JPS62296888A - Production of epoxide with optical activity - Google Patents

Abstract

PURPOSE:A microorganism selected from Micrococcus, capable of asymmetrically hydrolyzing epoxides, is allowed to act on an epoxide bearing aromatic rings to give an optically active epoxide, which is used as a medicine or agricultural chemical. CONSTITUTION:A microorganism capable of asymmetrically hydrolyzing epoxides, selected from the group consisting of Micrococcus, Arthrobacter, Micobacterium, Corynebacterium, Rhodococcus, Nocardia and Brevibacterium is allowed to act on a starting epoxide bearing aromatic rings such as styrene oxide, o-, m-, or p-chlorostyrene oxide. The cell bodies which are previously proliferated by culture may be brought into contact with the starting substance, or the microorganism may be cultured in another medium together with the starting substance. The optically active epoxide formed in the culture mixture is separated by a well-known method.

Description

Detailed Description of the Invention 3. Detailed Description of the Invention 1 The present invention relates to a method for producing an optically active epoxide from a racemic epoxide having an aromatic ring using microorganisms.

Epoxides having optical activity are widely used as raw materials or intermediates for producing various useful substances including medicines, agricultural chemicals, and ferroelectric crystals.

Traditionally, hydrolysis of epoxide has been carried out in humans, rats,
Epoxide hydrolase (E C3+) derived from pig liver microsomes
3.2°3), and it has also been reported that this epoxide hydrolase has broad substrate specificity and performs stereoselective hydrolysis (J.

[Journal of Biological Chemistry J (J, Biol, Che.
The above-mentioned epoxide hydrolase is not easy to obtain;
It is not practical to hydrolyze epoxides industrially.

On the other hand, trans-2,3-epoxysuccinate hydrolase (trans-2,3-epoxysuccinate hydrolase) is an epoxide hydrolase derived from microorganisms.
-Epoxy 5uccinaLe hydratas
e, E C4+2.1+37), and the hydrolase is derived from Pseudomonas spp.
It is known that it is produced by microorganisms belonging to the genus Aspergillus and Flavobaeterium.

Regarding the hydrolysis of cis-2,3-epoxysuccinic acid, Achromobacter tartarogenes (Achr.
omobacter tartaro enes) produced L-tartaric acid from the above epoxysuccinic acid, and Alcaltgenes repotartaricus (Alcaltgenes 1e
It has also been reported that D. votartaricus) produces D-tartaric acid.

However, these conventionally known epoxide hydrolases derived from microorganisms are enzymes that act only on epoxysuccinic acid, and cannot be used to hydrolyze other epoxides.

The present invention has been made in view of the above-mentioned problems regarding the hydrolysis of epoxides. An object of the present invention is to provide a method for producing an epoxide.

Another object of the present invention is to provide a method for producing an epoxide with high optical purity from an epoxide having an aromatic ring with low optical purity.

In the present invention, microorganisms that have the ability to asymmetrically hydrolyze epoxides having an aromatic ring have been discovered, and the above problems have been solved by allowing these microorganisms to act on the epoxides.

The present invention will be explained in detail below.

The structural feature of the present invention is that it has three dishes and a bottom.
ococcus), Arthrobacter (Arthro
genus Mycobacterium, Mycobacterium spp.
cterius+), Corynebacterium (Cory
genus nebacterim, Rhodococcus
coccus) genus, Nocardia
Genus and Brevibacterium
m) A microorganism having the ability to asymmetrically hydrolyze an epoxide selected from the group consisting of the genus 1 is allowed to act on an epoxide having an aromatic ring, and the optically active epoxide produced is separated and collected.

The microorganisms used in the present invention for decomposing L- have the ability to asymmetrically hydrolyze epoxides belonging to each genus of doffing, and examples of these microorganisms include the strains shown in Table 1. These strains are from the American Type Culture Collection.
es^TCC) under the following number and can be easily obtained.

In the present invention, epoxides having aromatic rings used as reaction substrates for producing optically active epoxides using each microorganism of Tamade include styrene oxide, o-, m-, or chlorostyrene oxide, o-, m-, or p-methylstyrene oxide,
Phenylglycidyl ether, 0-, TI-, or p-methylphenylglycidyl ether, 0-allylphenylglycidyl ether, O-allyloxyphenylglycidyl ether, benzylglycidyl ether, α-
Examples include naphthyl glycidyl ether.

