Synthesis And Application Of Fluorescent Probes Specific For Sulfite And Cysteine ​​/ Homocysteine ​​Detection

Small biological reactivce molecules including vitamin and inorganic salts (e.g. sulfite), amino acids (e.g. cysteine, homocysteine), hormone, etc. are an important part of homeostasis, which is known to play crucial roles in many physiological and pathological processes. More importantly, many studies have shown that these reactive species are interrelated in biological systems. However, the abnormal alteration of certain material contents may lead to serious diseases. Therefore, it is of great importance to develop techniques for sensing and imaging these species in live cells or tissues with simplicity, high sensitivity, and suitability.Fluorescence analysis has attracted much attention because of its good selectivity and high sensitivity. Currently, various florescent probes for small bioactive molecules have been reported in the literature, and this research area has attracted the attention of many researchers. Herein, two fluorescent probes for sulfite and cysteine/homocystein were designed and synthesized and their sensing behaviors were studies in detail.This thesis is divided into three chapters as follows:The first chapter summarized the research of the fluorescence analysis for detecting sulfite and Cys/Hcy. Based on the relevant research, the objective of this dissertation was proposed.In chapter 2, a ratiometric fluorescent probe for SO32- was designed and synthesized. We integrate the π-conjugation interruption reaction and the "quinone-phenol" transduction-activated ESIPT within a benzothiazole-containing rhodol derivative (1), and developed a ratiometric fluorescent probe for sulfite. Upon treating with sulfite, the π-conjugation system of 1 was interrupted and the "quinone-phenol" transduction occurred simultaneously, which leads to the ESIPT process between the phenolic OH and benzothiazole unit upon photoexcitation. The above conversions result in the decrease of the emission band at 578 nm along with a concomitant increase of a new fluorescence peak at 453 nm. The fluorescent intensity (I453/I578) increases linearly with sulfite concentration up to 8 μM with a detection of 0.22 μM (38). The proposed probe shows excellent selectivity toward sulfite over other common anions and nucleophiles. Moreover, the cellular imaging experiment indicated probe 1 possesses low cytotoxicity and desirable cell permeability for biological applications.In chapter 3, a fluorescein-based fluorescent probe for Cys/Hcy was designed and synthesized. The probe 2 was synthesized via the condensation of fluorescein and 2-chloro-3,5-dinitrobenzoic acid. The free probe 2 gives weakly emission buffered at pH 7.4 (phosphate buffer,20 mM,40% acetonitrile). However, upon treating with Cys/Hcy, the nucleophilic substitution between the -SH of Cys and the Ar-Cl of probe 2 occurs to give the corresponding thioether adduct. Then, the amino group of the thiother adduct would attack the C-S bond within the molecule and generated the thiol-containing product via intramolecular exchange reaction, and -SH will lead to the cleavage of the adjacent ester bond to release free fluorescein, which leads to the absorption and fluorescence intensity of reaction system increase dramaticlly. In the above sensing process, both the -SH and -NH2 of Cys/Hcy are involved. Thus, probe 2 exhibits excellent selectivity toward Cys/Hcy over other biological species. The limit detection of Cys was determined to be 5x10-7 M. The emission intensity of probe 2 was linearly proportional to Cys/Hcy concentrations in the range of 5-60 μM. Probe 2 has been applied for the detection of Cys content in fetal bovine serum samples.