CN114195855A

CN114195855A - Solid-phase synthesis method for polypeptide containing three pairs of disulfide bonds by introducing one pair of disulfide bonds

Info

Publication number: CN114195855A
Application number: CN202010980791.4A
Authority: CN
Inventors: 邹卫星; 尹传龙; 唐洋明; 余品香
Original assignee: Hybio Pharmaceutical Co Ltd
Current assignee: Hybio Pharmaceutical Co Ltd
Priority date: 2020-09-17
Filing date: 2020-09-17
Publication date: 2022-03-18

Abstract

The invention relates to a solid phase synthesis method for polypeptide containing three pairs of disulfide bonds by introducing a pair of disulfide bonds, and particularly discloses a method comprising the following steps: 1) synthesizing a conjugate of a first polypeptide fragment and a solid-phase synthetic resin by a solid-phase synthesis method, wherein the first polypeptide fragment comprises two cysteines forming a first pair of disulfide bonds; 2) removing thiol protecting groups of two cysteines of a first pair of disulfide bonds in the first polypeptide fragment, and oxidizing to form a first pair of disulfide bonds; 3) continuously synthesizing the residual fragments by a solid-phase synthesis method; 4) removing the sulfhydryl protecting group of cysteine of the third pair of disulfide bonds to form a third pair of disulfide bonds, and then removing the cysteine sulfhydryl protecting group of the second pair of disulfide bonds to form a second pair of disulfide bonds; 5) removing other protecting groups, and cracking the resin to obtain the polypeptide containing three pairs of disulfide bonds. The method of the invention can ensure that three pairs of polypeptides are selectively deprotected, thus greatly reducing the possibility of mismatching.

Description

Solid-phase synthesis method for polypeptide containing three pairs of disulfide bonds by introducing one pair of disulfide bonds

Technical Field

The invention belongs to the field of polypeptide synthesis, and particularly relates to a solid-phase synthesis method for polypeptides containing three pairs of disulfide bonds by introducing a pair of disulfide bonds.

Background

The protein and polypeptide drugs have the advantages of specific action sites, definite curative effect and the like, and in recent years, the research and development of the protein and polypeptide drugs have become a hotspot of the research in the field of biological medicine.

Disulfide bonds are S-S covalent bonds formed by oxidation of sulfhydryl (-SH) groups of Cys located at different peptide chains or at two different sites of the same peptide chain in a protein or polypeptide molecule. It can fold peptide chain into specific space structure, and plays an important role in maintaining space stereo structure of polypeptide and protein and biological activity determined thereby. The greater the number of disulfide bonds, the greater the stability of the protein molecule against the influence of external factors.

Among the polypeptide drugs currently on the market, there are several polypeptide drugs containing multiple disulfide bond pairs such as: procatide, ziconotide, linaclotide, and the like. Because of the abundance of multiple Cys residues, multiple isomers may form due to differences in disulfide bonding sites, e.g., 15 different combinations of disulfide bonds in a polypeptide containing 3 pairs of disulfide bonds. There are 14 different disulfide mismatch isoforms in addition to the drug itself. During drug development, the study of these disulfide mismatch isomer impurities is an indelible part of the quality studies of drug substances or preparations. However, how to synthesize the compound has been a troublesome problem.

Linaclotide is taken as an example. Linaclotide (Linaclotide) is a novel GC-C (uridylate cyclase C) receptor agonist. GC-C receptors on the apical surface of intestinal epithelial cells can be activated, resulting in an increase in intracellular and extracellular cyclic guanylic acid. It is used for treating adult slow transit constipation and constipation-predominant irritable bowel syndrome (IBS-C) patients by increasing the secretion of chlorine and bicarbonate into the intestinal lumen, which in turn results in increased fluid secretion and accelerated stool passage. It is composed of 14L-type amino acid residues, wherein 6 Cys residues are respectively connected by disulfide bonds. The structural sequence is：NH₂-Cys¹-Cys²-Glu-Tyr-Cys⁵-Cys⁶-Asn-Pro-Ala-Cys¹⁰-Thr-Gly-Cys¹³-Tyr-COOH (wherein Cys¹、Cys⁶，Cys²、Cys¹⁰And Cys⁵、Cys¹³Each linked by a disulfide bond).

Since linaclotide contains 6 cysteine residues, which are paired two by two to form 3 pairs of disulfide bonds, there are 14 other mismatched isoforms of disulfide bonds (see Table 1: its isoforms are designated as random IM-A, B … ….) in addition to linaclotide, due to the different disulfide bond binding sites.

Table 1: linaclotide and list of its disulfide mismatch isoforms

Name of	Peptide sequence of impurity
		Linaclotide	H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH(1-6，2-10，5-13)
IM-A	H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH(1-2，5-6，10-13)
		IM-B	H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH(1-5，2-6，10-13)
IM-C	H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH(1-6，2-5，10-13)
		IM-D	H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH(1-10，2-13，5-6)
IM-E	H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH(1-13，2-10，5-6)
		IM-F	H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH(1-2，5-13，6-10)
IM-G	H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH(1-2，5-10，6-13)
		IM-H	H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH(1-13，2-5，6-10)
IM-I	H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH(1-5，6-10，2-13)
		IM-J	H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH(1-10，2-5，6-13)
IM-K	H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH(1-5，2-10，6-13)
		IM-L	H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH(1-6，2-13，5-10)
IM-M	H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH(1-13，2-6，5-10)
		IM-N	H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH(1-10，2-6，5-13)

Benitez et al made a related report in 2010 (see Peptide Science,2011,96: 69-80). In this document, three methods were used to experimentally synthesize linaclotide.

The method comprises the following steps: all Cys residues adopt Trt or Mmt as side chain protecting groups of Cys, the synthesis of linear peptide is completed by Fmoc/tBu solid phase synthesis strategy, crude peptide is obtained by cracking, and then the target peptide is obtained by one-step oxidation by a liquid phase oxidation method.

The second method comprises the following steps: and (3) adopting Trt or Mmt and Acm as a Cys side chain protecting group (4Trt +2Acm or 4Acm +2Trt) for the sulfydryl of the Cys residue, obtaining a linear peptide with partial protection by using an Fmoc/tBu solid phase synthesis strategy, and then completing the synthesis of the disulfide bond by using a half-selection strategy.

The third method comprises the following steps: orthogonal protection is carried out by respectively adopting 3 different Cys protection strategies, such as three complete selectivity strategies of [2Mmt +2Acm +2Trt ], [2Acm +2Trt +2pMeOBzl ], [2StBu +2Trt +2pMeOBzl ] and the like to carry out the positioning synthesis of disulfide bonds.

For the synthesis of mismatched isomers of linaclotide, the above method is unsuitable. Although the synthesis procedure is simple, only one protecting group for Cys is used. Random oxidation, however, may yield many different isomers, but the disulfide bond formation of these isomers cannot be determined.

The second method is a semi-selective localized oxidation method, a pair of disulfide bonds is firstly localized and oxidized, and the other 4 Cys residues are freely combined into 3 possible isomers, the number of different formed isomers is reduced to 3, and theoretically, the isomers can be obtained by designing and positioning different disulfide bonds and then are compared and confirmed, but firstly, the workload is huge, and the accuracy of the disulfide bond structure of the mismatched isomer obtained by the second method cannot be judged.

The method for forming 3 pairs of disulfide bonds by completely orthogonal protection of Cys sulfydryl in the method III is a feasible strategy theoretically, but in actual operation, the conditions of poor orthogonality of protecting groups, incomplete removal of the protecting groups and the like exist, so that the conditions of multiple peaks, low yield and even incapability of distinguishing and identifying target products during synthesis occur.

Disclosure of Invention

In view of the above situation, the present invention provides a method for synthesizing mismatched isomers containing 3 pairs of disulfide bonds by using Fmoc/tBu solid phase synthesis strategy: a pair of disulfide bonds (two Cys which form the disulfide bond are adjacent or separated by one Cys residue) in a sequence to be synthesized is constructed firstly by an Fmoc/tBu solid phase method, and then a peptide fragment containing the disulfide bonds is integrally introduced into the synthesis of a peptide sequence by a fragment condensation method. And the other two pairs of disulfide bonds adopt Mmt or Trt and Acm as Cys side chain protecting groups, and the synthesis of mismatching isomers of 14 disulfide bonds is changed from 3 disulfide bonds to 2 disulfide bonds by the method, so that the technical difficulty of synthesis and purification is greatly reduced, the process yield is improved, and the disulfide bond structure of the mismatching isomers of the disulfide bonds is clear.

