Cyclometalated Ir(III) complexes have attracted significant attention in the field of two-photon photodynamic therapy (PDT) sensitizers due to their excellent luminescence property and good phototoxicity. Polypyridine derivatives are commonly chosen as ligands for iridium complexes. Considering that the introduction of thymine moiety not only enhances biocompatibility but also provides two easily modifiable amide-N groups, herein the 2,2':6',2''-terpyridine group was connected with thymine to prepare a novel polypyridine ligand (ttpy), and subsequently a binuclear iridium complex (Ir₂ttpy) was obtained, which containing divalent coordinating cation. Ir₂ttpy exhibited a strong intramolecular ligand to ligand charge transfer (ILCT) absorption peak between 250~350 nm. Due to strong spin-orbit coupling effect of Ir(III) center, the higher-energy excited singlet state undergo intersystem crossing to the lower-energy excited triplet state, and thus Ir₂ttpy displayed a very weak metal to ligand charge transfer (MLCT) band at 400~550 nm. Ir₂ttpy showed dual fluorescence peaks, with the emission at 350 nm originating from the ³LC excited state of the terpyridine group, while the emission at 600 nm coming from the ³MLCT excited state of the Ir(III) coordination cores. Since the introduction of thymine did not increase the conjugated system of the ligand ttpy and there was no weak interaction between the two Ir(III) centers in Ir₂ttpy, the fluorescence quantum yield and excited state lifetime of Ir₂ttpy were negligible (<1%, 100 ns), comparing with numerous reported long-lived and high-quantum-yield iridium complexes. When excited by a femtosecond laser at 650~1000 nm, the maximum two-photon absorption (TPA) cross-section of Ir₂ttpy was 24 GM (680 nm). Meanwhile, the two-photon excited fluorescence (TPEF) peak of Ir₂ttpy was located at 600 nm, which may also originate from the ³MLCT state. Ir₂ttpy showed certain phototoxicity against 4T1 tumor cells with phototoxicity index (PI) value up to 18. UV-Vis titration experiments indicated that Ir₂ttpy interacted with DNA to a certain extent. Autodock simulations showed that Ir₂ttpy was embedded into the major groove of DNA and binded to the bases DA-17 and DC-9 through hydrogen bonds. Extracellular tests indicated that singlet oxygen and superoxide anion counld be generated in the presence of Ir₂ttpy under visible light irradiation, and showed obvious catalytic oxidation effect on NADH. Confocal imaging results indicated that Ir₂ttpy could also generate reactive oxygen species (ROS) intracellularly. The antitumor activity of Ir₂ttpy is related to its induction of ROS generation and its interaction with DNA, and they act synergistically to cause tumor cell death. Due to the weak fluorescence of Ir₂ttpy in cells, co-staining with fluorescent reagents for mitochondria or lysosomes failed to clarify the localization selectivity of Ir₂ttpy for mitochondria or lysosomes. Although the potential of the complex Ir₂ttpy as a PDT photosensitizer is not ideal, the data in this paper provide certain theoretical base for the future development of novel photosensitizers.

Key words: thymine, binuclear Ir(III) complex, antitumor, reactive oxygen species (ROS), DNA