JP2003096029A - Optically active alcohol and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new optically active alcohol and optically active m-nitro- substituted-1-phenetyl alcohol derivative, which are important at raw materials of pharmaceuticals or agrochemicals and also to provide methods of producing the same. SOLUTION: The method of producing an optically active m-nitro- substituted-1-phenetyl alcohol derivative comprises contacting a m-nitro- substituted-2-halogen-substituted-acetophenone with microbacteria, culture supernatants or treated material thereof which can transform a m-nitro- substituted-acetophenone into a m-nitro-substituted-1-phenetyl alcohol derivative, and collecting the optically active m-nitro-substituted-1-phenetyl alcohol.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel optically active alcohol, an optically active m-nitro group-substituted-1-phenethyl alcohol derivative which is important as a raw material for medicines and agricultural chemicals, and a method for producing them.

[0002]

2. Description of the Related Art In the fields of medicine, agricultural chemicals, etc., it has been recognized that there are remarkable differences in physiological activity among optical isomers,
In recent years, the importance of optically active substances has been increasing. Under such circumstances, development of various optically active intermediates is desired. So far, racemic 2-halogen substitution-1
A production example of a-(m-nitrophenyl) alcohol derivative has been reported (CA53; 276a). However, there is no known synthetic example of the optically active substance. Further, Japanese Patent Application Laid-Open No. 9-289897 discloses a possibility of synthesizing an optically active p-position nitro group-substituted-1-phenethyl alcohol derivative, but the m-position nitro group-substituted product can be prepared by the method of the same publication. It was completely unknown whether it could be manufactured.

On the other hand, a method for obtaining an optically active alcohol from a ketone body by utilizing a microorganism, that is, a method for synthesizing an optically active intermediate using an enzyme asymmetric reduction technique has been previously studied. For example, microorganisms represented by baker's yeast are most often used in the above reaction. In recent years,
A method for synthesizing an optically active alcohol from various ketone bodies has been developed (for example, Journal of Organic Synthesis Vol. 59, 6).
59-669 (2001)). However, it cannot be said that the disclosed compounds are only limited compounds. Among them, Te
trahedron, Vol.54,13059-13072 (1998) has optical activity p
A synthetic method by enzymatic asymmetric reduction of a nitro group-substituted 1-phenethyl alcohol derivative has been reported. But m
Nothing is described about the substituted nitro group. That is, because of the extremely high substrate selectivity due to the nature of the enzymatic reaction, at present it was not known at all whether it could be applied to the m-position substitution product.

[0004]

Therefore, the present invention provides an important and novel optically active alcohol, an optically active m-nitro group-substituted-1-phenethyl alcohol derivative as a raw material for medicines and agricultural chemicals, and a method for producing them. The purpose is to do.

[0005]

Means for Solving the Problems As a result of earnest studies to develop a novel optically active alcohol, the present inventors have found that
The inventors have newly found that a novel optically active alcohol can be produced by enzymatic asymmetric reduction, and completed the present invention.

That is, according to the present invention, the general formula 1 (where X is
Represents a halogen atom), an (R) -2-halogen-substituted-1- (m-nitrophenyl) alcohol derivative,

[Chemical 5] And an optically active alcohol represented by the general formula 2 (wherein X represents a halogen atom), a (S) -2-halogen-substituted-1- (m-nitrophenyl) alcohol derivative,

[Chemical 6] Furthermore, the general formula 3

[Chemical 7] (In the formula, X represents a halogen atom), an m-nitro group-substituted acetophenone derivative represented by the general formula 4

