A method for the total liquid phase synthesis of a linatide intermediate and linatide

Abstract

The application discloses a method for synthesizing a linear linaclotide intermediate and linaclotide in a full liquid phase, and relates to the following process: at least two polypeptide fragments are designed and planned by segmenting a linear linaclotide; amino acid raw materials are selected according to the amino acid sequence order of the planned polypeptide fragments; the target polypeptide fragments are synthesized by using an activating agent one by one; the polypeptide fragments are sequentially connected by step-by-step liquid phase condensation to obtain a fully-protected straight-chain linaclotide; and finally, the side chain and the main chain of the fully-protected straight-chain linaclotide are removed to obtain the linear linaclotide, which is the linaclotide intermediate; and the linaclotide is prepared by further adopting an electrochemical one-step oxidation method. The application provides a method for synthesizing a linear linaclotide intermediate and linaclotide in a full liquid phase, can reduce production cost, improve the quality of linaclotide, is more suitable for industrialized production processes, meets environmental protection and safety requirements, and has better economic benefits and application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine synthesis, in particular to a method for synthesizing linaclotide intermediate and linaclotide in a full liquid phase. BACKGROUND

[0002] Linaclotide is a 14-amino acid polypeptide approved by FDA for the treatment of IBS-C, which can not only accelerate intestinal transport, but also improve abdominal pain. The drug belongs to the heat-stable enterotoxin analogue of Escherichia coli, which can activate the GC-C on the surface of intestinal epithelium, leading to the increase of cyclic guanosine monophosphate (cGMP) level. The increase of cGMP can activate the cystic fibrosis transmembrane conductance regulator (CFTR), and this activation can lead to the secretion of bicarbonate and chloride into the intestine, increase the secretion of fluid in the intestine, and accelerate intestinal transport. It has been found in animal models that linaclotide can increase the glycemic index and reduce intestinal pain, and linaclotide-induced increase of extracellular cGMP can reduce visceral pain and reduce pain-related neural activity. The structural formula of linear linaclotide is as follows:

[0003]

[0004] Before the present application, the main synthesis method of linear linaclotide in the prior art is solid-phase synthesis. In solid-phase synthesis, a large amount of expensive polypeptide resin, as well as a large amount of solvent and easily-toxic reagent, are needed, which not only brings environmental and cost pressure to large-scale production of enterprises, but also makes it impossible to separate the intermediate hetero-peptide on the solid-phase carrier, resulting in that the purity of the final product is not as good as that of liquid-phase synthesis, and the product must be purified by reliable separation means. Therefore, it is necessary to provide a process suitable for industrial production, which has high quality and is more in line with environmental protection and safety requirements.

[0005] Disulfide bond polypeptide drugs are a class of drugs containing at least one pair of disulfide bonds in the polypeptide sequence, which play an irreplaceable role in the treatment of metabolic, digestive system and other diseases. Studies have shown that the presence of disulfide bonds plays a crucial role in maintaining the structure and function of polypeptides. Disulfide bonds not only endow polypeptides with various biological activities, but also make the polypeptide structure be locked with strong chemical stability, enzyme stability and thermal stability. At present, dozens of chemically synthesized disulfide bond-containing polypeptides have been marketed for the treatment of tumors, diabetes, central diabetes insipidus, irritable bowel syndrome and other major diseases, and more disulfide bond-containing polypeptides are in the clinical research stage, and linaclotide is one of them.

[0006] Linaclotide is composed of 14 amino acids and contains three pairs of disulfide bonds (S-S), and the disulfide bond connection mode is Cys 1 -Cys 6 , Cys 2 -Cys 10Cys 5 -Cys 13 , molecular formula is C 59 H 79 N 15 O 21 S6, relative molecular mass 1526.8, CAS number is 851199-59-2, white to gray amorphous powder, slightly soluble in water and 0.9% sodium chloride aqueous solution, the specific structure sequence is as follows:

[0007]

[0008] The chemical structure of linaclotide is as follows:

[0009]

[0010] Prior to the present application, the existing technology of linaclotide three pairs of disulfide bond oxidation method is mainly step-by-step oxidation, this method not only complex process and various high-polluting reagent dosage, high cost, and due to the exchange of disulfide bond, the directional oxidation process will still form disulfide bond mismatched product, therefore, the step-by-step oxidation is not only complex operation, and low yield, not conducive to the scale-up production. In addition, for some conventional oxidation method, such as air oxidation method, the reaction time is long, and the mismatched product is easy to form. Therefore, it is urgent to develop a green and efficient oxidation method suitable for industrial production. SUMMARY

[0011] In view of the above technical problems existing in the prior art, the purpose of the present application is to provide a method for synthesizing linaclotide intermediate and linaclotide in whole liquid phase, which can reduce the production cost, improve the quality of linaclotide, and is more suitable for industrial production process, more in line with environmental protection and safety requirements, has better economic benefit and application prospect.

[0012] A method for synthesizing linaclotide intermediate in whole liquid phase, characterized in that it comprises the following steps:

[0013] In the first step, linear linaclotide is segmented to obtain at least two polypeptide fragments designed and planned; then the amino acid condensation reaction is carried out, the amino acid raw materials are selected according to the amino acid sequence order of the planned polypeptide fragments, and the target polypeptide fragments are synthesized by liquid phase condensation reaction through coupling with activator one by one, the polypeptide fragments have side chain protection group, and the amino terminal or carboxyl terminal has protection group;

[0014] In the second step, the polypeptide fragments synthesized in the first step are selected according to the polypeptide sequence of linear linaclotide, the protection groups of the amino terminal or carboxyl terminal of the polypeptide fragments are deprotected, and the polypeptide fragments are sequentially connected by step-by-step liquid phase condensation from N terminal to C terminal or from C terminal to N terminal, to obtain fully protected straight chain linaclotide;

[0015] In the third step, the protecting groups of the side chain and the main chain of the fully protected linear linifipotide are removed to obtain linear linifipotide, i.e. linifipotide intermediate.

[0016] The method for synthesizing linifipotide intermediate in whole liquid phase is characterized in that, in the first step, the liquid phase condensation reaction is carried out in solvent A, and amino acid or polypeptide fragment, base, activating agent and coupling agent are added in the solvent A to carry out the liquid phase condensation reaction.

[0017] Further, the solvent A is one or more of dichloromethane, ethyl acetate, N,N-dimethylformamide, acetonitrile, acetone, water and tetrahydrofuran, and the solvent A is preferably one or more of N,N-dimethylformamide, acetonitrile and tetrahydrofuran.

[0018] Further, the base is one of DIPEA, TEA, NMM, DMAP and potassium carbonate, and the base is preferably one of DIPEA, TEA, NMM and DMAP.

[0019] Further, the activating agent is one or more of HOBt, 6-NO2-HOBt, 6-CF3-HOBt, HOAt, 6-Cl-HOBt, 6-HOAt, 5-HOAt, 4-HOAt, HODhbt, HODhat, HODhad, HOSu, HONB, HOCt, HOPy, HOBI, HOI, 6-Cl-HOBI and Oxyma, and the activating agent is preferably one of HOBt, HOAt, HOSu, HONB and HOCt.

[0020] Further, the coupling agent is one or more of carbodiimide coupling agent, phosphonium salt coupling agent, tetramethylammonium salt coupling agent, ammonium salt coupling agent, triazine coupling agent, pyridine coupling agent; the carbodiimide coupling agent is one or more of DIC, DCC, EDCI, CIC, BMC, BEC, CPC, BDDC, PEC, PIC; the phosphonium salt coupling agent is one or more of BOP, BrOP, PyCloP, PyBrOP, CloP, PyBOP, AOP, PyAOP, PyOxm, PyNOP, PyFOP, PyFNBOP, PyCloK, PyPOP, PyTOP, PyDOP, PyDAOP; the tetramethylammonium salt coupling agent is one or more of N-HBTU, N-TBTU, TDTU, HDTU, TDATU, HDATU, TPTU, HPTU, TSTU, HSTU, TPFTU, HPFTU, N-CF3-HBTU, N-HATU, N-TATU, N-HATTU, HOTT, TOTU, HOTU, HTODC, HTODeC, HTOPC, TNTU, TPhTU; the ammonium salt coupling agent is one or more of PyCIU, HBPyU, HAPyU, HDPyU, HDAPyU, HPyOPfp, HPySPfp, HAPyTU, HPyONP, HPyOTCp, HBPipU, HAPipU, TOPPipU, CIP, HBMDU, HAMDU, CPP, HBMTU, HAMTU, HBPTU, HAPTU, HBM2PyU, HAM2PyU, HBM2PipU, HAM2PipU, HBE2PyU, HAE2PyU, HBE2PipU, HAE2PipU, HBTeU, DMCH, HDMB, HDMA, HDMC, 4-HDMA, 6-HDMFB, HDMPfp, HDMP, HDTMA, HDTMB; the triazine coupling agent is one or more of DMCT, DMTMM, TBCR1, TBCR2, TBCR3, TBCR4; the pyridine coupling agent is one or more of Mukaiyama’s Reagent, BEMT, BEP, FEP, BEPH, FEPH.

[0021] More preferably, the coupling agent is one or more of DIC, DCC, N-HATU, EDCI, N-HBTU, N-TBTU, BOP, PyBOP.

[0022] Further, the molar ratio of the amino acid or peptide, base, activating agent and coupling agent participating in the condensation reaction in the first step is 1:1.3-2.5:1-3:1-2, the reaction temperature is -10-30°C, and the reaction time is 0.5-24h.

[0023] Still further, the reaction temperature in the first step is preferably -10-20°C, and the reaction time is preferably 1-8h. The molar ratio of the amino acid or peptide, base, activating agent and condensing agent participating in the condensation reaction is preferably 1:1.5-2:1.1-2.5:1.2-1.8.

[0024] Further, the amino acid raw materials used in the first step are all carboxyl- or amino-terminally protected and side chain-protected, and specifically the following cases:

[0025] (1) the amino protecting group of the amino acid is one of Boc, Cbz, Fmoc or H;

[0026] (2) the carboxyl protecting group of the amino acid is one of methyl ester, tert-butyl ester, benzyl ester or H;

[0027] (3) the side chain protecting group of cysteine (Cys) is one of Trt, Bzl, Acm, Mmt, t Bu or H; the side chain protecting group of glutamic acid (Glu) is one of methyl ester, tert-butyl ester, benzyl ester or H; the side chain protecting group of tyrosine (Tyr) is one of Bzl, t Bu, Me or H; the side chain protecting group of threonine (Thr) is one of Bzl, t Bu, Me or H; the side chain protecting group of asparagine (Asn) is one of Trt, Xant, DMB or H. (H represents no protecting group).

[0028] The method for synthesizing a linatide intermediate in a full liquid phase is characterized in that linear linatide is fragmented in the first step, and the design is planned as 2-5 polypeptide fragments, and the number of amino acids in the polypeptide fragments is in the range of 2-6.

[0029] Further, linear linatide is fragmented in the first step, and the design is planned as 3-5 polypeptide fragments, and the number of amino acids in the polypeptide fragments is in the range of 2-6, and specifically one of the following three cases:

[0030] (1) the number of amino acids in the polypeptide fragments is in the design planning method of "6+4+4": the polypeptide fragments are 3, which are R 1 -Cys(R 2 )-Cys(R 2 )-Glu(R 4 )-Tyr(R 5 )-R3 , R 1 -Cys(R 2 )-Cys(R 2 )-Asn(R 7 )-Pro-R 3 and R 1 -Ala-Cys(R 2 )-Thr(R 6 )-Gly-Cys(R 2 )-Tyr(R 5 )-R 3 , the polypeptide fragments are sequentially connected by stepwise liquid phase condensation from N-terminus to C-terminus or from C-terminus to N-terminus to obtain a fully protected linear linifipotide;

[0031] (A) "6+4+4" fragment synthesis method, as follows:

[0032] Synthesis of polypeptide fragment A1: R 1 -Ala-Cys(R 2 )-Thr(R 6 )-Gly-Cys(R 2 )-Tyr(R 5 )-R 3 (as follows)

[0033]

[0034] Synthesis of polypeptide fragment A2: R 1 -Cys(R 2 )-Cys(R 2 )-Asn(R 7 )-Pro-R 3 (as follows)

[0035]

[0036] Synthesis of polypeptide fragment A3: R 1 -Cys(R 2 )-Cys(R 2 )-Glu(R 4 )-Tyr(R 5 )-R 3 (as follows)

[0037]

[0038] Synthesis of fully protected linear linifipotide (as follows):

[0039]

[0040] (2) The number of amino acids of the polypeptide fragments is in the design plan of "5+5+4": the polypeptide fragments are three, which are R 1 -Cys(R 2 )-Cys(R 2 )-Glu(R 4 )-Tyr(R 5 )-R 3 , R 1 -Cys(R 2 )-Cys(R 2 )-Asn(R 7 )-Pro-Ala-R 3 and R 1 -Cys(R 2 )-Thr(R 6 )-Gly-Cys(R 2 )-Tyr(R 5 )-R 3 , from N-terminal to C-terminal or from C-terminal to N-terminal, the polypeptide fragments are sequentially connected by step-by-step liquid phase condensation to obtain a fully protected linear linifrope;

