A heteromorphic bifunctional crosslinking agent, a preparation method and application thereof

Abstract

The application provides a heteromorphic bifunctional crosslinking agent and preparation and application thereof. The heteromorphic bifunctional crosslinking agent provided by the application is a bifunctional crosslinking agent containing N-hydroxysuccinimide (NHS) active ester and a dinitroimidazole functional group (DNIm), and can connect together a compound containing amino and a compound containing sulfydryl. The DNIm module of the crosslinking agent provided by the application is more stable than a maleimide module. In addition, the addition product of DNIm and sulfydryl is also more stable than the addition product of maleimide and sulfydryl. In a near neutral buffer, no side reaction product of the addition of DNIm and amino is found through LC-MS analysis, that is, no addition reaction with amino occurs under the condition. The crosslinking agent provided by the application can be applied to the construction of cyclic peptides of different sizes, and the selective single modification of sulfydryl or amino of proteins, including biotinylation, PEGylation or the introduction of fluorescent substances, and the double modification of a single protein molecule, including biotinylation and PEGylation.

Description

Technical Field

[0001] This invention belongs to the field of heterogeneous bifunctional crosslinking agents, specifically relating to a heterogeneous bifunctional crosslinking agent, its preparation method, and its application. Background Technology

[0002] Heterogeneous bifunctional crosslinking agents (SMCCs) are a class of bifunctional coupling agents containing N-hydroxysuccinimide (NHS) active esters and maleimide. They can bond compounds containing thiol and amino groups together, respectively, and act as linkers to connect antibodies and toxin molecules. They are widely used in immunoassays and radiolabeling for tumor imaging.

[0003] However, the maleimide module of SMCC is unstable and easily hydrolyzed; its addition product with thiol is also easily hydrolyzed; and in addition to mainly reacting with thiol, the module can also undergo side reactions with amino groups to some extent.

[0004] Therefore, a new solution needs to be developed to improve the above problems. Summary of the Invention

[0005] The present invention aims to provide a heterogeneous bifunctional crosslinking agent, its preparation method and application, to improve the problems of the maleimide module in the existing SMCC bifunctional crosslinking agent being unstable and easily hydrolyzed; its addition product with thiol is also easily hydrolyzed; and in addition to mainly reacting with thiol, the module also undergoes side reactions with amino groups to a certain extent.

[0006] On one hand, the present invention provides a heterogeneous bifunctional crosslinking agent, the bifunctional crosslinking agent being named DNIm-NHS, with the structural formula shown in Formula 1:

[0007]

[0008] Secondly, the present invention provides a method for preparing a heterogeneous bifunctional crosslinking agent, characterized by comprising the following steps:

[0009] Compound 2, condensing agent, and N-hydroxysuccinimide were sequentially added to the first solvent and stirred at room temperature for 15-16 h. After concentration and purification, the heterogeneous bifunctional crosslinking agent shown in Formula I was obtained; the compound 2 was 6-((1,4-dinitro-1H-imidazol-2-yl)methoxy)hexanoic acid.

[0010] Optionally, compound 2, condensing agent, and N-hydroxysuccinimide are added sequentially to the first solvent step, wherein the molar ratio of compound 2, condensing agent, and N-hydroxysuccinimide is 1:(1.0-1.2):(1.0-1.2).

[0011] Optionally, the condensing agent includes commonly used condensing agents in chemical synthesis, including dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

[0012] Optionally, the first solvent includes dichloromethane, chloroform, diethyl ether, tetrahydrofuran, ethyl acetate, N,N-dimethylformamide, N,N-dimethylacetamide, and dimethyl sulfoxide.

[0013] Optionally, the first solvent may be a pure solvent or a mixture of multiple alternative solvents.

[0014] Optionally, purification includes silica gel column chromatography.

[0015] Thirdly, the present invention provides an application of a heterogeneous bifunctional crosslinking agent in the construction of cyclic peptides.

[0016] Fourthly, the present invention provides an application of a heterogeneous bifunctional crosslinking agent in the chemical modification of proteins.

[0017] Optionally, the applications include protein biotinylation, protein PEGylation, and the introduction of fluorescent substances into proteins.

[0018] Optionally, the application includes the preparation of a modifying agent containing at least one of N-hydroxysuccinimide active ester and dinitroimidazole functional groups.

[0019] Fifthly, the present invention provides an application of a heterogeneous bifunctional crosslinking agent in drug preparation.

[0020] The beneficial effects of this invention include:

[0021] (1) The bifunctional crosslinking agent DNIm-NHS provided by the present invention has more stable chemical properties and a longer shelf life;

[0022] (2) The bifunctional crosslinking agent provided by the present invention can be applied to the construction of cyclic peptides, single modification and double modification of proteins, etc.

[0023] (3) The chemical properties of the cross-linking products generated by the bifunctional cross-linking agent provided by the present invention are more stable, which also makes the drug molecules prepared by using the cross-linking agent DNIm-NHS more stable.

