CN115403659A - A kind of fragment synthetic method of plecanatide - Google Patents

Abstract

The 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, adopting a solid-phase synthesis method according to the peptide sequence of plecanatide Obtain linear plecanatide resin; wherein, S5-S8 adopts tetrapeptide fragments, and all the other adopt corresponding protected amino acids or fragments; (2) the linear plecanatide resin obtained in step (1) is cracked to obtain linear plecanatide (3) cyclizing the linear plecanatide obtained in step (2) to obtain crude plecanatide peptide. The process of the present invention effectively improves the problem of difficult coupling caused by peptide resin shrinkage in the solid-phase synthesis process, reduces the generation of related missing peptide impurities, greatly reduces the difficulty of synthesis and purification of plecanatide, and the purity of the prepared plecanatide High, high yield, less impurities, is a kind of preparation method of plecanatide with wide practical value and application prospect.

Description

A kind of fragment synthesis method of plecanatide

technical field

The invention belongs to the field of polypeptide drug preparation, and specifically relates to a fragment synthesis method of plecanatide.

Background technique

Chronic idiopathic constipation (CIC) and irritable bowel syndrome with constipation (IBS-C) are two of the most common disorders affecting the gastrointestinal tract and are characterized by decreased stool frequency, straining, abdominal pain or discomfort. Chronic constipation among adults in my country, including CIC, functional defecation disorder and IBS-C, has a prevalence rate of 4%-6%. Chronic constipation can be secondary to mental and psychological disorders, and the degree of damage to the quality of life of patients is comparable to that of chronic diseases such as diabetes and heart failure.

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

In the prior art, the patent with the publication number CN104211777A adopts the Fmoc solid-phase synthesis method, and gradually couples to synthesize the plecanatide linear peptide resin. During the oxidation process of Kanatide, different deprotection strategies were selected according to the characteristics of the Cys side chain protecting group, and hydrogen peroxide and iodine were used for stepwise oxidation. However, according to experiments, it is found that the synthesis method will cause polycondensation of the peptide resin after the coupling of some amino acids, resulting in difficulties in the synthesis of linear peptides, low purity of crude products, and difficulties in purification. The patent with publication number CN110981939A uses Fmoc solid-phase synthesis method to synthesize plecanatide linear peptide resin step by step or fragment coupling, wherein Hmb/Dmb is used to protect Gly and Ala amino acids, and the linear peptide is obtained after cleavage, and then in the liquid phase In the stepwise oxidation with hydrogen peroxide and iodine, two pairs of disulfide bonds are formed to obtain plecanatide. Although this solution can improve the polycondensation problem of the resin, the Hmb/Dmb material is expensive, which is not conducive to cost control, and it is difficult to realize industrial production. The patent with the publication number CN109354607A uses octapeptide fragments and octapeptide resins to condense in the solid state in the presence of a condensing agent to obtain hexadepeptide resins, which are cracked to obtain linear peptides, and then oxidized step by step with hydrogen peroxide and iodine in the liquid phase to form two pairs of peptides. Disulfide bonds are used to obtain plecanatide. The total yield of plecanatide obtained by this method can reach 32.45% after purification, and the purity can reach more than 99%. The present invention can greatly shorten the synthesis cycle and avoid the problem of low efficiency of traditional solid-phase step-by-step coupling. However, the fully protected S8-S1 octapeptide fragment has large steric hindrance, difficult coupling, and poor solubility in DMF or DCM. It is not conducive to the reaction, the operation is relatively complicated, and the cost is high.

Contents of the invention

In order to overcome the phenomenon of difficult coupling, complex operation, and resin polycondensation of plecanatide during the synthesis process, and solve the problems of low purity and low yield after step-by-step coupling and cyclization, the invention provides a plecanatide The fragment synthesis method of that peptide mainly comprises the following steps:

(1) On the Fmoc-Leu-resin, a linear plecanatide resin is obtained by solid-phase synthesis according to the plecanatide peptide sequence; wherein, S5-S8 adopts tetrapeptide fragments, and the rest adopt corresponding protected amino acids or fragments;

(2) cracking the linear plecanatide resin obtained in step (1) to obtain linear plecanatide;

(3) Stepwise cyclization of the linear plecanatide obtained in step (2) to obtain crude plecanatide peptide.

Further, the linear plecanatide resin obtained in the step (1) is: R ₁ -Asn(R ₂ )-Asp(OtBu)-Glu(OtBu)-Cys(R ₃ )-Glu(OtBu)-Leu -Cys(R ₄ )-Val-Asn(Trt)-Val-Ala-Cys(R ₃ )-Thr(tBu)-Gly-Cys(R ₄ )-Leu-resin;

in:

R _is selected from Boc, Fmoc, Z;

R ₂ is selected from Trt, H;

_R3 and _R4 are selected from Trt, Acm, StBu or tBu, and _R3 and _R4 are different.

Further, the tetrapeptide fragment used in step (1) S5-S8 is Fmoc-Glu(OtBu)-Leu-Cys(R ₄ )-Val-OH; wherein, R ₄ is preferably Trt, Acm.

Further, the rest of the step (1) adopts corresponding fragments including S ₁ -S ₂ or S ₁ -S ₄ . The synthesized S ₁ -S ₂ fragment is R ₁ -Asn(R ₂ )-Asp(OtBu)-OH, and the S ₁ -S ₄ fragment is R ₁ -Asn(R ₂ )-Asp(OtBu)-Glu( OtBu)-Cys(R ₃ )-OH;

in:

R ₁ is preferably Fmoc, Boc;

R ₃ is preferably Trt, Acm.

In step (2), the linear plecanatide resin is cleaved while removing the resin and side chain protecting groups to obtain linear plecanatide: H-Asn-Asp-Glu-Cys(R ₃ )-Glu-Leu-Cys( R ₄ )-Val-Asn-Val-Ala-Cys(R ₃ )-Thr-Gly-Cys(R ₄ )-Leu-OH, wherein when the Cys side chain protecting group is Trt, it can be cleaved during the cutting process; As with other protecting groups, it cannot be cleaved.

In a preferred embodiment of the present invention, the cleavage and step-by-step cyclization in the steps (2) and (3) mainly include the following steps:

When R ₃ and R ₄ are selected from Trt, Acm, the linear plecanatide resin settles after the cracking reaction of the cleavage reagent to obtain a linear peptide containing a pair of Cys with Acm protection group in the peptide sequence; the linear peptide is dissolved, Adding an oxidizing agent and performing a cyclization reaction to obtain a cyclic peptide; removing the side chain protecting group Acm on Cys from the cyclic peptide in an iodine solution, and simultaneously cyclizing to obtain a crude peptide of plecanatide; wherein the oxidizing agent added is selected from hydrogen peroxide , DMSO or air.

When R ₃ and R ₄ were selected from Trt and StBu, the linear plecanatide resin was cleaved by a cleavage reagent and precipitated to obtain a linear peptide containing a pair of Cys with a StBu protecting group in the peptide sequence; the linear peptide was dissolved, Adding an oxidizing agent and performing a cyclization reaction to obtain a cyclic peptide; adding 2-mercaptoethanol to the cyclic peptide to remove the StBu protecting group; then adding an oxidizing agent to perform a second cyclization to obtain a crude plecanatide peptide. Wherein the oxidizing agent adopted in the second cyclization is selected from hydrogen peroxide, DMSO or air.

