Abstract Microbial resistance against current medications is on the rise, with the serious threat of bacteria becoming immune against all available drugs. There is no doubt that a renewed focus on anti-infective compounds is highly desired to prevent potential epidemic outbreaks of infectious diseases. Daptomycin is an FDA-approved antibiotic for the treatment of Gram-positive bacterial infections. It has a strict requirement for calcium to fulfill its antibiotic activity. Recent reports highlight the resistance of different strains against daptomycin. This urges the need for the development of next generation daptomycin antibiotics to circumvent resistance. However, the complexity of daptomycin’s chemical structure hinders modifying this antibiotic via traditional synthetic approaches. We have recently reported a novel chemoenzymatic method for the synthesis of specific daptomycin derivatives with stronger in vitro activity against daptomycin-susceptible and resistant bacteria. The new analogs, in contrast to the parent molecule, do not require calcium for antibacterial activity suggesting a new mechanism of action. The goal of this proposal is to study the new mechanism of the newly developed analogs in in vitro and in vivo models. We will use multidisciplinary approaches at the interface of chemistry and biology to provide more depth on the mechanisms and activity of the newly generated analogs. Specific Aim 1 will study the physicochemical and microscopic properties of our daptomycin derivatives to reveal the mechanisms of the newly synthesized compounds. Specific Aim 2 will study the new chemoenzymatically- synthesized derivatives in animal models to provide information on their in vivo activity and pharmacokinetics. This proposal emphasizes translational research and will lead to the development of stronger antibiotics that circumvent resistance. Hence this study will have a significant impact on multiple avenues that could lead to bridging these compounds to the clinic. Overall, the proposal will lay the groundwork for a research program that integrates in vivo activity, microbiology, physicochemical properties and mechanistic insights to access new routes to daptomycin biological diversity. The results obtained from this study will be extended to other lipopeptide antibiotics in terms of their microbial resistance and activity. This research will highlight the importance of chemoenzymatic approaches to complement synthetic ones to modify other bioactive compounds. This proposal will also align with my lab’s overall goal to address the constant need to expand the chemical space of small molecules to meet rising challenges of resistant microbes and improve their selectivity.