Positioning cyclization method for three pairs of disulfide bonds in polypeptide synthesis process

Abstract

The invention relates to the technical field of polypeptide synthesis, in particular to a method for positioning and cyclizing three pairs of disulfide bonds in a polypeptide synthesis process, namely solid-phase synthesis full-protection linear polypeptide resin, in which three pairs of cysteine side chains are protected by Trt, Acm and pMeOBz, and then protecting groups are removed step by step and the positioning and cyclizing of the disulfide bonds are completed step by step. According to the method disclosed by the invention, the complete deprotection of a pair of cysteine participating in cyclization independently in the deprotection process is ensured, and the cysteine protecting group not participating in cyclization is not removed at all, so that a perfect orthogonal protection strategy is provided, and the accurate positioning of the disulfide bond position in the cyclization process is ensured. In Acm deprotection, after the reaction liquid is optimized, the reaction time is shortened to 15 to 30 minutes; in pMeOBzl deprotection, TFA is used, and deprotection is completed by controlling the reaction temperature, so that the use of highly toxic products is avoided.

Description

Technical Field

[0001] The invention relates to the technical field of polypeptide synthesis, and in particular to a method for positioning cyclization of three pairs of disulfide bonds in a polypeptide synthesis process. Background Art

[0002] Disulfide bonds are SS covalent bonds formed by oxidation of sulfhydryl groups on a pair of cysteine ​​residues in a polypeptide sequence. They are important structures for maintaining the spatial structure of polypeptides and ensuring biological activity. In vivo, the formation of disulfide bonds is regulated by enzymes and can be accurately paired, but many problems are encountered in chemical synthesis. When there are two cysteine ​​residues in a peptide chain, intermolecular disulfide bonds or even multi-molecular polymerized impurities may also be generated. When the number of sulfhydryl groups that need to be paired increases, complex situations of disulfide mismatch or unpairing may occur only within the peptide chain. After the introduction of intermolecular disulfide bond pairing, the complexity immediately soars exponentially.

[0003] Patent CN1120174C, Patent CN110698553A, Patent CN110894225A, Patent CN116836255A, Patent CN118530327A, Patent CN113735955A, Patent CN104974237A, Patent CN109021087A, in the synthesis process, the 6 protected cysteines all use the same protective group, most of them use Trt, and some patents use Acm, tBu, after cleavage, a linear polypeptide without any protective group is uniformly obtained, and then various buffer salts, weak oxidizing solutions, or direct Air oxidation was then selected to allow the linear peptide to undergo disulfide cyclization through free folding to obtain the target product. However, the patents failed to provide an effective identification method for accurately identifying whether the position of the disulfide bond is the same as the spatial structure of the cono peptide, and some patents did not provide examples for the more difficult removal of the tBu protecting group. When we tried to use the tBu protecting group, we were unable to effectively remove it using high concentrations of TFA and high temperature heating. When we tried more acidic organic acids such as trifluoromethanesulfonic acid, degradation occurred and the purity of the crude peptide was significantly reduced. If the reaction time was further extended, the product completely disappeared.

[0004] Patent CN112279905A uses two different protecting groups, Trt and StBu, for the six protected cysteines during the synthesis process. The StBu protecting group is preferentially removed to complete the cyclization of the first pair of disulfide bonds, and then the Trt protecting group is removed to randomly complete the free cyclization of the other two pairs of disulfide bonds through spatially advantageous conformations. This method has been proven to not produce the correct conopeptide structure.

[0005] The three groups of protecting groups selected in the synthesis process of patent CN106496315A are Mtt, Trt, and Acm, and the synthesis is also completed by step-by-step deprotection and step-by-step cyclization. The removal method of Mtt protecting group is 0.5% TFA / DCM solution, and 6% TFA / DCM is used for Trt protecting group. When we repeated the experiment, we found that 0.5% TFA / DCM can only remove a very small part of Mtt protecting group. It is necessary to increase the TFA concentration to 5% to completely remove it, and some Trt protecting groups are removed synchronously during the removal process. Patent CN107216374A increases the reaction concentration of Mtt removal to 3% TFA / DCM. Although both methods can obtain ziconotide products, it is still difficult to avoid impurities produced by misplaced cyclization. It can be seen that Mtt and Trt protecting groups do not have a good orthogonal protection effect.

