Chemically responsive crosslinker and preparation method and application thereof

Abstract

The application discloses a chemical response type crosslinking agent and a preparation method and application thereof. The crosslinking agent is connected with two PBDA groups by a polyethylene glycol (PEG) chain, and a novel light response type chemical crosslinking agent bis-PDBA-PEGn is prepared; and then, an acid response deprotection group is applied to protect 2-buten-1,4-dialdehyde (BDA), and another novel acid response type chemical crosslinking agent bis-ABDA-PEGn is prepared. The crosslinking agent obtained by the application has a novel structure, is non-volatile, has low cytotoxicity, has high reaction yield with biological macromolecules, has stable products, can realize efficient crosslinking and cell fixation at a low concentration of the crosslinking agent, and has less influence on cell morphology and fluorescence imaging.

Description

Technical Field

[0001] This invention relates to the field of chemical crosslinking agents, specifically to a method for preparing an acid-responsive chemical crosslinking agent bis-ABDA-PEGn and a photoresponsive chemical crosslinking agent bis-PBDA-PEGn, and their applications in the preparation of biomedical materials and cell fixation. Background Technology

[0002] Protein cross-linking, modification, and labeling are commonly used techniques for studying protein structure and interactions. Among them, cross-linking proteins with small molecules and using cross-linking mass spectrometry to resolve cross-linking sites has gradually become an important method for studying protein structure and interactions, and its advantages are more intuitive than traditional biological methods. Protein cross-linking agents are a class of small molecule compounds with two or more reactive ends targeting specific groups (-NH2, -COOH, -HS, -OH, etc.), which can be coupled to two or more proteins respectively. In the 1970s, researchers generally used glutaraldehyde (GA) and paraformaldehyde (PFA) as protein cross-linking agents to link antibodies and indicators (such as enzymes), but their disadvantages are also very obvious: (1) volatility, toxicity, carcinogenicity, and harm to human health; (2) shrinkage after cell fixation, resulting in smaller volume, affecting cell morphology preservation and observation; (3) easy oxidation and acidification, affecting the cross-linking and fixation effect of cells; (4) cross-linking into clumps, leading to tissue hardening and brittleness. Recently, Ruedi Aebersold et al. used amino-specific bis(succinimide) octanoate (DSS) small molecule cross-linking agents to perform cross-linking mass spectrometry analysis on the protein phosphatase family, successfully resolving their interaction network (Science, 2012, 337, 1348-1352). This work is considered a classic example of using chemical cross-linking agents to study protein interactions. Subsequently, similar small molecule cross-linking agents emerged and were commercialized, including the DSS series, BS3, and DSSO (Anal Bioanal Chem, 2017, 409, 33-44). These small molecule cross-linking agents can achieve heterologous specific cross-linking by introducing different reactive groups; however, the cross-linking process is uncontrollable and can lead to many false positive results, especially in cases of protein self-cross-linking, which greatly limits their application.

[0003] Photoreaction, due to its spatiotemporal controllability, has been introduced into small molecule crosslinking agent systems, with crosslinking initiated by ultraviolet light. Currently, the commonly used photoresponsive groups applied to crosslinking agents are mainly aryl azides (Mol Biosyst, 2008, 4, 473-480), benzophenone (J Am Chem Soc, 2009, 131, 14240-14242.), and diacylpropidine (Nat Methods, 2005, 2, 261-267.), many of which have been commercialized. However, although this photoresponsive crosslinking achieves spatiotemporal controllability, the reaction mechanism is through the insertion reaction of free radicals (CH or NH), lacking selectivity for the groups and failing to clearly define the reaction site on the protein, thus losing the specificity of crosslinking. Furthermore, although most photoresponsive crosslinking agents have simple structures, the preparation and synthesis of their corresponding derivatives are difficult and unstable, making them expensive (e.g., diacylpropidine), and the crosslinking efficiency of the three traditional photoresponsive groups is not high. If benzophenone requires prolonged exposure to ultraviolet light, it may cause irreversible damage to biological samples.