[Chemical 8] (In the formula, X represents a halogen atom and * represents optical activity.) Microorganism cells or culture supernatants or those having the ability to be converted into an optically active m-nitro group-substituted-1-phenethyl alcohol derivative The optically active m-nitro for collecting the optically active m-nitro group-substituted-1-phenethyl alcohol derivative of the general formula 4 by contacting the treated product of the above with the m-nitro group-substituted 2-halogen-substituted acetophenone represented by the above general formula 3. It is a method for producing a group-substituted 1-phenethyl alcohol derivative.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.
(R) -2-halogen-substituted-1-in formula 1
(M-Nitrophenyl) alcohol derivative, (S) -2-halogen-substituted-1- (m-nitrophenyl) alcohol derivative represented by the general formula 2, and m-represented by the general formula 3.
In the nitro group-substituted acetophenone and the optically active m-nitro group-substituted-1-phenethyl alcohol derivative represented by the general formula 4, X represents a halogen atom, and specific examples thereof include a chlorine atom and a bromine atom. Further, in the optically active m-nitro group-substituted-1-phenethyl alcohol derivative represented by the general formula 4, * indicates that it is an optically active substance.

The m-nitro group-substituted acetophenone derivative represented by the general formula 3 as a raw material can be synthesized by halogenating m-nitroacetophenone by a conventional method (for example, J. Pharm. Sci., Vol. 56, 28. -32,1967,). A microorganism having the ability to asymmetrically reduce the m-nitro group-substituted acetophenone represented by the general formula 3 used in the present invention to the optically active 2-halogen-substituted-1- (m-nitrophenyl) alcohol derivative represented by the general formula 4. Is an optically active 2-halogen-substituted-1 represented by the general formula 4, which catalyzes this reaction.
The type and origin of the microorganism may be any, as long as it is a microbial cell having the ability to produce a-(m-nitrophenyl) alcohol derivative, a culture supernatant, or a treated product of these cells.

Generally, the microorganism which catalyzes this reaction can be found by the following method. Suitable medium such as glycerol 30 g / L, yeast extract 10 g / L, peptone 5 g / L, KH ₂ PO ₄ 11 g / L and K ₂ HPO _4.
After sterilizing a medium having a composition of 3 g / L or a potato dexulose medium (manufactured by Difco), planting a microorganism at 20 ° C
Incubate with shaking at 40 ° C for 2 days to 1 week. Thereafter, the cells are collected by a means such as centrifugation, suspended in a phosphate buffer containing 0.1-1.0% of m-nitro group-substituted acetophenone represented by the general formula 3, and then suspended for 2-3 times. React with shaking at 30 ° C for a day. At that time, the progress of the reaction can be promoted by using a reaction solution containing reduced nicotinamide adenine dinucleotide (NADH) and / or reduced nicotinamide adenine dinucleotide phosphate (NADPH) and / or glucose. .

Further, the reaction may also be promoted by using the collected bacterial cells dried by acetone treatment or vacuum drying. The reaction solution after completion of the reaction is extracted with an organic solvent such as ethyl acetate, and the produced optically active m-nitro group-substituted-1-phenethyl alcohol derivative of the general formula 4 is subjected to high performance liquid chromatography (column: Daicel Chemical Industries Ltd.). Company Chiralcel OD) or gas chromatography (column: Chrompack CP-Chirasi)
DEX CB 0.25mmID X 25M) to determine its optical purity and yield. By this method, it is possible to find microorganisms that catalyze this reaction.

<High-performance liquid chromatographic analysis conditions> Column: Chiralcel OD mobile bed manufactured by Daicel Chemical Industries, Ltd .; Isopropanol / n-hexane = 10: 90 Flow rate; 1.0 ml / min. Detection: 245 nm Elution order: R form and S form are eluted in this order.

<Gas chromatography analysis conditions> Column: CP-Chirasil DEX CB 0.25m manufactured by Chrompack
mID X 25M injection temperature; 230 ° C detection temperature; 230 ° C carrier gas; helium column temperature; 180 ° C detection; FID elution order; S form and R form are eluted in this order.

