基于NBO型二氟硼类荧光分子的硝基还原酶荧光探针及其传感行为

It is an important research trend in the fields of biotechnology and biosensors to design and develop the fluorescent probes for the real-time, non-destructive and visual monitoring the in-vivo activity of enzyme due to its important role in biological systems. Based on the status of fluorescent probes for nitroreductase and our research works on the design of the difluoroboron-based fluorescent probes for molecular recognition and fluorescent sensing, in this project, a series of novel nitroreductase fuolorescent probes with large stokes shift will be designed and synthesized by introducing the recognition units of nitroreductase to the skeleton of NBO-type difluoroboron compounds. The recognition and fluorescent sensing and imaging performance of the probes toward nitroreductase and various endogenous interferent species in organism will be studied systematically by using various spectroscopic techniques, such as fluorescence emission spectrum, UV-vis absorption spectrum, NMR, MS, etc., and laser scanning confocal fluorescence microscopy imaging system (LSCFMIS). The relation between the molecular structure of the probe and the performance of photochemistry and photophysics will be further explored to reveal the recognizing and sensing mechanism. The objective of this project is to develop new nitroreductase fuolorescent probes, which also provides an extended strategy for the design and application of the difluoroboron-derived fluorescent probes. A few new fluorescent sensing and imaging systems with high sensitivity and selectivity will be constructed for highly efficient monitoring of nitroreductase, then the detection kit for nitroreductase will be designed and used for real-time and quantitative determination of nitroreductase produced by Escherichia coli.

酶在细胞和生命活动中发挥重要作用，发展酶荧光探针及其对生物体中酶活性的实时、无损、可视化检测新技术新方法是当前生物传感领域重要研究方向。本项目针对硝基还原酶（NTR）荧光探针研究发展动态，并结合二氟硼类荧光探针的研究工作基础，通过在NBO型二氟硼类荧光分子骨架中引入NTR识别作用单元，设计合成系列新型具有大stokes位移的NTR荧光探针。利用荧光光谱、紫外可见光谱、核磁共振、质谱等谱学分析方法和激光扫描共聚焦显微活细胞成像系统，对探针分子进行结构和光化学光物理性能表征；系统评价各类探针分子对NTR以及生物体中内源性干扰物的识别作用和荧光传感与细胞成像性能；通过构效关系研究，阐明不同结构荧光探针分子的识别与荧光响应机制及其调控因素。筛选构建针对活细胞、大肠杆菌等生物体中NTR原位实时动态检测的高灵敏高选择性荧光传感与成像体系和NTR检测试剂盒，发展新型NTR荧光探针及其荧光分析检测技术。

生理病理过程密切相关的活性酶等生物标志物的实时、无损、可视化荧光检测与成像分析新方法新技术是当前生物传感领域重要研究课题。本项目以发展具有大Stokes位移和长波发射的硝基还原酶（NTR）荧光探针为目标，开展了系列NBO型二氟硼类荧光分子的设计合成及其NTR探针的识别与荧光传感研究，通过对NBO前体分子的结构优化和筛选，设计合成了系列具有大Stokes位移和长波发射的HBT/HBO类、NBD类、DFP类等荧光团，在荧光团不同位置引入识别反应单元，筛选制备了具有细胞线粒体靶向性、多功能性的OFF-ON型或比率型的系列荧光探针。利用荧光光谱、紫外可见光谱、核磁共振、质谱等谱学分析方法和激光扫描共聚焦显微活细胞成像分析技术，系统研究了各类探针分子对NTR等重要生物活性分子的识别与荧光传感行为、构效关系、传感机制以及作用体系的影响和调控因素。研究表明了NBO型二氟硼类荧光分子的结构稳定性和溶剂化效应等对构建NTR探针及其荧光传感体系的影响及其应用于含水体系中NTR荧光检测分析存在的局限性。研究揭示了HBT类荧光分子基于快速的激发态分子内质子转移（ESIPT）和分子内电荷传递（ICT）偶合过程形成具有大Stokes位移（>200 nm）的酮式长波发射（>600 nm）荧光响应和调控机制。筛选构建了针对NTR、NQO1酶以及H2O2、生物硫醇、HClO、HSO3-等重要生物标志物的快速、高灵敏、高选择性荧光传感检测体系，实现了对肿瘤细胞中外源和内生NTR等目标分析物的实时-动态-原位荧光成像分析以及对细胞缺氧状态的实时监测。通过本项目研究工作，发展了基于ESIPT-ICT偶合机制的荧光探针分子设计策略，构建了系列针对生物体中NTR等生物活性标志物的高灵敏高选择性OFF-ON荧光传感与成像分析方法，研究阐明了构效关系和作用机制，为进一步发展具有大Stokes 位移和长波发射的有机小分子荧光探针并建立复杂生物样品和生物体中活性酶等重要生物标志物的实时-动态-原位精准分析检测和荧光成像分析提供有效的解决方案和理论依据。