WO2021168987A1 - 手性二胺化合物的合成方法 - Google Patents

手性二胺化合物的合成方法 Download PDF

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WO2021168987A1
WO2021168987A1 PCT/CN2020/082594 CN2020082594W WO2021168987A1 WO 2021168987 A1 WO2021168987 A1 WO 2021168987A1 CN 2020082594 W CN2020082594 W CN 2020082594W WO 2021168987 A1 WO2021168987 A1 WO 2021168987A1
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transaminase
synthesis method
substrate
reaction
diamine compound
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PCT/CN2020/082594
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French (fr)
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洪浩
詹姆斯•盖吉
肖毅
张娜
李响
蒋相军
赵桐
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凯莱英医药集团（天津）股份有限公司
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Priority to JP2022551243A priority Critical patent/JP7345070B2/ja
Priority to US17/905,092 priority patent/US20230151396A1/en
Priority to EP20921866.8A priority patent/EP4112733A4/en
Priority to KR1020227033472A priority patent/KR20220157981A/ko
Publication of WO2021168987A1 publication Critical patent/WO2021168987A1/zh

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    • C—CHEMISTRY; METALLURGY
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/001—Amines; Imines
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
    • 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
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y206/00—Transferases transferring nitrogenous groups (2.6)
    • C12Y206/01—Transaminases (2.6.1)
    • 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
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    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to the field of synthesis of chiral diamine compounds, and in particular to a method for synthesizing chiral diamine compounds.
  • Chiral diamine compounds are widely found in many biologically active natural products and drug molecules, such as: ornithine, lysine, vitamin H, drug levamisole and so on.
  • Oxaliplatin (Oxaliplatin) molecule contains a diamine compound, which has certain anti-tumor activity.
  • Compounds with diamine structure are also widely used in organic synthesis. As a synthetic building block, they can effectively construct nitrogen heterocycles, and can also be used as ligands to complex with metals to generate highly reactive catalysts.
  • Diamine compounds are also widely used Used in liquid crystal materials, aviation materials, microelectronics and other fields. Chiral diamine compounds can also be used as chiral resolution reagents to resolve the enantiomers of aldehydes. Therefore, the synthesis of chiral diamines has always been a hot spot for chemists (Angew. Chem. Int. Ed. 1998, 37, 2580).
  • the synthesis methods of chiral diamine compounds include asymmetric halogen-containing compound (Chem. Rev., 2011, 111, 6947), asymmetric Michel addition (CN 105367427 A), and asymmetric ring opening of aziridine (CN 105753752 A). ) And other methods, but these methods have varying degrees of limitations in the scope and practicability of the substrate, and these methods are synthesizing 1,2-diamine compounds.
  • Asymmetric Michel addition and aziridine asymmetric ring opening usually require expensive metals and chiral catalysts, which greatly limits the application of the reaction system.
  • highly toxic azide compounds or cyanating reagents will be used in the reaction, and the three wastes of the chemical route are large and difficult to handle.
  • the main purpose of the present invention is to provide a method for synthesizing a chiral diamine compound to solve the problem that the method in the prior art has a limitation in the scope of application of the substrate.
  • the present invention provides a method for synthesizing a chiral diamine compound, the synthesis method comprising: using a transaminase to convert a substrate represented by formula I into a chiral diamine compound;
  • R group represents alkyl group, cycloalkyl group, heteroatom-containing alkyl group, heteroatom-containing cycloalkyl group, heteroatom-containing aryl group, residue of amide compound or residue of ether compound
  • the heteroatom is at least one of O, S and N
  • R1 and R2 are the same or different, and R1 and R2 are each independently hydrogen, C1-C3 alkyl or amino protecting group
  • the transaminase is derived from Chromobacterium violaceum DSM30191 (CVTA) (NCBI Reference Sequence: WP_011135573.1), Fonsecaea pedrosoi CBS 271.37 (NCBI Reference Sequence: XP_013286281.1), Klebsiella pneumoniae subsp.pneumoniae Mycobacterium (GenBank: CCN2 9541.1)0.1 (GenBank: good 9541.1) ), Paracoccus denitrificans (NCBI Reference Sequence: WP_01
  • transaminase has an amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.
  • amino protecting group is selected from any one of tert-butoxycarbonyl, benzyloxycarbonyl, formyl, trifluoroacetyl, benzyl, trityl and 9-fluorenylmethoxycarbonyl.
  • the substrate is selected from any one of the following: and
  • the synthesis method includes: mixing a phosphate buffer with an amino donor to obtain a first mixed liquid, adding a substrate represented by formula I to the first mixed liquid to obtain a second mixed liquid; A transaminase is added to obtain a reaction mixture; a chiral diamine compound is separated from the reaction mixture.
  • the synthesis method further includes: adjusting the pH of the first mixed solution to 7.0 to 9.0; preferably, adding transaminase to the second mixed solution, and adjusting the addition of the transaminase
  • the pH of the latter second mixed liquid is 7.0 to 9.0, and a reaction mixed liquid is obtained.
  • separating the chiral diamine compound from the reaction mixture includes: adjusting the acidity of the reaction mixture to denature the transaminase, preferably adjusting the pH of the reaction mixture to 1 to 2; filtering and removing the denatured transaminase to obtain a preliminary Filtrate; adjust the pH of the preliminary filtrate to alkaline to obtain an alkaline filtrate; extract the alkaline filtrate to obtain a chiral diamine compound; preferably, the pH of the alkaline filtrate is 12-13; preferably, The extraction is performed multiple times, more preferably 2 to 5 times, more preferably, dichloromethane is used for the first extraction, followed by dichloromethane or ethyl acetate for the remaining times.
  • the step of drying the organic phase obtained by the extraction is further included.
  • the organic phases obtained from each extraction are combined to obtain an extract; the extract is dried to obtain a dried organic phase product; and the dried organic phase product is dried. Concentrate under the conditions of temperature ⁇ 45°C and pressure ⁇ -0.08Mpa to no fractions to obtain chiral diamine compounds.
  • the mass ratio of transaminase to substrate is 0.4:1 to 1.0:1.
  • the concentration of the substrate is 60 g/L to 100 g/L.
  • amino donor is selected from isopropylamine, isopropylamine hydrochloride, alanine, phenethylamine or n-butylamine.
  • the specificity of the transaminase to the substrate shown in formula I can be used to effectively catalyze the conversion of such substrates into chiral diamine compounds, and the transaminase is effective for a variety of substrates shown in formula I.
  • the reaction selectivity and activity are both high. Therefore, the use of this biological enzyme to catalyze the synthesis of chiral diamine compounds is not only suitable for a wider range of substrates (the reactions in the prior art are all 1,2-diamine compounds, but the application can also synthesize 1,3 -Diamine, 1,4-diamine and other compounds), and the synthesis method has a short route, high product yield, greatly reduces production costs, and reduces organic solvents and three wastes.
  • Figure 1 shows the effect of different enzyme amounts on the conversion rate of the same amount of substrate in Example 11 of the present invention
  • Figure 2 shows the effect of the same enzyme amount on the conversion rate of different concentrations of substrate in Example 12 of the present invention
  • Figure 3 shows the influence of different amino donors on the conversion rate of the substrate in the same reaction in Example 13 of the present invention.
  • FIG. 4 shows that transaminase from different sources in Example 14 of the present invention all have conversion activity on the same substrate.
  • the method for synthesizing chiral diamine compounds has a limitation in the scope of application of the substrate.
  • the inventor of the present application tried to improve the existing synthesis route from the perspective of high efficiency and environmental protection.
