WO2021136121A1 - 含磺基丙氨酸的多肽的制备方法 - Google Patents

含磺基丙氨酸的多肽的制备方法 Download PDF

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WO2021136121A1
WO2021136121A1 PCT/CN2020/139807 CN2020139807W WO2021136121A1 WO 2021136121 A1 WO2021136121 A1 WO 2021136121A1 CN 2020139807 W CN2020139807 W CN 2020139807W WO 2021136121 A1 WO2021136121 A1 WO 2021136121A1
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resin
acid
preparation
cysteine
boc
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PCT/CN2020/139807
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English (en)
French (fr)
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王卫国
冒其昆
张藤
朱家培
贾辉军
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江苏金斯瑞生物科技有限公司
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Priority to CN202080075344.2A priority Critical patent/CN114616236A/zh
Publication of WO2021136121A1 publication Critical patent/WO2021136121A1/zh

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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
    • 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 present invention relates to the technical field of polypeptide synthesis, in particular to a method for preparing cysteic acid-containing polypeptides.
  • polypeptides containing cysteic acid are an important class of active molecules in the biological world.
  • cysteine instead of cysteine to synthesize polypeptides, and then obtain cysteine-containing polypeptides by oxidation.
  • Anatol Arendt (Protein and Peptide Letters, 2000, 7(6): 359-364) uses Fmoc-Cys(Mmt)-OH as the raw material to synthesize cysteine-containing polypeptides, and then remove mmt from the solid phase carrier. , Oxidation to obtain cysteic acid-containing protected peptides, and finally deprotection to obtain cysteic acid-containing peptides.
  • the problem of this method is that obtaining cysteic acid requires two steps of removal of mmt and oxidation, and the process is complicated.
  • the technical problem to be solved by the present invention is to provide a method for preparing cysteic acid-containing polypeptides. Further, the method for preparing cysteic acid-containing polypeptides provided by the present invention shortens the synthesis steps and simplifies The production process is reduced, the synthesis cost is reduced, and the purity and output of the product are improved.
  • the preparation method of the cysteine-containing polypeptide provided by the present invention uses S-tert-butyl sulfhydryl protected cysteine as one of the raw materials, and synthesizes the S-tert-containing polypeptide according to the amino acid sequence of the cysteic acid-containing polypeptide.
  • the peptide resin of cysteine protected by butyl sulfhydryl group is then used to convert the cysteine residues protected by the S-tert-butyl sulfhydryl group into cysteine residues.
  • the oxidant is a manganese oxidant or a peroxide Acid oxidant.
  • cysteine protected by the S-tert-butyl sulfhydryl group is Fmoc-Cys(StBu)-OH.
  • the manganese oxidant is manganese dioxide; the peroxy acid oxidant is selected from oxyformic acid, peroxyacetic acid, peroxybenzoic acid, peroxytrifluoroacetic acid, peroxy metachlorobenzoic acid, or peroxy P-Nitrobenzoic acid.
  • the synthesis of peptides includes solid-phase synthesis and liquid-phase synthesis.
  • the preparation method is a solid phase synthesis method.
  • the resin in the peptide resin is selected from Wang resin, Rink amide AM resin, and 2-chlorotrityl chloride resin.
  • the ratio of the amount of the oxidant and the resin is 5-10:1.
  • the reaction conditions for converting the cysteine residue protected by the S-tert-butyl sulfhydryl group into the cysteine residues are under the conditions of -4°C to 4°C, and the reaction time is 10 minutes to 60 minutes. .
  • the cysteine residue protected by the S-tert-butyl sulfhydryl group is converted into the cysteine residue by using an oxidizing agent, and the peptide resin is washed sequentially with DMF and DCM.
  • the peptide resin after the peptide resin is washed sequentially with DMF and DCM, it also includes the step of removing the resin and other side chain protecting groups.
  • the deprotection agent for removing resin and other side chain protecting groups is double distilled water, triethylsilane, 1,2 dithiothreitol, phenol, phenylmethyl sulfide, ammonium iodide, tri
  • the volume-to-mass ratio of the deprotection agent to the resin is 10:1.
  • the deprotection conditions are 0°C-60°C, and the reaction time is 1h-4h.
  • the method for preparing the polypeptide includes:
  • the oxidant is peroxyformic acid or peroxybenzoic acid
  • the deprotection reagent is composed of TFA, phenol, thioansole and EDT, wherein the volume ratio of TFA, phenol, thioansole and EDT is 87.5:5:2.5:5;
  • the deprotecting agent is composed of TFA, phenol, thioansole and water, wherein the volume ratio of TFA, phenol, thioansole and water is 87.5:5:2.5:5.
  • the method for preparing the polypeptide includes:
  • WAFEKRHXD where X is cysteic acid
  • the deprotection reagent is composed of TFA, phenol, thioansole and EDT, wherein the volume ratio of TFA, phenol, thioansole and EDT is 87.5:5:2.5:5.
  • the method for preparing the polypeptide includes:
  • the deprotection reagent is composed of TFA and water, wherein the volume ratio of TFA to water is 95:5.
  • the method for preparing the polypeptide includes:
  • the deprotection reagent is composed of TFA, water, thioansole and EDT, wherein the volume ratio of TFA, water, thioansole and EDT is 87.5:5:2.5:5.
  • the preparation method of the cysteine-containing polypeptide provided by the present invention takes S-tert-butyl sulfhydryl protected cysteine as one of the raw materials, and synthesizes the S-containing polypeptide according to the sequence of the amino acid sequence of the cysteic acid-containing polypeptide.
  • -Tert-butyl mercapto-protected cysteine peptide resin and then using an oxidizing agent to convert S-tert-butyl mercapto-protected cysteine residues into cysteine residues
  • the oxidizing agent is a manganese oxidant or Peroxidic acid oxidant.
  • the present invention uses low-cost and easily available Fmoc-Cys(StBu)-OH as a raw material, and the protective group can be removed while being oxidized. Therefore, the preparation method provided by the present invention simplifies the production process, reduces the synthesis cost, has fewer side reactions, and has higher yield and purity.
