WO2019218382A1 - 一种含丙氨酸的首尾环肽合成方法 - Google Patents

一种含丙氨酸的首尾环肽合成方法 Download PDF

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WO2019218382A1
WO2019218382A1 PCT/CN2018/087826 CN2018087826W WO2019218382A1 WO 2019218382 A1 WO2019218382 A1 WO 2019218382A1 CN 2018087826 W CN2018087826 W CN 2018087826W WO 2019218382 A1 WO2019218382 A1 WO 2019218382A1
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solid phase
fmoc
asp
synthetic resin
resin
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PCT/CN2018/087826
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French (fr)
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陈学明
曾驰
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
    • C07K1/10—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using coupling 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
    • 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 drug synthesis, and in particular relates to a method for synthesizing an alanine-containing head-to-tail cyclic peptide.
  • the chemical synthesis of the first and last cyclic peptides can be roughly divided into a liquid phase method and a solid phase method.
  • liquid phase method a fully protected peptide is prepared by solid phase first, then the liquid phase is looped, and finally the protecting group is removed.
  • the liquid phase method is used to synthesize the peptides in the first and last loops.
  • the reaction needs to be carried out in a highly diluted solution (10 -3 ⁇ 10 -4 M).
  • the reaction is slow, and the activated carboxyl groups can avoid decomposition reaction due to single molecule or solvent induced effects. .
  • its successful synthesis relies on orthogonally protected linear precursors, which require very complicated steps, which undoubtedly affect the yield of the peptide, and the requirements for the coupling reagent are also very high.
  • the solid phase method is a direct ring formation on a solid support.
  • the Asp ⁇ Glu main chain is generally protected by All, and the side chain carboxyl group is attached to the solid phase carrier. After the coupling is completed, the All protection is removed. The group completes the crude peptide synthesis by completing the amide bond ring formation on the solid phase.
  • Solid phase synthesis is much simpler than liquid phase synthesis, but in the currently reported methods, only amino acid residues containing a side chain group in the peptide sequence can be used in this way, and if there are no amino acid residues with side chains in the peptide sequence. It is difficult to use the solid phase method.
  • the present invention provides a novel and efficient synthesis method: firstly, Fmoc-Asp-OAll is attached to the resin; then coupled by peptide sequence Residual amino acid residues; after coupling, the solid phase removes All, followed by solid phase formation; finally, the cyclic peptide crude peptide is decarboxylated with L-aspartic acid- ⁇ decarboxylase to obtain alanine-containing head-end cyclic peptide .
  • the method is novel, the synthesis condition is mild, the process is simple, and the process is stable.
  • the synthetic route is as follows:
  • a method for synthesizing a first-tailed cyclic peptide comprising alanine comprising the steps of:
  • the intermediate cyclic peptide is subjected to decarboxylase to remove the side chain carboxyl group of the terminal aspartic acid to prepare an alanine-containing cyclic peptide.
  • the solid phase synthetic resin in the step 1) is a wang resin or a 2-chloro resin, and the degree of substitution of the resin is from 0.1 to 3.0 mmol/g, preferably from 0.5 to 2.5 mmol/g, more preferably from 1.0 to 1.5 mmol/g.
  • Step 2 The Fmoc-AA-OH form of the amino acid is not particularly limited, and a protecting group may be used or not, according to the conventional knowledge in the art, and a protecting group such as Trt, Boc, and tBu may be used.
  • the Fmoc/tBu solid phase synthesis strategy for coupling amino acids in step 2) includes the following steps:
  • reagent for removing Fmoc is 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 amide bond coupling agent in the step 2) of the present invention is a composition of DIC and Compound A or a combination 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, preferably a combination of DIC and Compound A.
  • DIA: A: B 2.0: 1.1: 1.0 in terms of a molar ratio.
  • the method of coupling Fmoc-Asp(OH)-OAll in the step 2) of the present invention is to condense and couple the side chain carboxyl group of Fmoc-Asp(OH)-OAll with a solid phase synthetic resin by an ester bond coupling agent, wherein
  • 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 carried out in a solid phase reaction column.
  • the solid phase reaction column is not particularly limited and may be any solid phase reaction column that can achieve this.
  • 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 2) preferably swells the resin prior to coupling, and the steps of washing and swelling are carried out in the art using any reagent which accomplishes 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, such as chromatography or chemical calibration, preferably using an agent that determines the endpoint of the reaction, 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.
  • Fmoc-AA-OH is a natural amino acid or a non-natural amino acid
  • the AAn is a peptide chain composed of n amino acids, wherein n ⁇ 1, and n is an integer.
  • Step 3 The catalyst for solid phase removal of the protecting group All is tetrakistriphenylphosphine palladium, the capturing agent is benzene silane, and the solvent used is DCM.
  • Step 4) The reagent used for solid phase cyclization is DIC+A or DIA+A+B, wherein A is HOBt or HOAt, and B is one or a combination of PyBOP, PyAOP, HATU, HBTU, TBTU.
  • the lysate of step 5 is a mixture of different ratios of TFA, H 2 O, PhOMe, and thioanisole.
  • TFA:H 2 O:PhOMe: thioanisole 90:5:4:1 (V:V).
  • the decarboxylase is L-aspartic acid- ⁇ decarboxylase;
  • the reaction temperature is 0-100 ° C, preferably 20-40 ° C; intermediate cyclic peptide crude peptide and decarboxylase mass ratio of 1000-50:1, preferably 100:1;
  • the invention also includes optionally step 7) a purification step.
  • Step 7) 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 present invention provides a novel method for preparing alanine-containing head and tail cyclic peptides.
  • the method has the advantages of simple operation, simplified process, environmental friendliness, high economic benefit and large-scale production.
  • the reaction column After adding 86.7 mL (500 mmol) of DIC for 3 min in an ice water bath, the mixture was added to the reaction column, and the reaction was carried out at room temperature. In the hour, 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). At the end of the reaction, 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 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 50.2 g, a purity of 99.5%, and a yield of 54.6%. MS [M+H]+ calcd. 876.1.
  • the reaction column After adding 86.7 mL (500 mmol) of DIC for 3 min in an ice water bath, the mixture was added to the reaction column, and the reaction was carried out at room temperature. In the hour, 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). At the end of the reaction, 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 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 48.1 g, a purity of 99.5%, and a yield of 57%. MS [M+H]+ calcd. 876.1.
  • the reaction column After adding 86.7 mL (500 mmol) of DIC for 3 min in an ice water bath, the mixture was added to the reaction column, and the reaction was carried out at room temperature. In the hour, 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). At the end of the reaction, 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 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 55.2 g of pure product, the purity was 99.6%, and the yield was 50.6%. MS [M+H]+ calcd for 1007.3.
  • the reaction column After adding 86.7 mL (500 mmol) of DIC for 3 min in an ice water bath, the mixture was added to the reaction column, and the reaction was carried out at room temperature. In the hour, 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). At the end of the reaction, 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 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 50.2 g, a purity of 99.6%, and a yield of 46.0%. MS [M+H]+ calcd for 1007.3.

