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Near infrared fluorescence probe substrate of carboxylesterase2 and application of substrate

A carboxylesterase and fluorescent probe technology, applied in the field of medicine, can solve the problems of large quantitative error, short emission wavelength, and large interference of biological matrix autofluorescence, and achieve high sensitivity, simple and easy synthesis process, and good fluorescence properties. Effect

Inactive Publication Date: 2017-08-25
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fluorescent detection methods are popular due to their advantages of high sensitivity, low invasiveness, rapidity and high throughput, but the emission wavelengths of existing fluorescent substrates are relatively short (λ em <630nm), which is greatly interfered by the autofluorescence of the biological matrix, and the quantitative error is large

Method used

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  • Near infrared fluorescence probe substrate of carboxylesterase2 and application of substrate
  • Near infrared fluorescence probe substrate of carboxylesterase2 and application of substrate
  • Near infrared fluorescence probe substrate of carboxylesterase2 and application of substrate

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Experimental program
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Effect test

Embodiment 1

[0040] Probe specificity evaluation

[0041] (1) Contains carboxylesterase 1, carboxylesterase 2, trypsin, pepsin, trypsin, carbonic anhydrase, a-chymotrypsin, paraoxonase 1, paraoxonase 2, a acid glycoprotein 198μL of phosphate of acetylcholinesterase (5μg / mL), butyrylcholinesterase (20U / L), human serum albumin (50μg / mL), bovine serum albumin (50μg / mL), at 37℃ Pre-incubate with shaking for 5 minutes;

[0042] (2) Add 2μL of 1,3-dichloro-7-hydroxy-9,9-dimethyl-2(9H)-acridone benzoyl ester (final concentration 5μM) to start the reaction, 37°C Shaking incubation;

[0043] (3) After 30 minutes, add 200 μL of acetonitrile and shake vigorously to terminate the reaction;

[0044] (4) Detection of hydrolysate (λ ex =600nm, λ em =662nm) the fluorescence intensity value at the collection wavelength (see figure 2 ).

Embodiment 2

[0046] Drawing of carboxylesterase 2 quantitative standard curve

[0047] (1) Using 5mg / mL carboxylesterase 2 (CE2) standard solution, dilute with phosphate buffer into different concentrations of single enzyme working solution (0,0.5,1,2,3,4,5,6 ,7,8,9,10μg / mL), pre-incubate at 37℃ for 5min;

[0048] (2) Add 2 μL of 1,3-dichloro-7-hydroxy-9,9-dimethyl-2(9H)-acridone benzoyl ester (198 μL) to each solution sample (198 μL) The final concentration is 10μM), incubate with shaking at 37°C for 15min, add 200μL of acetonitrile and shake vigorously for 15 seconds to stop the reaction;

[0049] (3) Detection of hydrolysate DDAO (λ ex =600nm, λ em =662nm) The fluorescence intensity value at the acquisition wavelength, the fluorescence intensity value of DDAO is linearly fitted to the CE2 concentration, and the quantitative standard curve of human carboxylesterase 2 is established; the curve equation is Y=2521*X–72.24, The square of the correlation coefficient is 0.997 (see image 3 ).

Embodiment 3

[0051] Loperamide inhibition test

[0052] (1) Recombinantly express 198μL of CE2 single enzyme (2μg / mL), human liver microsomes (4μg / mL), and human intestinal microsomes (4μg / mL), pre-incubate at 37℃ for 5 minutes with shaking;

[0053] (2) Add 1 μL of CE2 positive inhibitor loperamide final concentration (0-100 μM) to the reaction system, and incubate for 5 minutes;

[0054] (3) Add 2μL of 1,3-dichloro-7-hydroxy-9,9-dimethyl-2(9H)-acridone benzoyl ester (final concentration 5μM) to start the reaction;

[0055] (3) After 15 minutes, add 200 μL of acetonitrile and shake vigorously to terminate the reaction;

[0056] (4) Perform fluorescence detection (λ ex =600nm, λ em =662nm), calculate the inhibitory intensity of CE2 based on the ratio of the fluorescence intensity of each group at 662nm to the DMSO group (see Figure 4 ).

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Abstract

The invention provides a near infrared fluorescence probe substrate of carboxylesterase2 and application of the substrate. The near infrared fluorescence probe substrate is aromatic ester (DDAE for short) of 1,3-dichloro-7-hydroxy-9,9-dimethyl-2(9H)-acridinone (DDAO for short). The substrate can be used for rapidly and quantitatively detecting the activity of carboxylesterase2 (CE2) and rapidly screening and evaluating carboxylesterase2 inhibitor and activator. The process of rapidly and quantitatively detecting the activity of carboxylesterase2 includes the following steps of selecting a DDAE hydrolysis reaction as a probe reaction, adding the suitable probe substrate into a buffer solution system containing carboxylesterase2, and measuring the real activity of carboxylesterase2 in various in-vitro biological samples in a linear reaction interval through the generation amount of hydrolytic metabolite DDAO in the quantitative detection unit time. The probe has good selectivity, the fluorescence emission wavelength (662 nm) of the hydrolytic metabolite DDAO is in the near infrared area, background fluorescence of the biological samples can be remarkably reduced, sensitivity is improved, and the substrate has good practicability and application prospects.

Description

Technical field [0001] The invention belongs to the technical field of medicine, and specifically relates to a near-infrared fluorescent probe substrate of carboxylesterase 2 and its application. Background technique [0002] Carboxylesterases (CE) is an important phase I drug metabolizing enzyme, which is widely distributed in many mammalian cells. The enzyme participates in the detoxification and metabolism of various ester drugs, environmental toxicants and carcinogens, and can effectively catalyze the hydrolysis of exogenous substances containing carboxylic acid ester bonds, amide bonds, and thioester bonds. In humans, carboxylesterases mainly include two subtypes, carboxylesterase 1 (CE1) and carboxylesterase 2 (CE2), which have significant differences in tissue distribution and substrate specificity. CE2 is highly expressed in human intestines and tumor tissues, and plays a very important role in improving the oral bioavailability of many prodrugs, and its activity will al...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D219/06C07D405/12C07D409/12C09K11/06C12Q1/44
Inventor 杨凌葛广波金强冯磊崔京南王丹丹
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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