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WO2019011236A1 - 一种立体选择性的转氨酶在不对称合成手性胺中的应用 - Google Patents

一种立体选择性的转氨酶在不对称合成手性胺中的应用 Download PDF

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Publication number
WO2019011236A1
WO2019011236A1 PCT/CN2018/095148 CN2018095148W WO2019011236A1 WO 2019011236 A1 WO2019011236 A1 WO 2019011236A1 CN 2018095148 W CN2018095148 W CN 2018095148W WO 2019011236 A1 WO2019011236 A1 WO 2019011236A1
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polypeptide
amino acid
seq
compound
acid sequence
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English (en)
French (fr)
Inventor
程占冰
张涛
田振华
瞿旭东
丁少南
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Abiochem Biotechnology Co Ltd
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Abiochem Biotechnology Co Ltd
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Priority to CN201880046478.4A priority Critical patent/CN110914417B/zh
Priority to US16/630,297 priority patent/US11499172B2/en
Priority to EP18831049.4A priority patent/EP3650537A4/en
Publication of WO2019011236A1 publication Critical patent/WO2019011236A1/zh
Anticipated expiration legal-status Critical
Priority to US17/737,943 priority patent/US11965193B2/en
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
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    • C07B2200/07—Optical isomers

Definitions

  • the invention belongs to the field of biotechnology, and particularly relates to a highly active transaminase, and the use of the transaminase as a catalyst in asymmetric synthesis of chiral amines, and a method for producing chiral amines.
  • Chiral amines are important intermediates and active ingredients in many drugs, such as (3R)-3-amino-1-[3-(trifluoromethyl)-5,6-dihydro-1,2,4-triazole And [4,3-a]pyrazine-7(8H)-yl]-4-(2,4,5-trifluorophenyl)butan-1-one is an active ingredient for the treatment of type 2 diabetes, however
  • the organic synthesis method is not easy to obtain an optically pure product, and usually requires an asymmetrically synthesized metal catalyst, which is expensive, has a metal residue, and is difficult to handle. Therefore, there is an urgent need to find a green method for the production of chiral amines.
  • Transaminase also known as aminotransferase, is a type of enzyme that catalyzes the reversible transfer of an amino group between an amino compound and a carbonyl compound. It can be classified into a-transaminase and ⁇ -transaminase depending on the position of the substrate amino group. A-transaminase is more common and can only catalyze the transfer of a-amino group, while ⁇ -transaminase is rare.
  • ⁇ -transaminase is supplemented with pyridoxal phosphate, which does not require additional cofactor circulation during the catalytic reaction, which is beneficial to industrial production.
  • U.S. Patent No. 8,293,507 discloses Codexis' biocatalyst for the modification of Arthrobacter-derived ⁇ -transaminase instead of the ruthenium catalyst in the chemical process.
  • the transaminated product has an ee value of 99% and a substrate feed of 100 g/L.
  • One of the objects of the present invention is to provide a use of a polypeptide in the production of a chiral amine or a downstream product which is a precursor of a chiral amine.
  • Another object of the invention is to provide a process for the production of chiral amines.
  • Still another object of the present invention is to provide a method for constructing a chiral amine producing strain.
  • a polypeptide in the production of a chiral amine or a downstream product of a chiral amine as a precursor, the polypeptide being:
  • the chiral amine is formed by asymmetric synthesis of a prochiral carbonyl compound.
  • the prochiral carbonyl compound is selected from the group consisting of
  • the polypeptide (b1) is one or more, preferably 1-20, more preferably 1-15, more preferably 1- at either end of the amino acid sequence shown in SEQ ID NO: 1.
  • amino acid sequence of the polypeptide is set forth in SEQ ID NO: 1.
  • the polypeptide has an amino acid sequence of at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96 of the amino acid sequence set forth in SEQ ID NO:1. %, 97%, 98%, 99% or more of any polypeptide sequence of sequence identity.
  • the chiral amine comprises the following compound 1a-8a or a pharmaceutically acceptable salt thereof:
  • the downstream product of the chiral amine precursor comprises: sitagliptin (compound 5a), a chiral acid resolving agent or a chiral pharmaceutical intermediate (compound 1a), Dulutvir intermediate (compound 3a), chiral pharmaceutical intermediate starting material (compound 2a, compound 4a), sitagliptin intermediate (compound 6a, compound 7a, compound 8a).
  • the previous chiral carbonyl compound is a substrate, in the presence of a prosthetic group, under the catalysis of a stereoselective transaminase, the reaction shown in the reaction formula I is carried out to form a chiral amine;
  • the concentration of the prochiral carbonyl compound in the reaction system is from 1 g/L to 1000 g/L, preferably from 5 g/L to 500 g/L, more preferably 10 g/L. More preferably -100 g/L, still more preferably 5 g/L to 50 g/L, most preferably 20 g/L to 50 g/L.
  • the concentration of the stereoselective transaminase protein in the reaction system is from 1 to 500 mg/mL, preferably from 5 to 200 mg/mL, more preferably from 10 to 100 mg/mL, most The preferred condition is 20-80 mg/mL.
  • amino acid sequence of the polypeptide is set forth in SEQ ID NO: 1.
  • the polypeptide has an amino acid sequence of at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96 of the amino acid sequence set forth in SEQ ID NO:1. %, 97%, 98%, 99% or more of any polypeptide sequence of sequence identity.
  • the prochiral carbonyl compound is selected from the group consisting of
  • the prosthetic group is pyridoxal phosphate.
  • the concentration of the prosthetic group in the reaction system is from 1 to 200 mM, preferably from 5 to 150 mM, more preferably from 10 to 100 mM, most preferably from 5 to 50 mM.
  • the temperature is 5 to 50 ° C, preferably 10 to 45 ° C, more preferably 20 to 40 ° C, most preferably 25 to 35 ° C.
  • the pH is from 6 to 10, preferably from 7 to 10, more preferably from 7.5 to 8.5.
  • the polypeptide is one or more, preferably 1-20, more preferably 1-15, more preferably 1-10, at either end of the amino acid sequence shown in SEQ ID NO: 1. More preferably, the polypeptide derived from the polypeptide of the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1) is formed by the addition of 1-3, most preferably one amino acid residue.
  • amino acid sequence of the polypeptide is set forth in SEQ ID NO: 1.
  • the polypeptide has an amino acid sequence of at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96 of the amino acid sequence set forth in SEQ ID NO:1. %, 97%, 98%, 99% or more of any polypeptide sequence of sequence identity.
  • the production strain is a bacterium.
  • the bacterium is Escherichia coli.
  • the Escherichia coli is Escherichia coli BL21.
  • a method of producing a chiral amine comprising the steps of:
  • a chiral amine is isolated from the culture system of 1).
  • an appropriate amount of pyridoxal phosphate is added to step 1).
