新型硫烷硫类化合物生物发光探针的构建及其生物应用

Recent publications demonstrated important roles for sulfane sulfur, especially, persulfides and hydrogen polysulfides in H2S-mediated physiological functions, and suggested persulfides and (or) hydrogen polysulfides may be the actual signaling species. However, the underlying mechanism of persulfides and hydrogen polysulfide in biological functions, as well as the cross-talk between H2S and persulfides/ polysulfides, are still not fully understood. Therefore, new techniques for accurate detection of persulfides and hydrogen polysulfides levels are in high demand to provide valuable information to understand their functions. Bioluminescent imaging is a reliable, sensitive, and non-invasive imaging technique. To develop novel method for imaging sulfane sulfur (persulfides and hydrogen polysulfides), we designed a series of chemoselective bioluminescent probes for high sensitivity, nontoxicity, non-invasive and real-time imaging of persulfides and hydrogen polysulfides within living animals. We chose luciferin, amino luciferin and NIR luciferin analogues as substrate for luciferase. The groups, including benzyl bromide, 2,4-dicyano phenyl ether, and 2,4- dicyano azo, served as the reaction site. These probes are group-caged fierfly luciferin analogues that selectively react with persulfide and (or) hydrogen polysulfide to release luciferin, which triggers a bioluminescent response in the presence of luciferase. To investigate whether these probes has detection efficacy for persulfides and hydrogen polysulfides in vivo, living cells and animal imaging test will be conducted. Furthermore, these probes will be applied to the measurement of endogenous persulfides levels in cancer cell line and xenograft tumor, and investigated the role of endogenous persulfides in the pathogenesis of tumor. This project will provide effective and novel detection techniques for sulfane sulfur -related biological mechanism and signaling transduction research.

在硫化氢相关的生理过程中，硫烷硫类化合物，尤其是过硫化物和聚硫化氢发挥重要的调控作用，但机制未明。阐明上述问题的首要条件是建立准确、可靠的检测方法。硫烷硫类化合物的传统检测方法尚存较大的改进空间，仍缺乏无创实时的在体检测方法。本项目基于荧光素酶-荧光素的发光成像原理，构建用于硫烷硫类化合物（过硫化物和聚硫化氢）检测的生物发光探针，以苄溴、2,4-二氰基苯醚和2,4-二氰基偶氮作为新型的识别基团，采用荧光素及近红外荧光素作为荧光素酶的识别底物，实现目标物的高灵敏度、安全无毒、无创实时的在体检测。研究探针在细胞、活体中对过硫化物和聚硫化氢的成像性能；并运用探针检测肿瘤细胞与裸鼠移植瘤内过硫化物水平，研究内源性过硫化物在肿瘤发展过程中的作用。本项目将为硫烷硫类化合物的研究提供新型的可视化检测方法，有助于揭示它们在调节生理病理功能的作用与机制，有助于新靶点的发现，为医学研究提供有效新颖的技术支撑。

本项目开发了一系列小分子探针，用于检测生物体内聚硫化氢、硫化氢与硒半胱氨酸，实现了其可视化及原位检测。并应用开发的探针，研究内源性活性硫化物、活性硒化物与疾病发生的内在关系及相关机制。这对于揭示活性硫化物、活性硒化物在调节人体生理病理功能的作用与机制，寻找疾病标志物、揭示疾病的病因学机制、寻找新的药物靶点具有重要意义。.1. 新型聚硫化氢荧光探针.聚硫化氢是硫烷硫家族的重要成员之一。在硫化氢相关的生理活动中，聚硫化氢发挥重要的调控作用，可能是（至少部分是）实际的信号转导分子。（1）本项目制备了近红外聚硫化氢荧光探针NIR-CPS，该探针具有灵敏度高（检测限18 nM）、选择性好以及较大斯托克斯位移（100 nm）的优势，并实现了细胞和活体水平内源性聚硫化氢的荧光成像。（2）制备了基于ESIPT原理的聚硫化氢荧光探针HF-PS1，实现了细胞中内源性聚硫化氢的荧光成像。.2. 新型硫化氢荧光探针.硫化氢是一种重要的气体信号分子，发挥重要生理功能。本项目制备了近红外硫化氢荧光探针NIR-HS，不仅实现CBS（硫化氢的合成酶）高表达细胞中内源性硫化氢的荧光成像，还成功地实现了小鼠体内内源性硫化氢的荧光检测，并应用该探针检测了大鼠肾脏、肝脏组织中硫化氢的含量。.3. 硒半胱氨酸生物发光探针和荧光探针.硒半胱氨酸是硒在体内的主要存在形式之一，常位于硒蛋白的关键活性位点，从而参与多种生理功能。（1）本项目制备了识别硒半胱氨酸的生物发光探针BF-1，该探针具有选择性好、灵敏度高、检测限低(8 nM)及良好生物相容性等优点；探针BF-1实现了MCF-7-luc细胞、活体水平（FVB-Luc+转基因小鼠）的硒半胱氨酸生物发光成像；进一步应用该探针检测了裸鼠移植瘤模型中肿瘤组织内的硒半胱氨酸水平。（2）制备了能特异性识别硒半胱氨酸的近红外荧光探针Fsec-1，该探针具有较高的检测灵敏度（检测限10 nM）和较好的选择性，实现了活细胞和小鼠体内内源性硒半胱氨酸的荧光成像，进一步应用该探针检测了裸鼠移植瘤模型中肿瘤组织内的硒半胱氨酸水平。