Chemical response type cross-linking agent as well as preparation method and application thereof

Abstract

The invention relates to a chemical response type cross-linking agent as well as a preparation method and application thereof. According to the cross-linking agent, two PBDA groups are connected through a polyethylene glycol (PEG) chain, and the light response type chemical cross-linking agent bis-PDBA-PEGn with a novel structure is prepared; and protecting the 2-butene-1, 4-dialdehyde group (BDA) by using an acid response deprotected group to prepare another kind of acid response type chemical cross-linking agent bis-ABDA-PEGn with a novel structure. The cross-linking agent obtained by the invention is novel in structure; volatilization is avoided, and cytotoxicity is low; the reaction yield of the cross-linking agent and biological macromolecules is high, the product is stable, and efficient cross-linking and cell immobilization can be realized by the low-concentration cross-linking agent; the influence on cell morphology and fluorescence imaging is smaller.

Description

Technical Field

[0001] The present invention relates to the field of chemical crosslinking agents, and in particular to a preparation method of an acid-responsive chemical crosslinking agent bis-ABDA-PEGn and a light-responsive chemical crosslinking agent bis-PBDA-PEGn, as well as their applications in the preparation of biomedical materials and cell fixation. Background Art

[0002] Protein cross-linking, modification and labeling are currently common techniques for studying protein structure and interaction. Among them, protein cross-linking through small molecules and analyzing cross-linking sites using cross-linking mass spectrometry have gradually developed into an important method for studying protein structure and interaction. Its advantage is that it is more intuitive than traditional biological methods. Protein cross-linkers are a class of small molecule compounds with two or more reactive ends for special groups (-NH2, -COOH, -HS, -OH, etc.) that can be coupled to two or more proteins respectively. In the 1970s, researchers generally used glutaraldehyde (GA) and paraformaldehyde (PFA) as protein cross-linkers to connect antibodies and indicators (such as enzymes), but their disadvantages are also very obvious: (1) Volatile, toxic and carcinogenic, harmful to human health; (2) Cells shrink and become smaller after fixation, affecting the preservation and observation of cell morphology; (3) They are easily oxidized and acidified, affecting the cross-linking fixation effect of cells; (4) Cross-linking into blocks leads to tissue hardening and brittleness. Recently, Ruedi Aebersold et al. used the amino-specific disuccinimidyl suberate (DSS) small molecule crosslinker to perform crosslink mass spectrometry analysis on the protein phosphatase family and successfully analyzed its interaction network (Science, 2012, 337, 1348-1352.). This work can be regarded as a classic study of protein interactions using chemical crosslinkers. Subsequently, similar small molecule crosslinkers emerged and were commercialized, including DSS series, BS3, DSSO (Anal Bioanal Chem, 2017, 409, 33-44.). These small molecule crosslinkers can achieve heterologous specific crosslinking by introducing different reactive groups, but the crosslinking process is uncontrollable and will bring many false positive results, especially the self-crosslinking of proteins, which greatly limits its application.

[0003] Photoreaction is introduced into the small molecule crosslinker system due to its spatiotemporal controllability, and crosslinking is initiated by ultraviolet light. At present, the conventional photoresponsive groups commonly used in crosslinkers mainly include aryl azide (Mol Biosyst, 2008, 4, 473-480), benzophenone (J Am Chem Soc, 2009, 131, 14240-14242.), and diazirine (Nat Methods, 2005, 2, 261-267.), and many products have been commercialized. However, although this photoresponsive crosslinking realizes spatiotemporal controllability, the reaction mechanism is all through the insertion reaction of free radicals CH or NH, and there is no selectivity for the group, and the reaction site on the protein cannot be clearly defined, thereby losing the specificity of crosslinking. In addition, although most of the photoresponsive crosslinkers have simple structures, the preparation and synthesis of the corresponding derivatives are relatively difficult and unstable, making them expensive (such as diazirine), and the crosslinking efficiency of the three traditional photocrosslinking groups is not high. For example, the UV exposure time required for benzophenone is long, which may cause irreversible damage to biological samples.

[0004] The glutaraldehyde cell fixative (GA) (CN102533710A) commonly used by researchers in the 1970s, and the glyoxal (GO) cell fixative disclosed in recent years all require a high fixation concentration (CN109669032A), with more fluorescence quenching, easy oxidation and acidification resulting in poor fixation effect, and the formation of messy polymers resulting in low cross-linking efficiency. Therefore, Bunz, UHF et al. reported a photocaged glutaraldehyde cell fixative, which is a fixative obtained by protecting the two aldehyde groups of glutaraldehyde with lipophilic photocage protecting groups, and the fixative is non-toxic before photolysis. Cells are treated with the fixative, and ultraviolet irradiation releases active glutaraldehyde, cross-links intracellular proteins in situ, and realizes cell fixation. Compared with the free glutaraldehyde fixative, the tissue background fluorescence is lower after fixation. However, after light excitation, the active group of the photocaged glutaraldehyde fixative is still glutaraldehyde, and the cross-linking reaction with biological macromolecules is reversible. Therefore, a higher concentration is still required to achieve effective cross-linking and cell fixation (AngewChem Int Ed 2017, 56, 1396-1401.).

