A solid phase synthesis method of linaclotide

Abstract

The present invention discloses a solid-phase synthesis method of linaclotide, belonging to the technical field of peptide synthesis; the method adopts a solid-phase synthesis method, uses Fmoc-Tyr(tBu)-Wang resin as a starting material, and sequentially couples amino acids from the C-terminus to the N-terminus to obtain linaclotide peptide resin; then, after deprotection treatment, oxidation and cleavage, and purification treatment, linaclotide is obtained. The peptide sequence of linaclotide is as follows: H2N-Cys 1 ‑Cys 2 ‑Glu 3 ‑Tyr 4 ‑Cys 5 ‑Cys 6 ‑Asn 7 ‑Pro 8 ‑Ala 9 ‑Cys 10 ‑Thr 11 ‑Gly 12 ‑Cys 13 ‑Tyr 14 ‑OH (disulfide bridge: Cys 1 ‑Cys 6 &Cys 2 ‑Cys 10 &Cys 5 ‑Cys 13 The total yield of the linaclotide synthesized by the present invention is over 98%, and the purity is over 80.5%.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide synthesis, and particularly to a solid-phase synthesis method of linaclotide. Background Art

[0002] Currently, there are three main process routes for the purification of crude linaclotide peptide: The first method uses a one-step oxidation strategy to form three pairs of disulfide bonds. This method first synthesizes the linaclotide resin, then cleaves the linaclotide resin to remove all protecting groups and the resin solid phase carrier to obtain a crude linear linaclotide peptide, and finally uses an oxidation system to perform a one-step oxidation reaction on the crude linear linaclotide peptide to complete the construction of three pairs of disulfide bonds. Although this method is simple to operate and only uses one-step oxidation to form three pairs of disulfide bonds, it cannot avoid the occurrence of disulfide bond mismatches, resulting in a low final yield. The second method uses a synthetic method for the complete selective formation of three pairs of disulfide bonds. Its characteristics are the use of different protecting groups in solid-phase synthesis, cleavage to obtain crude linaclotide peptide, and finally all of them are formed in the liquid phase through a distributed removal and oxidation strategy to form three pairs of disulfide bonds. However, this method uses three different cysteine ​​side chain protecting groups and completes the three-step oxidation in the liquid phase. Iodine is used in the liquid phase oxidation process, which not only greatly increases the risk of chromatographic column contamination in the subsequent purification process, but also uses less solvent than the solid phase oxidation, while the liquid phase reaction consumes more solvent. The third method adopts a strategy that combines fragment condensation with step-by-step oxidation. First, a hexapeptide fragment is synthesized, cyclized to obtain a fragment containing a pair of disulfide bonds, and then condensed with another fragment peptide resin to obtain a linaclotide peptide resin containing a pair of disulfide bonds. After cleavage, a crude linaclotide peptide containing a pair of disulfide bonds is obtained. After step-by-step oxidation, the cyclized crude linaclotide is obtained, and then purified to obtain linaclotide. This method uses fragment condensation and step-by-step oxidation. Due to the exchange of disulfide bonds, products with mismatched disulfide bonds will still be formed during the directional oxidation process. Not only is the operation complicated and costly, but the yield is also low, making it unsuitable for scale-up production. In summary, the existing synthesis methods have significant deficiencies in product yield, process simplicity, and large-scale production. Therefore, developing a solid-phase synthesis process for linaclotide has important practical significance. Summary of the Invention

[0003] The object of the present invention is to provide a solid-phase synthesis method of linaclotide, which not only significantly improves the total yield of linaclotide by optimizing the solid-phase synthesis process, but also improves the purity of the crude linaclotide peptide by optimizing the structure and composition of the high-performance liquid chromatography column.

[0004] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are:

[0005] A solid-phase synthesis method for linaclotide comprises: using a solid-phase synthesis method, using Fmoc-Tyr(tBu)-Wang resin as a starting material, sequentially performing amino acid coupling reactions from the C-terminus to the N-terminus to obtain a linaclotide peptide resin; then subjecting the resin to a deprotecting agent treatment, oxidation, and cleavage treatment to obtain a crude linaclotide peptide; wherein the deprotecting agent is an N,N-dimethylformamide solution containing piperidine, and the mass of the piperidine is equivalent to 10-30 wt% of the volume of the N,N-dimethylformamide.

[0006] The present invention provides a method for synthesizing linaclotide by completing a directional three-step disulfide bond bridging on a solid phase. The method is simple to operate and significantly reduces the operational complexity of traditional synthesis methods; effectively reduces the generation of products such as mismatched disulfide bonds, and significantly improves the purity of the linaclotide product; at the same time, the overall process is economical, efficient, stable and reliable, with a high raw material utilization rate, significantly improving the total yield of linaclotide, being suitable for scale-up production, and having important economic value.

[0007] Preferably, in the synthesis of Fmoc-Tyr(tBu)-Wang resin, Fmoc-Tyr(tBu)-OH is mixed with an activator, first reacted with Wang resin under the action of N,N'-diisopropylcarbodiimide, and then reacted with a functional agent to obtain Fmoc-Tyr(tBu)-Wang resin.

[0008] More preferably, the activator comprises 1-hydroxybenzotriazole and 4-dimethylaminopyridine.

[0009] More preferably, the functional agent includes acetic anhydride, N,N-diisopropylethylamine and N,N-dimethylformamide.

[0010] Preferably, the order of amino acid coupling is Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(4-MeOBzl)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Bzh)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(4-MeOBzl)-OH, and Fmoc-Cys(Bzh)-OH.

[0011] More preferably, after the amino acid is coupled to the fifth amino acid Fmoc-Cys(Acm)-OH, iodine solution is added to complete the Cys 13 With Cys 5 The disulfide bond bridging cyclization; the iodine solution is a mixture of elemental iodine and N,N-dimethylformamide.

[0012] More preferably, after the amino acid is coupled to the second amino acid Fmoc-Cys(4-MeOBzl)-OH, an oxidant is added to complete the Cys 10 With Cys 2 The disulfide bond bridging cyclization is carried out; the oxidant is a mixture of N-iodosuccinimide and N,N-dimethylformamide.

