Ratio-type fluorescence probe for detecting alanine aminopeptidase as well as preparation method and application thereof

A technology of alanine aminopeptidase and alanine, applied in biochemical equipment and methods, fluorescence/phosphorescence, chemical instruments and methods, etc., can solve the problems of cumbersome kit operation, short analysis wavelength, and low detection sensitivity , to achieve the effect of easy promotion, simple operation and good solubility

Inactive Publication Date: 2017-06-13
INST OF CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the fluorescent probes currently used for the detection of alanine aminopeptidase have the disadvantages of short analysis wavelength and low detection sensitivity, and the operation of the kit is very cumbersome, therefore, it is necessary to develop a highly sensitive and rapid detection of alanine aminopeptidase Fluorescent probes have great application value
[0003] Cresyl violet has been widely used in biological analysis due to its advantages of long analysis wavelength, high quantum yield and good stability, but the long-wavelength ratiometric fluorescent probe based on cresyl violet for detection of alanine aminopeptidase has not been reported yet.

Method used

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  • Ratio-type fluorescence probe for detecting alanine aminopeptidase as well as preparation method and application thereof
  • Ratio-type fluorescence probe for detecting alanine aminopeptidase as well as preparation method and application thereof
  • Ratio-type fluorescence probe for detecting alanine aminopeptidase as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1, the preparation of the fluorescent probe (compound of formula I) that alanine aminopeptidase detects

[0050] according to figure 1 The chemical reaction scheme shown in the preparation was carried out.

[0051] Step 1): Dissolve 1mmol Boc-alanine, 1mmol HATU and 1mmol N-ethyldiisopropylamine in dichloromethane, and react for 30 minutes on ice. Then 1.2 mmol of cresyl violet was added, reacted overnight at room temperature, filtered, and then the filtrate was evaporated to dryness under reduced pressure to obtain a crude product. The compound represented by formula II was obtained by separation and purification by column chromatography.

[0052] Step 2): Dissolve 0.1 mmol of the compound represented by formula II in anhydrous dichloromethane, then add trifluoroacetic acid, stir for 2 hours under the protection of Ar gas, and then evaporate to dryness under reduced pressure to obtain a crude product. Separation by column chromatography to obtain the fina...

Embodiment 2

[0056] Embodiment 2, the application of fluorescent probe in detecting alanine aminopeptidase

[0057] 1) Weigh 3.33 mg of the compound of formula I obtained in Example 1 as a fluorescent probe, dissolve it in 10 mL of solvent dimethyl sulfoxide, and prepare a fluorescent probe stock solution (also known as probe mother solution, 1 mM).

[0058] 2) Make a standard curve:

[0059] Prepare standard concentration solutions of alanine aminopeptidase, the concentrations are 0, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 15, 30, 50 and 100ng / mL, and the solvent is pH 7.4 Phosphate buffer.

[0060] Take 25 μL of the probe mother solution and add it to 5 mL of standard solutions of alanine aminopeptidase at different concentrations, react at 37°C for 30 minutes, measure its fluorescence emission spectrum, the excitation wavelength is 525nm, and the obtained fluorescence spectrum is figure 2 .

[0061] Extract the fluorescence intensity at 575nm and 626nm, denoted as F 1 , F 2 , and calculate ...

Embodiment 3

[0065] Embodiment 3, the selectivity research of fluorescent probe

[0066] The histogram of the response of the fluorescent probe of the present invention to different substances is as follows: Figure 4 As shown, it can be seen that other substances have little response to the fluorescent probe. The detection substances are: KCl, 150mM; CaCl 2 , 2.5mM; MgCl 2 , 2.5mM; FeCl 3 , 100 μM; ZnSO 4 , 100 μM; CuSO 4 , 10μM; cysteine, 1mM; glutathione, 1mM; alanine, 1mM; arginine, 1mM; glucose, 1mM; vitamin C, 1mM; urea, 20mM; creatinine, 10mM; 2 o 2 , 100μM; Pyroglutamate aminopeptidase (PGP-1), 200ng / mL; Leucine aminopeptidase (LAP), 200ng / mL; γ-glutamyl transpeptidase (GGT), 200ng / mL; Alanine aminopeptidase (APN), 50 ng / mL; the concentration of the compound represented by formula I is 5 μM.

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Abstract

The invention provides a ratio-type fluorescence probe for detecting alanine aminopeptidase as well as a preparation method and application thereof. The fluorescence probe is a compound shown as a formula I. The fluorescence probe disclosed by the invention can be applied to detecting and imaging of the alanine aminopeptidase in a biological sample. The probe serves as a specific probe of the alanine aminopeptidase, alanine in the probe can be removed by hydrolysis under the condition of the presence of the alanine aminopeptidase only, a fluorescent substance with a novel fluorescence signal is generated, the fluorescence signal can be enhanced and can also have ratio change, and the alanine aminopeptidase can be measured and imaged. The structural formula is as shown in the specification.

Description

technical field [0001] The invention belongs to the field of molecular detection and molecular imaging, and in particular relates to a ratio-type fluorescent probe for detecting alanine aminopeptidase and its preparation method and application. Background technique [0002] Alanine aminopeptidase is a type II membrane-bound metalloprotease, which can specifically cleave the N-terminal alanine from the peptide chain, which is of great significance to the cycle of amino acids in cells. More importantly, alanine aminopeptidase plays a very important role in organisms, and is an important indicator for researching disease diagnosis and treatment. Alanine aminopeptidase is an important cancer marker, clinically urinary alanine aminopeptidase is also of certain value in the early diagnosis of kidney disease. Because the fluorescent probes currently used for the detection of alanine aminopeptidase have the disadvantages of short analysis wavelength and low detection sensitivity, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D265/38C09K11/06G01N21/64C12Q1/37
CPCC07D265/38C09K11/06C09K2211/1033C12Q1/37G01N21/6486G01N2333/948
Inventor 马会民史文贺新元
Owner INST OF CHEM CHINESE ACAD OF SCI
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