WO2015101176A1 - 一种环孢菌素的合成方法 - Google Patents

一种环孢菌素的合成方法 Download PDF

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WO2015101176A1
WO2015101176A1 PCT/CN2014/093969 CN2014093969W WO2015101176A1 WO 2015101176 A1 WO2015101176 A1 WO 2015101176A1 CN 2014093969 W CN2014093969 W CN 2014093969W WO 2015101176 A1 WO2015101176 A1 WO 2015101176A1
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meleu
ala
resin
cyclosporin
synthesizing
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PCT/CN2014/093969
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French (fr)
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粟武
房丽晶
武春雷
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深圳先进技术研究院
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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
    • 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
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • C07K7/645—Cyclosporins; Related peptides

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  • the invention belongs to the technical field of drug synthesis, and in particular relates to a method for synthesizing cyclosporin.
  • Cyclosporin (also known as cyclosporin A) is a group of cyclic undecapeptides discovered in the 1970s. Among them, cyclosporin A has been widely used as a highly effective immunosuppressant in organ transplantation in the world. The annual sales of the market has reached more than 10 billion US dollars. As a highly effective immunosuppressive agent, cyclosporin is widely used in organ transplantation. Its advent has caused a revolution in organ transplantation, which has improved the survival rate of cadaveric and live-derived kidney transplants from 53% and 78% respectively. By 80% and 90%, the 5-year survival rate of liver transplant patients has increased from 20% to 70%, and operations such as cardiopulmonary transplantation have been widely carried out.
  • cyclosporin In addition to immunosuppressive effects, cyclosporin also has a series of other biological activities, such as treatment of autoimmune diseases such as lupus erythematosus and psoriasis, inhibition of HIV-1 virus, and reversal of multidrug resistance of tumors.
  • cyclosporin contains a variety of N-methylated amino acids, it is difficult to synthesize with a general polypeptide condensation reagent.
  • the production process of cyclosporin is carried out by microbial fermentation, but only a few can be obtained by microbial fermentation.
  • Several cyclosporins make it difficult to construct a large library of polypeptide compounds required for drug screening.
  • the American chemist Danny Schiffsky team attempted to synthesize cyclosporin A in liquid phase conditions by isonitrile condensation reaction.
  • this method requires a plurality of condensation reagents, and the condensation is successful under microwave heating conditions.
  • the preparation process is harsh and difficult, and thus it is difficult to popularize the rapid preparation of the complete sequence of cyclosporin.
  • the object of the present invention is to provide a method for synthesizing cyclosporin, which aims to solve the prior art synthesis. Cyclosporin is harsh and difficult, and the problem of solid phase synthesis cannot be achieved.
  • the present invention is achieved by a method for synthesizing cyclosporin, comprising the steps of:
  • the method for synthesizing cyclosporin adopts triphosgene as a condensation reagent, and can directly convert the carboxyl group of the amino acid into a highly active acid chloride at room temperature, thereby enabling the N-methylated amino acid of cyclosporin to be
  • the amino group and the carboxyl group are condensed into an amide bond under very mild conditions, thereby realizing the complete sequence of solid phase synthesis of cyclosporin; meanwhile, the present invention uses an anthracene resin as a carrier resin for solid phase synthesis of cyclosporin, the resin a special linking functional group, such that the synthesized H 2 N-Ala-MeLeu-Val-MeLeu-MeGly-Abu-MeBmt-MeVal-MeLeu-MeLeu-D-Ala-indene resin can be cut while being in the oxidation activation treatment step
  • the amino group and the carboxyl group which are free at both ends of the polypeptide are directly
  • FIG. 1 is a schematic flow chart of a cyclosporin synthesis method according to an embodiment of the present invention
  • FIG. 2 is a process flow diagram of solid phase synthesis of cyclosporin provided by an embodiment of the present invention.
  • NBS N-bromosuccinimide
  • DIEA N,N'-diisopropylethylamine.
