Method for chemically synthesizing glycosylated insulin

Abstract

The invention belongs to the technical field of polypeptide preparation methods, and particularly relates to a method for chemically synthesizing glycosylated insulin. The preparation method comprises the following steps: preparing an insulin chain A by using a solid-phase polypeptide synthesis technology, selectively generating an intrachain disulfide bond A6-A11 in a post-treatment process of resin, preparing a glycosylated insulin chain B by using sugar amino acid, connecting the chain A and the chain B by forming an A20-B19 disulfide bond, then generating an A7-B7 disulfide bond, and preparing a glycosylated insulin chain B by using a solid-phase polypeptide synthesis technology. And the protecting group of the sugar is removed by using development conditions in the proper steps to obtain the glycosylated insulin. According to the method disclosed by the invention, the formation of disulfide bonds is accurately controlled, the product purity is higher, and the synthesis yield is higher. Moreover, the method also provides a sugar deprotection strategy with sialic acid methyl ester, so that the preparation of the glycosylated insulin is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide preparation methods, and particularly relates to a method for chemically synthesizing glycosylated insulin. Background Art

[0002] Insulin is an important hormone for maintaining blood glucose homeostasis. For patients with type 1 diabetes, insulin therapy has become a life-saving necessity, and this disease is characterized by immune-mediated depletion of β-cells in the pancreas, the site of insulin production. Insulin is also crucial for patients with type 2 diabetes, a chronic disease characterized by insulin resistance and eventual pancreatic β-cell dysfunction, which in some cases leads to persistently high blood glucose levels. It is estimated that there are 537 million people with diabetes globally, and this number is expected to increase to 643 million by 2030 and 783 million by 2045. In addition, it is estimated that diabetes caused more than 6.7 million deaths in 2021, with medical costs approaching $1 trillion.

[0003] Due to the important role of insulin in blood glucose control and diabetes treatment, over the past century, means including amino acid mutations have been used to modify insulin to obtain insulin drugs with better properties. In recent years, it has been reported that N-acetylgalactosamine (GalNAc), mannose (Man), dimannose (ManMan), or trimannose (ManManMan) is linked to ThrB27 in the B-chain of insulin, resulting in a higher anti-self-aggregation tendency (ACS.Chem.Biol.2018,13,73-81.).

[0004] Although this series of glycosylated insulin consists of only 51 amino acids, it contains an A-chain (21 amino acids) and a B-chain (30 amino acids), which are linked together by two disulfide bonds (A7-B7 and A20-B19), and the A-chain also has a third intra-chain disulfide bond (A6-A11). In addition, the attachment of sugars further increases the complexity of the synthesis, especially the compatibility issue between the removal of its protecting groups and the disulfide bonds. The intricate disulfide bond pattern and the difficulty of sugar deprotection are the main obstacles hindering the synthesis efficiency.

[0005] The synthetic strategy adopted in the above report is as follows: The insulin A-chain and B-chain are synthesized by solid-phase peptide synthesis (SPPS). Among them, cysteines at positions 6, 11, and 20 of the A-chain (hereinafter referred to as CysA6, CysA11, and CysA20 respectively) are protected with acetamidomethyl (Acm) groups, cysteine at position 7 of the A-chain (hereinafter referred to as CysA7) has no protecting group, cysteine at position 7 of the B-chain (hereinafter referred to as CysB7) is linked with 3-nitro-2-pyridine-sulfenyl (SNPy), cysteine at position 19 of the B-chain (hereinafter referred to as CysB19) is protected by Acm, and threonine at position 27 of the B-chain (hereinafter referred to as ThrB27) is linked with a sugar with a protecting group. The A-chain and B-chain are placed in a buffer solution of 8.0 M Gn·HCl, 0.1 M Tris·HCl, pH 8.0, and a disulfide bond will form between Cys at position 7 of the A-chain and Cys at position 7 of the B-chain. Subsequently, add I 2 for oxidation, remove the Acm groups of the remaining four Cys and form disulfide bonds. Finally, use NH 2 NH 2 solution to remove the protecting group on the sugar to obtain glycosylated insulin.

[0006]

[0007] Since the formation of disulfide bonds of the four Cys has not been effectively regioselectively controlled, by-products such as A-A chain connection, B-B chain connection, and A-B chain connection with incorrect disulfide bond pairing will be generated. This will not only lead to a messy reaction system and low product purity, but also a low synthesis yield. Among them, the yields of the monosaccharides GalNAc or Man are 4.2% or 2.6% respectively, the yield of the disaccharide ManMan is 2.1%, and the yield of the trisaccharide ManManMan is 0.8%. In addition, the sugar deprotection method in this scheme is only suitable for the case of only containing acetyl protecting groups and not suitable for the case of containing methyl ester protecting groups, while the commonly used protecting form of the carboxyl group in sugars containing sialic acid is methyl ester. Summary of the Invention

[0008] To solve the problems of low purity and low yield in the current synthesis of glycosylated insulin and the lack of a method for deprotecting sialic acid-containing groups, the present invention provides a method for chemically synthesizing glycosylated insulin, wherein the glycosylated insulin is formed by connecting a sugar to the hydroxyl group of threonine at position 27 of the B chain of human insulin. The insulin A chain is prepared using solid-phase polypeptide synthesis technology, and intramolecular disulfide bond A6-A11 is selectively formed during the resin post-treatment process. The glycosylated insulin B chain is prepared using sugar amino acids. The A chain and the B chain are connected by forming disulfide bond A20-B19, followed by the formation of disulfide bond A7-B7. The protecting group of the sugar is removed using the developed conditions in the above-mentioned appropriate steps to obtain glycosylated insulin. The following technical solutions can be specifically adopted:

[0009] Technical solution 1, the synthesis includes the following steps:

[0010] a1) Prepare the insulin A chain, which contains the intramolecular disulfide bond A6-A11, and the side chain of CysA7 is protected with acetamidomethyl;

[0011] a2) Prepare the insulin B chain, where the side chain of ThrB27 is connected to a sugar, the side chain of CysB7 is protected with acetamidomethyl, and the side chain of CysB19 is connected to 3-nitro-2-pyridylthiol;

[0012] a3) Place the A chain and the B chain in a buffer solution to form disulfide bond A20-B19, and then remove the acetamidomethyl groups on CysA7 and CysB7 and oxidize to form disulfide bond A7-B7;

[0013] a4) Use NH 2 NH 2 solution to remove the protecting group on the sugar to obtain glycosylated insulin.

