A method for preparing a plenatan peptide

Abstract

This invention relates to a method for preparing pulcanapeptide, comprising the following steps: Following the C-terminal to N-terminal peptide sequence of the pulcanapeptide backbone, using propylphosphoric anhydride (T3P) as a condensing agent, pulcanapeptide 7-16 fully protected peptide resin and pulcanapeptide 1-6 fully protected peptide resin are obtained respectively. The 1-6 fully protected peptide resin is cleaved under full protection and then linked to the 7-16 peptide resin to obtain pulcanapeptide peptide resin. After cleavage, a linear crude pulcanapeptide peptide is obtained, which is then cyclized sequentially to obtain monocyclic and bicyclic peptides. Purification is performed after each cyclization step, and finally, pulcanapeptide is obtained through elution, concentration, and precipitation. The preparation method of this invention reduces the difficulty of synthesis, improves coupling efficiency, and reduces the generation of impurities. It not only allows for directional cyclization, ensuring the correct cyclization sequence, but also performs purification during the two-step cyclization process, reducing the difficult-to-remove impurities generated during cyclization and lowering the purification difficulty, thereby improving the purity and yield of the crude peptide and the final product.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a method for preparing purcanapeptide. Background Technology

[0002] Chronic idiopathic constipation (CIC) is a lower gastrointestinal motility disorder. Its pathogenesis is related to colonic and anorectal motility, as well as psychological abnormalities. It is a common and frequently occurring disease affecting middle-aged and elderly people, impacting the work and quality of life of modern individuals to varying degrees. Epidemiological surveys show that the incidence of CIC varies significantly across countries and regions due to differences in dietary structures and lifestyles. According to incomplete statistics, the incidence rate in developed countries such as Europe and the United States ranges from 2.0% to 28.0%, while in northern and southern China it ranges from 9.0% to 20.3%.

[0003] Plecanatide, developed by Synergy Pharmaceuticals in the United States, is an analog of uroguanylin. It acts as a guanylate cyclase receptor agonist that promotes sodium excretion, regulates acid-base ions in the gastrointestinal tract, induces fluid transport into the gastrointestinal tract, and increases gastrointestinal motility. It is suitable for the treatment of chronic idiopathic constipation in adults.

[0004] Pucanatide is a cyclic polypeptide containing 16 amino acids linked by disulfide bonds. It is an agonist of guanylate cyclase-C peptide, and its amino acid sequence is as follows:

[0005] H-Asn 1 -Asp 2 -Glu 3 -Cys 4 -Glu 5 -Leu 6 -Cys 7 -Val 8 -Asn 9 -Val 10 -Ala 11 -Cys 12 -Thr 13 -Gly 14 -Cys 15 -Leu 16 -OH(Cys 4 and Cys 12 and Cys 7 and Cys 15 (Disulfide bonds exist between them).

[0006] Currently, the main methods for synthesizing pulcanapeptide are divided into one-step cyclization and stepwise cyclization. One-step cyclization involves the solid-phase stepwise splicing of amino acids, where both Cys groups are protected by a single group. The resin is cleaved, and cyclization is performed via oxidation to form two disulfide bonds (SS) in one step, yielding crude pulcanapeptide, which is then purified and lyophilized to obtain pulcanapeptide. Stepwise cyclization involves using different protecting groups on the two Cys groups, selectively constructing the two disulfide bonds (SS) stepwise, followed by purification and lyophilization to obtain pulcanapeptide. Regardless of whether it's one-step or stepwise cyclization, peptide synthesis is extremely difficult. The coupling efficiency of amino acids decreases with increasing chain length, generating a large number of impurities, increasing the difficulty of purification and impurity separation, and resulting in a low overall yield. Furthermore, one-step cyclization makes the directional selection of disulfide bond formation difficult, easily causing multiple impurity peaks, making it difficult to effectively determine the correct disulfide bond composition. Moreover, the purification processes reported so far yield samples with low purity, and most pulcanapeptide is obtained through lyophilization. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a method for synthesizing purcanapeptide. This method employs a 6+10 fragment synthesis strategy using T3P as a condensing agent, combined with a stepwise cyclization method to synthesize the purcanapeptide bicyclic peptide. This reduces the synthesis difficulty, improves coupling efficiency, and minimizes impurity generation.

[0008] T3P, also known as propylphosphonic anhydride, has a molecular weight of 318.18 and a molecular formula of C9H10⁻¹⁰. 21 O6P3 is a superior reagent for forming amide / peptide bonds. Compared with traditional condensing agents, T3P has excellent reaction selectivity and low epimerization, which can significantly improve coupling efficiency, reduce impurity generation, and is relatively safe to use and can be stably stored for a long time.