  • the embodiment of the invention provides a method for synthesizing cyclosporin, comprising the following steps, as shown in FIG. 1:
  • the oxime resin is a ruthenium-based carrier resin
  • the ruthenium resin is used as a solid phase synthesis carrier resin in the embodiment of the invention, and on the one hand, provides the active group required for solid phase synthesis of the polypeptide.
  • linear peptide H 2 N-Ala-MeLeu-Val-MeLeu-MeGly-Abu-MeBmt-MeVal-MeLeu-MeLeu-D-Ala-indene resin obtained by coupling with an anthracene resin is used in the following In the step of cleavage and cleavage, the amino group and the carboxyl group which are free at both ends of the polypeptide can be directly connected to form a cyclic peptide, thereby performing the cutting and ring closure reaction in one step.
  • the selection of the oxime resin is not limited, and the oxime resins commonly used in the art are all within the protection scope of the embodiments of the present invention.
  • the oxime resin may be selected from a benzoquinone resin.
  • the degree of substitution of the oxime resin has a certain influence on the coupling rate of the amino acid or amino acid fragment of the cyclosporin.
  • the substitution degree of the oxime resin is too high, the density of the reactive group on the resin is too large, and as the coupling occurs, the peptide chain of the polypeptide gradually increases, and therefore, the steric hindrance thereof is increased.
  • the interrupted peptide chain will be cleaved together with cyclosporin to become an impurity of the target product cyclosporin, and the types of impurities are various, which increases the difficulty of purification of cyclosporin;
  • the degree of substitution of the enamel resin in the examples of the present invention is 0.2 to 1.0 mmol/g.
  • the degree of substitution of the oxime resin is from 0.2 to 0.6 mmol/g.
  • the above-mentioned resin is subjected to a swelling treatment which is carried out using a dichloromethane reagent.
  • the swelling treatment method is not limited, and the methods commonly used in the art are all within the scope of protection of the embodiments of the present invention.
  • the oxime resin In order to avoid the chemical reaction failure of the above-mentioned oxime resin before the amino acid or amino acid fragment is coupled, the oxime resin should be protected by an Fmoc protecting group.
  • step S02 in the step of solid phase synthesis of H 2 N-Ala-MeLeu-Val-MeLeu-MeGly-Abu-MeBmt-MeVal-MeLeu-MeLeu-D-Ala-indene resin, in order to save production cost,
  • the Fmoc group-protected oxime resin can be coupled to the Fmoc group-protected D-Ala, MeLeu, Meleu, MeVal, MeBmt, Abu, MeGly, MeLeu, Val, Meleu, Ala linearly by coupling one by one. Peptide.
  • the synthetic amino acid fragment satisfying the cyclosporin sequence may be subjected to Fmoc protection according to actual needs, and then the Fmoc-protected amino acid fragment may be sequentially coupled to the Fmoc group-protected oxime resin.
  • the combination form of the above amino acid fragments and the number thereof are not limited, and only the amino acid sequence of cyclosporin is required.
  • each amino acid is coupled Before the amino acid fragment, the reactive group or the partially coupled amino acid peptide chain attached to the resin is subjected to a treatment for removing the Fmoc protecting group.
  • the Fmoc protecting group is removed.
  • the cyclosporin to be synthesized contains seven N-methylated amino acids
  • the general polypeptide condensation reagent is difficult to synthesize, and the liquid phase synthesis is difficult, and the polypeptide condensation reagent requires many types. Solid phase synthesis is even more difficult to achieve. Therefore, the synthesis of cyclosporin is stagnant in the microbial fermentation process. In view of this, it has been invented through repeated experiments and experiments to use triphosgene used as a liquid reaction medium as a condensation reagent for solid phase synthesis of the polypeptide of the present invention.
  • triphosgene can directly convert the carboxyl group of the amino acid into a highly active acid chloride at room temperature, so that the N-methylated amino acid of the cyclosporin can shrink the amino group and the carboxyl group under very mild conditions.
