Pure & Appi. Chern., Vol. 49, pp. 1135 1149. Pergamon Press, 1977. Printed in Great Britain.

CONFORMATIONS OF URONIC ACID CONTAINING POLYSACCHARIDES

EdwardD,T. Atkins

: H, 11. Wills Physics Laboratory, University of Bristol, Tyndall Avenue,Bristol BS8 1TL, U. K.

Abstract - Analyses of X-ray diffraction patterns obtained fromordered specimens of a variety of polyuronides have provided theopportunity to examine their molecular geometry in the condensedphase. A brief review is given of the simpler homopolysaccharidecomponents of alginic acid, followed by the connective tissuepolydisaccharides: hyaluronic acid, the chondroitin suiphates,dermatan sulphate, heparan sulphate and the blood anti-coagulantheparin. The interaction of these molecules with proteins and theirmolecular architecture in cartilage is mentioned. In addition someexamples of the bacterial capsular polysaccharides are examined.In particular the three serotypes K5, K57 and K8 from Klebsiella.

INTRODUCTION

During the past five years notable advances have been reported concerning the moleculargeometry of the uronic acid containing polysaccharides (polyuronides). The resultsobtained encompass the plant, animal and microbial domains with corresponding increasein the complexity of the repeating chemical sequences. Progress in this area hasemanated from the development of crystallization techniques, involving stress fields andannealing (Ref. 1, 2 & 3), to provide oriented, para-crystalline samples suitable for theapplication of X-ray diffraction.

X-ray diffraction methods are the central stereobate for the elucidation of thethree-dimensional shape and structure of molecules. The most detailed information is obtainedwhen the material is crystalline and in the case of long chain polymeric substances withessentially regular repeating chemical sequences, such as the polysaccharides discussedbelow, the best that can be usually achieved is to induce the molecular chains to orientin approximately parallel register and then to encourage disciplined molecular inter-connections between neighbouring chains (Ref. 1 & 2). Such an array is analogous to themolecular morphology of many natural and synthetic fibres. Indeed the study of the X-raydiffraction of fibres and in particular the polysaccharides is almost as old as the X-raydiffraction of crystals. It is with some considerable historical interest to recall,especially with regard to the venue of this Symposium, that as early as 1913, in Tokyo,Nishikawa and Ono (4) passed X-rays through the fibrous cellulosic substance asa -a kind of hemp.

Figures 1, 6, 8,15,17 & 27 are examples of X-ray diffraction patterns obtained frompolysaccharide fibres and oriented films. All the patterns exhibit diffracted intensityconfined to (approximately) horizontal layer lines which reflects the periodic nature alongthe chain direction. Thus measurement of the layer line spacing gives the pitch of thehelix. Further, for a helix with n units in one turn (i. e. per pitch (p)), diffracted intensityon the meridian (the vertical bisector of the pattern) only occurs on layer lines whoseinteger index lis a multiple of n. The quotient pJri yields the axial projection (h) whichcorresponds to the chemical repeat. Models of molecular helices incorporating theknown stereochemistry of the various saccharide units, together with the glycosidiclinkage geometries, can be computer generated while preserving the helix symmetry anddimensions as defined by the values of n and h. Additional information about themolecular structure involves the use of the full intensity data in a high resolution X-raydiffraction pattern together with more sophisticated chain packing procedures.

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I I 39 EDL'iVJ�D DJI VJIKII42

p

Conformations of uronic acid containing polysaccharides 1137

charges attendant on ionization form an electrostatic environment which modulates thebackbone conformation. Thus on formation of the univalent salt the conformation becomesa three fold left-handed helix (Fig. 3b) similar to 3, l—4 xylan (Ref. 14). Since theglycosidic bonds are close to and nearly parallel with the helix axis, the angulardistribution of side groups may be varied with little change in the axially projectedmonomer repeat. Thus although the fibre repeat alters drastically (Fig. 3b) to 1. 51 nmthe spacing of the meridional reflection 1. 51/3 = 0. 50 nm hardly changes (Fig. lb). Thisis a feature that will also be noticeable for the connective tissue polysaccharides.

b C

Fig. 3. Computer generated chain conformation of: (a) the two-foldpolymannuronic acid helix, in directions perpendicular and parallel tothe chain axis; and (b) corresponding projections for the three-fold sodiumsalt form. (c) Similar projections for the two-fold polyguluronic acidhelix.

