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8 Infrared and Raman Spectroscopy of Polysaccharides

Downloaded by UNIV LAVAL on July 14, 2014 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0045.ch008

JOHN BLACKWELL Department of Macromolecular Science, Case Western Reserve University, Cleveland, OH 44106

During the last 30 years, infrared spectroscopy has been used to obtain information about the physical structures and chain conformations of polysaccharides. In recent years, the Raman spectra have also been available, and have provided useful complementary data. These techniques have mainly been applied in conjunction with other structural methods, especially x-ray diffraction, where the vibrational data have often given information on hydrogen bonding networks and side-group orientations. This work for polysaccharides can be discussed in two general areas. Firstly, there are the direct structural investigations, which have utilized the identifiable group frequencies. The 0-H, C-H, and carboxyl stretching frequencies, as well as some of the amide modes ,can be identified and their infrared dichroisms determined. Hence, Marrinan and Mann (1) and subsequently Liang and Marchessault (2,3) showed that the four polymorphic forms of cellulose had different spectra in the 0-H stretching region, indicative of different hydrogen bonding in their crystal structures. Based on the dichroisms of the 0-H and C-H stretching bands, these authors discussed the possibilities for hydrogen bonding and selected what they considered the most likely structures. Similarly for chitin, (4,5) the orientation of the amide side chain relative to the fiber axis was determined from the dichroisms of the amide I and II bands. Secondly, known conformations of polysaccharides can often be differentiated by their I.R. and Raman spectra. Apart from the stretching frequencies listed above, most of the bands in polysaccharide spectra are due to complex molecular motions and structural interpretation of their dichroisms is not possible at this time. Nevertheless, despite this lack of understanding, changes in frequency or intensity can be used to follow polymorphic transitions. For example, the transition from cellulose I to cellulose II during mercerization has-been followed by monitoring four intensities in the1500-800cm range (6,7). -1

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In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.


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T h i s second aspect: i d e n t i f i c a t i o n of known conformations, i s probably the major area f o r p o t e n t i a l s t r u c t u r a l work on polysaccharides using t h i s technique. Raman spectroscopy and the r e c e n t l y developed F o u r i e r transform I.R. method, allow the s p e c t r a of polysaccharides i n s o l u t i o n t o be recorded at r e s o l u t i o n s comparable to the s o l i d s t a t e s p e c t r a . As a r e s u l t , i t i s p o s s i b l e to compare the s o l u t i o n s p e c t r a with those of known s o l i d s t a t e s t r u c t u r e s and hence a s s i g n a conformation to the p o l y saccharide i n s o l u t i o n or i n g e l s , i n a manner analogous to i d e n t i f i c a t i o n of polypeptide conformations i n s o l u t i o n using c i r c u l a r dichroism. In t h i s paper I w i l l review some of the progress we have made i n the l a s t few years i n a n a l y s i s o f a v a r i e t y of p o l y saccharide systems. Our i n i t i a l work on the polymorphic forms of amylose l e d on to s t u d i e s of the s p e c t r a of o r i e n t e d f i l m s of connective t i s s u e glycosaminoglycans and hence t o our present i n t e r e s t i n b a c t e r i a l polysaccharides i n s o l u t i o n . In a d d i t i o n , we have made t h e o r e t i c a l p r e d i c t i o n s of polysaccharide s p e c t r a using normal coordinate a n a l y s i s . Amylose Amylose (a(l,4)-D-glucan) i s the simplest polysaccharide which can be c r y s t a l l i z e d i n d i f f e r e n t chain conformations. P r e c i p i t a t i o n from organic s o l v e n t s leads to the s o - c a l l e d V-amylose s t r u c t u r e , (8,9) where the chains form compact h e l i c e s with s i x glucose residues per t u r n r e p e a t i n g i n 8.0Â. A v a r i e t y of chain packings are p o s s i b l e , depending on the degree of h y d r a t i o n of the presence of organic solvent molecules, but the b a s i c chain conformation i s b e l i e v e d t o be the same. When Vamylose i s maintained at high humidity f o r a p e r i o d of time, conv e r s i o n occurs to one or other of the s t r u c t u r e s found i n n a t i v e s t a r c h , A- and B-amylose, which again a r e b e l i e v e d t o be d i f f e r ent packings of a common chain conformation. The proposed conformation f o r B-amylose (10) i s a more extended 6^ h e l i x , repeating i n 10.4Â. Double h e l i c e s have a l s o been considered, t i l ) but such s t r u c t u r e s w i l l a l s o i n v o l v e more extended chains than occur i n V-amylose. The Raman spectrum o f V-amylose (12) i s shown i n Figure 1. The spectrum f o r B-amylose i s very s i m i l a r , except f o r four small but s i g n i f i c a n t d i f f e r e n c e s , which are shown i n Figure 2 : _ - l i n e s at 946 and 1263cm" f o r V-amylose s h i f t t o 936 and 1254cm~ r e s p e c t i v e l y i n the^B-form, and the r e l a t i v e i n t e n s i t i e s of l i n e s at 1334 and 2940cm are decreased with respect t o t h e i r neighbors (12). Based on our own C-H and 0-H deuterium exchange experiments, three of^the l i n e s i n question can be assigned as f o l l o w s . The 2040cm _ ^ i s probably a CH^ antisymmetric s t r e t c h i n g mode; those at 1334cm and 12£3cm are mixed -CHÔH deformation modes. For the mode at 946cm , from a study of tne s p e c t r a of glucose monomers and oligomers t h i s i s assigned as a l i n k a g e mode,



