Studies On The Design,Synthesis And Properties Of Biological Thiols Fluorescent Probes

Biological thiols such as cysteine?Cys?,homocysteine?Hcy?and glutathione?GSH?play important roles in transforming and transmitting signal,cell death regulation,protein synthesis,immune system regulation,maintaining physiological and redox balance in organisms,and the abnormal level of them may cause many diseases.Therefore,the development of rapid and accurate detection of biological thiol fluorescent probe has important scientific value and physiological significance.In recent years,fluorescence probes are focusing on researchers with the benefits of high sensitivity,good selectivity,low cost,convenient operation,real-time imaging and other advantages.The reactive fluorescence probes can generate fluorescence signal and achieve the purpose of detection based on specific binding of their own reaction sites and the analytes.In addition,they can be more accurate quantitative analysis owing to the fact that they have the least damage and lower interference to cells,and can accurately discern and mark the samples in organisms.Therefore,the reactive fluorescence probes were widely applied to life science,environmental science,photoelectric information science and other fields.Based on the current research upsurge,three reactive fluorescence probes?CN-NIR?Y-OH-NBD and F-OH-NBD?were designed and synthesized.The fluorescence properties and biological application of them were evaluated by the UV-Vis absorption spectrum,fluorescence spectrum,response time,selectivity,anti-interference ability,applicable pH,detection limit,toxicity and cell imaging.The main contents are as follows:Chapter 1:Literature reviewIn this chapter,the importance of biological thiols detection were elaborated,and the research progress of reactive fluorescent probes in the detection of biological thiols were reviewed.Chapter 2:Studies on the design,synthesis and properties of fluorescence probe CN-NIRIn this chapter,a reactive fluorescence probe CN-NIR was designed and synthesized for the detection of Cys using?E?-2-?aminomethyl?-3-?6-hydroxy-2,3-dihydro-1H-xanthan-4-yl?propene nitrile?CN-OH?as the fluorescence group and acrylate as the recognition group.The excitation wavelength was determined by UV-Vis absorption spectrum,and the reaction activity of the probe was detected by fluorescence spectrum in vitro.Upon addition of Cys,the fluorescent intensity was increased gradually,and the fluorescence intensity versus concentration of Cys gave an excellent linear relationship?R²=0.991?,along with significant changes in visible color?pink to purple?.According to 3?/k,the detection limit of CN-NIR for Cys was calculated to be as low as 1.03?M,which is lower than the normal content in cells?30-200?M?.Most importantly,the probe showed low cytotoxicity and applied to image intracellular Cys in HeLa cells.Chapter 3:Studies on the design,synthesis and properties of fluorescence probe Y-OH-NBDIn this chapter,we synthesized a fluorescence probe Y-OH-NBD which employed 4-?4-hydroxystyryl?-1-methylpyridinium iodide?Y-OH?as the fluorescence group and NBD as the response group.The performance of the probe was evaluated by UV-Vis absorption spectrum and fluorescence spectrum.After adding Cys,the fluorescence intensity increased gradually as the concentrations of cysteine increased,there was a good linear relationship?R²=0.990?between the fluorescence intensity and the Cys concentrations,which indicated that the probe could be used for detecting Cys qualitatively and quantitatively.The probe showed a rapid response?within 5 min?and low detection limit?1.88?M?to Cys.Most importantly,the problem of probe consumption caused by GSH was resolved.More broadly,the probe showed low cytotoxicity and can be conveniently applied to image intracellular Cys in living cells,indicating it is a promising tool for biological applications.Chapter 4:Studies on the design,synthesis and properties of fluorescence probe F-OH-NBDIn this chapter,we designed and synthesized a double emission channel fluorescence probe F-OH-NBD which used 1-?3-cyano-2-dicyanomethylen-5,5-dimethyl-2,5-dihydrofura n-4-yl?-2-?4-hydroxylphenyl?ethane?F-OH?as the fluorescent group and NBD as the response group.Based on the intramolecular charge transfer and aromatic nucleophilic substitution rearrangement,F-OH-NBD showed to be a selective turn-on fluorescence probe for Cys and GSH.And then,the structure of the probe F-OH-NBD was characterized by ¹H NMR,¹³CNMR and MS.Moreover,the fluorescence properties of the probe were studied by fluorescence spectrum.After adding Cys/Hcy,the fluorescent group F-OH was released through the aromatic nucleophilic substitution reaction,and a new fluorescent group was produced of NBD combined with Cys/Hcy by the intramolecular rearrangement.While after adding GSH,fluorescent group F-OH was observed only.The properties of F-OH-NBD can be used for detecting and distinguishing biological thiols by different fluorescence signals.In addition,the probe detected Cys and GSH with a rapid response time?Cys was 5 min and GSH was 10 min?and low detection limit.Most importantly,the problem of probe consumption caused by GSH was resolved.Moreover,the probe showed admirable imaging capability in living cells with low cytotoxicity.