A solid phase synthesis method for polypeptides containing three pairs of disulfide bonds by introducing a pair of disulfide bonds

Abstract

The present invention relates to a solid phase synthesis method for a polypeptide containing three pairs of disulfide bonds by introducing a pair of disulfide bonds, and specifically discloses the method comprising the following steps: 1) synthesizing a conjugate of a first polypeptide fragment and a solid phase synthesis resin by a solid phase synthesis method, wherein the first polypeptide fragment contains two cysteines forming a first pair of disulfide bonds; 2) removing the sulfhydryl protecting groups of the two cysteines of the first pair of disulfide bonds in the first polypeptide fragment, and oxidizing to form the first pair of disulfide bonds; 3) continuing to synthesize the remaining fragments by a solid phase synthesis method; 4) removing the sulfhydryl protecting groups of the cysteines of the third pair of disulfide bonds to form the third pair of disulfide bonds, and then removing the sulfhydryl protecting groups of the cysteine ​​of the second pair of disulfide bonds to form the second pair of disulfide bonds; 5) removing other protecting groups, cleaving the resin, and obtaining a polypeptide containing three pairs of disulfide bonds. The method of the present invention can selectively remove the protecting groups of the three pairs of polypeptides, greatly reducing the possibility of mismatching.

Description

Technical Field

[0001] The invention belongs to the field of polypeptide synthesis, and particularly relates to a solid phase synthesis method of a polypeptide containing three pairs of disulfide bonds by introducing a pair of disulfide bonds. Background Art

[0002] Protein and peptide drugs have the advantages of specific action sites and clear therapeutic effects. In recent years, the research and development of protein and peptide drugs has become a hot topic in the field of biomedicine.

[0003] Disulfide bonds are SS covalent bonds formed by oxidation of sulfhydryl groups (-SH) of Cys located at different peptide chains or two different sites of the same peptide chain in proteins or polypeptide molecules. It can fold the peptide chain into a specific spatial structure and plays an important role in maintaining the spatial structure of polypeptides and proteins and the biological activity determined by it. The more disulfide bonds there are, the greater the stability of the protein molecule against the influence of external factors.

[0004] Among the peptide drugs currently on the market, there are several peptide drugs with multiple pairs of disulfide bonds, such as plecanatide, ziconotide, linaclotide, etc. Because they are rich in multiple Cys residues, multiple isomers may be formed due to different disulfide bond connection sites. For example, a peptide containing 3 pairs of disulfide bonds has 15 different combinations of disulfide bonds. In addition to the drug itself, there are 14 other different disulfide mismatch isomers. In the process of drug development, the study of these disulfide mismatch isomer impurities is an indispensable part of the quality research of raw materials or preparations. But how to synthesize them has always been a headache.

[0005] Take linaclotide as an example. Linaclotide is a new type of GC-C (enterothelial cell uridylate cyclase C) receptor agonist. It can activate the GC-C receptor on the apical surface of intestinal epithelial cells, leading to an increase in intracellular and extracellular cyclic guanosine monophosphate. It increases the secretion of chloride and bicarbonate into the intestinal cavity, thereby increasing fluid secretion and accelerating stool passage. It is used to treat adult patients with slow-transmission constipation and constipation-type irritable bowel syndrome (IBS-C). It is composed of 14 L-type amino acid residues, of which 6 Cys residues are connected by disulfide bonds. Its structural sequence is: NH 2 -Cys 1 -Cys 2 -Glu-Tyr-Cys 5 -Cys 6 -Asn-Pro-Ala-Cys 10 -Thr-Gly-Cys 13 -Tyr-COOH (where Cys 1 、Cys 6 , Cys2 、Cys 10 and Cys 5 、Cys 13 connected by disulfide bonds).

[0006]

[0007] Since linaclotide contains 6 cysteine ​​residues, which are paired in pairs to form 3 pairs of disulfide bonds, in addition to linaclotide, there are 14 other different disulfide bond mismatch isomers due to different disulfide bond binding sites (see Table 1: the isomers are randomly named and represented by IM-A, B...).

[0008] Table 1: List of Linaclotide and its disulfide mismatched isomers

[0009] Nomenclature Impurity Peptide Sequence 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)

[0010] Benitez et al. reported on this in 2010 (see Peptide Science, 2011, 96: 69-80). The document used three methods to experimentally synthesize linaclotide.

[0011] Method 1: All Cys residues use Trt or Mmt as the side chain protecting group of Cys, and the linear peptide is synthesized using the Fmoc / tBu solid phase synthesis strategy. After cleavage to obtain the crude peptide, the target peptide is oxidized in one step using the liquid phase oxidation method.

[0012] Method 2: The thiol group of the Cys residue uses Trt or Mmt and Acm as the Cys side chain protecting group (4Trt+2Acm or 4Acm+2Trt), and the Fmoc / tBu solid phase synthesis strategy is used to obtain a partially protected linear peptide, and then the semi-selective strategy is used to complete the synthesis of the disulfide bond.

[0013] Method three: Three different Cys protection strategies were used for orthogonal protection, such as [2Mmt+2Acm+2Trt], [2Acm+2Trt+2pMeOBzl], and [2StBu+2Trt+2pMeOBzl], three completely selective strategies for the site-directed synthesis of disulfide bonds.

[0014] For the synthesis of linaclotide mismatch isomers, the above method 1 is not suitable. Although the synthesis steps are simple and only one Cys protecting group is used, random oxidation may result in many different isomers, but the disulfide bond formation mode of these isomers cannot be determined.

[0015] Method 2 is a semi-selective targeted oxidation method, which first targets and oxidizes a pair of disulfide bonds, and then the other four Cys residues are freely combined into three possible isomers. This method reduces the number of different isomers formed to three. Although in theory it is possible to obtain isomers by designing and positioning different disulfide bonds and then compare and confirm the obtained isomers, firstly, the workload is huge, and secondly, the mismatched isomers obtained in this way cannot determine the accuracy of their disulfide bond structure.

[0016] The method of using completely orthogonal protection of the Cys thiol group to form three pairs of disulfide bonds in method three is theoretically a feasible strategy. However, in actual operation, there are problems such as poor orthogonality of the protecting group and incomplete removal of the protecting group, which lead to many impurity peaks in the synthesis, low yield, and even inability to distinguish and identify the target product. Summary of the invention

[0017] In view of the above situation, the present invention provides a method for synthesizing mismatched isomers containing 3 pairs of disulfide bonds by using the Fmoc / tBu solid phase synthesis strategy: mainly, a pair of disulfide bonds in the sequence to be synthesized is first constructed by the Fmoc / tBu solid phase method (two Cys constituting the disulfide bond are adjacent or separated by a Cys residue), and then the peptide fragment containing the disulfide bond is introduced into the synthesis of the peptide sequence as a whole by the fragment condensation method. The other two pairs of disulfide bonds use Mmt or Trt and Acm as Cys side chain protecting groups. In this way, the synthesis of 14 mismatched isomers of disulfide bonds is changed from 3 pairs of disulfide bonds to 2 pairs of disulfide bonds, which greatly reduces the technical difficulty of synthesis and purification, improves the process yield, and the disulfide bond structure of the mismatched isomers of disulfide bonds is clear.

[0018] In one aspect, the present invention provides a method for synthesizing a polypeptide containing three pairs of disulfide bonds.

[0019] The polypeptide containing three pairs of disulfide bonds comprises a first pair of disulfide bonds, a second pair of disulfide bonds and a third pair of disulfide bonds, and at least one pair of disulfide bonds among the three pairs of 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;

[0020] The synthesis method comprises the following steps:

[0021] 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 contains two cysteines forming a first pair of disulfide bonds, one of the cysteines forming a second pair of disulfide bonds is between the two cysteines forming the first pair of disulfide bonds, or no cysteine ​​is 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 a thiol protecting group Trt or Mmt; and one of the cysteines forming the second pair of disulfide bonds is protected with a thiol protecting group Acm;

[0022] 2) removing the sulfhydryl protecting groups of the two cysteines of the first pair of disulfide bonds in the first polypeptide fragment, and oxidizing to form the first pair of disulfide bonds;

[0023] 3) Under solid phase conditions, continue to synthesize the remaining fragments by solid phase synthesis, wherein the remaining fragments contain cysteine ​​that forms the second pair of disulfide bonds and cysteine ​​that forms the third pair of disulfide bonds, wherein the sulfhydryl groups of the cysteine ​​that forms the second pair of disulfide bonds and the cysteine ​​that forms the third pair of disulfide bonds are both protected by sulfhydryl protecting groups; the sulfhydryl protecting groups of the two cysteines that form the second pair of disulfide bonds are the same, which is Acm; the sulfhydryl protecting groups of the cysteines that form the third pair of disulfide bonds are the same, which is Trt or Mmt;

[0024] 4) removing the thiol protecting group of cysteine ​​of the second pair of disulfide bonds to form the second pair of disulfide bonds, and then removing the thiol protecting group of cysteine ​​of the third pair of disulfide bonds to form the third pair of disulfide bonds; or

[0025] removing the thiol protecting group of cysteine ​​of the third pair of disulfide bonds to form the third pair of disulfide bonds, and then removing the thiol protecting group of cysteine ​​of the second pair of disulfide bonds to form the second pair of disulfide bonds;

[0026] 5) Removing other protecting groups, cleaving the resin, and obtaining a polypeptide containing three pairs of disulfide bonds.

[0027] A specific embodiment of the present invention provides a method for synthesizing a polypeptide containing three pairs of disulfide bonds.

