Preparation method of linaclotide

Abstract

The invention provides a preparation method of linaclotide, which comprises the following steps: cyclizing linear peptide of linaclotide to obtain linaclotide, wherein the cyclization refers to air oxidation in a phosphate and guanidine hydrochloride solution with the pH value of 7.5-8.5.

Description

Technical Field

[0001] This application belongs to the field of polypeptide synthesis technology. Specifically, this application provides a method for preparing linaclotide. Background Technology

[0002] Linaclotide is a guanylate cyclase C (GC-C) agonist. After entering the human body, it activates GC-C by binding to guanylate cyclase C receptors in the intestine, thereby increasing intestinal fluid secretion, accelerating gastrointestinal migration, and thus increasing defecation frequency. It can also reduce the sensitivity of pain nerves in the small intestine, and has visceral analgesic and secretory-promoting effects. It can relieve abdominal pain and constipation symptoms caused by constipation-predominant irritable bowel syndrome and is mainly used for the treatment of constipation-predominant irritable bowel syndrome.

[0003] Currently, the synthesis of linaclotide typically uses organic reagents, which leads to problems such as complex operation, low product yield, high production cost, and environmental pollution. Therefore, there is an urgent need to develop a linaclotide preparation method that is mild in reaction, easy to operate, low in cost, produces high-purity products, is environmentally friendly and efficient, and is suitable for large-scale production. Summary of the Invention

[0004] This application provides a method for preparing linaclotide, which includes cyclizing a linear linaclotide peptide to obtain linaclotide; wherein the cyclization is performed by air oxidation in a phosphate and guanidine hydrochloride solution with a pH of 7.5-8.5.

[0005] In one or more embodiments of this application, the method includes synthesizing a fully protected linaclotide linear peptide resin prior to the cyclization.

[0006] In one or more embodiments of this application, the method includes cleaving the fully protected linaclotide linear peptide resin.

[0007] In one or more embodiments of this application, the method further includes purifying and / or salt-replacing linaclotide. Attached Figure Description

[0008] Figure 1 This is the synthetic route diagram for linaclotin.

[0009] Figure 2 The purity results of crude linaclotide obtained by cyclizing the linear linaclotide under the following conditions: air oxidation for 48 h at room temperature in a buffer solution of 100 mM sodium dihydrogen phosphate and 10 mM guanidine hydrochloride at pH 8.0.

[0010] Figure 3The purity results of the linaclotide product obtained by cyclizing the linear linaclotide under the following conditions: air oxidation for 48 h at room temperature in a buffer solution of 100 mM sodium dihydrogen phosphate and 10 mM guanidine hydrochloride at approximately pH 8.0.

[0011] Figure 4 The purity results of crude linaclotide obtained by cyclizing the linear linaclotide under the following conditions: air oxidation for 24 h at room temperature in a buffer solution of 100 mM sodium dihydrogen phosphate and 10 mM guanidine hydrochloride at pH 7.5.

[0012] Figure 5 The purity results of crude linaclotide obtained by cyclizing the linear linaclotide under the following conditions: air oxidation for 24 h at room temperature in 50 mM Tris·HCl buffer at pH 7.5.

[0013] Figure 6 The purity results of crude linaclotide obtained by cyclizing the linear linaclotide under the following conditions: air oxidation at room temperature for 48 h in a buffer solution of 100 mM sodium dihydrogen phosphate and 10 mM guanidine hydrochloride at pH 7.5.

[0014] Figure 7 The purity results of crude linaclotide obtained by cyclizing the linear linaclotide under the following conditions: air oxidation at room temperature for 48 h in 100 mM sodium dihydrogen phosphate buffer at pH 7.5.

[0015] Figure 8 The purity results of crude linaclotide obtained by cyclizing the linear linaclotide under the following conditions: air oxidation at room temperature for 48 h in 10 mM guanidine hydrochloride buffer at pH 7.5.

[0016] Figure 9 The purity results are for crude linaclotide obtained by cyclizing the linear linaclotide peptide with DMSO. Detailed Implementation

[0017] The inventors of this application, in the step of obtaining crude linaclotide through linear peptide cyclization, utilized the principle that the natural molecular configuration of linaclotide is more stable. They selected an aqueous solution (e.g., phosphate and guanidine hydrochloride solution) as a solvent and, by adjusting the pH (e.g., to 7.5-8.5), allowed the linear linaclotide to oxidize naturally in air at room temperature, thus developing a new method for synthesizing linaclotide. This method reduces the use of organic reagents in the oxidation process and avoids the use of oxidizing agents, resulting in a milder reaction, higher purity of the crude product, and a higher final product yield, making it suitable for large-scale production.

