Protein / polypeptide-drug coupling method

Abstract

The invention discloses a protein / polypeptide-drug coupling method which comprises a method I or a method II. The method I comprises the following steps: carrying out mixed reaction on a salicylaldehyde derivative, a phenylboronic acid derivative and secondary amine, and then carrying out mixed reaction with lysine-containing polypeptide and / or protein to complete protein / polypeptide-drug coupling; the method II comprises the following steps: mixing a salicylaldehyde derivative, an aryne potassium trifluoroborate derivative and tryptophan-containing polypeptide and / or protein for reaction to complete protein / polypeptide-drug coupling; at least one of the salicylaldehyde derivative, the phenylboronic acid derivative and the aryne potassium trifluoroborate derivative comprises at least one drug molecule group capable of breaking and releasing drug molecules. The method is simple in condition, efficient in reaction and high in reaction site selectivity, coupling of various drugs and different polypeptides / proteins can be achieved, drug molecules can be released, and the pharmacodynamic activity of the drug molecules is reserved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a protein / peptide-drug coupling method. Background Art

[0002] With the rapid development of biotechnology, targeted therapy has received increasing attention in the treatment of complex diseases. As important means of targeted therapy, peptide-drug conjugates (PDCs) and antibody-drug conjugates (ADCs) have gradually become hot spots in research and clinical application due to their superior specificity and effectiveness. These technologies can significantly improve the specificity, targeting and effectiveness of the treatment of various diseases by combining the targeting specificity of peptides or antibodies with the efficacy of conventional drugs, while reducing toxicity to normal cells and enhancing the therapeutic effect of drugs. Currently, there are a variety of PDCs or ADCs in clinical trials, some of which have been approved on the market and used in clinical treatment, such as Mylotarg and Polivy. The successful launch of these drugs has demonstrated the potential of targeted therapy in the field of anti-tumor and has also promoted the further development of related conjugation technologies.

[0003] At present, the methods of protein / peptide-drug coupling mainly include protein engineering, biocoupling and chemical coupling. Among them, protein engineering methods can achieve precise design and site-specific labeling, but the process is usually complicated and the cost is high. The biocoupling method is highly specific and suitable for application in complex biological environments, but its effect is limited by enzyme stability and background interference. In contrast, chemical coupling methods have attracted widespread attention due to their simple operation and high efficiency, such as innovative methods for common cysteine ​​(alkylation and cross-linking) and lysine (acylation or alkylation) coupling, and expansion to coupling technologies based on carbonyl, amide, and azide (Chem. Soc. Rev. 2021, 50, 14801494; ChemBioChem 2023, 24, e202300254). However, due to factors such as drug structure and its release, many coupling technologies may face challenges of insufficient selectivity and poor stability, and the metal reagents used in some methods may have certain toxicity to organisms in some cases. It can be seen that the chemical coupling method still has broad room for development. Further optimization of coupling technology can promote the process of protein / peptide-drug coupling, improve coupling efficiency and selectivity, and achieve higher therapeutic effects. Summary of the invention

[0004] In view of the above-mentioned technical problems and the shortcomings in the art, the present invention provides a protein / peptide-drug coupling method with simple conditions, efficient reaction, and high reaction site selectivity. It can realize the coupling of various drugs with different polypeptides / proteins (such as aprotinin, etc.), and can release drug molecules while retaining the pharmacodynamic activity of drug molecules.

[0005] A protein / peptide-drug conjugation method, including method I or method II;

[0006] Method I: salicylaldehyde derivatives, phenylboronic acid derivatives and secondary amines are mixed and reacted, and then mixed and reacted with peptides and / or proteins containing lysine to complete protein / peptide-drug coupling; taking peptide as an example, the general reaction formula of method I is as follows: Fig.32 As shown, R 4 -NH-R 5 represents a secondary amine, aa represents an amino acid, and K represents lysine;

[0007] Method II: Mixing salicylaldehyde derivatives, potassium aryl trifluoroborate derivatives and tryptophan-containing peptides and / or proteins to complete protein / peptide-drug coupling;

[0008] The salicylaldehyde derivative has the following structure:

[0009]

[0010] The phenylboronic acid derivative has the following structure:

[0011]

[0012] The aralkynyl potassium trifluoroborate derivative has the structure shown below:

[0013]

[0014] R 1 , R 2 , R 3 Each independently represents H or one or more substituents (such as C1-C4 alkoxy, etc.), and R 1 , R 2 , R 3 One or more of the drug molecules contain at least one drug molecule group that can be cleaved to release the drug molecule.

