WO2019227612A1 - 一种含脯氨酸的首尾环肽合成方法 - Google Patents

一种含脯氨酸的首尾环肽合成方法 Download PDF

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WO2019227612A1
WO2019227612A1 PCT/CN2018/095427 CN2018095427W WO2019227612A1 WO 2019227612 A1 WO2019227612 A1 WO 2019227612A1 CN 2018095427 W CN2018095427 W CN 2018095427W WO 2019227612 A1 WO2019227612 A1 WO 2019227612A1
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fmoc
phase
solid
cyclic peptide
pro
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PCT/CN2018/095427
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French (fr)
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陈学明
朱艳婷
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
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • 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 synthesis, and particularly relates to a method for synthesizing a proline-containing head-to-tail cyclic peptide.
  • head and tail cyclic peptides can be roughly divided into liquid phase method and solid phase method.
  • liquid phase method a fully protected peptide is first prepared in the solid phase, then the liquid phase is cyclized, and then the protecting group is removed.
  • the liquid-phase method is used to synthesize peptides that form a loop.
  • the reaction needs to be performed in a highly diluted solution (10 -3 to 10 -4 M).
  • the reaction is relatively slow.
  • the activated carboxyl group can avoid decomposition reactions due to single molecule or solvent-induced effects .
  • its successful synthesis requires linear precursors that rely on orthogonal protection, and requires complicated steps, which undoubtedly affects the yield of the peptide, and also requires very high coupling reagents.
  • the solid phase method is to directly form a ring on a solid support.
  • the Asp ⁇ Glu main chain is generally protected by All, and the side chain carboxyl group is connected to the solid phase carrier. After the coupling is completed, All protection is removed. Group, complete the amide bond ring formation on the solid phase to complete the crude peptide synthesis.
  • Solid-phase synthesis is much simpler than liquid-phase synthesis, but in the currently reported methods, this method can only be used if the peptide sequence contains amino acid residues with side chain groups, and if there are no amino acid residues with side chains in the peptide sequence It is difficult to use solid phase method.
  • the present invention provides a new and efficient synthesis method: firstly connecting Fmoc-3-carboxy-Pro-OAll to the resin; and then pressing the peptide Other amino acid residues are coupled in sequence; after the coupling is completed, All is removed from the solid phase, followed by solid phase cyclization; finally, the crude peptide of the cyclic peptide is decarboxylated to obtain a proline-containing head-to-tail cyclic peptide.
  • the method is novel, the synthesis conditions are mild, the process is simple, and the process is stable.
  • the synthetic route is as follows:
  • the invention relates to a method for synthesizing a head-to-tail cyclic peptide containing alanine, which comprises the following steps: 1) selecting a solid-phase synthetic resin;
  • the solid-phase synthetic resin in step 1) is a wang resin or a 2-chloro resin, and the degree of resin substitution is 0.1-3.0 mmol / g, preferably 0.5-2.5 mmol / g, and more preferably 1.0-1.5 mmol / g.
  • Step 2 There is no particular limitation on the Fmoc-AA-OH form of the amino acid, and a protecting group can be used or not used in accordance with conventional knowledge in the art, and the protecting group that can be used is, for example, Trt, Boc, and tBu, etc.).
  • the Fmoc solid-phase synthesis strategy for coupling amino acids in step 2) includes the following steps:
  • the reagent for removing Fmoc is 20% piperidine / DMF solution (DBLK), that is, a mixed solution of piperidine: DMF (volume ratio) 1: 4.
  • DBLK piperidine / DMF solution
  • the amide bond-forming coupling agent in step 2) of the present invention is a composition of DIC and compound A or a composition of DIA and compound A and compound B, wherein compound A is HOBt or HOAt, and compound B is PyBOP, PyAOP, HATU, HBTU or TBTU is preferably a combination of DIC and compound A.
  • the method for coupling Fmoc-3-carboxy-Pro-OAll in step 2) of the present invention is to condense and couple the carboxyl group of Fmoc-3-carboxy-Pro-OAll with a solid-phase synthetic resin with an ester-forming coupling agent.
