JPS62175193A - Biochemical production of optically active 4-hydroxycyclopentenone - Google Patents

Abstract

PURPOSE:To industrially and advantageously obtain the titled compound useful as a raw material for agricultural chemicals, etc., by asymmetrically hydrolyzing a racemic cyclopentenone ester with a microbial enzyme, converting the hydrolyzate into an optically active ester using an aliphatic carboxylic acid and asymmetrically hydrolyzing the ester with a microbial enzyme. CONSTITUTION:A racemic cyclopentenone ester expressed by formula I (R' is alkyl or alkenyl which may be substituted by halogen; R is 1-7C straight- chain alkyl, alkenyl, etc.) is asymmetrically hydrolyzed with a microbial enzyme to isolate an optically active substance containing >=90% (R) isomer of 4- hydroxycyclopentenone expressed by formula II, which is reacted with an aliphatic carboxylic acid halide or acid anhydride to afford an optically active cyclopentenone ester expressed by formula III. The resultant ester expressed by formula III is then asymmetrically hydrolyzed with a microbial enzyme to give the aimed optically active 4-hydroxycyclopentenone containing >=97% (R) isomer of the compound expressed by formula II.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a biochemical method for producing optically active 11A4-hydroxycyclobentenones represented by formula (H).

More specifically, the present invention relates to a biochemical production method of (R)-form 4-hydroxycyclobentenones.

Prior Art and Problems 4-Hydroxycyclobentenones represented by the above formula DI), especially its (R) form, are useful as intermediates for non-f drugs, aromatic drugs, and pharmaceuticals, but they are particularly useful as prosthetic agents. Grandin '8f: 4-hydroxy-2-allyl-2-cyclobentenone, which is extremely important as an intermediate for the JE' form, and in which the substituted WR is an allyl group, was disclosed in JP-A No. 5
As described in Publication No. 8-41836, it is an extremely important compound as an intermediate to thiaprostaglandins that have pharmacological effects such as antiplatelet aggregation, and for that purpose, (R) It is necessary to obtain the compound with a high optical purity of 97% or more.

Conventionally, asymmetric hydrolysis methods using microbial enzymes have been known to obtain compounds similar to the 4-hydroxycyclobentenones of the present invention. (JP 60-12991) However, this asymmetric hydrolysis method using microbial enzymes is a superior method in that it does not require complicated steps or expensive reagents, but optical selection of microbial enzymes is Since the properties are not necessarily strict, it is difficult to obtain optically active 4-hydroxycyclobentenones with an optical purity of 97% or more in the (R) isomer content at an industrially advantageous yield. It was hard to become. That is, in order to obtain optically active 4-hydroxycyclobentenones with an (R) isomer content of 97% or more using a microbial enzyme whose optical selectivity is not strict, the hydrolysis rate during the asymmetric hydrolysis reaction must be adjusted. I needed to keep it low. Therefore, it is impossible to simultaneously satisfy two requirements: the optically active 4-hydroxycyclobentenones to be obtained have a (R) isomer content of 97% and can be produced at an industrially advantageous yield. Met.

Means for Solving the Problem The present inventors have developed an optically active 4-hydroxycyclobentenone that can produce optically active 4-hydroxycyclobentenones with a high optical purity and an (R) isomer content of 97% or more at an industrially advantageous yield. As a result of repeated research to find a biochemical production method for hydroxycyclobentenone, the racemic cyclobentenone ester represented by formula (I) was asymmetrically hydrolyzed using a microbial enzyme to produce (H). After producing optically active 4-hydroxycyclobentenones containing 90% or more of 4-hydroxycyclobentenones in the (R) form of the formula and isolating the optically active 4-hydroxycyclobentenones, the optically active compound represented by the formula (V) is obtained. By converting the optically active cyclobentenone esters into cyclobentenone esters and asymmetrically hydrolyzing the optically active cyclobentenone esters again using a microbial enzyme, 4-4- The present inventors have discovered that hydroxycyclopentenones can be produced with good yield, and have completed the present invention.

The present invention will be explained in detail below.

