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A fluorescent probe with aggregation-induced luminescent properties and its preparation method and application

A technology of aggregation-induced luminescence and fluorescent probes, which is applied in the field of drug screening and evaluation, can solve the problem of not being able to specifically identify SIRT1 protein AIE fluorescent probes, and achieve the effect of enhancing the fluorescence absorption intensity

Active Publication Date: 2016-07-06
ZHEJIANG WANBANG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there are no related reports on AIE fluorescent probes that can specifically recognize SIRT1 protein and detect its activity

Method used

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  • A fluorescent probe with aggregation-induced luminescent properties and its preparation method and application
  • A fluorescent probe with aggregation-induced luminescent properties and its preparation method and application
  • A fluorescent probe with aggregation-induced luminescent properties and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1 has the fluorescent probe synthesis of AIE characteristic

[0064] A kind of synthetic method of the fluorescent probe with AIE characteristic of this embodiment, its synthetic procedure is as follows figure 1 shown, including:

[0065] (1) Add 4-hydroxybenzophenone (1.9g, 10mmol), benzophenone (2.2g, 12mmol) and zinc powder (2.9g, 44mmol) into a 250ml three-necked flask, pump air, nitrogen, and repeat three times; Add 80ml THF (tetrahydrofuran), 0 ℃ ice-water bath for 30min; dropwise add titanium tetrachloride (2.4ml, 22mmol) under ice-water bath, reflux overnight, spin dry; add appropriate amount of dichloromethane and dilute hydrochloric acid for extraction, and remove the lower organic layer , dried with anhydrous magnesium sulfate, filtered, spin-dried, subjected to silica gel column chromatography, first washed with petroleum ether: ethyl acetate = 20:1 solvent, then washed with petroleum ether: ethyl acetate = 8:1 solvent, collected 8:1 The washed ...

Embodiment 2

[0071] Example 2 Application of Fluorescent Probe TPE-GK(Ac)YDD in Detection of SIRT1 Protein Activity

[0072] (1) Detection of SIRT1 protein activity by fluorescent probe TPE-GK(Ac)YDD

[0073] Sample group 1: add 50 μL SIRT1 (0.72 mg / mL), 12 μL TPE-GK(Ac)YDD (1 mM), 30 μL lysylendopeptidase (14 μg / mL) and 36 μL NAD + (50mM);

[0074] Sample group 2: Add 50 μL SIRT1 (0.72 mg / mL), 12 μL LTPE-GK(Ac)YDD (1 mM), 30 μL lysylendopeptidase (14 μg / mL), agonist SRT1720 (500 nM) and 36 μL NAD + (50mM);

[0075] Sample group 3: Add 50 μL SIRT1 (0.72 mg / mL), 12 μL TPE-GK(Ac)YDD (1 mM), 30 μL lysylendopeptidase (14 μg / mL), inhibitor EX527 (200 nM) and 36 μL NAD + (50mM);

[0076] Sample group 4: Add 12 μL TPE-GK(Ac)YDD (1 mM), 30 μL lysylendopeptidase (14 μg / mL) and 36 μL NAD + (50mM);

[0077] All four sample groups were incubated at 37°C for 3 hours. After the reaction, a JASCOFP-6500 spectrophotometer was used to measure the fluorescence spectrum from 400nm to 600nm under 320nm...

Embodiment 3

[0097] Example 3 Application of Fluorescent Probe TPE-GK(Ac)YDD in Screening SIRT1 Regulators

[0098] (1) Application of fluorescent probe TPE-GK(Ac)YDD in screening SIRT1 inhibitors

[0099] Take 2 μL fluorescent probe TPE-GK(Ac)YDD (1mM), add 10 μL SIRT1 (0.288 mg / ml), 10 μL lysyl endopeptidase (14 μg / mL), 6 μL NAD + (50mM), 10μL of different concentrations of EX527 mother solution (making the final concentration of EX527 0, 10, 25, 50, 100, 200, 500, 750, 1000, 2000nM), make up to 100μL with buffer solution Tris-HclpH8.8, incubate at 37℃ for 3h and use Tecan enzyme label instrument measurement, set E x 320nm (25nm), E m is 465 (25nm).

[0100] See the test results Figure 8 .

[0101] Depend on Figure 8 It can be seen that with the increase of the final concentration of EX527, the inhibitory effect of EX527 on SIRT1 protein also gradually increases; indicating that the fluorescent probe TPE-GK(Ac)YDD can better reflect the inhibition of SIRT1 protein and can be used...

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Abstract

The invention discloses a fluorescent probe having aggregation-induced luminescent property and a preparation method and application of the fluorescent probe. The fluorescent probe is formed by connection of polypeptide capable of specifically recognizing SIRT1 protein and aggregation-induced luminescent molecules, wherein the amino acid sequence of the polypeptide is glycine-acetylated lysine-tyrosine-aspartic acid-aspartic acid, and the aggregation-induced luminescent molecules are connected with the glycine of the polypeptide; as the polypeptide contains the acetylated lysine at the specific position, the polypeptide in the fluorescent probe can be recognized specifically by SIRT1 protein, the SIRT1 protein can perform deacetylation modification on the polypeptide; the fluorescent probe has no fluorescence absorption basically, but when the fluorescent probe loses the acetyl on the lysine, the fluorescent probe has remarkable fluorescence absorption; and furthermore, when the polypeptide is cut off at the position of the lysine by incision enzyme, the fluorescence absorption strength of the fluorescent probe is further enhanced and the activity of the SIRT1 protein can be detected specifically in real time.

Description

technical field [0001] The invention belongs to the field of drug screening and evaluation methods, and in particular relates to a fluorescent probe with aggregation-induced luminescent properties, a preparation method and application thereof. Background technique [0002] Histone deacetylation is an important covalent modification of histones, which plays a very important role in the regulation of gene expression. Histone deacetylation is mainly catalyzed by histone deacetylases. In addition to the classic zinc ion-dependent histone deacetylases, there is also a special class of histone deacetylases that have been discovered. Acetylase - silent information regulator 2 (silentinformationregulator2, Sir2) related protein. The Sir2-related protein family is a group of highly conserved nicotinamide adenine dinucleotide (NAD + )-dependent histone deacetylase widely exists in various species from lower organisms to humans. There are seven human Sirtuin family members discovere...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N21/64
CPCG01N21/6402G01N33/68
Inventor 程翼宇王毅赵筱萍
Owner ZHEJIANG WANBANG PHARMA
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