Method for synthesizing polypeptide or protein containing one or more non-proteinogenic amino acids

Abstract

Provided is a method for obtaining a polypeptide or protein containing one or more non-proteinogenic amino acids. The method mainly comprises the following steps: 1) activating a dipeptide of the following formula I under acidic or neutral conditions; 2) reacting the activated dipeptide with a polypeptide or protein; and 3) removing a protective group in situ; thereby obtaining a final polypeptide or protein. In the process of synthesizing the polypeptide, by means of activating the dipeptide under acidic or neutral conditions, the DeFmoc phenomenon in a long activation process can be avoided, thereby effectively controlling N-terminal His-Aib insertion peptide impurities, and reducing product loss. The activation of the dipeptide under acidic or neutral conditions provides an activated ester with good stability, resulting in a stable process with sufficient operating time during production, which is conducive to scaled-up industrial production. Moreover, fewer organic solvents such as DMF and acetonitrile are used, and water is used for dilution, thereby lowering costs.

Description

Methods for synthesizing polypeptides or proteins containing one or more non-protein-forming amino acids. Technical Field

[0001] This invention relates to the field of polypeptide or protein synthesis, and more particularly to a method for synthesizing polypeptides or proteins comprising one or more non-protein-forming amino acids. Background Technology

[0002] Natural peptides and proteins can have specific properties modified or enhanced by converting them into analogs and derived structures. Specifically, by strategically introducing non-natural amino acids (i.e., non-protein-derived amino acids) into the peptide chain or protein sequence as additions or replacements, their resistance to hydrolysis can be significantly improved; for example, effectively preventing GLP-1 peptides from being degraded by DPP-IV enzymes. Smegglutinin and Mazdutide are two typical examples of peptides that incorporate non-natural amino acids.

[0003] Semaglutide, developed by Novo Nordisk of Denmark, is a long-acting glucagon-like peptide-1 (GLP-1) analogue. The second amino acid position of semaglutide is changed from Ala to the non-natural amino acid aminoisobutyric acid (Aib), significantly improving the performance of the peptide drug and prolonging its duration of action in the body from several hours to several days. It is currently approved for marketing to lower blood sugar and reduce weight, with significant effects. The peptide sequence structure is as follows:

[0004] H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-Octadecanedioic acid)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH.

[0005] Mazdutide is an innovative drug developed by Innovent Biologics in collaboration with Eli Lilly and Company. It is a dual agonist of glucagon-like peptide-1 receptor (GLP-1R) / glucagon receptor (GCGR). It promotes insulin secretion, lowers blood glucose, and reduces weight by activating GLP-1R, while simultaneously increasing energy expenditure by activating GCGR, enhancing the weight loss effect, and improving hepatic lipid metabolism.

[0006] The peptide sequence structure is as follows:

[0007] In chemical synthesis, to reduce the formation of racemization byproducts, those skilled in the art typically couple one or more peptide fragments containing non-natural amino acids to the remaining peptide chain or protein matrix, while ensuring that the N-terminal amino group and all side chain amino groups of these peptide fragments are adequately protected. This method not only ensures the purity of the synthesized product but also improves the overall synthetic efficiency. CN110041399B discloses a method for using the dipeptide Fmoc-His(TFA)-Aib-OH, in which the dipeptide is activated using a phosphonium coupling agent during the preparation of smegglutinin. This condensation reagent requires the addition of a base TEA to adjust the pH to 8. However, the activation process requires the addition of triethylamine, and prolonged activation or poor temperature control can easily lead to the de-Fmoc generation of impurities, which in turn form impurities with structures similar to the target peptide. Patent CN115197312B discloses a liquid-phase method for preparing semaglutide. Arg34GLP-1(9-37) is first attached with a side chain, then reacted with the dipeptide Boc-His(Trt)-Aib-OH, followed by cleavage to obtain semaglutide. This route ultimately uses TFA cleavage and methyl tert-butyl ether precipitation, requiring a large amount of solvent and resulting in high costs. Patent CN115322250A discloses another liquid-phase method for preparing semaglutide. Arg34GLP-1(9-37) is first attached with a protecting side chain, then reacted with the dipeptide Boc-His(Trt)-Aib-OH, followed by cleavage to obtain semaglutide. This route also requires TFA cleavage and methyl tert-butyl ether precipitation, consuming a large amount of organic solvent and resulting in high costs. Summary of the Invention

