Biomolecular structures of naturally occurring carbohydrate polymers

Biomolecular structures of naturally occurring carbohydrate polymers Edward Atkins H.H. Wills Physics Laboratory, University of Bristol, Tyndall Avenue, Bristol BS8 ITL, UK

(Received 29 September 1986) The bonding mechanism in proteins and polysaccharides is compared and contrasted. Some of the structures for polyglucose are shown to mimic the classic fl-sheet, ~t-helix and collagen helices found in proteins. The effect of glycosidic linkage geometry on polysaccharide shape and structure is discussed together with the effect of changes in the geometry of the monomer units. Multistranded ropes and the effect of non-carbohydrate groups are mentioned briefly. Keywords: Carbohydratepolymers;bonding mechanism;helices;fl-sheets

Introduction Unlike their protein counterparts in the world of macromolecules the carbohydrate polymers utilize a wide variety of condensation linkage geometries. Figure 1 illustrates the basic types of linkages available in the proteins and those polysaccharides based on hexoses. The torsional angle pairs, 0, ~, of each swivel joint are influenced by interactions between atoms in the vicinity of the linkage. The proteins, with one type of condensation linkage (neglecting disulphide bridges and the like), are influenced and even obsequious in part to the amino acid sequence or R group appendage. Only a few regular structures are found naturally in the proteins: the fl-chain, stabilized by interchain hydrogen bonding and often occurring in sheets or stacks of sheets, the or-helix, stabilized by intrachain hydrogen bonding and forming a reasonably sturdy structure on its own, and finally the triple-strand rope of collagen where three helical chains (with threefold symmetry) drape around a common axis to create a robust structure, but one which demands glycine (R group being hydrogen) on every third residue. A large gap exists between these few regular structures and the complex folded globular protein structures, usually with a non-repeating amino acid sequence. Although the fl-chain and ~-helix can accommodate a variety of R groups, because both conformations dispose the R groups away from the backbone there is the inherent tendency for protein chains to be driven towards complex three-dimensional folded structures as the information content is increased, i.e. as the amino acid sequence becomes more diverse. Also there is little postpolymerization modification in proteins. This short review is about carbohydrate structures and I now wish to highlight just some of the many features of polysaccharide conformation against the backcloth of the proteins, which after all are their most conspicuous rivals in the world of naturally occurring macroPresented in part at BiologicallyEngineered Polymers Conference, Churchill College,Cambridge, 21-23 July 1986. 0141-8130/86/0603234)7503.00 © 1986 Butterworth& Co. (Publishers) Ltd

molecules. The variety of condensation linkages imparts an extra dimension to understanding the shapes and interactions of carbohydrate molecules. When this extra dimension of glycosidic linkage geometry is coupled with a variable chemical sequence of saccharides, chosen from a reservoir of some 30-40 different saccharide monomers, a wonderful world of shapes and architectures is generated. If the shape of the puckered six-membered ring is changed--by alteration in the chemistry of side groups for example--the spatial arrangement of glycosidic linkage bonds will change and the polymer conformation

R I ~ C ~o o

, "/N /c

N\

a (.~t0

CH2

b Figure I Schematic diagram of bonding. (a) Amino acids in proteins, with only one type of condensation linkage (R represents side group) and (b) saccharide units in standard 4C~ chair with various types and combinations of linkage geometry. Bonds directed approximately parallel to ring are called equatorial (e) and bonds directed approximately perpendicular to ring are called axial (a)

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Structures of carbohydrate polymers: E. Atkins will undergo a transformation with subsequent consequences on macromolecular interactions, properties and behaviour. Thus, a liaison exists between the chemistry, chemical modification and structure and interactions and an almost continuous spectrum of complexity in structure can exist from the simple to the exceedingly complex, the full appreciation of which is still over the horizon. Three conformations of polyglucose Let us consider three examples (and of course there are more!) of the chemically straightforward homopolymers of glucose which we can call the polyglucoses. The commonly occurring cellulose has glucose units linked diequatorially (see Figure 1) through carbon atoms at ring positions 1 and 4 (i.e. le-4e). This glycosidic linkage geometry generates a rather extended, gently undulating ribbon-like chain with twofold helical symmetry which usually interacts with adjacent parallel chains to form hydrogen-bonded sheets. Such sheets stack to form three-dimensional crystallites I (Figure 2a) and exhibit features closely similar to the classic flstructures found in particular in the protein fibrous silks. Bundles of cellulose chains in this stacked sheet arrangement form microfibrils of about 20nm in size which are the morphological units in plant cell wall surrounded by a less organized matrix of cellulose, xyloglucans or mixtures of other carbohydrate polymers to create a composite material with desirable, physical,