[0009] The first objective of this invention is to provide a method for preparing pulcanapeptide, comprising the following steps:

[0010] S1. Fmoc-Leu-OH was coupled with Wang Resin to obtain Fmoc-Leu-Wang Resin. The 7-16 fully protected peptide resin of pucanapeptide was obtained by using T3P as a condensing agent according to the peptide sequence from C-terminus to N-terminus of the pucanapeptide backbone.

[0011] Fmoc-Leu-OH was coupled with resin to obtain Fmoc-Leu-CTC Resin. The 1-6 fully protected peptide resin of purcanapeptide was obtained by using T3P as a condensing agent according to the C-terminus to N-terminus peptide sequence of the purcanapeptide backbone.

[0012] S2. The fully protected peptide resin of pucanatide 1-6 is fully cleaved and linked with the fully protected peptide resin of pucanatide 7-16 to obtain pucanatide peptide resin. The resin is then cleaved to obtain linear pucanatide.

[0013] S3. Cyclate the linear pulcanapeptide to obtain a monocyclic pulcanapeptide.

[0014] S4. Purify the pulcanapeptide monocyclic peptide, and then perform a second cyclization on the purified pulcanapeptide monocyclic peptide to obtain the pulcanapeptide bicyclic peptide, which is then purified.

[0015] S5. The purified purcanapeptide bicyclic peptide is eluted, concentrated, and precipitated to obtain the purcanapeptide.

[0016] Further, in step S1, Fmoc-Leu-OH is coupled with Wang Resin to obtain Fmoc-Leu-WangResin, and Fmoc-Cys(R1)-OH, Fmoc-Gly-OH, Fmoc-Thr(OtBu)-OH, Fmoc-Cys(R2)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, and Fmoc-Cys(R1)-OH are sequentially coupled to Fmoc-Leu-resin to obtain pulcanapeptide 7-16 fully protected peptide resin:

[0017] H2N-Cys(R1)-Val-Asn(Trt)-Val-Ala-Cys(R2)-Thr(tBu)-Gly-Cys(R1)-Leu-WangResin, wherein R1 and R2 are independently selected from the protecting groups Trt, Acm, Mmt or tBu, and R1 and R2 are different.

[0018] Furthermore, the solid support resin mentioned above is Wang Resin with a substitution degree of 0.5 to 1.2 mmol / g.

[0019] Further, in step S1, Fmoc-Leu-OH is coupled with CTC Resin to obtain Fmoc-Leu-CTCResin, and Fmoc-Glu(OtBu)-OH, Fmoc-Cys(R2)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, and Boc-Asn(Trt)-OH are sequentially coupled to Fmoc-Leu-CTC Resin to obtain the fully protected peptide resin of purcanapeptide 1-6: Boc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(R2)-Glu(OtBu)-Leu-CTC Resin, wherein R2 is selected from the protecting group of Trt, Acm, Mmt or tBu.

[0020] Furthermore, the solid-phase carrier resin mentioned above is CTC Resin with a substitution degree of 1.0 to 1.5 mmol / g.

[0021] Further, in step S2, a fully protected pyrolysis is performed using a trifluoroethanol / DCM mixed solution as the pyrolysis buffer.

[0022] Boc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(R2)-Glu(OtBu)-Leu-CTC Resin is cleaved into Boc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(R2)-Glu(OtBu)-Leu-OH.

[0023] The volume ratio of trifluoroethanol to DCM is 1:3 to 5, preferably 1:4.

[0024] Further, in step S2, using T3P as a condensing agent, the fully protected peptides 1-6 and 7-16 of pucanapeptide are linked to a resin to obtain...

[0025] Boc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(R2)-Glu(OtBu)-Leu-Cys(R1)-Val-Asn(Trt)-Val-Ala-Cys(R2)-Thr(tBu)-Gly-Cys(R1)-Leu-Wang Resin.

[0026] Further, in step S2, a lysis buffer of linear purcanapeptide is obtained by volume. The lysis buffer is mainly composed of 90-95% TFA, and the other components are one or more of the following: 1-5% triisopropylsilane, 1-5% EDT, 1-5% water and 1-5% phenol.

[0027] Further, in step S3, hydrogen peroxide is used as an oxidant to oxidize and obtain purcanapeptide monocyclic peptide.

[0028] Further, in step S4, Sepax PolyRP is used as the packing material, 1-5‰ trifluoroacetic acid is used as mobile phase A, and acetonitrile is used as mobile phase B to purify the purcanatatide monocyclic peptide.

[0029] Further, in step S4, iodine solution is added to the purcanatide monocyclic peptide for secondary cyclization.

[0030] Further, in step S4, the purification of the purcanatide bicyclic peptide includes two purification processes: primary purification using Sepax PolyRP as packing material, 1-5‰ trifluoroacetic acid as mobile phase A, and acetonitrile as mobile phase B; secondary purification using C18 as packing material, 1-5% triethylamine and 0.1-1% phosphoric acid to prepare a mixed solution (TEAP) with pH 7-8 as mobile phase A, and acetonitrile as mobile phase B.

