Project Summary/Abstract: Synthesis and Evaluation of aza-Novo29 as an Antibiotic Candidate New antibiotics are desperately needed against drug-resistant Gram-positive pathogens, such as methicillin- resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). This proposal seeks to synthesize and validate aza-Novo29 as a new antibiotic candidate against Gram-positive pathogens that is stable toward hydrolysis. The proposal is based on the findings that the recently discovered antibiotic Novo29 shows promising antibiotic activity but is hydrolytically unstable at physiological pH. The central hypothesis that will be tested in the current proposal, is that the macrolactam analogue of Novo29 — aza-Novo29 — will exhibit good activity against Gram-positive pathogens but will not suffer hydrolysis at physiological pH. The proposal builds upon studies of analogues of the related antibiotic teixobactin. These studies have established that a macrolactam analogue of teixobactin not only tolerates replacement of the macrolactone ring with a macrolactam ring, but actually exhibits 2–8-fold greater antibiotic activity. The proposal also builds upon the concept that lactams are far more resistant to hydrolysis than lactones. In combination, these observations suggest that aza-Novo29 will exhibit good antibiotic activity but resist hydrolysis. Novo29 contains a non-proteinogenic β-hydroxyasparagine residue. The stereochemistry of this β- hydroxyasparagine residue is not known. As part of the proposed studies, the stereochemistry of the β- hydroxyasparagine residue will be determined through chemical synthesis and correlation with authentic Novo29. There are three specific aims: (1) To develop a synthesis of Novo29 in order to assign its stereochemistry by spectroscopic comparison to authentic Novo29 and side-by-side comparison in antibiotic activity assays. (2) To develop a synthesis of aza-Novo29, the lactam analogue of Novo29. (3) To determine whether aza-Novo29 has good activity against Gram-positive pathogens, while having improved hydrolytic stability compared to Novo29. The expected outcome is the creation of aza-Novo29 as a promising antibiotic candidate for further drug development. It is expected that aza-Novo29 will exhibit good activity against MRSA and VRE, as well as resistance to hydrolysis under the conditions needed for intravenous administration. The results of this research will impact the field of antibiotics by developing and expanding upon the newly discovered antibiotic class that includes teixobactin and Novo29. The success of this project will pave the way for further development of aza- Novo29 as a preclinical antibiotic candidate.