  • a method of constructing a chiral amine producing strain comprising:
  • the strain is made to comprise an expression vector expressing a polypeptide or such that a gene expressing the following polypeptide is integrated into the genome of the strain, the polypeptide being:
  • the polypeptide of (b1) is one or more, preferably 1-20, more preferably 1-15, more preferably 1- at either end of the amino acid sequence shown in SEQ ID NO: 1.
  • amino acid sequence of the polypeptide is set forth in SEQ ID NO: 1.
  • the polypeptide has an amino acid sequence of at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96 of the amino acid sequence set forth in SEQ ID NO:1. %, 97%, 98%, 99% or more of any polypeptide sequence of sequence identity.
  • sequence of the gene is selected from the group consisting of:
  • the gene is constructed on an expression vector.
  • R 2 is selected from the group consisting of: none, halogen, -OH, -NH 2 , substituted or unsubstituted C 1 -C 8 alkyl, substituted or unsubstituted C 3 -C 8 cycloalkyl, substituted or unsubstituted C 1 -C 8 alkoxy, substituted or unsubstituted C 3 -C 8 cycloalkoxy;
  • X is selected from the group consisting of C or N;
  • Y is selected from the group consisting of C, N, O or S
  • R 1 is -NH 2 .
  • X is N.
  • Y is O.
  • the compound of Formula II includes the following compounds;
  • a seventh aspect of the invention the use of the compound of the sixth aspect of the invention, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, for the preparation of the sitagliptin intermediate Boc- (R)-3-Amino-4-(2,4,5-trifluorophenyl)butyric acid.
  • the compound of Formula II includes the following compounds;
  • a polypeptide (omega-transaminase) which is capable of efficiently converting a prochiral carbonyl compound into an R chiral amine as a stereoselective catalyst.
  • the ⁇ -transaminase of the present invention has strict R stereoselectivity and has a broad substrate spectrum, and has great application prospects in the production of chiral amines and chiral drugs. On the basis of this, the present invention has been completed.
  • the term “about” means that the value can vary by no more than 1% from the recited value.
  • the expression “about 100” includes all values between 99 and 101 and (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the terms "containing” or “including” may be open, semi-closed, and closed. In other words, the terms also include “consisting essentially of,” or “consisting of.”
  • reaction can be carried out and purified using the manufacturer's instructions for use of the kit, or in a manner well known in the art or as described in the present invention.
  • the above techniques and methods can generally be carried out according to conventional methods well known in the art, as described in the various summaries and more specific references cited and discussed in this specification.
  • group and its substituents can be selected by those skilled in the art to provide stable structural moieties and compounds.
  • substituent When a substituent is described by a conventional chemical formula written from left to right, the substituent also includes the chemically equivalent substituent obtained when the structural formula is written from right to left.
  • substituent -CH 2 O- is equivalent to -OCH 2 -.
  • C1-C6 alkyl refers to an alkyl group as defined below having a total of from 1 to 6 carbon atoms.
  • the total number of carbon atoms in the simplified symbol does not include carbon that may be present in the substituents of the group.
  • halogen means fluoro, chloro, bromo or iodo.
  • Haldroxy means an -OH group.
  • alkyl means a fully saturated straight or branched hydrocarbon chain group, It consists only of carbon atoms and hydrogen atoms, has, for example, 1 to 7 carbon atoms, and is linked to the rest of the molecule by a single bond, including, for example, but not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl Base, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, heptyl and the like.
  • polypeptide or “polypeptide of the invention” or “polypeptide of the invention” or “omega-transaminase” or “stereoselective transaminase” as used herein have the same meaning and are used interchangeably herein to mean catalyzed.
  • Prochiral carbonyl compounds produce chiral amine activity proteins. This polypeptide is naturally absent from E. coli and is an exogenous protein.
  • the polypeptide of the present invention may be: (a1) a polypeptide having the amino acid sequence shown in SEQ ID NO: 1; or (b1) passing one or more of the amino acid sequence shown in SEQ ID NO: 1. Formed by substitution, deletion or addition of 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-3, most preferably 1 amino acid residues, with (a1) A polypeptide derived from a polypeptide of the amino acid sequence set forth in SEQ ID NO: 1 functional of the polypeptide.
  • the polypeptide is one or more, preferably 1-20, more preferably 1-15, more preferably 1-10, at either end of the amino acid sequence set forth in SEQ ID NO: 1. More preferably, the polypeptide derived from the polypeptide of the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1) is formed by the addition of 1-3, most preferably one amino acid residue.
  • amino acid sequence of the polypeptide is set forth in SEQ ID NO: 1.
  • the polypeptide has an amino acid sequence of at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96 of the amino acid sequence set forth in SEQ ID NO:1. %, 97%, 98%, 99% or more of any polypeptide sequence of sequence identity.
  • polypeptide of the present invention represents a protein having the amino acid sequence of SEQ ID NO: 1, and the coding sequence thereof is set forth in SEQ ID NO: 2.
  • the polypeptide of the present invention comprises up to 20, preferably up to 10, more preferably up to 3, and even more preferably up to 2, compared to the polypeptide of the amino acid sequence of SEQ ID NO: 1.
  • polynucleotide encoding a polypeptide can be a polynucleotide comprising the polypeptide, or a polynucleotide further comprising additional coding and/or non-coding sequences.
  • the homology or sequence identity may be 80% or more, preferably 90% or more, more preferably 95% to 98%, and most preferably 99% or more.
  • Methods for determining sequence homology or identity include, but are not limited to, Computational Molecular Biology, Lesk, AM, Oxford University Press, New York, 1988; Biocomputing: Information Biocomputing: Informatics and Genome Projects, Smith, DW, Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM and Griffin, HG , Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux , J. M. Stockton Press, New York, 1991 and Carillo, H. and Lipman, D., SIAM J.
  • the preferred method of determining identity is to obtain the largest match between the sequences tested.
  • the method of determining identity is compiled in a publicly available computer program.
  • Preferred computer program methods for determining identity between two sequences include, but are not limited to, the GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. et al, 1990).
  • the BLASTX program is available to the public from NCBI and other sources (BLAST Handbook, Altschul, S. et al, NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al, 1990).
  • the well-known Smith Waterman algorithm can also be used to determine identity.
  • polypeptide of the present invention has the activity of a stereoselective aminotransferase ( ⁇ -transaminase) and can be used for the production of a chiral amine or a downstream product of a chiral amine as a precursor.
  • ⁇ -transaminase stereoselective aminotransferase
  • polypeptide is:
  • the chiral amine is formed by asymmetric synthesis of a prochiral carbonyl compound.
  • the prochiral carbonyl compound is selected from the prochiral carbonyl compounds corresponding to the amino donors in Table 2 and the amino acceptor compounds in Table 3.
  • the prochiral carbonyl compound is selected from the group consisting of
  • the polypeptide is one or more, preferably 1-20, more preferably 1-15, more preferably 1-10, at either end of the amino acid sequence set forth in SEQ ID NO: 1. More preferably, the polypeptide derived from the polypeptide of the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1) is formed by the addition of 1-3, most preferably one amino acid residue.
