t i c a c i d  ketene  (m/z  4 2 ) , methanol (m/z 32), methyl a c e t a t e  methyl a c e t a t e II.1  of  CHOAc  CHOAc  (m/z 104) o r acetoxymethyl  illustrates  acetate  (m/z 74), methoxy-  (m/z  the d i f f e r e n c e i n the mass s p e c t r a o f  acetyl-2,6-di-0-methyl-D-galactitol  and  (M/z 60),  132).  Figure  1,3,4,5-tetra-0-  1,2,5,6-tetra-0-acetyl-3,4-di-  O-methyl-D-glucitol.  II.4  SUGAR SEQUENCE  The  elucidation  involves  the i s o l a t i o n  fragments. techniques  Lindberg  of  the sequence and  and  o f sugars  characterization coworkers  5 8  have  i n a polysaccharide of  oligosaccharide  reviewed  the v a r i o u s  employed i n the s p e c i f i c 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 .  degradation  techniques  The  employed i n t h i s i n v e s t i g a t i o n a r e d i s c u s s e d i n  this section.  II.4.1  P e r i o d a t e o x i d a t i o n and Smith H y d r o l y s i s  O x i d a t i v e cleavage  o f the C-C bond  5 9  "  of v i c i n a l  6 4  diols  by  sodium  24  '  41  »f»° ei 7f tf Bf 4f 3f  r  if  61  III  I  Iff  r  'i i  ' • i-1 i i i i i | i i  1  ' iff  25f  r  | ri  3tf  ii i ii i i|i iii i i i i i |  3Sf  '»»  (a)  i«f *f if ii  te ** 4f If If If  _Lii II  f l | I "I 6f  iff  ?33 ,261 1 "1 1 '| I' V I ( I I I i I I' I I 1 I I I t I I I I I I II I| I I I I I I I I I | I I I I I I I I I | Iff Itf 4ff IH 2ff 2Bf 1  (b)  Figure II.1  Mass s p e c t r a o f (a) dimethylgalactitol. dimethylglucitol  1,3,4,5-tetra-0-acetyl-2,6(b)  1,2,5,6-tetra-0-acetyl-3,4-  25 -  metaperiodate i s  of  polysaccharides-^ technique  d  its  namely  Smith  the  structural  uses are two  fold.  degradation^.  i n aqueous media w i t h  oxidation about 4°C  in  u s i n g s m a l l amounts of m a t e r i a l and  technique out  a n  importance  may 60_  prevented  by p e r f o r m i n g  x;he  periodate  consumption  a n  d  the  results  terminal  side-chains  consumed  for  every  "polyaldehyde"  and  1-6  a  oxidizable  sugar  can  sodium b o r o h y d r i d e  glycol  are  conformation"''.  resistant Ebisu  /3-D-galactopyranosyl l e a v i n g the 1 — > In  a  similar  D-glucose, and  et.al.  residues  ion.  monitored  the number o f  exercise,  one  a  1 —>  periodate for  mole o f p e r i o d a t e i s  repeating  unit^.  The is  i n t o the p o l y o l . than  trans  o x i d a t i o n i f f i x e d i n an oxidized  i n the Pneumococcus S-14  selective  and  2  some  unfavourable  the  terminal  polysaccharide  oxidation  4 l i n k e d g a l a c t o s e over 1 — > was  spectro-  oxidation of a polysaccharide,  selectively  the  Over-  Except  4 l i n k e d /3-D-glucose u n i t s i n the main c h a i n  a c i d i n E. c o l i K34 For  to  as a p r e p a r a t i v e  are u s u a l l y c a r r i e d  be  C i s g l y c o l s are observed to o x i d i z e f a s t e r trans  analytical  polysaccharide.  in  of  the r e a c t i o n i n the dark at  l i n k e d sugars,  produced, on p e r i o d a t e  u s u a l l y reduced w i t h  secondly,  illustrate  s e n s i t i v e sugars per r e p e a t i n g u n i t i n  as an  metaperiodate  be  photometrically^O  First,  Oxidations  the water s o l u b l e  determination  of  the  linked  intact^ . 3  terminal glucuronic  achieved.  a n a l y t i c a l purpose, m e t h y l a t i o n  a n a l y s i s or sugar a n a l y s i s i s  mostly performed on s m a l l q u a n t i t i e s o f the p o l y o l . Smith oxidation  degradation devised  by  is Smith  an  important and  modification  co-workers  i n f o r m a t i o n on the sequencing of sugar  residues  .  It in  a  of gives  periodate valuable  polysaccharide.  - 26  Smith  degradation  hydrolysis  on  involves  on  leaving  glycosidic  or  poly-  the p o l y o l r e s u l t s  a p o l y o l obtained  II.4.2  Uronic  and  sugar  degradation  these  analysis  c a n be  carboxylic is  acid  degradation  fragments  functionality  esterified  base  proton  of  or  from  performing  a t C-5.  group and  When t h e m e t h y l a t e d  t h e j3-elimination  hex-4-eno-pyranosiduronate  degradation  are  5 8  •  6 5  •  to  6 6  >  6 7  acidic  enhances the polysaccharide  the p r o t o n  at  o f the 4 - 0 - s u b s t i t u e n t residues  o u t l i n e d as f o l l o w s :  '.  The  Selective  -  6 8  generate 5 8  '  '  6 6  .  residue  acidity  The  of  is a the  i s treated with C-5  is  w i t h the  main  6 5  defined  polysaccharide  esterified uronic acid thus  be  Scheme I I . 3 ) .  an a c i d i c p o l y s a c c h a r i d e  The  may  m i l d a c i d h y d r o l y s i s on  employed  i n an  acid  oligo-  saccharides  analysis.  (^-elimination)  c a n be  yields  o x i d a t i o n (see  o f the u r o n i c a c i d  on m e t h y l a t i o n .  poly-  acid  acetal linkages  Smith degradation or  mild  This mild  of the  methylation  (sodium m e t h y l - s u l f i n y l methanide),  f o l l o w e d by  cleavage  from s e l e c t i v e p e r i o d a t e  strong electron-withdrawing ring  o x i d a t i o n f o l l o w e d by  oligo-  a c h i e v e d by  Base c a t a l y z e d d e g r a d a t i o n oligosaccharide  i n the  linkages intact.  saccharides  c h a r a c t e r i z e d by Smith  periodate  l a r g e q u a n t i t i e s o f the r e s u l t a n t p o l y o l .  hydrolysis the  -  steps  removed formation of  this  - 27  ,— OH  1.  NaI0  C0.02M  4  3 hrs)  OH 3.  NaBH^  REDUCTION  hrs  CHjOH  CM OH 2  CHjOH  Scheme II.3  Selective Smith degradation of E. c o l i K34 polysaccharide  -  Aspinall conditions  and  The  o c c u r s and  h y d r o x y l group  alkylation  have  shown  in  their  experiments  that,  under  n o r m a l l y used f o r base d e g r a d a t i o n s , complete l o s s of  acid residues free  Rosell  28  that  the  acid hydrolysis  exposed a f t e r  is unnecessary . 6 8  /.-elimination,  can  be  i s analyzed  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 the  o f E. c o l i K34,  the  the  determined  comparing  by  s i t e o f attachment  t h a t o f the m e t h y l a t i o n  g.l.c.-m.s.  analysis.  of  by  uronic  iodide.  g.l.c.-m.s.  structure acid  The  l a b e l l e d by  w i t h methyl i o d i d e , e t h y l i o d i d e or t r i d e u t e r o m e t h y l  resultant alkylated oligosaccharide  uronic  of  elucidation unit  was  r e s u l t s o f the 6 - e l i m i n a t i o n  and  - 29 -  II.4.3  Bacteriophage degradation  Bacteriophages  ((}>)  m u l t i p l y i n g w i t h i n them. degrading  enzymes  glycanases ^  viruses,  Bacteriophages  which  include  and l y a s e s ! > _  infecting  their  hosts  carry host surface polysaccharide  and  carbohydrate  deacetylases , 6 9  For 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  there  an i n d i v i d u a l s p e c i f i c phage c o n t a i n i n g an endoglycanase -^.  Each  7  exists  are  7  7 2  7  enzyme  is  capable  of h y d r o l y z i n g a p a r t i c u l a r capsular polysaccharide  into oligosaccharide repeating non-carbohydrate coupled  with  F.A.B.-M.S.  units  substituents  its  intact^ .  acid  or  base  Bacteriophage  9  characterization etc.)  with  techniques  labile  degradation  (methylation  analysis,  may be used i n the sequencing o f sugar r e s i d u e s i n a  polysaccharide. Bacteriophages propagated  are  usually  isolated  from  sewage  and  may  on b a c t e r i a a c c o r d i n g to the s t a n d a r d p r o c e d u r e s o f A d a m s . 74  To d e p o l y m e r i z e 1 g o f a 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 , 1 0 ^ o f corresponding  bacteriophage  may  be  required .  degraded by i t s c o r r e s p o n d i n g b a c t e r i o p h a g e N.m.r.  and  methylation  i s shown i n F i g .  a n a l y s e s s t u d i e s on t h i s  K44  the g l u c o s e s ride.  respectively . 7 6  oligosaccharide  F.A.B.-mass  7 7  (see  K44  capsular  s p e c t r o m e t r y s t u d i e s on K44  o l i g o s a c c h a r i d e c o n f i r m e d the sequence o f given s t r u c t u r e  and  Thus g l u c u r o n i c a c i d i s l i n k e d t o one of  i n the main c h a i n o f K l e b s i e l l a  Recently  was  II.2.  r e v e a l e d t h a t g l u c u r o n i c a c i d and g l u c o s e were the n o n - r e d u c i n g end end  the  The s t r u c t u r e o f an  7 5  o l i g o s a c c h a r i d e s i n g l e r e p e a t i n g u n i t o b t a i n e d when K l e b s i e l l a  reducing  be  Figure I I . 2).  the  sugar  polysacchabacteriophage  residues  in  the  30  In  this  study  E,  coli  -  K31  and K34  polysaccharides  were  depolymerized u s i n g t h e i r corresponding bacteriophages.  ^  >G 1 c A ^ R h a i - ^ R h a ^ L J - G 1 c i - ^ G 1 c — > /3 a a p 16 a  I  OR R=H o r Ac  Figure II.2  II.5  S t r u c t u r e o f K l e b s i e l l a K44 b a c t e r i o p h a g e  oligosaccharide  DETERMINATION OF ANOMERIC LINKAGE  II.5.1  N u c l e a r magnetic resonance  II.5.1.1  Proton  N.m.r.  spectroscopy  magnetic  configurational  resonance  spectroscopy  is  widely  used  in  c o n f o r m a t i o n a l and s t r u c t u r 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  and t h e i r d e r i v a t i v e s . structural  spectroscopy  problems  ^H-n.m.r.  spectroscopy  was  first  applied  i n c a r b o h y d r a t e s by Lemieux and c o - w o r k e r s . 7 9  to The  OA introduction  of  Fourier-transform sensitivity spectrometer  of  magnets  based  technique ^ 8  ^H-n.m.r.  is essential  on  superconducting  have  spectroscopy. for ^H-n.m.r.  enhanced The  solenoids  the use  of  resolution a  and and  high-field  spectroscopy of polysaccharides  - 31 -  due  t o the h i g h v i s c o s i t y o f p o l y s a c c h a r i d e samples.  of  polysaccharides,  s i g n a l broadening times of  run  which i s l a r g e l y due  most p o l y s a c c h a r i d e at  Sharp s i g n a l s i n the serve  to  i n order  spectra  short  spin-spin  relaxation  A s u b s t a n t i a l enhancement i n the q u a l i t y  s p e c t r a can be  60°-90°  spectra  ambient temperature, a r e c h a r a c t e r i z e d by  o f the polymer p r o t o n s .  temperature  to  obtained  achieved  by  using  elevated  reduce v i s c o s i t y o f the sample. for bacterial  polysaccharides  as p r o o f o f the r e g u l a r r e p e a t i n g u n i t s p r e s e n t w i t h i n them.  dimensional  homo- and h e t e r o - n u c l e a r n.m.r. has been  enhancement and  at  ^H-n.m.r.  of  resolution  a s s i g n i n g chemical  shift  employed  as w e l l as o b t a i n i n g c o u p l i n g  Two  i n the  information  values . 8 3  I n t e r p r e t a t i o n o f a ^H-n.m.r.  spectrum  requires  measurement  of  c e r t a i n parameters.  (i)  Chemical  The factors, negativity nature  shift  chemical (a)  shifts  of  substitution,  effects  of  protons  orientation  neighbouring  and  o f the s o l v e n t can induce p r o t o n s  strengths.  Thus,  may  equatorial  depend  of  the f o l l o w i n g  the molecule,  distant  t o resonate  ring-hydrogen  on  electro-  groups, and (b) the at different  atoms have lower  than t h e i r a x i a l c o u n t e r p a r t s .  oligo-  o r p o l y - s a c c h a r i d e i s made up o f t h r e e main r e g i o n s namely:  anomeric  region  (5 4.5-5.5),  ( i i ) the  n.m.r.  chemical  shifts  the  The  field  ring  spectrum  proton  an (i)  region  3.0-4.5), and (c) the h i g h f i e l d  r e g i o n (S 1.15-2.5), where  CH3 o f 6-deoxy sugars,  0 - a c e t y l , N - a c e t y l e t c . c a n be  pyruvate,  of  (5  signals for observed  32 -  (see  Table  two,  that  I I . 1). is  The  signals  anomeric r e g i o n can be a r b i t r a r i l y d i v i d e d i n appearing  o f S 5.0  upfield  Q - l i n k a g e s and those d o w n f i e l d are a s s i g n e d  are  t o /3-linkages.  the l i n k a g e c o n f i g u r a t i o n (a and /!) can be determined measurements The  of  the  number o f sugar  number  chemical  shift  (ii)  d e s i g n a t e d by  using  from  agreement equation vicinal  spin-spin  coupling  J and are expressed  J  the  the  (see l a t e r ) .  integrals.  are  the  The r i n g  difficult.  Hall  8 4  constants  Nuclear  directly  the combined  a s o l u t i o n t o t h i s problem.  Coupling  spectrum,  from  r e s i d u e s p e r r e p e a t i n g u n i t can be deduced from  p r o t o n r e g i o n i s q u i t e complex and assignments  to  Furthermore  and c o u p l i n g c o n s t a n t  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  has proposed  assigned  magnitude  of  the spectrum.  Karplus with  gives  an  the  and  values  approximate  coupling constant  as H e r t z  over  (Hz).  coupling  these (see  (^J) and the  Figure  II.3). between  d i h e d r a l angle  2  - 0.28  9.5 c o s # - 0.28 2  0°^90°  90°£tel80°  bonds  first  are  order  can be measured  also  are  protons  8.5 c o s #  a  constants  values  relationship  three  In  C o u p l i n g c o n s t a n t s can  equation  observed  constants  be  predicted  usually The the  i n good Karplus  8 5  three-bond  (#) between the  33  Table II.1  Three r e g i o n s o f a carbohydrate  spectrum  Region  4.5  Anomeric  93 - 110  - 5.5  >5.0  ( J  <5.0  (J  1  1  2  >  2  1-3  Hz)  <101  7-9  Hz)  >101  3.0 - 4.5  Ring H-2  Man  H-5  GlcA  4.0 - 4.5  75 ± 5  2°C 2°C  60 - 85  80 - 85  (linked)  60 - 65  1°C  1.0 - 2.5  15 - 30  N-acetate  -2.03  -21  0-acetate  -2.15  -21  CH3  (6-deoxyhexose)  -1.33  -17  CH3  (pyruvic acid  High  field  axial  CH  ketal)  3  e q u a t o r i a l CH3  £  C o f C - 0 group -170 p.p.m.  -1.4  - -1.6 -18 -26  ( a c e t a t e , p y r u v a t e and u r o n i c a c i d ) appear  34  Figure II.3  Relationship between dihedral angle (^) and coupling constants  f o r a - and ^-D-hexoses  - 35 -  The v a l u e s are maximum when the d i h e d r a l  angle  (#) i s 0° or 180°,  the d e t e r m i n a t i o n o f  v a l u e s has been  and minimum when i t i s 90°. In carbohydrate used  to  establish  f o r pyranose,  chemistry,  c o n f i g u r a t i o n as w e l l as c o n f o r m a t i o n a l p r e f e r e n c e s  furanose and a c y c l i c  ( i i i ) R e l a t i v e i n t e n s i t i e s of the  The  relative  hydrogens  are  the s i g n a l s 6-deoxy  0 0  intensities  sugars.  signals  of  absorption  signals  for  different  equal to the r e l a t i v e numbers o f the hydrogens  .  The number o f anomeric  linkages,  relative  producing  amounts  of  sugars, 0 - a c e t y l , N - 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 s u b s t i t u e n t s  can be determined signals.  For  by  computing  oligo-  the  integral  of  their  corresponding  or p o l y - s a c c h a r i d e s , t h i s v a l u e p e r m i t s a r a p i d  q u a n t i t a t i v e a n a l y s i s o f the r a t i o o f a to ^ l i n k a g e s .  II.5.1.2  i J  The  C  n.m.r. s p e c t r o s c o p y  advent o f Q  modern  techniques  "7 '  Fourier-transform  have  enhanced  n.m.r.  the  spectroscopy  sensitivity  of  and  the  other  natural  abundance o f • C-n.m.r.  ^C-n.m.r. s p e c t r o s c o p y which i s r a p i d and non-  destructive  potential  LJ  has  great  in  the  study of p o l y s a c c h a r i d e s o f  b i o l o g i c a l o r i g i n where o n l y s m a l l amounts of m a t e r i a l are a v a i l a b l e f o r analysis.  In  the  study o f complex molecules  such as p o l y s a c c h a r i d e s ,  the amount o f i n f o r m a t i o n o b t a i n a b l e from lH-n.m.r. s p e c t r a  is  usually  - 36 limited  compared  spectroscopy  to  has  that  been  revealed  employed  by  -^C-n.m. r .  i n structural  8 8  .  2-D N.m.r.  studies  of  complex  carbohydrates ^•^0. 8  ^C-n  The  m  spectrum  r  of  a  polysaccharide  spectrum, can be c a t e g o r i z e d i n t o three r e g i o n s C-n.m.r.  the main  parameter  However, i f the s p e c t r a comparison  of  have  integrals  of  hydrogen atoms o f t e n y i e l d s amounts  of  components  a  carbon  appear  shielding effects. an  In  good  (s/n),  accurate  signal  to  noise  ratio  atoms c a r r y i n g the same number o f information  mixture^.  upfield  about  the  relative  An a r b i t r a r y d i v i s i o n o f the b u t c o n t r a r y t o the p.m.r. the  o f the /9-anomeric  and ^H s h i f t s a r e a f f e c t e d  carbons  due to  inversely^  because  2  i n c r e a s e d s h i e l d i n g o f a ^ C n u c l e u s i s accompanied by a decrease i n 3  the s h i e l d i n g o f the a t t a c h e d free  II.1).  shift.  anomeric r e g i o n a t 101 p.p.m. i s accepted a-anomeric  (see Table  the p.m.r.  used f o r assignment i s c h e m i c a l  carbon  in a  like  sugars  (reducing  proton.  S i g n a l s f o r anomeric  end) appear u p f i e l d ,  carbons  of  i n the r e g i o n 93-97 p.p.m.  1^  S i g n a l s f o r CO group ( a c e t a t e , p y r u v a t e and  uronic  acid)  and  C=C  group appear around 170 p.p.m. and 140-150 p.p.m. r e s p e c t i v e l y . Signals  due  carbon r e g i o n . signals  between  non-linked  to  C-2  t o C-6 o f sugar r e s i d u e s  Carbon atoms o f  60-65  by t h e i r  primary  alcohols  appear i n the r i n g  have  characteristic  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 or  chemical  shifts  (non-linked,  60-62  p.p.m.  when  l i n k e d , they a r e 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  —  of  secondary  O-glycosylation deshielded  alcohols  appear  at  75  5  p.p.m.,  b u t on  o r 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  (by 7-11 p.p.m.) as t o produce a s i g n a l w e l l  separated  from  - 37 -  other  ring  result  i n an or-effect t h a t i s the u p f i e l d s h i f t  carbon  carbons  (80 — 5 p.p.m.).  atom(s) i n v o l v e d .  a and /3  effects  o f the s i g n a l ( s ) o f the  However, carbons immediately a d j a c e n t  carbon w i l l be s l i g h t l y s h i e l d e d These  T h i s d e s h i e l d i n g phenomenon may  to t h a t  (1-2 p.p.m.) and t h i s i s the /3-effect.  must be  taken i n t o  consideration  -1 o  during  the  no  assignment o f - C s i g n a l s o f o l i g o - and p o l y LJ  saccharides^  .  1 3  • C Signals  f o r 6-deoxysugars (-17 p.p.m.), a c e t a t e  LJ  and  pyruvate  appear  i n the h i g h  f i e l d region.  the a c e t a l carbon i n p y r u v a t e s can be shift  of  their  methyl  group . 9 4  (-21  p.p.m.)  The s t e r e o c h e m i s t r y  differentiated  by  the  of  chemical  A x i a l methyl groups r e s o n a t e a t -18  p.p.m. and e q u a t o r i a l groups a t ~26 p.p.m.  II.5.2  Chromium t r i o x i d e o x i d a t i o n  The  anomeric  saccharides  c a n be  nature  of  sugar  residues  determined by s t u d y i n g  of  poly-  position anomer tion^ the  aldopyranoside, (g-linked)  w i t h an .  9 5  .  fully  A fully  i n which the a g l y c o n o c c u p i e s an e q u a t o r i a l  i s r e a d i l y o x i d i z e d by chromium  a x i a l aglycon (a-linked)  t r i o x i d e w h i l e the  i s quite r e s i s t a n t  However, s u b s t i t u t i o n i n o l i g o - and p o l y -  conformational  oligo-  the r e a c t i o n o f t h e i r  a c e t y l a t e d d e r i v a t i v e s w i t h chromium t r i o x i d e i n a c e t i c a c i d acetylated  or  equilibrium of a-fucosyl OA  thus making them s u s c e p t i b l e t o o x i d a t i o n -  3  .  saccharides  and a-rhamnosyl  t o oxidamay a l t e r residues  38  -  where, R = a l k y l group or sugar  The  o x i d i z e d product i s  converted  methylated a l d i t o l acetates In  this  study  and  alditol  analyzed  polysaccharide.  Sugar  analysis  used to determine  performed  on  the  K34  oxidized  a-linked.  Other t e c h n i q u e s  sugar  residues  the anomeric  i n o l i g o - and p o l y s a c c h a r i d e s .  i n v e s t i g a t i o n the anomeric c o n f i g u r a t i o n a s s i g n e d of  partially  i n Escherichia c o l i  t h a t i t was  O p t i c a l r o t a t i o n can be used to c o n f i r m of  or  g.l.c.-m.s.  sugar r e s i d u e s  p r o d u c t showed o n l y g l u c o s e thus p r o v i n g  II.5.3  by  acetates  chromium t r i o x i d e o x i d a t i o n was  the anomeric c o n f i g u r a t i o n o f the capsular  to  residue  Escherichia  coli  K34  capsular  chromium t r i o x i d e o x i d a t i o n data, From,  Hudson's I s o r o t a t o n R u l e s ^  oligo-  or p o l y -  model  methyl g l y c o s i d e s .  saccharides  using  was 7  one  For example, i n t h i s to the sugar  polysaccharide, confirmed  configuration  by  from  residues  n.m.r.  optical  and  rotation.  can p r e d i c t s p e c i f i c r o t a t i o n s of  the m o l e c u l a r r o t a t i o n v a l u e s  of  the  Normally, the o p t i c a l r o t a t i o n i s measured at  39  the sodium D - l i n e  (589 nm) and the  agree w i t h the v a l u e Enzymes,  such as e x o g l y c o s i d a s e s ,  technique  specific  rotation  should  p r e d i c t e d by Hudson's I s o r a t i o n R u l e s .  u n d e r g o i n g h y d r o l y s i s and f o r i t s classical  observed  are s p e c i f i c  anomeric  o f enzymic h y d r o l y s i s configurations.  f o r the sugar u n i t  configuration. 9 8  Thus,  can be used t o i n v e s t i g a t e  glycosidic  linkage  laboratory  have shown t h a t , enzymic h y d r o l y s i s on o l i g o s a c c h a r i d e  more s a t i s f a c t o r y r e s u l t s . oligosaccharide  was  Recent  observations  in  our gives  D u r i n g t h i s study, E. c o l i K34 b a c t e r i o p h a g e  incubated  (buffer  pH  a - D - g l u c o s i d a s e f o r two days and the h y d r o l y z a t e paper chromatography.  