A solid-liquid combined synthesis method of vasoactive intestinal peptide

Abstract

The application provides a solid-liquid combined synthesis method of fosoliptide, and relates to the technical field of polypeptide synthesis; the synthesis method splits fosoliptide with 39 amino acids into three short peptide fragments, respectively prepares each fragment by adopting a solid-phase synthesis method, then transfers the fragments to a liquid-phase system, and sequentially carries out t-butyl modification, fragment condensation, Fmoc protecting group removal, cleavage and settlement, and oxidation ring closure, so as to finally obtain a finished product of fosoliptide; the application combines the advantages of solid-phase synthesis and liquid-phase synthesis, optimizes core process parameters, avoids long peptide chain aggregation and side reactions, is simple and convenient to operate, has good stability, and is suitable for industrialized large-scale production of fosoliptide.

Description

Technical Field

[0001] This invention relates to the field of polypeptide synthesis technology, specifically to a solid-liquid phase combined synthesis method for vosolid peptide. Background Technology

[0002] Vosoritide (trade name Voxzogo) is a C-type natriuretic peptide (CNP) analogue, a once-daily chondrostimulant, and the world's first drug approved by the US FDA for the treatment of children with achondroplasia (ACH).

[0003] The vosol peptide sequence is as follows: H-Pro 1 -Gly-Gln-Glu-His-Pro-Asn-Ala-Arg-Lys 10 -Tyr-Lys-Gly-Ala-Asn-Lys-Lys-Gly-Leu-Ser 20 -Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg 30 -Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys-OH, its molecule contains Cys 23 With Cys 39 The disulfide bonds between them.

[0004] Achondroplasia is the most common form of short-limb dwarfism, caused by gain-of-function mutations in the fibroblast growth factor receptor 3 (FGFR3) gene, leading to negative regulation of intrachondral bone growth. Voxolitide can directly target the underlying pathophysiology of the disease by downregulating the FGFR3 signaling pathway, promoting intrachondral bone formation and effectively improving the growth rate of affected children. Since its approval by the FDA and the EU EC in 2021, voxolitide has been used clinically in many countries around the world. In August 2025, its marketing application in China was formally accepted by the CDE and included in the priority review, which will soon fill the gap in targeted therapy for achondroplasia in China and bring new hope for treatment to Chinese children and their families.

[0005] Currently, the mainstream preparation method for vosolid peptide is the solid-phase stepwise synthesis method. Although this process is a conventional route for peptide synthesis, it has a series of insurmountable technical defects for long-chain peptides like vosolid peptide containing 39 amino acids: as the peptide chain gradually lengthens, the steric hindrance increases significantly, leading to a sharp decrease in the coupling efficiency of subsequent amino acids, and easily generating a large number of impurities such as missing peptides and mismatched peptides, affecting the purity of the product; moreover, this method is difficult to purify, has complicated operation steps, and generates too much waste liquid, which not only increases environmental pressure, but is also difficult to adapt to industrial-scale production.

[0006] Currently, vosolidarene, as the only globally approved targeted therapy for achondroplasia, is experiencing a continuously increasing clinical demand. However, its high production cost keeps the drug price high (approximately $240,000 per year in the US market), severely limiting patient accessibility. The core limitations of existing stepwise solid-phase synthesis methods have become a key bottleneck restricting the large-scale production of vosolidarene and reducing the burden of medication costs for patients. Therefore, developing a novel synthetic method for vosolidarene that is impurity-controlled, highly pure, has excellent yield, low cost, is easy to operate, and suitable for industrial production is an urgent need to overcome the shortcomings of existing technologies and improve drug accessibility, possessing significant clinical value and industrial significance. Summary of the Invention

[0007] To address the shortcomings of existing technologies, a solid-phase-liquid-phase combined synthesis method for vosolidarine peptide is provided. This method involves splitting the 39-amino acid vosolidarine peptide into three short peptide fragments, preparing each fragment separately using solid-phase synthesis, and then transferring the fragments to a liquid-phase system for sequential tert-butyl modification, fragment condensation, Fmoc protecting group removal, cleavage and precipitation, and oxidative cyclization to finally obtain the vosolidarine peptide product. This synthesis method avoids the problems of poor purity and low yield in existing vosolidarine peptide preparations. It combines the advantages of solid-phase and liquid-phase synthesis, using solid-phase synthesis to prepare short peptide fragments, avoiding the drawbacks of direct synthesis of long peptide chains, and then using liquid-phase synthesis to achieve efficient fragment condensation, modification, and subsequent reactions. This ultimately achieves efficient, high-purity, and low-cost synthesis of vosolidarine peptide, meeting the requirements for large-scale industrial production.

[0008] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a solid-phase-liquid-phase combined synthesis method for vosolid peptide, the synthesis method comprising the following steps: S1. Using solid-phase synthesis, amino acids were sequentially coupled to prepare three fragments, including fragment 1, fragment 2, and fragment 3. The sequence of fragment 1 includes Fmoc-Pro-Gly-Gln(Trt)-Glu(OtBu)-His(Trt)-Pro-Asn(Trt)-Ala-Arg(Pbf)-Lys(Boc)-Tyr(tBu)-Lys(Boc)-Gly-OH; The sequence of fragment 2 includes Fmoc-Ala-Asn(Trt)-Lys(Boc)-Lys(Boc)-Gly-Leu-Ser(tBu)-Lys(Boc)-Gly-Cys(Trt)-Phe-Gly-OH; The sequence of fragment 3 includes Fmoc-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser-Gly-Leu-Gly-Cys(Trt)-OH; S2. The C-terminal carboxyl group of fragment 3 is modified by tert-butyl esterification, and then the Fmoc protecting group is removed to obtain fragment 4; the sequence of fragment 4 includes H-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser-Gly-Leu-Gly-Cys(Trt)-OtBu; S3. Fragment 4 and fragment 2 are condensed together. After condensation, the Fmoc protecting group is removed to obtain fragment 5. The sequence of fragment 5 includes... H-Ala-Asn(Trt)-Lys(Boc)-Lys(Boc)-Gly-Leu-Ser(tBu)-Lys(Boc)-Gly-Cys(Trt)-Phe-Gly-Leu -Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser-Gly-Leu-Gly-Cys(Trt)-OtBu; S4. Fragment 5 and fragment 1 are condensed together. After condensation, the Fmoc protecting group is removed to obtain fragment 6. The sequence of fragment 6 includes... H-Pro-Gly-Gln(Trt)-Glu(OtBu)-His(Trt)-Pro-Asn(Trt)-Ala-Arg(Pbf)-Lys(Boc)-Tyr(tBu)-Lys(Boc)-Gly-Ala-Asn(Trt)-Lys(Boc)-Lys(Boc)-Gly- Leu-Ser(tBu)-Lys(Boc)-Gly-Cys(Trt)-Phe-Gly-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser-Gly-Leu-Gly-Cys(Trt)-OtBu; S5. Fragment 6 was cleaved and precipitated to obtain crude vosolid peptide; S6. The unclosed crude vosolamide peptide is oxidized to complete the intramolecular disulfide bond cyclization, thus obtaining the finished vosolamide peptide product.

[0009] In some specific embodiments of the present invention, the specific method for sequentially coupling amino acids in step S1 is to use a solid-phase resin as a carrier to sequentially couple amino acids from the carbon terminus to the nitrogen terminus according to the amino acid sequence, and then deprotect, cut, and purify to obtain fragment 1, fragment 2, and fragment 3.

[0010] In some specific embodiments of the present invention, the solid-phase resin is chloro(o-chlorophenyl)diphenylmethane; the degree of substitution of the resin is 0.2~1.55 mmol / g.

