CN109824565B

CN109824565B - Light-responsive multifunctional chemical cross-linking agent and preparation method and application thereof

Info

Publication number: CN109824565B
Application number: CN201910225490.8A
Authority: CN
Inventors: 包春燕; 汪晨曦; 张姝雯; 项昌育; 林秋宁; 朱麟勇
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2019-03-25
Filing date: 2019-03-25
Publication date: 2022-10-25
Anticipated expiration: 2039-03-25
Also published as: CN109824565A

Abstract

The invention discloses a light-responsive multifunctional chemical cross-linking agent, which has a structure shown as a general formula I:

the definition of each substituent group in the formula is shown in the specification. The photoresponsive multifunctional chemical cross-linking agent has simple preparation method and low cost, is the first cross-linking agent based on o-nitrobenzyl alcohol photocrosslinking reaction, and cross-links protein by coupling reaction of photoproduction aldehyde group and amino: in the crosslinking agent, the photoreactive group pair contains-NH ₂ The amino acid residue has high crosslinking efficiency and selectivity; the heterofunctional cross-linking agent with photoresponse is obtained by combining with functional groups used in other traditional cross-linking agents, so that the specific cross-linking of heterologous proteins is realized, and the heterofunctional cross-linking agent has great potential application in proteomics research.

Description

Light-responsive multifunctional chemical cross-linking agent and preparation method and application thereof

Technical Field

The invention belongs to the technical field of protein and function research thereof, and particularly relates to a light-responsive multifunctional chemical cross-linking agent, and a preparation method and application thereof.

Background

Proteins are carriers of gene expression and directly influence the life process of organisms. In genomics, people find that the research on genes cannot meet the exploration requirement on life processes, and the research on proteins has an important role in deepening the understanding on the life processes. Therefore, proteomics is an important subject of research in the post-gene age. In recent years, the research on proteomics by using the traditional biological method is gradually unable to meet the requirements of scientists. With the maturity of chemical synthesis process, people find that chemical small molecules are an emerging tool and are important supplements for traditional biological methods (such as Western Blot, enzyme-linked immunosorbent Elisa and the like) and biological research tools (such as cryoelectron microscopy).

Protein cross-linking, modification and labeling are common techniques for studying protein structure and interactions in proteomics. The method gradually develops into an important research method for researching the structure and the interaction of the protein by carrying out protein crosslinking through small molecules and utilizing crosslinking mass spectrometry, and has more intuitive advantages compared with the traditional biological method. The protein cross-linking agent is a kind of small molecular compound with 2 or more specific groups (-NH) ₂ -COOH, -HS, -OH, etc.) can be coupled to 2 or more proteins, respectively. Glutaraldehyde was commonly used as a protein cross-linking agent to link antibodies and indicators (e.g., enzymes) in the 70 s, but it has the disadvantage that disordered polymers are easily formed because the cross-linking groups are random. Recently, ruedi Aebersold et al successfully resolved their interaction network by cross-linking mass spectrometry analysis of the protein phosphatase family using amino-specific bis-succinimidyl suberate (DSS), a classical small molecule cross-linker (Herzog, f., et al (2012).Science337 (6100):1348-1352.). This work is classically similar to the study of protein interactions with chemical crosslinkers. Subsequently, similar small molecule cross-linkers have emerged and commercialized, including DSS series, BS3, DSSO (a.sinz (2017).Anal Bioanal Chem409 (1): 33-44.), and the like. Although these small molecule cross-linking agents can achieve heterogeneous specific cross-linking by introducing different reactive groups, the cross-linking process is uncontrollable, and many false positive results are brought in the processing of protein samples,in particular, self-crosslinking of proteins, has largely limited their use in the study and further application of protein interactions.

The photoreaction is introduced into a micromolecule cross-linking agent system due to space-time controllability, and can firstly utilize unstable thermal reaction active groups to be cross-linked with one protein and then cross-link a second protein through ultraviolet light, so that the interference of protein self-cross-linking is avoided, and the limitation caused by the traditional chemical cross-linking is greatly reduced. The prior photoresponsive groups applied to the crosslinker are mainly phenyl azide (Tanaka, y., et al. (2008).Mol Biosyst4 (6): 473-480.), benzophenone (Majmudar, c.y., et al. (2009).J Am Chem Soc131 (40): 14240-14242.), bisazeocin (Suchanek, M., et al. (2005).Nat Methods2 (4): 261-267), many of which have also been commercialized. However, although this photoresponsive crosslinking achieves space-time controllability, the reaction mechanism is through the insertion reaction of free radicals C-H or N-H, there is no selectivity to groups, and the reaction sites on the protein cannot be defined, thereby losing the specificity of crosslinking. In addition, most photoresponsive crosslinking agents have simple structures, but the preparation and synthesis of corresponding derivatives are difficult, so that the price is high.

Therefore, a new light-responsive group which is easy to prepare and has specific reactivity is found and is introduced into a small-molecule chemical cross-linking agent system, and the defects of the existing cross-linking agent are expected to be overcome.

Disclosure of Invention

It is a first object of the present invention to provide a photo-responsive multifunctional chemical crosslinking agent.

The second object of the present invention is to provide a method for preparing the photo-responsive multifunctional chemical crosslinking agent.

It is a third object of the present invention to provide a use of the photo-responsive multifunctional chemical crosslinking agent.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the first aspect of the invention provides a photo-responsive multifunctional chemical crosslinking agent, which has a structure shown in a general formula I:

wherein R is ₁ 、R ₂ 、R ₃ 、R ₄ Each independently selected from hydrogen, halogen atoms, hydroxyl groups, mercapto groups, amine groups, nitro groups, cyano groups, aldehyde groups, ketone groups, ester groups, amide groups, phosphonate groups, sulfonic acid groups, sulfonate groups, sulfone groups, sulfoxide groups, aryl groups, heteroaryl groups, alkyl groups, alkoxy groups, alkylene groups or modified alkyl groups;

r' is a conventional chemically reactive group selected from one of the following structures:

r' is selected from biotin and derivatives thereof, alkyl alkene and derivatives thereof, alkyl alkyne and derivatives thereof and other groups or hydrogen atoms which can be used for the magnetic bead enrichment technology; further selected from fluorescein and its derivatives, rhodamine and its derivatives, 1,8-naphthalimide and other groups with fluorescence recognition function;

in particular, when R' is selected from hydrogen atoms, the photo-responsive multifunctional chemical cross-linking agent I is a photo-responsive bifunctional cross-linking agent.

In particular, when R' is selected from biotin, alkyl alkene and derivatives thereof, alkyl alkyne and derivatives thereof, and the like which can be used in the magnetic bead enrichment technology, the photo-responsive multifunctional chemical crosslinking agent I is a photo-responsive trifunctional crosslinking agent.

Wherein X and R ₁ 、R ₂ 、R ₃ 、R ₄ Is selected from the group consisting of aryl, heteroaryl, alkyl, alkylene, modified alkyl, modified alkylene, ether linkage, ester linkage, carbonate linkage, amide linkage, urea linkage, and combinations thereof.

The aryl is a 5-10 membered aromatic monocyclic ring or aromatic condensed bicyclic ring structure;

the heteroaryl is a 5-10 membered aromatic monocyclic ring or aromatic condensed bicyclic ring structure which contains at least one heteroatom selected from O, S, N or Si on the ring;

the alkyl is a saturated or unsaturated aliphatic straight chain or branched chain alkyl with 1 to 30 carbon atoms;

the alkylene group is a saturated or unsaturated aliphatic linear or branched alkylene group having 1 to 30 carbon atoms;

any carbon atom of the modified alkyl is selected from halogen atom, -OH, -SH, -NO ₂ -CN, -CHO, -COOH, ester group, aryl group-CO-, -O-, -S-, -SO ₂ -primary, secondary, tertiary or quaternary ammonium groups, said modified alkyl group having from 1 to 30 atoms, the carbon-carbon single bond of which may optionally be replaced by a carbon-carbon double or triple bond;

any carbon atom of the modified alkylene group being an alkylene group substituted by a halogen atom, -OH, -SH, -NO ₂ -CN, -CHO, -COOH, ester group, aryl group-CO-, -O-, -S-, -SO ₂ -primary, secondary, tertiary or quaternary ammonium groups, said modified alkylene group having from 1 to 30 atoms, the carbon-carbon single bond of which may optionally be replaced by a carbon-carbon double or triple bond;

the ether linkage is selected from one of the following structures:

-O(CH ₂ ) _x -、-(CH ₂ ) _x O(CH ₂ ) _y -、-(CH ₂ CH ₂ O) _x 、-(CH ₂ CH ₂ O) _x (CH ₂ ) _y -, where x and y are not less than 0 and are integers;

the ester linkage is selected from one of the following structures:

-COO(CH ₂ ) _x -、-OCO(CH ₂ ) _x -、-(CH ₂ ) _x COO(CH ₂ ) _y -、-(CH ₂ ) _x OCO(CH ₂ ) _y -, where x and y are not less than 0 and are integers;

the carbonate linkage is selected from one of the following structures:

-CO ₃ (CH ₂ ) _x -、-(CH ₂ ) _x CO ₃ (CH ₂ ) _y -, where x and y are not less than 0 and are integers;

the amide bond is selected from one of the following structures:

-NHCO(CH ₂ ) _x -、-CONH(CH ₂ ) _x -、-(CH ₂ ) _x NHCO(CH ₂ ) _y -、-(CH ₂ ) _x CONH(CH ₂ ) _y -, where x and y are not less than 0 and are integers;

the urea linkage is selected from one of the following structures:

-NHCONH(CH ₂ ) _x -、-(CH ₂ ) _x NHCONH(CH ₂ ) _y -, where x and y are not less than 0 and are integers;

the alkyl alkene and the derivative thereof are unsaturated aliphatic straight-chain or branched-chain alkyl with 1-30 carbon atoms, and non-specified points in the alkyl alkene contain unlimited number of carbon-carbon double bonds;

the alkyl alkynes and their derivatives are unsaturated aliphatic linear or branched alkyl groups having 1 to 30 carbon atoms, with an unlimited number of carbon-carbon triple bonds at non-specified points;

the fluorescein and the derivatives thereof are fluorescein, aminofluorescein, carboxyfluorescein and fluorescein isothiocyanate;

the rhodamine and the derivative thereof are rhodamine, aminorhodamine, carboxyl rhodamine and isothiocyanate rhodamine.