[0013] Such microorganisms are not particularly limited, but as a representative one, Absidia
Genus, Aspergillus genus, Aureobasidium genus, Blakesl
ea), Botryotinia, Caldariomyces, Cephaloascus, Chaetomium
Genus, Cunninghamella genus, Dipodascus genus, Endomyces (Endom
yces), galactomyces (Galactomyces), geotrichum (Geotrichum), Gibberella (Gibberella)
Genus, Helicostylum, Leucosporidium, Mortillera
rtierella genus, Mucor genus, Neurospora genus, Penicillium genus,
Phycomyces, Pythiosis
psis), Rhizopus, Scytalidium, and Synephalas
genus trum, Thermomyces genus, Trichoderma genus, Trichothesea
cium) genus, Vertillium (Verticillium) genus and digoringus (Zygorhynchus) genus and the like.

The microorganisms belonging to the genus Absidia include, for example, Absidia atrospora IFO 9471, Ab.
sidia glauca IFO 4002, Absidia glauca IFO 4003 and Absidia spinosa IFO 5873 etc.
Examples of microorganisms belonging to the genus pergillus include Asper
gillus flavus IFO 5324, Aspergillus niger IAM 3008
And Aspergillus sojae IAM 2703 and the like, as a microorganism belonging to the genus Aureobasidium (Aureobasidium), for example, Aureobasidium pullulans IFO 4464 and the like,
Examples of the microorganisms belonging to the genus Brakeserea (Blakeslea) include Blakesleatrispora IFO 5989 and the like, and examples of the microorganisms belonging to the genus Botryotinia include Botryotinia fuckeliana IAM 5126 and the like.
Examples of the microorganism belonging to the genus Caldariomyces include Caldariomyces fumago ATCC 1192.
Examples of microorganisms belonging to the genus Cephaloascus include Cephaloascus albidus IFO 3
Examples of the microorganisms belonging to the genus Chaetomium, such as 0596, include Chaetomium semispirale IFO 8
Examples of the microorganisms belonging to the genus Cunninghamella, such as 363, include Cunninghamella echin.
ulata var.elegans IFO 4446 and Cunninghamella ech
inulate IFO 6334 etc. are Dipodascus
Examples of microorganisms belonging to the genus include Dipodascus aggre
gatus IFO 10816, Dipodascus ambrosiae IFO 10801, D
ipodascus australiensis IFO 10805, Dipodascus capi
tatus IFO 10819, Dipodascus capitatus IFO 10820, D
ipodascus capitatus IFO 1197, Dipodascus geniculat
us IFO 10806, Dipodascus macrosporus IFO 10807, Di
podascus magnusii IFO 10808, Dipodascus magnusii IF
O 110, Dipodascus magnusii IFO4600, Dipodascus mag
nusii JCM 6360, Dipodascus magnusii JCM 6868, Dipo
dascus ovetensis IFO 1201, Dipodascus ovetensis JC
M 6358, Dipodascus spicifer IFO 10809 and Dipodas
cus tetrasperma IFO 10810 etc.
Examples of microorganisms belonging to the genus omyces) include Endomyce
s decipiens IFO 0102 etc.
Examples of the microorganism belonging to the genus omyces) include Galactom.
yces citri-aurantii IFO 10821, Galactomyces geotri
chum JCM 1945, Galactomyces geotrichum JCM 6359, G
alactomyces reessii IFO 10823 and Galactomyces re
Examples of microorganisms belonging to the genus Geotrichum, such as essii JCM 1942, include Geotrichum armillari.
ae JCM 2454, Geotrichumcandidum IFO 4597, Geotrich
um capitatum JCM 3908, Geotrichum eriense JCM391
2, Geotrichum fermentans JCM 2467, Geotrichum ferm
entans JCM 2468, Geotrichum fragrans JCM 1749, Geo
trichum klebahnii JCM 3913 and Geotrichum rectang
Examples of microorganisms belonging to the genus Gibberella, such as ulatum JCM 1750, include Gibberella fujikuroi I
FO 5268 and the like are, for example, Helicostylum nigri as microorganisms belonging to the genus Helicostylum.
The cans IFO 8091 etc.
Examples of microorganisms belonging to the genus (idium) include Leucospor
idium scotti IFO 9474 etc.
Examples of microorganisms belonging to the genus la) include Mortierella
humicola IFO 8188 and Mortierella isabellina IFO
7824 and the like are examples of microorganisms belonging to the genus Mucor, for example, Mucor javanicus IFO 4572, and examples of microorganisms belonging to the genus Neurospora, such as Neurospora crassa IFO 6067 and Neurospora sit.
Examples of microorganisms belonging to the genus Penicillium, such as ophila IFO 6069, include Penicillium caresc
ens IFO 7108 and Penicillium citrium IAM 7008 and the like, as microorganisms belonging to the genus Phycomyces, for example, Phycomyces nitens IFO 9422 and the like, as microorganisms belonging to the genus Pythiopsis, for example, Pythiopsis cymosa ATCC 26880 and the like. , Microorganisms belonging to the genus Rhizopus include, for example, Rhiz
opus microsporus IFO 4767 and Rhizopus microsporu
Examples of the microorganisms belonging to the genus Scytalidium, such as s IFO 4768, include Scytalidium flavob
runneum JCM 6268, Scytalidium infestans IFO 32370
And Scytalidium terminale IFO 6396 and the like, as a microorganism belonging to the genus Synephalastrum (Syncephalastrum), for example, Syncephalastrum racemosum IFO 4816 and the like, as a microorganism belonging to the genus Thermomyces (Thermomyces), for example, Thermomyces lanuginosus IFO 9738
Examples of the microorganisms belonging to the genus Trichoderma include Trichoderma longibrachiatum IFO.
4847 and Trichoderma viride IFO 5720 and the like are, for example, Trichothecium roseum IFO 4839 and the like as microorganisms belonging to the genus Trichothecium (Trichothecium), and microorganisms that belong to the genus Verticillium include, for example, Verticillium fungicola IFO 30616 and the like. , As a microorganism belonging to the genus Digoringus (Zygorhynchus), for example, Zygorhynchus exponens var.smithiiI
FO 6665 and the like are exemplified.