the  «= was  6.5,  37°C)  analyzed  by  with  40 -  CHAPTER I I I  RESULTS AND DISCUSSION  41 III.  III.l  RESULTS AND DISCUSSION  C o m p o s i t i o n and n . m . r .  E.  coli  was  K34  grown  p o l y s a c c h a r i d e p u r i f i e d as product  on  Mueller  described  later  monodispersed by g e l - p e r m e a t i o n ,  p o l y s a c c h a r i d e has value  studies  [a]n +24.2  H i n t o n agar and the (see  experimental).  weighed 940 mg.  w h i c h compares  the  glucose  Hydrolysis  and  glucuronic  acid.  a n a l y s i s of the h y d r o l y z a t e as glucose  and  reduction of acetylation  polysaccharide  alditol  the and  polysaccharide g.l.c. (see  analysis  gave  Table I I I . l ) .  The  by  g.l.c.  glucose,  indicated  M e t h a n o l y s i s and  hydrolysis,  reduction,  g l u c o s e and g a l a c t o s e i n molar These r e s u l t s  c o l i K34 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 s i s t s unit containing galactose,  by  galactose  the p o l y s a c c h a r i d e and  acetates  followed  chromato-  showed  g a l a c t o s e i n molar p r o p o r t i o n s o f 1:2.6.  p r o p o r t i o n s o f 2:2.7  r a t i o s of  of  calculated  Paper  7  The  This p u r i f i e d  very w e l l with  o f 25.7 mg u s i n g Hudson's R u l e o f I s o r o t a t i o n ^ .  graphy o f a n ' a c i d h y d r o l y z a t e of  acidic  suggested t h a t  of a pentasaccharide  E.  repeating  and g l u c u r o n i c a c i d r e s i d u e s  in  the  3:1:1. ^H and ^ C - n . m . r . s p e c t r a o f the K34 p o l y s a c c h a r i d e  indicated  the p r e s e n c e o f f i v e 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 , c o r r e s p o n d i n g to one a and  four ^ - l i n k a g e s .  deoxy s u g a r s , More  n.m.r. spectra  a c e t a t e s and p y r u v i c a c i d  precise  achieved  The  after  assignment sugar  of  the  analysis  -  ketals  signals of  showed the  in  the  chromium  (see  absence of  Table  I I I . 2).  n . m . r . s p e c t r a was trioxide  oxidized  -  42  -  Sugar analysis of K34 polysaccharide and derived  Table I I I . l  products  Sugars^  Mole ratio I  II  III  IV  V  Galactose  2.6  2.7  1.8  1.8  2.0  Glucose  1  2  (as alditol acetates)  0.6  Glyceraldehyde  1.1  VI  0.03 1  1.0  Using DB-17 column programmed for 180°C for 2 min, 5°C/min to 220°C I, original acid polysaccharide; ride;  III,  II, carboxyl reduced polysaccha-  oligosaccharide obtained from Smith degradation of  carboxyl reduced polysaccharide (P2);  IV, oligosaccharide  obtained from selective Smith degradation of acidic polysaccharide;  V, Product obtained from selective Smith  degradation of acidic polysaccharide;  VI, product obtained from  chromium trioxide oxidation of acidic polysaccharide  - 43 Table III.2  N.m.r. data for E. c o l l K34 capsular polysaccharide and derived products (see Appendix III) 13 C-n.m.r. data Assignment P-P m.d  Compound  ^H-n.m.r. data a Integral Assignment— (H)  K34 capsular polysaccharide  5.17  s  1  Q-GIC  4.71  b  1  /9-GlcA  102 90  /S-GlcA  4.57  b  3  P-Qal  103 44  /9-Gal  104 44  0-Gal  179 45  CO of Q-GlcA  Periodate oxidized K34 polysaccharide  5.17  s  1  o-Glc  4.71  b  1  /S-GlcA  4.57  8  3.1  0-Gal  4.53  8  2  98  71  Q-Glc  CHoi Gali-^Gal-O-  Gal  CH 0H 2  ^icA^Gall-^al— i 4.71  GlcA  4.57  Gal  £  Chemical s h i f t r e l a t i v e to internal acetone; 2.23 downfield from sodium 4,4-dimethyl-4-silapentane-l-sulfonate (D.S.S.)  —  Key: b - broad, unable to assign accurate coupling constant; s - singlet  £  For example, /9-Gal - proton on C - l of ^-linked-D-Gal residue  &  Chemical s h i f t i n p.p.m. downfield from Me^Si, r e l a t i v e to internal acetone; 31.07 p.p.m. downfield from D.S.S.  —  As f o r —, but for anomeric  £  Oligosaccharide (P2) obtained from Smith degradation of carbodiimide reduced polysaccharide  £  Polysaccharide obtained from selective Smith degradation of a c i d i c polysaccharide  nuclei  - 44  polysaccharide degradation  111.2  and  anomeric  polysaccharide fully  nature  degraded  was  glucuronic  products  obtained  from  Smith  (Table I I I . 2 ) .  deduced  sugar  residues,  chromium  trioxide  followed  i n K34 c a p s u l a r oxidation  o f the  by sugar a n a l y s i s .  s u r v i v e d and  other  sugar  G.l.c. residues  N.m.r. s t u d i e s (Table I I I . 2 ) showed t h a t  i n K34 p o l y s a c c h a r i d e t o be glucose  i s a - l i n k e d and t h i s  from t h i s g . l . c . r e s u l t .  a c i d and g a l a c t o s e r e s i d u e s  sugar  Thus, the  a r e /3-linked i n the p o l y s a c c h a -  chain.  111.3  Methylation  On  analysis  methylation,  hydrolysis,  o r i g i n a l capsular polysaccharide Table  the  polysaccharide,  o n l y one sugar r e s i d u e  ride  of  i n d i c a t e d t h a t o n l y glucose  residue  of  Oxidation  was determined by  acetylated  results  studies  (see l a t e r and Table I I I . 2 ) .  Chromium t r i o x i d e  The  were  n.m.r.  III.3  (column I ) .  gave  reduction, the  alditol  and  a c e t y l a t i o n the  acetates  i n Table  acetates  were  confirmed  that  III.3  analyzed  in  When the u r o n i c a c i d i n the m e t h y l a t e d polymer  was reduced b e f o r e h y d r o l y s i s , r e d u c t i o n , and a c e t y l a t i o n , acetates  shown  (column by  g.l.c.  the polysaccharide  I I ) were and under  obtained.  g.l.c.-m.s.  the  alditol  These  alditol  These  results  i n v e s t i g a t i o n consists of a  - 45 -  Table I I I . 3  M e t h y l a t i o n a n a l y s i s of K34  p o l y s a c c h a r i d e and d e r i v e d  products  M e t h y l a t e d sugar^-  Mole %^  (as a l d i t o l a c e t a t e ) 6  l£  II 0  III -  23.3  0  2,3,4,6-Glc  28.4  18.7  2,4,6-Gal  19.8  21.9  50.6  2,3,6-Gal  22.2  21.9  26.1  2,6-Gal  29.6  19.4  3,4-Glc  IV°-  58.3  V°-  47.5  40.9  12.6  18.1  2,3,4,6-Gal  41.7  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 m e t h y l g l u c i t o l e t c .  V a l u e s are c o r r e c t e d by use o f the e f f e c t i v e , carbon-response g i v e n by A l b e r s h e i m e t a l . ^ .  factors  7  U s i n g DB-17  column programmed f o r 180°C f o r 1 min,  2°C/min to 250°C  R e t e n t i o n times o f these p a r t i a l l y methylated sugar was confirmed by t h e i r a u t h e n t i c s t a n d a r d samples. I, o r i g i n a l a c i d p o l y s a c c h a r i d e ; II, reduction of uronic ester; I I I , p r o d u c t from / ^ - e l i m i n a t i o n and remethylation; IV, p r o d u c t from Smith d e g r a d a t i o n ; V, p r o d u c t from s e l e c t i v e Smith d e g r a d a t i o n  46  pentasaccharide  repeating  unit.  Methylation  analysis  i n d i c a t e d t h a t the p o l y s a c c h a r i d e has ( i ) a g a l a c t o s y l at  C - l , C-3 and C-4 as i t s branched p o i n t , and  t e r m i n a l non-reducing the  polysaccharide  permethylated  residue.  unit,  was  6 8  g.l.c.-m.s.  column  galactose  and  acid  The product was d i r e c t l y a l k y l a t e d  2 , 3 ,4, 6.-tetra-O-methylglucose that  the  in  w i t h methyl  reduced, and a c e t y l a t e d .  2,4,6-tri-O-methylgalactose,  deduced from these r e s u l t s  acid  degradation  a n a l y s i s o f the r e s u l t a n t a l d i t o l a c e t a t e s  I I I ) showed  as i t s  determined by s u b j e c t i n g the  i o d i d e and the i s o l a t e d r e s i d u e was h y d r o l y z e d , The  also linked  ( i i ) a glucose  polysaccharide to base-catalyzed uronic 6 7  residue  The l o c a t i o n o f the g l u c u r o n i c  repeating  (^-elimination) > .  results  (Table I I I ,  2,3,6-tri-0-methyl-  i n the r a t i o 2:1:1. I t was  glucuronic  acid  was  linked  to  branched g a l a c t o s e a t C-4.  III.4  P e r i o d a t e o x i d a t i o n - Smith h y d r o l y s i s  Carbodiimide-reduced periodate of  K34  (122 h) and reduced  p o l y s a c c h a r i d e was o x i d i z e d w i t h  w i t h sodium b o r o h y d r i d e .  p e r i o d a t e was monitored d u r i n g the course  I I I . l and I I I . 2 ) . consumed  and  agreed  with  the  theoretical  repeating value.  r e c o v e r e d a f t e r d i a l y s i s , was a n a l y z e d by n.m.r. s p e c t r o s c o p y III.2).  consumption  o f the r e a c t i o n (see F i g s .  About 4 moles o f p e r i o d a t e p e r  this  The  sodium  unit  were  The p o l y o l (see Table  The n.m.r. spectrum o b t a i n e d was b e t t e r than t h a t o f the parent  p o l y s a c c h a r i d e due t o the h i g h s o l u b i l i t y o f t h i s p o l y o l i n low v i s c o s i t y o f t h i s  sample.  p l u s the  Figure  III.2  P e r i o d a t e consumption by K34 r e s p e c t t o time  polysaccharide  with  - 48 -  This  polyol  was  s u b j e c t e d to Smith h y d r o l y s i s , d u r i n g which  a c e t a l l i n k a g e s o f the m o d i f i e d sugar hydrolyzed. of  protons  also  (Table I I I . 2 ) . (P ) 2  analysed  a t 8 = 4.53,  exhibits signal  saccharide  derived  n.m.r.  spectroscopy.  Sugar  the o l i g o -  two  the two  (P )  (Table  2  and  III.3,  saccharide  oligo-  glyceraldehyde analysis  column  IV)  showed an  (1,3,4 l i n k e d g a l a c t o s e and linked  to  derived  2  from  Smith  degradation  1,3 each  and m e t h y l a t i o n r e s u l t s t h e r e f o r e suggest  (P )  of  2,3,4,6-tetra-O-methylgalactose,  sugar r e s i d u e s  analysis  The  anomeric  derived  Methylation  l i n k e d g a l a c t o s e ) r e s i s t a n t to p e r i o d a t e o x i d a t i o n are other.  selectively  which i n t e g r a t e to  (Table I I I . l ) .  oligosaccharide  that  by  i n d i c a t e d the presence o f g a l a c t o s e and  2,4,6-tri-O-methyl-galactose indication  be  Sugar a n a l y s i s conducted on the  i n molar p r o p o r t i o n s o f 1.8:1 the  should  o l i g o s a c c h a r i d e ( P 2 ) i s o l a t e d by paper chromatography  The  the h y d r o l y z a t e , was  spectrum  residues  the  that  has  the  s t r u c t u r e below:  CHO GalL-iGal-L-O— CH OH 2  According  to the  saccharide a l s o has fragment  sugar  analysis  result,  o b t a i n e d from Smith d e g r a d a t i o n  the s t r u c t u r e shown above. from  the  oxidized  The  glucuronic  s p e c u l a t e d t h a t the main c h a i n o f E. c o l i has  the f o l l o w i n g s t r u c t u r e :  (Table  III.l),  the  oligo-  o f the n a t i v e p o l y s a c c h a r i d e  terminal glycerolaldehyde i s acid K34  residue. capsular  a  Thus, i t was polysaccharide  - 49 -  -^GlcAp^Galpi-^alp— P 13 P  T h i s s t r u c t u r e was native  III.5  confirmed  by  S e l e c t i v e Smith  rate  slowly  Observations  of  or  the  periodate  glycols, might  linked  glucuronic  The  is  oxidized  very  be  explained  by  native  and  according  to  the  t r a n s g l y c o l s are  oxidized  conformation . 