[0041] (B) The fragment synthesis method of "5+5+4" is as follows:

[0042] Synthesis of polypeptide fragment B1: R 1 -Cys(R 2 )-Thr(R 6 )-Gly-Cys(R 2 )-Tyr(R 5 )-R 3 (as shown below)

[0043]

[0044] Synthesis of polypeptide fragment B2: R 1 -Cys(R 2 )-Cys(R 2 )-Asn(R 7 )-Pro-Ala-R 3 (as shown below)

[0045]

[0046] Synthesis of polypeptide fragment B3: R 1 -Cys(R 2 )-Cys(R 2 )-Glu(R 4 )-Tyr(R 5 )-R 3 (as shown below)

[0047]

[0048] (3) The number of amino acids of the polypeptide fragments is in the design plan of "6+(2+2)+(2+2)": the polypeptide fragments are 5, which are R 1 -Cys(R 2 )-Cys(R 2 )-COOH, NH2-Glu(R 4 )-Tyr(R 5 )-R 3 , R 1 -Cys(R 2 )-Cys(R 2 )-COOH, NH2-Asn(R 7 )-Pro-R 3 and R 1 -Ala-Cys(R 2 )-Thr(R 6 )-Gly-Cys(R 2 )-Tyr(R 5 )-R 3 , sequentially connecting the polypeptide fragments by step-by-step liquid phase condensation from N-terminal to C-terminal or from C-terminal to N-terminal to obtain a fully protected linear linagliptin;

[0049] (C) The fragment synthesis method of "6+(2+2)+(2+2)", specifically as follows:

[0050] Synthesis of polypeptide fragment C1: R 1 -Ala-Cys(R 2 )-Thr(R 6 )-Gly-Cys(R 2 )-Tyr(R 5 )-R 3 (same as A1)

[0051] Synthesis of polypeptide fragment C2: R 1 -Cys(R 2 )-Cys(R 2 )-COOH (as shown below)

[0052]

[0053] Synthesis of polypeptide fragment C3: NH2-Asn(R 7 )-Pro-R 3 (as shown below)

[0054]

[0055] Synthesis of polypeptide fragment C4: NH2-Glu(R 4 )-Tyr(R 5 )-R 3 (as shown below)

[0056]

[0057] Synthesis of fully-protected linear linaclotide (as shown below):

[0058]

[0059] In the above-mentioned fragment synthesis, wherein R 1 is one of Boc, Cbz, Fmoc or H, the side chain protecting group R 2 of cysteine (Cys) is one of Trt, Bzl, Acm, Mmt, t Bu or H; the side chain protecting group R 4 of glutamic acid (Glu) is one of methyl ester, t-butyl ester, benzyl ester or H; the side chain protecting group R 5 of tyrosine (Tyr) is one of Bzl, t Bu, Me or H; the side chain protecting group R 6 of threonine (Thr) is one of Bzl, t Bu, Me or H; the side chain protecting group R 7 of asparagine (Asn) is one of Trt, Xant, DMB or H; R 3 is one of methyl ester, t-butyl ester, benzyl ester or H. (H represents no protecting group is used).

[0060] The method for synthesizing linaclotide intermediates of the present application has the following advantages over the prior art:

[0061] (1) In the synthesis of fully-protected linear linaclotide, the amino acids used in the present application are commercially available and are relatively inexpensive, greatly reducing the cost of synthesizing linear linaclotide;

[0062] (2) In the synthesis of polypeptide fragments, the polypeptide fragments can be subjected to recrystallization, greatly improving the purity of the obtained linear linaclotide;

[0063] (3) In the removal of the main chain and side chain protecting groups, the methods used are simple to operate and suitable for scale-up reactions, facilitating industrial production.

[0064] In summary, the present application has the advantages of simple operation, high purity of linaclotide intermediates, suitability for industrialization, and the like, and has greater practical value and application prospects than the prior art, and is a new method for synthesizing linaclotide intermediates.

[0065] The present application also provides a method for synthesizing linaclotide by electrochemical oxidation cyclization of linaclotide intermediate, comprising the following steps:

[0066] 1) The linaclotide intermediate is linear linaclotide, the linaclotide intermediate is dissolved in solvent B, an additive is added, and the reaction is carried out under the conditions of a certain current intensity, reaction concentration and electrode material for a certain time to obtain crude linaclotide; wherein the types of electrode anode material and cathode material are one of C(+)|C(-) (i.e. the electrode anode material and the cathode material are both carbon materials), C(+)|Pt(-) (i.e. the electrode anode material is a carbon material and the cathode material is a platinum material), Pt(+)|Pt(-) (i.e. the electrode anode material and the cathode material are both platinum materials), GC(+)|Pt(-) (i.e. the electrode anode material is a glassy carbon material and the cathode material is a platinum material), Carbon rod(+)|Pt(-) (i.e. the electrode anode material is a carbon rod material and the cathode material is a platinum material), Carbon cloth(+)|Pt(-) (i.e. the electrode anode material is a carbon cloth material and the cathode material is a platinum material), GF(+)|GF(-) (i.e. the electrode anode material and the cathode material are both graphite felt materials), GF(+)|Ni(-) (i.e. the electrode anode material is a graphite felt material and the cathode material is a metal nickel material); the electrode type is preferably one of C(+)|C(-), C(+)|Pt(-) and Pt(+)|Pt(-);

[0067] 2) The crude linaclotide obtained is purified by preparative liquid chromatography to obtain pure linaclotide.

[0068] Further, the solvent B is selected from one or a combination of any of the following: water, aromatic hydrocarbons, ethers, halogenated hydrocarbons, lower alcohols, esters, fatty acids, ketones, and other solvents; the other solvents are selected from at least one of N,N-dimethylformamide, dimethyl sulfoxide, hexamethylphosphoric triamide, and acetonitrile; the aromatic hydrocarbons are selected from at least one of benzene, toluene, and xylene; the ethers are selected from at least one of diethyl ether, dioxane, tetrahydrofuran, ethyl ether, and ethylene glycol dimethyl ether; the halogenated hydrocarbons are selected from at least one of dichloromethane, dichloroethane, chloroform, and carbon tetrachloride; the lower alcohols are selected from at least one of methanol, ethanol, isopropanol, butanol, tert-butanol, ethylene glycol, and hexafluoroisopropanol; the fatty acids are selected from at least one of formic acid and acetic acid; the esters are selected from at least one of ethyl acetate and methyl acetate; and the ketones are selected from at least one of acetone and methyl ethyl ketone.

[0069] The solvent B is preferably one or several of N,N-dimethylformamide, tetrahydrofuran, methanol, ethanol, hexafluoroisopropanol, acetonitrile, ethylene glycol dimethyl ether, butanol, chloroform, and dimethyl sulfoxide.

[0070] The concentration of the linaclotide intermediate in the solvent B is 0.1-10 mg / mL, preferably 0.4-3 mg / mL; the additive used in the electrochemical oxidation reaction is one or more of NaI, NaBr, NaCl, KI, KBr, KCl, NH4I, NH4Br, KPF6, NaPF6, NH4Cl, Et4NI, LiClO4, n Bu4NBF4, n Bu4NPF6, n Bu4NClO4, n Bu4NI, n Bu4NBr, n Bu4NCl, n Bu4NOAc, n Bu4NHSO4, Et4NClO4, Me4NI, Et4NBF4, Et4NPF6, one or more of them, preferably one or more of KI, NaI, LiClO4, n Bu4NBF4, Et4NI, NH4I, NH4Br, n Bu4NPF6, n Bu4NBr, n Bu4NCl, n Bu4NOAc. The concentration of the additive in the solvent B is 0.5-50 mg / mL, preferably 2-10 mg / mL.

[0071] Further, the current intensity of the electrochemical oxidation reaction is 2-15 mA, preferably 4-10 mA; the reaction time is 0.2-5 h, and the reaction time is preferably 0.5-4 h.

[0072] The method for synthesizing linaclotide by electrochemical oxidation ring formation of the present application has the following beneficial effects compared with the prior art:

[0073] 1) In the present application, linaclotide is prepared by electrochemical one-step oxidation during ring formation, avoiding the complexity of step-by-step oxidation.

[0074] 2) Compared with the traditional air oxidation method, the oxidation efficiency is greatly improved.

[0075] 3) Compared with the iodine oxidation method, the oxidation raw material used is less polluting, and the oxidation method is more clean, green and efficient.

[0076] 4) Compared with the hydrogen peroxide oxidation method, it is safer and more efficient, and less harmful to the human body.

[0077] In summary, the present application has the advantages of greenness, high efficiency, safety, suitability for industrialization, etc., and has more practical value and application prospect compared with the prior art, and is a new method for synthesizing linaclotide. DETAILED DESCRIPTION

[0078] The application will be further described in connection with the following specific examples, but the scope of the application is not limited thereto.

[0079] The English abbreviations of the substances appearing in the claims and the specification of the present application (partly) correspond to the Chinese names as shown in Table 1 (The amino acids referred to herein are all of L type, which will not be specifically described)

[0080] Table 1

[0081]

[0082]

[0083] First Part: Synthesis of Linatide Intermediates

[0084] Example 1:

[0085] (A) "6+4+4" fragment synthesis method:

[0086] Synthesis of polypeptide fragment A1: Fmoc-Ala-Cys(Trt)-Thr-Gly-Cys(Trt)-Tyr-OMe

[0087] 1) Synthesis of compound A1.1: NH2-Cys(Trt)-Tyr-OMe

[0088] MeO-Tyr-NH2 (10.0 mmol) and Fmoc-Cys(Trt)-COOH (10.0 mmol) were added to a round-bottom flask, DMF (15 mL) was added, then NMM (17.0 mmol) and HOBt (11.0 mmol) were added, followed by the addition of EDCI (12.0 mmol) at 0°C for 0.5 h, and the reaction was continued at room temperature. After TLC monitoring showed that the reaction was complete, water was added for quenching, and the above reaction solution was extracted with ethyl acetate, washed, dried and rotary evaporated to obtain white solid Fmoc-Cys(Trt)-Tyr-OMe (9.9 mmol) with a yield of 99%.

[0089] The above obtained solid was added to a round-bottom flask, dissolved in DCM (20 mL), and then DBU (10.9 mmol) was added. After TLC monitoring showed that the reaction was complete, the DCM solution was washed with water to remove unreacted DBU, and then the organic phase was adjusted to pH 2-3 with 5% potassium bisulfate aqueous solution, and Fmoc was removed by extraction with DCM, and the organic phase was discarded. The aqueous phase was adjusted to pH 7-8 with sodium bicarbonate solid, and then extracted with DCM. After washing, drying and rotary evaporation, white foamy solid NH2-Cys(Trt)-Tyr-OMe (9.6 mmol) was obtained with a yield of 97%.

[0090] 2) Synthesis of compound A1.2: NH2-Gly-Cys(Trt)-Tyr-OMe

[0091] MeO-Tyr-Cys(Trt)-NH2(9.6 mmol) and Boc-Gly-COOH (9.6 mmol) were added to a round bottom flask, DMF (15 mL) was added, followed by NMM (16.3 mmol) and HOBt (10.6 mmol), then EDCI (11.5 mmol) was added at 0 °C and reacted for 0.5 h, then the reaction was continued at room temperature. After TLC monitoring of the reaction completion, water was added for quenching, the above reaction solution was extracted with ethyl acetate, washed, dried and rotary evaporated to give a light yellow foamy solid Boc-Gly-Cys(Trt)-Tyr-OMe (9.5 mmol) with a yield of 99%.

[0092] The above obtained solid was taken in a round bottom flask, 4M HCl / 1,4-dioxane solution (20 mL) was added, the reaction was monitored by TLC, after the reaction was completed, it was concentrated under vacuum and reduced pressure to give a white solid MeO-Tyr-Cys(Trt)-Gly-NH2·HCl (9.3 mmol) with a yield of 98%.

[0093] 3) Synthesis of compound A1.3: NH2-Thr-Gly-Cys(Trt)-Tyr-OMe

[0094] MeO-Tyr-Cys(Trt)-Gly-NH2·HCl (9.3 mmol) and Fmoc-Thr-COOH (9.3 mmol) were added to a round bottom flask, DMF (15 mL) was added, followed by NMM (15.8 mmol) and HOBt (10.2 mmol), then EDCI (11.2 mmol) was added at 0 °C and reacted for 0.5 h, then the reaction was continued at room temperature. After TLC monitoring of the reaction completion, water was added for quenching, the above reaction solution was extracted with ethyl acetate, washed, dried and rotary evaporated to give a light yellow foamy solid Fmoc-Thr-Gly-Cys(Trt)-Tyr-OMe (9.2 mmol) with a yield of 99%.

[0095] The above obtained solid was added to a round bottom flask, dissolved in DCM (20 mL), then DBU (10.1 mmol) was added, TLC was used to monitor the completion of the reaction, then the DCM solution was washed with water to remove unreacted DBU, then the organic phase was adjusted to pH 2-3 with 5% potassium bisulfate aqueous solution, Fmoc was removed by extraction with DCM, the organic phase was discarded, then the water phase was adjusted to pH 7-8 with sodium bicarbonate solid, extracted with DCM, washed, dried and rotary evaporated to obtain white foamy solid NH2-Thr-Gly-Cys(Trt)-Tyr-OMe (8.9 mmol) with a yield of 97%.

