Special-shaped difunctional cross-linking agent as well as preparation method and application thereof

Abstract

The invention discloses a special-shaped difunctional cross-linking agent as well as a preparation method and application thereof. The heterotype bifunctional cross-linking agent provided by the invention is DNIm-N3, is a bifunctional cross-linking agent containing an azide module (N3) and a dinitroimidazole module (DNIm), and can be used for connecting cyclooctyne with a compound containing sulfydryl. According to the azide module disclosed by the invention, a DNIm-containing functional reagent can be constructed through an azide-alkyne cycloaddition reaction; in a nearly neutral buffer solution, the DNIm module selectively reacts with sulfydryl; the heterotype bifunctional cross-linking agent provided by the invention has good biological orthogonality and can be used for modifying protein, polypeptide and oligonucleotide.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of heteromorphic bifunctional crosslinking agents, and in particular to a heteromorphic bifunctional crosslinking agent and a preparation method and application thereof. Background Art

[0002] Heterobifunctional crosslinker 1e is a type of bifunctional crosslinker containing dinitroimidazole (DNIm) and a terminal alkyne group (the synthesis of 1e can be found in DOI:10.1038 / s41467-018-08010-2), which can connect compounds containing thiol groups with compounds containing azide groups. However, 1e cannot construct functional reagents containing DNIm through CuAAC reaction, such as Figure 2 As shown. For example, when compound 1e reacts with azide d2 by CuAAC reaction, compound ed2 is generated. Although the expected cycloaddition reaction occurs between the alkynyl group and the azide, it is accompanied by an unexpected side reaction - the departure of the nitro group at the N1 position of the imidazole ring. The dinitroimidazole part of the resulting product cannot react with the sulfhydryl group due to the departure of the nitro group at the N1 position, and cannot be used for protein modification; moreover, we cannot use the CuAAC reaction to prepare dinitroimidazole functional molecules connected with various biological probes. Although chemical modification of proteins can be achieved by changing the reaction order, that is, first reacting compound 1e with the sulfhydryl group of the protein and then reacting the resulting protein intermediate with the azide reagent, two consecutive reactions are required on the protein macromolecule, and the reaction process includes tedious operations such as ultrafiltration to remove excess small molecules.

[0003] Therefore, it is necessary to develop a new heterobifunctional cross-linker to improve the above problems. Summary of the invention

[0004] The present invention aims to provide a heterobifunctional crosslinking agent, which improves the problem that the heterobifunctional crosslinking agent in the prior art cannot prepare a functional reagent containing DNIm through a CuAAC reaction.

[0005] In one aspect, the present invention provides a heterobifunctional crosslinker, which is named DNIm-N3 and has a structural formula as shown in Formula I:

[0006]

[0007] In a second aspect, the present invention provides a method for preparing a heterobifunctional cross-linking agent, comprising the following steps:

[0008] S1. Under nitrogen protection, compound d1, DPPA and DBU are dissolved in a first solvent for reaction, and purified after the reaction to obtain compound d2; the first solvent comprises any one of tetrahydrofuran, ethyl acetate, toluene, 1,4-dioxane and dichloromethane;

[0009] S2, adding the compound d2 to a second solvent containing hydrochloric acid for reaction, and purifying after the reaction to obtain a crude compound d3; the second solvent comprises any one of 1,4-dioxane, ethyl acetate, toluene and dichloromethane;

[0010] S3, dissolving compound m1, DCC and NHS in a third solvent, and purifying after reaction to obtain compound m2; the third solvent comprises any one of dichloromethane, ethyl acetate, toluene and 1,4-dioxane;

[0011] S4. In an alkaline buffer system, the compound m2 is dissolved in a fourth solvent and then mixed with the crude compound d3, and the target compound, i.e., the heterobifunctional cross-linking agent DNIm-N3, is obtained by purification after reaction; the fourth solvent includes acetonitrile, methanol, ethanol, tetrahydrofuran, dimethyl sulfoxide and N,N-dimethylformamide.

[0012] In a third aspect, the present invention provides an application of a heterobifunctional crosslinker in the preparation of a protein modification reagent, the application comprising using the heterobifunctional crosslinker DNIm-N3 to connect with a cyclooctyne compound through an azide-alkyne cycloaddition reaction to obtain a protein modification reagent containing a DNIm functional group.

[0013] In a fourth aspect, the present invention provides an application of a heterobifunctional cross-linking agent in protein modification, wherein the application includes polyethylene glycol modification, biotin modification and fluorescent labeling.

