Method for catalytically synthesizing chiral amino alcohol compound and pyrazine compound by chemical-enzyme cascade method

Abstract

The invention relates to the field of biochemical engineering, in particular to a method for catalytically synthesizing a chiral amino alcohol compound and a pyrazine compound through a chemical-enzyme cascade method. Alcohol dehydrogenase is utilized to catalyze amino alcohol, kinetic resolution is performed to obtain chiral amino alcohol and an aminoketone compound, and the corresponding pyrazine compound is further obtained from the aminoketone compound under the catalysis of artificial flavin. In a catalytic synthesis system provided by the invention, the artificial flavin can promote condensation and oxidation of an aminoketone compound into a medical intermediate pyrazine while regenerating NAD (P) < + > required by alcohol dehydrogenase catalysis. Compared with traditional kinetic resolution, the method has the advantages that the resolution efficiency is improved, two valuable products are obtained, the atom utilization rate of the whole reaction is improved, and the method has the advantages of being efficient in reaction, easy to operate, green, mild and the like.

Description

Technical Field

[0001] The invention relates to the field of biochemical engineering, and in particular to a method for catalytically synthesizing chiral amino alcohol compounds and pyrazine compounds by a chemical-enzyme cascade method. Background Art

[0002] Chiral amino alcohols are widely used in the fields of medicine, materials, and fine chemicals. The traditional chemical synthesis of chiral amino alcohols has problems such as harsh reaction conditions, the use of toxic and expensive metal catalysts, and low selectivity. The strategy of biosynthesis of optically pure amino alcohols has the characteristics of high selectivity, mild reaction conditions, and environmental friendliness. However, there are still some defects in the enzymatic chiral synthesis process. For example, enzymes have strict requirements for potential chiral substrates, which limits the scope of their catalytic substrates; at the same time, some potential chiral substrates (amino ketones) will undergo spontaneous condensation to form by-products when used as substrates to synthesize chirality; potential chiral substrates are expensive, resulting in excessively high raw material costs; in addition, some enzymes exhibit strict stereoselectivity, and the other half of the enantiomer cannot be obtained through chiral synthesis.

[0003] In contrast, enzymatic chiral resolution is a good strategy for obtaining optically pure enantiomers. It can overcome the dilemma that the other half of the enantiomer cannot be prepared during the synthesis of chiral compounds, and can selectively synthesize the desired chiral isomers. At the same time, the raw materials (racemates) are relatively cheap and the cost is low. However, the enzymatic chiral resolution method still faces some challenges: (1) The resolution of amino alcohols mainly relies on the catalysis of amine dehydrogenases and transaminases, and requires a large amount of amino donors to promote the reaction in the forward direction; (2) It is easy to form by-products, which makes subsequent separation difficult; (3) The maximum yield of kinetic resolution of amino alcohols is only 50%, and the atomic utilization rate is low. Compared with amine dehydrogenases and transaminases, alcohol dehydrogenases have shown excellent performance and unique advantages in the preparation of chiral compounds: (1) No additional co-substrate is required during the reaction, which not only reduces unnecessary side reactions but also simplifies the synthesis steps; (2) The substrate spectrum of alcohol dehydrogenases is wider. However, the resolution of amino alcohols and the resolution of amino alcohols catalyzed by alcohol dehydrogenases have not been reported.