[0013] Preferably, the solid-phase synthesis method of linaclotide further comprises purification of crude linaclotide peptide; in the purification of crude linaclotide peptide, the crude linaclotide peptide is purified by high-performance liquid chromatography to obtain linaclotide; the chromatographic column of the high-performance liquid chromatography is filled with a prepolymer solution and then reacted in a water bath to obtain linaclotide.

[0014] More preferably, in the preparation of the prepolymerization solution, the alkenyl compound is mixed with 1-dodecanol and azobisisobutyronitrile.

[0015] More preferably, the alkenyl compound comprises methyl methacrylate and at least one of allyltriethoxysilane and allyl benzoate. The present invention optimizes and modulates the surface properties and spatial structure of the stationary phase by modifying the chromatographic column system, thereby increasing the specific surface area of ​​the stationary phase and enhancing its purification stability. Application of the modified chromatographic column system to the purification of crude linaclotide peptide significantly improves the purification efficiency of crude linaclotide, thereby increasing the purity of the final linaclotide product and providing reliable technical support for the industrial production of linaclotide.

[0016] Preferably, the synthesis of Fmoc-Tyr(tBu)-Wang resin is specifically as follows:

[0017] S1. Weigh Wang resin, add DMF and swell with nitrogen for 20-40 minutes to obtain swollen Wang resin.

[0018] S2. Weigh Fmoc-Tyr(tBu)-OH, HOBt, and DMAP, add DMF and stir to dissolve, precool to 0-5°C, add DIC to activate for 5-20 minutes, add all the reaction solution to the swollen Wang resin, react for 2-4 hours, remove the reaction solution, seal, and obtain an intermediate solution.

[0019] S3. Add acetic anhydride, DIEA and DMF to the intermediate solution and react for 12-36 hours. Wash with DMF 3-5 times, then wash with DCM 2-4 times, and finally shrink with methanol and dry to obtain Fmoc-Tyr(tBu)-Wang resin.

[0020] More preferably, the substitution degree of Wang resin in step S1 is 0.9-1.2 mmol / g.

[0021] More preferably, the ratio of Wang resin to DMF in step S1 is 1 mmol:5-10 mL.

[0022] More preferably, in step S2, the molar ratio of Fmoc-Tyr(tBu)-OH to HOBt is 1:1-2.

[0023] More preferably, in step S2, the molar ratio of Fmoc-Tyr(tBu)-OH to DMAP is 1:0.1-0.2.

[0024] More preferably, in step S2, the usage ratio of Fmoc-Tyr(tBu)-OH and DMF is 1 mmol:1-5 mL.

[0025] More preferably, in step S2, the molar ratio of Fmoc-Tyr(tBu)-OH to DIC is 1:1-2.

[0026] More preferably, in step S3, the volume ratio of acetic anhydride to DIEA is 1:0.5-1.

[0027] More preferably, in step S3, the volume ratio of acetic anhydride to DMF is 1:10-15.

[0028] Preferably, the synthesis of linaclotide peptide resin is specifically as follows:

[0029] S1. Wash the Fmoc-Tyr(tBu)-Wang resin 1-5 times with a detergent solution, then add DMF to swell for 20-40 minutes. Add a DMF solution containing Pip to remove the Fmoc group, and then wash it 4-6 times with a detergent solution to obtain Tyr(tBu)-Wang resin. Samples are taken and tested using the Kaiser assay. A positive result for Tyr(tBu)-Wang resin indicates the presence of free amines in the polypeptide, i.e., the amines are unprotected and can be used for subsequent amino acid coupling reactions.

[0030] S2. Weigh Fmoc-Cys(Acm)-OH and HOBt, dissolve them in DMF, add DIC to activate for 2-10 min in an ice-water bath at 0-5°C, add all the reaction solution to Tyr(tBu)-Wang resin and react for 1-4 h to obtain Fmoc-Cys(Acm)-Tyr(tBu)-Wang resin.

[0031] S3. Repeat steps S1 and S2, and sequentially couple Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(4-MeOBzl)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Bzh)-OH, and Fmoc-Cys(Acm)-OH until Fmoc-Cys(Acm)-Cys(Bzh)-Asn(Trt)-Pro-Ala-Cys(4-MeOBzl)-Thr(tBu)-Gly-Cys(Acm)-Tyr(tBu)-Wang resin is obtained.

[0032] S4. Weigh iodine (I2) and dissolve it in DMF to obtain an iodine solution. Add all the iodine solution to Fmoc-Cys(Acm)-Cys(Bzh)-Asn(Trt)-Pro-Ala-Cys(4-MeOBzl)-Thr(tBu)-Gly-Cys(Acm)-Tyr(tBu)-Wang resin and react for 1-2 hours. Wash with washing solution 4-6 times to obtain Fmoc-Cys-Cys(Bzh)-Asn(Trt)-Pro-Ala-Cys(4-MeOBzl)-Thr(tBu)-Gly-Cys-Tyr(tBu)-Wang resin (disulfide bridge: Cys 5 -Cys 13 ).

[0033] S5. Repeat steps S1 and S2 to sequentially couple Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, and Fmoc-Cys(4-MeOBzl)-OH until Fmoc-Cys(4-MeOBzl)-Glu(OtBu)-Tyr(tBu)-Cys-Cys(Bzh)-Asn(Trt)-Pro-Ala-Cys(4-MeOBzl)-Thr(tBu)-Gly-Cys-Tyr(tBu)-Wang resin (disulfide bridge: Cys 5 -Cys 13 ).

[0034] S6. Weigh NIS and DMF to obtain an oxidant, and add all of the oxidant to Fmoc-Cys(4-MeOBzl)-Glu(OtBu)-Tyr(tBu)-Cys-Cys(Bzh)-Asn(Trt)-Pro-Ala-Cys(4-MeOBzl)-Thr(tBu)-Gly-Cys-Tyr(tBu)-Wang resin (disulfide bridge: Cys 5 -Cys 13) for 1-2 h, and washed 4-6 times with washing solution to obtain Fmoc-Cys(4-MeOBzl)-Glu(OtBu)-Tyr(tBu)-Cys-Cys(Bzh)-Asn(Trt)-Pro-Ala-Cys(4-MeOBzl)-Thr(tBu)-Gly-Cys-Tyr(tBu)-Wang resin (disulfide bridge: Cys 2 -Cys 10 &Cys 5 -Cys 13 ).

