A ligand-free palladium-catalyzed cysteine ​​coupling method

Abstract

The present application discloses a palladium-catalyzed cysteine ​​coupling method without the assistance of a ligand, and relates to the technical field of protein labeling, including. The present invention has discovered a new and simple catalytic system through the screening of metal palladium reagents: a combination of palladium nitrate and sodium ascorbate, which can selectively label the cysteine ​​residues of proteins. The reaction system abandons complex and expensive organic ligands and uses highly water-soluble Pd (II) metal salts, which are reduced to Pd with catalytic ability under the action of a reducing agent, thereby catalyzing the reaction between the iodobenzene reaction group and the cysteine ​​residue. The cysteine ​​coupling probe prepared by the present invention uses the iodobenzene reaction group as a substrate, and performs a palladium-catalyzed direct coupling reaction of cysteine ​​residues without the assistance of a ligand in pure proteins and cell lysates, providing a new screening platform for the discovery of covalent inhibitors targeting cysteine ​​residues, and will be used in the development of chemical proteomics technology and covalent drug screening in the future.

Description

Technical Field

[0001] The invention relates to the technical field of protein labeling, and in particular to a palladium-catalyzed cysteine ​​coupling method without the assistance of a ligand. Background Art

[0002] Chemical proteomics is a new cross-technology platform that integrates proteomics and chemical probes. It can directly study the function of the proteome using chemical small molecules and analyze the target distribution of drug molecules. Therefore, one of the elements of chemical proteomics technology is small molecule active probes, including derivatized probes based on the structure of drug molecules and universal probes targeting specific amino acid residues. Among them, amino acid residues that can be specifically reacted by universal probes include cysteine, lysine, serine, etc. This type of probe is often used in the screening of covalent inhibitors or drugs, and therefore plays an important role in the drug discovery stage.

[0003] Chemical probe labeling methods for cysteine ​​can be divided into two categories: the first category of reactive groups includes electrophilic reagents such as α-haloamide, maleimide, α-halocarbonyl, 3-phenylpropionitrile or pyridyl disulfide. Although chemical probes represented by iodoacetamide reactive groups have been widely used in cysteine ​​acyl selective labeling, problems such as off-target lysine residues have also attracted the attention of researchers. Chemical probes represented by maleimide have a fast reaction rate, but the products after reaction with cysteine ​​residues are not stable in mass spectrometry detection, which also limits their application in mass spectrometry-based chemical proteomics. The second category is metal-catalyzed cysteine ​​residue-specific coupling methods. Palladium is a key transition metal in modern organic synthesis and plays an important role in many chemical reactions. The development of palladium-catalyzed cysteine ​​residue coupling reagents is a research hotspot.

[0004] However, the palladium-catalyzed cysteine ​​residue-specific coupling method has the following problems: 1. Expensive organic ligands need to be used to couple with palladium to jointly catalyze the reaction; 2. 2-10 times the equivalent of divalent palladium complexes need to be added to the reaction; 3. The reaction system is mostly a purified protein system (reference JACS 2020.142.20.9124-9129). If the coupling reaction is carried out under physiological conditions, such as in cell lysate, it must be completed with the assistance of a ligand (reference JACS2016.138.28.8678-8681).

[0005] Therefore, how to develop a catalytic method that does not require ligand assistance so that small molecule active probes can label target proteins under relatively mild conditions is a problem that needs to be solved at present. Summary of the invention

[0006] In order to solve the above problems, the present application aims to develop a palladium-catalyzed cysteine ​​coupling method that does not require the assistance of a ligand, and further expand the application of this method to complex cell lysate systems, which can label active cysteine ​​residues under endogenous physiological conditions, and provide a new chemical probe labeling strategy for cysteine ​​targeted drug screening.

[0007] The present invention provides the following technical solutions:

[0008] A cysteine-coupled probe, the structure of which is shown in formula (I):

[0009]

[0010] Wherein, X is Cl, Br or I.

[0011] In order to synthesize the above-mentioned cysteine-coupled probe, the present invention also provides a compound intermediate, whose structure is shown in formula (II):

[0012]

[0013] Wherein, X is Cl, Br or I.

