CN113912525A - Probe for modifying protein cysteine residue and preparation method thereof - Google Patents

Probe for modifying protein cysteine residue and preparation method thereof Download PDF

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CN113912525A
CN113912525A CN202111242530.3A CN202111242530A CN113912525A CN 113912525 A CN113912525 A CN 113912525A CN 202111242530 A CN202111242530 A CN 202111242530A CN 113912525 A CN113912525 A CN 113912525A
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李子刚
尹丰
万川
王蕊
杨冬燕
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Shenzhen Bay Laboratory Pingshan Biomedical R & D And Transformation Center
Peking University Shenzhen Graduate School
Shenzhen Bay Laboratory
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Peking University Shenzhen Graduate School
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    • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
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Abstract

The invention discloses a probe for modifying protein cysteine residues, which has the following structural general formula:
Figure DDA0003319729910000011
the invention also provides a preparation method of the probe for modifying protein cysteine residue, substrate halogenated beta-carbonyl compound I and thioether II are added into a reaction vessel, the structural formula of the halogenated beta-carbonyl compound I is shown as follows,
Figure DDA0003319729910000012
the structural formula of the thioether II is
Figure DDA0003319729910000013
The product is directly precipitated in the form of precipitate, or added with aether with the same volume of reaction solventAnd (4) precipitating a product. Experiments prove that the protein cysteine probe III is very safe to cells and can be used as a cysteine modified probe of living cells for development and exploration.

Description

Probe for modifying protein cysteine residue and preparation method thereof
Technical Field
The invention belongs to the field of biochemistry, and relates to a probe for modifying protein cysteine residues and a preparation method thereof, in particular to a beta-carbonyl sulfate derivative which can be used for modifying protein cysteine residues.
Background
The chemical modification of protein has important significance for the basic research of the structure and the function of proteome and the synthetic modification of biological macromolecular drugs. Electrophilic groups targeting the side chains of nucleophilic amino acids have been used in the manufacture of covalent ligands and drugs. Protein cysteine (Cys) is one of the essential amino acids in the organism and plays a crucial role in maintaining various physiological functions. The amino group of the cysteine side chain residue plays many important roles in the function of the protein. The sulfhydryl side chain is a good nucleophilic group, and chemical modification of the sulfhydryl side chain is expected to change the chemical structure of the protein and change the spatial structure of the protein, thereby improving the biological activity and function of the protein. Therefore, the chemical modification of cysteine in biological samples is an important means for researching the three-dimensional structure and physiological function of protein, is a method for directionally modifying the protein property, and has wide application prospect in protein engineering and omics research.
The sulfonium salt compound has good water solubility, is a common active functional group in a living organism, does not need other organic solvents when interacting with the living organism, and is environment-friendly and efficient. The sulfonium salt is used as a key structural unit of the probe, so that the sulfonium salt has better biocompatibility, lower biotoxicity, higher systemic property and higher selectivity, and the problem of high toxicity to cells of other amino acid probes such as iodoacetamide as cysteine probes can be solved, so that the sulfonium salt-containing protein cysteine probe has an important very wide application space in protein side chain chemical modification.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention provides a probe for modifying protein cysteine residues and a preparation method thereof, and the probe for modifying protein cysteine residues and the preparation method thereof aim to solve the technical problem of high toxicity of amino acid probes, such as iodoacetamide, as cysteine probes in the prior art.