[0035] S7, repeat steps S1 and S2, couple Fmoc-Cys(Bzh)-OH to obtain Fmoc-Cys(Bzh)-Cys(4-MeOBzl)-Glu(OtBu)-Tyr(tBu)-Cys-Cys(Bzh)-Asn(Trt)-Pro-Ala-Cys(4-MeOBzl)-Thr(tBu)-Gly-Cys-Tyr(tBu)-Wang resin (disulfide bridge: Cys 2 -Cys 10 &Cys 5 -Cys 13 ); then add DMF solution containing Pip to remove Fmoc group, wash with DMF 2-5 times, wash with DCM 2-5 times, finally shrink with methanol and dry to obtain linaclotide peptide resin, the sequence of which is: H2N-Cys(Bzh)-Cys(4-MeOBzl)-Glu(OtBu)-Tyr(tBu)-Cys-Cys(Bzh)-Asn(Trt)-Pro-Ala-Cys(4-MeOBzl)-Thr(tBu)-Gly-Cys-Tyr(tBu)-Wang resin (disulfide bridge: Cys 2 -Cys 10 &Cys 5 -Cys 13 ).

[0036] More preferably, in step S1, the washing liquid is DMF.

[0037] More preferably, in step S1, the usage ratio of Fmoc-Tyr(tBu)-Wang resin and DMF is 1 mmol:2-10 mL.

[0038] More preferably, in step S1, in the DMF solution containing Pip, the mass of Pip is equivalent to 10-30 wt% of the volume of DMF.

[0039] More preferably, in step S1, the usage ratio of Fmoc-Tyr(tBu)-Wang resin and DMF solution containing Pip is 1 mmol:2-10 mL.

[0040] More preferably, in step S2, the molar ratio of Fmoc-Cys(Acm)-OH to HOBt is 1:1-2.

[0041] More preferably, in step S2, the usage ratio of Fmoc-Cys(Acm)-OH to DMF is 1 mmol:2-10 mL.

[0042] More preferably, in step S2, the molar ratio of Fmoc-Cys(Acm)-OH to DIC is 1:1-2.

[0043] More preferably, in step S4, the usage ratio of I2 and DMF is 1 mmol:0.1-0.5 mL.

[0044] More preferably, in step S4, the washing liquid is DMF.

[0045] More preferably, in step S6, the usage ratio of NIS to DMF is 1 mmol:1-5 mL.

[0046] More preferably, in step S6, the washing liquid is DMF.

[0047] More preferably, in step S7, in the DMF solution containing Pip, the mass of Pip is equivalent to 10-30 wt % of the volume of DMF.

[0048] More preferably, in step S7, the usage ratio of Fmoc-Cys(Bzh)-OH to the DMF solution containing Pip is 1 mmol:2-10 mL.

[0049] Preferably, the synthesis of crude linaclotide peptide is specifically as follows:

[0050] TFA, DMSO, and H2O were measured and mixed to obtain a lysate, which was pre-cooled to below 0°C; linaclotide peptide resin was slowly added to the lysate, and the reaction was carried out in a water bath at 20-30°C for 2-5 hours. The mixture was filtered, and the filtrate was retained. The filtrate was slowly added to methyl tert-butyl ether pre-cooled to below 0°C, and the precipitated solid was collected. The solid was then washed with methyl tert-butyl ether 2-5 times, and the solid was vacuumed and dried at a constant temperature at 20-30°C for 12-24 hours to obtain a crude linaclotide peptide, the sequence of which is: H2N-Cys 1 -Cys 2 -Glu 3 -Tyr 4 -Cys 5 -Cys 6 -Asn 7-Pro 8 -Ala 9 -Cys 10 -Thr 11 -Gly 12 -Cys 13 -Tyr 14 -OH (disulfide bridge: Cys 1 -Cys 6 &Cys 2 -Cys 10 &Cys 5 -Cys 13 ).

[0051] More preferably, in the lysis solution, the volume ratio of DMSO to TFA is 1:5-10.

[0052] More preferably, in the lysis solution, the volume ratio of DMSO to H2O is 1:0.2-1.

[0053] More preferably, the usage ratio of linaclotide peptide resin to lysis solution is 1 g: 5-20 mL.

[0054] More preferably, the usage ratio of linaclotide peptide resin and methyl tert-butyl ether is 1 g: 5-20 mL.

[0055] Preferably, the solid phase synthesis method of linaclotide further comprises purification of crude linaclotide peptide, specifically,

[0056] The alkenyl compound is mixed with methanol, and 1-dodecanol and azobisisobutyronitrile are added to obtain a prepolymerization solution; the prepolymerization solution is ultrasonically mixed and degassed at 0-10°C for 5-30 minutes, and then purged with nitrogen for 20-40 minutes. The prepolymerization solution is injected into an empty tube chromatographic column with one end sealed. After the injection, the other end of the chromatographic column is sealed and vertically placed in a 50-60°C water bath for reaction for 12-24 hours. After the reaction is completed, the chromatographic column is removed and connected to a high performance liquid chromatograph; the crude linaclotide peptide solution is loaded into the chromatographic column using high performance liquid chromatography, and the mobile phase is started to elute to obtain linaclotide.

[0057] More preferably, the alkenyl compound includes methyl methacrylate and at least one of allyltriethoxysilane, allyl benzoate, and methylheptenol. The present invention further introduces methylheptenol into the chromatographic column system, which, through its hydrophobic properties, may significantly improve the separation selectivity and purification stability of the stationary phase and enhance its interaction with linaclotide, thereby achieving efficient purification of linaclotide and significantly improving the purity of the linaclotide final product.

[0058] More preferably, the mass ratio of methyl methacrylate to allyltriethoxysilane is 1:1.5-3.

[0059] More preferably, the mass ratio of methyl methacrylate to allyl formate is 1:0.2-1.

[0060] More preferably, the mass ratio of methyl methacrylate to methyl heptenol is 0.1-0.5.

[0061] More preferably, the ratio of the alkenyl compound to methanol is 1 g: 2-10 mL.

[0062] More preferably, the mass ratio of the alkenyl compound to 1-dodecanol is 1:0.01-0.02.