[0031] Further, in step S5, the purified purcanapeptide bicyclic peptide is loaded onto a Sepax PolyRP packed column and equilibrated with ammonium acetate buffer to replace the TEAP salt in the purified sample solution. The replaced ammonium acetate salt is then removed by equilibration with deionized water (ammonium acetate has a higher solubility in water than TEAP salt) to ensure complete desalting of purcanapeptide. Elution is then performed using 90% ethanol aqueous solution as the elution buffer.

[0032] Furthermore, in step S5, methyl tert-butyl ether is used for sedimentation. This invention employs an ethanol-water solution elution and concentration process followed by methyl tert-butyl ether sedimentation, which reduces solvent residues that cannot be removed during freeze-drying and provides a better particle size.

[0033] A second objective of this invention is to provide purcanatin obtained by the above method.

[0034] By means of the above-described solution, the present invention has at least the following advantages:

[0035] This invention uses T3P as a condensing agent and employs a 6+10 fragment synthesis strategy and a stepwise cyclization method to prepare purcanapeptide bicyclic peptides. This not only allows for directional cyclization, ensuring the correct cyclization sequence, but also enables purification during the two-step cyclization process, reducing the amount of difficult-to-remove impurities generated during cyclization and lowering the purification difficulty, thereby improving the purity and yield of the crude peptide and the finished product.

[0036] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the following describes the preferred embodiments of the present invention in conjunction with detailed drawings. Attached Figure Description

[0037] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0038] Figure 1 This is a schematic diagram of the process flow for preparing purcanapeptide according to the present invention;

[0039] Figure 2 The crude peptide map of purcanapeptide;

[0040] Figure 3 The monocyclic peptide map of purified purcanapeptide;

[0041] Figure 4 The bicyclic peptide map of purified purcanapeptide;

[0042] Figure 5 The image shows the purcanapeptide product obtained after elution, concentration, sedimentation and drying in Example 1;

[0043] Figure 6Image of the finished product from Example 2 of purcanapeptide;

[0044] Figure 7 Image of the finished product of Comparative Example 1 of purcanapeptide;

[0045] Figure 8 Image of the finished product of Comparative Example 2 of purcanapeptide;

[0046] Figure 9 The MS spectrum of the finished product of purcanapeptide in Example 1. Detailed Implementation

[0047] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0048] In this process, "substitution rate" refers to the amount of substance loaded per unit amount of resin, expressed in "mmol / g".

[0049] Ninhydrin test (Kaiser test): Place a small amount of resin in a small glass test tube, and add reagents a: 5% ninhydrin in anhydrous ethanol solution (w / v), b: phenol in anhydrous ethanol solution (4:1, w / v), and c: pyridine, 2 drops each. Heat at 105°C for 5 minutes. If the solution and resin are blue, dark blue, or brown, the ninhydrin test is positive; if the solution is clear and the resin is transparent and colorless, the ninhydrin test is negative.

[0050] The meanings of the abbreviations appearing in the text are as follows:

[0051] Table 1 Explanation of relevant terms in this invention

[0052] Fmoc 9-fluorenemethyloxycarbonyl Boc tert-Butoxycarbonyl tBu tert-butyl OtBu tert-butoxy Acm Acetamide methyl Mmt p-Methoxytriphenylmethyl StBu tert-butylthioalkyl Trt Triphenylmethyl T3P Propylphosphoanhydride DMF N,N-Dimethylformamide DIEA N,N-Diisopropylethylamine TFA Trifluoroacetic acid DBLK 20% hexahydropyridine / DMF solution Tis Triisopropylsilane DCM dichloromethane TFE Trifluoroethanol EDT 1,2-Ethylenedithiol

[0053] This invention provides a method for synthesizing purcanapeptide bicyclic peptide, specifically including the following steps:

[0054] Step 1: Fmoc-Leu-OH was modified with Wang Resin to obtain Fmoc-Leu-Wang Resin. The peptides were then sequentially coupled up to the 7th Cys position to complete deprotection and obtain fragment A.

[0055] H2N-Cys(R1)-Val-Asn(Trt)-Val-Ala-Cys(R2)-Thr(tBu)-Gly-Cys(R1)-Leu-WangResin;

[0056] Step 2: Fmoc-Leu-OH was modified with CTC Resin to obtain Fmoc-Leu-CTC Resin. The remaining amino acids were then gradually coupled to obtain Boc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(R2)-Glu(OtBu)-Leu-CTC Resin. After fully protected cleavage, fragment B was obtained.