  • the inventor discovered that a transaminase developed by the applicant has the general catalytic activity of catalyzing ketone compounds of the transaminase with PLP as the coenzyme, and also has the ability to catalyze a variety of different substrates. It is converted to the activity of chiral diamine compounds.
  • the transaminase when used to catalyze the synthesis of chiral diamine compounds, not only the types of substrates are wide, but the target product can be obtained through a one-step reaction. Due to the high selectivity of the biotransformation reaction using the transaminase, the ee value of the target product is greatly improved. Moreover, the use of biological enzymes to catalyze the reaction can increase the amount of substrates, greatly improve production efficiency, and reduce the production of organic solvents and three wastes.
  • a method for synthesizing a chiral diamine compound includes: using a transaminase to convert a substrate represented by formula I into a chiral diamine compound;
  • R group represents an alkyl group, a cycloalkyl group, a heteroatom-containing alkyl group, a heteroatom-containing cycloalkyl group, a heteroatom-containing aryl group, a residue of an amide compound or a residue of an ether compound
  • R1 and R2 are the same or different
  • R1 and R2 are each independently hydrogen, C1-C3 alkyl or amino protecting group.
  • the transaminase is derived from: Chromobacterium violaceum DSM30191 (CVTA) (NCBI Reference Sequence: WP_011135573.1), Fonsecaea pedrosoi CBS 271.37 (NCBI Reference Sequence: XP_013286281.1), Klebsiella pneumoniae subsp.pneumoniae Ecl8 (GenBank: CCN29541.1), Mycobacterium goodii (GenBank: AKS36000.1), Paracoccus 697 Penicillium brasilianum (GenBank: CEJ55334.1), Enterobacter sp.TL3 (NCBI Reference Sequence: WP_014885677.1), Aspergillus terreus NIH2624 (NCBI Reference Sequence: XP_001209325.1), Exophiala: Reference 01623sequence: Reference 01623Sequence geothermalis (strain DSM 11300) (NCBI Reference Sequence: WP_011530545.1),
  • the transaminase has an amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, which are respectively:
  • the above-mentioned transaminase of the present application has substrate specificity for the substrate represented by formula I, and can effectively catalyze the conversion of such substrates into chiral diamine compounds. Moreover, the transaminase has substrate specificity for a variety of substrates represented by formula I. The reaction selectivity and activity are both high.
  • this biological enzyme to catalyze the synthesis of chiral diamine compounds is not only suitable for a wider range of substrates (the reactions in the prior art are all 1,2-diamine compounds, but the application can also synthesize 1,3 -Diamine, 1,4-diamine and other compounds), and the synthesis method has a short route, high product yield, greatly reduces production costs, and reduces organic solvents and three wastes.
  • heteroatom in the "heteroatom-containing alkyl group, heteroatom-containing cycloalkyl group, and heteroatom-containing aryl group" in the above-mentioned substrate may be at least one of O, S, and N.
  • aminotransferases with the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 are all derived from ⁇ -transaminase, Chromobacterium violaceum DSM30191 (CVTA) .
  • amino protecting groups include, but are not limited to, tert-butoxycarbonyl, benzyloxycarbonyl, formyl, trifluoroacetyl, benzyl, trityl, or 9-fluorenylmethoxycarbonyl.
  • the substrate represented by formula I is selected from any one of the following:
  • the transaminase of the present application has catalytic activity and stereoselectivity to the substrate represented by the above formula I.
  • the above-mentioned synthesis method includes: mixing a phosphate buffer with an amino donor to obtain a first mixed solution; adding a substrate represented by formula I to the first mixed solution to obtain a second mixed solution; A transaminase is added to the second mixed liquid to obtain a reaction mixed liquid; and a chiral diamine compound is separated from the reaction mixed liquid.
  • the synthesis method reduces the reaction volume through the above-mentioned step-by-step reaction, so that the catalytic activity and efficiency of the transaminase on the substrate are higher, thereby obtaining a higher product yield and improving the production efficiency.
  • the synthesis method before adding the substrate of formula I to the first mixed solution, further comprises: adjusting the pH value of the first mixed solution to 7.0 to 9.0; A transaminase is added to the mixed liquid, and the pH of the second mixed liquid after the addition of the transaminase is adjusted to 7.0-9.0 to obtain a reaction mixed liquid.
  • the pH value of the first mixed solution is adjusted before adding the substrate of formula I to the first mixed solution, and the pH value of the enzyme reaction is adjusted to the optimum pH before adding the enzyme to prevent the enzyme from being added. Inactivated.
  • the pH value of the system is also adjusted, and its function is to ensure that the reaction proceeds at the optimum pH.
  • the above two pH adjustment ranges are best kept the same, for example, adjusted to 7.8 before, and also adjusted to 7.8 after adding transaminase.
  • separating the chiral diamine compound from the reaction mixture includes: adjusting the acidity of the reaction mixture to denature the transaminase, preferably adjusting the pH of the reaction mixture to 1 to 2; The transaminase is removed by filtration to obtain a preliminary filtrate; the pH of the preliminary filtrate is adjusted to alkaline to obtain an alkaline filtrate; the alkaline filtrate is extracted to obtain a chiral diamine compound; preferably, the pH of the alkaline filtrate is 12 ⁇ 13; Preferably, the extraction is multiple times, more preferably 2 to 5 times, more preferably, dichloromethane is used for the first extraction, followed by dichloromethane or ethyl acetate for the remaining times.
  • the method of adjusting acidity is used to denature the enzyme protein after the reaction. Compared with other denaturation methods (such as high temperature, reduction, or salting out), the denaturation is more thorough. Most of the products are stable under acidic conditions, and the subsequent operations are directly under acidic conditions. Advantages of extracting other impurities in the system.
  • the purpose of the first extraction with dichloromethane is to remove impurities that can be extracted under acidic conditions in the system. Compared with other extraction solvents (such as ethyl acetate, methyl tert-butyl ether or isopropyl acetate) for extraction, it has the advantage of higher extraction efficiency.
  • the extraction further includes a step of drying the extracted organic phase.
  • the organic phases obtained from each extraction are combined to obtain an extract; Dry to obtain a dry organic phase product; place the dried organic phase product at a temperature of ⁇ 45°C and a pressure of ⁇ -0.05Mpa to concentrate to no distillate ((The purpose of drying is to reduce the water content in the product, and the pressure affects the concentration speed).
  • the mass ratio of the transaminase to the substrate represented by formula I is 0.4:1 to 1.0:1, and the reaction is carried out according to the mass ratio, and the conversion efficiency of the substrate can reach more than 92%.
  • the concentration of the substrate represented by formula I is 60 g/L to 100 g/L.
  • biological enzymes are used for the reaction, and the conversion rate of the substrate is as high as 95%, which helps to improve the production efficiency and reduce the production of organic solvents and three wastes.
  • the amino donor is selected from isopropylamine, isopropylamine hydrochloride, alanine, phenethylamine or n-butylamine.
  • the room temperature in the following examples refers to a temperature in the normal temperature range of 10-25°C
  • the transaminase used in the following examples 1 to 15 is the transaminase shown in SEQ ID NO:4.
  • the combined organic phase was dried with anhydrous magnesium sulfate, and the organic phase was concentrated to no fraction under the conditions of T ⁇ 35°C and P ⁇ -0.06Mpa.
  • the purity is more than 98%, the ee value is more than 99%, and the yield is 90%.
  • the combined organic phase was dried with anhydrous magnesium sulfate, and the organic phase was concentrated to no fraction under the conditions of T ⁇ 45°C and P ⁇ -0.08Mpa.
  • the purity is more than 92%, the ee value is more than 99%, and the yield is 86%.