[0028] The polypeptide containing three pairs of disulfide bonds comprises a first pair of disulfide bonds, a second pair of disulfide bonds and a third pair of disulfide bonds, wherein the first pair of disulfide bonds among the three pairs of disulfide bonds is formed by two adjacent cysteines, and the first pair of disulfide bonds is closer to the C-terminus than the second pair of disulfide bonds and the third pair of disulfide bonds;

[0029] The synthesis method comprises the following steps:

[0030] 1) synthesizing a conjugate of a first polypeptide fragment and a solid phase synthesis resin by a solid phase synthesis method, wherein the first polypeptide fragment contains two cysteines forming a first pair of disulfide bonds, and there is no cysteine ​​between the two cysteines forming the first pair of disulfide bonds; and the thiol groups of the two cysteines forming the first pair of disulfide bonds are protected with a thiol protecting group Trt or Mmt;

[0031] 2) removing the sulfhydryl protecting groups of the two cysteines of the first pair of disulfide bonds in the first polypeptide fragment, and oxidizing to form the first pair of disulfide bonds;

[0032] 3) under solid phase conditions, the N-terminus of the first polypeptide fragment is continuously solid-phase coupled with the remaining fragment, wherein the remaining fragment contains the cysteine ​​forming the second pair of disulfide bonds and the cysteine ​​forming the third pair of disulfide bonds, wherein the sulfhydryl groups of the cysteine ​​forming the second pair of disulfide bonds and the cysteine ​​forming the third pair of disulfide bonds are both protected with sulfhydryl protecting groups; the sulfhydryl protecting groups of the two cysteines forming the second pair of disulfide bonds are the same, namely Acm; the sulfhydryl protecting groups of the cysteines forming the third pair of disulfide bonds are the same, namely Trt or Mmt;

[0033] 4) removing the thiol protecting group of cysteine ​​of the third pair of disulfide bonds to form the third pair of disulfide bonds, and then removing the thiol protecting group of cysteine ​​of the second pair of disulfide bonds to form the second pair of disulfide bonds;

[0034] 5) Removing other protecting groups, cleaving the resin, and obtaining a polypeptide containing three pairs of disulfide bonds.

[0035] A specific embodiment of the present invention, the second scheme, provides a method for synthesizing a polypeptide containing three pairs of disulfide bonds.

[0036] The polypeptide containing three pairs of disulfide bonds includes 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 is formed by two cysteines separated by one cysteine, and the first pair of disulfide bonds is closer to the C-terminus than the second pair of disulfide bonds and the third pair of disulfide bonds;

[0037] The synthesis method comprises the following steps:

[0038] 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 of the cysteines forming a second pair of disulfide bonds is located between the two cysteines forming the first pair of disulfide bonds; the sulfhydryl groups of the two cysteines forming the first pair of disulfide bonds are protected with a sulfhydryl protecting group Trt or Mmt; and one of the cysteines forming the second pair of disulfide bonds is protected with a sulfhydryl protecting group Acm;

[0039] 2) removing the sulfhydryl protecting groups of the two cysteines of the first pair of disulfide bonds in the first polypeptide fragment, and oxidizing to form the first pair of disulfide bonds;

[0040] 3) under solid phase conditions, the N-terminus of the first polypeptide fragment is continuously solid-phase coupled with the remaining fragment, wherein the remaining fragment contains a cysteine ​​forming a second pair of disulfide bonds and two cysteines forming a third pair of disulfide bonds, wherein the sulfhydryl groups of the cysteines forming the second pair of disulfide bonds and the cysteines forming the third pair of disulfide bonds are both protected with sulfhydryl protecting groups; the sulfhydryl protecting groups of the two cysteines forming the second pair of disulfide bonds are the same, namely Acm; the sulfhydryl protecting groups of the cysteines forming the third pair of disulfide bonds are the same, namely Trt or Mmt;

[0041] 4) removing the thiol protecting group of cysteine ​​of the third pair of disulfide bonds to form the third pair of disulfide bonds, and then removing the thiol protecting group of cysteine ​​of the second pair of disulfide bonds to form the second pair of disulfide bonds;

[0042] 5) Removing other protecting groups, cleaving the resin, and obtaining a polypeptide containing three pairs of disulfide bonds.

[0043] A specific embodiment of the present invention provides a method for synthesizing a polypeptide containing three pairs of disulfide bonds.

[0044] The polypeptide containing three pairs of disulfide bonds comprises a first pair of disulfide bonds, a second pair of disulfide bonds and a third pair of disulfide bonds, wherein the first pair of disulfide bonds among the three pairs of disulfide bonds is formed by two adjacent cysteines, and any one or more cysteines forming the second pair of disulfide bonds and the third pair of disulfide bonds are closer to the C-terminus than the two cysteines forming the first pair of disulfide bonds;

[0045] The synthesis method comprises the following steps:

[0046] 1) synthesizing a conjugate of a first polypeptide fragment and a solid phase synthesis resin by a solid phase synthesis method, wherein the first polypeptide fragment contains two cysteines forming a first pair of disulfide bonds, and there is no cysteine ​​between the two cysteines forming the first pair of disulfide bonds; the sulfhydryl groups of the two cysteines forming the first pair of disulfide bonds are protected with a sulfhydryl protecting group Trt or Mmt; and one of the cysteines forming a second pair of disulfide bonds is protected with a sulfhydryl protecting group Acm;

[0047] 2) removing the sulfhydryl protecting groups of the two cysteines of the first pair of disulfide bonds in the first polypeptide fragment, and oxidizing to form the first pair of disulfide bonds;

[0048] 3) synthesizing the remaining fragment by solid phase synthesis under solid phase conditions, wherein the remaining fragment contains the cysteine ​​forming the second pair of disulfide bonds and the cysteine ​​forming the third pair of disulfide bonds, wherein the thiol groups of the cysteine ​​forming the second pair of disulfide bonds and the cysteine ​​forming the third pair of disulfide bonds are both protected by thiol protecting groups; the thiol protecting groups of the two cysteines forming the second pair of disulfide bonds are the same, which is Acm; the thiol protecting groups of the cysteines forming the third pair of disulfide bonds are the same, which is Trt or Mmt; the remaining fragment is present at the N-terminus or C-terminus of the first polypeptide fragment, or a part of the remaining fragment is present at the N-terminus of the first polypeptide fragment and the other part is present at the C-terminus of the first polypeptide fragment, to form a polypeptide-resin conjugate;

[0049] 4) removing the thiol protecting group of cysteine ​​of the third pair of disulfide bonds to form the third pair of disulfide bonds, and then removing the thiol protecting group of cysteine ​​of the second pair of disulfide bonds to form the second pair of disulfide bonds;

[0050] 5) Removing other protecting groups, cleaving the resin, and obtaining a polypeptide containing three pairs of disulfide bonds;

[0051] Preferably, the solid phase synthesis resin used in step 1) is 2-CTC solid phase synthesis resin, and the solid phase synthesis resin used in step 3) to synthesize the remaining fragments is Wang solid phase synthesis resin.

[0052] A specific embodiment of the present invention, the fourth scheme, provides a method for synthesizing a polypeptide containing three pairs of disulfide bonds.

[0053] The polypeptide containing three 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 is formed by two cysteines separated by one cysteine, and any one or more cysteines forming the second pair of disulfide bonds and the third pair of disulfide bonds are closer to the C-terminus than the two cysteines forming the first pair of disulfide bonds;

[0054] The synthesis method comprises the following steps:

[0055] 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 of the cysteines forming a second pair of disulfide bonds is located between the two cysteines forming the first pair of disulfide bonds; the sulfhydryl groups of the two cysteines forming the first pair of disulfide bonds are protected with a sulfhydryl protecting group Trt or Mmt; and one of the cysteines forming the second pair of disulfide bonds is protected with a sulfhydryl protecting group Acm;

[0056] 2) removing the sulfhydryl protecting groups of the two cysteines of the first pair of disulfide bonds in the first polypeptide fragment, and oxidizing to form the first pair of disulfide bonds;

[0057] 3) Under solid phase conditions, continue to synthesize the remaining fragments by solid phase synthesis, wherein the remaining fragments contain cysteine ​​forming the second pair of disulfide bonds and cysteine ​​forming the third pair of disulfide bonds, wherein the thiol groups of the cysteine ​​forming the second pair of disulfide bonds and the cysteine ​​forming the third pair of disulfide bonds are both protected by thiol protecting groups; the thiol protecting groups of the two cysteines forming the second pair of disulfide bonds are the same, which is Acm; the thiol protecting groups of the cysteines forming the third pair of disulfide bonds are the same, which is Trt or Mmt; the remaining fragments are present at the N-terminus or C-terminus of the first polypeptide fragment, or a part of the remaining fragments are present at the N-terminus of the first polypeptide fragment and the other part is present at the C-terminus of the first polypeptide fragment, to form a polypeptide-resin conjugate;

[0058] 4) removing the thiol protecting group of cysteine ​​of the third pair of disulfide bonds to form the third pair of disulfide bonds, and then removing the thiol protecting group of cysteine ​​of the second pair of disulfide bonds to form the second pair of disulfide bonds;

[0059] 5) Removing other protecting groups, cleaving the resin, and obtaining a polypeptide containing three pairs of disulfide bonds;

[0060] Preferably, in step 2), the solid phase synthetic resin is removed while the thiol protecting group is removed;

[0061] Preferably, the solid phase synthesis resin used in step 1) is 2-CTC solid phase synthesis resin, and the solid phase synthesis resin used in step 3) to synthesize the remaining fragments is Wang solid phase synthesis resin.

[0062] In the technical solution of the present invention, the two adjacent cysteines refer to the two adjacent cysteines having no other cysteines between them, but containing other amino acids or not containing other amino acids.

