CN109824565A - A kind of optical Response multifunctional chemical crosslinking agent and the preparation method and application thereof - Google Patents

Abstract

The invention discloses a kind of optical Response multifunctional chemical crosslinking agents, and structure is as shown in general formula I:Each substituent group is defined in the specification in formula.Optical Response multifunctional chemical crosslinking agent preparation method of the invention is simple, low in cost, it is the first crosslinking agent based on o-nitro benzyl alcohol photo-crosslinking, coupling reaction occurs come crosslinking protein by photoproduction aldehyde radical and amino: in the crosslinking agent, photoreactive groups are to containing-NH₂Amino acid residue have very high cross-linking efficiency and selectivity；By being combined with functional group used in other conventional cross-linking agents, the exclusive-OR function crosslinking agent with photoresponse is obtained, the crosslinking of heterologous protein specificity is realized, there is great potential application in protein science research.

Description

A kind of optical Response multifunctional chemical crosslinking agent and the preparation method and application thereof

Technical field

The invention belongs to protein and its functional study technical field, in particular to a kind of optical Response multifunctional chemical is handed over Join agent and the preparation method and application thereof.

Background technique

Protein is the bearer of gene expression, directly affects the life process of biology.In genomics, Ren Menfa The demand of probing into life process is not now able to satisfy to the research of gene, and studies protein, then is deepened to life process Understanding has important role.Therefore, proteomics becomes important research object in rear era gene.In recent years, with tradition Biological method studies proteomics, is not able to satisfy the demand of scientific worker gradually.With chemical synthesis process Maturation, it has been found that chemical small molecule is (such as protein imprinted to traditional biological method as a kind of emerging tool Western Blot, Enzyme-linked Immunosorbent Assay Elisa etc.) and biological research tool (such as electron cryo-microscopy) important supplement.

To in the research of proteomics, protein-crosslinking, modification and label are study protein structure and interaction normal Use technology.Wherein, protein cross is carried out by small molecule and is developing progressively using interpretation of mass spectra is crosslinked as research protein The important research method of structure and interaction, advantage are more intuitive compared with traditional biological method.Protein cross agent is one Micromolecular compound has 2 or is more directed to specific groups (- NH₂,-COOH ,-HS ,-OH etc.) reactivity end End can be coupled respectively with 2 or more protein.People generally use glutaraldehyde as protein cross when the seventies Agent connects antibody and indicator (such as enzyme), but the disadvantage is that since crosslinked group is random, mixed and disorderly poly easy to form Body.Recently, Ruedi Aebersold etc. uses this classics of the double amber imide suberate (DSS) of amino specificity Small molecule crosslinking agent carries out crosslinking mass spectral analysis to protein phosphatase enzyme family, has successfully parsed its interactive network (Herzog,F.,et al.(2012).Science337(6100):1348-1352.).This work can be rated as chemical cross-linking agent The classics of Way for Studying Protein-Protein Interactions.Then, similar small molecule crosslinking agent is come into being, and realizes commercialization, including DSS Series matter, BS3, DSSO (A.Sinz (2017)Anal Bioanal Chem409 (1): 33-44.) etc..These small molecules are handed over Although connection agent can realize that heterologous specificity is crosslinked by introducing differential responses active group, cross-linking process is uncontrollable , processing protein sample in can bring many false positives as a result, the self-crosslinking of especially albumen, largely limits Its research and further application to protein-interacting.

Due to being introduced into small molecule crosslinking agent system with space-time controllability, it can be first with unstable for light reaction Thermal response active group be crosslinked with a kind of albumen, second of albumen is then crosslinked by ultraviolet light, to avoid egg The interference of white self-crosslinking greatly reduces traditional chemical crosslinking bring limitation.The existing light being applied on crosslinking agent is rung Answering group mainly has benzene nitrine (Tanaka, Y., et al. (2008)Mol Biosyst4 (6): 473-480.), benzophenone (Majmudar,C.Y.,et al.(2009).J Am Chem Soc131 (40): 14240-14242.), double a word used for translations third sting (Suchanek,M.,et al.(2005).Nat Methods2 (4): 261-267.) three kinds, wherein many products also achieve Commercialization.But although this photoresponse crosslinking realizes the controllability of space-time, reaction mechanism is all by free radical C- The intercalation reaction of H or N-H cannot define the reaction site on albumen, to lose the spy of crosslinking to group without selectivity It is anisotropic.Although the preparation synthesis of corresponding derivative is relatively difficult in addition, the crosslinking agent structure of most of photoresponse is simple, So that it is expensive.

Therefore, find it is a kind of new, easily prepared, there is the active optical Response group of specific reaction, and by its Small-molecule chemical cross linker system is introduced, is just expected to solve the defect of existing crosslinking agent.

Summary of the invention

The first purpose of the invention is to provide a kind of optical Response multifunctional chemical crosslinking agents.

A second object of the present invention is to provide a kind of preparation methods of optical Response multifunctional chemical crosslinking agent.

Third object of the present invention is to provide a kind of purposes of optical Response multifunctional chemical crosslinking agent.

To achieve the goals above, The technical solution adopted by the invention is as follows:

The first aspect of the invention provides a kind of optical Response multifunctional chemical crosslinking agent, and structure is as shown in general formula I:

Wherein, R₁、R₂、R₃、R₄Separately selected from hydrogen, halogen atom, hydroxyl, sulfydryl, amido, nitro, cyano, aldehyde radical, Ketone group, ester group, amide groups, phosphonic acid base, phosphonate group, sulfonic group, sulfonate group, sulfuryl, sulfoxide group, aryl, heteroaryl, alkane Base, alkoxy, alkylidene or modified alkyl；

R ' is traditional chemical reactive group, selected from one of flowering structure:

R ", which is selected from biotin and its derivative, alkyl alkene and its derivative, alkyl alkynes and its derivative etc., can be used for magnetic bead The group or hydrogen atom of beneficiation technologies；It is further selected from fluorescein and its derivative, rhodamine and its derivative, 1,8- naphthalene diformazan Acid imide etc. has the group of fluorescence identifying function；

Particularly, when R " is selected from hydrogen atom, optical Response multifunctional chemical crosslinking agent I is the difunctional crosslinking of photoresponse Agent.

Particularly, work as R " and be selected from biotin, alkyl alkene and its derivative, alkyl alkynes and its derivative etc. and can be used for magnetic bead richness When the group of collection technology, optical Response multifunctional chemical crosslinking agent I is three functional cross-link agent of photoresponse.

Wherein X and R₁、R₂、R₃、R₄In one connection, X be selected from aryl, heteroaryl, alkyl, alkylidene, modified alkyl, Modified alkylidene, ehter bond, ester bond, carbonic acid ester bond, amido bond, urea bond and independent assortment described above.

The aryl is 5~10 yuan of aromatic monocyclics or fragrant fused bicyclic structures；

The heteroaryl is on ring containing selected from heteroatomic 5~10 yuan of aromatic monocyclics of at least one of O, S, N or Si Or fragrant fused bicyclic structures；

The alkyl is the alkyl of saturation or unsaturated aliphatic linear chain or branched chain with 1~30 carbon atom；

The alkylidene is the alkylidene of saturation or unsaturated aliphatic linear chain or branched chain with 1~30 carbon atom；

The modified alkyl is that any carbon atom of alkyl is selected from halogen atom ,-OH ,-SH ,-NO₂、-CN、-CHO、- COOH, ester group, aryl ,-CO- ,-O- ,-S- ,-SO- ,-SO₂, primary amino group, secondary amine, tertiary amine groups or quaternary ammonium salt base substitution, institute Modified alkyl is stated with 1~30 atom, carbon-carbon single bond can be replaced arbitrarily by carbon-carbon double bond or carbon-carbon triple bond；

The modified alkylidene is that any carbon atom of alkylidene is selected from halogen atom ,-OH ,-SH ,-NO₂、-CN、- CHO ,-COOH, ester group, aryl ,-CO- ,-O- ,-S- ,-SO- ,-SO₂, primary amino group, secondary amine, tertiary amine groups or quaternary ammonium salt base replace Generation, the modified alkylidene have 1~30 atom, and carbon-carbon single bond can be replaced arbitrarily by carbon-carbon double bond or carbon-carbon triple bond；

The ehter bond is selected from one of flowering structure:

-O(CH₂)_x-、-(CH₂)_xO(CH₂)_y-、-(CH₂CH₂O)_x、-(CH₂CH₂O)_x(CH₂)_y, wherein x and y >=0 and to be whole Number；

