A method for preparing disulfide bond-containing polypeptides by solid-liquid combination

Abstract

The present invention relates to the field of polypeptide drug preparation, and discloses a method for preparing a disulfide bond-containing polypeptide by solid-liquid combination, which mainly includes the following steps: (1) using an amino resin as a starting resin to synthesize a linear peptide resin; (2) cracking the linear peptide resin obtained in step (1) to obtain a linear peptide Mpa (R1) XX1 XX2 XX3 Asn Cys (R2) Pro XX4 Gly NH2; (3) dissolving the linear peptide obtained in step (2) with an acetic acid aqueous solution, adding an iodine ethanol solution dropwise under stirring for oxidation, and obtaining a crude polypeptide product. The linear peptide obtained by this technical solution can be iodine-oxidized at a higher concentration, and the generation of dimer impurities is effectively controlled. While ensuring yield and purity, the present invention improves the linear peptide reaction concentration, reduces the oxidation reaction volume, greatly reduces the amount of waste liquid discharged, shortens the oxidation and purification cycle, and reduces the synthesis cost. It is a method for preparing a disulfide bond-containing polypeptide suitable for industrial production.

Description

Technical Field

[0001] The present invention belongs to the field of polypeptide drug preparation, and particularly relates to a method for preparing a disulfide bond-containing polypeptide by solid-liquid combination. Background Art

[0002] Disulfide bonds are a common post-translational modification in peptides and proteins, playing an important role in maintaining peptide conformation and certain biological activities. Many existing drugs, such as octreotide, nesiritide, desmopressin, and atosiban, require disulfide bonds for their activity. Therefore, disulfide bond oxidation methods have long been a research focus in the peptide industry.

[0003] There are two main methods for disulfide bond oxidation of peptides: solid-phase oxidation and liquid-phase oxidation, as follows: (1) Solid-phase oxidation: direct oxidation on the resin during solid-phase synthesis, and the oxidation product is obtained after cleavage, which is then purified; (2) Liquid-phase oxidation: solid-phase synthesis of linear peptide resin, resin cleavage to obtain linear crude peptide, the linear crude peptide is dissolved and liquid-phase oxidation is performed, and then purified, or the linear crude peptide is purified to improve the purity and then disulfide bond oxidation is performed to improve the purity of the oxidation product or reaction efficiency.

[0004] CN110759972A uses a dipeptide fragment of Mpa(R)-D-Tyr(Et) for solid-phase coupling to produce Mpa(R)-D-Tyr(Et)-Ile-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-Rink Amide MBHA Resin. This is then cleaved to yield atosiban linear peptide, which is then dissolved in acetic acid and oxidized with iodine dropwise to yield crude atosiban. The oxidized concentration of the atosiban linear peptide is 3 mg / mL.

[0005] Liquid-phase iodine oxidation of linear peptides typically uses low reaction concentrations, resulting in large oxidation volumes. This generates significant waste during scale-up, and large batch sizes require multiple oxidations, leading to long oxidation cycles. Increasing the reaction concentration can reduce the oxidation volume, but this increases impurities, particularly dimers, and significantly reduces yield. Summary of the Invention

[0006] In order to solve the above problems, the present invention provides a method for preparing a disulfide bond-containing polypeptide by solid-liquid combination, which mainly comprises the following steps:

[0007] (1) Using amino resin as the starting resin, linear peptide resin was synthesized;

[0008] (2) cleaving the linear peptide resin obtained in step (1) to obtain the linear peptide Mpa(R1)-XX1-XX2-XX3-Asn-Cys(R2)-Pro-XX4-Gly-NH2;

[0009] (3) dissolving the linear peptide obtained in step (2) in an aqueous acetic acid solution, and adding an ethanolic iodine solution dropwise under stirring for oxidation to obtain a crude polypeptide;

[0010] In step (2), XX1 is D-Tyr(Et), XX2 is Ile, XX3 is Thr, and XX4 is Orn; or XX1 is L-Tyr, XX2 is Phe, XX3 is Gln, and XX4 is D-Arg;

[0011] In the step (2), R1 and R2 are selected from tBu, H, and Acm, and one of them must be tBu, and R1 and R2 are different.

[0012] Preferably, the linear peptide resin in step (1) is Mpa(R3)-D-Tyr(Et)-Ile-Thr(R4)-Asn(R5)-Cys(R6)-Pro-Orn(R7)-Gly-Resin;

[0013] wherein R3 and R6 are selected from tBu or A, and R3 and R6 are different, one of which must be tBu, and A is selected from Trt, Acm, Tmob, and Mmt;

[0014] R4 is selected from H, tBu, Trt;

[0015] R5 is selected from H, Trt, Mtt, Xan;

[0016] R7 is selected from Boc, Mtt.

[0017] Preferably, the linear peptide resin in step (1) is Mpa(R3)-Tyr(R4)-Phe-Gln(R5)-Asn(R5)-Cys(R6)- Pro-D-Arg(R8)-Gly-Resin;

[0018] wherein R3 and R6 are selected from tBu or A, and R3 and R6 are different, one of which must be tBu, and A is selected from Trt, Acm, Tmob, and Mmt;

[0019] R4 is selected from H, tBu, Trt;

[0020] R5 is selected from H, Trt, Mtt, Xan;

[0021] R8 is selected from H, Pbf, Boc, Pmc, Mtr.