  • the present invention provides a method for preparing a cysteic acid-containing polypeptide, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters.
  • all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention.
  • the method and application of the present invention have been described through the preferred embodiments. It is obvious that relevant persons can make changes or appropriate changes and combinations to the methods and applications herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention.
  • Invent technology is obvious that relevant persons can make changes or appropriate changes and combinations to the methods and applications herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention.
  • test materials used in the present invention are all common commercially available products, all of which can be purchased in the market.
  • a protecting group includes a reactive group (ie, a protected group) attached to or configured to be attached to a molecule (eg, a peptide) such that the protecting group prevents or otherwise inhibits the protected group
  • a molecule eg, a peptide
  • Protection can be done by attaching a protecting group to the molecule. When the protecting group is removed from the molecule, for example, by a chemical transformation that removes the protecting group, deprotection can occur.
  • polypeptide or “peptide” has its ordinary meaning in the art and can refer to the loss of water from the carbonyl carbon of one aminocarboxylic acid molecule and the nitrogen atom of another aminocarboxylic acid molecule. Amides from two or more amino carboxylic acid molecules (same or different) that form a covalent bond.
  • amino acid also has its ordinary meaning in the art and may include proteinaceous amino acids and non-proteinaceous amino acids.
  • the abbreviation of amino acid residue in the present invention is the standard 3-letter and/or 1-letter code used in the art to refer to one of the 20 commonly used L-amino acids.
  • X or Xaa represents cysteic acid in the present invention.
  • the S-tert-butyl sulfhydryl group protects the sulfhydryl group of cysteine, and the amino group of cysteine can be protected by other protecting groups.
  • Fmoc to protect the amino group of cysteine.
  • eq is a molar equivalent, which is a unit used in chemistry or biological sciences to indicate the amount of a substance.
  • Fmoc-SPPS Fmoc strategy solid-phase peptide synthesis
  • Phenol Phenol
  • Trt Trityl
  • StBu S-tert-butyl mercapto group, its structural formula is:
  • Fmoc-Cys(StBu)-OH N-fluorenylmethyloxycarbonyl-S-tert-butylthio-L-cysteine, its structural formula is:
  • the scheme of the present invention uses S-tert-butyl mercapto-protected cysteine as a raw material, and the protective group can be removed at the same time as it is oxidized, which simplifies the production process, reduces the synthesis cost, and has fewer side reactions, resulting in a lower yield
  • the purity is relatively high. in particular:
  • the preparation method of the cysteine-containing polypeptide provided by the present invention uses S-tert-butyl sulfhydryl protected cysteine as one of the raw materials, and synthesizes the S-tert-containing polypeptide according to the amino acid sequence of the cysteic acid-containing polypeptide.
  • the peptide resin of cysteine protected by butyl sulfhydryl group is then used to convert the cysteine residues protected by the S-tert-butyl sulfhydryl group into cysteine residues.
  • the oxidant is a manganese oxidant or a peroxide Acid oxidant.
  • cysteine protected by the S-tert-butyl sulfhydryl group is Fmoc-Cys(StBu)-OH.
  • the present invention adopts suitable oxidants, which can not only convert cysteine into cysteic acid, but also remove the side chain protecting group on its sulfhydryl group at the same time, while the side chain protecting agent of other amino acids on the peptide chain is Will not be affected.
  • the manganese oxidant is manganese dioxide; the peroxyacid oxidant is selected from oxyformic acid, peroxyacetic acid, peroxybenzoic acid, peroxytrifluoroacetic acid, peroxy metachlorobenzoic acid, peroxy Nitrobenzoic acid.
  • the oxidant is peroxyformic acid, peroxybenzoic acid or manganese dioxide.
  • the "coupling" in the present invention refers to the process of adding new amino acids to the bound amino acids or peptides.
  • the preparation method of the present invention improves the coupling step of cysteic acid, and the coupling of other amino acids is carried out by methods well known in the art.
  • the position of the cysteic acid in the peptide chain is not limited.
  • the C-terminal, N-terminal or any other positions of the peptide chain can be used to achieve good results with the method provided by the present invention.
  • the synthesis of peptides includes solid-phase synthesis and liquid-phase synthesis. There are also reports using a combination of solid-phase and liquid-phase methods.
  • the coupling can be performed in a manner of coupling amino acid sequence by amino acid sequence, or multiple polypeptide fragments can be coupled in a segmented coupling manner according to the peptide sequence.
  • the preparation adopts In the solid phase method, coupling is one by one.
  • the resin in the peptide resin is selected from Wang resin, Rink amide AM resin, and 2-chlorotrityl chloride resin.
  • the amount of the oxidant in the oxidation, is 5-10 eq of the polypeptide. In some embodiments, the amount of the oxidizing agent is 5 times, 7 times, and 10 times that of the resin.
  • the oxidation conditions are -4°C to 4°C, and the reaction is 10min to 60min.
  • the oxidation is performed under ice bath conditions, and the reaction time is 10 min, 30 min, and 60 min.
  • the oxidant converts the cysteine residue protected by the S-tert-butyl sulfhydryl group into the cysteine residue, and then the peptide resin is washed with DMF and DCM in sequence.
  • the number of times of washing of the DMF is 6 times, and the number of times of washing of the DCM is 3 times.
  • the peptide resin after the peptide resin is washed sequentially with DMF and DCM, it also includes the step of removing the resin and other side chain protecting groups.
  • the deprotection agent for removing resin and other side chain protecting groups is double distilled water, triethylsilane, 1,2 dithiothreitol, phenol, phenylmethyl sulfide, ammonium iodide, tri A mixed liquid of at least one of isopropylsilane, anisole and ethanedithiol and trifluoroacetic acid; the volume fraction of trifluoroacetic acid in the mixed liquid is not less than 80%.
  • the deprotection refers to the removal of the side chain protecting group of the peptide chain.