In the present invention, these epoxides as starting materials are
They are used as reaction substrates either singly or as a mixture of two or more, or as a mixture with hydrocarbons other than epoxides such as saturated hydrocarbons and aromatic hydrocarbons. Furthermore, racemic or epoxides with low optical purity can also be used as starting materials and reaction substrates.

In the present invention, in order to cause the microorganism to act on the raw material epoxide, for example, (a method in which bacterial cells obtained by culturing and propagating all microorganisms in advance are brought into contact with raw epoxide, co) the above microorganism is reacted with the raw material epoxide. A method of culturing under aerobic conditions in a nutrient medium containing other carbon sources, nitrogen sources, inorganic salts, and, if necessary, growth-promoting substances can be applied.

The method of contacting and reacting the raw material epoxide with the growing microbial cells of (al) described above starts with carbohydrates such as glucose, sucrose, molasses, starch hydrolysates, cellulose hydrolysates, hydrocarbons such as propane, butane, etc. as carbon sources, Dodecane, tetradecane, and other substances with high bacterial growth effects such as acetic acid are used, and nitrogen such as ammonium chloride, ammonium sulfate, ammonium phosphate, ammonium nitrate, urea, aqueous ammonia, amino acids, and other assimilable organic nitrogen compounds are used. sources, inorganic salts such as potassium phosphate, sodium phosphate, magnesium sulfate, manganese sulfate, ferrous sulfate, ferric chloride, calcium chloride, manganese chloride, and optionally vitamins, yeast extract, and cornstarch. Inoculum of each of the above microorganisms is inoculated into a medium supplemented with a growth promoting substance such as Epliquor, and cultured under aerobic conditions to multiply the bacterial cells. Alternatively, a raw material epoxide is supplied to a suspension of bacterial cells isolated from the culture, or to an immobilized bacterial cell, and the raw material epoxide is reacted.

The reaction is carried out at pH 5-9, preferably 4;! 6-8(7) p.
The reaction is carried out at a temperature of 20 to 50°C, preferably 25 to 45°C, for several hours to several days in the HjJ range. During the reaction, the carbon source, nitrogen source, and other components used for bacterial growth are added as appropriate. By doing so, the activity of the reaction between the bacterial cells and the raw material epoxide can be maintained or increased.

The reaction can be carried out either batchwise or continuously.

In the case of a batch reaction method, the raw material epoxide can be supplied in its entirety at the start of the reaction, or it can also be supplied continuously or intermittently during the reaction.

The optically active epoxide produced in the reaction solution by the above reaction is separated and collected using known techniques such as phase separation, extraction, and distillation.

Next, in the above-mentioned culture method, a raw material epoxide is added during bacterial growth in the above-mentioned ta+ method, and an optically active epoxide is produced in one step. The culture conditions (PL+, temperature, pressure, etc.), culture method, and separation and collection of the optically active epoxide produced are as described above.
Reaction conditions, reaction methods, and separation and collection methods can be similarly used.

The optically active epoxide obtained by the present invention can be used in various conventionally known uses as mentioned above.

Example 1 The present invention will be explained in more detail with reference to Examples below.
BG medium (10g of Labrenco powder manufactured by Oxoid,
Bacteriological peptone 10g 1 glucose 10g
And add tap water to 5g of sodium chloride to make 11, I
N-Caustic soda water? Liquid culture medium adjusted to po 7.5 with '8 solution and then sterilized by heating at 120°C for 15 minutes in an autoclave) 10C1j! It was inoculated into a 500 ml volume Saka 1'' flask containing 100 ml of the following, and cultured with shaking at 30° C. for 48 hours.

10mj of the culture solution obtained above! , r+-hexadecane 10111 and 20 μl of racemic epoxide
The reaction mixture was placed in a 00 ml volume Sakaro flask and reacted by shaking at 30°C for 24 hours.Then, the reaction solution was centrifuged, the upper oil layer was taken, and the remaining epoxide was quantified by gas chromatography. Silicon 5R-30 ch
A gas chromatography system equipped with a 2raO column packed with 3% romosorb HAM 0MC3 and an ionization flame detector was used.

