Methods for modifying polypeptide side chains

Abstract

The present invention relates to a method for modifying a polypeptide side chain, comprising the following steps: S001, adding a polypeptide, an aryl iodide compound, Cu I, Ni(py) 4 Cl 2, 2,2'-bipiperazine and K 2 CO 3 into a dry threaded sealed tube; S002, adding an organic solvent for dissolution; S003, heating at a constant temperature of 100 ° C for 10 h under a nitrogen atmosphere; S004, adding a NaCl solution for quenching the reaction; S005, adding an organic extract for extraction to obtain an extract; S006, passing the extract through a short chromatography column equipped with anhydrous sodium sulfate, and after rotary evaporation to remove the solvent, column chromatography purification is performed to obtain the desired polypeptide product. This technical solution uses copper as a catalyst, which is not only cheap but also has low biological toxicity. In addition, in the synthesis route of this method, functional groups (such as fluorine-containing groups) can also be pre-set in the aryl compound, which is conducive to the further conversion of the target compound and expands the application range of the product.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide modification, and in particular to a method for modifying polypeptide side chains. Background Art

[0002] Peptide side chain modification is widely used in the biotechnology industry, mainly using precious metals (palladium, platinum, nickel) or special aryl sources (triaryl bismuth). Not only is the cost high, but metals such as palladium and platinum also have certain biological toxicity.

[0003] Based on this, the inventors proposed a method for modifying polypeptide side chains that can reduce costs and biological toxicity. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for modifying polypeptide side chains to solve the problem that the existing technology uses precious metals (palladium, platinum, nickel) or special aromatic sources (triaryl bismuth), which is not only costly but also has certain biological toxicity of metals such as palladium and platinum.

[0005] The present invention is achieved through the following technical solutions:

[0006] A method for modifying a polypeptide side chain comprises the following steps:

[0007] S001. Add peptide, aryl iodide, CuI, Ni(py)4Cl2, 2,2'-bipiperazine and K2CO3 into a dry screw-sealed tube;

[0008] S002, adding an organic solvent to dissolve;

[0009] S003, heating at 100°C for 10 h under nitrogen atmosphere;

[0010] S004, adding NaCl solution to quench the reaction;

[0011] S005, adding an organic extract to perform extraction to obtain an extract;

[0012] S006. The extract is passed through a short chromatography column filled with anhydrous sodium sulfate, the solvent is removed by rotary evaporation, and then the desired polypeptide product is purified by column chromatography.

[0013] It is further defined that in said S001:

[0014] The dosage of the peptide is: 0.05-0.1mmol, 1.0equiv;

[0015] The amount of aryl iodide compound used is: 0.25 mmol, 2.5 equiv;

[0016] The amount of CuI used is: 5.7 mg, 0.03 mmol, 30 mol%;

[0017] The amount of Ni(py)4Cl2 used is: 4.4 mg, 0.01 mmol, 10 mol%;

[0018] The amount of 2,2'-bipiperazine used is: 3 mg, 0.02 mmol, 20 mol%;

[0019] The amount of K2CO3 used is: 55.2 mg, 0.4 mmol, 4.0 equiv.

[0020] It is further defined that the organic solvent in S002 is a mixed solvent of 1,2-dichloroethane and acetonitrile, wherein the ratio and amount of 1,2-dichloroethane and acetonitrile are respectively: volume ratio 1:1, totaling 1.0 mL.

[0021] It is further defined that the organic extractant in S005 is ethyl acetate.

[0022] The beneficial effects of the present invention are:

[0023] This method for modifying polypeptide side chains uses copper as a catalyst, which is not only inexpensive but also has low biotoxicity. Furthermore, in this synthetic route, functional groups (such as fluorinated groups) can be pre-introduced into the aromatic compound, facilitating further conversion of the target compound and expanding the product's application range.

[0024] Other advantages, objects, and features of the present invention will be described in part in the following description and, in part, will be apparent to those skilled in the art upon examination of the following description or may be learned from practice of the present invention. The objects and other advantages of the present invention may be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 For the polypeptide product of Example 1 1 H NMR detection spectrum;

[0026] Figure 2 For the polypeptide product of Example 1 13 C NMR detection spectrum;

[0027] Figure 3 For the polypeptide product of Example 1 19 F NMR detection spectrum;

[0028] Figure 4 For the polypeptide product of Example 2 1 H NMR detection spectrum;

[0029] Figure 5 For the polypeptide product of Example 2 13C NMR detection spectrum;

[0030] Figure 6 For the polypeptide product of Example 2 19 F NMR detection spectrum;

[0031] Figure 7 For the polypeptide product of Example 3 1 H NMR detection spectrum;