[0063] In the technical solution of the present invention, the two cysteines separated by one cysteine ​​refer to a cysteine ​​between the two cysteines forming a disulfide bond. In addition, other amino acids may or may not be contained between the two cysteines forming a disulfide bond.

[0064] In the technical scheme of the present invention, in step 1), any method of coupling amino acids or peptides on a solid phase synthesis resin is used as a solid phase synthesis method, for example, an amino acid or peptide with an Fmoc-protected α-amino group is coupled on a solid phase synthesis resin, the Fmoc protecting group is removed, and then the amino acid or peptide with an Fmoc-protected α-amino group is coupled and the Fmoc protecting group is removed to form the couplings in sequence.

[0065] In the technical solution of the present invention, in step 1), the N-terminus of the conjugate of the first polypeptide fragment and the solid phase synthetic resin has an Fmoc protecting group.

[0066] In the technical solution of the present invention, in step 1), the two cysteines forming the first pair of disulfide bonds in the first polypeptide fragment contain 0-3 amino acids on both sides respectively, and do not contain cysteine, preferably 0, 1 or 2 amino acids.

[0067] In the technical scheme of the present invention, in step 2), the method of removing the thiol protecting groups of the two cysteines forming the first pair of disulfide bonds and oxidizing to form disulfide bonds is as follows: the first polypeptide fragment only contains the two cysteines forming the first pair of disulfide bonds, 1-20 equivalents of iodine are dissolved in an appropriate amount of DMF, and the thiol protecting groups are removed and disulfide bonds are formed at room temperature;

[0068] Alternatively, the first polypeptide fragment contains two cysteines forming the first pair of disulfide bonds and one cysteine ​​forming the second pair of disulfide bonds, and TFA:TIS:DCM=(1-5):(2-10):(97-85) is used to remove the two cysteine ​​sulfhydryl protecting groups of the first pair of disulfide bonds, and then 1-30 equivalents of H2 O 2 Oxidation is performed to form the first pair of disulfide bonds.

[0069] In the technical solution of the present invention, the first polypeptide fragment contains two cysteines or three cysteines.

[0070] In the technical scheme of the present invention, in step 3), the method for continuing to synthesize the remaining fragments under solid phase conditions is to use a solid phase synthesis method to synthesize and 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 contains 6 cysteines, of which 2 cysteines have formed disulfide bonds; the synthesis method includes but is not limited to adding amino acids to the N-terminus of the polypeptide fragment, synthesizing the polypeptide fragment by a solid phase synthesis method, and coupling each polypeptide fragment, or coupling a single added amino acid and a fragment. Preferably, it includes but is not limited to the following:

[0071] a. Continue solid phase coupling the remaining amino acids at the N-terminus of the first polypeptide fragment;

[0072] b. synthesizing a conjugate of the second polypeptide fragment and the solid phase synthetic resin under solid phase conditions, cleaving the solid phase synthetic resin on one of the first polypeptide fragment and the second polypeptide fragment, and coupling the first polypeptide fragment with the second polypeptide fragment to obtain a conjugate of the third polypeptide fragment and the solid phase synthetic resin;

[0073] c. synthesizing a conjugate of the second polypeptide fragment and the solid phase synthetic resin under solid phase conditions, continuing to couple amino acids at the N-terminus of the first polypeptide fragment and or at the N-terminus of the second polypeptide fragment, and then coupling the first polypeptide fragment or the second polypeptide fragment with the added amino acids to obtain a conjugate of the fourth polypeptide fragment and the solid phase synthetic resin;

[0074] d. In the three cases a, b, and c, the conjugates of the polypeptide fragment and the solid phase synthetic resin are obtained and then the amino acids are further coupled.

[0075] In the technical scheme of the present invention, the method of first removing the thiol protecting group of cysteine ​​and oxidizing to form a disulfide bond in step 4) is to use TFA:TIS:DCM=(1-5):(2-10):(97-85) to remove the two cysteine ​​thiol protecting groups Trt or Mmt of the disulfide bond, and then use 1-30 equivalents of H 2 O 2 The DMF solution is first solid-phase oxidized at room temperature to form a pair of disulfide bonds; then 1-20 equivalents of iodine are dissolved in an appropriate amount of DMF, the thiol protecting group Acm is removed at room temperature and a disulfide bond is formed.

[0076] In a preferred technical scheme of the present invention, in step 1), the number of 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.

[0077] In a preferred technical scheme of the present invention, in step 1), the number of cysteines contained in the first polypeptide fragment is three, the cysteines forming the first pair of disulfide bonds also contain one of the cysteines forming the second pair of disulfide bonds, the thiol protecting group of the cysteines forming the first pair of disulfide bonds is either Mmt or Trt, the thiol protecting group of the cysteines forming the second pair of disulfide bonds is Acm, and the thiol protecting group of the cysteines forming the third pair of disulfide bonds is either Mmt or Trt.

[0078] In the technical solution of the present invention, the method of cracking the resin and removing other protecting groups in step 5) is to remove them with 80-95% TFA solution.

[0079] In the technical scheme of the present invention, the solid phase synthesis method in step 1-3) is an Fmoc solid phase synthesis strategy, wherein an amino acid with Fmoc-protected amino group is coupled on a solid phase synthesis resin, and then the Fmoc protecting group is removed, and the amino acid with Fmoc-protected amino group is continued to be coupled until the synthesis of the polypeptide is completed.

[0080] In the technical solution of the present invention, the solid phase synthetic resin is selected from 2-CTC solid phase synthetic resin and Wang solid phase synthetic resin.

[0081] In the technical solution of the present invention, preferably, in the above-mentioned first solution and second solution, Wang solid phase synthetic resin is selected; in the above-mentioned third solution and fourth solution, 2-CTC solid phase synthetic resin is selected in step 1), and Wang solid phase synthetic resin is selected in step 3).

[0082] In the technical solution of the present invention, when the first pair of disulfide bonds, the second pair of disulfide bonds or the third pair of disulfide bonds are formed, the Fmoc protecting group at the end of the polypeptide is retained.

[0083] In the technical solution of the present 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 solid phase conditions.

[0084] Beneficial Effects

[0085] 1) The method of the present invention can prepare polypeptides containing three pairs of disulfide bonds, and the three pairs of polypeptides are selectively deprotected, which greatly reduces the possibility of mismatching. The combination of Mmt or Trt and Acm can achieve high orthogonal selectivity, simple removal conditions and high yield.

[0086] 2) The method of the present invention can selectively form disulfide bonds by using only two thiol protecting groups. The removal conditions of the two thiol protecting groups selected in the present invention are mild and will not affect the coupling of other amino acids.

[0087] 3) The disulfide bonds formed by the method of the present invention are formed under solid phase conditions as much as possible, rather than liquid phase conditions, which can maximize the purification efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 This is the crude peptide mass spectrum of the 6-peptide fragment containing a pair of disulfide bonds in Example 4 (Mw: 615).

[0089] Figure 2 This is the mass spectrum of the crude peptide sample containing a pair of disulfide bonds in Example 8 (Mw: 1672)

[0090] Figure 3 This is the mass spectrum of the crude peptide sample containing two pairs of disulfide bonds in Example 8 (Mw: 1670)

[0091] Figure 4 The mass spectrum of the crude peptide containing three pairs of disulfide bonds in Example 8 (Mw: 1526) DETAILED DESCRIPTION

[0092] To achieve the above objectives, the present invention adopts the following four technical solutions: (illustrated using linaclotide disulfide bond mismatch isomers)

[0093] Scheme 1: There is a disulfide bond between two adjacent Cys residues at the C-terminus (IM-A; IM-B; IM-C in Table 1)

[0094] These three disulfide mismatched isomers all contain Cys 10 -Cys 13 Disulfide bonds: Fmoc-Tyr(tBu)-Wang resin was selected as the starting resin. Then, each residue was coupled to Cys in sequence according to the Fmoc / tBu solid phase synthesis strategy. 10 or Asn 7 (Cys 10 or Asn 7 Any site in the 13 and Cys 10Using the same protecting group (Trt, Mmt or Acm), after the peptide resin fragment is coupled, 1-20 equivalents of iodine are weighed and dissolved in an appropriate amount of DMF, and the peptide resin fragment is oxidized at room temperature for 0.5-3h. After the reaction is completed, the peptide resin fragment is washed with DMF to clean the residual iodine. After the washing is completed, the subsequent residues are coupled in sequence according to the Fmoc / tBu solid phase synthesis strategy. According to the different binding modes of the disulfide bond, the four Cys residues are orthogonally protected with Trt or Mmt and Acm (preferably Mmt and Acm are orthogonal). After all the residues are coupled, the Mmt or Trt protecting group is removed with TFA:TIS:DCM=2:5:93 solution, and then 1-30 equivalents of H 2 O 2 The DMF solution is first solid-phase oxidized at room temperature to form a second pair of disulfide bonds, and the reaction time is 0.5-3h. Then, 1-20 equivalents of iodine in DMF solution are continued to be used to carry out a third pair of disulfide bond oxidation reaction on the peptide resin at room temperature, and the reaction time is 0.5-3h. After the reaction is completed, the peptide resin is washed and dried. After shrinkage and drying, the peptide resin is cleaved by TFA, and the crude peptide is precipitated and washed with tertiary methyl ether or ether, and then vacuum dried to obtain the crude peptide of the target peptide, which is purified by HPLC to obtain the refined peptide.

