CN113912532B - A probe that can be used for chemical modification of protein lysine residues and its preparation method - Google Patents

Abstract

The invention provides a probe for chemically modifying protein lysine residues, which has a structural general formula shown in a formula VI:the invention also provides a protein modification method, which comprises the steps of adding a protein to be modified, wherein the chemical modification site is protein lysine residue; probe VI, which can be used for chemical modification of lysine residues in proteins, is dosed from 0.1 to 200 equivalents of protein. According to the requirements of chemoselective modification technology and application of protein lysine residues, the invention provides a high-efficiency selective chemical modification method of protein lysine residues by taking pyridinium-containing salts as active functional groups.

Description

A probe that can be used for chemical modification of protein lysine residues and its preparation method

Technical field

The invention belongs to the field of biochemistry and relates to a probe that can be used for chemical modification of protein lysine residues and a preparation method thereof. Specifically, it is an ester derivative containing a pyridinium salt, which can be used to modify protein lysine. (Lys, K) residue.

Background technique

Nucleophilic amino acids make important contributions to protein function, including playing key roles in catalysis and serving as sites for post-translational modifications. Targeting electrophilic groups of amino acid nucleophiles has been used to create covalent ligands and drugs, but so far, this has been mainly limited to cysteine and serine. Protein lysine (Lys) is one of the essential amino acids for organisms and plays a vital role in maintaining various physiological functions. Lysine side chain residues that are amino groups play many important roles in protein function. The amino side chain is a good nucleophilic group. Chemical modification of it is expected to change the chemical structure of the protein and change its spatial structure to improve its biological activity and function. Therefore, the use of chemical methods to modify lysine in biological samples is an important means to study the three-dimensional structure and physiological function of proteins. It is also a method for directional modification of protein properties, and has broad application prospects in protein engineering and omics research.

Pyridinium salts have good water solubility and do not require the use of other organic solvents when interacting with living organisms. They are environmentally friendly and efficient. Using the pyridine ring as the key structural unit of the probe has better biocompatibility, lower biotoxicity, higher systemic properties and higher selectivity, and can solve the problems of other amino acid probes such as iodoacetamide as Cysteine probes have high toxicity to cells. Therefore, probes containing pyridinium salts that can be used for chemical modification of protein lysine residues have an important and very broad application space in the chemical modification of protein side chains.

Contents of the invention

In view of the above technical problems in the prior art, the present invention provides a probe that can be used for chemical modification of protein lysine residues and a preparation method thereof. The probe can be used for chemical modification of protein lysine residues. The probe and its preparation method are to solve the technical problem in the prior art of poor selectivity for chemical modification of protein lysine residues containing pyridinium salts as active functional groups.

The invention provides a probe that can be used for chemical modification of protein lysine residues, having a general structural formula shown in Formula VI:

Among them: R ¹ and R ² are respectively selected from: hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C2-C6 alkenyl, C2-C6 halo Alkenyl, C2-C6 alkynyl, C2-C6 haloalkynyl, hydroxyl, C3-C6 cycloalkyl, substituted piperidin-1-yl, substituted morpholin-1-yl, substituted tetrakis Hydropyrrol-1-yl, phenyl, halogen-substituted phenyl, C1-C6 alkyl-substituted phenyl or 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, pyridyl, halogen-substituted pyridyl, C1-C6 alkyl-substituted pyridyl, C1-C6 haloalkyl-substituted pyridyl, C3 -C6 cycloalkyl-substituted pyridyl, nitro-substituted pyridyl, C2-C6 alkenyl-substituted pyridyl, C2-C6 haloalkenyl-substituted pyridyl, C3-C6 cycloalkenyl-substituted pyridyl, C2-C6 alkynyl-substituted pyridyl, C2-C6 haloalkynyl-substituted pyridyl, C3-C6 cycloalkynyl-substituted pyridyl, pyrimidinyl, halogen-substituted pyrimidinyl, C1-C6 alkyl-substituted pyrimidine base, C1-C6 haloalkyl substituted pyrimidinyl, C3-C6 cycloalkyl substituted pyrimidinyl, nitro substituted pyrimidinyl, C2-C6 alkenyl substituted pyrimidinyl, C2-C6 haloalkenyl substituted pyrimidine base, C3-C6 cycloalkenyl substituted pyrimidinyl, C2-C6 alkynyl substituted pyrimidinyl, C2-C6 haloalkynyl substituted pyrimidinyl or C3-C6 cycloalkynyl substituted pyrimidinyl, substituted containing Five- or six-membered heteroaryl with 1 or 2 N atoms, substituted five- or six-membered heteroaryl with 1 or 2 S atoms, substituted with 1 or 2 O atoms Five- or six-membered heteroaryl, substituted five- or six-membered heteroaryl containing 1 N atom and 1 S atom, substituted 5- or 6-membered heteroaryl containing 1 N atom and 1 O atom One-membered heteroaryl, a substituted five- or six-membered heteroaryl containing 2 N atoms and 1 S atom, or a substituted five- or six-membered heteroaryl containing 2 N atoms and 1 O atom The above-mentioned five-membered or six-membered heteroaryl group is selected from: substituted furyl group, thienyl group, pyrrolyl group, imidazolyl group, pyrazolyl group, thiazolyl group, isothiazolyl group, oxazolyl group, isoxazolyl group, oxazolyl group Diazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, tetrazinyl, indolyl, benzothienyl, benzofuranyl, benzimidazolyl, Indazolyl, benzotriazolyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, isomerized quinolyl, isomerized isoquinolyl, phthalazinyl, quinolyl Silicon groups substituted by oxalinyl, quinazolinyl, cinnolinyl or naphthyridinyl, alkyl or alkenyl groups;

The halogen is fluorine, chlorine, bromine or iodine;

The alkyl group, alkenyl group or alkynyl group is a linear or branched alkyl group; the alkyl group itself or as part of other substituents is selected from methyl, ethyl, propyl, butyl, pentyl, hexyl and Its isomer, its isomer is selected from isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl or tert-amyl;

The haloalkyl group is selected from groups containing one or more identical or different halogen atoms, and the haloalkyl group is selected from CH ₂ Cl, CHCl ₂ , CCl ₃ , CH ₂ F, CHF ₂ , CF ₃ , CF ₃ CH ₂ , CH ₃ CF ₂ , CF ₃ CF ₂ or CCl ₃ CCl ₂ ;

The cycloalkyl itself or as part of other substituents is selected from cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl;

The alkenyl group itself or as part of other substituents is selected 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 itself or as part of other substituents is selected 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.

The

The alkyl acid radicals and substituted alkyl acid radicals are selected from C1-C6 alkyl acid radicals or C1-C6 haloalkylate radicals;

The halogen and its oxygen-containing acid radicals are selected from fluorine, chlorine, bromine or iodine, hypochlorite, chlorite, chlorate, perchlorate, hypobromite, bromate, bromate, perbromate, hypoiodide Acid radical, iodate radical, iodate radical or periodate radical;

The phosphate radical is selected from monohydrogen phosphate, dihydrogen phosphate, pyrophosphate, metaphosphate, hypophosphite, phosphite, polyphosphate, phosphate or hexafluorophosphate;

The sulfate is selected from the group consisting of sulfide anion, hydrogen sulfate, sulfate, bisulfite, sulfite, pyrosulfate, dithionate, thiosulfate, dithionite or persulfate;

The sulfonate radical is selected from triflate, methanesulfonate, phenylsulfonate or p-toluenesulfonate;

The borate radical is selected from borate radical or tetrafluoroborate radical;

Y is selected from: oxygen or sulfur.

The synthesis method of the probe VI that can be used for chemical modification of protein lysine residues of the present invention is as follows:

Among them, the substituent R ¹ is preferably from: phenyl, substituted phenyl, methyl; R ² is preferably from: methyl; X is preferably from: iodine, triflate; Y is preferably from: oxygen, sulfur .