[0014] The preparation method of the above cysteine-coupled probe comprises the following reaction:

[0015]

[0016] Compound II and Compound III are mixed and dissolved, and then stirred at room temperature for reaction. The reaction product is concentrated and purified by column chromatography to obtain Compound I.

[0017] Preferably, the molar ratio of compound II to compound III is 1 to 3:1.

[0018] The preparation method of the compound of formula (II) comprises the following reaction:

[0019]

[0020] Preferably, the molar ratio of compound V to compound IV is 1:10-30.

[0021] The preparation method of the compound of formula (II) also includes the following reaction:

[0022]

[0023] Preferably, the molar ratio of compound VII to compound VI is 1:0.8-1.1.

[0024] The above-mentioned cysteine ​​coupling probe is used for palladium-catalyzed cysteine ​​residue-specific coupling labeling, and the specific steps include:

[0025] S1, adding the compound I solution into the palladium nitrate aqueous solution and mixing;

[0026] S2, add DMSO to the mixture of S1 and mix well;

[0027] S3, adding sodium ascorbate aqueous solution to the mixture in S2 and mixing to obtain a labeling reagent;

[0028] S4, add the labeling reagent to the protein solution and shake the reaction in the dark;

[0029] S5. After the reaction is completed, add 3-mercaptopropionic acid to terminate the reaction to complete the labeling of cysteine ​​residues in the protein.

[0030] Preferably, the solvent of the Compound I solution is DMSO or phosphate buffer, and the protein solution is the supernatant of cell lysate centrifugation.

[0031] Preferably, the concentration of Compound I in the Compound I solution is 10-30 mM; the concentration of the aqueous palladium nitrate solution is 5-20 mM; the concentration of the aqueous sodium ascorbate solution is 100-900 mM; and the final concentration of the 3-mercaptopropionic acid is 0.02-0.1%.

[0032] Preferably, the volume ratio of the compound I solution to the palladium nitrate aqueous solution is 1:1-3; the volume of the compound I solution: DMSO: sodium ascorbate aqueous solution is 1-4:2-15:1; and the volume ratio of the labeling reagent to the protein solution is 1:25-50.

[0033] Preferably, the light-proof shaking reaction condition is a light-proof shaking reaction at room temperature for 0.5 to 2 hours.

[0034] Beneficial effects achieved by the present invention:

[0035] The present invention provides a novel and simple catalytic system by combining palladium nitrate and sodium ascorbate. The iodobenzene reaction group is used as a substrate to form a cysteine ​​labeling reaction system together with palladium nitrate and sodium ascorbate. The combination of palladium nitrate and sodium ascorbate can selectively label the cysteine ​​residues of proteins. The reaction system abandons complex and expensive organic ligands and uses highly water-soluble Pd(II) metal salts. Under the action of a reducing agent, the Pd is reduced to Pd with catalytic ability, thereby catalyzing the reaction of the iodobenzene reaction group with the cysteine ​​residues, realizing direct coupling reaction of the cysteine ​​residues under physiological conditions, providing a new screening platform for the discovery of covalent inhibitors targeting cysteine ​​residues, and will be applied to chemical proteomics technology development and covalent drug screening in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1Schematic diagram of the principle of palladium nitrate-catalyzed coupling of cysteine ​​residues.

[0037] Figure 2 This is the structure of the 5-TAMRA-IB probe.

[0038] Figure 3 This is the labeling signal diagram of pure protein by 5-TAMRA-IB probe.

[0039] Figure 4 This is a statistical diagram of amino acid residues labeled with 5-TAMRA-IB probe.

[0040] Figure 5 This is the labeling result diagram of 5-TAMRA-IB probe in the cell lysate system.

[0041] Figure 6 The synthetic route of the small molecule probe 5-TAMRA-IB for cysteine.

[0042] Figure 7 This is the 1HNMR spectrum of the small molecule probe 5-TAMRA-IB.

