Probe capable of being used for arylation cross-linking modification of protein nucleophilic amino acid residues and preparation method of probe

Abstract

The invention provides a probe capable of being used for arylation cross-linking modification of protein nucleophilic amino acid residues. The structural general formula of the probe is shown in the formula I. The invention further provides a preparation method of the probe. The invention also provides a method for carrying out cross-linking modification on the protein by adopting the probe. The modification sites of the method are cysteine and lysine residues of the protein. Aiming at application requirements of a protein chemical selective arylation crosslinking modification technology, the invention provides an efficient and selective protein crosslinking chemical modification method based on a multi-substituent pyridinium salt active functional group, and the method is suitable for crosslinking analysis of cysteine and lysine residues.

Description

Technical Field

[0001] The present invention belongs to the field of biochemistry, and specifically relates to a probe that can be used for arylation cross-linking modification of nucleophilic amino acid residues in proteins, a protein cross-linking modification method based on the probe, and its application in protein cross-linking mass spectrometry analysis. Background Art

[0002] Protein cross-linking plays an important role in chemical biology and pharmaceutical research. For example, it is used to stabilize protein conformation, study protein interactions, and develop novel bioconjugates or diagnostic tools. Therefore, developing efficient protein cross-linking methods is crucial for these biological and pharmaceutical applications. This method is not only a key tool for studying the three-dimensional structure and physiological function of proteins, but also an effective way to modify protein properties in a targeted manner, showing broad application prospects in protein engineering and proteomics research.

[0003] In recent years, aromatic nucleophilic substitution (S N Ar) reaction is widely used in site-specific modification of nucleophilic amino acid residues (such as cysteine ​​and lysine) in proteins due to its high reactivity and excellent chemical selectivity. Pyridinium salt compounds have excellent electrophilic reactivity (i.e., as S N Ar reaction electrophilic center). Therefore, halogenated pyridinium salt compounds are highly potential and efficient S N Ar reaction reagents. By rationally designing the reactivity of pyridinium salt molecules containing multiple leaving groups, the efficiency and reaction rate of chemical modification of nucleophilic amino acid residues in proteins can be effectively controlled. In addition, pyridinium salt compounds generally have good biocompatibility, low cytotoxicity, and excellent cell membrane permeability. Given these advantages, halogenated pyridinium salt compounds have significant potential in developing new protein arylation cross-linking modification tools. Summary of the Invention

[0004] To overcome the deficiencies of the prior art, the present invention provides a probe that can be used for the arylation cross-linking modification of nucleophilic amino acid residues in proteins. The probe that can be used for the arylation cross-linking modification of nucleophilic amino acid residues in proteins aims to address the lack or limitations of short rigid cross-linking reagents, enrichable cross-linking reagents, and cleavable cross-linking reagents in the prior art.

[0005] The present invention provides a probe that can be used for arylation cross-linking modification of nucleophilic amino acid residues in proteins, having the general structural formula shown in Formula I:

[0006]

[0007] in:

[0008] R1 and R2 are each independently selected from the group consisting of hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C1-C6 amide, C1-C6 haloalkoxy, C2-C6 alkenyl, C2-C6 haloalkenyl, C2-C6 alkynyl, C2-C6 haloalkynyl, hydroxyl, amino, C3-C6 cycloalkyl, phenyl, halogen, cyano, carboxyl, or aldehyde;

[0009] The halogen is fluorine, chlorine, bromine or iodine;

[0010] The alkyl, alkenyl or alkynyl group is a straight chain or branched chain; the alkyl, alkenyl or alkynyl group itself or as part of other substituents can be selected from:

[0011] Alkyl: methyl, ethyl, propyl, butyl, pentyl, hexyl or isomers thereof, wherein the isomers include isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl and tert-pentyl;

[0012] Haloalkyl: a group containing one or more identical or different halogen atoms selected from CH2Cl, CHCl2, CCl3, CH2F, CHF2, CF3, CF3CH2, CH3CF2, CF3CF2, CCl3CCl2;

[0013] Cycloalkyl: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl;

[0014] Alkenyl: vinyl, allyl, 1-propenyl, buten-2-yl, buten-3-yl, penten-1-yl, penten-3-yl, hexen-1-yl, 4-methyl-3-pentenyl;

[0015] Alkynyl: ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-2-yl, 1-methyl-2-butynyl, hexyn-1-yl, 1-ethyl-2-butynyl;

[0016] X is selected from: alkylate, substituted alkylate, halogen, halogen oxyacid, phosphate, sulfate, sulfonate or borate; wherein:

[0017] The alkyl acid radical or substituted alkyl acid radical is a C1-C6 alkyl acid radical or a C1-C6 halogenated alkyl acid radical;

[0018] The halogen or halogen oxygenate is selected from the group consisting of fluorine, chlorine, bromine, iodine, hypochlorite, chlorite, chlorate, perchlorate, hypobromite, bromite, bromate, perbromate, hypoiodite, iodite, iodate, and periodate;

[0019] The phosphate is selected from the group consisting of monohydrogen phosphate, dihydrogen phosphate, pyrophosphate, metaphosphate, hypophosphite, phosphite, polyphosphate, orthophosphate, and hexafluorophosphate;

[0020] The sulfate radical is selected from the group consisting of: sulfur anion (S 2- ), bisulfate, sulfate, bisulfite, sulfite, pyrosulfate, dithionate, thiosulfate, dithionite, persulfate;

[0021] The sulfonate group is selected from the group consisting of trifluoromethanesulfonate, methanesulfonate, benzenesulfonate, and p-toluenesulfonate;

[0022] The borate is selected from: borate or tetrafluoroborate;

[0023] Y is selected from fluorine, chlorine, bromine, iodine, methoxy, methylthio or methylsulfonyl.

