Cellulose Chemistry and Technology

5 X-Ray Diffraction by Bacterial Capsular Polysaccharides: Trial Conformations for Klebsiella Polyuronides K5, K57, and K8

Downloaded by UNIV LAVAL on July 11, 2016 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0048.ch005

E. D. T. ATKINS, Κ. H. GARDNER, and D. H. ISAAC H. H. Wills Physics Laboratory, University of Bristol, Tyndall Avenue, Bristol, BS8 1TL, UK I n t e r e s t i n t h e p o l y u r o n i d e s , i n t h i s l a b o r a t o r y , stems b a c k t o t h e e a r l y 1970's when we a p p l i e d x - r a y d i f f r a c t i o n methods t o elucidate the molecular structures of the plant polysaccharide a l g i n a t e components,polymannuronic a c i d and p o l y g u l u r o n i c a c i d (1,2). I n 1971 we t u r n e d o u r a t t e n t i o n t o t h e more c o m p l i c a t e d connective tissue l i n e a r polydisaccharides. S t a r t i n g with h y a l u r o n i c a c i d (3) we s y s t e m a t i c a l l y e x p l o r e d t h e c o n f o r m a t i o n s of t h e connective t i s s u e polyuronides ( 4 ) , a l s o i n c l u d i n g t h e c a p s u l a r b a c t e r i a l p o l y u r o n i d e pneumococcus t y p e I I I f o r compar ison and encompassing t h e h i g h l y s u l p h a t e d blood a n t i c o a g u l a n t h e p a r i n ( 5 , 6 ) . D u r i n g t h i s p e r i o d we d e v e l o p e d and e x t e n d e d o u r c o m p u t e r i s e d model b u i l d i n g p r o c e d u r e s and w i t h t h e s e i m p r o v e d a i d s have n a t u r a l l y become i n t e r e s t e d i n t h e even more complex m i c r o b i a l p o l y s a c c h a r i d e s . I n t h i s c o n t r i b u t i o n we w i s h t o r e p o r t on some s e l e c t e d complex p o l y u r o n i d e s f r o m t h e K l e b s i e l l a serotypes. B e c t e r i a o f t h e genus K l e b s i e l l a p r o d u c e a c a p s u l a r p o l y s a c c h a r i d e which i s a n t i g e n i c . Approximately e i g h t y d i f f e r e n t s e r o t y p e s a r e r e c o g n i z e d and Nimmich ( 7 , 8 ) has p r o v e d q u a l i t a t i v e a n a l y s e s o f t h e s e c a p s u l a r m a t e r i a l s . The c h e m i c a l c o v a l e n t r e p e a t i n g s e q u e n c e s o f a number o f t h e s e s e r o t y p e s i s a l r e a d y e s t a b l i s h e d ; others a r e i n t h e process o f being e l u c i d a t e d , w h i l e t h e r e m a i n d e r a r e o n l y p a r t i a l l y Known. We have i n d u c e d a number o f t h e s e s e r o t y p e s t o f o r m o r i e n t e d , c r y s t a l l i n e f i b r e s s u i t a b l e f o r x - r a y d i f f r a c t i o n a n a l y s i s . These x - r a y d a t a e n a b l e h e l i c a l m o l e c u l a r models t o be c o m p u t e r g e n e r a t e d w h i l e m a i n t a i n i n g s t a n d a r d bond l e n g t h s , bond a n g l e s , r i n g geo m e t r i e s and s i d e c h a i n c o n f o r m a t i o n s and a l s o a v o i d i n g any u n d e s i r a b l e s t e r e o - c h e m i c a l c l a s h e s , y e t p r e s e r v i n g t h e known h e l i x symmetry and r e p e a t i n g a x i a l d i m e n s i o n s . I n a d d i t i o n any s t a b i l i z i n g i n f l u e n c e s , p a r t i c u l a r l y i n t r a - c h a i n h y d r o g e n bonds a c r o s s g l y c o s i d i c l i n k a g e s , a r e m o n i t o r e d and i n c o r p o r a t e d i f a satisfactory solution i s indicated. We have c h o s e n t h e t h r e e s e r o t y p e s K 5 , K57 and K8 f r o m o u r s e l e c t i o n b e c a u s e t h e y a r e a l l p o l y u r o n i d e s and we have a r r a n g e d 56

Arthur; Cellulose Chemistry and Technology ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

5.

