JPH0155000B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a biochemical method for producing (S)-(-)-α-cyano-3-phenoxybenzyl alcohol. More specifically, esterases produced by microorganisms belonging to the genus Arthrobacter or Pseudomonas are treated at pH 7 or below.
-By acting on an organic carboxylic acid (saturated or unsaturated carboxylic acid having 1 to 18 carbon atoms) ester of cyano-3-phenoxybenzyl alcohol, (S)
By preferentially asymmetrically hydrolyzing the -(-)- alcohol ester, the (S)-(-)-α-
(S)- by obtaining the ester of cyano-3-phenoxybenzyl alcohol and its enantiomer
The present invention relates to a biochemical production method of (-)-α-cyano-3-phenoxybenzyl alcohol. α-Cyano-3-phenoxybenzyl alcohol is known as a novel alcohol component of a group of ester compounds called synthetic pyrethroids, which have excellent insecticidal activity. Synthetic pyrethroids, such as fuenvalerate, cypermethrin, and deltamethrin, which contain this alcohol as an alcohol component, have dramatically increased efficacy and are resistant to sunlight, so they are useful not only for household and epidemic prevention purposes but also for agricultural use. It is also attracting attention as an insecticide, and therefore the importance of α-cyano-3-phenoxybenzyl alcohol is increasing. Since the α-cyano-3-phenoxybenzyl alcohol has an asymmetric carbon at the α-position, two types of optical isomers exist, and the insecticidal efficacy as an ester is higher than that of the S-form alcohol. is known to be far superior to the corresponding R-isomer (Kosuke Yoshioka, Organic Synthetic Chemistry, Vol. 38, No. 12, 115-
1162 pages (1980)). Therefore, (R,S)-α-cyano-3 obtained by ordinary synthetic chemical production methods
- It has been desired to develop a technique for optically resolving phenoxybenzyl alcohol using an industrially advantageous method to obtain the S-form. By the way, the α-cyano-3-phenoxybenzyl alcohol is an unstable compound, and it is difficult to employ the conventional optical resolution method of alcohol via ester or half-ester with a normal optically active organic acid. Until now, (S)-(-)-α-cyano-
As a method for obtaining 3-phenoxybenzyl alcohol, (R,S)-α-cyano-3-phenoxybenzyl alcohol is converted to cis-2,2-dimethyl-3S-() in the presence of an acidic reagent. Dihydroxymethyl) cyclopropane is reacted with 1R-carboxylic acid lactone to form an ether compound, and the resulting two isomer mixtures are separated by physical means to form an ether compound containing an (S)-(-) alcohol component. A method for obtaining the desired (S)-(-)-α-cyano-3-phenoxybenzyl alcohol by hydrolyzing it in an acidic medium (Japanese Patent Application Laid-Open No. 109944-1989)
No.) or () Chiral cyclopropanecarboxylic acid (S)-(-)-α-cyano-3-phenoxybenzyl alcohol ester is reacted with boron halide and then water is reacted (S). −(−)−
Method for obtaining α-cyano-3-phenoxybenzyl alcohol (JP-A-56-12355). is only known. However, these methods also require complicated steps;
It requires an expensive optically active reagent and has a low total yield, and the method () requires an optically active ester of α-cyano-3-phenoxybenzyl alcohol in advance. However, this is not necessarily a sufficient method. The present inventors have conducted repeated research to overcome these problems and find an industrially advantageous method for producing (S)-(-)-α-cyano-3-phenoxybenzyl alcohol. , racemate i.e. (R,S)
-By using ester of α-cyano-3-phenoxybenzyl alcohol as a raw material and biochemically asymmetrically hydrolyzing it, (S)-(-)-α-cyano-3-phenoxybenzyl It was discovered that alcohol can be efficiently separated into the corresponding R-alcohol ester, and after further various studies, the present invention was completed. That is, the present invention provides an esterase produced by a microorganism belonging to the genus Arthrobacter or Pseudomonas at a pH of 7 or less.
-Cyano-3-phenoxybenzyl alcohol is reacted with an organic carboxylic acid (saturated or unsaturated carboxylic acid having 1 to 18 carbon atoms) ester to asymmetrically hydrolyze it to (S)-(- )-α-cyano-3
-(S)-(-)-α by splitting into phenoxybenzyl alcohol and its target ester
- A method for producing cyano-3-phenoxybenzyl alcohol is provided. Used as a raw material in the present invention (R,
Examples of the organic carboxylic acid component of the ester of S)-α-cyano-3-phenoxybenzyl alcohol include formic acid, acetic acid, propionic acid, butyric acid, antagonistic acid, isoantagonalic acid, caproic acid, caprylic acid, caproic acid, Examples include lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, etc., and esters of organic carboxylic acids having 2 to 18 carbon atoms are preferred from the viewpoint of ease of handling, optical purity of the reaction product, etc. Furthermore, ease of handling
Ester of acetic acid is more preferable from an economic point of view. These esters can be easily produced by a conventional method for producing esters, for example, by reacting (R,S)-α-cyano-3-phenoxybenzyl alcohol with an anhydride of an organic carboxylic acid or an organic carboxylic acid halide. can do. It can also be produced by reacting 3-phenoxybenzaldehyde with sodium cyanide and a halide of the organic carboxylic acid instead of (R,S)-α-cyano-3-phenoxybenzyl alcohol. The esterase that can be used in the present invention is a broadly defined esterase including lipase produced by a microorganism belonging to the genus Arthrobacter or Pseudomonas, and the microorganism is cultured in a liquid culture according to a conventional method, for example, in a sterilized liquid medium ( Inoculate microorganisms into a broth (broth medium) and store at 20-40°C for 2
Reciprocating shaking culture can be performed for ~3 days. Moreover, some of these microbial-derived esterases are commercially available. This commercially available enzyme can also be used for purposes of the present invention. Commercially available enzymes that can be used in the present invention include those shown below.

