Targeting Biofilms with a Novel Secondary Dithiol S-Nitrosothiol: Synthesis, Characterization, and Antibacterial Evaluation

Introduction: : Biofilm formation on biomedical surfaces complicates infection treatment by enhancing bacterial resistance to antibiotics and host defenses, necessitating effective broad-spectrum antibacterial strategies. Nitric oxide (NO) is a small, reactive molecule naturally produced by immune cells to combat infections by disrupting bacterial respiration, inducing oxidative stress, and dispersing biofilms. To mimic this biological defense mechanism, NO-releasing compounds have been developed, with S-nitrosothiols (RSNOs) emerging as a promising class. However, most studies have focused on primary and tertiary RSNOs, while secondary thiol-based NO donors remain largely unexplored due to challenges in achieving both stability and controlled NO release kinetics. This study synthesizes and characterizes a novel secondary thiol-based NO donor via a scalable route, confirmed by NMR, MS, and FTIR.

Materials and

Methods: : (2R,3S)-2,3-bis(nitrosothio)succinic acid (NO_DMSA) was synthesized via solubilizing dimercaptosuccinic acid (DMSA) in methanol and acidified with 1 M HCl and concentrated H₂SO₄ to protonate thiol groups. Further, sodium nitrite (NaNO2) 3 equivalent dissolved in DI water was added dropwise to initiate nitrosation, confirmed by a characteristic orange color. Upon completion of reaction the compound was extracted using chloroform. The organic layer was dried and subjected to rotary evaporation to isolate solid NO-DMSA. To confirm the formation of the novel secondary dithiol, UV-vis, FTIR, Ellman’s, NMR, and Mass spec were run. Antibacterial assays were conducted on E. coli, S. aureus, P. aeruginosa, and S. epidermidis using optical density-based minimum inhibitory concentration (MIC) determination in 96-well plates. Biofilm disruption was assessed on 48-hour matured bacterial films grown on Carbosil-coated substrates, treated with 3 mM NO_DMSA, and analyzed for colony-forming units (CFUs) after 24 hours.

Results, Conclusions, and Discussions:: The formation of NO_DMSA was validated using UV–vis spectroscopy, which confirmed RSNO formation through characteristic absorption between 320–430 nm, consistent with the π→π* transition of the –N═O bond. FTIR analysis detected NO-specific stretches (~1500 cm⁻¹), and Ellman’s assay quantified a >95% thiol conversion efficiency. NMR and ESI-MS further validated the compound’s structure. NO release was evaluated via chemiluminescence at 4–0.5 mM concentrations in PBS at 37°C, exhibiting measurable NO-release for 24 hours. The compound exhibited potent broad-spectrum antibacterial activity, with the lowest MIC values observed against Gram-positive S. aureus (2.5 mM) and Gram-negative P. aeruginosa (2.5 mM). The NO donor significantly enhanced biofilm dispersal and eradication in all tested Gram-positive and Gram-negative strains, achieving >94% bacterial reduction when applied to 48 h biofilms on polymer surfaces.

Acknowledgements and/or References (Optional):: Funding for this work was supported by the National Institutes of Health, USA, grant R01HL172496. Figures were created in part with BioRender.com.