[0032] Figure 8 For the polypeptide product of Example 3 13 C NMR detection spectrum;

[0033] Figure 9 For the polypeptide product of Example 3 19 F NMR detection spectrum;

[0034] Figure 10 For example four polypeptide products 1 H NMR detection spectrum;

[0035] Figure 11 For example four polypeptide products 13 C NMR detection spectrum;

[0036] Figure 12 For example four polypeptide products 19 F NMR detection spectrum;

[0037] Figure 13 For the polypeptide product of Example 5 1 H NMR detection spectrum;

[0038] Figure 14 For the polypeptide product of Example 5 13 C NMR detection spectrum;

[0039] Figure 15 For the polypeptide product of Example 5 19 F NMR detection spectrum;

[0040] Figure 16 For example six polypeptide product 1 H NMR detection spectrum;

[0041] Figure 17 For example six polypeptide product 13 C NMR detection spectrum;

[0042] Figure 18 For example six polypeptide product 19 F NMR detection spectrum;

[0043] Figure 19 For example seven polypeptide products1 H NMR detection spectrum;

[0044] Figure 20 For example seven polypeptide products 13 C NMR detection spectrum;

[0045] Figure 21 For example seven polypeptide products 19 F NMR detection spectrum;

[0046] Figure 22 For Example 8 polypeptide product 1 H NMR detection spectrum;

[0047] Figure 23 For Example 8 polypeptide product 13 C NMR detection spectrum;

[0048] Figure 24 For Example 8 polypeptide product 19 F NMR detection spectrum; DETAILED DESCRIPTION

[0049] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings herein can be arranged and designed in various different configurations.

[0050] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention as claimed, but rather merely represents selected embodiments of the present invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort shall fall within the scope of protection of the present invention.

[0051] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, it does not need to be further defined or explained in subsequent drawings.

[0052] In the above description of the present invention, it should be noted that the terms "one side," "the other side," and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, or the orientations or positional relationships in which the inventive product is typically placed when in use. These terms are intended solely to facilitate the description of the present invention and simplify the description, and are not intended to indicate or imply that the devices or components referred to must have, be constructed, or operate in a specific orientation. Therefore, they should not be construed as limitations on the present invention. Furthermore, the terms "first," "second," and the like are used solely for distinction and should not be construed as indicating or implying relative importance.

[0053] Furthermore, the term "identical" and similar terms do not necessarily require that the components be absolutely identical; slight variations are permitted. The term "perpendicular" simply refers to the positional relationship between components being more perpendicular than "parallel," not that the structure must be perfectly vertical; rather, it can be slightly tilted.

[0054] The present invention provides a technical solution: a method for modifying a polypeptide side chain, comprising the following steps:

[0055] S001. Add peptide, aryl iodide, CuI, Ni(py)4Cl2, 2,2'-bipiperazine and K2CO3 into a dry screw-sealed tube;

[0056] S002, adding an organic solvent to dissolve;

[0057] S003, heating at 100°C for 10 h under nitrogen atmosphere;

[0058] S004, adding NaCl solution to quench the reaction;

[0059] S005, adding an organic extract to perform extraction to obtain an extract;

[0060] S006. The extract is passed through a short chromatography column filled with anhydrous sodium sulfate, the solvent is removed by rotary evaporation, and then the desired polypeptide product is purified by column chromatography.

[0061] In said S001:

[0062] The dosage of the peptide is: 0.05-0.1mmol, 1.0equiv;

[0063] The amount of aryl iodide compound used is: 0.25 mmol, 2.5 equiv;

[0064] The amount of CuI used is: 5.7 mg, 0.03 mmol, 30 mol%;

[0065] The amount of Ni(py)4Cl2 used is: 4.4 mg, 0.01 mmol, 10 mol%;

[0066] The dosage of 2,2'-bipiperazine is: 3 mg, 0.02 mmol, 20 mol%;

[0067] The amount of K2CO3 used is: 55.2 mg, 0.4 mmol, 4.0 equiv.

[0068] The organic solvent in S002 is a mixed solvent of 1,2-dichloroethane and acetonitrile, wherein the ratio and amount of 1,2-dichloroethane and acetonitrile are respectively: volume ratio 1:1, totaling 1.0 mL.

[0069] The organic extractant in S005 is ethyl acetate.