  • the ester-forming coupling agent is selected from the group consisting of one or more of HOBt and DMAP.
  • the reaction of step 2) is performed in a solid-phase reaction column.
  • the solid-phase reaction column there is no particular limitation on the solid-phase reaction column, and it can be any solid-phase reaction column that can achieve this purpose.
  • the coupling reaction time of each amino acid is usually 1.5-4 hours, preferably 2-3 hours; the pressure is preferably normal pressure, and it can also be performed under an appropriately increased or reduced pressure; the temperature is preferably room temperature (that is, 20 ⁇ 5 ° C), can also be carried out at an appropriately increased or decreased temperature.
  • the reaction of step 2) preferably swells the resin before coupling.
  • the steps of washing and swelling can be performed in the art using any reagent for this purpose, including DMF, NMP, dichloromethane, etc., preferably DMF.
  • the detection method used in the reaction is any method known in the art to achieve this purpose, such as chromatography or chemical calibration, preferably using a reagent that can determine the end point of the reaction, preferably ninhydrin.
  • a reagent that can determine the end point of the reaction preferably ninhydrin.
  • ninhydrin is used If the resin is colored, it means that there is a free amine in the polypeptide, that is, there is no protective group on the amine.
  • Fmoc-AA-OH is a natural amino acid or an unnatural amino acid
  • AAn is a peptide chain composed of n amino acids, where n ⁇ 1, and n is an integer.
  • Step 3 The catalyst used for the solid phase removal of the protecting group All is tetratriphenylphosphine palladium, the capture agent is phenylsilane, and the solvent used is DCM.
  • Step 4) The reagent used for the solid-phase cyclization is DIC + A or DIA + A + B, where A is HOBt or HOAt and B is one or a combination of PyBOP, PyAOP, HATU, HBTU, and TBTU.
  • Step 6) The decarboxylating agent is silver nitrate and sodium persulfate; the solvent used is an acetonitrile aqueous solution, and the reaction temperature is 0-100 ° C, preferably 30-70 ° C; 1: 0.02: 2.
  • the invention also includes an optional step 7) purification step.
  • the purification step may use reversed-phase high-pressure liquid chromatography.
  • the reversed-phase high-pressure liquid chromatography method includes: using reversed octadecylsilane as a stationary phase, using a volume ratio of 0.1% aqueous acetic acid solution / acetonitrile as a mobile phase, and a mobile phase volume ratio of 0.1% aqueous acetic acid solution / acetonitrile.
  • the ratio is preferably 98: 2 to 50:50, more preferably 80:20 to 60:40, and most preferably 70:30.
  • the desired peak fractions were collected and concentrated to freeze-dry.
  • Example 1 100 g of Fmoc-3-carboxy-Pro-OAll-Wang resin with a substitution degree of 1.0 mmol / g prepared in Example 1 was weighed into a solid-phase reaction column, 150 mL of DMF was added, and nitrogen was bubbled to swell for 60 minutes; then 100 mL of DBLK was used. Deprotection was performed for 6 min + 8 min, and washed 6 times with 100 mL of DMF.
  • Example 1 100 g of Fmoc-3-carboxy-Pro-OAll-Wang resin with a substitution degree of 1.0 mmol / g prepared in Example 1 was weighed into a solid-phase reaction column, 150 mL of DMF was added, and nitrogen was bubbled to swell for 60 minutes; then 100 mL of DBLK was used. Deprotection was performed for 6 min + 8 min, and washed 6 times with 100 mL of DMF.
  • Example 10 80.2 g of the crude intermediate cyclic peptide obtained in Example 10 was added to a 500 ml reaction bottle, 300 ml of acetonitrile and 100 mL of water were added to dissolve it, and then 0.3 g of silver nitrate and 47.6 g of sodium persulfate were added and reacted at 60 ° C. After the reaction, the sample was directly loaded on a 10 cm ⁇ 25 cm preparative column for high-performance liquid purification. Reverse phase octadecylsilane was used as stationary phase, and volume ratio of 0.1% acetic acid aqueous solution / acetonitrile was used as mobile phase at a ratio of 70:30.