The ester bond is selected from one of flowering structure:

-COO(CH₂)_x-、-OCO(CH₂)_x-、-(CH₂)_xCOO(CH₂)_y-、-(CH₂)_xOCO(CH₂)_y, wherein x and y >=0 and For integer；

The carbonic acid ester bond is selected from one of flowering structure:

-CO₃(CH₂)_x-、-(CH₂)_xCO₃(CH₂)_y, wherein x and y >=0 and be integer；

The amido bond is selected from one of flowering structure:

-NHCO(CH₂)_x-、-CONH(CH₂)_x-、-(CH₂)_xNHCO(CH₂)_y-、-(CH₂)_xCONH(CH₂)_y, wherein x and y >=0 and be integer；

The urea bond is selected from one of flowering structure:

-NHCONH(CH₂)_x-、-(CH₂)_xNHCONH(CH₂)_y, wherein x and y >=0 and be integer；

The alkyl alkene and its derivative are the alkane of the unsaturated aliphatic linear chain or branched chain with 1~30 carbon atom Base, not limited number of carbon-carbon double bond is contained in nonspecific site wherein；

The alkyl alkynes and its derivative are the alkane of the unsaturated aliphatic linear chain or branched chain with 1~30 carbon atom Base, not limited number of triple carbon-carbon bonds are contained in nonspecific site wherein；

The fluorescein and its derivative are fluorescein, Aminofluorescein, Fluoresceincarboxylic acid, isothiocyanates fluorescein；

The rhodamine and its derivative are rhodamine, aminorhodamine, carboxyrhodamine, isothiocyanates rhodamine.

Currently preferred compound is the general structure of optical Response multifunctional chemical crosslinking agent are as follows:

R ' is traditional chemical reactive group, selected from one of flowering structure:

R " is hydrogen atom, and X is selected from one of flowering structure:

Wherein n₁It is 1~6 integer, n₂It is 1~10 integer, n₃It is 1~4 integer, n₄It is 1~6 integer, n₅It is 0 ~6 integer, above-mentioned optical Response multifunctional chemical crosslinking agent are photoresponse bi-functional cross-linking agent；

R " is selected from biotin and its derivative, and X is with flowering structure:

Wherein n is 2~6 integer, and above-mentioned optical Response multifunctional chemical crosslinking agent is three functional cross-link agent of photoresponse.

The preferred compound of the present invention is the general structure of optical Response multifunctional chemical crosslinking agent are as follows:

N is 1~6 integer in NBS-1, Sulfo-NBS-1,

N is 1~10 integer in NBS-2, Sulfo-NBS-2,

N is 0~6 integer in NBM, Sulfo-NBM,

N is 1~6 integer in NBNCO, and n is 1~4 integer in NBPM,

N is 2~6 integer in Tri-NB.

The most preferred compound of the present invention is that the structure of optical Response multifunctional chemical crosslinking agent is with one in flowering structure Kind:

The second aspect of the invention provides a kind of preparation method of optical Response multifunctional chemical crosslinking agent, packet Include following steps:

N is 1~6 integer in NBS-1, Sulfo-NBS-1,

N is 1~6 integer in NBNCO,

Molar ratio is 1:(3~5 by the first step): compound 4, potassium carbonate and the N-Boc bromine ethamine of (1.5~2.5) are dissolved in In solvent, back flow reaction filters to take filtrate after reaction, is spin-dried for removing solvent, column Chromatographic purification obtains compound 5；

Compound 5 prepared by the first step is dissolved in the mixed solution of solvent and excessive trifluoroacetic acid, room temperature by second step Reaction is spin-dried for removing solvent after reaction, and residue vacuum is drained 2h, the residue after draining is dissolved in solvent, slowly The molar ratio of solution of the dropwise addition containing double amber imide ester, double amber imide ester and compound 5 is (1~1.5): 1, it is added The triethylamine of catalytic amount, normal-temperature reaction are spin-dried for removing solvent, column Chromatographic purification obtains compound N BS-1 after reaction；

Or, the solution containing double amber imide ester sodium sulfonate is slowly added dropwise, double amber imide ester sodium sulfonate and chemical combination The molar ratio of object 5 is (1~1.5): 1, the triethylamine of catalytic amount is added, and normal-temperature reaction is spin-dried for removing solvent after reaction, Column Chromatographic purification obtains compound Sulfo-NBS-1；

Or, the solution containing isocyanates is slowly added dropwise, the molar ratio of isocyanates and compound 5 is (1~1.5): 1, The triethylamine of catalytic amount is added, normal-temperature reaction is spin-dried for removing solvent, column Chromatographic purification obtains compound after reaction NBNCO；

N is 1~10 integer in NBS-2, Sulfo-NBS-2,

Molar ratio is 1:(1.1~3 by the first step): compound 4, bromo butyric acid methyl ester and the potassium carbonate of (1.1~4) are dissolved in In solvent, back flow reaction filters to take filtrate after reaction, is spin-dried for removing solvent, ethyl alcohol recrystallization obtains compound 6；

Compound 6 prepared by the first step is dissolved in solvent, excessive potassium hydroxide normal-temperature reaction is added, instead by second step After answering, it is spin-dried for removing solvent, water dissolution adjusts pH until 4, generates yellow mercury oxide, filter to take filter cake, obtain compound 7；

Third step, the molar ratio by second step preparation are 1:(1~1.5): compound 7, the maloyl of (1~1.5) Imines and 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride are dissolved in solvent, normal-temperature reaction, after reaction, Methylene chloride and water extraction, take organic phase, are spin-dried for removing solvent, column Chromatographic purification obtains compound N BS-2；

Or, being 1:(1~1.5 by the molar ratio of second step preparation): compound 7, the hydroxysuccinimide of (1~1.5) Sodium sulfonate and 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride are dissolved in solvent, and normal-temperature reaction, reaction terminates Afterwards, methylene chloride and water extraction, take organic phase, are spin-dried for removing solvent, column Chromatographic purification obtains compound Sulfo-NBS-2；

N is 0~6 integer in NBM, Sulfo-NBM,

Molar ratio is 1:(1.1~3 by the first step): compound 4, dibromoalkane base and the potassium carbonate of (1.1~4) are dissolved in In solvent, back flow reaction filters to take filtrate after reaction, is spin-dried for removing solvent, column Chromatographic purification obtains compound 10；

Second step, the molar ratio by first step preparation are 1:(1.1~3): compound 10, the maleimide of (1.1~4) It is dissolved in solvent with potassium carbonate, back flow reaction, after reaction, filters to take filtrate, be spin-dried for removing solvent, column Chromatographic purification obtains To compound N BM；

Or, being 1:(1.1~3 by the molar ratio of first step preparation): compound 10, the maleimide sulfonic acid of (1.1~4) Sodium and potassium carbonate are dissolved in solvent, back flow reaction, after reaction, filter to take filtrate, be spin-dried for remove solvent, column Chromatographic purification, Obtain compound Sulfo-NBM；

N is 1~4 integer in NBPM,

Molar ratio is 1:(1.1~3 by the first step): compound 4, two bromo polycondensation ethylene glycol and the carbonic acid of (1.1~4) Potassium is dissolved in solvent, back flow reaction, after reaction, filters to take filtrate, is spin-dried for removing solvent, column Chromatographic purification obtains compound 11；

Second step, the molar ratio by first step preparation are 1:(1.1~3): compound 11, the maleimide of (1.1~4) It is dissolved in solvent with potassium carbonate, back flow reaction, after reaction, filters to take filtrate, be spin-dried for removing solvent, column Chromatographic purification obtains Compound N BPM；

N is 2~6 integer,

The first step, by molar ratio be 1:(1.1~3) compound N BS-2-4C, N-Boc amino acid be dissolved in solvent, often Temperature reaction is spin-dried for removing solvent, column Chromatographic purification obtains compound 12 after reaction；

Compound 12 prepared by the first step is dissolved in the mixed solution of solvent and excessive trifluoroacetic acid, room by second step Temperature reaction is spin-dried for removing solvent after reaction, and residue vacuum drains 2h, the residue after draining is dissolved in solvent, is added Enter succinimide biotin, the molar ratio of succinimide biotin and compound 12 is (1.1~3): 1, after reaction, It is spin-dried for removing solvent, column Chromatographic purification obtains compound 13；

Third step, the molar ratio by second step preparation are 1:(1.1~3): compound 13, the maloyl of (1.1~3) Imines and 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride are dissolved in solvent, normal-temperature reaction, after reaction, Methylene chloride and water extraction, take organic phase to be spin-dried for removing solvent, column Chromatographic purification obtains compound Tri-NB.

