WO2020034286A1 - 一种普卡那肽的制备方法 - Google Patents

一种普卡那肽的制备方法 Download PDF

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WO2020034286A1
WO2020034286A1 PCT/CN2018/104840 CN2018104840W WO2020034286A1 WO 2020034286 A1 WO2020034286 A1 WO 2020034286A1 CN 2018104840 W CN2018104840 W CN 2018104840W WO 2020034286 A1 WO2020034286 A1 WO 2020034286A1
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fmoc
cys
resin
protecting group
phase
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PCT/CN2018/104840
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French (fr)
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邹卫星
宓鹏程
陶安进
袁建成
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深圳翰宇药业股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention belongs to the field of polypeptide synthesis, and particularly relates to a method for preparing pukanatide.
  • Pukanatide is a guanylate cyclase C (GC-C) receptor agonist developed by Synergy Pharmaceuticals, Inc., under the trade name Trulance. The effect is similar to that of the natriuretic peptide uroguanylin, which can induce fluid secretion into the gastrointestinal tract, thereby increasing gastrointestinal motility and treating constipation. It was approved by the US Food and Drug Administration (FDA) on January 19, 2017.
  • FDA US Food and Drug Administration
  • pukanatide The English name of pukanatide is Plecanatide, and its CAS number is 467426-54-6. It consists of 16 L-type amino acid residues, of which Cys 4 , Cys 12 and Cys 7 , Cys 15 are connected by disulfide bonds. . Its peptide sequence is as follows:
  • CN103694320 A describes a method in which tBu and Acm are respectively used as protective groups for thiol groups on two pairs of disulfide bonds, and two pairs of disulfide bonds are formed by oxidation with H 2 O 2 and I 2 respectively.
  • CN104211777 A uses any two of StBu, Acm, Mmt, and Trt as the protective groups for the sulfhydryl groups on the two pairs of disulfide bonds of pukana peptide, and uses different oxidation methods to form the first pair and the second pair. Sulfur bond.
  • CN104628827A introduces a method for all-solid-phase synthesis to locate two disulfide bonds.
  • the biggest difference between this patent and the previous two patents lies in the choice of Cys (Cys4, Cys 12 ) thiol protecting group for the first pair of disulfide bonds. It is StBu and Mmt, and the Cys (Cys 7 , Cys 15 ) thiol protecting group of the second pair of disulfide bonds is Acm.
  • StBu is first removed on the solid phase with mercaptoethanol, then DTNP is added to react with the exposed thiol group, and then Mmt is removed with a certain concentration of TFA, and the first pair of disulfide bonds (Cys 4 , Cys 12 ), while the second pair of disulfide bonds (Cys 7 , Cys 15 ) is cyclized using I 2 oxidation. This completes the oxidation of two pairs of disulfide bonds on the solid phase.
  • CN107313870A and CN107313871A are both applied by Suzhou University of Science and Technology.
  • the former introduces the solid-phase synthesis of a peptide resin.
  • the linear crude peptide obtained after the peptide resin is cleaved is free to form a ring in the liquid phase.
  • the latter is a conventional all-solid-phase synthetic peptide resin strategy.
  • the patents CN103694320A, CN104211777A and CN104628827A are different experimental schemes for firstly synthesizing linear peptides by solid phase and then forming different disulfide bonding methods. Each of these schemes has a significant disadvantage: it ignores the difficulty of synthesizing the pukanatine linear peptide. According to the above scheme, the peptide linear peptide caused severe resin shrinkage due to the effect of its secondary structure, and the coupling was difficult. 40-55%, the yield of the final product obtained after linear peptide synthesis is completed and then the disulfide bonding is low, generally 20-25%.
  • the object of the present invention is to provide a Fmoc / tBu solid-phase synthesis route, which can greatly improve the swelling property of the resin, make the coupling easy, improve the purity of the peptide, and improve the yield.
  • the present invention adopts the following technical solutions:
  • Cys 7 and Cys 15 are protected by Mmt or Trt, Cys 4 and Cys 12 are protected by Acm, and peptides are synthesized by Fmoc / tBu solid-phase synthesis.
  • Mmt or Trt is removed using a certain concentration of TFA.
  • the protecting group is then solid-phase oxidized with H 2 O 2 to form a disulfide bond between Cys 7 and Cys 15 , and then the subsequent amino acid residues are coupled. After all the residues are coupled, I 2 is used. Oxidation of the second pair of disulfide bonds.
  • the reaction process is as follows:
  • a method for preparing pucanatide which includes the following steps:
  • Fmoc-Leu 6 -OH, Fmoc-Glu 5 (OtBu) -OH, Fmoc-Cys 4 (Acm) -OH, Fmoc-Glu 3 (OtBu) -OH, Fmoc are coupled according to the Fmoc solid-phase synthesis strategy.
  • step 1) and 4) Fmoc solid phase synthesis strategy coupling refers to coupling Fmoc protected amino acid residues, then removing the Fmoc protecting group, and then coupling the next Fmoc protected amino acid residue .
