Method for synthesizing (S)-1-tetrahydronaphthalene amine through in-vitro multi-enzyme cascade catalysis of tetrahydronaphthalene

Abstract

The invention belongs to the technical field of biochemical engineering, and particularly discloses a method for synthesizing (S)-1-tetrahydronaphthalene amine through in-vitro multi-enzyme cascade catalysis of tetrahydronaphthalene. The multi-enzyme cascade system is composed of non-specific peroxidase, galactose oxidase and omega-transaminase, a substrate is tetrahydronaphthalene, and a product is (S)-1-tetrahydronaphthylamine. The method comprises the following steps: firstly, catalyzing tetrahydronaphthalene and hydrogen peroxide by using non-specific peroxidase to generate tetrahydronaphthalene alcohol, generating tetrahydronaphthalene ketone under the action of galactose oxidase, and generating (S)-1-tetrahydronaphthalene amine under the action of omega-transaminase; compared with the prior art, the synthesis method provided by the invention provides a novel in-vitro multi-enzyme catalysis way without expensive cofactors for synthesizing (S)-1-tetrahydronaphthalene amine from tetrahydronaphthalene, the process is safe and green, the production cost can be remarkably reduced, a novel green way is developed for converting large-volume aromatic hydrocarbon into high-added-value amine organic compounds, and the synthesis method has wide application prospects. The method has important industrial application value.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis and biochemical engineering, and particularly relates to a method for generating (S)-1-tetrahydronaphthylamine by in vitro multi-enzyme cascade catalysis of tetralin. Background Art

[0002] (S)-1-Tetrahydronaphthylamine, also known as (S)-1,2,3,4-tetrahydro-1-naphthylamine, is an important intermediate for drug synthesis. It is often used as a chiral catalyst in organic synthesis. It can be used in asymmetric synthesis reactions, such as asymmetric reduction of chiral catalysts, asymmetric addition of chiral catalysts, etc. At the same time, it can also be used as a synthetic intermediate for naphthyridinone compounds.

[0003] The most common method for preparing S-1-tetrahydronaphthylamine is to synthesize it by direct reduction amination with a noble metal catalyst (Chemistry-A European Journal, 2014, 20(1): 245-252). This process requires the use of hydrogen and noble metal catalysts such as platinum, palladium or iridium, etc., which has high requirements on the reaction equipment and the optical purity of the product is not high. Another method is to first prepare the 1-tetrahydronaphthylamine racemate and then obtain (S)-1-tetrahydronaphthylamine by chiral separation (Bioorganic & Medicinal Chemistry, 2004, 12(15): 4189-4196), and the raw material utilization rate is low. Therefore, it is of great significance to develop a green and economical method for synthesizing (S)-1-tetrahydronaphthylamine.

[0004] The rapid development of biotechnology in recent years has brought new possibilities for the synthesis of chiral amines. The method of synthesizing chiral amines based on in vitro multi-enzyme catalysis has the advantages of being green and clean, and having mild reaction conditions. Multi-enzyme catalysis generally refers to cascade catalysis involving two or more enzymes, which has significant advantages in shortening reaction steps, reducing the accumulation of intermediates, and reducing the volume of equipment, and plays an increasingly important role in industrial biotechnology. Some reports have been made on the preparation of chiral amine substances by in vitro multi-enzyme catalytic cascade reactions using hydrocarbon compounds as substrates, and certain results have been achieved (Green Chemistry, 2023, 25, 3469-3474; Chem Sus Chem, 2019, 12 (4): 848-857; Angew. Chem. Int. Ed., 2024, e202407778). Although the methods reported can achieve the synthesis of chiral amines, they are mainly limited to small-volume chiral amine compounds. There are no reports on compounds such as tetrahydronaphthylamine with large steric hindrance. In addition, more expensive alcohols are usually used as substrates rather than hydrocarbon compounds. At the same time, existing multi-enzyme catalytic systems often require the use of expensive cofactors. In addition, most existing multi-enzyme catalytic methods focus on the conversion of aliphatic compounds, while there are few studies on the synthesis of (S)-1-tetrahydronaphthylamine directly using tetrahydronaphthyl as a raw material through a multi-enzyme cascade reaction. Therefore, the development of an in vitro multi-enzyme catalytic method that does not rely on cofactors for the synthesis of (S)-1-tetrahydronaphthylamine from tetrahydronaphthyl is of great significance in the field of green biomanufacturing. Summary of the invention

