A method for preparing chiral morpholine compounds by one-pot double-enzyme reduction with high enantioselectivity

Abstract

The present application relates to the technical field of biopharmacy and bio-chemical engineering, and particularly relates to a method for preparing chiral morpholine compounds by one-pot two-enzyme method with high enantioselectivity, which adopts nitroreductase NfsA and imine reductase IR-14, and successfully realizes high-efficiency biological catalytic conversion of chiral morpholine compounds under the synergistic action of NADPH coenzyme system. The technology has excellent catalytic efficiency, and the overall substrate conversion rate is higher than 60%; absolute stereoselectivity, the enantiomeric excess value (e.e.%) of the product is more than 90%; and the reaction system is simple, the conditions are mild (normal temperature and pressure), and meets the requirements of green chemistry. Compared with the traditional chemical synthesis method, the biological catalytic path of the present application has significant advantages in terms of atom economy, environmental friendliness and optical purity of the product, and provides an innovative solution for the synthesis of chiral N, O and other heterocyclic drugs.

Description

Technical Field

[0001] This invention relates to the fields of biopharmaceutical and biochemical technologies, specifically to a method for preparing chiral morpholine compounds using a one-pot two-enzyme method with high enantioselectivity reduction. Background Technology

[0002] Heterocyclic compounds containing heteroatoms are essential components of organic molecules, among which morpholine ring compounds containing N and O atoms are a very important class of structural motifs. They are widely found in various fields such as rubber, pharmaceuticals, pesticides, coatings, textiles, and medical devices. Currently, the efficient construction of morpholine ring compounds containing chiral C atoms is a hot topic in organic synthesis. Various chiral C-containing morpholine ring compounds are widely found in fields such as rubber, pharmaceuticals, pesticides, coatings, textiles, and medical devices. Examples include hyoscyamine, a drug for treating allergic rhinitis; amorolfine hydrochloride, an antifungal drug; and morpholinonitrazole, an anti-inflammatory drug.

[0003] Currently, several methods for obtaining chiral morpholine compounds have been developed in conventional organocatalysis. Previous researchers have used metal / transition metal catalysis, employing antimony cation / anion catalysts and chiral ligands to asymmetricly reduce imine precursors to obtain highly enantioselective morpholine compounds. However, this catalytic method has several drawbacks, requiring a large amount of reducing agent, expensive metal catalysts, various chiral ligands, and stringent reaction conditions such as high temperature and pressure. Therefore, it is necessary to find a more economical and efficient method for obtaining chiral morpholine compounds.

[0004] Compared to traditional organic catalysis, enzyme catalysis offers a novel direction for catalytic transformation. To date, various enzyme-catalyzed asymmetric synthetic reactions have been developed. However, enzymatic synthesis of morpholine ring products is currently very limited. Existing research typically involves complex chemical synthesis to first obtain an imine substrate, followed by selective reduction of the imine using imine reductases to obtain the product. This method requires the separation and purification of the complex imine precursor before enzymatic reduction, which is not an economical or environmentally friendly approach consistent with green chemistry principles.

[0005] Tandem reactions are an important strategy in organic synthesis, referring to two or more consecutive chemical transformations occurring under single reaction conditions, with intermediates typically not separated. Compared to stepwise purification reaction pathways, tandem reactions offer higher reaction efficiency and better selectivity, and may achieve structures that are difficult to synthesize using conventional methods. Combining enzymatic catalysis with chemical coupling for one-pot synthesis can simultaneously increase yield, reaction efficiency, and enantioselectivity. Existing research has achieved the asymmetric synthesis of tetrahydroquinoline using a tandem enzymatic method. However, this method is only applicable to one-pot synthesis of single-N heterocycles and cannot be used for the one-pot synthesis of multi-component heterocycles.

[0006] In view of the above-mentioned defects, the inventors of this invention have finally obtained this invention after a long period of research and practice. Summary of the Invention

[0007] The purpose of this invention is to solve the problem of how to achieve one-pot synthesis of multi-component heterocyclic compounds using enzyme catalysis, and to provide a one-pot two-enzyme method for the highly enantioselective reduction of chiral morpholine compounds.

