Glycoconjugate Journal (1998) 15: 251 — 263 Effect of shape, size, and valency of multivalent mannosides on their binding properties to phytohemagglutinins Rene ´ Roy 1 *, Daniel Page ´ 1 , Santiago Figueroa Perez 2 and Vicente Verez Bencomo 2 1 Department of Chemistry, University of Ottawa, Ottawa, ON, Canada, K1N 6N5 2 Laboratory of Synthetic Antigens, Facultad de Quimica Universidad de La Habana, Ciudad de la Habana, Cuba, 10400 Clusters of di-, tri-, and tetra-antennary -D-mannopyranosides were synthesized in good yields based on the coupling of amine-bearing mono- or trisaccharide {Man (16)[Man (1P3)]Man} haptens to poly-isocyanate or -isothiocyanate tethering cores. The relative binding properties of the resulting multivalent ligands were determined by turbidimetric and solid phase enzyme-linked lectin assays (ELLA) using plant lectins (phytohemagglutinins) Concanavalin A (Con A) and Pisum sativum (pea lectin) having four and two carbohydrate binding sites, respectively. Rapid and efficient cross-linking between tetravalent Con A and mannopyranosylated clusters were measured by a microtiter plate version of turbidimetric analyses. In inhibition of binding of the lectins to yeast mannan, the best tetravalent monosaccharide (30) and trisaccharide (31) inhibitors were shown to be 140 and 1155 times more potent inhibitors than monomeric methyl -D-mannopyranoside against pea lectin and Con A, respectively. Compounds 30 and 31 were thus 35- and 289-fold more potent than the reference monosaccharide based on their hapten contents. As a general observation, the ligands bearing the Man (1P6)[Man (1P3)]Man trimannoside structures were found to be more potent inhibitors for Con A than the ligands having single mannoside residues, whereas pea lectin could not discriminate between the two types of ligands. Keywords: mannoside clusters, Concanavalin A, pea lectin, cross-linking, valency Introduction The syntheses and development of potent carbohydrate inhibitors is of prime interest to understand the different factors involved in protein-carbohydrate interactions. Fea- tures such as structure, shape, size, geometry, and valency have proved to be determinant factors in influencing the generally weak binding properties of carbohydrate ligands (K  +0.1—1mM) [1]. To overcome these weak binding in- teractions, multivalent neoglycoconjugates [2, 3] ranging from clusters [4] and oligomers [5—7], to macromolecular polydispersed systems such as glycopolymers [8, 9] and glycodendrimers [10—13] have been generated to provide antiadhesins of higher affinity. A large number of these multivalent carbohydrate ligands demonstrated powerful inhibitory properties when tested against their specific ani- mal or plant lectins. For instance, a glycodendrimer carry- ing twelve terminal -sialoside residues was shown to be 182-fold more potent than its corresponding monomer when used in the inhibition of binding of the slug lectin * Author to whom correspondence should be addressed. Tel: #1(613)- 562-5800 ext 6055; Fax: (613)-562-5170; E-mail: RROY@SCIENCE. UOTTAWA.CA. ¸imax flavus to human orosomucoid [14]. Clusters contain- ing as few as three N-acetylgalactosamine (GalNAc) [15] or galactose (Gal) [16] residues represent classical examples of the ‘cluster effect’ [17] used in the study of hepatic asialo- glycoprotein receptors (ASGP-R). In these cases, thousand fold increases in binding affinity have been observed. Sim- ilar trends also prevail in the inhibition of influenza virus hemagglutination of human erythrocytes by polymers con- taining sialosides [8, 18]. Because of the potential toxicolo- gical and immunological drawbacks of glycopolymers for therapeutic interventions, smaller and better defined struc- tures are desirable. The sometime drastic increased inhibitory properties ob- served from these multivalent neoglycoconjugates are far from being clearly understood. As the higher associative forces that held these clusters to their reciprocally clustered receptors is not likely to originate from an intrinsic change in the individual binding affinities (K  ) (Roy R, Page´ D, Dimick S, Toone EJ, unpublished data), but rather from an overall cooperative binding forces (avidity), it becomes appealing to generate architecturally well defined ‘glyco- clusters’. One likely explanation for the improved binding of glycoclusters may reside in the overall structures of the cross-linked lattices which inevitably form when two 0282—0080  1998 Chapman & Hall