Probing Guanidino Pendant or Bridged Groups in Cyclic Antimicrobial Peptides Derived from Temporin L: A Strategy to Improve Efficacy against Gram-Negative Bacteria

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.抗生素耐药性问题日益严峻，凸显了开发新型抗菌剂的迫切需求。抗菌肽（Antimicrobial Peptides，AMPs）如 temporins，可通过独特机制发挥广谱抗菌活性，但常受限于细胞毒性与稳定性不佳的缺陷。为提升抗菌肽对革兰氏阴性菌的活性-毒性比，我们设计了一套聚焦型环状 temporin L（TL）类似物文库，其携带额外的带正电胍基基团，该基团可作为侧链悬挂基团或桥联功能基团引入。尽管胍基修饰在肽侧链修饰领域已有相关研究，但本研究首次将基于胍基的桥联策略应用于抗菌肽的设计中。在合成的所有化合物中，我们基于其综合抗菌活性与对人角质形成细胞的低细胞毒性，筛选出4种候选化合物（如编号2、6、7及12），它们是本次引入修饰后结构最具代表性的候选分子。进一步的表征分析全面揭示了这些化合物的生物学特性，凸显了基于胍基的环状 temporins 作为极具潜力的抗菌剂的应用价值，并为开发针对耐药菌及生物膜相关感染的下一代治疗药物提供了可行的研究框架。