One aspect of the present invention provides a method for synthesizing a polypeptide having 3 disulfide bonds,

wherein the 3 disulfide bond-containing polypeptide comprises a first pair of disulfide bonds, a second pair of disulfide bonds and a third pair of disulfide bonds, at least one of the 3 disulfide bonds is formed by two adjacent cysteines, or at least one pair of disulfide bonds is formed by two cysteines separated by one cysteine;

the synthesis method comprises the following steps:

1) synthesizing a conjugate of a first polypeptide fragment and a solid-phase synthetic resin by a solid-phase synthesis method, wherein the first polypeptide fragment comprises two cysteines forming a first pair of disulfide bonds, one cysteine forming a second pair of disulfide bonds is arranged between the two cysteines forming the first pair of disulfide bonds, or no cysteine is arranged between the two cysteines forming the first pair of disulfide bonds; the thiol groups of the two cysteines forming the first pair of disulfide bonds are protected with thiol protecting groups, Trt or Mmt; one of the cysteines forming the second pair of disulfide bonds is protected with a thiol protecting group, Acm;

2) removing thiol protecting groups of two cysteines of a first pair of disulfide bonds in the first polypeptide fragment, and oxidizing to form a first pair of disulfide bonds;

3) under the solid phase condition, continuously synthesizing the residual fragment by a solid phase synthesis method, wherein the residual fragment contains cysteine for forming a second pair of disulfide bonds and cysteine for forming a third pair of disulfide bonds, and the sulfydryl of the cysteine for forming the second pair of disulfide bonds and the sulfydryl of the cysteine for forming the third pair of disulfide bonds are protected by sulfydryl protecting groups; the thiol protecting groups of the two cysteines forming the second pair of disulfide bonds are the same and are Acm; the thiol protecting groups of the cysteines forming the third pair of disulfide bonds are the same and are Trt or Mmt;

4) removing the sulfhydryl protecting group of cysteine of the second pair of disulfide bonds to form a second pair of disulfide bonds, and then removing the cysteine sulfhydryl protecting group of a third pair of disulfide bonds to form a third pair of disulfide bonds; or

Removing the sulfhydryl protecting group of cysteine of the third pair of disulfide bonds to form a third pair of disulfide bonds, and then removing the cysteine sulfhydryl protecting group of the second pair of disulfide bonds to form a second pair of disulfide bonds;

5) removing other protecting groups, and cracking the resin to obtain the polypeptide containing three pairs of disulfide bonds.

In a first aspect, a specific embodiment of the invention provides a method for synthesizing a polypeptide having 3 disulfide bonds,

wherein the 3 disulfide bond-containing polypeptide comprises a first pair of disulfide bonds, a second pair of disulfide bonds and a third pair of disulfide bonds, the first pair of disulfide bonds of the 3 disulfide bonds being formed by two adjacent cysteines, and the first pair of disulfide bonds being closer to the C-terminus than the second and third pairs of disulfide bonds;

the synthesis method comprises the following steps:

1) synthesizing a conjugate of a first polypeptide fragment and a solid-phase synthetic resin by a solid-phase synthesis method, wherein the first polypeptide fragment comprises two cysteines forming a first pair of disulfide bonds, and no cysteine is contained between the two cysteines forming the first pair of disulfide bonds; the thiol groups of the two cysteines forming the first pair of disulfide bonds are protected with thiol protecting groups, Trt or Mmt;

3) under the solid phase condition, continuing solid phase coupling the remaining fragment at the N end of the first polypeptide fragment, wherein the remaining fragment comprises cysteine forming a second pair of disulfide bonds and cysteine forming a third pair of disulfide bonds, and the sulfydryl of the cysteine forming the second pair of disulfide bonds and the cysteine forming the third pair of disulfide bonds is protected by a sulfydryl protecting group; the thiol protecting groups of the two cysteines forming the second pair of disulfide bonds are the same and are Acm; the thiol protecting groups of the cysteines forming the third pair of disulfide bonds are the same and are Trt or Mmt;

4) removing the sulfhydryl protecting group of cysteine of the third pair of disulfide bonds to form a third pair of disulfide bonds, and then removing the cysteine sulfhydryl protecting group of the second pair of disulfide bonds to form a second pair of disulfide bonds;

In a second embodiment of the present invention, there is provided a method for synthesizing a polypeptide having 3 disulfide bonds,

wherein the 3-disulfide bond-containing polypeptide comprises a first pair of disulfide bonds, a second pair of disulfide bonds and a third pair of disulfide bonds, the first pair of disulfide bonds being formed by two cysteines separated by one cysteine, and the first pair of disulfide bonds being closer to the C-terminus than the second and third pairs of disulfide bonds;

the synthesis method comprises the following steps:

1) synthesizing a conjugate of a first polypeptide fragment and a solid-phase synthetic resin by a solid-phase synthesis method, wherein the first polypeptide fragment comprises two cysteines forming a first pair of disulfide bonds, and one cysteine forming a second pair of disulfide bonds is arranged between the two cysteines forming the first pair of disulfide bonds; the thiol groups of the two cysteines forming the first pair of disulfide bonds are protected with thiol protecting groups, Trt or Mmt; one of the cysteines forming the second pair of disulfide bonds is protected with a thiol protecting group, Acm;

3) under the solid phase condition, continuing solid phase coupling the remaining fragment at the N end of the first polypeptide fragment, wherein the remaining fragment comprises one cysteine forming a second pair of disulfide bonds and two cysteines forming a third pair of disulfide bonds, and the sulfydryl of the cysteine forming the second pair of disulfide bonds and the cysteine forming the third pair of disulfide bonds are protected by sulfydryl protecting groups; the thiol protecting groups of the two cysteines forming the second pair of disulfide bonds are the same and are Acm; the thiol protecting groups of the cysteines forming the third pair of disulfide bonds are the same and are Trt or Mmt;

The third embodiment of the present invention provides a method for synthesizing a polypeptide containing 3 disulfide bonds,

wherein the polypeptide containing 3 pairs of disulfide bonds comprises a first pair of disulfide bonds, a second pair of disulfide bonds and a third pair of disulfide bonds, the first pair of disulfide bonds of the 3 pairs of disulfide bonds are formed by two adjacent cysteines, and any one or more cysteines forming the second and third pairs of disulfide bonds are closer to the C-terminus than both cysteines forming the first pair of disulfide bonds;

the synthesis method comprises the following steps:

1) synthesizing a conjugate of a first polypeptide fragment and a solid-phase synthetic resin by a solid-phase synthesis method, wherein the first polypeptide fragment comprises two cysteines forming a first pair of disulfide bonds, and no cysteine is contained between the two cysteines forming the first pair of disulfide bonds; the thiol groups of the two cysteines forming the first pair of disulfide bonds are protected with thiol protecting groups, Trt or Mmt; one of the cysteines forming the second pair of disulfide bonds is protected with a thiol protecting group, Acm;

3) under the solid phase condition, synthesizing the rest fragment by a solid phase synthesis method, wherein the rest fragment comprises cysteine forming a second pair of disulfide bonds and cysteine forming a third pair of disulfide bonds, and the sulfydryl of the cysteine forming the second pair of disulfide bonds and the cysteine forming the third pair of disulfide bonds are protected by a sulfydryl protecting group; the thiol protecting groups of the two cysteines forming the second pair of disulfide bonds are the same and are Acm; the thiol protecting groups of the cysteines forming the third pair of disulfide bonds are the same and are Trt or Mmt; the remaining fragment is present at the N-terminus or C-terminus of the first polypeptide fragment, or a portion of the remaining fragment is present at the N-terminus and another portion is present at the C-terminus of the first polypeptide fragment, forming a polypeptide-resin conjugate;

5) removing other protecting groups, and cracking the resin to obtain polypeptide containing three pairs of disulfide bonds;

preferably, the solid phase synthetic resin used in step 1) is 2-CTC solid phase synthetic resin, and the solid phase synthetic resin for synthesizing the remaining fragments in step 3) is Wang solid phase synthetic resin.

In a fourth embodiment of the invention, there is provided a method of synthesizing a polypeptide having 3 disulfide bonds,

wherein the 3-disulfide bond-containing polypeptide comprises a first pair of disulfide bonds, a second pair of disulfide bonds and a third pair of disulfide bonds, the first pair of disulfide bonds being formed by two cysteines separated by one cysteine, and any one or more cysteines forming the second and third pairs of disulfide bonds being closer to the C-terminus than both cysteines forming the first pair of disulfide bonds;

the synthesis method comprises the following steps:

3) under the solid phase condition, continuously synthesizing the residual fragment by a solid phase synthesis method, wherein the residual fragment contains cysteine for forming a second pair of disulfide bonds and cysteine for forming a third pair of disulfide bonds, and the sulfydryl of the cysteine for forming the second pair of disulfide bonds and the sulfydryl of the cysteine for forming the third pair of disulfide bonds are protected by sulfydryl protecting groups; the thiol protecting groups of the two cysteines forming the second pair of disulfide bonds are the same and are Acm; the thiol protecting groups of the cysteines forming the third pair of disulfide bonds are the same and are Trt or Mmt; the remaining fragment is present at the N-terminus or C-terminus of the first polypeptide fragment, or a portion of the remaining fragment is present at the N-terminus and another portion is present at the C-terminus of the first polypeptide fragment, forming a polypeptide-resin conjugate;

preferably, the solid-phase synthetic resin is removed while the thiol protecting group is removed in step 2);

In the technical scheme of the invention, the two adjacent cysteines means that no other cysteine exists between the two adjacent cysteines, but other amino acids are contained or no other amino acids are contained.

In the technical scheme of the invention, the two cysteines separated by one cysteine means that one cysteine exists between the two cysteines forming the disulfide bond, and in addition, other amino acids or no other amino acids can be contained between the two cysteines forming the disulfide bond.

In the technical scheme of the invention, the solid phase synthesis method in the step 1) is any method for coupling amino acid or peptide fragments on solid phase synthesis resin, for example, the amino acid or peptide fragments of Fmoc protected alpha amino group are coupled on the solid phase synthesis resin, the Fmoc protecting group is removed, and then the amino acid or peptide fragments of Fmoc protected alpha amino group are coupled and sequentially coupled by the method of removing the Fmoc protecting group.

