CN113447464B - Protein labeling method for ethynyl sulfonium salt click reaction - Google Patents

Protein labeling method for ethynyl sulfonium salt click reaction Download PDF

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CN113447464B
CN113447464B CN202110708506.8A CN202110708506A CN113447464B CN 113447464 B CN113447464 B CN 113447464B CN 202110708506 A CN202110708506 A CN 202110708506A CN 113447464 B CN113447464 B CN 113447464B
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ethynyl
iaa
click reaction
sample
detected
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CN113447464A (en
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李子刚
尹丰
王跃娜
侯占峰
万川
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Shenzhen Bay Laboratory Pingshan Biomedical R & D And Transformation Center
Peking University Shenzhen Graduate School
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Shenzhen Bay Laboratory Pingshan Biomedical R & D And Transformation Center
Peking University Shenzhen Graduate School
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    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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Abstract

The invention provides a protein labeling method of ethynyl sulfonium click reaction, in particular to copper-free catalysis click reaction of cycloaddition of ethynyl sulfonium and azide in a water-soluble medium at room temperature, wherein a sample to be detected is modified by an azide reagent and then carries out click reaction with the ethynyl sulfonium. The ethynyl sulfur salt is synthesized by dimethyl sulfoxide, and the structural formula is as follows:
Figure DDA0003129556150000011
R1one selected from hydrogen, alkyl, phenyl, benzyl or substituted benzyl, R2And R3One selected from alkyl, phenyl, phenoxathiin, thianthrene or phenylphenothiazine. The method can be quickly carried out at room temperature without catalysis of copper, and solves the problems of overlarge probe volume, difficulty in synthesis, poor solubility and cell penetrability and the like in the prior art.

