Glycobiotogy vol. 4 no. 2 pp. 151-163, 1994

Insights into stereochemical features of sulphated carbohydrates:X-ray crystallographic and modelling investigations

Doriano Lamba1, Steven Glover2, William Mackie2,Abdul Rashid2, Bernard Sheldrick2 and Serge Pe"rez3-4

'Istituto di Struttunstica Chimica Giordano Giacomello, Area della Ricercadi Roma, CNR, CP No. 10, 1-00016 Monterotondo Stazione, Roma, Italy,2Department of Biochemistry and Molecular Biology, University of Leeds,Leeds LS2 9JT, UK and 3Institut National de la Recherche Agronomique,BP 527, 44026 Nantes C&lex 03, FranceCorresponding author

The three-dimensional structures of the 2-, 3-, 4- and6-monosulphates of methyl a-D-galactopyranoside havebeen determined by X-ray crystallography; the first two asthe sodium salt, the third as both the sodium and potassiumsalts, and the fourth as a potassium salt. These representthe principal sulphated monomers of the carrageenanpolysaccharides. The results extend our knowledge of thestereochemical features, such as ring conformation, sul-phate geometry, hydrogen bonding and cation co-ordination,which characterize sulphated monosaccharides. The stereo-chemical data have been used to derive a mean geometry ofthe 0-sulphate group and a set of force constants for use inmolecular mechanics calculations on sulphated monosac-charides. These may be used in an extrapolation of thepopulations of stable conformers of related oligo- and poly-saccharides.

Key words: crystal structures/molecular mechanics/O-sulphatedcarbohydrates/X-ray diffraction

Introduction

Sulphated carbohydrates are widely distributed in plants andanimals, occurring most often as building blocks of polysac-charides. These include the carrageenans from red algal cellwalls (Mackie and Preston, 1974) and the proteoglycan andglycosaminoglycan components (chondroitin, dermatan andkeratan sulphates, heparin and heparan sulphates) of the extra-cellular matrix and cells of many animal tissues (Muir, 1983;Kjellen and Lindahl, 1991). Sulphated monosaccharides alsooccur in brain and nerve tissue (Krusius etai, 1986, 1987;Finne, 1989; Schacher, 1989), hormonal (Green etai, 1986;Kamerling et al., 1988; Spiro and Bhoyroo, 1988), viral (Pinterand Compans, 1975; Nakamura and Compans, 1978), mucus(Mawhinney etai., 1987, 1992; Capon etai., 1989), enzyme(Freeze and Miller, 1981), immune (Rider, 1992) and albumin(Yamashita et al., 1983) glycoproteins, and also have beenidentified in tumour cells (Dennis et al., 1984; Roux et al.,1988; Sugahara et al., 1988; Sundblad etai, 1988a,b), lym-phocytes (Dahms and Hart, 1985) and in host-parasite inter-actions in plants (Lerouge et al., 1990; Truchet et al., 1991).Sulphated tetrasaccharides have also been indentified as specific

Data from this paper have been deposited in the Cambridge CrystallographicData Base.

ligands in the binding of L- and E-selectin molecules (Greenet al., 1992; Yuen etai., 1992). However, in most of thesecases, it is not obvious what the relationships between sulphatecontent and biological function are. For example, in thecarrageenan-glycosaminoglycan systems, many studies, byvarious techniques, suggest that the physical properties of theiraqueous solutions (viscosity, gel formation, etc.) are dependenton factors such as the number and location of sulphate groups,the nature of the counter ions and conformation of the poly-saccharide chain (Rees et al., 1982), which in turn determinetheir biological function of mechanical support in the cell walland extracellular matrix. However, as well as providing mech-anical support, sulphated carbohydrates appear to influenceother biological functions, including adhesion, mobility andproliferation of cells, on which subsequent differentiation andtissue morphogenesis depend. Various natural and syntheticpolysaccharides have been reported to elicit antibody responsesin which densely sulphated regions may be important deter-minants in some cases (Caterson et al., 1985; Carney and Muir,1988; Kongtawelert and Ghosh, 1990), while binding of hep-arin and related synthetic oligosaccharides to antithrombin HIrequires a specifically located tri-sulphated D-glucosamine unit(Petitou et al., 1988). All of these interactions are examplesof molecular recognition, but in no case is there a detailedmolecular description, in terms of the stereochemistry of theinteracting species, which explains the role of sulphate groups.Sulphated carbohydrates are also known to possess importantpharmaceutical properties such as anti-ulcer, anti-pepsin (Ochiet al., 1980) and anti-viral (Nakashima et al., 1987; Yamamotoet al., 1991; Uryu et al., 1992) activities, and may also beinvolved in the inhibition of cell migration during cancermetastases (Coombe et al., 1987).

The present study was undertaken to provide insight into thestereochemical features of carbohydrate sulphates (moleculargeometry, hydrogen bonding, cation co-ordination and chargedistribution) which could form a stereochemical basis for under-standing the origin of the role of sulphate groups in the variousbiological interactions described above. One approach to thedetermination of such stereochemical features is to study modelcompounds of low molecular weight. This is best achieved byX-ray crystallography. In the present study, the compoundsinvestigated were crystalline salts (sodium and potassium) ofthe 2-, 3-, 4- and 6-sulphates of methyl a-D-galactopyranosidessynthesized as model compounds by Rashid et al. (1990). Thecurrent investigation supplements previous crystallographicstudies on sucrose octasulphate (Nawata etai., 1981) andpotassium /3-D-glucopyranose 6-sulphate (Lamba et al., 1988).While this work was being prepared, a low-temperature determi-nation of the crystal structure of sodium methyl a-D-galactopyranoside 4-sulphate dihydrate was published (Kantersetai., 1991). The crystal structure of methyl a-D-galacto-pyranose 2,6-bis sodium sulphate has been reported (Lambaetai, 1993). The particular sulphated monosaccharides de-scribed below are most closely related to the carrageenanpolysaccharides. Nevertheless, their structural details, together

© Oxford University Press 151

D.Lamba el at.