[0043] Figure 8 This is the 13CNMR spectrum of the small molecule probe 5-TAMRA-IB. DETAILED DESCRIPTION

[0044] The specific implementation methods of the present application are described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present application.

[0045] Example 1: Synthesis of cysteine-coupled probe 5-TAMRA-IB:

[0046] The synthetic route is as follows:

[0047]

[0048] In this embodiment, X is iodine, such as Figure 6 As shown, the specific synthesis steps are as follows:

[0049] Compound VI (1.10 g, 9.8 mmol, 1.00 eq), 4-dimethylaminopyridine (DMAP, 0.12 g, 0.1 mmol, 0.10 eq) and compound VII, 4-iodobenzoic acid (2.48 g, 10.0 mmol, 1.00 eq), were added to a 100 mL eggplant-shaped bottle, and dichloromethane (50 mL) was added and stirred for 5 minutes. The mixture was cooled to 0°C in an ice-water bath, and dicyclohexylcarbodiimide (DCC, 2.07 g, 10.0 mmol, 1.00 eq) was added. After keeping the mixture warm for one hour, the ice-water bath was removed and the mixture was stirred at room temperature overnight. The mixture was diluted with DCM (30 mL), filtered, and the filtrate was dried by rotary evaporation to obtain compound V.

[0050] Compound V (3.45 g, 10 mmol, 1.00 eq) was dissolved in dichloromethane (DCM, 50 mL), and compound IV (29.50 g, 199.1 mmol, 20.00 eq) was added, and the mixture was stirred vigorously at room temperature overnight. The reaction solution was filtered, and the filtrate was washed with water (200 mL × 3), dried over anhydrous Na2SO4, and concentrated to obtain 1.97 g of compound II, with a yield of 57%.

[0051] Compound II (110 mg, 0.29 mmol, 2.10 eq) and compound III (75 mg, 0.14 mmol, 1.00 eq) were added to a 50 mL eggplant-shaped bottle, and DCM (15 ml) was added to dissolve, and the mixture was stirred at room temperature for 18 hours. After concentration, the crude compound I was purified by column chromatography (DCM / MeOH=15 / 1) to obtain 110 mg (containing 6% impurities, the impurities were compound II), and then purified by Pre-HPLC to obtain 65 mg of purple-red solid powder, namely compound I (5-TAMRA-IB).

[0052] Figure 7 The 5-TAMRA-IB probe 1 HNMR spectrum, Figure 8 The 5-TAMRA-IB probe 13CNMR spectrum. 1H NMR (400MHz, DMSO-d6) δ8.96(d,J=5.7Hz,1H),8.69(s,1H),8.59(t,J=5.5Hz,1H),8.30(d,J=8.0Hz,1H),7.83(d,J=7 .9Hz,2H),7.62(d,J=7.9Hz,2H),7.56(d,J=8.0Hz,1H),7.17–6.77(m,6H),3.56(q,J=11.1Hz,12H),3.25(s,12H).13C NMR(101MHz,DMSO-d6)δ166.09,165.21,137.61,136.38,134.25,130.96,129.62,99.22,96.81,70.08,70.04,69.29,69.24,40.95.LCMS calcd for C 38 H 39 IN4O7([M+H] + ):791.3,found:791.3.

[0053] Embodiment 2: The rest is the same as Embodiment 1, except that:

[0054] X is Cl.

[0055] The molar ratio of compound VII to compound VI is 1:0.8.

[0056] The molar ratio of compound V to compound IV is 1:10.

[0057] The molar ratio of compound II to compound III is 1:1.

[0058] Compound II and Compound III were stirred and reacted at room temperature for 5 hours.

[0059] The mass spectrum data of compound I prepared in this example are:

[0060] Chemical Formula:C 38 H 39 ClN4O7

[0061] Exact Mass:698.25

[0062] Molecular Weight:699.20

[0063] m / z: 698.25 (100.0%), 699.25 (41.1%), 700.25 (32.0%), 701.25 (13.1%), 700.26 (8.2%), 702.25 (2.6%), 699.25 (1.5%), 700.25 (1.4%)

[0064] Elemental Analysis: C, 65.28; H, 5.62; Cl, ​​5.07; N, 8.01; O, 16.02.

