JP2001204487A - Method for producing optically active pyrrolidinone derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing an optically active pyrrolidinone derivative conveniently in a good yield. SOLUTION: This method for producing the optically active pyrrolidinone derivative is to react 3-acetyl-1-benzyl-2-pyrrolidinone as a raw material in the presence of a specific microorganism and/or a bacterial cell processed product thereof in such a way as to perform stereoselective reduction.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing an optically active pyrrolidinone derivative from 3-acetyl-1-benzyl-2-pyrrolidinone using microorganism cells and / or a processed product of the cells.

[0002]

BACKGROUND OF THE INVENTION Optically active pyrrolidinone derivatives are useful substances as intermediate compounds for the synthesis of physiologically active or pharmacologically active ingredients (medicines, agricultural chemicals, etc.). Examples of the chemical production method of this optically active pyrrolidinone derivative include a method of producing 1-benzyl-2-pyrrolidinone by reacting acetaldehyde in the presence of n-butyllithium (JP-A-59-5189).
A method for asymmetric hydrogenation of acetyl-1-benzyl-2-pyrrolidinone using a complex formed of bidentate phosphine and ruthenium as a catalyst (Japanese Patent Laid-Open No. 10-204058), 2-[(1R) -1- [Tert-butyldimethylsilyloxyethyl] ethyl acrylate as a starting material
A method of manufacturing through steps (Japanese Patent Application Laid-Open No. 4-95067) has been reported.

[0003] However, these methods are difficult to say that they are practically industrial processes, for example, the number of steps is long, the operation is complicated, and expensive reagents must be used. On the other hand, as a biochemical production method, a method of producing 3-acetyl-1-benzyl-2-pyrrolidinone by allowing baker's yeast to act has been reported (JP-A-4-95067).
However, this method was hardly a practical production method.

[0004]

DISCLOSURE OF THE INVENTION The present inventors have conducted intensive studies to obtain an optically active pyrrolidinone derivative with high economical efficiency and a high yield by a simple method. Using pyrrolidinone as a raw material, it is found that an optically active pyrrolidinone derivative is efficiently produced and accumulated from 3-acetyl-1-benzyl-2-pyrrolidinone by reacting in the presence of a specific microorganism and / or a treated product of the microorganism, The present invention has been completed.

[0005]

DETAILED DESCRIPTION OF THE INVENTION
This is a method in which the carbonyl group of pyrrolidinone is stereoselectively reduced by a specific microbial cell and / or a treated product of the cell, and the optical isomer of the obtained optically active pyrrolidinone derivative is (3R, 1 ′S) Body, (3S, 1 '
There are four types: (S) form, (3R, 1'R) form, and (3S, 1'R) form. For example, when acted on 3-acetyl-1-benzyl-2-pyrrolidinone, (3R, 1'S)
-1-benzyl-3-[(1'-hydroxy) ethyl]
-2-pyrrolidinone, (3S, 1'S) -1-benzyl-3-[(1'-hydroxy) ethyl] -2-pyrrolidinone, (3R, 1'R) -1-benzyl-3-[(1 ′
-Hydroxy) ethyl] -2-pyrrolidinone and / or (3S, 1'R) -1-benzyl-3-[(1'-hydroxy) ethyl] -2-pyrrolidinone can be produced.

In the present invention, a specific microorganism can be used as the microorganism, for example, Candida genus (Candi).
da), the genus Pichia, the genus Kluyveromyces, the genus Issachenchia, the genus Trichosporonoides, the genus Filobasicoum, the bacterium of the genus Filobasicium, Micrococcus bacterium Genus (Corynebacterium), genus Bacillus, genus Rhodotora (Rhodoto)
rula), Yarrowia, Candida, Saccharomyces (S
acc., Machschnikovia, Kleechera (Kl)
oeckera), Hansenia spora (Hansen)
iaspora), Rhodosporium (Rhodos)
microorganisms selected from the group consisting of C. porium) can be used.

Among these microorganisms, they act mainly on 3-acetyl-1-benzyl-2-pyrrolidinone (3R,
1'S) -1-benzyl-3-[(1'-hydroxy)
Microorganisms having the ability to produce [ethyl] -2-pyrrolidinone include Candida, Pichia, and Kluyveromyces (Kluy).
veromyces), Isachenkia (Issat)
chenkia), Trichosporonoides (Tric)
hosporonoides, Filobasidium, Micrococcus (M
(micrococcus).