[0014] Technical solution 2, the synthesis includes the following steps:

[0015] b1) Prepare the insulin A chain, which contains the intramolecular disulfide bond A6-A11, the side chain of CysA7 is protected with acetamidomethyl, and the side chain of CysA20 is connected to 3-nitro-2-pyridylthiol;

[0016] b2) Prepare the insulin B chain, where the side chain of ThrB27 is connected to a sugar, and the side chain of CysB7 is protected with acetamidomethyl;

[0017] b3) Use NaOH solution to remove the protecting group on the sugar to obtain glycosylated B chain.

[0018] b4) Place the A chain and the B chain in a buffer solution to form disulfide bond A20-B19, and then remove the acetamidomethyl groups on CysA7 and CysB7 and oxidize to form disulfide bond A7-B7, thereby obtaining glycosylated insulin.

[0019] Technical solution three, the synthesis includes the following steps:

[0020] c1) Prepare insulin A chain, the A chain contains the intra-chain disulfide bond A6-A11, the side chain of CysA7 is protected with acetamidomethyl, and the side chain of CysA20 is connected to 3-nitro-2-pyridyl mercapto;

[0021] c2) Prepare insulin B chain, the side chain of ThrB27 of the B chain is connected to a sugar, and the side chain of CysB7 is protected with acetamidomethyl;

[0022] c3) Use NaOH solution and NH 2 NH 2 solution to remove the protecting group on the sugar to obtain the glycosylated B chain.

[0023] c4) Place the A chain and the B chain in a buffer solution to form the A20-B19 disulfide bond, then remove the acetamidomethyl on CysA7 and CysB7 and oxidize to form the A7-B7 disulfide bond, thereby obtaining glycosylated insulin.

[0024] Technical solution four, the synthesis includes the following steps:

[0025] d1) Prepare insulin A chain, the A chain contains the intra-chain disulfide bond A6-A11, the side chain of CysA7 is protected with acetamidomethyl, and the side chain of CysA20 is connected to 3-nitro-2-pyridyl mercapto;

[0026] d2) Prepare insulin B chain, the side chain of ThrB27 of the B chain is connected to a sugar, the side chain of CysB7 is protected with acetamidomethyl, and the side chain of LysB29 is protected with 1-(4,4-dimethyl-2,6-dioxocyclohexylidene methyl)-3-methylbutyl;

[0027] d3) Use NaOH solution and NH 2 NH 2 solution to remove the protecting group on the sugar to obtain the glycosylated B chain.

[0028] d4) Place the A chain and the B chain in a buffer solution to form the A20-B19 disulfide bond, then remove the acetamidomethyl on CysA7 and CysB7 and oxidize to form the A7-B7 disulfide bond, thereby obtaining glycosylated insulin.

[0029] Preferably, the step a1 includes:

[0030] Using Rink Amide AM resin as a solid-phase carrier, the insulin A chain was prepared by solid-phase peptide synthesis technology. Among them, CysA6, CysA7, ThrA8-SerA9, CysA11 and AsnA21 were respectively treated with Fmoc-Cys(STmp)-OH, Fmoc-Cys(Acm)-OH, Boc-Ser[Fmoc-Thr(tBu)]-OH, Fmoc-Cys(Mmt)-OH and Fmoc-Asp-OtBu. Subsequently, the STmp group was removed using 5% β-mercaptoethanol and 0.1 M N-methylmorpholine. 2,2'-Dithiobis(5-nitropyridine) was added to activate CysA6, and the Mmt group was selectively removed with 1% TFA to form the A6-A11 intrachain disulfide bond. TFA was added to remove the side-chain protecting groups of the polypeptide and cleave the polypeptide from the resin, obtaining the A chain InsA1, and the structure of the InsA1 was:

[0031] Among them, the Acm structure is acetylaminomethyl.

[0032] Preferably, the step b1, c1 or d1 includes:

[0033] Using Rink Amide AM resin as a solid-phase carrier, the insulin A chain was prepared by solid-phase peptide synthesis technology. Among them, CysA6, CysA7, ThrA8-SerA9, CysA11 and AsnA21 were respectively treated with Fmoc-Cys(STmp)-OH, Fmoc-Cys(Acm)-OH, Boc-Ser[Fmoc-Thr(tBu)]-OH, Fmoc-Cys(Mmt)-OH and Fmoc-Asp-OtBu. Subsequently, the STmp group was removed using 5% β-mercaptoethanol and 0.1 M N-methylmorpholine. 2,2'-Dithiobis(5-nitropyridine) was added to activate CysA6, and the Mmt group was selectively removed with 1% TFA to form the A6-A11 intrachain disulfide bond. The resin was treated with TFA containing 2,2'-dithiobis(5-nitropyridine), which could activate CysA20 while removing the side-chain protecting groups of the polypeptide and cleaving the polypeptide from the resin, obtaining the A chain InsA2, and the structure of the InsA2 was:

[0034] Among them, the Acm structure is acetylaminomethyl, and the SNPy structure is 3-nitro-2-pyridylthiol.

[0035] Preferably, the step a2 includes:

[0036] Using Fmoc-Thr(tBu)-2-ClTrt resin as the solid-phase carrier, the insulin B chain was prepared by solid-phase peptide synthesis technology. Among them, ThrB27 and CysB7 used glycoamino acid and Fmoc-Cys(Acm)-OH respectively. Adding TFA solution containing 2,2'-dithiobis(5-nitropyridine) to treat the resin can activate CysB19 while removing the protecting groups of the polypeptide side chains and cleaving the polypeptide from the resin, thus obtaining the B chain:

[0037] Among them, the Acm structure is acetylaminomethyl, the SNPy structure is 3-nitro-2-pyridylthiol, and R 1 The structure is a sugar with a protecting group.