  • Example 1 100 g of Fmoc-3-carboxy-Pro-OAll-Wang resin with a substitution degree of 1.0 mmol / g prepared in Example 1 was weighed into a solid-phase reaction column, 150 mL of DMF was added, and nitrogen was bubbled to swell for 60 minutes; then 100 mL of DBLK was used. Deprotection was performed for 6 min + 8 min, and washed 6 times with 100 mL of DMF.
  • 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.
  • the ninhydrin detected the resin to be colored.
  • Repeat the above coupling operation and continue coupling Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-D-Phe-OH, Fmoc-Ala-OH, Fmoc-Tyr (OtBu) -OH, Fmoc- Ala-OH, Fmoc-Met-OH.
  • Each amino acid, HOBT and DIC were fed 500 mmol each time.
  • the resin was shrunk with 200 mL of methanol and dried to obtain 240.1 g of a peptide resin.
  • Example 1 100 g of Fmoc-3-carboxy-Pro-OAll-Wang resin with a substitution degree of 1.0 mmol / g prepared in Example 1 was weighed into a solid-phase reaction column, 150 mL of DMF was added, and nitrogen was bubbled to swell for 60 minutes; then 100 mL of DBLK was used. Deprotection was performed for 6 min + 8 min, and washed 6 times with 100 mL of DMF.
  • Example 20 86.2 g of the crude intermediate cyclic peptide obtained in Example 20 was added to a 500 ml reaction bottle, 300 ml of acetonitrile and 100 mL of water were added to dissolve it, and then 0.35 g of silver nitrate and 48.6 g of sodium persulfate were added to react at 60 ° C. After the reaction, the sample was directly loaded on a 10 cm ⁇ 25 cm preparative column for high-performance liquid purification. Reverse phase octadecylsilane was used as stationary phase, and volume ratio of 0.1% acetic acid aqueous solution / acetonitrile was used as mobile phase at a ratio of 70:30.
  • the present invention provides a new and efficient synthesis method: firstly connecting Fmoc-3-carboxy-Pro-OAll to the resin; and then pressing the peptide Coupling other amino acid residues in sequence; after the coupling is completed, the solid phase is removed from All, followed by solid phase cyclization; finally, the crude cyclic peptide is decarboxylated with silver nitrate and sodium persulfate to obtain a proline head and tail cyclic peptide.