[0024] The synthesis method of the probe I that can be used for arylation cross-linking modification of nucleophilic amino acid residues of proteins of the present invention is as follows:

[0025]

[0026] Wherein, the substituents are as described above R 1 Preferably selected from: phenyl, substituted phenyl, alkoxy; R 2 Preferably selected from: methyl; X is preferably selected from: iodine, trifluoromethanesulfonate, tetrafluoroborate; Y is preferably selected from: halogen, oxygen, sulfur.

[0027] Furthermore, the probe comprises at least one leaving group, and the leaving group is located at the para position or meta position of the pyridine ring; wherein Y represents 1 to 3 leaving groups, and the leaving groups are the same or different.

[0028] Furthermore, the atom modified in the arylation cross-linking reaction is an oxygen atom, a sulfur atom or a selenium atom.

[0029] Furthermore, the probe is a six-membered nitrogen-containing onium salt compound.

[0030] Furthermore, the probe is used for cross-linking modification of cysteine ​​and lysine residues of proteins, and the dosage is 0.1 to 200 equivalents of the protein.

[0031] Furthermore, the cross-linking reaction is carried out in a buffer solution with a pH value of 5 to 8, and the buffer solution is weakly acidic, neutral or weakly alkaline.

[0032] Furthermore, the reaction solvent is selected from water, acetonitrile, methanol, ethanol, isopropanol, tert-butanol, ethylene glycol, glycerol, trifluoroethanol, hexafluoroisopropanol, dimethyl sulfoxide, N,N-dimethylformamide or a mixture thereof.

[0033] Furthermore, the reaction time is 0.1 hours to 100 hours.

[0034] Furthermore, the reaction temperature is -20°C to 50°C.

[0035] Furthermore, the method is used for cross-linking mass spectrometry analysis of proteins.

[0036] The present invention also discloses a method for preparing the above-mentioned probe that can be used for chemical modification of protein cysteine ​​residues, comprising the following steps:

[0037] Add compound III into a reaction vessel,

[0038]

[0039] and alkylating agent II R 2 X,

[0040] R1 and R2 are each independently selected from the group consisting of hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C1-C6 amide, C1-C6 haloalkoxy, C2-C6 alkenyl, C2-C6 haloalkenyl, C2-C6 alkynyl, C2-C6 haloalkynyl, hydroxyl, amino, C3-C6 cycloalkyl, phenyl, halogen, cyano, carboxyl, or aldehyde;

[0041] The halogen is fluorine, chlorine, bromine or iodine;

[0042] The alkyl, alkenyl or alkynyl group is a straight chain or branched chain; the alkyl, alkenyl or alkynyl group itself or as part of other substituents can be selected from:

[0043] Alkyl: methyl, ethyl, propyl, butyl, pentyl, hexyl or isomers thereof, wherein the isomers include isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl and tert-pentyl;

[0044] Haloalkyl: a group containing one or more identical or different halogen atoms selected from CH2Cl, CHCl2, CCl3, CH2F, CHF2, CF3, CF3CH2, CH3CF2, CF3CF2, CCl3CCl2;

[0045] Cycloalkyl: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl;

[0046] Alkenyl: vinyl, allyl, 1-propenyl, buten-2-yl, buten-3-yl, penten-1-yl, penten-3-yl, hexen-1-yl, 4-methyl-3-pentenyl;

[0047] Alkynyl: ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-2-yl, 1-methyl-2-butynyl, hexyn-1-yl, 1-ethyl-2-butynyl;

[0048] The reaction solvent is selected from: dichloromethane, toluene, acetonitrile, ether or tetrahydrofuran, and a probe that can be used for reversible chemical modification of protein cysteine ​​residues is obtained.

[0049]

[0050] The product is directly precipitated in the form of a precipitate, or an equal volume of ether is added to the reaction solvent to promote the precipitation of the product.

[0051] Furthermore, the alkylating agent II is selected from: iodomethane, methyl trifluoromethanesulfonate, ethyl trifluoromethanesulfonate, methyl benzenesulfonate or trimethyloxonium tetrafluoroborate.

[0052] Specifically, the structural formula of the probe is

[0053] The present invention also provides a method for modifying proteins using the above-mentioned probe for chemical modification of protein cysteine ​​residues. The modification sites of the method are cysteine ​​and lysine residues of proteins. The reaction process is as follows:

[0054]

[0055] The above-mentioned probes of the present invention can also be used in combination with other probes compatible with chemical biology (including commercial lysine probes, tyrosine probes, cysteine ​​probes, etc.). The combined application covers modified proteomics research at the polypeptide, protein and cellular levels, as well as applications in the biomedical field.

[0056] Compared with existing technologies, the present invention offers significant and positive technical benefits. Targeting protein cross-linking applications, the present invention provides a chemical modification method using a pyridinium salt containing a leaving group as a reactive functional group, suitable for cross-linking analysis of cysteine ​​and lysine residues. This method enables efficient and selective cross-linking of cysteine ​​and lysine residues in proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Schematic diagram of the reaction of two series of pyridinium salts with model peptide 1 (Melanotan I), used to screen the effects of pyridine ring substituents (R1: chlorine, methylaminoformyl, hydrogen, methoxy, or amino) on reaction efficiency. (a) Conversion of the two series of pyridinium salts with the peptide; (b) Liquid chromatography monitoring of the reaction of the two series of pyridinium salts with the peptide.

[0058] Figure 2Schematic diagram of the reaction of a series of pyridinium salts with model peptide 1 (Melanotan I), used to evaluate the regulatory effect of the pyridine ring leaving group on the reaction efficiency. (a) Conversion of the reaction of the series of pyridinium salts with the peptide; (b) Liquid chromatography monitoring of the reaction of the series of pyridinium salts with the peptide.