[0005] The purpose of the present invention is to provide a method for synthesizing (S)-1-tetrahydronaphthylamine from tetralin by multi-enzyme cascade catalysis based on the background of green biomanufacturing. The method uses tetralin as a raw material and obtains (S)-1-tetrahydronaphthylamine through a three-enzyme cascade catalytic pathway. The above-mentioned pathway provides a new idea for synthesizing amine organic compounds from large-volume aromatic hydrocarbon compounds.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The technical scheme of the present invention provides a method for generating (S)-1-tetrahydronaphthylamine from tetralin by multi-enzyme cascade catalysis. The steps are: adding (S)-1-tetrahydronaphthylamine synthesis-related enzymes to an aqueous solution system of tetralin, pyridoxal phosphate, hydrogen peroxide, an activator, and an amino donor, and synthesizing (S)-1-tetrahydronaphthylamine after catalytic reaction.

[0008] Furthermore, the (S)-1-tetrahydronaphthylamine synthesis-related enzymes (multi-enzyme cascade system) are composed of non-specific peroxidase, galactose oxidase and ω-transaminase.

[0009] Furthermore, the amino acid sequence of the non-specific peroxidase is shown in SEQ ID NO:1.

[0010] Furthermore, the amino acid sequence of the galactose oxidase is shown in SEQ ID NO:2.

[0011] Furthermore, the amino acid sequence of the ω-aminotransferase is shown in SEQ ID NO:3.

[0012] Furthermore, the non-specific peroxidase, galactose oxidase and ω-aminotransferase can be obtained by conventional methods, such as by prokaryotic expression and protein purification.

[0013] Furthermore, the host bacteria for prokaryotic expression in the present invention is preferably Escherichia coli; more preferably Escherichia coli C43 (DE3).

[0014] Furthermore, the protein purification can be performed by nickel column affinity chromatography.

[0015] Furthermore, the reaction system is a NaPi buffer system with a pH of 7.0-8.0.

[0016] Furthermore, the reaction system is: 20 μg / mL nonspecific peroxidase, 50 μg / mL galactose oxidase, 200 μg / mL ω-transaminase, 0.5-5 mM tetralin, 0.5-1 mM pyridoxal phosphate, 0.1-0.4 substrate equivalent hydrogen peroxide, 1-30 mM activator, and 0.5-2 M amino donor.

[0017] Furthermore, the amino donor is one or more of isopropylamine, β-alanine, L-alanine, 4-nitrophenylethylamine and o-xylylenediamine.

[0018] Furthermore, the galactose oxidase activator is one or more of potassium persulfate, sodium persulfate, potassium ferrocyanide and horseradish peroxidase.

[0019] Furthermore, the reaction temperature is 25-35°C; preferably 30°C.

[0020] Furthermore, the reaction speed is 600 rpm.

[0021] Furthermore, the reaction time is 24 hours.

[0022] Compared with the prior art, the advantages of the present invention are:

[0023] The reaction pathway of the synthesis method of the invention is based on the background of green biomanufacturing, realizes the multi-enzyme cascade catalysis of tetralin to synthesize (S)-1-tetralinamine, and develops a new pathway for synthesizing amine organic compounds from large-volume aromatic hydrocarbon compounds.

