Multidisciplinary Approaches to the Structures of Model Compounds

Chapter 3

Multidisciplinary Approaches to the Structures of Model Compounds for Cellulose II 1

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Bernard Henrissat , Serge Perez , Igor Tvaroska , and William T. Winter

Centre de Recherches sur les Macromolécules Végétales, Centre National de la Recherche Scientifique, B.P. 68, 38402 Saint Martin d'Hères Cedex, France

The 3-dimensional structural features of macromolecules such as cellulose are often preserved in oligomeric model compounds which provide the opportunity for more accurate characterization through the occurrence of larger crystalline domains. We have been examining cellobiose, methyl β-D-cellobioside and cellotrioside, and cellotetraose using X-ray and electron diffraction, CP-MAS NMR spectro scopy and computer modeling with the PCILO and MM2 algorithmns. The geometry of the pyranose ring in oligomers was shown by modeling to vary with linkage conformation. Thus, selecting either the cellobiose or methyl β-D-cellobioside conformation alters the geometry of the ring selected by the program to have minimal energy. When these different ring geometries are incorporated in the molecular mechanics program, they influence, in turn, the conformational energy surface, even altering the relative levels of minima. X-ray and electron diffraction studies have established the packing similarity of both methyl β-D-cellotrioside and cellotetraose to cellulose II. Finally, we have examined proposals that correlate chemical shift data from C CP-MAS NMR spectroscopy with conformations and with differences in the charge distribution in the individual conformers. 13

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Current address: Institut National de la Recherche Agronomique, rue de la Geraudière, F-44072 Nantes, France Correspondence should be addressed to this author. On a leave of absence from the Institute of Chemistry, Center of Chemical Research of the Slovak Academy of Sciences, 84238, Bratislava, Czechoslovakia On a leave of absence from the Department of Chemistry, Polytechnic University, 333 Jay Street, Brooklyn, NY 11201

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0097-6156/87/0340-0038$08.25/0 © 1987 American Chemical Society

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Model Compounds for Cellulose II

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Introduction Characteristics of macromolecules are often preserved in oligomeric model compounds. As one example, X-ray powder d i f f r a c t i o n p a t t e r n s (1) and t h e i n f r a r e d s p e c t r a (2) o f t h e c e l l o d e x t r i n s beginning with c e l l o t e t r a o s e are very s i m i l a r to those o f cellulose I I . Such a similarity implies a close r e l a t i o n s h i p i n t h e i r molecular structures. W h i l e t h e s e model compounds s h o u l d p e r m i t more a c c u r a t e c h a r a c t e r i z a t i o n because o f t h e i r m a c r o s c o p i c , s i n g l e - c r y s t a l domains ( 3 ) , c e l l o d e x t r i n s form non-centrosymmetric c r y s t a l s and a n a l y s i s o f t h e i r diffraction data i s a serious challenge. S i n c e s i n g l e - c r y s t a l d i f f r a c t i o n i s the o n l y method t h a t p r o v i d e s bond l e n g t h s , bond a n g l e s and the c h a r a c t e r i s t i c geometry, i t i s the u l t i m a t e s t r u c t u r a l method. On t h e o t h e r hand, polymer X-ray s t u d i e s depend on a p r o p o s e d model, and hence a r e n o t a b s o l u t e . Therefore, i t i s important to r e f e r to s i n g l e c r y s t a l s t u d i e s f o r d a t a on which t o base c e l l u l o s e models. The c r y s t a l and m o l e c u l a r s t r u c t u r e s o f c e l l o b i o s e (4) and m e t h y l 6 - D - c e l l o b i o s i d e (5) have been r e p o r t e d . We a l s o w i s h t o know the d e t a i l e d s t r u c t u r e s o f the h i g h e r o l i g o m e r s , which a r e more a n a l o g o u s t o c e l l u l o s e I I . A f t e r p i o n e e r i n g work on c e l l o t e t r a o s e ( 6 , 7 ) , P o p p l e t o n and Gatehouse (8) proposed a larger, triclinic unit cell, containing 2 m o l e c u l e s . The f u l l s t r u c t u r e i s n o t y e t d e t e r m i n e d . R e c e n t l y , new approaches such as m o l e c u l a r mechanics have been a p p l i e d t o 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 n a t i v e and r e g e n e r a t e d c e l l u l o s e s . Among t h e s e , t h e most p r o m i s i n g may be "magic a n g l e s p i n n i n g " f o r r e c o r d i n g t h e NMR s p e c t r a o f s o l i d c e l l u l o s e samples (9-17) and cellodextrins (10,18-19). The characteristics of spectra from cellulose I I were f o u n d t o be very s i m i l a r to o l i g o m e r s o f DP > 4 (10,11). In a d d i t i o n , c o n c l u s i o n s were drawn regarding the asymmetric unit in the crystalline region. Nevertheless, s e v e r a l p o i n t s are s t i l l obscure. The major aim o f the p r e s e n t work was t o c h a r a c t e r i z e c r y s t a l l i n e c e l l o d e x t r i n s u s i n g s e v e r a l methods i n o r d e r t o r e a c h a c o n s i s t e n t description o f the structural f e a t u r e s and to e s t a b l i s h the workability of each approach to the more complex case of cellulose. M a t e r i a l s and

Methods

Nomenclature. The numbering o f t h e atoms and t o r s i o n a n g l e s o f i n t e r e s t f o r c e l l o b i o s e i s shown i n F i g . 1. Numbering p r o c e e d s from the n o n r e d u c i n g end (unprimed) t o the r e d u c i n g end ( p r i m e d ) . The s i g n s o f the t o r s i o n a n g l e s agree w i t h t h e r u l e s o f the IUPAC-IUB Commission o f B i o c h e m i c a l Nomenclature ( 2 0 ) . The t o r s i o n a n g l e s o f i n t e r e s t a r e d e f i n e d as f o l l o w s : $ : 0 {05 - CI - 01 - C4'} V : 0 {CI - 01 - C4'- C5'} The c o n f o r m a t i o n o f t h e p r i m a r y h y d r o x y l group a t C6 (y) is r e f e r r e d t o as e i t h e r g a u c h e - t r a n s , gauche-gauche o r t r a n s - g a u c h e (21). In t h i s t e r m i n o l o g y , the t o r s i o n a n g l e : Q {05 - C5 - C6}06 i s s t a t e d f i r s t , t h e n t h e t o r s i o n a n g l e 0{C4 - C5 - C6 - 06}. The u n i t c e l l d i m e n s i o n s o f c e l l u l o s e I I , as d e t e r m i n e d from X-ray and electron diffraction s t u d i e s (22-25) on Rayon, Fortisan,

THE STRUCTURES OF CELLULOSE

F i g u r e 1. Schematic the atom l a b e l i n g and

representation of c e l l o b i o s e , along the t o r s i o n a l a n g l e s o f i n t e r e s t .

with

3. HENRISSAT ET AL.


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m e r c e r i z e d c o t t o n and s i n g l e c r y s t a l s a r e : a = 8.01 , b = 9.04 , c = 10.36 R and y = 117.1°. F o l l o w i n g the most accepted c r y s t a l l o g r a p h i c models (22, 24), the u n i t c e l l requires two c e l l u l o s e c h a i n s packed w i t h a n t i p a r a l l e l p o l a r i t y . Sample P r e p a r a t i o n . C e l l o b i o s e was p u r c h a s e d from F l u k a Company. Samples were d i s s o l v e d i n water and c r y s t a l l i z a t i o n r e s u l t e d from slow d i f f u s i o n o f e t h a n o l . Methyl 3 - D - c e l l o b i o s i d e , a g i f t from Dr. M. V i n c e n d o n (Grenoble, F r a n c e ) , was d i s s o l v e d i n h o t methanol and c r y s t a l l i z e d by s l o w l y c o o l i n g the s o l u t i o n . H e n d e c a - O - a c e t y l c e l l o t r i o s e and t e t r a d e c a - O - a c e t y l c e l l o t e t r a o s e were o b t a i n e d by acetolysis of cotton c e l l u l o s e according to Wolfrom e t a l . (26) and were p u r i f i e d by p r e p a r a t i v e HPLC. M e t h y l deca-O-acetyl3 - D - c e l l o t r i o s i d e was prepared a c c o r d i n g t o the method d e s c r i b e d by Takeo e t a l . (27). D e a c e t y l a t i o n o f the a c e t y l a t e d s u g a r s was a c h i e v e d i n methanol c o n t a i n i n g c a t a l y t i c amounts o f sodium m e t h y l a t e . The h i g h p u r i t y o f the samples and t h e i r anomeric c o n f i g u r a t i o n were c o n f i r m e d by h i g h - r e s o l u t i o n solution C NMR. S o l i d S t a t e NMR. The samples under i n v e s t i g a t i o n were o b t a i n e d from c r y s t a l s which were ground i n t o homogeneous powder. In each c a s e , wide a n g l e X-ray s c a t t e r i n g diagrams were r e c o r d e d i n o r d e r to c o n f i r m the u n i t c e l l d i m e n s i o n s p r e v i o u s l y f o u n d . Solid state C NMR s p e c t r a were r e c o r d e d a t 50.3 MHz with a B r u k e r CXP-200 s p e c t r o m e t e r e q u i p p e d w i t h a Doty p r o b e . P r o t o n 90° p u l s e w i d t h s were 4 us, and c r o s s - p o l a r i z a t i o n times were 1 ms. M a t c h i n g c o n d i t i o n s were checked w i t h an adamantane s t a n d a r d . The magic a n g l e was s e t by m o n i t o r i n g the Br NMR spectrum o f KBr (28) . Sample c o n t a i n e r s were made o f aluminum o x i d e w i t h K e l - F caps and were spun a t 2.5-3.5 KHz. C h a r a c t e r i s t i c a l l y , 200-10000 a c q u i s i t i o n s were o b t a i n e d p e r spectrum, w i t h r e c y c l e time o f 4 s. A l l peaks i n the s p e c t r a were r e f e r e n c e d t o the peak o f l i n e a r p o l y e t h y l e n e (33.6 ppm). A s m a l l amount o f p o l y e t h y l e n e was added t o each sample (29). 13