Incidentally, IFO, JCM, IAM and AT
The strains numbered by CC can be obtained from the Fermentation Research Institute, RIKEN microbial strain preservation facility, Institute of Molecular and Cellular Biology, University of Tokyo, and American Type Culture Collection. It is also possible to use genetically engineered microorganisms in which enzyme genes isolated from these microorganisms are introduced into various host vector systems.

In the present invention, as a medium for culturing these microorganisms, any medium can be used so long as these microorganisms can normally grow. As the carbon source, for example, glucose, sugars such as sucrose and maltose, organic acids such as acetic acid, citric acid and fumaric acid or salts thereof, alcohols such as ethanol and glycerol can be used. As the nitrogen source, for example, in addition to general natural nitrogen sources such as peptone, meat extract, yeast extract and amino acid, various inorganic and organic acid ammonium salts can be used. In addition, inorganic salts, trace metal salts, vitamins and the like are added as needed. In order to obtain high enzyme activity, it may be effective to add a compound having a ketone group or a carbonyl group such as acetophenone to the medium as an enzyme production inducer. The culture may be performed according to a conventional method, for example, aerobically culturing for 6 hours to 10 days at a pH of 4 to 10 and a temperature of 15 to 40 ° C. In addition, it may be possible to obtain a high enzyme activity by similarly culturing in static culture.

The microorganism thus obtained is a culture obtained by culturing in a medium as it is, or a microbial cell or a culture supernatant obtained by a harvesting operation such as centrifugation from the culture, and In the form of a treated product thereof is contacted with an m-nitro group-substituted 2-halogen-substituted acetophenone represented by the general formula 3 to collect an optically active m-nitro group-substituted-1-phenethyl alcohol derivative of the general formula 4. Thus, an optically active m-nitro group-substituted-1-phenethyl alcohol derivative can be produced.

