Method for synthesizing piperazine and derivatives thereof through double-enzyme cascade catalysis

Abstract

The invention discloses a method for synthesizing piperazine and derivatives thereof through double-enzyme cascade catalysis, which comprises the following steps: adding an amino alcohol compound, dehydrogenase, imine reductase and cofactors into a buffer solution to form a double-enzyme cascade system, and carrying out catalytic reaction to generate piperazine and derivatives thereof. The method disclosed by the invention is convenient to operate and relatively high in product yield, and has a relatively good industrial application prospect in preparation of piperazine and derivatives thereof through biological catalysis. According to the method for synthesizing the piperazine and the piperazine derivatives through double-enzyme cascade catalysis, the used cofactors can be recycled in the catalysis process, the cofactors do not need to be repeatedly added in the catalysis process, the use amount of the cofactors is reduced, and the production cost of the piperazine and the piperazine derivatives is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of biosynthesis, and in particular relates to a method for synthesizing piperazine and its derivatives by double enzyme cascade catalysis. Background Art

[0002] Piperazine and its derivatives were initially used mainly as pharmaceutical intermediates for quinolone antibiotics. With the continuous deepening of research and development of piperazine and its derivatives at home and abroad, new uses of piperazine and its derivatives continue to emerge, and the scope of application has gradually expanded to electronic chemicals, environmental chemicals, polymer materials, biopharmaceuticals and other fields.

[0003] At present, among the synthesis methods of piperazine, the chemical synthesis process is complex, the reaction time is long, and the reaction conditions are harsh. The biological method has mild reaction conditions, but the substrate cost is high. And there are few reports on the synthesis methods of piperazine derivatives. Therefore, finding new green methods to synthesize piperazine and its derivatives has important scientific value and social significance for promoting the green upgrade of existing chemical processes and strengthening environmental protection.

[0004] Amino alcohols and substituted amino alcohols are important chemical raw materials, which are easy to obtain and low in price. They are used as synthetic raw materials for piperazine and its derivatives with good economic benefits. Summary of the invention

[0005] Purpose of the invention: In view of the deficiencies in the prior art, the present invention provides a method for synthesizing piperazine and its derivatives by double enzyme cascade catalysis.

[0006] In order to solve the above technical problems, the present invention discloses a method for synthesizing piperazine and its derivatives by double enzyme cascade catalysis, which uses amino alcohol compounds as substrates and generates piperazine and its derivatives through dehydrogenase and imine reductase catalytic reactions. The specific technical scheme is as follows:

[0007] A method for synthesizing piperazine and its derivatives by double enzyme cascade catalysis, wherein an amino alcohol compound, a dehydrogenase, an imine reductase and a cofactor are added to a buffer to form a double enzyme cascade system, and piperazine and its derivatives are generated by catalytic reaction;

[0008] Wherein, the amino alcohol compound has a structural formula as shown in Formula I;

[0009] The piperazine and its derivatives have a structural formula as shown in Formula III;

[0010]

[0011] Wherein, R1 and R2 are independently selected from any one of H, Me, Et, Ph, CH(CH3)2, CH2CH2NH2, CH2CH2OH, COOCH3, COOCH2CH3 and COOH.

[0012] Wherein, the dehydrogenase and imine reductase are added simultaneously or stepwise to carry out the catalytic reaction.

[0013] When the stepwise addition is used to carry out the catalytic reaction, the method comprises the following steps:

[0014] S1: using the amino alcohol compound as a substrate, generating a pyrazine compound through a dehydrogenase-catalyzed reaction;

[0015] S2: using the pyrazine compound as a substrate, generating piperazine and its derivatives through a reaction catalyzed by the imine reductase;

[0016] Wherein, the structural formula of the pyrazine compound is shown in Formula II;

[0017]

[0018] Wherein, R1 and R2 are independently selected from any one of H, Me, Et, Ph, CH(CH3)2, CH2CH2NH2, CH2CH2OH, COOCH3, COOCH2CH3 and COOH.