  • amino acid sequence of the polypeptide is set forth in SEQ ID NO: 1.
  • the chiral amine comprises Compound 1a-8a or a pharmaceutically acceptable salt thereof
  • the downstream product of the chiral amine precursor comprises: sitagliptin (compound 5a), a chiral acid resolving agent or a chiral pharmaceutical intermediate (compound 1a), Dulutvir intermediate (compound 3a), chiral pharmaceutical intermediate (compound 2a, compound 4a), sitagliptin intermediate (compound 6a, compound 7a, compound 8a)
  • a strain expressing a polypeptide of the present invention is capable of stereoselectively catalyzing a reaction represented by the formula (I) to convert a prochiral carbonyl compound into a chiral amine (for example, an R-chiral amine).
  • the strain expresses the following polypeptide:
  • the polypeptide is one or more, preferably 1-20, more preferably 1-15, more preferably 1-10 at either end of the amino acid sequence set forth in SEQ ID NO:1.
  • amino acid sequence of the polypeptide is set forth in SEQ ID NO: 1.
  • the polypeptide has an amino acid sequence of at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95% of the amino acid sequence set forth in SEQ ID NO:1. 96%, 97%, 98%, 99% or more of any polypeptide sequence of sequence identity.
  • the production strain is a bacterium, preferably E. coli (e.g., E. coli BL21).
  • the stereoselective aminotransferase ( ⁇ -transaminase) can be used in various forms.
  • resting cells or wet cells expressing the ⁇ -transaminase of the present invention may be used, or various forms such as a crude enzyme solution, a pure enzyme or a crude enzyme powder may be used, or an immobilized enzyme may be used.
  • the method of producing a chiral amine comprises:
  • the previous chiral carbonyl compound is a substrate, in the presence of a prosthetic group, under the catalysis of a stereoselective transaminase, the reaction shown in the reaction formula I is carried out to form a chiral amine;
  • the method of producing a chiral amine comprises:
  • a chiral amine is isolated from the culture system of 1).
  • step 1 in the method for producing a chiral amine, an appropriate amount of pyridoxal phosphate is added to step 1).
  • a chiral amine producing strain having high conversion can be constructed by including the strain comprising an expression vector expressing the polypeptide of the present invention or integrating a gene expressing the polypeptide of the present invention into the genome of the strain.
  • the method further comprises determining the conversion of the resulting strain and/or chiral amine production to verify the resulting strain.
  • novel ⁇ transaminase disclosed by the present invention has strict R stereoselectivity and has a broad substrate spectrum, showing industrial application prospects in green production of chiral amines and unnatural amino acids.
  • Mycobacterium sp. NRRL B-3805 was purchased from the depository center, accession number: CP011022.1: 988057-989064, and PCR primers were designed based on the sequence of the Mycobacterium aureum aminotransferase gene (see Table 1).
  • PCR amplification was carried out using the genomic DNA of mycobacterial transaminase as a template.
  • the PCR system was: 10 ⁇ KOD-Plus PCR buffer 2 ⁇ L, 25 mM MgSO 4 1.2 ⁇ L, 2 mM dNTP 2 ⁇ L, KOD-Plus PCR high fidelity enzyme 0.3 ⁇ L, DNA template 0.5 ⁇ L (containing 0.1 ⁇ g of DNA template) and 13 ⁇ L of ddH 2 O were subjected to PCR amplification with 0.5 ⁇ L (10 mmol/L) of each of the gene primer upstream primer and the gene clone downstream primer in Table 1.
  • the PCR amplification steps are: (1) 95 ° C, pre-denaturation for 3 min; (2) 98 ° C, denaturation for 15 s; (3) annealing at 55 ° C for 30 s; (4) extension at 72 ° C for 1 min; steps (2) to (4) 30 times; (5) 72 ° C continued to extend for 10 min, cooled to 4 ° C.
  • the PCR product was purified by agarose gel electrophoresis.
  • the target band of 900-1200 bp was recovered by agarose gel DNA recovery kit to obtain a complete mycobacterial transaminase full-length gene sequence, which was sequenced by DNA sequencing and was 1008 bp in length.
  • MyTA the nucleotide sequence and the encoded amino acid sequence are shown as SEQ ID No: 2 and 1 in the Sequence Listing, respectively.
  • the transaminase gene of Example 1 was ligated into pET28a, and the enzyme cleavage site NdeI & HindIII was used to transform the host into a host E. coli BL21 competent cell.
  • the constructed strain was inoculated into TB medium, shaken at 200 rpm, and cultured at 30 ° C, IPTG concentration of 0.1 mM overnight, and collected.
  • the cells were resuspended in phosphate buffer (50 mM, pH 7.0), homogenized and disrupted, and the expression effect was analyzed by SDS-PAGE.
  • aqueous lithium hydroxide solution (2-3 eq lithium hydroxide, 3 times water) was prepared, and compound 6a was added thereto, and the mixture was heated to an external temperature of 95 ° C for about 16 hours.
  • the TLC showed basic reaction, and after cooling, the dichloromethane was extracted to recover the starting material.
  • the concentrated hydrochloric acid in the aqueous phase ice bath was slowly adjusted to pH 5.5-5.8 ( ⁇ 10 ° C), and washed with water to obtain a pale yellow solid (R)-3-amino-4-(2,4,5-trifluorophenyl). Butyric acid.