[0024] (4) The crosslinking agent provided by the present invention contains a DNIm module, which has a higher selectivity for thiol groups compared with maleimide modules. Therefore, the crosslinking agent DNIm-NHS of the present invention has better bioorthogonality for thiol and amino groups, which can avoid the generation of addition byproducts between DNIm modules and amino groups. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the preparation method of the heterogeneous bifunctional crosslinking agent in Example 1;

[0026] Figure 2 The crosslinking agent DNIm-NHS prepared in Example 1 1 H NMR spectrum;

[0027] Figure 3 The crosslinking agent DNIm-NHS prepared in Example 1 13 C NMR spectrum;

[0028] Figure 4 Compound 2 prepared in Example 1 1 H NMR spectrum;

[0029] Figure 5 The diagram shows the macrocyclization reaction process of polypeptide P1 with DNIm-NHS in Example 2, and the HPLC characterization results of the product.

[0030] Figure 6 The diagram shows the macrocyclization reaction process of peptide P2 with DNIm-NHS in Example 3, and the HPLC characterization results of the product.

[0031] Figure 7 The diagram shows the macrocyclization reaction process of peptide P6 with DNIm-NHS in Example 4, and the HPLC characterization results of the product.

[0032] Figure 8 The reaction equation and HPLC chromatogram of DNIm-NHS and biotin-NH2 in Example 5 are shown below.

[0033] Figure 9 Tandem mass spectrometry fragmentation analysis and HRMS / MS chromatogram of product 11a from the reaction of DNIm-NHS and biotin-NH2 in Example 5;

[0034] Figure 10 The reaction equation and HPLC chromatogram of DNIm-NHS and Dansyl-NH2 in Example 5 are shown.

[0035] Figure 11 Tandem mass spectrometry fragmentation analysis and HRMS / MS chromatogram of product 12a from the reaction of DNIm-NHS and Dansyl-NH2 in Example 5;

[0036] Figure 12 Tandem mass spectrometry fragmentation analysis and HRMS / MS plot of byproduct 13a from the reaction of DNIm-NHS and Dansyl-NH2 in Example 5.

[0037] Figure 13The reaction equation and HPLC chromatogram of DNIm-NHS and Furazan-NH2 in Example 5 are shown.

[0038] Figure 14 Tandem mass spectrometry fragmentation analysis and HRMS / MS chromatogram of product 14a from the reaction of DNIm-NHS and Furazan-NH2 in Example 5;

[0039] Figure 15 Tandem mass spectrometry fragmentation analysis and HRMS / MS chromatogram of byproduct 15a from the reaction of DNIm-NHS and Furazan-NH2 in Example 5;

[0040] Figure 16 The reaction process and HPLC chromatogram of DNIm-NHS with NH2-PEG-OH or SH-PEG-OH of different molecular weights in Example 5 are shown.

[0041] Figure 17 The MALDI-TOF diagrams of NH2-PEG-OH at 2000 Da in Example 5 and the MALDI-TOF diagram of product 16a after reaction with DNIm-NHS are shown.

[0042] Figure 18 The MALDI-TOF diagrams of NH2-PEG-OH with a capacity of 5000 Da and product 17a after reaction with DNIm-NHS are shown in Example 5.

[0043] Figure 19 The MALDI-TOF diagrams of SH-PEG-OH at 2000 Da and product 18a after reaction with DNIm-NHS in Example 5 are shown.

[0044] Figure 20 This is a schematic diagram and HPLC chromatogram of the reaction between the DNIm module of DNIm-NHS prepared in Example 1 and the thiol group of Ac-Cys.

[0045] Figure 21 Tandem mass spectrometry fragmentation analysis and high-resolution tandem mass spectrum of product 1a after the reaction of the DNIm module with the thiol group of Ac-Cys during performance verification.

[0046] Figure 22 Tandem mass spectrometry fragmentation analysis and high-resolution tandem mass spectrum of product 2a after the reaction of the DNIm module with the thiol group of Ac-Cys during performance verification.

[0047] Figure 23 The reaction diagram and HPLC chromatogram of the NHS module of DNIm-NHS prepared in Example 1 with the amino group of Ac-Lys are shown.

[0048] Figure 24 Tandem mass spectrometry fragmentation analysis and high-resolution tandem mass spectrum of product 3a after the reaction of the NHS module with the amino group of Ac-Lys during performance verification.

[0049] Figure 25 This is a schematic diagram illustrating the reaction of the modifier with BSA, 3CL, and Lysozyme during performance verification.

[0050] Figure 26 Characterization results of single-modified protein derivatives obtained from the reaction of modifiers with proteins during performance validation:

[0051] Figure 27 Characterization results of the dual-modified protein derivatives obtained from the reaction of the modifier with the protein in performance validation:

[0052] Figure 28 This is a schematic diagram illustrating the reaction process of applying DNIm-NHS to construct cyclic peptides and modify proteins. Detailed Implementation

[0053] The present invention will be further described in conjunction with the accompanying drawings and through the following embodiments.