  • the amide bond is synthesized so that the full phase of the solid phase synthesis of cyclosporin can be achieved.
  • the method of coupling the amino acid fragment or the single amino acid one by one on the Fmoc group-protected oxime resin is not limited, and the polypeptide solid phase synthesis method commonly used in the art is suitable for the embodiment of the present invention.
  • the above coupling reaction may be carried out by adding the Fmoc group-protected hydrazine resin to a solid phase reaction column, removing the Fmoc protecting group by using an Fmoc removal reagent, and then washing with a washing reagent to wash the reagent. Washing agents commonly used in the art, such as DMF, DCM, etc., may be employed.
  • the first reagent with a molar mass ratio of (1-3):1 to the ruthenium resin and a molar mass ratio of the ruthenium resin of (2-6):1 to the first amino acid or amino acid fragment with an Fmoc protecting group will be After removing the protecting group Fmoc from the above amino acid or amino acid fragment, it is dissolved in DIEA with triphosgene, added to a solid phase reaction column, and reacted at room temperature for 0.5-5 hours.
  • the coupling reaction time can be adjusted according to the actual situation, such as monitoring the reaction end point by the ninhydrin method until the resin is colorless and the reaction is terminated.
  • amino acid or amino acid fragment of cyclosporin was sequentially coupled in the same manner until linear H 2 N-Ala-MeLeu-Val-MeLeu-MeGly-Abu-MeBmt-MeVal-MeLeu-MeLeu-D-Ala-indene resin was obtained. .
  • the oxidation activation is performed during the oxidation activation treatment of the H 2 N-Ala-MeLeu-Val-MeLeu-MeGly-Abu-MeBmt-MeVal-MeLeu-MeLeu-D-Ala-indene resin.
  • the treated reagent is an organic solution of NBS, and the oxidation activation treatment method is: firstly oxidizing with NBS for 1-30 min, and then performing closed-loop cutting treatment with an organic solvent for 12-96 h.
  • the organic solvent is at least one of DCM and DMF.
  • the organic solvent is DCM.
  • the carrier resin for solid phase synthesis of the polypeptide is an oxime resin
  • the oxime resin can be detached from the peptide chain by cutting treatment, and on the other hand, the enamel resin is detached.
  • the latter linear peptide can form an end-loop of the amino group and the carboxyl group which are free at both ends of the polypeptide in the above oxidation activation system to form a cyclic peptide, thereby achieving automatic ring closure, thereby obtaining cyclosporin.
  • the synthesized cyclosporin can also be purified and lyophilized to obtain a cyclosporin powder injection having high purity and suitable for preservation.
  • the purification is purified by high performance liquid chromatography.
  • the high performance liquid chromatography is not limited.
  • the purification is purified by reverse-phase high performance liquid chromatography, and the column of the reversed-phase high performance liquid chromatography can be a commonly used column such as a C18 column or an amino column. , cyano column, etc.
  • the method for synthesizing cyclosporin adopts triphosgene (BTC) as a novel polypeptide synthesis condensation reagent, and develops a new process for synthesizing the complete sequence of cyclosporin by solid phase polypeptide synthesis technology.
  • BTC triphosgene
  • the triphosgene can directly convert the carboxyl group of the amino acid into a highly active acid chloride at room temperature, thereby being capable of condensing the amino group and the carboxyl group into an amide bond under very mild conditions, and further synthesizing the solid phase into the cyclosporine.
  • the full sequence of bacteriocin is possible.
  • the present invention uses a ruthenium resin as a solid phase synthetic carrier resin, and can directly form the cyclic peptide at the end of the solid phase synthesis by directly connecting the amino group and the carboxyl end of the polypeptide to form a cyclic peptide, thereby avoiding the polycondensation of the liquid phase in the process of condensation synthesis.
  • the reaction increases the purity and yield of the cyclosporin.
  • the synthesis condition of the process is mild, and only a polypeptide synthesis condensation reagent is needed to synthesize the complete sequence of cyclosporin by solid phase polypeptide synthesis technology at room temperature, and the number of by-products is small, and the synthesis step is simple, and the cyclospores are rapidly and economically constructed.
