A process for the preparation of linaclotide

Abstract

The application discloses a method for preparing linaclotide, and belongs to the field of polypeptide medicine synthesis, and particularly relates to a method for preparing linaclotide in an industrialized manner. In the method, the side chain of an amino acid Fmoc-Tyr-OtBu 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 carried out to prepare linaclotide. Compared with a traditional ester bond anchoring mode, the method adopts the ether bond to anchor the resin carrier, so that the stability of 2-CTC in the subsequent reaction is effectively improved, and the synthesis yield is not reduced due to the elongation of the peptide chain; meanwhile, the method reduces impurities such as missing peptides generated due to the instability of the carrier in the synthesis process. The production process in the method has high stability, the product quality level is high, and the method is suitable for large-scale linaclotide production.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of polypeptide medical synthesis, and particularly relates to a method for preparing linaclotide. BACKGROUND

[0002] Irritable bowel syndrome (IBS) is a common functional gastrointestinal disease in clinical practice, and the prevalence rate of the disease in the population is high and shows an upward trend, which has brought heavy economic burden to the society, family and individual. Constipation-predominant irritable bowel syndrome (IBS-C) is mainly characterized by difficult defecation and uncomfortable defecation, and the total number of IBS-C patients accounts for about 15.1% of the total number of IBS patients in China.

[0003] Linaclotide is a new type of oral intestinal epithelial cell guanylate cyclase-C (GC-C) agonist. In 2005, the first artificially synthesized GC-C agonist linaclotide proved that it could improve the stool hardness and quality in the I phase clinical trial, and was approved by the US FDA and the European EMA in 2012 for the treatment of adult IBS-C, and was listed in China in 2019. The results of a number of 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 (structure see Figure 1 ), containing three disulfide bonds. The stability of the tertiary structure is enhanced, and the affinity for receptor binding is also enhanced. Linaclotide lacks acid-sensitive residues, so its binding with GC-C in the gastrointestinal tract is not affected by pH. The special structure improves the pharmacodynamic and pharmacokinetic stability of linaclotide.

[0005] The structure of linaclotide is as follows:

[0006] .

[0007] Linaclotide has become a global drug for treating constipation-predominant irritable bowel syndrome.

[0008] The document Peptide Science, 2011, 96, 69-80 first reported that the Cys side chain was all protected by Trt protecting group, the linear peptide crude peptide was obtained by solid phase synthesis of the main chain anchored on the resin by amino acid Fmoc-Tyr(tBu)-OH, cutting, and then oxidized by one-step liquid phase oxidation to obtain linaclotide. The synthesis route and experimental results are based on small-scale level, and no purification and separation steps are involved. The process has not reached the level of commercial production process. In patent CN102875655A, the Cys side chain is protected by Mmt protecting group, the linear peptide crude peptide is obtained by solid phase synthesis of the main chain anchored on the resin by amino acid Fmoc-Tyr(tBu)-OH, cutting, and then oxidized by one-step liquid phase oxidation to obtain linaclotide crude peptide, and then purified to obtain linaclotide finished product at small-scale level. In patent CN104231051A, the Cys side chain is protected by Trt or Mmt protecting group, the linear peptide crude peptide is obtained by solid phase synthesis of the main chain anchored on the resin by amino acid Fmoc-Tyr(tBu)-OH, cutting, and then oxidized by one-step liquid phase oxidation to obtain linaclotide crude peptide, and then purified to obtain linaclotide finished product at pilot-scale level. In CN104628826A, CN104844693A, CN109311941A, CN105884864A, CN106008674A, CN109053863A, the linear peptide crude peptide is obtained by solid phase synthesis of the main chain anchored on the resin by amino acid Fmoc-Tyr(tBu)-OH, cutting, and then oxidized by one-step liquid phase oxidation to obtain linaclotide crude peptide, and then purified to obtain linaclotide finished product, with different yields.

[0009] CN103626849A, CN105017387A, CN106892968A, CN113956333A, CN104974229A, CN106831950A, CN113861274A, CN113754735A, etc. obtain linaclotide crude peptide by solid phase synthesis of the main chain anchored on the resin by amino acid Fmoc-Tyr(tBu)-OH, multi-step oxidation such as step-by-step solid phase oxidation or cutting followed by liquid phase oxidation, and then purify to obtain linaclotide finished product.

[0010] CN104163853A obtains linaclotide main chain by solid phase synthesis of polypeptide fragments with the main chain anchored on the resin by amino acid Fmoc-Tyr(tBu)-OH, liquid phase thioester exchange and S-N acyl shift of two polypeptide fragments, and then de-Fmoc protection, cutting to obtain linear peptide crude peptide, and then oxidized by one-step liquid phase oxidation to obtain linaclotide crude peptide, and then purified to obtain linaclotide finished product.

[0011] Each of the above routes or processes has its own innovative features, and some have reached pilot-scale production, providing substantial support for their respective claims. The above review also demonstrates that all patents, including those in the literature, utilize the carboxyl group of Fmoc-Tyr(tBu)-OH in the first anchoring step between the amino acid and the solid support. From a synthetic perspective, these patents employ essentially the same routes, demonstrating significant similarity.