biological and biochemical properties. The second example is amylose, a constituent of starch. In this case the linkage is la-4e. Although the glycosidic linkage occurs again through carbon atoms in ring positions 1 and 4 the axial disposition of the bond from C(1) affects the local stereochemistry and has a dramatic effect on the polymer conformation. Figure 2b illustrates the geometry of a particular polymorph of amylose 2. The polysaccharide chain traces out a hollow helical tube-like structure which is stabilized by intrachain hydrogen bonds between successive turns of the helix. This structure mimics the commonly occurring s-helix found in proteins. These self-stabilizing amylose tubes do not favour such strong interchain associations as are evident in cellulose and entirely different textures and properties are observed. The third and final example in this theme is provided by an extracellular microbial polysaccharide which is used as a gelling polymer and is known as curdlan. The glucose units are linked through ring atom positions 1 and 3 diequatorially, i.e. le-3e. The molecule consists of three chains with the same polarity twisting in unison, and 120° out of phase respectively, around a common axis. Thus the molecule is a three-strand, solid rope-like molecule 3 with individual chains fitting neatly together as illustrated in Figure 2c. A triad of interchain hydrogen bonds occurs along the core of the molecule at approximately 0.3 nm intervals yielding a very stable structure. The structure of curdlan bears a striking architectural resemblance to collagen.

,i

a

b

c

Figere 2 Threestructures for polyglucose. (a) Cellulose, le--4e linked polyglucose is a gentle undulating ribbon-like polymer. The conformation is similar to the/%conformation in proteins. (b) Amylose, 1a--4e linked polyglucose in the V form is a hollow helical tube. The conformation is stabilized by intrachain hydrogen bonds and resembles the ~t-helix in proteins. (c) Curdlan, le-3e linked polyglucose forms a triple-strand rope stabilized by buried hydrogen bonds in the core. The conformation bears a striking resemblance to the protein collagen

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Structures of carbohydrate polymers: E. Atkins Glycosidic linkage geometry versus chemistry In the three examples outlined in the previous section the chemistry remains constant while the glycosidic linkage geometry changes. What happens in situations where the chemistry of the saccharide units is changed while the glycosidic linkage is maintained? Consider the le-4e linkage geometry first (i.e. similar to cellulose). Poly-Nacetylglucosamine (chitin), mannan, polymannuronic acid, yield similar twofold extended conformations and patterns of behaviour ~-6. Curdlan has a le-3e linked xylan counterpart, which occurs in siphoneous green algae, and which also exists as an almost identical triplestrand rope. In fact the elucidation of the model was first undertaken on xylan ~'s and it was interesting to find that the change in chemistry, from xylose to glucose, did not change the glycosidic linkage geometry or the desirability to form a rope-like macromolecule. Scleroglucan, which is a polysaccharide similar to curdlan but decorated with a glucose side branch on every third residue in the backbone also exists as a three-strand rope. It is useful to

note that xylan in algae and curdlan from bacteria are biosynthesized in entirely different biological systems, yet both develop the same ingenious hierarchical structure. A similar control of polymer conformation by glycosidic linkage geometry is evident in comparisons between the connective tissue polydisaccharides hyaluronic acid, the chondroitin sulphates and the microbial extracellular polysaccharides from pneumococcus type III and Klebsiella K25, all of which exhibit threefold helical structures 9. These polymers have backbones linked le-3e and le-4e in an alternating manner although the chemistry of the saccharide units changes considerably and Klebsiella K25 has an additional disaccharide side appendage ~o. Although glycosidic linkage geometry has a strong influence and places constraints on polymer conformation it does not have complete authority. The polyelectrolytic nature of the connective tissue polysaccharides, for example, means that the conformation is sensitive and responsive to the environment these molecules find themselves in and many polymorphs have been observed ~1. Modifications to cellulose, either chemically induced by forming cellulose nitrate or hydroxypropylcellulose, or biosynthetically by the addition of a hefty trisaccharide side appendage on alternate glucose units (formula I) as in xanthan affects the conformation. Cellulose nitrate forms a 52 helix 12 as illustrated in Figure 3 and hydroxypropylcellulose becomes a threefold helix ~3 shown in Figure 4. The X-ray fibre diffraction pattern of xanthan gum (Figure 5) indicates a fivefold symmetry for the molecule. This perturbation from the twofold helical cellulosic backbone has important consequences for chain--chain interactions and a number of these polymers exhibit liquid-crystalline properties and behaviour t3.