[0005] Therefore, there is an urgent need and broad application prospects to find a responsive chemical cross-linking agent that has a novel structure, is easy to prepare, has a fast cross-linking speed, high cross-linking efficiency and high chemical selectivity. Summary of the invention

[0006] The purpose of the present invention is to provide a chemical responsive crosslinker and its preparation method and application in view of the defects of the current chemical crosslinkers such as low crosslinking efficiency, low chemical selectivity and high toxicity. The crosslinker connects two PBDA groups with a polyethylene glycol (PEG) chain to prepare a type of novel light-responsive chemical crosslinker bis-PDBA-PEGn; in addition, an acid-responsive deprotected group is used to protect 2-butene-1,4-dialdehyde (BDA) to prepare another type of novel acid-responsive chemical crosslinker bis-ABDA-PEGn. The crosslinker obtained by the present invention has a novel structure; it is non-volatile and has low cytotoxicity; the crosslinker has a high reaction yield with biomacromolecules, the product is stable, and a low concentration of the crosslinker can achieve efficient crosslinking and cell fixation; and the influence on cell morphology and fluorescence imaging is smaller.

[0007] The technical solution of the present invention is:

[0008] A chemical (acid) responsive crosslinker bis-ABDA-PEG n (Formula 1), its chemical structure is:

[0009]

[0010] Wherein, in formula (1), n=2-8.

[0011] The preparation method of the chemical (acid) responsive cross-linking agent comprises the following steps:

[0012] (1) Furfuryl alcohol, sodium bicarbonate and anhydrous methanol are first mixed to form a first mixed solution, and the first mixed solution is reacted with liquid bromine at -75 to -80°C for 1 to 2 hours; then a saturated sodium bicarbonate solution is added to neutralize the system to a pH of 8, the solvent is removed by rotary evaporation, and ABDA is obtained after dissolving with ethyl acetate, washing, drying, filtering and column chromatography;

[0013] Wherein, 1 mmol of furfuryl alcohol, 1.5-2 mmol of sodium bicarbonate, and 1.05-1.6 mmol of liquid bromine are added to every 1-10 mL of anhydrous methanol;

[0014] (2) dissolving the ABDA obtained in step (1) in a first mixed solvent and adding it dropwise to a THF suspension of NaH to form a second mixed solution, reacting the solution at 0°C for 0.5-3 hours, and then mixing the solution with dibromo-PEG n The mixture was reacted at 60-120°C for 12-72 hours; ice water was then added to quench the reaction, DMF and THF were removed by rotary evaporation, and then diluted with ethyl acetate, washed, dried, filtered, and subjected to column chromatography to obtain bis-ABDA-PEGn; wherein 1 mmol of ABDA, 2.5-3 mmol of NaH, and 0.4-0.6 mmol of dibromo-PEG were added to each 1-10 mL of the first mixed solvent.n ; The first mixed solvent is composed of THF:DMF, and the volume ratio of the two is 7:3;

[0015] Chemical (photo) responsive chemical crosslinker bis-PBDA-PEG n (Formula 2), its chemical structure is:

[0016]

[0017] Here, in formula (2), n=4-8.

[0018] The chemical (light) responsive crosslinker bis-PBDA-PEG n A preparation method comprising the following steps:

[0019] (1) Polyethylene glycol, tetrahydrofuran and NaH are mixed to form a fourth mixed solution, the fourth mixed solution is mixed with propargyl bromide and stirred, and the mixture is reacted at 10-25° C. for 1.5-2 hours; ice water is added to quench the reaction, tetrahydrofuran is removed by rotary evaporation, the mixture is dissolved and extracted with ethyl acetate, washed, dried, and the solvent is concentrated, and column chromatography is performed to obtain a yellow oily liquid propargyl-PEG n ;

[0020] Wherein, 1 mmol of polyethylene glycol, 5-6 mmol of NaH, and 5-10 mmol of propargyl bromide were added to every 5-10 mL of THF;

[0021] The polymerization degree of the polyethylene glycol is 4-8.

[0022] (2) The obtained step (1) Alkynyl-PEG n Mix with PBDA-N3, the second mixed solvent, copper sulfate pentahydrate, tris(3-hydroxypropyltriazolemethyl)amine (THPTA) and sodium ascorbate, and react at 10-25° C. for 1.5-2 hours under stirring; remove the solvent by rotary evaporation, dissolve and extract with dichloromethane, wash, dry, concentrate the solvent, purify on a thin layer chromatography plate and further purify by HPLC to obtain a light yellow solid bis-PBDA-PEG n .

[0023] Wherein, 1 mmol of Alkynyl PEG is added to every 5-300 mL of the second mixed solvent. n , 2.5-5 mmol of PBDA-N3, 2.5-5 mmol of copper sulfate pentahydrate, 2.5-5 mmol of tris(3-hydroxypropyltriazolemethyl)amine (THPTA), 5-10 mmol of sodium ascorbate;

[0024] The second mixed solvent is composed of CH3OH and H2O, and the volume ratio of the two is 2:1;

[0025] The stirring speed in both step (1) and step (2) is 300-600 rpm.

[0026] The chemical (acid) responsive crosslinker bis-ABDA-PEG n Applications include preparation of chitosan hydrogel materials in the field of cross-linking of biomacromolecules to prepare biomedical materials; and fixed staining fluorescence imaging of mitochondria in living cells in the field of cell fixation.

[0027] The chemical (light) responsive crosslinker bis-PBDA-PEG n Applications include preparation of chitosan gel materials in the field of cross-linking of biomacromolecules to prepare biomedical materials; and fixed staining fluorescence imaging of mitochondria in living cells in the field of cell fixation.

[0028] The essential features of the present invention are:

[0029] The present invention connects two protected BDA groups through polyethylene glycol (PEG) of different lengths, and is expected to be able to prepare chemical cross-linking agents with different stimulus response activities, achieve efficient and highly specific cross-linking of biomacromolecules containing amino groups, and be able to prepare biomedical materials (such as gels) and fix living cells at lower concentrations (suitable cross-linking agents that can replace traditional fixatives).

