Solid-phase preparation method for tirzepatide
By using a solid-phase synthesis method involving small peptide fragments and specific coupling agents, the problem of high coupling difficulty in the synthesis of telpoeptide has been solved, achieving high yield and high purity of telpoeptide, which is suitable for industrial production.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- FUJIAN GENOHOPE BIOTECH LTD
- Filing Date
- 2025-05-16
- Publication Date
- 2026-06-04
AI Technical Summary
In the existing technology for synthesizing telpoide, the long amino acid sequence and the presence of hydrophobic amino acids make it easy to form intramolecular hydrogen bonds, leading to resin condensation, which increases the difficulty of coupling, reduces coupling efficiency, and is costly and complex, making it unsuitable for industrial production.
A solid-phase synthesis method using small peptide fragments and specific coupling agents is employed. By sequentially coupling amino acids and small peptide fragments, the number of coupling steps is reduced, simplifying the production process. Filtering of specific precipitates is used instead of centrifugation, further simplifying the production steps and improving yield and purity.
It significantly improves the yield and purity of crude telpoeptide, reduces production costs, simplifies the industrial production process, and is suitable for large-scale production.
Smart Images
Figure CN2025095319_04062026_PF_FP_ABST
Abstract
Description
A solid-phase preparation method for telpoeptide Technical Field
[0001] This invention belongs to the technical field of polypeptide drug preparation methods, and specifically relates to a method for preparing telpolide. Background Technology
[0002] Tirzepatide is a dual agonist of both glucose-dependent insulinotropic peptide (GIP, also known as gastric inhibitory peptide) receptors and glucagon-like peptide-1 (GLP-1) receptors. Both GIP and GLP-1 are intestinal hormones that promote insulin secretion. Tirzepatide integrates the effects of these two insulin-stimulating mechanisms into a single molecule, representing a novel class of drugs for the treatment of type 2 diabetes. Tirzepatide demonstrates its efficacy by improving β-cell function and increasing insulin sensitivity.
[0003] The structure of telpoeptide is as follows: H-Tyr 1 -Aib 2 -Glu 3 -G1y 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Ser 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 - Lys 16 -Ile 17 -Ala 18 -GIn 19 -Lys 20 (AEEA-AEEA-γ-Glu-Eicosanedioicacid)-Ala 21 -Phe 22 -Val 23 -GIn 24 -Trp 25 -Leu 26 -Ile 27 -Ala 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 3 -Gly 34 -Ala 35 -Pro 36 -Pro37 -Pro 38 -Ser 39 -NH 2 .
[0004] Currently, the most efficient chemical synthesis method for peptide drugs is solid-phase synthesis. This involves starting from the carboxyl terminus of the peptide, attaching the first amino acid to a resin, removing the protecting group of the amino group, and then coupling the next amino acid according to the peptide sequence, removing the protecting group, and repeating this cycle until amino acid assembly is complete. The peptide is then cleaved from the resin while removing all protecting groups to obtain the crude peptide, which is then purified and lyophilized to obtain the peptide active pharmaceutical ingredient (API). In existing solid-phase synthesis of telpolide, due to the large number of amino acids and the presence of many hydrophobic amino acids in the sequence, intramolecular hydrogen bonds easily form during coupling, resulting in severe β-sheet formation and resin condensation. If stepwise coupling is used, it increases the difficulty of amino acid coupling, reduces coupling efficiency, and makes each subsequent coupling step extremely difficult, easily leading to the formation of numerous missing peptides. Synthesizing telpolide from long peptide fragments is costly, complex, and unsuitable for industrial production.
[0005] Therefore, designing and developing a low-cost, simple synthetic process and high-quality method for preparing telpoeptide has significant practical and industrial value. Summary of the Invention
[0006] To address the shortcomings of existing technologies, this invention provides a high-yield preparation method for telpolide suitable for industrial production. This method significantly reduces the number of coupling cycles and solvent usage. Furthermore, during the precipitation process after telpolide resin pyrolysis, centrifugation is unnecessary; crude telpolide can be obtained through simple filtration, resulting in a high yield and purity of the synthesized crude telpolide.
