WO2019085178A1 - 一种固相合成血管升压素受体肽激动剂selepressin的方法 - Google Patents

一种固相合成血管升压素受体肽激动剂selepressin的方法 Download PDF

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WO2019085178A1
WO2019085178A1 PCT/CN2017/116234 CN2017116234W WO2019085178A1 WO 2019085178 A1 WO2019085178 A1 WO 2019085178A1 CN 2017116234 W CN2017116234 W CN 2017116234W WO 2019085178 A1 WO2019085178 A1 WO 2019085178A1
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resin
reaction
allocam
solid phase
compound
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PCT/CN2017/116234
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English (en)
French (fr)
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陈学明
梁思思
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
    • 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
    • C07K1/06—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
    • C07K1/08—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 using activating agents
    • 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
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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 pharmaceutical technology, and in particular relates to a method for synthesizing Selepressin by a solid phase method.
  • Selepressin is a vasopressin receptor peptide agonist, a peptide drug developed by Huiling for the treatment of septic shock, currently in clinical stage III.
  • Selepressin is a synthetic nonapeptide compound with an amide at the C-terminus and a pair of disulfide bonds (1-6). The peptide sequence is shown in Formula I, Cas: 876296-47-8.
  • the patent CN1964732B has publicly reported a method for preparing Selepressin: using an amino resin, preparing a crude peptide by standard Fmoc solid phase peptide synthesis, and then oxidizing a disulfide bond in a liquid phase in a methanolic solution of acetic acid, a reverse high efficiency liquid The phase method is purified to obtain Selepressin phage.
  • the disadvantage of this method is that when the disulfide bond is oxidized by iodine, the impurities are more, the yield and the purity are not high, and the excess iodine is difficult to remove.
  • an object of the present invention is to provide an efficient and environmentally friendly Selepressin synthesis method.
  • the invention firstly synthesizes a linear peptide by solid phase reaction using an amino resin and an inexpensive amino acid raw material, and then catalyzes the oxidation of a disulfide bond in a solid phase by using the specific three-way catalytic oxidation system of the present invention, and is prepared by high-performance liquid phase purification.
  • Target peptide The method is novel, the synthesis condition is mild, the process is simple, and the process is stable.
  • One aspect of the present invention provides a method for synthesizing a vasopressin receptor peptide agonist Selepressin, which comprises the steps of:
  • the solid phase resin is sequentially coupled with an amino-protected amino acid to synthesize a polypeptide resin H-Cys(allocam)-Phe-Ile-Hgn(R 1 )-Asn(R 2 )-Cys(allocam)-Pro-Org(i- Pr)-Gly-resin;
  • the sulfhydryl group is a disulfide bond
  • R 1 in the step 1) is a side chain protecting group of Hgn, preferably Trt; and R 2 is a side of Asn
  • a chain protecting group preferably Trt.
  • step 2) removing the cysteine protecting group allocam and oxidizing the disulfide bond is the reaction condition in which the three-way catalytic oxidation system in the polypeptide resin obtained in the step 1) is a divalent palladium salt catalyst, a trapping agent and a solvent. reaction system.
  • the divalent palladium salt catalyst in the step 2) is selected from a combination of one or more of Pd(OAc) 2 , PdCl 2 , PdSO 4 , Pd(OTf) 2 , preferably Pd(OAc) 2 ;
  • the capture agent is selected from a combination of one or more of NMM, phenylsilane, triethylamine, preferably NMM;
  • Step 2) the reaction temperature is 0 ° C -100 ° C, preferably 20 ° C -40 ° C;
  • Step 2 The divalent palladium salt catalyst is used in an amount of from 0.5 to 10 equivalents, preferably from 1.0 to 1.5 equivalents.
  • step 2) of the present invention no iodine or iodine related reagent is used as a catalyst.
  • the resin in the step 1) is an amino resin, preferably Rink Amide resin, Rink Amide-AM resin, MBHA resin, PAL resin, and the degree of substitution of the resin is 0.1-2.0 mmol/g, preferably 0.5-1.5 mmol/g, more preferably 0.5-1.0 mmol/g.
  • the amino-protecting group in the amino-protected amino acid described in the step 1) is Fmoc or tBu, and the coupled amino acid is Gly, Org(i-Pr), Pro, Cys(Allocam), Asn(Trt), Hgn(Trt). , Ile, Phe, Cys (Allocam).
  • the solid phase polypeptide synthesis method comprises the following steps:
  • the reagent for removing Fmoc was a 20% piperidine/DMF solution (DBLK), that is, a mixed solution of piperidine:DMF (volume ratio) of 1:4.
