Preparation method of glycosylated GLP-1 (7-37) polypeptide

Abstract

The invention relates to the technical field of polypeptide synthesis, in particular to a preparation method of glycosylated GLP-1 (7-37) polypeptide. The method comprises the following steps: preparing a glycosylated GLP-1 (7-37) polypeptide chain by using amino acid and sugar amino acid as raw materials and adopting a solid-phase polypeptide synthesis method; dissolving the glycosylated GLP-1 (7-37) polypeptide chain in a methanol / water solution of NaOH to hydrolyze methyl ester; removing the acetyl group by using an aqueous solution of NH2.NH2; and finally, purifying to obtain the glycosylated GLP-1 (7-37) polypeptide. The preparation method of the glycosylated GLP-1 (7-37) polypeptide, provided by the invention, is short in synthesis period, simple in post-treatment, easy to operate, less in by-products, controllable in cost and easy to popularize. According to the method, the intermediate purification step is reduced, the synthesis efficiency is improved, and the preparation cost is reduced. In addition, the preparation method also realizes chemical synthesis of GLP-1 (7-37) polypeptide glycosylated by 11-site threonine, 13-site threonine or 18-site serine for the first time, and provides an important technology for research of the series of compounds.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide synthesis, and particularly relates to a method for preparing a glycosylated GLP-1(7-37) polypeptide. Background Art

[0002] Glucagon-like peptide-1 (GLP-1(7-37)) is a polypeptide hormone composed of 31 amino acids and has a wide range of pharmacological activities. Studies have found that GLP-1(7-37) reduces blood glucose concentration by stimulating insulin secretion in a blood glucose concentration-dependent manner, slowing gastric emptying, and inhibiting food intake. In addition, GLP-1(7-37) also has cardioprotective and neuroprotective effects, while reducing inflammation and apoptosis. These unique properties of GLP-1(7-37) have inspired a great deal of efforts to use it in clinical applications. However, GLP-1(7-37) is easily degraded and inactivated by the protease dipeptidyl peptidase-4 (DPP-4) widely present in the body, resulting in a half-life in vivo of only about 2 minutes. Relevant research reports have pointed out that by connecting sugars, the degradation of DPP-4 can be inhibited and the half-life of GLP-1 in vivo can be prolonged (J. Am. Chem. Soc. 2009, 131, 6237-6245.; J. Am. Chem. Soc. 2019, 141, 14210-14219.).

[0003] However, the above-mentioned research only connected simple monosaccharides to GLP-1(7-37) by solid-phase peptide synthesis (SPPS), and then prepared more complex polysaccharide (such as containing sialic acid) derivatives by chemoenzymatic methods. This method has a limited preparation scale and high cost, and cannot meet the needs of a large number of scientific research. Therefore, scientists need to develop a method for chemically synthesizing glycosylated GLP-1(7-37), especially derivatives containing complex structures such as sialic acid, in order to obtain more samples for subsequent research. The chemical method for preparing glycopeptides, especially glycopeptides containing sialic acid structures, usually requires more steps, including SPPS and post-treatment, the first round of purification, removal of protecting groups on sugars, the second round of purification, etc. Moreover, the deprotection of sugars is usually accompanied by side reactions, which is quite challenging. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for preparing a glycosylated GLP-1(7-37) polypeptide, wherein the glycosylated GLP-1(7-37) polypeptide is formed by the oxygen connection of a sugar to the side chain of threonine or serine of the human GLP-1(7-37) polypeptide, and the sequence of the human GLP-1(7-37) polypeptide is HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG. This method has a short synthesis cycle, easy post-treatment, few by-products, controllable costs, and is convenient for popularization and use.

[0005] To achieve the above technical objectives, the present invention adopts the following technical solutions:

[0006] Technical solution 1:

[0007] Step A1, solid-phase synthesis: Using sugar amino acids as raw materials, prepare a glycosylated GLP-1(7-37) polypeptide chain by solid-phase peptide synthesis method;

[0008] Step A2, deacetylation of sugar: Dissolve the synthesized glycosylated GLP-1(7-37) polypeptide chain in a methanol / aqueous solution of NaOH for reaction;

[0009] Step A3, purification: Purify the reaction solution obtained in the above step A2 to obtain a glycosylated GLP-1(7-37) polypeptide.

[0010] Technical solution 2:

[0011] Step B1, solid-phase synthesis: Using sugar amino acids as raw materials, prepare a glycosylated GLP-1(7-37) polypeptide chain by solid-phase peptide synthesis method;

[0012] Step B2, hydrolysis of methyl ester on sugar and partial deacetylation: Dissolve the synthesized glycosylated GLP-1(7-37) polypeptide chain in an aqueous solution of NaOH for reaction, add a termination solution, and lyophilize;

[0013] Step B3, deacetylation of sugar: Add an aqueous solution of NH2·NH2 to the lyophilized powder obtained in step B2 for reaction, and add a termination solution;

[0014] Step B4, purification: Purify the reaction solution obtained in the above step B3 to obtain a glycosylated GLP-1(7-37) polypeptide.