  • the combined organic phase was dried with anhydrous magnesium sulfate, and the organic phase was concentrated to no fraction under the conditions of T ⁇ 35°C and P ⁇ -0.06Mpa.
  • the purity is greater than 97%, the ee value is greater than 99%, and the yield is 78%.
  • the combined organic phase was dried with anhydrous magnesium sulfate, and the organic phase was concentrated to no fraction under the conditions of T ⁇ 35°C and P ⁇ -0.06Mpa.
  • the combined organic phase was dried with anhydrous magnesium sulfate, and the organic phase was concentrated to no fraction under the conditions of T ⁇ 45°C and P ⁇ -0.08Mpa.
  • the combined organic phase was dried with anhydrous magnesium sulfate, and the organic phase was concentrated to no fraction under the conditions of T ⁇ 45°C and P ⁇ -0.08Mpa.
  • the combined organic phase was dried with anhydrous magnesium sulfate, and the organic phase was concentrated to no fraction under the conditions of T ⁇ 45°C and P ⁇ -0.08Mpa.
  • the combined organic phase was dried with anhydrous magnesium sulfate, and the organic phase was concentrated to no fraction under the conditions of T ⁇ 45°C and P ⁇ -0.08Mpa.
  • the combined organic phase was dried with anhydrous magnesium sulfate, and the organic phase was concentrated to no fraction under the conditions of T ⁇ 45°C and P ⁇ -0.08Mpa.
  • amino donor Respectively, isopropylamine, isopropylamine hydrochloride, alanine, aniline, n-butylamine
  • the transaminase number TA1 derived from Chromobacterium violaceum DSM30191 (CVTA) (NCBI Reference Sequence: WP_011135573.1) will be derived from the transaminase of Fonsecaea pedrosoi CBS 271.37 (NCBI Reference Sequence: XP_013286281.1) Number TA2, number TA3 for the transaminase derived from Klebsiella pneumoniae subsp.pneumoniae Ecl8 (GenBank: CCN29541.1), and number TA4 for the transaminase derived from Mycobacterium goodii (GenBank: AKS36000.1), which will be derived from Paracoccus denitrificans (NCBI Reference Sequence) : WP_011746975.1) transaminase number TA5, the transaminase number TA6 derived from Penicillium brasilianum (GenBank: CE
  • the transaminase number TA11 will be derived from Pseudomonas putida KT2440 (NCBI Reference Sequence: WP_010954554.1)
  • the transaminase number TA12 will be derived from Lysinibacillus sphaericus (NCBI Reference Sequence: WP_024363741.
  • the transaminase number TA13, the transaminase number TA14 derived from Bacillus megaterium DSM 319 will be the transaminase number TA15 derived from Trichoderma harzianum (GenBank: KKP07030.1), which will be derived from Aspergillus fumigatus
  • the transaminase number TA16 of R-ATAs(AspFum) (NCBI Reference Sequence: XP_748821.1) will be derived from Geobacillus thermonitrificans subsp.thermodenitrificans DSM 465 (NCBI Reference Sequence: WP_008879436.1) transaminase number TA17, which will be derived from CaBS Cladophialophore
  • the transaminase number TA18 of 173.52 (NCBI Reference Sequence: XP_016617948.1) will be derived from the Bacillus
  • the transaminase number TA20 the transaminase number from Klebsiella pneumoniae subsp.pneumoniae MGH78578 (NCBI Reference Sequence: WP_002920226.1) will be derived from Geobacillus toebii (NCBI Reference Sequence: WP_06275389TA22, the transaminase number TA21) Transaminase number TA23 derived from Talaromyces cellulolyticus (GenBank: GAM37533.1), mutant TA1-V1 (corresponding sequence is SEQ ID NO : 1), TA1-V2 (corresponding sequence is SEQ ID NO: 2). TA1-V3 (corresponding sequence is SEQ ID NO: 3), TA1-V4 (corresponding sequence is SEQ ID NO: 4) for 10mg level screening reaction, the operation is as follows:
  • the 100mmol/L phosphate buffer, 5mol/L isopropylamine hydrochloride solution was prepared into a 50ml solution according to the volume ratio of 5:2, then 0.005g pyridoxal phosphate was added, and the mixture was stirred and mixed. Take 0.3ml of the above solution into 96-well plate, and add 10mg Then add 0.2ml of the above enzyme solution (10wt, the concentration of the enzyme solution is 0.5g/ml), the shaker rotates at 170 rpm, and the temperature is raised to 30°C for overnight reaction.
