CN114774382A - Method for stereoselectively preparing 6, 6-dimethyl-3-azabicyclo [3.1.0] hexene compound - Google Patents
Method for stereoselectively preparing 6, 6-dimethyl-3-azabicyclo [3.1.0] hexene compound Download PDFInfo
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Abstract
The invention provides a series of monoamine oxidases (PMAON) which are derived from penicillium and have high catalytic activity and strong chiral selectivity, and a method for stereoselectively preparing 6, 6-dimethyl-3-azabicyclo [3.1.0] hexene compounds by using the enzymes. The invention also provides a gene for coding the series of monoamine oxidases, a recombinant expression vector containing the series of genes, a recombinant expression transformant and a preparation method thereof.
Description
Technical Field
The invention relates to the technical field of medical intermediates, in particular to a biocatalysis method of a 6, 6-dimethyl-3-azabicyclo [3.1.0] hexene compound and a biocatalysis enzyme used in the method.
Background
Nemantyvir (Nirmatrelvir) is an antiviral drug developed by the company Peucervi, and is a novel coronavirus SARS-CoV-2-3CL protease inhibitor with oral activity. Nemadevir is a covalent inhibitor that binds directly to the cysteine catalytic residue (Cys145) of proteases. The compound preparation of the drug and ritonavir, which takes Paxovird (Paxlovid) as a trade name, has been approved by FDA as a first-line clinical treatment drug for treating new coronavirus, and the chemical structural formula is shown as follows:
boceprevir (Boceprevir) is a Hepatitis C Virus (HCV) protease inhibitor developed by pioneer and pauya, and is approved by FDA for treating adult chronic hepatitis c in combination with peginterferon alpha and ribavirin at 5/13 days of 2011, and has a chemical structural formula shown as follows:
it can be seen from the structural formulas of nemadefovir and boceprevir that the two have a common 6, 6-dimethyl-3-azabicyclo [3.1.0] hexane segment, and an intermediate (1R,2S,5S) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxylic acid methyl ester (hereinafter referred to as a compound of formula I) is a key intermediate for synthesizing the two products.
The preparation method of the compound of the formula I mainly comprises the following steps:
one, WO2004113295 reports a synthetic method for preparing the hydrochloride salt of the compound of formula I, the synthetic route is shown in scheme 1:
route 1 has multiple steps and long route, and the key is that the ee value of the final product is not high.
Secondly, WO2007075790 reports a synthetic method for obtaining the hydrochloride of the compound of formula I with ee value above 90%, but this method uses the diastereomer salt resolution of the final product, which is uneconomical, and the synthetic route is shown in scheme 2:
thirdly, in order to solve the above problems, WO2010008828 reports a synthetic method for stereoselectively preparing the compound of formula I by using a bio-enzyme catalysis method, wherein the synthetic route is shown in scheme 3:
Therefore, there is still a need in the art to find a new monoamine oxidase to solve the low productivity defect of the prior art.
Disclosure of Invention
The invention provides a series of monoamine oxidases from penicillium, which have high catalytic activity and strong hand shape selectivity, and the concentration of reaction substrates can reach 200g/L, thereby solving the problems of low yield and unsuitability for industrial production when the monoamine oxidase is used for enzyme catalytic oxidation in the prior art.
In a first aspect of the present invention a group of monoamine oxidases derived from Penicillium species is disclosed, characterized in that said monoamine oxidases are obtained from Penicillium species (Penicillium sp. art), Penicillium species (Penicillium polionium) and Penicillium species (Penicillium brasilinum).
Preferably, the amino acid sequence of the monoamine oxidase is SEQ ID No: 2. SEQ ID No: 4. SEQ ID No: 6. in another preferred embodiment, the sequence of such a fusion protein may be SEQ ID NO: 2. the amino acid sequence of SEQ ID NO: 4. SEQ ID NO: 6 and natural or artificial protein sequence with homology higher than 80% with the sequence.
Preferably, the mRNA sequence of monoamine oxidase is SEQ ID No: 1. SEQ ID No: 3. SEQ ID No: 5 and mRNA sequences with homology higher than 80% with the above sequences.
