Preparation method of tilpotide

Abstract

The invention relates to the technical field of preparation of tirpotide, in particular to a preparation method of tirpotide, which comprises the following steps: dividing 39 amino acid sequences of tirpotide into three fragments by adopting a segmented synthesis method, namely a fragment 1 at the 10th-39th site, a fragment 2 at the 5th-9th site and a fragment 3 at the 1st-4th site; respectively performing solid-phase synthesis on a fragment 1, a fragment 2 and a fragment 3; condensing the fragment 3 and the fragment 2 to obtain a fragment A which is the 1st-9th site; condensing the fragment A and the fragment 1 to obtain a full-protection peptide chain; and carrying out global deprotection, oxidative folding and purification on the fully protected peptide chain to obtain the tilpotide. According to the preparation method of the tilpotide, a segmented synthesis method is adopted, a protecting group system, fragment condensation conditions and a purification process are optimized, the yield and the purity of the tilpotide are remarkably improved, and the synthesis period is shortened.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of preparation of tirzepatide, in particular to a preparation method of tirzepatide. BACKGROUND

[0002] Tirzepatide is a new GLP-1 receptor agonist with good blood glucose-lowering and weight-reducing effects, and has broad application prospects in the fields of diabetes and obesity treatment. However, tirzepatide is composed of 39 amino acids, and the traditional linear solid-phase peptide synthesis (SPPS) method has problems such as long synthesis cycle, high cumulative defect rate, low yield, poor purity and the like, which limits the large-scale production and clinical application of tirzepatide.

[0003] In the prior art, the preparation of tirzepatide is mostly carried out by using the linear SPPS method, and in the synthesis process, defects such as missing sequences and racemization are prone to occur, and with the increase of the length of the peptide chain, these defects will continuously accumulate, resulting in the reduction of the yield and purity of the product. In addition, tirzepatide molecules contain multiple amino acid sites (such as Aib, Pro, etc.) that are difficult to couple and complex side chain modifications (such as lipid chains), which further increase the difficulty of synthesis.

[0004] Therefore, developing an efficient, stable and suitable tirzepatide preparation method for large-scale production has become an important direction of current research. SUMMARY

[0005] The application aims to provide a preparation method of tirzepatide, which adopts a segmented synthesis method, optimizes the protection group system, fragment condensation conditions and purification process, and significantly improves the yield and purity of tirzepatide and shortens the synthesis cycle.

[0006] To achieve the above-mentioned purpose, the application provides a preparation method of tirzepatide, comprising the following steps:

[0007] S1, using a segmented synthesis method, the 39 amino acid sequences of tirzepatide are divided into three fragments, fragment 1 is the 10th-39th position, fragment 2 is the 5th-9th position and fragment 3 is the 1st-4th position;

[0008] S2, solid-phase synthesis of fragment 1, fragment 2 and fragment 3 respectively;

[0009] S3, condensing fragment 3 and fragment 2 to obtain fragment A, which is the 1st-9th position;

[0010] S4, condensing fragment A and fragment 1 to obtain a fully protected peptide chain;

[0011] S5, globally deprotecting, oxidizing and folding the fully protected peptide chain, and obtaining tirzepatide.

[0012] Preferably, in S1, the amino acid sequence of fragment 1 is Fmoc-Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(AEEA-AEEA-γ-Glu(C20)-OtBu)-Ala-Phe-Val-Gln(Trt)-Trp(Pbf)-Leu-Ile-Ala-Gly-OH.

[0013] The amino acid sequence of fragment 2 is Fmoc-Gly-Ser(tBu)-Ser(tBu)-Pro-Gly-OH.

[0014] The amino acid sequence of fragment 3 is Fmoc-Pro-Pro-Pro-Ala-OH.

[0015] Preferably, in S2, the solid-phase synthesis is performed using ChemMatrix resin or Rink Amide MBHA resin as the solid support.

[0016] Preferably, in S2, the solid-phase synthesis of fragment 1 is performed using low-loading resin with a loading capacity of 0.3-0.5 mmol / g, and a stepwise loading method is adopted, i.e., the first 5 amino acids are coupled using 0.3 mmol / g low-loading resin, and the subsequent amino acids are coupled using 0.5 mmol / g low-loading resin, and the coupling reagent used is DIC / Oxyma Pure (diisopropyl carbodiimide / 2-cyano-2-(hydroxyimino) ethyl acetate coupling reagent).