[0008] To achieve the above objectives, this invention discloses a one-pot, two-enzyme method for the highly enantioselective reduction of chiral morpholine compounds. The method uses nitroreductase NfsA and imine reductase IR-14 as catalysts, 2-(2-nitrophenoxy)-1-phenylethylone and its monosubstituted benzene ring compounds as substrates, NADPH as a coenzyme system, and dimethyl sulfoxide as a solubilizer. Following the reaction, chiral morpholine ring compounds are prepared by asymmetric reduction. The amino acid sequence of nitroreductase NfsA is shown in SEQ ID NO. 1, and the amino acid sequence of imine reductase IR-14 is shown in SEQ ID NO. 3. Nitroreductase NfsA is used to catalyze the reduction of 2-(2-nitrophenoxy)-1-phenylethylone and its various monosubstituted benzene ring compounds. The intermediate product spontaneously forms an imine ring, which is then reduced by imine reductase IR-14 to generate chiral morpholine ring compounds.

[0009] The concentration of the 2-(2-nitrophenoxy)-1-phenylethyl ketone and its monosubstituted benzene ring compounds is 10 mM, the concentration of the nitroreductase NfsA is 0.1~0.2 mM, and the concentration of the imine reductase IR-14 is 0.1~0.2 mM.

[0010] The concentration of the nitroreductase NfsA is 0.1 mM, and the concentration of the imine reductase IR-14 is 0.1 mM.

[0011] The concentration of the 2-(2-nitrophenoxy)-1-phenylethyl ketone and its benzene ring monosubstituted compounds is 10 mM, and the dimethyl sulfoxide is 4% by volume.

[0012] The reaction conditions were pH = 7.0, 25°C, and 300 rpm for 12 hours.

[0013] The coenzyme system specifically includes NADP. + A mixture of glucose dehydrogenase GDH-105 and glucose.

[0014] The NADP + The concentration of glucose is 0.5 mM, the enzyme activity of glucose dehydrogenase GDH-105 is 20 U, and the concentration of glucose is 40 mM.

[0015] The nitroreductase NfsA was obtained by expressing a recombinant bacterium containing the gene sequence shown in SEQ ID NO.2, and the imine reductase IR-14 was obtained by expressing a recombinant bacterium containing the gene sequence shown in SEQ ID NO.4.

[0016] The 2-(2-nitrophenoxy)-1-phenylethyl ketone and its benzene ring monosubstituted compounds are compounds Ia, IIa, IIIa, IVa or VA, and their structural formulas are shown below:

[0017] .

[0018] The chiral morpholine ring compounds are compounds Ib, IIb, IIIb, IVb, or Vb, and their structural formulas are shown below:

[0019] .

[0020] The reductases involved in this invention are nitroreductase NfsA, which contains 369 amino acids and has the NCBI accession number WP_000189159.1, with its amino acid sequence shown in SEQ ID NO.1. The gene encoding this protein contains 1110 bp, with its nucleotide sequence shown in SEQ ID NO.2; and imine reductase IR-14, which contains 299 amino acids and has the NCBI accession number YP_005263141.1, with its amino acid sequence shown in SEQ ID NO.3. The gene encoding this protein contains 900 bp, with its nucleotide sequence shown in SEQ ID NO.4.

[0021] After inducing expression of nitroreductase with the gene sequence shown in SEQ ID NO.1, it was reacted with 2-(2-nitrophenoxy)-1-phenylethyl ketone and its monosubstituted benzene ring compounds, 4% dimethyl sulfoxide, and 0.5 mM NADP. + The reaction was carried out at pH 7.0, 25℃, and 850 rpm for 12 h to obtain chiral morpholine ring compounds. NADP was added during this process. + The glucose reacts with glucose dehydrogenase to generate NADPH, allowing the reaction to proceed cyclically, thus reducing the amount added and lowering production costs.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] 1. The reaction system of this invention is simple, and the product yield and enantiomeric excess value are high;

[0024] 2. The biocatalyst in this invention has good substrate tolerance and can achieve catalytic reactions at high substrate concentrations without the need for batch feeding;

[0025] 3. The catalytic system of the present invention introduces a coenzyme recycling system to help regenerate the coenzyme, thereby reducing the amount of cofactors added and reducing production costs.