In the technical scheme of the invention, the N end of the conjugate of the first polypeptide fragment and the solid-phase synthetic resin in the step 1) is provided with an Fmoc protecting group.

In the technical scheme of the invention, in the step 1), two sides of two cysteines forming the first pair of disulfide bonds in the first polypeptide fragment respectively comprise 0-3 amino acids, and do not comprise cysteines, and preferably 0, 1 or 2 amino acids are adopted.

In the technical scheme of the invention, in the step 2), the method for removing the sulfhydryl protecting groups of the two cysteines forming the first pair of disulfide bonds and oxidizing to form the disulfide bonds comprises the following steps: the first polypeptide fragment only contains two cysteines forming a first pair of disulfide bonds, 1-20 equivalents of iodine is dissolved in a proper amount of DMF, and the thiol protecting groups are removed and the disulfide bonds are formed under room temperature conditions;

or the first polypeptide fragment comprises two cysteines forming a first pair of disulfide bonds and one cysteine forming a second pair of disulfide bonds, the two cysteine sulfhydryl protecting groups of the first pair of disulfide bonds are removed by TFA, TIS, DCM (1-5), (2-10), (97-85), and then 1-30 equivalents of H are used₂O₂Oxidation to the first pair of disulfide bonds is performed.

In the technical scheme of the invention, the first polypeptide fragment comprises two cysteines or three cysteines.

In the technical scheme of the invention, in the step 3), the method for continuously synthesizing the residual fragment under the solid-phase condition is to synthesize by adopting a solid-phase synthesis method to form a conjugate of the polypeptide fragment and the solid-phase synthesis resin, wherein the conjugate of the polypeptide fragment and the solid-phase synthesis resin contains 6 cysteines, and 2 cysteines form a disulfide bond; synthetic methods include, but are not limited to, adding amino acids to the N-terminus of the polypeptide fragments, synthesizing the polypeptide fragments in solid phase synthesis, and coupling the polypeptide fragments, or coupling single added amino acids to the fragments for conjugation. Preferably, the following are included but not limited to:

a. continuing solid phase coupling of the remaining amino acids at the N-terminus of the first polypeptide fragment;

b. synthesizing a conjugate of a second polypeptide fragment and solid-phase synthetic resin under a solid-phase condition, cracking the solid-phase synthetic resin on one of the first polypeptide fragment and the second polypeptide fragment, and coupling the first polypeptide fragment and the second polypeptide fragment to obtain a conjugate of a third polypeptide fragment and the solid-phase synthetic resin;

c. synthesizing a conjugate of a second polypeptide fragment and solid-phase synthetic resin under a solid-phase condition, continuously coupling amino acids at the N end of the first polypeptide fragment and/or the N end of the second polypeptide fragment, and then coupling the first polypeptide fragment or the second polypeptide fragment added with the amino acids to obtain a conjugate of a fourth polypeptide fragment and the solid-phase synthetic resin;

d. and (c) obtaining the conjugate of the polypeptide fragment and the solid-phase synthetic resin in three conditions of a, b and c, and continuing to couple the amino acid.

In the technical scheme of the invention, the method for removing the sulfhydryl protecting group of the cysteine and oxidizing to form the disulfide bond in the step 4) comprises the steps of removing two cysteine sulfhydryl protecting groups Trt or Mmt of the disulfide bond by TFA, TIS, DCM (1-5), DCM (2-10), 97-85 and then using 1-30 equivalents of H₂O₂The DMF solution is firstly subjected to solid phase oxidation at room temperature to form a pair of disulfide bonds; then dissolving 1-20 equivalents of iodine in appropriate amount of DMF, and removing the sulfhydryl protecting group Acm and forming disulfide bond at room temperature.

In a preferred embodiment of the present invention, in step 1), the number of the cysteines contained in the first polypeptide fragment is two, the cysteines form a first pair of disulfide bonds, the thiol-protecting group of the cysteine forming the first pair of disulfide bonds is any one of Mmt, Trt or Acm, the thiol-protecting group of the cysteine forming the second pair of disulfide bonds is any one of Mmt or Trt, and the thiol-protecting group of the cysteine forming the third pair of disulfide bonds is Acm.

In a preferred embodiment of the present invention, in step 1), the number of the cysteines contained in the first polypeptide fragment is three, one of the cysteines forming the first pair of disulfide bonds is further contained between the cysteines forming the first pair of disulfide bonds, the thiol-protecting group of the cysteine forming the first pair of disulfide bonds is Mmt or Trt, the thiol-protecting group of the cysteine forming the second pair of disulfide bonds is Acm, and the thiol-protecting group of the cysteine forming the third pair of disulfide bonds is Mmt or Trt.

In the technical scheme of the invention, the method for cracking the resin and removing other protecting groups in the step 5) is to remove the protecting groups by using 80-95% TFA solution.

In the technical scheme of the invention, the solid-phase synthesis method in the step 1-3) is an Fmoc solid-phase synthesis strategy, amino acid with Fmoc protected amino group is coupled on solid-phase synthesis resin, then the Fmoc protecting group is removed, and the amino acid with Fmoc protected amino group is continuously coupled until the synthesis of polypeptide is completed.

In the technical scheme of the invention, the solid phase synthetic resin is selected from 2-CTC solid phase synthetic resin and Wang solid phase synthetic resin.

In the technical solution of the present invention, preferably, in the first and second solutions, Wang solid phase synthetic resin is selected; in the third and fourth embodiments described above, 2-CTC solid phase synthetic resin is selected in step 1) and Wang solid phase synthetic resin is selected in step 3).

In the technical scheme of the invention, the Fmoc protecting group at the tail end of the polypeptide is remained when the first pair of disulfide bonds, the second pair of disulfide bonds or the third pair of disulfide bonds are formed.

In the technical scheme of the invention, the formation of the first pair of disulfide bonds, the second pair of disulfide bonds or the third pair of disulfide bonds is carried out under the condition of a solid phase.

Advantageous effects

1) The method can prepare the polypeptide containing three pairs of disulfide bonds, and the three pairs of polypeptides are all selectively deprotected, thereby greatly reducing the possibility of mismatching. The combination of Mmt or Trt and Acm can realize high orthogonal selectivity, the removal condition is simple, and the yield is high.

2) The method can complete selective formation of disulfide bonds only by adopting two thiol protecting groups, and the two thiol protecting groups selected by the invention have mild removal conditions and cannot influence the coupling of other amino acids.

3) The disulfide bond formed by the method is performed under the solid phase condition as much as possible, but not under the liquid phase condition, so that the purification efficiency can be improved to the maximum extent.

Drawings

FIG. 1 is a mass spectrum (Mw: 615) of a crude peptide of the 6 peptide fragment containing a pair of disulfide bonds in example 4.

FIG. 2 is a mass spectrum of a small sample of the intermediate crude peptide of example 8 (Mw: 1672) containing a pair of disulfide bonds

FIG. 3 is a mass spectrum of a small sample of the intermediate crude peptide of example 8 containing two pairs of disulfide bonds (Mw: 1670)

FIG. 4 shows a mass spectrum of a crude peptide of example 8, which contains 3 pairs of disulfide bonds (Mw: 1526)

Detailed Description

In order to achieve the purpose, the invention adopts the following 4 groups of technical schemes: (illustrated by the mismatching isoform of the Linaclotide disulfide bond)

Scheme 1: disulfide bonds are present between adjacent Cys residues at the C-terminus (IM-A; IM-B; IM-C in Table 1)

All of the 3 disulfide mismatching isomers contain Cys¹⁰-Cys¹³Disulfide bond of (c): the starting resin was Fmoc-Tyr (tBu) -Wang resin. Then sequentially coupling each residue to Cys according to Fmoc/tBu solid phase synthesis strategy¹⁰Or Asn⁷(Cys¹⁰Or Asn⁷Any one of the sites). Wherein Cys¹³And Cys¹⁰After the peptide resin fragment is coupled by adopting the same protecting group (Trt, Mmt or Acm), weighing 1-20 equivalent of iodine to be dissolved in a proper amount of DMF, and carrying out oxidation reaction on the peptide resin fragment at room temperature for 0.5-3 h. And after the reaction is finished, washing the peptide resin fragment with DMF (dimethyl formamide), washing residual iodine, and continuously coupling each subsequent residue in sequence according to an Fmoc/tBu solid-phase synthesis strategy after the washing is finished. Wherein 4 Cys residues are orthogonally protected with Trt or Mmt to Acm (preferably Mmt is orthogonal to Acm) according to different binding modes of disulfide bonds. After all residues are coupled, Mmt or Trt protecting groups are removed by using TFA, TIS, DCM 2, 5 and 93 solution, and then 1-30 equivalents of H are used₂O₂The DMF solution is first solid-phase oxidized into the second pair of disulfide bonds at room temperature for 0.5-3 h. Then continuing to adopt 1-20 equivalent of iodine in DMF solution, and carrying out a third pair of disulfide bond oxidation reaction on the peptide resin at room temperature, wherein the reaction time is 0.5-3 h. And washing and drying the peptide resin after the reaction is finished. And (3) performing shrinkage drying on the peptide resin, then performing TFA cleavage, precipitating and washing the crude peptide by methyl tertiary ether or ethyl ether, then performing vacuum drying to obtain a crude target peptide product, and purifying by HPLC to obtain the refined peptide.

