A class of multi-targeted crosslinking agents and their preparation and application

Abstract

This application relates to the field of biotechnology, and in particular to a class of multi-target cross-linking agents and their preparation and application. This application provides a compound, and its salt or solvate, the chemical structure of which is shown in Formula I. The compound provided in this application employs a Michael receptor at one end and a bisacrylic acid group at the other end, exhibiting high reactivity. Furthermore, this compound, as a cross-linking agent, can further undergo click chemistry reactions, thereby enriching modified peptides.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a class of multi-targeted crosslinking agents and their preparation and application. Background Technology

[0002] Chemical cross-linking mass spectrometry (XL-MS) is a novel technique that covalently links closely spaced amino acid residues in proteins using cross-linking agents. The cross-linked proteins are then processed and analyzed using mass spectrometry to study protein structure and protein-protein interactions. Cross-linking agents are crucial in XL-MS studies. Currently, the most commonly used cross-linking agents are primarily bis(sulfosuccinimidyl) imide compounds (NHS) based on amino residues, including BS3 (bis(sulfosuccinimidyl)suberate), DSS (disuccinimidylsuberate), and DSSO (disuccinimidyl sulfoxide). These cross-linking agents have relatively limited coupling sites and exhibit poor stability in water.

[0003] Multi-target cross-linking agents can bind to a variety of amino acid residues, thereby increasing the coverage of protein cross-linking. The most typical multi-target cross-linking agents are those that can generate highly active carbene structures under light irradiation, including bis(acrylidine) groups, benzophenone, and azidobenzene, which produce nitrogen-carbenes or carbon-carbenes, theoretically capable of binding to any amino acid. However, while these structures yield a wealth of data, data analysis is extremely complex. Therefore, generally, cross-linking agents only employ this type of structure at one end, while using conventional NHS or similar materials at the other end. Summary of the Invention

[0004] In view of the shortcomings of the prior art described above, and to address the technical problem that multi-target crosslinking agents still need further development, the purpose of this application is to provide a class of multi-target crosslinking agents, their preparation and application, to solve the problems in the prior art. The crosslinking agent provided in this application is capable of mass spectrometry fragmentation, which can cleave crosslinked peptides into two parts under mass spectrometry conditions. Based on its characteristics, this can greatly improve the efficiency of data analysis and processing. The crosslinking agent of this application uses a Michael acceptor at one end and a bisacrylic acid group at the other end, exhibiting high reactivity. The crosslinking agent of this application can further undergo click chemistry reactions, thereby enriching the modified peptides.

[0005] To achieve the above and other related objectives, this application provides a compound, and its salt or solvate, the chemical structural formula of which is shown in Formula I:

[0006]

[0007] Where X is selected from one or more combinations of O, S, NH, and NCH3; R is selected from one or more combinations of C, CO, COO, CONH, and CONCH3; and n is a natural number from 1 to 10.

[0008] In any embodiment of this application, the compound is

[0009] Another aspect of this application provides a method for preparing the aforementioned compound and its salt or solvate, wherein 4-ethyl sulfate sulfone aniline reacts with a base to generate an intermediate product sulfone, and the sulfone undergoes a condensation reaction with 3-(3-(but-3-yn-1-yl)-3H-diazanaphth-3-yl)propionic acid to obtain a crosslinking agent.

[0010] This application also provides the use of the aforementioned compounds, and their salts or solvates, in the preparation of crosslinking agents.

[0011] Another aspect of this application provides a crosslinking agent, including the aforementioned compounds, and their salts or solvates.

[0012] This application also provides the use of the aforementioned compounds, their salts or solvates, or the aforementioned crosslinking agents in protein crosslinking.

[0013] This application also provides a method for studying protein structure or protein interactions, comprising adding the aforementioned compound, its salt or solvate, or the aforementioned cross-linking agent to a protein sample.

[0014] Compared with the prior art, the beneficial effects of this application are as follows:

[0015] 1. The compound of this application adopts a Michael acceptor at one end and a bisacrylidine group at the other end, and has high reactivity.

[0016] 2. The compound of this application, as a cross-linking agent, has the characteristic of being cleavable by mass spectrometry. Under mass spectrometry conditions, the cross-linked peptide can be cleaved into two parts. Based on this characteristic, the efficiency of data analysis and processing can be greatly improved.