[0012] In particular, in solid-phase synthesis processes using 2-CTC resin as a support, under alkaline conditions, the carboxyl group of Fmoc-Tyr(tBu)-OH ionizes and undergoes a nucleophilic substitution reaction with the C-Cl bond of the 2-CTC resin, anchoring the first amino acid to the 2-CTC resin via an ester bond. Due to the unique properties of the trityl ester in the 2-CTC resin structure, this ester bond is relatively unstable. This results in slow hydrolysis and degradation during each subsequent amino acid coupling reaction and post-processing, leading to the gradual loss of various intermediates and the accumulation of impurities, ultimately affecting product yield and quality. Furthermore, the literature Org. Process Res. Dev. 2021, 25, 250−261 reports the introduction of peptide impurities into 2-CTC resin.

[0013] Therefore, 2-CTC resin is generally not used for the synthesis of long peptides through solid phase synthesis via carboxyl anchoring. Summary of the Invention

[0014] The object 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 large-scale production.

[0015] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are:

[0016] A preparation method of amino acid ether bond-linked CTC resin comprises: mixing hydroxyl-containing amino acid with 2-CTC resin, reacting to generate ether bond-anchored CTC resin, i.e. amino acid ether bond-linked CTC resin.

[0017] Preferably, the hydroxyl-containing amino acid is Fmoc-Tyr(O-Resin)-OtBu.

[0018] Preferably, DCM and / or a base are also used in the preparation of amino acid ether-linked CTC resin.

[0019] More preferably, the base is DIEA, NMN, N(Et)3, etc., preferably DIEA.

[0020] Preferably, in the preparation of amino acid ether bonded CTC resin, the reaction temperature is 15-30°C, preferably 20-25°C.

[0021] Preferably, DCM, a base, Fmoc-Tyr-OtBu and 2-CTC resin are mixed to obtain Fmoc-Tyr(O-Resin)-OtBu after reaction.

[0022] Preferably, in the preparation of the ether-linked CTC resin, 2-CTC resin and Fmoc-Tyr-OtBu are mixed in DCM, DIEA-DCM solution is added at 20-30°C, and the reaction is continued for 2-9 hours, and then methanol is added to continue the reaction for 10-60 minutes. After the reaction is completed, the liquid is removed by filtration, and the resin is washed with DMF, DCM and MTBE in sequence and dried to obtain Fmoc-Tyr(O-Resin)-OtBu, i.e., the ether-linked CTC resin.

[0023] More preferably, in the preparation of the ether-linked CTC resin, the degree of substitution of the 2-CTC resin is 0.4-1.4 mmol / g.

[0024] More preferably, in the preparation of the ether-linked CTC resin, the molar amount of Fmoc-Tyr-OtBu used is 100-300% of the molar amount of active sites on the 2-CTC resin.

[0025] More preferably, in the preparation of ether-linked CTC resin, the amount of DCM used is 600-1000 wt % of the 2-CTC resin.

[0026] More preferably, in the preparation of the ether-linked CTC resin, the DIEA-DCM solution is prepared by mixing DIEA and DCM in a volume ratio of 1.36:1-5.

[0027] More preferably, in the preparation of the ether-linked CTC resin, the amount of DIEA-DCM solution used is 20-40 vol% of DCM.

[0028] More preferably, in the preparation of the ether-linked CTC resin, the amount of methanol used is 5-15 vol% of DCM.

[0029] More preferably, in the preparation of the ether-linked CTC resin, appropriate amounts of DMF, DCM and MTBE are used in the washing.

[0030] The invention discloses an amino acid ether bond-linked CTC resin, comprising: using 2-CTC resin as a solid phase carrier, connecting amino acids via ether bonds, wherein the amino acids have an amino protecting group, a carboxylic acid protecting group and a hydroxyl group forming an ether bond with the 2-CTC resin.

[0031] Preferably, the amino protecting group is an Fmoc group; or, the carboxylic acid protecting group is OtBu; or, the amino acid ether bond connecting the CTC resin is Fmoc-Tyr(O-Resin)-OtBu.

[0032] The present invention discloses a linaclotide-CTC resin, comprising: the above-mentioned amino acid ether bond connected to 2-CTC resin, and the amino protecting group of the amino acid is replaced by a linear polypeptide except Tyr.

[0033] 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, 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.

[0034] The present invention discloses a method for preparing linaclotide-CTC resin, comprising: the above-mentioned method for preparing CTC resin connected with an amino acid ether bond.

[0035] Preferably, in the preparation of linaclotide-CTC resin, Fmoc-Tyr(O-Resin)-OtBu is mixed with DMF and swelled for 10-60 minutes. After swelling is completed, the mixture is filtered, a deprotection solution is added to remove Fmoc, and 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, i.e., linaclotide-CTC resin.

[0036] More preferably, in the preparation of linaclotide-CTC resin, during the swelling treatment, DMF is allowed to submerge Fmoc-Tyr(O-Resin)-OtBu.

[0037] More preferably, in the preparation of linaclotide-CTC resin, the deprotection solution is prepared by mixing piperidine and DMF, the deprotection solution contains 10-20 vol% piperidine, and the deprotection solution is used to immerse the peptide resin.

