Design,Synthesis,and Application Of Bodipy-based Fluorescent Probes For Biothiols

As important biomolecules,biothiols such as cysteine?Cys?,homocysteine?Hcy?and glutathione?GSH?play vital roles in a wide range of physiological and pathological processes arising from their biological redox chemistry.Abnormal levels of these biothiols have been associated with various types of diseases.In addition,these biothiols play an important role in the normal life of lysosomes.It is believed that thiols are closely associated with proteolysis in the lysosome which reduces disulphide bonds.For example,stabilization of lysosome membranes may involve GSH,whereas Cys is an effective stimulator of albumin degradation in liver lysosomes.Therefore,it is significantly important to detect these thiols in biological cells and lysosome for various biochemical studies and clinical diagnosis-related diseases.Fluorescent detection has shown ever-increasing interest in recent years due to its simplicity,convenient,low cost,high sensitivity and capability of real-time detection and spatial imaging.Nevertheless,it is still a challenge to construct probes for discriminating one thiol species from another due to the similarity of the structures and the reaction activities of Cys,Hcy and GSH.Now,some impressive works have succeeded in differentiating GSH from Cys and Hcy,but the examples of distinguishing Cys from Hcy are much rarer because there are great similarities between them in terms of structure and reaction activity,the only difference is that Hcy has an additional methylene unit.Herein,based on the past research of our group and the literature investigation,we plan to design and synthesize BODIPY based fluorescent probes with different structures for selective detection of biothiols.We expect that the discriminating Cys from Hcy can be achieved by regulating the different structures of the probes,and their potential applications in biology can be further explored.In the second chapter,We first synthesized three new meso-chloro-BODIPY dyes?BDP-1,BDP-2,BDP-3?with long fluorescence emission wavelength.Next,we construct three new fluorescence enhancement probes?BDP-R-Me,BDP-Me and BDP-Ph?by utilizing BDP-1,BDP-2,BDP-3 respectively as fluorophore and 4-methoxythiophenol moiety as recognition unit.The 4-methoxythiophenol moiety was chosen because it functions not only as a good leaving group in the thiol-induced S_NAr substitution reaction,but also as a fluorescence quencher via the photo-induced electron transfer?PET?process to ensure a low fluorescence background.We expect that the discriminating Cys from Hcy can be achieved by regulating the different structures of the probes.Through the system solution test and the application of cell imaging of BDP-R-Me,BDP-Me and BDP-Ph,we successfully screened two fluorescent probes?BDP-R-Me and BDP-Me?for highly selective detection of Cys in living cells.Compared the responses of BDP-R-Me and BDP-Me to Cys,BDP-R-Me has a longer emission wavelength(?_em=608 nm),while BDP-Me has a faster reaction rate.Moreover,we also got BDP-Ph for selective detection of intracellular Cys and Hcy.In the third chapter,we further modified the BDP-Me which can be specifically and rapidly detects intracellular Cys with lysosome-targeting group to obtained Lyso-S with single morpholine group and Lyso-D with double morpholine group.It is expected that by comparing the localization effect of Lyso-S and Lyso-D in lysosome,a fluorescent probe with high lysosome-targeting ability was successfully screened for rapid detection of Cys in lysosome.By analyzing the system solution testing,cell imaging application studies,and lysosomal targeting ability test results of Lyso-S and Lyso-D,We found that Lyso-S with single morpholine and Lyso-D with double morpholine all have high lysosome-targeting capabilities,and there was no difference in the lysosomal targeting ability between Lyso-S and Lyso-D.Ultimately,we succeeded in obtaining two fluorescent probes?Lyso-S and Lyso-D?that can specifically and rapidly detect lysosomal Cys in vivo.