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Continuous preparation method of statin pharmaceutical intermediate namely (R)-3-hydroxyethyl glutarate

A technology of ethyl hydroxyglutarate and ethyl hydroxybutyrate, applied in the field of organic drug synthesis, can solve the problems of no nitrilase, difficult screening of biological catalysts, etc., achieve broad industrial production prospects, reduce reaction purification and separation steps, the effect of high reaction efficiency

Active Publication Date: 2015-02-25
NANJING NUOYUN BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the substrate 4-cyano-3-hydroxybutyric acid ethyl ester or its hydroxy-substituted derivatives all contain an ester group, and generally hydrolytic enzyme-containing microbial cells contain esterase or lipase that hydrolyzes the ester group, so The screening of this biocatalyst is also a big problem, and there has been no relevant report on the use of nitrilase in this reaction so far.

Method used

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  • Continuous preparation method of statin pharmaceutical intermediate namely (R)-3-hydroxyethyl glutarate
  • Continuous preparation method of statin pharmaceutical intermediate namely (R)-3-hydroxyethyl glutarate
  • Continuous preparation method of statin pharmaceutical intermediate namely (R)-3-hydroxyethyl glutarate

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Embodiment 1

[0023] Example 1: Such as figure 1 As shown, the preparation device of the present invention comprises a reaction tank 1 and a reaction tank 2, and a stirring device is provided in the reaction tank 1 and 2, and the reaction tank 1 and the reaction tank 2 are connected by a pipeline with a flow regulating valve 6, and the reaction Tank 1 is provided with substrate addition pipeline 1, pH control pipeline 2 and recombinant halohydrin dehalogenase addition pipeline 3, and reaction tank 2 is provided with recombinant nitrilase addition pipeline 4 and product outlet pipe.

Embodiment 2

[0024] Example 2: The actual implementation process of the preparation method of recombinant halohydrin dehalogenase HHDH and nitrilase NIT of the present invention is as follows:

[0025] Inoculate single colonies of recombinant Escherichia coli purchased on the market containing recombinant halohydrin dehalogenase and nitrilase genes into liquid LB medium containing kanamycin resistance, shake at 37°C and shake at 200rpm Cultivate for 7 hours, inoculate the cultured bacterial liquid into the liquid LB medium containing kanamycin resistance, and culture at 37°C with shaking at 200 rpm until OD 600 When the value reaches 1.0, add the inducer isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1mM, continue to cultivate at 28°C for 20 hours, collect the bacteria by centrifugation at 4°C, and add physiological saline to wash The cells were collected twice by centrifugation again, and the cells were suspended with 3 times the volume of triethanolamine buffer ...

Embodiment 3

[0026] Example 3: The actual implementation process of the method preparation method for the continuous preparation of statin drug intermediate (R)-3-hydroxyglutarate ethyl ester of the present invention is as follows:

[0027]Add 45g of substrate (S)-CHBE and 5g of 30% sodium cyanide phosphoric acid solution at pH 7.0 in reaction tank 1 with a volume of 500ml, add 230ml of deionized water, start stirring, and adjust the temperature to 50°C; in 500ml reaction tank 2 , the volume below the overflow port is 400ml, initially add 50ml of deionized water and 5g of crude nitrilase enzyme solution to the lowest stirring blade, start stirring, and adjust the reaction temperature to 25°C. Add 5g of halohydrin dehalogenase crude enzyme liquid into reaction tank 1 to start the reaction. During the reaction, use 30% NaCN solution to control the pH of the system to 7.0. After 6h, use gas chromatography (GC) to detect that the conversion rate of the substrate is 97.3 %, start to add the s...

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Abstract

The invention discloses a continuous preparation method of a statin pharmaceutical intermediate namely (R)-3-hydroxyethyl glutarate. The preparation method comprises the following technological steps: step one, converting (S)-4-chloro-3-hydroxyethyl butyrate ((S)-CHBE) into (R)-4-cyan-3-hydroxyethyl butyrate in the presence of a catalyst namely recombinant halohydrin dehalogenase (HHDH); step two, converting the (R)-4-cyan-3-hydroxyethyl butyrate into (R)-3-hydroxyethyl glutarate in the presence of a catalyst namely recombinant nitrilase (NIT); wherein the two catalytic reactions are both carried out in a water solution in a reaction tank. In the provided method, two steps of coupled bio-catalytic reactions are performed and specific recombinant halohydrin dehalogenase (HHDH) and recombinant nitrilase (NIT) are adopted to carry out continuous reactions so as to prepare a key intermediate of statin. The two green catalytic reactions are serially connected and carried out continuously, and the next reaction can be carried out without extracting the intermediate. The provided method has the advantages of continuous operation, high equipment utilization rate, low cost, and simple and convenient operation.

Description

technical field [0001] The invention relates to the field of organic drug synthesis, in particular to a method for preparing a statin intermediate (R)-3-hydroxyglutaric acid ethyl ester through two-step biocatalytic coupling. Background technique [0002] Statins are the lipid-lowering drugs with the largest sales volume and stable efficacy in the world. The most representative ones are Atorvastatin (Atorvastatin) and Rosuvastatin (Rosuvastatin), the former is the world's first drug with sales exceeding 10 billion U.S. dollars, while the latter has the best lipid-lowering effect so far Statins are also known as "super statins". Rosuvastatin was developed by AstraZeneca (English trade name Crestor), and was first approved in the Netherlands in November 2002. It was approved by the US FDA in August 2003, and its global sales in 2011 were US$6.62 billion. (R)-3-Hydroxyglutaric acid ethyl ester is an important chiral precursor for the synthesis of rosuvastatin. [0003] ...

Claims

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Application Information

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IPC IPC(8): C12P7/62C12R1/19
CPCC12P7/62
Inventor 陈令伟
Owner NANJING NUOYUN BIOLOGICAL TECH CO LTD
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