The monoamine oxidase sequences shown in SEQ ID No.2, 4, 6 were aligned with other known monoamine oxidases and showed identity of 67.7%, 63.2% and 51.4% respectively with the monoamine oxidase from Aspergillus terreus (a. niger) and less than 60% with the monoamine oxidase from Aspergillus niger (a. niger).
In a second aspect of the invention, nucleic acids encoding said monoamine oxidase are disclosed.
In a third aspect of the invention, recombinant expression vectors comprising said nucleic acids are disclosed.
Preferably, the plasmid used to construct the recombinant expression vector is pET28 a.
In a fourth aspect of the invention, recombinant expression transformants comprising said recombinant expression vector are disclosed.
The recombinant expression transformant is characterized in that the host cell is preferably Escherichia coli.
More preferably, the host cell is e.coli BL21(DE 3).
In a fifth aspect of the present invention, there is provided the use of monoamine oxidase (PMAON) according to the present invention in an asymmetric catalytic oxidation reaction, i.e. its use in catalyzing the asymmetric oxidation of a compound of formula II to produce the desired product, a compound of formula III, as shown below (formula 1):
in the catalytic oxidation reaction, the monoamine oxidase is 100 mg-10 g per liter; the dosage of catalase is 10 mg-300 mg; the aqueous solution is a buffer solution with the pH value of 5.0-8.0, and the oxidation reaction is carried out under the condition of oscillation or stirring; the reaction temperature of the oxidation reaction is 20-60 ℃.
The compound of formula III can be converted to the compound of formula I by the method of WO2008082508, examples 3-5, as shown below (equation 2):
the asymmetric catalytic oxidation reaction substrate of the formula II compound of the present invention can be prepared by the method of WO2008082508, example 1.
Compared with the prior art, the invention has the following advantages:
1. the invention is derived from monoamine oxidase in penicillium, has high catalytic activity and strong hand type selectivity;
2. the invention solves the defect of low productivity in the prior art, the concentration of the reaction substrate can reach 200g/L, and the method is suitable for industrial production.
Drawings
FIG. 1 is a diagram of the recombinant expression vector of monoamine oxidase of the present invention in example 2.
FIG. 2 is an agarose gel electrophoresis of the monoamine oxidase gene PCR product of the present invention in example 3.
FIG. 3 is a polyacrylamide gel electrophoresis chart of the crude enzyme solution of monoamine oxidase of the present invention in example 4.
FIG. 4 is a gas chromatogram showing substrate conversion activity of crude monoamine oxidase of the present invention in example 5 (product: 10.891min, substrate: 11.187 min).
Detailed Description
The invention is further illustrated by the following examples and figures, without however restricting the scope of protection of the invention to the following examples.
Example 1
Selecting 21 fungi (4 strains of Aspergillus niger, 1 strain of Aspergillus terreus and 16 strains of Penicillium) from a collected soil fungus sample library, selecting a single strain, culturing the single strain in 100mL of liquid YPD culture medium, ultrasonically breaking the wall, and detecting the activity of monoamine oxidase by using 6, 6-dimethyl-3-azabicyclo [3.1.0] hexane (a compound shown in a formula II) as a substrate, wherein 5 strains detect the substrate conversion activity and are respectively Aspergillus terreus, Aspergillus niger, Penicillium sp.ART, Penicillium policum and Penicillium bresillinium through identification. According to the genome sequencing results obtained previously, the mRNA (cDNA) sequences of the three active penicillium monoamine oxidases (PMAON) are respectively as follows: seq ID No: 1. 3, 5, and translated into protein sequences of: seq ID No: 2. 4 and 6.
Example 2
The invention also provides a recombinant expression vector containing the nucleotide sequence for coding monoamine oxidase. The nucleic acid sequences encoding the monoamine oxidases or mutants thereof of the present invention may be constructed by linking the sequences to various expression vectors by conventional methods of molecular biology. The expression vector may be any vector conventionally used in the art, such as a commercially available plasmid vector, and the like, and preferably the plasmid pET28 a.
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 expression vector pET28a are respectively double-digested by restriction endonucleases SacI and XhoI to form complementary cohesive ends, and the complementary cohesive ends are connected by T4-DNA ligase to form recombinant expression plasmid pET28a-His-PMAON recombinant expression plasmid containing three monoamine oxidase genes.