Table I. Crystallographic

Molecularformula

Crystal size (mm)Crystal systemSpace groupUnit cella (A) =b(A) =c(A) =00 =Volume (A3)ZFormula weightDc (Mg m"3)Absorption coefficent(mm"1)

Table I (continued). List

Atom co-ordinates with e

C-lC-2C-3C-4C-5C-6CMeO-lO-2O-3O-4O-5O-6S-lO-Sl0-S2O-S3

O-WlO-W2o-w3Na-1

data

C,H,3O,S-, Na+

3H2O

0.20 x 0.2O X 0.10MonoclinicP 2 ,

14.669(1)6.258(1)8.069(1)

103.36(1)720.8(1)

2350.27

1.61428.31

of atomic co-ordinates. la.

s.d.s and isotropic thermal

X/a

0.7943(2)0.7345(1)0.7651(1)0.7687(1)0.8308(2)0.8336(2)0.9435(2)0.8842(1)0.7421(1)0.7009(1)0.6742(1)0.7949(1)0.8996(1)0.6527(0)0.6791(1)0.5741(1)0.6436(1)

0.4456(1)0.4717(1)0.9286(1)0.5709(1)

C^HuO^", Na+

H2O

0.44 x 0.30 x 0.17OrthorhombicP 2 , 2 , 2,

6.895(2)12.926(3)13.676(5)-

1218.9(6)4

314.241.712

31.55

C^HuOjS", Na2H2O

0.32 x 0.12 xMonoclimcA 2

8.0561(5)5.8097(4)

28.9154(17)98.953(7)

1336.9(2)4

332.251.651

29.65

c^cvr, K+

H2O

0.09 0.26 x 0.15 x COrthorhombicP 2, 2, 2,

5.791(1)7.909(2)

28.116(3)-

1287.7(4)4

330.351.704

56.20

( Q H , , ^ - , K+),5H2O

1.12 0.30 x 0.10 x 0.03MonoclinicP 2 ,

11.385(6)8.606(6)

16.095(10)101.22(4)

1546.8(16)23.57.41.535

47.86

Methyl a-D-galactopyranoside 2-sulphate, sodium salt, trihydrate (a-MeGal-2S, Na, 3HjO)

parameters Ueq (A2) values for the non-hydrogen atoms

Y/b

-0.2660(5)-0.0663(4)

0.0655(5)-0.0773(5)-0.2701(5)-0.4225(6)-0.3796(7)-0.2029(4)

0.0704(4)0.2339(4)

-0.1421(4)-0.3867(3)-0.5913(4)

0.0741(2)0.2257(4)0.1388(4)

-0.1436(4)

0.0852(5)0.0442(4)0.0982(6)0.1807(0)

Z/c

0.8856(3)0.8842(2)1.0470(3)1.2009(3)1.1932(3)1.3404(3)0.8584(5)0.8756(2)0.7420(2)1.0454(2)1.2005(2)1.0363(2)1.3470(3)0.5843(0)0.4699(2)0.6523(2)0.5188(2)

0.2716(2)-0.1065(2)

0.6080(3)0.1580(1)

Ueq.

0.0288(6)0.0260(6)0.0263(6)0.0275(6)0.0293(6)0.0363(7)0.0596(10)0.0361(5)0 0305(4)0.0328(5)0.0302(5)0.0298(5)0.0438(6)0.0285(2)0.0405(6)0.0411(5)0.0388(6)

0.0430(6)0.0391(6)0.0648(9)0.0383(3)

with the parameterization of the sulphate group for inclusionin molecular mechanics calculations, have implications forsulphated sugars of other systems.

Results and discussion

Description of the crystal structures

The crystallographic data of the five different structures aresummarized in Table I. In four cases [methyl a-D-galacto-pyranoside 2-sulphate, sodium salt, trihydrate (a-MeGal-2S,Na), methyl a-D-galactopyranoside 3-sulphate, sodium salt,monohydrate (a-MeGal-3S, Na), methyl a-D-galactopyrano-side 4-sulphate, sodium salt, dihydrate (a-MeGaJ-4S, Na) andmethyl a-D-gaJactopyranoside 4-sulphate, potassium salt,monohydrate (a-MeGal-4S, K)] there is only one sulphatedmolecule per asymmetric unit and the reliability of these struc-tural determinations is high since the respective reliabilityfactors range from 0.02 to 0.06. Only in the case of the

152

potassium salt of a-MeGal-6S do two independent motifs occurin the asymmetric unit. Since the poor quality of this singlecrystal did not allow a satisfactory collection of experimentaldata, the final reliability factor is only 0.13. Consequently, thegeometrical features cannot be defined accurately.

Observed molecular geometries. The configurations, conforma-tions and atom numbering of the sulphated monosacchandes areshown in Figure 1. The final atomic co-ordinates for a-MeGal-2S, Na, a-MeGal-3S, Na, a-MeGal^S, Na, a-MeGal-4S, Kand a-MeGal-6S, K are listed as supplementary material (TableIS), as well as the bond lengths, valence angles and torsionalangles of interest (Table IIS). The present results establish thatfor the five sulphated monosacchandes the anomeric configura-tion is a.