[0054] On one hand, embodiments of the present invention provide a heterogeneous bifunctional crosslinking agent, named DNIm-NHS, with the structural formula shown in Formula I:

[0055]

[0056] Secondly, embodiments of the present invention provide a method for preparing a heterogeneous bifunctional crosslinking agent, characterized by comprising the following steps:

[0057] Compound 2, condensing agent, and N-hydroxysuccinimide were sequentially added to the first solvent, stirred at room temperature for 15-16 h, concentrated and purified to obtain the heterogeneous bifunctional crosslinking agent as shown in Formula 1; the compound 2 is 6-((1,4-dinitro-1H-imidazol-2-yl)methoxy)hexanoic acid.

[0058] In some embodiments, compound 2, condensing agent, and N-hydroxysuccinimide are added sequentially to the first solvent step, wherein the molar ratio of compound 2, condensing agent, and N-hydroxysuccinimide is 1:(1.0-1.2):(1.0-1.2).

[0059] Specifically, the first solvent includes at least one of dichloromethane, chloroform, diethyl ether, tetrahydrofuran, ethyl acetate, N,N-dimethylformamide, N,N-dimethylacetamide, and dimethyl sulfoxide.

[0060] Thirdly, embodiments of the present invention provide an application of a heterogeneous bifunctional crosslinking agent in the construction of cyclic peptides.

[0061] Fourthly, embodiments of the present invention provide an application of a heterogeneous bifunctional crosslinking agent in the chemical modification of proteins.

[0062] In some embodiments, the applications include protein biotinylation, protein PEGylation, and the introduction of fluorescent substances into proteins.

[0063] Specifically, the application includes the preparation of a modifying agent containing at least one of the following functional groups: N-hydroxysuccinimide active ester and dinitroimidazole.

[0064] Fifthly, the present invention provides an application of a heterogeneous bifunctional crosslinking agent in drug preparation.

[0065] Example 1

[0066] Example 1 of this invention provides a method for preparing a heterogeneous bifunctional crosslinking agent (DNIm-NHS), comprising the following steps:

[0067] S1. 130 mg (0.43 mmol) of compound 2, 97 mg (0.47 mmol) of DCC, and 54 mg (0.47 mmol) of N-hydroxysuccinimide (NHS) were added sequentially to a single-necked flask and stirred at 25 °C for 15.5 h.

[0068] S2. After stirring, concentrate the product using a rotary evaporator to obtain concentrate one.

[0069] The concentrate was purified by rapid silica gel column chromatography with a volume ratio of petroleum ether to ethyl acetate of 10:6.

[0070] After purification, 127 mg of a white amorphous solid was obtained, with a yield of 74%. High-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) identified the white amorphous solid as the heteromorphic bifunctional crosslinking agent shown in Formula I, namely target compound 1: DNIm-NHS; compound 2 is 6-((1,4-dinitro-1H-imidazol-2-yl)methoxy)hexanoic acid, and the structural formula of compound 2 and the reaction process in Example 1 are as follows. Figure 1 As shown;

[0071] target compound 1 1 H NMR spectrum as follows Figure 2 As shown, 13 C NMR spectrum as follows Figure 3 As shown, the characterization data is as follows:

[0072] 1H NMR (400MHz, MeOD) δ9.02 (s, 1H), 4.85 (s, 2H), 3.64 (t, J=6.3Hz, 2H), 2.83 (s, 4H), 2.63 (t, J=7.2Hz, 2H), 1.75 (m, 2H), 1.66 (m, 2H), 1.50 (m, 2H).

[0073] 13 C NMR (100MHz, MeOD) δ170.5, 168.9, 142.3, 142.2, 116.3, 70.9, 65.0, 30.1, 28.6, 25.1, 24.9, 24.0.

[0074] HRMS(ESI)m / z calcd.for C 14 H 17 N5NaO9 + [M+Na] + 422.0918, found 422.0910.

[0075] Before performing step S1, compound 2 needs to be synthesized, including the following steps:

[0076] S11. At 0°C, 1.15 g of compound m0 (ethyl 1H-imidazolium-2-carboxylate, 8.20 mmol) and 5.0 mL of concentrated H2SO4 (93 mmol) were added to a reaction flask; 1.0 mL of fuming HNO3 (24 mmol) was slowly added dropwise using a syringe; the mixture was stirred at 55°C for 6 h and then cooled to room temperature. The reaction mixture was then poured into ice, the resulting precipitate was filtered, and dried to give a white solid compound ml (0.91 g, 60% yield).

[0077] S12. Add 0.580 g of anhydrous K2CO3 (4.20 mmol) and 0.485 g of compound m1 (2.62 mmol) to a 25 mL two-necked round-bottom reaction flask equipped with a condenser. Under nitrogen protection, add 3.0 mL of dry DMF solution containing 0.39 mL of p-methoxybenzyl chloride PMBCl (2.9 mmol) using a syringe. Stir at 115 °C for 12 h, cool in an ice bath, filter, and wash the precipitate with ethyl acetate. After washing, combine the filtrates, concentrate, and purify by rapid silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 3, v / v) to give a white solid m2 (0.61 g, yield 76%), which was identified as the target compound by low-resolution mass spectrometry.