  • the method is novel, the synthesis conditions are mild, the process is simple, and the process is stable.
  • This synthesis method is also a protection point.
  • the invention provides a novel method for preparing a proline-containing head-to-tail cyclic peptide.
  • the method has the advantages of simple operation, simplified process, environmental friendliness, high economic benefits, and scaleable production.

Abstract

一种包含脯氨酸的首尾环肽合成方法，其包括以下步骤：1)选择固相合成树脂；2)首先偶联Fmoc-3-羧基-Pro-OAll，然后按照Fmoc固相合成策略，得到NH-(3-羧基-固相合成树脂)-Pro-OAll然后依次偶联肽序中的氨基酸残基Fmoc-AA-OH；3)固相脱除保护基All，得到NH 2-AAn-(3-羧基-固相合成树脂)-Pro-OH；4)固相首尾环化：偶联AAn上的氨基与Pro上的羧基；5)裂解固相合成树脂，制备中间体环肽粗肽；6)将中间体环肽粗肽在脱羧剂的作用下脱除末端Pro的3位羧基，制备含脯氨酸的环肽。该方法新颖、合成条件温和、工艺简单且工艺稳定。

Description

一种含脯氨酸的首尾环肽合成方法 技术领域
本发明属于药物合成领域，具体涉及一种含脯氨酸的首尾环肽合成方法。
背景技术
对于首尾酰胺键成环的肽，目前在自然界已经有很多发现。首尾环肽药物的研究也得到了非常快速的发展。随着研究的深入，环肽的合成已经引起了越来越多的关注，并且已经在许多研究中取得了一定的成果。
首尾环肽的化学合成大体可分为液相法与固相法。
液相法为先固相制备全保护肽，然后液相成环，最后再脱除保护基。采用液相方法合成首尾成环的肽，反应需要在高度稀释的溶液中(10 -3～10 -4M)进行，反应比较缓慢，活化的羧基可以避免因为单分子或溶剂诱导效应发生分解反应。但是，它的成功合成需要依赖于正交保护的线性前体，需要很繁杂的步骤，这样无疑影响了肽的产率，同时对偶联试剂的要求也非常高。
固相法为在固相载体上直接成环。对于肽序中含有Asp\Glu或者Asn\Gln残基的肽序，一般采用把Asp\Glu主链采用All保护，侧链羧基连接在固相载体上的方法，偶联完成后脱除All保护基团，在固相上完成酰胺键成环的方法完成粗肽合成。
固相合成要比液相合成简单得多，但目前报道的方法中，只有肽序中含有侧链基团的氨基酸残基，才能用此方法，而如果肽序中没有带侧链的氨基酸残基，很难用固相方法。
发明内容
对于肽序中不含带侧链氨基酸而有Pro的首尾环肽，本发明提供了一种全新的、高效的合成方法：首先在树脂上连接Fmoc-3-羧基-Pro-OAll；然后按肽序偶联其他氨基酸残基；偶联完毕后，固相脱除All，紧接着固相成环；最后环肽粗肽脱羧得到含脯氨酸的首尾环肽。该方法新颖、合成条件温和、工艺简单且工艺稳定。
合成路线如下：
Figure PCTCN2018095427-appb-000001
本发明涉及一种包含丙氨酸的首尾环肽合成方法，其包括以下步骤：1)选择固相合成树脂；
2)首先偶联Fmoc-3-羧基-Pro-OAll，然后按照Fmoc固相合成策略，得到NH-(3-羧基-固相合成树脂)-Pro-OAll然后依次偶联肽序中的氨基酸残基Fmoc-AA-OH得到NH 2-AAn-(3-羧基-固相合成树脂)-Pro-OAll；
3)固相脱除保护基All，得到NH 2-AAn-(3-羧基-固相合成树脂)-Pro-OH；
4)固相首尾环化：偶联AAn上的氨基与Pro上的羧基；
5)裂解固相合成树脂，制备中间体环肽粗肽；
6)将中间体环肽粗肽在脱羧剂的作用下脱除末端Pro的3位羧基，制备含脯氨酸的环肽。
步骤1)中所述固相合成树脂为wang树脂或2-氯树脂，树脂替代度为0.1-3.0mmol/g，优选0.5-2.5mmol/g，更优选1.0-1.5mmol/g。
步骤2)对于氨基酸的Fmoc-AA-OH形式没有特别限定，可以依照本领域的常规知识使用或者不使用保护基团，可使用的保护基团例如，Trt、Boc、和tBu等)。
步骤2)中Fmoc固相合成策略偶联氨基酸包括以下步骤：
2.1)脱除Fmoc，接着用溶剂洗涤树脂，直至用检测方法检测到完全脱除Fmoc为止；
2.2)将合适量的待偶联Fmoc-AA-OH和偶联剂在溶剂中溶解并活化后，一起加入到固相反应柱中，直至用检测方法检测到反应终止为止；