[0038] Preferably, the step b2, c2 or d2 includes:

[0039] The insulin B chain was prepared by solid-phase peptide synthesis technology. Among them, ThrB27 and CysB7 used glycoamino acid and Fmoc-Cys(Acm)-OH respectively. Adding TFA to remove the protecting groups of the polypeptide side chains and cleave the polypeptide from the resin, thus obtaining the B chain:

[0040] Among them, the Acm structure is acetylaminomethyl, and R 2 The structure is a sugar with a protecting group.

[0041] Preferably, the step a3, b4, c4 or d4 includes:

[0042] Dissolve the A chain and the B chain in a buffer solution to form the A20-B19 disulfide bond. Subsequently, add I 2 Remove the Acm protecting groups on CysA7 and CysB7 and oxidize to form the A7-B7 disulfide bond.

[0043] Preferably, the buffer solution in the step a3, b4, c4 or d4 is a buffer solution containing 6M urea and 0.2M NH 4 HCO 3 .

[0044] Preferably, the Acm removal solution in the step a3, b4, c4 or d4 is an acetic acid solution containing I 2 .

[0045] Preferably, the sugar structure of the first technical solution is

[0046]

[0047] Preferably, the sugar structure of the second technical solution is

[0048]

[0049] Preferably, the sugar structure in Technical Solution 3 or 4 is

[0050]

[0051] The beneficial effects of the present invention are as follows:

[0052] The present invention provides a method for chemically synthesizing glycosylated insulin. Since the formation of three disulfide bonds is precisely controlled in terms of regioselectivity, no by-products of disulfide bond mismatch are produced, the product purity is higher, and the synthesis yield is higher. Moreover, this method also provides a sugar deprotection strategy suitable for the case containing methyl sialic acid, enabling the preparation of such glycosylated insulin. Description of the Drawings

[0053] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments.

[0054] Figure 1 . Schematic diagram of Technical Solution 1.

[0055] Figure 2 . Schematic diagram of Technical Solution 2.

[0056] Figure 3 . Schematic diagram of Technical Solution 3.

[0057] Figure 4 . Schematic diagram of Technical Solution 4.

[0058] Figure 5 . UPLC and HRMS diagrams of product InsA1. Theoretical values: [M+2H] 2+ m / z = 1126.52, [M+3H] 3+ m / z = 818.01; Observed values: 1226.53, 818.01.

[0059] Figure 6 . UPLC and HRMS diagrams of product InsA2. Theoretical values: [M+2H] 2+ m / z = 1303.51, [M+3H] 3+ m / z = 869.34; Observed values: 1303.51, 869.34.

[0060] Figure 7 . UPLC and HRMS diagrams of product Ins1B. Theoretical values: [M+3H] 3+ m / z = 1328.28, [M+4H] 4+ m / z = 996.46, [M+5H] 5+m / z = 797.37; Observed values: 1328.29, 996.47, 797.37.

[0061] Figure 8 . UPLC and HRMS spectra of product Ins2B. Theoretical value: [M+3H] 3+ m / z = 1424.31, [M+4H] 4+ m / z = 1068.48, [M+5H] 5+ m / z = 854.99; Observed values: 1424.31, 1068.49, 854.99.

[0062] Figure 9 . UPLC and HRMS spectra of product Ins3B. Theoretical value: [M+3H] 3+ m / z = 1520.01, [M+4H] 4+ m / z = 1140.26, [M+5H] 5+ m / z = 912.41; Observed values: 1520.02, 1140.26, 912.41.

[0063] Figure 10 . UPLC and HRMS spectra of product Ins4B. Theoretical value: [M+3H] 3+ m / z = 1420.67, [M+4H] 4+ m / z = 1065.75, [M+5H] 5+ m / z = 852.80; Observed values: 1420.66, 1065.75, 852.80.

[0064] Figure 11 . UPLC and HRMS spectra of product Ins5B. Theoretical value: [M+3H] 3+ m / z = 1516.69, [M+4H] 4+ m / z = 1137.77, [M+5H] 5+ m / z = 910.42; Observed values: 1516.70, 1137.78, 910.43.

[0065] Figure 12 . UPLC and HRMS spectra of product Ins6B. Theoretical value: [M+3H] 3+ m / z = 1516.69, [M+4H] 4+ m / z = 1137.77, [M+5H] 5+ m / z = 910.42; Observed values: 1516.69, 1137.78, 910.42. Theoretical value for [M+4H] with two sugars missing in the mass spectrum 4+m / z = 947.46; Observed value: 947.46.

[0066] Figure 13 . UPLC and HRMS spectra of product Ins7B. Theoretical value: [M + 3H] 3+ m / z = 1660.41, [M + 4H] 4+ m / z = 1245.56, [M + 5H] 5+ m / z = 996.65; Observed values: 1660.39, 1245.56, 996.65.

[0067] Figure 14 . UPLC and HRMS spectra of product Ins6B+. Theoretical value: [M + 3H] 3+ m / z = 1585.40, [M + 4H] 4+ m / z = 1189.30, [M + 5H] 5+ m / z = 951.65; Observed values: 1585.40, 1189.30, 951.65.

[0068] Figure 15 . UPLC and HRMS spectra of product Ins7B+. Theoretical value: [M + 3H] 3+ m / z = 1729.12, [M + 4H] 4+ m / z = 1297.09, [M + 5H] 5+ m / z = 1037.87; Observed values: 1729.12, 1297.11, 1037.89.

[0069] Figure 16 . UPLC and HRMS spectra of product Ins4B′. Theoretical value: [M + 3H] 3+ m / z = 1331.97, [M + 4H] 4+ m / z = 999.23, [M + 5H] 5+ m / z = 799.59; Observed values: 1331.97, 999.24, 799.59. Theoretical value of losing one sugar in the mass spectrum: [M + 4H] 4+ m / z = 926.46; Observed value: 926.46.