  • the deprotection reagent is composed of TFA, phenol, thioansole and EDT, wherein the volume ratio of TFA, phenol, thioansole and EDT is 87.5:5:2.5:5;
  • the deprotecting agent is composed of TFA, phenol, thioansole and water, wherein the volume ratio of TFA, phenol, thioansole and water is 87.5:5:2.5:5.
  • the deprotection reagent is composed of TFA and water, where the volume ratio of TFA to water is 95:5.
  • the deprotection reagent is composed of TFA, water, thioansole and EDT, wherein the volume ratio of TFA, water, thioansole and EDT is 87.5:5:2.5:5.
  • the volume-to-mass ratio of the deprotection agent to the resin is 10:1.
  • the deprotection conditions are 0°C-60°C, and the reaction time is 1h-4h.
  • the temperature of the deprotection is 40°C, 25°C or 0°C; the reaction time is 1h, 2h or 4h.
  • the preparation method of the cysteic acid-containing polypeptide provided by the present invention includes the following steps:
  • Fmoc-SPPS strategy is adopted, that is, the ⁇ -amino protecting group of the amino acid used is Fmoc, and a solid-phase synthesis method is used to synthesize a peptide resin containing cysteine.
  • the protecting group of amino acid sulfhydryl is S-tert-butyl sulfhydryl;
  • oxidant is manganese dioxide or peroxyacid oxidant, including but not limited to peroxyformic acid, peroxyacetic acid, peroxybenzoic acid, peroxytrifluoroacetic acid, peroxym-chlorobenzoic acid, peroxyp-nitroperoxy Benzoic acid; the ratio of the amount of oxidant and resin is 5-10:1, and the reaction is 10min-60min under ice bath;
  • the deprotection agent is double distilled water, triethylsilane, 1,2 dithiothreitol, phenol, anisole, ammonium iodide, triisopropylsilane, anisole and ethylene disulfide
  • the method for preparing the polypeptide includes:
  • the oxidant is peroxyformic acid or peroxybenzoic acid
  • the deprotection reagent is composed of TFA, phenol, thioansole and EDT, wherein the volume ratio of TFA, phenol, thioansole and EDT is 87.5:5:2.5:5;
  • the deprotecting agent is composed of TFA, phenol, thioansole and water, wherein the volume ratio of TFA, phenol, thioansole and water is 87.5:5:2.5:5.
  • the method for preparing the polypeptide includes:
  • WAFEKRHXD where X is cysteic acid
  • the deprotection reagent is composed of TFA, phenol, thioansole and EDT, wherein the volume ratio of TFA, phenol, thioansole and EDT is 87.5:5:2.5:5.
  • the method for preparing the polypeptide includes:
  • the deprotection reagent is composed of TFA and water, wherein the volume ratio of TFA to water is 95:5.