[0006] There are also three groups of protecting groups, Trt, Acm and Mob. On the one hand, the reaction time of Acm deprotection is 2-5 hours, which is a long cycle. On the other hand, some Mob deprotection solutions use cleavage solutions containing trifluoromethanesulfonic acid, but the actual removal effect is not good, and most of the products are degraded. Some also use highly toxic controlled mercuric acetate, which is not conducive to subsequent amplification experiments. Summary of the invention

[0007] The purpose of the present invention is to provide a method for the positional cyclization of three pairs of disulfide bonds in the process of polypeptide synthesis, which can ensure that a pair of cysteines participating in the cyclization can be completely deprotected during the deprotection process, and the cysteine ​​protecting groups not participating in the cyclization are not completely removed, thereby ensuring the precise positioning of the disulfide bond positions during the cyclization process, and at the same time, the deprotection reaction cycle is short and safe.

[0008] To achieve the above object, the technical solution adopted by the present invention is: A method for positioning cyclization of three pairs of disulfide bonds in a polypeptide synthesis process comprises the following steps: S1. Solid phase synthesis of a fully protected linear polypeptide resin, wherein the fully protected linear polypeptide resin contains three pairs of cysteines, one pair of cysteine ​​side chains are protected by Trt, another pair of cysteine ​​side chains are protected by Acm, and another pair of cysteine ​​side chains are protected by pMeOBz; S2, adding the fully protected linear peptide resin to the lysate, removing all side chain protecting groups except Acm and pMeOBz while removing the resin, filtering out the resin, precipitating, washing and drying to obtain the first intermediate peptide; S3, dissolving the first intermediate peptide in a first acidic solution, and adding an iodine / methanol solution to carry out a first cyclization reaction; S4. After monitoring and confirming that the first pair of disulfide bond cyclization is completed, iodine / methanol solution is added to remove the Acm protecting group and perform a second cyclization reaction for 15 min-30 min; monitoring and confirming that the second pair of disulfide bond cyclization is completed, sodium sulfite solution is added to quench the iodine element; filtering, purifying and freeze-drying are performed to obtain the second intermediate peptide; S5, dissolving the second intermediate peptide of step S4 in TFA with a solubility of 25 mg / mL-35 mg / mL, adding triisopropylsilane as a scavenger, sealing, placing in a water bath at 80°C-100°C for heating reaction, monitoring to ensure complete removal of the pMeOBzl protecting group, then cooling, precipitating, washing and drying to obtain a third intermediate peptide; S6, dissolving the third intermediate peptide in step S5 in the second acidic solution, adding 1 equivalent of iodine / methanol solution to carry out a third cyclization reaction, monitoring and confirming the completion of the cyclization of the third pair of disulfide bonds, and purifying and lyophilizing to obtain a cyclic peptide containing three pairs of disulfide bonds.

[0009] Preferably, the resin is Rink Amide-AM Resin, Sieber Amide Resin or DEG-RinkAmide-AM Resin, and the degree of substitution thereof is 0.30 mmol / g-0.65 mmol / g.

[0010] Preferably, the monitoring adopts high performance liquid chromatography and MS mass spectrometry analysis.

[0011] Preferably, the purification in steps S3 and S6 is performed by semi-preparative HPLC column purification.

[0012] Preferably, the first acidic solution in step S3 and the second acidic solution in step S6 are both acetic acid:methanol:water=1-1.5:7.5-8:1, and the concentrations of the first intermediate peptide and the third intermediate peptide are 8 mg / mL-15 mg / mL.