0 2  was  s l o w l y by sodium p e r i o d a t e . the  repulsion  embarked upon i n t h i s study  polysaccharide  was  and  glucose  column V ) . M e t h y l a t i o n  between  r e s i d u e s over 1  was  treated  with  purified  by  gel  permeation  M  in  molar  proportions  4 —>  sodium  reduction.  The  chromatography  s u b j e c t e d to m i l d a c i d h y d r o l y s i s (0.5 M TFA) non-dialyzable  the  (see Scheme  0.02  f o r t h r e e hours f o l l o w e d by sodium b o r o h y d r i d e  LH-20)  residue,  Thus s e l e c t i v e o x i d a t i o n of 1 — >  48 hours. T o t a l sugar a n a l y s i s o f the galactose  varies  terminal glucopyranosyl  acid  r e s u l t a n t p o l y o l which was (Sephadex  Sugars h a v i n g  i o n and c a r b o x y l i c anion.  linked galactopyranosyl  periodate  oxidation  i n our l a b o r a t o r y have shown t h a t the u r o n i c a c i d  trans  periodate  II. 3).  of  not at a l l i f f i x e d i n an u n f a v o u r a b l e  this observation  2  degradation  degradation  c o n f i g u r a t i o n o f the g l y c o l s .  having  Smith  polysaccharide.  The  more  selective  material  o f 2.0:0.6 ( T a b l e  for  yielded III.l,  a n a l y s i s i n d i c a t e d t h a t the g a l a c t o s e r e s i d u e s i n  50 -  the  s e l e c t i v e Smith d e g r a d a t i o n p r o d u c t were 1 — >  l i n k e d galactopyranosides The  (Table I I I . 3 , column V ) .  ^H-n.m.r. spectrum o f the degraded p r o d u c t  anomeric protons  a t 8 4.71 (J\2  ~  8  therefore  consists  of  a  that this  a-linked  terminal  spectrum  8  glucopyranoside)  of  the  anc  degraded  native  and  also  the  8 4.71 and  t  n  e  s  e  product  Comparison o f polysaccharide  signal  ( f o r the  at  5 4.57  as compared t o t h r e e i n the n a t i v e  The o v e r a l l n.m.r. s t u d i e s t h e r e f o r e suggest  a t 8 5.17,  *  has no s i g n a l a t 8 5.17  n.m.r. spectrum  i n t e g r a t e d up t o two anomeric protons polysaccharide.  This  ~  trisaccharide repeating unit.  t h i s spectrum w i t h the n.m.r. indicates  showed s i g n a l s f o r  **z) and 8 4.57 (J\2  s i g n a l s i n t e g r a t e up t o t h r e e anomeric p r o t o n s .  signals  4 l i n k e d and 1 — > 3  8 4.57  can be  assigned to  that  the  a-glucose,  /9-glucuronic a c i d and ^ - g a l a c t o s e s r e s p e c t i v e l y .  III.6  Determination of the configuration (D or L) of the sugar  The coli  c o n f i g u r a t i o n (D o r L) o f the sugar r e s i d u e s , c o n t a i n e d  K34  capsular  polysaccharide,  was  determined  conveniently  c i r c u l a r d i c h r o i s m measurements a t 213 nm on t h e i r p a r t i a l l y alditol  acetates,  Glucose,  g l u c u r o n i c a c i d and g a l a c t o s e s  configuration  by  a l d i t o l acetates comparing  where  the  the  circular  (Table I I I . 4 ) .  acetoxy  group were  dichroism  acts  as  a chromophore.  shown  to  be  curves  of  the D  o f the c o r r e s p o n d i n g  These c o n f i g u r a t i o n s were  standards.  by  methylated  attained  the c i r c u l a r d i c h r o i s m measurements o f these a l d i t o l  w i t h those o f the a u t h e n t i c  i n E.  by  acetates  - 51 -  Table III.4  Configuration of sugar residues of E. c o l i K34 capsular polysaccharide  Sugar  Alditol  Configuration  -2-GlcA^-  3,4-D-O-methylglucitol  D  -^Gali3  2,6-Di-02methylgalactitol  D  -J±Gal±-  2,3,6-tri-O-methylgalactitol  D  —3-Gall-  2,4,6-tri-0-methylgalactitol  D  -J-Glc  2,3,4,6-tetra-0-methylglucitol  D  The D c o n f i g u r a t i o n o f treatment o f bacteriophage chromatographic  the terminal  glucose  was  confirmed  oligosaccharide with a-D-glucosidase.  a n a l y s i s o f t h e h y d r o l y z a t e from t h i s  enzymic  by  Paper reaction  i n d i c a t e d t h a t glucose has been h y d r o l y z e d b y the a-D-glucosidase.  III. 7  Isolation of bacteriophages (<^31 and ^34) and crossreactions  E. c o l i K 3 1 isolated No  bacteriophages  (<£31 a n d <£34)  f r o m V a n c o u v e r sewage w e r e p r o p a g a t e d  plaques  bacterial  a n d K34  on  their  originally  host  strains.  w e r e f o r m e d when E. c o l i K 3 4 b a c t e r i o p h a g e was s p o t t e d o n a  l a w n o f E. c o l i K 3 1 o r v i c e v e r s a .  Thus E. c o l i K 3 4 d o e s n o t  52 -  serologically group this of  of  c r o s s - r e a c t w i t h E. c o l i K31.  E.  coli  K34 must d i f f e r from t h a t o f E. c o l i K31.  i n v e s t i g a t i o n and p r e v i o u s  s t u d i e s have r e v e a l e d  E. c o l i K34 c a p s u l a r p o l y s a c c h a r i d e  E, c o l i K31  Hence the immunodeterminant In f a c t ,  t h a t the s t r u c t u r e  (K a n t i g e n ) d i f f e r s from t h a t of  (Appendix I ) .  E. c o l i K34 b a c t e r i o p h a g e d i d not g i v e c r o s s - a b s o r p t i o n f o l l o w i n g E, c o l i  strains,  -•  K28, K32 and K33.  Gali-^Glci-^-GlcrAi-^-Rhai^Rhal-  B K Antigen of E. c o l i  _3_G i 1_4_G 4 a c  1  1 c A  !_4 B  F u c  B  K31  !_  2  o r  a 3  I  Gal  0  A  c  K Antigen of E. c o l i K28  OAc 2 ^GlcLJlRhai-^Galit  Q  3  B  1 GlcA  K Antigen of E. c o l i K32  11  with  the  - 53 ^Iglc^GlcA^Fuc^ a 3 2 3 8  V  pyr  1 G a l + OAc  K A n t i g e n o f E. c o l i  K33  ^IgicA^Gal^GaliB 3 B 1 Gal 4  a  1 Glc  K A n t i g e n o f E. c o l i  The  immunodominant  sugars  of  l o c a t e d i n the s i d e c h a i n - ^ . 9  galactose  and  glucose  branched  K34  polysaccharides  E. c o l i K34 c a p s u l a r  i n i t s side chain.  are  usually  polysaccharide  However E. c o l i  has  s t r a i n s K33  and K28, which do n o t c r o s s - r e a c t w i t h E. c o l i K34,  have  the  From c r o s s - r e a c t i o n  side  chain  of t h e i r capsular polysaccharide.  galactose  r e s u l t s , we a r e l e d t o suggest t h a t the immunodominant sugar o f E. K34  c a p s u l a r p o l y s a c c h a r i d e may e i t h e r be the 1 — >  a c i d o r the t e r m i n a l g l u c o s e . acid  The o c c u r r e n c e  i n E. c o l i K34 c a p s u l a r 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 t o be r e p o r t e d i n b a c t e r i a l  o f 1,2  in  coli  2 linked glucuronic linked  i s the f i r s t  glucuronic  1 —>  polysaccharides.  2 linked  - 54 -  III.8  Depolymerization with E. c o l i K31 bacteriophage (<£31)  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 was in  Mueller  Hinton  broth.  c o n t i n u e d on an i n c r e a s i n g  scale  The r e s u l t s o f phage assays from tube  and f l a s k l y s i s are as t a b u l a t e d i n T a b l e I I I . 5.  After dialysis  lysis for  2 1 9  days,  the  concentration  of  plaque-forming u n i t s .  This  solution  K31  of  purified  the b a c t e r i o p h a g e s o l u t i o n was bacteriophage capsular  solution  polysaccharide  mixture i n c u b a t e d a t 37°C f o r a t o t a l o f 48 h. time,  the  indication product  reaction that  formed  mixture  appeared  depolymerization was  L  was  added  and  the r e a c t i o n  to  occurred.  l e s s v i s c o u s , an  The  depolymerized  s e p a r a t e d from the p o l y s a c c h a r i d e - p h a g e mixture by  d i a l y s i s and then p u r i f i e d by i o n exchange chromatography ( A m b e r l i t e 120  (H ) +  Propagation of Bacteriophage <j>31  II  (p.f.u./mL)£  Volume  Total  IR  resin).  Table III. 5  Titre  a  A f t e r 24 h o f i n c u b a t i o n  significantly  has  x 10 ^  1.8  4.95  -1.5  a  I,  b  p . f . u . = plaque forming u n i t .  test-tube l y s i s ;  10  1 0  5.5 x  30  (mL)  (p.f-u.)  x  x  III  10  1 0  180  100  10  5.5 x  I I , small f l a s k l y s i s ;  10  1 2  1.8  III, after  x  10  1 2  dialyzing  55  The coli  mixture K31  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 , a f t e r d e p o l y m e r i z a t i o n of E.  polysaccharide  w i t h phage #31,  components by g e l permeation The  reducing  Morrison's m e t h o d ^ . 0  nitrile, was the  end The  of  the  glucosidase  enzyme  that  occurs  47.2%  oligosaccharide  r e s u l t s showed  c l e a v e d by b a c t e r i o p h a g e - b o r n e of  then s e p a r a t e d i n t o pure  chromatography (85 mg,  r h a m n o n i t r i l e and g l u c i t o l  type  was  the  yield).  was  determined  presence  of  galactono-  i n d i c a t i n g t h a t the g l u c o s y l l i n k a g e glycanase in  (see T a b l e  E. c o l i K31  III.6).  Thus,  b a c t e r i o p h a g e has  activity.  Table I I I . 6  by  D e t e r m i n a t i o n of the r e d u c i n g end o f E . c o l i K31 o l i g o s a c c h a r i d e i s o l a t e d a f t e r b a c t e r i o p h a g e #31 degradation of E . c o l i K31 p o l y s a c c h a r i d e  Peracetylated derivative of  T Column a  (DB  17)  Rhamnonitrile  3.91  Galactononitrile  7.33  Glucitol  9.84  Column Temperature (180°C f o r 2 min,  5°C/min, to 220°C)  R e t e n t i o n times c o n f i r m e d by a u t h e n t i c s t a n d a r d samples  a  - 56 III.9  D e p o l y m e r i z a t i o n w i t h E . c o l i K34 b a c t e r i o p h a g e  E. ing  c o l i K34 b a c t e r i o p h a g e  scale  in  Mueller  of  the  was a l s o propagated on an i n c r e a s -  Hinton broth.  tube l y s i s and f l a s k l y s i s are concentration  (#34)  also  The r e s u l t s tabulated  bacteriophage  solution  f o r m i n g u n i t s a f t e r d i a l y s i n g f o r two d a y s . of  E.  coli  (#34)  o f phage assays from  in  Table  was  7.2 x 1 0 ^  Bacteriophage  indicated  that  fraction  repeating unit oligosaccharide Results and  after  Table I I I . 7  for ^H-n.m.r.  sodium  I  (see  reduction,  Propagation of bacteriophage  (p.f.u./mL)^  4.95 x 1 0  Volume (mL)  Total  are  -  I,  test-tube l y s i s ;  —  p.f.u.  1.48 x 1 0  II,  III.3)  resultant chromato-  was a double  1 0  III.8.  III  1.1 x 1 0  1 0  0.9 x  100  1 2  before  i n Table  II  10  1 1  10  1 2  80  1.1 x 1 0  small f l a s k l y s i s ;  - plaque forming u n i t .  shown  I,  #34  30  (p.f.u.)  Paper  s t u d i e s on f r a c t i o n  1=  Titre  degradation  (85.7 mg, 34% y i e l d ) .  spectroscopy  borohydride  Figure  The  plaque-  2  K34 c a p s u l a r 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 the  o l i g o s a c c h a r i d e was conducted as p r e v i o u s l y d e s c r i b e d . graphy  III.7.  