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C o n f o r m a t i o n a l A n a l y s i s C a l c u l a t i o n s . PCILP method : The s t a n d a r d v e r s i o n o f the s e m i e m p i r i c a l quantum-chemical method (30, 31) adopted for the optimization of the geometry according to Powell-Zangwill s (32, 33) a l g o r i t h m t h r o u g h bond l e n g t h s , bond angles and t o r s i o n a n g l e s was a p p l i e d . (PCILO : P e r t u r b a t i v e Configuration I n t e r a c t i o n with L o c a l i z e d O r b i t a l s ) . F o r c e F i e l d method : MM2CARB i s the MM2 ( M o l e c u l a r Mechanics) F o r c e F i e l d program (34) m o d i f i e d w i t h the J e f f r e y - T a y l o r a c e t a l segment p a r a m e t e r s (35) and i n t r a m o l e c u l a r hydrogen b o n d i n g (36) . I t was u t i l i z e d , f o l l o w i n g the s t r a t e g y o u t l i n e d by T v a r o s k a and P e r e z (37). To o b t a i n b e t t e r agreement between c a l c u l a t e d C-0 bond lengths and those observed by X-ray or neutron diffraction methods, the following equilibrium bond lengths for bond s t r e t c h i n g energy were used : Cl-05 from 1.396 t o 1.422 S, C l - 0 1 from 1.380 t o 1.388 8, C5-05 from 1.412 t o 1.420 ft , and C4-01 from 1.388 t o 1.415 &. " E m p i r i c a l " method; (PFOS : P o t e n t i a l F u n c t i o n s Oligosaccharide S t r u c t u r e s ) . The p o t e n t i a l energy i s c a l c u l a t e d by i n c l u d i n g the 1


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partitioned contributions arising from the van der Waals, t o r s i o n a l and hydrogen bond c o n t r i b u t i o n s . The van d e r Waals i n t e r a c t i o n s a r e e v a l u a t e d by u s i n g 6-12 p o t e n t i a l f u n c t i o n s w i t h t h e p a r a m e t e r s p r o p o s e d by S c o t t and Scheraga ( 3 8 ) . A t h r e e - f o l d p o t e n t i a l i s used f o r r o t a t i o n about t h e a g l y c o n bond 01-C4* w i t h a b a r r i e r o f 1.0 k c a l / m o l . F o r r o t a t i o n about g l y c o s i d i c bond C l - 0 1 , t h e i n t r a m o l e c u l a r mechanism r e s p o n s i b l e f o r exo-anomeric effect, i s taken into account u s i n g the p o t e n t i a l function p r o p o s e d by T v a r o s k a (39); a t h r e e - f o l d r o t a t i o n a l b a r r i e r o f 1.0 k c a l / m o l i s a l s o i n c l u d e d . Hydrogen bond e n e r g y i s computed by an empirical expression : V = 33.14 (R - 2.55) (R - 3.05) where R i s t h e d i s t a n c e between oxygen atoms which s h o u l d l i e between 2.55 and 3.05 A. No e l e c t r o s t a t i c i n t e r a c t i o n i s t a k e n i n t o a c c o u n t . The energy map i s c a l c u l a t e d as a f u n c t i o n o f $ and y a t i n t e r v a l s o f 5 ° . With respect t o the r e l a t i v e energy minimum, i s o - e n e r g y c o n t o u r s a r e drawn by interpolation at 1 kcal/mol increments. Helical arrangements are customarily d e s c r i b e d i n terms o f a s e t o f h e l i c a l p a r a m e t e r s ( n, h ) , n b e i n g the number o f r e s i d u e s p e r t u r n o f t h e h e l i x and h b e i n g t h e t r a n s l a t i o n a l o n g t h e h e l i x a x i s . These p a r a m e t e r s a r e c a l c u l a t e d f o l l o w i n g o u r a l g o r i t h m r e p o r t e d p r e v i o u s l y (40) . H B

D i f f r a c t i o n and r e l a t e d Methods. Methyl 8-D-cellotrioside was d i s s o l v e d i n water (10 m g . m l ) . C r y s t a l l i z a t i o n i n t o hexagonal p l a t e l e t s was a c h i e v e d by a slow d i f f u s i o n o f e t h a n o l . A c r y s t a l o f d i m e n s i o n s c a . 0.5 x 0.5 x 0.1 mm was mounted on a Nonius CAD4 d i f f r a c t o m e t e r . The r a d i a t i o n was n i c k e l - f i l t e r e d Cu Ka. The unit-cell d i m e n s i o n s were o b t a i n e d a s p a r t o f t h e a l i g n m e n t p r o c e s s on t h e d i f f r a c t o m e t e r by a l e a s t - s q u a r e s f i t t o t h e s e t t i n g o f 20 w e l l - c e n t e r e d r e f l e x i o n s . The m o n o c l i n i c space group P 2 was a s s i g n e d , b a s e d upon t h e s y s t e m a t i c absences : 0 k 0, k ^ 2n. 431C i n d e p e n d e n t r e f l e x i o n s were measured, o f which 616 were a s s i g n e d z e r o w e i g h t as t h e n e t c o u n t o f each was l e s s than 1.99 (I) where a (I) i s t h e s t a n d a r d d e v i a t i o n e s t i m a t e d from c o u n t i n g s t a t i s t i c s . Because o f t h e low v a l u e o f t h e a b s o r p t i o n coefficient, no a b s o r p t i o n c o r r e c t i o n was a p p l i e d . Scattering factors were obtained from International Tables f o r X-ray c r y s t a l l o g r a p h y ( 4 1 ) . The MULTAN computer program (42) was used f o r n o r m a l i z a t i o n and a t t e m p t s t o s o l v e t h e s t r u c t u r e t h r o u g h d i r e c t methods. _ 1

1

C r y s t a l l i z a t i o n o f c e l l o t e t r a o s e from aqueous s o l u t i o n was done by slow d i f f f u s i o n o f e t h a n o l . C r y s t a l s t h i n enough f o r e l e c t r o n diffraction were prepared by epitaxial crystallization of c e l l o t e t r a o s e onto VaIonia m i c r o f i b r i l s i n a s o l u t i o n c o n t a i n i n g 1% o f c e l l o t e t r a o s e i n a 3:2 m i x t u r e o f e t h a n o l i n water. Crystallization took place within a few weeks, at room temperature. A shish-kebab type o f s t r u c t u r e (43) was o b t a i n e d from which l a m e l l a r fragments o f c e l l o t e t r a o s e c r y s t a l s c o u l d be detached by m i l d sonication. The fragments were mounted on c a r b o n - c o a t e d g r i d s and were o b s e r v e d on a P h i l i p s EM 400T E l e c t r o n M i c r o s c o p e o p e r a t i n g a t 120 kV. A f u l l d e t e r m i n a t i o n o f the u n i t - c e l l p a r a m e t e r s was a c h i e v e d t h r o u g h a s e r i e s o f t i l t e x p e r i m e n t s and subsequent a n a l y s i s o f t h e r e s u l t i n g electron d i f f r a c t o g r a m s (Roche, E . ; u n p u b l i s h e d d a t a ) .

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About 1 cm o f h i g h l y c r y s t a l l i n e powdered c e l l o t e t r a o s e was used t o o b t a i n a n e u t r o n d i f f r a c t i o n p a t t e r n from t h e h i g h r e s o l u t i o n D1A machine a t t h e I n s t i t u t e Laue L a n g e v i n i n G r e n o b l e . S i n c e t h e m a t e r i a l c o u l d n o t be d e u t e r a t e d , t h e e f f e c t i v e a b s o r p t i o n was important, as was t h e " i n c o h e r e n t " background s c a t t e r i n g . In o r d e r t o o b t a i n good s t a t i s t i c s i n t h e background r e g i o n , o n l y a s i n g l e powder p a t t e r n was c o l l e c t e d i n t h e whole o f o u r a l l o c a t e d t h r e e d a y s . We had hoped t o be a b l e t o e x t r a c t a t l e a s t 50 Bragg intensities from such a p a t t e r n by u s i n g a r e l a t i v e l y long wavelength o f 3 A t o s p r e a d o u t t h e lower o r d e r s o v e r t h e whole a n g u l a r r a n g e . However, most o f t h e Bragg i n t e n s i t y goes i n t o s i x s t r o n g peaks, w i t h t h e r e m a i n d e r a l l v e r y s m a l l and b a r e l y above the background l e v e l . F u r t h e r m o r e , i t appears t h a t t h e w i d t h s o f the l i n e s may depend on t h e (h k 1) d i r e c t i o n i n t h e s t r u c t u r e : n e a r 2 0= 8 5 ° , n e i g h b o r i n g l i n e s have v e r y d i f f e r e n t w i d t h s . T h i s e f f e c t , presumably due t o d i r e c t i o n a l d i f f e r e n c e s i n s h o r t range crystalline order, complicates the a p p l i c a t i o n of profile r e f i n e m e n t methods t o r e s o l v e i n d i v i d u a l r e f l e x i o n s . Linked-Atom-Least-Squares Procedure. Molecular models having s t a n d a r d bond l e n g t h s , bond a n g l e s and r i n g c o n f o r m a t i o n s (43) were generated using the linked-atom-least-squares method p r e v i o u s l y d e s c r i b e d ( 4 4 ) . In t h e p r e s e n t a p p l i c a t i o n o f t h i s method f o r non-helical structures, the v a r i a b l e parameters include a l l the t o r s i o n angles and valence angles about g l y c o s i d i c l i n k a g e s , and t h o s e d e f i n i n g t h e orientations of r o t a t a b l e s u b s t i t u e n t s , n o t a b l y the primary h y d r o x y l groups. F o r each m o l e c u l e , two v a r i a b l e p a r a m e t e r s , u and v d e f i n e t h e p o i n t where the molecular axis intersects the orthogonal c r y s t a l l o g r a p h i c p l a n e o f t h e u n i t c e l l ; a d d i t i o n a l p a r a m e t e r s |i and define the o r i e n t a t i o n about, and r e l a t i v e translation p a r a l l e l t o t h a t a x i s . Any o r a l l o f t h e s e p a r a m e t e r s may be v a r i e d so as t o m i n i m i z e t h e f o l l o w i n g f u n c t i o n :