[0011] In some specific embodiments of the present invention, the coupling agent is one of HOBT / DIC, HOOBT / DIC, Oxyma / DIC, HOAT / DIC, HATU / DIEA, HBTU / DIEA, PYBOP / DIEA, Pyoxime / DIEA, TPTU / DIEA, TCFH / DIEA, and EDC·HCl / HOBT / DIEA. Furthermore, the coupling agent is HOBT / DIC, PYBOP / DIEA, or TPTU / DIEA; The amount of any one of HOBT / DIC, PYBOP / DIEA, and TPTU / DIEA is 1.5 to 4 times the molar amount of the solid resin; the coupling reaction time is 1 to 5 hours.

[0012] In some specific embodiments of the present invention, the reagent for removing Fmoc during the deprotection process is an organic base, and the organic base is one of piperidine, diethylamine, and DBU; The deprotection reaction time is 5-60 min.

[0013] In some specific embodiments of the present invention, the cutting is performed using 10%~40% HFIP / DCM or 1%~10% TFA / DCM, and the fragments are purified with ether solvents after cutting to obtain fragment 1, fragment 2 or fragment 3; The ether solvent is one of MTBE, diethyl ether, petroleum ether, and isopropyl ether.

[0014] In some specific embodiments of the present invention, in step S2, the tert-butyl esterification modification is performed using a tert-butylating agent, and the molar ratio of fragment 3 to the tert-butylating agent is 1:1~6; The tert-butylating agent is one of tert-butyltrichloroacetylimine ester, ditert-tert-butyl dicarbonate / DMAP, and DCC / DMAP, and the reaction solvent is one of DCM, THF, and DMF. In step S2, the removal of the Fmoc protecting group is performed using one or more of piperidine, diethylamine, and DBU.

[0015] In some specific embodiments of the present invention, in steps S3 and S4, the condensation reagent is selected from one of HOBT / DIC, HOOBT / DIC, Oxyma / DIC, HOAT / DIC, HATU / DIEA, HBTU / DIEA, PYBOP / DIEA, Pyoxime / DIEA, TPTU / DIEA, TCFH / DIEA, and EDC·HCl / HOBT / DIEA. Furthermore, the condensation reagent used in the condensation is selected from one of HOBT / DIC, PYBOP / DIEA, and TPTU / DIEA; In the condensation reaction, the molar ratio of fragment 4 to fragment 2 is 1:1~3, and the molar ratio of fragment 5 to fragment 1 is 1:1~3; the solvent for condensation is one or more of DCM, DMF, DMSO, THF, and acetone, and the condensation time is 0.5~6 h; In steps S3 and S4, the removal of the Fmoc protecting group is performed using one of the following: 20% Pip / DMF, 20% DEA / DMF, or 1%-10% DBU / DMF.

[0016] In some specific embodiments of the present invention, in step S5, the pyrolysis solution is a mixture of TFA, Tis, EDT, PhOH, and H2O, and the volume ratio of TFA, Tis, EDT, PhOH, and H2O is 80~87.5∶5~10∶2.5~5∶2.5~5∶2.5~5; The pyrolysis solution is precooled to 5~15℃, the pyrolysis temperature is 25~30℃, and the pyrolysis time is 2.5~3h.

[0017] In some specific embodiments of the present invention, in step S6, the specific method of oxidation is to use 0.1 mol / L methanol-iodine solution, add it dropwise until the solution turns a stable light brown color, continue stirring for 30 min, and then add 1% ascorbic acid aqueous solution dropwise until the solution becomes clear.

[0018] The beneficial effects achieved by this invention are as follows: This invention employs a solid-liquid phase combined synthesis strategy, combining the advantages of solid phase synthesis (simple operation and easy product separation) with the advantages of liquid phase synthesis (high fragment condensation efficiency and fewer side reactions), effectively solving the defects of single synthesis methods and improving synthesis efficiency and product purity. The synthesis method provided by this invention prepares short peptide fragments in the solid-phase synthesis stage. It adopts a segmented synthesis strategy to split the long peptide chain of 39 amino acids into 3 short peptide fragments, avoiding problems such as aggregation and incomplete coupling caused by direct synthesis of long peptide chains. The linear peptide resin yield is not less than 80%, and the purity and yield of each fragment are stable. The synthesis method provided by this invention optimizes process parameters such as modification, condensation, and pyrolysis in the liquid phase synthesis stage to reduce the occurrence of side reactions. The synthesis method provided by this invention is simple to operate, consumes reasonable amounts of reagents, has low cost, and is easy to separate and purify intermediate products. It is suitable for large-scale industrial production and has good prospects for industrial application. Attached Figure Description

[0019] To more clearly illustrate the technical solution of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 ESI-MS image of crude unclosed vosolamide peptide prepared in Example 1. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0022] The abbreviations and names of chemical reagents are as follows: HOBT / DIC: 1-Hydroxybenzotriazole / N,N'-Diisopropylcarbodiimide; HOOBT / DIC: 3-hydroxy-1,2,3-benzotriazine-4(3H)-one / N,N'-diisopropylcarbodiimide; Oxyma / DIC: Ethyl 2-oxime cyanoacetate / N,N'-diisopropylcarbodiimide; HOAT / DIC: 1-Hydroxy-7-azobenzotriazole / N,N'-Diisopropylcarbodiimide; HATU / DIEA: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate / N,N-diisopropylethylamine; HBTU / DIEA: Benzotriazole-N,N,N',N'-Tetramethylurea hexafluorophosphate / N,N-Diisopropylethylamine; PYBOP / DIEA: Benzotriazol-1-yl-oxytripyrrolidinylphosphine hexafluorophosphate / N,N-diisopropylethylamine; Pyoxime / DIEA: Cyano(hydroxyimino)ethyl acetate-O2]tri-1-pyrrolidinyl hexafluorophosphate / N,N-diisopropylethylamine; TPTU / DIEA: 2-(2-pyridone-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate / N,N-diisopropylethylamine; TCFH / DIEA: N,N,N',N'-Tetramethylchloromethamine hexafluorophosphate / N,N-Diisopropylethylamine; EDC·HCl / HOBT / DIEA: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride / 1-hydroxybenzotriazole / N,N-diisopropylethylamine; Pip / DMF: Piperidine / N,N-Dimethylformamide; DEA / DMF: Diethylamine / N,N-Dimethylformamide DBU / DMF: 1,8-diazabicyclo[5.4.0]undec-7-ene / N,N-dimethylformamide; HFIP: Hexafluoroisopropanol; TFA: Trifluoroacetic acid; Tis: Triisopropylsilane; EDT: 1,2-Ethylenedithiol; PhOH: Phenol; DMAP: 4-Dimethylaminopyridine; DCC: N,N'-Dicyclohexylcarbodiimide.

[0023] Example 1 Preparation of fully protected linear peptide fragment 1 (1) Weigh 100 g of CTC Resin with a degree of substitution of 1.0 mmol / g (i.e., 100 mmol) into a peptide synthesizer, add 800 mL of DCM and stir to swell, weigh 200 mmol of Fmoc-Gly-OH and 600 mmol of DIEA and add them to the above stirring system, stir at room temperature for 3 h, then add 100 mL of MeOH and continue stirring for 30 min to end up unreacted active sites, filter to remove liquid, and wash the resin 6 times with DMF, and dry after each wash.

[0024] (2) Add 800 mL of 20% diethylamine / DMF deprotection solution to the peptide synthesizer and deprotect twice at room temperature for 10 min and 20 min respectively. After deprotection, wash the resin with DMF 8 times and dry it after each wash.

[0025] (3) Weigh 300 mmol of Fmoc-Lys(Boc)-OH and add it to 600 mL of THF. Then add 150 mmol of PYBOP and stir until completely dissolved. Then add 150 mmol of DIEA. Add the activated solution to the peptide synthesizer and react with the resin at room temperature for 1 h. After the reaction is complete, remove the waste liquid and wash the resin 4 times with 1000 mL of DMF. After each wash, dry the resin.