  • the method for coupling Fmoc-protected amino acid residues is to synthesize Fmoc-AA-OH and a solid-phase synthetic resin or a polypeptide having the Fmoc-protected group-solid-phase synthesized under the action of a coupling reagent.
  • the resin is coupled.
  • the method of removing Fmoc protecting group is to remove Fmoc protecting group with 20% piperidine / DMF solution.
  • the coupling reagent used is a composition of DIPCDI and compound A or a composition of DIPEA and compound A and compound B, where compound A is HOAt or HOBt, and compound B is PyAOP, PyBOP, HATU, HBTU or TBTU, preferably DIPCDI and Composition of compound A.
  • Selected solvents include solvents such as tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetone, ether, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, dichloromethane, etc.
  • solvents such as tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetone, ether, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, dichloromethane, etc.
  • a 1: 1 mixed solution of dimethylformamide and dichloromethane is preferred.
  • the solid-phase synthetic resin is selected from wang resin and 2CTC resin.
  • the degree of resin substitution is 0.2-0.5 mmol / g, preferably 0.25-0.40 mmol / g.
  • the resin is swelled with a solvent before use.
  • the selected solvents include DMF, NMP, dichloromethane, etc., preferably DMF.
  • the AA in the Fmoc-AA-OH is various protected amino acids.
  • the coupling reaction time of each amino acid is usually 1.5-4 hours, preferably 2-3 hours; the temperature is preferably room temperature (ie, 20 ⁇ 5 ° C), and it can also be performed at a suitably elevated or reduced temperature.
  • the disulfide bonding reaction in step 3) uses H 2 O 2 for the bonding reaction, preferably 1-30 equivalents of H 2 O 2 of the reaction substrate mass and reacts with the peptide-solid phase synthetic resin for 0.5-3 hours. , More preferably 10 equivalents for 1 hour.
  • the reaction solvent in step 3) is selected from the group consisting of tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetone, ether, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, and dichloride.
  • the oxidation reaction is to use I 2 for the second pair of disulfide bonds.
  • the amount of I 2 is 0.8 to 10 equivalents, preferably 3 equivalents, based on the amount of the resin-polypeptide (product obtained in step 4).
  • the reaction time is 0.5 to 3 hours, preferably 1 hour.
  • the peptide resin is obtained by washing and shrinking. I 2 can remove the cysteine side chain protecting group and complete the disulfide bonding.
  • Step 5 The solvent is selected from tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dioxane, acetone, ether, acetonitrile, carbon tetrachloride, carbon disulfide, benzene, toluene, hexane, chloroform, and dichloromethane.
  • the purification step can be performed by reverse-phase high-pressure liquid chromatography.
  • the reversed-phase high-pressure liquid chromatography method includes: using reversed-phase octadecylsilane as a stationary phase and 0.1% trifluoroacetic acid aqueous solution / acetonitrile as a mobile phase, collecting the target peak fractions, and concentrating and lyophilizing.
  • the method of the present invention only changes the sequence of synthesis, the change of the secondary structure of the peptide sequence greatly enhances the swelling property of the resin, the difficulty of subsequent amino acid residue coupling is greatly reduced, and the purity of the final crude peptide is greatly improved. At the same time, the entire operation process is completed in the solid phase, and the operation is simple and feasible.
  • reaction solution was washed with DMF 4 times, and 140 mL of acetic anhydride, 120 mL of pyridine and an appropriate amount of DMF were added, and the reaction was mixed and blocked for more than 8 hours. After the reaction was completed, it was washed 4 times with DMF and 2 times with DCM. After the methanol was shrunk twice, the resin was drained to obtain 160.5 g of Fmoc-Leu-Wang resin, and the degree of substitution was 0.21 mmol / g.
  • the resin was washed 3 times with 150 mL of DMF, deprotected by adding 150 mL of DBLK for 6 min + 8 min, and the resin was washed 6 times with 150 mL of DMF. Ninhydrin detected that the resin was colored.
  • the resin was subjected to a de-Mmt treatment, and the resin was washed 6 times with DMF after completion.
  • Example 3 17.2 g of the crude peptide obtained in Example 3 was subjected to reversed-phase liquid phase preparation using octadecylsilane as a stationary phase and 0.1% trifluoroacetic acid aqueous solution / acetonitrile as a mobile phase, and the target peak fractions were collected, concentrated and lyophilized. . 6.8 g of spermeptide was obtained with a purity of 99.5%, and the overall yield of pucanatide was 40.4%.
  • the reaction time was 1 h. After the reaction was completed, deprotection was performed with 150 ml of DBLK for 5 min + 7 min, 150 mL of DMF was washed 5 times, and then DCM was washed 2 times, and then the resin was shrunk with 150 mL of methanol 2 times for 5 min each time, and the peptide resin was dried under reduced pressure to obtain 76.8 g of peptide resin.
  • Example 6 16.8 g of the crude peptide obtained in Example 6 was prepared and purified by reversed-phase liquid phase using octadecylsilane as a stationary phase and 0.1% trifluoroacetic acid aqueous solution / acetonitrile as a mobile phase. The target peak fractions were collected, concentrated and lyophilized. . 3.5 g of spermeptide was obtained with a purity of 99.3%, and the overall yield of pucanatide was 20.8%.