[0022] Preferably, the resin is selected from Rink Amide AM Resin, Rink Amide MBHA Resin, and Sieber Resin.

[0023] Preferably, the concentration of the linear peptide in step (3) is 1 to 30 mmol / L, more preferably 10 to 20 mmol / L.

[0024] Preferably, the proportion of acetic acid in the acetic acid aqueous solution in step (3) is 10%-60%, more preferably 30%-50%.

[0025] Preferably, the oxidation temperature during the oxidation process in step (3) is 20-40°C, more preferably 25-35°C.

[0026] Preferably, the concentration of the iodine ethanol solution in step (3) is 0.1-0.5 mol / L.

[0027] Preferably, the iodine equivalent in step (3) is 2-4 equivalents. Here, "equivalent" refers to the ratio of the amount of iodine to the amount of the linear peptide substance.

[0028] Preferably, the crude polypeptide obtained in step (3) is purified and lyophilized.

[0029] In the prior art synthesis of atosiban and desmopressin, the following methods can be used to directly oxidize and form a disulfide bond between MPa and Cys: (1) direct oxidation of Cys and MPa; (2) direct oxidation after only one -SH of Cys or MPa adopts a Trt or Acm protecting group; (3) direct oxidation after both -SH of Cys or MPa adopt an Acm or Trt protecting group. Those skilled in the art generally believe that if the -SH of Cys or MPa adopts tBu as a protecting group, it cannot be directly oxidized to form a disulfide bond, and the tBu protecting group must be removed before the disulfide bond can be formed. The applicant unexpectedly discovered that when one -SH of MPa or Cys adopts a tBu protecting group and the other -SH does not adopt a protecting group or adopts an Acm or Trt protecting group, it can be directly oxidized to form a disulfide bond without removing the tBu protecting group. Furthermore, it was unexpectedly discovered that the linear peptide obtained by this technical solution can be iodine-oxidized at a higher concentration and effectively controls the formation of dimer impurities. The present invention improves the reaction concentration of linear peptides, reduces the oxidation reaction volume, greatly reduces waste liquid discharge, shortens the oxidation and purification cycle, and reduces synthesis costs while ensuring yield and purity. The present invention is a method for preparing disulfide bond-containing polypeptides suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 HPLC chart of the crude atosiban prepared in Example 9;

[0031] Figure 2 HPLC chart of the crude atosiban prepared in Comparative Example 3. DETAILED DESCRIPTION

[0032] The present invention will be further described in detail below with reference to specific embodiments to facilitate further understanding of the present invention by those skilled in the art. The embodiments should not be construed as limiting the scope of protection.

[0033] The Chinese names corresponding to the English abbreviations involved in the present invention are shown in Table 1:

[0034] Table 1 Chinese names corresponding to the English abbreviations involved in the present invention

[0035]

[0036]

[0037] The technical solution of the present invention is further described in detail according to the following embodiments.

[0038] Example 1: Synthesis of Mpa(Trt)-D-Tyr(Et)-Ile-Thr-Asn(Trt)-Cys(tBu)-Pro-Orn(Boc)-Gly-Rink Amide AM Resin

[0039] Rink Amide AM resin (6.0 g, 5 mmol) with a degree of substitution of 0.84 mmol / g was weighed and added to a peptide solid-phase reactor. The resin was swollen with DMF for 30 min and washed three times. The Fmoc protecting group was removed by adding a 20% piperidine / DMF solution twice, first for 5 min and then for 15 min. The resin was washed with DMF six times. The ninhydrin test was positive.

[0040] Fmoc-Gly-OH (3.0 g, 10 mmol) and HOBt (1.6 g, 12 mmol) were weighed and dissolved in 45 mL of DMF. DIC (1.9 g, 15 mmol) was added for activation for 3 minutes. The activated amino acid solution was then added to the solid-phase reactor and stirred at room temperature for 2 hours. Ninhydrin was used to test the reaction, revealing a colorless resin and complete condensation. The resin was then washed four times with DMF. The Fmoc protecting group was removed twice by adding a 20% piperidine / DMF solution, first for 5 minutes and then for 15 minutes. The resin was then washed six times with DMF, and the ninhydrin test was positive.

[0041] According to the above coupling method, Fmoc-Orn(Boc)-OH, Fmoc-Pro-OH, Fmoc-Cys(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr-OH, Fmoc-Ile-OH, Fmoc-D-Tyr(Et)-OH and Mpa(Trt) were coupled in sequence.

[0042] After the coupling was completed, the resin was washed 4 times with DMF and then 3 times with DCM. The peptide resin was taken out and dried to obtain 14.5 g of peptide resin.

[0043] Example 2: Synthesis of Mpa(Acm)-D-Tyr(Et)-Ile-Thr(tBu)-Asn(Trt)-Cys(tBu)-Pro-Orn(Boc)-Gly-Rink Amide AM Resin

[0044] Rink Amide AM resin (6.0 g, 5 mmol) with a degree of substitution of 0.84 mmol / g was weighed and added to a peptide solid-phase reactor. The resin was swollen with DMF for 30 min and washed three times. The Fmoc protecting group was removed by adding a 20% piperidine / DMF solution twice, first for 5 min and then for 15 min. The resin was washed with DMF six times. The ninhydrin test was positive.