The invention provides a probe for modifying protein cysteine residues, which has the following structural general formula:
Figure BDA0003319729890000011
Figure BDA0003319729890000021
wherein R is1Selected from: hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C6-C6 alkoxy, C6-C6 haloalkoxy, C6-C6 alkenyl, C6-C6 haloalkenyl, C6-C6 alkynyl, C6-C6 haloalkynyl, hydroxy, C6-C6 cycloalkyl, substituted alkylamino, substituted phenylamino, substituted piperidin-1-yl, substituted morpholin-1-yl, substituted tetrahydropyrrole-1-yl, phenyl, halogen substituted phenyl, C6-C6 alkyl substituted phenyl, C6-C6 haloalkyl substituted phenyl, C6-C6 cycloalkyl substituted phenyl, nitro substituted phenyl, C6-C6 alkenyl substituted phenyl, C6-C6 haloalkenyl substituted phenyl, C6-C6 cycloalkenyl substituted phenyl, C6-C6 alkynyl substituted phenyl, C2-C6 haloalkynyl-substituted phenyl, C3-C6 cycloalkynyl-substituted phenyl, or pyridyl;
R2、R3are respectively provided withSelected from: C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 haloalkenyl, C2-C6 alkynyl, C2-C6 haloalkynyl, phenyl, halogen-substituted phenyl, C1-C6 alkyl-substituted phenyl, C1-C6 haloalkyl-substituted phenyl, C3-C6 cycloalkyl-substituted phenyl, nitro-substituted phenyl, C2-C6 alkenyl-substituted phenyl, C2-C6 haloalkenyl-substituted phenyl, C3-C6 cycloalkenyl-substituted phenyl, C2-C6 alkynyl-substituted phenyl, C2-C6 haloalkynyl-substituted phenyl, C3-C6 cycloalkynyl-substituted phenyl, or pyridyl;
the halogen is fluorine, chlorine, bromine or iodine;
the alkyl, alkenyl and alkynyl are straight-chain or branched-chain alkyl; alkyl is selected by itself or as part of another substituent from methyl, ethyl, propyl, butyl, pentyl, hexyl or an isomer selected from isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl or tert-pentyl;
the haloalkyl group is selected from the group consisting of one or more of the same or different halogen atoms, and the haloalkyl group is selected from CH2Cl、CHCl2、CCl3、CH2F、CHF2、CF3、CF3CH2、CH3CF2、CF3CF2Or CCl3CCl2；
The cycloalkyl group is selected by itself or as part of another substituent from cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl;
the alkenyl group is selected as such or as part of another substituent from vinyl, allyl, 1-propenyl, buten-2-yl, buten-3-yl, penten-1-yl, penten-3-yl, hexen-1-yl or 4-methyl-3-pentenyl;
the alkynyl group is selected by itself or as part of another substituent from ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-2-yl, 1-methyl-2-butynyl, hexyn-1-yl or 1-ethyl-2-butynyl.
X is selected from: alkyl acid radical, substituted alkyl acid radical, halogen and its oxygen acid radical, phosphate radical, sulfate radical, sulfonate radical or borate radical.
The alkyl acid radical, the substituted alkyl acid radical is selected from C1-C6 alkyl acid radical or C1-C6 halogenated alkyl acid radical;
the halogen and the oxygen-containing acid radical thereof are selected from fluorine, chlorine, bromine or iodine, hypochlorite, chlorite, chlorate, perchlorate, hypobromite, bromate, perbromite, hypoiodite, periodate, iodate or periodate;
the phosphate is selected from monohydrogen phosphate, dihydrogen phosphate, pyrophosphate, metaphosphate, hypophosphite, phosphite, polyphosphate, phosphate or hexafluorophosphate;
the sulfate radical is selected from sulfur negative ion, bisulfate radical, sulfate radical, bisulfite radical, sulfite radical, pyrosulfate radical, dithionate radical, thiosulfate radical, dithionite radical or persulfate radical;
the sulfonate is selected from trifluoromethanesulfonate, methanesulfonate, phenylsulfonate or p-methylbenzenesulfonate;
the borate is selected from borate or tetrafluoroborate.
Specifically, the R is1Preferably selected from: phenyl, substituted alkylamino, substituted phenylamino; r2、R3Are respectively and preferably selected from: C1-C6 alkyl, C3-C6 cycloalkyl, substituted phenyl; x is preferably selected from: bromine or tetrafluoroborate.
The invention also provides a preparation method of the probe for modifying the protein cysteine residue, which comprises the following steps:
adding substrates halogenated beta-carbonyl compound I and thioether II into a reaction vessel, wherein the structural formula of the halogenated beta-carbonyl compound I is shown in the specification,
Figure BDA0003319729890000031
the structural formula of the thioether II is
Figure BDA0003319729890000032
The reaction solvent is selected from: dichloromethane, toluene, acetonitrile, diethyl ether, tetrahydrofuran, the product being in the form of a precipitateDirectly separating out the product in the formula, or adding aether with the same volume of reaction solvent to promote the separation of the product, wherein the yield is 82-96%; the amount of compound III produced and the volume of the reaction vessel are scaled up or down accordingly.
In particular, the thioether II is selected from: dimethyl sulfide, tetrahydrothiophene, methylphenyl sulfide, diphenyl sulfide or dibenzyl sulfide.