1 2  III,  7.2 x  after  dialyzing  e l u t e d volume minus (mL) v o i d volume Fig.  III.3  S e p a r a t i o n o f the d e p o l y m e r i z a t i o n p r o d u c t s o f E . c o l l by g e l - p e r m e a t i o n chromatography  (Bio-Gel  P-2)  K34  58  Table I I I . 8  Proton n.m.r. data (400 MHz) f o r the o l i g o s a c c h a r i d e generated i n b a c t e r i o p h a g e d e p o l y m e r i z a t i o n o f the E. c o l i K34 c a p s u l a r p o l y s a c c h a r i d e  Fraction I Integral (H)  (p.p.m.)  Integral (H)  5.17  1.0  5.16  1.0  4.97  0.8  4.97  0.3  4.82  1.2  4.82  1.2  4.65  0.4  4.53  2.2  4.53  2.1  (p.p.m.)S  -  Fraction I (R)^  Chemical s h i f t r e l a t i v e t o i n t e r n a l acetone, 2.23 d o w n f i e l d from sodium 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 (DSS)  F r a c t i o n I a f t e r r e d u c t i o n w i t h sodium b o r o h y d r i d e  Fraction  I I I had  ^H-n.m.r. spectrum.  a  low  I  c o n t e n t as was judged from i t s  The r e s u l t s f o r the r e d u c i n g end  determination  on  1 0 ft  *  fraction  carbohydrate  by  Morrison's  results illustrated  method^  0  a r e shown i n T a b l e I I I . 9.  t h a t the E . c o l i K34 b a c t e r i o p h a g e - b o r n e  a ^-galactosidase activity.  enzyme  These has  59  Table I I I . 9  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 E. c o l i o l i g o s a c c h a r i d e i s o l a t e d a f t e r bacteriophage d e g r a d a t i o n o f E. c o l i K34 p o l y s a c c h a r i d e  K34  X—  Peracetylated d e r i v a t i v e of  Mole %  Column(DB 17)  Glucononitrile  7.73  Galactononitrile  7.88  74  10.04  19  Galactitol  £  Column Temperature,  (180°C f o r 2 min, 5°C/min, t o 220°C)  b  R e t e n t i o n times c o n f i r m e d by a u t h e n t i c s t a n d a r d  samples  CONCLUSION  This  study  revealed  that  E.  c o l i K34 d i d not c r o s s - r e a c t w i t h  o t h e r group 09 s t r a i n s l i k e K28, K31, K32, in  this  structure  investigation  revealed  and K33.  Studies  conducted  t h a t K a n t i g e n o f E. c o l i K34 has the  below:  ^-2-D - GlcApi-^-D - Galpi—2-D - G a l p i P 3 P P P 1 D-Galp 4 1 D-Glcp  - 60 A  comparison  E. c o l i  strains  significantly  o f t h i s s t r u c t u r e to the K a n t i g e n o f the above mentioned (see Appendix different.  antigen j u s t i f i e s polysaccharide  I ) , showed t h a t E. c o l i K34  The  i t s serological  is  novel  fact  that  E.  antigen  is  c o l i K34 has a unique K  differentiation.  i n t h a t , t h i s i s the f i r s t  g l u c u r o n i c a c i d has been r e p o r t e d i n b a c t e r i a l  K  E. c o l i K34 time 1 —>  polysaccharides.  capsular 2  linked  CHAPTER I V  EXPERIMENTAL  - 62 -  IV.  IV.1  EXPERIMENTAL  General methods  S o l u t i o n s were c o n c e n t r a t e d temperatures n o t exceeding dry i c e - a c e t o n e mixture Optical  rotation  40°C.  a  rotary  Frozen  measurements  were  evaporator  Unitrap  capacity  and  the  acetonitrile. The  infrared  Perkin  l e n g t h o f 0.1 cm.  appropriate The  alditol  c d . s p e c t r a were r e c o r d e d  in  model 457 spectrophotometer.  Whatman  No.  1  paper  cell  of  0.3  spectroscopic  (5  mL  grade  i n the range 210-240 run.  and  on a  The s o l v e n t used f o r sample tetrachloride.  A n a l y t i c a l paper chromatography was performed using  cell  d e r i v a t i v e s were r e c o r d e d  p r e p a r a t i o n was s p e c t r o s c o p i c grade carbon  method  dm  C d . samples were p r e p a r e d by  acetate  ( i . r . ) s p e c t r a o f methylated  Elmer  freeze-dryer.  were r e c o r d e d on a J a s c o J-500A  r e c o r d i n g spectropolarimeter, with a quartz  dissolving  II  model 141 p o l a r i m e t e r w i t h a 1  automatic  path  bath  conducted on aqueous s o l u t i o n s a t  C i r c u l a r dichroism spectra ( c d . )  a  with  s o l u t i o n s were o b t a i n e d u s i n g a  and l y o p h i l i z e d on a  20°i3° u s i n g a Perkin-Elmer mL) .  on  the  by  the  descending  f o l l o w i n g systems:  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 acid-water;  (2)  acetate-pyridine-water;  1-butanol-ethanol-  and, (3) upper phase o f 4:1:5  water.  P r e p a r a t i v e paper chromatography was c a r r i e d out  No.  paper and s o l v e n t system 1.  3  with a l k a l i n e s i l v e r being  sprayed  with  nitrate^  8:2:1  (1)  using  Chromatograms were e i t h e r  ethyl  Whatman developed  or by h e a t i n g a t 100°C f o r 10 min  p - a n i s i d i n e hydrochloride-*-^ i  n  aqueous  after  1-butanol.  - 63 -  Sugars  and o l i g o s a c c h a r i d e were d e t e c t e d by these methods. A B i o - G e l P-2 (400 mesh) column  preparative  gel-permeation  (2.5 x  100  chromatography.  The v o i d  column and the e f f i c i e n c y o f p a c k i n g were determined (0.2%).  The  was  used f o r  volume  using blue  o f the dextran  c o n c e n t r a t i o n o f the samples a p p l i e d t o t h e column ranged  from 40-100 mg/mL. approximately  6  Eluant mL/h.  was  distilled  m o l e c u l a r weight  water  at  F r a c t i o n s were c o l l e c t e d ,  and the e l u t i o n p r o f i l e was o b t a i n e d . large  cm)  a  flow  rate  of  f r e e z e - d r i e d , weighed  Sephadex LH-20 was used t o p u r i f y  carbohydrate m a t e r i a l that i s s o l u b l e i n organic  s o l v e n t , e.g. p e r m e t h y l a t e d o l i g o - and p o l y - s a c c h a r i d e s . A Hewlett-Packard ionization  detector  Hewlett-Packard Open  tubular  5890 i n s t r u m e n t  was  (capillary)  column  (DB-225-15N). dual  column  f o r analytical  columns The  columns  (DB-17-15N);  instrument  flow-rate  l°/min.  used  (B) f u s e d  fitted  with  silica  of  60  mL/min.  thermal mm)  This  the peak  areas.  carrier-gas  (A) f u s e d  silica  capillary  column  model  were  used  column  with  720  carrier-gas  was packed w i t h 3% o f  (100-200 mesh) and programmed from 175°C t o 240°C  with  a  Watson-Biemann  w i t h a V.C.  separator.  Micromass 12 Spectra  were  A and an i o n source  The columns used f o r the s e p a r a t i o n were (A) and ( B ) .  l C-n.m.r. and lH-n.m.r. s p e c t r a were r e c o r d e d on a Bruker 3  A  conductivity detectors.  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 100 200°.  flame-  g.l.c. separations.  were:  G.l.c.-m.s. a n a l y s e s were performed  i n s t r u m e n t equipped  at  dual  P r e p a r a t i v e g . l . c . was c a r r i e d out w i t h F & M  SP-2340 on S u p e l c o p o r t at  a  were used w i t h a h e l i u m  S t a i n l e s s - s t e e l columns (1.8 m x 6.3 helium  with  3392A i n t e g r a t o r was used t o q u a n t i f y  f l o w r a t e o f 48 mL/min. capillary  used  equipped  WH-400  - 64 -  instrument. (2.23  Acetone was used as an internal standard for both lH-n.m.r.  p.p.m.)  and  13  C-n.m.r.  (31.07 p.p.m.) spectroscopy.  ^H-n.m.r.  spectra were recorded at elevated temperature and chemical shift are  given  relative  to  that  of  pentanesulfonate (taken as zero).  values  external sodium-4,4-dimethyl-4-sila^H-n.m.r. samples  were  prepared by  dissolving in D2O and lyophilized three times from D2O solutions. samples were dissolved in D2O and submitted tubes.  in  5  These  mm diameter  n.m.r.  ""--X-n.m.r. spectra were recorded at ambient temperature. Samples  were dissolved in the minimum of D2O and submitted in n.m.r.  tubes  of  diameter sizes 5 mm or 10 mm. In  our  laboratory,  we generally  isolation of E. coli bacteriophages.  proceed  as  follows  for the  Large samples of sewage are  mixed  with concentrated Mueller Hinton broth and then with an actively growing culture of the bacteria for which a  virus  mixture  overnight.  is  then  incubated  (37°C)  shall  mixture are k i l l e d by adding chloroform and crude  bacteriophage  be  Isolated.  This  The bacteria in this  shaking vigorously.  The  solution is separated from the bacterial debris by  centrifugation. The bacterial lawn needed for bacteriophage assay is follows.  as  A Mueller Hinton agar plate was dried, upside down at 37°C in  an incubator for 2 hours. pipetted  prepared  Actively growing bacterial culture (3 mL) was  on to the agar surface and after 20 min at 37°C, excess liquid  was drained off.  The plate was then incubated at 37°C  for  1  hour  to  produce the bacterial lawn. An  assay for determining bacteriophage concentration is described  as follows.  0.3 mL portion  of  bacteriophage  suspension was  diluted  - 65 -  t e n - f o l d by adding 2.7 resultant  solution  ml o f s t e r i l e  was  broth.  10"  -  1  lO  s u s p e n s i o n was pipette  was  - 1 0  obtained.  to  a fine t i p .  of  plaque-forming  One  small lawn  the  drop by  of  means  range  bacteriophage of  a  sterile  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°C, the  units  (p.f.u.)  s u s p e n s i o n were c a l c u l a t e d based on solution applied,  of  ( i n a s i m i l a r manner)  number o f p l a q u e s observed f o r the h i g h e s t d i l u t i o n counts  portion  solutions u n t i l a d i l u t i o n  s p o t t e d on the b a c t e r i a l  drawn  mL  further diluted ten-fold  and the p r o c e s s r e p e a t e d on subsequent of  0.3  the  are  counted.  The  per  mL  o f u n d i l u t e d phage  volume  of  the  bacteriophage  the number o f plaques and the d i l u t i o n t h a t gave those  plaques. The  methods  bacteriophage  employed  in  building  Tube  was  on  p i c k e d up  bacterial  agar  and  c u l t u r e became t u r b i d  incubated  interval.  and  successive  (4 h o u r s ) .  37°C.  After  bacteria  i n c u b a t e d a t 37°C u n t i l S t e r i l e broth ( 5 x 5  then i n o c u l a t e d w i t h the b a c t e r i a l at  30  minutes  mL)  culture of  the in (0.5  incubation,  added to the t e s t - t u b e s c o n s e c u t i v e l y a t  Continued  the c l o u d y s o l u t i o n due  o b t a i n e d by  A c o l o n y o f t h i s a c t i v e l y growing  i n t o s t e r i l e b r o t h (5 mL)  b a c t e r i o p h a g e s u s p e n s i o n was minutes  of  lysis.  c u l t u r e o f E. c o l i was  plates.  c u l t u r e t e s t - t u b e s was mL)  concentration  lysis  An a c t i v e l y growing replating  the  to a l e v e l s u f f i c i e n t f o r d e g r a d i n g the p o l y s a c c h a r i d e i n  q u e s t i o n are tube l y s i s and f l a s k  (a)  up  30  i n c u b a t i o n r e s u l t s i n gradual c l e a r i n g of  to c e l l l y s i s .  