Q = I Wm ( oFm - Fm ) + S Z e. The f i r s t summation i s used t o o p t i m i z e agreement between o b s e r v e d s t r u c t u r e a m p l i t u d e s , oFm, w i t h t h o s e c a l c u l a t e d from t h e model, Fm a f t e r s c a l i n g by a f a c t o r 1/k. In c a l c u l a t i n g Fm, a l l atoms were assumed t o v i b r a t e i s o t r o p i c a l l y and 4 A was a s s i g n e d a s the v a l u e o f B i n t h e a t t e n u a t i o n f a c t o r , e x p ( - B s i n OA ) . U n i t w e i g h t s , Wm, were a s s i g n e d t o a l l measurable r e f l e c t i o n s w i t h i n a g i v e n r e s o l u t i o n s p h e r e . The second term i s used t o ensure t h e s t e r e o c h e m i c a l a c c e p t a b i l i t y o f t h e model by m i n i m i z i n g a q u a n t i t y , Z et which a p p r o x i m a t e s t h e non-bonded r e p u l s i o n e n e r g y . T h i s term i s a l s o used t o o p t i m i z e weak attractive i n t e r a c t i o n s such as hydrogen bonds. The p r e c i s e f o r m u l a t i o n o f e . and e m p i r i c a l c o n s t a n t s employed i n i t s e v a l u a t i o n have been d e s c r i b e d e l s e w h e r e ( 4 4 ) . The c a l c u l a t i o n s were p e r f o r m e d on an EC-1045.01 machine, a t t h e Computer C e n t r e o f the Slovak Academy o f S c i e n c e s , B r a t i s l a v a , and on a H o n e y w e l l - B u l l CII-HB68 mainframe a t t h e U n i v e r s i t y o f G r e n o b l e . The m o l e c u l a r drawings shown t h r o u g h o u t t h i s a r t i c l e were o b t a i n e d w i t h t h e a i d o f t h e PITMOS program ( 4 5 ) . The p e r s p e c t i v e drawing o f the three dimensional shape o f t h e m i r r o r image o f t h e c o n f o r m a t i o n a l energy w e l l o f c e l l o b i o s e ( F i g . 2) was o b t a i n e d w i t h t h e CARTOLAB program ( 4 6 ) . 2

2

f

P

F i g u r e 2. P e r s p e c t i v e d r a w i n g o f the t h r e e - d i m e n s i o n a l shape o f the m i r r o r image o f the c o n f o r m a t i o n a l energy w e l l f o r the f u l l a n g u l a r range f o r $ and Y. The volume was c o n s t r u c t e d u s i n g t h e f o l l o w i n g scheme: V( $ ¥ ) > 15 k c a l / m o l V ( $ , ¥) = 0 ; V( $ , < 15 k c a l / m o l : V ( $ , ¥ ) = - ( V ( $, ¥ ) - 1 5 ) . V b e i n g the energy e x p r e s s e d ^ r e l a t i v e t o t h e minimum. P r o c e e d i n g from t o p t o bottom o f t h e t h r e e - d i m e n s i o n a l shape, we note : t h e v e r y low e n e r g y r e g i o n (the arrows p o i n t towards the c o n f o r m a t i o n s o b s e r v e d f o r c r y s t a l l i n e c e l l o b i o s e and m e t h y l 3 - D - c e l l o b i c s i d e ) . The 5 k c a l / m o l t o the 10 k c a l / m o l e n e r g y c o n t o u r s c o r r e s p o n d t o t h e l i g h t g r e y r e g i o n o f t h e volume.

o3 § n r

^ H C H

3. HENRISSAT ET AL.


45

R e s u l t s and D i s c u s s i o n Conformational Analysis. Comparison o f r e s u l t s shows t h a t t h e l o c a t i o n s o f t h e energy minima on t h e ( $ , ¥ ) map a r e a l m o s t i n d e p e n d e n t o f t h e f u n c t i o n used f o r c a l c u l a t i n g energy, whereas the d i f f e r e n c e s i n energy levels o f t h e v a r i o u s minima a r e s t r o n g l y dependent on t h i s c h o i c e . The d i f f e r e n c e s a r e most pronounced i n t h e low-energy r e g i o n s . T h i s s u g g e s t s t h a t energy s u r f a c e s a r e v a l u a b l e i n r e f i n i n g t h e p e r t i n e n t p o r t i o n o f t h e map embodying " a l l o w e d " c o n f o r m a t i o n s , b u t r e l a t i v e energy values s h o u l d be t r e a t e d w i t h c a u t i o n . F i g u r e 2 shows t h e energy map f o r c e l l o b i o s e , as c a l c u l a t e d from PFOS method; t h e two d e e p e s t minima a r e i n d i c a t e d by a r r o w s . These two minima c o r r e s p o n d t o c r y s t a l l i n e conformations found f o r c e l l o b i o s e (4) and m e t h y l - 6 - D - c e l l o b i o s i d e (5) . F o r t h e s e models o f t h e two s t a b l e c o n f o r m a t i o n s , an i n t r a m o l e c u a l r hydrogen bond o f t h e t y p e 03*...05 i s p r e s e n t , w i t h 03 ...05 d i s t a n c e o f about 2.60 A, compared t o 2.76 A i n t h e c r y s t a l s t r u c t u r e s . We n e x t s t u d i e d t h e i n f l u e n c e o f t h e o r i e n t a t i o n o f t h e h y d r o x y l groups a t C6 on t h e c o n f o r m a t i o n s a t t h e g l y c o s i d i c junction. Because t h e t r a n s - g a u c h e ( t g ) p o s i t i o n o f 06 seldom o c c u r s , p r o b a b l y as t h e r e s u l t o f r e p u l s i v e i n t e r a c t i o n between 04 and 06 (21), we c o n s i d e r e d o n l y t h e gauche-gauche ( gg ) and g a u c h e - t r a n s ( g t ) c o n f o r m e r s . The r o l e o f t h e p r i m a r y h y d r o x y l group d i f f e r s depending on whether i t i s on t h e r e d u c i n g o r t h e n o n - r e d u c i n g r e s i d u e . F o r v a l u e s o f $ and V c o r r e s p o n d i n g t o t h e c r y s t a l l i n e conformations of either cellobiose (4) o r methyl 8-D-cellob i o s i d e ( 5 ) , a g t o r i e n t a t i o n o f t h e p r i m a r y h y d r o x y l group o f t h e non-reducing residue brings an 02-H...06 hydrogen bond. C r y s t a l l i n e m e t h y l c e l l o b i o s i d e (5) has t h i s bond; c e l l o b i o s e does not. P r e v i o u s work r e v e a l e d t h a t c e l l o b i o s e c a n e x i s t i n s e v e r a l s t a b l e c o n f o r m a t i o n s . Conformers c o r r e s p o n d i n g t o t h e e i g h t l o w e s t minima (Fig. 2) were used as s t a r t i n g p o i n t s f o r c a l c u l a t i o n s with MM2CARB. Seven conformers r e s u l t e d ; t h e i r r e l a t i v e e n e r g i e s a r e given i n Table I . In Table I I are s e l e c t e d c h a r a c t e r i s t i c s of ,

T a b l e I . R e l a t i v e e n e r g i e s (kcal/mol) o f s t a b l e c o n f o r m e r s f o r c e l l o b i o s e and m e t h y l B - D - c e l l o b i o s i d e c a l c u l a t e d by MM2CARB method w i t h and w i t h o u t hydrogen bond energy ( V ) .

Conformer

cellobiose w i t h o u t V, HB

CI C2 C3 C4 C5 C6 C7

1.33 0.00 3.42 1.52 4.24 6.39 2.88

methyl B - D - c e l l o b i o s i d e w i t h V, HB 0.93 0.50 0.00 1.75 2.11 2.94 2.24

w i t h o u t V, HB 1.45 0.00 1.60 2.20 4.48 2.69 3.34

with V

HB

0.84 0.36 0.00 1.68 2.09 3.04 1.67

II.

,

$

|i

H

; ^

4.3

112.7 107.2 117.1

= H1-C1-01-C4'

C(5)-0(5)-C(l) 0(5)-C(l)-0(l) C(l)-0(l)-C(4 )

1.4315 1.4264 1.3923 1.4289

63.6 62.9

X X'

,

= Cl-01-C4 -H4'

3.5

113.1 108.1 114.9

1.4289 1.4237 1.3997 1.4308

58.2 -64.1

49.9 16.7

32.8 -66.1

Y

31.58

C2

-66.3 -120.4

32.06

CI

4.3

112.9 108.8 114.9

1.4286 1.4240 1.3989 1.4283

61.0 61.2

161.4 5.8

48.2 -114.9

31.22

£3

4.9

112.3 109.4 117.3

1.4305 1.4221 1.3973 1.4328

64.3 68.0

44.5 171.7

-74.4 55.1

32.89

CA

5.9

112.6 102.6 115.6

1.4296 1.4273 1.3956 1.4269

64.2 63.7

-35.9 -26.9

-147.9 -145.0

33.31

C5

2.2

113.2 101.2 118.7

1.4299 1.4314 1.3963 1.4281

62.6 -67.2

-62.1 -60.6

-171.9 -176.0

34.26

£6

5.2

112.8 108.6 116.2

1.4280 1.4188 1.3944 1.4318

63.7 60.4

70.1 -164.3

-46.6 -83.0

32.89

C7

MM2CARB c a l c u l a t e d e n e r g i e s E ( k c a l / m o l ) , d i p o l e moments u (D), and s e l e c t e d g e o m e t r i c a l p a r a m e t e r s : bond l e n g t h s ( A ) , bond a n g l e s ( d e g ) , f o r m e t h y l 8 - D - c e l l o b i o s i d e c o n f o r m e r s .