Example 3
The invention also provides a recombinant expression transformant containing the recombinant expression vector. Can be prepared by transforming a recombinant expression vector of the present invention into a host cell. The host cell may be a host cell that is conventional in the art, as long as it is sufficient that the recombinant expression vector can stably replicate itself and the monoamine oxidase gene of the present invention carried therein can be efficiently expressed. Coli (e.coli) is preferred in the present invention, and e.coli BL21(DE3) is more preferred.
The genetic engineering strain preferred in the present invention, i.e., e.coli BL21(DE3)/pET28a-His-PMAON or a mutant thereof, can be obtained by transforming the aforementioned recombinant expression plasmid pET28a-His-PMAON or a mutant thereof into e.coli BL21(DE3) (see fig. 2 for results). The transformation method can be selected from conventional methods in the art, such as electrotransformation method, heat shock method, etc., preferably heat shock method.
Example 4
The invention provides a preparation method of recombinant monoamine oxidase, which comprises the steps of culturing the recombinant expression transformant and obtaining the recombinant monoamine oxidase from the culture. Wherein, the recombinant expression transformant is obtained by transforming the recombinant expression vector of the present invention into a host cell, as described above. The medium used in culturing the recombinant expression transformant may be any medium that is conventional in the art and that allows the transformant to grow and produce the monoamine oxidase of the present invention, and for the E.coli strain, LB medium (peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, pH 7.0) is preferred. The culture method and culture conditions are not particularly limited, and may be appropriately selected according to the ordinary knowledge in the art depending on the type of host, the culture method, and the like, as long as the transformant can grow and produce the monoamine oxidase of the present invention. Other specific procedures for culturing transformants may be performed as is conventional in the art.
For E.coli strains, the following method is preferably used for producing the enzyme by shake flask culture fermentation: inoculating the recombinant Escherichia coli (preferably E.coli BL21(DE3)/pET28a-His-PMAON or mutant thereof) related to the invention into LB culture medium containing the aminobenzomycin for culture, and when the optical density OD of the culture solution is600When the concentration reaches 0.5-0.7, isopropyl-beta-D-thiogalactopyranoside (IPTG) with the final concentration of 0.01-1.0 mmol/L is added for induction, the induction temperature is 20-40 ℃, and the culture is carried out for 10-18 h at 160-240 rpm, so that the recombinant monoamine oxidase can be efficiently expressed. After the culture is finished, the thalli are collected, homogenized or sonicated to obtain a crude enzyme solution, and polyacrylamide gel electrophoresis is used for detection (the result is shown in figure 3).
Example 5 enzyme-catalyzed reaction of monoamine oxidase PMAON
Adding a compound of formula II with a final concentration of 5-200g/L into 50mL of phosphate buffer solution (100mmol/L, pH7.5), introducing oxygen, stirring in a sealed container for 20-30 minutes, adding crude enzyme solution of monoamine oxidase prepared according to example 4 to a final protein concentration of 1g/L, simultaneously adding catalase with a final concentration of 1g/L, reacting at 30-50 ℃, monitoring the whole reaction process by thin gas chromatography, and determining that the reaction is finished when the substrate residue is less than 1%. After the reaction is finished, the pH is adjusted to 10.0, and the methyl tert-butyl ether is used for extraction to obtain a methyl tert-butyl ether solution of the compound shown in the formula III, which can be directly used for the next reaction. Or distilling off the solvent under reduced pressure to obtain the compound of the formula III, and detecting and analyzing by chiral GC, wherein the (1S,5R) -isomer is not detected.
The catalyst for catalyzing the oxidation-addition reaction of a chiral compound to form a chiral addition product in the present invention may be a culture of the transformant of the recombinant monoamine oxidase produced as described above, or a transformant cell obtained by centrifuging the culture or a product processed using the same. The "processed product" herein refers to an extract obtained from the transformed cells, or an isolated product obtained by isolating and/or purifying monoamine oxidase in the extract, or an immobilized enzyme preparation obtained by immobilization.
EXAMPLE 6 preparation of the hydrochloride salt of the Compound of formula I
To 670mL of 10% sodium bisulfite solution in a reaction flask at room temperature, 500mL of methyl t-butyl ether solution containing 70g of the compound of formula III obtained in example 5 was added dropwise, and the reaction temperature was controlled to be lower than 35 ℃. After dropping, stirring was continued at room temperature for 1 hour. After standing and separation, the aqueous phase was washed with 50mL of methyl t-butyl ether and used directly in the next reaction.
