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Method catalytically synthesizing (S)-N, N-dimethyl-3-hydroxy-(2-thiofuran)-1-propylamine((S)-DHTP) by aldehyde ketone reductase recombinant strain crude enzyme system

A technology of reductase and recombinant bacteria, which is applied in the field of biocatalytic asymmetric transformation, can solve problems such as affecting the space-time yield of catalytic reactions, restricting industrial applications, and hindering substrate interactions.

Active Publication Date: 2014-11-19
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This may be due to the limitation of the nature of its cell or intracellular functional enzyme and the membrane structure of the whole cell hindering the interaction between the substrate (DKTP) and the enzyme, thus affecting the space-time yield of the catalytic reaction, which greatly limits its industrial application.
Moreover, at present, there are few studies on duloxetine chiral intermediates in China, and the level is low, and there is no preparation of optically pure ( S )-DHTP report

Method used

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  • Method catalytically synthesizing (S)-N, N-dimethyl-3-hydroxy-(2-thiofuran)-1-propylamine((S)-DHTP) by aldehyde ketone reductase recombinant strain crude enzyme system
  • Method catalytically synthesizing (S)-N, N-dimethyl-3-hydroxy-(2-thiofuran)-1-propylamine((S)-DHTP) by aldehyde ketone reductase recombinant strain crude enzyme system

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Embodiment 1

[0044] The preparation of embodiment 1 recombinant bacteria crude enzyme system:

[0045] The composition of LB medium was tryptone 1%, yeast extract 0.5%, NaCl 1%, pH 7.0. When needed, add ampicillin (50 μg / mL) before use, and add 1.5% agar powder to the solid medium.

[0046] A single colony of the positive clone was picked and inoculated in 3 mL LB liquid medium containing 50 μg / mL ampicillin, and cultured overnight at 37 °C with shaking at 200 rpm. Take 1 mL of the culture solution and transfer it to 50 mL of LB liquid medium containing 50 μg / mL ampicillin, and culture at 37°C and 200 rpm until OD 600 About 0.6. The inducer IPTG was added to the culture to a final concentration of 1 mmol / L, and induced at a culture temperature of 17°C for 12 h.

[0047] The cultured recombinant E. coli cells were collected by centrifugation at 10,000 rpm for 10 min and washed three times with saline. The bacteria were resuspended in potassium phosphate buffer (pH 6.5, 0.1 M) to form a ...

Embodiment 2

[0048] The crude enzyme system catalyzes the asymmetric reduction reaction in the aqueous phase reaction system of embodiment 2:

[0049] 2 mL reaction system consists of 1 mL potassium phosphate buffer (pH 6.5, 0.1 M), 1 mL crude enzyme system (total soluble protein 15 mg), 10 g / L glucose, 5 g / L DKTP, 0.02 mM NADP + . The reaction mixture was shaken at 30° C. for 8 h, and the reaction mixture was extracted with 2 times the volume of ethyl acetate, and the product ( S )-DHTP has an optical purity of 99% e.e. and a yield of 69.4%.

Embodiment 3

[0050] The crude enzyme system catalyzes the asymmetric reduction reaction in the aqueous phase reaction system of embodiment 3:

[0051] The 2 mL reaction system consists of 1 mL potassium phosphate buffer (pH 6.5, 0.1 M), 1 mL crude enzyme system (total soluble protein 15 mg), 10 g / L lactose, 5 g / L DKTP, 0.02 mM NADP + . The reaction mixture was shaken at 30° C. for 8 h, and the reaction mixture was extracted with 2 times the volume of ethyl acetate, and the product ( S )-DHTP has an optical purity of 99% e.e. and a yield of 69.7%.

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Abstract

The invention discloses a method catalytically synthesizing (S)-N, N-dimethyl-3-hydroxy-(2-thiofuran)-1-propylamine((S)-DHTP) by aldehyde ketone reductase recombinant strain crude enzyme system, and belongs to the technical field of biological catalysis asymmetric conversion. An auxiliary substrate and original bulk coenzyme NADP<+> are added in the recombinant strain crude enzyme system to accelerate regeneration cycle of coenzyme NADPH in the system; the reaction substrate is N, N-dimethyl-3-ketone-(2-thiophene)-1-propylamine (DTKP); the recombinant strain is E. coliBL21(DE3)(pETCPAR4); aldehyde ketone reducing enzyme gene cpar4 comes from Candida parapsilosis; the gene coded aldehyde ketone reducing enzyme gene CPAR4 catalytically and asymmetrically reduces the DKTP into (S)-DHTP. The method utilizes the cell-free system to conduct catalytic reaction; coupling enzyme required by regeneration of the coenzyme is not extra added in the reaction system; direct acting efficiency of the enzyme and the substrate is improved; reaction time is shortened; the conversion effect is relatively good.

Description

technical field [0001] The present invention relates to a kind of catalyzed synthesis ( S )-N,N-Dimethyl-3-hydroxyl-3-(2-thiophene)-1-propanamine (( S )-DHTP), which belongs to the technical field of biocatalytic asymmetric transformation. Background technique [0002] ( S The chemical structure of )-DHTP is: [0003] [0004] Duloxetine (duloxetine), chemical name ( S )-N-methyl-3-(1-naphthyloxy)-3-(2-thienyl)-1-propanamine, the trade name is Cymbalta (羊百达), which is a kind of product developed by Eli Lilly Company of the United States Can effectively inhibit serotonin and norepinephrine reuptake inhibitors (SNRI). Its hydrochloride is used clinically to treat severe depression, diabetic peripheral nerve pain and female stress urinary incontinence. Of the two isomers with the same chemical composition as duloxetine, only ( S )-configuration has antidepressant pharmacological activity. [0005] At present, due to different starting materials, there are many method...

Claims

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Application Information

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IPC IPC(8): C12P17/00C12N15/53C12N15/70C12N1/21C12R1/19
Inventor 聂尧徐岩
Owner JIANGNAN UNIV
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