[0013] Preferably, the iodine / methanol solution in step S3 contains 1.5 eq≥equivalent≥1.0 eq of iodine; and the iodine / methanol solution in step S4 contains 10 eq≥equivalent≥5 eq of iodine.

[0014] Preferably, the fully protected linear polypeptide resin is pGlu-Gly-Cys(pMeOBzl)-Cys(Acm)-Asn(Trt)-Gly-Pro-Lys(Boc)-Gly-Cys(Trt)-Ser(tBu)-Ser(tBu)-Lys(Boc)-Trp(Boc)-Cys(pMeOBzl)-Arg(Pbf)-Asp(OtBu)-His(Trt)-Ala-Arg(Pbf)-Cys(Acm)-Cys(Trt)-resin.

[0015] Preferably, the lysate in step S2 is composed of trifluoroacetic acid, anisole, dithiothreitol, triisopropylsilane and water, the ratio of the fully protected linear polypeptide resin to the lysate is 1 g: 10 mL-20 mL, the reaction time is 60 min-180 min, and the reaction temperature is 30±5°C.

[0016] Preferably, the fully protected linear polypeptide resin is Cys(Acm)-Lys(Boc)-Gly-Lys(Boc)-Gly-Ala-Lys(Boc)-Cys(Trt)-Ser(tBu)-Arg(Pbf)-Leu-Met-Tyr(tBu)-Asp(OtBu)-Cys(pMeOBzl)-Cys(Acm)-Thr(tBu)-Gly-Ser(tBu)-Cys(Trt)-Arg(Pbf)-Ser(tBu)-Gly-Lys(Boc)-Cys(pMeOBzl)-resin.

[0017] Preferably, the lysate in step S2 is composed of trifluoroacetic acid, anisole, thioanisole, dithiothreitol, triisopropylsilane and water, the ratio of the fully protected linear polypeptide resin to the lysate is 1 g: 10 mL-20 mL, the reaction time is 60 min-180 min, and the reaction temperature is 30±5°C.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The three pairs of cysteine ​​side chains of the linear polypeptide of the present invention are protected by Trt, Acm and pMeOBz, the coupling positions of the three groups of amino acids can be arbitrarily exchanged, and then the protecting groups are gradually removed and the disulfide bond positioning cyclization is gradually completed. The present invention can ensure that a pair of cysteines participating in the cyclization are completely deprotected during the deprotection process, and the cysteine ​​protecting groups not participating in the cyclization are not completely removed, providing a perfect orthogonal protection strategy to ensure the precise positioning of the disulfide bond position during the cyclization process. In the Acm deprotection, the reaction time is shortened to 15-30 minutes by optimizing the reaction solution; in the pMeOBzl deprotection, TFA is used and the deprotection is completed by controlling the reaction temperature to avoid the use of highly toxic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1-1 This is the MS spectrum of the linear conopeptide of Example 1 of the present invention.

[0020] Figure 1-2 This is the HPLC spectrum of the linear conopeptide of Example 1 of the present invention.

[0021] Figure 2-1 This is the MS spectrum of the first pair of disulfide-cyclized conopeptides in Example 1 of the present invention.

[0022] Figure 2-2 This is the HPLC spectrum of the first pair of disulfide-cyclized conopeptides in Example 1 of the present invention.

[0023] Figure 3-1 This is the MS spectrum of the second pair of disulfide-cyclized conopeptides in Example 1 of the present invention.

[0024] Figure 3-2 This is the HPLC spectrum of the second pair of disulfide-cyclized conopeptides in Example 1 of the present invention.

[0025] Figure 4 This is the semi-preparative purification profile of the second pair of disulfide-cyclized conopeptides of Example 1 of the present invention.

[0026] Figure 5 This is the HPLC spectrum of the second pair of disulfide-cyclized cono peptides after purification in Example 1 of the present invention.

[0027] Figure 6-1 This is the MS spectrum of the conopeptide from Example 1 of the present invention after the pMeOBzl protecting group is removed.

[0028] Figure 6-2 This is the HPLC spectrum of the cono peptide from Example 1 of the present invention after the pMeOBzl protecting group is removed.