[0007] This invention provides a method for preparing telpolide, wherein the amino acid sequence of telpolide is H-Tyr. 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Ser 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Ile17 -Ala 18 -GIn 19 -Lys 20 (AEEA-AEEA-γ-Glu-Eicosanedioicacid)-Ala 21 -Phe 22 -Val 23 -GIn 24 -Trp 25 -Leu 26 -Ile 27 -Ala 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 3 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38 -Ser 39 -NH 2 The preparation method includes: using an amino resin as the starting resin, and synthesizing a telpoeptide resin by sequentially coupling amino acids and small peptide fragments according to the amino acid sequence of the telpoeptide using a solid-phase synthesis method; the telpoeptide resin is then cleaved and purified to obtain pure telpoeptide; wherein the small peptide fragments are: 1-4 tetrapeptide fragments Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH, 5-6 dipeptide fragments Fmoc-Thr(tBu)-Phe-OH, 7-8 dipeptide fragments Fmoc-Thr(tBu)-Ser(tBu)-OH, 10-11 dipeptide fragments Fmoc-Tyr(tBu)-Ser(tBu)-OH, 12-13 dipeptide fragments Fmoc-Ile-Aib-OH, 17-18 dipeptide fragments... The peptide fragments are Fmoc-Ile-Ala-OH, Fmoc-Lys(AEEA-AEEA-γ-Glu(α-OtBu)-Eicosanedioicacid(mon-tBu))-OH (20-side chain), Fmoc-Ala-Gly-Gly-OH (28-30-side chain), Fmoc-Ser(tBu)-Ser(tBu)-Gly-OH (32-34-side chain), and Fmoc-Pro-Pro-Pro-OH (36-38-side chain). The coupling agents for conjugating the amino acids and the small peptide fragments are selected from Oxyma and DIC, TPTU and TMP, or COMU and DIEA. The order of conjugation of the amino acids and the small peptide fragments is as follows:
[0008] Preferably, the coupling agent is: Oxyma and DIC for the amino acids with coupling sequences of 1, 3, 5 and 7-13; TPTU and TMP for the small peptides with coupling sequences of 2, 4 and 6; and COMU and DIEA for the amino acids and small peptides with coupling sequences of 14-25.
[0009] Preferably, the preparation method of telpoeptide of the present invention includes: (a) deprotecting the starting resin with a deprotecting agent; (b) adding the coupling agent to Fmoc-Ser(tBu)-OH to activate the amino acids; (c) adding the activated amino acids obtained in step (b) to the resin obtained in step (a) for coupling reaction to obtain Fmoc-Ser(tBu)-resin; (d) repeating the deprotection, amino acid activation and coupling reaction steps to sequentially couple the remaining amino acids and small peptide fragments to obtain telpoeptide resin; (e) adding the telpoeptide resin obtained in step (d) to a lysis buffer for lysis, adding a precipitate to the lysed solution for crystallization, filtering and drying to obtain crude telpoeptide; (f) purifying the crude telpoeptide obtained in step (e) using a chromatographic system, and performing ultrafiltration, salt exchange and lyophilization to obtain pure telpoeptide.
[0010] Preferably, the starting resin is Rink Amide resin, Rink Amide-AM resin, Rink Amide-MBHA resin, or Sieber resin, and the degree of substitution of the starting resin is 0.3 to 0.6 mmol / g.
[0011] Preferably, the deprotecting agent is a PIP / DMF solution, and the volume concentration of the PIP is 20% to 50%.
[0012] Preferably, the pyrolysis solution is a TFA / EDT / Tis / H2O / phenol pyrolysis solution, which is prepared by the following method: preparing a first solution with a volume ratio of TFA:EDT:Tis:H2O = 90:5:2.5:2.5, and then adding phenol to the first solution, wherein the mass of phenol is 2% of the mass of the first solution.
[0013] Preferably, the precipitate is selected from MTBE and EA, MTBE and IPAc, or diethyl ether and EA, wherein the volume ratio of the two components in MTBE and EA, MTBE and IPAc, or diethyl ether and EA is 1:1.
[0014] Preferably, in step (f), the crude telpolide obtained in step (e) is purified twice using a chromatographic system employing a reversed-phase C18 column. The purification includes: (f-1): dissolving the crude telpolide obtained in step (e) in a 30% acetonitrile aqueous solution, filtering, and collecting the filtrate; (f-2): performing a first purification on the filtrate, using mobile phase A as a phosphoric acid aqueous solution with pH 3 and mobile phase B as acetonitrile, collecting a qualified fraction with a purity greater than 96%; (f-3): performing a second purification on the qualified fraction collected in step (f-2), using mobile phase A as a sodium acetate solution with pH 8.5 and mobile phase B as acetonitrile, collecting a fraction with a purity greater than 99.5%.
[0015] Preferably, in the ultrafiltration salt replacement step of step (f), the filter membrane pore size is 1-2 nm and the pressure is set to 1.5 MPa.
