The conformations of the NeuAcα2(I)→3Galβ1(II)→4[Fucα1(III)→3]GlcNAc-O-CH3 tetrasaccharide (sLex), in aqueous solution and bound to E-, P-, and L-selectin have been determined using high resolution NMR spectroscopy. In the free ligand, the conformation of glycosidic linkage I is disordered with {ΦI, ΨI} sampling values close to {−60°, 0°}, {−100°, −50°}, and {180°, 0°}. The trisaccharide portion is rigid and characterized by {ΦII, ΨII; ΦIII, ΨIII} = {46°, 18°; 48°, 24°}. The measured dissociation rates and equilibrium binding constants, {k off, K D}, were {164 ± 24 s-1, 0.72 ± 0.4 mM}, {522 ± 166 s-1, 7.8 ± 1.0 mM}, and {1080 ± 167 s-1, 3.9 ± 0.6 mM} at 300 K for E-, P-, and L-selectin, respectively. The bound conformations of the ligand were calculated from the full relaxation matrix analysis of transferred-NOE spectra for E- and P-selectin or by using a two-spin approximation for the L-selectin complex. Both E- and P-selectin recognize the {−60°, 0°} conformation of sLex while the {−100°, −50°} conformer is probably recognized by L-selectin. The conformation of the branched trisaccharide portion in the bound state remains close to the conformation of the free ligand. In the E-, P-, and L-selectin complexes the GalH4 proton is in the vicinity of protein aromatic protons, most likely Tyr94 and/or Tyr48.

1 P. V. NIKRAD, H. BEIERBECK, and R. U. LEMIEUX. Can. J. Chem. 70, 241 (1992).The relative potencies of both the monodeoxy and mono-0-methyl derivatives of the Leb-OM^ tetrasaccharide (a-LFuc-(1 + 2)-P-D-Gal-(1 + 3)-[a-L-Fuc-(I + 4)]-P-D-GlcNAc-OMe) as inhibitors of the complexation of a Leb artifi; cia1 antigen by the lectin IV of Gr~ffonia sirnplicifolia are interpreted in terms of the X-ray crystal structure at 2.5 A resolution of the GS-IV . L , e b -O~e complex. Both kinds of derivatives maintain high potencies when the hydroxyl groups involved appear, in the crystal structure, to be in contact with the aqueous phase. Hydroxyl groups situated at the periphery of the combining site and hydrogen bonded to the protein can also be deoxygenated without important loss in potency. However, their methylation leads to a strong decrease in the stability of the complex, because the steric bulk of the introduced methyl group causes loss of complementarity. In contrast, the hydroxyl groups that form hydrogen bonds with the protein along the base of the shallow amphiphilic cleft of the combining site can neither be deoxygenated nor methylated without virtually complete loss of binding activity. Thus, the procedure can provide an appreciation of the various kinds of hydrogen bonds that are present in a protein. oligosaccharide complex. Hard-sphere calculations supported these contentions since an energetically favorable orientation was indicated for a methoxy group at any one of the five positions that were expected to remain in contact with the aqueous phase. However, the calculations, as expected, showed the introduction of strong destabilizing nonbonded interactions when the methylation involved hydroxyl groups that are hydrogen bonded to the protein in the complex. The results are in accord with the previously made rationalization of the near linear enthalpy-entropy compensation found for the active deoxy congeners.Key words: molecular recognition, lectin IV of Griffonia simplicifolia, 0-methyl derivatives of the Lewis b-OMe tetrasaccharide, detection of intermolecular hydrogen bonds, hard-sphere calculations. On a determine les efficacitks relatives des dCrivCs monodksoxy-et mono-0-mCthyle du tetrasaccharide Leb-OM^ (a-]-P-D-GICNAC-OM~) comme inhibiteurs de la complexation d'un antigene artificiel du Leb par la lectine IV du Grlffonia simnplicifolia; on interprete les rCsultats en fonction de la structure cristalline d'un complexe GS-IV . Leb-OM^, dCterminCe par diffraction des rayons-X, ti une rCsolution de 2,5 A. Les deux types de dCrivks maintiennent des efficacitks ClevCes lorsqu'il semble que, dans la structure cristalline, les groupes hydroxyles sont en contact avec la phase aqueuse. Lorsqu'on dksoxygkne les groupes hydroxyles qui sont situCs 2 la pCriphCrie du site qui se combine et qui sont lies a la protCine par des liaisons hydrogknes, il n'y a pas de perte importante de I'efficacitC. Toutefois, leur mkthylation conduit a une perte de complCmentaritC et a une baisse importante de la stabilitC du complexe qu...