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Raman spectrum of V -amylose in the region 1500-300 cm' (12) 1

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i . e . a complex mode i n v o l v i n g a s i g n i f i c a n t c o n t r i b u t i o n from motion of the g l y c o s i d i c C-O-C. The V - s t r u c t u r e has compact h e l i c e s i n which residues on successive turns ( i . e . residues i and i+6) are l i n k e d by i n t e r turn hydrogen bonds i n v o l v i n g the -CHÔH s i d e chains. On conversion to the B-form, the chain becomes more extended and these i n t e r t u r n hydrogen bonds w i l l be broken. T h i s w i l l probably l e a d to a r e o r i e n t a t i o n of the s i d e chains and the formation of other hydrogen bonds, e.g. to water molecules. Such changes would be l i k e l y to a f f e c t the frequency and i n t e n s i t y of the -CHÔH modes and would account f o r the changes seen. At the same time, expansion of the chain w i l l be e f f e c t e d by r o t a t i o n of the residues about the g l y c o s i d i c l i n k a g e s , which^would f i t i n with the observed frequency change f o r the 946cm linkage mode. In the s t r u c t u r e proposed f o r B-amylose, (11) the i n t e r turn bond i s broken and reformed through a water molecule, and the f i b e r repeat i s increased by r o t a t i o n of the residues about the g l y c o s e d i c bonds, which i s compatible with the observed Raman changes. Normal coordinate a n a l y s i s of the i s o l a t e d Vamylose chain p r e d i c t s complex deformation modes which are i n accord with the above assignments. (13) Increase i n the f i b e r repeat of the h e l i x to_J0.4Â reduces the frequency of the " l i n k a g e " mode by 4 cm . The above Raman c h a r a c t e r i s t i c s f o r V- and B- amylose can be used to i n t e r p r e t the s p e c t r a of t h i s polymer i n s o l u t i o n . Figure 3 shows the Raman spectrum of amylose i n deuterated DMSO.(12) Only a short region of the spectrum can be recorded, but the s p e c t r a l c h a r a c t e r i s t i c s are those of the B-form, with the observed frequency at 1254cm_^ and r e l a t i v e l y low r e l a t i v e i n t e n s i t y f o r the l i n e at 1334cm . These r e s u l t s are against the presence of the V - h e l i x i n s o l u t i o n , which i s i n t e r e s t i n g s i n c e the V - s t r u c t u r e i s formed when f i l m s are cast from t h i s solvent. This i s not to say that B - h e l i c e s are present i n s o l u t i o n s i n c e we b e l i e v e that random, s o l v a t e d amylose may show the same c h a r a c t e r i s t i c s . However, i t i s l i k e l y that the CHÔH groups are hydrogen bonded to solvent molecules r a t h e r than being involved i n i n t e r t u r n bonds on compact V - h e l i c e s . Glycosaminoglycans We are i n the process of extending t h i s type of work to the glycosaminoglycans of connective t i s s u e , each of which can be prepared as o r i e n t e d f i l m s i n a number of d i f f e r e n t chain conformations, depending on the r e l a t i v e humidity and type of counter ions. In c o l l a b o r a t i o n with E.D.T. Atkins and coworkers at U n i v e r s i t y of B r i s t o l , we have prepared c r y s t a l l i n e f i l m specimens of h y a l u r o n i c a c i d , c h o n d r o i t i n 4- and 6 - s u l f a t e s , and dermatan s u l f a t e . Raman s p e c t r a could not be obtained due to fluorescence of the specimens i n the l a s e r beam. However, using F o u r i e r transform techniques we have been able to record the