[0057] Boc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(R2)-Glu(OtBu)-Leu-OH;

[0058] Step 3: Dot fragment A and fragment B to obtain purcanapeptide peptide resin:

[0059] Boc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(R2)-Glu(OtBu)-Leu-Cys(R1)-Val-Asn(Trt)-Val-Ala-Cys(R2)-Thr(tBu)-Gly-Cys(R1)-Leu-Wang Resin;

[0060] Step four: The crude linear peptide of purcanapeptide with a pair of protecting groups is obtained by lysis, sedimentation, centrifugation, washing, and drying using a mixture of lysis reagents TFA, TIS, EDT, and water.

[0061] H-Asn-Asp-Glu-Cys-Glu-Leu-Cys(R)-Val-Asn-Val-Ala-Cys-Thr-Gly-Cys(R)-Leu-OH

[0062] Step 5: Dissolve the crude purcanapeptide in a mixed solution of water and acetonitrile, adjust the pH to 8-9, oxidize with hydrogen peroxide to obtain a purcanapeptide monocyclic peptide solution, adjust the pH to 5-6 to terminate the reaction, and purify to obtain a purified purcanapeptide monocyclic peptide solution; (Purification conditions: Sepax PolyRP packing material as the preparation medium, 1‰ TFA as mobile phase A, acetonitrile as mobile phase B)

[0063] Step 6: Concentrate the purified monocyclic peptide to a concentration of 1-2 mg / ml, add iodine solution for bicyclic polymerization, and terminate the polymerization with vitamin C to obtain a purcanapeptide bicyclic peptide solution. Purify the resulting solution to obtain the purified purcanapeptide bicyclic peptide solution. (Primary purification conditions: Sepax PolyRP packing material as the preparation medium, 1‰ TFA as mobile phase A, acetonitrile as mobile phase B; Secondary purification conditions: C18 as the preparation medium, TEAP (1.0% triethylamine, 0.5% phosphoric acid, pH=7.0) as mobile phase A, acetonitrile as mobile phase B)

[0064] Step 7: Load the purified purcanapeptide bicyclic peptide solution onto a Sepax PolyRP preparative column, equilibrate with ammonium acetate buffer for 10 min, equilibrate with deionized water for 10 min, and elute with 90% ethanol / water solution to obtain the desalted purcanapeptide solution.

[0065] Step 8: The eluted purcanapeptide solution is concentrated by rotary evaporation to form a large suspension. Methyl tert-butyl ether is added for precipitation, and a large amount of white solid is precipitated. The solid is collected by centrifugation and vacuum dried to obtain purcanapeptide.

[0066] Example 1

[0067] (1) Segment A:

[0068] Synthesis of NH2-Cys(Acm)-Val-Asn(Trt)-Val-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Acm)-Leu-Wang Resin

[0069] Synthesis of Fmoc-Leu-Wang Resin: Wang Resin (13.00 g, substitution = 1.0 mmol / g resin) was added to the reaction column and washed once with DMF (150 mL). Dichloromethane (DCM) (150 mL) was added to swell the resin for 30 minutes. Fmoc-Leu-OH (2.0 equivalents), N,N-diisopropylcarbodiimide (2.4 equivalents), 1-hydroxybenzotriazole (2.4 equivalents), and DMAP (0.1 equivalents) were added to DMF (120 mL) and stirred to dissolve. This solution was then added to the resin and reacted at 30-35 °C for 2 hours. After the reaction was completed, the solvent was removed and the resin was washed with DMF (3 x 120 mL). 10 equivalents of acetic anhydride, pyridine and DMF (120 mL) were added to the resulting resin for blocking for 2 h. After the blocking was completed, DMF (5 x 120 mL) was added to wash the resin, and anhydrous methanol (3 x 120 mL) was added to shrink the resin. The degree of substitution was measured to be 0.40 mmol / g after drying.

[0070] Add 5 mmol of Fmoc-Leu-Wang Resin to the reaction column and wash once with DMF (150 mL). Add dichloromethane (DCM) (150 mL) to swell for 30 minutes. Add DBLK (120 mL) (20% piperidine / DMF) to deprotect twice, for 5 min and 15 min respectively, washing the resin once with DMF (120 mL) in between. After deprotection, wash the resin with DMF (5 x 120 mL). Take a small amount of resin and confirm the completion of Fmoc deprotection by Kaiser colorimetry. Add Fmoc-Cys(Acm)-OH (3.0 equivalents), T3P (3.6 equivalents), and DIEA (9 equivalents) to DMF (120 mL) and stir to dissolve and activate. Add to the reaction column and react at 25-30℃ for 2-3 hours. Monitor the completion of the reaction by Kaiser colorimetry. After the reaction is complete, drain the solvent and wash the resin with DMF (3 x 120 mL). The same coupling and deprotection processes were performed on Fmoc-Gly-OH, Fmoc-Thr(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, and Fmoc-Cys(Acm)-OH to obtain fragment A:

[0071] NH2-Cys(Acm)-Val-Asn(Trt)-Val-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Acm)-Leu-Wang Resin.

