JPH02104295A - Production of optically active amine and its derivative - Google Patents

Abstract

PURPOSE:To simply obtain the title amine and its derivative in high optical purity by subjecting a specific mixture to microorganisms capable of asymmetrical hydrolysis of the amide linkage in said mixture. CONSTITUTION:Microorganisms belonging to Rhodococcus sp. is put to culture at pH3.9-9.5 and 20-45 deg.C for 1-5 days under a desired O2 concentration in a medium containing e.g., glucose to obtain a cultured product. This product is then added to 0.1-10wt.% solution of an enantiomer mixture of an acyl derivative of amine of formula I (X1-2 are each H, nitro, halogen, OH or alkyl; COR is acyl; n is 1-3) (e.g., N-acetyl-2-amino-4-phenylbutane) followed by reaction on agitation at pH3-9 and 10-60 deg.C for 1-120hr to obtain a reaction solution. Thence, this reaction solution is extracted and purified, thus separately obtaining the objective optically active (S) form of an amine of formula II (e.g., 2-amino-4- phenylbutane) and optically active (R) from of an amine acyl derivative of formula left.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for producing optically active amines and acyl derivatives of the amines. More specifically, general formula (I) (wherein x1 and x2 each represent a hydrogen atom, a halogen atom, a hydroxyl group, a nitro group, or an alkyl group, COR represents an acyl group, and n represents an integer of 1 to 3.) A mixture of enantiomers of an acyl derivative of an amine represented by the formula (II) is produced by reacting a microorganism or an enzyme capable of asymmetrically hydrolyzing the amide bond. is the same meaning as above) and the remaining optically active (R) form of the acyl derivative of the amine represented by the general formula (I) are separated and collected. The present invention relates to a method for producing optically active amines and derivatives thereof. These optically active compounds are important as synthetic intermediates for various medicines and agrochemicals.

[Problems to be solved by conventional techniques and inventions] Conventionally,
As a method for obtaining an optically active form of the amine derivative represented by the general formula (II), a method of optical resolution using an optical resolving agent according to the diastereomer method (Japanese Patent Laid-Open No. 10874-1989
9) and a method of synthesis from optically active 2-hydroxy-4-phenylbutane (JP-A-63-54351), but in the diastereomer method, equimolar amounts of different optically active compounds are synthesized. However, there is a problem in that it is difficult to obtain this optically active substance using the method of synthesis from 2-hydroxy-4-phenylbutane.

[Means to solve the problem]

In view of this situation, the present inventors have developed an optically active (S) form of the amine represented by the general formula (II) and an amine represented by the general formula (I) using an economically superior and simple method. As a result of intensive research to develop a method for producing the optically active (R) form of acyl derivatives, it was discovered that specific microorganisms or enzymes asymmetrically asymmetrically bonded the amide bond of the enantiomeric mixture of the acyl derivative of the amine represented by the general formula (I). When hydrolyzed, an optically active (S) form of an amine represented by formula (II) and an optically active (I form) of an acyl derivative of an amine represented by general formula (I) are produced. This discovery has led to the completion of the present invention.

That is, the present invention provides an enantiomeric mixture of the acyl derivative of the amine represented by the general formula (I) with the action of a microorganism or an enzyme capable of asymmetrically hydrolyzing the amide bond, thereby producing a compound of the general formula (II). An optically active amine characterized by separating and collecting the optically active (S) form of the amine represented by the formula (I) and the remaining optically active (R) form of the acyl derivative of the amine represented by the general formula (I). and a method for producing a derivative thereof.

In the acyl derivative of the amine represented by the general formula (I) used in the present invention, specific examples of the halogen atoms represented by χ1 and ×2 include CI and Br.

Specific examples of alkyl groups such as F, 1, etc. include methyl group,
Specific examples of the acyl group represented by COR include an ethyl group, a propyl group, an isopropyl group, a benzyl group, and an acetyl group, a propionyl group, a butyryl group, and the like.

Further, as a specific example of the acyl derivative of the amine represented by the general formula (I) that can be used in the present invention, N-acetyl-2-amino-4-phenylbutane and the like can be mentioned.