[0029] Figure 7 This is the semi-preparative purification map of the conopeptide with the third disulfide bond in Example 1 of the present invention.

[0030] Figure 8-1 This is the MS spectrum of the third pair of disulfide-cyclized cono peptides after purification in Example 1 of the present invention.

[0031] Figure 8-2 This is the HPLC spectrum of the third pair of disulfide-cyclized cono peptides after purification in Example 1 of the present invention.

[0032] Figure 8-3 This is the structural formula of the cyclic conopeptide of Example 1 of the present invention.

[0033] Figure 9-1 This is the MS spectrum of linear ziconotide in Example 2 of the present invention.

[0034] Figure 9-2 This is the HPLC spectrum of linear ziconotide of Example 2 of the present invention.

[0035] Figure 10-1 This is the MS spectrum of the first pair of disulfide-cyclized ziconotides in Example 2 of the present invention.

[0036] Figure 10-2 This is the HPLC spectrum of the first pair of disulfide-cyclized ziconotides of Example 2 of the present invention.

[0037] Figure 11-1 This is the MS spectrum of the second pair of disulfide-cyclized ziconotides of Example 2 of the present invention.

[0038] Figure 11-2 This is the HPLC spectrum of the second pair of disulfide-cyclized ziconotides of Example 2 of the present invention.

[0039] Fig.12 This is the semi-preparative purification profile of the second pair of disulfide-cyclized ziconotide of Example 2 of the present invention.

[0040] Fig.13 This is the HPLC spectrum of the second pair of disulfide-cyclized ziconotides after purification in Example 2 of the present invention.

[0041] Figure 14-1 This is the MS spectrum of ziconotide from Example 2 of the present invention after the pMeOBzl protecting group is removed.

[0042] Figure 14-2 This is the HPLC spectrum of ziconotide from Example 2 of the present invention after the pMeOBzl protecting group is removed.

[0043] Fig.15 This is the semi-preparative purification profile of ziconotide of the third pair of disulfide bonds in Example 2 of the present invention.

[0044] Figure 16-1 This is the MS spectrum of the third pair of disulfide-cyclized ziconotides after purification in Example 2 of the present invention.

[0045] Figure 16-2 This is the HPLC spectrum of the third pair of disulfide-cyclized ziconotides after purification in Example 2 of the present invention.

[0046] Figure 16-3 This is the structural formula of cyclic ziconotide according to Example 2 of the present invention. DETAILED DESCRIPTION

[0047] Example 1 This example provides cono peptide, the synthesis scale is 1.0 mmol, and the detailed steps of the synthesis method are as follows: Preparation of deprotection solution: Measure 250 mL of piperidine, add 750 mL of DMF, shake well and set aside.

[0048] Preparation of peptide resin: Weigh 2.86g of Rink Amide-AM Resin with a substitution degree of 0.35mmol / g and place it in a glass chromatography synthesis column. The synthesis scale is 1.0mmol. Add 30mL of dichloromethane to swell the resin for 15min, and drain the solvent. Add 30mL of deprotection solution to the swollen resin and react for 5min, and drain the solvent. Wash the resin 5 times with 30mL of DMF and twice with 30mL of DCM, and drain the solvent. Weigh 2.93g (5eq) of Fmoc-Cys (Trt) -OH and 0.71g (5eq) of Oxyma Pure in a reagent bottle, add 30mL of DMF to dissolve, then add 0.77mL (5eq) of DIC, shake well, and add to the synthesis column to react for 60min. After the reaction is completed, drain the reaction solution, wash the resin 5 times with 30mL of DMF and 30mL of The resin was washed twice with DCM and the solvent was drained; the above steps were repeated to sequentially couple the protected amino acids Fmoc-Cys(Acm)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-His(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Cys(pMeOBzl)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, and Fmoc-Gl y-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Cys(pMeOBzl)-OH, Fmoc-Gly-OH, pGlu-OH, to obtain 8.77g of fully protected peptide resin pGlu-Gly-Cys(pMeOBzl)-Cys(Acm)-Asn(Trt)-Gly-Pro- Lys(Boc)-Gly-Cys(Trt)-Ser(tBu)-Ser(tBu)-Lys(Boc)-Trp(Boc)-Cys(pMeOBzl)-Arg(Pbf)-Asp(OtBu)-His(Trt)-Ala-Arg(Pbf)-Cys(Acm)-Cys(Trt)-RinkAmide-AM Resin.

