The Synthesis And Characterization Of New Fluorescent Calcium Indicator Intermediates

Calcium is one of the most abundant elements in the human body. It plays an important role in regulating numerous physiological phenomena and serves as an intracellular second messenger and mediator. The measurement of cytosolic Ca²⁺ has become an important area of investigation in chemical, biological and medical research. Nowadays, the method of the measurement of cytosolic Ca²⁺is according to Tsien et al. There are several cytosolic Ca²⁺ indicators, such as Quin-2, Fluo-3. They have made cotributions and at the same time they have some drawbacks. In this paper, we try to design a novel cytosolic Ca²⁺ fluorescent probe with low affinity to calcium long and excitation wavelength. It should be noted that the optical properties of carbopyronine are very similar to those of the rhodamines. However, absorption and fluorescence are shifted remarkably towards longer wavelengths. Thus, a novel cytosolic Ca²⁺ fluorescent probe was designe.The dissertation is divided into three parts.1. Design and synthetic strategy of novel cytoplasmic calcium fluorescent probeThe thesis have summerized the properties and applications of some reported calcium fluorescent probe in view of the references. Then the research object has been presented. The structure and the optical properties of Carbopyronine is unique. Noteably, Carbopyronine fluorescence dye is hardly studied. In this paper, a novel method of synthesis of Carbopyronine fluorescence dye has been developed. At the same time, the synthetic route to the target compound is designed.2. Synthesis the intermediates of novel calcium fluorescent indicatorThrough a series of reactions, such as the reaction of esterification, amino-protected reaction, addtion of Grignard reagent, cycloreaction in the presence of non-proton solvent, an so on, more than twenty intermediates have been obtained, for example, N-Fomc-methyl-m-aminobenzoate,2,7-Bis(dimethylamino)-9,10-dihydro- 9, 9-dimehtylanthracene, ect. Several reaction conditions have been optimized and the results of the experiment laid solid theoretical foundation for further research work. 3. Study on the properties of novel calcium fluorescent indicator and some reaction condtions. Protection of amino group is important and frequently needed exercise in synthesis of the target compound. The properties and reaction conditions of four protection reagents, such as di-tert-butyl dicarbonate, benzyl bromide, are studied and the optimal protection reagent is located. Also, the protection mechanism is proposed.The bridged reaction and the cycloreaction also have been fully studied. It was found that the reported methods have some drawbacks, such as have long reaction route and complex reaction process. In order to solve the problems, we developed an efficient and practical method for the synthesis of the compound in the presence of non-protic Lewis acid. Mild conditions in a high yield were achieved. This research results have important directive to further research. To the best of our knowledge, it is the first time that the structure of 2-m- Dimethylaminophenylpropan-2-ol 3 and methylene-bridged alcohol 8 are confirmed by X-ray single crystal diffraction. The crystal data and structure refinement, some bond lengths and angles, and atomic coordinates and equivalent isotropic displacement parameters of 3 and 8 were disccussed. The results suggest that the structure and the synthetic route are correct. This paper is of certain directive significance to further study. This research is a good foundation for the synthesiz of Carbopyronine fluorescent indicator.