Design And Application Of Coumarin-based Thiol Fluorescent Probe

Since the birth of life on earth,it has operated with a highly complex and coordinated mechanism.In organisms,compounds such as proteins,nucleic acids,sugars and amino acids are the material basis of life activities.Sulfur-containing compounds refer to a class of compounds containing sulfur atoms,such as thiols(including cysteine(Cys),glutathione(GSH),homocysteine(Hcy),etc.),sulfur dioxide and hydrogen sulfide.They are important participants in life activities.Most of them exist in free and protein form in the synthesis and folding of peptide chains,and play a regulatory role in life processes such as cell signal transduction and apoptosis,redox reactions.Detection the real-time changes of these small molecules in the body is of great significance for the early prevention and monitoring of Alzheimer's disease,diabetes,chronic kidney disease,Down's syndrome and other diseases,as well as physiopathological investigation.Fluorescent probes have attracted attention due to their high sensitivity,specificity,low tissue damage,and even the ability to monitor in real time.And for fluorescent probes,its outstanding advantage is that it can achieve high space-time observation of biological analytes in living cells with a non-invasive manner under confocal laser scanning microscope.Among a wide variety of fluorophores,coumarin-based fluorophores have the advantages of easy modification,large Stokes shift,and stable optical properties,and have been research hotspots in recent years.In this paper,based on the recognition of small thiol molecules,two coumarin-based fluorescent probes were designed and synthesized based on steric effects.The optical properties and biological experiments of the probes were explored:1.Among the three thiols,Cys,GSH and Hcy,the molecular structure of Cys is the smallest.Due to this structural difference,a ratiometric cysteine(Cys)fluorescent probe L-1 was designed and synthesized which based on steric hindrance effect.The coumarin derivative was modified with orthotopic pinacol,and the steric hindrance effect was used to increase the specificity of the probe to Cys,thereby an ?,?-unsaturated ketone new Cys ratiometric fluorescent probe was designed.The nucleophilic-addition reaction between Cys and ?,?-unsaturated ketone shortened the original conjugate system of the probe,which causes the blue shift of the fluorescence emission peak.Probe L-1 has good water solubility and can reach reaction equilibrium in a short time.In addition,the results of bioimaging experiments proved that the probe can also detect exogenous and endogenous Cys in Hep G2 cells in a proportional manner.2.On the basis of probe L-1,the coumarin was used to modify the precursor to obtain a fluorescent probe L-2 with two reaction sites.By connecting coumarin with benzaldehyde through ?,?-unsaturated ketone,GSH can quickly react with ?,?-unsaturated ketone and aldehyde group,thereby emitting green fluorescence.H2 S first reacted with ?,?-unsaturated ketone,and then the sulfhydryl group underwent a second nucleophilic cyclization reaction with the aldehyde to form a five-membered ring,which was conjugated to the entire molecule and caused the system to emit yellow fluorescence.Therefore,highly selective discrimination of GSH and H2 S was achieved through two transmission channels.Moreover,RAW cell imaging results and mouse imaging experiments showed that the probe was suitable for detecting endogenous and exogenous GSH and H2 S.