[0004] Pyrazine derivatives are important raw materials and key precursors in the fields of food, medicine, materials, etc. Pyrazine derivatives are a class of nitrogen-containing aromatic compounds, usually with one or more substituents, and are widely distributed in plants, insects, fungi and bacteria. Due to the characteristics of high volatility, low aroma threshold and strong aroma potential, pyrazine derivatives are widely used as a flavoring substance in the food and flavor and fragrance industry. For example, 2,5-dimethylpyrazine (2,5-dimethylpyrazine, 2,5-DMP) is an important ingredient for some baked and fermented foods to have a unique flavor. It is clearly listed as a synthetic food flavoring allowed for use in my country's Food Additives Use Standard (GB2760-2014); tetramethylpyrazine is one of the important flavor ingredients for Chinese liquor to have a sauce-flavored flavor. Pyrazine derivatives are also widely used in the pharmaceutical field. For example, tetramethylpyrazine, also known as ligustrazine, has the functions of dilating blood vessels, improving microcirculation and inhibiting platelet aggregation, and is clinically used for the treatment of ischemic vascular diseases; 2,5-dimethylpyrazine is an important precursor of antihypertensive drugs such as acipimox and hypoglycemic and lipid-lowering drugs such as glipizide. In addition, pyrazine derivatives also have important applications in the field of materials. For example, tetraphenylpyrazine has easily adjustable excited state properties, good thermal stability and a clear luminescence mechanism, and is used to make optoelectronic and biomaterials. Therefore, the synthesis of pyrazine derivatives has practical application prospects. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for splitting alcohol amines of chiral amino alcohol compounds by using alcohol dehydrogenase to solve the defects of the prior art that chiral amino alcohol splitting easily forms by-products, subsequent separation is difficult and the yield is low. The method couples alcohol dehydrogenase with a bifunctional artificial flavin to construct a chemical enzyme cascade synergistic catalysis process mediated by artificial flavin.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for synthesizing chiral amino alcohol compounds and pyrazine compounds by chemical-enzyme cascade catalysis, wherein amino alcohol, alcohol dehydrogenase, coenzyme and artificial flavin are placed in a solvent for reaction to obtain chiral amino alcohol compounds and pyrazine compounds;

[0008] Wherein, the chiral amino alcohol compound is an R-configured chiral amino alcohol compound, and its structural formula is:

[0009]

[0010] Wherein, the pyrazine compound has the structural formula:

[0011]

[0012] Wherein, the R represents any one of methyl, ethyl, propyl, isopropyl, isobutyl, tert-butyl, phenyl and benzyl.

[0013] The amino alcohol is a racemic amino alcohol, including any one of a racemic aliphatic amino alcohol, a racemic aromatic amino alcohol, and a racemic cycloalkane amino alcohol.

[0014] Specifically, the aliphatic amino alcohol includes any one of 1-amino-2-butanol, 1-aminopentan-2-ol, 1-amino-3-methyl-butan-2-ol, 1-amino-4-methylpentane-2-ol, and 1-amino-3,3-dimethylbutane-2-ol; the aromatic amino alcohol is 2-amino-1-phenylethanol or 1-amino-3-phenylpropane-2-ol; and the cycloalkane amino alcohol is 2-aminocyclohexanol.

[0015] The specific process of the reaction is as follows: alcohol dehydrogenase is used to catalyze the oxidation and resolution of the racemic amino alcohol substrate to obtain chiral amino alcohol compounds of S and R configurations; the chiral amino alcohol compound of S configuration is oxidized by alcohol dehydrogenase and coenzyme NAD(P) + The catalytic process obtains amino ketone byproducts and NAD(P)H. Two molecules of amino ketone byproducts undergo intermolecular cyclization, and under the promotion of artificial flavin, pharmaceutical intermediate pyrazine compounds are synthesized.

[0016] Among them, the artificial flavin can not only promote the condensation oxidation of aminoketone byproducts, but also serve as NAD(P) + The regenerated catalyst reoxidizes NAD(P)H to generate NAD(P) required for alcohol dehydrogenase catalysis + , thus dependent on NAD(P) + The alcohol dehydrogenase catalyzes the oxidation reaction to form the coenzyme NAD(P) + A regeneration cycle system; wherein the molar percentage of artificial flavin in NAD(P)H is 0.1 to 5%, wherein NAD(P)H is oxidized to obtain NAD(P) + The conditions are pH 4-10 and temperature 30-70°C.

[0017] Specifically, the above reaction process does not rely on metal and base catalysts, and constructs a new, green chemical-enzyme-level reaction pathway for synthesizing chiral amino alcohol compounds and pyrazine compounds.

[0018] Specifically, the reaction pathway is shown as follows: wherein ADH refers to alcohol dehydrogenase, and F4 refers to artificial flavin.