[0026] (4) Repeat steps (2) and (3) above, and perform coupling reactions according to the amino acid sequences Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Pro-OH, Fmoc-His(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, and Fmoc-Pro-OH to finally obtain the linear peptide resin Fmoc-Pro-Gly-Gln(Trt)-Glu(OtBu)-His(Trt)-Pro-Asn(Trt)-Ala-Arg(Pbf)-Lys(Boc)-Tyr(tBu)-Lys(Boc)-Gly-CTC Resin; finally, wash the resin three times alternately with DCM and MeOH, and dry it under vacuum after each wash. Then, dry it in a vacuum drying oven at 35±5℃ for 20~24 h to obtain 342 g of linear peptide resin.

[0027] (5) Add 342 g of the dried linear peptide resin obtained in step (4) to 2.8 L of 20% HFIP / DCM solution, stir and react at room temperature for 1 h, filter to remove the resin, wash the resin three times with 150 mL of DCM, and combine all the filtrates; add the combined filtrate to 26 L of diethyl ether, stir evenly, centrifuge after the solid precipitates, after centrifugation, stir and slurry the solid with 2 L of diethyl ether, centrifuge again, after centrifugation, place the solid in a vacuum drying oven at 35±5℃ and dry under reduced pressure for 12~16 h to obtain fully protected linear peptide fragment 1 (Fmoc-Pro-Gly-Gln(Trt)-Glu(OtBu)-His(Trt)-Pro-Asn(Trt)-Ala-Arg(Pbf)-Lys(Boc)-Tyr(tBu)-Lys(Boc)-Gly-OH), weigh 231 g.

[0028] Preparation of fully protected linear peptide fragment 2 (1) Weigh 100 g of CTC Resin with a degree of substitution of 1.0 mmol / g (i.e., 100 mmol) into a peptide synthesizer, add 800 mL of DCM and stir to swell, weigh 200 mmol of Fmoc-Gly-OH and 600 mmol of DIEA and add them to the above stirring system, stir at room temperature for 3 h, then add 100 mL of MeOH and continue stirring for 30 min to end up unreacted active sites, filter to remove liquid, and wash the resin 6 times with DMF, and dry after each wash.

[0029] (2) Add 800 mL of 20% diethylamine / DMF deprotection solution to the peptide synthesizer and deprotect twice at room temperature for 10 min and 20 min respectively. After deprotection, wash the resin with DMF 8 times and dry it after each wash.

[0030] (3) Weigh 300 mmol of Fmoc-Phe-OH and add it to 600 mL of THF, then add 150 mmol of PYBOP and stir until completely dissolved. Then add 150 mmol of DIEA. Add the activated solution to the peptide synthesizer and react with the resin at room temperature for 1 h. After the reaction is complete, remove the waste liquid and wash the resin 4 times with 1000 mL of DMF each time. After each wash, dry the resin.

[0031] (4) Repeat steps (2) and (3) above, and perform coupling reactions according to the amino acid sequences Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, and Fmoc-Ala-OH to finally obtain the linear peptide resin Fmoc-Ala-Asn(Trt)-Lys(Boc)-Lys(Boc)-Gly-Leu-Ser(tBu)-Lys(Boc)-Gly-Cys(Trt)-Phe-Gly-OH-CTC Resin; finally, wash the resin three times alternately with DCM and MeOH, and dry it under vacuum after each wash. Then, dry it in a vacuum drying oven at 35±5℃ for 20~24 h to obtain 282 g of linear peptide resin.

[0032] (5) Add 282 g of the dried linear peptide resin obtained in step (4) to 2.3 L of 20% HFIP / DCM solution, stir and react at room temperature for 1 h, filter to remove the resin, wash the resin three times with 150 mL of DCM, and combine all the filtrates; add the combined filtrate to 22 L of diethyl ether, stir evenly, centrifuge after the solid precipitates, after centrifugation, stir and slurry the solid with 2 L of diethyl ether, centrifuge again, after centrifugation, place the solid in a vacuum drying oven at 35±5℃ and dry under reduced pressure for 12~16 h to obtain fully protected linear peptide fragment 2 (Fmoc-Ala-Asn(Trt)-Lys(Boc)-Lys(Boc)-Gly-Leu-Ser(tBu)-Lys(Boc)-Gly-Cys(Trt)-Phe-Gly-OH), weigh 173 g.

[0033] Preparation of fully protected linear peptide fragment 3 (1) Weigh 100 g of CTC Resin with a degree of substitution of 1.0 mmol / g (i.e., 100 mmol) into a peptide synthesizer, add 800 mL of DCM and stir to swell, weigh 200 mmol of Fmoc-Cys(Trt)-OH and 600 mmol of DIEA and add them to the above stirring system, stir at room temperature for 3 h, then add 100 mL of MeOH and continue stirring for 30 min to end up unreacted active sites, filter to remove liquid, and wash the resin 6 times with DMF, and dry after each wash.

[0034] (2) Add 800 mL of 20% diethylamine / DMF deprotection solution to the peptide synthesizer and deprotect twice at room temperature for 10 min and 20 min respectively. After deprotection, wash the resin with DMF 8 times and dry it after each wash.

[0035] (3) Weigh 300 mmol of Fmoc-Gly-OH and add it to 600 mL of DMF, then add 150 mmol of PYBOP and stir until completely dissolved. Then add 150 mmol of DIEA. Add the activated solution to the peptide synthesizer and react with the resin at room temperature for 1 h. After the reaction is complete, remove the waste liquid and wash the resin 4 times with 1000 mL of DMF each time. After each wash, dry the resin.

[0036] (4) Repeat steps (2) and (3) above, and perform coupling reactions according to the amino acid sequences Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser-OH, Fmoc-Met-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Leu-OH to finally obtain the linear peptide resin Fmoc-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser-Gly-Leu-Gly-Cys(Trt)-CTC. Resin; finally, wash the resin three times alternately with DCM and MeOH, and dry it under vacuum after each wash. Then, dry it in a vacuum drying oven at 35±5℃ for 20~24 h to obtain 303 g of linear peptide resin.

[0037] (5) Add 303 g of the dried linear peptide resin obtained in step (4) to 2.5 L of 20% HFIP / DCM solution, stir and react at room temperature for 1 h, filter to remove the resin, wash the resin three times with 150 mL of DCM, and combine all the filtrates; add the combined filtrate to 23.6 L of diethyl ether, stir evenly, centrifuge after the solid precipitates, after centrifugation, stir and slurry the solid with 2 L of diethyl ether, centrifuge again, after centrifugation, place the solid in a vacuum drying oven at 35±5℃ and dry under reduced pressure for 12~16 h to obtain the fully protected linear peptide fragment 3 (Fmoc-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser-Gly-Leu-Gly-Cys(Trt)-OH), weigh 171 g.

[0038] Preparation of linear peptide fragment 4 (tert-butyl esterification of fragment 3C end and Fmoc deprotection) (1) Add 23.7 g of fully protected linear peptide fragment 3 (10 mmol) to 230 mL THF, stir until completely dissolved, add 20 mmol of Boc2O and 2 mmol of DMAP, stir at room temperature for 5 h, and start post-processing when the remaining fragment 3 is ≤20% as detected by the intermediate control. Add 230 mL of 10% saline to the reaction system, stir evenly, separate the liquid and take the lower organic phase.

[0039] (2) After drying the lower organic phase with anhydrous sodium sulfate, add 60 mL of ethylenediamine and stir at room temperature for 1 h until the Fmoc protecting group is completely removed. Then, evaporate the reaction solution to obtain an oily substance.

[0040] (3) The above oily substance was purified by silica gel column chromatography to obtain linear peptide fragment 4 (H-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser-Gly-Leu-Gly-Cys(Trt)-OtBu), weighed to 14 g, and the purity was tested to be ≥90%.

[0041] Preparation of linear peptide fragment 5 (condensation of fragments 2 and 4 and deprotection of Fmoc) Weigh 10 g (4.44 mmol) of fragment 2 and add it to 200 mL of THF. Add 4.44 mmol of PYBOP / 8.88 mmol of DIEA and activate for 5 min. Then add 10 g (4.44 mmol) of fragment 4 and react at room temperature for 3 h. After the central control detects that fragment 4 has been completely consumed, add the reaction solution to 2000 mL of diethyl ether for precipitation. Centrifuge the suspension. The solid after centrifugation is stirred and slurried in 200 mL of diethyl ether for 10-15 min and centrifuged again. The solid after centrifugation is dried under reduced pressure in a vacuum drying oven at 35±5℃ for 20-24 h to obtain 15 g of white solid product. The yield of this step is 75%.