The following compounds are exemplified as racemic 4-hydroxycyclobentenones constituting the racemic cyclobentenone esters represented by formula (I), which are used as raw materials in the present invention.

2-ethyl-4-hydroxy-2-cyclobentenone, 2-n-propyl-4-hydroxy-2-cyclobentenone, 2-isopropyl-4-hydroxy-2-cyclobentenone, 2-n-butyl- 4-Hydroxy-2-cyclobentenone, 2-isobutyl-4-hydroxy-2-cyclobentenone, 2-n-pentyl-4-hydroxy-2-cylobentenone, 2-isopentyl-4-hydroxy-2-cyclobentenone , 2-n-hexyl-4-hydroxy-2-cyclobentenone, 2-n-heptyl-4-hydroxy-2-cyclobentenone, 2-allyl-4-hydroxy-2-cyclobentenone, 2-propargyl -4-hydroxy-2-cyclobentenone, 2-(2-cis-butenyl)-4-hydroxy-2-cyclobentenone, 4-hydroxy-2-(ω-butynyl)-2-cyclobentenone, 2 -(2--cis-pentenyl)-4-hytology 2
-cyclobentenone, 2-(2-1-lans-pentenyl)-1-hydrodi)
-cy2-cyclobentenone, 2-(3-cis-hexenyl)-4- and 1·'roxy-2-cyclobentenone, 2-(2-pentynyl)-4,-hydroxy-2-cyclopentenone.

Among these compounds, particularly useful ones are 2-
Allyl, 2-propargyl, l-(2-cis-butenyl), 2-(ω-butynyl), 2-(2-cis-pentenyl), 2-(2-trans-pentenyl), :2-
(3-cis-hexenyl) and 2-(2-pentynyl)-4-hydroxy-2-cyclobentenone.

Furthermore, examples of the aliphatic carboxylic acids that are the other component include the following compounds.

Acetic acid, propionic acid, acetic acid, caproic acid, caprylic acid,
Lauric acid, palmitic acid, chloroacetic acid, dichloroacetic acid.

For the esterification reaction between racemic 4-hydroxycyclobentenones and aliphatic carboxylic acids, a conventional method for producing esters is applied, and racemic 4-hydroxycyclobentenones are reacted with anhydrides of aliphatic carboxylic acids. Alternatively, the reaction is carried out by reacting aliphatic carboxylic acid halides using a catalyst in the presence or absence of a solvent.

In this reaction, when a solvent is used, the solvent may be an aliphatic or aromatic solvent such as tetrahydrofuran, ethyl ether, acetone, methyl ethyl ketone, toluene, benzene, chlorobene, dichloromethane, dichloroethane, chloroform, carbon tetrachloride, dimethylformamide, hexane, etc. Examples include solvents that are inert to the reaction, such as group hydrocarbons, ethers, and halogenated hydrocarbons, either alone or as a mixture. The amount used can be used without any particular restriction.

The aliphatic carboxylic acids used in the reaction are required in an amount of 1 equivalent or more corresponding to the racemic 4-hydroxy-2-cyclobentenone as a raw material, and there is no particular restriction on the upper limit, but preferably 1 to 4 equivalents. It is.

Examples of the catalyst include triethylamine, tri-n-butylamine, pyridine, picoline, sodium to lithium carbonate,
Examples include organic or inorganic basic substances such as sodium methylate and potassium hydrogen carbonate. The amount used is not particularly limited, but is usually 1 to 5 equivalents relative to racemic 4-hydroxycyclobentenone. When an organic amine is used as a solvent, the amine may act as a catalyst. Furthermore, acids such as toluenesulfonic acid, methanesulfonic acid, and sulfuric acid can also be used as catalysts.

The reaction temperature is usually -20 to 150'C, preferably -]0 to 120'C. There is no particular restriction on the reaction time.

Through such a reaction, the racemized cyclobentenone esters represented by (I)2 can be easily obtained in high yields, and these can be separated by conventional separation means such as extraction, liquid separation,
It can be easily isolated from the reaction mixture by concentration, distillation, etc.