[0008] In view of the shortcomings of the existing methods for synthesizing peptides or proteins containing non-protein-forming amino acids, and in order to overcome the problems of generating peptide-related impurities during the synthesis process, as well as the use of highly toxic solvents, large solvent volumes, and the generation of large amounts of waste liquid, this invention provides a technical solution for synthesizing peptides or proteins containing one or more non-protein-forming amino acids, mainly including the following steps:

[0009] Activation of dipeptides of formula I under acidic or neutral conditions

[0010] Wherein, R1 is H or Fmoc;

[0011] The activated dipeptide is reacted with a polypeptide or protein;

[0012] Remove the protective group in situ;

[0013] This yields the final polypeptide or protein.

[0014] When the dipeptide of Formula I is activated for a long time under alkaline conditions, the DeFmoc phenomenon occurs, generating H-His(R1)-Aib-OR2. This impurity reacts with Fmoc-His(R1)-Aib-OR2 to generate the impurity Fmoc-His(R1)-Aib-His(R)-Aib-OR2. After further reaction with P29C1, Fmoc is removed to form the impurity [Endo His-Aib]smegglutinin, with the following structure:

[0015] P29C1 is an intermediate of semaglutide, with the following structural formula:

[0016] The inventors have made a remarkable discovery that activation of dipeptides under acidic or neutral conditions can avoid the DeFmoc phenomenon during prolonged activation, thereby effectively controlling N-terminal His-Aib insertion peptide impurities and reducing product loss.

[0017] In some embodiments, the activation in step 1) uses a carbodiimide-type condensation reagent.

[0018] In some embodiments, the condensation reagent is selected from one or more of DCC, EDCI, DIC, TBEC, N,N'-di-tert-butylmethanediimine, and EDC.HCl.

[0019] In some embodiments, the activation in step 1) employs a combination of a condensation reagent and an activating reagent, wherein the activating reagent is selected from one or more of HOSu, HOPFP, HOBt, HOAt, HOPO, and Oxymapure.

[0020] In some embodiments, the activation in step 1) employs EDCI / HOPFP.

[0021] In some embodiments, the activation of the dipeptide in step 1) is as follows:

[0022] Where R1 is H or Fmoc, and R2 is selected from OBt, OPFP, OAT, OPO, and OSu.

[0023] In some embodiments, in step 2), the activated dipeptide is reacted with a polypeptide or protein in an aqueous medium, wherein the aqueous medium includes any water-based medium.

[0024] In some embodiments, the aqueous medium comprises one or more water-soluble organic solvents selected from the following: acetonitrile, DMF, NMP, THF.

[0025] In some embodiments, the pH of the aqueous medium is 8 to 10.

[0026] In some embodiments, the pH is adjusted by one or more bases selected from the group consisting of: DIEA, triethylamine, trimethylamine, carbonate, and phosphate.

[0027] In some embodiments, in step 3), the reagent used to remove the protecting group is selected from one or more of the following reagents: piperidine, piperazine, methylpiperazine, tert-butylamine.

[0028] In some embodiments, the polypeptide or protein that reacts with the dipeptide is immobilized on a solid phase.

[0029] In some embodiments, the reaction of the dipeptide with the polypeptide or protein is a liquid-phase synthesis.

[0030] In some embodiments, the dipeptide is reacted with the α-N-terminus of a polypeptide or protein.

[0031] In some embodiments, the polypeptide or protein that reacts with the dipeptide is a GLP-1 peptide.