[0019]

[0020] In some embodiments of the present invention, when the amino alcohol is any one of 1-amino-2-propanol, 1-amino-2-butanol, 1-aminopentan-2-ol, 1-amino-3-methyl-butan-2-ol, 1-amino-4-methylpentane-2-ol, 1-amino-3,3-dimethylbutane-2-ol, 2-amino-1-phenylethanol, and 1-amino-3-phenylpropane-2-ol, the structural formula of the chiral amino alcohol compound is:

[0021]

[0022] The structural formula of the pyrazine compound is:

[0023]

[0024] In some embodiments of the present invention, when the amino alcohol is 2-aminocyclohexanol, the structural formula of the chiral amino alcohol compound is:

[0025]

[0026] The structural formula of the pyrazine compound is:

[0027]

[0028] The alcohol dehydrogenase includes any one of alcohol dehydrogenase APDH, alcohol dehydrogenase Aa-ADH, alcohol dehydrogenase PpADH, alcohol dehydrogenase SyADH, alcohol dehydrogenase CoADH, and horse liver alcohol dehydrogenase HLADH.

[0029] Specifically, the alcohol dehydrogenase APDH is derived from 3-oxoacyl-[acyl carrier protein] reductase of Mycolicibacterium smegmatis MC2 155, and the NCBI accession number of its amino acid sequence is ABK71438.1; the alcohol dehydrogenase Aa-ADH is derived from aromaticumbacterium strain EbN1, and the NCBI accession number of its amino acid sequence is WP_011237148.1; the alcohol dehydrogenase PpADH is derived from Paracoccus pantotrophus, and the NCBI accession number of its amino acid sequence is WP_028710368.1; the alcohol dehydrogenase SyADH is derived from Sphingobium yanoikuyae DSM 6900, and the NCBI accession number of its amino acid sequence is 4BMV_A; the alcohol dehydrogenase CoADH is derived from Candida orthopsilosis (strain 90-125) (Yeast), the NCBI accession number of its amino acid sequence is CCG20495.1; the horse liver alcohol dehydrogenase HLADH is derived from Horse liver, and the NCBI accession number of its amino acid sequence is AAB26666.1.

[0030] In some embodiments of the present invention, the alcohol dehydrogenase is obtained by constructing the alcohol dehydrogenase gene into a plasmid vector, and transferring it into competent Escherichia coli cells, inducing expression and purifying it.

[0031] Specifically, the plasmid vector includes but is not limited to pET 28a(+) vector, and the Escherichia coli includes but is not limited to Escherichia coli BL 21(DE3).

[0032] Wherein, the coenzyme is NAD(P) + or NAD + Any one of .

[0033] Wherein, the artificial flavin is an artificial bridged flavin and its general structural formula is:

[0034]

[0035] Specifically, R1 and R2 are independently selected from: hydrogen, methyl, trifluoromethyl, methoxy, halogen atom, nitro or amino; R3 is selected from: hydrogen, C1-C5 alkyl, phenyl or benzyl; X- is selected from: halide ion, nitrate or trifluoromethanesulfonate ion.

[0036] Preferably, R1 includes but is not limited to hydrogen, methyl or halogen atom; R2 includes but is not limited to hydrogen, methyl, halogen atom or trifluoromethyl; R3 includes but is not limited to hydrogen, methyl; X- is a halide ion.

[0037] More preferably, the artificial bridged flavin includes but is not limited to 7-trifluoromethyl-1,10-ethylene isoalloxazine chloride (Compound I), 8-chloro-1,10-ethylene isoalloxazine chloride (Compound II), or 1,10-ethylene isoalloxazine chloride (Compound III).

[0038]

[0039] Most preferably, the artificial bridged flavin is 7-trifluoromethyl-1,10-ethyleneisoalloxazine chloride.

[0040] Wherein, the solvent is 50-200 mM CHES-NaOH buffer with a pH of 8.0-10.0.

