JPH0679558B2 - Biochemical production method of (R) -4-hydroxy-2-cyclopentenone - Google Patents

Description

TECHNICAL FIELD The present invention relates to a biochemical method for producing (R) -4-hydroxy-2-cyclopentenone.

The (R) -4-hydroxy-2-cyclopentenone obtained in the present invention is useful as an intermediate for pesticides, fragrances, pharmaceuticals and the like, and in particular, it has a prostrate having various pharmacological actions such as antiulcer action and thrombolytic action. It is extremely important as a raw material for Grandin. If the application is a pharmaceutical,
Particularly, a material having high optical purity is required. Therefore, development of a technique for obtaining (R) -4-hydroxy-2-cyclopentenone having high optical purity from racemic 4-hydroxy-2-cyclopentenone is desired.

<Prior Art> It has been conventionally reported that 4-acetoxy-2-cyclopentenone is hydrolyzed by plant-derived enzymes such as wheat germ lipase and citrus peel esterase (Japanese Patent Publication No.
55-34677). However, according to this method, when the racemic ester is used as a substrate, racemic 4-hydroxy-2-cyclopentenone is obtained, and the optically active 4
In order to obtain -hydroxy-2-cyclopentenone, it is necessary that the ester as a substrate is an optically active substance. Therefore, according to this method, racemic 4-hydroxy-2-cyclopentenone (R)-having high optical purity is obtained.
It is impossible to obtain 4-hydroxy-2-cyclopentenone.

<Structure of the Invention> The present inventors have found that an esterase derived from a microorganism belonging to the genus Alcaligenes asymmetrically hydrolyzes an organic carboxylic acid ester of racemic 4-hydroxy-2-cyclopentenone to give (R) -4. It was found that -hydroxy-2-cyclopentenone is preferentially produced, and various studies were added to this to complete the present invention.

The present invention has the general formula (I) [In the formula, R is an alkyl group having 1 to 18 carbon atoms and 2 carbon atoms.
It represents an alkenyl group having 1 to 18 or an alkynyl group having 2 to 18 carbon atoms. ] By allowing an esterase derived from a microorganism belonging to the genus Alcaligenes to act on an organic carboxylic acid ester of racemic 4-hydroxy-2-cyclopentenone represented by
Asymmetrically hydrolyzed to produce optically active 4-hydroxy-2-cyclopentenone containing 75% or more of (R) form, and optically active 4-hydroxy-2-cyclopentenone containing 75% or more of (R) form. After isolating and obtaining 2-cyclopentenone,
An organic carboxylic acid halide represented by the formula (II) or (II
I) Organic carboxylic acid anhydride represented by the formula (In the formula, R has the same meaning as described above, and X represents a chlorine atom or a bromine atom.) (In the formula, R has the same meaning as described above, and * represents an asymmetric carbon.) An optically active 4-hydroxy-2-cyclopentenone organic carboxylic acid ester represented by Asymmetrically hydrolyzed using
Isolation and acquisition by producing 90% or more of optically active 4-hydroxy-2-cyclopentenone, an organic carboxylic acid halide represented by the formula (II) or an organic carboxylic acid anhydride represented by the formula (III) And asymmetrically hydrolyzing the obtained optically active 4-hydroxy-2-cyclopentenone organic carboxylic acid ester using the above-mentioned microorganism-derived esterase, and the (R) content is 97% or more. The present invention relates to a biochemical production method of (R) -4-hydroxy-2-cyclopentenone, which comprises obtaining optically active 4-hydroxy-2-cyclopentenone.

In the present invention, the racemic 4-hydroxy-form used
Organic carboxylic acid esters of 2-cyclopentenone are known, and conventional methods for producing general esters, for example, racemic 4-hydroxy-2-cyclopentenone (II)
Organic carboxylic acid halide represented by the formula or (III)
It can be easily produced by reacting an anhydride of an organic carboxylic acid represented by the formula.

The esterase used in the present invention is a broad-sense esterase containing a lipase produced by a microorganism belonging to the genus Alcaligenes.

Some esterases of microbial origin are commercially available and can be easily obtained. Specific examples of commercially available esterases include lipases of the genus Alcaligenes (lipase "PL"; manufactured by Meito Sangyo Co., Ltd., Agricultural
and Biological Chemistry 46: 1159-1164 and 46: 1748-1750).

In carrying out the method of the present invention, the asymmetric hydrolysis of the cyclopentenone ester is carried out by a culture solution in which the above microorganism is cultured, bacterial cells separated from the culture solution, a culture filtrate containing esterase, or various enzyme separation methods. It is carried out by mixing the aqueous solution containing the crude esterase, the purified esterase and the esterase-containing extract or concentrate separated from the bacterial cells or the culture filtrate with the racemic cyclopentenone ester, and stirring or shaking. Also, immobilized cells or immobilized esterase can be used.