[0017] 3. The compounds in this application can further undergo click chemistry reactions to enrich the modified peptides.

[0018] 4. The compound of this application has high stability in aqueous crosslinking agent. Attached Figure Description

[0019] Figure 1 This invention demonstrates the crosslinking agent involved in the present invention. 1 H NMR spectrum;

[0020] Figure 2This invention demonstrates the crosslinking agent involved in the present invention. 13 C NMR spectrum;

[0021] Figure 3 The mass spectrum of the crosslinking agent involved in this invention is shown.

[0022] Figure 4 The mass spectrum of BSA after crosslinking with the crosslinking agent involved in this invention is shown.

[0023] Figure 5 This shows a mapping diagram on a crystal after the crosslinking agent involved in this invention crosslinks BSA. Detailed Implementation

[0024] To make the inventive objectives, technical solutions, and beneficial effects of this application clearer, the following description, in conjunction with embodiments, further illustrates this application. It should be understood that the embodiments described are for illustrative purposes only and are not intended to limit the scope of the application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this description.

[0025] The inventors of this application, through extensive research and exploration, discovered a class of multi-target crosslinking agents and their preparation and application, and completed this application based on this discovery.

[0026] The first aspect of this application provides a compound, and its salt or solvate, wherein the chemical structural formula of the compound is shown in Formula I:

[0027]

[0028] Where X is selected from one or more combinations of O, S, NH, and NCH3; R is selected from one or more combinations of C, CO, COO, CONH, and CONCH3; and n is a natural number from 1 to 10.

[0029] In this invention, the term "salt" generally refers to any salt that is physiologically tolerable when used in a suitable manner for treatment (particularly when applied or used in humans and / or mammals) (generally meaning that it is non-toxic, particularly as a result of counterions). These physiologically acceptable salts can be formed with cations or bases, and in the context of this invention, particularly when administered to humans and / or mammals, they should be understood as salts formed from at least one compound provided according to this invention, typically an acid (deprotonated), such as an anion, and at least one physiologically tolerable cation (preferably an inorganic cation). Specifically, in the context of this invention, this may include salts formed with alkali metals and alkaline earth metals, as well as salts formed with ammonium cations (NH4+). +The salts formed can specifically include, but are not limited to, salts formed with (mono) or (di) sodium, (mono) or (di) potassium, magnesium, or calcium. These physiologically acceptable salts can also be formed with anions or acids, and in the context of this invention, particularly when administered to humans and / or mammals, they should be understood as salts formed by at least one compound provided according to this invention, typically protonated (e.g., on nitrogen), such as a cation, and at least one physiologically tolerable anion. In the context of this invention, salts can specifically include salts formed with physiologically tolerable acids, i.e., salts formed by specific active compounds with physiologically tolerable organic or inorganic acids, specifically including, but not limited to, salts formed with hydrochloric acid, hydrobromic acid, hydroiodic acid; sulfuric acid, persulfate, pyrosulfonic acid; phosphoric acid, nitric acid; methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid; formic acid, acetic acid, acetoacetic acid, trifluoroacetic acid, pyruvic acid, propionic acid, butyric acid, valeric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid; benzoic acid, salicylic acid, 2- (4-Hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthylcarboxylic acid, nicotinic acid, pectic acid, 3-phenylpropionic acid, picric acid, terpentine; 2-hydroxyethanesulfonic acid, aminosulfonic acid, dodecyl sulfate, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid; citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, citric acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, aspartic acid, and salts formed from sulfosalicylic acid.

[0030] In this document, the term "solvent" refers to a compound of the present disclosure or a pharmaceutically acceptable salt thereof, contained in a stoichiometric or non-stoichiometric solvent in which non-covalent molecules are bound together by force. Preferred solvents are volatile and non-toxic, and can be administered to humans in very small doses. Examples of solvents, but not limited to, include water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. The term "hydrate" refers to a complex in which the solvent molecules are water.