[0045] Fmoc-Gly-OH (3.0 g, 10 mmol) and HOBt (1.6 g, 12 mmol) were weighed and dissolved in 45 mL of DMF. DIC (1.9 g, 15 mmol) was added for activation for 3 minutes. The activated amino acid solution was then added to the solid-phase reactor and stirred at room temperature for 2 hours. Ninhydrin was used to test the reaction, revealing a colorless resin and complete condensation. The resin was then washed four times with DMF. The Fmoc protecting group was removed by adding a 20% piperidine / DMF solution twice, first for 5 minutes and then for 15 minutes. The resin was then washed six times with DMF, and the ninhydrin test was positive.

[0046] According to the above coupling method, Fmoc-Orn(Boc)-OH, Fmoc-Pro-OH, Fmoc-Cys(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-D-Tyr(Et)-OH and Mpa(Acm) were coupled in sequence.

[0047] After the coupling was completed, the resin was washed 4 times with DMF and then 3 times with DCM. The peptide resin was taken out and dried to obtain 13.8 g of peptide resin.

[0048] Example 3: Synthesis of Mpa(tBu)-D-Tyr(Et)-Ile-Thr(tBu)-Asn(Mtt)-Cys(Trt)-Pro-Orn(Boc)-Gly-Rink Amide MBHA Resin

[0049] Rink Amide MBHA resin (6.0 g, 5 mmol) with a degree of substitution of 0.84 mmol / g was weighed and added to a peptide solid-phase reactor. The resin was swollen with DMF for 30 min and washed three times. The Fmoc protecting group was removed by adding a 20% piperidine / DMF solution twice, first for 5 min and then for 15 min. The resin was washed with DMF six times. The ninhydrin test was positive.

[0050] Fmoc-Gly-OH (3.0 g, 10 mmol) and HOBt (1.6 g, 12 mmol) were weighed and dissolved in 45 mL of DMF. DIC (1.9 g, 15 mmol) was added for activation for 3 minutes. The activated amino acid solution was then added to the solid-phase reactor and stirred at room temperature for 2 hours. Ninhydrin was used to test the reaction, revealing a colorless resin and complete condensation. The resin was then washed four times with DMF. The Fmoc protecting group was removed by adding a 20% piperidine / DMF solution twice, first for 5 minutes and then for 15 minutes. The resin was then washed six times with DMF, and the ninhydrin test was positive.

[0051] According to the above coupling method, Fmoc-Orn(Boc)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Mtt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-D-Tyr(Et)-OH and Mpa(tBu) were coupled in sequence.

[0052] After the coupling was completed, the resin was washed 4 times with DMF and then 3 times with DCM. The peptide resin was taken out and dried to obtain 14.1 g of peptide resin.

[0053] Example 4: Synthesis of Mpa(tBu)-D-Tyr(Et)-Ile-Thr(Trt)-Asn(Trt)-Cys(Acm)-Pro-Orn(Mtt)-Gly-Sieber Resin

[0054] Sieber resin (6.0 g, 5 mmol) with a degree of substitution of 0.84 mmol / g was weighed and added to a peptide solid-phase reactor. The resin was swollen with DMF for 30 min and washed three times. The Fmoc protecting group was removed by adding a 20% piperidine / DMF solution twice, first for 5 min and then for 15 min. The resin was washed with DMF six times. The ninhydrin test was positive.

[0055] Fmoc-Gly-OH (3.0 g, 10 mmol) and HOBt (1.6 g, 12 mmol) were weighed and dissolved in 45 mL of DMF. DIC (1.9 g, 15 mmol) was added for activation for 3 minutes. The activated amino acid solution was then added to the solid-phase reactor and stirred at room temperature for 2 hours. Ninhydrin was used to test the reaction, revealing a colorless resin and complete condensation. The resin was then washed four times with DMF. The Fmoc protecting group was removed by adding a 20% piperidine / DMF solution twice, first for 5 minutes and then for 15 minutes. The resin was then washed six times with DMF, and the ninhydrin test was positive.

[0056] According to the above coupling method, Fmoc-Orn(Mtt)-OH, Fmoc-Pro-OH, Fmoc-Cys(Acm)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-D-Tyr(Et)-OH and Mpa(tBu) were coupled in sequence.

[0057] After the coupling was completed, the resin was washed 4 times with DMF and then 3 times with DCM. The peptide resin was taken out and dried to obtain 14.5 g of peptide resin.

[0058] Example 5: Synthesis of Mpa-D-Tyr(Et)-Ile-Thr-Asn-Cys(tBu)-Pro-Orn-Gly-NH2

[0059] Prepare 150 mL of chilled lysate (TFA / TIS / H₂O / MPa = 87.5% / 5.0% / 5.0% / 2.5%) and add it to a 250 mL round-bottom flask. Add the atosiban linear peptide resin prepared in Example 1 to the lysate and allow to react at room temperature for 2 hours. Filter, wash the resin twice with a small amount of TFA, and add the combined filtrates to 1.5 L of cold isopropyl ether for precipitation. Wash by centrifugation to yield 6.2 g of a white solid.