  • DBLK 20% piperidine/DMF solution
  • the coupling agent in the step 2) of the present invention is a composition of DIC and Compound A, a combination of DIC and a compound of Russian A and Compound B, a composition of DIPCDI and Compound A, or a combination of DIPEA and Compound A and Compound B, Wherein compound A is HOBt or HOAt and compound B is PyBOP, PyAOP, HATU, HBTU or TBTU, preferably a combination of DIC and Compound A.
  • the solid phase reaction is carried out in a solid phase reaction column.
  • the solid phase reaction column of the present invention is not particularly limited, and may be any solid phase reaction column which can achieve this purpose.
  • the time for the coupling reaction of each amino acid is usually 1.5 to 4 hours, preferably 2 to 3 hours; the pressure is preferably atmospheric pressure, and may be carried out under a suitably increased or decreased pressure; the temperature is preferably room temperature (ie, 20 ⁇ 5 ° C) can also be carried out at a suitably elevated or lowered temperature.
  • step 1) preferably swells the resin prior to coupling, and the steps of washing and swelling can be carried out in the art using any reagent that accomplishes this, including DMF, NMP, dichloromethane, and the like, preferably DMF.
  • the reaction end point can be monitored by a detection method for each step of the reaction, and the detection method applied in the reaction is any method known in the art to achieve this purpose, such as chromatography or chemical calibration, preferably using a determinable reaction.
  • the reagent at the end point preferably ninhydrin, when ninhydrin is used, if the resin develops color, it means that there is a free amine in the polypeptide, that is, there is no protecting group on the amine.
  • the lysate of step 3)) is a mixture of different ratios of TFA, H 2 O, PhOMe and thioanisole; preferably, the ratio of each component is TFA: H 2 O: PhOMe: thioanisole 90: 5:4:1.
  • Step 4) The purification step is carried out by reverse phase high pressure liquid chromatography.
  • the reversed-phase high-pressure liquid chromatography comprises: using a reverse phase octadecylsilane as a stationary phase, a volume ratio of 0.1% aqueous acetic acid/acetonitrile as a mobile phase, and a mobile phase volume ratio of 0.1% aqueous acetic acid/acetonitrile.
  • the ratio is preferably from 98:2 to 50:50, more preferably from 80:20 to 60:40, most preferably 70:30.
  • the peak fraction of interest was collected and concentrated to freeze.
  • the method adopts a solid phase oxidation disulfide bond, and the reaction system adopts a special three-way catalytic oxidation system, and the reaction efficiency is high, and no iodine is used, thereby submitting a step of subsequent purification.
  • the utility model has the advantages of simple operation, simplified process, environmental friendliness, high economic efficiency and large-scale production.
  • Figure 1 is a synthetic route diagram of the present invention.
  • the reaction end point is detected by ninhydrin (if the resin is colorless and transparent, the reaction is terminated; if the resin develops color, the reaction is extended for 1 hour).
  • the resin was washed 3 times with 150 mL of DMF, deprotected by 150 ml of LDBLK for 6 min + 8 min, and washed with 150 mL of DMF for 6 times.
  • the ninhydrin detection resin was colored.
  • the mixture was added to the reaction column, and reacted at room temperature for 2 hours, and the reaction end point was detected with ninhydrin (if the resin was colorless and transparent, the reaction was terminated; if the resin developed color, the reaction was extended for 1 hour).
  • the resin was washed 3 times with 150 mL of DMF, deprotected by 150 ml of LDBLK for 6 min + 8 min, and washed with 150 mL of DMF for 6 times.
  • the ninhydrin detection resin was colored.
  • the reaction end point is detected by ninhydrin (if the resin is colorless and transparent, the reaction is terminated; if the resin develops color, the reaction is extended for 1 hour).
  • the resin was washed 3 times with 150 mL of DMF, deprotected by 150 ml of LDBLK for 6 min + 8 min, and washed with 150 mL of DMF for 6 times.
  • the ninhydrin detection resin was colored.
  • Example 8 46.1 g of the crude peptide obtained in Example 8 was added to a 1000 ml reaction flask, dissolved in 500 ml of an aqueous acetonitrile solution (1:1 by volume), and a preparative column of 15 cm ⁇ 25 cm was prepared for high-performance liquid phase purification.
  • the reverse phase octadecylsilane was used as the stationary phase, and the volume ratio was 0.1% acetic acid aqueous solution/acetonitrile as the mobile phase, the ratio was 70:30; the gradient elution preparation; the flow rate: 70-80 ml/min; the detection wavelength: 280 nm; The peak fraction of the target was collected, concentrated and lyophilized to obtain a pure product of 36.2 g, a purity of 99.5%, and a total yield of 69%.