  • the resin was washed 3 times with 150 mL of DMF, deprotected by adding 100 mL of DBLK for 6 min + 8 min, and the resin was washed 6 times with 100 mL of DMF. Ninhydrin detected that the resin was colored.
  • the reaction time was 1 h. After the reaction was completed, deprotection was performed with 150 ml of DBLK for 5 min + 7 min, 150 mL of DMF was washed 5 times, then DCM was washed 2 times, and then the resin was shrunk with 150 mL of methanol 2 times, each time for 5 min. The peptide resin was dried under reduced pressure, and 75.9 g of peptide resin was finally obtained.
  • Example 9 16.4 g of the crude peptide obtained in Example 9 was subjected to reversed-phase liquid phase preparation using octadecylsilane as a stationary phase and 0.1% trifluoroacetic acid aqueous solution / acetonitrile as a mobile phase, and the target peak fractions were collected, concentrated and lyophilized. . 3.5 g of spermeptide was obtained with a purity of 99.5%, and the overall yield of pucanatide was 24.8%.

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Abstract

一种普卡那肽的制备方法，其包括以下步骤：1)在固相合成树脂上按照Fmoc固相合成策略依次偶联第16-7位氨基酸；2)以脱除Fmoc保护基后，以脱Mmt保护基的试剂进行处理，脱除7位和15位Cys的侧链保护基Mmt；3)通过键合反应，形成7位和15位Cys之间的二硫键；4)按照Fmoc固相合成策略再依次偶联第6-1位氨基酸5)以脱除Fmoc保护基，通过氧化反应完成4位和12位Cys之间的二硫键；6)脱除所有侧链保护基，以裂解试剂裂解树脂，乙醚沉淀获得普卡那肽。该方法简单，效率高。

Description

一种普卡那肽的制备方法 技术领域
本发明属于多肽合成领域，具体涉及一种普卡那肽的制备方法。
背景技术
普卡那肽是药物协和股份有限公司(Synergy Pharmaceuticals,Inc)开发的一种鸟苷酸环化酶C(GC-C)受体激动剂，其商品名为Trulance。效应与利尿钠肽尿鸟苷素相似，可诱导液体分泌进入胃肠道，从而增加胃肠动力，从而治疗便秘。于2017年01月19日获得美国食品和药物监督管理局(FDA)的批准上市。
普卡那肽的英文名为Plecanatide，其CAS号为467426-54-6，它由16个L-型氨基酸残基组成，其中Cys 4、Cys 12及Cys 7、Cys 15分别由二硫键相连。其肽序如下：
Figure PCTCN2018104840-appb-000001
CN103694320 A及CN104211777 A均为本公司已授权申请专利，主要关于普卡那肽固液相结合定位成两对二硫键的不同方法的介绍。CN103694320 A介绍的是采用tBu及Acm分别作为两对二硫键上巯基的保护基团，通过H 2O 2及I 2分别氧化形成两对二硫键的方法。CN104211777 A则是采用StBu、Acm、Mmt、Trt四种中的任意两种作为普卡那肽两对二硫键上巯基的保护基团，通过不同的氧化方法成第一对及第二对二硫键。
CN104628827A介绍了一种全固相合成定位成两对二硫键的方法，此专利与前两篇专利最大的不同点在于第一对二硫键的Cys(Cys4、Cys 12)巯基保护基团选择的是StBu及Mmt，而第二对二硫键的Cys(Cys 7、Cys 15)巯基保护基团则是Acm。前期肽树脂合成完成后首先在固相上用巯基乙醇脱除StBu，然后加入DTNP与裸露出的巯基反应，然后用一定浓度的TFA脱除Mmt，同时进行第一对二硫键(Cys 4、Cys 12)的环化，而第二对二硫键(Cys 7、Cys 15)的环化则采用I 2氧化。这样即在固相上完成的两对二硫键的氧化。
CN107313870A与CN107313871A均为苏州科技大学申请，前者介绍的是先固相合成肽树脂，肽树脂经裂解后得到的线性粗肽在液相中自由成环。后者介绍是常规的全固相合成肽树脂策略。
