还原型辅酶I（NADH）荧光探针合成及生物应用

As an essential electron carrier, reduced coenzyme I (NADH) plays a vital role in cellular metabolism and energy systems. It has been suggested that the abnormal NADH level is closely related to the pathogenesis of many diseases. In this project, we have proposed two approaches to carry out the molecular design of novel fluorescent probes for the detection NADH via an electron transfer mechanism. Because NADH is generated primarily in the cytosol by glycolysis or in the mitochondria by the tricarboxylic acid cycle, and the NADH cannot traverse the mitochondrial membrane in all eukaryotic cells, it is thusly distinct metabolic mechanisms of NADH in the cytosol and mitochondria. Undoubtedly, it is very important to understand the metabolic mechanisms of NADH in the cytosol, mitochondria and mitochondrial membrane. Thus, the molecular design of the mitochondria-targeted and the mitochondral membrane-targeted fluorescent probes are carried out in this project to achieve the subcellular imaging and multicolor imaging. The mitochondria-targeted NADH probes can be realized by modification of the probes with triphenylphosphonium functional group, and the mitochondral membrane-targeted fluorescent NADH probes can be achieved by the paradigm of tuning the hydrophobic properties of the probes with alkyl groups or the combination both the tuning of the hydrophobic properties and the introduce of triphenylphosphonium functional group. The aims of this project are as follows: 1) obtaining 5-8 novel dyes and 4-5 mitochondria-targeted and mitochondral membrane-targeted fluorescent probes for cellular NADH detections via a direct electron transfer mechanism between the fluorescent signaling fluorophores and the NADH donor; 2) obtaining the key data which related to the metabolic mechanisms and pathogenic mechanisms of cellular NADH by detailed investigation of the fluorescent imaging and multicolor imaging by using these novel probes; and 3) establishing high signal-noise ratio NADH detection models by using these probes.

还原型辅酶I（NADH）作为细胞中重要的电子传递载体，对维持正常生命活动起着至关重要的作用，许多疾病的发病机制与其代谢紊乱密切相关，因此，建立快速、灵敏的NADH检测方法，实现生命活动中NADH代谢功能的准确评价一直是生命分析科学中的热点。本项目拟从设计可直接接受NADH传递电子的新型荧光探针切入，通过利用荧光探针和NADH之间直接发生电子转移而引起探针在新荧光发射波长下光学信号的增强，或探针在被还原前后双波长下荧光强度的比率变化的思路来发展NADH的荧光传感成像检测新模型；结合对探针的靶向修饰等途径实现细胞质、线粒体和线粒体膜等不同功能内源NADH的靶向荧光成像和通过不同荧光发射波长靶向探针分别对细胞质、线粒体和线粒体膜中NADH的多色荧光成像，获得对NADH的代谢功能、作用机制和致病机制等关键基础科学问题的深入认识；并建立细胞内高荧光信号-本底对比度的NADH荧光传感成像检测新模型。

还原型辅酶I，即NADH，是细胞中重要的的电子传递载体，对能量代谢活动具有重要的作用。研究发现NADH的代谢失衡与许多重大疾病的发病机制密切相关，因此，建立快速、灵敏的NADH检测方法，实现生命活动中NADH代谢功能的准确评价是非常有意义的工作。在国家自然科学基金委员会的支持资助下，本项目首先基于可直接接受NADH电子的检测原理，通过利用荧光探针在接受电子前后氧化-还原态转化而引起的光学信号变化的思路设计合成了5种新型NADH 的荧光传感成像检测新模型，系统研究了探针线粒体等细胞器的靶向标记、对NADH的识别性能和荧光成像性能。研究结果表明所设计合成的荧光探针对NADH成像呈现出高的选择性、高的检测灵敏度以及显著的荧光信号变化。所构建的具有双正电荷的双螺环荧光探针，是目前所报道的NADH荧光探针中时间响应最快的。荧光成像实验表明所设计的荧光探针具有良好的细胞穿透力、良好线粒体靶向定位能力和较小的细胞毒性，可实现细胞外源性和内源性NADH的荧光成像检测。也探索了探针在不同条件刺激应急损伤模型中NADH变化。同时，我们还设计合成了9中对细胞中活性物种具有识别检测功能的荧光探针，探索了其光谱识别性能和荧光成像检测性能。研究结果目前已在国外SCI刊物发表论文24 篇；申请发明专利2项，授权1项；协助培养博士研究生 3人，硕士研究生8人。