[0030] The beneficial effects of the present invention are:

[0031] Acid-responsive chemical crosslinker bis-ABDA-PEG n and the photoresponsive chemical crosslinker bis-PBDA-PEG n After treatment with acid and light, the generated bifunctional groups can react with amino groups quickly and selectively to obtain stable cross-linked products. This type of cross-linker has the characteristics of stimulus response, low toxicity, and can achieve rapid and high cross-linking with low concentrations (bis-ABDA-PEGn cleaved by 1M hydrochloric acid, neutralized with sodium bicarbonate, and the pH value of the mixture adjusted to neutral, and mixed with chitosan by oscillation, can quickly form a non-flowable acid-cured gel within a few minutes ( Figure 1 ); After the photoresponsive chemical crosslinker bis-PBDA-PEGn is mixed and shaken with chitosan, it is illuminated at 365nm for 5-30 minutes, and a non-flowable photocurable gel can be quickly formed within a few minutes to 2 hours ( Figure 2 ); The photoresponsive chemical crosslinker bis-PBDA-PEGn itself is non-cytotoxic. After irradiation at 365nm for 5-7 minutes, it can crosslink the biomacromolecules in the cell to achieve cell fixation ( Figure 4); 100-150μM bis-PBDA-PEGn can effectively fix living cells, and there is no obvious change in cell morphology after fixation. In contrast, traditional 4% paraformaldehyde and 2.5% glutaraldehyde fixation of cells not only causes cell shrinkage, but also increases cell granularity / internal complexity ( Figure 5 ); After fixing cells with bis-PBDA-PEGn, mitochondrial fluorescence staining had a good imaging effect and clear imaging, while the mitochondrial fluorescence staining imaging effect after fixing cells with paraformaldehyde was poor, and the mitochondrial fluorescence staining effect after fixing living cells with glutaraldehyde was extremely poor, and the fluorescence almost completely disappeared ( Figure 6 ). Therefore, bis-ABDA-PEG n and bis-PBDA-PEG n It has advantages and application prospects in the preparation of biomedical materials and living cell fixation staining imaging research. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a diagram showing the effect of preparing chitosan (CMCS) hydrogel using bis-ABDA-PEG3 in Example 3;

[0033] Figure 2 This is a diagram showing the effect of preparing chitosan (CMCS) hydrogel using bis-PBDA-PEG4 in Example 4;

[0034] Figure 3 Comparison of the efficiency of cross-linking histone H3 between the photoresponsive cross-linker bis-PBDA-PEGn and the traditional cross-linkers paraformaldehyde (PFA) and glutaraldehyde (GA).

[0035] Figure 4 Cytotoxicity of the photoresponsive crosslinker bis-PBDA-PEG4.

[0036] Figure 5 The effects of the photoresponsive crosslinker bis-PBDA-PEGn and traditional crosslinkers paraformaldehyde (PFA) and glutaraldehyde (GA) on cell morphology were compared.

[0037] Figure 6 For paraformaldehyde (PFA), glutaraldehyde (GA), bis-PBDA-PEG in Example 8 n Comparison of the effects of post-fixation staining and fluorescence imaging of mitochondria in living cells. DETAILED DESCRIPTION

[0038] The specific embodiments of the present invention are described in detail below. The specific embodiments described here are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0039] Dichloromethane (purchased from Tianjin Binhai New Area Guangshun Da Chemical Reagent Co., Ltd., item number A1040); sodium bicarbonate (purchased from Tianjin Binhai New Area Guangshun Da Chemical Reagent Co., Ltd., item number 2049); tetrahydrofuran (purchased from Beijing Huawei Ruike Chemical Co., Ltd., item number HWMT818767); dissolved acetonitrile (≤10ppm) (purchased from Hebei Dinaxingke Biotechnology Co., Ltd., item number R1012-4); DMSO (cell culture grade) (purchased from Tianjin Nankai District Shengfeier Biotechnology Service Center, item number FZ203); sodium chloride (purchased from Tianjin Chemical Reagent Supply and Marketing Company, item number 017); N,N-dimethylformamide, ultra dry solvent (purchased from Beijing Bailingwei Technology Co., Ltd., item number 983353-1L); methanol (purchased from Beijing =Purchased from Beijing Bailingwei Technology Co., Ltd., item number 980290-500ML); triethylamine (purchased from Beijing Inokai Company, item number T9710); furan (purchased from Grus (Tianjin) Technology Co., Ltd., item number F116393-500ml); n-butyl lithium (purchased from Grus (Tianjin) Technology Co., Ltd., item number B803316-500ml); ethylene oxide (purchased from Grus (Tianjin) Technology Co., Ltd., item number E808842-100ml); sodium hydride (the mass fraction of the active ingredient is 60%, dispersed in liquid paraffin) (purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd., item number S0481); imidazole (purchased from Tianjin Dingguo Biotechnology Co., Ltd., item number DH170-3*BI170-500 G); 2,3-dichloro-5,6-dicyano-1,4-benzoquinone 98+% (DDQ) (purchased from Beijing Inokai Company; item number A11879); N-bromosuccinimide (NBS) (Aladdin, item number B105057-100g); tert-butyldiphenylchlorosilane (TBDPSCl) (purchased from Beijing Bailingwei Technology Co., Ltd., item number SY002185); tetra-n-butylammonium fluoride (purchased from Tianjin Xiensiopude Technology Co., Ltd., item number T-21412); 2-azido-1,3-dimethylimidazolium hexafluorophosphate (ADMP) (purchased from Tianjin Chemical Industry Development Co., Ltd., item number A2457); 1,8-diazabicyclo[5.4.0] Undec-7-ene (DBU) (purchased from Gruss (Tianjin) Technology Co., Ltd., Catalog No. 75338C); copper sulfate pentahydrate (Shanghai MacLean Biochemical Technology Co., Ltd., C805356-100g); sodium ascorbate (Shanghai MacLean Biochemical Technology Co., Ltd., Catalog No. S817635-100g); tris (3-hydroxypropyltriazolemethyl) amine (THPTA) (purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd., Catalog No. T3171); furfuryl alcohol (purchased from Beijing Inotech Technology Co., Ltd., Catalog No. 119790010); liquid bromine (purchased from Amethyst, product number 931848); 1,2-bis(2-bromoethoxy)ethane (Bromo-PEG2-bromide) (purchased from Gruss (Tianjin) Technology Co., Ltd., product number BD760047); 1-bromo-2-(2-(2-(2-bromoethoxy)ethoxy)ethoxy)ethane (Bromo-PEG3-bromide) (purchased from Gruss (Tianjin) Technology Co., Ltd., product number BD760048) and other raw materials are commercially available. .