-87.6 178.9

C(5)-0(5) C(5)-C(l) C(l)-0(1) 0(1)-C(4')

* y

E

Conformers

Table

m p C

o n

pa n ^ 73 rn

=g

H

ON

3.

HENRISSAT ET AL.

47


m e t h y l 6 - D - c e l l o b i o s i d e . Note (Table 1) t h a t t h e i n c l u s i o n o f hydrogen b o n d i n g energy (V ) changes t h e r e l a t i v e s t a b i l i t y o f the c o n f o r m e r s . Because a l l possible intramolecular hydrogen bonds were c o n s i d e r e d , i t i s o f i n t e r e s t t o compare t h e v a r i o u s p a t t e r n s t h a t r e s u l t e d from t h e t h r e e b e s t c o n f o r m a t i o n s . The hydrogen b o n d i n g i n C2 c o r r e s p o n d s t o t h a t o f c r y s t a l l i n e o f m e t h y l 6 - D - c e l l o bioside, with 03'H...05 and 03'H...06 hydrogen bonds with i n t e r o x y g e n d i s t a n c e s o f about 2.77 and 2.80 A, r e s p e c t i v e l y . On t h e o t h e r hand, t h e C I c o n f o r m a t i o n does n o t have an 03'H...05 hydrogen bond. The i n t e r o x y g e n d i s t a n c e i s 3.0 A b u t t h e hydrogen atom on 03* i s n o t p o s i t i o n e d f a v o r a b l y . I n s t e a d i t forms a bond t o 06, t h e d i s t a n c e b e i n g 2.90 A. In t h e C3 conformer, 03'H...05 and 06'H...06 were formed. The parameters i n T a b l e I I demonstrate an i n t e r a c t i o n between t h e r i n g geometry and t h e c o n f o r m a t i o n around t h e g l y c o s i d i c and a g l y c o n bonds. The c a l c u l a t e d bond l e n g t h s agree w i t h t h e v a l u e s o b s e r v e d i n t h e c r y s t a l s t r u c t u r e s e x c e p t f o r 0 5 - C l and C l - 0 1 and for C5-05-C1 and C1-01-C4•. The C l - 0 1 bonds a r e s h o r t e r t h a n o b s e r v e d v a l u e s and v a r y w i t h $ and y . A t t h e same t i m e , t h e 05-C1-01 bond a n g l e s v a r y from 101.2 t o 109.5° and g l y c o s i d i c bond a n g l e s from 113 t o 1 2 0 ° . These v a r i a t i o n s o f i n t e r n a l geometry owing t o changes i n l i n k a g e t o r s i o n a n g l e a r e c o n s i s t e n t w i t h s t a t i s t i c a l a n a l y s i s o f carbohydrate c r y s t a l s t r u c t u r e s (47,48). D i p o l e moments range from 3.4 t o 6.0 D. When t h e t o r s i o n a n g l e s $ , y a r e i d e n t i c a l f o r a l l t h e p a i r s o f g l u c o s e r e s i d u e s a l o n g t h e c h a i n , t h e polymer c h a i n assumes a r e g u l a r h e l i c a l shape. From t h e g e o m e t r i c a l parameters defining the r e p e a t u n i t , i t i s p o s s i b l e t o c a l c u l a t e , f o r each $ , y t h e v a l u e s o f t h e h e l i c a l p a r a m e t e r s n, h . I n t u r n , t h e s e v a l u e s may be compared t o t h o s e d e r i v e d from X-ray d i f f r a c t i o n o f o r i e n t e d f i b r i l l a r m a t e r i a l which, i n t h e c a s e o f c e l l u l o s e c h a i n s a r e n = 2 and h = 5.15 A. None o f t h e s t a b l e c e l l o b i o s e c o n f o r m e r s as derived from e i t h e r e n e r g y m i n i m i z a t i o n o r c r y s t a l structure e l u c i d a t i o n s c a n g e n e r a t e such a h e l i c a l s t r u c t u r e . 1 3

Charge D e n s i t i e s - CP/MAS NMR S p e c t r o s c o p y . Recent C NMR s t u d i e s of s o l i d carbohydrates u s i n g the c r o s s - p o l a r i z a t i o n / m a g i c angle spinning (CP/MAS) technique (15) d e m o n s t r a t e s that NMR c h e m i c a l s h i f t s o f the C I , C4 and C6 c a r b o n atoms a r e c o n s i d e r a b l y d i s p l a c e d , up t o 10 ppm, depending on t h e p a r t i c u l a r s t r u c t u r e . A r e l a t i o n o f t h e s e s h i f t s t o c o n f o r m a t i o n would be v a l u a b l e i n l e a r n i n g whether a c o n f o r m a t i o n i s r e t a i n e d i n n o n - c r y s t a l l i n e o r solution states. A simple linear relationship h a s been p r o p o s e d between t h e chemical shift f o r C6 and t h e t o r s i o n angle X about t h e e x o - c y c l i c C5-C6 bond. The c h e m i c a l s h i f t s f e l l i n t o 3 groups o f 60-62 ppm, 62.5 ppm and 65.5-66.6 ppm f o r t h e gg, g t a n f t g c o n f o r m a t i o n s ( 1 8 ) . I n a more s o p h i s t i c a t e d e m p i r i c a l o b s e r v a t i o n , i t was p r o p o s e d that chemical s h i f t s i n a series of closely r e l a t e d m o l e c u l e s a r e r e l a t e d t o charge d e n s i t y (50,51). As a f i r s t s t e p i n u n d e r s t a n d i n g t h e r e l a t i o n s h i p o f c h e m i c a l shift t o c o n f o r m a t i o n , we c a l c u l a t e d charge densities fora and 6-D-glucose, c e l l o b i o s e , and m e t h y l 6 - D - c e l l o b i o s i d e , u s i n g the PCILO program. Charge d e n s i t i e s f o r C6 o f a - and 6-D-glucose

American Chemical Society. Library 1155 16th St., N.w. Washington. O.C. 20036

48

THE STRUCTURES OF CELLULOSE were, r e s p e c t i v e l y , 0.1195 and 0.1173 ( gg ) , 0.1189 and 0.1169 ( g t ) , and 0.1182 and 0.1164 ( t g ) . These v a l u e s c o r r e l a t e w e l l w i t h the o b s e r v e d c h e m i c a l s h i f t s , above. C a l c u l a t e d e l e c t r o n d e n s i t i e s on s e l e c t e d carbon and oxygen atoms of m e t h y l B - D - c e l l o b i o s i d e ( i n the c r y s t a l l i n e c o n f o r m a t i o n ) as a f u n c t i o n o f c o n f o r m a t i o n o f the p r i m a r y a l c o h o l groups a r e g i v e n i n T a b l e I I I . The results for cellobiose are s i m i l a r . While c o n f o r m a t i o n a l change results i n changed d e n s i t y w i t h i n the g l u c o s e r e s i d u e , t h e r e i s no e f f e c t on the o t h e r r e s i d u e . However, the c o r r e l a t i o n o f charge d e n s i t y and c h e m i c a l s h i f t observed above f o r g l u c o s e i s n o t o b s e r v e d i n t h i s c a s e . There a r e s e v e r a l p o s s i b l e e x p l a n a t i o n s . Owing t o the r a r i t y o f tc[ c o n f o r m a t i o n s , the c h e m i c a l s h i f t d a t a i s i n s u f f i c i e n t . (The v a l u e s r e p o r t e d a r e from c e l l u l o s e I , d e t e r m i n e d by f i b e r d i f f r a c t i o n , and may n o t be r e l i a b l e ) . A l s o , c h e m i c a l s h i f t s h o u l d be a c o n t i n u o u s f u n c t i o n o f torsional angle. Therefore, the proposed linear relationship cannot be c o r r e c t . Our u n d e r s t a n d i n g o f a r e l a t i o n s h i p between e l e c t r o n d e n s i t y and c h e m i c a l s f i f t may be v e r y a p p r o x i m a t e . Many more c a l c u l a t i o n s a r e needed, u s i n g f i n e i n c r e m e n t s o f t o r s i o n a l r o t a t i o n . Such attempts w i l l a l s o p r o v i d e i n f o r m a t i o n on c h e m i c a l s h i f t s o f u n s t a b l e conformers t h a t cannot be i s o l a t e d . P a c k i n g f e a t u r e s o f known o l i g o m e r s . X-ray d a t a on crystalline o l i g o m e r s p r o v i d e s i n f o r m a t i o n about some f e a t u r e s o f the p a c k i n g habits as well as on molecular conformation. For a full u n d e r s t a n d i n g o f any p a c k i n g arrangement, t h e energy between a reference molecule and a l l i t s neighbors i n the crystal is evaluated, taking into account the hydrogen bonds and the non-bonded i n t e r a c t i o n s ( 5 2 ) . These c a l c u l a t i o n s use the same p a r a m e t e r s as i n the c o n f o r m a t i o n a l a n a l y s i s o f s i n g l e m o l e c u l e s u s i n g the PFOS method. C o n t r i b u t i o n s t o the energy were b r o k e n down i n t o the pure, non-bonded p a r t and the i n t e r m o l e c u l a r hydrogen b o n d i n g . The p a c k i n g i n c r y s t a l s o f c e l l o b i o s e i s v e r y dense, each m o l e c u l e i s s u r r o u n d e d by 10 n e i g h b o r s t h a t o c c u r i n p a i r s ( F i g . 3a) . The s t r o n g e s t i n t e r a c t i o n s a r e c a l c u l a t e d f o r m o l e c u l e s r e l a t e d by p u r e t r a n s l a t i o n a l symmetry a l o n g t h e c r y s t a l l o g r a p h i c c a x i s (c = 5.091 A ) . The energy o f p a i r i n g o f p a r a l l e l m o l e c u l e s i s due o n l y t o van d e r Waals i n t e r a c t i o n s . The r e m a i n i n g p a c k i n g energy a r i s e s from m o l e c u l e s r e l a t e d by symmetry o p e r a t i o n s about t h e 2^ a x i s , w i t h a p p r o p r i a t e t r a n s l a t i o n s a l o n g the a and/or c axes. In these i n s t a n c e s , the i n t e r m o l e c u l a r a s s o c i a t i o n s a r e obtained through van d e r Waals and hydrogen b o n d i n g c o n t r i b u t i o n s . Some o f t h e s e same f e a t u r e s a r e a l s o found i n m e t h y l 6 - D - c e l l o b i o s i d e , which a l s o c r y s t a l l i z e s i n t h e P2 space group, each molecule has 10 p a i r e d neighbors (Fig. 3b). The strongest i n t e r m o l e c u l a r p a i r i n g i s a l s o obtained f o r molecules r e l a t e d to each o t h e r by p u r e t r a n s l a t i o n a l o n g the c r y s t a l l o g r a p h i c c a x i s (c = 4.496 £) . The p a i r i n g energy a g a i n a r i s e s from van d e r Waals f o r c e s . O t h e r pure t r a n s l a t i o n a l o p e r a t i o n s , such as - a o r - a c result i n strong cohesive energy, provided by comparable magnitudes o f van d e r Waals and hydrogen b o n d i n g . Here, the o n l y i n t e r a c t i o n s a l o n g the 2^ a x i s ( b = 25.532 K) o c c u r v i a the methanol molecule through hydrogen bonding with subsequent b r i d g i n g i n the d i s a c c h a r i d e m o l e c u l e s .

