This study focuses on the structure–response relationship of symmetrically substituted phenothiazinium
dyes. Four hydrophilic derivatives with the ability of additional hydrogen bonding (5, 6) or additional electrostaticinteraction (3, 4) were synthesized, photophysically characterized and compared to the parent
compound methylene blue (MB, 1) and a lipophilic derivative (2) without additional coordination sites.
Derivative 5 was most effective against Gram-positive Staphylococcus aureus and Gram-negative Escherichia
coli reaching a maximum photodynamic efficacy of >5log10 steps (≥99.999%) of bacteria killing in
10 minutes (5 μM, 30 J cm−2) without inherent dark toxicity after one single treatment with the incoherent
light source PDT1200 (λmax = 660 nm, 50 mW cm−2). Interestingly, one derivative with two additional
primary positive charges (3) showed selective killing of Escherichia coli (5 μM, 30 J cm−2, 4log10 steps
inactivation (≥99.99%)) and no antimicrobial effect on Staphylococcus aureus. This might allow the development
of a new generation of photosensitizers with higher antimicrobial efficacy and selectivity for
future applications.