[0038] More preferably, in the preparation of the Linatril-CTC resin, the amino acid reagent includes 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.

[0039] More preferably, in the preparation of the Linatril-CTC resin, the coupling order of the amino acid reagent 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 use molar amount of the amino acid reagent is consistent with the use molar amount of Fmoc-Tyr-OtBu in the preparation of the ether bond connected CTC resin.

[0040] More preferably, in the preparation of the Linatril-CTC resin, the condensation reagent includes HOBt, DIC and DMAP, the use molar amount of HOBt and DIC is consistent with the use molar amount of Fmoc-Tyr-OtBu in the preparation of the ether bond connected CTC resin, and the use molar amount of DMAP is 5-10% of the use molar amount of Fmoc-Tyr-OtBu in the preparation of the ether bond connected CTC resin.

[0041] More preferably, in the preparation of the Linatril-CTC resin, the solvent can use DMF, DCM or a mixture of the two.

[0042] The application discloses a preparation method of a linear Linatril crude peptide, comprising: the preparation method of the amino acid ether bond connected CTC resin or the preparation method of the Linatril-CTC resin.

[0043] Preferably, in the preparation of the Linatril crude peptide, the Linatril-CTC resin is dried and cleaved to obtain the linear Linatril crude peptide, then the linear Linatril crude peptide is taken and subjected to liquid phase oxidation of three pairs of disulfide bonds by DMSO, and HPLC purification to obtain the Linatril crude peptide.

[0044] The application discloses a preparation method of a linear Linatril crude peptide, comprising: the preparation method of the amino acid ether bond connected CTC resin or the preparation method of the Linatril-CTC resin.

[0045] Preferably, in the purification of crude linear linaclotide peptide, crude linear linaclotide peptide is added to an ammonium acetate solution, the pH is adjusted to 9.0-9.5 with aqueous ammonia, and then DMSO is added. The cyclization treatment is started at 25-35 ° C for 45-60 hours, filtered to obtain a crude linaclotide peptide solution, and purified by HPLC to obtain crude linaclotide peptide.

[0046] 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.

[0047] 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.

[0048] The cutting fluid 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.

[0049] The cutting solution for cutting the linaclotide-CTC resin in the present invention comprises TFA, EDT, and H₂O, with the volume ratio of TFA, EDT, and H₂O in the cutting solution being 1:0.27:0.07. The cutting solution is used in an appropriate amount. The cutting conditions are: cutting at a temperature of 5-25°C for 3 hours. After cutting, the cutting solution is transferred to MTBE and allowed to settle at a temperature of 5-35°C. After settling, the solid particles are centrifuged to obtain a solid particle product. The solid particle product is slurried with MTBE, washed, centrifuged, and finally dried to obtain a crude linear linaclotide peptide.

[0050] The present invention discloses an application of an amino acid ether bond-connected CTC resin in the preparation of linaclotide.

[0051] The invention discloses use of a linaclotide-CTC resin in the preparation of linaclotide.

[0052] The present invention also discloses a CTC resin solid phase synthesis of linaclotide. The steps include: mixing Fmoc-Tyr(tBu)-CTC Resin with DMF, swelling the mixture for 10-60 minutes, filtering the mixture after swelling, adding a deprotection solution for deprotection treatment, washing the mixture, adding an amino acid reagent and a condensation reagent for coupling, washing the mixture after coupling, and then repeating 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, the crude linaclotide peptide was cleaved to obtain a crude linear peptide of linaclotide; the crude linear peptide of linaclotide was then subjected to DMSO liquid phase oxidation of three pairs of disulfide bonds and purified by HPLC to obtain a crude linaclotide peptide.

[0053] Preferably, the degree of substitution of 2-CTC resin in Fmoc-Tyr(tBu)-CTC Resin is 0.4-1.4 mmol / g. During the preparation of Fmoc-Tyr(tBu)-CTC Resin, the molar amount of Fmoc-Tyr(tBu)-OH is 100-300% of the molar amount of active sites on the 2-CTC resin. 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. Condensation reagents include HOBt, DIC, and DMAP. The molar amounts of HOBt and DIC used are consistent with the molar amount of Fmoc-Tyr(tBu)-OH used. 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 also discloses a Wang resin solid-phase synthesis of linaclotide. The steps include: mixing Fmoc-Tyr(tBu)-Wang Resin with DMF, swelling the mixture for 10-60 minutes, filtering the mixture after swelling, adding a deprotection solution for deprotection treatment, washing the mixture, adding an amino acid reagent and a condensation reagent for coupling, washing the mixture after coupling, and then repeating 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, the crude linaclotide peptide was cleaved to obtain a crude linear peptide of linaclotide; the crude linear peptide of linaclotide was then subjected to DMSO liquid phase oxidation of three pairs of disulfide bonds and purified by HPLC to obtain a crude linaclotide peptide.