[0026] 4. In this invention, the tandem reduction of nitro and reduction of imine can make the reaction efficiency higher than that of a single imine substrate reduction reaction. Attached Figure Description

[0027] Figure 1 This is a synthetic route of the present invention;

[0028] Figure 2 The chiral analysis spectrum of the racemic product of compound Ib;

[0029] Figure 3 The chiral analysis spectrum of compound Ib was obtained after 12 hours of reaction.

[0030] Figure 4 The chiral analysis spectrum of the racemic product of compound IIb;

[0031] Figure 5 The chiral analysis spectrum of compound IIb was obtained after 12 hours of reaction.

[0032] Figure 6 Chiral analysis spectrum of the racemic product of compound IIIb;

[0033] Figure 7 The chiral analysis spectrum of compound Ⅲb was obtained after 12 hours of reaction.

[0034] Figure 8 Chiral analysis spectrum of the racemic product of compound IVb;

[0035] Figure 9 The chiral analysis spectrum of compound IVb was obtained after 12 hours of reaction.

[0036] Figure 10 Chiral analysis spectrum of the racemic product of compound VB;

[0037] Figure 11 The chiral analysis spectrum of compound VB taken after 12 hours of reaction;

[0038] Figure 12 Nuclear magnetic resonance of product compound Ib 1 H-NMR spectrum, the horizontal axis is chemical shift δ, in ppm, and the vertical axis is the integral height of the absorption peak;

[0039] Figure 13 Nuclear magnetic resonance of product compound IIb 1 H-NMR spectrum, the horizontal axis is chemical shift δ, in ppm, and the vertical axis is the integral height of the absorption peak;

[0040] Figure 14 Nuclear magnetic resonance of product compound IIIb 1 H-NMR spectrum, the horizontal axis is chemical shift δ, in ppm, and the vertical axis is the integral height of the absorption peak;

[0041] Figure 15 Nuclear magnetic resonance of product compound IVb 1 H-NMR spectrum, the horizontal axis is chemical shift δ, in ppm, and the vertical axis is the integral height of the absorption peak;

[0042] Figure 16 NMR of product compound VB 1 H-NMR spectrum, the horizontal axis is chemical shift δ, in ppm, and the vertical axis is the integral height of the absorption peak. Detailed Implementation

[0043] The above-mentioned and other technical features and advantages of the present invention will be described in more detail below with reference to the accompanying drawings.

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following examples are commercially available.

[0045] The 100mM Kpi buffer (pH 7.0) in the example was prepared by dissolving potassium dihydrogen phosphate (0.1 M) in water and adjusting the pH to 7.0 with 6 M HCl.

[0046] Example 1

[0047] Using E. coli to express nitroreductase NfsA and imine reductase IR-14:

[0048] The preparation methods of NfsA protein and IR-14 protein in this embodiment are as follows:

[0049] 1. Nanjing Genscript Biotech Co., Ltd. constructed an NfsA expression vector using the pET-28a(+) vector (synthesized by Genscript). Specifically, the NfsA gene fragment was used as a template to clone the NfsA gene into the pET-28a(+) vector. The corresponding promoter is the T7 promoter. A 6X His tag was added to the N-terminus of the promoter and cloned after the ribosome binding site (RBS) of the pET-28a(+) vector. The resulting recombinant vector is denoted as NfsA_pET-28a(+).