[0015] In the protein / peptide-drug coupling method, the polypeptide and / or protein containing lysine in method I and the polypeptide and / or protein containing tryptophan in method II can be independently one or more of free polypeptides and / or proteins, polypeptides and / or proteins connected to the resin. Further, the resin can include one or more of 2-Cl-Trt and Rink-Amide-MBHA. When the polypeptide and / or protein connected to the resin is used, it can be further cut by a mixed solution of trifluoroacetic acid (TFA) / triisopropylsilane (TIPS) / water to obtain a protein / peptide-drug conjugate. Furthermore, the volume ratio of trifluoroacetic acid, triisopropylsilane and water in the mixed solution of trifluoroacetic acid (TFA) / triisopropylsilane (TIPS) / water can be 95:1:4.

[0016] The protein / peptide-drug coupling method, method I, can be carried out in the first solvent.

[0017] The protein / peptide-drug coupling method, method II, can be carried out in a second solvent.

[0018] Furthermore, the first solvent and the second solvent can be independently selected from one or a mixture of acetonitrile, 1,2-dichloroethane, toluene, methanol, PBS buffer solution, N,N-dimethylformamide, dimethyl sulfoxide, 1,4-dioxane, and water.

[0019] The reaction temperatures of the protein / peptide-drug coupling method, method I and method II can be independently selected from room temperature to 120° C., such as 80° C.

[0020] In the protein / peptide-drug coupling method, the drug molecules released by breaking can be independently selected from at least one of ibuprofen, naproxen, fenbufen, indomethacin, diclofenac, etc.

[0021] In the protein / peptide-drug coupling method, the drug molecule group can release the drug molecule by breaking one or more of the ester bond and the amide bond. The ester bond and the amide bond are chemical bonds that are relatively easy to break, which is conducive to effectively releasing the drug molecule and taking effect.

[0022] In the protein / peptide-drug coupling method, the secondary amine can be one or more of 2-methyltetrahydropyrrole, diethylamine, dibutylamine, N-methylaniline, tetrahydropyrrole, 2,2'-dimethyltetrahydropyrrole, 2-(diphenylmethyl)pyrrolidine, prolineamide, prolinol, piperidine, 3,5-dimethylpiperidine, piperazine, morpholine, etc.

[0023] The protein / peptide-drug coupling method described is metal-free.

[0024] In the protein / polypeptide-drug coupling method, the lysine-containing polypeptide and / or protein in method I and the tryptophan-containing polypeptide and / or protein in method II can independently have one or more properties of transmembrane property and targeting specificity.

[0025] The protein / peptide-drug conjugation method, R 1 , R 2 , R 3 One or more of the may contain at least one group having fluorescent properties.

[0026] In the protein / peptide-drug coupling method, the salicylaldehyde derivative can be selected from any one or more of the following:

[0027]

[0028] In the protein / peptide-drug coupling method, the phenylboronic acid derivative can be selected from any one or more of the following:

[0029]

[0030] In the protein / peptide-drug coupling method, the aryl alkynyl potassium trifluoroborate derivative can be:

[0031]

[0032] In the protein / peptide-drug coupling method, the lysine-containing polypeptide in method I and the tryptophan-containing polypeptide in method II can each independently have 2 to 50 amino acids.