[0018] Unless otherwise specified, this application will be implemented using conventional peptide synthesis techniques in the art.

[0019] Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art.

[0020] Unless otherwise specified, all laboratory reagents are commercially available products.

[0021] definition

[0022] As used herein, the term "synthetic scale" refers to the number of moles of coupling sites available for the intermediate Fmoc-L-Tyr(tBu)-CTC resin at a given weight after the first amino acid is coupled to the resin.

[0023] As used herein, the term "about" refers to the possibility that the value in question may vary to some extent. This variation may arise from errors in the measuring instrument or method, individual differences, or inherent variability of the material. It typically refers to a value or range between ±1%, ±2%, ±2.1%, ±2.2%, ±2.3%, ±2.4%, ±2.5%, ±2.6%, ±2.7%, ±2.8%, ±2.9%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, ±10%, ±11%, ±12%, ±13%, ±14%, ±15%, ±16%, ±17%, ±18%, ±19%, and ±20% of the stated value. In one or more embodiments of this application, when used in conjunction with pH, ​​"about" refers to the value ±0.2; for example, the pH of the solution in this application is about 7.5, which is 7.5 ± 0.2.

[0024] This application provides a method for preparing linaclotide, which includes cyclizing a linear linaclotide peptide to obtain linaclotide; wherein the cyclization is performed by air oxidation in a phosphate and guanidine hydrochloride solution with a pH of 7.5-8.5.

[0025] In one or more embodiments of this application, the pH value is about 7.5 or 8.0. In one or more embodiments of this application, the pH value is 7.5 ± 0.2 or 8.0 ± 0.2. In one or more embodiments of this application, the pH value is adjusted using sodium hydroxide.

[0026] In one or more embodiments of this application, the phosphate in the phosphate and guanidine hydrochloride solution is sodium dihydrogen phosphate.

[0027] In one or more embodiments of this application, the cyclization is performed at room temperature. In some embodiments, the cyclization is performed at 10°C to 30°C; the cyclization is performed at 20°C to 30°C; the cyclization is performed at 15°C to 25°C; the cyclization is performed at 20°C to 25°C; or the cyclization is performed at 15°C, 20°C, or 25°C.

[0028] In one or more embodiments of this application, the circulation time is 24-72 hours, for example 24, 48 or 72 hours.

[0029] In one or more embodiments of this application, the concentration of phosphate in the phosphate and guanidine hydrochloride solution is 20-100 mM, for example 100 mM.

[0030] In one or more embodiments of this application, the concentration of guanidine hydrochloride in the phosphate and guanidine hydrochloride solution is 5-10 mM, for example, 10 mM.

[0031] In one or more embodiments of this application, the concentration of the linaclotide linear peptide is 5-10 mg / mL, for example 5, 7.5 or 10 mg / mL.

[0032] In one or more embodiments of this application, the method includes synthesizing a fully protected linaclotide linear peptide resin prior to the cyclization.

[0033] In one or more embodiments of this application, a fully protected linaclotide linear peptide resin is synthesized by a solid-phase synthesis method.

[0034] In one or more embodiments of this application, the resin is 2-chlorotriphenylmethyl chloride resin (2-CTCResin) or Wang resin.

[0035] In one or more embodiments of this application, the synthesis uses a protected amino acid, such as a Fmoc-amino acid with a protected side chain. In one or more embodiments of this application, the side chain of the protected amino acid may use any of the following protecting groups: triphenylmethyl (Trt), tert-butoxy (OtBu), tert-butyl (tBu), tert-butoxycarbonyl (Boc), and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf).

[0036] In one or more embodiments of this application, the following protected amino acids are condensed sequentially from the C-terminus to the N-terminus of linaclotide: Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-L-Ala-OH, Fmoc-L-Pro-OH, Fmoc-L-Asn(Trt)-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Cys(Trt)-OH, and Fmoc-L-Cys(Trt)-OH.