[0015] Preferably, the glycosylated GLP-1(7-37) polypeptide described in technical solution 1 is selected from

[0016]

[0017] wherein, X1, X2, X3 are selected from G1, G2 or G3, and the structural formulas of G1, G2, G3 are respectively:

[0018] Structural formula G1:

[0019] Structural formula G2:

[0020] Structural formula G3:

[0021] Among them, G1, G2 or G3 is covalently linked to the oxygen of the side chain of threonine at position 11, threonine at position 13 or serine at position 18 of GLP-1(7-37).

[0022] Preferably, the glycosylated GLP-1(7-37) polypeptide described in Technical Solution 2 is selected from

[0023]

[0024] Among them, X4, X5, X6 are selected from G4, G5, G6 or G7, and the structural formulas of the said G4, G5, G6, G7 are respectively:

[0025] Structural formula G4:

[0026] Structural formula G5:

[0027] Structural formula G6:

[0028] Structural formula G7:

[0029] Among them, G4, G5, G6 or G7 is covalently linked to the oxygen of the side chain of threonine at position 11, threonine at position 13 or serine at position 18 of GLP-1(7-37).

[0030] Preferably, the sugar amino acid in Step A1 is selected from F1, F2 or F3, and the structural formulas of the said F1, F2, F3 are respectively:

[0031] F1:

[0032] F2:

[0033] F3:

[0034] Among them, R = CH3 or H.

[0035] Preferably, the sugar amino acid in Step B1 is selected from F4, F5, F6 or F7, and the structural formulas of the said F4, F5, F6, F7 are respectively:

[0036] F4:

[0037] F5:

[0038] F6:

[0039] F7:

[0040] Among them, R = CH3 or H.

[0041] Preferably, the solid-phase synthesis described in step A1 or B1 includes the following steps:

[0042] Step C1: Add the support resin into a reactor, wash and swell it with DMF.

[0043] Step C2: Add a deprotection solution to remove the Fmoc protecting group.

[0044] Step C3: Add an Fmoc-protected amino acid or an Fmoc-protected glycoamino acid and a coupling reagent to carry out a coupling reaction.

[0045] Step C4: Repeat the deprotection-coupling cycle of steps C2 and C3 until the glycosylated GLP-1(7-37) polypeptide chain is synthesized.

[0046] Preferably, the support resin described in step C1 is selected from Fmoc-Gly-Wang resin or Fmoc-Gly-2-ClTrt resin.

[0047] Preferably, the coupling reagent described in step C3 is selected from HATU and DIEA, HBTU and DIEA, DIC and HOBt, or DIC and Oxyma.

[0048] Preferably, the reaction temperature of steps C2 and C3 is 50°C to 90°C.

[0049] Preferably, the methanol / water solution of NaOH in step A2 is a 40 mM NaOH methanol / water (1 / 1, v / v) solution.

[0050] Preferably, the aqueous solution of NaOH in step B2 is a 40 mM NaOH aqueous solution.

[0051] Preferably, the aqueous solution of NH2·NH2 in step B3 is an aqueous solution containing 5% to 15% NH2·NH2.

[0052] Preferably, the termination solution for steps B2 and B3 is 5% acetic acid.

[0053] The beneficial effects of the present invention are as follows:

[0054] The present invention provides a preparation method for synthesizing glycosylated GLP-1(7-37) polypeptide by a one-pot method, which reduces the intermediate purification steps, improves the synthesis efficiency, and reduces the preparation cost. This method realizes the continuous steps of hydrolysis of methyl ester on sugar and removal of acetyl group, without additional purification in the middle, further improving the synthesis efficiency. In addition, this preparation method also first realizes the chemical synthesis of glycosylated GLP-1(7-37) polypeptide formed by the oxygen connection of sugar with the side chains of threonine at position 11, threonine at position 13 or serine at position 18 of human GLP-1(7-37) polypeptide, providing an important technology for the research of this series of compounds. Description of the Drawings

[0055] Figure 1 . Structure, UPLC chromatogram and MS spectrum of product GLP1. Molecular formula: C 159 H 241 N 41 O 52 ; Theoretical value: [M + 3H] 3+ m / z = 1186.59, [M + 4H] 4+ m / z = 890.19, [M + 5H] 5+ m / z = 712.36; Observed value: 1186.59, 890.20, 712.35. Theoretical value of sugar loss in mass spectrum: [M + 4H] 4+ m / z = 839.42; Observed value: 839.42 (marked with *).