The double amber imide ester is double amber imide glutarate, double amber imide glutarate.

The double amber imide ester sodium sulfonate is double amber imide suberate sodium sulfonate, double amber imide penta 2 Acid esters sodium sulfonate.

The isocyanates is the pungent diisocyanate of 1,8-, hexamethylene diisocyanate.

The bromo butyric acid methyl ester is 4- bromo butyric acid methyl ester, 8- bromine methyl caprylate.

The dibromoalkane base is 1,4- dibromo-hexane, 1,4- dibromobutane.

The two bromos polycondensation ethylene glycol is the bromo- 2- of 1- (2- (2- (2- bromine oxethyl) ethyoxyl) ethyoxyl) ethane, 1, Bis- (2- bromine oxethyl) ethane of 2-.

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

The preparation method of the compound 4 the following steps are included:

Molar ratio is 1:(1.5~3 by the first step): vanillic aldehyde, potassium carbonate and the benzyl bromine of (1.1~1.5) are dissolved in solvent In, back flow reaction, reaction terminates filter, be spin-dried for, recrystallizes acquisition compound 1；

Second step, 1 grind into powder of compound prepared by the first step are slowly added to excessive nitric acid, and ice bath reacts, instead After answering, reaction solution is poured into agitation and filtration in ice water, is recrystallized to give compound 2；

Compound 2 prepared by second step is dissolved in excessive trifluoroacetic acid by third step, is reacted at room temperature, after reaction, It is spin-dried for removing solvent, residue is completely dissolved in sodium hydrate aqueous solution and ethyl acetate mixtures, tune pH value to faintly acid, Organic phase is extracted, is spin-dried for removing solvent, petroleum ether is added in residue, filters to take filter cake, is repeated 3 times, obtain compound 3；

Compound 3 prepared by third step is dissolved in solvent, is slowly added to 3 molal quantity 1.5~2.5 of compound by the 4th step Sodium borohydride again, room temperature reaction extract organic phase after reaction, adjust pH value to faintly acid, are spin-dried for removing solvent, must change Close object 4.

The solvent is dimethylformamide, acetonitrile, methylene chloride, ethyl alcohol, water, methanol, acetone.

It is mutual in protein that the third aspect of the invention provides a kind of optical Response multifunctional chemical crosslinking agent Active application.

The optical Response multifunctional chemical crosslinking agent protein interaction in application the following steps are included:

First method: the optical Response multifunctional chemical crosslinking agent is added in simple target protein solution and is incubated It educates, realizes the crosslinking to albumen with 365nm illumination, then carry out protein electrophoresis (SDS-PAGE), protein imprinted (Western Blot), protein cross mass spectrum (LC-MS/MS) is analyzed；

Second method: the optical Response multifunctional chemical crosslinking agent, which is added to one group, has specificity interaction Protein solution in be incubated for, with 365nm illumination realize crosslinking interaction albumen, capture protein interaction, then into Row protein electrophoresis (SDS-PAGE), protein imprinted (Western Blot), protein cross mass spectrum (LC-MS/MS) analysis；

Third method: the optical Response multifunctional chemical crosslinking agent is added in simple target protein solution and is incubated It educates, then dialyses, obtain the target protein with the optical Response multifunctional chemical crosslinking agent, will be rung with the light The target protein of answering property multifunctional chemical crosslinking agent interacts therewith albumen incubation, is realized with 365nm illumination mutual to albumen Then the crosslinking of effect pair carries out protein electrophoresis (SDS-PAGE), protein imprinted (Western Blot), protein cross matter Compose (LC-MS/MS) analysis；

Simple target albumen is Streptavidin (SA) as model protein in first method.

It is staphylococcus aureus protein A that one group, which has the albumen of specificity interaction, in second method (SPA) and mouse immune globulin G (IgG) is used as model protein.

Simple target albumen is staphylococcus aureus protein A (SPA) in the third method, interacts therewith egg White is mouse immune globulin G (IgG).

The present invention deprotects to obtain an intermediate using vanillic aldehyde as raw material by phenolic hydroxyl group protection, nitrification, phenolic hydroxyl group, The intermediate passes through and bromo compound reaction, can introduce the spacerarm with different length in phenolic hydroxyl group, then into one Step introduces traditional chemical group (succinimide, maleimide, isocyanates etc.) by reaction accordingly in the other end, i.e., It is provided with the effect of chemical cross-linking agent, by changing the conditions such as reaction raw materials feed ratio, reaction temperature, reaction dissolvent, is obtained Chemical cross-linking agent with different function, different length.

Present invention proposition utilizes o-nitro benzyl alcohol structure building optical Response multifunctional chemical crosslinking agent and for albumen The research of matter group, the structure have optical Response, can be to the trip on lysine side-chain on protein under the irradiation of 365nm light Specific reaction is carried out from amino, forms stable covalent cross-linking.Because of the optical Response of o-nitro benzyl alcohol, when which has Empty controllability may be implemented to interact to different proteins, carry out after introducing different brachiums, different chemical reaction groups The controllable analysis of space-time.

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

Optical Response multifunctional chemical crosslinking agent of the invention utilizes o-nitro benzyl alcohol structure, realizes the controllable amino of space-time Coupling label, compares other photoresponse groups, has higher efficiency and specific；The egg constructed using o-nitro benzyl alcohol structure White exclusive-OR function crosslinking agent, compared to traditional chemical crosslinking agent, with the advantage that space-time is controllable in proteomics, with albumen electricity Swimming and protein cross interpretation of mass spectra can more accurately capture protein interaction information；Using vanillic aldehyde as raw material, pass through A series of simple synthesis can prepare the molecule, and for comprehensive yied 50% or more, cost of material is cheap, and reaction is simple and efficient, It is commercially valuable.

Optical Response multifunctional chemical crosslinking agent preparation method of the invention is simple, low in cost, is the first based on adjacent nitre The crosslinking agent of base benzylalcohol photo-crosslinking occurs coupling reaction by photoproduction aldehyde radical and amino come crosslinking protein: being crosslinked described In agent, photoreactive groups are to containing-NH₂Amino acid residue have very high cross-linking efficiency and selectivity；By with other tradition The combination of functional group used in crosslinking agent, obtains the exclusive-OR function crosslinking agent with photoresponse, realizes the special sexual intercourse of heterologous protein Connection has great potential application in protein science research.

Detailed description of the invention

Fig. 1 is the schematic diagram of the Streptavidin photo-crosslinking electrophoresis in embodiment 18.

Fig. 2 is that the electrophoresis of the staphylococcus aureus protein A and mouse immune globulin G interaction in embodiment 19 shows It is intended to.

Fig. 3 is that the electrophoresis of the staphylococcus aureus protein A and mouse immune globulin G interaction in embodiment 20 shows It is intended to.

Specific embodiment

In order to illustrate more clearly of the present invention, below with reference to preferred embodiment, the present invention is described further.Ability Field technique personnel should be appreciated that following specifically described content is illustrative and be not restrictive, this should not be limited with this The protection scope of invention.

Synthesis is bought with source chemicals respectively in the resistance to Jilin Chemical of peace, lark prestige Science and Technology Ltd. and Ah in following embodiment The Reagent Companies such as Latin reagent Co., Ltd, the reagents of all purchases remove in addition illustrate without being further purified, it is straight after purchase Connect use；Reaction dissolvent such as dimethylformamide, methylene chloride, acetonitrile etc. is dried by distillation, and reaction process exists Implement under argon gas protective condition.

Embodiment 1

The first step, by vanillic aldehyde (50g, 328mmol), potassium carbonate (82.8g, 600mmol) and benzyl bromine (76.9g, It 450mmol) is dissolved in acetonitrile 1000ml, 80 DEG C of reflux 9h, point board monitoring (TLC monitoring reaction) to the end of reacting, filters to take filter Liquid is spin-dried for removing solvent, obtains pale yellow viscous liquid.Liquid is dissolved in ethyl alcohol, is heated to clear, a small amount of petroleum is added Ether is cooled to room temperature, and recrystallization overnight, is precipitated white crystal, filters to take filter cake, and obtaining 67g white crystal is compound 1, yield 84%.¹H NMR(400MHz,CDCl₃),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).¹³C NMR(100MHz,CDCl₃),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 C₁₅H₁₄O₃Na⁺[M+Na]⁺:265.2.Found:265.2.