[0019] Wherein, the dehydrogenase is any one or a combination of the dehydrogenases in Table 1 below, preferably ADH-A.

[0020] Table 1 Dehydrogenase

[0021]

[0022]

[0023] The imine reductase is any one or a combination of the imine reductases in Table 2 below, preferably R-IRED-Ms.

[0024] Table 2 Imine reductase

[0025]

[0026] Wherein, the buffer is 50-100mM Tris buffer with pH 7-9 or 50-100mM phosphate buffer with pH 6-8, preferably 50mM Tris buffer with pH 8.0 or 50mM phosphate buffer with pH 7.0.

[0027] Wherein, the cofactor is NAD + (oxidized coenzyme I) or NADP + (oxidized coenzyme II).

[0028] The initial concentration of the dehydrogenase in the dual enzyme cascade system is 1-5 U / mL, preferably 3 U / mL; the initial concentration of the imine reductase in the dual enzyme cascade system is 1-5 U / mL, preferably 3 U / mL.

[0029] The initial concentration of the amino alcohol compound in the dual enzyme cascade system is 1-10 mM, preferably 10 mM; the initial concentration of the cofactor in the dual enzyme cascade system is 0.1-2.0 mM, preferably 1 mM.

[0030] Wherein, the reaction temperature of the catalytic reaction is 25-37°C, preferably 37°C; the reaction time is 2-48h, preferably 24h.

[0031] The dehydrogenase and imine reductase described in the present invention can be purchased from the market or prepared according to conventional methods. Specifically, the preparation method is as follows:

[0032] The dehydrogenase and imine reductase can be catalyzed in the form of recombinant cells, crude enzyme solutions or pure enzymes.

[0033] When the dehydrogenase and imine reductase exist in the form of recombinant cells, the preparation method is: introduce a nucleic acid molecule capable of expressing the dehydrogenase or imine reductase into a host cell, and after inducing expression, obtain the recombinant cell.

[0034] Wherein, the host is a prokaryotic cell or a eukaryotic cell, the prokaryotic cell is preferably a bacterium, the eukaryotic cell is preferably a yeast cell, the bacterium is preferably Escherichia coli, and most preferably E. coli BL21 (DE3). The nucleic acid molecule capable of expressing dehydrogenase or imine reductase is introduced into the host cell in the form of a recombinant vector; the recombinant expression vector is transferred to a suitable microbial host by heat shock transformation, and the recombinant vector is a bacterial plasmid, bacteriophage, yeast plasmid or retroviral packaging plasmid carrying the dehydrogenase or imine reductase encoding gene. The induced expression is to add IPTG (isopropyl-β-D-thiogalactoside) inducer to the genetic engineering strain culture system to a concentration of 0.1-0.6mM, preferably 0.3mM, and induce culture at 20-35°C for 12-24h, preferably at 30°C for 20h.

[0035] When the dehydrogenase and imine reductase exist in the form of crude enzyme solution, the preparation method thereof is: lysing the recombinant cells to obtain the crude enzyme solution.

[0036] When the dehydrogenase and imine reductase exist in the form of pure enzymes, the preparation method thereof is: after centrifuging the crude enzyme solution, collecting the supernatant and performing resin purification to obtain the pure enzyme.

[0037] The present invention establishes a method for synthesizing piperazine and its derivatives by double enzyme cascade catalysis of dehydrogenase and imine reductase. Figure 1 The following is a schematic diagram of the reaction process for the synthesis of piperazine and its derivatives catalyzed by dehydrogenase coupled with imine reductase. Figure 1 As shown, the method for synthesizing piperazine and its derivatives provided by the present invention has a cofactor self-circulating system: while the dehydrogenase catalyzes the oxidation of amino alcohol compounds to pyrazine compounds, NAD + and NADP + They are reduced to form NADH and NADPH respectively, and then oxidized to form NAD in the process of imine reductase catalyzing pyrazine compounds to form piperazine and its derivatives. + and NADP + , and then re-participate in the process of dehydrogenase-catalyzed oxidation of amino alcohol compounds into pyrazine compounds.