The treated product is not particularly limited in its form of use as long as it exhibits an activity to catalyze the conversion reaction, and it is a dried bacterial cell, a bacterial cell treated with acetone or the like, a crushed bacterial cell, or a crushed bacterial cell. Cell-free extract, crude enzyme, purified enzyme and the like.

When the microbial cells, the cell culture solution, or a treated product thereof is subjected to the reaction, these can be immobilized on a suitable carrier before use. For example, it can be entrapped with a crosslinked acrylamide gel, a polysaccharide, or the like, or physically or chemically immobilized on a solid support such as an ion exchange resin, diatomaceous earth, or a ceramic. By immobilizing and using, not only the catalytic activity may increase,
Separation / recovery of the catalyst and its reuse after the reaction are facilitated. Furthermore, usually, one kind of these catalysts is used, but it is also possible to mix and use two or more kinds of catalysts having the same ability.

In the present invention, the production of the optically active 2-halogen-substituted-1- (m-nitrophenyl) alcohol derivative of the general formula 4 by the asymmetric reduction reaction can be carried out by the following method. If necessary, coenzymes (NADH, NAD
PH, NAD ⁺ and / or NADP ⁺ ) and / or m-nitro group-substituted acetophenone derivative of the general formula 3 in a reaction solvent such as water or a buffer solution in the presence of an energy source such as glucose, sucrose, ethanol or methanol. It can be carried out by contacting the above-mentioned microbial cells, microbial cell culture solution, treated microbial cells or enzymes produced by these microorganisms. Then, the reaction is performed while controlling the reaction temperature and, if necessary, the pH of the reaction solution. Depending on the case, the reaction substrate (m-nitro group-substituted acetophenone or a salt thereof) and / or the coenzyme and the energy source may be appropriately added during the reaction to continue the reaction. Coenzyme (NAD
In many cases, the yield of the target compound is improved by adding an energy source such as H, NADPH, NAD ⁺ , NADP ⁺ ) and / or glucose, sucrose, ethanol or methanol.

The substrate concentration of the reaction solution is not particularly limited within the range of 0.01 to 50% by mass, but it is 0.
05-30 mass% is preferable. The concentration of microbial cells and the like in the reaction solution is usually 0.001 to 20% by mass, preferably 0.005 to 10% by mass. The pH of the reaction solution is comprehensively determined in consideration of the optimum pH of the enzyme to be used,
There is no particular limitation, but it is generally in the range of pH 4 to 11, preferably pH 5 to 9. In addition, the pH may change as the reaction proceeds. In this case, a suitable pH adjusting agent, in this case, a suitable neutralizing agent, for example, sodium hydroxide, potassium hydroxide, hydrochloric acid, an aqueous sulfuric acid solution, etc. It is desirable to add to adjust the pH to optimum.

The reaction temperature is appropriately determined depending on the optimum temperature of the catalyst used and is not particularly limited, but is preferably 0 to 60 ° C, more preferably 5 to 50 ° C. As the reaction solvent, an aqueous medium such as ion-exchanged water or a buffer is usually used, but the reaction may be carried out even in a system containing an organic solvent or a surfactant to accelerate the dissolution of the m-nitro group-substituted acetophenone derivative or its salt. It can be carried out. As an organic solvent,
For example, methanol, ethanol, propanol, isopropanol, butanol, isobutanol, t-butyl alcohol, alcohol solvents such as t-amyl alcohol, pentane, hexane, heptane, aliphatic hydrocarbon solvents such as octane, benzene, toluene, Aromatic hydrocarbon solvents such as xylene, methylene chloride, chloroform,
Halogenated hydrocarbon solvents such as carbon tetrachloride and dichloroethane, ether solvents such as diethyl ether, diisopropyl ether, tetrahydrofuran and dioxane, ester solvents such as ethyl acetate, propyl acetate and butyl acetate, acetone, methyl ethyl ketone and methyl isobutyl ketone. Ketone-based solvent, such as acetonitrile, N,
N-dimethylformamide or the like can be used as appropriate.

