WO2018090929A1 - (1r,2s)-2-(3,4-二氟苯基)环丙胺d-扁桃酸盐(i)的生物制备方法 - Google Patents

(1r,2s)-2-(3,4-二氟苯基)环丙胺d-扁桃酸盐(i)的生物制备方法 Download PDF

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WO2018090929A1
WO2018090929A1 PCT/CN2017/111094 CN2017111094W WO2018090929A1 WO 2018090929 A1 WO2018090929 A1 WO 2018090929A1 CN 2017111094 W CN2017111094 W CN 2017111094W WO 2018090929 A1 WO2018090929 A1 WO 2018090929A1
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compound
formula
reaction system
coenzyme
preparation
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French (fr)
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张福利
倪国伟
陈少欣
鞠佃文
汤佳伟
谭支敏
邹杰
郭翔
王政文
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中国医药工业研究总院
上海医药工业研究院
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C209/00—Preparation of compounds containing amino groups bound to a carbon skeleton
    • C07C209/62—Preparation of compounds containing amino groups bound to a carbon skeleton by cleaving carbon-to-nitrogen, sulfur-to-nitrogen, or phosphorus-to-nitrogen bonds, e.g. hydrolysis of amides, N-dealkylation of amines or quaternary ammonium compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C209/00—Preparation of compounds containing amino groups bound to a carbon skeleton
    • C07C209/68—Preparation of compounds containing amino groups bound to a carbon skeleton from amines, by reactions not involving amino groups, e.g. reduction of unsaturated amines, aromatisation, or substitution of the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C231/00—Preparation of carboxylic acid amides
    • C07C231/02—Preparation of carboxylic acid amides from carboxylic acids or from esters, anhydrides, or halides thereof by reaction with ammonia or amines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/41—Preparation of salts of carboxylic acids
    • C07C51/412—Preparation of salts of carboxylic acids by conversion of the acids, their salts, esters or anhydrides with the same carboxylic acid part
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/30—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group
    • C07C67/333—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group by isomerisation; by change of size of the carbon skeleton
    • C07C67/343—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group by isomerisation; by change of size of the carbon skeleton by increase in the number of carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic

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  • the invention belongs to the technical field of medicine, and in particular relates to a preparation method of (1R, 2S)-2-(3,4-difluorophenyl)cyclopropylamine D-mandelic acid salt.
  • Ticagrelor chemical name [1S-[1 ⁇ , 2 ⁇ , 3 ⁇ (1S, 2R), 5 ⁇ ]]-3-[7-[2-(3,4-difluorophenyl)-cyclopropylamino ]-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)cyclopentane-1 , 2-diol, is an oral antiplatelet drug developed by AstraZeneca AB.
  • the drug can reversibly act on the ADP P2Y12 receptor, has a significant inhibitory effect on platelet aggregation caused by ADP, and has a rapid onset of action. It is clinically used to reduce the incidence of thrombotic cardiovascular events in patients with acute coronary syndrome.
  • ticagrelor The structural formula of ticagrelor is as follows:
  • (1R,2S)-2-(3,4-difluorophenyl)cyclopropylamine (II) is a key intermediate for the synthesis of ticagrelor. Because it is an oil, it is not conducive to preservation and quality control, so it is usually (1R, 2S)-2-(3,4-difluorophenyl)cyclopropylamine D-mandelate (I) was obtained as D-mandelic acid salt.
  • Patent WO 2008018822 discloses a method for preparing compound (II) as follows:
  • the catalyst S-2-methoxy-CBS-oxazol borane of the CBS reduction reaction in this method is unstable, uncommercial, and must be prepared in situ from trimethoxyborane and S-diphenylvalerol.
  • the operation process is increased, which is not conducive to industrial production; and the reducing agent borane-dimethyl sulfide complex releases malodorous dimethyl sulfide gas during post-treatment, which is not conducive to labor protection of production.
  • the compound (VI) is reduced with S-2-methoxy-CBS-oxazolidine borane and borane-dimethyl sulfide complex prepared in situ, and reacted at 40 ° C for 1 h to obtain a compound ( V), its ee value is only 76%.
  • the ee value of (1R,2S)-2-(3,4-difluorophenyl)cyclopropylamine (II) prepared by the method was 81%.
  • the CBS reduction reaction of the process is optimized, and the compound (VI) is obtained by reducing the compound (VI) by S-2-methyl-CBS-oxazolidine borane and borane-tetrahydrofuran complex ( V), increasing the ee value from 76% to 98-99%. Because the reaction control of the reaction conditions is more stringent, it is found that the product ee value is difficult to reach 99% and stabilized at 96-97% in the amplification experiment. When the reaction is quenched after the reaction, the system has a strong exotherm and there is obvious safety. Hidden dangers.
  • the above method is to obtain a chiral alcohol compound (V) by reducing a carbonyl group by borane under the catalysis of a chiral ligand.
  • the method has the following disadvantages: high ligand cost, sensitivity to oxygen and water, and therefore harsh process conditions. Such as nitrogen protection, anhydrous and oxygen-free; solvent system complex toluene, tetrahydrofuran mixed system.
  • biocatalysis is more green, environmentally friendly and economical. Stereoselection will be realized in biocatalysis theory
  • An enzyme that reduces a carbonyl group to a chiral hydroxyl group is called a carbonyl reductase.
  • the object of the present invention is to provide a process for the preparation of (1R,2S)-2-(3,4-difluorophenyl)cyclopropylamine D-mandelate biocatalyst.
  • a first aspect of the invention provides a process for the preparation of a compound of formula V, comprising the steps of:
  • the concentration of the compound of the formula VI in the reaction system is from 50 to 1000 g/L.
  • the concentration of the compound of the formula VI in the reaction system is from 60 to 700 g/L.
  • the concentration of the compound of the formula VI in the reaction system is from 80 to 600 g/L.
  • the concentration of the compound of the formula VI in the reaction system is from 20 to 500 g/L.
  • the concentration of the compound of the formula VI in the reaction system is from 50 to 200 g/L.
  • the concentration of the compound of the formula VI in the reaction system is from 100 to 150 g/L.
  • a co-substrate is also present in the reaction system.
  • the co-substrate is selected from the group consisting of isopropanol, glucose, ammonium formate, or a combination thereof.
  • the concentration of the co-substrate in the reaction system is 5-30%.
  • an enzyme for coenzyme regeneration is also present in the reaction system.
  • the enzyme for coenzyme regeneration is selected from the group consisting of alcohol dehydrogenase, formate dehydrogenase, glucose dehydrogenase, or a combination thereof.
  • the temperature is from 10 ° C to 50 ° C, preferably from 20 ° C to 40 ° C, more preferably from 25 ° C to 35 ° C.
  • the time is from 0.1 to 240 hours, preferably from 0.5 to 120 hours, more preferably from 1 to 72 hours, still more preferably from 3 to 10 hours.