[0041] In some embodiments of the present invention, the solvent is 50 mM CHES-NaOH buffer with a pH of 8.0-10.0.

[0042] The reaction system is as follows: an initial reaction concentration of 5 to 50 mM amino alcohol, an initial reaction concentration of 0.5 to 1 mM coenzyme, an initial reaction concentration of 0.5 to 1 mM artificial flavin, and an initial reaction addition amount of 0.5 to 6.0 mg / mL alcohol dehydrogenase APDH; the reaction conditions are: 25 to 37°C, 200 rpm, and reaction time of 8 to 12 hours.

[0043] In some embodiments of the present invention, the reaction system is: an initial reaction concentration of 5 mM amino alcohol, an initial reaction concentration of 1 mM coenzyme, an initial reaction concentration of 0.5 mM artificial flavin, and an initial reaction addition amount of 6.0 mg / mL alcohol dehydrogenase APDH; the reaction conditions are: 25-37°C, 200 rpm for 8 hours.

[0044] Beneficial effects: The present invention utilizes alcohol dehydrogenase to catalyze amino alcohols, and kinetically resolves chiral amino alcohols and amino ketone compounds. Furthermore, amino ketone compounds are further catalyzed by artificial flavin to obtain corresponding pyrazine compounds. At the same time, in the catalytic reaction system of the present invention, the bifunctional organic small molecule artificial flavin can regenerate the NAD(P) required for alcohol dehydrogenase catalysis. + At the same time, it promotes the condensation and oxidation of another product, aminoacetone, into the pharmaceutical intermediate pyrazine. Compared with the traditional kinetic resolution, this method not only improves the resolution efficiency and obtains two valuable products, but also improves the atomic utilization of the overall reaction. It has the advantages of high reaction efficiency, simple operation, and green and mild. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] 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.

[0046] Figure 1 Schematic diagram of the reaction pathway of the present invention.

[0047] Figure 2 This is the SDS-PAGE detection result of alcohol dehydrogenase. DETAILED DESCRIPTION

[0048] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0049] In the following examples, the amino alcohol derivatization reagent is prepared as follows: 8 mg of 2,4-dinitrofluorobenzene (FDNB) is weighed and dissolved in 2 mL of acetonitrile, gently shaken, and stored at low temperature and away from light for later use.

[0050] In the following examples, the analysis method of the chiral amino alcohol derivatives is as follows:

[0051] HPLC detection method: Agilent series high performance liquid chromatograph was used to analyze the derivatized solution, using CHIRALCELOD-H chiral column, phase A: chromatographic grade n-hexane, phase B: chromatographic grade isopropanol, mobile phase ratio: n-hexane / isopropanol = 1:10, column temperature 25°C, flow rate 0.2mL / min, wavelength 340nm. The calculation formula for the enantiomeric excess value of the substrate is: Where [R] and [S] represent the peak areas of the R and S enantiomers of the substrate in the liquid chromatogram, respectively.

[0052] In the following examples, the analysis method of pyrazine and its derivatives is as follows:

[0053] The reaction solution of pyrazine was analyzed using a 4600UPLC / Triple TOF liquid spectrometer (LC-MS), the chromatographic column model was Agilent Eclipse Plus C18 (250mm×4.6mm×5μm), phase A: 50mM sodium acetate solution, phase B: chromatographic grade acetonitrile; A:B=1:1, column temperature 25°C, flow rate 0.1mL / min, detection by UV detector, wavelength 254nm.

[0054] In the following examples, the 50 mM, pH 10.0 CHES-NaOH buffer solution is prepared as follows: (1) weigh 1.036 g CHES (MW = 207.3 g / mol) and add 40 mL pure water; (2) measure the pH value of 40 mL CHES aqueous solution to be 4.50 at a test temperature of 27°C; (3) add an appropriate amount of 1 M NaOH solution to adjust the pH value to 10.0; (3) add pure water to make up to 50 mL, transfer to a 50 mL sample bottle, and store at room temperature.