Scheme 2: disulfide bonds (IM-G; IM-J; IM-K in Table 1) exist with one Cys spaced by two Cys residues at the C-terminus

All of the 3 disulfide mismatching isomers contain Cys⁶-Cys¹³Disulfide bond of (c): the starting resin was Fmoc-Tyr (tBu) -Wang resin. Then sequentially coupling each residue to Cys according to Fmoc/tBu solid phase synthesis strategy⁶Wherein Cys is¹⁰Protected with Acm, Cys⁶And Cys¹³Mmt or Trt protection is adopted. After coupling of the peptide resin fragment was complete, Cys was removed using TFA: TIS: DCM ═ 2:5:93 solution⁶And Cys¹³Mmt or Trt protecting group, then using 1-30 equivalents of H₂O₂The DMF solution is first solid phase oxidized into the first pair of disulfide bonds at room temperature for 0.5-3 hr. After the reaction is completed, the subsequent residues are continuously coupled. Wherein the remaining 3 Cys residues in the peptide sequence are orthogonally protected with Trt or Mmt to Acm (preferably Mmt to Acm) according to different binding patterns of disulfide bonds. After all residues are coupled, Mmt or Trt protecting groups are removed by using TFA, TIS, DCM 2, 5 and 93 solution, and then 1-30 equivalents of H are used₂O₂The DMF solution is subjected to solid phase oxidation at room temperature to form a second pair of disulfide bonds, and the reaction time is 0.5-3 h. Then continuing to adopt 1-20 equivalent of iodine in DMF solution, and carrying out third pair of disulfide bond oxidation reaction on the peptide resin fragment at room temperature for 0.5-3 h. And washing and drying the peptide resin after the reaction is finished. And (3) performing shrinkage drying on the peptide resin, then performing TFA cleavage, precipitating and washing the crude peptide by methyl tertiary ether or ethyl ether, then performing vacuum drying to obtain a crude target peptide product, and purifying by HPLC to obtain the refined peptide.

Scheme 3: disulfide bonds are present between two adjacent Cys residues at other sites (IM-A; IM-B; IM-C; IM-D; IM-E; IM-F; IM-G; IM-H; IM-I; IM-J; in Table 1).

The mismatching disulfide isomers all contain adjacent CDisulfide bond between ys: cys is¹-Cys²；Cys²-Cys⁵；Cys⁵-Cys⁶Cys⁶-Cys¹⁰Disulfide bond of (a). Firstly, synthesizing a full-protection peptide fragment containing a disulfide bond, and then synthesizing a peptide sequence (Cys) from the full-protection peptide fragment by adopting a fragment condensation mode¹-Cys²、Cys⁵-Cys⁶Briefly, other equivalents):

3.1 Cys-containing¹-Cys²And (3) synthesis of fragments: coupling Cys with 2-CTC resin as initial resin²，Glu³，Tyr⁴The Fmoc protected amino acid corresponding to either residue. After the amino acid resin synthesis is completed, the following residues are coupled in sequence according to the peptide sequence to obtain the corresponding dipeptide, tripeptide and tetrapeptide fragment resin (the Fmoc group at the N terminal is kept). Then 1-20 equivalent of iodine DMF solution is adopted to carry out disulfide bond oxidation reaction between two adjacent Cys residues on the peptide resin fragment at room temperature, and the reaction time is 0.5-3 h. And washing and drying the peptide resin after the reaction is finished. The peptide resin is contracted, dried, cracked by 20% TFE/DCM for 1h, filtered, and the filtrate is decompressed and concentrated to obtain the product containing a pair of disulfide bonds (Cys)¹-Cys²) A fully protected peptide fragment of (1).

3.2 Cys-containing⁵-Cys⁶And (3) synthesis of fragments: coupling Cys with 2-CTC resin as initial resin⁶，Asn⁷，Pro⁸，Ala⁹The Fmoc protected amino acid corresponding to either residue. After the amino acid resin is synthesized, the subsequent residues are coupled in sequence according to the peptide sequence until Cys⁵，Tyr⁴，Glu³The Fmoc protected amino acid corresponding to either residue. The corresponding dipeptide to heptapeptide fragment resins were obtained (note retention of the N-terminal Fmoc group). Then, 1-20 equivalent of iodine DMF solution is adopted to carry out oxidation reaction of disulfide bonds between two adjacent Cys residues on the peptide resin fragment at room temperature, and the reaction time is 0.5-3 h. And washing and drying the peptide resin after the reaction is finished. The peptide resin is contracted, dried, cracked by 20% TFE/DCM for 1h, filtered, and the filtrate is decompressed and concentrated to obtain the product containing a pair of disulfide bonds (Cys)⁵-Cys⁶) A fully protected peptide fragment of (1).

3.3 Synthesis of peptide resin: the starting resin was Fmoc-Tyr (tBu) -Wang resin. And sequentially coupling the residues according to an Fmoc/tBu solid-phase synthesis strategy (coupling according to the sequence condition of the fully-protected fragment during sequential coupling, and simultaneously adopting Mmt or Trt and Acm orthogonal protection for thiol protecting groups in the other 4 Cys residues except the fully-protected fragment according to different combination modes of disulfide bonds). After coupling of the peptide resin fragment was complete, the Mmt or Trt protecting groups were removed using TFA, TIS, DCM 2:5:93 solution, followed by 1-30 equivalents of H₂O₂The DMF solution is subjected to solid phase oxidation at room temperature to form a second pair of disulfide bonds, and the reaction time is 0.5-3 h. Then continuing to adopt 1-20 equivalent of iodine in DMF solution, and carrying out oxidation reaction on the third pair of disulfide bonds on the peptide resin at room temperature for 0.5-3 h. And washing and drying the peptide resin after the reaction is finished. And (3) performing shrinkage drying on the peptide resin, then performing TFA cleavage, precipitating and washing the crude peptide by methyl tertiary ether or ethyl ether, then performing vacuum drying to obtain a crude target peptide product, and purifying by HPLC to obtain the refined peptide.

Scheme 4: the other sites have disulfide bonds with two Cys residues separated by one Cys (IM-B; IM-G; IM-I; IM-L; IM-M; IM-N; in Table 1)

These mismatching isoforms of disulfide bonds all contain a disulfide bond with two Cys residues separated by one Cys: cys is¹-Cys⁵；Cys²-Cys⁶；Cys⁵-Cys¹⁰Disulfide bond of (a). First, a disulfide bond-containing fully protected peptide fragment is also synthesized, and then the fully protected peptide fragment is introduced into the synthetic peptide sequence (Cys) by means of fragment condensation¹-Cys⁵；Cys²-Cys⁶Briefly, other equivalents):

4.1 Cys-containing¹-Cys⁵And (3) synthesis of fragments: coupling Cys with 2-CTC resin as initial resin⁵The corresponding Fmoc protected amino acids. After the synthesis of the amino acid resin is finished, the following residues are coupled in sequence according to the peptide sequence to obtain the pentapeptide fragment resin (note: Cys)¹，Cys⁵Protected by Mmt, Cys²With Acm protection, simultaneouslyLeaving the Fmoc group N-terminal). The resin was treated with TFA: TIS: DCM ═ 2:5:93 solution for 1-3 h. Removal of Cys¹And Cys⁵And (3) the Mmt group of (1), and cleaving the peptide fragment from the resin. Filtering, adjusting pH of the filtrate to weak alkalinity of 7.0-8.0 with DIPEA, and adding 1-30 equivalent of H₂O₂And stirring the mixture at room temperature for reaction for 0.5 to 3 hours. After the reaction is finished, adding H into the solution₂O₂Equivalent ascorbic acid, and concentrating the solution under reduced pressure to obtain crude peptide fragment. Purifying the crude peptide fragment to obtain the purity>95% of disulfide bond-containing 5 peptide fragment protien.

4.2 Cys-containing²-Cys⁶And (3) synthesis of fragments: coupling Cys with 2-CTC resin as initial resin⁶，Asn⁷，Pro⁸，Ala⁹The Fmoc protected amino acid corresponding to either residue. After the synthesis of the amino acid resin is finished, sequentially coupling each subsequent residue to Cys according to the peptide sequence²The corresponding Fmoc protected amino acids. Corresponding pentapeptide to octapeptide fragment resins were obtained (Note: Cys:. RTM.: Cys)²，Cys⁶Protected by Mmt, Cys⁵With Acm protection while retaining the N-terminal Fmoc group). The resin was treated with TFA: TIS: DCM ═ 2:5:93 solution for 1-3 h. Removal of Cys¹And Cys⁵And (3) the Mmt group of (1), and cleaving the peptide fragment from the resin. Filtering, adjusting pH of the filtrate to weak alkalinity of 7.0-8.0 with DIPEA, and adding 1-30 equivalent of H₂O₂And stirring the mixture at room temperature for reaction for 0.5 to 3 hours. After the reaction is finished, adding H into the solution₂O₂Equivalent ascorbic acid, and concentrating the solution under reduced pressure to obtain crude peptide fragment. Purifying the crude peptide fragment to obtain the purity>95% of the peptide fragment of the fully protected peptide containing disulfide bond.

