JPS5894389A - Biochemical preparation of (s)-(-)-alpha-cyano-3-phenoxybenzyl alcohol - Google Patents

Abstract

PURPOSE:To prepare the titled compound having high optical purity, by a biochemical means, by asymmetrically hydrolyzing the carboxylic acid ester of (R,S)-alpha-cyano-3-phenoxybenzyl alcohol with a specific esterase. CONSTITUTION:An organic carboxylic acid[1-18C (un)saturated carboxylic acid]ester of (R,S)-alpha-cyano-3-phenoxybenzyl alcohol is asymmetrically hydrolyzed with an esterase produced by microorganisms belonging to Arthrobacter, Alcaligenes, Achromobacter, Pseudomonas, Aspergillus, or Mucor genus or an esterase derived from the pancreas of an animal and capable of asymmetrically hydrolyzing said ester. The ester is resolved by this process into (S)-(-)-alpha-cyano- 3-phenoxy-benzyl alcohol and its enantiomorphic ester, from which the titled compound is separated. The S-isomer has much stronger insecticidal activity than R-isomer.

Description

Detailed Description of the Invention The present invention relates to the biochemistry of (8) -(-) -a-cyano-8-phenoxybenzyl alcohol'.
It is about law. In more detail, Arthrobacter genus, Alcaligenes paddy, Acroheptabacter genus, Pseudomonas genus, Aspergillus maple, Tahamkor genus, and (R,8)-α-cyano-3-phenoxybenzyl alcohol organic carboxylic acid (1 carbon number For esters/V of saturated or unsaturated organic carboxylic acids having ~18
Esterase produced by microorganisms with asymmetric hydrolysis ability or esterase derived from animal pancreas.

By acting on the ester at pH 7 or lower, the ester of the (S)-(-) alcohol is preferentially asymmetrically hydrolyzed to produce (S)-(-)-a-cyano-3-phenoxybenzyl with high optical purity. The present invention relates to a biochemical process for the production of C8) -(-) -α-cyano-8-phenoxybenzyl alcohol by means of a bump to obtain the ester of the leopard form of alcohol toso.

a-Cyano-8-phenoxybenzyl alcohol is known as a novel alcohol/L/component of a group of ester compounds called synthetic pyrethroids, which have excellent insecticidal activity. Synthetic pyrethroids, such as fenvalerate, cypermethrin, and dern-yumrin, which contain this alcohol as an alcohol/l/component, not only have dramatically enhanced efficacy but also have sunlight resistance.
It is attracting attention not only as an insecticide for household use and epidemic prevention, but also as an agricultural insecticide, and therefore, the popularity of α-cyano-8-phenoxybenzyl alcohol is increasing.

The a-cyano-3-phenoxybenzyl alcohol is
Because it has an asymmetric carbon at the α-position, two optical isomers exist, and the insecticidal activity of the Hs-form alcohol as an ester is superior to that of the corresponding R-form alcohol. (Kosuke Yoshioka, Synthetic Organic Chemistry, Vol. 88, No. 12, pp. 115-1162 (1980)).

Therefore, it can be obtained by ordinary synthetic chemical production methods (
It has been desired to develop a technique for optically resolving -a-cyano-8-phenoxybenzyl alcohol (R, 8) in an industrially advantageous manner to obtain S-unit.

By the way, 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. To (S)
-(-) -a-Cyano-3-phenoxybenzyl alcohol can be obtained by (1) (R18)-a-cyano-8 in the presence of a hydrating reagent.
- Phenoxybenzyl alcohol is reacted with cis-2,2-dimethyl-88-(dihydroxymethyfi7)cyclopropane-IR-carboxylic acid octonone to form an ether compound, and the resulting two isomer mixtures are separated by physical means. , (S) -(-) An ether compound containing an alcohol component is hydrolyzed in an acidic medium to form the desired (
S) Method for obtaining -(-)-α-cyano-3-phenoxybenzyl alcohol (Japanese Patent Application Laid-open No. 109944/1983)
or (II) chiral cyclopropanecarboxylic acid (8)-
(-)-α-cyano-8-phenoxybenzyl alcohol ester is reacted with halogenated boron and then water is reacted to form (S)-(-)-Q-77/-8-phenoxybenzyl alcohol. Method of obtaining (JP-A-56-1285
No. 5).

is only known. However, even with these methods, there are problems in that method (U) requires a large amount of plastic, an expensive optical viscosity reagent, and a low total yield. However, it is not necessarily an optical method because it requires an optically viscous ester of α-cyano-3-phenoxypenzyl alcohol in advance.

