Design,Synthesis And Bioimaging Applications Of New Fluorescence Probes Based On Coumarins

Fluorescent probes have advantages of simple operation,good selectivity,low cost and can be combined with biological imaging technology to detect enzymes and biological molecules in vivo.Therefore,fluorescent probes were widely used in medical and pathological research.In recent years,with the development of medical technology,researchers have increasingly demanded new fluorescent dyes,so many new fluorescent probes have been reported.Coumarin fluorescent dyes have large Stokes shifts,exce llent two-photon properties,and good optical stability.At the same time,the desired functional probes could be obtained by modifying the push-pull electron system of the coumarin structure.Therefore,the coumarin dyes were widely used in the design of new fluorescent probes.Studies have shown that bio-thiols?Cys,GSH,Hcy,and SO₂?are endogenous signaling molecules in the body,which may be related to some diseases.However,the physiological function of these bio-thiols has not been elucidated.Therefore,the development of specific and sensitive fluorescent probes is particularly important for the detection of bio-thiols.In this paper,we synthesized two-photon fluorescent probes for GSH,the NIR fluorescent probes for the detecting of GSH,Cys/Hcy and NAC and two-photon fluorescent probes for the detection SO₂,respectively.In chapter 2,a novel GSH fluorescent probe,Co-GSH,was designed and synthesized by using coumarin as the fluorescent nucleus and active olefin double bond as the recognition site of GSH.Compared with other active sulfur such as Cys,Hcy,SO₂,etc.,the probe showed high selectivity and sensitive response speed to GSH.Upon addition of GSH,a Michael-addition reaction took place,which interrupted the conjugated system of die thylamine to the active carbonyl group.The wavelength of the probe was blue-shifted and the color of the fluorescent light changed from red to blue.In bioimaging experiments,the probe was able to detect GSH in HeLa cells by single-photon and two-photon modes.In chapter 3,we described a multi-signaling GSH,Cys/Hcy,and NAC fluorescent probe Co-NA based on the coumarin hemicyanine structure.The emission wavelength of the probe was in the near-infrared region.After GSH was added,GSH firstly undergoed a nucleophilic substitution reaction with the carbon atom at the 3 position of the probe,and then took place the nucleophilic addition reaction with the quaternary ammonium salt,so the wavelength of the probe was blue-shifted.After the addition of Cys/Hcy,a series of nucleophilic substitution-rearrangement-cyclization reactions occured between the probe and Cys/Hcy,which caused the blue-shift of the probe's wavelength.Upon addition of NAC,the nucleophilic substitution reaction occured between the probe and NAC and the fluorescence intensity of the probe was reduced.In bio-imaging experiments,the probe achieved near-infrared and two-photon conversion imaging of Cys/Hcy and GSH in cells,tissues,and zebrafish,and achieved near-infrared imaging of NAC.In chapter 4,a red-shifted SO₂ fluorescent probe Co-SO₂ was further developed based on7-diethylamino-3-aldehyde-4-chlorocoumarin.The probe itself emitted green fluorescence.After the addition of Cys,the wavelength shifts blue-shifted.After the addition of SO₂,the probe undergoed a nucleophilic addition-substitution reaction with SO₂,which causes the wavelength of the probe to undergo a red shift.Upon addition of GSH,a red shift in the wavelength of probe occurred,but the fluorescence intens ity was lower than SO₂ and the rate of the reaction was slow.Therefore,the probe could detect SO₂ with its own good selectivity and fast response speed.In bioimaging experiments,SO₂ in cells,tissues and zebrafish was imaged by single-photon and two-photon modes.