[0016] Preferably, the solvent in the coupling reaction is at least one selected from DMF, DCM, DMAc, and NMP.
[0017] In this invention, the primary sequence of telpoeptide refers to the sequence of amino acids in the polypeptide chain. Specifically, the amino acid sequence of telpoeptide in this invention is H-Tyr. 1 -Aib 2 -G1u 3 -G1y 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Ser 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 - Lys 16 -Ile 17 -Ala 18 -GIn 19 -Lys 20 (AEEA-AEEA-γ-Glu-Eicosanedioicacid)-Ala 21 -Phe 22 -Val 23 -GIn 24 -Trp 25 -Leu 26 -Ile 27 -Ala 28 -Gly 29 -Gly30 -Pro 31 -Ser 32 -Ser 3 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38 -Ser 39 -NH 2 The small peptide fragments in this invention include dipeptide fragments, tripeptide fragments, and tetrapeptide fragments. The x-yN peptide fragments represent small molecule peptides containing amino acids numbered x, y, and N amino acids between x and y. For example, tetrapeptide fragments 1-4 refer to tetrapeptide fragments containing Tyr (numbered 1), Aib (numbered 2), G1u (numbered 3), Gly (numbered 4), and a protecting group. Specifically, the small peptide fragments include tetrapeptide fragments 1-4 (Boc-Tyr(tBu)-Aib-Glu(OtBu)-G1y-OH), dipeptide fragments 5-6 (Fmoc-Thr(tBu)-Phe-OH), dipeptide fragments 7-8 (Fmoc-Thr(tBu)-Ser(tBu)-OH), and fragments 10-11 (Fmoc-Thr(tBu)-Ser(tBu)-OH). The following peptide fragments are listed: Fmoc-Tyr(tBu)-Ser(tBu)-OH, Fmoc-Ile-Aib-OH (12–13), Fmoc-Ile-Ala-OH (17–18), Fmoc-Lys(AEEA-AEEA-γ-Glu(α-OtBu)-Eicosanedioicacid(mon-tBu))-OH (20), Fmoc-Ala-Gly-G1y-OH (28–30), Fmoc-Ser(tBu)-Ser(tBu)-Gly-OH (32–34), and Fmoc-Pro-Pro-Pro-OH (36–38).
[0018] Solid-phase synthesis differs from liquid-phase synthesis. In solid-phase synthesis, each step cannot achieve 100% complete coupling; the more coupling steps, the more impurities are generated, and the greater the decrease in yield. In the preparation method provided by this invention, amino acids and small peptide fragments are sequentially coupled according to the amino acid sequence of telpolide using a solid-phase synthesis method. By selecting specific small peptide fragments and specific coupling agents, the number of coupling reactions in the solid-phase synthesis method is greatly reduced, and the efficiency of the coupling reactions is improved, significantly increasing the yield and purity of crude telpolide. Furthermore, the preparation method of this invention reduces the use of solvents, and during the precipitation process after telpolide resin pyrolysis, centrifugation is unnecessary; crude telpolide can be obtained through simple filtration, simplifying the production process, reducing production costs, and making it more suitable for industrial production. Attached Figure Description
[0019] Figure 1 is the HPLC chromatogram of crude telpolide from Example 4; Figure 2 is the HPLC chromatogram of crude telpolide from Example 5; Figure 3 is the HPLC chromatogram of crude telpolide from Example 6; Figure 4 is the HPLC chromatogram of purified telpolide from Example 7; Figure 5 is the mass spectrum of purified telpolide from Example 7; Figure 6 is the HPLC chromatogram of purified telpolide from Example 8; Figure 7 is the HPLC chromatogram of purified telpolide from Example 9; Figure 8 is the HPLC chromatogram of crude telpolide from Comparative Example 1. Detailed Implementation
[0020] The technical solutions of the present invention will be further described below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the invention. Unless otherwise specified, the methods used in the present invention are conventional production methods; the raw materials used, unless otherwise specified, are conventional commercially available products. In the following embodiments, the small fragment peptides of the present invention were purchased from Sichuan Tongsheng Biomedical Co., Ltd.
