JPS62244396A - Biochemical production of (r)-4-hydroxy-2-cyclopentenone - Google Patents

Abstract

PURPOSE:To obtain the titled cyclopentenone at a low cost, by reacting a specific compound with an esterase of a microorganism of the genus Alcaligenes and reacting the resultant optically active cyclopentenone containing the (R)- isomer, etc., with an organic carboxylic acid anhydride, etc. CONSTITUTION:An esterase (ES) derived from a microorganism of the genus Alcaligenes is reacted with an organic carboxylic acid ester (CE) of a racemic 4-hydroxy-2-cyclopentenone (4HS) expressed by formula I (R is 1-18C alkyl, 2-18C alkenyl or 2-18C alkynyl) to carry out asymmetric hydrolysis and give an optically active 4HS containing >=75% (R)-isomer. An organic carboxylic acid halide expressed by formula II (X is Cl or Br) or organic carboxylic acid anhydride expressed by formula III to afford CE of optically active 4HS expressed by formula IV (mark * indicates asymmetric carbon atom), which is then reacted with the above-mentioned ES to give optically active 4HS containing >=90% (R)-isomer. The resultant optically active isomer is further reacted with the carboxylic acids expressed by formulas I-II, etc., to afford CE of the optically active 4HS, which is then reacted with the above-mentioned ES to provide the aimed (R)-4HS containing >=97% (R)-isomer.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a biochemical production method of (R)-4-hydroxy-2-cyclobentenone.

(R)-4-Hydroxy-2-cyclobentenone obtained in the present invention is useful as an intermediate for agricultural chemicals, fragrances, and pharmaceuticals, and is particularly useful as a prosthetic compound having various pharmacological effects such as anti-ulcer and thrombolytic effects. It is extremely important as a raw material for Grandin. When used as a pharmaceutical, products with particularly high optical purity are required. Therefore,
It is desired to develop a technique for obtaining (2)-4-hydroxy-2-cyclobentenone with high optical purity from racemic 4-hydroxy-2-cyclobentenone.

<Prior art> It has been previously reported that 4-acetoxy-2-cyclobentenone is hydrolyzed by plant-derived enzymes such as wheat germ lipase and citrus peel esterase (Japanese Patent Publication No. 84677/1984) 7 However, according to this method, when the racemic ester is used as a substrate, the racemic 4
-Hydroxy-2-cyclobentenone is obtained, and in order to obtain optically active 4-hydroxy-2-cyclobentenone, it is necessary that the ester serving as a substrate is an optically active substance. Therefore, by this method, racemic 4-hydroxy-2-cyclobentenone can be obtained with high optical purity (
2) It is impossible to obtain -4-hydroxy-2-cyclobentenone.

<Structure of the Invention> The present inventors have discovered that an esterase derived from a microorganism belonging to an alkaline earth metal inertly hydrolyzes an organic carboxylic acid ester of racemic 4-hydroxy-2-cyclobentenone, and (2) It was discovered that -4-hydroxy-2-cyclobentenone was preferentially produced, and after thorough investigation, the present invention was completed.

The present invention is based on the general formula (I) [wherein R is an alkyl group having 1 to 18 carbon atoms, an alkenyl group having 2 to 18 carbon atoms, or an alkenyl group having 2 to 18 carbon atoms]
8 represents an alkynyl group. ] An esterase derived from a microorganism belonging to an alkaline earth metal is allowed to act on the organic carboxylic acid ester of racemic 4-hydroxy-2-cyclobentenone shown in
Asymmetric hydrolysis produces optically active 4-hydroxy-2-cyclobentenone containing 75% or more of the (2) isomer; -After isolating and obtaining cyclobentenone (
1) Organic carboxylic acid nitride represented by the formula or (2)
What carboxylic acid anhydride RCOX is represented by the formula?
(1) -C (wherein, R has the same meaning as above, and X represents a chlorine atom or bromine atom), and the obtained av) formula (wherein, R has the same meaning as above) is reacted. The optically active organic carboxylic acid ester of 4-hydroxy-2-cyclobentenone represented by ) The optically active 4-hydroxy-2-cyclobentenone having a content of 90% or more is produced and isolated, and the organic carboxylic acid halide represented by the formula (I) or the organic carboxylic acid halide represented by the formula (2) is obtained. The optically active organic carboxylic acid ester of 4-hydroxy-2-cyclobentenone obtained by reacting the carboxylic acid anhydride was asymmetrically hydrolyzed using the above-mentioned microorganism-derived esterase. % or more of optically active 4-hydroxy-2-cyclobentenone @) Biochemistry of -4-hydroxy-2-cyclobentenone! 1! Regarding the manufacturing method.