专利CN103694320 A、CN104211777 A及CN104628827A均是先固相合成线性多肽然后围绕二硫键键合方式的不同形成的不同的实验方案。这几种方案均有一显著缺点：忽略 了普卡那肽线性肽的合成难度，按上述方案，此肽线性肽由于自身二级结构的影响导致树脂收缩严重，偶联困难，线性肽的纯度为40-55％，待线性肽合成完成后再进行二硫键的键合得到的终产品收率较低，一般为20-25％。
发明内容
鉴于上述问题，本发明的目的在于提供一条Fmoc/tBu固相合成路线，能大幅提高树脂的溶胀性，使偶联容易，提高肽的纯度，从而提高收率。为实现上述目的，本发明采取如下的技术方案：
通过Cys 7及Cys 15采用Mmt或Trt保护，而Cys 4及Cys 12采用Acm保护，采用Fmoc/tBu固相合成方法合成多肽，偶联完Cys 7后，采用一定浓度的TFA脱除Mmt或Trt保护基团，然后采用H 2O 2先进行固相氧化成Cys 7及Cys 15之间的二硫键，然后继续偶联后续的氨基酸残基，待所有残基偶联完毕后采用I 2进行第二对二硫键的氧化。由于二硫键的形成，导致肽序二级结构的改变大大增强了树脂的溶胀性，后续氨基酸残基偶联难度得到大大降低，最终粗肽的纯度大幅提高至80％以上，纯化后精肽的收率>40％。同时整个操作过程全部在固相完成，操作简单可行。
反应流程如下：
Figure PCTCN2018104840-appb-000002
本发明一个方面提供了一种普卡那肽的制备方法，其包括以下步骤：
1)在固相合成树脂上按照Fmoc固相合成策略依次偶联Fmoc-Leu 16–OH，Fmoc-Cys 15(Mmt)-OH，Fmoc-Gly 14-OH、Fmoc-Thr 13(tBu)-OH、Fmoc-Cys 12(Acm)-OH、Fmoc-Ala 11-OH、Fmoc-Val 10-OH、Fmoc-Asn 9(Trt)-OH、Fmoc-Val 8-OH、Fmoc-Cys 7(Mmt)-OH；
2)以脱除Fmoc保护基后，以脱Mmt保护基的试剂进行处理，脱除7位和15位Cys的侧链保护基Mmt；
3)通过键合反应，形成7位和15位Cys之间的二硫键；
4)按照Fmoc固相合成策略再依次偶联Fmoc-Leu 6-OH、Fmoc-Glu 5(OtBu)-OH、Fmoc-Cys 4(Acm)-OH、Fmoc-Glu 3(OtBu)-OH、Fmoc-Asp 2(OtBu)-OH、Fmoc-Asn 1(Trt)-OH；
5)通过氧化反应完成4位和12位Cys之间的二硫键，然后脱除Fmoc保护基，；
6)以裂解试剂裂解树脂同时脱除所有侧链保护基，乙醚沉淀获得普卡那肽；
任选地，7)反相色谱进行纯化。
在本发明的技术方案中，步骤1)和4)Fmoc固相合成策略偶联是指偶联Fmoc保护的氨基酸残基，然后脱除Fmoc保护基，再偶联下一个Fmoc保护的氨基酸残基。
在本发明的技术方案中，偶联Fmoc保护的氨基酸残基的方法为在偶联试剂的作用下将Fmoc-AA-OH和固相合成树脂或者已经脱除Fmoc保护基的多肽-固相合成树脂进行偶联。
脱除Fmoc保护基的方法为用20％的哌啶/DMF溶液的脱除Fmoc保护基
所用的偶联试剂为DIPCDI和化合物A的组合物或DIPEA和化合物A和化合物B的组合物，其中化合物A为HOAt或HOBt，化合物B为PyAOP、PyBOP、HATU、HBTU或TBTU，优选为DIPCDI和化合物A的组合物。进一步地，偶联剂中各成分与保护氨基酸(AA)的比例以摩尔比计为DIPCDI:A:AA＝1.3:1.2:1.0，DIPEA:A:B:AA＝2.0:1.2:1.0:1.0。
选用的溶剂包括四氢呋喃、二甲基亚砜、二甲基甲酰胺、二恶烷、丙酮、乙醚、乙腈、四氯化碳、二硫化碳、苯、甲苯、己烷、氯仿、二氯甲烷等溶剂，优选二甲基甲酰胺与二氯甲烷1:1的混合溶液。
在本发明的技术方案中，固相合成树脂选自wang树脂，2CTC树脂。树脂替代度为0.2-0.5mmol/g，优选0.25-0.40mmol/g。
树脂使用前用溶剂进行溶胀，所选溶剂包括DMF、NMP、二氯甲烷等，优选DMF。
所述的Fmoc-AA-OH中的AA为各种保护氨基酸。
每种氨基酸进行偶联反应的时间通常为1.5-4小时，优选2-3小时；温度优选为室温(即20±5℃)，也可在适当提高或降低的温度下进行。
步骤2)中脱除Mmt保护基团采用的方法是采用TFA:TIS:DCM＝(1-5):(2-10):(97-85)的混合溶液与树脂反应15-30次，每次1-5分钟，优选溶液比例为TFA:TIS:DCM＝2:5:93、反应次数为20次及每次2分钟。
步骤3)中二硫键的键合反应采用H 2O 2进行键合反应，优选为反应底物物质量的1-30当量的H 2O 2与多肽-固相合成树脂反应0.5-3小时，更优选10当量反应1小时。
步骤3)中反应溶剂选自四氢呋喃、二甲基亚砜、二甲基甲酰胺、二恶烷、丙酮、乙醚、乙腈、四氯化碳、二硫化碳、苯、甲苯、己烷、氯仿、二氯甲烷中的一种或多种的组合，优选二甲基甲酰胺。
