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A method for synthesizing (s)-n,n-dimethyl-3-hydroxyl-3-(2-thiophene)-1-propanamine catalyzed by the recombinant bacterial crude enzyme system of aldehyde and ketone reductase

A technology of reductase and recombinant bacteria, which is applied in the field of biocatalytic asymmetric transformation, can solve the problems of low level, limited industrial application, and affecting the space-time yield of catalytic reactions, etc.

Active Publication Date: 2016-10-19
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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  • A method for synthesizing (s)-n,n-dimethyl-3-hydroxyl-3-(2-thiophene)-1-propanamine catalyzed by the recombinant bacterial crude enzyme system of aldehyde and ketone reductase

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Effect test

Embodiment 1

[0043] The preparation of embodiment 1 recombinant bacteria crude enzyme system:

[0044] 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.

[0045] 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.

[0046] 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

[0047] The crude enzyme system catalyzes the asymmetric reduction reaction in the aqueous phase reaction system of embodiment 2:

[0048] 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

[0049] The crude enzyme system catalyzes the asymmetric reduction reaction in the aqueous phase reaction system of embodiment 3:

[0050] 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

Aldehyde and ketone reductase crude enzyme system catalytic synthesis of recombinant bacteria ( S )‑N,N‑Dimethyl‑3‑hydroxy‑3‑(2‑thiophene)‑1‑propylamine (( S )‑DHTP), which belongs to the technical field of biocatalytic asymmetric transformation. The present invention is to add auxiliary substrate and initial amount of coenzyme NADP in recombinant bacterial crude enzyme system + Promote the regeneration cycle of the coenzyme NADPH in the system, the reaction substrate is N,N-dimethyl-3-ketone-3-(2-thiophene)-1-propanamine (DTKP), and the recombinant bacteria express the aldehyde and ketone reductase gene of E. coli BL21(DE3)(pETCPAR4), aldehyde and ketone reductase gene cpar4 from Candida parapsilosis ( Candida parapsilosis ) CCTCC NO: M 203011, the gene encodes aldehyde and ketone reductase CPAR4, which catalyzes the asymmetric reduction of DKTP to ( S )‑DHTP. The invention utilizes the cell-free system to catalyze the reaction without additionally adding coupling enzymes required for coenzyme regeneration in the reaction system, improves the efficiency of the direct action of the enzyme and the substrate, shortens the reaction time, and obtains better conversion effects.

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 conversion. Background technique [0002] ( S The chemical structure of )-DHTP is: [0003] 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. [0004] At present, due to different starting materials, there are many methods for the asymme...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P17/00C12N15/53C12N15/70C12N1/21C12R1/19
Inventor 聂尧徐岩
Owner JIANGNAN UNIV
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