Toluidine Blue O Demethylated Photoproducts as Type Ii Photosensitizers

Toluidine blue O (TBO) is a type I–type II photosensitizer that has shown good efficacy and selectivity in antimicrobial and anticancer photodynamic therapy applications. However, its complex photochemistry with multiple photoproducts hinders its application as a photosensitizer. We have previously described the mechanism for photooxidative demethylation of TBO which in acetonitrile yields two main products: demethylated-TBO (d-TBO) and double-demethylated-TBO (dd-TBO). In the current work, we describe the photophysical properties of these two photoproducts. In acetonitrile and phosphate buffer, demethylation induces an hypsochromic shift in the absorption and fluorescence emission maxima. Fluorescence quantum yields increase slightly for the demethylated photoproducts, in agreement with the lengthening of the fluorescence lifetimes. Triplet excited states lifetimes in the presence of oxygen decreased slightly upon demethylation. However, the singlet oxygen quantum yield increased significantly reaching unity for the dd-TBO photoproduct. These results are interpreted in terms of the competing pathways of TBO photochemistry. For TBO, demethylation is the main pathway for deactivation of the excited state, while for d-TBO, demethylation and singlet oxygen generation are significant. For dd-TBO, singlet oxygen generation is the main deactivation pathway. Overall, TBO demethylated photoproducts demonstrate good potential as candidates for photodynamic therapy applications. © 2025 American Society for Photobiology.