As a condition for carrying out asymmetric hydrolysis of the racemic cyclopentenone ester, the reaction temperature is suitably 10 to 70 ° C, preferably 20 to 50 ° C.

The pH during the reaction is preferably pH 5-9. It also neutralizes the organic carboxylic acid produced by the hydrolysis reaction,
It is preferable to use a buffer solution to keep the pH constant, and a buffer solution of an inorganic acid salt such as sodium phosphate and potassium phosphate, and a buffer solution of an organic acid salt such as sodium acetate and sodium citrate can be used.

The concentration of the racemic 4-hydroxy-2-cyclopentenone organic carboxylic acid ester used as a raw material is 0.1 to 50% by weight, preferably 1 to 25% by weight, based on the reaction solution.

Since reaction for a long time may cause racemization of free 4-hydroxy-2-cyclopentenone, it is important to set the reaction time within 10 hours, preferably within 5 hours.

Here, the asymmetric hydrolysis reaction produces 4-hydroxy-
It is important to keep the content of 2-cyclopentenone in the (R) form within the range of not less than 75%.
It is carried out until the range of 10 to 45% is reached. The hydrolysis rate and reaction time can be adjusted by adjusting the amount of enzyme used.

After carrying out the asymmetric hydrolysis reaction in this way, the released 4-hydroxy-2-cyclopentenone and the unreacted ester are separated and recovered. In this separation and recovery, operations such as steam distillation, solvent extraction, fractional distillation, column chromatography and the like can be appropriately adopted. For example, when the reaction solution is first extracted with hexane, unreacted ester is isolated. Subsequent extraction with methyl isobutyl ketone isolates the free 4-hydroxy-2-cyclopentenone. In addition, the reaction solution was extracted with methyl isobutyl ketone, the extract was loaded on silica gel column chromatography (eluent; hexane / ethyl acetate = 10/1), and column chromatography separation was performed to obtain free 4-hydroxy-2.
It is also possible to isolate and obtain cyclopentenone.

Next, the optically active 4-hydroxy-2-cyclopentenone containing 75% or more of the obtained (R) form was esterified with the racemic 4-hydroxy-2-cyclopentenone described above. Similarly, it is esterified to be converted into an optically active organic carboxylic acid ester of 4-hydroxy-2-cyclopentenone. The optically active 4-obtained here
The organic carboxylic acid ester of hydroxy-2-cyclopentenone is again used with an esterase derived from a microorganism of the genus Alcaligenes under the same conditions as for the asymmetric hydrolysis of the organic carboxylic acid ester of racemic 4-hydroxy-2-cyclopentenone. Asymmetrically hydrolyzes. The asymmetric hydrolysis reaction at this time can be stopped within a range in which the content of (R) isomer of 4-hydroxy-2-cyclopentenone produced is not less than 90%, but usually the hydrolysis rate is in the range of 20 to 65%. Until it reaches. After completion of the reaction, 4-hydroxy-2-cyclopentenone produced from the reaction solution is separated in exactly the same manner as described above, and the content of the (R) isomer is 90% or more. -Obtain cyclopentenone. Furthermore, the optically active 4-hydroxy-2-cyclopentenone containing 90% or more of the (R) form obtained here,
Esterification is carried out in the same manner as the above-mentioned 4-hydroxy-2-cyclopentenone esterification of the racemate to obtain an optically active organic carboxylic acid ester of 4-hydroxy-2-cyclopentenone.

The optically active 4-hydroxy-2-cyclopentenone organic carboxylic acid ester thus obtained is further subjected to asymmetric hydrolysis of the racemic 4-hydroxy-2-cyclopentenone organic carboxylic acid ester. Under the same conditions, asymmetric hydrolysis is performed using an esterase derived from a microorganism of the genus Alcaligenes. In this case, the asymmetric hydrolysis reaction produces a content of (R) -form of 4-hydroxy-2-cyclopentenone of 97%.
The hydrolysis rate is usually maintained until the hydrolysis rate reaches 20 to 75%. After completion of the reaction, 4-
Hydroxy-2-cyclopentenone is separated to obtain (R) -4-hydroxy-2-cyclopentenone having high optical purity.

<Effects of the Invention> The present invention provides a method for obtaining (R) -4-hydroxy-2-cyclopentenone having high optical purity from racemic 4-hydroxy-2-cyclopentenone, which has not been known hitherto. The feature is that 1) it does not require expensive optically active reagents used in conventional organic synthetic chemical optical resolution methods, and 2) special equipment because the reaction operation can be performed at room temperature and atmospheric pressure. It is economically advantageous because it does not require.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Example 14.0 g of racemic 4-acetoxy-2-hydroxycyclopentenone, 20.0 g of lipase (lyase "PL"; manufactured by Meito Sangyo Co., Ltd.) of the genus Alcaligenes and 0.3 M phosphate buffer (pH 6.
5) 100 g was mixed and reacted at 40 ° C. for 3 hours with vigorous stirring. The hydrolysis rate at the end of the reaction was 44.7%. The hydrolysis rate was measured by gas chromatography analysis.