[0095]

[0096]

[0097] Scheme 2: There is a disulfide bond between two Cys residues at the C-terminus with one Cys between them (IM-G; IM-J; IM-K in Table 1)

[0098] These three disulfide mismatched isomers all contain Cys 6 -Cys 13 Disulfide bonds: Fmoc-Tyr(tBu)-Wang resin was selected as the starting resin. Then, each residue was coupled to Cys in sequence according to the Fmoc / tBu solid phase synthesis strategy. 6 , where Cys 10 Acm protection, Cys 6 and Cys 13 Mmt or Trt protection was used. After the peptide resin fragment was coupled, TFA:TIS:DCM = 2:5:93 solution was used to remove Cys 6 and Cys 13 The Mmt or Trt protecting group was then used with 1-30 equivalents of H 2 O 2The DMF solution is first solid-phase oxidized at room temperature to form the first pair of disulfide bonds, and the reaction time is 0.5-3h. After the reaction is completed, the subsequent residues are coupled. According to the different binding modes of the disulfide bonds, the remaining three Cys residues in the peptide sequence are orthogonally protected by Trt or Mmt and Acm (preferably Mmt and Acm are orthogonal). After all the residues are coupled, the Mmt or Trt protecting group is removed by using TFA:TIS:DCM=2:5:93 solution, and then 1-30 equivalents of H 2 O 2 The DMF solution is solid-phase oxidized at room temperature to form a second pair of disulfide bonds, and the reaction time is 0.5-3h. Then, 1-20 equivalents of iodine in DMF solution are used to carry out a third pair of disulfide bond oxidation reaction on the peptide resin fragment at room temperature, and the reaction time is 0.5-3h. After the reaction is completed, the peptide resin is washed and dried. After shrinkage and drying, the peptide resin is cleaved by TFA, and the crude peptide is precipitated with tertiary methyl ether or ether, washed, and then vacuum dried to obtain the crude peptide of the target peptide, which is purified by HPLC to obtain the refined peptide.

[0099]

[0100] Scheme 3: There is a disulfide bond 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).

[0101] These disulfide bond mismatch isomers all contain disulfide bonds between adjacent Cys: Cys 1 -Cys 2 ;Cys 2 -Cys 5 ;Cys 5 -Cys 6 Cys 6 -Cys 10 First, a fully protected peptide fragment containing a disulfide bond is synthesized, and then the fully protected peptide fragment is synthesized into a peptide sequence (with Cys 1 -Cys 2 、Cys 5 -Cys 6 Brief description, others are similar):

[0102] 3.1、Containing Cys 1 -Cys 2 Synthesis of fragments: 2-CTC resin was used as the starting resin, and Cys 2 Glu 3 , Tyr 4Fmoc protected amino acid corresponding to any residue. After the synthesis of the amino acid resin is completed, the subsequent residues are coupled in sequence according to the peptide sequence to obtain the corresponding dipeptide, tripeptide, and tetrapeptide fragment resins (note to retain the Fmoc group at the N-terminus). Then, a 1-20 equivalent of iodine DMF solution is used 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 0.5-3h. After the reaction is completed, the peptide resin is washed and dried. After shrinkage and drying, the peptide resin is cracked with 20% TFE / DCM for 1h and then filtered. The filtrate is concentrated under reduced pressure to obtain a product containing a pair of disulfide bonds (Cys 1 -Cys 2 )'s fully protected peptide fragment.

[0103] 3.2、Containing Cys 5 -Cys 6 Synthesis of fragments: 2-CTC resin was used as the starting resin, and Cys 6 , Asn 7 , Pro 8 , Ala 9 The Fmoc protected amino acid corresponding to any residue. After the amino acid resin synthesis is completed, the subsequent residues are coupled in sequence according to the peptide sequence until Cys 5 , Tyr 4 Glu 3 The Fmoc protected amino acid corresponding to any residue. The corresponding dipeptide to heptapeptide fragment resin is obtained (note to retain the Fmoc group at the N-terminus). Then, a 1-20 equivalent iodine DMF solution is used to carry out an oxidation reaction of the disulfide bond between two adjacent Cys residues on the peptide resin fragment at room temperature, and the reaction time is 0.5-3h. After the reaction is completed, the peptide resin is washed and dried. After shrinkage and drying, the peptide resin is cracked with 20% TFE / DCM for 1h and then filtered. The filtrate is concentrated under reduced pressure to obtain a product containing a pair of disulfide bonds (Cys 5 -Cys 6 )'s fully protected peptide fragment.

[0104] 3.3 Synthesis of peptide resin: Fmoc-Tyr(tBu)-Wang resin was selected as the starting resin. Then, each residue was coupled in sequence according to the Fmoc / tBu solid phase synthesis strategy (the sequential coupling was performed according to the sequence of the fully protected fragment, and at the same time, the sulfhydryl protecting groups in the other 4 Cys residues except the fully protected fragment were orthogonally protected by Mmt or Trt and Acm according to the different disulfide bond bonding modes). After the peptide resin fragment was coupled, the Mmt or Trt protecting group was removed using a TFA:TIS:DCM=2:5:93 solution, and then 1-30 equivalents of H 2 O 2The DMF solution is solid-phase oxidized at room temperature to form a second pair of disulfide bonds, and the reaction time is 0.5-3h. Then, 1-20 equivalents of iodine in DMF solution are used to carry out the third pair of disulfide bond oxidation reaction on the peptide resin at room temperature, and the reaction time is 0.5-3h. After the reaction is completed, the peptide resin is washed and dried. After shrinkage and drying, the peptide resin is cleaved by TFA, and the crude peptide is precipitated and washed with tertiary methyl ether or ether, and then vacuum dried to obtain the crude peptide of the target, and purified by HPLC to obtain the refined peptide.

[0105] Scheme 4: There is a disulfide bond between two Cys residues at other sites with one Cys between them (IM-B; IM-G; IM-I; IM-L; IM-M; IM-N in Table 1)

[0106] These disulfide bond mismatch isomers all contain two Cys residues separated by a Cys disulfide bond: Cys 1 -Cys 5 ;Cys 2 -Cys 6 ;Cys 5 -Cys 10 First, a fully protected peptide fragment containing a disulfide bond is synthesized, and then the fully protected peptide fragment is introduced into the synthetic peptide sequence by fragment condensation (with Cys 1 -Cys 5 ;Cys 2 -Cys 6 Brief description, others are similar):

[0107] 4.1、Containing Cys 1 -Cys 5 Synthesis of fragments: 2-CTC resin was used as the starting resin, and Cys 5 The corresponding Fmoc protected amino acid. After the amino acid resin is synthesized, the subsequent residues are coupled in sequence according to the peptide sequence to obtain the pentapeptide fragment resin (Note: Cys 1 , Cys 5 Using Mmt protection, Cys 2 Acm protection was used while retaining the Fmoc group at the N-terminus). The resin was treated with a TFA:TIS:DCM=2:5:93 solution for 1-3h. Cys was removed 1 and Cys 5 The Mmt group is removed and the peptide fragment is cleaved from the resin. Filter the filtrate, adjust the pH of the filtrate to a weakly alkaline pH of 7.0-8.0 with DIPEA, and then add 1-30 equivalents of H 2 O 2 The reaction was stirred at room temperature for 0.5-3h. After the reaction was completed, the solution was added with H 2 O 2The solution was concentrated under reduced pressure to obtain a crude peptide fragment, which was purified to obtain a 5-peptide fragment containing a disulfide bond with a purity of >95%.

[0108] 4.2、Containing Cys 2 -Cys 6 Synthesis of fragments: 2-CTC resin was used as the starting resin, and Cys 6 , Asn 7 , Pro 8 , Ala 9 Fmoc protected amino acid corresponding to any residue. After the amino acid resin synthesis is completed, the subsequent residues are coupled to Cys in sequence according to the peptide sequence. 2 The corresponding Fmoc protected amino acids were obtained by 5- to 8-peptide fragment resins (Note: Cys 2 , Cys 6 Using Mmt protection, Cys 5 Acm protection was used while retaining the Fmoc group at the N-terminus). The resin was treated with a TFA:TIS:DCM=2:5:93 solution for 1-3h. Cys was removed 1 and Cys 5 The Mmt group is removed and the peptide fragment is cleaved from the resin. Filter the filtrate, adjust the pH of the filtrate to a weakly alkaline pH of 7.0-8.0 with DIPEA, and then add 1-30 equivalents of H 2 O 2 The reaction was stirred at room temperature for 0.5-3h. After the reaction was completed, the solution was added with H 2 O 2 The solution was concentrated under reduced pressure to obtain a crude peptide fragment, which was purified to obtain a fully protected peptide fragment containing a disulfide bond with a purity of >95%.

[0109] 4.3 Synthesis of peptide resin: Fmoc-Tyr(tBu)-Wang resin was selected as the starting resin. Then, each residue was coupled in sequence according to the Fmoc / tBu solid phase synthesis strategy (the sequential coupling was performed according to the sequence of the fully protected fragment, and at the same time, the thiol protecting groups in the other three Cys residues except the fully protected fragment were orthogonally protected by two Mmt or Trt and one Acm according to the different binding modes of the disulfide bond.). After the peptide resin fragment was coupled, the Mmt or Trt protecting group was removed using a TFA:TIS:DCM=2:5:93 solution, and then 1-30 equivalents of H 2 O 2The DMF solution is solid-phase oxidized at room temperature to form a second pair of disulfide bonds, and the reaction time is 0.5-3h. Then, 1-20 equivalents of iodine in DMF solution are used to carry out the third pair of disulfide bond oxidation reaction on the peptide resin at room temperature, and the reaction time is 0.5-3h. After the reaction is completed, the peptide resin is washed and dried. The dried peptide resin is cleaved by TFA, and the filtrate is precipitated and washed with tertiary methyl ether or diethyl ether, and then vacuum dried to obtain the target peptide crude product, which is purified by HPLC to obtain the refined peptide.

