The chemical approach towards homogenous glycoprotein preparation and evaluation ABSTRACT – Our long-term goal is to uncover the unknown function of cellular and infectious prion protein by interrogating individual homogenous prion glycoform. The objective here, is to develop a class of efficient peptidyl coupling protocols utilizing strained molecules, such as β- lactone and β-thiolactone, for the construction of previously unattainable targets such as cyclic tetrapeptides and membrane prion protein. Our central hypothesis, derived from our published results and preliminary studies described below, is that chemical synthesis can provide the PrPC and PrPSc strains with high conformational fidelity, which allows prion biological studies to be carried out with faithful accuracy when interrogating synthetic homogenous glyco-PrPs. The flexibility of modifying protein construct furnishes previously unavailable tool for evaluation prion disease. The rationale for the proposed research objectives is that the β-thiolactone and β-lactone release of cyclic ester strain enables unprecedented rapid amide bond construction and connects two amino acid residues. Furthermore, the near-planar geometry of the cyclobutene ring will generate desired peptidyl adducts without epimerization by preventing the oxazolone formation. An analogous approach could be applied to construct homogenous membrane glycoproteins from β-thiolactone promoted protein ligation. We plan to test our central hypothesis and, thereby, accomplish the objectives of this application via the following goals: 1. Design and expansion of the range of constrained esters that can be utilized for peptide synthesis without epimerization. 2. Synthesis of homogeneous cellular prion glycoproteins and congeners. 3. Biological function and activities investigation of prepared peptide and proteins.

PROJECT NARRATIVE: The proposed research is relevant to the public health because cyclic peptides and glycoproteins exist ubiquitously in the pharmaceuticals and biologics. Despite significant advances in chemical studies of protein preparation, homogenous membrane glycoproteins are difficult to obtain. Upon conclusion, we will provide a powerful tool for the protein function elucidation and investigation.