[0055] Example 1: Construction and induced expression of alcohol dehydrogenase genetically engineered bacteria

[0056] Taking alcohol dehydrogenase APDH as an example, the gene of alcohol dehydrogenase APDH derived from Mycolicibacterium smegmatis was synthesized by General Biosystems (Anhui) Co., Ltd., and the NCBI accession number of its amino acid sequence is ABK71438.1.

[0057] The synthesized alcohol dehydrogenase APDH gene was cloned into the pET28a(+) vector to obtain the recombinant plasmid pET28a-APDH, and then the recombinant plasmid was transferred into the competent cells of Escherichia coli BL21(DE3), and then smeared on the LB solid medium plate containing 50μg / mL Kana resistance after shaking culture at 37℃ for 1h. After culturing at 37℃ for 16h, the single clone on the plate was picked and verified to construct the alcohol dehydrogenase gene engineering bacteria.

[0058] Select a single colony on the plate and inoculate it into 20 mL of LB medium containing 50 μg / mL Kana resistance. Cultivate it for about 12 hours as a seed solution. Inoculate it into 400 mL of LB medium containing 50 μg / mL Kana resistance at a 5% v / v inoculum. Cultivate it on a shaker at 37°C and 200 rpm until the OD reaches 0. 600= 0.6 to 0.8, add IPTG with a final concentration of 0.1mmol / L and induce at 25℃ for 12h. After induction, centrifuge the bacterial solution at 4℃ and 8000rpm for 5min to collect the bacterial cells, use 50mM, pH 7 KPi buffer (fill 0.8L of distilled water in a suitable container, add 2.829g of potassium dihydrogen phosphate to the solution, add 5.087g of potassium dihydrogen phosphate, add distilled water until the volume is 1L after complete dissolution) to clean the bacterial cells and store at 4℃. Resuspend the bacterial mud in 20mL of 50mM pH7.4 potassium phosphate buffer (KPi buffer), and crush the resuspended bacterial cells with an ultrasonic crusher on ice (on 3s, off 5s, power 300W) for 15 minutes. Then centrifuge (9000rpm, 4℃, 40min), collect the supernatant, and filter the supernatant with a 0.22μm water filter to obtain a crude alcohol dehydrogenase enzyme solution.

[0059] 2mL Ni-NTA agarose purification resin filler was loaded into a gravity protein purification column (purchased from Yisheng Bio), and the filler was balanced with KPi buffer three times, and then an appropriate amount of crude enzyme solution was slowly added, and gently shaken for about 5 minutes to promote the full adsorption of the enzyme and the filler. Use 20-500mM imidazole buffer (17.01g imidazole was weighed and dissolved in 500mL ultrapure water, 500mM imidazole buffer was obtained by dissolving, and other concentrations of imidazole buffer were obtained by dilution) for gradient elution. The specific process of gradient elution is as follows: the recombinant protein is recovered by gradient method, and the proportion of elution buffer is gradually increased until the recombinant protein is eluted. Specifically, 20mM imidazole buffer was added to rinse twice, and then 50mM imidazole buffer was used to rinse once to remove impurities; then the target protein was eluted with 200mM imidazole buffer, and the eluate was collected with a centrifuge tube; finally, 500mM imidazole buffer was used to remove the remaining protein in the purification column filler. The collected eluate was concentrated and desalted using a Millipore ultrafiltration tube to obtain a pure enzyme solution, which was then dispensed into 200 μL tubes and stored in an ultra-low temperature refrigerator at -80°C. Its purity and size were preliminarily verified using polyacrylamide gel electrophoresis (SDS-PAGE).

[0060] The construction and inducible expression of the genetically engineered bacteria of alcohol dehydrogenase Aa-ADH, alcohol dehydrogenase PpADH, alcohol dehydrogenase SyADH, alcohol dehydrogenase CoADH or horse liver alcohol dehydrogenase HLADH are the same as above.

[0061] The results are as follows Figure 2 As shown, the molecular weight of alcohol dehydrogenase APDH is 37KDa, the molecular weight of Aa-ADH is 31KDa, the molecular weight of PpADH is 36KDa, the molecular weight of SyADH is 40KDa, the molecular weight of HLADH is 39KDa, and the molecular weight of CoADH is 38KDa.

