Carbohydrates are crucial biomacromolecules found in organisms across all domains of life and play pivotal roles in biological processes. To better understand the biological function of glycans, structurally-defined glycopeptides/glycoproteins and glycans are highly demanded. In this dissertation, the study has been focusing on acquiring homogenous glycopeptides/glycoproteins and glycans which are good candidates for decoding their function.

Chapter 1 provides a brief introduction of function of glycosylation for proteins, significance of glycosylation for therapeutic protein and artificial glycosylation approaches; glycan synthetic approaches and glycan microarray and an overview of synthesis of oligosaccharides in large scale in vitro and in vivo.

Chapter 2 describes two techniques for synthesis of glycopeptide/glycoprotein. (1) A facile and efficient method for site-directed glycosylation of peptide/protein. The method contains two sequential steps: generation of a GlcNAc-O-peptide/protein, and subsequent ligation of a eukaryotic N-glycan to the GlcNAc moiety. (2) Peptides can be GlcNAcylated at one or two natural N-glycosylation sites via two-step enzymatic reactions catalyzed by an evolved N-glycosyltransferase (ApNGT Q469A) and a glucosamine N-acetyltransferase (GlmA), respectively. Then, the resulting GlcNAc-peptides were further modified by an endo-β-N-acetylglucosaminidase M mutant (EndoM N175Q) to generate glycopeptides carrying complex-type N-glycans.

In Chapter 3, two focused glycan libraries were synthesized which were N-glycans containing tandem epitopes and a new class of asymmetric N-glycans. (1) A focused library of 36 biantennary asparagine (Asn)-linked glycans with some presenting tandem glycan epitopes was synthesized and glycan microarray study showed that the internal epitopes and/or modifications of terminal epitopes influence the binding of glycans. (2) A class of asymmetric N-glycans was synthesized by coupling glycan-oxazoline and N-glycans. Branch-biased binding and spacial inhibition caused by bulky group on the other branch were observed in glycan protein interactions involving lectins and these glycans.

In Chapter 4, a microbial glycosyltrasferases Hp1,3FTα66 were readily expressed in Escherichia coli (E. coli) was biochemically characterized and applied as a diverse catalyst in the facile synthesis of fucosylated human milk oligosaccharides. In addition, multi-gram scale of fucosylated human milk oligosaccharides, lacto-N-fucopentaose (LNFP) III and lacto-N-neofucopentaose (LNnFP) V were synthesized by three major combined engineered E. coli strains.