WO2020103130A1 - 微波流动固相多肽合成方法及其系统 - Google Patents

微波流动固相多肽合成方法及其系统

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Publication number
WO2020103130A1
WO2020103130A1 PCT/CN2018/117193 CN2018117193W WO2020103130A1 WO 2020103130 A1 WO2020103130 A1 WO 2020103130A1 CN 2018117193 W CN2018117193 W CN 2018117193W WO 2020103130 A1 WO2020103130 A1 WO 2020103130A1
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amino acid
microwave
assisted
cycle
reaction
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PCT/CN2018/117193
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English (en)
French (fr)
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刘伟
贾豪俊
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刘伟
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Priority to CN201880099599.5A priority Critical patent/CN113056328B/zh
Priority to PCT/CN2018/117193 priority patent/WO2020103130A1/zh
Publication of WO2020103130A1 publication Critical patent/WO2020103130A1/zh

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/08—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/08—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor
    • B01J19/12—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor employing electromagnetic waves
    • 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
    • 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 relates to the technical field of polypeptide synthesis, in particular to a microwave solid-phase polypeptide synthesis method and system.
  • Polypeptide is a compound composed of a variety of amino acids combined by peptide bonds in a certain order. Its molecular structure is between amino acids and proteins. It is a biologically active substance that is related to various cellular functions in an organism.
  • Chinese patent application 201710135872.2 discloses a method for batch solid phase synthesis of peptides when preparing different derived peptides. This method uses a multi-channel peptide solid phase synthesis device to synthesize each peptide according to the coupling solution required for the next reaction The tubes are grouped, and the peptide synthesis tubes with the same coupling solution are grouped into the same group, thereby providing a basis for achieving batch reactions.
  • US Patent Document US2017 / 0266637A1 discloses a microwave-assisted peptide synthesis equipment and method, which uses microwave-assisted solid-phase peptide synthesis, in which new amino acids are added sequentially according to the peptide sequence in the same reactor, and repeated under microwave assistance Steps such as deprotection, activation, coupling and washing further improve the synthesis efficiency.
  • the present invention provides a microwave-assisted mobile solid-phase polypeptide synthesis method and system, which in principle establishes basic conditions for optimizing the conditions of amino acid reactions, which can reduce the synthesis of polypeptides The risk of failure, easy to control the quality of the production process, while saving synthesis time, increase speed and capacity.
  • the present invention provides a microwave-assisted mobile solid-phase polypeptide synthesis method, which includes at least two amino acid linking cycles.
  • the amino acid linking cycle the amino acid molecule and the reactive group on the resin carrier or the previously linked amino acid Molecular coupling; the at least two amino acid connection cycles are assisted by microwaves in a microwave-assisted environment, and each amino acid connection cycle includes an amino acid coupling step and a deprotection step; at least one amino acid connection in the at least two amino acid connection cycles
  • the circulating microwave-assisted environment is different from the microwave-assisted environment of other amino acid linking cycles.
  • the microwave-assisted environment may be determined by parameters including microwave radiation intensity and microwave radiation time; in the at least two amino acid linking cycles, each amino acid linking cycle The microwave-assisted environment remains unchanged, and the amino acid-containing resin product obtained in the previous amino acid linking cycle is transferred to another microwave-assisted environment for the next amino acid linking cycle; each of the at least two amino acid linking cycles is completing a batch After the second amino acid ligation cycle, continue to repeat the next batch of amino acid ligation cycles under the same microwave-assisted environment.
  • the above-mentioned amino acid coupling step and / or deprotection step can be assisted by microwave in a microwave assisted environment.
  • the reaction conditions of the reaction step included in at least one amino acid connection cycle of the above-mentioned at least two amino acid connection cycles are different from the reaction conditions of the reaction steps included in other amino acid connection cycles, and the reaction conditions of the reaction step may include raw materials
  • the parameters of the feed ratio, reaction time and reaction system temperature are determined.
  • the microwave-assisted environment of each amino acid linking cycle can be kept unchanged, and the reaction conditions of the amino acid coupling step and / or deprotection step can be kept unchanged.
  • the obtained amino acid-bearing resin product was transferred to another microwave-assisted environment for the next amino acid linking cycle.
  • Each of the above at least two amino acid linking cycles can continue in a microwave-assisted environment that remains unchanged after completing a batch of amino acid linking cycles, and can react in the amino acid coupling step and / or deprotection step When the conditions remain unchanged, the next batch of amino acid ligation cycles is repeated.
  • amino acids can be classified according to the degree of coupling difficulty. For each type of amino acid with different coupling difficulty, different microwave-assisted environments and reaction conditions can be set for the amino acid connection cycle.
  • amino acids According to the structure and properties of amino acids, amino acids can be divided into three categories according to the relative ease of coupling:
  • the first type of amino acids including glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, methionine, and Asparagine, glutamine, threonine, aspartic acid, glutamic acid and lysine;
  • the third type of amino acids including sterically hindered nitrogen methyl amino acids and ⁇ -methyl amino acids;
  • the coupling difficulty order of the three types of amino acids is: third type amino acids> second type amino acids> first type amino acids.
  • each amino acid can be further classified as a class. Furthermore, according to the difficulty of coupling between various types of amino acids and the amino acids to be connected, various types of amino acids can be further classified according to different amino acid connection objects.
  • the amino acid-bearing resin product obtained by the previous amino acid linking cycle When the amino acid-bearing resin product obtained by the previous amino acid linking cycle is moved into another microwave-assisted environment, the amino acid-bearing resin product can be moved into another microwave-assisted environment together with the container holding it.
  • Another aspect of the present invention also provides a microwave-assisted mobile solid-phase polypeptide synthesis system, which includes: at least two amino acid linking units for providing reaction accommodation spaces for different amino acid coupling steps and deprotection steps; at least two A microwave-assisted providing unit for respectively providing microwave assistance for different amino acid linking cycles; at least two reaction condition control units for separately controlling the reaction conditions of different amino acid linking cycles; the at least two amino acid connecting units are connected in series, the previous one The amino acid-bearing resin product obtained by the amino acid linking cycle in the amino acid linking unit moves to the next amino acid linking unit for the next amino acid linking cycle.
  • the microwave assisting providing unit provides microwave assistance for the amino acid coupling step and / or deprotection step in the amino acid linking cycle, while the reaction condition control unit performs reaction condition control on the amino acid coupling step and / or deprotection step in the amino acid linking cycle.
  • the above-mentioned microwave-assisted providing unit can provide microwave assistance for the amino acid connection cycle with the set microwave radiation intensity and microwave radiation time; and the reaction condition control unit can control each reaction with the set raw material feeding ratio, reaction time, and reaction system temperature Step reaction conditions.
  • the microwave-assisted mobile solid-phase polypeptide synthesis system may further include a container for directly containing an amino acid ligation reaction reagent and a reaction product, which is used for the band obtained by the circulative connection with the amino acid The amino acid resin products are moved into the next amino acid linking unit together for the next amino acid linking cycle.