Examples of the surface active agent include anionic surface active agents such as alkylbenzene sulfonates and alkyl sulfates, cationic surface active agents such as alkylpyridinium salts and dodecyltrimethylammonium chloride, polyoxyethylene alkyl (phenyl) ethers, Polyoxyethylene alkyl (phenyl) ester, sorbitan fatty acid ester (span surfactant), polyoxyethylene glycol sorbitan alkyl ester (tween surfactant), polyoxyethylene glycol p
-N-alkyl-N, N-dimethylammonium betaine, lecithin, phosphatidylethanolamine, lysolecithin, and other amphoteric interfaces such as -t-octylphenyl ether (triton-based surfactant) and sucrose fatty acid ester. Activators and the like can be used as appropriate.

It is also possible to add these organic solvents or surface active agents in excess of the solubility in water to carry out the reaction in a two-layer system. By allowing an organic solvent to coexist in the reaction system,
The selectivity, conversion rate, yield, etc. often improve. The reaction time is usually 1 hour to 10 days, preferably 3 hours to
It is one week, and it is preferable to select reaction conditions at which the reaction ends at such time.

The above substrate concentration, coenzyme concentration,
Regarding the enzyme concentration, pH, temperature, solvent, reaction time and other reaction conditions, the objective optically active 2-halogen-substituted-1- (m-nitrophenyl) alcohol derivative is most suitable in consideration of the reaction yield under the conditions. It is desirable to appropriately select the conditions that allow many samples to be collected.

The desired product can be isolated from the reaction-terminated mixed solution by sterilization, and then by a commonly known method such as concentration, extraction, distillation, column separation or crystallization. For example, after adjusting the pH as necessary, ethers such as diethyl ether and diisopropyl ether, esters such as ethyl acetate, hydrocarbons such as hexane, benzene and toluene, halogenated hydrocarbons such as methylene chloride and butanol. It can be separated by extraction with a general solvent such as an alcoholic solvent such as isobutanol, t-amyl alcohol and the like.

[0027]

EXAMPLES The present invention will be specifically described below with reference to examples. <Example 1> Glycerol 30 g / L, yeast extract 10 g / L
L, peptone 5 g / L, KH ₂ PO ₄ 11 g / L and K ₂ H
Dispense 10 ml of medium consisting of 3 g / L of PO ₄ into a test tube,
After heat sterilization at 121 ° C. for 15 minutes, the strains shown in Table 1 were inoculated and shake cultured at 30 ° C. for 2 to 7 days. After completion of the culture, the cells were collected by centrifugation.

[0028] 0.1M HEPES (4
-(2-Hydroxyethyl) -1-piperazineethanesulfonic acid) 1 ml, NADH 3.4 mg, 10% 2-
10 μl of an ethyl acetate solution containing chloro-m-nitroacetophenone was added, and the mixture was reacted at 30 ° C. for 24 hours.

After the reaction was completed, ethyl acetate was added to the same amount and 2
Extracted with -chloro-1- (m-nitrophenyl) ethanol. After drying the extract with Glauber's salt, the product 2-chloro-1- (m) was obtained by gas chromatography (column; CP-Chirasil DEX CB 0.25mmID X 25M, manufactured by Chrompack).
The optical purity of -nitrophenyl) ethanol was measured.
The results are shown in Table 1.

[0030]

[Table 1]

<Example 2> Potato dextrose medium (Di
10 ml (made by fco) was dispensed into a test tube, 121 ° C, 15
After heat sterilization for 30 minutes, inoculate the strains shown in Table 2 at 30 ° C
The cells were shake-cultured for 2 to 7 days. After completion of the culture, the cells were collected by centrifugation. Reaction and analysis were carried out in the same manner as in Example 1, and the optical purity of the product 2-chloro-1- (m-nitrophenyl) ethanol was measured. The results are shown in Table 2.