  • the pH is from 6 to 9, preferably from 6.5 to 8.5, more preferably from 7.0 to 7.5.
  • the carbonyl reductase in the reaction system, is an enzyme in a free form, an immobilized enzyme, or an enzyme in the form of a cell.
  • reaction system is a phosphate buffer salt system.
  • reaction system further contains a co-solvent.
  • the co-solvent is selected from the group consisting of dimethyl sulfoxide, methanol, ethanol, isopropanol, acetonitrile, toluene, acetone, or a combination thereof.
  • the concentration of the co-solvent is from 5 to 30%.
  • the separating comprises: adding isopropanol, centrifuging the cells, partially concentrating, extracting with a tertiary ether, and concentrating the organic layer.
  • the compound of the formula V in the step (b), in the reaction system after the reaction, has an ee value of ⁇ 90%, preferably ⁇ 95%, more preferably ⁇ 99%.
  • step (b) ⁇ 80% (preferably ⁇ 85%, more preferably ⁇ 90%) of the compound of formula VI is converted to a compound of formula V in the reacted reaction system .
  • the concentration of the compound of the formula V in the reaction system after the reaction is 50 to 1000 g/L.
  • the carbonyl reductase is selected from the group consisting of:
  • the coding gene sequence of the carbonyl reductase QNR is selected from the group consisting of:
  • the carbonyl reductase gene is constructed on an expression vector.
  • the coenzyme is selected from the group consisting of: a reduced coenzyme, an oxidative coenzyme, or a combination thereof.
  • the reducing coenzyme is selected from the group consisting of NADH, NADPH, or a combination thereof.
  • the oxidative coenzyme is selected from the group consisting of NAD, NADP, or a combination thereof.
  • the ratio of the amount of NAD to the amount of substrate used is from 0.01% to 1.0% (w/w), preferably from 0.01% to 0.5% (w/w).
  • the enzyme for coenzyme regeneration is an alcohol dehydrogenase, the gene of which is selected from the group consisting of:
  • the gene for the enzyme for coenzyme regeneration is constructed on an expression vector.
  • a second aspect of the invention provides a reaction system comprising:
  • the concentration of the compound of formula VI in the reaction system is from 50 to 1000 g/L.
  • a third aspect of the invention provides a process for the preparation of a compound of formula V, comprising the steps of: using the reaction system of the second aspect of the invention, enzymatically reacting under conditions suitable for enzymatic catalysis, thereby producing a compound of formula V :
  • the concentration of the compound of formula VI in the reaction system is from 50 to 1000 g/L.
  • a process for the preparation of a compound of the formula I wherein the compound of the formula V obtained by the process of the first aspect of the invention is subjected to the following reaction step.
  • Figure 1 shows the chiral map of the racemate of the compound of formula V.
  • Figure 2 shows a comparison of the resulting compound of formula V with a racemate catalyzed by a carbonyl reductase.
  • Figure 3 shows the ee value of the resulting compound of formula V catalyzed by a carbonyl reductase.
  • the inventors have unexpectedly discovered, for the first time, a preparation method of (1R,2S)-2-(3,4-difluorophenyl)cyclopropylamine D-mandelate (I) biocatalysis.
  • the present invention improves the key carbonyl reduction reaction in the preparation process of the existing (1R,2S)-2-(3,4-difluorophenyl)cyclopropylamine D-mandelate (I) to a carbonyl reductase Catalytic reduction, in the presence of a carbonyl reductase and a coenzyme, stereoselective reduction of a compound of formula VI to a compound of formula V (even at high concentrations of substrate, such as 50 to 1000 g/L, very efficiently Preparation of a compound of formula V having a stereoconfiguration).
  • the co-enzyme regeneration is coupled in the reaction system, thereby significantly increasing the reaction system to the organic solvent and the bottom.
  • Tolerance of the material which can greatly increase the substrate concentration, and can also be very efficiently prepared to obtain a compound of the formula V having a stereoconfiguration (ee value ⁇ 98%, 99%, or higher), thereby greatly improving production efficiency. ,reduce manufacturing cost.
  • the process of the present invention greatly simplifies subsequent processing and significantly reduces or eliminates the use of various types of contaminating chemicals as compared to chemical synthesis, thereby significantly reducing the risk of environmental pollution.
  • carbonyl reductase is an enzyme capable of stereoselectively catalyzing asymmetric reduction of a latent ketone to give a chiral alcohol.
  • the carbonyl reductase may be wild-type or mutant. In addition, it can be separated or recombined.
  • the carbonyl reductase useful in the present invention may be from a different species.
  • a carbonyl reductase from Microbacterium luteolum preferably from Microbacterium luteolum.
  • an enzyme having similar activity or homology e.g., ⁇ 80%, preferably ⁇ 90%, more preferably ⁇ 95%) to the above carbonyl reductase is also present in the present invention.
  • a representative carbonyl reductase is a carbonyl reductase of Microbacterium luteolum JCM 9174 (Microbacterium luteolum JCM 9174).
  • a typical carbonyl reductase has the amino acid sequence shown in SEQ ID No.: 2, and the coding gene thereof is shown in SEQ ID No.: 1.
  • the base sequence encoding the amino acid sequence shown by SEQ ID NO. 2 is not limited to SEQ ID NO.
  • Those skilled in the art can obtain homologs of the base sequence by appropriately introducing substitutions, deletions, alterations, insertions or additions, and the present invention encompasses such homologs as long as the recombinant enzyme expressed thereby retains the catalytic reduction activity of the compound of formula VI. can.
  • the homologue of the polynucleotide of the present invention can maintain the activity of the enzyme by one or more bases of the base sequence of SEQ ID NO. It is made by replacing, missing or adding in the circumference.
  • the carbonyl reductase of the present invention further comprises an amino acid sequence obtained by subjecting the amino acid sequence shown by SEQ ID NO. 2 to one or more amino acid substitutions, deletions, alterations, insertions or additions within the range of maintaining enzyme activity.
  • a recombinase resting cell, a wet cell, a crude enzyme solution, a pure enzyme or a crude enzyme powder constructed by the above-described carbonyl reductase can be used in the reaction system.
  • a crude enzyme solution In order to obtain higher conversion efficiency, it is preferred to use a crude enzyme solution.
  • the ratio of the amount of the carbonyl reductase to the amount of the substrate is preferably from 1% to 6% (w/w), or the ratio of the resting cell mass to the substrate mass is from 10 to 100%.
  • coenzyme means a coenzyme capable of realizing electron transport in a redox reaction.
  • the coenzyme of the invention is the reduced coenzyme NADH, NADPH or the oxidative coenzyme NAD + , NADP + . Since the cost of reducing coenzyme is expensive, it is preferred to select the oxidative coenzymes NAD + and NADP + .
  • oxidizing coenzyme When oxidizing coenzyme is selected, it is necessary to select a method for achieving coenzyme regeneration, which mainly includes three kinds of (1) glucose dehydrogenase and co-substrate glucose; (2) alcohol dehydrogenase and co-substrate isopropanol; (3) formic acid Hydrogenase and co-substrate ammonium formate.