[0070] Figure 17 . UPLC and HRMS spectra of product Ins5B′. Theoretical value: [M + 3H] 3+ m / z = 1385.99, [M + 4H] 4+ m / z = 1039.74, [M + 5H] 5+m / z = 832.00; Observed values: 1385.99, 1039.75, 831.99. Theoretical value for loss of 1 sugar (sialic acid unit) in the mass spectrum: [M+4H] 4+ m / z = 966.97; Observed value: 966.97. Theoretical value for loss of 1 sugar (galactose unit) in the mass spectrum: [M+5H] 4+ m / z = 799.59; Observed value: 799.59.

[0071] Figure 18 . UPLC and HRMS spectra of product Ins6B'. Theoretical value: [M+3H] 3+ m / z = 1385.99, [M+4H] 4+ m / z = 1039.74, [M+5H] 5+ m / z = 832.00; Observed values: 1385.99, 1039.75, 832.01. Theoretical value for loss of 1 sugar in the mass spectrum: [M+4H] 4+ m / z = 966.97; Observed value: 966.97.

[0072] Figure 19 . UPLC and HRMS spectra of product Ins7B'. Theoretical value: [M+3H] 3+ m / z = 1483.02, [M+4H] 4+ m / z = 1112.52, [M+5H] 5+ m / z = 890.22; Observed values: 1483.03, 1112.52, 890.23. Theoretical value for loss of 1 sugar in the mass spectrum: [M+4H] 4+ m / z = 1039.74; Observed value: 1039.75.

[0073] Figure 20 . UPLC and HRMS spectra of product Ins1AB. Theoretical value: [M+4H] 4+ m / z = 1534.19, [M+5H] 5+ m / z = 1227.56, [M+6H] 6+ m / z = 1023.13; Observed values: 1534.18, 1227.54, 1023.13.

[0074] Figure 21 . UPLC and HRMS spectra of product Ins2AB. Theoretical value: [M+4H] 4+ m / z = 1606.22, [M+5H] 5+ m / z = 1285.17, [M+6H] 6+m / z = 1071.15; Observed values: 1606.20, 1285.16, 1071.13.

[0075] Figure 22 . UPLC and HRMS spectra of product Ins3AB. Theoretical value: [M+4H] 4+ m / z = 1677.99, [M+5H] 5+ m / z = 1342.59, [M+6H] 6+ m / z = 1119.00; Observed values: 1678.00, 1342.60, 1119.01.

[0076] Figure 23 . UPLC and HRMS spectra of product Ins1. Theoretical value: [M+4H] 4+ m / z = 1502.69, [M+5H] 5+ m / z = 1202.35, [M+6H] 6+ m / z = 1002.13; Observed values: 1502.69, 1202.36, 1002.14.

[0077] Figure 24 . UPLC and HRMS spectra of product Ins2. Theoretical value: [M+4H] 4+ m / z = 1543.20, [M+5H] 5+ m / z = 1234.76, [M+6H] 6+ m / z = 1029.14; Observed values: 1543.21, 1234.77, 1029.14. Loss of sugar in mass spectrometry to become Ins1. Theoretical value: [M+6H] 6+ m / z = 1002.13; Observed value: 1002.14.

[0078] Figure 25 . UPLC and HRMS spectra of product Ins3. Theoretical value: [M+4H] 4+ m / z = 1593.97, [M+5H] 5+ m / z = 1275.38, [M+6H] 6+ m / z = 1062.98; Observed values: 1593.98, 1275.38, 1062.99.

[0079] Figure 26 . UPLC and HRMS spectra of product Ins4. Theoretical value: [M+4H] 4+ m / z = 1575.46, [M+5H] 5+ m / z = 1260.57, [M+6H] 6+m / z = 1050.64; Observed values: 1575.47, 1260.58, 1050.65.

[0080] Figure 27 . UPLC and HRMS spectra of product Ins5. Theoretical value: [M+4H] 4+ m / z = 1615.97, [M+5H] 5+ m / z = 1292.98, [M+6H] 5+ m / z = 1077.65; Observed values: 1615.98, 1292.99, 1077.66.

[0081] Figure 28 . UPLC and HRMS spectra of product Ins6. Theoretical value: [M+4H] 4+ m / z = 1615.97, [M+5H] 5+ m / z = 1292.98, [M+6H] 6+ m / z = 1077.65; Observed values: 1615.98, 1292.99, 1077.66. Theoretical value for loss of sugar in mass spectrometry to become Ins2: [M+5H] 5+ m / z = 1234.76, [M+6H] 6+ m / z = 1029.14; Observed values: 1234.77, 1029.14.

[0082] Figure 29 . UPLC and HRMS spectra of product Ins7. Theoretical value: [M+4H] 4+ m / z = 1688.75, [M+5H] 5+ m / z = 1351.20, [M+6H] 6+ m / z = 1126.17; Observed values: 1688.74, 1351.20, 1126.17. Theoretical value for loss of sugar in mass spectrometry to become Ins6: [M+5H] 5+ m / z = 1292.98, [M+6H] 6+ m / z = 1077.65; Observed values: 1292.98, 1077.65. Detailed implementation manners

[0083] The following is a further detailed description of the present invention in combination with specific embodiments. It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention, and is not used to limit the present invention described in detail in the claims. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0084] Example 1. Insulin solid-phase synthesis, purification, and identification process:

[0085] Swelling: Add 0.05 mmol of resin into the solid-phase synthesis reactor, wash the resin three times with DMF (3 mL × 3), and add 10 mL of DMF to swell for 5 minutes.

[0086] Deprotection: Add 3 mL of 20% 4-methylpiperidine dissolved in DMF. Use microwave heating to raise the temperature of the mixture to 50 °C and maintain it at 50 °C. Pass nitrogen gas from the bottom of the container (usually open for 2 seconds and closed for 3 seconds) for mixing. After reacting for 10 min, wash the resin four times with DMF (4 mL × 4).

[0087] Coupling: Sequentially add Fmoc-protected amino acid (0.2 M in DMF, 1.25 mL, 5 eq.), DIEA (0.5 M in DMF, 1 mL, 10 eq.), and HATU (0.25 M in DMF, 1 mL, 5 eq.) into the reaction vessel. After reacting at room temperature for 2 minutes, raise the temperature of the reaction mixture to 50 °C and maintain it at 50 °C with nitrogen gas mixing for 8 minutes. Among them, for amino acids containing β-branches in the sequence (Ile, Thr, and Val), Pro, and the residues after Fmoc-Arg(Pbf)-OH with large protecting groups, perform two couplings to ensure complete reaction.