Change in shape of saccharide unit

2.54 nm

It has been pointed out in the introduction how changes in the disposition of the bonds to the saccharide ring dramatically alter polymer conformation. Of course the ring itself is not a rigid body although in general for a particular chemistry (of side groups and their dispositions) the hexoses usually prefer a standard geometry. For glucose this is the 4C 1 chair 14 as illustrated in Figure 6. If the chemistry of the saccharide unit is changed the energetics of the puckered ring have to be reconsidered and the ring might flip into a different puckered arrangement, for example into the alternative

--~4)-fl-D-Glcp-(1~ 4)-fl-D-Glcp-(1--,

±

3 T 1

I

//-D-Manp-(1 --~4)-~-D-GlcAp-(1 --~2)
/\ 4

Figure 3 Projections of the 52 helix (fiveunits in two turns) of cellulose nitrate. Note the azimuthal rotation from the cellulosic two-fold helix is only 35°

6

\/ /\

C

CH a COOH formula I

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T

1.51 n m

_L

Figure 4 Threefold model of hydroxypropylcellulose. The irregular substitution of side groups suppresses side-to-side crystallization and imparts liquid-crystalline properties to the molecule (courtesy of Dr W. S. Fulton)

Alginate is a polyuronide found in brown algae and certain bacteria 15 and is extensively used industrially utilizing its characteristic gelling behaviour in the presence of divalent ions, particularly calcium. It was established by H~iug and coworkers 16 that alginic acid consists of blocks of fl-D-mannuronic acid (M) and C~-Lguluronic acid (G), diagrams of which are shown in Figure 7. The polymer is biosynthesized as polymannuronate and then sections of the polymer are epimerized with an enzyme which results in mannuronate being converted to guluronate such that runs of (-G-), occur in (-M-),. This is also an example where postpolymerization modification occurs in polysaccharides, other examples being in chondroitin sulphate, carrageenan and heparin. X-ray diffraction was used to establish the conformations of both polymannuronic acid 17 and polyguluronic acid is which enabled the gelation mechanism to be understood at atomic resolution. The mannuronate unit has one group, the hydroxyl group in the 2 position, axially disposed when in the normal 4C 1 chair (Figure 7a). The shape of the polymannuronic acid chain is similar to that expected for a le-4e linked homopolymer: a gentle rippling twofold helical ribbon (see Figure 8a) as mentioned previously. The epimerase moves the carboxylate group in the 5 position from an equatorial disposition to an axial disposition. The saccharide ring now has two groups axially placed: the carboxylate and the hydroxyl group at position 2. X-ray results ~8 show that the ring flips from the 4C~ chair to ~C4 chair so that both these groups become equatorially placed (Figure 7b). However the linking bonds at positions 1 and 4 change from diequatorial to diaxial with resultant change in the polymer shape, giving rise to a pronounced zig-zag appearance (Figure 8b) and a reduction of the chain periodicity of about 17 ~o. The X-ray structure refinement of polyguluronic acid 18 provides evidence for the incorporation of a water molecule into the largely 4

0

5

I

1

4

4C 1

lC4

Figure 6 The normal 4C~ and alternative ~C4 conformations of the pyranoid ring. The reference planes are indicated by dots and the carbon atoms numbered sequentially

OH

,coo Figure 5 X-ray fibre diffraction pattern of xanthan gum showing a meridional signal at spacing 0.95 nm on the fifth layer line indicating the fivefold symmetry of the molecule (courtesy of Dr K. Veluraja) ~C4 chair (see Figure 6) or various other shapes ~4. This occurs in a number of polysaccharides, but the obvious example to choose to illustrate the effect and subsequent consequences is alginate.