[0055] Preferably, the degree of substitution of Wang resin in Fmoc-Tyr(tBu)-Wang Resin is 0.4-1.4 mmol / g. During the preparation of Fmoc-Tyr(tBu)-Wang Resin, the molar amount of Fmoc-Tyr(tBu)-OH is 100-300% of the molar amount of active sites on the Wang resin. 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. Condensation reagents include HOBt, DIC, and DMAP. The molar amounts of HOBt and DIC used are consistent with the molar amount of Fmoc-Tyr(tBu)-OH used. 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.

[0056] The present invention, for the first time, utilizes the side chain phenolic hydroxyl group of Fmoc-Tyr-OtBu with 2-CTC resin under alkaline conditions to carry out a solid-phase anchoring reaction of the first amino acid. The phenoxide anion formed with the phenolic hydroxyl group under alkaline conditions undergoes a nucleophilic substitution reaction with the C-Cl bond of the 2-CTC resin, anchoring the first amino acid to the 2-CTC resin via an ether bond. Due to the excellent stability of the ether bond, the intermediate obtained using this method exhibits good stability in subsequent amino acid coupling reactions and post-processing operations, and is free of continuous degradation side reactions. This ensures a more stable production process for synthesizing linaclotide using 2-CTC resin, excellent yield, and higher purity. Therefore, the present invention provides a method for preparing linaclotide with high purity, good yield, low impurity peptide content, stable yield, and large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Schematic diagram of the synthesis of linaclotide.

[0058] Figure 2 HPLC chart. DETAILED DESCRIPTION

[0059] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0060] The following first describes the concepts involved in this application with reference to the accompanying drawings. It should be noted that the following description of each concept is intended only to make the content of this application easier to understand and does not limit the scope of protection of this application. At the same time, the embodiments and features in the embodiments of this application can be combined with each other unless there is a conflict. The following detailed description of this application will be made with reference to the accompanying drawings and in conjunction with the embodiments.

[0061] The present invention provides a method for preparing linaclotide, comprising the following steps:

[0062] (1) Under weak alkaline conditions, Fmoc-Tyr-OtBu is subjected to a substitution reaction with a 2-CTC resin support to obtain Fmoc-Tyr(O-Resin)-OtBu.

[0063] (2) Under the action of deprotection solution, Fmoc-Tyr(O-Resin)-OtBu removes the Fmoc protecting group to obtain H-Tyr(O-Resin)-OtBu.

[0064] (3) Under the 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.

[0065] (4) Repeat the above steps (2) and (3) and couple the remaining amino acids in the linaclotide sequence one by one from the carbon end to the nitrogen end. 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 was obtained.

[0066] (5) Under the action of deprotection solution, the Fmoc protecting group is removed from 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 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.

[0067] (6) Under the action of cutting solution, 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 removes all protecting groups and resin carrier to obtain crude linaclotide linear peptide H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH.

[0068] (7) The crude peptide H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH of linaclotide linear peptide was dissolved in ammonium sulfate solution and oxidized with DMSO to obtain the crude peptide solution of linaclotide H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH (1, 6 disulfide bonds, 2, 10 disulfide bonds, 5, 13 disulfide bonds).

[0069] (8) The crude linaclotide peptide solution was purified for the first time by preparative chromatography to obtain the first purified intermediate.

[0070] (9) The first purified intermediate of linaclotide is purified for a second time by preparative chromatography to obtain the second purified intermediate.

[0071] (10) The second purified intermediate of linaclotide is purified for the third time by preparative chromatography to obtain the third purified intermediate.

[0072] (11) The third purified intermediate of linaclotide was concentrated by preparative chromatography and freeze-dried to obtain the finished product of linaclotide.

[0073] in:

[0074] The degree of substitution of the 2-CTC resin used in step (1) is in the range of 0.4-1.4 mmol / g, preferably in the range of 0.9-1.1 mmol / g. The Fmoc-Tyr-OtBu anchoring reaction of the 2-CTC resin uses DCM as the solvent and DIEA or NMN as the base, preferably DIEA. The Fmoc-Tyr-OtBu feed ratio is 1.5-3.0 eq relative to the 2-CTC resin feed, preferably 2.0 eq. The base feed ratio is 3.0-6.0 eq relative to the 2-CTC resin feed, preferably 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 to carry out the 2-CTC resin end-capping reaction for 30 min, and then the resin is washed 4 times with DMF.

[0075] The deprotection solution in steps (2) and (5) is a DMF solution containing 15-25% piperidine, preferably a 20 vol% piperidine solution in DMF. The deprotection reaction temperature is 5-30°C, preferably 25°C. The reaction time is 1-4 hours, preferably 3 hours. After deprotection, the resin is washed six times with DMF.

[0076] The condensation reagent for the protected amino acid coupling in step (3) includes an activation system of one or more combinations of HOBt, 6-Cl-HOBt, DIC, PyBop, HBTU, HATU, HOAt, and DIEA. The HOBt / DIC system is preferred. The resin feed ratio: protected amino acid is between 1:2 and 1:3; protected amino acid: HOBt:DIC is 1:1:1. After the activation of the protected amino acid, it is added to the peptide resin obtained in the previous step to carry out 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. The condensation reagent also includes DMAP.

[0077] The protecting group strategies for protecting amino acids used in steps (3) and (4) are well known. The following strategies are preferred: 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, and Fmoc-Gly-OH.

