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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, reducing the reaction conversion rate, and high production costs

Active Publication Date: 2021-05-14
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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  • Method for preparing (S)-2-(3-pyridine)-pyrrolidine

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Experimental program
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Embodiment 1

[0037] Embodiment 1: the acquisition of highly expressed genetically engineered bacteria

[0038] The whole gene synthesis was completed by General Biosystems (Anhui) Co., Ltd.

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

[0040] Transform the prepared recombinant vector into Escherichia coli BL21, Rosetta or Origami by conventional methods to construct a genetically engineered bacterium in which the recombinant imine reductase exists in the bacterium in a soluble form, and screen out the successfully established genetically engineered bacteria, among which Escherichia coli Bacillus BL21 as the host bacterium recombinant bacteria protein expression is relat...

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 it for about 15 hours as a seed solution, inoculate it into 600ml of fermentation medium according to the inoculation amount of 1%, and cultivate it on a shaker at 37°C and 200rpm to OD 600 =0.6~0.8, add IPTG with a final concentration of 0.1mM to induce for more than 10h, collect the bacterial cells by centrifuging the culture solution at 8000rpm, and perform high-pressure crushing to obtain the 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 bacteria, 2eq glucose, 0.2mg / ml NADP + ,10mg GDH powder, substrate concentration 50mg / ml, react at 28°C, TLC plate to judge the reaction progress. After 12 hours, add saturated sodium hydroxide solution to adjust the pH to above 10, centrifuge to remove denatured protein, extract the supernatant with dichloromethane, dry, spin dry to collect the product, and detect 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, it can be seen that the buffer solution has a higher conversion rate at pH 7.0-9.0, especially at pH 7.5-8.0, the conversion rate effect is very significant.

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Abstract

The invention provides a method for preparing (S)-2-(3-pyridine)-pyrrolidine. The chiral 2-pyridine pyrrolidine compound is prepared by utilizing imine reductase (IRED) or engineering bacteria for expressing the imine reductase (IRED). A glucose dehydrogenase / glucose system is utilized to realize regeneration of coenzyme. More specifically, the invention provides a method for reducing a 2-pyridine-1-pyrroline compound into (S)-2-(3-pyridine)-pyrrolidine by utilizing the imine reductase derived from Myxococcus fulvus and a genetically engineered bacterium thereof, the imine reductase derived from Myxococcus fulvus is relatively high in activity, relatively high in reaction substrate concentration, relatively high in reaction yield and relatively high in optical purity of a product, In the reaction process, operation is easy, energy consumption is low, the green chemical requirement is met, and the method can be applied to biotransformation preparation of the (S)-2-(3-pyridine)-pyrrolidine compound in industrial production.

Description

technical field [0001] The invention belongs to the field of biocatalysis, and relates to a method of reducing 2-pyridine-1-pyrroline to generate (S)-2-( 3-pyridine)-pyrrolidine, the optical purity of the product is >98%. Background technique [0002] Chiral amines and their derivatives are an important branch of single-enantiomer drugs, and are the structural units of many pharmaceutical intermediates and agricultural chemicals. Currently, more than 70% of drugs are chiral amines and their derivatives, including neurological drugs. , 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)pyrrolidine is an important structural unit, which is commonly found in natural...

Claims

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Application Information

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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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