[0040] Example 1

[0041] Synthesis of acid-responsive chemical crosslinkers bis-ABDA-PEG3(5) and bis-ABDA-PEG4(6).

[0042] The synthesis steps are as follows:

[0043]

[0044] Step 1, preparation of compound ABDA (4): Under stirring (500 rpm. Same as in the following examples) and argon protection, 14.72 g (150 mmol, 1 eq.) of furfuryl alcohol (3) and 18.91 g of sodium bicarbonate (225.1 mmol, 1.5 eq.) were weighed into a round-bottom flask, 150 mL of dry methanol was added to dissolve, and the mixture was cooled to -78°C. 60 mL of a methanol solution of liquid bromine (containing 13.0 mL, 195.1 mmol, 1.3 eq.) was slowly added dropwise to the mixed solution, and the solution was completely added dropwise in about 45 minutes, and then the reaction was continued at -78°C for 2 hours. The reaction system was neutralized with a saturated sodium bicarbonate solution to a pH of 8, and the methanol was removed by rotary evaporation. 150 mL of ethyl acetate was added to dissolve the diluted product, and the product was washed with 150 mL of a saturated sodium bicarbonate solution and 150 mL of a saturated sodium chloride solution, respectively. The organic phase was dried over anhydrous sodium sulfate, the solvent was removed by rotary evaporation, and ethyl acetate / petroleum ether (v / v, 1:5) column chromatography was performed to obtain 22.3 g (139.2 mmol, yield 92.8 mol mass%) of light yellow oily liquid ABDA (4) (Acid-liable 2-Butene-1,4-dial, referred to as ABDA). Step 2, preparation of compound (bis-ABDA-PEG3): ABDA (4) (5.0 g, 31.22 mmol, 1 eq.) was taken into a round-bottom flask, dissolved in 190 mL of a mixed solution of THF and DMF (volume ratio of 7:3), and then added dropwise to a suspension of sodium hydride (containing 3.75 g, 93.66 mmol, 3 eq.) in tetrahydrofuran. The mixture was stirred at room temperature for 30 minutes. Dibromo-PEG2 (4.31 g, 15.61 mmol, 0.5 eq.) was then added, and the resulting solution was stirred at 80°C for 24 hours. The solvent was removed by rotary evaporation, and the mixture was diluted with 300 mL of ethyl acetate and washed twice with 300 mL of saturated NH4Cl solution and water, respectively. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The crude product was adsorbed on silica gel column chromatography and purified using CH2Cl2 / CH3OH (v / v, 100 / 1-50 / 1) as eluent to obtain 1.85 g (4.26 mmol, 13.6 mol mass%) of light yellow oily liquid bis-ABDA-PEG3(5).

[0045] 1 H NMR (400MHz,CDCl3)δ(ppm): 1 H NMR (400MHz, CDCl3) δ (ppm): 6.09-5.95 (m, 4H), 5.74-5.46 (s, 2H), 3.70-3.54 (m, 14H), 3.48-3.39 (m, 8H), 3.20-3.12 (s, 6H). 13C NMR (100.4MHz,CDCl3)δ(ppm): 13 C NMR(100.4MHz, CDCl3)(ppm):131.9,113.5,112.7,108.4,107.3,75.9,75.0,7 1.4,71.3,70.6,70.5,69.6,56.4,56.2,55.3,50.5,50.3,49.9.ESI-MS:[M+Na] + ,457.2050,found,457.2081.Through NMR and mass spectrometry identification, the target product, acid-responsive chemical cross-linker bis-ABDA-PEG3(5), was obtained.

[0046] In addition (parallel experiment of step 2), preparation of compound (bis-ABDA-PEG4): ABDA (5.0 g, 31.22 mmol, 1 eq.) was taken into a round-bottom flask, dissolved with 190 mL of a mixed solution of tetrahydrofuran and N,N-dimethylformamide (7:3), and then added dropwise to a suspension of sodium hydride (3.75 g, 93.66 mmol, 3 eq.) in tetrahydrofuran. The mixture was stirred at room temperature for 30 minutes. Dibromo-PEG3 (5.0 g, 15.61 mmol, 0.5 eq.) was then added, and the resulting solution was stirred at 80°C for 24 hours. The solvent was removed by rotary evaporation, and the diluted mixture was dissolved with 300 mL of ethyl acetate, and washed with 300 mL of saturated ammonium chloride solution and water, respectively. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The crude product was adsorbed on silica gel and purified by column chromatography using CH2Cl2 / CH3OH (v / v, 100 / 1-50 / 1) as eluent to obtain 1.65 g (3.45 mmol, 11.1 mol% ) of slightly yellowish oily liquid bis-ABDA-PEG4(6).