[0062] Example 2: Chemo-enzymatic synthesis of chiral amino alcohols and pyrazines from 1-aminopentan-2-ol (3a)

[0063] In 2 mL of 50 mM CHES-NaOH buffer, pH 10.0, 5 mM racemic 1-aminopentan-2-ol, 1 mM NAD + , 0.5 mM artificial flavin (7-trifluoromethyl-1,10-ethyleneisoxazine chloride), 6.0 mg / mL alcohol dehydrogenase APDH to a final concentration of 1.5 mg / mL, react at 37°C, 200 rpm for 8 h, take 200 μL of the reaction solution, add 200 μL of amino alcohol derivatization reagent system (20 μL of amino alcohol derivatization reagent, 20 μL of reaction solution, 20 μL of 1 M sodium bicarbonate and 140 μL DMSO mixed), react at 40°C for 2 h, centrifuge at 9000 rpm for 3 min, pass the supernatant through a 0.22 μm organic membrane to remove protein and other solid impurities in the reaction solution, and then perform HPLC detection to detect the yield and ee value of chiral amino alcohol derivatives, as well as the yield of pyrazine derivatives.

[0064] The product 2,5-dipropylpyrazine was synthesized using racemic 1-aminopentan-2-ol as a raw material. Its properties are yellow oily substances. The nuclear magnetic resonance characterization results are as follows: 1 H NMR(400MHz,MeOD)δ8.41(s,2H),2.78(s,4H),1.76(s,4H),0.98(t,6H); 13 C NMR (101MHz, MeOD) δ154.72, 143.14, 36.17, 22.47, 12.57. After data comparison, the retention time of (R)-1-aminopentan-2-ol was 30min, the retention time of (S)-1-aminopentan-2-ol was 35min, and the retention time of 2,5-dipropylpyrazine was 39min. After determination, the yield of the product chiral amino alcohol (R)-1-aminopentan-2-ol (3b) was 45%, and the ee value was 94%; the yield of the product 2,5-dipropylpyrazine (3d) was 44%.

[0065] Example 3: Chemo-enzymatic synthesis of chiral amino alcohols and pyrazines from 2-amino-1-phenylethanol (7a)

[0066] In 2 mL of 50 mM CHES-NaOH buffer, pH 10.0, 5 mM racemic 2-amino-1-phenylethanol, 1 mM NAD +, 0.5 mM artificial flavin (7-trifluoromethyl-1,10-ethylene isoalloxazine chloride), 6.0 mg / mL alcohol dehydrogenase APDH to a final concentration of 1.5 mg / mL, react at 37°C, 200 rpm for 8 h, take 200 μL of the reaction solution, add 200 μL of amino alcohol derivatization reagent system (20 μL of amino alcohol derivatization reagent, 20 μL of reaction solution, 20 μL of 1M sodium bicarbonate and 140 μL of DMSO mixed), react at 40°C for 2 h, centrifuge at 9000 rpm for 3 min, pass the supernatant through a 0.22 μm organic membrane to remove protein and other solid impurities in the reaction solution, and then perform HPLC detection to detect the yield and ee value of chiral amino alcohol derivatives, as well as the yield of pyrazine derivatives.

[0067] The product 2,5-diphenylpyrazine synthesized from racemic 2-amino-1-phenylethanol as a raw material is a light yellow oily substance. The characterization results are as follows: 1 H NMR (400MHz, DMSO) δ9.31 (s, 2H), 8.18 (s, 4H), 7.52 (s, 6H); 13 C NMR (101MHz, DMSO) δ150.17, 141.63, 136.18, 130.38, 129.56, 127.04. After data comparison, the retention time of (R)-2-amino-1-phenylethanol was 54min, the retention time of (S)-2-amino-1-phenylethanol was 64min, and the retention time of 2,5-diphenylpyrazine was 14min. After determination, the yield of the product chiral amino alcohol (R)-2-amino-1-phenylethanol (7b) was 50%, and the ee value was 100%; the yield of the product 2,5-diphenylpyrazine (7d) was 20%.

