PROJECT SUMMARY Peptide based pharmaceuticals are becoming increasingly prevalent in late-stage clinical trials and FDA approvals. Peptide synthesis is traditionally performed in the C to N direction on solid supports. Peptide synthesis in the N to C direction would enable new opportunities to improve peptide purity and yield because it will avoid side reactions that plague C to N SPPS and potentially alter the aggregation state of the peptide during its assembly. However, challenges such as oxazalone formation and diketopiperazine formation have long prevented the implementation of such an approach. The long-term objective of this program of research is to facilitate the synthesis of complex biologically active polypeptides. The objective of this application is to establish a platform for N to C SPPS that avoids the problematic hurdles of epimerization caused by oxazalone formation and peptide truncation due to diketopiperazine formation. We will achieve this objective by employing a mild carbonyl activation strategy that enable N to C SPPS without causing these undesirable side reactions. We will develop specialized methods to address slow reactions, challenging sequences, and unique functional groups that are important to the preparation of biologically active peptides. We will establish the compatibility of these methods with state-of-the-art SPPS technologies such as microwave and flow methods. Consistent with the mission of the NIH’s National Institute of General Medical Sciences, this basic research will ultimately facilitate developments in the study of biological processes. Furthermore, this research meets the objectives of the Focused Technology Research and Development Program because the specific aims focus on the technical challenges and milestones associated with implementing our innovative strategy for peptide synthesis. If successful, the proposed chemistry will advance biomedical research by positively impacting all fields where synthetic peptides are needed.