基于胸腺嘧啶三联吡啶的双核铱配合物的发光及抗肿瘤活性研究

doi:10.6023/A24120380

摘要/Abstract

环金属铱配合物因其优秀的发光性能和良好的光毒性, 在双光子光动力疗法(PDT)光敏剂研究领域备受关注. 多联吡啶衍生物常被选作铱配合物的配体, 考虑到胸腺嘧啶分子拥有两个便于修饰的酰胺基团, 将其引入配体有可能会提升生物相容性. 将2,2':6',2''-三联吡啶基团与胸腺嘧啶相连接以制备多联吡啶衍生物ttpy, 进而制备出含有正二价配位阳离子的双核铱配合物Ir₂ttpy. Ir₂ttpy在250~350 nm呈现出强配体内电荷转移(ILCT)吸收峰, 400~550 nm处呈现弱的金属到配体的荷移跃迁(MLCT)吸收肩峰. Ir₂ttpy表现出双重荧光发射, 350 nm处发射峰源自tpy基团³LC激发态, 而600 nm处的发射则来自配位中心³MLCT激发态. 配合物在680~800 nm飞秒激光激发下表现出一定的双光子荧光效应, 吸收截面约为24 GM. 配合物Ir₂ttpy针对4T1乳腺癌细胞表现出一定的光毒性, 光毒性指数(PI)值约为18. UV-Vis滴定实验表明配合物Ir₂ttpy与DNA有一定的相互结合作用, Autodock模拟显示配合物Ir₂ttpy嵌入DNA大沟, 并以氢键与DNA的碱基DA-17以及DC-9结合. 体外滴定实验表明配合物Ir₂ttpy在可见光照射下生成¹O₂和$\text{O}_{2}^{\centerdot }$, 能够光催化氧化烟酰胺腺嘌呤二核苷酸(NADH). 细胞成像结果也显示配合物Ir₂ttpy能在光照下生成活性氧物种(ROS). 配合物Ir₂ttpy的抗肿瘤活性与其诱导生成ROS以及与DNA相互结合有关, 二者协同作用而导致肿瘤细胞死亡.

关键词: 胸腺嘧啶, 双核铱配合物, 抗肿瘤, 活性氧物种(ROS), DNA

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

引用此文

于千水, 戚聪, 顾顺心, 杨欣达, 姜琴, 魏荣斌, 施鹏飞. 基于胸腺嘧啶三联吡啶的双核铱配合物的发光及抗肿瘤活性研究[J]. 化学学报, 2025, 83(6): 579-587.

Qianshui Yu, Cong Qi, Shunxin Gu, Xinda Yang, Qin Jiang, Rongbin Wei, Pengfei Shi. Study on the Luminescent and Antitumor Activities of Binuclear Iridium Complex Based on Thymidyl-Terpyridine[J]. Acta Chimica Sinica, 2025, 83(6): 579-587.

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图/表 13

图1. 配体ttpy与配合物Ir2ttpy的分子结构示意图

Figure 1. Molecular digrams for ligand ttpy and its Ir(III) complex Ir2ttpy

图2. 采用Gaussian 09软件包进行能量最低优化后的分子结构图(a为ttpy, b为Ir2ttpy. 去掉所有的H原子和阴离子PF6-, 以便更好地显示分子结构)

Figure 2. Molecular structure optimized for minimum energy using Gaussian 09 software package (a for ttpy and b for Ir2ttpy. All H atoms and PF6- anions were omitted for clarity)

图3. 配体ttpy与配合物Ir2ttpy的紫外-可见吸收光谱(虚线为模拟分峰的结果)

Figure 3. UV-Vis spectra of ligand ttpy and complex Ir2ttpy (the simulated peak splitting results indicated by dashed lines)

图4. 配体ttpy与配合物Ir2ttpy的紫外-可见吸收光谱指认结果(通过TD-DFT计算得到ttpy和Ir2ttpy优化几何结构的分子轨道. 使用TD-B3LYP方法和6-31G(d)基组计算跃迁能, 单位为eV)

Figure 4. Assignment for the UV-vis spectra of ligand ttpy and complex Ir2ttpy (Molecular orbitals for ttpy and Ir2ttpy were optimized from TD-DFT calculations. Transition energies were calculated using the TD-B3LYP method with 6-31G(d) basis sets, eV)

图5. 配体ttpy与配合物Ir2ttpy的荧光发射光谱

Figure 5. Fluorescence spectra of ligand ttpy and complex Ir2ttpy

图6. 配体ttpy与配合物Ir2ttpy在680 nm飞秒激光激发下的开孔Z-扫描结果及高斯拟合曲线(实线)

Figure 6. Open-aperture Z-scan experimental data and fitting curve for complex Ir2ttpy under a 680 nm femtosecond laser beam

图7. 配体ttpy与配合物Ir2ttpy双光子激发(680 nm, 飞秒)荧光发射谱

Figure 7. Two-photon excited fluorescence spectra of ligand ttpy and complex Ir2ttpy under a 680 nm laser beam

图8. ttpy (a)和Ir2ttpy (b)的紫外可见吸收光谱随DNA浓度递加的变化图(化合物的浓度为2.0×10-5 mol/L, 缓冲溶液组成: 5 mmol/L tris, 50 mmol/L NaCl, pH=7.42, r=[CT-DNA]/[ttpy or Ir2ttpy]=0~1.0.)

Figure 8. UV absorption spectra of ttpy (a) and Ir2ttpy (b) solution with increasing concentration of CT-DNA [buffer=5 mmol/L tris, 50 mmol/L NaCl, pH=7.42. The concentration of ttpy (or Ir2ttpy) is 2.0×10-5 mol/L, r=[CT-DNA]/[ttpy/or Ir2ttpy]=0~1.0]

图9. Autodock模拟的配体ttpy (a)以及配合物Ir2ttpy (b)与DNA分子进行相互对接作用的示意图

Figure 9. Docking interactions between the ligand ttpy (a) and complex Ir2ttpy (b) with DNA molecules, simulated by Autodock

图10. 二甲基亚砜(DMSO)溶液中ABDA紫外可见吸收光谱随Ir2ttpy浓度的递加的变化图

Figure 10. UV-Vis absorption spectra of ABDA in dimethyl sulfoxide (DMSO) solution changed with increasing concentration of Ir2ttpy

图11. DMSO溶液中Ir2ttpy敏化的DHR123荧光发射光谱变化

Figure 11. Fluorescence spectra of DHR123 sensitized by Ir2ttpy in DMSO solution

图12. 配合物Ir2ttpy催化氧化NADH的吸收光谱变化图

Figure 12. UV-vis absorption spectra of NADH during the photocatalytic oxidation by complex Ir2ttpy

图13. 未用或使用5 mmol/L的CellRoxs预处理30 min的HeLa细胞的共聚焦图像.

Figure 13. Confocal images of HeLa cells pretreated without or with 5 mmol/L of CellRoxs for 30 min

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