[0059] Figure 3 : Mass spectrometry analysis and circular dichroism (CD) characterization of the reaction of the preferred compound 2a with a model protein to verify the structural stability and conformational changes of the modified product. (a) General formula for the reaction of the preferred compound 2a with a model protein; (b) Mass spectrum of the reaction of compound 2a with myoglobin; (c) Mass spectrum of the reaction of compound 2a with carbonic anhydrase; (d) Mass spectrum of the reaction of compound 2a with lysozyme; (e) Circular dichroism spectrum of the reaction of compound 2a with myoglobin; (f) Circular dichroism spectrum of the reaction of compound 2a with carbonic anhydrase; (g) Circular dichroism spectrum of the reaction of compound 2a with lysozyme.

[0060] Figure 4 : Mass spectrometry analysis of the reaction of preferred compounds S1 and S10 with model proteins, comparing the modification efficiency of different leaving groups. (a) General formula for the reaction of preferred compounds S1 and S10 with model proteins; (b) Mass spectrum of the reaction of compound S1 with myoglobin; (c) Mass spectrum of the reaction of compound S1 with carbonic anhydrase; (d) Mass spectrum of the reaction of compound S1 with lysozyme; (e) Mass spectrum of the reaction of compound S10 with myoglobin; (f) Mass spectrum of the reaction of compound S10 with carbonic anhydrase; (g) Mass spectrum of the reaction of compound S10 with lysozyme.

[0061] Figure 5:The cross-linking efficiency of the dual-leaving group pyridinium salts PX1 / PX2 at the proteomic level (HeLa cell lysate) was evaluated to evaluate their labeling ability for multiple protein targets. (a) Chemical structures of the pyridinium salt cross-linkers (PX1 and PX2) and the control cross-linker; (b) HeLa cell lysate was labeled with a pyridinium salt probe and linked to a fluorescent dye (N3-TAMRA) via copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, followed by in-gel PAGE fluorescence scanning; (c) Concentration gradient labeling experiment of HeLa cell lysate with PX1; (d) Time gradient labeling experiment of HeLa cell lysate with PX1; (e) Temperature gradient labeling experiment of HeLa cell lysate with PX1; (f) Competition labeling experiment of HeLa cell lysate with PX1 in the presence of competitive electrophiles, including Ac-Cys-OH and Boc-Ser-OH , Boc-Tyr-OH, Boc-Arg-OH, Boc-Glu-OH, Boc-His-OH; (g) Competition labeling experiment of Hela cell lysate by PX1 in the presence of high concentration of competitive reagents, the competitive reagents are: sulfo-NHS acetate (Sulfo-NHS-Ac) and iodoacetamide (IAM); (H) Competition labeling experiment of Hela cell lysate by PX1 in the presence of sulfo-NHS acetate concentration gradient; (i) Experiment of adding reducing reagent (DTT or TCEP) after Hela cell lysate labeling; (j) Reaction of model protein BSA with cross-linker PX1 or CBDPS; (k) Reaction of model protein BSA with cross-linker PX1 or DFDNB.

[0062] Figure 6 : Study of the cross-linking efficiency of compound PX1 with various model proteins, validating the universality of pyridine ring-substituted substrates (R1 is trifluoromethyl, cyano, methoxycarbonyl, benzylcarbamoyl, naphthylcarbamoyl, chlorine, 2,6-dichloro, methoxy, amino, 2,4-dichloro, or 3,4-benzo). (a) Reaction of the model protein Iqah9.8E3 with the cross-linkers PX1, DFDNB, or CBDPS; (b) Reaction of the model protein STAT3 with the cross-linkers PX1, DFDNB, or CBDPS; (c) Reaction of the model protein SH2 Src with the cross-linkers PX1, DFDNB, or CBDPS; (d) Competition cross-linking experiment of the model protein Iqah9.8E3 with PX1, DFDNB, or CBDPS in the presence of high concentrations of competing reagents: sulfo-NHS-Ac and iodoacetamide (IAM); (e) Concentration gradient cross-linking experiment of the model protein BSA with DFDNB.

[0063] Figure 7:Comparison of the cross-linking efficiency of pyridinium salts PX3-PX13 containing double / tri-leaving groups with different protein models to screen for efficient cross-linker structures. (a) Chemical structure of pyridinium salt cross-linker (PX3-PX13); (b) Reaction of model protein Mpro with cross-linker PX3-PX13 (5 equivalents); (c) Reaction of model protein Mpro with cross-linker PX3-PX13 (10 equivalents); (d) Reaction of model protein Mpro with cross-linker PX3-PX13 (100 equivalents); (e) Concentration gradient cross-linking experiment of PX12 on model protein Mpro; (f) Reaction of model protein Iqah9.8E3 with cross-linker PX3-PX13 (20 equivalents);

[0064] Figure 8 : Hydrolytic stability and cytotoxicity tests of compound PX1 to evaluate its biocompatibility. (a) Hydrolytic stability of compound PX1; (b) Cytotoxicity of compound PX1 determined by MTT assay. DETAILED DESCRIPTION

[0065] The following describes in detail the implementation process of the protein arylation cross-linking method of the present invention through preparation examples, chemical characterization, and biological application examples. This is an illustrative description of the technical solution of the present invention and does not constitute a limitation on the scope of protection of the claims. Experimental operations must be carried out in a standard chemical fume hood, using dry glassware and inert gas protection (unless otherwise specified). The specific implementation steps are as follows:

[0066] Example 1: Preparation of cross-linked probe I

[0067]

[0068] To a 250 ml double-necked round-bottom flask, add 1.09 g of 2-methoxypyridine and 100 ml of dichloromethane, the reaction solvent. Add 1.65 g of methyl trifluoromethanesulfonate dropwise under ice. A solid precipitate or oily substance precipitates. Filter under reduced pressure or centrifuge the precipitate, wash it with a small amount of dichloromethane, and collect the solid precipitate or oily substance to obtain the product. The product is 2.36 g of a colorless oil, with a yield of 86%. The NMR and high-resolution characterization data of this compound are as follows:

[0069] Colorless oil, yield 86%.