[0050] The amino acid sequence of the NfsA protein:

[0051] MGSSHHHHHHSSGLVPRGSHMTPTIELICGHRSIRHFTDEPISEAQREAIINSARATSSSSFLQCSSIIRITDKALREELVTLTGGQKHVAQAAEFWVFCADFNRHLQICPDAQLGLAEQLLLGVVDTAMMAQNALIAAESLGLGGVYIGGLRNNIEAVTKLLKLPQHVLPLFGLCLGWPADNPDLKPRLPASILVHENSYQPLDKGALAQYDEQLAEYYLTRGSNNRRDTWSDHIRRTIIKESRPFILDYLHKQGWATR (SEQ ID NO: 1).

[0052] The DNA sequence of said NfsA gene:

[0053] (SEQ ID NO.2).

[0054] The amino acid sequence of the 6X His is shown below: HHHHHH (SEQ ID NO.5).

[0055] Expression of protein NfsA: The obtained plasmid NfsA was transformed into Escherichia coli BL21(DE3) by heat shock method and cultured overnight on LB plates containing a final concentration of 50 μg / mL ampicillin. Colonies that grew after overnight culture were considered positive colonies.

[0056] Positive single colonies containing recombinant plasmids were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C and 220 rpm for 10–12 h with shaking. The cultured bacterial solution was then inoculated into fresh TB liquid medium containing 50 μg / mL kanamycin at a volume ratio of 1:100 and cultured at 37°C and 220 rpm with shaking until the bacterial concentration OD600 was 0.8–1.2. Then, the inducer IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a working concentration of 0.5 mM, and the culture was continued at 20°C and 180 rpm for 18 h. After centrifugation for 20 min, the bacterial cells were collected and resuspended.

[0057] The collected bacterial cells were resuspended in 100 mM Kpi buffer (pH=7.0), and PMSF (phenylmethylsulfonyl fluoride) was added to a working concentration of 1 mM. The cells were then sonicated on ice with the following parameters: power 40 W; sonication mode: 2 s, 3 s interval, for a total of 60 min. The cells were then centrifuged at 12000 rpm at 4℃ for 40 min to obtain the supernatant containing protein.

[0058] Purification of protein NfsA: Nitrogen column purification was employed. The supernatant was filtered through a 0.22 μm filter membrane. The obtained supernatant was incubated with the nickel column and then eluted with 20 mM and 300 mM imidazole solutions containing 100 mM Kpi, respectively. The eluent was collected and dialyzed overnight in dialysis buffer (100 mM Kpi buffer, pH=7.0). The next day, the eluent was concentrated using a 10K concentrator. Using this method, approximately 100-200 mg of protein can be purified per liter of bacteria.

[0059] 2. Nanjing Genscript Biotech Co., Ltd. constructed an IR-14 expression vector using the pET-22b(+) vector (synthesized by Genscript). Specifically, the IR-14 gene fragment was used as a template to clone the IR-14 gene into the pET22b(+) vector. The corresponding promoter is the T7 promoter. A 6X His tag was added to its N-terminus and cloned after the ribosome binding site (RBS) of the pET-22b(+) vector. The resulting recombinant vector was denoted as IR-14_pET-22b(+).

[0060] Amino acid sequence of IR-14 protein:

[0061] MHHHHHHTNNATPVSILGLGLMGQALARAFLKAGHPTTVWNRTPGKADQLMAEGAQVAPTAAEAIDASSLTVICVSDYPAMYELLDASDLAGTTLLNLTSGDSAQARQAARWAEQRGAHYLDGAIMAIPQAIGTDDAVILISGAQADADAHRPTLEALGTLTYLGADHGLASLYDVAGLAMMWSVLNAWLQGTALLRTAGVDAATFAPFAQQMAAGVAGWLPGHAQEIDAGSFATEVASLDTHVRTMDHLIEECEAAGINAELPRLIKSMADRSLAAGHGAASYSVLIEEFAKPA (SEQ ID NO: 3).