The synthesis method of the protein cysteine probe III of the invention is as follows:
Figure BDA0003319729890000041
the invention also provides the beta-carbonyl sulfonium salt compound or the derivative thereof.
The invention also provides the application of the probe or the intermediate thereof in protein cysteine modification.
The invention also provides a method for modifying protein cysteine residues by using the probe, which is characterized in that the protein to be modified is added, and the chemical modification site is the protein cysteine residue; protein cysteine probe III is dosed from 0.1 to 200 equivalents of protein.
Further, the reaction solvent is water or a polar organic solvent: the solvent is selected from any one of water, acetonitrile, methanol, ethanol, isopropanol, tert-butanol, ethylene glycol, glycerol, trifluoroethanol, hexafluoroisopropanol, dimethyl sulfoxide or N, N-dimethylformamide or a mixed solvent of any two of the above.
Further, the reaction time is 0.1 to 100 hours.
Further, the reaction temperature is-20 to 50 ℃.
The invention provides a high-efficiency chemical modification method of protein cysteine aiming at the requirements of chemical selective modification technology and application of protein cysteine residue, and the modification method aims to solve the high-efficiency selective chemical modification of the protein cysteine residue with beta-carbonyl sulfonium salt as an active functional group.
The invention has the beneficial effects that: the protein cysteine probe III is subjected to synthesis research, and the chemical biological activity of the protein cysteine probe III is explored, including protein chemical modification, chemical proteomics research and the like. Compared with the prior art (mainly comprising iodoacetamide compounds and the like), the cell membrane has obvious advantages in the aspects of water solubility, cell penetrability, cytotoxicity and the like.
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FIG. 1 shows the reaction of protein cysteine probe III with bovine serum albumin.
FIG. 2 shows the comparison of protein cysteine Probe III with the reaction with cell lysates and the toxicity of commercial probes.
Detailed Description
The synthesis and biological application of protein cysteine probe III are more specifically illustrated by specific preparation and chemical biological examples, which are only used for specifically illustrating the invention and not limiting the invention, and particularly, the biological application is only used for illustrating and not limiting the patent, and the specific embodiments are as follows:
example 1: preparation of Compound III-1:
Figure BDA0003319729890000051
a100 ml single neck round bottom flask was charged with 1.2 g of I and 1.5 ml of tetrahydrothiophene. 30 ml of the reaction solvent dichloromethane were added. After the reaction was monitored by TLC, the product precipitated directly as a precipitate, and was 1.4 g of white powder with 88% yield. The nuclear magnetic data for this compound are as follows:1H NMR(400MHz,D2O)δ7.91(d,J＝8.9Hz,2H),7.12–7.05(m,2H),4.81(d,J＝2.3Hz,2H),3.62(dt,J＝13.2,6.5Hz,2H),3.46(dt,J＝12.5,5.5Hz,2H),2.90(t,J＝2.3Hz,1H),2.27(dhept,J＝14.0,6.4,5.7Hz,4H).
synthesis and characterization data for compounds of the same type:
Figure BDA0003319729890000052
white powder, yield 85%.1H NMR(400MHz,D2O)δ7.96–7.89(m,2H),7.73–7.64(m,1H),7.52(t,J＝7.9Hz,2H),2.94(s,6H).
Figure BDA0003319729890000053
White powder, yield 89%.1H NMR(400MHz,D2O)δ7.92(d,J＝7.5Hz,2H),7.69(t,J＝7.4Hz,1H),7.52(t,J＝7.7Hz,2H),3.64(dt,J＝13.1,6.0Hz,2H),3.48(h,J＝5.6,4.5Hz,2H),2.28(pd,J＝13.3,12.7,4.8Hz,4H).
Figure BDA0003319729890000054
White powder, yield 81%.1H NMR(400MHz,D2O)δ7.97–7.89(m,2H),7.74–7.64(m,1H),7.55–7.47(m,2H),3.59–3.48(m,2H),3.25(ddd,J＝12.8,9.1,3.1Hz,2H),2.10(dtt,J＝14.8,7.3,3.4Hz,2H),1.90(dtt,J＝15.7,9.0,3.4Hz,2H),1.77–1.54(m,2H).
Figure BDA0003319729890000055
White powder, yield 74%.1H NMR(400MHz,D2O)δ7.41–7.30(m,4H),7.26–7.14(m,1H),2.93(s,6H).