[0070] 1 H NMR (400MHz, D2O) δ8.39–8.26(m,2H),7.49(d,J=8.9Hz,1H),7.38(ddd,J=7.5,6.4,1.2Hz,1H),4.21(s,3H),3.96(s,3H).

[0071] 13C NMR (101MHz, D2O) δ160.76,147.84,143.15,118.39,110.68,58.79,41.24. 19 FNMR (376MHz, D2O)δ-78.87.

[0072] HRMS(ESI):[M + ]Calcd.m / z 124.0757, found m / z 124.0758.

[0073] Example 2:

[0074] A similar method to Example 1 was used to prepare the following using 4-methoxypyridine as a raw material:

[0075] White powder, yield 91%.

[0076] 1 H NMR(400MHz,D2O)δ8.48–8.40(m,2H),7.40–7.32(m,2H),4.11(s,3H),4.03(s,3H).

[0077] 13 C NMR(101MHz,D2O)δ171.04,146.20,113.18,57.56,46.16.

[0078] 19 F NMR (376 MHz, D2O) δ-78.87.

[0079] HRMS(ESI):[M + ]Calcd.m / z 124.0757, found m / z 124.0757.

[0080] Example 3:

[0081] A similar method to Example 1 was used to prepare the following using 2-methylthiopyrimidine as the raw material:

[0082]

[0083] Pale yellow powder, yield 69%.

[0084] 1 H NMR (400MHz, D2O) δ9.15(dd,J=4.8,2.0Hz,1H),8.95(dd,J=6.5,2.1Hz,1H),7.74(dd,J=6.4,4.6Hz,1H),4.15(s,3H),2.87(s,3H).

[0085] 13 C NMR(101MHz,D2O)δ170.20,163.13,153.81,117.44,44.60,14.85.

[0086] 19 F NMR (376 MHz, D2O) δ-78.72.

[0087] HRMS(ESI):[M + ]Calcd.m / z 141.0481, found m / z 141.0480.

[0088] Example 4:

[0089] A similar method to Example 1 was used to prepare the following using 2,6-dichloro-4-propargyloxypyridine as a raw material:

[0090] White powder, yield 88%.

[0091] 1 H NMR (400MHz, Methanol-d4) δ7.96 (s, 2H), 5.23 (d, J = 2.4Hz, 2H), 4.36 (s, 3H), 3.39 (t, J = 2.4Hz, 1H).

[0092] 13 C NMR (101MHz, Methanol-d4) δ169.52,149.65,114.73,79.27,74.82,59.14,42.44.

[0093] 19 F NMR(376MHz,Methanol-d4)δ-80.05.

[0094] 1 H NMR(400MHz,D2O)δ7.72(s,2H),5.04(s,2H),4.23(s,3H).

[0095] 13 C NMR(101MHz,D2O)δ169.10,149.46,114.82,59.06,42.73.

[0096] 19 F NMR (376 MHz, D2O) δ-78.87.

[0097] HRMS(ESI):[M +]Calcd.m / z 215.9977, found m / z 215.9978.

[0098] Example 5:

[0099] A similar method to that of Example 1 was used to prepare the following using 2,6-dichloro-4-propargylaminoformylpyridine as a raw material:

[0100] White powder, yield 75%.

[0101] 1 H NMR (400MHz, D2O) δ8.40 (s, 2H), 4.45 (s, 3H), 4.13 (d, J = 2.6Hz, 2H), 2.61 (t, J = 2.5Hz, 1H).

[0102] 13 C NMR (101MHz, D2O) δ162.42,150.65,149.29,126.38,78.33,72.56,45.00,29.77.

[0103] 19 F NMR (376 MHz, D2O) δ-78.87.

[0104] HRMS(ESI):[M + ]Calcd.m / z 243.0086, found m / z 243.0085.

[0105] Example 6:

[0106] A similar method to Example 1 was used to prepare the following using 2,6-dichloro-4-trifluoromethylpyridine as a raw material:

[0107] White powder, yield 70%.

[0108] 1 H NMR(400MHz,D2O)δ8.63(s,2H),4.52(s,3H).

[0109] 13 C NMR (101MHz, D2O) δ151.67,146.19,145.81,145.44,145.07,125.63,125.59,125.55,125.52,124.16,121.42,118.69,115.95,45.47.

[0110] 19F NMR(376MHz,D2O)δ-65.55,-78.86.

[0111] HRMS(ESI):[M + ]Calcd.m / z 229.9746, found m / z 229.9749.

[0112] Example 7:

[0113] A similar method to Example 1 was used to prepare the following using 2,6-dichloro-4-cyanopyridine as a raw material:

[0114] White powder, yield 72%.

[0115] 1 H NMR(400MHz,D2O)δ8.59(s,2H),4.49(s,3H).

[0116] 13 C NMR(101MHz,D2O)δ151.32,130.94,128.89,112.64,45.64.

[0117] 19 F NMR (376 MHz, D2O) δ-78.86.

[0118] HRMS(ESI):[M + ]Calcd.m / z 186.9824, found m / z 186.9826.