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Abstract

一种立体选择性的转氨酶在不对称合成手性胺中的应用。具体地，提供了一种多肽在生产手性胺或以手性胺为前体的下游产物中的用途。还提供了生产手性胺的方法，所述方法包括培养表达所述多肽的菌株，从而得到式手性胺。还提供了一种手性胺生产菌株，以及所述手性胺生产菌株的构建方法。立体选择性的转氨酶，具有宽广的底物谱，在手性胺的制备方面具有很大的应用潜力。

Description

一种立体选择性的转氨酶在不对称合成手性胺中的应用 技术领域
本发明属于生物技术领域，具体涉及一种高活性的转氨酶，以及该转氨酶作为催化剂在不对称合成手性胺中的应用，以及一种生产手性胺的方法。
背景技术
手性胺是许多药物的重要中间体及有效成分，例如(3R)-3-氨基-1-[3-(三氟甲基)-5,6-二氢-1,2,4-三唑并[4,3-a]吡嗪-7(8H)-基]-4-(2,4,5-三氟苯基)丁-1-酮是治疗II型糖尿病的有效成分，然而现有的有机合成方法不易得到光学纯的产物，而且通常需要不对称合成的金属催化剂，价格昂贵，金属残留，后处理困难。因此急需寻找手性胺生产的绿色方法。
转氨酶，又叫氨基转移酶，是催化氨基化合物和羰基化合物之间氨基可逆转移的一类酶，根据其底物氨基的位置，可以分为a-转氨酶和ω-转氨酶。a-转氨酶比较常见，只能催化a-氨基的转移，而ω-转氨酶较为罕见，除了能催化a-氨基的转移，也能催化非a-氨基的转移，具有更为宽广的底物谱和严格的立体选择性，能够在温和的反应条件下催化前手性羰基化合物来生产手性胺。另外ω-转氨酶以磷酸吡哆醛为辅基，催化反应过程中无需添加额外的辅因子循环，有利于工业化生产。
例如美国专利US8293507公开了Codexis公司对节杆菌来源的ω-转氨酶进行改造得到的生物催化剂代替了化学工艺中的铑催化剂，转氨化产物ee值达到99％，底物投料100g/L。
Figure PCTCN2018095148-appb-000001
由于酶的立体异构专一性，因此获得特定立体选择性的酶非常重要。有文献报道，ω-转氨酶中S选择性的酶较为常见，而R选择性的较为稀有，因此发掘具有工业应用前景的R型选择性的ω转氨酶对手性胺的生产具有重要的意义。
发明内容
本发明的目的之一是提供一种多肽在生产手性胺或以手性胺为前体的下游 产物中的用途。
本发明的另一目的是提供一种生产手性胺的方法。
本发明的又一目的是提供一种手性胺生产菌株。
本发明的再一目的是提供一种手性胺生产菌株的构建方法。
在本发明的第一方面，提供了一种多肽在生产手性胺或以手性胺为前体的下游产物中的用途，所述多肽是：
(a1)具有SEQ ID NO:1所示氨基酸序列的多肽；或
(b1)将SEQ ID NO:1所示氨基酸序列经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-3个、最优选1个氨基酸残基的取代、缺失或添加而形成的，具有(a1)所述多肽功能的由SEQ ID NO:1所示氨基酸序列的多肽衍生的多肽。
在另一优选例中，所述手性胺为前手性羰基化合物进行不对称合成反应形成。
在另一优选例中，所述前手性羰基化合物选自下组：
Figure PCTCN2018095148-appb-000002
在另一优选例中，(b1)所述多肽是在SEQ ID NO:1所示氨基酸序列的任一端经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-3个、最优选1个氨基酸残基的添加而形成的，具有(a1)所述多肽功能的由SEQ ID NO:1所示氨基酸序列的多肽衍生的多肽。
在另一优选例中，所述多肽的氨基酸序列如SEQ ID NO:1所示。
在另一优选例中，所述多肽的氨基酸序列为与SEQ ID NO:1所示氨基酸序列 具有至少70％，优选至少75％、80％、85％、90％，更优选至少95％、96％、97％、98％、99％以上的序列相同性的任一多肽序列。
在另一优选例中，所述手性胺包括以下化合物1a-8a或其药学上可接受的盐：
Figure PCTCN2018095148-appb-000003
在另一优选例中，所述以手性胺为前体的下游产物包括：西他列汀(化合物5a)，手性酸拆分剂或是手性药物中间体的原料(化合物1a)，度鲁特韦中间体(化合物3a)，手性药物中间体原料(化合物2a、化合物4a)，西他列汀中间体(化合物6a、化合物7a、化合物8a)。
在本发明的第二方面，提供了一种生产手性胺的方法，所述方法包括：
(a)在液态反应体系中，以前手性羰基化合物为底物，在辅基存在下，在立体选择性转氨酶催化下，进行反应式I所示的反应，从而形成手性胺；
Figure PCTCN2018095148-appb-000004
(b)任选地从所述上一步骤的反应后的反应体系中分离出手性胺。
在另一优选例中，所述反应体系中，所述前手性羰基化合物的浓度为1g/L-1000g/L，较佳地为5g/L-500g/L，更佳地为10g/L-100g/L，又更佳地为5g/L-50g/L，最佳地为20g/L-50g/L。
在另一优选例中，所述反应体系中，所述立体选择性转氨酶蛋白的浓度为1-500mg/mL，较佳地为5-200mg/mL，更佳地为10-100mg/mL，最佳地为20-80mg/mL。
在另一优选例中，所述立体选择性转氨酶：
(a1)具有SEQ ID NO:1所示氨基酸序列的多肽；或
(b1)将SEQ ID NO:1所示氨基酸序列经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-3个、最优选1个氨基酸残基的取代、缺失或添加而形成的，具有(a1)所述多肽功能的由SEQ ID NO:1所示氨基酸序列的多肽衍生的多肽。
在另一优选例中，所述多肽的氨基酸序列如SEQ ID NO:1所示。
在另一优选例中，所述多肽的氨基酸序列为与SEQ ID NO:1所示氨基酸序列具有至少70％，优选至少75％、80％、85％、90％，更优选至少95％、96％、97％、98％、99％以上的序列相同性的任一多肽序列。
在另一优选例中，所述前手性羰基化合物选自下组：
Figure PCTCN2018095148-appb-000005
在另一优选例中，所述的辅基为磷酸吡哆醛。
在另一优选例中，所述反应体系中，辅基的浓度为1-200mM，较佳地为5-150mM，更佳地为10-100mM，最佳地为5-50mM。
在另一优选例中，步骤(a)中，温度为5-50℃，较佳地为10-45℃，更佳地为20-40℃，最佳地为25-35℃。
在另一优选例中，步骤(a)中，pH为6-10，较佳地为7-10，更佳地为7.5-8.5。
在本发明的第三方面，提供了一种手性胺生产菌株，所述菌株表达以下多肽：
(a1)具有SEQ ID NO:1所示氨基酸序列的多肽；或
(b1)将SEQ ID NO:1所示氨基酸序列经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-3个、最优选1个氨基酸残基的取代、缺失或添加而形成的，具有(A)所述多肽功能的由SEQ ID NO:1所示氨基酸序列的多肽衍生的多肽。
在另一优选例中，所述多肽是在SEQ ID NO:1所示氨基酸序列的任一端经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-3个、最优选1个氨基酸残基的添加而形成的，具有(a1)所述多肽功能的由SEQ ID NO:1所示氨基酸序列的多肽衍生的多肽。
