A fragment synthesis method of plecanatide

Abstract

The present invention belongs to the field of polypeptide drug preparation and discloses a fragment synthesis method of plecanatide, which comprises the following steps: (1) on Fmoc-Leu-resin, a linear plecanatide resin is obtained by a solid-phase synthesis method according to the plecanatide peptide sequence; wherein S5-S8 adopts a tetrapeptide fragment, and the rest adopts corresponding protected amino acids or fragments; (2) the linear plecanatide resin obtained in step (1) is cracked to obtain a linear plecanatide; (3) the linear plecanatide obtained in step (2) is cyclized to obtain a crude plecanatide peptide. The process of the present invention effectively improves the problem of coupling difficulty caused by shrinkage of the peptide resin during solid-phase synthesis, reduces the generation of related missing peptide impurities, greatly reduces the synthesis and purification difficulty of plecanatide, and the prepared plecanatide has high purity, high yield, and few impurities. It is a plecanatide preparation method with broad practical value and application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of polypeptide drug preparation, and specifically relates to a method for synthesizing plecanatide fragments. Background Art

[0002] Chronic idiopathic constipation (CIC) and irritable bowel syndrome with constipation (IBS-C) are two of the most common gastrointestinal disorders, characterized by infrequent bowel movements, straining, and abdominal pain or discomfort. The prevalence of chronic constipation in adults in my country, including CIC, functional bowel dysfunction, and IBS-C, is 4%-6%. Chronic constipation can lead to secondary psychiatric and psychological disorders, and its impact on patients' quality of life is comparable to that of chronic diseases such as diabetes and heart failure.

[0003] Plecanatide is a guanylate cyclase C (GC-C) receptor agonist developed by Synergy Pharmaceuticals in the United States. It was approved by the FDA on January 19, 2017, under the trade name Trulance. Plecanatide is a cyclic polypeptide containing 16 amino acids that can regulate acid and base ions in the gastrointestinal tract, induce fluid transport into the gastrointestinal tract, and increase gastrointestinal motility. It is suitable for the treatment of chronic idiopathic constipation in adults. Its structural formula is as follows:

[0004]

[0005] In the prior art, the patent with publication number CN104211777A adopts the Fmoc solid phase synthesis method to gradually couple and synthesize the linear peptide resin of plecanatin, and obtain the linear peptide of plecanatin after cleavage, and then oxidize in a step-by-step manner in the liquid phase to obtain plecanatin. During the oxidation process, different deprotection strategies are selected according to the characteristics of the Cys side chain protecting group, and hydrogen peroxide and iodine are used for step-by-step oxidation. However, according to experiments, this synthesis method has the phenomenon of polycondensation of the peptide resin after coupling of some amino acids, resulting in difficulty in synthesizing the linear peptide, low crude product purity, and difficulty in purification. The patent with publication number CN110981939A adopts the Fmoc solid phase synthesis method to gradually or fragment-couple and synthesize the linear peptide resin of plecanatin, wherein Hmb / Dmb is used to protect Gly and Ala amino acids, and a linear peptide is obtained after cleavage, and then hydrogen peroxide and iodine are used in a liquid phase for step-by-step oxidation to form two pairs of disulfide bonds to obtain plecanatin. Although this scheme can improve the polycondensation problem of resin, Hmb / Dmb material is expensive, is unfavorable for cost control, and is difficult to realize industrialized production. Publication number is the patent of CN109354607A, adopts octapeptide fragment and octapeptide resin to obtain hexadecepeptide resin in the presence of condensing agent by solid phase condensation, cracking obtains linear peptide, then forms two pairs of disulfide bonds with hydrogen peroxide and iodine stepwise oxidation in liquid phase, obtains plecanatide, and the plecanatide obtained by this method has a total yield of up to 32.45% after purification, and purity can reach more than 99%. The present invention can greatly shorten the synthesis cycle, avoids the problem that traditional solid phase is progressively coupled to be inefficient, but the fully protected S8-S1 octapeptide fragment, steric hindrance is larger, coupling difficulty, and poor solubility in DMF or DCM is unfavorable for reaction and is carried out, and operation is relatively complicated, and cost is higher. Summary of the Invention

[0006] In order to overcome the coupling difficulties, complex operations, and resin polycondensation problems that occur during the synthesis of plecanatide, and to solve the problems of low purity and low yield after step-by-step coupling and cyclization, the present invention provides a fragment synthesis method of plecanatide, which mainly comprises the following steps:

[0007] (1) A linear plecanapeptide resin was obtained by solid-phase synthesis on Fmoc-Leu-resin according to the peptide sequence of plecanapeptide; wherein S5-S8 were tetrapeptide fragments, and the remaining were corresponding protected amino acids or fragments;

[0008] (2) cleaving the linear plecanatide resin obtained in step (1) to obtain linear plecanatide;

[0009] (3) The linear plecanatin obtained in step (2) is cyclized step by step to obtain crude plecanatin peptide.

[0010] Furthermore, the linear plecanatin resin obtained in step (1) is: R1-Asn(R2)-Asp(OtBu)-Glu(OtBu)-Cys(R3)-Glu(OtBu)-Leu-Cys(R4)-Val-Asn(Trt)-Val-Ala-Cys(R3)-Thr(tBu)-Gly-Cys(R4)-Leu-resin;

[0011] in:

[0012] R1 is selected from Boc, Fmoc, Z;

[0013] R2 is selected from Trt, H;

[0014] R3 and R4 are selected from Trt, Acm, StBu or tBu, and R3 and R4 are different.

[0015] Furthermore, the tetrapeptide fragment used in steps (1) S5-S8 is Fmoc-Glu(OtBu)-Leu-Cys(R4)-Val-OH; wherein R4 is preferably Trt or Acm.

[0016] Furthermore, the remaining corresponding fragments used in step (1) include S1-S2 or S1-S4. The synthesized S1-S2 fragment is R1-Asn(R2)-Asp(OtBu)-OH, and the S1-S4 fragment is R1-Asn(R2)-Asp(OtBu)-Glu(OtBu)-Cys(R3)-OH;

[0017] in:

[0018] R1 is preferably Fmoc or Boc;

[0019] R3 is preferably Trt or Acm.

[0020] In step (2), the linear plecanatin resin is cleaved and the resin and side chain protecting groups are removed simultaneously to obtain the linear plecanatin: H-Asn-Asp-Glu-Cys(R3)-Glu-Leu-Cys(R4)-Val-Asn-Val-Ala-Cys(R3)-Thr-Gly-Cys(R4)-Leu-OH, wherein when the Cys side chain protecting group is Trt, it can be cleaved during the cleavage process; if it is other protecting groups, it cannot be cleaved.

[0021] In a preferred embodiment of the present invention, the cleavage and stepwise cyclization in steps (2) and (3) mainly include the following steps:

[0022] When R3 and R4 are selected from Trt and Acm, the linear plecanatin resin is subjected to a cleavage reaction with a cleavage reagent and then precipitated to obtain a linear peptide containing a pair of Cys with Acm protecting groups in the peptide sequence; the linear peptide is dissolved, an oxidant is added, and a cyclization reaction is performed to obtain a cyclic peptide; the side chain protecting group Acm on the Cys of the cyclic peptide is removed in an iodine solution, and cyclization is performed to obtain a crude plecanatin peptide; the added oxidant is selected from hydrogen peroxide, DMSO or air.

[0023] When R3 and R4 are selected from Trt and StBu, a linear plecanatin resin is cleaved with a cleavage reagent and then precipitated to obtain a linear peptide containing a pair of Cys with StBu protecting groups in the peptide sequence; the linear peptide is dissolved, an oxidant is added, and a cyclization reaction is performed to obtain a cyclic peptide; 2-mercaptoethanol is added to the cyclic peptide to remove the StBu protecting group; and an oxidant is then added to perform a second cyclization to obtain a crude plecanatin peptide. The oxidant used in the second cyclization is selected from hydrogen peroxide, DMSO, or air.