  • the method for preparing the polypeptide includes:
  • the deprotection reagent is composed of TFA, water, thioansole and EDT, wherein the volume ratio of TFA, water, thioansole and EDT is 87.5:5:2.5:5.
  • RinkamideAM resin Take 1g of RinkamideAM resin (1.0mmol/g), and select Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Boc) for each amino acid.

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Abstract

含磺基丙氨酸的多肽的制备方法，以S-叔丁基巯基保护的半胱氨酸为原料之一，按照含磺基丙氨酸的多肽氨基酸序列合成含S-叔丁基巯基保护的半胱氨酸的肽树脂，然后采用氧化剂将S-叔丁基巯基保护的半胱氨酸残基转化成磺基丙氨酸残基，所述氧化剂为锰氧化剂或过氧化酸类氧化剂。采用成本低易得的Fmoc-Cys(StBu)-OH为原料，在氧化的同时即可脱除保护基。因此，提供的制备方法简化了生产工艺，降低了合成成本，且副反应较少，收率和纯度皆较高。

Description

含磺基丙氨酸的多肽的制备方法
本申请要求于2019年12月31日日提交中国专利局、申请号为201911405253.6、发明名称为“含磺基丙氨酸的多肽的制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及多肽合成技术领域，尤其涉及含磺基丙氨酸的多肽的制备方法。
背景技术
含有磺基丙氨酸(式I)的多肽是生物界中一类重要的活性分子。
Figure PCTCN2020139807-appb-000001
现有的关于这类多肽的合成方法报道较少，且存在一定的问题。现有的方法可以归为两种路线，一种是采用磺基丙氨酸原料直接合成多肽，如文献Toshio NAGASE(Tetrahedron Letters,1993,34(7):1173-1176)采用N-(9-Fluorenylmethoxycarbonyl)cysteic Acid的四丁基胺盐为原料替代N-(9-fluorenylmethoxycarbonyl)cysteic acid合成含磺基丙氨酸的多肽(纯化后产率为25％)，虽然解决了原料的溶解性问题，但是没有办法避免磺酸基不保护产生的副反应，另外该原料价格昂贵，缩合效率也较低，因而导致产物有较多的杂质。另一种方法是采用半胱氨酸替代磺基丙氨酸合成多肽，然后通过氧化的方法得到含磺基丙氨酸的多肽。如Anatol Arendt(Protein and Peptide Letters,2000,7(6):359-364)采用Fmoc-Cys(Mmt)-OH为原料合成含半胱氨酸的多肽，然后在固相载体上脱除mmt后，氧化得到含磺基丙氨酸的保护肽，最后再脱保护得到含磺基丙氨酸的多肽。该方法的问题是得到磺基丙氨酸需要脱除mmt和氧化两个步骤，工艺复杂。
可见，目前对含有磺基丙氨酸的多肽的合成方法复杂，且在合成过程中容易发生副反应。为了提高含有磺基丙氨酸的多肽的合成效率，降低合成成本，本领域技术人员仍在不断开发新的方法。
发明内容
有鉴于此，本发明要解决的技术问题在于提供含磺基丙氨酸的多肽的制备方法，进一步的，本发明提供的制备含磺基丙氨酸的多肽的方法缩短了合成的步骤，简化了生产工艺，降低了合成成本，提高了产物的纯度和产量。
本发明提供的含磺基丙氨酸的多肽的制备方法，以S-叔丁基巯基保护的半胱氨酸为原料之一，按照含磺基丙氨酸的多肽氨基酸序列合成含S-叔丁基巯基保护的半胱氨酸的肽树脂，然后采用氧化剂将S-叔丁基巯基保护的半胱氨酸残基转化成磺基丙氨酸残基，所述氧化剂为锰氧化剂或过氧化酸类氧化剂。
本发明中，所述S-叔丁基巯基保护的半胱氨酸为Fmoc-Cys(StBu)-OH。
本发明中，所述锰氧化剂为二氧化锰；所述过氧化酸类氧化剂选自氧甲酸、过氧乙酸、过氧苯甲酸、过氧三氟乙酸、过氧间氯苯甲酸、或过氧对硝基苯甲酸。
多肽的合成包括固相合成法、液相合成法。本发明实施例中，所述制备方法为固相合成法。
本发明实施例中，所述肽树脂中的树脂选自Wang树脂、Rink amide AM树脂、2-氯三苯甲基氯树脂。
本发明实施例中，所述氧化剂和树脂的物质的量的比例为5～10:1。
本发明实施例中，所述将S-叔丁基巯基保护的半胱氨酸残基转化成磺基丙氨酸残基的反应条件为的条件为-4℃～4℃，反应10min～60min。
本发明中，采用氧化剂将S-叔丁基巯基保护的半胱氨酸残基转化成磺基丙氨酸残基后肽树脂依次经DMF和DCM洗涤。
本发明中，肽树脂依次经DMF和DCM洗涤之后，还包括脱除树脂 和其他侧链保护基的步骤。
本发明中，所述脱除树脂和其他侧链保护基的脱保护剂为重蒸水、三乙基硅烷、1,2二硫苏糖醇、苯酚、苯甲硫醚、碘化铵、三异丙基硅烷、苯甲醚和乙二硫醇中的至少一种与三氟乙酸的混合液；所述的混合液中三氟乙酸的体积分数不小于80％。
本发明实施例中，所述脱保护剂与树脂的体积质量比为10：1。
本发明实施例中，所述脱保护的条件为0℃～60℃，反应1h～4h。
一些实施例中，多肽的制备方法包括：
制备树脂-Trp(Boc)-Ala-Phe-Glu(OtBu)-Lys(Boc)-Arg(Pbf)-His(Trt)-Cys(StBu)-Asp(OtBu)；然后加入5～7eq的氧化剂-4℃～4℃反应10min～30min，制得树脂-Trp(Boc)-Ala-Phe-Glu(OtBu)-Lys(Boc)-Arg(Pbf)-His(Trt)-X-Asp(OtBu)；
依次以DMF和DCM洗涤后，加入脱保护试剂0～25℃下反应2～4h，制得WAFEKRHXD；其中X为磺基丙氨酸；