[0056] Figure 2 . Structure, UPLC chromatogram and MS spectrum of product GLP2. Molecular formula: C 165 H 251 N 41 O 57 ; Theoretical value: [M + 3H] 3+ m / z = 1240.61, [M + 4H] 4+ m / z = 930.71, [M + 5H] 5+ m / z = 744.77; Observed value: 1240.61, 930.71, 744.77. Theoretical value of losing 1 sugar in mass spectrum to become GLP1: [M + 5H] 5+ m / z = 712.36; Observed value: 712.33 (marked with *). Theoretical value of losing 2 sugars in mass spectrum: [M + 4H] 4+ m / z = 839.42; Observed value: 839.42 (marked with *).

[0057] Figure 3 . Structure, UPLC chromatogram and MS spectrum of product GLP3. Molecular formula: C 173 H 264 N 42 O62 ; Theoretical value: [M + 3H] 3+ m / z = 1308.30, [M + 4H] 4+ m / z = 981.48, [M + 5H] 5+ m / z = 785.38; Observed values: 1308.30, 981.48, 785.38. Theoretical value of losing 1 sugar in the mass spectrum to become GLP2: [M + 4H] 4+ m / z = 930.71; Observed value: 930.71 (marked with *).

[0058] Figure 4 . Structure, UPLC chromatogram and MS spectrum of product GLP4. Molecular formula: C 170 H 258 N 42 O 60 ; Theoretical value: [M + 3H] 3+ m / z = 1283.62, [M + 4H] 4+ m / z = 962.97, [M + 5H] 5+ m / z = 770.58; Observed values: 1283.62, 962.97, 770.58. Theoretical value of losing 1 sugar in the mass spectrum to become GLP1: [M + 4H] 4+ m / z = 890.19; Observed value: 890.19 (marked with *).

[0059] Figure 5 . Structure, UPLC chromatogram and MS spectrum of product GLP5. Molecular formula: C 176 H 268 N 42 O 65 ; Theoretical value: [M + 3H] 3+ m / z = 1337.64, [M + 4H] 4+ m / z = 1003.48, [M + 5H] 5+ m / z = 802.99; Observed values: 1337.64, 1003.49, 802.99. Theoretical value of losing 1 sugar in the mass spectrum to become GLP2: [M + 4H] 4+ m / z = 930.71; Observed value: 930.71 (marked with *).

[0060] Figure 6 . Structure, UPLC chromatogram and MS spectrum of product GLP6. Molecular formula: C 176 H 268 N 42 O 65 ; Theoretical value: [M + 3H] 3+ m / z = 1337.64, [M + 4H] 4+m / z = 1003.48, [M+5H] 5+ m / z = 802.99; Observed values: 1337.64, 1003.49, 802.99. Theoretical value for losing one sugar in the mass spectrum to become GLP2: [M+4H] 4+ m / z = 930.71; Observed value: 930.71 (*marked). Theoretical value for losing two sugars in the mass spectrum to become GLP1: [M+4H] 4+ m / z = 890.19; Observed value: 890.19 (*marked).

[0061] Figure 7 . Structure, UPLC chromatogram and MS spectrum of product GLP7. Molecular formula: C 187 H 285 N 43 O 73 ; Theoretical value: [M+3H] 3+ m / z = 1434.67, [M+4H] 4+ m / z = 1076.26, [M+5H] 5+ m / z = 861.21; Observed values: 1434.67, 1076.26, 861.22. Theoretical value for losing one sugar in the mass spectrum to become GLP5 / GLP6: [M+4H] 4+ m / z = 1003.48, [M+5H] 5+ m / z = 802.99; Observed values: 1003.49, 802.99.

[0062] Figure 8 . Structure, UPLC chromatogram and MS spectrum of product GLP8. Molecular formula: C 159 H 241 N 41 O 52 ; Theoretical value: [M+3H] 3+ m / z = 1186.59, [M+4H] 4+ m / z = 890.19, [M+5H] 5+ m / z = 712.36; Observed values: 1186.59, 890.20, 712.36.

[0063] Figure 9 . Structure, UPLC chromatogram and MS spectrum of product GLP9. Molecular formula: C 165 H 251 N 41 O 57 ; Theoretical value: [M+3H] 3+ m / z = 1240.61, [M+4H] 4+ m / z = 930.71, [M+5H] 5+m / z = 744.77; Observed values: 1240.61, 930.71, 744.77. Theoretical value for GLP8 with one sugar removed in the mass spectrum: [M + 5H] 5+ m / z = 712.36; Observed value: 712.33 (marked with *). Theoretical value for GLP with two sugars removed in the mass spectrum: [M + 4H] 4+ m / z = 839.42; Observed value: 839.42 (marked with *).

[0064] Figure 10 . Structure, UPLC chromatogram and MS spectrum of product GLP10. Molecular formula: C 173 H 264 N 42 O 62 ; Theoretical value: [M + 3H] 3+ m / z = 1308.30, [M + 4H] 4+ m / z = 981.48, [M + 5H] 5+ m / z = 785.38; Observed values: 1308.30, 981.48, 785.38. Theoretical value for GLP9 with one sugar removed in the mass spectrum: [M + 4H] 4+ m / z = 930.71; Observed value: 930.71 (marked with *).