Next, microorganisms or enzymes that can be used in the present invention include:
-4 The amide bond of the enantiomeric mixture of the acyl derivative of the amine represented by formula (I) is asymmetrically hydrolyzed and the general formula (
Any can be used as long as it can produce the optically active (S) form of the amine represented by II) and the optically active (R) form of the acyl derivative of the amine represented by the general formula (I). However, specifically Rhodococcus
microorganisms belonging to the genus (us) or enzymes produced by the microorganisms, more specifically Rhodococcus erythropolis (
Rhodococcus erythropolis)
IFO12320, Rhodococcus erythropolis (R
hodococcuserythropolis)IF
O12682, Rhodococcus equi
ccus equi) IFO3730+ JCM 13
11. Rhodococcus fascians
Examples include microorganisms such as C. cus facians: JCM 1316, and enzymes produced by them.

Moreover, these mutant strains and the enzymes produced by them can also be used.

Among the above, the microorganisms with IFO numbers are Li5t of Cu- published by Fermentation Research Institute (RPO).
1tures, 8th Edition, Volume 1 (I98B), available from the IFO. Also JC
Microorganisms with M numbers are from the Microbial Strain Preservation Facility and Microbial Strain Catalog published by RIKEN.

It is described in the 3rd edition (I986) and is available from RIKEN.

The medium for culturing the microorganisms used in the present invention is not particularly limited as long as it contains a nutrient source in which the bacteria can grow. For example, carbon sources include sugars such as glucose and sucrose, alcohols such as ethanol and glycerol, organic acids such as acetic acid and propionic acid, hydrocarbons such as paraffin, or mixtures thereof; nitrogen sources include Inorganic and organic nitrogen-containing compounds such as ammonium sulfate, yeast extract, and urea are used. In addition, nutrient sources used in normal culture, such as inorganic salts, trace metal salts, and vitamins, can be appropriately mixed and used. In addition, factors that promote the growth of microorganisms or the pH of the medium may be added as necessary.
Substances effective for retention can also be added.

In addition, a substance effective for improving the production amount or enzyme activity of the desired asymmetric hydrolase may be added to the culture.

As for the culturing method, it is preferable to set the medium pH to 3.0 to 9.5, the culture temperature to 20 to 45°C, and to culture for 1 to 5 days under oxygen concentration conditions suitable for the growth of the microorganism.

In the present invention, the enzyme may be present in any form as long as the desired asymmetric hydrolysis reaction is substantially completed. That is, it is possible to use bacterial cell culture fluids, isolated bacterial cells, and treated bacterial cells that have the desired asymmetric hydrolytic activity, and enzymes that are separated from these and that have the desired asymmetric hydrolytic activity.

As for the acyl derivative of the amine used as the substrate, it is preferable to use a racemic form in terms of its price, but the enantiomeric mixing ratio is not particularly limited, and the present invention can be applied to any mixing ratio. .

In the present invention, methods for reacting microorganisms or enzymes with amine derivatives include (I) a method in which a bacterial cell culture solution is used as it is and an acyl derivative of amine is added to the culture solution;
2) A method in which bacterial cells separated by centrifugation or the like are resuspended in a buffer, water, etc., either as is or after being washed, and an acyl derivative of amine is added to the resulting suspension; (3) Bacterial cell disruption. (4) Method of immobilizing these microbial cells on a carrier; (5) Centrifuging the crushed microbial cells to obtain a crude enzyme solution. (6) Purifying the desired enzyme from this crude enzyme solution by a known method and reacting this enzyme with the acyl derivative of amine; (7) Various methods can be used, such as a method in which enzymes or purified enzymes are immobilized using known methods.

In these methods, the acyl derivative of the amine is used as it is, or dissolved in an organic solvent that does not affect the reaction, or dispersed in a surfactant, etc., and all or part of it is added from the beginning of the reaction. It may also be added. The reaction is preferably carried out at a temperature of 10 to 60°C at a pH in the range of 3 to 9, and is carried out under stirring for 1 to 120 hours. The concentration of the substrate is not particularly limited, but is preferably about 0.1 to 10%.

The optically active amine produced by the reaction and the remaining amine derivatives can be collected directly from the reaction solution, or after bacterial cell isolation, extraction with an organic solvent, column chromatography,
This can be easily carried out using ordinary purification methods such as distillation.

