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Specific fluorescence probe substrates of human carboxylesterase 2 and application thereof

A carboxylesterase and probe substrate technology, applied in the field of medicine, can solve problems such as toxic and side effects, and achieve the effects of high sensitivity, easy detection, and simple and easy synthesis process

Inactive Publication Date: 2014-10-29
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, some prodrugs administered intravenously, such as irinotecan, will be excreted into the intestinal tract through bile excretion, and then be hydrolyzed by CES2 in the intestinal tract to release active metabolites, which may cause local toxic side effects

Method used

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  • Specific fluorescence probe substrates of human carboxylesterase 2 and application thereof
  • Specific fluorescence probe substrates of human carboxylesterase 2 and application thereof
  • Specific fluorescence probe substrates of human carboxylesterase 2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The chemical synthesis of embodiment 1.C-4 hydroxynaphthalimide benzoyl ester

[0030] (1) Slowly add 0.6 mmol of benzoyl chloride (dissolved in 5 mL of in tetrahydrofuran), the temperature was controlled at 0°C;

[0031] (2) After mixing for 1 hour, heat the reaction solution to room temperature and react overnight (see Figure 8 );

[0032] (3) The solvent was removed from the reaction solution under reduced pressure, and the residual solid was purified by silica gel chromatography, and eluted with ethyl acetate-n-hexane (1:3 v / v), to obtain 62 mg of pure white solid powder;

[0033] (4) Using high-resolution mass spectrometry and NMR to characterize the structure of the compound;

Embodiment 2

[0034] Embodiment 2. In vitro quantitative determination of the enzyme activity of CES2 in the recombinant single enzyme

[0035] (1) Prepare 99 μl CES2 metabolic reaction system in advance, including PBS buffer (10mM) at pH 7.4 and recombinant human CES2 (2ug / ml), and pre-incubate with shaking at 37°C for 10 minutes;

[0036] (2) Add 1 μl of C-4 hydroxynaphthalimide benzoyl ester with a final concentration of 25 μM to the reaction system to initiate the reaction;

[0037] (3) After 30 minutes, add 100 μl of glacial acetonitrile, shake vigorously, and terminate the reaction;

[0038] (4) Use a high-speed refrigerated centrifuge at 4°C, 20,000×g, after high-speed centrifugation for 5 minutes, take the supernatant, and perform fluorescence detection (Ex=450nm, Em=560nm); calculate the maximum catalytic activity of recombinant human CES2 enzyme The rate was 2310±350 nmol / min / mg.

Embodiment 3

[0039] Example 3. Quantitative determination of the enzyme activity of CES2 in human intestinal microsomes in vitro

[0040](1) Prepare 99 μl human small intestinal microsome metabolic reaction system in advance, including PBS buffer (10mM) at pH 7.4 and human small intestinal microsome (20ug / ml), and pre-incubate at 37°C for 10 minutes;

[0041] (2) Add 1 μl of methylbenzoyl ester of C-4 hydroxynaphthalimide at a final concentration of 25 μM to the reaction system to initiate the reaction;

[0042] (3) After 30 minutes, add 100 μl of glacial acetonitrile, shake vigorously, and terminate the reaction;

[0043] (4) Use a high-speed refrigerated centrifuge at 4°C, 20,000×g, centrifuge at high speed for 5 minutes, take the supernatant, and perform fluorescence detection (Ex=450nm, Em=560nm); calculate the probe in the human small intestine The maximum catalytic rate of the compound is 642±34nmol / min / mg.

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Abstract

The invention provides a specific fluorescence probe substrates of human carboxylesterase 2 (CES2) and application thereof. The specific probe substrate is a benzoateb compound of a C4 hydroxyl naphthalimide, and is applicable to determine the enzyme activity of CES2 in a biological system. The CES2 enzyme activity determination flow comprises: selecting a hydrolysis benzoyl-removal reaction of the benzoate compound of the C4 hydroxyl naphthalimide as a probe reaction, and quantitatively determining the generation amount of a hydrolysis metabolite of the compound in a unit time, so as to determine the enzyme activity of CES2 in all biological samples, cells, bodies and integral organs. The probe is applicable to quantitative assessment of CES2 enzyme activity in biological samples of different species and different individual sources, and quantitative determination on CES2 enzyme activity in different sources of animal tissue cell culture fluids and cell preparation substances, so that the probe is expected to help to realize assessment on medicine disposal capability of important drug metablic enzyme CES2. Additionally, the probe also is applicable as an inhibitor for rapidly screening CES2 in vitro by means of the probe reaction.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a specific fluorescent probe substrate of human carboxylesterase 2 (CES2) and its application. Background technique [0002] Carboxylesterase (CES) is an important phase I hydrolysis metabolic enzyme in the body, which catalyzes the breakage of the ester bond of the compound, exposing the polar hydroxyl or carboxyl group, and then is catalyzed and metabolized by other metabolic enzymes such as CYP450 or UGTs in the body. It is excreted more effectively from the body. A variety of exogenous substances, such as irinotecan (CPT-11), oseltamivir (Tamiflu) and other ester-structured prodrugs, and other compounds containing ester bonds in their structures need to be catalyzed by carboxylesterase. undergo activation or metabolic elimination. [0003] There are currently two main families of carboxylesterases that mediate drug metabolism in the human body: CES1 and CES2, a...

Claims

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Application Information

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IPC IPC(8): C12Q1/44C07D455/04C12Q1/04
Inventor 杨凌刘兆明葛广波邹超王平杜逊甫
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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