Process for the Stereoselective Preparation of 6,6-Dimethyl-3-azabicyclo[3.1.0]hexene Compounds

Abstract

The present invention provides a series of monoamine oxidases (PMAON) with high catalytic activity and strong chiral selectivity, which are derived from Penicillium, as well as a method for stereoselectively preparing 6,6-dimethyl-3-azabicyclo[3.1.0]hexene compounds using the enzyme. The present invention also provides genes encoding the series of monoamine oxidases, recombinant expression vectors containing the series of genes, recombinant expression transformants and methods for preparing the same.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical intermediates, and specifically relates to a biocatalytic method for 6,6-dimethyl-3-azabicyclo[3.1.0]hexene compounds and the biocatalytic enzymes used in this method. Background Art

[0002] Nirmatrelvir is an antiviral drug developed by Pfizer and is an orally active SARS-CoV-2 3CL protease inhibitor. Nirmatrelvir is a covalent inhibitor that can directly bind to the cysteine catalytic residue (Cys145) of the protease. The compound preparation of this drug and ritonavir, under the trade name Paxlovid, has now been approved by the FDA as a first-line clinical treatment drug for the treatment of the novel coronavirus, and its chemical structural formula is shown as follows:

[0003]

[0004] Boceprevir is a hepatitis C virus (HCV) protease inhibitor developed by Schering-Plough Corporation and was approved by the FDA on May 13, 2011, for the combined treatment of adult chronic hepatitis C with peginterferon α and ribavirin, and its chemical structural formula is shown as follows:

[0005]

[0006] It can be seen from the structural formulas of nirmatrelvir and boceprevir that both have a common 6,6-dimethyl-3-azabicyclo[3.1.0]hexane fragment, and the intermediate methyl (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (hereinafter referred to as the compound shown in Formula I) is a key intermediate for the synthesis of these two products.

[0007]

[0008] The methods for preparing the compound shown in Formula I mainly include:

[0009] First, WO2004113295 reported a synthetic method for preparing the hydrochloride salt of the compound shown in Formula I, and the synthetic route is shown as Route 1:

[0010]

[0011] Route 1 has many steps and a long route. The key is that the ee value of the final product is not high.

[0012] II. WO2007075790 reported a synthesis method for the hydrochloride salt of the compound shown in Formula I with an ee value of over 90%. However, this method uses the diastereomeric salt resolution method of the final product, which is uneconomical. Its synthetic route is shown in Route 2:

[0013]

[0014] III. To solve the above problems, WO2010008828 reported a synthesis method for stereoselectively preparing the compound shown in Formula I using an enzymatic catalysis method. Its synthetic route is shown in Route 3:

[0015]

[0016] In the first step of Route 3, the enzymatic catalytic oxidation reaction can obtain an oxidation product with 100% ee value, (1R,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hex-2-ene (hereinafter referred to as the compound shown in Formula III). Subsequently, it is very easy to obtain the compound shown in Formula I with 100% ee value. Therefore, the catalytic oxidation reaction is particularly crucial in the preparation of the compound shown in Formula I, and monoamine oxidase is the most important factor in this reaction. The monoamine oxidase reported in WO2010008828 was obtained by gene construction after separation from Aspergillus oryzae and Aspergillus niger. The substrate concentration of this original sequence is relatively low (<10 g / L feeding amount), and the yield per batch is low, which is not suitable for large-scale industrial production.

[0017] Therefore, there is still a need in the art to find new monoamine oxidases to solve the defect of low productivity in the prior art. SUMMARY OF THE INVENTION

[0018] The present invention provides a series of monoamine oxidases derived from Penicillium, which have high catalytic activity, strong chiral selectivity, and the reaction substrate concentration can reach 200 g / L feeding amount, solving the problems of low yield and unsuitability for industrial production of the monoamine oxidases in the prior art when used in enzymatic catalytic oxidation.

[0019] The first aspect of the present invention discloses a monoamine oxidase, whose amino acid sequence is the amino acid sequence shown in SEQ ID No: 2, SEQ ID No: 4 or SEQ ID No: 6, or the amino acid sequence of the monoamine oxidase is an amino acid sequence having a homology of over 80% (more preferably over 95%) with the amino acid sequences shown in SEQ ID No: 2, SEQ ID No: 4 and SEQ ID No: 6.

[0020] The above-mentioned monoamine oxidase can be obtained from Penicillium sp. ART, Penicillium polonicum, and Penicillium brasilianum, respectively.

[0021] In some preferred specific embodiments of the present invention, the amino acid sequence of the monoamine oxidase is the amino acid sequence shown in Seq ID No: 6.

[0022] The second aspect of the present invention discloses a gene encoding the above-mentioned monoamine oxidase. The nucleotide sequence of the gene is the nucleotide sequence shown in SEQ ID No: 1, SEQ ID No: 3, or SEQ ID No: 5. Alternatively, the nucleotide sequence of the gene is a nucleotide sequence having a homology of more than 80% (more preferably more than 95%) with the nucleotide sequences shown in SEQ ID No: 1, SEQ ID No: 3, and SEQ ID No: 5.

[0023] When the monoamine oxidases with the amino acid sequences shown in SEQ ID NO.2, 4, and 6 of the present invention are aligned with other known monoamine oxidases, it is found that the identities between the amino acid sequences of the monoamine oxidases of the present invention and the amino acid sequence of the monoamine oxidase of Aspergillus terreus are 67.7%, 63.2%, and 51.4% respectively, and the identity with the amino acid sequence of the monoamine oxidase from Aspergillus niger is less than 60%.

[0024] The third aspect of the present invention discloses a recombinant expression vector containing the above-mentioned gene.

[0025] In some preferred specific embodiments of the present invention, the plasmid used to construct the recombinant expression vector is selected from the pET series vectors or the pQE series vectors. In some more preferred specific embodiments of the present invention, the plasmid used to construct the recombinant expression vector is selected from pET28a.

[0026] The fourth aspect of the present invention discloses a recombinant expression transformant containing the recombinant expression vector.

[0027] The recombinant expression transformant is prepared by transforming the recombinant expression vector into a host cell.

[0028] In some preferred specific embodiments of the present invention, the host cell is selected from Escherichia coli.

[0029] In some preferred specific embodiments of the present invention, the Escherichia coli is selected from E. coli BL21(DE3), E. coli Rosetta(DE3), and M15. In some more preferred specific embodiments of the present invention, the Escherichia coli is E. coli BL21(DE3).

[0030] The fifth aspect of the present invention provides the use of the above-mentioned monoamine oxidase (PMAON) in the asymmetric catalytic oxidation reaction of prochiral compounds.

