JP2002095495A - Method for producing optically active aminoalcohol derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently producing optically active aminoalcohol derivatives useful as raw materials or synthetic intermediates for medicines, agrochemicals, and the like. SOLUTION: This method for producing the optically active aminoalcohol derivatives comprises bringing racemic aminoalcohol derivatives into contact with a cultured liquid of microorganism cells having asymmetrically hydrolyzing ability, or a treated material thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing an optically active amino alcohol derivative useful as a raw material or an intermediate for pharmaceuticals, agricultural chemicals and the like.

[0002]

2. Description of the Related Art An optically active amino alcohol is produced by reducing optically active 2-methyl-4-aminobutyric acid with lithium aluminum hydride (J. Am.
Chem. Soc., 81, 4946-4951 (1959)), or a method of reducing the methyl ester of optically active 2-methyl-4-nitrobutyric acid with lithium aluminum hydride for synthesis. (J. Plant Growth Regul. 2 (1), 47-57
(1983)) However, the former method has a low yield, and the latter method has a problem in safety of nitromethane used as a raw material. Furthermore, the yield of the addition reaction is low, making it difficult to establish an industrial production method.

On the other hand, as a method for producing a racemic amino alcohol, for example, after obtaining methyl 3-cyanoisobutyrate from hydrocyanic acid and methyl methacrylate according to the method described in JP-A-8-291157, the alkali alcohol is dissolved in a suitable solvent. It can be obtained by reduction using a metal hydride, but there is no report on the optical resolution of this compound.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide an industrially advantageous method for producing an amino alcohol derivative which is useful as an intermediate for synthesizing optically active medicinal and agricultural chemicals.

[0005]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found a microorganism having an activity of optically selectively hydrolyzing a racemic amino alcohol derivative, and have achieved the present invention. completed. That is, the present invention provides a compound represented by the general formula (I):

Embedded image (Wherein R ₁ and R ₂ are substituted or unsubstituted carbon atoms having 1 to 1 carbon atoms)
A non-reacted enantiomer amino alcohol derivative is collected by contacting the racemic amino alcohol derivative represented by (12 hydrocarbon groups) with a microbial cell culture solution having asymmetric hydrolysis ability or the treated product. A method for producing an optically active amino alcohol derivative.

[0006]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.

Embedded image In the general formula (I), R ₁ and R ₂ are a substituted or unsubstituted hydrocarbon group having 1 to 12 carbon atoms. In particular,
1 carbon atom such as methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, n-hexyl group
An alkenyl group having 2 to 12 carbon atoms such as an ethene group, a propene group, an isopropene group, a butene group, an isobutene group and an n-hexene group; a carbon atom such as an ethynyl group, a propynyl group and a butynyl group; An alkynyl group having the number of 2 to 12; a cycloalkyl group having a carbon number of 3 to 12, preferably 3 to 7 such as a cyclohexyl group; a phenyl group, a tolyl group,
An aryl group such as a naphthyl group; an aralkyl group such as a benzyl group; Preferably, it is a hydrocarbon group having 1 to 4 carbon atoms. In these hydrocarbon groups, the hydrogen atom bonded to the carbon atom may be substituted with a substituent such as halogen.

The racemic amino alcohol derivative represented by the general formula (I) as a raw material is disclosed in, for example, the above-mentioned JP-A-8-108.
4-amino-2-methylbutane-
After synthesis of 1-ol, it can be obtained by reacting with a target acid halide or acid anhydride by a known method.

The asymmetric hydrolase used in the present invention is an asymmetric hydrolysis of a racemic amino alcohol derivative represented by the general formula (I) to produce an optically active amino alcohol derivative and an unreacted enantiomer thereof. Any enzyme and enzyme source can be used as long as the enzyme has the ability to do so. Among them, enzymes referred to as lipases, esterases, proteases and amidases are particularly effective. There is no limitation as long as it has the ability to asymmetrically hydrolyze the racemic amino alcohol derivative represented by the general formula (I) to produce an unreacted enantiomer amino alcohol derivative. Alternatively, it does not matter which of the 4-amides is subjected to asymmetric hydrolysis.

As the asymmetric hydrolase, for example, a microorganism having an ability to produce an unreacted enantiomer amino alcohol derivative by asymmetric hydrolysis of a racemic amino alcohol derivative represented by the general formula (I) is used. Can be used.