With the exception of the atoms to which the sulphate groupsare attached, all the bond lengths and valence angles conformto the tabulated values of carbohydrates. The anomeric bondlengths are in the normal range for the a glycosidic configura-tion. From the values of the ring parameters, it is clear that the

Stereochemical features of sulphated carbohydrates

Ib. Methyl a-D-galactopyranoside 3-sulphate, sodium salt, monohydrate (<x-MeGal-3S, Na, H,O)

Atom co-ordinates with e.s.d.s and isotropic thermal parameters Ueq (A2) values for the non-hydrogen atoms

X/a Y/b Z/c Ueq

C-lC-2C-3C-4C-5C-6CMe0-1O-20-3CMO-5O-6S-lo-siO-S2

O-S3

-0.1947(5)-0.1425(5)-0.1489(6)-0.3381(6)-0.3531(6)-0.5211(7)-0.0565(8)-0.0342(4)

0.0412(4)-0.1238(4)-0.4918(4)-0.3650(4)-0 7007(5)-0.0041(1)-0.0049(6)

0.1834(4)-0.1099(6)

0.0045(3)-0.1106(3)-0.1364(3)-0.1034(3)

0.0139(3)0.0678(3)0.1716(3)0.0636(2)

-0.1292(2)-0.2482(2)- 0 1588(2)

0.0324a)0.0338(3)

-0.2968(1)-0.4053(2)-0.2506(2)-0.2740(3)

0.5158(2)0.4995(2)0.3907(3)0 3451(3)0.3601(3)0.3136(3)0.5100(5)0.4856(2)0.5404(2)0.3867(2)0.3932(2)0.4648(2)0.3508(3)0.2977(1)0.3248(2)0.2996(3)0.2097(2)

C-lC-2C-3CAC-5C-6CMe0-1O-2O-3O-4O-5O-6S-lO-Sl

O-S2O-S3

O-WlO-W2Na-1Na-2

-0.1151(5)-0.1350(5)

0.0284(5)0.1731(5)0.1846(4)0.3323(5)

-0.1307(10)-0.1143(4)-0.2656(4)

0.0097(4)0.1335(3)0.0324(3)0.3401(5)0.2521(1)0.1992(4)0.2191(4)0.4242(3)

0.3921(4)0.4417(7)0.0000(0)0.5000(0)

0.0939(9)-0.1109(9)-0.2486(8)-0.0845(9)

0.0900(9)0.2551(10)0.1882(14)0.0106(7)

-0.2503(9)-0.4183(7)

0.0442(8)0.2228(7)0.3858(9)0.0386(5)0.2409(8)

-0.1727(8)0.0457(8)

-0.5544(9)0.2353(12)

-0.4457(0)-0 2871(7)

0.0586(9)0.0552(8)0.0581(9)0.0595(9)0.0616(10)0.0721(12)0.0840(15)0.0640(7)0.0641(7)0.0610(7)0.0693(8)0.0591(7)0.0845(11)0.0608(2)0.0821(10)0.0767(9)0.0830(11)

o-wiNa-l

Ic. Methyl a-D-gal

Atom co-ordinates

0.2888(11)-0.1872(2)

lactopyranoside 4-sulphate, sodium

with e.s.d.s and isotropic thermal

X/a

0.0918(4)-0.4758(1)

salt, dihydrate (<*-MeGal-4S,

parameters Ueq. (A2) values

Y/b

Na

for

0.6524(4)0 4794(1)

, 2H2O)

the non-hydrogen atoms

Z/c

0.1331(19)0 0753(5)

Ueq.

0.1508(1)0.1171(1)0 1188(1)0.1147(1)0.1541(1)0.1569(1)0.2288(2)0.1961(1)0.1285(1)0.0833(1)0.0709(1)0.1473(1)0.1984(1)0.0323(1)0.0054(1)0 0061(1)0.0558(1)

0 0573(1)0.2941(2)0.0000(0)0.0000(0)

0.0291(11)0.0301(11)0.0290(11)0.0289(11)0.0299(10)0.0377(13)0.0648(20)0.0361(9)0.0456(10)0.0359(9)0.0307(7)0.0308(8)0.0547(13)0.0258(2)0.0427(10)0.0388(10)0.0403(10)

0.0444(10)0.0746(19)0.0283(6)0.0490(8)

galactopyranose rings are in the expected 4C] conformation,albeit with some distortions from a perfect chair that are attri-buted to the sulphate substituents.

The primary hydroxyl groups adopt a gauche-trans (gt)conformation which is one of the two preferred non-eclipsedconformations found in the solid state for monosaccharideshaving the galacto configuration. However, in the case of a-MeGal-3S, Na the unfavoured gauche—gauche (gg) conforma-tion is found. As noted previously (Marchessault and Pe'rez,1979), such an energetically unstable arrangement is accom-panied by an intramolecular hydrogen bond between O-6 andO-4 (O-6. . .O-4 = 2.93 A, H-O6. . .O-4 = 2.42 A, O-6-H-O6. . .0-4 = 146°). A recent conformational study of salts ofbenzyl 6-0-sulpho-/3-D-glucopyranoside in solution concluded

that gt and gg conformations occurred in an approximate ratioof 2:3, and that neither sulphate nor counter ion had asignificant effect on this ratio (Meyer and Stuike-Prill, 1990).

The present work provides detailed experimental informationabout the geometry and conformation of the sulphate group incarbohydrate structures. The sulphate group is essentiallytetrahedral with O - S - 0 angles ranging from 100.6 to 115.5°.Substitution with sulphate results both in a significant length-ening of die C—O bond (mean = 1.460 A, range from 1.456to 1.472 A) and in the opening of the C-x-O-x-S angle (mean= 119.0°, range from 115.9 to 121.8°) at the point ofattachment of the sulphate moiety. At positions 2, 3 and 4, thebridging S-O bonds are eclipsed with the C-H bonds, whichdiminish steric repulsions of the sulphate groups with the

153

D.Lamba <

Id. Methyl

tt al.

a-D-galactopyranoside 4-sulphate, potassium salt,

Atom co-ordinates with e.s.d.s

C-lC-2C-3C-4C-5C-6CMeO-lO-2O-3O-4O-5O-6S-lO-SlO-S2O-S3

O-WlK-l

Ie. Methyl

monohydrate (a-MeGal-4S,

and isotropic thermal parameters Ueq. (A2) values for the

X/a

-0.0735(8)0.1229(8)0.2719(7)0.1183(8)