  • the coenzyme is NAD + and the coenzyme regeneration system is an alcohol dehydrogenase.
  • the alcohol dehydrogenase and the co-substrate isopropanol are preferred, and the coding sequence of the alcohol dehydrogenase is as shown in SEQ ID NO. Show (NCBI's accession number is AB213459).
  • the ratio of the amount of the oxidizing coenzyme NAD + to the substrate is 0.01% to 0.5% (w/w), and the amount of the isopropanol is 5% to 30% (v/v) of the buffer volume.
  • the buffer system was 0.1 mol/L phosphate buffer salt.
  • the pH of the buffer is 7.0-7.3.
  • a co-solvent may or may not be added to the reaction system.
  • solvent means a complex, a complex or a complex salt of a poorly soluble substance with a third substance added in a solvent to increase the solubility of a poorly soluble substance in a solvent. Solubility. This third substance is called a co-solvent.
  • the substrate compound (VI) is poorly soluble in water, and when the concentration of the substrate is increased, the reaction conversion rate is seriously affected. Therefore, it is necessary to improve the solubility of the substrate by adding a cosolvent to improve the reaction conversion.
  • a cosolvent are dimethyl sulfoxide, methanol, ethanol, isopropanol, acetonitrile, toluene, acetone, concentration It is preferably 5-30% (v/v), preferably dimethyl sulfoxide, methanol, ethanol, isopropanol.
  • the invention provides a preparation method of a compound of formula V, comprising the steps of:
  • the above reaction may or may not be coupled to a coenzyme regeneration system.
  • the above reaction is a coupling of a coenzyme regeneration system in the same system to further increase production efficiency, reduce production costs, and increase tolerance to substrates.
  • the present invention also provides a reaction system for use in the biological preparation method of the present invention.
  • a typical reaction system including:
  • a coenzyme regeneration system is also coupled to further increase production efficiency, reduce production costs, and increase resistance to substrates.
  • the invention also provides the use of the reaction system of the invention, that is, for preparing a compound of the formula V, which comprises the steps of: performing an enzymatic reaction under conditions suitable for enzyme catalysis using the reaction system; Compound of formula V:
  • reaction system and method of the present invention reduces or eliminates the use of various types of polluting chemicals, and does not require the use of expensive stereostructure separation reagents, it not only significantly reduces the risk of environmental pollution, but also is inexpensive.
  • the present invention is suitable for industrial production of a compound of formula V having high chemical purity and high optical purity, and then further performing subsequent reactions to produce a key intermediate of ticagrelor (1R, 2S)-2-(3,4-difluoro Phenyl) cyclopropylamine D-mandelate (I).
  • the method and the reaction system of the invention have high stereoselectivity, high catalytic activity, high tolerance to organic solvents, tolerance to substrates, and thus can be carried out at extremely high substrate concentrations. Scale production.
  • the present invention greatly improves production efficiency and reduces production costs.
  • the method of the present invention greatly simplifies the subsequent processing. Post-treatment requires only extraction and is easy to operate.
  • the invention has significant technical effects compared with the prior art, and is mainly embodied as follows:
  • the cost of preparing the compound (I) is mainly in the reduction step, and the enzyme catalysis cost has a significant advantage over the CBS reduction system for the key reduction step:
  • Table 1 shows the cost of preparing 1 kg of the compound of formula (V) as an example.
  • the carbonyl reductase (QNR) derived from Microbacterium luteolum JCM 9174 is obtained by commercialized whole-gene synthesis, and then the coding gene is constructed into an expression vector, introduced into a host strain, and induced to be expressed.
  • the alcohol dehydrogenase which realizes the regeneration of the coenzyme described above is constructed on the same plasmid pET28a(+) vector by the conventional technique in the art, and then introduced into the expression host Escherichia coli, and the expression of the host enzyme is obtained by inducing expression.
  • the cells can be obtained directly by centrifugation, or they can be broken to obtain a crude enzyme solution, and the crude enzyme powder is used for subsequent biotransformation reaction.
  • the present invention provides a method for preparing a compound (V) by catalytic reduction of a compound (VI) with a carbonyl reductase.
  • the reaction formula is as follows:
  • the biocatalytic system described therein comprises a carbonyl reductase, a coenzyme.
  • the carbonyl reductase-encoding gene sequence described in the present invention is SEQ ID NO. 1, and is derived from M. luteolum JCM 9174 (QNR), and the amino acid sequence of the carbonyl reductase is SEQ ID NO.
  • QNR M. luteolum JCM 9174
  • the amino acid sequence of the carbonyl reductase is SEQ ID NO. According to common general knowledge in the art, the above carbonyl reductase gene can be obtained by commercialized whole gene synthesis.
  • the preparation method is as follows: the substrate is sufficiently dissolved in a co-solvent such as dimethyl sulfoxide or isopropanol, and then added to the phosphate buffer, stirred uniformly, and then added to the cells.
  • a co-solvent such as dimethyl sulfoxide or isopropanol
  • the crude enzyme solution, crude enzyme powder or pure enzyme is added with coenzyme NAD + , and the substrate is isopropanol, maintained at 20 ° C ⁇ 40 ° C, monitored by TLC or HPLC, until the remaining material is ⁇ 2%, the reaction is terminated.
  • the organic solvent may be selected from methyl tert-butyl ether, toluene, ethyl acetate. Isopropyl acetate, dichloromethane, 2-methyltetrahydrofuran, n-butanol. The aqueous layer was extracted 2-3 times, and the organic phase was combined; washed with saturated brine for 2 to 3 times and concentrated to give a pale yellow oil.
  • the final concentration of the substrate compound (VI) in the system is from 50 to 200 g/L, preferably from 100 to 150 g/L, which meets industrial requirements (substrate concentration > 100 g/L).
  • the reaction temperature is 20-40 ° C
  • the rotation speed is 200 rpm / min
  • the reaction time is about 3-10 h
  • the change is according to the substrate concentration or the conversion of the raw material is monitored by HPLC, and the reaction is terminated when the raw material remains ⁇ 2%.
  • the invention also provides a (1R,2S)-2-(3,4-difluorophenyl)cyclopropylamine D-mandelate salt organism Catalytic preparation method.
  • the above-mentioned compound (V) obtained by the biocatalytic method is subjected to the reaction described later as follows:
  • the compound of the formula (V) obtained in the previous step is reacted with triethyl phosphonoacetate in toluene at 60 to 80 ° C under the action of a base to obtain a compound of the formula (IV) wherein the base is sodium hydride or sodium t-butoxide.
  • the compound of the formula (IV) is reacted with ammonia gas in methanol at 60 to 70 ° C under the action of sodium methoxide to obtain a compound of the formula (III).
  • the compound of the formula (III) is subjected to Hofmann degradation under the action of sodium hypochlorite and sodium hydroxide to obtain a compound of the formula (II), which is then salted with D-mandelic acid to prepare a compound of the formula (I).