When R ₃ and R ₄ were selected from Trt, tBu, the linear plecanatide resin was precipitated after the cleavage reaction of the cleavage reagent to obtain a linear peptide containing a pair of Cys with a tBu protecting group in the peptide sequence; the linear peptide was dissolved, Adding an oxidizing agent, cyclization reaction to obtain a cyclic peptide; adding TFA, diphenyl sulfoxide, trichloromethylsilane and anisole to the cyclic peptide to remove the tBu protecting group; then adding an oxidizing agent for the second cyclization , to obtain the crude peptide of plecanatide. Wherein the oxidizing agent adopted in the second cyclization is selected from hydrogen peroxide, DMSO or air.

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

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 obtained Fmoc-Leu-resin substitution range is 0.2-0.5mmol/g.

A fragment synthesis method of plecanatide proposed by the present invention, the corresponding protected amino acids and S5-S8 tetrapeptide fragments in the sequence are sequentially inserted on the Fmoc-Leu-resin by a solid-phase synthesis method, which can effectively improve the polycondensation of the resin phenomenon, greatly reducing the difficulty of synthesis of plecanatide, and the S5-S8 tetrapeptide fragments have better solubility in DMF or DCM, which can improve the purity of plecanatide and significantly increase its yield. The use of S1-S4 or S1-S2 fragments can reduce the problem of related missing peptide impurities due to coupling difficulties of amino acids at S1 or S4 positions. The purity of the pure plecanatide synthesized by the method of the invention can reach more than 99.5%, and the total yield can reach more than 45%. Compared with the existing technology, the process of the present invention effectively improves the problem of difficult coupling caused by the shrinkage of the peptide resin during the solid-phase synthesis process, reduces the generation of related missing peptide impurities, and greatly reduces the difficulty of synthesis and purification of plecanatide. The prepared plecanatide has high purity, high yield and less impurities, and has wide practical value and application prospect.

Description of drawings

Fig. 1 is the chromatogram of the crude peptide of plecanatide prepared in Example 22 of the present invention;

Fig. 2 is the chromatogram of the pure product of plecanatide prepared in Example 28 of the present invention;

Fig. 3 is the chromatogram of the plecanatide crude peptide that comparative example 3 of the present invention makes;

Fig. 4 is the chromatogram of the pure plecanatide prepared in Comparative Example 4 of the present invention.

Detailed ways

The present invention will be further described in detail through examples below, which are intended to illustrate the present invention rather than limit the present invention. It should be pointed out that those skilled in the art can make some improvements and modifications to the present invention without departing from the principle of the present invention, and these improvements and modifications also fall within the protection scope of the present invention.

Embodiment 1: Preparation of Fmoc-Leu-Wang resin with a substitution value of 0.20mmol/g

Take by weighing 303.00g (100mmol) of Wang resin with a substitution value of 0.33mmol/g, add it to a solid-phase reactor, wash it twice with DMF, and after swelling the resin with DMF for 60 minutes, take 70.64g Fmoc-Leu-OH, 27.13g HOBt, 25.35g DIC, and 2.06g DMAP were dissolved in DMF solution, added to a solid-phase reactor, and reacted at room temperature for 2 hours. After the reaction was completed, it was washed 3 times with DMF and 3 times with DCM. Then add a capping solution for capping for 30 minutes. The ratio of the capping solution is: acetic anhydride:DMF:DIEA=10:84:6 (V:V:V). After capping, it was washed 3 times with DMF, 3 times with DCM, shrunk and dried with methanol to obtain Fmoc-Leu-Wang resin, and the detected substitution value was 0.20 mmol/g.

Embodiment 2: Preparation of Fmoc-Leu-Wang resin with a substitution value of 0.50mmol/g

Take Wang resin 125.00g (100mmol) with a substitution value of 0.80mmol/g, add it to a solid-phase reactor, wash it twice with DMF, and after swelling the resin with DMF for 60 minutes, take 70.65g Fmoc-Leu-OH, 27.03g HOBt, 25.25g DIC, and 2.05g DMAP were dissolved in DMF solution, added to the solid-phase reactor, and reacted at room temperature for 2 hours. After the reaction was completed, it was washed 3 times with DMF and 3 times with DCM. Then add a capping solution for capping for 30 minutes. The ratio of the capping solution is: acetic anhydride:DMF:DIEA=10:84:6 (V:V:V). After capping, it was washed 3 times with DMF and 3 times with DCM, then shrank and dried with methanol to obtain Fmoc-Leu-Wang resin, and the detected substitution value was 0.50 mmol/g.

Embodiment 3: Preparation of Fmoc-Leu-2-chlorotrityl chloride resin with a substitution value of 0.30mmol/g

Weigh 200.02g (100mmol) of 2-chlorotrityl chloride resin with a substitution value of 0.50mmol/g, add it to a solid-phase reactor, wash it twice with DMF, and take 70.65g Fmoc-Leu-OH, 38.70g DIEA Dissolved in DMF solution, added to a solid-phase reactor, and reacted at room temperature for 2 hours. After the reaction was completed, it was washed 3 times with DMF and 3 times with DCM. Then add a capping solution for capping for 30 minutes, the ratio of the capping solution is: methanol:DMF:DIEA=1:10:20 (V:V:V). After capping, it was washed 4 times with DMF and 6 times with DCM to obtain Fmoc-Leu-2-chlorotrityl chloride resin with a detected substitution value of 0.30 mmol/g.

Example 4: Preparation of plecanatide S5-S8 fragment peptide Fmoc-Glu(OtBu)-Leu-Cys(Acm)-Val-OH

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

Weigh 111.11 g (100 mmol) of 2-chlorotrityl chloride resin with a degree of substitution of 0.90 mmol/g, add it to a solid-phase reaction column, wash it once with DMF, and wash the resin with DMF for 30 minutes after swelling the resin with DCM for 30 minutes. Once, get 50.91g Fmoc-Val-OH and dissolve it with DMF, add 49.6ml DIEA to the ice-water bath for activation, and then add it to the above-mentioned reaction column equipped with resin. =1:10:20 mixed solution was blocked overnight, alternately washed with DCM and MeOH, shrunk and dried to obtain Fmoc-Val-2-chlorotrityl chloride resin, and the detected substitution degree was 0.60 mmol/g.

(2) Synthesis of plecanatide S5-S8 fragment peptide peptide resin

Weigh 33.34g (20mmol) of the Fmoc-Val-2-chlorotrityl chloride resin with a degree of substitution of 0.60mmol/g obtained in step (1), add it to the solid-phase reaction column, wash it once with DMF, and wash it once with DMF After swelling the Fmoc-Val-2-chlorotrityl chloride resin for 30 minutes, remove the Fmoc protection with a mixed solution of DMF:piperidine volume ratio 4:1, then wash 6 times with DMF, and weigh 24.80g Fmoc-Cys Add (Acm)-OH and 8.90gHOBt into DMF solution to dissolve, add 14.2ml DIC under ice-water bath to activate, then add to the above-mentioned reaction column equipped with resin, react at room temperature for 2 hours, use ninhydrin detection to judge the reaction end point, if the resin If it is colorless and transparent, it means that the reaction is complete; if the resin develops color, the reaction is incomplete, and it needs to continue to react for another hour or re-dosing with a single amount or changing the condensation reagent for re-dosing. Ninhydrin detection is suitable for subsequent amino acid coupling reactions to determine the end point of the reaction. Repeat the steps of removing Fmoc protection and adding the corresponding amino acid for coupling, sequentially coupling Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, and directly shrinking the tetrapeptide resin without removing Fmoc after coupling the last amino acid Dry, wash with DCM for 6 times, and drain to obtain Fmoc-Glu(OtBu)-Leu-Cys(Acm)-Val-2-chlorotrityl chloride resin, which is ready for use.