Description

Protein labeling method for ethynyl sulfonium salt click reaction
Technical Field
The invention relates to the field of chemical biology and biotechnology, in particular to a protein labeling method of ethynyl sulfonium click reaction, and particularly relates to a protein labeling method of copper-free catalytic click reaction for cycloaddition of ethynylsulfonium and azide in a water-soluble medium at room temperature.
Background
The click reaction is one of the most efficient and widely used of bio-orthogonal reactions. This is a milestone in the field of chemical biology. Click-reactions were named by k.barry Sharpless in 1998 and published in 2001. Huisgen describes a click reaction as a [3+2] cycloaddition reaction between an azide and an alkyne. Sharpless and his colleagues overcome the high temperature barrier of cycloaddition by developing a Cu (I) catalyzed azide-to-alkyne cycloaddition reaction (CuAAC) that is readily performed at physiological temperatures and in a rich biological environment. Over the last two decades, there has been much development in the click reaction. The group of Bertozzi first reported a copper-free catalyzed click reaction in 2004 to overcome the cytotoxicity of the CuAAC reaction, also known as the tension-promoted azide-alkyne cycloaddition reaction (SPAAC). Subsequently, the jua and Folkin groups reported in 2005 ruthenium-catalyzed alkyne and azide cyclohexanediones. To date, dibenzocyclooctynes developed by the Bertozzi group have the highest reaction efficiency. It reacts faster with azide than any other reported compound, and this method has many applications in labeling cancer cells and surface glycans of zebrafish. This approach reduces cytotoxicity and improves the biocompatibility of the original copper-catalyzed click reaction.
On the other hand, cyclooctyne group is a probe having a large volume, and is a hydrophobic structure which is difficult to synthesize. Therefore, they may accumulate non-specifically on the cell surface and in other cell structures, thereby rendering the probes difficult to be taken up by the cells. Therefore, there is a need to develop probes with unobtrusive alkynyl size and better cell penetration. At the same time, the structure must also be easy to synthesize and react rapidly with azides in biological systems.
Therefore, the invention designs a simple synthesis method of the water-soluble ethynyl sulfide salt, successfully realizes the copper-free catalytic click reaction at room temperature, and is used for living cell marking and cell fluorescence imaging.
Disclosure of Invention
The invention provides a protein labeling method of ethynyl sulfonium click reaction, which comprises the following steps of modifying a sample to be detected with R azide, and carrying out click reaction with ethynyl sulfonium, wherein the chemical reaction formula is as follows:
Figure BDA0003129556130000021
wherein 2 is ethynyl sulfonium salt, 4 is a sample to be detected and modified by azide, and 3 is a generated 1, 5-triazole cycloaddition product;
wherein, the ethynyl sulfide is synthesized by dimethyl sulfoxide, and the structural general formula is shown in formula (I):
Figure BDA0003129556130000022
R1one selected from hydrogen, alkyl, phenyl, benzyl or substituted benzyl, R2And R3Is selected from one of alkyl, phenyl, phenoxathiin, thianthrene or phenylphenothiazine.
In the invention, the sample to be detected is one of pure albumin, a complex protein system or living cells.
In the invention, the azidation reagent is IAA-Az with the structural formula
Figure BDA0003129556130000023
In the invention, a sample to be detected is bovine albumin, an azidation reagent is IAA-Az, and after the reaction is carried out for one hour at room temperature, the sample to be detected is reacted with ethynyl sulfide salt in phosphate buffered saline at 37 DEG C
Figure BDA0003129556130000024
Incubate for 1 hour.
In the invention, a sample to be detected is cell lysate, an azidation reagent is IAA-Az, the sample is incubated with ethynyl sulfosalt ROX-AS for 1 hour at 25 ℃, and the chemical structural formula of the ethynyl sulfosalt ROX-AS is AS follows:
Figure BDA0003129556130000025
in the invention, a sample to be detected is a cell, the azide reagent is IAA-Az, the cell is incubated with the IAA-Az for 3 hours, then the cell is fixed and permeabilized, and the cell is incubated with ethinyl sulfonium salt ROX-AS for 2 hours.
The present invention utilizes dimethyl sulfoxide 1 to synthesize ethynylsulfonium salt 2, the chemical reaction formula of ethynylsulfonium salt 2 and azide 4 and the generated 1, 5-triazole cycloaddition product 3 according to the prior documents as follows:
Figure BDA0003129556130000031
the invention carries out systematic analysis on the influence of reactivity of the ethynyl sulfonium salt and the azide compound through careful Nuclear Magnetic Resonance (NMR) experiments, X-ray diffraction analysis and calculation research of Density Functional Theory (DFT). The results indicate that there is a correlation between the solution free energy (Δ Gsol) of the ethynyl sulfonium salt and the size of the alkyne bond angle and click reactivity.
The invention proves that the target protein can be efficiently modified based on the click reaction of the ethynyl sulfonium salt through a series of SDS-PAGE, In-gel fluorescence scanning, ESI-MS (MS/MS) and flow cytometry experiments.
The application of the ethynyl sulfonium salt-based click reaction in the aspect of living cell marking is proved through cell fluorescence imaging.
The invention has the beneficial effects that: the method of the invention can be rapidly carried out at room temperature without the catalysis of copper. Mainly solves the problems of overlarge probe volume, difficult synthesis, poor solubility and cell penetrability and the like in the prior art.
Drawings
FIG. 1 is a schematic representation of ethynyl sulfide salts for live cell labeling.
FIG. 2 is an X-ray diffraction analysis chart of 3g of a 1, 5-triazole cycloaddition single-crystal product of example 2.
FIG. 3 is the secondary mass spectrum identification of the use of ethynyl sulfide salt in protein labeling in example 3, wherein A is a reaction scheme, and B is a LC-MS/MS diagram.
FIG. 4 is the application of the click reaction of ethynyl sulfonium salt in example 4 in pure protein, complex protein system and living cell labeling, wherein, panel A is reaction schematic diagram, panels B and C are fluorescence scanning diagram, panel D is toxicity schematic diagram, and panel E is fluorescence intensity schematic diagram.
FIG. 5 shows the application of the click reaction of ethynyl sulfonium salt in cell fluorescence imaging in example 5.
Detailed Description
Example 1: and (3) synthesizing the ethynyl sulfonium salt and carrying out efficient click reaction with an azide compound to generate a 1, 5-triazole cycloaddition product.
Based on previous literature, we synthesized a series of ethynyl sulfonium salt substrates by a one-pot method. Synthesis of ethynyl sulfide from dimethyl sulfoxide
