Study Of The Synthesis Of α,β-Unsaturated Ester Substituted Fluorogenic Probes And Their Applications In Bioanalysis

The conjugate 1,4-addition of thiols to a, (3-unsaturated ketones to form thioethers is a key reaction in organic synthesis. The reaction is normally performed under drastic conditions, such as with bases or Lewis acids as catalysts, at elevated temperature, or with the use of organic solvents. However, Chakraborti and coworkers recently reported that this 1,4-addition could be conducted in water at room temperature without the use of any catalyst. Encouraged by these biologically compatible conditions, we envisioned that the 1,4-addition of thiols toα,β-unsaturated ketones could be employed as a platform for the construction of fluorescent thiol probes that may be potentially useful for thiol detection at physiological pH conditions. We developed a new fluorescent thiol probe based on the conjugate addition of thiols toα,β-unsaturated esters. The probe 1 and 3 were synthesized via the one-step reaction of HPBI or fluorescein with acryloyl chloride and their displayed high selectivity toward thiols in neutral buffer solutions. In probe 1 and 3, the a, P-unsaturated carbonyl moiety acted as a quencher of fluorophores'fluorescence. The chemosensing of fluorogenic probe 1 and 3 involved the conjugate addition of thiols toα,β-unsaturated carbonyl moiety to generate the correspondingβ-sulfido carbonyl adduct, thus eliminating the photoinduced electron transfer (PET) quenching of the fluorophore by the intramolecular carbon-carbon double bond and leading to a dramatic increase in fluorescence intensity simultaneously. The proposed method proves to be sensitive and selective and has been successfully used to detect biothiols in human urine samples.In chapter 1, several new fluorescent thiol probes based on the conjugate addition of thiols toα,β-unsaturated ketones were introduced, and the purposed of the present thesis was presented.In chapter 2, a sensitive fluorogenic probe 1 for biothiols was developed based on The Michael addition reaction. The probe 1 was readily synthesized via the reaction of 2-(4-hydroxyphenyl) benzimidazole (HPBI) with acryloyl chloride and shows weak fluorescence emission. Upon mixing with biothiols, the fluorescence of 1 is significantly enhanced due to the conjugate addition of thiols to the a,b-unsaturated carbonyl moiety, thus eliminating the photoinduced electron transfer (PET) quenching of the fluorophore by the intramolecular carbon-carbon double bond. Cysteine (Cys) was selected as the representative thiol in the spectral experiment. A good linear relationship was obtained from 1.0 to 30.0 mmol L-1 for Cys and the detection limit was 0.17 mmol L-L Furthermore, probe 1 was highly selective for biothiols without the interference of some biologically relevant analytes and has been applied to detecting biothiols in human urine samples.In chapter 3, a fluorescein-based fluorescent probe 3 for thiols was designed and synthesized. In this part, compound 3, compound 4 are weakly fluorescent compounds and were synthesized via the reaction of fluorescein with acryloyl chloride and cinnamoyl chloride, respectively. Upon mixing with biothiols under neutral aqueous conditions, the fluorescence of 3 is significantly enhanced due to the conjugate addition of thiols to theα,β-unsaturated carbonyl moiety, 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. As for compound 4, the addition of thiol toα,β-unsaturated carbonyl moiety of the probe is relatively difficult because of steric hindrance effect, which provides us some empirical rules on developing thiol-selective probes.In chapter 4, a fluorescein-based fluorescent probe 4 for thiophenol was designed and synthesized. In chapter 3, the reactivity of compound 4 toward thiols was studied and its shows lower reactivity toward thiols. Such as cysteine, homocysteine and glutathione. However, Upon mixing with thiophenol under neutral aqueous conditions, the fluorescence of 4 is significantly enhanced due to the conjugate addition of thiophenol to theα,β-unsaturated carbonyl moiety. It proved that the nucleophilic capability of the thiophenol was significantly higher than the others. Based on the above mechanism, a fluorogenic method for the determination of thiophenol was developed.