Cellulose Chemistry and Technology

Cellulose Chemistry and Technology

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

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

Cellulose Chemistry and Technology Jett C. Arthur, Jr., EDITOR Southern Regional Research Center, USDA

A symposium sponsored by the Cellulose, Paper and Textile Division at the 171st Meeting of the American Chemical Society, N e w York, N.Y., A p r i l 5-9, 1976.

ACS

SYMPOSIUM

AMERICAN

SERIES

CHEMICAL

SOCIETY

WASHINGTON, D. C. 1977

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

48

L i b r a r y o f Congress CIP D a t a Cellulose chemistry and technology. (ACS symposium series; 48 ISSN 0097-6156) Includes bibliographical references and index. 1. Cellulose—Congresses. I. Arthur, Jett C., 1918. II. American Chemical Society. Cellulose, Paper and Textile Division. III. Title. IV. Series: American Chemical Society. ACS symposium series; 48. TS933.C4S92 1976 676'01'54 77-6649 ISBN 0-8412-0374-1 ACSMC8 48 1-397

Copyright © 1977 American Chemical Society All Rights Reserved. N o part of this book may be reproduced or transmitted in any form or by any means—graphic, electronic, including photo­ copying, recording, taping, or information storage and retrieval systems—without written permission from the American Chemical Society. PRINTED IN T H E UNITED STATES OF AMERICA

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

ACS Symposium Series Robert F . G o u l d , Editor

Advisory Donald G. Crosby Jeremiah P. Freeman E. Desmond Goddard Robert A . Hofstader John L. Margrave Nina I. McClelland John B. Pfeiffer Joseph V . Rodricks Alan C. Sartorelli Raymond B. Seymour Roy L. Whistler Aaron Wold

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

FOREWORD The ACS SYMPOSIUM SERIES was founded in 1974 to provide a medium for publishing symposia quickly in book form. The format of the SERIES parallels that of the continuing ADVANCES IN CHEMISTRY S E R I E S except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. As a further means of saving time, the papers are not edited or reviewed except by the symposium chairman, who becomes editor of the book. Papers published in the ACS SYMPOSIUM S E R I E S are original contributions not published elsewhere in whole or major part and include reports of research as well as reviews since symposia may embrace both types of presentation.

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

PREFACE The centennial anniversary meeting of the American Chemical Society gave the Cellulose, Paper and Textile Division the opportunity to present a timely Symposium on International Developments in Cellulose, Paper, and Textiles. Research scientists from academia, industry, and government, representing more than sixteen countries, cooperated in presenting significant research accomplishments in paper, wood, and cellulose chemistry and in cotton, wool, and textile fiber chemistry. In this volume research advancements on structure and on properties and reactions in cellulose chemistry have been contributed by investigators from Australia Kingdom, the Union of Soviet Socialist Republics, and the United States. Two additional volumes, "Textile and Paper Chemistry and Technology" and "Cellulose and Fiber Science Developments: A World View," will include other contributed symposium manuscripts. I would like to thank the participants, presiding chairmen, and particularly P. Albersheim, D . F. Durso, C. T. Handy, B. Leopold, A. Sarko, L. Segal, and A. M . Sookne whose leadership made the 22 sessions of the symposium truly international in scope. In addition, Herman Mark kindly made significant remarks to open the symposium. New Orleans, L A

JETT C. ARTHUR, JR.

March 1, 1977

Organizing Chairman

ix

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

1 Crystal Structures of Oligocellulose Acetates and Cellulose Acetate II R. H. MARCHESSAULT Department of Chemistry, Université de Montréal, Montreal, Québec, Canada H. CHANZY Centre de recherches sur les macromolécules végétales, CNRS, Grenoble, France The crystal structure of a polysaccharide can be obtained by f i r s t determining the crystal structures of its oligosaccharide single crystals. By extrapolatio diagram, the structur the classical approach for polymer structures and some efforts along these lines using powder diffraction data (1) and a single crystal study on cellotetraose (2) have been reported for the cellulose system. Historically, it was extensive work on the cellulose oligosaccharides by Freudenberg (hydrolysis, optical rotation) which provided a proof of structure for cellulose (3) by showing that the behaviour of the oligomer molecules, for which the structure could be established rigorously, extrapolated to the observed properties of cellulose. The present approach aims at the same objective in the area of conformation and packing of the repeating anhydroglucose triacetate in the crystal structure of cellulose triacetate (4). The acetate oligosaccharides of cellulose which relate to the crystal structure of cellulose triacetate (CTA), were chosen for the following reasons: - pure oligosaccharide fractions are readily available; - single crystals of suitable size and perfection can be easily obtained from the acetylated oligomers compared to the unacetylated; - the absence of hydrogen bonding in the system was expected to lead to oligomers which have a conformation close to that of the polymer; - the oligomer crystal structures can be compared with data from a fiber diagram of outstanding quality: cellulose triacetate II; - lamellar single crystals of CTA II yield electron diffraction data which can be used to define the base plane projection of the CTA II crystal. The last argument may be a decisive one in all crystal structure work on oligosaccharides in the future. Ordinary fiber diagrams yield at best 5 to 10 equatorial (hkO) diffraction spots 3