[0031] In some preferred specific embodiments of the present invention, the prochiral compound is a compound represented by the following general formula VI:

[0032]

[0033] Wherein, n is an integer selected from 0 to 8, more preferably an integer from 0 to 5, and

[0034] R 1 and R 2 each independently selected from H, C 1 ~C 8 alkyl, more preferably C 1 ~C 5 alkyl.

[0035] In an aqueous solution, in the presence of catalase and O 2 the compound represented by the general formula VI forms a compound represented by the general formula VII under the catalysis of the monoamine oxidase, and the reaction formula is as follows

[0036]

[0037] Wherein, n is an integer selected from 0 to 8, more preferably an integer from 0 to 8, and

[0038] R 1 and R 2 each independently selected from H, C 1 ~C 8 alkyl, more preferably C 1 ~C 5 alkyl.

[0039] In some preferred specific embodiments of the present invention, in the reaction system, the concentration of the prochiral compound is 5-200 g / L.

[0040] In some preferred specific embodiments of the present invention, O 2 is continuously introduced.

[0041] In some preferred specific embodiments of the present invention, the pH of the reaction solution is 7.0-8.0.

[0042] In some preferred specific embodiments of the present invention, the reaction is carried out under oscillating or stirring conditions.

[0043] In some preferred specific embodiments of the present invention, the reaction temperature is 20 to 45 °C.

[0044] In some preferred specific embodiments of the present invention, it is 25 - 30 °C.

[0045] The fifth aspect of the present invention discloses a method for stereoselectively preparing 6,6 - dimethyl - 3 - azabicyclo[3.1.0]hexene compounds, and the method comprises the following steps:

[0046] In an aqueous solution, in the presence of O 2 and catalase, using the above - mentioned monoamine oxidase to catalytically oxidize 6,6 - dimethyl - 3 - azabicyclo[3.1.0]hexane (the compound shown in Formula II) to generate 6,6 - dimethyl - 3 - azabicyclo[3.1.0]hexene compounds (the compound shown in Formula III), and the reaction formula is as shown below (Reaction Formula 1):

[0047]

[0048] In some preferred specific embodiments of the present invention, in the said catalytic oxidation reaction, the amount of monoamine oxidase is 100 mg - 10 g per liter; the amount of catalase used is 10 mg - 300 mg; the said aqueous solution is a buffer solution with a pH range of 5.0 - 8.0, the said oxidation reaction is carried out under oscillating or stirring conditions; the reaction temperature of the said oxidation reaction is 20 - 60 °C.

[0049] In some more preferred specific embodiments of the present invention, the pH of the reaction solution is 7.0 - 8.0.

[0050] In some preferred specific embodiments of the present invention, in the reaction system, the concentration of 6,6 - dimethyl - 3 - azabicyclo[3.1.0]hexane is 5 - 200 g / L.

[0051] In some preferred specific embodiments of the present invention, during the reaction, O 2 is continuously introduced.

[0052] In some more preferred specific embodiments of the present invention, the reaction is carried out at 20 - 45 °C.

[0053] In some further more preferred specific embodiments of the present invention, the reaction is carried out at 25 - 30 °C.

[0054] In some more preferred specific embodiments of the present invention, 1000 g of crude enzyme solution prepared from 25 g of wet bacterial cells can catalyze 100 g of the compound shown in Formula II to the compound shown in Formula III.

[0055] The compound shown in Formula III can be converted into the compound shown in Formula I by the method of Examples 3-5 of WO2008082508, and the reaction formula is as follows (Reaction Formula 2):

[0056]

[0057] The compound shown in Formula II, which is the substrate of the asymmetric catalytic oxidation reaction of the present invention, can be prepared by the method of Example 1 of WO2008082508.

[0058] Compared with the prior art, the present invention has the following advantages:

[0059] 1. The monoamine oxidase of the present invention is derived from Penicillium, and has high catalytic activity and strong chiral selectivity;

[0060] 2. The present invention solves the defect of low production capacity in the prior art, has high catalytic efficiency, high yield and high ee value, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 It is a diagram of the recombinant expression vector of the monoamine oxidase of the present invention.

[0062] Figure 2 It is an agarose gel electrophoresis diagram of PCR verification of the recombinant expression vector of the monoamine oxidase of the present invention using the T7 / T7t universal primer pair. Among them, lanes 1 and 2 are the verification diagrams of pET28a-His-PMAON1; lanes 3 and 4 are the verification diagrams of pET28a-His-PMAON2; lanes 5 and 6 are the verification diagrams of pET28a-His-PMAON3, and lane 7 is Maker.

[0063] Figure 3 is a polyacrylamide gel electrophoresis diagram of the crude monoamine oxidase solution of the present invention. Among them Figure 3A is the PMAON1 protease; Figure 3B is the PMAON2 protease; Figure 3C is the PMAON3 protease.

[0064] Figure 4A 、 4B and 4C are respectively the gas chromatograms of the catalytic substrate reactions of the crude enzyme solutions of BL21PMAON1, BL21PMAON2, and BL21PMAON3 (product: about 11.1 min, substrate: about 11.5-11.7 min). Figure 5A is the detection result of adding 1 g / L substrate to the crude enzyme solution of BL21PMAON3 and reacting for 2 h, Figure 5B is the detection result of adding 2 g / L substrate to the crude enzyme solution of BL21PMAON3 and reacting for 4 h, Figure 5CThe detection results of adding 5 g / L of the substrate to the crude enzyme solution of BL21PMAON3 and reacting for 6 h are shown. (Product: approximately 10.9 min, Substrate: approximately 11.2 - 11.5 min)

[0065] Figure 6A The detection results after adding 1 g / L of the substrate to the crude enzyme solution of BL21PMAON3 for the first time and reacting for 2 h Figure 6B The detection results after adding 1 g / L of the substrate to the crude enzyme solution of BL21PMAON3 for the second time and reacting for 2 h Figure 6C The detection results after adding 1 g / L of the substrate to the crude enzyme solution of BL21PMAON3 for the third time and reacting for 3 h Figure 6D The detection results after adding 1 g / L of the substrate to the crude enzyme solution of BL21PMAON3 for the fourth time and reacting for 3 h Figure 6E The detection results after adding 1 g / L of the substrate to the crude enzyme solution of BL21PMAON3 for the fifth time and reacting for 3 h. (Product: approximately 10.8 - 10.9 min, Substrate: approximately 11.1 min) Detailed implementation manners

[0066] After extensive and in - depth research, the present inventors obtained three monoamine oxidases from Penicillium sp. ART, Penicillium polonicum, and Penicillium brasilianum for the first time. Using these three monoamine oxidases to catalyze prochiral compounds in asymmetric oxidation reactions has the characteristics of low cost and high yield, is suitable for industrial applications, and solves the problems of low reaction efficiency, low ee value, high cost, and unsuitability for industrial applications in existing methods. On this basis, the present invention was completed.