[0004] In the 1970s, researchers commonly used glutaraldehyde (GA) cell fixatives (CN102533710A), and the more recently published glyoxal (GO) cell fixatives (CN109669032A), both requiring high fixation concentrations. These fixatives suffer from significant fluorescence quenching, are prone to oxidation and acidification leading to poor fixation, and form disordered polymers resulting in low cross-linking efficiency. Therefore, Bunz, UHF, et al. reported a photocage-protected glutaraldehyde cell fixative, obtained by protecting the two aldehyde groups of glutaraldehyde with lipophilic photocage protecting groups. This fixative is non-toxic before photolysis. Cell treatment with this fixative releases active glutaraldehyde upon UV irradiation, which then cross-links intracellular proteins in situ, achieving cell fixation. Compared to free glutaraldehyde fixatives, the fixed tissue exhibits lower background fluorescence. However, the active group of the photocage glutaraldehyde fixative remains glutaraldehyde after photoexcitation, and its cross-linking reaction with biomacromolecules is reversible. Therefore, a relatively high 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 for finding a responsive chemical crosslinking agent with novel structure, easy preparation, fast crosslinking speed, high crosslinking efficiency and high chemical selectivity. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of current chemical crosslinking agents, such as low crosslinking efficiency, low chemical selectivity, and high toxicity, by providing a chemically responsive crosslinking agent, its preparation method, and its applications. This crosslinking agent connects two PBDA groups with a polyethylene glycol (PEG) chain to prepare a novel photoresponsive chemical crosslinking agent, bis-PDBA-PEGn. Additionally, by protecting the 2-buten-1,4-dialdehyde (BDA) group with an acid-responsive deprotection group, another novel acid-responsive chemical crosslinking agent, bis-ABDA-PEGn, is prepared. The crosslinking agents obtained by this invention have novel structures; are non-volatile; exhibit low cytotoxicity; demonstrate high reaction yields with biomacromolecules; produce stable products; and achieve efficient crosslinking and cell fixation even at low concentrations. Furthermore, they have minimal impact on cell morphology and fluorescence imaging.

[0007] The technical solution of this invention is as follows:

[0008] A chemically (acid) responsive crosslinking agent, bis-ABDA-PEG n (Formula 1), its chemical structural formula is:

[0009]

[0010] In equation (1), n ​​= 2 - 8.

[0011] The method for preparing the chemically (acid)-responsive crosslinking agent includes the following steps:

[0012] (1) Furfuryl alcohol, sodium bicarbonate and anhydrous methanol are mixed to form a first mixed solution. The first mixed solution is then reacted with liquid bromine at -75 to -80°C for 1-2 hours. Then, saturated sodium bicarbonate solution is added to neutralize the system to pH 8. The solvent is removed by rotary evaporation. The solution is dissolved in ethyl acetate, washed, dried, filtered, and column chromatography is used to obtain ABDA.

[0013] For every 1-10 mL of anhydrous methanol, add 1 mmol of furfuryl alcohol, 1.5-2 mmol of sodium bicarbonate, and 1.05-1.6 mmol of liquid bromine.

[0014] (2) Dissolve the ABDA obtained in step (1) in the first mixed solvent and add it dropwise to the NaH THF suspension to form the second mixed solution. React at 0°C for 0.5-3 hours, and then react it with dibromo-PEG. n The reaction was carried out at 60-120℃ for 12-72 hours; then the reaction was quenched with ice water, DMF and THF were removed by rotary evaporation, and the product was 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 every 1-10 mL of the first mixed solvent.n The first mixed solvent consists of THF:DMF, with a volume ratio of 7:3.

[0015] Chemically (photo) responsive chemical crosslinking agent bis-PBDA-PEG n (Equation 2), its chemical structural formula is:

[0016]

[0017] In equation (2), n = 4 - 8.