The present inventors have conducted research to overcome these problems and find an industrially advantageous method for producing (8)-(-)-α-cyano-8-phenoxybenzyl alcohol. <8) -(
-)-α-Cyano-8-phenoxybenzyl alcohol and the corresponding 8-alcohol ester were found to be able to be efficiently separated, and after further various studies, the present invention was completed.

That is, the present invention relates to Arthrobacter
C, ter) genus, Alcaligenes
es) &, Achromobacterium p-(Achromobac
cter) genus, Pseudomonas (Pseudomonas)
s) Genus, Aspergillus
(R,S)-α-cyano-8-phenoxybendiμ apcop organic carbon & (k%, number 1 to 18 saturated or unsaturated (8)
- (-) Esterases that have the ability to preferentially asymmetrically hydrolyze esters of monolithic alcohols (1) Esterases (in the present invention, esterases are broadly defined as lipases that are active against water-insoluble ester substrates) esterase) to asymmetrically hydrolyze the ester of the racemic alcohol at a pH of 7 or below to produce (S)
-(=)-[alpha]-cyano-8-phenoxybenzyl alcohol toso target o) A method for producing (8) -(-)-[alpha]-cyano-8-phenoxybenzyl alcohol by splitting into esters is provided.

(RlS)-α- used as a raw material in the present invention
Examples of the organic carbonic acid component of the ester of cyano-3-phenoxybenzyl alcohol include formic acid, acetic acid, propionic acid, butyric acid, subacillic acid, isosubilic acid, caproic acid,
caprylic acid, caproic acid, lauric acid, myristic acid,
Examples include valmitic acid, stearic acid, oleic acid, linoleic acid, etc., and esters with organic carbon atoms having 2 to 18 carbon atoms are preferred from the viewpoint of ease of handling and optical purity of the reaction product. Ester of acetic acid is more preferable from the viewpoint of ease and economy.

These esters can be easily produced by conventional methods for producing esters, for example, by reacting (R,8)-α-cyano-8-phenoxybenzyl alcohol with an anhydride of an organic carboxylic acid or a halide of an organic carboxylic acid. I can do it.

Also, instead of (8.R)-α-cyano-8-phenoxybenzyl alcohol, 8-phenoxybenzaldehyde, sodium cyanide, and the previous i-carboxylic acid carboxylic acid! --L Muiwa; It can also be manufactured by Kōshi.

Esterases that can be used in the present invention include esterases in a broad sense, including lipases produced by microorganisms belonging to the genus Arthrobacter, alkaline earth metals, Acrohepta bacterium, Pseudomonas or Mucor, and esterases derived from animal pancreas. Cultivate microorganisms in liquid culture using conventional methods! For example, microorganisms are inoculated into a sterilized liquid medium ('Ion medium for bacteria, malt extract, yeast extract medium for mold and tubes), and the microorganisms are usually
It is also possible to perform reciprocating recirculation culture at 0°C for 2 to 8 days,
Further, solid culture may be performed as necessary.

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.

When carrying out the method of the present invention, the asymmetric hydrolysis of the organic carboxylic acid ester of (R,8)-〇-cyano-8-phenoxypenzyl alcohol is carried out using a culture solution in which the microorganism is cultured, a culture solution, Mixing an esterase-containing substance such as an esterase extract or a concentrate, or an aqueous solution containing crude esterase, purified esterase, or animal pancreatic esterase with the organic carboxylic acid ester of (R,8) monoalcohol, and stirring or shaking the mixture. This is done by As needed.

Non-ester surfactants may also be added.

It is also possible to immobilize the enzyme and use it.

In addition, the appropriate reaction temperature at this time is 10 to 66°C, since the alcohol produced is relatively unstable to heat, and the reaction rate is slow at too low a temperature.
~60°C is preferred.

The reaction time is usually 8 to 48 hours, but the reaction time can be shortened by increasing the reaction temperature or using a highly active enzyme. In addition, since a-cyano-8-phenoxybenzyl alcohol is particularly easily decomposed by bases, it is necessary to control the pH of the water fg solution during the enzyme reaction.
It is heavy. That is, even if asymmetric hydrolysis by esterase proceeds in a basic medium, the alcohol produced will undergo denaturation, so it is important to carry out the enzymatic reaction at a pH of 7 or lower. Furthermore, since the enzyme is inactivated at too low a pH, it is preferable to keep the pH within the range of 14.5 to 6.5. Furthermore, in order to prevent the pH from decreasing due to organic acids such as acetic acid generated by hydrolysis, a buffer solution is used, etc.
It is desirable to control H constant. 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 can be determined per reaction solution.