[0067] The inventors selected 21 kinds of fungi from the collected soil fungus sample library (including 4 strains of Aspergillus niger, 1 strain of Aspergillus terreus, and 16 strains of Penicillium). Single colonies were picked and cultured in 100 mL of liquid YPD medium, and then ultrasonically disrupted. Using 6,6-dimethyl-3-azabicyclo[3.1.0]hexane (the compound shown in Formula II) as the substrate, the monoamine oxidase enzyme activity was detected. Among them, the substrate conversion activity was detected in 5 strains, which were identified as Aspergillus terreus, Aspergillus niger, Penicillium sp. ART, Penicillium polonicum, and Penicillium brasilianum respectively. According to the previously obtained genome sequencing results, the cDNA sequences of the above three active Penicillium monoamine oxidases (PMAON) were: Seq ID No: 1, Seq ID No: 3, Seq ID No: 5, and the amino acid sequences were: Seq ID No: 2, Seq ID No: 4, Seq ID No: 6.

[0068] The cDNA sequence of the monoamine oxidase (PMAON) derived from Penicillium sp. ART is as described in Seq ID No: 1:

[0069] atgacctctcgcgacggatttcaatggacaccacaggctggattggcccaaggggtgccatcaattggcgtaatctcgccatcaaccaacatttcttcaa

[0070] gcaatgggtcttgggatgtagtagttgttggaggtggctactccggtctcacagccacccgtgacctttgtgtcgcagggttcaaggttctcctcgttgag

[0071] gcgcgcgaccgtatcggtggccgatcgtggtcatcaaacattgaaggatacccatacgagatgggaggaacttgggtacactggggacagcctcatg

[0072] tctggcgtgagatatcgcgttaccaaatgcgcactgagttggaggaatcatttgacttcagtcgaggtgtgaaccactttcaactgcgcactgacaatgg

[0073] agatactgtaatgagccacgaagacgaagacgctcttttggctggcgcattggagaaatttgtaaacgtcgacggggatatggggcgcaaggtggtgc

[0074] cctatgcgcacgactccttccatgtcccagaggcacgcaaatatgatgagatgtcttgcaaagatcgcctactacagatcaacttatcccttacaccgaac

[0075] gagcgcgccggattggaaagtttcattctactctgcagctgcggcacgctggaaacaacaagcttttttgaatttctacattggtgggcattgtgcggctat

[0076] acctatcgtggctgtatggattctttaatctcatacaagttcaagggaggccagtcgtcatttgctatcaagtttttccaagaggcgctggctactgggaacc

[0077] tttcctatgtgtttagtaacccggtcaagtctatcacggaccaaggtgacagagttaccttgagcactcgaaatggtcaggattataccgcaaagcgcttg

[0078] gtctctaccattcctctcaacgtcctcaattcgatttcattcagcccacctttggattatcaccgagcctcggctacaaacattggccatgtcaaccagtgtgt

[0079] gaaggtccatgctgaggtttcaaacagagatatgcgatcctggactggaatttcatatccattcaataaattaatgtatgctattggtgatggtaccacgcct

[0080] gcgggaaatacccacatagtctgctttggagggaacaacaaccatatccaaccagaggagaatgtggacgagacgaagggagctgttgagagccta

[0081] gctcctggaaacatggacattcaaagattggttttccataactggtccaaggacgagtttgcgaaaggtgcttggttcttctcatcgccgaagttcttatcta

[0082] catctttggattcgttgcgcgcccgccatggaaatgtggtcttcgctaactcagattgggctgtcggctggcgtagcttcattgatggtgccattgaagaa

[0083] ggaactagagccgcgatgacagtgaaggaagaactggggcctatcgtcccgccccgggtccgtctgtag

[0084] The amino acid sequence of the monoamine oxidase derived from Penicillium sp. ART is as described in Seq ID No: 2:

[0085] MTSRDGFQWTPQAGLAQGVPSIGVISPSTNISSSNGSWDVVVVGGGYSGLTATRDLCVAGF

[0086] KVLLVEARDRIGGRSWSSNIEGYPYEMGGTWVHWGQPHVWREISRYQMRTELEESFDFSR

[0087] GVNHFQLRTDNGDTVMSHEDEDALLAGALEKFVNVDGDMGRKVVPYAHDSFHVPEARK

[0088] YDEMSCKDRLLQINLSLTPNERAGLESFILLCSCGTLETTSFFEFLHWWALCGYTYRGCMDS

[0089] LISYKFKGGQSSFAIKFFQEALATGNLSYVFSNPVKSITDQGDRVTLSTRNGQDYTAKRLVS

[0090] TIPLNVLNSISFSPPLDYHRASATNIGHVNQCVKVHAEVSNRDMRSWTGISYPFNKLMYAIG

[0091] DGTTPAGNTHIVCFGGNNNHIQPEENVDETKGAVESLAPGNMDIQRLVFHNWSKDEFAKG

[0092] AWFFSSPKFLSTSLDSLRARHGNVVFANSDWAVGWRSFIDGAIEEGTRAAMTVKEELGPIV

[0093] PPRVRL

[0094] The cDNA sequence of the monoamine oxidase derived from Penicillium polonicum is shown as Seq ID No: 3:

[0095] atgacttcccgcgacggtttccaatggacgcctgaaactggccttgcacaaggtgtgccttcaattggtgtcatttcgcctcctacaaaaatctcgacttca

[0096] tcagcaacgtatgacgttatcgttgtcggcggcggatatgctggtctcacggccactcgtgacttgactattgccggtcttcgtgttctcctggttgaggct

[0097] cgtgatcggattggtggacgttcttggtcctccaacattggcgactatccgtttgagatgggtggaacttgggtgcactggggtcagcctcacgtttggcg

[0098] cgagatatcgcgatacaacatgcgaaccgagctggaggaatcttttgatttctctcgcggagtaaaccacttccagttgcgcacaagccagggtgcttca

[0099] ataatgagccacgaagaggaggacactattcttgccgctggcctcgagagatttgtcaatgtcgatggtgatatgggcaggaaaatcattcctttccctca

[0100] cgacgcattccatgtccctgaggctcgccgctacgatgaaatgtccgcacaggaccgggtaaacgagattgcttcgtctcttaccccgaacgaacgtg

[0101] catcccttgagagttttatccttctttgcagctgcggaacattggaaacaaccagcttccttgaattacttcattggtgggctctatgtggctactcctatcgtg