  • the embodiment of the present invention saves the production cost while improving the purity of the cyclosporin by selecting a suitable substitute resin, and purifies the polysporin by high performance liquid chromatography, thereby improving the purification efficiency and Yield.
  • N-bromosuccinimide N-bromosuccinimide
  • N-bromosuccinimide N-bromosuccinimide

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Abstract

提供了一种环孢菌素的合成方法。该方法包括下述步骤：提供Fmoc基团保护的肼树脂；采用三光气作为缩合试剂，固相合成 H 2N-Ala-MeLeu-Val-MeLeu-MeGly-Abu-MeBmt-MeVal-MeLeu-MeLeu-D-Ala-肼树脂；将所述 H 2N-Ala-MeLeu-Val-MeLeu-MeGly-Abu-MeBmt-MeVal-MeLeu-MeLeu-D-Ala-肼树脂进行氧化活化处理，得到环孢菌素。

Description

一种环孢菌素的合成方法 技术领域
本发明属于药物合成技术领域，尤其涉及一种环孢菌素的合成方法。
背景技术
环孢菌素(又名环孢菌素A)是20世纪70年代发现的一组环状十一肽物质，其中的环孢菌素A已作为高效免疫抑制剂广泛应用于器官移植，在世界市场的年销售额已达到l0亿美元以上。环孢菌素作为高效免疫抑制剂广泛应用于器官移植术，它的问世引起了器官移植领域的一场革命，使尸体来源和活体来源肾移植一年的存活率分别从53％和78％提高到80％和90％，使肝移植患者5年的存活率从原来的20％提高到70％，并使心肺联合移植等手术得以广泛开展。除免疫抑制作用外，环孢菌素还有一系列其它生物活性，如治疗红斑狼疮、牛皮癣等自身免疫系统疾病，抑制HIV-1病毒，逆转肿瘤多药耐药等。
由于环孢菌素中含有多种N-甲基化氨基酸，难以用通用的多肽缩合试剂合成，目前环孢菌素的生产工艺均采用微生物发酵法，但是由微生物发酵法制备生产只能得到少数几种环孢菌素，难以构建药物筛选所需的大规模多肽化合物库。美国化学家丹尼雪夫斯基课题组在2010年尝试以异腈缩合反应在液相条件下合成环孢菌素A全序列。但是这种方法需要多种缩合试剂，并且在微波加热的条件下才能缩合成功，该制备工艺苛刻、难度高，因此难以推广应用于快速制备环孢菌素全序列。而目前还没有用固相多肽合成技术合成环孢菌素全序列的报道。
发明内容
本发明的目的在于提供一种环孢菌素的合成方法，旨在解决现有技术合成 环孢菌素条件苛刻、难度高，且无法实现固相合成的问题。
本发明是这样实现的，一种环孢菌素的合成方法，包括以下步骤：
(1)提供Fmoc基团保护的肼树脂；
(2)采用三光气作为缩合试剂，固相合成H2N-Ala-MeLeu-Val-MeLeu-MeGly-Abu-MeBmt-MeVal-MeLeu-MeLeu-D-Ala-肼树脂；
(3)将所述H2N-Ala-MeLeu-Val-MeLeu-MeGly-Abu-MeBmt-MeVal-MeLeu-MeLeu-D-Ala-肼树脂进行氧化活化处理，得到环孢菌素。