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

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w h i l e t h e e l e c t r o n d i f f r a c t o g r a m from t h e l a m e l l a r s i n g l e c r y s t a l s show a t l e a s t 5 t i m e s a s many hkO d i f f r a c t i o n d a t a . S i n c e hkO d i f f r a c t i o n corresponds t o the F o u r i e r transform o f the u n i t c e l l p r o j e c t e d down t h e f i b e r a x i s , e l e c t r o n d i f f r a c t o g r a m s such a s t h e s e w i l l h e l p i n s o l v i n g t h e p a c k i n g p r o b l e m f o r CTA I I ( 4 ) . F u r t h e r m o r e from t h i s a d d i t i o n a l i n f o r m a t i o n we may e x p e c t much g r e a t e r c e r t a i n t y i n the space group s e l e c t i o n and b e t t e r p r e c i ­ sion i n the c e l l dimensions. The method i s a p p l i c a b l e a l s o i n c a s e s where t h e p o l y s a c c h a r i d e i s h y d r a t e d s i n c e i t i s p o s s i b l e to o b s e r v e d i f f r a c t i o n f r o m t h e h y d r a t e d c r y s t a l even i n t h e vacuum o f t h e e l e c t r o n m i c r o s c o p e by u s i n g a c o o l i n g s t a g e ( 6 ) . The CTA c r y s t a l s t r u c t u r e b e a r s a c l o s e r e l a t i o n t o n a t i v e c e l l u l o s e m o r p h o l o g y . H e t e r o g e n e o u s a c e t y l a t i o n (7) l e a d s t o a p r e s e r v a t i o n o f the n a t i v e c e l l u l o s e m i c r o f i b r i l s and a u n i t c e l l r e f e r r e d t o a s CTA I . S i m p l e h e a t t r e a t m e n t o f t h e i s o l a t e d m i ­ c r o f i b r i l s l e a d s t o a t r a n s f o r m a t i o n i n t o CTA I I m i c r o f i b r i l s . I t has been o b s e r v e d (5 t r a n s f o r m a t i o n remains i n t r a m i c e l l a opment o f a s h i s h - k e b a b s t r u c t u r e . I n c r y s t a l l o g r a p h i c t e r m s i t i s t o be e x p e c t e d t h a t s u c h a t r a n s f o r m a t i o n i m p l i e s a n a n t i p a r a l ­ l e l c h a i n a r r a n g e m e n t i n CTA I I . S i n c e p r e v i o u s s t u d i e s o n t h i s s y s t e m have been i n c o n c l u s i v e (4) i n t h i s r e s p e c t , o n e o f o u r o b ­ j e c t i v e s i s t o s e t t l e the chain p o l a r i t y q u e s t i o n . F i n a l l y , i t has been shown i n a r e c e n t s t u d y (9) t h a t c o n ­ f o r m a t i o n a l a n a l y s i s o f p o l y s a c c h a r i d e c h a i n s i s most r e l i a b l e when c h a i n c o o r d i n a t e s a r e d e r i v e d from a s i n g l e c r y s t a l s t r u c t u ­ re o f t h e d i r e c t l y r e l a t e d d i m e r . T h e p r e s e n t a p p r o a c h w i l l p r o ­ v i d e s u c h d a t a . I t s h o u l d be a p p r e c i a t e d however t h a t c e l l o t r i o s e a c e t a t e (C40O27H54) has a m o l e c u l a r w e i g h t o f 966 w h i c h makes i t the l a r g e s t o l i g o s a c c h a r i d e s t r u c t u r e so f a r undertaken. Further­ more, i t w o u l d a p p e a r d e s i r a b l e t o s o l v e even l a r g e r o l i g o m e r s i n t h e s e r i e s i f o u r o b j e c t i v e s a r e t o be f u l l y a t t a i n e d . E x p e r i m e n t a l . T h e c r y s t a l s t r u c t u r e d e t e r m i n a t i o n o f β-cellobiose o c t a a c e t a t e (G2) h a s been r e p o r t e d ( 1 0 ) . Samples o f B - c e l l o t r i o s e u n d e c a a c e t a t e were o b t a i n e d f r o m D. H o r t o n ( O h i o S t a t e U n i v e r s i t y , Columbus, O h i o ) a n d a r e p a r t o f t h e m a t e r i a l p r e p a r e d by D i c k e y and W o l f r o m ( 1 1 ) . T h e y were d i s ­ s o l v e d i n h o t e t h a n o l w i t h a b o u t 5% w a t e r . A f t e r s l o w e v a p o r a ­ t i o n o f t h e s o l v e n t ( a b o u t two w e e k s ) , l o n g n e e d l e s s u i t a b l e f o r x - r a y s t u d y were o b t a i n e d . T h e s e c r y s t a l s were s i m i l a r i n s h a p e to t h o s e used f o r t h e c e l l o b i o s e o c t a a c e t a t e s t u d y . I n d e t a i l , t h e c r y s t a l s a r e p a r a l l e l e p i p e d s a n d t h e two l o n g e s t axes o f t h e p a r a l l e l e p i p e d are p e r p e n d i c u l a r t o the unique a x i s . The u n i t c e l l a n d s p a c e g r o u p d a t a f o r β - c e l l o b i o s e a c e t a t e and 3 - c e l l o t r i o s e a c e t a t e were d e r i v e d from W e i s s e n b e r g a n d p r e ­ c e s s i o n photographs. T h r e e d i m e n s i o n a l d i f f r a c t i o n was r e c o r d e d f o r β - c e l l o b i o s e a c e t a t e , 3013 d i f f r a c t i o n d a t a were r e c o r d e d a n d t h e s t r u c t u r e was r e s o l v e d by t h e d i r e c t method (10) f o l l o w e d b y a three dimensional refinement.

