Method for preparing linaclotide

Abstract

The invention discloses a method for preparing linaclotide, belongs to the field of polypeptide medicine synthesis, and particularly relates to a method for industrially preparing linaclotide. An amino acid Fmoc-Tyr-OtBu side chain is anchored to a 2-CTC resin carrier through an ether bond, and then subsequent solid-phase synthesis, cutting, oxidation, purification, concentration and freeze drying are performed to prepare linaclotide. Compared with a traditional ester bond anchoring mode, the method has the advantages that the ether bond anchoring resin carrier is adopted, so that the stability of 2-CTC in the subsequent reaction is effectively improved, and the synthesis yield is not reduced due to extension of a peptide chain; meanwhile, impurities such as deleted peptides generated due to unstable carriers in the synthesis process are reduced. The method disclosed by the invention has the advantages of high stability and high product quality level, and is suitable for large-scale linaclotide production.

Description

Technical Field

[0001] The present invention belongs to the field of polypeptide pharmaceutical synthesis, and particularly relates to a method for preparing linaclotide. Background Art

[0002] Irritable Bowel Syndrome (IBS) is a common functional gastrointestinal disease clinically, with a relatively high prevalence rate in the population and an upward trend, bringing a heavy economic burden to society, families and individuals. Among them, constipation-predominant irritable bowel syndrome (IBS-C) is mainly characterized by difficult defecation and discomfort during defecation. In China, IBS-C accounts for about 15.1% of the total number of IBS patients.

[0003] Linaclotide is a novel, orally administered guanylate cyclase-C (GC-C) agonist in intestinal epithelial cells. The first synthetic GC-C agonist, linaclotide, demonstrated its ability to improve fecal hardness and quality in a Phase I clinical trial in 2005. It was approved by the US FDA and the European EMA for the treatment of adult IBS-C in 2012 and was launched in China in 2019. The results of multiple clinical trials show that linaclotide treatment can significantly improve the symptoms of abdominal pain, abdominal discomfort and constipation in IBS-C patients, and improve the quality of life of patients.

[0004] Linaclotide is a polypeptide containing 14 amino acids (the structure is shown in Figure 1 ), containing 3 disulfide bonds. It enhances the stability of the tertiary structure and at the same time enhances the affinity for receptor binding. Since linaclotide lacks acid-sensitive residues, its binding to GC-C in the gastrointestinal tract is not affected by the pH value. The special structure improves the pharmacodynamic and pharmacokinetic stability of linaclotide.

[0005] The structure of linaclotide is shown as follows: .

[0006] Linaclotide has become a global drug for the treatment of constipation-predominant irritable bowel syndrome.

[0007] The literature "Peptide Science, 2011, 96, 69 - 80" first reported that all side chains of Cys were protected with Trt, and linear peptide crude peptides were obtained through solid-phase synthesis by anchoring the main chain of amino acid Fmoc-Tyr(tBu)-OH on the resin, followed by cleavage. Then, linaclotide was obtained through a one-step liquid-phase oxidation method. The synthesis route and experimental results were based on the small-scale level and did not involve purification and separation steps, and the process had not reached the level of commercial production technology. In patent CN102875655A, the side chain of Cys was protected with Mmt, and linear peptide crude peptides were obtained through solid-phase synthesis by anchoring the main chain of amino acid Fmoc-Tyr(tBu)-OH on the resin, followed by cleavage. Then, crude linaclotide was obtained through a one-step liquid-phase oxidation method, and the small-scale linaclotide finished product was obtained after purification. In patent CN104231051A, the side chain of Cys was protected with Trt or Mmt, and linear peptide crude peptides were obtained through solid-phase synthesis by anchoring the main chain of amino acid Fmoc-Tyr(tBu)-OH on the resin, followed by cleavage. Then, crude linaclotide was obtained through a one-step liquid-phase oxidation method, and the pilot-scale linaclotide finished product was obtained after purification. CN104628826A, CN104844693A, CN109311941A, CN105884864A, CN106008674A, CN109053863A also obtained linaclotide crude peptides through solid-phase synthesis by anchoring the main chain carboxyl group of amino acid Fmoc-Tyr(tBu)-OH on the resin, followed by cleavage, and then obtained linaclotide crude peptides through a one-step liquid-phase oxidation method, and the linaclotide finished products were obtained after purification, with varying yields.

[0008] CN103626849A, CN105017387A, CN106892968A, CN113956333A, CN104974229A, CN106831950A, CN113861274A, CN113754735A, etc. obtained linaclotide crude peptides through multi-step oxidation methods such as solid-phase synthesis by anchoring the main chain of amino acid Fmoc-Tyr(tBu)-OH on the resin, followed by stepwise solid-phase oxidation or liquid-phase oxidation after cleavage, and the linaclotide finished products were obtained after purification.

[0009] CN104163853A synthesized the main chain of linaclotide through solid-phase synthesis of polypeptide fragments by anchoring the main chain carboxyl group of amino acid Fmoc-Tyr(tBu)-OH on the resin, followed by thiolester exchange of two polypeptide fragments in the liquid phase and S-N acyl migration. Then, the Fmoc protecting group was removed and cleavage was carried out to obtain linear peptide crude peptides. Then, linaclotide crude peptides were obtained through a one-step liquid-phase oxidation method, and the linaclotide finished products were obtained after purification.

[0010] The above routes or processes all have their respective innovative points. Some of the processes have reached the pilot scale, which is of practical significance for their respective claims. As can be seen from the above review, all patents including the literature use the carboxyl group of Fmoc-Tyr(tBu)-OH to participate in the reaction for the first step of anchoring between the amino acid and the solid-phase carrier. From the synthetic routes, the routes adopted by these patents are basically the same and have great similarities.