[0017] Preferably, in S2, when coupling the Aib site in fragment 1, a pre-activated ester method is used, and Fmoc-Aib-OPfp is used in combination with a 10% DIEA / DMF (N,N-diisopropyl ethylamine / N,N-dimethyl formamide solution) system.

[0018] The modification of the Lys(AEEA-AEEA-γ-Glu-OtBu) site in fragment 1 is as follows: first, Fmoc-Lys(AEEA)-OH is coupled to the resin, and the terminal amino group of the side chain AEEA is protected with Alloc; after removing Alloc, the second AEEA unit is coupled, and finally γ-Glu(C20)-OtBu is introduced.

[0019] Preferably, in S2, the coupling reagent used for the solid phase synthesis of fragment 2 is a mixture of HBTU (benzotriazole-1-yl-oxytrispyrrolidino phosphonium hexafluorophosphate), HOBt (1-hydroxybenzotriazole) and DIEA (N,N-diisopropylethylamine), the molar ratio of Fmoc-protected amino acid:HATU:HOAt:DIEA is (0.8-1.2):(1-1.2):(1-1.2):(2.8-3.2).

[0020] Preferably, in S2, the coupling reagent used for the solid phase synthesis of fragment 3 is a mixture of HATU (2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate), HOAt (1-hydroxy-7-azabenzotriazole) and DIEA, the molar ratio of Fmoc-protected amino acid:HATU:HOAt:DIEA is (0.8-1.2):(1-1.2):(1-1.2):(2.8-3.2).

[0021] Preferably, in S3 and S4, the condensing reagent used for the condensation of fragments is a mixture of PyBOP (benzotriazole-1-yl-oxytrispyrrolidino phosphonium hexafluorophosphate), HOAt and DIEA, the molar ratio of PyBOP:HOAt:DIEA is (0.8-1.2):(0.8-1.2):(1.8-2.2).

[0022] The reaction solvent for the condensation of fragments is a mixture of DMF (dimethylformamide) and CH2Cl2, the molar ratio of DMF:CH2Cl2 is (0.8-1.2):(0.8-1.2).

[0023] Preferably, in S3, the condensation of fragment 3 and fragment 2 is carried out at 25°C for 2 hours.

[0024] In S4, the condensation of fragment A and fragment 1 is carried out at 37°C for 4 hours.

[0025] Preferably, in S5, the global deprotection is carried out by first reacting at 0°C for 1 hour, then warming to 25°C for 1 hour, the deprotection reagent used is a mixture of TFA (trifluoroacetic acid), TIS (triisopropylsilane), EDT (1,2-ethanedithiol) and anisole, the volume ratio of TFA:TIS:EDT:anisole is 92:2:3:3.

[0026] The oxidative folding is carried out by first carrying out primary folding in a phosphate buffer at a temperature of 25°C and pH=7.0 for 2 hours, then adding a mixture of GSH (reduced glutathione) and GSSG (oxidized glutathione) and carrying out oxidation at 37°C for 4 hours; finally, cooling to 15°C, adjusting the pH to 7.5 and maintaining for 12 hours for stabilization.

[0027] The purification is pre-purification by hydrophobic chromatography, refinement by RP-HPLC and removal of polymer by molecular exclusion chromatography.

[0028] Therefore, the application adopts the above-mentioned preparation method of telopeptide, adopts a segmented synthesis method, divides the 39 amino acids into three key fragments, reduces the cumulative defect rate of linear SPPS, improves the synthesis efficiency, optimizes the fragment condensation conditions and global deprotection system, reduces the occurrence of side reactions, improves the product yield, adopts a three-step folding method and combined purification technology, improves the correct folding rate and product purity.

[0029] The technical solutions of the application are further described below through examples. DETAILED DESCRIPTION

[0030] The application is further described below in combination with examples. Unless otherwise defined, the technical terms or scientific terms used in the application should be understood as the general meanings understood by persons having ordinary skills in the art to which the application belongs. The features mentioned in the application or the features mentioned in the specific examples can be combined arbitrarily, and these specific examples are only used to illustrate the application and not to limit the scope of the application.

[0031] The application provides a preparation method of telopeptide, including the following steps:

[0032] S1, a segmented synthesis method is adopted to divide the 39 amino acid sequences of telopeptide into three fragments, fragment 1 is the 10th-39th position, fragment 2 is the 5th-9th position and fragment 3 is the 1st-4th position;

[0033] S2, fragment 1, fragment 2 and fragment 3 are respectively synthesized by solid phase;

[0034] S3, fragment 3 is condensed with fragment 2 to obtain fragment A, which is the 1st-9th position;

[0035] S4, fragment A is condensed with fragment 1 to obtain a fully protected peptide chain;

[0036] S5, the fully protected peptide chain is subjected to global deprotection, oxidation folding and purification to obtain telopeptide.

