Study Of The Synthesis Of Electrophilic Substituted Fluorogenic Probes And Their Applications In Bioanalysis

Most of the biological molecules are nonfluorescent or weakly fluorescent compounds, Analytes are tagged with fluorescent tagging reagents and generated with covalent or non-covalent binding material which have high fluorescence. The detection limit is lowered. The fluorescent method is widely used in analytical chemistry, especially in biological analysis. So much attention has been attracted to study proteins, nucleic acids, metal ions and free radicals in biological systems.Intracellular thiols such as glutathione(GSH), cysteine(Cys) and homocysteine(Hcy) play an important role in maintaining balance between their oxidizing and reducing functions. Thiols deficiency in blood are associated with AIDS, heart disease, Alzheimer's and cardiovascular disease. The glutathione S-transferases (GSTs) are phaseâ…¡detoxification enzymes that catalyze the nucleophilic attack of glutathione (GSH) to a wide range of hydrophobic electrophilic compounds, so as to achieve detoxification purpose. Fluorimetric analysis have many advantages such as high sensitivity, high selectivity, real-time detection. In recent years, the detection of biological thiols based on fluorescence techniques has received much attention. In this paper, coumarin and fluorescein-based fluorescent probes which have high selectivity for thiols and GST were designed and synthesized. This dissertation consists of five chapters summarized as follows:In chapter 1, the research progress on fluorescent probes containing electrophilic moiety for thiols and GST were summarized. Based on the literatures reported, the objective of this dissertation was proposed.In chapter 2, a 4-methylumbelliferyl-based thiols-selective fluorescent probe was designed and synthesized. The fluorescent probe is a nonfluorescent compound and was synthesized via the one-step reaction of 4-methylumbelliferone (4-MU) with 1-chlorine-2,4, 6-trinitrobenzene. Upon mixing with biothiols under neutral aqueous conditions, the 2,4, 6-trinitrophenyl group of compound 1 is efficiently removed and the emissive free dye 4-MU was released, hence leading to a dramatic increase in fluorescence emission of the reaction mixture. Based on the above mechanism, a fluorogenic method for the determination of biothiols in human serum samples was developed.In chapter 3, a fluorescein-based fluorescent probe 3 for thiols was designed and synthesized. In this part, compound 3, compound 4 or compound 5 are weakly fluorescent compounds and were synthesized via the reaction of fluorescein with 1-chlorine-2,4, 6-trinitrobenzene, 1-chlorine-2,4, dinitrobenzene and 1-chlorine-4-nitrobenzene, respectively. Upon mixing with biothiols under neutral aqueous conditions, the 2,4,6-trinitrophenyl group of probe 3 was efficiently removed and results in the release of fluorescein, hence leading to a dramatic increase in fluorescence emission of the reaction mixture. Based on the above mechanism, a fluorogenic method for the determination of biothiols was developed. In addition, the reactivity of compound 3 and 4 toward thiols were tested, and it was observed that compound 4' reactivity toward thiols was much lower than that of 3, whereas for compound 5, it exhibits no response toward thiols. The above results indicate that the reactivity of the fluorescent probe can be rationally modulated.In chapter 4, a fluorescein-based fluorescent probe 4 for Glutathione S-transferase(GST) was designed and synthesized. In chapter 3 compound 4'reactivity toward thiols was studied and its shows lower reactivity toward thiols. However, under the catalysis of the GST, the nucleophilic capabilities of reduced glutathione was significantly enhanced. Upon introducing GST to the mixture of GSH and 4 under neutral aqueous conditions, the 2,4-dinitrophenyl group of probe 4 was efficiently removed and fluorescein was released, hence leading to a dramatic increase in fluorescence emission of the reaction mixture. Based on the above mechanism, a fluorogenic method for the determination of GST was developed.Apart from the above work, a new optical chemosensor for Zn2+ was designed and synthesized, details as follows:In chapter 5, a coumarin-based fluorescent chemosensor 6 for Zn2+ was designed and synthesized. Compound 6 exhibits lower background fluorescence due to intramolecular photoinduced electron transfer. However, upon mixing with Zn2+ in 30%(v/v)aqueous ethanol, a "turn-on" fluorescence emission is observed due to the formation of a 1:1 complex 6-Zn2+, and hence the intramolecular photoinduced electron transfer of compound 6 is alleviated. Based on the above mechanism, a fluorogenic method for the determination of Zn2+was developed. This probe is highly selective for Zn2+ over other metal ions.