NEW METHODS FOR THE CONVERGENT GLYCOLIGATION OF PEPTIDES

Thesis (Ph.D.), Washington State UniversityThe attachment of oligosaccharides (glycans) to proteins is an important co- and post-translational modification which greatly expands the functional and structural diversity of a proteome. As glycosylation is integral to many biological processes, studies in this area provide valuable and far reaching insights. Herein is described the chemoselective Cu(II)-HOBt promoted chemical ligation of glycosylamines and peptide thioacids to give N-glycosylated peptides.The method is distinguished from other chemical approaches to peptide N-glycosylation in that: (1) it can be employed in the presence of unprotected N-terminal and Lys sidechain amines, (2) it is remarkably fast, going to completion in under 30 minutes, and (3) it produces glycopeptide without attendant aspartimide formation. The N-glycoligation is exemplified by the coupling of chitobiosylamine and an unprotected heptapeptide containing an aspartyl thioacid side-chain to produce an N-linked glycopeptide corresponding to a truncated segment of a human glycoprotein hormone. The N-glycoligation provides a simple and convergent means for attaching a carbohydrate to a specific Asp residue in an unprotected peptide. Full realization of the benefits of site specific N-glycoligation requires practical methods for the synthesis of unprotected aspartic thioacids containing peptides. The development of a direct solid phase peptide synthesis of unprotected aspartic thioacids is described. This method involves the novel use of a silyl ester as a carboxylate surrogate for mild peptide bond formation in the presence of a reactive aspartyl thioester. Analysis and quantification of aspartimide formation is provided using known aspartimide-prone sequences. The resulting peptide thioacids may be used in N-glycoligation and other thioacid-mediated conjugation reactions.Department of Chemistry, Washington State Universit