ATKINS

E T

A L .

Bacterial

Capsular

Polysaccharides

57

them i n o r d e r o f i n c r e a s i n g c o m p l e x i t y . Our i n t e n t i o n i n t h i s s h o r t paper i s t w o - f o l d . F i r s t we w i s h t o e s t a b l i s h a c o n t i n u u m between o u r p r e v i o u s s t u d i e s on t h e c o n n e c t i v e t i s s u e p o l y u r o n i d e s and s e c o n d l y we w i s h t o g a i n an u n d e r s t a n d i n g o f t h e r u l e s which govern t h e m o l e c u l a r geometry o f t h e p o l y s a c c h a r i d e s as we a d v a n c e up t h e h i e r a r c h y o f c o m p l e x i t y .


K l e b s i e l l a K5 Of t h e t h r e e K l e b s i e l l a s e r o t y p e s t o be d i s c u s s e d i n t h i s p a r t i c u l a r c o n t r i b u t i o n t h e K5 p o l y s a c c h a r i d e h a s t h e l e a s t complicated chemical covalent repeat. I ti s a l i n e a r polyt r i s a c c h a r i d e o f t h e f o r m C-A-B-C- ) , and t h e d e t a i l e d c h e m i c a l c o n s t i t u t i o n has been r e p o r t e d by Duïton and Mo-Tai Yang ( 9 ) , a s shown s c h e m a t i c a l l y i n F i g u r e 1. The e s s e n t i a l b a c k b o n e s t r u c t u r e c o n s i s t s o f two n e u t r a l s u g a r s , a 1 , 3 - l i n k e d $-Dmannose and a 1 , 4 - l i n k e d 3 - D - g l u c o s e , s a n d w i c h i n g a 1,4 - l i n k e d β-D-glucuronic a c i d r e s i d u e . T h e r e a r e two a p p u r t e n a n c e s w o r t h m e n t i o n i n g : p y r u v i c a c i d i s l i n k e d t o t h e D-mannopyranose a s a 4,6 a c e t a l , and an 0 - a c e t a t e g r o u p i s a t t a c h e d t o t h e 2 - p o s i t i o n of t h e glucopyranose r i n g . Thus t h e r e p e a t i n g s e q u e n c e c o n t a i n s two c h a r g e d c a r b o x y l a t e g r o u p s and t h e g l y c o s i d i c l i n k a g e g e o m e t r y i s i l l u s t r a t e d i n F i g u r e 1. We w o u l d e x p e c t a l l t h r e e mono s a c c h a r i d e s t o e x i s t i n t h e n o r m a l 4C1 c h a i r c o n f o r m a t i o n r e s u l t i n g i n a p a i r o f 1+4 - d i e q u a t o r i a l g l y c o s i d i c l i n k a g e s t o g e t h e r w i t h a s i n g l e 1->3 - d i e q u a t o r i a l l i n k a g e . B o t h t h e s e l i n k a g e g o e m e t r i e s a r e common t o t h e s i m p l e r p l a n t and a n i m a l polyuronides. I f t h e v e c t o r s between e a c h g l y c o s i d i c oxygen atom were t o a l i g n p r e c i s e l y t h e maximum t h e o r e t i c a l e x t e n s i o n , using s t a n d a r d i z e d c o o r d i n a t e s (10), p e r c o v a l e n t repeat would be 1.56nm. Of c o u r s e i t i s e x t r e m e l y u n l i k e l y t h a t a s t e r e o c h e m i c a l a c c e p t a b l e model c o u l d e x i s t w i t h s u c h a r e p e a t b u t a t l e a s t i t g i v e s us an i d e a o f t h e u p p e r l i m i t t h a t we s h o u l d expect. F o r t h e s i m p l e r p o l y u r o n i d e s such as t h e a l g i n a t e s and c o n n e c t i v e t i s s u e p o l y u r o n i d e s o u r o b s e r v e d a x i a l l y p r o j e c t e d r e p e a t s i n t h e s o l i d s t a t e were w i t h i n 18% o f t h e t h e o r e t i c a l l i m i t and t y p i c a l l y e x t e n d e d c o n f o r m a t i o n s gave r e p e a t s c e n t r e d a r o u n d v a l u e s 1 0 % l e s s t h a n t h e maximum. The x - r a y d i f f r a c t i o n p a t t e r n o b t a i n e d f r o m t h e s o d i u m s a l t o f K5 i s shown i n F i g u r e 2. The l a y e r l i n e s p a c i n g i s 2.70 nm and m e r i d i o n a l r e l f e c t i o n s o c c u r o n l y on even l a y e r l i n e s suggesting a two-fold h e l i c a l conformation of the molecule. The a x i a l l y p r o j e c t e d r e p e a t o f 2.70/2 = 1.35 nm c o r r e l a t e s w e l l w i t h t h e c o v a l e n t t r i s a c c h a r i d e r e p e a t and i s O n l y 14% b e l o w t h e t h e o r e t i c a l l i m i t s u g g e s t i n g an e x t e n d e d c h a i n c o n f o r m a t i o n . A t t h i s s t a g e i t i s w o r t h w h i l e t o s y s t e m a t i c a l l y examine t h e t h r e e t y p e s o f g l y c o s i d i c l i n k a g e g e o m e t r y u s i n g Ramachandran t y p e h a r d s p h e r e p l o t s ( 1 1 ) . These maps ( F i g u r e 3) show t h e combination o f g l y c o s i d i c t o r s i o n angles t h a t c o u l d produce a s t e r e o c h e m i c a l ^ a l l o w a b l e l i n k a g e geometry. I n a d d i t i o n , t h e