[0031] In a specific embodiment of this application, the compound is: The compound in this application has a Michael receptor on the left. The right side is a bis-acrylidine group. In this paper, the broken bonds indicated by the wavy lines show the connection points between the illustrated group and other parts of the molecule. The diazirine group is a reactive group containing two nitrogen atoms in an unsaturated three-membered ring, capable of rapidly generating carbene and nitrogen gas under ultraviolet light irradiation or heating (Science 2019, 366, 875–878). The resulting carbene intermediate exhibits high reactivity, efficiently undergoing insertion reactions with CH bonds in various adjacent chemical environments, thereby achieving chemical cross-linking. Michael acceptors are a class of groups that can undergo Michael addition with nucleophilic groups on the side chains of protein amino acids to form stable covalent bonds. The cross-linking agent in this application is multi-targeted, meaning that the Michael acceptor can react with any nucleophilic amino acid, and the diazirine group can theoretically react with any amino acid. The compound in this application uses a Michael acceptor at one end and a diazirine group at the other, exhibiting high reactivity. The compound in this application, as a cross-linking agent, has the characteristic of being mass spectrometrically cleavable in the Michael receptor moiety. Under mass spectrometry conditions, the cross-linked peptide can be cleaved into two parts. Based on this characteristic, the efficiency of data analysis and processing can be greatly improved.

[0032] The second aspect of this application provides a method for preparing the aforementioned compound and its salt or solvate, wherein 4-ethyl sulfate sulfone aniline reacts with a base to generate an intermediate product sulfone, and the sulfone undergoes a condensation reaction with 3-(3-(but-3-yn-1-yl)-3H-diazanaphth-3-yl)propionic acid to obtain a crosslinking agent.

[0033] In the preparation method provided in this application, the alkali is selected from sodium hydroxide, potassium hydroxide, magnesium hydroxide, or calcium hydroxide. In a specific embodiment of this application, the alkali is sodium hydroxide. The reaction time of 4-ethyl sulfate sulfone aniline with the alkali is 6–18 h, specifically, it can be 6–10 h, 10–12 h, or 12–18 h, etc. The reaction temperature of 4-ethyl sulfate sulfone aniline with the alkali is room temperature. The molar ratio of alkali to 4-ethyl sulfate sulfone aniline is 2–4:1, specifically, it can be 2–3:1, 3–3.5:1, or 3.5–4:1, etc. After the reaction of 4-ethyl sulfate sulfone aniline with the alkali, the intermediate product sulfone is separated by C-18 reversed-phase chromatography (CH3CN / H2O = 15%).

[0034] In the preparation method provided in this application, the reaction time for the condensation reaction between sulfone and 3-(3-(but-3-yn-1-yl)-3H-diazanaphth-3-yl)propionic acid is 0.5–12 h, specifically, it can be 0.5–h, 2–10 h, or 10–12 h, etc. The molar ratio of sulfone to 3-(3-(but-3-yn-1-yl)-3H-diazanaphth-3-yl)propionic acid is 0.5–2:1, specifically, it can be 0.5–1:1, 1–1.5:1, or 1.5–2:1, etc. The condensation reaction temperature is room temperature. After the condensation reaction, the crosslinking agent is separated by C-18 reversed-phase chromatography (CH3CN / H2O = 35%). Processing aids are also included during the condensation reaction, including tetramethylchlorourea hexafluorophosphate and N-methylimidazole.

[0035] The third aspect of this application provides the use of the aforementioned compounds, and their salts or solvates, in the preparation of crosslinking agents.

[0036] A fourth aspect of this application provides a crosslinking agent, including the aforementioned compound and its salts or solvates. The crosslinking agent provided in this application also includes a buffer solution. The buffer solution is not particularly limited, and may be, for example, PBS buffer (phosphate buffered saline solution). The crosslinking agent of this application can further undergo click chemistry reactions to enrich modified peptides.

[0037] The fifth aspect of this application provides the use of the aforementioned compounds, their salts or solvates, or the aforementioned crosslinking agents in protein crosslinking.

[0038] The sixth aspect of this application provides a method for studying protein structure or protein interactions, comprising adding the aforementioned compound, its salt or solvate, or the aforementioned cross-linking agent to a protein sample. The protein may be, for example, a common protein well known to those skilled in the art, such as bovine serum albumin or Escherichia coli lysate.