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Abstract

提供了包含丙氨酸环肽的首尾环化固相合成方法，包括以下步骤：1）选择固相合成树脂；2）将Fmoc-Asp(OH)-OAll与固相合成树脂偶联，然后按照Fmoc/tBu固相合成策略，得到NH 2-Asp-(固相合成树脂)-OAll，并依次偶联肽序列中的氨基酸残基得到NH 2-AAn-Asp-(固相合成树脂)-OAll；3）脱除保护基All，得到NH 2-AAn-Asp-(固相合成树脂)-OH；4）将步骤3)得到的多肽长链的首尾氨基酸进行首尾缩合环化；5）将步骤4)得到的产物从树脂上裂解下来，得到中间体环肽粗肽；6）将中间体环肽粗肽在L-天冬氨酸-β脱羧酶的作用下脱除天冬氨酸的侧链羧基，得到成品。

Description

一种含丙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，具体涉及一种含丙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，就很难用固相方法。
发明内容
对于肽序中不含带侧链氨基酸、而有Ala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-Asp-OAll；然后按肽序偶联剩余氨基酸残基；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L-天冬氨酸-β脱羧酶催化脱羧得到含丙氨酸的首尾环肽。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 PCTCN2018087826-appb-000001
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-Asp(OH)-OAll，然后按照Fmoc/tBu固相合成策略，得到NH 2-Asp-(固相合成树脂)-OAll然后依次偶联肽序中的氨基酸残基得到NH 2-AAn-Asp-(固相合成树脂)-OAll；
3)固相脱除保护基All，得到NH 2-AAn-Asp-(固相合成树脂)-OH；
4)固相首尾环化；
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酶的作用下脱除末端天冬氨酸的侧链羧基，制备含丙氨酸的环肽。
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/tBu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)将合适量的待偶联氨基酸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，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-Asp(OH)-OAll的方法为以成酯键偶联剂将Fmoc-Asp(OH)-OAll的侧链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、苯甲硫醚的不同比例混合物。优选为TFA:H 2O:PhOMe:苯甲硫醚＝90:5:4:1(V:V)。
步骤6)所述脱羧酶为L-天冬氨酸-β脱羧酶；所用溶剂为磷酸钠的缓冲液(pH＝2-9)，pH优选5-7；反应温度为0-100℃，优选20-40℃；中间体环肽粗肽与脱羧酶的质量比为1000-50：1，优选100：1；
本发明还包括可选地步骤7)纯化步骤。步骤7)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。
Figure PCTCN2018087826-appb-000002
Figure PCTCN2018087826-appb-000003
有益效果
本发明提供了一种制备含丙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。
附图说明 具体实施方式
环肽[Pro-Tyr-Ala-Phe-leu-Ile-Val-Ala]环八肽的合成
实施例1 Fmoc-β-Asp-(Wang树脂)-OAll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500mLDMF，氮气鼓泡溶胀60分钟；称取Fmoc-Asp-OAll 39.5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-β-Asp-(Wang树脂)-OAll树脂130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-β-Asp-(Wang树脂)-OAll树脂100克于固相反应柱中，加入500mLDMF，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-Pro-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190.2克。
实施例3 固相脱除All
将实施例2得到的树脂190.2g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285.0g。
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285.2g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)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80.1克。
实施例6 含丙氨酸环肽的制备
将实施例5得到的中间体环粗肽80.1克加入到1000ml反应瓶中，加入磷酸钠的缓冲液(pH＝6.0)800ml溶解，然后加入L-天冬氨酸-β脱羧酶0.8g，37℃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50.2g，纯度为99.5％，收率54.6％。MS[M+H]+理论值876.1，实验值876.1。
环肽[D-Pro-Tyr-Ala-Phe-leu-Ile-Val-Ala]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-β-Asp-(Wang树脂)-OAll100克于固相反应柱中，加入500mLDMF，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-D-Pro-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189.1克。
实施例8：固相脱除All
将实施例2得到的树脂189.1g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284.0g。