步骤5)中氧化反应是采用I 2进行第二对二硫键的键合。I 2的用量为树脂-多肽(步骤4 所得产物)物质的量的0.8-10当量，优选3当量。反应时间为0.5-3小时，优选1小时。反应完成后经洗涤、收缩得到肽树脂。I 2能够脱除半胱氨酸侧链保护基同时完成二硫键键合。
步骤5)溶剂选择四氢呋喃、二甲基亚砜、二甲基甲酰胺、二恶烷、丙酮、乙醚、乙腈、四氯化碳、二硫化碳、苯、甲苯、己烷、氯仿、二氯甲烷中的一种或多种的组合，优选二甲基甲酰胺。
步骤6)中所用裂解液为TFA、TIS、H 2O、苯甲醚、苯甲硫醚、苯酚一种或多种的混合物；优选为TFA，TIS和H 2O的混合物，更优选为TFA:TIS:H 2O＝92:4:4。
步骤7)所述纯化步骤可采用反相高压液相色谱法。进一步地，所述反相高压液相色谱法包括：以反相十八烷基硅烷为固定相，以0.1％三氟乙酸水溶液/乙腈为流动相，收集目的峰馏分，浓缩冻干。
缩写及英文含义
Figure PCTCN2018104840-appb-000003
Figure PCTCN2018104840-appb-000004
有益效果
本发明的方法仅仅改变了合成的顺序，肽序二级结构的改变大大增强了树脂的溶胀性，后续氨基酸残基偶联难度得到大大降低，最终粗肽的纯度大幅提高。同时整个操作过程全部在固相完成，操作简单可行。
具体实施方式
提供下述实例是为了更好地进一步理解本发明，并不局限于所述实施方式，不对本发明的内容和保护范围构成限制，任何人在本发明的启示下或是将本发明与其他现有技术的特征进行组合而得出的任何与本发明相同或相近似的方法，均落在本发明的保护范围之内。
实施例1：Fmoc-Leu-Wang Resin的制备
称取替代度为1.03mmol/g的Wang树脂150g于固相反应柱中，用DMF洗涤2次后用DMF溶胀树脂30分钟，取37.5g Fmoc-Leu-OH、15.6g HOBt、1.29gDMAP溶于400ml体积比为1:1的DCM和DMF混合溶液，于冰盐水浴中搅拌，控制温度0-10℃时滴加17.4gDIPCDI后活化2-3min，将上述溶液加入固相反应柱中室温反应1.5h。反应结束后用DMF洗涤4次后加入140mL醋酸酐和120mL吡啶及适量DMF，混合封闭反应8小时以上。反应结束后用DMF洗涤4次，DCM洗2次。甲醇收缩2次后抽干树脂，得到Fmoc-Leu-Wang树脂160.5g，检测替代度为0.21mmol/g。
实施例2：肽树脂的制备
称取实施例1制备的替代度为0.21mmol/g的Fmoc-Leu-Wang树脂47.6g(10mmol)于固相反应柱中，用DMF洗涤2次后用DMF溶胀树脂30分钟，然后用150ml的DBLK脱保护 5min+7min，150mLDMF洗涤6次。
称取30.8g(50mmol)Fmoc-Cys(Mmt)-OH和8.1g(60mmol)HOBt用100mL的DMF/DCM(1:1)溶解，冰水浴下加入10mL(65mmol)DIPCDI活化2-3min后，将混合液加入到反应柱中，室温反应2小时，以茚三酮检测反应终点(如树脂无色透明则终止反应；如树脂显色则延长反应1小时)。反应结束，用150mLDMF洗涤树脂3次，加入150mLDBLK脱保护6min+8min，150mLDMF洗涤树脂6次，茚三酮检测树脂有颜色。
重复上述偶联操作，按肽序列顺序继续依次偶联Fmoc-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(Acm)-OH、Fmoc-Ala-OH、Fmoc-Val-OH、Fmoc-Asn(Trt)-OH、Fmoc-Val-OH、Fmoc-Cys(Mmt)-OH各保护氨基酸均投料50mmol，其它物料投料比与保护氨基酸对应。
脱Fmoc保护结束DMF洗涤树脂6次后，肽树脂继续用DCM洗涤3次并抽干，然后采用TFA:TIS:DCM＝2:5:93、反应次数为20次及每次2分钟来对肽树脂进行脱Mmt处理，结束后树脂用DMF洗涤6次。
量取30％的双氧水10.2ml(100mmol)加入到150ml的DMF中，加入到固相反应柱中与树脂进行第一对二硫键的键合反应，鼓气反应1h，反应完毕后继续DMF洗涤6次。
重复上述偶联操作，按肽序列顺序继续依次偶联剩余的氨基酸残基：Fmoc-Leu-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Cys(Acm)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Asn(Trt)-OH。各保护氨基酸均投料50mmol，其它物料投料比与保护氨基酸对应。
上述残基偶联完毕后进行第二对二硫键的键合。称取7.6g(30mmol)的I 2溶解于150ml的DMF后加入反应柱中。反应时间为1h。反应完成后用150ml的DBLK脱保护5min+7min，150mLDMF洗涤5次后DCM洗涤2次，然后用150mL甲醇收缩树脂2次，每次5min，减压抽干肽树脂，最终得到肽树脂78.6克。
实施例3：肽树脂的裂解