[0070] Example 1

[0071] Into a dry 8 mL screw-sealed tube, dipeptide 1 (0.1 mmol, 1.0 equiv.), aryl iodide 2 (0.25 mmol, 2.5 equiv.), CuI (5.7 mg, 0.03 mmol, 30 mol%), Ni(py)4Cl2 (4.4 mg, 0.01 mmol, 10 mol%), 2,2'-bipiperazine (3 mg, 0.02 mmol, 20 mol%), and K2CO3 (55.2 mg, 0.4 mmol, 4.0 equiv.) were added, followed by a mixed solvent of 1,2-dichloroethane and acetonitrile (volume ratio 1:1, total 1.0 mL);

[0072] The mixture was heated at 100 °C for 10 h under a nitrogen atmosphere, and the reaction progress was monitored by thin layer chromatography;

[0073] The reaction was quenched by adding saline solution, followed by extraction with ethyl acetate. The resulting organic solution was passed through a short chromatography column filled with anhydrous sodium sulfate. The solvent was removed by rotary evaporation and then purified by column chromatography to obtain the desired dipeptide product 3 in a yield of 75% as a light yellow solid with a melting point of 52.2-53.6°C.

[0074] Among them, the reaction chemical formula is:

[0075]

[0076] Peptide product detection:

[0077] 1 H NMR (such as Figure 1 shown):

[0078] (400MHz, CDCl3) δ7.90(dd,J=7.7,1.1Hz,1H),7.64(td,J=7.6,1.3Hz,1H),7.57-7.54(m,1H),7.50(td,J=7.2,0.6Hz,1 H),7.42(d,J=7.5Hz,1H),7.14(d,J=4.2Hz,1H),7.13(d,J=4.4Hz,1H),7.08(s,1H),7.05(td,J=5.1,2.0Hz,1H),6.65( d,J=7.4Hz,1H),5.59(s,1H),4.98(s,1H),4.94(dd,J=13.0,5.5Hz,1H),4.15(s,1H),3.71(s,3H),3.39-3.36(m,2H),2 .10-2.06(m,2H),1.37(s,9H),1.35-1.32(m,4H),1.30(d,J=4.8Hz,3H),1.03(s,3H),0.98(s,3H),0.87(t,J=7.1,3H);

[0079] 13 C NMR (such as Figure 2 shown):

[0080] (100MHz, CDCl3) δ200.61 (q, J = 3.9Hz), 172.46, 172.12, 165.30, 155.37, 137.92, 137 .85,132.77,131.16,130.68,128.73,128.32,127.98,127.70,123.26(q,J=272Hz),1 22.74,120.20,118.88,110.52,110.31,104.24,102.23(q,J=33.7Hz),79.99,79.76, 52.92,52.51,50.24,29.40,28.29,27.60,26.71,26.00,25.87,22.21,18.39,13.81;

[0081] 19 F NMR (such as Figure 3 (shown): (376MHz,CDCl3)δ=-64.07(s);

[0082] HRMS-ESI(m / z):[M+Na] + calcd.for C 38 H 46 F3N3NaO7 +,736.3180;found,736.3171;

[0083] IR(KBr,cm -1 ):ν3325,2978,1715,1601,1497,1293,742;

[0084] Example 2

[0085] Into a dry 8 mL screw-sealed tube were added dipeptide 4 (0.1 mmol, 1.0 equiv.), aryl iodide 2 (0.25 mmol, 2.5 equiv.), CuI (5.7 mg, 0.03 mmol, 30 mol%), Ni(py)4Cl2 (4.4 mg, 0.01 mmol, 10 mol%), 2,2'-bipiperazine (3 mg, 0.02 mmol, 20 mol%), and K2CO3 (55.2 mg, 0.4 mmol, 4.0 equiv.), followed by a mixed solvent of 1,2-dichloroethane and acetonitrile (volume ratio 1:1, total 1.0 mL);

[0086] The mixture was heated at 100 °C for 10 h under a nitrogen atmosphere, and the reaction progress was monitored by thin layer chromatography;

[0087] The reaction was quenched by adding saline solution, followed by extraction with ethyl acetate. The resulting organic solution was passed through a short chromatography column filled with anhydrous sodium sulfate. The solvent was removed by rotary evaporation and then purified by column chromatography to obtain the desired dipeptide product 5 in a 50% yield as a light yellow solid with a melting point of 50.4-51.1°C.