[0033] The present invention also provides a protein / peptide-functional molecule coupling method, wherein the functional molecule may be a molecule with fluorescent properties, etc., similar to method I and method II, wherein R 1 , R 2 , R 3 It is not necessary that one or more of them contain at least one drug molecule group that can be cleaved to release the drug molecule. 1 , R 2 , R 3 One or more of them contain at least one functional molecular group, such as a molecular group with fluorescent properties, etc., of course R 1 , R 2 , R 3 One or more of them may also contain at least one drug molecule group that can be broken to release drug molecules. The further specific selection of the protein / peptide-functional molecule coupling method, technical scheme and parameter conditions can refer to the protein / peptide-drug coupling method introduced above.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The method of the present invention has simple conditions, efficient reactions, and high reaction site selectivity, and can realize the coupling of various drugs, functional molecules and different polypeptides / proteins (such as aprotinin, etc.), and can release drug molecules while retaining the pharmacodynamic activity of drug molecules and the original functions of functional molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the liquid chromatography (LC) chart of the product iii-1 of Example 1.

[0037] Figure 2 This is the mass spectrum (MS) of the product iii-1 in Example 1.

[0038] Figure 3 This is the LC chart of the product iii-2 in Example 2.

[0039] Figure 4 This is the MS chart of product iii-2 in Example 2.

[0040] Figure 5 This is the LC chart of product iii-3 of Example 3.

[0041] Figure 6 This is the MS chart of product iii-3 in Example 3.

[0042] Figure 7 This is the LC chart of product iii-4 of Example 4.

[0043] Figure 8 This is the MS chart of product iii-4 in Example 4.

[0044] Fig. 9 This is the LC chart of the product iii-5 of Example 5.

[0045] Fig.10 This is the MS chart of product iii-5 in Example 5.

[0046] Fig.11 This is the LC chart of product iii-6 from Example 6.

[0047] Fig.12 This is the MS chart of product iii-6 in Example 6.

[0048] Fig.13 This is the LC chart of product iii-7 of Example 7.

[0049] Fig.14 This is the MS chart of product iii-7 in Example 7.

[0050] Fig.15 This is the LC chart of the product v-1 of Example 8.

[0051] Fig.16 This is the MS chart of the product v-1 in Example 8.

[0052] Fig.17 This is the LC chart of the product v-2 of Example 9.

[0053] Fig.18 This is the MS chart of the product v-2 in Example 9.

[0054] Fig.19 This is the LC chart of the product v-3 of Example 10.

[0055] Fig. 20 This is the MS chart of the product v-3 in Example 10.

[0056] Fig.21 This is the LC chart of the product v-4 of Example 11.

[0057] Fig. 22 This is the MS chart of the product v-4 in Example 11.

[0058] Fig.23 This is the LC chart of the product iii-8 of Example 12.

[0059] Fig.24 This is the MS chart of the product iii-8 in Example 12.

[0060] Fig.25 This is the LC chart of the product iii-9 in Example 13.

[0061] Fig.26 This is the MS chart of product iii-9 in Example 13.

[0062] Fig. 27 This is the LC chart of the product iii-10 of Example 14.

[0063] Fig.28 This is the MS chart of product iii-10 in Example 14.

[0064] Fig.29 This is the LC chart of the product iii-11 of Example 15.

[0065] Fig.30 This is the MS chart of product iii-11 in Example 15.

[0066] Fig.31 This is the MS chart of product vi-1 in Example 16.

[0067] Fig.32 The figure is a schematic diagram of the general reaction formula of the protein / polypeptide-drug coupling method I of the present invention taking polypeptide as an example. DETAILED DESCRIPTION

[0068] The present invention will be further described below in conjunction with the accompanying drawings and specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention. The operating methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0069] Example 1

[0070]

[0071] 4-Methoxyphenylboronic acid (ii-1) (4.6 mg, 0.03 mmol), acetonitrile (1 mL), salicylaldehyde (i-1) (2.4 mg, 0.02 mmol), and 2-methylpyrrolidine (2.6 mg, 0.03 mmol) were added to a reaction tube equipped with a magnetic bar in sequence, and stirred at 80°C for 2 hours. Then, the peptide (AcHN-Lys-Leu-Pro-Val-Met-CONH2) (amino acid sequence KLPVM, SEQ ID NO: 1) (0.01 mmol) was added, and stirring was continued at 80°C for 20 hours. After the reaction was completed, it was purified by preparative liquid separation with a yield of 70%. The LC and MS test results of the product iii-1 are as follows: Figure 1 , Figure 2 shown.