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

1.

MARCHESSAULT

AND

CHANZY

Crystal

Structures

5

F o r β - c e l l o t r i o s e a c e t a t e (G3) t h e 3396 r e c o r d e d d i f f r a c t i o n d a t a a l l o w e d s o l u t i o n b y t h e d i r e c t method b u t t h e l a t t e r was mo­ d i f i e d (12) t o t a k e a d v a n t a g e o f t h e known c o n f o r m a t i o n o f β-cel1 o b i o s e a c e t a t e and t h e o b v i o u s r e l a t i o n between t h e v a l u e s o f t h e m o l e c u l a r l e n g t h o f t h e two m o l e c u l e s a s d e r i v e d f r o m t h e c dimension o f the u n i t c e l l ( c f . below). T h i s approach i n v o l v e d the g e n e r a t i o n o f c o o r d i n a t e s f o r β-cellotriose a c e t a t e by u s i n g t h e c o o r d i n a t e s o f β - c e l l o b i o s e a c e t a t e and a d d i n g a n o t h e r u n i t t o t h e n o n - r e d u c i n g end w i t h t h e same g e o m e t r y a s t h a t o f t h e nonr e d u c i n g u n i t i n β-cellobiose a c e t a t e . I n t h i s way t h e d i r e c t method was b i a s e d by t h e a c c u m u l a t e d s t e r e o c h e m i c a l knowledge w h i c h we have f o r t h i s s y s t e m and t h e c r y s t a l l o g r a p h i c s o l u t i o n o f a l a r g e r s t r u c t u r e by t h e d i r e c t method was h e l p e d by s o l u t i o n o f t h e p r e v i o u s s t r u c t u r e o f t h e homologous s e r i e s . D e n s i t i e s were m e a s u r e d by t h e c l a s s i c a l f l o a t a t i o n method. R e s u l t s . S i n c e polymer o f making t h e £ a x i s p a r a l l e o u r o l i g o m e r d a t a i n a s l i g h t l y n o n - c o n v e n t i o n a l way f r o m a c r y s t a l l o g r a p h i c p o i n t o f view. T h i s u l t i m a t e l y w i l l permit an e a s i e r c o m p a r i s o n between t h e s i n g l e c r y s t a l d a t a on o l i g o m e r s and t h e f i b e r X-ray data. The n e e d l e shaped c r y s t a l s o f t h e t h r e e o l i g o m e r s seem t o have a s i m i l a r m o r p h o l o g y and m o l e c u l a r o r i e n t a t i o n i n t h e c r y s ­ t a l . Remembering t h a t c i s t h e u n i q u e a x i s i n t h e p o l y m e r s y s t e m , t h e r e l a t i v e s h a p e and o r i e n t a t i o n o f t h e m o l e c u l a r {c) a x i s i n t h e n e e d l e s and l a m e l l a r p o l y m e r s i n g l e c r y s t a l s a r e shown i n F i g . 1. T h e s e d a t a were d e r i v e d f r o m t h e s i n g l e c r y s t a l s t u d i e s o f β-cellobiose a c e t a t e ( 1 0 ) , β-cellotriose a c e t a t e ( 1 2 ) , from e l e c ­ t r o n d i f f r a c t i o n o f CTA I I s i n g l e c r y s t a l s (8) and f i b e r d i f f r a c ­ t i o n d a t a ( 1 4 ) . A c t u a l l y , t h e m o l e c u l a r a x i s f o r G2 i s n o t q u i t e p a r a l l e l t o c_ and d a t a f o r β - c e l l o t r i o s e a c e t a t e a r e n o t s u f f i ­ c i e n t y e t t o e s t a b l i s h whether o r not t h e r e i s a p e r f e c t p a r a l l e l i t y . I n f a c t t h e d i f f e r e n c e between t h e c d i m e n s i o n s f o r G2 and G3 i s 5.4 A w h i c h i s s i g n i f i c a n t l y g r e a t e r t h a n t h e v a l u e o f 5.2 - 5.3 A d e r i v e d f r o m f i b e r d i f f r a c t i o n hence i t a p p e a r s t h a t o n l y f o r t h e s t r u c t u r e o f G3 o r G4 w i l l o n e p r o b a b l y have a s u i t a b l e e q u i v a l e n c e between o l i g o m e r and p o l y m e r . The p e r t i n e n t c r y s t a l s t r u c t u r e d a t a a r e summarized i n T a b l e I . The £ a x i s i n c r e a s e s i n g o i n g f r o m G2 t o G3 by an amount r e l a t e d t o t h e i n c r e m e n t f o r one added monomer, b u t i t i s s l i g h ­ t l y t o o h i g h compared t o t h e f i b e r r e p e a t . T h e b d i m e n s i o n i s r e l a t i v e l y c o n s t a n t and f r o m t h e c r y s t a l s t r u c t u r e o f G2 i t i s known t h a t t h i s i s t h e t h i c k n e s s o f t h e r i b b o n - l i k e G2 m o l e c u l e . The a d i m e n s i o n i s r e l a t e d t o t h e w i d t h o f t h e r i b b o n - l i k e molecule. The t h e o r e t i c a l d e n s i t y f o r G3 i s c l o s e t o what i s o b s e r v e d f o r t h e polymer hence i t may be assumed t h a t t h e same s o r t o f i n t e r c h a i n f o r c e s a r e p r e s e n t i n t h e o l i g o m e r and p o l y m e r . T h e s p a c e g r o u p f o r G3 i s P2] a n d t h e r e a r e two m o l e c u l e s p e r u n i t

