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A fluorescent probe substrate for human intestinal carboxylesterase activity detection and its application

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

Active Publication Date: 2017-12-01
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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  • A fluorescent probe substrate for human intestinal carboxylesterase activity detection and its application
  • A fluorescent probe substrate for human intestinal carboxylesterase activity detection and its application
  • A fluorescent probe substrate for human intestinal carboxylesterase activity detection and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

[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 reaction solution was decompressed to remove the solvent, 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 (synthetic see route Figure 9 ). 1 H NMR (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...

Embodiment 2

[0050] Selectivity among recombinantly expressed human single enzymes

[0051] (1) Prepare 99 μL metabolic reaction system in advance, including PBS buffer (10 mM) at pH 7.4, recombinantly expressed human hCE1 (10 μg / mL) / human hCE2 (10 μg / mL) / serum albumin (10 μg / mL) / acetylcholine Esterase (10μg / mL) / butyrylcholinesterase (1.5U / L) / paraoxonase 1 (10μg / mL) / paraoxonase 2 (10μg / mL) / phosphate buffer at 37°C Shake pre-incubation for 10 minutes under the condition;

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

[0053] (3) After 30 minutes, add 100 μL of glacial acetonitrile, shake vigorously, and terminate the reaction;

[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 Reaction in Recombinant Single Enzyme

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

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

[0059] (3) After 30 minutes, add 100 μL of glacial acetonitrile, shake vigorously, and terminate the reaction;

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

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Abstract

A fluorescent probe substrate for detecting the activity of human intestine carboxylesterase and uses thereof, which relate to the technical field of pharmaceuticals. The specific probe substrate is an aromatic ester (TCFE for short) of (E)-2-(4-(4-hydroxybenzene vinyl)-3-cyan-5,5-dimethylfuran-2(5H)-subunit)malononitrile (TCF for short). The substrate can be used for quick quantitative detection of the activity of the human intestine carboxylesterase and for quick screening and estimation of an inhibitor of the human intestine carboxylesterase. The process of the quick quantitative detection of the activity of the human intestine carboxylesterase comprises: selecting a TCFE hydrolysis reaction as a probe reaction, adding a proper substrate into a buffer system comprising hCE2, and determining the actual activity of hCE2 enzyme in each in-vitro organism sample by quantitatively detecting the production of a hydrolysis metabolism product TCF in a unit time in a linear reaction interval. The probe has good selectivity, the hydrolysis product TCF has a long fluorescence emission wavelength (612 nm), and the probe can reduce background fluorescence of the organism sample and increase the sensitivity, and has good practicability and application prospect.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a fluorescent probe substrate for detecting human intestinal carboxylesterase activity and application thereof. Background technique [0002] Carboxylesterase (CE) is an important phase I metabolic enzyme in the body. It can catalyze the breakage of the ester bond of various endogenous and exogenous ester compounds, and release a relatively polar alcohol after hydrolysis. Molecules and carboxylic acid molecular fragments, which are then catalyzed by other metabolic enzymes such as cytochrome P450 (CYP450) or uridine diphosphate glucuronosyltransferase (UGTs) in the body, so that drug molecules can be excreted more effectively. A variety of drug molecules containing ester bonds in their structures, such as irinotecan hydrochloride (irinotecan, CPT-11), oseltamivir (Tamiflu), clopidogrel, beinolate, etc., are all activated by carboxylesterase catalysis or metabolic el...

Claims

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Application Information

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Patent Type & Authority Patents(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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