[0088] Peptide chain elongation: Repeat the above deprotection and coupling steps until the sequence is completed. The Fmoc group on the last amino acid is removed through the deprotection step.

[0089] Cleavage: Transfer the obtained resin to a glass polypeptide synthesis reactor, wash the resin three times alternately with DMF and DCM (5 mL each time). Add freshly prepared cleavage solution (4 mL), and gently stir at room temperature for 2 hours. Filter and collect the cleavage solution with a 50 mL centrifuge tube.

[0090] Collection: Add 40 mL of ice-cold diethyl ether to precipitate the crude peptide. Centrifuge (5800 g, 4 °C, 5 min) to collect the precipitate, and then carefully pour out the liquid from the centrifuge tube. Dissolve the residue in 15 mL of MeCN / H 2 O (1 / 1, v / v).

[0091] UPLC-HRMS analysis: Use a Waters Acquity ultra-high performance liquid chromatography (UPLC) system for chromatographic separation. The separation column used is Acquity UPLC BEH 300 C4, 1.7 μm column. The flow rate is 0.3 mL / min, and the detection wavelength is 214 nm. Phase A uses H 2For phase O and B, MeCN containing 0.1% formic acid was used and eluted with a linear gradient (20%-40%) for a total of 6 minutes. After each run, the column was washed with 95% (B%) for 2 min and re-equilibrated with 5% (B%) for 2 min. Mass spectrometry analysis was performed using an Exactive Plus Orbitrap mass spectrometer. An electrospray ionization (ESI) source was used in the positive ion mode, with a scanning range of m / z 300 - 4500 and a spray voltage of 3 kV. Depending on the concentration, the sample loading volume varied from 1 μL to 10 μL.

[0092] High-performance liquid chromatography (HPLC) purification: Using a C18, 10×250 mm, 5 μm, separation column. The column was eluted with 95% (B%) for 15 min and equilibrated with 5% (B%) for 15 min. The filtered solution was injected into the column and eluted with a linear gradient. The eluate was monitored at wavelengths of 214 nm and 275 nm. The desired product was collected according to the UPLC-MS analysis results and obtained as a pure product after lyophilization.

[0093] Example 2. Synthesis of InsA1:

[0094] According to Technical Solution 1 ( Figure 1 ), Rink Amide AM resin was used as the carrier, and the resin would be coupled with the β-carboxyl group of Fmoc-Asp-OtBu. After resin cleavage, Asp was converted to the natural Asn residue at A21. Except that CysA6, CysA7, ThrA8-SerA9, CysA11, and AsnA21 used Fmoc-Cys(STmp)-OH, Fmoc-Cys(Acm)-OH, Boc-Ser[Fmoc-Thr(tBu)]-OH, Fmoc-Cys(Mmt)-OH, and Fmoc-Asp-OtBu respectively, the remaining steps were the same as those described in Example 1.

[0095] After SPPS was completed, 5 mL of DMF containing 5% β-mercaptoethanol and 0.1 M N-methylmorpholine was added to the resin, and stirred at room temperature for 5 min, repeated 3 times. After washing the resin with DCM, 4 mL of DCM and 10 equivalents of 2,2′-dithiobis(5-nitropyridine) were added and stirred for 1 hour. After washing the resin, 4 mL of a DCM solution containing 1% TFA and 5% TIS was added and stirred for 2 min, repeated 5 times. The resin was washed again, DCM was added, and stirred for 1 hour. The DCM was removed, and freshly prepared TFA / TIPS / H 2 O (92.5 / 5 / 2.5, v / v / v, 5 mL) cleavage solution was added and stirred at room temperature for 2 hours. The solution was filtered and collected into a 50 mL centrifuge tube, and 40 mL of cold Et2 At O(+4 °C), a white precipitate was formed. After centrifugation (5800 g, 4 °C, 5 min), the liquid was carefully decanted. The crude polypeptide was dissolved in 10 mL of MeCN / H 2 O (1 / 1, v / v). 50 μL of the solution was taken and diluted with 0.5 mL of MeCN / H 2 O / TFA (1 / 1 / 0.05%, v / v / v) and filtered through a 0.22 μm membrane to obtain the LCMS sample. The remaining solution was lyophilized for 2 days to obtain the crude polypeptide. The crude polypeptide was dissolved in MeCN / H 2 O (2 / 8, v / v) to a concentration of 4 mg / mL and filtered through a 0.22 μm membrane. In each operation, 8 mg (2 mL) of the sample was loaded onto a C18 column (10 × 250 mm, 5 μm, ) and washed with a 20% to 40% gradient for 40 min. The target fraction was collected and lyophilized to obtain the desired InsA1 product. InsA1 contains O-acyl isopeptide units and an Acm protecting group at the A7 position. The yield was 21% based on the starting resin loading. Its LC-MS details are shown in Figure 5 .

[0096] Example 3. Synthesis of InsA2:

[0097] According to Technical Scheme 2 or 3 or 4 ( Figure 2 , Figure 3 , Figure 4 ), the synthesis and effect of InsA2 were basically the same as those of InsA1, except that 10 equivalents of 2,2′-dithiobis(5-nitropyridine) were added in the cleavage step. Its LC-MS details are shown in Figure 6 .

[0098] Example 4. Solid-phase synthesis of the B chain:

[0099] For the synthesis of the B chain, a cheap resin, Fmoc-Thr(tBu)-2-ClTrt resin completely based on polystyrene material, was used, and glycosyl groups were introduced into the B chain using Fmoc-protected glycoamino acid (FPGA). For the condensation reaction of Fmoc-protected glycoamino acid, a DMF solution containing 0.75 mL of 0.1 M FPGA (1.5 eq.), 0.3 mL of 0.5 M DIC (3 eq.), and 0.3 mL of 0.25 M Oxyma (1.5 eq.) was added to the reaction vessel in sequence. The reaction was carried out at room temperature for 2 minutes, then the temperature was raised to 50 °C and the reaction was continued at 50 °C for 18 minutes while mixing was carried out by bubbling nitrogen. This condensation reaction was carried out 2 times in total.