[0011] Especially in the solid-phase synthesis process using 2-CTC resin as the carrier, under alkaline conditions, after the carboxyl group of Fmoc-Tyr(tBu)-OH is ionized, it undergoes a nucleophilic substitution reaction with the C-Cl bond of 2-CTC resin, and the first amino acid is anchored to the 2-CTC resin through an ester bond. Due to the particularity of the trityl ester in the structure of 2-CTC resin, the stability of this ester bond is poor, resulting in the slow hydrolysis and dropping of this ester bond during each subsequent amino acid coupling reaction and post-treatment process, leading to the gradual loss of each intermediate product and the continuous increase of impurities, and ultimately affecting the yield and quality of the product. In addition, the literature Org. Process Res. Dev. 2021, 25, 250−261 reported that 2-CTC Resin introduced impurity peptides.

[0012] Therefore, the solid-phase synthesis of 2-CTC resin by carboxyl anchoring is generally not used for the synthesis of long peptides. Summary of the Invention

[0013] The purpose of the present invention is to provide a method for preparing linaclotide with high purity, good yield, low content of impurity peptides, stable yield, and capable of large-scale production.

[0014] The technical solution adopted by the present invention to achieve the above purpose is as follows: A preparation method of an amino acid ether bond-linked CTC resin, comprising: mixing a hydroxyl-containing amino acid with 2-CTC resin, and reacting to generate an ether bond-anchored CTC resin, that is, an amino acid ether bond-linked CTC resin.

[0015] Preferably, the hydroxyl-containing amino acid is Fmoc-Tyr(O-Resin)-OtBu.

[0016] Preferably, in the preparation of the amino acid ether bond-linked CTC resin, DCM and / or a base are also used.

[0017] More preferably, the base is DIEA or NMN, N(Et)3, etc., and preferably DIEA.

[0018] Preferably, in the preparation of the amino acid ether bond-linked CTC resin, the reaction temperature is 15-30 °C, and preferably 20-25 °C.

[0019] Preferably, DCM, a base, Fmoc-Tyr-OtBu and 2-CTC resin are mixed, and after reaction, Fmoc-Tyr(O-Resin)-OtBu is obtained.

[0020] Preferably, in the preparation of the CTC resin linked by an ether bond, 2-CTC resin and Fmoc-Tyr-OtBu are mixed in DCM, and a DIEA-DCM solution is added at 20-30 °C and reacted for 2-9 h, then methanol is added and the reaction continues for 10-60 min. After the reaction is completed, the liquid is removed by suction filtration, and washed successively with DMF, DCM and MTBE, and dried to obtain Fmoc-Tyr(O-Resin)-OtBu, that is, the CTC resin linked by an ether bond.

[0021] More preferably, in the preparation of the CTC resin linked by an ether bond, the substitution degree of 2-CTC resin is 0.4-1.4 mmol / g.

[0022] More preferably, in the preparation of the CTC resin linked by an ether bond, the molar amount of Fmoc-Tyr-OtBu used is 100-300% of the molar amount of the active sites on 2-CTC resin.

[0023] More preferably, in the preparation of the CTC resin linked by an ether bond, the amount of DCM used is 600-1000 wt% of 2-CTC resin.

[0024] More preferably, in the preparation of the CTC resin linked by an ether bond, the DIEA-DCM solution is prepared by mixing DIEA and DCM in a volume ratio of 1.36:1-5.

[0025] More preferably, in the preparation of the CTC resin linked by an ether bond, the amount of the DIEA-DCM solution used is 20-40 vol% of DCM.

[0026] More preferably, in the preparation of the CTC resin linked by an ether bond, the amount of methanol used is 5-15 vol% of DCM.

[0027] More preferably, in the preparation of the CTC resin linked by an ether bond, DMF, DCM and MTBE are used in appropriate amounts during washing.

[0028] The present invention discloses an amino acid CTC resin linked by an ether bond, including: using 2-CTC resin as a solid-phase carrier, an amino acid is linked through an ether bond, and the amino acid has an amino protecting group, a carboxylic acid protecting group and a hydroxyl group that forms an ether bond with 2-CTC resin.

[0029] Preferably, the amino protecting group is an Fmoc group; or, the carboxylic acid protecting group is an OtBu; or, the amino acid CTC resin linked by an ether bond is Fmoc-Tyr(O-Resin)-OtBu.

[0030] The present invention discloses a linaclotide-CTC resin, comprising: the above-mentioned amino acid ether bond-linked 2-CTC resin, wherein the amino protecting group of the amino acid is replaced with a linear polypeptide other than Tyr.

[0031] Preferably, the linear polypeptide other than Tyr is H-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt); or, the linaclotide-CTC resin is H-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(O-CTC Resin)-OtBu.

[0032] The present invention discloses a preparation method of a linaclotide-CTC resin, comprising: the preparation method of the above-mentioned amino acid ether bond-linked CTC resin.

[0033] Preferably, in the preparation of the linaclotide-CTC resin, Fmoc-Tyr(O-Resin)-OtBu is mixed with DMF and swollen for 10 - 60 min. After swelling, suction filtration is carried out, and a deprotection solution is added to remove Fmoc. After washing, an amino acid reagent and a condensation reagent are added for coupling. The deprotection and coupling of the amino acid reagent are repeated according to the peptide sequence of linaclotide to obtain H-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)- Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(O-Resin)-OtBu, that is, the linaclotide-CTC resin.

[0034] More preferably, in the preparation of the linaclotide-CTC resin, in the swelling treatment, it is only necessary to make DMF submerge Fmoc-Tyr(O-Resin)-OtBu.

[0035] More preferably, in the preparation of the linaclotide-CTC resin, the deprotection solution is composed of piperidine and DMF, and the deprotection solution contains 10 - 20 vol% of piperidine. In the use of the deprotection solution, it is only necessary to submerge the peptide resin.

[0036] More preferably, in the preparation of linaclotide-CTC resin, the amino acid reagents include Fmoc-Cys(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH.

[0037] More preferably, in the preparation of linaclotide-CTC resin, the coupling order of the amino acid reagents is: Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH. The molar amount of the amino acid reagents used is the same as that of Fmoc-Tyr-OtBu used in the preparation of the ether bond-linked CTC resin.