[0042] 16 g of the white solid product obtained in step (1) was added to 160 mL of 20% diethylamine / DMF and stirred for 2 h until the Fmoc protecting group was completely removed. The reaction solution was added to 1600 mL of diethyl ether for precipitation, and the suspension was centrifuged. The centrifuged solid was stirred and slurried in 200 mL of diethyl ether for 10-15 min, then centrifuged again. The centrifuged solid was dried under reduced pressure in a vacuum drying oven at 35±5℃ for 20-24 h to obtain 12.9 g of the product. The yield of this linear peptide fragment 5 (H-Ala-Asn(Trt)-Lys(Boc)-Lys(Boc)-Gly-Leu-Ser(tBu)-Lys(Boc)-Gly-Cys(Trt)-Phe-Gly-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser-Gly-Leu-Gly-Cys(Trt)-OtBu) was 84.9%.

[0043] Preparation of linear peptide fragment 6 (condensation of fragment 1 and fragment 5 and deprotection of Fmoc) (1) Weigh 9.68 g (3.23 mmol) of fragment 1 and add it to 200 mL of THF. Add 3.23 mmol of PYBOP / 6.46 mmol of DIEA and activate for 5 min. Then add 13.8 g (3.23 mmol) of fragment 5 and react at room temperature for 3 h. After the fragment 5 is completely consumed, add the reaction solution to 2000 mL of diethyl ether for precipitation. Centrifuge the suspension. Stir the solid after centrifugation with 200 mL of diethyl ether for 10-15 min and centrifuge again. Dry the solid after centrifugation in a vacuum drying oven at 35±5℃ for 20-24 h under reduced pressure to obtain 21.6 g of white solid product. The yield of this step is 92.2%.

[0044] (2) Add 16g of the white solid product obtained in step (1) to 160 mL of 20% diethylamine / DMF and stir for 2 h until the Fmoc protecting group is completely removed; add the reaction solution to 1600 mL of diethyl ether for precipitation, centrifuge the suspension, and then stir and beat the solid with 200 mL of diethyl ether for 10-15 min, centrifuge again, and then dry the solid under reduced pressure in a vacuum drying oven at 35±5℃ for 20-24 h to obtain 21.1 The yield of this linear peptide fragment 6 (H-Pro-Gly-Gln(Trt)-Glu(OtBu)-His(Trt)-Pro-Asn(Trt)-Ala-Arg(Pbf)-Lys(Boc)-Tyr(tBu)-Lys(Boc)-Gly-Ala-Asn(Trt)-Lys(Boc)-Lys(Boc)-Gly-Leu-Ser(tBu)-Lys(Boc)-Gly-Cys(Trt)-Phe-Gly-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser-Gly-Leu-Gly-Cys(Trt)-OtBu) was 97.7%.

[0045] Preparation of crude unclosed vortexilide Weigh 21.1 g of linear peptide fragment 6 and add it to 210 mL of lysis buffer pre-cooled to 5-15℃ (lysis buffer formula: TFA:Tis:EDT:PhOH:H2O=80:5:5:5:5). React at 25-30℃ for 3 h. Add the reaction solution to 2100 mL of diethyl ether for precipitation. Centrifuge the suspension. After centrifugation, stir and homogenize the solid with 200 mL of diethyl ether for 10-15 min, then centrifuge again. Repeat this homogenization-centrifugation step 3 times. Dry the solid under reduced pressure in a vacuum drying oven at 35±5℃ for 20-24 h to obtain 15.1 g of crude unclosed voxori peptide. Figure 1As shown, ESI-MS analysis revealed that the characteristic multicharged ion peaks were [M+3H]. 3+ =1368.9, [M+4H] 4+ =1027.1, [M+5H] 5+ =821.8, [M+6H] 6+ =685.1, [M+7H] 7+ =587.5, [M+8H] 8+ =514.1, which is completely consistent with the theoretical molecular weight of 4104.79.

[0046] Preparation of vosolide finished product Weigh 15.1 g of unclosed crude voxolipid peptide and add it to a mixture of 200 mL acetic acid and 200 mL purified water. Stir at room temperature until completely dissolved. Then, slowly add 0.1 mol / L methanol-iodine solution until the solution turns a stable light brown color. Continue stirring for 30 min, then add 1% ascorbic acid (VC) aqueous solution until the solution becomes clear again. Filter the solution through a 0.45 μm filter and collect the clear filtrate, which is the finished voxolipid peptide solution. Lyophilize the solution under reduced pressure to obtain 8.2 g of finished solid voxolipid peptide. The yield of the oxidative cyclization step is 54.3% based on the unclosed crude product.

[0047] Example 2 Preparation of fully protected linear peptide fragment 1 (1) Weigh 100 g of CTC Resin with a degree of substitution of 1.0 mmol / g (i.e., 100 mmol) into a peptide synthesizer, add 800 mL of DCM and stir to swell, weigh 200 mmol of Fmoc-Gly-OH and 600 mmol of DIEA and add them to the above stirring system, stir at room temperature for 3 h, then add 100 mL of MeOH and continue stirring for 30 min to end up unreacted active sites, filter to remove liquid, and wash the resin 6 times with DMF, and dry after each wash.

[0048] (2) Add 800 mL of 20% Pip / DMF deprotection solution to the peptide synthesizer and deprotect twice at room temperature for 5 min and 15 min respectively. After deprotection, wash the resin with DMF 8 times and dry it after each wash.

[0049] (3) Weigh 300 mmol of Fmoc-Lys(Boc)-OH and add it to 600 mL of DMF, then add 300 mmol of HOBT and stir until completely dissolved. Then add 300 mmol of DIC. Add the activated solution to the peptide synthesizer and react with the resin at room temperature for 2 h. After the reaction is complete, remove the waste liquid and wash the resin 4 times with 1000 mL of DMF each time. After each wash, dry the resin.

[0050] (4) Repeat steps (2) and (3) above, and perform coupling reactions according to the amino acid sequences Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Pro-OH, Fmoc-His(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, and Fmoc-Pro-OH to finally obtain the linear peptide resin Fmoc-Pro-Gly-Gln(Trt)-Glu(OtBu)-His(Trt)-Pro-Asn(Trt)-Ala-Arg(Pbf)-Lys(Boc)-Tyr(tBu)-Lys(Boc)-Gly-CTC Resin; finally, wash the resin three times alternately with DCM and MeOH, and dry it under vacuum after each wash. Then, dry it in a vacuum drying oven at 35±5℃ for 20~24 h to obtain 350 g of linear peptide resin.

[0051] (5) Add 350 g of the dried linear peptide resin obtained in step (4) to 2.8 L of 1% TFA / DCM solution, stir and react at room temperature for 1 h, filter to remove resin, wash the resin three times with 150 mL of DCM, and combine all filtrates; add the combined filtrate to 26 L of MTBE, stir evenly, centrifuge after the solid precipitates, after centrifugation, stir and slurry the solid with 2 L of MTBE, centrifuge again, after centrifugation, place the solid in a vacuum drying oven at 35±5℃ and dry under reduced pressure for 12~16 h to obtain fully protected linear peptide fragment 1 (Fmoc-Pro-Gly-Gln(Trt)-Glu(OtBu)-His(Trt)-Pro-Asn(Trt)-Ala-Arg(Pbf)-Lys(Boc)-Tyr(tBu)-Lys(Boc)-Gly-OH), weigh 235 g.