[0063] More preferably, the mass ratio of the alkenyl compound to azobisisobutyronitrile is 1:0.015-0.03.

[0064] More preferably, in the preparation of the crude linaclotide peptide solution, the crude linaclotide peptide is dissolved in DMF to obtain the crude linaclotide peptide solution, and the amount ratio of the crude linaclotide peptide to DMF is 1 g: 5-20 mL.

[0065] The present invention utilizes a linaclotide synthesis method that performs a three-step disulfide bridge on a solid phase to obtain crude linaclotide peptide, followed by optimization of the HPLC column system. This method offers the following beneficial effects: The solid-phase synthesis method for linaclotide provided by the present invention is not only simple to operate, significantly reducing the operational complexity of traditional synthesis methods, but also effectively reduces the formation of products such as mismatched disulfide bonds, significantly increasing the overall yield of linaclotide, reaching 98.3-99.4%. Furthermore, by optimizing the HPLC column system, the present invention significantly improves the purification stability of the column, resulting in a relative standard deviation of 0.44-1.04% for column purification, thereby significantly increasing the purity of the purified crude linaclotide peptide to 89.7-98.6%. Therefore, the present invention provides a solid-phase synthesis method for linaclotide with high overall yield and purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 Flow chart of the solid phase synthesis method of linaclotide.

[0067] Figure 2 The crude peptide yield of linaclotide prepared in Example 1-2. DETAILED DESCRIPTION

[0068] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0069] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are commercially available unless otherwise specified.

[0070] Table 1 shows the English abbreviations and Chinese meanings of the reagents used in the present invention.

[0071] Table 1 English abbreviations and Chinese meanings of reagents used in the present invention

[0072]

[0073] Example 1:

[0074] A solid-phase synthesis method of linaclotide includes the synthesis of Fmoc-Tyr(tBu)-Wang resin, the synthesis of linaclotide peptide resin and the synthesis of linaclotide crude peptide.

[0075] The synthesis of Fmoc-Tyr(tBu)-Wang resin includes:

[0076] S1. Weigh Wang resin, add DMF and swell it with nitrogen for 30 minutes to obtain swollen Wang resin; the substitution degree of Wang resin is 1.2 mmol / g, and the usage ratio of Wang resin and DMF is 1 mmol:7 mL.

[0077] S2. Weigh Fmoc-Tyr(tBu)-OH, HOBt, and DMAP, add DMF, stir and dissolve, precool to 0°C, add DIC to activate for 10 minutes, add the entire reaction solution to the swollen Wang resin, react for 2.5 hours, remove the reaction solution, seal, and obtain an intermediate solution. The molar ratio of Fmoc-Tyr(tBu)-OH to HOBt is 1:1.2, the molar ratio of Fmoc-Tyr(tBu)-OH to DMAP is 1:0.1, the amount ratio of Fmoc-Tyr(tBu)-OH to DMF is 1 mmol:2.5 mL, and the molar ratio of Fmoc-Tyr(tBu)-OH to DIC is 1:1.2.

[0078] S3. Add acetic anhydride, DIEA, and DMF to the intermediate solution and allow to react for 24 hours. Wash the mixture four times with DMF, then three times with DCM, shrink it with methanol, and dry it to obtain Fmoc-Tyr(tBu)-Wang resin. The volume ratio of acetic anhydride to DIEA is 1:0.875; the volume ratio of acetic anhydride to DMF is 1:10.625.

[0079] Synthesis of linaclotide peptide resin, including,

[0080] S1. Wash the Fmoc-Tyr(tBu)-Wang resin twice with a washing solution, then add DMF to swell for 30 minutes, add a DMF solution containing Pip to remove the Fmoc group, and then wash it six times with a washing solution to obtain Tyr(tBu)-Wang resin. Take a sample and test it using the Kaiser test method. The Tyr(tBu)-Wang resin is positive. The washing solution is DMF; the ratio of Fmoc-Tyr(tBu)-Wang resin to DMF is 1mmol:5mL; in the DMF solution containing Pip, the mass of Pip is equivalent to 20wt% of the volume of DMF; the ratio of Fmoc-Tyr(tBu)-Wang resin to the DMF solution containing Pip is 1mmol:5mL.

[0081] S2. Weigh Fmoc-Cys(Acm)-OH and HOBt, dissolve them in DMF, and activate them with DIC in a 0°C ice-water bath for 5 minutes. Add the entire reaction solution to Tyr(tBu)-Wang resin and react for 2 hours to obtain Fmoc-Cys(Acm)-Tyr(tBu)-Wang resin. The molar ratio of Fmoc-Cys(Acm)-OH to HOBt is 1:1; the ratio of Fmoc-Cys(Acm)-OH to DMF is 1 mmol:5 mL; and the molar ratio of Fmoc-Cys(Acm)-OH to DIC is 1:1.

[0082] S3. Repeat steps S1 and S2, and sequentially couple Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(4-MeOBzl)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Bzh)-OH, and Fmoc-Cys(Acm)-OH until Fmoc-Cys(Acm)-Cys(Bzh)-Asn(Trt)-Pro-Ala-Cys(4-MeOBzl)-Thr(tBu)-Gly-Cys(Acm)-Tyr(tBu)-Wang resin is obtained.

[0083] S4. Weigh iodine (I2) and dissolve it in DMF to obtain an iodine solution. Add all the iodine solution to Fmoc-Cys(Acm)-Cys(Bzh)-Asn(Trt)-Pro-Ala-Cys(4-MeOBzl)-Thr(tBu)-Gly-Cys(Acm)-Tyr(tBu)-Wang resin and react for 1 hour. Wash with washing solution 6 times to obtain Fmoc-Cys-Cys(Bzh)-Asn(Trt)-Pro-Ala-Cys(4-MeOBzl)-Thr(tBu)-Gly-Cys-Tyr(tBu)-Wang resin (disulfide bridge: Cys 5-Cys 13 The ratio of I2 to DMF was 1 mmol:0.25 mL; the washing liquid was DMF.

