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High-selectivity ratio-type fluorescent probe and application thereof

A technology selected from, compounds, applied in the field of fluorescent probes, can solve the problem of not being able to use adjacent sulfhydryl groups of proteins

Active Publication Date: 2016-04-06
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Therefore, the above two types of probes only focus on the development of a protein labeling strategy, and cannot be used for in situ detection of endogenous protein adjacent sulfhydryl groups in living cells.

Method used

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  • High-selectivity ratio-type fluorescent probe and application thereof
  • High-selectivity ratio-type fluorescent probe and application thereof
  • High-selectivity ratio-type fluorescent probe and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] The synthesis of target probes using 7-aminocoumarin and 1,8-naphthoimide as energy donors and acceptor fluorophores is as follows:

[0070]

[0071] PAO: p-aminophenylarsine (10.85g, 50mmol) was dissolved in methanol, heated to reflux to make the solution clear, then phenylhydrazine (10.30mL, 100mmol) was added dropwise, and the addition was completed in 10 minutes. A large amount of nitrogen gas was generated, and when the nitrogen gas became low, the reflux was continued for 1 hour. The reaction solution was concentrated at 90°C, 85mL of water and 0.1M aqueous sodium hydroxide solution (85mL) were added, washed with diethyl ether (2×75mL), and aqueous ammonium chloride solution (1M, 40mL) was added to the aqueous phase, placed in a refrigerator at 0°C to crystallize a large amount of White needle-like crystals of p-aminophenylarsine oxide were vacuum-dried to obtain 5.54 g of the product (yield 53%). Stored in KOH filled with Ar gas.

[0072] VTAF1: VTA1 (129mg,...

Embodiment 2

[0080] Embodiment 2: target probe pH titration test

[0081] pH titration experiments with target probes. Test solution pH with SartoriusbasicpH-MeterPB-20, take probe stock solution 10 -3 M200μL was diluted into 20mL of ultrapure aqueous solution to obtain 10 -5M probe aqueous solution. Use 2N hydrochloric acid and 1N NaOH solution to adjust the pH, and take a reading after the value in the pH meter is stable for 30 seconds. Then take 3mL solution in the cuvette with VarianCary100 UV-visible spectrophotometer and VarianCaryEclipse fluorescence photometer for absorption and emission spectrum testing. The pH titration curve of the target probe was obtained by fitting the maximum absorption value and emission value to the pH value in origin. From this it is possible to read out the optimal pH window where the probe is suitable for the application. see results figure 2 , under physiological conditions (pH7.4 or so), the probe can not be affected by external conditions and ...

Embodiment 3

[0083] Using fluorescence ratio detection technology and electrophoresis to carry out the in vitro test of adjacent thiol proteins, using reduced bovine serum albumin (rBSA) as a model protein, and the probe VTAF synthesized in the examples as a small molecule fluorescent probe, the selective , concentration gradient and kinetic tests.

[0084] 1. Test the selectivity of the probe for adjacent thiol proteins

[0085] Using 40 μM microcuvettes, the probe was diluted to 1 μM, and various chemicals and proteins containing different thiol forms were added. The test temperature was controlled at 37° C. by water bath circulation, and the excitation wavelength was 405 nm. Use the Scan module in the VarianCaryEclipse fluorescence photometer for spectral testing, and the resulting fluorescence data can be fitted and calculated in origin as needed. The result is as Figure 4 shown. It can be seen from the figure that the probe VTAF does not respond to various amino acids, especially...

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Abstract

The invention relates to a high-selectivity ratio-type fluorescent probe and application thereof and particularly relates to a compound represented by a formula I, a fluorescent probe represented by a formula II designed based on the formula I, and a detection composition and a detection kit containing the fluorescent probe. The invention also relates to a method for detecting or positioning vicinal-dithiol-containing proteins (VDPs) by using fluorescent molecules or the detection kit. The formula I and the formula II have structural formulae shown in the description, and the definition of all groups is as stated in the text.

Description

technical field [0001] The invention belongs to the field of fluorescent probes, and in particular relates to a high-selectivity ratio fluorescent probe and its application in the quantitative detection of adjacent sulfhydryl groups of living cell proteins and the fluorescence in situ imaging method of living cells. Background technique [0002] Changes in the intracellular redox environment profoundly affect various physiological functions in cells. The adjacent thiol protein, as the apex of the reducing end in the redox balance, is closely involved in the regulation of various redox, and plays a key role in the regulation of enzyme activity, cell signal transduction, and disease progression. Since the sulfhydryl modification of proteins basically has no spectroscopic properties, it cannot be directly measured, and the general detection method often needs to destroy the cell and then detect it by chemical methods, and it is usually impossible to obtain information about the...

Claims

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Application Information

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IPC IPC(8): C07F9/80C09K11/06G01N21/64
Inventor 朱维平黄楚森徐玉芳唐孟方钱旭红
Owner EAST CHINA UNIV OF SCI & TECH
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