Design, Synthesis, and Antimicrobial Evaluation of Short Hybrid Peptides Incorporating Β- And Γ-Amino Acids for Membrane-Targeted Activity Against Multidrug-Resistant Bacteria

The rapid emergence of multidrug-resistant (MDR) bacteria poses a critical challenge to current antimicrobial therapy, necessitating the development of novel agents with unique mechanisms of action. Antimicrobial peptides (AMPs) are promising candidates due to their broad-spectrum activity and membrane-targeted bactericidal effects; however, their clinical translation is often hindered by proteolytic instability and high production costs. In this study, we designed and synthesized a series of short lipidated hybrid peptides (P1–P8) incorporating cationic residues (Orn/Lys) with β,β-disubstituted β-amino acids and mono/disubstituted γ-amino acids to improve protease resistance, membrane selectivity, and pharmacokinetic properties. The β3,3-Ac6c and β3,3-Pip(Ac) building blocks, along with Boc-γ4-L-Phe-OH and Boc-γ4-L-Leu-OH, were synthesized following established literature protocols. Peptides were assembled via stepwise EDC/HOBt coupling, lipidated with lauric acid to enhance membrane affinity, purified by silica gel chromatography, and structurally confirmed by ^1H/^13C NMR and HRMS. Antimicrobial activity, evaluated using CLSI broth microdilution, revealed that P2, P3, P4, and P5 exhibited potent bactericidal effects against both Gram-positive (S. aureus, B. subtilis) and Gram-negative (P. aeruginosa, E. coli, S. typhimurium, K. pneumoniae) MDR strains, with minimum inhibitory concentrations (MICs) as low as 6.25 μM. Time–kill kinetics demonstrated rapid bacterial eradication within 2 h at MIC levels, with P4 showing the fastest killing rate. Membrane permeabilization assays using propidium iodide staining confirmed significant disruption of bacterial membranes, while hemolytic evaluation indicated low cytotoxicity toward human erythrocytes, with HC50 values exceeding 500 μM for P4, suggesting excellent selectivity. These findings highlight the potential of β- and γ-amino acid-based hybrid peptides as promising candidates for combating MDR bacterial infections through membrane-targeted mechanisms.