[0032]

[Table 2]

<Example 3> Glycerol 30 g / L, yeast extract 10 g / L, peptone 5 g / L, KH ₂ PO ₄ 11 g /
10 ml of a medium consisting of L and K ₂ HPO ₄ 3 g / L was dispensed into a test tube, sterilized by heat at 121 ° C. for 15 minutes, inoculated with the strains shown in Table 3, and shake cultured at 30 ° C. for 2 to 7 days. did. After completion of the culture, the cells were collected by centrifugation. The bacterial cells were washed three times with acetone to give acetone-treated bacterial cells.

5.32 mg of 2-chloro-m-nitroacetophenone, 100 mM NADH solution 17.7 mg
And 15 mg of acetone-treated cells shown in Table 1 were suspended in 1 ml of 0.1 M HEPES buffer (pH 7),
The reaction was carried out at ℃ for 24 hours. Analysis was performed in the same manner as in Example 1 to measure the optical purity of the product 2-chloro-1- (m-nitrophenyl) ethanol. The results are shown in Table 3.

[0035]

[Table 3]

<Example 4> The same culture medium 200 as in Example 3
Geotrichum candidum IFO 4597 was cultured in 0 ml at 30 ° C. for 48 hours with shaking. After completion of the culture, the cells were collected by centrifugation.
50 m of the wet cells containing the same mass of 0.04% 2-mercaptoethanol and 0.1 mM dithiothreitol
The cells were suspended in MHEPES buffer (pH 7) and crushed twice with a French press (2000 kg / cm2).
The disrupted cell suspension was centrifuged and the supernatant was collected. The same amount of 4% protamine sulfate 50 mM HEPES buffer solution was added to the supernatant, and the mixture was stirred at 2 to 5 ° C overnight. The next day, the resulting insoluble matter was removed by centrifugation, ammonium sulfate was added to 70% saturation, and the mixture was left overnight at 2 to 5 ° C. The obtained precipitate was recovered by centrifugation, dissolved in the 50 mM HEPES buffer solution, and then dissolved in 10 mM phosphate buffer solution (pH 6.5).
It was dialyzed in ~ 5 ° C. Ammonium sulfate fractionated solution after dialysis is 5'AM
It was applied to a P Sepharose 4B (Amersham Pharmacia Biotech, dry mass 0.25 g) column. 10m
After washing the column with M phosphate buffer (pH 6.5), the column was eluted with a solution containing 0.2 mM of NAD ⁺ in the phosphate buffer to elute the active fraction as an enzyme solution.

5.32 mg of 2-chloro-m-nitroacetophenone, 100 mM NADH solution 17.7 mg
And 0.1M HEPES buffer (pH 7) 0.9m
0.1 ml of the obtained enzyme solution was added to 1 liter, and the mixture was added at
Reacted for hours. When the optical purity of the product 2-chloro-1- (m-nitrophenyl) ethanol was measured by performing the analysis in the same manner as in Example 1, the R isomer was 99% ee or more.

<Example 5> The same medium 200 as in Example 3
Dipodascus magnusii JCM 6360 was shake-cultured in 0 ml at 30 ° C. for 48 hours. After completion of the culture, dialysis was performed in the same manner as in Example 4 to obtain an ammonium sulfate fractionation solution. The same reaction was performed except that the ammonium sulfate fractionation solution was used in place of the enzyme solution of Example 4,
When the optical purity of 2-chloro-1- (m-nitrophenyl) ethanol was measured, it was R form 99% ee or more.

<Example 6> Di prepared in the same manner as in Example 3
100m of acetone powder of podascus magnusii JCM 6360 strain
15 mg of NADH and 30 mg of NADH were suspended and dissolved in 3 ml of 0.1 M HEPS buffer (pH 7).
2-chloro-m-nitroacetophenone dissolved in 90 μl of methyl alcohol was added, and the mixture was mixed at 30 ° C. for 3 days.
Reacted for days. After completion of the reaction, extraction was performed with 1.5 ml of ethyl acetate. This extract was analyzed in the same manner as in Example 1 to determine the optical purity of the product 2-chloro-1- (m-nitrophenyl) ethanol and its concentration. -2-chloro-1- (m
It was confirmed that 9.3 mg of -nitrophenyl) ethanol was present.

