JPH0661271B2 - Process for producing optically active 2-hydroxy-4-phenylbutyric acid - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing optically active 2-hydroxy-4-phenylbutyric acid.

Optically active 2-hydroxy-4-phenylbutyric acid is useful as a raw material for various pharmaceuticals. For example, that (R)
The body's ethyl ester is a raw material for cilazapril, which is an effective antihypertensive agent as an angiotensin-exchangerase inhibitor (J. Chem. Soc., Perkin TranS.I, 1011 (1
986)).

<Prior Art> Conventionally, as a method for producing optically active 2-hydroxy-4-phenylbutyric acid, it has been previously organically synthesized to prepare a racemic compound.
After synthesizing 4-hydroxy-4-phenylbutyric acid, l-
Method of inducing menthol ester and performing optical resolution (An
n.chim., 20, 97 (1933)) and also racemic 2-hydroxy-4-phenylbutyric acid with optically active borniamine (Chem, Ber., 89 , 671).
(1956)) and the like are known.

<Problems to be Solved by the Invention> However, the optical resolution method using these resolving agents is complicated in operation and cannot be said to be an industrially advantageous method.

<Means and Actions for Solving the Problems> As a result of various investigations on the method for producing 2-hydroxy-4-phenylbutyric acid, the present inventors utilized 2-oxo-4-phenyl by utilizing the reducing power of microorganisms. The inventors have found that butyric acid can be advantageously converted to optically active 2-hydroxy-4-phenylbutyric acid, and completed the present invention. Optically active 2-hydroxy-4-from 2-oxo-4-phenylbutyric acid using microorganisms
Accumulation of phenylbutyric acid has hitherto been neither known nor practiced.

The present invention uses 2-oxo-4-phenylbutyric acid as an optically active compound.
-Hydroxy-4-phenylbutyric acid can be converted to the genus Apiotrichum, Candida (Ca
ndida, Torulopsis, Rodotorula
genus hodotorula, genus Trichosporon, genus Rhodosporidium, Pichia
Genus, Klebsiella, Rhodococcus
Coccus), Paracoccus (Paracoccus), Corynebacterium (Corynebacterium) and Nocardioides (Nocardioides) at least one microorganism selected from the microorganism, a bacterial cell or a treated product thereof, Optically characterized by reacting with oxo-4-phenylbutyric acid to produce and accumulate optically active 2-hydroxy-4-phenylbutyric acid and isolating and collecting optically active 2-hydroxy-4-phenylbutyric acid from the reaction solution. A method for producing active 2-hydroxy-4-phenylbutyric acid.

Hereinafter, the configuration of the present invention will be described in detail.

In the present invention, the compound has the ability to convert 2-oxo-4-phenylbutyric acid into optically active 2-hydroxy-4-phenylbutyric acid, and includes Apiotricum genus, Candida genus, Torlopsis genus, Rhodotorula genus, Trichopolone genus, Rhodosporidi. At least one microorganism selected from the microorganisms belonging to the genera Urum, Pichia, Klebsiella, Rhodococcus, Paracoccus, Corynebacterium and Nocardioides is used.

Specific examples of such microorganisms include, for example, Apiotricum, Humicola ATCC36992, Candida.
Parapsilosis ATCC 10232, Torrlopsis Candida ATCC 36584, Rhodotorula minuta A
TCC10658, Trichosporon Kutaneum ATC
C36993, Rhodosporidium toluroides AT
CC10788, Pichia halophila ATCC2424
0, Klebsiella oxytoca FERM P-1099
7, Rhodococcus erythropolis IFO12540,
Paracoccus denitrificans FERM P-10
098, Corynebacterium glutamicum ATCC2
1651, Nocardioides Albus ATCC2
7980 can be mentioned.

For culturing these microorganisms, a medium in which an organic and inorganic carbon source, a nitrogen source, vitamins, minerals and the like that can be assimilated by these bacteria are appropriately mixed is usually used. The pH of the medium is
It is slightly different depending on the microbial cells, and is usually pH 2 to 8 for yeast and pH 3 to 9 for bacteria. The temperature is usually 20 to 40 ° C., and the bacterium may be cultivated aerobically or anaerobically for 4 to 20 days.