所述氧化剂为过氧甲酸或过氧苯甲酸；
所述脱保护试剂由TFA、phenol、thioansole和EDT组成，其中TFA、phenol、thioansole和EDT的体积比为87.5：5：2.5：5；
或者脱保护剂由TFA、phenol、thioansole和水组成，其中TFA、phenol、thioansole和水的体积比为87.5：5：2.5：5。
一些具体实施例中，多肽的制备方法包括：
制备树脂-Trp(Boc)-Ala-Phe-Glu(OtBu)-Lys(Boc)-Arg(Pbf)-His(Trt)-Cys(StBu)-Asp(OtBu)；然后加入5eq的过氧甲酸-4℃～4℃反应10min，制得树脂-Trp(Boc)-Ala-Phe-Glu(OtBu)-Lys(Boc)-Arg(Pbf)-His(Trt)-X-Asp(OtBu)；
依次以DMF和DCM洗涤后，加入脱保护试剂25℃下反应2h，制得WAFEKRHXD；其中X为磺基丙氨酸；
所述脱保护试剂由TFA、phenol、thioansole和EDT组成，其中TFA、phenol、thioansole和EDT的体积比为87.5：5：2.5：5。
一些具体实施例中，多肽的制备方法包括：
制备树脂-Gly-Val-Leu-Cys(StBu)-Gly-Phe-Lys(Boc)-Lys(Boc)；然后加入7eq的过氧苯甲酸4℃～4℃反应30min，制得树脂-Gly-Val-Leu-X-Gly-Phe-Lys(Boc)-Lys(Boc)；
依次以DMF和DCM洗涤后，加入脱保护试剂0℃下反应1h，制得GVLXGFKK；其中X为磺基丙氨酸；
所述脱保护试剂由TFA和水组成，其中TFA与水的体积比为95:5。
一些具体实施例中，多肽的制备方法包括：
制备树脂-Cys(StBu)-Glu(OtBu)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Phe-Phe；然后加入10eq的过氧苯甲酸4℃～4℃反应60min，树脂-X-Glu(OtBu)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Phe-Phe；
依次以DMF和DCM洗涤后，加入脱保护试剂40℃下反应4h，制得XEEKKFF；其中X为磺基丙氨酸；
所述脱保护试剂由TFA、水、thioansole和EDT组成，其中TFA、水、thioansole和EDT的体积比为87.5：5：2.5：5。
本发明提供的含磺基丙氨酸的多肽的制备方法，以S-叔丁基巯基保护的半胱氨酸为原料之一，按照含磺基丙氨酸的多肽氨基酸序列顺序逐一合成含S-叔丁基巯基保护的半胱氨酸的肽树脂，然后采用氧化剂将S-叔丁基巯基保护的半胱氨酸残基转化成磺基丙氨酸残基，所述氧化剂为锰氧化剂或过氧化酸类氧化剂。本发明采用成本低易得的Fmoc-Cys(StBu)-OH为原料，在氧化的同时即可脱除保护基。因此，本发明提供的制备方法简化了生产工艺，降低了合成成本，且副反应较少，收率和纯度皆较高。
具体实施方式
本发明提供了含磺基丙氨酸的多肽的制备方法，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明采用的试材皆为普通市售品，皆可于市场购得。
除非另有定义，本文使用的所有科技术语具有本领域普通技术人员所理解的相同含义。
本发明所述“保护基团”或“保护基”具有其在本领域中的普通含义。保护基团包括连接至或被配置成连接至分子(例如，肽)内的反应性基团(即，受保护的基团)以使得所述保护基团阻止或以其他方式抑制受保护的基团参与反应的化学部分。可通过将保护基团连接至分子来进行保护。当将保护基团从分子中除去时，例如，通过除去保护基团的化学转化，可发生去保护。
本发明中，所述“多肽”或“肽”具有其在本领域中的普通含义并且可指通过由一个氨基羧酸分子的羰基碳与另一个氨基羧酸分子的氮原子形式上失去水而形成共价键之来自两个或更多个氨基羧酸分子(相同或不同)的酰胺。“氨基酸残基”也具有其在本领域中的普通含义并且指氨基酸(作为单个氨基酸或作为肽的一部分)在其与肽、另一个氨基酸或氨基酸残基组合之后的组合物。通常，当氨基酸与另一个氨基酸或氨基酸残基组合时，除去水，并且保留下来的氨基酸被称为氨基酸残基。术语“氨基酸”也具有其在本领域中的普通含义并且可包括蛋白质氨基酸和非蛋白质氨基酸。本发明中氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。其中，X或Xaa在本发明中表示磺基丙氨酸。所述S-叔丁基巯基保护的半胱氨酸中，S-叔丁基巯基保护的是半胱氨酸的巯基，半胱氨酸的氨基可以采用其他保护基团进行保护，本发明中，采用Fmoc对半胱氨酸的氨基保护。
本发明中，eq为摩尔当量，是一个在化学或生物科学中使用的单位，用于表示物质的量。
另外，本发明中一些常用的缩写具有如下含义：
Fmoc:芴甲氧羰酰基
Fmoc-SPPS：Fmoc策略固相多肽合成
TFA：三氟醋酸
EDT：乙二硫醇
Phenol：苯酚
Thioanisole：苯甲硫醚
TES：三乙基硅烷
TIS：三异丙基硅烷
DTT：1,2二硫苏糖醇
Anisole：苯甲醚
NH 4I：碘化铵
DCM：二氯甲烷
DMF：N,N-二甲基甲酰胺
X：磺基丙氨酸
Mmt：4-甲氧基三苯甲基
Pbf：2,2,4,6,7-五甲基二氢苯并呋喃-5-磺酰基
Trt：三苯甲基
Boc：叔丁氧羰基
OtBu：叔丁酯
StBu：S-叔丁基巯基，其结构式为：
Figure PCTCN2020139807-appb-000002
Fmoc-Cys(StBu)-OH：N-芴甲氧羰基-S-叔丁硫基-L-半胱氨酸，其结构式为：
Figure PCTCN2020139807-appb-000003
本发明的方案以S-叔丁基巯基保护的半胱氨酸为原料，在氧化的同时即可脱除保护基，简化了生产工艺，降低了合成成本，且副反应较少，收率和纯度皆较高。具体而言：