[0047] 1 H NMR (400MHz, CDCl3) δ (ppm): 6.10-6.01 (m, 4H), 5.79-5.51 (s, 2H), 3.73-3.63 (m, 18H), 3.51-3.43 (s, 8H), 3.22-3.16 (s, 6H). 13 C NMR (100.4MHz, CDCl3) δ (ppm) 132.5, 113.4, 112.6, 108.3, 107.2, 75.7, 74.8, 71.3, 71.2, 70.5, 70.4, 56.1, 55.1, 50.1, 49.7.ESI-MS: [M+Na] +,501.2312,found,501.2346.(The target product, acid-responsive chemical crosslinker bis-ABDA-PEG4(6), was obtained through NMR and mass spectrometry identification.)

[0048] Example 2

[0049] Synthesis of photoresponsive chemical cross-linkers bis-PBDA-PEG4 and bis-PBDA-PEG8.

[0050] The synthesis steps are as follows:

[0051]

[0052] Step 1, preparation of compound Alkynyl PEG4 (8): Weigh NaH (containing 1.05 mg, 25.75 mmol, 5.0 eq. of sodium hydride) and place it in a round-bottom flask, add 15 mL of dry tetrahydrofuran, mix well and cool to 0°C. Dissolve tetraethylene glycol (n represents the degree of polymerization of ethylene glycol or the length of the ethylene glycol chain. The common name of polyethylene glycol with n=4 is tetraethylene glycol) (1.0 g, 5.15 mmol, 1.0 eq.) in 15 mL of dry tetrahydrofuran, and then add it dropwise to the above-mentioned tetrahydrofuran suspension of NaH in an argon atmosphere. After the addition is completed (10 minutes), warm to room temperature and continue stirring for 30 minutes. Then add propargyl bromide (2.22 mL, 25.75 mmol, 5.0 eq.) to the above reaction system and stir at room temperature for 5 hours. Add water to quench the reaction, remove tetrahydrofuran by rotary evaporation, add ethyl acetate to dissolve and extract, and wash with saturated sodium chloride solution. The organic phases were combined and dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (EtOAc / PE=1 / 4-1 / 3-1 / 2) to obtain 1.25 g (4.62 mmol, yield 90 mol mass %) of Alkynyl PEG4(8). f =0.35 (EtOAc / PE=1 / 2).

[0053] 1 H NMR (400MHz, CDCl3) δ (ppm): 4.13 (s, 4H), 3.61 (s, 8H), 3.59 (s, 8H), 2.43 (s, 2H). 13 C NMR (100.4MHz, CDCl3) (ppm): 79.3, 74.3, 70.2, 70.0, 68.7, 57.9.

[0054] Step 2, preparation of compound bis-PBDA-PEG4 (10): Compound 8 (40 mg, 148.1 μmol, 1.0 eq.) synthesized in the previous step was dissolved in 20 mL CH3OH and 10 mL H2O, 207.8 mg (370.3 μmol, 2.5 eq.) of PBDA-N3 (9), 93.0 mg (370.3 μmol, 2.5 eq.) of copper sulfate pentahydrate, 160.9 mg (370.3 μmol, 2.5 eq.) of tris(3-hydroxypropyltriazolemethyl)amine (THPTA) and 146.7 mg (740.5 μmol, 5 eq.) of sodium ascorbate were added, and the mixture was stirred at room temperature overnight. The solvent was removed by rotary evaporation, CH2Cl2 was added to dissolve and extract, saturated NaCl solution was added to wash, the organic phases were combined and dried, and CH2Cl2 was removed by concentration, and then separated. The light yellow solid bis-PBDA-PEG4(10) 1.25 g (4.62 mmol, yield 90 mol% was obtained by high pressure preparative liquid separation. Liquid phase conditions: C18, 5 μm, 21.2 mm×250 mm, flow rate 10 mL / min. Mobile phase A: water; B: acetonitrile. 10 min, B: 5%-30%, 30 min, 30%-55%, 10 min, 55%-75%. RT = 52-54 min.

[0055] 1 H NMR (400MHz, CDCl3) δ (ppm): 7.74 (s, 2H), 7.70 (s, 2H), 7.65 (s, 1H), 7.21-7.17 (m, 4H), 6.26 (s, 2H), 5.97 (s, 2H), 5.82(s,2H),5.09-4.67(m,16H),4.00(s,12H),3.94(s,6H),3.88(s,6H),3.65(m,16H),2.63(m,2H),2.55(m,2H). 13 C NMR(100.4MHz, CDCl3)(ppm):153.6,153.3,147.4,147.1,144.9,138.7,138.6,132.2,131.8,130.7,129.2,122.6,1 12.1,109.6,109.4,107.5,107.2,106.0,70.2,69.4,67.7,64.3,61.9,56.2,56.0,55.9,45.4,39.4.ESI-MS:[M+Na] + ,cal.1415.4779,found 1415.4797.(The target product, photoresponsive chemical crosslinker bis-PBDA-PEG4(10), was obtained by NMR and mass spectrometry identification.)