-0.0613(7)-0.2231(10)

0.1584<12)0.0167(6)0.2647(7)0.4394(5)

-0.0050(6)-0.1986(6)-0.3655(8)-0.0005(2)-0.1767(8)

0.2278(6)-0.0642(7)

0.3137(10)0.5331(2)

a-D-galactopyranoside 6-sulphate, potassium salt,

Atom co-ordinates with e.s.d.s

C-lC-2C-3C-4C-5C-6CMeO-lO-2O-3O-4O-5O-6S-lO-SlO-S2O-S3K-lO-WlO-W2O-W3O-W4O-W5

X/a

1 038(4)0.963(3)0.892(3)0.811(4)0.900(3)0.822(4)1.219(6)1.121(2)1.048(2)0.818(2)0.732(2)0.969(2)0.890(2)0.824(1)0.901(4)0.775(4)0.733(5)0.929(1)0.3515(2)0.4369(7)0.3509(5)1.1327(4)0.3911(4)

for the non-hydrogen atoms

Y/b

-0.431(0)-0.509(7)-0.639(7)-0.569(8)-0.509(8)-0.451(9)-0.479(12)-0.537(7)-0.551(7)-0.707(7)-0.466(7)-0.382(7)-0.474(7)-0.454(6)-0.469(8)-0.316(9)-0.564(10)-0.787(6)

0.9538(7)0 2797(10)0.5561(10)

-0.6305(9)0.2560(9)

Y/b

0.2901(5)0.2812(5)0.1249(5)

-0.0324(5)-0.0114(5)-0.1584(6)

0.3578(8)0.3292(4)0.4266(4)0 1108(5)

- 0 0461(3)0.1351(4)

-0.1291(5)-0.2182(1)-0.1881(5)- 0 2363(4)- 0 3532(4)

-0.3700(7)-0.4942(1)

pentahydrate ((a-MeGal-6S,

Z/c

0.624(3)0.686(2)0.643(2)0.560(3)0.507(2)0.422(3)0.577(4)0.614(1)0.763(1)0.698(1)0.583(2)0.547(2)0.362(2)0.267(1)0.232(2)0.248(3)0.254(3)0.132(1)0.2498(1)0.6862(4)0.3707(3)0.2065(3)0.2556(3)

c-rC-2'C-3'C-4'C-5'C-6'CMe'

o-rO-2'O-3'O-4'O-5'O-6'S-6'

o-srO-S2'O-S3'K-r

K, H2O)

non-hydrogen atoms

Z/c

0.1056(1)0.1419(1)0.1349(1)0.1326(1)0.0937(1)0.0875(2)0.0261(2)0.0610(1)0.1390(1)0.1717(1)0.1776(1)0 1047(1)0.0474(1)0.2073(1)0.2425(1)0.2254(2)0 1749(1)

0 0455(2)0.2296(1)

K)2, 5H2O)

X/a

0.658(2)0.717(2)0.649(4)0.512(3)0.471(3)0.342(3)0.648(4)0.689(2)0.836(2)0.692(2)0.490(2)0.537(2)0.2984(2)0.161(1)0.155(2)0.115(2)0 122(2)

- 0 065(1)

Ueq

0.0375(12)0.0369(11)0.0349(11)0.0359(11)0.0349(11)0.0447(13)0.0596(18)0.0434(8)00506(11)0.0459(10)0.0356(7)0.0359(8)0.0575(12)0 0343(3)0.0542(12)0.0604(12)0.0502(12)

0.0814(18)0.0497(3)

Y/b Z/c

-0.0268(7) 0.047(2)-0.0050(7) -0.030(2)

0.1248(8) -0.081(2)0.0941(7) -0.100(2)0.0702(7) -0.018(2)0.0349(8) -0.035(2)0.0905(9) 0.182(3)0.0932(7) 0.100(1)0.0215(7) -0.011(1)0.1509(7) -0.159(1)

-0.0336(7) -0.155(2)-0.0596(7) 0.030(1)

0 0348(7) 0.048(1)0.0475(6) 0.041(1)0.0403(7) 0.128(2)

-0.0862(7) -0.005(2)0.1934(7) 0.002(2)0.2013(6) -0.139(1)

neighbours; the torsion angle H - C - O - S being -12°(2),-27°(2), 7°(3) and -8°(3) for a-MeGal-2S, Na, a-MeGal-3S, Na, a-MeGal-4S, Na and a-MeGal-4S, K, respectively.For the same reason, sulphate groups in a-MeGal-4S, Na anda-MeGal-4S, K deviate significantly ( -20° and 10°, respect-ively) from the ideal staggered conformation with respect to theC-4-O-4 ester bond.

In order to rationalize these observations, the potential energysurfaces were calculated for rotations of the sulphate groupin a-MeGal-2S, Na, a-MeGal-3S, Na, a-MeGal-4S, Na,a-MeGal^S, K and j8-Glc-6S, K (Figure 2b, c, d and f,respectively). These calculations were performed using theinformation derived from the crystal structures. The generalfeatures of the maps arise from a combination of restricted

154

motions around the fi angle (rotation around the C—O esterbond), and a large region of allowed values for v (rotationaround the O—S bond). The restricted angular variations of /tare —80-100°, except in the case where the sulphate group isat the C-6 position. The torsional angles v can take values overthe whole angular range from —180° to 180°. The energyminima are located at v = 180°, -60° and 60°. Theorientations of the sulphate group observed in the fivecrystalline structures do not depart from perfect staggering andfall into the low-energy domains of the corresponding fi, v iso-energy maps. These calculations were conducted withoutconsidering the contributions arising from electrostatic inter-actions. The agreement between calculated and observedconformations indicates that the orientations taken in the crystal

Stereochemical features of sulphated carbohydrates

o-si

O-IS2

Fig. 1. Atom labelling, molecular conformation of: (a) methyl a-D-galactopyranoside 2-sulphate; (b) methyl a-D-galactopyranoside 3-sulphate; (c) methyla-D-galactopyranoside 4-sulphate; (d) methyl a-D-galactopyranoside 4-sulphate; (e) methyl a-D-gaJactopyranoside 6-sulphate; (f) 0-D-glucopyranose 6-sulphate;(g) methyl a-D-galadopyranoside 2,6-sulphate.

structures correspond to the arrangements where non-bondedinteractions are the most favoured, although it is possible forother orientations about the O-S bond (such as the eclipsedones) to occur. These are —5 kcal/mol higher in energy thanthe staggered ones, and such a difference could be easilycompensated for by favourable interactions with neighbouringcations.

