JPH0153039B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention is represented by the following formula () (±)-4
This invention relates to a biochemical optical resolution method of -hydroxy-3-methyl-2-2'-propynyl-2-cyclopentenone. For more information, see Arthrobacter spp.
The organic carboxylic acid (±)-4-hydroxy-3-methyl-2-2'-propynyl-2-cyclopentenone (±)-4-hydroxy-3-methyl-2-2'-propynyl-2-cyclopentenone ( Asymmetrically hydrolyzing esters of saturated or unsaturated carboxylic acids having 1 to 18 carbon atoms to produce optically active 4-hydroxy-3-methyl-2-2'-propynyl-2- with high optical purity. Industrially advantageous 4-hydroxy-3 to obtain esters of cyclopentenone and its enantiomer
-Relates to a biochemical optical resolution method of methyl-2-2'-propynyl-2-cyclopentenone. 4-Hydroxy-3-methyl-2-2'-propynyl-2-cyclopentenone (hereinafter abbreviated as propynylcyclopentenolone), which is the subject of the present invention, is a so-called synthetic compound with excellent insecticidal activity. It is known as one of the important alcohol components of a group of ester compounds called pyrethroids. For example, 2 of the propynylcyclopentenolone,
The compound represented by the following formula (), which is an ester with 2,3,3-titramethylcyclopropanecarboxylic acid, is an excellent insecticide with extremely strong knock-down and lethal effects.
Publication No. 15843). Since propynylcyclopentenolone has an asymmetric carbon at the 4-position, there are two types of optical isomers, and the activities of these esters usually differ greatly.
For example, among the esters represented by the above formula (), the insecticidal efficacy of (+)-propynylcyclopentenolone ester is several times superior to that of the corresponding (-)-propynylcyclopentenolone ester. Therefore, there is a need for an industrially advantageous technique for optically resolving (±)-propynylcyclopentenolone obtained by conventional production methods. As a method for producing optically active propynylcyclopentenolone, (±)-propynylcyclopentenolone is made into a half ester of phthalic acid, and then this is reacted with an optically active amine to form a diastereomeric salt of optically active propynylcyclopentenolone. A known method is to produce optically active propynylcyclopentenolone by separating it, recovering it from the used amine as a half ester, and hydrolyzing the obtained half ester (Japanese Unexamined Patent Application Publication No. 1989-1999).
-2929 Publication). However, this method is not necessarily sufficient because it has a low total yield, requires complicated steps, and requires expensive optically active reagents. The present inventors have conducted extensive research to overcome these problems and establish an industrially more advantageous optical resolution method for (±)-propynylcyclopentenolone. Esterase produced by microorganisms belonging to the genus Achromobacter, Alcaligenes, or Trichoderma acts on organic carboxylic acid (saturated or unsaturated organic carboxylic acid having 1 to 18 carbon atoms) ester of (±)-propynylcyclopentenolone. The inventors have discovered that optically active propynylcyclopentenolone and its enantiomer ester with high optical purity can be obtained by this process, and have completed the present invention by conducting various studies on this. Next, the present invention will be explained in detail. The organic carboxylic acid (saturated organic carboxylic acid having 1 to 18 carbon atoms) ester of (±)-propynylcyclopentenolone used as a raw material in the method of the present invention can be produced by a conventional method for producing esters, such as (±)-propynylcyclopentenolone. -Propynyl It can be easily produced by reacting cyclopentenolone with an anhydride of an organic carboxylic acid or by reacting an organic carboxylic acid chloride in the presence of an organic base. The esterase-producing microorganism used in the present invention is a microorganism that belongs to the genus Arthrobacter, Pseudomonas, Achromobacter, Alcaligenes, or Trichoderma and that produces esterase in a broad sense including lipase. . Representative strain names belonging to each of these genera are illustrated below, but the microorganisms of the present invention are not limited to these examples. (1) Arthrobacter simplex IFO 3530 (2) Psecudomonas fluorescens IFO 3081 (3) Alcaligenes faecalis IFO 12669 (4) Trichoderma viride
viride) IFO 4847 All of these strains are American Type
It is preserved at the Culture Collection (ATCC) or the Institute of Fermentation (IFO) in Osaka City, and can be obtained from these institutions. For culturing the above-mentioned microorganisms, a culture solution is usually obtained by performing liquid culture according to a conventional method. For example, a sterilized liquid medium [for mold and yeast, malt extract/yeast extract medium (5.0 g of peptone in 1 part water,
Dissolve 10.0 g of glucose, 3.0 g of malt extract, and 3.0 g of yeast extract and adjust the pH to 6.5). For bacteria, use sweetened bouillon medium (10.0 g of glucose in 1 part water,
Dissolve 5.0 g of peptone, 5.0 g of meat extract, and 3.0 g of NaCl to give a pH of 7.2)] and inoculate it with microorganisms.
Carry out reciprocal shaking culture at 40°C for 1 to 3 days. Further, solid culture may be performed as necessary. Furthermore, some of these microbial-derived esterases are commercially available and can be easily obtained. Specific examples of commercially available esterases include Pseudomonas lipase (manufactured by Amano Pharmaceutical);