Second step,

Compound 1 (8g, 33mmol) grind into powder prepared by the first step, is slowly added to the round bottom containing 30mL nitric acid In flask, emerge wait react rufous gas when, to bath on the rocks is reacted, put board monitoring.After reaction, reaction solution is poured into It is stirred in 1.5L ice water, filters to take filter cake, ethyl alcohol recrystallization obtains 8g yellow crystals compound 2, yield 84%.¹H 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).¹³C NMR(100MHz,CDCl₃),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 C₁₅H₁₄NO₅ ⁺[M+H]⁺:288.2.Found:288.2.

Third step,

Compound 2 (10g, 35mmol) prepared by second step is dissolved in 100mL trifluoroacetic acid, 9h, contact plate are reacted at room temperature Monitoring.After reaction, it is spin-dried for removing solvent.Residue is completely dissolved in sodium hydrate aqueous solution and ethyl acetate mixtures, It adjusts pH value to faintly acid, extracts organic phase.It is spin-dried for removing solvent, a small amount of petroleum ether is added in residue, filters to take filter cake, repeats 3 times, obtain 6.5g khaki powder compound 3, yield 95%.¹H 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).¹³C NMR(100MHz,DMSO-d₆),δ (ppm):188.3,151.7,150.9,143.7,123.3,111.0,110.5,56.3.MS(ESI):m/z:Calcd.for C₈H₇NO₅Na⁺[M+Na]⁺:220.0.Found:220.0.

4th step,

Compound 3 (10g, 51mmol) prepared by third step is dissolved in methylene chloride 100ml and methanol 400ml mixed solution In, it is slowly added to sodium borohydride (3.8g, 100mmol), reacts at room temperature 15min.After reaction, organic phase is extracted, pH value is adjusted To faintly acid, it is spin-dried for removing solvent, obtains 10g yellow powder compound 4, yield 99%.¹H NMR(400MHz,DMSO-d₆)δ 9.92(s,1H),7.56(s,1H),7.34(s,1H),4.79(s,2H),3.90(s,3H).¹³C NMR(101MHz,DMSO-d₆) δ153.30,145.42,138.84,132.89,111.71,110.42,60.62,56.43.MS(ESI):m/z:Calcd.for C₈H₉NO₅Na⁺[M+Na]⁺:222.1.Found:222.1.

5th step,

Compound 4 (5g, 25mmol), potassium carbonate (13.8g, 100mmol) and N-Boc bromine ethamine prepared by the 4th step (11.2g, 50mmol) is dissolved in acetonitrile, 80 DEG C of reflux 9h, puts board monitoring.After reaction, filtrate is filtered to take, is spin-dried for removing molten Agent, column Chromatographic purification (v methylene chloride: v methanol=1000:5), obtains 8g yellow powder compound 5, yield 93%.¹H NMR (400MHz,CDCl₃), 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)¹³C NMR(100MHz,CDCl₃),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 C₁₅H₂₃N₂O₇ ⁺[M+H]⁺:343.2.Found:343.2.

6th step,

Compound 5 (5g, 14.6mmol) prepared by the 5th step is dissolved in the mixed solution of methylene chloride and trifluoroacetic acid In 100ml (v:v=10:1), 1h is reacted at room temperature, is spin-dried for removing solvent after reaction, residue vacuum is drained 2h, will be drained Residue afterwards is dissolved in methylene chloride 50ml, is slowly added dropwise containing double amber imide suberate (7.4g, 20mmol) Excess of triethylamine, normal-temperature reaction 3h is added in dichloromethane solution 50ml.After reaction, it is spin-dried for removing solvent, column Chromatographic purification (v methylene chloride: v methanol=1000:1), obtains 6g pale yellow powder compound N BS-1-8C, yield 83%.¹H NMR (400MHz,CDCl₃), 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).¹³C NMR(100MHz,CDCl₃),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 C₂₂H₃₀N₃O₁₀ ⁺[M+H]⁺:496.2.Found: 496.2.

Embodiment 2

Compound 5 (5g, 14.6mmol) in embodiment 1 is dissolved in the mixed solution 100ml of methylene chloride and trifluoroacetic acid (v:v=10:1) in, 1h is reacted at room temperature.It is spin-dried for removing solvent after reaction, residue vacuum drains 2h.It is surplus after draining Excess is dissolved in methylene chloride 50ml, and the dichloromethane containing double amber imide glutarate (6.5g, 20mmol) is slowly added dropwise Excess of triethylamine, normal-temperature reaction 3h is added in alkane 100ml solution.After reaction, it is spin-dried for removing solvent, column Chromatographic purification (v bis- Chloromethanes: v methanol=1000:1), obtain 5.8g pale yellow powder compound N BS-1-5C, yield 88%.¹H NMR(400MHz, CDCl₃), 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)¹³C NMR(100MHz,CDCl₃),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 C₁₉H₂₄N₃O₁₀ ⁺[M+ H]⁺:454.1.Found:454.1.

Embodiment 3

Compound 5 (5g, 14.6mmol) in embodiment 1 is dissolved in the mixed solution 100ml of methylene chloride and trifluoroacetic acid (v:v=10:1) in, 1h is reacted at room temperature, is spin-dried for removing solvent after reaction, residue vacuum drains 2h, surplus after draining Excess is dissolved in methylene chloride 50ml, is slowly added dropwise containing double amber imide suberate sodium sulfonate (11.4g, 20mmol) Excess of triethylamine, normal-temperature reaction 3h is added in methylene chloride 50ml solution.After reaction, it is spin-dried for removing solvent, column Chromatographic purification (v methylene chloride: v methanol=1000:5), obtains 6g pale yellow powder compound Sulfo-NBS-1-8C, yield 69%.¹H NMR (400MHz,CDCl₃), 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)¹³C NMR(100MHz,CDCl₃),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 C₂₂H₂₈N₃O₁₃S^-[M-Na]^-:574.1.Found:574.1.

Embodiment 4

Compound 5 (5g, 14.6mmol) in embodiment 1 is dissolved in the mixed solution 100ml of methylene chloride and trifluoroacetic acid (v:v=10:1) in, 1h is reacted at room temperature.It is spin-dried for removing solvent after reaction, residue vacuum drains 2h.It is surplus after draining Excess is dissolved in methylene chloride 50ml, is slowly added dropwise containing double amber imide glutarate sodium sulfonate (10.6g, 20mmol) Excess of triethylamine, normal-temperature reaction 3h is added in methylene chloride 50ml solution.After reaction, it is spin-dried for removing solvent, column Chromatographic purification (v methylene chloride: v methanol=1000:5), obtains 5.5g pale yellow powder compound Sulfo-NBS-1-5C, yield 68%.¹H NMR(400MHz,CDCl₃), 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).¹³C NMR(100MHz,CDCl₃),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 C₁₉H₂₂N₃O₁₃S^-[M-Na]^-:532.1.Found:532.1.

Embodiment 5

The first step, by compound 4 (5g, 20mmol), 4- bromo butyric acid methyl ester (5.43g, 30mmol) and carbonic acid in embodiment 1 Potassium (6.9g, 50mmol) is dissolved in acetonitrile 250ml, 80 DEG C of reflux 9h, puts board monitoring.After reaction, filtrate is filtered to take, is spin-dried for Solvent is removed, ethyl alcohol recrystallization obtains 4.8g yellow powder compound 6-1, yield 85%.¹H NMR(400MHz,CDCl₃),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)¹³C NMR(100MHz,CDCl₃),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 C₁₃H₁₇NO₇Na[M+Na]⁺:322.1.Found:322.1.

Compound 6-1 (5g, 17.5mmol) prepared by the first step is dissolved in the ethanol water that concentration is 90% by second step In 100ml, it is added potassium hydroxide (1.4g, 25mmol), normal-temperature reaction 15min.After reaction, it is spin-dried for removing solvent, it is water-soluble Solution adjusts pH until 4, generates yellow mercury oxide, filters to take filter cake, obtain 4g yellow powder compound 7-1, yield 84%.¹H 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)¹³C 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 C₁₂H₁₅NO₇K[M+K]⁺:324.0.Found:324.1.

Third step, by second step preparation compound 7-1 (5g, 18.5mmol), hydroxysuccinimide (2.3g, 20mmol) and 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (3.8g, 20mmol) is dissolved in methylene chloride In 100ml, normal-temperature reaction 2h.After reaction, methylene chloride and water extraction, take organic phase, are spin-dried for removing solvent, column chromatography mentions Pure (methylene chloride) obtains 5.5g yellow powder compound N BS-2-4C, yield 81%.¹H NMR(400MHz,CDCl₃),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).¹³C NMR(100MHz,CDCl₃),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 C₁₆H₁₉N₂O₉[M+H]⁺:383.1.Found:383.2.