The preferable compound of the invention is that the structure general formula of the photoresponsive multifunctional chemical crosslinking agent is as follows:

r' is a hydrogen atom, and X is selected from one of the following structures:

wherein n is ₁ Is an integer of 1 to 6, n ₂ Is an integer of 1 to 10, n ₃ Is an integer of 1 to 4, n ₄ Is an integer of 1 to 6, n ₅ Is an integer of 0 to 6, and the light-responsive multifunctional chemical cross-linking agent is a light-responsive bifunctional cross-linking agent;

r' is selected from biotin and derivatives thereof, and X is the following structure:

wherein n is an integer of 2 to 6, and the photoresponsive multifunctional chemical crosslinking agent is a photoresponsive trifunctional crosslinking agent.

More preferred compounds of the present invention are compounds having the general structural formula:

in NBS-1 and Sulfo-NBS-1, n is an integer of 1 to 6,

in NBS-2 and Sulfo-NBS-2, n is an integer of 1 to 10,

in NBM and Sulfo-NBM, n is an integer of 0 to 6,

n in NBNCO is an integer of 1 to 6, n in NBPM is an integer of 1 to 4,

n in Tri-NB is an integer from 2 to 6.

The most preferred compound of the present invention is a photo-responsive multifunctional chemical cross-linking agent having one of the following structures:

the second aspect of the present invention provides a method for preparing the photo-responsive multifunctional chemical crosslinking agent, comprising the steps of:

in NBS-1 and Sulfo-NBS-1, n is an integer of 1 to 6,

in NBNCO, n is an integer of 1 to 6,

firstly, dissolving a compound 4, potassium carbonate and N-Boc bromoethylamine with the molar ratio of 1 (3-5) (1.5-2.5) in a solvent, carrying out reflux reaction, filtering to obtain filtrate after the reaction is finished, removing the solvent by spin drying, and carrying out column chromatography purification to obtain a compound 5;

secondly, dissolving the compound 5 prepared in the first step in a mixed solution of a solvent and excessive trifluoroacetic acid, reacting at room temperature, performing spin-drying to remove the solvent after the reaction is finished, performing vacuum pumping on the residue for 2 hours, dissolving the pumped residue in the solvent, slowly dropwise adding a solution containing bis-succinimide ester, wherein the molar ratio of the bis-succinimide ester to the compound 5 is (1-1.5) to 1, adding a catalytic amount of triethylamine, reacting at room temperature, performing spin-drying to remove the solvent after the reaction is finished, and performing column chromatography purification to obtain a compound NBS-1;

or slowly dropwise adding a solution containing sodium disuccinimidyl ester sulfonate, wherein the molar ratio of the sodium disuccinimidyl ester sulfonate to the compound 5 is (1-1.5): 1, adding a catalytic amount of triethylamine, reacting at normal temperature, removing the solvent by spin drying after the reaction is finished, and purifying by column chromatography to obtain a compound Sulfo-NBS-1;

or slowly dropwise adding a solution containing isocyanate, wherein the molar ratio of the isocyanate to the compound 5 is (1-1.5): 1, adding a catalytic amount of triethylamine, reacting at normal temperature, removing the solvent by spin drying after the reaction is finished, and purifying by column chromatography to obtain a compound NBNCO;

in NBS-2 and Sulfo-NBS-2, n is an integer of 1 to 10,

firstly, dissolving a compound 4, methyl bromobutyrate and potassium carbonate in a molar ratio of 1 (1.1-3) to 1.1-4 in a solvent, carrying out reflux reaction, filtering to obtain filtrate after the reaction is finished, removing the solvent by spin drying, and recrystallizing ethanol to obtain a compound 6;

secondly, dissolving the compound 6 prepared in the first step in a solvent, adding excessive potassium hydroxide for reaction at normal temperature, after the reaction is finished, removing the solvent by spinning, dissolving in water, adjusting the pH value until 4 to generate yellow precipitate, and filtering to obtain a filter cake to obtain a compound 7;

thirdly, dissolving the compound 7, the hydroxysuccinimide and the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride which are prepared in the second step according to the molar ratio of 1 (1-1.5) to 1 (1-1.5) in a solvent, reacting at normal temperature, extracting dichloromethane and water after the reaction is finished, taking an organic phase, removing the solvent by spin drying, and purifying by column chromatography to obtain a compound NBS-2;

or, dissolving the compound 7, the hydroxysuccinimide sodium sulfonate and the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride which are prepared in the second step according to the molar ratio of 1 (1-1.5) to 1 (1-1.5) in a solvent, reacting at normal temperature, extracting dichloromethane and water after the reaction is finished, taking an organic phase, removing the solvent by spin drying, and purifying by column chromatography to obtain a compound Sulfo-NBS-2;

in NBM and Sulfo-NBM, n is an integer of 0 to 6,

firstly, dissolving a compound 4, dibromoated alkyl and potassium carbonate in a molar ratio of 1 (1.1-3) to 1.1-4 in a solvent, carrying out reflux reaction, filtering to obtain a filtrate after the reaction is finished, removing the solvent by spin drying, and purifying by column chromatography to obtain a compound 10;

secondly, dissolving the compound 10, maleimide and potassium carbonate which are prepared in the first step and have the molar ratio of 1 (1.1-3) to 1.1-4 in a solvent, carrying out reflux reaction, filtering to obtain filtrate after the reaction is finished, removing the solvent by spin drying, and carrying out column chromatography purification to obtain a compound NBM;

or dissolving the compound 10, the sodium maleimide sulfonate and the potassium carbonate which are prepared in the first step and have the molar ratio of 1 (1.1-3) to 1.1-4 in a solvent, carrying out reflux reaction, filtering to obtain filtrate after the reaction is finished, removing the solvent by spin drying, and carrying out column chromatography purification to obtain a compound Sulfo-NBM;

in NBPM, n is an integer of 1 to 4,

firstly, dissolving a compound 4, dibromo-condensed ethylene glycol and potassium carbonate in a molar ratio of 1 (1.1-3) to 1.1-4 in a solvent, carrying out reflux reaction, filtering to obtain a filtrate after the reaction is finished, removing the solvent by spin drying, and purifying by column chromatography to obtain a compound 11;

secondly, dissolving the compound 11, maleimide and potassium carbonate which are prepared in the first step and have the molar ratio of 1 (1.1-3) to 1.1-4 in a solvent, carrying out reflux reaction, filtering and taking filtrate after the reaction is finished, removing the solvent by spin drying, and carrying out column chromatography purification to obtain a compound NBPM;

n is an integer of 2 to 6,

step one, mixing a mixture of 1: (1.1-3) dissolving NBS-2-4C, N-Boc amino acid in a solvent, reacting at normal temperature, removing the solvent by spin drying after the reaction is finished, and purifying by column chromatography to obtain a compound 12;

and secondly, dissolving the compound 12 prepared in the first step in a mixed solution of a solvent and excess trifluoroacetic acid, reacting at room temperature, after the reaction is finished, removing the solvent by rotary drying, performing vacuum pumping on the residue for 2 hours, dissolving the pumped residue in the solvent, and adding succinimide biotin into the mixture, wherein the molar ratio of the succinimide biotin to the compound 12 is (1.1-3): 1, after the reaction is finished, spin-drying to remove the solvent, and purifying by column chromatography to obtain a compound 13;

and step three, the molar ratio of the second step preparation is 1: (1.1-3): and (1.1-3) dissolving the compound 13, hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in a solvent, reacting at normal temperature, extracting dichloromethane and water after the reaction is finished, taking an organic phase, spin-drying to remove the solvent, and purifying by column chromatography to obtain the compound Tri-NB.

The bis-succinimide ester is bis-succinimide glutarate or bis-succinimide glutarate.

The sodium disuccinimidyl ester sulfonate is sodium disuccinimidyl suberate sulfonate and sodium disuccinimidyl glutarate sulfonate.

The isocyanate is 1,8-octanedionate, 1,6-hexanedionate.

The methyl bromobutyrate is 4-methyl bromobutyrate and 8-methyl bromooctanoate.

The dibromoalkyl is 1,4-dibromohexane, 1,4-dibromobutane.

The dibromo polyglycol is 1-bromo-2- (2- (2- (2-bromoethoxy) ethoxy) ethane and 1,2-bis (2-bromoethoxy) ethane.

The N-Boc amino acid is N-Boc lysine.