(3) Preparation of plecanatide S5-S8 fragment peptide

Weigh 20mmol 51.50g of plecanatide S5-S8 fragment peptide resin dried in step (2) in 30% TFE DCM solution, the material volume ratio is 1g peptide resin 10ml solution. After the solution is mixed evenly, add the above peptide resin, react at room temperature for 2 hours, filter, concentrate the filtrate to 1/4 volume under reduced pressure, add 8 times the amount of ice isopropyl ether to settle for 1 hour, centrifuge, and wash with isopropyl ether for 4 Once, dry to obtain plecanatide S5-S8 fragment peptide Fmoc-Glu(OtBu)-Leu-Cys(Acm)-Val-OH17.10g.

Example 5: Preparation of plecanatide S5-S8 fragment peptide Fmoc-Glu(OtBu)-Leu-Cys(Trt)-Val-OH

(1) Synthesis of plecanatide S5-S8 fragment peptide peptide resin

Weigh 33.35g (20mmol) Fmoc-Val-2-chlorotrityl chloride resin with a degree of substitution of 0.60mmol/g obtained in step (1) of Example 4, add it to the solid-phase reaction column, and wash once with DMF , after 30 minutes with DMF swelling Fmoc-Val-2-chlorotrityl chloride resin, with DMF: the mixed solution of piperidine volume ratio 4:1 takes off Fmoc protection, washes 6 times with DMF then, weighs 27.50g Add Fmoc-Cys(Trt)-OH, 8.60gHOBt into DMF solution to dissolve, add 14.2ml DIC under ice-water bath to activate, then add to the above-mentioned reaction column equipped with resin, react at room temperature for 2 hours, use ninhydrin to detect and judge 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 needs to continue to react for another hour or re-dosing with a single amount or changing the condensation reagent. Ninhydrin detection is suitable for subsequent amino acid coupling reactions to determine the end point of the reaction. Repeat the steps of removing Fmoc protection and adding the corresponding amino acid for coupling, sequentially coupling Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, and directly shrinking the tetrapeptide resin without removing Fmoc after coupling the last amino acid Dry, wash with DCM for 6 times, and drain to obtain Fmoc-Glu(OtBu)-Leu-Cys(Trt)-Val-2-chlorotrityl chloride resin, which is ready for use.

(2) Preparation of plecanatide S5-S8 fragment peptide

Weigh 20mmol 51.50g of plecanatide S5-S8 fragment peptide resin dried in step (2) in DCM solution of 10% TFE, the material volume ratio is 10ml solution of 1g peptide resin. After the solution is mixed evenly, add the above peptide resin, react at room temperature for 2 hours, filter, concentrate the filtrate to 1/4 volume under reduced pressure, add 10 times the amount of ice isopropyl ether to settle for 1 hour, centrifuge, and wash with isopropyl ether for 4 Once, dry to obtain plecanatide S5-S8 fragment peptide Fmoc-Glu(OtBu)-Leu-Cys(Trt)-Val-OH17.05g.

Embodiment 6: Preparation of plecanatide S1-S4 fragment peptide Fmoc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(Trt)-OH

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

Weigh 55.55 g (500 mmol) of 2-chlorotrityl chloride resin with a degree of substitution of 0.90 mmol/g, add it to a solid-phase reaction column, wash it once with DMF, and wash the resin with DMF for 30 minutes after swelling the resin with DCM for 30 minutes. Once, get 58.55g Fmoc-Cys(Trt)-OH and dissolve with DMF, add 24.2ml DIEA in the ice-water bath after activation and add in the above-mentioned reaction column that resin is housed, after reacting for 2 hours, wash 3 times with DMF, use DIEA: MeOH:DMF=1:10:20 mixed solution was blocked overnight, DCM and MeOH were alternately washed, shrunk and dried to obtain Fmoc-Cys(Trt)-2-chlorotrityl chloride resin, and the detected substitution degree was 0.60mmol/g.

(2) Synthesis of plecanatide S1-S4 fragment peptide peptide resin

Take by weighing 33.34g (20mmol) the degree of substitution that step (1) makes is the Fmoc-Cys(Trt)-2-chlorotrityl chloride resin of 0.60mmol/g, join in the solid-phase reaction column, wash with DMF Once, 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 volume ratio 4:1, and then wash 6 times with DMF, Weigh 25.53g Fmoc-Glu(OtBu)-OH, 8.92g HOBt and add DMF solution to dissolve, add 14.1ml DIC under ice-water bath to activate, add to the above-mentioned reaction column equipped with resin, and react at room temperature for 2 hours, then use ninhydrin Detect and judge the end point of the reaction. If the resin is colorless and transparent, it means that the reaction is complete; if the resin develops color, the reaction is incomplete. You need to continue to react for another hour or re-dosing with a single dose or replace the condensation reagent. Ninhydrin detection is suitable for subsequent amino acid coupling reactions to determine the end point of the reaction. Repeat the above steps of removing Fmoc protection and adding the corresponding amino acid for coupling, sequentially coupling Fmoc-Asp(OtBu)-OH, Fmoc-Asn(Trt)-OH, after coupling the last amino acid without removing Fmoc, the tetrapeptide The peptide resin was shrunk and dried, washed 6 times with DCM, and dried to obtain Fmoc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(Trt)-2-chlorotrityl chloride resin, which was ready for use.

(3) Preparation of plecanatide S1-S4 fragment peptide

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

Example 7: Preparation of plecanatide S1-S4 fragment peptide Fmoc-Asn-Asp(OtBu)-Glu(OtBu)-Cys(Acm)-OH

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

Weigh 55.56 g (500 mmol) of 2-chlorotrityl chloride resin with a degree of substitution of 0.90 mmol/g, add it to a solid-phase reaction column, wash it once with DMF, and wash the resin with DMF for 30 minutes after swelling the resin with DCM for 30 minutes. Once, get 58.55g Fmoc-Cys(Acm)-OH and dissolve with DMF, add 24.2ml DIEA activation in the ice-water bath and add in the above-mentioned reaction column that resin is equipped with, after reacting for 2 hours, wash 3 times with DMF, use DIEA: MeOH:DMF=1:10:20 mixture was sealed overnight, DCM and MeOH were alternately washed, shrunk and dried to obtain Fmoc-Cys(Acm)-2-chlorotrityl chloride resin, and the detected substitution degree was 0.60mmol/g.