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OH

o:, OH

a

b

Figure 7 Repeating monosaccharides in alginate (a) fl-Dmannuronic acid in normal 4C 1 chair. Glycosidic linkages are diequatorial via 1 and 4 positions. (b) a-L-guluronic acid in alternative 1C4 chair. Glycosidic linkages are diaxial via 1 and 4 positions

Structures of carbohydrate polymers: E. Atkins

tim

tim

ingredient of the gelation mechanism and used to account for the change in optical rotation. An alternative model based on side-by-side association of two carrageenan chains has been proposed 26 to overcome the topological concern although such a model is not supported by X-ray diffraction studies on oriented fibres. Gellan gum is a recently discovered extracellular polysaccharide. The native material contains approximately 6 wt % of O-acetyl groups. It forms weak gels whilst the deacetylated polysaccharide forms stiff, brittle gels. The primary structure 27'28 has a linear tetrasaccharide repeat (formula II). The X-ray diffraction patterns of oriented native and deacetylated samples show the same general features 29 although the latter is much more crystalline. 3)-fl-D-Glcp-(l ---*4)-fl-D-GlcpA -(1 ---,4)-fl-D-Glcp-(1 ---*4)-Ct-L-Rhap-(l ---,

formula II

a

b

Figure 8 Projections of the chain conformations of alginate in directions perpendicular (top) and parallel (bottom) to chain axis. (a) Twofold helix for polymannuronic acid. (b) Twofold helix for polyguluronic acid. Note the pronounced zig-za'gshape compared with that of (a), and the contraction of the molecular dimensions from 1.04 to 0.87 nm

hydrophobic pocket created between juxtapositioned zigzag chains. This feature of the structure has been used to speculatively propose 2° the so-called 'egg-box' model for incorporating divalent counterions such as calcium into the guluronate rich parts of alginate and creating junction zones in the gel as illustrated schematically in Figure 9.

The first meridional diffraction signal occurs at a spacing of 0.94 nm which is less than half the expected value for a fully extended tetrasaccharide repeat. Although the model still needs to be refined to obtain a better fit with the intensity data a threefold double-helix, with the chains displaced by half the pitch relative to each other is the model preferred 29, a projection of which to illustrate the general features, is shown in Figure 10. A double-helix model has also been proposed for the native polymorphs of amylose 3° which can be converted into a single helix 2 in such reagents as DMSO; however this is apparently a non-reversible process. Clearly multistrand ropes exist in polysaccharides and I suspect more will be found, especially amongst the new microbial polysaccharides now being investigated. It would appear that Nature biosynthesizes multistrand ropes in an attempt to create more robust, less flexible molecules. Certainly we know on general grounds that the stiffer the macromolecule--the more rigid the rod in solution--the lower is the concentration for the transition from isotropic liquid to the liquid-crystalline phase. Since

Intertwining double helices Multistranded ropes in polysaccharides were discovered 21, their proper characters established 7'a and their details analysed using good quality X-ray fibre diffraction patterns in the 1960s. Double-strand ropes were proposed for carrageenan based on X-ray fibre diffraction patterns 22'23 and although the measurement of the vital intensity data essential to confirm a model is irritated by reported statistical disorder 2a in the patterns used for the analyses, which therefore adds an extra degree of uncertainty, a double helix with two similar chains axially displaced by half the pitch is favoured on Xray diffraction grounds, at least for iota-carrageenan. The untwining, or more to the point, the retwining of chains as a function of temperature to form junction zones in gelation 24 is difficuB to visualize from a topological point of view. Although the original rather naive model for gelation has been replaced with aggregation* of doublehelix molecules 25 as the basic mechanism for gelation, the single chain to double helix transition is still an * J u n c t i o n zones based o n a g g r e g a t i o n is a n old concept and is a k i n to the 'fringed micelle' m o d e l found in m a n y texts on synthetic polymers.

Figure 9 Schematic representation of a guluronate junction zone in alginate, the egg-box model. The filled circles represent calcium ions

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Structures of carbohydrate polymers: E. Atkins interaction between the chains increases locking the molecule into a stiff double-strand rod which will (at that concentration) interact and aggregate to form gels. It is interesting to note, for example in the case ofcarrageenan, that single chains have never been captured (with regular conformations) in the solid state and therefore a mechanism of loosely bound to tightly bound doublestrand ropes might offer a solution.