[0032] In some embodiments, the obtained polypeptide is selected from:

[0033] [Aib8, Arg34]GLP-1-(7-37) peptide; or

[0034] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Gl u-Phe-Val-Glu-Trp-Leu-Leu-Xaa28-Gly-Gly-Pro-Ser-Ser-Gly, where Xaa2 is Aib; Xaa28 is Glu or Ser;

[0035] In some embodiments, the obtained polypeptide is selected from:

[0036] His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-Octadecanedioic acid)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly;

[0037] or

[0038] His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr 10 -Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys 20 (AEEA-AEEA-γGlu-Eicosanedioic acid)-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-Gly-Gly 30 -Pro-Ser-Ser-Gly 34 -NH2.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] The present invention uses dipeptide activation under acidic or neutral conditions during peptide synthesis, which avoids the DeFmoc phenomenon during prolonged activation, thereby effectively controlling N-terminal His-Aib insertion peptide impurities and reducing product loss. Activation of the dipeptide under acidic or neutral conditions results in better stability of the activated ester, stable process, and sufficient operating time during production, which is conducive to industrial-scale production. Furthermore, it uses less organic solvents such as DMF and acetonitrile, and is diluted with water, greatly reducing costs. Attached Figure Description

[0041] Figure 1 is the HPLC chromatogram of crude smegglutinin peptide from Example 2 of the present invention.

[0042] Figure 2 is the HPLC chromatogram of the crude smegglutinin peptide from Example 3 of the present invention.

[0043] Figure 3 is the HPLC chromatogram of the crude smegglutinin peptide from Example 4 of the present invention.

[0044] Figure 4 is the HPLC chromatogram of the crude smegglutinin peptide from Example 5 of the present invention.

[0045] Figure 5 is the HPLC chromatogram of the crude smegglutinin peptide from Example 6 of the present invention.

[0046] Figure 6 is the HPLC chromatogram of the crude smegglutinin peptide from Example 7 of the present invention.

[0047] Figure 7 shows the HPLC chromatogram of crude smegglutinin from Comparative Example 1.

[0048] Figure 8 shows the HPLC chromatogram of crude smegglutinin from Comparative Example 2. Detailed Implementation

[0049] The present invention will be further described in detail below through embodiments, which are intended to illustrate the invention and not limit it. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications also fall within the protection scope of the present invention.

[0050] The abbreviations used in this invention have the following meanings:

[0051] DCC: N,NCC dicyclohexylcarbodiimide

[0052] DIC: N,NIC diisopropylcarbodiimide

[0053] EDCI: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0054] TBEC: 1-tert-butyl-3-ethylcarbodiimide

[0055] N,N'-di-tert-butylmethanediimine: N,N'-di-tert-butylcarbodiimine

[0056] HOSu: N-hydroxysuccinimide

[0057] HOPFP: Pentafluorophenol

[0058] HOBt: 1-Hydroxybenzotriazole

[0059] HOAt: N-hydroxy-7-azabenzotriazole

[0060] HOPO: 2-hydroxypyridine-N-oxide

[0061] Oxymapure: Ethyl 2-oxime cyanoacetate

[0062] DMF: N,N-dimethylformamide

[0063] THF: Tetrahydrofuran

[0064] ACN: Acetonitrile

[0065] NMP: N-methylpyrrolidone

[0066] DIEA: N,N-Diisopropylethylamine

[0067] Et3N: Triethylamine

[0068] Example 1: Synthesis of P29C1

[0069] Weigh P29 (50g, 15.7mmol, 1.0eq), add acetonitrile (250ml) and water (750ml), stir well, add DIEA to adjust the pH to 11±0.5, weigh DC18-γGlu-ADOADO-OSu (14.39g, 17.3mmol, 1.1eq), add acetonitrile (210ml), stir well, and slowly add dropwise to the P29 solution. After the addition is complete, react for 1.0h. The reaction solution is concentrated, diluted with water, and the pH is adjusted to 4.5±0.5 with glacial acetic acid. A white solid precipitates, centrifuges, washes the solid twice with water, and vacuum dries to obtain 59.35g of white solid, with a yield of 96.86% and a purity of 94.88%.