在另一优选例中，所述多肽的氨基酸序列如SEQ ID NO:1所示。
在另一优选例中，所述多肽的氨基酸序列为与SEQ ID NO:1所示氨基酸序列具有至少70％，优选至少75％、80％、85％、90％，更优选至少95％、96％、97％、98％、99％以上的序列相同性的任一多肽序列。
在另一优选例中，所述生产菌株为细菌。
在另一优选例中，所述细菌大肠杆菌。
在另一优选例中，所述大肠杆菌为大肠杆菌BL21。
在本发明的第四方面，提供了一种生产手性胺的方法，所述方法包括步骤：
1)采用生产条件培养本发明的第三方面所述的生产菌株，从而得到手性胺；
2)任选地，从1)的培养体系中分离获得手性胺。
在另一优选例中，所述方法中，步骤1)中加入适量的磷酸吡哆醛。
在本发明的第五方面，提供了一种手性胺生产菌株的构建方法，所述方法包括：
使得所述菌株包含表达以下多肽的表达载体或使得所述菌株的基因组中整合有表达以下多肽的基因，所述多肽是：
(a1)具有SEQ ID NO:1所示氨基酸序列的多肽；或
(b1)将SEQ ID NO:1所示氨基酸序列经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-3个、最优选1个氨基酸残基的取代、缺失或添加而形成的，具有(a1)所述多肽功能的由SEQ ID NO:1所示氨基酸序列的多肽衍生的多肽。
在另一优选例中，(b1)所述多肽是在SEQ ID NO:1所示氨基酸序列的任一端经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-3个、最优选1个氨基酸残基的添加而形成的，具有(a1)所述多肽功能的由SEQ ID NO:1所示氨基 酸序列的多肽衍生的多肽。
在另一优选例中，所述多肽的氨基酸序列如SEQ ID NO:1所示。
在另一优选例中，所述多肽的氨基酸序列为与SEQ ID NO:1所示氨基酸序列具有至少70％，优选至少75％、80％、85％、90％，更优选至少95％、96％、97％、98％、99％以上的序列相同性的任一多肽序列。
在另一优选例中，所述基因的序列选自下组：
(i)SEQ ID NO:2所示的序列；
(ii)与(i)限定的序列互补的多核苷酸；或
(iii)与(i)限定的序列具有至少70％(优选至少75％、80％、85％、90％，更优选至少95％、96％、97％、98％、99％)以上的序列一致性的任一多核苷酸或互补序列。
在另一优选例中，所述基因构建在表达载体上。
在本发明的第六方面，提供了一种式Ⅱ所示化合物、或其药学上可接受的盐、或其立体异构体：
Figure PCTCN2018095148-appb-000006
式中，
R 1选自下组：无、氧代(＝O)、卤素、-OH、-NH 2、取代或未取代的C 1-C 8烷基、取代或未取代的C 3-C 8环烷基、取代或未取代的C 1-C 8烷氧基、取代或未取代的C 3-C 8环烷氧基；
R 2选自下组：无、卤素、-OH、-NH 2、取代或未取代的C 1-C 8烷基、取代或未取代的C 3-C 8环烷基、取代或未取代的C 1-C 8烷氧基、取代或未取代的C 3-C 8环烷氧基；
X选自下组：C或N；
Y选自下组：C、N、O或S
其中，当Y为O或S时，R 2为无。
在另一优选例中，R 1为氧代(＝O)。
在另一优选例中，R 1为-NH 2。
在另一优选例中，X为N。
在另一优选例中，Y为O。
在另一优选例中，所述式Ⅱ所示化合物包括以下化合物；
Figure PCTCN2018095148-appb-000007
在本发明的第七方面，提供了本发明的第六方面所述的化合物、或其药学上可接受的盐、或其立体异构体的用途，用于制备西他列汀中间体Boc-(R)-3-氨基-4-(2,4,5-三氟苯基)丁酸。
在另一优选例中，所述式Ⅱ所示化合物包括以下化合物；
Figure PCTCN2018095148-appb-000008
Figure PCTCN2018095148-appb-000009
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
具体实施方式
发明人经过广泛而深入的研究，出乎意料地发现一种多肽(ω-转氨酶)，其能够作为立体选择性催化剂，将前手性羰基化合物高效地转化为R手性胺。本发明所述的ω-转氨酶具有严格的R立体选择性，并且具有宽广的底物谱，在手性胺以及手性药物的生产中具有极大的应用前景。在此基础上，完成了本发明。
术语定义
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。
如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
除非另有定义，否则在说明书和权利要求书中所使用的下述术语具有的含义为所属领域技术人员通常理解的涵义。除非另有说明，本文全文引用的所有专利、专利申请、公开材料通过引用方式整体并入本文。
应理解，上述简述和下文的详述为示例性且仅用于解释，而不对本发明主题作任何限制。在本申请中，除非另有具体说明，否则使用单数时也包括复数。必须注意，除非文中另有清楚的说明，否则在本说明书和权利要求书中所用的单数形式包括所指事物的复数形式。还应注意，除非另有说明，否则所用“或”、“或者”表示“和/或”。此外，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
可在参考文献(包括Carey and Sundberg"ADVANCED ORGANIC CHEMISTRY 4TH ED."Vols.A(2000)and B(2001),Plenum Press,New York)中找到对标准化学术语的定义。除非另有说明，否则采用本领域技术范围内的常规方法，如质谱、NMR、IR和UV/VIS光谱法和药理学方法。除非提出具体定义，否则本文在分析化学、有机合成化学以及药物和药物化学的有关描述中采用的术语是本领域已知的。可在化学合成、化学分析、药物制备、制剂和递送，以及对患者的治疗中使用标准技术。例如，可利用厂商对试剂盒的使用说明，或者按照本领域公知的方式或本发明的说明来实施反应和进行纯化。通常可根据本说明书中引用和讨论的多个概要性和较具体的文献中的描述，按照本领域熟知的常规方法实施上述技术和方法。在本说明书中，可由本领域技术人员选择基团及其取代基以提供稳定的结构部分和化合物。
当通过从左向右书写的常规化学式描述取代基时，该取代基也同样包括从右向左书写结构式时所得到的在化学上等同的取代基。举例而言，-CH 2O-等同于-OCH 2-。
本文所用的章节标题仅用于组织文章的目的，而不应被解释为对所述主题的限制。本申请中引用的所有文献或文献部分包括但不限于专利、专利申请、文章、书籍、操作手册和论文，均通过引用方式整体并入本文。
在本文中定义的某些化学基团前面通过简化符号来表示该基团中存在的碳原子总数。例如，C1-C6烷基是指具有总共1至6个碳原子的如下文所定义的烷基。简化符号中的碳原子总数不包括可能存在于所述基团的取代基中的碳。
除前述以外，当用于本申请的说明书及权利要求书中时，除非另外特别指明，否则以下术语具有如下所示的含义。
在本申请中，术语“卤素”是指氟、氯、溴或碘。
“羟基”是指-OH基团。