[0052] Preparation of fully protected linear peptide fragment 2 (1) Weigh 100 g of CTC Resin with a degree of substitution of 1.0 mmol / g (i.e., 100 mmol) into a peptide synthesizer, add 800 mL of DCM and stir to swell, weigh 200 mmol of Fmoc-Gly-OH and 600 mmol of DIEA and add them to the above stirring system, stir at room temperature for 3 h, then add 100 mL of MeOH and continue stirring for 30 min to end up unreacted active sites, filter to remove liquid, and wash the resin 6 times with DMF, and dry after each wash.

[0053] (2) Add 800 mL of 20% Pip / DMF deprotection solution to the peptide synthesizer and deprotect twice at room temperature for 5 min and 15 min respectively. After deprotection, wash the resin with DMF 8 times and dry it after each wash.

[0054] (3) Weigh 300 mmol of Fmoc-Phe-OH and add it to 600 mL of DMF, then add 300 mmol of HOBT and stir until completely dissolved. Then add 300 mmol of DIC. Add the activated solution to the peptide synthesizer and react with the resin at room temperature for 2 h. After the reaction is complete, remove the waste liquid and wash the resin 4 times with 1000 mL of DMF each time. After each wash, dry the resin.

[0055] (4) Repeat steps (2) and (3) above, and perform coupling reactions according to the amino acid sequences Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, and Fmoc-Ala-OH to finally obtain the linear peptide resin Fmoc-Ala-Asn(Trt)-Lys(Boc)-Lys(Boc)-Gly-Leu-Ser(tBu)-Lys(Boc)-Gly-Cys(Trt)-Phe-Gly-OH-CTC Resin; finally, wash the resin three times alternately with DCM and MeOH, and dry it under vacuum after each wash. Then, dry it in a vacuum drying oven at 35±5℃ for 20~24 h to obtain 290 g of linear peptide resin.

[0056] (5) Add 290 g of the dried linear peptide resin obtained in step (4) to 2.3 L of 1% TFA / DCM solution, stir and react at room temperature for 1 h, filter to remove resin, wash the resin three times with 150 mL of DCM, and combine all filtrates; add the combined filtrate to 22 L of MTBE, stir evenly, centrifuge after the solid precipitates, after centrifugation, stir and slurry the solid with 2 L of MTBE, centrifuge again, after centrifugation, place the solid in a vacuum drying oven at 35±5℃ and dry under reduced pressure for 12~16 h to obtain fully protected linear peptide fragment 2 (Fmoc-Ala-Asn(Trt)-Lys(Boc)-Lys(Boc)-Gly-Leu-Ser(tBu)-Lys(Boc)-Gly-Cys(Trt)-Phe-Gly-OH), weigh 179 g.

[0057] Preparation of fully protected linear peptide fragment 3 (1) Weigh 100 g of CTC Resin with a degree of substitution of 1.0 mmol / g (i.e., 100 mmol) into a peptide synthesizer, add 800 mL of DCM and stir to swell, weigh 200 mmol of Fmoc-Cys(Trt)-OH and 600 mmol of DIEA and add them to the above stirring system, stir at room temperature for 3 h, then add 100 mL of MeOH and continue stirring for 30 min to end up unreacted active sites, filter to remove liquid, and wash the resin 6 times with DMF, and dry after each wash.

[0058] (2) Add 800 mL of 20% Pip / DMF deprotection solution to the peptide synthesizer and deprotect twice at room temperature for 5 min and 15 min respectively. After deprotection, wash the resin with DMF 8 times and dry it after each wash.

[0059] (3) Weigh 300 mmol of Fmoc-Gly-OH and add it to 600 mL of DMF, then add 300 mmol of HOBT and stir until completely dissolved. Then add 300 mmol of DIC. Add the activated solution to the peptide synthesizer and react with the resin at room temperature for 2 h. After the reaction is complete, remove the waste liquid and wash the resin 4 times with 1000 mL of DMF each time. After each wash, dry the resin.

[0060] (4) Repeat steps (2) and (3) above, and perform coupling reactions according to the amino acid sequences Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser-OH, Fmoc-Met-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Leu-OH to finally obtain the linear peptide resin Fmoc-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser-Gly-Leu-Gly-Cys(Trt)-CTC. Resin; finally, wash the resin three times alternately with DCM and MeOH, and dry it under vacuum after each wash. Then, dry it in a vacuum drying oven at 35±5℃ for 20~24 h to obtain 310 g of linear peptide resin.

[0061] (5) Add 310 g of the dried linear peptide resin obtained in step (4) to 2.5 L of 1% TFA / DCM solution, stir and react at room temperature for 1 h, filter to remove resin, wash the resin three times with 150 mL of DCM, and combine all filtrates; add the combined filtrate to 23.6 L of MTBE, stir evenly, centrifuge after the solid precipitates, after centrifugation, the solid is stirred and slurried with 2 L of MTBE, centrifuged again, after centrifugation, the solid is placed in a vacuum drying oven at 35±5℃ and dried under reduced pressure for 12~16 h to obtain fully protected linear peptide fragment 3, namely (Fmoc-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser-Gly-Leu-Gly-Cys(Trt)-OH), weighing 179 g.

[0062] Preparation of linear peptide fragment 4 (tert-butyl esterification of fragment 3C end and Fmoc deprotection) (1) Add 23.7 g of fully protected linear peptide fragment 3 (10 mmol) to 230 mL of DCM, stir until completely dissolved, add 50 mmol of tert-butyltrichloroacetylimine ester, stir at room temperature for more than 12 h, and start post-processing when the remaining fragment 3 is ≤20% as detected by the intermediate control; add 230 mL of 10% saline to the reaction system, stir evenly, separate the liquid and take the lower organic phase.

[0063] (2) After drying the lower organic phase with anhydrous sodium sulfate, add 60 mL of piperidine and stir at room temperature for 1 h until the Fmoc protecting group is completely removed. Then, evaporate the reaction solution to obtain an oily substance.

[0064] (3) The above oily substance was purified by silica gel column chromatography to obtain linear peptide fragment 4 (H-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser-Gly-Leu-Gly-Cys(Trt)-OtBu), weighed to 15 g, and the purity was tested to be ≥90%.

[0065] Preparation of linear peptide fragment 5 (condensation of fragments 2 and 4 and deprotection of Fmoc) (1) Weigh 10 g (4.44 mmol) of fragment 2 and add it to 200 mL of DMF. Add 4.44 mmol HOBT / 8.88 mmol DIC and activate for 5 min. Then add 10 g (4.44 mmol) of fragment 4 and react at room temperature for 3 h. After the central control detects that fragment 4 has been completely consumed, add the reaction solution to 2000 mL of MTBE for sedimentation. Centrifuge the suspension. Stir and beat the solid after centrifugation with 200 mL of MTBE for 10-15 min and centrifuge again. Dry the solid after centrifugation in a vacuum drying oven at 35±5℃ for 20-24 h under reduced pressure to obtain 16 g of white solid product. The yield of this step is 80%.

[0066] (2) Add 16 g of the white solid product obtained in step (1) to 160 mL of 20% Pip / DMF and stir for 2 h until the Fmoc protecting group is completely removed; add the reaction solution to 1600 mL of MTBE for precipitation, and centrifuge the suspension; after centrifugation, the solid is stirred and slurried with 200 mL of MTBE for 10-15 min, then centrifuged again, and the solid is dried under reduced pressure in a vacuum drying oven at 35±5℃ for 20-24 h to obtain 13.8 g of the product. The yield of this linear peptide fragment 5 (H-Ala-Asn(Trt)-Lys(Boc)-Lys(Boc)-Gly-Leu-Ser(tBu)-Lys(Boc)-Gly-Cys(Trt)-Phe-Gly-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser-Gly-Leu-Gly-Cys(Trt)-OtBu) was 90.79%.