[0060] Example 6: Synthesis of Mpa(Acm)-D-Tyr(Et)-Ile-Thr-Asn-Cys(tBu)-Pro-Orn-Gly-NH2

[0061] Prepare 150 mL of chilled lysate (TFA / TIS / H₂O / MPa = 87.5% / 5.0% / 5.0% / 2.5%) and add it to a 250 mL round-bottom flask. Add the atosiban linear peptide resin prepared in Example 2 to the lysate and allow to react at room temperature for 2 hours. Filter, wash the resin twice with a small amount of TFA, and add the combined filtrates to 1.5 L of cold isopropyl ether for precipitation. Wash by centrifugation to yield 6.3 g of a white solid.

[0062] Example 7: Synthesis of Mpa(tBu)-D-Tyr(Et)-Ile-Thr-Asn-Cys-Pro-Orn-Gly-NH2

[0063] Prepare 150 mL of chilled lysate (TFA / TIS / H₂O / MPa = 87.5% / 5.0% / 5.0% / 2.5%) and add it to a 250 mL round-bottom flask. Add the atosiban linear peptide resin prepared in Example 3 to the lysate and allow to react at room temperature for 2 hours. Filter, wash the resin twice with a small amount of TFA, and add the combined filtrates to 1.5 L of cold isopropyl ether for precipitation. Wash by centrifugation to yield 6.1 g of a white solid.

[0064] Example 8: Synthesis of Mpa(tBu)-D-Tyr(Et)-Ile-Thr-Asn-Cys(Acm)-Pro-Orn-Gly-NH2

[0065] Prepare 150 mL of chilled lysate (TFA / TIS / H₂O / MPa = 87.5% / 5.0% / 5.0% / 2.5%) and add it to a 250 mL round-bottom flask. Add the atosiban linear peptide resin prepared in Example 4 to the lysate and allow to react at room temperature for 2 hours. Filter, wash the resin twice with a small amount of TFA, and add the combined filtrates to 1.5 L of cold isopropyl ether for precipitation. Wash by centrifugation to yield 6.2 g of a white solid.

[0066] Example 9: Synthesis of crude atosiban

[0067] The linear peptide of atosiban (6.2 g, 5 mmol) prepared in Example 5 was weighed and dissolved in 250 mL of acetic acid. 250 mL of water was added to a reaction concentration of 10 mmol / L. 4.0 equivalents of 0.5 mol / L iodine ethanol solution (40 ml) was added under stirring. The mixture was reacted at 35° C. for 3 h. A small amount of ascorbic acid was added until the solution faded to obtain a crude atosiban product with a synthesis yield of 78% and an HPLC purity of 84.65%. The HPLC spectrum is shown in FIG. Figure 1 The HPLC spectrum data are shown in Table 2.

[0068] Table 2 HPLC spectrum characteristic peak retention time and peak area detection results of atosiban prepared in Example 9

[0069]

[0070]

[0071] Example 10: Synthesis of crude atosiban

[0072] The linear peptide of atosiban (6.3 g, 5 mmol) prepared in Example 6 was weighed and dissolved in 500 mL of acetic acid. 4.5 L of water was added to a reaction concentration of 1 mmol / L. 2.0 equivalents of a 0.1 mol / L iodine-ethanol solution (100 mL) was added with stirring. The mixture was reacted at 20°C for 2 h. A small amount of ascorbic acid was added until the solution faded to obtain crude atosiban with a synthesis yield of 75% and an HPLC purity of 82%. HPLC spectrum and Figure 1 similar.

[0073] Example 11: Synthesis of crude atosiban

[0074] The linear peptide of atosiban (6.1 g, 5 mmol) prepared in Example 7 was weighed and dissolved in 75 mL of acetic acid. 175 mL of water was added to a reaction concentration of 20 mmol / L. 3.0 equivalents of 0.5 mol / L iodine-ethanol solution (30 mL) were added with stirring. The mixture was reacted at 25°C for 4 h. A small amount of ascorbic acid was added until the solution faded to obtain crude atosiban with a synthesis yield of 67% and an HPLC purity of 80.65%. HPLC spectrum and Figure 1 similar.

[0075] Example 12: Synthesis of crude atosiban

[0076] The linear peptide of atosiban (6.2 g, 5 mmol) prepared in Example 8 was weighed and dissolved in 100 ml of acetic acid. 65 mL of water was added to a reaction concentration of 30 mmol / L. 4.0 equivalents of 0.3 mol / L iodine-ethanol solution (67 mL) was added with stirring. The mixture was reacted at 40°C for 5 h. A small amount of ascorbic acid was added until the solution faded to obtain crude atosiban with a synthesis yield of 65% and an HPLC purity of 75.12%. HPLC spectrum and Figure 1 similar.

[0077] Example 13: Synthesis of Mpa-Tyr-Phe-Gln(Trt)-Asn(Trt)-Cys(tBu)-Pro-D-Arg(Pbf)-Gly-Rink Amide AM Resin

[0078] Rink Amide AM resin (6.0 g, 5 mmol) with a degree of substitution of 0.84 mmol / g was weighed and added to a peptide solid-phase reactor. The resin was swollen with DMF for 30 min and washed three times. The Fmoc protecting group was removed by adding a 20% piperidine / DMF solution twice, first for 5 min and then for 15 min. The resin was washed with DMF six times. The ninhydrin test was positive.