[0014] In a fifth aspect, the present invention provides an application of a heterobifunctional cross-linking agent in oligonucleotide functionalization, wherein the application includes polyethylene glycol modification, preparation of oligonucleotide-polypeptide and preparation of oligonucleotide-protein conjugates.

[0015] In a sixth aspect, the present invention provides an application of a heterobifunctional cross-linking agent in drug preparation.

[0016] The beneficial effects of the present invention include:

[0017] (1) The heterobifunctional cross-linking agent provided by the present invention can be used to prepare a variety of functional reagents containing DNIm modules through ring strain-promoted azide-alkyne cycloaddition reaction (SPAAC);

[0018] (2) The functional reagent prepared by the heterobifunctional cross-linking agent of the present invention can modify proteins, including polyethylene glycol modification, biotin modification and fluorescent labeling;

[0019] (3) The heterobifunctional cross-linking agent provided by the present invention can modify oligonucleotides and is applied in the field of oligonucleotide functionalization, including polyethylene glycol modification, preparation of oligonucleotide-polypeptide and preparation of oligonucleotide-protein conjugates. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the structural formula of the heterobifunctional cross-linking agent DNIm-N3;

[0021] Figure 2 It is the CuAAC reaction of heterobifunctional cross-linker 1e and azide reagent;

[0022] Figure 3 is the compound d2 obtained in Example 1 1 H NMR spectrum;

[0023] Figure 4 is the compound m2 obtained in Example 1 1 H NMR spectrum;

[0024] Figure 5 is the compound m2 obtained in Example 1 13 C NMR spectrum;

[0025] Figure 6 is the cross-linking agent DNIm-N3 obtained in Example 1 1 H NMR spectrum;

[0026] Figure 7 is the cross-linking agent DNIm-N3 obtained in Example 1 13 C NMR spectrum;

[0027] Figure 8 This is a flow chart of preparing a functional modification reagent containing DNIm by SPAAC reaction in Example 2;

[0028] Fig. 9 The schematic diagram and HPLC characterization diagram of the reaction between DNIm-N3 and BCN-biotin in Example 2;

[0029] Fig.10 The schematic diagram and HPLC characterization diagram of the reaction of DNIm-N3 and BCN-Dansyl in Example 2;

[0030] Fig.11 The schematic diagram and HPLC characterization diagram of the reaction of DNIm-N3 and BCN-Furazan in Example 2;

[0031] Fig.12 Schematic diagram of the reaction between DNIm-N3 and 5000Da BCN-PEG in Example 2;

[0032] Fig.13 is the compound ed2 prepared in Comparative Example 1 1 H NMR spectrum;

[0033] Fig.14 The schematic diagram and HPLC characterization diagram of the reaction between the DNIm module and the cysteine ​​(Cys) derivative in the cross-linking agent;

[0034] Fig.15 The schematic diagram and HPLC characterization diagram of the reaction between the DNIm module and the DBCO derivative in the cross-linking agent;

[0035] Fig.16 The schematic diagram and HPLC characterization diagram of the reaction between the DNIm module and DBCO derivative II in the cross-linking agent;

[0036] Fig.17 The schematic diagram of the reaction between the DNIm module in the cross-linking agent and the BCN derivative and the HPLC characterization diagram;

[0037] Fig.18 To verify the protein modification effect of the modification reagent prepared in Example 2;

[0038] Fig.19 Mass spectrometry characterization of the products obtained by modifying BSA with some of the modification reagents prepared in Example 2;

[0039] Fig. 20 To verify the protein modification effect of the modification reagent prepared in Example 2;

[0040] Fig.21 This is to verify the modification effect of the modification reagent prepared in Example 2 on oligonucleotides. DETAILED DESCRIPTION

[0041] The present invention will be further described through the following embodiments in conjunction with the accompanying drawings.

[0042] The present invention aims to provide a heterobifunctional crosslinking agent, which improves the problem that the heterobifunctional crosslinking agent in the prior art cannot prepare a functional reagent containing DNIm through a CuAAC reaction.