[0110]

[0111] Step 1: According to the Fmoc / tBu solid phase synthesis strategy, a resin or fully protected peptide fragment containing a pair of disulfide bond fragments is synthesized.

[0112] Step 2: If the resin synthesized in step 1 contains a pair of disulfide bond fragments, then continue to sequentially couple the Fmoc-AA-OH corresponding to each subsequent residue to the peptide resin to complete the coupling according to the peptide sequence. If the fully protected peptide fragment synthesized in step 1 contains a pair of disulfide bonds, then follow the Fmoc / tBu solid phase synthesis strategy to sequentially couple the Fmoc-AA-OH corresponding to each other residue in the peptide sequence; the fully protected peptide containing disulfide bonds is coupled in a fragment condensation manner to complete the synthesis of the peptide resin. The thiol protecting group of the Cys residue is orthogonally protected by Mmt, Trt and Acm.

[0113] Step 3: Use dilute acid solution to remove the Mmt or Trt protecting group, and then use H 2 O 2 The exposed sulfhydryl groups are oxidized by oxidants such as 4-hydroxy-2-nitropropene to form a second disulfide bond.

[0114] Step 4: Finally adopt I 2 Oxidation is performed to bond the third disulfide bond. After the reaction is completed, the residual I 2 , shrink and dry the peptide resin.

[0115] Step 5: Prepare a certain concentration of TFA cleavage solution, cleave the peptide resin, precipitate with ether or tert-methyl ether and wash to obtain the crude peptide.

[0116] Step 6: Purify the crude peptide by reverse phase chromatography and freeze-dry to obtain refined peptide.

[0117] In step 1, the synthesis is carried out according to the Fmoc / tBu solid phase peptide synthesis strategy. The starting resin needs to select one of WangResin and 2CTC Resin according to different situations. If the synthesized peptide fragment containing disulfide bonds is selected, Wang Resin is selected; if the synthesized peptide fragment containing disulfide bonds is fully protected, 2CTC Resin is selected. The resin substitution degree range is 0.20-0.75mmol / g. The synthesis of amino acid resin (Wang Resin, 2CTC Resin) is a conventional synthesis method. Coupling of subsequent residues: 1) the resin is swollen in a solid phase reaction column with a solvent for 15-60 min, and the selected solvent includes DMF, NMP, dichloromethane, etc., preferably DMF; 2) the Fmoc protecting group is removed with a 20% piperidine / DMF solution, and the treatment time and number are: 5-10 min / time*2-3 times, and the resin is repeatedly washed 6-7 times with an appropriate amount of solvent; 3) after dissolving and activating an appropriate amount of the amino acid to be coupled and the coupling agent in a solvent, they are added together to the solid phase reaction column until the reaction is terminated by the ninhydrin detection method; 4) 2) and 3) are repeated. The coupling agent in the above 3) is a combination of DIPCDI and compound A or a combination of DIPEA, compound A and compound B, wherein compound A is HOAt or HOBt, and compound B is PyAOP, PyBOP, HATU, HBTU or TBTU, preferably a combination of DIPCDI and compound A. Furthermore, the ratio of each component in the coupling agent to the protected amino acid (AA) is calculated as DIPCDI:A:AA=1.3:1.2:1.0, DIPEA:A:B:AA=2.0:1.2:1.0:1.0 in terms of molar ratio. In addition, the coupling reaction time of each amino acid is usually 1.0-4h, and the reaction end point is determined by the ninhydrin detection method; the temperature is room temperature (i.e., 20±5°C), and it can also be carried out at an appropriately increased or decreased temperature. After the coupling is completed, the resin is oxidized or the fully protected peptide fragment is prepared. Different methods are generally used according to different situations: one is to directly use 1-20 equivalents of iodine in DMF solution to oxidize the peptide resin fragment at room temperature for 0.5-3h. After the reaction is completed, the fragment resin containing disulfide bonds is obtained by washing. If the fragment resin is cleaved with 20% TFE / DCM (2CTC Resin is the starting resin), the corresponding fully protected peptide fragment is obtained; the other is to use TFA:TIS:DCM=2:5:93 solution to remove the Mmt or Trt protecting group, and then use 1-30 equivalents of H 2 O 2The DMF solution is solid-phase oxidized at room temperature to form a second pair of disulfide bonds. The reaction time is 0.5-3h. After the reaction is completed, the fragment resin containing disulfide bonds is obtained by washing. The third method is to treat the resin with a solution of TFA:TIS:DCM=2:5:93 for 1-3h. The Mmt group is removed and the peptide fragment is cut off from the resin. Filter, adjust the pH of the filtrate to a weakly alkaline pH of 7.0-8.0 with DIPEA, and then add 1-30 equivalents of H 2 O 2 The reaction was stirred at room temperature for 0.5-3h. After the reaction was completed, the solution was added with H 2 O 2 The solution was concentrated under reduced pressure to obtain a crude peptide fragment. The crude peptide fragment was purified to obtain a disulfide bond-containing fully protected peptide fragment with a purity of >95% for standby use. The fully protected peptide fragment needs to retain the terminal Fmoc protecting group.

[0118] In step 2, the subsequent protected amino acid residues are coupled in sequence according to the Fmoc / tBu solid phase synthesis method, and the coupling method is as described in step 1. It should be noted that in some cases, when it is necessary to couple a fully protected peptide containing a pair of disulfide bond fragments by fragment condensation, the selected solvent includes DMF, NMP, dichloromethane, etc., preferably DCM (containing a small amount of DMF) to increase the solubility of the fully protected peptide and inhibit the racemization during activation and coupling. The coupling system is preferably a combination of DIPCDI and HOBt. After the synthesis is completed, a peptide resin containing a pair of disulfide bond intermediates is obtained.

[0119] In step 3, the method for removing the Mmt protecting group is to use a mixed solution of TFA:TIS:DCM=(1-5):(2-10):(97-85) to react with the resin for 15-30 times, each time for 1-5 minutes, preferably the solution ratio is TFA:TIS:DCM=2:5:93, the number of reactions is 20 times and each time for 2 minutes; and the disulfide bond is formed by using 1-30 equivalents of H 2 O 2 The resin is reacted for 0.5-3h, preferably 10 equivalents for 1h, and the reaction solvent is the same as listed in step 1, preferably dimethylformamide. The resin can be detected with 0.1-0.2mmol / l DTNB solution in an alkaline environment. If the resin is colorless, it means that the disulfide cyclization is complete.

[0120] Step 4: Wash the resin in step 3, and then use I 2 To bond the third disulfide bond, the solvent is selected as listed in step 1, preferably dimethylformamide, and I 2 The dosage is 1-10 equivalents of the resin substance. The reaction time is 0.5-3h. After the reaction is completed, the peptide resin is obtained by washing, shrinking and drying.

[0121] The lysate used in step 5 is TFA, H 2 A mixture of different proportions of TFA:TIS:H O, TIS, anisole, thioanisole, phenol, etc. Note that the crude peptide obtained in this step is cyclized, so components such as EDT and DTT that can reduce disulfide bonds should be avoided in the lysate. The lysate ratio is: TFA:TIS:H 2 O=(80-90):(0-10):(0-10) mixed solution; the preferred ratio is TFA:TIS:H 2 O=92:4:4, the reaction temperature is room temperature, the reaction time is 2-5h, preferably 3h. The amount of lysate used is preferably 10 times the volume of the resin weight. After the reaction is completed, filter, precipitate the obtained filtrate with ether or methyl tert-butyl ether and centrifuge, and continue to wash the obtained precipitate with ether or methyl tert-butyl ether for 3 times and then vacuum dry to obtain a crude isomer peptide containing 3 pairs of disulfide bonds.

[0122] The purification step in step 6 may be performed by reverse phase high pressure liquid chromatography. Further, the reverse phase high pressure liquid chromatography comprises: using reverse phase octadecylsilane as the stationary phase, using 0.1% trifluoroacetic acid aqueous solution / acetonitrile as the mobile phase, collecting the target peak fraction, concentrating and freeze drying.

[0123] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the specific implementation modes of the present invention are described in detail below, but it should not be understood as limiting the applicable scope of the present invention.

[0124] Abbreviations and English meanings

[0125]

[0126]

[0127] Example 1: Preparation of Fmoc-Tyr(tBu)-Wang Resin

[0128] Weigh 100g (103mmol) of Wang resin with a substitution degree of 1.03mmol / g into a solid phase reaction column, wash it twice with an appropriate amount of DMF, and then swell the resin with DMF for 30 minutes. Weigh 45.9g of Fmoc-Tyr(tBu)-OH (100mmol); 16.2g (120mmol) of HOBt, 1.2g (10mmol) of DMAP, dissolve it with an appropriate amount of DMF / DCM (1:1), add 20.3mL DIC (120mmol) in a 0℃ ice water bath, activate for 3-5min, add the activated solution to the reaction column and react for 2.5 hours. After the reaction is completed, wash it with DMF 5 times, add 70mL of acetic anhydride and 60mL of pyridine, and add an appropriate amount of DMF to ensure that the resin is stirred evenly. After mixing and sealing for more than 8 hours, the resin was washed with DMF 5 times, DCM 3 times, and methanol shrunk 2 times, and then the resin was dried to obtain 142g of Fmoc-Tyr(tBu)-WangResin, and the substitution degree was 0.55mmol / g.