[0065] Embodiment 3: The rest is the same as Embodiment 1, except that:

[0066] X is Br.

[0067] The molar ratio of compound VII to compound VI is 1:1.1.

[0068] The molar ratio of compound V to compound IV is 1:30.

[0069] The molar ratio of compound II to compound III is 3:1.

[0070] Compound II and Compound III were stirred and reacted at room temperature for 24 hours.

[0071] The mass spectrum data of compound I prepared in this example are:

[0072] Chemical Formula:C 38 H 39 BrN4O7

[0073] Exact Mass:742.20

[0074] Molecular Weight:743.66

[0075] m / z:742.20(100.0%),744.20(97.3%),743.20(41.1%),745.20(40.0%),746.20(8.0%),744.21(5 .1%),744.21(3.1%),743.20(1.5%),744.20(1.4%),745.20(1.4%),746.20(1.4%),747.21(1.0%)

[0076] Elemental Analysis: C, 61.37; H, 5.29; Br, 10.74; N, 7.53; O, 15.06.

[0077] Example 4: Pure protein system study on the labeling effect of the 5-TAMRA-IB probe prepared in Example 1 on cysteine ​​residues

[0078] Ligand-assisted metal-catalyzed cysteine ​​coupling methods often use iodobenzene substituents as substrate molecules. During the course of this study, the applicant discovered a new and simple catalytic system through screening of metal palladium reagents: a combination of palladium nitrate and sodium ascorbate, which can selectively label the cysteine ​​residues of proteins ( Figure 1 ). This reaction system abandons the complex and expensive organic ligands and uses highly water-soluble Pd(II) metal salts, which are reduced to catalytic Pd under the action of a reducing agent, thereby catalyzing the reaction of iodobenzene reactive groups with cysteine ​​residues. In subsequent experiments, the applicant verified the above coupling method in pure protein and cell lysate systems.

[0079] Palladium-catalyzed direct coupling reaction of 5-TAMRA-IB with cysteine

[0080] Add DTT with a final concentration of 20mM to the BSA sample, react at 90℃ for 5min, cool to room temperature, and divide the sample into two equal parts. Add IAA with a final concentration of 25mM to one part, and react at room temperature in the dark for 30min. Add an equal amount of 1×PBS to the other part without any treatment. After the reaction, precipitate the sample with methanol and chloroform to remove the excess DTT and IAA samples. Then add 5μL of 10mM palladium nitrate aqueous solution to 2.5μL of 20mM 5-TAMRA-IB DMSO solution; then add 7.5μL DMSO and mix; then add 1μL sodium ascorbate (500mM) to the above solution and mix; take 3.6μL of the above solution and add it to 116.4μL of protein solution, shake at 300rpm in the dark at room temperature for 1h. Vortex mixing is required immediately after each reaction reagent is added. After the reaction, 0.07% 3-mercaptopropionic acid was added to terminate the reaction, and finally 35 μL 5× Loading buffer was added, and the metal bath (95°C, 5 min) was cooled to room temperature, and 10 uL of sample was taken, 10% SDS-PAGE, electrophoresis parameters: 150V, 1h. The gel fluorescent bands were scanned using ChemiDoc MP (Bio Rad), and then the gel was stained with Coomassie Brilliant Blue to prove that the amount of protein in each lane was equal.

