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.

Fig. 1. A schematic diagram depicting the step-by-step process for synthesizing dyes D1–D4.

Scheme 1. The synthetic pathway for the dyes D1–D4 and their corresponding structures.

Fig. 2. The stacked FT-IR spectra for synthetic dyes D1–D4.

Fig. 3. UV-vis spectra for the synthetic dyes D1–D4 in ethanol.

Fig. 4. (a) Experimental ¹H-NMR spectrum of dye D2. (b) The expanded aromatic region from ¹H-NMR spectrum of dye D2. (c) The expanded aromatic region from the ¹H-NMR spectrum of dye D2 in the presence of D₂O. (d) Computed ¹H-NMR spectrum of dye D2 in DMSO. (e) HMQC spectrum of dye D2.

Fig. 5. Graphical representation of HOMO, LUMO, and MEP maps for the synthesized D1–D4 dyes.

Fig. 6. HOMO–LUMO energy gap for the synthesized azo-dyes D1–D4.

Fig. 7. Growth curves of the MRSA clinical isolate in the absence or presence of different concentrations of D4. Bacterial inocula were adjusted and allowed to grow without or with varying concentrations of D4 equivalent to 0.5, 0.25, and 0.125 MIC. At different time intervals, the bacterial count was determined.

Fig. 8. Biofilm inhibition assay of dye D4 against the strong biofilm former MRSA isolates. Bacterial inocula were allowed to form biofilm in the absence or the presence of different concentrations of D4. After incubation, biofilm was quantified, and the percent biofilm formation was plotted against concentrations of D4 expressed as μg mL⁻¹ in (a) or as MIC equivalent in (b).

Fig. 9. D4 significantly reduces SaeS and SaeR two-component regulatory system expression. MRSA clinical isolate was allowed to grow in the presence or absence of 0.5 MIC of D4. After 24 hours, RNA was extracted, and SaeS and SaeR mRNA were quantified using qPCR relative to the gyrB housekeeping gene. Fold changes were normalized relative to solvent-treated control. Student's t-test was used to analyze fold changes. (*, p < 0.05; **, p < 0.01).