[0119] Example 8:

[0120] A similar method to Example 1 was used to prepare the following using 2,6-dichloro-4-methoxyformylpyridine as a raw material:

[0121]

[0122] White powder, yield 61%.

[0123] 1 H NMR(400MHz,D2O)δ8.60(s,2H),4.49(s,3H),3.99(s,3H).

[0124] 13 C NMR (101MHz, D2O) δ162.14,150.89,145.61,127.76,107.60,54.49,45.20. 19 FNMR (376MHz, D2O)δ-78.88.

[0125] HRMS(ESI):[M + ]Calcd.m / z 219.9927, found m / z 219.9930.

[0126] Example 9:

[0127] A similar method to Example 1 was used to prepare the following using 2,4,6-trichloropyridine as a raw material:

[0128] White powder, yield 66%.

[0129] 1 H NMR(400MHz,D2O)δ8.32(s,2H),4.39(s,3H).

[0130] 13 C NMR(101MHz,D2O)δ154.65,149.68,128.68,44.29.

[0131] 19 F NMR (376 MHz, D2O) δ-78.79.

[0132] HRMS(ESI):[M + ]Calcd.m / z 195.9482, found m / z 195.9480.

[0133] Example 10:

[0134] A similar method to Example 1 was used to prepare the following using 2,6-dichloropyridine as a raw material:

[0135] White powder, yield 83%.

[0136] 1 H NMR (400MHz, D2O) δ8.37(t,J=8.3Hz,1H),8.10(d,J=8.3Hz,2H),4.42(s,3H).

[0137] 13 C NMR(101MHz,D2O)δ149.50,146.84,128.49,44.54.

[0138] 19 F NMR (376 MHz, D2O) δ-78.84.

[0139] HRMS(ESI):[M +]Calcd.m / z 161.9872, found m / z 161.9871.

[0140] Example 11:

[0141] A similar method to Example 1 was used to prepare the following using 2,6-dichloro-4-methoxypyridine as a raw material:

[0142]

[0143] White powder, yield 81%.

[0144] 1 H NMR(400MHz,D2O)δ7.65(s,2H),4.24(s,3H),4.08(s,3H).

[0145] 13 C NMR(101MHz,D2O)δ171.18,149.22,114.16,58.64,42.51.

[0146] 19 F NMR (376 MHz, D2O) δ-78.85.

[0147] HRMS(ESI):[M + ]Calcd.m / z 191.9977, found m / z 191.9980.

[0148] Example 12:

[0149] A similar method to Example 1 was used to prepare the following using 2,4-dichloropyridine as a raw material:

[0150] White powder, yield 77%.

[0151] 1 H NMR (400MHz, D2O) δ8.77(d,J=6.8Hz,1H),8.27(d,J=2.3Hz,1H),7.97(dd,J=6.8,2.4Hz,1H),4.26(s,3H).

[0152] 13 C NMR (101MHz, D2O) δ154.90,148.44,147.74,129.81,126.79,47.02.

[0153] 19 F NMR (376 MHz, D2O) δ-78.88.

[0154] HRMS(ESI):[M + ]Calcd.m / z 161.9872, found m / z 161.9874.

[0155] Example 13:

[0156] A similar method to Example 1 was used to prepare the following using 2,4,6-trifluoropyridine as a raw material:

[0157]

[0158] White powder, yield 67%.

[0159] 1 H NMR (400MHz, D2O) δ6.10 (ddd, J=7.7, 5.9, 2.6Hz, 1H), 6.02 (ddd, J=10.5, 2.6, 1.5Hz, 1H), 3.31 (d, J=3.1Hz, 3H).

[0160] 13 C NMR(101MHz,D2O)δ173.40,173.22,170.77,170.59,164.20,163.99,159.01,158.80,156.36,156.14,1 24.29,121.14,117.98,114.83,97.73,97.68,97.53,97.48,84.50,84.23,84.18,83.92,28.55,28.48.

[0161] 19 F NMR(376MHz,D2O)δ-79.00,-87.40,-87.41,-87.45,-87.46,-92.84,-92.87,-92.89,-92.91,-92.94.

[0162] HRMS(ESI):[M + ]Calcd.m / z 148.0369, found m / z 148.0367.

[0163] Example 2: Identification and Analysis of the Reaction of Pyridinium Salts with Lysine

[0164]

[0165] To a 5 ml centrifuge tube, add 0.56 g of 2-chloro-N-methylpyridine, 0.25 g of Boc-lysine, 0.32 g of sodium phosphate, and 1 ml of deionized water. The centrifuge tube was shaken at room temperature for 30 minutes on a thermostatic oscillator. The reaction solution was filtered and the product was directly isolated by preparative liquid chromatography. The fraction containing the product was lyophilized in a freeze dryer. The product was a colorless oil (0.12 g) with a yield of 33%. The NMR and high-resolution characterization data of this compound are as follows:

[0166] Colorless oil, yield 33%.

[0167] 1 H NMR (400MHz, Methanol-d4) δ8.07(dd,J=6.7,1.6Hz,1H),8.01(ddd,J=8.9,7.1,1.6Hz,1H),7.26(d,J=9.1Hz,1H),6.95(td,J=6.9,1.2Hz,1H),4.13( dd,J=9.0,4.8Hz,1H),3.89(s,3H),3.52(t,J=7.2Hz,2H),1.91(dh,J=13.7 ,5.1Hz,1H),1.75(tt,J=14.4,8.2Hz,3H),1.63–1.51(m,2H),1.47(s,9H).

[0168] 13 C NMR (101MHz, Methanol-d4) δ174.76,156.83,153.26,142.94,141.49,112.47,110.85,79.17,53.27,42.71,40.98,31.07,27.53,27.39,22.74.