  • the total yield calculation formula is 36.2 ⁇ (50 ⁇ 0.001 ⁇ 1048) ⁇ 100%.
  • the reaction end point is detected by ninhydrin (if the resin is colorless and transparent, the reaction is terminated; if the resin develops color, the reaction is extended for 1 hour).
  • the resin was washed 3 times with 150 mL of DMF, deprotected by adding 150 ml of LDBLK for 6 min + 8 min, 150 mL of DMF. The resin was washed 6 times, and the ninhydrin detection resin was colored.
  • Example 15 26.2 g of the crude peptide obtained in Example 15 was added to a 1000 ml reaction flask, dissolved in 500 ml of an aqueous acetonitrile solution (1:1 by volume), and a preparative column of 15 cm ⁇ 25 cm was prepared for high-performance liquid phase purification.
  • the reverse phase octadecylsilane was used as the stationary phase, and the volume ratio was 0.1% acetic acid aqueous solution/acetonitrile as the mobile phase, the ratio was 70:30; the gradient elution preparation; the flow rate: 70-80 ml/min; the detection wavelength: 280 nm; The peak fraction of the target was collected, concentrated and lyophilized to obtain a pure product of 14.0 g, a purity of 99.5%, and a total yield of 27%.
  • the total yield calculation formula is 14.0 / (50 ⁇ 0.001 ⁇ 1048) ⁇ 100%.
  • the mixture was added to the reaction column, and reacted at room temperature for 2 hours, and the reaction end point was detected with ninhydrin (if the resin was colorless and transparent, the reaction was terminated; if the resin developed color, the reaction was prolonged for 1 hour).
  • the resin was washed 3 times with 150 mL of DMF, deprotected by 150 ml of LDBLK for 6 min + 8 min, and washed with 150 mL of DMF for 6 times.
  • the ninhydrin detection resin was colored.
  • Example 19 25.1 g of the crude peptide obtained in Example 19 was placed in a 1000 ml reaction flask, dissolved in 500 ml of an aqueous acetonitrile solution (1:1 by volume), and a preparative column of 15 cm ⁇ 25 cm was prepared for high-performance liquid phase purification.
  • the reverse phase octadecylsilane was used as the stationary phase, and the volume ratio was 0.1% acetic acid aqueous solution/acetonitrile as the mobile phase, the ratio was 70:30; the gradient elution preparation; the flow rate: 70-80 ml/min; the detection wavelength: 280 nm; The peak fraction of the target was collected, concentrated and lyophilized to obtain a pure product of 13.9 g, a purity of 99.5%, and a total yield of 26.5%.