[0078] S13. A single-necked round-bottom flask was cooled in an ice bath. 0.305 g of compound m2 (1.0 mmol) and 6 mL of dry tetrahydrofuran (THF) were added. With rapid stirring, 0.057 g of lithium aluminum hydride (LiAlH4) (1.5 mmol) was added in portions. After 2 h, the reaction was quenched successively with 2.0 mL of ice water and 3.0 mL of saturated NaHCO3 at 0 °C. The resulting bright red viscous liquid was filtered with diatomaceous earth assistance, washed with ethyl acetate, and the filtrates were combined. The filtrate was dried over anhydrous sodium sulfate, concentrated, and purified by rapid silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 1, v / v) to give a white solid m3 (0.146 g, yield 56%). NMR was performed. 1 H NMR identified it as the target analyte;

[0079] S14. Under ice bath conditions, add 1600 mg of compound m9 (ethyl 6-hydroxyhexanoate, 10.0 mmol), 2 mL of triethylamine TEA (15.0 mmol), and 20.0 mL of dichloromethane (DCM) to a single-necked flask; stir vigorously, then carefully add 10.0 mL of DCM solution containing 2286 mg of p-toluenesulfonyl chloride (TsCl, 12 mmol); react at room temperature for 5 h; concentrate the reaction solution and purify it by rapid silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1, v / v) to obtain a clear oily liquid m8 (2170 mg, yield 69%).

[0080] S15. At room temperature, 130 mg of compound m3 (0.50 mmol) and 18 mg of NaH (0.75 mmol) were added to a two-necked flask. Under argon (Ar2) protection, 1.0 mL of dry DMF solution and 1 mL of DMF solution containing 235 mg of compound m8 (0.75 mmol) were added sequentially via syringe. The reaction was continued for 22 h. The mixture was diluted with 50 mL of DCM and neutralized with saturated NH4Cl solution. The aqueous layer was extracted with DCM (20 mL × 2). The organic layers were combined, washed with saturated brine (5 mL × 2), dried over anhydrous Na2SO4, filtered, and concentrated. The mixture was purified by rapid silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 7, v / v) to give compound m4 as a yellow-green oil (105 mg, yield 52%). Low-resolution mass spectrometry identified it as the target compound.

[0081] S16. 80 mg of compound m4 (0.20 mmol), 4.0 mL of trifluoroacetic acid (TFA), and 0.4 mL of anisole were added sequentially to a single-necked flask. The mixture was reacted at 95 °C for 2.5 h. Thin-layer chromatography analysis showed that the substrate reaction was complete. The mixture was concentrated and purified by rapid silica gel column chromatography (petroleum ether / ethyl acetate / MeOH = 10 / 5 / 0.2, v / v / v) to give compound m5 as a transparent oil (37 mg, yield 66%). Low-resolution mass spectrometry identified it as the target analyte.

[0082] S17. Add 37 mg of compound m5 (0.13 mmol), 1.0 mL of THF, 16 mg of LiOH·H2O (0.35 mmol), and 1 mL of water to a single-necked flask in sequence; stir at room temperature for 4 h, then neutralize with diluted NaHCO3 solution; wash the aqueous layer with diethyl ether (4 mL × 2), acidify with formic acid, then extract with ethyl acetate (15 mL × 3), combine the organic layers, dry with anhydrous Na2SO4, filter, concentrate, and dry under vacuum to give compound m6 as a white solid (30 mg, yield 90%).

[0083] S18. Under ice bath conditions, 2 mL of CH3COOH, 130 μL of Ac2O (1.33 mmol), and 50 μL of fuming HNO3 (1.10 mmol) were added sequentially to a single-necked flask. The mixture was stirred at room temperature for 2 h, and then 30 mg of compound m6 (0.12 mmol) was added at 0 °C. The mixture was then transferred to room temperature and stirred for another 8 h until the starting material was completely consumed. Dichloromethane (20 mL × 3) and 10 mL of water were added to the reaction flask. The organic phase was dried over anhydrous Na2SO4, filtered, concentrated, and dried under vacuum to give a pale yellow solid (30 mg, yield 83%). The solid was analyzed by NMR. 1 H NMR identified it as compound 2;

[0084] The compounds obtained in steps S11-S18 are consistent with those obtained by methods in known literature (Dinitroimidazoles as bifunctional bioconjugation reagents for protein functionalization and peptide macrocyclization).

[0085] Compound 2 1 H NMR spectrum as shown Figure 4 As shown, the characterization data is as follows:

[0086] 1 H NMR (400MHz, CDCl3) δ8.54 (s, 1H), 4.83 (s, 2H), 3.58 (t, J=6.4Hz, 2H), 2.34 (t, J=7.4Hz, 2H), 1.67-1.57 (m, 4H), 1.39 (m, 2H).