Figure BDA0003319729890000061
White powder, yield 82%.1H NMR(400MHz,D2O)δ7.42–7.31(m,4H),7.26–7.15(m,1H),3.67–3.42(m,4H),2.38–2.16(m,4H).
Figure BDA0003319729890000062
White powder, yield 90%.1H NMR(400MHz,D2O)δ4.37(dd,J＝5.0,2.8Hz,1H),4.25(q,J＝7.1Hz,2H),2.94(s,6H),1.23(t,J＝7.1Hz,3H).
Figure BDA0003319729890000063
White powder, yield 91%.1H NMR(400MHz,D2O)δ4.33–4.20(m,3H),3.69–3.57(m,2H),3.57–3.46(m,2H),2.39–2.18(m,4H),1.24(t,J＝7.2Hz,3H).
Figure BDA0003319729890000064
White powder, yield 89%.1H NMR(400MHz,D2O)δ7.93–7.84(m,2H),7.06–6.97(m,2H),3.83(s,3H),3.67–3.56(m,2H),3.52–3.37(m,2H),2.37–2.17(m,4H).
Figure BDA0003319729890000065
Pale yellow powder, yield 88%.1H NMR(400MHz,D2O)δ8.35–8.27(m,2H),8.15–8.07(m,2H),3.73–3.62(m,2H),3.57–3.46(m,2H),2.41–2.20(m,4H).
Figure BDA0003319729890000066
Pale yellow powder, yield 81%.1H NMR(400MHz,D2O)δ8.19(dd,J＝8.3,1.1Hz,1H),7.85(td,J＝7.6,1.2Hz,1H),7.75(ddd,J＝8.3,7.6,1.5Hz,1H),7.59(dd,J＝7.6,1.5Hz,1H),3.74–3.63(m,2H),3.58–3.47(m,2H),2.39–2.22(m,4H).
Figure BDA0003319729890000067
White powder, yield 92%.1H NMR(400MHz,D2O)δ8.01–7.91(m,2H),7.26–7.16(m,2H),3.61(dt,J＝13.6,6.9Hz,2H),3.45(dt,J＝11.8,5.7Hz,2H),2.39–2.15(m,4H).
Figure BDA0003319729890000071
White powder, yield 94%.1H NMR(400MHz,D2O)δ7.90–7.82(m,2H),7.55–7.47(m,2H),3.62(dt,J＝13.6,6.8Hz,2H),3.46(dt,J＝13.8,6.6Hz,2H),2.35–2.16(m,4H).
Figure BDA0003319729890000072
White powder, yield 90%.1H NMR(400MHz,D2O)δ7.85–7.78(m,2H),6.92–6.84(m,2H),3.60(dt,J＝13.9,6.7Hz,2H),3.43(dt,J＝13.7,5.9Hz,2H),2.35–2.15(m,4H).
Figure BDA0003319729890000073
White powder, yield 83%.1H NMR(400MHz,D2O)δ7.96(dd,J＝5.0,1.1Hz,1H),7.90(dd,J＝3.9,1.1Hz,1H),7.21(dd,J＝5.0,4.0Hz,1H),3.68–3.55(m,2H),3.54–3.43(m,2H),2.38–2.17(m,4H).
Figure BDA0003319729890000074
White powder, yield 80%.1H NMR(400MHz,D2O)δ7.96–7.88(m,2H),7.13–7.05(m,2H),4.81(d,J＝2.2Hz,2H),2.92(s,6H),2.89(t,J＝2.4Hz,1H).
Figure BDA0003319729890000075
White powder, yield 74%.1H NMR(400MHz,D2O)δ7.54–7.46(m,2H),7.46–7.37(m,2H),4.51(s,2H),3.46(s,1H),2.97(s,6H).
Figure BDA0003319729890000076
White powder, yield 75%.1H NMR(400MHz,D2O)δ7.53–7.47(m,2H),7.43–7.37(m,2H),4.42(s,2H),3.67–3.60(m,2H),3.52(dt,J＝12.0,5.9Hz,2H),3.46(s,1H),2.38–2.24(m,4H).
