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CALB mutant used for preparing (R)-3-substituted monoalkyl glutarate compound through non-aqueous catalysis

A mutant and substrate technology, applied in the field of bioengineering, can solve the problem of low selectivity of R-type products, and achieve the effects of high atom utilization, high optical purity and mild reaction conditions

Active Publication Date: 2015-07-01
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using the mutant enzyme as a catalyst to catalyze the synthesis of R-type products can solve the problem of low selectivity of existing enzymes to R-type products

Method used

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  • CALB mutant used for preparing (R)-3-substituted monoalkyl glutarate compound through non-aqueous catalysis
  • CALB mutant used for preparing (R)-3-substituted monoalkyl glutarate compound through non-aqueous catalysis
  • CALB mutant used for preparing (R)-3-substituted monoalkyl glutarate compound through non-aqueous catalysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Put the airtight 10ml shaking flask into a constant temperature shaker, add 3-tert-butyldimethylsilyl glutaric anhydride (substrate) 80g / L, the molar concentration ratio of methanol and substrate is 3:1, biological The catalyst is a CALB mutant, mainly including A141S, Q106H, S201D, I189V, T138S, S47N, D134S, and the mass of biological free enzyme added is 0.60g. After adding, fix the temperature at 25°C and react for 24 hours. Chromatographic monitoring, after the reaction is completed, post-treatment, removal of organic solvent and unreacted methanol, purification treatment to obtain colorless crystal (R)-3-tert-butyldimethylsilyl glutaric acid monomethyl ester.

[0050] Experimental results such as figure 1 , compared with the control group, all mutants ee R The values ​​were all increased, and the effect of A141S and S201D mutants was the most obvious. A141S mutant ee R Value up to 8.7%, (R)-3-tert-butyldimethylsilyl glutaric acid monomethyl ester (R-J 6 ) yield...

Embodiment 2

[0052] Put the airtight 10ml shaker flask into a constant temperature shaker, add 3-tert-butyldimethylsilyl glutaric anhydride (substrate) 40g / L, the molar concentration ratio of methanol and substrate is 3:1, biological The catalyst is a CALB mutant, including T138S-I189V, S47N-D134S-S201D, A141S-A283V, S47N-I189V, S47N-Q106H, CALB-Lost, T138S-S47N-S201D, T138S-S47N, S47N-S201D, biological free enzyme The added mass is 0.60g. After the addition is completed, the temperature is fixed at 25°C and reacted for 24 hours. During the period, it is monitored by liquid chromatography. After the reaction is completed, the post-treatment removes the organic solvent and unreacted methanol, and purifies to obtain colorless crystals. (R)-3-tert-Butyldimethylsilyl glutaric acid monomethyl ester.

[0053] Enzyme-catalyzed synthesis of R-J by multi-site mutants of CALB gene 6 The effect is as figure 2 , A141S-A283V and CALB-Lost mutants are more effective in catalyzing the synthesis of R-t...

Embodiment 3

[0055] Put the airtight 10ml shaking flask into a constant temperature shaker, add 3-tert-butyldimethylsilyl glutaric anhydride (substrate) 80g / L, the molar concentration ratio of methanol and substrate is 3:1, biological Catalyst is A141S-A283V mutant, the quality that biological free enzyme adds is 0.60g, adds the triethylamine (organic base) of different concentration (5-30% v / v) in the system, investigates the effect of organic base on esterification reaction ee R It is worthy of influence, the addition is completed, and the reaction is carried out for 24 hours. During the period, it is monitored by liquid chromatography. After the reaction is completed, the post-treatment is performed to remove the organic solvent and unreacted methanol, and the purification process obtains colorless crystal (R)-3-tert-butyl dimethyl Monomethyl silyl glutarate. A141S-A283V Catalyzed Synthesis of R-J by Adding Different Volumes of Triethylamine 6 The impact of the results such as image...

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Abstract

The invention discloses a CALB mutant used for preparing a (R)-3-substituted monoalkyl glutarate compound through non-aqueous catalysis, and relates to the technical field of biological engineering. According to the invention, a mutant enzyme is adopted as a catalyst, a 3-substituted glutaric acid compound and an organic alcohol are adopted as raw materials. Under the effect of the biological enzyme, a reaction is carried out in an organic solvent under a temperature of 4-50 DEG C, such that the (R)-3-substituted monoalkyl glutarate compound is prepared. The preparation method provided by the invention has mild reaction conditions. The output of the (R)-3-substituted monoalkyl glutarate compound can be higher than 32.8g / L, the yield can be higher than 82.0%, and the eeR value can be higher than 99%. Post-treatment is simple, atom utilization degree is high, and the requirements of green chemistry is met. With the method, cost can be greatly reduced, and the social demand of the (R)-3-substituted monoalkyl glutarate compound can be satisfied.

Description

technical field [0001] The invention relates to a CALB mutant for preparing (R)-3-substituted glutaric acid monoalkyl ester compounds by non-aqueous phase catalysis, and belongs to the technical field of bioengineering. Background technique [0002] The chiral monomer (R)-3-substituted glutaric acid monoalkyl ester compound (I) is an important intermediate for the synthesis of statins and their analogues. The chemical structural formula of (R)-3-substituted monoalkyl glutaric acid compounds is as follows: [0003] [0004] Statins are currently the drug of choice for the treatment of hyperlipidemia. This type of drug has become the mainstream drug in lipid-lowering drugs due to its advantages of safety, efficacy, good tolerance, and long-lasting effect. Successive reports of new clinical applications have aroused the interest and attention of the medical community. It is reported that statins also have anti-AIDS virus (HIV) effect, and there are other potential applica...

Claims

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Application Information

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IPC IPC(8): C12N9/20C12P7/62C07F7/08C07C69/675C07C69/708C07C67/08A61P3/06
CPCA61K31/22C12N9/20C12P7/62A61K2300/00
Inventor 刘立明王鸿江
Owner JIANGNAN UNIV
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