The X-ray fibre diffraction pattern of polyguluronic acid exhibits a layer line spacing of0. 87 nm with meridional reflections on even layer lines only. These dimensions andhelical symmetry can be obtained by placing the O(-L-guluronic acid residue in the alternate1C4 chair conformation (Fig. 2b) so that we have a l-. 4 diaxially linked polymer similar tosodium pectate (Ref. 15). The resulting polymeric shape (Fig. 3c) is substantially alteredhaving a pronounced zig-zag molecular geometry. -guluronic acid is obtained by C(5)epimerization of -mannuronic acid and there is good evidence to suggest that this occursat the polymer level (Ref. 16). The block-like structure in alginate enables the G-blocks toassociate and provide junction zones for the well known gelation behaviour (Ref. 17).Further details of the crystalline structure of the alginate components have been reportedpreviously (Ref. 18).

CONNECTIVE TISSUE POLYURONIDES

Hyaluronate, the chondroitin sulphates and dermatan sulphate are essentially poly-disaccharides with alternating l— 3 and 1—4 glycosidic linkages. Heparan sulphate and theblood anti-coagulant heparin also have a polydisaccharide backbone but the glycosidiclinkages for these biopolymers are all 1—4.

0. 8 nm

1138 EDWARD D.T. ATKINS

HluronatesHyaluronic acid is a high molecular weight glycosaminoglycan (about 1,000,000 Daltons)found in soft connective tissues such as umbilical cord, synovial fluid and vitreous humour.The chemical and physical properties of hyaluronate have been discussed by Laurent (19).X-ray diffraction patterns obtained from oriented hyaluronate films have been analysed andmolecular models constructed (Ref. 20-25). The first X-ray pattern of sodium hyaluronatesuggested a highly extended three-.fold helix with an axially projected repeat of 0. 95 nm(Ref. 20) which correlates with the disaccharide repeat (Fig. 4) and a computer generatedstructure is illustrated in Fig. 5a. Recently a detailed structure refinement has beenreported (Ref. 25). By changing the experimental conditions such as pH, ionic strength,relative humidity, etc. other distinct conformations have been observed. For example,on lowering the pH to below 2. 5 a two-fold conformation is observed with a slight increasein the projected axial disaccharide repeat (h) from 0. 95 mu to 0. 98 nm (Ref. 20). Also ahighly extended four-fold helix with h 0. 93 nm has been reported (Ref. 22, 26 & 27), amodel of which is shown in Fig. Sb. A further contracted form with a value of h = 0.84 nm(Ref. 21 & 22) was originally thought to be a double-helix (Ref. 21) but more detailedcalculations have favoured a áontracted single stranded conformation (Ref. 24). TypicalX-ray diffraction patterns obtained from oriented hyaluronate films are illustrated in Fig. 6.

CO CH,OH OH NHCOCH, co

OH NHCOCH3 CO CH,OH OH

Fig. 4. Repeating sequence of hyaluronic acid.

Fig. S. Projections of hyaluronate conformations (a) a three-fold helix,(b) a four-fold helix.

Thus hyaluronate exhibits a variety for molecular conformations depending on the localenvironment during crystallization. Such results offer scope for monitoring theconformation in the condensed phase as a function of ionic strength, cationic species etc.

Chondroitin sulphateThe two types of chondroitin, 1. e. chondroitin 4-sulphate and 6-sulphate differ fromhyaluronic acid by replacement. of N-acetylglucosamine with sulphated

bø

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IIHCOCH3 CO5 HO CH5205

OH OH klHCOCH3 CO5

9 OH 14HCOCH3 O5 O2O CH5OH

0201 OH IIHCOCH3CO_

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C0Ut0LWt0U2 O flLOUIC crq cOururIJ boJAeccpLrqss 113

1140

a b

EDWARD D.T. ATKINS

Fig. 9. Projections of the 2helices for: (a) chondroitin 4-sulphate, and (b) chondroitin 6-sulphate.

