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Human intestinal tract carboxylesterase activity detection fluorescent probe substrate and use thereof

A technology of human intestinal carboxylate and intestinal carboxylate, which is applied in the field of medicine to achieve high sensitivity, enhanced sensitivity, and good application prospects

Active Publication Date: 2016-03-30
ZHANGJIAGANG IND TECH RES INST CO LTD DALIAN INST OF CHEM PHYSICS CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, some intravenously administered prodrugs, such as irinotecan hydrochloride, are excreted into the intestine through biliary excretion, and then hydrolyzed by hCE2 in the intestine to release the toxic metabolite SN-38, which is considered to be The main cause of delayed diarrhea side effect of irinotecan

Method used

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  • Human intestinal tract carboxylesterase activity detection fluorescent probe substrate and use thereof
  • Human intestinal tract carboxylesterase activity detection fluorescent probe substrate and use thereof
  • Human intestinal tract carboxylesterase activity detection fluorescent probe substrate and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Synthesis of (E)-2-(4-(4-benzoyloxystylenyl)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene)malononitrile method

[0046] (1) To 10 mL of (E)-2-(4-(4-hydroxystyryl)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene) containing 0.5 mmol In the N,N dimethylformamide solution of malononitrile and 0.625mmol triethylamine, slowly add 0.6mmol of p-benzoyl chloride (dissolved in 5mL of N,N dimethylformamide) dropwise, and control the temperature at 0℃;

[0047] (2) After stirring in an ice bath for 1 h, the solution was naturally warmed to room temperature and stirred overnight;

[0048] (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 30.6 mg of orange solid powder (synthesized See the route Figure 9 ). 1 HNMR(400MHz,DMSO)δ8.16(d,J=7.9Hz,2H),8.05(d,J=8.5Hz,2H),7.97(d,J=16.5Hz,1H),7.78(t,J= 7.2Hz, 1H), 7.63(t, J=7.7Hz, 2H), 7.48(d, J...

Embodiment 2

[0050] Selectivity in recombinantly expressed human single enzymes

[0051] (1) Prepare 99 μL metabolic reaction system in advance, including pH7.4 PBS buffer (10 mM), recombinantly expressed human hCE1 (10 μg / mL) / human hCE2 (10 μg / mL) / serum albumin (10 μg / mL) / Acetylcholinesterase (10μg / mL) / butyrylcholinesterase (1.5U / L) / paraoxonase 1 (10μg / mL) / paraoxonase 2 (10μg / mL) / phosphate buffer at 37 Pre-incubate with shaking at ℃ for 10 minutes;

[0052] (2) Add 1 μL of (E)-2-(4-(4-benzoyloxystyryl)-3-cyano-5,5-dimethylfuran with a final concentration of 20 μM to the reaction system -2(5H)-ylidene)malononitrile starting reaction;

[0053] (3) After 30 minutes, 100 μL of ice acetonitrile was added, and the reaction was terminated after vigorous shaking;

[0054] (4) Fluorescence detection (E x =560nm, E m =612nm); calculate the fluorescence intensity in each system (see Figure 5 ).

Embodiment 3

[0056] Protein concentration of hCE2-catalyzed linear reactions in recombinant single enzymes

[0057] (1) Prepare 99 μL of hCE2 metabolic reaction system in advance, including pH 7.4 PBS buffer (10 mM), recombinant human hCE2 (0-15 μg / mL), and pre-incubate with shaking at 37°C for 10 minutes;

[0058] (2) Add 1 μL of (E)-2-(4-(4-benzoyloxystyryl)-3-cyano-5,5-dimethylfuran with a final concentration of 20 μM to the reaction system -2(5H)-ylidene)malononitrile starting reaction;

[0059] (3) After 30 minutes, 100 μL of ice acetonitrile was added, and the reaction was terminated after vigorous shaking;

[0060] (4) Fluorescence detection (E x =560nm, E m =612nm); calculate the fluorescence intensity in each system (see Image 6 ); the standard curve is obtained as follows: Y=101.9*X-97.05, where Y represents the fluorescence intensity of the product, X represents the hCE2 concentration, the unit is μg / mL, and the range of X is 0-15 μg / mL.

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Abstract

The invention relates to a human intestinal tract carboxylesterase activity detection fluorescent probe substrate and a use thereof and belongs to the technical field of medicine. The specific probe substrate is an aromatic ester (TCFE) of (E)-2-(4-(4-hydroxystyryl)-3-cyano-5, 5-dimethylfuran-2(5H)-ylidene)malononitrile (TCF). The substrate can be used for fast quantitative detection of human intestinal tract carboxylesterase activity and fast screening and assessment of a human intestinal tract carboxylesterase inhibitor. The fast quantitative determination process of human intestinal tract carboxylesterase activity comprises selecting a TCFE hydrolysis reaction as a probe reaction, adding an appropriate substrate into a buffer system containing hCE2, and determining real activity of hCE2 enzyme in various external biological samples through quantitative determination of a hydrolysis metabolite TCF generation amount in unit time in a linear reaction zone. The probe has good selectivity and the hydrolysate TCF has long fluorescence emission wavelength of 612nm. The human intestinal tract carboxylesterase activity detection fluorescent probe substrate can reduce biological sample background fluorescence and improve sensitivity and has good practicality and application prospect.

Description

technical field [0001] The invention belongs to the technical field of medicine, and particularly relates to a fluorescent probe substrate for detecting the activity of human intestinal carboxylesterase and its application. Background technique [0002] Carboxylesterase (CE) is an important phase I metabolic enzyme in the body. It can catalyze the cleavage of ester bonds in a variety of endogenous and exogenous ester compounds, and release the more polar alcohol after hydrolysis. molecules and fragments of carboxylic acid molecules, which in turn are further catalyzed and metabolized by other metabolic enzymes such as cytochrome P450 (CYP450) or uridine diphosphate glucuronyltransferases (UGTs) in the body, allowing drug molecules to be excreted more efficiently. A variety of drug molecules containing ester bonds in their structures, such as irinotecan hydrochloride (irinotecan, CPT-11), oseltamivir (Tamiflu), clopidogrel, benotelate, etc., are all activated by carboxylester...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C09K11/06C07D307/68G01N21/64C12Q1/44
CPCC07D307/68C09K11/06C12Q1/04C12Q1/48
Inventor 杨凌崔京南冯磊葛广波刘兆明宁静
Owner ZHANGJIAGANG IND TECH RES INST CO LTD DALIAN INST OF CHEM PHYSICS CHINESE ACADEMY OF SCI
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