[0024] When R3 and R4 are selected from Trt and tBu, a linear plecanatin resin is cleaved with a cleavage reagent and then precipitated to obtain a linear peptide containing a pair of Cys with tBu protecting groups in the peptide sequence; the linear peptide is dissolved, an oxidant is added, and a cyclization reaction is performed to obtain a cyclic peptide; TFA, diphenyl sulfoxide, trichloromethylsilane, and anisole are added to the cyclic peptide to remove the tBu protecting groups; an oxidant is then added to perform a second cyclization to obtain a crude plecanatin peptide. The oxidant used in the second cyclization is selected from hydrogen peroxide, DMSO, or air.

[0025] In a preferred embodiment of the present invention, the cleavage reagent in step (2) is selected from trifluoroacetic acid and other components, and the other components are selected from 1-4 of water, phenol, 3-mercaptopropionic acid, and triisopropylsilane, wherein trifluoroacetic acid accounts for more than 90% and the other components each account for 1%-5%.

[0026] In a preferred embodiment of the present invention, the resin in step (1) is selected from Wang resin or 2-chlorotrityl chloride resin, and the substitution degree of the obtained Fmoc-Leu-resin is in the range of 0.2-0.5 mmol / g.

[0027] The present invention proposes a fragment synthesis method for plecanatide. The corresponding protected amino acids and the S5-S8 tetrapeptide fragments in the sequence are sequentially added to an Fmoc-Leu resin via solid-phase synthesis. This effectively improves the resin's polycondensation and significantly reduces the difficulty of plecanatide synthesis. Furthermore, the S5-S8 tetrapeptide fragment has good solubility in DMF or DCM, thereby improving the purity and yield of plecanatide. The use of S1-S4 or S1-S2 fragments can reduce the problem of related deletion peptide impurities generated by coupling difficulties of amino acids at the S1 or S4 positions. Pure plecanatide synthesized using the present method can achieve a purity exceeding 99.5% and an overall yield exceeding 45%. Compared with the prior art, the present process effectively alleviates the problem of peptide resin shrinkage during solid-phase synthesis leading to coupling difficulties, reduces the generation of related deletion peptide impurities, and significantly reduces the difficulty of synthesizing and purifying plecanatide. The prepared plecanatide has high purity, high yield, and few impurities, and has broad practical value and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a chromatogram of the crude plecanatide peptide prepared in Example 22 of the present invention;

[0029] Figure 2 This is a chromatogram of the pure plecanatide prepared in Example 28 of the present invention;

[0030] Figure 3 This is a chromatogram of the crude plecanatide peptide prepared in Comparative Example 3 of the present invention;

[0031] Figure 4 This is a chromatogram of the pure plecanatide prepared in Comparative Example 4 of the present invention. DETAILED DESCRIPTION

[0032] The present invention will be further described in detail below by way of examples, which are intended to illustrate the present invention but not to limit the present invention. It should be noted that those skilled in the art may make several improvements and modifications to the present invention without departing from the principles of the present invention, and such improvements and modifications also fall within the scope of protection of the present invention.

[0033] Example 1: Preparation of Fmoc-Leu-Wang resin with a substitution value of 0.20 mmol / g

[0034] 303.00 g (100 mmol) of Wang resin with a substitution value of 0.33 mmol / g was weighed and added to a solid-phase reactor. The resin was washed twice with DMF and swollen with DMF for 60 minutes. 70.64 g of Fmoc-Leu-OH, 27.13 g of HOBt, 25.35 g of DIC, and 2.06 g of DMAP were dissolved in DMF and added to the solid-phase reactor. The reaction was allowed to proceed at room temperature for 2 hours. After the reaction, the resin was washed three times with DMF and three times with DCM. A blocking solution was then added for 30 minutes. The blocking solution ratio was 10:84:6 (V:V:V) of acetic anhydride:DMF:DIEA. After blocking, the resin was washed three times with DMF and three times with DCM. The resin was then dried with methanol and evaporation to obtain Fmoc-Leu-Wang resin with a substitution value of 0.20 mmol / g.

[0035] Example 2: Preparation of Fmoc-Leu-Wang resin with a substitution value of 0.50 mmol / g

[0036] 125.00 g (100 mmol) of Wang resin with a substitution value of 0.80 mmol / g was weighed and added to a solid-phase reactor. The resin was washed twice with DMF and swollen with DMF for 60 minutes. 70.65 g of Fmoc-Leu-OH, 27.03 g of HOBt, 25.25 g of DIC, and 2.05 g of DMAP were dissolved in DMF and added to the solid-phase reactor. The reaction was allowed to proceed at room temperature for 2 hours. After the reaction, the resin was washed three times with DMF and three times with DCM. A blocking solution was then added for 30 minutes. The blocking solution ratio was acetic anhydride: DMF: DIEA = 10:84:6 (V:V:V). After blocking, the resin was washed three times with DMF and three times with DCM. The resin was then condensed and drained with methanol to obtain Fmoc-Leu-Wang resin with a substitution value of 0.50 mmol / g.

[0037] Example 3: Preparation of Fmoc-Leu-2-chlorotrityl chloride resin with a substitution value of 0.30 mmol / g

[0038] 200.02 g (100 mmol) of 2-chlorotrityl chloride resin with a substitution value of 0.50 mmol / g was weighed and added to a solid-phase reactor. The resin was washed twice with DMF. 70.65 g of Fmoc-Leu-OH and 38.70 g of DIEA were dissolved in DMF and added to the solid-phase reactor. The reaction was allowed to react at room temperature for 2 hours. After the reaction, the resin was washed three times with DMF and three times with DCM. A blocking solution was then added for 30 minutes. The blocking solution ratio was methanol:DMF:DIEA = 1:10:20 (V:V:V). After blocking, the resin was washed four times with DMF and six times with DCM to obtain Fmoc-Leu-2-chlorotrityl chloride resin with a substitution value of 0.30 mmol / g.

[0039] Example 4: Preparation of Plecanapeptide S5-S8 Fragment Peptide Fmoc-Glu(OtBu)-Leu-Cys(Acm)-Val-OH

[0040] (1) Synthesis of Fmoc-Val-2-chlorotrityl chloride resin with a degree of substitution of 0.60 mmol / g.

[0041] 111.11 g (100 mmol) of 2-chlorotrityl chloride resin with a degree of substitution of 0.90 mmol / g was weighed and added to a solid phase reaction column. The resin was washed once with DMF. The resin was swelled with DCM for 30 minutes and then washed three times with DMF. 50.91 g of Fmoc-Val-OH was dissolved in DMF, activated with 49.6 ml of DIEA in an ice-water bath, and then added to the reaction column containing the resin. After reacting for 2 hours, the resin was washed three times with DMF and blocked overnight with a mixture of DIEA:MeOH:DMF = 1:10:20. The resin was washed alternately with DCM and MeOH, shrunk and dried to obtain Fmoc-Val-2-chlorotrityl chloride resin with a detection degree of substitution of 0.60 mmol / g.

[0042] (2) Synthesis of Plecanapeptide S5-S8 Fragment Peptide Resin

[0043] Weigh 33.34g (20mmol) of Fmoc-Val-2-chlorotrityl chloride resin having a degree of substitution of 0.60mmol / g obtained in step (1), add it to a solid phase reaction column, wash it once with DMF, swell the Fmoc-Val-2-chlorotrityl chloride resin with DMF for 30 minutes, remove the Fmoc protection with a mixed solution of DMF: piperidine in a volume ratio of 4:1, and then wash it 6 times with DMF. Weigh 24.80g Fmoc-Cys(Acm)-OH and 8.90g HOBt and dissolve them in DMF solution. After activation by adding 14.2ml DIC under an ice-water bath, add it to the reaction column containing the resin. After reacting for 2 hours at room temperature, use ninhydrin to detect the reaction end point. If the resin is colorless and transparent, it indicates that the reaction is complete. If the resin develops color, the reaction is incomplete and it is necessary to continue to react for another hour or re-invest in a single amount or replace the condensation reagent and re-invest. Ninhydrin test is suitable for subsequent amino acid coupling reaction to determine the reaction endpoint. Repeat the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and sequentially couple Fmoc-Leu-OH and Fmoc-Glu(OtBu)-OH. After coupling the last amino acid, do not remove Fmoc. Directly shrink and dry the tetrapeptide resin, wash with DCM 6 times, and drain to obtain Fmoc-Glu(OtBu)-Leu-Cys(Acm)-Val-2-chlorotrityl chloride resin for use.