  • HPLC conditions phenomenex Gemini 5u C18 110A, 250 x 4.6 mm, 5 ⁇ m; flow rate: 1 ml/min; mobile phase gradient as follows; UV detection wavelength: 260 nm; column temperature: 30 ° C; sample concentration: 10 mg/ml; injection volume 10 ⁇ l.
  • the retention time of S-39 was 12.3 min.
  • the QNR gene and alcohol dehydrogenase gene were entrusted to a commercial company for whole-gene synthesis, cloned into pET28a(+) vector, transferred into E. coli DH5 ⁇ competent cells, plated, picked positive transformant single colonies and extracted plasmid sequencing After confirmation, the recombinant plasmid was extracted, introduced into BL21 (DE3) strain, and cultured in LB to obtain a genetically engineered bacteria capable of inducing expression of recombinant carbonyl reductase and alcohol dehydrogenase.
  • the genetically engineered bacteria stored in the glycerol in the previous step were inoculated into LB liquid medium containing kanamycin, and cultured at 37 ° C, 220 rpm for 13 hours to obtain a seed culture medium, and the seed culture solution was inoculated at a ratio of 1.5%.
  • a liquid medium containing 50 ug/ml kanamycin resistance then incubated at 37 ° C, 220 rpm to an OD 600 value of > 2.0, adding a final concentration of 1.0% lactose, cooling to 25 ° C, continuing to culture for 3 h, adding the final concentration to 0.5% lactose, cultured for 20 hours, placed in a can, centrifuged to obtain bacteria, ready for biotransformation.
  • the fermentation formula is as follows:
  • the compound (VI) (1 g) was dissolved in isopropanol (12 ml), 0.1 M phosphate buffer (40 ml) was added, NAD + (0.1 g) was added, and the above-mentioned fermented cells (0.5 g) were added.
  • the reaction was carried out at 25 ° C, 220 rpm, shaken for 24 h, and the reaction conversion was monitored by HPLC to 50%, and the reaction was terminated.

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Abstract

提供了一种(1R,2S)-2-(3,4-二氟苯基)环丙胺D-扁桃酸盐(I)的生物制备方法，包括：(a)在液态反应体系中，以式(VI)化合物为底物，在辅酶存在下，在羰基还原酶催化下，进行不对称还原反应，从而形成式(V)化合物；(b)式(V)化合物与膦酰基乙酸三乙酯反应得式(IV)化合物；(c)式(IV)化合物经氨解、霍夫曼降解后与D-扁桃酸成盐制得式(I)化合物；所述反应体系包括：(i)水性溶剂；(ii)底物，所述底物为式(VI)化合物；(iii)辅酶；(iv)羰基还原酶；(v)共底物；和(vi)用于辅酶再生的酶。

Description

(1R,2S)-2-(3,4-二氟苯基)环丙胺D-扁桃酸盐(Ⅰ)的生物制备方法 技术领域
本发明属于医药技术领域，尤其涉及一种(1R,2S)-2-(3,4-二氟苯基)环丙胺D-扁桃酸盐的制备方法。
背景技术
替卡格雷(ticagrelor)，化学名[1S-[1α,2α,3β(1S,2R),5β]]-3-[7-[2-(3,4-二氟苯基)-环丙胺基]-5-(丙硫基)-3H-1,2,3-三唑并[4,5-d]嘧啶-3-基]-5-(2-羟乙氧基)环戊烷-1,2-二醇，是由阿斯利康公司(AstraZeneca AB)研发的一种口服抗血小板药物。该药能可逆地作用于ADP P2Y12受体，对ADP引起的血小板聚集有明显的抑制作用，且口服起效迅速，临床用于降低急性冠状动脉综合征患者的血栓性心血管事件发生率。
替卡格雷的结构式如下：
Figure PCTCN2017111094-appb-000001
(1R,2S)-2-(3,4-二氟苯基)环丙胺(Ⅱ)是合成替卡格雷的关键中间体，因其是油状物，不利于保存及质控，故通常将其成D-扁桃酸盐制得(1R,2S)-2-(3,4-二氟苯基)环丙胺D-扁桃酸盐(Ⅰ)。
Figure PCTCN2017111094-appb-000002
专利WO 2008018822公开了一种制备化合物(Ⅱ)的如下方法：
Figure PCTCN2017111094-appb-000003
以邻二氟苯为起始原料，与氯乙酰氯发生傅-克酰化反应得化合物(Ⅵ)，化合物(Ⅵ)在S-2-甲氧基-CBS-噁唑硼烷(须由三甲氧基硼烷和S-二苯基脯氨醇原位制备)催化下与硼烷-二甲硫醚络合物进行CBS不对称还原得化合物(Ⅴ)，化合物(Ⅴ)与膦酰基乙酸三乙酯经环丙烷化反应得化合物(Ⅳ)，化合物(Ⅳ)经氨解、霍夫曼降解制得替卡格雷关键中间体(1R,2S)-2-(3,4-二氟苯基)环丙胺(Ⅱ)。