  • the total yield calculation formula is 13.9 / (50 ⁇ 0.001 ⁇ 1048) ⁇ 100%.

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Abstract

提供了一种固相合成血管升压素受体肽激动剂SELEPRESSIN的方法，其包括如下步骤：1)固相树脂依次偶联氨基保护的氨基酸，合成多肽树脂H-Cys(allocam)-Phe-Ile-Hgn(Trt)-Asn(Trt)-Cys(allocam)-Pro-Org(i-Pr)-Gly-树脂；2)固相条件下，以三元催化氧化体系脱除半胱氨酸的保护基allocam并氧化巯基为二硫键；3)裂解液脱除树脂及并Hgn和Asn侧链保护基，并纯化。本发明的方法没有使用碘催化剂，极大的降低了后续处理的问题，具有操作简单、工艺简化、环境友好、经济效益高、可规模化生产等优点。

Description

一种固相合成血管升压素受体肽激动剂SELEPRESSIN的方法 技术领域
本发明本发明属于制药技术领域，尤其是涉及一种固相法合成Selepressin的方法。
背景技术
Selepressin，即FE-202158，是一种血管升压素受体肽激动剂，由辉凌公司开发的一种治疗化脓性休克的多肽类药物，目前处于临床III期。Selepressin为人工合成的九肽化合物，C端为酰胺，含有一对二硫键(1-6)，肽序见式I，Cas：876296-47-8。
Figure PCTCN2017116234-appb-000001
到目前为止，专利CN1964732B公开报道了制备Selepressin的方法：使用氨基树脂，通过标准的Fmoc固相肽合成制备粗肽，然后在液相中在醋酸甲醇溶液中碘氧化二硫键，反向高效液相法纯化得到Selepressin精肽。此方法的缺点在于用碘氧化二硫键时，杂质较多，产率与纯度不高，同时过量的碘很难除掉。
发明内容
为了解决上述问题，本发明的目的是提供一种高效环保的Selepressin合成方法。本发明先使用氨基树脂和便宜的氨基酸原料，通过Fmoc/tBu策略固相合成线性肽，然后使用本发明特定的三元催化氧化体系在固相中催化氧化二硫键，高效液相纯化制备得到目标肽。该方法新颖、合成条件温和、工艺简单且工艺稳定。
本发明一个方面提供了一种血管升压素受体肽激动剂Selepressin的合成方法，其包括如下步骤：
1)固相树脂依次偶联氨基保护的氨基酸，合成多肽树脂H-Cys(allocam)-Phe-Ile-Hgn(R1)-Asn(R2)-Cys(allocam)-Pro-Org(i-Pr)-Gly-树脂；
2)固相条件下，以三元催化氧化体系脱除半胱氨酸的保护基allocam并氧
化巯基为二硫键；
3)裂解液脱除树脂及并Hgn和Asn侧链保护基，并纯化；
其中，步骤1)中的R1为Hgn的侧链保护基，优选为Trt；R2为Asn的侧
链保护基，优选为Trt。
其中，步骤2)脱除半胱氨酸的保护基allocam并氧化二硫键的反应条件为步骤1)所得多肽树脂中的三元催化氧化体系为二价钯盐催化剂、捕获剂以及溶剂形成的反应体系。
步骤2)中所述二价钯盐催化剂选自Pd(OAc)2、PdCl2、PdSO4、Pd(OTf)2中的一种或多种的组合，优选Pd(OAc)2；
所述捕获剂选自NMM、苯硅烷、三乙胺中的一种或多种的组合，优选为NMM；
步骤2)中所用溶剂为乙酸与二甲亚砜的混合液，优选比例为：乙酸：二甲亚砜＝1:100—1:1(体积比)，更优选1:10—1:70；最优选1:18-1:20。
步骤2)反应温度为0℃-100℃，优选20℃-40℃；
步骤2)二价钯盐催化剂的用量为0.5-10当量，优选1.0-1.5当量。
本发明步骤2)中不使用碘或碘相关试剂作催化剂。
步骤1)中所述树脂为氨基树脂，优选为Rink Amide树脂、Rink Amide-AM树脂、MBHA树脂、PAL树脂，树脂替代度为0.1-2.0mmol/g，优选0.5-1.5mmol/g，更优选0.5-1.0mmol/g。
步骤1)中所述的氨基保护的氨基酸中氨基保护基为Fmoc或tBu，偶联氨基酸依次为Gly、Org(i-Pr)、Pro、Cys(Allocam)、Asn(Trt)、Hgn(Trt)、Ile、Phe、Cys(Allocam)。
所述固相多肽合成方法包括以下步骤：
i)脱除氨基保护，至完全；
ii)将待偶联的氨基保护的氨基酸和偶联剂溶解并活化后，一起加入到固相反应柱中，直至反应完全；
iii)重复i)和ii)直至形成完整的多肽树脂。
其中，脱除Fmoc的试剂为20％的哌啶/DMF溶液(DBLK)，即哌啶：DMF(体积比)为1:4的混合溶液。
本发明步骤2)中的偶联剂为DIC和化合物A的组合物、DIC和化合物俄A和化合物B的组合物、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HOBt或HOAt，化合物B为PyBOP、PyAOP、HATU、HBTU或TBTU，优选为DIC和化合物A的组合物。