CELLULOSE

CHEMISTRY

A N D TECHNOLOGY

CH3-C-COOH -3)-jB-D^an*-(l-4)-p-g-GlcUA-(l-4)-^-D-Glc-(l-


(Q)

Figure 1.

2-OAc

K l e b s i e l l a serotype K5: (a) chemical repeat, (b) schematic. Note the chair shape of the pyruvic acid.

Figure 2. X-Ray fibre diffraction pattern from the sodium salt K5 polysaccharide. This shows a layer line spacing of 2.70 nm with meridional reflections on even layer lines only indicating a two-fold conformation for the molecule.


ATKINS

Bacterial

E T A L .

Capsular

59

Folysaccharides


β-η-Μan-11-4 I-/M>--(;ΐ-Μηη linkaj-

C.lc()!2)-ManO3 d i e q u a t o r i a l g l y c o s i d i c l i n k a g e i s such t h a t the c a r b o x y l group o f t h e p y r u v a t e i s p l a c e d a t t h e maximum d i s t a n c e f r o m t h e h e l i x a x i s and t h e a x i a l 0(2) on t h e mannopyranose r e s i d u e a l l o w s f o r m a t i o n o f an 0(2) - - - 0(2) s t a b i l i z i n g i n t r a - c h a i n h y d r o g e n bond. I f t h e p y r u v a t e d r e s i d u e had been o t h e r t h a n manupyranose t h i s l i n k a g e c o n f o r m a t i o n w o u l d have been s t e r i c a l l y d i s a l l o w e d . The o t h e r i n t e r e s t i n g f e a t u r e o f t h e K5 p o l y s a c c h a r i d e i s t h e p r e s e n c e o f an 0 - a c e t y l s u b s t i t u e n t on t h e C(2) o f t h e g l u c o p y r a n o s e r e s i d u e . The 0 - a c e t y l g r o u p was p o s i t i o n e d f o l l o w i n g t h e a r r a n g e m e n t d e t e r m i n e d f r o m model compounds ( 1 4 ) . I t has l o n g been known t h a t n o n c a r b o h y d r a t e c o n s t i t u e n t s s u c h as 0a c e t y l g r o u p s and p y r u v a t e may f u n c t i o n as a n t i g e n i c d e t e r m i n a n t s . We f i n d t h a t i n t h e t h r e e d i m e n s i o n a l s t r u c t u r e f o r K5 t h e 0a c e t y l and p y r u v a t e a r e i n c l o s e p r o x i m i t y ( s e e F i g u r e 4) and c o u l d w e l l r e p r e s e n t the determinant s i t e . Klebsiella

K57

K57 i s a p o l y t e t r a s a c c h a r i d e c o n s i s t i n g o f t h r e e s u g a r r e s i d u e s i n t h e b a c k b o n e and w i t h one s i n g l e r e s i d u e i n t h e s i d e c h a i n . The r e p e a t i s t h e r e f o r e o f t h e f o r m :


ATKINS

E T A L .