The aqueous phase was transferred to a reaction flask and 35g of sodium cyanide were added in portions, the reaction temperature being controlled below 25 ℃. After the addition was complete, stirring was continued at room temperature for 1 hour. 300mL of methyl tert-butyl ether was added and stirred for 5 minutes. After standing and separation, the organic phase was washed twice with 100mL of 20% NaCl aqueous solution and used directly in the next reaction.
The organic phase was added dropwise to 350mL of 28% methanolic hydrogen chloride solution at room temperature, the reaction temperature being controlled below 30 ℃. After dropping, the mixture was heated to reflux for 5 hours. The solvent was distilled off under reduced pressure, and 500mL of methyl t-butyl ether was added to the residue. Cooled to 0-5 deg.C, and 500mL water is added with stirring to clarify. The pH was adjusted to 9-10 with aqueous NaOH and stirring was continued for 15 minutes. The mixture was allowed to stand, the layers were separated, and the aqueous phase was extracted twice with 200mL of methyl tert-butyl ether. The organic phases were combined and washed with 100mL of 20% aqueous NaCl solution. The organic phase was dried over anhydrous sodium sulfate, and the solvent was recovered by distillation under reduced pressure.
To the residue was added 70mL of isopropyl alcohol to dissolve. Cooling to 0-5 deg.C, and adding 25-28% methanol solution of hydrogen chloride until the pH of the reaction mixture is 4-5. After dropping, stirring was continued for 30 minutes. 200mL of methyl tert-butyl ether was added dropwise, and a white solid gradually precipitated during the addition. After dripping, keeping the temperature for crystallization for 4 hours. Filtering, leaching the filter cake with a small amount of methyl tert-butyl ether, and drying in vacuum at 40 ℃ to obtain 95g of a white solid compound hydrochloride of the formula I with an ee value of 100%.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.
Sequence listing
<110> Yangzhou Union Australia biomedical Co Ltd
Guangdong province Zollin pharmaceuticals Co Ltd
<120> method for stereoselectively producing 6, 6-dimethyl-3-azabicyclo [3.1.0] hexene compound
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<213> Artificial Sequence (Artificial Sequence)
<400> 4
Met Thr Ser Ala Ala Gly Pro Gly Thr Thr Pro Gly Thr Gly Leu Ala
1 5 10 15
Gly Gly Val Pro Ser Ile Gly Val Ile Ser Pro Pro Thr Leu Ile Ser
20 25 30
Thr Ser Ser Ala Thr Thr Ala Val Ile Val Val Gly Gly Gly Thr Ala
35 40 45
Gly Leu Thr Ala Thr Ala Ala Leu Thr Ile Ala Gly Leu Ala Val Leu
50 55 60
Leu Val Gly Ala Ala Ala Ala Ile Gly Gly Ala Ser Thr Ser Ser Ala
65 70 75 80
Ile Gly Ala Thr Pro Pro Gly Met Gly Gly Thr Thr Val His Thr Gly
85 90 95
Gly Pro His Val Thr Ala Gly Ile Ser Ala Thr Ala Met Ala Thr Gly
100 105 110
Leu Gly Gly Ser Pro Ala Pro Ser Ala Gly Val Ala His Pro Gly Leu
115 120 125
Ala Thr Ser Gly Gly Ala Ser Ile Met Ser His Gly Gly Gly Ala Thr
130 135 140
Ile Leu Ala Ala Gly Leu Gly Ala Pro Val Ala Val Ala Gly Ala Met