[0072] (2) Fragment B:

[0073] Synthesis of Boc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(Trt)-Glu(OtBu)-Leu-OH

[0074] Synthesis of Fmoc-Leu-CTC Resin: 30.00 g of 2-ClTrt resin (substitution = 1.0 mmol / g resin) was added to a reaction column and washed once with DMF (400 ml). Dichloromethane (DCM) (400 ml) was added to the reaction column to swell for 30 minutes. Fmoc-Leu-OH (2.0 equivalents), DIPEA (6.0 equivalents), and DMF (300 ml) were added to the resin and stirred until homogeneous. The reaction was carried out at 25-30 °C for 2 hours under nitrogen bubbling and gentle stirring. The reaction mixture was then drained and the resin was washed with DMF (3 x 300 ml). DCM (255 ml), anhydrous methanol (30 ml), and DIPEA (15 ml) were added to the resin and stirred until homogeneous. The mixture was then blocked for 10 minutes. The blocking process was repeated. After blocking, the resin was dried under vacuum and washed with DMF (4 x 300 ml).

[0075] Add DBLK (300 mL) (20% piperidine / DMF) to the resin obtained above for deprotection twice, for 5 min and 15 min respectively, washing the resin once with DMF (500 mL) in between. After deprotection, wash the resin with DMF (5 x 300 mL). Take a small amount of resin and confirm the completion of Fmoc deprotection by Kaiser color test. Add Fmoc-Glu(OtBu)-OH (3.0 equivalent), T3P (3.6 equivalent), and DIEA (9 equivalent) to DMF (300 mL) and stir to dissolve and activate. Add to the reaction column and react at 25-30℃ for 2-3 hours. Monitor the completion of the reaction by Kaiser color test. After the reaction is completed, drain the solvent and wash the resin with DMF (3 x 300 mL). Then, the same coupling and Fmoc deprotection processes were performed on Fmoc-Cys(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, and Boc-Asn(Trt)-OH to obtain peptide chain resins:

[0076] Boc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(Trt)-Glu(OtBu)-Leu-CTC Resin.

[0077] After drying the peptide chain resin obtained in the above steps, trifluoroethanol (120 ml) and DCM (480 ml) were added for fully protected cleavage. The reaction was carried out at room temperature for 2 h. The resin was filtered to obtain a fragment B solution containing the fully protected peptide. The fragment B was then rotary evaporated in a 30°C water bath. When a white solid precipitated, DCM (3 x 200 mL) was added until the solution was evaporated to dryness to obtain fragment B.

[0078] Boc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(Trt)-Glu(OtBu)-Leu-OH 25.30g.

[0079] (3) Synthesis of Pulcanapeptide Resin

[0080] Fragment B:

[0081] Boc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(Trt)-Glu(OtBu)-Leu-OH (3.0 equivalents), T3P (3.6 equivalents), and DIEA (9 equivalents) were added to DMF (150 mL) and stirred to dissolve and activate the resin. The solution was then added to a reaction column and reacted at 25-30°C for 2-3 hours. The reaction was monitored for completion using a Kaiser colorimetric assay. After the reaction was complete, the solvent was drained, and the resin was washed with DMF (3 x 150 mL). Anhydrous methanol (3 x 150 mL) was added to shrink the resin, and the resin was dried to obtain the pucana peptide resin.

[0082] Boc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(Trt)-Glu(OtBu)-Leu-Cys(Acm)-Val-Asn(Trt)-Val-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Acm)-Leu-Wang Resin 26.10g.

[0083] (4) Preparation of crude purcanapeptide

[0084] Prepare a lysis buffer (TFA:TIS:EDT:water) of 90:5:2.5:2.5 (v / v, 200 mL), pre-cool to below 0°C, add the obtained peptide resin to the lysis buffer, and lyse at 25-30°C for 2.5 h. Filter the resin, and add the filtrate to 1600 mL of methyl tert-butyl ether (MTBE) below 0°C while stirring. After standing, centrifuge, wash three times with MTBE, and dry to obtain 9.85 g of crude peptide with a purity of 81.17% (linear crude peptide with Acm protecting group).

[0085] Crude peptide map of purcanapeptide can be found Figure 2 As can be seen from the figure, the crude peptides cleaved using this method are clearly linear peptides.

