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

Production of optically active amine and its derivative

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Publication number
JPH02104295A
JPH02104295A JP25538488A JP25538488A JPH02104295A JP H02104295 A JPH02104295 A JP H02104295A JP 25538488 A JP25538488 A JP 25538488A JP 25538488 A JP25538488 A JP 25538488A JP H02104295 A JPH02104295 A JP H02104295A
Authority
JP
Japan
Prior art keywords
optically active
formula
amine
general formula
phenylbutane
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
JP25538488A
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Japanese (ja)
Other versions
JP2698627B2 (en
Inventor
Nobumi Kunishige
国重 悦己
Akikazu Matsuyama
彰収 松山
Yoshinori Kobayashi
良則 小林
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Daicel Corp
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Daicel Chemical Industries Ltd
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Publication of JPH02104295A publication Critical patent/JPH02104295A/en
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Publication of JP2698627B2 publication Critical patent/JP2698627B2/en
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  • Preparation Of Compounds By Using Micro-Organisms (AREA)

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

【発明の詳細な説明】 〔産業上の利用分野〕 本発明は光学活性なアミン及びそのアミンのアシル誘導
体の製造方法に関する。さらに詳しくは、一般式(I) (式中、×1、×2はそれぞれ水素原子、ハロゲン原子
、水酸基、ニトロ基又はアルキル基、CORはアシル基
、nは1〜3の整数を示す。)で表されるアミンのアシ
ル誘導体のエナンチオマー混合物に、そのアミド結合を
不斉加水分解する能力を有する微生物或いは酵素を作用
させ、生成する一般式(II) ×1 (式中、X1% X2及びnは前記と同義)で表される
アミンの光学活性な(S)体と残存する一般式(I)で
表されるアミンのアシル誘導体の光学活性な(R)体と
を分離採取することを特徴とする光学活性アミン及びそ
の誘導体の製造方法に関する。これらの光学活性化合物
は各種医農薬の合成中間体として重要である。
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.

〔従来の技術及び発明が解決しようとする課題〕従来、
一般式(II)で表されるアミンの誘導体の光学活性体
を得る方法としてはジアステレオマー法により光学分割
剤を用いて光学分割する方法(特開昭59−10874
9号公報)、光学活性な2−ヒドロキシ−4−フェニル
ブタンから合成する方法(特開昭63−54351号公
報)等が知られているが、ジアステレオマー法では等モ
ルの別種光学活性化合物が必要であるという問題があり
、2−ヒドロキシ−4−フェニルブタンから合成する方
法では本光学活性体を得るのが困難であるという問題点
が有る。
[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.

〔課題を解決するための手段〕[Means to solve the problem]

係る状況に鑑み、本発明者らは経済的に優れ、かつ簡便
な方法で一般式(II)で表されるアミンの光学活性な
(S)体及び一般式(I)で表されるアミンのアシル誘
導体の光学活性な(R)体の製造法を開発すべく鋭意研
究した結果、特定の微生物或いは酵素が一般式(I)で
表されるアミンのアシル誘導体のエナンチオマー混合物
のアミド結合を不斉加水分解し、−船式(II)で表さ
れるアミンの光学活性な(S)体及び一般式(I)で表
されるアミンのアシル誘導体の光学活性な(Iり体を生
成しうろことを見出し本発明を完成したものである。
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.

即ち、本発明は、一般式(I)で表されるアミンのアシ
ル誘導体のエナンチオマー混合物に、そのアミド結合を
不斉加水分解する能力を有する微生物或いは酵素を作用
させ、生成する一般式(II)で表されるアミンの光学
活性な(S)体と残存する一般式(I)で表されるアミ
ンのアシル誘導体の光学活性な(R)体とを分離採取す
ることを特徴とする光学活性アミン及びその誘導体の製
造方法を提供するものである。
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.

本発明に用いられる一般式(I)で表されるアミンのア
シル誘導体に於いて、χ1、×2で表されるハロゲン原
子の具体例としては、CI、 Br。
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.

