Functional inhibition of wheat germ agglutinin by glycodendrimers: Interplay of affinity, architecture, and temperature.

Multivalent interactions between lectins and glycans are crucial for biological recognition; however, predicting functional inhibition based on binding affinity remains challenging. Herein, we investigated a series of structurally defined N-acetylglucosamine (GlcNAc)-functionalized dendrimers (1a-1c and 2a-2c) to examine how spatial orientation and temperature influenced the inhibition of wheat germ agglutinin (WGA). Using enzyme-linked lectin assays (ELLAs), we observed biphasic inhibition profiles for all the dendrimers, characterized by an initial enhancement of WGA binding at low concentrations, followed by effective inhibition at higher concentrations. Notably, compounds 2a and 1b exhibited greater inhibitory potency at a lower temperature (4 °C) compared to that at ambient temperature (37 °C) despite having comparable dissociation constants (K) in the nanomolar range. Complementary dynamic light scattering (DLS) analysis revealed temperature-dependent assembly behaviors, with 2a and 2b forming larger and more heterogeneous complexes at 37 °C, and more stable aggregates at 4 °C. These results reveal that the inhibitory efficacy arises from a complex interplay between binding affinity, dendrimer architecture, and the dynamic behavior of lectins across varying thermal conditions. Our findings emphasize the limitations of affinity-based predictions and underscore the need to integrate structural and kinetic perspectives when designing multivalent inhibitors targeting glycan-binding proteins.