[0018] The chemically (photo-)responsive crosslinking agent bis-PBDA-PEG n A method for preparing [the substance], the method comprising the following steps:

[0019] (1) Polyethylene glycol, tetrahydrofuran, and NaH were mixed to form a fourth mixed solution. The fourth mixed solution was mixed with propargyl bromide and stirred. The reaction was carried out at 10-25°C for 1.5-2 hours. The reaction was quenched with ice water, the tetrahydrofuran was removed by rotary evaporation, the solution was dissolved and extracted with ethyl acetate, washed, dried, and the solvent was concentrated. The solution was then subjected to column chromatography to obtain a yellow oily liquid propargylated polyethylene glycol Alkynyl-PEG. n ;

[0020] For every 5-10 mL of THF, add 1 mmol of polyethylene glycol, 5-6 mmol of NaH, and 5-10 mmol of propargyl bromide.

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

[0022] (2) The result obtained in step (1) Alkynyl-PEG n The mixture was prepared with PBDA-N3, a second mixed solvent, copper sulfate pentahydrate, tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), and sodium ascorbate. The mixture was reacted with stirring at 10-25°C for 1.5-2 hours. The solvent was removed by rotary evaporation, and the mixture was dissolved and extracted with dichloromethane. The solution was washed, dried, and the solvent concentrated. The mixture was then purified by thin-layer chromatography and further purified by HPLC to obtain a pale yellow solid, bis-PBDA-PEG. n .

[0023] In this process, 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-hydroxypropyltriazolylmethyl)amine (THPTA), and 5-10 mmol of sodium ascorbate;

[0024] The second mixed solvent consists of CH3OH and H2O in a volume ratio of 2:1.

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

[0026] The chemically (acid) responsive crosslinking agent bis-ABDA-PEG n Its applications include the preparation of chitosan hydrogel materials in the field of cross-linking biomacromolecules to prepare biomedical materials; and its use in the field of cell fixation for live cell mitochondrial fixation, staining, and fluorescence imaging.

[0027] The chemically (photo-)responsive crosslinking agent bis-PBDA-PEG n Its applications include the preparation of chitosan gel materials in the field of cross-linking biomacromolecules to prepare biomedical materials; and its use in the field of cell fixation for live cell mitochondrial fixation, staining, and fluorescence imaging.

[0028] The essential features of this invention are:

[0029] This invention connects two protected BDA groups with polyethylene glycol (PEG) of different lengths, which is expected to enable the preparation of chemical crosslinking agents with different stimuli-related activities, achieving efficient and highly specific crosslinking of amino-containing biomacromolecules. This will enable the preparation of biomedical materials (e.g., gels) and live cell fixation (suitable crosslinking agents that can replace traditional fixatives) at lower concentrations.

[0030] The beneficial effects of this invention are:

[0031] Acid-responsive chemical crosslinking agent bis-ABDA-PEG n and photoresponsive chemical crosslinking agent bis-PBDA-PEG n After treatment with acid and light respectively, the resulting bifunctional groups can react rapidly and selectively with amino groups to obtain stable cross-linked products. This type of cross-linking agent is stimuli-responsive, has low toxicity, and can achieve rapid and high cross-linking even at low concentrations (bis-ABDA-PEGn cleaved with 1M hydrochloric acid, neutralized with sodium bicarbonate, and the pH of the mixture adjusted to neutral, can rapidly form a non-flowing acid-cured gel within minutes after being shaken and mixed with chitosan). Figure 1 The photoresponsive chemical crosslinking agent bis-PBDA-PEGn, after being mixed and shaken with chitosan, can rapidly form a non-flowing photocurable gel within minutes to 2 hours after being irradiated at 365nm for 5-30 minutes. Figure 2 The photoresponsive chemical cross-linking agent bis-PBDA-PEGn itself is non-cytotoxic. After irradiation at 365nm for 5-7 minutes, it can cross-link intracellular biomolecules, achieving cell fixation. Figure 4Effective fixation of live cells can be achieved using 100-150 μM bis-PBDA-PEGn, with no significant changes in cell morphology after fixation. In contrast, traditional fixation with 4% paraformaldehyde and 2.5% glutaraldehyde not only leads to cell shrinkage but also increases cell granularity / internal complexity. Figure 5 Mitochondrial fluorescence staining after cell fixation with bis-PBDA-PEGn showed good and clear imaging results, while mitochondrial fluorescence staining after cell fixation with paraformaldehyde showed poor imaging results, and mitochondrial fluorescence staining after fixation of live cells with glutaraldehyde showed extremely poor results, with fluorescence almost completely disappearing. Figure 6 Therefore, bis-ABDA-PEG n and bis-PBDA-PEG n It has advantages and promising applications in the fields of preparing biomedical materials and live cell fixation, staining, and imaging research. Attached Figure Description

