基于半花菁的新型比率荧光探针的构建及其线粒体靶向成像研究

The species and concentration of intracellular reactive species are not the same in distinct cellular organelles, in order to detailly understand these differences, it is urgent to develop bifunctional fluorescent probes which can be targeted to specific organelles and can quantitatively report the distribution of the object. Mitochondria are not only the principal energy-producing compartments in most cells, but also closely related to many pathological processes. As one of the most important cellular organelles, mitochondria are always the focus of researches. In this project, we will use the characteristics of hemicyenine with positive charge which can localize to mitochondria, and select the quinoline with the nature of ratiometric measurement as the donor for the first time to design and synthesize a series of mitochondria-targeted ratiometric fluorescent probes. The structure will be modified by organic chemical synthesis method to extend the emission wavelength to near-infrared range, and the novel design concept can be used as a general pattern to synthesize a series of near-infrared ratiometric fluorescent probes based on hemicyenine for the detection of different reactive species in mitochondria. Therefore, this investigation not only riches the design method of ratiometric fluorescent probe based on hemicyenine, but also has very important scientific value and guiding significance in the fields of biological research, disease diagnosis and development of mitochondria-targeted drugs through accurate and rapid detection of the concentration of reactive species in mitochondria.

不同细胞器内的活性物种的种类和浓度不尽相同，为了详尽了解这些差异，迫切需要开发出既具有细胞器定位功能又能够定量反映客体分布的双功能荧光探针。线粒体不仅是细胞的能量工厂，而且还与许多病理学过程密切相关，是一种重要的细胞器，一直是研究的热点。本项目拟利用半花菁自身含有季铵盐能精确定位线粒体的特性，首次选用具有比率测量性质的喹啉作为供体，设计和合成一系列线粒体靶向的比率型荧光探针。通过对其结构进行修饰，将其发射波长延伸至近红外光区，而且该新颖的设计理念可作为通用模式合成一系列基于半花菁的检测线粒体内不同活性物种的近红外比率荧光探针。本项目的研究一方面将丰富基于半花菁的比率荧光探针的设计方法；另一方面精确快速地检测线粒体内活性物种的浓度变化对生物学研究、疾病诊断和线粒体靶向药物开发都具有非常重要的科学价值和指导意义。

本项目基于分析物与荧光探针分子的特异性化学反应，通过有机化学的合成方法，开发了含特定识别基团的新型荧光探针，构建了对半胱氨酸、铜离子和生物硫醇等具有高选择性、高灵敏度响应的荧光探针分子，并将性能优良的荧光探针分子应用于活细胞成像研究。具体研究成果包括:（1）基于吡啶盐与吡啶的性质差异改变半花菁电子受体吸电子能力差异，实现其光物理化学性能的调控，开发了含线粒体定位功能吡啶盐的半花菁为荧光团，丙烯酰基为识别基团的半胱氨酸比率荧光探针，该设计原理可作为一种通用模式设计比率型荧光探针，而且首次报道将丙烯酰基衍生成苄溴衍生物为半胱氨酸识别基团；（2）通过对半花菁供体的电子调控，合成了铜离子比率荧光探针；（3）基于质子活化共振转移，通过对6-二甲氨基喹啉4位氧原子保护和去保护，设计了生物硫醇荧光探针；（4）基于分子内电荷转移原理，首次构建了以Boranil为荧光团的半胱氨酸、铜离子和生物硫醇荧光探针以及6-乙酰基-2-羟基萘为荧光团的铜离子荧光探针。本项目研究为荧光探针在疾病诊断和药物开发等领域的研究提供参考。相关研究成果已发表学术论文11篇，申请国家发明专利8项，获授权国家发明专利2项。