Bacterial

Capsular

61

Folysaccharides


5.

Figure

4.

Projections

of proposed molecular conformation of the K5 Hydrogen bonds are indicated by dotted lines.

polysaccharide.



62

CELLULOSE

CHEMISTRY

AND

TECHNOLOGY

S (- A - Β - C -) , where S i s an α - D - mannose residue. The d e t a i l e d c h e m i c a l c o v a l e n t r e p e a t has been e s t a b l i s h e d by K a m e r l i n g e t a l . ( 1 5 ) and i s g i v e n i n F i g u r e 5. As i n t h e c a s e o f t h e K5 s e r o t y p e t h e b a c k b o n e c o n s i s t s o f two n e u t r a l s u g a r s and a u r o n i c a c i d r e s i d u e . T h i s 1,3 - l i n k e d - α - D g a l a c t u r o n i c a c i d r e s i d u e i s a t t a c h e d t o a 1,2 - l i n k e d - α - D mannose r e s i d u e f o l l o w e d by a 1,3 - l i n k e d 3 - D - g a l a c t o s e r e s i d u e . The s i d e g r o u p (S) i s a t t a c h e d t o t h e u r o n i c a c i d r e s i d u e . A g a i n we w o u l d a n t i c i p a t e t h a t a l l t h e s u g a r r e s i d u e s e x i s t i n t h e n o r m a l 4C1 c h a i r c o n f o r m a t i o n r e s u l t i n g i n one 1+3 - d i e q u a t o r i a l g l y c o s i d i c l i n k a g e a 1+2 - d i a x i a l l i n k a g e and one 1 ax +3eq l i n k a g e ( s e e , F i g u r e 5 ) . I n a d d i t i o n t h e mannopyranose a p p u r t e n a n c e i s 1+4 d i a x i a l l y a t t a c h e d . A priori t h i s s t r u c t u r e p r e s e n t s us w i t h some n o v e l g l y c o s i d i c l i n k a g e g e o m e t r i e s t o examine and w i t h t h e added c o m p l i c a t i o n o f a s m a l l s i d e c h a i n . The maximum t h e o r e t i c a l e x t e n s i o n f o r t h e c h e m i c a l r e p e a t , f o l l o w i n g t h e method d e s c r i b e d f o r K5, i s 1.27 nm. The x - r a y d i f f r a c t i o n p a t t e r n f r o m t h e K57 p o l y s a c c h a r i d e i s shown i n F i g u r e 6. The l a y e r l i n e s p a c i n g was measured t o be 3.429 nm w i t h m e r i d i o n a l r e f l e c t i o n s p r e s e n t o n l y on l a y e r l i n e s 1 = 3n. The s i m p l e s t i n t e r p r e t a t i o n o f t h i s p a t t e r n i s t h a t t h e p o l y s a c c h a r i d e backbone f o r m s a t h r e e - f o l d h e l i x w i t h a p r o j e c t e d a x i a l r e p e a t o f 1.143 nm. T h i s v a l u e , 10% l e s s t h a n t h e maximum p e r m i s s i b l e , c o r r e l a t e s w i t h a s i n g l e c o v a l e n t r e p e a t and s u g g e s t a f a i r l y extended s t r u c t u r e . T r i a l m o d e l s have been c o m p u t e r g e n e r a t e d c o n f o r m i n g t o t h e h e l i c a l p a r a m e t e r s . B o t h l e f t and r i g h t - h a n d e d m o d e l s have been g e n e r a t e d u s i n g t h e t e c h n i q u e s and c r i t e r i a o u t l i n e d e a r l i e r . A t t e m p t s were made t o f o r m t h e maximum number o f i n t r a - c h a i n h y d r o g e n bonds. I t was f o u n d t h a t no model c o u l d be c o n s t r u c t e d t h a t i n c l u d e d h y d r o g e n bonds a c r o s s a l l t h r e e backbone g l y c o s i d i c linkages. O n l y a l e f t - h a n d e d h e l i x a l l o w e d t h e f o r m a t i o n o f two i n t r a - c h a i n h y d r o g e n bonds i n t h e backbone:α - D - GalUA - 0(2) 0(3) - D - Man and α - D - Man 0(5) - - - Η - 0(2) 3-D - G a l . The t o r s i o n a n g l e s a t t h e g l y c o s i d i c l i n k a g e s o f t h i s model a r e marked on t h e s t e r i c maps i n F i g u r e 7. Computer drawn p r o j e c t i o n s o f t h e t h r e e - f o l d s t r u c t u r e a r e shown i n F i g u r e 8. I t i s i n t e r e s t i n g t o n o t e t h e 1 +3 - d i e q u a t o r i a l g l y c o s i d i c l i n k a g e a d j a c e n t t o t h e a t t a c h m e n t o f t h e mannopyranose s i d e c h a i n a p p e a r s u n a b l e t o f o r m a s u i t a b l e i n t r a - c h a i n h y d r o g e n bond. The i n t r o d u c t i o n o f t h e mannopyranose s i d e c h a i n p r e v e n t s t h e f o r m a t i o n o f a GalUA - 0 (4) - - - G a l 0(5) i n t r a - c h a i n h y d r o g e n bond t h a t i s p o s s i b l e i n t h e K8 p o l y s a c c h a r i d e ( s e e b e l o w ) . Thus t h e s i d e c h a i n may have a c o n t r o l l i n g i n f l u e n c e on t h e backbone geo m e t r y . A t t h e l i n k a g e t o t h e s i d e g r o u p , namely α - D - Man (1+4) - α - D - GalUA, no h y d r o g e n bonds a r e p o s s i b l e , i r r e s p e c t i v e o f t h e b a c k b o n e c o n f o r m a t i o n , and s i n c e t h e t o r s i o n a n g l e s a t t h i s l i n k a g e do not a f f e c t t h e h e l i c a l p a r a m e t e r s a c o n f o r m a t i o n was α