In the present invention, the racemic 4-hydroxy-
Organic carboxylic acid esters of 2-cyclobentenone are known and can be prepared by conventional methods for the preparation of esters, e.g. racemic 4-hydroxy-2-cyclobentenone <1).
It can be easily produced by reacting an organic carboxylic acid halide represented by the formula or an anhydride of an organic carboxylic acid represented by the formula (2).

The esterase used in the present invention is a broad maple esterase containing lipase produced by a microorganism belonging to alkaline earth metals.

Some of these microbial-derived esterases are commercially available and can be easily obtained. Specific examples of commercially available esterases include alkaline earth metal lipase (Lipase 1-PL, manufactured by Yatsuto Sangyo Co., Ltd., Agric Co., Ltd.
Ultra and Biological Che
mistry magazine vol. 46 pages 1159-1164 and same magazine 4
6, pp. 1748-1750).

When carrying out the method of the present invention, the asymmetric hydrolysis of the cyclobentenone ester is performed using a culture solution in which the above-mentioned microorganism is cultured, bacterial cells isolated from the culture solution, a culture filtrate containing esterase, or various enzyme separation methods. The racemic cyclobentenone ester is mixed with an aqueous solution containing crude esterase, purified esterase, and esterase-containing extract or concentrate separated from bacterial cells or culture filtrate, and the mixture is stirred for 4 hours or heated to 4Mt for 5 hours. It will be done. Furthermore, immobilized bacterial cells or immobilized esterase can also be used.

As conditions for asymmetric hydrolysis of the racemic cyclobentenone 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. In addition, it neutralizes the organic carboxylic acid produced by the hydrolysis reaction [J,
In order to keep the oH constant during the reaction, it is preferable to use a buffer solution, and it is preferable to use a buffer solution of an inorganic acid salt such as sodium phosphate or potassium phosphate, or a buffer solution of an organic acid salt such as sodium acetate or sodium citrate. can.

The concentration of the organic carboxylic acid ester of racemic 4-hydroxy-2-cyclobentenone used as a raw material is 0.1 to 50% of the reaction solution! lit%, preferably 1-2
It is 5% by weight.

Since prolonged reaction may cause racemization of liberated 4-hydroxy-2-cyclobentenone, it is important to keep the reaction time within 10 hours, preferably within 5 hours.

Here, the asymmetric hydrolysis reaction produces 4-hydroxy-
It is important to keep the (2)-isolate ratio of 2-cyclobentenone within a range of not less than 75%, and the hydrolysis is usually carried out until the hydrolysis ratio reaches a range of 10 to 45%. 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 manner, the liberated 4-hydroxy-2-cyclobentenone and unreacted ester are separated and recovered. In this separation and recovery, operations such as steam distillation, solvent extraction, fractional distillation, column chromatography, etc. can be appropriately employed. For example, if the reaction solution is first extracted with hexane, unreacted ester is isolated. Subsequent extraction with methyl isobutyl ketone isolates free 4-hydroxy-2-cyclobentenone. In addition, the reaction solution was extracted with methyl isobutyl ketone, and the extract was loaded onto silica gel column chromatography (eluent: hexane/ethyl acetate = 10/1) to perform column chromatographic separation. It is also possible to isolate and obtain cyclopentenone.