本发明提供的环孢菌素的合成方法，采用三光气作为缩合试剂，能在室温条件下将氨基酸的羧基直接转变为高活性的酰氯，从而使得环孢菌素的N-甲基化氨基酸能够在非常温和的条件下将氨基和羧基缩合成酰胺键，从而使固相合成环孢菌素全序列得以实现；同时，本发明采用肼树脂作为环孢菌素固相合成的载体树脂，该树脂的特殊连接官能团，使得合成的H2N-Ala-MeLeu-Val-MeLeu-MeGly-Abu-MeBmt-MeVal-MeLeu-MeLeu-D-Ala-肼树脂在氧化活化处理步骤中，可在切割的同时、直接将多肽两端游离的氨基和羧基首尾相连形成环肽，从而一步实现切割和闭环反应，简化了操作程序，同时避免了液相缩合成环过程中的多聚缩合副反应。
附图说明
图1是本发明实施例提供的环孢菌素合成方法流程示意图；
图2是本发明实施例提供的固相合成环孢菌素的工艺流程图。
具体实施方式
为了使本发明要解决的技术问题、技术方案及有益效果更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
本发明实施例使用的英文缩写的含义如下：
BCT：三光气；
DCM：二氯甲烷；
Fmoc：2,2,4,6,7-五甲基苯并呋喃；
NBS：N-溴代丁二酰亚胺；
DMF：N,N-二甲基甲酰胺；
DIEA：N,N’-二异丙基乙胺。
本发明实施例提供了一种环孢菌素的合成方法，包括以下步骤，如图1所示：
S01.提供Fmoc基团保护的肼树脂；
S02.线形肽的合成：采用三光气作为缩合试剂，固相合成H2N-Ala-MeLeu-Val-MeLeu-MeGly-Abu-MeBmt-MeVal-MeLeu-MeLeu-D-Ala-肼树脂；
S03.环孢菌素的合成：将所述H2N-Ala-MeLeu-Val-MeLeu-MeGly-Abu-MeBmt-MeVal-MeLeu-MeLeu-D-Ala-肼树脂进行氧化活化处理，得到环孢菌素。
具体地，上述步骤S01中，所述肼树脂为含有肼基的载体树脂，本发明实施例以肼树脂作为固相合成的载体树脂，一方面，提供了多肽固相合成所需的活性基团，更重要的是，采用肼树脂偶联得到的线性肽H2N-Ala-MeLeu-Val-MeLeu-MeGly-Abu-MeBmt-MeVal-MeLeu-MeLeu-D-Ala-肼树脂，在进行下述步骤的裂解切割中，能直接将多肽两端游离的氨基和羧基首尾相连形成环肽，从而一步实现切割和闭环反应。
所述肼树脂的选择不受限制，本领域内常用的肼树脂均在本发明实施例的保护范围内。作为优选实施例，所述肼树脂可选用苯肼树脂。
上述步骤S01中，所述肼树脂的替代度对后期环孢菌素组成氨基酸或氨基酸片段的偶联率会有一定的影响。当肼树脂的替代度过高时，反应基团在树脂上的密度过大，随着偶联的发生，多肽肽链逐渐增长，因此，其空间位阻会越 来越大，这将增加后续氨基酸或氨基酸片段的偶联难度，导致部分肽链的偶联中断，从而降低了多肽合成的合成效率，浪费了原料，更重要的是，在后续的裂解切割过程中，这些被中断的肽链将连同环孢菌素一起切割下来，成为目的产物环孢菌素的杂质，且杂质类型多种多样，增加了环孢菌素的纯化难度；当肼树脂的替代度过低时，合成一定量的环孢菌素所需要的数值量将增加，从而提高了生产成本。为了同时解决上述问题，经过发明人反复研究发现，本发明实施例所述肼树脂的替代度为0.2～1.0mmol/g。作为进一步优选实施例，所述肼树脂的替代度为0.2～0.6mmol/g。
在进行步骤S02前，上述肼树脂需要进行溶胀处理，所述溶胀处理采用二氯甲烷试剂进行。溶胀处理方法不受限制，本领域常用方法均在本发明实施例保护范围内。