[0169] HRMS(ESI):[M + ]Calcd.m / z 338.2074, found m / z 338.2074.

[0170] Using the above method, the reaction product of 4-chloro-N-methylpyridine and Boc-lysine is:

[0171] Colorless oil, yield 27%.

[0172] 1H NMR(400MHz,D2O)δ7.85(d,J=7.2Hz,1H),7.74–7.67(m,1H),6.67(t,J=5.6Hz,2H),3.96(dt,J=12.3,6.1Hz,1H),3.76(s,3H),3.2 1(t,J=6.8Hz,2H),1.75(t,J=6.9Hz,1H),1.67–1.59(m,1H),1.56(dq,J=7.5,3.8,3.2Hz,2H),1.36(d,J=8.0Hz,2H),1.29(s,9H).

[0173] 13 C NMR (101MHz, D2O) δ176.75,157.76,156.87,143.86,141.68,110.56,105.16,81.39,53.50,44.21,42.03,30.24,27.55,26.92,22.32.

[0174] HRMS(ESI):[M + ]Calcd.m / z 338.2074, found m / z 338.2075.

[0175] Using the above method, the reaction product of 2-methylthio-N-methylpyrimidine and Boc-lysine is:

[0176] Colorless oil, yield 11%.

[0177] 1 H NMR (400MHz, Methanol-d4) δ8.88(dd,J=4.4,2.2Hz,1H),8.48(dd,J=6.6,2.2Hz,1H),7.05(dd,J=6.6,4.4Hz,1H),4.11(dd,J=9.0, 4.9Hz,1H),3.85(s,3H),3.71(td,J=7.2,1.7Hz,2H),1.97–1.85(m,1H),1.83–1.67(m,3H),1.54(q,J=8.5,7.8Hz,2H),1.47(s,9H).

[0178] 13 C NMR (101MHz, Methanol-d4) δ174.83,165.35,156.82,154.47,151.10,109.69,79.17,53.34,42.01,40.82,31.03,27.87,27.40,22.68.

[0179] HRMS(ESI):[M + ]Calcd.m / z 339.2027, found m / z 339.2022.

[0180] Example 3: Reaction of pyridinium salt probe with polypeptide

[0181] Dissolve the peptide (model peptide 1 (commercially available Melanotan I, purchased from Wuxi Yipeptide)) in deionized water to prepare a 0.1 mM peptide solution. Accurately weigh the pyridinium salt probe and prepare a reaction solution of the appropriate concentration in PBS buffer or distilled water. Incubate the peptide solution with 1 mM probe in PBS at 37°C for 60 minutes. Filter the reaction solution and analyze it directly using liquid chromatography-mass spectrometry. The peptide is a biologically active peptide containing a single lysine residue without a protecting group.

[0182] Figure 1 and Figure 2 The conversion rates of pyridinium salts 2 (2a-2e) and the S series (S1-S11) with model peptide 1 (commercial Melanotan I) are shown, along with liquid chromatography monitoring of the reactions between the pyridinium salts and peptides. This analysis was used to screen the effects of pyridine ring substituents and leaving groups on the reaction efficiency. The results indicate that increasing the electron-withdrawing nature of the ring substituent increases the reaction efficiency, and that halogens or methylsulfonyl groups are highly efficient leaving groups.

[0183] Example 4: Primary mass spectrometry and circular dichroism studies of the reaction between pyridinium salt probe and protein

[0184] Dissolve the protein in deionized water and prepare a 50 μM protein solution. Accurately weigh the pyridinium salt probe and prepare a reaction solution of the corresponding concentration using PBS buffer or distilled water. Incubate the protein solution with 1000 μM probe in PBS solution at 37°C for 60 minutes. Use an ultrafiltration tube to directly filter out salts and small molecules in the reaction solution. The resulting pure protein solution is subjected to ESI primary mass spectrometry and circular dichroism analysis. The protein can be bovine serum albumin (BSA), horse serum albumin (HSA), lysozyme, insulin, carbonic anhydrase, myoglobin, ribonuclease A (RNaseA), etc.

[0185] Figure 3 and Figure 4Shown are the primary mass spectra of the labeling and cross-linking reactions of proteins with pyridinium salt probes (2a, S1, and S10). The results demonstrate the high reactivity of the dihalogenated pyridinium salt cross-linking probes. Circular dichroism analysis reveals that the proteins undergo no significant changes before and after the labeling reaction, indicating that neither the cross-linking nor the labeling reaction significantly affects the secondary structure of the protein.

[0186] Example 5: In-gel fluorescence analysis of the reaction between pyridinium salt probe and protein

[0187] The protein was dissolved in deionized water to prepare a 5 μM protein solution. The pyridinium salt probe was accurately weighed and the reaction solution of the appropriate concentration was prepared in PBS buffer or distilled water. The protein solution and 50 μM probe were incubated in PBS at 37°C for 60 minutes. The proteins were then fluorescently labeled using a "click" reaction. The reaction system was added with 1 mM CuSO4, 1 mM TCEP, 100 μM TBTA, and 100 μM 5-TAMRA-N3. The reaction was incubated at 25°C for 2 hours. After completion of the reaction, SDS-PAGE electrophoresis was performed to visualize the in-gel fluorescence signal and stain the protein bands with Coomassie Brilliant Blue. Proteins included bovine serum albumin (BSA), horse serum albumin (HSA), lysozyme, insulin, carbonic anhydrase, myoglobin, ribonuclease A (RNase A), and HeLa cell lysate.

[0188] Figure 5 The cross-linking and labeling efficiency of the two-leaving group pyridinium salts PX1 and PX2 in the proteome are studied. The results show that PX1 has a higher proteome labeling efficiency.