[0008] Specifically, Candida spp.
da): Candida Boidini (Candida)
bodinini), Candida zelanoides Pichia: Pichia angusta (Pi)
chia angusta, Pichia subpeliculosa Kluyveromyces:
Kluyveromyces thermotolerance (Kluyver
Omyces thermotolerans Isatchenkia: Isatchenkiat
erricola), Isatchenchia orientalis (Trichosporono)
ides): Trichosporonoides oedose
pharis), Trichosporonoides megagiensis (Trichosporonoides mega)
chiliensis Filobasidium: Filobasidium capsiligenum (Filabasid)
ium capsuligenum Micrococcus: Micrococcus morr
huae).

Further, it acts on 3-acetyl-1-benzyl-2-pyrrolidinone to mainly convert (3S, 1'S) -1-benzyl-3-[(1'-hydroxy) ethyl] -2-pyrrolidinone. Examples of microorganisms having the ability to produce include microorganisms belonging to the genus Bacillus. Specifically, Bacillus: Bacillus thurgensis
ingensis).

Further, it acts on 3-acetyl-1-benzyl-2-pyrrolidinone to mainly convert (3R, 1'R) -1-benzyl-3-[(1'-hydroxy) ethyl] -2-pyrrolidinone. Microorganisms having the ability to produce include the genus Rhodotorula and the genus Yarrowia (Y
arrowia, Candid genus (Candid)
a), the genus Trichosporonoides
onoides), Saccharomyces (Sacchar)
omyces), the genus Pichia, the genus Metschnikowia, the genus Filobasidium, the genus Kloeckera, the genus Hancenia spora (H)
anseniaspora, Rhodosporidium (R
For example, microorganisms belonging to H. sp.

Specifically, Rhodotorula (Rhodot)
orula: Rhodotorula auranteiaka (Rho)
dotorula aurantiaca), Rhodotorula rubra,
Rhodotorula
glutinis Genus Yarrowia: Yarrowia lipolytica Candida: Candida parapsilosis
is), Candida tropicalis (Candida)
tropicalis), Candida intermedia (Candida intermedia) Trichosporonoides
ides): Trochosporonoides oedose
phalis), Trichosporonoides megagiensis (Trichosporonoides mega)
chiliensis Saccharomyces: Saccharomyces
s cerevisiae Pichia: Pichia angasta (Pi)
chia angusta), Pichia capsulate (Pichia capsulate), Pichia guillermonde (Pichia guillermond)
ii) Metschnikowia
a): Metoshnikovia Bicasp Data (Mets)
chnikowia bicuspidata Filobasidium: Filobasidium capsiligenum (Filabasid)
ium capsuligenum, Filobasidium uniguturatam (Filobasidium un)
igutulatatum Kloeckera: Kloeckera corticis Hanseniaspora:
Hanseniaspora Ubalam
ra uvarum Rhodosporidiu (Rhodosporidiu)
m): Rhodosporium toluroides (Rhodos)
poridium tolloides).

Also, it acts on 3-acetyl-1-benzyl-2-pyrrolidinone and (3S, 1'R) -1-benzyl-
Examples of microorganisms having the ability to produce 3-[(1′-hydroxy) ethyl] -2-pyrrolidinone include microorganisms belonging to the genus Filobasidium and Corynebacterium. Specifically, the genus Filobasidium: Filobasidium uniguturatum
ttulatum) Corynebacterium (Corynebacterium)
m): Corynebacterium glutamicum (Coryn)
ebacterium glutamicum). As described above, in order to produce the target isomer, an appropriate microorganism can be selected, and if necessary, a mutant of the microorganism, or genetics such as a cell fusion or genetic recombination method. Any strain such as a recombinant strain induced by a genetic technique may be used. The above-mentioned strains are all known strains, and can be easily obtained from the Fermentation Research Institute (IFO), Central Bureau Forschmel Cultures (CBS), and American Type Culture Collection (ATCC), respectively. it can.

In the production method of the present invention, one or more of the above microorganisms are used as cells and / or treated cells. Specifically, the cells obtained by culturing the microorganisms as they are or those obtained by culturing the cells obtained by a known method, that is, those treated with acetone, those subjected to freeze-drying, A treated product of cells, such as one obtained by physically or enzymatically crushing cells, can be used. Further, an enzyme fraction having the ability to act on 3-acetyl-1-benzyl-2-pyrrolidinone and convert it into an optically active pyrrolidinone derivative is used as a crude product or a purified product from these cells or processed cells. It is also possible. Furthermore, the cells obtained in this manner, the treated cells, enzyme fractions and the like are used using a usual immobilization technique, that is, those obtained by immobilizing on a carrier such as polyacrylamide and carrageenan gel are used. It is also possible. Therefore, in this specification, the term "microbial cells and / or processed cells" is used as a concept including all of the above-mentioned microbial cells, processed microbial cells, enzyme fractions, and immobilized products thereof.