为了避免上述肼树脂在偶联氨基酸或氨基酸片段前，其反应基团发生化学反应失效，应采用Fmoc保护基团将所述肼树脂进行保护。
上述步骤S02中，所述固相合成H2N-Ala-MeLeu-Val-MeLeu-MeGly-Abu-MeBmt-MeVal-MeLeu-MeLeu-D-Ala-肼树脂的步骤中，为了节省生产成本，在所述Fmoc基团保护的肼树脂上可采用逐一偶联的方式依次偶联Fmoc基团保护的D-Ala、MeLeu、Meleu、MeVal、MeBmt、Abu、MeGly、MeLeu、Val、Meleu、Ala合成线性肽。当然，也可以根据实际需要，将合成得到的满足环孢菌素序列的氨基酸片段进行Fmoc保护后，再在所述Fmoc基团保护的肼树脂上依次偶联Fmoc保护的氨基酸片段。上述氨基酸片段的组合形式及其个数不受限制，只需符合环孢菌素的氨基酸序列即可。本发明实施例中，为了降低环孢菌素的合成成本，优选采用逐一偶联氨基酸的方式合成H2N-Ala-MeLeu-Val-MeLeu-MeGly-Abu-MeBmt-MeVal-MeLeu-MeLeu-D-Ala-肼树脂。本发明实施例中，上述固相合成H2N-Ala-MeLeu-Val-MeLeu-MeGly-Abu-MeBmt-MeVal-MeLeu-MeLeu-D-Ala-肼树脂的步骤中，在偶联每个氨基酸或氨基酸片段前，需将连在树脂上的反应 基团或已部分偶联的氨基酸肽链进行去除Fmoc保护基团的处理，本发明实施例中，所述Fmoc保护基团的去除试剂为含体积分数为20％的哌啶的DMF。所述处理方式不受限制，本领域常用的去除Fmoc保护基团的处理均能用于本发明实施例。
本发明实施例中，由于所要合成的环孢菌素中含有7个N-甲基化的氨基酸，通用的多肽缩合试剂难以合成，其液相合成难度巨大，且多肽缩合试剂需要的种类多，固相合成更是难以企及。因此，环孢菌素的合成停滞在微生物发酵法。有鉴于此，发明了经过反复的实验、摸索，将用作液相反应介质的三光气作为本发明实施例多肽固相合成的缩合试剂。本发明实施例中，三光气能在室温条件下将氨基酸的羧基直接转变为高活性的酰氯，从而使得环孢菌素的N-甲基化氨基酸能够在非常温和的条件下将氨基和羧基缩合成酰胺键，从而使固相合成环孢菌素全序列得以实现。
上述固相合成步骤中，在所述Fmoc基团保护的肼树脂上逐一偶联氨基酸片段或单个氨基酸的方法不受限制，本领域内常用的多肽固相合成方法适用于本发明实施例。作为具体实施例，上述偶联反应的方法可以为将所述Fmoc基团保护的肼树脂加入到固相反应柱中，采用Fmoc去除试剂脱除Fmoc保护基后，用洗涤试剂进行清洗，洗涤试剂可采用本领域常用的洗涤试剂，如DMF、DCM等。取与肼树脂摩尔质量比为(1-3)：1的偶合试剂三光气、与肼树脂摩尔质量比为(2-6)：1的第一个带Fmoc保护基的氨基酸或氨基酸片段，将上述氨基酸或氨基酸片段去除保护基团Fmoc后，与三光气溶于DIEA中，加入固相反应柱，于室温下反应0.5-5小时。所述偶联反应时间可以根据实际情况进行调节，如以茚三酮法监测反应终点，直至树脂呈无色时终止反应。采用同样的方法依次偶联环孢菌素的氨基酸或氨基酸片段，直至获得线性H2N-Ala-MeLeu-Val-MeLeu-MeGly-Abu-MeBmt-MeVal-MeLeu-MeLeu-D-Ala-肼树脂。
上述步骤S03中，将所述 H2N-Ala-MeLeu-Val-MeLeu-MeGly-Abu-MeBmt-MeVal-MeLeu-MeLeu-D-Ala-肼树脂进行氧化活化处理的过程中，所述氧化活化处理的试剂为NBS的有机溶液，所述氧化活化处理方法为：先采用NBS氧化1-30min后，再用有机溶剂进行闭环切割处理12-96h。作为优选实施例，所述有机溶剂为DCM、DMF中的至少一种，为了提高氧化活化处理效果，作为优选实施例，所述有机溶剂为DCM。