Example 2: preparation of Compound III-2:
Figure BDA0003319729890000081
at 100 mmA liter Single neck round bottom flask was charged with 1.2 g of I, 2.5 ml of methyl phenyl sulfide and 1.9 g of silver tetrafluoroborate AgBF4. 30 ml of the reaction solvent dichloromethane were added. After TLC monitoring reaction is completed, using diatomite to carry out vacuum filtration, combining organic phases, carrying out vacuum concentration to remove excessive solvent, and purifying residues through 100-200-mesh silica gel column chromatography to obtain a compound III, wherein an eluent is dichloromethane to methanol with a volume ratio of 30:1, a product is 1.7 g of colorless oily matter, and the yield is 44%. The nuclear magnetic data for this compound are as follows:1H NMR(400MHz,Methanol-d4)δ8.10(d,J＝7.8Hz,2H),8.06–8.00(m,2H),7.81–7.71(m,3H),7.20–7.12(m,2H),5.11(s,2H),3.35(s,3H),2.83(s,1H).
synthesis and characterization data for compounds of the same type:
Figure BDA0003319729890000082
white powder, yield 61%.1H NMR(400MHz,MeOD)δ8.18–8.11(m,2H),8.11–8.03(m,2H),7.89–7.70(m,4H),7.60(t,J＝7.8Hz,2H),3.41(s,3H).
Figure BDA0003319729890000083
White powder, yield 71%.1H NMR(400MHz,MeOD)δ8.17–8.08(m,6H),7.88–7.72(m,7H),7.67–7.59(m,2H).
Example 3: reaction of the protein cysteine Probe III of the invention with an isolated protein:
to verify the reactivity of the protein cysteine probe covalently bound to cysteine at the protein label level, the protein was solubilized with distilled water and configured into a5 μ M to 100 μ M protein solution. Accurately weighing the protein cysteine probe III, and preparing a reaction solution with a corresponding concentration by using PBS buffer solution or distilled water. The protein solution was incubated with the probe in PBS solution at 37 ℃. Then, a 'click' reaction is used for marking a fluorescent label on the protein, and the specific method is to add CuSO into the reaction system4，TECP，TBTA，5-TAMRA-N3The reaction was terminated by incubation at 25 ℃. And finally, running SDS-PAGE protein gel and observing fluorescence in the gel. The protein may beBovine Serum Albumin (BSA), Horse Serum Albumin (HSA).
From the labeling experiments of bovine serum albumin in fig. 1a and 1b, we observe that the sulfur salt probe has stronger fluorescence, and CP1, CP2 and CP3 all show stronger fluorescence labeling capability, especially the high fluorescence labeling capability of CP2 exceeds that of the commercial cysteine probe IAA-alkyne, so we select CP2 for subsequent protein labeling reaction. From FIG. 1c, it is found that the CP2 probe has a certain fluorescence intensity after reacting with protein for 10min, and the fluorescence intensity of the reaction with protein is increased along with the extension of the reaction time, which proves that the reaction speed of the protein cysteine probe III of the present invention with bovine serum albumin is faster. At the reaction dose (FIG. 1d), the probe concentration of 5. mu.M reacted with bovine serum albumin to show a certain fluorescence, while the fluorescence gradually increased with the increase of CP2 concentration, and the probe concentration had an important influence on the fluorescence intensity.
The commercial cysteine probe IAA-alkyne is used as a control to research the specific labeling of the probe of the invention on the cysteine. IAA is a commercially available blocking reagent for cysteine, CP-B is a probe of the present invention for cysteine blocking. From FIG. 1e, it is shown that the reaction of bovine serum albumin with CP2 probe is competed by IAA and CP-B, and the fluorescence is gradually blocked with increasing concentration of IAA and CP-B, indicating that the label can be blocked by commercial cysteine blocking reagent, and the trend of the fluorescence change of the probe CP2 of the present invention is consistent with that of commercial cysteine probe IAA-alkyne, thus proving that the protein cysteine probe III of the present invention selectively acts on protein cysteine residue.
Example 4: reaction of the protein cysteine probe III of the invention with cell lysate:
harvested cells were lysed by sonication and concentrated. Accurately weighing the protein cysteine probe III, and preparing a reaction solution with a corresponding concentration by using PBS buffer solution or distilled water. Cell lysates were diluted to appropriate concentrations and incubated with probes in PBS solution at 37 ℃. Then, a 'click' reaction is used for marking a fluorescent label on the protein, and the specific method is to add CuSO into the reaction system4，TECP，TBTA，5-TAMRA-N3The reaction was terminated by incubation at 25 ℃. And finally, running SDS-PAGE protein gel and observing fluorescence in the gel. The cell can be A549, 293T, Hela, MCF-7 and the like.
