Construction Of Fluorescent Probes For Specifical Recognition Of Enzymes

Recently,fluorescent probes have attracted great interests as a newly developed detection method.The classic fluorescent probes are small molecules which are capable of transforming chemical information into a fluorescent signal.Based on its several advantages,fluorescent probes have been widely used in the detection of interest in vitro and in vivo.Enzymes are important biomarkers which play essential role in various biological and physiological events.Thus,developing an effective fluorescent probe that can specifically monitoring the enzyme in vivo is of great value.To further explore potential applications of fluorescent molecular probes in monitoring biological enzyme activity.We have developed several fluorescent probes in this paper,and evaluated its application in living samples.(1)We have designed a novel BODIPY-based fluorescent probe(BODIPY-C-Leu)that can monitor leucine aminopeptidase(LAP)both in vitro and in vivo with an turn-on and ratiometric model.The fluorescent probe(BODIPY-C-Leu)contains an asymmetric BODIPY dye as a fluorescence reportor and a cysteine-leucine dipeptide(Cys-Leu)as the enzyme-active trigger.As LAP can selectively catalyze the hydrolysis of the N-terminus in leucine of proteins and peptides.Therefore,the cysteine-leucine dipeptide(Cys-Leu)conjugated on BODIPY dye can be hydrolyzed by LAP,and the sulfur-substituted BODIPY would convert to be amino-substituted BODIPY by a S?N conversion after the free amino group generated,resulting in a dramatic fluorescence change and realizing the specific recognition of LAP.In addition,BODIPY-C-Leu have been successfully used to monitor LAP activity in different cancer cells,indicating that HeLa cells have higher levels of LAP activity than A549 cells.Furthermore,we also evaluated its ability of real-time monitoring drug-induced LAP change in zebrafish model.(2)We have developed a fluorescein-based fluorescent probe(Fluorescein-Ph)for the detection of tyrosinase(TYR).Fluorescein-Ph contains a 3-hydroxyphenyl units which can specifically react with TYR.The mechanism is mainly based on that the 3-hydroxyphenyl group was converted into a diphenol structure by TYR,and a further 1,6-rearrangement elimination reaction occured to release fluorescein,a bright green fluorophore,and then a typical"turn-on" fluorescence response allowed the specific recognition of TYR.In addition,Fluorescein-Ph have been successfully used for the detection of TYR in living cells and zebrafish model.(3)A coumarin-based fluorescent probe(Coumarin-Gal)for monitoring ?-galactosidase(p-Gal)with a turn-on and ratiometric model have also been constructed.The probe was synthesized by a simple nucleophilic reaction between coumarin dye and 2,3,4,6-Tetra-O-acetyl-alpha-D-galactopyranosyl bromide,and the pyranose was served as the recognition moiety.After exposuring to ?-Gal,a red shift and significant fluorescence enhancement were observed,realizing the detection of ?-Gal.