The invention also discloses a method for preparing the above-mentioned probe that can be used for chemical modification of protein lysine residues, which includes the following steps:

1) A step for preparing compound III:

Add compound I, carboxylic acid substrate and esterification condensation agent into a reaction vessel. The structural formula of compound I is: The compound I is selected from: 2-hydroxypyridine, 3-hydroxypyridine, 4-hydroxypyridine, 2-mercaptopyridine, 3-mercaptopyridine or 4-mercaptopyridine, and the structural formula of the carboxylic acid substrate is/> The reaction solvent is selected from: dichloromethane, toluene or acetonitrile. After the reaction is complete, the excess solvent is removed by concentration under reduced pressure. The residue is purified by 100-200 mesh silica gel column chromatography to obtain compound III. The structural formula of compound III is/> The eluent is petroleum ether at 60 to 90 degrees Celsius: ethyl acetate. Depending on the product, the volume ratio of petroleum ether to ethyl acetate is 10:1-6:1, and the yield is 76-91%; Compound III is prepared The volume of the reaction vessel is expanded or reduced accordingly.

2) Preparation of compound VI:

Add compound III and alkylating reagent IV R2X to another reaction vessel. The alkylating reagent IV is selected from: methyl iodide, methyl triflate, ethyl triflate, and benzenesulfonic acid. Methyl ester or trimethyloxonium tetrafluoroborate, the reaction solvent is selected from: dichloromethane, toluene, acetonitrile, diethyl ether or tetrahydrofuran, to obtain a probe that can be used for chemical modification of protein lysine residues The product precipitates directly in the form of a precipitate, or an equal volume of ether as the reaction solvent is added to promote the precipitation of the product, with a yield of 83%-95%; the amount of compound VI prepared and the volume of the reaction vessel are expanded or reduced accordingly.

The present invention also provides the above-mentioned pyridinium salt-containing ester compounds that can be used as probes for chemical modification of protein lysine residues and their derivatives.

The present invention also provides the use of the above-mentioned probe that can be used for chemical modification of protein lysine residues and its intermediates in protein lysine modification.

The invention also provides a protein modification method, which involves adding a protein that needs to be modified, and the chemical modification site is a protein lysine residue; the probe VI that can be used for chemical modification of a protein lysine residue is fed into the protein 0.1 equivalent to 200 equivalent.

Further, the reaction solvent is water or a polar organic solvent: the solvent is selected from water, acetonitrile, methanol, ethanol, isopropanol, tert-butanol, ethylene glycol, glycerin, trifluoroethanol, and hexafluoroisopropanol. , any one of dimethyl sulfoxide, N,N-dimethylformamide or a mixed solvent of any two of them.

Further, the reaction time is 0.1 hour to 100 hours.

Further, the reaction temperature is -20 to 50 degrees Celsius.

The present invention conducts synthesis research on probe VI that can be used for chemical modification of protein lysine residues, and explores the chemical biological activity of probe VI that can be used for chemical modification of protein lysine residues.

The present invention discloses the general structural formula, synthesis method and use of the above-mentioned compound as a probe for chemical modification of protein lysine residues, which are consistent with chemically biologically acceptable probes and commercial cysteine probes. The combination of needles, tyrosine probes and lysine probes is used in modified proteomics and biomedicine at the polypeptide, protein and cell levels and their preparation methods.

Compared with the prior art, the technical progress of the present invention is significant. According to the chemical selective modification technology and application requirements of protein lysine residues, the present invention provides an efficient and selective chemical modification method of protein lysine residues using a pyridinium salt as an active functional group.

Picture description:

Figure 1 shows the reaction between Probe VI that can be used for chemical modification of protein lysine residues and the protein and the secondary mass spectrometry analysis.

Figure 2 can be used for the reaction and proteomic analysis of probe VI chemically modified with protein lysine residues and cell lysates.