[0043] In one aspect, the present invention provides a heterobifunctional crosslinker, which is named DNIm-N3 and has a structural formula as shown in Formula I:

[0044]

[0045] In a second aspect, the present invention provides a method for preparing a heterobifunctional crosslinking agent, the reaction formula of which is shown below, comprising the following steps:

[0046]

[0047] S1. Under nitrogen protection, compound d1, DPPA and DBU are dissolved in a first solvent for reaction, and purified after the reaction to obtain compound d2; the first solvent comprises any one of tetrahydrofuran, ethyl acetate, toluene, 1,4-dioxane and dichloromethane;

[0048] S2, adding the compound d2 to a second solvent containing hydrochloric acid for reaction, and purifying after the reaction to obtain a crude compound d3; the second solvent comprises any one of 1,4-dioxane, ethyl acetate, toluene and dichloromethane;

[0049] S3, dissolving compound m1, DCC and NHS in a third solvent, and purifying after reaction to obtain compound m2; the third solvent comprises any one of dichloromethane, ethyl acetate, toluene and 1,4-dioxane;

[0050] S4. In an alkaline buffer system, the compound m2 is dissolved in a fourth solvent and then mixed with the crude compound, and the target compound, i.e., the heterobifunctional cross-linking agent DNIm-N3, is obtained by purification after reaction; the fourth solvent includes acetonitrile, methanol, ethanol, tetrahydrofuran, dimethyl sulfoxide and N,N-dimethylformamide.

[0051] In a third aspect, the present invention provides an application of a heterobifunctional crosslinker in the preparation of a protein modification reagent, the application comprising using the heterobifunctional crosslinker DNIm-N3 to connect with a cyclooctyne compound through an azide-alkyne cycloaddition reaction to obtain a protein modification reagent containing a DNIm functional group.

[0052] In a fourth aspect, the present invention provides an application of a heterobifunctional cross-linking agent in protein modification, wherein the application includes polyethylene glycol modification, biotin modification and fluorescent labeling.

[0053] In a fifth aspect, the present invention provides an application of a heterobifunctional cross-linking agent in oligonucleotide functionalization, characterized in that the application includes polyethylene glycol modification, preparation of oligonucleotide-polypeptide and preparation of oligonucleotide-protein conjugates.

[0054] In a sixth aspect, the present invention provides an application of a heterobifunctional cross-linking agent in drug preparation.

[0055] Example 1

[0056] Example 1 of the present invention provides a method for preparing a heterogeneous bifunctional crosslinking agent DNIm-N3, the reaction formula is as follows, comprising the following steps:

[0057]

[0058] S1. In a nitrogen atmosphere and under an ice bath, 240 mg of compound d1 (1.0 mmol) in 2 mL of dry tetrahydrofuran (THF) solution, 0.3 mL of diphenylphosphoryl azide (DPPA, 1.66 mmol) and 0.15 mL of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 1.15 mmol) in 8 mL of dry THF solution were added into a two-necked reaction bottle with a syringe. The mixture was stirred for 18 h and then concentrated in vacuo. The concentrated product was purified by rapid silica gel column chromatography, first eluted with petroleum ether and ethyl acetate in a volume ratio of 20:1, and then eluted with petroleum ether and ethyl acetate in a volume ratio of 10:1, to obtain 214 mg of a white solid with a yield of 82%, which was identified as compound d2 by nuclear magnetic resonance (NMR). Figure 3 The characterization data are as follows:

[0059] 1 H NMR (600MHz, CDCl3) δ7.33–7.28(m,4H),4.95(s,1H),4.33(s,4H),1.48(s,9H).;

[0060] S2, dissolving 16.5 mg of compound d2 (63 μmol) in 2 mL of 1,4-dioxane, slowly adding 0.55 mL of 4 mol / L HCl 1,4-dioxane solution at 0°C, stirring for 12 h, and analyzing by thin layer chromatography (TLC). When no raw material remained, concentrating to obtain a crude compound d3;

[0061] S3. The precursor compound m1 was synthesized by the method in reference (DOI: 10.1038 / s41467-018-08010-2), and then compound m2 was synthesized using m1 as a substrate: 130 mg of compound m1 (0.43 mmol), 97 mg of dicyclohexylcarbodiimide (DCC, 0.47 mmol), 54 mg of N-hydroxysuccinamide (NHS, 0.47 mmol), and 10 mL of dry dichloromethane (DCM) were added to a single-necked flask in sequence, and the mixture was stirred at 25° C. for 15.5 h and then concentrated in vacuo. After concentration, the mixture was purified by rapid silica gel column chromatography (the eluent was petroleum ether and ethyl acetate in a volume ratio of 10:6) to obtain 127 mg of a white solid with a yield of 74%, which was identified as compound m2 by NMR, as shown in FIG. Figures 4 to 5 The characterization data are as follows:

[0062] 1H NMR (500MHz, CDCl3) δ8.53 (s, 1H), 4.84 (s, 2H), 3.59 (t, J = 6.3Hz, 2H), 2.83 (s,4H),2.60(t,J=7.3Hz,2H),1.78–1.70(m,2H),1.63(m,2H),1.46(m,2H).

