Probing GuanidinoPendant or Bridged Groups in CyclicAntimicrobial Peptides Derived from Temporin L: A Strategy to ImproveEfficacy against Gram-Negative Bacteria

Abstract The rise of antibiotic resistance underscores the urgent need for new antimicrobial agents. Antimicrobial peptides (AMPs), such as temporins, offer broad-spectrum activity through unique mechanisms but are often limited by cytotoxicity and poor stability. To improve the Gram-negative activity-to-toxicity ratio, we designed a focused library of cyclic Temporin L (TL) analogues bearing an additional positively charged guanidino group, introduced either as a side-chain pendant or as a bridged functionality. While guanidino modifications have been studied in side-chain peptide contexts, this represents the first application of guanidino-based bridging in AMP design. Among the synthesized compounds, four (e.g., 2, 6, 7 and 12) were selected based on their combined antimicrobial potency and low cytotoxicity toward human keratinocytes, emerging as the most structurally representative candidates of the introduced modifications. Further characterization provided an integrated view of their biological properties, highlighting guanidino-based cyclic temporins as attractive agents and a framework for developing next-generation therapeutics against resistant and biofilm-associated infections.