Design And Biological Application Of Reactive Fluorescent Probe For Detecting Cytochrome P450

Cytochrome P450 enzymes(P450 or CYPs)are a type of heme-containing monooxygenase,which is responsible for the oxidative metabolism of various endogenous substances and foreign chemical substances.As important members of P450,CYP3A5 and CYP2D6 are closely related to the oxidative metabolism of various drugs and carcinogens.Due to the influence of genetic polymorphism and environmental factors,the expression of their enzyme activities in different individuals is significantly different.The individual difference greatly limits the understanding of enzymes in drug metabolism,and many diseases and cancers are related to changes in enzyme activity.Therefore,it is necessary to develop an effective method to detect CYP3A5 and CYP2D6 enzyme activity,explore its role in drug metabolism and related disease pathology,and provide technical support for individualized treatment and related drug development.The fluorescence analysis method using fluorescent probes as molecular tools has attracted the attention of researchers due to its unique advantages such as good selectivity,high sensitivity,and simple operation.Cyanine dyes have a strong intramolecular charge transfer(ICT)effect,easy structure modification,excellent photophysical properties and good water solubility,so they are selected as fluorophores for water-soluble fluorescent probes.In addition,near-infrared(NIR)luminescent fluorescent probes have good biocompatibility and deep tissue penetration,and the hemicyanine dye emits in the NIR region,which can be used as an ideal molecular tool for in vivo imaging,receiving more and more attention.In this thesis,based on molecular design strategies two different series of reactive fluorescent probes were designed and synthesized using cyanine dye as the fluorophore,which were used for the specific detection of CYP3A5 and CYP2D6 activity in living system.The main research contents are as follows:1).Using hemicyanine derivatives as the fluorophore,two near-infrared ratio fluorescent probes(E)-2-(2-(6-((4-(2-bromoethoxy)benzyl)oxy)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-1-ethyl-3,3-dimethyl-3H-indol-1-ium iodide(Hcy-Br)and(E)-2-(2-(6-((4-(2-chloroethoxy)benzyl)oxy)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-1-ethyl-3,3-dimethyl-3H-indol-1-ium iodide(Hcy-Cl)have been developed for detecting CYPs activity.Among them,Hcy-Br showed good specificity,high sensitivity(LOD=0.049n M)and near-infrared ratio fluorescence response to CYP3A5-mediated catalytic debromoethylation.In the simulated enzyme-catalyzed reaction system,the fluorescence emission of Hcy-Br located at 670 nm.Upon addition of CYP3A5 to the system,the fluorescence intensity gradually decreased at 670 nm and generated a new fluorescence emission peak at 713 nm,reaching the maximum within 30 min.Studies on the catalytic mechanism shown that Hcy-Br was catalyzed by CYP3A5 to remove the bromoethyl group and the self-immolative linker could perform 1,6-elimination reaction to release the fluorophore,leading to a strong NIR fluorescence signal.Finally,by virtue of the NIR emission and low cytotoxicity,Hcy-Br had been successfully applied in living cells and tumor-bearing mice for monitoring endogenous CYP3A5activity in real time.Moreover,it is worth noting that Hcy-Br has outstanding mitochondria-targeted ability in living cells due to its quaternary amine N~+.Therefore,Hcy-Br is a potential excellent molecular tool for the detection of CYP3A5 enzyme-related diseases.2).Using anthocyanin as the fluorophore,three"off-on"fluorescent probes(E)-1-ethyl-2-(4-methoxystyryl)-3,3-dimethyl-3H-indol-1-ium iodide(Cy-Me),(E)-1-ethyl-2-(4-((4-methoxybenzyl)oxy)styryl)-3,3-dimethyl-3H-indol-1-ium iodide(Bn Cy-Me),and(E)-2-(4-((4-(2-chloroethoxy)benzyl)oxy)styryl)-1-ethyl-3,3-dimethyl-3H-indol-1-ium iodide(Bn Cy-Cl)were synthesized by introducing different alkoxy groups on their characteristic hydroxyl groups to screen out a fluorescent probe that specifically responds to the target enzyme.The results of the study showed that the probe Cy-Me displayed good specificity for CYP2D6-mediated catalytic demethylation.The probe Cy-Me itself hardly fluoresces.Under simulated physiological conditions(p H=7.4,37°C),the probe substrate was catalyzed by CYP2D6 to demethylate and release the fluorophore(Cy-OH),making the fluorescence intensity of the sample at 553 nm increased significantly.Meanwhile,it was about 9 times that before the reaction,and the color of the solution system changed from yellow to slightly pink,which can be distinguished by the naked eye.In addition,the linear response range of Cy-Me towarding CYP2D6 concentration is 0-18 n M,and the theoretical detection limit is as low as 0.052 n M.At the same time,the probe Cy-Me can be used to detect the activity of CYP2D6 in human liver microsomes(HLM),and can be used to screen CYP2D6inhibitors.Cy-Me has low cytotoxicity(50?M,cell survival rate>80%),which has been successfully used in the living cells and mice to monitor endogenous CYP2D6activity in real time.