新型增强型比率半胱氨酸荧光探针的合成及特异性识别机理

To overcome the problem that it is hard to specially recognize cysteine which is important substance containing sulfhydryl in vivo using fluorescent probes, this study focuses on the synthesis of novel fluorescent probes and the mechanism of specific recognizing cysteine. Based on obtained turn-on radiometric fluorescent probe and its specific action with cysteine before, we design and synthesize new probes through introducing coumarin and cyanine dyes as fluorophores into the structure of the probe Fluor-A1 for improving the sensitivity of detection, and then study the fluorescence quantum yield of the probe and fluorescence spectroscopy. Futhermore, we introduce enal structure as a specific recognizing amino group and mercapto group with designed reaction pathway as below. The amino group reacts with aldehyde group condense firstly, and then mercapto group reacts with the double bond to form a ring. Identifying the structure of reaction products and measuring the secondary reaction rate is to differentiate cysteine over homocysteine and glutathione. Based on obtaining the novel turn-on radiometric probes, we develop a fast method to monitor cysteine specifically with high sensitivity. The available results will be useful for discoverying potential targets and studying pesticide targeting mechanism.

本项目针对目前利用荧光探针难以特异性识别生物体内重要巯基类物质半胱氨酸的问题，而展开新型荧光探针的合成及特异性识别半胱氨酸机理的研究。项目在前期获得荧光探针Fluor-A1且能够检测半胱氨酸的基础上，拟设计将香豆素和花青染料等作为荧光基团分别引入到探针Fluor-A1的结构中，可望提高探针的荧光量子产率和扩大荧光光谱，提升检测半胱氨酸的灵敏度；并设计利用烯醛结构对氨基和巯基的特异性反应，氨基与醛基先缩合，巯基再与双键加成成环反应，通过对反应产物的结构表征以及测定其二级反应速率，探究合成的荧光探针能够从高半胱氨酸和谷胱甘肽中特异性识别半胱氨酸的机理。研究可获得新型增强型比率荧光探针，建立一种可以快速、灵敏、特异性检测生物体内半胱氨酸的方法，研究结果对于探索新的农药作用靶标，研究农药靶向作用机制具有重要意义。

本项目针对目前利用荧光探针难以特异性识别生物体内重要巯基类物质半胱氨酸的现状，而开展新型荧光探针的合成及特异性识别半胱氨酸的机理研究。项目在前期获得荧光探针Fluor-A1且能够检测半胱氨酸的基础上，设计、合成7种具有不同荧光母体结构、识别基团和荧光识别机理的荧光探针，其中3种新型荧光探针可以特异性识别半胱氨酸，不受高半胱氨酸和谷胱甘肽的干扰。完成了全部荧光探针性能的测试，包括荧光探针的紫外光谱、荧光光谱和斯托克斯位移等。探究加入半胱氨酸前后荧光探针溶液体系荧光强度的变化，明确荧光探针对半胱氨酸识别的有效性。进一步探索和优化了测试条件，考察温度、pH值、温育时间以及有机溶剂浓度、其他分析物等因素对特异性识别半胱氨酸的影响。利用NMR、LC-HRMS、DFT等手段，分析荧光探针和生物硫醇的反应产物结构，通过分别测定其二级反应速率对三种不同氨基酸进行区分，明确了荧光探针特异性识别半胱氨酸的机理。并成功利用荧光探针识别细胞和斑马鱼体内的半胱氨酸，完成生物成像。建立一种可以快速、灵敏、特异性检测生物体内半胱氨酸的方法，研究结果对于探索新的农药作用靶标，研究农药靶向作用机制具有重要意义。