[0044] (3) Preparation of Plecanapeptide S5-S8 Fragment Peptide

[0045] Weigh 51.50 g of the dried 20 mmol of the plecanatin S5-S8 fragment peptide resin in step (2) in a 30% TFE-DCM solution, with the volume ratio of 1 g of the peptide resin to 10 ml of the solution. After the solution is evenly mixed, add the above-mentioned peptide resin, react at room temperature for 2 hours, filter, and concentrate the filtrate under reduced pressure to 1 / 4 volume. Add 8 times the amount of ice isopropyl ether and let it settle for 1 hour. Centrifuge, wash with isopropyl ether by centrifugation 4 times, and dry to obtain 17.10 g of the plecanatin S5-S8 fragment peptide Fmoc-Glu(OtBu)-Leu-Cys(Acm)-Val-OH.

[0046] Example 5: Preparation of Plecanapeptide S5-S8 Fragment Peptide Fmoc-Glu(OtBu)-Leu-Cys(Trt)-Val-OH

[0047] (1) Synthesis of Plecanapeptide S5-S8 Fragment Peptide Resin

[0048] Weigh 33.35g (20mmol) of Fmoc-Val-2-chlorotrityl chloride resin having a degree of substitution of 0.60mmol / g obtained in step (1) of Example 4, add it to a solid phase reaction column, wash it once with DMF, swell the Fmoc-Val-2-chlorotrityl chloride resin with DMF for 30 minutes, remove the Fmoc protection with a mixed solution of DMF:piperidine in a volume ratio of 4:1, and then wash it 6 times with DMF. Weigh 27.50g Fmoc-Cys(Trt)-OH and 8.60g HOBt and dissolve them in DMF solution. After activation by adding 14.2ml DIC under ice-water bath, add it to the reaction column containing the resin, react at room temperature for 2 hours, and use ninhydrin to detect the reaction end point. If the resin is colorless and transparent, it indicates that the reaction is complete; if the resin develops color, the reaction is incomplete and it is necessary to continue to react for another hour or re-add a single dose or replace the condensation reagent and re-add. Ninhydrin test is suitable for subsequent amino acid coupling reaction to determine the reaction endpoint. Repeat the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and sequentially couple Fmoc-Leu-OH and Fmoc-Glu(OtBu)-OH. After coupling the last amino acid, do not remove Fmoc. Directly shrink and dry the tetrapeptide resin, wash with DCM 6 times, and drain to obtain Fmoc-Glu(OtBu)-Leu-Cys(Trt)-Val-2-chlorotrityl chloride resin for use.

[0049] (2) Preparation of Plecanapeptide S5-S8 Fragment Peptide

[0050] Weigh 51.50 g of the dried 20 mmol of the plecanatin S5-S8 fragment peptide resin in step (2) in a 10% TFE DCM solution, with the volume ratio of 1 g of the peptide resin to 10 ml of the solution. After the solution is evenly mixed, add the above-mentioned peptide resin, react at room temperature for 2 hours, filter, and concentrate the filtrate under reduced pressure to 1 / 4 volume. Add 10 times the amount of ice isopropyl ether and let it settle for 1 hour. Centrifuge, wash with isopropyl ether by centrifugation 4 times, and dry to obtain 17.05 g of the plecanatin S5-S8 fragment peptide Fmoc-Glu(OtBu)-Leu-Cys(Trt)-Val-OH.

[0051] Example 6: Preparation of Plecanapeptide S1-S4 Fragment Peptide Fmoc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(Trt)-OH

[0052] (1) Synthesis of Fmoc-Cys(Trt)-2-chlorotrityl chloride resin with a degree of substitution of 0.60 mmol / g.

[0053] 55.55 g (500 mmol) of 2-chlorotrityl chloride resin with a degree of substitution of 0.90 mmol / g was weighed and added to a solid phase reaction column. The resin was washed once with DMF. The resin was swelled with DCM for 30 minutes and then washed three times with DMF. 58.55 g of Fmoc-Cys(Trt)-OH was dissolved in DMF, activated with 24.2 ml of DIEA in an ice-water bath, and then added to the reaction column containing the resin. After reacting for 2 hours, the resin was washed three times with DMF and blocked overnight with a mixture of DIEA:MeOH:DMF = 1:10:20. The resin was washed alternately with DCM and MeOH, shrunk and dried to obtain Fmoc-Cys(Trt)-2-chlorotrityl chloride resin with a detected degree of substitution of 0.60 mmol / g.

[0054] (2) Synthesis of Plecanapeptide S1-S4 Fragment Peptide Resin

[0055] Weigh 33.34 g (20 mmol) of Fmoc-Cys (Trt) -2-chlorotrityl chloride resin with a substitution degree of 0.60 mmol / g obtained in step (1) and add it to a solid phase reaction column. Wash it once with DMF. After swelling the Fmoc-Cys (Trt) -2-chlorotrityl chloride resin with DMF for 30 minutes, remove the Fmoc protection with a mixed solution of DMF: piperidine in a volume ratio of 4:1, and then wash it with DMF 6 times. Weigh 25.53 g of Fmoc-Glu (OtBu) -OH and 8.92 g of HOBt and dissolve them in DMF solution. After adding 14.1 ml of DIC for activation under ice-water bath, add it to the reaction column containing the resin. After reacting for 2 hours at room temperature, use ninhydrin to detect the reaction end point. If the resin is colorless and transparent, it means the reaction is complete. If the resin shows color, the reaction is incomplete and needs to be continued for another hour or repeated with a single dose or a different condensation reagent. Ninhydrin test is suitable for subsequent amino acid coupling reaction to determine the reaction endpoint. Repeat the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and sequentially couple Fmoc-Asp(OtBu)-OH and Fmoc-Asn(Trt)-OH. After coupling the last amino acid, do not remove Fmoc. Directly shrink and dry the tetrapeptide resin, wash with DCM 6 times, and drain to obtain Fmoc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(Trt)-2-chlorotrityl chloride resin for use.

[0056] (3) Preparation of Plecanapeptide S1-S4 Fragment Peptide

[0057] Weigh 58.51 g of the dried 20 mmol of the peptide S1-S4 fragment peptide resin from step (2) in a 30% TFE / DCM solution, with the volume ratio of 1 g of the peptide resin to 10 ml of the solution. After the solution is evenly mixed, add the above-mentioned peptide resin, react at room temperature for 2 hours, filter, and concentrate the filtrate under reduced pressure to 1 / 4 volume. Add 8 times the amount of ice isopropyl ether and let it settle for 1 hour. Centrifuge, wash with isopropyl ether by centrifugation 4 times, and dry to obtain 24.10 g of Fmoc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(Trt)-OH.

[0058] Example 7: Preparation of Plecanapeptide S1-S4 Fragment Peptide Fmoc-Asn-Asp(OtBu)-Glu(OtBu)-Cys(Acm)-OH

[0059] (1) Synthesis of Fmoc-Cys(Acm)-2-chlorotrityl chloride resin with a degree of substitution of 0.60 mmol / g.

[0060] 55.56 g (500 mmol) of 2-chlorotrityl chloride resin with a degree of substitution of 0.90 mmol / g was weighed and added to a solid phase reaction column. The resin was washed once with DMF. The resin was swelled with DCM for 30 minutes and then washed three times with DMF. 58.55 g of Fmoc-Cys(Acm)-OH was dissolved in DMF and activated with 24.2 ml of DIEA in an ice-water bath. The resin was then added to the reaction column containing the resin. After reacting for 2 hours, the resin was washed three times with DMF and blocked overnight with a mixture of DIEA:MeOH:DMF = 1:10:20. The resin was washed alternately with DCM and MeOH, shrunk and dried to obtain Fmoc-Cys(Acm)-2-chlorotrityl chloride resin with a detected degree of substitution of 0.60 mmol / g.

