Ultrafast Excited-State Dynamics and Two-Photon Near-Infrared Induced Photodynamic Therapy Performance of 5‑Phenylethynyl-4-thiouridine.

5-Phenylethynyl-4-thiouridine (5ph-4TU), featuring both π-conjugated phenylethynyl and carbonyl-sulfur substitutions, overcomes the UVA absorption limitations of conventional thio-nucleosides by extending absorption into visible and potentially longer-wavelength regions under single or two photon excitationan essential advancement for enhancing nucleosides' photoactivity toward deep-tissue cancer photodynamic therapy (PDT). However, the ultrafast excited-state dynamics and synergistic effects of these dual modifications remain unexplored, despite their critical role in generating unique excited-state characters inaccessible for canonical nucleosides. Here, we employ ultrafast spectroscopy combined with QM-(MS-CASPT2//CASSCF)/MM calculations to map out the unusual excited-state pathways of 5ph-4TU. We reveal that the C5-phenylethynyl group can effectively suppress C5C6 bond twisting to incur a high barrier of 0.8 eV from the S2 (1ππ*)min to access S2 (1ππ*)/S1 (1nπ*) conical intersection. This hinders internal conversion from S2 (1ππ*) to S1 (1nπ*), resulting in a long-lived S2 (1ππ*)min state (∼600 ps in CH3CN) from which enhanced fluorescence occurs (ΦFL = 0.11 ± 0.03). Interestingly, once reaching S1 (1nπ*)min, the triplet manifold can be effectively populated through the S1 (1nπ*)/T2 (3ππ*)/T1 (3nπ*) triple quasi-degeneracy region with significant spin-orbit coupling (SOC = 123 cm-1). Furthermore, we find that the extended π-conjugation enhances two-photon absorption into the near-infrared range while preserving the same excited-state dynamics as single-photon excitation. In vitro experiments confirm excellent biocompatibility and efficient singlet oxygen (1O2) generation, inducing cancer cell death under 760-770 nm two-photon irradiation, underscoring its potential for deep-tissue PDT. These findings provide a new paradigm for designing next-generation biophotosensitizers with combined imaging and therapeutic functionalities.