Next, remove the oil layer by stirring the epoxide to a concentration of 5 to 0.5 ffig, transfer it to a Pyrex 206+ A-capacity ampoule, add 4 ml of isopropanol and 2 ml of isopropylamine, and seal the tube at 80°C for 4 hours. After the heated 0 reaction was completed, the package was opened and the solvent was removed.
I to benzene? 8 solved lN-1 (C120ni I
After extraction twice with I, 20tsl of 6N-NaOH was added to the aqueous layer, and 2kj! of benzene was added. Extracted with. After drying benzene with Naz304, drying and transferring the residue to a 1wr 1 volume vial, 100 μl of bis(trimethylsilyl) 1
-Lifluoro-acetamide (bis(trimeLh)
ylsilyl) Lr1f Iuo-ro-acet
amide] and heated at 60°C for 15 minutes. After cooling, N
-heptafluorobutyryl-L-prolyl chloride (
N-heptaf Iuorobutyryl-L-p
Add 100 µl of a 1M methylene chloride solution of
The analysis was performed using a 60n+ glass capillary column designated as V225.

By using the above method, the optical purity of the remaining epoxide can be determined from the area ratio on gas chromatography, and the absolute configuration of the remaining epoxide can be determined from comparison with a sample of optically active epoxide treated in the same way. Can be done.

The amount of residual epoxide, the absolute configuration of the residual epoxide, and the optical purity of the residual epoxide determined as above are shown in Table 2 using styrene oxide as the substrate, and Table 3 shows the results using phenyl glycidyl ether as the substrate. Table 4 shows the results using benzyl glycidyl ether and benzyl glycidyl ether as substrates.

In addition, in Table 2, the absolute configuration is shown assuming that the absolute configuration of phenyl glycidyl ether having an optical rotation of (+) is (S). Moreover, in Tables 2 to 4, reaction medium Il! is used instead of the culture solution. ! (KtHPO-11,74g/
1; Mg5O171h011.5g/l;
The results obtained using FeFe5057HzO15080 are shown as a reference example.

Example 2 Corynebacterium fudiokens was grown in a manner similar to that described in Example 1.
(fu■le standard product) ATCC21496 culture solution Loo
ml was prepared, 200 μl of racemic styrene oxide was added thereto, and the mixture was reacted by shaking at 30° C. for 24 hours. Next, the reaction solution was extracted three times with 200 na l of ether, the ether solution was dried with NaSO4, and the ether was distilled off, leaving a residue of 30 m j! of water was added. After washing the aqueous layer once with 50 ml of hexane, the aqueous layer was extracted three times with 60 ml of ether. After drying the ether solution, the ether was distilled off to obtain 100 mg of white crystals. IR of this crystal
The spectrum and mass spectrum were consistent with standard styrene glycol.

Example 3 Corynebacterium fudiokens prepared by the method described in Example 1
fu几蛯凱憇) ATCC21496 culture solution 10n
+ 12, the solvent IQm17 listed in Table 5 below, and
Add 20μl of phenylglycidyl ether to each
The reaction was carried out by shaking in a 500 ml shaking flask at 30° C. for 24 hours.

The amount of residual epoxide and the optical purity of the residual epoxide determined by the method described in Example 1 are shown in Table 5.

Table 5 shows the results of using the reaction medium described in Example 1 instead of the bacterial solution, as well as examples of reactions conducted without adding a solvent and reactions conducted with the air in the flask replaced with nitrogen gas. Reaction examples are also described. In a reaction example performed without adding a solvent, after the reaction was completed, the remaining epoxide was
Extracted with ether.

Example 4 14-Hexadecane Low I was added to the culture film of Corynebacterium fudiokens (Normal)8TCC21496 prepared by the method described in Example 1 as shown in Table 6 below. Add 20 μl of each racemic epoxide as Jil,
The reaction was carried out at 30° C. for 24 hours with shaking in a Sakaro flask. Table 6 shows the amount of residual epoxide, the absolute configuration of the residual epoxide, and the optical purity determined by the method described in Example 1 and Katsura's method.

Table 6 also shows the results using the reaction medium described in Example 1 instead of the culture solution. Further, among the absolute configurations, the absolute configurations of 0- and p-methylphenylglycidyl ether are described assuming that those having an optical rotation of (+) are (s).

Claims (1)

[Claims] (1) Micrococcus genus,
Genus Arthrobacter, Genus Mycobacterium,
Corynebacterium
A microorganism having the ability to asymmetrically hydrolyze an epoxide selected from the group consisting of the genus Rhodococcus, the genus Nocardia, and the genus Brevibacterium is made to act on an epoxide having an aromatic ring to produce the epoxide. 1. A method for producing an optically active epoxide, which comprises separating and collecting the optically active epoxide.