[0065] Figure 11 . Structure, UPLC chromatogram and MS spectrum of product GLP11. Molecular formula: C 170 H 258 N 42 O 60 ; Theoretical value: [M + 3H] 3+ m / z = 1283.62, [M + 4H] 4+ m / z = 962.97, [M + 5H] 5+ m / z = 770.58; Observed values: 1283.62, 962.97, 770.58. Theoretical value for GLP8 with one sugar removed in the mass spectrum: [M + 4H] 4+ m / z = 890.19; Observed value: 890.19 (marked with *).

[0066] Figure 12 . Structure, UPLC chromatogram and MS spectrum of product GLP12. Molecular formula: C 176 H 268 N 42 O 65 ; Theoretical value: [M + 3H] 3+ m / z = 1337.64, [M + 4H] 4+ m / z = 1003.48, [M + 5H] 5+m / z = 802.99; Observed values: 1337.64, 1003.49, 802.99. Theoretical value of losing one sugar in the mass spectrum to become GLP9: [M+4H] 4+ m / z = 930.71; Observed value: 930.71 (marked with *).

[0067] Figure 13 . Structure, UPLC chromatogram and MS spectrum of product GLP13. Molecular formula: C 176 H 268 N 42 O 65 ; Theoretical value: [M+3H] 3+ m / z = 1337.64, [M+4H] 4+ m / z = 1003.48, [M+5H] 5+ m / z = 802.99; Observed values: 1337.64, 1003.49, 802.99. Theoretical value of losing one sugar in the mass spectrum to become GLP9: [M+4H] 4+ m / z = 930.71; Observed value: 930.71 (marked with *). Theoretical value of losing two sugars in the mass spectrum to become GLP8: [M+4H] 4+ m / z = 890.19; Observed value: 890.19 (marked with *).

[0068] Figure 14 . Structure, UPLC chromatogram and MS spectrum of product GLP14. Molecular formula: C 187 H 285 N 43 O 73 ; Theoretical value: [M+3H] 3+ m / z = 1434.67, [M+4H] 4+ m / z = 1076.26, [M+5H] 5+ m / z = 861.21; Observed values: 1434.68, 1076.26, 861.21. Theoretical value of losing one sugar in the mass spectrum to become GLP12 / GLP13: [M+4H] 4+ m / z = 1003.48, [M+5H] 5+ m / z = 802.99; Observed values: 1003.49, 802.99.

[0069] Figure 15 . Structure, UPLC chromatogram and MS spectrum of product GLP15. Molecular formula: C 159 H 241 N 41 O 52 ; Theoretical value: [M+3H] 3+ m / z = 1186.59, [M+4H]4+ m / z = 890.19, [M + 5H] 5+ m / z = 712.36; Observed values: 1186.59, 890.20, 712.36. Theoretical value of sugar loss in mass spectrometry: [M + 4H] 4+ m / z = 839.42; Observed value: 839.42 (marked with *).

[0070] Figure 16 . Structure, UPLC chromatogram and MS spectrum of product GLP16. Molecular formula: C 165 H 251 N 41 O 57 ; Theoretical value: [M + 3H] 3+ m / z = 1240.61, [M + 4H] 4+ m / z = 930.71, [M + 5H] 5+ m / z = 744.77; Observed values: 1240.61, 930.71, 744.77. Theoretical value of losing 1 sugar in mass spectrometry to become GLP15: [M + 5H] 5+ m / z = 712.36; Observed value: 712.34 (marked with *). Theoretical value of losing 2 sugars in mass spectrometry: [M + 4H] 4+ m / z = 839.42; Observed value: 839.42 (marked with *).

[0071] Figure 17 . Structure, UPLC chromatogram and MS spectrum of product GLP17. Molecular formula: C 173 H 264 N 42 O 62 ; Theoretical value: [M + 3H] 3+ m / z = 1308.30, [M + 4H] 4+ m / z = 981.48, [M + 5H] 5+ m / z = 785.38; Observed values: 1308.30, 981.48, 785.39. Theoretical value of losing 1 sugar in mass spectrometry to become GLP16: [M + 4H] 4+ m / z = 930.71; Observed value: 930.71 (marked with *). Theoretical value of losing 2 sugars in mass spectrometry to become GLP15: [M + 4H] 4+ m / z = 890.19; Observed value: 890.19 (marked with *).

[0072] Figure 18 . Structure, UPLC chromatogram and MS spectrum of product GLP18. Molecular formula: C 170 H 258 N 42 O 60; Theoretical value: [M + 3H] 3+ m / z = 1283.62, [M + 4H] 4+ m / z = 962.97, [M + 5H] 5+ m / z = 770.58; Observed values: 1283.62, 962.97, 770.58.

