线粒体靶向性和双色比率定量双功能硫化氢荧光探针的设计合成及性能研究

Hydrogen sulfide has emerged as the third gastransmitter, and it has been recognized to serve important physiological functions.Abnormal hydrogen sulfide levels are associated with many diseases, such as Down syndrome and Alzheimer's diseases.However,traditional methods for the detection of hydrogen sulfide require complicated process,destruction of samples or are limited to extracellular detection.The development of fluorescent probes for the detection of hydrogen sulfide has become a hot research field. This strategy will provide a strong research tool for hydrogen sulfide research in biological systems as their excellent cell permeability and high sensitivity.Recently,we reported two generation fluorescent probes for hydrogen sulfide detection. We has sucessfully used these probes to detect hydrogen sulfide in biological tissues samples and intracelluar H2S. However, Fluorescent probes that can locate the special parts of hydrogen sulfide metabolism in mitochondrial, real-time quantitative detection, and visual imaging are still rarely reported at home and abroad.This project will be an on-going study based on the preliminary work.Through the innovative design of the probe structure,we will introduce the targeted positioning group, two-color ratio quantitative fluorescence group, and rapid reaction of hydrogen sulfide identify group into the molecular structure of the probe.Then, the novel fluorescent probes with the dual functional of mitochondrial targeting and two-color ratio quantitative can be obtained.These probes will provide novel visualization tools for hydrogen sulfide quantitative detection and visual imaging in mitochondrial parts.The accomplishment of this project will promote the development of biology of hydrogen sulfide, as well as the progress of early diagnose and treatment of related diseases.

硫化氢，作为第三种气体信号分子，具有重要的生物学功能。它的含量异常与唐氏综合征、阿尔茨海默氏病等多种疾病相关联。而传统检测硫化氢的方法操作复杂，会导致生物样品破坏或局限于细胞外检测。荧光探针具有良好的细胞通透性和高效选择性，它们的开发将为硫化氢研究提供新型高效的检测工具。本课题组近期报道的两代硫化氢探针可在体外组织样品和活体细胞内检测硫化氢。构建新型硫化氢探针分子已成为目前研究的热点，但能够同时定位于硫化氢代谢特殊部位（线粒体）、实时定量、可视化检测成像的探针，迄今国内外尚鲜见报道。本项目将以前期工作为基础，通过探针结构的创新性设计，将细胞器靶向定位基团、双色比率定量基团和快速反应硫化氢选择识别基团引入探针分子结构中，构建出新型的线粒体靶向性和双色比率定量双功能的硫化氢荧光探针，以实现线粒体内硫化氢定位定量及可视化示踪成像。本项目的实施将有望推动硫化氢生物学的发展和相关疾病前期诊疗的进步。

硫化氢是人体重要的气体信号分子，在生命过程中起着重要作用，研发选择灵敏性的硫化氢荧光分子探针具有重要的科学意义和实际应用价值。本项目采用化学合成获得新颖的荧光分子探针，利用探针结构表征、体外荧光性质测试、生物样品检测、细胞和活体模式动物水平上成像造影等研究手段，筛选出NAP-1、SFP-GR、NJ1等针对气体信号分子硫化氢进行高选择性、高灵敏性检测的荧光分子探针，可能为硫化氢的生物学相关研究提供简便快捷的检测技术方法。本项目首先利用naphthalimide作为荧光基团, 叠氮官能团作为硫化氢的反应基团，构建了双色比例型硫化氢荧光分子探针,NAP-1。该探针表现出良好的选择性和灵敏度，在生物缓冲液和血清体系中均表现出优良的线性关系，检测限可达100 nM水平，实现了硫化氢的定量检测。利用该探针实现了在血浆和小鼠海马组织内检测内源性硫化氢，及外源性刺激（如LPS）引发的硫化氢水平变化，并实现了活体细胞中内源性硫化氢的比例成像。同时，通过将硫化氢反应性基团引入到新颖的荧光基团dicyanoisophorone中，获得了性能更加优异的双色比例型探针分子SFP-GR, 此探针分子自身具有绿色荧光，与硫化氢作用后，其荧光发射光谱发生红移进而发出红色荧光。该探针分子表现出良好的水溶性，选择性和灵敏度，低细胞毒性。利用该探针不仅实现了活细胞中硫化氢的比例造影，也实现了在活体斑马鱼体内的示踪成像。最后还基于“开-关-开”的策略构建了新颖的探针分子NJ1，该探针实现了对Cu2+和H2S的双向检测，并成功实现了在活体细胞中对Cu2+和H2S的选择性示踪成像，这些研究成果将为工业生产、环境以及生物中硫化氢的监测应用奠定基础。