[0067] Preparation of linear peptide fragment 6 (condensation of fragment 1 and fragment 5 and deprotection of Fmoc) (1) Weigh 9.68 g (3.23 mmol) of fragment 1 and add it to 200 mL of DMF. Add 3.23 mmol HOBT / 6.46 mmol DIC and activate for 5 min. Then add 13.8 g (3.23 mmol) of fragment 5 and react at room temperature for 3 h. After the central control detects that fragment 5 has been completely consumed, add the reaction solution to 2000 mL of MTBE for sedimentation. Centrifuge the suspension. After centrifugation, stir and slurry the solid in 200 mL of MTBE for 10-15 min and centrifuge again. Dry the solid under reduced pressure in a vacuum drying oven at 35±5℃ for 20-24 h to obtain 22.3 g of white solid product. The yield of this step is 95.2%.

[0068] (2) Add 16 g of the white solid product obtained in step (1) to 160 mL of 20% Pip / DMF and stir for 2 h until the Fmoc protecting group is completely removed; add the reaction solution to 1600 mL of MTBE for precipitation, centrifuge the suspension, and then stir and beat the solid with 200 mL of MTBE for 10-15 min, centrifuge again, and then dry the solid under reduced pressure in a vacuum drying oven at 35±5℃ for 20-24 h to obtain 21.4 g of the product. The yield of this linear peptide fragment 6 (H-Pro-Gly-Gln(Trt)-Glu(OtBu)-His(Trt)-Pro-Asn(Trt)-Ala-Arg(Pbf)-Lys(Boc)-Tyr(tBu)-Lys(Boc)-Gly-Ala-Asn(Trt)-Lys(Boc)-Lys(Boc)-Gly-Leu-Ser(tBu)-Lys(Boc)-Gly-Cys(Trt)-Phe-Gly-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser-Gly-Leu-Gly-Cys(Trt)-OtBu) was 99.03%.

[0069] Preparation of crude unclosed vortexilide Weigh 21.4 g of linear peptide fragment 6 and add it to 210 mL of lysis buffer pre-cooled to 5-15℃ (lysis buffer formula: TFA:Tis:EDT:PhOH:H2O=87.5:5:2.5:2.5:2.5), and react at 25-30℃ for 2.5 h. Add the reaction solution to 2100 mL of MTBE for sedimentation, centrifuge the suspension, and then stir and homogenize the solid with 200 mL of MTBE for 10-15 min, followed by centrifugation. Repeat this homogenization-centrifugation step 3 times. Dry the solid under reduced pressure in a vacuum drying oven at 35±5℃ for 20-24 h to obtain 15.4 g of crude unclosed vosolid peptide.

[0070] Preparation of vosolide finished product Weigh 15.4 g of unclosed crude voxolipid and add it to a mixture of 200 mL acetic acid and 200 mL purified water. Stir at room temperature until completely dissolved. Then, slowly add 0.1 mol / L methanol-iodine solution until the solution turns a stable light brown color. Continue stirring for 30 min, then add 1% ascorbic acid (VC) aqueous solution until the solution becomes clear again. Filter the solution through a 0.45 μm filter and collect the clear filtrate, which is the finished voxolipid solution. Freeze-dry the solution under reduced pressure to obtain 8.8 g of voxolipid solid product. The yield of the oxidative cyclization step is 57.1% based on the unclosed crude product.

[0071] Example 3 Preparation of fully protected linear peptide fragment 1 (1) Weigh 100 g of CTC Resin with a degree of substitution of 1.0 mmol / g (i.e., 100 mmol) into a peptide synthesizer, add 800 mL of DCM and stir to swell, weigh 200 mmol of Fmoc-Gly-OH and 600 mmol of DIEA and add them to the above stirring system, stir at room temperature for 3 h, then add 100 mL of MeOH and continue stirring for 30 min to end up unreacted active sites, filter to remove liquid, and wash the resin 6 times with DMF, and dry after each wash.

[0072] (2) Add 800 mL of 1% DBU / DMF deprotection solution to the peptide synthesizer and deprotect twice at room temperature for 20 min and 40 min respectively. After deprotection, wash the resin with DMF 8 times and dry it after each wash.

[0073] (3) Weigh 300 mmol of Fmoc-Lys(Boc)-OH and add it to 600 mL of DCM, then add 400 mmol of TPTU and stir until completely dissolved. Then add 400 mmol of DIEA. Add the activated solution to the peptide synthesizer and react with the resin at room temperature for 5 h. After the reaction is complete, remove the waste liquid and wash the resin 4 times with 1000 mL of DMF each time. After each wash, dry the resin.

[0074] (4) Repeat steps (2) and (3) above, and perform coupling reactions according to the amino acid sequences Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Pro-OH, Fmoc-His(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, and Fmoc-Pro-OH to finally obtain the linear peptide resin Fmoc-Pro-Gly-Gln(Trt)-Glu(OtBu)-His(Trt)-Pro-Asn(Trt)-Ala-Arg(Pbf)-Lys(Boc)-Tyr(tBu)-Lys(Boc)-Gly-CTC Resin; finally, wash the resin three times alternately with DCM and MeOH, and dry it under vacuum after each wash. Then, dry it in a vacuum drying oven at 35±5℃ for 20~24 h to obtain 347 g of linear peptide resin.

[0075] (5) Add 347 g of the dried linear peptide resin obtained in step (4) to 2.8 L of 10% TFA / DCM solution, stir and react at room temperature for 1 h, filter to remove the resin, wash the resin three times with 150 mL of DCM, and combine all the filtrates; add the combined filtrate to 26 L of petroleum ether, stir evenly, centrifuge after the solid precipitates, after centrifugation, stir and slurry the solid with 2 L of petroleum ether, centrifuge again, after centrifugation, place the solid in a vacuum drying oven at 35±5℃ and dry under reduced pressure for 12~16 h to obtain fully protected linear peptide fragment 1 (Fmoc-Pro-Gly-Gln(Trt)-Glu(OtBu)-His(Trt)-Pro-Asn(Trt)-Ala-Arg(Pbf)-Lys(Boc)-Tyr(tBu)-Lys(Boc)-Gly-OH), weigh 233 g.

[0076] Preparation of fully protected linear peptide fragment 2 (1) Weigh 100 g of CTC Resin with a degree of substitution of 1.0 mmol / g (i.e., 100 mmol) into a peptide synthesizer, add 800 mL of DCM and stir to swell, weigh 200 mmol of Fmoc-Gly-OH and 600 mmol of DIEA and add them to the above stirring system, stir at room temperature for 3 h, then add 100 mL of MeOH and continue stirring for 30 min to end up unreacted active sites, filter to remove liquid, and wash the resin 6 times with DMF, and dry after each wash.

[0077] (2) Add 800 mL of 1% DBU / DMF deprotection solution to the peptide synthesizer and deprotect twice at room temperature for 20 min and 40 min respectively. After deprotection, wash the resin with DMF 8 times and dry it after each wash.

[0078] (3) Weigh 300 mmol of Fmoc-Phe-OH and add it to 600 mL of DCM, then add 400 mmol of TPTU and stir until completely dissolved. Then add 400 mmol of DIEA. Add the activated solution to the peptide synthesizer and react with the resin at room temperature for 5 h. After the reaction is complete, remove the waste liquid and wash the resin 4 times with 1000 mL of DMF each time. After each wash, dry the resin.

[0079] (4) Repeat steps (2) and (3) above, and perform coupling reactions according to the amino acid sequences Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, and Fmoc-Ala-OH to finally obtain the linear peptide resin Fmoc-Ala-Asn(Trt)-Lys(Boc)-Lys(Boc)-Gly-Leu-Ser(tBu)-Lys(Boc)-Gly-Cys(Trt)-Phe-Gly-OH-CTC Resin; finally, wash the resin three times alternately with DCM and MeOH, and dry it under vacuum after each wash. Then, dry it in a vacuum drying oven at 35±5℃ for 20~24 h to obtain 288 g of linear peptide resin.