[0079] Fmoc-Gly-OH (3.0 g, 10 mmol) and HOBt (1.6 g, 12 mmol) were weighed and dissolved in 45 mL of DMF. DIC (1.9 g, 15 mmol) was added for activation for 3 minutes. The activated amino acid solution was then added to the solid-phase reactor and stirred at room temperature for 2 hours. Ninhydrin was used to test the reaction, revealing a colorless resin and complete condensation. The resin was then washed four times with DMF. The Fmoc protecting group was removed by adding a 20% piperidine / DMF solution twice, first for 5 minutes and then for 15 minutes. The resin was then washed six times with DMF, and the ninhydrin test was positive.

[0080] According to the above coupling method, Fmoc-D-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Cys(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH, Fmoc-Tyr-OH and Mpa were coupled in sequence.

[0081] After the coupling was completed, the resin was washed 4 times with DMF and then 3 times with DCM. The peptide resin was taken out and dried to obtain 14.4 g of peptide resin.

[0082] Example 14: Synthesis of Mpa(Acm)-Tyr(tBu)-Phe-Gln(Trt)-Asn(Trt)-Cys(tBu)-Pro-D-Arg(Pbf)-Gly-Rink Amide AM Resin

[0083] Rink Amide AM resin (6.0 g, 5 mmol) with a degree of substitution of 0.84 mmol / g was weighed and added to a peptide solid-phase reactor. The resin was swollen with DMF for 30 min and washed three times. The Fmoc protecting group was removed by adding a 20% piperidine / DMF solution twice, first for 5 min and then for 15 min. The resin was washed with DMF six times. The ninhydrin test was positive.

[0084] Fmoc-Gly-OH (3.0 g, 10 mmol) and HOBt (1.6 g, 12 mmol) were weighed and dissolved in 45 mL of DMF. DIC (1.9 g, 15 mmol) was added for activation for 3 minutes. The activated amino acid solution was then added to the solid-phase reactor and stirred at room temperature for 2 hours. Ninhydrin was used to test the reaction, revealing a colorless resin and complete condensation. The resin was then washed four times with DMF. The Fmoc protecting group was removed by adding a 20% piperidine / DMF solution twice, first for 5 minutes and then for 15 minutes. The resin was then washed six times with DMF, and the ninhydrin test was positive.

[0085] According to the above coupling method, Fmoc-D-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Cys(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH, Fmoc-Tyr(tBu)-OH and Mpa(Acm) were coupled in sequence.

[0086] After the coupling was completed, the resin was washed 4 times with DMF and then 3 times with DCM. The peptide resin was taken out and dried to obtain 14.6 g of peptide resin.

[0087] Example 15: Synthesis of Mpa(tBu)-Tyr(tBu)-Phe-Gln(Mtt)-Asn(Trt)-Cys(Trt)-Pro-D-Arg(Pmc)-Gly-Rink Amide MBHA Resin

[0088] Rink Amide MBHA resin (6.0 g, 5 mmol) with a degree of substitution of 0.84 mmol / g was weighed and added to a peptide solid-phase reactor. The resin was swollen with DMF for 30 min and washed three times. The Fmoc protecting group was removed by adding a 20% piperidine / DMF solution twice, first for 5 min and then for 15 min. The resin was washed with DMF six times. The ninhydrin test was positive.

[0089] Fmoc-Gly-OH (3.0 g, 10 mmol) and HOBt (1.6 g, 12 mmol) were weighed and dissolved in 45 mL of DMF. DIC (1.9 g, 15 mmol) was added for activation for 3 minutes. The activated amino acid solution was then added to the solid-phase reactor and stirred at room temperature for 2 hours. Ninhydrin was used to test the reaction, revealing a colorless resin and complete condensation. The resin was then washed four times with DMF. The Fmoc protecting group was removed by adding a 20% piperidine / DMF solution twice, first for 5 minutes and then for 15 minutes. The resin was then washed six times with DMF, and the ninhydrin test was positive.

[0090] According to the above coupling method, Fmoc-D-Arg(Pmc)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Mtt)-OH, Fmoc-Phe-OH, Fmoc-Tyr(tBu)-OH and Mpa(tBu) were coupled in sequence.

[0091] After the coupling was completed, the resin was washed 4 times with DMF and then 3 times with DCM. The peptide resin was taken out and dried to obtain 14.5 g of peptide resin.

[0092] Example 16: Synthesis of Mpa(tBu)-Tyr(tBu)-Phe-Gln(Trt)-Asn(Xan)-Cys(Acm)-Pro-D-Arg(Pbf)-Gly-Sieber Resin

[0093] Sieber resin (6.0 g, 5 mmol) with a degree of substitution of 0.84 mmol / g was weighed and added to a peptide solid-phase reactor. The resin was swollen with DMF for 30 min and washed three times. The Fmoc protecting group was removed by adding a 20% piperidine / DMF solution twice, first for 5 min and then for 15 min. The resin was washed with DMF six times. The ninhydrin test was positive.