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i n f r a r e d s p e c t r a o f the same o r i e n t e d f i l m s (14) as were prepared f o r x-ray work. Figure 4 shows the p o l a r i z e d i n f r a r e d s p e c t r a of c h o n d r o i t i n 6 - s u l f a t e , prepared i n the 8^ h e l i c a l conformation. T h i s polymer approximates t o a repeating d i s a c c h a r i d e of N - a c e t y l D-galactosamine 6 - s u l f a t e and D-glucuronic a c i d , with a l t e r n a t i n g 8(1,4) and 8(1,3) l i n k a g e s , and~has e i g h t d i s a c c h a r i d e s r e p e a t i n g i n three turns, with a r i s e per residue of 9.8Â (15). The s p e c t r a i n Figure 4 show perpendicular dichroism f o r the amide I and I I modes a t 1650 and 1560cm r e s p e c t i v e l y , i n d i c a t i n g that the plane of the amide group i s approximately perpendicular t o the chain a x i s . S i m i l a r l y the antisymmetric and symmetric carboxyl s t r e t c h i n g frequencies a t 1620 and 1420cm respectively, both have s l i g h t perpendicular dichroism and the plane of the carboxyl group i s more n e a r l y perpendicular t o the c h j i n a x i s . The bands with p a r a l l e l dichroism i n the 1200-lOOOcm range are complex C-0 and C-C s t r e t c h i n g modes. The dichroism i s analogous to that f o r c e l l u l o s e i n the same range, and i s c h a r a c t e r i s t i c of extended chain polysaccharides. We have a l s o prepared c h o n d r o i t i n 4 - s u l f a t e and dermatan s u l f a t e , each i n the 3- conformation, (16,17) and two forms of h y a l u r o n i c a c i d , both 4^ conformations with d i f f e r e n t f i b e r repeats (18,19). These give s i m i l a r r e s u l t s t o those f o r c h o n d r o i t i n 6 - s u l f a t e f o r the amide o r i e n t a t i o n . For the two forms of h y a l u r o n i c a c i d , and c h o n d r o i t i n 4 - s u l f a t e however, the carboxyl symmetric s t r e t c h i n g band has p a r a l l e l dichroism. These conformations a r e l e s s extended than the 8^ form of C6S, and the C-C0Ô bond can be o r i e n t e d so that i t i s more n e a r l y p a r a l l e l t o the chain a x i s . The same band has perpendicular dichroism f o r dermatan s u l f a t e , which i s c o n s i s t a n t with the CI chain f o r the L - i d u r o n i c a c i d residue of t h i s polysaccharide (17). So f a r we have only examined h y a l u r o n i c a c i d prepared i n two d i f f e r e n t conformations, both 4^ with d i f f e r e n t f i b e r repeats. These specimens do not show any s p e c t r a l d i f f e r e n c e s which can be a s c r i b e d t o the d i f f e r e n c e i n conformation. T h i s i s disappoint i n g , but such d i f f e r e n c e s are more l i k e l y when there are l a r g e r d i f f e r e n c e s i n conformation, e.g. between 3-, 8^, and 4- h e l i c e s . These i n v e s t i g a t i o n s are continuing, and w i l l be a p p l i e d t o s o l u t i o n s i f the d i f f e r e n t conformations can be s u c c e s s f u l l y differentiated. Bacterial