Figure BDA0003129556130000032
The reaction equation is as follows:
Figure BDA0003129556130000041
in a 100 ml three-necked flask, sulfoxide (5 mmol, 1.0 eq) was dissolved in 40 ml of dichloromethane under nitrogen, cooled to-50 ℃, trifluoromethanesulfonic anhydride (5 mmol, 1.0 eq) was added dropwise and stirred at this temperature for 1 hour, and trimethylsilylalkynyl (5 mmol, 1 eq) was dissolved in 5 ml of dichloromethane and added dropwise to the reaction mixture. Slowly heating to-15 ℃, stirring for 6 hours, after the reaction is finished, carrying out vacuum spin-drying on the solvent, and recrystallizing the crude product to obtain the product 2a-2 n.
Under the same experimental conditions, taking dimethyl sulfoxide derivatives 1a-1n as reactants respectively to obtain ethynyl sulfhydrate 2a-2n, wherein the structural formula and the yield are as follows:
Figure BDA0003129556130000042
we next started the click reaction with benzyl azide and 2b as substrates. The chemical reaction equation is as follows:
Figure BDA0003129556130000051
we have found that the 1, 5-triazole cycloaddition product can be detected in 50% yield by mixing benzyl azide with 2b in dichloromethane at room temperature for 2 hours. We then tested the suitability of the reaction in different solvents and found that the reaction can be carried out in PBS in water.
We hypothesize for ethynyl sulfonium salt that different R1Groups influence the reactivity of the reaction and thus replace 5 different Rs1The groups were separately reacted with benzyl azide to give products 2a-2e, and no significant increase in activity was observed.
We believe that the different sulphite centres have a greater influence on the reactivity. We designed six different sulfur salt centers 2f/2g/2j/2k/2i/2m to compare these structures, respectively with benzyl azide reaction, the product was 3f/3g/3j/3k/3i/3 m. We found 2g of 5- (alkynyl) dibenzothiophene to have the highest reactivity. Methylphenylalkynylthio salt 2j is most soluble in water. We hypothesize that molecules with strong electron action and low steric hindrance favor this click reaction (3g, 3j, 3 m). Finally we also tried to further improve nucleophilicity by fine tuning the structure of the sulfur salts. We found that compounds 2k and 2l did not have significantly increased reactivity. While the less basic phenyl-substituted nitrogen compound 2m proved to be 93% yield.
Under the same experimental conditions, the click reaction between different ethynyl sulfites 2a to 2n and benzyl azide to obtain products 3a to 3n (wherein 3o is the product of the 2j reaction), and the structural formula and the yield respectively correspond to the following:
Figure BDA0003129556130000061
example 2: x-ray diffraction analysis of 3g of a 1, 5-triazole cycloaddition single-crystal product.
To fully elucidate the structure of the product, we performed careful Nuclear Magnetic Resonance (NMR) experiments and X-ray diffraction analysis of 3g of single crystals of 3g of the product prepared as described in example 1, as shown in FIG. 2. The distances of the C-N, C-C and N-N bonds in the 1, 5-triazole cycloaddition structure are consistent with the results for the five-membered ring.
Example 3: and (3) identifying the application of the ethinyl sulfate in protein labeling by using a secondary mass spectrum.
After confirming that ethynylthio salts are susceptible to click reactions with azides, we performed protein labeling experiments. In this labeling reaction, 2.5mg/mL bovine albumin (BSA) was incubated with 1mM azide alkylating reagent IAA-Az at room temperature for 1 hour to install a bioorthogonal N3Groups, then incubated with ethynyl sulfide 2f for 1 hour only in phosphate buffered saline PBS (pH 7.4) at 37 ℃. As shown in FIG. 3, LC-MS/MS results show that ethynyl sulfide 2f can undergo a highly efficient click reaction with azide-modified proteins.
Example 4: the click reaction of the ethynyl sulfonium salt is applied to pure protein, complex protein systems and living cell markers.
To explore the kinetics and stoichiometry of the click reaction of ethynyl sulfide, we next performed protein labeling experiments with ethynylsulfide ROX-AS with a rhodamine fluorophore, 10uM BSA was saturated with 100uM ROX-AS in PBS (pH 7.4) at 37 ℃ for 1 hour; under the same reaction condition, 10uM BSA and ROX-AS with different concentrations react for one hour in a PBS solution, and the reaction efficiency is continuously enhanced along with the increase of the concentration of the ROX-AS. The azide-modified BSA click reactions with ROX-AS were labeled with fluorophores so that the reactions could be monitored by SDS-PAGE fluorescence scans. The experimental results show that the concentration-dependent click reaction can react quickly within 1 hour.
To confirm the high labeling efficiency of this click reaction in a complex cellular environment, IAA-Az treated MCF7 cell lysate (100 μ g) was incubated with ethinyl sulfite ROX-AS for 1 hour at 25 ℃. Fluorescence scanning in the gel revealed many single clear bands, indicating effective labeling of MCF7 cell lysates with ethinyl sulfate ROX-AS. We tested the cytotoxicity of the three methods by MTT assay. The cyclooctyne structure (DBCO) is the least cytotoxic, followed by the ethynylthio structure (ROX-AS), which is less toxic than the copper-catalyzed click reaction.
After the efficiency of this click reaction for labeling proteins in complex lysates was successfully demonstrated, we next compared ROX-AS with other copper-free catalyzed cyclooctyne structure DBCO to label viable cells. MCF7 cells were cultured in medium containing 50. mu.M IAA-Az for 3 hours to install azide groups on live cell proteins. Next, the cells were treated with ROX-AS/DBCO (10, 20 or 30. mu.M) for 2h and analyzed by flow cytometry. The fluorescence intensity of ROX-AS is obviously stronger than DBCO, and the ROX-AS is further determined to have good cell permeability compared with DBCO, so that living cells can be efficiently marked.
As shown in FIG. 4, in which, panel A is a reaction diagram, panels B and C are fluorescence scanning diagrams, panel D is a toxicity diagram, and panel E is a fluorescence intensity diagram.
Example 5: the application of the click reaction of the ethynyl sulfonium salt in the aspect of cell fluorescence imaging.
To trace proteins in living cells, we performed a cell fluorescence imaging experiment as shown in fig. 1. Viable MCF-7 cells were incubated with IAA-Az for 3 hours, then fixed and permeabilized. Subsequently, the samples were incubated with 50. mu.M ROX-AS in PBS solution (pH 7.4) for 2 hours at room temperature. As shown in fig. 5, the results show that both the red fluorescence of rhodamine and the blue fluorescence of DAPI are clearly visible, whereas the control sample is only slightly background fluorescence. These results indicate that ethynylthiolate click reactions provide a more efficient tool to follow proteins in living cells.