Lipase of the genus Alcaligenes (Lipase PL (manufactured by each sugar industry)), lipase of the genus Achromobacter (Lipase AL (manufactured by each sugar industry)), lipase of the genus Arthrobacter (Lipase Joint BSL (Joint Sake Refining)), etc. Can be mentioned. When carrying out the method of the present invention, the asymmetric hydrolysis of organic carboxylic acid (saturated or unsaturated carboxylic acid having 1 to 18 carbon atoms) ester of (±)-propynylcyclopentenolone is carried out using a culture of the above-mentioned microorganisms. liquid, bacterial cells isolated from culture liquid, culture filtrate containing esterase, crude esterase, purified esterase, and esterase-containing extract or concentrate separated from bacterial cells or culture filtrate by various enzyme separation methods (±) - by mixing the organic carboxylic acid ester of propynylcyclopentenolone and stirring or shaking. Furthermore, immobilized bacterial cells or immobilized esterase can also be used. The appropriate reaction temperature for asymmetric hydrolysis of the organic carboxylic acid ester of (±)-propynylcyclopentenolone is 10 to 70°C. 50-65℃ for thermostable esterases or 20℃ for esterases that are not particularly thermostable.
~50°C is preferred. The reaction time is usually 3 to 48 hours, but the reaction time can be shortened by raising the reaction temperature or increasing the amount of enzyme. The pH during the reaction is preferably PH8 to 11 for alkaline esterase from a culture of an alkalophilic microorganism, and PH5 to 8 for a culture of a microorganism that is not alkalophilic or an esterase that does not have alkali resistance. It also neutralizes organic carboxylic acids produced by hydrolysis,
In order to keep the pH constant during the reaction, it is preferable to use a buffer, and use an inorganic acid salt buffer such as sodium phosphate or potassium phosphate, or an organic acid salt buffer such as sodium acetate or sodium citrate. be able to. The concentration of the organic carboxylic acid ester of (±)-propynylcyclopentenolone, which is the substrate, is 1 to 50 wt%, preferably 5 to 50 wt%, based on the reaction solution.
It is 25wt%. Next, after carrying out the asymmetric hydrolysis reaction in this manner, the liberated optically active propynylcyclopentenolone and the unreacted enantiomer ester are separated and recovered. For this separation and recovery, operations such as steam distillation, solvent extraction, fractional distillation, column chromatography, etc. can be appropriately employed. For example, the reaction solution is steam-distilled and the distillate is extracted with ether, or the reaction solution is directly extracted with an organic solvent such as ether, ethyl acetate, or benzene, and this extract is fractionally distilled to produce optically active propynylcyclopentenolone and its opposite compound. The optically active organic carboxylic acid ester of propynylcyclopentenolone is first isolated by separating and obtaining the ester of the optically active propynylcyclopentenolone, or by subjecting the extract to column chromatography on silica gel and eluting with a toluene-ethyl acetate (5:1) solution. was separated and then toluene-ethyl acetate (2:
1) Free propynylcyclopentenolone, its enantiomer, is separated by elution with a solution. Furthermore, the ester of optically active propynylcyclopentenolone separated as described above can be easily led to optically active propynylcyclopentenolone by further deacylation.
For example, water and an equivalent amount of sodium hydrogen carbonate are added to the organic carboxylic acid ester of propynylcyclopentenolone, and then 10
After adjusting the pH to 5 by adding % HCl, propynylcyclopentenolone is easily separated by refluxing with stirring for 8 hours. As explained in detail above, the optical resolution of (±)-propynylcyclopentenolone by the method of the present invention is a simpler process than the conventionally known organic synthetic chemical optical resolution method, and also requires the use of expensive optically active reagents. It is economically advantageous because it does not require Furthermore, the optically active propynylcyclopentenolone obtained has a high yield and optical purity, and is extremely advantageous industrially. Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto. Examples 1 to 4 1.0 g of (±)-propynylcyclopentenolone acetate and each esterase listed in Table 1
20mg buffer (PH7.0, McIlvaine buffer or PH
9.5, 0.2M concentration of _{Na2CO3 − NaHCO3} buffer) 10
ml and reacted at 30°C with vigorous stirring using a stirrer. After reacting for 24 hours, the reaction product was extracted with ethyl acetate. The extract was analyzed by gas chromatography (5% DEGS, 1.1m, 180°C), and the hydrolysis rate was calculated from the peak area ratio of propynylcyclopentenolone acetate and propynylcyclopentenolone, and the following results were obtained. . The extract was concentrated and subjected to silica gel column chromatography, eluted with a toluene-ethyl acetate (5:1) solution to separate and obtain unreacted propynylcyclopentenolone acetate, and then toluene-ethyl acetate (2:1). 1) Free propynylcyclopentenolone was obtained by elution with the solution. 10 mg of the free propynylcyclopentenolone obtained here was dissolved in 1 ml of toluene, 0.2 ml of pyridine, (-)-α-methoxy-α-trifluoromethylphenylacetic acid chloride ((-)-
Add 45 mg of MTPA chloride) and heat under reflux for 1 hour to obtain (-)- of propynylcyclopentenolone.
Optical isomer analysis was performed using gas chromatography (silicon DCQF-1, 30m capillary column, 180℃) as MTPA diastereomer.
The optical isomer type and optical purity of free propynylcyclopentenolone were determined from the peak area ratio of the diastereomer of (+)-propynylcyclopentenolone and the diastereomer of (-)-propynylcyclopentenolone. Meanwhile, 5 ml of water and an equivalent amount of sodium hydrogen carbonate to the ester were added to the unreacted ester obtained by column chromatography, and then 10% HCl was added.
After adjusting the pH to 5 by adding water, the mixture was refluxed under stirring for 8 hours to perform deacylation. After cooling the reaction solution, ethyl acetate, water and salt were added for extraction. After distilling off the solvent, the resulting oil was subjected to silica gel column chromatography and eluted with a toluene-ethyl acetate (2:1) solution to obtain optically active propynyl. Cyclopentenolone was isolated and obtained. The optical isomer ratio of this propynylcyclopentenolone was analyzed by gas chromatography in the same manner as above to determine the optical isomer type and optical purity of the unreacted ester. The results are shown in Table 1.