Detailed ways:

The present invention further illustrates the synthesis and biological application of probe VI that can be used for chemical modification of protein lysine residues through specific preparation and chemical biology examples. The examples are only used to specifically illustrate the present invention and do not limit the present invention. , especially biological applications are only examples, rather than limiting this patent, the specific implementation is as follows:

Example 1: Preparation of compound III-1:

In a 250 ml double-neck round bottom flask, add 2.4 g of 4-hydroxypyridine I, 4.4 g of 4-propargyloxybenzoic acid and 4.8 g of 1-ethyl-(3-dimethylaminopropyl)carbodioxide Amine hydrochloride. Add 100 ml of reaction solvent methylene chloride. After TLC monitors the completion of the reaction, concentrate under reduced pressure to remove excess solvent. The residue is purified by 100-200 mesh silica gel column chromatography to obtain compound III. The eluent is petroleum ether:ethyl acetate at 60-90 degrees Celsius, and the volume ratio is 6: 1. The product is 5.0 grams of white powder, and the yield is 78%. The NMR data of this compound are as follows: 1H NMR (500MHz, CDCl3) δ8.66 (dd, J＝4.7, 1.6Hz, 2H), 8.21–8.10 (m, 2H), 7.23 (dd, J＝4.7, 1.6Hz, 2H), 7.13–7.01 (m, 2H), 4.79 (d, J = 2.4Hz, 2H), 2.57 (t, J = 2.4Hz, 1H). Synthesis and characterization data of similar compounds:

White powder, yield 91%. ¹ H NMR (500MHz, CDCl ₃ ) δ8.66 (dd, J＝4.8, 1.3Hz, 2H), 8.21–8.13 (m, 2H), 7.69–7.63 (m, 1H) ,7.51(t,J＝7.9Hz,2H),7.24(dd,J＝4.7,1.6Hz,2H).

Yellow powder, yield 84%. ¹ H NMR (400MHz, CDCl ₃ ) δ8.70 (d, J = 4.5Hz, 2H), 8.03 (d, J = 7.8Hz, 2H), 7.66 (t, J = 7.2 Hz,1H),7.60–7.42(m,4H).

Yellow powder, yield 81%. ¹ H NMR (400MHz, CDCl ₃ ) δ8.74–8.66 (m, 1H), 8.07–8.00 (m, 2H), 7.79 (td, J = 7.6, 1.8Hz, 1H) ,7.74(d,J＝7.8Hz,1H),7.65–7.60(m,1H),7.50(t,J＝7.7Hz,2H),7.34(ddd,J＝7.2,4.9,1.2Hz,1H).

Yellow powder, yield 65%. ¹ H NMR (500MHz, CDCl ₃ ) δ8.66 (d, J = 5.8Hz, 2H), 8.00 (d, J = 8.8Hz, 2H), 7.46 (dd, J = 4.7 ,1.3Hz,2H),7.06(d,J＝9.0Hz,2H),4.78(d,J＝2.4Hz,2H),2.57(t,J＝2.4Hz,1H).

Yellow powder, yield 54%. ¹ H NMR (400MHz, CDCl ₃ ) δ8.74–8.68 (m, 1H), 8.06 (d, J = 8.9Hz, 2H), 7.87–7.73 (m, 2H), 7.37 (ddd,J＝7.3,4.9,1.1Hz,1H),7.09(d,J＝8.9Hz,2H),4.81(d,J＝2.3Hz,2H),2.61(t,J＝2.4Hz,1H) .

Example 2: Preparation of compound VI-1:

Add 1.3 g of III and 1.5 ml of methyl triflate to a 100 ml single-neck round bottom flask. Add 30 ml of reaction solvent methylene chloride. After TLC monitored the completion of the reaction, an equal volume of diethyl ether as the reaction solvent was added to promote the precipitation of the product. The product was 1.8 g of white powder, with a yield of 89%. The NMR data of this compound are as follows: 1H NMR (500MHz, MeOD) δ8.93 (d, J = 7.1Hz, 2H), 8.23–8.17 (m, 2H), 8.10 (d, J = 7.4Hz, 2H), 7.21 –7.15(m,2H),4.88(d,J＝2.4Hz,2H),4.37(s,3H),3.04(t,J＝2.4Hz,1H).