After the reaction was completed, sodium hydroxide was added to the reaction solution and extraction was performed with methyl isobutyl ketone. The methyl isobutyl ketone extract was concentrated, and the concentrated residue was subjected to column chromatography separation with a mixed solvent of hexane / ethyl acetate = 10/1 (volume ratio) to obtain 7.35 g of unreacted 4-acetoxy-2-cyclopentenone. 4.2 g of released 4-hydroxy-2-cyclopentenone was separated and obtained. After derivatizing 50 mg of 4-hydroxy-2-cyclopentenone obtained here to an ester of (+)-p-chlorophenylisobutyric acid, high performance liquid chromatography [column: Lichrosorb Sl-100, eluent: hexane] / Isopropanol = 500/1] and diastereomer separation was performed, and the result of measuring the optical isomer ratio was
The (R) body content was 77.5% [R body: S body = 77.5: 22.
Five〕. 4.0 g of 4-hydroxy-2-cyclopentenone obtained here was dissolved in 10 g of toluene, 0.54 g of acetic anhydride and 0.05 g of sulfuric acid were added, and the mixture was stirred at 40 ° C. for 3 hours to carry out an acetylation reaction. After the reaction, 20 g of water was added to the reaction solution for extraction and separation, the water-soluble components were removed, and the toluene solution was recovered and concentrated to obtain 5.4 g of 4-acetoxy-2-cyclopentenone as a concentrated residue. 4-acetoxy-2 obtained here
-Cyclopentenone 5.4 g and Alcaligenes lipase 8.
An asymmetric hydrolysis reaction was carried out by mixing 0 g and 50 g of 0.3 M phosphate buffer with vigorous stirring at 40 ° C. for 2 hours. The hydrolysis rate at the end of the reaction was 53.8%. After completion of the reaction, concentration and column chromatographic separation were carried out in the same manner as described above to obtain 4-hydroxy-2-cyclopentenone as a hydrolysis product.
Obtained 1.9g. 4-hydroxy-2-obtained here
As a result of measuring the optical isomer ratio of cyclopentenone in the same manner as described above, the content of the (R) isomer was 91.9%.
[R form: S form = 91.9: 8.1] 1.8 g of 4-hydroxy-2-cyclopentenone obtained here was acetylated in the same manner as above to obtain 2.5 g of 4-acetoxy-2-cyclopentenone. . This 4-acetoxy-2-cyclopentenone
2.5 g and 4.0 g of Alcaligenes lipase and 0.3 M buffer 20
g was mixed, and the asymmetric hydrolysis reaction was carried out with vigorous stirring at 40 ° C. The hydrolysis rate at the end of the reaction was 53.2%. After the reaction was completed, sodium hydroxide was added to the reaction solution and extraction was performed with methyl isobutyl ketone. Thereafter, concentration and column chromatographic separation were carried out in the same manner as above to obtain 0.9 g of 4-hydroxy-2-cyclopentenone produced. Obtained 4-
As a result of measuring the optical isomer ratio of hydroxy-2-cyclopentenone in the same manner as above, the content ratio of the (R) isomer was 9
It was 7.7%. [R body: S body = 97.7: 2.3]

Claims (1)

[Claims] 1. A general formula (I) [In the formula, R is an alkyl group having 1 to 18 carbon atoms and 2 carbon atoms.
It represents an alkenyl group having 1 to 18 or an alkynyl group having 2 to 18 carbon atoms. ] By allowing an esterase derived from a microorganism belonging to the genus Alcaligenes to act on an organic carboxylic acid ester of racemic 4-hydroxy-2-cyclopentenone represented by
Asymmetrically hydrolyzed to produce optically active 4-hydroxy-2-cyclopentenone containing 75% or more of (R) form, and optically active 4-hydroxy-2-cyclopentenone containing 75% or more of (R) form. After isolating and obtaining 2-cyclopentenone,
An organic carboxylic acid halide represented by the formula (III) or (II
I) Organic carboxylic acid anhydride represented by the formula (In the formula, R has the same meaning as described above, and X represents a chlorine atom or a bromine atom.) (In the formula, R has the same meaning as described above, and * represents an asymmetric carbon.) An optically active 4-hydroxy-2-cyclopentenone organic carboxylic acid ester represented by Asymmetrically hydrolyzed using
Isolation and acquisition by producing 90% or more of optically active 4-hydroxy-2-cyclopentenone, an organic carboxylic acid halide represented by the formula (II) or an organic carboxylic acid anhydride represented by the formula (III) And asymmetrically hydrolyzing the obtained optically active 4-hydroxy-2-cyclopentenone organic carboxylic acid ester using the above-mentioned microorganism-derived esterase, and the (R) content is 97% or more. And a method for producing (R) -4-hydroxy-2-cyclopentenone, which comprises obtaining optically active 4-hydroxy-2-cyclopentenone