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Method for synthesizing pregabalin chiral intermediate through regio-selective and stereo-selective bio-catalytic synthesis

A stereoselective, chiral intermediate technology, applied in fermentation and other directions, can solve the problems of many by-products, cumbersome product purification process, difficult to obtain both yield and optical purity, etc., and achieves simple operation, improved yield and optical purity. Effects with high purity, regio- and stereoselectivity

Active Publication Date: 2018-10-30
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, a challenging problem in the biocatalytic process of nitrilase is that there are many by-products, the product purification process is tedious, and it is difficult to achieve both yield and optical purity. For example, patent CN1942587B uses NIT-101, NIT-102, NIT-103 and pseudo The yield and optical purity of preparing (S)-3-cyano-5-methylhexanoic acid from isobutylsuccinonitrile catalyzed by A. thaliana nitrilase correspond to: yield 34.2%, optical purity 96.3% e.e.; Yield 38.6%, Optical Purity 91.1% e.e.; Yield 35.5%, Optical Purity 95.5% e.e. and Yield 17.5%, Optical Purity 98.5% e.e.

Method used

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  • Method for synthesizing pregabalin chiral intermediate through regio-selective and stereo-selective bio-catalytic synthesis
  • Method for synthesizing pregabalin chiral intermediate through regio-selective and stereo-selective bio-catalytic synthesis
  • Method for synthesizing pregabalin chiral intermediate through regio-selective and stereo-selective bio-catalytic synthesis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1 constructs recombinant nitrilase engineering bacterium

[0045] According to the report in NCBI, the turnip (Brassica rapa) nitrilase BrNIT (ABM55734.1), BrNIT2 (BAG72074), Arabidopsis thaliana (Arabidopsis thaliana) nitrilase AtNIT (NP 851011) and alpine Arabidopsis (Arabis alpine) nitrile The gene sequence was fully synthesized by the hydrolase AaNIT (KFK44999), and restriction sites XhoI and XbaI were designed at both ends, and cloned into the empty vector pET28b to obtain a recombinant plasmid, which was recovered and transformed into Escherichia coli BL21 (DE3), and the positive bacteria were cultured to obtain Recombinant nitrilase gene engineering bacteria BL21(DE3) / pET28b-BrNIT, BL21(DE3) / pET28b-BrNIT2, BL21(DE3) / pET28b-AtNIT and BL21(DE3) / pET28b-AaNIT.

Embodiment 2

[0046] Embodiment 2 constructs recombinant amidase engineering bacterium

[0047] According to the reported amidase genes Pa-Ami (WP_008109374), Cc-Ami (WP_027015397) and Dt-Ami (KP943494) derived from Pantoea sp.YR343, Comamonas composti and D.tsuruhatensis ZJB-05174, their gene sequences were total synthesis. Design upstream and downstream primers containing EcoRI and NcoI sites, insert the gene into pET28b after amplifying the gene, construct the expression vector, transform into BL21(DE3) competent cells by heat shock, and obtain amidase genetically engineered bacteria BL21(DE3) / pET28b- Pa-Ami, BL21(DE3) / pET28b-Cc-Ami and BL21(DE3) / pET28b-Dt-Ami.

Embodiment 3

[0048] The expression culture of embodiment 3 genetically engineered bacteria

[0049] Pick a single colony and inoculate it into 5 mL of liquid LB medium, and the final concentration of kanamycin is 50 mg / L. The culture conditions are 37° C., 200 rpm, and cultured for 6-8 hours. The above seed solution was transferred to fresh LB liquid medium containing 50mg / L kanamycin at a volume ratio of 2%, and cultivated at 37°C and 150rpm until the cell OD 600 At about 0.6-0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG, final concentration: 0.1mM) to the above LB liquid medium, induce culture at 28°C, 150rpm for 10-12h, The fermented broth was collected and centrifuged at 4°C and 8000rpm for 10 minutes, then the cells were washed once with physiological saline, and the centrifuged cells were stored in a -20°C refrigerator for use in the following examples.

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Abstract

The invention discloses a preparation method for synthesizing a pregabalin chiral intermediate through regio-selective and stereo-selective bio-catalytic synthesis. The method comprises the followingsteps: reacting racemic isobutyl butyronitrile (IBSN) in a reaction medium at a temperature of 20-50 DEG C in presence of nitrilase and amidase catalysis for 4-10 hours, thereby obtaining (S)-3-cyano-5-methyl hexanoate. The method is mild in reaction conditions, simple and feasible in operation and excellent in regio-selectivity and stereo-selectivity, the conversion rate of the pregabalin key chiral intermediate, namely (S)-3-cyano-5-methyl hexanoate, synthesized from the racemic substrate IBSN through a one-pot process can reach 49.5%, the value e.e. is 99.5%, and the conversion rate is close to a theoretical conversion rate of 50%. Meanwhile, the optical purity exceeds 99%, and the method has obvious application values for simplifying the production process steps of pregabalin and reducing the production cost of the pregabalin.

Description

technical field [0001] The invention relates to the field of biochemical industry, specifically, the invention relates to a nitrilase, amidase two-step enzymatic region, stereoselective biocatalyzed synthesis of racemic 3-cyano-5-methylcapronitrile (IBSN) A method for the key chiral intermediate of pregabalin (S)-3-cyano-5-methylhexanoic acid. Background technique [0002] Pregabalin, chemical name (S)-(+)-3-aminomethyl-5-methylhexanoic acid (I), is a new type of γ-aminobutyric acid (γ-aminobutyric acid, GABA ) receptor agonist, can effectively block the voltage-dependent calcium channel, reduce the release of neurotransmitters, and have good therapeutic effects on anxiety and neuropathic pain (Angew.Chem.Int.Ed., 2008,47:3500-3504 ). [0003] [0004] Compared with traditional drugs, pregabalin has the advantages of low dose, less frequency, less toxic and side effects, long duration, and strong tolerance, and is considered to be one of the most promising antiepileptic...

Claims

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Application Information

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IPC IPC(8): C12P41/00C12P13/00
CPCC12P13/002C12P41/00
Inventor 郑仁朝郑裕国张琴汤晓玲卢夏锋
Owner ZHEJIANG UNIV OF TECH
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