The New <sup> Of Ca 2 + </ Sup> Fluorescent Probe Design, Synthesis And Intracellular Messenger Role Of Research

Ca²⁺ acts as a universal messenger in a variety of cells. Numerous functions of all types of cells are regulated by calcium to a greater or lesser degree. Serving as an intracellular second messenger and mediator,Ca²⁺ plays a very important role in intracellular signal transduction as well as extracellular. Consequently, the measurement of cytosolic Ca²⁺ has become an important investigation area in chemical, biological and medical research.Fluorescent indicators with the structure of EGTA,BAPTA were developed by Tsien and his colleagues in the 1980's for cytosolic Ca²⁺ detection. Fluorescence probes, which are sensitive, fast-responding and capable of affording high spatial resolution via microscopic imaging, provide a new method in the research of intracellular calcium. Therefore, the design and synthesis of new fluorescence probes for cytosolic Ca²⁺ have arisen much more attention. Among the fluorescent Ca²⁺ probes synthesized, fluorescein-based calcium fluorescent probes and fluorescent probes with an indole structure, which are excitated by visible light and UV light respectively, have been widely used. Nevertheless, the fluorescence probes for Ca²⁺ which can be used widely and commercially are quite limited nowadays.In order to test the Ca²⁺ in different concentration, improve the biological affinity to Ca²⁺and the fluorescence intensity, the investigations on the design, synthesis and function test of new fluorescence probes for Ca²⁺ are carried out, mainly including the following several aspects:1.According to the analysis of the structure of BAPTA,five kinds of novel derivates of BAPTA were synthesized successfully. Furthermore, the structures of these compounds were all confirmed.2.Have designed and synthesized three novel fluorescein-based calcium fluorescent probes. The methyl ester forms of the new probes were confirmed. The fluorescent spectrum of the novel compounds were reported.3.Have designed and synthesized five novel fluorescein-based calcium fluorescent probes linked chlorine. The methyl ester forms of the new probes were confirmed. The fluorescent spectrum of the novel compounds were reported. Simultaneously, very useful results about designing of the new probes were obtained from theoretic calculation. 4.Have designed and synthesized five novel calcium fluorescent probes with an indole structure.The methyl ester forms of the new probes were confirmed. The fluorescent spectrum of the novel compounds were reported.5.The fluorescent spectrum of the novel fluorescein-based calcium fluorescent probe Fluo-3M were investigated. The experimental result shows that the fluorescence intensity enhanced obviously after bonded with Ca²⁺.The fluorescence excitation and emission wavelength maxima for Fluo-3M are nearly the same as those reported for Fluo-3. Thus, fluorescence microscopy applications are applicable for detection of Fluo-3M as well.6.Have calculated the K_d of the novel fluorescent probe Fluo-3M. The data shows that Fluo-3M has a relatively high affinity to calcium. It can be used to measure the Ca²⁺ distributed near the membrane of the cell.7.Fluo-3M was injected into HEK-293 cells and CHO cells, the Olympusâ…¨71 fluorescent microscope was used for detecting the intracellular calcium and the imaging of the cell. The experimental results show that Fluo-3M which has a high fluorescence intensity and biological affinity can be used in vivo.