[0189] Example 6: Coomassie Brilliant Blue Staining Analysis of the Reaction of Pyridinium Salt Probe with Protein

[0190] Dissolve the protein in deionized water and prepare a 5 μM protein solution. Accurately weigh the pyridinium salt probe and prepare the corresponding concentration of the reaction solution in PBS buffer or distilled water. Incubate the protein solution with 50 μM probe in PBS solution at 37°C for 60 minutes, and stain the protein bands with Coomassie Brilliant Blue. Proteins include bovine serum albumin (BSA), horse serum albumin (HSA), lysozyme, insulin, carbonic anhydrase, myoglobin, ribonuclease A (RNaseA), etc.

[0191] Figure 6 and Figure 7It was shown that the rigid structure short cross-linking probes based on pyridinium salts PX1 and PX3-PX13 were mostly difficult to undergo intermolecular cross-linking reactions, so it can be inferred that the cross-linking reaction mainly occurred within the protein molecules.

[0192] Example 7: Hydrolytic Stability Test of Pyridinium Salt Probe

[0193] The pyridinium salt probe (PX1) was dissolved in water to a final concentration of 100 μM and incubated at 37°C with constant shaking (200 rpm). Samples were collected at 0 h, 1 h, 2 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 168 h, filtered through a 0.22 μm filter, and the peak area ratio of the intact probe was determined by high-performance liquid chromatography (HPLC). The residual rate was calculated, with the initial peak area (0 h) as 100%.

[0194] Figure 8 a shows that the residual rate of pyridinium salt PX1 in aqueous solution is still more than 80% after 168 h, indicating that the probe has good stability in the aqueous phase system.

[0195] Example 8: Cytotoxicity Detection of Pyridinium Salt Probes

[0196] HeLa cells in good proliferation state were seeded into 96-well plates (approximately 5000 cells per well). After 24 hours, a series of concentrations of pyridinium salt probes were added to the cells and incubated for 24 hours and 48 hours, respectively, with three replicates for each concentration. After the incubation, 20 μL of MTT solution (5 mg / mL) was added to each well and incubated for another 4 hours. The culture medium was discarded, and 200 μL of DMSO was added to each well to dissolve the blue-purple formazan crystals. The absorbance at 570 nm was measured using a microplate reader, and the relative cell viability (compared with the control group) was calculated. A dose-effect curve was drawn with the probe concentration as the horizontal axis and the cell survival rate as the vertical axis.

[0197] Figure 8 b shows that after incubation with pyridinium salt PX1 at a concentration of 100 μM for 24 hours, the cell survival rate is still higher than 50%, confirming that the probe has good biocompatibility.

Claims

1. A probe that can be used for arylation cross-linking modification of nucleophilic amino acid residues in proteins, characterized in that: Having the structure shown in formula I: in: R1 and R2 are each independently selected from the group consisting of hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C1-C6 amide, C1-C6 haloalkoxy, C2-C6 alkenyl, C2-C6 haloalkenyl, C2-C6 alkynyl, C2-C6 haloalkynyl, hydroxyl, amino, C3-C6 cycloalkyl, phenyl, halogen, cyano, carboxyl, or aldehyde; The halogen is fluorine, chlorine, bromine or iodine; The alkyl, alkenyl or alkynyl group is a straight chain or branched chain; the alkyl, alkenyl or alkynyl group itself or as part of other substituents can be selected from: Alkyl: methyl, ethyl, propyl, butyl, pentyl, hexyl or isomers thereof, wherein the isomers include isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl and tert-pentyl; Haloalkyl: a group containing one or more identical or different halogen atoms selected from CH2Cl, CHCl2, CCl3, CH2F, CHF2, CF3, CF3CH2, CH3CF2, CF3CF2, CCl3CCl2; Cycloalkyl: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl; Alkenyl: vinyl, allyl, 1-propenyl, buten-2-yl, buten-3-yl, penten-1-yl, penten-3-yl, hexen-1-yl, 4-methyl-3-pentenyl; Alkynyl: ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-2-yl, 1-methyl-2-butynyl, hexyn-1-yl, 1-ethyl-2-butynyl. X is selected from: alkylate, substituted alkylate, halogen, halogen oxyacid, phosphate, sulfate, sulfonate or borate; wherein: The alkyl acid radical or substituted alkyl acid radical is a C1-C6 alkyl acid radical or a C1-C6 halogenated alkyl acid radical; The halogen or halogen oxygenate is selected from the group consisting of fluorine, chlorine, bromine, iodine, hypochlorite, chlorite, chlorate, perchlorate, hypobromite, bromite, bromate, perbromate, hypoiodite, iodite, iodate, and periodate; The phosphate is selected from the group consisting of monohydrogen phosphate, dihydrogen phosphate, pyrophosphate, metaphosphate, hypophosphite, phosphite, polyphosphate, orthophosphate, and hexafluorophosphate; The sulfate group is selected from the group consisting of sulfide, bisulfate, sulfate, bisulfite, sulfite, pyrosulfate, dithionate, thiosulfate, dithionite, and persulfate; The sulfonate group is selected from the group consisting of trifluoromethanesulfonate, methanesulfonate, benzenesulfonate, and p-toluenesulfonate; The borate is selected from: borate or tetrafluoroborate; Y is selected from fluorine, chlorine, bromine, iodine, methoxy, methylthio or methylsulfonyl.

2. A probe for arylation and cross-linking modification of nucleophilic amino acid residues in proteins according to claim 1, characterized in that: The probe comprises at least one leaving group, and the leaving group is located at the para position or meta position of the pyridine ring; wherein Y represents 1 to 3 leaving groups, and the leaving groups are the same or different.