Embodiment 6

By compound 7-1 (5g, 18.5mmol) in embodiment 5, hydroxysuccinimide sodium sulfonate (4.3g, 20mmol) and 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (3.8g, 20mmol) is dissolved in methylene chloride 100ml, room temperature React 2h.It is spin-dried for removing solvent, ethyl alcohol recrystallization obtains 8g yellow powder compound Sulfo-NBS-2-4C, yield 92%.¹H NMR(400MHz,CDCl₃), 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).¹³C NMR(100MHz,CDCl₃),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 C₁₆H₁₇N₂O₁₂S^-[M-Na]^-:461.1.Found:461.2.

Embodiment 7

The first step, by compound 4 (5g, 20mmol), 8- bromine methyl caprylate (7.1g, 30mmol) and carbonic acid in embodiment 1 Potassium (6.9g, 50mmol) is dissolved in acetonitrile 100ml, 80 DEG C of reflux 9h, puts board monitoring.After reaction, filtrate is filtered to take, is spin-dried for Solvent is removed, ethyl alcohol recrystallization obtains 5.8g yellow powder compound 6-2, yield 85%.¹H NMR(400MHz,CDCl₃),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)¹³C NMR(100MHz,CDCl₃),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 C₁₇H₂₅NO₇Na[M+Na]⁺: 378.2.Found:378.1.

Compound 6-2 (5g, 14.6mmol) prepared by the first step is dissolved in the ethanol water that concentration is 90% by second step In 100ml, it is added potassium hydroxide (1.4g, 25mmol), normal-temperature reaction 15min.After reaction, it is spin-dried for removing solvent, it is water-soluble Solution adjusts pH until 4, generates yellow mercury oxide, filters to take filter cake, obtain 4.2g yellow powder compound 7-2, yield 89%.¹H 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)¹³C 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 C₁₆H₂₄NO₇[M+H]⁺: 342.2.Found:342.1.

Third step, by second step preparation compound 7-2 (4.9g, 15mmol), hydroxysuccinimide (2.3g, 20mmol) and 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (3.8g, 20mmol) is dissolved in methylene chloride In 100ml, normal-temperature reaction 2h.After reaction, methylene chloride water extracts, and takes organic phase, is spin-dried for removing solvent, column Chromatographic purification (methylene chloride) obtains 5.5g yellow powder compound N BS-2-8C, yield 87%.¹H NMR(400MHz,CDCl₃),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).¹³C NMR(100MHz,CDCl₃),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 C₂₀H₂₇N₂O₉[M+H]⁺:439.2.Found:439.2.

Embodiment 8

By compound 7-2 (4.9g, 15mmol) in embodiment 7, hydroxysuccinimide sodium sulfonate (4.3g, 20mmol) and 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (3.8g, 20mmol) is dissolved in methylene chloride 100ml, room temperature React 2h.It is spin-dried for removing solvent, ethyl alcohol recrystallization obtains 6.8g yellow powder compound Sulfo-NBS-2-8C, yield 87%.¹H NMR(400MHz,CDCl₃), 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).¹³C NMR(100MHz,CDCl₃),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 C₂₀H₂₅N₂O₁₂S^-[M-Na]^-:517.1.Found:517.2.

Embodiment 9

The first step, by compound 4 (5g, 20mmol), Isosorbide-5-Nitrae-dibromobutane (8.6g, 40mmol) and carbonic acid in embodiment 1 Potassium (6.9g, 50mmol) is dissolved in acetonitrile 100ml, 80 DEG C of reflux 9h, puts board monitoring.After reaction, filtrate is filtered to take, is spin-dried for Remove solvent, column Chromatographic purification (methylene chloride).Obtain 6g yellow powder compound 10-1, yield 71%.¹H NMR(400MHz, CDCl₃), 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)¹³C NMR(100MHz,CDCl₃),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 C₁₂H₁₇NO₅Br⁺[M+H]⁺:334.0.Found:334.1.

Second step, by the first step preparation compound 10-1 (5g, 15mmol), maleimide (1.9g, 20mmol) and Potassium carbonate (4.1g, 30mmol) is dissolved in dimethylformamide 100ml, 50 DEG C of reflux 3h, puts board monitoring.After reaction, mistake Leaching filtrate is spin-dried for removing solvent, and column Chromatographic purification (v methylene chloride: v methanol=1000:1) obtains 4g pale yellow powder chemical combination Object NBM-4C, yield 76%.¹H NMR(400MHz,CDCl₃),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).¹³C NMR(100MHz,CDCl₃),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 C₁₆H₁₉N₂O₇ ⁺[M+H]⁺:351.1.Found:351.1.

Embodiment 10

The first step, by compound 4 (5g, 20mmol), Isosorbide-5-Nitrae-dibromo-hexane (9.7g, 40mmol) and carbonic acid in embodiment 1 Potassium (6.9g, 50mmol) is dissolved in acetonitrile 100ml, 80 DEG C of reflux 9h, puts board monitoring.After reaction, filtrate is filtered to take, is spin-dried for Solvent is removed, column Chromatographic purification (methylene chloride) obtains 6.5g yellow powder compound 10-2, yield 90%.¹H NMR (400MHz,CDCl₃), 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)¹³C NMR(100MHz,CDCl₃),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 C₁₄H₂₁NO₅Br⁺[M+H]⁺:362.1.Found:362.2.

Second step, by the compound 10-2 (5.4g, 15mmol) of first step preparation, maleimide (1.9g, 20mmol) It is dissolved in dimethylformamide 100ml with potassium carbonate (4.1g, 30mmol), 50 DEG C of reflux 3h, puts board monitoring.After reaction, Filtrate is filtered to take, is spin-dried for removing solvent, column Chromatographic purification (v methylene chloride: v methanol=1000:1) obtains 4.5g pale yellow powder Compound N BM-6C, yield 79%.¹H NMR(400MHz,CDCl₃),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).¹³C NMR(100MHz,CDCl₃),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 C₁₈H₂₃N₂O₇ ⁺[M+H]⁺:379.2.Found:379.1.

Embodiment 11

By compound 10-1 (5g, 15mmol), maleimide sodium sulfonate (4g, 20mmol) and potassium carbonate in embodiment 9 (4.1g, 30mmol) is dissolved in dimethylformamide 100ml, 50 DEG C of reflux 3h, puts board monitoring.After reaction, filter is filtered to take Liquid is spin-dried for removing solvent, and column Chromatographic purification (v methylene chloride: v methanol=1000:5) obtains 5g pale yellow powder compound Sulfo-NBM-4C, yield 74%.¹H NMR(400MHz,CDCl₃),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).¹³C NMR(100MHz,CDCl₃),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 C₁₆H₁₇N₂O₁₀S^-[M-Na]^-:429.1.Found:429.2.

Embodiment 12

By compound 10-2 (5.4g, 15mmol), maleimide sodium sulfonate (4g, 20mmol) and carbonic acid in embodiment 10 Potassium (4.1g, 30mmol) is dissolved in dimethylformamide 100ml, 50 DEG C of reflux 3h, puts board monitoring.After reaction, it filters to take Filtrate is spin-dried for removing solvent, and column Chromatographic purification (v methylene chloride: v methanol=1000:5) obtains 5.5g pale yellow powder compound Sulfo-NBM-6C, yield 76%.¹H NMR(400MHz,CDCl₃),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).¹³C NMR(100MHz,CDCl₃),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 C₁₆H₁₇N₂O₁₀S^-[M-Na]^-:429.1.Found:429.2.

Embodiment 13

Compound 5 (5.1g, 15mmol) in embodiment 1 is dissolved in the mixed solution 100ml of methylene chloride and trifluoroacetic acid (v:v=10:1) in, 1h is reacted at room temperature.It is spin-dried for removing solvent after reaction, residue vacuum drains 2h.It is surplus after draining Excess is dissolved in methylene chloride 50ml, is slowly added dropwise containing 1, the methylene chloride of hexamethylene-diisocyanate (3.4g, 20mmol) is molten Excess of triethylamine, normal-temperature reaction 3h is added in liquid 50ml.After reaction, it is spin-dried for removing solvent, column Chromatographic purification (v dichloromethane Alkane: v methanol=1000:1), obtain 5g pale yellow powder compound N BNCO-6C, yield 70%.¹H NMR(400MHz,CDCl₃),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)¹³C NMR(100MHz,CDCl₃),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 C₁₈H₂₆N₄O₇Na⁺[M+Na]⁺: 433.2.Found:433.1.

