Development of a Nucleoside Photosensitizer Efficiently Activated by One- or Two-Photon Absorption in the Optical...

Developing DNA/RNA nucleoside prodrugs that absorb in the red-to-near-infrared is crucial for applications requiring deep-tissue penetration. This study reports the rational design and synthesis of a novel thiocarbonyl analog, 5-(5-(4-(dimethylamino)phenyl)thiophen-2-yl)-2',3',5'-tri-O-benzoyl-6-aza-2,4-dithiouridine (DATU), efficiently activated through one- and two-photon absorption in the red-to-near-infrared spectrum. We report DATU's electronic structure and its photophysical and triplet-state properties under both excitation, comparing these results to its carbonyl counterpart, 5-(5-(4-(dimethylamino)phenyl)thiophen-2-yl)-2',3',5'-tri-O-benzoyl-6-azauridine (DAU). DATU exhibits strong visible one-photon absorption, with ε₅₂₂ = (0.73 ± 0.02) × 10⁴ M^-1 cm^-1 in aqueous solution, extending to ∼750 nm. Additionally, it features a triplet-state lifetime of 1.7 ± 0.1 μs and a singlet-oxygen quantum yield of 56% in benzene. Photoexcited DATU generates both singlet oxygen and hydroxyl radicals in solvents of varying polarity (water, methanol, acetonitrile, and benzene), functioning as both a type I and type II photosensitizer depending on the cellular environment. Notably, replacing carbonyl with thiocarbonyl groups increases the two-photon absorption cross section at 800 nm 3-fold, from 55 ± 5 GM for DAU to 160 ± 20 GM for DATU, the largest reported for a nucleoside analog. In vitro investigations using 4T1 murine mammary carcinoma cells confirm that DATU exhibits significant photocytotoxicity under one-photon (525 nm, 6 μM) and two-photon (800 nm, 25 μM) activation, with negligible dark toxicity. Together, these findings position DATU as the most promising nucleoside photosensitizer for deep-tissue photodynamic therapy (PDT) to date, laying the foundation for future mechanistic/in vivo studies while broadly guiding the development of next-generation thiocarbonyl photosensitizers for PDT, bioimaging, and photocatalysis.