Funding Opportunity Announcement (FOA) Number: PA-20-272 Equipment request for Maximizing Investigators' Research Award R35 136365 (Project Title: Chemical Biology Studies of the Dynamics and Inhibition of Peptidoglycan Biosynthesis) PROJECT SUMMARY This equipment request is being made to significantly enhance our research capabilities in support of our R35 MIRA award. Our funded MIRA application represents the fusion of two complementary research programs that are, broadly described, directed at the urgent public health threat posed by antibiotic resistance. Our MIRA award seeks to capitalize on our discovery of fluorescent D-amino acids (FDAAs) that have provided unprecedented, and heretofore unavailable, tools for the visualization of bacterial cell wall peptidoglycan (PG) dynamics in real time and in live bacterial cells. We have initiated additional studies to elucidate the details of bacterial cell division and cell separation in Bacillus subtilis, and we are seeking to develop new classes of fluorogenic (i.e., “turn- on”) probes that will enable the study of PG synthesis and dynamics in real-time and in live bacterial cells. We will also continue our effort directed at the synthesis and mechanistic study of cyclic depsipeptide antibiotics that inhibit PG biosynthesis. Furthermore, we have recently uncovered data that suggest the cyclic depsipeptides may have a second mechanism of action that involves inhibition of lipid recycling, an essential activity in the PG biosynthesis pathway. The lipid recycling pathway remains to be clearly elucidated and, when coupled with dual-mode activity that may be inherent to these cyclic depsipeptides under study, very promising new avenues for the identification of new antibacterial targets and development of new antibacterial agents are likely to emerge from this research effort. Research in each of these areas requires routine access to HPLC instrumentation with high resolution capabilities with multiple mods of detection. Our current HPLC system is approximately twenty years old and is no longer supported by the manufacturer. In addition, the computer system required to run the instrument has become obsolete and is no longer supported by our university. As a result, this instrument has a significant amount of ‘down time’ that adversely impacts research progress under the parent R35 award. In this application, we are seeking funds for the purchase of an Agilent 1290 Infinity Flex Series HPLC/UHPLC instrument that has the capability for routine purity analysis of amino acids, cyclic peptides, and the like as well as UHPLC capability to enable analysis of more complex samples, such as PG fragments. This instrument will replace our aging and unrepairable system, significantly enhance our analytical capabilities, and have a profoundly positive impact on our activities under the parent R35 award.