Method for preparing L-tertiary leucine

A technology of tert-leucine and leucine dehydrogenase, which is applied in the field of high-efficiency preparation of L-tert-leucine with single optical purity, can solve the problems of serious environmental pollution, harsh reaction conditions, and complicated reaction process, and achieve high Effects of economic value, low cost, and cost reduction of coenzymes

Inactive Publication Date: 2013-01-23
陈依军
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, asymmetric synthesis has many disadvantages: complex reaction process, harsh reaction conditions (-78°C), high production cost, and the need to use a variety of toxic reagents in the production process, serious environmental pollution (see: J. Am. Chem . Soc. 1992, 114,

Method used

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  • Method for preparing L-tertiary leucine
  • Method for preparing L-tertiary leucine
  • Method for preparing L-tertiary leucine

Examples

Experimental program
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Example Embodiment

[0036] Example one

[0037] In a 10 mL reaction system, add the substrates trimethylpyruvate, ammonium formate, ammonium formate-ammonia buffer, NAD in sequence + , Leucine dehydrogenase, formate dehydrogenase, the reaction system contains: substrate trimethylpyruvate 1.0M (130g / L), ammonium formate 1.0M (63g / L), 0.1 M (pH 8.0) Ammonium formate-ammonia buffer, NAD + 0.01mM (0.007 g / L), leucine dehydrogenase 2.0 U / mL, formate dehydrogenase 2.0 U / mL, maintain the pH with ammonia and hydrochloric acid, agitate the reaction at room temperature (25°C) for 3 hours, and after the reaction, Heat to 80°C for 30 minutes to fully denature the protein in the reaction solution, centrifuge for 15 minutes to remove the denatured protein, remove the solvent, and filter to obtain the L-tert-leucine product.

[0038] The conversion rate of the substrate was analyzed by high performance liquid chromatography. The results showed that the substrate was completely converted into the product L-tert-leu...

Example Embodiment

[0040] Example two

[0041] A 10 mL reaction system was prepared, the reaction system including: substrate trimethylpyruvate 2.0M (260 g / L), ammonium formate 3.0M (189g / L), 0.1 M (pH 8.5) ammonium chloride-ammonia Buffer, NAD + 0.04mM (0.03 g / L), leucine dehydrogenase 3.0 U / mL, formate dehydrogenase 4.6 U / mL, shaking at room temperature for 24 hours. Other operations are the same as in Example 1 to obtain the white product L-tert-leucine, and the substrate conversion is complete.

Example Embodiment

[0042] Example three

[0043] A 100 mL reaction system was prepared, the reaction system including: substrate trimethylpyruvate 1.5M (195 g / L), ammonium formate 3.0M (189g / L), 0.1 M (pH 8.5) Tris-HCl buffer , NAD + 0.1mM (0.07 g / L), leucine dehydrogenase 3.0 U / mL, formate dehydrogenase 4.6 U / mL, shaking at room temperature for 24 hours. Other operations are the same as in Example 1 to obtain the white product L-tert-leucine, and the substrate conversion is complete.

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Abstract

The invention discloses a technological method for preparing single optically pure L-tertiary leucine by using leucine dehydrogenase as a biocatalyst, specifically comprising the following steps of: adding substrates trimethylpyruvic acid, ammonium formate and leucine dehydrogenase as well as a hydrogenlyase-mediated coenzyme cyclic regeneration system into a reaction solution, oscillating or stirring, heating the reaction system after the reaction, filtering or centrifuging to remove denatured protein, removing a solvent, filtering to obtain the product L-tertiary leucine and a filtrate containing a small amount of L-tertiary leucine, and recycling the filtrate for the next round of reaction, wherein the reaction temperature is 15-50 DEG C. By the use of leucine dehydrogenase and with the cooperation of the coenzyme cyclic regeneration system, L-tertiary leucine is prepared by a recycling technology. The concentration of the substrate reaches up to 2.0 M. The required coenzyme NAD+ or NADH has low concentration and can be recycled for many times so as to ignore the cost of the coenzyme. The method provided by the invention has an important application value.

Description

technical field [0001] The invention belongs to the field of preparation of chiral pharmaceutical intermediates, and relates to a method for asymmetrically preparing chiral pharmaceutical intermediates by a biocatalytic method, in particular to a method using trimethylpyruvate as a substrate and leucine dehydrogenase as a A biocatalyst, a technological method for realizing the high-efficiency preparation of L-tert-leucine with a single optical purity. Background technique [0002] L-tert-leucine, that is, L-2-amino-3,3-dimethylbutyric acid, the molecular formula is C 6 h 13 NO 2 , the molecular weight is 131.17, and the CAS number is 20859-02-3. L-tert-leucine is a non-protein-derived chiral amino acid. Due to the large steric hindrance of the hydrophobic tert-butyl side chain, it can well control the molecular conformation in the synthesis of compounds. Therefore, this amino acid As an important pharmaceutical intermediate, it is widely used in the synthesis of biologic...

Claims

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Application Information

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IPC IPC(8): C12P13/04
Inventor 陈依军江金鹏吴旭日
Owner 陈依军
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