[0102] gctgcatggcctccctgatctcgtacaagttcaagggaggtcaatcgaccttcgcaatcaagttcttcatagaggcactcgccactggaagactctcgtat

[0103] gtgttcaatagccctgtcagctcaatcagtgatcgaggtgataaagtcactcttaccactcgtgatggtcatcagtataccggtgctcgtctggtctctacta

[0104] ttcctctcaatgttcttaattccgtatctttcgatcctcctctgggcacccagcgagcgacagccaccaatatcggtcatgtcaaccaatgtgtgaaggtag

[0105] acgcggaaatttccagcaaggacatgcgttcgtggactggagtctcctatcccttcaacaagctcatgtatggcattggtgatggaactacaccctcagg

[0106] aaacacccacatcgtttgctttggcggttctagcaaccacattcatccggaggaagatattaacgaaaccaagaaggccgtcgagagcatggctcccg

[0107] gtaacatggatgttaagcgattggtttttcataattggagcaaagatgagttttccaagggcgcctggttcttctcgcctccccggcttttgtccacatcattg

[0108] gatgcgatgcgggctcggcatggaaacattgtctttgcaagctctgattgggctattggctggcgtggcttcattgatggtgctattgaagagggtacacg

[0109] ggcggctatgacggtgaagggggagcttcaacctgctcctgtcccgcgttcttatctatag

[0110] The amino acid sequence of the monoamine oxidase derived from Penicillium polonicum is shown in Seq ID No: 4:

[0111] MTSRDGFQWTPETGLAQGVPSIGVISPPTKISTSSATYDVIVVGGGYAGLTATRDLTIAGLR

[0112] VLLVEARDRIGGRSWSSNIGDYPFEMGGTWVHWGQPHVWREISRYNMRTELEESFDFSRG

[0113] VNHFQLRTSQGASIMSHEEEDTILAAGLERFVNVDGDMGRKIIPFPHDAFHVPEARRYDEM

[0114] SAQDRVNEIASSLTPNERASLESFILLCSCGTLETTSFLELLHWWALCGYSYRGCMASLISYK

[0115] FKGGQSTFAIKFFIEALATGRLSYVFNSPVSSISDRGDKVTLTTRDGHQYTGARLVSTIPLNV

[0116] LNSVSFDPPLGTQRATATNIGHVNQCVKVDAEISSKDMRSWTGVSYPFNKLMYGIGDGTTP

[0117] SGNTHIVCFGGSSNHIHPEEDINETKKAVESMAPGNMDVKRLVFHNWSKDEFSKGAWFFSP

[0118] PRLLSTSLDAMRARHGNIVFASSDWAIGWRGFIDGAIEEGTRAAMTVKGELQPAPVPRSYL

[0119] The cDNA sequence of the monoamine oxidase (PMAON) derived from Penicillium brasilianum is shown as Seq ID No: 5:

[0120] atgaccaatcaaccaaaaagccgcgacggctatcagtggaccaaggccacgggcctagtccaaggagttccaactctcggtctgatccaaccaccca

[0121] ctcacttcacaagggggaagaactacgacgtaattgtaatcggaggcggatacgccggcataaccacgtgtcgggatctcacccttgccggcaacaa

[0122] cgtgctactcgtcgaagcgcgagaccgtattggtgggcggtcatggtcatctaatattgacggatacccctatgagatgggtggaacgtgggtgcattg

[0123] gcatcaaccttttgtgtggcgggagttgagacggtacgggatggtcgatcaattggagatttcgccaaggaagaatgttgagggcggggcgagagtta

[0124] ctgttaacttggatggagagatgaagcatctgacgcatgacgatgaggatgccattgtcgagtccgcattcaagaaatttatcaatgtggacggccaat

[0125] tcggccgcactgtggttcccttcccgcacgatatcgaattacacatggcgggcgtagaggagtacgaccacatgtctatggccgaccgcatggtacaa

[0126] gttgcacctcacttgacaccattggaaaagaacatgttcgagggattcctatccatcacgcacggcggaaaatgggaagaagcatctttcttcgagctgc

[0127] tgcggtggtgggcattgatggattacaatctccccaacttcatggaacttggactcatgtacaagatccgtgatggtcagtctgcgctggcgagacggat

[0128] tttcgatgaagctgtcagcacgggtagattggactatactttctctacgccggtcaaggacgtaattgatcatggacacggagttgaagttatcgctcgaa

[0129] gtggaggggaggttttcaaggcgagacgtttggtctgcactgtgccgttgaatgtcttgcataccttggccttttcgccaaagctgccgactctcaagacg

[0130] gaagcttctctagctggtcatgtcaacaaagtcgtcaaatgtcatgccgaagttgcgaatcccgagatgcgctcgctcggggcaacgaactacccccac

[0131] ggcagactcacctacactttcggtgatggaactacgcccgcgggaaatactcacctcgttgcctttggcagctcccttcctggagttcacttggatcccg

[0132] agcaggatattgaagtcactaagaaggcctttgaggccttccacccgggcatgaatgtgcagaagttggtgttccacaactggcacaaggatgaatttg

[0133] cccagggtgcgtgggagtggttgcggccgggtatgacaacaaagtacctgaaggcgctgcgagagcgacatggaaatgtgttctttgccagctcgga

[0134] ttcttcctttggatggagaggattcatcgatggagcgatggatgacggcgggagaatcgcgaagctcgttcatgatgagttgaaggacgtgcgtcccatt

[0135] gcttctaagctttga

[0136] The amino acid sequence of the monoamine oxidase derived from Penicillium brasilianum is shown in Seq ID No: 6:

[0137] MTNQPKSRDGYQWTKATGLVQGVPTLGLIQPPTHFTRGKNYDVIVIGGGYAGITTCRDLTL

[0138] AGNNVLLVEARDRIGGRSWSSNIDGYPYEMGGTWVHWHQPFVWRELRRYGMVDQLEISP

[0139] RKNVEGGARVTVNLDGEMKHLTHDDEDAIVESAFKKFINVDGQFGRTVVPFPHDIELHMA

[0140] GVEEYDHMSMADRMVQVAPHLTPLEKNMFEGFLSITHGGKWEEASFFELLRWWALMDY

[0141] NLPNFMELGLMYKIRDGQSALARRIFDEAVSTGRLDYTFSTPVKDVIDHGHGVEVIARSGG

[0142] EVFKARRLVCTVPLNVLHTLAFSPKLPTLKTEASLAGHVNKVVKCHAEVANPEMRSLGAT

[0143] NYPHGRLTYTFGDGTTPAGNTHLVAFGSSLPGVHLDPEQDIEVTKKAFEAFHPGMNVQKL

[0144] VFHNWHKDEFAQGAWEWLRPGMTTKYLKALRERHGNVFFASSDSSFGWRGFIDGAMDD

[0145] GGRIAKLVHDELKDVRPIASKL

[0146] Due to the degeneracy of codons, the nucleotide sequences encoding the amino acid sequences shown in Seq ID No: 2, 4, and 6 are not limited to only: Seq ID No: 1, 3, and 5. Those skilled in the art can obtain homologs of the nucleotide sequence by appropriately introducing substitutions, deletions, alterations, insertions, or additions. The present invention encompasses these homologs as long as the recombinant enzymes expressed therefrom maintain oxidative catalytic activity. The homologs of the above nucleotide sequences of the present invention can be prepared by substituting, deleting, altering, inserting, or adding one or more bases in Seq ID No: 1, 3, or 5 within the range of maintaining enzyme activity.