在本申请中，作为基团或是其它基团的一部分(例如用在卤素取代的烷基 等基团中)，术语“烷基”是指完全饱和的直链或支链的烃链基，仅由碳原子和氢原子组成、具有例如1至7个碳原子，且通过单键与分子的其余部分连接，例如包括但不限于甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基、2-甲基丁基、2,2-二甲基丙基、正己基、庚基等。
多肽
本文所用的术语“多肽”或“本发明多肽”或“本发明的多肽”或“ω-转氨酶”或“立体选择性转氨酶”具有相同的意义，在本文可以互换使用，均是指具有催化前手性羰基化合物产生手性胺活性的蛋白。大肠杆菌中天然不存在此多肽，其属于外源性蛋白。
基于现有技术的知识，本领域普通技术人员不难知晓，在多肽的某些区域，例如非重要区域改变少数氨基酸残基基本上不会改变生物活性，例如，适当替换某些氨基酸得到的序列并不会影响其活性(可参见Watson等，Molecular Biology of The Gene，第四版，1987，The Benjamin/Cummings Pub.Co.P224)。因此，本领域普通技术人员能够实施这种替换并且确保所得分子仍具有所需生物活性。
因此，在具体的实施方式中，本发明的多肽可以是：(a1)具有SEQ ID NO:1所示氨基酸序列的多肽；或(b1)将SEQ ID NO:1所示氨基酸序列经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-3个、最优选1个氨基酸残基的取代、缺失或添加而形成的，具有(a1)所述多肽功能的由SEQ ID NO:1所示氨基酸序列的多肽衍生的多肽。
在优选的实施方式中，所述多肽是在SEQ ID NO:1所示氨基酸序列的任一端经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-3个、最优选1个氨基酸残基的添加而形成的，具有(a1)所述多肽功能的由SEQ ID NO:1所示氨基酸序列的多肽衍生的多肽。
在优选的实施方式中，所述多肽的氨基酸序列如SEQ ID NO:1所示。
在另一优选例中，所述多肽的氨基酸序列为与SEQ ID NO:1所示氨基酸序列具有至少70％，优选至少75％、80％、85％、90％，更优选至少95％、96％、97％、98％、99％以上的序列相同性的任一多肽序列。
在具体的实施方式中，本发明多肽表示氨基酸序列如SEQ ID NO:1所示的 蛋白质，其编码序列如SEQ ID NO:2所示。
在本发明中，本发明多肽包括与氨基酸序列SEQ ID NO:1所示的多肽相比，有至多20个、较佳地至多10个，再佳地至多3个，更佳地至多2个，最佳地至多1个氨基酸被性质相似或相近的氨基酸所替换而形成的突变体。这些保守性变异的突变体可根据，例如下表所示进行氨基酸替换而产生。
初始残基 代表性的取代残基 优选的取代残基
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还提供了编码本发明多肽的多核苷酸。术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
因此，本文所用的“含有”，“具有”或“包括”包括了“包含”、“主要由……构成”、“基本上由……构成”、和“由……构成”；“主要由……构成”、“基本上由……构成”和“由……构成”属于“含有”、“具有”或“包括”的下位概念。
在具体的实施方式中，所述同源性或序列相同性可以是80％以上，优选90％以上，更优选95％-98％，最优选99％以上。
本领域普通技术人员公知的测定序列同源性或相同性的方法包括但不限于：计算机分子生物学(Computational Molecular Biology)，Lesk，A.M.编，牛津大学出版社，纽约，1988；生物计算：信息学和基因组项目(Biocomputing:Informatics and Genome Projects)，Smith，D.W.编，学术出版社，纽约，1993；序列数据的计算机分析(Computer Analysis of Sequence Data)，第一部分，Griffin，A.M.和Griffin，H.G.编，Humana Press，新泽西，1994；分子生物学中的序列分析(Sequence Analysis in Molecular Biology)，von Heinje，G.，学术出版社，1987和序列分析引物(Sequence Analysis Primer)，Gribskov，M.与Devereux，J.编M Stockton Press，纽约，1991和Carillo，H.与Lipman，D.，SIAM J.Applied Math.，48:1073(1988)。测定相同性的优选方法要在测试的序列之间得到最大的匹配。测定相同性的方法编译在公众可获得的计算机程序中。优选的测定两条序列之间相同性的计算机程序方法包括但不限于：GCG程序包(Devereux，J.等，1984)、BLASTP、BLASTN和FASTA(Altschul，S，F.等，1990)。公众可从NCBI和其它来源得到BLASTX程序(BLAST手册，Altschul，S.等，NCBI NLM NIH Bethesda，Md.20894；Altschul，S.等，1990)。熟知的Smith Waterman算法也可用于测定相同性。
多肽的用途
本发明人出乎意料的发现本发明多肽具有立体选择性转氨酶(ω-转氨酶)的活性，能够用于生产手性胺或以手性胺为前体的下游产物。
在具体的实施方式中，所述多肽是：
(a1)具有SEQ ID NO:1所示氨基酸序列的多肽；或
(b1)将SEQ ID NO:1所示氨基酸序列经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-3个、最优选1个氨基酸残基的取代、缺失或添加 而形成的，具有(a1)所述多肽功能的由SEQ ID NO:1所示氨基酸序列的多肽衍生的多肽。
在优选的实施方式中，所述手性胺为前手性羰基化合物进行不对称合成反应形成。
在优选的实施方式中，所述前手性羰基化合物选自表2中氨基供体对应的前手性羰基化合物和表3中的氨基受体化合物。
典型地，所述前手性羰基化合物选自下组：
Figure PCTCN2018095148-appb-000010
在优选的实施方式中，所述多肽是在SEQ ID NO:1所示氨基酸序列的任一端经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-3个、最优选1个氨基酸残基的添加而形成的，具有(a1)所述多肽功能的由SEQ ID NO:1所示氨基酸序列的多肽衍生的多肽。
典型地，所述多肽的氨基酸序列如SEQ ID NO:1所示。
在优选的实施方式中，所述手性胺包括化合物1a-8a或其药学上可接受的盐
Figure PCTCN2018095148-appb-000011
在优选的实施方式中，所述以手性胺为前体的下游产物包括：西他列汀(化合物5a)，手性酸拆分剂或是手性药物中间体的原料(化合物1a)，度鲁特韦中间体(化合物3a)，手性药物中间体原料(化合物2a、化合物4a)，西他列汀中间体(化合物6a、化合物7a、化合物8a)
手性胺生产菌株
本发明人出乎意料的发现表达本发明多肽的菌株能够立体选择性地催化反应式(I)所示的反应，将前手性羰基化合物转化为手性胺(例如R-手性胺)。
在具体的实施方式中，所述菌株表达以下多肽：
(a1)具有SEQ ID NO:1所示氨基酸序列的多肽；或