F、 1等、アルキル基の具体例としては、メチル基、
エチル基、プロピル基、イソプロピル基、ベンジル基等
を、また、CORで表されるアシル基の具体例としては
、アセチル基、プロピオニル基、ブチリル基等を挙げる
ことができる。
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.

また、本発明に於いて使用できる一般式(I)で表され
るアミンのアシル誘導体の具体例としてはN−アセチル
−2−アミノ−4−フェニルブタン等を挙げることがで
きる。
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.

次に本発明で使用できる微生物、或いは酵素としては、
−4式(I)で表されるアミンのアシル誘導体のエナン
チオマー混合物のアミド結合を不斉加水分解し一般式(
II)で表されるアミンの光学活性な(S)体及び一般
式(I)で表されるアミンのアシル誘導体の光学活性な
(R)体を生成しうるちのであればいずれも使用可能で
あるが、具体的にはロドコッカス(Rhodococc
us)属に属する微生物、或いはその微生物が生産する
酵素、更に具体的にはロドコッカス・エリスロポリス(
Rhodococcus erythropolis)
IFO12320、ロドコッカス・エリスロポリス(R
hodococcuserythropolis)IF
O12682、ロドコッカス・エクイ(Rhodoco
ccus equi)IFO3730+ JCM 13
11、ロドコッカス・ファシアンス(Rhodococ
cus facians:JCM 1316等の微生物
、或いはそれらの生産する酵素を挙げることができる。
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.

また、これらの変異株及びそれらの生産する酵素も用い
ることができる。
Moreover, these mutant strains and the enzymes produced by them can also be used.

尚、上記のうち、IFO番号の付された微生物は、(財
)醗酵研究所(rpo)発行のLi5t of Cu−
1tures、第8版、第1巻(I98B)に記載され
ており、該IFOから入手することができる。またJC
M番号の付された微生物は、理化学研究所発行の微生物
系統保存施設、微生物株カタログ。
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.

第3版(I986)に記載されており、理化学研究所か
ら入手することができる。
It is described in the 3rd edition (I986) and is available from RIKEN.

本発明に於いて用いられる微生物を培養する際の培地は
菌が増殖しうる栄養源を含む培地であれば、特に制限は
無い。例えば、炭素源としては、グルコース、シュクロ
ース等の糖類、エタノール、グリセロール等のアルコー
ル類、酢酸、プロピオン酸等の有機酸類、パラフィン等
の炭化水素類、またはこれらの混合物等、窒素1 源と
しては、硫酸アンモニウム、酵母エキス、尿素等の無機
及び有機含窒素化合物等が用いられる。この他、無機塩
、微量金属塩、ビタミン類等、通常の培養に用いられる
栄養源を適宜、混合して用いることができる。また必要
に応じて微生物の増殖を促進する因子或いは培地のpH
保持に有効な物質も添加できる。
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.

また、目的とする不斉加水分解酵素の生産量、或いは酵
素活性を向上させるのに有効な物質を添加して培養して
も良い。
In addition, a substance effective for improving the production amount or enzyme activity of the desired asymmetric hydrolase may be added to the culture.

培養方法としては、培地pHを3.0〜9,5、培養温
度を20〜45°Cの範囲とし、その微生物の生育に適
した酸素濃度条件下1〜5日間培養するのが好ましい。
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.

本発明に於いては、目的とする不斉加水分解反応が実質
的に完遂されるならば、酵素の存在形態はいかなるもの
でも良い。即ち、目的とする不斉加水分解活性を有する
菌体培養液、分離菌体、菌体処理物、及びこれらから分
離した、目的とする不斉加水分解活性を有する酵素を使
用することができる。
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.

基質として用いられるアミンのアシル誘導体は、その価
格の面でラセミ体を用いることが好ましいが、そのエナ
ンチオマー混合比率は特に限定されるものではなく、本
発明は該混合比率がいかなるものにも適用できる。
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. .