[0063] 13 C NMR (125MHz, CDCl3) δ169.19,168.51,141.50,115.10,71.42,64.98,30.81,28.84,25.59,25.15,24.23.;

[0064] S4, dissolving the crude compound d3 (0.063 mmol) in 10 mL of HEPES buffer at pH = 7.5 and a concentration of 0.2 mol / L, stirring vigorously, adding 1 mL of compound m2 (0.063 mmol) dissolved in acetonitrile, mixing and stirring at 25° C. for 1 h;

[0065] After stirring, 60 mL of dichloromethane was used for the first extraction, and after the extraction was completed, 20 mL of dichloromethane was used for the second extraction;

[0066] After the extraction was completed, the organic layer was washed three times with a saturated NaCl solution, using 3 mL of NaCl solution each time; after washing, it was dried with anhydrous sodium sulfate, filtered, and concentrated in vacuo. The concentrated product was purified by rapid silica gel column chromatography (the eluent was petroleum ether, ethyl acetate, and methanol in a volume ratio of 100:50:1). After purification, 25 mg of a white solid was obtained with a yield of 89%. It was identified as a heterobifunctional cross-linking agent DNIm-N3 by high-resolution mass spectrometry (HRMS) and NMR. Figure 6 to Figure 7 The characterization data are as follows:

[0067] 1 H NMR (400MHz, CDCl3) δ8.52(s,1H),7.31–7.27(m,4H),5.87(s,1H),4.83(s,2H),4.44(d,J=5.8Hz,2 H),4.32(s,2H),3.59(t,J=6.3Hz,2H),2.23(t,J=7.5Hz,2H),1.70–1.61(m,4H),1.43–1.37(m,2H).

[0068] 13C NMR (100MHz, CDCl3) δ172.73,141.64,138.64,134.62,128.60,128.27,115.06,71.63,65.05,54.44,43.20,36.50,29.72,29.06,25.59,25.20.

[0069] HRMS (ESI) m / z calcd.for C 18 H 23 N8O6 + [M+H] + 447.1735, found 447.1732.

[0070] Example 2

[0071] Example 2 of the present invention provides a method for preparing a protein modification reagent containing a DNIm functional group by a ring strain-promoted azide-alkyne cycloaddition reaction (SPAAC), the process is as follows: Figure 8 As shown, the following steps are included:

[0072] The crosslinker DNIm-N3 and the compound BCN-biotin were mixed in a HEPES buffer with a pH of 7.0 and reacted at 25°C for 14 h to obtain the modified reagent 3a containing the DNIm functional group. The reaction process is as follows: Fig. 9 As shown in A, the two-step reaction HPLC characterization is as follows Fig. 9 As shown in B;

[0073] Compound 3a was characterized by HRMS, and the characterization data are shown below:

[0074] HRMS (ESI) m / z calcd.for C 41 H 56 N 12 NaO 10 S + [M+Na] + 931.3855, found 931.3861.;

[0075] The crosslinker DNIm-N3 and the compound BCN-Dansyl were dissolved in methanol buffer and reacted at 25°C for 18 h to obtain the modified reagent 4a containing the DNIm functional group. The reaction process is as follows: Fig.10 As shown in A, the two-step reaction HPLC characterization is as follows Fig.10 As shown in B;

[0076] Compound 4a was characterized by HRMS, and the characterization data are as follows:

[0077] HRMS (ESI) m / z calcd.for C 43 H 55 N 11 O 10 S 2+ [M+2H] 2+ 458.6922, found 458.6920.;

[0078] The crosslinker DNIm-N3 and the compound BCN-Furazan were dissolved in methanol buffer and reacted at 25°C for 16 h to obtain the modified reagent 5a containing the DNIm functional group. The reaction process is as follows: Fig.11 As shown in A, the two-step reaction HPLC characterization is as follows Fig.11 As shown in B;

[0079] Compound 5a was characterized by HRMS, and the characterization data are shown below:

[0080] HRMS (ESI) m / z calcd.for C 39 H 50 N 13 O 11 S + [M+H] + 908.3468, found 908.3472.;

[0081] The crosslinking agent DNIm-N3 and the compound BCN-PEG were mixed and dissolved in a HEPES buffer with a pH of 7.0 and reacted at 25°C for 24 h to obtain a modification reagent 6a containing a DNIm functional group. The reaction process is as follows: Fig.12 As shown;

[0082] The above-mentioned BCN ring-containing compounds BCN-biotin, BCN-dansyl, BCN-furazan and BCN-PEG are intermediates, which are prepared by mixing the compound BCN-NHS with the corresponding primary amine substances biotin-NH2, Dansyl-NH2, Furazan-NH2, NH2-PEG-OH (5000Da) in weakly alkaline methanol and reacting at 25°C for 6h, 3.5h, 7h, and 21h respectively;

