In our initial report on the synthesis and stereochemical determination of clovibactin, we observed through X-ray crystallography of a clovibactin epimer that the residues Phe1, D-Leu2, Leu7, and Leu8 align on one side of the molecule to create a hydrophobic surface. We postulated that this hydrophobic surface may be important to interactions with the bacterial cell membrane. Solid-state NMR studies by Weingarth and coworkers would later determine the specific contacts that D-Leu2, Ala6, Leu7, and Leu8 make with lipid II and the bacterial cell membrane. We then reported an alanine scan of clovibactin which determined the importance of each side chain to the antibiotic activity of clovibactin. The sequential mutation of Phe1, D-Leu2, Leu7, and Leu8 to alanine had resulted in an 8-256-fold decrease in antibiotic activity -suggesting that these hydrophobic side chains are very important for antibiotic activity. Collectively, the X-ray crystallographic, NMR, and SAR studies suggest that the hydrophobic interactions of D-Leu2, Leu7, and Leu8 with lipid II and the bacterial cell membrane are especially important in the exceptional antibiotic activity of clovibactin.