[0094] Fmoc-Gly-OH (3.0 g, 10 mmol) and HOBt (1.6 g, 12 mmol) were weighed and dissolved in 45 mL of DMF. DIC (1.9 g, 15 mmol) was added for activation for 3 minutes. The activated amino acid solution was then added to the solid-phase reactor and stirred at room temperature for 2 hours. Ninhydrin was used to test the reaction, revealing a colorless resin and complete condensation. The resin was then washed four times with DMF. The Fmoc protecting group was removed by adding a 20% piperidine / DMF solution twice, first for 5 minutes and then for 15 minutes. The resin was then washed six times with DMF, and the ninhydrin test was positive.

[0095] According to the above coupling method, Fmoc-D-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Cys(Acm)-OH, Fmoc-Asn(Xan)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH, Fmoc-Tyr(tBu)-OH and Mpa(tBu) were coupled in sequence.

[0096] After the coupling was completed, the resin was washed 4 times with DMF and then 3 times with DCM. The peptide resin was taken out and dried to obtain 14.4 g of peptide resin.

[0097] Example 17: Synthesis of Mpa-Tyr-Phe-Gln-Asn-Cys(tBu)-Pro-D-Arg-Gly-NH2

[0098] Prepare 150 mL of chilled lysate (TFA / TIS / H₂O / MPa = 87.5% / 5.0% / 5.0% / 2.5%) and add it to a 250 mL round-bottom flask. Add the desmopressin linear peptide resin prepared in Example 13 to the lysate and allow to react at room temperature for 2 h. Filter, wash the resin twice with a small amount of TFA, and add the combined filtrates to 1.5 L of cold isopropyl ether for precipitation. Wash by centrifugation to yield 6.2 g of a white solid.

[0099] Example 18: Synthesis of MPa(Acm)-Tyr-Phe-Gln-Asn-Cys(tBu)-Pro-D-Arg-Gly-NH2

[0100] Prepare 150 mL of chilled lysate (TFA / TIS / H₂O / MPa = 87.5% / 5.0% / 5.0% / 2.5%) and add it to a 250 mL round-bottom flask. Add the desmopressin linear peptide resin prepared in Example 14 to the lysate and allow to react at room temperature for 2 h. Filter, wash the resin twice with a small amount of TFA, and add the combined filtrates to 1.5 L of cold isopropyl ether for precipitation. Wash by centrifugation to yield 6.1 g of a white solid.

[0101] Example 19: Synthesis of Mpa(tBu)-Tyr-Phe-Gln-Asn-Cys-Pro-D-Arg-Gly-NH2

[0102] Prepare 150 mL of chilled lysate (TFA / TIS / H₂O / MPa = 87.5% / 5.0% / 5.0% / 2.5%) and add it to a 250 mL round-bottom flask. Add the desmopressin linear peptide resin prepared in Example 15 to the lysate and allow to react at room temperature for 2 hours. Filter, wash the resin twice with a small amount of TFA, and add the combined filtrates to 1.5 L of cold isopropyl ether for precipitation. Wash by centrifugation to yield 6.3 g of a white solid.

[0103] Example 20: Synthesis of Mpa(tBu)-Tyr-Phe-Gln-Asn-Cys(Acm)-Pro-D-Arg-Gly-NH2

[0104] Prepare 150 mL of chilled lysate (TFA / TIS / H₂O / MPa = 87.5% / 5.0% / 5.0% / 2.5%) and add it to a 250 mL round-bottom flask. Add the desmopressin linear peptide resin prepared in Example 16 to the lysate and allow to react at room temperature for 2 h. Filter, wash the resin twice with a small amount of TFA, and add the combined filtrates to 1.5 L of cold isopropyl ether for precipitation. Wash by centrifugation to yield 6.2 g of a white solid.

[0105] Example 21: Synthesis of crude desmopressin

[0106] The desmopressin linear peptide (6.2 g, 5 mmol) obtained in Example 17 was weighed and dissolved in 250 mL of acetic acid. 250 mL of water was added to a reaction concentration of 10 mmol / L. 4.0 equivalents of a 0.5 mol / L iodine-ethanol solution (40 mL) was added with stirring. The mixture was reacted at 35° C. for 3 h. A small amount of ascorbic acid was added until the solution faded to obtain crude desmopressin with a synthesis yield of 76% and an HPLC purity of 85.36%.

[0107] Example 22: Synthesis of crude desmopressin

[0108] The desmopressin linear peptide (6.1 g, 5 mmol) obtained in Example 18 was weighed and dissolved in 500 mL of acetic acid. 4.5 L of water was added to a reaction concentration of 1 mmol / L. 2.0 equivalents of a 0.1 mol / L iodine-ethanol solution (100 mL) was added with stirring. The mixture was reacted at 20° C. for 2 h. A small amount of ascorbic acid was added until the solution faded to obtain crude desmopressin with a synthesis yield of 74% and an HPLC purity of 81.23%.

[0109] Example 23: Synthesis of crude desmopressin

[0110] The desmopressin linear peptide (6.3 g, 5 mmol) obtained in Example 19 was weighed and dissolved in 75 mL of acetic acid. 175 mL of water was added to a reaction concentration of 20 mmol / L. 3.0 equivalents of a 0.5 mol / L iodine-ethanol solution (30 mL) was added with stirring. The mixture was reacted at 25° C. for 4 h. A small amount of ascorbic acid was added until the solution faded to obtain crude desmopressin with a synthesis yield of 66% and an HPLC purity of 79.35%.