145 150 155 160
Gly Ala Leu Ile Ile Pro Pro Pro His Ala Ala Pro His Val Pro Gly
165 170 175
Ala Ala Ala Thr Ala Gly Met Ser Ala Gly Ala Ala Val Ala Gly Ile
180 185 190
Ala Ser Ser Leu Thr Pro Ala Gly Ala Ala Ser Leu Gly Ser Pro Ile
195 200 205
Leu Leu Cys Ser Cys Gly Thr Leu Gly Thr Thr Ser Pro Leu Gly Leu
210 215 220
Leu His Thr Thr Ala Leu Cys Gly Thr Ser Thr Ala Gly Cys Met Ala
225 230 235 240
Ser Leu Ile Ser Thr Leu Pro Leu Gly Gly Gly Ser Thr Pro Ala Ile
245 250 255
Leu Pro Pro Ile Gly Ala Leu Ala Thr Gly Ala Leu Ser Thr Val Pro
260 265 270
Ala Ser Pro Val Ser Ser Ile Ser Ala Ala Gly Ala Leu Val Thr Leu
275 280 285
Thr Thr Ala Ala Gly His Gly Thr Thr Gly Ala Ala Leu Val Ser Thr
290 295 300
Ile Pro Leu Ala Val Leu Ala Ser Val Ser Pro Ala Pro Pro Leu Gly
305 310 315 320
Thr Gly Ala Ala Thr Ala Thr Ala Ile Gly His Val Ala Gly Cys Val
325 330 335
Leu Val Ala Ala Gly Ile Ser Ser Leu Ala Met Ala Ser Thr Thr Gly
340 345 350
Val Ser Thr Pro Pro Ala Leu Leu Met Thr Gly Ile Gly Ala Gly Thr
355 360 365
Thr Pro Ser Gly Ala Thr His Ile Val Cys Pro Gly Gly Ser Ser Ala
370 375 380
His Ile His Pro Gly Gly Ala Ile Ala Gly Thr Leu Leu Ala Val Gly
385 390 395 400
Ser Met Ala Pro Gly Ala Met Ala Val Leu Ala Leu Val Pro His Ala
405 410 415
Thr Ser Leu Ala Gly Pro Ser Leu Gly Ala Thr Pro Pro Ser Pro Pro
420 425 430
Ala Leu Leu Ser Thr Ser Leu Ala Ala Met Ala Ala Ala His Gly Ala
435 440 445
Ile Val Pro Ala Ser Ser Ala Thr Ala Ile Gly Thr Ala Gly Pro Ile
450 455 460
Ala Gly Ala Ile Gly Gly Gly Thr Ala Ala Ala Met Thr Val Leu Gly
465 470 475 480
Gly Leu Gly Pro Ala Pro Val Pro Ala Ser Thr Leu
485 490
<210> 5
<211> 1500
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atgaccaatc aaccaaaaag ccgcgacggc tatcagtgga ccaaggccac gggcctagtc 60
caaggagttc caactctcgg tctgatccaa ccacccactc acttcacaag ggggaagaac 120
tacgacgtaa ttgtaatcgg aggcggatac gccggcataa ccacgtgtcg ggatctcacc 180
cttgccggca acaacgtgct actcgtcgaa gcgcgagacc gtattggtgg gcggtcatgg 240
tcatctaata ttgacggata cccctatgag atgggtggaa cgtgggtgca ttggcatcaa 300
ccttttgtgt ggcgggagtt gagacggtac gggatggtcg atcaattgga gatttcgcca 360
aggaagaatg ttgagggcgg ggcgagagtt actgttaact tggatggaga gatgaagcat 420
ctgacgcatg acgatgagga tgccattgtc gagtccgcat tcaagaaatt tatcaatgtg 480
gacggccaat tcggccgcac tgtggttccc ttcccgcacg atatcgaatt acacatggcg 540
ggcgtagagg agtacgacca catgtctatg gccgaccgca tggtacaagt tgcacctcac 600
ttgacaccat tggaaaagaa catgttcgag ggattcctat ccatcacgca cggcggaaaa 660
tgggaagaag catctttctt cgagctgctg cggtggtggg cattgatgga ttacaatctc 720
cccaacttca tggaacttgg actcatgtac aagatccgtg atggtcagtc tgcgctggcg 780
agacggattt tcgatgaagc tgtcagcacg ggtagattgg actatacttt ctctacgccg 840
gtcaaggacg taattgatca tggacacgga gttgaagtta tcgctcgaag tggaggggag 900
gttttcaagg cgagacgttt ggtctgcact gtgccgttga atgtcttgca taccttggcc 960
ttttcgccaa agctgccgac tctcaagacg gaagcttctc tagctggtca tgtcaacaaa 1020
gtcgtcaaat gtcatgccga agttgcgaat cccgagatgc gctcgctcgg ggcaacgaac 1080
tacccccacg gcagactcac ctacactttc ggtgatggaa ctacgcccgc gggaaatact 1140
cacctcgttg cctttggcag ctcccttcct ggagttcact tggatcccga gcaggatatt 1200
