Chemoenzymatic Synthesis and Functional Studies of O-Acetylated Sialoglycans and N-Glycan Derivatives

This thesis explores the structural diversity, biological functions, and synthetic accessibility of O-acetylated sialic acids and related complex glycans. It focuses especially on the development of practical chemical and chemoenzymatic methods for producing structurally defined O-acetylated sialoglycans and N-glycan derivatives, thereby enabling the investigation of their roles in viral recognition, innate immune sensing, and early N-glycosylation. In chapter 1, an overview was provided of the structures, biological roles, and synthetic strategies of O-acetylated sialic acids. In Chapter 2, a chemoenzymatic methodology is described that provides well-defined 7-O-, 9-O-, and 7,9-di-O-acetylated GD3 gangliosides. All O-acetylated GD3 patterns are fully characterized by NMR and HRMS. We further demonstrate that these glycosphingolipids can be used to remodel cell surfaces. LC-IM-MS analysis confirms stable insertion into the plasma membrane with preservation of the installed acetyl-ester pattern. As a ready-to-use tool, O-acetylated GD3 modified erythrocytes provide an off-the-shelf platform to study viral protein interactions in a native membrane environment. In Chapter 3, a chemo-enzymatic route is presented for generating the first K1 antigen (polysialic acid, PSA) fragments bearing uniformly 7- or 9-O-acetylated Neu5Ac. The approach combines chemical assembly of the oligosaccharide backbone with late-stage O-acetyl editing using coronavirus hemagglutinin-esterases. These chemically well-defined K1 PSAs are used to interrogate interactions with human L-ficolins; notably, L-ficolin preferentially recognizes 7-O-acetylated sialosides over their 9-O-acetylated and non-acetylated counterparts. Saturation-transfer difference NMR (STD-NMR), together with molecular docking, shows that the 7-O-acetyl methyl aligns with hydrophobic residues in the L-ficolin binding pocket, rationalizing the observed selectivity and revealing a pattern-recognition mechanism by which innate immunity discriminates subtle PSA O-acetylation states. Chapter 4 focuses on the biochemical study of early N-glycosylation by providing practical access to dolichol-linked oligosaccharide substrates. A concise route is developed to obtain the Man₃GlcNAc₂ core glycan through enzymatic trimming of sialoglycopeptide, followed by conversion into phosphate or phosphorothioate donors and coupling to synthetic dolichol phosphate. This strategy provides access to both native and enzymatically stabilized LLO analogs in quantities suitable for biochemical analysis, thereby facilitating mechanistic investigations of the RFT1 flippase and oligosaccharyltransferase.