[0070] The structure of P29 C1 is as follows:

[0071] Example 2: Synthesis of Smegglutinin

[0072] Weigh out Fmoc-His-Aib-OH (2.38 g, 5.14 mol, 4.0 eq) and HOPFP (0.96 g, 5.14 mmol, 4.0 eq), add 15 mL of DMF, maintain the temperature in an ice-water bath at 0–10 °C, add EDCI (0.99 g, 5.14 mmol, 4.0 eq) while stirring, and continue the reaction for 1.0–3.0 h.

[0073] Weigh P29C1 (5.0g, 1.28mmol, 1.0q), add DMF (70ml) and water (30ml) in sequence, stir well, control the temperature at 10-30℃, adjust the pH value to 9-10 with DIEA, stir until dissolved, slowly add the activated dipeptide to the solution, and stir until P29C1 has reacted completely.

[0074] Piperidine was added to the reaction solution to remove the Fmoc protecting group for 1.0–3.0 h. After deprotection, the pH was adjusted to 8.0 ± 0.5 with glacial acetic acid, diluted with water, and filtered to obtain a crude smegglutinin solution with a molar yield of 92.53%. The HPLC chromatogram and data of the crude smegglutinin are shown in Figure 1 and Table 1, respectively.

[0075] Table 1: Statistics of [EndoHis-Aib]semaglutide impurities in the crude peptide of Example 2

[0076] Example 3: Synthesis of Smegglutinin

[0077] Weigh out Fmoc-His-Aib-OH (2.38 g, 5.14 mol, 4.0 eq) and HOBt (0.69 g, 5.14 mmol, 4.0 eq), add 15 mL of DMF, maintain the temperature in an ice-water bath at 0–10 °C, add EDCI (0.99 g, 5.14 mmol, 4.0 eq) with stirring, and continue the reaction for 1.0–3.0 h.

[0078] Weigh P29C1 (5.0g, 1.28mmol, 1.0q), add DMF (70ml) and water (30ml) in sequence, stir well, control the temperature at 10-30℃, adjust the pH value to 9-10 with DIEA, stir until dissolved, slowly add the activated dipeptide to the solution, and stir until P29C1 has reacted completely.

[0079] Piperidine was added to the reaction solution to remove the Fmoc protecting group for 1.0–3.0 h. After deprotection, the pH was adjusted to 8.0 ± 0.5 with glacial acetic acid, diluted with water, and filtered to obtain a crude smegglutinin solution with a molar yield of 93.05%. The HPLC chromatogram and data of the crude smegglutinin are shown in Figure 2 and Table 2, respectively.

[0080] Table 2: Statistics of [EndoHis-Aib]semaglutide impurities in the crude peptide of Example 3

[0081] Example 4: Synthesis of Smegglutinin

[0082] Weigh out Fmoc-His-Aib-OH (2.38 g, 5.14 mol, 4.0 eq) and HOPO (0.57 g, 5.14 mmol, 4.0 eq), add 15 mL of DMF, maintain the temperature in an ice-water bath at 0–10 °C, add TBEC (0.63 g, 5.14 mmol, 4.0 eq) with stirring, and continue the reaction for 1.0–3.0 h.

[0083] Weigh out P29C1 (5.0g, 1.28mmol, 1.0q), add DMF (70ml) and water (30ml) in sequence, stir well, control the temperature at 10-30℃, adjust the pH value to 9-10 using Et3N, stir until dissolved, slowly add the activated dipeptide to the solution, and stir until P29C1 has reacted completely.

