PROJECT SUMMARY/ABSTRACT The objective in this application is to exploit stereochemically robust thioamides, at any point in the peptide se- quence, as biophysical probes to address current barriers in peptide synthesis, folding, and drug discovery. There is a perception that current methods to incorporate thioamides into peptides are sufficient. However, as an example, the rapid racemization of the alpha-carbon stereochemistry of thioamide residues during synthesis belies that perception. Indeed, a survey of reported peptides containing thioamides points to these limitations. The majority of ‘successful’ sequences incorporate the thioamide close to the N-terminus, where exposure to synthetic reagents is necessarily minimized during Fmoc solid-phase peptide synthesis (SPPS) procedures. Thus, the instability of thioamides during Fmoc SPPS present a significant barrier to the synthesis and evalua- tion of thioamide peptides, and restricts the sequence space in which thioamides can be employed. Anecdotal reports indicate that many labs have wished to employ thioamides in a variety of peptide studies, but a lack of documented pitfalls and synthetic options leads to intractable peptide products and, ultimately, abandonment of such ventures. The approach in this proposal is to protect the thioamide, in analogy to the protection of the functional groups of amino acid side chains, in order to preserve the thioamide moiety during peptide elonga- tion. The rationale for this approach is that thioamide protection can be easily included within the standard SPPS work-flow to enable novel applications in peptide synthesis, backbone modification, and protein-drug interactions. The research plans of this project will exploit thioamides to probe protein folding and site-selective insertion other chemistries. Thioamides will be employed in previously uncharted sequence space to address fundamental questions in protein folding. We will also develop methods to transform thioamides into functional groups that will unlock new constrained peptide scaffolds. Peptides with persistent structure hold tremendous promise as therapeutics to bridge the performance gap between small molecules and biologics. Finally, this work will identify strategies to interrogate and target therapeutically relevant protein-protein interfaces. Interac- tions between hydrogen bond donors and acceptors of the main-chain of a peptide and a protein binding target are underutilized in drug design. Based on structural bioinformatics, new strategies to identify underutilized in- teractions at protein-protein interfaces (PPIs) will assist in the design of more potent inhibitors. Other work will also develop new tools to interrogate PPIs for which very little structural information may be available to inform future experimental design. The proposed research is innovative because it represents a substantive departure from the status quo by developing and employing new methods to preserve thioamide stabi...