[0073] Figure 19 . Structure, UPLC chromatogram and MS spectrum of product GLP19. Molecular formula: C 176 H 268 N 42 O 65 ; Theoretical value: [M + 3H] 3+ m / z = 1337.64, [M + 4H] 4+ m / z = 1003.48, [M + 5H] 5+ m / z = 802.99; Observed values: 1337.64, 1003.49, 802.99. Theoretical value of losing 1 sugar in the mass spectrum to become GLP16: [M + 4H] 4+ m / z = 930.71; Observed value: 930.71 (marked with *).

[0074] Figure 20 . Structure, UPLC chromatogram and MS spectrum of product GLP20. Molecular formula: C 176 H 268 N 42 O 65 ; Theoretical value: [M + 3H] 3+ m / z = 1337.64, [M + 4H] 4+ m / z = 1003.48, [M + 5H] 5+ m / z = 802.99; Observed values: 1337.64, 1003.49, 802.99. Theoretical value of losing 1 sugar in the mass spectrum to become GLP16: [M + 4H] 4+ m / z = 930.71; Observed value: 930.71 (marked with *). Theoretical value of losing 2 sugars in the mass spectrum to become GLP15: [M + 4H] 4+ m / z = 890.19; Observed value: 890.19 (marked with *).

[0075] Figure 21 . Structure, UPLC chromatogram and MS spectrum of product GLP21. Molecular formula: C 187 H 285 N 43 O 73 ; Theoretical value: [M + 3H] 3+ m / z = 1434.67, [M + 4H] 4+m / z = 1076.26; Observed values: 1434.67, 1076.26. Theoretical value of losing one sugar in the mass spectrum to become GLP19 / GLP20: [M+4H] 4+ m / z = 1003.48; Observed value: 1003.49 (marked with *). Theoretical value of losing two sugars in the mass spectrum to become GLP18: [M+4H] 4+ m / z = 962.97; Observed value: 962.97 (marked with *). Theoretical value of losing two sugars in the mass spectrum to become GLP16: [M+4H] 4+ m / z = 930.71; Observed value: 930.71 (marked with *).

[0076] In the above figures: AU is the absorbance unit; Time (min) is the retention time in UPLC and its unit is minute; m / z is the mass-to-charge ratio. Detailed implementation manners

[0077] To make the objectives, features and beneficial effects of the present invention clearer and more understandable, the following will describe in detail the specific implementation manners of the present invention in combination with the technical solutions. It should be noted that the content described in the following embodiments is only for explaining and interpreting the present invention, and the present invention is not limited to these embodiments. Any modification or equivalent replacement of the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention shall be covered by the protection scope of the present invention.

[0078] The following table 1 shows the explanations of some English abbreviations used in the present invention and their meanings:

[0079] Table 1:

[0080]

[0081]

[0082] The naming and structure of the glycosylated GLP-1(7-37) polypeptide involved in the present invention are as follows:

[0083] Structural formula GLP1:

[0084] Structural formula GLP2:

[0085] Structural formula GLP3:

[0086] Structural formula GLP4:

[0087]

[0088] Structural formula GLP5:

[0089]

[0090] Structural formula GLP6:

[0091]

[0092] Structural formula GLP7:

[0093]

[0094] Structural formula GLP8: Structural formula GLP9:

[0095] Structural formula GLP10:

[0096] Structural formula GLP11:

[0097]

[0098] Structural formula GLP12:

[0099]

[0100] Structural formula GLP13:

[0101]

[0102] Structural formula GLP14:

[0103]

[0104] Structural formula GLP15: Structural formula GLP16: Structural formula GLP17: Structural formula GLP18:

[0105] Structural formula GLP19:

[0106] Structural formula GLP20:

[0107]

[0108] Structural formula GLP21:

[0109]

[0110] Among them, the sugar in GLP1-GLP7 is covalently linked to the oxygen of the side chain of threonine at position 11 of GLP-1(7-37); the sugar in GLP8-GLP14 is covalently linked to the oxygen of the side chain of threonine at position 13 of GLP-1(7-37), and the sugar in GLP15-GLP21 is covalently linked to the oxygen of the side chain of serine at position 18 of GLP-1(7-37).

[0111] Example 1. Preparation of GLP1.