[0100] Synthesis of Ins1B, Ins2B and Ins3B: According to Technical Solution 1 ( Figure 1 ), after SPPS, the resin was placed in a peptide synthesis reaction tube, and a cleavage solution of TFA / TIPS / H 2 O (92.5 / 5 / 2.5, v / v / v, 5 mL) containing 10 equivalents of 2,2′-dithiobis(5-nitropyridine) was added, and the mixture was stirred at room temperature for 2 h. The solution was filtered and collected into a 50 mL centrifuge tube, and 40 mL of cold Et 2 O (+4 °C) was added to form a white precipitate. After centrifugation (5800 g, 4 °C, 5 min), the liquid was carefully decanted. The crude peptide was dissolved in 10 mL of MeCN / H 2 O (1 / 1, v / v). 50 μL of the solution was taken, diluted with 0.5 mL of MeCN / H 2 O / TFA (1 / 1 / 0.05%, v / v / v), and filtered through a 0.22 μm membrane to obtain an LCMS sample. The remaining solution was lyophilized for 2 days to obtain the crude peptide. The crude peptide was dissolved in MeCN / H 2 O (2 / 8, v / v) to a concentration of 4 mg / mL and filtered through a 0.22 μm membrane. In each operation, 8 mg (2 mL) of the sample was loaded onto a C18 column (10 × 250 mm, 5 μm, ), and eluted with a 30% to 50% gradient for 40 min. The target fractions were collected and lyophilized to obtain Ins1B, Ins2B and Ins3B, with yields of 28%, 20% and 26% respectively. These intermediates will be used for subsequent synthesis, and their structures are as follows:

[0101]

[0102] Their LC-MS details are shown in Figures 7 - 9 .

[0103] Synthesis of Ins4B, Ins5B, Ins6B and Ins7B: According to Technical Solution 2 or 3 ( Figure 2 , Figure 3 ), after SPPS, the resin was placed in a peptide synthesis reaction tube, and a cleavage solution of TFA / TIPS / H 2 O (92.5 / 5 / 2.5, v / v / v, 5 mL) was added, and the mixture was stirred at room temperature for 2 h. The solution was filtered and collected into a 50 mL centrifuge tube, and 40 mL of cold Et 2 O (+4 °C) was added to form a white precipitate. After centrifugation (5800 g, 4 °C, 5 min), the liquid was carefully decanted. The crude peptide was dissolved in 10 mL of MeCN / H 2 O (1 / 1, v / v). 50 μL of the solution was taken, diluted with 0.5 mL of MeCN / H 2Dilute with O / TFA (1 / 1 / 0.05%, v / v / v) and filter through a 0.22 μm membrane to obtain the LCMS sample. The remaining solution was lyophilized for 2 days to obtain the crude polypeptide. Dissolve the crude polypeptide in MeCN / H 2 O (2 / 8, v / v) to a concentration of 4 mg / mL and filter through a 0.22 μm membrane. In each operation, load 8 mg (2 mL) of the sample onto a C18 column (10×250 mm, 5 μm, ) and elute with a 30% to 50% gradient for 40 min. Collect the target fraction and lyophilize to obtain Ins4B, Ins5B, Ins6B, and Ins7B, with yields of 25%, 24%, 25%, and 19% respectively. These intermediates will be used for subsequent synthesis, and their structures are as follows:

[0104]

[0105] The LC-MS details of them are shown in Figures 10 - 13 .

[0106] Synthesis of Ins6B+ and Ins7B+: According to Technical Scheme Four ( Figure 4 ), in SPPS, Fmoc-Lys(ivDde)-OH was used at position 29. After SPPS, place the resin in a polypeptide synthesis reaction tube, add TFA / TIPS / H 2 O (92.5 / 5 / 2.5, v / v / v, 5 mL) cleavage solution and stir at room temperature for 2 h. Filter and collect the solution into a 50 mL centrifuge tube, add 40 mL of cold Et 2 O (+4 °C) to form a white precipitate. After centrifugation (5800 g, 4 °C, 5 min), carefully pour off the liquid. Dissolve the crude polypeptide in 10 mL of MeCN / H 2 O (1 / 1, v / v), take 50 μL of the solution, dilute it with 0.5 mL of MeCN / H 2 O / TFA (1 / 1 / 0.05%, v / v / v) and filter through a 0.22 μm membrane to obtain the LCMS sample. The remaining solution was lyophilized for 2 days to obtain the crude polypeptide. Dissolve the crude polypeptide in MeCN / H 2 O (2 / 8, v / v) to a concentration of 4 mg / mL and filter through a 0.22 μm membrane. In each operation, load 8 mg (2 mL) of the sample onto a C18 column (10×250 mm, 5 μm, ) and elute with a 30% to 50% gradient for 40 min. Collect the target fraction and lyophilize to obtain Ins6B+ and Ins7B+, with yields of 24% and 18% respectively. These intermediates will be used for subsequent synthesis, and their structures are as follows:

[0107]

[0108] The LC-MS of them is shown in detail in Figures 14 - 15 .

[0109] Example 5. Synthesis of fully naked B chain

[0110] Synthesis of Ins4B′ and Ins5B′: According to Technical Solution 2 ( Figure 2 ), 5 mg of Ins4B or Ins5B was dissolved in 5 mL of 30 mM sodium hydroxide solution at 4 °C, and stirred at 4 °C for 5 h. The pH of the reaction mixture was adjusted to 5 with 10% acetic acid solution, filtered through a 0.45 μm membrane, loaded onto a C18 column (10×250 mm, 5 μm, ), and eluted with a 25% to 40% gradient for 40 min. The target components were collected and freeze-dried to obtain Ins4B′ or Ins5B′, with yields of 37% and 33% respectively. These intermediates will be used for subsequent synthesis, and their structures are as follows:

[0111]

[0112]

[0113] The LC-MS of them is shown in detail in Figures 16 - 17 .