该方法中CBS还原反应的催化剂S-2-甲氧基-CBS-噁唑硼烷性质不稳定，未商品化，必须由三甲氧基硼烷和S-二苯基脯氨醇原位制备，增加了操作工序，不利于工业化生产；而还原剂硼烷-二甲硫醚络合物则在后处理时放出恶臭的二甲硫醚气体，不利于生产的劳动保护。参照该方法操作，用原位制备的S-2-甲氧基-CBS-噁唑硼烷和硼烷-二甲硫醚络合物还原化合物(Ⅵ)，于40℃反应1h制得化合物(Ⅴ)，其ee值仅为76％。参照该法制得的(1R,2S)-2-(3,4-二氟苯基)环丙胺(Ⅱ)的ee值为81％。
在发明人申请专利CN201410139006中，对该工艺CBS还原反应进行了优化，改进后为S-2-甲基-CBS-噁唑硼烷和硼烷-四氢呋喃络合物还原化合物(Ⅵ)得化合物(Ⅴ)，将ee值由76％提高至98-99％。由于该反应对反应条件的控制较为严苛，在放大实验中发现产物ee值较难达到99％，稳定在96-97％，反应后淬灭反应时，体系出现剧烈放热，存在明显的安全隐患。
上述方法是在手性配体催化下，经硼烷还原羰基制得手性醇化合物(Ⅴ)，该法存在以下不足：配体成本较高，对氧气，水敏感，因此工艺操作条件严苛，如氮气保护，无水无氧；溶剂体系复杂甲苯，四氢呋喃混合体系等。相较化学法，生物催化法更加绿色，环保，经济。生物催化理论中将能够实现立体选择 性还原羰基为手性羟基的酶称为羰基还原酶。由于酶具有高的立体选择性，底物特异性特点，对于本申请中提及的特定底物化合物(Ⅵ)，能立体选择性的将其还原的羰基还原酶，文献报道较少,仅在CN201610051136.4中，吴中柳等人报道ChKRED20羰基还原酶，可立体选择性还原本发明所述底物。Applied and Environmental Microbiology 2013,79(4),p1378-1384中报道来源于Microbacterium luteolum JCM 9174羰基还原酶QNR对于奎宁环酮表现良好活性，但对于类似底物脂肪环酮则表现出较低的催化活性，因此仍未有相关文献报道将其用于芳香酮底物的还原，包括对于本发明中涉及的底物式(Ⅵ)化合物。
发明内容
针对现有制备方法存在的上述问题，本发明目的是提供了一种(1R,2S)-2-(3,4-二氟苯基)环丙胺D-扁桃酸盐生物催化的制备方法。
本发明的第一方面提供了一种式Ⅴ化合物的制备方法，包括步骤：
(a)在液态反应体系中，以式Ⅵ化合物为底物，在辅酶存在下，在羰基还原酶催化下，进行不对称还原反应，从而形成式Ⅴ化合物；
Figure PCTCN2017111094-appb-000004
和
(b)任选地从所述上一步骤的反应后的反应体系中分离出式Ⅴ化合物。
在另一优选例中，所述反应体系中，所述式Ⅵ化合物的浓度为50～1000g/L。
在另一优选例中，所述反应体系中，所述式Ⅵ化合物的浓度为60～700g/L。
在另一优选例中，所述反应体系中，所述式Ⅵ化合物的浓度为80～600g/L。
在另一优选例中，所述反应体系中，所述式Ⅵ化合物的浓度为20～500g/L。
在另一优选例中，所述反应体系中，所述式Ⅵ化合物的浓度为50～200g/L。
在另一优选例中，所述反应体系中，所述式Ⅵ化合物的浓度为100～150g/L。
在另一优选例中，所述的反应体系中，还存在共底物。
在另一优选例中，所述的共底物选自下组：异丙醇、葡萄糖、甲酸铵、或其组合。
在另一优选例中，所述的反应体系中，共底物的浓度为5-30％。
在另一优选例中，所述的反应体系中，还存在用于辅酶再生的酶。
在另一优选例中，所述的用于辅酶再生的酶选自下组：醇脱氢酶，甲酸脱氢酶、葡萄糖脱氢酶、或其组合。
在另一优选例中，步骤(a)中，温度为10℃-50℃，较佳地20℃-40℃，更佳地25℃-35℃。
在另一优选例中，步骤(a)中，时间为0.1-240小时，较佳地0.5-120小时，更佳地1-72小时，又更佳地3-10小时。
在另一优选例中，步骤(a)中，pH为6-9，较佳地6.5-8.5，更佳地7.0-7.5。
在另一优选例中，所述的反应体系中，羰基还原酶为游离形式的酶、固定化酶、或菌体形式的酶。
在另一优选例中，所述的反应体系为磷酸缓冲盐体系。
在另一优选例中，所述的反应体系还含有助溶剂。
在另一优选例中，所述的助溶剂选自下组：二甲基亚砜，甲醇，乙醇，异丙醇，乙腈，甲苯、丙酮、或其组合。
在另一优选例中，所述助溶剂的浓度为5～30％。
在另一优选例中，在步骤(b)中，所述的分离包括：加入异丙醇，离心菌体，部分浓缩，甲叔醚萃取，浓缩有机层。
在另一优选例中，在步骤(b)中，所述反应后的反应体系中，式Ⅴ化合物的ee值≥90％，较佳地≥95％，更佳地≥99％。
在另一优选例中，在步骤(b)中，所述反应后的反应体系中，≥80％(较佳地≥85％，更佳地≥90％)式Ⅵ化合物被转化为式Ⅴ化合物。
在另一优选例中，在步骤(b)中，所述反应后的反应体系中，式Ⅴ化合物的浓度50～1000g/L。
在另一优选例中，所述羰基还原酶选自下组：
(i)来源于M.luteolum JCM 9174的羰基还原酶QNR，其氨基酸序列如SEQ ID NO.2所示；
(ii)对SEQ ID NO.2所示的氨基酸序列在保持酶活性范围内，进行一个或多个氨基酸的替换、缺失、改变、插入或增加，所得到的氨基酸序列。
在另一优选例中，所述的羰基还原酶QNR的编码基因序列选自下组：
(a)SEQ ID NO.1所示的序列(NCBI的登录号为AB733448)；
(b)与(a)限定的序列互补的多核苷酸；或
(c)与(a)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，所述羰基还原酶基因构建在表达载体上。
在另一优选例中，所述的辅酶选自：还原性辅酶、氧化性辅酶，或其组合。
在另一优选例中，所述还原性辅酶选自NADH、NADPH、或其组合。
在另一优选例中，所述氧化性辅酶选自NAD、NADP、或其组合。
在另一优选例中，所述NAD用量与底物用量比率为0.01％～1.0％(w/w)、较佳地0.01％～0.5％(w/w)。
在另一优选例中，所述用于辅酶再生的酶为醇脱氢酶，其基因选自：
(d)SEQ ID NO.3所示的序列(NCBI的登录号为AB213459)；
(e)与(d)限定的序列互补的多核苷酸；或
(f)与(d)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，所述用于辅酶再生的酶的基因构建在表达载体上。
本发明第二方面提供了一种反应体系，所述反应体系包括：
(i)水性溶剂；
(ii)底物，所述底物为式VI化合物；
Figure PCTCN2017111094-appb-000005
(iii)辅酶；
(iv)羰基还原酶；
(v)共底物；和
(vi)用于辅酶再生的酶。
在另一优选例中，在所述的反应体系中，式VI化合物浓度为50～1000g/L。