[0039] In some embodiments, when studying protein structure or protein interactions, the cross-linking agent of this application can be added to a protein sample, reacted in a buffer well-known to those skilled in the art, and then the sample and data can be processed and analyzed. The processing method includes: first, reduction with dithiothreitol (DTT), followed by alkylation with iodoacetamide. Then, sequencing-grade trypsin is added to enzymatically digest the cross-linked peptides into peptides. After digestion, formic acid is added to inactivate the enzyme. The peptides are then enriched with titanium dioxide microspheres, and non-cross-linked peptides are removed. The enriched peptides are then desalted to obtain the cross-linked peptides. Utilizing the mass spectrometry-based fragmentation and multi-targeting characteristics of the cross-linking agent of this application, it can cross-link with a variety of proteins, which is beneficial for obtaining more data results in routine protein structure or protein interaction studies.

[0040] The beneficial effects of this invention are:

[0041] The compound provided by this invention employs a Michael acceptor at one end and a bisacrylidine group at the other, exhibiting high reactivity. As a cross-linking agent, the Michael acceptor portion of the compound possesses the characteristic of mass spectrometry-based fragmentation, allowing for the cleavage of cross-linked peptides into two parts under mass spectrometry conditions. This characteristic significantly improves data analysis and processing efficiency. The compound provided in this application can further undergo click chemistry reactions, thereby enriching the modified peptides. The compound of this application exhibits high stability in aqueous solutions as a cross-linking agent.

[0042] The present application will be further illustrated by the following examples, but these examples do not limit the scope of the present application.

[0043] Example 1

[0044] Synthesis of Reagent 4

[0045]

[0046] Synthesis of Compound 2

[0047] 500 mg (1.78 mmol) of ethyl sulfone aniline 4-sulfate was added to (1 M) aqueous sodium hydroxide solution (4 mL, 4 mmol, maintaining pH 12). The reaction was carried out at room temperature for 12 hours. The mixture was separated by C-18 reversed-phase chromatography (CH3CN / H2O = 15%) to obtain a white solid compound 2, totaling 221 mg, with a yield of 68%. (ESI-HRMS calculated for C8H9NO2S[(M+H)]) + ]:183.0354,found:183.0354.

[0048] Synthesis of Compound 4

[0049] Compound 2 (37 mg, 0.20 mmol) was dissolved in acetonitrile (2 mL) in a 25 mL flask. 3-(3-(but-3-yn-1-yl)-3H-diazanaphth-3-yl)propionic acid (3, 32 mg, 0.20 mmol), tetramethylchlorourea hexafluorophosphate (TCFH, 59 mg, 0.21 mmol), and N-methylimidazolium (NMI, 54.6 μL, 0.62 mmol) were added to the flasks separately, and the reaction was carried out at room temperature for 2 hours. Compound 4 (yDVSP) was obtained as a white solid (35 mg) by C-18 reversed-phase chromatography (CH3CN / H2O = 35%), with a yield of 52%.

[0050] The results are as follows Figures 1-3 As shown. 1H NMR (500MHz, DMSO-d6) δ10.43(s,1H),7.84–7.76(m,4H),7.05(dd,J=16.4,9.8Hz,1H),6.27(d,J=16.4Hz,1H),6.14(d,J=9.8Hz, 1H), 2.83 (t, J=2.6Hz, 1H), 2.20 (t, J=7.6Hz, 2H), 2.01 (td, J=7.4, 2.7Hz, 2H), 1.77 (dd, J=8.3, 6.9Hz, 2H), 1.60 (t, J=7.4Hz, 2H). 13 C NMR (126MHz, DMSO-d6)δ12.7,27.5,28.2,30.6,31.5,71.8,83.2,119.0,127.7,128.8,132.8,139.0,143.9,170.6.ESI-HRMS calculated forC 16 H 17 N3O3S[(M+H) + ]:331.0991,found:331.0987.