3.8842 3.8837 3.8798 3.8839 3.8834 3.8795 3.8840 3.8835 3.8795

*0(6")

"0(6)

6.1672 6.1674 6.1670 6.1666 6.1668 6.1666 6.1680 6.1678 6.1683

*C(6)

3.8857 3.8857 3.8856 3.8846 3.8846 3.8846 3.8852 3.8852 3.8852

*C(5)

3.8979 3.8981 3.8980 3.8979 3.8981 3.8980 3.8940 3.8942 3.8941

(see t e x t f o r e x p l a n a t i o n ) .

6.1692 6.158C 6.1657 6.1691 6.1578 6.1658 6.1691 6.1574 6.1661

respectively

3.8851 3.8837 3.8847 3.8851 3.8837 3.8847 3.8850 3.8837 3.8846

*C(6')

g and t s t a n d f o r gauche and t r a n s ,

f

gt, tg tg gt gg*gt gt,tg tg,tg ggrtg gt,gg tg,gg gg^gg

*C(5»)

T a b l e I I I . PCILO c a l c u l a t e d e l e c t r o n d e n s i t i e s f o r s e l e c t e d c a r b o n and oxygen atoms as a f u n c t i o n o f h y d r o x y m e t h y l g r o u p s o r i e n t a t i o n s o f m e t h y l β-D-cellobioside i n c r y s t a l c o n f o r m a t i o n .

Ι

ε-

9 I

70

m ζ


F i g u r e 3. Stereoscopic representations of s u r r o u n d i n g found i n t h e c r y s t a l l i n e s t a t e o f : a C e l l o b i o s e (4) b M e t h y l β-D-cellobioside ( 5 ) .

the

molecular

3.

HENRISSAT ET AL.

51


M e t h y l β-D-cellotrioside. Fig. 4 shows a typical hexagonal crystalline platelet o f m e t h y l β-D-cellotrioside, which upon r e d u c t i o n t o powder g i v e s r i s e t o t h e d i f f r a c t i o n p a t t e r n o f F i g u r e 5. The u n i t c e l l d a t a a r e i n T a b l e IV. From t h e o b s e r v e d

T a b l e IV.

C r y s t a l d a t a f o r m e t h y l β-D

C

a =

8.005 (2) A

β = 116.39 (5) °

19

H

34

°16

; monoclinic ;

= 518.46

Mr

; b = 76.403 (9) Κ Ρ2

χ

cellotrioside.

;

c =

;

Ζ = 8

8.995 (2) A

d e n s i t y o f 1.50 Mg.m , t h e asymmetric unit contains four independent molecules. S o l v i n g a non-centrosymmetrical s t r u c t u r e w i t h 140 non-hydrogen atoms from o n l y 3694 r e f l e c t i o n s i s a formidable challenge. Preliminary attempts to resolve the s t r u c t u r e w i t h c o n v e n t i o n a l d i r e c t methods were u n s u c c e s s f u l ; the work i s s t i l l b e i n g p u r s u e d i n our l a b o r a t o r y . The c r y s t a l morphology ( F i g . 4) and the d i m e n s i o n o f the u n i t c e l l b parameter o f 76.403 A l e a d t o the c o n c l u s i o n s t h a t : -the m o l e c u l e s a r e i n extended c o n f o r m a t i o n s and a r e p l a c e d p a r a l l e l t o the b a x e s . In some r e s p e c t , t h e y form a pseudochain structure. -the base p l a n e p a r a m e t e r s ( a , c and β ) resemble those o f c e l l u l o s e I I and o f c e l l o t e t r a o s e . Fig. 6 summarizes t h e p o s s i b l e m o l e c u l a r f e a t u r e s i n terms o f p a r a l l e l and a n t i p a r a l l e l arrangements c o m p a t i b l e w i t h t h e u n i t cell d a t a f o r m e t h y l β-D-cellotrioside. By analogy with the a n t i p a r a l l e l t y p e o f p a c k i n g p r o p o s e d f o r c e l l u l o s e I I and f o r c e l l o t e t r a o s e , one would f a v o r t y p e d i n F i g . 6. Owing t o i t s h i g h c r y s t a l l i n i t y and the s i m i l a r i t y o f i t s X-ray d i f f r a c t o g r a m w i t h t h a t o f c e l l o t e t r a o s e , m e t h y l β-D-cellotrioside was used as a model f o r c e l l u l o s e I I i n a s t u d y u s i n g C NMR s p e c t r o s c o p y . I t was thought t h a t the a g l y c o n i c m e t h y l group would a l s o be u s e f u l i n p r o v i d i n g i n f o r m a t i o n about t h e v a r i e t y o f l i n k a g e c o n f o r m a t i o n s i n t h e s t r u c t u r e . F i g . 7 shows t h e s p e c t r a , a l o n g w i t h some r e l a t e d o l i g o m e r s . The spectra are similar, e s p e c i a l l y the c h e m i c a l s h i f t s f o r C4, which o c c u r a t lower f i e l d than i n the lower o l i g o m e r s . The r e s o n a n c e a t t r i b u t e d t o the C6 n u c l e i g i v e s r i s e t o a s i n g l e b r o a d peak c e n t e r e d a t 64.2 ppm; t h r e e s i g n a l s appear a t 108.3, 106.0 and 104.0 ppm f o r t h e 3 CI atoms. At h i g h f i e l d , t h e m e t h y l groups produce two peaks, a t 58.4 and 56.7 ppm. T h i s i m p o r t a n t o b s e r v a t i o n s i g n i f i e s t h a t t h e r e a r e two main magnetic e n v i r o n m e n t s , and t h e r e f o r e , c o n f o r m a t i o n s , f o r the a g l y c o n i c m o i e t i e s . Moreover, t h e s e two peaks are themselves composites o f two o r t h r e e peaks, c o n s i s t e n t w i t h t h e l a r g e u n i t c e l l . T h i s s u p p o r t s model d, which has b o t h "head-to-head" and " h e a d - t o - t a i l " environments f o r the m e t h y l g r o u p s . By comparing


F i g u r e 4. Typical β-D-cellotrioside.

Figure 5.

X-ray

β-D-cellotrioside.

hexagonal

powder

crystalline

diffraction

platelet

of

pattern

of

methyl

methyl

HENRISSAT ET AL.


Φ © ©

©

©

© © Φ ©

©

© ©

© © © Φ

©

©

Φ ©

Θ

©

©

© Φ

©

©

Φ © ©

©

F i g u r e 6. Schematic drawings of the possible molecular arrangements f o r c r y s t a l l i n e m e t h y l 0 - D - c e l i o t r i o s i d e c o n s i s t e n t w i t h a P2^ space group symmetry. The 4 i n d e p e n d e n t m o l e c u l e s a r e numbered from 1 t o 4; t h e arrows p o i n t towards t h e m e t h y l g r o u p s . F o r o b v i o u s c r y s t a l l o g r a p h i c r e a s o n s t h e 2^ a x i s o f symmetry cannot c o i n c i d a t e w i t h any o f t h e m o l e c u l a r a x i s .

54


£(ppm) 1 3

F i g u r e 7. CP/MAS C NMR s p e c t r a o f some c e l l u l o s e o l i g o m e r s r e c o r d e d a t 50.36 MHz : a / c e l l c b i o s e , b / m e t h y l β-D-cello b i o s i d e , c / c e l l o t e t r a o s e and d/ m e t h y l β-D-cellotrioside.

3.

55


HENRISSAT E T A L .

this spectrum with t h a t o f methyl β-D-cellobioside we could a t t r i b u t e t h e s i g n a l a t 58.1 ppm t o the a g l y c o n i c m e t h y l group; the s i g n a l a t 51.6 ppm t h e n a r i s e s from t h e methanol m o l e c u l e t h a t c o - c r y s t a l l i z e s w i t h m e t h y l β-D-cellobioside. Cellotetraose. D e s p i t e s e v e r a l y e a r s o f s t e a d y e f f o r t , no s i n g l e c r y s t a l adequate f o r c o n v e n t i o n a l X-ray c r y s t a l l o g r a p h y c o u l d be grown. F i g 8. shows a powder d i f f r a c t i o n p h o t o g r a p h ; t h e wide a n g l e n e u t r o n d i f f r a c t o g r a m i s i n F i g . 9. C r y s t a l d a t a a r e i n T a b l e V. F i g . 10 shows b o t h a l a m e l l a r fragment o f c e l l o t e t r a o s e

T a b l e V.

