Design And Synthesis Of SO₂/biothiol Dual-function Fluorescent Probe And Its Application In Fluorescence Imaging In Living Cells

Sulfur dioxide and biothiols?glutathione,cysteine and homocysteine,etc?play important roles in a variety of physiological processes in living organisms.Hence,it is of practical significance to establish rapid,accurate and simple analytical methods for monitoring them in the organisms.Compared with traditional chromatographic and electrochemical based methods,fluorescence spectroscopy shows a series of merits,such as easy processing,high speed,low cost,low detection limit and simple operation.In the thesis,novel fluorescent probes were designed and synthesised,which indicated many excellent fluorescent characteristics with large Stokes shift,high fluorescence quantum yield,high stability and biocompatibility,as well as simultaneous detection of sulfur dioxide and biothiol.The work was desribed in detail as below:1)In chapter 1,we reviewed the physiological function of sulfur dioxide and biothiols,design strategies and research progress of fluorescent probes and their advantages and disadvantages for sulfur dioxide and biothiols detecting,as well as a simple introduction about benzopyran.2)In chapter 2,a series of fluorescent probes were designed and prepared.Benzopyran?BPOH?was selected as chromophore to modify the thiol reaction sites on the phenol ether sites in its molecular structure,including 2,4-dinitrobenzenesulfonyl,2,4-dinitrobenyl,substituted pyridine,pyrazine and other strong electron-absorbing groups.Totally 12compounds were obtained including BPO-Py-diNO₂,BPO-Py-3-NO₂,BPO-Py-5-NO₂,BPO-DNP and BPO-DNSP etc.Their structures were characterized by ¹H NMR,¹³C NMR and HRMS.3)In chapter 3,the UV-Vis and fluorescence optical properties of the above-mentioned5 compounds?BPO-Py-3-NO₂,BPO-Py-5-NO₂,BPO-Py-diNO₂,BPO-DNP and BPO-DNSP?were detailedly investigated by UV spectroscopy and fluorescence spectroscopy.The results indicated that the sensitivity of the probe for sulfur dioxide was affected to some extent by the different strong electron-absorbing groups,the different positions of nitro groups on the same strong electron-absorbing groups and the number of nitro groups.Meanwhile BPO-Py-diNO₂ and BPO-DNSP also exhibited specific responds to biothiols.Moreover,they all had large Stokes shift?for biothiols,Stokes shift was 105 nm,for biothiols,it was109 nm?,and two channels fluorescence shift was 170 nm.It meant the both of them had high possibility for simultaneous detection of sulfur dioxide and biothiols.All evidences suggested that the protection group of phenol ether,the reaction site of biothiols,not only played an important role in the selective detection of biothiols,but also regulated the charge distribution of carbon-carbon double bond?sulfur dioxide reaction site?which further affected tthe sensitivity and selectivity for SO₂.4)In chapter 4,the cytotoxicity of the five compounds were firstly investigated by CCK-8 method.The results indicated all of them had some cytotoxicity but in general,all still had favorable biocompatibility.Among those,the probes with pyridine substituted group showed a higher cytotoxicity,while the lipid-soluble groups on heterocyclic rings were less.And then,BPO-Py-diNO₂ and BPO-DNSP were used to simultaneously detect sulfur dioxide and biothiols in HeLa cells with laser scanning confocal microscopy.The results indicated that both probes can penetrate into cell and targetly enrich in lysosome.The study provided one novel tool for fluorescence imaging of sulfur dioxide and biothiols in the lysosome,which would be helpful to further understanding the physiological functions of sulfur dioxide and biothiols and their relationship.