Protein labeling method for acetenyl sulfosalt 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: 2021-09-28
PEKING UNIV SHENZHEN GRADUATE SCHOOL +1
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  • 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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  • Protein labeling method for acetenyl sulfosalt click reaction
  • Protein labeling method for acetenyl sulfosalt click reaction
  • Protein labeling method for acetenyl sulfosalt click reaction

Examples

Experimental program
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Effect test

example 1

[0028] Example 1: Synthesis of ethynylsulfur salt and efficient click reaction with azide compound to generate 1,5-triazole cycloaddition product.

[0029] Based on previous literature, we synthesized a series of ethynylsulfonate substrates via 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 equivalent) was dissolved in 40 ml of dichloromethane with nitrogen gas exchange, cooled to -50 ° C, and trifluoromethanesulfonic anhydride (5 mmol, 1.0 equivalent) was stirred at this temperature for 1 hour, trimethylsilyl alkynyl (5 mmol, 1 equivalent) was dissolved in 5 ml of dichloromethane, and added dropwise to the reaction solution. Slowly raise the temperature to -15°C and stir for 6 hours. After the reaction, the solvent is spin-dried in vacuo, and the crude product is recrystallized to obtain products 2a-2n.

[0032] Under the same experiment...

example 2

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

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

example 3

[0043] Example 3: Identification of acetylene sulfide salts in protein labeling by secondary mass spectrometry.

[0044] After confirming that ethynyl sulfide easily undergoes a click reaction with azide, 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 at room temperature for 1 hr to install a bioorthogonal N 3 group, and then incubated with ethynylsulfonate 2f only in phosphate-buffered saline PBS (pH 7.4) at 37°C for 1 hr. Such as image 3 As shown, the LC-MS / MS results show that ethynylsulfonate 2f can undergo efficient click reaction with azide-modified proteins.

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Abstract

The invention provides a protein labeling method for an ethynyl sulfosalt click reaction, and particularly relates to a method for carrying out a copper-free catalytic click reaction of cycloaddition of ethynyl sulfosalt and azide in a water-soluble medium at room temperature, and carrying out a click reaction on a sample to be detected and ethynyl sulfosalt after the sample to be detected is modified by an azide reagent. The ethynyl sulfur salt is synthesized by dimethyl sulfoxide, and the structural formula is as follows: R1 is selected from one of hydrogen, alkyl, phenyl, benzyl or substituted benzyl, and R2 and R3 are selected from one of alkyl, phenyl, phenoxathiane, thianthrene or phenyl phenothiazine. The method does not need catalysis of copper, can be rapidly carried out at room temperature, and solves the problems that in the prior art, a probe is too large in size, difficult to synthesize, poor in solubility and cell penetrability and the like.

Description

technical field [0001] The present invention relates to the field of chemical biology and biotechnology, and relates to the characterization and application of a protein modification method, in particular to a protein labeling method of acetylene sulfide click reaction, especially a method for acetylene sulfide in a water-soluble medium at room temperature. Copper-free catalyzed "click" reaction for protein labeling by salt and azide cycloaddition. 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. The click reaction was named by K.Barry Sharpless in 1998 and published in 2001. Huisgen described click reactions as [3+2] cycloaddition reactions between azides and alkynes. Sharpless and co-workers overcame the high-temperature barrier of cycloaddition reactions by developing Cu(I)-catalyzed cycloaddition of azides to alkynes (CuAAC), which is facile at p...

Claims

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Application Information

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Patent Type & Authority Applications(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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