C r y s t a l data f o r c e l l o t e t r a o s e .

C

a =

8.963

(3) A

α = 94.98

(10)

triclinic

;

° PI

24

H

41

°21

; b =

Μ

'

8.033

; β = 89.34

Γ

(3) A (10)

°

=

6

6

5

·

5

7

; c = 22.473 ; γ =116.13

(7) A (10)

0

; Ζ = 2

and the corresponding electron d i f f r a c t o g r a m . The base plane p a t t e r n has t w o - d i m e n s i o n a l p i symmetry which i s c o n s i s t e n t w i t h both triclinic and monoclinic space groups. The fact that r e c o r d i n g d i f f r a c t i o n d a t a from t h e base p l a n e r e q u i r e d t i l t i n g the c r y s t a l by 9-11° i n d i c a t e d t h a t the c o r r e c t c h o i c e was the t r i c l i n i c space g r o u p . F o r a c h i r a l m o l e c u l e t h i s c o u l d o n l y be P I . The u n i t c e l l p a r a m e t e r s agreed w i t h p r e v i o u s v a l u e s ( 8 ) . We a l s o confirmed t h a t t h i s t r i c l i n i c c e l l f i t s the neutron d a t a . Observed s t r u c t u r e f a c t o r s were k i n d l y p r o v i d e d by P o p p l e t o n . We found that only 869 of 2951 measured reflections could be c o n s i d e r e d as " o b s e r v e d " (1/ σ (I) > 1.99). S i n c e t h e r e a r e two tetramers per cell, determination of the structure requires location of 90 non-hydrogen atoms with only 869 observed reflections. Preliminary calculations produced a negative temperature f a c t o r , i n d i c a t i n g an u n r e l i a b l e d a t a s e t , as i f from a damaged c r y s t a l . The c r y s t a l used by P o p p l e t o n was o n l y 0.01 mm t h i c k , and i t r e q u i r e d 900 hours o f e x p o s u r e t o a h i g h - i n t e n s i t y beam t o r e c o r d t h e d a t a . Because the d a t a were n o t l i k e l y t o be a c c u r a t e enough f o r c o n v e n t i o n a l methods, we d e c i d e d t o a t t t e m p t to s o l v e the structure with one of the "Real-Space Crystal Structure Resolution" procedures with the 70 reflections corresponding to 3 A r e s o l u t i o n . The Linked-Atom L e a s t - S q u a r e s (LALS) p r o c e d u r e (44) was used t o g e n e r a t e m o l e c u l a r models o f c e l l o t e t r a o s e . The g l u c o s e r e s i d u e s were k e p t i n the s t a n d a r d C c o n f o r m a t i o n ; a l l bond a n g l e s and bond l e n g t h s were f i x e d a t s t a n d a r d v a l u e s ( 4 3 ) . The c o n s t r a i n e d model o f the c r y s t a l s t r u c t u r e was o p t i m i z e d a g a i n s t b o t h X-ray d a t a and n o n - c o v a l e n t i n t e r a t o m i c i n t e r a c t i o n s , as d e s c r i b e d by Smith and A r n o t t (44). h

1

56


F i g u r e 8.

X-ray powder d i f f r a c t i o n p a t t e r n o f c e l l o t e t r a o s e .

COUNT

1

1

1

1

1

I

!

2200 L-

1

1700

1+

Li

ι 50

.

.

.

.

ι 100

.

—•

F i g u r e 9. Wide cellotetraose.

angle

neutron

powder

.

.

.

ι 150

2 THETA

diffractogram

of

HENRISSAT ET AL.


F i g u r e 10. Electron micrograph of microcrystals of cellotetraose. Insert : corresponding electron diffraction diagram p r o p e r l y o r i e n t e d .


58

The p r e l i m i n a r y m o l e c u l a r models were t r e a t e d as r i g i d b o d i e s and the r e l a t i v e o r i e n t a t i o n s about and t r a n s l a t i o n a l o n g the l o n g crystal axis were v a r i e d i n increments. At each step, the magnitude o f s t e r i c i n t e r f e r e n c e was c a l c u l a t e d . The v a l u e s o f packing parameters c o r r e s p o n d i n g t o l o c a l minima were used as s t a r t i n g p o i n t s f o r r e f i n e m e n t t h a t v a r i e d some o f the m o l e c u l a r and p a c k i n g parameters a l o n g w i t h t h e X-ray s c a l e f a c t o r . We c o n c l u d e d a t t h i s s t a g e t h a t the s t r u c t u r e c o n t a i n s a n t i p a r a l l e l molecules. F u r t h e r refinement v a r i e d m o l e c u l a r parameters such as t o r s i o n a n g l e s of the p r i m a r y h y d r o x y l groups and the t o r s i o n and v a l e n c e a n g l e s a t the g l y c o s i d i c l i n k a g e s . The f i n a l R was 0.21; the o b s e r v e d and c a l c u l a t e d s t r u c t u r e f a c t o r s a r e i n T a b l e V I . Atomic c o o r d i n a t e s a r e i n T a b l e V I I ; l a b e l i n g o f the atoms p r o c e e d s from the n o n - r e d u c i n g r e s i d u e ( a and e i n t h e f i r s t and second molecules, r e s p e c t i v e l y ) t o the r e d u c i n g r e s i d u e ( d and h respectively). O n l y the main f e a t u r e s can be a s s e s s e d w i t h o u t a m b i g u i t y a t 3 A r e s o l u t i o n . The c h i e f one i s the a n t i p a r a l l e l arrangement o f the two independent molecules i n the unit cell. F i g . 11 is a stereoscopic drawing of the structure, Fig. 12 shows the p r o j e c t i o n o n t o the a, b base p l a n e . T h i s p r o j e c t i o n i s c o n s i s t e n t w i t h the p l a t e l e t morphology o f the c r y s t a l s shown i n F i g . 10. Because a l l g l u c o s e r e s i d u e s were t r e a t e d as r i g i d b o d i e s i n the r e f i n e m e n t , t h e v a r i a b l e l i n k a g e t o r s i o n a n g l e s and glycosidic bond a n g l e s had t o accomodate a l l v a r i a t i o n s r e q u i r e d t o produce t h e b e s t s t r u c t u r e . They may therefore suffer some l o s s of a c c u r a c y . A l l t h e ( Φ, Ψ) a n g l e s , however, f a l l i n t o t h e v e r y low-energy r e g i o n o f the map (see F i g . 1 3 ) . G o i n g from r e s i d u e t o residue, φ and ψ were not similar enough to suggest local pseudo-symmetry. The glycosidic angles ranged from 117.2 to 121.8 . D i s t a n c e s between 03 and 05 on t h e p r e c e d i n g u n i t ranged from 2.47 to 3.02 A, i n d i c a t i n g hydrogen bonding. P r o c e e d i n g from the n o n - r e d u c i n g end, d i s t a n c e s s p a n n i n g d i s a c c h a r i d e r e s i d u e s a r e 10.43, 10.55 and 10.38 A ( m o l e c u l e 1) and 10.36, 10.48, and 10.41 A (molecule 2 ) . While t h e r e i s s c a t t e r i n the d i s t r i b u t i o n of φ and ψ, p a r t l y due t o t h e low a c c u r a c y , some o f the conformations a r e near the C l conformer (methyl β-D-cellobioside) and some a r e near C2 ( c e l l o b i o s e ) . C

Conclusion The m u l t i d i s c i p l i n a r y approaches d e s c r i b e d i n t h e p r e s e n t work a r e all aimed a t describing, and understanding the structural features of c e l l u l o s e oligomers a t a molecular l e v e l . Intimate details are indeed taken i n t o account i n the conformational a n a l y s i s c a l c u l a t i o n s . The utilization of h i g h l y parametrized molecular mechanics programs, including the contribution of i n t r a m o l e c u l a r hydrogen bonding t o the t o t a l energy, produced c o n f o r m a t i o n s i n agreement w i t h o b s e r v e d c r y s t a l s t r u c t u r e s . The v e r y same method was a l s o very powerful i n demonstrating the interactions between ring geometry and rotations about the glycosidic and aglycon linkages. When the hydrogen bond i s c o n s i d e r e d , i t appears t h a t t h e p y r a n o s e r i n g undergoes s m a l l

3.

HENRISSAT ET AL.

Table VI.