[0083] The obtained intermediates, except the polymer BCN-PEG, were characterized by HRMS, and the results were as follows:

[0084] BCN-biotin: HRMS(ESI)m / z calcd.for C 23 H 34 N4NaO4S + [M+Na] +485.2193, found 485.2198;

[0085] BCN-Dansyl:HRMS(ESI)m / z calcd.for C 25 H 31 N3NaO4S + [M+Na] + 492.1927, found 492.1929;

[0086] BCN-Furazan: HRMS(ESI)m / z calcd.for C 21 H 28 N5O5S + [M+H] + 462.1806, found 462.1812.

[0087] Comparative Example 1

[0088] Comparative Example 1 of the present invention provides a CuAAC reaction of 1e and azide compound d2, comprising the following steps:

[0089] D1, dissolve the crosslinker 1e and the azide compound d2 in 50mmol / L neutral HEPES buffer, the final concentration of 1e and d2 is 1mmol / L; add CuSO4 (0.5mmol / L), THPTA (0.5mmol / L) and VcNa (1mmol / L) in sequence, mix and stir for 5h;

[0090] D2. After the reaction was complete, ethyl acetate was added for extraction, and after washing with saturated NaCl, anhydrous Na2SO4 was added for drying and filtration, and the filtrate was concentrated in vacuo; the obtained residue was purified by rapid silica gel column chromatography (eluent: DCM and MeOH in a volume ratio of 50:1) to obtain 8 mg of light brown solid, which was identified as compound ed2 by HRMS and NMR, with a yield of 93%. 1 H NMR spectrum Fig.13 As shown;

[0091] HRMS and NMR characterization data are as follows:

[0092] 1 H NMR (400MHz, CDCl3) δ7.84(s,1H),7.46(s,1H),7.30(d,J=8.0Hz,2H),7.24(d,J=8.1Hz,2H),5.5 0(s,2H),5.12(s,1H),4.95(s,1H),4.72(s,2H),4.68(s,2H),4.31(d,J=5.3Hz,2H),1.45(s,9H).

[0093] HRMS (ESI) m / z calcd.for C 20 H 25 N7NaO5 + [M+Na] + 466.1809, found 466.1808.

[0094] See also Fig.13 The H signal of the imidazole ring in cross-linker 1 was located at 8.5 ppm, while the H signal of the imidazole ring in compound ed2 appeared at 7.84 ppm, indicating that -NO2 left the N1 position of the imidazole ring, resulting in the destruction of the dinitroimidazole ring and the inability to perform thiol-selective protein modification, which was consistent with the HRMS results.

[0095] Performance Verification

[0096] 1. Verification of the orthogonality of the cross-linking agent prepared in Example 1

[0097] (1) Reaction of DNIm module with thiol group

[0098] In a neutral HEPES buffer, the crosslinker DNIm-N3 prepared in Example 1 was reacted with L-cysteine ​​(Cys, compound c1). The reaction was characterized by HPLC and HRMS to generate compound mc1. The reaction process is as follows: Fig.14 As shown in A in Figure 1, the characterization data of mc1 are as follows:

[0099] HRMS (ESI) m / z calcd.for C 21 H 29 N8O6S + [M+H] + 521.1925, found 521.1929.;

[0100] In a neutral buffer, the cross-linking agent DNIm-N3 prepared in Example 1 was reacted with a cysteine-containing polypeptide (compound c2, AcNH-CWHKM-CONH2, purchased from Jiangsu Shenlang Biotechnology Co., Ltd.), and the reaction generated compound mc2 as characterized by HPLC and HRMS. The reaction process is as follows: Fig.14 As shown in B, the characterization data of mc2 is as follows:

[0101] HRMS (ESI) m / z calcd.for C 51 H 71 N 17 O 10 S2 2+ [M+2H] 2+572.7500, found 572.7497.;

[0102] The HPLC spectrum of the reaction between DNIm module and thiol group is as follows Fig.14 As shown in C, the characterization data of the by-product compound 2 during the reaction are as follows:

[0103] HRMS (ESI) m / z calcd.for C 18 H 24 N7O4 + [M+H] + 402.1884, found 402.1881.;

[0104] Only the DNIm module in the cross-linker reacts with the sulfhydryl group in the peptide, and the azido (N3) module in the cross-linker does not participate in the reaction.