Next, the production method of the present invention will be specifically described. In the present invention, an optically active pyrrolidinone derivative is produced by using 3-acetyl-1-benzyl-2-pyrrolidinone as a raw material and allowing the cells of the above-mentioned specific microorganism and / or a treated product thereof to act on the 3-acetyl-1-benzyl-2-pyrrolidinone. . In the production method of the present invention, the microorganism is usually used after being cultured, and this culture can be performed according to a standard method. The medium used for culturing the present microorganism contains a carbon source, a nitrogen source, inorganic ions, and the like which can be utilized by the present microorganism. As a carbon source, glucose, fructose, carbohydrates such as saccharose, glycerol, mannitol, xylitol, polyalcohols such as ribitol,
Organic acids and the like are used as appropriate. NZ as a nitrogen source
Amines, tryptose, yeast extract, polypeptone, meat extract, organic nitrogen sources such as soybean extracts, or ammonium sulfate salts, inorganic nitrogen sources such as ammonium nitrate salts,
Others are appropriately used. As the inorganic ion, a phosphate ion, a magnesium ion, an iron ion, a manganese ion, a molybdenum ion and others are appropriately used as needed. Furthermore, it is effective to add inositol, pantothenic acid, nicotinamide and other vitamins as necessary. Lactones are appropriately used as an enzyme inducer. Culture conditions can be appropriately selected depending on the microorganism to be used, but usually, under aerobic conditions, a pH of about 3 to 11,
While controlling the temperature within a suitable range of about 4 to 50 ° C, 1 to 100
Do time.

The reaction format for producing the optically active pyrrolidinone derivative is as follows. The present microorganism is cultured, and the obtained cells and / or the treated cells are treated with 3-acetyl-1.
-A method of obtaining an optically active pyrrolidinone derivative by adding and reacting benzyl-2-pyrrolidinone, a method of adding 3-acetyl-1-benzyl-2-pyrrolidinone to the medium and simultaneously performing the culture and the reaction, or 3- A method in which acetyl-1-benzyl-2-pyrrolidinone is added and the reaction is further performed can be used as appropriate.

The reaction is carried out at a temperature of 4 to 70 ° C., preferably 20 to 70 ° C.
The reaction is carried out in the range of 50 ° C., and the pH is 2 to 11, preferably 6 to
Perform in the range of 9. The concentration of 3-acetyl-1-benzyl-2-pyrrolidinone is preferably in the range of 0.0001 to 80% by weight, and more preferably 0.01 to 50% by weight. -2-Pyrrolidinone can be supplementarily added.

As a method for collecting the optically active pyrrolidinone derivative obtained by culturing and reaction, the reaction solution obtained by removing solids such as microorganisms by a usual separation device such as centrifugation, filter press, ultrafiltration or the like is treated with an organic solvent. The optically active substance can be obtained by subjecting it to separation and purification means such as extraction, crystallization, column chromatography, concentration and distillation, and the separation and purification means can be used alone or in combination of a plurality of means. Examples of the organic solvent include alcohols such as butanol, hydrocarbons such as hexane, cyclohexane, and toluene, halogenated hydrocarbons such as chloroform and methylene chloride, esters such as ethyl acetate and methyl acetate, ketones, and ethers. And a mixed solvent thereof can be used.

[0018]

EXAMPLES The present invention will be described below in detail with reference to examples, but the present invention is not limited to these examples. The determination of 3-acetyl-1-benzyl-2-pyrrolidinone and the optically active pyrrolidinone derivative or its isomer in the reaction solution was performed by high performance liquid chromatography [column: S
pherisoeb, column temperature: 40 ° C., detection wavelength:
254 nm, mobile phase: acetonitrile / water = 1/1, flow rate 0.4 ml / min). The optical purity of the generated optically active pyrrolidinone derivative was determined by HPLC (column: Chiralpak AD, mobile phase: hexane / isopropyl alcohol / trifluoroacetic acid = 90/10/0.
1. Column temperature: 25 ° C., detection wavelength: 254 nm, flow rate: 1.0 ml / min.