[0037] In one or more embodiments of this application, Fmoc-L-Tyr(tBu)-OH and 2-CTC resin are reacted in dichloromethane (DCM) under the action of N,N-diisopropylethylamine (DIPEA) or N-methylmorpholine (NMM) to obtain Fmoc-L-Tyr(tBu)-CTC resin. In one or more embodiments of this application, the molar ratio of Fmoc-L-Tyr(tBu)-OH to DIPEA is 1:2. In one or more embodiments of this application, the molar ratio of Fmoc-L-Tyr(tBu)-OH to NMM is 1:2.

[0038] In one or more embodiments of this application, the deFmoc protecting agent used in the synthesis is a mixture of piperidine and N,N-dimethylformamide (DMF). In one or more embodiments of this application, the volume ratio of piperidine to DMF is 2:8.

[0039] In one or more embodiments of this application, the condensing agent used in the synthesis is Oxyma Pure, a mixture of 1-hydroxybenzotriazole (HOBt) or 1-hydroxy-7-azobenzotriazole (HOAt) with N,N'-diisopropylcarbodiimide (DIC), or a mixture of (3H-1,2,3-triazolo[4,5-b]pyridin-3-oxy)tri-1-pyrrolidinyl hexafluorophosphate (PyAop) or benzotriazole-1-yl-oxytripyrrolidinyl hexafluorophosphate (PyBop) with an organic base (e.g., N-methylmorpholine or N,N-diisopropylethylamine), or a mixture of O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU) or 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) with an organic base (e.g., N-methylmorpholine or N,N-diisopropylethylamine).

[0040] In one or more specific embodiments of this application, the condensing agent used in the synthesis is a mixture of HOBt and DIC.

[0041] In one or more embodiments of this application, the amount of the protected amino acid fed is 2-3 equivalents of the synthesis scale, for example, 2 equivalents.

[0042] In one or more embodiments of this application, the protected amino acid is in the same molar amount as each reagent in the condensing agent, for example, the protected amino acid: HOBt:DIC = 1:1:1 (molar ratio).

[0043] In one or more embodiments of this application, the method includes cleaving the fully protected linaclotide linear peptide resin, for example, by acid cleaving (cutting) the fully protected linaclotide linear peptide resin.

[0044] In one or more embodiments of this application, the reagent used for pyrolysis comprises one or more of the following: trifluoroacetic acid (TFA), triisopropylsilane (TIS), 1,2-ethylenedithiol (EDT), H2O, anisole, methyl sulfide, benzoyl sulfide, and phenol. In one or more embodiments of this application, the reagent used for pyrolysis includes TFA, TIS, EDT, and H2O.

[0045] In one or more specific embodiments of this application, the reagent used for lysis consists of TFA, TIS, EDT and H2O.

[0046] In one or more embodiments of this application, the volume ratio of TFA, TIS, EDT and H2O is 85:5:5:5.

[0047] In one or more embodiments of this application, the ratio of the reagent used for lysis to the fully protected linaclotide linear peptide resin is (5-15):1 (mL / g), for example 5:1 (mL / g), 10:1 (mL / g), or 15:1 (mL / g).

[0048] In one or more embodiments of this application, the method further includes purifying and / or salt-replacing linaclotide.

[0049] In one or more embodiments of this application, purification is performed using reversed-phase high-performance liquid chromatography. In one or more embodiments of this application, an aqueous solution of ammonium acetate is used as mobile phase A, and acetonitrile is used as mobile phase B.

[0050] In one or more embodiments of this application, reversed-phase high-performance liquid chromatography (RP-HPLC) is used for salt replacement. In one or more embodiments of this application, glacial acetic acid aqueous solution is used as mobile phase A, and acetonitrile is used as mobile phase B.

[0051] In one or more embodiments of this application, the method for preparing linaclotide uses side-chain protected Fmoc-amino acids and 2-CTC resin as starting materials, and obtains a fully protected linaclotide linear peptide resin by sequentially coupling amino acids from the C-terminus to the N-terminus of the peptide; then, the fully protected linaclotide linear peptide resin is acid-cracked (e.g., using a lysis buffer including TFA, TIS, EDT and H2O) to obtain linaclotide linear peptide; the linaclotide linear peptide is then oxidized in air in a weakly alkaline phosphate and guanidine hydrochloride solution (e.g., a sodium dihydrogen phosphate and guanidine hydrochloride solution with a pH of 7.5-8.5) to obtain crude linaclotide; the crude linaclotide is purified by reversed-phase high-performance liquid chromatography, salt is replaced, and then freeze-dried to obtain the linaclotide product.