The preparation method of the compound 4 comprises the following steps:

firstly, dissolving vanillin, potassium carbonate and benzyl bromide with the molar ratio of 1 (1.5-3) to 1.1-1.5 in a solvent, carrying out reflux reaction, filtering after the reaction is finished, spin-drying and recrystallizing to obtain a compound 1;

secondly, grinding the compound 1 prepared in the first step into powder, slowly adding excessive nitric acid, carrying out ice bath reaction, pouring the reaction liquid into ice water after the reaction is finished, stirring and filtering, and recrystallizing to obtain a compound 2;

dissolving the compound 2 prepared in the second step in excessive trifluoroacetic acid, reacting at room temperature, after the reaction is finished, spin-drying to remove the solvent, completely dissolving the remainder in a mixed solution of sodium hydroxide aqueous solution and ethyl acetate, adjusting the pH value to weak acidity, extracting an organic phase, spin-drying to remove the solvent, adding petroleum ether into the remainder, filtering to obtain a filter cake, and repeating the steps for 3 times to obtain a compound 3;

and step four, dissolving the compound 3 prepared in the step three in a solvent, slowly adding sodium borohydride with the mole number of 1.5-2.5 times that of the compound 3, reacting at room temperature, extracting an organic phase after the reaction is finished, adjusting the pH value to weak acidity, and removing the solvent by spin drying to obtain a compound 4.

The solvent is dimethylformamide, acetonitrile, dichloromethane, ethanol, water, methanol and acetone.

The third aspect of the present invention provides a use of the photo-responsive multifunctional chemical cross-linking agent in protein interaction.

The application of the photo-responsive multifunctional chemical cross-linking agent in protein interaction comprises the following steps:

the first method comprises the following steps: adding the light-responsive multifunctional chemical cross-linking agent into a single target protein solution for incubation, using 365nm illumination to realize cross-linking of protein, and then carrying out protein electrophoresis (SDS-PAGE), western Blot (Western Blot) and protein cross-linking mass spectrometry (LC-MS/MS) analysis;

the second method comprises the following steps: adding the light-responsive multifunctional chemical cross-linking agent into a group of protein solutions with specific interaction for incubation, illuminating by 365nm to realize cross-linking of the proteins with interaction, capturing protein interaction, and then performing protein electrophoresis (SDS-PAGE), western Blot (Western Blot) and protein cross-linking mass spectrometry (LC-MS/MS) analysis;

the third method comprises the following steps: adding the light-responsive multifunctional chemical cross-linking agent into a single target protein solution for incubation, then dialyzing to obtain target protein with the light-responsive multifunctional chemical cross-linking agent, incubating the target protein with the light-responsive multifunctional chemical cross-linking agent and interacting protein thereof, realizing cross-linking of protein interaction pairs by 365nm illumination, and then carrying out protein electrophoresis (SDS-PAGE), protein imprinting (Western Blot) and protein cross-linking mass spectrometry (LC-MS/MS) analysis;

in the first method, the single target protein is Streptavidin (SA) as a model protein.

The second method is one in which a group of proteins having specific interactions are Staphylococcus aureus protein A (SPA) and mouse immunoglobulin G (IgG) as model proteins.

In the third method, the single target protein is staphylococcus aureus protein A (SPA), and the interacting protein is mouse immunoglobulin G (IgG).

The invention takes vanillin as raw material, obtains an intermediate through phenolic hydroxyl protection, nitration and phenolic hydroxyl deprotection, and the intermediate can introduce a spacer arm with different length into phenolic hydroxyl through reaction with bromo-compound, and further introduces traditional chemical groups (succinimide, maleimide, isocyanate and the like) into the other end through corresponding reaction, thus having the function of chemical cross-linking agent.

The invention provides a multifunctional chemical cross-linking agent with photoresponse, which is constructed by utilizing an o-nitrobenzyl alcohol structure and used for researching proteomics, wherein the structure has photoresponse, and can perform specific reaction on free amino on a lysine side chain on protein under 365nm light irradiation to form stable covalent cross-linking. Because of the photoresponse of the o-nitrobenzyl alcohol, the structure has space-time controllability, and after different arm lengths and different chemical reaction groups are introduced, the interaction of different proteins can be realized, and the space-time controllable analysis can be carried out.

Due to the adoption of the technical scheme, the invention has the following advantages and beneficial effects:

the photoresponsive multifunctional chemical cross-linking agent realizes the time-space controllable amino coupling marking by utilizing the structure of the o-nitrobenzyl alcohol, and has higher efficiency and specificity compared with other photoresponsive groups; compared with the traditional chemical cross-linking agent, the protein heterofunctional cross-linking agent constructed by the structure of the o-nitrobenzyl alcohol has the advantage of space-time controllability in proteomics, and can more accurately capture protein interaction information by using protein electrophoresis and protein cross-linking mass spectrometry; the molecule can be prepared by taking vanillin as a raw material through a series of simple synthesis, the comprehensive yield is over 50 percent, the raw material price is low, the reaction is simple and efficient, and the commercial value is high.

The photoresponsive multifunctional chemical cross-linking agent has simple preparation method and low cost, is the first cross-linking agent based on o-nitrobenzyl alcohol photocrosslinking reaction, and cross-links protein by coupling reaction of photoproduction aldehyde group and amino: in the crosslinking agent, the photoreactive group pair contains-NH ₂ The amino acid residue has high crosslinking efficiency and selectivity; the heterofunctional cross-linking agent with photoresponse is obtained by combining with functional groups used in other traditional cross-linking agents, so that the specific cross-linking of heterologous proteins is realized, and the heterofunctional cross-linking agent has great potential application in proteomics research.

Drawings

FIG. 1 is a schematic diagram of streptavidin photocrosslinking electrophoresis in example 18.

FIG. 2 is a schematic diagram of the electrophoresis of the interaction of Staphylococcus aureus protein A and mouse immunoglobulin G in example 19.

FIG. 3 is a schematic diagram of the electrophoresis of the interaction of Staphylococcus aureus protein A and mouse immunoglobulin G in example 20.

Detailed Description

In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.

In the following examples, raw materials for synthesis were purchased from An Naiji chemical, welfare science and technology ltd, and reagents ltd, such as alatin reagent ltd, and all the purchased reagents were used as they were without further purification unless otherwise specified; the reaction solvent such as dimethylformamide, dichloromethane, acetonitrile and the like is distilled and dried, and the reaction process is carried out under the protection of argon.

Example 1

In the first step, vanillin (50g, 328mmol), potassium carbonate (82.8 g, 600 mmol) and benzyl bromide (76.9g, 450mmol) were dissolved in acetonitrile 1000ml, refluxed at 80 ℃ for 9h, monitored by dot plate (reaction monitored by TLC), and after the reaction was completed, the filtrate was filtered and the solvent was removed by spin-drying to give a pale yellow viscous liquid. Dissolving the liquid in ethanol, heating to be clear and transparent, adding a small amount of petroleum ether, cooling to room temperature, recrystallizing overnight, separating out white crystals, filtering to obtain a filter cake, and obtaining 67g of white crystals as the compound 1 with the yield of 84%. ¹ H NMR(400MHz,CDCl ₃ ),δ(ppm):9.81(s,1H)；7.45-7.37(m,7H)；7.01(d,J＝8.6Hz,1H)；5.24(s,2H)；3.94(s,3H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):190.9,153.6,150.1,136.0,130.3,128.8,128.2,127.2,126.6,112.4,109.3,70.8,56.1.MS(ESI):m/z:Calcd.for C ₁₅ H ₁₄ O ₃ Na ⁺ [M+Na] ⁺ :265.2.Found:265.2.

In the second step, the first step is that,

compound 1 (8g, 33mmol) prepared in the first step was ground to a powder, slowly added to a round-bottomed flask containing 30mL of nitric acid, and when the reaction bubbled out a red-brown gas, the reaction was placed in an ice bath and monitored on a point-and-plate basis. After the reaction, the reaction solution was poured into 1.5L of ice water and stirred, the filter cake was filtered and recrystallized from ethanol to obtain 8g of yellow crystal compound 2 with a yield of 84%. ¹ H NMR(400MHz,CDCl3),δ(ppm):10.44(s,1H),7.67(s,1H),7.45-7.36(m,6H),5.26(s,2H),4.01(s,3H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):187.8,153.7,151.4,134.8,128.8,127.6,125.7,110.0,108.8,71.5,56.7.MS(ESI):m/z:Calcd.for C ₁₅ H ₁₄ NO ₅ ⁺ [M+H] ⁺ :288.2.Found:288.2.

In the third step, the first step is,

compound 2 (10g, 35mmol) prepared in the second step was dissolved in 100mL of trifluoroacetic acid, and reacted at room temperature for 9 hours, followed by monitoring on a dot plate. After the reaction was completed, the solvent was removed by spin-drying. The residue was completely dissolved in a mixture of aqueous sodium hydroxide and ethyl acetate, the pH was adjusted to weak acidity, and the organic phase was extracted. Spin-drying to remove solvent, adding a small amount of petroleum ether into the residue, filtering to obtain filter cake, and repeating for 3 times to obtain 6.5g of yellowish brown powder compound 3 with a yield of 95%. ¹ H NMR(400MHz,DMSO-d6),δ(ppm):11.12(s,1H),10.16(s,1H),7.51(s,1H),7.36(s,1H),3.95(s,3H). ¹³ C NMR(100MHz,DMSO-d ₆ ),δ(ppm):188.3,151.7,150.9,143.7,123.3,111.0,110.5,56.3.MS(ESI):m/z:Calcd.for C ₈ H ₇ NO ₅ Na ⁺ [M+Na] ⁺ :220.0.Found:220.0.

