Maleimide-to-polypeptide site selective alkylate and method

Abstract

The invention provides a maleimide-to-polypeptide site selective alkylate and a method, and belongs to the technical field of biomedical material manufacturing. The preparation method comprises the following steps: dissolving palladium salt, maleimide and phenylalanine polypeptide in a solvent to form a mixed solution; reacting the mixed solution under a heating condition, and separating out a reaction product after the reaction is completed, so as to obtain the polypeptide containing the benzoquinone structure. According to the scheme, endogenous methionine in the polypeptide is used as a guiding group, so that tedious steps such as pre-installation and subsequent removal of the guiding group in the reaction process are avoided, and the atom economy and the reaction efficiency of the reaction process are improved.

Description

Technical Field

[0001] The present application relates to a product and method for selective alkylation of polypeptide sites by maleimide, and belongs to the technical field of biomedical material manufacturing. Background Art

[0002] The information disclosed in this background section is intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Peptides are important drug candidates and are mainly used to treat metabolic diseases, cancer, allergies and immunity, and cardiovascular diseases. At the same time, they are also key tools for regulating protein-protein interactions (PPIs). Due to the lack of structural diversity or enzymatic degradation, natural peptides often have poor pharmacological properties. Studies have shown that chemically modified non-natural peptides have higher binding affinity to the target and can significantly improve their pharmacokinetic properties, improve the stability of the molecule and cell permeability.

[0004] Transition metal-catalyzed directed CH functionalization has become a useful strategy for peptide modification, allowing precise editing of peptide molecules at previously inaccessible sites. Among them, modification of aliphatic residues without obvious functional handles in peptides has received extensive attention. For example, Yu's group found that the C-terminal carboxylic acid group or the adjacent amide of a dipeptide can serve as an auxiliary group to achieve β-C(sp 3 )-H arylation (Site-selective C(sp 3 )-H functionalization of di-, tri-, and tetrapeptides at the N-terminus. J. Am. Chem. Soc. Wei Gong, Guofu Zhang, Tao Liu, Ramesh Giri, and Jin-Quan Yu. 2014, 136, 16940-16946). Recently, the Ackermann and Weng groups used the side chains of aspartic acid or asparagine as the directing group to achieve the arylation of the alanine residue at the N-terminus of the peptide (Nikolaos Kaplaneris, Meijie Zhu, Hantao Chen, Zhuo Chen, Lutz Ackermann. Peptide late-stage C (sp 3)-H arylation by native asparagineassistance without exogenous directing groups.Chem.Sci.Yiyi Weng,XingxingDing,Xiaobin Xu,2020,11,9290-9295,Palladium-catalyzed C(sp 3 )-Harylation and alkynylation of peptides directed by aspartic acid (Asp). ACS Catal. Zhuo Chen, Meijie Zhu, Mengwei Cai, Lulu Xu, and Yiyi Weng. 2021, 11, 7401-7410.). However, to the best of our knowledge, the current methods for directing the inert side chain C (sp 3 There are no reports on alkylation of )-H. Summary of the invention

[0005] In view of this, the present application first provides a product of site-selective alkylation of a polypeptide with maleimide, which is a polypeptide containing a quinone structure, and realizes the alkylation of alanine β-C (sp 3 )-H coupling with maleimide.

[0006] Specifically, the present application is implemented through the following scheme:

[0007] A product of site-selective alkylation of a polypeptide with maleimide, characterized in that the structural formula of the product satisfies: Among them, R 1 Any one selected from Me, Ph, Et, i-Pr, tBu, R 2 Any one selected from OMe, OBn, polypeptide fragment, and glutamic acid residue.

[0008] Preferably, the product is:

[0009]

[0010] Any of .

[0011] The second object of the applicant is to provide a method for selectively alkylating the above-mentioned product with maleimide to a polypeptide site, the steps of which are as follows:

[0012] Step 1: dissolving palladium salt, maleimide and alanine-containing polypeptide in a solvent to form a mixed solution.

[0013] The alanine-containing polypeptide structural formula is:

[0014] Step 2: The mixed solution obtained in step 1 is reacted under heating conditions, and after the reaction is completed, the reaction product is separated, which is the site-selective alkylation product of the polypeptide by maleimide.