ATKINS

Bacterial

E T A L .

Capsular

Polysaccharides

cx-Q-Man

%

(α)


-3)-cx-D-GaluA-(l-2)-o(-Q-Man-(l-3)-/9-B-Gal-(l-

Figure

5.

Klebsiella

serotype K57: (a) chemical repeat, (b) schematic.

Figure 6. X-ray fibre diffraction pattern of K57 polysaccharide. This shows a hyer line spacing of 3.429 nm with meridional re flections on hyer lines (b) governed by l=3n. The simphst interpretation is a three-fold helix.


CELLULOSE

64

CHEMISTRY AND

TECHNOLOGY

a - D - M a n - ( l - 3 ) - # - D - C , a l linkage

a - D - G a l U A - < 1-2) -«-n-Man linkage

Man()(5)-GalO(2

-120

;AO(2)-ManO(3l


θ

J

9

J

L

I

I

L

-120

120

Θ,

(b)

(Q)

β-Ώ-Gal- (1-3) - a - D - G a l U A

α-D-Man- (1-4) - « - » - ( î a l l ' A linkage

linkage

ManO(5)-GalUAO(3) GalO(2)-GalUAO(2)

-120 h

-12o|—

J

h

Θ,

[GalO(5)-GairA()(4)]

J

L

-120

L

Θ,

(d)

(C)

Figure 7. Steric maps and possible hydrogen bonds for the K57 polysaccharide. In each case the final position of our trial model is marked (o). The bracketed hydrogen bond cannot be formed in the specific case of K57 since the GalUA-0(4) atom is glycosidically linked to the Man side chain.

(a) ^ = C f 2 ) - C ( J ) - 0 - C f 2 ) ; θ = C(l) - Ο - C(2) ~ C(l). 2

(b) Θ, = 0(5) - C ( l ) - 0 - C(3); e = C ( l ) - 0 - C(3) - C(2). (c) Θ, = C(2) - C ( l ) - 0 - C(3) e = C ( l ) - 0 - C(3) - C(2). (d) Θ, = C(2) - C ( l ) - 0 - C(4); e = C ( l ) - 0 - C(4) - C(3). 2

;

2

2



ATKINS

E T AL.