进一步地，偶联剂中各成分的比例以摩尔比计为DIC:A＝1.2:1.1、DIC:A:B＝2.0:1.1:1.0、DIPEA:A＝1.2:1.1、DIPEA:A:B＝2.0:1.1:1.0。
在本发明中，固相反应在固相反应柱中进行。本发明对固相反应柱无特别限制，可为可实现此目的的任意固相反应柱。
此外，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；压力优选为常压，也可在适当提高或降低的压力下进行；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1)的反应优选将树脂在偶联之前进行溶胀，所述洗涤和溶胀的步骤本领域可采用实现该目的的任何试剂进行，包括DMF、NMP、二氯甲烷等，优选DMF。
在本发明中，每一步反应均可以采用检测方法监测反应终点，反应中应用的检测方法是本领域已知的可实现此目的的任意方法，例如色谱法或化学标定法，优选使用可判定反应终点的试剂，优选茚三酮，当使用茚三酮时，若树脂显色则说明多肽中有游离的胺，即胺上无保护基。
步骤3))所述的裂解液为TFA、H2O、PhOMe、苯甲硫醚的不同比例混合物；优选地，各组分的比例为TFA:H2O:PhOMe:苯甲硫醚90:5:4:1。
步骤4)所述纯化步骤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，以体积比0.1％醋酸水溶液/乙腈为流动相，流动相体积比0.1％醋酸水溶液/乙腈的比例优选为98:2至50:50，更优选80:20至60:40，最优选70:30。收集目的峰馏分，浓缩冻干。
在本发明中，本发明的英文缩写及中文含义，如下表所示。
Figure PCTCN2017116234-appb-000002
Figure PCTCN2017116234-appb-000003
有益效果
本方法采用固相氧化二硫键，反应体系采用特殊的三元催化氧化体系，反应效率高，没有使用碘，从而提交了后续纯化的步骤。具有操作简单、工艺简化、环境友好、经济效益高、可规模化生产等优点。
附图说明
图1为本发明的合成路线图。
具体实施方式
下面通过具体实施案例对本发明做进一步说明，本发明的技术方案和保护范围不受下述实施案例的任何限制。
实施例1 多肽树脂的制备
称取替代度为1.0mmol/g的Rink Amide树脂50g于固相反应柱中，加入50mLDMF，氮气鼓泡溶胀60分钟；然后用50mL DBLK脱保护6min+8min，100mLDMF洗涤6次。称取74.3g(250mmol)Fmoc-Gly-OH和33.8g(250mmol)HOBT用150mL DMF溶解，冰水浴下加入31.5g(210mmol)DIC活化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，按照肽序继续依次偶联Fmoc-Org(i-Pr)-OH、Fmoc-Pro-OH、Fmoc-Cys(Allocam)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Hgn(Trt)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Cys(Allocam)-OH。每种氨基酸、HOBT及DIC每次分别投料210mmol。反应结束后，用DMF洗涤6次，每次100mL，然后用200mL甲醇收缩树脂，抽干，得到多肽树脂127.6克。肽树脂增重88.8克，增重率为98％。计算公式：88.8/(50×1812×0.001)×100％。
实施例2 多肽树脂的制备
称取替代度为1.0mmol/g的Rink Amide-AM树脂50g于固相反应柱中，加入50mLDMF，氮气鼓泡溶胀60分钟；然后用50mL DBLK脱保护6min+8min，100mLDMF洗涤6次。称取74.3g(250mmol)Fmoc-Gly-OH和33.8g(250mmol)HOBT用150mL DMF溶解，冰水浴下加入31.5g(210mmol) DIC活化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，按照肽序继续依次偶联Fmoc-Org(i-Pr)-OH、Fmoc-Pro-OH、Fmoc-Cys(Allocam)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Hgn(Trt)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Cys(Allocam)-OH。每种氨基酸、HOBT及DIC每次分别投料210mmol。反应结束后，用DMF洗涤6次，每次100mL，然后用200mL甲醇收缩树脂，抽干，得到多肽树脂113.6克。肽树脂增重73.4克，增重率为81％。计算公式：73.4/(50×1812×0.001)×100％。
实施例3 多肽树脂的制备