  • the microwave-assisted flow solid phase peptide synthesis method allows resin-containing amino acids to be queued and coupled to connect new amino acids in the flow.
  • This new flow synthesis method makes the resins with amino acids continuously transfer and line up. After the first batch of resins takes the first step to complete the coupling in the first module, the second batch of resins crosses into the first module again, with resin amino acids The second batch is only one working module step slower than the first batch.
  • the resin-containing amino acids are always transferred and queued forward, and each forward step is added with an amino acid. This is also the biggest advantage of the present invention that distinguishes it from existing methods such as performing all reactions in the same reactor.
  • the present invention can also standardize the process and optimize the conditions for each step of the reaction.
  • This standard modular sequencing can more easily achieve the optimal synthesis results, achieve faster speed, higher yield, better activity, and save Time and environmental protection, and greatly reduce the probability of large-scale errors, that is, reduce the incidence of errors, and fundamentally increase productivity, at least to improve the synthesis of peptides to achieve clinical-grade dosage.
  • FIG. 1 is a schematic diagram illustrating the principle of a mobile solid-phase polypeptide synthesis method according to an embodiment of the present invention.
  • FIG. 2 is a schematic block diagram illustrating a microwave flowing solid-phase polypeptide synthesis system according to an embodiment of the present invention.
  • FIG. 3 is a schematic diagram illustrating a solid phase peptide synthesis process using a microwave flowing solid phase peptide synthesis system according to an embodiment of the present invention.
  • FIG. 4 is an HPLC chart of an exemplary crude peptide product synthesized by a microwave flow solid phase polypeptide synthesis method according to an embodiment of the present invention.
  • FIG. 5 is an ESI-MS spectrum of an exemplary crude peptide product synthesized by a microwave flow solid-phase polypeptide synthesis method according to an embodiment of the present invention.
  • Amino acids are compounds in which hydrogen atoms on the carbon atoms of carboxylic acids are replaced with amino groups.
  • Amino acid molecules contain two functional groups, amino and carboxyl. Similar to hydroxy acids, amino acids can be divided into ⁇ -, ⁇ -, ⁇ -... w-amino acids according to the different positions of the amino groups connected to the carbon chain, and most of the amino acids constituting proteins are ⁇ -amino acids. There are twenty kinds of amino acids that make up human protein.
  • the twenty natural amino acids are glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, Twenty kinds of serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine, They are the basic units that make up proteins.
  • the microwave flow solid phase polypeptide synthesis of the present invention first optimizes and standardizes the optimal process conditions for specific amino acid pairings, optimizes and fixes the coupling process conditions for specific amino acid pairings through modularization, and realizes the polypeptides by amino acid resin transfer technology According to the amino acid sequence, different modules are arranged and combined into a flow synthesis production line to realize the flow sequencing synthesis of modular sequencing design, thereby improving the high-purity and high-activity capacity of large-scale synthesis, and ensuring batch repetition Sex and unity.
  • the applicant of the present invention classifies and modularizes amino acids according to the structure and properties of various amino acids, such as the particularity and difficulty of pairing coupling of amino acids in polypeptide synthesis, screening and Optimize the optimal coupling and deprotection conditions, standardize and modularize each amino acid connection cycle.
  • amino acids that are more prone to side reactions such as cysteine, arginine, and histidine in solid-phase peptide synthesis, such as cysteine, arginine, and histidine. Based on this, the applicant of the present invention classifies amino acids into the following three categories:
  • the first category glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, methionine, asparagine, glutamine Amide, threonine, aspartic acid, glutamic acid, lysine.
  • the second category cysteine, arginine, histidine.
  • the third category unnatural amino acids, mainly including sterically hindered nitrogen methyl amino acids and ⁇ -methyl amino acids.
  • a First amino acid Second amino acid The third amino acid Microwave coupling Medium radiation ⁇ 75 °C Low radiation ⁇ 35 °C High radiation ⁇ 95 °C Coupling time 2min 6min 4min Main solvent DMF DMF DMF Coupling agent DIC / Oxyma DIC / Oxyma DIC / Oxyma Resin / amino acid feed ratio 1: 5 1: 5 1: 8
  • a First amino acid Second amino acid The third amino acid Deprotection temperature Medium radiation ⁇ 75 °C Low radiation ⁇ 35 °C Medium radiation ⁇ 95 °C Deprotection time 2min 3min 1min Deprotection reagent 20% piperidine / DMF 20% piperidine / DMF 20% piperidine / DMF
  • the classification of the above-mentioned amino acids in this specification and the coupling conditions and deprotection conditions in the above table are only exemplary examples, not limitations.
  • the optimal coupling and deprotection conditions are set in different amino acid connection modules as above, and the resin with amino acids is continuously transferred and queued to flow in several serially connected amino acid connection modules, in order Each type of amino acid linking cycle is performed.
  • each amino acid linking module continuously performs the same amino acid linking cycle, and the entire amino acid linking cycle keeps running uninterrupted. That is to say, after the first batch of resin took the first step to complete the coupling in the first module, the second batch of resin stepped into the first module again, the second batch with resin amino acid was only one working module step slower than the first batch In this way, the amino acid with resin has been transferred and lined up, and every step forward, an amino acid is added.
  • each step of peptide synthesis can be standardized and optimized, which not only greatly reduces the risk of peptide synthesis failure, but also establishes basic conditions for optimizing the amino acid reaction in principle, and is easier to carry out
  • the quality control of the production process reduces the incidence of errors, while also saving synthesis time, improving synthesis efficiency, and fundamentally increasing speed and productivity.
  • FIG. 1 is a schematic diagram illustrating the principle of a mobile solid-phase polypeptide synthesis method according to an embodiment of the present invention.
  • "Library" in Figure 1 represents the amino acid library, library A, library Q, library Y, library F represent alanine library, glutamine library, tyrosine library, phenylalanine library; W represents resin; WG Represents a glycine-linked resin.
  • W-G resin flows continuously along the peptide synthesis route, and in each amino acid coupling module, amino acid coupling is performed according to the set optimal reaction conditions. After completing an amino acid connection, the amino acid-bearing resin continues to enter the next amino acid coupling module to connect the next amino acid.
  • WGA After WG resin is connected to alanine in the amino acid coupling module 1, WGA enters the next amino acid connection cycle, that is, the glutamine connection is performed in the amino acid coupling module 2, and this continues continuously. 4. Connect tyrosine and phenylalanine in sequence to obtain the WGAQYF chain, and then use the conventional method to cut to obtain the GAQYF peptide chain.
  • each amino acid coupling module completes the amino acid connection of a batch of resin, as soon as the amino acid resin is removed from the module, the next batch of the same amino acid is added from the amino acid library to perform the next batch of amino acid connection
  • the same reaction conditions are maintained and the same coupling reaction is performed.