[0084] Piperidine was added to the reaction solution to remove the Fmoc protecting group for 1.0–3.0 h. After deprotection, the pH was adjusted to 8.0 ± 0.5 with glacial acetic acid, diluted with water, and filtered to obtain a crude smegglutinin solution with a molar yield of 91.89%. The HPLC chromatogram and data of the crude smegglutinin are shown in Figure 3 and Table 3, respectively.

[0085] Table 3: Statistics of [EndoHis-Aib]semaglutide impurities in the crude peptide of Example 4

[0086] Example 5: Synthesis of P29C2

[0087] Weigh out Fmoc-His-Aib-OH (2.38 g, 5.14 mol, 4.0 eq) and HOBt (0.69 g, 5.14 mmol, 4.0 eq), add 15 mL of DMF, maintain the temperature in an ice-water bath at 0–10 °C, add DCC (1.06 g, 5.14 mmol, 4.0 eq) while stirring, and continue the reaction for 1.0–3.0 h.

[0088] Weigh out P29C1 (5.0g, 1.28mmol, 1.0q), add DMF (70ml) and water (30ml) in sequence, stir well, control the temperature at 10-30℃, adjust the pH value to 9-10 using Et3N, stir until dissolved, slowly add the activated dipeptide to the solution, and stir until P29C1 has reacted completely.

[0089] Piperidine was added to the reaction solution to remove the Fmoc protecting group for 1.0–3.0 h. After deprotection, the pH was adjusted to 8.0 ± 0.5 with glacial acetic acid, diluted with water, and filtered to obtain a crude smegglutinin solution with a molar yield of 92.06%. The HPLC chromatogram and data of the crude smegglutinin are shown in Figure 4 and Table 4, respectively.

[0090] Table 4: Statistics of [EndoHis-Aib]semaglutide impurities in the crude peptide of Example 5

[0091] Example 6: Synthesis of Smegglutinin

[0092] Weigh out Fmoc-His(Fmoc)-Aib-OH (3.52 g, 5.14 mol, 4.0 eq) and HOAt (0.70 g, 5.14 mmol, 4.0 eq), add 15 mL of THF, maintain the temperature in an ice-water bath at 0–10 °C, add N,N'-di-tert-butylcarbodiimide (0.78 g, 5.14 mmol, 4.0 eq) with stirring, and continue the reaction for 1.0–3.0 h.

[0093] Weigh out P29C1 (5.0g, 1.28mmol, 1.0q), add THF (70ml) and water (30ml) in sequence, stir well, control the temperature at 10-30℃, adjust the pH value to 9-10 using Et3N, stir until dissolved, slowly add the activated dipeptide to the solution, and stir until P29C1 has reacted completely.

[0094] Piperidine was added to the reaction solution to remove the Fmoc protecting group for 1.0–3.0 h. After deprotection, the pH was adjusted to 8.0 ± 0.5 with glacial acetic acid, diluted with water, and filtered to obtain a crude smegglutinin solution with a molar yield of 93.46%. The HPLC chromatogram and data of the crude smegglutinin are shown in Figure 5 and Table 5, respectively.

[0095] Table 5: Statistics of [EndoHis-Aib]semaglutide impurities in the crude peptide of Example 6

[0096] Example 7: Synthesis of Smegglutinin

[0097] Weigh out Fmoc-His-Aib-OH (2.38 g, 5.14 mol, 4.0 eq) and Oxymapure (0.73 g, 5.14 mmol, 4.0 eq), add 15 mL of NMP, maintain the temperature in an ice-water bath at 0–10 °C, add DIC (0.65 g, 5.14 mmol, 4.0 eq) with stirring, and continue the reaction for 1.0–3.0 h.

[0098] Weigh P29C1 (5.0g, 1.28mmol, 1.0q), add NMP (70ml) and water (30ml) in sequence, stir well, control the temperature at 10-30℃, adjust the pH value to 9-10 with DIEA, stir until dissolved, slowly add the activated dipeptide to the solution, and stir until P29C1 has reacted completely.