Bacterial

Capsular

Polysaccharides

3.43 nm

Figure 8. Projections of proposed molecular conformation of the K57 polysaccharide. Hydrogen bonds are indicated by dotted lines.


66

CELLULOSE

c h o s e n t o be n e a r t h e c e n t r e o f t h e a l l o w e d (Figure 7(d)).


Klebsiella

CHEMISTRY

AND

TECHNOLOGY

r e g i o n as marked

K8

The c o v a l e n t c h e m i c a l r e p e a t i n g s e q u e n c e has been e s t a b l i s h e d by S u t h e r l a n d (16) and i s g i v e n i n F i g u r e 9. I t i s s i m i l a r to t h e K57 p o l y s a c c h a r i d e w i t h a p o l y t r i s a c c h a r i d e b a c k b o n e and a s i n g l e m o n o s a c c h a r i d e c h a i n . B o t h s t r u c t u r e s have a u r o n i c a c i d i n t h e r e p e a t , however, i n K57 t h e u r o n i c a c i d i s i n c o r p o r a t e d i n t h e b a c k b o n e and t h e s i d e c h a i n i s a n e u t r a l s u g a r . The c o n v e r s e i s t r u e i n K8 w h i c h has an u n c h a r g e d b a c k b o n e and an α - JD - g l u c u r o n i c a c i d and s i d e c h a i n . The b a c k b o n e c o n s i s t s o f t h r e e commonly o c c u r r i n g n e u t r a l s u g a r s : a 1,3 - l i n k e d 3 D - g a l a c t o s e f o l l o w e d by a 1,3 - l i n k e d 3 - Ό - g a l a c t o s e and f i n a l l y a 1,3 - l i n k e d α - D - g l u c o s e . A l l f o u r monosaccharides a r e e x p e c t e d t o e x i s t i n t h e n o r m a l 4C1 c h a i r c o n f o r m a t i o n and a s c h e m a t i c diagram r e p r e s e n t i n g the g l y c o s i d i c l i n k a g e geometry i s shown i n F i g u r e 9. Thus two 1+3 - d i e q u a t o r i a l g l y c o s i d i c l i n k a g e s s t r a d d l e a 1 ax+3 e q - g l y c o s i d i c l i n k a g e i n t h e b a c k b o n e . The maximum e x t e n s i o n f o r t h e c h e m i c a l r e p e a t , f o l l o w i n g t h e method d e s c r i b e d p r e v i o u s l y , i s 1.38 nm f a l l i n g p a r t w a y between t h e v a l u e s f o r K5 and K57. The x - r a y d i f f r a c t i o n p a t t e r n f o r t h e s o d i u m s a l t o f t h e K8 p o l y s a c c h a r i d e i s shown i n F i g u r e 10. The m a t e r i a l i s h i g h l y o r i e n t e d and c r y s t a l l i n e and f r o m t h e s y s t e m a t i c a b s e n c e s o f odd o r d e r m e r i d i o n a l r e f l e c t i o n s i t can be s e e n t h a t t h e m o l e c u l e f o r m s a Zj h e l i x . However t h e l a y e r l i n e s p a c i n g o f 5.078 nm i s f a r t o o l a r g e f o r a r e p e a t o f two asym metric units. In f a c t t h i s value i s very c l o s e to the t h e o r e t i c a l maximum e x t e n s i o n f o r f o u r c o m p l e t e c o v a l e n t r e p e a t s , i . e . 4 χ 1.38 = 5.52 nm. Thus t h e o b s e r v e d r e p e a t i s o n l y 10% l e s s t h a n maximum e x t e n s i o n . F o r p r e l i m i n a r y model b u i l d i n g i t a p p e a r e d r e a s o n a b l e t o assume t h a t t h e s t r u c t u r e o f t h e i s o l a t e d m o l e c u l e i s a p e r f e c t f o u r f o l d h e l i x w i t h an a x i a l a d v a n c e p e r c o v a l e n t r e p e a t 1.27 nm. P e r t u r b a t i o n s f r o m an i d e l i z e d f o u r - f o l d h e l i x w o u l d be e x p e c t e d t o r e s u l t i n a l o w e r symmetry and c o n s e q u e n t l y t h e p a c k i n g o f t h e m o l e c u l a r c h a i n s i n an o r t h o r h o m b i c r a t h e r t h a n a t e t r a g o n a l u n i t cell. T h i s phenomenon has a l s o been o b s e r v e d i n h y a l u r o n i c a c i d (13) . M o d e l s were c o n s t r u c t e d i m p o s i n g t h e e x p e r i m e n t a l l y d e t e r m i n e d f i b r e r e p e a t and t h e a s s u m p t i o n t h a t t h e i s o l a t e d m o l e c u l e i s a four-fold helix. A t t e m p t s w e r e made t o c o n s t r u c t a s t e r e o c h e m i c a l ^ a c c e p t a b l e s t r u c t u r e i n c o r p o r a t i n g t h e maximum number o f i n t r a m o l c u l a r h y d r o g e n b o n d s . B o t h r i g h t - h a n d e d and l e f t handed s t r u c t u r e s w e r e c o n s i d e r e d . I t was i m p o s s i b l e t o c o n s t r u c t a model w h i c h i n c o r p o r a t e d h y d r o g e n bonds a t a l l t h r e e b a c k b o n e linkages. O n l y a l e f t - h a n d e d h e l i x a l l o w e d t h e f o r m a t i o n o f two h y d r o g e n bonds i n t h e b a c k b o n e . The g l y c o s i d i c t o r s i o n a n g l e s t h a t g e n e r a t e t h i s model a r e i n d i c a t e d on t h e s t e r i c maps i n