3. A probe for arylation and cross-linking modification of nucleophilic amino acid residues in proteins according to claim 1, characterized in that: The atom modified in the arylation cross-linking reaction is an oxygen atom, a sulfur atom or a selenium atom.

4. A probe for arylation and cross-linking modification of nucleophilic amino acid residues in proteins according to claim 1, characterized in that: The probe is a six-membered nitrogen-containing onium salt compound.

5. A probe for arylation and cross-linking modification of nucleophilic amino acid residues in proteins according to claim 1, characterized in that: The probe is used for cross-linking modification of cysteine ​​and lysine residues of proteins, and the feeding amount is 0.1 to 200 equivalents of the protein.

6. A probe for arylation and cross-linking modification of nucleophilic amino acid residues in proteins according to claim 5, characterized in that: The cross-linking reaction is carried out in a buffer solution with a pH value of 5 to 8, and the buffer solution is weakly acidic, neutral or weakly alkaline.

7. A probe for arylation and cross-linking modification of nucleophilic amino acid residues in proteins according to claim 5, characterized in that: The reaction solvent is selected from water, acetonitrile, methanol, ethanol, isopropanol, tert-butanol, ethylene glycol, glycerol, trifluoroethanol, hexafluoroisopropanol, dimethyl sulfoxide, N,N-dimethylformamide or a mixture thereof.

8. The probe for arylation and cross-linking modification of nucleophilic amino acid residues in proteins according to claim 5, characterized in that: The reaction time is 0.1 hour to 100 hours.

9. The probe for arylation and cross-linking modification of nucleophilic amino acid residues in proteins according to claim 5, characterized in that: The reaction temperature is -20°C to 50°C.

10. The probe for arylation and cross-linking modification of nucleophilic amino acid residues in proteins according to claim 5, characterized in that: The method is used for cross-linking mass spectrometry analysis of proteins.

11. The probe for arylation and cross-linking modification of nucleophilic amino acid residues in proteins according to claim 5, characterized in that: Its structural formula is 12. The method for preparing a probe for chemical modification of protein cysteine ​​residues according to claim 1, characterized in that The following steps are involved: Add compound III into a reaction vessel, and alkylating agent II R 2 X, R1 and R2 are each independently selected from the group consisting of hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C1-C6 amide, C1-C6 haloalkoxy, C2-C6 alkenyl, C2-C6 haloalkenyl, C2-C6 alkynyl, C2-C6 haloalkynyl, hydroxyl, amino, C3-C6 cycloalkyl, phenyl, halogen, cyano, carboxyl, or aldehyde; The halogen is fluorine, chlorine, bromine or iodine; The alkyl, alkenyl or alkynyl group is a straight chain or branched chain; the alkyl, alkenyl or alkynyl group itself or as part of other substituents can be selected from: Alkyl: methyl, ethyl, propyl, butyl, pentyl, hexyl or isomers thereof, wherein the isomers include isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl and tert-pentyl; Haloalkyl: a group containing one or more identical or different halogen atoms selected from CH2Cl, CHCl2, CCl3, CH2F, CHF2, CF3, CF3CH2, CH3CF2, CF3CF2, CCl3CCl2; Cycloalkyl: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl; Alkenyl: vinyl, allyl, 1-propenyl, buten-2-yl, buten-3-yl, penten-1-yl, penten-3-yl, hexen-1-yl, 4-methyl-3-pentenyl; Alkynyl: ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-2-yl, 1-methyl-2-butynyl, hexyn-1-yl, 1-ethyl-2-butynyl; X is selected from: alkylate, substituted alkylate, halogen, halogen oxyacid, phosphate, sulfate, sulfonate or borate; wherein: The alkyl acid radical or substituted alkyl acid radical is a C1-C6 alkyl acid radical or a C1-C6 halogenated alkyl acid radical; The halogen or halogen oxygenate is selected from the group consisting of fluorine, chlorine, bromine, iodine, hypochlorite, chlorite, chlorate, perchlorate, hypobromite, bromite, bromate, perbromate, hypoiodite, iodite, iodate, and periodate; The phosphate is selected from the group consisting of monohydrogen phosphate, dihydrogen phosphate, pyrophosphate, metaphosphate, hypophosphite, phosphite, polyphosphate, orthophosphate, and hexafluorophosphate; The sulfate group is selected from the group consisting of sulfide, bisulfate, sulfate, bisulfite, sulfite, pyrosulfate, dithionate, thiosulfate, dithionite, and persulfate; The sulfonate group is selected from the group consisting of trifluoromethanesulfonate, methanesulfonate, benzenesulfonate, and p-toluenesulfonate; The borate is selected from: borate or tetrafluoroborate; Y is selected from fluoro, chloro, bromo, iodo, methoxy, methylthio or methylsulfonyl; The reaction solvent is selected from: dichloromethane, toluene, acetonitrile, ether or tetrahydrofuran, and a probe that can be used for reversible chemical modification of protein cysteine ​​residues is obtained. ， The product is directly precipitated in the form of a precipitate, or an equal volume of diethyl ether is added to the reaction solvent to promote the precipitation of the product.

13. A method for preparing a probe for chemical modification of protein cysteine ​​residues according to claim 11, characterized in that: The alkylating agent II is selected from: iodomethane, methyl trifluoromethanesulfonate, ethyl trifluoromethanesulfonate, methyl benzenesulfonate or trimethyloxonium tetrafluoroborate.

14. A method for modifying a protein using the probe for chemical modification of protein cysteine ​​residues according to claim 1, characterized in that: The modification sites of the method are cysteine ​​and lysine residues of proteins, and the reaction process is as follows:

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