[0072] Example 2

[0073]

[0074] 4-Methoxyphenylboronic acid (ii-1) (4.6 mg, 0.03 mmol), acetonitrile (1 mL), salicylaldehyde (i-1) (2.4 mg, 0.02 mmol), and 2-methyltetrahydropyrrole (2.6 mg, 0.03 mmol) were added to a reaction tube equipped with a magnetic bar in sequence, and stirred at 80°C for 2 hours. Then the polypeptide (AcHN-Met-Pro-Phe-Arg-Trp-Phe-Lys-Pro-Val-CONH2) (amino acid sequence MPFRWFKPV, SEQ ID NO: 2) (0.01 mmol) was added, and stirring was continued at 80°C for 20 hours. After the reaction was completed, the product was purified by preparative liquid separation with a yield of 77%. The LC and MS test results of the product iii-2 are shown as follows: Figure 3 , Figure 4 shown.

[0075] Example 3

[0076]

[0077] 4-Methoxyphenylboronic acid (ii-1) (4.6 mg, 0.03 mmol), salicylaldehyde derivative (i-2) (7.4 mg, 0.02 mmol), acetonitrile (1 mL), 2-methyltetrahydropyrrole (2.6 mg, 0.03 mmol) were added to a reaction tube equipped with a magnetic bar in sequence, and stirred at 80°C for 2 hours. Then the polypeptide (AcHN-Lys-Leu-Pro-Val-Met-CONH2) (amino acid sequence KLPVM, SEQ ID NO: 1) (0.01 mmol) was added, and stirring was continued at 80°C for 20 hours. After the reaction was completed, it was purified by preparative liquid phase separation with a yield of 68%. The LC and MS test results of the product iii-3 are as follows: Figure 5 , Figure 6 shown.

[0078] Example 4

[0079]

[0080] 4-Methoxyphenylboronic acid (ii-1) (4.6 mg, 0.03 mmol), salicylaldehyde derivative (i-3) (7.8 mg, 0.02 mmol), acetonitrile (1 mL), 2-methyltetrahydropyrrole (2.6 mg, 0.03 mmol) were added to a reaction tube equipped with a magnetic bar in sequence, and stirred at 80°C for 2 hours. Then the polypeptide (AcHN-Lys-Leu-Pro-Val-Met-CONH2) (amino acid sequence KLPVM, SEQ ID NO: 1) (0.01 mmol) was added, and stirring was continued at 80°C for 20 hours. After the reaction was completed, it was purified by preparative liquid separation with a yield of 56%. The LC and MS test results of the product iii-4 are as follows: Figure 7 , Figure 8 shown.

[0081] Example 5

[0082]

[0083] 4-Methoxyphenylboronic acid (ii-1) (4.6 mg, 0.03 mmol), salicylaldehyde derivative (i-4) (8.3 mg, 0.02 mmol), acetonitrile (1 mL), 2-methyltetrahydropyrrole (2.6 mg, 0.03 mmol) were added to a reaction tube equipped with a magnetic bar in sequence, and stirred at 80°C for 2 hours. Then the polypeptide (AcHN-Lys-Leu-Pro-Val-Met-CONH2) (amino acid sequence KLPVM, SEQ ID NO: 1) (0.01 mmol) was added, and stirring was continued at 80°C for 20 hours. After the reaction was completed, it was purified by preparative liquid phase separation with a yield of 53%. The LC and MS test results of the product iii-5 are as follows: Fig. 9 , Fig.10 shown.