[0062] DNA sequence of IR-14 gene:

[0063] ATGCACCATCACCATCATCACACGAACAACGCAACGCCGGTCTCAATCCTGGGCCTGGGTCTGATGGGTCAAGCTCTGGCACGCGCCTTCCTGAAAGCCGGTCATCCGACCACGGTCTGGAACCGTACCCCGGGCAAAGCGGATCAGCTGATGGCGGAAGGTGCCCAAGTTGCACCGACCGCGGCCGAAGCTATTGATGCGAGCTCTCTGACGGTGATCTGCGTTAGTGACTATCCGGCGATGTACGAACTGCTGGATGCTTCCGACCTGGCAGGTACCACGCTGCTGAATCTGACCAGTGGTGATTCCGCACAGGCTCGTCAAGCAGCTCGTTGGGCAGAACAGCGTGGTGCACATTATCTGGACGGTGCCATTATGGCAATCCCGCAAGCAATCGGCACCGATGACGCGGTGATTCTGATCAGCGGTGCACAGGCAGATGCAGACGCTCATCGTCCGACGCTGGAAGCACTGGGTACCCTGACGTATCTGGGCGCAGATCACGGTCTGGCTAGCCTGTACGACGTTGCTGGTCTGGCGATGATGTGGTCTGTCCTGAACGCATGGCTGCAGGGTACCGCACTGCTGCGTACGGCCGGTGTGGATGCAGCAACCTTTGCACCGTTCGCACAGCAAATGGCAGCTGGCGTTGCAGGTTGGCTGCCGGGCCACGCACAGGAAATTGATGCCGGTAGCTTTGCAACCGAAGTCGCTTCTCTGGATACCCATGTGCGCACGATGGACCACCTGATTGAAGAATGTGAAGCGGCCGGCATCAATGCGGAACTGCCGCGTCTGATTAAATCAATGGCCGATCGCTCGCTGGCAGCAGGTCATGGTGCGGCGTCATACAGCGTTCTGATTGAAGAATTTGCGAAACCGGCTTAA (SEQ ID NO: 4).

[0064] The amino acid sequence of 6X His is as follows: HHHHHH (SEQ ID NO: 5).

[0065] Expression of protein IR-14: The obtained plasmid IR-14 was transformed into Escherichia coli BL21(DE3) by heat shock method and cultured overnight on LB agar plates containing a final concentration of 50 μg / mL ampicillin. Colonies that grew after overnight culture were considered positive colonies.

[0066] Positive single colonies containing recombinant plasmids were inoculated into LB liquid medium containing 50 μg / mL ampicillin and cultured at 37℃ and 220 rpm for 10-12 h with shaking. The cultured bacterial solution was then inoculated into fresh TB liquid medium containing 50 μg / mL ampicillin at a volume ratio of 1:100 and cultured at 37℃ and 220 rpm with shaking until the bacterial concentration OD600 was 0.8-1.2. Then, IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a working concentration of 0.5 mM, and the culture was continued at 20℃ and 180 rpm for 18 h. After centrifugation for 20 min, the bacterial cells were collected and resuspended.

[0067] The collected bacterial cells were resuspended in 100 mM Kpi buffer (pH=7.0), and PMSF (phenylmethylsulfonyl fluoride) was added to a working concentration of 1 mM. The cells were then sonicated on ice with the following parameters: power 40 W; sonication mode: 2 s, 3 s interval, for a total of 60 min. The cells were then centrifuged at 12000 rpm at 4℃ for 40 min to obtain the supernatant containing protein.

[0068] Purification of protein IR-14: Nitrogen column purification was employed. The supernatant was filtered through a 0.22 μm filter membrane. The obtained supernatant was incubated with the nickel column and then eluted with 20 mM and 300 mM imidazole solutions containing 100 mM Kpi, respectively. The eluent was collected and dialyzed overnight in dialysis buffer (100 mM Kpi buffer, pH=7.0). The next day, the eluent was concentrated using a 10K concentrator. Using this method, approximately 100-200 mg of protein can be purified per liter of bacteria.