  • transaminase to carry out the biotransformation reaction can directly obtain the desired target compound in one step reaction.
  • the substrate type is applicable to a wide range. It can not only synthesize patents but also synthesize 1,3-diamine, 1,4-diamine and other compounds. Synthesis of 1,3-diamine, 1,4-diamine and other compounds.
  • transaminase for the biotransformation reaction has high selectivity, and the improved chiral purity of the product greatly improves the ee value of the product.
  • the substrate concentration can reach 100g/L, which greatly improves the production efficiency.
  • transaminase for biocatalysis does not require the use of heavy metal catalysts, azide compounds and cyanation reagents. Realize green chemistry.
  • the transaminase has high catalytic efficiency, small reaction volume, short synthesis route, high product yield, greatly reduces the three wastes, and saves production costs.

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Abstract

本发明提供了一种手性二胺化合物的合成方法。该合成方法包括：利用转氨酶将式I所示底物转化为式I的手性二胺化合物，其中n＝1～10，R基表示烷基、环烷基、含杂原子的烷基、含杂原子的环烷基、含杂原子的芳基、酰胺类化合物残基或醚类化合物残基，杂原子为O、S和N种的至少一种；R1、R2相同或不相同，R1和R2各自独立地为氢、C1-C3烷基或氨基保护基；转氨酶来源于多个菌种。这些转氨酶对多种式1所示底物的反应选择性和活性均较高。利用该生物酶催化合成手性二胺化合物，不仅底物更广泛，且路线短，产品收率高，大大降低了生产成本，减少了有机溶剂和三废产生。

Description

手性二胺化合物的合成方法 技术领域
本发明涉及手性二胺化合物的合成领域，具体而言，涉及一种手性二胺化合物的合成方法。
背景技术
手性二胺化合物广泛存在于许多具有生物活性的天然产物及药物分子中，如：鸟氨酸，赖氨酸，维生素H，药物左旋咪唑等。奥沙利铂(Oxaliplatin)分子中存在二胺化合物，具有一定的抗肿瘤活性。具有二胺结构的化合物也被广泛应用于有机合成，其作为合成砌块，可以有效构建氮杂环，也可以作为配体与金属络合生成具有高度反应活性的催化剂，二胺化合物还被广泛应用于液晶材料，航空材料，微电子等领域。手性二胺化合物也可以作为手性拆分试剂对醛的对映异构体进行拆分。因此，手性二胺的合成一直是化学家们研究的热点(Angew.Chem.Int.Ed.1998,37,2580)。
目前，手性二胺化合物的合成方法有不对称strecker反应(Chem.Rev.,2011,111，6947)，不对称Michel加成(CN 105367427 A)、吖丙啶不对称开环(CN 105753752 A)等方法，但这些方法在底物的适用范围和实用性上均存在不同程度的局限性，且这些方法合成的均为1,2-二胺类化合物。不对称Michel加成及吖丙啶不对称开环中通常需要昂贵的金属及手性催化剂，这极大地限制了反应体系的应用。此外，反应中会使用剧毒的叠氮化合物或氰基化试剂，化学路线三废量较大，不好处理。
因此，如何改进现有方法，以拓展对底物的适用范围便成了一个亟待解决的技术问题。
发明内容
本发明的主要目的在于提供一种手性二胺化合物的合成方法，以解决现有技术中的方法对底物的适用范围存在局限性的问题。
为了实现上述目的，本发明提供了一种手性二胺化合物的合成方法，该合成方法包括：利用转氨酶将式I所示底物转化为手性二胺化合物；
Figure PCTCN2020082594-appb-000001
其中，n＝1～10，R基表示烷基、环烷基、含杂原子的烷基、含杂原子的环烷基、含杂原子的芳基、酰胺类化合物残基或醚类化合物残基，其中，杂原子为O、S和N种的至少一种；R1、R2相同或不相同，R1和R2各自独立地为氢、C1-C3烷基或氨基保护基；转氨酶来源于 Chromobacterium violaceum DSM30191(CVTA)(NCBI Reference Sequence:WP_011135573.1)、Fonsecaea pedrosoi CBS 271.37(NCBI Reference Sequence:XP_013286281.1)、Klebsiella pneumoniae subsp.pneumoniae Ecl8(GenBank:CCN29541.1)、Mycobacterium goodii(GenBank:AKS36000.1)、Paracoccus denitrificans(NCBI Reference Sequence:WP_011746975.1)、Penicillium brasilianum(GenBank:CEJ55334.1)、Enterobacter sp.TL3(NCBI Reference Sequence:WP_014885677.1)、Aspergillus terreus NIH2624(NCBI Reference Sequence:XP_001209325.1)、Exophiala spinifera(NCBI Reference Sequence:XP_016233821.1)、Deinococcus geothermalis(strain DSM 11300)(NCBI Reference Sequence:WP_011530545.1)、Geomyces destructans 20631-21(GdTA)in E.coli(GenBank:ELR05573.1)、Pseudomonas putida KT2440(NCBI Reference Sequence:WP_010954554.1)、Lysinibacillus sphaericus(NCBI Reference Sequence:WP_024363741.1)、Bacillus megaterium DSM 319(NCBI Reference Sequence:WP_013082219.1)、Trichoderma harzianum(GenBank:KKP07030.1)、Aspergillus fumigatus R-ATAs(AspFum)(NCBI Reference Sequence:XP_748821.1)、Geobacillus thermodenitrificans subsp.thermodenitrificans DSM 465(NCBI Reference Sequence:WP_008879436.1)、Cladophialophora bantiana CBS 173.52(NCBI Reference Sequence:XP_016617948.1)、Bacillus megaterium(NCBI Reference Sequence:WP_016763026.1)、Burkholderia thailandensis MSMB121(BtS-TA)(GenBank:AGK49399.1)、Klebsiella pneumoniae subsp.pneumoniae MGH 78578(NCBI Reference Sequence:WP_002920226.1)、Geobacillus toebii(NCBI Reference Sequence:WP_062753894.1)和Talaromyces cellulolyticus(GenBank:GAM37533.1)。
进一步地，转氨酶具有SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3或SEQ ID NO：4所示的氨基酸序列。