  • the amino acid coupling module 1 always connects alanine under the same microwave-assisted environment and constant reaction conditions, and the amino acid coupling module 2 always under the same microwave-assisted environment and the same Glutamine linkage is carried out under reaction conditions.
  • microwave-assisted solid-phase peptide synthesis is a well-known technology in the art, and there are currently relatively mature microwave peptide synthesizers that are commercially sold and used in large-scale applications.
  • existing microwave peptide synthesizers use microwaves to realize all amino acid connections in the same reactor (microwave radiation chamber), so the synthesis efficiency is low, the productivity is low, and the risk of synthesis errors is high.
  • the invention creatively connects multiple microwave radiation chambers in series to realize different amino acid connection cycles in different coupling modules (reactors), and the coupling conditions and deprotection conditions of various types of amino acids are optimized and kept unchanged.
  • each amino acid coupling module continuously and stably connects the same type of amino acids, thereby achieving the effects of high efficiency, high productivity, and high synthesis accuracy.
  • each amino acid linking unit 41, 42, 43 completes a class of amino acid linking cycles, including an amino acid coupling step and a deprotection step after coupling.
  • the amino acid coupling step includes introducing the corresponding activated amino acid from the amino acid activation unit 20, then under the action and control of the microwave assisted supply unit 31 and the reaction condition control unit (not shown in the figure) Under the protection of nitrogen, the amino acid coupling reaction was carried out.
  • the microwave assistance providing unit 31 can provide microwave assistance for the amino acid connection cycle with the microwave radiation intensity and microwave radiation time parameters set as required.
  • the reaction condition control unit can control the reaction conditions of each reaction step with the raw material feeding ratio, reaction time, and reaction system temperature set as required.
  • the structures, functions, and operation methods of the microwave-assisted providing unit 31 and the reaction condition control unit belong to well-known technologies in the art, and thus will not be described in detail.
  • the microwave auxiliary providing unit 31 and the reaction condition control unit may be independent unit modules, or they may be integrated together and controlled by the same control unit.
  • the amino acid activation unit 20 shown in FIG. 2 is schematically connected to the amino acid linking units 41, 42, 43.
  • the amino acid activation unit 20 may include a plurality of amino acid activation subunits, each of which is used for one amino acid, and can be controlled as needed
  • the specific amino acid in the specific amino acid activator unit is added to the specific amino acid linking unit at a specific time, and the amount and speed of addition of the amino acid can be controlled.
  • the structure, function and operation method of the above-mentioned amino acid activation unit are existing technologies in the art, as disclosed in the US Patent Document US2017 / 0266637A1, and will not be repeated here.
  • the deprotecting agent providing unit 10 shown in FIG. 2 is schematically connected to the amino acid linking units 41, 42, 43, and is used to provide a necessary deprotecting agent, such as DMF, for the deprotecting step after the amino acid coupling step.
  • the deprotecting agent in the deprotecting agent providing unit 10 can be controlled to add a specific amino acid linking unit at a specific time as needed, and the amount and rate of addition of the deprotecting agent can be controlled.
  • the deprotection step can also be performed under the protection and function of the microwave-assisted supply unit 31 and the reaction condition control unit, and under the protection of nitrogen provided by the nitrogen source 60.
  • the amino acid connecting units 41, 42, 43 ..., n are sequentially connected in series, and are respectively used to provide reaction accommodation spaces for different amino acid coupling steps and deprotection steps. After completing the amino acid connection of a batch of resins in each amino acid linking unit 41, 42, 43 ..., n, after washing and filtering the resin with amino acids, it is moved to the next amino acid linking unit 42, 43 ..., n + 1. Carry out the next amino acid linking cycle, and continue until all the amino acid links are completed to form the desired polypeptide sequence. In addition, once the amino acid-containing resin is removed from a certain amino acid linking unit, the next batch of the same amino acid is added from the corresponding amino acid library of the unit. The same reaction conditions are maintained in the coupling module, and the same coupling and deprotection reactions are carried out. The next batch of amino acids is connected.
  • the above amino acid linking units 41, 42, 43 ..., n are only schematic illustrations, after completing a batch of amino acid linking, the resin with amino acids can be directly moved out to the next amino acid linking unit, or in the amino acid linking unit A dedicated container (not shown in the figure) is provided, and the container is used to transfer the amino acid-bearing resin together with the container to the next amino acid connecting unit to perform the next batch of amino acid connection.
  • the waste generated in each step of the reaction can be filtered through a filter and discharged to the waste liquid collection unit 50 through a connecting pipe.
  • FIG. 3 is a schematic diagram illustrating a solid phase peptide synthesis process using a microwave flowing solid phase peptide synthesis system according to an embodiment of the present invention.
  • W-G resin flows continuously along the peptide synthesis route, and in each amino acid coupling module, amino acid coupling is performed with the corresponding activated amino acid according to the set optimal reaction conditions. After completing an amino acid connection, the amino acid-bearing resin continues to enter the next amino acid coupling module to connect the next amino acid.
  • the WGA is deprotected, and then enters the next amino acid connection cycle, that is, the coupling module 2 is connected with glutamine, and then the WGA
  • the deprotection is carried out continuously, and tyrosine and phenylalanine are sequentially connected in the coupling modules 3 and 4, respectively, to obtain a WGAQYF chain, which is then cut by a conventional method to obtain a GAQYF peptide chain.
  • the microwave mobile solid-phase peptide synthesis according to the present invention is based on the standardization, modularization, and fluidization coupling sequencing on the basis of microwave chemistry, adding new amino acids through sequencing flow of resin amino acids in multiple modules, and arranging them reasonably and efficiently through mathematical sequencing combinations
  • the full-coupling (coupling) process of amino acids realizes continuous flow of chemical synthesis of peptides and the realization of multi-module fluidized large-scale synthesis capabilities, which solves the difficulties of large-scale synthetic production technology in traditional solid-phase peptide synthesis.
  • the following polypeptide sequences were synthesized using the conventional microwave solid phase peptide synthesis method and the mobile microwave peptide synthesis method of the present invention: Fmoc-Ile-Pro-His-Gln-Ser-Phe-Phe (molecular weight: 1096).
  • the traditional microwave solid-phase peptide synthesis method uses an existing commercial synthesizer (model: 336X, produced by CSBio) to synthesize according to the conditions recommended by the manufacturer.
  • the experimental conditions of the mobile microwave peptide synthesis method of the present invention are as follows:
  • Microwave conditions The radiant power is about 1000W, which is automatically adjusted by temperature.
  • Irradiation time coupling each amino acid for 2-6 minutes, deprotecting each amino acid for 1-5 minutes.
  • Deprotection reaction conditions a) Deprotection reagent: 20% piperidine / DMF. b) Feeding ratio of raw materials: a large excess of deprotection solution, the dosage is 20mL of deprotection solution per millimole of resin. c) Temperature of the reaction system: 35-95 ° C. The details are shown in Table 3 below.