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

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

[0034] Figure 3 Comparison of the efficiency of the photoresponsive crosslinking agent bis-PBDA-PEGn with that of the traditional crosslinking agents paraformaldehyde (PFA) and glutaraldehyde (GA) in crosslinking histone H3.

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

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

[0037] Figure 6 Paraformaldehyde (PFA), glutaraldehyde (GA), and bis-PBDA-PEG in Example 8 n Comparison of the effects of staining and fluorescence imaging after fixation of mitochondria in live cells. Detailed Implementation

[0038] The specific embodiments of the present invention will be described in detail below. The specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0039] Dichloromethane (purchased from Tianjin Binhai New Area Guangshunda Chemical Reagent Co., Ltd., item number A1040); Sodium bicarbonate (purchased from Tianjin Binhai New Area Guangshunda Chemical Reagent Co., Ltd., item number 2049); Tetrahydrofuran (purchased from Beijing Huawi Ruike Chemical Co., Ltd., item number HWMT818767); Acetonitrile (≤10ppm) (purchased from Hebei Dina Xingke 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 Co., Ltd., 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 Co., Ltd., item number T9710); Furan (purchased from Gruss (Tianjin) Technology Co., Ltd., item number F116393-500ml); n-Butyllithium (purchased from Gruss (Tianjin) Technology Co., Ltd., item number B803316-500ml); Ethylene oxide (purchased from Gruss (Tianjin) Technology Co., Ltd., item number E808842-100ml); Sodium hydride (60% active ingredient by mass, dispersed in liquid paraffin) (purchased from TISA (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 Xiens Opd Technology Co., Ltd., item number T-21412); 2-azido-1,3-dimethylimidazolium hexafluorophosphate (ADMP) (purchased from TISA (Shanghai) 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., item number 75338C); Copper sulfate pentahydrate (Shanghai Maclean Biochemical Technology Co., Ltd., item number C805356-100g); Sodium ascorbate (Shanghai Maclean Biochemical Technology Co., Ltd., item number S817635-100g); Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) (purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd., item number T3171); Furfuryl alcohol (purchased from Beijing Inokai Technology Co., Ltd., item number... The following raw materials are commercially available: 119790010); liquid bromine (purchased from Amethyst, catalog number 931848); 1,2-bis(2-bromoethoxy)ethane (Bromo-PEG2-bromide) (purchased from Gruss (Tianjin) Technology Co., Ltd., catalog number BD760047); 1-bromo-2-(2-(2-(2-bromoethoxy)ethoxy)ethoxy)ethane (Bromo-PEG3-bromide) (purchased from Gruss (Tianjin) Technology Co., Ltd., catalog number BD760048).

