Synthesis of n-linked glycans, glycopeptides and glycoproteins and their functional applications

N-linked glycosylation, perhaps the most ubiquitous posttranslational modification, has wide ranging significance in nature and great promise in therapeutic development. In order to fulfill their delicate and complex functions, nature endows N-linked glycans with enormous structural diversity. This thereby generates enormous synthetic challenges in the production of well-defined glycans that can be utilized to unveil the coding between specific structures of N-linked glycans and their associated functions. Although substantial advances have been achieved, significant difficulties still remain in the production homogenous N-linked glycans and glycoconjugates. The research described in this dissertation was undertaken to overcome some of these difficulties and to better understand the effects of N-linked glycosylation on the biological functions of glycosylated peptides and proteins.;To accomplish this, a reliable method to obtain homogenous human N-linked glycans by using glycosylation deficient yeast and further in vitro enzymatic reactions was developed. This gave access to relatively large quantities of homogeneous N-linked glycans which were utilized to develop synthetic methods to produce N-linked glycoconjugates. Utilizing the N-linked glycans obtained from yeast, an efficient method for synthesis of N-linked glycosylated Asparagine on solid phase was developed that allows the convenient incorporation of various chemical handles. This allowed the production of a series of labeled N-linked glycans which can be utilized in glycobiology studies. Similar chemical methods were applied to the production of different glycoforms of glycosylated gold nanoparticles, which have been utilized to study binding of the lectins Concanavalin A (ConA) and Wheat germ agglutinin (WGA), and adhesion of type 1 Fimbriated Escherichia coli.;The N-linked glycans obtained from yeast have also been utilized in the comprehensive study of on-resin convergent synthesis of N-linked glycopeptides containing a large high mannose N-linked oligosaccharide. This approach was applied to the solid phase synthesis of glycosylated forms of the 34 amino acid HIV-1 gp41 C34 glycopeptide, which is an HIV-1 entry inhibitor. These glycopeptide synthesis methods were also applied to the semi-synthesis of glycoproteins using native chemical ligation. Glycopeptide thioesters were synthesized using an on-resin convergent strategy and coupled to a 225 amino acid glycoprotein, IgG1 Fc. Utilizing this approach independent control of multiple N-linked glycosylation sites on the 225 amino acid glycoprotein was achieved. Native chemical ligation was also utilized to ligate a series of cyclic-RGD peptides to the IgG1 Fc glycoprotein to optimize the chemically modified antibody fragment for cancer cell targeting. Finally, we have site-specifically incorporated a large N-linked high mannose oligosaccharide onto several glucagon-related peptide hormones. In vitro tests have been performed to investigate the influence of the N-linked glycosylation on different aspects of these peptides, including solubility, structure, stability and receptor agonist activity. Ongoing studies are being utilized to assess and refine the in vivo pharmacodynamics of these glycosylated glucagon and GLP-1 analogs.