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Near-infrared fluorescent probe for detecting CYP 1A1 enzyme

A CYP1A1, fluorescent probe technology, applied in the field of near-infrared fluorescent probes, can solve the problems of low selectivity and biological interference, and achieve the effect of wide application range, high selectivity and good biocompatibility

Active Publication Date: 2021-11-02
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Purpose of the invention: The present invention provides a near-infrared fluorescent probe for detecting CYP 1A1 enzymes, aiming at the problems of interference by organisms and low selectivity during detection caused by the green light of the CYP 1A1 enzyme fluorescent probe in the prior art, By modifying the structure of the fluorescent probe, the molecular structure can realize the specific detection of CYP 1A1 enzyme. At the same time, the fluorescence wavelength emitted by the fluorescent group in the fluorescent probe is in the near-infrared region, so it can avoid being affected by the green light of the organism. interference

Method used

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  • Near-infrared fluorescent probe for detecting CYP 1A1 enzyme
  • Near-infrared fluorescent probe for detecting CYP 1A1 enzyme
  • Near-infrared fluorescent probe for detecting CYP 1A1 enzyme

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Fluorescent probe of the present invention 2-((6-((4-(2-bromoethoxy)benzyl)oxy)-2,3-dihydro-1H-xanthin-4-yl)methylene) Malononitrile (BrDXMM) is prepared as follows:

[0031] 1mmol of HDXMM (HDXMM of the present invention is prepared by the synthetic method disclosed in the following documents: Sensors&Actuators: B.Chemical 273 (2018) 167-175), 2mmol of potassium carbonate and 1.2mmol of 4-(2-bromoethoxy ) benzyl bromide was placed in a 25mL two-necked bottle, and then 8mL of N,N-dimethylformamide was added to the two-necked bottle, and the reaction material was heated to 90°C under nitrogen protection, and the reaction was stirred for 1 hour; after the reaction After cooling to room temperature, the reaction solution was added to cold water, stirred vigorously, and a large amount of dark red solid was precipitated, filtered, and the filter cake was separated by column chromatography (developing solvent: ethyl acetate:petroleum ether=5:1, v:v ), to obtain a dark red so...

Embodiment 2

[0032] Example 2: Determination of the selectivity of the fluorescent probe BrDXMM to each subtype of CYP450

[0033]Preparation of phosphate buffered saline solution (PBS): weigh sodium dihydrogen phosphate dihydrate (0.7410g), disodium hydrogen phosphate dodecahydrate (7.2495g) and magnesium chloride hexahydrate (0.2030g) with an analytical balance and place them in a 100mL beaker , add twice distilled water (total 200mL) to the beaker in 4 times to fully dissolve, let it stand still, transfer to a 250mL volumetric flask, and add twice distilled water to make up the volume, fully shake evenly, and then test the pH of the buffer with a pH meter . Prepare PBS buffer (0.1M, pH 7.4, c(MgCl 2 )=4.0mM), store at 4°C.

[0034] Configuration of the reaction substrate solution: weigh BrDXMM and dissolve it in dimethyl sulfoxide to prepare a mother solution with a concentration of 5 mM. Take 40 μL of the mother solution and add it to 4.96 mL of PBS buffer to prepare a 40 μM reactio...

Embodiment 3

[0037] Embodiment 3: the chemical inhibition experiment of fluorescent probe BrDXMM

[0038] Add 100 μL of the above glucose-6-phosphate solution, 50 μL of β-nicotinamide adenine dinucleoside phosphate solution, 5 μL of glucose-6-phosphate dehydrogenase solution, and 5 μL of CYP 1A1 ( Final concentration 50nM), resveratrol (final concentration 25μM, CYP 1A1 specific inhibitor), after mixing, add 10μL, 40μM BrDXMM (final concentration 10μM), and finally dilute to the corresponding concentration with PBS buffer. Then the above mixture was mixed and incubated in a constant temperature water bath at 37°C for 60 minutes. At the end of the incubation, dimethyl sulfoxide (200 μL) was added to the reaction system to terminate the reaction and mixed well. The mixture was centrifuged at low temperature (13,300×g, 4°C) for 20 minutes, and the supernatant was taken for fluorescence analysis by image 3 It can be seen that the fluorescent probe BrDXMM of the present invention does produc...

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Abstract

The invention discloses a near-infrared fluorescent probe for detecting CYP 1A1 enzyme. According to the near-infrared fluorescent probe, a molecular structure is modified, so the molecular structure can realize specific detection on the CYP 1A1 enzyme, and the near-infrared fluorescent probe has high selectivity; meanwhile, the wavelength of fluorescence emitted by a fluorophore in the fluorescent probe is in a near-infrared region, so the interference of green light emitted by an organism can be avoided during fluorescence detection; BrDXMM is very weak in fluorescence performance, and HDXMM has good fluorescence emission spectrum characteristics (590-800 nm) under the same condition, so the BrDXMM and the HDXMM can be distinguished well, and the fluorescent probe has high signal-to-noise ratios in vivo and in vitro; the fluorescent probe also has good biocompatibility, and cell activity is still greater than 85% when cells are incubated for 24 hours by using a culture medium containing BrDXMM (100 [mu]M); and the fluorescent probe disclosed by the invention can be used for determining the activity of recombinant single enzyme in a solution and CYP 1A1 enzyme in living cells, in-vitro tissues, living zebra fish and tumor-bearing nude mouse, and is wide in an application range.

Description

technical field [0001] The invention relates to a near-infrared fluorescent probe for detecting CYP 1A1 enzyme. Background technique [0002] Cytochrome P450 enzymes (CYP450) are a superfamily of heme-containing monooxygenases and are the most important metabolic enzymes in organisms. CYP450 enzymes participate in the biotransformation of carcinogens, chemicals, drugs, and many endogenous compounds, and play an important role in maintaining the normal physiological functions of organisms. The phase I reaction catalyzed by CYP450 enzymes is a key step in the metabolism of exogenous and endogenous compounds in the human body. It is usually the rate-limiting step for drug clearance from the body, and is crucial to the half-life and clearance rate of compounds. However, there are great individual differences in the distribution of CYP 1A1, and the expression of CYP 1A1 enzymes is closely related to factors such as genetics, gender, age, disease, environment, and co-administered...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D311/82C09K11/06G01N21/359G01N21/64
CPCC07D311/82C09K11/06G01N21/6428G01N21/6486G01N21/359C09K2211/1088G01N2021/6439
Inventor 祁争健代艳鹏薛珂赵鑫鑫
Owner SOUTHEAST UNIV
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