[0021] The English abbreviations and Chinese meanings of the reagents used in this invention are shown in Table 1: Table 1
[0022] This invention, by using a certain number of small peptide fragments and a specific coupling agent, significantly reduces the coupling steps compared to synthesizing individual amino acids one by one. This reduces the use of solvents and other raw materials, decreases impurity generation, simplifies the production process, lowers production costs, and also reduces the probability of generating severe β-sheets and missing peptides, thus improving coupling efficiency. Specifically, the preparation method of this invention includes deprotecting the starting resin with a deprotecting agent; adding a coupling agent to Fmoc-Ser(tBu)-OH to activate the amino acids; adding the activated amino acids to the deprotected resin for a coupling reaction to obtain Fmoc-Ser(tBu)-resin; repeating the deprotection, amino acid activation, and coupling reaction steps to sequentially couple the remaining amino acids and small peptide fragments to obtain telpoeptide resin; adding the telpoeptide resin to a lysis buffer for lysis; adding a precipitate to the lysed solution for crystallization; filtering and drying to obtain crude telpoeptide; purifying the crude telpoeptide using a chromatographic system, followed by ultrafiltration, salt exchange, and lyophilization to obtain pure telpoeptide.
[0023] In a preferred embodiment of the present invention, the coupling agents used in each coupling step of the synthesis of telpoeptide are not entirely the same. The choice of coupling agent is related to the amino acid site. TPTU is suitable for the condensation of racemic fragments, while COMU has good catalytic coupling effect and inhibits racemization. Depending on the carboxyl and amino components, the condensation reaction varies in ease and speed. For example, Val, which has high steric hindrance, and Pro, which has a secondary amino group, are amino acids that are difficult to condense, requiring enhanced conditions, such as the addition of a high-multiplicity excess of the carboxyl component, a highly active condensing agent, and extending the reaction time or appropriately increasing the reaction temperature. In addition to the influence of the structure of the amino acids involved in the reaction, factors such as the length of the assembled peptide chain and the degree of hydrophobicity of the peptide chain also have an important impact on the condensation reaction. The present invention improves the efficiency of the coupling reaction by selecting specific coupling agents in each coupling step, and can significantly improve the yield and purity of crude telpoeptide. In a preferred embodiment, the solvent in the coupling reaction is at least one selected from DMF, DCM, DMAc, and NMP.
[0024] The amino acids, small peptide fragments, coupling agents, and their amounts used in each synthesis step of the present invention are shown in Tables 2 and 3 below.
[0025] In a preferred embodiment of the present invention, the starting resin is Rink Amide resin, Rink Amide-AM resin, Rink Amide-MBHA resin, or Sieber resin, and the degree of substitution of the starting resin is 0.3–0.6 mmol / g. In a preferred embodiment of the present invention (trans), the deprotecting agent is a PIP / DMF solution, wherein the volume concentration of PIP is 20%–50%.
[0026] In a preferred embodiment of the present invention, the lysis buffer used is a TFA / EDT / Tis / H2O / phenol lysis buffer, which is prepared by the following method: A first solution with a volume ratio of TFA:EDT:Tis:H2O = 90:5:2.5:2.5 is prepared, and then phenol is added to the first solution, wherein the mass of phenol is 2% of the mass of the first solution. For example, if 90 mL of solution is prepared according to the TFA / EDT / Tis / H2O = 90 / 5 / 2.5 / 2.5 volume ratio, and the weight is 100 grams, then 2 grams of phenol are added. Phenol acts as an antioxidant to prevent the oxidation of Tyr and Trp.
[0027] In a preferred embodiment of the present invention, the precipitate is selected from MTBE and EA, MTBE and IPAc, or diethyl ether and EA, wherein the volume ratio of the two components in MTBE and EA, MTBE and IPAc, or diethyl ether and EA is 1:1. By using the above precipitate, crude thiopeptide can be obtained directly by filtration without centrifugation, simplifying the production steps and saving costs.
[0028] In a preferred embodiment of the present invention, the crude telpolide is purified twice using a chromatographic system employing a reversed-phase C18 column. The purification includes: First purification: dissolving the crude telpolide in a 30% acetonitrile aqueous solution, filtering, and collecting the filtrate; Second purification: subjecting the filtrate to the first purification, using mobile phase A as a phosphoric acid aqueous solution with pH 3 and mobile phase B as acetonitrile, collecting a qualified fraction with a purity greater than 96%; and Second purification of the collected qualified fraction, using mobile phase A as a sodium acetate solution with pH 8.5 and mobile phase B as acetonitrile, collecting a fraction with a purity greater than 99.5%.
[0029] In a preferred embodiment of the present invention, in the ultrafiltration salt exchange step, the filter membrane used has a pore size of 1-2 nm and the pressure is set to 1.5 MPa.
