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Biological preparation method of 6-cyano-(3R, 5R)-dihydroxyhexanoate

A technology of tert-butyl hydroxyhexanoate and tert-butyl hexanoate is applied in the field of biological preparation of tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate, which can solve the problem of large amount of enzyme and substrate Low concentration, high production cost and other problems, to achieve the effect of reducing enzyme dosage, increasing substrate concentration and reducing production cost

Active Publication Date: 2013-04-03
ENZYMEWORKS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, there are problems such as low substrate concentration and large enzyme dosage in this process. For example, in the example of preparing the product in this patent, the initial substrate concentration is 300mg / mL, and the enzyme powder dosage is 1.2% of the substrate. The amount of coenzyme is 0.05% of the substrate, which makes the production cost higher

Method used

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  • Biological preparation method of 6-cyano-(3R, 5R)-dihydroxyhexanoate

Examples

Experimental program
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Effect test

Embodiment 1

[0021] Embodiment one: reaction monitoring method

[0022] A LC-MS detection method was developed for the determination of tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate to tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate transform. For detection, take 50 μL of reaction solution at different time points, add 950 μL of methanol, mix well, filter with 0.45 μm microporous membrane, and then inject for detection (injection volume 1 μL). Chromatographic conditions: chromatographic column SB-C182.1×50mm, 3.5μm; mobile phase A water (10mM HCOONH4-0.1%HCOOH), B acetonitrile; flow rate 0.3mL / min; mobile phase gradient 0-0.5min 22%B, 0.5 -0.6min 22%B-100%B, 0.6-1.0min 100%B, 1.0-1.1min 100%B, 1.1-5.0min 22%B. The retention time of 6-cyano-(5R)-hydroxy-3-oxohexanoic acid tert-butyl ester is 2.7min, and the retention time of 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl ester is 1.8 min. Mass spectrometry conditions: dry gas flow rate 12L / min; sheath gas pressure 40PSI; dry gas tempe...

Embodiment 2

[0023] Embodiment two: product content detection method

[0024] Preparation of reference substance solution: Weigh 125 mg of 6-cyano-(5R)-hydroxy-3-oxohexanoic acid tert-butyl ester reference substance, and weigh 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl Butyl ester reference substance 125mg was transferred to a 50mL volumetric flask, dissolved in methanol to volume. Take 3mL, 4mL, 5mL, 6mL, 7mL from the mother liquor and add it into a 10mL volumetric flask, set the volume to the mark, filter to the liquid phase vial, and inject 8μL. Preparation of sample solution: Weigh 100 mg of tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate and 100 mg of tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate to 50mL volumetric flask, dissolved in methanol to volume. Filter into a liquid phase vial and inject 8 μL. The content was calculated according to the external standard method. Chromatographic conditions: chromatographic column eclipse XDB-C18, 4.6×150mm, 5μm; mobile phase A water ...

Embodiment 3

[0025] Embodiment three: Gram-level preparation process

[0026] Add 8.0g of glucose and 10.00mL of buffer solution (100mM, pH=7.00 triethanolamine hydrochloride solution) to a 100.0mL three-neck flask in sequence, and place the mixed system on a 30°C water bath with magnetic stirring to dissolve it. , drop 6.83g of the substrate, stir evenly, adjust the pH to 7.0 with 2.0M NaOH solution, then add 0.15g of glucose dehydrogenase lyophilized powder and 0.05g of ketoreductase-101 lyophilized powder at a stirring speed of 900rpm, Adjust the pH to 7.0, and then add 5 mg of NADP freeze-dried powder; at the same time, use 2.0M NaOH solution to maintain the pH at 7.0 to start the reaction. Sampling of the control reaction every 2-3 hours, after 24 hours, LC-MS showed that the conversion rate was >97%, and the reaction was terminated, and then the content of the product was detected by the method described in Example 2.

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Abstract

The invention relates to a biological preparation method of 6-cyano-(3R, 5R)-dihydroxyhexanoate. According to the biological preparation method, 6-cyano-(6R)-hydroxyl-3-oxo-tertiary butyl hydroperoxide is used as a substrate; under the condition that a biocatalyst, a cofactor and a cofactor regeneration system exist, the substrate performs the reduction reaction to generate 6-cyano-(3R, 5R)-dihydroxyhexanoate; and the reduction reaction is carried out in water-phase buffer solution with pH of 6 to 8. According to the invention, by optimizing the process condition and mainly optimizing the pH of the reduction reaction, the concentration of the substrate is improved and the enzyme dosage is reduced, so that production cost is reduced and the high-efficiency biological conversion process is realized.

Description

Technical field: [0001] The invention belongs to the fields of biopharmaceuticals and green chemistry, and specifically relates to a biological preparation method of tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate. Background technique: [0002] Cholesterol-lowering statins are currently the world's best-selling drug class. The world's best-selling drug in 2010, Pfizer's Lipitor (trade name Lipitor, with annual sales of 11.9 billion US dollars) has an effective activity of atorvastatin (Nature 2012, 485:185-194). tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate is a key chiral intermediate used in the synthesis of this class of cholesterol-lowering statins. At present, it can be prepared by chemical method or enzymatic method (Angew. Chem. Int. Ed. 2005, 44:362-365). [0003] The current chemical process to produce tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate is to use borane derivatives to convert 6-cyano-(5R)-hydroxyl- Reduction of tert-butyl 3-oxohexanoate. This process r...

Claims

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Application Information

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IPC IPC(8): C12P13/00
CPCY02P20/584
Inventor 乐庸堂鞠鑫梁长辉庄继昌
Owner ENZYMEWORKS
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