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 in

Method used

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

Examples

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Example Embodiment

[0021] Example 1: Reaction monitoring method

[0022] Developed an LC-MS detection method for the determination of 6-cyano-(5R)-hydroxy-3-oxohexanoate tert-butyl to 6-cyano-(3R,5R)-dihydroxyhexanoate tert-butyl Conversion. When testing, take 50μL of the reaction solution at different time points, add 950μL of methanol, mix well, filter with a 0.45μm microporous membrane, and then inject the sample (injection volume 1μL). Chromatographic conditions: 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-oxohexanoate tert-butyl ester is 2.7min, and the retention time of 6-cyano-(3R,5R)-dihydroxyhexanoate tert-butyl ester is 1.8 min. Mass spectrometry conditions: drying gas flow rate 12L / min; sheath gas pressure 40PSI; drying gas temperature 350°C; capillary voltag...

Example Embodiment

[0023] Example 2: Product content detection method

[0024] Preparation of reference substance solution: Weigh 125 mg of 6-cyano-(5R)-hydroxy-3-oxohexanoate reference substance, and weigh 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert The butyl ester reference substance 125mg to a 50mL volumetric flask, dissolved in methanol to a constant volume. Take 3mL, 4mL, 5mL, 6mL, 7mL from the mother liquor and add it to a 10mL volumetric flask, dilute to the mark, filter to a liquid phase vial, and inject 8μL. Sample solution preparation: Weigh 100mg of 6-cyano-(5R)-hydroxy-3-oxohexanoate tert-butyl and 6-cyano-(3R,5R)-dihydroxyhexanoate tert-butyl sample to A 50mL volumetric flask, dissolved in methanol to a constant volume. Filter to liquid phase vial, and inject 8μL. Calculate the content according to the external standard method. Chromatographic conditions: eclipse XDB-C18 column, 4.6×150mm, 5μm; mobile phase A water (0.1% HCOOH), B acetonitrile; 0-10min 25% B; ELSD detector conditio...

Example Embodiment

[0025] Example 3: Gram-level preparation process

[0026] Add 8.0g glucose and 10.00mL buffer solution (100mM, pH=7.00 triethanolamine hydrochloride solution) to a 100.0mL three-necked flask, and place the mixed system on a 30℃ water bath with magnetic stirring. , Drop in 6.83g substrate, stir well and adjust the pH to 7.0 with 2.0M NaOH solution, then add 0.15g glucose dehydrogenase freeze-dried powder and 0.05g ketoreductase-101 freeze-dried powder under 900rpm stirring speed, Adjust the pH to 7.0, and then add 5 mg of NADP lyophilized powder; at the same time maintain the pH at 7.0 with 2.0M NaOH solution to start the reaction. Sampling the control reaction situation every 2-3 hours, after 24 hours LC-MS shows the conversion rate> 97%, the reaction was completed, and 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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