[0030] High-purity telpoeptide was obtained through two purification processes using a chromatographic system, followed by further purification using ultrafiltration with salt exchange. Example 1: Preparation of telpoeptide resin
[0031] Step 1: Place 30g of Rink Amide-AM resin (degree of substitution 0.41mmol / g) in a solid-phase reactor, add DMF solution to swell for 30 minutes, filter, add 20% PIP / DMF (PIP:DMF volume ratio of 20%:80%) to protect for 10 minutes, filter, add 20% PIP / DMF again to protect for 10 minutes, filter, wash with DMF 6 times, stirring for 3 minutes each time.
[0032] Add 300 mL of DMF to another reaction flask (hereinafter referred to as "amino acid activation reaction flask"), add 14.15 g of Fmoc-Ser(tBu)-OH and 6.3 g of Oxyma to the amino acid activation reaction flask and stir to dissolve. Slowly add 6.8 mL of DIC (Suzhou Haofan Biotechnology Co., Ltd.) and stir for at least 5 min to activate the amino acids.
[0033] The activated amino acid solution was added to a solid-phase reactor and reacted for 2 hours. A small amount of resin ninhydrin was tested, and the resin was colorless. The resin was filtered and washed twice with DMF to obtain Fmoc-Ser(tBu)-amide resin.
[0034] Step 2: Add 20% PIP / DMF to the resin obtained in Step 1 for protection for 10 minutes, filter, add 20% PIP / DMF again for protection for 10 minutes, filter, wash with DMF 6 times, stirring for 3 minutes each time. In the amino acid activation reaction flask, add 300 mL of a DCM / DMF (1 / 1 volume ratio) mixed solvent, add 13.08 g of Fmoc-Pro-Pro-Pro-OH, add 7.31 g of TPTU (Suzhou Haofan Biotechnology Co., Ltd.) to dissolve and clarify, cool to below 0℃, add 3.57 mL of TMP (Annegi (Shanghai) Pharmaceutical Chemical Co., Ltd.), stir and activate at below 0℃ for at least 10 minutes, then add to the solid-phase reactor and stir for 2 hours. Take a small amount of resin for ninhydrin testing; the resin is colorless. Filter, wash twice with DMF to obtain Fmoc-Pro-Pro-Pro-Ser(tBu)-amide resin.
[0035] The remaining amino acids and small peptide fragments were coupled sequentially. Fmoc-Ser(tBu)-Ser(tBu)-Gly-OH and Fmoc-Ala-Gly-Gly-OH were coupled using the amino acid coupling method described in step two, while the rest were coupled using the method described in step one. The order of the coupled amino acids and small peptide fragments, the coupling agents used, and their amounts are shown in Table 2 below: Table 2
[0036] Step Fourteen: Add a mixed solution of DMAc / DMF / DCM = 1 / 1 / 1 to the amino acid activation reaction flask, add 30.12g Fmoc-Lys(AEEA-AEEA-γ-Glu(α-OtBu)-Eicosanedioic acid(mon-tBu))-OH and 10.54g COMU (Suzhou Haofan Biotechnology Co., Ltd.) to dissolve, cool to below 0℃, add 4.72ml DIEA (Annaiji (Shanghai) Pharmaceutical Chemical Co., Ltd.) dropwise for activation for at least 5 minutes, add to the solid-phase reactor and stir for 5 hours, take a sample to detect the endpoint of the reaction.
[0037] Repeat the above coupling operation to sequentially couple the remaining amino acids and small peptide fragments. The coupling order, coupling agent, and dosage of the amino acids and small peptide fragments are shown in Table 3 below. After all amino acids and small peptide fragments are coupled, methanol is added for contraction three times, each time for 10 minutes. The mixture is then filtered, vacuum dried for 4 hours, and 130g of the target peptide resin is collected and weighed.