[0024] The invention reduces the addition amount of the co-substrate hydrogen peroxide by introducing the hydrogen peroxide circulation system, thereby greatly reducing the reaction cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The schematic diagram of the catalytic pathway for the synthesis of (S)-1-tetrahydronaphthylamine from tetralin catalyzed by multi-enzymes in vitro;

[0026] Figure 2 The effect of different activators on the catalytic activity of galactose oxidant;

[0027] Figure 3 The effect of different equivalents of hydrogen peroxide on the catalytic activity of nonspecific peroxidase and galactose oxidase cascades;

[0028] Figure 4 The product analysis result of (S)-1-tetrahydronaphthylamine by gas chromatography in the present invention;

[0029] Figure 5 This is the standard curve of (S)-1-tetrahydronaphthylamine. DETAILED DESCRIPTION

[0030] To make the present invention easy to understand, the present invention will be described in detail below in conjunction with the accompanying drawings. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terms used herein are only for describing specific embodiments and are not intended to be limiting.

[0031] Unless otherwise defined, all terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. The reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The experimental methods in the following examples where specific experimental conditions are not specified are generally based on conventional experimental conditions. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0032] The present invention develops a method for synthesizing (S)-1-tetrahydronaphthylamine from tetralin by using a multi-enzyme cascade system in vitro. The method uses tetralin as a substrate and synthesizes (S)-1-tetrahydronaphthylamine by using a multi-enzyme cascade system. Figure 1 shown.

[0033] The method for synthesizing (S)-1-tetrahydronaphthylamine by multi-enzyme cascade catalysis of tetralin is constructed by the present invention. The method uses tetralin as a raw material and utilizes three enzymes, namely, non-specific peroxidase, galactose oxidase and ω-aminotransferase, to catalyze the synthesis of (S)-1-tetrahydronaphthylamine. The method specifically comprises: (1) utilizing non-specific peroxidase to catalyze the synthesis of 1-tetral alcohol from tetralin and hydrogen peroxide; (2) utilizing galactose oxidase to catalyze the synthesis of corresponding 1-tetralone from 1-tetralone substrate and oxygen; (3) utilizing ω-aminotransferase to catalyze the synthesis of (S)-1-tetrahydronaphthylamine from 1-tetralone and amino donor isopropylamine (see Figure 1 ).

[0034] Example 1

[0035] Construction of recombinant bacteria expressing nonspecific peroxidase, galactose oxidase and ω-aminotransferase:

[0036] The gene sequences of nonspecific peroxidase, galactose oxidase (sequence from the document DOI: 10.1021 / acscatal.3c03427) and ω-aminotransferase (sequence from the document DOI: 10.1002 / anie.202212555) were handed over to Shanghai Biotechnology Co., Ltd. for codon optimization and synthesis, and were inserted into the corresponding sites of pET-28a plasmid at EcoR I site and Sac I site, respectively. Enzyme digestion analysis and identification were performed to obtain the corresponding recombinant plasmids.

[0037] Nonspecific peroxidase (SEQ ID NO.1):

[0038]

[0039] Galactose oxidase (SEQ ID NO.2):

[0040]

[0041] ω-aminotransferase (SEQ ID NO.3):

[0042]

[0043] Example 2

[0044] Obtaining nonspecific peroxidase, galactose oxidase and ω-aminotransferase:

[0045] The recombinant plasmid was introduced into the competent E. coli cells by heat shock method, and after recovery, it was spread on LB solid medium containing 30 μg / mL kanamycin. After inverted culture at 37°C, single clones were picked and identified to obtain the corresponding recombinant bacteria.

[0046] The recombinant bacterial strain was inoculated into LB liquid culture medium and cultured overnight at 37°C and 180rpm. The recombinant strain culture liquid was then transferred to 1L ZYM-5052 auto-induction liquid culture medium containing 30μg / mL kanamycin at a 1% inoculum amount and cultured at 16°C for 3 days. The bacteria were collected by centrifugation at 8000rpm for 5min, resuspended with pH 8 buffer (20mM Tris-HCl), and ultrasonically disrupted at 350W (working 3s, resting 5s, 20min), and centrifuged (12000rpm, 10min) to collect the soluble supernatant expression components (crude enzyme solution).