(2) Synthesis of plecanatide S1-S4 fragment peptide peptide resin

Weigh 33.33g (20mmol) of the Fmoc-Cys(Acm)-2-chlorotrityl chloride resin with a degree of substitution of 0.60mmol/g prepared in step (1), add it to the solid-phase reaction column, and wash with DMF Once, 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 volume ratio 4:1, and then wash 6 times with DMF, Weigh 25.53g Fmoc-Glu(OtBu)-OH, 8.92g HOBt and add DMF solution to dissolve, add 14.1ml DIC under ice-water bath to activate, add to the above-mentioned reaction column equipped with resin, and react at room temperature for 2 hours, then use ninhydrin Detect and judge the end point of the reaction. If the resin is colorless and transparent, it means that the reaction is complete; if the resin develops color, the reaction is incomplete. You need to continue to react for another hour or re-dosing with a single dose or replace the condensation reagent. Ninhydrin detection is suitable for subsequent amino acid coupling reactions to determine the end point of the reaction. Repeat the above steps of removing Fmoc protection and adding corresponding amino acid coupling, sequentially coupling Fmoc-Asp(OtBu)-OH, Fmoc-Asn-OH, after coupling the last amino acid without removing Fmoc, directly shrink the tetrapeptide resin Dry, wash with DCM for 6 times, and drain to obtain Fmoc-Asn-Asp(OtBu)-Glu(OtBu)-Cys(Acm)-2-chlorotrityl chloride resin, which is ready for use.

(3) Preparation of plecanatide S1-S4 fragment peptide

Weigh 20mmol 50.50g of plecanatide S1-S4 fragment peptide peptide resin dried in step (2) in 10% TFE DCM solution, the material volume ratio is 10ml solution of 1g peptide resin. After the solution is mixed evenly, add the above peptide resin, react at room temperature for 2 hours, filter, concentrate the filtrate to 1/4 volume under reduced pressure, add 9 times the amount of methyl tert-butyl ether to settle for 1 hour, centrifuge, and methyl tert-butyl ether The base ether was centrifuged and washed 4 times, and dried to obtain 1.50 g of Fmoc-Asn-Asp(OtBu)-Glu(OtBu)-Cys(Acm)-OH.

Example 8: Preparation of plecanatide S1-S2 fragment peptide Fmoc-Asn(Trt)-Asp(OtBu)-OH

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

Weigh 55.55 g (50 mmol) of 2-chlorotrityl chloride resin with a degree of substitution of 0.90 mmol/g, add it to a solid-phase reaction column, wash it once with DMF, and wash it with DMF for 30 minutes after swelling the resin with DCM. Once, get 41.15g Fmoc-Asp(OtBu)-OH and dissolve with DMF, add 24.8ml DIEA activation in the ice-water bath and add in the above-mentioned reaction column that resin is equipped with, after reacting for 2 hours, wash 3 times with DMF, use DIEA: MeOH:DMF=1:10:20 mixed solution was blocked overnight, DCM and MeOH were alternately washed, shrunk and dried to obtain Fmoc-Asp(OtBu)-2-chlorotrityl chloride resin, and the detected substitution degree was 0.60mmol/g.

(2) Synthesis of plecanatide S1-S2 fragment peptide peptide resin

Take by weighing 32.34g (20mmol) the degree of substitution that step (1) makes is the Fmoc-Asp(OtBu)-2-chlorotrityl chloride resin of 0.60mmol/g, join in the solid-phase reaction column, wash with DMF Once, after swelling Fmoc-Asp(OtBu)-2-chlorotrityl chloride resin with DMF for 30 minutes, remove Fmoc protection with a mixed solution of DMF:piperidine volume ratio 4:1, then wash 6 times with DMF, Weigh 35.80g of Fmoc-Asn(Trt)-OH and 8.91g of HOBt into DMF solution to dissolve, add 14.1ml of DIC in an ice-water bath to activate, then add to the above-mentioned reaction column equipped with resin, react at room temperature for 2 hours, and detect with ninhydrin Judging the end point of the reaction, if the resin is colorless and transparent, 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-dosing with a single dose or replace the condensation reagent. Ninhydrin detection is suitable for subsequent amino acid coupling reactions to determine the end point of the reaction. After coupling, without removing Fmoc, the dipeptide peptide resin was shrink-dried directly, washed with DCM for 6 times, and drained to obtain Fmoc-Asn(Trt)-Asp(OtBu)-2-chlorotrityl chloride resin, which was ready for use.

(3) Preparation of plecanatide S1-S2 fragment peptide

Weigh the dried 20mmol 44.50g plecanatide S1-S2 fragment peptide peptide resin in 20% TFE DCM solution, the material volume ratio is 1g peptide resin 10ml solution. After the solution is mixed evenly, add the above peptide resin, react at room temperature for 2 hours, filter, concentrate the filtrate under reduced pressure to 1/4 volume, add 8 times the amount of ether to settle for 1 hour, centrifuge, wash with ether for 4 times, and dry to obtain Fmoc -Asn(Trt)-Asp(OtBu)-OH 13.10 g.

Example 9: Preparation of plecanatide S1-S2 fragment peptide Boc-Asn-Asp(OtBu)-OH

(1) Synthesis of plecanatide S1-S2 fragment peptide peptide resin

Take by weighing 33.33g (20mmol) the Fmoc-Asp(OtBu)-2-chlorotrityl chloride resin that the degree of substitution prepared in embodiment 8 step (1) is 0.60mmol/g, join in the solid phase reaction column, Wash once with DMF, use DMF to swell the Fmoc-Asp(OtBu)-2-chlorotrityl chloride resin for 30 minutes, remove Fmoc protection with a mixed solution of DMF:piperidine volume ratio 4:1, and then wash with DMF 6 times, weigh 13.90g Boc-Asn-OH, 8.91g HOBt, add DMF solution to dissolve, add 14.1ml DIC under ice-water bath to activate, add to the above reaction column with resin, react at room temperature for 2 hours, use ninhydrin Detect and judge the end point of the reaction. If the resin is colorless and transparent, it means that the reaction is complete; if the resin develops color, the reaction is incomplete. You need to continue to react for another hour or re-dosing with a single dose or replace the condensation reagent. Ninhydrin detection is suitable for subsequent amino acid coupling reactions to determine the end point of the reaction. After coupling, without removing Fmoc, the dipeptide resin was shrink-dried directly, washed with DCM for 6 times, and drained to obtain Boc-Asn-Asp(OtBu)-2-chlorotrityl chloride resin, which was ready for use.

(2) Preparation of plecanatide S1-S2 fragment peptide

Weigh the above dried 20mmol 35.51g plecanatide S1-S2 fragment peptide peptide resin in 10% TFE DCM solution, the material volume ratio is 1g peptide resin 10ml solution. After the solution is mixed evenly, add the above peptide resin, react at room temperature for 2 hours, filter, concentrate the filtrate to 1/4 volume under reduced pressure, add 10 times the amount of ice isopropyl ether to settle for 1 hour, centrifuge, and wash with isopropyl ether for 4 times and dried to obtain 11.80 g of Boc-Asn-Asp(OtBu)-OH.