4.3 Synthesis of peptide resin: the starting resin was Fmoc-Tyr (tBu) -Wang resin. And sequentially coupling the residues according to an Fmoc/tBu solid-phase synthesis strategy (coupling according to the sequence condition of the fully-protected fragment during sequential coupling, and simultaneously adopting 2Mmt or Trt and 1 Acm for orthogonal protection of thiol protecting groups in the other 3 Cys residues except the fully-protected fragment according to different combination modes of disulfide bonds). After coupling of the peptide-resin fragment was complete, Mm was removed with TFA: TIS: DCM ═ 2:5:93 solutiont or Trt protecting group, then using 1-30 equivalents of H₂O₂The DMF solution is subjected to solid phase oxidation at room temperature to form a second pair of disulfide bonds, and the reaction time is 0.5-3 h. Then continuing to adopt 1-20 equivalent of iodine in DMF solution, and carrying out oxidation reaction on the third pair of disulfide bonds on the peptide resin at room temperature for 0.5-3 h. And washing and drying the peptide resin after the reaction is finished. And (3) cracking the dried peptide resin by TFA, precipitating the filtrate by methyl tertiary ether or diethyl ether, washing, drying in vacuum to obtain a crude target peptide product, and purifying by HPLC to obtain the refined peptide.

Step 1: resin or fully protected peptide fragments containing a pair of disulfide bond fragments were synthesized according to the Fmoc/tBu solid phase synthesis strategy.

Step 2: continuing to couple Fmoc-AA-OH corresponding to each subsequent residue in sequence to completion of peptide resin coupling as synthesized in step 1 as a resin containing a pair of disulfide bond fragments, and coupling Fmoc-AA-OH corresponding to each other residue in sequence of the peptide in sequence according to Fmoc/tBu solid phase synthesis strategy as synthesized in step 1 as a fully protected peptide fragment containing a pair of disulfide bonds; the synthesis of the peptide resin is completed by coupling the disulfide bond-containing fully-protected peptide in a fragment condensation mode. Wherein the sulfhydryl protecting group of Cys residue forms orthogonal protection with Mmt, Trt and Acm.

And step 3: removing Mmt or Trt protecting group by dilute acid solution, and then using H₂O₂And oxidizing the exposed sulfhydryl groups into a second pair of disulfide bonds by using an oxidant.

And 4, step 4: finally, using I₂Oxidation proceeds to bond the third pair of disulfide bonds. Washing off residual I after the reaction is finished₂The peptide resin was shrunk and dried.

And 5: preparing TFA lysate with a certain concentration, cleaving peptide resin, precipitating with diethyl ether or methyl tertiary ether, and washing to obtain crude peptide.

Step 6: purifying the crude peptide by reverse phase chromatography, and freeze-drying to obtain the refined peptide.

In step 1Synthesizing according to Fmoc/tBu solid phase polypeptide synthesis strategy, selecting one of WangResin and 2CTC Resin according to different conditions for initial Resin, and selecting Wang Resin if synthesized fragment peptide Resin containing disulfide bond; 2CTC Resin was selected if a disulfide bond-containing fully protected peptide fragment was synthesized. The resin substitution range is 0.20-0.75 mmol/g. The synthesis of amino acid resins (Wang Resin, 2CTC Resin) is a conventional synthesis method. Coupling of subsequent residues: 1) swelling the resin with solvent in solid phase reaction column for 15-60min, wherein the solvent includes DMF, NMP, dichloromethane, etc., preferably DMF; 2) fmoc protection removal with 20% piperidine/DMF solution, treatment time and frequency: washing the resin with appropriate amount of solvent repeatedly for 6-7 times at a rate of 2-3 times at 5-10 min/time; 3) dissolving and activating appropriate amount of amino acid to be coupled and coupling agent in solvent, adding into solid phase reaction column together until reaction termination is detected by ninhydrin detection method; 4) repeat 2) and 3). The coupling agent in the step 3) is a composition of DIPCDI and a compound A or a composition of DIPEA and a compound A and a compound B, wherein the compound A is HOAt or HOBt, the compound B is PyAOP, PyBOP, HATU, HBTU or TBTU, and the combination of DIPCDI and the compound A is preferred. Further, the ratio of each component in the coupling agent to the protected Amino Acid (AA) is, in terms of molar ratio, DIPCDI: a: AA ═ 1.3:1.2:1.0, DIPEA: a: B: AA ═ 2.0:1.2:1.0: 1.0. In addition, the time for each amino acid to carry out the coupling reaction is usually 1.0-4h, and the reaction end point is judged by a ninhydrin detection method; the temperature is room temperature (i.e., 20. + -. 5 ℃ C.), and it can also be carried out at an appropriately elevated or reduced temperature. After the coupling is complete, the resin is subjected to oxidation or preparation of fully protected peptide fragments. According to different situations, different methods are generally adopted: firstly, directly adopting 1-20 equivalent of DMF solution of iodine to carry out oxidation reaction on a peptide Resin fragment at room temperature, wherein the reaction time is 0.5-3h, washing after the reaction is finished to obtain a fragment Resin containing a disulfide bond, and if the fragment Resin is cracked by 20% TFE/DCM (2CTC Resin is used as an initial Resin), obtaining a corresponding fully-protected peptide fragment; in another, the Mmt or Trt protecting group is removed using a TFA, TIS, DCM 2:5:93 solution, followed by 1-30 equivalents of H₂O₂Is subjected to solid phase oxidation at room temperature to form a second pair of DMF solutionsDisulfide bond, the reaction time is 0.5-3h, and the fragment resin containing disulfide bond is obtained by washing after the reaction is finished. The 3 rd was resin treated with TFA TIS DCM ═ 2:5:93 solution for 1-3 h. The Mmt group is removed and the peptide fragment is cleaved from the resin. Filtering, adjusting pH of the filtrate to weak alkalinity of 7.0-8.0 with DIPEA, and adding 1-30 equivalent of H₂O₂And stirring the mixture at room temperature for reaction for 0.5 to 3 hours. After the reaction is finished, adding H into the solution₂O₂Equivalent ascorbic acid, and concentrating the solution under reduced pressure to obtain crude peptide fragment. Purifying the crude peptide fragment to obtain the purity>95% of the whole protected peptide fragment fine peptide containing disulfide bond is ready for use. Fully protected peptide fragments require retention of the terminal Fmoc protecting group.

And 2, sequentially coupling each subsequent protected amino acid residue according to an Fmoc/tBu solid-phase synthesis method, wherein the coupling method is as described in the step 1. It is noted that in some cases it may be desirable to couple the fully protected peptide containing a pair of disulfide bond fragments by fragment condensation using solvents selected from DMF, NMP, dichloromethane, etc., preferably DCM (containing a small amount of DMF) to increase the solubility of the fully protected peptide and to inhibit racemization during activation and coupling. The coupling system is preferably a combination of DIPCDI and HOBt. After the synthesis is completed, the peptide resin containing a pair of disulfide bond intermediates is obtained.

The Mmt protective group is removed in the step 3 by adopting a method of reacting a mixed solution of TFA, TIS, DCM (1-5) and (2-10) with resin for 15-30 times and 1-5 minutes each time, wherein the preferable solution ratio is TFA, TIS, DCM (2: 5: 93), the reaction times are 20 times and 2 minutes each time; while disulfide bond formation is carried out using 1-30 equivalents of H₂O₂The reaction with the resin is carried out for 0.5 to 3 hours, preferably 10 equivalents for 1 hour, and the reaction solvent is the same as that listed in step 1, preferably dimethylformamide. The resin can be detected by 0.1-0.2mmol/l DTNB solution in alkaline environment, and if the resin is colorless, the completion of disulfide bond cyclization is indicated.

Step 4, washing the resin in the step 3, and adopting I after washing₂Bonding of a third pair of disulfide bonds is carried out using a solvent selected from those listed in step 1, preferably dimethylformamide, and I₂The amount of (B) is 1 to 10 equivalents based on the amount of the resin substance. The reaction time is 0.5-3 h. Reaction ofAfter completion, the peptide resin was obtained by washing, shrinking and drying.

The lysis solution used in the step 5 is TFA and H₂O, TIS anisole, thioanisole, phenol, etc. Note that in this step, the cyclized crude peptide is obtained, so that components capable of reducing disulfide bonds, such as EDT and DTT, are avoided in the lysate fraction. The ratio of the cracking liquid is as follows: TFA TIS H₂Mixed solution of (80-90): (0-10): 0-10); the preferred ratio is TFA to TIS to H₂O92: 4:4, reaction temperature room temperature, reaction time 2-5h, preferably 3 h. The amount of the lysis solution is preferably 10 times by volume based on the weight of the resin. Filtering after the reaction is finished, precipitating the obtained filtrate by using diethyl ether or methyl tert-butyl ether, centrifuging, continuously washing the obtained precipitate for 3 times by using diethyl ether or methyl tert-butyl ether, and drying in vacuum to obtain the isomer crude peptide containing the 3-pair disulfide bonds.

Step 6 the purification step may be performed by reverse phase high pressure liquid chromatography. Further, the reversed-phase high-pressure liquid chromatography comprises: using reverse octadecylsilane as stationary phase and 0.1% trifluoroacetic acid water solution/acetonitrile as mobile phase, collecting target peak fraction, concentrating, and lyophilizing.

In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below, but the present invention is not to be construed as being limited to the implementable range thereof.

