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Method for asymmetric synthesis of duloxetine intermediate by carbonyl reductase

A carbonyl reductase, asymmetric technology, applied in the field of biocatalysis, can solve the problems of low level, low efficiency, and limited industrial application

Active Publication Date: 2016-07-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, when C.tropicalis and C.viswanathii catalyze 1g / LDKTP to asymmetrically generate (S)-DHTP, the conversion rate is about 80%, and the enantiomeric excess value is >99%, but the reaction time is as long as 60h, and the efficiency is low, which greatly limits industrial application
At present, there are few studies on chiral intermediates of duloxetine in China, and the level is low

Method used

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  • Method for asymmetric synthesis of duloxetine intermediate by carbonyl reductase
  • Method for asymmetric synthesis of duloxetine intermediate by carbonyl reductase
  • Method for asymmetric synthesis of duloxetine intermediate by carbonyl reductase

Examples

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Embodiment 1

[0049] Example 1: Expression and purification of recombinant carbonyl reductase

[0050] LB medium (g / L): yeast extract 5.0, peptone 10.0, NaCl 10.0, pH 7.0 (20.0 agar added to solid medium). Sterilization condition of medium: 1×10 5 Pa, sterilize for 30min.

[0051] Recombinant bacteria construction: the carbonyl reductase pure enzyme gene cr2 (amino acid sequence genebank: AB183149) from Candidamacedoniensis AKU4588 was obtained from the full-length DNA fragment by PCR based on the gene sequence, and the carbonyl reductase gene cr2 was respectively treated by NdeI and XhoI double enzyme digestion DNA fragment and expression vector pET21c, using ligase to connect the cohesive ends of the DNA fragment of the target gene and the vector fragment, obtain the recombinant plasmid pET21c-cr2, and further transform the competent Escherichia coli E.coliBL21 to obtain the recombinant strain E.coliBL21 / pET21c-cr2 .

[0052] Pick a single colony of E.coliBL21 / pET21c-cr2 and inoculate ...

Embodiment 2

[0054] Embodiment 2: the optimal pH value of recombinant carbonyl reductase CR2

[0055] Enzyme activity assay system: 100μL system, 0.5mmol / L NAD(P)H, 5mmol / L substrate, 30°C constant temperature for 3min, finally add 20U / L pure enzyme solution and mix well, start scanning the change of absorbance value at 340nm.

[0056] The relative enzymatic activity of recombinant carbonyl reductase CR2 was determined under different pH gradients (6.0-9.0) ( figure 1 ), the buffer used is 0.1mol / L phosphate buffered saline (PBS) (pH6.0-8.0), 0.1mol / L Tris-HCl buffer (pH7.0-8.5) and 0.1mol / L triethanolamine buffer ( TEA) (pH7.5-9.0). The research results show that the pH value has a greater impact on the enzyme activity of CR2 in the range of less than 7.0; between pH7.5-9.0, the relative enzyme activity of CR2 is above 50%; at pH8.4 (0.1mol / LTEA) CR2 has the highest catalytic activity of DKTP.

Embodiment 3

[0057] Embodiment 3: the optimum temperature of recombinant carbonyl reductase CR2:

[0058] Enzyme activity assay system: 100μL system, 0.1mol / L Tris-HCl buffer (pH7.5), 0.5mmol / L NAD(P)H, 5mmol / L substrate, kept at different temperatures for 3min, and finally added 20U / L pure enzyme After the liquid is mixed evenly, start to scan the change of absorbance value at 340nm.

[0059] The relative enzymatic activity of recombinant carbonyl reductase CR2 was determined under different temperature gradients (20-80°C) ( figure 2 ). The study found that in the range of 25-40°C, the relative activity of CR2 was higher and remained above 80%; in the range of 20-35°C, as the temperature increased, the activity of CR2 continued to increase, and at 35°C The activity reached the maximum; when the temperature was higher than 40°C, the activity of CR2 began to decrease, and when the temperature was higher than 55°C, it was completely inactivated.

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Abstract

The invention belongs to the technical field of biocatalysis and discloses a method for asymmetric synthesis of duloxetine intermediate by carbonyl reductase.In a pure enzyme catalytic system, by control of reaction pH, reaction temperature and metal ions, biocatalysis performance is improved.N,N-dimethyl-3-keto-3-(2-thienyl)-1-propylamine (DKTP) serves as a reaction substrate, a recombinant strain refers to E.coli BL21 / Pet21c-cr2 expressing an aldo-keto reductase gene, a carbonyl reductase gene cr2 is derived from Candida macedoniensis AKU4588 and codes carbonyl reductase CR2, and catalysis of asymmetric reduction of DKTP is realized to obtain (S)-DHTP.By pure enzyme catalytic reaction and optimal control of the reaction pH, the reaction temperature and the metal ions, properties and functions of DKTP catalyzed by CR2 can be known, highly-stereoselective DKTP catalysis is realized, and optically-pure duloxetine key intermediate (S)-DHTP is prepared through asymmetric conversion reaction.

Description

technical field [0001] The invention relates to a method for asymmetrically synthesizing duloxetine intermediates by using carbonyl reductase, and belongs to the technical field of biocatalysis. Background technique [0002] The chemical structure of (S)-DHTP is: [0003] [0004] Duloxetine, English name (R)-Duloxetine, chemical name (S)-N-methyl-3-(1-naphthyloxy)-3-(2-thienyl)-1-propanamine, trade name Xin Pictet, is a dual serotonin and norepinephrine reuptake inhibitors (SNRIs). Clinical application of its hydrochloride treatment of depression, stress urinary incontinence, depression associated with chronic pain, diabetic peripheral neuropathic pain. Of the two isomers with the same chemical composition of duloxetine, only the (S)-form has the aforementioned pharmacological activity. Currently, there are many methods for the asymmetric synthesis of chiral duloxetine. Through retrosynthetic analysis, it can be found that (S)-N,N-dimethyl-3-hydroxyl-3-(2-thiophene)-...

Claims

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Application Information

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IPC IPC(8): C12P17/00
CPCC12P17/00
Inventor 聂尧徐岩王越
Owner JIANGNAN UNIV
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