Effect of Chirality and Amphiphilicity on the Antimicrobial Activity of Tripodal Lysine-Based Peptides

A series of tripodal (three-arm) lysine-based peptides were designed and synthesized and their self-assembly properties in aqueous solution and antimicrobial activity were investigated. We compare the behaviors of homochiral tripodal peptides (KKY)₃K and a homologue containing the bulky aromatic fluorenylmethoxycarbonyl (Fmoc) group Fmoc-(KKY)₃K, and heterochiral analogues containing k (d-Lys), (kkY)₃K and Fmoc-(kkY)₃K. The molecular conformation and self-assembly in aqueous solutions were probed using various spectroscopic techniques, along with small-angle X-ray scattering (SAXS) and cryogenic-transmission electron microscopy (cryo-TEM). In cell viability assays using fibroblast cell lines, the tripodal peptides without Fmoc were observed to be noncytotoxic over the concentration range studied, and the Fmoc functionalized tripodal peptides were only cytotoxic at the highest concentrations (above the critical aggregation concentration of the lipopeptides). The molecules also show good hemocompatibility at sufficiently low concentration, and antimicrobial activity was assessed via MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) assays. These revealed that the Fmoc-functionalized tripodal peptides had significant activity against both Gram-negative and Gram-positive bacteria, and in the case of Gram-positive Staphylococcus aureus, the antimicrobial activity for Fmoc-(kkY)₃K was improved compared to polymyxin B. The mechanism of the antimicrobial assay was found to involve rupture of the bacterial membrane as evident from fluorescence microscopy live/dead cell assays, and scanning electron microscopy images.