Parameterizing the sulphate group

The three-dimensional structures of the 2-, 3-, 4-monosulphatesof methyl a-D-galactopyranosides and the /3-D-glucopyranose6-sulphate constituted the set from which a mean geometry forthe sulphate group has been derived. The corresponding bonddistances and angles are given in Table II.

155

D.Lamba et al.

M =

v =

O-Sl

C-x

C-x O-x

O-S3

9T6O-S2

i_O-.\_S-l

S-1 O-Sl

O-\

-1 v *

Methyl a-D-gakctoprranoside 2 sulphate

Methyl a-D-galactopyranoside 3 sulphate Methyl a-D-galactopyranoside 4 sulphate

cu = O-5

v = C-x

03

- C - 5 .

O-x_

C-6

tC-5

C-6.

s-i_

)o-5

O-6

O-Sl

O-6

O-S3,—^

\

V

O-S2

O-Sl

308

Fig. 2. Potential energy surface of the sulphate group as a function of the n.v torsion angles. Iso-energy contours are drawn at intervals of 1 kcal/mol withrespect to the absolute minimum, (a) Sulphate group torsion angles (ji,v). (b) Methyl a-D-galactopyranoside 2-sulphate as in the crystalline sodium salttrihydrate (~jf). (c) Methyl a-D-galactopyranoside 3-sulphate as in the crystaline sodium salt, monohydrate (if), (d) Methyl a-D-galactopyranoside4-sulphate, as in the crystalline sodium salt dihydrate (~jf) and as in the potassium salt monohydrate ( © ) . (e) Sulphate group torsion angles O,u) at theprimary hydroxyl location or in the case of (1-6) linkage, (f) |3-D-glucopyranose 6-sulphate,calculated for a gauche—trans conformation of the primaryhydroxyl group as in the crystalline potassium salt ( ^ ) . The occurrences of the low-energy conformations found in methyl a-D-galactopyranoside o^sulphateand in methyl a-D-galactopyranoside 2,6-sulphate are indicated by • and V, respectively.

156

Stereochemlcal features of sulphated carbohydrates

Table n. Geometry for the O-sulphate group

Mean geometry MM3 geometry

c-o (A)o-s (A)s-o(S) (A)C-O-S (°)O-S-O(S) (°)O(S)-S-O(S)

1.464(17)1.589(15)1.439(11)

119.0(20)105.2(2.5)113 0(1.9)

1.4681.5901 440

119.4105.5113.1

A set of stretching, bending and dipole parameters specific tothe Cx—Ox—Sx—O(S)J was derived for incorporation intoMM3. As the O-S bond is more highly polarized than any otherbond in the molecule, it has an effect on the length of theadjoining C-O bond. This necessitated the use of a polarizationcorrection term ^ which is set to the value of 0.0401 A. Thisgives an average MM3 calculated value for l<;_o of 1.464 Acompared with 1.461 A for the mean of the crystal structures.The values for the bond dipoles O--S and O(S)-S were setarbitrarily to 2.8 D and 3.2 D, respectively, by analogywith the charge dipoles for the sulphone group and to allow,in effect, for the attenuation of electrostatic potential thatwould occur in solution or in the crystalline state. Torsionalparameters were set to zero. The set of parameters to be usedin MM3 for O-sulphated monosaccharides is given in Table HI.

The predictive capability of this set of parameters was veri-fied by testing on the crystal structure of the sodium salt ofmethyl a-D-galactopyranose 2,6-bis sulphate. Omitting thehydrogen atoms, the root-means-square error between theMM3 optimized and the observed structure is 0.29 A; it shouldbe noted that most of the deviations arise from small rotationsof the sulphate groups about n and v. This is to be expectedsince no environmental effect was taken into account during theenergy optimization. Similarly, the crystalline conformation ofthe two independent a-MeGal-6S molecules was refined,yielding a satisfactory description of their geometrical features.

Cation co-ordination

The Na and K ion co-ordinations are shown in Figure 3, andtheir geometrical data are available from supplementary mater-ial (Table HIS). In a-MeGal-2S, Na, the sodium ion is sur-rounded by six oxygen atoms from two hydroxyl groups, onesulphate group and three water molecules, and has an octa-hedral co-ordination. The average Na—O distance is 2.44 A.There are two symmetry-related anions in the co-ordinationshell (Figure 3a). In a-MeGal-3S, Na, the sodium ion has asquare-trigonal anti-prismatic co-ordination comprising sevenoxygen atoms from three hydroxyl groups, one sulphate group,one ring oxygen, one glycosidic oxygen and one water molec-ule. The average Na—O distance is 2.53 A. There are threesymmetry-related anions in the co-ordination shell (Figure 3b).In a-MeGal-4S, Na, the sodium ions lie at two crystallographicspecial positions having an octahedral co-ordination andcomprising six oxygen atoms. Two hydroxyl oxygen atoms andfour oxygen atoms from sulphates participate in the formationof the Nal ion shell with an average Na—O distance of 2.39A. In the Na2 ion shell, two water molecules and four oxygenatoms from sulphate groups are found. The average Na—Odistance is 2.46 A. There are four symmetry-related anions inthe co-ordination shell of the Nal ion, but only two in Na2. TheO-S2 sulphate oxygen atom bridges the two octahedra (Figure

Table ID. MM3 parameterization for 0-sulphated

Stretching parametersO-SS-CKS)

Dipole parametersO-SS-O(S)

Bending parametersC-O-SO-S-O(S)O(S)-S-O(S)

Dielectric constant: e

U.o) (A)1.5911 44

monosaccharides

k(s)2.02.0

Electronegativity correction to L(o) (A)C-O 0.0405

Moment (Debye)2.83 2

B(0) (°)110.1103 8114.9

3

k(b)0.60.20.15

3c). The structural features are essentially the same as reportedin the low-temperature X-ray crystallographic study of thesodium salt of a-methyl-D-galactopyranoside 4-sulphate(Kanters etai, 1991).

