Design And Application Of Near-Infrared Fluorescent Probes For Cytochrome P450 Specific Recognition

Cytochrome P450(CYPs)are a class of heme-containing monooxygenases,of which CYP1A1 and CYP2J2 are important members of the CYP1A and CYP2J subfamilies,respectively,and can participate in the metabolism of endogenous compounds in the body and catalyze carcinogenic chemicals.Generated,so it can be used as a new biomarker and a potential target for human cancer diagnosis and treatment.Fluorescence probe imaging technology has been applied in the fields of cell biology,pharmacology,disease diagnosis and so on.Among them,near-infrared(NIR)fluorescent probes have become the focus of attention and research because of their advantages of low biological background interference,sensitive and noninvasive imaging effects,and good tissue penetration depth.Dicyanoisophorone derivatives with strong intramolecular charge transfer(ICT)effects,large Stokes shifts,tunable photophysical properties,and facile synthesis have become one of the hotspots in fluorescence imaging probes.In this paper,the dicyanoisophorone derivative 2-(3-(4-hydroxystyryl)-5,5-dimethylcyclohexyl-2-enyl)malononitrile(DPOH)is used as the maternal fluorophore,designed and synthesized two series of near-infrared fluorescent probes to achieve the specific detection of CYP1A1 and CYP2J2 enzymes in vivo and in vitro.The main research contents are as follows:1.The methoxy,isopropoxy and chloroethoxy groups were covalently grafted onto the aromatic ring skeleton of the dicyanoisophorone derivative,and under simulated physiological conditions(pH=7.4),their response behavior of subfamily enzymes.The results show that 2-(3-(4-(2-chloroethoxy)styryl)-5,5-dimethylcyclohex-yl-2-enyl)malononitrile(DPCl)can be highly selectively catalyzed by the CYP1A1enzyme and dechlorinated to produce the reduction product DPOH.In a weakly alkaline environment,the phenolic hydroxyl group of DPOH is deprotonated to form a phenoxide anion,fluorescence signal at 673 nm(NIR region),Stokes shift is about118 nm,and the color and luminescence change of the solution can be observed with the naked eye.In the CYP1A1 enzyme concentration range of 0-14 n M,the fluorescence intensity of enzymatic hydrolysis product of probe DPCl showed a good linear relationship with the enzyme concentration(R~2=0.9932),and the detection limit was 0.026 n M.The probe DPCl can be used to quantitatively detect the catalytic activity of CYP1A1 in human liver microsomes(HLM),and has low cytotoxicity(50?M,survival rate>80%).It can monitor the distribution status of CYP1A1 in living cells and organisms and concentration expression information.2.In view of the narrow and long shape of the substrate channel of CYP2J2enzyme and its active cavity,p-hydroxybenzyl group was introduced into the parent fluorophore as a linking group to regulate the probe structure so that it can enter the CYP2J2 active cavity,and six kinds of NIR fluorescent probes were prepared by valence grafting alkyl groups with different spatial structures and volumes as recognition groups.Under simulated physiological conditions(pH=7.4),screening identified 2-(3-(4-(4-methoxyphenyl)styrene)-5,5-dimethylcyclohexyl-2-enyl)malono-nitrile(DPBM)can be highly selectively demethylated by CYP2J2 enzyme and trigger 1,6-elimination of p-hydroxybenzyl,releasing the metabolite DPOH.In a weakly alkaline environment,a NIR fluorescence signal of 673 nm is generated.In the CYP2J2 enzyme concentration range of 0-20 n M,the fluorescence intensity of enzymatic hydrolysis product of probe DPBM showed a good linear relationship with the enzyme concentration(R~2=0.9982),and the detection limit was 0.090 n M.The probe DPBM can be used for quantitative detection of CYP2J2 activity in HLM,and has low cytotoxicity(50?M,survival rate>85%),and can monitor the distribution status and concentration expression information of CYP2J2 in living cells.