[0080] (5) Add 288 g of the dried linear peptide resin obtained in step (4) to 2.3 L of 10% TFA / DCM solution, stir and react at room temperature for 1 h, filter to remove resin, wash the resin three times with 150 mL of DCM, and combine all filtrates; add the combined filtrate to 22 L of petroleum ether, stir evenly, centrifuge after the solid precipitates, after centrifugation, stir and slurry the solid with 2 L of petroleum ether, centrifuge again, after centrifugation, place the solid in a vacuum drying oven at 35±5℃ and dry under reduced pressure for 12~16 h to obtain fully protected linear peptide fragment 2 (Fmoc-Ala-Asn(Trt)-Lys(Boc)-Lys(Boc)-Gly-Leu-Ser(tBu)-Lys(Boc)-Gly-Cys(Trt)-Phe-Gly-OH), weigh 180 g.

[0081] Preparation of fully protected linear peptide fragment 3 (1) Weigh 100 g of CTC Resin with a degree of substitution of 1.0 mmol / g (i.e., 100 mmol) into a peptide synthesizer, add 800 mL of DCM and stir to swell, weigh 200 mmol of Fmoc-Cys(Trt)-OH and 600 mmol of DIEA and add them to the above stirring system, stir at room temperature for 3 h, then add 100 mL of MeOH and continue stirring for 30 min to end up unreacted active sites, filter to remove liquid, and wash the resin 6 times with DMF, and dry after each wash.

[0082] (2) Add 800 mL of 1% DBU / DMF deprotection solution to the peptide synthesizer and deprotect twice at room temperature for 20 min and 40 min respectively. After deprotection, wash the resin with DMF 8 times and dry it after each wash.

[0083] (3) Weigh 300 mmol of Fmoc-Gly-OH and add it to 600 mL of DCM, then add 400 mmol of TPTU and stir until completely dissolved. Then add 400 mmol of DIEA. Add the activated solution to the peptide synthesizer and react with the resin at room temperature for 5 h. After the reaction is complete, remove the waste liquid and wash the resin 4 times with 1000 mL of DMF each time. After each wash, dry the resin.

[0084] (4) Repeat steps (2) and (3) above, and perform coupling reactions according to the amino acid sequences Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser-OH, Fmoc-Met-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Leu-OH to finally obtain the linear peptide resin Fmoc-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser-Gly-Leu-Gly-Cys(Trt)-CTC. Resin; finally, wash the resin three times alternately with DCM and MeOH, and dry it under vacuum after each wash. Then, dry it in a vacuum drying oven at 35±5℃ for 20~24 h to obtain 308 g of linear peptide resin.

[0085] (5) Add 308 g of the dried linear peptide resin obtained in step (4) to 2.5 L of 10% TFA / DCM solution, stir and react at room temperature for 1 h, filter to remove resin, wash the resin three times with 150 mL of DCM, and combine all filtrates; add the combined filtrate to 23.6 L of petroleum ether, stir evenly, centrifuge after the solid precipitates, after centrifugation, stir and slurry the solid with 2 L of petroleum ether, centrifuge again, after centrifugation, place the solid in a vacuum drying oven at 35±5℃ and dry under reduced pressure for 12~16 h to obtain fully protected linear peptide fragment 3 (Fmoc-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser-Gly-Leu-Gly-Cys(Trt)-OH), weigh 177 g.

[0086] Preparation of linear peptide fragment 4 (tert-butyl modification of fragment 3 and Fmoc deprotection) (1) Add 23.7 g of fully protected linear peptide fragment 3 (10 mmol) to 230 mL of DMF and stir until completely dissolved. Then add 30 mmol of DCC and 3 mmol of DMAP and stir at room temperature for more than 6 h. When the remaining fragment 3 is ≤20% as detected by the central control, start the post-processing. Add 230 mL of 10% saline to the reaction system, stir evenly, separate the liquid and take the lower organic phase.

[0087] (2) After drying the lower organic phase with anhydrous sodium sulfate, add 10 mL of DBU and stir at room temperature for 1 h until the Fmoc protecting group is completely removed. Then, evaporate the reaction solution to obtain an oily substance.

[0088] (3) The above oily substance was purified by silica gel column chromatography to obtain linear peptide fragment 4 (H-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser-Gly-Leu-Gly-Cys(Trt)-OtBu), which weighed 14.4 g and was found to have a purity of ≥90%.

[0089] Preparation of linear peptide fragment 5 (condensation of fragments 2 and 4 and deprotection of Fmoc) (1) Weigh 10 g (4.44 mmol) of fragment 2 and add it to 200 mL of DCM. Add 4.44 mmol TPTU / 8.88 mmol DIEA and activate for 5 min. Then add 10 g (4.44 mmol) of fragment 4 and react at room temperature for 3 h. After the central control detects that fragment 4 has been completely consumed, add the reaction solution to 2000 mL of petroleum ether for precipitation. Centrifuge the suspension. Stir the solid after centrifugation with 200 mL of petroleum ether for 10-15 min and centrifuge again. Dry the solid after centrifugation in a vacuum drying oven at 35±5℃ for 20-24 h under reduced pressure to obtain 16.2 g of white solid product. The yield of this step is 81%.

[0090] (2) Add 16 g of the white solid product obtained in step (1) to 160 mL of 1% DBU / DMF and stir for 3 h until the Fmoc protecting group is completely removed; add the reaction solution to 1600 mL of petroleum ether for precipitation, and centrifuge the suspension; after centrifugation, the solid is stirred and slurried with 200 mL of petroleum ether for 10-15 min, and then centrifuged again. The solid after centrifugation is dried under reduced pressure in a vacuum drying oven at 35±5℃ for 20-24 h to obtain 14 g of the white solid product obtained in step (1). The yield of this linear peptide fragment 5 (H-Ala-Asn(Trt)-Lys(Boc)-Lys(Boc)-Gly-Leu-Ser(tBu)-Lys(Boc)-Gly-Cys(Trt)-Phe-Gly-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser-Gly-Leu-Gly-Cys(Trt)-OtBu) was 92.11%.

[0091] Preparation of linear peptide fragment 6 (condensation of fragment 1 and fragment 5 and deprotection of Fmoc) (1) Weigh 9.68 g (3.23 mmol) of fragment 1 and add it to 200 mL of DCM. Add 3.23 mmol TPTU / 6.46 mmol DIEA and activate for 5 min. Then add 13.8 g (3.23 mmol) of fragment 5 and react at room temperature for 3 h. After the central control detects that fragment 5 has been completely consumed, add the reaction solution to 2000 mL of petroleum ether for precipitation. Centrifuge the suspension. Stir the solid after centrifugation with 200 mL of petroleum ether for 10-15 min and centrifuge again. Dry the solid after centrifugation in a vacuum drying oven at 35±5℃ for 20-24 h under reduced pressure to obtain 22.5 g of white solid product. The yield of this step is 96.1%.

[0092] (2) Add 16 g of the white solid product obtained in step (1) to 160 mL of 10% DBU / DMF and stir for 3 h until the Fmoc protecting group is completely removed; add the reaction solution to 1600 mL of petroleum ether for precipitation, centrifuge the suspension, and then stir and slurry the solid with 200 mL of petroleum ether for 10-15 min, centrifuge again, and then dry the solid under reduced pressure in a vacuum drying oven at 35±5℃ for 20-24 h to obtain 21.5 g of the product. The yield of this linear peptide fragment 6 (H-Pro-Gly-Gln(Trt)-Glu(OtBu)-His(Trt)-Pro-Asn(Trt)-Ala-Arg(Pbf)-Lys(Boc)-Tyr(tBu)-Lys(Boc)-Gly-Ala-Asn(Trt)-Lys(Boc)-Lys(Boc)-Gly-Leu-Ser(tBu)-Lys(Boc)-Gly-Cys(Trt)-Phe-Gly-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser-Gly-Leu-Gly-Cys(Trt)-OtBu) was 99.49%.

[0093] Preparation of crude unclosed vortexilide Weigh 21.5 g of linear peptide fragment 6 and add it to 210 mL of lysis buffer pre-cooled to 5-15℃ (lysis buffer formula: TFA:Tis:EDT:PhOH:H2O=80:10:3:3:4). React at 25-30℃ for 2.5 h. Add the reaction solution to 2100 mL of petroleum ether for precipitation. Centrifuge the suspension. After centrifugation, stir and slurry the solid with petroleum ether for 10-15 min, then centrifuge again. Repeat the slurry-centrifugation step 3 times. Dry the solid after centrifugation in a vacuum drying oven at 35±5℃ for 20-24 h under reduced pressure to obtain 15.6 g of crude unclosed vosolid peptide.