将实施例2中得到的78.6g(10mmol)肽树脂(1)全部置于裂解反应器中，以10ml/g肽树脂的比例加入780ml的裂解试剂(TFA:TIS:H 2O＝92:4:4(V/V))，室温搅拌2h。反应物用砂芯漏斗过滤，过滤结束后加入少量TFA洗涤树脂，收集并合并滤液后减压浓缩至一定体积。加入冰冻的无水乙醚(100ml/g肽树脂)使溶液沉淀，离心、去除上清液后将沉淀用无水乙醚洗涤3次，真空干燥得到白色固体粉末的粗肽17.2g。粗肽纯度为81.3％(HPLC检测条件：Waters C18 300A 1.7um 2.0*100mm；缓冲液：50mM磷酸铵缓冲液(磷酸调pH＝6.2)；A相：乙腈:缓冲液＝10:90(V:V)；B相：缓冲液＝50:50(V:V)、梯度：5-45％(20min)/45.1-95％(10min)；流速：0.4ml/min检测波长：220nm。下同)。
实施例4：普卡那肽的制备
将实施例3中获得的17.2g粗肽经以十八烷基硅烷为固定相，以0.1％三氟乙酸水溶液/乙腈为流动相进行反相液相制备纯化，收集目的峰馏分，浓缩冻干。得到精肽6.8g，纯度99.5％，普卡那肽总体收率40.4％。
实施例5：肽树脂的制备(常规对比方法1)
称取实施例1制备的替代度为0.21mmol/g的Fmoc-Leu-Wang树脂47.6g(10mmol)于固相反应柱中，用DMF洗涤2次后用DMF溶胀树脂30分钟，然后用150ml的DBLK脱保护5min+7min，150mLDMF洗涤6次。称取30.8g(50mmol)Fmoc-Cys(Mmt)-OH和8.1g(60mmol)HOBt用100mL的DMF/DCM(1:1)溶解，冰水浴下加入10mL(65mmol)DIPCDI活化2-3min后，将混合液加入到反应柱中，室温反应2小时，以茚三酮检测反应终点(如树脂无色透明则终止反应；如树脂显色则延长反应1小时)。反应结束，用150mLDMF洗涤树脂3次，加入100mLDBLK脱保护6min+8min，100mLDMF洗涤树脂6次，茚三酮检测树脂有颜色。重复上述偶联操作，按照上文步骤2中所述按肽序列顺序继续依次偶联Fmoc-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(Acm)-OH、Fmoc-Ala-OH、Fmoc-Val-OH、Fmoc-Asn(Trt)-OH、Fmoc-Val-OH、Fmoc-Cys(Mmt)-OH、Fmoc-Leu-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Cys(Acm)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Asn(Trt)-OH。各保护氨基酸均投料50mmol，其它物料投料比与保护氨基酸对应。
上述残基偶联完毕后采用适量的TFA:TIS:DCM＝2:5:93、反应次数为20次及每次2分钟来对肽树脂进行脱Mmt处理，结束后树脂用DMF洗涤6次。量取30％的双氧水10.2ml(100mmol)加入到150ml的DMF中，加入到固相反应柱中与树脂进行第一对二硫键的键合反应，鼓气反应1h后继续DMF洗涤6次，接着进行第二对二硫键的键合。称取7.6g(30mmol)的I 2溶解于150ml的DMF后加入反应柱中。反应时间为1h。反应完成后用150ml的DBLK脱保护5min+7min，150mLDMF洗涤5次后DCM洗涤2次，然后用150mL甲醇收缩树脂2次，每次5min，减压抽干肽树脂，最终得到肽树脂76.8克。
实施例6：肽树脂的裂解(常规对比方法1)
将实施例5中得到的76.8g(10mmol)肽树脂全部置于裂解反应器中，以10ml/g肽树脂的比例加入770ml的裂解试剂(TFA:TIS:H 2O＝92:4:4(V/V))，室温搅拌2h。反应物用砂芯漏斗过滤，过滤结束后加入少量TFA洗涤树脂，收集并合并滤液后减压浓缩至一定体积。加入冰冻的无水乙醚(100ml/g肽树脂)使溶液沉淀，离心、去除上清液后将沉淀用无水乙醚洗涤3次，真空干燥得到白色固体粉末的粗肽16.8g。粗肽纯度为42.3％。
实施例7：普卡那肽的制备(常规对比方法1)
将实施例6中获得的16.8g粗肽经以十八烷基硅烷为固定相，以0.1％三氟乙酸水溶液/乙腈为流动相进行反相液相制备纯化，收集目的峰馏分，浓缩冻干。得到精肽3.5g，纯度99.3％，普卡那肽总体收率20.8％。
实施例8：肽树脂的制备(常规对比方法2)
称取实施例1制备的替代度为0.21mmol/g的Fmoc-Leu-Wang树脂47.6g(10mmol)于固相反应柱中，用DMF洗涤2次后用DMF溶胀树脂30分钟，然后用150ml的DBLK脱保护5min+7min，150mLDMF洗涤6次。称取20.7g(50mmol)Fmoc-Cys(Acm)-OH和8.1g(60mmol)HOBt用100mL的DMF/DCM(1:1)溶解，冰水浴下加入10mL(65mmol)DIPCDI活化2-3min后，将混合液加入到反应柱中，室温反应2小时，以茚三酮检测反应终点(如树脂无色透明则终止反应；如树脂显色则延长反应1小时)。反应结束，用150mLDMF洗涤树脂3次，加入100mLDBLK脱保护6min+8min，100mLDMF洗涤树脂6次，茚三酮检测树脂有颜色。重复上述偶联操作，按照上文步骤2中所述按肽序列顺序继续依次偶联Fmoc-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(Mmt)-OH、Fmoc-Ala-OH、Fmoc-Val-OH、Fmoc-Asn(Trt)-OH、Fmoc-Val-OH、Fmoc-Cys(Acm)-OH、Fmoc-Leu-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Cys(Mmt)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Asn(Trt)-OH。各保护氨基酸均投料50mmol，其它物料投料比与保护氨基酸对应。