[0086] (5) Preparation of purcanapeptide monocyclic peptide

[0087] The crude peptide was dissolved in 10% acetonitrile water to a concentration of approximately 1 mg / mL. The pH of the solution was adjusted to 8-9 using ammonia water, and the mixture was stirred until completely dissolved. 10 equivalents of 30% H₂O₂ solution were added to the crude peptide solution. The reaction was stirred at room temperature, and the reaction progress was monitored by HPLC. After the reaction was complete, the pH of the reactants was adjusted to 5-6 using TFA to terminate the reaction. The crude monocyclic peptide solution was obtained by filtration (cyclization at positions 4 and 12 occurred first). The resulting solution was purified under the following conditions:

[0088] Column: Sepax PolyRP packing material, 10μm, 300A, 150mm x 250mm; column temperature: 35℃; wavelength: 215nm; flow rate: 400ml / min

[0089] Mobile phase A: 1‰ TFA, Mobile phase B: Acetonitrile

[0090] 0 90 10 7 90 10 8 80 20 48 60 40

[0091] Collect samples with a purity ≥80%, and process the other fractions according to the above conditions. Concentrate the collected samples and analyze related substances using analytical RP-HPLC; the chromatograms are shown below. Figure 3 It can be seen that the purity of the main peak has increased significantly, and the yield of one-ring purification reaches 95%.

[0092] (6) Preparation of purcanapeptide bicyclic peptide

[0093] The monocyclic peptide obtained above was concentrated to a concentration of 1 mg / ml, and 1.5% iodine / acetonitrile (2 equivalents) was added until the yellow color of the solution persisted. The cyclization temperature was below 20°C. The reaction progress was monitored by HPLC, and a new peak was observed (removal of the Acm protecting group led to cyclization at positions 7 and 15). After the reaction was completed, ascorbic acid was added to terminate the reaction until the yellow solution became clear. The pH was adjusted to 5 with acetic acid, and the crude bicyclic peptide filtrate was obtained by filtration. The resulting solution was purified under the following conditions:

[0094] Yi Chun:

[0095] Column: Sepax PolyRP packing material 10μm, 300A, 150mm*250mm; column temperature: 35℃; wavelength: 215nm; flow rate: 400ml / min

[0096] Mobile phase A: 1‰ TFA, Mobile phase B: Acetonitrile

[0097] 0 90 10 7 90 10 8 80 20 48 60 40

[0098] Collect samples with a purity of ≥80% and process the other fractions according to the above conditions.

[0099] Erchun:

[0100] Column: Kromasil C18 10μm, 100A, 150mm*250mm, column temperature: 35℃, wavelength: 215nm, flow rate: 400ml / min

[0101] Mobile phase A: TEAP (1.0% triethylamine, 0.5% phosphoric acid, pH = 7.0), Mobile phase B: Acetonitrile

[0102] 0 80 10 7 80 10 8 80 10 48 70 30

[0103] Collect samples with a purity ≥99%, and process the other fractions according to the above conditions. Perform desalting on the collected samples and analyze related substances using analytical RP-HPLC; the chromatograms are shown below. Figure 4 This shows that this method has a significant effect on removing impurities.

[0104] (7) Desalting with purcanapeptide solution

[0105] The obtained purcanapeptide bicyclic peptide solution was subjected to desalting and elution.

[0106] Column: Sepax PolyRP packing material, 10μm, 300A, 150mm x 250mm; column temperature: 35℃; wavelength: 215nm; flow rate: 400ml / min

[0107] Mobile phase A: Ammonium acetate buffer, deionized water, 90% ethanol / water solution; Mobile phase B: Acetonitrile

[0108]

[0109]

[0110] Collect samples with a purity of ≥99%.

[0111] (8) Preparation of purcanapeptide samples

[0112] The eluted purcanapeptide solution was concentrated by rotary evaporation to form a large suspension. Methyl tert-butyl ether was added for precipitation, resulting in a large amount of white solid. The solid was collected by centrifugation and vacuum dried to obtain 2.91 g of purcanapeptide product (total yield 34.60%, purity 99.70%, maximum single impurity 0.20%). The Ms spectrum is shown below. Figure 9

[0113] ([M+2H)) 2+ =841.3[M+H] + =1681.2), theoretical molecular weight 1681.9, actual measured 1681.2, indicating that the target substance is correct.

[0114] Example 2

[0115] (1) The synthesis of the purcanapeptide peptide resin is the same as in Example 1, except that in Example 1, Cys(Acm) and Cys(Trt) are used at positions 7 / 15 and 4 / 12, respectively, while in Example 2, Cys(Mmt) and Cys(Acm) are used at positions 7 / 15 and 4 / 12, respectively. That is, fragment A is: NH2-Cys(Mmt)-Val-Asn(Trt)-Val-Ala-Cys(Acm)-Thr(tBu)-Gly-Cys(Mmt)-Leu-Wang Resin, and fragment B is:

[0116] Boc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(Acm)-Glu(OtBu)-Leu-OH.

[0117] The peptide resin for Pucanapeptide is: Boc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(Acm)-Glu(OtBu)-Leu-Cys(Mmt)-Val-Asn(Trt)-Val-Ala-Cys(Acm)-Thr(tBu)-Gly-Cys(Mmt)-Leu-WangResin.