[0015] The above scheme is to use methionine-directed palladium to catalyze the coupling of alanine and maleimide in the polypeptide, that is, to apply the methionine-directed strategy, use palladium as a catalyst, and use the polypeptide of the above structure as a reaction raw material to achieve the later selective alkylation modification of the polypeptide. At the same time, by introducing different groups into the polypeptide containing alanine, a series of maleimide alkylation modifications of the polypeptide are successfully achieved. In the present invention, maleimide is used as an alkylating agent to selectively alkylate alanine β-C (sp 3 The advantage of the synthetic strategy of the present invention is that the endogenous methionine in the polypeptide is used as the guiding group, thereby avoiding the tedious steps of pre-installation and subsequent removal of the guiding group during the reaction process, thereby improving the atom economy and reaction efficiency of the reaction process.

[0016] Further, as a preference:

[0017] In step one,

[0018] In the mixed solution, the molar ratio of palladium salt, maleimide and alanine-containing polypeptide is 1:5:10 to 1:50:100.

[0019] The solvent is any one of 1,2-dichloroethane, dichloromethane, hexafluoroisopropanol and methanol.

[0020] The palladium salt is any one of tetrakistriphenylphosphine palladium (Pd(PPh3)4), palladium acetate (Pd(OAc)2), palladium trifluoroacetate (Pd(TFA)2), and palladium chloride (PdCl2). The functions of the palladium salt include: Pd(II) coordinates with the S of methionine in the polypeptide, and then forms a six-membered palladium ring intermediate to react with the alanine δ-C (sp 3 )-H for activation. Then maleimide coordinates with palladium through the double bond and double bond insertion, and finally undergoes reductive elimination to form the final alkylation product.

[0021] In step 2,

[0022] The reaction under heating conditions refers to the reaction at 50-120°C for 4-10 hours, and preferably at 80°C for 5 hours.

[0023] The separation refers to separation of the reaction product by column chromatography, and the eluent is petroleum ether (bp 60-90° C.)-ethyl acetate.

[0024] In the above scheme, silver salts such as silver carbonate (Ag2CO3), silver acetate (AgOAc), silver phosphate (Ag3PO4), and silver nitrate (AgNO3) can also be added as oxidants in step one to achieve smooth catalytic cycle.

[0025] The present invention uses endogenous methionine Met in a polypeptide as a guiding group and maleimide as an alkylating agent to achieve site-selective modification of alanine residues in a polypeptide molecule under palladium catalysis. The present invention makes full use of the guiding factors inside the polypeptide, greatly improving the atomic economy and reaction efficiency of the reaction. The advantages of the method for site-selective alkylation of polypeptides of the present invention are:

[0026] (1) Selectively achieve alkylation of alanine residues in polypeptide molecules;

[0027] (2) Shows good compatibility with polypeptide molecules;

[0028] (3) Having good separation yield;

[0029] (4) The modified molecules have potential biological activity against tumor cells.

[0030] The above scheme shows excellent compatibility with polypeptide sequences, good separation yield and potential inhibitory activity on tumor cells. Therefore, the third purpose of the applicant is to provide the application of the above product, that is, the product of the selective alkylation of polypeptide sites by maleimide is used in the preparation of anti-tumor drugs. This further shows that the present application has potential application value in the later selective modification of polypeptides and proteins.

[0031] Compared with the prior art, the present invention has the following beneficial and unique effects:

[0032] (1) In the present invention, palladium salt is used as a catalyst, and a polypeptide containing alanine of a specific structure is used as a reaction raw material to efficiently perform site-selective alkylation on alanine residues in the polypeptide. At the same time, different groups are introduced into the alanine-containing polypeptide to successfully achieve selective modification of polypeptides of different sequences and lengths.

[0033] (2) The present invention uses maleimide as an alkylating agent to successfully achieve the selective functionalization of alanine residues in polypeptides. Importantly, the reaction process does not require the participation of an external directing group, and fully utilizes the endogenous methionine in the polypeptide as a directing group to achieve alkylation modification of the N-terminal alanine residue of the polypeptide via a six-membered palladium ring intermediate. The present invention is easy to operate, has good chemical selectivity, and has high compatibility with polypeptides, indicating that the reaction strategy has potential application value in the later selective modification of polypeptides and proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1 is the NMR spectrum of the product of Example 1;

[0036] Figure 2 is the NMR spectrum of the product of Example 2;

[0037] Figure 3 is the NMR spectrum of the product of Example 3;

[0038] Figure 4 The NMR spectrum of the product of Example 4 is shown in FIG. DETAILED DESCRIPTION

[0039] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application clearer, the technical solutions in the embodiments of this application will be further described in detail below in conjunction with the drawings in the embodiments of this application. It should be understood that the specific embodiments described herein are only used to explain this application and are not used to limit the technical solutions of this application. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0040] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. The reagents or raw materials used in the present invention can be purchased through conventional channels. If not otherwise specified, the reagents or raw materials used in the present invention are used in a conventional manner in the art or in accordance with the product instructions. In addition, any method and material similar to or equivalent to the described content can be applied to the method of the present invention. The preferred implementation methods and materials described in the present invention are for demonstration purposes only. The present invention is further described according to the accompanying drawings and specific embodiments of the specification.