[0038] More preferably, in the preparation of linaclotide-CTC resin, the condensation reagents include HOBt, DIC and DMAP. The molar amounts of HOBt and DIC used are the same as that of Fmoc-Tyr-OtBu used in the preparation of the ether bond-linked CTC resin, and the molar amount of DMAP used is 5-10% of that of Fmoc-Tyr-OtBu used in the preparation of the ether bond-linked CTC resin.

[0039] More preferably, in the preparation of linaclotide-CTC resin, the solvent can be DMF, DCM or a mixture of the two.

[0040] The present invention discloses a method for preparing linaclotide linear crude peptide, including: the above-mentioned method for preparing an amino acid ether bond-linked CTC resin; or, the above-mentioned method for preparing linaclotide-CTC resin.

[0041] Preferably, in the preparation of linaclotide crude peptide, the linaclotide-CTC resin is dried and then cleaved to obtain linaclotide linear crude peptide, and then the linaclotide linear crude peptide is taken and oxidized with DMSO in liquid phase to form three pairs of disulfide bonds and purified by HPLC to obtain linaclotide crude peptide.

[0042] The present invention discloses a method for preparing linaclotide, including: the above-mentioned method for preparing an amino acid ether bond-linked CTC resin; or, the above-mentioned method for preparing linaclotide-CTC resin.

[0043] Preferably, in the purification of linaclotide linear crude peptide, linaclotide linear crude peptide is added into an ammonium acetate solution, the pH is adjusted to 9.0 - 9.5 with ammonia water, then DMSO is added, and cyclization treatment is started at 25 - 35 °C for 45 - 60 h, followed by filtration to obtain a linaclotide crude peptide solution, which is purified by HPLC to obtain linaclotide crude peptide.

[0044] In the present invention, the deprotection solution is a DMF solution containing 15 - 25% piperidine or a DMF solution containing 0.1 - 2% DBU or a mixed solution of DBU and piperidine. The reaction temperature is 5 - 30 °C, preferably 15 - 25 °C.

[0045] In the present invention, the condensation reagent includes HOBt / DIC, PyBop / DIEA, TBTU / HOBt / DIEA, HBTU / HOBt / DIEA or HATU / HOAt / DIEA. The HOBt / DIC system is preferred. The activation reaction temperature is 0 - 20 °C, preferably 0 - 15 °C. The coupling reaction temperature is 20 - 35 °C.

[0046] The cleavage solution is a combination of several components such as TFA, EDT, DODT, TIS, phenol or H2O, preferably a combination of TFA, EDT and H2O. The reaction temperature is 5 - 30 °C, preferably 15 - 25 °C.

[0047] In the present invention, the cleavage solution for cleaving linaclotide - CTC resin includes TFA, EDT and H2O, and the volume ratio of TFA, EDT and H2O in the cleavage solution is 1:0.27:0.07. The cleavage solution is used in an appropriate amount. The cleavage conditions are: cleavage treatment is carried out at a temperature of 5 - 25 °C for 3 h. After cleavage is completed, the cleavage solution is transferred to MTBE, and sedimentation is carried out at a temperature of 5 - 35 °C. After sedimentation, centrifugal separation is carried out to obtain a solid particle product. The solid particle product is slurried, washed and centrifuged with MTBE, and finally dried to obtain linaclotide linear crude peptide.

[0048] The present invention discloses the use of an amino acid ether - linked CTC resin in the preparation of linaclotide.

[0049] The present invention discloses the use of a linaclotide - CTC resin in the preparation of linaclotide.

[0050] The present invention also discloses the solid-phase synthesis of linaclotide on CTC resin. Fmoc-Tyr(tBu)-CTC Resin is mixed with DMF and swollen for 10 - 60 min. After swelling, suction filtration is carried out. A deprotection solution is added for deprotection treatment. After washing, an amino acid reagent and a coupling reagent are added for coupling. After coupling is completed, washing is carried out. Then, the steps of deprotection, coupling, and washing after coupling are repeated according to the peptide sequence of linaclotide to obtain H-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-CTC Resin. After drying, cleavage treatment is carried out to obtain the linear crude peptide of linaclotide. Then, the linear crude peptide of linaclotide is used to oxidize three pairs of disulfide bonds in DMSO by liquid phase, and purified by HPLC to obtain the crude peptide of linaclotide.

[0051] Preferably, the substitution degree of 2-CTC resin in Fmoc-Tyr(tBu)-CTC Resin is 0.4 - 1.4 mmol / g. When preparing Fmoc-Tyr(tBu)-CTC Resin, the molar amount of Fmoc-Tyr(tBu)-OH used is 100 - 300% of the molar amount of active sites on 2-CTC resin, and the molar amount of Fmoc-Tyr(tBu)-OH used is consistent with the molar amount of the amino acid reagent used in subsequent coupling. The coupling reagent includes HOBt, DIC, and DMAP. The molar amounts of HOBt and DIC used are consistent with the molar amount of Fmoc-Tyr(tBu)-OH used, and the molar amount of DMAP used is 5 - 10% of the molar amount of Fmoc-Tyr(tBu)-OH used. The solvent can be DMF, DCM, or a mixture of the two.

[0052] The present invention also discloses the solid-phase synthesis of linaclotide. Fmoc-Tyr(tBu)-Wang Resin is mixed with DMF and swollen for 10 - 60 min. After swelling, suction filtration is performed, and deprotection treatment is carried out by adding a deprotection solution. After washing, an amino acid reagent and a condensation reagent are added for coupling. After the coupling is completed, washing is carried out, and then the steps of deprotection, coupling, and washing after coupling are repeated according to the peptide sequence of linaclotide to obtain H-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-Wang Resin. After drying, cleavage treatment is carried out to obtain the linear crude peptide of linaclotide; then, the linear crude peptide of linaclotide is used to oxidize three pairs of disulfide bonds in DMSO by liquid phase, and purified by HPLC to obtain the crude peptide of linaclotide.