[0099] Piperidine was added to the reaction solution to remove the Fmoc protecting group for 1.0–3.0 h. After deprotection, the pH was adjusted to 8.0 ± 0.5 with glacial acetic acid, diluted with water, and filtered to obtain a crude smegglutinin solution with a molar yield of 91.66%. The HPLC chromatogram and data of the crude smegglutinin are shown in Figure 6 and Table 6, respectively.

[0100] Table 6: Statistics of [EndoHis-Aib]semaglutide impurities in the crude peptide of Example 7

[0101] Comparative Example 1: Synthesis of Smegglutinin

[0102] Weigh out Fmoc-His-Aib-OH (2.38 g, 5.14 mol, 4.0 eq) and DIEA (1.30 g, 10.2 mmol, 8.0 eq), add 15 mL of NMP, maintain the temperature in an ice-water bath at 0–10 °C, add PyBop (2.62 g, 5.14 mmol, 4.0 eq) with stirring, and continue the reaction for 0.5–1.0 h.

[0103] Weigh P29C1 (5.0g, 1.28mmol, 1.0q), add NMP (70ml) and water (30ml) in sequence, stir well, control the temperature at 10-30℃, adjust the pH value to 9-10 with DIEA, stir until dissolved, slowly add the activated dipeptide to the solution, and stir until P29C1 has reacted completely.

[0104] Piperidine was added to the reaction solution to remove the Fmoc protecting group. The deprotection time was 1.0 to 3.0 h. After deprotection was completed, the pH value was adjusted to 8.0 ± 0.5 with glacial acetic acid, diluted with water, and filtered to obtain a crude smegglutinin solution with a molar yield of 83.11%. The HPLC chromatogram and data of the crude smegglutinin are shown in Figure 7 and Table 7, respectively.

[0105] Table 7: Statistics of [EndoHis-Aib]semaglutide impurities in the crude peptide of Comparative Example 1

[0106] Comparative Example 2: Synthesis of Smegglutinin

[0107] Weigh out Fmoc-His(Fmoc)-Aib-OH (3.44 g, 5.14 mol, 4.0 eq) and Et3N (1.02 g, 10.2 mmol, 8.0 eq), add 15 mL of NMP, maintain the temperature in an ice-water bath at 0–10 °C, add PyBop (2.62 g, 5.14 mmol, 4.0 eq) with stirring, and continue the reaction for 0.5–1.0 h.

[0108] Weigh out P29C1 (5.0g, 1.28mmol, 1.0q), add NMP (70ml) and water (30ml) in sequence, stir well, control the temperature at 10-30℃, adjust the pH value to 9-10 with Et3N, stir until dissolved, slowly add the activated dipeptide to the solution, and stir until P29C1 has reacted completely.

[0109] Piperidine was added to the reaction solution to remove the Fmoc protecting group. The deprotection time was 1.0 to 3.0 h. After deprotection was completed, the pH value was adjusted to 8.0 ± 0.5 with glacial acetic acid, diluted with water, and filtered to obtain a crude smegglutinin solution with a molar yield of 79.03%. The HPLC chromatogram and data of the crude smegglutinin are shown in Figure 8 and Table 8, respectively.

[0110] Table 8: Statistics of [EndoHis-Aib]semaglutide impurities in the crude peptide of Comparative Example 2

[0111] As can be seen from the HPLC chromatograms and data in Examples 2-7 and the comparative examples, the preparation method of the present invention greatly inhibits / reduces the generation of [Endo His-Aib] smegglutinin impurities.

[0112] In the crude peptides of the comparative examples, the content of [Endo His-Aib]smegglutinin was relatively high, exceeding 6%. However, surprisingly, in the crude peptides of Examples 2-7, the content of [Endo His-Aib]smegglutinin did not exceed 1.05%, a decrease of at least 83.15% compared to the comparative examples.

