The Design,Synthesis And Application Of Turn-on Near-Infrared Fluorescent Probes For Hydrolytic Enzyme

Enzymes,very important biological macromolecules,participate in various physiological processes and play a very important role in organisms.Furthermore,Enzymes are associated with various diseases,forexample,abnormally elevated levels of alkaline phosphatase?ALP?are generally considered to be associated with various diseases such as bone diseases,hepatic diseases,heart disease and cancer;?-galactosidase??-gal?is a biomarker of ovarian cancer and also been found that it is closely associated with the senescence of cells.Therefore,the detection of enzymes is of great significance in biological research and disease diagnosis.This article aims to design and synthesize near-infrared?NIR?fluorescent probes with excellent photophysical properties for the detection of biological enzymes?such as ALP and?-gal?.According to the design mechanism of organic fluorescent probes,we have designed and synthesized a series of NIR fluorescent probes with high selectivity,high sensitivity and good biocompatibility for the detection in biological systems.The main research contents are as follows:1.A NIR turn-on fluorescent probe named DXMP was designed and synthesized for detection of endogenous ALP in vivo.DXMP was composed of an enzyme-triggered moiety?phosphate moiety?and a fluorophore?DXM-OH?.In the presence of ALP,phosphate moiety was specifically cleaved lead to DXMP was transformed into DXM-OH following strong fluorescence intensity enhancement which achieved detection of ALP.These experiments demonstrated that DXMP was able to detect ALP sensitively with a low detection limit for ALP in the test system.Moreover,the MTT assay results indicated that DXMP has minimal toxicity and enjoy superior biocompatibility toward living cells.These imaging results indicated that ALP was overexpressed in HepG2cells compared to LO2 cells.More importantly,it has been successfully used to detect and image endogenous ALP in zebrafish.2.A NIR turn-on fluorescent probe?DXM-?gal?was designed and synthesized for detection of endogenous?-gal based on the connection of ?-D-galactopyranoside with DXM-OH.In the presence of?-gal,?-D-galactopyranoside group of the DXM-?gal was hydrolyzed and the hydroxyl group was released,which leaded to fluorescence recovery. According to the change of fluorescence intensity quantitative detection of?-gal can be realized.Furthermore,DXM-?gal had a significant color change from red to purple after incubated with?-gal,indicating DXM-?gal had the ability to detect?-gal by“naked eye”.These results indicated that DXM-?gal can detect?-gal by colorimetric and fluorescent dual signals.More importantly,DXM-?gal has been successfully used to detect and image endogenous?-gal in living cells and zebrafish.3.We have designed and synthesized a NIR turn-on fluorescent probe?DCM-CHO-?gal?with large Stokes shift for detecting?-gal.Upon addition of?-gal to the solution of the probe,the reaction mechanism was that?-gal-catalyzed cleavage?-galactosidic bond in DCM-CHO-?gal prompted the transformation of DCM-CHO-?gal into DCM-CHO-OH with a significant color change from yellow to red.These results indicated that DCM-CHO-?gal can detect?-gal by colorimetric and fluorescent dual signals.Furthermore,DCM-CHO-?gal shows a good NIR characteristic(?_abs/?_em=500/665 nm)with a large Stokes shift?165 nm?,which effectively reduces self-quenching of fluorescence and the interference caused by excitation light and scattered light.Thus,the new probe is more suitable for detecting endogenous?-gal activity avoiding possible interference with living systems and the probe has been successfully applied to visualize?-gal activity in living cells and zebrafish.