[0096] 4) Synthesis of compound A1.4: NH2-Cys(Trt)-Thr-Gly-Cys(Trt)-Tyr-OMe

[0097] MeO-Tyr-Cys(Trt)-Gly-Thr-NH2 (8.9 mmol) and Fmoc-Cys(Trt)-COOH (8.9 mmol) were added to a round bottom flask, DMF (15 mL) was added, then NMM (15.1 mmol) and HOBt (9.8 mmol) were added, then EDCI (10.7 mmol) was added at 0°C for 0.5 h, and the reaction was continued at room temperature. After TLC was used to monitor the completion of the reaction, water was added to quench the reaction, then the above reaction solution was extracted with ethyl acetate, washed, dried and rotary evaporated to obtain light yellow foamy solid Fmoc-Cys(Trt)-Thr-Gly-Cys(Trt)-Tyr-OMe (8.8 mmol) with a yield of 99%.

[0098] The above obtained solid was added to a round bottom flask, dissolved in DCM (20 mL), then DBU (10.1 mmol) was added, TLC was used to monitor the completion of the reaction, then the DCM solution was washed with water to remove unreacted DBU, then the organic phase was adjusted to pH 2-3 with 5% potassium bisulfate aqueous solution, Fmoc was removed by extraction with DCM, the organic phase was discarded, then the water phase was adjusted to pH 7-8 with sodium bicarbonate solid, extracted with DCM, washed, dried and rotary evaporated to obtain white foamy solid NH2-Thr-Gly-Cys(Trt)-Tyr-OMe (8.9 mmol) with a yield of 97%.

[0099] 5) Synthesis of compound A1: Fmoc-Ala-Cys(Trt)-Thr-Gly-Cys(Trt)-Tyr-OMe

[0100] MeO-Tyr-Cys(Trt)-Gly-Thr-Cys(Trt)-NH2(8.5 mmol) and Fmoc-Ala-COOH (8.5 mmol) were added to a round bottom flask, DMF (15 mL) was added, followed by NMM (14.5 mmol) and HOBt (9.4 mmol), then EDCI (10.2 mmol) was added at 0 °C for 0.5 h, and the reaction was continued at room temperature. After TLC monitoring of the reaction completion, water was added for quenching, and the above reaction solution was extracted with ethyl acetate, washed, dried and rotary evaporated to give a light yellow foamy solid Fmoc-Ala-Cys(Trt)-Thr-Gly-Cys(Trt)-Tyr-OMe (8.4 mmol) with a yield of 99%.

[0101] Synthesis of polypeptide fragment A2: Boc-Cys(Trt)-Cys(Trt)-Asn-Pro-OMe

[0102] 1) Synthesis of compound A2.1: NH2-Asn-Pro-OMe

[0103] MeO-Pro-NH2 (10 mmol) and Cbz-Asn-COOH (10 mmol) were added to a round bottom flask, DMF (15 mL) was added, followed by NMM (17 mmol) and HOBt (11 mmol), then EDCI (12 mmol) was added at 0 °C for 0.5 h, and the reaction was continued at room temperature. After TLC monitoring of the reaction completion, water was added for quenching, and the above reaction solution was extracted with ethyl acetate, washed, dried and rotary evaporated to give a white solid Cbz-Asn-Pro-OMe (9.9 mmol) with a yield of 99%.

[0104] The above obtained white solid was added to a round bottom flask, dissolved in methanol (30 mL), Pd / C (374 mg, 10% of palladium content) was added, and catalytic hydrogenation was carried out with a hydrogen balloon (i.e. the H2 pressure for catalytic hydrogenation was normal pressure), the reaction was monitored by TLC, and the reaction was completed after about 12 h, and then the reaction was filtered with diatomite, and concentrated under vacuum to give a white solid NH2-Asn-Pro-OMe (9.4 mmol) with a yield of 95%.

[0105] 2) Synthesis of compound A2.2: NH2-Cys(Trt)-Asn-Pro-OMe

[0106] MeO-Pro-Asn-NH2(9.4 mmol) and Boc-Cys(Trt)-COOH (9.4 mmol) were added to a round bottom flask, DMF (15 mL) was added, followed by NMM (16.0 mmol) and HOBt (10.3 mmol), then EDCI (11.3 mmol) was added at 0 °C and the reaction was allowed to proceed for 0.5 h, then the reaction was allowed to proceed at room temperature. After the reaction was complete as monitored by TLC, the reaction was quenched with water, the reaction was extracted with ethyl acetate, washed, dried and concentrated to yield a light yellow foamy solid, Boc-Cys(Trt)-Asn-Pro-OMe (9.3 mmol) in 99% yield.

[0107] The product from above was added to a round bottom flask, dissolved in DCM (30 mL), then trifluoroacetic acid (10 mL) was added, the reaction was monitored by TLC, the solvent was removed by vacuum rotary evaporation, the product was precipitated by the addition of diethyl ether, and the product was isolated by suction filtration to yield a white solid, NH2-Cys(Trt)-Asn-Pro-OMe (9.0 mmol) in 97% yield.

[0108] 3) Synthesis of Compound A2: Boc-Cys(Trt)-Cys(Trt)-Asn-Pro-OMe

[0109] MeO-Pro-Asn-Cys(Trt)-NH2(9.0 mmol) and Boc-Cys(Trt)-COOH (9.0 mmol) were added to a round bottom flask, DMF (15 mL) was added, followed by NMM (15.3 mmol) and HOBt (9.9 mmol), then EDCI (10.8 mmol) was added at 0 °C and the reaction was allowed to proceed for 0.5 h, then the reaction was allowed to proceed at room temperature. After the reaction was complete as monitored by TLC, the reaction was quenched with water, the reaction was extracted with ethyl acetate, washed, dried and concentrated to yield a light yellow foamy solid, Boc-Cys(Trt)-Cys(Trt)-Asn-Pro-OMe (8.9 mmol) in 99% yield.

[0110] Synthesis of Polypeptide Fragment A3: Cbz-Cys(Trt)-Cys(Trt)-Glu(OMe)-Tyr-O t Bu

[0111] 1) Synthesis of Compound A3.1: Cbz-Cys(Trt)-Cys(Trt)-COOH

[0112] MeO-Cys(Trt)-NH2(10.0 mmol) and Cbz-Cys(Trt)-COOH (10.0 mmol) were added to a round bottom flask, DMF (15 mL) was added, followed by NMM (17.0 mmol) and HOBt (11.0 mmol), then EDCI (12.0 mmol) was added at 0 °C and reacted for 0.5 h, then the reaction was continued at room temperature. After TLC monitoring of the reaction completion, water was added to quench the reaction, and the above reaction solution was extracted with ethyl acetate, washed, dried and rotary evaporated to obtain a white foamy solid Cbz-Cys(Trt)-Cys(Trt)-OMe (9.9 mmol) with a yield of 99%.

[0113] The above obtained solid was dissolved in methanol (20 mL), tetrahydrofuran (20 mL) and water (10 mL), and sodium hydroxide (29.7 mmol) was added, and the reaction end point was monitored by TLC. After the reaction was completed, the solvent was removed by vacuum rotary evaporation, 1M aqueous HCl was added to adjust the pH to 2-3, and then extracted with ethyl acetate, the organic phase was combined, washed, dried and vacuum concentrated to obtain a white solid Cbz-Cys(Trt)-Cys(Trt)-COOH (9.4 mmol) with a yield of 95%.

[0114] 2) Synthesis of compound A3.2: Cbz-Cys(Trt)-Cys(Trt)-Glu(OMe)-COOH

[0115] The above obtained product was added to a round bottom flask, dissolved in DCM (20 mL), and then trifluoroacetic acid (10 mL) was added, and the reaction end point was monitored by TLC. The solvent was removed by vacuum rotary evaporation, and ice ethyl ether and n-hexane were added to precipitate a white solid Cbz-Cys(Trt)-Cys(Trt)-Glu(OMe)-COOH (8.9 mmol) with a yield of 95%. t BuO-Glu(OMe)-NH2(9.5 mmol) and Cbz-Cys(Trt)-Cys(Trt)-COOH (9.5 mmol) were added to a round bottom flask, DMF (15 mL) was added, followed by NMM (16.2 mmol) and HOBt (10.5 mmol), then EDCI (11.4 mmol) was added at 0 °C and reacted for 0.5 h, then the reaction was continued at room temperature. After TLC monitoring of the reaction completion, water was added to quench the reaction, and the above reaction solution was extracted with ethyl acetate, washed, dried and rotary evaporated to obtain a white foamy solid Cbz-Cys(Trt)-Cys(Trt)-Glu(OMe)-O t Bu (9.4 mmol) with a yield of 99%.

[0116] The above obtained product was added to a round bottom flask, dissolved in DCM (20 mL), and then trifluoroacetic acid (10 mL) was added, and the reaction end point was monitored by TLC. The solvent was removed by vacuum rotary evaporation, and ice ethyl ether and n-hexane were added to precipitate a white solid Cbz-Cys(Trt)-Cys(Trt)-Glu(OMe)-COOH (8.9 mmol) with a yield of 95%.

[0117] 3) Synthesis of compound A3: Cbz-Cys(Trt)-Cys(Trt)-Glu(OMe)-Tyr-O t Bu

[0118] To a round bottom flask was added compound A2 (8.9 mmol) and methanol (20 mL), tetrahydrofuran (20 mL) and water (10 mL) were added to dissolve the compound, then sodium hydroxide (26.7 mmol) was added, TLC was used to monitor the reaction end point. After the reaction was completed, the solvent was removed by vacuum rotary evaporation, 1 M aqueous HCl was added to adjust the pH to 2-3, then ethyl acetate was added to extract the product, the organic phase was combined and washed, dried and concentrated by vacuum to obtain a white solid, Boc-Cys(Trt)-Cys(Trt)-Asn-Pro-COOH (8.4 mmol), with a yield of 94%. t BuO-Tyr-NH2 (8.9 mmol) and Cbz-Cys(Trt)-Cys(Trt)-Glu(OMe)-COOH (8.9 mmol) were added to a round bottom flask, DMF (15 mL) was added, then NMM (15.1 mmol) and HOBt (9.8 mmol) were added, followed by the addition of EDCI (10.7 mmol) at 0°C, and the reaction was continued for 0.5 h, then the reaction was continued at room temperature. After the reaction was completed by TLC monitoring, water was added to quench the reaction, the above reaction solution was extracted with ethyl acetate, washed, dried and rotary evaporated to obtain a white foamy solid, Cbz-Cys(Trt)-Cys(Trt)-Glu(OMe)-Tyr-O t Bu (8.8 mmol) with a yield of 99%.

[0119] Synthesis of linear linaclotide:

[0120] 1) Synthesis of fully protected linear linaclotide: removal of Fmoc protecting group of peptide chain fragment A1: Compound A1 (8.5 mmol) was taken in a round bottom flask, dissolved in DCM (20 mL), then DBU (9.4 mmol) was added, and the reaction was monitored by TLC until completion. The DCM solution was washed with water to remove unreacted DBU, then the organic phase was adjusted to pH 2-3 with 5% aqueous potassium hydrogen sulfate solution, and the Fmoc was removed by extraction with DCM, the organic phase was discarded, and the aqueous phase was adjusted to pH 7-8 with sodium bicarbonate solid, extracted with DCM, washed, dried and rotary evaporated to obtain a white solid, NH2-Ala-Cys(Trt)-Thr-Gly-Cys(Trt)-Tyr-OMe (8.2 mmol), with a yield of 96%.

[0121] Removal of methyl ester of peptide chain fragment A2: Compound A2 (8.9 mmol) was taken in a round bottom flask, dissolved in methanol (20 mL), tetrahydrofuran (20 mL) and water (10 mL), then sodium hydroxide (26.7 mmol) was added, and the reaction was monitored by TLC until completion. After the reaction was completed, the solvent was removed by vacuum rotary evaporation, 1 M aqueous HCl was added to adjust the pH to 2-3, then ethyl acetate was added to extract the product, the organic phase was combined and washed, dried and concentrated by vacuum to obtain a white solid, Boc-Cys(Trt)-Cys(Trt)-Asn-Pro-COOH (8.4 mmol), with a yield of 94%.

[0122] The product of the above two steps was condensed: MeO-Tyr-Cys(Trt)-Gly-Thr-Cys(Trt)-Ala-NH2(8.0 mmol) and Boc-Cys(Trt)-Cys(Trt)-Asn-Pro-COOH (8.0 mmol) were added to a round bottom flask, DMF (30 mL) was added, followed by NMM (13.6 mmol) and HOBt (8.8 mmol), then EDCI (9.6 mmol) was added at 0 °C and reacted for 0.5 h, then the reaction was continued at room temperature. After the reaction was completed by TLC monitoring, water was added to quench the reaction, and the above reaction solution was extracted with ethyl acetate, washed and dried, and then rotary evaporated to obtain a white solid Boc-Cys(Trt)-Cys(Trt)-Asn-Pro-Ala-Cys(Trt)-Thr-Gly-Cys(Trt)-Tyr-OMe (7.9 mmol), with a yield of 99%.