本发明提供的含磺基丙氨酸的多肽的制备方法，以S-叔丁基巯基保护的半胱氨酸为原料之一，按照含磺基丙氨酸的多肽氨基酸序列合成含S-叔丁基巯基保护的半胱氨酸的肽树脂，然后采用氧化剂将S-叔丁基巯基保护的半胱氨酸残基转化成磺基丙氨酸残基，所述氧化剂为锰氧化剂或过氧化酸类氧化剂。
本发明中，所述S-叔丁基巯基保护的半胱氨酸为Fmoc-Cys(StBu)-OH。
并且，本发明采用适宜的氧化剂，不仅可以使半胱氨酸转换为磺基丙氨酸，还可以同时去除其巯基上的侧链保护基团，而肽链上其他氨基酸的侧链保护剂则不会受到影响。本发明中，所述锰氧化剂为二氧化锰；所述过氧化酸类氧化剂选自氧甲酸，过氧乙酸，过氧苯甲酸，过氧三氟乙酸，过氧间氯苯甲酸，过氧对硝基苯甲酸。一些实施例中，所述氧化剂为过氧甲酸、过氧苯甲酸或二氧化锰。
本发明中所述“偶联”是指将新的氨基酸添加至结合的氨基酸或肽的过程。本发明所述的制备方法针对磺基丙氨酸的偶联步骤进行改进，对其他氨基酸的偶联采用本领域熟知的方法进行。本发明提供的方法中，对肽链中磺基丙氨酸的位置没有限定，磺基丙氨酸在肽链的C端、N端或其他任意位置都能够采用本发明提供的方法实现良好的制备效果。多肽的合成包括固相合成法、液相合成法，也有报道采用固相和液态结合的方式进行。本领域中，所述偶联可采用逐一氨基酸顺序偶联的方式进行，也可根据肽序采用分段偶联的方式对多个多肽片段进行偶联，本发明实施例中，所述制备采用固相法，偶联为逐个偶联。
本发明实施例中，所述肽树脂中的树脂选自Wang树脂、Rink amide AM树脂、2-氯三苯甲基氯树脂。
本发明实施例中，所述氧化中，氧化剂的用量为多肽的5～10eq。一些实施例中，氧化剂的物质的量为树脂的5倍、7倍、10倍。
本发明实施例中，所述氧化的条件为-4℃～4℃，反应10min～60min。具体实施例中，所述氧化的在冰浴条件下进行，反应时间为10min、30min、60min。
本发明中，所述氧化剂将S-叔丁基巯基保护的半胱氨酸残基转化成磺基丙氨酸残基后肽树脂依次经DMF和DCM洗涤。所述DMF洗涤的次数为6次，所述DCM的洗涤次数为3次。
本发明中，肽树脂依次经DMF和DCM洗涤之后，还包括脱除树脂和其他侧链保护基的步骤。
本发明中，所述脱除树脂和其他侧链保护基的脱保护剂为重蒸水、三乙基硅烷、1,2二硫苏糖醇、苯酚、苯甲硫醚、碘化铵、三异丙基硅烷、苯甲醚和乙二硫醇中的至少一种与三氟乙酸的混合液；所述的混合液中三氟乙酸的体积分数不小于80％。在此步骤中，所述脱保护是指脱除肽链的侧链保护基。
一些实施例中，所述脱保护试剂由TFA、phenol、thioansole和EDT组成，其中TFA、phenol、thioansole和EDT的体积比为87.5：5：2.5：5；
或者脱保护剂由TFA、phenol、thioansole和水组成，其中TFA、phenol、thioansole和水的体积比为87.5：5：2.5：5。
或者脱保护试剂由TFA和水组成，其中TFA与水的体积比为95:5。
或者脱保护试剂由TFA、水、thioansole和EDT组成，其中TFA、水、thioansole和EDT的体积比为87.5：5：2.5：5。
本发明实施例中，所述脱保护剂与树脂的体积质量比10：1。
本发明实施例中，所述脱保护的条件为0℃～60℃，反应1h～4h。一些实施例中，所述脱保护的温度为40℃、25℃或0℃；反应时间为1h、2h或4h。
实施例中，本发明提供的含磺基丙氨酸多肽的制备方法，包括以下步骤：
(1)制备含半胱氨酸的肽树脂：采用Fmoc-SPPS策略，即所用氨基酸的α氨基保护基为Fmoc，并采用固相合成的方法合成含半胱氨酸的肽树脂，其中半胱氨酸巯基的保护基为S-叔丁基巯基；
(2)制备含磺基丙氨酸的肽树脂：向步骤(1)得到的肽树脂中加入氧化剂，氧化结束后，加入DMF洗涤树脂6遍，再用DCM洗涤树脂3 遍，并过滤掉洗涤液。所述的氧化剂为二氧化锰或过氧酸类氧化剂，包括但不仅限于过氧甲酸，过氧乙酸，过氧苯甲酸，过氧三氟乙酸，过氧间氯苯甲酸，过氧对硝基苯甲酸；氧化剂和树脂的物质的量的比例为5～10：1，冰浴下反应10min-60min；
(3)制备含磺基丙氨酸的多肽：向步骤(2)得到的含磺基丙氨酸的肽树脂中加入脱保护剂，脱除多肽侧链保护基，得到含磺基丙氨酸的多肽。所述的脱保护剂为为重蒸水、三乙基硅烷、1,2二硫苏糖醇、苯酚、苯甲硫醚、碘化铵、三异丙基硅烷、苯甲醚和乙二硫醇中的至少一种与三氟乙酸的混合液，所述的混合液中三氟乙酸的体积比不小于80％；脱保护反应的温度为0℃～40℃，反应时间为1小时～4小时。反应结束后，过滤，加入乙醚将滤液沉淀，进行质谱鉴定。
一些实施例中，多肽的制备方法包括：
制备树脂-Trp(Boc)-Ala-Phe-Glu(OtBu)-Lys(Boc)-Arg(Pbf)-His(Trt)-Cys(StBu)-Asp(OtBu)；然后加入5～7eq的氧化剂-4℃～4℃反应10min～30min，制得树脂-Trp(Boc)-Ala-Phe-Glu(OtBu)-Lys(Boc)-Arg(Pbf)-His(Trt)-X-Asp(OtBu)；
依次以DMF和DCM洗涤后，加入脱保护试剂0～25℃下反应2～4h，制得WAFEKRHXD；其中X为磺基丙氨酸；
所述氧化剂为过氧甲酸或过氧苯甲酸；
所述脱保护试剂由TFA、phenol、thioansole和EDT组成，其中TFA、phenol、thioansole和EDT的体积比为87.5：5：2.5：5；
或者脱保护剂由TFA、phenol、thioansole和水组成，其中TFA、phenol、thioansole和水的体积比为87.5：5：2.5：5。
一些具体实施例中，多肽的制备方法包括：
制备树脂-Trp(Boc)-Ala-Phe-Glu(OtBu)-Lys(Boc)-Arg(Pbf)-His(Trt)-Cys(StBu)-Asp(OtBu)；然后加入5eq的过氧甲酸-4℃～4℃反应10min，制得树脂-Trp(Boc)-Ala-Phe-Glu(OtBu)-Lys(Boc)-Arg(Pbf)-His(Trt)-X-Asp(OtBu)；