进一步地，氨基保护基选自叔丁氧羰基，苄氧羰基，甲酰基，三氟乙酰基，苄基，三苯甲基及9-芴基甲氧基羰基中的任意一种。
进一步地，底物选自如下任意一种：
Figure PCTCN2020082594-appb-000002
Figure PCTCN2020082594-appb-000003
Figure PCTCN2020082594-appb-000004
Figure PCTCN2020082594-appb-000005
及
Figure PCTCN2020082594-appb-000006
进一步地，合成方法包括：将磷酸缓冲液与氨基供体混合，得到第一混合液，向第一混合液中添加式I所示的底物，得到第二混合液；向第二混合液中添加转氨酶，得到反应混合液；从反应混合液中分离得到手性二胺化合物。
进一步地，在向第一混合液中添加底物之前，合成方法还包括：将第一混合液的pH值调节至7.0～9.0；优选地，向第二混合液中添加转氨酶，并调节加入转氨酶后的第二混合液的pH为7.0～9.0，得到反应混合液。
进一步地，从反应混合液中分离得到手性二胺化合物包括：调节反应混合液的酸度使转氨酶变性，优选调节反应混合液的pH值为1～2；将变性后的转氨酶过滤除去，得到初步滤液；将初步滤液的pH值调至碱性，得到碱性滤液；对碱性滤液进行萃取，得到手性二胺类化合物；优选地，碱性滤液的pH值为12～13；优选地，萃取为多次，更优选为2～5次，更优选采用二氯甲烷进行第一次萃取，后续采用二氯甲烷或乙酸乙酯萃取剩余次数。
进一步地，萃取之后还包括对萃取所得有机相进行干燥的步骤，优选地，将每次萃取所得有机相合并，得到萃取物；对萃取物进行干燥，得到干燥有机相产物；将干燥有机相产物置于温度＜45℃，压力≤-0.08Mpa的条件下浓缩至无馏分，得到手性二胺类化合物。
进一步地，转氨酶与底物的质量比为：0.4：1～1.0：1。
进一步地，底物的浓度为60g/L～100g/L。
进一步地，氨基供体选自异丙胺、异丙胺的盐酸盐、丙氨酸、苯乙胺或正丁胺。
应用本发明的技术方案，采用对转氨酶对式I所示底物具有特异性，能够有效催化此类底物转化为手性二胺化合物，而且，该转氨酶对多种式I所示底物的反应选择性和活性均较高。因此，利用该生物酶催化合成手性二胺化合物，不仅适用于更广泛的底物(现有技术反应得到的都是1，2-二胺类化合物，而本申请中还可以合成1，3-二胺，1，4-二胺等化合物)，而且该合成方法路线短，产品收率高，大大降低了生产成本，减少了有机溶剂和三废产生。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1示出了本发明的实施例11中不同酶量对相同量的底物的转化率的影响；
图2示出了本发明的实施例12中相同酶量对不同浓度的底物的转化率的影响；
图3示出了本发明的实施例13中不同的氨基供体对同一反应中的底物的转化率的影响；以及
图4示出了本发明的实施例14中不同来源的转氨酶对同一底物均具有转化活性。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将结合实施例来详细说明本发明。
如背景技术所提到的，现有技术中，手性二胺化合物的合成方法对底物的适用范围存在局限性的问题。为了改善这一状况，本申请的发明人尝试从高效、绿色环保的角度来改进现有合成路线。在研究过程中，发明人发现，申请人自行研制的一种转氨酶除了具有以PLP为 辅酶的转氨酶所具有的催化酮类化合物的通用催化活性外，还具有催化多种不同的底物，并将其转化为手性二胺化合物的活性。并进一步研究发现采用该转氨酶催化合成手性二胺化合物时，不仅底物类型广泛，而且可以通过一步反应获得目标产物。由于使用该转氨酶进行生物转化反应选择性很高，大大提升了目标产物的ee值。而且，采用生物酶催化反应，能够提高底物用量，大大提高了生产效率，减少了有机溶剂和三废产生。
在上述研究结果的基础上，申请人提出了本申请的技术方案。在本申请一种典型的实施方式中，提供了一种手性二胺化合物的合成方法，该合成方法包括：利用转氨酶将式I所示底物转化为手性二胺化合物；
Figure PCTCN2020082594-appb-000007
其中，n＝1～10，R基表示烷基、环烷基、含杂原子的烷基、含杂原子的环烷基、含杂原子的芳基、酰胺类化合物残基或醚类化合物残基，R1、R2相同或不相同，R1和R2各自独立地为氢、C1-C3烷基或氨基保护基，该转氨酶来源于：Chromobacterium violaceum DSM30191(CVTA)(NCBI Reference Sequence:WP_011135573.1)、Fonsecaea pedrosoi CBS 271.37(NCBI Reference Sequence:XP_013286281.1)、Klebsiella pneumoniae subsp.pneumoniae Ecl8(GenBank:CCN29541.1)、Mycobacterium goodii(GenBank:AKS36000.1)、Paracoccus denitrificans(NCBI Reference Sequence:WP_011746975.1)、Penicillium brasilianum(GenBank:CEJ55334.1)、Enterobacter sp.TL3(NCBI Reference Sequence:WP_014885677.1)、Aspergillus terreus NIH2624(NCBI Reference Sequence:XP_001209325.1)、Exophiala spinifera(NCBI Reference Sequence:XP_016233821.1)、Deinococcus geothermalis(strain DSM 11300)(NCBI Reference Sequence:WP_011530545.1)、Geomyces destructans 20631-21(GdTA)in E.coli(GenBank:ELR05573.1)、Pseudomonas putida KT2440(NCBI Reference Sequence:WP_010954554.1)、Lysinibacillus sphaericus(NCBI Reference Sequence:WP_024363741.1)、Bacillus megaterium DSM 319(NCBI Reference Sequence:WP_013082219.1)、Trichoderma harzianum(GenBank:KKP07030.1)、Aspergillus fumigatus R-ATAs(AspFum)(NCBI Reference Sequence:XP_748821.1)、Geobacillus thermodenitrificans subsp.thermodenitrificans DSM 465(NCBI Reference Sequence:WP_008879436.1)、Cladophialophora bantiana CBS 173.52(NCBI Reference Sequence:XP_016617948.1)、Bacillus megaterium(NCBI Reference Sequence:WP_016763026.1)、Burkholderia thailandensis MSMB121(BtS-TA)(GenBank:AGK49399.1)、Klebsiella pneumoniae subsp.pneumoniae MGH 78578(NCBI Reference Sequence:WP_002920226.1)、Geobacillus toebii(NCBI Reference Sequence:WP_062753894.1)和Talaromyces cellulolyticus(GenBank:GAM37533.1)。
优选地，该转氨酶具有SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3或SEQ ID NO：4所示的氨基酸序列，分别为：
Figure PCTCN2020082594-appb-000008
如上述，本申请的上述转氨酶对式I所示底物具有底物特异性，能够有效催化此类底物转化为手性二胺化合物，而且，该转氨酶对多种式I所示底物的反应选择性和活性均较高。因此，利用该生物酶催化合成手性二胺化合物，不仅适用于更广泛的底物(现有技术反应得到的都是1，2-二胺类化合物，而本申请中还可以合成1，3-二胺，1，4-二胺等化合物)，而且该合成方法路线短，产品收率高，大大降低了生产成本，减少了有机溶剂和三废产生。
上述底物中的“含杂原子的烷基、含杂原子的环烷基、含杂原子的芳基”中的杂原子可以是O、S及N中的至少一种。
上述具有SEQ ID NO:1、SEQ ID NO：2、SEQ ID NO：3和SEQ ID NO：4所示氨基酸序列的转氨酶，均来源于ω-转氨酶(ω-transaminase)，Chromobacterium violaceum DSM30191(CVTA)。
上述氨基保护基包括但不限于叔丁氧羰基、苄氧羰基、甲酰基、三氟乙酰基、苄基、三苯甲基或9-芴基甲氧基羰基。
在一种优选的实施例中，式I所示底物选自如下任意一种：
