A method for synthesizing (s)-1-tetralin amine by in vitro multi-enzyme cascade catalysis of tetralin

Abstract

The application belongs to the technical field of bio-chemical industry, and particularly discloses a method for synthesizing (S)-1-tetralinamine by in-vitro multi-enzyme cascade catalysis of tetralin. The multi-enzyme cascade system is composed of non-specific peroxidase, galactose oxidase and ω-transaminase, the substrate is tetralin, and the product is (S)-1-tetralinamine. First, the non-specific peroxidase is used to catalyze tetralin and hydrogen peroxide to generate tetralin alcohol, the tetralin alcohol is subjected to the action of the galactose oxidase to generate tetralin ketone, and the tetralin ketone is subjected to the action of the ω-transaminase to generate (S)-1-tetralinamine. Compared with the prior art, the synthesis method provides a new in-vitro multi-enzyme catalysis way for synthesizing (S)-1-tetralinamine from tetralin without expensive cofactors, the process is safe and green, the production cost can be significantly reduced, a new green way is developed for converting large-volume aromatic hydrocarbons into high-value amine organic compounds, and the method has important industrial application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biocatalysis and bio-chemical industry, and particularly relates to a method for generating (S)-1-tetralin amine by in vitro multi-enzyme cascade catalysis of tetralin BACKGROUND

[0002] (S)-1-tetralin amine, also known as (S)-1,2,3,4-tetrahydro-1-naphthalenamine, is an important drug synthesis intermediate, which 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 of naphthyridinone compounds.

[0003] The most common method for preparing S-1-tetralin amine is to synthesize it by direct reduction amination through 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, and has high requirements for reaction equipment, and the optical purity of the product is not high. Another method is to first prepare 1-tetralin amine racemate and then obtain (S)-1-tetralin amine by chiral resolution (Bioorganic & Medicinal Chemistry, 2004, 12(15): 4189-4196), which has low utilization rate of raw materials. Therefore, it is of great significance to develop a green and economical method for synthesizing (S)-1-tetralin amine.

[0004] In recent years, the rapid development of biotechnology 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 green and clean, mild reaction conditions, etc. Multi-enzyme catalysis generally refers to the cascade catalysis of two or more enzymes, which has significant advantages in shortening reaction steps, reducing intermediate accumulation, and reducing equipment size, and plays an increasingly important role in industrial biotechnology. There have been some reports on the preparation of chiral amines by in vitro multi-enzyme catalytic cascade reaction using hydrocarbon compounds as substrates, and some achievements have been made (Green Chemistry, 2023, 25, 3469-3474; ChemSusChem, 2019, 12(4): 848-857; Angew. Chem. Int. Ed., 2024, e202407778). Although the reported methods can achieve the synthesis of chiral amines, they are mainly limited to small-volume chiral amine compounds, and there is currently no related report on compounds such as tetrahydronaphthalene amine with large steric hindrance. In addition, the existing multi-enzyme catalytic system often needs to use expensive co-factors. In addition, most of the existing multi-enzyme catalytic methods focus on the conversion of aliphatic compounds, and there are few studies on the synthesis of (S)-1-tetrahydronaphthalene amine by multi-enzyme cascade reaction directly using tetrahydronaphthalene as raw material. Therefore, it is of great significance to develop an in vitro multi-enzyme catalytic method independent of co-factors for synthesizing (S)-1-tetrahydronaphthalene amine from tetrahydronaphthalene in the field of green biological manufacturing. SUMMARY

[0005] The purpose of the present application is to provide a multi-enzyme cascade catalytic method for synthesizing (S)-1-tetrahydronaphthalene amine from tetrahydronaphthalene based on the background of green biological manufacturing. The method uses tetrahydronaphthalene as raw material and obtains (S)-1-tetrahydronaphthalene amine 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 application is achieved by the following technical solutions:

[0007] The technical scheme of the present application provides a multi-enzyme cascade catalytic method for synthesizing (S)-1-tetrahydronaphthalene amine from tetrahydronaphthalene. The steps are as follows: adding (S)-1-tetrahydronaphthalene amine synthesis related enzymes into the system of tetrahydronaphthalene, pyridoxal phosphate, hydrogen peroxide, and an aqueous solution of activator and amino donor, and catalyzing the reaction to synthesize (S)-1-tetrahydronaphthalene amine.

[0008] Further, the (S)-1-tetrahydronaphthalene amine synthesis related enzyme (multi-enzyme cascade system) is composed of non-specific peroxidase, galactose oxidase, and ω-transaminase.

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

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

[0011] Further, the amino acid sequence of the omega-transaminase is shown as SEQ ID NO: 3.

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

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

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

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

[0016] Further, the reaction system is: 20 μg / mL non-specific peroxidase, 50 μg / mL galactose oxidase, 200 μg / mL omega-transaminase, 0.5-5 mM tetrahydronaphthalene, 0.5-1 mM pyridoxal phosphate, 0.1-0.4 substrate equivalent hydrogen peroxide, 1-30 mM activator, 0.5-2 M amino donor.