[0087] Example 2

[0088] Example 2 of this invention provides an application of DNIm-NHS in the construction of cyclic peptide 4a, comprising the following steps:

[0089] Take a certain volume of concentrated peptide P1 and add it to 1 mL of 100 mM HEPES solution with pH = 8.0 to make the final concentration of peptide P1 1 mM.

[0090] The DNIm-NHS prepared in Example 1 was dissolved in acetonitrile to prepare a concentrated solution. An appropriate volume of the concentrated solution was added to the HEPES buffer solution to make the final concentration of DNIm-NHS 1mM.

[0091] The mixture was shaken at 25°C and reacted for 0.5 h to obtain cyclic peptide 4a with a yield of 90%.

[0092] Example 2: The reaction process for constructing the cyclic peptide is as follows Figure 5 As shown in Figure A; the reaction of DNIm-NHS with peptide P1 and its high-performance liquid chromatography (HPLC) chromatogram with peptide P1 as the control group are shown in Figure A. Figure 5 As shown in B;

[0093] Furthermore, peptide P1 and cyclic peptide 4a were characterized by HRMS, and the data are as follows:

[0094] Peptide P1: HRMS(ESI) m / z calcd.for C 26 H 46 N 10 O 10 SK + [M+K] + 729.2751, found 729.2725.

[0095] Cyclic peptide 4a: HRMS(ESI) m / z calcd.for C 36 H 57 N 13 O 14 SK + [M+K] + 966.3500, found966.3476.

[0096] Example 3

[0097] Example 3 of this invention provides an application of DNIm-NHS in the construction of cyclic peptide 5a, which differs from Example 2 in that the polypeptide used in the construction is polypeptide P2; polypeptide P2 is used as the control group.

[0098] Example 3: The reaction process for constructing the cyclic peptide is as follows Figure 6 As shown in Figure A; the HPLC chromatograms of the reaction between DNIm-NHS and polypeptide P2 and its control experiment are shown in Figure A. Figure 6 As shown in B; polypeptide P2 was ordered from Jiangsu Shenlang Biotechnology Co., Ltd.

[0099] Cyclic peptide 5a was characterized by HRMS, and the data are as follows: HRMS(ESI) m / z calcd.for C 43 H 60 N 13 O 10 S2 + [M+H] + 982.4022, found 982.4040.

[0100] Example 4

[0101] Example 4 of this invention provides an application of DNIm-NHS in the construction of cyclic peptide 10a, which differs from Example 2 in that the polypeptide used in the construction is polypeptide P6;

[0102] Example 4: The reaction process for constructing the cyclic peptide is as follows Figure 7 As shown in Figure A; the reaction of DNIm-NHS with peptide P6 and its HPLC chromatogram with peptide P6 as a control are shown in Figure A. Figure 7 As shown in B;

[0103] Furthermore, peptide P6 and cyclic peptide 10a were characterized by HRMS, and the data are as follows:

[0104] Peptide P6: HRMS(ESI) m / z calcd.for C 59 H 80 N 17 O 18 S + [M+H] + 1346.5582, found 1346.5575.

[0105] Cyclic peptide 10a: HRMS(ESI)m / z calcd.for C 69 H 91 N 20 O 22 S + [M+H] + 1583.6332, found1583.6338.

[0106] Example 5

[0107] Example 5 of this invention provides the preparation and characterization of a functionally modified reagent containing DNIm or NHS active esters, comprising the following steps:

[0108] 1. Preparation of modifying reagents containing DNIm functional groups:

[0109] D1. Dissolve DNIm-NHS in biotin-NH2, fluorescent amine Dansyl-NH2, fluorescent amine Furazan-NH2, 2000 Da polyethylene glycol amine NH2-PEG-OH, and 5000 Da polyethylene glycol amine NH2-PEG-OH in HEPES buffer at pH 7.5 and react at 25°C for 2 h. The final concentrations of DNIm-NHS, biotin-NH2, Dansyl-NH2, Furazan-NH2, and NH2-PEG-OH in the reaction system are all 1 mM.

[0110] D2. After the reaction is completed, in reaction one of DNIm-NHS and biotin-NH2, a modifying reagent 11a containing DNIm functional group and biotin group is prepared; in reaction two of DNIm-NHS and fluorescein-NH2, a modifying reagent 12a containing DNIm functional group and fluorescein-NH2 is prepared, and byproduct 13a is generated; in reaction three of DNIm-NHS and fluorescein-NH2, a modifying reagent 14a containing DNIm functional group and fluorescein-NH2 is prepared, and byproduct 15a is generated; in reaction four of DNIm-NHS and 2000 Da NH2-PEG-OH, a modifying reagent 16a containing DNIm functional group and PEG molecule is prepared; in reaction five of DNIm-NHS and 5000 Da NH2-PEG-OH, a modifying reagent 17a containing DNIm functional group and PEG molecule is prepared.