[0049] Cleavage: Prepare 100 mL of cleavage solution, the ratio of which is trifluoroacetic acid: anisole: dithiothreitol: triisopropylsilane: water = 90 mL: 2 mL: 5 g: 1 mL: 2 mL. Add the peptide resin obtained in the previous step to the cleavage solution and stir to react for 120 min, filter out the resin, add 500 mL of ice methyl tert-butyl ether (≤ 0°C) to the filtrate to precipitate, resuspend and wash twice, and dry under reduced pressure at room temperature to obtain 2.45 g of the first intermediate peptide (linear cono peptide containing Acm and pMeOBzl protective groups), with an HPLC purity of 67.55%, and the MS detection results are as follows Figure 1-1 , HPLC detection spectrum see Figure 1-2 .

[0050] HPLC analysis method: First pair of disulfide bond cyclization: Dissolve the above 2.45g sample in a solvent of acetic acid / methanol / water = 25mL / 200mL / 25mL (1:8:1); weigh 262mg (1eq) of iodine and dissolve it in 5mL of methanol; add the iodine / methanol solution dropwise to the peptide solution under stirring, react for 5min, and take a sample. The MS test results are as follows: Figure 2-1 , HPLC test results are shown in Figure 2-2 .

[0051] Second disulfide bond cyclization: Weigh 2.35 g (9 eq) of iodine, dissolve it in 20 mL of methanol, and continue to add it dropwise to the peptide solution in the previous step under stirring. After the addition is complete, continue the reaction for 15 minutes and take a sample. The MS test results are as follows: Figure 3-1 , HPLC test results are shown in Figure 3-2 .

[0052] A sodium sulfite aqueous solution was added dropwise to the above reaction solution until the brown-yellow color of the solution disappeared, thereby quenching the reaction.

[0053] The reaction solution was diluted with 500 mL of water, filtered through a 0.45 μm micropore, purified by semi-preparative HPLC column, and the collected cyclic peptide solution containing the pMeOBzl protective group was lyophilized to obtain the second intermediate peptide as 980 mg solid powder with an HPLC purity of 97.26%. The semi-preparative HPLC purification pattern is shown in Figure 4 , the HPLC detection spectrum after purification is shown in Figure 5 .

[0054] Removal of pMeOBzl protecting group: 980 mg of the above solid powder was dissolved in 30 mL of TFA, 296 mg (5 eq) of triisopropylsilane was added dropwise, the reaction vessel was sealed, and heated in a water bath at 80°C for 60 min. Samples were taken, and the MS test results were as follows: Figure 6-1 , HPLC test results are shown in Figure 6-2The reaction solution was cooled to room temperature, and 200 mL of icy methyl tert-butyl ether (≤ 0°C) was added. The obtained precipitate was washed with methyl tert-butyl ether and dried to obtain the third intermediate peptide as 807 mg of solid powder.

[0055] The third pair of disulfide bond cyclization: dissolve the above 807 mg sample in a solvent of acetic acid / methanol / water = 10 mL / 80 mL / 10 mL, with a concentration of ≈10 mg / mL; weigh 86 mg (1 eq) of iodine and dissolve it in 2 mL of methanol; add the iodine / methanol solution dropwise to the peptide solution under stirring, and continue the reaction for 5 min to obtain a crude cono peptide solution. The above reaction solution was diluted with 200 mL of water, filtered through a 0.45 um microporous filter, purified by semi-preparative HPLC column, and the collected cono peptide solution was freeze-dried to obtain 395 mg of solid powder with an HPLC purity of 99.02% and a total yield of 16.63%. See the semi-preparative HPLC purification map. Figure 7 , MS test results are as follows Figure 8-1 , the HPLC detection spectrum after purification is shown in Figure 8-2 , the structure of conopeptide is shown in Figure 8-3 .

