We hypothesized that increasing the hydrophobicity of residues 2, 7, and 8 may increase the antibiotic activity of D-Thr5-clovibactin. To test this hypothesis, we sequentially mutated leucines 2, 7, and 8 to the more hydrophobic homologue cyclohexylalanine. Cyclohexylalanine is commercially available, and it contains three more carbon atoms than leucine, making it substantially more hydrophobic (DCLogP = +1.2, Figure ). We thus prepared the three Cha analogues (D-Cha2,D-Thr5clovibactin, Cha7,D-Thr5-clovibactin, and Cha8,D-Thr5-clovibactin; Figure ) by solid-phase peptide synthesis and assessed their antibiotic activity against MRSA, VRE, and other Gram-positive bacteria through minimum inhibitory concentration (MIC) assays. 80), which is expected to help prevent the adsorption of the peptides the 96-well polystyrene plates that are used for the assays. Leu2, Leu7, and Leu8 promote important interactions between clovibactin and the bacterial cell membrane. The cyclohexylalanine analogues exhibit modest activity (8-32 μg/mL) against the Gram-negative bacterium E. coli. This activity is greater than that of D-Thr5-clovibactin (>32 μg/mL) and may reflect a greater propensity of the more lipophilic analogues to interact with a variety of types of cells.