  • HPLC profile of the exemplary crude peptides synthesized according to the above examples of the present invention (HPLC conditions: 40% B solution-70% B solution 0-5min, 70% B solution-100% B solution 5-6.5min (A solution: water Contains 0.1% TFA (trifluoroacetic acid), liquid B: 80% acetonitrile 20% water contains 0.1% TFA)) and ESI-MS spectra are shown in Figure 4 and Figure 5, respectively.

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Abstract

提供一种利用微波辅助的流动固相多肽合成方法及其系统，其利用微波对氨基酸连接循环进行辅助，所述氨基酸连接循环的微波辅助环境不同，并且各个氨基酸连接循环的微波辅助环境保持不变，在先氨基酸连接循环所得到的产物移入另一微波辅助环境，各个氨基酸连接循环在完成一个批次的氨基酸连接循环后，继续在保持不变的微波辅助环境下重复进行下一批次的氨基酸连接循环。能够降低多肽合成的失败风险，有利于生产过程的质量控制，同时节省了合成的时间，增加速度和产能。

Description

微波流动固相多肽合成方法及其系统 技术领域
本发明涉及多肽合成技术领域，特别涉及一种微波固相多肽合成方法及系统。
背景技术
多肽是由多种氨基酸按照一定的排列顺序通过肽键结合而成的化合物，它的分子结构介于氨基酸和蛋白质之间，是一种与生物体内各种细胞功能都相关的生物活性物质。
1963年，Merrifield首次提出了固相多肽合成(Solid phase peptide synthesis，SPPS)方法，由于其合成方便，迅速，成为多肽合成的首选方法。传统的固相多肽合成采用单一反应器模式，即合成过程中不进行带树脂氨基酸的转移，在同一反应器中不断重复进行脱保护、活化、耦合和冲洗等步骤，氨基酸一个个按顺序地加入并进行耦合，所有的氨基酸耦合都发生在同一个反应器中。待一批多肽合成完成后，进行树脂切割和转移，然后再进行下一批多肽的合成。例如，如果是10个氨基酸序列的肽，根据多肽的序列依次加入第1个、第2个，重复进行脱保护、活化、耦合和冲洗等步骤，直到第10个，然后进行树脂切割和转移，得到含10个氨基酸的肽。固相多肽合成的优点主要表现在最初的反应物和产物都是连接在固相载体上，因此可以在一个反应容器中进行所有的反应。
在多肽固相合成中，氨基酸的极性以及空间位阻和耦合位置都会对多肽的合成难易程度造成影响。中国专利200910136132.6公开了一种多肽固相合成法，其依据多肽固相合成反应中待反应的氨基酸的极性参数、空间位阻和耦合位置来确定耦合反应时间和温度，按照所得到的反应温度及时间进行多肽的合成，提升了产物收率，并降低了副反应发生率。
中国专利申请201710135872.2公开了一种能够在制备不同的衍生 肽时批量进行多肽固相合成的方法，该方法使用多通道多肽固相合成装置，根据进行下一步反应所需要的耦合溶液对各多肽合成管进行分组，将需要的耦合溶液相同的多肽合成管归为同一组，从而为实现批量反应提供基础。
美国专利文献US2017/0266637A1公开了一种微波辅助的多肽合成设备及方法，其利用微波辅助进行固相多肽合成，在同一反应器中根据多肽序列按顺序地添加入新氨基酸，在微波辅助下重复进行脱保护、活化、耦合和冲洗等步骤，进一步提高了合成效率。
虽然固相多肽合成方法发展至今已取得了长足的进步，但现有的固相多肽合成方法仍存在以下问题，即由于每一步耦合所使用的试剂以及耦合反应时间和温度等条件都不尽相同，在同一反应器中进行多步耦合反应时，需频繁转换反应条件，这会造成各耦合步骤之间衔接间断，降低效率；另外，必须在加完了所有氨基酸以后才能得出最后结果，如果其中某个氨基酸的耦合或者由于脱保护没做好，可能就会出现整批多肽合成严重错误，甚至全部作废。这造成现有的固相多肽合成法速度慢、产率低、失败风险较大，容易发生质量控制事故，造成极大的浪费。
需要说明的是，以上背景技术部分所公开的信息仅用于增强对本发明背景的理解，因此其可能包含不构成对本领域技术人员已知的现有技术的信息。
发明内容
为了解决现有技术中存在的问题，本发明提供一种利用微波辅助的流动固相多肽合成方法和系统，该方法和系统从原理上给优化氨基酸反应的条件建立基本的条件，能够降低多肽合成的失败风险，容易进行生产过程的质量控制，同时节省合成的时间，增加速度和产能。
本发明在一方面提供一种利用微波辅助的流动固相多肽合成方法，其包括至少两个氨基酸连接循环，在氨基酸连接循环中，氨基酸分子与树脂载体上的反应基团或者前一已连接氨基酸分子耦合；所述至少两个氨基酸连接循环在微波辅助环境下利用微波进行辅助，并且每个氨基酸连接循环包括氨基酸耦合步骤和脱保护步骤；所述至少两 个氨基酸连接循环中的至少一个氨基酸连接循环的微波辅助环境与其他氨基酸连接循环的微波辅助环境不同，所述微波辅助环境可以由包括微波辐射强度、微波辐射时间的参数确定；所述至少两个氨基酸连接循环中，各个氨基酸连接循环的微波辅助环境保持不变，将在先氨基酸连接循环所得到的带氨基酸树脂产物移入另一微波辅助环境，进行下一个氨基酸连接循环；所述至少两个氨基酸连接循环中的每一个在完成一个批次的氨基酸连接循环后，继续在保持不变的微波辅助环境下重复进行下一批次的氨基酸连接循环。
上述氨基酸耦合步骤以及/或者脱保护步骤可以在微波辅助环境下利用微波进行辅助。
上述所述至少两个氨基酸连接循环中的至少一个氨基酸连接循环所含的反应步骤的反应条件与其他氨基酸连接循环所含的反应步骤的反应条件不同，所述反应步骤的反应条件可以由包括原料投料比、反应时间、反应体系温度的参数确定。
上述至少两个氨基酸连接循环中，可以使各个氨基酸连接循环的微波辅助环境保持不变，并且使所述氨基酸耦合步骤以及/或者脱保护步骤的反应条件保持不变，将在先氨基酸连接循环所得到的带氨基酸树脂产物移入另一微波辅助环境，进行下一个氨基酸连接循环。
上述至少两个氨基酸连接循环中的每一个在完成一个批次的氨基酸连接循环后，可以继续在保持不变的微波辅助环境下，并且可以在所述氨基酸耦合步骤以及/或者脱保护步骤的反应条件保持不变的情况下，重复进行下一批次的氨基酸连接循环。
