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A kind of method for preparing (s)-2-(3-pyridine)-pyrrolidine

A pyrrolidine and pyridine technology, applied in the field of biocatalysis, can solve the problems of increasing the amount of coenzyme NAD, high production cost, and reduced reaction conversion rate

Active Publication Date: 2022-06-24
SHANDONG JINCHENG PHARMACCUTICAL CHEM CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] However, due to the unique properties of amine substrates, the substrate concentration cannot be too high during the reduction process, otherwise the reaction conversion rate will be significantly reduced. In order to increase the substrate concentration, it is necessary to increase the amount of expensive coenzyme NAD(P)H, and the production higher cost

Method used

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  • A kind of method for preparing (s)-2-(3-pyridine)-pyrrolidine
  • A kind of method for preparing (s)-2-(3-pyridine)-pyrrolidine
  • A kind of method for preparing (s)-2-(3-pyridine)-pyrrolidine

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Experimental program
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Effect test

Embodiment 1

[0037] Example 1: Acquisition of high-expressing genetically engineered bacteria

[0038] Whole gene synthesis was performed by General Biosystems (Anhui) Co., Ltd.

[0039] According to the imine reductase MsIR1 (WP_074958336.1) of bacteria (Myxococcus fulvus), it was codon-optimized to enable the gene to be expressed in the E. coli expression host. Nde I and EcoR I restriction sites were added to both ends of the gene to construct into pET-28a(+) vector to obtain genetically engineered bacteria M1.

[0040] The prepared recombinant vector is transformed into Escherichia coli BL21, Rosetta or Origami by conventional methods to construct genetically engineered bacteria in which the recombinant imine reductase exists in the bacteria in a soluble form, and screened out the genetically engineered bacteria that have been established successfully. The recombinant bacteria with Bacillus BL21 as the host bacteria expressed relatively good protein of interest. The engineering bacter...

Embodiment 2

[0041] The cultivation of embodiment 2 genetically engineered bacteria and the preparation of crude enzyme liquid

[0042] Pick a single colony on the plate and inoculate it into 5ml of fermentation medium containing corresponding antibiotics, cultivate for about 15h as seed liquid, inoculate into 600ml of fermentation medium according to 1% of the inoculum, and cultivate at 37°C, 200rpm on a shaking table to OD 600 =0.6-0.8, add IPTG with a final concentration of 0.1 mM for induction for more than 10 hours, centrifuge the culture solution at 8000 rpm to collect bacterial cells, and perform high-pressure crushing to obtain a crude enzyme solution of imine reductase.

Embodiment 3

[0043] Example 3 Whole cell catalytic synthesis of (S)-2-(3-pyridine)-pyrrolidine using imine reductase

[0044] 10ml phosphate buffer (pH7.5), 30mg / ml cells, 2eq glucose, 0.2mg / ml NADP + , 10mg GDH powder, substrate concentration 50mg / ml, 28 ℃ reaction, TLC spot plate to judge the progress of the reaction. After 12 hours, a saturated sodium hydroxide solution was added to adjust the pH value to above 10, and the denatured protein was removed by centrifugation. The supernatant was extracted with dichloromethane, dried, and the product was collected by spin-drying and detected by HPLC.

[0045] serial number pH Conversion rate ee 1 6.0 26.1 99.7 2 6.5 48.9 99.8 3 7.0 84.6 99.7 4 7.5 98.7 99.8 5 8.0 89.1 99.8 6 8.5 80.3 99.7 7 9.0 72.6 99.7 8 9.5 55.8 99.7

[0046] According to the above table, the pH of the buffer solution is 7.0-9.0, and the conversion rate is relatively high, especially when the pH is...

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Abstract

The present invention provides a method for preparing (S)-2-(3-pyridine)-pyrrolidine, using imine reductase (IRED) or engineering bacteria expressing the enzyme to prepare chiral 2-pyridine pyrrolidine compounds. Coenzyme regeneration is achieved using a glucose dehydrogenase / glucose system. More specifically, the present invention provides a method to reduce 2-pyridine-1-pyrroline compound into (S)-2-(3-pyridine)-pyrrolidine by using imine reductase derived from Myxococcus fulvus and its genetically engineered bacteria According to the method, the imine reductase derived from Myxococcus fulvus has high activity, high reaction substrate concentration, reaction yield and product optical purity, simple operation in the reaction process, low energy consumption, and meets the requirements of green chemistry. It is applied to the biotransformation preparation of (S)‑2‑(3‑pyridine)‑pyrrolidine compounds in industrial production.

Description

technical field [0001] The invention belongs to the field of biocatalysis, and relates to an imine reductase derived from Myxococcus as a biocatalyst and NADP (H) as a coenzyme to reduce 2-pyridine-1-pyrroline to generate (S)-2-( 3-Pyridine)-pyrrolidine method, product optical purity >98%. Background technique [0002] Chiral amines and their derivatives are important branches of single enantiomer drugs, and are the structural units of many pharmaceutical intermediates and agrochemicals. Currently, more than 70% of drugs are chiral amines and their derivatives, including neural , antihypertensive and cardiovascular and cerebrovascular drugs [Mitsukura K, Kuramoto T, Yoshida T, et al. [J]. Appl Microbiol Biotechnol, 2013, 97: 8079-8086.]. [0003] [0004] X = C, O, N, S; [0005] R=Cl,F,Br,I,CH 3 ,OCH 3 ,OH,NO 2 [0006] [0007] X=C,N; [0008] R=Cl,F,Br,I,CH 3 ,OCH 3 ,OH,NO 2 [0009] Optically pure 2-aryl(hetero)ylpyrrolidines are important building bl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P17/16
CPCC12P17/165
Inventor 李家全魏庚辉孟宪强
Owner SHANDONG JINCHENG PHARMACCUTICAL CHEM CO LTD
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