[0114] Synthesis of Ins6B′ and Ins7B′: According to Technical Solution 3 or 4 ( Figure 3 , Figure 4 ), 6 mg of Ins6B, Ins7B, Ins6B+ or Ins7B+ was dissolved in 3 mL of 30 mM sodium hydroxide solution at 4 °C, and stirred at 4 °C for 1 h. The pH of the reaction mixture was adjusted to 5 with 10% acetic acid solution, filtered through a 0.45 μm membrane, loaded onto a C18 column (10×250 mm, 5 μm, ), and eluted with a 30% to 40% gradient for 40 min. After HPLC, the fractions containing the intermediate products were collected and freeze-dried. 6 mL of 15% NH 2 NH 2 solution and 1.5 mL of TCEP·HCl (4.0 mg / mL) were added to the above freeze-dried powder. After 30 seconds of vortex mixing, a clear solution was obtained. The reaction mixture was kept at room temperature for 2 h. The pH of the reaction mixture was adjusted to 5 with 10% acetic acid solution, diluted with 2 mL of water, filtered through a 0.45 μm membrane, loaded onto a C18 column (10×250 mm, 5 μm, ) It was eluted at a gradient of 25% to 40% for 40 min. The target components were freeze-dried to obtain Ins6B′ or Ins7B′, with yields of 43% and 62% (from Ins6B and Ins6B+ respectively) and 32% and 53% (from Ins7B and Ins7B+ respectively). These intermediates will be used for subsequent synthesis, and their structures are as follows:

[0115]

[0116] The details of their LC-MS are shown in Figures 18 - 19 .

[0117] Example 6. Synthesis of insulin:

[0118] Synthesis of Ins1AB, Ins2AB and Ins3AB: According to Technical Solution 1 ( Figure 1 ), InsA1 (4 mg, 1.63 μmol, 1 eq.) and Ins1B, Ins2B or Ins3B (1.75 μmol, 1.07 eq.) were dissolved in 1 mL of a buffer solution containing 6 M urea and 0.2 M NH 4 HCO 3 (pH 8.03), and the reaction was carried out at room temperature for 5 minutes. 1.9 mL of an acetic acid solution containing I 2 (10.3 mg, 40.8 μmol, 25 eq.) was added, and the reaction was carried out at room temperature for 10 min. An appropriate amount of 1 M ascorbic acid was added until the color of iodine disappeared, and it was filtered through a 0.45 μm membrane and loaded onto a C18 column (10 × 250 mm, 5 μm, ), and eluted at a gradient of 20% to 50% for 40 min. The target components were freeze-dried to obtain Ins1AB, Ins2AB or Ins3AB, with yields of 39%, 36% and 35% respectively. The details of their structures and LC-MS are shown in Figures 20 - 22 .

[0119] Synthesis of Ins1, Ins2 and Ins3: 3 mg of Ins1AB was dissolved in 3 mL of 5% NH 2 NH 2 solution and stirred for 0.5 h. The pH of the reaction mixture was adjusted to 5 with 10% acetic acid and filtered through a 0.45 μm membrane, and loaded onto a C18 column (10 × 250 mm, 5 μm, ), and eluted at a gradient of 20% to 50% for 40 min. The target component was freeze-dried to obtain Ins1, with a yield of 80%. For Ins2AB and Ins3AB, Ins2 and Ins3 could be obtained by the same method, with yields of 88% and 82% respectively. The details of their structures and LC-MS are shown in Figures 23 - 25 .

[0120] Synthesis of Ins4, Ins5, Ins6 and Ins7: By performing intermolecular ligation of Ins4B′, Ins5B′, Ins6B′ or Ins7B′ with InsA2, the synthesis of Ins4, Ins5, Ins6 and Ins7 can be completed according to the method for synthesizing Ins1AB, Ins2AB and Ins3AB previously, with yields of 47%, 35%, 31% and 37% respectively. Their structures and LC-MS are shown in detail in Figures 26 - 29 .

Claims

1. A method for chemically synthesizing glycosylated insulin, wherein the glycosylated insulin is formed by the connection of a sugar to the hydroxyl group of threonine at position 27 of the B chain of human insulin. Characterized in that, The synthesis includes the following steps: a1) Prepare the insulin A chain, which contains the intra-chain disulfide bond A6-A11, and the side chain of CysA7 is protected with acetamidomethyl. a2) Prepare the insulin B chain, wherein the side chain of ThrB27 is connected to a sugar, the side chain of CysB7 is protected with acetamidomethyl, and the side chain of CysB19 is connected to 3-nitro-2-pyridylthiol. a3) Place the A chain and the B chain in a buffer solution to form the A20-B19 disulfide bond, and then remove the acetamidomethyl groups on CysA7 and CysB7 and oxidize to form the A7-B7 disulfide bond. a4) Use NH 2 NH 2 to remove the protecting groups on the sugar to obtain glycosylated insulin.

2. A method for chemically synthesizing glycosylated insulin, wherein the glycosylated insulin is formed by the connection of a sugar to the hydroxyl group of threonine at position 27 of the B chain of human insulin. Characterized in that, The synthesis includes the following steps: b1) Prepare the insulin A chain, which contains the intra-chain disulfide bond A6-A11, the side chain of CysA7 is protected with acetamidomethyl, and the side chain of CysA20 is connected to 3-nitro-2-pyridylthiol. b2) Prepare the insulin B chain, wherein the side chain of ThrB27 is connected to a sugar, and the side chain of CysB7 is protected with acetamidomethyl. b3) Use a NaOH solution to remove the protecting group on the sugar to obtain the glycosylated B chain. b4) Place the A chain and the B chain in a buffer solution to form the A20-B19 disulfide bond, and then remove the acetamidomethyl groups on CysA7 and CysB7 and oxidize to form the A7-B7 disulfide bond, thereby obtaining glycosylated insulin.