[0037] Preferably, in S1, the amino acid sequence of fragment 1 is Fmoc-Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(AEEA-AEEA-g-Glu(C20)-OtBu)-Ala-Phe-Val-Gln(Trt)-Trp(Pbf)-Leu-Ile-Ala-Gly-OH.

[0038] The amino acid sequence of fragment 2 is Fmoc-Gly-Ser(tBu)-Ser(tBu)-Pro-Gly-OH.

[0039] The amino acid sequence of fragment 3 is Fmoc-Pro-Pro-Pro-Ala-OH.

[0040] The present application divides the 39 amino acids of telopeptide into three key fragments, reduces the cumulative defect rate of linear SPPS, and improves the synthesis efficiency.

[0041] Preferably, in S2, the solid-phase synthesis uses ChemMatrix resin or Rink Amide MBHA resin as the solid-phase carrier.

[0042] The ChemMatrix resin used in the present application has a unique macroporous structure and high swelling property, which can effectively reduce steric hindrance in long-chain peptide synthesis, reduce the occurrence rate of side reactions, and significantly improve the purity and yield of the target product. The Rink Amide MBHA resin used in the present application can realize C-terminal amidation under mild conditions through a pre-activated carbamate linker, avoid the destruction of the peptide chain structure under harsh reaction conditions, simplify the synthesis steps, and improve the preparation efficiency.

[0043] Preferably, in S2, the solid-phase synthesis of fragment 1 uses low-loading resin with a loading capacity of 0.3-0.5 mmol / g, and adopts a stepwise loading method, i.e., the first five amino acids are synthesized using 0.3 mmol / g low-loading resin, and the subsequent amino acids are synthesized using 0.5 mmol / g low-loading resin. The coupling reagent used is DIC / Oxyma Pure (diisopropyl carbodiimide / 2-cyano-2-(hydroxyimino) ethyl acetate coupling reagent).

[0044] The stepwise loading method used in the present application reduces steric hindrance and improves synthesis efficiency, and the coupling reagent used can reduce racemization.

[0045] Preferably, in S2, when coupling the Aib site in fragment 1, a pre-activated ester method is used, employing Fmoc-Aib-OPfp in combination with a 10% DIEA / DMF (N,N-diisopropylethylamine / N,N-dimethylformamide solution) system to improve coupling efficiency.

[0046] The modification of the Lys(AEEA-AEEA-γ-Glu-OtBu) site in fragment 1 is as follows: first, Fmoc-Lys(AEEA)-OH is linked to the resin, and the terminal amino group of the AEEA side chain is protected with Alloc; after removing Alloc, a second AEEA unit is linked, and finally γ-Glu(C20)-OtBu is introduced to improve the lipid chain linkage efficiency.

[0047] Preferably, in S2, the coupling reagent used for solid-phase synthesis of fragment 2 is a mixture of HBTU (benzotriazole-1-yl-oxotripyrrolylphosphine hexafluorophosphate), HOBt (1-hydroxybenzotriazole), and DIEA (N,N-diisopropylethylamine), and the molar ratio of Fmoc protected amino acid: HATU: HOAt: DIEA is (0.8-1.2): (1-1.2): (1-1.2): (2.8-3.2).

[0048] In this invention, the synergistic effect of HBTU and HOBt efficiently activates carboxyl groups, significantly improves amino acid coupling efficiency, reduces racemization, and ensures the accuracy of peptide chain synthesis. DIEA, as an organic base, not only neutralizes the acid generated during the reaction and maintains pH stability, but also promotes nucleophilic substitution reactions. Precisely controlled molar ratios allow each reagent to fully exert its effect, ensuring the reaction rate while effectively reducing side reactions, improving the purity and yield of the target peptide fragment, and enhancing the quality of the prepared thiopeptide.

[0049] Preferably, in S2, the coupling reagent used for solid-phase synthesis of fragment 3 is a mixture of HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate), HOAt (1-hydroxy-7-azabenzotriazole), and DIEA, and the molar ratio of Fmoc protected amino acid:HATU:HOAt:DIEA is (0.8-1.2):(1-1.2):(1-1.2):(2.8-3.2).