2.3)重复1)和2)。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。
本发明所述步骤2)中的成酰胺键偶联剂为DIC和化合物A的组合物或DIA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，成酰胺键偶联剂中各成分的比例以摩尔比计为DIC:A＝1.2:1.1，DIA:A:B＝2.0:1.1:1.0。
在本发明步骤2)中偶联Fmoc-3-羧基-Pro-OAll的方法为以成酯键偶联剂将Fmoc-3-羧基-Pro-OAll的3位羧基与固相合成树脂缩合偶联，其中所述的成酯键偶联剂选自HOBt、DMAP一种或多种的组合物。
步骤2)的反应在固相反应柱中进行。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2)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。
所述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2)中Fmoc-AA-OH为天然氨基酸或非天然氨基酸，所述AAn为由n个氨基酸组成的肽链，其中n≥1，且n为整数。
步骤3)固相脱除保护基All所用催化剂为四三苯基膦钯，捕获剂为苯硅烷，所用溶剂为DCM。
步骤4)固相首尾环化所用试剂为DIC+A或者DIA+A+B，其中A为HOBt或HOAt，B为PyBOP、PyAOP、HATU、HBTU、TBTU其中一种或几种的组合。
步骤5)所述的裂解液为TFA、H 2O、PhOMe、苯甲硫醚的混合物。优选为TFA:H 2O:PhOMe:苯甲硫醚＝90:5:4:1(V:V)。
步骤6)所述脱羧剂为硝酸银和过硫酸钠；所用溶剂为乙腈水溶液，反应温度为0-100℃，优选30-70℃；粗环肽与硝酸银、过硫酸钠物质的量比为1：0.02：2。
本发明还包括可选地步骤7)纯化步骤。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。
具体实施方式
环肽[Ala-Tyr-Ala-Phe-leu-Ile-Val-Pro]环八肽的合成
实施例1 Fmoc-3-羧基-Pro-OAll-Wang树脂的制备
称取替代度为1.5mmol/g的Wang树脂100g于固相反应柱中，加入DMF，氮气鼓泡溶胀60分钟；称取Fmoc-3-羧基-Pro-OAll 42.1克(100mmol)、HOBt16.2克(120mmol)、DMAP 1.2克(10mmol)，用DMF溶解，0℃下加 入20.3mLDIC,活化5分钟，加入反应柱。反应两小时后，抽掉溶液，用DMF洗涤六次、DCM洗涤三次，甲醇收缩后，抽干树脂，得到Fmoc-3-羧基-Pro-OAll-Wang树脂129克，检测替代度为1.0mmol/g。
实施例2 肽树脂的制备
称取实施例1制备的替代度为1.0mmol/g的Fmoc-3-羧基-Pro-OAll-Wang树脂100克于固相反应柱中，加入150mLDMF，氮气鼓泡溶胀60分钟；然后用100mL DBLK脱保护6min+8min，100mLDMF洗涤6次。称取169.7g(500mmol)Fmoc-Val-OH和78.1g(500mmol)HOBT用150mL DMF溶解，冰水浴下加入86.7mL(500mmol)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-Ile-OH、Fmoc-Leu-OH、Fmoc-Phe-OH、Fmoc-Ala-OH、Fmoc-Tyr(OtBu)-OH、Fmoc-Ala-OH。每种氨基酸、HOBT及DIC每次分别投料500mmol。偶联结束，用200mL甲醇收缩树脂，抽干，得到肽树脂192.2克。
实施例3 固相脱除All
将实施例2得到的树脂192.2g中，加入300mLDMF溶胀2小时，抽掉溶剂，加入DCM300mL,再依次加入四三苯基膦钯50mmol、苯硅烷2000mmol,室温反应1小时，反应结束后，抽掉溶液，用DCM洗三次(300mL×3)、DMF洗三次(300mL×3)，得中间体肽树脂。
实施例4 固相环化
在实施例3得到的中间体肽树脂中依次加入DMF300ml、78.1g(500mmol)HOBT和86.7mL(210mmol)DIC，室温反应4小时。反应结束后，抽掉溶液，DMF洗涤六次(300mL×6),二氯甲烷洗涤六次(300mL×6)，甲醇收缩2次(150mL×2)，真空干燥，得中间体环肽树脂287.0g。
实施例5 中间体环肽制备
将实施例4得到的中间体环肽树脂287.0g加入到5l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)3L，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 30L冰乙醚中沉淀，离心，冰乙醚5L洗涤5次，减压干燥得到中间体环粗肽81.2克。