Example 7 The 2-chloro-1- (m-nitrophenyl) ethanol obtained in Example 6 was subjected to Mosher method using 1 H-NMR (JA Dale, HS Mosher, J. Am. C.
hem. Soc., 95, 512 (1973)), the absolute configuration was determined.
The procedure and results are shown below. The sample was divided into two, and one of them was converted into an R-MTPA ester by using a commercially available chiral derivatization reagent (+)-R-methoxytrifluoromethylphenylacetic acid chloride ((+)-R-MTPA · Cl). The other of the two divided samples was made into an S-MTPA ester by using a commercially available chiral derivatization reagent (-)-S-methoxytrifluoromethylphenylacetic acid chloride ((-)-S-MTPA.Cl). Dissolve R-MTPA ester and S-MTPA ester in deuterated chloroform, and
Fourier transform nuclear magnetic resonance (FT-NMR) device UNITY manufactured by rian
INOVA 500 ⁽¹ H resonance frequency 499.818MHz) ¹ at room temperature H
The NMR spectrum was measured. CH2 and CH were observed as follows.

[(+)-R-MTPA ester 1H NMR (499.818MH
z, CDCl3, RT) δ 6.156ppm (dd, J = 4Hz, 8Hz, 1H, C
H), 3.836 (dd, J = 8Hz, 12Hz, 1H, CH 2), 3.762 (d
d, J = 4Hz, 12Hz, 1H, CH ₂ )]

[(-)-S-MTPA ester 1H NMR (499.818MH
z, CDCl3, RT) δ 6.208ppm (dd, J = 5Hz, 7Hz, 1H, C
H), 3.818 (dd, J = 7Hz, 12Hz, 1H, CH 2), 3.756 (d
d, J = 5Hz, 12Hz, 1H, CH ₂ )]

¹ H-NMR of R-MTPA ester and S-MTPA ester
As a result of comparing the chemical shifts of the spectra, it was confirmed that the sample 2-chloro-1- (m-nitrophenyl) ethanol was the R-form.

[0044]

INDUSTRIAL APPLICABILITY The present invention provides a novel optically active alcohol, an optically active m-nitro group-substituted-1-phenethyl alcohol derivative important as a raw material for medicines and agricultural chemicals, and a method for producing them.

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI Theme Coat (Reference) C07M 7:00 F Term (Reference) 4B064 AE01 CA05 CB18 CD12 DA01 DA11 4H006 AA01 AC41 AC81

Claims (5)

[Claims] 1. (R) -2-halogen-substituted-1- (m-) represented by formula 1 (wherein X represents a halogen atom):
Nitrophenyl) alcohol derivative. [Chemical 1] 2. The X in the general formula 1 is a chlorine atom.
The (R) -2-halogen-substituted-1- (m-nitrophenyl) alcohol derivative described. 3. (S) -2-halogen-substituted-1- (m-) represented by the general formula 2 (wherein X represents a halogen atom).
Nitrophenyl) alcohol derivative. [Chemical 2] 4. The X in the general formula 2 is a chlorine atom.
The (S) -2-halogen-substituted-1- (m-nitrophenyl) alcohol derivative described. 5. General formula 3 The acetophenone substituted with m-nitro group represented by the formula (wherein X represents a halogen atom) is represented by the following general formula 4 (In the formula, X represents a halogen atom and * represents optical activity.) A microbial cell or a culture supernatant having the ability to be converted into an optically active m-nitro group-substituted-1-phenethyl alcohol derivative or a culture supernatant thereof The treated product is brought into contact with the m-nitro group-substituted 2-halogen-substituted acetophenone represented by the general formula 3 to collect the optically active m-nitro group-substituted-1-phenethyl alcohol of the above general formula 4.
-A method for producing a nitro group-substituted-1-phenethyl alcohol derivative.