. Can. J. Chem. 68, 1116 (1990).The lectin IV of Griffonia simplicifolia (GS4) specifically binds the terminal tetrasaccharide unit of the Lewis b human blood group determinant (~e~) .The single crystal X-ray analysis of the complex with L~~-o M~ has demonstrated that the binding site on the lectin is a shallow depression with a negatively charged aspartate side chain at the bottom of the cavity. In addition to this aspartate, a serine and an asparagine side chain provide the polar groups that hydrogen bond to the three hydroxyl groups of Leb, which has been termed the key polar grouping for complex formation. A notable characteristic of the binding site is that five aromatic amino acid side chains (one Phe, two Tyr, and two Trp residues) surround these polar interactions and make van der Waals contacts with the tetrasaccharide. Thus, as predicted from previous solution binding studies, extensive nonpolar interactions are involved, which contribute importantly both to the specificity of the reaction and the stability of the noncovalent complex that is formed. These results represent the first structural example of the molecular recognition of a human blood group determinant by the receptor site of a protein. Extensive sequence homology exists between GS4 and the concanavalin A (Con A), pea, and favin lectins. The main hydrophilic groups of the carbohydrate-binding site of GS4 and Con A are aspartate, asparagine, and serine residues; the homology suggests that the serine is replaced by asparagine in the case of the pea and favin lectins. It appears probable that these two latter lectins possess very similar, if not identical, specificities.Key words: lectin, carbohydrate, molecular recognition, binding.Lours T. J. DELBAERE, MARGARET VANDONSELAAR, LATA PRASAD, J. WILSON QUAIL, JOYCE R. PEARLSTONE, MICHAEL R. CARPENTER, LAWRENCE B. SMILLIE, PANDURANG V. NIKRAD, ULRIKE SPOHR et RAYMOND U. LEMIEUX. Can. J. Chem. 68, 1116 (1990).La lectine IV du Grrffonia simplicifolia (GS4) se lie sptcifiquement a l'unitt tktrasaccharide terminale du determinant du groupe sanguin humain Lewis b (Leb). Une analyse par diffraction des rayons-X effectute sur un cristal unique de son complexe avec le Leb-OM^ a perrnis de dtmontrer que le site de liaison de la lectine est une ltgtre dkpression au fond de laquelle on retrouve une chaine lattrale d'aspartate chargke nkgativement. En plus de cet aspartate, des chaines lattrales de strine et d'asparagine fournissent des groupements polaires qui foment des points hydrogenes avec les trois groupements hydroxyles du ~e~; il s'agit du groupement polaire clt pour la formation de complexes. Le fait que cinq chaines lattrales formtes d'acides amints arornatiques entourent ces interactions dipolaires et qu'elles ttablissent des contacts de van der Waals avec le tktrasaccharide est une caracteristique notable de ce site de liaison. Tel que prkdit 2 partir d'ttudes anttrieures de liaisons en solution, il existe donc de nornbreuses interactions non-polaires qui apportent une contribution importante tant...

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Dinitroarenes containing substituents such as hydroxyl and amine groups could be conveniently reduced with 3 molar equivalents of hydrazine hydrate in presence of Raney nickel catalyst in ethanol/1,2-dichloroethane solvent mixture to give a product wherein one of the two nitro groups was reduced to the amino group. The yields of the partial reduction products are good. Under similar conditions alkoxyl substituents in the o,p-position to the nitro groups were displaced by the hydrazino group to give 2,4-dinitrophenylhydrazine as the main product. The details of the reduction reaction are described.

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