[0078] The cutting liquid in step (6) is a combination of several components selected from TFA, EDT, DODT, or H2O. Calculated by volume, the TFA content is 70-80%; the content of either EDT or DODT is 15-25%; and the content of H2O is 3-10%. The composition and volume ratio of the cutting liquid are preferably: TFA:EDT:H2O=75:20:5. The cutting liquid feed ratio is: peptide resin feed amount (g): cutting liquid volume (ml) = 1 g:8 ml to 1 g:12 ml, preferably: peptide resin feed amount (g): cutting liquid volume (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.

[0079] Step (7) A linaclotide linear peptide solution is prepared at a concentration of 0.5-1.5 g / L using a 4-10 g / L aqueous solution of ammonium sulfate, ammonium acetate, or guanidine hydrochloride, preferably ammonium acetate. After the solution is prepared, the pH is adjusted to 8-10, preferably pH 9, with aqueous ammonia. DMSO is then added at 4-10% of the solution volume, preferably 5%. The reaction temperature is 15-35°C, preferably 30°C. The reaction is carried out for 45-60 hours, preferably 50 hours.

[0080] Step (8) The first purification preparative chromatography purification filler is C18 filler, and the detection wavelength is 210-220 nm. The chromatographic purification mobile phase adopts the well-known TFA solution-acetonitrile system, and the TFA solution concentration ranges from 0.05-0.15%, preferably 0.1%.

[0081] Step (9) The second purification preparative chromatography packing is C18 packing, and the detection wavelength is 210-220 nm. The chromatographic purification mobile phase adopts the well-known triethylammonium phosphate solution (TEAP)-acetonitrile system. The pH of the triethylammonium phosphate solution is 2.0-3.0, preferably pH 2.5.

[0082] In step (10), the third chromatographic purification is carried out using C18 filler and a detection wavelength of 210-220 nm. The chromatographic purification mobile phase is a known acetic acid-ammonium acetate solution-acetonitrile system. The acetic acid concentration in the acetic acid-ammonium acetate solution is in the range of 0.1-0.5 wt %, preferably 0.3 wt %; the ammonium acetate concentration in the acetic acid-ammonium acetate solution is in the range of 2-5 wt %, preferably 3.1 wt %.

[0083] The fourth chromatographic purification step (11) was performed using C18 filler and a detection wavelength of 210-220 nm. The chromatographic purification mobile phase was a water-acetonitrile system. The sample loading amount was 300-500 g / injection, preferably 400 g / injection, based on linaclotide.

[0084] In step (12), the intermediate is concentrated by column distillation under reduced pressure according to known conditions, with the temperature of the intermediate solution 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.

[0085] After the cleavage reaction is completed, the cleavage liquid is filtered and the resulting filtrate is added to an ether solvent for precipitation. The resulting precipitate is subjected to solid-liquid separation and drying to obtain crude linear linaclotide peptide. 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. During the precipitation process, the ratio of the volume of the cleavage liquid to the volume of the ether solvent is: 1:8 to 1:12, preferably 1:10.

[0086] After solid-liquid separation, the precipitate is dried under reduced pressure at a temperature of 30-50° C., preferably 50° C. After drying, crude linear linaclotide peptide is obtained.

[0087] The crude linear linaclotide peptide synthesized using the method described herein has an HPLC purity of 80-90% and a peptide content of 65-75%. The crude linaclotide peptide solution achieves a purity of 70-75%. After purification, concentration, and freeze-drying, the purity reaches over 99.8%, with a maximum single impurity content of no more than 0.10% and an overall yield of 25-35%. The production batch size can reach 500-600 g / batch, fully meeting the requirements of commercial production in terms of both quantity and quality.

[0088] Example 1: A method for preparing linaclotide

[0089] CTC resin solid phase synthesis of linaclotide: Fmoc-Tyr(tBu)-CTC Resin was mixed with DMF and swelled for 30 minutes. After swelling, the mixture was filtered and deprotected by adding a deprotection solution. After washing, an amino acid reagent and a condensation reagent were added for coupling. After coupling, the mixture was washed. The deprotection, coupling, and post-coupling washing steps 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(tBu)-CTC Resin. After drying, the peptide was cleaved to obtain a crude linear peptide of linaclotide, H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH. The crude linear peptide was then subjected to liquid-phase oxidation of three disulfide bonds using DMSO and purified by HPLC to obtain crude linaclotide. The degree of substitution of 2-CTC resin in the Fmoc-Tyr(tBu)-CTC resin was 0.625 mmol / g, and the amount of 2-CTC resin used was 3.20 g. During the preparation of the Fmoc-Tyr(tBu)-CTC resin, the molar amount of Fmoc-Tyr(tBu)-OH was 4 mmol, which was consistent with the molar amount of the amino acid reagent used in the subsequent coupling reaction. The condensation reagents include HOBt, DIC, and DMAP. The molar amounts of HOBt and DIC used are consistent with those of Fmoc-Tyr(tBu)-OH. The molar amount of DMAP used is 10% of the molar amount of Fmoc-Tyr(tBu)-OH. The solvent can be DMF, DCM, or a mixture of the two.

