Design And Synthesis Of Fluorescent Probes For Detecting Intracellular Biothiols And Thiophenol

Cys teine(Cys),homocysteine(Hcy)and glutathione(GSH)are important small molecule thiols in cells that play an important role in maintaining intracellular redox homeostasis,biocatalysis,metal binding and protein expression modification.Thiophenol has an important role in organic synthesis and is widely used in the preparation of agrochemicals,pharmaceuticals and various industrial products.However,thiophenol and its derivatives are highly toxic,and long-term exposure to thiophenol can cause the damage of the central nervous system or other nervous system,leading to difficulty in breathing,muscle weakness,paralysis of the hind legs,coma,and even death.Therefore,the detection of small molecule biothiols in cells and thiophenols invading into cells is of great significance.In recent years,fluorometry has been widely used for the qualitative and quantitative detection of active substances in cells due to its rapid detection,low cost and relatively simple operation.Accordingly,we designed and synthesized reaction-based fluorescent probes for sensing mitochondrial biothiols and thiophenol in cells especially in mitochondria.The main contents are summarized as follows:(1)The probe NP-SH,composed of 2,5-diaminoterephthalic acid diethyl ester as the fluorophore and 2,4-dinitrobenzenesulfonamide as the reactive group,was designed and synthesized based on the intermolecular charge transfer(ICT)mechanism.The probe has the characteristics of easy preparation,good water solubility,specific response to biothiol,large Stokes shift(154 nm)and good biocompatibility.Additionally,the probe was successfully applied to detect biothiol in HeLa cells.(2)Considering that the fluorophore has the advantages of easy preparation,good spectral properties,easy modification and two-photon excitation,we used the skeleton to construct the probe DMP-SH based on the nucleophilic attack of thiophenol on electrophilic aromatic rings.The probe achieves a specific response to thiophenol with a detection limit as low as 65 nM.Two-photon fluorescence imaging experiments show that the probe could be excited by near-infrared light with a wavelength of 780 nm,and was successfully applied for monitoring mitochondrial thiophenol in HeLa cells.(3)Employing the skeleton of 4-(4-hydroxyphenyl)buta-1,3-dien-1-yl)-1-methylpyridin-1-ium,we designed and synthesized a mitochondria-targeted two-photon fluorescent probe DP-SH,which is capable of specifically detecting biothiols.After the probe was reacted with cysteine,it quickly reached equilibrium with a Stokes shift of 165 nm and a detection limit as low as 127 nM.Two-photon fluorescence imaging experiments suggest that the probe can be excited by near-infrared light with a wavelength of 790 nm,and was successfully applied for sensing mitochondrial thiophenol in HeLa cells.