[0118] (2) The preparation of crude purcanapeptide is the same as in Example 1, except that the lysis buffer is selected as TFA:TIS:EDT:phenol:water (90:2.5:2.5:2.5:2.5, volume ratio).

[0119] (3) The preparation of the cyclopeptide purcanapeptide is the same as in Example 1, except that the cyclization order is different. Cyclopeptide is first formed at positions 7 and 15, and then at positions 4 and 12.

[0120] 2.84 g of purcanapeptide was obtained by the purification method in Example 1 (total yield 33.81%, purity 99.60%, maximum single impurity 0.30%).

[0121] Comparative Example 1

[0122] (1) Preparation of purcanapeptide peptide resin

[0123] Wang Resin (13.00 g, substitution = 1.0 mmol / g resin) was added to the reaction column and washed once with DMF (150 mL). Dichloromethane (DCM) (150 mL) was added to swell the resin for 30 minutes. Fmoc-Leu-OH (2.0 equivalents), N,N-diisopropylcarbodiimide (2.4 equivalents), 1-hydroxybenzotriazole (2.4 equivalents), and DMAP (0.1 equivalents) were added to DMF (120 mL) and stirred to dissolve. This solution was then added to the resin, and the reaction was carried out at 30-35 °C for 2 hours. After the reaction was complete, the solvent was removed, and the resin was washed with DMF (3 x 120 mL). 10 equivalents of acetic anhydride:pyridine and DMF (120 mL) were added to the resulting resin for blocking for 2 hours. After the blocking was completed, DMF (5 x 120 mL) was added to wash the resin, and anhydrous methanol (3 x 120 mL) was added to shrink the resin. The degree of substitution was measured to be 0.42 mmol / g after drying.

[0124] Add 5 mmol of Fmoc-Leu-Wang Resin to the reaction column and wash once with DMF (150 mL). Add dichloromethane (DCM) (150 mL) to swell for 30 minutes. Add DBLK (120 mL) (20% piperidine / DMF) to deprotect twice, for 5 min and 15 min respectively, washing the resin once with DMF (120 mL) in between. After deprotection, wash the resin with DMF (5 x 120 mL). Take a small amount of resin and confirm the completion of Fmoc deprotection by Kaiser colorimetry. Add Fmoc-Cys(Acm)-OH (3.0 equivalents), T3P (3.6 equivalents), and DIEA (9 equivalents) to DMF (120 mL) and stir to dissolve and activate. Add to the reaction column and react at 25-30℃ for 2-3 hours. Monitor the completion of the reaction by Kaiser colorimetry. After the reaction is complete, drain the solvent and wash the resin with DMF (3 x 120 mL). The same coupling and deprotection process was performed on Fmoc-Gly-OH, Fmoc-Thr(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Cys(Acm)-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, and Boc-Asn(Trt)-OH, and the resulting product was dried to obtain purcanapeptide peptide resin.

[0125] Boc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(Trt)-Glu(OtBu)-Leu-Cys(Acm)-Val-Asn(Trt)-Val-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Acm)-Leu-Wang Resin 22.10g.

[0126] (2) Preparation of crude pucanatide peptide

[0127] Prepare a lysis buffer (TFA:TIS:EDT:water) of 90:5:2.5:2.5 (v / v, 200 mL), pre-cool to below 0°C, add the obtained peptide resin to the lysis buffer, and lyse at 25-30°C for 2.5 h. Filter the resin, and add the filtrate to 1600 mL of methyl tert-butyl ether (MTBE) below 0°C while stirring. After standing, centrifuge, wash three times with MTBE, and dry to obtain 7.85 g of crude peptide (linear crude peptide with Acm protecting group).

[0128] (3) Purification of crude pucanatide peptide

[0129] 1.65 g of purcanapeptide was obtained by purification using the method described in Example 1 (total yield 19.62%, purity 98.68%, maximum single impurity 0.84%).

[0130] Comparative Example 2

[0131] (1) 9.65 g of crude purcanapeptide with a purity of 81.20% was prepared by the synthesis method in Example 1.

[0132] (2) Purification of crude pucanatide peptide

[0133] The crude peptide was dissolved in 10% acetonitrile water to a concentration of approximately 1 mg / mL. The pH of the solution was adjusted to 8-9 using ammonia water, and the mixture was stirred until completely dissolved. 10 equivalents of 30% H₂O₂ solution was added to the crude peptide solution. The reaction was stirred at room temperature, and the reaction progress was monitored by HPLC. After the reaction was complete, the pH of the reactants was adjusted to 5-6 using TFA to terminate the reaction. The pH was then adjusted to 3-4 using acetic acid, and 1.5% iodine / acetonitrile (2 equivalents) was added until the yellow color of the solution persisted. The cyclization temperature was below 20°C. HPLC monitoring showed the formation of new peaks (de-Acm protecting group removal leading to cyclization at positions 7 and 15). After the reaction was complete, ascorbic acid was added to terminate the reaction until the yellow solution became clear. The solution was then filtered to obtain the crude bicyclic peptide filtrate.