[0056] Example 2 This example provides ziconotide, with a synthesis scale of 0.5 mmol, and the detailed steps of the synthesis method are as follows: Preparation of deprotection solution: Measure 250 mL of piperidine, add 750 mL of DMF, shake well and set aside.

[0057] Preparation of peptide resin: Weigh 0.93g of DEG-Rink Amide-AM Resin with a substitution degree of 0.54mmol / g and place it in a glass chromatography synthesis column. The synthesis scale is 0.5mmol. Add 10mL of dichloromethane to swell the resin for 15min, and drain the solvent. Add 10mL of deprotection solution to the swollen resin and react for 5min, and drain the solvent. Wash the resin 5 times with 10mL DMF and twice with 10mL DCM, and drain the solvent. Weigh 1.16g (5eq) Fmoc-Cys(pMeOBzl)-OH and 0.34g (5eq) HoBt in a reagent bottle, add 10mL DMF to dissolve, then add 0.39mL (5eq) DIC, shake well, add to the synthesis column and react for 30min. After the reaction, drain the reaction solution, wash the resin 5 times with 10mL DMF and 10mL The resin was washed twice with DCM and the solvent was drained; the above steps were repeated to sequentially couple the protected amino acids Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc c-Cys(Acm)-OH, Fmoc-Cys(pMeOBzl)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Met- OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)- OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Acm)-OH to obtain 3.22 g of fully protected peptide resin Cys(Acm)-Lys(Boc)-Gly-Lys(Boc)-Gly-Ala-Lys(Boc)-Cys(Trt) -Ser(tBu)-Arg(Pbf)-Leu-Met-Tyr(tBu)-Asp(OtBu)-Cys(pMeOBzl)-Cys(Acm)-Thr(tBu)-Gl y-Ser(tBu)-Cys(Trt)-Arg(Pbf)-Ser(tBu)-Gly-Lys(Boc)-Cys(pMeOBzl)-DEG-RinkAmide-AM Resin.

[0058] Cleavage: Prepare 35 mL of cleavage solution, the ratio of which is trifluoroacetic acid: anisole: thioanisole: dithiothreitol: triisopropylsilane: water = 28 mL: 1.75 mL: 1.75 mL: 1.75 g: 0.35 mL: 1.4 mL. Add the peptide resin obtained in the previous step to the cleavage solution and stir to react for 120 min, filter out the resin, add 200 mL of ice methyl tert-butyl ether (≤ 0°C) to the filtrate to precipitate, resuspend and wash twice, and dry under reduced pressure at room temperature to obtain 1.63 g of the first intermediate peptide (linear ziconotide containing Acm and pMeOBzl protective groups), with an HPLC purity of 66.86%, and the MS detection results are as follows Figure 9-1 , HPLC detection spectrum see Figure 9-2 .

[0059] HPLC analysis method: The first pair of disulfide bond cyclization: Dissolve the above 1.63g sample in a solvent of acetic acid / methanol / water = 20mL / 160mL / 20mL (1:8:1); weigh 134mg (1eq) of iodine and dissolve it in 5mL of methanol; add the iodine / methanol solution dropwise to the peptide solution under stirring, react for 5min, and take a sample. The MS test results are as follows: Figure 10-1 , HPLC test results are shown in Figure 10-2 .

[0060] Second disulfide bond cyclization: Weigh 1.21 g (9 eq) of iodine, dissolve it in 20 mL of methanol, and continue to add it dropwise to the peptide solution in the previous step under stirring. After the addition is complete, continue the reaction for 15 min and take a sample. The MS test results are as follows: Figure 11-1 , HPLC test results are shown in Figure 11-2 .