gaagtcacta agaaggcctt tgaggccttc cacccgggca tgaatgtgca gaagttggtg 1260
ttccacaact ggcacaagga tgaatttgcc cagggtgcgt gggagtggtt gcggccgggt 1320
atgacaacaa agtacctgaa ggcgctgcga gagcgacatg gaaatgtgtt ctttgccagc 1380
tcggattctt cctttggatg gagaggattc atcgatggag cgatggatga cggcgggaga 1440
atcgcgaagc tcgttcatga tgagttgaag gacgtgcgtc ccattgcttc taagctttga 1500
<210> 6
<211> 499
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Met Thr Ala Gly Pro Leu Ser Ala Ala Gly Thr Gly Thr Thr Leu Ala
1 5 10 15
Thr Gly Leu Val Gly Gly Val Pro Thr Leu Gly Leu Ile Gly Pro Pro
20 25 30
Thr His Pro Thr Ala Gly Leu Ala Thr Ala Val Ile Val Ile Gly Gly
35 40 45
Gly Thr Ala Gly Ile Thr Thr Cys Ala Ala Leu Thr Leu Ala Gly Ala
50 55 60
Ala Val Leu Leu Val Gly Ala Ala Ala Ala Ile Gly Gly Ala Ser Thr
65 70 75 80
Ser Ser Ala Ile Ala Gly Thr Pro Thr Gly Met Gly Gly Thr Thr Val
85 90 95
His Thr His Gly Pro Pro Val Thr Ala Gly Leu Ala Ala Thr Gly Met
100 105 110
Val Ala Gly Leu Gly Ile Ser Pro Ala Leu Ala Val Gly Gly Gly Ala
115 120 125
Ala Val Thr Val Ala Leu Ala Gly Gly Met Leu His Leu Thr His Ala
130 135 140
Ala Gly Ala Ala Ile Val Gly Ser Ala Pro Leu Leu Pro Ile Ala Val
145 150 155 160
Ala Gly Gly Pro Gly Ala Thr Val Val Pro Pro Pro His Ala Ile Gly
165 170 175
Leu His Met Ala Gly Val Gly Gly Thr Ala His Met Ser Met Ala Ala
180 185 190
Ala Met Val Gly Val Ala Pro His Leu Thr Pro Leu Gly Leu Ala Met
195 200 205
Pro Gly Gly Pro Leu Ser Ile Thr His Gly Gly Leu Thr Gly Gly Ala
210 215 220
Ser Pro Pro Gly Leu Leu Ala Thr Thr Ala Leu Met Ala Thr Ala Leu
225 230 235 240
Pro Ala Pro Met Gly Leu Gly Leu Met Thr Leu Ile Ala Ala Gly Gly
245 250 255
Ser Ala Leu Ala Ala Ala Ile Pro Ala Gly Ala Val Ser Thr Gly Ala
260 265 270
Leu Ala Thr Thr Pro Ser Thr Pro Val Leu Ala Val Ile Ala His Gly
275 280 285
His Gly Val Gly Val Ile Ala Ala Ser Gly Gly Gly Val Pro Leu Ala
290 295 300
Ala Ala Leu Val Cys Thr Val Pro Leu Ala Val Leu His Thr Leu Ala
305 310 315 320
Pro Ser Pro Leu Leu Pro Thr Leu Leu Thr Gly Ala Ser Leu Ala Gly
325 330 335
His Val Ala Leu Val Val Leu Cys His Ala Gly Val Ala Ala Pro Gly
340 345 350
Met Ala Ser Leu Gly Ala Thr Ala Thr Pro His Gly Ala Leu Thr Thr
355 360 365
Thr Pro Gly Ala Gly Thr Thr Pro Ala Gly Ala Thr His Leu Val Ala
370 375 380
Pro Gly Ser Ser Leu Pro Gly Val His Leu Ala Pro Gly Gly Ala Ile
385 390 395 400
Gly Val Thr Leu Leu Ala Pro Gly Ala Pro His Pro Gly Met Ala Val
405 410 415
Gly Leu Leu Val Pro His Ala Thr His Leu Ala Gly Pro Ala Gly Gly
420 425 430
Ala Thr Gly Thr Leu Ala Pro Gly Met Thr Thr Leu Thr Leu Leu Ala
435 440 445
Leu Ala Gly Ala His Gly Ala Val Pro Pro Ala Ser Ser Ala Ser Ser
450 455 460
Pro Gly Thr Ala Gly Pro Ile Ala Gly Ala Met Ala Ala Gly Gly Ala
465 470 475 480
Ile Ala Leu Leu Val His Ala Gly Leu Leu Ala Val Ala Pro Ile Ala
485 490 495
Ser Leu Leu
Claims (12)
1. A group of monoamine oxidases from Penicillium species, characterized in that they are obtained from Penicillium species (Penicillium sp. art), Penicillium species (Penicillium polonicum) and Penicillium species (Penicillium brasiliinum).