[0111] D3. To perform MALDI-TOF analysis on the products of reactions four and five, they were first separated and purified by preparative HPLC, and all product peaks were collected together to obtain a collection solution. Compound 7,7,8,8-tetracyanoquinone dimethane was selected as the matrix and co-incubated with the collection solution for MALDI-TOF analysis. NH2-PEG-OH at 2000 Da and NH2-PEG-OH at 5000 Da were used as controls and were also analyzed by MALDI-TOF.

[0112] D4. The prepared modifying reagents and reaction byproducts were characterized by high performance liquid chromatography (HPLC) and high resolution tandem mass spectrometry (HRMS / MS) or matrix-assisted laser desorption / ionization-time mass spectrometry (MALDI-TOF):

[0113] The reaction process of reaction one is as follows: Figure 8 As shown in Figure A and the HPLC characterization results are as follows: Figure 8 As shown in B, the tandem mass spectrometry fragmentation analysis of product 11a is as follows: Figure 9 As shown in Figure A, HRMS / MS characterization is as follows: Figure 9 As shown in B;

[0114] The reaction process of reaction two is as follows: Figure 10 As shown in Figure A and the HPLC characterization results are as follows: Figure 10 As shown in B, the tandem mass spectrometry fragmentation analysis of product 12a is as follows. Figure 11 As shown in Figure A, HRMS / MS characterization is as follows: Figure 11 As shown in B; tandem mass spectrometry fragmentation analysis of byproduct 13a is as follows. Figure 12 As shown in Figure A, HRMS / MS characterization is as follows: Figure 12 As shown in B;

[0115] The reaction process of reaction three is as follows: Figure 13 As shown in Figure A and the HPLC characterization results are as follows: Figure 13 As shown in B, the fragmentation analysis of product 14a by tandem mass spectrometry is as follows. Figure 14 As shown in Figure A, HRMS / MS characterization is as follows: Figure 14 As shown in B; tandem mass spectrometry fragmentation analysis of byproduct 15a is as follows. Figure 15 As shown in Figure A, HRMS / MS characterization is as follows: Figure 15 As shown in B;

[0116] The reaction processes of reactions four and five are as follows: Figure 16 As shown in Figure A and the HPLC characterization results are as follows: Figure 16 As shown in Figure B, the MALDI-TOF diagram of NH2-PEG-OH at 2000 Da is shown in Figure 17A, and the MALDI-TOF diagram of product 16a from reaction four is shown in Figure 17A. Figure 17 As shown in B; the MALDI-TOF plot of NH2-PEG-OH at 5000 Da is shown in 18A; the MALDI-TOF plot of product 17a from reaction 5 is shown in... Figure 18 As shown in B.

[0117] 2. Preparation of modifying reagents containing NHS active ester functional groups

[0118] F1. DNIm-NHS and 2000 Da polyethylene glycol SH-PEG-OH containing thiol (-SH) were dissolved in HEPES buffer at pH 7.0, mixed thoroughly at 25°C, and reacted for 0.5 h to prepare the modifying reagent 18a containing NHS active ester functional groups.

[0119] F2. The prepared modification reagents were characterized by high performance liquid chromatography (HPLC) and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF).

[0120] F3. To perform MALDI-TOF analysis on product 18a, it was first separated and purified by preparative HPLC, and the product peak was collected to obtain a collection solution. Compound 7,7,8,8-tetracyanoquinone dimethane was selected as the matrix and co-incubated with the collection solution for MALDI-TOF analysis. SH-PEG-OH at 2000 Da was used as a control and was also analyzed by MALDI-TOF.

[0121] The reaction process of the modifying reagent 18a is as follows: Figure 16 As shown in Figure A and the HPLC characterization results are as follows: Figure 16 As shown in B; the MALDI-TOF plot of SH-PEG-OH at 2000 Da is shown in 19A, and the MALDI-TOF plot at 18a is shown in... Figure 19 As shown in B.

[0122] Performance verification

[0123] 1. Verification of the bioorthogonality of the crosslinking agent DNIm-NHS prepared in Example 1:

[0124] J1. DNIm-NHS was reacted with N-acetyl-L-cysteine ​​(Ac-Cys) in HEPES buffer at pH 6.5 or 7.4 for 2.5 min; HPLC and HRMS / MS analysis showed that the module involved in the reaction was DNIm; the reaction process is as follows. Figure 20 As shown in Figure A; tandem mass spectrometry fragmentation analysis and fragment molecular weights of reaction product 1a are shown in Figure A. Figure 21 As shown in A, the high-resolution tandem mass spectrum results are shown in 21B.

[0125] The reaction process of compound 3, which lacks the NHS ester functional group, with Ac-Cys in HEPES buffer at pH 7.4 is as follows: Figure 20 As shown in B; tandem mass spectrometry fragment analysis and fragment molecular weights of reaction product 2a are shown in Figure B. Figure 22 As shown in A, the high-resolution tandem mass spectrum results are shown in 22B.