[0010] Such microorganisms are not particularly limited, but typical microorganisms include the genera Pichia, Rhodotorula, and S.
accharopolyspora, Streptomyces, Actinomucor
Genus, Aspergillus, Bacillus, Candida, Cladospo
genus rium, genus Fusarium, genus Geotrichum, Acinetobacter
Genus, Gibberella, Kluyveromyces, Microsporum,
Brevibacillus, Mucor, Actinomadura, Coprinus
Genus, Agaricus, Rhodococcus, Rhizomucor, Rhiz
Examples include microorganisms belonging to the genus opus and the genus Commomonas.

As microorganisms belonging to the genus Pichia, for example,
Pichia abadieae (ATCC 22263) and the like, as microorganisms belonging to the genus Rhodotorula, for example, Rhodotorula mucilagin
osa (ATCC 20129) and the like, as microorganisms belonging to the genus Saccharopolyspora, for example, Saccharopolyspora hirsuta (ATC
C 27875) and the like, as microorganisms belonging to the genus Streptomyces, for example, Streptomyces coelicolor (ATCC 10147), S
treptomyces rimosus (ATCC 10970), Streptomyces roch
ei (ATCC 10739), Streptomyces albus (ATCC 3004), etc., as microorganisms belonging to the genus Actinomucor, for example, Act
inomucor elegans (ATCC 6476) and the like, as microorganisms belonging to the genus Aspergillus, for example, Aspergillus alliaceus (AT
CC 1024), Aspergillus nidulans (ATCC 11267), Aspe
rgillus ochraceus (ATCC 1008), Aspergillus foetidu
s (ATCC 10254), Aspergillus niger (ATCC 9029), Aspe
rgillus oryzae (ATCC 26850) and the like, as microorganisms belonging to the genus Bacillus, for example, Bacillus subtlis (ATCC 605
1), Bacillus cereus (ATCC 11778), Bacillus amylol
iequifaciens (ATCC 23842) and the like, as microorganisms belonging to the genus Candida, for example, Candida antarctica (ATCC 2832
3), Candida oregonensis (ATCC42268), Candida cylind
racea (ATCC 14830), Candida tropicalis (ATCC 13803)
As a microorganism belonging to the genus Cladosporium, for example,
Cladosporium herbarum (ATCC 28987) and the like, as microorganisms belonging to the genus Fusarium, for example, Fusarium oxysporum
(IFO 7156), Fusarium oxysporum (ATCC 7601), etc.
Examples of microorganisms belonging to the genus trichum include Geotrichum
amycelicum (ATCC 24658) and the like, as a microorganism belonging to the genus Acinetobacter, for example, Acinetobacter calcoaceticu
s (ATCC 14987) and the like, as a microorganism belonging to the genus Gibberella, for example, Gibberella fujikuroi (ATCC 14842) and the like,
Examples of microorganisms belonging to the genus Kluyveromyces include Kluy
Veromyces lactis (ATCC 2628) and the like, as microorganisms belonging to the genus Microsporum, for example, Microsporum gypseum (ATCC
As a microorganism belonging to the genus Brevibacillus, for example, Brevibacillus parabrevis (ATCC 8185) and the like, M
Examples of microorganisms belonging to the genus ucor include Mucor rouxii (A
TCC 24905) and the like, as a microorganism belonging to the genus Actinomadura, for example, Actinomadura citrea (ATCC 27887) and the like, Cop
As microorganisms belonging to the genus rinus, for example, Coprinus sp.
(ATCC 16789) and the like, as microorganisms belonging to the genus Agaricus, for example, Agaricus campestris (ATCC 26815) and the like, Rhod
Microorganisms belonging to the genus ococcus include, for example, Rhodococcu
s rhodochrous (ATCC 12674) and the like, as microorganisms belonging to the genus Rhizomucor, for example, Rhizomucor pusillus (ATCC 5
6683) and the like, as microorganisms belonging to the genus Rhizopus, for example, Rhizopus oryzae (ATCC 22580), Rhizopus stolonif
er (ATCC 6227b) and the like, as microorganisms belonging to the genus Commomonas, for example, Commomonas testosterone (ATCC 11996)
Etc. are exemplified.

[0012] These microorganisms can be easily obtained from the Institute of Fermentation (IFO) and the American Type Culture Collection (ATCC). Also,
It is also possible to use genetically engineered microorganisms in which enzyme genes isolated from these microorganisms have been introduced into various host vector systems by a usual method.