The microorganism that produces the esterase used in the present invention acts on racemic cyclobentenone esters represented by the formula (T), preferentially producing the (R) cyclobentenone ester represented by (TI>). Any microorganism may be used as long as it produces esterase having the ability to produce esterase, and is not particularly limited (Esterase in the present invention means nisrase in a broad sense including lipase).Specific examples of such microorganisms Examples include microorganisms belonging to the following genera.

Pseudomonas s
p,) Arthrobacter sp.
r sp,) Chromobacterium (Chromobacterium sp.
cterium sp,) alkaline earth metals (Δl
Ca11. (:eneS 5p-) Some of these microbial-derived esterases are commercially available and can be easily obtained.

Specific examples of commercially available esterases include Pseudomonas fluorescens.
) lipase (Lipase “P”; manufactured by Hitoyori Seiyaku, Bioc
hemica etBiophysica Acta
vol. 488, pp. 353-358), Arthrobacter ureafaciens knob var (8 rthrobac
ter ureafaciens nov, var,)
lipase (Shin Nippon Kagaku Preparation), Kurobacterium
Hiscozam (Cbromobacterium vis
cosum) lipase (Lipase “LP”; manufactured by Toyo Jojo Co., Ltd., 8glycultural and Bi
oroHical Chemistry a,t
Volume 37, pages 999-1005), alkaline earth metal lipase (Lipase "PL"; manufactured by Najo 1 Sangyo A)
(Bricalturaland [lic, logi
cal Chemistry magazine vol. 46, 11.59-1
164 pages and the same magazine, Vol. 46, pages 1743-1750).

When carrying out the method of the present invention, the asymmetric hydrolysis of the cyclobentenone esters is performed using a culture solution in which the above-mentioned microorganisms are cultured, bacterial cells isolated from the culture solution, a culture filtrate containing esterase, or various enzyme separation methods. This is carried out by mixing the racemic cyclobentenone ester with an aqueous solution containing crude esterase, produced esterase, and esterase-containing extract or concentrate separated from bacterial cells or culture filtrate, and stirring or shaking the mixture. Furthermore, immobilized bacterial cells or immobilized esterase can also be used.

The conditions for carrying out the asymmetric hydrolysis reaction of the racemic cyclobentenone esters include a reaction temperature of 10 to 70°.
C is suitable, usually 20-50°C.

The pH during the reaction is p118-11. For esterases that are not alkalophilic, a pH of 5 to 8 is preferred. In addition, it is preferable to use a buffer solution in order to neutralize the organic carboxylic acid produced by the hydrolysis reaction and keep the pl+ constant during the reaction.
Buffer solutions of inorganic acid salts such as sodium phosphate and potassium phosphate,
Sodium acetate, citrate) IJ column organic acid salt buffers can be used.

The concentration of the racemic cyclobentenone ester as a substrate is 0.1 to 50% by weight, preferably 0.5 to 25% by weight, based on the reaction solution. The concentration of esterase used is 0.1 to 50% by weight based on the weight of the substrate.
It is.

Here, it is important to stop the asymmetric hydrolysis reaction within a range where the (R) isomer content of the produced 4-hydroxycyclobentenone does not become less than 90%, and usually the hydrolysis rate is 40%.
This is done until a range of ~60% is reached.

After the reaction is complete, in order to separate the hydrolysis product and hydrolysis residue from the reaction solution, the hydrolysis solution is extracted with a solvent such as methyl isobutyl ketone, ethyl acetate, or ethyl ether, and the solvent is distilled from the organic layer. After the concentration residue is further distilled or treated with column chromatography, the (R) isomer, which is a hydrolysis product, is separated from the 90%
% or more of optically active 4-hydroxycyclobentenones and unreacted cyclobentenone esters remaining from hydrolysis can be separated.