[0088] Among them, the reaction chemical formula is:

[0089]

[0090] Peptide product detection:

[0091] 1 H NMR (such as Figure 4 shown):

[0092] (400MHz, CDCl3) δ7.90(d,J=7.6Hz,1H),7.71(s,1H),7.64(t,J=7.6Hz,1H),7.50(t,J=7.6Hz,1H),7 .45(d,J=7.8Hz,1H),7.18-7.13(m,3H),7.10-7.03(m,1H),6.39(s,1H),5.61(s,1H),5.30(s,1H),4. 54(s,1H),3.94(td,J=18.1,5.2Hz,1H),3.86(d,J=17.6Hz,1H),3.66(s,3H),3.47-3.15(m,2H),2.13 -2.04(m,2H),1.44(s,9H),1.39-1.28(m,4H),1.06(s,3H),1.02(s,3H),0.87(td,J=7.0,2.7Hz,3H);

[0093] 13 C NMR (such as Figure 5 shown):

[0094] (100MHz, CDCl3) δ200.64 (q, J = 4.1Hz), 171.90, 169.94, 165.16, 155.56, 138.14, 137.92,132.80,131.24,130.49,128.70,128.19,127.95,123.62(q,J=272Hz),12 2.81,120.31,119.16,111.29,110.17,104.23,102.31(q,J=33.6Hz),80.24,79. 80,52.29,52.28,41.26,29.79,29.42,28.38,26.78,26.73,26.01,22.21,13.79;

[0095] 19 F NMR (such as Figure 6 (shown): (376MHz,CDCl3)δ=-64.08(s);

[0096] HRMS-ESI(m / z):[M+Na] + calcd.for C 37 H 44 F3N3NaO7 + ,722.3024;found,722.3038;

[0097] IR(KBr,cm -1):ν3310,2932,1714,1667,1496,1293,1116,741;

[0098] Example 3

[0099] Into a dry 8 mL screw-sealed tube were added dipeptide 6 (0.1 mmol, 1.0 equiv.), aryl iodide 2 (0.25 mmol, 2.5 equiv.), CuI (5.7 mg, 0.03 mmol, 30 mol%), Ni(py)4Cl2 (4.4 mg, 0.01 mmol, 10 mol%), 2,2'-bipiperazine (3 mg, 0.02 mmol, 20 mol%), and K2CO3 (55.2 mg, 0.4 mmol, 4.0 equiv.), followed by a mixed solvent of 1,2-dichloroethane and acetonitrile (volume ratio 1:1, total 1.0 mL);

[0100] The mixture was heated at 100 °C for 10 h under a nitrogen atmosphere, and the reaction progress was monitored by thin layer chromatography;

[0101] The reaction was quenched by adding saline solution, followed by extraction with ethyl acetate. The resulting organic solution was passed through a short chromatography column filled with anhydrous sodium sulfate. The solvent was removed by rotary evaporation and then purified by column chromatography to obtain the desired dipeptide product 7 in a yield of 72% as a white solid with a melting point of 53.2-54.5°C.

[0102] Among them, the reaction chemical formula is:

[0103]

[0104] Peptide product detection:

[0105] 1 H NMR (such as Figure 7 shown):

[0106] (400MHz, CDCl3) δ7.91(dd,J=7.7,1.1Hz,1H),7.70(s,1H),7.61(td,J=7.6,1.1Hz,1H),7.48(t,J= 7.6Hz,1H),7.44(d,J=7.5Hz,1H),7.17-7.11(m,3H),7.08-7.04(m,1H),6.49(s,1H),5.54(s,1H), 5.19(s,1H),4.52(s,1H),4.37(ddd,J=8.3,4.5Hz,1H),3.40-3.22(m,2H),2.11-2.05(m,3H),1.43 (s,9H),1.40(s,9H),1.36-1.27(m,4H),0.94(s,6H),0.86(t,J=7.1Hz,3H),0.83(d,J=6.2Hz,6H);

[0107] 13 C NMR (such as Figure 8 shown):

[0108] (100MHz, CDCl3)δ200.60(q,J=3.3Hz),171.46,170.60,165.32,155.62,138.08,137.91 ,132.67,131.34,130.57,128.71,128.28,127.86,127.84,123.64(q,J=273Hz),122.74, 120.23,119.17,111.26,110.20,104.24,102.17(q,J=33.6Hz),81.98,81.97,79.69,57. 66,57.65,31.46,29.40,28.37,28.07,26.76,26.02,25.83,22.21,18.77,17.78,13.79;

[0109] 19 F NMR (such as Figure 9 (shown): (376MHz,CDCl3)δ=-64.08(s);

[0110] HRMS-ESI(m / z):[M+Na] + calcd.for C 43 H 56 F3N3NaO7 + ,806.3963;found,806.3983;

[0111] IR(KBr,cm -1):ν3326,2968,2932,1716,1665,1496,1292,1116,742;

