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Method for preparing chiral intermediate of atorvastatin

A technology of cyano, tert-butyl hydroxycaproate, applied in microorganism-based methods, biochemical equipment and methods, introduction of foreign genetic material using carriers, etc., can solve the complex preparation process of crude enzyme liquid and the catalytic efficiency of crude enzyme liquid. low cost, hindering large-scale production, etc., to achieve the effect of low price, simplified process, and increased maximum substrate loading

Inactive Publication Date: 2014-07-09
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in actual industrial production, there are many obstacles in the catalytic process, such as the need to add high-cost nicotinamide coenzyme NADPH, the low catalytic efficiency of the crude enzyme solution and the complicated preparation process of the crude enzyme solution, etc. A series of problems hindering mass production, such as poor economy and low production efficiency

Method used

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  • Method for preparing chiral intermediate of atorvastatin
  • Method for preparing chiral intermediate of atorvastatin
  • Method for preparing chiral intermediate of atorvastatin

Examples

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Effect test

Embodiment 1

[0033] Construction of Gene Recombinant E. Coli

[0034]The carbonyl reductase gene cr1 from Saccharomyces cerevisiae and the glucose dehydrogenase gene gdh from Bacillus megaterium were fully synthesized, and restriction enzyme endonuclease sites were inserted in the upstream and downstream of the gene sequence: co-expression vector pETDeut-1 No. 1 multiple The restriction sites used to insert foreign genes on the cloning site (MCS1) are Nco I and BamH I sites; the restriction sites used to insert foreign genes on the No. 2 multiple cloning site (MCS2) are Nde I and Xho I. The carbonyl reductase gene and the glucose dehydrogenase gene were respectively inserted into the No. 1 and No. 2 multiple cloning sites (MCS) of the prokaryotic co-expression vector by using the enzyme cutting-ligation method familiar to those skilled in the art. After verification and identification, the positive plasmids were divided into two schemes according to the different gene insertion sites. Sch...

Embodiment 2

[0036] Preparation of Fermentation Broth of Gene Recombinant E.Coli

[0037] The two genetically recombinant E. coli obtained in Example 1 were respectively cultured in 50 mL of LB medium (containing 100 μg / mL ampicillin) at 37° C. overnight with shaking. Add 20 mL of overnight cultured bacterial liquid to 250 mL of LB medium (containing 100 μg / mL ampicillin), and shake at 37°C until the culture medium is OD 600 When = 0.8±0.1, add IPTG with a final concentration of 0.5mM, continue shaking culture at 15-35°C for 16h, and obtain the recombinant E. Coli fermentation broth.

Embodiment 3

[0039] Co-expression of Carbonyl Reductase and Glucose Dehydrogenase at Different Temperatures

[0040] The bacterial cell fermentation broth in Example 2 was centrifuged at 5000*g for 15 minutes to collect the bacterial cells, wherein 8 g / L of wet bacteria was obtained at 15 °C, 11 g / L of wet bacteria at 25 °C, and 13 g / L of wet bacteria at 20 °C. After the cells were collected, potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer was added to resuspend the cells, and the cells were crushed by high pressure at 35kpsi. Centrifuge the crushed bacteria at 15000*g for 20 minutes to obtain the crude enzyme solution. Using 15% SDS-PAGE to test the crude enzyme solution, the two enzymes are normally co-expressed in pGC and pCG, and the molecular weight of the carbonyl reductase CR1 monomer is about 35.5kDa, and the molecular weight of the glucose dehydrogenase GDH monomer is about 28.3kDa .

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Abstract

The invention discloses a method for preparing 6-cyano-(3R, 5R)-dihydroxyl tert-butyl caproate by using genetically engineered bacteria as a whole-cell biocatalyst. The method specifically comprises the following steps of designing and optimizing a tandem co-expression policy of a carbonyl reductase and a glucose dehydrogenase according to the expression characteristics of the carbonyl reductase and the glucose dehydrogenase, establishing a brand-new biological catalysis system in which cyclic regeneration of coenzymes is matched with the reduction of the 6-cyano-(3R, 5R)-dihydroxyl tert-butyl caproate, thus realizing in-situ biological synthesis of the 6-cyano-(3R, 5R)-dihydroxyl tert-butyl caproate, namely a chiral side chain synthesis precursor of atorvastatin. By optimizing expression conditions and reaction conditions and under the conditions that a cosolvent is 5% dimethyl sulfoxide, a reaction solution pH is 7.0, the temperature is 20 DEG C, and a ratio of glucose to a substrate is 1.2: 1, the concentration of the substrate for the in-situ biological synthesis of the 6-cyano-(3R, 5R)-dihydroxyl tert-butyl caproate can be 35g / L, and meanwhile, the addition of an exogenous coenzyme is completely avoided, and therefore, the method has wide application prospect.

Description

technical field [0001] The invention belongs to the field of preparation of chiral pharmaceutical intermediates, and relates to a method for the asymmetric preparation of chiral pharmaceutical intermediates by a biocatalytic method, in particular to a 6-cyano-(5R)-hydroxy-3-carbonylhexanoic acid tertiary Butyl ester is used as a substrate, and Escherichia coli co-expressing carbonyl reductase and glucose dehydrogenase is used as a biocatalyst to realize the process method of tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate with single optical purity . Background technique [0002] Statins were first discovered in secondary metabolites of fungi, and their molecular structure consists of a rigid hydrophobic core and side chains of (3R,5S / R)-dihydroxy ester structure. Due to the structural similarity between the side chain structure and hydroxymethylglutaryl, statins have a strong competitive inhibitory effect on hydroxymethylglutaryl-CoA reductase. This inhibitory effect can bl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/00C12N1/21C12N15/70C12R1/19
Inventor 陈依军苟旭东吴旭日
Owner CHINA PHARM UNIV
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