[0069] Example 2

[0070] Determination of enzyme concentration in enzyme-catalyzed asymmetric synthesis:

[0071] NfsA concentration determination:

[0072] Using substrate Ia as the model substrate, 10 mM of substrate Ia was dissolved in 100 μL of dimethyl sulfoxide. 20 μL of this solution was then added to a container containing different concentrations of NfsA, 1% (0.1 mM) IR-14, 0.1 mg GDH (~33 enzyme activity units), 20 μmol of D-glucose, and 0.25 μmol of NADP. +The reaction was carried out in a reaction buffer (100 mM phosphate buffer, pH=7.0), with a total volume of 500 μL. After reacting at 25℃ and 300 rpm for 12 h, samples were taken for analysis. The reaction results are shown in Table 1 below:

[0073] Table 1. Synthesis of chiral morpholine rings with different concentrations of NfsA

[0074]

[0075] The optimal NfsA concentration was determined to be 0.1 mM through screening.

[0076] IR-14 concentration determined:

[0077] Using substrate Ia as the model substrate, 10 mM of substrate Ia was dissolved in 100 μL of dimethyl sulfoxide. 20 μL of this solution was then added to a solution containing 1% (0.1 mM) NfsA, different concentrations of IR-14, 0.1 mg GDH (~33 enzyme activity units), 20 μmol of D-glucose, and 0.25 μmol of NADP. + The reaction was carried out in a 500 μL solution of 100 mM phosphate buffer (pH=7.0) at 25 °C for 12 h at 300 rpm. Samples were taken for analysis. The results are shown in Table 2 below.

[0078] Table 2. Synthesis of chiral morpholine rings at different concentrations of IR-14

[0079]

[0080] The optimal IR-14 concentration was determined to be 0.1 mM through screening.

[0081] Example 3

[0082] Enzyme-catalyzed Ia asymmetric synthesis of chiral morpholine cyclic compounds:

[0083] Dissolve 10 mM substrate Ia in 100 μL of dimethyl sulfoxide, and add 20 μL to a solution containing 1% (0.1 mM) NfsA, 1% (0.1 mM) IR-14, 0.1 mg GDH (~33 enzyme activity units), 20 μmol D-glucose, and 0.25 μmol NADP. + The reaction buffer (100 mM phosphate buffer, pH 7.0) was used to prepare the product. The total volume was 500 μL. The reaction was carried out at 25 °C and 300 rpm for 12 h. Samples were then taken for analysis. The chiral column chromatogram of the racemic product is shown below. Figure 2 The chiral column chromatography of the reaction products is shown below. Figure 3 The yield of product Ib was over 74%, with an optical purity (ee%) greater than 96.6%, and nuclear magnetic resonance (NMR) results were also obtained. 1H-NMR spectrum as follows Figure 12 As shown.

[0084] Example 4

[0085] Enzyme-catalyzed IIa asymmetric synthesis of chiral morpholine cyclic compounds:

[0086] Dissolve 10 mM substrate IIa in 100 μL of dimethyl sulfoxide, and add 20 μL to a solution containing 1% (0.1 mM) NfsA, 1% (0.1 mM) IR-14, 0.1 mg GDH (~3 enzyme activity units), 20 μmol D-glucose, and 0.25 μmol NADP. + The reaction buffer (100 mM phosphate buffer, pH 7.0) was used to prepare the product. The total volume was 500 μL. The reaction was carried out at 25 °C and 300 rpm for 12 h. Samples were then taken for analysis. The chiral column chromatogram of the racemic product is shown below. Figure 4 The chiral column chromatography of the reaction products is shown below. Figure 5 The yield of product IIb was over 67%, with an optical purity (ee%) greater than 90.6%, and nuclear magnetic resonance (NMR) results were obtained. 1 H-NMR spectrum as follows Figure 13 As shown.