[0038] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0039] 1. The present invention provides a new green biosynthetic pathway for piperazine and its derivatives, using amino alcohol compounds as raw materials to synthesize piperazine and its derivatives through double enzyme cascade catalysis. The present invention is easy to operate and has a high product yield, and has good industrial application prospects in the biocatalytic preparation of piperazine and its derivatives.

[0040] 2. In the method for synthesizing piperazine and its derivatives by dual enzyme cascade catalysis of the present invention, the cofactors used can be recycled in the catalytic process, and there is no need to repeatedly add cofactors in the catalytic process, which reduces the amount of cofactors used and reduces the production cost of piperazine and its derivatives. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0042] Figure 1 The present invention is a schematic diagram of the reaction process of dehydrogenase-coupled imine reductase catalyzing the synthesis of piperazine and its derivatives. DETAILED DESCRIPTION

[0043] The present invention is further described below based on the following examples. It should be understood that the following embodiments are only used to illustrate the present invention, but not to limit the present invention.

[0044] The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0045] The substrates 1-amino-2-butanol and 2-amino-1-phenylethanol used in the following examples were purchased from Beijing Bailingwei Technology Co., Ltd.; the cofactor NADP used was + Purchased from Manton Biotechnology (Shenzhen) Co., Ltd.

[0046] HPLC quantitative analysis of the product: High performance liquid chromatography was carried out on an Agilent system with an automatic sampler. The chromatographic column was a C18 column (5 μm, 4.6×250 mm). The analytes were detected at a wavelength of 261 nm using a diode array detector (DAD). The mobile phase was pure water and methanol in a volume ratio of 3:1. The elution was isocratic with a flow rate of 0.5 mL / min. The test temperature was 20°C and the detection wavelength was 270 nm.

[0047] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0048] The calculation method of product yield is: actual product yield / theoretical product yield × 100%

[0049] Example 1 Dehydrogenase ADH-A and imine reductase R-IRED-Ms dual enzyme cascade catalyzes the production of 2,5-dimethylpiperazine from 1-amino-2-propanol

[0050] The pure enzymes of dehydrogenase ADH-A and imine reductase R-IRED-Ms catalyze the production of 2,5-dimethylpiperazine from 1-amino-2-propanol in the following steps:

[0051] 1. Add 50 mM Tris hydrochloric acid buffer (pH 8.0), 10 mM 1-amino-2-propanol, and 1 mM NADP to the reaction vessel in sequence. + , the pure enzyme of dehydrogenase ADH-A with an initial concentration of 3U / mL and the pure enzyme of imine reductase R-IRED-Ms with an initial concentration of 3U / mL, constitute a dual enzyme cascade catalytic reaction system. After the reaction system is reacted at 37°C for 24 hours, a reaction solution is obtained.

[0052] 2. Mix 400 μL of ethyl acetate and 200 μL of the reaction solution thoroughly, then centrifuge at 10,000 rpm for 1 min, collect the supernatant and filter it with a filter membrane. The filtrate was tested by HPLC. The results showed that the yield of 2,5-dimethylpiperazine reached 38%, indicating that dehydrogenase ADH-A and imine reductase R-IRED-Ms can cascade catalyze 1-amino-2-propanol to generate 2,5-dimethylpiperazine.

[0053] Example 2 Dehydrogenase ADH-A and imine reductase R-IRED-Ms dual enzyme cascade catalyzes the production of 2,5-diethylpiperazine from 1-amino-2-butanol

[0054] The pure enzymes of dehydrogenase ADH-A and imine reductase R-IRED-Ms catalyze the production of 2,5-diethylpiperazine from 1-amino-2-butanol in the following steps:

[0055] 1. Add 50 mM Tris hydrochloric acid buffer (pH 8.0), 10 mM 1-amino-2-butanol, and 1 mM NADP to the reaction vessel in sequence. + , the pure enzyme of dehydrogenase ADH-A with an initial concentration of 3U / mL and the pure enzyme of imine reductase R-IRED-Ms with an initial concentration of 3U / mL, constitute a dual enzyme cascade catalytic reaction system. After the reaction system is reacted at 37°C for 24 hours, a reaction solution is obtained.

