Development of Novel Molecular Probes of Endogenous Nitric Oxide Detection and Imaging

内源性一氧化氮检测和成像新型分子探针的研制

• 批准号: 12450339
• 负责人: KATAYAMA Yoshiki
• 金额: $ 3.2万
• 依托单位: Kyushu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12450339/
• 关键词: Nitric oxide; NO; Molecular probe; Fluorescent probe; ESR; Spin trapper; スピントラップ剤; スピントラツプ剤; 蛍光分析; 細胞情報伝達; イメージング; 生体分析

Firstly, various types of dithiocarbamate-iron(II) were synthesized and those sensitivities of NO detection were examined. These experiments proposed the structure-sensitivity relationship of dithiocarbamate-iron(II) type complexes in NO detection. Then, a novel concept of NO-detection, radical-exchange, was found using the dithiocarbamate-iron(II) complex and TEMPOL, which is a stable organic radical. In this system, NO bound to the centered iron in the complex to release the TEMPOL, which is a stable organic radical. In this system, NO bound to the centered iron in the complex to release the TEMPOL radical, which can be detected with high-sensitivity. This molecular system could detect NO with 25 times higher sensitivity than that in ordinary dithiocarbamate-iron(II)-type NO-spin traps. As a next stage, we applied this new strategy to make a fluorescent probe of NO. Thus, a dithiocarbamate-iron(II) complex and acridine-labeled TEMPO were used to construct new type of NO-fluorescent probe. This probe could monitor the NO production from a spontaneous NO-releasing agent using the fluorescence decrease of acridine due to the release of the acridine-TEMPO by NO. Finaly, we designed and synthesized a dual fluorescent-labeled probe system using iron (II) complex of a coumaline-labeled cyclam and a fluorescamine-labeled TEMPO. This probe gave the fluorescence based on the fluorescamine when the coumaline was excited due to the fluorescence energy transfer. In this system, NO bound to the iron(II) to release the fluorescamine-TEMPO. The fluorescence in the released fluorescamine-TEMPO was quenched by the newly produced TEMPO radical. This cancelled the fluorescence energy transfer to increase the fluorescence intensity of the coumaline. Thus the probe can monitor the NO production independent on the background fluorescence in biological samples using ratiometry. After all, we have established the basic concept to design a highly sensitive NO probe successfully.

首先，各种类型的二硫代氨基甲酸铁（II）的合成和NO检测的灵敏度进行了检查。这些实验提出了二硫代氨基甲酸铁（II）型配合物在NO检测中的结构-灵敏度关系。然后，一个新的概念NO-检测，自由基交换，发现使用二硫代氨基甲酸铁（II）配合物和TEMPO，这是一个稳定的有机自由基。在该体系中，NO与络合物中心的铁结合，释放出稳定的有机自由基TEMPOL。在该系统中，NO结合到络合物中的中心铁以释放TEMPOL自由基，其可以以高灵敏度检测。该分子系统可以检测NO的灵敏度比普通的二硫代氨基甲酸铁（II）型NO自旋陷阱高25倍。作为下一阶段，我们应用这种新的策略，使NO的荧光探针。因此，二硫代氨基甲酸铁（II）配合物和吖啶标记的克里思被用来构建新型的NO-荧光探针。该探针可以监测NO的产生，从一个自发的NO释放剂使用吖啶的荧光减少，由于由NO释放的吖啶-TEMPO。最后，我们设计和合成了一个双荧光标记的探针系统，使用铁（II）的配合物的香豆碱标记cyclam和荧光胺标记克里思。当由于荧光能量转移而激发香豆素时，该探针基于荧光胺给出荧光。在该系统中，NO与铁（II）结合以释放荧光胺-TEMPO。释放的荧光胺-TEMPO中的荧光被新产生的克里思自由基猝灭。这抵消了荧光能量转移，从而增加了Couponin的荧光强度。因此，该探针可以监测NO的生产独立的背景荧光生物样品中使用比率。总之，我们已经建立了基本的概念，成功地设计了一个高灵敏度的NO探针。