From the labeling experiments of the cell lysates in fig. 2a and 2b, we observed that the carbonyl sulfide probe CP2 has a good fluorescent labeling effect on cells, and the CP2 probe has a certain fluorescence intensity after reacting with the cell lysate for 10min, and simultaneously the fluorescence intensity of the reaction with the protein increases with the extension of the reaction time, and the fluorescence tends to be balanced within 3-6 h. In the reaction dose (FIG. 2b), the probe reacts with the cell lysate at a CP2 concentration of 5. mu.M to show a certain fluorescence, and the fluorescence gradually increases with the increase of the CP2 concentration, so that the probe has a good labeling effect at a low concentration.
A commercial cysteine probe IAA-alkyne is also adopted in the cell lysate as a control, a commercial cysteine blocking reagent IAA is used for blocking cysteine, and CP-B is used for the cysteine-blocked probe. From FIG. 2d, it is found that the reaction between the cell lysate and the CP2 probe can be competed by IAA and CP-B, the fluorescence is gradually blocked with the increase of the concentration of IAA and CP-B, and the fluorescence labeling effect of the probe CP2 on the cell lysate is consistent with the trend of the commercial cysteine probe IAA-alkyne, thereby further showing that the cysteine probe III of the present invention can selectively act on protein cysteine residues from the cell lysate level.
Example 5: toxicity study of the protein cysteine Probe III of the present invention:
the protein cysteine probe III and the cytotoxicity determination method specifically comprise the following steps: in a 96-well plate, approximately 5000 cells per well, protein lysine probe III was incubated with the cells for 20 hours, and then MTT reagent was added to continue the incubation for 4 hours. After removing the medium, water-insoluble blue-violet formazan was dissolved by adding DMSO, and the absorbance was measured by a microplate reader, and the relative cell viability was calculated as compared with a control.
Meanwhile, compared with the commercial probe IAA-alkyne, the CP2 probe is safer to cells. From FIG. 2c, the survival rate of cells treated with CP2 decreased slowly with increasing concentration of the drug, but decreased rapidly with commercial IAA-alkyne, and the survival rate of cells treated with CP2 at 100. mu.M was close to 80%, while the survival rate of cells treated with commercial IAA-alkyne at this concentration was only 30%, demonstrating that the protein cysteine probe III of the present invention is very safe for cells and can be developed and explored as a cysteine modified probe for living cells.

Claims (10)

1. A probe for modifying protein cysteine residues, which is characterized by having the following structural general formula:
Figure FDA0003319729880000011
wherein:
R1selected from: hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C6-C6 alkoxy, C6-C6 haloalkoxy, C6-C6 alkenyl, C6-C6 haloalkenyl, C6-C6 alkynyl, C6-C6 haloalkynyl, hydroxy, C6-C6 cycloalkyl, substituted alkylamino, substituted phenylamino, substituted piperidin-1-yl, substituted morpholin-1-yl, substituted tetrahydropyrrole-1-yl, phenyl, halogen substituted phenyl, C6-C6 alkyl substituted phenyl, C6-C6 haloalkyl substituted phenyl, C6-C6 cycloalkyl substituted phenyl, nitro substituted phenyl, C6-C6 alkenyl substituted phenyl, C6-C6 haloalkenyl substituted phenyl, C6-C6 cycloalkenyl substituted phenyl, C6-C6 alkynyl substituted phenyl, C2-C6 haloalkynyl-substituted phenyl, C3-C6 cycloalkynyl-substituted phenyl, or pyridyl;
R2、R3are respectively selected from: C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 haloalkenyl, C2-C6 alkynyl, C2-C6 haloalkynyl, phenyl, halogen-substituted phenyl, C1-C6 alkyl-substituted phenyl, C1-C6 haloalkyl-substituted phenyl, C3-C6 cycloalkyl-substituted phenyl, nitro-substituted phenyl, C2-C6 alkenyl-substituted phenyl, C2-C6 haloalkenyl-substituted phenyl, C3-C6 cycloalkenyl-substituted phenyl, C2-C6 alkynyl-substituted phenyl, C2-C6 haloalkynyl-substituted phenyl, C3-C6 cycloalkynyl-substituted phenyl, or pyridyl;
the halogen is fluorine, chlorine, bromine or iodine;
the alkyl, alkenyl and alkynyl are straight-chain or branched-chain alkyl; alkyl is selected by itself or as part of another substituent from methyl, ethyl, propyl, butyl, pentyl, hexyl or an isomer selected from isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl or tert-pentyl;
the haloalkyl group is selected from the group consisting of one or more of the same or different halogen atoms, and the haloalkyl group is selected from CH2Cl、CHCl2、CCl3、CH2F、CHF2、CF3、CF3CH2、CH3CF2、CF3CF2Or CCl3CCl2；
The cycloalkyl group is selected by itself or as part of another substituent from cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl;
the alkenyl group is selected as such or as part of another substituent from vinyl, allyl, 1-propenyl, buten-2-yl, buten-3-yl, penten-1-yl, penten-3-yl, hexen-1-yl or 4-methyl-3-pentenyl;
the alkynyl group is selected by itself or as part of another substituent from ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-2-yl, 1-methyl-2-butynyl, hexyn-1-yl or 1-ethyl-2-butynyl.