ATKINS

Bacterial

E T AL.

Capsular

Polysaccharides

(Χ-ρ-GlcUA

(α)

4s. -3)-/9-D-Gal-(l-3)-a-D-Gal-(l-3)-/9-B-Glc-(l-

GlcUA COO' Gal OH

Gal

Glc

0


(b)

Figure 9.

K l e b s i e l l a serotype K8: (a) chemical repeat, (b) sche matic

Figure 10. X-Ray fibre diffraction pattern of K8 polysaccharide. This shows a hyer line spacing of 5.078 nm with meridional re flections on even hyer lines only. Such a spacing is compatible with four covalent repeats, and we interpret this as a slightly perturbed four-fold helix.



68

CELLULOSE

CHEMISTRY

AND

TECHNOLOGY

F i g u r e 11. P r o j e c t i o n s o f t h i s l e f t - h a n d e d h e l i c a l model a r e shown i n F i g u r e 12. The s t r u c t u r e c o n t a i n s h y d r o g e n bonds 0(5) 0 ( 4 ) and 0(5) 0(2) a t t h e 3-D - G a l - (1+3) - α D - G a l and α - D - G a l - (1+3) - 3 - β"- G l c l i n k a g e s respectively. I t was f o u n d t o be i m p o s s i b l e t o f o r m a f o u r - f o l d h e l i x ( w i t h the observed e x t e n s i o n ) which c o n t a i n e d a hydrogen bond a t t h e 3 - D - G l c - (1+3) - 3 - D - Gal g l y c o s i d i c l i n k a g e . This l i n k a g e adjacent to the attachment s i t e of the u r o n i c a c i d s i d e c h a i n . T h i s f e a t u r e i s a n a l o g o u s t o t h e s i t u a t i o n i n K57. The s t e r i c map f o r t h e g l y c o s i d i c l i n k a g e t h a t d e f i n e s t h e o r i e n t a t i o n o f t h e s i d e c h a i n i s shown i n F i g u r e 1 1 ( d ) . Lacking any i n f o r m a t i o n t o d e f i n e t h e p o s i t i o n o f t h e s i d e c h a i n , t h e g l y c o s i d i c t o r s i o n a n g l e s were s e t a t v a l u e s c o r r e s p o n d i n g t o t h e c e n t r e o f t h e a l l o w e d r e g i o n on t h e s t e r i c map. One p o s s i b l e e x p l a n a t i o n f o r the t w o - f o l d r a t h e r than f o u r - f o l d nature of the molecule i n the c r y s t a l l i n e s t a t e i s that adjacent covalent r e p e a t s have t h e s i d e c h a i n i n d i f f e r e n t o r i e n t a t i o n s . Discussion We have p r e s e n t e d e x a m p l e s o f t r i a l model b u i l d i n g on a s m a l l s e l e c t i o n of the l a r g e v a r i e t y of m i c r o b i a l p o l y s a c c h a r i d e s . T h i s has r e c e n t l y become p o s s i b l e w i t h t h e c r y s t a l l i z a t i o n o f these molecules i n a form s u i t a b l e f o r x-ray d i f f r a c t i o n s t u d i e s . I t i s only through these x-ray s t u d i e s t h a t the h e l i c a l para m e t e r s n e c e s s a r y f o r m e a n i n g f u l model b u i l d i n g can be o b t a i