[0050] In the present application, HATU is used as a high-efficiency condensing agent, which can quickly activate the carboxyl group to form an active intermediate, greatly shortening the reaction time; HOAt is used as an additive, which can effectively inhibit the racemization side reaction and ensure the high optical purity of the synthesis product; DIEA is used as an organic base, which can not only neutralize the acidic by-products generated in the reaction process, but also promote the smooth progress of the nucleophilic addition reaction. The synergistic effect of the three can realize a fragment coupling efficiency of more than 95% under the optimized molar ratio, significantly reduce the generation of by-products, and have good compatibility with Fmoc-protected amino acids of different structures.

[0051] Preferably, in S3 and S4, the condensing reagent for fragment condensation is a mixture of PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate), HOAt and DIEA, and the molar ratio of PyBOP:HOAt:DIEA is (0.8-1.2):(0.8-1.2):(1.8-2.2).

[0052] The reaction solvent for fragment condensation is a mixed solvent of DMF (dimethylformamide) and CH2Cl2, and the molar ratio of DMF:CH2Cl2 is (0.8-1.2):(0.8-1.2).

[0053] In the present application, PyBOP can promote the rapid progress of the peptide bond formation reaction; HOAt can enhance the activity of PyBOP, reduce the occurrence of racemization side reactions, and ensure the stereochemical purity of the product; DIEA can effectively neutralize the acid generated in the reaction process, maintain the acid-base balance of the reaction system, promote the forward progress of the reaction, and by combining PyBOP, HOAt and DIEA, the activity and selectivity of the fragment condensation reaction can be significantly improved.

[0054] In the present application, DMF has good solubility for various organic compounds, which can fully dissolve the reactants and condensing reagents, so that the reaction can be carried out in a homogeneous system, improving the mass transfer efficiency and reaction rate; CH2Cl2 has a low boiling point and good volatility, which is conducive to the separation of the product after the reaction and the recovery of the solvent. By mixing them in a molar ratio of (0.8-1.2):(0.8-1.2), an appropriate polar environment can be provided for the fragment condensation reaction, the reaction kinetics can be optimized, the smooth progress of the reaction can be promoted, and the occurrence of side reactions can be reduced, thereby improving the purity and yield of the product.

[0055] Preferably, in S3, the condensation of fragment 3 and fragment 2 is carried out at 25℃ for 2 hours.

[0056] In S4, the condensation of fragment A and fragment 1 is carried out at 37℃ for 4 hours.

[0057] Preferably, in S5, the global deprotection is first reacted at 0℃ for 1 hour, then warmed to 25℃ for 1 hour, and the deprotection reagent used is a mixture of TFA (trifluoroacetic acid), TIS (triisopropylsilane), EDT (1,2-ethanedithiol), and anisole, and the volume ratio of TFA:TIS:EDT:anisole is 92:2:3:3.

[0058] In the present application, the step-by-step temperature control for global deprotection can effectively reduce the reactivity and reduce side reactions caused by excessive reaction, thereby improving the selectivity of the deprotection reaction; subsequent warming to 25℃ for 1 hour continues the reaction, which promotes the deprotection reaction under mild conditions, ensuring the integrity and efficiency of the reaction. And through the synergistic effect of specific mixed deprotection reagents, TFA as a strong acid reagent can quickly destroy the protecting group; TIS and EDT have antioxidant properties, which can inhibit the possible oxidative side reactions during the reaction, protecting the structure of the target product stable; anisole as a stable carbocation intermediate reagent can prevent the rearrangement or other side reactions of the intermediate, ensuring that the deprotection process is accurate and efficient, ultimately improving the yield and purity of the deprotection step in the preparation of telopeptide.

[0059] Preferably, in S5, the oxidative folding is first carried out primary folding in a phosphate buffer at 25℃ and pH 7.0 for 2 hours, then a mixture of GSH (reduced glutathione) and GSSG (oxidized glutathione) is added, and the oxidation is carried out at 37℃ for 4 hours; finally, the temperature is lowered to 15℃, the pH is adjusted to 7.5, and the stability is maintained for 12 hours.

[0060] In the present application, by 25℃ primary folding, the peptide chain can be induced to form the correct conformation under mild conditions, reducing the risk of misfolding; by adding GSH and GSSG at 37℃ oxidation stage, the physiological environment is simulated to accelerate the formation of disulfide bond, improving the folding efficiency; the low temperature stability step at 15℃ combined with pH adjustment can effectively inhibit the aggregation and degradation of the peptide chain, ultimately improving the purity of telopeptide product by 15%-20%, and the activity recovery rate is increased to more than 90%, while reducing the production cost, enhancing the process stability and repeatability.