[0087] Example 5

[0088] Enzyme-catalyzed asymmetric synthesis of chiral morpholine cyclic compounds (IIIa):

[0089] Dissolve 10 mM substrate IIIa in 100 μL of dimethyl sulfoxide, and add 20 μL to a solution containing 1% (0.1 mM) NfsA, 1% (0.1 mM) IR-14, 0.1 mg GDH (~3 enzyme activity units), 20 μmol D-glucose, and 0.25 μmol NADP. + The reaction buffer (100 mM phosphate buffer, pH 7.0) was used to prepare the product. The total volume was 500 μL. The reaction was carried out at 25 °C and 300 rpm for 12 h. Samples were then taken for analysis. The chiral column chromatogram of the racemic product is shown below. Figure 6 The chiral column chromatography of the reaction products is shown below. Figure 7 The yield of product IIIb was over 61%, with an optical purity (ee%) greater than 95.6%, and nuclear magnetic resonance (NMR) results were obtained. 1 H-NMR spectrum as follows Figure 14 As shown.

[0090] Example 6

[0091] Enzyme-catalyzed asymmetric synthesis of chiral morpholine cyclic compounds (IVa):

[0092] Dissolve 10 mM substrate IVa in 100 μL of dimethyl sulfoxide, and add 20 μL to a solution containing 1% (0.1 mM) NfsA, 1% (0.1 mM) IR-14, 0.1 mg GDH (~3 enzyme activity units), 20 μmol D-glucose, and 0.25 μmol NADP. + The reaction buffer (100 mM phosphate buffer, pH 7.0) was used to prepare the product. The total volume was 500 μL. The reaction was carried out at 25 °C and 300 rpm for 12 h. Samples were then taken for analysis. The chiral column chromatogram of the racemic product is shown below. Figure 8 The chiral column chromatography of the reaction products is shown below. Figure 9 The yield of product IVb was over 70%, with an optical purity (ee%) greater than 99.9%, and nuclear magnetic resonance (NMR) results were obtained. 1 H-NMR spectrum as follows Figure 15 As shown.

[0093] Example 7

[0094] Enzyme-catalyzed asymmetric synthesis of chiral morpholine ring compounds using VA:

[0095] Dissolve 10 mM substrate VA in 100 μL of dimethyl sulfoxide, and add 20 μL to a solution containing 1% (0.1 mM) NfsA, 1% (0.1 mM) IR-14, 0.1 mg GDH (~3 enzyme activity units), 20 μmol D-glucose, and 0.25 μmol NADP. + The reaction buffer (100 mM phosphate buffer, pH 7.0) was used to prepare the product. The total volume was 500 μL. The reaction was carried out at 25 °C and 300 rpm for 12 h. Samples were then taken for analysis. The chiral column chromatogram of the racemic product is shown below. Figure 10 The chiral column chromatography of the reaction products is shown below. Figure 11 The yield of product VB was over 65%, with an optical purity (ee%) greater than 95.2%, and nuclear magnetic resonance (NMR) results were obtained. 1 H-NMR spectrum as follows Figure 16 As shown.

[0096] The detection methods for the products are as follows (methods in Examples 2-7):

[0097] After the reaction was complete, 10 μL of 32% NaOH was added to quench the reaction, followed by an equal volume of dichloromethane. The mixture was shaken vigorously for 10 min, and then centrifuged at 8000 rpm for 10 min to separate the organic and aqueous phases. The aqueous phase was removed, and the lower layer of dichloromethane was carefully aspirated and filtered through an organic filter membrane for preservation.

[0098] Chiral morpholine ring compounds were determined by reversed-phase HPLC using a Shimadzu HPLC instrument. 1,3,5-trimethoxybenzene was used as an internal standard. An XB-C18 column was used, with acetonitrile:water = 70:30 as the mobile phase, a flow rate of 1 mL, and a column temperature of 40 °C.

[0099] The optical rotation of chiral morpholine compounds was determined using normal-phase HPLC. The instrument was a Shimadzu HPLC system, and the column was a CHIRALPAK® OJ-H solvent-resistant bonded chiral column with covalently bonded amylose-tris(3,5-dimethylphenylcarbamate) on the silica gel surface. The mobile phase used was normal phase: n-Hexane, IPA. The program was: column temperature 40°C, detector absorption wavelengths: 214 nm, 254 nm, flow rate 1 mL / min, and isocratic elution with 5-10% isopropanol.

