Unveiling the structural aspects of novel azo-dyes with promising anti-virulence activity against MRSA: a deep dive into the spectroscopy via integrated experimental and computational approaches

Abstract

A novel series of azo dyes was successfully synthesized by combining amino benzoic acid and amino phenol on the same molecular framework via azo linkage. The structural elucidation of these dyes was carried out using various spectroscopic techniques, including UV-vis, FT-IR, NMR spectroscopy, and HRMS. Surprisingly, the aromatic proton in some dyes exhibited exchangeability in D₂O, prompting a 2D NMR analysis to confirm this phenomenon. Furthermore, comprehensive density functional theory (DFT) calculations were conducted to unravel synthetic dyes' geometrical and electronic properties. Meanwhile, the reactivity of various sites was further investigated through Frontier Molecular Orbitals (FMOs) analysis and molecular electrostatic potential mapping. Besides, the experimental NMR spectra were interpreted by incorporating theoretically computed NMR spectrum and reduced density gradient (RDG) function. These computations revealed a pronounced intramolecular hydrogen bond through O–H⋯N interaction that significantly influenced the proton chemical shift. The dyes were assessed for their antimicrobial activities using agar diffusion, micro broth dilution, and biofilm inhibition assays. Interestingly, one of the synthetic dyes showed promising antibacterial effects against S. aureus (ATCC-6538) as well as against a multidrug-resistant MRSA clinical isolate with a MIC (minimum inhibitory concentration) of 78.12 μg mL⁻¹. Moreover, that dye inhibited biofilm formation of the strong biofilm former clinical MRSA isolate with a concentration as low as 0.25 MIC (19.53 μg mL⁻¹). Indeed, our qPCR data suggest that inhibiting the SaeS/SaeR system is another potential mechanism by which D4 exerts its antibacterial and anti-virulence effects. Altogether, this shows these synthetic azo dyes' promising antibacterial and anti-virulence activities concerning MRSA clinical infections.