[0126] HPLC analysis results of the reactions of DNIm-NHS and compound 3 with Ac-Cys are as follows: Figure 20 As shown in C;

[0127] J2. DNIm-NHS was reacted with Nα-acetyl-L-lysine (Ac-Lys) in HEPES buffer at pH 7.4 or 8.0 for 30 min; HPLC and HRMS / MS analysis showed that the reacting module was NHS; the reaction process and results are as follows. Figure 23 As shown in Figure A; tandem mass spectrometry fragmentation analysis and fragment molecular weights of reaction product 3a are shown in Figure A. Figure 24 As shown in A, the high-resolution tandem mass spectrum results are shown in 24B.

[0128] Compound 3, which lacks the NHS functional group, was reacted with Ac-Lys in HEPES buffer at pH 7.4 or 8.0 for 30 minutes. No reaction occurred. Figure 23 As shown in B;

[0129] HPLC analysis results of the reactions of DNIm-NHS and compound 3 with Ac-Lys are as follows: Figure 23 As shown in C.

[0130] 2. Verification of the protein-modifying effect of the protein modifier prepared in Example 5 includes the following steps:

[0131] (1) Single modification of proteins:

[0132] G1. Modifiers 11a, 12a, 14a, 16a, and 17a containing the functional group DNIm prepared in Example 5 were respectively incubated with bovine serum albumin (BSA) at 25°C in neutral HEPES buffer solution for 1 hour; the reaction process of 12a, 14a, 16a, and 17a is as follows. Figure 25 As shown; the reaction process and results of 11a are illustrated and demonstrated in the double modification of proteins;

[0133] G2. Modifiers 12a and 14a containing the functional group DNIm prepared in Example 5 were respectively incubated with the SARS-CoV-2 main protease (3CL) in neutral HEPES buffer solution at 25°C for 1 hour. The reaction process is as follows. Figure 25 As shown;

[0134] G3. The NHS-containing modifier 18a prepared in Example 5 was incubated with lysozyme and BSA in HEPES buffer at pH 7.5 for 2.5 h at 25 °C; the reaction process is as follows. Figure 25 As shown;

[0135] The corresponding proteins, including BSA, 3CL, and Lysozyme, were added to HEPES buffer at the same final concentration as the corresponding treatment group, serving as a blank control group.

[0136] G4 and modifier 11a contain a biotin group. Western blot analysis was performed, and the results are as follows: Figure 19 As shown in lanes A (8 and 9); the results all show the presence of a biotin group, indicating that a biotin group was successfully introduced into the BSA molecule;

[0137] Modifier 12a contains a Dansyl fluorescent group. Analysis was performed using SDS-PAGE and 302nm excitation imaging. The results are as follows: Figure 26 As shown in A and 26C;

[0138] The results demonstrate that BSA successfully introduced the Dansyl fluorescent group; and for the 3CL protein, the fluorescence intensity increased with increasing concentration of the modifying reagent.

[0139] The 14a molecule contains a Furazan fluorescent group. SDS-PAGE and blue light (440-485 nm) excitation imaging analysis were performed, and the results are as follows: Figure 26 As shown in B and 26D, compared with the corresponding blank control group, it shows that the Furazan fluorescent group was successfully introduced; and for the 3CL protein, the fluorescence intensity increased with the increase of the concentration of the modifying agent.

[0140] 16a and 17a contain 2000 Da and 5000 Da of PEG, respectively. SDS-PAGE analysis was performed, and the results are as follows: Figure 26 As shown in F, compared with the corresponding blank control group, the results indicate that PEG was successfully introduced;

[0141] SDS-PAGE technology was used to identify and analyze the characteristic PEG groups contained in the modifying reagent 18a. The results are as follows: Figure 26 As shown in E and F;

[0142] In the diagram, "++" indicates a high concentration of the modifier added; "+" indicates a low concentration of the modifier added; and "-" indicates no modifier added.

[0143] The reaction conditions are shown in Table 1, and all concentrations are final concentrations in the reaction system:

[0144] Table 1 Final concentrations of each substance in the protein monomodification reaction system

[0145]

[0146] (2) Dual modification of proteins:

[0147] H1. Mix BSA and 11a at a molar ratio of 1:10 in HEPES buffer at pH 8.0 for 30 min to obtain mixed solution one;

[0148] H2. Add HEPES buffer solution (pH 7.0) containing 18a to mixed solution 1, where the molar ratio of 18a to BSA added in H1 is 10:1. Continue the reaction for 2.5 h; the final BSA concentration is 25 μM. The reaction process is as follows: Figure 27 As shown in A;

[0149] H3. After the reaction was completed, all samples were analyzed by Western blot, and the results are as follows: Figure 27 As shown in lane 5 of swimming pool B;

[0150] H4 Figure 27The difference between lane 6 and lane 5 in swimmer B is that the order in which 11a and 18a are added is reversed.

[0151] Figure 27 The difference between lane 7 and lane 5 in swimming pool B is that lanes 11a and 18a are reduced to a molar ratio of 5:1 with BSA.