Example 1 [Cell culture medium]-For yeast-1% by weight of glucose-1% by weight of yeast extract-2% by weight of CSL-pH 6.0-For bacteria-1.5% by weight of polypeptone-0.3% of yeast extract % K ₂ HPO ₄ 0.3% by weight NaCl 0.2% by weight pH 7.0

50 ml of the above-mentioned medium for cell preparation is
After sterilization at 121 ° C. for 15 minutes in a Erlenmeyer flask, one platinum loop of each of the microorganisms shown in Table 1 was inoculated.
Shaking culture was performed at a temperature of 1 to 2 days. After completion of the culture, the cells were separated by a centrifuge. Then, a 50 ml Erlenmeyer flask containing 0.1% M containing 2% by weight glucose and 0.5% by weight of 3-acetyl-1-benzyl-2-pyrrolidinone.
10 ml of a potassium phosphate buffer solution (pH 7.0) and the above-prepared cells were put therein, and a rotational shaking reaction was carried out at 30 ° C. for 20 hours.

After completion of the reaction, the reaction mixture was removed by centrifugation, and the supernatant was appropriately diluted.
-Benzyl-2-pyrrolidinone and the reaction product (3R, 1'S) -1-benzyl-3-[(1'-hydroxy) ethyl] -2-pyrrolidinone were quantified. The reaction product in the supernatant was extracted with ethyl acetate, and the obtained ethyl acetate layer was distilled off under reduced pressure, analyzed by high performance liquid chromatography, and the yield and optical purity were measured. Table 1 shows the obtained results.

Example 2 The microorganisms shown in Table 2 were cultured in the medium for preparing bacterial cells shown in Example 1 and reacted in the same manner as in Example 1. As a reaction product, (3S, 1'R)-
1-benzyl-3-[(1′-hydroxy) ethyl]-
2-Pyrrolidinone was obtained. Table 2 shows the results.

Example 3 Using the microorganisms shown in Table 3, cell preparation and reaction were carried out in the same manner as in Example 2. As a reaction product, (3S, 1 ′
S) -1-Benzyl-3-[(1'-hydroxy) ethyl] -2-pyrrolidinone was obtained. Table 3 shows the results.

Example 4 Using the microorganisms shown in Table 4, cell preparation and reaction were carried out in the same manner as in Example 1. As a reaction product, (3R, 1 ′
R) -1-Benzyl-3-[(1'-hydroxy) ethyl] -2-pyrrolidinone was obtained. Table 4 shows the results.

[0025]

[Table 1]

[0026]

[Table 2]

[0027]

[Table 3]

[0028]

[Table 4]

[0029]

[Table 5]

Example 5 50 ml of the yeast cell preparation medium shown in Example 1 was
After sterilization in a 0 ml Erlenmeyer flask at 121 ° C. for 15 minutes, Candida boidini IFO10035
One platinum loop of (Candida boidiniii) was inoculated and shake-cultured at 27 ° C. for 48 hours. After completion of the culture, the cells were separated by a centrifuge. Then, in a 50 ml Erlenmeyer flask, 10 m of a 0.1 M potassium phosphate buffer solution (pH 7.0) containing 2% by weight of glucose and 0.5% by weight of 3-acetyl-1-benzyl-2-pyrrolidinone was added.
1 and the bacterial cells prepared above were added, and a rotational shaking reaction was performed at 30 ° C. for 20 hours. The reaction product in the supernatant was extracted with ethyl acetate, the obtained ethyl acetate layer was distilled off under reduced pressure, and the residue was subjected to chromatography using silica gel, followed by n-hexane and ethyl acetate (3: 2). After elution, the mixture was concentrated under reduced pressure to give (3R, 1'S) -1-benzyl-3-.
[(1′-hydroxy) ethyl] -2-pyrrolidinone 3
0 mg was obtained. The optical purity was 99.5% ee.

Example 6 50 ml of the medium for preparing bacterial cells shown in Example 1
Was placed in a 500 ml Erlenmeyer flask, sterilized at 121 ° C. for 15 minutes, and then Corynebacterium glutamicum AT
CC13032 (Corynebacterium g
lutamicum) and inoculated with a platinum loop and incubated at 30 ° C for 24 hours.
Shaking culture was performed for a time. After completion of the culture, the cells were separated by a centrifuge. Then, in a 50 ml Erlenmeyer flask, 10 ml of a 0.1 M potassium phosphate buffer solution (pH 7.0) containing 2% by weight of glucose and 0.5% by weight of 3-acetyl-1-benzyl-2-pyrrolidinone and the cells prepared above Was added, and a rotary shaking reaction was performed at 30 ° C. for 20 hours. The reaction product in the supernatant was extracted with ethyl acetate, the obtained ethyl acetate layer was distilled off under reduced pressure, and the residue was subjected to chromatography using silica gel, followed by n-hexane and ethyl acetate (3: 2). After elution, the mixture was concentrated under reduced pressure.
5 mg of S, 1'R) -1-benzyl-3-[(1'-hydroxy) ethyl] -2-pyrrolidinone were obtained. The optical purity was 72.7% ee.