Next, optically active 4-hydroxy-2-cyclobentenone containing 75% or more of the (R) form obtained here,
Esterification is carried out in the same manner as in the above-described esterification of racemic 4-hydroxy-2-cyclobentenone to produce an optically active organic carboxylic acid ester of 4- and droxy-2-cyclobentenone. The optically active organic carboxylic acid ester of 4-hydroxy-2-cyclobentenone thus obtained was again converted into the racemic 4-hydroxy-2-cyclobentenone.
- Asymmetric hydrolysis is carried out using an esterase derived from Alcaligenes microorganisms under the same conditions as for the asymmetric hydrolysis of organic carboxylic acid ester of cyclobentenone. The asymmetric hydrolysis reaction at this time is kept within a range in which the content of the (2) isomer in the produced 4-hydroxy-2-cyclobentenone does not become less than 90%, but usually the hydrolysis rate is in the range of 20 to 65%. is carried out until it reaches.

After the reaction is completed, the 4-hydroxy-2-cyclobentenone produced from the reaction solution is separated in exactly the same manner as described above, and the optically active 4-hydroxy-2-cyclobentenone with a content of (2) isomer is 90% or more.
-Hydroxy-2-cyclobentenone is obtained. Furthermore, the optically active 4-hydroxy-2-cyclobentenone containing 90% or more of the (support) isomer obtained here is esterified with the racemic 4-hydroxy-2-cyclobentenone. Similarly, esterified and optically active 4
- Derivatizing hydroxy-2-cyclobentenone to an organic carboxylic acid ester.

Here, the excellent optically active carboxylic acid ester of 4-hydroxy-2-cyclopentenone is further asymmetrically hydrolyzed of the racemic organic carboxylic acid ester of 4-hydroxy-2-cyclopentenone. Asymmetric hydrolysis is performed using esterase derived from the Alcaligenes falcon microorganism under the same conditions as . In this asymmetric hydrolysis reaction, the (R) form at of 4-hydroxy-2-cyclobentenone is 97
Although the hydrolysis rate should not be less than 20%, it is usually carried out until the hydrolysis rate reaches a range of 20 to 75%. After the reaction is completed, 4-hydroxy-2-cyclobentenone produced from the reaction solution is separated in exactly the same manner as above, and (R)-4-hydroxy-2 with high optical purity is separated from the reaction solution.
- Obtain cyclobentenone.

<Effects of the Invention> The present invention produces a highly optically pure (2
)-4-Hydroxy-2-cyclobentenone, its characteristics are l) it does not require expensive optically active reagents used in ordinary organic synthetic chemical optical resolution methods; , 2) Since the reaction operation can be carried out at room temperature and pressure, no special equipment is required, which is economically advantageous.

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

Example 14.0Q of racemic 4-acetoxy-2-hydroxycyclobentenone, 20.0g of alkaline earth metal lipase (Lysase l'-PLJ; manufactured by Octosu Sangyo) and 0.0Q.
8M phosphate buffer (pH 6,5) mix 10 (1') and 4
The reaction was carried out at 0°C for 3 hours with vigorous stirring. The hydrolysis rate during reaction path r was 44.7%. 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 condensed, and the condensation residue was diluted with hexane/ethyl acetate = 10/1.
Column chromatography separation was performed using a mixed solvent of (volume ratio) of unreacted 4-acetoxy-2-cyclobentenone 7.
851 and liberated 4-hydroxy-2-cyclobentenone 4.2f were separated and obtained. Of the 4-hydroxy-2-cyclobentenone obtained here, 50sy was (+)-
After derivatization into an ester of p-chlorophenylisobutyric acid,
High performance liquid chromatography [Column: Lichro
Diastereomer separation was performed using sorb Sl-100, eluent: hexane/isopropanol = 500/I], and the optical isomer ratio was measured. As a result, the (9) white stone ratio was 77.5% [ 2 bodies: S body = 77.5: 2
2.51. 4-hydroxy-2-cyclobentenone obtained here. Dissolve Of in 10f of toluene, add 0.549f of acetic anhydride and 0.05f of sulfuric acid, and heat at 40°C for 8
The mixture was stirred for an hour to carry out an acetylation reaction. After the reaction, 20 g of water was added to the reaction solution for extraction and separation, water-soluble components were removed, and the toluene solution was collected and condensed to obtain 5.4 flz of 4-acetoxy-2-cyclobentenone, which is a condensation residue. . The obtained mercury-acetoxy-2-cyclobentenone 5
．． 41 and Alcaligenes 1m lipase 8.0g and 0.
50 g of 8M phosphate buffer was mixed and an asymmetric hydrolysis reaction was carried out with vigorous stirring at 40°C for 2 hours.