[0084] S5. Repeat steps S1 and S2 to sequentially couple Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, and Fmoc-Cys(4-MeOBzl)-OH until Fmoc-Cys(4-MeOBzl)-Glu(OtBu)-Tyr(tBu)-Cys-Cys(Bzh)-Asn(Trt)-Pro-Ala-Cys(4-MeOBzl)-Thr(tBu)-Gly-Cys-Tyr(tBu)-Wang resin (disulfide bridge: Cys 5 -Cys 13 ).

[0085] S6. Weigh NIS and DMF to obtain an oxidant, and add all of the oxidant to Fmoc-Cys(4-MeOBzl)-Glu(OtBu)-Tyr(tBu)-Cys-Cys(Bzh)-Asn(Trt)-Pro-Ala-Cys(4-MeOBzl)-Thr(tBu)-Gly-Cys-Tyr(tBu)-Wang resin (disulfide bridge: Cys 5 -Cys 13 ) for 1 h, and washed 6 times with washing solution to obtain Fmoc-Cys(4-MeOBzl)-Glu(OtBu)-Tyr(tBu)-Cys-Cys(Bzh)-Asn(Trt)-Pro-Ala-Cys(4-MeOBzl)-Thr(tBu)-Gly-Cys-Tyr(tBu)-Wang resin (disulfide bridge: Cys 2 -Cys 10 &Cys 5 -Cys 13 The ratio of NIS to DMF was 1 mmol:3 mL; the washing solution was DMF.

[0086] S7, repeat steps S1 and S2, couple Fmoc-Cys(Bzh)-OH to obtain Fmoc-Cys(Bzh)-Cys(4-MeOBzl)-Glu(OtBu)-Tyr(tBu)-Cys-Cys(Bzh)-Asn(Trt)-Pro-Ala-Cys(4-MeOBzl)-Thr(tBu)-Gly-Cys-Tyr(tBu)-Wang resin (disulfide bridge: Cys 2 -Cys 10 &Cys 5 -Cys 13); DMF solution containing Pip was then added to remove the Fmoc group, and the mixture was washed three times with DMF and three times with DCM, and finally shrunk with methanol and dried to obtain linaclotide peptide resin, the sequence of which is: H2N-Cys(Bzh)-Cys(4-MeOBzl)-Glu(OtBu)-Tyr(tBu)-Cys-Cys(Bzh)-Asn(Trt)-Pro-Ala-Cys(4-MeOBzl)-Thr(tBu)-Gly-Cys-Tyr(tBu)-Wang resin (disulfide bridge: Cys 2 -Cys 10 &Cys 5 -Cys 13 In the DMF solution containing Pip, the mass of Pip is equivalent to 20 wt % of the volume of DMF; the usage ratio of Fmoc-Cys(Bzh)-OH to the DMF solution containing Pip is 1 mmol:5 mL.

[0087] Synthesis of crude linaclotide peptide, including:

[0088] TFA, DMSO, and H2O were measured and mixed to obtain a lysate, which was precooled to -20°C; linaclotide peptide resin was slowly added to the lysate, and the reaction was carried out in a water bath at 25°C for 3 hours. The mixture was filtered, and the filtrate was retained. The filtrate was slowly added to methyl tert-butyl ether precooled to -20°C, and the precipitated solid was collected. The solid was then washed three times with methyl tert-butyl ether, and the solid was vacuumed and dried at a constant temperature at 25°C for 24 hours to obtain a crude linaclotide peptide, the sequence of which is: H2N-Cys 1 -Cys 2 -Glu 3 -Tyr 4 -Cys 5 -Cys 6 -Asn 7 -Pro 8 -Ala 9 -Cys 10 -Thr 11 -Gly 12 -Cys 13 -Tyr 14 -OH (disulfide bridge: Cys 1 -Cys 6 &Cys 2 -Cys 10 &Cys 5 -Cys 13In the lysis buffer, the volume ratio of DMSO to TFA was 1:8.5, and the volume ratio of DMSO to H2O was 1:0.5; the amount ratio of linaclotide peptide resin to lysis buffer was 1 g:10 mL; and the amount ratio of linaclotide peptide resin to methyl tert-butyl ether was 1 g:10 mL.

[0089] Example 2:

[0090] A solid-phase synthesis method of linaclotide includes the synthesis of Fmoc-Tyr(tBu)-Wang resin, the synthesis of linaclotide peptide resin and the synthesis of linaclotide crude peptide.

[0091] The synthesis of Fmoc-Tyr(tBu)-Wang resin was performed in the same manner as in Example 1, except that the Wang resin used in step S1 was changed to a Wang resin with a substitution degree of 0.9 mmol / g. Other conditions were the same as in Example 1.

[0092] The synthesis of linaclotide peptide resin was performed in the same manner as in Example 1, except that the Fmoc-Tyr(tBu)-Wang resin in step S1 was replaced with the Fmoc-Tyr(tBu)-Wang resin prepared in this example.

[0093] The synthesis of crude linaclotide peptide was carried out under the same conditions as in Example 1, except that the linaclotide peptide resin was replaced with the linaclotide peptide resin prepared in this example.

[0094] Example 3:

[0095] A solid-phase synthesis method of linaclotide includes the synthesis of Fmoc-Tyr(tBu)-Wang resin, the synthesis of linaclotide peptide resin, the synthesis of crude linaclotide peptide and the purification of crude linaclotide peptide.

[0096] The synthesis of Fmoc-Tyr(tBu)-Wang resin was the same as in Example 1.

[0097] The synthesis of linaclotide peptide resin is the same as in Example 1.

[0098] The synthesis of crude linaclotide peptide was the same as in Example 1.

[0099] Purification of crude linaclotide peptide, including,

[0100] Methyl methacrylate, allyltriethoxysilane, allyl benzoate and methanol were mixed, and 1-dodecanol and azobisisobutyronitrile were added to obtain a prepolymerization solution. The prepolymerization solution was ultrasonically mixed and degassed at 4°C for 15 minutes, and then purged with nitrogen for 30 minutes. The prepolymerization solution was injected into an empty tube chromatographic column with one end sealed. After the injection, the other end of the chromatographic column was sealed and placed vertically in a 60°C water bath for reaction for 12 hours. After the reaction was completed, the chromatographic column was removed and connected to a high performance liquid chromatograph. Using high performance liquid chromatography, the crude linaclotide peptide solution was loaded into the chromatographic column, and the mobile phase was started to elute to collect linaclotide. The mass ratio of methyl methacrylate to allyltriethoxysilane is 1:3; the mass ratio of methyl methacrylate to allyl benzoate is 1:1; the amount ratio of the alkenyl compound to methanol is 1 g:5 mL; the mass ratio of the alkenyl compound to 1-dodecanol is 1:0.01; the mass ratio of the alkenyl compound to azobisisobutyronitrile is 1:0.015; in the preparation of the crude linaclotide peptide solution, the crude linaclotide peptide is dissolved in DMF to obtain the crude linaclotide peptide solution, and the amount ratio of the crude linaclotide peptide to DMF is 1 g:10 mL.

