Design, Synthesis Of Novel Fluorescein-Based Cytoplasmic Calcium Fluorescent Probes And Their Applications In Signal Transduction

Calcium 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.Calcium serves as an intracellular second messenger and mediator which play a very important role in intracellular signal transduction as well as in extracellular signal transduction. Consequently,the measurement of cytosolic Ca²⁺has become an important area of investigation in chemical,biological and medical research;it also arise attention of organic synthesis chemist.Fluorescence probes are excellent sensors for biomolecules, being sensitive,fast-responding and capable of affording high spatial resolution via microscopic imaging.The use of fluorescence probes in the measurement of cytosolic Ca²⁺provides a new method in the research of intracellular calcium.Fluorescein derivatives are prized for their high extinction coefficient,long excitation and emission wavelength,high fluorescence quantum yield and low cytotoxicity.Their fluorescence characteristics are tailored for a certain application. Recently,many fluorescein-based fluorescence probes have been developed.They have been used in the research of a variety of protein,nucleic acid,cell,and immunology. Fluorescent calcium probes are developed for cytosolic Ca²⁺detection in recent years. Among the fluorescent calcium probes,fluorescein-based calcium fluorescent probes have been widely used.Most fluorescent calcium probes have the advantage of long excitation wavelength but share a drawback in their high affinity to calcium(low constant of K_d).This kind of fluorescent calcium probe is prone to be overloaded,which has a deficiency in the measurement of cytosolic calcium.In this thesis,a series of novel fluorescein-based calcium fluorescent probes have been designed and synthesized.One of the probes was investigated for its application in intracellular signal transduction.1.The popular used fluorescent probe Fluo-3 was synthesized commercially.The synthesis conditions were studied and optimized.By the same method,Fluo-4 and Fluo-2 were synthesized successfully.The structure of these compounds was determined by ¹HNMR,elemental analysis and MS.2.Five kinds of novel fluorescein-based calcium fluorescent probes have been designed.Three intermediates of calcium fluorescent probes were synthesized.It provides a novel method for synthesis of calcium fluorescent probes.3.Five novel fluorescein-based calcium fluorescent probes were synthesized.The structures of all these compounds were confirmed.The fluorescent spectrum and the calcium titration curve of the five novel compounds were reported for the first time.The synthesis of fluorescein-based calcium fluorescent probes was researched.4.The calcium binding affinity of two novel fluorescein-based calcium fluorescent probes has been calculated.The experiment shows that these two probes have a low affinity to calcium,which avoids the disadvantages of high calcium binding affinity probes,and are very useful for high concentration of calcium.5.One of the novel probes Fluo-2FFM was transfected into Chinese Hamster Ovary(CHO)cells,the Olympusâ…¨71 fluorescent microscope was used for detecting the intracellular calcium and the imaging of the cell.It was found that the novel fluorescein-based calcium fluorescent probes are excellent green fluorescent probes that are less of cytotoxicity and can be used in vivo.