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

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ζ \ · The four models a r e : P ^ p a r a l l e l chains o r i e n t e d "up" with a s h i f t of ^c/4; P ~ p a r a l l e l chains o r i e n t e d "down" with a s h i f t of ^c/4; a - - a n t i p a r a l l e l chains with an "up" chain a t (0,0) and a down chain a t (1/2,1/2), with a s h i f t of ^-c/4; and a - a n t i p a r a l l e l chains a f

1 1

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U

ρ / ς

W

C

W

2

2

Refinement Each of the above s t r u c t u r e s i s d e f i n e d by parameters determining the p o s i t i o n and o r i e n t a t i o n of the r i g i d chains and t h e i r pendant -CH 0H groups. The l e a s t squares procedure r e f i n e s these parameters to give the best agreement between the observed and c a l c u l a t e d s t r u c t u r e amplitudes. The seven r e f i n a b l e para­ meters f o r each model a r e : 1) SHIFT, the stagger of the center chain along c with respect to the chain a t the o r i g i n ; 2) φ , the r o t a t i o n of the o r i g i n chain about i t s h e l i x a x i s ; 3) φ , the r o t a t i o n of the center chain about i t s h e l i x a x i s ; 4) χ, the o r i e n t a t i o n of the -CH^OH groups of the o r i g i n c h a i n ; 5) χ , the o r i e n t a t i o n of the -Cfl^OH groups of the center chain; 6) K, a s c a l e f a c t o r f o r comparison of the observed and c a l c u l a t ­ ed s t r u c t u r e amplitudes, and 7) B, the i s o t r o p i c temperature f a c t o r . The refinement w i l l be discussed i n terms of the r e s i d u a l s , which give a measure of the agreement between the observed and c a l c u l a t e d s t r u c t u r e amplitudes. These are d e f i n e d : ?

1

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where F and F a r e the observed and c a l c u l a t e d s t r u c t u r e amplitudes and w i s a weigh assigned t o each observed s t r u c t u r e amplitude. C

Cellulose I The i n i t i a l refinement was done f o r models where both chains had the same o r i e n t a t i o n f o r the CH^OH groups i . e . χ = χ . In l a t e r work i t was found tjiat models with a l l but very small !