依次以DMF和DCM洗涤后，加入脱保护试剂25℃下反应2h，制得 WAFEKRHXD；其中X为磺基丙氨酸；
所述脱保护试剂由TFA、phenol、thioansole和EDT组成，其中TFA、phenol、thioansole和EDT的体积比为87.5：5：2.5：5。
一些具体实施例中，多肽的制备方法包括：
制备树脂-Gly-Val-Leu-Cys(StBu)-Gly-Phe-Lys(Boc)-Lys(Boc)；然后加入7eq的过氧苯甲酸4℃～4℃反应30min，制得树脂-Gly-Val-Leu-X-Gly-Phe-Lys(Boc)-Lys(Boc)；
依次以DMF和DCM洗涤后，加入脱保护试剂0℃下反应1h，制得GVLXGFKK；其中X为磺基丙氨酸；
所述脱保护试剂由TFA和水组成，其中TFA与水的体积比为95:5。
一些具体实施例中，多肽的制备方法包括：
制备树脂-Cys(StBu)-Glu(OtBu)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Phe-Phe；然后加入10eq的过氧苯甲酸4℃～4℃反应60min，树脂-X-Glu(OtBu)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Phe-Phe；
依次以DMF和DCM洗涤后，加入脱保护试剂40℃下反应4h，制得XEEKKFF；其中X为磺基丙氨酸；
所述脱保护试剂由TFA、水、thioansole和EDT组成，其中TFA、水、thioansole和EDT的体积比为87.5：5：2.5：5。
下面结合实施例，进一步阐述本发明：
实施例1：制备含磺基丙氨酸的多肽WAFEKRHXD
(1)制备含半胱氨酸的肽树脂W(Boc)AFE(OtBu)K(Boc)R(Pbf)H(Trt)C(StBu)D(OtBu)
取1gWang树脂(1.0mmol/g)，各氨基酸分别选用Fmoc-Trp(Boc)-OH，Fmoc-Ala-OH，Fmoc-Phe-OH，Fmoc-Glu(OtBu)-OH，Fmoc-Lys(Boc)-OH，Fmoc-Arg(Pbf)-OH，Fmoc-His(Trt)-OH，Fmoc-Cys(StBu)-OH，Fmoc-Asp(OtBu)-OH，氨基酸与树脂物质的量比为3：1，采用Fmoc固相多肽合成策略，按照多肽序列，合成含半胱氨酸的肽树脂W(Boc)AFE(OtBu)K(Boc)R(Pbf)H(Trt)C(StBu)D(OtBu)。
(2)制备含磺基丙氨酸的肽树脂W(Boc)AFE(OtBu)K(Boc) R(Pbf)H(Trt)XD(OtBu)
向含半胱氨酸的肽树脂W(Boc)AFE(OtBu)K(Boc)R(Pbf)H(Trt)C(StBu)D(OtBu)中加入5mmol的氧化剂过氧甲酸，冰浴下反应10min，反应完毕后，过滤。然后加入DMF洗涤树脂六遍，再加入DCM洗涤树脂3遍，过滤。
(3)制备含磺基丙氨酸的多肽WAFEKRHXD
向上述保护的含磺基丙氨酸的肽树脂中加入10ml脱保护试剂(按体积百分含量计，TFA:phenol:thioansole:EDT＝87.5％:5％:2.5％:5％)，0℃下反应4h。反应结束后，过滤，加入乙醚将滤液沉淀，干燥后称量得到0.73g粗品，产率73.6％。取粗品进行质谱分析和高效液相分析，分子量为1239.3，纯度为75％。
实施例2：制备含磺基丙氨酸的多肽WAFEKRHXD
(1)制备含半胱氨酸的肽树脂W(Boc)AFE(OtBu)K(Boc)R(Pbf)H(Trt)C(StBu)D(OtBu)
取1gRinkamideAM树脂(1.0mmol/g)，各氨基酸分别选用Fmoc-Trp(Boc)-OH，Fmoc-Ala-OH，Fmoc-Phe-OH，Fmoc-Glu(OtBu)-OH，Fmoc-Lys(Boc)-OH，Fmoc-Arg(Pbf)-OH，Fmoc-His(Trt)-OH，Fmoc-Cys(StBu)-OH，Fmoc-Asp(OtBu)-OH，氨基酸与树脂物质的量比为4：1，采用Fmoc固相多肽合成策略，按照多肽序列，合成含半胱氨酸的肽树脂W(Boc)AFE(OtBu)K(Boc)R(Pbf)H(Trt)C(StBu)D(OtBu)。
(2)制备含磺基丙氨酸的肽树脂W(Boc)AFE(OtBu)K(Boc)R(Pbf)H(Trt)XD(OtBu)
向含半胱氨酸的肽树脂W(Boc)AFE(OtBu)K(Boc)R(Pbf)H(Trt)C(StBu)D(OtBu)中加入7mmol的氧化剂过氧苯甲酸，冰浴下反应30min，反应完毕后，过滤。然后加入DMF洗涤树脂六遍，再加入DCM洗涤树脂3遍，过滤。
(3)制备含磺基丙氨酸的多肽WAFEKRHXD
向上述保护的含磺基丙氨酸的肽树脂中加入10ml脱保护试剂(按体积百分含量计，TFA:phenol:thioansole:水＝87.5％:5％:2.5％:5％)，25℃ 下反应2h。反应结束后，过滤，加入乙醚将滤液沉淀，干燥后称量得到0.85g粗品，产率85.7％。取粗品进行质谱分析和高效液相分析，分子量为1238.6，纯度为84％。
实施例3：制备含磺基丙氨酸的多肽WAFEKRHXD
(1)制备含半胱氨酸的肽树脂W(Boc)AFE(OtBu)K(Boc)R(Pbf)H(Trt)C(StBu)D(OtBu)
取1g2-氯三苯甲基氯树脂(1.0mmol/g)，各氨基酸分别选用Fmoc-Trp(Boc)-OH，Fmoc-Ala-OH，Fmoc-Phe-OH，Fmoc-Glu(OtBu)-OH，Fmoc-Lys(Boc)-OH，Fmoc-Arg(Pbf)-OH，Fmoc-His(Trt)-OH，Fmoc-Cys(StBu)-OH，Fmoc-Asp(OtBu)-OH，氨基酸与树脂物质的量比为3：1，采用Fmoc固相多肽合成策略，按照多肽序列，合成含半胱氨酸的肽树脂W(Boc)AFE(OtBu)K(Boc)R(Pbf)H(Trt)C(StBu)D(OtBu)。
(2)制备含磺基丙氨酸的肽树脂W(Boc)AFE(OtBu)K(Boc)R(Pbf)H(Trt)XD(OtBu)