[0051] Example 2

[0052] Cross-linking with proteins

[0053] Crosslinking with bovine serum albumin (BSA)

[0054] Bovine serum albumin (BSA) (purchased from Shanghai Bioengineering Co., Ltd.) was dissolved in PBS (pH=7.0) buffer to prepare a 10 mg / mL solution. Compound 4 prepared in Example 1 was dissolved in DMSO to prepare a 10 mM solution. 50 μL of BSA solution, 22.5 μL of compound 4 solution, and 428 μL of PBS (pH=7.4) buffer were added to a 1.5 mL centrifuge tube. After co-incubation at 37°C for 12 h, the solution was added to a Nanosep filter (10K, Pall Corporation) and centrifuged at 10,000 rpm for 5 min to remove excess small molecule compounds. The solution was washed with 200 μL of PBS (pH=7.0) buffer and centrifuged three times to ensure complete removal of excess small molecules. Finally, 1 mL of PBS (pH=7.0) buffer was added to dissolve the BSA from the filter membrane. The solution was then transferred to a clean 12-well plate and irradiated with 365 nm UV for 5 min to concentrate the protein solution to 2 mg / mL.

[0055] Example 3

[0056] Preparation of Escherichia coli lysate

[0057] Collect the grown *E. coli* bacteria and centrifuge at 1000g, 4°C, for 5 minutes. After removing the supernatant, resuspend the *E. coli* in 10mL of PBS and centrifuge again at 1000g, 4°C, for 5 minutes, removing the supernatant once more. Then add 5mL of pre-chilled RIPA lysis buffer and 50μL of protease inhibitor, resuspend the *E. coli*, and incubate on ice for 10 minutes. After incubation, centrifuge at 10000g, 4°C, for 15 minutes. The collected supernatant is the *E. coli* lysis buffer.

[0058] Cross-linking with E. coli lysate

[0059] Take 63.86 μL of E. coli lysate (7.83 mg / mL) into a 5 mL centrifuge tube, add 2 μL of the compound 4 solution prepared in Example 1 and 186.1 μL of PBS (pH=7.0) buffer, and incubate at 37 °C for 12 h. Add the solution to a Nanosepfilter (10K, Pall Corporation), centrifuge at 4000 rpm for 20 min to remove excess small molecule compounds, wash with 250 μL of PBS (pH=7.0) buffer, and repeat centrifugation three times to ensure complete removal of excess small molecules. Finally, add 500 μL of PBS (pH=7.0) buffer to dissolve BSA from the filter membrane, transfer to a clean 12-well plate, irradiate with 365 nm UV for 10 min, and concentrate the protein solution to 2 mg / mL.

[0060] Example 4

[0061] Protein enrichment after modification

[0062] Biotin-azide (CAS No.: 908007-17-0, Xi'an Kangfuno Biotechnology Co., Ltd.), a compound containing azide and enrichment handle, was dissolved in distilled water to prepare a 20 mM solution. Sodium ascorbate, {4-[(bis{[1-(2-methyl-2-propyl)-1H-1,2,3-triazol-4-yl]methyl}amino)methyl]-1H-1,2,3-triazol-1-yl}acetic acid (BTTAA), and anhydrous copper sulfate were each dissolved in distilled water to prepare 80 mM solutions. These solutions were then added to the protein solutions obtained in Example 2 or Example 3 to bring the final concentration of the compound to 1 mM, the final concentrations of sodium ascorbate, BTTAA, and anhydrous copper sulfate to 4 mM, and the final protein concentration to 1 mg / mL. The solutions were incubated at 37°C for 2 hours. Subsequently, 10 mM EDTA-2Na (pH=7.0) solution was added to complex copper ions, and the reaction was allowed to proceed at room temperature for 5 minutes. The solution was then added to a Nanosep filter (10K, Pall Corporation) and centrifuged at 10,000 rpm for 10 minutes to remove excess small molecule compounds. The solution was washed with PBS (pH=7.0) buffer and centrifuged three times to ensure that excess small molecules were completely removed. Finally, Tris-HCl (100 mM, pH=8) was added to dissolve the compounds, and the solution was collected for subsequent experiments.