American Chemical Society Library

1155 16th St., N.W. Washington, D.C. 20036 In Cellulose Chemistry and Technology; Arthur, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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d i f f e r e n c e s between χ and χ are not compatible with the x-ray data f o r stereochemical requirements. These small d i f f e r e n c e s do not give s i g n i f i c a n t improvement i n the f i t between the observed and c a l c u l a t e d s t r u c t u r e amplitudes, and hence i n the s t r u c t u r e s described below, χ = χ , and the refinement i s f o r 6 v a r i a b l e s . When the models were r e f i n e d against the 36 observed r e f l e c t i o n s only, the r e s u l t i n g R values were R =0.207, R =0.249, R =0.179, and R =0.202. In a l l four r e f i n e d models, the 'planes of the pyranose r i n g s are approximately i n the a c_ plane and the value of SHIFT staggers the g l y c o s i d i c oxygens by ^c/4. The χ value f o r models a^, a^ and P- places the -CH 0H groups near the t g p o s i t i o n , such that 0(2 ;-Η··-0(6) i n t r a m o l e c u l a r and reasonable i n t r a m o l e c u l a r hydrogen bonds can be formed. For model p and χ value places the CH 0H group intermediate between the t£ and _gt p o s i t i o n s , which does not allow f o r hydrogen bond­ i n g of the 0(2)-H groups. S t a t i s t i c a l t e s t s (15) i n d i c a t e the model p^ gives s i g n i f i c a n t l data than model p « Mode a^ on the same b a s i s . Thus models a and p were the most l i k e l y a n t i p a r a l l e l and p a r a l l e l chain models and were considered f o r f u r t h e r refinement. At t h i s stage the unobserved r e f l e c t i o n s were i n c l u d e d i n the refinement where the c a l c u l a t e d s t r u c t u r e amplitude was l a r g e r than the t h r e s h o l d value, under these circumstances. A weighting scheme of w=l f o r observed and w=l/2 f o r unobserved r e f l e c t i o n s was used at t h i s p o i n t . The f i n a l r e s i d u a l s were R =0.233, R =0.299, R" =0.215 and R" =0.270. A p p l i c a t i o n o f the Hamilton s t a t u l t i c a l t e s t t l 6 ) t o these data i n d i c a t e a preference f o r the p a r a l l e l chain model (p-) at a s i g n i f i c a n c e l e v e l of 0.5%, i . e . , the p a r a l l e l model i s p r e f e r e d by a f a c t o r o f more than 200 to 1. The ab and ac p r o j e c t i o n s of the s t r u c t u r e are shown i n F i g u r e 4. The s t r u c t u r e has no bad contacts on the b a s i s of accepted stereochemical c r i t e r i a . The r e f i n e d value of φ i s 0.4° from that of φ , and the Hamilton t e s t i n d i c a t e s that the c o n s t r a i n e d model with φ = φ i s i n as good agreement with the data as the model w i t h φ as a separate v a r i a b l e . The f i n a l value of φ=19.4° ( a r b i t r a r y o r i g i n ) p l a c e s the chains so that the "planes" of the r i n g s are approximately i n the ac plane (see F i g u r e 4). The r e f i n e d value of SHIFT=0.266c:. T h i s d e v i a t i o n from a p e r f e c t quarter stagger i s not unexpected: the weak 002 m e r i d i o n a l would be absent f o r a stagger of 0.25c. The r e f i n e d value of χ=80.3° p l a c e s the CH 0H groups w i t h i n ^20° of the t£ p o s i t i o n (χ=60°). T h i s o r i e n t a t i o n d i d not s h i f t s i g n i f i c a n t l y from that r e f i n e d f o r the observed r e f l e c t i o n s only. The i s o t r o p i c temperature f a c t o r i s B=2.50. 1

2

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1

9

2

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2 >

2

2

f

f

P

1

1

1

2

Hydrogen Bonding i n C e l l u l o s e I The hydrogen bonding network i n c e l l u l o s e I i s shown i n Figure 4c. A l l the hydroxyl groups form hydrogen bonds with acceptable bond lengths and angles. In a d d i t i o n t o the

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•rS .