[0123] The solid obtained in the previous step (7.9 mmol) was taken in a round bottom flask, dissolved in DCM (30 mL), and then trifluoroacetic acid (10 mL) was added. The reaction end point was monitored by TLC, and the solvent was removed by vacuum rotary evaporation. Ice ethyl ether was added to precipitate a white solid NH2-Cys(Trt)-Cys(Trt)-Asn-Pro-Ala-Cys(Trt)-Thr-Gly-Cys(Trt)-Tyr-OMe (7.7 mmol), with a yield of 97%.

[0124] The tert-butyl protecting group of the peptide chain A3 was removed: Compound A3 (8.8 mmol) was taken in a round bottom flask, dissolved in DCM (20 mL), and then trifluoroacetic acid (10 mL) was added. The reaction end point was monitored by TLC, and the solvent was removed by vacuum rotary evaporation. Ice ethyl ether and n-hexane were added to precipitate a white solid Cbz-Cys(Trt)-Cys(Trt)-Glu(OMe)-Tyr-COOH (8.5 mmol), with a yield of 97%.

[0125] Condensation of the product of the above two steps with the fully protected linear linifrope: NH2-Cys(Trt)-Cys(Trt)-Asn-Pro-Ala-Cys(Trt)-Thr-Gly-Cys(Trt)-Tyr-OMe (7.7 mmol) and Cbz-Cys(Trt)-Cys(Trt)-Glu(OMe)-Tyr-COOH (7.7 mmol) were added to a round bottom flask, DMF (30 mL) was added, followed by NMM (13.1 mmol) and HOBt (23.1 mmol), and then EDCI (9.2 mmol) was added at 0 °C and the reaction was allowed to proceed. After the reaction was complete as monitored by TLC, the reaction was quenched with water, and the reaction was extracted with ethyl acetate, washed, dried, and concentrated to yield Cbz-Cys(Trt)-Cys(Trt)-Glu(OMe)-Tyr-Cys(Trt)-Cys(Trt)-Asn-Pro-Ala-Cys(Trt)-Thr-Gly-Cys(Trt)-Tyr-OMe (7.6 mmol) as a white solid in 99% yield.

[0126] 2) Removal of the C-terminal and glutamic acid side chain methyl ester protecting groups: The fully protected linear linifrope (5 mmol) was taken in a round bottom flask, dissolved in methanol (30 mL), tetrahydrofuran (30 mL), and water (15 mL), and sodium hydroxide (25.0 mmol) was added. The reaction was monitored by TLC until the reaction was complete. After the reaction was complete, the solvent was removed by vacuum rotary evaporation, and the reaction was acidified to pH 2-3 with 1 M aqueous HC1. The reaction was extracted with ethyl acetate, the organic layers were combined, washed, dried, and concentrated to yield Cbz-Cys(Trt)-Cys(Trt)-Glu-Tyr-Cys(Trt)-Cys(Trt)-Asn-Pro-Ala-Cys(Trt)-Thr-Gly-Cys(Trt)-Tyr-COOH (4.4 mmol) as a white solid in 94% yield.

[0127] Cbz-Cys(Trt)-Cys(Trt)-Glu-Tyr-Cys(Trt)-Cys(Trt)-Asn-Pro-Ala-Cys(Trt)-Thr-Gly-Cys(Trt)-T yr-COOH (4.4 mmol), in 94% yield.

[0128] One step removal of the N-terminal Cbz protecting group and the cysteine side chain Trt protecting groups: The solid product from the previous step (4.4 mmol) was taken in a round bottom flask, and HBr / HOAc (20 mL, 30% mass fraction) was added. The reaction was allowed to proceed overnight, and the product was concentrated by vacuum rotary evaporation. The product was crystallized from ether, and the product was filtered to yield linear linifrope intermediate NH2-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-COOH (4.0 mmol) in 92% yield.

[0129] Example 2:

[0130] (B) "5+5+4" fragment synthesis method

[0131] Synthesis of polypeptide fragment B1 : Fmoc-Cys(Trt)-Thr-Gly-Cys(Trt)-Tyr-OMe

[0132] Synthesis method same as compound A1.1-A1.4, total yield 86%.

[0133] Synthesis of polypeptide fragment B2: Boc-Cys(Trt)-Cys(Trt)-Asn-Pro-Ala-OMe

[0134] Take compound A2 (10.0 mmol) in a round-bottom flask, add methanol (20 mL), tetrahydrofuran (20 mL) and water (10 mL) to dissolve, add sodium hydroxide (30.0 mmol), TLC monitor reaction end point. After the reaction is completed, the solvent is removed by vacuum rotary evaporation, add 1M aqueous HCl to adjust the pH to 2-3, then extract with ethyl acetate, combine the organic phase, wash and dry, concentrate under vacuum to give white solid Boc-Cys(Trt)-Cys(Trt)-Asn-Pro-COOH (9.0 mmol), total yield 94%.

[0135] Boc-Cys(Trt)-Cys(Trt)-Asn-Pro-COOH (8.5 mmol) and MeO-Ala-NH2 (8.5 mmol) are added to a round-bottom flask, DMF (15 mL) is added, then NMM (14.5 mmol) and HOBt (9.4 mmol) are added, followed by the addition of EDCI (10.2 mmol) at 0°C for 0.5 h, and the reaction is continued at room temperature. After TLC monitoring of the complete reaction, water is added to quench the reaction, and the above reaction solution is extracted with ethyl acetate, washed, dried and rotary evaporated to give a light yellow foamy solid MeO-Ala-Pro-Asn-Cys(Trt)-Cys(Trt)-Boc (8.4 mmol), yield 99%.

[0136] Synthesis of polypeptide fragment B3: Cbz-Cys(Trt)-Cys(Trt)-Glu(OMe)-Tyr-O t Bu (synthesis method same as A3)

[0137] Synthesis of linear linaclotide:

[0138] 1) Synthesis of fully protected linear linaclotide:

[0139] Remove the Fmoc protecting group of B1 : synthesis method same as A1.4;

[0140] Removal of the methyl ester protecting group of B2: Take compound B2 (8.0 mmol) in a round bottom flask, add methanol (20 mL), tetrahydrofuran (20 mL) and water (10 mL) to dissolve, add sodium hydroxide (24.0 mmol), monitor the reaction end point by TLC. After the reaction is completed, remove the solvent by rotary evaporation under vacuum, add 1 M aqueous HC1 to adjust the pH to 2-3, then extract with ethyl acetate, combine the organic phases, wash, dry and concentrate under vacuum to obtain white solid Boc-Cys(Trt)-Cys(Trt)-Asn-Pro-Ala-COOH (7.4 mmol) with a total yield of 92%.

[0141] Condensation of the product of the above two steps: Take MeO-Tyr-Cys(Trt)-Gly-Thr-Cys(Trt)-NH2 (5.0 mmol) and Boc-Cys(Trt)-Cys(Trt)-Asn-Pro-Ala-COOH (5.0 mmol) in a round bottom flask, add DMF (30 mL), then add NMM (8.5 mmol) and HOBt (15.0 mmol), then add EDCI (6.0 mmol) at 0 °C, continue the reaction at 0 °C. After the reaction is completed, monitor by TLC, quench with water, extract the reaction mixture with ethyl acetate, wash, dry and concentrate under vacuum to obtain white solid Boc-Cys(Trt)-Cys(Trt)-Asn-Pro-Ala-Cys(Trt)-Thr-Gly-Cys(Trt)-Tyr-OMe (4.9 mmol) with a yield of 99%.

[0142] Take the solid obtained in the previous step (4.9 mmol) in a round bottom flask, add DCM (30 mL) to dissolve, then add trifluoroacetic acid (10 mL), monitor the reaction end point by TLC, remove the solvent by rotary evaporation under vacuum, precipitate by adding ice ethyl ether to obtain white solid NH2-Cys(Trt)-Cys(Trt)-Asn-Pro-Ala-Cys(Trt)-Thr-Gly-Cys(Trt)-Tyr-OMe (4.8 mmol) with a yield of 97%.

[0143] Removal of the tert-butyl protecting group of the peptide chain B3: Take compound B3 (8.8 mmol) in a round bottom flask, add DCM (20 mL) to dissolve, then add trifluoroacetic acid (10 mL), monitor the reaction end point by TLC, remove the solvent by rotary evaporation under vacuum, precipitate by adding ice ethyl ether and n-hexane to obtain white solid Cbz-Cys(Trt)-Cys(Trt)-Glu(OMe)-Tyr-COOH (8.5 mmol) with a yield of 97%.

[0144] Condensation of the product of the above two steps with the fully protected linear linifrope: NH2-Cys(Trt)-Cys(Trt)-Asn-Pro-Ala-Cys(Trt)-Thr-Gly-Cys(Trt)-Tyr-OMe (4.5 mmol) and Cbz-Cys(Trt)-Cys(Trt)-Glu(OMe)-Tyr-COOH (4.5 mmol) were added to a round bottom flask, DMF (30 mL) was added, followed by NMM (7.7 mmol) and HOBt (13.5 mmol), and then EDCI (5.4 mmol) was added at 0 °C and the reaction was allowed to proceed. After the reaction was complete as monitored by TLC, the reaction was quenched with water, and the reaction was extracted with ethyl acetate, washed, dried, and concentrated to yield Cbz-Cys(Trt)-Cys(Trt)-Glu(OMe)-Tyr-Cys(Trt)-Cys(Trt)-Asn-Pro-Ala-Cys(Trt)-Thr-Gly-Cys(Trt)-Tyr-OMe (4.4 mmol) as a white solid in 99% yield.

[0145] 2) Removal of the C-terminal and glutamic acid side chain methyl ester protecting groups: The fully protected linear linifrope (5 mmol) was taken in a round bottom flask, dissolved in methanol (30 mL), tetrahydrofuran (30 mL), and water (15 mL), and sodium hydroxide (25.0 mmol) was added. The reaction was monitored by TLC until the reaction was complete. After the reaction was complete, the solvent was removed by vacuum rotary evaporation, and the reaction was acidified to pH 2-3 with 1 M aqueous HC1. The reaction was extracted with ethyl acetate, the organic layers were combined, washed, dried, and concentrated to yield Cbz-Cys(Trt)-Cys(Trt)-Glu-Tyr-Cys(Trt)-Cys(Trt)-Asn-Pro-Ala-Cys(Trt)-Thr-Gly-Cys(Trt)-Tyr-COOH (4.4 mmol) as a white solid in 94% yield.

[0146] Cbz-Cys(Trt)-Cys(Trt)-Glu-Tyr-Cys(Trt)-Cys(Trt)-Asn-Pro-Ala-Cys(Trt)-Thr-Gly-Cys(Trt)-T yr-COOH (4.4 mmol) in 94% yield.

[0147] One step removal of the N-terminal Cbz protecting group and the cysteine side chain Trt protecting groups: The solid product from the previous step (4.4 mmol) was taken in a round bottom flask, and HBr / HOAc (20 mL, 30% mass fraction) was added. The reaction was allowed to proceed overnight, and the product was concentrated by vacuum rotary evaporation. The product was crystallized from ether, and the product was filtered to yield linear linifrope intermediate NH2-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-COOH (4.0 mmol) in 92% yield.

[0148] Example 3:

[0149] (C) "6+(2+2)+(2+2)" fragment synthesis method:

[0150] Synthesis of polypeptide fragment C1 : Fmoc-Ala-Cys(Trt)-Thr-Gly-Cys(Trt)-Tyr-OMe (synthesis method same as Al)

[0151] Synthesis of polypeptide fragment C2: Boc-Cys(Trt)-Cys(Trt)-COOH

[0152] Boc-Cys(Trt)-COOH (10.0 mmol) and MeO-Cys(Trt)-NH2 (10.0 mmol) were added to a round-bottom flask, DMF (15 mL) was added, followed by NMM (17.0 mmol) and HOBt (11.0 mmol), then EDCI (12.0 mmol) was added at 0°C and reacted for 0.5 h, and then the reaction was continued at room temperature. After TLC monitoring showed that the reaction was complete, water was added to quench the reaction, and the above reaction solution was extracted with ethyl acetate, washed, dried, and then rotary evaporated to obtain white foamy solid Boc-Cys(Trt)-Cys(Trt)-OMe (9.9 mmol) with a yield of 99%.

[0153] The above obtained solid (9.9 mmol) was taken in a round-bottom flask, methanol (20 mL), tetrahydrofuran (20 mL) and water (10 mL) were added for dissolution, sodium hydroxide (29.7 mmol) was added, and TLC monitoring was performed to determine the end point of the reaction. After the reaction was completed, the solvent was removed by vacuum rotary evaporation, 1M aqueous HCl was added to adjust the pH to 2-3, and then extracted with ethyl acetate, the organic phases were combined, washed, dried, and vacuum concentrated to obtain white solid Boc-Cys(Trt)-Cys(Trt)-COOH (9.4 mmol) with a yield of 95%.