本发明第三方面提供了一种制备式Ⅴ化合物的方法，包括步骤：使用本发明第二方面所述的反应体系，在适合酶催化的条件下，进行酶促反应，从而制得式Ⅴ化合物：
Figure PCTCN2017111094-appb-000006
在另一优选例中，在所述的反应体系中，式VI化合物浓度为50～1000g/L。
本发明第四方面提供了一种式Ⅰ化合物扁桃酸盐的制备方法，通过本发明第一方面所述的方法所制得式Ⅴ化合物进行如下的反应步骤制得。
Figure PCTCN2017111094-appb-000007
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。
附图说明
图1显示了式V化合物的消旋体的手性图谱。
图2显示了羰基还原酶催化所得式V化合物与消旋体的比较。
图3显示了羰基还原酶催化所得式V化合物的ee值。
具体实施方式
本发明人经过广泛而深入的研究，首次意外地发现一种(1R,2S)-2-(3,4-二氟苯基)环丙胺D-扁桃酸盐(I)生物催化的制备方法。具体地，本发明将现有(1R,2S)-2-(3,4-二氟苯基)环丙胺D-扁桃酸盐(I)制备方法中的关键的羰基还原反应改进为羰基还原酶催化还原，即在羰基还原酶和辅酶的存在下，将式Ⅵ化合物立体选择性的还原为式Ⅴ化合物(即使在高浓度的底物，如50～1000g/L条件下，也能够非常高效地制备获得具有立体构象的式Ⅴ化合物)。然后以式Ⅴ化合物为底物，进行后续反应。本发明仅需萃取操作，操作简单， 成本低廉，绿色环保，更适合工业化生产具有高化学纯度和高光学纯度式Ⅴ化合物，然后进一步进行后续反应，生产替卡格雷的关键中间体(1R,2S)-2-(3,4-二氟苯基)环丙胺D-扁桃酸盐(Ⅰ)，以用于替卡格雷等药品的制备。在此基础上完成了本发明。
在一具体优选例中，本发明人利用来源于Microbacterium luteolum JCM 9174羰基还原酶作为代表性的羰基还原酶时，通过在反应体系中偶联辅酶再生，从而显著提高了反应体系对有机溶剂、底物的耐受性，这样可以大幅提高了底物浓度，也能够非常高效地制备获得具有立体构象的式Ⅴ化合物(ee值≥98％、99％、或更高)，从而极大地提高生产效率，降低生产成本。此外，本发明方法大幅简化了后续处理，而且与化学合成法相比，显著减少或消除了各类污染性化学品的使用，从而显著降低了环境污染风险。
术语
羰基还原酶
本发明中，“羰基还原酶”是能够立体选择性催化潜手性酮不对称还原得到手性醇的酶。
在本发明中，所述的羰基还原酶可以是野生型的或突变型的。此外，可以分离的，也可以是重组的。
可用于本发明的羰基还原酶可以来自不同物种。例如，来自微杆菌属，更佳地来自Microbacterium luteolum的羰基还原酶。此外，与上述羰基还原酶有类似活性或同源性(如≥80％，较佳地≥90％，更佳地较佳地≥95％)的酶(包括来自其他物种的酶)也在本发明范围之内。
在本发明中，一种代表性的羰基还原酶为微杆菌属luteolum JCM 9174(Microbacterium luteolum JCM 9174)的羰基还原酶。
一种典型的羰基还原酶的氨基酸序列如SEQ ID No.:2所示，其编码基因如SEQ ID No.:1所示。
由于密码子的简并性，编码SEQ ID NO.2所示的氨基酸序列的碱基序列不仅仅局限于SEQ ID NO.1。本领域技术人员可以通过适当引入替换、缺失、改变、插入或增加来获得该碱基序列的同系物，本发明涵盖这些同系物，只要其表达的重组酶保持对式Ⅵ化合物的催化还原活性即可。本发明中多聚核苷酸的同系物可以通过对碱基序列SEQ ID NO.1的一个或多个碱基在保持酶活性范 围内进行替换、缺失或增加来制得。
本发明的羰基还原酶还包括对SEQ ID NO.2所示的氨基酸序列在保持酶活性范围内，进行一个或多个氨基酸的替换、缺失、改变、插入或增加，所得到的氨基酸序列。
根据本领域的一般常识，反应体系中可以使用上述羰基还原酶构建的重组酶静息细胞、湿菌体、粗酶液、纯酶或者粗酶粉等。为获得较高的转化效率，优选使用粗酶液。羰基还原酶用量与底物用量比率优选为1％～6％(w/w)，或者静息细胞质量与底物质量比率为10-100％。
辅酶
本发明中，“辅酶”是指能够实现氧化还原反应中电子传递的辅酶。
典型地，本发明的辅酶为还原性辅酶NADH、NADPH或氧化性辅酶NAD+、NADP+。由于还原性辅酶的价格成本昂贵，优选选择氧化性辅酶NAD+、NADP+。
当选择氧化性辅酶时，需要选择实现辅酶再生的方法，主要包括三种(1)葡萄糖脱氢酶与共底物葡萄糖；(2)醇脱氢酶与共底物异丙醇；(3)甲酸脱氢酶与共底物甲酸铵。
在一个优选实施例中，辅酶为NAD+，辅酶再生体系为醇脱氢酶，本发明优选醇脱氢酶与共底物异丙醇，醇脱氢酶的编码基因序列如SEQ ID NO.3所示(NCBI的登录号为AB213459)。
氧化性辅酶NAD+用量与底物用量比率为0.01％～0.5％(w/w)，异丙醇的用量为缓冲液体积的5％-30％(v/v)。缓冲体系为0.1mol/L磷酸缓冲盐。缓冲液的pH为7.0-7.3。
助溶剂
本发明中，可以在反应体系中添加或不添加助溶剂。
如本文所用，术语“助溶剂”是指难溶性物质与加入的第三种物质在溶剂中形成可溶性分子间的络合物、缔合物或复盐等，以增加难溶性物质在溶剂中的溶解度。这种第三种物质称为助溶剂。
本发明中，底物化合物(Ⅵ)难溶于水，当底物浓度增大时，严重影响反应转化率。因此，需通过加入助溶剂提高底物溶解性，以改善反应转化情况。可选的助溶剂为二甲基亚砜，甲醇，乙醇，异丙醇，乙腈，甲苯、丙酮，浓度 优选为5-30％(v/v)，优选为二甲基亚砜，甲醇，乙醇，异丙醇。
生物制备方法
本发明提供了一种式Ⅴ化合物的制备方法，包括步骤：
(a)在液态反应体系中，以式Ⅵ化合物为底物，在辅酶存在下，在羰基还原酶催化下，进行不对称还原反应，从而形成式Ⅴ化合物；
Figure PCTCN2017111094-appb-000008
和
(b)任选地从所述上一步骤的反应后的反应体系中分离出式Ⅴ化合物。
在本发明中，上述的反应可以偶联或不偶联辅酶再生体系。
优选地，上述反应是在同一体系中偶联辅酶再生系统，从而进一步提高生产效率，降低生产成本并提高对底物的耐受性。
反应体系
本发明还提供了一种用于本发明所述生物制备方法的反应体系。
一种典型的一种反应体系，包括：
(i)水性溶剂；
(ii)底物，所述底物为式VI化合物；
Figure PCTCN2017111094-appb-000009
(iii)辅酶；
(iv)羰基还原酶；
(v)共底物；和
(vi)用于辅酶再生的酶。
在本发明的优选反应体系中，还偶联有辅酶再生系统，从而进一步提高生产效率，降低生产成本并提高对底物的耐受性。
此外，在本发明第一方面中所述的各类优选特征或其组合，同样适用于本发明所述的反应体系。
本发明还提供了所述本发明反应体系的应用，即用于制备式Ⅴ化合物，它包括步骤：使用所述的反应体系中，在适合酶催化的条件下，进行酶促反应，从而制得式Ⅴ化合物：