[0147] The present invention also provides a recombinant expression vector comprising the nucleotide sequence encoding monoamine oxidase of the present invention. Generally, those skilled in the art can construct it by ligating the nucleic acid sequence encoding monoamine oxidase or its mutant of the present invention to various expression vectors by conventional molecular biology methods. The expression vectors can be various conventional vectors in the art, such as commercially available plasmid vectors, etc., and plasmid pET28a(+) is preferred.

[0148] In some preferred embodiments of the present invention, the recombinant expression vector of the present invention can be prepared by a method similar to the following: The nucleic acid product obtained by DNA synthesis and the expression vector pET28a are respectively double-digested with restriction endonucleases SacⅠ and XhoⅠ to form complementary sticky ends, and then ligated with T4-DNA ligase to form recombinant expression plasmids (i.e., recombinant expression vectors) pET28a-His-PMAON1, pET28a-His-PMAON2, and pET28a-His-PMAON3 containing the three monoamine oxidase genes of the present invention.

[0149] The present invention also provides a recombinant expression transformant comprising the recombinant expression vector of the present invention. The recombinant expression transformant can be prepared by transforming the recombinant expression vector of the present invention into a host cell. The host cell can be a conventional host cell in the art as long as it can satisfy that the recombinant expression vector can stably replicate itself and the monoamine oxidase gene carried by it can be effectively expressed. In some preferred embodiments of the present invention, the host cell is Escherichia coli. In some preferred embodiments of the present invention, the Escherichia coli is selected from E. coli BL21(DE3), E. coli Rosetta(DE3), M15. In some more preferred embodiments of the present invention, the Escherichia coli is selected from E. coli BL21(DE3).

[0150] In a preferred embodiment of the present invention, the aforementioned recombinant expression vectors pET28a-His-PMAON1, pET28a-His-PMAON2, and pET28a-His-PMAON3 or their mutants are respectively transformed into E. coli BL21(DE3), and recombinant expression transformants (i.e., the genetically engineered strains of the present invention), namely E. coli BL21(DE3) / pET28a-His-PMAON1, E. coli BL21(DE3) / pET28a-His-PMAON2, and E. coli BL21(DE3) / pET28a-His-PMAON3 or their mutants, can be obtained. The transformation method can be selected from conventional methods in the art, such as electroporation, heat shock method, etc. Preferably, the heat shock method is selected for transformation.

[0151] The present invention provides a method for preparing a recombinant monoamine oxidase, including culturing the recombinant expression transformant of the present invention to induce the expression of the target protein, and obtaining the recombinant monoamine oxidase from the culture. Among them, the aforementioned recombinant expression transformant can be obtained by transforming the recombinant expression vector of the present invention into a host cell. The medium used for culturing the recombinant expression transformant can be any conventional medium in the art that can enable the transformant to grow and produce the monoamine oxidase of the present invention. For E. coli strains, LB medium (for example, peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH = 7.0) is preferably used. There are no special restrictions on the culture method and culture conditions, and appropriate selection can be made according to factors such as the host type and culture method according to the common knowledge in the art, as long as the transformant can grow and produce the monoamine oxidase of the present invention. Other specific operations for culturing the transformant can be carried out according to the conventional operations in the art.

[0152] For E. coli strains, the following method is preferably used for shake flask culture fermentation to produce the enzyme: The recombinant E. coli (recombinant expression transformant) (preferably E. coli BL21(DE3) / pET28a-His-PMAON or its mutant) involved in the present invention is inoculated into LB medium containing ampicillin and cultured. When the optical density OD 600 of the culture solution reaches 0.5 - 0.7, isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.01 - 1.0 mmol / L is added for induction, and the induction temperature is 20 - 40°C, and the culture is carried out at 160 - 240 rpm for 10 - 18 h, and the recombinant monoamine oxidase of the present invention can be highly expressed. After the culture is completed, the cells are collected, homogenized or sonicated to obtain a crude enzyme solution, and detected by polyacrylamide gel electrophoresis.

[0153] The present invention also provides the use of the above-mentioned monoamine oxidase PMAON in the asymmetric oxidation reaction of prochiral compounds. In some preferred embodiments of the present invention, a compound shown in Formula II with a final concentration of 5-200 g / L is added to 50 mL of phosphate buffer (100 mmol / L, pH 7.5), oxygen is introduced, and the mixture is stirred in a sealed container for 20-30 minutes. Then, the crude enzyme solution of the above-mentioned monoamine oxidase is added until the final protein concentration is 1 g / L, and the reaction is carried out at 30-50 °C. The whole reaction process is monitored by thin-layer gas chromatography. When the remaining substrate is less than 1%, the reaction is considered to be completed. After the reaction is completed, the pH is adjusted to 10.0, and the mixture is extracted with methyl tert-butyl ether to obtain a methyl tert-butyl ether solution of the compound shown in Formula III, which can be directly used for the next reaction. Alternatively, the solvent can be evaporated under reduced pressure to obtain the compound shown in Formula III, and the chiral GC detection analysis shows that no (1S,5R)-isomer is detected.

[0154] In the present invention, the catalyst for catalyzing the oxidation-addition reaction of chiral compounds to form chiral addition products can be the culture of the transformant of the above-mentioned recombinant monoamine oxidase, or the transformant cells obtained by centrifuging the culture or the products processed therefrom. Here, the "products processed therefrom" refers to the extracts obtained from the transformant cells, or the separated products obtained by separating and / or purifying the monoamine oxidase in the extracts, or the immobilized enzyme preparations obtained by immobilization.

[0155] On the basis of conforming to the common knowledge in the art, the above various conditions can be arbitrarily combined to obtain the embodiments of the present invention.

[0156] Unless otherwise specified, the reagents and raw materials used in the present invention are all commercially available.

