Project Summary Advances in organic synthesis have enabled the construction of increasingly complex peptide drugs and natural products. However, access to many important peptides is still hindered or prevented by limitations in current methods. Accordingly, the objective of this project is to discover new synthetic methods and apply them to constructing bioactive peptide natural products. The hypothesis is that the proposed methods will enable efficient construction of peptides that will be used to accelerate advances in medicinal chemistry and chemical biology. The rationale for this idea is that pushing back the frontiers of peptide synthesis will unlock access to unexplored areas of peptide chemical space, providing valuable new tools to medicinal chemists and chemical biologists in the fight against disease. The hypothesis will be tested by pursuing three Specific Aims. Aim 1 involves devising a universal method of synthesizing ,-dehydroamino acids and streamlining their incorporation into peptides. Eliminations of -nitroamino acids will be explored for this purpose. The process will first be optimized in solution with model tripeptide substrates. Then, on-resin eliminations will be developed in the context of a library of tunicyclin B analogs. Aim 2 entails determining the structure of the anticancer peptide prethioviridamide through synthesis of the candidate diastereomers. A convergent approach involving late-stage coupling of macrocycles and thioamide-rich hexapeptides will be pursued. The macrocycles will be synthesized in solution, and the hexapeptides will be assembled on solid support. Aim 3 consists of developing a new serine ligation protocol. The combination of carboxylate alkylation and O-to-N acyl transfer will be used to ligate peptide segments and achieve macrocyclizations, with both processes occurring without epimerization. Various electrophiles and N-methylated nucleophiles will be studied. Cyclic peptides ranging from four to eight residues will be targeted by the macrocyclization investigation. The approach is innovative because it features the development of novel synthetic methods such as eliminations of resin-bound -nitroamino acids, convergent couplings of macrocyclic -thioenamides with linear polythioamides, and ligations that use the carboxylate as a nucleophile instead of an electrophile. The significance of the proposed research lies in its ability to facilitate the synthesis of peptides that can be used to solve important medicinal chemistry and chemical biology problems. Such studies could include the use of ,-dehydroamino acids to impart rigidity and proteolytic stability to bioactive peptides, the design and synthesis of potent and stable analogs of prethioviridamide as anticancer agents, and the synthesis of medicinally relevant peptides via epimerization-free serine ligations. This project is envisioned to create new synthetic methods that will facilitate exciting advances in medicinal chemistry and c...