Abbreviations and English meanings

Example 1: preparation of Fmoc-Tyr (tBu) -Wang Resin

100g (103mmol) of Wang resin with a degree of substitution of 1.03mmol/g was weighed into a solid phase reaction column, washed 2 times with an appropriate amount of DMF and the resin was swollen with DMF for 30 minutes. 45.9 g of Fmoc-Tyr (tBu) -OH (100mmol) were weighed; HOBt16.2 g (120mmol), DMAP1.2 g (10mmol), dissolved in DMF/DCM (1:1) in appropriate amount, added with 20.3mL DIC (120mmol) in ice-water bath at 0 deg.C, activated for 3-5min, and added to the reaction column for reaction for 2.5 h. After the reaction is finished, washing the reaction product with DMF for 5 times, adding 70mL of acetic anhydride and 60mL of pyridine, and supplementing a proper amount of DMF to ensure that the resin is uniformly stirred. Mixing and sealing for more than 8h, washing with DMF for 5 times, washing with DCM for three times, shrinking with methanol for 2 times, and draining to obtain Fmoc-Tyr (tBu) -Wang Resin with 142g total, and detection substitution degree of 0.55mmol/g

Example 2: preparation of Fmoc-Cys (Mmt) -2CTC Resin

100g (120mmol) of 2CTC resin with a degree of substitution of 1.20mmol/g was weighed into a solid phase reaction column, washed 2 times with an appropriate amount of DMF and the resin was swollen with DMF for 30 minutes. 61.6 g of Fmoc-Cys (Mmt) -OH (100mmol) was dissolved in DMF/DCM (1:1) and the solution was cooled to 0 ℃ and added to the reaction column while adding 31.0g of DIPEA (61mL, 360mmol) in 2 portions and reacted for 1.5 hours. After the reaction is finished, adding 90mL of methanol and 30mL of DIPEA into the reaction column, continuing the reaction for 30min, washing the reaction column for 5 times by using DMF after the reaction is finished, washing the reaction column for three times by using DCM, and draining the Resin after the methanol shrinks for 2 times to obtain 151g of Fmoc-Cys (Mmt) -2CTC Resin, wherein the detection substitution degree is 0.65 mmol/g.

Example 3: preparation of Fmoc-Gly-2CTC Resin

50g (60mmol) of 2CTC resin with a degree of substitution of 1.20mmol/g was weighed into a solid phase reaction column, washed 2 times with an appropriate amount of DMF and the resin was swollen with DMF for 30 minutes. 14.9 g of Fmoc-Gly-OH (50mmol) were dissolved in DMF and the solution was loaded onto a reaction column and 15.5g of DIPEA (30.5mL, 180mmol) was added in 2 portions and reacted for 1.5 h. After the reaction is finished, adding 60mL of methanol and 20mL of DIPEA into the reaction column, continuing the reaction for 30min, washing the reaction column for 5 times by using DMF after the reaction is finished, washing the reaction column for three times by using DCM, and draining the Resin after the methanol shrinks for 2 times to obtain 59g of Fmoc-Gly-2CTC Resin in total, wherein the detection substitution degree is 0.71 mmol/g.

Example 4: synthesis of a peptide resin fragment containing a disulfide bond between a pair of two adjacent Cys (S-S: Cys)^10-13)

18.2 g of Fmoc-Tyr (tBu) -Wang Resin obtained in example 1 (Sub ═ 0.55mmol/g, 10 m) were weighed outmol) in a solid phase reaction column, washing with DMF for 2 times, and swelling with DMF for 30 minutes. It was then deprotected 2 times with DBLK (5min +7min), washed 6 times with additional DMF and 1 time with DCM. 18.5g of Fmoc-Cys (Mmt) -OH (30mmol) and 4.9g of HOBt (36mmol) are dissolved in 50mL of DMF/DCM (1:1), 6mL (39mmol) of DIPCDI is added under an ice-water bath to activate for 2-3min, the mixture is added into a reaction column and reacted for 2h at room temperature, and the reaction end point is detected by ninhydrin (the reaction is stopped if the resin is colorless and transparent; the reaction is prolonged for 1h if the resin is colored). At the end of the reaction, the resin was washed 3 times with 80ml DMF, then deprotected 2 times with DBLK (5min +7min), followed by 6 additional washes with DMF and 1 wash with DCM. Ninhydrin detection the resin was coloured. Repeating the coupling operation, continuing to couple Fmoc-Gly-OH, Fmoc-Thr (tBu) -OH, Fmoc-Cys (Mmt) -OH and Fmoc-Ala-OH in sequence, feeding 30mmol of each protected amino acid, and feeding the other materials in a ratio corresponding to the protected amino acid. After the coupling of the above residues was completed, the residue was washed 6 times with DMF. 7.6g of iodine (30mmol) was weighed into the reactor, and 70mL of DMF was added simultaneously, and the reaction was allowed to proceed for 1h at room temperature by bubbling. After the reaction was complete, the resin was washed 3 times with 80ml DMF, then deprotected 2 times with DBLK (5min +7min), followed by 6 times with DMF and 1 time with DCM. Namely to obtain NH₂-Ala-Cys¹⁰-Thr(tBu)-Gly-Cys¹³-Tyr(tBu)-Wang Resin(S-S:Cys^10-13) A fragment peptide resin containing a pair of disulfide bonds.

Example 5: synthesis of a pair of inter-Cys disulfide peptide resin fragments separated by one Cys (S-S: Cys)^6-13)

18.2 g of Fmoc-Tyr (tBu) -Wang Resin (Sub ═ 0.55mmol/g, 10mmol) obtained in example 1 were weighed out in a solid phase reaction column, washed 2 times with DMF and swollen with DMF for 30 minutes. It was then deprotected 2 times with DBLK (5min +7min), washed 6 times with additional DMF and 1 time with DCM. 18.5g of Fmoc-Cys (Mmt) -OH (30mmol) and 4.9g of HOBt (36mmol) are dissolved in 50mL of DMF/DCM (1:1), 6mL (39mmol) of DIPCDI is added under an ice-water bath to activate for 2-3min, the mixture is added into a reaction column and reacted for 2h at room temperature, and the reaction end point is detected by ninhydrin (the reaction is stopped if the resin is colorless and transparent; the reaction is prolonged for 1h if the resin is colored). After the reaction was complete, the resin was washed 3 times with 80mL DMF, then deprotected 2 times with DBLK (5min +7min), followed by 6 additional DMF washes and DCM washesWashing for 1 time. Ninhydrin detection the resin was coloured. The coupling operation is repeated, the coupling operation is continued to couple Fmoc-Gly-OH, Fmoc-Thr (tBu) -OH, Fmoc-Cys (Acm) -OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn (Trt) -OH and Fmoc-Cys (Mmt) -OH, each protected amino acid is fed by 30mmol, and the feeding ratio of other materials corresponds to the protected amino acid. After the coupling of the above residues was completed, the residue was washed 6 times with DMF. The peptide resin was then subjected to a demamt treatment with TFA TIS DCM 2:5:93, 20 times and 2 min each, after which the resin was washed 6 times with 1% DIPEA in DMF. Measuring 30% hydrogen peroxide 3mL (30mmol), adding into 80mL DMF, adding into solid phase reaction column, blowing for reaction for 1h, washing resin with 80mLDMF for 3 times after reaction, deprotecting with DBLK for 2 times (5min +7min), continuing DMF washing for 6 times, and washing with DCM for 1 time. Namely to obtain NH₂-Cys⁶-Asn(Trt)-Pro-Ala-Cys(Acm)¹⁰-Thr(tBu)-Gly-Cys¹³-Tyr(tBu)-Wang Resin(S-S:Cys^6-13) A fragment peptide resin containing a pair of disulfide bonds.

Example 6: synthesis of a fully protected fragment containing a pair of disulfide bonds (S-S: Cys)^6-10)

42.2 g of Fmoc-Gly-2CTC Resin obtained in example 3 (Sub ═ 0.71mmol/g, 30mmol) were weighed out into a solid phase reaction column, washed 2 times with DMF and swollen with DMF for 30 minutes. It was then deprotected 2 times with DBLK (5min +7min) and washed 6 times with DMF. 35.8g of Fmoc-Thr (tBu) -OH (90mmol) and 14.6g of HOBt (108mmol) are weighed and dissolved in 120mL of DMF/DCM (1:1), 18mL (117mmol) of DIPCDI is added under an ice-water bath for activation for 2-3min, and the mixture is added into a reaction column and reacted for 2h at room temperature, and the reaction end point is detected by ninhydrin (the reaction is stopped if the resin is colorless and transparent; and the reaction is prolonged for 1h if the resin is colored). At the end of the reaction, the resin was washed 3 times with 160ml DMF, then deprotected 2 times with DBLK (5min +7min), followed by 6 additional washes with DMF and 1 wash with DCM. Ninhydrin detection the resin was coloured. The coupling operation is repeated, the Fmoc-Cys (Mmt) -OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn (Trt) -OH and Fmoc-Cys (Mmt) -OH are coupled in sequence, each protected amino acid is fed by 90mmol, and the feeding ratio of other materials corresponds to the protected amino acid. After the coupling of the above residues was completed, the residue was washed 6 times with DMF. 22.9g of iodine (90mmol) were weighed into the reactorWhile adding 150mL of DMF, the reaction was carried out by bubbling at room temperature for 1 hour. Washing the resin with DMF 4 times, washing with DCM 2 times, shrinking with methanol, drying to obtain 61.4g of fragment peptide resin, cracking the peptide resin with 20% TFE/DCM for 1h, filtering, and concentrating the filtrate under reduced pressure to obtain 28.4g of product, i.e. Fmoc-Cys⁶-Asn(Trt)-Pro-Ala-Cys¹⁰-Thr(tBu)-Gly-OH(S-S:Cys^6-10) A pair of disulfide-bond containing fully protected peptide fragments.

