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。

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.