Method for preparing (S)-chiral amine through imine reductase catalysis

Abstract

The invention relates to a method for preparing (S)-chiral amine through asymmetric catalysis of imine reductase, and belongs to the technical field of biological pharmacy. Comprising the following steps that imine reductase catalyzes imine to be subjected to a reductive hydrogenation reaction under the conditions of coenzyme and a coenzyme regeneration system, and the (S)-chiral amine compound is generated. According to the method, imine reductase is utilized to catalyze imine asymmetric reduction to prepare the innovative drug (S)-chiral amine compound, and compared with a traditional chemical synthesis method, the method has the advantages of being high in reaction efficiency, good in stereoselectivity, mild in reaction condition, environmentally friendly and the like, and is a method meeting the green production requirement.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for preparing (S)-chiral amine by catalysis of imine reductase. Background Art

[0002] Chiral amines are present in many biologically active substances and are important chiral auxiliary agents. They are also key intermediates for the synthesis of natural products and chiral drugs. Among the top 200 drugs in terms of retail sales in 2019, more than 30% contain chiral amine structures. Therefore, it is very important to develop efficient and convenient methods for synthesizing chiral amine compounds. Optically pure (S)-chiral amines are a class of pharmaceutical intermediates with great value and are used in the synthesis of many innovative drugs. The efficient preparation of (S)-chiral amines has always been a research hotspot. For example, patent WO2009095253A1 discloses that the drug is an mGluR5 allosteric modulator drug with the potential to treat central nervous system diseases such as Parkinson's disease, anxiety, depression, fragile X syndrome and drug addiction. Among them, a large number of chiral amine compounds are involved. See pages 413 to 454 of the international application publication.

[0003] Currently, the methods for preparing (S)-chiral amines mainly include chemical methods and biocatalytic methods.

[0004] Chemical methods mainly include chemical asymmetric hydrogenation reduction and chemical splitting. These methods have disadvantages such as poor selectivity, high cost and harsh reaction conditions.

[0005] Compared with chemical methods, biocatalytic methods for preparing (S)-chiral amines offer advantages such as high reaction efficiency, good stereoselectivity, mild reaction conditions, and environmental friendliness. Therefore, the use of biocatalysis can effectively overcome the shortcomings of chemical methods, such as poor selectivity and high costs.

[0006] Currently reported enzymes that can be used to prepare chiral amines include lipases, transaminases, monoamine oxidases, amine dehydrogenases, imine reductases, reductive amination enzymes, and lyases. Further research and development is needed to obtain enzymes that can act on substrates in a targeted manner to catalyze the preparation of (S)-chiral amine compounds. Summary of the Invention

[0007] In response to the importance of (S)-chiral amines and the challenges of chemical preparation processes, the present invention provides a method for preparing (S)-chiral amines represented by Formula II using imine reductase. In the presence of a coenzyme and a coenzyme regeneration system, the imine reductase is used to catalyze the reduction and hydrogenation reaction of the raw material represented by Formula I to produce the (S)-chiral amine represented by Formula II. The reaction formula is as follows:

[0008]

[0009] Wherein, R is an alkyl group; X and Y are the same or different and are C or N atoms.

[0010] Preferably, R is a methyl group, X and Y are C atoms, and the reaction formula is:

[0011]

[0012] Preferably, R is a methyl group, X and Y are nitrogen atoms, and the reaction formula is:

[0013]

[0014] In the present invention, the imine reductase is derived from Amycolatopsis decaplanina (DSM 44594), the amino acid sequence of which is shown in SEQ ID NO.1, and the nucleotide sequence encoding the imine reductase is shown in SEQ ID NO.2.

[0015] In the present invention, the mass ratio of the imine reductase enzyme solution to the raw material represented by formula I in the reaction system is (0.1-10):1.

[0016] In the present invention, during the imine reduction reaction, the preferred coenzyme is NADP + , the coenzyme regeneration system consists of glucose and glucose dehydrogenase.

[0017] In the present invention, the coenzyme is NADP + The amount of the coenzyme used can be a conventional amount, for example, 0.1-1 mM.

[0018] In the present invention, the coenzyme regeneration system consists of glucose and glucose dehydrogenase.

[0019] In the present invention, the reaction system is a buffered saline solution, and the pH of the reaction system is controlled by the buffer salt. Commonly used buffers include, but are not limited to, phosphate buffer, triethanolamine-hydrochloric acid buffer, Tris-HCl buffer, glycine-sodium hydroxide buffer, etc. Preferably, the reaction system has a pH of 4-9, more preferably 5-6.

[0020] In the present invention, the temperature of the imine reduction reaction is 20°C-60°C, preferably 30°C.

[0021] In the present invention, the time of the imine reduction reaction can be determined by the amount of raw materials charged, and can usually be 0.1-120 hours.