Example 7: synthesis of a pair of inter-Cys disulfide peptide resin fragments separated by one Cys (S-S: Cys)^1-5)

46.2 g of Fmoc-Cys (Mmt) -2CTC Resin (Sub ═ 0.65mmol/g, 30mmol) obtained in example 2 were weighed into a solid phase reaction column, washed 2 times with DMF and swollen with DMF for 30 minutes. It was then deprotected 2 times with DBLK (5min +7min) and washed 6 times with DMF. 41.3g of Fmoc-Tyr (tBu) -OH (90mmol) and 14.6g of HOBt (108mmol) are weighed and dissolved in 140mL of DMF/DCM (1:1), 18mL (117mmol) of DIPCDI is added under an ice-water bath for activation for 2-3min, the mixture is added into a reaction column and reacted for 2h at room temperature, and the reaction end point is detected by ninhydrin (the reaction is stopped if the resin is colorless and transparent; the reaction is prolonged for 1h if the resin is colored). At the end of the reaction, the resin was washed 3 times with 160ml DMF, then deprotected 2 times with DBLK (5min +7min), followed by 6 additional washes with DMF and 1 wash with DCM. Ninhydrin detection the resin was coloured. The above coupling procedure was repeated. The subsequent steps are coupled with Fmoc-Tyr (tBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Cys (Acm) -OH and Fmoc-Cys (Mmt) -OH, each protected amino acid is fed by 90mmol, and the feeding ratios of other materials correspond to the protected amino acid. After coupling of the above residue was completed, the peptide was washed 4 times with DMF, 2 times with DCM, after methanol contraction and dried to give 82.3g of a fragment peptide resin, which was stirred at room temperature for 1h with 800mL of TFA, TIS, DCM 2:5: 93. Removal of Cys¹And Cys⁵And (3) the Mmt group of (1), and cleaving the peptide fragment from the resin. After the reaction is finished, filtering, adjusting the pH of the filtrate to be alkalescent 7.0-8.0 by using DIPEA, then measuring 9mL (90mmol) of 30% hydrogen peroxide, stirring and reacting for 0.5-1h at room temperature, and judging the reaction end point by using 0.1-0.2mmol/l DTNB solution. After the reaction is finished, adding H into the solution₂O₂Concentrating the solution under reduced pressure to obtain tablet29.5g of crude peptide fragment. The crude peptide fragment was purified by HPLC to obtain the purity>95% of refined peptide 23.1 g. I.e. Fmoc-Cys¹-Cys(Acm)²-Glu(OtBu)-Tyr(tBu)-Cys⁵-OH(S-S:Cys^1-5) A pair of disulfide-bond containing fully protected peptide fragments.

Example 8: synthesis of peptide resin (1)

The NH obtained in example 4 was reacted₂-Ala-Cys¹⁰-Thr(tBu)-Gly-Cys¹³-Tyr(tBu)-Wang Resin(S-S:Cys^10-13) The fragment peptide resin containing a pair of disulfide bonds of (1) was subsequently coupled and synthesized with the sequence of IM-B in the present invention. The subsequent residues were coupled in sequence as described above in step 2: Fmoc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc-Cys (Acm) -OH, Fmoc-Cys (Mmt) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Cys (Acm) -OH and Fmoc-Cys (Mmt) -OH, each protected amino acid being charged at 30mmol, and the other charge ratios corresponding to the protected amino acids. After the coupling of the above residues was completed, the residue was washed 6 times with DMF. The peptide resin was then subjected to a demamt treatment with TFA TIS DCM 2:5:93, 20 times and 2 min each, after which the resin was washed 6 times with 1% DIPEA in DMF. 3mL (30mmol) of 30% hydrogen peroxide is weighed and added into 120mL of DMF, the mixture is added into a solid phase reaction column and is aerated for reaction for 1h, after the reaction is finished, 120mL of DMF is used for washing resin for 5 times, 7.6g of iodine (30mmol) is weighed and added into a reactor, and simultaneously 120mL of DMF is added, and the mixture is aerated for reaction for 1h at room temperature. After the reaction, the resin was washed 3 times with 120ml DMF, then deprotected 2 times with DBLK (5min +7min), washed 4 times with DMF, 2 times with DCM, and dried after methanol shrinkage to give 38.5g of IM-B peptide resin.

Example 9: synthesis of peptide resin (2)

The NH obtained in example 5 was reacted₂-Cys⁶-Asn(Trt)-Pro-Ala-Cys(Acm)¹⁰-Thr(tBu)-Gly-Cys¹³-Tyr(tBu)-Wang Resin(S-S:Cys^6-13) The fragment peptide resin containing a pair of disulfide bonds of (1) was subsequently coupled and synthesized with the sequence of IM-G in the present invention. The subsequent residues were coupled in sequence as described above in step 2: Fmoc-Cys (Acm) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Cys (Mmt) -OH, Fmoc-Cys (Mmt) -OH, each protected amino acid is fed by 30mmol, and the feeding ratio of other materials corresponds to the protected amino acid. After the coupling of the above residues was completed, the residue was washed 6 times with DMF. The peptide resin was then subjected to a demamt treatment with TFA TIS DCM 2:5:93, 20 times and 2 min each, after which the resin was washed 6 times with 1% DIPEA in DMF. 3mL (30mmol) of 30% hydrogen peroxide is weighed and added into 120mL of DMF, the mixture is added into a solid phase reaction column and is aerated for reaction for 1h, after the reaction is finished, 120mL of DMF is used for washing resin for 5 times, 7.6g of iodine (30mmol) is weighed and added into a reactor, and simultaneously 120mL of DMF is added, and the mixture is aerated for reaction for 1h at room temperature. After the reaction, the resin was washed 3 times with 120ml DMF, then deprotected 2 times with DBLK (5min +7min), washed 4 times with DMF, washed 2 times with DCM, shrunk with methanol and dried to obtain 37.3G of peptide resin IM-G

Example 10: synthesis of peptide resin (3)

18.2 g of Fmoc-Tyr (tBu) -Wang Resin (Sub ═ 0.55mmol/g, 10mmol) obtained in example 1 were weighed out in a solid phase reaction column, washed 2 times with DMF and swollen with DMF for 30 minutes. It was then deprotected 2 times with DBLK (5min +7min), washed 6 times with additional DMF and 1 time with DCM. 12.4g of Fmoc-Cys (Acm) -OH (30mmol) and 4.9g of HOBt (36mmol) are dissolved in 50mL of DMF/DCM (1:1), 6mL (39mmol) of DIPCDI is added under an ice-water bath to activate for 2-3min, the mixture is added into a reaction column and reacted for 2h at room temperature, and the reaction end point is detected by ninhydrin (the reaction is stopped if the resin is colorless and transparent; the reaction is prolonged for 1h if the resin is colored). At the end of the reaction, the resin was washed 3 times with 80ml DMF, then deprotected 2 times with DBLK (5min +7min), followed by 6 additional washes with DMF and 1 wash with DCM. Ninhydrin detection the resin was coloured. The peptide fragment obtained in example 6: Fmoc-Cys⁶-Asn(Trt)-Pro-Ala-Cys¹⁰-Thr(tBu)-Gly-OH(S-S:Cys^6-10) A total of 28.4g (24mmol) was dissolved in 60mL of DCM/DMF (9:1) with the addition of 4.1g of HOBt (30 mmol). Adding 6mL (39mmol) of DIPCDI under ice water bath for activation for 2-3min, adding the mixed solution into a reaction column, reacting for 3h at room temperature, detecting the reaction endpoint with ninhydrin (stopping the reaction if the resin is colorless and transparent; washing with DMF for 5 times if the resin is colored, adding 21mL of acetic anhydride, 18mL of pyridine and a proper amount of DMF, and carrying out end-capping treatment on the N end for 2 h.). At the end of the reaction, the resin was washed 3 times with 80ml DMF, then deprotected 2 times with DBLK (5min +7min), followed by 6 additional washes with DMF and 1 wash with DCM. Ninhydrin detection the resin was coloured. The above coupling procedure was repeated with reference to the sequence of IM-F above, and the subsequent coupling procedure was continued with Fmoc-Cys (Acm) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Cys (Mmt) -OH, each protected amino acid was dosed 30mmol, and the other dosing ratios corresponded to the protected amino acids. After the coupling of the above residues was completed, the residue was washed 6 times with DMF. The peptide resin was then subjected to a demamt treatment with TFA TIS DCM 2:5:93, 20 times and 2 min each, after which the resin was washed 6 times with 1% DIPEA in DMF. 3mL (30mmol) of 30% hydrogen peroxide is weighed and added into 120mL of DMF, the mixture is added into a solid phase reaction column and is aerated for reaction for 1h, after the reaction is finished, 120mL of DMF is used for washing resin for 5 times, 7.6g of iodine (30mmol) is weighed and added into a reactor, and simultaneously 120mL of DMF is added, and the mixture is aerated for reaction for 1h at room temperature. After the reaction, the resin was washed 3 times with 120ml DMF, then deprotected 2 times with DBLK (5min +7min), washed 4 times with DMF, 2 times with DCM, shrunk with methanol and dried to obtain 35.3g of peptide resin IM-F