n e d . Even t h o u g h t h e examples we have c h o s e n have s u b s t a n t i a l l y d i f f e r e n t primary s t r u c t u r e s , c e r t a i n s a l i e n t p o i n t s are apparent. For example, a l l the s t r u c t u r e s presented e x i s t i n extended conformations. T h i s f e a t u r e a p p e a r s t o be i n d e p e n d e n t o f t h e h e l i c a l symmetry e x h i b i t e d by t h e m o l e c u l e . We have p r e s e n t e d p r e l i m i n a r y s t r u c t u r a l models computed on the b a s i s of a search f o r s t a b i l i s i n g i n t r a c h a i n hydrogen bonds. This approach i s a u s e f u l s t a r t i n determining h e l i c a l s t r u c t u r e s for f u r t h e r refinement. The i n v e s t i g a t i o n o f c o n f o r m a t i o n a l maps and p o t e n t i a l h y d r o g e n bonds g i v e s us i n s i g h t i n t o t h e m o l e c u l e s c o n c e r n e d and c o n c u r r e n t l y p r o v i d e s us w i t h v a r i o u s s t a r t i n g models f o r f u r t h e r r e f i n e m e n t u s i n g t e r m s a p p r o x i m a t i n g more c l o s e l y t o p o t e n t i a l e n e r g y f u n c t i o n s , ( s e e e.g.Guss e t a l . ( 1 3 ) ) . By s e l e c t i n g t h e models c o n t a i n i n g t h e maximum number o f h y d r o g e n bonds c o n s i s t e n t w i t h t h e s t e r e o c h e m i s t r y we a r e c h o o s i n g t h e most l i k e l y s t r u c t u r e a t t h i s f i r s t l e v e l h a r d s p h e r e a p p r o x i m a t i o n . As we i n c l u d e more complex i n t e r a c t i o n t e r m s we w o u l d hope t h a t t h e model w i l l not change s i g n i f i c a n t l y . I f t h i s indeed turns o u t t o be t h e c a s e , as o u r c o n t i n u i n g c a l c u l a t i o n s have so f o r i n d i c a t e d , t h e n s u c h a s i m p l e a p p r o a c h c o u l d be e x t r e m e l y u s e f u l i n model b u i l d i n g . C l e a r l y , the q u a l i t y of d i f f r a c t i o n photographs i s going to determine the eventual d e t a i l of the r e f i n e d s t r u c t u r e s . In the c a s e o f K8 f o r w h i c h a h i g h l y c r y s t a l l i n e p a t t e r n has been


5.

ATKINS

Bacterial

E T A L .

Capsular

Polysaccharides

0 - D - G a l - (1-3) -α-D-Gal linkage

GalO(2)-Gal()(2)

69

a - D - G a l - i l - 3 ) - / ? - D - G l c linkage

Gal()(5)-GlcO(2) r

GalO(2)-GlcO(4)


GalOl5)-GalO(4>

J

120

L

J

-120

I

120

Θ,

(a)

(b)

0-D-Gle- ( 1 -3 ) - 0 - D - G a l linkage

L -120

Θ,

a-D-GlcUA-(l-4)-y3-D-Gal

linkage

Gle()(2)-GalO(2)

[GleO

Cellulose Chemistry and Technology

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