[0090] The yield of the crude linear peptide of linaclotide prepared in Example 1 of the present invention was 1.84 g, with a yield of 60.45%, and the purity after HPLC purification was 67.68%.

[0091] The HPLC purity of the crude linaclotide peptide prepared in Example 1 of the present invention was 41.49%.

[0092] The present invention analyzed the crude linear peptide of linaclotide prepared in Example 1, and the results are shown in Table 1 below.

[0093] Table 1 Analysis results of crude linear peptide content of linaclotide

[0094]

[0095] The yields of the peptide resin product and linear linaclotide obtained in Example 1 of the present invention were significantly lower than the theoretical values ​​because the ester bond formed between the carbon-terminal Tyr and the 2-CTC resin during solid-phase synthesis was unstable, resulting in the peptide chain easily falling off the resin during amino acid coupling. Impurities inserted into the first amino acid Tyr and missing one or more carbon-terminal amino acids were detected in the crude linear peptide.

[0096] Example 2: A method for preparing linaclotide

[0097] Wang resin solid phase synthesis of linaclotide: Fmoc-Tyr(tBu)-Wang Resin was mixed with DMF and swelled for 30 minutes. After swelling, the mixture was filtered and deprotected by adding a deprotection solution. After washing, an amino acid reagent and a condensation reagent were added for coupling. After coupling, the mixture was washed. The steps of deprotection, coupling, and post-coupling washing 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(tBu)-Wang Resin. After drying, the peptide was cleaved to obtain the crude linear peptide H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH. The crude linear peptide was then subjected to liquid-phase oxidation of three disulfide bonds using DMSO and purified by HPLC to obtain the crude peptide. The degree of substitution of Wang resin in Fmoc-Tyr(tBu)-Wang Resin was 0.48 mmol / g, and the amount of Wang resin used was 4.15 g. During the preparation of Fmoc-Tyr(tBu)-Wang Resin, the molar amount of Fmoc-Tyr(tBu)-OH was 4 mmol, which was consistent with the molar amount of amino acid reagent used in the subsequent coupling reaction. The condensation reagents include HOBt, DIC, and DMAP. The molar amounts of HOBt and DIC used are consistent with those of Fmoc-Tyr(tBu)-OH. The molar amount of DMAP used is 10% of the molar amount of Fmoc-Tyr(tBu)-OH. The solvent can be DMF, DCM, or a mixture of the two.

[0098] The yield of the crude linear peptide of linaclotide prepared in Example 2 of the present invention was 2.76 g, with a yield of 90.20%, and the purity after HPLC purification was 68.51%.

[0099] The HPLC purity of the crude linaclotide peptide prepared in Example 2 of the present invention was 43.75%.

[0100] The present invention analyzed the crude linear peptide of linaclotide prepared in Example 2, and the results are shown in Table 2 below.

[0101] Table 2 Analysis results of linaclotide crude linear peptide content

[0102]

[0103] The peptide resin and linear peptide yields finally obtained in Example 2 of the present invention are close to the theoretical values. Impurities with two amino acids missing from the carbon terminal are also found in the crude linear peptide, which may be due to the drop of dione piperazine formed during the coupling process. At the same time, due to the difficulty in coupling and incomplete reaction during the amino acid coupling process, the impurity content in the crude linear peptide is relatively high. The HPLC purity of the crude linear peptide is not much different from that in Example 1.

[0104] Example 3: A method for preparing linaclotide

[0105] Preparation of ether-linked CTC resin: 2-CTC resin and Fmoc-Tyr-OtBu were mixed in DCM. DIEA-DCM solution was added and the reaction was allowed to proceed at 25°C for 4.5 hours. Methanol was then added and the reaction continued for 30 minutes. After completion of the reaction, the liquid was removed by filtration, washed sequentially with DMF, DCM, and MTBE, and dried to obtain Fmoc-Tyr(O-Resin)-OtBu, the ether-linked CTC resin. The degree of substitution of 2-CTC resin was 1.02 mmol / g. The amount of 2-CTC resin used was 30 g. The molar amount of Fmoc-Tyr-OtBu was 200% of the molar amount of active sites on the 2-CTC resin. The amount of DCM used was 867 wt% of the 2-CTC resin. The DIEA-DCM solution was prepared by mixing DIEA and DCM in a volume ratio of 1.36:2, with the DIEA-DCM solution amounting to 30.77 vol% of the DCM, and the methanol amount being 11.54 vol% of the DCM. DMF, DCM and MTBE were used in appropriate amounts during washing.

[0106] Preparation of linaclotide-CTC resin: Fmoc-Tyr(O-Resin)-OtBu was mixed with DMF and swelled for 30 minutes. After swelling, the mixture was filtered and a deprotection solution was added to remove Fmoc. After washing, an amino acid reagent and a condensation reagent were added for coupling. The deprotection and coupling of the 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, i.e., linaclotide-CTC resin. During the swelling treatment, DMF is sufficient to submerge Fmoc-Tyr(O-Resin)-OtBu; the deprotection solution is a mixture of piperidine and DMF, containing 20 vol% piperidine, and the peptide resin can be submerged in the deprotection solution; the coupling order of the amino acid reagent 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 reagent used should be consistent with the molar amount of Fmoc-Tyr-OtBu used in the preparation of the ether-linked CTC resin. The condensation reagents include HOBt, DIC, and DMAP. The molar amounts of HOBt and DIC used should be consistent with the molar amounts of Fmoc-Tyr-OtBu used in the preparation of the ether-linked CTC resin. The molar amount of DMAP used should be 10% of the molar amount of Fmoc-Tyr-OtBu used in the preparation of the ether-linked CTC resin. The solvent can be DMF, DCM, or a mixture of the two.

