Method for preparing rosuvastatin intermediate

A liquid, ampicillin technology, applied in the field of biological preparation of 3-hydroxyglutaric acid monomethyl ester, can solve the problem of not meeting green chemistry and atom economy, not having industrial application value, low substrate dosage, etc. problems, to achieve the effect of mild conditions, easy operation and high reaction efficiency

Active Publication Date: 2013-10-23
CODEXIS INC
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Problems solved by technology

Although this method can overcome the shortcomings of the chemical method, the target product obtained after the hydrolysis of the racemate has only a theoretical yield of 50%, which means that half of the substrate is wasted, which does not meet the principles of green chemistry and atom economy. And the substrate charging amount of this method is relatively low, only has 20mmol / L, does not have the value of industrial application

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  • Method for preparing rosuvastatin intermediate
  • Method for preparing rosuvastatin intermediate
  • Method for preparing rosuvastatin intermediate

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Experimental program
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Effect test

Embodiment 1

[0027] The preparation of embodiment 1 recombinant nitrilase

[0028] Inoculate a single colony of recombinant Escherichia coli containing the nitrilase gene from a glycerol tube or transformation plate into 4 mL of liquid LB medium containing ampicillin resistance and activate overnight (37°C, 200rpm). Transfer 100mL liquid LB medium containing ampicillin resistance from the overnight culture at 1 / 100 inoculum size, culture at 37°C, 200rpm shaking until OD 600 When the value reaches 0.6-0.8, add IPTG and continue culturing overnight at 30°C. Cells were collected by centrifugation, and suspended in 10 mL of phosphate buffer (2 mM, pH 7.0). The cell suspension was ultrasonically broken in an ice bath for 10 minutes, centrifuged, the supernatant was pre-frozen overnight, and freeze-dried for 24h-48h to obtain the recombinant nitrilase in the form of freeze-dried powder.

Embodiment 2

[0029] The preparation of embodiment 2 gram level (R)-3-hydroxyglutaric acid monomethyl ester

[0030] Add 85ml of phosphate buffer (0.1M, pH8.0) to a 250mL round bottom flask, add 450mg of recombinant nitrilase; weigh 15g of (R)-methyl 4-cyano-3-hydroxybutyrate and use Add a constant current pump to the system, stir the reaction at 30°C for 24 hours, then take a sample for LC-MS analysis, the conversion rate is >99%, stop the reaction, adjust the pH of the reaction solution to acidic (pH=2~3) with hydrochloric acid, Filter through diatomaceous earth, rinse the filter residue with ethyl acetate; extract the filtrate with ethyl acetate, combine the organic phases; dry over anhydrous sodium sulfate, and concentrate to obtain 14g of a yellow-brown oily substance (R)-3-hydroxyglutaric acid mono Methyl ester, yield 81.4%, GC purity 88.2%.

Embodiment 3

[0031] Preparation of 3 kilograms of embodiment (R)-3-hydroxyglutaric acid monomethyl ester

[0032] Add 8.5L phosphate buffer (0.1M, pH8.0) in the reactor, then add 45g recombinant nitrilase; Weigh 1.5Kg (R)-4-cyano-3-hydroxybutyrate methyl ester and adopt A constant flow pump was added to the system, and the reaction was mechanically stirred at 30°C for 24 hours, and then a sample was taken for LC-MS analysis. The conversion rate was >99%, and the reaction was terminated. Adjust the pH of the reaction solution to acidity (pH=2~3) with hydrochloric acid, filter with diatomaceous earth, wash the filter residue with ethyl acetate; extract the filtrate with ethyl acetate, combine the organic phases; dry with anhydrous sodium sulfate, and concentrate to obtain about 1.4Kg The yellow-brown oil is (R)-3-hydroxyglutaric acid monomethyl ester, the yield is 75%, and the GC purity is 88%.

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Abstract

The invention relates to a method for preparing a rosuvastatin intermediate which is (R)-3-hydroxy-monomethyl glutarate and hydroxyl-protected (R)-3-hydroxy- monomethyl glutarate. According to the method, (R)-4-cyano-3-hydroxy-methyl butyrate which servers as a substrate is subjected to reaction under the action of an enzyme to generate the (R)-3-hydroxy- monomethyl glutarate, and particularly the enzyme is the recombinant nitrilase, and the reaction is carried out in a aqueous-phase buffering solution with the pH value of 7.0-9.0 at the temperature of 25-35 DEG C. Because the specific recombinant nitrilase is adopted by the method, the problems of ultralow concentration of the substrate and low conversion rate in the existing method for preparing the rosuvastatin intermediate through biological catalysis are solved. The method has the characteristics of mild reaction conditions and high reaction efficiency, is easy and convenient to operate and has important industrial application value.

Description

technical field [0001] The invention belongs to the technical fields of biological pharmacy and biochemical industry, and in particular relates to a biological preparation method of (R)-3-hydroxyglutaric acid monomethyl ester. Background technique [0002] Cardiovascular and cerebrovascular diseases are one of the most common and serious diseases that endanger human health (especially middle-aged and elderly people). Dyslipidemia is an important risk factor for atherosclerosis, coronary heart disease and other cardiovascular and cerebrovascular diseases. According to the survey of Chinese residents' nutrition and health conducted nationwide by the Ministry of Health in recent years, the overall prevalence rate is 18.6%, and the total number of patients in the country reaches 160 million. [0003] Lipid-lowering drugs can effectively reduce the incidence and mortality of such diseases, and have a positive and far-reaching impact on the prevention and treatment of cardiovascu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/62C12N9/78C12R1/19
CPCC12P7/62
Inventor 陶军华唐苏苏李斌刘根
Owner CODEXIS INC
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