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284.0g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)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78.2克。
实施例11：含丙氨酸环肽的制备
将实施例10得到的中间体环粗肽78.2克加入到1000ml反应瓶中，加入磷酸钠的缓冲液(pH＝6.0)800ml溶解，然后加入L-天冬氨酸-β脱羧酶0.8g，37℃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48.1g，纯度为99.5％，收率57％。MS[M+H]+理论值876.1，实验值876.1。
环肽[Met-Pro-Tyr-Ala-Phe-leu-Ile-Val-Ala]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-β-Asp-(Wang树脂)-OAll100克于固相反应柱中，加入500mLDMF，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-Pro-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240.2克。
实施例13 固相脱除All
将实施例2得到的树脂240.2g中，加入400mLDMF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315.0g。
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315.0g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)3.5L，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35L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85.1克。
实施例16 含丙氨酸环肽的制备
将实施例15得到的中间体环粗肽85.1克加入到1000ml反应瓶中，加入磷酸钠的缓冲液(pH＝6.0)850ml溶解，然后加入L-天冬氨酸-β脱羧酶0.85g，37℃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55.2g，纯度为99.6％，收率50.6％。MS[M+H]+理论值1007.3，实验值1007.3。
环肽[Met-Pro-Tyr-Ala-D-Phe-leu-Ile-Val-Ala]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-β-Asp-(Wang树脂)-OAll100克于固相反应柱中，加入500mLDMF，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-Pro-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242.2克。
实施例18 固相脱除All
将实施例2得到的树脂242.2g中，加入400mLDMF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318.0g。
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315.0g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)3.5L，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35L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86.1克。
实施例21 含丙氨酸环肽的制备
将实施例20得到的中间体环粗肽86.1克加入到1000ml反应瓶中，加入磷酸钠的缓冲液(pH＝6.0)850ml溶解，然后加入L-天冬氨酸-β脱羧酶0.86g，37℃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50.2g，纯度为99.6％，收率46.0％。MS[M+H]+理论值1007.3，实验值1007.3。

Claims (10)

  1. 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)首先将Fmoc-Asp(OH)-OAll与固相合成树脂偶联，然后按照Fmoc/tBu固相合成策略，得到NH 2-Asp-(固相合成树脂)-OAll，并依次偶联肽序中的氨基酸残基得到NH 2-AAn-Asp-(固相合成树脂)-OAll；
    3)固相脱除保护基All，得到NH 2-AAn-Asp-(固相合成树脂)-OH；
    4)固相条件下，步骤3)得到的多肽长链的首尾氨基酸进行首尾缩合环化；
    5)将步骤4)得到的产物裂解固相合成树脂，制备中间体环肽粗肽；
    6)将中间体环肽粗肽在脱羧酶的作用下脱除末端天冬氨酸的侧链羧基，制备包含丙氨酸的首尾环肽。
  2. 根据权利要求1所述的合成方法，其中，步骤6)中所述脱羧酶为L-天冬氨酸-β脱羧酶。
  3. 根据权利要求1所述的合成方法，步骤6)中中间体环肽粗肽与脱羧酶的质量比为1000-50：1，优选80-150：1。
  4. 根据权利要求1所述的合成方法，步骤2)中偶联Fmoc-Asp(OH)-OAll的方法为以成酯键偶联剂将Fmoc-Asp(OH)-OAll的侧链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所述的合成方法，步骤2)中Fmoc/tBu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)将待偶联氨基酸Fmoc-AA-OH和偶联剂在溶剂中溶解并活化后，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5所述的合成方法，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所述的合成方法，步骤3)中固相脱除保护基All所用催化剂为四三苯基膦钯，捕获剂为苯硅烷。
  8. 根据权利要求1所述的合成方法，步骤5)中固相环化所用试剂为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。
  9. 根据权利要求1所述的合成方法，步骤5)所述的裂解液为TFA、H 2O、PhOMe、苯甲硫醚的混合物。
  10. 根据权利要求1所述的合成方法，还包括可选地步骤7)纯化步骤，步骤7)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，流动相为0.1％醋酸水溶液和乙腈(优选0.1％醋酸水溶液和乙腈的体积比为98:2至50:50，更优选80:20至60:40，最优选70:30)。
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