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Fluorescent probe substrate for testing activity dipeptidyl peptidase IV and application of fluorescent probe substrate

A dipeptidyl peptidase and fluorescent probe technology, applied in the field of biomedicine, can solve the problems of large quantitative error, low selectivity of single enzyme, false positive, etc. The effect of sensitivity

Active Publication Date: 2016-11-23
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

The known fluorescent substrates are all off-on probes, the single enzyme selectivity is not high and is easily interfered by the biological matrix, the quantitative error is relatively large, and it is easy to get false when applied (such as the screening of DPP-IV inhibitors). positive or false negative results

Method used

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  • Fluorescent probe substrate for testing activity dipeptidyl peptidase IV and application of fluorescent probe substrate
  • Fluorescent probe substrate for testing activity dipeptidyl peptidase IV and application of fluorescent probe substrate
  • Fluorescent probe substrate for testing activity dipeptidyl peptidase IV and application of fluorescent probe substrate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Synthesis of N-butyl-4-(glycine-proline)-amino-1,8-naphthalimide (GPAN)

[0042] The synthetic route of N-butyl-4-(glycine-proline)-amino-1,8-naphthalimide (GPAN) is as follows figure 2 shown. (1) Synthesis of Compound 1

[0043] At room temperature, add n-butylamine (1.10g, 15mmol) into a solution of 4-nitro-1,8-naphthalene anhydride (2.43g, 10mmol) in acetic acid (50mL), react at 100-110°C overnight, then filter while hot, The filter cake was washed with acetic acid and dried in vacuo to obtain compound 1 as a beige solid with a yield of 45-55%.

[0044] (2) Synthesis of compound 2

[0045] At room temperature, add N-butyl-4-nitro-1,8-naphthalimide (1.49g, 5mmol) and tin dichloride dihydrate (6.77g, 30mmol) into the ethanol (50mL) solution in turn, and stir After uniformity, concentrated hydrochloric acid (10 mL) was slowly added dropwise, and the reaction was completed at room temperature for 30 min. Quench the reaction with 10% sodium carbonate solution (40mL)...

Embodiment 2

[0053] In Vitro Determination of the Selectivity of Human Recombinant DPP-IV Single Enzyme

[0054] (1) Prepare 198 μL DPP-IV metabolic reaction system in advance, including PBS buffer (50 mM) at pH 7.4, recombinant human DPP-IV single enzyme (1 μg / mL), carbonic anhydrase (CA, 10 μg / mL), trypsin (trypsin, 10μg / mL), pepsin (pepsin, 10μg / mL), butyrylcholinesterase (BChE, 10μg / mL), acetylcholinesterase (AChE, 10μg / mL), carboxylesterase (hCE1, hCE2, 10 μg / mL), bovine serum albumin (BSA, 10 μg / mL), human serum albumin (HSA, 10 μg / mL) were shaken and pre-incubated at 37°C for 3 minutes;

[0055] (2) Add 2 μL of GPAN with a concentration of 10 mM to the reaction system to initiate the reaction;

[0056] (3) After 30 minutes, add 200 μL of glacial acetonitrile, shake vigorously, and terminate the reaction;

[0057] (4) Use a high-speed refrigerated centrifuge at 4°C, under the condition of 20,000×g, after high-speed centrifugation for 20 minutes, take the supernatant, and perform fl...

Embodiment 3

[0059] Determination of DPP-IV time standard curve

[0060] The experiment was carried out on a microplate reader using a 96-well plate, GPAN 100 μM, DPP-IV single enzyme 0.1 μg, PBS buffer solution of pH 7.4 50 mM, a total volume of 200 μL, incubated at 37°C for 30 min, and the microplate reader every 5 minutes Analysis, the ratio of the fluorescence intensity of the product to the fluorescence intensity of the substrate and the incubation time to make a standard curve, the R of each standard curve 2 >0.99, indicating that the standard curve has a wide linear range and can accurately quantify the content of DPP-IV ( Figure 5 ).

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Abstract

The invention provides a fluorescent probe substrate for testing of activity dipeptidyl peptidase IV and application of the fluorescent probe substrate and belongs to the technical field of biomedicine. The fluorescent probe substrate is a C-4 amide derivative GPAN of naphthalimide and can be used for testing enzyme activity of DPP-IV in different biological systems. A process for testing enzyme activity of DPP-IV includes: selecting GPAN amide hydrolysis reaction as probe reaction; quantitatively detecting generation quantity of a GPAN dipeptidyl-removed metabolism product to test activity of DPP-IV in various biological samples. The fluorescent probe substrate can be used for quantitatively testing enzyme activity of DPP-IV in biological samples different in species and individual source and enzyme activity of DPP-IV in animal tissue cell culture liquid and cell preparation products different in source, and is expected to realize quantitative evaluation on activity of DPP-IV which is a metabolic enzyme important to human body. In addition, with the help of probe reaction, the fluorescent probe substrate can be used for quickly screening inhibitors or inducers of DPP-IV and evaluating inhibiting or inducing capability thereof.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a specific fluorescent probe substrate for measuring dipeptidyl peptidase IV and its application. Background technique [0002] Dipeptidyl peptidase IV (Dipeptidyl peptidase IV, DPP-IV, EC 3.4.14.5) is a transmembrane serine protease in dimer form, widely distributed in mammalian kidney, liver, gastrointestinal, pancreatic epithelial cells and Vascular endothelial cells can also exist in plasma and cerebrospinal fluid in a dissolved form. DPP-IV can specifically catalyze the hydrolytic cleavage of the peptide bond of the amino acid residue proline (Pro) or alanine (Ala) at the second position of the N-terminus of the polypeptide chain, that is, the hydrolysis of two amino acid residues Xa-Pro and Xa -Ala (Xa is any amino acid except proline), which participates in the activation of various biologically active polypeptides in the body, and partially or completely ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K5/062C12Q1/37
Inventor 杨凌邹立伟葛广波钱星凯冯磊王平
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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