[0053] Preferably, the substitution degree of Wang Resin in Fmoc-Tyr(tBu)-Wang Resin is 0.4 - 1.4 mmol / g. When preparing Fmoc-Tyr(tBu)-Wang Resin, the molar amount of Fmoc-Tyr(tBu)-OH used is 100 - 300% of the molar amount of active sites on Wang Resin, and the molar amount of Fmoc-Tyr(tBu)-OH used is consistent with the molar amount of the amino acid reagent used in the subsequent coupling. The condensation reagent includes HOBt, DIC, and DMAP. The molar amounts of HOBt and DIC used are consistent with the molar amount of Fmoc-Tyr(tBu)-OH used, and the molar amount of DMAP used is 5 - 10% of the molar amount of Fmoc-Tyr(tBu)-OH used. The solvent can be DMF, DCM, or a mixture of the two.

[0054] The present invention first uses the side-chain phenolic hydroxyl group of Fmoc-Tyr-OtBu to carry out the solid-phase anchoring reaction of the first amino acid with 2-CTC resin under alkaline conditions. The phenoxide anion formed by the phenolic hydroxyl group under alkaline conditions undergoes a nucleophilic substitution reaction with the C-Cl bond of 2-CTC resin, and the first amino acid is anchored to 2-CTC resin through an ether bond. Due to the excellent stability of the ether bond, the intermediate obtained by this method shows good stability in the subsequent amino acid coupling reaction and post-treatment operation, and no continuous degradation side reactions occur. It ensures a more stable production process, excellent yield, and higher purity for the synthesis of linaclotide using 2-CTC resin. Therefore, the present invention is a method for preparing linaclotide with high purity, good yield, low content of impurity peptides, stable yield, and can be produced on a large scale. Description of the Drawings

[0055] Figure 1 It is a schematic diagram for the synthesis of linaclotide.

[0056] Figure 2 It is an HPLC chart. Detailed implementation manners

[0057] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0058] First, the concepts involved in the present application will be described in conjunction with the accompanying drawings. It should be noted here that the following descriptions of each concept are only for making the content of the present application easier to understand, and do not represent a limitation on the protection scope of the present application; at the same time, without conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in conjunction with the embodiments.

[0059] The present invention provides a method for preparing linaclotide, which includes the following steps: (1) Under weakly alkaline conditions, Fmoc-Tyr-OtBu is subjected to a substitution reaction with a 2-CTC resin carrier to obtain Fmoc-Tyr(O-Resin)-OtBu.

[0060] (2) Under the action of a deprotection solution, Fmoc-Tyr(O-Resin)-OtBu removes the Fmoc protecting group to obtain H-Tyr(O-Resin)-OtBu.

[0061] (3) Under an activation system, the protected amino acid Fmoc-Cys(Trt)-OH is coupled with H-Tyr(O-Resin)-OtBu to obtain Fmoc-Cys(Trt)-Tyr(O-Resin)-OtBu.

[0062] (4) Repeat the operations in the above steps (2) and (3), and couple them one by one in sequence according to the remaining amino acids of the linaclotide sequence from the carboxyl terminus to the amino terminus. Fmoc-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(O-Resin)-OtBu is obtained.

[0063] (5) Under the action of the deprotection solution, the Fmoc group of Fmoc-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(O-Resin)-OtBu is removed to obtain H-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(O-Resin)-OtBu.

[0064] (6) Under the action of the cleavage solution, all the protecting groups and the resin carrier of H-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(O-Resin)-OtBu are removed to obtain the crude linear peptide of linaclotide H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH.

[0065] (7) After the crude linear peptide of linaclotide H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH is dissolved in the ammonium sulfate solution, it is oxidized by DMSO, and after the reaction, a crude peptide solution of linaclotide H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH (1,6 disulfide bond, 2,10 disulfide bond, 5,13 disulfide bond) is obtained.

[0066] (8) The crude peptide solution of linaclotide is first purified by preparative chromatography to obtain the first purified intermediate.

[0067] (9) The first purified intermediate of linaclotide is secondarily purified by preparative chromatography to obtain the second purified intermediate.

[0068] (10) The second purified intermediate of linaclotide is tertiarily purified by preparative chromatography to obtain the third purified intermediate.

[0069] (11) The third purified intermediate of linaclotide is concentrated by preparative chromatography and freeze-dried to obtain the finished product of linaclotide.

[0070] Among them: For the 2-CTC resin used in step (1), the substitution degree ranges from 0.4 to 1.4 mmol / g, and the preferred range is 0.9 to 1.1 mmol / g. For the reaction of Fmoc-Tyr-OtBu anchored to 2-CTC resin, the solvent is DCM, and the bases used are DIEA or NMN, etc., preferably DIEA. Relative to the feeding amount of 2-CTC resin, the feeding ratio of Fmoc-Tyr-OtBu is 1.5 - 3.0 eq, and the preferred ratio is 2.0 eq. Relative to the feeding amount of 2-CTC resin, the feeding ratio of the base is 3.0 - 6.0 eq, and the preferred ratio is 4.5 - 6.0 eq. The reaction temperature is 15 - 30 °C, preferably 25 °C. The reaction time is 2 - 6 h, preferably 4 h. After the anchoring reaction is completed, methanol is added for the capping reaction of 2-CTC resin for 30 min, and then the resin is washed 4 times with DMF.

[0071] For steps (2) and (5), the deprotection solution is a DMF solution containing 15 - 25% piperidine, preferably a DMF solution of 20 vol% piperidine. The deprotection reaction temperature is 5 - 30 °C, preferably 25 °C. The reaction time is 1 - 4 h, preferably 3 h. After the deprotection is completed, the resin is washed 6 times with DMF.

[0072] The condensation reagents for the coupling of protected amino acids in step (3) include one or a combination of activation systems such as HOBt, 6-Cl-HOBt, DIC, PyBop, HBTU, HATU, HOAt, DIEA. The preferred is the HOBt / DIC system. Among them, the resin feeding amount : protected amino acid = between 1:2 and 1:3; protected amino acid : HOBt : DIC = 1:1:1. After the activation of the protected amino acid is completed, it is added to the peptide resin obtained in the previous step for the coupling reaction. The reaction temperature is 20 - 35 °C, preferably 30 °C, and the reaction time is 1 - 4 h, preferably 3 h. After the reaction is completed, the resin is washed 4 times with DMF. DMAP is also included in the condensation reagent.