称取替代度为1.0mmol/g的MBHA树脂50g于固相反应柱中，加入50mLDMF，氮气鼓泡溶胀60分钟；然后用50mL DBLK脱保护6min+8min，100mLDMF洗涤6次。称取74.3g(250mmol)Fmoc-Gly-OH和33.8g(250mmol)HOBT用150mL DMF溶解，冰水浴下加入31.5g(210mmol)DIC活化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，按照肽序继续依次偶联Fmoc-Org(i-Pr)-OH、Fmoc-Pro-OH、Fmoc-Cys(Allocam)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Hgn(Trt)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Cys(Allocam)-OH。每种氨基酸、HOBT及DIC每次分别投料210mmol。反应结束后，用DMF洗涤6次，每次1000mL，然后用200mL甲醇收缩树脂，抽干，得到全保护多肽树脂128.9克。计算公式：90.6/(50×1812×0.001)×100％。
实施例4 固相氧化二硫键
将按照实施例3制得的128.9克肽树脂中，依次加入1.5L二甲亚砜、80mL乙酸、75mmol醋酸钯、1.5mol N-甲基吗啉，然后25℃反应。反应结束后，用DMF洗6次，每次1000mL，然后用200mL甲醇收缩树脂，抽干，得到环肽树 脂121.9克。肽树脂增重71.9克，增重率为92％。计算公式：71.9/(50×1578×0.001)×100％。
实施例5 固相氧化二硫键
将按照实施例3制得的128.9克肽树脂中，依次加入1.5L二甲亚砜、80mL乙酸、75mmol氯化钯、1.5mol N-甲基吗啉，然后25℃反应。反应结束后，用DMF洗6次，每次1000mL，然后用200mL甲醇收缩树脂，抽干，得到环肽树脂120.2克。肽树脂增重70.2克，增重率为89％。计算公式：70.2/(50×1578×0.001)×100％。
实施例6 固相氧化二硫键
将按照实施例3制得的128.9克肽树脂中，依次加入1.5L二甲亚砜、80mL乙酸、75mmol硫酸钯、1.5molN-甲基吗啉，然后25℃反应。反应结束后，用DMF洗6次，每次1000mL，然后用200mL甲醇收缩树脂，抽干，得到环肽树脂118.9克。肽树脂增重68.9克，增重率为87％。计算公式：68.9/(50×1578×0.001)×100％。
实施例7 固相氧化二硫键
将按照实施例3制得的128.9克肽树脂中，依次加入1.5L二甲亚砜、80mL乙酸、75mmol醋酸钯、1.5mol苯硅烷，然后25℃反应。反应结束后，用DMF洗6次，每次1000mL，然后用200mL甲醇收缩树脂，抽干，得到环肽树脂120.9克。肽树脂增重70.9克，增重率为90％。计算公式：70.9/(50×1578×0.001)×100％。
实施例8 粗肽的制备
将实施例4得到的环肽肽树脂121.9g加入到3L三口瓶中，加入预先配置好的TFA:H2O:PhOMe:苯甲硫醚＝90:5:4:1(V:V)1200mL，室温反应2小时，减压过滤树脂，收集滤液。用少量TFA洗涤树脂，合并滤液。将滤液缓慢加入12L冰乙醚中沉淀，离心，冰乙醚5L洗涤5次，减压干燥得到粗肽46.1克，收率88％(收率计算公式：46.1/(50×0.001×1048)×100％)，纯度87％。
实施例9 粗肽的制备
将实施例5得到的环肽肽树脂120.2g加入到3L三口瓶中，加入预先配置好的TFA:H2O:PhOMe:苯甲硫醚＝90:5:4:1(V:V)1200mL，室温反应2小时，减压过滤树脂，收集滤液。用少量TFA洗涤树脂，合并滤液。将滤液缓慢加入 12L冰乙醚中沉淀，离心，冰乙醚5L洗涤5次，减压干燥得到粗肽40.3克,收率77％(收率计算公式：40.3/(50×0.001×1048)×100％)，纯度82％。
实施例10 粗肽的制备
将实施例6得到的环肽肽树脂118.9g加入到3L三口瓶中，加入预先配置好的TFA:H2O:PhOMe:苯甲硫醚＝90:5:4:1(V:V)1200mL，室温反应2小时，减压过滤树脂，收集滤液。用少量TFA洗涤树脂，合并滤液。将滤液缓慢加入12L冰乙醚中沉淀，离心，冰乙醚5L洗涤5次，减压干燥得到粗肽38.1克,收率73％(收率计算公式：38.1/(50×0.001×1048)×100％)，纯度78％。
实施例11 粗肽的制备
将实施例7得到的环肽肽树脂120.9g加入到3L三口瓶中，加入预先配置好的TFA:H2O:PhOMe:苯甲硫醚＝90:5:4:1(V:V)1200mL，室温反应2小时，减压过滤树脂，收集滤液。用少量TFA洗涤树脂，合并滤液。将滤液缓慢加入12L冰乙醚中沉淀，离心，冰乙醚5L洗涤5次，减压干燥得到粗肽42.1克,收率80％(收率计算公式：42.1/(50×0.001×1048)×100％)，纯度70％。
实施例12 Selepressin的制备
将实施例8得到的粗肽46.1克加入到1000ml反应瓶中，加入乙腈水溶液(体积比1:1)500ml溶解，上样15cm×25cm制备柱高效液相纯化制备。以反相十八烷基硅烷为固定相，以体积比0.1％醋酸水溶液/乙腈为流动相，比例为70:30；等梯度洗脱制备；流速：70-80ml/min；检测波长：280nm；收集目的峰馏分，浓缩冻干，得纯品36.2g，纯度为99.5％，总收率69％。总收率计算公式36.2÷(50×0.001×1048)×100％。MS[M+H]+理论值1048.5，实验值1048.5。