[0105] (2) Reaction of N3 module with cyclooctyne compounds

[0106] In a 20% dimethyl sulfoxide aqueous solution, the crosslinking agent DNIm-N3 prepared in Example 1 was reacted with a DBCO derivative (compound d4), as follows: Fig.15 As shown, Fig.15 A in the figure is a schematic diagram of the reaction process; Fig.15 B in the figure is the HPLC spectrum of the reaction, in which md1 has isomers, so two peaks with similar integrated areas appear;

[0107] Through HRMS characterization, the reaction generated compound md1, and the characterization data are as follows:

[0108] HRMS (ESI) m / z calcd.for C 37 H 37 N9NaO9 + [M+Na] + 774.2606, found 774.2607.;

[0109] It was proved that under this condition, only the azido N3 module in the cross-linker reacted, and the DNIm module did not participate in the reaction;

[0110] In a neutral HEPES buffer, the cross-linking agent DNIm-N3 prepared in Example 1 was reacted with DBCO derivative II (compound d5), as follows: Fig.16 As shown, Fig.16 A in the figure is a schematic diagram of the reaction process; Fig.16 C in is the reaction HPLC spectrum;

[0111] The reaction generated compound 2a through HRMS characterization, and the characterization data are as follows:

[0112] HRMS (ESI) m / z calcd.for C 46 H 53 N 12 9S + [M+H] + 949.3774, found 949.3784.;

[0113] Further, 1,4-dinitroimidazole (DNIm) was incubated with compound d4 containing DBCO functional group in neutral buffer, and no reaction occurred. Fig.16 B in the figure is a schematic diagram of the reaction process; Fig.16 D in the figure is the HPLC spectrum of the reaction.

[0114] It was proved that under this condition, only the azido N3 module in the cross-linker reacted with the alkyne group of the DBCO derivative, and the DNIm module did not participate in the reaction, among which the reaction product 2a was also a modification reagent;

[0115] In a neutral HEPES buffer, the cross-linking agent DNIm-N3 prepared in Example 1 was reacted with a BCN derivative (compound d6), as follows: Fig.17 As shown, Fig.17 A in the figure is a schematic diagram of the reaction process; Fig.17 B in the figure is the reaction HPLC spectrum;

[0116] Through HRMS characterization, the reaction generated compound md2, and the characterization data are as follows:

[0117] HRMS (ESI) m / z calcd.for C 28 H 36 N8NaO7 + [M+Na] + 619.2599, found 619.2601.;

[0118] It was proved that under this condition, only the azido N3 module in the cross-linker reacted with the alkyne group of the BCN derivative, and the DNIm module did not participate in the reaction;

[0119] The bioorthogonality experiment verified that the two modules of the cross-linker DNIm-N3 prepared in Example 1 of the present invention, DNIm and azido N3, respectively reacted with Cys and cyclooctyne compounds (DBCO and BCN), showing good orthogonality. Therefore, the cross-linker prepared in Example 1 of the present invention is an excellent heterobifunctional cross-linker.

[0120] 2. Effects of the modification reagents 3a, 4a, 5a, 6a prepared in Example 2 and the modification reagent 2a prepared during verification in protein modification

[0121] In HEPES neutral buffer, the modification reagent was incubated with protein bovine serum albumin (BSA) or recombinant novel coronavirus main protease (3CL) for 2 h. The reaction process was as follows: Fig.18 A and B in Fig. 20 As shown in A, the concentrations of various substances in the reaction system are shown in Table 1;

[0122] Table 1 Final concentrations of modification reagents and proteins in the reaction system

[0123]

[0124] BSA contains one free cysteine ​​thiol group, and 3CL contains 12 cysteine ​​thiol groups. The amino acid sequence of 3CL is:

[0125] SGFRKMAFPSGKVEGCMVQVTCGTTTLNGLWLDDVVYCPRHVICTSEDML

[0126] NPNYEDLLIRKSNHNFLVQAGNVQLRVIGHSMQNCVLKLKVDTANPKTPKY

[0127] KFVRIQPGQTFSVLACYNGSPSGVYQCAMRPNFTIKGSFLNGSCGSVGFNIDY

[0128] DCVSFCYMHHMELPTGVHAGTDLEGNFYGPFVDRQTAQAAGTDTTTITVNVL

[0129] AWLYAAVINGDRWFLNRFTTTLNDFNLVAMKYNYEPLTQDHVDILGPLSAQTGIAVLDMCASLKELLQNGMNGRTILGSALLEDEFTPFDVVRQCSGVTFQ.

