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Bacterial resistance to current ß-lactam antibiotics necessitates new therapeutic approaches. Silver-based drugs offer a promising alternative because of their antibacterial properties and minimal bacterial resistance. However, many silver-based antibacterial drugs rapidly release silver ions, limiting the bioavailability of silver ions for a longer period of time. Recently, N-heterocyclic carbenes (NHC) containing silver complexes have become a popular subject of research due to their versatility and the slow release of silver ions under biological conditions. To further enhance the efficacy of silver-NHC drugs, we fused redox-active substituents such as naphthoquinone and ferrocenes to NHCs. Both naphthoquinone and ferrocenes have been shown to increase reactive oxygen species (ROS) under biological conditions that may augment the antibacterial action of redox-active silver-NHC complexes. Herein, we report synthesis and characterization of several redox-active silver-NHC complexes, 1-(ferrocenylmethyl)-3-mesityl-imidazol-2-ylidene silver chloride [(2)AgCl] (compound 6), 1,3-di (ferrocenylmethyl)imidazol-2-ylidene silver chloride [(3)AgCl] (compound 7), bis (1-benzyl-3-mesitylimidazol-2-ylidene) silver chloride [(1)2Ag][Cl], (compound 9), Bis (1-(ferrocenylmethyl)-3-mesityl-imidazol-2-ylidene) silver chloride [(2)2Ag][Cl] (compound 10), Bis (1,3 di(ferrocenylmethyl)imidazole-2-ylidene) silver chloride [(3)2Ag][Cl] (compound 11), and bis (1,3-dimesityl-4,5-naphthoquino-imidazol-2-ylidene) silver chloride [(4)2Ag][Cl] (compound 12). Complexes were identified and confirmed via various analytical techniques, including 1H NMR, 13C NMR, infrared spectroscopy, UV-vis spectroscopy, and single crystal X-ray crystallography.A series of cyclic and differential pulse voltammetry experiments were conducted on the complexes to investigate their electrochemical properties. Complexes [(2)AgCl], [(3)AgCl], [(2)2Ag][Cl], [(2)2Ag][Cl], [(3)2Ag][Cl], and [(4)2Ag][Cl] exhibited redox activity attributed to the presence of ferrocene and naphthoquinone moiety. Their antibacterial properties were tested against gram-negative bacterial strains (Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa) and showed excellent antibacterial properties that are comparable to previously reported silver-NHC complexes. All silver-NHC complexes detailed in this report exhibit MIC and MBC values in the range of 2-12.5 µg/mL. Further mechanistic studies are necessary to elucidate the manner in which these complexes affect bacterial cells.

The T box mechanism is a gene expression controlling mechanism in various Gram-positive bacteria species. Recognition of the T box antiterminator and the tRNA is an essential step for the gene expression, which might function as a potential drug target for the development of antibacterial drugs. Libraries of ligands, includingaminoglycosides, triazoles and oxazolididones, were tested for targeting effects on the T box antiterminator and inhibition effects on the formation of the tRNA-antiterminator complex. Mg2+ and spermidine, as cofactors for the T box antitermination, were also investigated for their effects on the recognition of tRNA and the T box antiterminator model.Fluorescence screening assay, fluorescence anisotropy screening assay and fluorescence binding assay were developed and/or optimized for testing ligand interactions with the T box antiterminator model and also ligand inhibition effects on the formation of the tRNA-antiterminator complex. Four ligands show high potentials for the drug discovery purpose in reference to the combinational results of the three assays: IAB-3, IAB-4, RLB-42 and RLB-38.Fluorescence techniques, including fluorescence binding and fluorescence anisotropy were applied for monitoring the tRNA-T box antiterminator interaction at variable Mg2+/spermidine conditions. Thermodynamic characterization of the Mg2+ and spermidine effects on the tRNA-antiterminator complex formation was performed by the Isothermal Titration Calorimeter (ITC). ITC results indicated that under in vitro condition, different Mg2+ levels can facilitate the formation of the discriminator base pair between the tRNA and the antiterminator model and induce conformational changes in the complex, which might provide thermodynamic explanation for the antitermination initiated by the tRNA-T box antiterminator recognition in the T box mechanism. Application of spermidine might serve as a solution to lower the high Mg2+ requirement in the in vitro research on the T box antitermination mechanism.