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

首先，合成了各种类型的二硫代氨基甲酸铁(II)，并考察了它们对NO检测的灵敏度。这些实验提出了二硫代氨基甲酸-铁(II)型络合物在NO检测中的结构与灵敏度关系。然后，利用二硫代氨基甲酸-铁(II)络合物和稳定的有机自由基Tempoll发现了一个新的无检测的概念--自由基交换。在这个体系中，NO结合到中心铁的络合物中释放出坦普尔，这是一种稳定的有机自由基。在该体系中，NO结合到络合物中的中心铁来释放Temol自由基，可以高灵敏度地检测到它。该分子体系检测NO的灵敏度是普通二硫代氨基甲酸铁(II)型非自旋捕捉器的25倍。作为下一步，我们应用这一新策略制作了NO的荧光探针。因此，我们利用二硫代氨基甲酸-铁(II)络合物和吖啶标记的TEMPO构建了一种新型的无荧光探针。该探针可以监测自发NO释放剂产生的NO，它利用了由于NO释放吖啶-TEMPO而导致的吖啶的荧光减弱。最后，我们设计并合成了一个双荧光标记的探针体系，它与香豆碱标记的Cyclam和荧光胺标记的TEMPO形成铁(II)络合物。当香豆碱由于荧光能量转移而被激发时，该探针给出了基于荧光胺的荧光。在这个体系中，没有结合的铁(II)能释放出荧光胺-Tempo。释放的荧光胺-TEMPO中的荧光被新产生的TEMPO自由基猝灭。这取消了荧光能量的转移，从而增加了香豆素的荧光强度。因此，该探针可以使用比率计量法独立于生物样品中的背景荧光来监测NO的产生。毕竟，我们已经建立了成功设计高灵敏度NO探头的基本概念。

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.