[0052] In one or more specific embodiments of this application, the method for preparing the linaclotide includes the following steps:

[0053] (1) Synthesis of Fmoc-L-Tyr(tBu)-CTC resin; (2) Using Fmoc-L-Tyr(tBu)-CTC resin as raw material, a fully protected linaclotide linear peptide resin 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 was synthesized; (3) Using the fully protected linaclotide linear peptide resin as raw material, the linaclotide linear peptide H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH was obtained by cleavage (e.g., acid cleavage using a cleavage buffer including TFA, TIS, EDT and H2O); (4) Using H-fully protected linear linaclotide as raw material, crude linaclotide is obtained by air oxidation in a weakly alkaline phosphate and guanidine hydrochloride solution (e.g., sodium dihydrogen phosphate and guanidine hydrochloride solution with a pH of 7.5-8.5); (5) Using crude linaclotide as raw material, linaclotide product is obtained after purification, salt replacement and freeze drying.

[0054] In one or more embodiments of this application, the linaclotide preparation method of this application has at least one of the following advantages:

[0055] (1) The three pairs of disulfide bonds in linaclotide can be linked in one step;

[0056] (2) No organic reagents are required;

[0057] (3) No oxidizing agent is required;

[0058] (4) The purity of the obtained crude peptide product can reach over 70%;

[0059] (5) The purity of the finished product can reach over 99%;

[0060] (6) Product yield can reach over 30%;

[0061] (7) Easy to operate;

[0062] (8) Low production cost;

[0063] (9) Less environmental pollution; and

[0064] (10) Suitable for large-scale production.

[0065] It should be understood that the above detailed description is only intended to provide a clearer understanding of the contents of this application to those skilled in the art, and is not intended to limit in any way. Those skilled in the art can make various modifications and variations to the described embodiments.

[0066] Example

[0067] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0068] Example 1. Cyclization production of linaclotide in sodium dihydrogen phosphate and guanidine hydrochloride solution via air oxidation

[0069] The preparation method includes the following steps:

[0070] (1) Synthesis of Fmoc-L-Tyr(tBu)-CTC resin: Fmoc-L-Tyr(tBu)-OH and 2-CTC resin were reacted in dichloromethane (DCM) under the action of N,N-diisopropylethylamine (DIPEA) to obtain Fmoc-L-Tyr(tBu)-CTC resin. 4.5g of Fmoc-L-Tyr(tBu)-OH was added for every gram of 2-CTC resin. The feed ratio of Fmoc-L-Tyr(tBu)-OH to DIPEA was 1:2 (molar ratio).

[0071] (2) Synthesis of fully protected linaclotide linear peptide resin (synthetic scale 10 mmol): Using Fmoc-L-Tyr(tBu)-CTC resin as raw material, and a mixture of piperidine and DMF as the de-Fmoc group removal agent; under the action of a condensing agent, using N,N-dimethylformamide (DMF) as solvent, according to Fmoc-L-Cys(Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Cys(Trt)-OH, Fmoc The fully protected linaclotide linear peptide resin was obtained by sequentially coupling Fmoc-L-Pro-OH, Fmoc-L-Asn(Trt)-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Cys(Trt)-OH, and Fmoc-L-Cys(Trt)-OH. The piperidine / DMF mixture ratio was 2:8 (V / V); the condensing agent was a mixture of HOBt and DIC; the starting material amount of Fmoc-AA-OH was 2 equivalents of the synthesis scale, and the actual feeding amounts are shown in Table 1 below; the molar ratio of Fmoc-AA-OH:HOBt:DIC was 1:1:1. Fmoc-AA-OH represents the above-mentioned protected amino acid.

[0072] Table 1. Starting material input amount

[0073]

[0074] (3) Synthesis of linear linaclotide: Using fully protected linear linaclotide resin as raw material, linear linaclotide was obtained by acid cleavage under the action of a mixture of TFA, TIS, EDT and H2O. The volume ratio of TFA, TIS, EDT and H2O was 85: 5: 5: 5, and the amount of mixture used was 10 mL / g peptide resin.

[0075] (4) Synthesis of crude linaclotide: Using linear linaclotide as raw material, the crude linaclotide was obtained by stirring in a buffer solution of 100 mM sodium dihydrogen phosphate (NaH2PO4) and 10 mM guanidine hydrochloride (Gdn·HCl) at pH 8.0 and allowing it to oxidize naturally at room temperature for 48 h. The pH was adjusted with sodium hydroxide, and the concentration of linear linaclotide was 5 mg / mL.