[0130] The DNIm active functional groups in the modification reagent can react with the sulfhydryl groups of the two proteins to generate corresponding modification products, and each modification product is characterized; the modification products containing biotin groups are analyzed by Western Blot or SDS-PAGE, and the modification products containing fluorescent groups are analyzed by SDS-PAGE and 302nm or blue light (440-485nm) excitation imaging; the modification products containing PEG are analyzed by SDS-PAGE:

[0131] (1) Characterization of modification products of modification reagents 2a and 3a

[0132] The modification reagent 2a and modification reagent 3a molecules contain biotin groups, and Western Blot analysis, such as Fig.18 As shown in C, D, E, and F, lanes 1-5 are the analysis results of BSA protein modification products, and lanes 6-9 are the analysis results of 3CL protein modification products:

[0133] Lane 1 represents the BSA modification product of adding a cross-linking agent first and then adding BCN-biotin, lane 2 represents the BSA modification product of modification reagent 3a, lane 3 represents the control group, i.e., BSA protein, lane 4 represents the BSA modification product of modification reagent 2a, and lane 5 represents the control group, i.e., BSA protein. The bands show that all contain biotin groups; and the relevant protein modification products are further identified by mass spectrometry: Fig.19 A is the deconvoluted mass spectrum of raw BSA protein. Fig.19 B is the deconvoluted mass spectrum of the intermediate of the reaction between BSA and DNIm-N3. Fig.19 C is the deconvoluted mass spectrum of the products of the reaction of BSA with DNIm-N3 and BCN-biotin in sequence. Fig.19 D is the deconvoluted mass spectrum of BSA (measured for the second time), Fig.19 E is the deconvoluted mass spectrum of the product of the reaction between BSA and 3a. Fig.19 F is the deconvoluted mass spectrum of the product of the reaction between BSA and 2a; these modification reagents all showed high modification yields for BSA and selectively modified the thiol group. However, Fig.19 After the thiol group of a small amount of BSA in F reacts with the modification reagent 2a, the amino group continues to react with 2a (the mass spectrometry signal marked by the asterisk in the figure), which may be due to the addition of a certain proportion of organic solvent DMSO in the reaction (the reaction solution contains 3% DMSO); these results show that the modification reagent provided by the present invention can biotinylate BSA protein;

[0134] Lane 6 represents the 3CL modification product of modification reagent 2a, lane 7 represents the control group, i.e., 3CL protein, lane 8 represents the 3CL modification product of modification reagent 3a, and lane 9 represents the control group, i.e., 3CL protein;

[0135] right Fig.18 The products in lanes 8 and 9 of F were analyzed by SDS-PAGE. Fig.18 As shown in G in FIG. 1 , it further illustrates that the method provided by the present invention can perform biotinylation modification on 3CL protein.

[0136] (2) Characterization of modification products of modification reagents 4a, 5a, and 6a

[0137] The Dansyl fluorescent group in the protein modification product of 4a was analyzed by SDS-PAGE and 302nm ultraviolet excitation imaging analysis technology; the Furazan fluorescent group in the protein modification product of 5a was analyzed by SDS-PAGE and blue light (440-485nm) excitation imaging analysis technology; the PEG group in the protein modification product of 6a was analyzed by SDS-PAGE; in the figure, "++" indicates the addition of a high concentration of modification reagent, "+" indicates the addition of a low concentration of modification reagent, and "-" indicates no addition of modification reagent, and the control group is the group without the addition of modification reagent;

[0138] See also Fig. 20 In B and C, compared with the control group, the bands indicate that the Dansyl fluorescent group was successfully introduced into the BSA protein and the 3CL protein, and for the 3CL protein, the fluorescence intensity increased with the increase of the concentration of the modification reagent; in the light excitation imaging analysis, Fig. 20 As shown in C, the molecular weight of the fluorescent band in the frame is larger, which proves that more fluorescent groups are connected to the 3CL protein; the 4a modification reagent provided by the present invention can successfully modify the BSA protein and the 3CL protein.

[0139] See also Fig. 20 In D and E, compared with the control group, the bands indicate that the Furazan fluorescent group was successfully introduced into the BSA protein and the 3CL protein, and for the 3CL protein, the fluorescence intensity increased with the increase in the concentration of the modification reagent; in the light excitation imaging analysis, Fig. 20 As shown in E in the figure, the molecular weight of the fluorescent band in the frame is larger, which proves that more Furazan fluorescent groups are connected to the 3CL protein; the 5a modification reagent provided by the present invention can successfully modify the BSA protein and the 3CL protein.

[0140] See also Fig. 20 In F and G, compared with the control group, the addition of modification reagent 6a introduced PEG into the protein molecules, but the yield was lower; Fig. 20 As shown in G, the yield increased after increasing the concentration of the modification reagent, but not significantly.

