Method for preparing (R)-2-bromo-1-(4-nitrophenyl) ethanol

A technology of nitrophenyl and nitroacetophenone, which is applied in the field of bioengineering, can solve the problems of low conversion rate of substrate, low optical purity of product, recycling of coenzyme, etc., and achieve the effect of reducing production cost

Inactive Publication Date: 2014-03-19
JIANGNAN UNIV
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Problems solved by technology

[0004] Therefore, in the current technology of using biocatalysis to prepare (R)-2-bromo-1-(4-nitrophenyl)ethanol, the substrate conversion rate is low, the product optical purity is low and the coenzyme cycle regeneration (control cost), etc. The problem needs to be further studied and solved

Method used

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  • Method for preparing (R)-2-bromo-1-(4-nitrophenyl) ethanol

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Embodiment 1

[0017] The determination of embodiment 1 enzyme activity

[0018] Carbonyl reductase (CBR) activity measurement: the reaction system is 250μl 0.1mol / L potassium phosphate buffer (pH7.0), 0.2mM NAD(P)H, 5mM substrate and appropriate amount of crude enzyme solution. Immediately after adding the enzyme solution, start scanning the change of the absorbance of the reaction system at 340nm. Enzyme activity definition: the amount of enzyme required to oxidize 1 μmol NAD(P)H per minute is one enzyme activity unit (U).

[0019] Determination of glucose dehydrogenase (GDH) activity: the reaction is 250μl 0.1mol / L potassium phosphate buffer (pH7.0), 0.2mM NADP + , 0.1M substrate glucose and appropriate amount of crude enzyme solution. Immediately after adding the enzyme solution, start scanning the change of the absorbance of the reaction system at 340nm. Enzyme activity definition: reduce 1 μmol NADP per minute + The required amount of enzyme is one enzyme activity unit (U). At the...

Embodiment 2

[0020] Embodiment 2 recombinant bacteria BL21 (pET28a-cbr), the fermentation of BL21 (pET28a-gdh)

[0021] Inoculate the recombinant bacteria BL21(pET28a-cbr) and BL21(pET28a-gdh), in 2ml of LB liquid medium containing 100μg / ml kanamycin, shake culture at 37°C, 220r / min for 12h. Transfer 300ul of the culture solution to 30ml of LB liquid medium containing 100μg / ml kanamycin and culture at 37°C with shaking at 220r / min until OD 600 When the concentration is 0.6-0.8, add IPTG to the culture to a final concentration of 1.0mmol / L, shake at 25°C and 220r / min for 10h, and collect the bacteria by centrifugation at 4°C for later use.

Embodiment 3

[0022] The detection method of embodiment 3 products

[0023] Get the thallus of Example 2 and wash twice with potassium phosphate buffer (100mmol / L, pH7.0), fix and weigh 0.5g (wet weight) of recombinant Escherichia coli BL21 (pET28a-cbr) thalline, add 2 times the amount The specific E. coli BL21 (pET28a-gdh) cells were suspended in 10 ml of pH 7.0 potassium phosphate buffer (containing 500 μl of dimethyldimethylamine). Add 0.1M glucose, 10g / L 2-bromo-4-nitroacetophenone, NAD(P)H0.5mmol / L, 37°C, 200rpm, the reaction is over for 12h, and the product (R)2-bromo-1-(4 The productive rate of -nitrophenyl)ethanol is 9.72g / L, and its productive rate is determined to be 97.2%, and the optical purity ee% is 99.9%.

[0024] After the water / organic solvent two-way system reaction was completed, 5ml of ethyl acetate was added, shaken vigorously for 5min, and left to separate the organic layer and the aqueous layer. Aspirate the upper ethyl acetate phase, add an appropriate amount of an...

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Abstract

The invention discloses a method for biological preparation of (R)-2-bromo-1-(4-nitrophenyl) ethanol by using recombinant escherichia coli to express carbonyl reductase, and an application of the method in preparation of (R)-2-bromo-1-(4-nitrophenyl) ethanol through asymmetric reduction. Escherichia coli BL21 (pET28a-cbr) for expressing genes of carbonyl reductase (CBR) is used for coupling glucose dehydrogenase (GDH) to ensure that escherichia coli BL21 (pET28a-gdh) is recombined into a catalyst, and a small amount of NAD(P)H is added to realize asymmetric reduction of 2-bromo-4-nitro-acetophenone in a two-phase reaction system of an organic solvent namely dimethyl dimethylamine and water so as to efficiently prepare (R)-2-bromo-1-(4-nitrophenyl) ethanol, wherein the molar conversion rate is higher than 95%, and the enantiomeric excess (e.e.) of the product is more than 99%.

Description

technical field [0001] The invention relates to a method for preparing (R)-2-bromo-1-(4-nitrophenyl)ethanol by catalyzing biological enzymes, belonging to the technical field of bioengineering. Background technique [0002] (R)-2-bromo-1-(4-nitrophenyl)ethanol molecular formula is C 8 h 8 BrNO 3 , English name R-2-bromo-1-(4-nitrophenyl)ethanol, and (S)-2-bromo-1-(4-nitrophenyl)ethanol are enantiomers of each other, is an important The chiral compound is an important intermediate in the synthesis of β-adrenergic receptor blocker-(R)-nifevolol. [0003] The preparation methods of this kind of compound include resolution method and synthesis method. The resolution method includes chemical resolution and biological resolution, but the maximum yield does not exceed 50%; the synthesis method includes chemical synthesis and biosynthesis, and the yield of the chemical synthesis method is 70-90%. The (ee) value is 50-80%, the catalyst used is expensive, the optical purity of th...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P7/22C12R1/19
Inventor 邬敏辰余涛董运海李剑芳
Owner JIANGNAN UNIV
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