[0068] Example 4: Chemo-enzymatic synthesis of chiral amino alcohols and pyrazines from 2-aminocyclohexanol (9a)

[0069] In 2 mL of 50 mM CHES-NaOH buffer, pH 10.0, 5 mM racemic 2-aminocyclohexanol, 1 mM NAD +, 0.5 mM artificial flavin (7-trifluoromethyl-1,10-ethyleneisoxazine chloride), 6.0 mg / mL alcohol dehydrogenase APDH to a final concentration of 1.5 mg / mL, react at 37°C, 200 rpm for 8 h, take 200 μL of the reaction solution, add 200 μL of amino alcohol derivatization reagent system (20 μL of amino alcohol derivatization reagent, 20 μL of reaction solution, 20 μL of 1 M sodium bicarbonate and 140 μL DMSO mixed), react at 40°C for 2 h, centrifuge at 9000 rpm for 3 min, pass the supernatant through a 0.22 μm organic membrane to remove protein and other solid impurities in the reaction solution, and then perform HPLC detection to detect the yield and ee value of chiral amino alcohol derivatives, as well as the yield of pyrazine derivatives.

[0070] According to data comparison, the retention time of (R)-2-aminocyclohexanol is 30min, the retention time of (S)-2-aminocyclohexanol is 33min, and the retention time of octahydrophenazine is 35min. According to the determination, the yield of the chiral amino alcohol (R)-2-aminocyclohexanol (9b) is 50%, and the ee value is 100%; the yield of the product octahydrophenazine (9d) is 30%.

[0071] Example 5: Chemo-enzymatic synthesis of chiral amino alcohols and pyrazines from 1-amino-2-propanol, 1-amino-2-butanol, 1-amino-3-methyl-butan-2-ol, 1-amino-4-methylpentan-2-ol or 1-amino-3,3-dimethylbutan-2-ol

[0072] The 1-aminopentan-2-ol (2a) in Example 2 was replaced by 1-amino-2-propanol (1a), or 1-amino-2-butanol (2a), or 1-amino-3-methyl-butan-2-ol (4a), or 1-amino-4-methylpentane-2-ol (5a), or 1-amino-3,3-dimethylbutane-2-ol (6a), and other conditions remained unchanged to synthesize the corresponding chiral amino alcohols and pyrazines (1b and 1d, 2b and 2d, 4b and 4d, 5b and 5d, 6b and 6d), respectively.

[0073] Example 6: Chemo-enzymatic synthesis of chiral amino alcohols and pyrazines from 2-amino-1-phenylethanol

[0074] The 2-amino-1-phenylethanol (7a) in Example 3 was replaced by 1-amino-3-phenylpropane-2-ol (8a), and other conditions remained unchanged to synthesize the corresponding chiral amino alcohols and pyrazines (8b and 8d).

[0075] Specifically, the amino alcohol substrates described in Examples 2 to 6, the corresponding synthetic products and characterization data are shown in Table 1.

[0076] Table 1 Amino alcohol substrates described in Examples 2 to 6, corresponding synthetic products and characterization data

[0077]

[0078]

[0079] The present invention provides a concept and method for chemical-enzymatic cascade catalytic synthesis of chiral amino alcohol compounds and pyrazine compounds. 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 chiral amino alcohol compounds and pyrazine compounds by chemical-enzymatic cascade catalysis, characterized in that: placing amino alcohol, alcohol dehydrogenase, coenzyme and artificial flavin in a solvent for reaction to obtain a chiral amino alcohol compound and a pyrazine compound; Wherein, the chiral amino alcohol compound is an R-configured chiral amino alcohol compound, and its structural formula is: Wherein, the pyrazine compound has the structural formula: Wherein, the R represents any one of methyl, ethyl, propyl, isopropyl, isobutyl, tert-butyl, phenyl and benzyl.