[0141] 3. Verification of the chemical modification effect of the cross-linking agent prepared in Example 1 on oligonucleotides

[0142] Select an oligonucleotide with 22 bases and a DBCO group at the 5' end (o1);

[0143] In neutral HEPES buffer, o1 was reacted with the cross-linking agent DNIm-N3 via SPAAC to prepare the intermediate DNA-D-DNIm;

[0144] The reaction products of DNA-D-DN Im and sulfhydryl-containing compounds were analyzed by Urea-PAGE, such as Fig.21 As shown in lanes 1-4, lane 1 is the analysis band of o1, which is the control group; lane 2 is the band of the intermediate DNA-D-DNIm;

[0145] Lane 3: The intermediate DNA-D-DNIm reacts with SH-PEG-OH (2000 Da) containing a thiol group, and the product is oligonucleotide-PEG;

[0146] Lane 4: The intermediate DNA-D-DNIm was conjugated with the cysteine-containing peptide AcNH-CWHKM-CONH2 (c2), and the product was an oligonucleotide-peptide conjugate;

[0147] Urea-PAGE analysis revealed that o1 was successfully coupled with PEG or peptide;

[0148] The coupling products of oligonucleotide and BSA protein were analyzed by SDS-PAGE. Fig.21 As shown in lanes 5-9; Lane 5 is BSA protein, i.e. the control group; Lane 6 is the reaction of BSA protein and cross-linking agent 1, and the product bands are shown in the figure;

[0149] Lane 7: After o1 reacted with DNIm-N3 crosslinker, BSA was added to react, and the product band is shown in the figure;

[0150] Lane 8: After the BSA protein reacted with the DNIm-N3 cross-linker, o1 was added for reaction, and the product band is shown in the figure;

[0151] Lane 9: Oligonucleotide-BSA conjugate was prepared by reacting BSA, o1, and DNIm-N3 in a one-pot method;

[0152] The SDS-PAGE results show that the results of changing the reaction order are basically the same, and the conversion rate is about 50%, which proves that the cross-linking agent provided by the present invention can modify oligonucleotides.

[0153] Although the embodiments of the present invention are described in detail above, it is obvious 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 are within the scope and spirit of the present invention as described in the claims. Moreover, the present invention described herein may have other embodiments and may be implemented or realized in a variety of ways.

Claims

1. A heterobifunctional crosslinking agent, characterized in that: The heterobifunctional crosslinker is named DNIm-N3, and its structural formula is shown in Formula I:

2. The method for preparing a heterobifunctional crosslinking agent according to claim 1, characterized in that: The following steps are involved: S1. Under nitrogen protection, compound d1, DPPA and DBU are dissolved in a first solvent for reaction, and purified after the reaction to obtain compound d2; the first solvent comprises any one of tetrahydrofuran, ethyl acetate, toluene, 1,4-dioxane and dichloromethane; S2, adding the compound d2 to a second solvent containing hydrochloric acid for reaction, and purifying after the reaction to obtain a crude compound d3; the second solvent comprises any one of 1,4-dioxane, ethyl acetate, toluene and dichloromethane; S3, dissolving compound m1, DCC and NHS in a third solvent, and purifying after reaction to obtain compound m2; the third solvent comprises any one of dichloromethane, ethyl acetate, toluene and 1,4-dioxane; S4. In an alkaline buffer system, the compound m2 is dissolved in a fourth solvent and then mixed with the crude compound d3, and the target compound, i.e., the heterobifunctional cross-linking agent DNIm-N3, is obtained by purification after reaction; the fourth solvent includes acetonitrile, methanol, ethanol, tetrahydrofuran, dimethyl sulfoxide and N,N-dimethylformamide.

3. Use of the heterobifunctional cross-linking agent according to claim 1 in preparing a protein modification reagent, characterized in that: The application includes using the heterobifunctional cross-linking agent DNIm-N3 to connect with cyclooctyne compounds through an azide-alkyne cycloaddition reaction to prepare a protein modification reagent containing a DNIm functional group.

4. Use of the heterobifunctional cross-linking agent according to claim 1 in protein modification, characterized in that: The applications include polyethylene glycol modification, biotin modification and fluorescent labeling.

5. Use of the heterobifunctional cross-linking agent according to claim 1 in oligonucleotide functionalization, characterized in that: The applications include polyethylene glycol modification, preparation of oligonucleotide-polypeptide and preparation of oligonucleotide-protein conjugates.

6. Use of the heterobifunctional cross-linking agent as claimed in claim 1 in drug preparation.

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