本发明实施例中，由于多肽固相合成的载体树脂为肼树脂，因此，在进行上述裂解切割的同时，一方面，肼树脂经过切割处理能从肽链中脱落，另一方面，脱落肼树脂后的线性肽能在上述氧化活化体系将多肽两端游离的氨基和羧基首尾相连形成环肽、实现自动闭环，从而得到环孢菌素。这样，经过上述处理，能够一步实现切割和闭环反应，简化了操作程序，同时避免了液相缩合成环过程中的多聚缩合副反应。
本发明实施例中，还可对合成得到的环孢菌素进行纯化、冻干处理，从而得到纯度高、适于保存的环孢菌素粉针剂。作为优选实施例，为了获得纯度高、收率高的环孢菌素样品，所述纯化采用高效液相色谱法纯化。所述高效液相色谱法不受限制。作为进一步优选实施例，为了进一步提高纯化效率，所述纯化采用反相高效液相色谱法纯化，所述反相高效液相色谱法的色谱柱可采用常用的色谱柱，如C18柱、氨基柱、氰基柱等。
本发明实施例提供的环孢菌素的合成方法，采用三光气(BTC)作为新型多肽合成缩合试剂，首次开发了以固相多肽合成技术合成环孢菌素全序列的新工艺。本发明实施例中，所述三光气能够在室温下将氨基酸的羧基直接转变为高活性的酰氯，因而能够在非常温和的条件下将氨基和羧基缩合成酰胺键，进一步使固相合成环孢菌素全序列成为可能。
其次，本发明以肼树脂作为固相合成载体树脂，可以在固相合成结束时直接将多肽的氨基和羧基端首尾相连而形成环肽，避免了液相缩合成环过程中的多聚缩合副反应，提高了环孢菌素的纯度和产率。
本工艺合成条件温和，只需要一种多肽合成缩合试剂就可在室温下以固相多肽合成技术合成环孢菌素全序列，且副产物少，合成步骤简单，是快速和经济地构建环孢菌素药物筛选化合物库、以及大规模生产环孢菌素的有效途径。
此外，本发明实施例通过选用合适替代度的肼树脂，在提高环孢菌素纯度的同时，节约了生产成本；且采用高效液相色谱法进行多孢菌素的纯化，提高了纯化效率和收率。
下面结合具体实施例进行说明。
实施例1 一种环孢菌素的合成方法
环孢菌素的合成：称量200毫克2,2,4,6,7-五甲基苯并呋喃(Fmoc)保护的肼树脂(Hydrazine Resin 0.15mmol/g)于手动固相多肽合成器中。依次用Fmoc保护的D-Ala，MeLeu，Meleu，MeVal，MeBmt，Abu，MeGly，MeLeu，Val，Meleu，Ala为基本氨基酸合成原料，以三光气(BCT)为多肽缩合剂，按照标准Fmoc策略合成化合物。用N-溴代丁二酰亚胺(NBS)氧化切割得到粗产物，经制备高效液相色谱纯化后冻干得到纯产物，总收率为18％。
实施例1的固相合成环孢菌素A的工艺流程图如图2所示。
实施例2 一种环孢菌素的合成方法
环孢菌素的合成：称量200毫克2,2,4,6,7-五甲基苯并呋喃(Fmoc)保护的肼树脂(Hydrazine Resin 0.45mmol/g)于手动固相多肽合成器中。依次以Fmoc保护的D-Ala，MeLeu，Meleu，MeVal，MeBmt，Abu，MeGly，MeLeu，Val，Meleu，Ala为基本氨基酸片段合成原料，以三光气(BCT)为多肽缩合剂，按照标准Fmoc策略合成化合物。用N-溴代丁二酰亚胺(NBS)氧化5分钟之后，室温下加入DCM切割72小时得到粗产物，经制备高效液相色谱纯化后冻干得到纯产物，总收率为17％。
实施例3 一种环孢菌素的合成方法