[0152] Figure 27 The difference between lanes 8, 9 and 3 in B and lane 5 is that BSA only reacts with 11a, which is a single protein modification and only biotinylation modification occurs.

[0153] Figure 27 B, the frame line section, and Figure 27 The arrows in C indicate that BSA is modified not only by biotinylation but also by PEGylation.

[0154] Results Analysis

[0155] See Figure 5-7 The heteromorphic bifunctional crosslinking agent DNIm-NHS prepared in Example 1 of this invention can be applied to the construction of cyclic peptides;

[0156] See Figure 8-16 This invention provides a method for preparing a functional modification reagent containing DNIm or NHS active esters; characterization results confirm that the functional modification reagent prepared by this invention includes the functional group DNIm or the functional group NHS active esters, and can be used for subsequent protein modification. Figure 16 HPLC analysis of the reactions of polyethylene glycol in component B with the crosslinking agent also showed several asymmetric broad peaks, which may be related to the fact that the raw material polyethylene glycol is a polymer with low homogeneity.

[0157] See Figure 17-19 Compared with the corresponding starting material PEG, the strongest (intermediate) peak of the product (part B of each figure) shifts slightly to the right (increases by about 250 m / z), indicating that a PEG product containing DNIm or NHS active esters was generated.

[0158] See Figure 20-24This invention connects two reaction modules, DNIm and NHS, with a suitable oxoaliphatic chain. One end of the chain is connected to the C2 atom of DNIm, and the other end is connected to the carboxyl carbon atom of NHS. The two reaction modules are separated by 7 atoms, forming a crosslinking agent, DNIm-NHS. When DNIm-NHS reacts with Ac-Cys in HEPES buffer at pH 6.5 or 7.4, high-performance liquid chromatography (HPLC) and high-resolution tandem mass spectrometry (HRMS / MS) analysis show that only the DNIm module reacts with the thiol group of Ac-Cys, while the NHS module is unaffected. When the crosslinking agent reacts with Ac-Lys in HEPES buffer at pH 7.4 or 8.0, HPLC and HRMS / MS analysis show that only the NHS module reacts with the amino group of Ac-Lys, while the DNIm module is unaffected. This indicates that the two modules of the crosslinking agent—DNIm and NHS—exhibit good bioorthogonality when reacting with thiol or amino groups, respectively.

[0159] See Figure 25-27 BSA contains one free cysteine ​​thiol group, and 3CL contains 12 cysteine ​​thiol groups; 11a, 12a, 14a, 16a, and 17a all contain the DNIm active functional group, which can react with the thiol groups of BSA and 3CL to generate corresponding modified products; the modified 3CL protein in the examples only shows the results of 12a and 14a modification; 18a contains the NHS active ester reaction group, Lysozyme contains 6 lysine residues, and BSA contains 59 lysine residues; 18a can react with the amino groups on the side chains of the lysine residues of Lysozyme or BSA to generate corresponding protein modified products;

[0160] In summary, this invention links DNIm and NHS active esters through organic synthesis to obtain the heterogeneous bifunctional crosslinking agent DNIm-NHS, such as... Figure 28 As shown, the DNIm-NHS prepared by this invention can be applied to the construction of cyclic peptides of different sizes, as well as the selective single modification of proteins by thiol or amino groups, including biotinylation, PEGylation, or the introduction of fluorescent substances, and the dual modification of a single protein molecule: biotinylation and PEGylation.

[0161] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A heterogeneous bifunctional crosslinking agent, characterized in that, The bifunctional crosslinking agent is named DNIm-NHS, and its structural formula is shown in Formula I: 。 2. The method for preparing a heterogeneous bifunctional crosslinking agent as described in claim 1, characterized in that, Includes the following steps: Compound 2, the condensing agent, and N-hydroxysuccinimide were sequentially added to the first solvent, stirred at room temperature for 15-16 h, and then concentrated and purified to obtain the heterogeneous bifunctional crosslinking agent shown in Formula I; the compound 2 is 6-((1,4-dinitro-1H-imidazol-2-yl)methoxy)hexanoic acid; the first solvent includes one or more of dichloromethane, chloroform, diethyl ether, tetrahydrofuran, ethyl acetate, N,N-dimethylformamide, N,N-dimethylacetamide, or dimethyl sulfoxide; the molar ratio of the compound 2, the condensing agent, and N-hydroxysuccinimide is 1:(1.0-1.2):(1.0-1.2).

3. The application of the heterobifunctional crosslinking agent as described in claim 1 in the construction of cyclic peptides.

4. The application of the heterobifunctional crosslinking agent as described in claim 1 in the chemical modification of proteins.

5. The application according to claim 4, characterized in that, The applications include protein biotinylation, protein PEGylation, and the introduction of fluorescent substances into proteins.

6. The application according to claim 4, characterized in that, The application includes the preparation of a modifying agent containing at least one of the following functional groups: N-hydroxysuccinimide active ester and dinitroimidazole.

7. The application of the heterogeneous bifunctional crosslinking agent as described in claim 1 in drug preparation.

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