2. The method according to claim 1, characterized in that The amino alcohol is a racemic amino alcohol, including any one of a racemic aliphatic amino alcohol, a racemic aromatic amino alcohol, and a racemic cycloalkane amino alcohol.

3. The method according to claim 2, characterized in that The aliphatic amino alcohol includes any one of 1-amino-2-butanol, 1-aminopentan-2-ol, 1-amino-3-methyl-butan-2-ol, 1-amino-4-methylpentane-2-ol, and 1-amino-3,3-dimethylbutane-2-ol; the aromatic amino alcohol is 2-amino-1-phenylethanol or 1-amino-3-phenylpropane-2-ol; and the cycloalkane amino alcohol is 2-aminocyclohexanol.

4. The method according to claim 3, characterized in that (1) When the amino alcohol is any one of 1-amino-2-butanol, 1-aminopentan-2-ol, 1-amino-3-methyl-butan-2-ol, 1-amino-4-methylpentane-2-ol, 1-amino-3,3-dimethylbutane-2-ol, 2-amino-1-phenylethanol, and 1-amino-3-phenylpropane-2-ol, the structural formula of the chiral amino alcohol compound is: The structural formula of the pyrazine compound is: (2) When the amino alcohol is 2-aminocyclohexanol, the structural formula of the chiral amino alcohol compound is: The structural formula of the pyrazine compound is:

5. The method according to claim 1, characterized in that The alcohol dehydrogenase includes any one of alcohol dehydrogenase APDH, alcohol dehydrogenase Aa-ADH, alcohol dehydrogenase PpADH, alcohol dehydrogenase SyADH, alcohol dehydrogenase CoADH, and horse liver alcohol dehydrogenase HLADH; Among them, the NCBI accession number of the amino acid sequence of the alcohol dehydrogenase APDH is ABK71438.1; the NCBI accession number of the amino acid sequence of the alcohol dehydrogenase Aa-ADH is WP_011237148.1; the NCBI accession number of the amino acid sequence of the alcohol dehydrogenase PpADH is WP_028710368.1; the NCBI accession number of the amino acid sequence of the alcohol dehydrogenase SyADH is 4BMV_A; the NCBI accession number of the amino acid sequence of the alcohol dehydrogenase CoADH is CCG20495.1; and the NCBI accession number of the amino acid sequence of the horse liver alcohol dehydrogenase HLADH is AAB26666.

1.

6. The method according to claim 5, characterized in that The alcohol dehydrogenase is obtained by constructing the alcohol dehydrogenase gene into a plasmid vector, transferring the gene into competent Escherichia coli cells, inducing expression and purifying the cells.

7. The method according to claim 1, characterized in that The coenzyme is NAD(P) + or NAD + Any one of .

8. The method according to claim 1, characterized in that The artificial flavin is an artificial bridged flavin, and its general structural formula is: Wherein, R1 and R2 are independently selected from: hydrogen, methyl, trifluoromethyl, methoxy, halogen atom, nitro or amino; R3 is selected from: hydrogen, C1-C5 alkyl, phenyl or benzyl; X- is selected from: halide ion, nitrate or trifluoromethanesulfonate ion.

9. The oxidation method according to claim 8, characterized in that The artificial bridged flavin is 7-trifluoromethyl-1,10-ethylene isoalloxazine chloride, 8-chloro-1,10-ethylene isoalloxazine chloride or 1,10-ethylene isoalloxazine chloride.

10. The method according to claim 1, characterized in that The solvent is a CHES-NaOH buffer with a pH of 8.0 to 10.0 and a concentration of 50 to 200 mM. The reaction system is as follows: an amino alcohol with an initial reaction concentration of 5 to 50 mM, a coenzyme with an initial reaction concentration of 0.5 to 1 mM, an artificial flavin with an initial reaction concentration of 0.5 to 1 mM, and an alcohol dehydrogenase with an initial reaction addition amount of 0.5 to 6.0 mg / mL. The reaction conditions are as follows: 25 to 37° C. and 200 rpm for 8 to 12 hours.

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