[0111] Example 24: Synthesis of crude desmopressin

[0112] The desmopressin linear peptide (6.2 g, 5 mmol) prepared in Example 20 was weighed and dissolved in 100 mL of acetic acid. 65 mL of water was added to a reaction concentration of 30 mmol / L. 4.0 equivalents of a 0.3 mol / L iodine-ethanol solution (67 mL) was added with stirring. The mixture was reacted at 40°C for 5 h. A small amount of ascorbic acid was added until the solution faded to obtain crude desmopressin with a synthesis yield of 62% and an HPLC purity of 74.52%. Comparative Example 1: Synthesis of MPa(Trt)-D-Tyr(Et)-Ile-Thr-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-Rink Amide AM Resin

[0113] Rink Amide AM resin (6.0 g, 5 mmol) with a degree of substitution of 0.84 mmol / g was weighed and added to a peptide solid-phase reactor. The resin was swollen with DMF for 30 min and washed three times. The Fmoc protecting group was removed by adding a 20% piperidine / DMF solution twice, first for 5 min and then for 15 min. The resin was washed with DMF six times. The ninhydrin test was positive.

[0114] Fmoc-Gly-OH (3.0 g, 10 mmol) and HOBt (1.6 g, 12 mmol) were weighed and dissolved in 45 mL of DMF. DIC (1.9 g, 15 mmol) was added for activation for 3 minutes. The activated amino acid solution was then added to the solid-phase reactor and stirred at room temperature for 2 hours. Ninhydrin was used to test the reaction, revealing a colorless resin and complete condensation. The resin was then washed four times with DMF. The Fmoc protecting group was removed by adding a 20% piperidine / DMF solution twice, first for 5 minutes and then for 15 minutes. The resin was then washed six times with DMF, and the ninhydrin test was positive.

[0115] According to the above coupling method, Fmoc-Orn(Boc)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr-OH, Fmoc-Ile-OH, Fmoc-D-Tyr(Et)-OH and Mpa(Trt) were coupled in sequence.

[0116] After the coupling was completed, the resin was washed 4 times with DMF and then 3 times with DCM. The peptide resin was taken out and dried to obtain 14.3 g of peptide resin.

[0117] Comparative Example 2: Synthesis of Mpa-D-Tyr(Et)-Ile-Thr-Asn-Cys-Pro-Orn-Gly-NH2

[0118] Prepare 150 mL of chilled lysate (TFA / TIS / H₂O / MPa = 87.5% / 5.0% / 5.0% / 2.5%) and add it to a 250 mL round-bottom flask. Add the atosiban linear peptide resin prepared in Comparative Example 1 to the lysate and allow to react at room temperature for 2 hours. Filter, wash the resin twice with a small amount of TFA, and add the combined filtrates to 1.5 L of cold isopropyl ether for precipitation. Wash by centrifugation to yield 6.2 g of a white solid.

[0119] Comparative Example 3: Synthesis of crude atosiban

[0120] The linear peptide of atosiban (6.2 g, 5 mmol) prepared in Comparative Example 2 was weighed and dissolved in 250 mL of acetic acid. 250 mL of water was added to a reaction concentration of 10 mmol / L. 4.0 equivalents of 0.5 mol / L iodine ethanol solution (40 mL) was added under stirring. The solution turned yellow and reacted at 35° C. for 1 h. A small amount of ascorbic acid was added until the solution faded to obtain crude atosiban with a synthesis yield of 54% and an HPLC purity of 66.11%. The HPLC spectrum is as follows: Figure 2 The HPLC spectrum data are shown in Table 3.

[0121] Table 3 HPLC spectrum characteristic peak retention time and peak area test results of atosiban prepared in Comparative Example 3

[0122]

[0123]

[0124]

[0125] Comparative Example 4: Synthesis of Mpa-Tyr-Phe-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-D-Arg(Pbf)-Gly-Rink Amide AM Resin

[0126] Rink Amide AM resin (6.0 g, 5 mmol) with a degree of substitution of 0.84 mmol / g was weighed and added to a peptide solid-phase reactor. The resin was swollen with DMF for 30 min and washed three times. The Fmoc protecting group was removed by adding a 20% piperidine / DMF solution twice, first for 5 min and then for 15 min. The resin was washed with DMF six times. The ninhydrin test was positive.

[0127] Fmoc-Gly-OH (3.0 g, 10 mmol) and HOBt (1.6 g, 12 mmol) were dissolved in 45 mL of DMF. DIC (1.9 g, 15 mmol) was added for activation for 3 minutes. The activated amino acid solution was then added to the solid-phase reactor and stirred at room temperature for 2 hours. Ninhydrin was used to monitor the reaction, revealing a colorless resin and complete condensation. The resin was then washed four times with DMF. The Fmoc protecting group was removed by adding a 20% piperidine / DMF solution twice, first for 5 minutes and then for 15 minutes. The resin was then washed six times with DMF, and the ninhydrin test was positive.

[0128] According to the above coupling method, Fmoc-D-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH, Fmoc-Tyr-OH and Mpa were coupled in sequence.

[0129] After the coupling was completed, the resin was washed 4 times with DMF and then 3 times with DCM. The peptide resin was taken out and dried to obtain 14.7 g of peptide resin.