Example 7 50 ml of the medium for preparing bacterial cells shown in Example 1
Was placed in a 500 ml Erlenmeyer flask and sterilized at 121 ° C. for 15 minutes, followed by Bacillus thuringiensis IFO39.
51 (Bacillus thuringiensi
s) was inoculated with one platinum loop and shake-cultured at 30 ° C. for 24 hours. After completion of the culture, the cells were separated by a centrifuge. Then, in a 50 ml Erlenmeyer flask, 10 ml of a 0.1 M potassium phosphate buffer solution (pH 7.0) containing 2% by weight of glucose and 0.5% by weight of 3-acetyl-1-benzyl-2-pyrrolidinone and the cells prepared above Was added, and a rotary shaking reaction was performed at 30 ° C. for 20 hours. The reaction product in the supernatant was extracted with ethyl acetate, the obtained ethyl acetate layer was distilled off under reduced pressure, and the residue was subjected to chromatography using silica gel, followed by n-hexane and ethyl acetate (3: 2). After elution, concentrate under reduced pressure to obtain (3S, 1'S)-
1-benzyl-3-[(1′-hydroxy) ethyl]-
20 mg of 2-pyrrolidinone were obtained. Optical purity is 85.1
% Ee.

Example 8 50 ml of the medium for preparing yeast cells shown in Example 1 was
After sterilizing in a 0 ml Erlenmeyer flask at 121 ° C. for 15 minutes, Rhodotorula auranteiaca IFO0754
One platinum loop of (Rhodotorura aurantiaca) was inoculated and shake-cultured at 27 ° C. for 48 hours. After completion of the culture, the cells were separated by a centrifuge. Then, 2 wt% glucose, 0.5 wt.
0.1M potassium phosphate buffer solution containing 3% by weight of 3-acetyl-1-benzyl-2-pyrrolidinone (pH 7.
0) Add 10 ml of the cells prepared above and add
A rotation shaking reaction was performed for 0 hours. The reaction product in the supernatant was extracted with ethyl acetate, the obtained ethyl acetate layer was distilled off under reduced pressure, and the residue was subjected to chromatography using silica gel, followed by n-hexane and ethyl acetate (3: 2). After elution, the mixture was concentrated under reduced pressure to obtain 40 mg of (3R, 1′R) -1-benzyl-3-[(1′-hydroxy) ethyl] -2-pyrrolidinone. The optical purity was 99.3% ee.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:84) C12R 1:84) (C12P 17/10 (C12P 17/10 C12R 1: 865) C12R 1 : 865) (C12P 17/10 (C12P 17/10 C12R 1:78) C12R 1:78) (C12P 17/10 (C12P 17/10 C12R 1: 645) C12R 1: 645) (C12P 17/10 (C12P 17/10 C12R 1: 265) C12R 1: 265) (C12P 17/10 (C12P 17/10 C12R 1:15) C12R 1:15) (C12P 17/10 (C12P 17/10 C12R 1:07) C12R 1 : 07) F term (reference) 4B050 CC01 DD02 DD03 DD04 DD05 LL01 LL05 4B064 AE49 CA01 CA02 CA05 CA06 CA21 CB17 CB18 CC01 CD08 CD12 CD27 CE08 CE10 CE20 DA01

Claims (1)

[Claims] 1. 3-acetyl-1 represented by the formula (1)
In producing an optically active pyrrolidinone derivative represented by the formula (2) from -benzyl-2-pyrrolidinone, Candida, Pichia (Pic)
ia), Kluyveromyces (Kluyveromy)
ces), the genus Isachenkia
a) Trichosporonides (Trichospor)
onoides), Filobasidium (Filoba)
sidium), Micrococ
cus), Corynebacterium (Corynebac)
terium), the genus Bacillus, the genus Rhodotorula, the genus Yarrowia, the genus Candid (Candid)
a), Saccharomyces
s), the genus Metschnikow
ia), a microorganism selected from the group consisting of the genus Kloeckera, the genus Hanseniaspora, and the genus Rhodosporium, and / or reacted in an aqueous medium in the presence of a microorganism and / or a treated product thereof. A method for producing an optically active pyrrolidinone derivative, comprising producing and accumulating an optically active pyrrolidinone derivative in an aqueous medium, and then collecting the optically active pyrrolidinone derivative from the aqueous medium.