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Compound and method for in-situ detecting o-sulfhydryl protein in organism

A compound and protein technology, which is applied in the field of in situ detection of adjacent thiol proteins in organisms, can solve the problems of not being able to detect adjacent thiol proteins in situ, and achieve good redox tolerance and good fat solubility.

Active Publication Date: 2012-12-05
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the above two types of probes only focus on developing a protein labeling strategy and cannot be used for in situ detection of endogenous adjacent thiol proteins.

Method used

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  • Compound and method for in-situ detecting o-sulfhydryl protein in organism
  • Compound and method for in-situ detecting o-sulfhydryl protein in organism
  • Compound and method for in-situ detecting o-sulfhydryl protein in organism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] The synthetic route of the target probe using naphthalimide as fluorophore is as follows:

[0073]

[0074] VTA1: p-aminophenylarsine (10.85g, 50mmol) was dissolved in methanol, heated to reflux to make the solution clear, and 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, and 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.

[0075] VTA2: VTA1 (1.24g, 6.43mmol) was dissolved in anhydr...

Embodiment 2

[0082] The synthesis route of the target probe using NBD as the fluorophore is as follows:

[0083]

[0084] S6: VTA4 (122 mg, 0.328 mmol) was dissolved in 10 mL of dichloromethane, triethylamine (75 μL) was added, after stirring, NBD-Cl (78.3 mg, 0.393 mmol) was dissolved in 8 mL of dichloromethane and slowly added dropwise into the S5 solution and stirred overnight at room temperature. The reaction solution turned from light yellow to dark brown. Spin-dry solvent column chromatography, dichloromethane / methanol = 20 / 1 to 10 / 1 gradient elution to obtain 58 mg of tan solid (yield 33%). 1 H NMR (DMSO-d 6 , 400MHz) δ1.40(q, 2H), 1.66(m, 4H), 2.31(t, 2H, J=7.2Hz), 3.47(t, 2H, J=6.0Hz,), 6.42(d, 1H, J=9.2Hz), 7.00(t, 1H, J=7.2Hz), 7.26(t, 2H, J=7.6Hz), 7.55(d, 2H, J=7.6Hz), 8.49(d, 1H, J= 8.8Hz).

Embodiment 3

[0086] The target probe synthesis using fluorescein as the fluorophore is as follows:

[0087]

[0088] S8: Fluorescein (3.32g, 10mmol), N-hydroxysuccinimide (1.41g, 12mmol) and DCC (3.15g, 15mmol) were dissolved in 10mL of dry DMF, heated to 70 ~80°C, react for 1 hour. Cooled in an ice bath, a large amount of crystals precipitated, filtered, and the by-product dicyclohexyl urea (white crystals) was filtered off, the filter cake was washed with acetone until white, discarded; the filtrate was a deep red solution, concentrated, and directly went to Next do the next reaction.

[0089] S9: S8 (2.49 g, 5.77 mmol) was dissolved in 10 mL of DMF, piperazine (0.99 g, 11.55 mmol) and triethylamine (2.4 mL, 17.32 mmol) were added. The reaction solution container was covered with aluminum foil to avoid light, and stirred at room temperature for 14 hours under the protection of argon. Neutralize with acetic acid. The solvent was spin-dried to obtain a dark red oil; column chromatog...

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Abstract

The invention belongs to the technical field of biological detection, and specifically relates to a compound and a method for in-situ detecting o-sulfhydryl protein in an organism. A kind of micromolecular fluorescent probes capable of specifically detecting o-sulfhydryl of protein are designed and synthesized, and a method for in-situ fluorescence labeling and detection of endogenous o-sulfhydryl protein in the organism is built by combining fluorescence polarization technology, protein fluorescent electrophoresis and confocal fluorescence microtechnique.

Description

technical field [0001] The invention relates to a compound and a method for in-situ detection of adjacent thiol proteins in organisms, in particular to an in-situ fluorescence detection method for adjacent thiol proteins using naphthalimide compounds as fluorescent probes. Background technique [0002] Changes in the intracellular redox environment profoundly affect various physiological functions in cells. Adjacent sulfhydryl proteins, as the vertex of the reducing end in the redox balance, are closely involved in the regulation of various redox, and play a very important 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 distribution of adjacent thi...

Claims

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Application Information

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IPC IPC(8): C07F9/86C07F9/80C09K11/06G01N21/64G01N33/52
CPCY02P20/55
Inventor 朱维平杨弋黄楚森尹琴徐玉芳钱旭红陈孟杰王新
Owner EAST CHINA UNIV OF SCI & TECH
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