[0129] Example 2: Preparation of Fmoc-Cys(Mmt)-2CTC Resin

[0130] Weigh 100g (120mmol) of 2CTC resin with a substitution degree of 1.20mmol / g in a solid phase reaction column, wash it twice with an appropriate amount of DMF, and then swell the resin with DMF for 30 minutes. Weigh 61.6 grams of Fmoc-Cys(Mmt)-OH (100mmol) and dissolve it with an appropriate amount of DMF / DCM (1:1). After cooling the solution to 0°C, add it to the reaction column, and add 31.0g and 15.5g of DIPEA (61mL, 360mmol) twice and react for 1.5 hours. After the reaction is completed, add 90mL of methanol and 30mL of DIPEA to the reaction column, continue to react for 30min, wash it with DMF 5 times, wash it with DCM three times, shrink it with methanol twice, and then drain the resin to obtain a total of 151g of Fmoc-Cys(Mmt)-2CTC Resin, and the substitution degree is detected to be 0.65mmol / g.

[0131] Example 3: Preparation of Fmoc-Gly-2CTC Resin

[0132] Weigh 50g (60mmol) of 2CTC resin with a substitution degree of 1.20mmol / g in a solid phase reaction column, wash twice with an appropriate amount of DMF, and then swell the resin with DMF for 30 minutes. Weigh 14.9 grams of Fmoc-Gly-OH (50mmol) and dissolve it with an appropriate amount of DMF, add the solution to the reaction column, and add 15.5g and 7.7g of DIPEA (30.5mL, 180mmol) twice and react for 1.5 hours. After the reaction is completed, add 60mL of methanol and 20mL of DIPEA to the reaction column, continue to react for 30min, wash with DMF 5 times, wash three times with DCM, shrink the methanol twice, and drain the resin to obtain a total of 59g of Fmoc-Gly-2CTC Resin, and the detection degree of substitution is 0.71mmol / g.

[0133] Example 4: Synthesis of a peptide resin fragment containing a pair of disulfide bonds between two adjacent Cys (SS:Cys 10-13 )

[0134] Weigh 18.2 g of Fmoc-Tyr(tBu)-Wang Resin (Sub=0.55 mmol / g, 10 mmol) obtained in Example 1 into a solid phase reaction column, wash twice with DMF, and then swell with DMF for 30 minutes. Then deprotect with DBLK twice (5min+7min), continue to wash with DMF 6 times, and wash with DCM once. Weigh 18.5 g of Fmoc-Cys(Mmt)-OH (30 mmol) and 4.9 g of HOBt (36 mmol) and dissolve them in 50 mL of DMF / DCM (1:1). Add 6 mL (39 mmol) of DIPCDI in an ice water bath to activate for 2-3 minutes, then add the mixed solution to the reaction column, react at room temperature for 2 hours, and detect the end point of the reaction with ninhydrin (if the resin is colorless and transparent, terminate the reaction; if the resin is colored, extend the reaction for 1 hour). After the reaction is completed, wash the resin 3 times with 80mL DMF, then deprotect with DBLK 2 times (5min+7min), continue to wash with DMF 6 times, and wash with DCM once. The resin is colored when detected by ninhydrin. Repeat the above coupling operation, and continue to couple Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Mmt)-OH, and Fmoc-Ala-OH in sequence. Each protected amino acid is fed with 30mmol, and the feed ratio of other materials corresponds to the protected amino acid. After the above residues are coupled, wash with DMF 6 times. Weigh 7.6g of iodine (30mmol) and add it to the reactor. At the same time, add 70mL of DMF and react at room temperature for 1h. After the reaction is completed, wash the resin 3 times with 80mL DMF, then deprotect with DBLK 2 times (5min+7min), continue to wash with DMF 6 times, and wash with DCM once. That is, NH 2-Ala-Cys 10 -Thr(tBu)-Gly-Cys 13 -Tyr(tBu)-Wang Resin(SS:Cys 10-13 ) containing a pair of disulfide bonds.

[0135] Example 5: Synthesis of a pair of disulfide bond peptide resin fragments (SS:Cys 6-13 )

[0136] Weigh 18.2 g of Fmoc-Tyr(tBu)-Wang Resin (Sub=0.55 mmol / g, 10 mmol) obtained in Example 1 into a solid phase reaction column, wash twice with DMF, and then swell with DMF for 30 minutes. Then deprotect with DBLK twice (5min+7min), continue to wash with DMF 6 times, and wash with DCM once. Weigh 18.5 g of Fmoc-Cys(Mmt)-OH (30 mmol) and 4.9 g of HOBt (36 mmol) and dissolve them in 50 mL of DMF / DCM (1:1). Add 6 mL (39 mmol) of DIPCDI in an ice water bath to activate for 2-3 minutes, then add the mixed solution to the reaction column, react at room temperature for 2 hours, and detect the end point of the reaction with ninhydrin (if the resin is colorless and transparent, terminate the reaction; if the resin is colored, extend the reaction for 1 hour). After the reaction is completed, the resin is washed 3 times with 80 mL DMF, then deprotected with DBLK 2 times (5 min + 7 min), and then washed with DMF 6 times and DCM 1 time. The resin is colored when detected by ninhydrin. Repeat the above coupling operation, and continue to sequentially 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 with 30 mmol, and the feed ratio of other materials corresponds to the protected amino acid. After the above residues are coupled, wash with DMF 6 times. Then use TFA:TIS:DCM=2:5:93, the number of reactions is 20 times and each time is 2 minutes to remove Mmt from the peptide resin. After the end, the resin is washed 6 times with a DMF solution containing 1% DIPEA. Measure 30% hydrogen peroxide 3mL (30mmol) and add it to 80mL DMF, and then add it to the solid phase reaction column to react for 1h. After the reaction is completed, wash the resin 3 times with 80mL DMF, then deprotect with DBLK 2 times (5min+7min), continue to wash with DMF 6 times, and wash with DCM once. NH 2 -Cys 6 -Asn(Trt)-Pro-Ala-Cys(Acm)10 -Thr(tBu)-Gly-Cys 13 -Tyr(tBu)-Wang Resin(SS:Cys 6-13 ) containing a pair of disulfide bonds.

[0137] Example 6: Synthesis of a fully protected fragment containing a pair of disulfide bonds (SS:Cys 6-10 )

[0138] Weigh 42.2 g of Fmoc-Gly-2CTC Resin (Sub = 0.71 mmol / g, 30 mmol) obtained in Example 3 into a solid phase reaction column, wash twice with DMF, and then swell with DMF for 30 minutes. Then deprotect with DBLK twice (5min + 7min), and continue to wash with DMF for 6 times. Weigh 35.8 g of Fmoc-Thr(tBu)-OH (90 mmol) and 14.6 g of HOBt (108 mmol) and dissolve them in 120 mL of DMF / DCM (1:1). Add 18 mL (117 mmol) of DIPCDI in an ice water bath to activate for 2-3 minutes, then add the mixed solution to the reaction column, react at room temperature for 2 hours, and detect the end point of the reaction with ninhydrin (if the resin is colorless and transparent, terminate the reaction; if the resin is colored, extend the reaction for 1 hour). After the reaction is completed, the resin is washed 3 times with 160 mL of DMF, then deprotected with DBLK 2 times (5 min + 7 min), washed with DMF 6 times, and washed with DCM 1 time. The resin is colored when detected by ninhydrin. Repeat the above coupling operation, and continue to sequentially couple Fmoc-Cys (Mmt) -OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc-Cys (Mmt) -OH, each protected amino acid is fed with 90 mmol, and the feed ratio of other materials corresponds to the protected amino acid. After the above residues are coupled, wash with DMF 6 times. Weigh 22.9 g of iodine (90 mmol) and add it to the reactor, add 150 mL of DMF at the same time, and react at room temperature for 1 hour. After the reaction was completed, the resin was washed with DMF 4 times and DCM 2 times, and then dried after methanol shrinkage to obtain 61.4 g of fragment peptide resin. The peptide resin was cleaved by 20% TFE / DCM for 1 h and then filtered. The filtrate was concentrated under reduced pressure to obtain 28.4 g of product, namely Fmoc-Cys 6 -Asn(Trt)-Pro-Ala-Cys 10 -Thr(tBu)-Gly-OH(SS:Cys 6-10 ) containing a pair of disulfide bonds.

[0139] Example 7: Synthesis of a pair of disulfide bond peptide resin fragments (SS:Cys1-5 )

[0140] Weigh 46.2 g of Fmoc-Cys(Mmt)-2CTC Resin (Sub=0.65 mmol / g, 30 mmol) obtained in Example 2 into a solid phase reaction column, wash twice with DMF, and then swell with DMF for 30 minutes. Then deprotect with DBLK twice (5min+7min), and continue to wash with DMF for 6 times. Weigh 41.3 g of Fmoc-Tyr(tBu)-OH (90 mmol) and 14.6 g of HOBt (108 mmol) and dissolve them in 140 mL of DMF / DCM (1:1). Add 18 mL (117 mmol) of DIPCDI to activate for 2-3 min in an ice water bath, then add the mixed solution to the reaction column, react at room temperature for 2 h, and detect the end point of the reaction with ninhydrin (if the resin is colorless and transparent, terminate the reaction; if the resin is colored, extend the reaction for 1 h). After the reaction is completed, wash the resin 3 times with 160 mL of DMF, then deprotect with DBLK 2 times (5 min + 7 min), continue to wash with DMF 6 times, and wash with DCM once. The resin is colored when detected by ninhydrin. Repeat the above coupling operation. Continue to sequentially couple Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Acm)-OH, and Fmoc-Cys(Mmt)-OH. Each protected amino acid is fed with 90 mmol, and the feed ratio of other materials corresponds to the protected amino acid. After the above residues are coupled, wash with DMF 4 times and DCM 2 times. After shrinking with methanol, dry to obtain 82.3 g of fragment peptide resin. Stir the peptide resin with 800 mL of TFA:TIS:DCM=2:5:93 at room temperature for 1 hour. Remove Cys 1 and Cys 5 The Mmt group is removed and the peptide fragment is cut off from the resin. After the reaction is completed, filter the filtrate and adjust the pH of the filtrate to a weakly alkaline pH of 7.0-8.0 with DIPEA. Then, add 9 mL (90 mmol) of 30% hydrogen peroxide and stir at room temperature for 0.5-1 h. The end point of the reaction is determined by a 0.1-0.2 mmol / l DTNB solution. After the reaction is completed, add the solution with H 2 O 2 After adding an equivalent amount of ascorbic acid, the solution was concentrated under reduced pressure to obtain 29.5 g of crude peptide fragment. The crude peptide fragment was purified by HPLC to obtain 23.1 g of refined peptide with a purity of >95%. 1 -Cys(Acm) 2 -Glu(OtBu)-Tyr(tBu)-Cys 5 -OH(SS:Cys 1-5 ) containing a pair of disulfide bonds.