[0040] Example 1

[0041] Synthesis of acid-responsive chemical crosslinking agents 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, the same applies to the following examples) and argon protection, 14.72 g (150 mmol, 1 eq.) of furfuryl alcohol (3) and 18.91 g (225.1 mmol, 1.5 eq.) were weighed into a round-bottom flask, dissolved in 150 mL of dry methanol, and the mixture was cooled to -78 °C. 60 mL of a methanol solution containing 13.0 mL (195.1 mmol, 1.3 eq.) of liquid bromine was slowly added dropwise to the mixture over approximately 45 minutes, and the reaction was continued at -78 °C for 2 hours. The reaction system was neutralized to pH 8 with saturated sodium bicarbonate solution, and methanol was removed by rotary evaporation. 150 mL of ethyl acetate was added to dissolve and dilute the product, and the product was washed with 150 mL of saturated sodium bicarbonate solution and 150 mL of saturated sodium chloride solution, respectively. The organic phase was dried over anhydrous sodium sulfate, the solvent was removed by rotary evaporation, and the solution was subjected to column chromatography with ethyl acetate / petroleum ether (v / v, 1:5) to give 22.3 g (139.2 mmol, yield 92.8 mol%) of a pale yellow oily liquid ABDA(4) (Acid-liable 2-Butene-1,4-dial, abbreviated as ABDA). Step 2: Preparation of compound (bis-ABDA-PEG3): ABDA(4) (5.0 g, 31.22 mmol, 1 eq.) was placed in a round-bottom flask and dissolved in 190 mL of a mixed solution of THF and DMF (volume ratio 7:3). This solution was then added dropwise to a tetrahydrofuran suspension containing sodium hydride (3.75 g, 93.66 mmol, 3 eq.). The mixture was stirred at room temperature for 30 minutes. Then, dibromo-PEG2 (4.31 g, 15.61 mmol, 0.5 eq.) was added, and the resulting solution was stirred at 80 °C for 24 hours. The solvent was removed by rotary evaporation, and the mixture was dissolved and 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 with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The crude product was adsorbed onto silica gel for column chromatography and purified with CH2Cl2 / CH3OH (v / v, 100 / 1-50 / 1) as eluent to obtain 1.85 g (4.26 mmol, 13.6 mol%) of pale yellow oily liquid bis-ABDA-PEG3(5).

[0045] 1 ¹H 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. The target product, acid-responsive chemical crosslinking agent bis-ABDA-PEG3(5), was obtained by identification by nuclear magnetic resonance and mass spectrometry.

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

[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 crosslinking agent bis-ABDA-PEG4(6), was obtained by NMR and mass spectrometry identification.)

[0048] Example 2

[0049] Synthesis of photoresponsive chemical crosslinking agents 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) into 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 for 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 NaH tetrahydrofuran suspension under an argon atmosphere. After the addition is complete (10 minutes), heat 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. Quench the reaction with water, remove tetrahydrofuran by rotary evaporation, dissolve and extract with ethyl acetate, 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 give Alkynyl PEG4(8) 1.25 g (4.62 mmol, yield 90 mol%). 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) was added, along with 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-hydroxypropyltriazolylmethyl)amine (THPTA) and 146.7 mg (740.5 μmol, 5 eq.) of sodium ascorbate. The mixture was stirred overnight at room temperature. The solvent was removed by rotary evaporation, and the mixture was dissolved and extracted with CH2Cl2. The mixture was washed with saturated NaCl solution, and the organic phases were combined and dried. CH2Cl2 was concentrated to remove the organic phases, and the mixture was then separated. 1.25 g (4.62 mmol, 90 mol% yield) of a pale yellow solid bis-PBDA-PEG4(10) was obtained by preparative liquid-phase separation under high pressure. 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 crosslinking agent 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 one): 270.0 mg (6.75 mmol, 5 eq.) of NaH was weighed into a round-bottom flask, 15 mL of dry tetrahydrofuran was added, and the mixture was cooled to 0 °C in an ice bath. 500.0 mg (1.35 mmol, 1.0 eq.) of octaethylene glycol was dissolved in 15 mL of dry THF, and then added dropwise to the above NaH tetrahydrofuran suspension. After the addition was complete, the mixture was stirred for 15 minutes, and then heated to room temperature and stirred for another 30 minutes. Propylbromine (581.9 μL, 6.75 mmol, 5.0 eq.) was added to the above reaction system, and the mixture was stirred at room temperature for 5 hours. The product was quenched with H2O, tetrahydrofuran was removed by rotary evaporation, dissolved in ethyl acetate, washed with saturated sodium chloride solution, the organic phases were combined and dried, the solvent was concentrated, and separated by column chromatography (EtOAc / PE = 1 / 4-1 / 3-1 / 2) to obtain product Alkynyl PEG8(12) 517.7 mg (1.16 mmol, yield 86 mol%). 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 two): 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. 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 overnight at room temperature. The solvent was removed by rotary evaporation, dissolved and extracted with CH2Cl2, washed with saturated NaCl solution, and the organic phases were combined and dried. The solvent was concentrated for separation. High-pressure preparative liquid chromatography (HPLC) C18 column separation yielded a pale yellow liquid bis-PBDA-PEG8(13)119.5 mg (76.19 μmol, yield 85 mol / kg).