[0073] The protecting group strategies of the protected amino acids used in steps (3) and (4) are within the known range. The preferred strategies are as follows: Fmoc-Cys(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH.

[0074] In step (6), the cutting fluid is a combination of several components among TFA, EDT, DODT, or H2O. Among them, calculated by volume, the content of TFA is 70 - 80%; either EDT or DODT is selected, and the content is 15 - 25%; the content of H2O is 3 - 10%. The composition and volume ratio of the cutting fluid are preferably: TFA:EDT:H2O = 75:20:5. The feeding ratio of the cutting fluid is: the feeding amount of peptide resin (g): the volume of the cutting fluid (ml) = 1 g:8 ml to 1 g:12 ml, and the preferred ratio is: the feeding amount of peptide resin (g): the volume of the cutting fluid (ml) = 1 g:10 ml. The cutting reaction temperature is 5 - 30°C, preferably 25°C. The cutting reaction time is 1 - 4 h, preferably 3 h.

[0075] In step (7), the concentration of the linaclotide linear peptide solution is 0.5 - 1.5 g / L, and the solvent is an aqueous solution of ammonium sulfate, ammonium acetate, or guanidine hydrochloride at 4 - 10 g / L, preferably an ammonium acetate solution. After the solution is prepared, the pH value is adjusted to 8 - 10 with ammonia water, preferably pH 9. Then, 4 - 10% of DMSO, preferably 5%, is added to the volume of the solution. The reaction temperature is 15 - 35°C, preferably 30°C. The reaction is carried out for 45 - 60 h, preferably 50 h.

[0076] In step (8), the chromatographic purification packing material for the first purification is C18 packing material, and the detection wavelength is 210 - 220 nm. The mobile phase for chromatographic purification uses the well-known TFA solution - acetonitrile system, and the concentration range of the TFA solution is 0.05 - 0.15%, preferably 0.1%.

[0077] In step (9), the chromatographic purification packing material for the second purification is C18 packing material, and the detection wavelength is 210 - 220 nm. The mobile phase for chromatographic purification uses the well-known triethylamine phosphate solution (TEAP) - acetonitrile system. The pH of the triethylamine phosphate solution is 2.0 - 3.0, preferably pH 2.5.

[0078] In step (10), the packing material for the third chromatographic purification is C18 packing material, and the detection wavelength is 210 - 220 nm. The mobile phase for chromatographic purification uses the well-known acetic acid - ammonium acetate solution - acetonitrile system. The concentration range of acetic acid in the acetic acid - ammonium acetate solution is 0.1 - 0.5 wt%, preferably 0.3 wt%; the concentration range of ammonium acetate in the acetic acid - ammonium acetate solution is 2 - 5 wt%, preferably 3.1 wt%.

[0079] In step (11), the packing material for the fourth chromatographic purification is C18 packing material, and the detection wavelength is 210 - 220 nm. The mobile phase for chromatographic purification uses the water - acetonitrile system. The sample loading amount is 300 - 500 g / needle calculated based on linaclotide, preferably 400 g / needle.

[0080] In step (12), the column concentration intermediate is concentrated by vacuum distillation under known conditions. The temperature of the intermediate solution is controlled below 25°C and concentrated to 20 - 40% of the original volume. The concentrated product is freeze-dried under known freeze-drying conditions to obtain the finished product of linaclotide.

[0081] After the cleavage reaction is completed, the cleavage solution is filtered. The obtained filtrate is added to an ether solvent for precipitation. The obtained precipitate is separated by solid-liquid separation and dried to obtain the linear crude peptide of linaclotide. The ether can be ethyl ether, isopropyl ether or tert-butyl methyl ether, preferably tert-butyl methyl ether. The precipitation temperature is below 40°C. The volume ratio of the cleavage solution to the ether solvent during the precipitation process is: cleavage solution volume: ether solvent volume = 1:8 to 1:12, preferably cleavage solution volume: ether solvent volume = 1:10.

[0082] After the precipitate is separated by solid-liquid separation, it is dried under reduced pressure. The drying temperature is 30 - 50°C, preferably 50°C. The linear crude peptide of linaclotide is obtained after drying.

[0083] The HPLC purity of the linear crude peptide of linaclotide synthesized by the method of the present invention is 80 - 90%, and the peptide content is 65 - 75%. The purity of the crude peptide solution of linaclotide reaches 70 - 75%. After purification, concentration and freeze-drying, the purity reaches more than 99.8%, the maximum single impurity does not exceed 0.10%, and the total yield is 25 - 35%. The production batch can reach 500 - 600 g / batch, and the product batch and quality fully meet the requirements of commercial production.

[0084] Example 1: A method for preparing linaclotide Solid-phase synthesis of linaclotide on CTC resin: Mix Fmoc-Tyr(tBu)-CTC Resin with DMF and swell for 30 min. After swelling, filter by suction, add deprotection solution for deprotection treatment. After washing, add amino acid reagent and condensation reagent for coupling. After coupling, wash, and then repeat the steps of deprotection, coupling, and washing after coupling according to the peptide sequence of linaclotide to obtain H-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-CTC Resin. After drying, perform cleavage treatment to obtain the linear crude peptide of linaclotide, H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH; then take the linear crude peptide of linaclotide and oxidize three pairs of disulfide bonds in DMSO liquid phase, and purify by HPLC to obtain the crude peptide of linaclotide. The substitution degree of 2-CTC resin in Fmoc-Tyr(tBu)-CTC Resin is 0.625 mmol / g, the amount of 2-CTC resin used is 3.20 g. When preparing Fmoc-Tyr(tBu)-CTC Resin, the molar amount of Fmoc-Tyr(tBu)-OH used is 4 mmol, and the molar amount of Fmoc-Tyr(tBu)-OH used is consistent with the molar amount of amino acid reagent used in subsequent coupling. The condensation reagent includes HOBt, DIC, and DMAP. The molar amounts of HOBt and DIC used are consistent with the molar amount of Fmoc-Tyr(tBu)-OH used, and the molar amount of DMAP used is 10% of the molar amount of Fmoc-Tyr(tBu)-OH used. The solvent can be DMF, DCM, or a mixture of both.

