花菁类小分子在靶向清除线粒体ROS的设计合成与抗辐射作用研究

In recent years, mitochondrial ROS has been widely recognized as a crucial role in low-dose-induced radiation injury. To date, there is never a clinically available radioprotective drug which could be used to specifically removing the mitochondrial ROS. Based on our previous findings on a new kind of mitochondria-targeted cyanine dyes, this project will propose to introduce TEMPO into their chromophore skeleton through formation of different conjugated chemical bonds. On the one hand, this design can realize the targeted removal of mitochondrial ROS by using the classic antioxidant TEMPO. On the other hand, with a nitrogen oxygen moiety in the chromophore of mitochondria-targeted cyanine dyes, the fluorescence emission of cyanine dyes was quenched because of intramolecular photoinduced electron transfer (PET). When ROS reacts with nitrogen oxygen moiety of TEMPO, the PET effect would be inhibited and fluorescence emission occurs. The change in fluorescent intensity can be utilized to monitor the real-time level of mitochondrial ROS, which may avoid or reduce the side effects caused by excessive oxidation. The project will begin with chemical modification of cyanine molecules in their N- alkyl side chains, for design and synthesis of new derivatives of cyanine dyes with different degrees of lipophilic cationic properties. Then, TEMPO will be chemically conjugated to the new synthesized cyanine dyes through different coupled bonds. Finally, the abilities of targeting mitochondria, monitoring ROS level, radioprotective effect and underlying mechanism would be investigated through in vitro and in vivo experiments. This project may provide a theoretical basis to further design and develop new mitochondria-targeted radioprotective agents with high efficacy and low side effects.

线粒体ROS在低剂量辐射诱导损伤被广泛证实起着关键作用，然而临床上尚无可特异性清除线粒体ROS的抗辐射药物。本项目在前期发现一类新型线粒体靶向花菁分子的基础上，提出通过各种共轭化学键将抗氧化剂TEMPO分子，引入至该类花菁分子中的荧光发射基团中，以实现靶向清除线粒体ROS。同时由于TEMPO分子中的氮氧基与ROS反应后，导致其原本与荧光发射团发生的分子内光诱导电子转移（PET）被抑制，从而通过光学改变同步实现实时监测线粒体ROS水平，为避免或减轻过度抗氧化导致的副作用提供可能性。本项目拟首先通花菁分子N-烷基侧链的化学修饰，化学设计与合成亲脂性阳离子特性不同程度增强的新衍生物，然后将TEMPO化学偶联至上述花菁分子骨架中，接着在细胞和动物水平上评价其线粒体靶向、近红外监测ROS水平、抗辐射作用及机制等研究，为进一步设计和发展高效低毒的线粒体靶向抗辐射分子提供实验依据。

线粒体作为细胞的能源工厂，在细胞生存、凋亡和自噬等方面都发挥着重要作用。近年来，线粒体ROS被证实在辐射诱导凋亡起关键作用，然而临床上尚无可特异性清除线粒体ROS的抗辐射药物。在前期发现一类线粒体靶向七甲川花菁分子的基础上，申请人通过共价连接抗氧化剂TEMPO分子，成功构建同时具有线粒体靶向和ROS清除能力的花菁小分子，实现靶向清除线粒体ROS。本项目通过对不同N-烷基侧链和电荷数花菁分子的修饰，优化分子骨架的脂水分配系数，获得线粒体靶向优异的多个花菁小分子；在此基础上，进一步与抗氧化基团TEMPO通过不同取代方式的共价偶联，得到一系列不同取代类型的TEMPO类花菁抗辐射小分子。通过细胞水平上的效应评价，筛选获得具有线粒体靶向清除ROS能力且辐射防护效应优于临床药物氨磷汀的新型花菁小分子（IR-84 和IR-82）。最后，在动物水平证实IR-84和IR-82均具有较好的辐射防护作用，并对其机制开展研究，为进一步设计和发展高效低毒的线粒体靶向抗辐射分子提供实验依据。