The optically active (S) form of the amine represented by the general formula (II) and the optically active (I' form) of the acyl derivative of the amine represented by the general formula (I) thus obtained are Alternatively, it can be used as various synthetic intermediates after performing various chemical transformations by known methods depending on the purpose.

In addition, the optically active (R) form of the acyl derivative of the amine represented by general formula (I) is chemically hydrolyzed by a conventional method (
The optically active (R) form of the amine represented by the general formula (II) can also be obtained by refluxing in an acid or alkaline aqueous solution.

(Examples) Hereinafter, the present invention will be specifically explained using Examples, but the present invention is not limited thereto.

In the examples, quantitative analysis of amines and amine derivatives was performed using a high performance liquid cucomatography C column: Uni
sil Pack 5C+a-25OA (manufactured by Gas Chloé Industries), solvent: 40mM phosphate buffer (p++3.5
)/acetonitrile 6;4]. In addition, the optical purity of these compounds can be determined by acetylation using a standard method in the case of the amine produced in the reaction, or by high-performance liquid chromatography using an optical resolution column (Column: Chiralcel OB (Daicel)). (manufactured by Kagaku Kogyo ■), solvent: n-hexane/isopropanol (95:5).

Example 1 Glucose 1%, yeast extract 0.5%, polypeptone 0
．． 5%, sodium chloride 0.3%, N-acetyl-2-
A culture medium 50- containing 0.1% of the racemic form of amino-4-phenylbutane was placed in a 500-Yosaka flask;
After sterilization, Rhodococcus ae IJ 7! it: lbo IJ
7. IF012320 was inoculated and cultured with reciprocal shaking at 30°C for 2 days.

After the culture was completed, the bacterial cells were separated by centrifugation and washed once with physiological saline to obtain viable bacterial cells.

Next, 50 mm of distilled water was put into a 500 mZ Sakaro flask, and the above-mentioned bacterial cells were suspended therein.

Thereafter, 0.5 g of racemic N-acetyl-2-amino-4-phenylbutane was added, and the mixture was shaken back and forth at 30"C to react for 24 hours.

After the reaction, the bacterial cells were removed by centrifugation, and the resulting supernatant contained (S)-2-amino-4-phenylbutane and the remaining (R)-N-acetyl-2-amino- 4-phenylbutane was quantitatively analyzed to determine the reaction yield.

In addition, rhohexane 10- was added to the supernatant 1- (R).
-N-acetyl-2-amino-4-phenylbutane was extracted, and its optical purity was determined using Chiralcel OB. Also,
After adding a caustic soda aqueous solution to this aqueous layer to make it alkaline, 10 m7 of n-hexane was added in the same manner (S) -2-
Amino-4-phenylbutane was extracted. Thereafter, the obtained (S)-2-amino-4-phenylbutane was acetylated with acetyl chloride according to a conventional method, and the optical purity was determined in the same manner as above.

The results obtained are shown in Table 1.

Table 1 Example 2 Pre-culture solution 100@ of Rhodococcus erythropolis IF012682 cultured in the same manner as in Example 1 in a 51-jar fermentor containing medium 22 used in Example 1
1 was inoculated and cultured with aeration at 30''C for 30 hours at a stirring speed of 400 rp+n and an aeration amount of 0.5 vvm.

After the culture was completed, the bacteria were collected by centrifugation, and the cells were washed with water 500 @Z. Thereafter, the cells were placed in a 21-volume Erlenmeyer flask, and 500 mm of 50mM phosphate buffer was added to suspend the cells. After that, N-acetyl-2-amino-
Add 15g of racemic 4-phenylbutane and heat to 30"C.
The mixture was shaken and reacted for 24 hours.

After the reaction was completed, the bacterial cells were separated by centrifugation, and the supernatant was concentrated using a rotary evaporator and closed at 200 ml.

Next, ethyl acetate 200@1 was added and shaken to perform an extraction operation. After performing this operation twice, the ethyl acetate layers were combined, and then the solvent was removed using a rotary evaporator to obtain 6.1 g of (R)-N-acetyl-2-amino-4-phenylbutane in the form of an oil. When this sample was analyzed by the liquid chromatography method described above, the chemical purity was 97.7% and the optical purity was 99.8%e,e.