[0085] In Example 1 of the present invention, the yield of the linear crude peptide of linaclotide prepared is 1.84 g, the yield is 60.45%, and the purity after purification by HPLC is 67.68%.

[0086] The HPLC purity of the crude peptide of linaclotide prepared in Example 1 of the present invention is 41.49%.

[0087] The present invention analyzes the linear crude peptide of linaclotide prepared in Example 1, and the results are shown in Table 1 below.

[0088] Table 1 Analysis results of the content of the linear crude peptide of linaclotide

[0089] In Example 1 of the present invention, the yields of the peptide resin product and linear linaclotide finally obtained are much lower than the theoretical values because the ester bond formed between the C-terminal Tyr and 2-CTC resin is unstable during the solid-phase synthesis stage, and the peptide chain is prone to fall off from the resin end during the amino acid coupling process. Impurities with the first amino acid Tyr inserted and impurities lacking one or more amino acids at the C-terminus were detected in the linear crude peptide of the present invention.

[0090] Example 2: A method for preparing linaclotide Solid-phase synthesis of linaclotide on Wang resin: Mix Fmoc-Tyr(tBu)-Wang Resin with DMF and swell for 30 min. After swelling, filter by suction, add deprotection solution for deprotection treatment, wash, then add amino acid reagent and condensation reagent for coupling. After coupling, wash, and then repeat the steps of deprotection, coupling, and washing after coupling according to the peptide sequence of linaclotide to obtain H-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(tBu)-Wang Resin. After drying, perform cleavage treatment to obtain the linear crude peptide of linaclotide H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH; then take the linear crude peptide of linaclotide and oxidize the three pairs of disulfide bonds by DMSO in liquid phase and purify by HPLC to obtain the crude peptide of linaclotide. The substitution degree of Wang resin in Fmoc-Tyr(tBu)-Wang Resin is 0.48 mmol / g, the amount of Wang resin used is 4.15 g. When preparing Fmoc-Tyr(tBu)-Wang Resin, the molar amount of Fmoc-Tyr(tBu)-OH used is 4 mmol, and the molar amount of Fmoc-Tyr(tBu)-OH used is consistent with the molar amount of amino acid reagent used in the subsequent coupling. The condensation reagent includes HOBt, DIC, and DMAP. The molar amounts of HOBt and DIC used are consistent with the molar amount of Fmoc-Tyr(tBu)-OH used, and the molar amount of DMAP used is 10% of the molar amount of Fmoc-Tyr(tBu)-OH used. The solvent can be DMF, DCM, or a mixture of the two.

[0091] The yield of the linear crude peptide of linaclotide prepared in Example 2 of the present invention is 2.76 g, the yield is 90.20%, and the purity is 68.51% after purification by HPLC.

[0092] The HPLC purity of the crude linaclotide peptide prepared in Example 2 of the present invention is 43.75%.

[0093] The present invention analyzes the linear crude linaclotide peptide prepared in Example 2, and the results are shown in Table 2 below.

[0094] Table 2 Analysis results of the content of linear crude linaclotide peptide

[0095] In the present invention, the yields of the peptide resin and the linear peptide finally obtained in Example 2 are close to the theoretical values. Impurities lacking the two amino acids at the carbon terminus are also found in the linear crude peptide, which may be due to the formation of piperazine dione dropping during the coupling process; at the same time, due to difficulties in coupling and incomplete reactions during the amino acid coupling process, the impurity content in the linear crude peptide is also relatively large, and the HPLC purity of the linear crude peptide is not much different from that of Example 1.

[0096] Example 3: A method for preparing linaclotide Preparation of CTC resin linked by ether bond: Mix 2-CTC resin with Fmoc-Tyr-OtBu in DCM, add DIEA-DCM solution at 25 °C and react for 4.5 h, then add methanol and continue to react for 30 min. After the reaction is completed, filter off the liquid, wash successively with DMF, DCM and MTBE, and dry to obtain Fmoc-Tyr(O-Resin)-OtBu, that is, CTC resin linked by ether bond. The substitution degree of 2-CTC resin is 1.02 mmol / g, the amount of 2-CTC resin used is 30 g, the molar amount of Fmoc-Tyr-OtBu used is 200% of the molar amount of the active sites on 2-CTC resin, the amount of DCM used is 867 wt% of 2-CTC resin, the DIEA-DCM solution is composed of DIEA and DCM mixed at a volume ratio of 1.36:2, the amount of DIEA-DCM solution used is 30.77 vol% of DCM, and the amount of methanol used is 11.54 vol% of DCM. Appropriate amounts of DMF, DCM and MTBE are used in the washing.

[0097] Preparation of linaclotide-CTC resin: Fmoc-Tyr(O-Resin)-OtBu was mixed with DMF and swollen for 30 min. After swelling, suction filtration was carried out. Deprotecting solution was added to remove Fmoc. After washing, amino acid reagent and condensing reagent were added for coupling. Deprotection and coupling of amino acid reagent were repeated according to the peptide sequence of linaclotide to obtain H-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(O-Resin)-OtBu, namely linaclotide-CTC resin. During the swelling treatment, it was only necessary to submerge Fmoc-Tyr(O-Resin)-OtBu with DMF; the deprotecting solution was composed of piperidine and DMF, and the deprotecting solution contained 20 vol% piperidine. During the use of the deprotecting solution, it was only necessary to submerge the peptide resin; the coupling sequence of amino acid reagents was: Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH. The molar amount of amino acid reagent used was consistent with the molar amount of Fmoc-Tyr-OtBu used in the preparation of CTC resin connected by ether bond. The condensing reagents included HOBt, DIC and DMAP. The molar amounts of HOBt and DIC used were consistent with the molar amount of Fmoc-Tyr-OtBu used in the preparation of CTC resin connected by ether bond, and the molar amount of DMAP used was 10% of the molar amount of Fmoc-Tyr-OtBu used in the preparation of CTC resin connected by ether bond. The solvent could be DMF, DCM or a mixture of both.