The fourth step is that the first step is that,

compound 3 (10g, 51mmol) prepared in the third step was dissolved in a mixed solution of 100ml of methylene chloride and 400ml of methanol, and sodium borohydride (3.8g, 100mmol) was slowly added thereto, and the reaction was carried out at room temperature for 15min. After the reaction, the organic phase was extracted, the pH was adjusted to weak acidity, and the solvent was removed by spin-drying to obtain 10g of compound 4 as a yellow powder with a yield of 99%. ¹ H NMR(400MHz,DMSO-d ₆ )δ9.92(s,1H),7.56(s,1H),7.34(s,1H),4.79(s,2H),3.90(s,3H). ¹³ C NMR(101MHz,DMSO-d ₆ )δ153.30,145.42,138.84,132.89,111.71,110.42,60.62,56.43.MS(ESI):m/z:Calcd.for C ₈ H ₉ NO ₅ Na ⁺ [M+Na] ⁺ :222.1.Found:222.1.

In the fifth step, the first step is carried out,

compound 4 (5g, 25mmol), potassium carbonate (13.8g, 100mmol) and N-Boc bromoethylamine (11.2g, 50mmol) prepared in the fourth step were dissolved in acetonitrile, refluxed at 80 ℃ for 9h and monitored by spotting. After the reaction was completed, the filtrate was filtered, the solvent was removed by spin-drying, and column chromatography purification (v dichloromethane: v methanol = 1000). ¹ H NMR(400MHz,CDCl ₃ ),δ(ppm):7.71(s,1H),7.21(s,1H),4.97(s,2H),4.13(t,J＝5.3Hz,2H),3.99(s,3H),3.60(q,J＝5.4Hz,2H),1.45(s,9H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):155.87,154.31,146.89,139.54,132.98,111.14,110.19,79.74,69.08,62.70,56.36,39.86,28.39.MS(ESI):m/z:Calcd.for C ₁₅ H ₂₃ N ₂ O ₇ ⁺ [M+H] ⁺ :343.2.Found:343.2.

In the sixth step, the first step is carried out,

dissolving the compound 5 (5 g,14.6 mmol) prepared in the fifth step in 100ml of a mixed solution of dichloromethane and trifluoroacetic acid (v: v = 10. After the reaction was completed, the solvent was removed by spin-drying, and column chromatography purification (v dichloromethane: v methanol =1000 1) was carried out to obtain 6g of a pale yellow powder compound NBS-1-8C with a yield of 83%. ¹ H NMR(400MHz,CDCl ₃ ),δ(ppm):7.71(s,1H),7.26(s,1H),6.23(t,J＝5.8Hz,1H),4.97(s,2H),4.15(t,J＝5.0Hz,2H),3.99(s,3H),3.72(q,J＝5.3Hz,2H),2.84(d,J＝4.2Hz,4H),2.57(t,J＝7.2Hz,2H),2.22(t,J＝7.4Hz,2H),1.68(dp,J＝14.0,7.1Hz,4H),1.39(ddt,J＝20.1,14.2,6.8Hz,4H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):173.75,169.36,168.62,154.24,146.73,139.49,133.30,111.05,110.31,68.67,62.55,56.42,38.67,36.30,30.85,28.41,28.21,25.61,25.23,24.36.MS(ESI):m/z:Calcd.for C ₂₂ H ₃₀ N ₃ O ₁₀ ⁺ [M+H] ⁺ :496.2.Found:496.2.

Example 2

The compound 5 (5 g,14.6 mmol) in example 1 was dissolved in 100ml of a mixed solution of dichloromethane and trifluoroacetic acid (v: v = 10. And (4) after the reaction is finished, removing the solvent by spin-drying, and vacuumizing the residue for 2h. The residue after being dried by suction is dissolved in 50ml of dichloromethane, 100ml of dichloromethane solution containing disuccinimidyl glutarate (6.5g, 20mmol) is slowly dripped, excess triethylamine is added, and the reaction is carried out for 3 hours at normal temperature. After the reaction was completed, the solvent was removed by spin-drying, and column chromatography purification (v dichloromethane: v methanol =1000 1) was carried out to obtain 5.8g of a pale yellow powder compound NBS-1-5C with a yield of 88%. ¹ H NMR(400MHz,CDCl ₃ ),δ(ppm):7.72(s,1H),7.25(s,1H),6.20(t,J＝5.8Hz,1H),4.87(s,2H),4.05(t,J＝5.0Hz,2H),3.99(s,3H),3.62(q,J＝5.3Hz,2H),2.74(d,J＝4.2Hz,4H),2.47(t,J＝7.2Hz,2H),2.32(t,J＝7.4Hz,2H),1.88(dp,J＝14.0,7.1Hz,2H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):173.75,169.36,168.62,154.24,146.73,139.49,133.30,111.05,110.31,68.67,62.55,56.42,38.67,36.30,30.85,28.21,24.36.MS(ESI):m/z:Calcd.for C ₁₉ H ₂₄ N ₃ O ₁₀ ⁺ [M+H] ⁺ :454.1.Found:454.1.

Example 3

The compound 5 (5 g,14.6 mmol) in example 1 was dissolved in 100ml (v: v =10The solvent is removed by spin drying, the residue is vacuumized for 2h, the vacuumized residue is dissolved in 50ml of dichloromethane, 50ml of dichloromethane solution containing disuccinimidyl suberate sodium sulfonate (11.4g, 20mmol) is slowly dropped, excess triethylamine is added, and the reaction is carried out for 3h at normal temperature. After the reaction was completed, the solvent was removed by rotary drying and purified by column chromatography (v dichloromethane: v methanol =1000 5), to obtain 6g of sulfol-NBS-1-8C as a pale yellow powder compound in 69% yield. ¹ H NMR(400MHz,CDCl ₃ ),δ(ppm):7.71(s,1H),7.26(s,1H),6.23(t,J＝5.8Hz,1H),4.97(s,2H),4.25(t,J＝5.0Hz,1H),4.15(t,J＝5.0Hz,2H),3.99(s,3H),3.72(q,J＝5.3Hz,2H),3.32(m,1H),3.02(m,1H),2.57(t,J＝7.2Hz,2H),2.22(t,J＝7.4Hz,2H),1.68(dp,J＝14.0,7.1Hz,4H),1.39(ddt,J＝20.1,14.2,6.8Hz,4H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):173.75,169.36,168.62,154.24,146.73,139.49,133.30,111.05,110.31,68.67,62.55,59.01,56.42,38.67,36.30,30.85,28.41,28.21,25.61,24.36,20.23.MS(ESI):m/z:Calcd.for C ₂₂ H ₂₈ N ₃ O ₁₃ S ^- [M-Na] ^- :574.1.Found:574.1.

Example 4

The compound 5 (5 g,14.6 mmol) in example 1 was dissolved in 100ml of a mixed solution of dichloromethane and trifluoroacetic acid (v: v = 10. And (4) after the reaction is finished, removing the solvent by spin-drying, and vacuumizing the residue for 2h. The residue after being drained is dissolved in 50ml of dichloromethane, 50ml of dichloromethane solution containing disuccinimidyl glutarate sodium sulfonate (10.6 g, 20mmol) is slowly dripped, excess triethylamine is added, and the reaction is carried out for 3 hours at normal temperature. After the reaction was completed, the solvent was removed by spin-drying and purified by column chromatography (v dichloromethane: v methanol = 1000). ¹ H NMR(400MHz,CDCl ₃ ),δ(ppm):7.72(s,1H),7.25(s,1H),6.20(t,J＝5.8Hz,1H),4.87(s,2H),4.25(t,J＝5.0Hz,1H),4.05(t,J＝5.0Hz,2H),3.99(s,3H),3.62(q,J＝5.3Hz,2H),3.32(m,1H),3.02(m,1H),2.47(t,J＝7.2Hz,2H),2.32(t,J＝7.4Hz,2H),1.88(dp,J＝14.0,7.1Hz,2H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):173.75,169.36,168.62,154.24,146.73,139.49,133.30,111.05,110.31,68.67,62.55,59.01,56.42,38.67,36.30,30.85,24.36,20.23.MS(ESI):m/z:Calcd.for C ₁₉ H ₂₂ N ₃ O ₁₃ S ^- [M-Na] ^- :532.1.Found:532.1.

Example 5

In the first step, compound 4 (5 g, 20mmol) of example 1, methyl 4-bromobutyrate (5.43g, 30mmol) and potassium carbonate (6.9g, 50mmol) were dissolved in acetonitrile 250ml, refluxed at 80 ℃ for 9h, and spotted on a plate for monitoring. After the reaction is finished, filtering to obtain filtrate, spin-drying to remove the solvent, and recrystallizing by ethanol to obtain 4.8g of yellow powder compound 6-1 with the yield of 85%. ¹ H NMR(400MHz,CDCl ₃ ),δ(ppm):7.70(s,1H),7.18(s,1H),4.96(s,2H),4.13(t,J＝6.2Hz,2H),3.98(s,3H),3.71(s,3H),2.57(t,J＝7.2Hz,2H),2.27–2.11(m,2H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):173.43,154.29,147.13,139.56,132.49,111.10,109.48,68.26,62.77,56.43,51.76,30.38,24.25.MS(ESI):m/z:Calcd.for C ₁₃ H ₁₇ NO ₇ Na[M+Na] ⁺ :322.1.Found:322.1.

