Fluorescent labeled molecule with alkenyl sulfonyl groups and method for labeling protein by virtue of fluorescent labeled molecule

A technology of alkenylsulfonyl group and fluorescent labeling, which is applied in peptide preparation methods, chemical instruments and methods, luminescent materials, etc., can solve the problems of poor stability of linking molecules or protein conjugates, and achieve the effect of improving stability

Active Publication Date: 2015-06-24
上海中科润达精准医学检验有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It aims to solve the technical problem of poor stability of protein fluorescently labeled linker molecules or protein conjugates in the prior art

Method used

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  • Fluorescent labeled molecule with alkenyl sulfonyl groups and method for labeling protein by virtue of fluorescent labeled molecule
  • Fluorescent labeled molecule with alkenyl sulfonyl groups and method for labeling protein by virtue of fluorescent labeled molecule
  • Fluorescent labeled molecule with alkenyl sulfonyl groups and method for labeling protein by virtue of fluorescent labeled molecule

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1: the synthesis of compound (Ia)

[0056]

[0057] The synthetic route is as follows:

[0058]

[0059] Concrete synthetic steps are as follows:

[0060] (1) Compound 2a (i.e. coumarin 3-carboxylate) (1.9g, 10mmol) was dissolved in 15mL SOCl 2 , the reaction solution was refluxed for 1 hour, and the remaining SOCl 2 , the obtained product compound 4a was directly subjected to the next reaction without purification.

[0061] (2) Add compound 4a prepared above to a solution of compound 3a (3g, 12mmol) and triethylamine (2mL, 15mmol) in dichloromethane under ice bath, and the reaction solution was stirred at room temperature for 10 minutes. Add NaHCO 3 The reaction was quenched with saturated solution and extracted with dichloromethane, and the organic phase was once washed with NaHCO 3 Saturated solution, 1M HCl, and water were washed, dried over anhydrous sodium sulfate, spin-dried, and passed through a column (petroleum ether / ethyl acetate=2:3) to...

Embodiment 2

[0064] Embodiment 2: the synthesis of compound (IIa)

[0065]

[0066] 2-Chloroethanesulfonyl chloride (209 μL, 2 mmol) was added dropwise to a solution of 7-aminocoumarin (134 mg, 0.83 mmol) and triethylamine (0.3 mL, 2 mmol) in 5 mL of dichloromethane, and reacted at room temperature for 5 minutes. Add water to quench the reaction, then extract with dichloromethane, anhydrous Na 2 SO 4 After drying, it was passed through the column to obtain the product Compound (IIa) (164 mg, 79% purification yield) as a colorless liquid. m / z[M+H] + =252.

Embodiment 3

[0067] Embodiment 3: the synthesis of compound (IIIa)

[0068]

[0069] The synthetic route is as follows:

[0070]

[0071] Concrete synthetic steps are as follows:

[0072] (1) Add DCC (412mg, 2mmol) to the ethyl acetate solution of 7-aminocoumarin (compound 8a) (322mg, 2mmol) and compound 9a (494mg, 2mmol), stir at room temperature for one hour, filter, and pass The column gave the product compound 10a (585 mg, 75% purified yield) m / z [M+H] + =363.

[0073] (2) Compound 10a (585mg, 1.79mmol) was dissolved in TFA, the reaction was stirred for 30 minutes, the solvent was spun out, and washed with ether to obtain the product Compound 11a (508mg, 98%) m / z[M+H] + =263.

[0074] (3) Add 2-chloroethanesulfonyl chloride (183 μL, 1.75 mmol) dropwise to a solution of compound 11a (508 mg, 1.75 mmol) and triethylamine (0.25 mL, 1.75 mmol) in 5 mL of dichloromethane, and react at room temperature for 5 minutes . Add water to quench the reaction, then extract with dichlorome...

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Abstract

The invention discloses a fluorescent labeled molecule with alkenyl sulfonyl groups and a method for labeling protein by virtue of the fluorescent labeled molecule. The fluorescent labeled molecule is prepared by linking alkenyl sulfonyl amide with fluorescent groups and is coupled with protein by virtue of Michael addition reaction between alkenyl of the fluorescent labeled molecule and amide of amino acid in protein. According to the fluorescent labeled molecule, by virtue of alkenyl sulfonyl amide, the solubility of the fluorescent labeled molecule can be improved, furthermore, alkenyl sulfonyl amide has very high stability in water, and alkenyl in alkenyl sulfonyl amide can cause the Michael addition reaction with amide of protein at a relatively mild condition, for instance, in a phosphate buffer solution and a Tris buffer solution, and after the fluorescent labeled molecule is coupled with protein, the stability of the fluorescent labeled molecule in human serum is greatly improved, so that the fluorescent labeled molecule has very good application prospect in fluorescent labeling of protein.

Description

technical field [0001] The invention belongs to the technical field of protein fluorescent labeling, and in particular relates to a fluorescent labeling molecule with an alkenylsulfonyl group and a protein labeling method thereof. Background technique [0002] There are many ways to use certain substances to label proteins to detect and analyze them. Commonly used methods include isotope labeling and fluorescent labeling. Isotope labeling is more harmful to the human body and requires special equipment, so this method is seldom used at present; but it can achieve the purpose of quantitative detection and analysis. More widely used is the use of fluorescein to label proteins. [0003] In the current methods for labeling proteins with fluorescein, the linking molecules used are mainly succinimide esters and isothiocyanates. Succinimide ester is a relatively reliable group for modifying proteins, and it is easy to store, but the linking group is also easy to react with the su...

Claims

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Application Information

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IPC IPC(8): C07D311/12C07D311/16C07D493/10C09K11/06C07K1/13
CPCC07D311/12C07D311/16C07D493/10C07K1/13C09K11/06C09K2211/1088
Inventor 姜标曹刚杨建平
Owner 上海中科润达精准医学检验有限公司
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