Effect of acetylation

i~~....

i~i

///I

Figure 10 Trial model structure of the double-helix model for geilan gum. The chains are displaced relative to each other by half the pitch (5.64 nm) in order to cancel completely odd-order layer lines of this spacing resulting in layer lines which are orders of 2.82 nm

for long-chain molecules liquid-crystalline formation and gelation can be different facets of the same phenomenon (gels being created due to chains connecting through from one mesophase domain to others), stiffer molecular ropes will interact at lower concentrations giving rise to gelation with lower levels of polymer present. On general, perhaps speculative, grounds a loosely coupled doublestrand rope could be flexible in solution, yet when conditions change, say by lowering the temperature, the

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Two polysaccharides, the capsular polysaccharide produced by Klebsiella aerogenes serotype K54 and Enterobacter (NC1B 11870), known also as XM6, have similar chemical structures 31'32. The structure for K54 is given by formula III and consists of an octasaccharide repeat. K54 is similar except it does not have the O-acetyl group attached to every other fucose unit and so the formal chemical repeat reduces to a tetrasaccharide repeat. The X-ray fibre diffraction patterns are shown in Figure 11. As can be seen a considerable difference exists: the deacetylated K54 (similar to XM6) pattern is highly crystalline while the K54 pattern exhibits rather less crystallinity. In collaboration with Drs V. J. Morris and M.J. Miles at the Food Research Institute, Norwich my research student Philip Attwool and I have established 33 that deacetylation of K54 in the solid state converts the K54 X-ray pattern into a highly crystalline pattern similar to XM6. It is known that XM6 will disperse in water to form a viscous solution. Addition of inorganic salts to raise the ionic strength leads to thermoreversible gelation. K54 will also disperse in water to form a viscous solution but raising the ionic strength does not lead to gelation. Thus the presence of O-acetyl groups in K54 inhibits gelation. When K54 is deacetylated it will gel on the addition of ions. X-ray diffraction results suggest that the gelation of XM6 results from aggregation (crystallization) of segments of helices. In the previous section I illustrated the effect of acetylation on gellan gum. These examples illustrate the importance of non-carbohydrate substituents on polysaccharide interactions. X-ray diffraction is a useful method to monitor the changes, both in terms of conformation and interactions, created by removing these decorating appendages.

--~ 4)-~t-D-GlcpA-(1--* 3)-~-L-Fucp-(I --~ 3)-fl-D-Glcp-(1--* 4 T

1

fl-o-Glcp 4)-~t-D-GlcpA-(1--* 3)-~t-L-Fucp-(1--~ 3)-fl-D-Glcp(1--~ 4 4 (2 or 4) T T 1 OAc fl-D-Glcp K54 and XM6 have identical glycosyl sequences. The only difference is that K54 contains one O-acetyl group attached to a fucosyl residue on every alternate tetrasaccharide. formula III

Structures o f carbohydrate polymers: E. Atkins

i~%~i!~!i~ii~iii%i~iiii!i~ii~ii~i~iiiiiii~iii~iii®!!~iiiii~iiiiii~iii~iiiiii!~iii~i~iii~i!ii!iii~i!~iiiii~i~iiii~iiiii!~!~ii~ ~iiiii I~?!~~ii!ii~ /

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

a

b

Figure 11 (a) X-ray fibre diffraction pattern of native K54 polysaccharide. (b) X-ray pattern of deacetylated K54 (in the solid state) which bears a close resemblance to the pattern obtained for XM633 (courtesy of Mr P. T. Attwooi)

Summary I n such a short review it has been possible to t o u c h o n only a few of the features a n d p r o b l e m s still unsolved which involve the m o l e c u l a r biology of c a r b o h y d r a t e polymers. It is hoped however that just those glimpses of c o n f o r m a t i o n a n d architecture will be sufficient to highlight the potential a d v a n t a g e s to be gained from a fuller u n d e r s t a n d i n g of the relationship between structure, interaction, f u n c t i o n a n d properties of these macromolecules.

11 12 13 14 15 16

Acknowledgements

17

I wish to t h a n k Philip A t t w o o l for allowing m e to show some results, in press b u t as yet u n p u b l i s h e d , o n X M 6 a n d K 5 4 a n d Drs M. J. Miles a n d V. J. Morris for useful discussions. I t h a n k the A F R C a n d S E R C for s u p p o r t i n g this work.