Synthesis and characterization data of similar compounds:

Off-white powder, yield 91%. ¹ H NMR (500MHz, MeOD) δ8.99 (d, J＝7.1Hz, 2H), 8.25 (dt, J＝8.5, 1.5Hz, 2H), 8.15 (d, J＝ 7.3Hz,2H),7.78(tt,J＝7.3,1.3Hz,1H),7.69–7.57(m,2H),4.41(s,3H).

White powder, yield 94%. ¹ H NMR (400MHz, MeOD) δ9.00 (d, J＝7.1Hz, 2H), 8.32–8.25 (m, 2H), 8.17 (d, J＝7.2Hz, 2H) ,7.86–7.77(m,1H),7.70–7.61(m,2H),4.44(s,3H).

Yellow powder, yield 92%. ¹ H NMR (400MHz, MeOD) δ8.93 (d, J = 6.7Hz, 2H), 8.39 (d, J = 6.7Hz, 2H), 8.16–8.09 (m, 2H) ,7.86–7.77(m,1H),7.70–7.62(m,2H),4.46(s,3H).

Yellow powder, yield 89%. ¹ H NMR (500MHz, MeOD) δ8.06 (d, J = 7.3Hz, 1H), 8.01–7.96 (m, 2H), 7.66–7.55 (m, 2H), 7.42 ( dt,J＝28.7,7.5Hz,3H),6.85(t,J＝6.1Hz,1H),3.96(s,3H).

White powder, yield 86%. ¹ H NMR (500MHz, MeOD) δ8.93 (d, J = 6.9 Hz, 2H), 8.20 (d, J = 9.0 Hz, 2H), 8.10 (d, J = 7.1 Hz ,2H),7.18(d,J＝9.0Hz,2H),4.88(d,J＝2.2Hz,2H),4.38(s,3H),3.04(t,J＝2.3Hz,1H).

Yellow powder, yield 89%. ¹ H NMR (400MHz, MeOD) δ8.90 (d, J = 6.7Hz, 2H), 8.37 (d, J = 6.8Hz, 2H), 8.16–8.08 (m, 2H) ,7.27–7.20(m,2H),4.93(d,J＝2.4Hz,2H),4.44(s,3H),3.10(t,J＝2.4Hz,1H).

Yellow powder, yield 85%. ¹ H NMR (400MHz, MeOD) δ9.28 (d, J＝6.1Hz, 1H), 8.68 (td, J＝7.9, 1.2Hz, 1H), 8.51–8.44 (m, 1H),8.27–8.19(m,1H),8.15–8.07(m,2H),7.30–7.22(m,2H),4.95(d,J＝2.3Hz,2H),4.51(s,3H),3.11 (t,J＝2.4Hz,1H).

Example 3: Reaction results between the probe VI of the present invention that can be used for chemical modification of protein lysine residues and isolated proteins:

In order to verify the reactivity of the probe that can be used for chemical modification of protein lysine residues and lysine (Lys) covalent binding at the protein labeling level, the protein was dissolved in distilled water and prepared into a 7.5 μM protein solution. Accurately weigh the probe VI that can be used for chemical modification of protein lysine residues and use PBS buffer or distilled water to prepare a reaction solution of corresponding concentration. The protein solution was incubated with 50 μM probe in PBS solution at 37°C for 2 hours. Then use the "click" reaction to label the protein with a fluorescent tag. The specific method is to add CuSO ₄ (1mM), TECP (1mM), TBTA (100μM), 5-TAMRA-N ₃ (100μM) to the reaction system, and incubate at 25°C. After 2 hours, the reaction was completed. Finally, run SDS-PAGE protein gel and observe the fluorescence in the gel. The protein can be bovine serum albumin (BSA), horse serum albumin (HSA), lysozyme (Lysozyme), insulin (Insulin), carbonic anhydrase (CA), myoglobin (Myoglobin), ribonuclease A (RNaseA), etc. .