In a-MeGal-4S, K, the potassium ion has a distorted square-trigonal anti-prismatic co-ordination involving seven oxygenatoms from two hydroxyl groups and five oxygen atoms fromsulphate groups. The average K—O distance is 2.86 A. Fivesymmetry-related anions are involved in the co-ordination shellfrom which the water molecule is absent (Figure 3d). In a-MeGal-6S, K, two independent potassium ions are found perasymmetric unit; one is surrounded by four oxygen atoms,whereas the other co-ordination comprises five oxygen atoms.One hydroxyl oxygen is shared by these two co-ordinationpolyhedrons (Figure 3e).

The co-ordination numbers and geometries of the cations areof the type to be expected, but no definite rules can be formula-ted with regard to which of the oxygen atoms of the sulphates,hydroxyl, sugar ring and waters contribute to the co-ordination.However, it is noticeable that water does not contribute to theco-ordination of potassium.

The preceding description of cation co-ordination does notallow any prediction to be made concerning the oxygen ligandswhich may contribute to the co-ordination ion shell in othermolecular interactions involving sulphated carbohydrates.

Crystal packing

It is not proposed to discuss hydrogen bonding schemes andpacking features in detail (Figure 4). In each structure, how-ever, an intricate network of hydrogen bonds is found involvingthe oxygen atoms of the sulphate groups, each oxygen atom ofthe hydroxyl groups, occasionally the ring oxygen atoms O-5,and all of the water molecules. The hydrogen network char-acteristics are listed in the supplementary material (Table IVS).Furthermore, the packing patterns are governed by cationco-ordinations that serve as cohesive elements. The galacto-pyranoside rings are stacked along the shortest axis, and ina-MeGal-2S, Na, a-MeGal-3S, Na and a-MeGal-4S, K areconnected via H-O-3. . .0-5 hydrogen bonds along the b, a andb axes, respectively, whereas in a-MeGal-4S, Na they areconnected via H-O-4. . .O-S2 hydrogen bonds along the a axis.

In a-MeGal-2S, Na, hydroxyl oxygens O-3 and O-4 actboth as donors and acceptors, whereas the primary hydroxylO-6 acts only as acceptor. The water molecules O-Wl and

157

D.Larnba et al.

ois,.

O W L O6,

O W I

OWI OIS,.

O2S

OSS

O2S*

OJS,,

OW'j,

OIS,,

O1S,O3.

Fig. 3. Cation co-ordination of: (a) sodium methyl a-D-galactopyranoside 2-sulphate, 3H2O. , = (I), t = 0—c), e = (II + a), where I and II are thesymmetry equivalent positions of the space group, and a, b and c are unit cell translations, (b) Sodium methyl a-D-galactopyranoside 3-sulphate, H2O„ = (I), j, = (II—b + c); c = (II —a—b + c), where I and II are the symmetry equivalent positions of the space group, and a, b and c are unit celltranslations, (c) Sodium methyl a-D-galactopyranoside 4-sulphate, 2H2O. „ = (I), k = (II), e = Q-b); d = (II + a), , = (D-b), where I and II are thesymmetry equivalent positions of the space group, and a, b and c are unit cell translations, (d) Potassium methyl a-r>-galactopyranoside 4-sulphate, H3O.„ = (I). t = (I + a), c = (I-b), 4 = (TV-b), , = (TV + a -b) , where I, II and IV are the symmetry equivalent positions of the space group, and a, band c are unit cell translations, (e) Potassium methyl a-D-galactopyranoside 6-sulphate, 5H2O. , = (I). b = (I-b), r = ( I I - a -b ) , d = ( I I - 2 a - b - c ) ., = (1—a + b—c), s = (I—a), where I and II are the symmetry equivalent positions of the space group, and a, b and c are unit cell translations.

158

Stereochemical features of sulphated carbohydrates

Fig. 4. Perspective drawings of the crystal packing of: (a) sodium methyl a-D-galactopyranoside 2-sulphale, 3H2O; (b) sodium methyl a-D-galactopyranoside3-sulphate, H2O; (c) sodium methyl a-D-galactopyranoside 4-sulphate, 2H2O; (d) potassium methyl a-D-galactopyranoside 4-sulphate, H2O; (e) potassiummethyl a-D-galactopyranoside 6-sulphate, 5H2O.

159

D.Lamba et al.

O-W2 co-ordinate the Na ions, and are involved in hydrogenbonds to symmetry-related sulphated groups. An interestingfeature of the hydrogen bond network concerns the watermolecules O-W2 and O-W3. O-W2 donates a single bond to thesulphate oxygen O-S2 and a bifurcated bond to 0-3 and O-4 ofneighbouring galactopyranoside molecules. Likewise, O-W3donates a single bond to 0-6 and a bifurcated bond to 0-1 and0-2 of neighbouring galactopyranoside molecules.

In a-MeGal-3S, Na, hydroxyl oxygen O-4 acts both as adonor and acceptor, whereas the hydroxyls O-2 and 0-6 actonly as donors. The hydroxyl 0-2 is involved in a bifurcatedbond to 0-3 and 0-4 of symmetry-related galactopyranosidemolecules. The water molecule O-Wl co-ordinates the Na ionand is involved in hydrogen bonding to symmetry-related sul-phated groups.