18

References 1 2 3 4 5 6 7 8 9 10

Gardner, K. H. and Blackwell,J. Biopolymer$ 1974, 13, 1975 Sarko, A. in 'Structure of Fibrous Biopolymers', (Eds E. D. T. Atkins and A. Keller), Butterworths, London, p. 335 Fulton, W. S. and Atkins, E. D. T. in 'Fibre Diffraction Methods', (Eds A. D. French and K. H. Gardner), ACS Symposium Series 141, 1980, 385 Gardner, K. H. and Blackwell,J. Biopolymers 1975, 14, 1581 Frei, E.andPreston, R.D. Proc. R. Soc. Lond.(B)1968,169,127 Atkins, E. D. T., Mackie, W., Parker, K. D. and Smolko, E. E. J. Polym. Sci. 1971, Bg, 311 Atkins, E. D. T., Parker, K. D. and Preston, R. D. Proc. R. Soc. Lond. B. 1969, 173, 209 Atkins, E. D. T. and Parker, K. D. J. Polym.Sci. (C) 1969.28, 69 Atkins, E. D. T. Proc. Int. Symp. Biomol. Struct. Interactions, Suppl. J. Biosci. 1985, 8 Elloway,H. F., Isaac, D. H. and Atkins, E. D. T. in 'Fiber

19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

Diffraction Methods', (Eds A. D. French and K. H. Gardner), ACS Symposium Series, 1980, 141,429 Sheehan,J. K. and Atkins, E. D. T. Int. J. Biol. Macromol. 1983, 5, 215 Meader,D., Atkins, E. D. T. and Happey, F. Polymer 1978, 19, 1371 Atkins, E. D. T., Fulton, W. S. and Miles, M. J. TAPPI Conference Papers: 5th Int. Dissolvino Pulps Symp. 1980, p. 208 Stoddart, J. F. 'Stereochemistry of Carbohydrates', WileyInterscience, New York, 1971 Percival,E. E. and McDowell, R. H. in 'Chemistry and Enzymologyof Marine Algae Polysaccharides', AcademicPress, New York, 1967, p. 107 Hiiug,A., Larsen, B. and Baardseth, E. Proc. 6th Int. Seaweed Symp. Santiago, Spain, 1969, p. 443 Atkins, E. D. T., Nieduszynski,I, A., Mackie, W., Parker, K. D. and Smoko, E. E. Biopolymers 1973, 12, 1865 Atkins, E. D. T., Nieduszynski,I. A., Mackie, W., Parker, K. D. and Smolko, E. E. Biopolymers 1973, 12, 1879 H~iug,A. and Larsen, B. Carbohydr. Res. 1971, 17, 345 Grant, G. T., Morris, E. R., Rees, D. A., Smith, P. ~I. C. and Thorn, D. FEBS Lett. 1973, 32, 195 Frei, E. and Preston, R. D. Proc. R. Soc. Lond. (B) 1964, 160, 293 Anderson, N. S., Campbell, J. W., Harding, M. M., Rees, D. A. and Samuel, J. W. B. J. Mol. Biol. 1969, 45, 85 Arnott, S., Fulmer, A., Scott, W. E., Dea, I. C. M., Moorhouse, R. and Rees, D. A. J. Mol. Biol. 1974, 90, 269 Rees,D. A. Biochem. J. 1972, 126, 257 Rees,D. A. Pure Appl. Chem. 1981, 53, 1 Smidsmd, O. and Grasdalen, H. Carbohydr. Polym. 1982,2, 270 O'Neill, M. A., Selvendran, R. R. and Morris, V. J. Carbohydr. Res. 1983, 124, 123 Jansson, P-E., Lindberg, B. and Sandford, P. A. Carbohydr. Res. 1983, 124, 135 Upstill, C., Atkins, E. D. T. and Attwool, P. T. Int. J. Biol. Macromol. 1986, g, 275 Wu, H. H. and Sarko, A. Carbohydr. Res. 1978, 61, 7 O'Neill, M. A., Morris, V. J., Selvendran, R. R., Suthedand, I. W. and Taylor, I. T. Carbohydr. Res. 1986, 148, 63 Dutton, G. G. S. and Merrifield, E. H. Carbohydr. Res. 1982, 105, 189 Atkins, E. D. T., Attwool, P. T., Miles, V. J., Morris, V. J., O'Neill, M. A. and Sutherland, I. W. to be published

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