[0056] In addition, the preparation of compound Alkynyl PEG8 (12) (parallel experiment of step 1): weigh 270.0 mg (6.75 mmol, 5 eq.) of NaH in a round-bottom flask, add 15 mL of dry tetrahydrofuran, and cool to 0°C in an ice bath. Dissolve 500.0 mg (1.35 mmol, 1.0 eq.) of octaethylene glycol in 15 mL of dry THF, and then add dropwise to the above suspension of NaH in tetrahydrofuran. After the addition is complete, stir for 15 minutes, then warm to room temperature and continue stirring for 30 minutes. Add propargyl bromide (581.9 μL, 6.75 mmol, 5.0 eq.) to the above reaction system and stir at room temperature for 5 hours. Add H2O to quench, remove tetrahydrofuran by rotary evaporation, add ethyl acetate to dissolve, wash with saturated sodium chloride solution, combine and dry the organic phases, concentrate the solvent, and separate by column chromatography (EtOAc / PE=1 / 4-1 / 3-1 / 2) to obtain 517.7 mg (1.16 mmol, yield 86 mol mass%) of the product Alkynyl PEG8(12). f =0.2 (EtOAc / PE=1 / 2).

[0057] 1 H NMR (400MHz, CD3OD) δ (ppm): 4.19 (d, J = 1.73Hz, 4H), 3.65 (s, 10H), 3.63 (s, 12H), 2.93 (s, 2H). 13 C NMR (100.4MHz, CD3OD) (ppm):79.7,75.1,70.3,70.1,68.8,57.8,48.5,48.3,48.1,47.9,47.7,47.5,47.2.

[0058] Preparation of compound bis-PBDA-PEG8 (13) (parallel experiment of step 2): 1240 mg (89.64 μmol, 1.0 eq.) of the compound synthesized in the previous step was dissolved in 20 mL CH3OH and 10 mL H2O, and 130.8 mg (224.10 μmol, 2.5 eq.) of PBDA-N3, 56.0 mg (224.10 μmol, 2.5 eq.) of CuSO4·5H2O, 97.4 mg (224.10 μmol, 2.5 eq.) of THPTA and 88.8 mg (448.20 μmol, 5.0 eq.) of sodium ascorbate were added, and stirred at room temperature overnight. The solvent was removed by rotary evaporation, CH2Cl2 was added to dissolve and extract, and saturated NaCl solution was added to wash, the organic phases were combined and dried, and the solvent was concentrated for separation. The bis-PBDA-PEG8(13) of slightly pale yellow liquid (119.5 mg, 76.19 μmol, yield 85 mol mass%) was obtained by separation using a high pressure preparative liquid C18 column.

[0059] 1 H NMR (400MHz, CDCl3) δ (ppm): 7.65 (s, 4H), 7.48 (s, 2H), 7.20 (s, 2H), 7.17 (s, 2H), 6.24 (s, 2H), 5.94 (s, 2H), 5.80 (s, 2H), 5.06-5. 02(m,2H),4.98-4.74(m,6H),4.67-4.61(m,8H),3.98(s,12H),3.93(s,6H),3.87(s,6H),3.63(m,32H),2.61(m,2H),2.51(m,2H). 13 C NMR(100.4MHz, CDCl3)(ppm):153.7,153.4,147.5,147.2,145.0,138.8,138.7,132.3,131.9,130.8,129.2,122.7,112 .2,109.6,109.5,107.6,107.3,106.1,70.3,69.5,67.8,64.4,62.0,56.3,56.1,56.0,45.5,39.5,29.5.ESI-MS:[M+Na] + ,cal.1591.5828,found 1591.5887.(The target product, photoresponsive chemical crosslinker bis-PBDA-PEG8(13), was obtained by NMR and mass spectrometry.)

[0060] Example 3

[0061] Acid-responsive chemical crosslinker bis-ABDA-PEGn Application in the preparation of chitosan (CMCS) hydrogel.

[0062] Taking bis-ABDA-PEG3 as an example, the preparation steps are as follows:

[0063] Take 100 μL of bis-ABDA-PEG3 DMSO solution (51.4 mg / mL), add 1 mL of 1 M HCl, and leave at room temperature for 2 hours to remove the acid-responsive protecting group on ABDA. Then add saturated sodium bicarbonate solution to neutralize to neutrality.

[0064] Another 50 mg of chitosan (CMCS) was dissolved in 0.9 mL of H2O and shaken for 1 hour to prepare a 50 mg / mL chitosan aqueous solution.

[0065] The above two solutions were mixed and incubated at 37°C for 20 minutes to obtain 5% (w / v) chitosan hydrogel ( Figure 1 ). Figure 1 It shows that the acid-responsive chemical crosslinker bis-ABDA-PEG3 cannot form a gel after being directly mixed with chitosan. After bis-ABDA-PEGn is acid-lyzed with HCl and neutralized with NaHCO3, it can cross-link with chitosan and form a non-flowable acid-cured gel in 0.5-30 minutes.

[0066] Example 4

[0067] Photoresponsive chemical crosslinker bis-PBDA-PEG n Application in the preparation of chitosan (CMCS) hydrogel.

[0068] Taking bis-PBDA-PEG4 as an example, the preparation steps are as follows:

[0069] Take 50 mg of chitosan (CMCS) and dissolve it in 1 mL of H2O.

[0070] Separately, 100 μL of a DMSO solution of bis-PBDA-PEG4 (50 mg / mL) was prepared.

[0071] After the two solutions were mixed evenly by oscillation, they were irradiated with ultraviolet light (365 nm, 7 mW cm -2 ) for 5 minutes to remove the photoresponsive protective group on PBDA. After incubation at 37°C for 2 hours, a 5% (w / v) chitosan hydrogel ( Figure 2 ). Figure 2It shows that the photoresponsive chemical crosslinker bis-PBDA-PEG4 cannot form a gel after direct oscillation mixing with chitosan. After being irradiated with ultraviolet light at 365nm for 5-60 minutes, it can be photo-crosslinked with chitosan and a non-flowable photocurable gel can be formed in 0.5-120 minutes.