Embodiment 10: Preparation 1 of plecanatide peptide resin

Take the Fmoc-Leu-Wang resin 50.05g (10mmol) with a degree of substitution of 0.20mmol/g prepared in Example 1 of the present invention, add it to the solid-phase reaction column, wash once with DMF, and swell the Fmoc-Leu-Wang resin with DMF Resin after 30 minutes, with DMF: the mixed solution of piperidine volume ratio 4: 1 takes off Fmoc protection, washes 6 times with DMF then, weighs 12.37g Fmoc-Cys(Acm)-OH, 4.01g HOBt adds DMF solution to dissolve, After activation by adding 4.67ml DIC in an ice-water bath, add it to the above-mentioned reaction column equipped with resin. After reacting at room temperature for 2 hours, use ninhydrin to detect the end point of the reaction. If the resin is colorless and transparent, it means that the reaction is complete; the resin develops color , the reaction is not complete, and it is necessary to continue the reaction for another hour or re-dosing with a single dose or replace the condensation reagent. Ninhydrin detection is suitable for subsequent amino acid coupling reactions to determine the end point of the reaction. Repeat the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and insert 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)-OH prepared in Example 4 OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH and Fmoc-Asn(Trt)-OH, to obtain plecanatide 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 73.50 g. The volume shrinkage of the peptide resin did not occur during the coupling process, and the coupling was easy. The resin was colorless and transparent by the ninhydrin method for 1.5 hours of each reaction.

Embodiment 11: Preparation 2 of plecanatide peptide resin

Take the Fmoc-Leu-Wang resin 19.99g (10mmol) with a degree of substitution of 0.50mmol/g prepared in Example 2 of the present invention, add it to the solid phase reaction column, wash once with DMF, and swell the Fmoc-Leu-Wang with DMF After 30 minutes of the resin, remove Fmoc protection with a mixed solution of DMF:piperidine volume ratio 4:1, then wash 6 times with DMF, weigh 12.37g Fmoc-Cys(Trt)-OH, 4.01gHOBt, add DMF solution to dissolve, ice-water bath After adding 4.67ml of DIC to activate it, add it to the above-mentioned reaction column equipped with resin, react at room temperature for 2 hours, use ninhydrin to detect and judge the end of the reaction, if the resin is colorless and transparent, it means that the reaction is complete; if the resin develops color, the reaction is complete. Incomplete, you need to continue to react for another 1 hour or re-dosing with a single dose or replace the condensation reagent and re-dosing. Ninhydrin detection is suitable for subsequent amino acid coupling reactions to determine the end point of the reaction. Repeat the above steps of removing Fmoc protection and adding corresponding amino acid coupling, and insert 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(OtBu)-OH, and Fmoc-Asn(Trt)-Asp(OtBu)-OH that embodiment 8 makes, obtains 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. The volume shrinkage of the peptide resin did not occur during the coupling process, and the coupling was easy. The resin was colorless and transparent by the ninhydrin method for each reaction of 2 hours.

Embodiment 12: Preparation 3 of plecanatide peptide resin

Take the Fmoc-Leu-2-chlorotrityl chloride resin 33.33g (10mmol) with a degree of substitution 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, and use After DMF swells Fmoc-Leu-Wang resin for 30 minutes, remove the Fmoc protection with a mixed solution of DMF: piperidine volume ratio 4:1, then wash 6 times with DMF, weighing 12.37g Fmoc-Cys(Trt)-OH, 4.01gHOBt Add DMF solution to dissolve, add 4.67ml DIC under ice-water bath to activate, then add to the above-mentioned reaction column equipped with resin, react at room temperature for 2 hours, use ninhydrin to detect the end of the reaction, if the resin is colorless and transparent, it means the reaction is complete ; If the resin develops color, the reaction is not complete, and it is necessary to continue to react for another hour or re-dosing with a single amount or replace the condensation reagent and re-dosing. Ninhydrin detection is suitable for subsequent amino acid coupling reactions to determine the end point of the reaction. Repeat the above steps of removing Fmoc protection and adding corresponding amino acid coupling, and insert 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 produced in Example 7 -Asn-Asp(OtBu)-Glu(OtBu)-Cys(Acm)-OH, to obtain plecanatide 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, weighed It is 65.10g. The volume shrinkage of the peptide resin did not occur during the coupling process, and the coupling was easy. The resin was colorless and transparent by the ninhydrin method for 1.5 hours of each reaction.

Embodiment 13: Preparation of plecanatide peptide resin 4

Take the Fmoc-Leu-Wang resin 20.02g (10mmol) with a degree of substitution of 0.50mmol/g prepared in Example 2 of the present invention, add it to the solid-phase reaction column, wash once with DMF, and swell the Fmoc-Leu-Wang with DMF After 30 minutes of the resin, remove the Fmoc protection with a mixed solution of DMF:piperidine volume ratio 4:1, then wash 6 times with DMF, weigh 12.37g Fmoc-Cys(Acm)-OH, 4.01gHOBt, add DMF solution to dissolve, and ice-water bath After adding 4.67ml of DIC to activate it, add it to the above-mentioned reaction column equipped with resin, react at room temperature for 2 hours, use ninhydrin to detect and judge the end of the reaction, if the resin is colorless and transparent, it means that the reaction is complete; if the resin develops color, the reaction is complete. Incomplete, you need to continue to react for another 1 hour or re-dosing with a single dose or replace the condensation reagent and re-dosing. Ninhydrin detection is suitable for subsequent amino acid coupling reactions to determine the end point of the reaction. Repeat the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and insert 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)-OH prepared in Example 4 OH, Fmoc-Glu(OtBu)-OH and the Boc-Asn-Asp(OtBu)-OH that embodiment 9 makes, obtains plecanatide 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, called It weighs 46.55g. The volume shrinkage of the peptide resin did not occur during the coupling process, and the coupling was easy. The resin was colorless and transparent by the ninhydrin method for each reaction of 2 hours.

Embodiment 14: Preparation of plecanatide peptide resin 5

Take the Fmoc-Leu-Wang resin 20.03g (10mmol) with a degree of substitution of 0.50mmol/g prepared in Example 2 of the present invention, add it to the solid phase reaction column, wash once with DMF, and swell the Fmoc-Leu-Wang with DMF After 30 minutes of the resin, remove the Fmoc protection with a mixed solution of DMF:piperidine volume ratio 4:1, then wash 6 times with DMF, weigh 12.37g Fmoc-Cys(Acm)-OH, 4.01gHOBt, add DMF solution to dissolve, and ice-water bath After adding 4.67ml of DIC to activate it, add it to the above-mentioned reaction column equipped with resin, react at room temperature for 2 hours, use ninhydrin to detect and judge the end of the reaction, if the resin is colorless and transparent, it means that the reaction is complete; if the resin develops color, the reaction is complete. Incomplete, you need to continue to react for another 1 hour or re-dosing with a single dose or replace the condensation reagent and re-dosing. Ninhydrin detection is suitable for subsequent amino acid coupling reactions to determine the end point of the reaction. Repeat the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and insert Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Ala-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, the Fmoc-Glu(OtBu)-Leu-Cys(Acm)-Val-OH prepared in Example 4 and the Fmoc produced in Example 6 -Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(Trt)-OH, to obtain plecanatide 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, weighed as 46.60g. The volume shrinkage of the peptide resin did not occur during the coupling process, and the coupling was easy. The resin was colorless and transparent by the ninhydrin method for 1.5 hours of each reaction.