Further, in the case of the asymmetric hydrolysis reaction, a microorganism culture solution obtained by culturing the above microorganisms in an appropriate medium or the treated product can be used. As the treated product, a culture supernatant obtained by a cell collection operation such as centrifugation from a microorganism culture solution, a microbial cell, a microbial cell treated with acetone or toluene, a crushed product of the microbial cell, Examples of the obtained cell-free extract, immobilized cells, purified purified enzyme, immobilized enzyme and the like can be given. The unreacted enantiomer amino alcohol derivative can also be produced by asymmetric hydrolysis of the racemic amino alcohol derivative shown in (I) in the presence of the biocatalyst.

In the present invention, an asymmetric hydrolysis reaction is usually carried out using one kind of the above-mentioned microorganisms, a culture solution of the microorganism cells, a processed product of the microorganism cells, and the like, but two or more types having the same ability are mixed. It is also possible to carry out the reaction.

Further, in the production method of the present invention, microbial cells, purified from the viewpoint of separation of the hydrolyzate of the optically active amino alcohol derivative after the reaction and its unreacted enantiomer, separation and recovery of the enzyme, and reuse. It is preferable to use the enzyme or the like immobilized on a suitable carrier.

In the present invention, any medium can be used as a medium for culturing these microorganisms as long as these microorganisms can normally grow. As the carbon source, for example, sugars such as glucose, sucrose and maltose, organic acids such as acetic acid, citric acid and fumaric acid or salts thereof, and alcohols such as ethanol and glycerol can be used. As a nitrogen source, for example, peptone,
In addition to general natural nitrogen sources such as meat extract, yeast extract and amino acids, 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. Also, to obtain high enzyme activity,
It is also effective to add an amino alcohol derivative represented by the general formula (I), a compound having an ester bond or an amide bond, or the like as an enzyme-producing inducer to the medium.

The cultivation of the microorganism may be carried out according to a conventional method.
For example, the cells are cultured aerobically for 6 to 96 hours at a pH of 4 to 10 and a temperature of 15 to 40C.

In the present invention, the enantiomer amino alcohol derivative can be produced by optically selective hydrolysis of the amino alcohol derivative represented by the general formula (I) by the following method. An amino alcohol derivative represented by the general formula (I) as a substrate is dissolved or suspended in a reaction solvent. Before or after the addition of the substrate to the reaction solvent, a culture of the above-mentioned microorganism having the ability to perform asymmetric hydrolysis as a catalyst or the treated product is added. Then, while controlling the reaction temperature and, if necessary, the pH of the reaction solution, the general formula (I)
The reaction is continued until about half of the amino alcohol derivative represented by the formula is hydrolyzed. In some cases, the reaction may be interrupted at an early stage of the reaction, or the reaction may be excessively performed.

The concentration of the substrate in the reaction solution is not particularly limited in the range of 0.1 to 80% by mass, but is preferably 1 to 50% by mass in consideration of productivity and the like.

The concentration of the enzyme in the reaction solution is usually 0.01 to 10% by mass, preferably 0.05 to 5% by weight.

The pH of the reaction solution depends on the optimum pH of the enzyme to be used, but is generally in the range of pH 4 to 11. It is preferable to carry out the reaction at pH 5 to 9 from the viewpoint that a decrease in optical purity and a decrease in yield due to a chemical hydrolysis reaction can be suppressed. When the ester bond is asymmetrically hydrolyzed, the pH decreases as the reaction proceeds.In this case, a suitable neutralizing agent such as sodium hydroxide or aqueous potassium hydroxide is added. It is desirable to adjust the pH to the optimum.
Even when the amide bond is asymmetrically hydrolyzed, the pH is expected to change as the reaction proceeds. In this case as well, it is desirable to adjust the pH to an optimum pH with a suitable neutralizing agent.

The reaction temperature is preferably 5 to 70 ° C., and 10 to 50 ° C.
Is more preferred.

As a reaction solvent, an aqueous medium such as ion-exchanged water or a buffer is usually used, but the reaction can be carried out in a system containing an organic solvent. Examples of the organic solvent include alcohol solvents such as methanol, ethanol, propanol, isopropanol, butanol, isobutanol, t-butyl alcohol, and t-amyl alcohol; and aliphatic hydrocarbon solvents such as pentane, hexane, heptane, and octane. Benzene, toluene, xylene and other aromatic hydrocarbon solvents, methylene chloride, chloroform, carbon tetrachloride, dichloroethane and other halogenated hydrocarbon solvents, diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane and other ether solvents, acetic acid Ester solvents such as ethyl, propyl acetate and butyl acetate, ketone solvents such as acetone, methyl ethyl ketone and methyl isobutyl ketone, acetonitrile, N, N-dimethylformamide and the like can be appropriately used.