In the second step, compound 6-1 (5 g,17.5 mmol) prepared in the first step was dissolved in 100ml of 90% aqueous ethanol solution, and potassium hydroxide (1.4 g, 25mmol) was added thereto to conduct a reaction at ordinary temperature for 15min. After the reaction is finished, the solvent is removed by spin drying, the solvent is dissolved by water, the pH is adjusted until 4, yellow precipitate is generated, and filter cakes are obtained by filtration, 4g of yellow powdery compound 7-1 is obtained, and the yield is 84%. ¹ H NMR(400MHz,DMSO),δ(ppm):12.20(s,1H),7.67(s,1H),7.39(s,1H),4.83(s,2H),4.07(t,J＝6.5Hz,2H),3.93(s,3H),2.41(t,J＝7.3Hz,2H),1.97(p,J＝6.9Hz,2H). ¹³ C NMR(100MHz,DMSO),δ(ppm):174.00,153.68,145.98,138.25,134.23,109.61,108.82,67.83,60.08,56.03,29.91,24.01.MS(ESI):m/z:Calcd.for C ₁₂ H ₁₅ NO ₇ K[M+K] ⁺ :324.0.Found:324.1.

In the third step, the compound 7-1 (5 g,18.5 mmol), hydroxysuccinimide (2.3 g, 20mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (3.8g, 20mmol) prepared in the second step were dissolved in 100ml of dichloromethane and reacted at normal temperature for 2 hours. After the reaction, dichloromethane and water were extracted, and the organic phase was taken, dried by spinning to remove the solvent, and purified by column chromatography (dichloromethane), to obtain 5.5g of a yellow powdery compound NBS-2-4C in a yield of 81%. ¹ H NMR(400MHz,CDCl ₃ ),δ(ppm):7.72(s,1H),7.27(s,1H),4.96(s,2H),4.19(t,J＝6.0Hz,2H),3.99(s,3H),2.96–2.78(m,6H),2.37–2.23(m,2H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):169.10,168.16,154.41,146.98,139.65,132.64,111.29,109.93,67.49,62.89,27.56,25.60,24.17.MS(ESI):m/z:Calcd.for C ₁₆ H ₁₉ N ₂ O ₉ [M+H] ⁺ :383.1.Found:383.2.

Example 6

In 100ml of methylene chloride, the compound 7-1 (5 g,18.5 mmol) obtained in example 5, sodium hydroxysuccinimide hydrochloride (4.3 g, 20mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (3.8g, 20mmol) were dissolved and reacted at ordinary temperature for 2 hours. The solvent was removed by rotary drying and recrystallized from ethanol to give 8g of Sulfo-NBS-2-4C as a yellow powder with a yield of 92%. ¹ H NMR(400MHz,CDCl ₃ ),δ(ppm):7.72(s,1H),7.27(s,1H),4.96(s,2H),4.25(t,J＝5.0Hz,1H),4.19(t,J＝6.0Hz,2H),3.99(s,3H),3.32(m,1H),3.02(m,1H),2.96–2.78(m,2H),2.37–2.23(m,2H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):169.43,168.56,154.07,146.65,139.54,132.33,111.22,109.65,67.78,62.45,59.78,27.78,25.34,24.11.MS(ESI):m/z:Calcd.for C ₁₆ H ₁₇ N ₂ O ₁₂ S ^- [M-Na] ^- :461.1.Found:461.2.

Example 7

In the first step, compound 4 (5 g, 20mmol) in example 1, methyl 8-bromooctanoate (7.1g, 30mmol) and potassium carbonate (6.9g, 50mmol) were dissolved in acetonitrile 100ml, refluxed at 80 ℃ for 9h, and spotted on a plate for monitoring. After the reaction is finished, filtering to obtain filtrate, spin-drying to remove the solvent, and recrystallizing by ethanol to obtain 5.8g of yellow powder compound 6-2 with the yield of 85%. ¹ H NMR(400MHz,CDCl ₃ ),δ(ppm):7.70(s,1H),7.18(s,1H),4.96(s,2H),4.13(t,J＝6.2Hz,2H),3.98(s,3H),3.81(s,3H),2.67(t,J＝7.2Hz,2H),2.32–2.02(m,10H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):173.43,154.29,147.13,139.56,132.49,111.10,109.48,68.26,62.77,56.43,51.76,30.38,29.45,26.78,25.34,24.78,24.25.MS(ESI):m/z:Calcd.for C ₁₇ H ₂₅ NO ₇ Na[M+Na] ⁺ :378.2.Found:378.1.

In the second step, 6-2 (5 g,14.6 mmol) of the compound prepared in the first step was dissolved in 100ml of 90% aqueous ethanol, and potassium hydroxide (1.4 g, 25mmol) was added thereto to carry out a reaction at ordinary temperature for 15min. After the reaction was completed, the solvent was removed by spin-drying, dissolved in water, adjusted to pH 4 to give a yellow precipitate, and the filtrate was filtered to obtain 4.2g of compound 7-2 as yellow powder with a yield of 89%. ¹ H NMR(400MHz,DMSO),δ(ppm):7.70(s,1H),7.18(s,1H),4.96(s,2H),4.13(t,J＝6.2Hz,2H),3.98(s,3H),2.67(t,J＝7.2Hz,2H),2.32–2.02(m,10H). ¹³ C NMR(100MHz,DMSO),δ(ppm):174.00,153.68,145.98,138.25,134.23,109.61,108.82,67.83,60.08,56.03,29.91,29.45,26.78,25.34,24.78,24.01.MS(ESI):m/z:Calcd.for C ₁₆ H ₂₄ NO ₇ [M+H] ⁺ :342.2.Found:342.1.

In the third step, the compound 7-2 (4.9g, 15mmol) prepared in the second step, hydroxysuccinimide (2.3g, 20mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (3.8g, 20mmol) were dissolved in 100ml of dichloromethane and reacted at normal temperature for 2 hours. After the reaction, dichloromethane was extracted with water, the organic phase was taken, the solvent was removed by spin-drying, and column chromatography purification (dichloromethane) was carried out to obtain 5.5g of a yellow powdery compound NBS-2-8C with a yield of 87%. ¹ H NMR(400MHz,CDCl ₃ ),δ(ppm):7.72(s,1H),7.27(s,1H),4.96(s,2H),4.19(t,J＝6.0Hz,2H),3.99(s,3H),2.96–2.78(m,6H),2.45–2.01(m,10H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):169.10,168.16,154.41,146.98,139.65,132.64,111.29,109.93,67.49,62.89,29.91,29.45,27.56,26.78,25.60,24.78,24.17.MS(ESI):m/z:Calcd.for C ₂₀ H ₂₇ N ₂ O ₉ [M+H] ⁺ :439.2.Found:439.2.

Example 8

In 100ml of methylene chloride, the compound 7-2 (4.9g, 15mmol) in example 7, sodium hydroxysuccinimide hydrochloride (4.3g, 20mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (3.8g, 20mmol) were dissolved and reacted at ordinary temperature for 2 hours. The solvent was removed by rotary drying and recrystallized from ethanol to give 6.8g of sulfol-NBS-2-8C as a yellow powder with a yield of 87%. ¹ H NMR(400MHz,CDCl ₃ ),δ(ppm):7.72(s,1H),7.27(s,1H),4.96(s,2H),4.25(t,J＝5.0Hz,1H),4.19(t,J＝6.0Hz,2H),3.99(s,3H),3.32(m,1H),3.02(m,1H),2.96–2.78(m,2H),2.37–2.23(m,10H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):169.43,168.56,154.07,146.65,139.54,132.33,111.22,109.65,67.78,62.45,59.78,29.91,29.45,27.56,26.78,25.60,24.78,24.17.MS(ESI):m/z:Calcd.for C ₂₀ H ₂₅ N ₂ O ₁₂ S ^- [M-Na] ^- :517.1.Found:517.2.

Example 9

In the first step, compound 4 (5 g, 20mmol) in example 1, 1,4-dibromobutane (8.6 g, 40mmol) and potassium carbonate (6.9g, 50mmol) were dissolved in acetonitrile 100ml, refluxed at 80 ℃ for 9h, and spotted on a plate for monitoring. After the reaction is finished, filtering to obtain filtrate, removing the solvent by spin drying, and purifying by column chromatography (dichloromethane). 6g of compound 10-1 are obtained as a yellow powder with a yield of 71%. ¹ H NMR(400MHz,CDCl ₃ ),δ(ppm):7.70(s,1H),7.17(s,1H),4.96(s,2H),4.12(t,J＝5.9Hz,2H),3.99(s,3H),3.51(t,J＝6.3Hz,2H),2.15–1.99(m,4H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):154.28,147.25,139.67,132.32,111.24,109.42,68.49,62.89,56.45,33.21,29.33,27.58.MS(ESI):m/z:Calcd.for C ₁₂ H ₁₇ NO ₅ Br ⁺ [M+H] ⁺ :334.0.Found:334.1.

In the second step, compound 10-1 (5 g, 15mmol) prepared in the first step, maleimide (1.9g, 20mmol) and potassium carbonate (4.1g, 30mmol) were dissolved in 100ml of dimethylformamide, refluxed at 50 ℃ for 3 hours, and spotted on a plate for monitoring. After the reaction was completed, the filtrate was filtered, and the solvent was removed by spin-drying and purified by column chromatography (v dichloromethane: v methanol =1000: 1), to obtain 4g of a pale yellow powder compound NBM-4C with a yield of 76%. ¹ H NMR(400MHz,CDCl ₃ ),δ(ppm):7.68(s,1H),7.16(s,1H),6.71(s,2H),4.95(s,2H),4.09(t,J＝6.0Hz,2H),3.98(s,3H),3.62(t,J＝6.7Hz,2H),1.94–1.72(m,4H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):170.83,154.30,147.29,139.70,134.14,132.26,111.28,109.50,68.71,62.92,56.43,37.42,26.22,25.20.MS(ESI):m/z:Calcd.for C ₁₆ H ₁₉ N ₂ O ₇ ⁺ [M+H] ⁺ :351.1.Found:351.1.