[0157] In the description of the present invention, "C 1 ~C 8 alkyl" refers to an alkyl group with 1-8 carbon atoms, and "C 1 ~C 5 alkyl" refers to an alkyl group with 1-5 carbon atoms.

[0158] The following specific examples are used to further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions mentioned in the following examples are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturers. Unless otherwise stated, the percentages and parts are weight percentages and weight parts.

[0159] Example 1 Construction of recombinant expression vector:

[0160] The required primers are as follows:

[0161] Forward primer P1: tgggtcgcggatccgaattcATGACGAGCCGCGATGGCTTT

[0162] Reverse primer P2: tcgagtgcggccgcaagcttTTACAGATAGCTGCGCGGCACC

[0163] Forward primer P3: tgggtcgcggatccgaattcATGACGAGCCGTGATGGTTTTCAG

[0164] Reverse primer P4: tcgagtgcggccgcaagcttTTATTACAGGCGCACGCGCGGCGG

[0165] Forward primer P5: tgggtcgcggatccgaattcATGACCAATCAGCCGAAAAGC

[0166] Reverse primer P6: tcgagtgcggccgcaagcttTTACAGTTTGCTCGCAATCGG

[0167] The expression vector pET28a(+)(see Figure 1 ) was digested with the restriction endonucleases EcoRI and HindIII in a double digestion system: 84 μL of pET28a(+)(about 30 μg), 3 μL of EcoRI, 3 μL of HindIII. After digestion at 37 °C for 1.5 h, agarose gel electrophoresis was performed. The target band was at the position of 5.3 kb, and the digested fragment was recovered using a DNA recovery kit.

[0168] The coding sequences of three monoamine oxidases (PMAON): Seq ID No:1, Seq ID No:3, Seq ID No:5 were synthesized by Suzhou GeneCreate Biotech Co., Ltd. Using the synthesized gene of Seq ID No:1 as a template, PCR amplification was performed with primers P1 / P2. After agarose gel electrophoresis, a band of about 1.5 k was separated; using the synthesized gene of Seq ID No:3 as a template for PCR amplification, PCR amplification was performed with primers P3 / P4. After agarose gel electrophoresis, a band of about 1.5 k was separated; using the synthesized gene of Seq ID No:5 as a template, PCR amplification was performed with primers P5 / P6. After agarose gel electrophoresis, a band of about 1.5 k was separated. The three groups of PCR amplification products were recovered using a DNA recovery kit.

[0169] The vector linearized by double digestion with EcoRI and HindIII was ligated with the PCR amplification product using a seamless cloning kit to construct a recombinant expression vector containing the three monoamine oxidase genes of the present invention. The ligation was carried out according to the following reaction system: 50 ng of linearized vector pET28a(+), 100 ng of the amplification product of primers P1 / P2, 5 μL of 2* seamless cloning buffer, incubated at 50 °C for 20 min to obtain the recombinant expression vector, and verified by pcr with T7 / T7t universal primer pairs (see Figure 2 ) and sequencing analysis. After verification, it was named pET28a-His-PMAON1. In the same way, recombinant expression vectors pET28a-His-PMAON2 and pET28a-His-PMAON3 were obtained.

[0170] Example 2 Obtaining of recombinant expression transformants

[0171] The aforementioned recombinant expression vectors pET28a-His-PMAON1, pET28a-His-PMAON2, and pET28a-His-PMAON3 were respectively transformed into E. coli BL21(DE3) to obtain the preferred genetic engineering strains of the present invention. The transformation method used heat shock: The competent cells were taken out from the -80 °C refrigerator and placed on ice. After adding about 200 ng - 500 ng of the expression vector, they were ice-bathed for 15 min, heat-shocked at 42 °C for 90 s, ice-bathed for 3 min, added with 500 μL of LB culture medium, incubated at 37 °C and 220 rpm for 45 min. 50 uL was taken and evenly spread on the LB solid medium containing 50 μg / ml, and cultured overnight in an inverted manner to obtain monoclonal colonies. After amplification culture, they were stored with 20% final concentration glycerol at -80 °C. After verification by sequencing analysis, they were recombinant expression transformants, named BL21PMAON1, BL21PMAON2, and BL21PMAON3.

[0172] Example 3 Shake flask culture and fermentation

[0173] The recombinant expression transformants (BL21PMAON1, BL21PMAON2, BL21PMAON3) prepared in Example 2 were respectively inoculated into 3 - 5 mL of LB liquid medium containing 50 μg / mL Kan, and cultured at 37 °C and 220 rpm for 16 h. Then, inoculated into 100 ml of LB liquid medium containing 50 μg / ml Kan according to the ratio of bacterial liquid: medium = 1:100 (v:v), and cultured at 37 °C and 220 rpm until OD 600 was approximately 0.8, and isopropyl thiogalactoside (IPTG) was added to a final concentration of 0.5 mM, and induced at 30 °C for 20 h. Each of the three transformants was fermented for 3 L.

[0174] After cultivation, the cells were collected by centrifugation at 5000 rpm at 4°C, resuspended in 50 mL of 50 mM Tris-HCl buffer at pH 8.0, sonicated, and the supernatant was collected by centrifugation, clarified with a 0.45 μm filter membrane, and then subjected to affinity chromatography using an AKTA protein purification system.

[0175] The mobile phase for affinity chromatography was: 1×NI-NTA Bind Buffer (1L): 50 mM NaH 2 PO 4 300 mM NaCl, 10 mM imidazole, adjusted to pH 8.0 with NaOH; 1×NI-NTA Wash Buffer (1L): 50 mM NaH 2 PO 4 300 mM NaCl, 20 mM imidazole, adjusted to pH 8.0 with NaOH; 1×NI-NTA Elution Buffer (1L): 50 mM NaH 2 PO 4 300 mM NaCl, 200 mM imidazole, adjusted to pH 8.0 with NaOH.

[0176] 1×NI-NTA Bind Buffer was used for equilibration, 1×NI-NTA Wash Buffer was used for washing away impurities, and 1×NI-NTA Elution Buffer was used for elution. The results of SDS-PAGE detection of the eluates of the three enzymes are shown in Figure 3, where Figure 3A is PMAON1 protease (corresponding amino acid sequence Seq ID No: 2); Figure 3B is PMAON2 protease (corresponding amino acid sequence Seq ID No: 4); Figure 3C is PMAON3 protease (corresponding amino acid sequence Seq ID No: 6).

[0177] The crude enzyme solutions of the three monoamine oxidases in Example 4 were used for the catalytic oxidation of the compound shown in Formula II

[0178] According to the method described in Example 3, the cells of the recombinant expression transformants (BL21PMAON1, BL21PMAON2, BL21PMAON3) were obtained, and the supernatant was sonicated. This sonicated supernatant was the crude enzyme solution of monoamine oxidase.