It is also possible to add these organic solvents to a solvent having a solubility in water or higher and to carry out the reaction in a two-layer system. Coexistence of an organic solvent in the reaction system often improves selectivity, conversion, yield, and the like. The reaction time is generally 1 hour to 1 week, preferably 1 to 72 hours, and it is preferable to select reaction conditions under which the reaction is completed.

The substrate concentration, enzyme concentration, p
As for H, temperature, solvent, reaction time, and other reaction conditions, it is desirable to appropriately select conditions under which the target optically active compound can be collected most, taking into account the reaction yield, optical yield, and the like under those conditions.

By the above reaction, the amino alcohol derivative represented by the general formula (I) is asymmetrically hydrolyzed to produce a hydrolyzate of an optically active amino alcohol derivative and an unreacted enantiomer amino alcohol derivative. Isolation of the produced optically active amino alcohol derivative hydrolyzate and unreacted enantiomer amino alcohol derivative from the reaction mixture can be carried out by a known isolation method such as extraction, distillation, and column separation.

When the resulting hydrolyzate of the optically active amino alcohol derivative has the 4-amide moiety asymmetrically hydrolyzed, for example, after adjusting the pH to near neutral, ethers such as diethyl ether and diisopropyl ether; Unreacted enantiomers can be extracted and separated with common solvents such as esters such as ethyl; hydrocarbons such as hexane, octane, benzene, and toluene; and halogenated hydrocarbons such as methylene chloride.

On the other hand, when the resulting hydrolyzate of an optically active amino alcohol derivative has a 1-ester portion asymmetrically hydrolyzed, for example, it can be similarly extracted and separated with a common low-polarity organic solvent.

Furthermore, the hydrolyzate of the optically active amino alcohol derivative and the unreacted enantiomer can hydrolyze the 1-ester portion and / or the 4-amide portion by a usual method while maintaining the optical activity. The hydrolyzate of the optically active amino alcohol derivative can be esterified / amidated by a usual method while maintaining the optical activity. Therefore, any three-dimensional configuration can be obtained according to the purpose.

Furthermore, the amino alcohol derivative amidated and esterified by the above-mentioned method can be used as a substrate of the enzyme, and the reaction can be repeated a plurality of times, so that a target compound having higher optical purity can be obtained. Similarly, a target compound having high optical purity can be obtained by repeating the reaction by arbitrarily combining enzymes having different optical selectivities (reverse).

[0031]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to examples, but the scope of the present invention is not limited to the examples.

Examples 1 to 39 A liquid medium having a composition consisting of 20 g of glucose, 5 g of peptone, 5 g of yeast extract, 5 g of NaCl, 5 g of K ₂ HPO ₄ and 1 L of distilled water (pH 7.0) was prepared. Was dispensed in 30 ml portions into 250 ml Erlenmeyer flasks, steam-sterilized at 120 ° C for 15 minutes, and butyl acetate was added to 6 ml.
μL was added, and the microorganisms shown in Table 1 were inoculated and cultured at 28 ° C. with shaking. Next, the cells were collected from the culture solution in each flask by centrifugation, and washed three times with a 50 mM phosphate buffer (pH = 7.0) (10 ° C. or lower). Bacterial cells in 50 mM phosphate buffer (pH = 7.0)
It was suspended in 10 ml. The amount of racemic 1-acetoxy-N-acetyl-4-amino-2-methylbutane shown in Table 1 was added to the cell suspension, and reacted at 28 ° C. with shaking for 24 hours.

[0033] To the reaction solution, 15 ml of ethyl acetate was added and stirred. Optical activity 1-acetoxy contained in ethyl acetate layer
-N-acetyl-4-amino-2-methylbutane was subjected to liquid chromatography equipped with an optical resolution column (Chiral Cell OD column (0.46 cm ID × 25 cm); manufactured by Daicel Corporation), n-hexane: 2-propanol = 95. : 5 and analyzed. Table 1 shows the culture time, the amount of substrate added, and the results of the reaction solution analysis.