Next, add 10% caustic soda solution to the remaining aqueous layer to adjust the pH.
10, extraction was performed with ethyl acetate in the same manner as described above to obtain 5.3 g of oily (S)-2-amino-4-phenylbutane. When this sample was analyzed by liquid chromatography in the same manner as in the previous case, the chemical purity was 96.8%, and the optical purity was 99.5%e, e.
,Met.

Example 3 After culturing Rhodococcus erythropolis IF012682 in the same manner as in Example 2, bacterial cells were obtained by centrifugation. 20g of this wet bacterial body was added to 50mM phosphate buffer (p
H7,0) The enzyme was extracted by suspending it in 50aI and subjecting it to ultrasonic disruption treatment under water cooling. Next, fragments of bacterial cells were removed by centrifugation to obtain 40 m/w of crude enzyme solution. 2 g of racemic N-acetyl-2-amino-4-phenylbutane was added to this crude enzyme solution, and the mixture was shaken rotary at 30°C and allowed to react for 24 hours.

Extraction was performed from this reaction solution using ethyl acetate in the same manner as in Example 2, resulting in (S)-2-amino-4
0.72 g of -phenylbutane and 0.85 g of remaining (R)-N-acetyl-2-amino-4-phenylbutane were obtained. The chemical purity of the obtained (S)-2-amino-4-phenylbutane was 96.8%, and the optical purity was 99.5%.
, e. Moreover, the chemical purity of (R)-N-acetyl-2-amino-4-phenylbutane was 97.6%, and the optical purity was 99.3%e,e.

Example 4 5.5 g of (R)-N-acetyl-2-amino-4-phenylbutane obtained in Example 2 was dissolved in 55 m7 of 6M hydrochloric acid and refluxed for 20 hours at a pot temperature of 109 to 110"C. .

As a result, (R)-N-acetyl-2-amino-4-phenylbutane was 89% hydrolyzed.

Extraction was performed from this hydrolyzate using ethyl acetate in the same manner as in Example 2, and (R)-2-amino-4-
3.5 g of phenylbutane was obtained.

The chemical purity of the obtained (R)-2-amino-4-phenylbutane was 97.2%, and the optical purity was 99.5%e,e.

Met.

〔Effect of the invention〕

The method for producing optically active amines and amine derivatives by an asymmetric hydrolysis method using microorganisms or enzymes produced by the present invention makes it possible to easily produce these compounds with high optical purity, and is suitable for industrial use. This is extremely advantageous as a manufacturing method.

Claims (1)

[Claims] 1. General formula (I) ▲ Numerical formula, chemical formula, table, etc.▼... (I) (In the formula, X_1 and X_2 are each a hydrogen atom, a halogen atom, a hydroxyl group, a nitro group, or an alkyl group, COR is an acyl group, and n is an integer of 1 to 3.) is treated with a microorganism capable of asymmetrically hydrolyzing the amide bond,
Generated general formula (II) ▲There are mathematical formulas, chemical formulas, tables, etc.▼...(II) (In the formula, X_1, X_2 and n have the same meanings as above) Optically active (S) form of amine and the remaining general formula (I
) Optically active (R) of the acyl derivative of amine represented by
1. A method for producing optically active amines and derivatives thereof, which comprises separating and collecting the amines and their derivatives. 2. General formula (I) ▲There are mathematical formulas, chemical formulas, tables, etc.▼... (I) (In the formula, X_1 and X_2 are hydrogen atoms, halogen atoms, hydroxyl groups, nitro groups, or alkyl groups, respectively, and COR is an acyl group. , n is an integer of 1 to 3) is reacted with an enzyme capable of asymmetrically hydrolyzing the amide bond to produce the general formula (II) ▲ There are mathematical formulas, chemical formulas, tables, etc.▼...(II) (In the formula, X_1, X_2 and n are the same as above) The optically active (S) form of the amine and the remaining general formula (I
) Optically active (R) of the acyl derivative of amine represented by
1. A method for producing optically active amines and derivatives thereof, which comprises separating and collecting the amines and their derivatives. 3. The microorganism is Rhodococcus
The manufacturing method according to claim 1, wherein the microorganism belongs to the genus. 4. The production method according to claim 2, wherein the enzyme is an enzyme produced by a microorganism belonging to the genus Rhodococcus. 5. The amine represented by general formula (II) is 2-amino-4-
The manufacturing method according to claim 1 or 2, wherein the phenylbutane is phenylbutane.