Example 10

In the first step, compound 4 (5 g, 20mmol) of example 1, 1,4-dibromohexane (9.7g, 40mmol) and potassium carbonate (6.9g, 50mmol) were dissolved in acetonitrile 100ml, refluxed at 80 ℃ for 9 hours, and spotted on a plate for monitoring. After the reaction is finished, filtrate is obtained by filtration, solvent is removed by spin drying, and column chromatography purification (dichloromethane) is carried out, so that 6.5g of yellow powder compound 10-2 is obtained, and the yield is 90%. ¹ H NMR(400MHz,CDCl ₃ ),δ(ppm):7.70(s,1H),7.17(s,1H),4.96(s,2H),4.12(t,J＝5.9Hz,2H),3.99(s,3H),3.51(t,J＝6.3Hz,2H),2.15–1.99(m,8H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):154.28,147.25,139.67,132.32,111.24,109.42,68.49,62.89,56.45,33.21,29.33,27.58,25.41,24.92.MS(ESI):m/z:Calcd.for C ₁₄ H ₂₁ NO ₅ Br ⁺ [M+H] ⁺ :362.1.Found:362.2.

In the second step, compound 10-2 (5.4 g, 15mmol) prepared in the first step, maleimide (1.9g, 20mmol) and potassium carbonate (4.1g, 30mmol) were dissolved in 100ml of dimethylformamide, refluxed at 50 ℃ for 3 hours, and spotted on a plate for monitoring. After the reaction was completed, the filtrate was filtered, the solvent was removed by spin-drying, and column chromatography purification (v dichloromethane: v methanol =1000: 1) was performed to obtain 4.5g of a pale yellow powder compound NBM-6C with a yield of 79%. ¹ H NMR(400MHz,CDCl ₃ ),δ(ppm):7.68(s,1H),7.16(s,1H),6.71(s,2H),4.95(s,2H),4.09(t,J＝6.0Hz,2H),3.98(s,3H),3.62(t,J＝6.7Hz,2H),1.94–1.72(m,8H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):170.83,154.30,147.29,139.70,134.14,132.26,111.28,109.50,68.71,62.92,56.43,37.42,26.22,25.20,25.41,24.92.MS(ESI):m/z:Calcd.for C ₁₈ H ₂₃ N ₂ O ₇ ⁺ [M+H] ⁺ :379.2.Found:379.1.

Example 11

Compound 10-1 (5g, 15mmol) of example 9, sodium maleimide sulfonate (4g, 20mmol) and potassium carbonate (4.1g, 30mmol) were dissolved in 100ml of dimethylformamide, refluxed at 50 ℃ for 3 hours, and spotted on a plate for monitoring. After the reaction was completed, the filtrate was filtered, and the solvent was removed by rotary drying and purified by column chromatography (v dichloromethane: v methanol = 1000) to obtain 5g of a pale yellow powder compound, sulfo-NBM-4C, in 74% yield. ¹ H NMR(400MHz,CDCl ₃ ),δ(ppm):8.21(s,1H),7.68(s,1H),7.16(s,1H),4.95(s,2H),4.09(t,J＝6.0Hz,2H),3.98(s,3H),3.62(t,J＝6.7Hz,2H),1.94–1.72(m,4H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):170.83,154.30,147.29,139.70,134.14,132.26,111.28,109.50,68.71,62.92,59.05,56.43,37.42,26.22.MS(ESI):m/z:Calcd.for C ₁₆ H ₁₇ N ₂ O ₁₀ S ^- [M-Na] ^- :429.1.Found:429.2.

Example 12

Compound 10-2 (5.4g, 15mmol) from example 10, sodium maleimide sulfonate (4g, 20mmol) and potassium carbonate (4.1g, 30mmol) were dissolved in 100ml of dimethylformamide, refluxed at 50 ℃ for 3 hours, and spotted on a plate for monitoring. After the reaction was completed, the filtrate was filtered, and the solvent was removed by rotary drying and purified by column chromatography (v dichloromethane: v methanol = 1000). ¹ H NMR(400MHz,CDCl ₃ ),δ(ppm):8.21(s,1H),7.68(s,1H),7.16(s,1H),4.95(s,2H),4.09(t,J＝6.0Hz,2H),3.98(s,3H),3.62(t,J＝6.7Hz,2H),1.94–1.72(m,8H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):170.83,154.30,147.29,139.70,134.14,132.26,111.28,109.50,68.71,62.92,59.05,56.43,37.42,26.22,25.41,24.92.MS(ESI):m/z:Calcd.for C ₁₆ H ₁₇ N ₂ O ₁₀ S ^- [M-Na] ^- :429.1.Found:429.2.

Example 13

Compound 5 (5.1g, 15mmol) in example 1 was dissolved in 100ml of a mixed solution of dichloromethane and trifluoroacetic acid (v: v = 10. And after the reaction is finished, the solvent is removed by spin drying, and the residue is vacuumized and dried for 2 hours. The residue after the air drying was dissolved in 50ml of dichloromethane, 50ml of a dichloromethane solution containing 1,6-hexamethylene diisocyanate (3.4g, 20mmol) was slowly added dropwise thereto, and an excess amount of triethylamine was added thereto to conduct a reaction at room temperature for 3 hours. After the reaction was completed, the solvent was removed by spin-drying and purified by column chromatography (v dichloromethane: v methanol =1000 1), to obtain 5g of a pale yellow powder compound NBNCO-6C with a yield of 70%. ¹ H NMR(400MHz,CDCl ₃ ),δ(ppm):7.72(s,1H),7.25(d,J＝13.5Hz,1H),6.20(t,J＝5.8Hz,1H),6.03(s,2H),4.87(s,2H),4.05(t,J＝5.0Hz,2H),3.99(s,3H),3.62(q,J＝5.3Hz,2H),3.41(t,J＝5.1Hz,2H),3.04(t,J＝5.0Hz,2H),1.73-1.45(m,8H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):160.82,156.21,148.83,140.31,129.75,127.49,120.40,110.23,69.45,59.31,56.29,44.31,40.32,40.01,32.45,29.36,25.40,25.01.MS(ESI):m/z:Calcd.for C ₁₈ H ₂₆ N ₄ O ₇ Na ⁺ [M+Na] ⁺ :433.2.Found:433.1.

Example 14

Compound 5 (5.1g, 15mmol) in example 1 was dissolved in 100ml of a mixed solution of dichloromethane and trifluoroacetic acid (v: v = 10. And after the reaction is finished, the solvent is removed by spin drying, and the residue is vacuumized and dried for 2 hours. The residue after draining was dissolved in 50ml of methylene chloride, 50ml of a methylene chloride solution containing 1,8-octanedionate (4 g, 20mmol) was slowly added dropwise thereto, and thenAdding excessive triethylamine, and reacting for 3h at normal temperature. After the reaction was completed, the solvent was removed by spin-drying and purified by column chromatography (v dichloromethane: v methanol =1000 1), to obtain 6.5g of a pale yellow powdery compound NBNCO-8C with a yield of 83%. ¹ H NMR(400MHz,CDCl ₃ ),δ(ppm):7.72(s,1H),7.25(d,J＝13.5Hz,1H),6.20(t,J＝5.8Hz,1H),6.03(s,2H),4.87(s,2H),4.05(t,J＝5.0Hz,2H),3.99(s,3H),3.62(q,J＝5.3Hz,2H),3.41(t,J＝5.1Hz,2H),3.04(t,J＝5.0Hz,2H),1.93-1.32(m,12H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):160.82,156.21,148.83,140.31,129.75,127.49,120.40,110.23,69.45,59.31,56.29,44.31,40.32,40.01,32.45,29.36,25.40,25.01,24.84,24.32.MS(ESI):m/z:Calcd.for C ₂₀ H ₃₀ N ₄ O ₇ Na ⁺ [M+Na] ⁺ :461.2.Found:461.3.

Example 15

In the first step, the compound 4 (5 g, 20mmol) in example 1, 1-bromo-2- (2- (2- (2-bromoethoxy) ethoxy) ethane (9.6 g, 30mmol) and potassium carbonate (4.1g, 30mmol) were dissolved in acetonitrile 100ml, refluxed at 80 ℃ for 9 hours, and spotted on a plate for monitoring. After the reaction was completed, the filtrate was collected by filtration, and the solvent was removed by spin-drying and purified by column chromatography (v dichloromethane: v methanol = 1000) to obtain 7g of a yellow transparent liquid compound 11-1 with a yield of 80%. ¹ H NMR(400MHz,CDCl ₃ ),δ(ppm):7.71(s,1H),7.20(s,1H),5.31(s,1H),4.96(s,2H),4.26–4.19(m,2H),3.94(s,3H),3.98–3.87(m,2H),3.78–3.62(m,12H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):154.24,146.79,139.00,133.38,110.38,109.74,72.57,70.79,70.65,70.20,69.46,68.85,62.23,56.31,53.50,32.13.MS(ESI):m/z:Calcd.for C ₁₆ H ₂₅ O ₈ NBr ⁺ [M+H] ⁺ :438.1.Found:438.2.