[0101] Example 4:

[0102] A solid-phase synthesis method of linaclotide includes the synthesis of Fmoc-Tyr(tBu)-Wang resin, the synthesis of linaclotide peptide resin, the synthesis of crude linaclotide peptide and the purification of crude linaclotide peptide.

[0103] The synthesis of Fmoc-Tyr(tBu)-Wang resin was the same as in Example 1.

[0104] The synthesis of linaclotide peptide resin is the same as in Example 1.

[0105] The synthesis of crude linaclotide peptide was the same as in Example 1.

[0106] The purification of crude linaclotide peptide was carried out under the same conditions as in Example 3 except that the mass ratio of methyl methacrylate to allyltriethoxysilane was changed to 1:1.5.

[0107] Example 5:

[0108] A solid-phase synthesis method of linaclotide includes the synthesis of Fmoc-Tyr(tBu)-Wang resin, the synthesis of linaclotide peptide resin, the synthesis of crude linaclotide peptide and the purification of crude linaclotide peptide.

[0109] The synthesis of Fmoc-Tyr(tBu)-Wang resin was the same as in Example 1.

[0110] The synthesis of linaclotide peptide resin is the same as in Example 1.

[0111] The synthesis of crude linaclotide peptide was the same as in Example 1.

[0112] The purification of crude linaclotide peptide was carried out under the same conditions as in Example 3 except that the mass ratio of methyl methacrylate to allyl benzoate was changed to 1:0.2.

[0113] Example 6:

[0114] A solid-phase synthesis method of linaclotide includes the synthesis of Fmoc-Tyr(tBu)-Wang resin, the synthesis of linaclotide peptide resin, the synthesis of crude linaclotide peptide and the purification of crude linaclotide peptide.

[0115] The synthesis of Fmoc-Tyr(tBu)-Wang resin was the same as in Example 1.

[0116] The synthesis of linaclotide peptide resin is the same as in Example 1.

[0117] The synthesis of crude linaclotide peptide was the same as in Example 1.

[0118] Purification of crude linaclotide peptide, including,

[0119] Methyl methacrylate, allyltriethoxysilane, allyl benzoate, methylheptenol and methanol were mixed, and 1-dodecanol and azobisisobutyronitrile were added to obtain a prepolymerization solution. The prepolymerization solution was ultrasonically mixed and degassed at 4°C for 15 minutes, and then purged with nitrogen for 30 minutes. The prepolymerization solution was injected into an empty tube chromatographic column with one end sealed. After the injection, the other end of the chromatographic column was sealed and placed vertically in a 60°C water bath for reaction for 12 hours. After the reaction was completed, the chromatographic column was removed and connected to a high performance liquid chromatograph. The crude linaclotide peptide solution was loaded into the chromatographic column using high performance liquid chromatography, and the mobile phase was started to elute to collect linaclotide. The mass ratio of methyl methacrylate to allyltriethoxysilane is 1:3; the mass ratio of methyl methacrylate to allyl benzoate is 1:1; the mass ratio of methyl methacrylate to methylheptenol is 1:0.5; the mass ratio of the alkenyl compound to methanol is 1 g:5 mL; the mass ratio of the alkenyl compound to 1-dodecanol is 1:0.01; the mass ratio of the alkenyl compound to azobisisobutyronitrile is 1:0.015; in the preparation of the crude linaclotide peptide solution, the crude linaclotide peptide is dissolved in DMF to obtain the crude linaclotide peptide solution, and the mass ratio of the crude linaclotide peptide to DMF is 1 g:10 mL.

[0120] Example 7:

[0121] A solid-phase synthesis method of linaclotide includes the synthesis of Fmoc-Tyr(tBu)-Wang resin, the synthesis of linaclotide peptide resin, the synthesis of crude linaclotide peptide and the purification of crude linaclotide peptide.

[0122] The synthesis of Fmoc-Tyr(tBu)-Wang resin was the same as in Example 1.

[0123] The synthesis of linaclotide peptide resin is the same as in Example 1.

[0124] The synthesis of crude linaclotide peptide was the same as in Example 1.

[0125] The purification of crude linaclotide peptide was carried out under the same conditions as in Example 6 except that the mass ratio of methyl methacrylate to methylheptenol was changed to 1:0.1.

[0126] Comparative Example 1:

[0127] A solid-phase synthesis method of linaclotide includes the synthesis of Fmoc-Tyr(tBu)-Wang resin, the synthesis of linaclotide peptide resin, the synthesis of crude linaclotide peptide and the purification of crude linaclotide peptide.

[0128] The synthesis of Fmoc-Tyr(tBu)-Wang resin was the same as in Example 1.

[0129] The synthesis of linaclotide peptide resin is the same as in Example 1.

[0130] The synthesis of crude linaclotide peptide was the same as in Example 1.

[0131] The purification of crude linaclotide peptide was carried out under the same conditions as in Example 3 except that allyltriethoxysilane and allyl benzoate were not used.

[0132] Comparative Example 2:

[0133] A solid-phase synthesis method of linaclotide includes the synthesis of Fmoc-Tyr(tBu)-Wang resin, the synthesis of linaclotide peptide resin, the synthesis of crude linaclotide peptide and the purification of crude linaclotide peptide.

[0134] The synthesis of Fmoc-Tyr(tBu)-Wang resin was the same as in Example 1.

[0135] The synthesis of linaclotide peptide resin is the same as in Example 1.

[0136] The synthesis of crude linaclotide peptide was the same as in Example 1.

[0137] The purification of crude linaclotide peptide was carried out under the same conditions as in Example 3 except that allyltriethoxysilane was not used.