[0041] In the following examples, the reaction equation for the selective alkylation of polypeptides is shown below:

[0042]

[0043] Example 1

[0044] The product of this example is an alkylated modified dipeptide, and the specific process is as follows:

[0045] (1) The peptide Phth-Ala-Met-OMe (1.0 mmol), 1,2-dichloroethane (10 mL), palladium acetate (10 mol%), N-methylmaleimide (2.0 mmol), and silver carbonate (2.0 mmol) were added to a 100 mL pressure bottle and heated in an oil bath at 80°C for 5 h.

[0046] (2) After the reaction was completed, the reaction mixture was concentrated under reduced pressure using a rotary evaporator at a speed of 120 rps, a water bath temperature of 45° C., and a water pump vacuum of 0.1 MPa. The product was then separated by silica gel column chromatography using petroleum ether-ethyl acetate as the eluent for gradient elution to obtain a quinone-modified polypeptide compound represented by formula A with a yield of 86%.

[0047] The alkylated modified dipeptide constructed in this example was subjected to nuclear magnetic resonance detection, and the detection results were: 1 H NMR (500MHz, CDCl3) δ7.89-7.84(m,2H),7.79-7.75(m,2H),7.25(d,J=8.2Hz,1H),5.2 3(dd,J=9.9,4.5Hz,1H),4.70(dt,J=6.5,4.7Hz,1H),3.50-3.42(m,3H),3.03-2.99(m, 1H),2.90-2.83(m,2H),2.82-2.78(m,3H),2.61-2.51(m,2H),2.45(t,J=7.1Hz,2H),2 .06(ddd,J=14.9,7.4,2.6Hz,1H), 1.99(d,J=1.6Hz,3H), 1.84(dd,J=14.1,7.0Hz,1H).

[0048] Figure 1 is the alkylated modified dipeptide prepared in this example ( 1 HNMR, 500M, solvent CDCl3) nuclear magnetic resonance spectrum.

[0049]

[0050] Example 2

[0051] The product of this example is an alkylation-modified dipeptide, and the specific process is as follows:

[0052] (1) The peptide Phth-Ala-Met-OMe (1.0 mmol), dichloromethane (10 mL), palladium acetate (10 mol%), N-phenylmaleimide (2.0 mmol), and silver phosphate (2.0 mmol) were added to a 100 mL pressure tube and heated in an oil bath at 80° C. for 5 h.

[0053] (2) After the reaction is completed, the reaction mixture is concentrated under reduced pressure using a rotary evaporator at a speed of 120 rps, a water bath temperature of 45° C., and a water pump vacuum of 0.1 MPa. The product is then separated by silica gel column chromatography using petroleum ether-ethyl acetate as the eluent for gradient elution to obtain the alkylated polypeptide product represented by formula B with a yield of 80%.

[0054] The alkylated dipeptide synthesized in this example was subjected to nuclear magnetic resonance detection, and the detection results were: 1 H NMR (500MHz, CDCl3) δ7.87-7.81(m,2H),7.77-7.72(m,2H),7.40(dd,J=10.4,4.8Hz,2H),7.35-7.32(m,1 H),7.20(dd,J=5.2,3.4Hz,2H),7.09(dd,J=14.6,7.9Hz,1H),5.28-5.21(m,1H),4.72(td,J=7.6,4.9Hz, 1H),3.59(d,J=13.6Hz,3H),3.14(ddd,J=12.9,7.9,4.7Hz,1H),3.01-2.92(m,2H),2.74-2.66(m,2H),2. 46(dd,J=9.2,5.2Hz,2H),2.11(tdd,J=14.3,9.7,4.8Hz,1H),1.99(s,3H),1.92(dd,J=14.3,7.1Hz,1H).

[0055] Figure 2 This example prepares the alkylated dipeptide ( 1 HNMR, 500M, solvent CDCl3) nuclear magnetic resonance spectrum.