Fig. 11. Projections of the 85 helixfor dermatan sulphate.

chair conformation for -glucuronic acid appears quite satisfactory with all the sideappendages favourably positioned equatorially. On the other hand, -iduronic acid has alarge carboxyl group axially positioned (Fig. lOb) and serious consideration must be givento the alternate 1C4 chair conformation. This bears a resemblance to the situation foundin the alginates discussed above. The effect of converting the -iduronic acid residue tothe alternate 1C4 chair conformation would introduce a diaxially linked unit substantiallyaltering the shape of the backbone. X-ray diffraction patterns obtained from dermatansulphate (Ref. 31 & 32) yield values of h = 0. 93 nm (8 helix), 0. 95 nm (2 helix) and0. 98 nm (21 helix). These spacings are not compatible with the jd-iduronic acid residuein the alternate 1C4 chair conformation (Ref. 32). Computer projections of the 8 helixare shown in Fig. 11.

ProteogylcansUnlike hyaluronic acid, the chondroitin and dermatan sulphate chains are of lowermolecular weight (about 50, 000 Daltons) and occur in vivo covalently linked to a proteincore. The resulting proteoglycan is similar to a laboratory test tube brush. Recentbiochemical studies (Ref. 33) have established a specific interaction between hyaluronicacid and the proteoglycans in the intercellular matrix in cartilage. The essential featuresof the proposed model (Fig. 12) are that many proteoglycans are able to bind along theentire length of a hyaluronic acid chain and.that at saturation there is one proteoglycanbound to each region of hyaluronic acid of about twenty disaccharide units long (about2Onm). Thus for a hyaluronic acid chain of molecular weight 500, 000 Daltons there areabout forty proteoglycans regularly distributed. Each proteoglycan can bind to only onehyaluronic acid chain so the system does not readily form a network or gel by aninteraction of the form HA-PG-HA. These proteoglycan aggregates containing hyaluronicacid interact electrostatically through the charges on the polysaccharide side chains withcollagen to form the molecular organization in cartilage (Fig. 13). X-ray diffractionpatterns from such proteoglycan aggregates have been reported (Ref. 34).

\\Aç

Conformations of uronic acid containing polysaccharides 1141

HO4.-)\OH bOFig. 10. Schematic representation of: (a) /3 --g1ucuronic acid, and(b) o(-L-iduronic acid.

Fig. 13. Model for connective

tissue.

Fig. 14. Projections of the two—fold helix for heparan sulphate.(a) with no sulphate groups included, (b) with NSO groups on theglucosamine residues (black).

a

PG MW. 25x10'HA MW. 05x10'

colLagen

l200nm

Fig. 12. Proteoglycan-hyaluronicacid complex.

bundles of collagen being held

together by proteogLycan matrix

a b

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9 p

Conformations of uronic acid containing polysaccharides 1143

CH3C-COOH4/\6

..u.3).Man..(1..4)..p..Q-GIcUA-(1..4)--Q-GC-(1-.

2-OAc

Fig. 19. Chemical sequence of

serotype K5.

o-Q-Mn.

ii

...3)..Q.GaIUA-(1..2)--Q-Man -(13) -p-Q-GaI-(1-

Fig. 22. Chemical sequence ofserotype K57

o-QGIcUA

3)-fl-R-GaI-

Fig. 25. Chemical sequence ofserotype K8.

Fig. 20. Schematic representation for repeat of K5.

Man

Gal

H0Fig. 23. Schematic representation for repeat of K57.

nm

Fig. 18. Projections of a two-fold helical conformation ofheparin.