[0061] Preferably, in S5, the purification is carried out by hydrophobic chromatography pre-purification, RP-HPLC refining, and molecular exclusion chromatography to remove polymers.

[0062] In the present application, the three-step purification process synergizes, the hydrophobic chromatography pre-purification realizes preliminary separation through the difference in protein hydrophobicity, effectively removes most of the impurities, and reduces the subsequent refining pressure; the RP-HPLC refining is based on the principle of reversed-phase chromatography, can realize high-resolution separation, and accurately obtain the target product; the molecular exclusion chromatography utilizes the difference in molecular size, removes polymers specifically, and ensures the purity of the product. The purification method not only can improve the purity of telopeptide, but also has mild conditions in each step, can maximize the retention of product biological activity, reduces the use of organic solvents, reduces the production cost, and is suitable for industrial large-scale production.

[0063] In some embodiments of the present application, the oxidative folding phosphate buffer contains 1 mM EDTA.

[0064] In some embodiments of the present application, the stationary phase of the hydrophobic chromatography pre-purification is C4 column (model Sepax GP), the mobile phase is 0.1% TFA / acetonitrile system, and the gradient elution condition is 10%-80% acetonitrile, eluted for 60 minutes.

[0065] In some embodiments of the present application, the stationary phase of the RP-HPLC refining is C18 column (model BEH300), the mobile phase is 0.05% TFA / acetonitrile system, and the gradient elution condition is 10%-80% acetonitrile, eluted for 60 minutes.

[0066] In some embodiments of the present application, the stationary phase of the molecular exclusion chromatography is a gel with a porous structure (dextran gel or agarose gel), and the mobile phase is a phosphate buffer, and the composition of the mobile phase is unchanged during elution, and an isocratic elution is adopted.

[0067] Example 1

[0068] The present application provides a preparation method of telopeptide, comprising the following steps:

[0069] S1, using a segmented synthesis method, the 39 amino acid sequence of telopeptide is divided into 3 fragments, fragment 1 is 10-39, the amino acid sequence is Fmoc-Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(AEEA-AEEA-γ-Glu(C20)-OtBu)-Ala-Phe-Val-Gln(Trt)-Trp(Pbf)-Leu-Ile-Ala-Gly-OH; fragment 2 is 5-9, the amino acid sequence is Fmoc-Gly-Ser(tBu)-Ser(tBu)-Pro-Gly-OH; fragment 3 is 1-4, the amino acid sequence is Fmoc-Pro-Pro-Pro-Ala-OH.

[0070] S2, solid phase synthesis of fragment 1, fragment 2 and fragment 3 respectively;

[0071] S2.1, solid phase synthesis of fragment 1

[0072] Resin pretreatment: take 1.0g ChemMatrix resin (0.3mmol / g) and place it in a solid phase synthesis column, soak it in DMF (10mL) for 30 minutes, stir every 10 minutes during the period, make sure the resin is fully swollen (volume expansion to 2.5 times the original volume), then add 20% piperidine / DMF solution (8mL), shake at room temperature for 5 minutes, then discard, repeat the operation once (the second reaction is 10 minutes), completely remove the Fmoc group.

[0073] Initial amino acid connection: dissolve Fmoc-Boc-Tyr(tBu)-OH (1.5mmol), DIC (1.5mmol), OxymaPure (1.5mmol) in DMF (5mL), activate for 5 minutes, then add to the pretreated resin, shake at 30°C for 2 hours. Ninhydrin test is colorless (negative), indicating complete connection.

[0074] Ladder load adjustment and sequence extension: maintain a low load of 0.3mmol / g resin, use DIC / OxymaPure coupling reagent (1.5mmol:1.5mmol), DMF solvent, react at room temperature for 2 hours, sequentially couple amino acids 2-5 to extend the sequence. Change to a low load of 0.5mmol / g resin, use microwave-assisted activation (50°C, 25W), DIC / OxymaPure system reaction for 15 minutes, sequentially couple amino acids 6 and later.

[0075] Where coupling Aib site: Fmoc-Aib-OPfp (1.8 mmol) was mixed with 10% DIEA / DMF solution (5 mL) and added to the reaction system for 90 minutes, and ninhydrin test was negative.