Example 11: synthesis of peptide resin (4)

18.2 g of Fmoc-Tyr (tBu) -Wang Resin (Sub ═ 0.55mmol/g, 10mmol) obtained in example 1 were weighed out in a solid phase reaction column, washed 2 times with DMF and swollen with DMF for 30 minutes. It was then deprotected 2 times with DBLK (5min +7min), washed 6 times with additional DMF and 1 time with DCM. 12.4g of Fmoc-Cys (Acm) -OH (30mmol) and 4.9g of HOBt (36mmol) are dissolved in 50mL of DMF/DCM (1:1), 6mL (39mmol) of DIPCDI is added under an ice-water bath to activate for 2-3min, the mixture is added into a reaction column and reacted for 2h at room temperature, and the reaction end point is detected by ninhydrin (the reaction is stopped if the resin is colorless and transparent; the reaction is prolonged for 1h if the resin is colored). At the end of the reaction, the resin was washed 3 times with 80ml DMF, then deprotected 2 times with DBLK (5min +7min), followed by 6 additional washes with DMF and 1 wash with DCM. Ninhydrin detection the resin was coloured. Repeating the above coupling procedure according to the sequence of IM-I above, continuing the coupling procedure to Fmoc-Gly-OH, Fmoc-Thr (tBu) -OH, Fmoc-Cys (Mmt) -OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc-Cys (Mmt) -OH, each protecting amino groupThe acid is fed by 30mmol, and the feeding ratio of other materials corresponds to the protected amino acid. After the coupling of the above residues was completed, the residue was washed 6 times with DMF. Wash 1 time with DCM. Ninhydrin detection the resin was coloured. The peptide fragment obtained in example 7: Fmoc-Cys¹-Cys(Acm)²-Glu(OtBu)-Tyr(tBu)-Cys⁵-OH(S-S:Cys^1-5) A total of 23.1g (about 23mmol) of the refined peptide was dissolved in 60mL of DCM/DMF (9:1) with the addition of 4.1g of HOBt (30 mmol). After 6mL (39mmol) of DIPCDI was added under ice water bath to activate for 2-3min, the mixture was added to a reaction column and reacted at room temperature for 3 h. The reaction was complete, washed 6 times with DMF and 1 time with DCM. The peptide resin was then subjected to a demamt treatment with TFA TIS DCM 2:5:93, 20 times and 2 min each, after which the resin was washed 6 times with 1% DIPEA in DMF. 3mL (30mmol) of 30% hydrogen peroxide is weighed and added into 120mL of DMF, the mixture is added into a solid phase reaction column and is aerated for reaction for 1h, after the reaction is finished, 120mL of DMF is used for washing resin for 5 times, 7.6g of iodine (30mmol) is weighed and added into a reactor, and simultaneously 120mL of DMF is added, and the mixture is aerated for reaction for 1h at room temperature. After the reaction, the resin was washed 3 times with 120ml DMF, then deprotected 2 times with DBLK (5min +7min), washed 4 times with DMF, 2 times with DCM, shrunk with methanol and dried to obtain 34.1g of IM-I peptide resin

Example 12: cleavage of peptide resins

The peptide resins obtained in examples 8, 9, 10 and 11 were cleaved. The cleavage conditions were such that a cleavage reagent (TFA: TIS: H) was added at a ratio of 10mL/g peptide resin₂O90: 5:5(V/V)), and stirred at room temperature for 3 h. After the reaction was completed, the resin was filtered and washed with a small amount of TFA, and the filtrates were collected and combined and concentrated under reduced pressure to a certain volume. Adding into frozen methyl tert-butyl ether (100mL/g peptide resin), precipitating, centrifuging, removing supernatant, washing the precipitate with methyl tert-butyl ether for 3 times, and vacuum drying to obtain corresponding crude peptide. The purity of the crude peptide was determined by HPLC as follows (HPLC assay conditions: Waters symmetry shield RP18(5 μm) (250X 4.6mM), phase A: 50mM/l ammonium dihydrogen phosphate buffer (pH 3.0 adjusted by phosphoric acid), phase B: acetonitrile, gradient: 15-45% (30min), flow rate: 0.5mL/min assay wavelength: 215nm, the same applies here):

table 2: crude peptides of group 4 Linaclotide disulfide mismatching isomers and purity list

Numbering	Name of peptide	Weight of peptide resin (g)	Crude peptide weight (g)	Purity of crude peptide
					1	IM-B	38.5	12.5	57.6％
2	IM-G	37.3	11.9	59.4％
					3	IM-F	35.3	11.1	52.1％
4	IM-I	34.1	10.8	48.5％

Example 13: purification preparation of 4-group Linaclotide disulfide bond mismatching isomer

The 4 groups of crude peptides obtained in example 12 above were subjected to reverse phase liquid phase preparative purification using octadecylsilane as a stationary phase and 50mM/l ammonium dihydrogen phosphate buffer/acetonitrile as a mobile phase, and the desired peak fractions (> 95%) were collected, followed by desalting treatment, and concentration and freeze-drying of the fractions. Specific data are shown in the following table.

Table 3: group 4 Linaclotide disulfide mismatch isoform purification data

Numbering	Name of peptide	Weight of Fine peptide (g)	Purity of	Weight yield
					1	IM-B	3.1	97.2％	20.3％
2	IM-G	3.4	98.5％	22.3％
					3	IM-F	2.7	96.3％	17.7％
4	IM-I	2.4	97.5％	15.7％

Claims

1. A method for synthesizing polypeptide containing 3 pairs of disulfide bonds,

the synthesis method comprises the following steps:

2. The method of claim 1, wherein in step 2), the thiol protecting groups of the two cysteines forming the first pair of disulfide bonds are removed and oxidized to form disulfide bonds by: the first polypeptide fragment only contains two cysteines forming a first pair of disulfide bonds, 1-20 equivalents of iodine is dissolved in a proper amount of DMF, and the thiol protecting groups are removed and the disulfide bonds are formed under room temperature conditions;

3. The method of any one of claims 1-2, wherein the step 3) of continuing to synthesize the remaining fragments under solid phase conditions comprises synthesizing by solid phase synthesis to form a conjugate of the polypeptide fragment and a solid phase synthetic resin, wherein the conjugate of the polypeptide fragment and the solid phase synthetic resin comprises 6 cysteines, and wherein 2 cysteines have formed disulfide bonds; the synthesis method comprises adding amino acids to the N-terminal of the polypeptide fragment, synthesizing the polypeptide fragment by a solid phase synthesis method, and coupling each polypeptide fragment, or coupling single added amino acids and the fragment for combination.

4. The synthesis method according to any one of claims 1 to 3, wherein the thiol-protecting group of cysteine is removed and oxidized to form a disulfide bond in step 4) by removing two cysteine thiol-protecting groups Trt or Mmt from the disulfide bond using TFA, TIS, DCM (1-5), (2-10), (97-85), and then using 1-30 equivalents of H₂O₂The DMF solution is firstly subjected to solid phase oxidation at room temperature to form a pair of disulfide bonds; then dissolving 1-20 equivalents of iodine in appropriate amount of DMF, and removing the sulfhydryl protecting group Acm and forming disulfide bond at room temperature.

5. The method of synthesis according to any one of claims 1 to 4, wherein the Fmoc protecting group at the end of the polypeptide remains when the first, second or third pair of disulfide bonds are formed.

6. The method of any one of claims 1-5, wherein the number of cysteines contained in the first polypeptide fragment in step 1) is two, the cysteines form a first pair of disulfide bonds, the thiol-protecting group of the cysteines forming the first pair of disulfide bonds is any one of Mmt, Trt, or Acm, the thiol-protecting group of the cysteines forming the second pair of disulfide bonds is any one of Mmt or Trt, and the thiol-protecting group of the cysteines forming the third pair of disulfide bonds is Acm.

7. The method of any one of claims 1-6, wherein the first polypeptide fragment in step 1) comprises three cysteines, one of the cysteines forming the first pair of disulfide bonds is further comprised between the cysteines forming the first pair of disulfide bonds, the thiol-protecting group of the cysteine forming the first pair of disulfide bonds is either Mmt or Trt, the thiol-protecting group of the cysteine forming the second pair of disulfide bonds is Acm, and the thiol-protecting group of the cysteine forming the third pair of disulfide bonds is either Mmt or Trt.

8. The method of any one of claims 1 to 7, wherein in step 3), the method for continuing the synthesis of the remaining fragments under solid phase conditions is:

9. The synthesis method according to any one of claims 1 to 8, wherein the cleavage of the resin and the removal of other protecting groups in step 5) are performed by 80-95% TFA solution.

10. The method of synthesis according to any one of claims 1 to 9, wherein the solid phase synthesis in step 1-3) is an Fmoc solid phase synthesis strategy, coupling the Fmoc-protected amino acid on a solid phase synthesis resin, then removing the Fmoc protecting group, and continuing to couple the Fmoc-protected amino acid until completion of the polypeptide synthesis.