[0061] (2) Synthesis of Plecanapeptide S1-S4 Fragment Peptide Resin

[0062] Weigh 33.33 g (20 mmol) of Fmoc-Cys(Acm)-2-chlorotrityl chloride resin with a substitution degree of 0.60 mmol / g obtained in step (1) and add it to a solid phase reaction column. Wash it once with DMF. After swelling the Fmoc-Cys(Acm)-2-chlorotrityl chloride resin with DMF for 30 minutes, remove the Fmoc protection with a mixed solution of DMF:piperidine in a volume ratio of 4:1. Then wash it with DMF 6 times. Weigh 25.53 g of Fmoc-Glu(OtBu)-OH and 8.92 g of HOBt and dissolve them in DMF solution. After adding 14.1 ml of DIC for activation under ice-water bath, add it to the reaction column containing the resin. After reacting for 2 hours at room temperature, use ninhydrin to detect the reaction end point. If the resin is colorless and transparent, it means the reaction is complete. If the resin shows color, the reaction is incomplete and needs to be continued for another hour or repeated with a single dose or a different condensation reagent. Ninhydrin test is suitable for subsequent amino acid coupling reaction to determine the reaction endpoint. Repeat the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and sequentially couple Fmoc-Asp(OtBu)-OH and Fmoc-Asn-OH. After coupling the last amino acid, do not remove Fmoc. Directly shrink and dry the tetrapeptide resin, wash with DCM 6 times, and drain to obtain Fmoc-Asn-Asp(OtBu)-Glu(OtBu)-Cys(Acm)-2-chlorotrityl chloride resin for use.

[0063] (3) Preparation of Plecanapeptide S1-S4 Fragment Peptide

[0064] Weigh 50.50 g of the dried 20 mmol of the peptide resin (50.50 g) of the S1-S4 fragment of plecanatin obtained in step (2) in a 10% TFE / DCM solution, with the volume ratio of 1 g of the peptide resin to 10 ml of the solution. After the solution is evenly mixed, add the peptide resin, react at room temperature for 2 hours, filter, and concentrate the filtrate under reduced pressure to 1 / 4 volume. Add 9 times the amount of methyl tert-butyl ether and let it settle for 1 hour. Centrifuge, wash the methyl tert-butyl ether by centrifugation 4 times, and dry to obtain 21.50 g of Fmoc-Asn-Asp(OtBu)-Glu(OtBu)-Cys(Acm)-OH.

[0065] Example 8: Preparation of Plecanapeptide S1-S2 Fragment Peptide Fmoc-Asn(Trt)-Asp(OtBu)-OH

[0066] (1) Synthesis of Fmoc-Asp(OtBu)-2-chlorotrityl chloride resin with a degree of substitution of 0.60 mmol / g

[0067] 55.55 g (50 mmol) of 2-chlorotrityl chloride resin with a degree of substitution of 0.90 mmol / g was weighed and added to a solid phase reaction column. The resin was washed once with DMF. The resin was swelled with DCM for 30 minutes and then washed three times with DMF. 41.15 g of Fmoc-Asp(OtBu)-OH was dissolved in DMF, activated with 24.8 ml of DIEA in an ice-water bath, and then added to the reaction column containing the resin. After reacting for 2 hours, the resin was washed three times with DMF and blocked overnight with a mixture of DIEA:MeOH:DMF = 1:10:20. The resin was washed alternately with DCM and MeOH, shrunk and dried to obtain Fmoc-Asp(OtBu)-2-chlorotrityl chloride resin with a detected degree of substitution of 0.60 mmol / g.

[0068] (2) Synthesis of Plecanapeptide S1-S2 Fragment Peptide Resin

[0069] Weigh 32.34 g (20 mmol) of Fmoc-Asp (OtBu) -2-chlorotrityl chloride resin with a substitution degree of 0.60 mmol / g obtained in step (1) and add it to a solid phase reaction column. Wash it once with DMF. After swelling the Fmoc-Asp (OtBu) -2-chlorotrityl chloride resin with DMF for 30 minutes, remove the Fmoc protection with a mixed solution of DMF: piperidine in a volume ratio of 4:1, and then wash it with DMF 6 times. Weigh 35.80 g of Fmoc-Asn (Trt) -OH and 8.91 g of HOBt and dissolve them in DMF solution. After adding 14.1 ml of DIC for activation under ice-water bath, add it to the reaction column containing the resin. After reacting for 2 hours at room temperature, use ninhydrin to detect the reaction end point. If the resin is colorless and transparent, it means the reaction is complete. If the resin shows color, the reaction is incomplete and needs to be continued for another hour or repeated with a single dose or a different condensation reagent. The ninhydrin assay is suitable for subsequent amino acid coupling reactions to determine the reaction endpoint. After coupling, without removing Fmoc, the dipeptide resin was directly shrunk to dryness, washed with DCM six times, and drained to obtain Fmoc-Asn(Trt)-Asp(OtBu)-2-chlorotrityl chloride resin for future use.

[0070] (3) Preparation of Plecanapeptide S1-S2 Fragment Peptide

[0071] Weigh 44.50 g of the dried 20 mmol plecanapeptide S1-S2 fragment peptide resin in a 20% TFE / DCM solution, with a volume ratio of 1 g of peptide resin to 10 ml of solution. Once the solution is evenly mixed, add the peptide resin and react at room temperature for 2 hours. Filter and concentrate the filtrate under reduced pressure to 1 / 4 volume. Add 8 times the volume of diethyl ether and let settle for 1 hour. Centrifuge and wash with diethyl ether four times by centrifugation. Dry to obtain 13.10 g of Fmoc-Asn(Trt)-Asp(OtBu)-OH.

[0072] Example 9: Preparation of Plecanapeptide S1-S2 Fragment Peptide Boc-Asn-Asp(OtBu)-OH

[0073] (1) Synthesis of Plecanapeptide S1-S2 Fragment Peptide Resin

[0074] 33.33 g (20 mmol) of Fmoc-Asp(OtBu)-2-chlorotrityl chloride resin with a degree of substitution of 0.60 mmol / g obtained in step (1) of Example 8 was weighed and added to a solid phase reaction column, washed once with DMF, and after swelling the Fmoc-Asp(OtBu)-2-chlorotrityl chloride resin with DMF for 30 minutes, the Fmoc protection was removed with a mixed solution of DMF:piperidine in a volume ratio of 4:1, and then washed 6 times with DMF. 13.90 g of Boc-Asn-OH and 8.91 g of HOBt were weighed and dissolved in DMF solution. After activation by adding 14.1 ml of DIC in an ice-water bath, the resin was added to the reaction column containing the resin. After reacting for 2 hours at room temperature, the reaction endpoint was determined by ninhydrin detection. If the resin was colorless and transparent, the reaction was complete; if the resin showed color, the reaction was incomplete and it was necessary to continue the reaction for another hour or re-add a single dose or replace the condensation reagent and re-add. The ninhydrin assay is suitable for subsequent amino acid coupling reactions to determine the reaction endpoint. After coupling, without removing Fmoc, the dipeptide resin was directly shrunk to dryness, washed with DCM six times, and drained to obtain Boc-Asn-Asp(OtBu)-2-chlorotrityl chloride resin for later use.

[0075] (2) Preparation of Plecanapeptide S1-S2 Fragment Peptide

[0076] Weigh 35.51 g of the dried 20 mmol plecanapeptide S1-S2 fragment peptide resin in a 10% TFE / DCM solution, with a volume ratio of 1 g of peptide resin to 10 ml of solution. Once the solution is evenly mixed, add the peptide resin and react at room temperature for 2 hours. Filter and concentrate the filtrate under reduced pressure to 1 / 4 volume. Add 10 times the volume of icy isopropyl ether and let settle for 1 hour. Centrifuge and wash with isopropyl ether four times by centrifugation. Dry to obtain 11.80 g of Boc-Asn-Asp(OtBu)-OH.