[0154] Synthesis of polypeptide fragment C3: NH2-Asn-Pro-OMe (synthesis method same as A2.1)

[0155] Synthesis of polypeptide fragment C4: NH2-Glu(OMe)-Tyr-O t Bu

[0156] Cbz-Glu(OMe)-COOH (10.0 mmol) and MeO-Cys(Trt)-NH2 (10.0 mmol) were added to a round-bottom flask, DMF (15 mL) was added, followed by NMM (17.0 mmol) and HOBt (11.0 mmol), then EDCI (12.0 mmol) was added at 0°C and reacted for 0.5 h, and then the reaction was continued at room temperature. After TLC monitoring showed that the reaction was complete, water was added to quench the reaction, and the above reaction solution was extracted with ethyl acetate, washed, dried, and then rotary evaporated to obtain white foamy solid Boc-Cys(Trt)-Cys(Trt)-OMe (9.9 mmol) with a yield of 99%. tBuO-Tyr-NH2(10.0 mmol) was added to a round bottom flask, DMF (15 mL) was added, followed by NMM (17.0 mmol) and HOBt (11.0 mmol), then EDCI (12.0 mmol) was added at 0 °C and the reaction was allowed to proceed for 0.5 h, then the reaction was allowed to proceed at room temperature. After the reaction was complete as monitored by TLC, the reaction was quenched with water, and the reaction was extracted with ethyl acetate, washed, dried and concentrated to yield Cbz-Glu(OMe)-Tyr-O t Bu (9.9 mmol) in 99% yield.

[0157] The white solid obtained above was added to a round bottom flask, dissolved in methanol (30 mL), and Pd / C (484 mg, 10% Pd) was added. The Cbz protecting group was removed by catalytic hydrogenation using a hydrogen balloon at atmospheric pressure. The reaction was monitored by TLC and was complete after about 12 h. The reaction was filtered through celite and concentrated under vacuum to yield NH2-Glu(OMe)-Tyr-O t Bu (9.4 mmol) in 95% yield.

[0158] Synthesis of linear linaclotide:

[0159] 1) The compound C1 (8.5 mmol) obtained above was added to a round bottom flask, dissolved in DCM (20 mL), and DBU (9.4 mmol) was added. The reaction was monitored by TLC and was complete after which the DCM solution was washed with water to remove unreacted DBU, then the organic phase was acidified to pH 2-3 using 5% aqueous potassium hydrogen sulfate solution, and Fmoc was removed by extraction with DCM, which was discarded. The aqueous phase was then basified to pH 7-8 using sodium bicarbonate solid, and extracted with DCM, which was washed, dried and concentrated to yield NH2-Ala-Cys(Trt)-Thr-Gly-Cys(Trt)-Tyr-OMe (8.2 mmol) as a white solid in 96% yield.

[0160] 2) Synthesis of peptide chain fragment Boc-Cys(Trt)-Cys(Trt)-Asn-Pro-COOH

[0161] C4: Boc-Cys(Trt)-Cys(Trt)-Glu(OBu)-Tyr-OMe (8.9 mmol) was obtained in 99% yield.

[0162] Removal of the Boc-Cys(Trt)-Cys(Trt)-Glu(OBu)-Tyr-OMe methyl ester protecting group: The above obtained solid (8.9 mmol) was taken in a round bottom flask, methanol (20 mL), tetrahydrofuran (20 mL) and water (10 mL) were added to dissolve, sodium hydroxide (26.7 mmol) was added, and the reaction end point was monitored by TLC. After the reaction was completed, the solvent was removed by vacuum rotary evaporation, 1 M aqueous HC1 was added to adjust the pH to 2-3, and then extracted with ethyl acetate, the organic phase was combined, washed, dried and concentrated under vacuum to obtain a white solid Boc-Cys(Trt)-Cys(Trt)-Glu(OBu)-Tyr-COOH (8.4 mmol) with a yield of 94%.

[0163] 3) Synthesis of peptide chain fragment Boc-Cys(Trt)-Cys(Trt)-Glu(O t Bu)-Tyr-COOH

[0164] C4: Boc-Cys(Trt)-Cys(Trt)-Glu(OBu)-Tyr-OMe (8.9 mmol) was obtained in 99% yield. t Bu)-Tyr-OMe (9.0 mmol) in a round bottom flask, DMF (15 mL) was added, followed by the addition of NMM (15.3 mmol) and HOBt (9.9 mmol), and then EDCI (10.8 mmol) was added at 0°C for 0.5 h, and the reaction was continued at room temperature. After the reaction was completed as monitored by TLC, it was quenched with water, and the above reaction solution was extracted with ethyl acetate, washed, dried and rotary evaporated to obtain a white solid Boc-Cys(Trt)-Cys(Trt)-Glu(O t Bu)-Tyr-OMe (8.9 mmol) with a yield of 99%.

[0165] Removal of the Boc-Cys(Trt)-Cys(Trt)-Glu(OBu)-Tyr-OMe methyl ester protecting group: The above obtained solid (8.9 mmol) was taken in a round bottom flask, methanol (20 mL), tetrahydrofuran (20 mL) and water (10 mL) were added to dissolve, sodium hydroxide (26.7 mmol) was added, and the reaction end point was monitored by TLC. After the reaction was completed, the solvent was removed by vacuum rotary evaporation, 1 M aqueous HC1 was added to adjust the pH to 2-3, and then extracted with ethyl acetate, the organic phase was combined, washed, dried and concentrated under vacuum to obtain a white solid Boc-Cys(Trt)-Cys(Trt)-Glu(OBu)-Tyr-COOH (8.4 mmol) with a yield of 94%. tMethyl ester protecting group of Boc-Cys(Trt)-Cys(Trt)-Glu(O t Bu)-Tyr-OMe (8.0 mmol) was dissolved in a round bottom flask with methanol (20 mL), tetrahydrofuran (20 mL) and water (10 mL), sodium hydroxide (24.0 mmol) was added, and the reaction was monitored by TLC until the reaction was complete. After the reaction was complete, the solvent was removed by rotary evaporation under vacuum, and the reaction was acidified to pH 2-3 with 1 M aqueous HCl. The reaction was extracted with ethyl acetate, and the organic phase was washed, dried, and concentrated under vacuum to obtain a white solid of Boc-Cys(Trt)-Cys(Trt)-Glu(O t Bu)-Tyr-COOH (7.4 mmol) with a yield of 92%.

[0166] 4) Condensation of the final product obtained in 1) and 2) above

[0167] MeO-Tyr-Cys(Trt)-Gly-Thr-Cys(Trt)-Ala-NH2 (8.0 mmol) and Boc-Cys(Trt)-Cys(Trt)-Asn-Pro-COOH (8.0 mmol) were added to a round bottom flask, and DMF (30 mL) was added. NMM (13.6 mmol) and HOBt (8.8 mmol) were added, and the reaction was carried out at 0°C for 0.5 h, and then the reaction was continued at room temperature. After the reaction was complete, as monitored by TLC, the reaction was quenched with water, and the reaction was extracted with ethyl acetate. The organic phase was washed, dried, and concentrated under vacuum to obtain a white solid of Boc-Cys(Trt)-Cys(Trt)-Asn-Pro-Ala-Cys(Trt)-Thr-Gly-Cys(Trt)-Tyr-OMe (7.9 mmol) with a yield of 99%.

[0168] The solid obtained in the previous step (7.9 mmol) was dissolved in a round bottom flask with DCM (30 mL), and trifluoroacetic acid (10 mL) was added. The reaction was monitored by TLC until the reaction was complete, and the solvent was removed by rotary evaporation under vacuum. The reaction was precipitated with diethyl ether to obtain a white solid of NH2-Cys(Trt)-Cys(Trt)-Asn-Pro-Ala-Cys(Trt)-Thr-Gly-Cys(Trt)-Tyr-OMe (7.7 mmol) with a yield of 97%.

[0169] 5) Condensation of the final product obtained in 3) and 4) above to obtain a fully protected linear linifrope

[0170] NH2-Cys(Trt)-Cys(Trt)-Asn-Pro-Ala-Cys(Trt)-Thr-Gly-Cys(Trt)-Tyr-OMe (7.7 mmol) and Boc-Cys(Trt)-Cys(Trt)-Glu(O t Bu)-Tyr-COOH (7.7 mmol) was added to a round bottom flask, DMF (30 mL) was added, followed by NMM (13.1 mmol) and HOBt (23.1 mmol), then EDCI (9.2 mmol) was added at 0 °C and the reaction was allowed to proceed. After the reaction was complete as monitored by TLC, the reaction was quenched with water, extracted with ethyl acetate, washed, dried and concentrated to yield the fully protected linear linaclotide solid Boc-Cys(Trt)-Cys(Trt)-Glu(O t Bu)-Tyr-Cys(Trt)-Cys(Trt)-Asn-Pro-Ala-Cys(Trt)-Thr-Gly-Cys(Trt)-Tyr-OMe (7.6 mmol) in 99% yield.

[0171] 6) Removal of the C-terminal methyl ester protecting group: The fully protected linear linaclotide (5 mmol) was taken in a round bottom flask, methanol (30 mL), tetrahydrofuran (30 mL) and water (15 mL) were added to dissolve the solid, sodium hydroxide (25.0 mmol) was added and the reaction was monitored by TLC until completion. After the reaction was complete, the solvent was removed by vacuum rotary evaporation, the reaction was acidified to pH 2-3 by the addition of 1 M aqueous HC1 and extracted with ethyl acetate. The organic layers were combined, washed, dried and concentrated to yield Boc-Cys(Trt)-Cys(Trt)-Glu(O t Bu)-Tyr-Cys(Trt)-Cys(Trt)-Asn-Pro-Ala-Cys(Trt)-Thr-Gly-Cys(Trt)-Tyr-COOH (4.4 mmol) in 94% yield.

[0172] One pot removal of the N-terminal Boc protecting group, the glutamic acid side chain tert-butyl ester and the cysteine side chain Trt protecting groups: The solid product from the previous step (4.4 mmol) was taken in a round bottom flask, trifluoroacetic acid (20 mL) was added and triisopropylsilane (4.8 mmol) was slowly added dropwise at 0 °C. The reaction was allowed to proceed overnight, the product was concentrated by vacuum rotary evaporation and was crystallized from ether. The product was filtered to yield the linear linaclotide intermediate NH2-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-COOH (4.0 mmol) in 92% yield.

[0173] Second Part: Synthesis of Linaclotide

[0174] Example 4

[0175] Preparation of crude Linatide:

[0176] Take the linear Linatide substrate (5 mg) obtained in Example 3, additive KI (25 mg), acetonitrile (50 mL), add 100 mL of reaction bottle, take C (+) | C (-) as electrode (i.e. the electrode anode material and cathode material are both carbon materials), constant current of 7 mA, stirring at room temperature for 2 hours, remove acetonitrile by reducing pressure concentration, filter with 0.45 μm filter membrane, obtain crude Linatide, ready for purification, use mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile in a volume ratio of 1:1 for liquid phase purification chromatography, get a yield of 89.0%.

[0177] Example 5

[0178] Preparation of crude Linatide:

[0179] Take the linear Linatide substrate (10 mg) obtained in Example 3, additive KI (50 mg), acetonitrile (50 mL), add 100 mL of reaction bottle, take C (+) | C (-) as electrode (i.e. the electrode anode material and cathode material are both carbon materials), constant current of 7 mA, stirring at room temperature for 2 hours, remove acetonitrile by reducing pressure concentration, filter with 0.45 μm filter membrane, obtain crude Linatide, ready for purification, use mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile in a volume ratio of 1:1 for liquid phase purification chromatography, get a yield of 89.3%.

[0180] Example 6

[0181] Preparation of crude Linatide:

[0182] Take the linear Linatide substrate (20 mg) obtained in Example 3, additive KI (100 mg.), acetonitrile (50 mL), add 100 mL of reaction bottle, take C (+) | C (-) as electrode (i.e. the electrode anode material and cathode material are both carbon materials), constant current of 7 mA, stirring at room temperature for 2 hours, remove acetonitrile by reducing pressure concentration, filter with 0.45 μm filter membrane, obtain crude Linatide, ready for purification, use mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile in a volume ratio of 1:1 for liquid phase purification chromatography, get a yield of 94.3%.

[0183] Example 7

[0184] Preparation of crude Linatide:

[0185] Take the linear linearity of the example 3 obtained substrate (40mg), additive KI (200mg), acetonitrile (50ml), add 100ml reaction bottle, with C (+) | C (-) as electrode (i.e. electrode anode material and cathode material are carbon material), with 7ma constant current stirring at room temperature for 2 hours, remove acetonitrile under reduced pressure, filter with 0.45um filter membrane, get crude linearity, purification for use, with mobile phase for 0.1% FA / H2O and 0.1% FA / acetonitrile of volume ratio 1:1, liquid phase purification chromatography, get yield of 94.5%.

[0186] Example 8

[0187] Preparation of crude linearity:

[0188] Take the linear linearity of the example 3 obtained substrate (50mg), additive KI (250mg), acetonitrile (50ml), add 100ml reaction bottle, with C (+) | C (-) as electrode (i.e. electrode anode material and cathode material are carbon material), with 7ma constant current stirring at room temperature for 2 hours, remove acetonitrile under reduced pressure, filter with 0.45um filter membrane, get crude linearity, purification for use, with mobile phase for 0.1% FA / H2O and 0.1% FA / acetonitrile of volume ratio 1:1, liquid phase purification chromatography, get yield of 95.0%.

