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Preparation method of 2,4-difluorobenzylamine

A technology of difluorobenzylamine and difluorobenzonitrile, which is applied in the field of biocatalysis, can solve the problems of low production yield and environmental pollution, and achieve the effect of safe raw materials and environmentally friendly production process

Pending Publication Date: 2021-02-26
ABIOCHEM BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the technical problems of low production yield of 2,4-difluorobenzylamine and environmental pollution caused by traditional chemical catalytic methods in the prior art, the invention provides a preparation method of 2,4-difluorobenzylamine

Method used

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  • Preparation method of 2,4-difluorobenzylamine
  • Preparation method of 2,4-difluorobenzylamine
  • Preparation method of 2,4-difluorobenzylamine

Examples

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Embodiment 1

[0037] The preparation of embodiment 1 nitrile reductase

[0038] Composition of LB liquid medium: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, dissolved in deionized water and then constant volume, sterilized at 121°C for 20min, ready for use.

[0039] TB liquid medium composition: peptone 10g / L, yeast powder 18g / L, glycerin 4mL / L, KH 2 PO 4 2.31g / L, K2 HPO 4 2H 2 O 16.43g / L, dissolved in deionized water to constant volume, sterilized at 121°C for 20min, ready for use.

[0040] See Table 1 below for the amino acid and nucleotide sequence information of nitrile reductase. Suzhou Jinweizhi Biotechnology Co., Ltd. (No. 211 Pubin Road, Yanchuang Park, Jiangbei New District, Nanjing, Jiangsu Province) synthesized the nitrile reductase (Enz.1~Enz.3) gene (SEQ IDNO:1-3) from the whole gene, digested with enzymes The sites NdeI and HindIII were connected into the vector pET28a. The synthetic nitrile reductase gene is transformed into host E. coli BL21 (DE3) competent cells to ...

Embodiment 2

[0051] The preparation of embodiment 2 glucose dehydrogenase

[0052] According to the glucose dehydrogenase (GDH) gene sequence derived from Bacillus subtilis 168 (NCBI accession number NP_388275.1), Suzhou Jinweizhi Biotechnology Co., Ltd. (Pubin, Jiangbei New District, Nanjing, Jiangsu Province) Road No. 211) whole gene synthesis of glucose dehydrogenase (GDH) gene, restriction site NdeI, HindIII, connected into vector pET28a. The synthetic GDH gene was transformed into host E.coli BL21 (DE3) competent cells to obtain engineering strains containing the GDH gene.

[0053] After the engineered bacteria containing the GDH gene were activated by streaking on the plate, a single colony was picked and inoculated into 5 mL LB liquid medium containing 50 μg / mL kanamycin, and cultured with shaking at 37 °C for 4 h. Transfer to 150mL fresh TB liquid medium containing 50μg / mL kanamycin at 1v / v% inoculum amount, shake culture at 37°C until OD600 reaches about 0.8, add IPTG to its fina...

Embodiment 3

[0058] Example 3 Nitrile reductase catalyzes 2,4-difluorobenzonitrile to prepare 2,4-difluorobenzylamine

[0059]

[0060] Add 50mL of pure water, 6.5g of glucose, NADP+0.5mg to the reaction system, add 20mL of isopropanol, 5g of 2,4-difluorobenzonitrile, stir and dissolve in a water bath at 30°C, and dissolve with 20% aqueous sodium carbonate (mass volume ratio) to fine-tune the pH to 7.2-7.5, and finally add 20 mL of the liquid nitrile reductase obtained in Example 1 (2.5 g of bacteria slime plus buffer homogeneously obtained) and 10 mL of GDH prepared in Example 2. The reaction was continued in a water bath at 30°C, pH 7.4, 200 rpm. During the reaction, the conversion rate of the substrate was detected by HPLC. The results are shown in Table 3 below.

[0061]

[0062] The 20 hour conversion of nitrile reductase Enz.03 as shown in the table above was 70%. In this example, the reaction system or reaction conditions are adjusted, for example, when the reaction temperatu...

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Abstract

The invention discloses a preparation method of 2,4-difluorobenzylamine, which comprises the following steps: contacting 2,4-difluorobenzonitrile with nitrile oxidoreductase, and reducing 2,4-difluorobenzonitrile into 2,4-difluorobenzylamine. The nitrile oxidoreductase is nitrile oxidoreductase with an NCBI (National Center of Biotechnology Information) login number of WP_004867005.1. The invention also discloses an application of the nitrile oxidoreductase in preparation of dolutegravir or an intermediate of dolutegravir. According to the preparation method provided by the invention, the conversion rate of can reach 70%, the raw materials are safe, the production process is environmentally friendly, and the preparation method is suitable for large-scale industrial production.

Description

technical field [0001] The invention relates to the field of biocatalysis, in particular to a method for preparing a dolutegravir intermediate, namely 2,4-difluorobenzylamine. Background technique [0002] Dolutegravir (Dologevir, Dolutegravir, Tivicay) is a new anti-HIV drug owned by GlaxoSmithKline (GSK), which was approved by the US Food and Drug Administration (FDA) on August 12, 2013 Or treatment-naïve HIV-1 adults and children aged 12 and over weighing at least 40 kg. Due to its advantages of high efficiency and low toxicity, the market sales have risen rapidly in recent years, and the momentum is unanimously optimistic. [0003] [0004] 2,4-Difluorobenzylamine is the key intermediate of dolutegravir, and its production method is very important. At present, the production method of 2,4-difluorobenzylamine mainly adopts the method of reducing 2,4-difluorobenzonitrile, such as the method of reducing hydrogen and Raney nickel in US5068371, but the productive rate is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/00
CPCC12P13/001
Inventor 冯庆梅王舒田振华孙传民程占冰
Owner ABIOCHEM BIOTECH CO LTD
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