Specific fluorescent probe for UDP-glucuronosyltransferase UGT1A1 and application thereof

A glucuronic acid and fluorescent probe technology is applied in the field of specific fluorescent probe substrates for glucuronyltransferase UGT1A1, which can solve the problems of low detection sensitivity, low single-enzyme selectivity, poor chemical stability, etc. The effect of high sensitivity, simple synthesis process and low detection cost

Active Publication Date: 2015-02-11
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

Although bilirubin has high single-enzyme selectivity, it has poor chemical stability and low detection sensitivity
However, the single-enzyme selectivity of estradiol and etoposide is not high, and expensive analytical instruments such as LC-MS / MS are needed to achieve quantitative analysis of the products

Method used

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  • Specific fluorescent probe for UDP-glucuronosyltransferase UGT1A1 and application thereof
  • Specific fluorescent probe for UDP-glucuronosyltransferase UGT1A1 and application thereof
  • Specific fluorescent probe for UDP-glucuronosyltransferase UGT1A1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The synthetic route of embodiment 1.N-(3-carboxyalkyl)-4-hydroxyl-1,8-naphthalimide

[0036] (1) Synthesis of compound 1

[0037] Add 4.2mmol of 4-aminobutyric acid to 50ml of ethanol solution containing 1g (3.61mmol) of 4-bromo-1,8 naphthalene anhydride, react overnight at 70-80°C, add 200ml of water, precipitate a large amount of solid, filter and dry in vacuo N-(3-carboxypropyl)-4-bromo-1,8-naphthalimide was obtained as a beige solid with a yield of 80-90%.

[0038] (2) Synthesis of compound 2

[0039] Put 800mg of compound 1 and 2.54g of potassium carbonate in a 100ml single-necked bottle, add 30ml of methanol, react at 60-70°C overnight, cool, adjust the pH to acidic with 1M hydrochloric acid, a large amount of yellow solid precipitates, filter, wash with a large amount of water, Vacuum drying gave yellow solid N-(3-carboxypropyl)-4-methoxy-1,8-naphthalimide with a yield of 80-90%.

[0040] (3) Synthesis of compound 3

[0041] Put 300mg of compound 2 in a 25ml ...

Embodiment 2

[0044] Embodiment 2. In vitro determination of the selectivity of human recombinant UGT single enzyme

[0045] (1) Prepare 95μl UGT metabolic reaction system in advance, including Tris-Hcl buffer (50mM) at pH 7.4, each single enzyme of recombinant human UGT (0.06mg / ml), N-(3-carboxypropyl)-4- The final concentration of hydroxy-1,8-naphthoimide is 10 μM, and it is shaken and pre-incubated at 37°C for 3 minutes;

[0046] (2) Add 5 μl of UDPGA with a concentration of 40 mM (final concentration 2 mM) to the reaction system to initiate the reaction;

[0047] (3) After 30 minutes, add 100 μl of glacial acetonitrile, shake vigorously, and terminate the reaction;

[0048] (4) Use a high-speed refrigerated centrifuge at 4°C, 20,000×g, after high-speed centrifugation for 20 minutes, take the supernatant, and perform fluorescence detection (Ex=362nm, Em=450nm); the selectivity of recombinant human UGT1A1 enzyme is the highest It is about 25 times that of other single enzymes ( Figure...

Embodiment 3

[0049] Example 3. Determination of IC of human liver microsomes and UGT1A1 by in vitro inhibition test 50

[0050] (1) Prepare 190 μl human liver microsomes and UGT1A1 metabolic reaction system in advance, including Tris-Hcl buffer (50mM) at pH 7.4, human liver microsomes (0.25mg / ml), UGT1A1 (0.06mg / ml), N The final concentration of -(3-carboxypropyl)-4-hydroxy-1,8-naphthalimide was 10 μM, and different concentrations of protopanaxatriol were shaken and pre-incubated at 37°C for 3 minutes;

[0051] (2) Add 10 μl of UDPGA with a concentration of 40 mM to the reaction system to initiate the reaction;

[0052] (3) After 30 minutes, add 200 μl of glacial acetonitrile and shake vigorously to terminate the reaction;

[0053] (4) Use a high-speed refrigerated centrifuge at 4°C, 20,000×g, after high-speed centrifugation for 20 minutes, take the supernatant, and perform fluorescence detection (Ex=362nm, Em=450nm); calculate its effect on human liver microsomes and IC of UGT1A1 50 T...

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Abstract

Disclosed is the use of a compound as shown by formula (1) for a specific fluorescent probe of glucuronosyltransferase UGT1A1.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a specific fluorescent probe substrate of glucuronosyltransferase UGT1A1 and an application thereof. Background technique [0002] The glucuronosyltransferase (Uridine diphosphate-glucuronosyltransferase, UGT) superfamily is the most important phase II drug metabolizing enzyme in the body. N 2 The reaction mechanism catalyzes the combination of lipophilic compounds with the glucuronic acid (GA) of the cofactor uridine diphosphate glucuronic acid (UDPGA), thereby increasing the hydrophilicity of the substrate so that it can be more efficiently removed from urine or bile excreted. Usually the glucuronide conjugation reaction mediated by UGT enzyme is an important detoxification process of the body. Many endogenous compounds, mutagens, drugs, and their metabolites are substrates of UGTs, such as endogenous substances such as bilirubin and estradiol, and exogenous s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C07D221/14A61K49/00
CPCC12Q1/48C09B57/08G01N2333/91097A61K49/0021C07D221/14C09K11/06C09K2211/1029G01N2333/91102
Inventor 杨凌崔京南葛广波吕侠冯磊刘兆明
Owner ZHANGJIAGANG IND TECH RES INST CO LTD DALIAN INST OF CHEM PHYSICS CHINESE ACADEMY OF SCI
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