环孢菌素的合成：称量200毫克2,2,4,6,7-五甲基苯并呋喃(Fmoc)保护的肼树脂(Hydrazine Resin 0.8mmol/g)于手动固相多肽合成器中。依次Fmoc保 护的D-Ala，MeLeu，Meleu，MeVal，MeBmt，Abu，MeGly，MeLeu，Val，Meleu，Ala为基本氨基酸合成原料，以三光气(BCT)为多肽缩合剂，按照标准Fmoc策略合成化合物。用N-溴代丁二酰亚胺(NBS)氧化7分钟之后，室温下加入DCM切割48小时得到粗产物，经制备高效液相色谱纯化后冻干得到纯产物，总收率为12％。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种环孢菌素的合成方法，包括以下步骤：
    (1)提供Fmoc基团保护的肼树脂；
    (2)采用三光气作为缩合试剂，固相合成
    H2N-Ala-MeLeu-Val-MeLeu-MeGly-Abu-MeBmt-MeVal-MeLeu-MeLeu-D-Ala-肼树脂；
    (3)将所述
    H2N-Ala-MeLeu-Val-MeLeu-MeGly-Abu-MeBmt-MeVal-MeLeu-MeLeu-D-Ala-肼树脂进行氧化活化处理，得到环孢菌素。
  2. 如权利要求1所述的环孢菌素的合成方法，其特征在于，在步骤(2)所述固相合成
    H2N-Ala-MeLeu-Val-MeLeu-MeGly-Abu-MeBmt-MeVal-MeLeu-MeLeu-D-Ala-肼树脂的步骤中，在所述Fmoc基团保护的肼树脂上采用逐一偶联的方式依次偶联Fmoc基团保护的D-Ala、MeLeu、Meleu、MeVal、MeBmt、Abu、MeGly、MeLeu、Val、Meleu、Ala。
  3. 如权利要求1所述的环孢菌素的合成方法，其特征在于，在步骤(2)所述固相合成
    H2N-Ala-MeLeu-Val-MeLeu-MeGly-Abu-MeBmt-MeVal-MeLeu-MeLeu-D-Ala-肼树脂的步骤中，在所述Fmoc基团保护的肼树脂上依次偶联Fmoc保护的氨基酸片段。
  4. 如权利要求1～3任一所述的环孢菌素的合成方法，其特征在于，所述氧化活化处理方法为：先采用NBS氧化1-30min后，再用有机溶剂进行闭环切割处理12-96h。
  5. 如权利要求1～3任一所述的环孢菌素的合成方法，其特征在于，所述肼树脂为苯肼树脂；
    优选地，所述肼树脂的替代度为0.2～1.0mmol/g，更优选为0.2～0.6mmol/g；
    优选地，在进行步骤(2)之前，先将肼树脂进行溶胀处理，所述溶胀处理采用二氯甲烷试剂进行。
  6. 如权利要求2或3所述的环孢菌素的合成方法，其特征在于，在偶联每个氨基酸或每个氨基酸片段前，将连在树脂上的反应基团或已部分偶联的氨基酸肽链进行去除Fmoc保护基团的处理，所述Fmoc保护基团的去除试剂为含体积分数为20％的哌啶的DMF。
  7. 如权利要求4所述的环孢菌素的合成方法，其特征在于，所述有机溶剂为DCM、DMF中的至少一种；优选地，所述有机溶剂为DCM。
  8. 如权利要求1～3任一所述的环孢菌素的合成方法，其特征在于，还包括将合成得到的环孢菌素进行纯化、冻干处理。
  9. 如权利要求8所述的环孢菌素的合成方法，其特征在于，所述纯化采用高效液相色谱法纯化。
  10. 如权利要求8所述的环孢菌素的合成方法，其特征在于，所述纯化采用反相高效液相色谱法纯化。
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