Polysaccharides

More r e c e n t l y we have examined the b a c t e r i a l polysaccharide xanthan, working with specimens obtained from Drs. A. Jeanes and P.A. Sandford at U.S.D.A., P e o r i a . T h i s polysaccharide i s b e l i e v e d t o be a repeating p o l y s a c c h a r i d e , the backbone i s a 8 ( l , 4 ) - g l u c a n with a l t e r n a t i n g residues having a t r i s a c c h a r i d e of mannose 6-acetate, g l u c u r o n i c a c i d , and mannose; approximately 50% of the t e r m i n a l mannose residues have a peruvate residue attached at the 4 and 6 p o s i t i o n s .


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Figure S. Raman spectrum of amylose in deuterated DMSO solution (Me SO — d ) in the 1500-1200 cm' re gion. ( ) indicates the approximate base line for scattering by the solvent 2


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Polarized infrared spectra for the 8 conformation of chondroitin 6-sulfate. ( )A ;(—)A (U). S

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This p o l y s a c c h a r i d e has a very i n t e r e s t i n g property i n that the v i s i o s i t y of an aqueous s o l u t i o n undergoes a sudden i n c r e a s e as the temperature r i s e s (20). I t i s argued that the polysacchar i d e has a compact conformation at low temperatures and undergoes a t r a n s i t i o n to an expanded form at a s p e c i f i c temperature, reported at 55°C. This t r a n s i t i o n has been followed by Rees and coworkers (21) by N.M.R., which i n d i c a t e s an ordered conformation below 55°C and a d i s o r d e r e d random c o i l at higher temperatures. These workers have a l s o followed the change by c i r c u l a r dichroism spectroscopy . We have used F o u r i e r transform i n f r a r e d spectroscopy to i n v e s t i g a t e these thermal changes (22). The specimens were d i a l i z e d thoroughly against d i s t i l l e d water p r i o r to r e c o r d i n g the s p e c t r a . The s p e c t r a of a 1% xanthan s o l u t i o n at d i f f e r e n t temperatures are shown i n F i g u r e 5. No obvious d i f f e r e n c e s are seen i n the frequencies of the observed bands, but there i s a general broadening at higher temperatures, i n d i c a t i n g development of a l e s s ordered s t a t e . T h i s broadening can be quantized i n a v a r i e t y of ways; one convenient method i s to measure the areas of the peaks above the unresolved background. P l o t s of these " i n t e n s i t i e s " against temperature f o r three of the bands are shown i n Figure 6. A l l three show a sigmoidal t r a n s i t i o n , with midpoint at 40°C, i n d i c a t i n g development of a more random conformation above t h i s temperature. A d d i t i o n of s a l t s to the xanthan s o l u t i o n i s known to p r e vent the t r a n s i t i o n i n the v i s c o s i t y (20). F i g u r e 7 shows the i n f r a r e d s p e c t r a of a 1% xanthan s o l u t i o n i n 1% KC1 over the same temperature range as i n Figure 5. The c o n t r a s t between Figures"5 and 7 i s q u i t e s t r i k i n g i n that the s p e c t r a of the s a l t s o l u t i o n s show very l i t t l e change with temperature. Our observations of a t r a n s i t i o n at 40°C i s p u z z l i n g s i n c e other workers have reported 55°C. We have a l s o performed v i s c o s i t y and CD measurements on s o l u t i o n s of t h i s polysacchar i d e , and observe t r a n s i t i o n s with midpoints of 38° and 40°. I t i s p o s s i b l e that our specimen of xanthan i s d i f f e r e n t from those used by other workers, perhaps due to mutation or degradation, or that we have achieved a lower i o n i c s t r e n g t h when the specimen was d i a l y s e d against water. Acknowlegements T h i s work was supported by N.S.F. Grant No. GB 32405. am indebeted to my c o l l a b o r a t o r s i n Cleveland and B r i s t o l , e x p e c i a l l y J . J . C a e l , J . Southwick, and J.L. Koenig f o r t h e i r part i n the work described above.