[0179] Since the substrate is poorly soluble in water but soluble in DMSO, mix the substrate with DMSO in equal volumes. Pipette 3 mL of crude enzyme solution of BL21PMAON1, crude enzyme solution of BL21PMAON2, and crude enzyme solution of BL21PMAON3 respectively, and add 6 μL of the substrate and DMSO mixture (the final concentration of the substrate is 1 g / L). After vortexing and mixing evenly, react in a water bath at 25 °C for 1 h. Take 200 μL of the reaction solution, add 200 μL of acetonitrile and mix to terminate the enzyme reaction, and perform gas phase detection with an injection volume of 1 μL. The results are shown in Figure 4, Figure 4A , Figure 4B , Figure 4C which are the reaction results of the crude enzyme solution of BL21PMAON1 (monoamine oxidase containing the amino acid sequence shown in Seq ID No: 2), the crude enzyme solution of BL21PMAON2 (monoamine oxidase containing the amino acid sequence shown in Seq ID No: 4), and the crude enzyme solution of BL21PMAON3 (monoamine oxidase containing the amino acid sequence shown in Seq ID No: 6) respectively. It can be clearly seen from these figures that the oxidation catalytic reaction effect of the crude enzyme solution of BL21PMAON3 is the best.

[0180] Example 5 Catalytic oxidation of the compound shown in Formula II using the crude enzyme solution of BL21PMAON3

[0181] First, optimize the substrate addition concentration: add 1 g / L, 2 g / L, and 5 g / L to the crude enzyme solution of BL21PMAON3 respectively, and perform gas phase detection after reacting for 2 - 6 h, as shown in Figure 5A , 5B and 5C. Figure 5A is the detection result of adding 1 g / L substrate to the crude enzyme solution of BL21PMAON3 and reacting for 2 h, Figure 5B is the detection result of adding 2 g / L substrate to the crude enzyme solution of BL21PMAON3 and reacting for 4 h, Figure 5C is the detection result of adding 5 g / L substrate to the crude enzyme solution of BL21PMAON3 and reacting for 6 h. According to the detection results, it is inferred that the greater the substrate concentration, the stronger the inhibitory effect on the enzyme catalytic reaction. During the reaction process, the substrate concentration should not be too high. The substrate distillation addition method is used to keep the substrate at a low concentration level for the catalytic oxidation of the compound shown in Formula II.

[0182] Therefore, according to Figures 5A - 5C the detection results, 1 g / L substrate distillation addition is preferred here. Take 10 mL of the crude enzyme solution, add 1 g / L substrate for the first time, and then sample and detect every 2 - 3 h. When the substrate is almost completely reacted, add 1 g / L substrate again until the 1 g / L substrate added for the fifth time is completely reacted and the distillation addition ends. Figure 6A is the detection result 2 h after adding 1 g / L substrate for the first time, Figure 6B is the detection result 3 h after adding 1 g / L substrate for the second time, Figure 6CThe detection results 3 hours after the third addition of 1 g / L substrate, Figure 6D The detection results 3 hours after the fourth addition of 1 g / L substrate, Figure 6E The detection results 3 hours after the fifth addition of 1 g / L substrate. According to the detection results, after each addition of substrate, the substrate is continuously consumed and the product continuously increases. However, after the fifth addition of substrate, the increment amplitude of the product begins to decrease. Therefore, the distillation addition method can effectively increase the feeding amount of the catalytic reaction substrate of the compound shown in II.

[0183] Example 6 Fermentation culture of recombinant expression transformant BL21PMAON3 in a fermenter

[0184] The specific process of fermenting and culturing the recombinant expression transformant BL21PMAON3 in a fermenter is as follows:

[0185] (1) Absorb the bacteria preserved in the glycerol tube into the shake flask according to an inoculation amount of 0.1%. The shake flask medium is LB liquid medium containing 50 μg / mL Kan. The shake flask is placed in a shaker at 37°C and 220 rpm for a culture period of 7 - 10 h. When the OD 600 reaches 4 - 9, inoculate the above shake flask seed liquid into the seed tank according to an inoculation amount of 1%.

[0186] (2) The seed tank medium is LB liquid medium containing 50 μg / mL Kan. The culture temperature of the seed tank is 37 ± 2°C, the tank pressure is 0.02 - 0.06 Mpa, the rotation speed is 200 - 400 rpm, and the flow rate is 0.5 - 1.0 vvm. When the OD of the seed tank 600 reaches 2 - 4, transfer it to the fermenter for culture according to an inoculation amount of 10%.

[0187] (3) The fermenter medium is LB liquid medium containing 50 μg / mL Kan. The initial culture temperature of the fermenter is 37 ± 2°C, the tank pressure is 0.02 - 0.06 Mpa, the rotation speed is 200 - 400 rpm, and the flow rate is 0.5 - 1.0 vvm.

[0188] (4) During the fermentation culture for 4 - 8 hours, when the pH drops to 6.7 - 7.0, add ammonia water to control the pH at 6.7 - 7.0; during the culture for 8 - 12 hours, when the dissolved oxygen rises rapidly, add a glucose solution to control the dissolved oxygen at 20 - 50%; during the culture for 12 - 16 hours, when the OD 600 reaches 40 - 90, add an α-lactose solution for induction culture at a ratio of 1%, and the temperature is lowered to 35 ± 2°C for culture during induction.

[0189] (5) When the fermentation culture reaches about 24 h and the OD of the bacteria 600 reaches 100 - 150, end the culture.

[0190] The specific process of the crude enzyme solution of BL21PMAON3 is as follows:

[0191] The fermentation broth obtained from the above fermenter was centrifuged to obtain wet cells. The wet cells were dissolved in an aqueous solution of sodium dihydrogen phosphate at 50 mM with a pH value of 7.4 - 7.8. 25 g of wet cells were added to 1 L of the aqueous sodium dihydrogen phosphate solution. Resuspension and cell wall breaking treatment were carried out by adding 3 - 5 g / L of lysozyme according to 25 g / L of cells. The cell wall breaking temperature was room temperature. After uniformly stirring for 4 - 10 h, the obtained resuspended cell wall breaking solution was the crude enzyme solution.

[0192] Example 7 Using the crude enzyme solution of the monoamine oxidase in Example 6 to catalytically oxidize the compound shown in Formula II to obtain the compound shown in Formula III and Formula IV.