[0038] Table 3 Example 2: Preparation of telpoeptide resin
[0039] Except for using Rink Amide-MBHA resin (substitution degree 0.47 mmol / g) as the starting resin, all other steps were the same as in Example 1, yielding 138 g of the target peptide resin. Example 3: Preparation of Thilborpeptide Resin
[0040] Except for using Sieber resin (substitution degree 0.45 mmol / g) as the starting resin, all other steps were the same as in Example 1, yielding 133 g of the target peptide resin. Example 4: Preparation of crude telpoeptide
[0041] The lysis buffer was prepared as follows: a first solution with a volume ratio of TFA:EDT:Tis:H2O = 90:5:2.5:2.5 was prepared, and then phenol was added to the first solution, the mass of which was 2% of the mass of the first solution. The target peptide resin prepared in Example 1 was added to the lysis buffer, the amount of which was 10 times the weight of the target peptide resin prepared in Example 1. The mixture was stirred for 3 hours, filtered, and the filtrate was collected. The filtrate was cooled to below 0°C, and then slowly added to a mixture with a volume ratio of MTBE / EA = 1 / 1 to precipitate the resin. The amount of which was 60 times the weight of the target peptide resin prepared in Example 1 was used. The mixture was filtered, the filter cake was washed with MTBE, and dried in a solid oven for 4 hours to obtain 62g of crude telpoeptide. The yield of the crude product was 105%, and the HPLC purity of the crude product was 74.00%, where purity is the area ratio in the HPLC chromatogram. The yield was calculated as crude product mass / (resin degree of substitution x resin feed amount x product molecular weight), and the product molecular weight was 4813.5. The following chromatographic detection method was used: Liquid Chromatograph: Waters 2695 Separations Module; Model: UV (ALLLANCE E2695 / ACQUITY Arc); Column: Kinetex 2.6μm C18 150x4.6mm; Column temperature: 30℃; Detection wavelength: 210nm; Injection volume: 10μL; Flow rate: 0.7mL / min; Mobile phase A: Weigh 18.4g of ammonium dihydrogen phosphate, add 1600g of ultrapure water to dissolve, adjust the pH to 3.6 with phosphoric acid, then add 150g of acetonitrile and 200g of ultrapure water, and mix well;
[0042] Mobile phase B: Weigh 930g of acetonitrile, 310g of isopropanol, and 400g of ultrapure water, and mix well;
[0043] Example 5: Preparation of crude telpoeptide resin
[0044] The lysis buffer was the same as that used in Example 4. The target peptide resin prepared in Example 2 was added to the lysis buffer, and the amount of lysis buffer was 10 times the weight of the target peptide resin prepared in Example 2. The mixture was stirred for 3 hours, filtered, and the filtrate was collected. The filtrate was cooled to below 0°C, and the filtrate was slowly added to a mixture with a volume ratio of MTBE / IPAc = 1 / 1 to precipitate the resin. The amount of the mixture was 60 times the weight of the target peptide resin prepared in Example 2. The mixture was filtered, the filter cake was washed with MTBE, and dried in a solid oven for 4 hours to obtain 69 g of crude telpoeptide. The yield of the crude product was 102%, and the HPLC purity of the crude product was 75.74%. Example 6: Preparation of crude telpoeptide resin
[0045] The lysis buffer was the same as that used in Example 4. The target peptide resin prepared in Example 3 was added to the above lysis buffer, and the amount of lysis buffer was 10 times the weight of the target peptide resin prepared in Example 3. The mixture was stirred for 3 hours, filtered, and the filtrate was collected. The temperature was lowered to below 0°C, and the filtrate was slowly added to a mixture with a volume ratio of ether / EA = 1 / 1 to precipitate the peptide. The amount of the mixture was 60 times the weight of the target peptide resin prepared in Example 3. The mixture was filtered, the filter cake was washed with ether, and dried in a solid oven for 4 hours to obtain 68 g of crude telpoeptide. The yield of crude product was 105%, and the HPLC purity of crude product was 73.95%. Example 7: Purification of Crude Telpoeptide
[0046] The crude telpolide obtained in Example 4 was dissolved in acetonitrile-water with a volume concentration of 30% and filtered. Purification was performed twice using a high-performance liquid chromatography (HPLC) system. The HPLC instrument used in the purification process and its parameters are shown in Table 4 below: Table 4
[0047] For the first purification, mobile phase A was an aqueous phosphoric acid solution with pH 3, and mobile phase B was acetonitrile. The elution gradient is shown in Table 5 below: Table 5
[0048] Collect qualified fractions with a purity greater than 96% for secondary purification.
[0049] For the second purification, mobile phase A was sodium acetate solution at pH 8.5, and mobile phase B was acetonitrile. The elution gradient is shown in Table 6 below: Table 6
[0050] Collect qualified fractions with a purity greater than 99.5%.
