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A method for enzymatically synthesizing (s)-1,2,4-butanetriol

An enzymatic synthesis, butanetriol technology, applied in the directions of oxidoreductase, fermentation, bulk chemical production, etc., can solve the problems of unfavorable industrialization, high cost, large pollution, etc., and achieves small amount of organic solvent, mild reaction conditions, The effect of green and environmental protection of the reaction system

Active Publication Date: 2022-05-17
江苏惠利生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Among them, methods 1 and 2 apply relatively harsh production conditions, and the raw materials require chiral malic acid, which has high cost and relatively large pollution; method 3 adopts the principle of synthetic biology and fermentation methods, and the current output is still low and the cost is high. , not conducive to industrialization

Method used

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  • A method for enzymatically synthesizing (s)-1,2,4-butanetriol
  • A method for enzymatically synthesizing (s)-1,2,4-butanetriol
  • A method for enzymatically synthesizing (s)-1,2,4-butanetriol

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Preparation of recombinant ketoreductase:

[0038] The gene sequence of the whole gene synthesis is shown as SEQ ID No.1.

[0039] The resulting gene fragment was double-digested with restriction enzymes NdeI and HindIII for 12h; the pET30a(+) vector was double-digested with restriction enzymes NdeI and HindIII for 12h to obtain a linear pET30a(+) vector;

[0040] The double digested gene fragment was ligated with the linear pET30a(+) vector with DNA ligase overnight at 16°C, and the ligated product was transformed into Escherichia coli JM109 competent cells;

[0041] The positive recombinants were screened on the resistance plate containing kanamycin, and the recombinant expression vector was obtained after the positive transformants were picked and sequenced and identified;

[0042] After the recombinant expression vector containing the target gene is transferred into Escherichia coli BL21 (DE3) competent cells, the genetically engineered bacteria that can induce the...

Embodiment 2

[0045] Preparation of (S)-1,2,4-butanetriol:

[0046] Add 100g 1,4-dihydroxy-2-butanone, 40g wet bacteria containing recombinant ketoreductase, 0.2g coenzyme NAD+, 4g glucose dehydrogenase enzyme powder, 120g Anhydrous glucose, dilute to 1L. The reduction reaction was carried out in a water bath at 30°C. During the reaction, 3 mol / L sodium hydroxide solution was added to maintain the pH of the reaction solution at 6.5. TLC detected that the raw material point disappeared, that is, the reaction was complete.

[0047] After the reaction solution was kept at 80°C for 1 hour, it was left to cool in ice water and a large amount of solid precipitated out, which was filtered under reduced pressure.

[0048] The solid was washed twice with 500ml of absolute ethanol, the filtrate and the washings were combined, and the solvent was distilled off under reduced pressure to obtain 118g of a yellow-brown oily product, which was the crude product of (S)-1,2,4-butanetriol.

[0049] The crud...

Embodiment 3

[0051] Preparation of (S)-1,2,4-butanetriol:

[0052] Add 150g 1,4-dihydroxy-2-butanone, 60g wet bacteria containing recombinant ketoreductase, 0.3g coenzyme NAD+, 6g glucose dehydrogenase enzyme powder, 180g anhydrous glucose, dilute to 1L. Carry out the reduction reaction in a water bath at 30°C;

[0053] During the reaction process, 3 mol / L sodium hydroxide solution was added to maintain the pH of the reaction solution at 6.5, and TLC detected that the raw material point disappeared, that is, the reaction was completed.

[0054] After the reaction solution was kept at 80°C for 1 hour, it was left to cool in ice water and a large amount of solid precipitated out, which was filtered under reduced pressure.

[0055] The solid was washed twice with 500ml of absolute ethanol, the filtrate and washings were combined, and the solvent was distilled off under reduced pressure to obtain 166g of a yellow-brown oily product, which was the crude product of (S)-1,2,4-butanetriol.

[0...

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Abstract

The invention discloses a method for enzymatically synthesizing (S)-1,2,4-butanetriol, which comprises adding 1,4-dihydroxy-2-butanediol to a potassium phosphate buffer solution with a pH of 6.0-7.0 Butanone, hydrogen donor, coenzyme, and ketoreductase form a reaction mixture, and react at 25-35°C for 16-24 hours to generate (S)-1,2,4-butanetriol. The reaction conversion rate of the method of the invention is greater than 95%, the product yield is up to 89%, and the chiral purity of the product reaches 99.3% ee. The invention has mild reaction conditions, a small amount of organic solvent, a more environmentally friendly reaction system, a high conversion rate, greatly improves the preparation efficiency of (S)-1,2,4-butanetriol, and has good industrial application prospects.

Description

technical field [0001] The invention belongs to the technical field of pharmaceutical intermediate synthesis, and in particular relates to a method for enzymatically synthesizing (S)-1,2,4-butanetriol. Background technique [0002] (S)-1,2,4-Butanetriol can be used as an important synthetic building block in the synthesis of many natural products, drugs, intermediates, etc. and a synthetic precursor of various chiral compounds, such as cationic lipids for drug delivery The synthetic precursor of plastid, the intermediate of type 2 diabetes drug Empagliflozin, the intermediate of non-small cell lung cancer drug Afatinib, and can also be used for the cholesterol-lowering drug Lipitor and the drug hydroxyeicosan for the treatment of skin diseases Manufacture of various important drugs such as tetraenoic acid (12-HETE). [0003] At present, there are three main methods for the synthesis of (S)-1,2,4-butanetriol that have been reported: [0004] (1) The reduction reaction of L-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/18C12N9/02C12N15/70
CPCC12P7/18C12N9/0008C12N15/70Y02P20/55
Inventor 高新星张鑫郭宏明
Owner 江苏惠利生物科技有限公司
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