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0(3)-Η···0(5 ) hydrogen bond o f l e n g t h 2.75Â d e f i n e d i n the model, there i s a second i n t r a m o l e c u l a r bond: 0(2 )-Η···06 of l e n g t h 2.87Â. These i n t r a m o l e c u l a r bonds run on both s i d e s of the c e l l u l o s e chain. In a d d i t i o n , there i s an i n t e r c h a i n hydrogen bond between 0(6)-H and 0(3) of the next chain along the a a x i s ; t h i s bond i s 2.79Â i n length. No hydrogen bonding occurs along the u n i t c e l l d i a g o n a l s , r a t h e r the hydrogen bonding i s a l l i n the 020 planes, and the s t r u c t u r e i s seen as a s e r i e s of hydrogen bonded sheets o f chains, where s u c c e s s i v e sheets are staggered and a l l the chains have the same sense. !

Cellulose I I For c e l l u l o s e I I , study of molecular models i n d i c a t e d that the two chains probably have d i f f e r e n t conformations f o r the -CH^OH groups, and hence a l l seven v a r i a b l e s were considered i n the refinement. Refinemen gave r e s i d u a l s of R ^0.254 Of these four modell, only a^ ?s s t e r e o c h e m i c a l ^ acceptable, and t h i s gives the lowest r e s i d u a l . Model P^ contains four bad contacts and models p and a^ c o n t a i n two each (The worst o f these contact d i s t a n c e s are nonbonded oxygen-oxygen d i s t a n c e s of 2.17Â, 2.05Â and 2.11Â i n p^, p and a r e s p e c t i v e l y , which a r e t o t a l l y unacceptable). E f f o r t s t o remove these contacts by i n c o r p o r a t i o n o f non-bonded c o n s t r a i n t s were not s u c e s s f u l : the R values increased t o R =0.272, R =0.219 and R=0.230, but although the contact d i s t a n c e s werl lengthened, the stereochemical c r i t e r i a were s t i l l not s a t i s f i e d . A l l four models were then r e f i n e d against the t o t a l observed and unobserved data, as was done f o r c e l l u l o s e I . The bad contacts f o r models p^, p and a^ were not removed and these s t r u c t u r e s remain unacceptable. For model a , a short oxygenoxygen contact o f 2.49Â was introduced, but t h i s was e r r a d i c a t e d with an a p p r o p r i a t e nonbonded c o n s t r a i n t . The f i n a l R values were R =0.219 and R"=0.167. Thus an a n t i p a r a l l e l chain model i s proposed f o r c e l l u l o s e I I . The ab and ac p r o j e c t i o n s o f the s t r u c t u r e are shown i n F i g u r e 5a and 5b. The r e f i n e d values o f φ and φ o r i e n t the chains so that the r i n g s a r e almost p a r a l l e l t o the ac planes, although not q u i t e so c l o s e as f o r c e l l u l o s e I. The r e l a t i v e stagger o f the chains i s 0.216c. The s i d e chains have d i f f e r e n t conformations f o r the corner "up" chains (through 0,0), χ=186.3°, p l a c i n g the -CH^OH group c l o s e t o the £t p o s i t i o n (χ=180°), f o r the center "down" chains (through 1/2,1/2), = 7 0 . 2 ° , p l a c i n g these -CH 0H groups c l o s e t o the t £ p o s i t i o n (χ=60°). The r e f i n e d i s o t r o p i c temperature f a c t o r i s B=19.96. 2

2

2

2

2

?

f

1

f

x

2

Hydrogen Bonding i n C e l l u l o s e I I The hydrogen bonding network i n c e l l u l o s e I I i s more complex

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than i n c e l l u l o s e I , and i s shown i n F i g u r e 5b-e. A l l o f the hydroxyl groups form hydrogen bonds with acceptable bond lengths and angles. Each chain has the 0(3)-Η···0(5') i n t r a m o l e c u l a r bond o f l e n g t h 2.69Â, as d e f i n e d i n the model. The -CI^OH groups of the center "down" chains a r e c l o s e t o the t& p o s i t i o n and these chains have a second i n t r a m o l e c u l a r 0(2)-Η···0(6) bond o f l e n g t h 2.73Â. The 0(6)-H group of t h i s chain a l s o forms an i n t e r m o l e c u l a r 0(6)-Η···0(3) bond of l e n g t h 2.67Â t o the next ("down") chain along the a a x i s , with a r e s u l t that the "down" chains form hydrogen bonded sheets i n the 020 planes s i m i l a r to those i n c e l l u l o s e I. The sheet of down chains i s shown i n Figure 5c. For the "up" corner chains the -CH^OH groups a r e c l o s e t o the £t p o s i t i o n , and form 0(6)-Η···0(2) i n t e r m o l e c u l a r bond of length 2.73Â to the next chain along the a a x i s , again i n the 020 plane. The sheet o f "up" chains i s shown i n Figure 5d. For the _gt_ conformation th molecular bond, but i 0(2)-Η···0(2 ) bond o f l e n g t h 2.77Â t o the next "down" chain on the d i a g o n a l along the 110 plane, as shown i n F i g u r e 5e. Hence the c e l l u l o s e I I s t r u c t u r e i s an a r r a y o f staggered hydrogen bonded sheets. The chain sense i s the same w i t h i n the sheets, but the sheets have a l t e r n a t i n g p o l a r i t i e s and are hydrogen bonded together along the long diagonal o f the u n i t c e l l . ?