Next, after performing the asymmetric hydrolysis reaction in this manner, the liberated (8)-(-)-α-cyano-8-phenoxybenzyl alcohol and the unreacted enantiomer ester are separated and recovered. For this separation and recovery, operations such as solvent extraction, fractional distillation, column chromatography, etc. can be employed as appropriate. 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 (8) -(-) -(t -cyano-8-phenoxybenzyl alcohol and its normal compound). Free (8)-(-)-α-cyano-8-phenoxybenzyl alcohol can be obtained by separating the extract from Nisdel, or by separating the extract by column chromatography using silica glu or the like. .

Similarly, the unreacted ester separated and removed in this way is converted into shrimp 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 1-4 Acetic acid ester of (R,5)-a-cyano-8-phenoxybenzyl alcohol/L/1. OF and each esterase 20q listed in Table 1 below were mixed with acetic acid at a concentration of 0.2M.
The mixture was added to a liquid (pus, o) totw and reacted at 30° C. with vigorous stirring using a stirrer. After carrying out the reaction for 24 hours, the reaction product was extracted with toluene. The extract was subjected to high performance liquid chromatography (HPLO) [Lich
rosurbRP-13, Menu-Water (6:4), 2
54 nm nv detection], and the acetate ester of α-cyano-8-phenoxybenzyl alcohol and α-fusi ratio were calculated. It was confirmed from the IPLc analysis results that 8-phenogysibenzaldehyde was produced as a by-product during the reaction under the above conditions.

The extract was concentrated, subjected to silica gel column chromatography, and cyclohexane-ethyfVx-? lv(94:
'6) Unreacted α-cyano-8- eluted with solution 1B
After separating and removing the acetate ester of phenoxybenzyl alcohol, it was further eluted with methanol containing a small amount (10%) of para-toluenesulfonic acid, and the free a-cyano-8-
Phenoxybenzyl alcohol was obtained 3, and its structure was confirmed by RPLC, NMR and IR measurements.

Next, a part of the hindlimb alcohol from which the elution solvent had been distilled off was taken and its specific rotation was measured in chloroform.
μti(-) body, i.e. (8)-(-)-El-
Cyano-8-phenoxybenzyl alcohol8
18 magazines were published.

In addition, 1OIlv of the obtained free ll1a-cyano-8-phenoxybenzyl alcohol was dissolved in toluene l-, and the equivalent amount of (8)-(+)-2-(4-chloroform)V)-isovaleric m Taloride and pyridine were added and reacted to form (8) -(Q -2-(4-chlorophenyl)) of a-cy7no-8-phenoxybenzyl alcohol.
- Diastereomer of isokychi monoacid and analyzed by gas chromatography (IQ%DcQl'-t, 2.5 μm, 250
The optical isomer ratio was analyzed using T ).

The results are shown in Table 1 below.

It was also found that the specific 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 [αII)=-8,5(C=
1.6)).

Examples 5 to 11 In Examples 1 to 4, the amount of each esterase was varied from 20 to 4
σ■, and the buffer solution was 0.2M acetate buffer (
p) 15.0) to 0.1 M concentration of phosphate buffer (
As the reaction progresses, I
The enzymatic reaction and separation of reactants were carried out in the same manner as in Examples 1 to 4, except that the MM concentration NaOH aqueous solution was made into minute water droplets using a drop controller, and then carefully added, and the pH was controlled to a constant level using a p controller. Got it (8)
The specific rotation and hydrolysis rate of -(-)-α-cyano-8-phenoxybenzyl alcohol were calculated.

-The results are shown in Table 2.

Table 2 Examples 11-20 Capric acid ester of (R,8)-α-cyano-phenoxybenzyl alcohol 1.5? 30wIg of each esterase listed in Table 8 below was added to 0.2M acetate buffer (pH 5,4) 10-, and reacted at 80° C. with vigorous stirring for 24 hours. Thereafter, separation and analysis were performed in the same manner as in Examples 1 to 4.

The results are shown in Table 8.

Table 8 Examples 21 to 26 In Examples 1 to 4, the amount of each esterase was varied from 20 to 8.
At 0w9, the buffer was added to 0.2M concentrated acetate buffer (pH
5,0) to 0.1M 731 degree acetate buffer (PH5
, 8), and as the reaction progresses, 1MII
While carefully adding the Na0J1 aqueous solution of WL into minute water droplets using a drop controller, the pH was controlled constant using a pH controller, and the (R18)-a of the substrate was added.
-The usage value of the acetate ester of cyano-8-phenoxybenzyl alcohol is 1. Enzyme reaction, separation, and analysis were performed in exactly the same manner as in Examples 1 to 4, except that the amount of OF was changed to the amount shown in Table 4 below, and the hydrolysis rate, specific optical rotation, and optical isomer ratio by gas chromatography were determined. was measured.

The results are shown in Table 4.