[0107] Preparation of crude linaclotide peptide: The linaclotide-CTC resin is dried and then cleaved to obtain crude linear linaclotide peptide H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH. The crude linear linaclotide peptide is then subjected to liquid phase oxidation of three pairs of disulfide bonds using DMSO and purified by HPLC to obtain crude linaclotide peptide.

[0108] The yield of the crude linear peptide of linaclotide prepared in Example 3 of the present invention was 2.84 g, with a yield of 97.28%, and the purity after HPLC purification was 82.31%.

[0109] The HPLC purity of the crude linaclotide peptide prepared in Example 3 of the present invention was 75.38%.

[0110] No significant impurity peptides related to insertions and deletions of amino acids were detected in the crude linear linaclotide peptide prepared in Example 3 of the present invention. The yields of the peptide resin product and crude linear linaclotide peptide were close to the theoretical values. The crude linear linaclotide peptide of the present invention had a high HPLC purity.

[0111] The present invention performed a cleavage tolerance test and a thermal tolerance test 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. The results are shown in Table 3.

[0112] Table 3 Stability test results

[0113]

[0114] In the tolerance test of the present invention, the results of the cleavage tolerance test show that the ether-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. The stability under cleavage and heat conditions is better than that of the prior art, and the yield and purity are significantly improved, and it can be used for industrial production. The active structure of Wang resin is hydroxyl. Therefore, the hydroxyl group on Wang resin is difficult to form an ether bond with the Fmoc-Tyr-OtBu used in the present invention and 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 by the present invention cannot be formed.

[0115] The method of the present invention is at least applicable to the synthesis of a peptide whose first amino acid is Tyr.

[0116] The method of the present invention can also be applied to the synthesis of peptides containing a hydroxyl group in the first amino acid.

[0117] Example 4: A method for preparing linaclotide

[0118] Preparation of ether-linked CTC resin: 2-CTC resin and Fmoc-Tyr-OtBu were mixed in DCM, DIEA-DCM solution was added at 25°C and the reaction was continued for 4.5 hours, and then methanol was added to continue the reaction for 30 minutes. After the reaction was completed, the liquid was removed by filtration, and the resin was washed with DMF, DCM and MTBE in sequence and dried to obtain Fmoc-Tyr(O-Resin)-OtBu, i.e., ether-linked CTC resin. The degree of substitution of 2-CTC resin was 0.98 mmol / g, and the amount of 2-CTC resin used was 3061 g. The molar amount of Fmoc-Tyr-OtBu was 200% of the molar amount of active sites on the 2-CTC resin. The amount of DCM used was 867 wt % of the 2-CTC resin. The DIEA-DCM solution was prepared by mixing DIEA and DCM in a volume ratio of 1.36:2, with the amount of DIEA-DCM solution being 30.77 vol % of the DCM. The amount of methanol was 11.54 vol % of the DCM. DMF, DCM, and MTBE were used in appropriate amounts during the washes.

[0119] Preparation of linaclotide-CTC resin: Fmoc-Tyr(O-Resin)-OtBu was mixed with DMF and swelled for 30 minutes. After swelling, the mixture was filtered and a deprotection solution was added to remove Fmoc. After washing, an amino acid reagent and a condensation reagent were added for coupling. The deprotection and coupling of the 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, i.e., linaclotide-CTC resin. During the swelling treatment, DMF is sufficient to submerge Fmoc-Tyr(O-Resin)-OtBu; the deprotection solution is a mixture of piperidine and DMF, containing 20 vol% piperidine, and the peptide resin can be submerged in the deprotection solution; the coupling order of the amino acid reagent 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 reagent used should be consistent with the molar amount of Fmoc-Tyr-OtBu used in the preparation of the ether-linked CTC resin. The condensation reagents include HOBt, DIC, and DMAP. The molar amounts of HOBt and DIC used should be consistent with the molar amounts of Fmoc-Tyr-OtBu used in the preparation of the ether-linked CTC resin. The molar amount of DMAP used should be 10% of the molar amount of Fmoc-Tyr-OtBu used in the preparation of the ether-linked CTC resin. The solvent can be DMF, DCM, or a mixture of the two.

[0120] Preparation of crude linaclotide peptide: The linaclotide-CTC resin is dried and then cleaved to obtain crude linear linaclotide peptide H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH. The crude linear linaclotide peptide is then subjected to liquid phase oxidation of three pairs of disulfide bonds using DMSO and purified by HPLC to obtain crude linaclotide peptide.