[0047] The soluble supernatant expression components after the above-mentioned disruption were separated and purified by nickel column affinity chromatography to obtain non-specific peroxidase, galactose oxidase and ω-aminotransferase. The specific operation is as follows: balance the medium with 5 column volumes of equilibration buffer (pH7.5 20mM NaPi, 40mM imidazole, 500mM NaCl); filter the crude enzyme solution obtained after crushing with a 0.45μm filter head; load the sample at a flow rate of 1mL / min; after loading, wash the impurities with 10-20 column volumes of buffer (pH7.5 20mM NaPi, 100mM imidazole, 500mM NaCl); then elute with 5-10 column volumes of elution buffer (pH7.5 20mM NaPi, 250mM imidazole, 500mM NaCl), and collect the eluate; dilute the eluate with 50mM pH8.0 NaPi buffer, concentrate by ultrafiltration (10KDa), repeat 5-10 times to remove imidazole; and determine the concentration of each target protein obtained after ultrafiltration by the BCA method.

[0048] Example 3

[0049] Effects of different activators on the synthesis of 1-tetralone catalyzed by galactose oxidase:

[0050] The effects of different activators on the catalytic synthesis of 1-tetralone by galactose oxidase were investigated. The specific steps were as follows: the reaction system contained 1 mM tetral alcohol, pH 7.5 buffer (500 mM NaPi), different activators (potassium persulfate K2S2O8, potassium ferrocyanide K3[Fe(CN)6], horseradish peroxidase HRP), and galactose oxidase was added at a final concentration of 50 μg / mL. The reaction time was 12 h, and the yield was as follows: Figure 2 shown.

[0051] Example 4

[0052] Effects of different amino donors on the synthesis of (S)-1-tetrahydronaphthylamine catalyzed by ω-aminotransferase:

[0053] The effects of different amino donors on the synthesis of (S)-1-tetrahydronaphthylamine catalyzed by ω-aminotransferase were investigated. The specific steps were as follows: the reaction system contained 1 mM tetralone, pH 7.5 buffer (500 mM NaPi), 0.5 mM pyridoxal phosphate and different amino donors (isopropylamine, β-alanine, L-alanine, 4-nitrophenylethylamine, o-xylylenediamine), and galactose oxidase was added at a final concentration of 150 μg / mL. When o-xylylenediamine was used as the amino donor, the product yield was 10.24% after 12 hours of reaction.

[0054] Example 5

[0055] Effect of initial hydrogen peroxide equivalent on the cascade catalytic synthesis of 1-tetralone by nonspecific peroxidase and galactose oxidase:

[0056] The effect of initial hydrogen peroxide equivalent on the cascade catalysis of tetralin by nonspecific peroxidase and galactose oxidase to synthesize 1-tetralone was investigated. The specific steps were as follows: the reaction system contained 1 mM tetralin, 20 mM potassium persulfate, pH 7.5 buffer (500 mM NaPi), different equivalents of hydrogen peroxide (specifically 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 equiv.), nonspecific peroxidase with a final concentration of 20 μg / mL and 50 μg / mL galactose oxidase were added, the reaction time was 12 h, and the yield was as follows: Figure 3 shown.

[0057] Example 6

[0058] The three-enzyme cascade catalyzes the production of (S)-1-tetrahydronaphthylamine from tetralin:

[0059] A multi-enzyme cascade catalytic reaction was carried out in a Na2HPO4-NaH2PO4 buffer at pH 7.5 with a final concentration of 20 μg / mL nonspecific peroxidase, 50 μg / mL galactose oxidase, 200 μg / mL ω-transaminase, 1 mM tetralin, 20 mM sodium persulfate, 1 mM hydrogen peroxide, 1 mM pyridoxal phosphate and 1 M isopropylamine at 30°C and 600 rpm for 24 h. The gas chromatography detection of the final product (S)-1-tetralinamine is shown in Figure 4 .