Embodiment 14

Compound 5 (5.1g, 15mmol) in embodiment 1 is dissolved in the mixed solution 100ml of methylene chloride and trifluoroacetic acid (v:v=10:1) in, 1h is reacted at room temperature.It is spin-dried for removing solvent after reaction, residue vacuum drains 2h.It is surplus after draining Excess is dissolved in methylene chloride 50ml, and the dichloromethane solution containing the pungent diisocyanate of 1,8- (4g, 20mmol) is slowly added dropwise Excess of triethylamine, normal-temperature reaction 3h is added in 50ml.After reaction, it is spin-dried for removing solvent, column Chromatographic purification (v methylene chloride: v Methanol=1000:1), obtain 6.5g pale yellow powder compound N BNCO-8C, yield 83%.¹H NMR(400MHz,CDCl₃),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)¹³C NMR(100MHz,CDCl₃),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 C₂₀H₃₀N₄O₇Na⁺[M+Na]⁺:461.2.Found:461.3.

Embodiment 15

The first step, by compound 4 (5g, 20mmol), the bromo- 2- of 1- (2- (2- (2- bromine oxethyl) ethyoxyl) in embodiment 1 Ethyoxyl) ethane (9.6g, 30mmol) and potassium carbonate (4.1g, 30mmol) is dissolved in acetonitrile 100ml, 80 DEG C of reflux 9h, contact plate Monitoring.After reaction, filter to take filtrate, be spin-dried for remove solvent, column Chromatographic purification (v methylene chloride: v methanol=1000:5), Obtain 7g yellow transparent liquid compound 11-1, yield 80%.¹H NMR(400MHz,CDCl₃),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).¹³C NMR(100MHz,CDCl₃),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 C₁₆H₂₅O₈NBr⁺[M+H]⁺:438.1.Found:438.2.

Second step, by the first step preparation compound 11-1 (5g, 11mmol), maleimide (1.9g, 20mmol) and Potassium carbonate (4.1g, 30mmol) is dissolved in dimethylformamide 100ml, 50 DEG C of reflux 3h, puts board monitoring.After reaction, mistake Leaching filtrate is spin-dried for removing solvent, and column Chromatographic purification (v methylene chloride: v methanol=1000:1) obtains 4g pale yellow powder chemical combination Object NBPM-4C, yield 80%.¹H NMR(400MHz,CDCl₃),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).¹³C NMR(100MHz,CDCl₃),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 C₂₀H₂₇O₁₀N₂ ⁺[M+H]⁺:455.2.Found:455.2.

Embodiment 16

The first step, by bis- (2- bromine oxethyl) ethane of compound 4 (5g, 20mmol), 1,2- in embodiment 1 (8.3g, It 30mmol) is dissolved in acetonitrile 100ml with potassium carbonate (4.1g, 30mmol), 80 DEG C of reflux 9h, puts board monitoring.After reaction, mistake Leaching filtrate is spin-dried for removing solvent, and column Chromatographic purification (v methylene chloride: v methanol=1000:5) obtains 6.7g yellow transparent liquid Compound 11-2, yield 85%.¹H NMR(400MHz,CDCl₃),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).¹³C NMR(100MHz,CDCl₃),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 C₁₄H₂₁O₇NBr⁺[M+ H]⁺:394.1.Found:394.2.

Second step, by the first step preparation compound 11-2 (5g, 13mmol), maleimide (1.9g, 20mmol) and Potassium carbonate (4.1g, 30mmol) is dissolved in dimethylformamide 100ml, 50 DEG C of reflux 3h, puts board monitoring.After reaction, mistake Leaching filtrate is spin-dried for removing solvent, and column Chromatographic purification (v methylene chloride: v methanol=1000:1) obtains 4.7g pale yellow powder Close object NBPM-3C, yield 89%.¹H NMR(400MHz,CDCl₃),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).¹³C NMR(100MHz,CDCl₃),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 C₁₈H₂₃O₉N₂ ⁺[M+H]⁺:411.1.Found:411.2.

Embodiment 17

The first step, compound N BS-2-4C (5g, 13mmol) prepared by embodiment 5 and N-Boc lysine (4.9g, It 20mmol) is dissolved in dimethylformamide 250ml, point board monitoring reaction, after reaction, is spin-dried for removing solvent, column chromatography mentions Pure (v methylene chloride: v methanol: acetic acid=1000:50:10), obtains 4g yellow liquid compound 12-1, yield 60%.¹H NMR (400MHz,CDCl₃),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).¹³C NMR(100MHz,CDCl₃),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 C₂₃H₃₆N₃O₁₀ ⁺[M+H]⁺:514.2.Found:514.2.

Compound 12-1 (5g, 10mmol) prepared by the first step is dissolved in the mixed of methylene chloride and trifluoroacetic acid by second step It closes in solution 100ml (v:v=10:1), reacts at room temperature 1h.It is spin-dried for removing solvent after reaction, residue vacuum drains 2h. Residue after draining is dissolved in dimethylformamide 100ml, is added NHS- biotin (5.1g, 15mmol), board monitoring is put. After reaction, it is spin-dried for removing solvent, column Chromatographic purification (v methylene chloride: v methanol: acetic acid=1000:80:10) obtains 5g yellow Solid powder chemical combination 13-1, yield 78%.¹H NMR(400MHz,CDCl₃),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).¹³C NMR(100MHz,CDCl₃),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 C₂₈H₄₂N₅O₁₀S⁺[M+H]⁺:640.3.Found:640.3.

Third step, by second step preparation compound 13-1 (5g, 7.8mmol), hydroxysuccinimide (2.3g, 20mmol) and 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (3.8g, 20mmol) is dissolved in methylene chloride In 100ml, normal-temperature reaction 2h.After reaction, methylene chloride water extracts, and takes organic phase, is spin-dried for removing solvent, column Chromatographic purification (v methylene chloride: v methanol :=1000:80), obtains 5g yellow powder compound Tri-lys-NB, yield 87%.¹H NMR (400MHz,CDCl₃),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).¹³C NMR (100MHz,CDCl₃),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 C₃₂H₄₅N₆O₁₂S⁺[M+H]⁺:737.3.Found:737.3.

Optical Response multifunctional chemical crosslinking agent is applied to proteome analysis, specific such as following embodiment:

Embodiment 18

Application in protein interaction:

1) it chooses Streptavidin (SA) and is used as model protein, compound N BS-2-4C prepared by embodiment 5 is dissolved in two Solution (52mg/mL, 136mM) is configured in methyl sulfoxide, commercialization crosslinking agent double amber imide suberate (DSS) is molten It is configured in dimethyl sulfoxide solution (50mg, 136mM), SA solution (1mg/mL) phosphate buffer (10mM, pH= 7.0) it prepares；Sample protein solution, corresponding swimming lane SA.

2) solution that the solution (10uL) (the 1st) of compound N BS-2-4C is formulated is added in SA solution (1mL)), Shaking table, which is protected from light, is incubated for 2h (37 DEG C, 150rad/min), samples protein solution, corresponding swimming lane SA+NBS-2-4C；In SA solution The solution that DSS solution (10uL) (the 1st) is formulated is added in (1mL)), shaking table, which is protected from light, is incubated for 2h (37 DEG C, 150rad/min)；It takes Sample protein solution, corresponding swimming lane SA+DSS.

3) by all of above sampling protein solution in 365nm wavelength, 10mw/cm²Lower illumination 2min, subsequent shaking table, which is protected from light, incubates Educate 2h (37 DEG C, 150rad/min).After, routine SDS-PAGE is carried out, applied sample amount is every swimming lane 10uL, dense using separation gel Degree is 15%.It as a result, therefrom can be with as shown in Figure 1, Fig. 1 is the schematic diagram of the Streptavidin photo-crosslinking electrophoresis in embodiment 18 Find out, Dimer 30KDa is the dimer band that Streptavidin generates under crosslinking action, and Monomer15KDa is strepto- parent With plain monomer band, compound N BS-2-4C prepared by embodiment 5, which is able to achieve, is crosslinked four subunits of Streptavidin Dimer is obtained, effect same with cross liner DS S is commercialized can be reached.