[0084] Example 6

[0085]

[0086] 4-Methoxyphenylboronic acid (ii-1) (4.6 mg, 0.03 mmol), salicylaldehyde derivative (i-5) (10.4 mg, 0.02 mmol), acetonitrile (1 mL), 2-methyltetrahydropyrrole (2.6 mg, 0.03 mmol) were added to a reaction tube equipped with a magnetic bar in sequence, and stirred at 80°C for 2 hours. Then the polypeptide (AcHN-Lys-Leu-Pro-Val-Met-CONH2) (amino acid sequence KLPVM, SEQ ID NO: 1) (0.01 mmol) was added, and stirring was continued at 80°C for 20 hours. After the reaction was completed, it was purified by preparative liquid phase separation with a yield of 45%. The LC and MS test results of the product iii-6 are as follows: Fig.11 , Fig.12 shown.

[0087] Example 7

[0088]

[0089] The phenylboronic acid derivative (ii-2) (12.5 mg, 0.03 mmol), acetonitrile (1 mL), salicylaldehyde (i-1) (2.4 mg, 0.02 mmol), and 2-methyltetrahydropyrrole (2.6 mg, 0.03 mmol) were added to a reaction tube equipped with a magnetic bar in sequence, and stirred at 80°C for 2 hours. Then the polypeptide (AcHN-Lys-Leu-Pro-Val-Met-CONH2) (amino acid sequence KLPVM, SEQ ID NO: 1) (0.01 mmol) was added, and stirring was continued at 80°C for 20 hours. After the reaction was completed, it was purified by preparative liquid separation with a yield of 55%. The LC and MS test results of the product iii-7 are as follows: Fig.13 , Fig.14 shown.

[0090] Example 8

[0091]

[0092] Potassium phenylethynyl trifluoroborate (iv-1) (4.2 mg, 0.02 mmol), salicylaldehyde derivative (i-2) (7.4 mg, 0.02 mmol), polypeptide (H2N-Tyr-Pro-Trp-Phe-CONH2) (amino acid sequence YPWF, SEQ ID NO: 3) (0.01 mmol), acetonitrile (1 mL) were added to a reaction tube equipped with a magnetic bar in sequence and stirred at 80°C for 20 hours. After the reaction was completed, the product was purified by preparative liquid phase separation with a yield of 35%. The LC and MS test results of the product v-1 are as follows: Fig.15 , Fig.16 shown.

[0093] Example 9

[0094]

[0095] Potassium phenylethynyl trifluoroborate (iv-1) (4.2 mg, 0.02 mmol), salicylaldehyde derivative (i-3) (7.8 mg, 0.02 mmol), polypeptide (H2N-Tyr-Pro-Trp-Phe-CONH2) (amino acid sequence YPWF, SEQ ID NO: 3) (0.01 mmol), acetonitrile (1 mL) were added to a reaction tube equipped with a magnetic bar, and stirred at 80°C for 20 hours. After the reaction was completed, the product was purified by preparative liquid separation with a yield of 33%. The LC and MS test results of the product v-2 were as follows: Fig.17 , Fig.18 shown.

[0096] Example 10

[0097]

[0098] Potassium phenylethynyl trifluoroborate (iv-1) (4.2 mg, 0.02 mmol), salicylaldehyde derivative (i-4) (8.3 mg, 0.02 mmol), polypeptide (H2N-Tyr-Pro-Trp-Phe-CONH2) (amino acid sequence YPWF, SEQ ID NO: 3) (0.01 mmol), acetonitrile (1 mL) were added to a reaction tube equipped with a magnetic bar in sequence and stirred at 80°C for 20 hours. After the reaction was completed, the product was purified by preparative liquid phase separation with a yield of 45%. The LC and MS test results of the product v-3 are shown as follows: Fig.19 , Fig. 20 shown.

[0099] Embodiment 11

[0100]

[0101] Potassium phenylethynyl trifluoroborate (iv-1) (4.2 mg, 0.02 mmol), salicylaldehyde derivative (i-5) (10.4 mg, 0.02 mmol), polypeptide (H2N-Tyr-Pro-Trp-Phe-CONH2) (amino acid sequence YPWF, SEQ ID NO: 3) (0.01 mmol), acetonitrile (1 mL) were added to a reaction tube equipped with a magnetic bar, and stirred at 80°C for 20 hours. After the reaction was completed, the product was purified by preparative liquid separation with a yield of 29%. The LC and MS test results of the product v-4 are as follows: Fig.21 , Fig. 22 shown.