Next, the optically active 4-hydroxycyclobentenone containing 90% or more of the (R) form obtained here is esterified in the same manner as in the esterification of the racemic 4-hydroxycyclobentenone described above. It is esterified and induced into optically active cyclobentenone esters. This optically active cyclobentenone ester is again asymmetrically hydrolyzed using a microbial esterase under the same conditions as the asymmetric hydrolysis reaction of the racemic cyclobentenone ester. The asymmetric hydrolysis reaction at this time is stopped within a range where the (R) isomer content of the 4-hydroxycyclobentenones produced does not become less than 97%, but usually the hydrolysis rate is 7%.
This is done until the percentage reaches 0-95%.

After the reaction is completed, the hydrolysis product and the hydrolysis residue are separated from the reaction solution in exactly the same manner as above, and here,
Optically active 4-hydroxycyclobentenones with a (R) content of 97% or more are obtained.

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

Example 1 68.6 g of racemic 2-allyl-4-acetoxy-2-cyclobentenone, 140 w of Arsulochakudu lipase (manufactured by Shin Nihon Kagaku) and 0.6 g of racemic 2-allyl-4-acetoxy-2-cyclobentenone, 140 w of Arsulochakudu lipase (manufactured by Shin Nihon Kagaku), and 0.6 g of racemic 2-allyl-4-acetoxy-2-cyclobentenone. 1M phosphate buffer (pH
6,5) 28'Og was mixed and an asymmetric hydrolysis reaction was carried out with vigorous stirring at 45'C for 20 hours. The hydrolysis rate at the end of the reaction was 43.4%. In addition,
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 extract was concentrated, and the concentrated residue was purified by column chromatography using a mixed solvent of ethyl acetate=toluene-3:5 (weight ratio) to obtain 2-allyl-4-acetoxy-2-cyclobentenone, which is a hydrolysis residue. 3
8.8 g and 22.4 g of 2-allyl-4-hydroxyl 2-cyclobentenone, which is a hydrolysis product, were obtained. The 2-allyl-4-hydroxy-2-cyclobentenone obtained here was converted into an ester of (+)-α-methoxy-α-(trifluoromethyl)-phenylacetic acid, and then diaphragm was obtained by high-performance liquid chromatography. As a result of separating the stereomers and measuring the optical isomer ratio, the (R) content was 91.8%.
[R form: 3 forms - 91.8:8.2]. 22.4 g of 2-allyl-4-hydroxy-2-cyclobentenone obtained here was dissolved and carbonized in methyl isobutyl ketone, and 1.8.8 g of acetic anhydride and 0.09 g of sulfuric acid were added.
The mixture was stirred at 0°C for 3 hours to carry out an acetylation reaction. After the reaction, 100 g of water was added to the reaction solution for extraction and separation, water-soluble components were removed, and a methyl isobutyl ketone solution was recovered. This methyl isobutyl ketone solution was concentrated to give 2-allyl-4-acetoxy-2-cyclobentenone 29 as a concentrated residue.

2g was obtained. 29.2 g of the obtained 2-allyl-4-amothoxy-2-cyclobentenone, 60 μg of Arthrobacter lipase (manufactured by Shin Nippon Chemical) and 100 g of 0.1 M phosphate buffer were mixed, and the mixture was heated at 45°C for 24 hours. The asymmetric hydrolysis reaction was carried out with vigorous stirring for hours. The hydrolysis rate at the end of the reaction was 73%. After the reaction was completed, Glauber's salt was added to the reaction solution, and extraction was performed with methyl isobutyl quinone.

After that, do the same as above? After column chromatography and purification, the hydrolyzed product 2-aryl)'
v-4. -H)-Waxy 2-cyclobentenone 16.

3g was obtained. Regarding the 2-allyl-4-hydroxy-2-cyclobentenone obtained here, the optical isomer ratio was measured in the same manner as above, and the (R) isomer content was 9.
It was 8.5% [R-isomer=S-isomer=98.5:1.5].

Moreover, the yield was 61%. (Here, the yield refers to racemic 2-allyl-4-acetoxy-2-cyclobentenone used as a raw material, (R) = 2-allyl-4
- refers to the molar ratio of produced (R)-2-allyl-4-hydroxy-2-cyclobentenone to acetoxy-2-cyclobentenone. ) Reference example 68.6 g of racemic 2-allyl-4-acetoxy-2-cyclobentenone, 140 g of Arthrobacter lipase (Shin Nippon Chemical Type) and 280 g of 0.1 M phosphate buffer
The asymmetric hydrolysis reaction was carried out with vigorous stirring at 45° C. for 5 hours.