X is selected from: alkyl acid radical, substituted alkyl acid radical, halogen and its oxygen acid radical, phosphate radical, sulfate radical, sulfonate radical or borate radical.
The alkyl acid radical, the substituted alkyl acid radical is selected from C1-C6 alkyl acid radical or C1-C6 halogenated alkyl acid radical;
the halogen and the oxygen-containing acid radical thereof are selected from fluorine, chlorine, bromine or iodine, hypochlorite, chlorite, chlorate, perchlorate, hypobromite, bromate, perbromite, hypoiodite, periodate, iodate or periodate;
the phosphate is selected from monohydrogen phosphate, dihydrogen phosphate, pyrophosphate, metaphosphate, hypophosphite, phosphite, polyphosphate, phosphate or hexafluorophosphate;
the sulfate radical is selected from sulfur negative ion, bisulfate radical, sulfate radical, bisulfite radical, sulfite radical, pyrosulfate radical, dithionate radical, thiosulfate radical, dithionite radical or persulfate radical;
the sulfonate is selected from trifluoromethanesulfonate, methanesulfonate, phenylsulfonate or p-methylbenzenesulfonate;
the borate is selected from borate or tetrafluoroborate.
2. The probe for modifying cysteine residues of a protein according to claim 1, wherein R is1Selected from: phenyl, substituted alkylamino, or substituted phenylamino; r2、R3Are respectively selected from: C1-C6 alkyl, C3-C6 cycloalkyl, or substituted phenyl; x is selected from: bromine or tetrafluoroborate.
3. The method for preparing a probe for modifying a cysteine residue of a protein according to claim 1, comprising the steps of:
adding substrates halogenated beta-carbonyl compound I and thioether II into a reaction vessel, wherein the structural formula of the halogenated beta-carbonyl compound I is shown in the specification,
Figure FDA0003319729880000021
the structural formula of the thioether II is
Figure FDA0003319729880000022
The reaction solvent is selected from: dichloromethane, toluene, acetonitrile, diethyl ether or tetrahydrofuran, and the product is precipitated directly or is promoted to precipitate by adding the same volume of diethyl ether as the reaction solvent.
4. The method according to claim 3, wherein the thioether II is selected from the group consisting of: dimethyl sulfide, tetrahydrothiophene, methylphenyl sulfide, diphenyl sulfide or dibenzyl sulfide.
5. A β -carbonyl sulfonium salt compound or a derivative thereof as defined in claim 1.
6. Use of the probe of claim 1 or an intermediate thereof for cysteine modification of a protein.
7. The method for modifying protein cysteine residues by using the probe of claim 1, wherein a protein to be modified is added, and the chemical modification site is a protein cysteine residue; protein cysteine probe III is dosed from 0.1 to 200 equivalents of protein.
8. The method for modifying cysteine residues in proteins using the probe according to claim 7, wherein the reaction solvent is water or a polar organic solvent: the solvent is selected from any one of water, acetonitrile, methanol, ethanol, isopropanol, tert-butanol, ethylene glycol, glycerol, trifluoroethanol, hexafluoroisopropanol, dimethyl sulfoxide or N, N-dimethylformamide or a mixed solvent of any two of the above.
9. The method of claim 7, wherein the reaction time is 0.1 to 100 hours.
10. The method for modifying cysteine residues in a protein according to claim 7, wherein the reaction temperature is from-20 to 50 ℃.
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