The unusual d-l-l-d-d-l-l pattern of stereochemistry in residues 1-7 of the peptide antibiotic teixobactin is critical to its extraordinary antibiotic activity, creating an unusual amphiphilic β-sheetlike structure that is essential to its mechanism of action. The current study sought to replace the three d-amino acids in the tail with l-amino acids while maintaining amphiphilicity. We find that swapping residues d-Gln4 and d-allo-Ile5 in O-acyl isopeptide prodrugs of teixobactin permits the introduction of l-stereochemistry with retention of antibiotic activity. Nevertheless, modifying the N-terminal stereochemistry results in a loss of antibiotic activity.

Our laboratory reported the chemical synthesis and stereochemical assignment of the recently discovered peptide antibiotic clovibactin. The current paper reports an improved, gram-scale synthesis of the amino acid building block Fmoc-(2R,3R)-3-hydroxyasparagine-OH that enables structure-activity relationship studies of clovibactin. An alanine scan reveals that residues Phe1, d-Leu2, Ser4, Leu7, and Leu8 are important for antibiotic activity. The side-chain amide group of the rare d-Hyn5 residue is not essential to activity and can be replaced with a methyl group with a moderate loss of activity. An acyclic clovibactin analogue reveals that the macrolactone ring is essential to antibiotic activity. The enantiomer of clovibactin is active, albeit somewhat less so than clovibactin. A conformationally constrained clovibactin analogue retains moderate antibiotic activity, while a backbone N-methylated analogue is almost completely inactive. X-ray crystallography of these two analogues reveals that the macrolactone ring adopts a crown-like conformation that binds anions.