Thereafter, in the same manner as in Example 1, the hydrolysis product 2-
Allyl-4-hydroxy-2-cyclobentenone], 5
．． 4g was isolated. As a result of measuring the optical isomer ratio of the obtained 2-allyl-4-hydroxy-2-cyclobentenone in the same manner as in Example 1, the (R) isomer content was 9.
It was 3.5% [9 bodies:8 bodies-93,5:6.5].

Also, the yield was 55%.

Examples 2-4 Racemized 2-allyl-4-acetoxy-2-cyclobentenone 30.0 g, Arthrobacter lipase 0
．． 24g and 0.1M phosphate buffer (pH 6,5) 1
00 g were mixed and an asymmetric hydrolysis reaction was carried out at 40° C. with vigorous stirring (hydrolysis rate 42.0%). After the reaction was completed, the same operation as in Example 1 was carried out to obtain 9.67 g of 2-allyl-4-hydroxy-2-cyclobentenone (9 bodies: 8 bodies - 92,5:1.5 ), which was then induced to 2-4-acetoxy-2-cyclobentenone (yield: 112.5 g). 1 g of 2-allyl-4-ace1-xy-2-cyclobentenone obtained here, each lipase listed in Table 1 and 0.1. 10 g of M phosphate buffer (pH 6.5) was mixed and an asymmetric hydrolysis reaction was carried out at 40° C. for 8 hours with vigorous stirring. Thereafter, in the same manner as in Example I, optically active 2-allyl-4-
Hydroxy-2-cyclobentenone was obtained and the results shown in Table 1 were obtained.

\\, ゛≧Hemi Table 1 Example 5 Racemic 2-propargyl-4-acetoxy-2-cyclobentenone 69.0g, Pseudomonas lipase (manufactured by Hitoyori Seiyaku) 300■ and 0゜1M phosphate buffer (p
280 g of H6,5) were mixed and an asymmetric hydrolysis reaction was carried out with vigorous stirring at 30° C. for 20 hours. The hydrolysis rate at the end of the reaction was 55%. Note that the hydrolysis rate was determined 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. Concentrate the extract? The agricultural shrinkage residue was purified by column chromatography using a mixed solvent of acetic acid and toluene in a ratio of 3:5 (weight ratio) to obtain 2-propargyl-4-acetoxy-2-cyclobentenone, a hydrolysis residue.
0.7g and the hydrolysis product 2-propargyl-4
-Hydroxy-2-cyclobentenone 28.2! I got g. 2-propargyl-4-hydroxy- obtained here
Collect 10 mg of 2-cyclobentenone, (+)-α
-Methoxy-α-(trifluoromethyl)-phenylacetic acid was converted into an ester, the diastereomers were separated by high performance liquid chromatography, and the optical isomer ratio was measured. As a result, the (R) isomer content was 90. 5% [9 bodies: 8 bodies -90,
5:9.5).

Furthermore, 28; 2 g of 2-propal-4-hydroxy-2-cyclobentenone was dissolved in 30 g of methyl isobutyl ketone, and 24.0 g of acetic anhydride and 0.11 g of sulfuric acid were added.
The mixture was dissolved at 40° C. for 3 hours to perform an acetylation reaction. After the reaction, 100 g of water was added to the reaction solution for extraction and separation, water-soluble components were removed, and a methyl isobutyl ketone solution was recovered.