[0081] The applicant first took serum protein (BSA) as an example and used 5-TAMRA-IB probe ( Figure 2) is used to label cysteine ​​residues. The probe contains an iodobenzene reactive group and a rhodamine fluorescent reporter group, which facilitates the subsequent detection of the labeling through fluorescent signals. First, dithiothreitol (DTT) is used to open the disulfide bonds of the BSA protein to expose the cysteine ​​sulfhydryl group as much as possible. At the same time, iodoacetamide (IAA) is used to reduce the sulfhydryl group as a control experiment. The treated samples are then precipitated with methanol and chloroform to remove excess DTT. Finally, palladium nitrate at a final concentration of 100 μM, 1 mM sodium ascorbate, and 100 μM 5-TAMRA-IB are added to the sample in sequence, and gel electrophoresis is performed after the reaction at room temperature for 1 hour. The labeling results are shown in the figure below. Figure 3 As shown in the figure, the fluorescence signal of the BSA sample treated with IAA is significantly lower than that of the control group. Since IAA is a thiol-specific reaction reagent, the BSA sample was pre-blocked with IAA, resulting in the loss of the 5-TAMRA-IB probe labeling signal. This experimental result shows that the 5-TAMRA-IB probe can specifically label cysteine ​​residues under this condition.

[0082] Example 5: Palladium-catalyzed direct coupling reaction of 5-TAMRA-IB with cysteine ​​in a cell lysate system

[0083] 22RV1 cells were ultrasonically lysed in 1×PBS phosphate buffer, and the supernatant protein was obtained by centrifugation after lysis. Then, 5μL 10mM palladium nitrate aqueous solution was added to 2.5μL 20mM 5-TAMRA-IB DMSO solution; 7.5μL DMSO was added and mixed; then 1μL sodium ascorbate (500mM) was added to the above solution and mixed; 3.6μL of the above solution was added to 116.4μL protein solution, and the same volume of DMSO was added to the control group, and the reaction was shaken at 300rpm at room temperature in the dark for 1h. Vortex mixing was required immediately after each reaction reagent was added. After the reaction was completed, 3-mercaptopropionic acid was added to a final concentration of 0.07% to terminate the reaction, and finally 35μL 5×Loading buffer was added, and the metal bath (95℃, 5min) was cooled to room temperature, and 10uL sample was taken, 10% SDS-PAGE, electrophoresis parameters: 150V, 1h. The gel was scanned using ChemiDoc MP (Bio Rad) to visualize fluorescent bands, and then stained with Coomassie Brilliant Blue to demonstrate that the amount of protein in each lane was equal.

[0084] At present, the reported ligand-assisted target catalytic coupling labeling methods are generally applied to polypeptide or pure protein systems, and the reaction system is relatively simple. However, many drug targets, such as G protein-coupled receptors, ion channel proteins, etc., have complex purification processes, which limits the application of the above labeling methods in drug discovery for such targets. The combination of palladium nitrate and sodium ascorbate used in this study has good water solubility and a simple catalytic mechanism. It has demonstrated high reactivity and selectivity for cysteine ​​residues in BSA experiments. Therefore, the applicant further expanded the labeling method to cell lysate to label cysteine ​​residues in endogenous proteins under physiological conditions. The applicant ultrasonically lysed 22RV1 cells in phosphate buffer PBS, took the supernatant after centrifugation, added palladium nitrate, sodium ascorbate and 5-TAMRA-IB respectively, reacted at room temperature for a period of time, and electrophoretically separated the probe-labeled protein group and detected the fluorescence signal. The results are as follows: Figure 5 Compared with the control group, under this experimental condition, 5-TAMRA-IB can significantly label the proteome, and the bands are evenly distributed, indicating a high coverage of the entire proteome, providing opportunities for drug discovery for more targets.

[0085] Embodiment 6: The rest is the same as Embodiment 5, except that:

[0086] S1, adding the compound I solution into the palladium nitrate aqueous solution and mixing;

[0087] S2, add DMSO to the mixture of S1 and mix well;

[0088] S3, adding sodium ascorbate aqueous solution to the mixture in S2 and mixing to obtain a labeling reagent;

[0089] S4, adding the labeling reagent to the protein solution at a volume ratio of 1:25, and reacting at room temperature in a dark place with shaking for 0.5 h;

[0090] S5. After the reaction is completed by shaking in the dark, add 3-mercaptopropionic acid with a final concentration of 0.02% to terminate the reaction to complete the cysteine ​​labeling.