向含半胱氨酸的肽树脂W(Boc)AFE(OtBu)K(Boc)R(Pbf)H(Trt)C(StBu)D(OtBu)中加入10mmol的氧化剂二氧化锰，冰浴下反应60min，反应完毕后，过滤。然后加入DMF洗涤树脂六遍，再加入DCM洗涤树脂3遍，过滤。
(3)制备含磺基丙氨酸的多肽WAFEKRHXD
向上述保护的含磺基丙氨酸的肽树脂中加入10ml脱保护试剂(按体积百分含量计，脱保护试剂(按体积百分含量计，TFA:水:thioansole:TIS＝87.5％:5％:2.5％:5％)，40℃下反应1h。反应结束后，过滤，加入乙醚将滤液沉淀，干燥后称量得到0.55g粗品，产率55.5％。取粗品进行质谱分析和高效液相分析，分子量为1239.3，纯度为51％。
实施例4：制备含磺基丙氨酸的多肽GVLXGFKK
(1)制备含半胱氨酸的肽树脂GVLC(StBu)GFK(Boc)K(Boc)
取1gWang树脂(1.0mmol/g)，各氨基酸分别选用Fmoc-Gly-OH，Fmoc-Val-OH，Fmoc-Leu-OH，Fmoc-Cys(StBu)-OH，Fmoc-Lys(Boc)-OH，氨基酸与树脂物质的量比为3：1，采用Fmoc固相多肽合成策略，按照 多肽序列，合成含半胱氨酸的肽树脂GVLC(StBu)GFK(Boc)K(Boc)；
(2)制备含磺基丙氨酸的肽树脂GVLXGFK(Boc)K(Boc)
向含半胱氨酸的肽树脂GVLC(StBu)GFK(Boc)K(Boc)中加入7mmol的氧化剂过氧苯甲酸，冰浴下反应30min，反应完毕后，过滤。然后加入DMF洗涤树脂六遍，再加入DCM洗涤树脂3遍，过滤。
(3)制备含磺基丙氨酸的多肽GVLXGFKK
向上述保护的含磺基丙氨酸的肽树脂复合物中加入10ml脱保护试剂(按体积百分含量计，TFA:水＝95％:5％)，0℃下反应1h。反应结束后，过滤，加入乙醚将滤液沉淀。干燥后称量得到0.68g粗品，产率75.6％。取粗品进行质谱分析和高效液相分析，分子量为899.3，纯度为81％。
实施例5：制备含磺基丙氨酸的多肽XEEKKFF
(1)制备含半胱氨酸的肽树脂C(StBu)E(OtBu)E(OtBu)K(Boc)K(Boc)FF
取1gWang树脂(1.0mmol/g)，各氨基酸分别选用Fmoc-Cys(StBu)-OH，Fmoc-Glu(OtBu)-OH，Fmoc-Glu(OtBu)-OH，Fmoc-Lys(Boc)-OH，Fmoc-Lys(Boc)-OH，Fmoc-Phe-OH，Fmoc-Phe-OH，氨基酸与树脂物质的量比为3：1，采用Fmoc固相多肽合成策略，按照多肽序列，合成含半胱氨酸的肽树脂C(StBu)E(OtBu)E(OtBu)K(Boc)K(Boc)FF。
(2)制备含磺基丙氨酸的肽树脂XE(OtBu)E(OtBu)K(Boc)K(Boc)FF
向含半胱氨酸的肽树脂C(StBu)E(OtBu)E(OtBu)K(Boc)K(Boc)FF中加入10mmol的氧化剂二氧化锰，冰浴下反应60min，反应完毕后，过滤。然后加入DMF洗涤树脂六遍，再加入DCM洗涤树脂3遍，过滤。
(3)制备含磺基丙氨酸的多肽XEEKKFF
向上述保护的含磺基丙氨酸的肽树脂复合物中加入10ml脱保护试剂(按体积百分含量计，TFA:水:thioansole:EDT＝87.5％:5％:2.5％:5％)，40℃下反应4h。反应结束后，过滤，加入乙醚将滤液沉淀。干燥后称量得到0.54g粗品，产率56.0％。取粗品进行质谱分析和高效液相分析，分子量为978.6，纯度为53％。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (12)

  1. 含磺基丙氨酸的多肽的制备方法，其特征在于，包括以下步骤：以S-叔丁基巯基保护的半胱氨酸为原料之一，按照含磺基丙氨酸的多肽的氨基酸序列合成含S-叔丁基巯基保护的半胱氨酸的肽树脂，然后采用氧化剂将S-叔丁基巯基保护的半胱氨酸残基转化成磺基丙氨酸残基，所述氧化剂为锰氧化剂或过氧化酸类氧化剂。
  2. 根据权利要求1所述的制备方法，其特征在于，所述S-叔丁基巯基保护的半胱氨酸为Fmoc-Cys(StBu)-OH。
  3. 根据权利要求1或2所述的制备方法，其特征在于，所述锰氧化剂为二氧化锰。
  4. 根据权利要求1或2所述的制备方法，其特征在于，所述过氧化酸类氧化剂为氧甲酸、过氧乙酸、过氧苯甲酸、过氧三氟乙酸、过氧间氯苯甲酸或过氧对硝基苯甲酸。
  5. 根据权利要求1所述的制备方法，其特征在于，所述肽树脂中的树脂选自Wang树脂、Rink amide AM树脂、2-氯三苯甲基氯树脂。
  6. 根据权利要求1～5任一项所述的制备方法，其特征在于，所述氧化剂和树脂的物质的量的比例为5～10：1。
  7. 根据权利要求1～6任一项所述的制备方法，其特征在于，所述将S-叔丁基巯基保护的半胱氨酸残基转化成磺基丙氨酸残基的反应条件为-4℃～4℃，反应10min～60min。
  8. 根据权利要求1～7任一项所述的制备方法，其特征在于，所述采用氧化剂将S-叔丁基巯基保护的半胱氨酸残基转化成磺基丙氨酸残基后，肽树脂依次经DMF和DCM洗涤。
  9. 根据权利要求8所述的制备方法，其特征在于，肽树脂依次经DMF和DCM洗涤之后，还包括脱除树脂和其他侧链保护基的步骤。
  10. 根据权利要求9所述的制备方法，其特征在于，所述脱除树脂和其他侧链保护基的脱保护剂为重蒸水、三乙基硅烷、1,2二硫苏糖醇、苯酚、苯甲硫醚、碘化铵、三异丙基硅烷、苯甲醚和乙二硫醇中的至少一种 与三氟乙酸的混合液；所述的混合液中三氟乙酸的体积分数不小于80％。
  11. 根据权利要求10所述的制备方法，其特征在于，所述脱保护剂与树脂的体积质量比为10：1。
  12. 根据权利要求10或11所述的制备方法，其特征在于，所述脱保护的条件为0℃～60℃，反应1h～4h。
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