基于模块化设计的香豆素-查尔酮探针用于谷胱甘肽的可逆比率检测

Coumarin-chalcone Probes with Modular Design for Reversible Ratiometric Detection of Glutathione

摘要

基于模块化设计策略， 以7-氨基香豆素为荧光母核， 2′-羟基查尔酮为谷胱甘肽（GSH）的反应中心， 并通过在查尔酮苯环5-位引入不同取代基(硝基、 甲氧基)作为调控模块， 系统调控探针的光物理性质及其与GSH反应的动力学特性， 开发了一系列香豆素-查尔酮类可逆型比率荧光探针(探针1~3). 实验结果表明， 所有探针均在生理浓度(0~10 mmol/L)范围内对GSH有良好的比率荧光响应， 其解离常数(K_d=4.61~8.25 mmol/L)适用于细胞内的GSH检测. 其中， 探针1的硝基与酮羰基的协同吸电子效应使其不仅具有最高的灵敏度与最显著的比率信号变化， 对GSH的响应也最迅速， 约5 min即可达平衡， 反应速率常数为2.54 L·mol⁻¹·s⁻¹. 可逆实验结果证实探针1对GSH响应具有突出的可逆循环特性； 荧光成像研究验证了其对活细胞内GSH比率成像的可行性. 本研究通过理性的调控模块设计， 为活细胞GSH动态变化的荧光成像研究提供了性能优异的工具.

Abstract

Based on a modular design strategy， this study developed a series of coumarin-chalcone-based reversible ratiometric fluorescent probes（Probes 1~3）， using 7-aminocoumarin as the fluorophore and 2´-hydroxychalcone as the glutathione（GSH） reaction center. Different substituents（nitro， methoxy） were introduced at the 5-position of the chalcone phenyl ring as modulating modules to systematically tune the photophysical properties and reaction kinetics of the probes towards GSH. The results demonstrated that all probes exhibited good ratiometric fluorescence responses within the physiological concentration range of GSH（0—10 mmol/L）， with dissociation constants（K _d=4.61—8.25 mmol/L） suitable for intracellular GSH detection. Among them， Probe 1， by virtue of the synergistic electron-withdrawing effect of its nitro group and ketone carbonyl， not only displayed the highest sensitivity and the most significant ratiometric signal change， but also responded to GSH most rapidly， reaching equilibrium in approximately 5 min with a reaction rate constant of 2.54 L·mol⁻¹·s⁻¹. Reversibility experiments confirmed that Probe 1 also possessed the most prominent reversible cycling characteristics. Fluorescence imaging studies demonstrated the feasibility of ratiometric fluorescent imaging of GSH within live cells. The rational modular design in this work provides a high-performance tool for fluorescent imaging studies of dynamic GSH changes in living cells.

关键词

荧光探针 / 香豆素 / 查尔酮 / 谷胱甘肽

Key words

Fluorescent probe / Coumarin / Chalcone / Glutathione（GSH）

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基金资助

国家自然科学基金(22174090)

陕西省自然科学基础研究计划项目(2024JC-YBMS-138)