[Table] When carrying out the method of the present invention, (R,S)-α-
Asymmetric hydrolysis of organic carboxylic acid esters of cyano-3-phenoxybenzyl alcohol is carried out using esterase-containing materials such as the culture solution, culture filtrate, esterase extract, or concentrate obtained by culturing the microorganism, or crude esterase or purified esterase. This is carried out by mixing an aqueous solution containing an esterase and an organic carboxylic acid ester of the (R,S)-alcohol, and stirring or shaking the mixture. As needed,
Non-ester surfactants may also be added. It is also possible to immobilize the enzyme and use it. Further, the reaction temperature at this time is suitably 10 to 65°C, and preferably 20 to 50°C since the produced alcohol is relatively unstable to heat and the reaction rate is slow at too low a temperature. The reaction time is usually 3 to 48 hours, but the reaction time can be shortened by increasing the reaction temperature or using a highly active enzyme. Furthermore, since α-cyano-3-phenoxybenzyl alcohol is particularly easily decomposed by bases, it is important to control the pH of the aqueous solution during the enzymatic reaction. That is, even if asymmetric hydrolysis by esterase proceeds in a basic medium, the resulting alcohol will be denatured, so it is important to carry out the enzymatic reaction at a pH of 7 or below. Furthermore, since enzyme deactivation occurs at too low a pH, it is preferable to carry out the reaction within the range of PH4.5 to PH6.5. Furthermore, in order to prevent the pH from decreasing due to organic acids such as acetic acid produced by hydrolysis, it is desirable to control the pH to a constant level by using a buffer solution. As the buffer solution, either an inorganic acid salt buffer or an organic acid salt buffer can be used. The concentration of the ester substrate used is in the range of 1 to 80 W/W%, preferably 5 to 35%, based on the reaction solution, and even high substrate concentrations can be used. Next, after performing the asymmetric hydrolysis reaction in this way, the liberated (S)-(-)-α-cyano-3-
Phenoxybenzyl alcohol and unreacted enantiomer ester are separated and recovered. For this separation and recovery, operations such as solvent extraction, fractional distillation, column chromatography, etc. can be appropriately employed. For example, the reaction solution is extracted with an organic solvent such as ether, benzene, or toluene, and this extract is fractionally distilled under reduced pressure to produce (S)-(-)-α-cyano-3-phenoxybenzyl alcohol and its opposite compound. Free (S)-(-)-α-cyano-3 can be obtained by separating the ester from the body or by separating the extract by column chromatography using silica gel or the like.
- Phenoxybenzyl alcohol can be obtained. The unreacted ester thus separated and removed can be epitomized by contacting with a basic compound such as ammonia, pyridine, triethylamine, etc., and can be used again as a raw material in the method of the present invention. Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto. Examples 1-2 1.0 g of acetic acid ester of (R,S)-α-cyano-3-phenoxybenzyl alcohol and Table 1 below
20 mg of each esterase described in 1 was added to 10 ml of 0.2 M acetate buffer (PH5.0), and the mixture was reacted at 30°C with vigorous stirring using a stirrer. After reacting for 24 hours, the reaction product was extracted with toluene. High-performance liquid chromatography (HPLC) of the extract
[Lichrosorb RP-18, MeOH-water (6:4),
α-cyano-3-
The hydrolysis rate was calculated from the peak area ratio of acetic ester of phenoxybenzyl alcohol and α-cyano-3-phenoxybenzyl alcohol. It was confirmed from the HPLC analysis results that there was no by-product of 3-phenoxybenzaldehyde during the reaction under the above conditions. The extracted product was concentrated, subjected to silica gel column chromatography, and cyclohexane-
After eluting with ethyl ether (94:6) solution and separating and removing unreacted acetic ester of α-cyano-3-phenoxybenzyl alcohol, a trace amount (10 ^-5 %) of para-toluenesulfonic acid is also included. Elution with methanol yielded free α-cyano-3-phenoxybenzyl alcohol. Its structure is
Confirmed by HPLC, NMR and IR measurements. Next, after distilling off the elution solvent, a portion of the alcohol was taken and its specific rotation was measured in chloroform. As a result, the α-cyano-3-phenoxybenzyl alcohol obtained here was (-) It was confirmed to be (S)-(-)-α-cyano-3-phenoxybenzyl alcohol. In addition, 10 mg of the obtained free α-cyano-3-phenoxybenzyl alcohol was dissolved in 1 ml of toluene, and an equimolar amount of (S)-(+)-2-(4-chlorophenyl)-isovaleric acid chloride was added. was reacted with pyridine to form a diastereomer of (S)-(+)-2-(4-chlorophenyl)-isovaleric acid of α-cyano-3-phenoxybenzyl alcohol. DCQF-1,
The optical isomer ratio was analyzed at 2.5 m and 250°C. The results are shown in Table 1 below. It was found that the specific optical rotation of the unreacted ester separated and obtained after the enzymatic reaction showed a negative value in chloroform (for example, in the case of Example 1 [α] _D = -
8.5 (C=1.5).

