Design,Synthesis And Biological Application Of Biologically Active Sulfur Molecule Fluorescent Probe

Bioactive sulfur are a class of important molecules in biological systems that can regulate redox homeostasis.Abnormal concentrations of bioactive sulfur in the body may cause a number of diseases such as Alzheimer’s disease,cancer and coronary heart disease.Due to the advantages of easy handling,fast response,good selectivity and the possibility of real-time imaging,fluorescent probes are nowadays widely used to detect bioactive sulfur.Therefore,the use of fluorescent probes for the detection of bioactive sulfur is of great importance for the diagnosis of related diseases.This paper aims to develop fluorescent probes that can specifically detect bioactive sulfur,to realize real-time monitoring of bioactive sulfur,and to apply fluorescent probes to bioimaging.The details of the study are as follows.Chapter 1:The importance of bioactive sulfur and the study of fluorescent probes are reviewed.First,the important role of bioactive sulfur in living organisms is introduced.Then,the concept,classification and composition of fluorescent probes are briefly described,and the research progress is reviewed focusing on fluorescent probes that specifically detect biothiols and fluorescent probes that distinguish between detecting bioactive sulfur.Chapter 2:By combining coumarin derivatives with NBD,a dual-site fluorescent probe was designed and synthesized,which can distinguish and detect Cys/Hcy and SO₂through two emission channels.The fluorescence intensity of free probe was negligible.After the reaction with Cys/Hcy,the ether bond of the probe was broken by NBD and green fluorescence was released at 540 nm,while SO₃^2-reacted with the carbonyl group of probe and orange fluorescence was released at 590 nm.It can be seen that the probe with two potential reaction sites can distinguish Cys/Hcy and SO₂.The limit of detection for Cys/Hcy and SO₂were 0.25μM,0.14μM and 0.05μM.And,the probe was successfully applied for Cys/Hcy and SO₂imaging in Hep G-2 cells and zebrafish.Chapter 3:A dual-site fluorescent probe with low detection limits was designed and synthesized by using 2-（3,3,5-trimethylcyclohexylidene）malononitrile as the fluorescent group and SBD-Cl as the recognition group.Free probe had almost no fluorescence.When reacting with Cys/Hcy,the sulfhydryl group on Cys/Hcy underwent substitution-rearrangement reaction with chlorine atom,releasing yellow fluorescence at555 nm.When the probe reacted with SO₃^2-,SO₃^2-broke the ether bond of the probe,releasing near-infrared fluorescence of precursor at 660 nm.Thus,the probe can quickly identify and distinguish between Cys/Hcy and SO₂by two emission channels with a fluorescence signal spacing large enough（105 nm）to avoid signal interference between the two channels.The limit of detection for Cys/Hcy and SO₂were 0.12μM,0.07μM and 11 n M.And,the probe was successfully applied to image Cys/Hcy and SO₂in Hep G-2 cells and zebrafish.Chapter 4:A near-infrared turn-on fluorescent probe specifically recognizing Cys was synthesized using 2-（3,3,5-trimethylcyclohexylidene）malononitrile and4-diethylaminosalicylaldehyde as fluorophores and SBD-Cl as the recognition group.Free probe had almost no fluorescence.The probe reacted with Cys,which broke the ether bond of the probe and releases near-infrared fluorescence at 655 nm.The response mechanism was verified by ¹H NMR titration experiments and HRMS experiments.The probe had fast response to Cys,high specificity,low detection limit（0.16μM）and large Stokes shift（155 nm）.And,the probe was able to visualize endogenous and exogenous Cys in He La cells and zebrafish.