Discussion A p a r a l l e l chain s t r u c t u r e f o r c e l l u l o s e I e f f e c t i v e l y r u l e s out chain f o l d i n g during s y n t h e s i s o f c e l l u l o s e m i c r o f i b r i l s . Native m i c r o f i b r i l s are t h e r e f o r e shown t o be extended-chain polymer s i n g l e c r y s t a l s , which are h i g h l y d e s i r a b l e s t r u c t u r e s i n terms o f mechanical p r o p e r t i e s . For c e l l u l o s e I I , the chains are a n t i p a r a l l e l , which i s c e r t a i n l y compatible with f o l d e d chains, although there i s no d e f i n i t e evidence f o r such a c r y s t a l l i z a t i o n process. C e l l u l o s e I I i s the s t a b l e polymorphic form, i n that i t i s p o s s i b l e to convert form I t o form I I , but not v i c a v e r s a . The c e l l u l o s e I I s t r u c t u r e contains the a t t r a c t i v e f e a t u r e o f a hydrogen bond between adjacent sheets of chains, which may account f o r t h i s s t a b i l i t y . In a d d i t i o n the hydrogen bonds have an average length o f 2.72Â i n c e l l u l o s e I I , as compared t o 2.80Â i n c e l l u l o s e I. The r e s o l u t i o n a t t a i n a b l e i n the x-ray r e f i n e ments i s not s u f f i c i e n t t o determine i n d i v i d u a l bond l e n g t h s , but t h i s d i f f e r e n c e i n the average bond lengths i s probably s i g n i f i cant, and r e f l e c t s a t i g h t e r chain packing i n c e l l u l o s e I I , c o n s i s t a n t with the higher s t a b i l i t y o f t h i s form. Of the v a r i o u s p o s s i b i l i t i e s f o r the chain p o l a r i t i e s i n the two forms, the p a r a l l e l I - a n t i p a r a l l e l I I s o l u t i o n seems t o be the most reasonable. R e s u l t s from x-ray and packing analyses by Sarko and Muggli (17) a l s o favor these p o l a r i t i e s . I f

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Figure 5. Projections of the antiparallel chain model for cellulose II. (a) Projection perpendicular to the ac plane. The center "down" chains (dark) are staggered with respect to the corner "up" chains, (b) Projection perpendicular to the a b plane along the fiber axis. The 0(2)-Η· · -0(2') hydrogen bond along the 110 planes is indicated.

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Figure 5. (c) Hydrogen bonding network in the 020 plane for the center "down" chains. These sheets are very simihr to those for cellulose I. (d) Hydrogen bonding network in the 020 plane for the corner "up" chains, (e) Hydrogen bonding between antiparallel chains in the 110 plane.

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c e l l u l o s e I was an array o f a n t i p a r a l l e l chains, i t i s d i f f i c u l t to see why these would not adopt the c e l l u l o s e I I l a t t i c e . A consequence o f the p a r a l l e l chain s t r u c t u r e , however, i s that i t r e q u i r e s a r e l a t i v e l y complex b i o s y n t h e s i s mechanism w i t h p o l y m e r i z a t i o n followed very c l o s e l y by c r y s t a l l i z a t i o n . I f the two steps were to be w e l l separated then a r a y o n - l i k e s t r u c t u r e would be produced. The r e s u l t s f o r c e l l u l o s e I I were obtained f o r rayon. There i s no reason to b e l i e v e they do not apply to mercerized c e l l u l o s e , although we are c u r r e n t l y r e i n v e s t i g a t i n g the l a t t e r s t r u c t u r e . The m e r c e r i z a t i o n process i n v o l v e s s w e l l i n g i n c a u s t i c soda s o l u t i o n and i s accompanied by only a s m a l l change i n l e n g t h . Chanzy e t al.(18) have r e c e n t l y shown that shish-kabob s t r u c t u r e s of low molecular weight c e l l u l o s e with the form I I l a t t i c e w i l l e p i t a x i a l l y c r y s t a l l i z e on c e l l u l o s e I f i b e r s . Such e p i t a x i a l c r y s t a l l i z a t i o n i s to be expected s i n c e h a l f of the sheets i n c e l l u l o s e I I a r e the sam proceeds showly and neve c e l l u l o s e I could maintain the f i b e r dimensions and serve as a template f o r c r y s t a l l i z a t i o n of c e l l u l o s e I I . Acknowledgements This work was supported by N.S.F. Grant No. DMS 75-01028 and N.I.H. Career Development Award No. AM 70642 (to J.B.).