59


Structure factor table resolution.

f o r cellotetraose

at 3 A

h

k

1

F-calc

F-obs

h

k

1

F-calc

F-obs

0 1 -1 0 0 1 0 0 -1 0 1 0 0 -1 1 0 -1 0 1 -1 2 2 2 1 -1 -1 0 2 -2 1 2 -1 1 -2 0

0 0 0 0 1 -1 -1 1 0 -1 -1 1 0 0 0 1 0 -1 0 -1 -1 -1 1 1 1 -1 1 -1 1 1 0 2 -2 0 -1

2 0 1 3 0 0 1 1 2 2 2 2 4 3 3 3 4 4 4 1 0 1 1 1 4 2 4 2 2 2 0 0 1 1 5

41.03 12.12 22.48 28.67 127.23 22.69 45.58 29.38 49.74 48.73 30.88 19.88 30.80 33.77 21.08 19.42 75.26 30.20 91.85 52.42 243.12 83.26 31.31 31.12 15.41 56.27 31.47 62.66 51.92 39.63 256.80 20.21 21.00 38.89 21.25

19.56 14.28 14.62 20.42 101.05 22.91 48.63 16.32 40.51 99.01 17.84 27.46 34.60 40.05 25.08 23.04 101.92 32.20 97.10 43.09 224.51 78.40 43.04 36.83 31.30 54.58 34.21 50.98 54.34 51.39 245.83 20.66 25.12 34.66 34.86

1 -1 2 1 1 1 -2 -2 2 0 0 -2 2 2 0 0 -2 -1 1 1 1 0 -2 0 2 -1 -1 0 0 -1 0 1 -2 -2 -1

-1 0 0 -1 -2 0 0 1 0 0 2 0 -2 -2 -2 2 1 0 -1 -2 1 _2

3 5 1 5 2 5 2 3 2 6 0 3 0 1 2 1 4 6 6 4 4 3 2 2 3 5 6 6 4 4 3 5 5 5 7

23.45 35.84 33.76 27.01 12.17 15.93 23.48 34.10 56.59 11.54 17.05 18.07 45.81 42.48 21.73 22.37 14.20 25.14 33.34 16.96 78.75 24.16 34.27 41.33 29.25 59.45 68.13 40.29 14.84 52.79 42.20 57.56 28.68 20.49 36.51

38.39 28.27 25.37 39.77 28.07 25.74 23.81 27.95 49.18 23.65 22.46 23.20 34.62 32.61 31.18 23.81 22.05 26.39 35.35 20.34 112.36 42.68 40.22 50.74 28.65 87.40 81.51 45.66 30.40 64.98 58.31 56.06 28.89 21.60 34.90

2 2 -2 -1 1 1 -2 2 2 -2 1 0 0

60


Table VII.

a-Cl a-01 a-C2 a-02 a-C3 a-03 a-C4 a-04 a-C5 a-05 a-C6 a-06 b-Cl b-01 b-C2 b-02 b-C3 b-03 b-C4 b-C5 b-05 b-C6 b-06 c-Cl c-01 c-C2 c-02 c-C3 c-03 c-C4 c-C5 c-05 c-C6 c-06 d-Cl d-01 d-C2 d-02 d-C3 d-03 d-C4 d-C5 d-05 d-C6 d-06

0.0624 -0.0243 -0.0468 -0.0767 0.0350 -0.0765 0.0815 0.1785 0.1832 0.0932 0.2214 0.0741 0.0717 0.1568 0.1687 0.1738 0.0906 0.1931 0.0699 -0.0206 0.0658 -0.0319 -0.0047 0.0701 -0.0125 -0.0286 -0.0320 0.0462 -0.0580 0.0653 0.1579 0.0743 0.1678 0.2222 0.0209 0.0931 0.0242 -0.0755 -0.0366 -0.0159 0.0596 0.0569 0.1204 0.1630 0.2370

F r a c t i o n a l c o o r d i n a t e s o f C and Ο atoms f o r ο c e l l o t e t r a o s e model o b t a i n e d a t 3 A resolution.

0.4084 0.3739 0.2672 0.0851 0.3112 0.1891 0.5126 0.5616 0.6429 0.5914 0.8440 0.8651 0.4925 0.5298 0.6525 0.8202 0.6126 0.7544 0.4234 0.2739 0.3239 0.0856 0.0078 0.4546 0.4329 0.2848 0.1235 0.3099 0.1586 0.4931 6535 6168 8368 9753 6793 7968 7979 8914 6784 0.7945 0.5642 0.4580 0.5846 0.3547 0.3757

-0.9037 -0.8510 -0.9534 -0.9403 -1.0134 -1.0600 -1.0241 -1.0764 -0.9709 -0.9170 -0.9766 -0.9869 -0.6679 -0.6134 -0.7057 -0.6771 -0.7683 -0.8049 -0.7960 -0.7537 -0.6967 -0.7766 -0.7298 -0.4334 -0.3798 -0.4767 -0.4538 -0.5383 -0.5798 -0.5596 -0.5122 -0.4565 -0.5287 -0.4791 -0.2070 -0.1557 -0.2568 -0.2411 -0.3159 -0.3630 -0.3290 -0.2756 -0.2228 -0.2840 -0.3412

e-Cl e-01 e-C2 e-02 e-C3 e-03 e-C4 e-04 e-C5 e-05 e-C6 e-06 f-Cl f-Ol f-C2 f-02 f-C3 f-03 f-C4 f-C5 f-05 f-C6 f-06 g-Cl g-01 g-C2 g-02 g-C3 g-03 g-C4 g-C5 g-05 g-C6 g-06 h-Cl h-01 h-C2 h-02 h-C3 h-03 h-C4 h-C5 h-05 h-C6 h-06

0.5370 0.5944 0.5992 0.5292 0.5548 0.6289 0.6160 0.5549 0.5550 0.6065 0.6234 0.7946 0.5693 0.5000 0.5307 0.6074 0.5898 0.5368 0.5207 0.5576 0.4931 0.4791 0.3077 0.5378 .6127 .5783 .5071 .5148 .5694 0.5780 0.5395 0.6085 0.6124 0.5230 0.5775 0.5081 0.6044 0.7217 0.6632 0.6705 0.5456 0.5194 0.4604 0.3921 0.2414

0.4817 0.4398 0.4067 0.2092 0.4662 0.4108 0.6764 0.7298 0.7400 0.6800 0.9497 1.0352 0.5068 0.5000 0.6391 0.8210 0.6400 0.7490 0.4420 0.3177 0.3243 0.1155 0.0227 0.3450 0.3303 0.2381 .0459 .2663 .1817 .4732 0.5702 0.5379 0.7792 0.8576 0.3976 0.4129 0.5657 0.7304 0.5464 0.6950 0.3604 0.2014 0.2320 0.0144 0.0203

-0.4833 -0.5375 -0.4342 -0.4440 -0.3731 -0.3275 -0.3655 -0.3117 -0.4181 -0.4733 -0.4157 -0.4306 -0.7178 -0.7735 -0.6748 -0.6945 -0.6115 -0.5710 -0.5933 -0.6404 -0.6979 -0.6273 -0.6455 -0.9522 -1.0055 -0.9073 -0.9282 -0.8459 -0.8032 -0.8269 -0.8757 -0.9311 -0.8615 -0.8918 -1.1852 -1.2382 -1.1419 -1.1656 -1.0808 -1.0392 -1.0587 -1.1061 -1.1613 -1.0888 -1.0704

HENRISSAT ET AL.


F i g u r e 11. S t e r e o s c o p i c r e p r e s e n t a t i o n o f the u n i t c e l l of c r y s t a l l i n e cellotetraose.

F i g u r e 12. Projection of a,b base p l a n e .

the

content

c e l l o t e t r a o s e s t r u c t u r e onto

the


F i g u r e 13. Calculated linear cellodextrins.

and

observed

(Φ,Ψ)

conformations

of

A : c a l c u l a t e d c o n f o r m a t i o n s from M o l e c u l a r Mechanics l a b e l e d from 1 t o 7 and r e f e r t o c o n f o r m e r s CI t o C7 l i s t e d i n Table I I . Observed

( Φ , ψ) conformations :

m : cellobiose m e t h y l β-D-cellobioside Ο : cellotetraose, unit a to unit b to unit c to unit e to unit f to unit g to

b; c; d; f; g; h;

1 2 3 4 5 6

-73.3 -88.9 -90 -103 -68 -80 -94 -83

-132.3 -160.7 -141 -138 -129 -156 -150 -131

The e x t e r n a l c o n t o u r c o r r e s p o n d s t o 10 k c a l / m o l e x p r e s s e d r e l a t i v e t o t h e minimum.

3.

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d i s t o r s i o n s . The p r e s e n t r e s u l t s o f f e r a p i c t u r e which i s a compromise between the extreme d i s t o r s i o n s mentioned by Melberg and Rasmussen (53) on t h e one hand, and the lack of such d i s t o r s i o n s r e p o r t e d by P i z z i and E a t o n (54) on t h e o t h e r hand. C o n v e n t i o n a l X-ray c r y s t a l l o g r a p h y on o l i g o m e r s i s i n f a c t t h e u l t i m a t e method s i n c e , upon c o m p l e t i o n o f a 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 , t h e c o o r d i n a t e s o f a l l t h e atoms i n a m o l e c u l e are d e t e r m i n e d w i t h a h i g h a c c u r a c y . T h i s , s i m p l y , r e s u l t s from t h e f a c t t h a t t h e r e i s u s u a l l y a f a r g r e a t e r number o f o b s e r v a b l e s , i. e. d i f f r a c t i o n intensities, t h a n p a r a m e t e r s t o be found. U n f o r t u n a t e l y , such a s i t u a t i o n i s n o t found i n the c a s e o f cellulose o l i g o m e r s w i t h a DP h i g h e r t h a n two. N e i t h e r 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 o f c e l l o t e t r a o s e , nor t h a t o f m e t h y l B-Dcellotrioside have been s o l v e d and refined t o an acceptable a c c u r a c y . N e v e r t h e l e s s , c r u c i a l s t r u c t u r a l i n f o r m a t i o n has been gained since the antiparallel orientation of cellotetraose m o l e c u l e s has been d e t e r m i n e d . A l s o , o u r r e s u l t s s t r o n g l y s u g g e s t t h a t s e v e r a l d i s t i n c t c o n f o r m a t i o n s about the g l y c o s i d i c bonds a r e found i n the c e l l o t e t r a o s e molecules. To what e x t e n t such a 1 3 b e h a v i o u r may e x p l a i n t h e s p l i t t i n g o f t h e C NMR resonance o f the CI atoms i s s t i l l n o t w e l l e s t a b l i s h e d . Whereas i t was w e l l known t h a t c e l l o t e t r a o s e was a good model f o r c e l l u l o s e I I , we have c l e a r l y shown t h a t m e t h y l β-D-cellotrioside i s a l s o q u i t e an adequate model. Such an adequacy i s based on t h e similarity between the h i g h r e s o l u t i o n s o l i d s t a t e NMR s p e c t r a t o g e t h e r w i t h the dimensions of the crystal unit cell. Crystal structure elucidation of methyl β-D-cellotrioside represents quite a c h a l l e n g e , b u t a t l e a s t , s i n g l e c r y s t a l s good enough f o r X-ray a n a l y s i s can be grown i n a r e p r o d u c i b l e manner, which i s n o t t h e case y e t f o r c e l l o t e t r a o s e . The high resolution solid state spectrum o f m e t h y l β-D-cellotrioside e x h i b i t s a d i s t i n c t s p l i t t i n g of t h e CI atoms r e s o n a n c e s and t h i s may r e f l e c t t h e o c c u r e n c e o f d i s t i n c t c o n f o r m a t i o n s a t t h e g l y c o s i d i c l i n k a g e s . T h i s v e r y same spectrum a l s o shows how the m e t h y l groups o f m e t h y l g l y c o s i d e s may be used as c o n v e n i e n t p r o b e s i n e s t a b l i s h i n g gross structural f e a t u r e s . N e v e r t h e l e s s , drawing d e f i n i t e c o n c l u s i o n s about any direct correlation between an observed splitting and known s t r u c t u r a l f e a t u r e s as d e r i v e d from X-ray i n v e s t i g a t i o n s h o u l d be h a n d l e d w i t h c a u t i o n . Whereas i t appears t h a t t h e o c c u r r e n c e o f s p l i t r e s o n a n c e s r e f l e c t s c o n f o r m a t i o n a l inhomogeneity (55), the converse i s not n e c e s s a r i l y t r u e . T h i s statement is illustrated by t h e m e t h y l β-D-cellobioside c a s e , f o r which two distinct c o n f o r m a t i o n s o f the C6 c a r b o n atoms a r e o b s e r v e d i n the c r y s t a l s t r u c t u r e whereas o n l y one resonance a t 63.1 ppm can be a s s i g n e d to the c o r r e s p o n d i n g atoms i n t h e . C CP /MAS NMR spectrum. Our c a l c u l a t i o n s , a l t h o u g h s t i l l p r i m i t i v e , appear t o e x p l a i n such a behavior. D e s p i t e the l a c k o f h i g h a c c u r a c y , some c o n s i s t e n t f e a t u r e s can be drawn from the s t u d y o f c e l l u l o s e o l i g o m e r s t h r o u g h our a p p r o a c h . The r e s u l t s i n d i c a t e c l e a r l y t h a t the g l y c o s i d i c l i n k a g e s i n c e l l u l o s e o l i g o m e r s have d i f f e r e n t c o n f o r m a t i o n s . I t i s a l s o c l e a r t h a t t h e g l y c o s i d i c l i n k a g e does n o t e x i s t i n a conformation c o n s i s t e n t w i t h a " t w o - f o l d " h e l i x symmetry. These c o n c l u s i o n s which a r e e s s e n t i a l l y r e a c h e d through conformational analysis c a l c u l a t i o n s a r e s u p p o r t e d by t h e appearence o f t h e s o l i d s t a t e 1 3