[0112] Example 4

[0113] Into a dry 8 mL screw-sealed tube were added dipeptide 8 (0.1 mmol, 1.0 equiv.), aryl iodide 2 (0.25 mmol, 2.5 equiv.), CuI (5.7 mg, 0.03 mmol, 30 mol%), Ni(py)4Cl2 (4.4 mg, 0.01 mmol, 10 mol%), 2,2'-bipiperazine (3 mg, 0.02 mmol, 20 mol%), and K2CO3 (55.2 mg, 0.4 mmol, 4.0 equiv.), and then a mixed solvent of 1,2-dichloroethane and acetonitrile (volume ratio 1:1, total 1.0 mL);

[0114] The mixture was heated at 100 °C for 10 h under a nitrogen atmosphere, and the reaction progress was monitored by thin layer chromatography;

[0115] The reaction was quenched by adding saline solution, followed by extraction with ethyl acetate. The resulting organic solution was passed through a short chromatography column filled with anhydrous sodium sulfate. The solvent was removed by rotary evaporation and then purified by column chromatography to obtain the desired dipeptide product 9 in a yield of 72% as a white solid with a melting point of 53.5-54.2°C.

[0116] Among them, the reaction chemical formula is:

[0117]

[0118] Peptide product detection:

[0119] 1 H NMR (such as Figure 10 shown):

[0120] 400MHz, CDCl3) δ7.91(dd,J=7.7,1.1Hz,1H),7.69(s,1H),7.63(td,J=7.7,1.3Hz,1H),7.49(t,J=7.4Hz, 1H),7.44(d,J=7.8Hz,1H),7.17-7.12(m,3H),7.08-7.06(m,1H),6.53(s,1H),5.56(s,1H),5.21(s,1H), 4.49-4.46(m,2H),3.61(s,3H),3.39-3.17(m,2H),2.11-2.03(m,2H),1.78(s,1H),1.44(s,9H),1.37-1. 28(m,6H),0.99(s,3H),0.94(s,3H),0.87(t,J=6.9Hz,3H),0.83(t,J=7.3Hz,3H),0.76(d,J=6.6Hz,3H);

[0121] 13 C NMR (such as Figure 11 shown):

[0122] (100MHz, CDCl3) δ200.61 (q, J = 3.8Hz), 171.83, 171.44, 165.28, 155.62, 138.05, 137.93 ,132.71,131.36,130.57,128.62,128.21,127.91,127.78,123.63(q,J=272Hz),122.77, 120.27,119.14,111.29,110.18,104.22,102.19(q,J=33.6Hz),80.23,79.70,56.65,56. 64,52.04,37.94,29.40,28.36,26.74,26.01,25.83,25.16,22.20,15.27,13.79,11.56;

[0123] 19 F NMR (such as Figure 12 (shown): (376MHz,CDCl3)δ=-64.09(s);

[0124] HRMS-ESI(m / z):[M+Na] + calcd.for C 41 H 52 F3N3NaO7 + ,778.3650;found,778.3667;

[0125] IR(KBr,cm -1 ):ν3323,2964,1715,1496,1293,1117,739;

[0126] Example 5

[0127] Into a dry 8 mL screw-sealed tube were added dipeptide 10 (0.1 mmol, 1.0 equiv.), aryl iodide 2 (0.25 mmol, 2.5 equiv.), CuI (5.7 mg, 0.03 mmol, 30 mol%), Ni(py)4Cl2 (4.4 mg, 0.01 mmol, 10 mol%), 2,2'-bipiperazine (3 mg, 0.02 mmol, 20 mol%), and K2CO3 (55.2 mg, 0.4 mmol, 4.0 equiv.), followed by a mixed solvent of 1,2-dichloroethane and acetonitrile (volume ratio 1:1, total 1.0 mL);

[0128] The mixture was heated at 100 °C for 10 h under a nitrogen atmosphere, and the reaction progress was monitored by thin layer chromatography;

[0129] The reaction was quenched by adding saline solution, followed by extraction with ethyl acetate. The resulting organic solution was passed through a short chromatography column filled with anhydrous sodium sulfate. The solvent was removed by rotary evaporation and then purified by column chromatography to obtain the desired dipeptide product 11 in a yield of 75% as a white solid with a melting point of 54.6-56.2°C.