[0112] Solid-phase peptide synthesis (SPPS) of the glycosylated GLP-1(7-37) polypeptide GLP1 was carried out using a CEM Liberty Blue automatic microwave peptide synthesizer. The specific steps are as follows: First, weigh 0.05 mmol of Fmoc-Gly-2-ClTrt resin, add it to the reactor, and add 10 mL of DMF to swell for 5 min. After removing the DMF solution, add 3 mL of 4-methylpiperidine / DMF solution (20 / 80, v / v), and microwave heat at 50 °C for 5 min to remove the Fmoc protecting group. Wash the resin four times with DMF (4 mL × 4). After removing the DMF solution, add 0.2 M Fmoc-protected amino acid (5.0 equivalents) dissolved in DMF, 0.5 M DIC (10 equivalents) dissolved in DMF, and 0.25 M Oxyma (5.0 equivalents) dissolved in DMF, and microwave heat at 50 °C for 10 min for the coupling reaction. For the amino acids (Gly10, Phe12, Asp15, Phe28, Leu32, Gly35) after the amino acids with β-side chain branching (Thr, Ile, Val) and the amino acids with large steric hindrance side chain protecting groups (Arg), two coupling reactions are required. The coupling reaction of Arg needs to be carried out at room temperature for 25 min, and a total of two rounds are carried out. In addition, the coupling of the sugar amino acid F1 (0.2 M concentration, 4.5 equivalents) can be completely reacted by microwave heating at 50 °C for 20 min. After the coupling of the amino acid or the sugar amino acid F1 is completed, wash the resin four times with DMF (4 mL × 4). Repeat the above coupling-deprotection cycle until the synthesis of the GLP1 glycopeptide chain is completed.

[0113] After solid-phase synthesis, transfer the resin from the reactor to a peptide synthesis reaction tube, and wash it three times alternately with DMF and DCM (5 mL each time). Then add 3.5 mL of TFA / TIPS / H2O / DODT (90 / 5 / 2.5 / 2.5, v / v / v / v) cleavage solution, and stir at 38 °C for 40 min. Collect the cleavage solution with a 50 mL centrifuge tube, and then add 40 mL of methyl tert-butyl ether at 4 °C to precipitate the polypeptide, and centrifuge (9000 g, 7 min, 4 °C). After discarding the supernatant, dissolve the precipitate in 10 mL of MeCN / H2O (1 / 1, v / v), and obtain 104 mg of crude peptide after freeze-drying.

[0114] Weigh 6.03 mg of the above-mentioned crude peptide, add it to 3 mL of a methanol / water (1 / 1, v / v) solution of 40 mM NaOH, stir at room temperature until all acetyl groups are removed (2 - 3 h), and then neutralize with 5% acetic acid. After filtering the reaction solution through a 0.2 μm filter membrane, purify it by HPLC using a C18 column (10x250 mm, 5 μm, ), mobile phase A is water containing 0.05% TFA, mobile phase B is acetonitrile containing 0.05% TFA, perform linear elution with 20% to 40% (B%) for 40 minutes at a flow rate of 4 mL / min, and the detection wavelength is 214 nm. During the purification process, use LC-MS (Waters SQD2-ACQUITY UPLC, mobile phase A is water containing 0.1% formic acid, mobile phase B is acetonitrile containing 0.1% formic acid, gradient is 20% to 40% (B%), flow rate is 0.3 mL / min, detection wavelength is 214 nm) to identify the collected eluate, combine the eluate containing the product, and obtain 2.08 mg of the product GLP1 after lyophilization, with a yield of 20%. The product GLP1 is characterized by LC-MS, and its structure is correct. The results are as shown in the attached Figure 1 figure.

[0115] Example 2. Preparation of GLP2, GLP3, GLP8 - GLP10 and GLP15 - GLP17.

[0116] The preparation of GLP2, GLP3, GLP8 - GLP10 and GLP15 - GLP17 is basically the same as that in Example 1, except that the glycoamino acids they use are different. The corresponding relationships between the synthesized glycopeptides and glycoamino acids are listed in the following table:

[0117] Table 2: Corresponding relationships between glycopeptides and the glycoamino acids used in their synthesis.

[0118] Glycopeptide Glycoamino acid GLP8, GLP15 F1 GLP2, GLP9, GLP16 F2 GLP3, GLP10, GLP17 F3

[0119] The structures of the synthesized GLP2, GLP3, GLP8 - GLP10 and GLP15 - GLP17 are all correct. Their LC-MS characterization results are as shown in the attached Figure 2 , 3 , 8 - 10 and 15 - 17. Their synthesis yields are as shown in the following table:

[0120] Table 3: Statistical results of the synthesis yields of GLP1 - GLP3, GLP8 - GLP10 and GLP15 - GLP17.

[0121] Yield Yield Yield GLP1 20％ GLP8 21％ GLP15 26％ GLP2 27％ GLP9 26％ GLP16 23％ GLP3 19％ GLP10 20％ GLP17 19％

[0122] Example 3. Preparation of GLP4.