A second step ofCompound 11-1 (5 g, 11mmol), maleimide (1.9g, 20mmol) and potassium carbonate (4.1g, 30mmol) prepared in the first step were dissolved in 100ml of dimethylformamide, refluxed at 50 ℃ for 3 hours, and monitored by a dot plate. After the reaction was completed, the filtrate was filtered, and the solvent was removed by spin-drying and purified by column chromatography (v dichloromethane: v methanol =1000: 1), to obtain 4g of a pale yellow powdery compound NBPM-4C with a yield of 80%. ¹ H NMR(400MHz,CDCl ₃ ),δ(ppm):7.71(s,1H),7.20(s,1H),6.71(s,2H),5.31(s,1H),4.96(s,2H),4.26–4.19(m,2H),3.94(s,3H),3.98–3.87(m,2H),3.78–3.62(m,12H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):170.50,154.24,146.79,139.00,136.43,133.38,110.38,109.74,72.57,70.79,70.65,70.20,69.46,68.85,62.23,56.31,53.50,40.31.MS(ESI):m/z:Calcd.for C ₂₀ H ₂₇ O ₁₀ N ₂ ⁺ [M+H] ⁺ :455.2.Found:455.2.

Example 16

In the first step, compound 4 (5g, 20mmol) of example 1, 1,2-bis (2-bromoethoxy) ethane (8.3g, 30mmol) and potassium carbonate (4.1g, 30mmol) were dissolved in acetonitrile 100ml, refluxed at 80 ℃ for 9h, and monitored by dot plate. After the reaction was completed, the filtrate was collected by filtration, and the solvent was removed by spin-drying and purified by column chromatography (v dichloromethane: v methanol = 1000) to obtain 6.7g of a yellow transparent liquid compound 11-2 with a yield of 85%. ¹ H NMR(400MHz,CDCl ₃ ),δ(ppm):7.71(s,1H),7.20(s,1H),5.31(s,1H),4.96(s,2H),4.26–4.19(m,2H),3.94(s,3H),3.98–3.87(m,2H),3.78–3.62(m,8H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):154.24,146.79,139.00,133.38,110.38,109.74,72.57,70.79,70.65,70.20,69.46,62.23,56.31,32.13.MS(ESI):m/z:Calcd.for C ₁₄ H ₂₁ O ₇ NBr ⁺ [M+H] ⁺ :394.1.Found:394.2.

In the second step, compound 11-2 (5g, 13mmol) prepared in the first step, maleimide (1.9g, 20mmol) and potassium carbonate (4.1g, 30mmol) were dissolved in 100ml of dimethylformamide, refluxed at 50 ℃ for 3 hours, and spotted on a plate for monitoring. After the reaction was completed, the filtrate was collected by filtration, and the solvent was removed by spin-drying and purified by column chromatography (v dichloromethane: v methanol =1000: 1), whereby 4.7g of a pale yellow powdery compound NBPM-3C was obtained in 89% yield. ¹ H NMR(400MHz,CDCl ₃ ),δ(ppm):7.71(s,1H),7.20(s,1H),6.71(s,2H),5.31(s,1H),4.96(s,2H),4.26–4.19(m,2H),3.94(s,3H),3.98–3.87(m,2H),3.78–3.62(m,8H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):170.50,154.24,146.79,139.00,136.43,133.38,110.38,109.74,70.79,70.20,69.46,68.85,62.23,56.31,53.50,40.31.MS(ESI):m/z:Calcd.for C ₁₈ H ₂₃ O ₉ N ₂ ⁺ [M+H] ⁺ :411.1.Found:411.2.

Example 17

In the first step, the compound NBS-2-4C (5g, 13mmol) prepared in example 5 and N-Boc lysine (4.9g, 20mmol) were dissolved in 250ml of dimethylformamide, and the reaction was monitored by a dot plate, and after the completion of the reaction, the solvent was removed by spin-drying, and column chromatography purification (v dichloromethane: v methanol: acetic acid =1000: 10) was carried out to obtain 12-1 as a yellow liquid compound in a yield of 60%. ¹ H NMR(400MHz,CDCl ₃ ),δ(ppm):12.66(s,1H),7.82(m,1H),7.71(s,1H),7.42(d,1H),7.20(s,1H),5.31(s,1H),4.96(s,2H),4.52(d,1H),4.26–4.19(m,2H),3.94(s,3H),3.98–3.87(m,4H),1.52–2.22(m,6H),1.42(s,9H),1.25(m,2H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):174.71,172.66,155.95,154.64,148.56,140.31,129.24,127.11,113.24,79.54,68.67,60.23,57.44,56.11,39.22,32.81,30.53,29.76,28.44,24.98,22.77.MS(ESI):m/z:Calcd.for C ₂₃ H ₃₆ N ₃ O ₁₀ ⁺ [M+H] ⁺ :514.2.Found:514.2.

In the second step, compound 12-1 (5 g,10 mmol) prepared in the first step was dissolved in 100ml (v: v = 10) of a mixed solution of dichloromethane and trifluoroacetic acid and reacted at room temperature for 1h. And (4) after the reaction is finished, removing the solvent by spin-drying, and vacuumizing the residue for 2h. The residue after draining was dissolved in 100ml of dimethylformamide, NHS-biotin (5.1g, 15mmol) was added, and the plate was monitored. After the reaction was finished, the solvent was removed by spin-drying, and column chromatography purification (v dichloromethane: v methanol: acetic acid = 1000. ¹ H NMR(400MHz,CDCl ₃ ),δ(ppm):12.66(s,1H),10.86(s,2H),7.82(m,1H),7.71(s,1H),7.42(d,1H),7.20(s,1H),5.31(s,1H),4.96(s,2H),4.61(m,2H),4.52(d,1H),4.26–4.19(m,2H),3.94(s,3H),3.98–3.87(m,4H),3.30–2.85(m,3H),1.52–2.22(m,12H),1.25(m,4H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):174.71,173.92,172.65,164.74,154.92,148.65,140.22,129.23,127.12,113.25,68.22,63.77,62.45,60.43,56.44,56.11,55.65,41.11,39.22,36.55,32.83,30.61,29.77,28.44,25.22,24.99,24.44,22.77.MS(ESI):m/z:Calcd.for C ₂₈ H ₄₂ N ₅ O ₁₀ S ⁺ [M+H] ⁺ :640.3.Found:640.3.

In the third step, compound 13-1 (5 g, 7.8mmol) prepared in the second step, hydroxysuccinimide (2.3 g, 20mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (3.8g, 20mmol) were dissolved in 100ml of dichloromethane and reacted at room temperature for 2 hours. After the reaction, dichloromethane was extracted with water, the organic phase was taken, the solvent was removed by spin-drying, and column chromatography purification (v dichloromethane: v methanol: = 1000. ¹ H NMR(400MHz,CDCl ₃ ),δ(ppm):10.86(s,2H),7.82(m,1H),7.71(s,1H),7.42(d,1H),7.20(s,1H),5.31(s,1H),4.96(s,2H),4.61(m,2H),4.52(d,1H),4.26–4.19(m,2H),3.94(s,3H),3.98–3.87(m,4H),3.30–2.85(m,3H),2.64(s,4H),1.52–2.22(m,12H),1.25(m,4H). ¹³ C NMR(100MHz,CDCl ₃ ),δ(ppm):174.71,173.92,172.65,169.11,164.74,154.92,148.65,140.22,129.23,127.12,113.25,68.22,63.77,62.45,60.43,56.44,56.11,55.65,41.11,39.22,36.55,32.83,30.61,29.77,28.44,25.68,25.22,24.99,24.44,22.77.MS(ESI):m/z:Calcd.for C ₃₂ H ₄₅ N ₆ O ₁₂ S ⁺ [M+H] ⁺ :737.3.Found:737.3.

The light-responsive multifunctional chemical cross-linking agent is applied to proteomics analysis, and is specifically shown in the following examples:

example 18

Use in protein interactions:

1) Selecting Streptavidin (SA) as a model protein, dissolving the compound NBS-2-4C prepared in example 5 in dimethyl sulfoxide to prepare a solution (52 mg/mL,136 mM), dissolving a commercial cross-linking agent bis-succinimide suberate (DSS) in dimethyl sulfoxide to prepare a solution (50mg, 136mM), and preparing an SA solution (1 mg/mL) with a phosphate buffer (10mM, pH = 7.0); the protein solution was sampled, corresponding to lane SA.

2) Adding a solution (10 uL) of a compound NBS-2-4C (No. 1) into the SA solution (1 mL), incubating for 2h (37 ℃,150 rad/min) in a shaking table in the dark, and sampling a protein solution, wherein the lane corresponds to SA + NBS-2-4C; adding DSS solution (10 uL) (the solution prepared in the 1 st) into SA solution (1 mL), and incubating for 2h (37 ℃,150 rad/min) in a shaking table in the dark; the protein solution was sampled, corresponding to lane SA + DSS.

3) All the above sampled protein solutions were mixed at 365nm wavelength and 10mw/cm ² The plates were illuminated for 2min and subsequently incubated in the dark for 2h (37 ℃ C., 150 rad/min). After completion, conventional SDS-PAGE was performed at a loading of 10uL per lane using a gel concentration of 15%. As shown in FIG. 1, FIG. 1 is a schematic diagram of the streptavidin photocrosslinking electrophoresis in example 18, from which it can be seen that Dimer 30kDa is a Dimer band generated by streptavidin under the action of a crosslinking agent, monomer15kDa is a streptavidin Monomer band, and the compound NBS-2-4C prepared in example 5 can crosslink four subunits of streptavidin to obtain a Dimer,can achieve the same effect as the commercial cross-linking agent DSS.