[0130] Among them, the reaction chemical formula is:

[0131]

[0132] Peptide product detection:

[0133] 1 H NMR (such as Figure 13 shown):

[0134] (400MHz, CDCl3)δ7.91(d,J=7.6Hz,1H),7.70(s,1H),7.61(t,J=7.2Hz,1H),7.48t,J=7.5Hz,1H),7 .44(s,1H),7.17-7.11(m,3H),7.09-7.05(m,1H),6.35(s,1H),5.55(s,1H),5.17(s,1H),4.50(s,1 H),4.45(dd,J=13.3,8.0Hz,1H),3.32-3.28(m,2H),2.10-2.15(m,2H),1.64-1.65(m,2H),1.57-1. 50(m,1H)1.43(s,9H),1.40(s,9H),1.35-1.28(m,4H),0.99(s,3H),0.95(m,3H),0.88-0.85(m,9H);

[0135] 13 C NMR (such as Figure 14 shown):

[0136] (100MHz, CDCl3)δ200.60(q,J=4.8Hz),171.75,171.18,165.32,155.58,138.08,137.87,1 32.68,131.33,130.54,128.67,128.34,127.92,127.86,123.63(q,J=272Hz),122.73,120 .25,119.20,111.19,110.19,104.23,102.17(q,J=33.7Hz),81.87,80.12,79.69,51.52,5 1.50,41.98,29.40,28.36,28.01,26.75,26.02,25.83,24.82,22.77,22.21,22.14,13.80;

[0137] 19 F NMR (such as Figure 15 (shown): (376MHz,CDCl3)δ=-64.08(s);

[0138] HRMS-ESI(m / z):[M+Na] + calcd.for C 44 H 58 F3N3NaO7 + ,820.4119;found,820.4140;

[0139] IR(KBr,cm-1 ):ν3337,2961,1716,1666,1496,1293,1117,741

[0140] Example 6

[0141] Into a dry 8 mL screw-sealed tube were added tripeptide 30 (0.1 mmol, 1.0 equiv.), aryl iodide 2 (0.25 mmol, 2.5 equiv.), CuI (5.7 mg, 0.03 mmol, 30 mol%), Ni(py)4Cl2 (4.4 mg, 0.01 mmol, 10 mol%), 2,2'-bipiperazine (3 mg, 0.02 mmol, 20 mol%), and K2CO3 (55.2 mg, 0.4 mmol, 4.0 equiv.), followed by a mixed solvent of 1,2-dichloroethane and acetonitrile (volume ratio 1:1, total 1.0 mL);

[0142] The mixture was heated at 100 °C for 10 h under a nitrogen atmosphere, and the reaction progress was monitored by thin layer chromatography;

[0143] The reaction was quenched by adding saline solution, followed by extraction with ethyl acetate. The resulting organic solution was passed through a short chromatography column filled with anhydrous sodium sulfate. The solvent was removed by rotary evaporation and then purified by column chromatography to obtain the desired tripeptide product 31 in a yield of 65% as a light white solid with a melting point of 67.8-69.5°C.

[0144] Among them, the reaction chemical formula is:

[0145]

[0146] Peptide product detection:

[0147] 1 H NMR (such as Figure 16 shown):

[0148] (400 MHz, CDCl3) δ 7.89 (d, J = 7.5 Hz, 1H), 7.62 (t, J = 7.3 Hz, 1H), 7.55 (m, 1H), 7.49 - 7.45 (m, 1H), 7.13 - 7.09 (m, 3H), 7.05 - 7.03 (m, 1H), 6.92 (s, 1H), 6.64 (s, 1H), 5.54 (s, 1H), 5.57 (s, 1H), 4.89 (dd, J = 12.0, 6.6 Hz, 1H), 4.41 (s, 1H), 4.11 - 4.08 (m, 1H), 3.68 (s, 3H), 3.40 - 3.27 (m, 4H), 2.09 - 2.02 (m, 4H), 1.84 - 1.75 (m, 2H), 1.42 (s, 9H), 1.35 - 1.24 (m, 7H), 1.00 (s, 3H), 0.95 (s, 3H), 0.85 (t, J = 6.9 Hz, 3H);

[0149] 13 13C NMR (as Figure 17 shown):

[0150] (100 MHz, CDCl3) δ 200.58 (q, J = 4.2 Hz), 172.12, 171.97, 171.74, 165.32, 155.85, 137.96, 137.85, 132.81, 131.22, 130.58, 128.79, 128.27, 127.89, 127.68, 123.63 (q, J = 273 Hz), 122.70, 120.24, 118.86, 110.66, 110.27, 104.25, 102.21 (q, J = 34.4 Hz), 80.60, 79.72, 59.87, 52.97, 52.47, 48.96, 47.15, 29.39, 28.38, 28.13, 27.60, 26.70, 26.00, 25.82, 24.63, 22.20, 17.58, 13.79;

[0151] 19 19F NMR (as Figure 18 shown): (376 MHz, CDCl3) δ = -64.08 (s);

[0152] HRMS - ESI (m / z): [M + Na] + calcd. for C 43 H 53 F3N4NaO 10 + , 833.3708; found, 833.3731;

[0153] IR(KBr,cm -1 ):ν3306,2933,1693,1498,1292,1118,910,734;