[0123] Solid-phase peptide synthesis (SPPS) of glycosylated GLP-1(7-37) polypeptide GLP4 was carried out using a CEM Liberty Blue automatic microwave peptide synthesizer. The specific steps are as follows: First, weigh 0.05 mmol of Fmoc-Gly-2-ClTrt resin, add it to the reactor, and add 10 mL of DMF to swell for 5 min. After removing the DMF solution, add 3 mL of 4-methylpiperidine / DMF solution (20 / 80, v / v), and microwave heat at 50 °C for 5 min to remove the Fmoc protecting group. Wash the resin four times with DMF (4 mL × 4). After removing the DMF solution, add 0.2 M Fmoc-protected amino acid (5.0 equivalents) dissolved in DMF, 0.5 M DIC (10 equivalents) dissolved in DMF, and 0.25 M Oxyma (5.0 equivalents) dissolved in DMF, and microwave heat at 50 °C for 10 min for the coupling reaction. For the amino acids (Gly10, Phe12, Asp15, Phe28, Leu32, Gly35) after the amino acids with β-side chain branching (Thr, Ile, Val) and amino acids with large steric hindrance side chain protecting groups (Arg), two coupling reactions are required. The coupling reaction of Arg needs to be carried out at room temperature for 25 min, and two rounds are carried out in total. In addition, the coupling of the sugar amino acid F4 (0.2 M concentration, 4.5 equivalents) can be completed by microwave heating at 50 °C for 20 min. After the coupling of the amino acid or the sugar amino acid F4 is completed, wash the resin four times with DMF (4 mL × 4). Repeat the above coupling-deprotection cycle until the synthesis of the GLP4 glycopeptide chain is completed.

[0124] After solid-phase synthesis, transfer the resin from the reactor to a peptide synthesis reaction tube, and wash it three times alternately with DMF and DCM (5 mL each time). Then add 3.5 mL of TFA / TIPS / H2O / DODT (90 / 5 / 2.5 / 2.5, v / v / v / v) cleavage solution, and stir at 38 °C for 40 min. Collect the cleavage solution with a 50 mL centrifuge tube, and then add 40 mL of methyl tert-butyl ether at 4 °C to precipitate the polypeptide, and centrifuge (9000 g, 7 min, 4 °C). After discarding the supernatant, dissolve the precipitate in 10 mL of MeCN / H2O (1 / 1, v / v), and obtain 96 mg of crude peptide after freeze-drying.

[0125] Weigh 5.19 mg of the above crude peptide, add 2.5 mL of an aqueous solution of 40 mM NaOH, stir at room temperature until most of the acetyl groups and all of the methyl esters are removed (0.5 h), and then add 5% acetic acid for neutralization. After freeze-drying the reaction solution, continue to add 1.6 mL of an aqueous solution of 10% NH2·NH2, stir at room temperature for 2 h to remove the remaining acetyl groups. After the reaction is completed, add 5% acetic acid for neutralization. After filtering the reaction solution through a 0.2 μm filter membrane, purify it by HPLC using a C18 column (10x250mm, 5μm, ) The mobile phase A is water containing 0.05% TFA, and the mobile phase B is acetonitrile containing 0.05% TFA. Linear elution is carried out at 20% to 40% (B%) for 40 minutes, the flow rate is 4 mL / min, and the detection wavelength is 214 nm. During the purification process, LC-MS (Waters SQD2-ACQUITY UPLC, the mobile phase A is water containing 0.1% formic acid, the mobile phase B is acetonitrile containing 0.1% formic acid, the gradient is 20% to 40% (B%), the flow rate is 0.3 mL / min, and the detection wavelength is 214 nm) is used to identify the collected eluate. The eluates containing the product are combined and lyophilized to obtain 1.54 mg of the product GLP4, and the yield is 15%. The product GLP4 is characterized by LC-MS, and its structure is correct. The results are as shown in the appendix Figure 4 shown.

[0126] Example 4. Preparation of GLP5-GLP7, GLP11-GLP14 and GLP18-GLP21.

[0127] The preparation of GLP5-GLP7, GLP11-GLP14 and GLP18-GLP21 is basically the same as that in Example 3, except that the glycoamino acids used are different. The corresponding relationships between the synthesized glycopeptides and glycoamino acids are listed in the following table:

[0128] Table 4: Corresponding relationships between glycopeptides and the glycoamino acids used in their synthesis.

[0129] Glycopeptide Glycoamino acid GLP11, GLP18 F4 GLP5, GLP12, GLP19 F5 GLP6, GLP13, GLP20 F6 GLP7, GLP14, GLP21 F7

[0130] The structures of the synthesized GLP4-GLP7, GLP11-GLP14 and GLP18-GLP21 are all correct. Their LC-MS characterization results are as shown in the appendix Figures 5 - 7 、11-14 and 18-21. Their synthesis yields are shown in the following table:

[0131] Table 5: Statistical results of the synthesis yields of GLP4-GLP7, GLP11-GLP14 and GLP18-GLP21.

[0132] Yield Yield Yield GLP4 15％ GLP11 16％ GLP18 26％ GLP5 13％ GLP12 13％ GLP19 16％ GLP6 14％ GLP13 14％ GLP20 14％ GLP7 13％ GLP14 14％ GLP21 13％ .