[0091] The concentration of Compound I in the Compound I solution is 10 mM; the concentration of the aqueous palladium nitrate solution is 5 mM, and the concentration of the aqueous sodium ascorbate solution is 100 mM;

[0092] The volume ratio of the compound I solution to the palladium nitrate aqueous solution is 1:1; the volume of the compound I solution: DMSO: sodium ascorbate aqueous solution is 1:2:1.

[0093] Embodiment 7: The rest is the same as Embodiment 5, except that:

[0094] S1, adding the compound I solution into the palladium nitrate aqueous solution and mixing;

[0095] S2, add DMSO to the mixture of S1 and mix well;

[0096] S3, adding sodium ascorbate aqueous solution to the mixture in S2 and mixing to obtain a labeling reagent;

[0097] S4, adding the labeling reagent to the protein solution at a volume ratio of 1:50, and shaking the solution at room temperature in the dark for 2 h;

[0098] S5. After the reaction is completed by shaking in the dark, add 3-mercaptopropionic acid with a final concentration of 0.1% to terminate the reaction to complete the cysteine ​​labeling.

[0099] The concentration of Compound I in the Compound I solution is 30 mM; the concentration of the palladium nitrate aqueous solution is 20 mM, and the concentration of the sodium ascorbate aqueous solution is 900 mM.

[0100] The volume ratio of the compound I solution to the palladium nitrate aqueous solution is 1:3; the volume of the compound I solution: DMSO: sodium ascorbate aqueous solution is 4:15:1.

[0101] Example 8: LC-MS / MS detection of 5-TAMRA-IB probe modification sites

[0102] Mass spectrometry sample preparation

[0103] The BSA sample labeled with 5-TAMRA-IB probe was subjected to protein gel electrophoresis in a mass spectrometry electrophoresis tank. After the electrophoresis, the gel was carefully removed, and an appropriate volume of Coomassie Brilliant Blue dye was added. The target protein band was stained at room temperature until the target protein band could be clearly seen, and then high-purity water was added for decolorization. Subsequently, all the target protein bands were cut out with a scalpel, and cut into 1 cubic meter small pieces, transferred to a 1.5mL centrifuge tube, and decolorized with a special decolorizing solution until colorless. After decolorization, acetonitrile was added and rotated at room temperature for 10 minutes, and the acetonitrile was removed. After the micelles were dried, the reducing solution was added, and the mixture was incubated at 55°C for 45 minutes. The reducing solution was removed, and the washing solution was added for rinsing, and then acetonitrile was added for rinsing at room temperature for 10 minutes. The acetonitrile was removed, and after the micelles were dried, the blocking solution was added, and the micelles were left to stand at room temperature for 30 minutes. The blocking solution was removed, and the washing solution was added to wash the micelles. After washing, acetonitrile was added for rinsing at room temperature for 10 minutes. After the micelles are dried, add the corresponding enzymatic solution, let stand at 4°C for 1h, then remove the excess enzymatic solution, add a double volume of enzymatic solution washing solution, and incubate at 37°C for 16h. After the incubation, centrifuge quickly, collect the supernatant in a new centrifuge tube, add an appropriate amount of extract to the remaining micelles, shake at 37°C, 1200rpm for 25min, centrifuge quickly, and collect the supernatant in a new centrifuge tube. After the sample is spin-dried, desalt it, and the sample is spin-dried again for mass spectrometry detection.

[0104] LC-MS / MS mass spectrometry analysis

[0105] The mass spectrometry acquisition conditions are: positive ion mode, Orbitrap mass analyzer, primary spectrum scanning range 350 to 1800Da, resolution 60000, profile format, secondary spectrum data acquisition mode is data dependent, centroid format, the 20 ion peaks with the highest intensity are selected for secondary HCD (high-energy collision induced dissociation) fragmentation, and the secondary spectrum resolution is 15000. Other parameters that need to be set include isolation window, 1.6m / z units; default charge, 2+; normalized collision energy, 28%; maximum IT, 50ms; dynamic exclusion, 30.0s.