(b1)将SEQ ID NO:1所示氨基酸序列经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-3个、最优选1个氨基酸残基的取代、缺失或添加而形成的，具有(A)所述多肽功能的由SEQ ID NO:1所示氨基酸序列的多肽衍生的多肽。
在另一优选的实施方式中，所述多肽是在SEQ ID NO:1所示氨基酸序列的任一端经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-3个、最优选1个氨基酸残基的添加而形成的，具有(a1)所述多肽功能的由SEQ ID NO:1所示氨基酸序列的多肽衍生的多肽。
在另一优选的实施方式中，所述多肽的氨基酸序列如SEQ ID NO:1所示。
在另一优选的实施方式中，所述多肽的氨基酸序列为与SEQ ID NO:1所示氨基酸序列具有至少70％，优选至少75％、80％、85％、90％，更优选至少95％、96％、97％、98％、99％以上的序列相同性的任一多肽序列。
在另一优选的实施方式中，所述生产菌株是细菌，优选大肠杆菌(例如大肠杆菌BL21)。
生产手性胺的方法
在本发明中，所述的立体选择性转氨酶(ω-转氨酶)可以各种形式使用。例如，可使用表达本发明所述ω-转氨酶的静息细胞或湿菌体，也可以使用粗酶液、纯酶或者粗酶粉等各种不同形式，或者使用固定化酶。
在优选的实施方式中，所述生产手性胺的方法包括：
(a)在液态反应体系中，以前手性羰基化合物为底物，在辅基存在下，在立体选择性转氨酶催化下，进行反应式I所示的反应，从而形成手性胺；
Figure PCTCN2018095148-appb-000012
(b)任选地从所述上一步骤的反应后的反应体系中分离出手性胺。
在另一优选的实施方式中，所述生产手性胺的方法包括：
1)采用生产条件培养本发明的手性胺生产菌株，从而得到手性胺；
2)任选地，从1)的培养体系中分离获得手性胺。
在另一优选的实施方式中，所述生产手性胺的方法中，步骤1)中加入适量的磷酸吡哆醛。
手性胺生产菌株的构建方法
发明人出乎意料地发现通过使得所述菌株包含表达本发明多肽的表达载体或使得所述菌株的基因组中整合有表达本发明多肽的基因，可构建具有高转化率的手性胺生产菌株。
在另一具体的实施方式中，所述方法还包括测定所得菌株的转化率和/或手性胺产量以验证所得菌株。
本发明的主要优点：
(1)本发明公布的新ω转氨酶具有严格的R立体选择性，并且具有宽广的底物谱，在手性胺和非天然氨基酸的绿色生产中显示出工业应用前景。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
本发明所用试剂和原料均市售可得。除非另有定义，本文所用的所有技术 和科学术语与本发明所属领域普通技术人员通常理解的意义相同。虽然可利用与本文所述相似或等价的任何方法和材料来实施或检验本发明，但优选本文所述的方法和材料。
实施例1 小金色分支杆菌转氨酶
从保藏中心购买到小金色分支杆菌Mycobacterium sp.NRRL B-3805，馆藏号：CP011022.1:988057-989064，以小金色分支杆菌转氨酶基因序列为依据，设计PCR引物(见表1)。
表1：引物序列表
Figure PCTCN2018095148-appb-000013
以分支杆菌转氨酶基因组DNA为模板，进行PCR扩增，PCR体系为：10×KOD-Plus PCR buffer 2μL，25mM MgSO 4 1.2μL，2mM dNTP 2μL，KOD-Plus PCR高保真酶0.3μL，DNA模板0.5μL(含DNA模板0.1μg)，ddH 2O 13μL，以表1中基因克隆上游引物与基因克隆下游引物各0.5μL(10mmol/L)进行PCR扩增。PCR扩增步骤为：(1)95℃，预变性3min；(2)98℃，变性15s；(3)55℃退火30s；(4)72℃延伸1min；步骤(2)～(4)重复30次；(5)72℃继续延伸10min，冷却至4℃。PCR产物经琼脂糖凝胶电泳纯化，利用琼脂糖凝胶DNA回收试剂盒回收900～1200bp区间的目标条带，获得一条完整的分支杆菌转氨酶全长基因序列，经DNA测序，全长1008bp，命名为MyTA，其核苷酸序列与编码的氨基酸序列分别如序列表中SEQ ID No：2和1所示。
SEQ ID No：1
Figure PCTCN2018095148-appb-000014
SEQ ID No：2
Figure PCTCN2018095148-appb-000015
Figure PCTCN2018095148-appb-000016
实施例2 酶的表达
将实施例1中转氨酶基因酶连pET28a，酶切位点NdeI & HindIII，将酶连好的载体，转化宿主大肠杆菌BL21感受态细胞。将构建好的菌种接种TB培养基，200rpm摇床，30℃，IPTG浓度0.1mM诱导过夜，收菌。用磷酸盐缓冲液(50mM，pH7.0)重悬细胞，均质破碎，SDS-PAGE分析表达效果。
实施例3 化合物6，7，8的合成
将2,2-二甲基-5-[2-(2,4,5-三氟苯基)乙酰基]-[1,3]二氧六环-4,6-二酮(购自江苏八巨药业有限公司)悬浮于3倍甲苯中，搅拌下慢慢加入吗啉(1eq)，室温搅拌反应1h，氮气保护下升温至110℃回流反应16h。TLC显示反应完全，HPLC显示原料<1％。冷却后浓缩得化合物6。
经测试： 1HNMR(400MHz,DMSO-D6)δ7.52-7.45(m，1H)，7.40-7.33(m，1H)，3.91(s，2H)，3.76(s，2H)，3.53-3.51(t，4H)，3.44-3.42(d，2H)，3.41-3.32(t，2H)。
Figure PCTCN2018095148-appb-000017
同样合成方法将吗啉换成N-甲基哌嗪或哌嗪分别合成了化合物7和8。
实施例4 转氨酶氨基供体底物谱
反应式：
1-(3-(三氟甲基)-5,6-二氢-[1,2,4]三唑并[4,3-a]吡嗪-7(8H)-基)-4-(2,4,5-三氟苯基)丁-1,3-二酮+氨基供体→(3R)-3-氨基-1-[3-(三氟甲基)-5,6-二氢-1,2,4-三唑并[4,3-a]吡嗪-7(8H)-基]-4-(2,4,5-三氟苯基) 丁-1-酮+氨基供体脱氨后的羰基化合物
反应条件：
1ml 0.2M TEA-HCl缓冲液中，pH 8.0，加入DMSO 0.1ml，20mM磷酸吡哆醛0.1ml，1-(3-(三氟甲基)-5,6-二氢-[1,2,4]三唑并[4,3-a]吡嗪-7(8H)-基)-4-(2,4,5-三氟苯基)丁-1,3-二酮25mg，氨基供体20mg，转氨酶(蛋白浓度20mg/mL)0.5ml，于45℃反应6小时后，用乙腈终止反应。检测反应体系中氨基供体的转化率(见表2)。
表2：不同氨基供体的转化率
氨基供体 转化率
α-苯乙胺 31％
β-氨基丁酸 2％
甘氨酸 0
丙氨酸 3.6％
异丙胺 35％
实施例5 转氨酶氨基受体底物谱
反应式：
异丙胺+氨基受体→丙酮+氨基受体加氨后的氨基化合物
反应条件：
1ml 0.2M TEA-HCl缓冲液中，pH 8.0,加入DMSO 0.1ml，磷酸吡哆醛20mM，0.1ml，pH 8.0的异丙胺4M 0.2ml，氨基受体20mg，转氨酶(蛋白浓度20mg/mL)0.5ml，于45℃反应6小时后，用乙腈终止反应。检测反应中氨基受体的转化率(见表3)。
表3：不同氨基受体时的转化率
Figure PCTCN2018095148-appb-000018