Embodiment 19

Application in protein interaction:

1) it chooses staphylococcus aureus protein A (SPA) and mouse immune globulin G (IgG) is used as model protein, SPA It is the known one group albumen with specificity interaction with IgG.Compound N BS-2-4C prepared by embodiment 5 is dissolved in two Solution (52mg/mL, 136mM) is configured in methyl sulfoxide, commercialization crosslinking agent double amber imide suberate (DSS) is molten It is configured in dimethyl sulfoxide solution (50mg, 136mM), SPA (1mg/mL) solution and IgG (1mg/mL) solution use phosphoric acid Salt buffer (10mM, pH=7.0) is prepared.

2) isometric SPA solution and the mixing of IgG solution are taken, shaking table, which is protected from light, is incubated for 2h (37 DEG C, 150rad/min), sampling Protein solution, corresponding SPA+IgG swimming lane；Mixed liquor 100uL is respectively taken, NBS-2-4C solution and each 10uL of DSS solution are separately added into, Protein solution is sampled, SPA+IgG+NBS-2-4C, SPA+IgG+DSS swimming lane are respectively corresponded.

3) by all of above sampling protein solution in 365nm wavelength, 10mw/cm²Lower illumination 2min, subsequent shaking table, which is protected from light, incubates Educate 2h (37 DEG C, 150rad/min).After, routine SDS-PAGE is carried out, applied sample amount is every swimming lane 10uL, dense using separation gel Degree is 10%.As a result as shown in Fig. 2, Fig. 2 is staphylococcus aureus protein A and mouse immune globulin G in embodiment 19 The electrophoresis schematic diagram of interaction, there it can be seen that Cross-link chain is Staphylococcus aureus under crosslinking action The band that mycoprotein A and mouse immune globulin G crosslinking generates, IgG-Heavy chain 50KDa is mouse immune globulin G Heavy chain band, SPA 33.4KDa is staphylococcus aureus protein A band, and IgG-light chain 25KDa is that mouse is exempted from The light chain bands of epidemic disease Lysozyme, compound N BS-2-4C prepared by embodiment 5 can be crosslinked the albumen of interaction, capture albumen Matter interaction, and the effect of close commercialization cross liner DS S.

Embodiment 20

Application in protein interaction:

1) it chooses staphylococcus aureus protein A (SPA) and mouse immune globulin G (IgG) is used as model protein, SPA It is the known one group albumen with specificity interaction with IgG.Compound N BS-2-4C prepared by embodiment 5 is dissolved in two Solution (52mg/mL, 136mM) is configured in methyl sulfoxide, commercialization crosslinking agent double amber imide suberate (DSS) is molten It is configured in dimethyl sulfoxide solution (50mg, 136mM), SPA (1mg/mL) solution and IgG (1mg/mL) solution use phosphoric acid Salt buffer (10mM, pH=7.0) is prepared.

2) take SPA solution 1mL that compound N BS-2-4C solution 100uL prepared by embodiment 5 is added, shaking table, which is protected from light, is incubated for 2h (37 DEG C, 150rad/min).After, with phosphate buffer dialysis 12h, obtain SPA (NBS-2-4C) solution.It takes isometric saturating SPA (NBS-2-4C) solution and the mixing of IgG solution after analysis, shaking table, which is protected from light, is incubated for 2h (37 DEG C, 150rad/min), and sampling albumen is molten Liquid, corresponding SPA (NBS-2-4C)+IgG (NO hv) swimming lane；Protein solution is sampled, in 365nm wavelength, 10mw/cm²Lower illumination 2min, subsequent shaking table, which is protected from light, is incubated for 2h (37 DEG C, 150rad/min), corresponding SPA (NBS-2-4C)+IgG swimming lane, by embodiment 19 Method, prepare SPA+IgG+NBS-2-4C swimming lane.

3) routine SDS-PAGE is carried out, it is 10% using resolving gel concentration that applied sample amount, which is every swimming lane 10uL,.As a result such as Fig. 3 Shown, Fig. 3 is the electrophoresis signal of the staphylococcus aureus protein A and mouse immune globulin G interaction in embodiment 20 Figure, there it can be seen that Cross-link chain is staphylococcus aureus protein A and mouse immune ball under crosslinking action The band that Protein G crosslinking generates, IgG-Heavy chain 50KDa is the heavy chain band of mouse immune globulin G, SPA/SPA (NBS-2-4C) 33.4KDa is the band that staphylococcus aureus protein A is crosslinked after agent label, IgG-light chain 25KDa is the light chain bands of mouse immune globulin G, and compound N BS-2-4C prepared by embodiment 5 can be crosslinked interaction Albumen, capture protein interaction；By such technical method, may be implemented light to the IgG for being not involved in protein-interacting Chain reduces false positive cross-linking result without crosslinking with this.

The above is only presently preferred embodiments of the present invention, is not intended to limit the present invention in any form, though So the present invention has been disclosed as a preferred embodiment, and however, it is not intended to limit the invention, any technology people for being familiar with this patent Member without departing from the scope of the present invention, when the technology contents using above-mentioned prompt make it is a little change or be modified to The equivalent embodiment of equivalent variations, but anything that does not depart from the technical scheme of the invention content, it is right according to the technical essence of the invention Any simple modification, equivalent change and modification made by above embodiments, in the range of still falling within the present invention program.

Claims (10)

1. a kind of optical Response multifunctional chemical crosslinking agent, it is characterised in that: structure is as shown in general formula I:

Wherein, R₁、R₂、R₃、R₄Separately selected from hydrogen, halogen atom, hydroxyl, sulfydryl, amido, nitro, cyano, aldehyde radical, ketone group, Ester group, amide groups, phosphonic acid base, phosphonate group, sulfonic group, sulfonate group, sulfuryl, sulfoxide group, aryl, heteroaryl, alkyl, alcoxyl Base, alkylidene or modified alkyl；

R ' is selected from one of flowering structure:

R " is selected from biotin and its derivative, alkyl alkene and its derivative, alkyl alkynes and its derivative or hydrogen atom；

Wherein X and R₁、R₂、R₃、R₄In a connection, X is selected from aryl, heteroaryl, alkyl, alkylidene, modified alkyl, modified sub- Alkyl, ehter bond, ester bond, carbonic acid ester bond, amido bond, urea bond and independent assortment described above.

2. optical Response multifunctional chemical crosslinking agent according to claim 1, it is characterised in that: the more function of optical Response The general structure of energy chemical cross-linking agent are as follows:

R ' is selected from one of flowering structure:

R " is hydrogen atom, and X is selected from one of flowering structure:

Wherein n₁It is 1~6 integer, n₂It is 1~10 integer, n₃It is 1~4 integer, n₄It is 1~6 integer, n₅It is 0~6 Integer；

Or, R " is selected from biotin and its derivative, X is with flowering structure:

Wherein n is 2~6 integer.

3. optical Response multifunctional chemical crosslinking agent according to claim 2, it is characterised in that: the more function of optical Response The general structure of energy chemical cross-linking agent are as follows:

N is 1~6 integer in NBS-1, Sulfo-NBS-1,

N is 1~10 integer in NBS-2, Sulfo-NBS-2,

N is 0~6 integer in NBM, Sulfo-NBM,

N is 1~6 integer in NBNCO, and n is 1~4 integer in NBPM,

N is 2~6 integer in Tri-NB.

