Organelle-targeted NIR Fluorescent Probes For Specific Recognition Of Biothiols And Bioimaging Applications

Biothiols such as cysteine(Cys),homocysteine(Hcy),and glutathione(GSH)play key roles in biological processes.Moreover,the concentration and distribution of each biothiols affect a variety of physiological processe,which are closely related to many diseases.Cells are composed of many organelles,such as nucleus,mitochondria,lysosome,endoplasmic reticulum and so on.Accurate detection of the concentration and distribution of different biothiols in different organelles has an active role in guiding physiological and pathological studies related to biothiols.This article aimed to develop these NIR fluorescent probes that specifically targeted or distinguished biological thiols in organelles,which provided visualization tools to reveal the intrinsic link between biothiols in organelles and physiopathology.Details of the study are as follows:1.NIR fluorescent probe 2-1 targeting lysosomes which specific recognition of Cys was designed and synthesized based on conjugated dicyanoisophorone and coumarin.Probe 2-1 contained acrylate group which can be regarded as a reaction site and morpholine as the lysosomal target group.It could specifically detect Cys at pH 7.4 in the physiological environment and pH 5.0 in the lysosomal environment.When probe 2-1reacted with Cys,molecule emitted a specific NIR fluorescence emission(680 nm at pH 7.4,650 nm at pH 5.0)and had large Stokes shift(> 140 nm).The results of co-staining with Lyso Tracker Green DND-26 and cell imaging showed that probe 2-1 could target lysosomes(Pearson Correlation Coefficient = 0.88),indicating that probe 2-1 was able to enter lysosomes and specifically detect lysosomal Cys in the living cells.It may be an effective tool for detecting Cys in lysosomes.2.Based on the vinyl-conjugated coumarin and tricyanofuran NIR fluorophore,a lysosomal-targeted NIR fluorescent probe 3-1 for distinguishing and recognizing Cys and GSH was designed and synthesized.Probe 3-1 which had morpholine as lysosomal target group,acrylate and active C=C as reaction sites distinguished Cys and GSH.The reaction with Cys emitted NIR fluorescence at 670 nm,and the reaction with GSH emitted NIR fluorescence at 688 nm and green fluorescence at 506 nm,respectively,which was capable of distinguishing between Cys and GSH by a dual-channel fluorescent signal.The results of co-staining with Lyso Tracker Blue DND-22 showed that the probe 3-1 could target lysosomes(Pearson Correlation Coefficient = 0.81).It was successfully used to detect Cys and GSH in living cells and zebrafish.3.A NIR fluorescent probe 4-1 targeting the endoplasmic reticulum for the differentiated recognition of Cys/Hcy and GSH was designed and synthesized based on vinyl-conjugated julonidine and tricyanofuran.Probe4-1 emitted NIR fluorescence at 684 nm which had twisted N,N-diethylamino group as endoplasmic reticulum targeting group,acrylate and active C=C as reaction sites distinguished Cys/Hcy and GSH.The NIR fluorescence was quenched by the reaction of Cys/Hcy with probe 4-1 at the acrylate site.GSH was quenched by the reaction of GSH with probe 4-1 at the acrylate site and the active C=C,meanwhile,the NIR fluorescence emitted blue fluorescence at 468 nm,which realized the detection of Cys/Hcy and GSH.The results of co-staining with ER-Tracker Green showed that probe 4-1 could target endoplasmic reticulum(Pearson Correlation Coefficient = 0.94/0.95).Probe 4-1 was successfully used to detect Cys/Hcy and GSH in living cells and zebrafish.