[0063] Cross-linked protease digestion

[0064] First, the collected solution (i.e., the cross-linked protein sample) was added to a Nanosep filter (10K, Pallcorporation), centrifuged at 10,000 rpm for 5 minutes, and then Tris-HCl (100 mM, pH = 8) was added. DTT was added for reduction (final concentration 20 mM), and the mixture was incubated at 56°C for 30 minutes. Then, it was centrifuged at 10,000 rpm for 10 minutes. Next, Tris-HCl (100 mM, pH = 8) and iodoacetamide (final concentration 50 mM) were added, and the mixture was reacted in the dark for 30 minutes. After reduction and alkylation, the sample was washed three times with Tris-HCl (100 mM, pH = 8). Then, it was dissolved in Tris-HCl (100 mM, pH = 8), and trypsin was added. The mixture was digested at 37°C for 12 hours (protein:enzyme ratio 50:1). Then, formic acid was added to acidify the solution to pH=3 to inactivate the enzyme. The solution was centrifuged at 10000 rpm for 10 minutes to obtain the enzyme-digested peptides. The peptides were desalted using a C-18 solid-phase extraction column (Hypersep C-18, 100mg Thermo Scientific) and then freeze-dried to obtain peptide powder.

[0065] Next, two solutions were prepared: Washing buffer: 80% acetonitrile, 0.5% trifluoroacetic acid; Loading buffer: 1M glycolic acid dissolved in 80% acetonitrile, 0.5% trifluoroacetic acid. 4 mg of titanium dioxide powder was weighed into a 1.5 mL centrifuge tube, washed once with 200 μL of 0.5% ammonia, then washed three times with 200 μL of acetonitrile, and once with 200 μL of loading buffer. The enzyme-digested peptide solution was then added, and the mixture was incubated together at room temperature for 20 min. After centrifugation, the supernatant was discarded, and the mixture was washed once with 200 μL of loading buffer, then three times with 200 μL of washing buffer. The enriched peptides were eluted from the titanium dioxide powder with 180 μL of 0.5% ammonia. After elution, formic acid was added to acidify the solution to pH 3. The enriched cross-linked peptides were then desalted using a C-18 solid-phase extraction column (Hypersep C-18, 100mg Thermo Scientific) and lyophilized to obtain peptide powder.

[0066] Example 5

[0067] Protein sample mass spectrometry data analysis

[0068] The peptide powder samples prepared in Example 4 were redissolved in 2% formic acid and then analyzed using liquid chromatography-mass spectrometry (LC-MS / MS). A three-stage mass spectrometry strategy was employed to analyze the cross-linked peptides: first, a full scan of MS1 mass spectrometry was performed; then, the ten most intense ions with trivalent or higher charges were fragmented using low-energy CID and detected using a high-resolution orbital ion trap. Database searches identified two peptide sequences for all cross-linked peptides.

[0069] The results are as follows Figures 4-5 As shown.

[0070] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit this application. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in this invention should still be covered by the claims of this application.

Claims

1. A compound and its salt, characterized in that, The chemical structural formula of the compound is shown below: 。 2. The method for preparing the compound and its salt as described in claim 1, characterized in that, 4-ethyl sulfate sulfonyl aniline reacts with a base to produce an intermediate product sulfone. The sulfone then undergoes a condensation reaction with 3-(3-(but-3-yn-1-yl)-3H-diazanaphth-3-yl)propionic acid to obtain the compound.

3. The method for preparing the compound and its salt as described in claim 2, characterized in that, The alkali is selected from sodium hydroxide, potassium hydroxide, magnesium hydroxide, or calcium hydroxide; Alternatively, the reaction time of 4-ethyl sulfate sulfone aniline with alkali is 6~18 h; Alternatively, the reaction temperature of 4-ethyl sulfate sulfone aniline with a base is room temperature; Alternatively, the molar ratio of the base to 4-ethyl sulfate sulfone aniline is 2~4 :

1.

4. The method for preparing the compound and its salt as described in claim 2, characterized in that, The reaction time for the condensation reaction is 0.5~12 h; Alternatively, the condensation reaction is performed at room temperature; Alternatively, the condensation reaction may also include a processing aid selected from tetramethylchlorourea hexafluorophosphate and N-methylimidazole; Alternatively, the molar ratio of sulfone to 3-(3-(but-3-yn-1-yl)-3H-diazanaphth-3-yl)propionic acid is 0.5~2 :

1.

5. A crosslinking agent comprising the compound as claimed in claim 1, and a salt thereof.

6. The crosslinking agent as described in claim 5, characterized in that, It also includes buffer solutions.

7. The use of the compound of claim 1, its salt, or the crosslinking agent of any one of claims 5-6 in protein crosslinking.

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