Figure PCTCN2020082594-appb-000009
Figure PCTCN2020082594-appb-000010
及
Figure PCTCN2020082594-appb-000011
本申请的转氨酶对上述式I所示底物均具有催化活性和立体选择性。
采用本申请的转氨酶对式I所示底物进行催化转氨基反应的具体步骤与现有转氨反应类似。在一种优选的实施例中，上述合成方法包括：将磷酸缓冲液与氨基供体混合，得到第一混合液；向第一混合液中添加式I所示底物，得到第二混合液；向第二混合液中添加转氨酶，得到反应混合液；从反应混合液中分离得到手性二胺化合物。
该合成方法通过上述分步反应，减少了反应体积，使得转氨酶对底物的催化活性和效率较高，从而获得较高的产品收率，提高了生产效率。
在一种优选的实施例中，在向第一混合液中添加式I所示底物之前，合成方法还包括：将第一混合液的pH值调节至7.0～9.0；优选地，向第二混合液中添加转氨酶，并调节加入转氨酶后的第二混合液的pH为7.0～9.0，得到反应混合液。
上述优选实施例中，通过在在向第一混合液中添加式I所示底物之前，对第一混合液进行pH值调节，加入酶之前调节到酶反应最适pH，防止酶加入过程中失活。而在向第二混合液中添加转氨酶后也对体系的pH值进行调节，其作用是确保反应在最适pH下进行。上述两次pH值调节的范围最好保持一致，比如，前面调节至7.8，加入转氨酶后也同样调节至7.8。
上述从反应后的混合液中分离得到目的产物的步骤采用已知的分离、提纯方法进行即可。在一种优选的实施例中，从反应混合液中分离得到手性二胺化合物包括：调节反应混合液的酸度使转氨酶变性，优选调节反应混合液的pH值为1～2；将变性后的转氨酶过滤除去，得到初步滤液；将初步滤液的pH值调至碱性，得到碱性滤液；对碱性滤液进行萃取，得到手性二胺化合物；优选地，碱性滤液的pH值为12～13；优选地，萃取为多次，更优选为2～5次，更优选采用二氯甲烷进行第一次萃取，后续采用二氯甲烷或乙酸乙酯萃取剩余次数。
采用调节酸度的方式使反应后的酶蛋白变性，相比其他变性方式(比如高温、调减、或盐析)有变性比较彻底，产物大多在酸性条件下稳定，且之后的操作直接在酸性条件下可将体系其他杂质萃取优势。采用二氯甲烷进行首次萃取的目的在于除去体系中酸性条件可被萃 取的杂质。相比比采用其他萃取溶剂(比如乙酸乙酯、甲基叔丁基醚或乙酸异丙酯)进行萃取有萃取效率较高的优势。
为进一步提高纯度和收率，在一种优选的实施例中，萃取后还包括对萃取有机相进行干燥的步骤，优选地，将每次萃取所得有机相合并，得到萃取物；对萃取物进行干燥，得到干燥有机相产物；将干燥有机相产物置于温度＜45℃，压力≤-0.05Mpa的条件下浓缩至无馏分((干燥的目的在于减少产品中的含水量，而压力大小影响浓缩速度)。
在一种优选的实施例中，转氨酶与式I所示底物的质量比为：0.4:1～1.0:1，按照该质量比进行反应，底物的转化效率能够达到92％以上。
在一种优选的实施例中，式I所示底物的浓度为60g/L～100g/L。在该浓度范围下采用生物酶进行反应，底物的转化率高达95％以上，有助于提高生产效率，减少有机溶剂和三废的产生。
在一种优选的实施例中，氨基供体选自异丙胺、异丙胺的盐酸盐、丙氨酸、苯乙胺或正丁胺。
下面将结合具体的实施例来进一步说明本申请的有益效果。需要说明的是，以下实施例中的室温是指10～25℃的常温范围内的温度，以下实施例1至15中所用到的转氨酶为SEQ ID NO：4所示的转氨酶。
实施例1
室温向250mL四口瓶内加入50mL 100mmol/L磷酸盐缓冲液(5vol)，24ml 5mol/L异丙胺盐酸盐溶液(2.4vol，3eq)，调pH＝7.5～8.0。再加入0.1g磷酸吡哆醛(1wt％)，10g
Figure PCTCN2020082594-appb-000012
搅拌均匀，再加入转氨酶酶液10ml(0.5wt，0.5g/ml)调pH＝7.5～8.0。升温至30℃搅拌反应过夜。反应完全后，将体系调酸至pH＝1，变性蛋白。过滤后滤液用50mL二氯甲烷萃取。水相调pH＝12，再用50ml二氯甲烷萃取三次。合并有机相用无水硫酸镁干燥后有机相于T＜35℃，P≤-0.06Mpa条件下浓缩至无馏分。得到目标产物
Figure PCTCN2020082594-appb-000013
经HPLC检测，纯度＞98％，ee值＞99％，收率90％。
实施例2
室温向250mL四口瓶内加入60mL 100mmol/L磷酸盐缓冲液(6vol)，18ml 5mol/L异丙胺盐酸盐溶液(1.8vol，3eq)，调pH＝8.0～8.5。再加入0.1g磷酸吡哆醛(1wt％)，10g
Figure PCTCN2020082594-appb-000014
搅拌均匀，再加入转氨酶酶液10ml(0.5wt，0.5g/ml)，调pH＝8.0～8.5。调温至20℃搅拌反应过夜。反应完全后，将体系调酸至pH＝2，变性蛋白。过滤后滤液用50mL二氯甲烷萃取。水相调pH＝13，再用50ml乙酸乙酯萃取三次。合并有机相用无水硫酸镁干燥后有机相于T＜45℃，P≤-0.08Mpa条件下浓缩至无馏分。得到目标产物
Figure PCTCN2020082594-appb-000015
经HPLC检测，纯度＞92％，ee值＞99％，收率86％。
实施例3
室温向250mL四口瓶内加入70mL 100mmol/L磷酸盐缓冲液(7vol)，13ml 5mol/L异丙胺盐酸盐溶液(1.3vol，3eq)，调pH＝7.0～7.5。再加入0.1g磷酸吡哆醛(1wt％)，10g
Figure PCTCN2020082594-appb-000016
搅拌均匀，再加入转氨酶酶液10ml(0.5wt，0.5g/ml)升温至50℃搅拌反应过夜。反应完全后，将体系调酸至pH＝1，变性蛋白。过滤后滤液用50mL二氯甲烷萃取。水相调pH＝13，再用50ml二氯甲烷萃取三次。合并有机相用无水硫酸镁干燥后有机相于T＜35℃，P≤-0.06Mpa条件下浓缩至无馏分。得到目标产物
Figure PCTCN2020082594-appb-000017
经HPLC检测，纯度大于97％，ee值＞99％，收率78％。
实施例4
室温向250mL四口瓶内加入50mL 100mmol/L磷酸盐缓冲液(5vol)，38ml 5mol/L异丙胺盐酸盐溶液(3.8vol，3eq)，调pH＝8.5～9.0。再加入0.1g磷酸吡哆醛(1wt％)，10g
Figure PCTCN2020082594-appb-000018
搅拌均匀，再加入转氨酶酶液10ml(0.5wt，0.5g/ml)，调pH＝8.5～9.0。调温至10℃搅拌反应过夜。反应完全后，将体系调酸至pH＝2，变性蛋白。过滤后滤液用50mL二氯甲烷萃取。水相调pH＝13，再用50ml二氯甲烷萃取三次。合并有机相用无水硫酸镁干燥后有机相于T＜35℃，P≤-0.06Mpa条件下浓缩至无馏分。得到目标产物
Figure PCTCN2020082594-appb-000019
经HPLC检测，纯度＞95％，ee值＞99％，收率：79％。
实施例5
室温向250mL四口瓶内加入70mL 100mmol/L磷酸盐缓冲液(7vol)，17ml 5mol/L异丙胺盐酸盐溶液(1.7vol，3eq)，调pH＝7.5～8.0。再加入0.1g磷酸吡哆醛(1wt％)，10g
Figure PCTCN2020082594-appb-000020
搅拌均匀，再加入转氨酶酶液10ml(0.5wt，0.5g/ml)，调pH＝7.5～8.0。升温至29℃搅拌反应过夜。反应完全后，将体系调酸至pH＝1，变性蛋白。过滤后滤液用50mL二氯甲烷萃取。水相调pH＝12，再用50ml乙酸乙酯萃取三次。合并有机相用无水硫酸镁干燥后有机相于T＜45℃，P≤-0.08Mpa条件下浓缩至无馏分。得到目标产物
Figure PCTCN2020082594-appb-000021
经HPLC检测，纯度＞98％，ee值＞99％，收率：87％。
实施例6
室温向500mL四口瓶内加入140mL 100mmol/L磷酸盐缓冲液(7vol)，50ml 5mol/L异丙胺盐酸盐溶液(1.7vol，3eq)，调pH＝7.5～8.0。再加入0.2g磷酸吡哆醛(1wt％)，20g
Figure PCTCN2020082594-appb-000022
搅拌均匀，再加入转氨酶酶液20ml(0.5wt，0.5g/ml)，调pH＝7.5～8.0。升温至32℃搅拌反应过夜。反应完全后，将体系调酸至pH＝1，变性蛋白。过滤后滤液用100mL二氯甲烷萃取。水相调pH＝12，再用100ml乙酸乙酯萃取三次。合并有机相用无水硫酸镁干燥后有机相于T＜45℃，P≤-0.08Mpa条件下浓缩至无馏分。得到目标产物
Figure PCTCN2020082594-appb-000023
经HPLC检测，纯度＞97％，ee值＞99％，收率：84％。
实施例7
室温向500mL四口瓶内加入220mL 100mmol/L磷酸盐缓冲液(11vol)，50ml 5mol/L异丙胺盐酸盐溶液(2.5vol，3eq)，调pH＝7.5～8.0。再加入0.2g磷酸吡哆醛(1wt％)，20g
Figure PCTCN2020082594-appb-000024
