Redox-controlled Site-specific Enzymatic Glycosylation

Sialylation and fucosylation are ubiquitous modifications of various complex naturaloccurring glycans.α2,6-Sialylated and α1,3-fucosylated glycans play essential roles in many physiological and pathological processes.Biosynthesis of sialylated glycans or fucosylated glycans involves multiple sialyltransferases or fucosyltransferases that introduce the corresponding monosaccharide to glycan backbones in a non-site-specific manner,generating heterogeneous and incomplete sialylated or fucosylated mixtures.To decipher their important biological functions,novel methods for site-selectively introducing α2,6-sialic acid and α1,3fucose moieties to glycans with multiple glycosylation sites are highly desired.This thesis focuses on the site-specific synthesis of complex α2,6-sialylated and α1,3fucosylated glycans with a redox-controlled strategy.The study contains the following parts.To achieve site-specific α2,6-sialylation on acceptor substrates containing multiple sialylation sites,a novel redox-controlled site-specific α2,6-sialylation strategy was developed.Galactose oxidase was utilized to mask the unwanted sialylation sites by selectively oxidizing the C6-hydroxyl group of non-reducing terminal Gal or GalNAc unit to aldehyde so that it cannot be recognized by Photobacterium damselae α2,6-sialyltransferase(Pd2,6ST).Meanwhile,α2,6-sialylation was carried out on the intact Gal and/or GalNAc units.The aldehyde groups can be easily reduced by NaBH4 to afford a variety of αa2,6-sialosides with defined sialylation patterns.Seven enzyme modules were designed to construct seven different types of glycosidic linkages.The construction of corresponding glycosidic linkages was achieved from simple monosaccharides including Gal,GalNAc,Sia,and GlcNAc.The precise enzymatic synthesis of a2,6-sialosides with different sialylation degrees and different sialic acid types(Neu5Ac,Neu5Gc,and Kdn)on glycans comprising common disaccharide units including LacNAc,LNB,LacDiNAc,and GNB was realized by this redox-controlled substrate engineering strategy.The redox-controlled substrate engineering strategy was also used to realize the sitespecific α1,3-fucosylation of complex glycans containing multiple fucosylation sites.The GlcNAc residues of LacNAc units are potential fucosylation sites.The oxidation module containing galactose oxidase was used to selectively oxidize the C6-hydroxyl group of Gal of non-reducing terminal LacNAc units into aldehyde to temporarily block the fucosylation sites on the LacNAc units.Meanwhile,α1,3-fucosylation was carried out directly on the intact LacNAc units by using Helicobacter pylori αl,3-fucosyltransferase(Hpα1,3FucT),and then NaBH4 was used to reduce the aldehyde groups back to hydroxyl groups to provide a variety of a1,3-fucosides with defined fucosylation patterns.Three enzyme modules were designed to introduce three different types of glycosyl units including β1,4-galactose,β1,3-Nacetylglucosamine,and α1,3-fucose.The substrate specificity of Hpα1,3FucT was investigated by using HILIC-ELSD.This strategy provides easy access to the site-specificα1,3-fucosylation of poly-LacNAc glycans,O-GalNAc glycans,and N-glycans.By incorporating the concept and strategy of organic synthesis with the enzymatic synthesis,a novel redox-controlled site-specific glycosylation strategy was developed,which overcome the limitation of glycosyltransferase availability and substrate specificity.This strategy provides a general solution for the precise manipulation and regulation of sialyltransferase and fucosyltransferase-catalyzed glycosylation.The preparative scale of sitespecific α2,6-sialosides and α1,3-fucosides with important biological functions were systematically synthesized.