[0017] Further, the amino donor is one or more of isopropylamine, beta-alanine, L-alanine, 4-nitrophenylethylamine and o-xylylene diamine.

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

[0019] Further, the reaction temperature is 25-35℃; preferably 30℃.

[0020] Further, the rotation speed of the reaction is 600 rpm.

[0021] Further, the reaction time is 24 h.

[0022] Compared with the prior art, the application has the following advantages:

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

[0024] The present application reduces the amount of co-substrate hydrogen peroxide by introducing a hydrogen peroxide recycling system, thereby greatly reducing the cost of the reaction. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 A schematic diagram of the catalytic pathway for the synthesis of (S)-1-tetralinamine by in vitro multi-enzyme catalysis of tetralin;

[0026] Figure 2 Effect of different activators on the catalytic activity of galactose oxidase;

[0027] Figure 3 Effect of different amounts of hydrogen peroxide on the catalytic activity of the non-specific peroxidase and galactose oxidase cascade;

[0028] Figure 4 Product analysis results of (S)-1-tetralinamine by gas chromatography in the present application;

[0029] Figure 5 (S)-1-tetralinamine standard curve. DETAILED DESCRIPTION

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

[0031] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The reagents, methods, and equipment employed in the present application are those conventional in the art. The test methods in the following examples, unless otherwise noted, were typically conducted according to conventional experimental conditions. Unless specifically indicated otherwise, the reagents and raw materials used in the present application are commercially available.

[0032] The present application develops a method for the synthesis of (S)-1-tetralinamine by in vitro multi-enzyme cascade catalysis of tetralin. The method uses tetralin as a substrate and synthesizes (S)-1-tetralinamine through a multi-enzyme cascade system, as shown in Figure 1

[0033] ​The method for synthesizing (S)-1-tetralylamine by the multi-enzyme cascade catalysis of tetralin constructed by the application is to use non-specific peroxidase, galactose oxidase and omega-transaminase to catalyze the synthesis of (S)-1-tetralylamine from tetralin; the method specifically comprises the following steps: (1) using non-specific peroxidase to catalyze the synthesis of 1-tetralyl alcohol from tetralin and hydrogen peroxide; (2) using galactose oxidase to catalyze the synthesis of 1-tetralyl ketone from 1-tetralyl alcohol and oxygen; and (3) using omega-transaminase to catalyze the synthesis of (S)-1-tetralylamine from 1-tetralyl ketone and isopropylamine (see Figure 1 ).

[0034] Example 1

[0035] Construction of recombinant bacteria expressing non-specific peroxidase, galactose oxidase and omega-transaminase

[0036] The gene sequences of non-specific peroxidase, galactose oxidase (the sequence is from the literature DOI: 10.1021 / acscatal.3c03427) and omega-transaminase (the sequence is from the literature DOI: 10.1002 / anie.202212555) were codon-optimized and synthesized by Shengong Biotech (Shanghai) Co., Ltd., and were inserted into the corresponding sites of pET-28a plasmid through EcoR I and Sac I sites, respectively, and were identified by enzyme digestion analysis to obtain the corresponding recombinant plasmids.

[0037] Non-specific peroxidase (SEQ ID NO. 1):

[0038]

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

[0040]

[0041] Omega-transaminase (SEQ ID NO. 3):

[0042]

[0043] Example 2

[0044] Obtaining non-specific peroxidase, galactose oxidase and omega-transaminase

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

[0046] The recombinant strain was inoculated into LB liquid medium and cultured at 37°C, 180 rpm overnight, then the recombinant strain was inoculated into 1L ZYM-5052 self-induction liquid medium containing 30 μg / mL kanamycin at an inoculation amount of 1%, and cultured at 16°C for 3 days, and then centrifuged at 8000 rpm for 5 min to collect the bacterial cells, resuspended the bacterial cells with a buffer (20 mM Tris-HCl) at pH 8, and subjected to ultrasonic disruption at 350 W (working for 3 s, intermittent for 5 s, 20 min), and then centrifuged (12000 rpm, 10 min) to collect the soluble supernatant expression components (crude enzyme solution).

[0047] The soluble supernatant expression components after disruption were separated and purified by nickel column affinity chromatography to obtain non-specific peroxidase, galactose oxidase and ω-transaminase. The specific operation was as follows: the medium was equilibrated with 5 times the column volume of equilibration buffer (pH 7.5 20 mM NaPi, 40 mM imidazole, 500 mM NaCl); the crude enzyme solution obtained after disruption was filtered with a 0.45 μm filter; the sample was loaded at a flow rate of 1 mL / min; after loading, the buffer (pH 7.5 20 mM NaPi, 100 mM imidazole, 500 mM NaCl) was used to wash the impurities for 10-20 times the column volume; then elution buffer (pH 7.5 20 mM NaPi, 250 mM imidazole, 500 mM NaCl) was used for elution for 5-10 times the column volume, and the eluate was collected; the eluate was diluted with 50 mM NaPi buffer at pH 8.0, ultrafiltration concentrated (10 KDa), repeated 5-10 times, and the imidazole was removed; the concentration of each target protein obtained after ultrafiltration was determined by BCA method.

