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':2'',6''-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 exhibits a strong 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 displays a very weak ³MLCT band at 400-550 nm. Ir₂ttpy shows 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 does not increase the conjugated system of the ligand ttpy and there is no weak interaction between the two Ir(III) center in Ir₂ttpy, the fluorescence quantum yield and excited state lifetime of Ir₂ttpy are 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 TPA cross-section of Ir₂ttpy is 24 GM (680 nm). Meanwhile, the TPEF peak of Ir₂ttpy is located at 600 nm, which may also originate from the ³MLCT state. Ir₂ttpy shows certain phototoxicity against 4T1 tumor cells with PI value up to 18. UV-Vis titration experiments indicate that Ir₂ttpy interacts with DNA to a certain extent. Autodock simulations show that Ir₂ttpy is embedded into the major groove of DNA and binds to the bases DA-17 and DC-9 through hydrogen bonds. Extracellular tests indicate that singlet oxygen and superoxide anion can be generated in the presence of Ir₂ttpy under visible light irradiation, and shows obvious catalytic oxidation effect on NADH. Confocal imaging results indicated that Ir₂ttpy can also generate 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, ROS, DNA