Design, Synthesis And Imaging Study Of Fluorescent Probes For Active Species H₂S_n And ¹O₂

Hydrogen polysulfide（H₂S_n,n>1）is an important active sulfur molecule in organisms,mainly produced by 3-thiopyruvate catalyzed by 3-mercaptothiopyruvate thiotransferase,and easily combined with cysteine residues to produce protein persulfide.Normal levels of H₂S_nhas multiple biological functions.However,excess H₂S_nalso appears to be involved in a variety of diseases,including cancer,ethylmalachic acid encephalopathy,Parkinson’s disease and Huntington’s disease.Singlet oxygen（¹O₂）is a special reactive oxygen species that plays an important role in a variety of physical,chemical,biological and medical events.But excess¹O₂could diffuse and oxidize biomolecules such as proteins,lipids and DNA,thereby triggering chronic diseases such as aging,cardiovascular disease and cancer.Given the importance of H₂S_nand ¹O₂,it is of great interest to develop methods to visualize them in vivo.Small molecule fluorescent probes have become a powerful imaging tool due to their low cost,simple operation,high selectivity,high sensitivity and non-invasiveness.In this paper,two small molecule fluorescent probes,NR-BS4 and BPC,were designed and synthesized based on Nile Red scaffold to detect H₂S_nand ¹O₂in vivo,respectively.The paper is divided into three chapters,as follows:Chapter one:introduction.Firstly,the composition,design principle and luminescence mechanism of fluorescent probe are introduced.Secondly,the research progress of fluorescent probes based on Nile Red dye are reviewed.Then,the research status of H₂S_nand ¹O₂fluorescent probes are discussed through the classification of recognition sites.On this basis,the research contents of this paper are briefly described.Chapter two:the design,synthesis and imaging study of H₂S_nfluorescent probes were carried out.Inspired by the recognition group 2,4-dinitrobenzenesulfonyl of biological thiols and considering the strong nucleophilic properties of H₂S_n,this chapter explored the effect of the changes of substituents and their positions on the benzene ring of benzenesulfonyl on the response of biothiols and H₂S_n.A series of fluorescent probes,NR-BS,were constructed using2-or 3-hydroxyl Nile Red as fluorophore scaffold and modified with different benzenesulfonyl derivatives.After the evaluation of fluorescence properties and DFT calculations,the optimized probe NR-BS4 is based on the fluorescence response mechanism of the PET process.In addition to its fast response（<10 min）,high sensitivity（0.14μM）,no interference from biological thiols and wide linear range（0～350μM）,probe NR-BS4 can also function in the pH=4～10 range with good biocompatibility,allowing visualization of endogenous H₂S_nlevels in live cells and zebrafish.Chapter three:a novel ¹O₂fluorescent probe BPC was developed based on 2-hydroxy Nile Red skeleton and Boc-substitution modification.Due to the high reactivity of ¹O₂,there is an obvious fluorescence enhancement phenomenon at 495 nm after adding it to the BPC solution.The spectral properties indicate that the probe BPC has the advantages of high sensitivity（LOD=92 n M）,wide linear range（0～300μM）,high selectivity and large Stokes shift（149nm）.Most importantly,the probe BPC has low cytotoxicity and successfully applied to the selective imaging of PMA-induced endogenous ¹O₂in Hep G2 cells to observe intracellular¹O₂levels.