[0034]

[Table 1]

[0035]

According to the present invention, optically active amino alcohol derivatives useful as raw materials for pharmaceuticals, agricultural chemicals and the like or synthetic intermediates can be efficiently and industrially produced.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:01) C12R 1:01) (C12P 41/00 (C12P 41/00 H C12R 1:03) C12R 1:03) (C12P 41/00 (C12P 41/00 H C12R 1:07) C12R 1:07) (C12P 41/00 (C12P 41/00 H C12R 1: 085) C12R 1: 085) (C12P 41 / 00 (C12P 41/00 H C12R 1: 465) C12R 1: 465) (C12P 41/00 (C12P 41/00 H C12R 1:47) C12R 1:47) (C12P 41/00 (C12P 41/00 H C12R) 1: 645) C12R 1: 645) (C12P 41/00 (C12P 41/00 H C12R 1:66) C12R 1:66) (C12P 41/00 (C12P 41/00 H C12R 1: 685) C12R 1: 685 ) (C 2P 41/00 (C12P 41/00 H C12R 1:69) C12R 1:69) (C12P 41/00 (C12P 41/00 H C12R 1:72) C12R 1:72) (C12P 41/00 (C12P 41 / 00 H C12R 1:74) C12R 1:74) (C12P 41/00 (C12P 41/00 H C12R 1:77) C12R 1:77) (C12P 41/00 (C12P 41/00 H C12R 1: 785) C12R 1: 785) (C12P 41/00 (C12P 41/00 H C12R 1:84) C12R 1:84) (C12P 41/00 (C12P 41/00 H C12R 1: 845) C12R 1: 845) (C12N 1 / 14 (C12N 1/14 A C12R 1: 645) C12R 1: 645) (C12N 1/14 (C12N 1/14 A C12R 1:66) C12R 1:66) (C12N 1/14 (C12N 1/14 A C12R) 1: 685) C12R 1: 685) (C12N 1/14 (C12N 1/14 A C12R 1:69) C12R 1:69) (C12N 1/14 (C12N 1/14 A C12R 1:77 (C12R 1:77) (C12N 1/14 (C12N 1/14 A C12R 1: 785) C12R 1: 785) (C12N 1/14 (C12N 1/14 A C12R 1: 845) C12R 1: 845) (C12N 1 / 16 (C12N 1/16 A C12R 1:72) C12R 1:72) (C12N 1/16 (C12N 1/16 A C12R 1:74) C12R 1:74) (C12N 1/16 (C12N 1/16 A C12R 1:84) C12R 1:84) (C12N 1/20 (C12N 1/20 A C12R 1:01) C12R 1:01) (C12N 1/20 (C12N 1/20 A C12R 1:03) C12R 1: 03) (C12N 1/20 (C12N 1/20 A C12R 1:07) C12R 1:07) (C12N 1/20 (C12N 1/20 A C12R 1: 085) C12R 1: 085) (C12N 1/20 ( C12N 1/20 A C12R 1: 465) C12R 1: 465) (C12N 1/20 (C12N 1/20 A C12R 1:47) C12R 1:47) F term (reference) 4B064 AE02 CA02 CA04 CA05 CA06 CB05 CC03 CD12 CD27 CE10 DA16 4B065 AA04X AA05X AA15X AA16X AA19X AA45X AA50X AA58X AA60X AA62X AA63X AA65X AA66X AA69X AA71X AA72X AA73X CA47A AAX47

Claims (2)

[Claims] 1. A process for producing an amino alcohol derivative, comprising the steps of: (Wherein R ₁ and R ₂ are substituted or unsubstituted carbon atoms having 1 to 1 carbon atoms)
A non-reacted enantiomer amino alcohol derivative is collected by contacting the racemic amino alcohol derivative represented by (12 hydrocarbon groups) with a microbial cell culture solution having asymmetric hydrolysis ability or the treated product. A method for producing an optically active amino alcohol derivative. 2. Microorganisms having asymmetric hydrolysis ability include Pichia, Rhodotorula, Saccharopolyspora, Streptomyces, and Actinomucol.
nomucor), Aspergillus, Bacillus, Candida, Cladosporium, Fusarium
m) genus, genus Geotrichum, genus Acinetobacter, Gibberella
Genus, Kluyveromyces, Microsporum, Brevibacillus
cillus, Mucor, Actinomadra (A
genus ctinomadura), genus Coprinus, genus Agaricus, Rhodococcus
Genus, Rhizomucor, Rhizops
The method for producing an optically active amino alcohol derivative according to claim 1, which is at least one member selected from the group consisting of genus pus) and genus Commomonas.