3. A method for chemically synthesizing glycosylated insulin, wherein the glycosylated insulin is formed by the connection of a sugar to the hydroxyl group of threonine at position 27 of the B chain of human insulin. Characterized in that, The synthesis includes the following steps: c1) Prepare the insulin A chain, which contains the intra-chain disulfide bond A6-A11, the side chain of CysA7 is protected with acetamidomethyl, and the side chain of CysA20 is connected to 3-nitro-2-pyridylthiol. c2) Prepare the insulin B chain, wherein the side chain of ThrB27 is connected to a sugar, and the side chain of CysB7 is protected with acetamidomethyl. c3) Using NaOH solution and NH 2 NH 2 solution to remove the protecting group on the sugar to obtain the glycosylated B chain; c4) Place the A chain and the B chain in a buffer solution to form the A20-B19 disulfide bond, and then remove the acetamidomethyl groups on CysA7 and CysB7 and oxidize to form the A7-B7 disulfide bond, thereby obtaining glycosylated insulin.

4. A method for chemically synthesizing glycosylated insulin, wherein the glycosylated insulin is formed by the connection of a sugar to the hydroxyl group of threonine at position 27 of the B chain of human insulin. Characterized in that, The synthesis includes the following steps: d1) Prepare the insulin A chain, which contains the intra-chain disulfide bond A6-A11, the side chain of CysA7 is protected with acetamidomethyl, and the side chain of CysA20 is connected to 3-nitro-2-pyridylthiol. d2) Prepare the insulin B chain, where the side chain of ThrB27 of the B chain is linked to a sugar, the side chain of CysB7 is protected with acetamidomethyl, and the side chain of LysB29 is protected with 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl; d3) Use NaOH solution and NH 2 NH 2 solution to remove the protecting group on the sugar and obtain the glycosylated B chain; d4) Place the A chain and the B chain in a buffer solution to form the A20-B19 disulfide bond, and then remove the acetamidomethyl groups on CysA7 and CysB7 and oxidize them to form the A7-B7 disulfide bond, thereby obtaining glycosylated insulin.

5. A method for chemically synthesizing glycosylated insulin according to claim 1, characterized in that, the step a1 includes: Using Rink Amide AM resin as a solid-phase carrier, prepare the insulin A chain by solid-phase peptide synthesis technology, where CysA6, CysA7, ThrA8-SerA9, CysA11 and AsnA21 are respectively Fmoc-Cys(STmp)-OH, Fmoc-Cys(Acm)-OH, Boc-Ser[Fmoc-Thr(tBu)]-OH, Fmoc-Cys(Mmt)-OH and Fmoc-Asp-OtBu; Subsequently, use 5% β-mercaptoethanol and 0.1 M N-methylmorpholine to remove the STmp group, add 2,2'-dithiobis(5-nitropyridine) to activate CysA6, and selectively remove the Mmt group with 1% TFA to form the A6-A11 intra-chain disulfide bond. Add TFA to remove the protecting groups of the polypeptide side chains and cleave the polypeptide from the resin to obtain the A chain InsA1, and the structure of the InsA1 is: Wherein the Acm structure is acetamidomethyl.

6. A method for chemically synthesizing glycosylated insulin according to claims 2 to 4, characterized in that, the step b1, c1 or d1 includes: Using Rink Amide AM resin as a solid-phase carrier, prepare the insulin A chain by solid-phase peptide synthesis technology, where CysA6, CysA7, ThrA8-SerA9, CysA11 and AsnA21 are respectively Fmoc-Cys(STmp)-OH, Fmoc-Cys(Acm)-OH, Boc-Ser[Fmoc-Thr(tBu)]-OH, Fmoc-Cys(Mmt)-OH and Fmoc-Asp-OtBu; Subsequently, use 5% β-mercaptoethanol and 0.1 M N-methylmorpholine to remove the STmp group, add 2,2'-dithiobis(5-nitropyridine) to activate CysA6, and selectively remove the Mmt group with 1% TFA to form the A6-A11 intra-chain disulfide bond; Add TFA containing 2,2'-dithiobis(5-nitropyridine) to treat the resin, which can activate CysA20 while removing the protecting groups of the polypeptide side chains and cleaving the polypeptide from the resin to obtain the A chain InsA2, and the structure of the InsA2 is: Among them, the Acm structure is acetylaminomethyl, and the SNPy structure is 3-nitro-2-pyridyl mercapto.

7. A method for chemically synthesizing glycosylated insulin according to claim 1, characterized in that, the step a2 includes: The insulin B chain was prepared by solid-phase peptide synthesis technology, in which sugar amino acid and Fmoc-Cys(Acm)-OH were used for ThrB27 and CysB7 respectively; adding TFA containing 2,2′-dithiobis(5-nitropyridine) to treat the resin can activate CysB19 while removing the protecting groups of the polypeptide side chain and cleaving the polypeptide from the resin, thereby obtaining the B chain: Among them, the Acm structure is acetylaminomethyl, the SNPy structure is 3-nitro-2-pyridylthiol, and R 1 structure is a sugar with a protecting group.

8. A method for chemically synthesizing glycosylated insulin according to claims 2 to 4, characterized in that the step b2, c2 or d2 includes: The insulin B chain was prepared by solid-phase peptide synthesis technology, in which sugar amino acid and Fmoc-Cys(Acm)-OH were used for ThrB27 and CysB7 respectively; adding TFA to remove the protecting groups of the polypeptide side chain and cleave the polypeptide from the resin, thereby obtaining the B chain: Among them, the Acm structure is acetylaminomethyl, and R 2 The structure is a sugar with a protecting group.

9. A method for chemically synthesizing glycosylated insulin according to claims 1 to 4, characterized in that the step a3, b4, c4 or d4 includes: Dissolve chain A and chain B in a buffer to form the A20-B19 disulfide bond; then add I 2 Remove the Acm protecting groups on CysA7 and CysB7 and oxidize to form the A7-B7 disulfide bond.

10. A method for chemically synthesizing glycosylated insulin according to claim 1, characterized in that the sugar structure is 11. A method for chemically synthesizing glycosylated insulin according to claim 2, characterized in that the sugar structure is 12. A method for chemically synthesizing glycosylated insulin according to claims 3 to 4, characterized in that the sugar structure is

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