搅拌均匀，再加入转氨酶酶液40ml(1.0wt，0.5g/ml)，调pH＝7.5～8.0。升温至32℃搅拌反应过夜。反应完全后，将体系调酸至pH＝1，变性蛋白。过滤后滤液用100mL二氯甲烷萃取。水相调pH＝12，再用100ml乙酸乙酯萃取三次。合并有机相用无水硫酸镁干燥后有机相于T＜45℃，P≤-0.08Mpa条件下浓缩至无馏分。得到目标产物
Figure PCTCN2020082594-appb-000025
经HPLC检测，纯度＞95％，ee值＞99％，收率：85％。
实施例8
室温向500mL四口瓶内加入180mL 100mmol/L磷酸盐缓冲液(12vol)，36ml 5mol/L异丙胺盐酸盐溶液(2.4vol，3eq)，调pH＝7.5～8.0。再加入0.15g磷酸吡哆醛(1wt％)，15g
Figure PCTCN2020082594-appb-000026
搅拌均匀，再加入转氨酶酶液30ml(1.0wt，0.5g/ml)，调pH＝7.5～8.0。升温至35℃搅拌反应过夜。反应完全后，将体系调酸至pH＝1，变性蛋白。过滤后滤液用100mL二氯甲烷萃取。水相调pH＝12，再用100ml乙酸乙酯萃取三次。合并有机相用无水硫酸镁干燥后有机相于T＜45℃，P≤-0.08Mpa条件下浓缩至无馏分。得到目标产物
Figure PCTCN2020082594-appb-000027
经HPLC检测，纯度＞96％，ee值＞99％，收率：91％。
实施例9
室温向500mL四口瓶内加入180mL 100mmol/L磷酸盐缓冲液(12vol)，26ml 5mol/L异丙胺盐酸盐溶液(1.7vol，3eq)，调pH＝7.5～8.0。再加入0.15g磷酸吡哆醛(1wt％)，15g
Figure PCTCN2020082594-appb-000028
搅拌均匀，再加入转氨酶酶液30ml(1.0wt，0.5g/ml)，调pH＝7.5～8.0。升温至40℃搅拌反应过夜。反应完全后，将体系调酸至pH＝1，变性蛋白。过滤后滤液用100mL二氯甲烷萃取。水相调pH＝12，再用100ml乙酸乙酯萃取三次。合并有机相用无水硫酸镁干燥后有机相于T＜45℃，P≤-0.08Mpa条件下浓缩至 无馏分。得到目标产物
Figure PCTCN2020082594-appb-000029
经HPLC检测，纯度＞97％，ee值＞99％，收率：90％。
实施例10
室温向500mL四口瓶内加入110mL 100mmol/L磷酸盐缓冲液(11vol)，23ml 5mol/L异丙胺盐酸盐溶液(2.3vol，3eq)，调pH＝7.5～8.0。再加入0.1g磷酸吡哆醛(1wt％)，10g
Figure PCTCN2020082594-appb-000030
搅拌均匀，再加入转氨酶酶液10ml(0.5wt，0.5g/ml)，调pH＝7.5～8.0。升温至40℃搅拌反应过夜。反应完全后，将体系调酸至pH＝2，变性蛋白。过滤后滤液用50mL二氯甲烷萃取。水相调pH＝12，再用50ml乙酸乙酯萃取三次。合并有机相用无水硫酸镁干燥后有机相于T＜45℃，P≤-0.08Mpa条件下浓缩至无馏分。得到目标产物
Figure PCTCN2020082594-appb-000031
经HPLC检测，纯度＞95％，ee值＞99％，收率：83％。
实施例11
以实施例2为例，对反应的酶量进行优化具体操作如下：室温向10mL小瓶内加入1.2mL 100mmol/L磷酸盐缓冲液(6vol)，0.36ml 5mol/L异丙胺盐酸盐溶液(1.8vol，3eq)，调pH＝8.0～8.5。再加入0.002g磷酸吡哆醛(1wt％)，0.2g
Figure PCTCN2020082594-appb-000032
混匀，分别加入转氨酶酶液0.04ml，0.08ml，0.12ml，0.16ml，0.2ml，0.24ml，0.28ml，0.32ml，0.36ml，0.4ml，0.44ml，0.48ml，0.52ml，0.56ml，0.6ml(酶液浓度为0.5g/ml，对应质量比分别为0.1wt，0.2wt，0.3wt，0.4wt，0.5wt，0.6wt，0.7wt，0.8wt，0.9wt，1.0wt，1.1wt，1.2wt，1.3wt，1.4wt，1.5wt)， 调pH＝8.0～8.5。摇床转速170rpm，升温至29℃条件下反应过夜。转化率结果如图1所示，证明0.5～1.0wt底物可转化率大于99％，酶量继续增大也并无显著效果。
实施例12
以实施例2为例，对反应的底物浓度进行优化具体操作如下：室温向10～50mL小瓶内(根据反应体积选择合适的摇瓶)分别加入0.58mL，0.66ml，0.78ml，0.9ml，1.06ml，1.24ml，1.44ml，1.74ml，2.1ml，2.56ml，3.24ml，4.24ml，5.84ml，9.24ml 100mmol/L磷酸盐缓冲液(分别为2.9vol，3.3vol，3.9vol，4.5vol，5.3vol，6.2vol，7.2vol，8.7vol，10.5vol，12.8vol，16.2vol，21.2vol，29.2vol，46.2vol)，加入0.36ml 5mol/L异丙胺盐酸盐溶液(1.8vol，3eq)，调pH＝8.0～8.5。再加入0.002g磷酸吡哆醛(1wt％)，0.2g(1vol,0.2ml)
Figure PCTCN2020082594-appb-000033
混匀，加入转氨酶酶液0.2ml(1vol,0.5wt，酶液浓度为0.5g/ml)，调pH＝8.0～8.5。摇床转速170rpm，升温至29℃条件下反应过夜。如图2所示，证明60～100g/L浓度下，底物可转化率大于99％。继续降低底物浓度会导致反应体积增加，三废量增加，且转化率并无进一步显著提高的效果。
实施例13
以实施例2为例，对反应的氨基供体进行优化具体操作如下：室温向10mL小瓶内加入1.2mL 100mmol/L磷酸盐缓冲液(6vol)，分别使用3eq氨基供体的体系(氨基供体分别为异丙胺、异丙胺盐酸盐、丙氨酸、苯胺，正丁胺)，调pH＝8.0～8.5。再加入0.002g磷酸吡哆醛(1wt％)，0.2g
Figure PCTCN2020082594-appb-000034
混匀，加入转氨酶酶液0.2ml(0.5wt，酶液浓度为0.5g/ml)，调pH＝8.0～8.5。摇床转速170rpm，升温至29℃条件下反应过夜。转化率结果如图3所示，证明异丙胺、异丙胺盐酸盐、丙氨酸、正丁胺作为氨基供体，底物可转化率大于99％。而苯胺作为氨基供体在此反应条件下转化率仅为89％。
实施例14
以实施例1底物为例，将来源于Chromobacterium violaceum DSM30191(CVTA)(NCBI Reference Sequence:WP_011135573.1)的转氨酶编号TA1，将来源于Fonsecaea pedrosoi CBS 271.37(NCBI Reference Sequence:XP_013286281.1)的转氨酶编号TA2，将来源于Klebsiella pneumoniae subsp.pneumoniae Ecl8(GenBank:CCN29541.1)的转氨酶编号TA3，将来源于Mycobacterium goodii(GenBank:AKS36000.1)的转氨酶编号TA4，将来源于Paracoccus denitrificans(NCBI Reference Sequence:WP_011746975.1)的转氨酶编号TA5，将来源于Penicillium brasilianum(GenBank:CEJ55334.1)的转氨酶编号TA6，将来源于Enterobacter sp.TL3(NCBI Reference Sequence:WP_014885677.1)的转氨酶编号TA7， 将来源于Aspergillus terreusNIH2624(NCBI Reference Sequence:XP_001209325.1)的转氨酶编号TA8，将来源于Exophiala spinifera(NCBI Reference Sequence:XP_016233821.1)的转氨酶编号TA9，从Deinococcus geothermalis(strain DSM 11300)(NCBI Reference Sequence:WP_011530545.1)的转氨酶编号TA10，将来源于Geomyces destructans 20631-21(GdTA)in E.coli(GenBank:ELR05573.1)的转氨酶编号TA11，将来源于Pseudomonas putida KT2440(NCBI Reference Sequence:WP_010954554.1)的转氨酶编号TA12，将来源于Lysinibacillus sphaericus(NCBI Reference Sequence:WP_024363741.1)的转氨酶编号TA13，将来源于Bacillus megaterium DSM 319(NCBI Reference Sequence:WP_013082219.1)的转氨酶编号TA14，将来源于Trichoderma harzianum(GenBank:KKP07030.1)的转氨酶编号TA15，将来源于Aspergillus fumigatus R-ATAs(AspFum)(NCBI