根据本发明进一步的实施方式，可以按照耦合难易程度对氨基酸进行分类，对于耦合难度不同的每一类氨基酸，可以分别对其氨基酸连接循环设定不同的微波辅助环境和反应条件。
可以根据氨基酸的结构和性质，按照相对耦合难易程度将氨基酸分为三类：
(1)第一类氨基酸：包括甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸以及赖氨酸；
(2)第二类氨基酸：包括半胱氨酸、精氨酸以及组氨酸；
(3)第三类氨基酸：包括位阻较大的氮甲基氨基酸、α-甲基氨基酸；
其中，所述三类氨基酸的耦合难度顺序为：第三类氨基酸>第二类氨基酸>第一类氨基酸。
另外，还可以进一步将每一种氨基酸作为一类。并且，还可以进一步根据各类氨基酸与其所要连接的氨基酸之间的耦合难度，将各类氨基酸按照不同的氨基酸连接对象进一步分类。
将在先氨基酸连接循环所得到的带氨基酸树脂产物移入另一微波辅助环境时，可以将所述带氨基酸树脂产物与盛装其的容器一同移入另一微波辅助环境。
本发明另一方面还提供一种利用微波辅助的流动固相多肽合成系统，其包括：至少两个氨基酸连接单元，用于为不同氨基酸耦合步骤及脱保护步骤提供反应容置空间；至少两个微波辅助提供单元，用于分别为不同氨基酸连接循环提供微波辅助；至少两个反应条件控制单元，用于分别控制不同氨基酸连接循环的反应条件；所述至少两个氨基酸连接单元串联连接，前一氨基酸连接单元中的氨基酸连接循环所得到的带氨基酸树脂产物移入下一氨基酸连接单元进行下一个氨基酸连接循环。
上述微波辅助提供单元为氨基酸连接循环中的氨基酸耦合步骤以及/或者脱保护步骤提供微波辅助，而上述反应条件控制单元对氨基酸连接循环中的氨基酸耦合步骤以及/或者脱保护步骤进行反应条件控制。
上述微波辅助提供单元能够以设定的微波辐射强度、微波辐射时间为氨基酸连接循环提供微波辅助；而所述反应条件控制单元能够以设定的原料投料比、反应时间、反应体系温度控制各反应步骤的反应条件。
根据本发明的一个实施方式，本发明提供的利用微波辅助的流动固相多肽合成系统还可以包括用于直接盛装氨基酸连接反应试剂及反应产物的容器，其用于与氨基酸连接循环所得到的带氨基酸树脂产物一同移入下一氨基酸连接单元进行下一个氨基酸连接循环。
本发明所提供的利用微波辅助的流动固相多肽合成方法，使带树 脂氨基酸在流动中排队耦合连接新氨基酸。这种全新的流动合成方式使带氨基酸树脂不停地转移排队，第一批树脂跨出第一步在第一模块中完成耦合后，第二批树脂再跨进第一模块中，带树脂氨基酸第二批只比第一批慢一个工作模块步骤，带树脂氨基酸就这样一直转移着排队向前，而每向前一步，都添加一个氨基酸。这也是本发明区别于现有方法如在同一反应器中进行所有反应的最大优点。本发明还可以对每一步反应进行工艺的标准化和条件优化，这种标准模块化排序能更容易达到最优化的合成结果，实现更快的速度，更高的产率，更好的活性，节约时间且环保，而且大大降低了大规模发生错误的概率，即降低错误的发生率，并且根本上增加了产能，至少使多肽合成提高到实现临床级用量以上。
附图说明
以下将详细参考附图示出的特定示例性实施例，对本发明的上述和其他特征进行说明，所述示例性实施例在下文中仅以说明的方式给出，因此并不限制本发明，其中：
图1是说明根据本发明的一个实施例的流动固相多肽合成方法的原理示意图。
图2是说明根据本发明的一个实施例的微波流动固相多肽合成系统的示意性框图。
图3是说明利用本发明的一个实施例的微波流动固相多肽合成系统进行固相多肽合成过程的示意图。
图4是根据本发明实施例利用微波流动固相多肽合成方法合成的示例性多肽粗品的HPLC图谱。
图5是根据本发明实施例利用微波流动固相多肽合成方法合成的示例性多肽粗品的ESI-MS图谱。
具体实施方式
以下通过具体实施例对本发明进行详细描述，以使本领域技术人员能够容易地根据本说明书的公开内容实施本发明。以下所描述的实施例仅是本发明的一部分实施例，而不是全部。基于本发明中的实施 例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例，都属于本发明保护的范围。
氨基酸是羧酸碳原子上的氢原子被氨基取代后的化合物，氨基酸分子中含有氨基和羧基两种官能团。与羟基酸类似，氨基酸可按照氨基连在碳链上的不同位置而分为α-，β-，γ-...w-氨基酸，组成蛋白质的氨基酸大部分为α-氨基酸。组成人体蛋白质的氨基酸共二十种，这二十种天然氨基酸是指甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸和组氨酸这二十种，它们是构成蛋白质的基本单位。
本发明的微波流动固相多肽合成首先把针对特定氨基酸配对的最佳工艺条件进行优化并实现标准化，通过模块化把特定氨基酸配对的耦合工艺条件优化和固定下来，通过带氨基酸树脂转移技术实现多肽的流动化排序合成，再根据氨基酸序列将不同的模块排列组合成流动合成生产线，实现模块化排序设计的流动多肽合成，从而提高大规模合成的高纯度和高活性的能力，而且保证批量的重复性和统一性。
首先，根据配对耦合氨基酸的特性，本发明的申请人根据各种不同氨基酸的结构和性质，例如氨基酸在多肽合成中的配对耦合的特殊性和难点，对氨基酸进行分类和模块化处理，筛选和优化最佳的耦合和脱保护条件，将每个氨基酸连接循环标准化和模块化。
例如，二十种天然氨基酸中，在固相多肽合成中有比较容易发生消旋化等副反应的氨基酸，如半胱氨酸、精氨酸、组氨酸等，有不易消旋的天然氨基酸，本发明申请人据此将氨基酸分为以下三类：
第一类：甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸。
第二类：半胱氨酸、精氨酸、组氨酸。
第三类：非天然氨基酸，主要包括位阻较大的氮甲基氨基酸、α-甲基氨基酸。
通过对不同种类型的氨基酸的区分，针对不同类型的氨基酸，优化设计不同的氨基酸耦合条件。例如，不同的氨基酸耦合条件如下表1 所示。
表1 不同类型氨基酸的耦合条件
  第一类氨基酸 第二类氨基酸 第三类氨基酸
微波耦合 中量辐射<75℃ 低量辐射<35℃ 高量辐射<95℃
耦合时间 2min 6min 4min
主溶剂 DMF DMF DMF
耦合剂 DIC/Oxyma DIC/Oxyma DIC/Oxyma
树脂/氨基酸投料比 1:5 1:5 1:8
并且，针对不同类型的氨基酸，优化设计不同的耦合后脱保护基本条件。例如，不同的脱保护条件如下表2所示。
表2 不同类型氨基酸的脱保护条件
  第一类氨基酸 第二类氨基酸 第三类氨基酸
脱保护温度 中量辐射<75℃ 低量辐射<35℃ 中量辐射<95℃
脱保护时间 2min 3min 1min
脱保护试剂 20％哌啶/DMF 20％哌啶/DMF 20％哌啶/DMF