From the protein labeling experiments in Figures 1a and 1b, we observed that the pyridinium salt probe KP2 (structural formula VI) has strong fluorescence. The high fluorescence activity of KP2 attracted our attention and deserves further in-depth research, so we selected KP2 Perform subsequent protein labeling reactions. From Figure 1b, it is found that the KP2 probe has a certain fluorescence intensity after reacting with the protein for 5 minutes, and the fluorescence intensity increases as the reaction time increases. In terms of reaction dose, as the concentration of KP2 increases, the fluorescence gradually increases, and the probe concentration has an important influence on the fluorescence intensity. This protein has a large number of lysine residues, and a complete reaction requires about 450 μM of probe. In order to further explore the labeling efficiency of the KP2 probe-labeled protein reaction under different pH states, we performed the protein reaction in a buffer solution of pH 3-11 and observed the fluorescence intensity. The study found that the fluorescence gradually increased as the pH increased, but at too high a pH, the protein was unstable and easily destroyed by alkali. Therefore, the fluorescence became weaker at pH 11 and 12, and the CBB bands of the protein also became dispersed. The optimal reaction pH is 7-10.

Commercial NHS-Ac is a commonly used reagent in protein labeling, mainly used for blocking lysine residues, IAA is a commercial blocking reagent for cysteine, and KP-B of the present invention is used for blocking lysine. probe. From Figure 1c, it is found that BSA and KP2 probes can be competed by NHS-Ac and KP-B and will not be affected by the concentration of IAA. As the concentrations of NHS-Ac and KP-B increase, the fluorescence is gradually blocked. However, there is no significant change in fluorescence as the IAA concentration increases, indicating that the label can be blocked by commercial lysine blocking reagent, proving that the probe VI in this study, which can be used for chemical modification of protein lysine residues, selectively acts on Protein lysine residues.

In order to test the specific labeling of protein lysine by the KP2 probe, secondary mass spectrometry was used to study the protein. The specific steps were: react the protein with the probe in PBS buffer and run SDS-PAGE protein gel. After running and cutting the gel for mass spectrometry sample preparation, first decolorize and centrifuge, then reduce alkylation (optional), trypsin digestion, peptide extraction and enrichment, and prepare for secondary mass spectrometry analysis.

From Figure 1d, sequence fragments modified with protein lysine can be detected in the fragment peak, and corresponding b, y signals can be detected from the fragment peak, proving that the probe of the present invention is modified on K (Lys) of BSA. Secondary mass spectrometry experiments further prove that the main reaction site of the probe VI that can be used for chemical modification of protein lysine residues with proteins is lysine, and can be used as a highly selective lysine modification probe for further research. .

Example 4: Reaction results between the probe VI of the present invention that can be used for chemical modification of protein lysine residues and cell lysate:

The specific steps are: lyse the harvested cells through ultrasonic lysis and determine the concentration. Accurately weigh the probe VI that can be used for chemical modification of protein lysine residues and use PBS buffer or distilled water to prepare a reaction solution of corresponding concentration. Dilute the cell lysate to the appropriate concentration and incubate with 50 μM probe in PBS solution at 37°C for 2 hours. Then use the "click" reaction to label the protein with a fluorescent tag. The specific method is to add CuSO ₄ (1mM), TECP (1mM), TBTA (100μM), 5-TAMRA-N ₃ (100μM) to the reaction system, and incubate at 25°C. After 2 hours, the reaction was completed. Finally, run SDS-PAGE protein gel and observe the fluorescence in the gel. The cells can be A549, 293T, Hela, MCF-7, etc.

From the cell lysate labeling experiments in Figures 2a and 2b, we observed that the pyridinium salt probe KP2 has strong fluorescence, so KP2 was selected to continue the subsequent cell lysate labeling reaction. From Figure 2b, it is found that the KP2 probe has a certain fluorescence intensity after reacting with the cell lysate for 10 minutes. At the same time, the fluorescence intensity of the reaction with the protein increases as the reaction time prolongs, and reaches its best effect at 1 hour. In terms of reaction dose (Figure 2a), as the concentration of KP2 increases, the fluorescence gradually increases, and the probe can have a good labeling effect at low concentrations.