[0102] Example 12

[0103]

[0104] The phenylboronic acid derivative (ii-3) (11.1 mg, 0.03 mmol), salicylaldehyde derivative (i-2) (7.4 mg, 0.02 mmol), acetonitrile (1 mL), and 2-methyltetrahydropyrrole (2.6 mg, 0.03 mmol) were added to a reaction tube equipped with a magnetic bar in sequence, and stirred at 80°C for 2 hours. Then, the solid phase peptide (AcHN-Lys-Leu-Pro-Val-Met-CONH-Resin) (amino acid sequence KLPVM, SEQ ID NO: 1) (50 mg) was added, and stirring was continued at 80°C for 20 hours. After the reaction was completed, the filtered solid was cut with TFA / TIPS / H2O (volume ratio 95 / 1 / 4), dried, and purified by preparative liquid phase separation with a yield of 44%. The LC and MS test results of the product iii-8 are shown as follows: Fig.23 , Fig.24 shown.

[0105] Embodiment 13

[0106]

[0107] The phenylboronic acid derivative (ii-3) (11.1 mg, 0.03 mmol), salicylaldehyde derivative (i-3) (7.8 mg, 0.02 mmol), acetonitrile (1 mL), and 2-methyltetrahydropyrrole (2.6 mg, 0.03 mmol) were added to a reaction tube equipped with a magnetic bar in sequence, and stirred at 80°C for 2 hours. Then the polypeptide (AcHN-Lys-Leu-Pro-Val-Met-CONH-Resin) (amino acid sequence KLPVM, SEQ ID NO: 1) (50 mg) was added, and stirring continued at 80°C for 20 hours. After the reaction was completed, the filtered solid was cut with TFA / TIPS / H2O (volume ratio 95 / 1 / 4), dried, and purified by preparative liquid phase separation with a yield of 40%. The LC and MS test results of the product iii-9 are shown as follows: Fig.25 , Fig.26 shown.

[0108] Embodiment 14

[0109]

[0110] The phenylboronic acid derivative (ii-3) (11.1 mg, 0.03 mmol), salicylaldehyde derivative (i-4) (8.3 mg, 0.02 mmol), acetonitrile (1 mL), and 2-methyltetrahydropyrrole (2.6 mg, 0.03 mmol) were added to a reaction tube equipped with a magnetic bar in sequence, and stirred at 80°C for 2 hours. Then the polypeptide (AcHN-Lys-Leu-Pro-Val-Met-CONH-Resin) (amino acid sequence KLPVM, SEQ ID NO: 1) (50 mg) was added, and stirring continued at 80°C for 20 hours. After the reaction was completed, the filtered solid was cut with TFA / TIPS / H2O (volume ratio 95 / 1 / 4), dried, and purified by preparative liquid phase separation with a yield of 47%. The LC and MS test results of the product iii-10 are shown as follows: Fig. 27 , Fig.28 shown.

[0111] Embodiment 15

[0112]

[0113] The phenylboronic acid derivative (ii-4) (11.1 mg, 0.03 mmol), salicylaldehyde derivative (i-5) (10.4 mg, 0.02 mmol), acetonitrile (1 mL), and 2-methyltetrahydropyrrole (2.6 mg, 0.03 mmol) were added to a reaction tube equipped with a magnetic bar in sequence, and stirred at 80°C for 2 hours. Then the polypeptide (AcHN-Lys-Leu-Pro-Val-Met-CONH-Resin) (amino acid sequence KLPVM, SEQ ID NO: 1) (50 mg) was added, and stirring continued at 80°C for 20 hours. After the reaction was completed, the filtered solid was cut with TFA / TIPS / H2O (volume ratio 95 / 1 / 4), dried, and purified by preparative liquid phase separation with a yield of 47%. The LC and MS test results of the product iii-11 are shown as follows: Fig.29 , Fig.30 shown.