[0189] Example 9

[0190] Preparation of crude linearity:

[0191] Take the linear linearity of the example 3 obtained substrate (100mg), additive KI (500mg.), acetonitrile (50ml), add 100ml reaction bottle, with C (+) | C (-) as electrode (i.e. electrode anode material and cathode material are carbon material), with 7ma constant current stirring at room temperature for 2 hours, remove acetonitrile under reduced pressure, filter with 0.45um filter membrane, get crude linearity, purification for use, with mobile phase for 0.1% FA / H2O and 0.1% FA / acetonitrile of volume ratio 1:1, liquid phase purification chromatography, get yield of 94.2%.

[0192] Example 10

[0193] Preparation of crude linearity:

[0194] Take the linear linearity of the example 3 obtained substrate (150mg), additive KI (750mg), acetonitrile (50ml), add 100ml of reaction bottle, take C (+) | C (-) as electrode (i.e. electrode anode material and cathode material are carbon materials), with 7ma constant current stirring at room temperature for 2 hours, remove acetonitrile under reduced pressure, filter with 0.45um filter membrane, get crude linearity, purification for use, adopt 0.1% FA / H2O and 0.1% FA / acetonitrile as mobile phase, carry out liquid phase purification chromatography, get yield of 93.4%.

[0195] Example 11

[0196] Preparation of crude linearity:

[0197] Take the linear linearity of the example 3 obtained substrate (200mg), additive KI (1000mg.), acetonitrile (50ml), add 100ml of reaction bottle, take C (+) | C (-) as electrode (i.e. electrode anode material and cathode material are carbon materials), with 7ma constant current stirring at room temperature for 2 hours, remove acetonitrile under reduced pressure, filter with 0.45um filter membrane, get crude linearity, purification for use, adopt 0.1% FA / H2O and 0.1% FA / acetonitrile as mobile phase, carry out liquid phase purification chromatography, get yield of 92.1%.

[0198] Example 12

[0199] Preparation of crude linearity:

[0200] Take the linear linearity of the example 3 obtained substrate (250mg), additive KI (1250mg), acetonitrile (50ml), add 100ml of reaction bottle, take C (+) | C (-) as electrode (i.e. electrode anode material and cathode material are carbon materials), with 7ma constant current stirring at room temperature for 2 hours, remove acetonitrile under reduced pressure, filter with 0.45um filter membrane, get crude linearity, purification for use, adopt 0.1% FA / H2O and 0.1% FA / acetonitrile as mobile phase, carry out liquid phase purification chromatography, get yield of 89.0%.

[0201] Example 13

[0202] Preparation of crude linearity:

[0203] Take the linear linearity of the example 3 obtained substrate (400mg), additive KI (2000mg), acetonitrile (50ml), add 100ml reaction bottle, with C (+) |C (-) as electrode (i.e. electrode anode material and cathode material are carbon material), with 7ma constant current stirring at room temperature for 2 hours, remove acetonitrile under reduced pressure, filter with 0.45um filter membrane, get crude linearity, purification for use, with mobile phase for 0.1% FA / H2O and 0.1% FA / acetonitrile of volume ratio 1:1, liquid phase purification chromatography, get yield of 85.4%.

[0204] Example 14

[0205] Preparation of crude linearity:

[0206] Take the linear linearity of the example 3 obtained substrate (500mg), additive KI (2500mg), acetonitrile (50ml), add 100ml reaction bottle, with C (+) |C (-) as electrode (i.e. electrode anode material and cathode material are carbon material), with 7ma constant current stirring at room temperature for 2 hours, remove acetonitrile under reduced pressure, filter with 0.45um filter membrane, get crude linearity, purification for use, with mobile phase for 0.1% FA / H2O and 0.1% FA / acetonitrile of volume ratio 1:1, liquid phase purification chromatography, get yield of 82.1%.

[0207] Example 15

[0208] Preparation of crude linearity:

[0209] Take the linear linearity of the example 3 obtained substrate (50mg), additive KI (250mg), acetonitrile (50ml), add 100ml reaction bottle, with C (+) |C (-) as electrode (i.e. electrode anode material and cathode material are carbon material), with 2ma constant current stirring at room temperature for 2 hours, remove acetonitrile under reduced pressure, filter with 0.45um filter membrane, get crude linearity, purification for use, with mobile phase for 0.1% FA / H2O and 0.1% FA / acetonitrile of volume ratio 1:1, liquid phase purification chromatography, get yield of 82.3%.

[0210] Example 16

[0211] Preparation of crude linearity:

[0212] The linear linifipotide substrate (50 mg) obtained in Example 3, additive KI (250 mg), acetonitrile (50 mL) were added into a 100 mL reaction bottle, C (+) | C (-) was used as the electrode (i.e. the anode material and the cathode material of the electrode were both carbon materials), constant current of 3 mA was used for stirring at room temperature for 2 hours, acetonitrile was removed by concentration under reduced pressure, and the crude linifipotide was obtained by filtration with a 0.45 μm filter membrane, which was ready for purification. Liquid phase purification chromatography was performed with mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile in a volume ratio of 1:1, and the yield was 86.2%.

[0213] Example 17

[0214] Preparation of crude linifipotide:

[0215] The linear linifipotide substrate (50 mg) obtained in Example 3, additive KI (250 mg), acetonitrile (50 mL) were added into a 100 mL reaction bottle, C (+) | C (-) was used as the electrode (i.e. the anode material and the cathode material of the electrode were both carbon materials), constant current of 4 mA was used for stirring at room temperature for 2 hours, acetonitrile was removed by concentration under reduced pressure, and the crude linifipotide was obtained by filtration with a 0.45 μm filter membrane, which was ready for purification. Liquid phase purification chromatography was performed with mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile in a volume ratio of 1:1, and the yield was 90.3%.

[0216] Example 18

[0217] Preparation of crude linifipotide:

[0218] The linear linifipotide substrate (50 mg) obtained in Example 3, additive KI (250 mg), acetonitrile (50 mL) were added into a 100 mL reaction bottle, C (+) | C (-) was used as the electrode (i.e. the anode material and the cathode material of the electrode were both carbon materials), constant current of 6 mA was used for stirring at room temperature for 2 hours, acetonitrile was removed by concentration under reduced pressure, and the crude linifipotide was obtained by filtration with a 0.45 μm filter membrane, which was ready for purification. Liquid phase purification chromatography was performed with mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile in a volume ratio of 1:1, and the yield was 93.3%.

[0219] Example 19

[0220] Preparation of crude linifipotide:

[0221] The linear linifipotide substrate (50 mg) obtained in Example 3, additive KI (250 mg), acetonitrile (50 mL) were added into a 100 mL reaction flask, C (+) | C (-) as electrode (i.e. the anode material and cathode material of the electrode are both carbon materials), constant current of 8 mA, stirred at room temperature for 2 hours, acetonitrile was removed by concentration under reduced pressure, filtered with a 0.45 μm filter, to obtain a crude linifipotide, which was purified and used for liquid phase purification chromatography with a mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile in a volume ratio of 1:1, to obtain a yield of 92.8%.

[0222] Example 20

[0223] Preparation of crude linifipotide:

[0224] The linear linifipotide substrate (50 mg) obtained in Example 3, additive KI (250 mg), acetonitrile (50 mL) were added into a 100 mL reaction flask, C (+) | C (-) as electrode (i.e. the anode material and cathode material of the electrode are both carbon materials), constant current of 50 mA, stirred at room temperature for 2 hours, acetonitrile was removed by concentration under reduced pressure, filtered with a 0.45 μm filter, to obtain a crude linifipotide, which was purified and used for liquid phase purification chromatography with a mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile in a volume ratio of 1:1, to obtain a yield of 91.3%.

[0225] Example 21

[0226] Preparation of crude linifipotide:

[0227] The linear linifipotide substrate (50 mg) obtained in Example 3, additive KI (250 mg), acetonitrile (50 mL) were added into a 100 mL reaction flask, C (+) | C (-) as electrode (i.e. the anode material and cathode material of the electrode are both carbon materials), constant current of 11 mA, stirred at room temperature for 2 hours, acetonitrile was removed by concentration under reduced pressure, filtered with a 0.45 μm filter, to obtain a crude linifipotide, which was purified and used for liquid phase purification chromatography with a mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile in a volume ratio of 1:1, to obtain a yield of 89.2%.

[0228] Example 22

[0229] Preparation of crude linifipotide:

[0230] The linear linifipotide substrate (50 mg) obtained in Example 3, additive KI (250 mg), acetonitrile (50 mL) were added into a 100 mL reaction flask, C(+)-C(-) as electrode (i.e. the anode material and cathode material of the electrode are both carbon materials), constant current of 12 mA, stirred at room temperature for 2 hours, acetonitrile was removed by concentration under reduced pressure, filtered with a 0.45 μm filter, to obtain a crude linifipotide, which was purified and used for liquid phase purification chromatography with a mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile in a volume ratio of 1:1, to obtain a yield of 85.2%.

[0231] Example 23

[0232] Preparation of a crude linifipotide:

[0233] The linear linifipotide substrate (50 mg) obtained in Example 3, additive KI (250 mg), acetonitrile (50 mL) were added into a 100 mL reaction flask, C(+)-C(-) as electrode (i.e. the anode material and cathode material of the electrode are both carbon materials), constant current of 12 mA, stirred at room temperature for 2 hours, acetonitrile was removed by concentration under reduced pressure, filtered with a 0.45 μm filter, to obtain a crude linifipotide, which was purified and used for liquid phase purification chromatography with a mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile in a volume ratio of 1:1, to obtain a yield of 85.2%.

[0234] Example 24

[0235] Preparation of a crude linifipotide:

[0236] The linear linifipotide substrate (50 mg) obtained in Example 3, additive KI (250 mg), acetonitrile (50 mL) were added into a 100 mL reaction flask, C(+)-C(-) as electrode (i.e. the anode material and cathode material of the electrode are both carbon materials), constant current of 12 mA, stirred at room temperature for 2 hours, acetonitrile was removed by concentration under reduced pressure, filtered with a 0.45 μm filter, to obtain a crude linifipotide, which was purified and used for liquid phase purification chromatography with a mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile in a volume ratio of 1:1, to obtain a yield of 85.2%.

[0237] Example 25

[0238] Preparation of a crude linifipotide:

[0239] The linear linaclotide substrate (50 mg) obtained in Example 3, additive LiClO4 (160 mg), and acetonitrile (50 mL) were added to a 100 mL reaction flask. Using C(+)-C(-) as the electrode (i.e., both the anode and cathode materials are carbon materials), the mixture was stirred at room temperature for 2 hours with a constant current of 7 mA. The acetonitrile was removed by concentration under reduced pressure. The mixture was filtered through a 0.45 μm filter membrane to obtain crude linaclotide, which was purified for later use. Liquid chromatography was performed using a mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile (v / v), yielding a yield of 91.4%.

[0240] Example 26

[0241] Preparation of crude linaclotide:

[0242] Take 50 mg of the linear linaclotide substrate obtained in Example 3, and the additives... n Bu4NBF4 (494 mg) and acetonitrile (50 mL) were added to a 100 mL reaction flask. Using C(+)-C(-) electrodes (i.e., both the anode and cathode materials were carbon materials), the mixture was stirred at room temperature for 2 hours with a constant current of 7 mA. The mixture was then concentrated under reduced pressure to remove acetonitrile. The solution was filtered through a 0.45 μm filter membrane to obtain crude linaclotide. After purification, the crude product was purified by liquid chromatography using a mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile (v / v), yielding a yield of 96.3%.

[0243] Example 27

[0244] Preparation of crude linaclotide:

[0245] The linear linaclotide substrate (50 mg) obtained in Example 3, the additive Et4NI (386 mg), and acetonitrile (50 mL) were added to a 100 mL reaction flask. Using C(+)-C(-) as the electrode (i.e., both the anode and cathode materials are carbon materials), the mixture was stirred at room temperature for 2 hours with a constant current of 7 mA. The acetonitrile was removed by concentration under reduced pressure, and the mixture was filtered through a 0.45 μm filter membrane to obtain crude linaclotide. The purified product was then purified by liquid chromatography using a mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile at a volume ratio of 1:1, yielding a yield of 93.2%.

[0246] Example 28

[0247] Preparation of crude linaclotide:

[0248] The linear linaclotide substrate (50 mg) obtained in Example 3, additives KI (125 mg) and LiClO4 (80 mg), and acetonitrile (50 mL) were added to a 100 mL reaction flask. Using C(+)-C(-) as the electrode (i.e., both the anode and cathode materials are carbon materials), the mixture was stirred at room temperature for 2 hours with a constant current of 7 mA. The acetonitrile was removed by concentration under reduced pressure, and the mixture was filtered through a 0.45 μm filter membrane to obtain crude linaclotide. The purified product was then purified by liquid chromatography using a mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile at a volume ratio of 1:1, yielding a yield of 91.3%.

[0249] Example 29

[0250] Preparation of crude linaclotide:

[0251] Take 50 mg of the linear linaclotide substrate obtained in Example 3, and the additives... n Bu4NOAc (452 ​​mg) and acetonitrile (50 mL) were added to a 100 mL reaction flask. Using C(+)-C(-) electrodes (i.e., both the anode and cathode materials were carbon materials), the mixture was stirred at room temperature for 2 hours with a constant current of 7 mA. The mixture was then concentrated under reduced pressure to remove acetonitrile. The solution was filtered through a 0.45 μm filter membrane to obtain crude linaclotide. After purification, the crude product was purified by liquid chromatography using a mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile (v / v), yielding a yield of 90.2%.

