JPH0728755B2 - Biochemical production method of optically active 4-hydroxy-2-cyclopentenone - Google Patents

Description

TECHNICAL FIELD The present invention relates to a biochemical production method of optically active 4-hydroxy-2-cyclopentenone. More specifically, 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. ] The racemic 4-hydroxy-2-cyclopentenone of the organic carboxylic acid ester asymmetrically hydrolyzed to produce an optically active 4-hydroxy-2-cyclopentenone, Achromobacter, Alcaligenes Genus, Arthrobacter, Candida,
An esterase derived from a microorganism belonging to the genus Chromobacterium, the genus Pseudomonas or the genus Humicola (in the present invention, esterase means a broadly defined esterase including lipase) is allowed to act on the ester represented by the general formula (I). Asymmetrically hydrolyzed to produce optically active 4
The present invention relates to a biochemical method for producing optically active 4-hydroxy-2-cyclopentenone, which comprises separately obtaining -hydroxy-2-cyclopentenone.

The optically active 4-hydroxy-2-cyclopentenone obtained in the present invention is useful as a raw material for prostaglandin, which has various pharmacological actions such as pharmaceuticals, particularly antiulcer action and thrombolytic action. Those having an R configuration are particularly useful for this purpose, but those having an S configuration have recently been found. Therefore, development of a technique for optically activating racemic 4-hydroxy-2-cyclopentenone is desired.

<Prior Art> It has been conventionally reported that 4-hydroxy-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, it is impossible to obtain optically active 4-hydroxy-2-cyclopentenone from racemic 4-hydroxy-2-cyclopentenone by this method. Further, prior to the present invention, it was not known at all that the carboxylic acid ester of 4-hydroxy-2-cyclopentenone could be asymmetrically hydrolyzed.

<Structure of the Invention> The present inventors have found that racemic 4-hydroxy-2-cyclopentenone organic carboxylic acid ester is a genus of Achromobacter, Alcaligenes, Arthrobacter, Candida, Chromobacterium. The inventors have found that they can be asymmetrically hydrolyzed by an esterase derived from a microorganism belonging to the genus Pseudomonas or the genus Humicola, and have completed the present invention.

In the present invention, 4 of the racemate of the general formula (I) used
The organic carboxylic acid ester of -hydroxy-2-cyclopentenone is known, and a general method for producing a general ester, for example, racemic 4-hydroxy-2-cyclopentenone to an organic carboxylic acid anhydride or organic carboxylic acid is used. It can be easily produced by reacting with an acid halide.

According to the present invention, a racemic 4-hydroxy-2-cyclopentenone organic carboxylic acid ester of the general formula (I) is added to Achromobacter, Alcaligenes, Arthrobacter, Candida, Chromobacter. Umm,
An optically active 4-hydroxy-2-cyclopentenone can be produced by asymmetric hydrolysis by using an esterase derived from a microorganism belonging to the genus Pseudomonas or the genus Humicola.

The esterase to be used is derived from a microorganism belonging to the genus Achromobacter, Alcaligenes, Arthrobacter, Candida, Chromobacterium, Pseudomonas or Humicola, and is a racemic 4-formula of the general formula (I). There is no particular limitation as long as it can asymmetrically hydrolyze an organic carboxylic acid ester of hydroxy-2-cyclopentenone. There are commercially available esterases derived from microorganisms belonging to such Achromobacter, Alcaligenes, Arthrobacter, Candida, Chromobacterium, Pseudomonas or Humicola, and are easily commercially available. Can be obtained. Specific examples of commercially available esterases include lipases of the genus Achromobacter (lipase "AL": manufactured by Meito Sangyo), lipases of the genus Alcaligenes (lipase "PL": Meito Sangyo, Agricu
ltural and Biological Chemistry 46: 1159-1164 and 46: 1743-1750), Arthrobacter ureafas (Arthrobacter ureafas)
iens nov.var.) lipase (Nippon Kagaku), Caudida cylindracea lipase (lipase “MY”; Meito Sangyo, Biochimicaet B)
iophisica Acta, Vol. 231, 15-22, Chromobacterium viscosum lipase (lipase "LP"; Toyo Brewing, Agricultural and B)
Iological Chemistry, Vol. 37, 999-1005), Pseudomonas aeruginosa lipase (lipase "AMANO-3"; Amano Pharmaceutical Co., Ltd., JP 58
−104792), Pseudomonas fluorescens (Pseu
domonas fluorescens) lipase (lipase "P";
Amano Pharmaceutical Co., Biochemica et Biophysica Acta 488 Vol. 35
3 to 358), Humicola lipase (lipase "CE";
Amano Pharmaceutical Co., Ltd.) and the like.

