Chemoenzymatic synthesis of homogeneous glycopeptides and glycoproteins for functional studies

Functional and structural studies on glycoproteins are hampered by lack of homogeneous structures due to the inherent structural heterogeneity in the pendant glycans which makes separation by routine chromatographic techniques extremely difficult. To address this problem, we used a chemoenzymatic method to make homogeneous glycopeptides and glycoproteins, which relies on the glycosynthase-based transglycosylation of a GlcNAc-peptide/protein using synthetic glycan oxazoline as the enzyme substrate. The thesis research presented here consists of three projects.;First, we synthesized selectively fluorinated homogeneous Man3GlcNAc 2-RNase glycoproteins for binding studies to the concanavalin A (con A) lectin. From SPR analysis, we observed that fluorine substitution at the C6-hydroxyl of the alpha-1,6-branched mannose residue of the Man 3GlcNAc2 negated lectin binding suggesting the importance of that hydroxyl group in binding interactions. We found that the fluorinated glycan oxazoline corresponding to the Man3GlcNAc core could serve as an excellent substrate for the Endo-A enzyme during transglycosylation and a structural modeling analysis of the Endo-A enzyme binding pocket suggested favorable hydrophobic interactions between a suitably located tyrosine and the fluorine. This suggest that the selectively fluorinated glycoproteins can be used to probe specific protein-carbohydrate interactions.;Secondly, we synthesized homogeneous HIV-1 V3 glycopeptides for characterizing the epitope of a recently discovered, potent anti-HIV-1 broadly neutralizing antibody (bnAb) PGT121 and related antibody 1074. SPR binding analysis indicated that antibody PGT121 recognizes complex type N-glycan while the PGT121-like antibody 10-1074 binds to high-mannose type N-glycan (Man9GlcNAc 2) in the context of the V3 domain. Interestingly, these antibodies did not bind to free N-glycans or non-glycosylated V3 polypeptides under the same conditions. This study has facilitated the characterization of the glycan specificity of the two anti-HIV antibodies.;Finally, we designed and synthesized a novel multifunctional BALT tag for separation of N-glycans for functional glycomics applications. It consists of an N-methylhydroxylamine group for attachment to reducing ends of glycans with preservation of ring structure, free primary amine for immobilization to microarrays, tyrosine group providing a chromophore and hydrophobicity during chromatographic separation and biotin for binding to streptavidin. The tag was successfully used for separation and recovery of individual N-glycans from a mixture of four N-glycans released from the glycoprotein bovine fetuin.