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Method for synthesizing chiral bisaryl alcohol compound

A technology of biaryl alcohol and compound, applied in the field of synthesis of chiral biaryl alcohol compound, can solve the problems of low efficiency, unreported species source, low yield and the like

Active Publication Date: 2019-06-04
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the efficiency of these whole-cell transformation methods is low, and there is no report on the amino acid sequence of the relevant enzyme
In 2007, Truppo et al. reported the research on the synthesis of (S)-(4-chlorophenyl)pyridine-2-methanol by using a commercial carbonyl reductase (ketoreductase, KRED) to asymmetrically reduce prochiral ketones, but the ee value was only 60 %, the sequence of the enzyme protein is unknown, and the source of the species has not been reported (Truppo et al., Org. Lett., 2007, 9(2):335-338)
[0006] From the above data, it can be seen that the preparation of chiral (S)-(4-chlorophenyl)pyridine-2-methanol by biological asymmetric reduction method not only has a low yield, but also the stereoselectivity does not meet the requirements of industrialization, and further development of high-efficiency alcohols is needed. Dehydrogenase

Method used

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  • Method for synthesizing chiral bisaryl alcohol compound
  • Method for synthesizing chiral bisaryl alcohol compound
  • Method for synthesizing chiral bisaryl alcohol compound

Examples

Experimental program
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Effect test

Embodiment 1

[0057] Embodiment 1, the preparation of the engineering bacteria of carbonyl reductase SSCR and alcohol dehydrogenase TbSADH gene or its mutant

[0058] 1. Preparation of wild-type genetically engineered strains of carbonyl reductase SSCR and alcohol dehydrogenase TbSADH genes

[0059] The carbonyl reductase SSCR gene derived from Sporobolomyces Salmonicolor and the thermoanaerobacter brockii alcohol dehydrogenase TbSADH were codon-optimized using Escherichia coli as the host cell, and optimized by Twist Bioscience (USA ) or GenScript (Nanjing) whole gene synthesis, the optimized sequences are SEQ ID No.1 and SEQ ID No.3 respectively.

[0060] The two target genes were respectively connected into the pET22b(+) plasmid through NdeI and HindIII restriction sites to obtain a recombinant vector. Electrotransform the recombinant vector pET22b-SSCR or pET22b-TbSADH into E.coli BL21(DE3) competent cells, culture them upside down on LB solid plates containing ampicillin (Amp) resista...

Embodiment 2

[0085] Example 2, Expression of carbonyl reductase SSCR and alcohol dehydrogenase TbSADH genes or their mutants and preparation of crude enzyme powder

[0086] Pick the transformants of recombinant bacteria E.coli BL21 (recombinant plasmids of carbonyl reductase SSCR or alcohol dehydrogenase TbSADH genes or their mutants) into 5mL LB liquid medium containing 50μg / mL ampicillin, shake at 37°C and 220rpm Overnight for 12-16 hours, according to the inoculation amount of 1% (volume percentage content), inoculate them into TB liquid medium containing 50 μg / mL ampicillin, and culture at 37°C until OD 600 When it was 0.7, add IPTG with a final concentration of 0.1mmol / L, induce expression at 20°C and 220rpm for 18h, then centrifuge at 4°C and 4000rpm for 10min to collect the bacteria. The collected bacteria were resuspended with potassium phosphate buffer (100 mM, pH 7.0) for use. Cells were sonicated in an ice bath. Centrifuge the sonicated sample at 4°C, 12000 rpm, for 10 min, an...

Embodiment 3

[0087] Example 3. Carbonyl reductase SSCR / alcohol dehydrogenase TbSADH and its mutants catalyze (4-chlorophenyl)pyridine-2-methanone to generate (S)-(4-chlorophenyl)pyridine-2-methanol

[0088] Carbonyl reductase SSCR / alcohol dehydrogenase TbSADH catalyzes the reduction of (4-chlorophenyl)pyridine-2-methanone to generate (S)-(4-chlorophenyl)pyridine-2-methanol. figure 1 shown.

[0089] Determination of the catalytic effect of carbonyl reductase SSCR or alcohol dehydrogenase TbSADH asymmetric reduction of prochiral bisaryl ketone (4-chlorophenyl) pyridine-2-methanone, wherein the substrate concentration is 10mmol / L, recombinant carbonyl reduction The dosage of enzyme SSCR or alcohol dehydrogenase TbSADH is 10g / L (crude enzyme powder), the dosage of isopropanol is 10% of the total volume, NAD(P) +The dosage is 1 mmol / L, the concentration of the phosphate buffer is 100 mmol / L, and the pH is 7.0. The temperature of the asymmetric reduction reaction was 30° C., and the reaction w...

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Abstract

The invention discloses a method for synthesizing a chiral bisaryl alcohol compound. The method provided by the present invention specifically uses a mutant of a carbonyl reductase SSCR derived from Sporobolomyces Salmonicolor and a mutant of alcohol dehydrogenase TbSADH derived from Thermoanaerobacter brockii to catalyze asymmetric reduction of (4-chlorophenyl)pyridine-2-ketone to form (S)-(4-chlorophenyl) pyridine-2-methanol. The experiment proves that the method provided by the invention has high yield and high ee value, can reduce the addition amount of coenzyme, and does not need to add enzyme such as glucose alcohol dehydrogenase for cofactor circulation, the reaction condition is mild, and the operation is simple.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a synthesis method of a chiral bisaryl alcohol compound, in particular to two alcohol dehydrogenases, including a carbonyl reductase SSCR derived from Sporobolomyces Salmonicolor and A mutant of alcohol dehydrogenase TbSADH derived from Thermoanaerobacter brockii, and the application of the corresponding mutant as a catalyst in the preparation of optical bisaryl secondary alcohols by asymmetric reduction. Background technique [0002] Chiral bisaryl secondary alcohols are an important class of chiral compounds. Many drugs have a block structure of bisaryl secondary alcohols. Among them, (S)-(4-chlorophenyl)pyridine-2 -Methanol is a synthetic chiral precursor of antihistamines, which can be used to synthesize antihistamines carbinoxamine (Barouh et al., J.Med.Chem.,1971,14(9):834-836) and bepotastine besilate (Takahashi et al., Clin. Exp. Dermatol., 2004, 29(5):526-532). Moreover, the...

Claims

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Application Information

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IPC IPC(8): C12P17/12C12N9/04C12N15/53
Inventor 孙周通刘保艳刘贝贝曲戈
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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