Synthesis of the Novel N-(2-Hexadecynoyl)-l-Homoserine Lactone and Evaluation of Its Antiquorum Sensing Activity in Chromobacterium violaceum

Abstract

Chromobacterium violaceum is commonly found in soil and freshwater within tropical and subtropical regions.Although not a common occurrence, this bacterium has the potential to cause severe diseases in humans and animals, such as liver and lung abscesses and septicemia. Herein we report the synthesis of novel N-acyl homoserine lactones (HSLs) to evaluate their effectiveness as antiquorum sensing (anti-QS) agents in C. violaceum. The HSLs were prepared through three synthetic approaches, where hexanoic acid, decanoic acid, 6-decynoic acid, or 2-hexadecynoic acid (2-HDA) was treated with commercially available L-homoserine lactone (HSL) hydrobromide in either dichloromethane or tetrahydrofuran in the presence of EDC and DMAP. The effectiveness of HSLs as anti-QS agents was assessed through susceptibility tests and violacein quantification. The most effective anti-QS inhibitor among all N-acyl-HSLs tested was the N-(2-hexadecynoyl)-L-homoserine lactone (HSL 4). Further experimental approaches, such as quantification of acyl-homoserine lactones and biofilm inhibitory tests, were carried out to determine the effect of HSL 4 on these QS-dependent mechanisms. These experiments showed that HSL 4 was highly effective at inhibiting the production of HSLs and biofilm in C. violaceum at 0.25, 0.50, and 1 mg/mL. In addition, the cytotoxicity activity was evaluated against Vero cells to determine the selectivity of HSL 4 as a nontraditional antibacterial agent. HSL 4 was not toxic against Vero cells at concentrations ranging from 0.0039 to 1 mg/mL. Molecular docking experiments were conducted to study the interactions between novel HSLs and CviR (PDB ID 3QP5), a receptor that plays a significant role in C. violaceum QS. In silico studies indicate that HSL 4 exhibits better interactions with Leu 72 and Gln 95 of the CviR binding pocket when compared to the other analogs. These results validate previous in vitro studies, such as susceptibility tests and violacein production assays. The findings above indicate that novel acetylenic HSLs may potentially be agents that combat bacterial communication and biofilm formation. However, further investigation is necessary to expand the spectrum of bacterial strains capable of resisting antibiotics through QS and evaluate the compounds' cytotoxicity in other cell lines.