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 an aqueous solution containing a crude esterase, a purified esterase and an esterase-containing extract or concentrate separated from the bacterial cells or the culture filtrate with the racemic cyclopentenone ester, and stirring or shaking the mixture. .
Also, immobilized cells or immobilized esterase can be used.

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

The pH during the reaction is 6 to 10 for the culture solution of alkaline bacterium and alkaline esterase, and 4 to 8 for normal esterase.
Is preferred.

In addition, it is preferable to use a buffer solution to neutralize the organic carboxylic acid generated by the hydrolysis reaction and keep the pH constant during the reaction. Sodium phosphate, a buffer solution of an inorganic acid salt such as potassium phosphate, sodium acetate, A buffer of an organic acid salt such as sodium citrate can be used.

The concentration of the racemic cyclopentenone ester used as a substrate is 0.1 to 50% by weight, preferably 0.1 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. 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.

<Effects of the Invention> The present invention provides a method for obtaining optically active 4-hydroxy-2-cyclopentenone from previously unknown racemic 4-hydroxy-2-cyclopentenone. Its features are that it is economically free, such as 1) the reaction using an enzyme does not require expensive optically active reagents, and 2) the steps and equipment are simple.

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

Example 1 1.4 g of racemic 4-acetoxy-2-hydroxycyclopentenone, 3.0 g of lipase of the genus Alcaligenes (lipase "PL"; manufactured by Meito Sangyo) and 0.1 M phosphate buffer (pH 6.
5) 100 g was mixed and reacted at 40 ° C. for 2 hours with vigorous stirring. The hydrolysis rate at the end of the reaction was 36.0%. The hydrolysis rate was measured by gas chromatography analysis.

After the reaction was completed, Glauber's salt 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 give 0.85 g of unreacted 4-acetoxy-2-cyclopentenone. 0.34 g of released 4-hydroxy-2-cyclopentenone
Obtained separately.

The 4-hydroxy-2-cyclopentenone obtained here was derivatized into an ester of (+)-p-chlorophenylisobutyric acid and then subjected to high performance liquid chromatography [column: Lichros
orb Sl-100, eluent: hexane / isopropanol = 5
As a result of performing diastereomer separation in [00/1] and measuring the optical isomer ratio, the content of the (R) isomer was 80.1% [R isomer: S isomer = 80.1: 19.9].

Examples 2 to 8 The same operation as in Example 1 was carried out except that the amounts described in Table 1 were used for each esterase described in Table 1, and the results in Table 1 were obtained.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication (C12P 41/00 C12R 1:06) (C12P 41/00 C12R 1:72) (C12P 41/00 C12R 1:01) (C12P 41/00 C12R 1:38)

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. ] An asymmetric hydrolysis of an organic carboxylic acid ester of 4-hydroxy-2-cyclopentenone represented by the following formula to produce an optically active 4-hydroxy-2-cyclopentenone, Achromobacter, Alcaligenes Asterase derived from a microorganism belonging to the genus Genus, Arthrobacter, Candida, Chromobacterium, Pseudomonas or Humicola is asymmetrically hydrolyzed by acting on an ester represented by the general formula (I). A biochemical method for producing optically active 4-hydroxy-2-cyclopentenone, which comprises separately obtaining the optically active 4-hydroxy-2-cyclopentenone.