[0056] 2. 400 μL of ethyl acetate and 200 μL of the reaction solution were thoroughly mixed, then centrifuged at 10,000 rpm for 1 min, the supernatant was collected and filtered with a filter membrane. The filtrate was tested by HPLC. The results showed that the yield of 2,5-diethylpiperazine reached 25%, indicating that dehydrogenase ADH-A and imine reductase R-IRED-Ms can cascade catalyze 1-amino-2-butanol to produce 2,5-diethylpiperazine.

[0057] Example 3 Dehydrogenase ADH-A and imine reductase R-IRED-Ms dual enzyme cascade catalyzes 2-amino-1-phenylethanol to produce 2,5-diphenylpiperazine.

[0058] 1. Add 50 mM Tris hydrochloric acid buffer (pH 8.0), 10 mM 2-amino-1-phenylethanol, and 1 mM NADP to the reaction vessel in sequence. + , the pure enzyme of dehydrogenase ADH-A with an initial concentration of 3U / mL and the pure enzyme of imine reductase R-IRED-Ms with an initial concentration of 3U / mL, constitute a dual enzyme cascade catalytic reaction system. After the reaction system is reacted at 37°C for 24 hours, a reaction solution is obtained.

[0059] 2. 400 μL of ethyl acetate and 200 μL of the reaction solution were thoroughly mixed, then centrifuged at 10,000 rpm for 1 min, the supernatant was collected and filtered with a filter membrane. The filtrate was tested by HPLC. The results showed that the yield of 2,5-diphenylpiperazine reached 21%, indicating that dehydrogenase ADH-A and imine reductase R-IRED-Ms can cascade catalyze 2-amino-1-phenylethanol to generate 2,5-diphenylpiperazine.

[0060] Example 4 Optimization of substrate type, substrate concentration and reaction temperature

[0061] According to the catalytic effects of dehydrogenase ADH-A and imine reductase R-IRED-Ms on the substrate, the substrate type, substrate concentration and reaction temperature were optimized, and the addition amount of other substances in the dual enzyme cascade catalytic reaction system and other reaction conditions were the same as in Example 1. The results are shown in Table 3. It can be seen from Table 3 that when 1-amino-2-propanol is used as the substrate, when the substrate concentration is 10mM and the reaction temperature is 37°C, the yield of the product is the highest, which is 38%.

[0062] Table 3 Optimization experiment of substrate concentration, reaction temperature and reaction time

[0063]

[0064] The present invention provides a dual enzyme cascade catalytic synthesis of piperazine and its derivatives. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for synthesizing piperazine and its derivatives by double enzyme cascade catalysis, It is characterized in that An amino alcohol compound, a dehydrogenase, an imine reductase and a cofactor are added to a buffer to form a double enzyme cascade system, and piperazine and its derivatives are generated through a catalytic reaction; Wherein, the amino alcohol compound has a structural formula as shown in Formula I; The piperazine and its derivatives have a structural formula as shown in Formula III; Among them, R 1 and R 2 are independently selected from H, Me, Et, Ph, CH(CH 3 ) 2 , CH 2 CH 2 NH 2 , CH 2 CH 2 OH, COOCH 3 、COOCH 2 CH 3 and COOH.

2. The method according to claim 1, It is characterized in that The dehydrogenase and imine reductase are added simultaneously or stepwise to carry out the catalytic reaction.

3. The method according to claim 2, It is characterized in that When the stepwise addition is used to carry out the catalytic reaction, the method comprises the following steps: S1: using the amino alcohol compound as a substrate, generating a pyrazine compound through a dehydrogenase-catalyzed reaction; S2: using the pyrazine compound as a substrate, generating piperazine and its derivatives through a reaction catalyzed by the imine reductase; Wherein, the structural formula of the pyrazine compound is as shown in Formula II; Among them, R 1 and R 2 are independently selected from H, Me, Et, Ph, CH(CH 3 ) 2 , CH 2 CH 2 NH 2 , CH 2 CH 2 OH, COOCH 3 、COOCH 2 CH 3 and COOH.