Examples 27-82 Bouillon medium (water 1/
Peptone 5.0? , meat extract 5.0PNap/a,
In Examples 27 to 29, 2? of tributyrin was added, and in Examples 80 to 82, 2P olive oil was added, and Na2HPO4 and KH2PO
4 step I 7.2 and le. ) 200- to sterilize the culture, two platinum loops of each microorganism listed in Table 5 below were inoculated from the slant culture, and cultured with reciprocating shaking at 80° C. for 48 hours. Next, centrifugation is performed, and acetone is added to the aqueous solution layer until the concentration is 85%. Immediately after filtration, the resulting precipitate is washed with water and added with 0.2 M 1 l of acetate buffer (pH 5, 2).
5-, and in Examples 27 to 29 (R,8)
-a-cyano-8-phenoxybenzyl alcohol butyric acid ester 1.6P was added, and in Examples 80 to 82, (R18)-α-cyano-8-phenoxypene 4
) lauric acid ester of alcohol8. Add OF,
Both were reacted for 26 hours at -8°C with vigorous stirring. Thereafter, separation and analysis were performed in the same manner as in Examples 1 to 4.
The hydrolysis rate and specific rotation were determined.

The results are shown in Table 5.

Procedural Amendment (Showa! 74! July 74, 1939) Haruki Shimada, Commissioner of the Japan Patent Office 1 Indication of the case Showa σ, Patent Application No. 1F/, MO No. 2 Name of the Invention (S) - (-) - α- Cyano-3-phenoxy (Biochemical production method of Nriruano V Cool 3 Relationship with the case of the person making the amendment Patent applicant address 5-1 Kitahama, Higashi-ku, Osaka-shi Akiji name (209)
) Representative of Sumitomo Chemical Co., Ltd. Hijikata - Takeshi 4 Agent Address 6, Kitahama 5-1, Akiji, Higashi-ku, Osaka, Detailed description of the invention in the specification to be amended, Column 0, 6, Contents of the amendment (1) Details In the 4th line of page 4 of the book, the phrase ``S unite'' has been corrected to ``eight bodies.''

(2) In the 6th line of page 8, correct r(8,R)j to r(R,8)J.

(3) In the table on page 9, on the fourth line from the bottom, replace "Lipase PL266J" with "Lipase P LA 266J".
I am corrected.

(4) In the table on page 9, in the 8th line from the bottom, [Lipase PL679J] is corrected to [Lipase PLA679J.

Correct the statement to "1-80/w%".

(6) In the table on page 16, the bottom line shows [Riverzem G66
J is corrected as [Lipase PLA2661].

(7) In Table 2 on page 18, in the row of Example No. 6, [Lipase A679J] is corrected to [Lipase PLA679J.

(8) In Table 8 on page 19, in the row of Example number 18, [
Lipase A266J [Lipase PLjll! 2
Corrected to 66J.

(9) In Table 8 on page 19, in the row of Example No. 14, [Lipase A679J] was corrected to [Lipase PLA679J.

αQ In Table 4 on page 21, in the row of Example No. 25, [Lipase A266j] is corrected to [Lipase PL4266J.

(Insert the following after Table 5 on page 28 of 10.

Example 88 Acetate ester of (R,8)-α-cyano-8-phenoxybenzyl alcohol 8. Og and Arthrobacter Toki esterase (lipase combination B8L) 160■ 0.1M
Add to 2 m of concentrated acetate buffer (pH 4,5) and incubate at 40°
The reaction was carried out at C with vigorous stirring using a stirrer. At this time, during the reaction, an NaOH aqueous solution having an IM concentration was carefully added as minute water droplets using a drop controller, and the pH was controlled to be constant using a pH controller. After reacting for 24 hours, the reaction product was extracted with toluene.

Thereafter, the same operations as in Examples 1 to 4 were performed, and the results shown in Table 6 were obtained.

Table 6 ” that's all

Claims (1)

[Claims] Arthrobacter p-(Arthrobacter)
Paulownia, 7. Al caligenes
) genus, Achromobacter (Achromobacter)
r ), Pseudomonas (Psr, -uiiomon
as) M, Aspergillus (A!Ipergi 1l
us) Kaede or Mucor (Mucor) % Bi, (R,8) -1]-carboxylic acid (saturated or unsaturated carbonic acid having 1 to 18 carbon atoms) ester of -cyano-3-phenoxybenzyl alcohol ni”I=JL
Asymmetric hydrolysis An esterase produced by a microorganism that produces hE or an esterase derived from animal pancreas is allowed to act on the esterase/l/[at a pH of 7 or lower to asymmetrically hydrolyze it, <s')-(-)- Patented for the separation of α-cyano-3-phenoxybenzyl alcohol and its phantom ester (8) -(-) -a-cyano-3
-Biogenesis of phenoxybenzyl alcohol? - IJ base system.