[Table] Examples 5 to 7 0.65 g of capric acid ester of (±)-propynylcyclopentenolone and 10 mg of each esterase listed in Table 2 were added to 5 ml of buffer solution, and the mixture was stirred vigorously using a stirrer at 30°C. The reaction was allowed to proceed for 24 hours. Thereafter, the same operations as in Examples 1 to 4 were performed to obtain the results shown in Table 2.

[Table] Examples 8 to 9 Liquid medium in a 500ml shoulder flask [For molds and yeasts (Example 9), malt extract, yeast extract medium (1 part water to 5.0 g peptone, 10.0 g glucose, 3.0 g malt extract, Dissolve 3.0g of yeast extract, pH6.5
shall be. ) For bacteria (Example 8), sweetened bouillon medium (10.0 g glucose, peptone in 1 part water)
Dissolve 5.0g, meat extract 5.0g, NaCl 3.0g, pH
7.2. )] After sterilization, two platinum loops of each microorganism listed in Table 4 were inoculated from the slant culture, and cultured with reciprocating shaking at 30°C for 48 hours. Next, 7 g of (±)-propynylcyclopentenolone acetate was added to this culture solution, and after shaking the mixture reciprocally at 30° C. for 24 hours, the reaction solution was steam distilled, and the distillate was extracted with ether. The extract was concentrated, and free propynylcyclopentenolone was obtained by the same column chromatography operation as in Examples 1 to 4. This free propynylcyclopentenolone was prepared from Examples 1 to 2.
Optical isomer analysis was performed in the same manner as in 4. The results are shown in Table 3.

[Table] Example 10 Culture solution 1 of Trichoderma viride IFO4847 prepared in the same manner as in Example 9 was filtered to obtain a culture filtrate. Concentrate 30ml of this culture filtrate 3 times,
1.5 g of formic acid ester of (±)-propynylcyclopentenolone was added, and the mixture was reacted at 30° C. for 30 hours with vigorous stirring using a stirrer. Hereinafter Examples 1 to 4
The same operation as above was performed to obtain the results shown in Table 4.

【table】

Claims (1)

[Claims] 1 Esterase produced by a microorganism belonging to the genus Arthrobacter, Pseudomonas, Achromobacter, Alcaligenes or Trichoderma is (±)-4-hydroxy-3-methyl-2-
An optically active 4- Hydroxy-3-
(±)-4-hydroxy-3-methyl- characterized by splitting into esters of methyl-2-2'-propynyl-2-cyclopentenone and its enantiomer
Biochemical optical resolution method of 2-2'-propynyl-2-cyclopentenone.