[0048] Example 3

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

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

[0051] Example 4

[0052] Effect of different amino donors on the catalytic synthesis of (S)-1-tetralamine by ω-transaminase:

[0053] ​The effect of different amino donors on the synthesis of (S)-1-tetralinamine catalyzed by ω-transaminase was explored. 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-xylylene diamine), and the final concentration of galactose oxidase was 150 μg / mL. When o-xylylene diamine was used as the amino donor, the yield of the product was 10.24% after 12 h of reaction.

[0054] Example 5

[0055] Effect of initial hydrogen peroxide equivalent on the synthesis of 1-tetralin catalyzed by non-specific peroxidase and galactose oxidase cascade:

[0056] The effect of initial hydrogen peroxide equivalent on the synthesis of 1-tetralin catalyzed by non-specific peroxidase and galactose oxidase cascade was explored. 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.), and the final concentrations of non-specific peroxidase and galactose oxidase were 20 μg / mL and 50 μg / mL, respectively. The reaction time was 12 h, and the yield was shown in Figure 3 .

[0057] Example 6

[0058] Three-enzyme cascade catalysis of tetralin to (S)-1-tetralinamine:

[0059] The final concentrations of non-specific peroxidase, galactose oxidase, and ω-transaminase were 20 μg / mL, 50 μg / mL, and 200 μg / mL, respectively. The reaction system contained 1 mM tetralin, 20 mM sodium persulfate, 1 mM hydrogen peroxide, 1 mM pyridoxal phosphate, and 1 M isopropylamine in a pH 7.5 Na2HPO4-NaH2PO4 buffer. The multi-enzyme cascade catalytic reaction was carried out 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] Detection method of product:

[0062] (S)-1-tetralin was detected in Example 5 using gas chromatography (GC) with the following detection conditions: Agilent cp7502 column; nitrogen flow rate of 1 mL / min; temperature program of 100 °C for 2 min, 25 °C / min to 130 °C for 5 min, 2 °C / min to 140 °C for 5 min, 5 °C / min to 150 °C for 3 min, 25 °C / min to 180 °C for 3 min. The (S)-1-tetralin concentration standard curve is shown in FIG. 1. Figure 5 .

[0063] It should be noted that the foregoing examples have been provided merely for the purposes of illustration and are not intended to limit the application thereof in any way. The application has been described in reference to the exemplary embodiments thereof, but it is understood that the words which have been used herein are words of description, and not words of limitation. Changes can be made within the purview of the appended claims, as presently set forth, and as amended, without departing from the scope and spirit of the application in its aspects. Although the application has been described herein with reference to particular methods, materials, and embodiments, the application is not intended to be limited to the particulars disclosed herein; rather, changes can be made within the scope and spirit of the application, as those skilled in the art are aware.

Claims

1. A method for in vitro multi-enzyme cascade catalysis of tetralin to (S)-1- tetralinamine, characterized in that, A conversion of tetrahydronaphthalene to (S)-1-tetrahydronaphthalenamine is realized by a multi-enzyme cascade system consisting of a non-specific peroxygenase, a galactose oxidase and an omega-transaminase in the presence of hydrogen peroxide, pyridoxal phosphate, an activator and an amino donor, wherein the amino acid sequence of the non-specific peroxygenase is shown as SEQ ID NO:1, the amino acid sequence of the galactose oxidase is shown as SEQ ID NO:2, the amino acid sequence of the omega-transaminase is shown as SEQ ID NO:3, the activator is one or more of potassium persulfate, sodium persulfate, potassium ferricyanide and horseradish peroxidase, and the amino donor is one or more of isopropylamine, beta-alanine, L-alanine, 4-nitrophenylethylamine and o-xylylene diamine.

2. The method of claim 1, wherein, The non-specific peroxygenase is added in an amount of 10-20 μg / mL, the galactose oxidase is added in an amount of 50-100 μg / mL, and the omega-transaminase is added in an amount of 150-200 μg / mL.

3. The method of claim 1, wherein, The non-specific peroxygenase, the galactose oxidase and the omega-transaminase are all obtained by expressing recombinant bacteria of the non-specific peroxygenase, the galactose oxidase and the omega-transaminase.

4. The method of claim 1, wherein, The substrate tetrahydronaphthalene, pyridoxal phosphate, hydrogen peroxide, an activator, an amino donor, a non-specific peroxygenase, a galactose oxidase, an omega-transaminase are reacted at 25-35°C, pH 7.0-8.0, and a rotation speed of 600 rpm for 24 h, and the product obtained after the reaction is (S)-1-tetrahydronaphthalenamine.

5. The method of claim 4, wherein, In the reaction system, the concentration of the tetrahydronaphthalene is 0.5-5 mM, the concentration of the pyridoxal phosphate is 0.5-2 mM, the concentration of the 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.

Images