Man jç c2i

'3

I I 44 EDWARD D .T. ATKINS

MICROBIAL POLYURONIDES

In this section some examples have been taken from the capsular polysaccharides of thegenus Klebsiella. They have been chosen in order of increasing complexity and an overallmodel building procedure has been adopted described as follows.

Molecular models were generated using a procedure similar to the linked-atom description(Ref.49&50).Bond lengths, bond angles and sugar residue conformations were held constant.Only the torsion angles at the various backbone glycosidic linkages were allowed to vary.Pendant groups were placed in stereochemically reasonable positions after the chainconformation had been determined. A conformation was considered possible when itsimultaneously met two criteria: adherence to the appropriate helix pitch and symmetrywhile having no unacceptable non..bonded contacts. The presence (or absence) of

unacceptable interatomic contacts was defined using steric criteria (Ref. 51). In caseswhere alternative models were possible the model which contained the maximum numberof intramolecular hydrogen bonds were favoured. To aid in following the course of themodel building, and to assess the possibility of hydrogen bonding across each of theglycosidic linkages, steric maps were calculated for each glycosidic linkage.

Klebsiella serotypesThe genus Klebsiellabelongs to the family of Enterobacteriacae and are found in theintestines of man and animals. In general Klebsiella bacteriaproduce extracellularpolysaccharides which surround the bacterium as an additional outer layer. These

capsules act as a visoelastic barrier against mechanical damage and protect the cellsagainst desiccation and phagocytosis. The capsular materials also exhibit specificantigenic properties, and the serological classification of Kiebsiella is based onimmunological reactions of these capsular or K antigens (Ref. 52). To date about eightyserologically different strains have been recognized, the variety of which presumably hasevolved due to the host defence mechanisms. In all cases known the isolated capsular

material consists of heteropolysaccharides with a regular repeating unit of three to sixsaccharides, which are chemically different for each strain. The repeating unit is oftenbranched being a mono or disaccharide as a side appendage. The presence of a uronicacid, pyruvate or both seems to be a characteristic feature of these glycans (Ref. 53).

A number of these serotypes have been induced to form oriented, crystalline filmssuitable for X-ray diffraction analysis. The three serotypes K5, K57 and K8 have beenchosen from our collection because they are all polyuronides and they are arrangedin order, of increasing complexity.

Serotype K5Of the three Klebsiella serotypes to be discussed the K5 polysaccharide has the least

complicated chemical covalent repeat. It is a linear polytrisaccharide of the form

(-A-B-C-)n, and the detailed chemical constitution has been reported by Dutton andMo-Tai Yang (54) as shown schematically in Fig. 19. The essential backbone structureconsists of two neutral sugars, a 1, 3-linked-/3 --mannose and a 1, 4-linked /3-a-glucose, sandwiching a 1, 4-linked /3 --glucuronic acid residue. There are twoappurtenances worth mentioning: pyruvic acid is linked to the -mannopyranose as a4, 6 acetal, and an 0-acetate group is attached to the 2-position of the glucopyranosering. Thus the repeating sequence contains two charged carboxylate groups and theglycosidic linkage geometry is illustrated in Fig. 20. All three saccharide units wouldbe expected to exist in the normal 4C1 chair conformation resulting in a pair of 1 -4diequatorial glycosidic linkages together with a single 1—3 diequatorial linkage. Boththese linkage geometries are common to the simpler plant and animal polyuronidesdiscussed above. If the vectors between each glycosidic oxygen atom were to alignprecisely the maximum theoretical extension per chemical repeat would be 1. 56 nm. Ofcourse it is extremely unlikely that a stereochemical acceptable model could exist withsuch a repeat but at least it indicates an upper limit. For the simpler polyuronides suchas the alginates and connective tissue polyuronides the reported axially projected repeatsin the condensed phase are all within 18% of the theoretical limit and typically highlyextended conformations gave repeats centred around value 10% less than the maximum.