Example 19

Use in protein interactions:

1) Staphylococcus aureus protein A (SPA) and mouse immunoglobulin G (IgG) were chosen as model proteins, SPA and IgG being a known group of proteins with specific interactions. The compound NBS-2-4C prepared in example 5 was dissolved in dimethyl sulfoxide to prepare a solution (52 mg/mL,136 mM), and the commercial crosslinking agent bis-succinimidyl suberate (DSS) was dissolved in dimethyl sulfoxide to prepare a solution (50mg, 136mM), and both the SPA (1 mg/mL) solution and the IgG (1 mg/mL) solution were prepared using a phosphate buffer (10mM, pH = 7.0).

2) Mixing the equal volume of SPA solution and IgG solution, incubating in a shaking table for 2h (37 ℃,150 rad/min) in the dark, and sampling protein solution, wherein the protein solution corresponds to SPA + IgG lane; 100uL of each mixed solution was taken, 10uL of each of NBS-2-4C solution and DSS solution was added, and the protein solution was sampled and corresponded to the SPA + IgG + NBS-2-4C, SPA + IgG + DSS lanes, respectively.

3) All the above sampled protein solutions were mixed at 365nm wavelength and 10mw/cm ² The plates were illuminated for 2min and subsequently incubated in the dark for 2h (37 ℃ C., 150 rad/min). After completion, conventional SDS-PAGE was performed at a loading of 10uL per lane using a 10% gel. As shown in FIG. 2, FIG. 2 is a schematic diagram of the electrophoresis of the interaction between Staphylococcus aureus protein A and mouse IgG in example 19, from which it can be seen that Cross-link chain is a Cross-linked band of Staphylococcus aureus protein A and mouse IgG under the action of a Cross-linking agent, igG-Heavy chain 50kDa is a Heavy chain band of mouse IgG, SPA 33.4kDa is a Staphylococcus aureus protein A band, igG-light chain 25kDa is a light chain band of mouse IgG, and the compound NBS-2-4C prepared in example 5 is capable of Cross-linking the interacting proteins, capturing the protein interaction, and approximating the effect of the commercial Cross-linking agent DSS.

Example 20

Use in protein interactions:

2) 100uL of NBS-2-4C solution of the compound prepared in example 5 was added to 1mL of SPA solution, and the mixture was incubated in a shaker for 2 hours (37 ℃ C., 150 rad/min) in the absence of light. After the completion of the reaction, the mixture was dialyzed against phosphate buffer for 12 hours to obtain SPA (NBS-2-4C) solution. Mixing equal volume of dialyzed SPA (NBS-2-4C) solution and IgG solution, incubating in a shaking table away from light for 2h (37 ℃,150 rad/min), and sampling protein solution, which corresponds to SPA (NBS-2-4C) + IgG (NO hv) lane; sampling the protein solution at a wavelength of 365nm and 10mw/cm ² The column was illuminated under light for 2min, and then incubated with shaking light for 2h (37 ℃ C., 150 rad/min) corresponding to SPA (NBS-2-4C) + IgG lane, SPA + IgG + NBS-2-4C lane was prepared as described in example 19.

3) Routine SDS-PAGE was performed with loading of 10uL per lane using 10% gel concentration. As shown in FIG. 3, FIG. 3 is a schematic diagram of the electrophoresis of the interaction between Staphylococcus aureus protein A and mouse IgG in example 20, from which it can be seen that Cross-link chain is a Cross-linked band of Staphylococcus aureus protein A and mouse IgG under the action of a Cross-linking agent, igG-Heavy chain 50kDa is a Heavy chain band of mouse IgG, SPA/SPA (NBS-2-4C) 33.4kDa is a Cross-linked band of Staphylococcus aureus protein A labeled with a Cross-linking agent, igG-light chain 25kDa is a light chain band of mouse IgG, and NBS-2-4C compound prepared in example 5 is capable of Cross-linking and capturing protein interactions; by the technical method, the IgG light chain which does not participate in protein interaction can be not crosslinked, so that the false positive crosslinking result is reduced.

Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A photo-responsive multifunctional chemical cross-linking agent which is one of the following compounds:

in NBS-1 and Sulfo-NBS-1, n is an integer of 1 to 6,

in NBS-2 and Sulfo-NBS-2, n is an integer of 1 to 10,

in NBM and Sulfo-NBM, n is an integer of 0 to 6,

n in NBNCO is an integer of 1 to 6, n in NBPM is an integer of 1 to 4, and n in Tri-NB is an integer of 2 to 6.

2. A photo-responsive multifunctional chemical cross-linking agent according to claim 1, wherein: the light-responsive multifunctional chemical cross-linking agent is one of the following compounds:

3. a method for preparing a photo-responsive multifunctional chemical crosslinking agent according to claim 1 or 2, comprising the steps of:

in NBS-1 and Sulfo-NBS-1, n is an integer of 1 to 6,

in NBNCO, n is an integer of 1 to 6,

secondly, dissolving the compound 5 prepared in the first step in a mixed solution of a solvent and excessive trifluoroacetic acid, reacting at room temperature, after the reaction is finished, spin-drying to remove the solvent, vacuum-drying the remainder, dissolving the dried remainder in the solvent, slowly dropwise adding a solution containing bis-succinimidyl ester, wherein the molar ratio of the bis-succinimidyl ester to the compound 5 is (1-1.5): 1, adding a catalytic amount of triethylamine, reacting at room temperature, after the reaction is finished, spin-drying to remove the solvent, and purifying by column chromatography to obtain a compound NBS-1; or slowly dropwise adding a solution containing sodium disuccinimidyl ester sulfonate, wherein the molar ratio of the sodium disuccinimidyl ester sulfonate to the compound 5 is (1-1.5): 1, adding a catalytic amount of triethylamine, reacting at normal temperature, removing the solvent by spin drying after the reaction is finished, and purifying by column chromatography to obtain a compound Sulfo-NBS-1;

or the like, or, alternatively,

in NBS-2 and Sulfo-NBS-2, n is an integer of 1 to 10,

in NBM and Sulfo-NBM, n is an integer of 0 to 6,

in NBPM, n is an integer of 1 to 4,

n is an integer of 2 to 6,

and secondly, dissolving the compound 12 prepared in the first step in a mixed solution of a solvent and excess trifluoroacetic acid, reacting at room temperature, performing spin-drying to remove the solvent after the reaction is finished, performing vacuum pumping on the residue for 2 hours, dissolving the drained residue in the solvent, and adding succinimide biotin into the mixture, wherein the molar ratio of the succinimide biotin to the compound 12 is (1.1-3): 1, after the reaction is finished, spin-drying to remove the solvent, and purifying by column chromatography to obtain a compound 13;

4. The production method according to claim 3, characterized in that: wherein the bis-succinimide ester is bis-succinimide glutarate, bis-succinimide glutarate;

the sodium disuccinimidyl ester sulfonate is sodium disuccinimidyl suberate sulfonate and sodium disuccinimidyl glutarate sulfonate;

the isocyanate is 1,8-octanedionate, 1,6-hexanedionate;

the methyl bromobutyrate is 4-methyl bromobutyrate and 8-methyl bromooctanoate;

the dibromoalkyl is 1,4-dibromohexane, 1,4-dibromobutane;

the dibromo-condensed polyethylene glycol is 1-bromo-2- (2- (2- (2-bromoethoxy) ethoxy) ethane, 1,2-bis (2-bromoethoxy) ethane;

the N-Boc amino acid is N-Boc lysine.

5. The production method according to claim 3, characterized in that: wherein the preparation method of the compound 4 comprises the following steps:

secondly, grinding the compound 1 prepared in the first step into powder, slowly adding excessive nitric acid, carrying out ice-bath reaction, pouring the reaction liquid into ice water after the reaction is finished, stirring and filtering, and recrystallizing to obtain a compound 2;

and step four, dissolving the compound 3 prepared in the step three in a solvent, slowly adding sodium borohydride with the mole number of 1.5-2.5 times that of the compound 3, reacting at room temperature, extracting an organic phase after the reaction is finished, adjusting the pH value to subacidity, and removing the solvent by spin drying to obtain a compound 4.

6. The production method according to claim 3, characterized in that: wherein the solvent is dimethylformamide, acetonitrile, dichloromethane, ethanol, water, methanol, acetone or ethyl acetate.

7. A use of the photo-responsive multifunctional chemical cross-linking agent of claim 1 or 2 in protein interaction.

8. Use according to claim 7, characterized in that: the application comprises the following steps:

the first method comprises the following steps: adding the light-responsive multifunctional chemical cross-linking agent into a single target protein solution for incubation, using 365nm illumination to realize cross-linking of protein, and then carrying out protein electrophoresis, protein imprinting and protein cross-linking mass spectrometry;

or, the second method: adding the light-responsive multifunctional chemical cross-linking agent into a group of protein solutions with specific interaction for incubation, using 365nm illumination to realize cross-linking interaction of proteins, capturing protein interaction, and then performing protein electrophoresis, protein imprinting and protein cross-linking mass spectrometry;

or, a third method: adding the light-responsive multifunctional chemical cross-linking agent into a single target protein solution for incubation, then dialyzing to obtain target protein with the light-responsive multifunctional chemical cross-linking agent, incubating the target protein with the light-responsive multifunctional chemical cross-linking agent and the interaction protein thereof, realizing the cross-linking of the protein interaction pair by 365nm illumination, and then carrying out protein electrophoresis, protein imprinting and protein cross-linking mass spectrometry.