In a-MeGal-4S, Na, the hydroxyl oxygens O-2 and 0-6 actboth as donors and acceptors, whereas hydroxyl 0-3 acts onlyas donor. The water molecule O-Wl donates two hydrogenbonds to symmetry-related sulphated groups, accepts an 0-2Hbond and co-ordinates the Na-2 ion. The water molecule 0-W2donates a single bond to 0-6 and a bifurcated bond to O-l and0-2 of neighbouring galactopyranoside molecules.

In a-MeGal^S, K, the hydroxyl oxygens 0-2, O-3 and O-6show the same hydrogen bonding pattern as the one found ina-MeGal-4S, Na. The water molecule O-Wl likewise donatesa single bond to 0-6 and a bifurcated bond to O-1 and 0-2 ofneighbouring galactopyranoside molecules.

The poor accuracy of the structural determination ofa-MeGal-6S, K does not provide enough information to char-acterize unambiguously the hydrogen bonding scheme of thestructure. Only inter-atomic distances between atoms belongingto putative hydrogen bonds are presented along with other shortcontacts.

It is worth noting that the sulphate groups, as already ob-served in the crystal and molecular structure of /3-Glc-6S, K,also in a-MeGal-4SK, are not involved in hydrogen bonds witheither water molecules or hydroxyl groups, but do indeed parti-cipate only to the K-co-ordination sphere.

Biological implications

The introductory survey indicates the extent to which thesulphated sugars are known to be distributed in biologicalmolecules. In addition to the well-established occurrence ofesters in the carrageenan and glycosaminoglycan poly-saccharides, heed must be taken of their occurrence in a widerange of glycoproteins. For example, the recent reports describ-ing the high-affinity binding of L- and E-selectin molecules tosulphated Le° and Le* determinants indicates a specific role forsulphate groups in the modulation of these particular molecularrecognition interactions (Green et al., 1992; Yuen et al., 1992),as must also be the case of binding of synthetic heparin oligo-saccharides to antithrombin III (Petitou et al., 1988). Thus, itis essential that, in attempts to understand the biological rolesof sulphate groups, cognizance is taken of their stereochemistryand charge distributions. Herein lies the importance of thepresent protocol for parameterizing the sulphate group forapplication in modelling studies.

In conclusion, this work provides details of the structuralfeatures of sulphated monosaccharides which are found innaturally occurring sulphated carbohydrates. Substitution withsulphate slightly affects the chair conformation, and results bothin a lengthening of the C-O bond and the opening of the

C-O-S angle at the point of attachment of the sulphate moiety.The results obtained show that the conformation of the sulphategroup does not depend on environmental factors such asposition, cation co-ordination, hydrogen bonding and packing.Furthermore, these observations are supported by energy cal-culations in which the van der Waals interactions alone accountfor the spatial orientation of the sulphate group. Accordingly,the force field which has been derived in the present study mayhave applications in the structural investigations of more com-plex cases, such as sulphated polysaccharides and glyco-conjugates.

Materials and methods

Preparation and crystallization of monosulphated monosaccharides

Crystalline compounds studied in the present investigations were: methyl a-D-galactopyranoside 2-sulphate, sodium salt, trihydrate (a-MeGal-2S, Na); methyla-D-galactopyranoside 3-sulphate, sodium salt, monohydrate (a-MeGal-3S,Na); methyl or-r>galactopyranoside 4-sulphate, sodium salt, dihydrate (or-MeGal-4S, Na); methyl a-D-galactopyranoside 4-sulphate, potassium salt, mono-hydrate (a-MeGal-4S, K); and methyl a-D-galactopyranoside 6-sulphate,potassium salt, pentahydrate (a-MeGal-6S, K). These compounds were syn-thesized according to the procedure described by Rashid el al. (1990) and werecrystallized from a slow concentration of aqueous isopropanol solutions at roomtemperature. The crystal structures of jS-D-glucopyranose 6-sulphate, potassiumsalt (/3-MeGlc-6S, K) (Lamba el al., 1988) and of methyl a-D-galactopyranose2,6-bis (sodium sulphate) (Lamba et al., 1993) have been reported.

X-ray crystallography

Details of the pertinent crystal data are reported in Table VS. Intensity data werecollected al room temperature, with a 20-OJ scan mode on an Enraf-NoniusCAD-4F diffractometer (CuKa radiation, Nickel filtered). Accurate unit-cellparameters were determined by a least-squares fit of the setting angles at high26 values. Lorentz and polarization corrections were applied, but no correctionwas made for absorption.

The structures were determined by direct methods and refined by full-matrixleast-squares methods minimizing Eu( |F o | - |F j ) 2 , where u is derived fromstatistical counting (Sheldriclc, 1976, 1986, 1990; Nardelli, 1983). The hydro-gen positions were found by difference-Fourier methods. The reliability indicesrange from 0.027 to 0.059. The atomic scattering factors used were taken fromthe International Tables for X-Ray Crystallography (1974). The details of thedata collection, solution and refinement of the structures are summarized inTable IV. Because of the poor quality of the single crystals of methyl a-D-galactopyranoside 6-sulphate, potassium salt, pentahydrate (a-MeGal-6S, K),the structure could not be highly refined.

Energy calculations and parameterization

Nomenclature. The labelling of the atoms is indicated in Figure 1. The recom-mendations and symbols proposed by the IUPAC-IUB (1971) are used through-out this paper. The two prochiral hydrogen atoms at C-6, H-C-6R and H-C-6Sare differentiated using the rule proposed by Hanson (1986).

The orientation of the hydroxymethyl group is described by a> =O-5-C-5-C-6-O-6 and o^ = H-5-C-5-C-6-O-6, and is referred to as eithergauche—trans (gt), gauche—gauche (gg) or trans—gauche (tg), corresponding toai values of 60°, -60° and 180°, respectively (Marchessault and Pfrez, 1979).