[0113] The crude peptides in the comparative examples had a molar yield of no more than 83%, while the crude peptides in Examples 2-7 had a molar yield of over 91%, representing an improvement of at least 9.6% compared to the comparative examples. The crude peptides in the comparative examples had a purity of no more than 78%, while the crude peptides in Examples 2-7 had a purity of over 86%, representing an improvement of at least 10% compared to the comparative examples.

[0114] This invention avoids the use of highly toxic TFA and the consumption of large amounts of organic solvents, and instead uses less organic solvents such as DMF and acetonitrile, and uses water for dilution, which greatly reduces costs.

[0115] The method of this invention has the advantages of good synthesis effect, high purity, few impurities, high yield and low cost, which reduces the synthesis cost and is conducive to large-scale industrial production.

Claims

1. A method for synthesizing a polypeptide or protein comprising one or more non-protein-forming amino acids, wherein the method mainly comprises the following steps: 1) Activation of dipeptides such as I under acidic or neutral conditions. in, Where R1 is H or Fmoc; 2) React the activated dipeptide with a polypeptide or protein; 3) Remove the protecting group in situ; This yields the final polypeptide or protein.

2. The method according to claim 1, characterized in that, The activation in step 1) uses a carbodiimide-type condensation reagent.

3. The method according to claim 2, characterized in that, The condensation reagent is selected from one or more of DCC, EDCI, DIC, TBEC, N,N'-di-tert-butylmethanediimine, and EDC.HCl.

4. The method according to claim 1, characterized in that, The activation in step 1) uses a combination of condensation reagent and activating reagent, wherein the activating reagent is selected from one or more of HOSu, HOPFP, HOBt, HOAt, HOPO, and Oxymapure.

5. The method according to claim 1, characterized in that, The activation in step 1) uses EDCI / HOPFP.

6. The method according to any one of claims 1, characterized in that, The activation of the dipeptide in step 1) is as follows: Where R1 is H or Fmoc, and R2 is selected from OBt, OPFP, OAT, OPO, and OSu.

7. The method according to claim 1, characterized in that, In step 2), the activated dipeptide is reacted with a polypeptide or protein in an aqueous medium, wherein the aqueous medium includes any water-based medium.

8. The method according to claim 7, characterized in that, The aqueous medium contains one or more water-soluble organic solvents selected from the following: acetonitrile, DMF, NMP, THF.

9. The method according to claim 7, characterized in that, The pH of the aqueous medium is 8 to 10.

10. The method according to claim 9, characterized in that, The pH is adjusted by one or more bases selected from the following: DIEA, triethylamine, trimethylamine, carbonate, phosphate.

11. The method according to claim 1, characterized in that, In step 3), the reagent used to remove the protecting group is selected from one or more of the following reagents: piperidine, piperazine, methylpiperazine, tert-butylamine.

12. The method according to any one of claims 1, characterized in that, The polypeptide or protein that reacts with the dipeptide is immobilized on a solid phase.

13. The method according to any one of claims 1, characterized in that, The reaction of the dipeptide with the polypeptide or protein is a liquid-phase synthesis.

14. The method according to any one of claims 1-13, characterized in that, The dipeptide is reacted with the α-N-terminus of the polypeptide or protein.

15. The method according to any one of claims 1-13, characterized in that, The polypeptide or protein that reacts with the dipeptide is a GLP-1 peptide.

16. The method according to any one of claims 1-13, characterized in that, The obtained polypeptide is selected from: [Aib8, Arg34]GLP-1-(7-37) peptide; or His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys-Gl u-Phe-Val-Glu-Trp-Leu-Leu-Xaa28-Gly-Gly-Pro-Ser-Ser-Gly, wherein Xaa2 is Aib; Xaa28 is Glu or Ser.

17. The method according to any one of claims 1-13, characterized in that, The obtained polypeptide is selected from: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-Octadecanedioic acid)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly; or His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr 10 -Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala- Lys 20 (AEEA-AEEA-γGlu-Eicosanedioic acid)-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-Gly-Gly 30 -Pro-Ser-Ser-Gly 34 -NH2。

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