Figure PCTCN2018095148-appb-000019
实施例6 化合物6a进一步合成西他列汀
化合物6a测试： 1HNMR(400MHz,DMSO-D6)δ7.46-7.38(m，2H)，3.53-3.49(m，4H)，3.40-3.37(t，4H)，3.24-3.21(t，1H)，2.66-2.52(d，1H)，2.54-2.52(d，1H)，2.31-2.30(d，2H)，1.56(s，2H)。
配制氢氧化锂水溶液(2-3eq氢氧化锂，3倍水)，加入化合物6a，加热至外温95℃，反应约16h。TLC显示基本反应完，冷却后二氯甲烷萃取，回收原料。水相冰浴下浓盐酸慢慢调pH 5.5-5.8(<10℃)，过滤水洗烘干得淡黄色固体(R)-3-氨基-4-(2,4,5-三氟苯基)丁酸。
(R)-3-氨基-4-(2,4,5-三氟苯基)丁酸溶于20倍的氢氧化钠水溶液中(10eq NaOH)，控制温度25±3℃下滴加boc酸酐(3eq)，25-30℃条件下继续滴加约(0.3eq)使原料反应完，HPLC跟踪原料<0.5％。5倍的二氯甲烷萃取杂质两次，二氯甲烷合并后水洗两次，水相合并后10-20℃条件下1N的盐酸调pH至2.5-3.0。过滤后50倍水打浆，HPLC检测单杂<0.1％，20-25℃真空干燥，得产品Boc-(R)-3-氨基-4-(2,4,5-三氟苯基)丁酸。
Figure PCTCN2018095148-appb-000020
以上对本发明做了详尽的描述，其目的在于让熟悉此领域的技术人员能够了解本发明的内容并加以实施，并不能以此限制本发明的保护范围，凡根据本发明的精神实质所做的等效变化或修饰，都应涵盖在本发明的保护范围之内。
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单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (14)

  1. 一种多肽在生产手性胺或以手性胺为前体的下游产物中的用途，其特征在于，所述多肽是：
    (a1)具有SEQ ID NO:1所示氨基酸序列的多肽；或
    (b1)将SEQ ID NO:1所示氨基酸序列经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-3个、最优选1个氨基酸残基的取代、缺失或添加而形成的，具有(a1)所述多肽功能的由SEQ ID NO:1所示氨基酸序列的多肽衍生的多肽。
  2. 如权利要求1所述的用途，其特征在于，(b1)所述多肽是在SEQ ID NO:1所示氨基酸序列的任一端经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-3个、最优选1个氨基酸残基的添加而形成的，具有(a1)所述多肽功能的由SEQ ID NO:1所示氨基酸序列的多肽衍生的多肽。
  3. 如权利要求1所述的用途，其特征在于，所述多肽的氨基酸序列如SEQ ID NO:1所示。
  4. 如权利要求1-3中任一项所述的用途，其特征在于，所述手性胺包括以下化合物1a-8a或其药学上可接受的盐：
    Figure PCTCN2018095148-appb-100001
  5. 如权利要求1-3中任一项所述的用途，其特征在于，所述以手性胺为前体的下游产物包括：西他列汀(化合物5a)，手性酸拆分剂或是手性药物中间体的原料(化合物1a)，度鲁特韦中间体(化合物3a)，手性药物中间体原料(化合物2a、化合物4a)，西他列汀中间体(化合物6a、化合物7a，化合物8a)。
  6. 一种生产手性胺的方法，其特征在于：所述方法包括：
    (a)在液态反应体系中，以前手性羰基化合物为底物，在辅基存在下，在立体选择性转氨酶催化下，进行反应式I所示的反应，从而形成手性胺；
    Figure PCTCN2018095148-appb-100002
    (b)任选地从所述上一步骤的反应后的反应体系中分离出手性胺。
  7. 一种手性胺生产菌株，其特征在于，所述菌株表达以下多肽：
    (a1)具有SEQ ID NO:1所示氨基酸序列的多肽；或
    (b1)将SEQ ID NO:1所示氨基酸序列经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-3个、最优选1个氨基酸残基的取代、缺失或添加而形成的，具有(A)所述多肽功能的由SEQ ID NO:1所示氨基酸序列的多肽衍生的多肽。
  8. 一种生产手性胺的方法，其特征在于，所述方法包括步骤：
    1)采用生产条件培养权利要求7所述的生产菌株，从而得到手性胺；
    2)任选地，从1)的培养体系中分离获得手性胺。
  9. 一种手性胺生产菌株的构建方法，其特征在于，所述方法包括：
    使得所述菌株包含表达以下多肽的表达载体或使得所述菌株的基因组中整合有表达以下多肽的基因，所述多肽是：
    (a1)具有SEQ ID NO:1所示氨基酸序列的多肽；或
    (b1)将SEQ ID NO:1所示氨基酸序列经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-3个、最优选1个氨基酸残基的取代、缺失或添加而形成的，具有(a1)所述多肽功能的由SEQ ID NO:1所示氨基酸序列的多肽衍生的多肽。
  10. 根据权利要求9所述的制备方法，其特征在于，所述基因的序列选自下组：
    (i)SEQ ID NO:2所示的序列；
    (ii)与(i)限定的序列互补的多核苷酸；或
    (iii)与(i)限定的序列具有至少70％，优选至少75％、80％、85％、90％，更优选至少95％、96％、97％、98％、99％以上的序列一致性的任一多核苷酸或互补序列。
  11. 一种式Ⅱ所示化合物、或其药学上可接受的盐、或其立体异构体：
    Figure PCTCN2018095148-appb-100003
    式中，
    R 1选自下组：无、氧代(＝O)、卤素、-OH、-NH 2、取代或未取代的C 1-C 8烷基、取代或未取代的C 3-C 8环烷基、取代或未取代的C 1-C 8烷氧基、取代或未取代的C 3-C 8环烷氧基；
    R 2选自下组：无、卤素、-OH、-NH 2、取代或未取代的C 1-C 8烷基、取代或未取代的C 3-C 8环烷基、取代或未取代的C 1-C 8烷氧基、取代或未取代的C 3-C 8环烷氧基；
    X选自下组：C或N；
    Y选自下组：C、N、O或S；
    其中，当Y为O或S时，R 2为无。
  12. 根据权利要求11所述的化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，所述式Ⅱ所示化合物包括以下化合物；
    Figure PCTCN2018095148-appb-100004
  13. 权利要求11所述的式Ⅱ所示化合物、或其药学上可接受的盐、或其立体异构体的用途，其特征在于，用于制备西他列汀中间体Boc-(R)-3-氨基-4-(2,4,5-三氟苯基)丁酸。
  14. 根据权利要求13所述的用途，其特征在于，所述式Ⅱ所示化合物包括以下化合物；
    Figure PCTCN2018095148-appb-100005
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