[0077] Example 10: Preparation of Plecanapeptide Peptide Resin 1

[0078] Take 50.05g (10mmol) of Fmoc-Leu-Wang resin with a degree of substitution of 0.20mmol / g prepared in Example 1 of the present invention, add it to a solid phase reaction column, wash it once with DMF, swell the Fmoc-Leu-Wang resin with DMF for 30 minutes, remove the Fmoc protection with a mixed solution of DMF: piperidine volume ratio of 4:1, then wash it 6 times with DMF, weigh 12.37g Fmoc-Cys(Acm)-OH and 4.01g HOBt and dissolve them in DMF solution. After adding 4.67ml DIC activation under ice-water bath, add it to the reaction column equipped with the resin, react at room temperature for 2 hours, and use ninhydrin to detect the reaction end point. If the resin is colorless and transparent, it means that the reaction is complete; if the resin develops color, the reaction is incomplete and it is necessary to continue to react for another hour or re-do the single dose or replace the condensation reagent and re-do. The ninhydrin detection is applicable to subsequent amino acid coupling reactions to determine the reaction end point. Repeat the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and sequentially add Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(OtBu)-Leu-Cys(Acm)-Val-OH prepared in Example 4, Fmoc-Cys(Trt)-OH, Fmoc-Glu The peptide resin Fmoc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(Trt)-Glu(OtBu)-Leu-Cys(Acm)-Val-Asn(Trt)-Val-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Acm)-Leu-Wang resin was obtained by coupling with Fmoc-Asn(Trt)-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, and Fmoc-Asn(Trt)-OH. The resin weighed 73.50 g. No volume shrinkage of the peptide resin occurred during the coupling process, indicating easy coupling. The resin was colorless and transparent as detected by the ninhydrin method after 1.5 h of reaction.

[0079] Example 11: Preparation of Plecanapeptide Peptide Resin 2

[0080] 19.99 g (10 mmol) of Fmoc-Leu-Wang resin with a degree of substitution of 0.50 mmol / g prepared in Example 2 of the present invention was added to a solid phase reaction column and washed once with DMF. After swelling the Fmoc-Leu-Wang resin with DMF for 30 minutes, the Fmoc protection was removed with a mixed solution of DMF:piperidine in a volume ratio of 4:1, and then washed 6 times with DMF. 12.37 g of Fmoc-Cys(Trt)-OH and 4.01 g of HOBt were added to the DMF solution and dissolved. After activation with 4.67 ml of DIC under an ice-water bath, the resin was added to the reaction column containing the resin. After reacting for 2 hours at room temperature, the reaction endpoint was determined using the ninhydrin assay. If the resin was colorless and transparent, the reaction was complete. If the resin developed color, the reaction was incomplete and required further reaction for another hour or repeated addition of a single dose or replacement of the condensation reagent. The ninhydrin assay was applicable to subsequent amino acid coupling reactions to determine the reaction endpoint. Repeat the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and sequentially add Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(StBu)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(OtBu)-Leu-Cys(Trt)-Val-OH, Fmoc-Cys(StBu)-OH, Fmoc-Glu prepared in Example 5, and (OtBu)-OH, and Fmoc-Asn(Trt)-Asp(OtBu)-OH prepared in Example 8, to obtain the plecanatide peptide resin Fmoc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(StBu)-Glu(OtBu)-Leu-Cys(Trt)-Val-Asn(Trt)-Val-Ala-Cys(StBu)-Thr(tBu)-Gly-Cys(Trt)-Leu-Wang resin, weighing 46.70 g. No volume shrinkage of the peptide resin occurred during the coupling process, and the coupling was easy. The resin was colorless and transparent when tested by the ninhydrin method for 2 hours each reaction.

[0081] Example 12: Preparation of Plecanapeptide Peptide Resin 3

[0082] Take 33.33g (10mmol) of Fmoc-Leu-2-chlorotrityl chloride resin with a substitution degree of 0.30mmol / g prepared in Example 3 of the present invention, add it to a solid phase reaction column, wash it once with DMF, swell the Fmoc-Leu-Wang resin with DMF for 30 minutes, remove the Fmoc protection with a mixed solution of DMF:piperidine in a volume ratio of 4:1, and then wash it with DMF 6 times. Weigh 12.37g Fmoc-Cys(Trt)-OH and 4.01g HOBt and dissolve them in DMF solution. Add 4.67ml DIC to activate it under an ice-water bath, and add it to the reaction column containing the resin. After reacting for 2 hours at room temperature, use the ninhydrin test to determine the reaction endpoint. If the resin is colorless and transparent, it indicates that the reaction is complete; if the resin develops color, the reaction is incomplete and it is necessary to continue the reaction for another hour or re-do the single dose or replace the condensation reagent and re-do it. The ninhydrin test is applicable to subsequent amino acid coupling reactions to determine the reaction endpoint. Repeat the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and sequentially add Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(OtBu)-Leu-Cys(Trt)-Val-OH prepared in Example 5, and Fmoc-Asn(Trt)-OH prepared in Example 7 according to the sequence of plecanatide. The peptide resin Fmoc-Asn-Asp(OtBu)-Glu(OtBu)-Cys(Acm)-Glu(OtBu)-Leu-Cys(Trt)-Val-Asn(Trt)-Val-Ala-Cys(Acm)-Thr(tBu)-Gly-Cys(Trt)-Leu-2-chlorotrityl chloride resin was obtained by coupling with n-Asp(OtBu)-Glu(OtBu)-Cys(Acm)-OH, weighing 65.10 g. No volume shrinkage of the peptide resin occurred during the coupling process, indicating easy coupling. The resin was colorless and transparent as determined by the ninhydrin method after 1.5 h of reaction.

[0083] Example 13: Preparation of Plecanapeptide Peptide Resin 4

[0084] Take 20.02g (10mmol) of Fmoc-Leu-Wang resin with a degree of substitution of 0.50mmol / g prepared in Example 2 of the present invention, add it to a solid phase reaction column, wash it once with DMF, swell the Fmoc-Leu-Wang resin with DMF for 30 minutes, remove the Fmoc protection with a mixed solution of DMF:piperidine volume ratio of 4:1, and then wash it with DMF 6 times. 12.37g Fmoc-Cys(Acm)-OH and 4.01g HOBt are added to the DMF solution to dissolve. After adding 4.67ml DIC for activation under an ice-water bath, add it to the reaction column containing the resin. After reacting for 2 hours at room temperature, use ninhydrin to detect the reaction end point. If the resin is colorless and transparent, it means that the reaction is complete; if the resin develops color, the reaction is incomplete and it is necessary to continue to react for another hour or re-do the single dose or replace the condensation reagent and re-do. The ninhydrin detection is applicable to subsequent amino acid coupling reactions to determine the reaction end point. Repeat the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and sequentially add Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(OtBu)-Leu-Cys(Acm)-Val-OH, Fmoc-Cys(Trt) ... The peptide resin Boc-Asn-Asp(OtBu)-Glu(OtBu)-OH and Boc-Asn-Asp(OtBu)-OH prepared in Example 9 were used to obtain the peptide resin Boc-Asn-Asp(OtBu)-Glu(OtBu)-Cys(Trt)-Glu(OtBu)-Leu-Cys(Acm)-Val-Asn(Trt)-Val-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Acm)-Leu-Wang resin, weighing 46.55 g. No volume shrinkage of the peptide resin occurred during the coupling process, indicating easy coupling. The resin was colorless and transparent when tested by the ninhydrin method after 2 hours of reaction.

[0085] Example 14: Preparation of Plecanapeptide Peptide Resin

[0086] Take 20.03g (10mmol) of Fmoc-Leu-Wang resin with a degree of substitution of 0.50mmol / g prepared in Example 2 of the present invention, add it to a solid phase reaction column, wash it once with DMF, swell the Fmoc-Leu-Wang resin with DMF for 30 minutes, remove the Fmoc protection with a mixed solution of DMF:piperidine in a volume ratio of 4:1, and then wash it with DMF 6 times. 12.37g Fmoc-Cys(Acm)-OH and 4.01g HOBt are added to the DMF solution to dissolve. After adding 4.67ml DIC for activation under an ice-water bath, add it to the reaction column containing the resin. After reacting for 2 hours at room temperature, use ninhydrin to detect the reaction end point. If the resin is colorless and transparent, it means that the reaction is complete; if the resin develops color, the reaction is incomplete and it is necessary to continue to react for another hour or re-do the single dose or replace the condensation reagent and re-do. The ninhydrin detection is applicable to subsequent amino acid coupling reactions to determine the reaction end point. The above steps of removing Fmoc protection and adding the corresponding amino acid coupling were repeated, and Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(OtBu)-Leu-Cys(Acm)-Val-OH prepared in Example 4 and Fmoc-Asn(Trt)-OH prepared in Example 6 were sequentially added according to the sequence of plecanatide. The peptide resin Fmoc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(Trt)-OH was obtained by the reaction of plecanapeptide with Fmoc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(Trt)-Glu(OtBu)-Leu-Cys(Acm)-Val-Asn(Trt)-Val-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Acm)-Leu-Wang resin, weighing 46.60 g. No volume shrinkage of the peptide resin occurred during the coupling process, indicating easy coupling. The resin was colorless and transparent as detected by the ninhydrin method after each reaction lasted 1.5 h.