In the reaction of the present invention, cultures of these microorganisms, cells or treated products thereof are used. A bacterial cell suspension or a treated bacterial cell product is preferably used. The bacterial cell suspension here is obtained by centrifugation of the bacterial cells obtained by culturing,
Examples of the treated product of cells include those obtained by subjecting cells obtained by culturing to ultrasonic treatment and those immobilized on an acrylamide gel carrier by a known method.

An energy source is usually added to the reaction system of the present invention.

As an energy source, depending on the strain used,
Generally, sugars such as glucose, fructose, sucrose, glycerol and sorbitol are used.

The concentration of 2-oxo-4-phenylbutyric acid, which is a reaction substrate, in the reaction solution can be usually about 0.1 to 5%. The addition method may be either batch or divided addition.

The reaction temperature is usually 20 to 40 ° C., preferably 25 to 35.
℃. The pH of the reaction solution is usually maintained at 4.0 to 8.5, preferably 6.0 to 8.0. The reaction time varies depending on the reaction temperature, but is usually 30 to 90 hours at 30 ° C.

As a reaction method, a substrate is added to the culture-finished solution and shaken aerobically, and a sugar is added as an energy source to the bacterial cell suspension or the treated bacterial cell product, and then the substrate is added, There is a method of shaking aerobically, both of which can be adopted, but the latter gives better results.

Thus, according to the reaction of the present invention, 2-oxo-4-phenylbutyric acid is asymmetrically reduced to give optically active 2-hydroxy-4-hydroxy-4-phenylbutyric acid.
Phenylbutyric acid is produced. Usually, (R) -2-hydroxy-4-phenylbutyric acid is produced as optically active 2-hydroxy-4-phenylbutyric acid.

In order to isolate the optically active 2-hydroxy-4-phenylbutyric acid thus produced from the reaction solution, a general separation and purification method may be used. For example, after removing insoluble substances such as bacterial cells from the reaction solution by centrifugation, the pH of the reaction solution
Is acidified, extracted with ethyl acetate or the like, dehydrated, dried under reduced pressure or recrystallized to isolate and collect the desired product.

<Example> Hereinafter, the present invention will be specifically described with reference to Examples.
The invention is not limited to these examples only.

In the examples, the production amount of 2-hydroxy-4-phenylbutyric acid and the ratio of R form and S form were analyzed by high performance liquid chromatography (HPLC). ODS column was used for the analysis of production amount, and the ratio of R-form and S-form was determined by Sumika column
PAK OA-3000 (Sumitomo Chemical Co., Ltd.) was used.

In addition, in the Examples, the yield was decreased.
Expressed in mol% of hydroxy-4-phenylbutyric acid, R%
Represents the ratio of the (R) form in the produced 2-hydroxy-4-phenylbutyric acid.

Example 1 A liquid medium consisting of 5% glucose and 5% corn steep liquor was adjusted to pH 6.0 with a caustic soda aqueous solution, and 18-mmφ was used.
5 ml each was dispensed into a test tube and sterilized by heating in an autoclave at 120 ° C. for 20 minutes. 1 platinum loop of each of the bacteria shown in Table 1 was inoculated from the slant culture medium at 28 ° C. for 63 hours,
The culture was performed aerobically on a shaker. After that, the cells are separated by centrifugation and washed once with water to adjust the cells.
Add this fungus body to a -mmφ test tube, add glucose to a concentration of 5%, add 2-oxo-4-phenylbutyric acid to a concentration of 1 wt%, and add 2 ml in total.
Then, the mixture was shaken aerobically at 28 ° C. for 17 hours at pH 7.0.

The results of HPLC analysis of the reaction solution thus obtained are shown in Table 1.

