JPH1075798A - Production of (r)-2-hydroxy-4-phenyl-3-butenoic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for industrially and advantageously producing (R)-2-hydroxy-4-phenyl-3-butenoic acid useful as an intermediate, etc., for a medicine by acting a microorganism belonging to Leuconostoc oenos species and having asymmetrically reducing ability of 2-oxo-4-phenyl-3-butenoic acid on the 2-oxo-4-phenyl-3-butenoic acid. SOLUTION: The objective (R)-2-hydroxy-4-phenyl-3-butenoic acid useful as an important intermediate, etc., for various medicines and an optically and pharmaceutically active material, its derivative, etc., is industrially and advantageously produced by acting a microorganism (e.g. Leuconostoc oenos ATCC 27310) selected from microorganisms belonging to Leuconostoc oenos species having an ability capable of asymmetrically reducing 2-oxo-4-phenyl-3-butenoic acid to (R)-2-hydroxy-4-phenyl-3-butenoic acid on the 2-oxo-4 phenyl-3-butenoic acid and collecting the oroduct.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing (R) -2-hydroxy-4-phenyl-3-butenoic acid. (R) -2-hydroxy-4-phenyl-3-butenoic acid is an important intermediate such as various pharmaceuticals, optically active physiologically active substances, and derivatives thereof.

[0002]

2. Description of the Related Art Conventionally, as a method for producing (R) -2-hydroxy-4-phenyl-3-butenoic acid, a racemic form of the acid is converted to a mixture of bornylamine and a diastereomer. And optical splitting (Chem. Ber. 89.671-677 (19).
56)) and an optical resolution method by liquid chromatography using a simple substance to which a metal salt of an optically active amino acid is bound as a filler (Japanese Patent Application Laid-Open No. 61-87640), but the former is complicated in operation. It is difficult to say that it is industrially superior, and a simple and economical method has been desired. In addition, 2-oxo-4-phenyl-
A method for obtaining (R) -2-hydroxy-4-phenyl-3-butenoic acid from 3-butenoic acid is disclosed in JP-A-2-23876.
And JP-A-2-100688 are known, but it is hard to say that they are industrially superior in terms of performance.

[0003]

DISCLOSURE OF THE INVENTION The present invention provides a simple method and high optical purity of (R) -2-hydroxy-4-.
As a method for obtaining phenyl-3-butenoic acid, the present inventors focused on an asymmetric reduction method using microorganisms, and searched for microorganisms suitable for this purpose. Heading, the present invention has been completed. That is, the present invention relates to 2-oxo-4-
Phenyl-3-butenoic acid is converted to (R) -2-hydroxy-
A microorganism selected from the group of microorganisms belonging to the species Leuconostoc oenos having the ability to asymmetrically reduce to 4-phenyl-3-butenoic acid, or a treated product thereof is treated with 2-oxo-4
-Phenol-3-butenoic acid to produce (R)
A method for producing (R) -2-hydroxy-4-phenyl-3-butenoic acid, comprising collecting -2-hydroxy-4-phenyl-3-butenoic acid.

The microorganism used in the present invention is a microorganism belonging to the species Leuconostoc oenos and is 2-oxo-
Any substance having the ability to asymmetrically reduce 4-phenyl-3-butenoic acid to (R) -2-hydroxy-4-phenyl-3-butenoic acid can be used,
Specifically, Leuconostoc Oenos (Leucon
ostoc oenos) ATCC 27310, AT
CC 27311, DSM 20252, NRRC 1
088.

[0005] The microorganisms with ATCC numbers are Am
Catalogu issued by erican Type Culture Collection (ATCC)
e of Bacteria Phages rDNA Vectors, 18th edition (199
2) and can be obtained from the ATCC. Microorganisms with DSM are Deutsche Sammlun
g von Mikrrorganismen und Zellkulturen GebH (DSM), published in the fourth edition of the Catalog of Strains (1989), which can be obtained from the DSM. Also N
Microorganisms with RRC numbers are Cu
lture Collection of NODAI NO.1 (1985) and can be obtained from Tokyo University of Agriculture. These mutants can also be used.

[0006] The medium used in the present invention is not particularly limited as long as the medium can grow bacteria. For example, glucose, sugars such as sucrose, ethanol, alcohols such as glycerol, acetic acid, organic acids such as propionic acid, hydrocarbons such as paraffin, other carbon sources, or mixtures thereof, ammonium sulfate as a nitrogen source, yeast Nutrient sources used in ordinary culture media, such as inorganic organic nitrogen-containing compounds such as extracts and urea, as well as inorganic salts and vitamins, can be appropriately mixed and used. If necessary, a factor for promoting the growth of microorganisms or a compound for maintaining the pH of the medium may be added. As a culture method, it is preferable to culture aerobically or anaerobically for 1 to 5 days at a medium pH of 3.0 to 9.5 and a medium temperature of 20 to 45 ° C.