The orientation of the sulphate group attached at position x is described bythe torsional angles \i = C-x +1-C-x-O-x-S and u, = C-x-O-x-S-O-S 1 (uj= C-x-O-x-S-O-S2) (u3 = C-x-O-x-S-O-S3) (see Figure 2a).

Molecular mechanics calculations. The investigation of the preferred low-energy conformations of the sulphate group was conducted using the programPFOS (Tvaroska and Pe'rez, 1986). The conformational energy is evaluated byincluding the partitioned contributions arising from van der Waals interactionsand torsional potential. The geometry is considered to be invariant andhydroxylic hydrogen atoms are not taken into account. The torsional angles(ji.v) describing the orientations of the sulphate groups were increasedin increments of 5° in the range -180° to 180°. The iso-contour levelswere drawn at intervals of 1 kcal/mol with respect to the global minimum.The low-energy region was limited by the iso-energy contour corresponding to10 kcal/mol above the minimum.

160

Stereochemkal features of sulphated carbohydrates

Table IV. Data collection

Empirical

Data collectionDiffractometer usedRadiation (A)Temperature (K)Max. theta (°)Index ranges: h

k1

Reflections collectedIndependent reflectionsRmergeObserved reflections[Fo>4.0 a(Fo)]

Solution and refinementSolutionRefinement methodQuantity minimizedHydrogen atoms

Weighting scheme[w"1 = (r

!(Fo)+gFo2] g =Final RFinal wRLargest and mean shift/aData-to-parameter ratioLargest diff. peak (eA"3)Largest diff. hole (eA~3)

, structure resolution

C^H.jO^"Na+ '3H,0

CAD-4F

29370-17 to 17Oto 7- 9 to 9

309928760.0431472

Diff. Founer

=0.0026940 0270.0300 042, 0.0915.6:1+0.19-0.31

and refinement

C,H,AS-Na+

H2O

CAD^F

293700 to 8- 1 5 to 15- 1 6 to 16

501045300.0482213

Diff. Founer

0.0055380.0560.0730.169, 0.1089.5 1+0.42-0.78

C7HI3O,S-Na+I3

2H2O

CAD-4FCuKa(1.5418)

293700 to 6- 8 to 8-31 to 31

278125710.0201341

Direct methodsFull-matnx least-squaresEu)(Fo-Fc)2

Diff. Founer

0.0079770 0590.0640 141, 0.0619.7:1+ 1.00-1.03

C^H.jCLS-K+ 'H2O

CAD-4F

293700 to 7- 9 to 9-34 to 34

537046470.0372243

Diff. Founer

0.0O4O700.0440.0460 210, 0.0765.4:1+0.82- 0 48

C H,,O,S-K

Siemens R3m/V

29870Oto 12- 9 to 2-17 to 17

24702253-1252

Rigid modelFixed isotropic

0.00790.1300.1730.010, 0.0027.6:11 19-0.83

The parameterization of the O-sulphate group was made for incorporation inthe MM3 molecular mechanics program (Allinger el al., 1989) The total energytakes into account the stretching, bending, stretch—bending, torsional anddipolar contributions, and the van der Waals interactions. The MM3 potentialfunction does not use lone pairs. It has been parameterized to account forhydrogen bonding; the parameterization for the anomeric effect is preliminary.

Protocol for parameterization. The aim of the parameterization was toinvestigate how molecular mechanics calculations can best reproduce thegeometrical and conformational features of sulphated monosaccharide units.There is only one report in the literature of parameters dealing with O-sulphategroups for use with the MM2 type of molecular mechanics calculations (Ragazziet al., 1986). Unfortunately, this set of parameters does not account for someof the geometrical features found in the crystal structures investigated in thepresent work. Therefore, a set of torsional, stretching, bending and dipoleparameters specific for the Cx-Ox-Sx-O(S)3 ~~ sequence had to be derived.

For this purpose, calculations were carried out on a training set whichconsisted of all but one sulphated monosaccharide structures. Both the geometryparameters and the force constants were adjusted to reproduce the moleculargeometry, but also the degree of variance in the parameters. All sulphatedihedral angles were permitted to rotate freely, i.e. no additional torsional forceconstants were added to the natural van der Waals barriers. When a suitable setof parameters was obtained (after -50-60 runs of MM3), its predictivecapability was verified by testing on the crystal structure of a sulphated moleculewhich was not used in the training set. This iterative procedure yielded theparameters listed in Table m.

Acknowledgements

We thank the University of Leeds Computing Service, Centre de Recherches,INRA (Nantes) and the CNR (Research Area of Rome) for facilities. Theprovision of financial supports by AFRC to S.G. and A.R., by CNR (ProgrettoFinalizzato Chimica Fine H) to D.L., and by Ministere de la Recherche et dela Technologie to S.P. are also gratefully acknowledged. The authors are

indebted to Mr Edwin Yates who contributed to the work on (a-MeGal 6S,K),, 5H2O and to Dr A.Imberty for her critical reading of the manuscript.

Abbreviations

gg, gauche—gauche; gt, gauche-trans; tg, trans-gauche; or-MeGal-2S, Na,methyl a-D-galactopyranoside 2-sulphate, sodium salt, trihydrate, a-MeGal-3S, Na, methyl a-D-galactopyranoside 3-sulphate, sodium salt, monohydrate,a-MeGal-4S, Na, methyl a-D-galactopyranoside 4-sulphate, sodium salt, di-hydrate; a-MeGal-4S, K, methyl a-D-galactopyranoside 4-sulphate, potassiumsalt, monohydrate; a-MeGal-6S, K, methyl a-D-galactopyranoside 6-sulphate,potassium salt, pentahydrate.

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Received on July 7, 1993; revised on November 17, 1993; accepted onNovember 18, 1993

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