2. The monoamine oxidase of claim 1, wherein the amino acid sequence of the monoamine oxidase is SEQ ID No: 2. SEQ ID No: 4. SEQ ID No: 6.
3. the monoamine oxidase of claim 1, wherein the mRNA sequence of the monoamine oxidase is SEQ ID No: 1. SEQ ID No: 3. SEQ ID No: 5.
4. a nucleic acid encoding the monoamine oxidase of any of claims 1-3.
5. A recombinant expression vector comprising the nucleic acid of claim 4.
6. The recombinant expression vector of claim 5, wherein the plasmid used to construct the recombinant expression vector is pET28 a.
7. A recombinant expression transformant containing the recombinant expression vector according to claim 5 to 6.
8. The recombinant expression transformant according to claim 7, wherein the host cell is Escherichia coli.
9. The recombinant expression transformant according to claim 7, wherein the host cell is E.coli BL21(DE 3).
10. Use of a monoamine oxidase as claimed in any one of claims 1 to 3, in aqueous solution, to catalyze the oxidation of 6, 6-dimethyl-3-azabicyclo [3.1.0] hexane in the presence of catalase to give (1R,5S) -6, 6-dimethyl-3-azabicyclo [3.1.0] hex-2-ene.
11. The use according to claim 10, wherein the monoamine oxidase is present in an amount of 100 mg to 10g per liter; the amount of catalase is 10 mg-300 mg.
12. The use according to claim 10, wherein the aqueous solution is a buffer solution having a pH ranging from 5.0 to 8.0, and the oxidation reaction is carried out under shaking or stirring conditions; the reaction temperature of the oxidation reaction is 20-60 ℃.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210365498.6A CN114774382A (en) | 2022-04-07 | 2022-04-07 | Method for stereoselectively preparing 6, 6-dimethyl-3-azabicyclo [3.1.0] hexene compound |
| CN202211614411.0A CN115851644A (en) | 2022-04-07 | 2022-12-13 | Method for stereoselectively preparing 6, 6-dimethyl-3-azabicyclo [3.1.0] hexene compound |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210365498.6A CN114774382A (en) | 2022-04-07 | 2022-04-07 | Method for stereoselectively preparing 6, 6-dimethyl-3-azabicyclo [3.1.0] hexene compound |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114774382A true CN114774382A (en) | 2022-07-22 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210365498.6A Withdrawn CN114774382A (en) | 2022-04-07 | 2022-04-07 | Method for stereoselectively preparing 6, 6-dimethyl-3-azabicyclo [3.1.0] hexene compound |
| CN202211614411.0A Pending CN115851644A (en) | 2022-04-07 | 2022-12-13 | Method for stereoselectively preparing 6, 6-dimethyl-3-azabicyclo [3.1.0] hexene compound |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211614411.0A Pending CN115851644A (en) | 2022-04-07 | 2022-12-13 | Method for stereoselectively preparing 6, 6-dimethyl-3-azabicyclo [3.1.0] hexene compound |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN114774382A (en) |
-
2022
- 2022-04-07 CN CN202210365498.6A patent/CN114774382A/en not_active Withdrawn
- 2022-12-13 CN CN202211614411.0A patent/CN115851644A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| CN115851644A (en) | 2023-03-28 |
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