[0134] The above solution was purified using the bicyclic peptide purification method of Example 1 to obtain 2.35 g of purcanapeptide (overall yield 27.94%, purity 99.51%, maximum single impurity 0.36%).

[0135] Test case

[0136] The purity data of the purcanapeptide products prepared in the above embodiments and comparative examples were tested, and the yield of the products was calculated. The results are shown in the table below.

[0137] Example 1 99.70 0.20 34.60 Example 2 99.60 0.24 33.81 Comparative Example 1 98.68 0.84 19.62 Comparative Example 2 99.50 0.36 27.94

[0138] Under this synthesis and purification method, both Example 1 and Example 2 showed good purity and yield. In Comparative Example 1, the stepwise coupling method was used, which significantly reduced the purity of the crude peptide. The yield and purity of the finished product were much lower than those of Example 1. In Comparative Example 2, the two-step cyclization process was not purified, which resulted in a decrease in the yield and purity of the final product.

[0139] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing pulcanapeptide, characterized in that, Includes the following steps: S1. Fmoc-Leu-WangResin was prepared by coupling Fmoc-Leu-OH with WangResin. Fmoc-Cys(R1)-OH, Fmoc-Gly-OH, Fmoc-Thr(OtBu)-OH, Fmoc-Cys(R2)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, and Fmoc-Cys(R1)-OH were coupled to Fmoc-Leu-WangResin in sequence from the C-terminus to the N-terminus of the purcanapeptide backbone. Propylphosphohydrin (T3P) was used as the condensing agent to obtain the purcanapeptide 7-16 fully protected peptide resin. Fmoc-Leu-CTCResin was prepared by coupling Fmoc-Leu-OH with 2-CTCResin. Fmoc-Glu(OtBu)-OH, Fmoc-Cys(R2)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, and Boc-Asn(Trt)-OH were sequentially coupled to Fmoc-Leu-CTCResin according to the C-terminus to N-terminus peptide sequence of the purcanapeptide backbone. Propylphosphoanhydride (T3P) was used as the condensing agent to obtain the purcanapeptide 1-6 fully protected peptide resin. Among them, R1 and R2 are independently selected from Trt, Acm or Mmt protecting groups, and R1 and R2 are different; S2. The fully protected peptide resin of pucanatide 1-6 is subjected to fully protected cleavage. Using propylphosphonic anhydride as a condensing agent, the fully protected peptides of pucanatide 1-6 are linked with the fully protected peptide resin of pucanatide 7-16 to obtain pucanatide peptide resin. The peptide resin is then cleaved to obtain linear pucanatide. The cleavage buffer used for fully protected cleavage is prepared by mixing trifluoroethanol and dichloromethane at a volume ratio of 1:3 to 5. The cleavage buffer for the cleavage peptide resin includes 90 to 95% TFA, and also includes one or more of the following: 1 to 5% triisopropylsilane, 1 to 5% EDT, 1 to 5% water, and 1 to 5% phenol. S3. Cyclate the linear pulcanapeptide to obtain a monocyclic pulcanapeptide peptide. The cyclization solution is hydrogen peroxide. S4. Purify the monocyclic peptide of pulcanapeptide. Perform a second cyclization on the purified monocyclic peptide of pulcanapeptide to obtain a bicyclic peptide of pulcanapeptide, and then purify it. Use Sepax PolyRP as packing material, 1-5‰ trifluoroacetic acid as mobile phase A, and acetonitrile as mobile phase B to purify the monocyclic peptide of pulcanapeptide. S5. The purified purcanapeptide bicyclic peptide is eluted, concentrated, and precipitated to obtain the purcanapeptide.

2. The method according to claim 1, characterized in that: In step S4, iodine solution is added to the purcanatide monocyclic peptide for secondary cyclization.

3. The method according to claim 1, characterized in that: In step S4, the purification of the purcanatide bicyclic peptide includes two purification processes: primary purification using Sepax PolyRP as packing material, 1-5‰ trifluoroacetic acid as mobile phase A, and acetonitrile as mobile phase B; secondary purification using C18 as packing material, a mixed solution of 1-5% triethylamine and 0.1-1% phosphoric acid with a pH of 7-8 as mobile phase A, and acetonitrile as mobile phase B.

4. The method according to claim 1, characterized in that: In step S5, the purified purcanatatide bicyclic peptide is loaded onto a Sepax PolyRP column and equilibrated with ammonium acetate buffer to replace the TEAP salt in the purified sample solution. The replaced ammonium acetate salt is then removed by equilibration with deionized water, and elution is performed using 90% ethanol aqueous solution as the elution buffer.

5. The method according to claim 1, characterized in that: In step S5, methyl tert-butyl ether is used for sedimentation.

Images