[0087] Example 15: Preparation of Plecanapeptide Peptide Resin

[0088] 19.99 g (10 mmol) of Fmoc-Leu-Wang resin with a degree of substitution of 0.50 mmol / g prepared in Example 2 of the present invention was added to a solid phase reaction column and washed once with DMF. After swelling the Fmoc-Leu-Wang resin with DMF for 30 minutes, the Fmoc protection was removed with a mixed solution of DMF:piperidine in a volume ratio of 4:1, and then washed 6 times with DMF. 12.37 g of Fmoc-Cys(Trt)-OH and 4.01 g of HOBt were added to the DMF solution and dissolved. After activation with 4.67 ml of DIC under an ice-water bath, the resin was added to the reaction column containing the resin. After reacting for 2 hours at room temperature, the reaction endpoint was determined using the ninhydrin assay. If the resin was colorless and transparent, the reaction was complete. If the resin developed color, the reaction was incomplete and required further reaction for another hour or repeated addition of a single dose or replacement of the condensation reagent. The ninhydrin assay was applicable to subsequent amino acid coupling reactions to determine the reaction endpoint. Repeat the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and sequentially add Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(tBu)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(OtBu)-Leu-Cys(Trt)-Val-OH, Fmoc-Cys(tBu)-OH, Fmoc-Glu prepared in Example 5, and (OtBu)-OH, and Fmoc-Asn(Trt)-Asp(OtBu)-OH prepared in Example 8, to obtain the plecanatide peptide resin Fmoc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(tBu)-Glu(OtBu)-Leu-Cys(Trt)-Val-Asn(Trt)-Val-Ala-Cys(tBu)-Thr(tBu)-Gly-Cys(Trt)-Leu-Wang resin, weighing 44.50 g. No volume shrinkage of the peptide resin occurred during the coupling process, and the coupling was easy. The resin was colorless and transparent when tested by the ninhydrin method for 1.5 h per reaction.

[0089] Example 16: Preparation of linear plecanatide 1

[0090] 73.50 g of the linear peptide resin of plecanatin prepared in Example 10 of the present invention was added to a 1000 ml three-necked round-bottom flask, and 730 ml of the lysis solution was prepared at a volume ratio of 94:3:3 TFA:Tis:Mpr. After mixing evenly, the above-mentioned peptide resin was added and reacted at room temperature for 2 hours. The mixture was filtered and the filtrate was added to 8 times the amount of ice isopropyl ether and precipitated for 1 hour. The mixture was centrifuged and washed with isopropyl ether four times by centrifugation. The mixture was dried to obtain 16.90 g of a white solid linear crude peptide with an Acm protecting group at the Cys (7, 15) position of plecanatin.

[0091] Example 17: Preparation of linear plecanatide 2

[0092] 46.70 g of the linear peptide resin of plecanatin prepared in Example 11 of the present invention was added to a 1000 ml three-necked round-bottom flask, and 470 ml of the lysis solution was prepared at a volume ratio of TFA:Tis:Mpr:Phenol:H2O of 90:3:3:3:1. After mixing evenly, the above-mentioned peptide resin was added and reacted at room temperature for 2 hours. The mixture was filtered and the filtrate was added to 9 times the amount of ice isopropyl ether and precipitated for 1 hour. The mixture was centrifuged and washed with isopropyl ether four times by centrifugation. The mixture was dried to obtain 17.03 g of a white solid linear crude peptide with a StBu protecting group at the Cys (4, 12) position of plecanatin.

[0093] Example 18: Preparation of linear plecanatide 3

[0094] 65.10 g of the linear peptide resin of plecanatin prepared in Example 12 of the present invention was added to a 1000 ml three-necked round-bottom flask, and 650 ml of the lysis solution was prepared at a volume ratio of 92:4:4 TFA:Tis:H2O. After mixing evenly, the above-mentioned peptide resin was added and reacted at room temperature for 2 hours. The mixture was filtered and the filtrate was added to 10 times the amount of icy petroleum ether and allowed to settle for 1 hour. The mixture was centrifuged and washed with petroleum ether four times by centrifugation. The mixture was dried to obtain 16.91 g of a white solid linear crude peptide with an Acm protecting group at the Cys (4, 12) position of plecanatin.

[0095] Example 19: Preparation of linear plecanatide 4

[0096] 46.55 g of the linear peptide resin of plecanatin prepared in Example 13 of the present invention was added to a 1000 ml three-necked round-bottom flask, and 460 ml of the lysis solution was prepared at a volume ratio of 95:5 TFA:Tis. After mixing evenly, the above-mentioned peptide resin was added and reacted at room temperature for 2 hours. The mixture was filtered and the filtrate was added to 9 times the amount of ice methyl tert-butyl ether and allowed to settle for 1 hour. The mixture was centrifuged and washed with methyl tert-butyl ether 4 times. The mixture was dried to obtain 16.80 g of a white solid linear crude peptide with an Acm protecting group at the Cys (7, 15) position of plecanatin.

[0097] Example 20: Preparation of linear plecanatide 5

[0098] 46.60 g of the linear peptide resin of plecanatide prepared in Example 14 of the present invention was added to a 1000 ml three-necked round-bottom flask, and 460 ml of the lysis solution was prepared at a volume ratio of 94:2:2:2 TFA:Tis:Mpr:H2O. After mixing evenly, the above-mentioned peptide resin was added and reacted at room temperature for 2 hours. The mixture was filtered and the filtrate was added to 8 times the amount of ice isopropyl ether and precipitated for 1 hour. The mixture was centrifuged and washed with isopropyl ether four times. The mixture was dried to obtain 16.75 g of a white solid linear crude peptide with an Acm protecting group at the Cys (7, 15) position of plecanatide.

[0099] Example 21: Preparation of linear plecanatide 6

[0100] 44.50 g of the linear peptide resin of plecanatin prepared in Example 15 of the present invention was added to a 1000 ml three-necked round-bottom flask, and 450 ml of the lysis solution was prepared at a volume ratio of 90:3:3:3:1 TFA:Tis:Mpr:Phenol:H2O. After mixing evenly, the above-mentioned peptide resin was added and reacted at room temperature for 2 hours. The mixture was filtered and the filtrate was added to 9 times the amount of ice isopropyl ether and precipitated for 1 hour. The mixture was centrifuged and washed with isopropyl ether four times by centrifugation. The mixture was dried to obtain 15.93 g of a white solid linear crude peptide with a tBu protecting group at the Cys (4, 12) position of plecanatin.

[0101] Example 22: Preparation of Pulcanatide Cyclic Peptide 1

[0102] Take 16.90g of the crude linear peptide of pulcanatin prepared in Example 16 of the present invention, prepare the crude peptide into a 1g / L aqueous solution, adjust the pH of the solution to alkaline with dilute ammonia water, add 400μL of 10% hydrogen peroxide in a single amount, and cyclize for 1.5 hours to obtain a cyclic peptide at the Cys (4, 12) position of pulcanatin; adjust the pH of the cyclic peptide solution to acidic with acetic acid, add 1.90g of iodine in a double amount, dissolve it with ethanol, and add it dropwise to the cyclic peptide solution after the iodine is completely dissolved. After cyclization for 2 hours, use Vc to remove excess iodine residues to achieve disulfide cyclization at the Cys (7, 15) position of pulcanatin, and finally obtain a crude peptide solution with two pairs of cyclized disulfide bonds of pulcanatin. The purity of the crude peptide is measured to be 67.88%, and the yield is 65%. The chromatogram of the crude peptide of pulcanatin is shown as follows: Figure 1 As shown,

[0103] Example 23: Preparation of Plecanatide Cyclic Peptide 2

[0104] 17.03 g of crude linear peptide of plecanatin prepared in Example 17 of the present invention was taken, and the crude peptide was prepared into a 1 g / L aqueous solution. The pH of the solution was adjusted to alkaline with dilute ammonia water, and a single amount of 10% hydrogen peroxide (400 μL) was added. Cyclization took place for 1.5 hours, and lyophilization was performed to obtain a cyclic peptide at the Cys (7, 15) position of plecanatin. The cyclic peptide was reacted with 1 g / L of 20% mercaptoethanol in NMP and 0.1 M N-methylmorpholine at room temperature for 16 hours to remove the StBu protecting group of the Cys side chain. The same oxidation method was used to realize the disulfide bond cyclization at the Cys (4, 12) position of plecanatin, and finally a crude peptide solution of two pairs of cyclized disulfide bonds of plecanatin was obtained. The purity of the crude peptide was measured to be 65.58%, and the yield was 63%. The chromatogram of the crude peptide of plecanatin is similar to that of the crude peptide of plecanatin. Figure 1 similar.