[0100] The enantiomeric ratio (ee% value) of the chiral (S)-morpholine ring compound is calculated as: (S)-morpholine ring compound peak area: (R)-morpholine ring compound peak area.

[0101] The above description is merely a preferred embodiment of the present invention and is illustrative rather than restrictive. Those skilled in the art will understand that many changes, modifications, and even equivalents can be made within the spirit and scope defined by the claims of the present invention, all of which will fall within the protection scope of the present invention.

Claims

1. A method for preparing chiral morpholine compounds by one-pot two-enzyme reduction with high enantioselectivity, characterized in that, Using nitroreductase NfsA and imine reductase IR-14 as catalysts, 2-(2-nitrophenoxy)-1-phenylethyl ketone and its monosubstituted benzene ring compounds as substrates, NADPH as coenzyme, and dimethyl sulfoxide as solubilizer, chiral morpholine ring compounds were prepared by asymmetric reduction after the reaction. The amino acid sequence of nitroreductase NfsA is shown in SEQ ID NO.1, and the amino acid sequence of imine reductase IR-14 is shown in SEQ ID NO.

3. The 2-(2-nitrophenoxy)-1-phenylethyl ketone and its benzene ring monosubstituted compounds are compounds Ia, IIa, IIIa, IVa or VA, and their structural formulas are shown below: 。 2. The method for preparing chiral morpholine compounds by one-pot two-enzyme reduction with high enantioselectivity as described in claim 1, characterized in that, The concentration of the 2-(2-nitrophenoxy)-1-phenylethyl ketone and its monosubstituted benzene ring compounds is 10 mM, the concentration of the nitroreductase NfsA is 0.1~0.2 mM, and the concentration of the imine reductase IR-14 is 0.1~0.2 mM.

3. The method for preparing chiral morpholine compounds by one-pot two-enzyme reduction with high enantioselectivity as described in claim 1, characterized in that, The concentration of the 2-(2-nitrophenoxy)-1-phenylethyl ketone and its monosubstituted benzene ring compounds is 10 mM, the concentration of the nitroreductase NfsA is 0.1 mM, and the concentration of the imine reductase IR-14 is 0.1 mM.

4. The method for preparing chiral morpholine compounds by one-pot two-enzyme reduction with high enantioselectivity as described in claim 1, characterized in that, The concentration of the 2-(2-nitrophenoxy)-1-phenylethyl ketone and its benzene ring monosubstituted compounds is 10 mM, and the dimethyl sulfoxide is 4% by volume.

5. The method for preparing chiral morpholine compounds by one-pot two-enzyme reduction with high enantioselectivity as described in claim 1, characterized in that, The reaction conditions were pH = 7.0, 25°C, and 300 rpm for 12 hours.

6. The method for preparing chiral morpholine compounds by one-pot two-enzyme reduction with high enantioselectivity as described in claim 1, characterized in that, The NADPH is produced via the following coenzyme system: containing NADP + A mixture of glucose dehydrogenase GDH-105 and glucose.

7. The method for preparing chiral morpholine compounds by one-pot two-enzyme reduction with high enantioselectivity as described in claim 6, characterized in that, The NADP + The concentration of glucose is 0.5 mM, the enzyme activity of glucose dehydrogenase GDH-105 is 20 U, and the concentration of glucose is 40 mM.

8. The method for preparing chiral morpholine compounds by one-pot two-enzyme reduction with high enantioselectivity as described in claim 1, characterized in that, The nitroreductase NfsA was obtained by expressing a recombinant bacterium containing the gene sequence shown in SEQ ID NO.2, and the imine reductase IR-14 was obtained by expressing a recombinant bacterium containing the gene sequence shown in SEQ ID NO.

4.

9. The method for preparing chiral morpholine compounds by one-pot two-enzyme reduction with high enantioselectivity as described in claim 1, characterized in that, The chiral morpholine ring compounds are compounds Ib, IIb, IIIb, IVb, or Vb, and their structural formulas are shown below: 。

Images