[0076] Where coupling Lys site: Fmoc-Lys(AEEA-Alloc)-OH (1.8 mmol), PyBOP (1.8 mmol), HOAt (1.8 mmol), DIEA (3.6 mmol) were dissolved in DMF / CH2Cl2(1:1, 5 mL) and reacted at room temperature for 3 hours to ensure stable connection of the main chain. Pd(PPh3)4(0.15 mmol) and PhSiH3(1.5 mmol) in CH2Cl2solution (5 mL) were added and reacted at room temperature for 30 minutes to remove the Alloc protecting group and connect the second AEEA. Fmoc-AEEA-OH (1.8 mmol) was activated with PyBOP / HOAt and added, and reacted for 2 hours. Fmoc protection was removed by adding 20% piperidine / DMF, and γ-Glu(C20)-OtBu (1.5 mmol), DIC (1.5 mmol), OxymaPure (1.5 mmol) were added and reacted at 40°C in DMF for 4 hours to complete the lipid chain side chain modification, and then added to the reaction system.

[0077] After the coupling was completed, the resin was washed with DMF (5 times, 5 minutes each time) and DCM (5 times, 5 minutes each time) to remove residual reagents and by-products; finally, it was dried in a vacuum drying oven at 40°C under reduced pressure for 12 hours to obtain fragment 1.

[0078] S2.2, solid phase synthesis of fragment 2

[0079] Rink Amide MBHA resin was chosen as the solid phase synthesis resin, and the resin was pretreated (step S2.1). Fmoc-Gly-OH was mixed with the condensing agent HBTU, HOBt, DIEA in DMF at a molar ratio of 1:1.2:1.2:3 for 15 minutes, then added to the resin system, and the coupling reaction was carried out at 25°C for 2 hours. The successful coupling of Fmoc-Gly-OH to the resin was confirmed by ninhydrin detection. After removing the Fmoc protecting group of Fmoc-Ser(tBu)y-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH (treated with 20% piperidine / DMF solution for 10 minutes, repeated 2 times), the above-mentioned activation-coupling-detection process was repeated, and the subsequent amino acids were introduced in turn until the synthesis of fragment 2 was completed. After the synthesis was completed, the resin was washed with DMF and DCM alternately for 5 times, 5 minutes each time, and finally dried at 40°C under vacuum for 8 hours to obtain fragment 2.

[0080] S2.3, solid phase synthesis of fragment 3

[0081] Rink Amide MBHA resin was chosen as the solid phase synthesis resin, and the resin was pretreated (step S2.1). Fmoc-Pro-OH was mixed with the condensing agent HATU, HOAt, DIEA in DMF at a molar ratio of 1:1.1:1.1:3 for 20 minutes, then added to the resin system, and the coupling reaction was carried out at 30°C for 2.5 hours. The successful coupling of Fmoc-Pro-OH was confirmed by ninhydrin detection. Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH were deprotected in turn (treated with 20% piperidine / DMF solution for 10 minutes, repeated 2 times), and the above-mentioned activation-coupling-detection steps were followed to gradually introduce amino acids to complete the synthesis of fragment 3. The resin was washed with DMF and DCM for 5 times, 5 minutes each time, to remove impurities; finally dried in a vacuum drying oven at 40°C for 8 hours to obtain fragment 3.

[0082] S3, fragment 3 is condensed with fragment 2 to obtain fragment A;

[0083] Fragment 3 and fragment 2 were added to the reaction bottle at a molar ratio of 1:1.2, and a mixture of PyBOP, HOAt, DIEA (molar ratio 1:1:2) was used as the condensing agent, and a mixture of DMF / CH2Cl2 (molar ratio 1:1) was used as the solvent, and the reaction was carried out at 25°C for 2 hours. After the reaction was completed, washing with DMF and DCM obtained fragment A.

[0084] S4, fragment A is condensed with fragment 1 to obtain a fully protected peptide chain;

[0085] Fragment A and Fragment 1 were added into the reaction bottle at a molar ratio of 1:1.2, a mixture of PyBOP, HOAt, DIEA (molar ratio of 1:1:2) was used as condensation reagent, and a mixture of DMF / CH2Cl2(molar ratio of 1:1) was used as solvent, and the reaction was carried out at 37℃ for 4 hours. During the reaction, samples were taken every 30 minutes for HPLC analysis to ensure that the condensation degree was >99%. After the reaction was completed, washing with DMF and DCM was performed to obtain the fully protected peptide chain.

[0086] S5, the fully protected peptide chain was subjected to global deprotection, oxidative folding and purification to obtain tirzepatide.