[0061] A sodium sulfite aqueous solution was added dropwise to the above reaction solution until the brown-yellow color of the solution disappeared, thereby quenching the reaction.

[0062] The reaction solution was diluted with 200 mL of water, filtered through a 0.45 μm micropore, purified by semi-preparative HPLC column, and the collected cyclic peptide solution containing the pMeOBzl protective group was lyophilized to obtain the second intermediate peptide as 421 mg solid powder with an HPLC purity of 90.67%. The semi-preparative HPLC purification pattern is shown in Fig.12 , the HPLC detection spectrum after purification is shown in Fig.13 .

[0063] Removal of pMeOBzl protecting group: 421 mg of the above solid powder was dissolved in 15 mL of TFA, 115 mg (5 eq) of triisopropylsilane was added dropwise, the reaction vessel was sealed, and heated in a water bath at 85°C for 60 min. Samples were taken, and the MS test results were as follows: Figure 14-1 , HPLC test results are shown in Figure 14-2 The reaction solution was cooled to room temperature, 100 mL of icy methyl tert-butyl ether (≤ 0°C) was added, the resulting precipitate was washed with methyl tert-butyl ether, and dried to obtain the third intermediate peptide as a solid powder (239 mg).

[0064] The third pair of disulfide bond cyclization: Dissolve the above 239 mg sample in a solvent of acetic acid / methanol / water = 4 mL / 32 mL / 4 mL, with a concentration of ≈10 mg / mL; weigh 23 mg (1 eq) of iodine and dissolve it in 2 mL of methanol; add the iodine / methanol solution dropwise to the peptide solution under stirring, and continue the reaction for 5 min to obtain a crude ziconotide peptide solution. The above reaction solution was diluted with 100 mL of water, filtered through a 0.45 um microporous filter, purified by semi-preparative HPLC column, and the collected ziconotide solution was freeze-dried to obtain 167 mg of solid powder with an HPLC purity of 97.16% and a total yield of 12.66%. See the semi-preparative HPLC purification map. Fig.15 , MS test results are as follows Figure 16-1 , the HPLC detection spectrum after purification is shown in Figure 16-2 , Ziconotide structural formula see Figure 16-3 .

[0065] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for illustrating the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which shall fall within the scope of the present invention to be protected. The scope of protection of the present invention shall be defined by the attached claims and their equivalents.

Claims

1. A method for the localized cyclization of three pairs of disulfide bonds in a polypeptide synthesis process, characterized in that: The specific steps include: S1. Solid phase synthesis of a fully protected linear polypeptide resin, wherein the fully protected linear polypeptide resin contains three pairs of cysteines, one pair of cysteine ​​side chains are protected by Trt, another pair of cysteine ​​side chains are protected by Acm, and another pair of cysteine ​​side chains are protected by pMeOBz; S2, adding the fully protected linear peptide resin to the lysate, removing all side chain protecting groups except Acm and pMeOBz while removing the resin, filtering out the resin, precipitating, washing and drying to obtain the first intermediate peptide; S3, dissolving the first intermediate peptide in a first acidic solution, and adding an iodine / methanol solution to carry out a first cyclization reaction; S4. After monitoring and confirming that the first pair of disulfide bond cyclization is completed, iodine / methanol solution is added to remove the Acm protecting group and perform a second cyclization reaction for 15 min-30 min; monitoring and confirming that the second pair of disulfide bond cyclization is completed, sodium sulfite solution is added to quench the iodine element; filtering, purifying and freeze-drying are performed to obtain the second intermediate peptide; S5, dissolving the second intermediate peptide of step S4 in TFA with a solubility of 25 mg / mL-35 mg / mL, adding triisopropylsilane as a scavenger, sealing, placing in a water bath at 80°C-100°C for heating reaction, monitoring to ensure complete removal of the pMeOBzl protecting group, then cooling, precipitating, washing and drying to obtain a third intermediate peptide; S6, dissolving the third intermediate peptide in step S5 in the second acidic solution, adding 1 equivalent of iodine / methanol solution to carry out a third cyclization reaction, monitoring and confirming the completion of the cyclization of the third pair of disulfide bonds, and purifying and lyophilizing to obtain a cyclic peptide containing three pairs of disulfide bonds.

