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A kind of protein labeling method of ethynyl sulfate click reaction

A click reaction and protein labeling technology, applied in the fields of chemical biology and biology, can solve the problem that probes are difficult to be absorbed by cells

Active Publication Date: 2022-07-12
PEKING UNIV SHENZHEN GRADUATE SCHOOL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, they may accumulate non-specifically on the cell surface and in other cellular structures, resulting in difficult uptake of probes by cells

Method used

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  • A kind of protein labeling method of ethynyl sulfate click reaction
  • A kind of protein labeling method of ethynyl sulfate click reaction
  • A kind of protein labeling method of ethynyl sulfate click reaction

Examples

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example 1

[0028] Example 1: Synthesis of ethynyl sulfur salts and efficient click reactions with azides to generate 1,5-triazole cycloaddition products.

[0029] Based on previous literature, we synthesized a series of ethynyl sulfate substrates by a one-pot method. Synthesis of Ethynyl Sulfide from Dimethyl Sulfoxide The reaction equation is as follows:

[0030]

[0031] In a 100-mL three-necked flask, sulfoxide (5 mmol, 1.0 equiv.) was dissolved in 40 mL of dichloromethane with nitrogen ventilation, cooled to -50°C, and trifluoromethanesulfonic anhydride (5 mmol, 1.0 equiv.) was added dropwise. equiv.) was stirred at this temperature for 1 hour, and trimethylsilylalkynyl (5 mmol, 1 equiv.) was dissolved in 5 mL of dichloromethane and added dropwise to the reaction solution. The temperature was slowly raised to -15° C. and stirred for 6 hours. After the reaction was completed, the solvent was revolved to dryness in vacuo, and the crude product was recrystallized to obtain product...

example 2

[0041] Example 2: X-ray diffraction analysis of 1,5-triazole cycloaddition single crystal product 3g.

[0042] In order to fully elucidate the product structure, we carried out careful nuclear magnetic resonance (NMR) experiments and X-ray diffraction analysis of the 3g single crystal of the product 3g prepared by the method of Example 1, as shown in figure 2 shown. The distances of the C-N, C-C and N-N bonds in the cycloaddition structure of 1,5-triazole are consistent with the results for the five-membered ring.

example 3

[0043] Example 3: Secondary Mass Spectrometry Identification of the Use of Ethynyl Sulfate for Protein Labeling.

[0044] After confirming that ethynyl sulfur salts readily click-react with azides, we performed protein labeling experiments. In this labeling reaction, 2.5 mg / mL bovine albumin (BSA) was incubated with 1 mM azide alkylating reagent IAA-Az for 1 h at room temperature to install a bioorthogonal N 3 group, and then only incubated with ethynyl sulfate 2f in phosphate buffered saline PBS (pH 7.4) at 37°C for 1 hour. like image 3 As shown, the LC-MS / MS results showed that ethynyl sulfate 2f could undergo an efficient click reaction with azide-modified proteins.

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Abstract

The invention provides a protein labeling method for acetylene sulfur salt click reaction, especially a copper-free catalyzed "click" reaction in which acetylene sulfur salt and azide undergo cycloaddition in a water-soluble medium at room temperature. After the sample is modified with an azide reagent, a click reaction occurs with ethynyl sulfate. Ethynyl sulfur salt is synthesized from dimethyl sulfoxide, the structural formula is: R 1 One selected from hydrogen, alkyl, phenyl, benzyl or substituted benzyl, R 2 with R 3 One selected from alkyl, phenyl, phenothiazine, thianthrene or phenylphenothiazine. The invention does not need the catalysis of copper, can be rapidly carried out at room temperature, and solves the problems of the probes in the prior art that are too large in size, difficult to synthesize, and have poor solubility and cell penetration.

Description

technical field [0001] The invention relates to the fields of chemical biology and biotechnology, and relates to the characterization and application of a protein modification method, in particular to a protein labeling method for an ethynyl sulfate click reaction, in particular to a method for ethynyl sulfur in a water-soluble medium at room temperature A copper-free method for protein labeling of catalyzed "click" reactions by cycloaddition of salts and azides. Background technique [0002] The click reaction is one of the most efficient and widely used bioorthogonal reactions. This is a milestone in the field of chemical biology. Click React was named by K. Barry Sharpless in 1998 and published in 2001. Huisgen describes the click reaction as a [3+2] cycloaddition reaction between an azide and an alkyne. Sharpless and co-workers overcome the high temperature barrier of cycloaddition by developing Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) that is facile at phys...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
CPCG01N21/6486
Inventor 李子刚尹丰王跃娜侯占峰万川
Owner PEKING UNIV SHENZHEN GRADUATE SCHOOL
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