[0114] Example 16

[0115]

[0116] 4-Methoxyphenylboronic acid (ii-1) (23.4 mg, 0.15 mmol), salicylaldehyde derivative (i-3) (39.2 mg, 0.1 mmol), acetonitrile (1 mL), 2-methyltetrahydropyrrole (12.8 mg, 0.15 mmol) were added to a reaction tube equipped with a magnetic bar in sequence, and stirred at 80°C for 2 hours. Aprotinin (CAS No. 9087-70-1) (0.001 mmol) and PBS solution (1 mL) were added, and stirred at room temperature for 3 days. After the reaction was completed, the product was analyzed by LCMS. The MS test results of product vi-1 are as follows: Fig.31 shown.

[0117] In addition, it should be understood that after reading the above description of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A protein / peptide-drug conjugation method, characterized in that: Including Method I or Method II; Method I: salicylaldehyde derivatives, phenylboronic acid derivatives and secondary amines are mixed and reacted, and then mixed and reacted with peptides and / or proteins containing lysine to complete protein / peptide-drug coupling; Method II: Mixing salicylaldehyde derivatives, potassium aryl trifluoroborate derivatives and tryptophan-containing peptides and / or proteins to complete protein / peptide-drug coupling; The salicylaldehyde derivative has the following structure: The phenylboronic acid derivative has the following structure: The aralkynyl potassium trifluoroborate derivative has the structure shown below: R 1 , R 2 , R 3 Each independently represents H or one or more substituents, and R 1 , R 2 , R 3 One or more of the drug molecules contain at least one drug molecule group that can be cleaved to release the drug molecule.

2. The protein / peptide-drug conjugation method according to claim 1, characterized in that: The polypeptide and / or protein containing lysine in method I and the polypeptide and / or protein containing tryptophan in method II are independently one or more of free polypeptide and / or protein and polypeptide and / or protein connected to a resin; The resin includes one or more of 2-Cl-Trt and Rink-Amide-MBHA; The lysine-containing polypeptide in method I and the tryptophan-containing polypeptide in method II each independently have 2 to 50 amino acids.

3. The protein / peptide-drug conjugation method according to claim 1, characterized in that: Method I is carried out in a first solvent; Method II is carried out in a second solvent; The first solvent and the second solvent are independently selected from acetonitrile, 1,2-dichloroethane, toluene, methanol, PBS buffer solution, N,N-dimethylformamide, dimethyl sulfoxide, 1,4-dioxane, water, or a mixture of the two or more thereof.

4. The protein / peptide-drug conjugation method according to claim 1, characterized in that: The reaction temperature of method I and method II is independently selected from room temperature to 120°C.

5. The protein / peptide-drug conjugation method according to claim 1, characterized in that: The drug molecules released by rupture are independently selected from at least one of ibuprofen, naproxen, fenbufen, indomethacin and diclofenac.

6. The protein / peptide-drug conjugation method according to claim 1, characterized in that: The drug molecule group releases the drug molecule by breaking one or more of an ester bond and an amide bond.

7. The protein / peptide-drug conjugation method according to claim 1, characterized in that: The secondary amine is one or more of 2-methyltetrahydropyrrole, diethylamine, dibutylamine, N-methylaniline, tetrahydropyrrole, 2,2'-dimethyltetrahydropyrrole, 2-(diphenylmethyl)pyrrolidine, prolineamide, prolinol, piperidine, 3,5-dimethylpiperidine, piperazine, and morpholine.

8. The protein / peptide-drug conjugation method according to claim 1, characterized in that: The protein / peptide-drug coupling method does not involve metals; The lysine-containing polypeptide and / or protein described in method I and the tryptophan-containing polypeptide and / or protein described in method II independently have one or more properties of transmembrane property and targeting specificity.

9. The protein / peptide-drug conjugation method according to claim 1, characterized in that: R 1 , R 2 , R 3 One or more of the compounds contain at least one group having fluorescent properties.

10. The protein / peptide-drug conjugation method according to claim 1, characterized in that: The salicylaldehyde derivative is selected from any one or more of the following: The phenylboronic acid derivative is selected from any one or more of the following: The aralkynyl potassium trifluoroborate derivative is:

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