实施例6 含脯氨酸环肽的制备
将实施例5得到的中间体环粗肽81.2克加入到500ml反应瓶中，加入300ml乙腈和100mL水溶解，然后加入硝酸银0.3g、过硫酸钠47.6g，60℃反应。反应结束后，直接上样10cm×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52.8g，纯度为99.5％，收率60％。MS[M+H]+理论值876.1，实验值876.1。
环肽[Ala-Tyr-Ala-D-Phe-leu-Ile-Val-Pro]环八肽的合成
实施例7 肽树脂的制备
称取实施例1制备的替代度为1.0mmol/g的Fmoc-3-羧基-Pro-OAll-Wang树脂100克于固相反应柱中，加入150mLDMF，氮气鼓泡溶胀60分钟；然后用100mL DBLK脱保护6min+8min，100mLDMF洗涤6次。称取169.7g(500mmol)Fmoc-Val-OH和78.1g(500mmol)HOBT用150mL DMF溶解，冰水浴下加入86.7mL(500mmol)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-Ile-OH、Fmoc-Leu-OH、Fmoc-D-Phe-OH、Fmoc-Ala-OH、Fmoc-Tyr(OtBu)-OH、Fmoc-Ala-OH。每种氨基酸、HOBT及DIC每次分别投料500mmol。偶联结束，用300mL甲醇收缩树脂，抽干，得到肽树脂190.1克。
实施例8 固相脱除All
将实施例7得到的树脂190.1g中，加入300mLDMF溶胀2小时，抽掉溶剂，加入DCM300mL,再依次加入四三苯基膦钯50mmol、苯硅烷2000mmol,室温反应1小时，反应结束后，抽掉溶液，用DCM洗三次(300mL×3)、DMF洗三次(300mL×3)，得中间体肽树脂。
实施例9 固相环化
在实施例8得到的中间体肽树脂中依次加入DMF300ml、78.1g(500mmol)HOBT和86.7mL(210mmol)DIC，室温反应4小时。反应结束后，抽掉溶液，DMF洗涤六次(300mL×6),二氯甲烷洗涤六次(300mL×6)，甲醇收缩2次(150mL×2)，真空干燥，得中间体环肽树脂285.0g。
实施例10 中间体环肽制备
将实施例9得到的中间体环肽树脂285.0g加入到5l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)3L，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30L冰乙醚中沉淀，离心，冰乙醚5L洗涤5次，减压干燥得到中间体环粗肽80.2克。
实施例11 含脯氨酸环肽的制备
将实施例10得到的中间体环粗肽80.2克加入到500ml反应瓶中，加入300ml乙腈和100mL水溶解，然后加入硝酸银0.3g、过硫酸钠47.6g，60℃反应。反应结束后，直接上样10cm×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51.6g，纯度为99.5％，收率58.6％。MS[M+H]+理论值876.1，实验值876.1。
环肽[Met-Ala-Tyr-Ala-Phe-leu-Ile-Val-Pro]环九肽的合成
实施例12 肽树脂的制备
称取实施例1制备的替代度为1.0mmol/g的Fmoc-3-羧基-Pro-OAll-Wang树脂100克于固相反应柱中，加入150mLDMF，氮气鼓泡溶胀60分钟；然后用100mL DBLK脱保护6min+8min，100mLDMF洗涤6次。称取169.7g(500mmol)Fmoc-Val-OH和78.1g(500mmol)HOBT用150mL DMF溶解，冰水浴下加入86.7mL(500mmol)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-Ile-OH、Fmoc-Leu-OH、Fmoc-D-Phe-OH、Fmoc-Ala-OH、Fmoc-Tyr(OtBu)-OH、Fmoc-Ala-OH、Fmoc-Met-OH。每种氨基酸、HOBT及DIC每次分别投料500mmol。偶联结束，用200mL甲醇收缩树脂，抽干，得到肽树脂240.1克。
实施例13 固相脱除All
将实施例12得到的树脂240.1g中，加入500mLDMF溶胀2小时，抽掉溶剂，加入DCM300mL,再依次加入四三苯基膦钯50mmol、苯硅烷2000mmol,室温反应1小时，反应结束后，抽掉溶液，用DCM洗三次(300mL×3)、DMF洗三次(300mL×3)，得中间体肽树脂。
实施例14 固相环化