[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 crosslinking agent bis-PBDA-PEG8(13), was obtained by NMR and mass spectrometry identification.)

[0060] Example 3

[0061] Acid-responsive chemical crosslinking agent bis-ABDA-PEGn Applications in the preparation of chitosan (CMCS) hydrogels.

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

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

[0064] Separately, 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 two solutions were mixed and incubated at 37°C for 20 minutes to obtain a 5% (w / v) chitosan hydrogel. Figure 1 ). Figure 1 The results show that the acid-responsive chemical crosslinking agent bis-ABDA-PEG3 cannot form a gel when directly mixed with chitosan. However, after bis-ABDA-PEGn is acid-hydrolyzed with HCl and neutralized with NaHCO3, it can crosslink with chitosan and form a non-flowing acid-cured gel in 0.5-30 minutes.

[0066] Example 4

[0067] Photoresponsive chemical crosslinking agent bis-PBDA-PEG n Applications in the preparation of chitosan (CMCS) hydrogels.

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

[0069] Dissolve 50 mg of chitosan (CMCS) in 1 mL of H2O.

[0070] In addition, prepare 100 μL of DMSO solution (50 mg / mL) of bis-PBDA-PEG4.

[0071] After thoroughly mixing the two solutions by shaking, irradiate them with ultraviolet light (365nm, 7mW cm⁻¹). -2 5 minutes to remove the photoresponsive protective groups on PBDA. Incubate at 37°C for 2 hours to obtain 5% (w / v) chitosan hydrogel. Figure 2 ). Figure 2The results show that the photoresponsive chemical crosslinking agent bis-PBDA-PEG4 cannot form a gel when directly mixed with chitosan by oscillation. However, after being irradiated with ultraviolet light at 365 nm for 5-60 minutes, it can undergo photocrosslinking with chitosan, and a non-flowing photocurable gel can be formed in 0.5-120 minutes.

[0072] Example 5

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

[0074] Different concentrations of photoresponsive chemical cross-linking agents bis-PBDA-PEG4, bis-PBDA-PEG8, paraformaldehyde (PFA), glutaraldehyde (GA), and histone H3 (65 μM) were mixed in a buffer solution (20 mM PBS, pH 7.4, 50 mM NaCl). After irradiation at 360 nm for 10 minutes, 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 cross-linking agent to histone H3 to obtain the desired results. Figure 3 . Figure 3 The results show that the photoresponsive chemical crosslinking agent bis-PBDA-PEGn (n=4 and 8) has a significantly higher crosslinking efficiency for histone H3 after photoexcitation than the traditional crosslinking agents paraformaldehyde (PFA) and glutaraldehyde (GA).

[0075] Example 6: Cytotoxicity of the photoresponsive chemical crosslinking agent bis-PBDA-PEG4.

[0076] Ten thousand HeLa cells were seeded into cell culture plates (24-96 wells) and cultured overnight at 37°C in a 5% CO2 incubator for 24 hours.

[0077] Different concentrations of bis-PBDA-PEG4 were added to HeLa cells and the cells were incubated at 37°C for 10-12 hours.