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Abstract Progress in several areas is described in the application of vibrational spectroscopy to investigate the structure and confor mation of polysaccharides. Infrared and Raman spectroscopy pro vides information on the orientation of side groups and the type of hydrogen bonds formed in crystalline polysaccharide structures. In addition, spectral characteristics of polysaccharides prepared in different known crystal structures can be used to investigate the conformation in solution. These methods have been applied to investigations of amylose, where differences in the Raman spectra of the V- and B- forms have been interpreted in terms of the change in conformation, and indicate that the V-conformation is not present in solution. Fourier transform infrared spectra of oriented crystalline films of the connective tissue glycos aminoglycans have been used to determine the orientation of the amide and carboxyl groups for the various crystal structures. Finally, infrared spectra of xanthan in solution show that an order-disorder transition occurs as the temperature is increased, which is correlated with the sharp increase in viscosity in the same temperature range. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Marinan, H.J. and Mann, J., J. Polymer Sci. (1958), 32, 357. Liang, C.Y. and Marchessault, R.H., J. Polymer Sci., (1959), 37, 385. Marchessault, R.H. and Liang, C.Y., J. Polymer Sci., (1960), 43, 31. Darmon, S.E. and Rudall, K.M., Disc. Farad. Soc., (1950), 9, 215. Carlstrom, D., J. Biophys. Biochem. Cytol.,(1957),3,669. McKenzie, A.W. and Higgins, H.G., Svensk Papperstidn., (1958) 61, 893. Hurtubise, F.G. and Krassig, Η., Anal. Chem., (1960), 32, 177. Rundle, R.F. and French, D., J. Amer. Chem. Soc., (1943), 65, 558. Zobel, H.F., French, A.D., and Hinkle, M.E., Biopolymers, (1967), 5, 837. Blackwell, J., Sarko, Α., and Marchessault, R.H., J. Molec. Biol., (1969), 42, 379. Kainuma, K. and French, D., Biopolymers, (1972), 11, 2241. Cael, J . J . , Koenig, J.L., and Blackwell, J., Carbohydrate res., (1973), 29, 123. Cael, J . J . , Koenig, J.L., and Blackwell, J., Biopolymers, (1975), 14, 1885. Cael, J . J . , Isaac, D.H., Blackwell, J., Koenig, J.L., Atkins, E.D.T., and Sheehan, J.K., Carbohydrate Res. in press. Arnott, S, Guss, J.M., Hukins, D.M., and Mathews, M.B., Science, (1975), 180, 743.


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Isaac, D.H. and Atkins, E.D.T., Nature (London), New Biol. (1973), 244, 252. 17. Atkins, E.D.T. and Isaac, D.H., J . Molec. B i o l . , (1973), 80, 773. 18. Dea, I.C.M., Moorhouse, R., Rees, D.A., Guss, J.M., and Balazs, E.A., Science, (1973), 179, 560. 19. Guss, J.M., Hukins, D.W., Smith, P.J.C., Winter, W.T., Arnott, S., Moorhouse, R., and Rees, D.A., J . Molec. B i o l . , (1975), 95, 359. 20. Jeanes, Α., Pittsley, J . E . , and Senti, Α., J . Appl. Polymer S c i . , (1961), 17, 519. 21. Rees, D.A. and Morris, Ε., (in press). 22. Southwick, J., Koenig, J.L., and Blackwell, J., (in press).


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