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Method for preparing L-homophenylalanine

A technology of high phenylalanine and high phenylalanine dehydrogenase, applied in chemical recovery, fermentation and other directions, can solve the problems of complex operation of membrane reactor, harsh reaction conditions, complicated reaction process, etc. The effect of low cost and cost reduction of coenzyme

Inactive Publication Date: 2012-03-14
陈依军
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The former utilizes a chiral resolution reagent to resolve the racemate, but the maximum yield generally does not exceed 50% (see: Chem.Pharm.Bull.1989, 37:280-283); the asymmetric synthesis method utilizes a chiral catalyst Catalysis directly obtains enantiomers, but there are many shortcomings: complex reaction process, harsh reaction conditions (-60 ° C), high production costs, and serious environmental pollution (see: J.Am.Chem.Soc.1988, 110: 1547 -1557)
Another more general method is the method that amino acid aminotransferase catalyzes the corresponding keto acid transamination, such as the Chinese invention patent with the patent number 200710021346.X and the use of Lo et al. Aspartate aminotransferase and U.S. Patent No. 6,146,859 use tryptophan aminotransferase to successfully synthesize L-homophenylalanine, obtaining a yield greater than 95% and high stereoselectivity, but the amino acid donor amino acid used in the raw material of this method increases the production cost , and transaminase activity is usually inhibited at high substrate concentrations
In addition, the method of reducing and aminating 2-oxo-4-phenylbutyric acid with homophenylalanine dehydrogenase is also used to prepare L-homophenylalanine. The conversion rate of this method is 99.1%, and the ee value Greater than 99.1%, but the method is restricted by factors such as relatively low (200mM) reaction substrate concentration (200mM), large coenzyme input amount (10mM) causing cost too high, membrane reactor operation complexity (referring to: J.Org.Chem.1990 , 55:5567-5571; Biotechnol. Adv.2009, 27:286-296)

Method used

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  • Method for preparing L-homophenylalanine
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  • Method for preparing L-homophenylalanine

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Embodiment 1

[0036] A method for preparing L-homophenylalanine, the schematic diagram of the preparation process is shown in figure 1 , including the following steps: in a 5mL reaction system, sequentially add substrate 2-oxo-4-phenylbutyric acid, ammonium formate, phosphate buffer, NAD + , homophenylalanine dehydrogenase, formate dehydrogenase, the reaction system contains: substrate 2-oxo-4-phenylbutyric acid 2g / L (11mM), ammonium formate 11~110mM, 0.1M ( pH 8.0) phosphate buffer, NAD + 0.5mM, homophenylalanine dehydrogenase 5U / mL, formate dehydrogenase 5.6U / ml, maintain pH with concentrated potassium hydroxide and phosphoric acid, stir at room temperature for 15-20 hours, after the reaction, filter to obtain L- High phenylalanine, substrate conversion and product optical purity were analyzed by HPLC. The substrate conversion rate was 99.9%.

[0037] Described homophenylalanine dehydrogenase and formate dehydrogenase can be obtained by the following method: the escherichia coli of the...

Embodiment 2

[0040] A method for preparing L-homophenylalanine, comprising the steps of: preparing a 1mL reaction system, said reaction system comprising: substrate ethyl 2-oxo-4-phenylbutyrate 5g / L (25mM, 50μl ethanol for dissolution), ammonium formate 25~250mM, 0.1M (pH 8.5) phosphate buffer, NAD + 0.5mM, homophenylalanine dehydrogenase 5U / mL, formate dehydrogenase 5.6U / ml, shake at room temperature for 20 hours. After the reaction was completed, L-homophenylalanine was obtained by filtration, and the conversion rate of the substrate and the optical purity of the product were analyzed by high performance liquid chromatography. The substrate conversion rate was 99.9%.

Embodiment 3

[0042] The preparation of the substrate stock solution includes the following steps: dissolving the ammonium formate and the substrate in a buffer solution or water with a pH value of 7.0 to 9.0 at a molar ratio of 1:1 to 10:1, dissolving at room temperature or slightly heating, and maintaining the substrate with an alkaline solution. The pH of the solution is 7.0-9.0, and a mixed solution of the substrate and ammonium formate is prepared, and the concentration of the substrate is less than or equal to 850mM.

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Abstract

The invention discloses a process method for preparing single optical pure L-homophenylalanine by using L-homophenylalanine dehydrogenase as a biological catalyst. The method particularly comprises the following steps of: adding a substrate, ammonium formate, the L-homophenylalanine dehydrogenase and a formate dehydrogenase mediated coenzyme recycling and regenerating system into reaction liquid; after reacting under an oscillating or stirring condition, filtering from the reaction system to directly obtain an L-homophenylalanine product; and circulating the filtrate for the next reaction, wherein the reaction temperature is between 15 and 40 DEG C. The L-homophenylalanine is prepared by coordinating the L-homophenylalanine dehydrogenase with the coenzyme recycling and regenerating system and by adopting a simple filtering circulation process, wherein the substrate concentration reaches 560 mM; the NAD+ concentration of the required coenzyme is low; the coenzyme can be recycled and reused for many times, so that the cost of the coenzyme can be ignored; the yield excesses 95 percent; and important application value is achieved.

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 biocatalysis, in particular to a method using 2-oxo-4-phenylbutyric acid and its esters as substrates , using homophenylalanine dehydrogenase as a biocatalyst to realize a process for the efficient preparation of L-homophenylalanine with single optical purity. Background technique [0002] L-homophenylalanine, namely (S)-2-amino-4-phenylbutyric acid, molecular formula C 10 h 13 NO 2 , the molecular weight is 179.22, and the CAS number is 943-73-7. L-homophenylalanine is a non-natural chiral α-amino acid, and this type of amino acid and its ester are important raw materials for preparing angiotensin (ACE) inhibitor drugs. It is the common intermediate of about 20 new antihypertensive drugs in the world. Captopril (Captopril), Temocapril, Cilazapril (Silazapril), et...

Claims

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Application Information

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IPC IPC(8): C12P13/04
CPCY02P20/582
Inventor 吴旭日陈依军张洁琳
Owner 陈依军
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