[0252] Example 30

[0253] Preparation of crude linaclotide:

[0254] Take 50 mg of the linear linaclotide substrate obtained in Example 3, and the additives... n Bu4NCl (417 mg) and acetonitrile (50 mL) were added to a 100 mL reaction flask. Using C(+)-C(-) electrodes (i.e., both the anode and cathode materials were carbon materials), the mixture was stirred at room temperature for 2 hours with a constant current of 7 mA. The mixture was then concentrated under reduced pressure to remove acetonitrile. The solution was filtered through a 0.45 μm filter membrane to obtain crude linaclotide. After purification, the crude product was purified by liquid chromatography using a mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile (v / v), yielding a yield of 91.2%.

[0255] Example 31

[0256] Preparation of crude linaclotide:

[0257] Take 50 mg of the linear linaclotide substrate obtained in Example 3, and the additives... nBu4NHSO4(509 mg), acetonitrile (50 mL), added into a 100 mL reaction flask, C(+)-C(-) as electrodes (i.e. both anode material and cathode material of the electrode are carbon materials), stirred at a constant current of 7 mA for 2 hours at room temperature, acetonitrile was removed by concentration under reduced pressure, filtered with a 0.45 μm filter membrane to obtain a crude product of linaclotide, ready for purification, liquid phase purification chromatography was performed with mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile at a volume ratio of 1:1, and the yield was 83.2%.

[0258] Example 32

[0259] Preparation of a crude product of linaclotide:

[0260] Take the linear linaclotide substrate (50 mg) obtained in Example 3, add additives n Bu4NHSO4(509 mg), acetonitrile (50 mL), added into a 100 mL reaction flask, C(+)-C(-) as electrodes (i.e. both anode material and cathode material of the electrode are carbon materials), stirred at a constant current of 7 mA for 2 hours at room temperature, acetonitrile was removed by concentration under reduced pressure, filtered with a 0.45 μm filter membrane to obtain a crude product of linaclotide, ready for purification, liquid phase purification chromatography was performed with mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile at a volume ratio of 1:1, and the yield was 83.2%.

[0261] Example 33

[0262] Preparation of a crude product of linaclotide:

[0263] Take the linear linaclotide substrate (50 mg) obtained in Example 3, add additives n Bu4NHSO4(509 mg), acetonitrile (50 mL), added into a 100 mL reaction flask, C(+)-C(-) as electrodes (i.e. both anode material and cathode material of the electrode are carbon materials), stirred at a constant current of 7 mA for 2 hours at room temperature, acetonitrile was removed by concentration under reduced pressure, filtered with a 0.45 μm filter membrane to obtain a crude product of linaclotide, ready for purification, liquid phase purification chromatography was performed with mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile at a volume ratio of 1:1, and the yield was 83.2%.

[0264] Example 34

[0265] Preparation of a crude product of linaclotide:

[0266] Take the linear linaclotide substrate (50 mg) obtained in Example 3, add additives nBu4NBF4(494 mg), tetrahydrofuran (50 mL), added into a 100 mL reaction flask, C(+)-C(-) as electrodes (i.e. both anode material and cathode material of the electrode are carbon materials), stirred at a constant current of 7 mA at room temperature for 2 hours, removed tetrahydrofuran under reduced pressure, filtered with a 0.45 μm filter membrane to obtain a crude product of linaclotide, ready for purification, purified by liquid chromatography with a mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile at a volume ratio of 1:1, to obtain a yield of 90.1%.

[0267] Example 35

[0268] Preparation of a crude product of linaclotide:

[0269] Take the linear linaclotide substrate (50 mg) obtained in Example 3, add additives n Bu4NBF4(494 mg), tetrahydrofuran (50 mL), added into a 100 mL reaction flask, C(+)-C(-) as electrodes (i.e. both anode material and cathode material of the electrode are carbon materials), stirred at a constant current of 7 mA at room temperature for 2 hours, removed tetrahydrofuran under reduced pressure, filtered with a 0.45 μm filter membrane to obtain a crude product of linaclotide, ready for purification, purified by liquid chromatography with a mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile at a volume ratio of 1:1, to obtain a yield of 90.1%.

[0270] Example 36

[0271] Preparation of a crude product of linaclotide:

[0272] Take the linear linaclotide substrate (50 mg) obtained in Example 3, add additives n Bu4NBF4(494 mg), tetrahydrofuran (50 mL), added into a 100 mL reaction flask, C(+)-C(-) as electrodes (i.e. both anode material and cathode material of the electrode are carbon materials), stirred at a constant current of 7 mA at room temperature for 2 hours, removed tetrahydrofuran under reduced pressure, filtered with a 0.45 μm filter membrane to obtain a crude product of linaclotide, ready for purification, purified by liquid chromatography with a mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile at a volume ratio of 1:1, to obtain a yield of 90.1%.

[0273] Example 37

[0274] Preparation of a crude product of linaclotide:

[0275] Take the linear linaclotide substrate (50 mg) obtained in Example 3, add additives nBu4NBF4(494 mg), methanol (6 mL), chloroform (4 mL), added into a 100 mL reaction flask, C(+)-C(-) as electrodes (i.e. both anode material and cathode material of the electrode are carbon materials), stirred at a constant current of 7 mA for 2 hours at room temperature, removed the solvent by concentration under reduced pressure, filtered by 0.45 μm filter membrane to obtain the crude linaclotide, ready for purification, purified by liquid chromatography with mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile (1:1, by volume), the yield was 93.2%.

[0276] Example 38

[0277] Preparation of crude linaclotide:

[0278] Take the linear linaclotide substrate (50 mg) obtained in Example 3, add additives n Bu4NBF4(494 mg), methanol (6 mL), chloroform (4 mL), added into a 100 mL reaction flask, C(+)-C(-) as electrodes (i.e. both anode material and cathode material of the electrode are carbon materials), stirred at a constant current of 7 mA for 2 hours at room temperature, removed the solvent by concentration under reduced pressure, filtered by 0.45 μm filter membrane to obtain the crude linaclotide, ready for purification, purified by liquid chromatography with mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile (1:1, by volume), the yield was 93.2%.

[0279] Example 39

[0280] Preparation of crude linaclotide:

[0281] Take the linear linaclotide substrate (50 mg) obtained in Example 3, add additives n Bu4NBF4(494 mg), methanol (6 mL), chloroform (4 mL), added into a 100 mL reaction flask, C(+)-C(-) as electrodes (i.e. both anode material and cathode material of the electrode are carbon materials), stirred at a constant current of 7 mA for 2 hours at room temperature, removed the solvent by concentration under reduced pressure, filtered by 0.45 μm filter membrane to obtain the crude linaclotide, ready for purification, purified by liquid chromatography with mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile (1:1, by volume), the yield was 93.2%.

[0282] Example 40

[0283] Preparation of crude linaclotide:

[0284] Take the linear linaclotide substrate (50 mg) obtained in Example 3, add additives nBu4NBF4 (494 mg) and ethanol (50 mL) were added to a 100 mL reaction flask. Using C(+)|Pt(-) as the electrode (i.e., both the anode and cathode materials are carbon materials), the mixture was stirred at room temperature for 2 hours with a constant current of 7 mA. The ethanol was removed by vacuum concentration, and the mixture was filtered through a 0.45 μm filter membrane to obtain crude linaclotide. The purified product was then purified by liquid chromatography using a mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile (v / v), yielding a yield of 93.2%.

[0285] Example 41

[0286] Preparation of crude linaclotide:

[0287] Take 50 mg of the linear linaclotide substrate obtained in Example 3, and the additives... n Bu4NBF4 (494 mg) and ethanol (50 mL) were added to a 100 mL reaction flask. Using Pt(+)|Pt(-) as electrodes (i.e., both the anode and cathode materials were platinum), the mixture was stirred at room temperature for 2 hours with a constant current of 7 mA. The ethanol was removed by vacuum concentration, and the mixture was filtered through a 0.45 μm filter membrane to obtain crude linaclotide. The purified product was then purified by liquid chromatography using a mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile (v / v), yielding a yield of 92.1%.

[0288] Example 42

[0289] Preparation of crude linaclotide:

[0290] Take 50 mg of the linear linaclotide substrate obtained in Example 3, and the additives... n Bu4NBF4 (494 mg) and ethanol (50 mL) were added to a 100 mL reaction flask. Using GC(+)|Pt(-) as the electrode (i.e., glassy carbon as the anode material and platinum as the cathode material), the mixture was stirred at room temperature for 2 hours with a constant current of 7 mA. The ethanol was removed by vacuum concentration, and the mixture was filtered through a 0.45 μm filter membrane to obtain crude linaclotide. The purified product was then purified by liquid chromatography using a mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile (v / v), yielding a yield of 89.1%.

[0291] Example 43

[0292] Preparation of crude linaclotide:

[0293] Take 50 mg of the linear linaclotide substrate obtained in Example 3, and the additives... nBu4NBF4(494 mg), ethanol (50 mL), added to a 100 mL reaction flask, with C (+) | Pt (-) as electrodes (i.e. the anode material of the electrode is carbon fiber cloth material, and the cathode material is platinum material), stirred at a constant current of 7 mA at room temperature for 2 hours, concentrated to remove ethanol under reduced pressure, filtered with a 0.45 μm filter membrane, to obtain a crude linaclotide product, ready for purification, liquid phase purification chromatography was performed with a mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile at a volume ratio of 1:1, to obtain a yield of 83.2%.

[0294] Example 44

[0295] Preparation of crude linaclotide:

[0296] Take the linear linaclotide substrate (50 mg) obtained in Example 3, add additives n Bu4NBF4(494 mg), ethanol (50 mL), added to a 100 mL reaction flask, with C (+) | Pt (-) as electrodes (i.e. the anode material of the electrode is carbon fiber cloth material, and the cathode material is platinum material), stirred at a constant current of 7 mA at room temperature for 2 hours, concentrated to remove ethanol under reduced pressure, filtered with a 0.45 μm filter membrane, to obtain a crude linaclotide product, ready for purification, liquid phase purification chromatography was performed with a mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile at a volume ratio of 1:1, to obtain a yield of 83.2%.

[0297] Example 45

[0298] Preparation of crude linaclotide:

[0299] Take the linear linaclotide substrate (50 mg) obtained in Example 3, add additives n Bu4NBF4(494 mg), ethanol (50 mL), added to a 100 mL reaction flask, with C (+) | Pt (-) as electrodes (i.e. the anode material of the electrode is carbon fiber cloth material, and the cathode material is platinum material), stirred at a constant current of 7 mA at room temperature for 2 hours, concentrated to remove ethanol under reduced pressure, filtered with a 0.45 μm filter membrane, to obtain a crude linaclotide product, ready for purification, liquid phase purification chromatography was performed with a mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile at a volume ratio of 1:1, to obtain a yield of 83.2%.

[0300] Example 46

[0301] Preparation of crude linaclotide:

[0302] Take the linear linaclotide substrate (50 mg) obtained in Example 3, add additives nBu4NBF4(494 mg), ethanol (50 mL), added to a 100 mL reaction flask, C(+)-C(-) as electrodes (i.e. both anode and cathode materials of the electrodes are carbon materials), stirred at a constant current of 7 mA at room temperature for 0.5 hours, removed ethanol by concentration under reduced pressure, filtered with a 0.45 μm filter membrane to obtain a crude linaclotide, ready for purification, purified by liquid chromatography with a mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile at a volume ratio of 1:1 to obtain a yield of 90.2%.

[0303] Example 47

[0304] Preparation of crude linaclotide:

[0305] Take the linear linaclotide substrate (50 mg) obtained in Example 3, add additives n Bu4NBF4(494 mg), ethanol (50 mL), added to a 100 mL reaction flask, C(+)-C(-) as electrodes (i.e. both anode and cathode materials of the electrodes are carbon materials), stirred at a constant current of 7 mA at room temperature for 0.5 hours, removed ethanol by concentration under reduced pressure, filtered with a 0.45 μm filter membrane to obtain a crude linaclotide, ready for purification, purified by liquid chromatography with a mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile at a volume ratio of 1:1 to obtain a yield of 90.2%.

[0306] Example 48

[0307] Preparation of crude linaclotide:

[0308] Take the linear linaclotide substrate (50 mg) obtained in Example 3, add additives n Bu4NBF4(494 mg), ethanol (50 mL), added to a 100 mL reaction flask, C(+)-C(-) as electrodes (i.e. both anode and cathode materials of the electrodes are carbon materials), stirred at a constant current of 7 mA at room temperature for 0.5 hours, removed ethanol by concentration under reduced pressure, filtered with a 0.45 μm filter membrane to obtain a crude linaclotide, ready for purification, purified by liquid chromatography with a mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile at a volume ratio of 1:1 to obtain a yield of 90.2%.

