The cluster glycoside effect - the observation that multivalent glycosides bind to their polyvalent protein receptors with apparent affinities greater than those that can be rationalized solely on the basis of valency - is by now a well established phenomenon. As part of a continuing effort to provide a molecular basis for the cluster glycoside effect, we report here the synthesis of two series of mannosylated dendritic ligands and their performance in a range of competitive and non-competitive binding assays, including hemeagglutination inhibition (HIA), enzyme-linked lectin assays (ELLA) and isothermal titration microcalorimetry (ITC). The first series of ligands contained a semi-rigid glycylglycine spacer and showed no significant performance enhancement in any binding studies. The second series of ligands contained a flexible tetraethylene glycol spacer; these ligands showed marked enhancements at tetravalent and hexavalent levels in both HIA (IC =3 and<0.8 μM, respectively) and ITC (K(A)=6.2x10 and 1.5x10 M , respectively) studies. In all cases, the thermodynamic parameters of association are consistent with non-specific aggregation rather than enhanced lectin-ligand affinity. This conclusion is reinforced by the lack of enhancements in ligand activity observed in ELLA studies. Copyright (C) 2000 Elsevier Science Ltd. 50 4 6 -1