需要说明的是，本说明书中上述氨基酸的分类以及上表中的耦合条件和脱保护条件仅为示例性的举例，而非限制。例如，可以在以上氨基酸分类的基础上、或独立于上述氨基酸的分类方法，对氨基酸进行其他方式的分类。举例来说，可以根据需要将各种氨基酸分别作为一个类别；或者，根据各类氨基酸与其所要连接的氨基酸之间的耦合难度，将各类氨基酸按照不同的氨基酸连接对象进一步分类，即在二十种氨基酸的基础上考虑其连接二十种氨基酸的可能性，进一步细分为20×20＝400种类型。在此基础上，本领域技术人员在本发明的思想指导下，可以根据氨基酸的不同分类，通过实验和计算改进耦合条件和脱保护条件。需要说明的是，所有这些改进和变化都应解释为本发明技术方案的具体实施方式，均处于本发明的保护范围内。
进而，针对不同类型的氨基酸，在不同的氨基酸连接模块中如上设定最佳耦合及脱保护条件，并使带氨基酸树脂在若干个串联的氨基酸连接模块中连续不停地转移排队流动，依序进行每种类型的氨基酸连接循环。同时，每个氨基酸连接模块持续不断地进行同样的氨基酸连接循环，整个氨基酸连接循环保持不间断运行。也就是说，第一批 树脂跨出第一步在第一模块中完成耦合后，第二批树脂再跨进第一模块中，带树脂氨基酸第二批只比第一批慢一个工作模块步骤，带树脂氨基酸就这样一直转移着排队向前，而每向前一步，都添加一个氨基酸。
通过这样的设计和安排，可以对多肽合成的每一步进行工艺的标准化和条件优化，从而不仅大大降低多肽合成的失败风险，从原理上给优化氨基酸反应的条件建立基本的条件，而且更容易进行生产过程的质量控制，降低错误的发生率，同时也节省合成的时间，提高合成效率，从根本上增加速度和产能。
以下结合附图对本发明进行更详细的说明，应当理解，以下详细说明仅为使本领域技术人员更容易理解本发明的思想而作的举例性说明，并非构成对本发明的任何限制。
图1是说明根据本发明的一个实施例的流动固相多肽合成方法的原理示意图。图1中的“库”代表氨基酸库，A库、Q库、Y库、F库分别代表丙氨酸库、谷氨酰胺库、酪氨酸库、苯丙氨酸库；W代表树脂；W-G代表连接了甘氨酸的树脂。如图1中所示，W-G树脂沿多肽合成路线连续流动，在每个氨基酸耦合模块中，分别按照设定的最优反应条件进行氨基酸耦合。在完成一个氨基酸连接后，带氨基酸树脂继续进入下一个氨基酸耦合模块，进行下一个氨基酸的连接。例如，W-G树脂在氨基酸耦合模块1中连接了丙氨酸之后，W-G-A进入下一个氨基酸连接循环，即在氨基酸耦合模块2中进行谷氨酰胺的连接，如此不断延续，分别在氨基酸耦合模块3、4中依次连接酪氨酸、苯丙氨，得到W-G-A-Q-Y-F链，然后采用常规方法进行切割，即可得到G-A-Q-Y-F肽链。
在上述合成过程中，各个氨基酸耦合模块在完成一批树脂的氨基酸连接之后，一俟带氨基酸树脂移出该模块，即由该氨基酸库中加入下一批同样的氨基酸，进行下一批的氨基酸连接，该耦合模块中保持同样的反应条件，进行同样的耦合反应。例如，氨基酸耦合模块1中始终在不变的微波辅助环境下和不变的反应条件下进行丙氨酸的连接，氨基酸耦合模块2中始终在另一不变的微波辅助环境下和不变的反应条件下进行谷氨酰胺的连接。
利用微波辅助固相多肽合成已是本领域公知的技术，目前已有相对成熟的微波多肽合成仪在进行商业化销售和得到规模化应用。但现有微波多肽合成仪均利用微波在同一反应器(微波辐射腔)中实现所有的氨基酸连接，因而合成效率低下、产能低、合成错误风险高。而本发明创造性地将多个微波辐射腔串联连接，实现不同氨基酸连接循环在不同的耦合模块(反应器)中进行，各类氨基酸的耦合条件及脱保护条件均经优化后各自保持不变，且每个氨基酸耦合模块持续不间断地进行同类氨基酸的稳定连接，从而达到了高效率、高产能、合成准确率高的效果。
图2是说明根据本发明一个实施例的微波流动固相多肽合成系统的示意性框图。图2所示的实施例中，氨基酸连接单元41、42、43用于为不同氨基酸耦合步骤及脱保护步骤提供反应容置空间。在微波辅助固相多肽合成过程中，在每个氨基酸连接单元41、42、43中完成一个类别的氨基酸的连接循环，包括氨基酸耦合步骤和耦合之后的脱保护步骤。其中，氨基酸耦合步骤包括从氨基酸活化单元20引入相应经活化的氨基酸，然后在微波辅助提供单元31及反应条件控制单元(图中未示出)的作用和控制下，并在氮气源60提供的氮气保护下，进行氨基酸耦合反应。微波辅助提供单元31能够以按需设定的微波辐射强度、微波辐射时间参数为氨基酸连接循环提供微波辅助。反应条件控制单元能够以按需设定的原料投料比、反应时间、反应体系温度控制各反应步骤的反应条件。微波辅助提供单元31和反应条件控制单元的结构、功能及操作方法属于本领域公知技术，因此不再赘述。微波辅助提供单元31和反应条件控制单元可以是相互独立的单元模块，也可以将二者集成在一起，由同一控制单元进行统一操控。
图2中所示氨基酸活化单元20示意性连接至氨基酸连接单元41、42、43，该氨基酸活化单元20可以包括多个氨基酸活化子单元，每个子单元用于一种氨基酸，并且可以根据需要控制特定氨基酸活化子单元中的特定氨基酸在特定时间加入特定的氨基酸连接单元，并且可以控制氨基酸的加入量和加入速度。上述氨基酸活化单元的结构、功能及操作方法是本领域的现有技术，如美国专利文献US2017/0266637A1中所公开的，在此不再赘述。
图2中所示脱保护剂提供单元10示意性连接至氨基酸连接单元41、42、43，其用于为氨基酸耦合步骤之后的脱保护步骤提供必须的脱保护剂，例如DMF。可以根据需要控制脱保护剂提供单元10中的脱保护剂在特定时间加入特定的氨基酸连接单元，并且可以控制脱保护剂的加入量和加入速度。同样，脱保护步骤也可以在微波辅助提供单元31及反应条件控制单元的作用和控制下，并在氮气源60提供的氮气保护下进行。
本发明将氨基酸连接单元41、42、43……、n依次串联连接，分别用于为不同氨基酸耦合步骤及脱保护步骤提供反应容置空间。在各氨基酸连接单元41、42、43……、n中完成一批树脂的氨基酸连接之后，将带氨基酸的树脂经清洗过滤后，即移入到下一氨基酸连接单元42、43……、n+1，进行下一个氨基酸连接循环，如此不断连续，直至完成所有氨基酸的连接，形成所需的多肽序列。另外，一俟带氨基酸树脂移出某氨基酸连接单元，即由该单元对应的氨基酸库中加入下一批同样的氨基酸，该耦合模块中保持同样的反应条件，进行同样的耦合及脱保护反应，进行下一批的氨基酸连接。
以上氨基酸连接单元41、42、43……、n仅为示意性说明，在其中完成一个批次的氨基酸连接后，带氨基酸树脂可以直接移出到下一氨基酸连接单元，也可以在氨基酸连接单元中设置专用的容器(图中未示出)，利用该容器将带氨基酸树脂连同容器一起移送到下一个氨基酸连接单元，进行下一批的氨基酸连接。
各步反应所产生的废弃物可以通过过滤器滤出，通过连接管道排出到废液收集单元50。