[0022] The sequence information of the imine reductase of the present invention is as follows:

[0023] SEQ ID NO.1:

[0024] MITLIGLGPMGQAMVRVLLENGHGVTVWNRTAARADGVVAAGAVRAGTPADAVAASELVLLSL

[0025] TDYAAMYDILGKAEDALAGKVIVNLSSDTPEKTREAADWVKARGGRFVAGGVMVPAELVGKE

[0026] EAYVFYSGPADVFEKHRETLALIGRPDFLGEDVRLAQLFYQAQLDIFLTSLSVFMHASALVRSAG

[0027] VPVEKFVPYAKDNFKMMDFYLDAAAEQIEKGEHPGDDANVTMMGATADHIVQASRDAGVDV

[0028] VLPEAVKSHYDRAIAAGHGRSSWTSLFEIIKADRK

[0029] SEQ ID NO.2:

[0030] ATGATCACACTGATCGGGCTCGGTCCGATGGGACAGGCCATGGTCCGGGTACTCCTGGAGAA

[0031] CGGCCACGGAGTGACGGTCTGGAACCGCACGGCCGCCCGCGCGGACGGCGTCGTCGCCGC

[0032] GGGCGCCGTGCGCGCCGGGACACCGGCGGACGCGGTGGCGGCATCGGAGCTGGTGTTGCT

[0033] GAGTCTCACCGACTACGCGGCGATGTACGACATTCTCGGCAAGGCCGAGGATGCCCTGGCG

[0034] GGCAAGGTGATCGTCAACCTCAGCTCGGACACTCCGGAGAAGACCCGTGAAGCCGCCGACT

[0035] GGGTCAAGGCCCGGGGCGGGCGGTTCGTCGCGGGCGGCGTGATGGTGCCTGCCGAGTTGGT

[0036] GGGCAAGGAGGAGGCGTATGTCTTCTACAGCGGCCCGGCCGACGTGTTCGAGAAGCACCGC

[0037] GAGACCCTGGCGCTGATCGGGCGGCCGGACTTCCTCGGTGAGGACGTCCGGCTGGCACAGC

[0038] TGTTCTACCAGGCGCAGCTGGACATCTTCCTGACCTCGCTTTCGGTGTTCATGCACGCGAGC

[0039] GCGCTGGTGCGTTCGGCGGGTGTGCCGGTGGAGAAGTTCGTCCCGTACGCCAAGGACAACT

[0040] TCAAGATGATGGATTTCTACCTGGACGCCGCGGCCGAGCAGATCGAAAAGGGCGAGCACCC

[0041] CGGCGACGACGCCAACGTGACCATGATGGGTGCGACGGCCGACCACATCGTCCAGGCGAGC

[0042] CGGGACGCCGGGGTGGACGTGGTGTTGCCGGAAGCGGTCAAATCGCACTACGACCGGGCGA

[0043] TCGCCGCGGGGCACGGGCGCAGCTCGTGGACCAGCCTTTTCGAAATCATCAAGGCGGACCG

[0044] CAAGTAG BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 is a liquid chromatogram showing the conversion rate of the (S)-chiral amine of Formula II prepared catalyzed by imine reductase in Example 5;

[0046] Figure 2 This is a chiral liquid chromatogram of the imine reductase-catalyzed preparation of the (S)-chiral amine of Formula II in Example 5. DETAILED DESCRIPTION

[0047] To further understand the present invention, the following detailed description of the method for preparing (S)-chiral amines catalyzed by imine reductase is provided in conjunction with the following examples. It should be understood that these examples are intended only to further illustrate the features of the present invention and are not intended to limit the scope of the present invention or the claims.

[0048] Example 1: Construction of recombinant Escherichia coli expressing imine reductase

[0049] The entire gene was artificially synthesized with the nucleotide sequence shown in SEQ ID NO. 2 and inserted into the multiple cloning site of plasmid pET21a to obtain recombinant plasmid pET21a-AdIRED. The recombinant plasmid pET21a-AdIRED was transformed into competent Escherichia coli BL21(DE3) cells, which were then plated onto LB solid medium containing ampicillin and cultured overnight at 37°C in a biochemical incubator. The next day, a single colony was picked from the LB solid medium and inoculated into LB liquid medium, cultured at 37°C, 200 rpm for 12 hours, and a glycerol stock was prepared and stored in a -80°C freezer to obtain recombinant Escherichia coli BL21(DE3)-pET21a-AdIRED for heterologous expression of imine reductase.

[0050] Example 2: Preparation of Imine Reductase Enzyme Solution

[0051] The recombinant Escherichia coli BL21 (DE3) -pET21a -AdIRED glycerol bacteria obtained in Example 1 were inoculated into LB liquid medium containing ampicillin and cultured overnight at 37 ° C and 200 rpm to obtain a seed solution. The seed solution was transferred to fresh LB liquid medium at a 1% (v / v) inoculation amount, cultured at 37 ° C and 200 rpm for 3 hours, and then induced with an inducer. After induction at 25 ° C and 200 rpm for 20 hours, a fermentation broth was obtained. The fermentation broth was centrifuged, the supernatant was removed to collect the bacteria, and then the bacteria were resuspended in 0.1M (pH 7.0) phosphate buffer 5 times the mass of the bacteria to obtain a bacterial suspension. The cells were disrupted using a cell ultrasonic disruptor to obtain a crude imine reductase enzyme solution.