对比实验：利用碘固相氧化二硫键制备Selepressin
实施例13 多肽树脂的制备
称取替代度为1.0mmol/g的MBHA树脂50g于固相反应柱中，加入50mLDMF，氮气鼓泡溶胀60分钟；然后用50mL DBLK脱保护6min+8min，100mLDMF洗涤6次。称取74.3g(250mmol)Fmoc-Gly-OH和33.8g(250mmol)HOBT用150mL DMF溶解，冰水浴下加入31.5g(210mmol)DIC活化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，按照肽序继续依次偶联Fmoc-Org(i-Pr)-OH、Fmoc-Pro-OH、Fmoc-Cys(Trt)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Hgn(Trt)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Cys(Trt)-OH。每种氨基酸、HOBT及DIC每次分别投料210mmol。反应结束后，用DMF洗涤6次，每次1000mL，然后用200mL甲醇收缩树脂，抽干，得到全保护多肽树脂151.1克。肽树脂增重101.1克，增重率为98％。计算公式：101.1/(50×2064×0.001)×100％。
实施例14 固相氧化二硫键
将按照实施例13制得的151.1克肽树脂中，依次加入1.5LDMF、12.7g碘，然后25℃反应。反应结束后，用DMF洗6次，每次1000mL，然后用200mL甲醇收缩树脂，抽干，得到环肽肽树脂119.2克。肽树脂增重69.2克，增重率为88％。计算公式：69.2/(50×1578×0.001)×100％。
实施例15 粗肽的制备
将实施例14得到的环肽肽树脂119.2g加入到3L三口瓶中，加入预先配置好的TFA:H2O:PhOMe:苯甲硫醚＝90:5:4:1(V:V)1200mL，室温反应2小时，减压过滤树脂，收集滤液。用少量TFA洗涤树脂，合并滤液。将滤液缓慢加入12L冰乙醚中沉淀，离心，冰乙醚5L洗涤5次，减压干燥得到粗肽26.2克,收率50％(收率计算公式：26.2/(50×0.001×1048)×100％)，纯度55％。
实施例16 Selepressin的制备
将实施例15得到的粗肽26.2克加入到1000ml反应瓶中，加入乙腈水溶液(体积比1:1)500ml溶解，上样15cm×25cm制备柱高效液相纯化制备。以反相十八烷基硅烷为固定相，以体积比0.1％醋酸水溶液/乙腈为流动相，比例为70:30；等梯度洗脱制备；流速：70-80ml/min；检测波长：280nm；收集目的峰馏分，浓缩冻干，得纯品14.0g，纯度为99.5％，总收率27％。总收率计算公式14.0/(50×0.001×1048)×100％。
实施例17 多肽树脂的制备
称取替代度为1.0mmol/g的MBHA树脂50g于固相反应柱中，加入50mLDMF，氮气鼓泡溶胀60分钟；然后用50mL DBLK脱保护6min+8min，100mLDMF洗涤6次。称取74.3g(250mmol)Fmoc-Gly-OH和33.8g(250mmol)HOBT用150mL DMF溶解，冰水浴下加入31.5g(210mmol)DIC 活化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，按照肽序继续依次偶联Fmoc-Org(i-Pr)-OH、Fmoc-Pro-OH、Fmoc-Cys(Acm)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Hgn(Trt)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Cys(Acm)-OH。每种氨基酸、HOBT及DIC每次分别投料210mmol。反应结束后，用DMF洗涤6次，每次1000mL，然后用200mL甲醇收缩树脂，抽干，得到全保护多肽树脂135.2克。肽树脂增重85.2克，增重率为99％。计算公式：85.2/(50×1722×0.001)×100％
实施例18 固相氧化二硫键
将按照实施例17制得的135.2克肽树脂中，依次加入1.5LDMF、12.7g碘,然后25℃反应。反应结束后，用DMF洗6次，每次1000mL,然后用200mL甲醇收缩树脂，抽干，得到环肽肽树脂119.2克。肽树脂增重68.5克，增重率为87％。计算公式：68.5/(50×1578×0.001)×100％。
实施例19 粗肽的制备
将实施例18得到的环肽肽树脂119.2g加入到3L三口瓶中，加入预先配置好的TFA:H2O:PhOMe:苯甲硫醚＝90:5:4:1(V:V)1200mL，室温反应2小时，减压过滤树脂，收集滤液。用少量TFA洗涤树脂，合并滤液。将滤液缓慢加入12L冰乙醚中沉淀，离心，冰乙醚5L洗涤5次，减压干燥得到粗肽25.1克，收率48％(收率计算公式：25.1/(50×0.001×1048)×100％)，纯度59％。
实施例20 Selepressin的制备