图3是说明利用本发明的一个实施例的微波流动固相多肽合成系统进行固相多肽合成过程的示意图。如图3所示，W-G树脂沿多肽合成路线连续流动，在每个氨基酸耦合模块中，分别按照设定的最优反应条件与活化的相应氨基酸进行氨基酸耦合。在完成一个氨基酸连接后，带氨基酸树脂继续进入下一个氨基酸耦合模块，进行下一个氨基酸的连接。例如，经过脱保护的W-G树脂在耦合模块1中连接了丙氨酸之后，对W-G-A进行脱保护，之后进入下一个氨基酸连接循环，即在耦合模块2中进行谷氨酰胺的连接，然后对W-G-A进行脱保护，如 此不断延续，分别在耦合模块3、4中依次连接酪氨酸、苯丙氨，得到W-G-A-Q-Y-F链，然后采用常规方法进行切割，即可得到G-A-Q-Y-F肽链。
根据本发明的微波流动固相多肽合成基于微波化学基础上的标准化、模块化，和流动化耦合排序，通过带树脂氨基酸在多模块中排序流动进行新氨基酸添加，通过数学排序组合而合理高效安排氨基酸全耦合(耦合)过程，实现连续流动的多肽化学合成，并且实现多模块流动化大规模的合成能力，解决了传统固相多肽合成中大规模合成生产技术的难点。
实施例：两种多肽合成方法的对比
分别利用传统的微波固相多肽合成方法和本发明的流动微波多肽合成法合成以下多肽序列：Fmoc-Ile-Pro-His-Gln-Ser-Phe-Phe(分子量：1096)。传统微波固相多肽合成法利用现有商业化合成仪(型号：336X，CSBio公司生产)按照生产厂家推荐的条件进行合成，本发明的流动微波多肽合成法实验条件如下：
使用树脂：Fmoc-Phe-Wang Resin
微波条件：辐射功率约1000W左右，由温度自动调节。辐射时间：耦合每个氨基酸2-6分钟，脱保护每个氨基酸1-5分钟。
脱保护反应条件：a)脱保护试剂：20％哌啶/DMF。b)原料投料比：脱保护液大幅过量，用量为每毫摩尔树脂使用20mL脱保护液。c)反应体系温度：35-95℃。具体如下表3所示。
表3 本发明流动微波多肽合成法中各氨基酸连接循环的实验条件
  Phe Ser Gln His Pro Ile
微波辐射强度 1000W 1000W 1000W 1000W 1000W 1000W
辐射时间 2min 2min 2min 6min 2min 2min
耦合温度 75℃ 75℃ 75℃ 35℃ 75℃ 75℃
脱保护试剂 200mL 200mL 200mL 200mL 200mL 200mL
脱保护时间 2min 2min 2min 3min 2min 2min
脱保护温度 75℃ 75℃ 75℃ 35℃ 75℃ 75℃
根据本发明以上实施例合成的示例性多肽粗品的HPLC图谱(HPLC条件：40％B液-70％B液0-5min，70％B液-100％B液5-6.5min(A液：水含0.1％TFA(三氟乙酸)，B液:80％乙腈20％水含0.1％TFA))和ESI-MS图谱分别见图4和图5。
两种方法所用时间及所得结果见下表4。
表4 传统平行合成与本发明的流动微波多肽合成法的对比
Figure PCTCN2018117193-appb-000001
由上表4可见，合成10mmol该多肽所需总时间，传统的平行合成方法需要约73小时(单通道合成规模为0.5mmol，3通道同时合成总规模为1.5mmol。为合成总规模为10mmol的该多肽，需要总时间(10mmol/1.5mmol)*11小时＝约73小时)，而本发明的流动微波多肽合成仪为3.5小时。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被完整引用至本说明书作为参考。
此外应理解，在阅读了本发明的上述说明内容之后，本领域技术人员可以对本发明做出各种改动或修改，这些等同形式同样落入本发明的保护范围。

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  1. 一种利用微波辅助的流动固相多肽合成方法，其包括至少两个氨基酸连接循环，在所述氨基酸连接循环中，氨基酸分子与树脂载体上的反应基团或者前一已连接氨基酸分子耦合，所述至少两个氨基酸连接循环在微波辅助环境下利用微波进行辅助，并且每个氨基酸连接循环包括氨基酸耦合步骤和脱保护步骤，其特征在于：
    所述至少两个氨基酸连接循环中的至少一个氨基酸连接循环的微波辅助环境与其他氨基酸连接循环的微波辅助环境不同，所述微波辅助环境由包括微波辐射强度、微波辐射时间的参数确定；
    所述至少两个氨基酸连接循环中，各个氨基酸连接循环的微波辅助环境保持不变，将在先氨基酸连接循环所得到的带氨基酸树脂产物移入另一微波辅助环境，进行下一个氨基酸连接循环；
    所述至少两个氨基酸连接循环中的每一个在完成一个批次的氨基酸连接循环后，继续在保持不变的微波辅助环境下重复进行下一批次的氨基酸连接循环。
  2. 根据权利要求1所述的利用微波辅助的流动固相多肽合成方法，其特征在于，所述氨基酸耦合步骤以及/或者脱保护步骤在微波辅助环境下利用微波进行辅助。
  3. 根据权利要求1所述的利用微波辅助的流动固相多肽合成方法，其特征在于，所述至少两个氨基酸连接循环中的至少一个氨基酸连接循环所含的反应步骤的反应条件与其他氨基酸连接循环所含的反应步骤的反应条件不同，所述反应步骤的反应条件由包括原料投料比、反应时间、反应体系温度的参数确定。
  4. 根据权利要求3所述的利用微波辅助的流动固相多肽合成方法，其特征在于，所述至少两个氨基酸连接循环中，各个氨基酸连接循环的微波辅助环境保持不变，并且所述氨基酸耦合步骤以及/或者脱保护步骤的反应条件保持不变，将在先氨基酸连接循环所得到的带氨 基酸树脂产物移入另一微波辅助环境，进行下一个氨基酸连接循环。
  5. 根据权利要求4所述的利用微波辅助的流动固相多肽合成方法，其特征在于，所述至少两个氨基酸连接循环中的每一个在完成一个批次的氨基酸连接循环后，继续在保持不变的微波辅助环境下，并且在所述氨基酸耦合步骤以及/或者脱保护步骤的反应条件保持不变的情况下，重复进行下一批次的氨基酸连接循环。
  6. 根据权利要求1所述的利用微波辅助的流动固相多肽合成方法，其特征在于，按照耦合难易程度对氨基酸进行分类，对于耦合难度不同的每一类氨基酸，分别对其氨基酸连接循环设定不同的微波辅助环境和反应条件。
  7. 根据权利要求6所述的利用微波辅助的流动固相多肽合成方法，其特征在于，根据氨基酸的结构和性质，按照相对耦合难易程度将氨基酸分为三类：
    (1)第一类氨基酸：包括甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸以及赖氨酸；
    (2)第二类氨基酸：包括半胱氨酸、精氨酸以及组氨酸；