[0130] Comparative Example 5: Synthesis of Mpa-Tyr-Phe-Gln-Asn-Cys-Pro-D-Arg-Gly-NH2

[0131] Prepare 150 mL of chilled lysate (TFA / TIS / H₂O / MPa = 87.5% / 5.0% / 5.0% / 2.5%) and add it to a 250 mL round-bottom flask. Add the desmopressin linear peptide resin prepared in Comparative Example 4 to the lysate and allow to react at room temperature for 2 hours. Filter, wash the resin twice with a small amount of TFA, and add the combined filtrates to 1.5 L of cold isopropyl ether for precipitation. Wash by centrifugation to yield 6.0 g of a white solid.

[0132] Comparative Example 6: Synthesis of crude desmopressin

[0133] The desmopressin linear peptide (6.0 g, 5 mmol) prepared in Comparative Example 5 was weighed and dissolved in 250 mL of acetic acid. 250 mL of water was added to a reaction concentration of 10 mmol / L. 4.0 equivalents of a 0.5 mol / L iodine-ethanol solution (40 mL) was added with stirring. The solution turned yellow and was reacted at 35° C. for 1 h. A small amount of ascorbic acid was added until the solution faded to obtain crude desmopressin with a synthesis yield of 52% and an HPLC purity of 63.11%.

[0134] The crude yield of atosiban obtained in Example 9 reached 78%, with a purity of 84.65%, and the dimer impurity (RT=11.533min, with a relative retention time of 1.33 to the main peak) was 0.51%. Compared with the method provided in Comparative Example 3, the yield and purity of the crude peptide of atosiban increased by 44.44% and 28.04%, respectively, and the dimer impurity decreased by 90.74%. The crude yield of desmopressin obtained in Example 21 reached 76%, with a purity of 85.36%. Compared with Comparative Example 6, the yield and purity of desmopressin increased by 46.15% and 35.26%, respectively. It can be seen from this that the method for preparing disulfide bond-containing polypeptides by solid-liquid combination provided by the present invention can be iodine-oxidized at a higher linear peptide concentration, effectively controlling the generation of dimer impurities, and reducing the oxidation reaction volume under conditions of yield and purity, greatly reducing waste liquid discharge, shortening oxidation and purification cycles, reducing synthesis costs, and being suitable for industrial production.

Claims

1. A method for preparing a disulfide bond-containing polypeptide by solid-liquid combination, characterized in that: The method consists of the following steps: (1) Using amino resin as the starting resin, synthesize linear peptide resin; (2) cleaving the linear peptide resin obtained in step (1) to obtain the linear peptide Mpa(R1)-XX1-XX2-XX3-Asn-Cys(R2)-Pro-XX4-Gly-NH2; (3) The linear peptide obtained in step (2) is dissolved in an aqueous acetic acid solution, and an ethanolic iodine solution is added dropwise under stirring to oxidize the peptide to obtain a crude peptide; In step (2), XX1 is D-Tyr (Et), XX2 is Ile, XX3 is Thr, and XX4 is Orn; or XX1 is L-Tyr, XX2 is Phe, XX3 is Gln, and XX4 is D-Arg; In step (2), R1 and R2 are selected from tBu, H, and Acm, and one of them must be tBu, and R1 and R2 are different; In step (3), the concentration of the iodine ethanol solution is 0.1-0.5 mol / L, the iodine equivalent is 2-4 equivalents, and the oxidation temperature is 20-40°C.

2. The method according to claim 1, characterized in that The linear peptide resin in step (1) is Mpa(R3)-D-Tyr(Et)-Ile-Thr(R4)-Asn(R5)-Cys(R6)-Pro-Orn(R7)-Gly-Resin; wherein R3 and R6 are selected from tBu or A, and R3 and R6 are different, one of which must be tBu, and A is selected from Trt, Acm, Tmob, and Mmt; R4 is selected from H, Trt; R5 is selected from H, Trt, Mtt, Xan; R7 is selected from Boc, Mtt.

3. The method according to claim 1, characterized in that The linear peptide resin in step (1) is Mpa(R3)-Tyr(R4)-Phe-Gln(R5)-Asn(R5)-Cys(R6)-Pro-D-Arg(R8)-Gly-Resin; wherein R3 and R6 are selected from tBu or A, and R3 and R6 are different, one of which must be tBu, and A is selected from Trt, Acm, Tmob, and Mmt; R4 is selected from H, Trt; R5 is selected from H, Trt, Mtt, Xan; R8 is selected from H, Pbf, Boc, Pmc, Mtr.

4. The method according to any one of claims 1 to 3, characterized in that: The resin is selected from Rink Amide AM Resin, Rink Amide MBHA Resin, and Sieber Resin.

5. The method according to claim 1, wherein The linear peptide concentration in step (3) is 1 to 30 mmol / L.

6. The method according to claim 5, characterized in that The linear peptide concentration is 10-20 mmol / L.

7. The method according to claim 1, characterized in that The proportion of acetic acid in the acetic acid aqueous solution in step (3) is 10%-60%.

8. The method according to claim 7, characterized in that The acetic acid ratio is 30%-50%.

9. The method according to claim 1, characterized in that The oxidation temperature is 25-35°C.

10. The method according to claim 1, characterized in that The crude polypeptide obtained in step (3) is purified and freeze-dried.

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