Investigating protein - carbohydrate interactions with glycomimetics and oligovalent mannosides

To explore the molecular features responsible for the specificity of carbohydrate-binding proteins, we have investigated mono- and multivalent ligands targeted toward rat mannose binding protein A (MBP-A) and concanavalin A (Con A), respectively. We have developed methods for the solid-supported parallel synthesis of non-carbohydrate-based analogues of mannose utilizing the carbocycle shikimic acid as a scaffold. The α-β unsaturated carboxylic acid moiety of shikimic acid allowed for the incorporation of diversifying elements onto the carbocyclic scaffold by sequential amide formation and conjugate addition of thiols. The reaction conditions were tailored to the FlexChem 96-well combinatorial block and 192 compounds were prepared in multi-milligram quantities. The ability of the compounds to block the binding of the C-type lectin MBP-A to a multivalent mannosylated surface was assessed in a novel bead elution assay. Results of the assay identified ten library members with inhibitory activity approximately equal to that of α-methylmannopyranoside. The results suggest that shikimic acid can be utilized to develop libraries of non-carbohydrate-containing molecules targeted toward the C-type lectin family. To investigate the structural features of multivalent carbohydrate ligands that influence carbohydrate-protein binding, the interaction between a small multivalent ligand and lectin concanavalin A was investigated. The small multivalent ligand investigated contains three mannose residues appended to a rigid macrocyclic backbone. Inhibition assays performed with the trivalent ligand against Con A on a surface plasmon resonance instrument containing a mannose-derivatized sensor surface suggest that the small multivalent ligand enhances the interaction between Con-A and the surface. In contrast, a monovalent control ligand inhibits the binding of Con-A to the surface. Fluorescence resonance energy transfer experiments, performed with fluorescently labeled Con A and either the tri- or monovalent ligands, indicate that the trivalent ligand is capable of clustering the lectin in solution whereas the monovalent ligand does not. This solution clustering occurs without precipitation and its implications on carbohydrate-protein binding are discussed.