This methylisobutylkene solution was concentrated to obtain 34.7 g of 2-propargyl-4-acetoxy-2-cyclobentenone as a concentrated residue. 2-propargyl-4-acetoxy-2-cyclobentenone 34 obtained here
．． 7g and Pseudomonas lipase (manufactured by Hitoyori Seiyaku) 17
Mix 0Ing and 100g of O,1,M phosphate buffer,
The asymmetric hydrolysis reaction was carried out at 30° C. with vigorous stirring for 24 hours. The hydrolysis rate at the end of the reaction was 73%. After the reaction was completed, Glauber's salt was added to the reaction solution, and extraction was performed with methyl isobutyl quinone. Thereafter, concentration and column chromatography purification were performed in the same manner as above to obtain 19.3 g of 2-propargyl-4-hydroxy-2-cyclobentenone, a hydrolysis product.

2-propargyl-4-hydroxy-2 obtained here
- Regarding cyclobentenone, the optical isomer ratio was measured in the same manner as above, and the result was that R-isomer: S-isomer = 99. sho, 5
There was. The yield was 68.5%. (Here, the yield refers to the amount of (R) 2-propargyl-4-acetoxy-2-cyclobentenone contained in the racemic 2-propargyl-4-acetoxy-2-cyclobentenone used as a raw material. Produced (R)-2-allyl-4-hydroxy-2-
Means the molar ratio of cyclobentenone. ) Examples G to 8 30.0 g of racemic 2-propargyl-4-acetoxy-2-cyclobentenone, 200 g of Pseudomonas lipase and 0.1 M phosphate buffer (pH 6.5)
100 g were mixed and an asymmetric hydrolysis reaction was carried out at 40°C with vigorous stirring (hydrolysis rate 53.5%).

After the reaction was completed, the same operation as in Example 5 was carried out to obtain the hydrolysis product 2-propargyl-4-hydroxy-2-
cyclobentenone] 1.6 g (R form = S form -91,0:
After obtaining 9゜0, 2-propargyl-4-ace1hexy ~
2-cyclobentenone (yield: 114.6 g) was derived. 1 g of the obtained 2-propargyl-4-acetoxy-2-cyclobentenone, each lipase listed in Table 2, and 0.1M phosphate buffer (pH 6).

5) Mix 10g and heat at 40°C with vigorous stirring.
The asymmetric hydrolysis reaction was carried out for several hours. Thereafter, optically active 2-propargyl-4-hydroxy-2-cyclobentenone was obtained in the same manner as in Example 5, and the results shown in Table 2 were obtained.

Table 2 complete Procedural amendment (voluntary) June 30, 1986

Claims (1)

[Claims] (I) Formula ▲ Numerical formula, chemical formula, table, etc. ▼ (I) (In the formula, R' is an alkyl group or alkenyl group that may be substituted with halogen, and R has 1 carbon number. The racemic cyclopentenone esters represented by 7) are asymmetrically hydrolyzed using a microbial enzyme to form the formula (II) ▲ mathematical formula, chemical formula, table. etc. ▼ (II) (In the formula, R has the same meaning as above, and the * mark indicates an asymmetric carbon.) 90% or more of the (R) form of 4-hydroxysillopentenones After producing the optically active 4-hydroxycyclopentenone containing the (R) form and isolating and obtaining the optically active 4-hydroxycyclopentenone containing 90% or more of the (R) form, R'COX (III) in the aliphatic carboxylic acid halide or formula (IV) ▲ Numerical formulas, chemical formulas, tables, etc. are available ▼ (IV) (In the formula, R' has the same meaning as above, and X is a chlorine atom Alternatively, it represents a bromine atom.) The aliphatic carboxylic acid anhydride shown is reacted, and the obtained formula (V) ▲ Numerical formulas, chemical formulas, tables, etc. are available ▼ (V) (In the formula, R and R' are the same as above. The optically active cyclopentenone esters represented by (*) represent an asymmetric carbon having the same absolute configuration as above) are asymmetrically hydrolyzed using a microbial enzyme to form a compound represented by formula (II). Biochemistry of optically active 4-hydroxycyclopentenones characterized by obtaining optically active 4-hydroxycyclopentenones having an (R) form content of 97% or more. (2) The microbial enzyme is an enzyme produced by a microorganism selected from microorganisms belonging to the genus Arthrobacter, the genus Alcaligenes, the genus Chromobacterium, or the genus Pseudomonas. Manufacturing method described in Section 1