Reference Sequence:XP_748821.1)的转氨酶编号TA16，将来源于Geobacillus thermodenitrificans subsp.thermodenitrificans DSM 465(NCBI Reference Sequence:WP_008879436.1)的转氨酶编号TA17，将来源于Cladophialophora bantiana CBS 173.52(NCBI Reference Sequence:XP_016617948.1)的转氨酶编号TA18，将来源于Bacillus megaterium(NCBI Reference Sequence:WP_016763026.1)的转氨酶编号TA19，将来源于Burkholderia thailandensis MSMB121(BtS-TA)(GenBank:AGK49399.1)的转氨酶编号TA20，将来源于Klebsiella pneumoniae subsp.pneumoniae MGH78578(NCBI Reference Sequence:WP_002920226.1)的转氨酶编号TA21，将来源于Geobacillus toebii(NCBI Reference Sequence:WP_062753894.1)的转氨酶编号TA22，将来源于Talaromyces cellulolyticus(GenBank:GAM37533.1)的转氨酶编号TA23，以Chromobacterium violaceum DSM30191(CVTA)(NCBI Reference Sequence:WP_011135573.1)来源的转氨酶进化得到的突变体TA1-V1(对应序列为SEQ ID NO：1)，TA1-V2(对应序列为SEQ ID NO：2)。TA1-V3(对应序列为SEQ ID NO：3)，TA1-V4(对应序列为SEQ ID NO：4)进行10mg级别筛选反应，操作如下：
将100mmol/L磷酸盐缓冲液，5mol/L异丙胺盐酸盐溶液按照体积比5：2配置成50ml溶液，再加入0.005g磷酸吡哆醛，搅拌混匀。取上述溶液0.3ml分别加入96孔板中，并加入10mg
Figure PCTCN2020082594-appb-000035
再分别加入上述酶液0.2ml(10wt，酶液浓度为0.5g/ml)，摇床转速170rpm，升温至30℃条件下反应过夜。
筛选结果如图4所示：证明所有来源的酶对该底物均有催化活性，以Chromobacterium violaceum DSM30191(CVTA)来源的转氨酶最好，以Chromobacterium violaceum DSM30191(CVTA)来源的转氨酶进行突变，得到具有转化率更好的四个突变体转化率均大于99％。
从以上的描述中，可以看出，本发明上述的实施例实现了如下技术效果：
1)使用转氨酶进行该生物转化反应可以一步反应直接得到所需目标化合物，底物类型适用广泛，不仅可以合成专利也可以合成1，3-二胺，1，4-二胺等化合物，还可以合成1，3-二胺，1，4-二胺等化合物。
2)使用转氨酶进行该生物转化反应选择性很高，产品手性纯度提高大大提升了产品ee值。
3)使用自行提取的前述转氨酶，底物浓度可达到100g/L，大大提高了生产效率。
4)使用转氨酶进行生物催化，不需要使用重金属催化剂及叠氮化合物和氰基化试剂。实现绿色化学。
5)该转氨酶催化效率高，反应体积少，合成路线短，产品收率高，大大降低了三废，节约生产成本。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (17)

  1. 一种手性二胺化合物的合成方法，其特征在于，所述合成方法包括：
    利用转氨酶将式I所示底物转化为所述手性二胺化合物；
    Figure PCTCN2020082594-appb-100001
    其中，n＝1～10，
    R基表示烷基、环烷基、含杂原子的烷基、含杂原子的环烷基、含杂原子的芳基、酰胺类化合物残基或醚类化合物残基，其中，所述杂原子为O、S和N种的至少一种；
    R1、R2相同或不相同，R1和R2各自独立地为氢、C1-C3烷基或氨基保护基；
    所述底物选自如下任意一种：
    Figure PCTCN2020082594-appb-100002
    Figure PCTCN2020082594-appb-100003
    所述转氨酶来源于Chromobacterium violaceum DSM30191(CVTA)、Fonsecaea pedrosoi CBS 271.37、Klebsiella pneumoniae subsp.pneumoniae Ecl8、Mycobacterium goodii、Paracoccus denitrificans、Penicillium brasilianum、Enterobacter sp.TL3、Aspergillus terreus NIH2624、Exophiala spinifera、Deinococcus geothermalis(strain DSM 11300)、Geomyces destructans 20631-21(GdTA)in E.coli、Pseudomonas putida KT2440、Lysinibacillus sphaericus、Bacillus megaterium DSM 319、Trichoderma harzianum、Aspergillus fumigatus R-ATAs(AspFum)、Geobacillus thermodenitrificans subsp.thermodenitrificans DSM 465、Cladophialophora bantiana CBS 173.52、Bacillus megaterium、Burkholderia thailandensis MSMB121(BtS-TA)、Klebsiella pneumoniae subsp.pneumoniae MGH 78578、Geobacillus toebii和Talaromyces cellulolyticus。
  2. 根据权利要求1所述的合成方法，其特征在于，所述转氨酶具有SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3或SEQ ID NO：4所示的氨基酸序列。
  3. 根据权利要求1所述的合成方法，其特征在于，所述氨基保护基选自叔丁氧羰基，苄氧羰基，甲酰基，三氟乙酰基，苄基，三苯甲基及9-芴基甲氧基羰基中的任意一种。
  4. 根据权利要求1所述的合成方法，其特征在于，所述合成方法包括：
    将磷酸缓冲液与氨基供体混合，得到第一混合液，
    向所述第一混合液中添加所述式I所示的底物，得到第二混合液；
    向所述第二混合液中添加所述转氨酶，得到反应混合液；
    从所述反应混合液中分离得到所述手性二胺化合物。
  5. 根据权利要求4所述的合成方法，其特征在于，在向所述第一混合液中添加所述底物之前，所述合成方法还包括：将所述第一混合液的pH值调节至7.0～9.0。
  6. 根据权利要求5所述的合成方法，其特征在于，
    向所述第二混合液中添加所述转氨酶，并调节加入所述转氨酶后的所述第二混合液的pH为7.0～9.0，得到所述反应混合液。
  7. 根据权利要求6所述的合成方法，其特征在于，从所述反应混合液中分离得到所述手性二胺化合物包括：
    调节所述反应混合液的酸度使所述转氨酶变性；
    将变性后的所述转氨酶过滤除去，得到初步滤液；
    将所述初步滤液的pH值调至碱性，得到碱性滤液；
    对所述碱性滤液进行萃取，得到所述手性二胺类化合物。
  8. 根据权利要求7所述的合成方法，其特征在于，调节所述反应混合液的pH值为1～2。
  9. 根据权利要求7所述的合成方法，其特征在于，所述碱性滤液的pH值为12～13。
  10. 根据权利要求7所述的合成方法，其特征在于，所述萃取为多次。
  11. 根据权利要求7所述的合成方法，其特征在于，所述萃取为2～5次。
  12. 根据权利要求7所述的合成方法，其特征在于，采用二氯甲烷进行第一次所述萃取，后续采用二氯甲烷或乙酸乙酯萃取剩余次数。
  13. 根据权利要求7所述的合成方法，其特征在于，所述萃取之后还包括对萃取所得有机相进行干燥的步骤。
  14. 根据权利要求7所述的合成方法，其特征在于，
    将每次萃取所得有机相合并，得到萃取物；
    对所述萃取物进行干燥，得到干燥有机相产物；
    将所述干燥有机相产物置于温度＜45℃，压力≤-0.08Mpa的条件下浓缩至无馏分，得到所述手性二胺类化合物。
  15. 根据权利要求1至14中任一项所述的合成方法，其特征在于，所述转氨酶与所述底物的质量比为：0.4：1～1.0：1。
  16. 根据权利要求1至14中任一项所述的合成方法，其特征在于，所述底物的浓度为60g/L～100g/L。
  17. 根据权利要求4所述的合成方法，其特征在于，所述氨基供体选自异丙胺、异丙胺的盐酸盐、丙氨酸、苯乙胺或正丁胺。
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