2. The method for positioning cyclization of three pairs of disulfide bonds in the polypeptide synthesis process according to claim 1, characterized in that: The resin is Rink Amide-AM Resin, Sieber Amide Resin or DEG-Rink Amide-AM Resin, and its substitution degree is 0.30 mmol / g-0.65 mmol / g.

3. The method for positioning cyclization of three pairs of disulfide bonds in the polypeptide synthesis process according to claim 1, characterized in that: The monitoring adopts high performance liquid chromatography and MS mass spectrometry analysis.

4. The method for positional cyclization of three pairs of disulfide bonds in the polypeptide synthesis process according to claim 1, characterized in that: The purification in steps S3 and S6 is performed by semi-preparative HPLC column purification.

5. The method for positional cyclization of three pairs of disulfide bonds in the polypeptide synthesis process according to claim 1, characterized in that: The first acidic solution in step S3 and the second acidic solution in step S6 are both acetic acid:methanol:water=1-1.5:7.5-8:1, and the concentrations of the first intermediate peptide and the third intermediate peptide are 8 mg / mL-15 mg / mL.

6. The method for positional cyclization of three pairs of disulfide bonds in the polypeptide synthesis process according to claim 1, characterized in that: The iodine / methanol solution in step S3 contains iodine element: 1.5eq≥equivalent≥1.0eq; the iodine / methanol solution in step S4 contains iodine element: 10eq≥equivalent≥5eq.

7. The method for positional cyclization of three pairs of disulfide bonds in a polypeptide synthesis process according to any one of claims 1 to 6, characterized in that: The fully protected linear polypeptide resin is pGlu-Gly-Cys(pMeOBzl)-Cys(Acm)-Asn(Trt)-Gly-Pro-Lys(Boc)-Gly-Cys(Trt)-Ser(tBu)-Ser(tBu)-Lys(Boc)-Trp(Boc)-Cys(pMeOBzl)-Arg(Pbf)-Asp(OtBu)-His(Trt)-Ala-Arg(Pbf)-Cys(Acm)-Cys(Trt)-resin.

8. The method for positional cyclization of three pairs of disulfide bonds in the polypeptide synthesis process according to claim 7, characterized in that: The lysis solution in step S2 is composed of trifluoroacetic acid, anisole, dithiothreitol, triisopropylsilane and water, the ratio of the fully protected linear polypeptide resin to the lysis solution is 1 g: 10 mL-20 mL, the reaction time is 60 min-180 min, and the reaction temperature is 30±5°C.

9. The method for positional cyclization of three pairs of disulfide bonds in a polypeptide synthesis process according to any one of claims 1 to 6, characterized in that: The fully protected linear polypeptide resin is Cys(Acm)-Lys(Boc)-Gly-Lys(Boc)-Gly-Ala-Lys(Boc)-Cys(Trt)-Ser(tBu)-Arg(Pbf)-Leu-Met-Tyr(tBu)-Asp(OtBu)-Cys(pMeOBzl)-Cys(Acm)-Thr(tBu)-Gly-Ser(tBu)-Cys(Trt)-Arg(Pbf)-Ser(tBu)-Gly-Lys(Boc)-Cys(pMeOBzl)-resin.

10. The method for positional cyclization of three pairs of disulfide bonds in the polypeptide synthesis process according to claim 9, characterized in that: The lysis solution in step S2 is composed of trifluoroacetic acid, anisole, thioanisole, dithiothreitol, triisopropylsilane and water. The ratio of the fully protected linear polypeptide resin to the lysis solution is 1 g: 10 mL-20 mL. The reaction time is 60 min-180 min and the reaction temperature is 30±5°C.

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