Figure PCTCN2017111094-appb-000010
由于本发明的反应体系和方法减少了或消除了各类污染性化学品的使用，也不必使用昂贵的立体结构分拆试剂，因此不仅显著降低了环境污染风险，而且成本低廉。
本发明的主要优点在于：
(1)利用来源于羰基还原酶(如Microbacterium luteolum JCM 9174的羰基还原酶)的新型催化反应体系，通过生物催化还原高效进行生产。
(2)本发明适合工业化生产具有高化学纯度和高光学纯度的式Ⅴ化合物，然后进一步进行后续反应，生产替卡格雷的关键中间体(1R,2S)-2-(3,4-二氟苯基)环丙胺D-扁桃酸盐(Ⅰ)。
(3)本发明方法和反应体系有高立体选择性、高催化活性、以及对有机溶剂的高耐受性、对底物的耐受性，从而可以在极高的底物浓度下，进行大规模生产。
(4)本发明极大地提高生产效率，降低生产成本。
(5)本发明方法大幅简化了后续处理。后处理仅需萃取操作，操作简单，。
(6)绿色环保。与化学合成法相比，本发明显著减少或消除了各类污染性化学品的使用，从而显著降低了环境污染风险。
本发明与现有技术比较具有显著的技术效果，主要体现如下：
1.制备式化合物(Ⅰ)的成本主要在还原步骤，对于关键还原步骤，酶催化成本较CBS还原体系具有明显优势：
表一制备1kg式(Ⅴ)化合物的成本为例
Figure PCTCN2017111094-appb-000011
注：两种催化方法中，反应的转化率近定量，因此底物成本等同。
由上表知羰基还原酶催化体系较CBS催化体系成本显著高于生物催化体系。
表二关键还原步骤反应条件的比较
Figure PCTCN2017111094-appb-000012
由上表比较知，羰基还原酶催化体系较CBS还原催化体系操作上得到了极大的简化，降低操作安全风险，减少人力成本，更加环保，更适于生产应用。
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。本发明中所涉及的实验材料如无特殊说明均可从市售渠道获得。
材料
化合物(Ⅵ)，消旋体外购自上海翰鸿化工有限公司，基因全合成由上海百利格完成。
来源于Microbacterium luteolumJCM 9174的羰基还原酶(QNR)，通过商业化的全基因合成得到编码基因，然后将编码基因构建入表达载体，导入宿主菌，诱导表达得到。
方法
1.酶的制备方法
通过本领域常规技术，将上述实现辅酶再生的醇脱氢酶与目的基因构建在同一质粒pET28a(+)载体上，然后导入表达宿主大肠杆菌，通过诱导表达，获得含有目的双酶的菌体。可直接使用离心获得菌体，也可将其破壁获得粗酶液，粗酶粉用于后续的生物转化反应。
2.生物催化还原化合物(Ⅵ)制备化合物(Ⅴ)的方法
本发明提供了一种羰基还原酶催化还原化合物(Ⅵ)制备化合物(Ⅴ)的方法。反应式如下：
Figure PCTCN2017111094-appb-000013
其中所述的生物催化体系包括有羰基还原酶，辅酶。本发明中所述的羰基还原酶编码基因序列为SEQ ID NO.1，来源于M.luteolumJCM 9174(QNR)，羰基还原酶的氨基酸序列为SEQ ID NO.2。根据本领域一般常识，上述羰基还原酶基因可通过商业化的全基因合成得到。
根据上述优选体系，所述制备方法的实施过程如下：将底物充分溶解在助溶剂，如二甲基亚砜或异丙醇，然后加入到磷酸缓冲液中，搅拌均匀后，加入菌体、粗酶液、粗酶粉或纯酶，加入辅酶NAD+，共底物异丙醇，维持在20℃～40℃，TLC或HPLC监控，至原料剩余<2％，终止反应。向反应液中加入异丙醇，离心或过陶瓷膜，除去菌体，取上清液，上清液用有机溶剂萃取，有机溶剂可选为甲基叔丁基醚、甲苯、乙酸乙酯、乙酸异丙脂、二氯甲烷，2-甲基四氢呋喃、正丁醇。萃取水层2-3次，合并有机相；用饱和食盐水洗涤2～3次，浓缩后得到浅黄色油状物。
体系中底物化合物(Ⅵ)的终浓度为50-200g/L，优选为100-150g/L，满足工业要求(底物浓度>100g/L)。反应温度为20-40℃，转速为200rpm/min，反应时间约3-10h，根据底物浓度而有所变化或通过HPLC监控原料转化情况，当原料剩余<2％，终止反应。
3.(1R,2S)-2-(3,4-二氟苯基)环丙胺D-扁桃酸盐生物催化的制备方法
本发明还提供了一种(1R,2S)-2-(3,4-二氟苯基)环丙胺D-扁桃酸盐生物 催化的制备方法。将上述通过生物催化法制得的化合物(Ⅴ)经过后续描述的反应如下：
Figure PCTCN2017111094-appb-000014
在碱的作用下，上一步所得式(Ⅴ)化合物与膦酰基乙酸三乙酯在甲苯中于60～80℃反应，以获得式(Ⅳ)化合物，其中碱为氢化钠或叔丁醇钠。
Figure PCTCN2017111094-appb-000015
在甲醇钠的作用下，式(Ⅳ)化合物与氨气在甲醇中于60～70℃反应，以获得式(Ⅲ)化合物。
Figure PCTCN2017111094-appb-000016
式(Ⅲ)化合物在次氯酸钠和氢氧化钠的作用下经霍夫曼降解制得式(Ⅱ)化合物，再与D-扁桃酸成盐制得式(Ⅰ)化合物。
4.2-氯-1-S-(3,4-二氟苯基)-乙醇的手性正相监测方法：
HPLC条件：Daicel IC-3(250×4.6mm，3μm)；流速0.8ml/min；流动相：正己烷:异丙醇＝99:1；紫外检测波长260nm；柱温25℃；样品溶于甲醇，浓度10mg/ml；进样体积2μl。
5.2-氯-1-S-(3,4-二氟苯基)-乙醇的反相监测方法：
HPLC条件：phenomenex Gemini 5u C18 110A，250×4.6mm,5μm；流速：1ml/min；流动相梯度如下表；紫外检测波长：260nm；柱温：30℃；样品浓度：10mg/ml；进样体积10μl。S-39的保留时间为12.3min。
流动相梯度：
时间(min) H2O-0.1％TFA(％) ACN-0.1％TFA(％)
0 80 20
15 20 80
35 20 80
35.1 80 20
40 20 80
实施例1、羰基还原酶工程菌的构建
将QNR目的基因，醇脱氢酶基因委托商业化公司进行全基因合成，克隆入pET28a(+)载体，转入大肠杆菌DH5α感受态细胞，平板培养，挑取阳性转化子单菌落并提取质粒测序确定后，提取重组质粒，导入BL21(DE3)菌株中，LB培养，获得可以诱导表达重组羰基还原酶与醇脱氢酶的基因工程菌。
实施例2、重组羰基还原酶，醇脱氢酶的制备
将上一步保存于甘油中的基因工程菌，接种到含卡那霉素的LB液体培养基中，37℃，220rpm，培养13h，得到种子培养基，将种子培养液按1.5％的比例接种到含50ug/ml卡那霉素抗性的液体培养基上，然后37℃，220rmp培养至OD600值>2.0，加入终浓度为1.0％乳糖，降温至25℃，继续培养3h，加入终浓度为0.5％的乳糖，培养20h，放罐，离心得菌体，为生物转化做准备。
发酵配方如下：