[Table] Example 3 In Example 1, the amount of eselase was changed from 20 mg to 40 mg.
mg and also change the buffer from 0.2M acetate buffer (PH5.0) to 0.1M phosphate buffer (PH6.0).
As the reaction progresses, the concentration of 1M is increased.
Enzyme reaction and reaction product separation were carried out in the same manner as in Example 2, except that the NaOH aqueous solution was made into minute water droplets using a drop controller and carefully added, while the PH was controlled to be constant using a controller. (S) The specific rotation and hydrolysis rate of -(-)-α-cyano-3-phenoxybenzyl alcohol were measured. The results are shown in Table 2.

[Table] Examples 4 to 5 1.5 g of capric acid ester of (R,S)-α-cyano-3-phenoxybenzyl alcohol and 30 mg of each esterase listed in Table 3 below were added to a 0.2M acetate buffer solution. (PH5.4) and reacted for 24 hours at 30°C with vigorous stirring. Thereafter, separation and analysis were performed in the same manner as in Examples 1 and 2. The results are shown in Table 3.

[Table] Examples 6 to 9 In Examples 1 to 2, the amount of each esterase was 20 mg.
From 30mg to 0.2M acetate buffer (PH5.0) to 0.1M acetate buffer (PH5.3)
As the reaction progresses, the concentration of 1M is increased.
While carefully adding the NaOH aqueous solution into minute water droplets using a drop controller, the PH was controlled constant using a PH controller, and the substrate (R,
Examples 1 to 2 except that the amount of acetic ester of S)-α-cyano-3-phenoxybenzyl alcohol was changed from 1.0 g to the amount shown in Table 4 below.
The enzymatic reaction, separation, and analysis were performed in exactly the same manner as above, and the hydrolysis rate, specific rotation, and optical isomer ratio were measured by gas chromatography. The results are shown in Table 4.

[Table] Examples 10 to 13 In a 1000ml Erlenmeyer flask, add bouillon medium (1 part water)
5.0 g of peptone, 5.0 g of meat extract and 3.0 g of NaCl were dissolved in the solution, to which 2 g of triptyline was added in Examples 10-11, and 2 g of olive oil was added in Examples 12-13, and Na ₂ HPO ₄ and KH ₂ Set the pH to 7.2 with PO ₄ . ) After sterilizing 200 ml, inoculate 2 platinum loops of each microorganism listed in Table 5 below from the slant culture.
Culture was performed at ℃ for 48 hours with reciprocal shaking. Next, centrifugation is performed, acetone is added to the aqueous solution layer until the concentration is 85%, and immediately filtered. The resulting precipitate is washed with water and then dissolved in 15 ml of 0.2 M acetate buffer (PH5.2). In Examples 10 to 11, (R,S)-α-
1.5 g of butyric acid ester of cyano-3-phenoxybenzyl alcohol was added, and in Examples 12-13, 3.0 g of lauric acid ester of (R,S)-α-cyano-3-phenoxybenzyl alcohol.
was added, and the mixture was reacted for 26 hours at 33°C with vigorous stirring. Thereafter, separation and analysis were performed in the same manner as in Examples 1 and 2, and the hydrolysis rate and specific rotation were determined. The results are shown in Table 5.

[Table] Example 14 8.0 g of acetate ester of (R,S)-α-cyano-3-phenoxybenzyl alcohol and Arthrobacter esterase (total lipase BSL)
160mg in 2ml of 0.1M acetate buffer (PH4.5)
In addition, the reaction was carried out at 40°C with vigorous stirring using a stirrer. At this time, during the reaction, a 1M NaOH aqueous solution was carefully added as minute water droplets using a drop controller, and the pH was controlled at a constant level using a PH controller. After reacting for 24 hours, the reaction product was extracted with toluene. Thereafter, the same operations as in Example 1 were performed, and the results shown in Table 6 were obtained.

【table】

Claims (1)

[Claims] 1. Esterase produced by microorganisms belonging to the genus Arthrobacter or Pseudomonas was treated with an organic carboxylic acid (carbon Number 1~
(S)-
(S)-(-)-α-cyano-3-phenoxybenzyl alcohol characterized by its separation into (-)-α-cyano-3-phenoxybenzyl alcohol and its enantiomer ester. Biochemical manufacturing method.