Project Summary Peptides are now widely used to treat human diseases and represent a fast-growing class of therapeutics in the biopharmaceutical market. In general, peptides have the advantages of high specificity and potency but the disadvantages of being susceptible to aggregation and proteolytic degradation. Because of these drawbacks, therapeutic peptides are often administered through injections and, in order to maintain sufficient levels of circulating biologically active peptides, such injections are often frequent. This makes the treatment process expensive, inconvenient, and occasionally dangerous to patients, particularly during long-term treatment of chronic diseases. To address these issues, many attempts have been made to develop less invasive routes for peptide administration. By selectively retarding drug release in the acidic environment of the stomach through the use of pH sensitive polymers, peptides can now be efficiently conveyed to the small intestine, where the majority of drug absorption occurs after oral delivery. The absorption of peptides in the small intestine is not, however, free of challenges. For example, self-association and degradation by proteases located in the small intestinal lumen can significantly lower the bioavailability of peptides. Recently, we have demonstrated that almost all of the relevant physical properties of peptides can be altered by attaching particular glycans to flexible or fragile regions. Based on these results, we hypothesize that better guidelines of using glycans in peptide engineering can be developed by deeply examining the effects of glycosylation on two representative therapeutic peptides: human insulin and glucagon-like peptide-1 (GLP-1). The objective of our proposed research is to test this hypothesis through chemical synthesis and biological characterization of collections of differently O-glycosylated insulin and GLP-1 variants. We will begin our study by investigating how O-linked glycans modulate various properties of insulin. This will be achieved by designing, quantifying and then comparing the properties of a library of synthetically prepared, pure, and homogeneous insulin glycoforms with systematic variations in glycosylation patterns (glycosylation sites, glycan sizes and structures) and/or amino acid sequences. In the second part of the study, we will use a similar strategy to evaluate the effects of O-glycosylation on the properties of GLP-1. The proposed study is one of the first attempts to develop a rational approach to glycoengineer therapeutic peptides with a focus on O-glycosylation. The results of the proposed research are expected to lead to a better understanding of peptide glycoengineering, O-...