[0121] In the purification of crude linear peptide of linaclotide, crude linear peptide of linaclotide was added to ammonium acetate solution, the pH was adjusted to 9.0 with ammonia water, and then DMSO was added. The cyclization treatment was started at 25°C for 60 hours, and the crude peptide solution of linaclotide was obtained by filtration. The crude peptide was purified by HPLC to obtain crude peptide of linaclotide.

[0122] The yield of crude linaclotide peptide prepared in Example 4 of the present invention was 595 g, with a total yield of 26.90%. After HPLC purification, the purity was 99.89%, the total impurities were 0.11%, and the maximum single impurity was 0.07%. The HPLC chromatogram is shown in FIG. Figure 1 shown.

[0123] Example 4 of the present invention is a method for scaled-up production. By scaling up the preparation method of linaclotide at the same ratio, linaclotide with excellent purity and yield can also be obtained.

[0124] The cutting solution used to cut the linaclotide-CTC resin in the present invention comprises TFA, EDT, and H₂O, with the volume ratio of TFA, EDT, and H₂O in the cutting solution being 1:0.27:0.07. The cutting solution is used in an appropriate amount. The cutting conditions are: cutting at 25°C for 3 hours. After cutting, the cutting solution is transferred to MTBE and allowed to settle at 25°C. After settling, the solid particles are centrifuged to obtain a solid particle product. The solid particle product is then slurried with MTBE, washed, centrifuged, and finally dried to obtain a crude linear linaclotide peptide.

[0125] In the HPLC purification of the present invention, the crude peptide solution of linaclotide is converted into 400 g of linaclotide per needle and loaded onto the chromatography system for primary purification. The product of the primary purification is then loaded onto the chromatography system for secondary purification. The product of the secondary purification is then loaded onto the chromatography system for tertiary purification. The product of the tertiary purification is then loaded onto the chromatography system for quadruple purification.

[0126] The gradient elution program for the primary purification was as follows:

[0127]

[0128] The primary purification preparative chromatography packing material was C18 packing material, and the detection wavelength was 210-220 nm.

[0129] The gradient elution procedure for the secondary purification was as follows:

[0130]

[0131] The secondary purification preparative chromatography packing was C18 packing, and the detection wavelength was 210-220 nm.

[0132] The gradient elution program for the three purifications was as follows:

[0133]

[0134] The filler for the three-stage chromatography purification was C18 filler, and the detection wavelength was 210-220 nm.

[0135] The gradient elution procedures for the four purifications were as follows:

[0136]

[0137] The filler for the four chromatographic purifications was C18, and the detection wavelength was 210-220 nm.

[0138] In the present invention, the product solution obtained after four purifications is transferred to a rotary evaporation system for vacuum distillation and concentration. The solution temperature is controlled below 25° C., concentrated to 20-40% of the original volume of the product solution, and freeze-dried to obtain crude linaclotide peptide. In the examples of the present invention, Examples 3 and 4 were purified four times. The HPLC purity given in Example 3 is data from a single purification. Due to poor purity and yield, Examples 1 and 2 were purified only once. That is, the HPLC purity given in Examples 1 and 2 is data from a single purification.

[0139] The HPLC chromatogram of linaclotide in Example 4 of the present invention is as follows: Figure 2 As shown, wherein the results of HPLC chromatography are shown in Table 4 below.

[0140] Table 4 HPLC chromatographic results

[0141]

[0142] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and do not impose any form of limitation on the implementation methods of the technology of the present invention. Any person skilled in the art may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0143] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression, there are objectively infinite specific structures. For ordinary technicians in this technical field, without departing from the principles of this application, they can also make several improvements, modifications or changes, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A method for preparing an amino acid ether bond-linked 2-CTC resin, characterized in that: include: The hydroxyl-containing amino acid is mixed with 2-CTC resin to react and generate an ether bond anchored 2-CTC resin, that is, the amino acid ether bond is connected to the 2-CTC resin; the hydroxyl-containing amino acid is Fmoc-Tyr-OtBu.

2. An amino acid ether bond-linked 2-CTC resin, characterized in that: include: 2-CTC resin is used as a solid phase carrier, and an amino acid is connected via 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. The amino acid is Fmoc-Tyr-OtBu. The amino acid ether bond connected to the 2-CTC resin is Fmoc-Tyr(O-Resin)-OtBu.

3. A linaclotide linear peptide-CTC resin, characterized in that include: The amino acid ether bond of claim 2 is connected to 2-CTC resin, and the amino protecting group of the amino acid is replaced by a linear polypeptide other than Tyr.

4. The linaclotide linear peptide-CTC resin according to claim 3, characterized in that: 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 linear peptide-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.

5. A method for preparing linaclotide linear peptide-CTC resin, characterized in that: include: The method for preparing the amino acid ether bond-linked 2-CTC resin according to claim 1.

6. A method for preparing a crude linear peptide of linaclotide, characterized in that: include: The method for preparing an amino acid ether-linked 2-CTC resin according to claim 1; or the method for preparing a linaclotide linear peptide-CTC resin according to claim 5.

7. A method for preparing linaclotide, characterized in that: include: The method for preparing an amino acid ether bond-linked 2-CTC resin according to claim 1; Or, the preparation method of the linaclotide linear peptide-CTC resin according to claim 5.

Images