[0094] Preparation of vosolide finished product Weigh 15.6 g of unclosed crude vosolamide and add it to a mixture of 200 mL acetic acid and 200 mL purified water. Stir at room temperature until completely dissolved. Then, slowly add 0.1 mol / L methanol-iodine solution until the solution turns a stable light brown color. Continue stirring for 30 min, then add 1% ascorbic acid (VC) aqueous solution until the solution becomes clear again. Filter the solution through a 0.45 μm filter and collect the clear filtrate, which is the finished vosolamide solution. Freeze-dry the solution under reduced pressure to obtain 8.5 g of vosolamide solid product. The yield of the oxidative cyclization step is 54.5% based on the unclosed crude product.

[0095] The above specific embodiments further illustrate the purpose, technical solution and beneficial effects of this application. It should be understood that the above are only specific embodiments of this application and are not intended to limit the scope of protection of this application. Any modifications, equivalent substitutions, improvements, etc., made on the basis of the technical solution of this application should be included within the scope of protection of this application.

Claims

1. A solid-phase-liquid-phase combined synthesis method for vosolid peptide, characterized in that, The synthesis method includes the following steps: S1. Using solid-phase synthesis, amino acids were sequentially coupled to prepare three fragments, including fragment 1, fragment 2, and fragment 3. The sequence of fragment 1 includes Fmoc-Pro-Gly-Gln(Trt)-Glu(OtBu)-His(Trt)-Pro-Asn(Trt)-Ala-Arg(Pbf)-Lys(Boc)-Tyr(tBu)-Lys(Boc)-Gly-OH; The sequence of fragment 2 includes Fmoc-Ala-Asn(Trt)-Lys(Boc)-Lys(Boc)-Gly-Leu-Ser(tBu)-Lys(Boc)-Gly-Cys(Trt)-Phe-Gly-OH; The sequence of fragment 3 includes Fmoc-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser-Gly-Leu-Gly-Cys(Trt)-OH; S2. The C-terminal carboxyl group of fragment 3 is modified by tert-butyl esterification, and then the Fmoc protecting group is removed to obtain fragment 4; the sequence of fragment 4 includes H-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser-Gly-Leu-Gly-Cys(Trt)-OtBu; S3. Fragment 4 and fragment 2 are condensed together. After condensation, the Fmoc protecting group is removed to obtain fragment 5. The sequence of fragment 5 includes... H-Ala-Asn(Trt)-Lys(Boc)-Lys(Boc)-Gly-Leu-Ser(tBu)-Lys(Boc)-Gly-Cys(Trt)-Phe-Gly-Leu -Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser-Gly-Leu-Gly-Cys(Trt)-OtBu; S4. Fragment 5 and fragment 1 are condensed together. After condensation, the Fmoc protecting group is removed to obtain fragment 6. The sequence of fragment 6 includes... H-Pro-Gly-Gln(Trt)-Glu(OtBu)-His(Trt)-Pro-Asn(Trt)-Ala-Arg(Pbf)-Lys(Boc)-Tyr(tBu)-Lys(Boc)-Gly-Ala-Asn(Trt)-Lys(Boc)-Lys(Boc)-Gly- Leu-Ser(tBu)-Lys(Boc)-Gly-Cys(Trt)-Phe-Gly-Leu-Lys(Boc)-Leu-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ser(tBu)-Met-Ser-Gly-Leu-Gly-Cys(Trt)-OtBu; S5. Fragment 6 was cleaved and precipitated to obtain crude vosolid peptide; S6. The unclosed crude vosolamide peptide is oxidized to complete the intramolecular disulfide bond cyclization, thus obtaining the finished vosolamide peptide product.

2. The synthesis method according to claim 1, characterized in that, The specific method for sequentially coupling amino acids in step S1 is to use a solid-phase resin as a carrier to sequentially couple amino acids from the carbon terminus to the nitrogen terminus according to the amino acid sequence, and then deprotect, cleave, and purify to obtain fragment 1, fragment 2, and fragment 3.

3. The synthesis method according to claim 2, characterized in that, The solid resin is chloro(o-chlorophenyl)diphenylmethane; the degree of substitution of the resin is 0.2~1.55 mmol / g.

4. The synthesis method according to claim 2, characterized in that, The coupling agent is one of HOBT / DIC, HOOBT / DIC, Oxyma / DIC, HOAT / DIC, HATU / DIEA, HBTU / DIEA, PYBOP / DIEA, Pyoxime / DIEA, TPTU / DIEA, TCFH / DIEA, and EDC·HCl / HOBT / DIEA. Furthermore, the coupling agent is HOBT / DIC, PYBOP / DIEA, or TPTU / DIEA; The amount of any one of HOBT / DIC, PYBOP / DIEA, and TPTU / DIEA is 1.5 to 4 times the molar amount of the solid resin; the coupling reaction time is 1 to 5 hours.

5. The synthesis method according to claim 2, characterized in that, The reagent used to remove Fmoc during the deprotection process is an organic base, which is one of piperidine, diethylamine, and DBU. The deprotection reaction time is 5-60 min.

6. The synthesis method according to claim 2, characterized in that, The cleavage is performed using 10%~40% HFIP / DCM or 1%~10% TFA / DCM, and the cleavage is purified with ether solvents to obtain fragment 1, fragment 2 or fragment 3; The ether solvent is one of MTBE, diethyl ether, petroleum ether, and isopropyl ether.

7. The synthesis method according to claim 1, characterized in that, In step S2, the tert-butyl esterification modification is performed using a tert-butylating agent, and the molar ratio of fragment 3 to the tert-butylating agent is 1:1~6; The tert-butylating agent is one of tert-butyltrichloroacetylimine ester, ditert-tert-butyl dicarbonate / DMAP, and DCC / DMAP, and the reaction solvent is one of DCM, THF, and DMF. In step S2, the removal of the Fmoc protecting group is performed using one or more of piperidine, diethylamine, and DBU.

8. The synthesis method according to claim 1, characterized in that, In steps S3 and S4, the condensation reagent used for condensation is selected from one of HOBT / DIC, HOOBT / DIC, Oxyma / DIC, HOAT / DIC, HATU / DIEA, HBTU / DIEA, PYBOP / DIEA, Pyoxime / DIEA, TPTU / DIEA, TCFH / DIEA, and EDC·HCl / HOBT / DIEA. Furthermore, the condensation reagent used in the condensation is selected from one of HOBT / DIC, PYBOP / DIEA, and TPTU / DIEA; In the condensation reaction, the molar ratio of fragment 4 to fragment 2 is 1:1~3, and the molar ratio of fragment 5 to fragment 1 is 1:1~3; the solvent for condensation is one or more of DCM, DMF, DMSO, THF, and acetone, and the condensation time is 0.5~6 h; In steps S3 and S4, the removal of the Fmoc protecting group uses one of the following: 20% Pip / DMF, 20% DEA / DMF, or 1%-10% DBU / DMF.

9. The synthesis method according to claim 1, characterized in that, In step S5, the pyrolysis solution is a mixture of TFA, Tis, EDT, PhOH, and H2O, and the volume ratio of TFA, Tis, EDT, PhOH, and H2O is 80~87.5∶5~10∶2.5~5∶2.5~5∶2.5~5; The pyrolysis solution is precooled to 5~15℃, the pyrolysis temperature is 25~30℃, and the pyrolysis time is 2.5~3h.

10. The synthesis method according to claim 1, characterized in that, In step S6, the specific method of oxidation is to use 0.1 mol / L methanol-iodine solution, add it dropwise until the solution turns a stable light brown color, continue stirring for 30 min, and then add 1% ascorbic acid aqueous solution dropwise until the solution becomes clear.

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