A f t e r the l a s t tube had  cleared  - 66 (about 5 hours after the f i r s t addition of bacteriophage) a few drops of chloroform were added to each tube to  prevent  last  bacteriophage  two  tubes  were  combined  and  bacterial  growth.  The  separated from the  b a c t e r i a l debris by centrifugation.  (b)  Flask l y s i s  This technique i s quite similar to large  the  tube  volumes of bacteriophage are produced.  Mueller Hinton broth, i n s i x inoculated  with  1  ml  i n t e r v a l s , bacteriophage otherwise)  was  of  Erlenmeyer actively  suspension  consecutively added  flasks  mL  except  that  48 ml aliquots of s t e r i l e  growing (1  lysis  (125  ml),  culture. from  the  to the f l a s k s .  were  At  each  30 minute  tube  lysis  or  The procedure  was  then continued as described for tube l y s i s .  IV.2  I s o l a t i o n and p u r i f i c a t i o n of E. c o l i K34 capsular polysaccharide  The medium used for the growth of the bacteria was Mueller agar:  Hinton  beef extract (3.0 g), acid hydrolyzate of Casein (17.5 g), starch  (1.5 g), and agar glassware  and  (12.0  Mueller  g)  per  liter  of  International  K34  Sterilization  Hinton medium was done i n a American  model 57-CR for 15 minutes at 121° and 15-20 E. c o l i  water.  culture  Escherichia  was  obtained  Center,  of  Sterilizer  p.s.i. from  Dr.  Copenhagen).  Ida  Orskov  Actively  (WHO  growing  colonies of E. c o l i K34 were propagated by replating several times  onto  - 67 -  P e t r i dishes  (layered with s t e r i l e Mueller Hinton agar); a s i n g l e  b e i n g s e l e c t e d each overnight  of  time  bacteria  (100 mL)  was  hours.  Actively  the on  bacteria  were  be  P e t r i d i s h e s a t 37°C was  i n o c u l a t e d w i t h E. c o l i K34 growing  to  E.  coli  plated.  bacteria  incubated were  s t e r i l e , M u e l l e r H i n t o n agar medium ( i n a metal t r a y 60 i n c u b a t e d f o r f o u r days a t 37°C.  The  K34  was  The  mixture  hours  at  15°  on  x  40  cm)  and  from  the  s t i r r e d a t 4°C  Beckmann  coloured  the  supernatant  dead  was  s t r i n g y p r e c i p i t a t e was  dissolved in  dissolved  4M  other L3-50  with  bromide).  NaCl  The  solution,  viscous  ethanol.  debris Ultra-  treated  precipitated  with  into  Sugar analysis and  Cetavlon  acid  2 7  complex  days).  reThe  lyophiliza-  f u r t h e r p u r i f i e d by g e l permeation cm x 2.5  cm).  composition  H y d r o l y s i s o f a sample (20 trifluoroacetic  honey-  ethanol,  i s o l a t e d as a s t y r o f o a m - l i k e m a t e r i a l , by  i s o l a t e d p o l y s a c c h a r i d e was  the  The r e s u l t a n t  Cetavlon-polysaccharide  chromatography u s i n g a B i o - G e l P2 column (100  IV.3  The  i n water and d i a l y z e d a g a i n s t d i s t i l l e d water (two  p o l y s a c c h a r i d e was t i o n . The  precipitated  cells.  d i s s o l v e d i n water and  (cetyltrimethylammonium was  bacterial  5  for  c e n t r i f u g e u s i n g r o t o r 45 T i a t 31000 r.p.m. or 80000 g) to s e p a r a t e p o l y s a c c h a r i d e from  4  poured onto a  o f p o l y s a c c h a r i d e , b a c t e r i a l c e l l s and  ultracentrifuged (for 4  Broth for  b a c t e r i a were s c r a p e d  agar s u r f a c e , d i l u t e d w i t h 1% phenol s o l u t i o n and hours.  Growth  sufficient.  b a c t e r i a and  K34  colony  (TFA)  mg)  of  K34  polysaccharide  f o r 20 h a t 95°C, removal o f excess  with acid  2M by  - 68 c o e v a p o r a t i o n w i t h water, f o l l o w e d by paper chromatography ( s o l v e n t showed The  glucose,  galactose,  g l u c u r o n i c a c i d and an a l d o b i o u r o n i c a c i d .  sugars r e l e a s e d were reduced  hours) and the mixture was The  r e a c t i o n mixture was  (sodium b o r o h y d r i d e  steam  f i l t e r e d , e v a p o r a t e d to dryness and  A  The  o f K34  an i . r . lamp, was refluxed  p o l y s a c c h a r i d e (13 mg),  t r e a t e d with methanolic  overnight  on  a  steam-bath  resultant  precipitate. o b t a i n e d was  mixture The  hour.  water.  2 hours  to  220°C  at  column 1.  d r i e d i n vacuo and under and  under anhydrous c o n d i t i o n s .  The  chloride  n e u t r a l i z e d with lead to  remove  carbonate. chloride  evaporated to dryness and the  residue  n e u t r a l i z e d w i t h A m b e r l i t e IR-120 (H )  mixture was  borate  f i l t e r e d , the f i l t r a t e  ion.  The  methanol  r e s i d u e was  The h y d r o l y z a t e was  a t room temperature.  cation-exchange  resin  +  (5  The  mL)  in  removed by  to on a  codistillation  chromatography. borohydride  r e s i d u e ( a l d i t o l ) was  chromatography and c o d i s t i l l a t i o n  after  order  h y d r o l y z e d w i t h 2 M TFA  sodium  The  e v a p o r a t e d to dryness  a n a l y z e d by paper  sugars r e l e a s e d were reduced w i t h aqueous for  f o r 1 hour on a  180°C  hydrogen  steam-bath (20 hours) a f t e r which the TFA was with  The  reduced w i t h sodium b o r o h y d r i d e i n anhydrous methanol.  The  the  codistilled  the l e a d  and c o d i s t i l l e d w i t h t h r e e p o r t i o n s o f remove  resin.  (3%)  centrifuged  s u p e r n a t a n t was  r e a c t i o n mixture was 1  was  4  resultant a l d i t o l acetates  (column A, programmed from  excess a c i d i n the r e a c t i o n mixture was The  The  g . l . c . r e s u l t s are shown i n T a b l e I I I . l ,  sample  for  i n o r d e r to remove the b o r a t e i o n .  under anhydrous c o n d i t i o n s .  were a n a l y z e d by g . l . c .  water, +  t r e a t e d w i t h a c e t i c a n h y d r i d e - p y r i d i n e (1:1)  bath  5°C/min).  in  n e u t r a l i z e d w i t h A m b e r l i t e IR-120 (H )  w i t h p o r t i o n s o f methanol (5 mL) r e s i d u e was  (1))  with  The  solution  purified  methanol.  by The  - 69 alditols  were  acetylated using acetic anhydride-pyridine  i n anhydrous c o n d i t i o n s f o r 1 dissolved  i n chloroform,  Table I I I . l ,  polysaccharide. recorded  were  The  resultant  alditol  acetates,  a n a l y z e d by g . l . c . u s i n g column A (see  column I I ) .  N.m.r. s p e c t r o s c o p y  are  hour.  (1:1) a t 95°C  (^H and ^C)  was  performed  The p r i n c i p a l s i g n a l s f o r both  i n Table  on  the  original  ^C-n.m.r,  ^H-n.m.r. and  I I I . 2 (see Appendix I I I f o r the  reproduction  of  these n.m.r. s p e c t r a ) .  IV.4  Chromium t r i o x i d e oxidation  A  sample (10 mg) o f the 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 formamide  (5 ml) and t r e a t e d w i t h a c e t i c anhydride  (1  overnight  a c e t y l a t e d m a t e r i a l (12 mg) was  at  room  temperature.  The  r e c o v e r e d by d i a l y s i s and f r e e z e - d r y i n g . dissolved  and  The water.  recovered m a t e r i a l .  IV.5  and  pyridine  The a c e t y l a t e d  material Sugar  was  recovered  analysis  was  (1  mL)  polysaccharide  i n a c e t i c a c i d was t r e a t e d w i t h chromium t r i o x i d e  50°C f o r 2 hours. chloroform  mL)  (100 mg) a t  by  partition  between  then  performed  on the  G . l . c . r e s u l t s a r e shown i n T a b l e I I I . l ,  column V I .  Methylation analysis  The  capsular  polysaccharide  (30  mg)  i n the  free-acid  form,  o b t a i n e d by p a s s i n g the sodium s a l t through a column o f A m b e r l i t e  IR-120  - 70 -  (H ) +  r e s i n , was  methylated  d i s s o l v e d i n anhydrous d i m e t h y l  by  5 3  treatment w i t h 5 mL  and then w i t h 10 mL m e t h y l i o d i d e saccharide  was  recovered  d i s t i l l e d water o v e r n i g h t . by  partition  permeation i.r.  between  by  for  followed  1  dialysis  A p o r t i o n (15 mg) for  20 hours.  ml)  methylated  and  poly-  c u t o f f 13,500) a g a i n s t  polysaccharide and  water,  was  as  purified  well  D r y i n g i n vacuo  as  and  spectroscopic analysis indicated  gel under  complete  and 3200-3500 cm"-*-).  o f t h i s p r o d u c t was  The  The  (M.W.  dichloromethane  m e t h y l a t i o n (no a b s o r p t i o n s a t 3625 cm'*-  95°C  hour.  The m e t h y l a t e d  by i . r .  (5  d i m e t h y l s u l f i n y l a n i o n f o r 4 hours  chromatography (Sephadex LH 20).  lamp,  sulfoxide  excess a c i d was  hydrolyzed with 2 M  TFA  at  removed by c o d i s t i l l a t i o n w i t h  water and the h y d r o l y z a t e a n a l y z e d by paper chromatography ( s o l v e n t  (3)  developed  was  with  p-anisidine  hydrochloride).  The  c o n v e r t e d t o a l d i t o l a c e t a t e s by sodium b o r o h y d r i d e by  acetylation  with  1:1  acetic  hydrolyzate  reduction  anhydride-pyridine.  followed  These  alditol  a c e t a t e s were a n a l y z e d by g . l . c . and g.l.c.-m.s. u s i n g columns A and G.l.c.  and  g.l.c.-m.s.  (see mass-spectra  with excess  r e s u l t s are as shown i n T a b l e I I I . 3, column I,  o f the a l d i t o l a c e t a t e s i n Appendix I I I ) .  A p o r t i o n o f the m e t h y l a t e d  polysaccharide  lithium  in  aluminum  hydride  l i t h i u m aluminum h y d r i d e was  precipitate  formed  was  dissolved  refluxing  analyzed  by  in  10%  20  hours.  Reduction  of  hydrolyzate  was  The  The  ethanol.  The  a c i d and  r e s i d u e was  The m e t h y l a t e d  with  reduced  overnight.  hydrochloric  h y d r o l y z e d w i t h 2 M TFA the  mg)  oxolane  (3x).  i n f r a - r e d spectroscopy.  reduced p o l y s a c c h a r i d e was  (15  decomposed by adding  p r o d u c t r e c o v e r e d by c h l o r o f o r m e x t r a c t i o n and  B.  dried  and c a r b o x y l -  on a steam sodium  the  bath  for  borohydride,  - 71 -  f o l l o w e d by a c e t y l a t i o n g.l.c.-m.s.  analyses  (with  acetic  (column  A  anhydride-pyridine), g.l.c.  programmed  from  180°C  to  and  250°C a t  2°C/min) gave the d a t a i n T a b l e I I I . 3 , column I I .  IV.6  Uronic a c i d d e g r a d a t i o n  A sample (20 mg) then  with  a  trace  6 6  o f m e t h y l a t e d K34 p o l y s a c c h a r i d e of  p-toluenesulfonic  dimethylsulfoxide-2,3-dimethoxypropane under  nitrogen.  (12 mL)  and  dissolved i n  19:1  was  sealed  added and a l l o w e d to  Methyl i o d i d e  (3 mL)  was  added  the c o o l e d r e a c t i o n mixture and s t i r r i n g c o n t i n u e d f o r an hour.  methylated, chloroform  degraded and  water.  product  2M  TFA  a c e t a t e s were g.l.c.-m.s. f o r 2°C/min.  for  was  isolated  The p r o d u c t was  chromatography (Sephadex LH-20). with  dried  and the f l a s k was  D i m e t h y l s u l f i n y l a n i o n (5 mL)  r e a c t f o r 18 hours a t room temperature. to  a c i d , was  was  by  partition  between  then p u r i f i e d by g e l permeation  The degraded  product  was  hydrolyzed  8 hours a t 95°C and the p a r t i a l l y m e t h y l a t e d  prepared  as  described  The  earlier.  