[0138] Comparative Example 3:

[0139] A solid-phase synthesis method of linaclotide includes the synthesis of Fmoc-Tyr(tBu)-Wang resin, the synthesis of linaclotide peptide resin, the synthesis of crude linaclotide peptide and the purification of crude linaclotide peptide.

[0140] The synthesis of Fmoc-Tyr(tBu)-Wang resin was the same as in Example 1.

[0141] The synthesis of linaclotide peptide resin is the same as in Example 1.

[0142] The synthesis of crude linaclotide peptide was the same as in Example 1.

[0143] The purification of crude linaclotide peptide was carried out under the same conditions as in Example 3 except that allyl benzoate was not used.

[0144] Comparative Example 4:

[0145] A solid-phase synthesis method of linaclotide includes the synthesis of Fmoc-Tyr(tBu)-Wang resin, the synthesis of linaclotide peptide resin, the synthesis of crude linaclotide peptide and the purification of crude linaclotide peptide.

[0146] The synthesis of Fmoc-Tyr(tBu)-Wang resin was the same as in Example 1.

[0147] The synthesis of linaclotide peptide resin is the same as in Example 1.

[0148] The synthesis of crude linaclotide peptide was the same as in Example 1.

[0149] The purification of crude linaclotide peptide was carried out under the same conditions as in Example 6 except that allyltriethoxysilane and allyl benzoate were not used.

[0150] Experimental example:

[0151] 1. Substitution test of Fmoc-Tyr(tBu)-Wang resin

[0152] The Fmoc-Tyr(tBu)-Wang resins prepared in Example 1 and Example 2 were collected, and the substitution degrees of the Fmoc-Tyr(tBu)-Wang resins prepared in Example 1 and Example 2 were determined using an ultraviolet spectrophotometer.

[0153] The substitution degree of the Fmoc-Tyr(tBu)-Wang resin prepared in Example 1 is 0.62 mmol / g, and the substitution degree of the Fmoc-Tyr(tBu)-Wang resin prepared in Example 2 is 0.58 mmol / g.

[0154] 2. Yield

[0155] Figure 1 The solid-phase synthesis process flow chart for linaclotide is shown below. The actual yields of the linaclotide peptide resin and crude linaclotide peptide prepared in Examples 1-2 were weighed, and the crude linaclotide peptide yield was calculated: Yield (%) = Actual crude linaclotide peptide yield / Theoretical yield × 100%.

[0156] The actual yield of the linaclotide peptide resin prepared in Example 1 was 65.39 g, and the actual yield of the crude linaclotide peptide was 4.59 g; the actual yield of the linaclotide peptide resin prepared in Example 2 was 66.83 g, and the actual yield of the crude linaclotide peptide was 4.64 g. Figure 2 is the crude peptide yield of linaclotide prepared in Example 1-2. The crude peptide yield of linaclotide prepared in Example 1 is 98.3%, and the crude peptide yield of linaclotide prepared in Example 2 is 99.4%.

[0157] 3. Purity

[0158] The purity of the crude linaclotide prepared in Examples 1-2, as well as the linaclotide prepared in Examples 3-7 and Comparative Examples 1-3, was determined by high performance liquid chromatography. The results are shown in Table 2. Table 2 shows the purity (%).

[0159] Table 2 Purity (%)

[0160]

[0161] As shown in Table 2, the purity of Examples 3-5 of the present invention is higher than that of Examples 1-2. This is because Examples 3-5 use high-performance liquid chromatography to purify crude linaclotide peptide to obtain linaclotide, while Examples 1-2 do not purify the crude linaclotide peptide. This shows that the use of high-performance liquid chromatography to purify crude linaclotide peptide can effectively improve the purity of linaclotide.

[0162] The purity of Examples 3-5 was higher than that of Comparative Example 1 because, in the purification of crude linaclotide peptide, Comparative Example 1 used a prepolymerization solution prepared by mixing methyl methacrylate, methanol, 1-dodecanol, and azobisisobutyronitrile to fill the chromatographic column, which was then used for purification of crude linaclotide peptide after a water bath reaction; while Examples 3-5 also added allyltriethoxysilane and allyl benzoate to the prepolymerization solution. The purity of Example 3 was higher than that of Examples 4 and 5 because the amounts of allyltriethoxysilane and allyl benzoate used were different. The purity of Examples 3-5 was higher than that of Comparative Examples 2 and 3 because Examples 3-5 used allyltriethoxysilane and allyl benzoate in combination, while Comparative Example 2 used only allyl benzoate and Comparative Example 3 used only allyltriethoxysilane. This shows that compared with the use of allyltriethoxysilane and allyl benzoate alone, the synergistic use of allyltriethoxysilane and allyl benzoate to prepare a prepolymer solution to fill the chromatographic column and then use it for the purification of crude linaclotide peptide after water bath reaction can effectively improve the purification effect of crude linaclotide peptide and enhance the purity of linaclotide.

[0163] The higher purity of Examples 6-7 than that of Example 3 is due to the further introduction of methylheptenol into the prepolymerization solution in Examples 6-7; the higher purity of Example 6 than that of Example 7 is due to the different amounts of methylheptenol used; and the higher purity of Examples 6-7 than that of Comparative Example 4 is due to the fact that Comparative Example 4 only introduced methylheptenol into the prepolymerization solution without using allyltriethoxysilane and allyl benzoate. This indicates that further introducing methylheptenol into the prepolymerization solution, then filling the prepolymerization solution into a chromatographic column, and then using it for purification of crude linaclotide after a water bath reaction can further improve the purity of linaclotide.

[0164] 4. Purification stability of chromatographic columns

[0165] To evaluate the stability of the chromatographic columns used in the purification of crude linaclotide peptide in Examples 3-7 and Comparative Examples 1-4, crude linaclotide peptide prepared in Example 1 was used as the test sample. The crude linaclotide peptide was injected 10 times according to the purification methods described in Examples 3-7 and Comparative Examples 1-4, respectively. The resulting linaclotide was collected and tested for purity. Dead time was calibrated using the solvent peak. The relative standard deviation (%) of the column retention factor was calculated as follows: standard deviation of retention factor / arithmetic mean of retention factor × 100%. Retention factor = (retention time of target compound - dead time) / dead time. The results are shown in Table 3. Table 3 shows the relative standard deviation (%).