[0076] (5) Preparation of linaclotide product: Crude linaclotide was used as raw material and purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The stationary phase was octadecylsilane-bonded silica gel. After loading the crude product solution, ammonium acetate aqueous solution was used as mobile phase A and acetonitrile as mobile phase B for elution. The resulting product solution was subjected to salt replacement by reversed-phase HPLC with the same stationary phase, using glacial acetic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B for gradient elution. The eluent was collected and lyophilized to obtain the linaclotide product.

[0077] The purity of the crude linaclotide obtained in step (4) was determined by HPLC. The reaction solution was diluted 5 times with purified water before analysis. The results are as follows: Figure 2 As shown, the purity is 71.44%.

[0078] The purity of the linaclotide product obtained in step (5) was determined by HPLC, and the results are as follows: Figure 3 As shown, the purity is 99.8317%.

[0079] Synthesis yield = (Weight of synthesized peptide resin - Weight of Fmoc-L-Tyr(tBu) - Weight of CTC resin) / (Synthetic scale × Molecular weight of fully protected peptide sequence × 10) -3 )×100%

[0080] Cleavage yield = Weight of crude product obtained from cleavage / (Synthetic scale × peptide sequence molecular weight × weight of peptide resin used for cleavage / weight of peptide resin obtained from synthesis × 10) -3 )×100%

[0081] Purification yield = Weight of purified product / Weight of crude product used for purification

[0082] Product yield = Synthesis yield × Cutting yield × Purification yield

[0083] The yield of linaclotide was calculated to be 32%.

[0084] Example 2. Cyclation production of linaclotide using different oxidation buffers

[0085] Except for the buffer system and oxidation time used in step (4) of Example 1, all other steps are the same.

[0086] The following four buffer systems were used to study the linear peptide oxidation of linaclotin:

[0087] Dissolve 100 mM sodium dihydrogen phosphate and 10 mM guanidine hydrochloride in 1 L of purified water and adjust the pH to approximately 7.5 using NaOH.

[0088] Dissolve 50 mM Tris·HCl in 1 L of purified water and adjust the pH to approximately 7.5 with HCl.

[0089] Dissolve 50 mM NaCl in 1 L of purified water and adjust the pH to approximately 7.5 using NaOH.

[0090] Dissolve 50 mM NH4Cl in 1 L of purified water and adjust the pH to approximately 7.5 using ammonia.

[0091] The experimental results are shown in Table 2 below.

[0092] Table 2

[0093]

[0094] Example 3. Comparison of the effects of cyclization production of linaclotide using sodium dihydrogen phosphate, guanidine hydrochloride, and combinations of sodium dihydrogen phosphate and guanidine hydrochloride.

[0095] Except for the buffer system used in step (4) of Example 1, all other steps are the same.

[0096] The following three buffer systems were used to study the linear peptide oxidation of linaclotin:

[0097] ① Dissolve 100mM sodium dihydrogen phosphate and 10mM guanidine hydrochloride in 1L of purified water, and adjust the pH to approximately 7.5 using NaOH.

[0098] ② Dissolve 100mM sodium dihydrogen phosphate in 1L of purified water and adjust the pH to approximately 7.5 using NaOH.

[0099] ③ Dissolve 10mM guanidine hydrochloride in 1L of purified water and adjust the pH to approximately 7.5 using NaOH.

[0100] Experimental results are as follows Figures 6 to 8 As shown, the purities of crude linaclotide obtained using the three oxidation buffer systems ① to ③ were 70.76%, 67.54%, and 61.57%, respectively. It is evident that using a 100mM sodium dihydrogen phosphate and 10mM guanidine hydrochloride buffer system can achieve higher crude linaclotide purity, demonstrating a synergistic effect.

[0101] Example 4. Cyclization production of linaclotide using DMSO oxidation

[0102] Except for step (4) in Example 1, all other steps are the same.

[0103] Linaclotide linear peptide was used as raw material. It was dissolved in PBS solution (pH 7.0) to a concentration of 10 mg / mL, and then an equal volume of DMSO was added. After reacting at room temperature for 48 h, crude linaclotide was obtained.

[0104] The purity of the crude linaclotide obtained in step (4) was tested, and the results are as follows: Figure 9 As shown, the purity is 55.81%.