在实施例13得到的中间体肽树脂中依次加入DMF500ml、78.1g(500mmol)HOBT和86.7mL(210mmol)DIC，室温反应4小时。反应结束后，抽掉溶液，DMF洗涤六次(300mL×6),二氯甲烷洗涤六次(300mL×6)，甲醇收缩2次(150mL×2)，真空干燥，得中间体环肽树脂325.0g。
实施例15 中间体环肽制备
将实施例14得到的中间体环肽树脂325.0g加入到5l三口瓶中，加入预先配置好的TFA:H 2O:PhOMe:苯甲硫醚＝90:5:4:1(V:V)4L，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40L冰乙醚中沉淀，离心，冰乙醚5L洗涤5次，减压干燥得到中间体环粗肽85.2克。
实施例16 含脯氨酸环肽的制备
将实施例15得到的中间体环粗肽85.2克加入到500ml反应瓶中，加入300ml乙腈和100mL水溶解，然后加入硝酸银0.35g、过硫酸钠48.6g，60℃反应。反应结束后，直接上样10cm×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60.6g，纯度为99.5％，收率55.6％。MS[M+H]+理论值1007.3，实验值1007.3。
Cyclo[D-Met-Ala-Tyr-Ala-Phe-leu-Ile-Val-Pro]环九肽的合成
实施例17 肽树脂的制备
称取实施例1制备的替代度为1.0mmol/g的Fmoc-3-羧基-Pro-OAll-Wang树脂100克于固相反应柱中，加入150mLDMF，氮气鼓泡溶胀60分钟；然后用100mL DBLK脱保护6min+8min，100mLDMF洗涤6次。称取169.7g(500mmol)Fmoc-Val-OH和78.1g(500mmol)HOBT用150mL DMF溶解，冰水浴下加入86.7mL(500mmol)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-Ile-OH、Fmoc-Leu-OH、Fmoc-D-Phe-OH、Fmoc-Ala-OH、Fmoc-Tyr(OtBu)-OH、Fmoc-Ala-OH、Fmoc-D-Met-OH。每种氨基酸、HOBT及DIC每次分别投料500mmol。偶联结束，用300mL甲醇收缩树脂，抽干，得到肽树脂245.2克。
实施例18 固相脱除All
将实施例17得到的树脂245.2g中，加入500mLDMF溶胀2小时，抽掉溶剂，加入DCM300mL,再依次加入四三苯基膦钯50mmol、苯硅烷2000mmol,室温反应1小时，反应结束后，抽掉溶液，用DCM洗三次(300mL×3)、DMF洗三次(300mL×3)，得中间体肽树脂。
实施例19 固相环化
在实施例18得到的中间体肽树脂中依次加入DMF500ml、78.1g(500mmol)HOBT和86.7mL(210mmol)DIC，室温反应4小时。反应结束后，抽掉溶液，DMF洗涤六次(300mL×6),二氯甲烷洗涤六次(300mL×6)，甲醇收缩2次(150mL×2)，真空干燥，得中间体环肽树脂329.0g。
实施例20 中间体环肽制备
将实施例19得到的中间体环肽树脂329.0g加入到5l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)4L，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40L冰乙醚中沉淀，离心，冰乙醚5L洗涤5次，减压干燥得到中间体环粗肽86.2克。
实施例21 含脯氨酸环肽的制备
将实施例20得到的中间体环粗肽86.2克加入到500ml反应瓶中，加入300ml乙腈和100mL水溶解，然后加入硝酸银0.35g、过硫酸钠48.6g，60℃反应。反应结束后，直接上样10cm×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61.6g，纯度为99.5％，收率56.5％。MS[M+H]+理论值1007.3，实验值1007.3。
对于肽序中不含带侧链氨基酸而有Pro的首尾环肽，本发明提供了一种全新的、高效的合成方法：首先在树脂上连接Fmoc-3-羧基-Pro-OAll；然后按肽序偶联其他氨基酸残基；偶联完毕后，固相脱除All，紧接着固相成环；最后环肽粗肽使用硝酸银与过硫酸钠脱羧得到含脯氨酸的首尾环肽。该方法新颖、合成条件温和、工艺简单且工艺稳定。
本合成方法也为保护点。
本发明提供了一种制备含脯氨酸的首尾环肽全新的方法。本方法具有操作简单、工艺简化、环境友好、经济效益高、可规模化生产等优点。
1)Contemporary strategies for peptide Macrocyclization.《Nature Chemistry》，2011，3(7)：509-524
2)Persulfate/Silver Ion De羧基ation of羧基ic Acid.Preparation of Alkanes,Alkenes，and Alcohols.J.Org.Chem.,1983,40(20)，3575-3577.
