Monoamine oxidase and application thereof in synthesis of chiral azabicyclic compounds

A technology for monoamine oxidase and oxidase activity, applied in the field of bioengineering, can solve the problems of low corresponding selectivity, incomplete reaction, low yield, etc.

Active Publication Date: 2016-03-30
ABIOCHEM BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0035] The technical problem to be solved by the present invention is, low yield, high raw material cost, incomplete reaction, and poor corresponding selectivity in the reaction of the reported preparation of boceprevir intermediate fragment A and telaprevir intermediate fragment B. High or unfriendly to the environment, a monoamine oxidase with high catalytic activity, strong enantioselectivity, and good substrate tolerance is provided for enzymatic synthesis of (1S,2S,5R)-6,6-dimethyl -2-substituted-3-azabicyclo[3.1.0]hexane or (1S,3aR,6aS)-1-substituted-octahydrocyclopenta[c]pyrrole, further synthesis of (1S,2S,5R)- 6,6-Dimethyl-3-azabicyclo[3.1.0]hexane-2-carbonitrile and its hydrolysis product (1S,2S,5R)-6,6-dimethyl-3-azabicyclo[ 3.1.0] Hexane-2-methyl carboxylate, or further enzymatic-chemical synthesis of (1S,3aR,6aS)-octahydrocyclopenta[c]pyrrole-1-carbonitrile

Method used

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  • Monoamine oxidase and application thereof in synthesis of chiral azabicyclic compounds
  • Monoamine oxidase and application thereof in synthesis of chiral azabicyclic compounds
  • Monoamine oxidase and application thereof in synthesis of chiral azabicyclic compounds

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Experimental program
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Effect test

Embodiment 1

[0076] The enzyme activity assay of embodiment 1 monoamine oxidase

[0077] Dissolve 5mmol / L benzylamine in 50mmol / L phosphate buffer to obtain a substrate solution, take 1mL and add 10μL enzyme solution, react at 30°C for 20min, add 400μL dinitrophenylhydrazine (1mmol / L, dissolved in 3M HCl), and then add 2.4mL1.25MNaOH, react for 5min, and detect the absorbance at a wavelength of 450nm.

[0078]Optical purity: Agilent1260 series HPLC, chiral column is ChiralpakAD-H (4.6×250mm, Diacel ChemicalInd.Ltd.), analyzed at 25°C, mobile phase is 90% n-heptane, 10% ethanol, 0.1% trifluoroacetic acid, flow rate It is 1mL / min, and the detection wavelength is 220nm. The retention time of the S-configuration product was 7.4min, and the retention time of the R-configuration product was 8.9min.

Embodiment 2

[0079] Embodiment 2 screening monoamine oxidase

[0080] Soil sample DNA (ChromaSpinTE-1000, Clontech Laboratories, Inc.USA) was collected from the campus of Wuhan University, partially digested with Sau3AI, and 1-4kb fragments were collected by electrophoresis, recovered and connected to the BamHI site of pUC19 to obtain a plasmid library; Transform the library into E.coliDH10b, smear it on an LB plate containing 100 μg / mL ampicillin, select positive clones and transfer it to a 96 deep-well plate with 500 μg / mL LB (100 μg / mL ampicillin), culture at 37°C for 4 hours, then add 1 mmol / LIPTG Induction, continue culturing overnight at 30°C, then transfer 10 μL of deep well culture to a new 96-well plate containing 50mmol / L sodium phosphate buffer (pH7.5), freeze and thaw repeatedly at -80°C to lyse the bacteria, add 5mmol / L L benzylamine, incubate at 30°C for 30 minutes, then add 400μL of dinitrophenylhydrazine (1mmol / L, dissolved in 3M HCl), then add 2.4mL of 1.25M NaOH, react fo...

Embodiment 3

[0081] Construction and expression of embodiment 3 monoamine oxidase recombinant bacteria

[0082] Adopt primer PF (nucleotide sequence is SEQIDNO:3) and PR (nucleotide sequence is SEQIDNO:4) PCR amplification monoamine oxidase full-length gene (results see figure 1 ), the PCR product was digested with NdeI / XhoI and connected to pET28, and transformed into E.coliBL21(DE3), cultivated on an LB plate containing kanamycin (50 μg / mL), and selected positive colonies into 100mL liquid LB medium For cultivation, the overnight culture was transferred to fresh 1 LLB culture medium and cultivated to OD 600 To 0.6 ~ 0.8, add IPTG200μM to induce protein expression, lower the temperature to 30°C and continue to culture for 24 hours. The bacteria were collected by centrifugation at 5000rpm, washed once with 0.2M pH7.0 sodium phosphate buffer, 1g of bacteria was resuspended in the above 5mL phosphate buffer, after ultrasonic disruption, the expression level was tested by SDS-PAGE.

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Abstract

The invention provides a monoamine oxidase with high catalysis activity, strong enantioselectivity and good substrate survivability, and an enzyme-chemical synthesis method using the above enzyme to carry out enzyme catalyzed synthesis of (1S,2S,5R)-6,6-dimethyl-2-substituted-3-azabicyclo[3.1.0]hexane or (1S,3aR,6aS)-1-substituted-octahydrocyclopenta[c]pyrrole in order to further synthesize (1S,2S,5R)-6,6-dimethyl-2-3-azabicyclo[3.1.0]hexane-2-nitrile and its hydrolysate methyl (1S,2S,5R)-6,6-dimethyl-2-3-azabicyclo[3.1.0]hexane-2-carboxylate or further synthesize (1S,3aR,6aS)-1-octahydrocyclopenta[c]pyrrole-1nitrile The invention also provides a gene for coding the monoamine oxidase, a recombinant expression vector containing the gene, a recombinant expression transformant containing the gene, and efficient preparation methods of the vector and the transformant. The monoamine oxidase can respectively react with 3-5 and 5-5 azacyclopentamine systems with different sizes to generate corresponding imides; the imides undergo cyanide addition, so a cyanide is added to an enzyme catalysis system, and the above obtained addition product can be directly alcoholyzed in a hydrochloric acid-alcohol solution to obtain hydrochloride of amino acid methyl ester, so one-pot feeding is realized, the reaction technology is simplified, and industrial production is facilitated.

Description

technical field [0001] The invention belongs to the field of bioengineering, and specifically relates to a monoamine oxidase, a recombinant expression vector and a recombinant expression transformant containing the gene encoding the enzyme, the expressed recombinant enzyme and the preparation method of the recombinant enzyme, and the monoamine oxidase as a catalyst in the synthesis of bocecept Application in Wei intermediates. Background technique [0002] Boceprevir and telaprevir are hepatitis C virus (HCV) protease inhibitors, and their chemical structural formulas are as follows: [0003] [0004] The phase III SPRINT-2 study showed that, compared with standard treatment, 24 weeks of standard treatment with boceprevir can improve the SVR rate in untreated patients with HCV genotype 1. The antiviral efficacy of standard treatment with boceprevir for 24 weeks was similar to that of standard treatment with boceprevir for 44 weeks. [0005] Fragment A is one of the key ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/70C12N1/21C12P17/10
Inventor 罗煜丁时澄瞿旭东钱龙
Owner ABIOCHEM BIOTECH CO LTD
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