Claims (6)

1. A protein marking method of ethynyl sulfonium salt click reaction is characterized in that: the method comprises the following steps of modifying a sample R to be detected by an azide reagent, and then carrying out click reaction with ethynyl sulfonium salt, wherein the chemical reaction formula is as follows:
Figure DEST_PATH_IMAGE002
，
wherein 2 is ethynyl sulfonium salt, 4 is a sample to be detected and modified by azide, and 3 is a generated 1, 5-triazole cycloaddition product; the ethynyl sulfide salt 2 is selected from one of 2a-2n, and the specific structure of 2a-2n is shown as follows:
Figure DEST_PATH_IMAGE004
、
Figure DEST_PATH_IMAGE006
、
Figure DEST_PATH_IMAGE008
、
Figure DEST_PATH_IMAGE010
、
Figure DEST_PATH_IMAGE012
、
Figure DEST_PATH_IMAGE014
、
Figure DEST_PATH_IMAGE016
、
Figure DEST_PATH_IMAGE018
、
Figure DEST_PATH_IMAGE020
、
Figure DEST_PATH_IMAGE022
、
Figure DEST_PATH_IMAGE024
、
Figure DEST_PATH_IMAGE026
、
Figure DEST_PATH_IMAGE028
、
Figure DEST_PATH_IMAGE030
。
2. the method of claim 1, wherein: the sample to be detected is selected from one of pure albumin, a complex protein system or living cells.
3. The method of claim 1, wherein: the azidation reagent is IAA-Az, and the structural formula is as follows:
Figure 404858DEST_PATH_IMAGE031
。
4. the method of claim 3, wherein: the sample to be detected is bovine albumin, the azidation reagent is IAA-Az, and the bovine albumin and the azidation reagent IAA-Az react for one hour at room temperature and then react with ethynyl sulfide salt in phosphate buffered saline with the temperature of 37 ℃ and the pH value of 7.4
Figure DEST_PATH_IMAGE032
Incubate for 1 hour.
5. The method of claim 3, wherein: the sample to be detected is cell lysate, the azidation reagent is IAA-Az, the cell lysate modified by the azidation reagent IAA-Az is incubated with ethynyl sulfosalt ROX-AS for 1 hour at 25 ℃ and pH 7.4 phosphate buffered saline, and the structural formula of the ethynyl sulfosalt ROX-AS is AS follows:
Figure 400627DEST_PATH_IMAGE033
。
6. the method of claim 3, wherein: the sample to be detected is a cell, the azide reagent is IAA-Az, and the cell is incubated with the IAA-Az for 3 hours and then incubated with ethynyl sulfate ROX-AS for 2 hours at 25 ℃ and pH 7.4.
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CN110257051A (en) * 2019-06-10 2019-09-20 武汉大学 A kind of preparation method of the DNA functional quantum point based on click chemistry and its application in biomarker and detection

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