* HPB = 2-hydroxy-4-phenylbutyric acid Example 2 The same liquid medium as in Example 1 was added to 10 Sakaguchi flasks.
It was dispensed by 00 ml and sterilized by heating in an autoclave at 120 ° C for 20 minutes. One platinum loop of Rhodotorula minuta (ATCC 10658) was inoculated from the slant culture medium, shaken at 28 ° C. for 63 hours, and cultured aerobically. After that, the bacterial cells of 1 minute of the culture solution are separated by centrifugation, washed once with water to adjust the bacterial cells, and the bacterial cells are added to the Erlenmeyer flask of 5 so that the concentration becomes 5%. Glucose is added to the mixture to give 2-oxo-
4-phenylbutyric acid was added to give a total volume of 500 ml, 28
It was shaken aerobically at pH 7.0 for 30 hours at ℃. next,
After removing the bacterial cells from the reaction solution by centrifugation and concentrating the reaction solution of 500 ml to about 100 ml, the pH was adjusted to 2.0, extraction operation was performed 3 times with 100 ml of ethyl acetate, and dehydration was performed with anhydrous magnesium sulfate. The specific optical rotation ▲ [α] ²⁰ _D ▼ -8.46 by drying under reduced pressure and recrystallizing with toluene.
1.83 g of (R) -2-hydroxy-4-phenylbutyric acid having (C = 1 E + OH) was obtained (isolated yield 83.
8%).

Example 3 Glucose 4%, Polypeptone 2%, Yeast extract 0.5
%, Potassium dihydrogen phosphate 0.5%, a liquid medium was adjusted to pH 7.0 with a caustic soda aqueous solution, dispensed in 5 ml portions into 18-mmφ test tubes, and sterilized by heating in an autoclave at 120 ° C. for 20 minutes. Each of the various strains shown in Table 2 was inoculated into each platinum loop from the slant culture medium, and cultured aerobically on a shaker at 28 ° C for 48 hours. Then, the cells are separated by centrifugation, washed once with water to adjust the cells, and 18-
Add the cells to a mmφ test tube, add glucose to a concentration of 5%, add 2-oxo-4-phenylbutyric acid to a concentration of 1 wt%, and add 2 ml in total.
Then, the mixture was shaken aerobically at 28 ° C. for 40 hours at pH 7.0.

The results of HPLC analysis of the reaction solution thus obtained are shown in Table 2.

Example 4 The same liquid medium as in Example 3 was added to 10 Sakaguchi flasks.
Dispense each by 00 ml and in an autoclave at 120 ° C for 20
Heat sterilized for a minute. 1 platinum loop of Rhodococcus erythropolis (ATCC21035) was inoculated from the slant medium, and cultured aerobically on a shaker at 28 ° C. for 48 hours.
After that, the cells of 1 minute of the culture solution are separated by centrifugation,
The cells were adjusted by washing once with water, and the cells were added to an Erlenmeyer flask of 5 and further glucose was added so that the concentration became 5%. -Oxo-4-phenylbutyric acid was added to give a total amount of 5
The mixture was shaken aerobically at 00 ml for 80 hours at pH 7.0. Next, isolation was carried out in the same manner as in Example 2, and the specific rotation was ▲
1.2 g of (R) -2-hydroxy-4-phenylbutyric acid having [α] ²⁰ _D ▼ -8.4 (C = 1 E + OH)
Obtained (isolated yield 85%).

<Effects of the Invention> According to the present invention, optically active 2-hydroxy-4-phenylbutyric acid can be directly obtained from 2-oxo-4-phenylbutyric acid by an asymmetric reduction reaction using a microorganism, and a complicated operation can be performed. It can be manufactured industrially advantageously without the need.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication (C12P 7/42 C12R 1:88) (C12P 7/42 C12R 1:84) (C12P 7/42 C12R 1:22) (C12P 7/42 C12R 1:01) (C12P 7/42 C12R 1:15)

Claims (1)

[Claims] 1. A compound having the ability to convert 2-oxo-4-phenylbutyric acid into optically active 2-hydroxy-4-phenylbutyric acid, which belongs to the genus Apiotrichum, Candida (Ca).
ndida, Torulopsis, Rodotorula
genus hodotorula, genus Trichosporon, genus Rhodosporidium, Pichia
Genus, Klebsiella, Rhodococcus
Coccus), Paracoccus (Paracoccus), Corynebacterium (Corynebacterium) and Nocardioides (Nocardioides) at least one microorganism selected from the microorganism, a bacterial cell or a treated product thereof, Optically characterized by reacting with oxo-4-phenylbutyric acid to produce and accumulate optically active 2-hydroxy-4-phenylbutyric acid and isolating and collecting optically active 2-hydroxy-4-phenylbutyric acid from the reaction solution. Process for producing active 2-hydroxy-4-phenylbutyric acid.