The X-ray diffraction results from the sodium salt of KS (Ref. 55) yielded a layer linespacing of 2.70 nm and with meridional reflections only occurring on even layer linessuggesting a two-fold helical conformation of the molecule. The value for h of 2. 70/2 =

OL I<2ET I. jo-oq PGITX

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2

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sy x-' qicoubUGLu ow 2GLo%bG ir

1146 EDWARD D.T. ATKINS

1. 35 nm correlates well with the trisaccharide repeat and is only 14% below the theoreticallimit suggesting an extended chain conformation.

Stereochemical acceptable structures with the required helical parameters have beencomputer generated and it has been found possible to form intra-chain hydrogen bondsacross all three glycosidic linkages and fit the experimentally recorded helical symmetryand dimensions. Computer drawn projections are shown in Fig. 21. The structureincorporates an 0(5). . . 11-0(3) hydrogen bond at both the f3--Man.(l-..4)-ft-Q-GlcUA andthe /3 --.GlcUA.-(l--.. 4) /3 --Glc glycosidic linkages. This hydrogen bond is present inmany homopolysaccharides such as cellulose (Ref. 11) and the connective tissuepolydisaccharide structures (Ref. 24). It is gratifying to find it also appearing in thismore complex structure. At the third glycosidic linkage (ft--Glc-(l—..3)-fl ..D..Man)there is an 0(2). . . 0(2) hydrogen bond.

Serotype K57K57 isapolytetrasaccharide consisting of three saccharide units in the backbone and witha single saceharide unit ( o-- mannose) in the side chain. The detailed chemical repeathas been established by Kamerling, Lindberg, Lonngren and Nimmich (56) and is given inFig. 22. As in the case of the K5 serotype the backbone consists of two neutral sugarsand a uronic acidresidue. This 1,3 linked x-..galacturonic acid residue is attached toa 1, 2 linked o-D-mannose residue followed by a 1, 3 linked/3 ..-galactose residue. Theside appendage is attached to the uronic acid residue. Again it would be anticipated thatall saccharide units exist in the normal 4C1 chair conformation resulting in one 1 3diequatorial glycosidic linkage, a l.-.2 diaxial linkage and one lax —3eq linkage (Fig. 23).In addition the mannopyranose side chain is l—.4 diaxially attached. This structureintroduces some novel glycosidic linkage geometries together with the added complicationof a side chain. The maximum theoretical extension for the chemical repeat, followingthe method described previously, is 1. 27 nm.

The X-ray diffraction patterns show layer lines with a measured spacing of 3. 43 rim withmeridional reflections present only on layer lines with index 1 = 3n (Ref. 57). Thesimplest interpretation of this diffraction pattern is that the polysaccharide backbone formsa three-fold helix with the value of h = 1.143 nm. This value, 10% less than the maximumpermissible, correlates with the chemical repeat and indicates a highly extendedconformation.

Trial models have been generated conforming to the helical symmetry and dimensions.Both left-handed and right-handed models have been generated using the techniques andcriteria outlined above. Attempts were made to form the maximum number of intra-chain hydrogen bonds. It was found that no model could be constructed that includedhydrogen bonds across all three backbone glycosidic linkages. Only a left-handed helixallowed the formation of two intra-chain hydrogen bonds in the backbone: o. --GalUA-0(2).. 0(3)-c-D-Man and oC--Man-0(5).. .H-0(2)- G -a-Gal. Computer drawnprojections of this conformation are illustrated in Fig. 24.

Serotype KBThe covalent chemical repeating sequence has been established by Sutherland (58) and isgiven in Fig. 25. It is similar to the KS? polysaccharide having a polytrisaccharidebackbone and a single saccharide unit for the side chain. Both structures have a uronicacid in the repeat, however, in K57 the uronic acid is incorporated in the backbone andthe side chain is a neutral sugar. The converse is true in serotype K8 which hasan uncharged backbone and an of.--glucuronic acid side chain. All four saccharide unitsare expected to exist in the normal 4C1 chaii conformation and a schematic diagramrepresenting the glycosidic linkage geometry is shown in Fig. 26.

