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

A technology of tert-butyl hydroxycaproate and tert-butyl hexanoate, applied in the field of biopharmaceuticals, can solve the problems of complicated operation steps and high cost, and achieve the effects of simplifying operation steps, reducing production process and low raw material price

Inactive Publication Date: 2014-04-02
HUANGGANG HUAYANG PHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When the applicant realized the present invention, he found that the raw material used in the process was (5R)-6-cyano-5-hydroxyl-3-oxohexanoic acid tert-butyl ester, and the operation steps were cumbersome and costly.

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: the composite enzyme dry powder shake flask production process of recombinant ketoreductase and glucose dehydrogenase

[0026] Inoculate a single colony of Escherichia coli carrying a ketoreductase gene and a glucose dehydrogenase gene from a glycerol tube or transformation plate into 10ml of liquid LB medium containing double resistance to ampicillin and kalamycin, at 37°C Activation on a shaking table for 12 hours (150rpm), inoculate the above culture into 100ml liquid LB medium containing double resistance to ampicillin and kalamycin at 1 / 100 inoculum amount, and expand the culture on a shaking table at 37°C ( 150rpm), to OD 600When the value reaches 1, add isopropyl-β-D-thiogalactoside, continue to incubate at 37°C for 6 hours, centrifuge, collect the precipitate, add 10ml of triethanolamine hydrochloride buffer (0.1M, pH=7.0) Suspension, put the suspension in an ice-water bath for 15 minutes to ultrasonically break the wall, then centrifuge, pre-free...

Embodiment 2

[0027] Embodiment 2: production process of dehalogenase dry powder shake flask

[0028] Inoculate 10 ml of ampicillin-containing liquid LB medium from a glycerol tube or a single colony on a transformation plate (a single colony of recombinant Escherichia coli carrying a dehalogenase gene), and activate it on a shaker at 37°C for 12 hours (150rpm). The culture was inoculated into 100ml liquid LB medium containing ampicillin at 1 / 100 inoculum, and expanded on a shaking table (150rpm) at 37°C until OD 600 When the value reaches 1, add isopropyl-β-D-thiogalactoside, continue to incubate at 37°C for 6 hours, centrifuge, collect the precipitate, add 10ml of triethanolamine hydrochloride buffer (0.1M, pH=7.0) Suspension, the suspension is placed in an ice-water bath for 15 minutes to ultrasonically break the wall, and then centrifuged, the supernatant is pre-frozen until the temperature drops to -20°C, and then freeze-dried for 24 hours to obtain a dry powdered dehalogenase.

Embodiment 3

[0030] In the reaction vessel, 50 ml of 0.1M triethanolamine hydrochloride buffer solution with a pH of 7 was first added, followed by adding 0.15 g of recombinant ketoreductase and glucose dehydrogenase compound enzyme (prepared in Example 1), and dehalogenase (prepared in Example 2) ) 0.15g, 10mg NAD, 15g glucose and 10g (5R)-6-chloro-5-hydroxy-3-oxohexanoic acid tert-butyl ester, stir well, and maintain pH6.8~7.2 with 30% NaCN aqueous solution , stirred at room temperature for 12 hours and the reaction was completed. Ethyl acetate was added for multiple extractions. The organic phases were combined and the solvent was evaporated under reduced pressure to obtain 8.91 g of (3R,5R)-6-cyano-3,5-dihydroxyhexyl Acid tert-butyl ester, a light yellow oily liquid.

[0031] product structure 1 H NMR, 13 C NMR and ESI-HRMS detection, the results are as follows: 1 H NMR (400MHz, CDCl 3 ):δ=4.25(bs, 1H), 4.34-4.23(m, 1H), 4.21(bt, 1H), 3.96(bd, 1H), 2.55(dd, J = 5.5, 11.1 Hz, 2H),...

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Abstract

The embodiment of the invention provides a biological preparation method of (3R, 5R)-6-cyano-3,5-dyhydroxytert-butylhexanoate and belongs to the field of biological pharmacy. The preparation method comprises the following steps: by taking (5R)-6-chloro-5-hydroxy-3-tert-butyloxohexanoate as a raw material in a triethanolamine hydrochloride buffer solution, maintaining the pH value to be 6-8 under the actions of a complex enzyme of recombinant ketoreductase and glucose dehydrogenase, dehalogenase, a cofactor and glucose by using a cyaniding reagent, and reacting to obtain the (3R, 5R)-6-cyano-3,5-dyhydroxytert-butylhexanoate at room temperature. According to the preparation method provided in the invention, the (5R)-6-chloro-5-hydroxy-3-tert-butyloxohexanoate is taken as the raw material, a reduction reaction and a cyano substitution reaction are finished to obtain the product through a one-pot method under the catalysis of the complex enzyme of recombinant ketoreductase and glucose dehydrogenase and the dehalogenase, and the method is simple in process and low in production cost.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to a biological preparation method of atorvastatin intermediate (3R,5R)-6-cyano-3,5-dihydroxyhexanoic acid tert-butyl ester. Background technique [0002] Statins reduce low-density lipoprotein cholesterol (LDL-C) levels by inhibiting HMG-CoA reductase in the cholesterol synthesis pathway, and have a good effect on hyperlipidemia mainly caused by elevated cholesterol. Widely used lipid-regulating drugs. Atorvastatin (Atorvastatin) has become a new third-generation, fully synthetic, highly purified, and highly selective HMG-CoA reductase inhibitor due to its advantages of rapid onset of action, strong lipid-lowering effect, and long action time. The most promising statin drugs in the market. And (3R,5R)-6-cyano-3,5-dihydroxyhexanoic acid tert-butyl ester is the most important intermediate of atorvastatin, therefore, the development of (3R,5R)-6-cyano-3 , The synthetic...

Claims

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Application Information

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IPC IPC(8): C12P13/00
Inventor 张龑熊进军葛石平
Owner HUANGGANG HUAYANG PHARMA
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