[0193]

[0194] In a 2 L four-necked reaction flask, about 1000 ml of the crude enzyme solution of monoamine oxidase prepared according to the method of Example 6 was added, 0.1 g of defoamer and 10 g of catalase were added. The temperature of the reaction solution was 25 - 27 °C. A solution prepared from 100 g (0.9611 mol) of sodium bisulfite, 500 g of water and 100 g (0.8994 mol) of the compound shown in Formula II was added dropwise. During the addition process, oxygen was introduced into the reaction flask and 15% sodium hydroxide was added dropwise to control the pH of the reaction solution at 7.3 - 7.9. After the addition of the substrate preparation solution was completed, the reaction was continued for about 30 minutes to obtain an adduct product of the compound shown in Formula III and sodium bisulfite (i.e., the compound shown in Formula IV). The reaction solution was directly used for the next reaction without treatment.

[0195] Example 8 Preparation of the hydrochloride salt of the compound shown in Formula I

[0196]

[0197] At room temperature in a reaction flask, the reaction solution containing the compound shown in Formula IV (0.8994 mol) obtained in Example 7 was added, and the reaction temperature was controlled at 5 - 15 °C. Aqueous solution prepared by adding 53 g of sodium cyanide (1.0816 mol) to 200 g was added dropwise, and the reaction temperature was 10 - 20 °C. After the addition was completed, the reaction was continued with stirring for 1 hour. 800 mL of methyl tert-butyl ether was added, and the mixture was stirred for 5 - 10 minutes. It was allowed to stand and layer. The organic phase was washed with 300 mL of 20% aqueous NaCl solution, dried with 50 g of anhydrous sodium sulfate for 1 - 2 hours, and then filtered.

[0198] Cool the organic phase by 5 - 10 °C, and add dropwise 420 g of 25% hydrogen chloride methanol (2.8767 mol) solution while controlling the temperature below 30 °C during the addition. After the addition is complete, gradually heat to reflux and reflux for 5 hours. When the reaction ends, distill off the recovered solvent under reduced pressure. Add 800 mL of methyl tert-butyl ether to the residue. Cool to 0 - 5 °C, and add 500 mL of water with stirring to make it clear. Adjust the pH to 9 - 10 with an aqueous NaOH solution and continue stirring for 15 minutes. Let it stand and separate the layers. Extract the aqueous phase with 400 mL of methyl tert-butyl ether. Combine the organic phases and wash with 200 ml of 20% aqueous NaCl solution. Add 50 g of anhydrous sodium sulfate to dry the organic phase, and distill off the solvent under reduced pressure for recovery.

[0199] Add 200 mL of isopropanol to the residue to dissolve it. Cool to 0 - 10 °C, and add dropwise 110 g of 25% hydrogen chloride methanol solution until the pH of the reaction mixture is 4 - 5. After the addition is complete, continue stirring for 30 minutes. Add dropwise 600 mL of methyl tert-butyl ether, and a white solid gradually precipitates during the addition. After the addition is complete, keep the temperature for crystal precipitation for 4 hours. Filter, wash the filter cake with a small amount of methyl tert-butyl ether, and dry in vacuo at 40 °C to obtain 142 g of the hydrochloride of the compound shown in formula I. Based on the compound shown in formula II, the total yield is 77% and the ee value is 100%.

[0200] All the documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. Use of monoamine oxidase in the asymmetric oxidation reaction of prochiral compounds, wherein the amino acid sequence of the monoamine oxidase is the amino acid sequence shown in SEQ ID No: 2, SEQ ID No: 4 or SEQ ID No: 6, The prochiral compound is the compound shown in the following formula II, which is oxidized to form the compound shown in formula III, 。 2. The use according to claim 1, characterized in that, The compound shown by Formula II forms a compound shown by Formula III under the catalysis of the monoamine oxidase in an aqueous solution with a pH value of 7.0 - 8.0 in the presence of catalase, O 2 and sodium bisulfite, and the reaction formula is as follows: 。 3. The use according to claim 2, characterized in that, the pH of the reaction solution is 7.3 to 7.9, and / or in the reaction system, the concentration of the prochiral compound is 5 - 200 g / L, and / or the reaction temperature is 20 to 45 °C.

4. The use according to claim 1, characterized in that, the amino acid sequence of the monoamine oxidase is the amino acid sequence shown in SeqID No:

6.

5. The use according to claim 1, characterized in that, the nucleotide sequence of the gene encoding the amino acid sequence shown in SEQ ID No: 2, SEQ ID No: 4 or SEQ ID No: 6 is the nucleotide sequence shown in SEQ ID No: 1, SEQ ID No: 3 or SEQ ID No:

5.

6. A method for stereoselectively preparing 6,6-dimethyl-3-azabicyclo[3.1.0]hexene compound, characterized in that, the method comprises the following steps: In an aqueous solution with a pH value of 7.0 - 8.0, in the presence of catalase, O 2 and sodium bisulfite, 6,6-dimethyl-3-azabicyclo[3.1.0]hexane is catalytically oxidized by monoamine oxidase to produce a 6,6-dimethyl-3-azabicyclo[3.1.0]hexene compound, and the reaction formula is as follows: ， the amino acid sequence of the monoamine oxidase is the amino acid sequence shown in SEQ ID No: 2, SEQ ID No: 4 or SEQ ID No:

6.

7. The method according to claim 6, characterized in that, the amino acid sequence of the monoamine oxidase is the amino acid sequence shown in Seq ID No:

6.

8. The method according to claim 6, characterized in that, the pH of the reaction solution is 7.3 to 7.9, and / or in the reaction system, the concentration of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane is 5 - 200 g / L, and / or the reaction temperature is 20 to 45 °C.

9. A monoamine oxidase, characterized in that, the amino acid sequence of the monoamine oxidase is the amino acid sequence shown in SEQ ID No:

2.

10. A gene encoding the monoamine oxidase according to claim 9, characterized in that, the nucleotide sequence of the gene is the nucleotide sequence shown in SEQ ID No:

1.

11. A recombinant expression vector, characterized in that, the recombinant expression vector contains the gene according to claim 10.

12. The recombinant expression vector according to claim 11, characterized in that, the plasmid used to construct the recombinant expression vector is selected from pET series vectors or pQE series vectors.

13. A recombinant expression transformant containing the recombinant expression vector according to any one of claims 11 - 12.

14. The recombinant expression transformant according to claim 13, characterized in that, the recombinant expression transformant is prepared by transforming the recombinant expression vector into a host cell.

15. The recombinant expression transformant according to claim 14, wherein, the host cell is Escherichia coli.

16. The recombinant expression transformant according to claim 15, wherein, The Escherichia coli is selected from E.coli BL21 (DE3), E.coli Rosetta(DE3) or M15.

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