Claims

1. A method for preparing a glycosylated GLP-1(7-37) polypeptide, wherein the glycosylated GLP-1(7-37) polypeptide is formed by an oxygen linkage between a sugar and the oxygen of the threonine or serine side chain of the human GLP-1(7-37) polypeptide, and the human GLP-1(7-37) polypeptide sequence is HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG, characterized in that, It includes the following steps: Step A1, solid-phase synthesis: Using sugar amino acids as raw materials, prepare a glycosylated GLP-1(7-37) polypeptide chain by solid-phase peptide synthesis method; Step A2, deacetylation of sugar: Dissolve the synthesized glycosylated GLP-1(7-37) polypeptide chain in a methanol / water solution of NaOH for reaction; Step A3, purification: Purify the reaction solution obtained in the above Step A2 to obtain a glycosylated GLP-1(7-37) polypeptide.

2. A method for preparing a glycosylated GLP-1(7-37) polypeptide, wherein the glycosylated GLP-1(7-37) polypeptide is formed by the oxygen connection of a sugar to the oxygen of the threonine or serine side chain of the human GLP-1(7-37) polypeptide, and the sequence of the human GLP-1(7-37) polypeptide is HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG, characterized in that, It includes the following steps: Step B1, solid-phase synthesis: Using sugar amino acids as raw materials, prepare a glycosylated GLP-1(7-37) polypeptide chain by solid-phase peptide synthesis method; Step B2, hydrolysis of methyl ester on sugar and partial deacetylation: Dissolve the synthesized glycosylated GLP-1(7-37) polypeptide chain in an aqueous solution of NaOH for reaction, add a termination solution, and freeze-dry; Step B3, deacetylation of sugar: Add an aqueous solution of NH2·NH2 to the freeze-dried powder obtained in Step B2 for reaction, and add a termination solution; Step B4, purification: Purify the reaction solution obtained in the above Step B3 to obtain a glycosylated GLP-1(7-37) polypeptide.

3. The preparation method of the glycosylated GLP-1(7-37) polypeptide according to claim 1, wherein, The glycosylated GLP-1(7-37) polypeptide is selected from wherein, X1, X2, X3 are selected from G1, G2 or G3, and the structural formulas of G1, G2, G3 are respectively: Structural formula G1: Structural formula G2: Structural formula G3: wherein, G1, G2 or G3 is covalently linked to the oxygen of the side chain of threonine at position 11, threonine at position 13 or serine at position 18 of GLP-1(7-37).

4. The preparation method of the glycosylated GLP-1(7-37) polypeptide according to claim 2, wherein, The glycosylated GLP-1(7-37) polypeptide is selected from wherein, X4, X5, X6 are selected from G4, G5, G6 or G7, and the structural formulas of G4, G5, G6, G7 are respectively: Structural formula G4: Structural formula G5: Structural formula G6: Structural formula G7: wherein, G4, G5, G6 or G7 is covalently linked to the oxygen of the side chain of threonine at position 11, threonine at position 13 or serine at position 18 of GLP-1(7-37).

5. The preparation method of the glycosylated GLP-1(7-37) polypeptide according to claim 1, characterized in that, The sugar amino acid in Step A1 is selected from F1, F2 or F3, and the structural formulas of F1, F2, F3 are respectively: F1： F2： F3： wherein, R = CH3 or H.

6. The preparation method of a glycosylated GLP-1(7-37) polypeptide according to claim 2, wherein The sugar amino acid in Step B1 is selected from F4, F5, F6 or F7, and the structural formulas of F4, F5, F6, F7 are respectively: F4： F5： F6： F7： wherein, R = CH3 or H.

7. The preparation method of the glycosylated GLP-1(7-37) polypeptide according to any one of claims 1 to 2, characterized in that, The solid-phase synthesis includes the following steps: Step C1: Add the carrier resin into the reactor, wash and swell with DMF; Step C2: Add a deprotection solution to remove the Fmoc protecting group; Step C3: Add an Fmoc-protected amino acid or an Fmoc-protected sugar amino acid and a condensation reagent for coupling reaction; Step C4: Repeat the deprotection-coupling cycle of Step C2 and C3 until a glycosylated GLP-1(7-37) polypeptide chain is synthesized.

8. The preparation method of the glycosylated GLP-1(7-37) polypeptide according to claim 1, characterized in that, The methanol / water solution of NaOH in Step A2 is a 40 mM NaOH methanol / water (1 / 1, v / v) solution.

9. The preparation method of the glycosylated GLP-1(7-37) polypeptide according to claim 2, characterized in that, The aqueous solution of NaOH in Step B2 is a 40 mM NaOH aqueous solution.

10. The preparation method of the glycosylated GLP-1(7-37) polypeptide according to claim 2, characterized in that, The aqueous solution of NH2·NH2 in Step B3 is an aqueous solution containing 5% - 15% NH2·NH2.

11. The preparation method of the glycosylated GLP-1(7-37) polypeptide according to claim 2, wherein, The termination solution in Steps B2 and B3 is 5% acetic acid.

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