[0072] Example 5

[0073] Application of photoresponsive chemical crosslinker bis-PBDA-PEGn in protein crosslinking.

[0074] Different concentrations of photoresponsive chemical crosslinkers bis-PBDA-PEG4, bis-PBDA-PEG8, paraformaldehyde (PFA), glutaraldehyde (GA) were mixed with histone H3 (65 μM) in a buffer solution (20 mM PBS, pH 7.4, 50 mM NaCl). After irradiation at 360 nm for 10 minutes, the cells were incubated at 37 ° C for 2 hours. 15% SDS-PAGE gel analysis was performed, and the percentage of remaining uncrosslinked histone H3 was plotted against the equivalent ratio of crosslinker to histone H3 to obtain Figure 3 . Figure 3 The results showed that the photoresponsive chemical crosslinker bis-PBDA-PEGn (n=4 and 8) had a significantly higher crosslinking efficiency for histone H3 than the traditional crosslinkers paraformaldehyde (PFA) and glutaraldehyde (GA) after light excitation.

[0075] Example 6 Cytotoxicity of photoresponsive chemical crosslinker bis-PBDA-PEG4.

[0076] Ten thousand Hela cells were seeded in a cell culture plate (24-96 well plate) and cultured overnight in a 37°C, 5% CO2 incubator for 24 hours;

[0077] Different concentrations of bis-PBDA-PEG4 were added to Hela cells and incubated at 37°C for 10-12 h;

[0078] In addition, different concentrations of bis-PBDA-PEG4 were irradiated at 365 nm for 7 min and then added to Hela cells, and the cells were incubated at 37 °C for 10-12 h;

[0079] CCK-8 kit was used to detect cell viability ( Figure 4 ). Figure 4It shows that for the non-illumination group, as the concentration of bis-PBDA-PEG4 gradually increases, the cell survival rate is not affected, indicating that bis-PBDA-PEG4 itself is non-cytotoxic. For the illumination group, as the concentration of bis-PBDA-PEG4 gradually increases, the cell survival rate gradually decreases. When the concentration is around 150μM, the cell survival rate is less than 20%. This shows that after light activation, 150μM bi-PBDA-PEGn can efficiently cross-link the biomacromolecules in the cell, leading to cell death.

[0080] Example 7

[0081] Effect of photoresponsive chemical crosslinker bis-PBDA-PEGn on cell morphology after cell fixation.

[0082] HeLa cells (2 million) were cultured overnight in 12-well tissue culture plates (Nest). Subsequently, cells were fixed using one of the following methods: 4% (w / v) paraformaldehyde fixation for 15 minutes, 2% (w / v) glutaraldehyde fixation for 20 minutes, or bis-PBDA-PEG4 / bis-PBDA-PEG8 (130 μM, irradiated for 5 minutes and incubated for 2 hours). After fixation, cells were collected by EDTA-free trypsin digestion. The fixed cells were washed twice with 2 mL of pre-cooled 1× PBS (centrifuged at 4000 rpm for 5 minutes at 4°C). After discarding the PBS, the cells were washed once with 1 mL of binding buffer and resuspended in 500 μL of binding buffer. 500 μL of cell suspension was transferred to a 5 mL flow tube, 5 μL of Annexin V-FITC was added and incubated for 5 minutes, followed by 5 μL of PI. The sample was gently pipetted to mix, incubated at room temperature in the dark for 5 minutes, and immediately analyzed using a flow cytometer (BD, USA) ( Figure 5 ).

[0083] High concentrations of PFA (4%, w / v, about 1.33 M) and GA (2.5%, w / v, about 0.25 M) are usually used for cell fixation. Figure 5 As shown, 4% PFA (paraformaldehyde) induced cell shrinkage, while 2.5% GA (glutaraldehyde) not only caused cell shrinkage but also increased cell granularity / internal complexity. In contrast, fixation with 130 μM photoexcited bis-PBDA-PEG4 and bis-PBDA-PEG8 did not significantly change cell size and intracellular structure, even when the concentration of bis-PBDA-PEG4 was increased to 1300 μM.

[0084] Example 8

[0085] The photoresponsive chemical crosslinker bis-PBDA-PEGn was used for fluorescence imaging of living cells after fixation.

[0086] The steps for fixing mitochondria in living cells are as follows:

[0087] (1) Hela cells were seeded in a confocal dish and cultured overnight in a 37°C, 5% CO2 incubator for 24 hours; (2) The culture medium was removed and 1 mL of PBS was added to each well to wash the cells twice, each time for 5 minutes; (3) The cells were stained for mitochondria by adding 1 mL of freshly prepared mitochondrial red dye (100 nM) to each well and incubating the cells in an incubator for 30 minutes; (4) The mitochondrial red dye was removed and the cells were washed three times with PBS, each time for 5 minutes; (5) 1 mL of 4% paraformaldehyde (1.2 M) was added to the confocal dish to fix the cells for 20 minutes; 1 mL of 2% glutaraldehyde (200 mM) was added to fix the cells for 30 minutes; and 1 mL of Bis-PBDA-PEG was added to the confocal dish to fix the cells for 20 minutes. n The cells were fixed with photolysis buffer (final concentration 150 μM, 350 nm illumination for 7 min) for 1 h; (6) paraformaldehyde, glutaraldehyde and di-PBDA-PEGn (n=4 and 8) were discarded respectively, and the cells were washed three times with PBS; (7) the cells were immediately placed under a laser confocal microscope for live cell imaging (excitation light 579 nm, emission light 599 nm) ( Figure 6 ). Figure 6 The comparison of fluorescence intensity before and after fixation of mitochondria in living cells by 4% paraformaldehyde, 2.5% glutaraldehyde, photocleaved bis-PBDA-PEG4 and bis-PBDA-PEG8 is shown. The experimental results show that the concentration of photocleaved bis-PBDA-PEG4 and bis-PBDA-PEG8 used to fix mitochondria in living cells is lower, 100-150μM can achieve cell fixation, and the fluorescence intensity of mitochondrial imaging is stronger, with less fluorescence quenching; while the molar concentrations of 4% paraformaldehyde and 2-2.5% glutaraldehyde commonly used in biology and medicine are higher, corresponding to 1.2M and 200mM, respectively, but the fluorescence intensity of cell mitochondrial fluorescence imaging is weaker, with more fluorescence quenching.