The theoretical maximum extension for the chemical repeat is 1. 38 nm falling partwaybetween the values, for KS and K57. The X-ray diffraction pattern for the sodium salt ofthe K8 polysaccharide is shown in Fig. 27. The material is highly oriented andcrystalline and from the systematic absences of odd order meridional reflections it canbe seen that the molecule is a 2 helix. However the layer line spacing of 5. 078 nm isfar too large for a repeat of two asymmetric units. In fact it is very close to thetheoretical maximum extension for four complete covalent repeats, i. e.4 x 1 .38 5. 52nm.Thus the observed repeat is only some 10% less than the maximum permissible extension.

Conformations of uronic acid containing polysaccharides 1147

From preliminary model building it appears reasonable to assume that the structure of theisolated model is a perfect four-fold helix with an axial advance per covalent repeat (h)1. 27 nm. Perturbations from an idealized four-fold helix would be expected to result ina lower symmetry and consequently the packing of the molecular chains in an orthorhombicrather than a tetragonal unit cell (Ref. 59). The phenomenon has also been observed inhyaluronic acid (Ref. 24).

Stereochemically feasible models were constructed to fit a four-fold helical symmetry andincorporating the maximum number of intra molecular hydrogen bonds. It was foundimpossible to construct a model which incorporated hydrogen bonds at all three backbonelinkages. Only a left-handed helix allowed the formation of two hydrogen bonds in thebackbone. Projections of this structure are shown in Fig. 28. The conformation containshydrogen bonds 0(5). . . 0(4) and 0(5). . . 0(2) at the , --Gal-(l—3) - o--Gal and x--Gal-(l—-.3)- -Q-Glc glycosidic linkages respectively. It was found not possible to forma four-fold helix (with the observed extension) which contained a hydrogen bond at the(.3 --Glc-(l--. 3)- 8 -a-Gal glycosidic linkage. This linkage is adjacent to the

attachment site of the uronic acid side chain.

Lacking any information to define the position, of the side chain in the isolated molecule,other than that it must be in a :stereochemically allowed position, it was necessary to useX-ray intensity information to determine the orientation of the pendant residue (Ref. 60).

DISCUSSION

Examples have been briefly reviewed covering the simpler plant homopolyuronides, theuronic acid containing connective tissue polydisaccharides and the more complexbacterial capsular polyuronides from Klebsiella. Conformations have been derived fromthe X-ray diffraction evidence in an attempt to provide a general outlook on the behaviourof these macromolecules in the condensed phase. This has recently become possiblewith the crystallization of these macromolecules in a form suitable for X-ray diffractionstudies. It is only through these X-ray studies that the helical parameters necessary formeaningful model building can be obtained. One noticeable feature of the resultspresented is that all the conformations are highly extended, all that is except for thecontracted four-fold of hyaluronic acid (Ref. 24). No simple explanation appearsavailable to explain this puzzling phenomenon.

We have witnessed an avalanche of molecular information relevant to the uronic acidcontaining polysaccharides which will naturally take time to be completely digested andfully understood, yet we have probably only scratched the surface. Much work is stillto be done in refining the molecular conformations outlined in this report. However, oneinstinctively feels that this is an exciting period since not only has the crystallization ofthese complex substances been achieved, but a whole variety of molecular conformationsare evident as a function of the chemic al and thermodynamical environment. The ability -to monitor detailed molecular geometries, even in the condensed phase, will greatlyfacilitate our general understanding of their properties and molecular biology.

Acknowledgements - I am indebted to my colleagues Drs. K. H. Gardner,D. H. Isaac, W. Mackie, I. A. Nieduszynski and J.K. Sheehan, and Ms.H. F, Elloway, for many valuable discussions and for allowing me toreproduce some of their results. My thanks also to the manycollaborators who have generously supplied material which made muchof this work possible. I am grateful to Professors F, C. Frank, FRSand A. Keller FRS for their constant support and encouragement.

This work was financed by the Science Research Council and in part bythe Arthritis and Rheumatism Research Council.

I I 48 EDWARD D .T . ATKINS

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