Understanding the effects of multivalent glycopolymer structure on molecular recognition
The Graduate School, Stony Brook University: Stony Brook, NY.
Carbohydrate-protein interactions are involved in a large number of important physiological and pathological processes. The weak binding affinity of a monovalent carbohydrate is compensated by multivalency in which multimeric recognition elements interact with a cell surface display of two or more receptors. In order to study lectin-carbohydrate interactions and find analytical and diagnostic applications, a large number of synthetic glycoconjugates have been investigated in the last decade. In order to explore the effects of structural features of glycopolymers on the binding events, a series of polymers were synthesized. Multivalent ligands with pendant saccharide moieties were prepared from two different types of backbones via ruthenium catalyzed ring-opening metathesis polymerization (ROMP). Several functional groups and neutral and charged spacers were introduced onto the backbone to explore binding of glycopolymers to cholera toxin B subunit (CT B5) The interactions of cholera toxin and the polymers were determined using the intrinsic fluorescence of the Trp 88 residue in the cholera toxin binding site. As sugar epitopes in polymers bind to CT B5, a variable decrease in fluorescence was observed. The improvement in inhibition over glycopolymers was also observed using competitive ELISA experiments. The interesting insight we found was that self-assembly of glycopolymers was involved in lectin binding events. Since glycopolymers have hydrophobic backbone and hydrophilic sugar moieties, glycopolymers form micelles and aggregates which derive from hydrophobic interactions of polymer backbone and inter- or/and intra-molecular hydrogen bonding of sugars. Further studies to identify polymer behaviors in aqueous solution were carried out by measuring their critical micelle concentration (CMC) and determining their size and morphology by dynamic light scattering (DLS) and transmission electron microscopy (TEM). These studies revealed that norbornene-based and cyclobutene-based polymers containing sugar moieties self-assemble into micelles and vesicles. The polymer particles were spherical and their size was heterogeneous. Binding of the cholera toxin B5 protein did not induce further aggregation. In glycopolymer particles, hydrophilic sugar moieties are located on the surface and hydrophobic backbones which composed of backbones of polymers are inside. In the recognition of lectin by synthetic glycopolymers, the binding affinity of glycopolymer was significantly inversely correlated with the self-assembled polymeric structure.