[0087] Global deprotection is to cut the fully protected peptide chain from the resin, and a mixture of TFA:TIS:EDT:anisole (volume ratio of 92:2:3:3) is added, and the reaction is carried out at 0℃ for 1 hour, and then the temperature is raised to 25℃ for 1 hour. After the reaction is completed, the reaction solution is poured into ice ethyl ether for precipitation, centrifuged to collect the precipitate, washed with ice ethyl ether for several times, and dried to obtain the crude peptide.

[0088] Oxidative folding is to dissolve the crude peptide in a pH 7.0 phosphate buffer (containing 1mM EDTA) at a concentration of 1mg / mL, and the reaction is carried out at 25℃ for 2 hours. Then GSH / GSSG (molar ratio of 5:1, total concentration of 10mM) is added, and the reaction is carried out at 37℃ for 4 hours. Finally, the temperature is lowered to 15℃, and the pH is adjusted to 7.5 with NaOH solution, and maintained for 12 hours.

[0089] Purification is a three-step purification, which specifically includes:

[0090] Hydrophobic chromatography pre-purification: the folded solution is loaded onto a Sepax GPC4 column, 0.1% TFA / acetonitrile is used as the mobile phase, gradient elution (10%-80%, 60 minutes) is carried out, the target peak is collected, and rotary evaporation is performed to concentrate to obtain concentrated solution one.

[0091] RP-HPLC refining: concentrated solution one is loaded onto a BEH300 C18 column (3.5μm), elution is carried out with 0.05% TFA / acetonitrile as the mobile phase, the target peak is collected, and after concentration, concentrated solution two is obtained.

[0092] Molecular exclusion chromatography removes polymers: concentrated solution two is loaded onto a molecular exclusion chromatography column to remove polymers, the target peak is collected, and freeze-drying is performed to obtain pure tirzepatide.

[0093] Comparative Example 1

[0094] Traditional linear SPPS method was used to prepare tirzepatide, and 39 amino acids were coupled one by one, and other reagents and conditions were consistent with Example 1.

[0095] Comparative Example 2

[0096] The preparation procedure is basically the same as that of Example 1, except that the stepwise loading method is not used in the solid-phase synthesis of fragment 1, and 0.5 mmol / g low-loading resin is used throughout.

[0097] Comparative Example 3

[0098] The preparation procedure is basically the same as that of Example 1, except that the one-step oxidation folding method is used, i.e., the reaction is carried out at 37°C in a phosphate buffer (containing 1 mM EDTA and GSH / GSSG (5:1, total concentration 10 mM)) at pH = 7.5 for 18 hours.

[0099] Performance detection

[0100] The products prepared in Example 1 and Comparative Examples 1-3 are detected for purity by HPLC; the condensation efficiency of fragment 1 is monitored by HPLC, and LCMS detection is carried out every 5 amino acids; the proportion of missing sequences is detected by LCMS, and the racemization rate is monitored by chiral HPLC; and the polymer content is detected by molecular exclusion chromatography. The results are shown in Table 1.

[0101] Table 1 Performance of telopeptides prepared by different methods

[0102]

[0103] As can be seen from Table 1, the HPLC purity of Example 1 is significantly higher than that of Comparative Examples 1-3, indicating that the use of the segmented synthesis strategy, stepwise loading method and three-step oxidation folding process can effectively reduce the generation of impurities and improve the purity of the target product. The condensation efficiency of fragment 1 in Example 1 is higher than that in Comparative Example 2, indicating that the stepwise loading method can reduce steric hindrance and improve the condensation efficiency of long-chain fragments; the cumulative defect rate of Comparative Example 1 using the traditional linear SPPS method is as high as 12.3%, while the segmented synthesis of Example 1 significantly reduces the defect risk, reflecting the advantage of the segmented strategy in reducing the cumulative error of long-chain synthesis. The racemization rate of Example 1 is <0.5%, which is much lower than the 3.1% of Comparative Example 1, indicating that the use of coupling reagents (such as DIC / OxymaPure) and pre-activated ester method in the present application can effectively inhibit racemization; the polymer content of Example 1 is <0.5%, which is significantly lower than that of other comparative examples, indicating that the combination of purification techniques (hydrophobic chromatography + RP-HPLC + molecular exclusion chromatography) can accurately remove polymer impurities.

[0104] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit them. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can still be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present application.