[0078] In addition, different concentrations of bis-PBDA-PEG4 were irradiated with 365nm light for 7 minutes, and then added to HeLa cells and incubated at 37°C for 10-12 hours.

[0079] Cell viability was detected using the CCK-8 assay kit. Figure 4 ). Figure 4The results showed that, in the non-light-exposed group, cell viability was unaffected by increasing bis-PBDA-PEG4 concentration, indicating that bis-PBDA-PEG4 itself is non-cytotoxic. In the light-exposed group, cell viability gradually decreased with increasing bis-PBDA-PEG4 concentration. At a concentration of approximately 150 μM, cell viability was less than 20%. This indicates that after light activation, 150 μM of bi-PBDA-PEGn can efficiently cross-link intracellular biomolecules, leading to cell death.

[0080] Example 7

[0081] Effects of photoresponsive chemical crosslinking agent bis-PBDA-PEGn on cell morphology after cell fixation.

[0082] Two million HeLa cells were cultured overnight in 12-well tissue culture plates (Nest). Cells were then fixed using one of the following methods: 4% (w / v) paraformaldehyde for 15 min, 2% (w / v) glutaraldehyde for 20 min, or bis-PBDA-PEG4 / bis-PBDA-PEG8 (130 μM, irradiated for 5 min followed by incubation for 2 h). After fixation, cells were collected by trypsin digestion with EDTA-free enzymes. The fixed cells were washed twice with 2 mL of pre-chilled 1×PBS (centrifuged at 4000 rpm for 5 min 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 the cell suspension was transferred to a 5 mL flow cytometry tube, 5 μL of Annexin V-FITC was added, and the cells were incubated for 5 min, followed by 5 μL of PI. The sample was gently mixed by pipetting and incubated at room temperature in the dark for 5 min. The cells were then immediately analyzed using a flow cytometer (BD, USA). Figure 5 ).

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

[0084] Example 8

[0085] The photoresponsive chemical crosslinking agent bis-PBDA-PEGn is used for fluorescence imaging after live cell fixation.

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

[0087] (1) Seed HeLa cells in confocal dishes and incubate overnight at 37°C in a 5% CO2 incubator for 24 hours; (2) Remove the culture medium and wash the cells twice with 1 mL PBS per well for 5 minutes each time; (3) Stain the cells for mitochondria by adding 1 mL of freshly prepared mitochondrial red staining agent (100 nM) to each well and incubating the cells for 30 minutes in an incubator; (4) Remove the mitochondrial red staining solution and wash the cells three times with PBS for 5 minutes each time; (5) Fix the cells in the confocal dishes with 1 mL of 4% paraformaldehyde (1.2 M) for 20 minutes; 1 mL of 2% glutaraldehyde (200 mM) for 30 minutes; and 1 mL of Bis-PBDA-PEG. n (6) Fix cells for 1 hour with photolysis buffer (final concentration 150 μM, 350 nm light for 7 minutes); (7) Discard paraformaldehyde, glutaraldehyde and di-PBDA-PEGn (n=4 and 8) respectively, and wash cells 3 times with PBS; (8) Immediately place cells under a laser confocal microscope for live cell imaging (excitation light 579 nm, emission light 599 nm) Figure 6 ). Figure 6 The image shows a comparison of fluorescence intensity before and after fixation of live cell mitochondria using 4% paraformaldehyde, 2.5% glutaraldehyde, and photolyzed bis-PBDA-PEG4 and bis-PBDA-PEG8. The results indicate that photolyzed bis-PBDA-PEG4 and bis-PBDA-PEG8 require lower concentrations for fixation of live cell mitochondria, achieving cell fixation at 100-150 μM, and producing stronger fluorescence intensity and less fluorescence quenching in mitochondrial imaging. In contrast, the commonly used biological and medical 4% paraformaldehyde and 2-2.5% glutaraldehyde, at higher molar concentrations (1.2 M and 200 mM respectively), produce weaker fluorescence intensity and more fluorescence quenching in mitochondrial imaging.