[0105] Example 24: Preparation of Plecanatide Cyclic Peptide 3

[0106] Take 16.78g of crude linear peptide of pulcanatide prepared in Example 18 of the present invention, prepare the crude peptide into a 1g / L aqueous solution, adjust the pH of the solution to alkaline with dilute ammonia water, add 1.69mL of 10% DMSO, and cyclize for 4.0 hours to obtain a cyclic peptide at the Cys (7, 15) position of pulcanatide; adjust the pH of the cyclic peptide solution to acidic with acetic acid, add 3.80g of iodine in 5 times the amount, dissolve it with ethanol, and add it dropwise to the cyclic peptide solution after the iodine is completely dissolved. After cyclization for 2 hours, use Vc to remove excess iodine residues to achieve disulfide cyclization at the Cys (4, 12) position of pulcanatide, and finally obtain a crude peptide solution with two pairs of cyclized disulfide bonds of pulcanatide. The purity of the crude peptide is measured to be 66.05%, and the yield is 64%. The chromatogram of the crude peptide of pulcanatide is consistent with that of the crude peptide of pulcanatide. Figure 1 similar.

[0107] Example 25: Preparation of Plecanatide Cyclic Peptide 4

[0108] Take 16.80g of crude linear peptide of Plecanapeptide prepared in Example 19 of the present invention, prepare the crude peptide into a 1g / L aqueous solution, adjust the pH of the solution to alkaline with dilute ammonia water, and stir the reaction in air for more than 12 hours to obtain a cyclic peptide at the Cys (4, 12) position of Plecanapeptide; adjust the pH of the cyclic peptide solution to acidic with acetic acid, then add 1.90g of iodine (2.5 times the amount), dissolve it with ethanol, and after the iodine is completely dissolved, add it dropwise to the cyclic peptide solution. After cyclization for 2 hours, use Vc to remove excess iodine residues to achieve disulfide cyclization at the Cys (7, 15) position of Plecanapeptide, and finally obtain a crude peptide solution with two pairs of cyclized disulfide bonds of Plecanapeptide. The purity of the crude peptide is measured to be 66.55%, and the yield is 65%. The chromatogram of the crude peptide of Plecanapeptide is consistent with that of the crude peptide of Plecanapeptide. Figure 1 similar.

[0109] Example 26: Preparation of Plecanatide Cyclic Peptide 5

[0110] Take 16.75g of crude linear peptide of pulcanatin prepared in Example 20 of the present invention, prepare the crude peptide into a 1g / L aqueous solution, adjust the pH of the solution to alkaline with dilute ammonia water, add 200μL of 20% hydrogen peroxide in a single amount, and cyclize for 1.5 hours to obtain a cyclic peptide at the Cys (4, 12) position of pulcanatin; adjust the pH of the cyclic peptide solution to acidic with acetic acid, add 1.90g of iodine in a 3.5-fold amount, dissolve it with ethanol, and add it dropwise to the cyclic peptide solution after the iodine is completely dissolved. After cyclization for 2 hours, remove the excess iodine residue with Vc to realize the disulfide bond cyclization at the Cys (7, 15) position of pulcanatin, and finally obtain a crude peptide solution with two pairs of cyclized disulfide bonds of pulcanatin. The purity of the crude peptide is measured to be 66.25%, and the yield is 65%. The chromatogram of the crude peptide of pulcanatin is consistent with that of the crude peptide of pulcanatin. Figure 1 similar.

[0111] Example 27: Preparation of Plecanatide Cyclic Peptide

[0112] Take 15.93g of crude linear peptide of plecanatin prepared in Example 21 of the present invention, prepare the crude peptide into a 1g / L aqueous solution, adjust the pH of the solution to alkaline with dilute ammonia water, add 400μL of 10% hydrogen peroxide in a single amount, cyclize for 1.5 hours, and lyophilize to obtain a cyclic peptide at the Cys (7, 15) position of plecanatin; dissolve the cyclic peptide with 1g / L TFA, 10 equivalents of diphenyl sulfoxide, 100 equivalents of trichloromethylsilane, and 100 equivalents of anisole, react for 30min, remove the tBu protecting group of the Cys side chain, and use the same oxidation method to realize the disulfide bond cyclization at the Cys (4, 12) position of plecanatin, and finally obtain a crude peptide solution with two pairs of cyclized disulfide bonds of plecanatin. The purity of the crude peptide is measured to be 65.85%, and the yield is 64%. The chromatogram of the crude peptide of plecanatin is the same as that of the crude peptide of plecanatin. Figure 1 similar.

[0113] Example 28: Purification of crude plecanatide 1

[0114] The crude peptide solution of plecanatin prepared in Example 22 of the present invention was prepared using an RP-PLC preparative liquid phase system with a wavelength of 220 nm, a reversed-phase C18 column, and a mobile phase of TEAP pH 6.8 / acetonitrile in a volume ratio of 75:25. After RP-PLC purification and salt conversion, the target peptide fraction was collected, concentrated by rotary evaporation, and lyophilized to obtain pure plecanatin. The purity of the pure plecanatin was 99.7% and the total yield was 48%. The chromatogram of the pure plecanatin prepared by the present invention is as follows: Figure 2 shown.

[0115] Example 29: Purification of crude plecanatide 2

[0116] The crude peptide solution of plecanatin prepared in Example 23 of the present invention was prepared using an RP-PLC preparative liquid phase system with a wavelength of 220 nm, a reversed-phase C18 column, and a mobile phase of TEAP pH 6.8 / acetonitrile in a volume ratio of 75:25. After RP-PLC purification and salt conversion, the target peptide fraction was collected, concentrated by rotary evaporation, and lyophilized to obtain pure plecanatin. The purity of the pure plecanatin was 99.5% and the total yield was 48%. The chromatogram of the pure plecanatin prepared by the present invention is similar to that of the pure plecanatin prepared by the present invention. Figure 2 similar.

[0117] Example 30: Purification of crude plecanatide peptide 3

[0118] The crude peptide solution of plecanatin prepared in Example 24 of the present invention was prepared using an RP-PLC preparative liquid phase system with a wavelength of 220 nm, a reversed-phase C18 column, and a mobile phase of TEAP pH 6.8 / acetonitrile in a volume ratio of 75:25. After RP-PLC purification and salt conversion, the target peptide fraction was collected, concentrated by rotary evaporation, and lyophilized to obtain pure plecanatin. The purity of the pure plecanatin was 99.6% and the total yield was 47%. The chromatogram of the pure plecanatin prepared by the present invention is similar to that of the pure plecanatin prepared by the present invention. Figure 2 similar.

[0119] Example 31: Purification of crude plecanatide 4

[0120] The crude peptide solution of plecanatin prepared in Example 25 of the present invention was prepared using an RP-PLC preparative liquid phase system with a wavelength of 220 nm, a reversed-phase C18 column, and a mobile phase of TEAP pH 6.8 / acetonitrile in a volume ratio of 75:25. After RP-PLC purification and salt conversion, the target peptide fraction was collected, concentrated by rotary evaporation, and lyophilized to obtain pure plecanatin. The purity of the pure plecanatin was 99.5% and the total yield was 48%. The chromatogram of the pure plecanatin prepared by the present invention is similar to that of the pure plecanatin prepared by the present invention. Figure 2 similar.