4. The method according to any one of claims 1 to 3, It is characterized in that The dehydrogenase is any one or a combination of the following a1 to a9; a1, HLADH dehydrogenase from Equus caballus (Horse), the UniParc of amino acid sequence is P00327.1; a2, dehydrogenase LkADH from Lentilactobacillus kefiri, the amino acid sequence of which is Q6WVP7 in UniProtKB; a3, dehydrogenase YsADH from Yokenella sp. WZY002, the amino acid sequence of which is W6CX26 in UniProtKB; a4, dehydrogenase AaADH from Aromatoleum aromaticum, with the amino acid sequence of UniProtKB as Q5P5I4; a5, dehydrogenase BsADH from Geobacillus stearothermophilus, the amino acid sequence of UniProtKB is P42328; a6, dehydrogenase ADH-A from Rhodococcus ruber, the amino acid sequence of UniProtKB is Q8KLT9; a7, dehydrogenase PpADH from Paracoccus pantotrophus, with the amino acid sequence of UniProtKB as B9U358; a8, dehydrogenase TeSADH from Thermoanaerobacter ethanolicus, with the amino acid sequence of UniProtKB as P77990; a9, dehydrogenase ScADH from Saccharomyces cerevisiae, the amino acid sequence of which is P00331 in UniProtKB.

5. The method according to any one of claims 1 to 3, It is characterized in that The imine reductase is any one or a combination of the following b1 to b11; b1, imine reductase AoIRED from Amycolatopsis orientalis, the UniParc of amino acid sequence is R4SNK4; b2, imine reductase R-IRED-Ms from Myxococcus stipitatus, the amino acid sequence of which is L7U9F5 in UniProtKB; b3, reductaminase AspRedAm from Aspergillus oryzae, the amino acid sequence of which is Q2TW47 in UniProtKB; b4, imine reductase AbIRED from Aspergillus bombycis, with the amino acid sequence of UniProtKB A0A1F7ZK02; b5, imine reductase AnoIRED from Aspergillus nomius NRRL 13137, the amino acid sequence of UniProtKB is A0A0L1J820; b6, imine reductase Sp(S)-IRED from Streptomyces sp. GF3546, the amino acid sequence of which is M4ZS15 in UniProtKB; b7, imine reductase Sp(R)-IRED from Streptomyces sp. GF3587, the amino acid sequence of which is M4ZRJ3 in UniProtKB; b8, imine reductase Sk(R)-IRED from Streptomyces kanamyceticus, with the amino acid sequence of UniProtKB as Q1EQE0; b9, S-selective imine reductase S-IRED-Ms from Myxococcus smegmatis, with the amino acid sequence of UniProtKB A0R5X0; b10, imine reductase Nh-IRED from Nocardiopsis halophila, with the amino acid sequence of UniProtKB A0A0J9X1X6; b11, imine reductase Pp(R)-IRED from Pseudomonas putida KT2440, the amino acid sequence of which is Q88J51 in UniProtKB.

6. The method according to any one of claims 1 to 3, It is characterized in that The buffer is a Tris buffer of 50-100 mM and pH 7-9 or a phosphate buffer of 50-100 mM and pH 6-8.

7. The method according to any one of claims 1 to 3, It is characterized in that The cofactor is NAD + or NADP + .

8. The method according to any one of claims 1 to 3, It is characterized in that The initial concentration of the dehydrogenase in the dual enzyme cascade system is 1 to 5 U / mL; the initial concentration of the imine reductase in the dual enzyme cascade system is 1 to 5 U / mL.

9. The method according to any one of claims 1 to 3, It is characterized in that The initial concentration of the amino alcohol compound in the dual enzyme cascade system is 1-10 mM; the initial concentration of the cofactor in the dual enzyme cascade system is 0.1-2.0 mM.

10. The method according to any one of claims 1 to 3, It is characterized in that The reaction temperature of the catalytic reaction is 25-37° C., and the reaction time is 2-48 hours.

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