[0088] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

[0089] Matters not covered by the present invention are known technologies.

Claims

1. A chemical (acid) responsive cross-linking agent, characterized in that the chemical structural formula of the cross-linking agent is: in, In formula (1), n=2-8.

2. The method for preparing a chemical (acid) responsive cross-linking agent as claimed in claim 1, characterized in that the method comprises the following steps: (1) Furfuryl alcohol, sodium bicarbonate and anhydrous methanol are first mixed to form a first mixed solution, and the first mixed solution is reacted with liquid bromine at -75 to -80°C for 1 to 2 hours; then a saturated sodium bicarbonate solution is added to neutralize the system to a pH of 8, and the system is subjected to rotary evaporation, dissolved in ethyl acetate, washed, dried, filtered, and subjected to column chromatography to obtain ABDA; Wherein, 1 mmol of furfuryl alcohol, 1.5-2 mmol of sodium bicarbonate, and 1.05-1.6 mmol of liquid bromine are added to every 1-10 mL of anhydrous methanol; (2) dissolving the ABDA obtained in step (1) in a first mixed solvent and adding it dropwise to a THF suspension of NaH to form a second mixed solution, reacting the solution at 0°C for 0.5-3 hours, and then mixing the solution with dibromo-PEG n React at 60-120°C for 12-72 hours; then add ice water to quench the reaction, rotary evaporate, dilute with ethyl acetate, wash, dry, filter, and column chromatograph to obtain bis-ABDA-PEGn; Wherein, 1 mmol of ABDA, 2.5-3 mmol of NaH, and 0.4-0.6 mmol of dibromo-PEG are added to every 1-10 mL of the first mixed solvent. n ; The composition of the first mixed solvent is THF:DMF, and the volume ratio of the two is 7:

3.

3. A chemical (light) responsive crosslinking agent, characterized in that: The structural formula of the crosslinking agent is: Here, in formula (2), n=4-8.

4. The method for preparing the light-responsive chemical crosslinking agent according to claim 3, characterized in that: The method comprises the following steps: (1) Polyethylene glycol, tetrahydrofuran and NaH are mixed to form a fourth mixed solution, the fourth mixed solution is mixed with propargyl bromide and stirred, and reacted at 10-25° C. for 1.5-2 hours; ice water is added to quench the reaction, tetrahydrofuran is removed by rotary evaporation, ethyl acetate is used for dissolution and extraction, washing, drying, concentrating the solvent, and column chromatography is performed to obtain a yellow oily liquid propargyl-PEG n ; Wherein, 1 mmol of polyethylene glycol, 5-6 mmol of NaH, and 5-10 mmol of propargyl bromide were added to every 5-10 mL of THF; (2) The Alkynyl-PEG obtained in step (1) n Mix with PBDA-N3, the second mixed solvent, copper sulfate pentahydrate, tris(3-hydroxypropyltriazolemethyl)amine (THPTA) and sodium ascorbate, and react at 10-25° C. for 1.5-2 hours under stirring; remove the solvent by rotary evaporation, dissolve and extract with dichloromethane, wash, dry, concentrate the solvent, purify on a thin layer chromatography plate and further purify by HPLC to obtain a light yellow solid bis-PBDA-PEG n ; Wherein, 1 mmol of Alkynyl PEG is added to every 5-300 mL of the second mixed solvent. n , 2.5-5 mmol of PBDA-N3, 2.5-5 mmol of copper sulfate pentahydrate, 2.5-5 mmol of tris(3-hydroxypropyltriazolemethyl)amine (THPTA), 5-10 mmol of sodium ascorbate; The second mixed solvent is composed of CH3OH and H2O, and the volume ratio of the two is 2:

1.

5. The method for preparing the light-responsive chemical crosslinking agent according to claim 3, characterized in that: The stirring speed in both step (1) and step (2) is 300-600 rpm.

6. The method for preparing the light-responsive chemical crosslinking agent according to claim 3, characterized in that: The polymerization degree of the polyethylene glycol is 4-8.

7. The use of the chemically responsive cross-linking agent as claimed in claim 1, characterized in that: It is used in the preparation of chitosan hydrogel materials in the field of cross-linking of biomacromolecules to prepare biomedical materials; in the field of cell fixation, it is used for fixation staining and fluorescence imaging of mitochondria in living cells.

8. The use of the photoresponsive chemical crosslinking agent as claimed in claim 3, characterized in that: It is used in the preparation of chitosan gel materials in the field of cross-linking of biomacromolecules to prepare biomedical materials; in the field of cell fixation, it is used for fixation staining and fluorescence imaging of mitochondria in living cells.

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