本発明に於いて微生物或いは酵素とアミン誘導体を反応
させる方法としては、(I)菌体培養液をそのまま用い
、該培養液にアミンのアシル誘導体を添加する方法、(
2)遠心分離等により分離した菌体をそのまま、或いは
洗浄した後、緩衝液、水等に再懸濁し、得られた懸濁液
にアミンのアシル誘導体を添加する方法、(3)菌体破
砕物、アセトン処理、凍結乾燥等による菌体処理物を用
いる方法、(4)これらの菌体を担体に固定化して用い
る方法、(5)菌体破砕物を遠心分離等にかけ、粗酵素
液を調製しこれにアミンのアシル誘導体を添加する方法
、(6)この粗酵素液から目的の酵素を公知の方法によ
り精製し、この酵素とアミンのアシル誘導体とを反応さ
せる方法、(7)この粗酵素或いは精製酵素を公知の方
法で固定化して用いる方法等、種々の方法が用いられる
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.

これらの方法においてはアミンのアシル誘導体は、その
まま、或いは、反応に影響を与えないような有機溶媒に
溶解したり、界面活性剤等に分散させたりして、反応の
始めから全て、あるいは分割して添加しても良い。反応
はpH3〜9の範囲で10〜60°Cの温度で行うのが
好ましく、1〜120時間、撹拌下で行う。基質の濃度
は特に制限されないが、0.1〜10%程度が好ましい
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%.

反応によって生成した光学活性なアミン及び残存するア
ミン誘導体の採取は、反応液から直接、あるいは菌体分
離後、有機溶媒で抽出し、カラムクロマトグラフィー、
蒸溜等の通常の精製方法を用いれば容易に行うことがで
きる。
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.

このようにして得られた一般式(II)で表されるアミ
ンの光学活性な(S)体及び一般式(I)で表されるア
ミンのアシル誘導体の光学活性な(I’り体はそのまま
或いは、目的により公知方法により種々の化学変換を行
った後、種々の合成中間体として利用することができる
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.

また一般式(I)で表されるアミンのアシル誘導体の光
学活性な(R)体を常法により化学的に加水分解する(
酸あるいはアルカリ水溶液中でリフラックスする)こと
により、一般式(II)で表されるアミンの光学活性な
(R)体を得ることもできる。
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.

(実施例) 以下、実施例にて本発明を具体的に説明するが、本発明
はこれらに限定されるものではない。
(Examples) Hereinafter, the present invention will be specifically explained using Examples, but the present invention is not limited thereto.

なお、実施例に於ける、アミン及びアミン誘導体の定量
分析は高速液体クコマドグラフィーCカラム: Uni
sil Pack 5C+a−25OA(ガスクロエ業
■製)、溶媒: 40mMリン酸緩衝液(p++3.5
)/アセトニトリルー6;4〕により行った。またこれ
らの化合物の光学純度は、反応で生成したアミンの場合
は定法によりアセチル化した後、残存するアミン誘導体
の場合はそのまま、光学分割カラムを用いた高速液体ク
ロマトグラフィー〔カラム;キラルセルOB (ダイセ
ル化学工業■製)、溶媒:n−ヘキサン/イソプロパノ
−ルー95:5)により求めた。
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).

実施例1 グルコース1%、酵母エキス0.5%、ポリペプトン0
.5%、塩化ナトリウム0.3%、N−アセチル−2−
アミノ−4−フェニルブタンのラセミ体0.1%より成
る組成の培地50−を50〇−容坂ロフラスコに入れ、
滅菌後、ロドコッカス−エIJ 7!i t:lボIJ
 7. IF012320を植菌し、30°Cで2日間
往復振盪培養を行った。
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.

培養終了後、遠心分離により菌体を分離、生理食塩水で
一回洗浄し生菌体を得た。
After the culture was completed, the bacterial cells were separated by centrifugation and washed once with physiological saline to obtain viable bacterial cells.

次に、500 mZ容坂ロフラスコに蒸溜水50−を入
れ、これに上記の菌体を懸濁させた。
Next, 50 mm of distilled water was put into a 500 mZ Sakaro flask, and the above-mentioned bacterial cells were suspended therein.

