Design,Synthesis And Biological Application Of Fluorescent Probes For Protein Thiols/Disulfides

Protein thiols are an important part of biological thiols,which play an essential role in maintaining the structures and functions of proteins.Cysteine residues in proteins exhibit unique reactivity and spatial arrangement,and different redox states of protein thiols(such as protein thiols,protein vicinal dithiols(PVD)and protein disulfides)also directly affect the redox homeostasis of organisms.Meanwhile,with the development of fluorescence imaging technology in the fields of chemical biology,clinical diagnosis and drug discovery,fluorescent probes have been paid more attentions as tools with excellent sensitivity,simple operation and high biocompatibility.In order to specifically detect different redox states of protein thiols,we designed and synthesized some novel fluorescent probes in this dissertation.With these probes and a variety of experimental methods,we realised the fluorescence detection of protein thiols under physiological or pathological conditions,and these probes would be more efficient and convenient tools in the subsequent biological studies.The contents of this dissertation are as follows:Chapter 1.The redox changes of protein thiols in organisms and the mechanisms and types of environment-sensitive fluorophores were introduced.Then common methods and fluorescent probes or alkylation/rebridging reagents of detecting reduced or oxidized states of protein thiols were summarized.Chapter 2.We reported a red-emitting and environment-sensitive probe(FM-red)for detecting and labeling protein thiols.The emission signal of the probe was exclusively switched on by binding to protein sulfhydryl groups through the twisted intramolecular charge transfer mechanism,while negligible fluorescence was observed when FM-red reacted with low molecular weight thiols.The fast response and red-emitting character of the probe enable it to image protein thiols in live cells and in vivo.Moreover,this probe was applied to determine the redox states of thioredoxin as a mass tag,and disclosed that a significant decrease of protein thiols and accumulation of oxidized thioredoxin in a cellular model of Parkinson's disease.Chapter 3.We developed a turn-on monoarsenical fluorescent probe(NEP)based on the intramolecular charge transfer mechanism.Naphthalimide was chosen as a fluorophore and linked with cyclic dithiarsolane via carbamate segment.In the presence of PVD,NEP displays a strong green fluorescence signal produced by the cyclic dithiarsolane cleavage and subsequent intramolecular cyclization to liberate fluorophore.Furthermore,NEP exhibits high selectivity toward PVD and fluorescence enhancement.The favorable properties of NEP enable it readily to detect PVD in live cells and in vivo.In addition,a remarkable decrease of PVD in parkinsonism was disclosed for the first time.Chapter 4.Based on the thiols/disulfides exchange reaction,a series of potential fluorescent probes for detecting protein disulfides was designed and synthesized by connecting mercaptoethylamine with multiple environment-sensitive fluorophores.Through spectroscopic tests,S6 was screened for detecting protein disulfides and exhibited strong specificity and high fluorescence signal increment,while negligible fluorescence was observed when S6 reacted with low molecular weight disulfides.The probe S6 was covalently binded to the protein through the formation of new disulfide bonds after the exchange of its sulfydryl and protein disulfides.Moreover,probe S6 was used to label protein disulfides.Furthermore,reversible fluorescence imaging of protein disulfids was realized in live cells by probe S6.Chapter 5.Based on the aggregation-induced emission(AIE)mechanism and above-mentioned works,using tetraphenylethylene as the fluorophore,two novel fluorescent probes TPE-PAO-EDT and TPE-SH were designed and synthesized for detecting PVD and protein disulfides,respectively.The two probes exhibited typical AIE properties and realized fluorescence detection and labeling of PVD and protein disulfides,which would provid guidances for the development of tools with well performance and wide application.Chapter 6.We summarized the research work,and put forward the prospect of detecting protein sulfhydryl groups in the future.