[0098] Preparation of crude linaclotide: The linaclotide-CTC resin was dried and then cleaved to obtain the linear crude linaclotide H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH. Then, the linear crude linaclotide was taken and the three pairs of disulfide bonds were oxidized by DMSO in liquid phase, and purified by HPLC to obtain the crude linaclotide.

[0099] In Example 3 of the present invention, the yield of the prepared linear crude linaclotide was 2.84 g, the recovery rate was 97.28%, and the purity was 82.31% after purification by HPLC.

[0100] The HPLC purity of the crude linaclotide peptide prepared in Example 3 of the present invention is 75.38%.

[0101] No obvious impurity peptides with inserted or deleted amino acids were detected in the linear crude linaclotide peptide prepared in Example 3 of the present invention, and the yields of the peptide resin product and the linear crude linaclotide peptide are close to the theoretical values. The HPLC purity of the linear crude linaclotide peptide in the present invention is relatively high.

[0102] The present invention conducted cleavage tolerance tests and thermal tolerance tests on the Fmoc-Tyr(tBu)-CTC Resin prepared in Example 1, the Fmoc-Tyr(tBu)-Wang Resin prepared in Example 2, and the Fmoc-Tyr(O-Resin)-OtBu prepared in Example 3, and the results are shown in Table 3.

[0103] Table 3 Results of stability tests

[0104] In the tolerance test of the present invention, the results of the cleavage tolerance test show that the ether bond-linked Fmoc-Tyr(O-Resin)-OtBu prepared by the method of Example 3 of the present invention has good cleavage stability and good heat stability, and its stability under cleavage and heat conditions is better than that of the prior art, and the yield and purity are significantly improved, which can be used for industrial production. The active structure of Wang resin is a hydroxyl group. Therefore, it is difficult for the hydroxyl group on Wang resin to form an ether bond with Fmoc-Tyr-OtBu used in the present invention, and it cannot be used for the subsequent synthesis of linaclotide. The first amino acid in the present invention needs to be an amino acid with a hydroxyl group. If it does not have a hydroxyl group, the ether bond required in the present invention cannot be formed.

[0105] The method of the present invention can be applied at least to the synthesis of peptides with Tyr as the first amino acid.

[0106] The method of the present invention can also be applied to the synthesis of peptides containing a hydroxyl group in the first amino acid.

[0107] Example 4: A method for preparing linaclotide Preparation of Ether Bond-Linked CTC Resin: Mix 2-CTC resin with Fmoc-Tyr-OtBu in DCM, add DIEA-DCM solution at 25 °C and react for 4.5 h, then add methanol and continue to react for 30 min. After the reaction is completed, filter to remove the liquid, wash successively with DMF, DCM and MTBE, and dry to obtain Fmoc-Tyr(O-Resin)-OtBu, that is, ether bond-linked CTC resin. The substitution degree of 2-CTC resin is 0.98 mmol / g, the amount of 2-CTC resin used is 3061 g, the molar amount of Fmoc-Tyr-OtBu used is 200% of the molar amount of active sites on 2-CTC resin, the amount of DCM used is 867 wt% of 2-CTC resin, the DIEA-DCM solution is prepared by mixing DIEA and DCM in a volume ratio of 1.36:2, the amount of DIEA-DCM solution used is 30.77 vol% of DCM, and the amount of methanol used is 11.54 vol% of DCM. Appropriate amounts of DMF, DCM and MTBE are used in the washing.

[0108] Preparation of linaclotide-CTC resin: Fmoc-Tyr(O-Resin)-OtBu was mixed with DMF and swollen for 30 min. After swelling, suction filtration was carried out. Deprotecting solution was added to remove Fmoc. After washing, amino acid reagents and condensation reagents were added for coupling. The deprotection and coupling of amino acid reagents were repeated according to the peptide sequence of linaclotide to obtain H-Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr(O-Resin)-OtBu, namely linaclotide-CTC resin. During the swelling treatment, it was only necessary to immerse Fmoc-Tyr(O-Resin)-OtBu with DMF; the deprotecting solution was composed of piperidine and DMF, and the deprotecting solution contained 20 vol% of piperidine. During the use of the deprotecting solution, it was only necessary to immerse the peptide resin; the coupling sequence of amino acid reagents was: Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH. The molar amount of amino acid reagents used was consistent with the molar amount of Fmoc-Tyr-OtBu used in the preparation of ether bond-linked CTC resin. The condensation reagents included HOBt, DIC and DMAP. The molar amounts of HOBt and DIC used were consistent with the molar amount of Fmoc-Tyr-OtBu used in the preparation of ether bond-linked CTC resin. The molar amount of DMAP used was 10% of the molar amount of Fmoc-Tyr-OtBu used in the preparation of ether bond-linked CTC resin. Solvents such as DMF, DCM or a mixture of both could be used.

[0109] Preparation of crude linaclotide peptide: The linaclotide-CTC resin was dried and cleaved to obtain the linear crude linaclotide peptide H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH. Then, the linear crude linaclotide peptide was used to oxidize three pairs of disulfide bonds by DMSO liquid phase oxidation and purified by HPLC to obtain the crude linaclotide peptide.

[0110] In the purification of linaclotide linear crude peptide, linaclotide linear crude peptide was taken and added to an ammonium acetate solution, and the pH was adjusted to 9.0 with ammonia water. Then DMSO was added, and cyclization treatment was started at 25 °C for 60 h. After filtration, a linaclotide crude peptide solution was obtained, and it was purified by HPLC to obtain linaclotide crude peptide.

[0111] In Example 4 of the present invention, the yield of the prepared linaclotide crude peptide was 595 g, the total yield was 26.90%, the purity after HPLC purification was 99.89%, the total impurities were 0.11%, and the maximum single impurity was 0.07%. The HPLC chromatogram is as Figure 1 shown.