[0154] Example 7

[0155] Into a dry 8 mL screw-sealed tube were added tetrapeptide 59 (0.05 mmol, 1.0 equiv.), aryl iodide 2 (0.25 mmol, 2.5 equiv.), CuI (5.7 mg, 0.03 mmol, 30 mol%), Ni(py)4Cl2 (4.4 mg, 0.01 mmol, 10 mol%), 2,2'-bipiperazine (3 mg, 0.02 mmol, 20 mol%), and K2CO3 (55.2 mg, 0.4 mmol, 4.0 equiv.), followed by a mixed solvent of 1,2-dichloroethane and acetonitrile (volume ratio 1:1, total 1.0 mL);

[0156] The mixture was heated at 100 °C for 10 h under a nitrogen atmosphere, and the reaction progress was monitored by thin layer chromatography;

[0157] The reaction was quenched by adding saline solution, and then extracted with ethyl acetate. The resulting organic solution was passed through a short chromatography column filled with anhydrous sodium sulfate. The solvent was removed by rotary evaporation and then purified by column chromatography to obtain the desired tetrapeptide product 60 in a yield of 57% as a light yellow solid at 91.0-92.2°C.

[0158] Among them, the reaction chemical formula is:

[0159]

[0160] Peptide product detection:

[0161] 1 H NMR (such as Figure 19 shown):

[0162] (400 MHz, CDCl3) δ 7.91 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.62 - 7.56 (m, 4H), 7.50 - 7.40 (m, 4H), 7.16 - 7.04 (m, 8H), 6.93 (s, 2H), 6.40 (s, 1H), 5.60 - 5.70 (m, 2H), 5.18 - 5.15 (m, 1H), 4.91 (dd, J = 12.0, 5.6 Hz, 1H), 4.39 - 4.37 (m, 1H), 4.30 (m, 1H), 4.26 - 4.24 (m, 1H), 3.71 (s, 3H), 3.45 - 3.36 (m, 2H), 3.27 - 3.17 (m, 2H), 2.10 - 2.05 (m, 4H), 1.38 (s, 9H), 1.36 - 1.26 (m, 14H), 1.12 (s, 6H), 1.00 (s, 6H), 0.86 (t, J = 7.0 Hz, 6H);

[0163] 13 13C NMR (as Figure 20 shown):

[0164] (100 MHz, CDCl3) δ 200.63 (q, J = 3.4 Hz), 200.61 (q, J = 4.0 Hz), 172.20, 172.19, 172.00, 171.65, 165.40, 165.13, 156.01, 138.05, 137.98, 137.91, 137.80, 132.87, 132.71, 131.36, 131.23, 130.62, 130.44, 128.68, 128.62, 128.40, 128.08, 128.03, 127.85, 127.80, 127.58, 123.64 (q, J = 272 Hz), 123.61 (q, J = 272 Hz), 122.99, 122.65, 120.40, 120.16, 119.05, 118.97, 111.16, 110.68, 110.31, 110.23, 104.29, 104.16, 102.35 (q, J = 34.0 Hz), 102.21 (q, J = 33.2 Hz), 80.81, 79.78, 79.76, 55.58, 53.05, 52.48, 49.55, 49.17, 29.41, 28.29, 27.56, 26.75, 26.01, 25.89, 22.20, 17.70, 13.80; <00​​​​(shown): (376MHz,CDCl3)δ=-64.05(s);

[0166] HRMS-ESI(m / z):[M+Na] + calcd.for C 70 H 80 F6N6NaO 11 + ,1317.5681; found,1317.5692;

[0167] IR(KBr,cm -1 ):ν3287,2932,1714,1634,1497,1292,1116,738;

[0168] Example 8

[0169] Into a dry 8 mL screw-sealed tube were added pentapeptide 48 (0.05 mmol, 1.0 equiv.), aryl iodide 2 (0.25 mmol, 2.5 equiv.), CuI (5.7 mg, 0.03 mmol, 30 mol%), Ni(py)4Cl2 (4.4 mg, 0.01 mmol, 10 mol%), 2,2'-bipiperazine (3 mg, 0.02 mmol, 20 mol%), and K2CO3 (55.2 mg, 0.4 mmol, 4.0 equiv.), followed by a mixed solvent of 1,2-dichloroethane and acetonitrile (volume ratio 1:1, total 1.0 mL);

[0170] The mixture was heated at 100 °C for 10 h under a nitrogen atmosphere, and the reaction progress was monitored by thin layer chromatography;

[0171] The reaction was quenched by adding saline solution, followed by extraction with ethyl acetate. The resulting organic solution was passed through a short chromatography column filled with anhydrous sodium sulfate. The solvent was removed by rotary evaporation and then purified by column chromatography to obtain the desired tetrapeptide product 49 in a 49% yield as a white solid with a melting point of 109.2-110.5°C.

