Clovibactin kills bacteria by binding lipid II and related cell wall precursors and inhibiting cell-wall biosynthesis. Clovibactin is able to evade bacterial resistance because it binds to the immutable pyrophosphate group of lipid II and its precursors. In the current study, we set out to develop potent analogues of clovibactin in which Dhydroxyasparagine (D-Hyn) is replaced with the commercially available amino acid D-threonine (D-Thr, Figure ). Replacement of D-Hyn with D-Thr eliminates the need to prepare D-Hyn through a multistep synthesis and opens the possibility of large-scale preparation of clovibactin analogues for preclinical studies. Our laboratory recently reported a series of structure-activity relationship (SAR) studies of clovibactin in which D-Thr5-clovibactin (NB-018) -an analogue pioneered by NovoBiotic Pharmaceuticals -was introduced. This study demonstrated that replacement of D-Hyn5 in clovibactin with D-Thr results in a 4-32-fold decrease in activity. Herein, we report potent analogues of clovibactin containing D-Thr that are similar in in vitro antibiotic activity to clovibactin.