G.l.c.  alditol  analysis  and  were conducted u s i n g column A programmed from 180°C to 250°C G . l . c . and g.l.c.-m.s.  results  are  as  shown  in  Table  I I I . 3, column I I I .  IV. 7  C a r b o d i i m i d e r e d u c t i o n o f K34 p o l y s a c c h a r i d e ^ 2  A  portion  (120 mg)  o f K34 p o l y s a c c h a r i d e ( H  +  form) was  dissolved  72 -  in  water  (30  mL).  l-Cyclohexyl-3-(2-morpholinoethyl)-carbodiimide  metho-p-toluenesulfonate solution. ions),  As  hours  dropwise. ml  was  423  mg)  proceeded  maintained  a t 4.75  was  an  sodium  consumption  ions  c o n t r o l l e d by c o n s t a n t  borohydride  at  about  pH  solution  7  ceased,  by  was  the  titrating  The model  Smith h y d r o l y s i s - ^  consumption o f p e r i o d a t e was at  223  M.  The  the  mmoles  following  ( i i ) The  obtained  of  NaI04  then weighed  of carboxyl  reduced  (Figure  f o r monitoring  periodate  ( i ) IO^'/IC^" s o l u t i o n s c o n t a i n i n g  were  absorbance o f these  spectrophotometer.  The  against  m o n i t o r e d u s i n g spectrophotometer  c a l i b r a t i o n curve  o b t a i n e d as f o l l o w s .  was  solution  dialyzed  l y o p h i l i z e d p r o d u c t was  consumption was  a  Approximately  polysaccharide  240  0.075.  added  mg).  P e r i o d a t e o x i d a t i o n and K34  was  2  with hydrochloric acid  water f o r two  IV.8  (3M)  reaction  distilled  ( y i e l d = 124  (0.1M)  added over a p e r i o d o f 2  A f t e r c o n c e n t r a t i n g , the p o l y s a c c h a r i d e was The  hydrogen  approximately  stirring.  solution.  days.  of  by adding h y d r o c h l o r i c a c i d  Throughout the base a d d i t i o n , the pH o f  maintained  polysaccharide  aqueous s o l u t i o n o f sodium b o r o h y d r i d e  Foaming was of  added to the  (with  When consumption o f hydrogen  later,  hours.  reaction  the pH was  dropwise.  100  the  (CMC,  prepared;  s o l u t i o n s was  0.30,  0.225, 0.15  and  measured by means o f  ( i i i ) A p l o t o f absorbance v e r s u s  concentration  III.l).  c a r b o x y l reduced  polysaccharide  (120 mg)  was  dissolved  in  30  73 -  mL  water  and 20 mL 0.06M sodium metaperiodate was added.  was conducted a t room temperature  and i n the dark.  Aliquots  the r e a c t i o n s o l u t i o n were withdrawn p e r i o d i c a l l y , a n a l y z e d on the spectrophotometer. plateau  after  about  122  hours  The r e a c t i o n (0.1 M)  d i l u t e d 250 times and  The p e r i o d a t e consumption (see F i g u r e  I I I . 2).  reached  The  i n absorbance  a f t e r 122 hours.  a  number o f  m i l l i m o l e s consumed was o b t a i n e d from the c a l i b r a t i o n curve knowing change  of  the  A p p r o x i m a t e l y 4 moles 10^" were  consumed p e r r e p e a t i n g u n i t .  The  excess  periodate  adding  mL) and the p r o d u c t was d i a l y z e d o v e r n i g h t  ethylene  glycol  (1  against d i s t i l l e d  water.  Sodium b o r o h y d r i d e  solution  was l e f t  overnight.  was  (0.5 g) was added  +  The  L  of  dried. on  distilled  f o r 48 h.  water.  freeze-dried  on  product,  using  solvent  B.  to  Hakomori's  Sugar a n a l y s i s  procedure- -'. 3  chromatography  was  column I I I ) . The  P2  methylated  was a n a l y z e d 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 by g . l . c .  and g.l.c.-m.s.  (column A programmed from 180°C t o 250°C a t 2°C/min).  Smith d e g r a d a t i o n was a l s o conducted on form).  against  The s l o w i n g moving  P2 as p r e v i o u s l y d e s c r i b e d (Table I I I . l ,  was m e t h y l a t e d a c c o r d i n g product  of  The d i a l y z a t e was c o n c e n t r a t e d and f r e e z e -  compound P2 was a n a l y z e d by ^H-n.m.r. s p e c t r o s c o p y . performed  portions  The p r o d u c t was d i a l y z e d  Two compounds were i s o l a t e d by p r e p a r a t i v e paper  the  and the  d e r i v e d p o l y a l c o h o l was d i s s o l v e d i n 10 mL 0.5 M TFA and  s t i r r e d a t room temperature 2  by  The s o l u t i o n was d e i o n i z e d w i t h A m b e r l i t e  IR 120 [ H ] r e s i n and the p r o d u c t c o d i s t i l l e d w i t h s e v e r a l methanol.  destroyed  K34  polysaccharide  (Na  +  - 74 IV.9  Selective Smith degradation  A  solution  of K34 polysaccharide (20 mg)  0.02 M NalO^ (20 temperature.  mL)  and  Ethylene  kept  glycol  in  the  dark  (0.2  mL)  was  i n water was mixed with for  3  hours  added,  s t i r r e d f o r 1 hour and the polyaldehyde formed was  at  room  the mixture  dialyzed  was  overnight.  This polyaldehyde was reduced to the polyol by adding sodium borohydride to  a  concentrated  hydrolysis  was  solution  effected  of  by  the  non-dialyzable  treating  the  1  L  of  distilled  water.  Smith  product with 0.5 M TFA and  s t i r r i n g for 24 hours at room temperature. against  residue.  The  product  was  dialyzed  The substance that remained i n the  d i a l y z i n g sac was freeze dried and analyzed  by  ^H-n.m.r.  spectroscopy  (Table III.2) . This  degraded  procedure .  The  53  methylated  product methylated  alditol  acetates  was  methylated  product by  was  g.l.c.  according analyzed  and  programmed from 180°C to 250°C at 2°C/min).  to Hakomori's as  partially  g.l.c.-m.s.  Methylation  (column  analysis  A  data  are given i n Table III.3. The  degraded  product,  dried  in  vacuo and under i . r . lamp, was  treated with methanolic hydrogen chloride (3%) and refluxed overnight on a steam bath under anhydrous conditions. The excess acid was removed by treatment of the reaction centrifugation.  The  mixture  supernatant  converted to a l d i t o l acetates. g.l.c.  and  5°C/min).  g.l.c.-m.s.  with  lead  carbonate,  followed  was concentrated and the residue was  These a l d i t o l acetates were analyzed  (column  by  programmed  from  180°C  Sugar analysis data are shown i n Table I I I . l .  to  by  220°C at  -  IV.10  75  D e t e r m i n a t i o n of the c o n f i g u r a t i o n (D or L) of the  The p r o d u c t from individual g.l.c.  the  partially  methylation  methylated  analysis  alditol  was  acetates  separated  using  (column SP 2340 programmed from 1 7 5 ° C to 2 4 0 ° C at  partially  methylated  t e c h n i q u e are dissolved  alditol  in  Comparison o f the c d . their  authentic  standards  preparative  l°C/min).  The  Each o f these a l d i t o l a c e t a t e s  acetonitrile  spectra  into  a c e t a t e s i s o l a t e d by the above mentioned  shown i n Table I I I . 4 .  separately  sugars  and t h e i r c d .  spectra  o f these a l d i t o l a c e t a t e s  with  was  recorded. that  of  showed t h a t a l l the sugar r e s i d u e s have the D  configuration. K34 b a c t e r i o p h a g e sodium  oligosaccharide  a c e t a t e b u f f e r (pH -  7.0)  mg i n 1 mL b u f f e r ) was added.  (3 mg) was d i s s o l v e d i n 1 mL  and a s o l u t i o n o f a - D - g l u c o s i d a s e  The mixture was i n c u b a t e d f o r 2  37°C,  then  acid.  The p r o d u c t i s o l a t e d by l y o p h i l i z a t i o n ,  was  examined  (0.5  days  the r e a c t i o n was t e r m i n a t e d by a d d i n g a t r a c e o f 50%  of  at  acetic  by  paper  chromatograph.  IV.11  Serological  E.  coli  K34  cross-reactions  bacteriophage  i s o l a t e d from Vancouver Sewage. strains  K28,  K31,  By means o f a s t e r i l e  and  E.  c o l i K31 b a c t e r i o p h a g e  The b a c t e r i a l  lawn  for  the  E.  were coli  K32, K33 and K34 were made as p r e v i o u s l y d e s c r i b e d . f i n e l y drawn p i p e t t e ,  was s p o t t e d on the b a c t e r i a l  E.  coli  lawns f o r the E . c o l i  K34  strains  bacteriophage K28, K31, K33  - 76 -  and K34.  E. coli K31 bacteriophage was also  bacterial  lawn.  spotted  on E . coli K34  After overnight incubation of these plates (containing  bacterial lawn spotted with bacteriophages) the plates  were  inspected  for plaques formed.  IV.12  Bacteriophage depolymerization of E. c o l i K31 capsular polysaccharide  E. coli K31 bacteriophage (#31) isolated from Vancouver sewage was propagated on E. coli K31 bacteria according to standard methods of Adam .  #31 was propagated using tube lysis and flask lysis (see Table  III. 5).  #31 solution (5.5 x 10 ) was dialyzed (cut off 3500) against  74  buffer days.  12  pH -  7  (ammonium acetate and ammonium carbonate buffer) for 2  After concentrating by evaporation under  reduced pressure,  the  concentration of #31 was 1.8 x l O ^ p . f . u . 2  Stirm  bacteriophages  degrade  1 g of the corresponding  bacterial  are required  to  capsular polysaccharide.  75  had shown that l O ^  3  This concentrated #31 solution was  added to E. coli K31 capsular polysaccharide (180 mg; prepared by Dr. E. Altman  according  to  the procedure  described in IV.2) solution. The  depolymerization was conducted in an incubator at 37°C for 48 hours. Molisch  test^l^  a n c  \ paper chromatography of the crude reaction mixture  indicated the presence of an oligosaccharide. concentrated (3x). The  and dialyzed  The reaction mixture was  (M.W. cut off 3500) against d i s t i l l e d water  The dialyzate collected each time was combined and crude  A  concentrated.  depolymerized product was subjected to Amberlite IR 120 (H ) +  cationic of  exchange treatment and f r e e z e - d r i e d .  concentrated  A  solution  the p u r i f i e d d e p o l y m e r i z e d p r o d u c t was p l a c e d on a column o f  P2 (400  mesh)  and e l u t e d at  6.8  ml/h.  Fractions  (2  mL  Bio-Gel  each)  were  c o l l e c t e d and f r e e z e - d r i e d .  IV.13  Bacteriophage  d e p o l y m e r i z a t i o n o f E . c o l i K34  capsular  polysaccharide  E.  coli  K34 b a c t e r i o p h a g e  (#34)  was a l s o i s o l a t e d from Vancouver  sewage and propagated on E . c o l i K34 b a c t e r i a methods o f A d a m ^ .  #34  7  (Table I I I . 7 ) .  #34  3500)  buffer  against  buffer)  f o r 3 days.  pressure, trated (250  mg,  solution. for  48  s o l u t i o n (1.1 pH  -  x 10 )  was  13  prepared  o f #34  was 7.2  evaporation  x 10^  2  to  the  as  Paper chromatography  o f an o l i g o s a c c h a r i d e . previously  described.  under  p.f.u.  procedure  The d e p o l y m e r i z a t i o n was conducted i n an  the presence  flask lysis off  7 (ammonium a c e t a t e and ammonium carbonate  A f t e r c o n c e n t r a t i n g by  according  standard  d i a l y z e d (M.W. cut  s o l u t i o n was added to E . c o l i K34 c a p s u l a r  hours.  