Embodiment 15: Preparation of plecanatide peptide resin 6

Take the Fmoc-Leu-Wang resin 19.99g (10mmol) with a degree of substitution of 0.50mmol/g prepared in Example 2 of the present invention, add it to the solid phase reaction column, wash once with DMF, and swell the Fmoc-Leu-Wang with DMF After 30 minutes of the resin, remove Fmoc protection with a mixed solution of DMF:piperidine volume ratio 4:1, then wash 6 times with DMF, weigh 12.37g Fmoc-Cys(Trt)-OH, 4.01gHOBt, add DMF solution to dissolve, ice-water bath After adding 4.67ml of DIC to activate it, add it to the above-mentioned reaction column equipped with resin, react at room temperature for 2 hours, use ninhydrin to detect and judge the end of the reaction, if the resin is colorless and transparent, it means that the reaction is complete; if the resin develops color, the reaction is complete. Incomplete, you need to continue to react for another 1 hour or re-dosing with a single dose or replace the condensation reagent and re-dosing. Ninhydrin detection is suitable for subsequent amino acid coupling reactions to determine the end point of the reaction. Repeat the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and insert 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(OtBu)-OH, and Fmoc-Asn(Trt)-Asp(OtBu)-OH that embodiment 8 makes, obtains 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. The volume shrinkage of the peptide resin did not occur during the coupling process, and the coupling was easy. The resin was colorless and transparent by the ninhydrin method for 1.5 hours of each reaction.

Example 16: Preparation 1 of linear plecanatide

Take 73.50 g of the plecanatide linear peptide resin prepared in Example 10 of the present invention, add it to a 1000 ml three-necked round-bottomed flask, configure 730 ml of lysate according to TFA:Tis:Mpr with a volume ratio of 94:3:3, and wait to mix evenly , add the above-mentioned peptide resin, react at room temperature for 2 hours, filter, add the filtrate to 8 times the amount of ice isopropyl ether to settle for 1 hour, centrifuge, wash 4 times with isopropyl ether, and dry to obtain plecanatide as a white solid 16.90 g of linear crude peptide with Acm protecting group at Cys (7, 15) position.

Example 17: Preparation of linear plecanatide 2

Take 46.70 g of the plecanatide linear peptide resin prepared in Example 11 of the present invention, and add it to a 1000 ml three-necked round-bottomed flask. TFA:Tis:Mpr:Phenol:H ₂ O to prepare 470ml of lysate, mix well, add the above peptide resin, react at room temperature for 2 hours, filter, add the filtrate to 9 times the amount of ice isopropyl ether to settle for 1 hour, centrifuge, wash with isopropyl ether for 4 times, After drying, 17.03 g of plecanatide linear crude peptide with a StBu protecting group at the Cys (4, 12) position was obtained as a white solid.

Example 18: Preparation of linear plecanatide 3

Take 65.10 g of the plecanatide linear peptide resin prepared in Example 12 of the present invention, add it to a 1000 ml three-necked round-bottomed flask, and prepare 650 ml of lysate according to TFA:Tis:H ₂ O with a volume ratio of 92:4:4. Mix evenly, add the above-mentioned peptide resin, react at room temperature for 2 hours, filter, add the filtrate to 10 times the amount of ice petroleum ether to settle for 1 hour, centrifuge, wash 4 times with petroleum ether, and dry to obtain plecanatide as a white solid 16.91 g of linear crude peptide with Acm protecting group at Cys (4, 12) position.

Example 19: Preparation of linear plecanatide 4

Take 46.55 g of the plecanatide linear peptide resin prepared in Example 13 of the present invention, add it to a 1000 ml three-necked round-bottomed flask, and prepare 460 ml of lysate according to TFA:Tis with a volume ratio of 95:5. After mixing evenly, add the above peptide The resin was reacted at room temperature for 2 hours, filtered, and the filtrate was added to 9 times the amount of glacial methyl tert-butyl ether to settle for 1 hour, centrifuged, washed with methyl tert-butyl ether for 4 times, and dried to obtain a white solid. 16.80 g of linear crude peptide with Acm protecting group at Cys (7, 15) position of that peptide.

Example 20: Preparation of linear plecanatide 5

Take 46.60 g of the plecanatide linear peptide resin prepared in Example 14 of the present invention, add it to a 1000 ml three-necked round-bottomed flask, and crack according to the configuration of TFA:Tis:Mpr:H ₂ O with a volume ratio of 94:2:2:2 Liquid 460ml, after mixing evenly, add the above peptide resin, react at room temperature for 2 hours, filter, add the filtrate to 8 times the amount of ice isopropyl ether to settle for 1 hour, centrifuge, wash 4 times with isopropyl ether, and dry to obtain a white solid 16.75 g of linear crude peptide with Acm protecting group at Cys (7, 15) position of plecanatide.

Example 21: Preparation of linear plecanatide 6

Take 44.50 g of the plecanatide linear peptide resin prepared in Example 15 of the present invention, and add it to a 1000 ml three-necked round-bottomed flask, TFA:Tis:Mpr:Phenol:H with a volume ratio of 90:3:3:3:1 ₂ O to prepare 450ml of lysate, mix well, add the above peptide resin, react at room temperature for 2 hours, filter, add the filtrate to 9 times the amount of ice isopropyl ether to settle for 1 hour, centrifuge, wash with isopropyl ether for 4 times, After drying, 15.93 g of plecanatide linear crude peptide with a tBu protecting group at the Cys (4, 12) position was obtained as a white solid.

Example 22: Preparation 1 of plecanatide cyclic peptide

Take 16.90 g of the plecanatide linear crude peptide prepared in Example 16 of the present invention, prepare the crude peptide into a 1 g/L aqueous solution, adjust the pH of the solution to alkaline with dilute ammonia water, and add a single amount of 10% hydrogen peroxide 400 μL, cyclization for 1.5 hours, to obtain the monocyclic peptide at the Cys (4, 12) position of plecanatide; adjust the pH of the monocyclic peptide solution to acidic with acetic acid, then add 1.90 g of iodine twice the amount, dissolve with ethanol, After the iodine is completely dissolved, it is added dropwise into the one-ring peptide solution, and after cyclization for 2 hours, the excess iodine residue is removed with Vc to realize the disulfide bond formation at Cys (7, 15) position of plecanatide, and finally obtain The crude peptide solution of two pairs of cyclized disulfide bonds of plecanatide was measured to have a purity of 67.88% and a yield of 65%. The chromatogram of plecanatide crude peptide is shown in Figure 1,

Example 23: Preparation 2 of plecanatide cyclic peptide

Take 17.03 g of the plecanatide linear crude peptide prepared in Example 17 of the present invention, prepare the crude peptide into a 1 g/L aqueous solution, adjust the pH of the solution to alkaline with dilute ammonia water, and add a single amount of 10% hydrogen peroxide 400 μL, cyclized for 1.5 hours, and freeze-dried to obtain a cyclic peptide at the Cys (7, 15) position of plecanatide; the cyclic peptide was mixed with 1 g/L 20% mercaptoethanol NMP, 0.1M N-methylmorpholine React at room temperature for 16 hours to remove the Cys side chain StBu protecting group, and use the same oxidation method to realize the disulfide bond formation at the Cys (4, 12) position of plecanatide, and finally obtain two pairs of cyclized disulfide bonds in plecanatide The crude peptide solution, the measured purity of the crude peptide is 65.58%, and the yield is 63%. The chromatogram of the crude peptide of plecanatide is similar to that in Figure 1.