[0106] The MS data were searched and quantitatively analyzed using Thermo Proteomo Discoverer 2.5 with the following parameters:

[0107]

[0108] In order to further confirm the selectivity of the 5-TAMRA-IB probe for cysteine ​​residues, the applicant cut out the corresponding bands of BSA in the two groups of samples, performed in-gel digestion, and submitted them for high-resolution mass spectrometry detection. Data processing found that multiple 5-TAMRA-IB-modified cysteine ​​residues were detected in samples that were not treated with IAA, as shown in Table 1, including P02769-C590, P02769-C223, etc., while no modified cysteine ​​sites were detected in samples treated with IAA. In order to further explore the reaction selectivity of the 5-TAMRA-IB probe, the applicant investigated eight amino acid residues with potential reaction activity, including Lys, Asp, Glu, His, Ser, Thr, Trp, and Tyr. However, no 5-TAMRA-IB modification was found in the data processing results. The results are as follows: Figure 4 The above experimental data further verified that under the reaction conditions, the 5-TAMRA-IB probe is highly selective for cysteine ​​residues, and the modification can be stably detected in mass spectrometry, laying the foundation for subsequent application in drug screening.

[0109] Table 1 Mass spectrometry characterization results of cysteine ​​labeling

[0110]

[0111] The present application is described in detail above in conjunction with specific implementation methods and exemplary examples, but these descriptions cannot be understood as limiting the present application. Those skilled in the art understand that, without departing from the spirit and scope of the present application, a variety of equivalent replacements, modifications or improvements can be made to the technical solution of the present application and its implementation methods, all of which fall within the scope of the present application. The scope of protection of the present application shall be subject to the attached claims.

Claims

1. A cysteine-coupled probe, characterized in that: Its structure is shown in formula (I): Wherein, X is Cl, Br or I.

2. A method for preparing a cysteine-coupled probe as claimed in claim 1, characterized in that: The steps include: Compound II and Compound III are mixed and dissolved, and then stirred at room temperature for reaction. The reaction product is concentrated and purified by column chromatography to obtain Compound I.

3. The method for preparing the cysteine-coupled probe according to claim 2, characterized in that: The molar ratio of the compound II to the compound III is 1 to 3:

1.

4. A palladium-catalyzed cysteine ​​coupling method, characterized in that: The cysteine-coupled probe described in claim 1 is used for palladium-catalyzed cysteine ​​residue-specific coupling labeling, and the specific steps include: S1, adding compound I into a palladium nitrate aqueous solution and mixing; S2, add DMSO to the mixture of S1 and mix well; S3, adding sodium ascorbate aqueous solution to the mixture in S2 and mixing to obtain a labeling reagent; S4, add the labeling reagent to the protein solution and shake the reaction in the dark; S5. Add 3-mercaptopropionic acid to terminate the reaction and complete the labeling of cysteine ​​residues in the protein.

5. The palladium-catalyzed cysteine ​​coupling method according to claim 4, characterized in that: The compound I is dissolved in DMSO or phosphate buffer to form a solution of compound I, and the protein solution is the supernatant of cell lysate centrifugation.

6. The palladium-catalyzed cysteine ​​coupling method according to claim 5, characterized in that: The concentration of Compound I in the Compound I solution is 10-30 mM.

7. The palladium-catalyzed cysteine ​​coupling method according to claim 4, characterized in that: The concentration of the palladium nitrate aqueous solution is 5-20 mM, and the concentration of the sodium ascorbate aqueous solution is 100-900 mM.

8. The palladium-catalyzed cysteine ​​coupling method according to claim 4, characterized in that: The final concentration of the 3-mercaptopropionic acid is 0.02-0.1%.

9. The palladium-catalyzed cysteine ​​coupling method according to claim 5, characterized in that: The volume ratio of the solution of compound I to the aqueous palladium nitrate solution is 1:1-3; the volume of the solution of compound I: DMSO: sodium ascorbate aqueous solution is 1-4:2-15:1; the volume ratio of the labeling reagent to the protein solution is 1:25-50.

10. The palladium-catalyzed cysteine ​​coupling method according to claim 5, characterized in that: The light-proof shaking reaction condition is to shake the reaction at room temperature in the dark for 0.5 to 2 hours.

Images