Construction Of Reaction-based Fluoescence Probes For Reactive Sulfur And Reactive Oxygen Species And Their Applications In Biological Imaging

Small molecules such as reactive sulfur and oxygen species in living organisms play an important role in maintaining the redox homeostasis of the organism,and their fluctuating levels are closely related to many physiological health diseases,thus realizing real-time detection of them is of great importance for biological and medical research.Fluorescent probe technology can provide in situ,real-time,dynamic optical imaging of target bioactive molecules in organisms in a non-invasive form,which helps to better understand the pathogenesis of relevant diseases at the molecular level and is an important contribution to the early diagnosis and treatment of diseases.Among them,reactive fluorescent probes are capable of specific organic chemical reactions with the substances to be measured,exhibit high selectivity,and have begun to be widely applied to the imaging detection of biologically relevant substances,becoming a research hotspot in recent years.Although many fluorescent probes have been developed for imaging,however,there are still some problems such as poor water solubility,long time required for detection,and short excitation wavelength.These have limited the further application of probe molecules.Based on this,this thesis selected SO₂ derivatives(SO₃^2-/HSO₃^-),biothiols（Cys,Hcy,and GSH）,hypochlorous acid（HClO）in reactive sulfur and oxygen species that can affect the redox balance of organisms as detection targets,and designed and synthesized a series of reactive organic small molecule fluorescent probes,evaluated their detection performance in vitro,and applied to biofluorescence imaging.The work in the third and fourth chapters of this thesis was only used for cellular and zebrafish imaging due to the short excitation wavelength of the product,which would cause damage to the tissue;subsequently,in order to make the study more in-depth,the long-emitting test halocin dye was selected as the fluorophore in the fifth and sixth chapters to further apply the probe to the mouse live imaging level.The related contents are as follows:A ratiometric two-photon fluorescent probe DRQ was constructed using the nucleophilic addition mechanism of SO₂derivatives(SO₃^2-/HSO₃^-)to the double bond.This probe has a deep red fluorescence at 613 nm,and the long conjugation system is interrupted by the addition of SO₃^2-/HSO₃^-to release the two-photon absorbing benzopyranquinoline structure at 514 nm,thus enabling the ratiometric detection of SO₂derivatives.The probe is capable of rapid response（within 5 s）to SO₂ derivatives in pure aqueous solution,accompanied by high selectivity and sensitivity（red channel:103 n M;green channel:17 n M）.In addition,the probe can be used for two-photon ratio imaging of SO₂ derivatives in cellular mitochondria and zebrafish.Two probes with multiple reaction sites,NTPC and PMCI,were constructed using the nucleophilic nature of biothiols and the cycloaddition reaction to aldehyde groups.Here,benzopyrone（coumarin）was selected as the fluorophore,and different strong electron-absorbing groups,4-nitrophenyl thioether bond and 4-pyridinyl methyl thioether bond,were introduced at its 4-position,respectively,based on the retention of the aldehyde group at the 3-position,to synthesize these two probes.Due to the photo-induced electron transfer effect（d-PET）between the strongly electron-absorbing thioether part of the probe structure and coumarin,the probes themselves are without background fluorescence.The reaction with different biothiols results in different fluorescence（Cys/Hcy:Green;GSH:Red）,thus achieving the distinguish Cys/Hcy and GSH.Subsequently,the study focused on PMCI with excellent water solubility.It exhibited high selectivity and sensitivity for biothiols（Cys:132 n M;Hcy:105 n M;GSH:62 n M）.In addition,the probe has good cell membrane penetration and low cytotoxicity,allowing simultaneous differentiated imaging detection of cellular and zebrafish endogenous and exogenous Cys/Hcy and GSH by different fluorescent signals.A series of HClO fluorescent probes were constructed using the oxidative cleavage amide mechanism.Taking advantage of the obvious difference in optical properties between oxidized and reduced test halide dyes,a series of HClO fluorescent probes with lactam structures（RECl O-1—RECl O-6）were synthesized by introducing different acyl groups on the nitrogen atoms of the reduced state to interrupt the original long conjugated structure of test halide.This series of fluorescent probes is able to complete the response to HClO rapidly（<50 s）,accompanied by high selectivity and sensitivity.One of the representative probes,RECl O-6,was used for fluorescence imaging detection of HClO in cells and zebrafish.It was furthermore used for fluorescence imaging of endogenous HClO in mouse models of joint inflammation.The HClO fluorescent probe RESCl O was constructed using an oxidative cleavage thiocarbamate mechanism.In this part,the thiocarbamate structure was selected as the recognition group,and a HClO-specific triggered self-degradation probe RESCl O was synthesized by attaching it to the nitrogen atom of the reduced test halogenated dyes through self-degradation linker arms（p-toluenequinone and carbonate）.The probe itself has no background fluorescence,and in the presence of HClO,the probe first deletes thiocarbamoyl to release phenolic compounds,followed by a rapid spontaneous p-quinone methanone elimination and decarboxylation reaction to release the strongly fluorescent oxidized test halogenated dyes.The fluorescent probe was able to accomplish a rapid response to HClO（<10 s）,accompanied by high selectivity and sensitivity.It has also been used for fluorescence imaging of endogenous HClO in mouse joint inflammation.