purified  was propagated u s i n g tube l y s i s and  the c o n c e n t r a t i o n  #34  a c c o r d i n g to the  The  A  concen-  polysaccharide  described  in  incubator  o f the r e a c t i o n mixture  The r e s u l t a n t  reduced  IV.2)  at  37°C  indicated  degraded p r o d u c t  concentrated  solution  of  p u r i f i e d d e p o l y m e r i z e d p r o d u c t was p l a c e d on a column o f B i o - G e l P2 mesh)  and e l u t e d at  freeze-dried.  6.6  mL/h.  Fractions  was the (400  (2 mL each) were c o l l e c t e d and  The e l u t i o n p r o f i l e i s shown i n F i g .  III.3.  - 78 -  N.m.r.  study  A s o l u t i o n o f E . c o l i K34 b a c t e r i o p h a g e (Fraction  I)  was  cation-exchanged acidic.  reduced  with  sodium  w i t h A m b e r l i t e IR 120  generated borohydride  (H )  resin  +  The e l u a n t o b t a i n e d was c o n c e n t r a t e d  for  ^H-n.m.r.  was  also  until  min)  the  pH  (3x).  The  and was  borate reduced  mg) was exchanged t h r e e times w i t h D 2 O and s u b m i t t e d  spectroscopy.  exchanged  spectroscopy. Table  (23  (45  to dryness and the  formed was removed by c o d i s t i l l a t i o n w i t h methanol oligosaccharide  oligosaccharide  three  Bacteriophage times  (#34)  oligosaccharide  w i t h D 2 O and s u b m i t t e d f o r  N . m . r . data f o r these o l i g o s a c c h a r i d e s  are  as  (20  mg)  lH-n.m.r. shown  in  III.8.  D e t e r m i n a t i o n o f the r e d u c i n g e n d  NaBH^ (30 5 ml o f  H2O).  borohydride  i U 0  mg) was added to a s o l u t i o n o f o l i g o s a c c h a r i d e After  stirring  for  5  hours  was removed by c a t i o n - e x c h a n g e  and c o e v a p o r a t i o n w i t h m e t h a n o l . w i t h 2M t r i f l u o r o a c e t i c  the  excess  chromatography  The reduced  material  ( 1 0 mg i n of  sodium  (IR 1 2 0 ( H ) ) +  was  hydrolyzed  a c i d on a steam b a t h f o r 2 0 hours and the  excess  a c i d removed by c o e v a p o r a t i o n w i t h w a t e r . A hydroxylamine H C 1 s t o c k 0.5  g  of  solution  was  prepared  hydroxylamine H C 1 i n 2 0 ml o f p y r i d i n e .  by  0 . 5 ml o f h y d r o x y l -  amine-HC1 s t o c k s o l u t i o n was added to the h y d r o l y z a t e and mixture  was  heated  on  dissolving  a steam b a t h f o r 15 m i n u t e s .  the  reaction  A c e t i c anhydride  (0.5 mL) was reaction mixture acetates  added to  mixture of  isolated  cool  refluxed  peracetylated  was  p r o d u c t was  was  the  reaction on  a  mixture  and  the  resultant  steam b a t h f o r 45 minutes.  aldononitriles  and  peracetylated  alditol  by p a r t i t i o n between water and c h l o r o f o r m .  a n a l y z e d by g . l . c . and  g.l.c.-m.s.  column programmed from 180°C to 220°C a t 5°C/min.  using  DB-17  The  The  capillary  - 80 -  BIBLIOGRAPHY  81 -  BIBLIOGRAPHY  1.  G . O . A s p i n a l l , i n G . O . 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Stirm, Pure and A p p l . Chem., 55, 1983, 637-653.  106.  L. Beynon,  107.  D.P. Sweet, R.H. S h a p i r o and P. A l b e r s h e i m , 1975, 217-225.  108.  I.A. M o r r i s o n ,  109.  C E . B a l l o u , P.N. Lepke and W.C 1974, 461-467.  110.  Z. D i s c h e ,  111.  V. Smirnyagin, C T . Bishop and F.P. Copper, 1965, 3109.  112.  R. Morona, J . Tommassen and U. Henning, ( 1 ) , 1985, 161-169.  V i r o l o g y , 2, 1956, 719-736. A c t a Biochem. Polon.,  J . Gen.  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. Carbohydr. Res., 40,  J . Chromatogr., 108, 1975, 361-364. Raschke,  J . B a c t e r i o l . , 117.  Methods i n Carbohydr. Chem., V o l . 1, pp. 478. Can. J . Chem., 43,  E u r . J . Biochem., 150  -  ti /  APPENDICES  APPENDIX I THE KNOWN STRUCTURES OF THE ESCHERICHIA COLI K ANTIGENS (as of August 1, 1985)  - 88 -  E. COLI K ANTIGENS  NANA5AC  2  —  a  E. c o l l KI  ^_  P _4  Gal L-2 Gly U21...,.? _ 5  G  a  l  f  1_2  G l y  H31  }  E. c o l i K2  A  GlcA  GlcNAc 1 -  P  E. c o l i K5  _2  R  i  b  f  1_2 P  R  i  b  f  1_7 P  2—  ^  a  -3. R i b 2 P 1 Rib  f  i - I KDO 2—  0  f  E.  c o l i 6a  E. c o l l K6  3- ManNAcA - —^ Glc - — 1  1  OAc E. c o l i K7 and K56  /9  - 89 Rha I - - Rha i—5—KDO — a ct 7/8 P 2  2  I  OAc E. c o l i K12 and K82  -i Rib  f  l~l KDO 2— P U P  I  OAc E. c o l i K13  —& GalNAc i - - KDO 2_ 8 B 5  tt  I  OAc E. c o l i K14  GlcNAc ^-5- KDO 2_  E. c o l i K15  A Rib 5,  f  L_Z KDO 2—  OAc E. c o l l K20  90  Rib  E.  A  2_  i _ Z KDO  B  coli  G l c LA 3  f  B  K23  GlcA  F  O  u  c  i .  Q  C  Q  1  Gal  E.  coli  G l c LA 3 a  K27  GlcA LA  Fuc -A—  o  /3  Q  2 or  I  1  Gal  3  OAc E.  coli  K28  Man L-l G l c A- - GlcA i - - G a l 3  3  a 1  G l c J-- - Man 2  4  6 b  \ / pyr  E.  M  a  coli  n  3  i_3  K29  G  a  l  a GlcA L-l G a l  0  E.  coli  1_  B  Q  K30  91  Gal i - - Glc i - - GlcA i - ^ Rha i - - Rha 2 2  3  2  P  E. c o l l K31  OAc  2  1-4  Glc  Rha i - - Gal 3  P  1 GlcA E. c o l i K32  _3 Glc  GlcA i - - Fuc 3 2 P 3 V pyr Gal 4  °  E. coli K33  -•—2- GlcA  Gal I- - Gal 13 /* 3  1 Gal 4 a 1 Glc E. coli K34  OAc  - 92  _3  G  a  i  1_3 a  G  a  l  1_3  A  F  a  u  1_  c  a  E. c o l l K42  0  A3 G a l A1 0 - P - 0 1  2 Fru  E.  +  OH OAc + OPr  c o l i K52  GlcA Uh. GlcA LA  M  a  n  i_3  M  a  n  1_3  G  l  c  N  A  1_  c  1 2 Rha  M  a  n  1_3  M  a  n  1_3  G  Rha  E.  ->-A GlcA LA  c o l l K85  FucNAc i - - GlcNAc I- - G a l L 4 3  6  1 Glc  E.  c o l l K87  -2 NANA5AC "L-l NANA5Ac  E.  2-OAc  c o l i K92  L.  l  c  N  A  c  1_  93  0 —  Rib  i - - ribitol b 2  f r  0 —  I  P OH  E. c o l i K100  —  - 94 -  References KI  E . J . McGuire and S . B . B i n k l e y ,  Biochemistry,  K2  K. J a n n , B. Jann and A . M . 1108-1115.  K5  W . F . Vann, M . A . Schmidt, B. J a n n , B i o c h e m . , 116, 1981, 359-364.  K6  P. Messner and F . M . Unger, 1980, 1003-1010.  K6a  H . J . J e n n i n g s , K . - G . R o s e l l and K . G . Johnson, 105. 1982, 45-56.  K7-K56  F . - P . T s u i , R . A . Boykins and W. Egan, 1982, 263-271.  K12-K82  M . A . Schmidt, B. Jann and K. J a n n , 1982, 69-74.  K13  W . F . Vann and K. J a n n , I n f e c t . Immun., 25, 1979, 85-92. W.F. Vann, T . Soderstrom, W. Egan, F . - P . T s u i , R. Schneerson, I . Orskov and F . Orskov, I n f e c t . Immun., 39, 1983, 623-929.  K14  B . J a n n , P. Hofmann and K. J a n n , P r o g . A l l e r g y , 33, 1983, 53-79.  K15  W. Vann, u n p u b l i s h e d r e s u l t s . A l l e r g y , 33, 1983, 53-79.  K20.K23  W . F . Vann, T . Soderstrom, W. Egan, F . - P . T s u i , R. Schneerson, I . Orskov and F . Orskov, I n f e c t . Immun., 39, 1983, 623-629.  K27  K . J a n n , B. J a n n , K . F . S c h n e i d e r , F . Orskov and I . O r s k o v , E u r . J . B i o c h e m . , 5, 1968, 456-465.  K28  E . Altman and G . G . S .  K29  Y . - M . Choy, F . Fehmel, N . Frank and S. S t i r m , 1975, 581-590.  K30  A . K . C h a k r a b o r t y , H . F r i e b o l i n and S. S t i r m , 141. 1980, 971-972.  K31  K. Jann, unpublished r e s u l t s . Jann and K . J a n n , Bacteriol.  K32  E . Altman,  Schmidt,  J.  Bact.,  3,  1964,  143,  and K. J a n n ,  247-251.  1980,  Eur. J .  Biochem. B i o p h y s . R e s . Commun.,  dutton,  96.  Carbohydr. R e s . ,  C a r b o h y d r . R e s . , 102,  FEMS M i c r o b i o l .  Lett.,  14,  (from K . Jann and B. J a n n ) ,  (From K . Jann and B . J a n n ) ,  Prog.  Carbohydr. R e s . , i n p r e s s . J. Virol.,  J.  16,  Bacteriol.,  (From I . O r s k o v , F . O r s k o v , B. R e v . , 41 (1977) 667-710).  unpublished r e s u l t s .  - 95 -  K33  B . A . Lewis,  unpublished  results.  K34  G . G . S . Dutton and A . Kuma-Mintah, u n p u b l i s h e d  K42  H . Niemann, A . K . Chakraborty, H . F r i e b o l i n and S. S t i r m , B a c t e r i o l . , 133, 1978, 390-391.  K52  P. Hofmann, B. Jann and K. J a n n , 12th 1984 A b s t r a c t s , p . 367.  K85  K. J a n n , B. J a n n , 1966, 368-385.  K87  L . Tarcsay, 505-514.  K92  W. Egan, T . - Y . L u i , D. Dorow, J . S . Cohen, J . D . R o b b i n s , E . C . G o t s c h l i c h and J . B . Robbins, B i o c h e m i s t r y , 16, 1977, 3687-3692.  K100  W. Egan, F . P . T s u i , Chem., i n p r e s s .  I n t . Symp. Carbohydr.  F . Orskov and I . Orskov,  B. Jann and K. J a n n ,  results  Biochem.  Eur. J . Biochem.,  R. Schneerson and J . B . R o b b i n s ,  J.  Chem.,  Z . , 346.  23,  J.  1971,  Biol.  -  96 -  APPENDIX I I  MASS SPECTRA  SPECTRUM NO. 1 HEXITOL HEXAACETATE MASS SPECTRUM  100  43  in  115  90  145  80  187  70  217  60  05  50 40  73  259  289  30 20 10 0  LU  rt 50  100  rr  rVrri ii i i r i i i i  rt 150  200  250  300  r i i i i i i i i TI i I i i i i | i i i f i 350  400  I I |I I I I I I I I I | 450 500  SPECTRUM NO. 2 1,2,5,6-TETRA-0-ACETYL-3,4-DI-O-METHYLGLUCITOL MASS SPECTRUM  129  100 90 80 70 60 50  189  40 30 20  .87 43  10 71  0  l"'l '| I "| I  50  TT"I  I..  ..i.l  13  1  100  U51  r"r v i i-i-i '| 1 i i f i i 150  233  I | I I l"l I I I I I | I I  200  250  261  "TT 300  i  r  r  r  350  400  SPECTRUM N O . 3 1,3,4,5-TETRA-0-ACETYL-2,6-DI-METHYLGA1ACTITOL MASS SPECTRUM  100 90»  '  43  i  117  1  VO  80 70 60 50 40 30  87  20  58  305  185  143  231  74  10  Ii. ..lilll.i, ...Li  0  r r j r  50  100  tVi'fe 150  TT-  'i i y "i 200  r  I  I"I  T"  I "I  250  I  I I I  I  T  300  I  I I I  I I | I  350  I  I I I  I I I  |  400  SPECTRUM NO. 4 1,3,5-TRI-0-ACETYL-2,4,6-TRI-METHYLGALACTITOL MASS SPECTRUM  o o  117 90  43  80 70 60 50 161  4Vl 30 '•' 20  233  10  58 17,1  0  T T  50  i  r r  100  I T  T  |  I  ilk  150  I I I I I I | I I 2 7 7  'i i | i i • 200  II  i  T T  250  300  I I I I | I  350  I I I I  400  SPECTRUM NO. 5 1,4,5-TRI-0-ACETYL-2,3,6-TRI-METHYLGALACTITOL MASS SPECTRUM  43  117  233  50  "i r'"|'"i "i' 100  ,142  161 I I I  T T  150  I  I  I  I T T T T  200  I  I I  |  I  250  I  I  I  I I'  I  |  I I I I  300  I  I  I  |  I I I  350  400  SPECTRUM NO. 6 1, 5-DI-0-ACETYL-2,3,4,6-TETRA-O-METHYLGLUCITOL MASS SPECTRUM  100  i0i  43  161  90 80 70  129  60 50 40  71  20 10  0  205  87  30  4 50  IT 100  1  1  " " | ' ' I "I l  150  i "I  I  II  IJ  "I" I I' I I I i I I  200  j  I I 'I  250  I  I I I I I  (  . ,  300  l  l l l l I I | I I I I I  350  I I I  400  - 103 -  APPENDIX I I I  1  H AND C-N.M.R. SPECTRA 13  K34 Polysaccharide  Spectrum No. 1  ^-N.m.r. 400 MHz,  95°  T  4  r 5  T 4  K34 Polysaccharide 13  Spectrum No. 3  C-N.m.r.  100.6 MHz,  90°  acetone 31.071  I  i i  K34 Polysaccharide (Selective Smith degraded) ^-N.m.r. 400 MHz,  95° HOD  Spectrum No. 4  

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