Design and synthesis of visualization sensor molecules for bio-imaging

激酶和磷酸酶通过各种生物分子的磷酸化和去磷酸化来调节信号转导。激酶和磷酸酶活性的检测技术对于阐明信号转导机制至关重要。荧光比率成像是一种灵敏、非破坏性和准确的方法，用于检测活细胞内的这种酶反应。在本研究中，我们设计了一种荧光探针，它可以特异性地与磷酸酶反应，改变磷酸酶的荧光特性。（1）探针应该具有开关功能，通过该开关功能，探针的激发光谱在与磷酸酶反应时改变，以及（2）探针中的反应位点的结构可以容易地改变，这样探针就能特异性地与另一种磷酸酶反应，而这种磷酸酶能识别不稳定位点的独特结构。首先，我们选择7-羟基香豆素作为 关于我们 探针的基本结构。重要的是，我们的初步实验表明，羟基的pK_a是由空间位置接近羟基的阴离子基团控制的，并影响化合物的激发光谱。因此，我们通过脂肪族酯键将磷酸基团引入羟基的近端位置，使得磷酸酶去除磷酸基团导致羟基的pKa改变，从而导致探针的激发光谱改变。此外，脂肪族连接基允许修饰接近磷酸基团的结构。结果表明，该探针能与酸性磷酸酶发生特异性反应，改变了酸性磷酸酶的激发光谱。此外，激发光谱的变化大到足以进行比率测量。这些结果为荧光比率探针的设计提供了有价值的信息，我们的策略在生物成像领域具有很大的应用潜力。少

Kinases and phosphatases regulate signal transduction through phosphorylation and dephosphorylation of various biological molecules. The techniques for detecting the activity of kinases and phosphatases are essential to the elucidation of signal transduction mechanism. The fluorescence ratio imaging is a sensitive, undestructive and accurate method for the detection of such enzymatic reactions inside living calls. In this research, we created fluorescent probes, which specifically react with phosphatase and change its fluorescence properties.Our design strategy is based on the following requirements; (1) The probes should have a switch function, by which the excitation spectrum of the probe changes upon the reaction with phosphatase, and (2) the structure of the reactive site in the probe can be easily modified, so that the probe specifically reacts with a different kind of phosphatase, which recognizes a distinctive structure of the restive site. First, we chose 7-hydr xycoumarin as … More a basic structure of the probe. Importantly, our preliminary experiments show that the pK_a of the hydroxyl group is controlled by the anionic group sterically close to the hydroxyl group and affects excitation spectrum of the compound. We, therefore, introduced phosphate group into the proximal position of the hydroxyl group via the aliphatic ester linkage, so that removal of the phosphate group by phosphatase results in the alteration of pKa of the hydroxyl group, leading to the change of the excitation spectrum of the probe. In addition, the aliphatic linker allows modification of the structure close to the phosphate group. It was demonstrated that the probe specifically reacted with acid phosphatase and changed its excitation spectrum. Furthermore, the change of the excitation spectrum was large enough to conduct ratio measurements. These results provides valuable information about design of fluorescent ratio probes and our strategy has great potential toward application in the field of bio-imaging. Less