[0172] Among them, the reaction chemical formula is:

[0173]

[0174] Peptide product detection:

[0175] 1 H NMR (such as Figure 22 shown):

[0176] (400 MHz, Acetone-D6) δ 7.90 (d, J = 7.8 Hz, 1H), 7.84 - 7.70 (m, 4H), 7.64 - 7.56 (m, 3H), 7.23 - 7.10 (m, 9H), 7.03 - 7.00 (m, 1H), 6.30 (d, J = 6.1 Hz, 1H), 5.65 - 5.61 (m, 1H), 4.81 (dd, J = 13.1, 7.6 Hz, 1H), 4.71 - 4.64 (m, 1H), 4.58 (s, 1H), 4.45 - 4.41 (m, 1H), 4.16 - 4.04 (m, 1H), 3.70 (s, 3H), 3.41 - 3.22 (m, 2H), 3.20 - 3.12 (m, 1H), 3.07 - 2.84 (m, 3H), 2.55 - 2.42 (m, 2H), 2.14 - 2.09 (m, 2H), 2.02 (s, 3H), 1.93 - 1.85 (m, 1H), 1.46 - 1.39 (m, 5H), 1.37 (s, 9H), 1.34 - 1.29 (m, 4H), 1.02 (s, 3H), 0.99 (s, 3H), 0.91 (t, J = 7.6 Hz, 6H), 0.87 - 0.85 (m, 9H);

[0177] 13 13C NMR (as Figure 23 shown):

[0178] (100 MHz, Acetone-D6) δ 200.48 (q, J = 3.6 Hz), 173.58, 171.70, 171.51, 171.26, 171.10, 165.02, 156.14, 138.61, 138.37, 137.82, 133.54, 131.47, 131.30, 130.00, 129.49, 129.17, 128.83, 128.55, 128.46, 127.03, 124.45 (q, J = 273 Hz), 122.92, 120.39, 119.83, 112.72, 110.43, 105.26, 102.23 (q, J = 33.3 Hz), 79.59, 79.42, 57.40, 55.70, 54.66, 54.33, 52.99, 51.85, 41.68, 37.76, 37.65, 32.43, 30.40, 28.43, 28.05, 26.85, 26.25, 25.92, 25.64, 25.10, 23.01, 22.48, 22.00, 15.76, 15.01, 13.76, 11.70;

[0179] 19 19F NMR (as Figure 24 (shown): (376MHz,CDCl3)δ=-64.06(s);

[0180] HRMS-ESI(m / z):[M+Na] + calcd.for C 61 H 81 F3N6NaO 10 S + ,1169.5579; found,1169.5596;

[0181] IR(KBr,cm -1 ):ν3289,2960,1716,1635,1497,1117,742.

[0182] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for modifying a polypeptide side chain, characterized in that: The steps are as follows: S001. Add a polypeptide, an aryl iodide compound, CuI, Ni(py)4Cl2, 2,2'-bipiperazine and K2CO3 into a dry screw-sealed tube. Any one of: S002, adding an organic solvent to dissolve; S003, heating at 100°C for 10 h under nitrogen atmosphere; S004, adding NaCl solution to quench the reaction; S005, adding an organic extract to perform extraction to obtain an extract; S006. The extract is passed through a short chromatography column filled with anhydrous sodium sulfate, the solvent is removed by rotary evaporation, and then the desired polypeptide product is purified by column chromatography.

2. The method for modifying a polypeptide side chain according to claim 1, wherein: In said S001: The dosage of the peptide is: 0.05-0.1mmol, 1.0equiv; The amount of aryl iodide compound used is: 0.25 mmol, 2.5 equiv; The amount of CuI used is: 5.7 mg, 0.03 mmol, 30 mol%; The amount of Ni(py)4Cl2 used is: 4.4 mg, 0.01 mmol, 10 mol%; The dosage of 2,2'-bipiperazine is: 3 mg, 0.02 mmol, 20 mol%; The amount of K2CO3 used is: 55.2 mg, 0.4 mmol, 4.0 equiv.

3. The method for modifying a polypeptide side chain according to claim 2, wherein: The organic solvent in S002 is a mixed solvent of 1,2-dichloroethane and acetonitrile, wherein the ratio and amount of 1,2-dichloroethane and acetonitrile are respectively: volume ratio 1:1, totaling 1.0 mL.

4. The method for modifying a polypeptide side chain according to claim 1, wherein: The organic extractant in S005 is ethyl acetate.

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