Example 24: Preparation of plecanatide cyclic peptide 3

Take 16.78 g of the plecanatide linear crude peptide prepared in Example 18 of the present invention, prepare the crude peptide into a 1 g/L aqueous solution, adjust the pH of the solution to alkaline with dilute ammonia, add 1.69 mL of 10% DMSO, and After 4.0 hours, the monocyclic peptide at the Cys (7, 15) position of plecanatide was obtained; the pH of the monocyclic peptide solution was adjusted to acidity with acetic acid, and then 3.80 g of iodine was added in 5 times the amount, and dissolved in ethanol until the iodine was completely After dissolving, add it dropwise to a cyclic peptide solution, and after cyclization for 2 hours, use Vc to remove excess iodine residue to realize the disulfide bond formation at Cys (4, 12) position of plecanatide, and finally obtain plecanatide The crude peptide solution of two pairs of cyclized disulfide bonds has a purity of 66.05% and a yield of 64%. The chromatogram of the crude peptide of plecanatide is similar to that in Figure 1.

Example 25: Preparation of plecanatide cyclic peptide 4

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

Example 26: Preparation of plecanatide cyclic peptide 5

Take 16.75 g of the crude peptide of plecanatide linear peptide prepared in Example 20 of the present invention, prepare the crude peptide into a 1 g/L aqueous solution, adjust the pH of the solution to alkaline with dilute ammonia water, and add a single amount of 20% of Hydrogen peroxide 200 μL, cyclization for 1.5 hours, to obtain the monocyclic peptide at the Cys (4, 12) position of plecanatide; adjust the pH of the monocyclic peptide solution to acidic with acetic acid, then add 3.5 times the amount of iodine 1.90 g, dissolve with ethanol , after the iodine is completely dissolved, it is added dropwise into a cyclic peptide solution, and after cyclization for 2 hours, the excess iodine residue is removed with Vc, and the disulfide bond at the Cys (7, 15) position of plecanatide is formed into a ring, and finally A crude peptide solution of two pairs of cyclized disulfide bonds of plecanatide was obtained, the purity of the crude peptide was measured to be 66.25%, and the yield was 65%. The chromatogram of the crude peptide of plecanatide is similar to that in Figure 1.

Example 27: Preparation of plecanatide cyclic peptide 6

Take 15.93 g of the plecanatide linear crude peptide prepared in Example 21 of the present invention, prepare the crude peptide into a 1 g/L aqueous solution, adjust the pH of the solution to alkaline with dilute ammonia water, and add a single amount of 10% hydrogen peroxide 400 μL, cyclized for 1.5 hours, freeze-dried to obtain a cyclic peptide at the Cys (7, 15) position of plecanatide; dissolve the cyclic peptide with 1 g/L TFA, 10 times the equivalent of diphenylsulfoxide, 100 times Equivalent trichloromethylsilane, 100 times the equivalent of anisole, reacted for 30 minutes, removed the Cys side chain tBu protecting group, and used the same oxidation method to realize the disulfide bond formation at the Cys (4, 12) position of plecanatide ring, and finally obtain a crude peptide solution of two pairs of cyclized disulfide bonds of plecanatide. The purity of the crude peptide was measured to be 65.85%, and the yield was 64%. The chromatogram of the crude peptide of plecanatide is similar to that in Figure 1.

Example 28: Purification of plecanatide crude peptide 1

Take the plecanatide crude peptide solution prepared in Example 22 of the present invention, and use RP-PLC to prepare a liquid phase system with a wavelength of 220nm. The chromatographic column is a reversed-phase C18 column, and the volume ratio TEAP pH6.8/acetonitrile is the mobile phase, and the ratio for 75:25. Purified by RP-PLC, converted to salt, collected target peptide fractions, concentrated by rotary evaporation, and freeze-dried to obtain pure plecanatide. The purity of pure plecanatide was detected to be 99.7%, and the total yield could reach 48%. The chromatogram of the pure plecanatide prepared by the present invention is shown in Figure 2.

Example 29: Purification of plecanatide crude peptide 2

Take the plecanatide crude peptide solution prepared in Example 23 of the present invention, and use RP-PLC to prepare a liquid phase system with a wavelength of 220nm. The chromatographic column is a reversed-phase C18 column, and the volume ratio TEAP pH6.8/acetonitrile is the mobile phase, and the ratio for 75:25. Purified by RP-PLC, converted to salt, collected target peptide fractions, concentrated by rotary evaporation, and freeze-dried to obtain pure plecanatide. The purity of pure plecanatide was detected to be 99.5%, and the total yield could reach 48%. The chromatogram of the pure plecanatide prepared by the present invention is similar to that shown in Figure 2.

Example 30: Purification of plecanatide crude peptide 3

Take the plecanatide crude peptide solution prepared in Example 24 of the present invention, and use RP-PLC to prepare a liquid phase system with a wavelength of 220nm. The chromatographic column is a reversed-phase C18 column, and the volume ratio TEAP pH6.8/acetonitrile is the mobile phase, and the ratio for 75:25. Purified by RP-PLC, converted to salt, collected target peptide fractions, concentrated by rotary evaporation, and freeze-dried to obtain pure plecanatide. The purity of pure plecanatide was detected to be 99.6%, and the total yield could reach 47%. The chromatogram of the pure plecanatide prepared by the present invention is similar to that shown in Figure 2.

Example 31: Purification of plecanatide crude peptide 4

Take the plecanatide crude peptide solution prepared in Example 25 of the present invention, and use RP-PLC to prepare a liquid phase system with a wavelength of 220nm. The chromatographic column is a reversed-phase C18 column, and the volume ratio TEAP pH6.8/acetonitrile is the mobile phase, and the ratio for 75:25. Purified by RP-PLC, converted to salt, collected target peptide fractions, concentrated by rotary evaporation, and freeze-dried to obtain pure plecanatide. The purity of pure plecanatide was detected to be 99.5%, and the total yield could reach 48%. The chromatogram of the pure plecanatide prepared by the present invention is similar to that shown in Figure 2.

Example 32: Purification of Plecanatide Crude Peptide 5

Take the plecanatide crude peptide solution prepared in Example 26 of the present invention, and use RP-PLC to prepare a liquid phase system with a wavelength of 220nm. The chromatographic column is a reversed-phase C18 column, and the volume ratio TEAP pH6.8/acetonitrile is the mobile phase, and the ratio for 75:25. Purified by RP-PLC, converted to salt, collected target peptide fractions, concentrated by rotary evaporation, and freeze-dried to obtain pure plecanatide. The purity of pure plecanatide was detected to be 99.5%, and the total yield could reach 48%. The chromatogram of the pure plecanatide prepared by the present invention is similar to that shown in Figure 2.

Example 33: Purification of Plecanatide Crude Peptide 6

Take the plecanatide crude peptide solution prepared in Example 27 of the present invention, and use RP-PLC to prepare a liquid phase system with a wavelength of 220nm. The chromatographic column is a reversed-phase C18 column, and the volume ratio TEAP pH6.8/acetonitrile is the mobile phase, and the ratio for 75:25. Purified by RP-PLC, converted to salt, collected target peptide fractions, concentrated by rotary evaporation, and freeze-dried to obtain pure plecanatide. The purity of pure plecanatide was detected to be 99.5%, and the total yield could reach 47%. The chromatogram of the pure plecanatide prepared by the present invention is similar to that shown in Figure 2.