[0166] Table 3 Relative standard deviation (%)

[0167]

[0168] As shown in Table 3, the relative standard deviations of Examples 3-5 are lower than those of Comparative Example 1. This is because, in the purification of crude linaclotide peptide, Comparative Example 1 uses a prepolymer solution prepared by mixing methyl methacrylate, methanol, 1-dodecanol, and azobisisobutyronitrile to fill the chromatographic column, which is then used for purification of crude linaclotide peptide after a water bath reaction; while Examples 3-5 also add allyltriethoxysilane and allyl benzoate to the prepolymer solution. The relative standard deviation of Example 3 is lower than that of Examples 4 and 5 due to the different amounts of allyltriethoxysilane and allyl benzoate used. The relative standard deviations of Examples 3-5 are lower than those of Comparative Examples 2 and 3 because Examples 3-5 use allyltriethoxysilane and allyl benzoate in combination, while Comparative Example 2 uses only allyl benzoate and Comparative Example 3 uses only allyltriethoxysilane. This shows that compared with the use of allyltriethoxysilane and allyl benzoate alone, the synergistic use of allyltriethoxysilane and allyl benzoate to prepare a prepolymer solution to fill the chromatographic column and then use it for the purification of crude linaclotide peptide after water bath reaction can effectively improve the purification stability of the chromatographic column for crude linaclotide peptide, thereby achieving a better purification effect for crude linaclotide peptide.

[0169] The relative standard deviations of Examples 6-7 were lower than those of Example 3 because Examples 6-7 further introduced methylheptenol into the prepolymerization solution; the relative standard deviation of Example 6 was lower than that of Example 7 due to the different amounts of methylheptenol used; and the relative standard deviations of Examples 6-7 were lower than those of Comparative Example 4 because Comparative Example 4 only introduced methylheptenol into the prepolymerization solution without using allyltriethoxysilane and allyl benzoate. This suggests that further introducing methylheptenol into the prepolymerization solution, then filling the prepolymerization solution into a chromatographic column, and then using it for purification of crude linaclotide peptide after a water bath reaction can further improve the chromatographic column's stability in purifying crude linaclotide peptide, thereby further enhancing the purification efficiency of crude linaclotide peptide.

[0170] The conventional operations in the operating steps of the present invention are well known to those skilled in the art and will not be described in detail here.

[0171] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above descriptions are only specific embodiments of the present invention and are not intended to limit the present invention. Any changes and modifications made within the scope of the principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A solid phase synthesis method of linaclotide, characterized in that: The method comprises: using a solid-phase synthesis method, using Fmoc-Tyr(tBu)-Wang resin as a starting material, sequentially performing amino acid coupling reactions from the C-terminus to the N-terminus to obtain a linaclotide peptide resin; then subjecting the resin to a deprotection agent treatment, oxidation, and cleavage treatment to obtain a crude linaclotide peptide; and purifying the crude linaclotide peptide by high-performance liquid chromatography to obtain linaclotide. In the solid phase synthesis of linaclotide peptide resin, after the amino acid is coupled to the fifth amino acid Fmoc-Cys(Acm)-OH, iodine solution is added to complete the Cys 13 With Cys 5 The disulfide bond bridged cyclization, the amino acid was coupled to the second amino acid Fmoc-Cys(4-MeOBzl)-OH, and the oxidant was added to complete the Cys 10 With Cys 2 disulfide bond bridging cyclization; the iodine solution is a mixture of elemental iodine and N,N-dimethylformamide; the oxidant is a mixture of N-iodosuccinimide and N,N-dimethylformamide; The synthesis steps of the crude linaclotide peptide are as follows: trifluoroacetic acid, dimethyl sulfoxide, and water are measured and mixed to obtain a lysate, and the lysate is pre-cooled to below 0°C; linaclotide peptide resin is slowly added to the lysate, reacted in a water bath at 20-30°C for 2-5 hours, filtered, and the filtrate is retained. The filtrate is slowly added to methyl tert-butyl ether pre-cooled to below 0°C, the precipitated solid is collected, and the solid is washed with methyl tert-butyl ether for 2-5 times, the solid is vacuumed, and dried at a constant temperature at 20-30°C for 12-24 hours to obtain the crude linaclotide peptide, whose sequence is: H2N-Cys 1 -Cys 2 -Glu 3 -Tyr 4 -Cys 5 -Cys 6 -Asn 7 -Pro 8 -Ala 9 -Cys 10 -Thr 11 -Gly 12 -Cys 13 -Tyr 14 -OH, where the disulfide bridge is Cys 1 -Cys 6 &Cys 2 -Cys 10 &Cys 5 -Cys 13 ; The chromatographic column of the high-performance liquid chromatography is filled with a prepolymerization solution and then reacted in a water bath; the deprotecting agent is an N,N-dimethylformamide solution containing piperidine, and the mass of piperidine is equivalent to 10wt%-30wt% of the volume of N,N-dimethylformamide; in the preparation of the prepolymerization solution, an alkenyl compound is mixed with 1-dodecanol and azobisisobutyronitrile to obtain the alkenyl compound; the alkenyl compound includes methyl methacrylate and at least one of allyltriethoxysilane, allyl benzoate and methylheptenol.

2. The solid phase synthesis method of linaclotide according to claim 1, characterized in that: In the synthesis of the Fmoc-Tyr(tBu)-Wang resin, Fmoc-Tyr(tBu)-OH is mixed with an activator, first reacted with the Wang resin under the action of N,N'-diisopropylcarbodiimide, and then reacted with a functional agent to obtain the Fmoc-Tyr(tBu)-Wang resin; the functional agent includes acetic anhydride, N,N-diisopropylethylamine and N,N-dimethylformamide.

3. The solid phase synthesis method of linaclotide according to claim 2, characterized in that: The activators include 1-hydroxybenzotriazole and 4-dimethylaminopyridine.

4. The solid phase synthesis method of linaclotide according to claim 1, characterized in that: The order of amino acid coupling is Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(4-MeOBzl)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Bzh)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(4-MeOBzl)-OH, and Fmoc-Cys(Bzh)-OH.

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