[0060] Example 7

[0061] Product detection method:

[0062] The (S)-1-tetrahydronaphthylamine in Example 5 was detected by gas chromatography (GC) under the following detection conditions: the chromatographic column was Agilent cp7502; the nitrogen flow rate was 1 mL / min, and the temperature program was: starting from 100°C, holding for 2 minutes, increasing to 130°C at 25°C / min, holding for 5 minutes, increasing to 140°C at 2°C / min, holding for 5 minutes, increasing to 150°C at 5°C / min, holding for 3 minutes, increasing to 180°C at 25°C / min, holding for 3 minutes. The (S)-1-tetrahydronaphthylamine concentration standard curve is shown in Figure 5 .

[0063] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein, on the contrary, the present invention can be extended to all other having the same functions.

Claims

1. A method for producing (S)-1-tetrahydronaphthylamine by in vitro multi-enzyme cascade catalysis of tetralin, characterized in that: With tetralin as a substrate, the conversion of tetralin to (S)-1-tetralinamine is achieved through an in vitro multi-enzyme cascade system which is independent of cofactors and can recycle hydrogen peroxide in the system; the multi-enzyme cascade system consists of a non-specific peroxidase, a galactose oxidase and an ω-aminotransferase.

2. The method for producing (S)-1-tetrahydronaphthylamine by in vitro multi-enzyme cascade catalysis of tetralin according to claim 1, characterized in that: The amino acid sequence of the non-specific peroxidase is shown in SEQ ID NO:

1.

3. The method for producing (S)-1-tetrahydronaphthylamine by in vitro multi-enzyme cascade catalysis of tetralin according to claim 1, characterized in that: The amino acid sequence of the galactose oxidase is shown in SEQ ID NO:

2.

4. The method of producing (S)-1-tetrahydronaphthylamine by in vitro multi-enzyme cascade catalysis of tetralin according to claim 1, characterized in that: The amino acid sequence of the ω-aminotransferase is shown in SEQ ID NO:

3.

5. The method for producing (S)-1-tetrahydronaphthylamine by in vitro multi-enzyme cascade catalysis of tetralin according to claim 1, characterized in that: The addition amount of the non-specific peroxidase is 10-20 μg / mL, the addition amount of the galactose oxidase is 50-100 μg / mL, and the addition amount of the ω-aminotransferase is 150-200 μg / mL.

6. The method of producing (S)-1-tetrahydronaphthylamine from tetralin by in vitro multi-enzyme cascade catalysis according to claim 1, characterized in that: The non-specific peroxidase, galactose oxidase and ω-aminotransferase are all obtained through recombinant bacteria expressing the non-specific peroxidase, galactose oxidase and ω-aminotransferase.

7. The method of producing (S)-1-tetrahydronaphthylamine from tetralin by in vitro multi-enzyme cascade catalysis according to claim 1, characterized in that: The substrate tetralin, pyridoxal phosphate, hydrogen peroxide, an activator, an amino donor, a nonspecific peroxidase, a galactose oxidase and an ω-transaminase are reacted at 25-35° C., a pH of 7.0-8.0 and a rotation speed of 600 rpm for 24 hours to complete the reaction and obtain the product (S)-1-tetralinamine.

8. The method for producing (S)-1-tetrahydronaphthylamine from tetralin by in vitro multi-enzyme cascade catalysis according to claim 7, characterized in that: In the reaction system, the concentration of tetralin is 0.5-5 mM, the concentration of pyridoxal phosphate is 0.5-2 mM, the concentration of hydrogen peroxide is 0.1-1 substrate equivalent, the concentration of the activator is 1-30 mM, and the concentration of the amino donor is 0.5-2 M.

9. The method for producing (S)-1-tetrahydronaphthylamine from tetralin by in vitro multi-enzyme cascade catalysis according to claim 7, characterized in that: In the reaction system, the activator is one or more of potassium persulfate, sodium persulfate, potassium ferrocyanide and horseradish peroxidase.

10. The method for producing (S)-1-tetrahydronaphthylamine from tetralin by in vitro multi-enzyme cascade catalysis according to claim 7, characterized in that: In the reaction system, the amino donor is one or more of isopropylamine, β-alanine, L-alanine, 4-nitrophenylethylamine and o-xylylenediamine.

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