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C NMR spectra o f both cellotetraose and m e t h y l β-D-cello t r i o s i d e , and c e l l u l o s e I I as w e l l . D e s p i t e a l a r g e d i f f e r e n c e between t h e u n i t c e l l c o n t e n t s o f m e t h y l β-D-cellotrioside and c e l l o t e t r a o s e , t h e i r r e s p e c t i v e base p l a n e d i m e n s i o n s , i . e . normal to the d i r e c t i o n p a r a l l e l t o the molecular a x i s , a r e s t r i k i n g l y comparable ; t h i s must r e f l e c t s t r o n g p a c k i n g h a b i t s , o f n o t o n l y l o n g c e l l u l o s e c h a i n s b u t c e l l u l o s e - l i k e c h a i n s as c e l l o t e t r a o s e or m e t h y l β-D-cellotrioside (at the exclusion o f metastable arrangements resulting from biosynthetic pathways and organizations). There has been c o n s i d e r a b l e d i s c u s s i o n r e g a r d i n g t h e c r y s t a l s t r u c t u r e o f c e l l u l o s e I I and two models have been p r o p o s e d ; t h e y b o t h a r e b a s e d upon a m o n o c l i n i c P2^ space g r o u p . I n one, t h e u n i t c e l l i s p o s t u l a t e d t o c o n t a i n two i n d e p e n d e n t c h a i n s (22-24) (model A ) , w h i l e i n t h e o t h e r , t h e c h a i n c o n f o r m a t i o n i s thought to be such t h a t a c e l l o b i o s y l u n i t does i n d e e d c o n s t i t u t e t h e r e p e a t i n g u n i t (17) (model B) . In model A, t h e t w o - f o l d h e l i c a l conformation i s assumed, i . e . a l l atoms ( i n c l u d i n g C I c a r b o n atoms) a r e c o n f o r m a t i o n a l l y i d e n t i c a l w i t h r e s p e c t t o t h e polymer a x i s . S i n c e t h e c h a i n s a r e l o c a t e d on t h e c r y s t a l l o g r a p h i c axes o f symmetry t h e y may have d i f f e r e n t c o n f o r m a t i o n s . Model A h a s an antiparallel arrangement, i n agreement with our finding on c e l l o t e t r a o s e . T h e r e f o r e , e a c h atom i n a c h a i n would have a d i f f e r e n t p a c k i n g environment than t h e c o r r e s p o n d i n g atom i n t h e n e i g h b o r i n g c h a i n s . Can such f e a t u r e s , i n v o l v i n g d i f f e r e n c e s i n d i s t a n c e s g r e a t e r than 3 A, e x p l a i n t h e s i g n i f i c a n t s p l i t t i n g o f the C I r e s o n a n c e s on t h e NMR s p e c t r a ? Why i s s i m i l a r s p l i t t i n g not observed f o r the other carbon atoms ? In model B, a c e l l o b i o s y l u n i t c o n s t i t u t e s t h e r e p e a t i n g u n i t (11), i n agreement w i t h t h e r e s u l t s o f c o n f o r m a t i o n a l a n a l y s i s p e r f o r m e d on i s o l a t e d molecules. Therefore, two p a i r s o f g l y c o s i d i c torsion angles a l t e r n a t e a l o n g t h e polymer c h a i n , t h e r e b y p r o v i d i n g a r a t i o n a l e x p l a n a t i o n f o r t h e o b s e r v e d s p l i t t i n g o f t h e C I r e s o n a n c e s . No l o n g e r c a n t h e m a c r o m o l e c u l a r c h a i n have a t w o - f o l d helical symmetry and hence t h e c o i n c i d e n c e between polymer a x i s and c r y s t a l l o g r a p h i c t w o - f o l d a x i s i s f o r b i d d e n . To keep m o n o c l i n i c symmetry, t h e c e l l u l o s e c h a i n s would have t o be l o c a t e d between the c r y s t a l l o g r a p h i c axes; t h e c h a i n s a r e no l o n g e r i n d e p e n d e n t and have t o be i n a p a r a l l e l r e g i s t e r which i s n o t c o n s i s t e n t w i t h the arrangement found f o r cellotetraose and p o s t u l a t e d f o r m e t h y l - β-D c e l l o t r i o s i d e . The o n l y way would be t o c o n s i d e r t h e triclinic space group, w i t h two i n d e p e n d e n t , antiparallel c h a i n s t r a v e r s i n g t h e u n i t c e l l . T h i s would be c o n s i s t e n t w i t h a l l the f e a t u r e s d i s p l a y e d by t h e c e l l u l o s e o l i g o m e r s . T h i s would a l s o e x p l a i n why t h e (0 0 1) r e f l e c t i o n i s n o t a b s e n t from t h e X-ray f i b e r d i f f r a c t i o n p a t t e r n o f c e l l u l o s e I I as i t s h o u l d r e a l l y be i n a m o n o c l i n i c P2^ space g r o u p . Addendum : Subsequent t o t h e p r e s e n t a t i o n o f t h i s work, a r e p o r t by D.L. V a n d e r H a r t ( J . Chem. Phys. (1986) 84, 1196) e s t a b l i s h e d a f i e l d dependency f o r C NMR c h e m i c a l s h i f t s o f p o l y e t h y l e n e i n t h e s o l i d s t a t e . A t t h e 50.3 MHz f r e q u e n c y u s e d i n t h e p r e s e n t work, the c o r r e c t v a l u e i s 32.9 ppm n o t 33.6 and hence a l l c h e m i c a l s h i f t s s h o u l d be d e c r e a s e d by 0.7 ppm from t h e v a l u e s r e p o r t e d above. 1 3

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Acknowledgments The a u t h o r s a r e much i n d e b t e d t o D r . H. Chanzy o f t h i s I n s t i t u t e f o r i n i t i a t i n g some p a r t s o f t h e work r e p o r t e d h e r e and h i s u n t i r i n g s u p p o r t . A p p r e c i a t i o n i s e x t e n d e d t o D r . E. O h l e y e r f o r h i s e x p e r t i s e i n the p r e p a r a t i o n o f c o n s i d e r a b l e amounts o f the cellodextrins, and t o D r . E. Roche who made a v a i l a b l e some u n p u b l i s h e d r e s u l t s on e l e c t r o n d i f f r a c t i o n o f c e l l o t e t r a o s e . D r . B. P o p p l e t o n , Commonwealth S c i e n t i f i c and I n d u s t r i a l R e s e a r c h O r g a n i z a t i o n , Melbourne, A u s t r a l i a , k i n d l y s u p p l i e d a l i s t o f measured i n t e n s i t i e s on c e l l o t e t r a o s e . The h e l p o f D r . A. Hewatt, Institut Laue L a n g e v i n , G r e n o b l e , F r a n c e , was invaluable f o r r e c o r d i n g t h e n e u t r o n powder d i f f r a c t i o n p a t t e r n o f c e l l o t e t r a o s e . Dr. R. H. Marchessault, Xerox R e s e a r c h C e n t r e , Mississauga, Canada, gave us a c c e s s t o t h e high resolution s o l i d state NMR spectrometer. G r a n t s f o r s u p p o r t i n g t h e s a b b a t i c a l s t a y s o f two o f us ( I . T. and W. T. W.) were s u p p l i e d by t h e C e n t r e N a t i o n a l de l a Recherche Scientifique.

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