It is predicted that the molecular weight of plecanatide is 1681.88. According to mass spectrometry, the molecular weight of all plecanatide prepared in the present invention is 1681.69, which can meet expectations, which proves that the present invention has successfully prepared plecanatide.

Comparative example 1: Preparation of plecanatide peptide resin

Take the Fmoc-Leu-Wang resin 50.08g (10mmol) with a degree of substitution of 0.20mmol/g prepared in Example 1 of the present invention, add it to the solid phase reaction column, wash once with DMF, and swell the Fmoc-Leu-Wang with DMF After 30 minutes of the resin, remove Fmoc protection with a mixed solution of DMF:piperidine volume ratio 4:1, then wash 6 times with DMF, weigh 12.45g Fmoc-Cys(Acm)-OH, 4.05g HOBt and add DMF solution to dissolve, After activation by adding 4.67ml DIC in an ice-water bath, add it to the above-mentioned reaction column equipped with resin. After reacting at room temperature for 2 hours, use ninhydrin to detect the end point of the reaction. If the resin is colorless and transparent, it means that the reaction is complete; the resin color develops , the reaction is not complete, and it is necessary to continue the reaction for another hour or re-dosing with a single dose or replace the condensation reagent. Ninhydrin detection is suitable for subsequent amino acid coupling reactions to determine the end point of the reaction. Repeat the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and 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-Asn(Trt)-OH to obtain the plecanatide 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 70.50g, during the linear peptide coupling process, the volume of the peptide resin shrinks significantly when coupling S9-S1 residues, and the coupling is difficult when coupling S7 residues, and the reaction takes 2.5h The resin was light blue as detected by the ninhydrin method, and there was no obvious improvement by re-introduction.

Comparative Example 2: Preparation of linear plecanatide

Take 70.50 g of the plecanatide linear peptide resin prepared in Comparative Example 1, add it to a 1000 ml three-necked round-bottomed flask, prepare 730 ml of lysate according to TFA:Tis:Mpr with a volume ratio of 94:3:3, and mix well, add The above-mentioned peptide resin was reacted at room temperature for 2 hours, filtered, and the filtrate was added to 8 times the amount of ice isopropyl ether to settle for 1 hour, centrifuged, washed 4 times with isopropyl ether, and dried to obtain plecanatide Cys ( 7, 15) 14.91 g of linear crude peptide with Acm protecting group at position.

Comparative Example 3: Preparation of plecanatide cyclic peptide

Take 14.91 g of the plecanatide linear crude peptide prepared in Comparative Example 2, and prepare the crude peptide into a 1 g/L aqueous solution. In the 6 L solution, adjust the pH of the solution to alkaline with dilute ammonia water, and add a single amount of 10% 400 μL of hydrogen peroxide, cyclized for 1.5 hours, to obtain a cyclic peptide at the Cys (4, 12) position of plecanatide; adjust the pH of the cyclic peptide solution to acidity with acetic acid, then add 1.90 g of iodine twice the amount, and use ethanol to Dissolve, after the iodine is completely dissolved, add it dropwise into a cyclic peptide solution, and after cyclization for 2 hours, use Vc to remove excess iodine residues to realize the disulfide bond formation at Cys (7, 15) position of plecanatide, Finally, a crude peptide solution of two pairs of cyclized disulfide bonds of plecanatide was obtained, the purity of the crude peptide was 63.46%, and the yield was 55%. The crude peptide chromatogram of plecanatide is shown in Figure 3.

Comparative Example 4: Purification of crude peptide of plecanatide

Get the crude peptide solution of plecanatide prepared in Comparative Example 3, and use RP-PLC to prepare a liquid phase system with a wavelength of 220nm. The chromatographic column is a reversed-phase C18 column, and the volume ratio TEAP pH 6.8/acetonitrile is the mobile phase, and the ratio is 75: 25. Purified by RP-PLC, converted to salt, collected target peptide fractions, concentrated by rotary evaporation, and freeze-dried to obtain pure plecanatide. The purity of the pure product was 97.12% and the total yield was 39%. The chromatogram of pure plecanatide is shown in Figure 4.

Claims (9)

1. a fragment synthesis method of plecanatide, is characterized in that, mainly comprises the following steps: (1) On the Fmoc-Leu-resin, a linear plecanatide resin is obtained by solid-phase synthesis according to the plecanatide peptide sequence; wherein, S5-S8 adopts tetrapeptide fragments, and the rest adopt corresponding protected amino acids or fragments; (2) cracking the linear plecanatide resin obtained in step (1) to obtain linear plecanatide; (3) Cyclizing the linear plecanatide obtained in step (2) to obtain crude plecanatide peptide. 2. The fragment synthesis method of plecanatide according to claim 1, characterized in that: the linear plecanatide resin obtained in the step (1) is: R ₁ -Asn(R ₂ )-Asp(OtBu )-Glu(OtBu)-Cys(R ₃ )-Glu(OtBu)-Leu-Cys(R ₄ )-Val-Asn(Trt)-Val-Ala-Cys(R ₃ )-Thr(tBu)-Gly- Cys(R ₄ )-Leu-resin; in: R _is selected from Boc, Fmoc, Z; R ₂ is selected from Trt, H; _R3 and _R4 are selected from Trt, Acm, StBu or tBu, and _R3 and _R4 are different. 3. the fragment synthesis method of plecanatide according to claim 1, is characterized in that: the tetrapeptide fragment that described step (1) S5-S8 adopts is Fmoc-Glu(OtBu)-Leu-Cys(R ₄ )-Val-OH; Wherein, R ₄ is selected from Trt, Acm. 4. The fragment synthesis method of plecanatide according to claim 1, characterized in that: the remaining corresponding fragments used in the step (1) include S1-S2 or S1-S4. 5. The fragment synthesis method of plecanatide according to claim 4, characterized in that: the S1-S2 fragment is R ₁ -Asn(R ₂ )-Asp(OtBu)-OH, and the S1-S4 fragment is R ₁ -Asn(R ₂ )-Asp(OtBu)-Glu(OtBu)-Cys(R ₃ )-OH; in: R ₁ is selected from Fmoc, Boc; R ₃ is selected from Trt, Acm. 6. The fragment synthesis method of plecanatide according to claim 1, characterized in that: in the step (1), the resin is selected from Wang resin or 2-chlorotrityl chloride resin. 7. The fragment synthesis method of plecanatide according to claim 1, characterized in that: the step (1) Fmoc-Leu-resin substitution range is 0.2-0.5mmol/g. 8. the fragment synthesis method of plecanatide according to claim 1 is characterized in that: in the described step (2) cracking step, cracking reagent is selected from trifluoroacetic acid and other components, and other components are selected from water, Phenol, 3-mercaptopropionic acid, and triisopropylsilane, among which trifluoroacetic acid accounts for more than 90%, and other components each account for 1%-5%. 9. The fragment synthesis method of plecanatide according to claim 1, is characterized in that: the crude peptide of plecanatide obtained in the step (3) can be further obtained through reverse-phase high performance liquid chromatography purification and freeze-drying Plecanatide Refined Peptide.

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