一氧化氮荧光探针的构建及其生物细胞成像应用

Nitric oxide is considered as one of the three endogenous gaseous signaling compound (the others are hydrogen sulfide and carbon monoxide).Although the half-life of nitric oxide is only several seconds at low concentrations in biological systems ,it plays important roles in biologicl systems. It is involved in many critical biological processes including signal transduction, smooth muscle relaxation, peristalsis, immune system control, neurotransmission, blood pressure modulation, learning, and memory.However,misregulation of nitric oxide production is implicated with various diseases ranging from stroke, heart disease,hypertension, neurodegeneration, erectile dysfunction, to gastrointestinal distress. Although a wide variety of techniques have been developed to detect nitric oxide, ?uorescence sensing has become the gold standard due to its high sensitivity and simplicity. Thus, the construction of small-molecule ?uorescent probes suitable for speci?c nitric oxide detection in living systems has received great attention.Based on the literatures, we designed series of reaction based nitric oxide fluorescent probes for the imaging of endogenously produced nitric oxide in cells. 1. Based on the intramolecular charge-transfer and fluorescence resonance energy transfer sensing mechanisms,fluorescence ratiometric probes for nitric oxide are designed. 2.Targetable fluorescent probes for imaging nitric oxide in organelles of living cells are designed. 3. Probes with near-infrared emission for nitric oxide detection were designed based on BODIPY fluorophore, which are more suitable for imaging nitric oxide in living cells compared with the traditional probes with emission wavelength located in visable region. 4.A new fluorescent dye with emission wavelength located in near-infrared region was explored and applicated for the design of new fluorescent probes for nitric oxide sensing.

一氧化氮是除一氧化碳和硫化氢之外的另一生物气体信使分子，在生命活动中扮演了重要的角色，对一氧化氮的选择性识别与传感在环境监测、疾病诊断等方面具有重要的意义。在各种识别和传感方法中，由于荧光法具有高灵敏度、设备简单、即时监测的优点，因而被化学家和生物学家广泛采用。本项目设计了基于一氧化氮独特化学反应性的荧光探针，拟在一氧化氮传感和生物细胞成像方面展开研究。本项目的创新点主要体现在四个方面：1.设计了基于分子内电荷转移机理以及荧光共振能量转移机理调控的荧光比率一氧化氮探针；2.设计包含有靶向细胞位点的荧光探针，实现细胞器内一氧化氮的定点检测；3. 开发出基于BODIPY荧光团的近红外一氧化氮荧光探针，使其更适合于生物学应用； 4.开发出基于螺环"开-关"原理调控的具有比率性质的近红外荧光染料，并将其应用于一氧化氮荧光探针的设计。

一氧化氮(NO)是除一氧化碳和硫化氢之外的另一生物气体信使分子，在生命活动中扮演了重要的角色，对一氧化氮的选择性识别与传感在环境监测、疾病诊断等方面具有重要的意义。在各种识别和传感方法中，由于荧光法具有高灵敏度、可视化、非侵入性的优点，因而被化学家和生物学家广泛采用。在过去的十多年间，尽管有许多一氧化氮荧光探针被报道，然而构建能够快速响应一氧化氮同时兼具高选择性、高灵敏度的荧光探针仍然是该研究领域的难点之一。针对一氧化氮荧光探针领域存在的科学问题和挑战，本项目设计合成了系列基于一氧化氮特异性化学反应的荧光探针，并在一氧化氮传感和生物细胞成像方面展开研究。具体包括如下工作：（1）利用BODIPY为母体荧光团，2-氨基-3‘-二甲氨基联苯作为识别单元设计合成了基于光诱导电子转移（PET）机理的一氧化氮荧光探针Bod-1，并将其应用到近红外荧光探针的开发,合成了Bod-2，Bod-3。探针Bod-1可以高选择性识别传感一氧化氮，检测限达到纳摩尔级别，探针对生物细胞具有良好的兼容性，能够用于影像细胞内的一氧化氮。（2）首次利用富电子芳香二级胺的亚硝化反应结合光诱导电子转移机理设计合成了基于BODIPY荧光团的新型一氧化氮荧光探针Bod-4，该探针主要由一氧化氮的的识别单元N-苄基-4-羟基苯胺与荧光信号单元BODIPY组成，对一氧化氮展现出快速的 “关-开”荧光响应变化，同时具有极高的灵敏性和选择性。进一步，以探针Bod-4为基础，通过向其分子骨架中引入三苯基膦阳离子基团合成出具有靶向线粒体功能的荧光探针Bod-5。探针Bod-5不仅展现出优良的选择性和灵敏性，而且还具有靶向细胞线粒体特性，结合激光共聚焦显微镜可实现对RAW264.7细胞线粒体内源一氧化氮的实时监测与影像，并且能够监测内皮细胞氧糖剥夺（OGD）条件下内源性一氧化氮的产生。（3）将吡罗红染料与邻苯二胺基团结合，构建出具有靶向线粒体功能的一氧化氮荧光探针Rod-1,探针同样体现出良好的选择性和高的灵敏性。更有趣的是，探针与一氧化氮作用后的产物能够进一步与半胱氨酸以及谷胱甘肽作用，分别显示出绿色荧光和近红外荧光。基于上述实验结论，探针Rod-1可以通过双荧光通道实现对生物细胞内一氧化氮的检测与影像。本项目的实施将为深层次研究NO的生理病理功能奠定重要的化学基础。