[0141] Example 8: Synthesis of peptide resin (1)

[0142] The NH obtained in Example 4 2 -Ala-Cys 10 -Thr(tBu)-Gly-Cys 13 -Tyr(tBu)-Wang Resin(SS:Cys 10-13 ) is used to carry out subsequent coupling with a fragment peptide resin containing a pair of disulfide bonds, and the sequence of IM-B in the present invention is used for synthesis. According to the operation in the above step 2, the subsequent residues are coupled in sequence: 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, Fmoc-Cys(Mmt)-OH, each protected amino acid is fed 30mmol, and the feed ratio of other materials corresponds to the protected amino acid. After the above residues are coupled, they are washed with DMF for 6 times. Then, TFA:TIS:DCM=2:5:93, the reaction number is 20 times and each time is 2 minutes to remove Mmt from the peptide resin. After the end, the resin is washed 6 times with a DMF solution containing 1% DIPEA. 30% hydrogen peroxide 3mL (30mmol) was added to 120mL DMF, and then added to the solid phase reaction column for aeration reaction for 1h. After the reaction was completed, the resin was washed 5 times with 120mL DMF, and 7.6g of iodine (30mmol) was weighed and added to the reactor, and 120mL DMF was added at the same time, and the reaction was aerated for 1h at room temperature. After the reaction was completed, the resin was washed 3 times with 120mL DMF, and then deprotected with DBLK 2 times (5min+7min), and then washed with DMF 4 times, washed with DCM 2 times, and dried after shrinking with methanol to obtain 38.5g of IM-B peptide resin.

[0143] Example 9: Synthesis of peptide resin (2)

[0144] The NH obtained in Example 5 2 -Cys 6 -Asn(Trt)-Pro-Ala-Cys(Acm) 10 -Thr(tBu)-Gly-Cys 13 -Tyr(tBu)-Wang Resin(SS:Cys 6-13) is used to carry out subsequent coupling with a fragment peptide resin containing a pair of disulfide bonds, and the sequence of IM-G in the present invention is used for synthesis. According to the operation in the above step 2, the subsequent residues are coupled in sequence: Fmoc-Cys(Acm)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Mmt)-OH, and Fmoc-Cys(Mmt)-OH. Each protected amino acid is fed with 30 mmol, and the feed ratio of other materials corresponds to the protected amino acid. After the above residues are coupled, they are washed with DMF for 6 times. Then, TFA:TIS:DCM=2:5:93, the reaction number is 20 times, and each time is 2 minutes to remove Mmt from the peptide resin. After the end, the resin is washed 6 times with a DMF solution containing 1% DIPEA. 30% hydrogen peroxide 3mL (30mmol) was added to 120mL DMF, and then added to the solid phase reaction column for aeration reaction for 1h. After the reaction was completed, the resin was washed 5 times with 120mL DMF, and 7.6g of iodine (30mmol) was weighed and added to the reactor, and 120mL DMF was added at the same time, and the reaction was aerated for 1h at room temperature. After the reaction was completed, the resin was washed 3 times with 120mL DMF, and then deprotected with DBLK 2 times (5min+7min), and then washed with DMF 4 times, washed with DCM 2 times, and dried after shrinking with methanol to obtain 37.3g of IM-G peptide resin

[0145] Example 10: Synthesis of peptide resin (3)

[0146] Weigh 18.2 g of Fmoc-Tyr(tBu)-Wang Resin (Sub=0.55 mmol / g, 10 mmol) obtained in Example 1 into a solid phase reaction column, wash twice with DMF, and then swell with DMF for 30 minutes. Then deprotect with DBLK twice (5min+7min), continue to wash with DMF 6 times, and wash with DCM once. Weigh 12.4 g of Fmoc-Cys(Acm)-OH (30 mmol) and 4.9 g of HOBt (36 mmol) and dissolve them in 50 mL of DMF / DCM (1:1). Add 6 mL (39 mmol) of DIPCDI in an ice water bath to activate for 2-3 minutes, then add the mixed solution to the reaction column, react at room temperature for 2 hours, and detect the end point of the reaction with ninhydrin (if the resin is colorless and transparent, terminate the reaction; if the resin is colored, extend the reaction for 1 hour). After the reaction was completed, the resin was washed 3 times with 80 mL DMF, then deprotected with DBLK 2 times (5 min + 7 min), washed 6 times with DMF, and washed once with DCM. The resin was colored when detected by ninhydrin. The peptide fragment obtained in Example 6: Fmoc-Cys 6 -Asn(Trt)-Pro-Ala-Cys 10-Thr(tBu)-Gly-OH(SS:Cys 6-10 ) A total of 28.4g (24mmol) was dissolved in 60mL of DCM / DMF (9:1), and 4.1g of HOBt (30mmol) was added at the same time. After adding 6mL (39mmol) of DIPCDI to activate for 2-3min under ice-water bath, the mixture was added to the reaction column and reacted at room temperature for 3h. The reaction end point was detected by ninhydrin (if the resin is colorless and transparent, the reaction is terminated; if the resin is colored, it is washed with DMF 5 times and then 21mL of acetic anhydride, 18mL of pyridine and appropriate amount of DMF are added to cap the N-terminus for 2h.). After the reaction is completed, the resin is washed 3 times with 80mL of DMF, and then deprotected with DBLK 2 times (5min+7min), and then washed with DMF 6 times and DCM once. The resin is colored when ninhydrin is detected. Repeat the above coupling operation with reference to the sequence of IM-F above, and continue to sequentially couple Fmoc-Cys(Acm)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Mmt)-OH, and Fmoc-Cys(Mmt)-OH. Each protected amino acid is fed 30 mmol, and the feed ratio of other materials corresponds to the protected amino acid. After the above residues are coupled, wash with DMF 6 times. Then, use TFA:TIS:DCM=2:5:93, the reaction number is 20 times, and each time for 2 minutes to remove Mmt from the peptide resin. After the end, the resin is washed 6 times with a DMF solution containing 1% DIPEA. 30% hydrogen peroxide 3mL (30mmol) was added to 120mL DMF, and then added to the solid phase reaction column for aeration reaction for 1h. After the reaction was completed, the resin was washed 5 times with 120mL DMF, and 7.6g of iodine (30mmol) was weighed and added to the reactor, and 120mL DMF was added at the same time, and the reaction was aerated for 1h at room temperature. After the reaction was completed, the resin was washed 3 times with 120mL DMF, and then deprotected with DBLK 2 times (5min+7min), and then washed with DMF 4 times, washed with DCM 2 times, and dried after shrinking with methanol to obtain 35.3g of IM-F peptide resin.

[0147] Example 11: Synthesis of peptide resin (4)

[0148] Weigh 18.2 g of Fmoc-Tyr(tBu)-Wang Resin (Sub=0.55 mmol / g, 10 mmol) obtained in Example 1 into a solid phase reaction column, wash twice with DMF, and then swell with DMF for 30 minutes. Then deprotect with DBLK twice (5min+7min), continue to wash with DMF 6 times, and wash with DCM once. Weigh 12.4 g of Fmoc-Cys(Acm)-OH (30 mmol) and 4.9 g of HOBt (36 mmol) and dissolve them in 50 mL of DMF / DCM (1:1). Add 6 mL (39 mmol) of DIPCDI in an ice water bath to activate for 2-3 minutes, then add the mixed solution to the reaction column, react at room temperature for 2 hours, and detect the end point of the reaction with ninhydrin (if the resin is colorless and transparent, terminate the reaction; if the resin is colored, extend the reaction for 1 hour). After the reaction is completed, the resin is washed 3 times with 80 mL DMF, then deprotected with DBLK 2 times (5 min + 7 min), and then washed with DMF 6 times and DCM once. The resin is colored when detected by ninhydrin. Repeat the above coupling operation according to the sequence of IM-I above, and continue to sequentially couple Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, and Fmoc-Cys(Mmt)-OH. Each protected amino acid is fed with 30 mmol, and the feed ratio of other materials corresponds to that of the protected amino acid. After the above residues are coupled, wash with DMF 6 times. , and wash with DCM once. The resin is colored when detected by ninhydrin. The peptide fragment obtained in Example 7: Fmoc-Cys 1 -Cys(Acm) 2 -Glu(OtBu)-Tyr(tBu)-Cys 5 -OH(SS:Cys 1-5) was dissolved in 60 mL of DCM / DMF (9:1), and 4.1 g of HOBt (30 mmol) was added. After adding 6 mL (39 mmol) of DIPCDI for activation for 2-3 min in an ice-water bath, the mixture was added to the reaction column and reacted at room temperature for 3 h. After the reaction was completed, the column was washed with DMF 6 times and DCM once. Then, the peptide resin was de-Mmt treated with TFA:TIS:DCM=2:5:93, 20 reactions and 2 minutes each time. After the reaction, the resin was washed 6 times with a DMF solution containing 1% DIPEA. 30% hydrogen peroxide 3mL (30mmol) was added to 120mL DMF, and then added to the solid phase reaction column for aeration reaction for 1h. After the reaction was completed, the resin was washed 5 times with 120mL DMF, and 7.6g of iodine (30mmol) was weighed and added to the reactor, and 120mL DMF was added at the same time, and the reaction was aerated for 1h at room temperature. After the reaction was completed, the resin was washed 3 times with 120mL DMF, and then deprotected with DBLK 2 times (5min+7min), and then washed with DMF 4 times, washed with DCM 2 times, and dried after shrinking with methanol to obtain 34.1g of IM-I peptide resin