[0056]

[0057] Example 3

[0058] The product of this example is an alkylation-modified tripeptide, and the specific process is as follows:

[0059] (1) The peptide Phth-Ala-Met-Val-OMe (1 mmol), 1,2-dichloroethane (10 mL), palladium acetate (5 mol%), N-methylmaleimide (2.0 mmol), and silver carbonate (2.0 mmol) were added to a 100 mL pressure tube and heated in an oil bath at 80°C for 5 h.

[0060] (2) After the reaction is completed, the reaction mixture is concentrated under reduced pressure using a rotary evaporator at a speed of 120 rps, a water bath temperature of 45° C., and a water pump vacuum of 0.1 MPa. The product is then separated by silica gel column chromatography using petroleum ether-ethyl acetate as the eluent for gradient elution to obtain the alkylated tripeptide product shown in formula C with a yield of 79%.

[0061] The alkylated tripeptide constructed in this example was subjected to nuclear magnetic resonance detection, and the detection results were: 1 H NMR (500MHz, CDCl3) δ7.88(dd,J=5.1,3.1Hz,2H),7.78(dd,J=5.4,3.0Hz,2H),7.15(d,J=7.3Hz,1H),6.84(d,J= 5.2Hz,1H),5.14(dd,J=10.1,4.7Hz,1H),4.69(q,J=6.9Hz,1H),4.42-4.35(m,1H),3.71(s,3H),2.91(dd,J=10. 6,4.9Hz,1H),2.85(s,3H),2.78(ddd,J=11.6,9.9,5.2Hz,2H),2.59(dt,J=10.6,5.3Hz,3H),2.45(dd,J=18.0,5 .3Hz,1H),2.17(dd,J=12.5,6.4Hz,1H),2.06(s,3H),2.00(dd,J=13.6,6.9Hz,2H),0.91(dd,J=10.1,6.9Hz,6H).

[0062] Figure 3 is the alkylated tripeptide prepared in this example ( 1 HNMR, 500M, solvent CDCl3) nuclear magnetic resonance spectrum.

[0063]

[0064] Example 4

[0065] The product of this example is an alkylation-modified tripeptide, and the specific process is as follows:

[0066] (1) The peptide Phth-Ala-Met-Leu-OMe (1.0 mmol), 1,2-dichloroethane (10 mL), palladium acetate (5 mol%), N-methylmaleimide (2.0 mmol), and silver carbonate (2.0 mmol) were added to a 100 mL pressure tube and heated in an oil bath at 80°C for 6 h.

[0067] (2) After the reaction is completed, the reaction mixture is concentrated under reduced pressure using a rotary evaporator at a speed of 120 rps, a water bath temperature of 45° C., and a water pump vacuum of 0.1 MPa. The product is then separated by silica gel column chromatography using petroleum ether-ethyl acetate as the eluent for gradient elution to obtain the polypeptide alkylation product represented by formula D, with a yield of 75%.

[0068] The alkylated polypeptide constructed in this example was subjected to nuclear magnetic resonance detection, and the detection results were: 1 H NMR (500MHz, CDCl3) δ7.88(dd,J=5.4,3.0Hz,2H),7.78(dd,J=5.5,3.0Hz,2H),7.12(d,J=7.4Hz,1H),6.77(d,J=7. 7Hz, 1H), 5.13 (dd, J=10.1, 4.7Hz, 1H), 4.65 (q, J=6.8Hz, 1H), 4.49 (td, J=8.6, 4.9Hz, 1H), 3.69 (s, 3H), 2.91 (dd, J= 8.9,5.6Hz,1H),2.85(s,3H),2.82-2.73(m,2H),2.58(ddd,J=9.9,7.4,2.9Hz,3H),2.44(dd,J=18.0,5.2Hz,1H),2. 06(d,J=2.6Hz,3H),2.03-1.99(m,2H),1.67-1.61(m,2H),1.57(dd,J=9.1,7.5Hz,1H),0.92(dd,J=9.6,6.0Hz,6H).

[0069] Figure 4 is the alkylated polypeptide prepared in this example ( 1 HNMR, 500M, solvent CDCl3) nuclear magnetic resonance spectrum.