上述残基偶联完毕后采用适量的TFA:TIS:DCM＝2:5:93、反应次数为20次及每次2分钟来对肽树脂进行脱Mmt处理，结束后树脂用DMF洗涤6次。量取30％的双氧水10.2ml(100mmol)加入到150ml的DMF中，加入到固相反应柱中与树脂进行第一对二硫键的键合反应，鼓气反应1h后继续DMF洗涤6次，接着进行第二对二硫键的键合。称取7.6g(30mmol)的I 2溶解于150ml的DMF后加入反应柱中。反应时间为1h。反应完成后用150ml的DBLK脱保护5min+7min，150mLDMF洗涤5次后DCM洗涤2次，然后用150mL甲醇收缩树脂2次，每次5min，减压抽干肽树脂，最终得到肽树脂75.9克。
实施例9：肽树脂的裂解(常规对比方法2)
将实施例8中得到的75.9g(10mmol)肽树脂全部置于裂解反应器中，以10ml/g肽树脂的比例加入760ml的裂解试剂(TFA:TIS:H 2O＝92:4:4(V/V))，室温搅拌2h。反应物用砂芯漏斗过滤，过滤结束后加入少量TFA洗涤树脂，收集并合并滤液后减压浓缩至一定体积。加入冰冻的无水乙醚(100ml/g肽树脂)使溶液沉淀，离心、去除上清液后将沉淀用无水乙醚洗涤3次，真空干燥得到白色固体粉末的粗肽16.4g。粗肽纯度为44.7％。
实施例10：普卡那肽的制备(常规对比方法1)
将实施例9中获得的16.4g粗肽经以十八烷基硅烷为固定相，以0.1％三氟乙酸水溶液/乙腈为流动相进行反相液相制备纯化，收集目的峰馏分，浓缩冻干。得到精肽3.5g，纯度99.5％，普卡那肽总体收率24.8％。

Claims (9)

  1. 一种普卡那肽的制备方法，其包括以下步骤：
    1)在固相合成树脂上按照Fmoc固相合成策略依次偶联Fmoc-Leu 16–OH，Fmoc-Cys 15(Mmt)-OH，Fmoc-Gly 14-OH、Fmoc-Thr 13(tBu)-OH、Fmoc-Cys 12(Acm)-OH、Fmoc-Ala 11-OH、Fmoc-Val 10-OH、Fmoc-Asn 9(Trt)-OH、Fmoc-Val 8-OH、Fmoc-Cys 7(Mmt)-OH；
    2)以脱除Fmoc保护基后，以脱Mmt保护基的试剂进行处理，脱除7位和15位Cys的侧链保护基Mmt；
    3)通过键合反应，形成7位和15位Cys之间的二硫键；
    4)按照Fmoc固相合成策略再依次偶联Fmoc-Leu 6-OH、Fmoc-Glu 5(OtBu)-OH、Fmoc-Cys 4(Acm)-OH、Fmoc-Glu 3(OtBu)-OH、Fmoc-Asp 2(OtBu)-OH、Fmoc-Asn 1(Trt)-OH；
    5)通过氧化反应完成4位和12位Cys之间的二硫键，然后脱除Fmoc保护基；
    6)以裂解试剂裂解树脂同时脱除所有侧链保护基，乙醚沉淀获得普卡那肽；任选地，7)反相色谱进行纯化。
  2. 根据权利要求1所述的制备方法，其中，步骤2)中脱除Mmt保护基团采用的方法是采用TFA:TIS:DCM＝(1-5):(2-10):(97-85)的混合溶液与树脂反应15-30次，每次1-5分钟，优选溶液比例为TFA:TIS:DCM＝2:5:93、反应次数为20次及每次2分钟。
  3. 根据权利要求1所述的制备方法，步骤3)中二硫键的键合反应采用H 2O 2进行键合反应，优选为1-30当量的H 2O 2与多肽-固相合成树脂反应0.5-3小时，更优选10当量反应1小时。
  4. 根据权利要求1所述的制备方法，步骤5)中氧化反应是采用I 2进行第二对二硫键的键合；I 2的用量为树脂物质的量的0.8-10当量，优选3当量。
  5. 根据权利要求1所述的制备方法，步骤1)和4)Fmoc固相合成策略偶联是指偶联Fmoc保护的氨基酸残基，然后脱除Fmoc保护基，再偶联下一个Fmoc保护的氨基酸残基。
  6. 根据权利要求1所述的制备方法，偶联Fmoc保护的氨基酸残基的方法为在偶联试剂的作用下将Fmoc-AA-OH和固相合成树脂或者已经脱除Fmoc保护基的多肽-固相合成树脂进行偶联；优选地，所用的偶联试剂为DIPCDI和化合物A的组合物或DIPEA和化合物A和化合物B的组合物，其中化合物A为HOAt或HOBt，化合物B为PyAOP、PyBOP、HATU、HBTU或TBTU，优选为DIPCDI和化合物A的组合物，更优选地，偶联剂中各成分与保护氨基酸的比例以摩尔比计为DIPCDI:A:AA＝1.3:1.2:1.0，DIPEA:A:B:AA＝2.0:1.2:1.0:1.0。
