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

High Resolution Image Download MS PowerPoint Slide 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 S 2 ( 1 ππ*) min to access S 2 ( 1 ππ*)/S 1 ( 1 nπ*) conical intersection. This hinders internal conversion from S 2 ( 1 ππ*) to S 1 ( 1 nπ*), resulting in a long-lived S 2 ( 1 ππ*) min state (∼600 ps in CH 3 CN) from which enhanced fluorescence occurs (Φ FL = 0.11 ± 0.03). Interestingly, once reaching S 1 ( 1 nπ*) min, the triplet manifold can be effectively populated through the S 1 ( 1 nπ*)/T 2 ( 3 ππ*)/T 1 ( 3 nπ*) 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 ( 1 O 2 ) 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.