将实施例19得到的粗肽25.1克加入到1000ml反应瓶中，加入乙腈水溶液(体积比1:1)500ml溶解，上样15cm×25cm制备柱高效液相纯化制备。以反相十八烷基硅烷为固定相，以体积比0.1％醋酸水溶液/乙腈为流动相，比例为70:30；等梯度洗脱制备；流速：70-80ml/min；检测波长：280nm；收集目的峰馏分，浓缩冻干，得纯品13.9g，纯度为99.5％，总收率26.5％。总收率计算公式13.9/(50×0.001×1048)×100％。
由以上实验结果可以得知，采用传统的Fmoc-Cys(Trt)-OH、Fmoc-Cys(Acm)-OH为原料，固相碘氧化制备Selepressin，产率较低(总收率 分别为27％和26.5％)，且产品颜色发黄；而采用Fmoc-Cys(Allocam)-OH为原料，并以本发明所述的三元催化体系固相氧化制备Selepressin，产率和纯度较高，总收率接近70％。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。

Claims (10)

  1. 一种血管升压素受体肽激动剂Selepressin的合成方法，其包括如下步骤：
    1)固相树脂依次偶联氨基保护的氨基酸，合成多肽树脂H-Cys(allocam)-Phe-Ile-Hgn(R1)-Asn(R2)-Cys(allocam)-Pro-Org(i-Pr)-Gly-树脂；
    2)固相条件下，以三元催化氧化体系脱除半胱氨酸的保护基allocam并氧化巯基为二硫键；
    3)裂解液脱除树脂及并Hgn和Asn侧链保护基，并纯化；
    其中，步骤1)中的R1为Hgn的侧链保护基，优选为Trt；R2为Asn的侧链保护基，优选为Trt；
    其中，步骤2)脱除半胱氨酸的保护基allocam并氧化二硫键的反应条件为步骤1)所得多肽树脂中的三元催化氧化体系为二价钯盐催化剂、捕获剂以及溶剂形成的反应体系。
  2. 根据权利要求1所述的合成方法，其中，步骤2)中所述二价钯盐催化剂选自Pd(OAc)2、PdCl2、PdSO4、Pd(OTf)2中的一种或多种的组合，优选Pd(OAc)2。
  3. 根据权利要求1或2所述的合成方法，其中，所述捕获剂选自NMM、苯硅烷、三乙胺中的一种或多种的组合，优选为NMM。
  4. 根据权利要求1-3任一项所述的合成方法，其中，步骤2)反应温度为0℃-100℃，优选20℃-40℃。
  5. 根据权利要求1-4任一项所述的合成方法，其中，固相多肽合成方法包括以下步骤：
    i)脱除氨基保护，至完全；
    ii)将待偶联的氨基保护的氨基酸和偶联剂溶解并活化后，一起加入到固相反应柱中，直至反应完全；
    iii)重复i)和ii)直至形成完整的多肽树脂；
    优选地，步骤1)中所述树脂为氨基树脂，优选为Rink Amide树脂、Rink Amide-AM树脂、MBHA树脂、PAL树脂，树脂替代度为0.1-2.0mmol/g，优选0.5-1.5mmol/g，更优选0.5-1.0mmol/g。
  6. 根据权利要求5所述的合成方法，其中，步骤1)中所述的氨基保护的氨基酸中氨基保护基为Fmoc或tBu，偶联氨基酸依次为Gly、Org(i-Pr)、Pro、 Cys(Allocam)、Asn(Trt)、Hgn(Trt)、Ile、Phe、Cys(Allocam)。
  7. 根据权利要求5所述的合成方法，其中，脱除Fmoc的试剂为20％的哌啶/DMF溶液(DBLK)，即哌啶：DMF(体积比)为1:4的混合溶液。
  8. 根据权利要求5所述的合成方法，本发明步骤2)中的偶联剂为DIC和化合物A的组合物、DIC和化合物俄A和化合物B的组合物、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HOBt或HOAt，化合物B为PyBOP、PyAOP、HATU、HBTU或TBTU，优选为DIC和化合物A的组合物；
    进一步地，偶联剂中各成分的比例以摩尔比计为DIC:A＝1.2:1.1、DIC:A:B＝2.0:1.1:1.0、DIPEA:A＝1.2:1.1、DIPEA:A:B＝2.0:1.1:1.0。
  9. 根据权利要求1-8任一项所述的合成方法，步骤3))所述的裂解液为TFA、H2O、PhOMe、苯甲硫醚的不同比例混合物；优选地，各组分的比例为TFA:H2O:PhOMe:苯甲硫醚90:5:4:1。
  10. 根据权利要求1-9任一项所述的合成方法，步骤4)所述纯化步骤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，以体积比0.1％醋酸水溶液/乙腈为流动相，流动相体积比0.1％醋酸水溶液/乙腈的比例为98:2至50:50，收集目的峰馏分，浓缩冻干；更优选体积比0.1％醋酸水溶液/乙腈为80:20至60:40，最优选体积比0.1％醋酸水溶液/乙腈为70:30。
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CN1964732A (zh) * 2004-08-11 2007-05-16 辉凌公司 血管升压素受体肽激动剂
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CN1964732A (zh) * 2004-08-11 2007-05-16 辉凌公司 血管升压素受体肽激动剂
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