  7. 根据权利要求1所述的制备方法，脱除Fmoc保护基的方法为用20％的哌啶/DMF溶液的脱除Fmoc保护基。
  8. 根据权利要求1所述的制备方法，步骤6)中所用裂解液为TFA、H 2O、苯甲醚、苯甲硫醚、苯酚中的一种或多种的混合物。
  9. 根据权利要求1所述的制备方法，步骤7)所述纯化步骤可采用反相高压液相色谱法；优选地，所述反相高压液相色谱法包括：以反相十八烷基硅烷为固定相，以0.1％三氟乙酸水溶液/乙腈为流动相，收集目的峰馏分，浓缩冻干。
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CN113444150B (zh) * 2021-08-10 2023-02-28 礼济生物医药科技(南京)有限公司 一种普卡那肽的固相制备方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103694320A (zh) * 2013-12-11 2014-04-02 深圳翰宇药业股份有限公司 一种普利卡那肽的制备方法
CN104628827A (zh) * 2015-03-10 2015-05-20 南京工业大学 一种普利卡那肽的制备方法
CN105764916A (zh) * 2013-06-05 2016-07-13 辛纳吉制药公司 鸟苷酸环化酶c的超纯激动剂、制备和使用所述激动剂的方法
CN107383171A (zh) * 2017-08-15 2017-11-24 苏州科技大学 一种通过二次环化固相合成普卡那肽的方法
CN108003222A (zh) * 2017-12-31 2018-05-08 江苏诺泰澳赛诺生物制药股份有限公司 一种普利卡那肽的固相合成方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150273013A1 (en) * 2014-03-25 2015-10-01 Synergy Pharmaceuticals, Inc. Agonists of guanylate cyclase useful for the treatment of gastrointestinal disorders, inflammation, cancer and other disorders
CN105504012A (zh) * 2014-09-30 2016-04-20 深圳翰宇药业股份有限公司 一种多肽的制备方法
CN105017387B (zh) * 2015-07-31 2018-09-14 济南康和医药科技有限公司 一种制备利那洛肽的方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105764916A (zh) * 2013-06-05 2016-07-13 辛纳吉制药公司 鸟苷酸环化酶c的超纯激动剂、制备和使用所述激动剂的方法
CN103694320A (zh) * 2013-12-11 2014-04-02 深圳翰宇药业股份有限公司 一种普利卡那肽的制备方法
CN104628827A (zh) * 2015-03-10 2015-05-20 南京工业大学 一种普利卡那肽的制备方法
CN107383171A (zh) * 2017-08-15 2017-11-24 苏州科技大学 一种通过二次环化固相合成普卡那肽的方法
CN108003222A (zh) * 2017-12-31 2018-05-08 江苏诺泰澳赛诺生物制药股份有限公司 一种普利卡那肽的固相合成方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
FANG, G.M. ET AL.: "Discovery, structure, and chemical synthesis of disulfide-rich peptide toxins and their analogs", CHINESE CHEMICAL LETTERS, vol. 29, no. 7, 1 July 2018 (2018-07-01), pages 1033 - 1042, XP055686664, ISSN: 1001-8417, DOI: 10.1016/j.cclet.2018.02.002 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114573662A (zh) * 2021-09-30 2022-06-03 深圳翰宇药业股份有限公司 一种普卡那肽铵盐的制备方法
CN114573662B (zh) * 2021-09-30 2024-06-11 深圳翰宇药业股份有限公司 一种普卡那肽铵盐的制备方法
CN114044813A (zh) * 2022-01-13 2022-02-15 浙江湃肽生物有限公司南京分公司 一种普卡那肽的制备及纯化方法
CN114044813B (zh) * 2022-01-13 2022-04-15 浙江湃肽生物有限公司南京分公司 一种普卡那肽的制备及纯化方法

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