The hydrolysis rate at the end of the reaction was 58.8%.

After completion of the reaction, 1-condensation and column chromatography separation were performed in the same manner as above, and the hydrolysis product 4-hydroxy-2
-1.91 of cyclobentenone was obtained. The optical isomer ratio of the 4-hydroxy-2-cyclobentenone obtained here was measured in the same manner as described above, and the (2)-isomer ratio was 91.9%. [2 bodies: S body = 91.9:
8.11 4-hydroxy-2-cyclobentenone 1.82 obtained in step 0 was acetylated in the same manner as above,
2.5 g of 4-acetoxy-2-cyclobentenone was obtained. This 4-acetoxy-2-cyclobentenone 2.
5g and Alcaligenes Nene Lipase 4.0g and 0.8M
Mix 20g of buffer solution and shake vigorously at 40'O.
An asymmetric hydrolysis reaction was performed. The hydrolysis rate at the end of the reaction was 53.2%. After the reaction was completed, Glauber's salt was added to the reaction solution, and extraction was performed with methyl isobutyl ketone. From then on,
Perform acondensation and column chromatography in the same manner as above,
4-hydroxy-2-cyclobentenone produced 0.9
I got an F. The optical isomer ratio of the obtained 4-hydroxy-2-cyclobentenone was measured in the same manner as described in IMJ, and the (t) isomer content was 97.7%. [2 bodies: S body = 97.7: 2j31

Claims (1)

[Claims] 1) General formula (I) ▲There are numerical formulas, chemical formulas, tables, etc.▼(I) [In the formula, R is an alkyl group having 1 to 18 carbon atoms, an alkenyl group having 2 to 18 carbon atoms, or Represents 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 the organic carboxylic acid ester of racemic 4-hydroxy-2-cyclopentenone shown in
Asymmetric hydrolysis produces optically active 4-hydroxy-2-cyclopentenone containing 75% or more of the (R) form, and optically active 4-hydroxy-2-cyclopentenone containing 75% or more of the (R) form.
After isolating and obtaining hydroxy-2-cyclopentenone,
Organic carboxylic acid halide represented by formula (II) or (III
) Organic carboxylic acid anhydride RCOX (II) ▲ Numerical formulas, chemical formulas, tables, etc. are available ▼ (III) (In the formula, R has the same meaning as above, and X represents a chlorine atom or a bromine atom. ) is reacted, and the obtained formula (IV) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (IV) (In the formula, R has the same meaning as above, and the * symbol represents an asymmetric carbon.) An optically active organic carboxylic acid ester of 4-hydroxy-2-cyclopentenone is asymmetrically hydrolyzed using the above-mentioned microorganism-derived esterase to produce an optically active 4-hydroxy having an (R) isomer content of 90% or more. -2-Cyclopentenone is produced, isolated, and reacted with an organic carboxylic acid halide represented by formula (II) or an organic carboxylic acid anhydride represented by formula (III), resulting in an optically active 4 The organic carboxylic acid ester of -hydroxy-2-cyclopentenone is asymmetrically hydrolyzed using the above-mentioned microorganism-derived esterase, and an optically active 4-hydroxy-2- (R)-4-hydroxy-2 characterized by obtaining cyclopentenone
-Production method of cyclopentenone