しかる後、N−アセチル−2−アミノ−4−フェニルブ
タンのラセミ体を0.5 g添加し30”Cで往復振盪
し、24時間反応させた。
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.

反応終了後、遠心分離にて菌体を除去し、得られた上澄
液について生成した(S) −2−アミノ−4−フェニ
ルブタンと残存する(R)−N−アセチル−2−アミノ
−4−フェニルブタンを定量分析し反応収率を求めた。
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.

また、上澄液の1−にローヘキサン10−を加え(R)
−N−アセチル−2−アミノ−4−フェニルブタンを抽
出し、キラルセルOBにより光学純度を求めた。また、
この水層にカセイソーダ水溶液を加えアルカリ性にした
後、同様にn−ヘキサン10m7を加え(S) −2−
アミノ−4−フェニルブタンを抽出した。しかる後、得
られた (S)−2−アミノ−4−フェニルブタンを定
法により塩化アセチルによりアセチル化した後、上記と
同様に光学純度を求めた。
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.

得られた結果を表1に示した。The results obtained are shown in Table 1.

表      1 実施例2 実施例1で使用した培地22を含む51ジャーファーメ
ンタ−に、実施例1と同様にして培養したロドコッカス
・エリスロポリスIF012682の前培養液100@
lを植菌し、30″Cで撹拌速度400rp+n 、通
気量0.5vvmにて30時間通気撹拌培養した。
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.

培養終了後、遠心分離にて集菌し、水500 @Zにて
菌体を洗浄した。しかる後、この菌体を21容三角フラ
スコに入れ、50mMリン酸緩衝液50〇−を加え菌体
を懸濁させた。しかる後、N−アセチル−2−アミノ−
4−フェニルブタンのラセミ体を15g添加し30“C
で回転振盪し、24時間反応させた。
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.

反応終了後、遠心分離にて菌体を分離し、この上澄液を
ロータリーエバポレーターにて濃縮し200−とじた。
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.

次いで酢酸エチル200 @1を加え振盪し抽出操作を
行った。この操作を2回行った後、酢酸エチル層を合わ
せ、次いでロータリーエバポレーターにて脱溶剤しオイ
ル状の(R)−N−アセチル−2−アミノ−4−フェニ
ルブタン6.1gを得た。このサンプルを前記の液体ク
ロマトグラフィー法により分析したところ、化学純度は
97.7%、光学純度は99.8%e、e、であった。
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.

次に残りの水層に10%カセイソーダ水溶液を加えpH
10にした後、前記の方法と同様に酢酸エチルで抽出操
作を行いオイル状の(S) −2−アミノ−4−フェニ
ルブタン5.3gを得た。このサンプルを前記の場合と
同様に液体クロマトグラフィー法により分析したところ
、化学純度は96.8%、光学純度は99.5%e、e
、であった。
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.

実施例3 実施例2の場合と同様にロドコッカス・エリスロポリス
IF012682を培養した後、遠心分離により、菌体
を得た。この湿菌体20gを50mMリン酸緩衝液(p
H7,0) 50aIに懸濁し水冷下、超音波破砕処理
を行い酵素の抽出を行った。次いで、遠心分離により菌
体の破片を除き粗酵素液40m/ヲ得た。この粗酵素液
にN−アセチル−2−アミノ−4−フェニルブタンのラ
セミ体を2g添加し30℃で回転振盪し、24時間反応
させた。
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.

この反応液から実施例2の場合と同様に酢酸エチルを用
いて抽出操作を行い、生成した(S)−2−アミノ−4
−フェニルブタン0.72gと残存した(R)−N−ア
セチル−2−アミノ−4−フェニルブタン0.85gを
得た。得られた(S) −2−アミノ−4−フェニルブ
タンの化学純度は96.8%、光学純度は99.5%e
、e、であった。また、(R)−N−アセチル−2−ア
ミノ−4−フェニルブタンの化学純度は97.6%、光
学純度は99.3%e、e、であった。
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.