[0149] Example 12: Cleavage of peptide resin

[0150] The peptide resins obtained in Examples 8, 9, 10, and 11 were cleaved. The cleavage conditions were as follows: 10 mL / g of peptide resin was added with a cleavage reagent (TFA:TIS:H 2 O=90:5:5 (V / V)), stirred at room temperature for 3h. After the reaction is completed, filter and add a small amount of TFA to wash the filtered resin, collect and combine the filtrate and concentrate it to a certain volume under reduced pressure. Add it to frozen methyl tert-butyl ether (100mL / g peptide resin) for precipitation, centrifuge, remove the supernatant, wash the precipitate with methyl tert-butyl ether 3 times, and vacuum dry to obtain the corresponding crude peptide. The purity of the crude peptide was detected by HPLC, and the results are as follows (HPLC detection conditions: Waters symmetryshield RP18 (5μm) (250×4.6mm); Phase A: 50mM / l diammonium phosphate buffer (pH adjusted to 3.0 with phosphoric acid); Phase B: acetonitrile, gradient: 15-45% (30min); flow rate: 0.5mL / min detection wavelength: 215nm. Same below):

[0151] Table 2: List of crude peptides and purity of four groups of Linaclotide disulfide mismatched isomers

[0152] serial number Peptide name Peptide resin weight (g) Crude peptide weight (g) Crude peptide purity 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％

[0153] Example 13: Purification and preparation of four groups of disulfide mismatched isomers of linaclotide

[0154] The four groups of crude peptides obtained in Example 12 above were subjected to reverse phase liquid phase preparative purification using octadecylsilane as the stationary phase and 50 mM / l ammonium dihydrogen phosphate buffer / acetonitrile as the mobile phase, and the target peak fractions (>95%) were collected, and then desalted, and the fractions were concentrated and freeze-dried. Specific data are shown in the table below.

[0155] Table 3: Purification data of four groups of Linaclotide disulfide mismatched isomers

[0156] serial number Peptide name Weight of refined peptide (g) purity 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 a polypeptide containing three pairs of disulfide bonds, in, The polypeptide containing three pairs of disulfide bonds comprises a first pair of disulfide bonds, a second pair of disulfide bonds and a third pair of disulfide bonds, wherein at least one pair of disulfide bonds among the three pairs of 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 contains two cysteines forming a first pair of disulfide bonds, one of the cysteines forming a second pair of disulfide bonds is between the two cysteines forming the first pair of disulfide bonds, or no cysteine ​​is 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 a thiol protecting group Trt or Mmt; and one of the cysteines forming the second pair of disulfide bonds is protected with a thiol protecting group Acm; 2) removing the sulfhydryl protecting groups of the two cysteines of the first pair of disulfide bonds in the first polypeptide fragment, and oxidizing to form the first pair of disulfide bonds; 3) Under solid phase conditions, continue to synthesize the remaining fragments by solid phase synthesis, wherein the remaining fragments contain cysteine ​​that forms the second pair of disulfide bonds and cysteine ​​that forms the third pair of disulfide bonds, wherein the sulfhydryl groups of the cysteine ​​that forms the second pair of disulfide bonds and the cysteine ​​that forms the third pair of disulfide bonds are both protected by sulfhydryl protecting groups; the sulfhydryl protecting groups of the two cysteines that form the second pair of disulfide bonds are the same, which is Acm; the sulfhydryl protecting groups of the cysteines that form the third pair of disulfide bonds are the same, which is Trt or Mmt; 4) removing the thiol protecting group of cysteine ​​of the second pair of disulfide bonds to form the second pair of disulfide bonds, and then removing the thiol protecting group of cysteine ​​of the third pair of disulfide bonds to form the third pair of disulfide bonds; or removing the thiol protecting group of cysteine ​​of the third pair of disulfide bonds to form the third pair of disulfide bonds, and then removing the thiol protecting group of cysteine ​​of the second pair of disulfide bonds to form the second pair of disulfide bonds; 5) Removing other protecting groups, cleaving the resin, and obtaining a polypeptide containing three pairs of disulfide bonds.

2. The synthesis method according to claim 1, characterized in that In step 2), the method of removing the thiol protecting groups of the two cysteines forming the first pair of disulfide bonds and oxidizing to form disulfide bonds is as follows: the first polypeptide fragment only contains the two cysteines forming the first pair of disulfide bonds, 1-20 equivalents of iodine are dissolved in an appropriate amount of DMF, and the thiol protecting groups are removed and disulfide bonds are formed at room temperature; Alternatively, the first polypeptide fragment contains two cysteines forming a first pair of disulfide bonds and one cysteine ​​forming a second pair of disulfide bonds, and TFA:TIS:DCM=(1-5):(2-10):(97-85) is used to remove the two cysteine ​​thiol protecting groups of the first pair of disulfide bonds, and then 1-30 equivalents of H2O2 are used to oxidize the first pair of disulfide bonds.

3. The synthesis method according to any one of claims 1 to 2, characterized in that In step 3), the method for continuing to synthesize the remaining fragments under solid phase conditions is to synthesize them using a solid phase synthesis method to form a conjugate of the polypeptide fragment and a solid phase synthesis resin, wherein the conjugate of the polypeptide fragment and the solid phase synthesis resin contains 6 cysteines, of which 2 cysteines have formed disulfide bonds; the synthesis method includes adding amino acids to the N-terminus of the polypeptide fragment, synthesizing the polypeptide fragment by a solid phase synthesis method, and coupling the polypeptide fragments, or coupling a single added amino acid and a fragment.

4. The synthesis method according to any one of claims 1 to 2, characterized in that In step 4), the method of first removing the thiol protecting group of cysteine ​​and oxidizing to form a disulfide bond is to use TFA:TIS:DCM=(1-5):(2-10):(97-85) to remove the two cysteine ​​thiol protecting groups Trt or Mmt of the disulfide bond, and then use 1-30 equivalents of H2O2 in DMF solution to perform solid phase oxidation at room temperature to form a pair of disulfide bonds; then 1-20 equivalents of iodine are dissolved in an appropriate amount of DMF, and the thiol protecting group Acm is removed at room temperature to form a disulfide bond.

5. The synthesis method according to any one of claims 1 to 2, characterized in that When the first pair of disulfide bonds, the second pair of disulfide bonds or the third pair of disulfide bonds are formed, the Fmoc protecting group at the polypeptide terminal is retained.

6. The synthesis method according to any one of claims 1 to 2, characterized in that In step 1), the number of 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.

7. The synthesis method according to any one of claims 1 to 2, characterized in that In step 1), the number of cysteines contained in the first polypeptide fragment is three, and the cysteines forming the first pair of disulfide bonds also contain one of the cysteines forming the second pair of disulfide bonds, the thiol protecting group of the cysteines forming the first pair of disulfide bonds is either Mmt or Trt, the thiol protecting group of the cysteines forming the second pair of disulfide bonds is Acm, and the thiol protecting group of the cysteines forming the third pair of disulfide bonds is either Mmt or Trt.

8. The synthesis method according to any one of claims 1 to 2, characterized in that In step 3), the method for continuing to synthesize the remaining fragments under solid phase conditions is: a. Continue solid phase coupling the remaining amino acids at the N-terminus of the first polypeptide fragment; b. synthesizing a conjugate of the second polypeptide fragment and the solid phase synthetic resin under solid phase conditions, cleaving the solid phase synthetic resin on one of the first polypeptide fragment and the second polypeptide fragment, and coupling the first polypeptide fragment with the second polypeptide fragment to obtain a conjugate of the third polypeptide fragment and the solid phase synthetic resin; c. synthesizing a conjugate of the second polypeptide fragment and the solid phase synthetic resin under solid phase conditions, continuing to couple amino acids at the N-terminus of the first polypeptide fragment and or at the N-terminus of the second polypeptide fragment, and then coupling the first polypeptide fragment or the second polypeptide fragment with the added amino acids to obtain a conjugate of the fourth polypeptide fragment and the solid phase synthetic resin; d. In the three cases a, b, and c, the conjugates of the polypeptide fragment and the solid phase synthetic resin are obtained and then the amino acids are further coupled.

9. The synthesis method according to any one of claims 1 to 2, characterized in that In step 5), the resin is cleaved and other protecting groups are removed by using 80-95% TFA solution.

10. The synthesis method according to any one of claims 1 to 2, characterized in that: The solid phase synthesis method in steps 1)-3) is the Fmoc solid phase synthesis strategy, coupling the amino acid with Fmoc protected amino group on the solid phase synthesis resin, then removing the Fmoc protecting group, and continuing to couple the amino acid with Fmoc protected amino group until the synthesis of the polypeptide is completed.

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