Abstract The crystal structures of native and regenerated celluloses have been determined using x-ray diffraction and least squares refinement techniques. Both structures have monoclinic unit cells containing sections of two chains with 2 screw axes. Models containing both parallel and antiparallel chains were refined in each case by comparison with the x-ray intensities for Valonia cellulose I and rayon cellulose II. For native cellulose, the results show a preference for a system of parallel chains (i.e. all the chains have the same sense). The refinement orients the -CHOH groups close to the tg conformation such that an 0(6)···Η-0(2') intramolecular hydrogen bond is formed. The structure also contains an 0(3)-Η···0(3) intermolecular bond along the a axis. All these bonds lie in the 020 plane, and the native structure is an array of staggered hydrogen bonded sheets. In contrast, for regenerated cellulose the only acceptable structure contains antiparallel chains (i.e. the chains have alternating sense). The CHOH groups of the corner chain are oriented close to the gt position; those of the center chain are close to the tg position. Both center and corner chains have the 0(3)-Η···0(5') intramolecular bond and the center chain also has an 0(2')-Η···0(6) intramolecular bond. Intermolecular hydrogen bonding occurs along the 020 planes: 0-(6)-Η···0(2) 1

2

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bonds for the corner chains and 0(6)-Η···0(3) bonds for the center chains, and also along the 110 planes, with a hydrogen bond between 0(2)-H of the corner chain and 0(2') of the center chain. The major consequence of these structures is that native cellulose is seen as extended chain polymer single crytals. The cellulose II structure is compatible with regular chain folding, although there is no direct evidence for such folding. Literature Cited 1. Meyer, K.H., and Misch, L. Helv. Chim. Acta. (1937) 20, 232-244. 2. Wellard, N.J., J. Polymer Sci. (1954) 13, 471-476. 3. Jones, D.W., J. Polymer Sci. (1958) 32, 371-394. 4. Jones, D.W., J. Polymer Sci. (1960) 42, 173-188. 5. Liang, C.Y. and Marchessault, R.H., J. Polymer Sci. (1959) 37, 385-395. 6. Frey-Wyssling, Α., 7. Hermann, P.H., DeBooys 102, 169-180. 8. Rao, V.S.R., Sundararajan, P.R., Ramakrisnan, C., and Ramachandan, G.N., in Conformation of Biopolymers, (G.N. Ramachandran, ed.), (1967), Vol. 2, p. 271. Academic Press, New York. 9. Arnott, S., and Wonacott, A.J., Polymer (1966) 7, 157-166. 10. Gardner, K.H., and Blackwell, J., Biopolymers (1974) 14, 1975-2001. 11. Kolpak, F.J., and Blackwell, J. (1976) 9, 273-278. 12. Cella, R.J., Lee, Β., and Hughes, R.E. Acta Cryst. (1970) A26, 118-124. 13. Honjo, G., and Watanabe, Μ., Nature (1958) 181, 326-328. 14. Arnott, S., and Scott, W.E., J. Chem. Soc. Perkin. Trans. II (1972) 324-335. 15. Sundaratingham, Μ., Biopolymers (1966) 6, 189-213. 16. Hamilton, W.C., Acta Cryst. (1965) 18, 502-510. 17. Sarko, Α., and Muggli, R. Macromolecules (1974) 7, 486-494. 18. Chanzy, Η., Roche, Ε., and Vuong, R. Appl. Polymer Sci. Symposia (in press).

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

5 X-Ray Diffraction by Bacterial Capsular Polysaccharides: Trial Conformations for Klebsiella Polyuronides K5, K57, and K8 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 Interest i nthepolyuronides t o t h e e a r l y 1970's whe 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

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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 β-D-glucuronic a c i d r e s i d u e 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

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

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

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.

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

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 :

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

5.

ATKINS

Figure

4.

E T A L .

Projections

Bacterial

Capsular

61

Folysaccharides

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

polysaccharide.

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

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 maximu r e p e a t , f o l l o w i n g t h e metho 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

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

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.

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

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

L

9

-12

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

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

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.

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

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 glucose A l l f o u r monosaccharides are expected to e x i s t i schematic diagram r e p r e s e n t i n 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

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

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.

Klebsiell

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.

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

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 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

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

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

-120

Θ,

Θ,

(a)

(b)

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

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

linkage

Gle()(2)-GalO(2)

[GleO