Claims

1. A method for preparing telopeptides, characterized in that, The method comprises the following steps: S1, using a segment synthesis method, the 39 amino acid sequence of telopeptide is divided into three segments, segment 1 is the 10th-39th, segment 2 is the 5th-9th and segment 3 is the 1st-4th; S2, solid-phase synthesis of segment 1, segment 2 and segment 3 respectively; S3, segment 3 is condensed with segment 2 to obtain segment A, which is the 1st-9th; S4, segment A is condensed with segment 1 to obtain a fully protected peptide chain; S5, global deprotection, oxidation folding and purification are performed on the fully protected peptide chain to obtain telopeptide.

2. A process for the preparation of a thymopentin according to claim 1, characterized in that: In S1, the amino acid sequence of segment 1 is Fmoc-Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(AEEA-AEEA-γ-Glu(C20)-OtBu)-Ala-Phe-Val-Gln(Trt)-Trp(Pbf)-Leu-Ile-Ala-Gly-OH; The amino acid sequence of segment 2 is Fmoc-Gly-Ser(tBu)-Ser(tBu)-Pro-Gly-OH; The amino acid sequence of segment 3 is Fmoc-Pro-Pro-Pro-Ala-OH.

3. A process for the preparation of a thymopentin according to claim 1, characterized in that: In S2, solid-phase synthesis is performed by using ChemMatrix resin or Rink Amide MBHA resin as a solid-phase carrier.

4. A process for the preparation of a thymopentin according to claim 1, characterized in that: In S2, low-loading resin is used for solid-phase synthesis of segment 1, the loading capacity is 0.3-0.5mmol / g, and a stepwise loading method is used, the first five amino acids are synthesized by using 0.3mmol / g low-loading resin, and the subsequent amino acids are synthesized by using 0.5mmol / g low-loading resin, and the coupling reagent used is DIC / Oxyma Pure.

5. A process for the preparation of a thymopentin according to claim 4, characterized in that: In S2, when the Aib site in segment 1 is coupled, a pre-activated ester method is used, Fmoc-Aib-OPfp is used in combination with a 10% DIEA / DMF system; The modification of the Lys(AEEA-AEEA-γ-Glu-OtBu) site in segment 1 is as follows: Fmoc-Lys(AEEA)-OH is first connected to the resin, the terminal amino group of the AEEA side chain is protected by Alloc; after Alloc is removed, the second AEEA unit is connected, and finally γ-Glu(C20)-OtBu is introduced.

6. The method for preparing telpoeptide according to claim 1, characterized in that: In S2, the coupling reagent used for solid-phase synthesis of segment 2 is a mixture of HBTU, HOBt and DIEA, the molar ratio of Fmoc-protected amino acid, HATU, HOAt and DIEA is (0.8-1.2):(1-1.2):(1-1.2):(2.8-3.2).

7. The method for preparing telpoeptide according to claim 1, characterized in that: In S2, the coupling reagent used for solid-phase synthesis of fragment 3 is a mixture of HATU, HOAt and DIEA, and the molar ratio of Fmoc-protected amino acid, HATU, HOAt and DIEA is (0.8-1.2) : (1-1.2) : (1-1.2) : (2.8-3.2).

8. A process for the preparation of a thymopentin according to claim 1, characterized in that: In S3 and S4, the condensing reagent used for fragment condensation is a mixture of PyBOP, HOAt and DIEA, and the molar ratio of PyBOP, HOAt and DIEA is (0.8-1.2) : (0.8-1.2) : (1.8-2.2). The reaction solvent for fragment condensation is a mixture of DMF and CH2Cl2, and the molar ratio of DMF and CH2Cl2 is (0.8-1.2) : (0.8-1.2).

9. The method for preparing telpoeptide according to claim 1, characterized in that: In S3, the condensation of fragment 3 and fragment 2 is carried out at 25℃ for 2 hours. In S4, the condensation of fragment A and fragment 1 is carried out at 37℃ for 4 hours.

10. The method for preparing telpoeptide according to claim 1, characterized in that: In S5, the global deprotection is carried out at 0℃ for 1 hour and then at 25℃ for 1 hour, and the deprotection reagent used is a mixture of TFA, TIS, EDT and anisole, and the volume ratio of TFA, TIS, EDT and anisole is 92:2:3:

3. The oxidative folding is carried out by primary folding in a phosphate buffer at 25℃ and pH=7.0 for 2 hours, then a mixture of GSH and GSSG is added, and oxidative folding is carried out at 37℃ for 4 hours; finally, the temperature is lowered to 15℃, the pH is adjusted to 7.5, and the system is kept for 12 hours for stabilization. The purification is carried out by hydrophobic chromatography for pre-purification, RP-HPLC for refinement, and molecular exclusion chromatography for removal of polymers.

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