[0052] Example 3: Reaction test of imine reductase to different imine raw materials

[0053]

[0054] The enzyme catalytic system composition and reaction conditions are as follows: 1 mL of the reaction system contains 0.01 g of the imine starting material (compound of Formula I), 0.03 g of glucose, 0.5 mg of NADP+, 0.1 mL of imine reductase enzyme solution, 0.02 mL of glucose dehydrogenase enzyme solution, and 0.88 mL of phosphate buffer (0.1 M, pH 5.5). The system is placed on a thermomixer and reacted at 30°C and 800 rpm for 24 hours. Samples are then collected and analyzed for conversion and chirality by HPLC.

[0055]

[0056] Example 4: Imine reduction reaction (50 mL system)

[0057]

[0058] Weigh 0.13g of dipotassium hydrogen phosphate trihydrate and 0.51g of potassium dihydrogen phosphate into a 250mL round-bottom flask, add 43.5mL of water, stir thoroughly for 10min, and then use phosphoric acid to adjust the pH of the solution to 5.0; weigh 1g of the raw material shown in formula I (feed concentration 20g / L) and 3g of glucose into the round-bottom flask, stir thoroughly for 10min; then add 0.5mL of 5% NADP + The reaction was started by adding 1 mL of glucose dehydrogenase solution and 5 mL of crude imine reductase solution. The pH of the reaction solution was adjusted to 5.0 with phosphoric acid or sodium hydroxide solution every 1 h. After reacting at 30°C for 24 h, an appropriate amount of the reaction solution was taken and analyzed for conversion and chirality using liquid chromatography. The conversion rate was 97.98% and the ee value was 99.45%.

[0059] Example 5: Imine reduction reaction (200 mL system)

[0060]

[0061] Weigh 0.52g of dipotassium hydrogen phosphate trihydrate and 2.04g of potassium dihydrogen phosphate into a 500mL round-bottom flask, add 174mL of water, stir thoroughly for 10min, and then adjust the pH of the solution to 5.0 with phosphoric acid; weigh 4g of the raw material shown in formula I (feed concentration 20g / L) and 12g of glucose into the round-bottom flask, stir thoroughly for 10min; then add 2mL of 5% NADP + The reaction was started by adding 4 mL of glucose dehydrogenase solution and 20 mL of imine reductase crude enzyme solution. The pH of the reaction solution was adjusted to 5.0 with phosphoric acid or sodium hydroxide solution every 1 h. After reacting at 30 ° C for 24 h, an appropriate amount of the reaction solution was taken and analyzed by liquid chromatography for conversion rate and chirality, such as Figure 1As shown, the retention time of the (S)-chiral amine of formula II is 10.107 min, the retention time of the imine raw material of formula I is 10.478 min, and the conversion rate is 99.61%; Figure 2 As shown, the retention time of the (S)-chiral amine of formula II is 10.581 min, the retention time of its enantiomer is 5.372 min, and the ee value is 99.56%.

[0062] In summary, the above examples are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing (S)-chiral amines catalyzed by imine reductase, characterized in that: Imine reductase catalyzes a reduction hydrogenation reaction of the raw material shown in formula I in the presence of a coenzyme and a coenzyme regeneration system to generate a product (S)-chiral amine shown in formula II. The reaction formula of the method is as follows: Wherein, R is an alkyl group; X and Y are the same or different and are C or N atoms.

2. The method according to claim 1, characterized in that R is a methyl group, X and Y are C atoms, 3. The method according to claim 1, characterized in that R is a methyl group, X and Y are nitrogen atoms, 4. The method according to any one of claims 1 to 3, characterized in that: The amino acid sequence of the imine reductase is shown in SEQ ID NO.1, and the nucleotide sequence encoding the imine reductase is shown in SEQ ID NO.

2.

5. The method according to any one of claims 1 to 3, characterized in that: The reaction conditions are coenzyme and coenzyme regeneration system, in which the coenzyme is NADP + ; The coenzyme regeneration system consists of glucose and glucose dehydrogenase.

6. The method according to any one of claims 1 to 3, characterized in that: The reaction temperature is 20°C-60°C.

7. The method according to any one of claims 1 to 3, characterized in that: The pH of the reaction is 4-9.

8. The method according to any one of claims 1 to 3, characterized in that: The imine reductase catalyzes the reaction, and the reaction conversion rate is above 97%, and the product ee value is above 99%.

9. The method according to any one of claims 1 to 3, characterized in that: The imine reductase participates in the reaction in the form of enzyme solution.

10. The method according to any one of claims 1 to 3, characterized in that: The imine reductase is derived from Amycolatopsis decaplanina (DSM 44594).

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