Figure PCTCN2018095427-appb-000002
Figure PCTCN2018095427-appb-000003

Claims (9)

  1. 一种包含脯氨酸的首尾环肽合成方法，其包括以下步骤：1)选择固相合成树脂；
    2)首先偶联Fmoc-3-羧基-Pro-OAll，然后按照Fmoc固相合成策略，得到NH-(3-羧基-固相合成树脂)-Pro-OAll然后依次偶联肽序中的氨基酸残基Fmoc-AA-OH得到NH 2-AAn-(3-羧基-固相合成树脂)-Pro-OAll；
    3)固相脱除保护基All，得到NH 2-AAn-(3-羧基-固相合成树脂)-Pro-OH；
    4)固相首尾环化：偶联AAn上的氨基与Pro上的羧基；
    5)裂解固相合成树脂，制备中间体环肽粗肽；
    6)将中间体环肽粗肽在脱羧剂的作用下脱除末端Pro的3位羧基，制备含脯氨酸的环肽。
  2. 根据权利要求1所述的包含脯氨酸的首尾环肽合成方法，步骤6)所述脱羧剂为硝酸银和过硫酸钠的混合物。
  3. 根据权利要求1所述的包含脯氨酸的首尾环肽合成方法，步骤6)所用溶剂为乙腈水溶液，反应温度为0-100℃，优选30-70℃；粗环肽与硝酸银、过硫酸钠物质的量比为1：0.02：2。
  4. 根据权利要求1所述的包含脯氨酸的首尾环肽合成方法，步骤2)中偶联Fmoc-3-羧基-Pro-OAll的方法为以成酯键偶联剂将Fmoc-3-羧基-Pro-OAll的3位羧基与固相合成树脂缩合偶联，其中所述的成酯键偶联剂选自HOBt、DMAP一种或多种的组合物。
  5. 根据权利要求1所述的包含脯氨酸的首尾环肽合成方法，步骤2)中Fmoc固相合成策略偶联氨基酸包括以下步骤：
    2.1)脱除Fmoc，接着用溶剂洗涤树脂，直至用检测方法检测到完全脱除Fmoc为止；
    2.2)将合适量的待偶联Fmoc-AA-OH和偶联剂在溶剂中溶解并活化后，一起加入到固相反应柱中，直至用检测方法检测到反应终止为止；
    2.3)重复1)和2)。其中脱除Fmoc的试剂为20％的哌啶/DMF溶液。
  6. 根据权利要求1所述的包含脯氨酸的首尾环肽合成方法，所述步骤2)中的成酰胺键偶联剂为DIC和化合物A的组合物或DIA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。
  7. 根据权利要求1所述的包含脯氨酸的首尾环肽合成方法，步骤4)固相首尾环化所用试剂为DIC+A或者DIA+A+B，其中A为HOBt或HOAt，B为PyBOP、PyAOP、HATU、HBTU、TBTU其中一种或几种的组合。
  8. 根据权利要求1所述的包含脯氨酸的首尾环肽合成方法，步骤5)所述的裂解液为TFA、H 2O、PhOMe、苯甲硫醚的混合物；优选为TFA:H 2O:PhOMe:苯甲硫醚＝90:5:4:1。
  9. 根据权利要求1所述的包含脯氨酸的首尾环肽合成方法，还包括可选地步骤7)纯化步骤，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。
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