[0051] Subsequently, the qualified fraction was subjected to salt exchange using a multifunctional membrane experimental device (Hangzhou Ruina Membrane Engineering Co., Ltd., RNM-18G), with a filter membrane pore size of 1-2 nm and a pressure of 1.5 MPa. The concentrate was collected and lyophilized to obtain pure telpolide. A total of 38 g of pure telpolide was obtained, with a yield of 64.2% and an HPLC purity of 99.88%. The obtained pure telpolide was analyzed by mass spectrometry using a liquid chromatography-mass spectrometry (LC-MS) instrument. The instrument and its parameters were as follows: Instrument name: Ultra-high performance liquid chromatography (UHPLC); Brand and model: ACQUIIY UPLC H-CLASSPIUS; Supplier: Waters Technology (Shanghai) Co., Ltd.; Column: 1.7μm PEPTIDE XB-C18 10C LC Column 150x2.1nm; Column temperature: 30℃; Injection volume: 10μL; Flow rate: 0.4mL / min; Phase A: 0.1% formic acid aqueous solution; Phase B: acetonitrile; Example 8: Purification of crude telpopeptide
[0052] The crude telpolide obtained in Example 5 was purified using the same method as in Example 7 to obtain pure telpolide. A total of 41 g of pure telpolide was obtained, with a yield of 60.4% and an HPLC purity of 99.61%. Example 9: Purification of Crude Telpolide.
[0053] The crude telpoeptide obtained in Example 6 was purified using the same method as in Example 7 to obtain pure telpoeptide. A total of 40g of pure telpoeptide was obtained, with a yield of 61.5% and an HPLC purity of 99.66%.
[0054] Comparative Example 1: Using 30g of Rink Amide-AM resin (degree of substitution 0.41mmol / g) as the starting resin, telpoide resin was prepared by sequentially coupling single amino acids to the resin. Except for the amino acids and coupling agents used, all other steps were the same as in Example 1. The amino acids, coupling agents, and their amounts used in the coupling process are shown in Table 7 below: Table 7
[0055]
[0056] After drying, 126g of the target peptide resin was obtained. It was cleaved in the same manner as in Example 4 to obtain 42g of crude telpoeptide, with a yield of 70.9% and an HPLC purity of 45.48%.
[0057] Comparative Example 2: A fragment synthesis method was used to synthesize telpoeptide, comprising fragment 1 (30-39 AA), fragment 2 (14-29 AA), and fragment 3 (1-13 AA). The peptide resin obtained from the reaction of fragments 2 and 3 was cleaved and then reacted with the cleavage product of fragment 1 using liquid-phase synthesis to obtain a protected API. After deprotection and precipitation, telpoeptide was obtained. Fragment 1 was prepared by using 5.0 g of Sieber resin (0.81 mmol / g as the starting resin), sequentially coupling amino acids to obtain the peptide resin. The peptide resin was then cleaved and dried to obtain 4.6 g of fragment 1. The amino acids and coupling agents used and their amounts are shown in Table 8 below: Table 8
[0058] Fragment 2 was prepared by starting with 11.0 g of 2-CTC resin (0.74 mmol / g degree of substitution), sequentially coupling amino acids to obtain peptide resin, and then pyrolyzing and drying the peptide resin to obtain 30.6 g of fragment 2. The amino acids, coupling agents, and their amounts used are shown in Table 9 below: Table 9
[0059] Fragment 3 was prepared by starting with 12g of 2-CTC resin (substitution degree 0.74mmol / g), sequentially coupling amino acids to obtain peptide resin, and then pyrolyzing and drying the peptide resin to obtain 4.1g of fragment 3. The amino acids, coupling agents, and their amounts used are shown in Table 10 below: Table 10
[0060] Solid-phase synthesis of fragment 3 and fragment 2: The reaction performance was poor when Oxyma / DIC was chosen as the coupling agent. Using COMU / DIC as the coupling agent resulted in a reaction time of 4-6 hours, and fragment 2 required a 2-fold equivalent, leading to higher costs.
[0061] As can be seen from the above examples and comparative examples, Comparative Example 1 uses a single amino acid as the material, which involves multiple coupling steps, is time-consuming, and results in low purity. Using large peptide fragments, such as in Comparative Example 2, for coupling leads to problems such as poor reaction efficiency, long reaction time, high raw material consumption, and high cost. The preparation method of the present invention has fewer steps, is simpler to operate, improves the reaction yield, and yields a higher crude product, making it more suitable for industrial production.
[0062] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.