実施例4 実施例2で得られた(R)−N−アセチル−2−アミノ
−4−フェニルブタン5.5gを6Mの塩酸55m7に
溶解し、缶温109〜110″Cで20時間還流した。
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. .

その結果、(R)−N−アセチル−2−アミノ−4−フ
ェニルブタンは89%加水分解された。
As a result, (R)-N-acetyl-2-amino-4-phenylbutane was 89% hydrolyzed.

この加水分解液から実施例2の場合と同様に酢酸エチル
を用いて抽出操作を行い、(R) −2−アミノ−4−
フェニルブタン3.5gを得た。
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.

得られた(R) −2−アミノ−4−フェニルブタンの
化学純度は97.2%、光学純度は99.5%e、e。
The chemical purity of the obtained (R)-2-amino-4-phenylbutane was 97.2%, and the optical purity was 99.5%e,e.

であった。Met.

〔発明の効果〕〔Effect of the invention〕

本発明の微生物あるいはその生産する酵素を用いた不斉
加水分解法による光学活性なアミン及びアミン誘導体の
製造方法は光学純度の高いこれらの化合物を簡便に製造
できることを可能にさせるものであり、工業的製造方法
として掻めて有利である。
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)

【特許請求の範囲】 1、一般式( I ) ▲数式、化学式、表等があります▼・・・( I ) (式中、X_1、X_2はそれぞれ水素原子、ハロゲン
原子、水酸基、ニトロ基又はアルキル基、CORはアシ
ル基、nは1〜3の整数を示す。)で表されるアミンの
アシル誘導体のエナンチオマー混合物に、そのアミド結
合を不斉加水分解する能力を有する微生物を作用させ、
生成する一般式(II) ▲数式、化学式、表等があります▼・・・(II) (式中、X_1、X_2及びnは前記と同義)で表され
るアミンの光学活性な(S)体と残存する一般式( I
)で表されるアミンのアシル誘導体の光学活性な(R)
体とを分離採取することを特徴とする光学活性アミン及
びその誘導体の製造方法。 2、一般式( I ) ▲数式、化学式、表等があります▼・・・( I ) (式中、X_1、X_2はそれぞれ水素原子、ハロゲン
原子、水酸基、ニトロ基又はアルキル基、CORはアシ
ル基、nは1〜3の整数を示す。)で表されるアミンの
アシル誘導体のエナンチオマー混合物に、そのアミド結
合を不斉加水分解する能力を有する酵素を作用させ、生
成する一般式(II) ▲数式、化学式、表等があります▼・・・(II) (式中、X_1、X_2及びnは前記と同義)で表され
るアミンの光学活性な(S)体と残存する一般式( I
)で表されるアミンのアシル誘導体の光学活性な(R)
体とを分離採取することを特徴とする光学活性アミン及
びその誘導体の製造方法。 3、微生物がロドコッカス(Rhodococcus)
属に属する微生物である請求項1記載の製造方法。 4、酵素がロドコッカス(Rhodococcus)属
に属する微生物が生産する酵素である請求項2記載の製
造方法。 5、一般式(II)で表されるアミンが2−アミノ−4−
フェニルブタンである請求項1又は2記載の製造方法。
[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.
JP25538488A 1988-10-11 1988-10-11 Method for producing optically active amines and derivatives thereof Expired - Fee Related JP2698627B2 (en)

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JPH02104295A true JPH02104295A (en) 1990-04-17
JP2698627B2 JP2698627B2 (en) 1998-01-19

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0569555U (en) * 1992-02-26 1993-09-21 日立化成工業株式会社 Automatic bath equipment
WO1996027022A1 (en) * 1995-03-02 1996-09-06 Bayer Aktiengesellschaft Process for preparing optically active amines

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4984925B2 (en) * 2007-01-31 2012-07-25 住友化学株式会社 Aminoacylase gene

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0569555U (en) * 1992-02-26 1993-09-21 日立化成工業株式会社 Automatic bath equipment
WO1996027022A1 (en) * 1995-03-02 1996-09-06 Bayer Aktiengesellschaft Process for preparing optically active amines

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