[0070]

[0071] In the above-mentioned examples 1 to 4, different groups were introduced into the alanine-containing polypeptides, and the selective modification of polypeptides of different sequences and lengths was successfully achieved. From the experimental results, it can be seen that in the reaction system of the present application, maleimide and amino acid-containing polypeptides can obtain alkylation products with a higher yield, and the guiding effect of methionine can be used to achieve the selective modification of polypeptides. Comparison of Examples 1 and 2 shows that when the polypeptide structure is the same, maleimide alkane of different structures matches the corresponding catalyst and solvent, and the yield can be basically the same as that of Example 1. This also indirectly proves that in this reaction system, the reaction activity of N-methylmaleimide is slightly weaker than that of N-phenylmaleimide; and when the polypeptide structure is different (comparative examples 3 and 4), the same maleimide still obtains a good yield, proving that the reaction shows good compatibility with polypeptide molecules.

[0072] The cytotoxicity of the alkylated modified polypeptide prepared in the above example was determined by the MTT method to verify the anti-tumor activity of the prepared alkylated product. The detection process is as follows:

[0073] U87MG and Hela cells were routinely cultured in DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin at 37°C and 5% CO2. Cells were seeded in 96-well plates at a density of 10,000 cells / well and cultured overnight. The next day, cells were treated with serial 2-fold dilutions of compounds ranging from 200 to 3.125 μM. After incubation at 37°C and 5% CO2 for 24 h, 50 μL of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h-tetrazolium bromide) and alkylated products were added to each well and incubated at 37°C for 4 h. After incubation for 4 h, the supernatant was removed, 150 μL of dimethyl sulfoxide (DMSO) was added, and the plates were shaken for 10 min and placed in a microplate reader (Spectra Max M2e, Molecular Devices, USA) to measure the absorbance (A490) value at 490 nm. Relative viability was calculated by dividing the mean absorbance value of duplicate wells containing treated cells by the absorbance value of wells containing untreated cells (100%). All experiments were performed three times independently.

[0074] Table 1: Cytotoxicity of alkylated products (IC 50 , μM)

[0075] Alkylation products U87MG(μM) Hela(μM) Example 1 >42±3 >100 Example 2 30±6 >100 Example 3 >100 37±4 Example 4 26±2 35±5

[0076] It can be seen from Table 1 that the alkylation products corresponding to Examples 1 and 2 have significant cytotoxicity to U87MG, the alkylation product prepared in Example 3 exhibits high cytotoxicity to Hela cells, and the alkylation product prepared in Example 4 exhibits high activity to both U87MG and Hela cells. Therefore, according to different usage requirements, the polypeptides given in this application are reacted with maleimide to obtain products with different anti-tumor effects.

[0077] Example 5

[0078] The product of this example is an alkylation-modified dipeptide, and the specific process is as follows:

[0079] (1) The peptide Phth-Ala-Met-OMe (1 mmol), 1,2-dichloromethane (10 mL), palladium acetate (10 mol%), and N-methylmaleimide (2.0 mmol) were added to a 100 mL pressure tube and heated in an oil bath at 80°C for 5 h.

[0080] (2) After the reaction is completed, the reaction mixture is concentrated under reduced pressure using a rotary evaporator at a speed of 120 rps, a water bath temperature of 45° C., and a water pump vacuum of 0.1 MPa. The product is then separated by silica gel column chromatography using petroleum ether-ethyl acetate as the eluent for gradient elution to obtain the dipeptide alkylation product represented by formula A with a yield of 36%.

[0081] Compared with Example 1, under the same conditions, when no silver salt is added as an oxidant, the catalytic cycle of the catalyst cannot be carried out, resulting in a significantly lower yield in this example than in Example 1.

[0082] Example 6

[0083] The product of this example is an alkylation-modified tripeptide, and the specific process is as follows:

[0084] (1) The peptide Phth-Ala-Met-OMe (1 mmol), acetonitrile (10 mL), palladium acetate (10 mol%), N-methylmaleimide (2.0 mmol), and silver carbonate (2.0 mmol) were added to a 100 mL pressure tube and heated in an oil bath at 100° C. for 5 h.

[0085] (2) After the reaction is completed, the reaction mixture is concentrated under reduced pressure using a rotary evaporator at a speed of 120 rps, a water bath temperature of 45° C., and a water pump vacuum of 0.1 MPa. The product is then separated by silica gel column chromatography using petroleum ether-ethyl acetate as the eluent for gradient elution to obtain the polypeptide alkylation product represented by formula A with a yield of 23%.

[0086] Compared with Example 1, under the same conditions, when the common solvent acetonitrile is selected as the solvent, the yield of the alkylation product is significantly lower than the yield corresponding to the solvent selected in the present application. This also shows that the reaction system provided in the present application has specific requirements for the solvent, and not any solvent can be applied to the reaction system of the present application and obtain the corresponding high yield.

