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Method for preparing dapoxetine intermediate by using carbonyl reductase

A technology of dapoxetine and reductase is applied in the field of preparing dapoxetine intermediates by using carbonyl reductase, and can solve the problems of cheap preparation of unfavorable intermediates, complicated steps, low product yield and the like

Inactive Publication Date: 2020-07-07
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, chemical synthesis is the main method at present, but the method has cumbersome steps, low product yield and high pollution, and is not suitable for large-scale production (CN103396320)
The current biological preparation method of (R)-3-chlorophenylpropanol needs to add an additional expensive coenzyme NADH or use glucose dehydrogenase to regenerate the coenzyme in the catalytic process, which is not conducive to the cheap preparation of this intermediate

Method used

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  • Method for preparing dapoxetine intermediate by using carbonyl reductase
  • Method for preparing dapoxetine intermediate by using carbonyl reductase
  • Method for preparing dapoxetine intermediate by using carbonyl reductase

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1: Synthesis of gene encoding carbonyl reductase

[0031] According to the original gene sequence of carbonyl reductase (NCBI Reference Sequence: WP_011906790.1), the codon optimization of the original gene sequence was carried out according to the codon analysis table of Escherichia coli and the total synthesis of the target gene was carried out. Shanghai) Co., Ltd., the obtained coding gene sequence is shown in SEQ ID NO.1, and the amino acid sequence of the translated carbonyl reductase is shown in SEQ ID NO.2. Both ends of the synthesized coding gene were cut with BamI and XhoI sites respectively and connected to pET30a(+) to obtain the recombinant expression vector pET30a(+)-Nov (see figure 2 ).

Embodiment 2

[0032] Example 2: Expression of carbonyl reductase

[0033] LB plate: peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, 1.5% agar, solvent is deionized water, pH7.2.

[0034] LB culture medium: peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, solvent is deionized water, pH7.2.

[0035] The recombinant expression vector pET30a(+)-Nov constructed in Example 1 was transformed into Escherichia coli BL21(DE3) by transformation method to obtain recombinant Escherichia coli BL21(DE3)-Nov. Spread onto LB plates containing 50 μg / mL kanamycin and culture overnight at 37°C. On the next day, pick a single colony, inoculate the single colony into 5 mL of LB culture solution supplemented with 50 μg / mL kanamycin, and cultivate overnight at 37° C. with shaking. The next day, take 1 mL of the bacterial liquid and add it to 100 mL of LB culture solution containing 50 μg / mL kanamycin, and culture it with shaking at 37 °C until the OD600 reaches 3.0, then add IPTG to a final co...

Embodiment 3

[0036] Example 3: Kinetic parameters of carbonyl reductase

[0037] Under standard conditions (20° C., 101 KPa), the enzyme activity was measured by changing the concentration of the substrate in the reaction system of Example 4, and the corresponding kinetic constants were calculated according to the double reciprocal plotting method. The substrates used in the calculation of kinetic constants and their concentrations are as follows: (3)-chloro-(1)-propiophenone (0-50.0 mM). The apparent kinetic parameters of the corresponding substrates are shown in Table 1. Each unit of enzyme can catalyze 4.945mM substrate, and it only takes 0.11 seconds to catalyze 1 molecule of substrate.

[0038] Table 1 Apparent kinetic parameters of carbonyl reductase Nov

[0039]

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Abstract

The invention discloses a method for preparing dapoxetine intermediate by using carbonyl reductase. In the method, a crushed mixed solution of wet cells obtained by induction culture of engineered bacteria containing the carbonyl reductase gene after ultrasonication is used as a catalyst, and 3-chlorophenylacetone is used as a substrate, and NADH and isopropanol are added, and a phosphate buffer with a pH of 6.5. -7.5 is used as a reaction medium to constitute a reaction system. The reaction system is complete with magnetic stirring at 30-40 DEG C. The reaction solution is separated and purified to obtain (R)-3-chlorophenylpropanol. The invention adopts a new type of carbonyl reductase to catalyze a reduction reaction, has the advantages of small amount of biocatalyst, mild reaction conditions (30-40 DEG C), short reaction time (6-12 hours), 95.2% of yield coefficient, and good optical purity (greater than 99), and the like.

Description

[0001] (1) Technical field [0002] The invention relates to the technical field of biopharmaceuticals, in particular to a method for preparing a dapoxetine intermediate by using carbonyl reductase. [0003] (2) Background technology [0004] Dapoxetine (Dapoxetine, trade name Priligy), chemical name S-(+)-N,N-dimethyl-a-[2-(naphthyloxy)ethyl]benzylamine hydrochloride (N,N-dimethyl-a-[2-(naphthalenyloxy)ethyl]benyenemethanamine hydrochloride), a fast-acting and metabolized selective serotonin reuptake inhibitor (SSRI), is a drug for the treatment of 16 Drugs for premature ejaculation in men up to 84 years old. Dapoxetine was first developed by Eli Lilly and Company in the United States, and was sold to Johnson & Johnson in 2003, and was submitted to the US Food and Drug Administration for approval in 2004 as a drug for the treatment of premature ejaculation. [0005] The chemical structural formula of dapoxetine is as follows: [0006] [0007] (R)-3-Chlorophenylpropanol ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/22C12N15/53C12N15/70
CPCC12P7/22C12N9/0006C12Y101/01184C12N15/70
Inventor 邵泽辉王逸峰杨胜利
Owner ZHEJIANG UNIV OF TECH
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