Figure PCTCN2017111094-appb-000017
实施例3、2-氯-1-S-(3,4-二氟苯基)-乙醇(化合物Ⅴ)的生物催化制备
Figure PCTCN2017111094-appb-000018
取化合物VI(20g)，溶解于异丙醇(120ml)中，加入0.1M的磷酸盐缓冲液(400ml)，加入NAD+(0.2g)，加入上述发酵所得菌体(10g)，25℃，220rpm，摇床反应，HPLC监控反应转化率>98％时，终止反应。
加入异丙醇(100ml)，离心，取上清液，部分浓缩异丙醇，甲叔醚(400ml)萃取，有机溶剂(100ml×2)萃取水层，合并有机层，饱和食盐水洗，无水硫酸钠干燥，过滤，浓缩得浅黄色油状物18.8g，收率93.0％，ee值100％。
对比实施例：QNR还原结构类似物
Figure PCTCN2017111094-appb-000019
取化合物(Ⅵ)(1g)，溶解于异丙醇(12ml)中，加入0.1M的磷酸盐缓冲液(40ml)，加入NAD+(0.1g)，加入上述发酵所得菌体(0.5g)，25℃，220rpm，摇床反应，反应24h,HPLC监控反应转化率50％，终止反应。
实施例4、(1R,2S)-2-(3,4-二氟苯基)环丙胺D-扁桃酸盐(化合物Ⅰ)的制备
参见发明人申请专利CN201410139006中所述制备方法
Figure PCTCN2017111094-appb-000020
以上实施例仅用以说明本发明的技术方案，而非对其进行限制；尽管参照前述实施例对本发明进行了详细的说明，对于本领域的普通技术人员来说，依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或替换，并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。
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 (24)

  1. 一种式Ⅴ化合物的制备方法，其特征在于，包括步骤：
    (a)在液态反应体系中，以式Ⅵ化合物为底物，在辅酶存在下，在羰基还原酶催化下，进行不对称还原反应，从而形成式Ⅴ化合物；
    Figure PCTCN2017111094-appb-100001
    和
    (b)任选地从所述上一步骤的反应后的反应体系中分离出式Ⅴ化合物。
  2. 根据权利要求1所述的制备方法，其特征在于，所述的反应体系中，羰基还原酶为游离形式的酶、固定化酶、或菌体形式的酶。
  3. 根据权利要求1所述的制备方法，其特征在于，所述羰基还原酶选自下组：
    (i)来源于M.luteolum JCM 9174的羰基还原酶QNR，其氨基酸序列如SE Q ID NO.2所示；
    (ii)对SEQ ID NO.2所示的氨基酸序列在保持酶活性范围内，进行一个或多个氨基酸的替换、缺失、改变、插入或增加，所得到的氨基酸序列。
  4. 根据权利要求3所述的制备方法，其特征在于，所述的羰基还原酶QNR的编码基因序列选自下组：
    (a)SEQ ID NO.1所示的序列；
    (b)与(a)限定的序列互补的多核苷酸；或
    (c)与(a)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。
  5. 根据权利要求1-3中任一所述的制备方法，其特征在于，所述的辅酶选自：还原性辅酶、氧化性辅酶，或其组合。
  6. 根据权利要求5所述的制备方法，其特征在于，所述氧化性辅酶选自NAD、NADP、或其组合。
  7. 根据权利要求6所述的制备方法，其特征在于，所述NAD用量与底物用量比率为0.01％～1.0％(w/w)、较佳地0.01％～0.5％(w/w)。
  8. 根据权利要求1-3中任一所述的制备方法，其特征在于，所述的反应体系中，还存在用于辅酶再生的酶。
  9. 根据权利要求8所述的制备方法，其特征在于，所述的用于辅酶再生的酶选自下组：醇脱氢酶，甲酸脱氢酶、葡萄糖脱氢酶、或其组合。
  10. 根据权利要求8所述的制备方法，其特征在于，所述用于辅酶再生的酶为醇脱氢酶，其基因选自：
    (d)SEQ ID NO.3所示的序列；
    (e)与(d)限定的序列互补的多核苷酸；或
    (f)与(d)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. 根据权利要求1所述的制备方法，其特征在于，所述的反应体系中，还存在共底物。
  12. 根据权利要求11所述的制备方法，其特征在于，所述的反应体系中，共底物的浓度为5-30％。
  13. 根据权利要求11所述的制备方法，其特征在于，所述的共底物选自下组：异丙醇、葡萄糖、甲酸铵、或其组合。
  14. 根据权利要求1所述的制备方法，其特征在于，所述的反应体系还含有助溶剂。
  15. 根据权利要求14所述的制备方法，其特征在于，所述的助溶剂选自下组：二甲基亚砜，甲醇，乙醇，异丙醇，乙腈，甲苯、丙酮、或其组合。
  16. 根据权利要求14所述的制备方法，其特征在于，所述助溶剂的浓度为5～30％。
  17. 根据权利要求1所述的制备方法，其特征在于，所述的反应体系为磷酸缓冲盐体系。
  18. 根据权利要求1所述的制备方法，其特征在于，所述反应体系中，所述式Ⅵ化合物的浓度为50～200g/L。
  19. 根据权利要求1所述的制备方法，其特征在于，在步骤(b)中，所述反应后的反应体系中，式Ⅴ化合物的ee值≥90％，较佳地≥95％，更佳地≥99％。
  20. 根据权利要求1所述的制备方法，其特征在于，在步骤(b)中，所述反应后的反应体系中，≥80％(较佳地≥85％，更佳地≥90％)式Ⅵ化合物被转化为式Ⅴ化合物。
  21. 一种反应体系，其特征在于，所述反应体系包括：
    (i)水性溶剂；
    (ii)底物，所述底物为式VI化合物；
    Figure PCTCN2017111094-appb-100002
    (iii)辅酶；
    (iv)羰基还原酶；
    (v)共底物；和
    (vi)用于辅酶再生的酶。
  22. 根据权利要求21所述的反应体系，其特征在于，在所述的反应体系中，式VI化合物浓度为50～1000g/L。
  23. 一种制备式Ⅴ化合物的方法，其特征在于，包括步骤：使用如权利要求21所述的反应体系，在适合酶催化的条件下，进行酶促反应，从而制得式Ⅴ化合物：
    Figure PCTCN2017111094-appb-100003
  24. 一种式Ⅰ化合物扁桃酸盐的制备方法，其特征在于通过权利要求1所述的方法所制得式Ⅴ化合物进行如下的反应步骤制得：
    Figure PCTCN2017111094-appb-100004
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