Claims
1. A solid-phase preparation method for telpolide, wherein the amino acid sequence of telpolide is H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Ser 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Ile 17 -Ala 18 -GIn 19 -Lys 20 (AEEA-AEEA-γ-Glu-Eicosanedioicacid)-Ala 21 -Phe 22 -V2l 23 -GIn 24 -Trp 25 -Leu 26 -Ile 27 -Ala 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 3 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38 -Ser 39 -NH 2 Its characteristics are, The preparation method includes: Using amino resin as the starting resin, telpoeptide resin is synthesized by sequentially coupling amino acids and small peptide fragments according to the amino acid sequence of the telpoeptide using a solid-phase synthesis method. The telpoeptide resin is then cleaved and purified to obtain pure telpoeptide. The small peptide fragments are: 1-4 tetrapeptide fragments Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH; 5-6 dipeptide fragments Fmoc-Thr(tBu)-Phe-OH; 7-8 dipeptide fragments Fmoc-Thr(tBu)-Ser(tBu)-OH; 10-11 dipeptide fragments Fmoc-Tyr(tBu)-Ser(tBu)-OH; 12-13 dipeptide fragments Fmoc-Ile-Aib-OH; 17-18 dipeptide fragments Fmoc-Ile-Ala-OH; and 20 side chain peptides Fmoc-Lys(AEEA-AEEA-γ-Glu(α-OtBu)-Eicosanedioic The tripeptide fragments Fmoc-Ala-Gly-Gly-OH (28–30), Fmoc-Ser(tBu)-Ser(tBu)-Gly-OH (32–34), and Fmoc-Pro-Pro-Pro-OH (36–38) are also present. The coupling agent for conjugating the amino acid and the small peptide fragment is selected from Oxyma and DIC, TPTU and TMP, or COMU and DIEA; The sequence of coupling the amino acid and the small peptide fragment is as follows:
2. The preparation method according to claim 1, characterized in that, The coupling agents are as follows: Oxyma and DIC are used for the amino acids with coupling sequences of 1, 3, 5 and 7-13; TPTU and TMP are used for the small peptides with coupling sequences of 2, 4 and 6; and COMU and DIEA are used for the amino acids and small peptides with coupling sequences of 14-25.
3. The preparation method according to claim 1, characterized in that, The preparation method includes: (a) Deprotecting the starting resin with a deprotecting agent; (b) The coupling agent is added to Fmoc-Ser(tBu)-OH to activate the amino acids; (c) The activated amino acid obtained in step (b) is added to the resin obtained in step (a) for coupling reaction to obtain Fmoc-Ser(tBu)-resin; (d) Repeat the deprotection, amino acid activation and coupling reaction steps to sequentially couple the remaining amino acids and small peptide fragments to obtain telpoeptide resin. (e) The telpoeptide resin obtained in step (d) is added to the lysis buffer for lysis, and the precipitate is added to the lysed solution to induce crystallization. After filtration and drying, crude telpoeptide is obtained. (f) The crude telpoeptide obtained in step (e) is purified by a chromatographic system, and subjected to ultrafiltration, salt exchange, and lyophilization to obtain pure telpoeptide.
4. The preparation method according to claim 1, characterized in that, The starting resin is Rink Amide resin, Rink Amide-AM resin, Rink Amide-MBHA resin, or Sieber resin, and the degree of substitution of the starting resin is 0.3 to 0.6 mmol / g.
5. The preparation method according to claim 3, characterized in that, The deprotectant is a PIP / DMF solution, and the volume concentration of the PIP is 20% to 50%.
6. The preparation method according to claim 3, characterized in that, The pyrolysis solution is a TFA / EDT / Tis / H2O / phenol pyrolysis solution, which is prepared by the following method: preparing a first solution with a volume ratio of TFA:EDT:Tis:H2O = 90:5:2.5:2.5, and then adding phenol to the first solution, wherein the mass of phenol is 2% of the mass of the first solution.
7. The preparation method according to claim 3, characterized in that, The precipitate is selected from MTBE and EA, MTBE and IPAc, or diethyl ether and EA, wherein the volume ratio of the two components in MTBE and EA, MTBE and IPAc, or diethyl ether and EA is 1:
1.
8. The preparation method according to claim 3, characterized in that, In step (f), the crude telpoeptide obtained in step (e) is purified twice using a chromatographic system employing a reversed-phase C18 column. The purification includes: (f-1): Dissolve the crude telpoeptide obtained in step (e) in a 30% acetonitrile aqueous solution, filter, and collect the filtrate; (f-2): The filtrate is purified for the first time. Mobile phase A is an aqueous solution of phosphoric acid with pH 3, and mobile phase B is acetonitrile. The qualified fraction with a purity greater than 96% is collected. (f-3): The qualified fraction collected in step (f-2) is purified a second time. Mobile phase A is sodium acetate solution with pH 8.5 and mobile phase B is acetonitrile. The fraction with a purity greater than 99.5% is collected.
9. The preparation method according to claim 3, characterized in that, In the ultrafiltration salt replacement step of step (f), the filter membrane pore size is 1-2 nm and the pressure is set to 1.5 MPa.
10. The preparation method according to claim 3, characterized in that, The solvent in the coupling reaction is at least one selected from DMF, DCM, DMAc, and NMP.