基于trityl自由基的超氧自旋捕捉剂的分子设计、合成及性能研究

Superoxide anion radical is one of the most important reactive oxygen species and has been closely associated with development and progression of various diseases. Electron paramagnetic resonance (EPR, or electron spin resonance, ESR)-spin trapping technique is the most reliable and specific method for the detection of superoxide but its biomedical applications were limited by slow reaction of the currently available spin traps with superoxide as well as low stability of the corresponding spin adducts. Recently, EPR coupled with the use of tetrathiatriarylmethyl (trityl) radicals showed great potential for the detection of superoxide. However, their applications were still limited due to low trapping rates, cell-impermeability and relatively low specificity. To address these problems, this proposal consists of three research aims: 1) theoretical calculation will be used for molecular design of trityl parent compounds with high reactivity and specificity to superoxide; these parent compounds will be synthesized and used to investigate the substituent effect on the trapping efficiency for superoxide. 2) Trityl parent compounds will be further modified with PEG chains, low molecular weight protamine and targeting groups to afford highly effective, specific and cell-permeable superoxide spin traps. Then, dual-modal EPR/fluorescent detection and imaging method will be developed for subcellular and living tissue superoxide. 3) The application potential of these spin traps for biomedical imaging of superoxide will be tested in cell models of hypoxia reoxygenation and mouse models of myocardial ischemia-reperfusion injury. Overall, the superoxide spin traps developed in this proposal will show great potential to investigate the mechanism of the oxidative stress-related diseases, determine the efficacy of potential therapies and develop new therapies.

超氧阴离子自由基是最重要的活性氧物种之一，与诸多重大疾病的发生发展密切相关。电子顺磁共振（即电子自旋共振）联合自旋捕捉技术是最可靠和最特异的超氧检测方法，但其生物医学应用却因现有自旋捕捉剂与超氧反应速率低及生成加合物不稳定等因素受到限制。近来，三苯甲基（trityl）自由基在超氧检测方面表现出很强的应用潜质，但在捕捉速率、专一性及透膜性等方面仍有不足。鉴于此，本项目拟：1）以理论计算为指导，设计合成对超氧具有高反应性和专一性的trityl母体化合物，研究取代基对捕捉性能的影响；2）功能衍生化母体化合物，构建高效、专一且生物膜可透的新型超氧自旋捕捉剂，并建立电子顺磁共振和荧光双模态检测方式，实现亚细胞到活体组织内超氧的检测与成像；3）以细胞缺氧再给氧和小鼠心肌缺血再灌注为模型考察自旋捕捉剂检测与成像超氧的生物应用潜质，为其在氧化应激相关疾病的发病机制研究及其诊治方面的应用提供直接的实验依据。

超氧自由基的精准检测对于阐释其生理学及病理学意义具有至关重要的作用。本项目的主体目标是构建trityl双自由基作为高灵敏、高选择、生物相容的电子顺磁共振超氧探针，并探索其生物医学应用前景。为此，我们首先设计合成了全硫取代trityl自由基PST及其树枝状分子包裹的衍生物PST-NA；PST-NA对超氧自由基表现出高度的反应性和选择性，已成功用于细胞体系中超氧自由基的检测，是用于构建本项目中双自由基超氧探针的理想反应基团。在此基础上，将反应基团PST-NA与对不同氧化还原性物质均惰性的报告基团相偶联，实现了双自由基探针的构建。此外，我们在trityl自由基的氧化还原性质研究及新型trityl自由基研发方面也取得了一些成果：1）揭示了trityl衍生物可在巯基介导下还原溶解氧产生超氧自由基，该研究颠覆了trityl 自由基对GSH等生物巯基小分子稳定的传统观点，也为trityl 类氧化还原探针的设计及应用提出了警示；2）发展了高效的trityl自由基PEG衍生化方法，详尽阐述了PEG修饰对trityl自由基理化性质的影响，并探索了该类trityl自由基用于活体EPR测氧的应用潜质；3）设计合成了新型水溶性trityl自由基TFO，该分子具有OX063相似的理化性质，将有效拓展trityl自由基在磁共振领域的应用；4）基于trityl碳正离子的反应机制合成了系列单取代trityl衍生物，研究了取代基对trityl自由基氧化还原性质影响。在本项目支持下，我们在超氧刺激响应的一氧化氮供体研究、过氧化氢和过氧亚硝酸等活性氧分子探针开发以及基于trityl-nitroxide 的动态核极化试剂研发等方面也开展了系列工作，并取得了一定成绩。在该项目支持下，目前共发表学术论文9篇（其中，共同通讯作者8篇），申请国内专利2项，参加国内国际会议10次。