[0088] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

[0089] Matters not covered in this invention are common knowledge.

Claims

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

2. The method for preparing the chemically acid-responsive crosslinking agent as described in claim 1, characterized in that the method comprises the following steps: (1) Furfuryl alcohol, sodium bicarbonate and anhydrous methanol are mixed to form a first mixed solution. The first mixed solution is then reacted with liquid bromine at -75~-80℃ for 1-2 hours. Then, saturated sodium bicarbonate solution is added to neutralize the system to pH 8. After rotary evaporation, the solution is dissolved in ethyl acetate, washed, dried, filtered, and column chromatography is performed to obtain ABDA. For every 1-10 mL of anhydrous methanol, add 1 mmol of furfuryl alcohol, 1.5-2 mmol of sodium bicarbonate, and 1.05-1.6 mmol of liquid bromine. (2) Dissolve the ABDA obtained in step (1) in the first mixed solvent and add it dropwise to the NaH THF suspension to form the second mixed solution. React at 0°C for 0.5-3 hours, and then react it with dibromo-PEG. n The reaction was carried out at 60-120℃ for 12-72 hours; then ice water was added to quench the reaction, and after rotary evaporation, the mixture was diluted with ethyl acetate, washed, dried, filtered, and column chromatography was performed to obtain bis-ABDA-PEGn, i.e., compound (1); In this mixture, 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 first mixed solvent consists of THF:DMF, with a volume ratio of 7:

3.

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

4. The method for preparing the photoresponsive chemical crosslinking agent as described in claim 3, characterized in that, The method includes the following steps: (1) Polyethylene glycol is mixed with tetrahydrofuran and NaH to form a fourth mixed solution. The fourth mixed solution is mixed with propargyl bromide and stirred. The reaction is carried out at 10-25℃ for 1.5-2 hours. The reaction is quenched with ice water, tetrahydrofuran is removed by rotary evaporation, ethyl acetate is used for dissolution and extraction, the mixture is washed, dried, and the solvent is concentrated. The solution is then subjected to column chromatography to obtain a yellow oily liquid propargylated polyethylene glycol Alkynyl-PEG. n ; For every 5-10 mL of THF, add 1 mmol of polyethylene glycol, 5-6 mmol of NaH, and 5-10 mmol of propargyl bromide. (2) The Alkynyl-PEG obtained in step (1) n The mixture was prepared with PBDA-N3, a second mixed solvent, copper sulfate pentahydrate, tris(3-hydroxypropyltriazolylmethyl)amine, and sodium ascorbate. The mixture was stirred and reacted at 10-25°C for 1.5-2 hours. The solvent was removed by rotary evaporation, and the mixture was dissolved and extracted with dichloromethane. The solution was washed, dried, and the solvent concentrated. The solution was then purified by thin-layer chromatography and further purified by HPLC to obtain a pale yellow solid, bis-PBDA-PEG. n That is, the compound of formula (2); In this process, 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-hydroxypropyltriazolylmethyl)amine, and 5-10 mmol of sodium ascorbate; The second mixed solvent consists of CH3OH and H2O in a volume ratio of 2:1; wherein PBDA-N3 is... N V for .

5. The method for preparing the photoresponsive chemical crosslinking agent as described in claim 4, characterized in that, The stirring speed in both steps (1) and (2) is 300-600 rpm.

6. The method for preparing the photoresponsive chemical crosslinking agent as described in claim 4, characterized in that, The degree of polymerization of the polyethylene glycol is 4-8.

7. The application of the chemically acid-responsive crosslinking agent as described in claim 1, characterized in that, Chitosan hydrogel materials are used to prepare biomedical materials through cross-linking of biological macromolecules.

8. The application of the photoresponsive chemical crosslinking agent as described in claim 3, characterized in that, Chitosan gel materials are used in the cross-linking of biological macromolecules to prepare biomedical materials; and in the field of cell fixation, they are used to prepare fluorescent imaging agents for fixing and staining mitochondria in live cells.

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