[0087] Example 7

[0088] The product of this example is an alkylation-modified tripeptide, and the specific process is as follows:

[0089] (1) The peptide Phth-Ala-Met-OMe (1 mmol), 1,2-dichloroethane (10 mL), palladium acetate (5 mol%), N-methylmaleimide (2.0 mmol), and silver carbonate (2.0 mmol) were added to a 100 mL pressure tube and heated in an oil bath at 80°C for 5 h.

[0090] (2) After the reaction is completed, the reaction mixture is concentrated under reduced pressure using a rotary evaporator at a speed of 120 rps, a water bath temperature of 45° C., and a water pump vacuum of 0.1 MPa. The product is then separated by silica gel column chromatography using petroleum ether-ethyl acetate as the eluent for gradient elution to obtain the dipeptide alkylation product represented by formula A with a yield of 53%.

[0091] In the reaction system of the present application, the role of the palladium salt is mainly manifested as follows: first, Pd(II) coordinates with the S of methionine in the polypeptide, and then forms a six-membered ring palladium intermediate to react with the alanine δ-C (sp 3 )-H is activated. Then maleimide coordinates with palladium through double bonds and double bond insertion, and finally undergoes reduction elimination to form the final alkylation product. Therefore, the yield will also decrease with the reduction of the catalyst dosage (Comparative Example 1 and Example 7).

[0092] From the above experimental process, it can be seen that among the solvents suitable for the alkylation system in this case (hexafluoroisopropanol, methanol, 1,2-dichloroethane and dichloromethane have the same performance, and the specific experiments are not listed one by one), 1,2-dichloroethane and dichloromethane are preferred, and 1,2-dichloroethane is the best. The oxidant is suitable for silver carbonate, silver acetate, silver phosphate, and silver nitrate, and silver carbonate is the best.

[0093] The above-mentioned embodiments only express several feasible implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention, and the embodiments are not used to limit the scope of protection in the claims of the present invention. For ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, and all equivalent implementations or changes that do not depart from the present invention should be included in the technology of the present invention.

Claims

1. A product of site-selective alkylation of a polypeptide by maleimide, It is characterized in that The structural formula of the product satisfies: Among them, R 1 Any one selected from Me, Ph, Et, i-Pr, tBu, R 2 Any one selected from OMe, OBn, polypeptide fragment, and glutamic acid residue.

2. A product of site-selective alkylation of polypeptides with maleimide according to claim 1, It is characterized in that The product is: Any of .

3. A method for site-selective alkylation of polypeptides by maleimide of the product of claim 1, It is characterized in that Here are the steps: Step 1: dissolving palladium salt, maleimide and alanine-containing polypeptide in a solvent to form a mixed solution. The alanine-containing polypeptide structural formula is: Step 2: The mixed solution obtained in step 1 is reacted under heating conditions, and after the reaction is completed, the reaction product is separated, which is the site-selective alkylation product of the polypeptide by maleimide.

4. A method for site-selective alkylation of polypeptides with maleimide according to claim 3, Features: In the mixed solution, the molar ratio of palladium salt, maleimide and alanine-containing polypeptide is 1:5:10 to 1:50:

100.

5. A method for site-selective alkylation of polypeptides with maleimide according to claim 3, Features: The solvent is any one of 1,2-dichloroethane, dichloromethane, hexafluoroisopropanol and methanol.

6. A method for site-selective alkylation of polypeptides with maleimide according to claim 3, Features: The palladium salt is any one of tetrakistriphenylphosphine palladium, palladium acetate, palladium trifluoroacetate and palladium chloride.

7. A method for site-selective alkylation of polypeptides with maleimide according to claim 3, Features: The reaction under heating conditions refers to the reaction under 50-120° C. for 4-10 hours.

8. A method for site-selective alkylation of polypeptides with maleimide according to claim 3, Features: The separation refers to separation of the reaction products by column chromatography, with the eluent being petroleum ether-ethyl acetate.

9. A method for site-selective alkylation of polypeptides with maleimide according to any one of claims 3 to 8, Features: In step one, silver carbonate, silver acetate, silver phosphate or silver nitrate is also added.

10. An application of the alkylation product according to claim 1, Features: The maleimide-selective alkylation product of polypeptide sites is used for the preparation of anti-tumor drugs.

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