    (3)第三类氨基酸：包括位阻较大的氮甲基氨基酸、α-甲基氨基酸；
    其中，所述三类氨基酸的耦合难度顺序为：第三类氨基酸>第二类氨基酸>第一类氨基酸。
  8. 根据权利要求6所述的利用微波辅助的流动固相多肽合成方法，其特征在于，将每一种氨基酸作为一类。
  9. 根据权利要求6所述的利用微波辅助的流动固相多肽合成方法，其特征在于，根据各类氨基酸与其所要连接的氨基酸之间的耦合难度，将各类氨基酸按照不同的氨基酸连接对象进一步分类。
  10. 根据权利要求1-9中任一项所述的利用微波辅助的流动固相多肽合成方法，其特征在于，所述将在先氨基酸连接循环所得到的带氨基酸树脂产物移入另一微波辅助环境的步骤中，将所述带氨基酸树脂产物与盛装其的容器一同移入另一微波辅助环境。
  11. 一种利用微波辅助的流动固相多肽合成系统，其特征在于，包括：
    至少两个氨基酸连接单元，用于为不同氨基酸耦合步骤及脱保护步骤提供反应容置空间；
    至少两个微波辅助提供单元，用于分别为不同氨基酸连接循环提供微波辅助；
    至少两个反应条件控制单元，用于分别控制不同氨基酸连接循环的反应条件；
    所述至少两个氨基酸连接单元串联连接，前一氨基酸连接单元中的氨基酸连接循环所得到的带氨基酸树脂产物移入下一氨基酸连接单元进行下一个氨基酸连接循环。
  12. 根据权利要求11所述的利用微波辅助的流动固相多肽合成系统，其特征在于，所述微波辅助提供单元为氨基酸连接循环中的氨基酸耦合步骤以及/或者脱保护步骤提供微波辅助，所述反应条件控制单元对氨基酸连接循环中的氨基酸耦合步骤以及/或者脱保护步骤进行反应条件控制。
  13. 根据权利要求12所述的利用微波辅助的流动固相多肽合成系统，其特征在于，所述微波辅助提供单元能够以设定的微波辐射强度、微波辐射时间为氨基酸连接循环提供微波辅助。
  14. 根据权利要求13所述的利用微波辅助的流动固相多肽合成系统，其特征在于，所述反应条件控制单元能够以设定的原料投料比、反应时间、反应体系温度控制各反应步骤的反应条件。
  15. 根据权利要求11所述的利用微波辅助的流动固相多肽合成系统，其特征在于，还包括用于直接盛装氨基酸连接反应试剂及反应产物的容器，其用于与氨基酸连接循环所得到的带氨基酸树脂产物一同移入下一氨基酸连接单元进行下一个氨基酸连接循环。
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007065807A2 (en) * 2005-12-06 2007-06-14 F. Hoffmann-La Roche Ag Automated solid phase synthesis
CN101531699A (zh) * 2009-04-30 2009-09-16 昆明积大制药有限公司 一种多肽固相合成法
CN106317163A (zh) * 2016-08-23 2017-01-11 安徽天达谱申生物科技有限公司 一种高效自动化的多肽合成方法
CN106892959A (zh) * 2017-03-08 2017-06-27 河南工程学院 多通道多肽固相合成方法
CN107056879A (zh) * 2017-03-08 2017-08-18 河南工程学院 多通道多肽固相合成装置
US20170266637A1 (en) * 2003-06-23 2017-09-21 Cem Corporation Microwave-assisted peptide synthesis
CN108752416A (zh) * 2018-06-29 2018-11-06 烟台智本知识产权运营管理有限公司 氨基酸多肽的制备方法

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3631662A1 (de) * 1986-09-17 1988-03-24 Biotechnolog Forschung Gmbh Verfahren zur simultanen synthese mehrerer peptide an fester phase
EP0385433A3 (en) * 1989-02-28 1991-08-14 Ceskoslovenska Akademie Ved Method and apparatus for the continuous polymer synthesis on a solid carrier
CN1298732C (zh) * 2005-05-17 2007-02-07 南京工业大学 多肽微波固相合成法
GB0712922D0 (en) * 2007-07-03 2007-08-15 Swedish Biomimetics 3000 Ltd Solid phase reaction method
CN101538310A (zh) * 2009-01-13 2009-09-23 杭州中肽生化有限公司 微波辅助连续流大规模多肽合成方法与装置
CN103788184A (zh) * 2013-12-13 2014-05-14 东北农业大学 含有两个亮氨酸重复单元的抗菌肽及制备方法和应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170266637A1 (en) * 2003-06-23 2017-09-21 Cem Corporation Microwave-assisted peptide synthesis
WO2007065807A2 (en) * 2005-12-06 2007-06-14 F. Hoffmann-La Roche Ag Automated solid phase synthesis
CN101531699A (zh) * 2009-04-30 2009-09-16 昆明积大制药有限公司 一种多肽固相合成法
CN106317163A (zh) * 2016-08-23 2017-01-11 安徽天达谱申生物科技有限公司 一种高效自动化的多肽合成方法
CN106892959A (zh) * 2017-03-08 2017-06-27 河南工程学院 多通道多肽固相合成方法
CN107056879A (zh) * 2017-03-08 2017-08-18 河南工程学院 多通道多肽固相合成装置
CN108752416A (zh) * 2018-06-29 2018-11-06 烟台智本知识产权运营管理有限公司 氨基酸多肽的制备方法

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