[0309] Example 49

[0310] Preparation of crude linaclotide:

[0311] Take the linear linaclotide substrate (50 mg) obtained in Example 3, add additives nBu4NBF4(494 mg), ethanol (50 mL), added to a 100 mL reaction flask, C(+)-C(-) as electrodes (i.e. both anode and cathode materials of the electrodes are carbon materials), stirred at a constant current of 7 mA at room temperature for 3 hours, removed ethanol by concentration under reduced pressure, filtered with a 0.45 μm filter membrane to obtain a crude linaclotide, ready for purification, purified by liquid chromatography with a mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile at a volume ratio of 1:1 to obtain a yield of 94.2%.

[0312] Example 50

[0313] Preparation of crude linaclotide:

[0314] Take the linear linaclotide substrate (50 mg) obtained in Example 3, add additives n Bu4NBF4(494 mg), ethanol (50 mL), added to a 100 mL reaction flask, C(+)-C(-) as electrodes (i.e. both anode and cathode materials of the electrodes are carbon materials), stirred at a constant current of 7 mA at room temperature for 4 hours, removed ethanol by concentration under reduced pressure, filtered with a 0.45 μm filter membrane to obtain a crude linaclotide, ready for purification, purified by liquid chromatography with a mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile at a volume ratio of 1:1 to obtain a yield of 92.1%.

[0315] Example 51

[0316] Preparation of crude linaclotide:

[0317] Take the linear linaclotide substrate (50 mg) obtained in Example 3, add additives n Bu4NBF4(494 mg), ethanol (50 mL), added to a 100 mL reaction flask, C(+)-C(-) as electrodes (i.e. both anode and cathode materials of the electrodes are carbon materials), stirred at a constant current of 7 mA at room temperature for 5 hours, removed ethanol by concentration under reduced pressure, filtered with a 0.45 μm filter membrane to obtain a crude linaclotide, ready for purification, purified by liquid chromatography with a mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile at a volume ratio of 1:1 to obtain a yield of 86.4%.

[0318] Example 52

[0319] Preparation of crude linaclotide:

[0320] Take the linear linaclotide substrate (1 g) obtained in Example 3, add additives nBu4NBF4(494mg), ethanol (1000ml), added into 2000ml reaction flask, C(+)-C(-) as electrode (i.e. both electrode anode material and cathode material are carbon material), constant current 7ma, stirred at room temperature for 15 hours, removed ethanol under reduced pressure, filtered by 0.45μm filter membrane to obtain crude linaclotide, ready for purification, purified by liquid chromatography with mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile (1:1, by volume), to obtain linaclotide with a yield of 88.3%.

[0321] Example 53

[0322] Purification of crude linaclotide:

[0323] The final product obtained in Examples 4-52 was purified by preparative liquid chromatography with mobile phase of 0.1% FA / H2O and 0.1% FA / acetonitrile (1:1, by volume). The crude product was loaded onto a chromatographic column, and the mobile phase was started to elute. The main peak was collected and the purity was monitored by analytical liquid chromatography. The main peak solution was combined to obtain a linaclotide solution, which was concentrated under reduced pressure to obtain the final linaclotide product.

[0324] The content described in the specification is only a list of implementation forms of the inventive concept, and the protection scope of the present application should not be regarded as limited to the specific forms stated in the examples.

Claims

1. A method for synthesizing linaclotide, characterized in that... Includes the following steps: The first step is to segment the linear linaclotin into fragments, specifically in one of the following three ways: (1) The design and planning method of the number of amino acids in the polypeptide fragment is "6 + 4 + 4": There are 3 polypeptide fragments, namely R 1 -Cys(R 2 )-Cys(R 2 )-Glu(R 4 )-Tyr(R 5 )-R 3 R 1 -Cys(R 2 )-Cys(R 2 )-Asn(R 7 )-Pro-R 3 and R 1 -Ala-Cys(R 2 )-Thr(R 6 )-Gly-Cys(R 2 )-Tyr(R 5 )-R 3 The polypeptide fragments are sequentially linked by stepwise liquid-phase condensation from the N-terminus to the C-terminus or from the C-terminus to the N-terminus to obtain a fully protected linear linaclotin. (2) The design and planning method of the number of amino acids in the polypeptide fragment is "5 + 5 + 4": There are 3 polypeptide fragments, namely R 1 -Cys(R 2 )-Cys(R 2 )-Glu(R 4 )-Tyr(R 5 )-R 3 R 1 -Cys(R 2 )-Cys(R 2 )-Asn(R 7 )-Pro-Ala-R 3 and R 1 -Cys(R 2 )-Thr(R 6 )-Gly-Cys(R 2 )-Tyr(R 5 )-R 3 The polypeptide fragments are sequentially linked by stepwise liquid-phase condensation from the N-terminus to the C-terminus or from the C-terminus to the N-terminus to obtain a fully protected linear linaclotin. (3) The design and planning method for the number of amino acids in the polypeptide fragment is "6 + (2 + 2) + (2 + 2)": There are 5 polypeptide fragments, namely R 1 -Cys(R 2 )-Cys(R 2 )-COOH, NH2-Glu(R 4 )-Tyr(R 5 )-R 3 R 1 -Cys(R 2 )-Cys(R 2 )-COOH, NH2-Asn(R 7 )-Pro-R 3 and R 1 -Ala-Cys(R 2 )-Thr(R 6 )-Gly-Cys(R 2 )-Tyr(R 5 )-R 3 The polypeptide fragments are sequentially linked by stepwise liquid-phase condensation from the N-terminus to the C-terminus or from the C-terminus to the N-terminus to obtain a fully protected linear linaclotin. Among them, R 1 R is a side-chain protecting group of cysteine ​​(Cys) selected from Boc, Cbz, Fmoc, or H. 2 For Trt, Bzl, Acm, Mmt, t One of Bu or H; the side-chain protecting group R of glutamic acid Glu. 4 It is one of methyl ester, tert-butyl ester, benzyl ester, or H; the side chain protecting group R of tyrosine Tyr 5 For Bzl, t One of Bu, Me, or H; the side-chain protecting group R of threonine Thr. 6 For Bzl, t One of Bu, Me, or H; the side-chain protecting group R of asparagine Asn. 7 It is one of Trt, Xant, DMB, or H; R 3 It is one of methyl ester, tert-butyl ester, benzyl ester, or H; choosing H indicates that no protecting group is used. The second step involves selecting the polypeptide fragments synthesized in the first step according to the polypeptide sequence of the linear linaclotide, deprotecting the amino-terminal or carboxyl-terminal protecting groups of the polypeptide fragments, and sequentially linking the polypeptide fragments by stepwise liquid-phase condensation from the N-terminus to the C-terminus or from the C-terminus to the N-terminus to obtain a fully protected linear linaclotide. The third step involves removing the protecting groups from the side chains and main chain of the fully protected linear linaclotin to obtain the linear linaclotin, which is the linaclotin intermediate. Then, the linaclotide intermediate is electrochemically oxidized to synthesize linaclotide, including the following steps: 1) The linaclotide intermediate is a linear linaclotide. The linaclotide intermediate is dissolved in solvent B, an additive is added, and the reaction is carried out for a certain time under certain current intensity, reaction concentration and electrode material conditions to obtain crude linaclotide. The electrode anode and cathode materials used are one of C(+)|C(-), C(+)|Pt(-), Pt(+)|Pt(-), GC(+)|Pt(-), Carbon rod(+)|Pt(-), Carbon cloth(+)|Pt(-), GF(+)|GF(-), GF(+)|Ni(-). The additives used in the electrochemical oxidation reaction are KI, NaI, and LiClO4. n Bu4NBF4, Et4NI, NH4I, NH4Br, n Bu4NPF6, n Bu4NBr、 n Bu4NCl、 n One or more of Bu4NOAc; Solvent B is N , N - One or more of dimethylformamide, tetrahydrofuran, methanol, ethanol, hexafluoroisopropanol, acetonitrile, ethylene glycol dimethyl ether, butanol, chloroform, and dimethyl sulfoxide; the concentration of the linaclotide intermediate in solvent B is 0.1~10 mg / mL, and the concentration of the additive in solvent B is 0.5~50 mg / mL; The electrochemical oxidation reaction current intensity is 2~15 mA, and the reaction time is 0.2~5 h; 2) The crude linaclotide obtained was purified by liquid chromatography to obtain pure linaclotide.

2. The method as described in claim 1, characterized in that... In the first step, the liquid-phase condensation reaction is carried out in solvent A, where amino acids or polypeptide fragments, bases, activators and coupling agents are added to solvent A to carry out the liquid-phase condensation reaction. Solvent A is dichloromethane, ethyl acetate, N , N - One or more of dimethylformamide, acetonitrile, acetone, water, and tetrahydrofuran; The alkali is one of DIPEA, TEA, NMM, DMAP, or potassium carbonate; The activator is one or more of the following: HOBt, 6-NO2-HOBt, 6-CF3-HOBt, HOAt, 6-Cl-HOBt, 6-HOAt, 5-HOAt, 4-HOAt, HODhbt, HODhat, HODhad, HOSu, HONB, HOCt, HOPy, HOBI, HOI, 6-Cl-HOBI, and Oxyma. The coupling agent is one or more of the following: carbodiimide coupling agents, phosphate salt coupling agents, tetramethylammonium salt coupling agents, ammonium salt coupling agents, triazine coupling agents, and pyridine coupling agents; the carbodiimide coupling agent is selected from one or more of DIC, DCC, EDCI, CIC, BMC, BEC, CPC, BDDC, PEC, and PIC; the phosphate salt coupling agent is selected from one or more of BOP, BrOP, PyCloP, PyBrOP, CloP, PyBOP, AOP, PyAOP, PyOxm, PyNOP, PyFOP, PyFNBOP, PyCloK, PyPOP, PyTOP, PyDOP, and PyDAOP; the tetramethylammonium salt coupling agent is selected from... N -HBTU、 N -TBTU, TDTU, HDTU, TDATU, HDATU, TPTU, HPTU, TSTU, HSTU, TPFTU, HPFTU, N -CF3-HBTU N -HATU、 N -TATU、 N -HATTU, HOTT, TOTU, HOTU, HTODC, HTODeC, HTOPC, TNTU, TPhTU or more of these; the ammonium salt coupling agent is selected from PyCIU, HBPyU, HAPyU, HDPyU, HDAPyU, HPyOPfp, HPySPfp, HAPyTU, HPyONP, HPyOTCp, HBPipU, HAPipU, TOPPipU, CIP, HBMDU, HAMDU, CPP, HBMTU, HAMTU, HBPTU, HAPTU, HBM2P The following are selected from the following: yU, HAM2PyU, HBM2PipU, HAM2PipU, HBE2PyU, HAE2PyU, HBE2PipU, HAE2PipU, HBTeU, DMCH, HDMB, HDMA, HDMC, 4-HDMA, 6-HDMFB, HDMPfp, HDMP, HDTMA, HDTMB; the triazine coupling agent is selected from the following: DMCT, DMTMM, TBCR1, TBCR2, TBCR3, TBCR4; the pyridine coupling agent is selected from the following: Mukaiyama's Reagent, BEMT, BEP, FEP, BEPH, FEPH. In the first step, the molar ratio of the amino acid or polypeptide fragment, base, activator and coupling agent participating in the condensation reaction is 1:1.3~2.5:1~3:1~2; In the first step, the reaction temperature is -10 to 30 ℃, and the reaction time is 0.5 to 24 h.

3. The method as described in claim 2, characterized in that... Solvent A is N , N - One or more of dimethylformamide, acetonitrile, and tetrahydrofuran; The base is one of DIPEA, TEA, NMM, or DMAP; The activator is one of HOBt, HOAt, HOSu, HONB, and HOCt; The coupling agents are DIC, DCC, N-HATU, EDCI, N -HBTU、 N -One or more of TBTU, BOP, and PyBOP; In the first step, the molar ratio of the amino acid or polypeptide fragment, base, activator and coupling agent participating in the condensation reaction is 1:1.5~2:1.1~2.5:1.2~1.8; the reaction temperature in the first step is -10~20 ℃; and the reaction time is 1~8 h.

4. The method as described in claim 1, characterized in that... The amino acid raw materials used are all carboxyl-terminally or amino-terminally protected and side-chain protected, specifically as follows: (1) The amino protecting group of the amino acid is one of Boc, Cbz, Fmoc or H; (2) The carboxyl protecting group of the amino acid is one of methyl ester, tert-butyl ester, benzyl ester or H; (3) The side-chain protecting groups of cysteine ​​are Trt, Bzl, Acm, Mmt, t One of Bu or H; the side chain protecting group of glutamic acid is one of methyl ester, tert-butyl ester, benzyl ester or H; the side chain protecting group of tyrosine is Bzl, t One of Bu, Me, or H; the side chain protecting group of threonine is Bzl, t One of Bu, Me, or H; the side chain protecting group of asparagine is one of Trt, Xant, DMB, or H; Choosing H indicates that no protecting base is used.

5. The method as described in claim 1, characterized in that... The concentration of linaclotide intermediate in solvent B is 0.4~3 mg / mL; the concentration of additive in solvent B is 2~10 mg / mL.

6. The method as described in claim 1, characterized in that... The electrochemical oxidation reaction current intensity is 4~10 mA; the reaction time is 0.5~4 h.

7. The method as described in claim 1, characterized in that... The electrode type is one of C(+)|C(-), C(+)|Pt(-), or Pt(+)|Pt(-).

Images