[0105] It is understood that although the inventions described in this application are in the specific forms described above, these inventions are not limited to the specific content described in these specific forms. It will be apparent to those skilled in the art that various equivalent changes can be made to the technical features contained in the inventions described herein without departing from the spirit of the inventions described herein, and all such changes should fall within the scope of the inventions.

Claims

1. A method for preparing linaclotide, comprising cyclizing a linear linaclotide peptide to obtain linaclotide; wherein the cyclization is performed by air oxidation in a phosphate and guanidine hydrochloride solution with a pH of 7.5-8.

5.

2. The method of claim 1, wherein The pH value is 7.5 ± 0.2 or 8.0 ± 0.2; optionally, the pH value is adjusted using sodium hydroxide. The phosphate in the phosphate and guanidine hydrochloride solution is sodium dihydrogen phosphate; The cyclization is performed at room temperature; and / or The circulation time is 24-72 hours; preferably, the circulation time is 24, 48 or 72 hours.

3. The method as described in claim 1 or 2, wherein The concentration of phosphate in the phosphate and guanidine hydrochloride solution is 20-100 mM, preferably 100 mM; and / or The concentration of guanidine hydrochloride in the phosphate and guanidine hydrochloride solution is 5-10 mM, preferably 10 mM.

4. The method according to any one of claims 1-3, wherein The method includes synthesizing a fully protected linaclotide linear peptide resin prior to the cyclization. Preferably, the fully protected linaclotide linear peptide resin is synthesized by a solid-phase synthesis method; More preferably, the resin is a 2-chlorotriphenylmethyl chloride (2-CTC) resin or a Wang resin; and / or the synthesis uses a protected amino acid, such as a Fmoc-amino acid with a protected side chain.

5. The method of claim 4, wherein the following protected amino acids are condensed sequentially from the C-terminus to the N-terminus of the linaclotide: Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-L-Ala-OH, Fmoc-L-Pro-OH, Fmoc-L-Asn(Trt)-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Cys(Trt)-OH, and Fmoc-L-Cys(Trt)-OH.

6. The method of claim 5, wherein The deFmoc protecting agent used in the synthesis is a mixture of piperidine and N,N-dimethylformamide (DMF); preferably, the volume ratio of piperidine to DMF is 2:

8. The condensing agent used in the synthesis was Oxyma A mixture of Pure, 1-hydroxybenzotriazole (HOBt) or 1-hydroxy-7-azobenzotriazole (HOAt) with N,N'-diisopropylcarbodiimide (DIC), or a mixture of (3H-1,2,3-triazolo[4,5-b]pyridin-3-oxy)tri-1-pyrrolidinyl hexafluorophosphate (PyAop) or benzotriazole-1-yl-oxytripyrrolidinyl hexafluorophosphate (PyBop) with an organic base (e.g., N-methylmorpholine or N,N-diisopropylethylamine), or a mixture of O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU) or 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) with an organic base (e.g., N-methylmorpholine or N,N-diisopropylethylamine), preferably a mixture of HOBt and DIC; The amount of the protected amino acid fed is 2-3 equivalents of the synthesis scale; and / or The protected amino acid is present in the same molar amount as each reagent in the condensing agent.

7. The method of claim 5 or 6, comprising cleaving the fully protected linaclotide linear peptide resin; Preferably, the pyrolysis is acid pyrolysis; More preferably, the reagent used for cleavage comprises one or more of the following: trifluoroacetic acid (TFA), triisopropylsilane (TIS), 1,2-ethylenedithiol (EDT), H2O, anisole, methyl sulfide, anisole, and phenol. Most preferably, the reagents used for lysis include TFA, TIS, EDT and H2O.

8. The method of claim 7, wherein The volume ratio of TFA, TIS, EDT, and H2O is 85:5:5:5; and / or The ratio of the reagent used for lysis to the fully protected linaclotide linear peptide resin is (5-15):1 (mL / g).

9. The method according to any one of claims 1-8, further comprising purifying and / or salt-replacing linaclotide.

10. The method of claim 9, wherein Purification was performed using reversed-phase high-performance liquid chromatography; preferably, an aqueous solution of ammonium acetate was used as mobile phase A, and acetonitrile was used as mobile phase B; and / or Salt replacement was performed using reversed-phase high-performance liquid chromatography; preferably, glacial acetic acid aqueous solution was used as mobile phase A and acetonitrile was used as mobile phase B.

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