[0112] Example 4 of the present invention is a method for scaled-up production. In the method for preparing linaclotide with the same proportion, linaclotide with excellent purity and yield can also be obtained.

[0113] In the present invention, the cleavage solution for cleaving linaclotide-CTC resin includes TFA, EDT and H2O, and the volume ratio of TFA, EDT and H2O in the cleavage solution is 1:0.27:0.07. The cleavage solution is used in an appropriate amount. The cleavage conditions are as follows: cleavage treatment is carried out at a temperature of 25 °C for 3 h. After cleavage is completed, the cleavage solution is transferred to MTBE, and sedimentation is carried out at a temperature of 25 °C. After sedimentation, centrifugal separation is carried out to obtain a solid particle product. The solid particle product is slurried, washed and centrifuged with MTBE, and finally dried to obtain linaclotide linear crude peptide.

[0114] In the HPLC purification of the present invention, a linaclotide crude peptide solution was taken, converted to 400 g / needle of linaclotide for loading, loaded onto a chromatographic system for primary purification, the product of primary purification was loaded onto the chromatographic system for secondary purification, the product of secondary purification was loaded onto the chromatographic system for tertiary purification, and the product of tertiary purification was loaded onto the chromatographic system for quaternary purification.

[0115] The gradient elution program for primary purification is as follows:

[0116] The preparative chromatographic purification packing for primary purification is C18 packing, and the detection wavelength is 210 - 220 nm.

[0117] The gradient elution program for secondary purification is as follows:

[0118] The preparative chromatographic purification packing for secondary purification is C18 packing, and the detection wavelength is 210 - 220 nm.

[0119] The gradient elution program for tertiary purification is as follows:

[0120] The packing material for three - stage chromatographic purification is C18 packing material, and the detection wavelength is 210 - 220 nm.

[0121] The gradient elution procedure for four - stage purification is as follows:

[0122] The packing material for four - stage chromatographic purification is C18 packing material, and the detection wavelength is 210 - 220 nm.

[0123] In the present invention, the product solution obtained after four - stage purification is transferred to a rotary evaporation system for vacuum distillation and concentration. The solution temperature is controlled below 25°C, and it is concentrated to 20 - 40% of the original volume of the product solution, followed by freeze - drying to obtain crude linaloceptide. In the examples of the present invention, both Example 3 and Example 4 were subjected to four - stage purification. The HPLC purity given in Example 3 is the data for one - stage purification. Since the purity and yield of Example 1 and Example 2 were poor, only one - stage purification was carried out, that is, the HPLC purity given in Example 1 and Example 2 is the data for one - stage purification.

[0124] The HPLC chromatogram of linaloceptide in Example 4 of the present invention is as Figure 2 shown, and the results of the HPLC chromatogram are shown in Table 4 below.

[0125] Table 4 HPLC chromatogram results

[0126] The above - mentioned examples and / or embodiments are only used to illustrate the preferred examples and / or embodiments for implementing the technology of the present invention, and do not impose any formal restrictions on the implementation of the technology of the present invention. Any person skilled in the art, without departing from the scope of the technical means disclosed in the content of the present invention, may make some changes or modifications to other equivalent embodiments, but should still be regarded as the same technology or embodiment as the present invention in essence.

[0127] Specific examples are used in this article to elaborate on the principles and implementation methods of the present application. The description of the above examples is only used to help understand the method and its core idea of the present application. The above is only the preferred implementation method of the present application. It should be noted that due to the limitations of written expression and objectively existing infinite specific structures, for those of ordinary skill in the art, without departing from the principle of the present application, several improvements, refinements or changes can be made, or the above - mentioned technical features can be combined in an appropriate manner; these improvements, refinements, changes or combinations, or directly applying the concept and technical solution of the invention to other occasions without improvement, should all be regarded as the protection scope of the present application.

Claims

1. Preparation method of amino acid ether bond-linked 2-CTC resin, comprising: Mix the hydroxy - containing amino acid with 2 - CTC resin and react to form an ether - bond - anchored 2 - CTC resin, that is, the amino acid is ether - bond - linked to the 2 - CTC resin.

2. An amino acid ether bond-linked 2-CTC resin, comprising: Using 2 - CTC resin as a solid - phase carrier, an amino acid is linked through an ether bond. The amino acid has an amino - protecting group, a carboxylic - acid - protecting group, and a hydroxyl group that forms an ether bond with the 2 - CTC resin.

3. An amino acid ether bond-linked 2-CTC resin according to claim 2, characterized in that: The amino - protecting group is an Fmoc group; or, the carboxylic - acid - protecting group is OtBu; or, the amino - acid ether - bond - linked 2 - CTC resin is Fmoc - Tyr(O - Resin)-OtBu.

4. A linaclotide-CTC resin, comprising: For the amino - acid ether - bond - linked 2 - CTC resin described in claim 2, the amino - protecting group of the amino acid is replaced with a linear polypeptide other than Tyr.

5. A linaclotide-CTC resin according to claim 4, wherein: The linear polypeptide other than Tyr is H - Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro - Ala - Cys(Trt)-Thr(tBu)-Gly - Cys(Trt); or, the linaclotide - CTC resin is H - Cys(Trt)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Trt)-Asn(Trt)-Pro - Ala - Cys(Trt)-Thr(tBu)-Gly - Cys(Trt)-Tyr(O - CTC Resin)-OtBu.

6. A preparation method of linaclotide-CTC resin, comprising: A method for preparing an amino - acid ether - bond - linked 2 - CTC resin according to claim 1.

7. A method for preparing a linaclotide linear crude peptide, comprising: A method for preparing an amino - acid ether - bond - linked 2 - CTC resin according to claim 1; or, a method for preparing a linaclotide - CTC resin according to claim 6.

8. A preparation method of linaclotide, comprising: A method for preparing an amino - acid ether - bond - linked 2 - CTC resin according to claim 1; or, a method for preparing a linaclotide - CTC resin according to claim 6.

9. Use of an amino - acid ether - bond - linked 2 - CTC resin in the preparation of linaclotide.

10. Use of a linaclotide - CTC resin in the preparation of linaclotide.

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