Due to the increasing prevalence of antimicrobial resistance and the paucity of antibiotics in the pipeline, there is a pressing need for new methods to bolster the armamentarium of antimicrobial therapeutics. The goal of our NIH R35 award is to develop antibiotic candidates using as templates natural and synthetic molecules that inhibit the bacterial ribosome. These candidates, accessible through novel chemical approaches, have the potential to overcome the limitations of previous classes of protein synthesis inhibitors. During ≤2 years of support, we have: • Developed the first synthesis of seco-lankacidinols and reassigned the structure of 2,18-secolankacidinol B (Angew. Chem. Int. Ed 2018, 57, 13551-13554) • Designed an improved, organotin-free synthesis of streptogramin antibiotics (Tetrahedron 2019, 75, 3309-3318) • Reassigned the structure of iso-lankacidinol by means of a modular, fully synthetic route (submitted) • Synthesized >100 streptogramin analogs, tested each of them in >20 strains of bacteria, characterized mechanisms with CryoEM, and discovered analogs that overcome resistance (submitted, see preprint at ChemRXiv, doi: 10.26434/chemrxiv.8346107.v2) • Developed a late-stage C–H amination strategy to epi-lankacidins (manuscript prepared, submission anticipated April 2020) • Prepared >20 candidates for binding-induced hybridization All of the primary goals of our R35 award are reliant on a workhorse LCMS system that can accurately and rapidly analyze small molecules in reaction mixtures and in crude biological samples.