Commercial NHS-Ac and IAA were also used in the cell lysate for blocking of lysine and cysteine respectively, and KP-B of the present invention was used for blocking of lysine. From Figure 2c, it is found that the reaction between cell lysate and KP2 probe can be competed by NHS-Ac and KP-B and will not be affected by the concentration of IAA. As the concentrations of NHS-Ac and KP-B increase, the fluorescence It was gradually blocked, but the increase in IAA concentration had no significant change in fluorescence. From the cell lysate level, it also showed that the lysine probe VI in this study can selectively act on protein lysine residues.

Example 5: Toxicity study of probe VI of the present invention that can be used for chemical modification of protein lysine residues:

At the same time, compared with commercial probes NHS-ph and NHS, the KP2 probe is safer for MCF-7 cells. From Figure 2d, in the case of KP2 at 200 μM, the cell survival rate is close to 100%, while the cell survival rate of commercial NHS-ph and NHS drops rapidly at this concentration, proving that KP2 is very safe for cells at this concentration and can Develop and explore as a lysine modification probe for living cells.

Example 6: Proteomic research results of the probe VI of the present invention that can be used for chemical modification of protein lysine residues:

Incubate the cells and probes for 3 hours, perform a click reaction, and centrifuge directly to pellet at 4°C 14,000 rpm for 5 min. Pre-cool the cells with ice acetone or ice methanol at -80°C, and wash the pellet. Reconstitute with PBS containing 1.2% SDS by sonication, observe in real time, and sonicate until no precipitation occurs) and dilute to 0.2% SDS. Incubate with a certain volume of SA beads at 29°C for 3 hours. The beads were washed with water and PBS and resuspended in 500uL of 50mM Tris solution containing 6M urea at pH 8.5. Add DTT and IAA in sequence, react, centrifuge after the reaction, add trypsin working solution, and add trypsin for digestion overnight. Centrifuge, add acid to the supernatant to terminate the reaction, remove salt, and perform LC-MS/MS analysis. Beads were washed with PBS and water and subjected to light or acid cutting. The supernatant was collected by centrifugation, desalted and analyzed by LC-MS/MS.

From Figure 2e, it is found that the KP2 probe can detect KP2-lysine-modified reaction sites in proteomic studies of cells of different cell lines. Among them, 4424 KP2-lysine modifications can be found in Jurkat cells. Of the modification sites, 5338 KP2-to-lysine modification sites can be identified for MCF7 cells, 5969 KP2-to-lysine modification sites can be identified for A549 cells, and 3699 KP2-to-lysine modification sites can be identified for Hela cells. Acid modification sites. These experimental results prove the broad-spectrum applicability of the probe VI of the present invention that can be used for chemical modification of protein lysine residues, and can be widely used in proteomics research on various cell lines. The probe of the present invention has high selectivity for modifying protein lysine residues, and has extremely important application value for selective modification of protein lysine residues.

Claims (6)

1. A probe useful for chemical modification of lysine residues in proteins, characterized by the following structural formula:

or->

2. Use of a probe according to claim 1 for the chemical modification of protein lysine residues for protein lysine modification.

3. A method for modifying a protein by using the probe according to claim 1, wherein a protein to be modified is added, and the chemical modification site is a protein lysine residue; the probe dosage for chemical modification of lysine residues of proteins according to claim 1, wherein the amount of the probe is 0.1 to 200 equivalents of the protein.

4. A method of modifying a protein according to claim 3, wherein the reaction solvent is water or a polar organic solvent: the solvent is selected from any one of water, acetonitrile, methanol, ethanol, isopropanol, tertiary butanol, ethylene glycol, glycerol, trifluoroethanol, hexafluoroisopropanol, dimethyl sulfoxide and N, N-dimethylformamide or a mixed solvent of any two of the solvents.

5. A method of modifying a protein according to claim 3, wherein the reaction time is from 0.1 hours to 100 hours.

6. A method of modifying a protein according to claim 3, wherein the reaction temperature is from-20 to 50 ℃.