As a reaction method, (1) a method in which a culture solution is used as it is (2) cells are separated by a centrifuge,
A method of adding 2-oxo-4-phenyl-3-butenoic acid to a suspension resuspended in a buffer solution, water, or the like as it is or after washing, and then reacting the suspension. (4) A method in which a substance subjected to a treatment such as freeze-drying is used in place of a viable cell (4) A method in which these are immobilized on a single substance and used can be applied. The yield is often improved by adding a carbon source such as glucose to the reaction system as an energy source. 2-oxo-4-phenyl-3-butenoic acid may be dissolved as it is or in an organic solvent that does not affect the reaction, or dispersed in a surfactant or the like,
It may be added all at once or dividedly from the beginning of the reaction. Further, 2-oxo-4-phenyl-3-butenoic acid can be used as a sodium salt, a potassium salt, a calcium salt, an ammonium salt and the like.

The reaction is preferably carried out at a temperature in the range of 3 to 9 at a temperature of 10 to 60 ° C., for 1 to 120 hours under stirring or standing. The concentration of the substrate used is not particularly limited, but is preferably about 0.1 to 10%.

2-hydroxy-4 formed by the reaction
-Phenyl-3-butenoic acid can be easily obtained by using an ordinary purification method such as column chromatography, distillation, or the like, directly or from the reaction solution, after acidifying the cells, extracting with an organic solvent, and then extracting with an organic solvent.

[0010]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to only these Examples.

The optical purity in the examples was determined by extracting the reaction product with an organic solvent and then performing high-performance liquid chromatography using an optical resolution column (column: Chiralpak WH (copper salt of L-proline manufactured by Daicel Chemical Industries, Ltd.) Column packed with silica gel to which is bonded 4.6 mm ID x 25
0 mm, moving layer: 0.5 mM CuSO ₄ / acetonitrile = 4: 1, detection: 254 nm, flow rate 1.5 ml / min). (Retention time: (S) 21 minutes, (R) 2
4 minutes). The yield was determined by high performance liquid chromatography using a reversed phase column (column: nucleosyl 10C18,
(4.6 mm ID × 250 mm), mobile phase: 40 mM potassium phosphate, pH 3.0 / acetonitrile = 4: 1, detection: 254 nm, flow rate 1.0 ml / min).

(Example 1) Glucose 2.0%, yeast extract 1.0%, peptone 1.0%, MnSO4 10pp
m, a medium 100 having a composition of 1.0% calcium carbonate
ml was placed in a 500 ml Erlenmeyer flask, sterilized, and each of the strains shown in Table 1 was inoculated with one platinum wire, and cultivated with rotation and shaking at 30 ° C. for 48 hours. After completion of the culture, the cells were separated by centrifugation and washed once with physiological saline to obtain viable cells. 500
50 ml of distilled water was placed in a ml Erlenmeyer flask, the viable cells were suspended therein, and 5 g of glucose was added. 1 at 30 ° C
After shaking for 0 minutes, 2-oxo-4-phenyl-3-
0.5 g of potassium butenoate was added, and the mixture was subjected to a rotational shake reaction at 30 ° C. for 40 hours. After the completion of the reaction, sulfuric acid was added to adjust the pH to 1 or less, followed by extraction with 100 ml of ethyl acetate. After the solvent was removed from the extract, quantification of the generated 2-hydroxy-4-phenyl-3-butenoic acid and measurement of optical purity were performed using high performance liquid chromatography.

[0013]

[Table 1] (Example 2) Leuconostoc oenos A was added to a 5 L jar fermenter containing 2 L of the medium used in Example 1.
Inoculate TCC 27310 and stir at 30 ° C. 100 rpm
For 40 hours. After completion of the culture, the cells were collected by centrifugation and washed with 1 L of water. Thereafter, the cells were suspended in 500 ml of water, and 2-oxo-4-phenyl-3 was added.
-5 g of potassium butenoate, 50 g of glucose and 5 g of calcium carbonate were added and reacted at 30 ° C with stirring at 100 rpm for 48 hours. After completion of the reaction, the mixture was adjusted to pH 1 with sulfuric acid, and extracted twice with an equal amount of ethyl acetate. After dehydrating the ethyl acetate layer with anhydrous sodium sulfate, the solvent was removed under reduced pressure to give 2-hydroxy-4.
4.0 g of crude crystals of -phenyl-3-butenoic acid were obtained. When this was recrystallized from ethanol, 3.8 g of crystals of (R) -2-hydroxy-4-phenyl-3-butenoic acid were obtained. (Optical purity 100% ee, yield 93%)

[0014]

The process for producing (R) -2-hydroxy-4-phenyl-3-butenoic acid by the asymmetric reduction method using the microorganism of the present invention provides a highly optically pure (R) -2-hydroxy- This makes it possible to easily produce 4-phenyl-3-butenoic acid, and is extremely advantageous as an industrial production method.

Claims (1)

[Claims] 1. Leuconostoc having the ability to asymmetrically reduce 2-oxo-4-phenyl-3-butenoic acid to (R) -2-hydroxy-4-phenyl-3-butenoic acid.
A microorganism selected from the group of microorganisms belonging to the species Leuconostoc oenos or a processed product thereof is
(R) -2-hydroxy-4 by reacting -oxo-4-phenyl-3-butenoic acid and collecting (R) -2-hydroxy-4-phenyl-3-butenoic acid produced. -A method for producing phenyl-3-butenoic acid.