BEYOND SINGLET OXYGEN: HYDROPHOBICITY-DRIVEN ANTIMICROBIAL PHOTODYNAMIC ACTIVITY OF ASYMMETRIC PHENOTHIAZINIUM DYES

Antimicrobial Photodynamic Therapy (aPDT) employs light-activated photosensitizers (PS) to generate reactive oxygen species that cause extensive oxidative damage to multiple microbial targets. This multitarget mode of action minimizes the potential for acquired resistance, making aPDT a promising alternative against multidrug-resistant pathogens. However, the molecular determinants of PS efficacy remain poorly understood. Singlet oxygen quantum yield (ΦΔ) alone often fails to predict biological performance, suggesting that factors such as bacterial association play a decisive role. To investigate these relationships, a library of asymmetric phenothiazinium derivatives was synthesized via the Strekowski procedure and evaluated for singlet oxygen generation, Photodynamic Minimum Inhibitory Concentrations (PD-MICs), and bactericidal activity against methicillin-resistant Staphylococcus aureus, extended-spectrum β-lactamase-producing Escherichia coli, Pseudomonas aeruginosa, and Neisseria gonorrhoeae. Compounds 4, 6, and 10 displayed ΦΔ values comparable to Methylene Blue, whereas piperazine-bearing derivatives (5, 8, and 9) exhibited lower values, likely due to electron-transfer quenching by tertiary amines. Importantly, ΦΔ did not correlate directly with antimicrobial activity, as several highly active derivatives showed moderate singlet oxygen production. PD-MIC assays revealed substantially enhanced activity relative to MB, with compound 5 reducing the PD-MIC against P. aeruginosa by 64-fold and compounds 9 and 11 by at least 32- and 64-fold, respectively, against N. gonorrhoeae. These effects translated into pronounced bactericidal activity, with compound 5 achieving a 7.66-log₁₀ reduction against P. aeruginosa, compound 11 a 6.18-log₁₀ reduction against N. gonorrhoeae (>160,000-fold greater efficacy than MB), and compounds 6 and 11 >3-log₁₀ reductions against MRSA. Notably, derivative 11 exhibited the broadest activity, retaining efficacy against all four pathogens. These findings demonstrate that asymmetric substitution of the phenothiazinium scaffold can decouple ΦΔ from antimicrobial efficacy, highlighting bacterial association as a key determinant of photodynamic killing. This structural insight may guide the development of phenothiazinium derivatives with enhanced activity against multidrug-resistant Gram-negative pathogens.