4. optical Response multifunctional chemical crosslinking agent according to claim 3, it is characterised in that: the more function of optical Response The structure of energy chemical cross-linking agent is with one of flowering structure:

5. a kind of preparation method of the described in any item optical Response multifunctional chemical crosslinking agents of Claims 1-4, feature exist In: the following steps are included:

N is 1~6 integer in NBS-1, Sulfo-NBS-1,

N is 1~6 integer in NBNCO,

Molar ratio is 1:(3~5 by the first step): compound 4, potassium carbonate and the N-Boc bromine ethamine of (1.5~2.5) are dissolved in solvent In, back flow reaction filters to take filtrate after reaction, is spin-dried for removing solvent, column Chromatographic purification obtains compound 5；

Compound 5 prepared by the first step is dissolved in the mixed solution of solvent and excessive trifluoroacetic acid by second step, and room temperature is anti- It answers, is spin-dried for removing solvent after reaction, residue vacuum is drained, the residue after draining is dissolved in solvent, is slowly added dropwise The molar ratio of solution containing double amber imide ester, double amber imide ester and compound 5 is (1~1.5): 1, catalysis is added The triethylamine of amount, normal-temperature reaction are spin-dried for removing solvent, column Chromatographic purification obtains compound N BS-1 after reaction；Or, slow Slow that the solution containing double amber imide ester sodium sulfonate is added dropwise, double amber imide ester sodium sulfonate and the molar ratio of compound 5 are (1~1.5): 1, the triethylamine of catalytic amount is added, normal-temperature reaction is spin-dried for removing solvent, column Chromatographic purification obtains after reaction To compound Sulfo-NBS-1；

Or, the solution containing isocyanates is slowly added dropwise, the molar ratio of isocyanates and compound 5 is (1~1.5): 1, it is added The triethylamine of catalytic amount, normal-temperature reaction are spin-dried for removing solvent, column Chromatographic purification obtains compound N BNCO after reaction；

Or,

N is 1~10 integer in NBS-2, Sulfo-NBS-2,

Molar ratio is 1:(1.1~3 by the first step): compound 4, bromo butyric acid methyl ester and the potassium carbonate of (1.1~4) are dissolved in solvent In, back flow reaction filters to take filtrate after reaction, is spin-dried for removing solvent, ethyl alcohol recrystallization obtains compound 6；

Compound 6 prepared by the first step is dissolved in solvent by second step, and excessive potassium hydroxide normal-temperature reaction, reaction knot is added Shu Hou is spin-dried for removing solvent, and water dissolution adjusts pH until 4, generates yellow mercury oxide, filter to take filter cake, obtain compound 7；

Third step, the molar ratio by second step preparation are 1:(1~1.5): compound 7, the hydroxysuccinimide of (1~1.5) It is dissolved in solvent with 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride, normal-temperature reaction, after reaction, dichloro Methane and water extraction, take organic phase, are spin-dried for removing solvent, column Chromatographic purification obtains compound N BS-2；

Or, being 1:(1~1.5 by the molar ratio of second step preparation): compound 7, the hydroxysuccinimide sulfonic acid of (1~1.5) Sodium and 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride are dissolved in solvent, normal-temperature reaction, after reaction, two Chloromethanes and water extraction, take organic phase, are spin-dried for removing solvent, column Chromatographic purification obtains compound Sulfo-NBS-2；

N is 0~6 integer in NBM, Sulfo-NBM,

Molar ratio is 1:(1.1~3 by the first step): compound 4, dibromoalkane base and the potassium carbonate of (1.1~4) are dissolved in solvent In, back flow reaction filters to take filtrate after reaction, is spin-dried for removing solvent, column Chromatographic purification obtains compound 10；

Second step, the molar ratio by first step preparation are 1:(1.1~3): compound 10, maleimide and the carbon of (1.1~4) Sour potassium is dissolved in solvent, back flow reaction, after reaction, filters to take filtrate, is spin-dried for removing solvent, column Chromatographic purification is changed Close object NBM；

Or, being 1:(1.1~3 by the molar ratio of first step preparation): the compound 10 of (1.1~4), maleimide sodium sulfonate and Potassium carbonate is dissolved in solvent, back flow reaction, after reaction, filters to take filtrate, is spin-dried for removing solvent, column Chromatographic purification obtains Compound Sulfo-NBM；

N is 1~4 integer in NBPM,

Molar ratio is 1:(1.1~3 by the first step): the compound 4 of (1.1~4), two bromo polycondensation ethylene glycol and potassium carbonate are molten In solvent, back flow reaction filters to take filtrate after reaction, is spin-dried for removing solvent, column Chromatographic purification obtains compound 11；

Second step, the molar ratio by first step preparation are 1:(1.1~3): compound 11, maleimide and the carbon of (1.1~4) Sour potassium is dissolved in solvent, back flow reaction, after reaction, filters to take filtrate, is spin-dried for removing solvent, column Chromatographic purification obtains chemical combination Object NBPM；

N is 2~6 integer,

The first step, by molar ratio be 1:(1.1~3) compound N BS-2-4C, N-Boc amino acid be dissolved in solvent, room temperature is anti- It answers, after reaction, is spin-dried for removing solvent, column Chromatographic purification obtains compound 12；

Compound 12 prepared by the first step is dissolved in the mixed solution of solvent and excessive trifluoroacetic acid by second step, and room temperature is anti- It answers, is spin-dried for removing solvent after reaction, residue vacuum drains 2h, the residue after draining is dissolved in solvent, and amber is added The molar ratio of amber acid imide biotin, succinimide biotin and compound 12 is (1.1~3): 1, after reaction, it is spin-dried for Solvent is removed, column Chromatographic purification obtains compound 13；

Third step, the molar ratio by second step preparation are 1:(1.1~3): compound 13, the hydroxysuccinimide of (1.1~3) It is dissolved in solvent with 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride, normal-temperature reaction, after reaction, dichloro Methane and water extraction, take organic phase to be spin-dried for removing solvent, column Chromatographic purification obtains compound Tri-NB.

6. the preparation method of optical Response multifunctional chemical crosslinking agent according to claim 5, it is characterised in that: described double Succinimide ester is double amber imide glutarate, double amber imide glutarate；

The double amber imide ester sodium sulfonate is double amber imide suberate sodium sulfonate, double amber imide glutarate Sodium sulfonate；

The isocyanates is the pungent diisocyanate of 1,8-, hexamethylene diisocyanate；

The bromo butyric acid methyl ester is 4- bromo butyric acid methyl ester, 8- bromine methyl caprylate；

The dibromoalkane base is 1,4- dibromo-hexane, 1,4- dibromobutane；

The two bromos polycondensation ethylene glycol is the bromo- 2- of 1- (2- (2- (2- bromine oxethyl) ethyoxyl) ethyoxyl) ethane, 1,2- is bis- (2- bromine oxethyl) ethane；；

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

7. the preparation method of optical Response multifunctional chemical crosslinking agent according to claim 5, it is characterised in that: describedization Close object 4 preparation method the following steps are included:

Molar ratio is 1:(1.5~3 by the first step): vanillic aldehyde, potassium carbonate and the benzyl bromine of (1.1~1.5) are dissolved in solvent, are returned Stream reaction, reaction terminate filter, be spin-dried for, recrystallize acquisition compound 1；

Second step, 1 grind into powder of compound prepared by the first step are slowly added to excessive nitric acid, ice bath reaction, reaction knot Reaction solution is poured into agitation and filtration in ice water, is recrystallized to give compound 2 by Shu Hou；

Compound 2 prepared by second step is dissolved in excessive trifluoroacetic acid by third step, is reacted at room temperature, after reaction, is spin-dried for Solvent is removed, residue is completely dissolved in sodium hydrate aqueous solution and ethyl acetate mixtures, adjusts pH value to faintly acid, extraction Organic phase is spin-dried for removing solvent, petroleum ether is added in residue, filters to take filter cake, is repeated 3 times, obtain compound 3；

Compound 3 prepared by third step is dissolved in solvent, is slowly added to 1.5~2.5 times of 3 molal quantity of compound by the 4th step Sodium borohydride, room temperature reaction extract organic phase after reaction, adjust pH value to faintly acid, are spin-dried for removing solvent, obtain compound 4。

8. the preparation method of optical Response multifunctional chemical crosslinking agent according to claim 5 or 7, it is characterised in that: institute Stating solvent is dimethylformamide, acetonitrile, methylene chloride, ethyl alcohol, water, methanol, acetone, ethyl acetate.

9. a kind of described in any item optical Response multifunctional chemical crosslinking agents of Claims 1-4 are in protein interaction Application.

10. application of the optical Response multifunctional chemical crosslinking agent according to claim 9 in protein interaction, It is characterized in that: the following steps are included:

First method: the optical Response multifunctional chemical crosslinking agent being added in simple target protein solution and is incubated for, and is used The crosslinking to albumen is realized in 365nm illumination, then carries out protein electrophoresis, protein imprinted, protein cross mass spectral analysis；

Or, second method: the optical Response multifunctional chemical crosslinking agent, which is added to one group, has specificity interaction Protein solution in be incubated for, with 365nm illumination realize crosslinking interaction albumen, capture protein interaction, then into Row protein electrophoresis, protein imprinted, protein cross mass spectral analysis；

Or, third method: the optical Response multifunctional chemical crosslinking agent is added in simple target protein solution and is incubated for, Then it dialyses, obtains the target protein with the optical Response multifunctional chemical crosslinking agent, the photoresponse will be had Property multifunctional chemical crosslinking agent target protein interact therewith albumen incubation, with 365nm illumination realize to albumen phase interaction With pair crosslinking, then carry out protein electrophoresis, protein imprinted, protein cross mass spectral analysis.