[0121] Example 32: Purification of crude plecanatide peptide

[0122] The crude peptide solution of plecanatin prepared in Example 26 of the present invention was prepared using an RP-PLC preparative liquid phase system with a wavelength of 220 nm, a reversed-phase C18 column, and a mobile phase of TEAP pH 6.8 / acetonitrile in a volume ratio of 75:25. After RP-PLC purification and salt conversion, the target peptide fraction was collected, concentrated by rotary evaporation, and lyophilized to obtain pure plecanatin. The purity of the pure plecanatin was 99.5% and the total yield was 48%. The chromatogram of the pure plecanatin prepared by the present invention is similar to that of the pure plecanatin prepared by the present invention. Figure 2 similar.

[0123] Example 33: Purification of crude plecanatide peptide

[0124] The crude peptide solution of plecanatin prepared in Example 27 of the present invention was prepared using an RP-PLC preparative liquid phase system with a wavelength of 220 nm, a reversed-phase C18 column, and a mobile phase of TEAP pH 6.8 / acetonitrile in a volume ratio of 75:25. After RP-PLC purification and salt conversion, the target peptide fraction was collected, concentrated by rotary evaporation, and lyophilized to obtain pure plecanatin. The purity of the pure plecanatin was 99.5% and the total yield was 47%. The chromatogram of the pure plecanatin prepared by the present invention is similar to that of the pure plecanatin prepared by the present invention. Figure 2 similar.

[0125] It was predicted that the molecular weight of plecanatide was 1681.88. After mass spectrometry detection, the molecular weight of all plecanatide prepared by the present invention was 1681.69, which was in line with the expectation, proving that the present invention successfully prepared plecanatide.

[0126] Comparative Example 1: Preparation of Plecanapeptide Peptide Resin

[0127] 50.08 g (10 mmol) of the Fmoc-Leu-Wang resin prepared in Example 1 of the present invention with a degree of substitution of 0.20 mmol / g was added to a solid-phase reaction column and washed once with DMF. After swelling the Fmoc-Leu-Wang resin with DMF for 30 minutes, the Fmoc protection was removed with a mixed solution of DMF:piperidine in a volume ratio of 4:1. The resin was then washed six times with DMF. 12.45 g of Fmoc-Cys(Acm)-OH and 4.05 g of HOBt were weighed and dissolved in the DMF solution. After activation with 4.67 ml of DIC in an ice-water bath, the resin was added to the reaction column containing the resin. After reacting at room temperature for 2 hours, the reaction endpoint was determined using the ninhydrin assay. If the resin was colorless and transparent, the reaction was complete; if the resin developed color, the reaction was incomplete and required further reaction for another hour or repeated addition of a single dose or a different condensation reagent. The ninhydrin assay is suitable for determining the reaction endpoint in subsequent amino acid coupling reactions. Repeat the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and sequentially insert Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Cys(Acm)-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH and Fmoc-Gly-OH according to the sequence of plecanatide. Moc-Asn(Trt)-OH was used to obtain the peptide resin Fmoc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(Trt)-Glu(OtBu)-Leu-Cys(Acm)-Val-Asn(Trt)-Val-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Acm)-Leu-Wang resin, weighing 70.50 g. During the linear peptide coupling process, the volume of the peptide resin shrank significantly when coupling the S9-S1 residues, and the coupling was difficult when coupling the S7 residue. The resin was light blue when detected by the ninhydrin method after 2.5 hours of reaction, and no significant improvement was achieved by repeated injection.

[0128] Comparative Example 2: Preparation of linear plecanatide

[0129] 70.50 g of the linear peptide resin of plecanatin prepared in Comparative Example 1 was added to a 1000 ml three-necked round-bottom flask, and 730 ml of the lysis solution was prepared at a volume ratio of 94:3:3 TFA:Tis:Mpr. After mixing evenly, the above peptide resin was added and reacted at room temperature for 2 hours. The mixture was filtered and the filtrate was added to 8 times the amount of ice isopropyl ether and precipitated for 1 hour. The mixture was centrifuged and washed with isopropyl ether 4 times. The mixture was dried to obtain 14.91 g of a white solid linear crude peptide with an Acm protecting group at the Cys (7, 15) position of plecanatin.

[0130] Comparative Example 3: Preparation of Pulcanatide Cyclic Peptide

[0131] Take 14.91g of crude linear peptide of Plecanapeptide prepared in Comparative Example 2, prepare the crude peptide into a 1g / L aqueous solution, adjust the pH of the solution to alkaline with dilute ammonia water in 6L solution, add 400μL of 10% hydrogen peroxide in a single amount, and cyclize for 1.5 hours to obtain a cyclic peptide at the Cys (4, 12) position of Plecanapeptide; adjust the pH of the cyclic peptide solution to acidic with acetic acid, add 1.90g of iodine in a double amount, dissolve it with ethanol, and add it dropwise to the cyclic peptide solution after the iodine is completely dissolved. After cyclization for 2 hours, remove the excess iodine residue with Vc to realize the disulfide bond cyclization at the Cys (7, 15) position of Plecanapeptide, and finally obtain a crude peptide solution with two pairs of cyclized disulfide bonds of Plecanapeptide. The purity of the crude peptide is 63.46% and the yield is 55%. The chromatogram of crude peptide of Plecanapeptide is as follows: Figure 3 shown.

[0132] Comparative Example 4: Purification of crude plecanatide peptide

[0133] The crude peptide solution of plecanatin prepared in Comparative Example 3 was prepared using an RP-PLC preparative liquid phase system with a wavelength of 220 nm, a reversed-phase C18 column, and a mobile phase of TEAP pH 6.8 / acetonitrile in a volume ratio of 75:25. After RP-PLC purification and salt transfer, the target peptide fraction was collected, concentrated by rotary evaporation, and lyophilized to obtain pure plecanatin. The purity of the pure product was 97.12% and the total yield was 39%. The chromatogram of pure plecanatin is shown in Figure 2. Figure 4 shown.

Claims

1. A fragment synthesis method of plecanatide, characterized in that: The main steps include: (1) On Fmoc-Leu-resin, a linear plecanapeptide resin was obtained by solid-phase synthesis according to the peptide sequence of plecanapeptide; wherein S5-S8 were tetrapeptide fragments, and the rest were corresponding protected amino acids or fragments; (2) cleaving the linear plecanatide resin obtained in step (1) to obtain linear plecanatide; (3) cyclizing the linear plecanatin obtained in step (2) to obtain crude plecanatin peptide; Wherein, the tetrapeptide fragment used in steps (1) S5-S8 is Fmoc-Glu(OtBu)-Leu-Cys(R4)-Val-OH, and R4 is selected from Trt and Acm; Wherein, the linear plecanatin resin obtained in step (1) is: R1-Asn(R2)-Asp(OtBu)-Glu(OtBu)-Cys(R3)-Glu(OtBu)-Leu-Cys(R4)-Val-Asn(Trt)-Val-Ala-Cys(R3)-Thr(tBu)-Gly-Cys(R4)-Leu-resin; in: R1 is selected from Boc, Fmoc; R2 is selected from Trt, H; R3 and R4 are selected from Trt and Acm, and R3 and R4 are different; The remaining corresponding segments used in step (1) include S1-S2 or S1-S4; The S1-S2 fragment is R1-Asn(R2)-Asp(OtBu)-OH, and the S1-S4 fragment is R1-Asn(R2)-Asp(OtBu)-Glu(OtBu)-Cys(R3)-OH; in: R1 is selected from Fmoc, Boc; R3 is selected from Trt, Acm.

2. The method for synthesizing a fragment of plecanatide according to claim 1, wherein: In the step (1), the resin is selected from Wang resin or 2-chlorotrityl chloride resin.

3. The method for synthesizing a fragment of plecanatide according to claim 1, wherein: The substitution degree of Fmoc-Leu-resin in step (1) is in the range of 0.2-0.5 mmol / g.

4. The method for synthesizing a fragment of plecanatide according to claim 1, wherein: In the cleavage step (2), the cleavage reagent is selected from trifluoroacetic acid and other components, and the other components are selected from water, phenol, 3-mercaptopropionic acid, and triisopropylsilane, wherein trifluoroacetic acid accounts for more than 90% and the other components each account for 1%-5%.

5. The method for synthesizing a fragment of plecanatide according to claim 1, wherein: The crude plecanatin peptide obtained in step (3) can be further purified by reverse-phase high performance liquid chromatography and lyophilized to obtain refined plecanatin peptide.

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