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KR100688770B1 - Method for preparing an optically active R-2-amino-1-butanol by enzymatic method - Google Patents

Method for preparing an optically active R-2-amino-1-butanol by enzymatic method Download PDF

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KR100688770B1
KR100688770B1 KR1020020071243A KR20020071243A KR100688770B1 KR 100688770 B1 KR100688770 B1 KR 100688770B1 KR 1020020071243 A KR1020020071243 A KR 1020020071243A KR 20020071243 A KR20020071243 A KR 20020071243A KR 100688770 B1 KR100688770 B1 KR 100688770B1
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김종근
조남륜
전선영
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에스케이 주식회사
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Abstract

본 발명은 효소적 방법에 의한 광학활성 (R)-2-아미노-1-부탄올 ((R)-2-amino-1-butanol)의 제조방법에 관한 것으로, 좀 더 상세하게는 수용액상에서 라세믹체로 존재하는 2-아미노-1-부탄올의 에스테르 화합물을 가수분해 효소를 이용하여 선택적으로 가수분해하는 효소적 방법에 의한 광학활성 (R)-2-아미노-1-부탄올의 제조방법에 관한 것이다. 본 발명의 제조 방법은 가수분해 효소를 사용하여 간단한 방법으로 높은 광학순도를 갖는 광학활성 화합물을 제조할 수 있어 산업상 매우 유용하다.The present invention relates to a method for preparing optically active (R) -2-amino-1-butanol ((R) -2-amino-1-butanol) by enzymatic method, more specifically racemic in aqueous solution The present invention relates to a method for producing optically active (R) -2-amino-1-butanol by an enzymatic method of selectively hydrolyzing an ester compound of 2-amino-1-butanol present in a sieve using a hydrolase. The production method of the present invention is very useful in the industry because it is possible to produce an optically active compound having a high optical purity by a simple method using a hydrolase.

2-아미노-1-부탄올, 라세믹체, 가수분해, 가수분해 효소, 프로테아제, 에스테라제, 리파제, L-(+)-타르타르산2-amino-1-butanol, racemic body, hydrolysis, hydrolase, protease, esterase, lipase, L-(+)-tartaric acid

Description

효소적 방법에 의한 광학활성 (R)-2-아미노-1-부탄올의 제조방법{Method for preparing an optically active (R)-2-amino-1-butanol by enzymatic method}Method for preparing an optically active (R) -2-amino-1-butanol by enzymatic method}

본 발명은 효소적 방법에 의한 광학활성 (R)-2-아미노-1-부탄올의 제조방법에 관한 것으로, 좀 더 상세하게는 라세믹 2-아미노-1-부탄올의 에스테르 화합물을 버퍼(buffer)용액에 용해시킨 후, 미생물 유래의 가수분해 효소를 작용시켜 하나의 에스테르를 선택적으로 가수분해시키는 효소적 방법에 의한 광학활성 (R)-2-아미노-1-부탄올의 제조방법에 관한 것이다. The present invention relates to a method for preparing optically active (R) -2-amino-1-butanol by enzymatic method, and more particularly, to buffer an ester compound of racemic 2-amino-1-butanol. After dissolving in a solution, it relates to a method for producing optically active (R) -2-amino-1-butanol by an enzymatic method of selectively hydrolyzing one ester by reacting a hydrolytic enzyme derived from a microorganism.

라세믹 2-아미노-1-부탄올은 (S)-2-아미노-1-부탄올과 (R)-2-아미노-1-부탄올이 반반씩 존재하고 있다. 이중 (S)-2-아미노-1-부탄올은 결핵치료제인 에탐부톨(Ethambutol)의 중간체로 사용되고 있으며, (R)-2-아미노-1-부탄올 또한 의약용 중간체로 유용할 것으로 판단된다. In racemic 2-amino-1-butanol, (S) -2-amino-1-butanol and (R) -2-amino-1-butanol are present in half and half. Of these, (S) -2-amino-1-butanol is used as an intermediate of Ethambutol, an anti-tuberculosis drug, and (R) -2-amino-1-butanol may also be useful as a pharmaceutical intermediate.

라세믹 2-아미노-1-부탄올로부터 (S)-2-아미노-1-부탄올을 제조하는 방법은 많이 알려져 있다. 예를 들어, 아메리칸 시아나미드(American Cyanamid)사가 1976년 발표한 미국특허 제3,944,619호에는 L-(+)-타르타르산을 이용하여 라세믹 2-아미노-1-부탄올로부터 (S)-2-아미노-1-부탄올을 제조하는 방법이 기재되어 있다. 그 러나 이 방법은 재결정 방법이 복잡할 뿐만 아니라, 수율이 낮고, L-(+)-타르타르산과 (S)-2-아미노-1-부탄올 타르타르산염의 용해도 차가 크지 않아 두 물질을 분리하기 어려운 단점이 있다. 또한, 타르타르산염은 흡습성이 매우 강하여 다루기 어려운 단점이 있다. There are many known methods for producing (S) -2-amino-1-butanol from racemic 2-amino-1-butanol. For example, U.S. Patent No. 3,944,619, published in 1976 by American Cyanamid, describes the use of L-(+)-tartaric acid from (S) -2-amino from racemic 2-amino-1-butanol. A method for preparing -1-butanol is described. However, this method is not only difficult to recrystallize, but also has a low yield, and it is difficult to separate the two materials due to the low solubility difference between L-(+)-tartaric acid and (S) -2-amino-1-butanol tartarate. have. In addition, tartarate has a disadvantage in that it is very hygroscopic and difficult to handle.

니폰 소다(Nippon Soda)사의 영국 공개특허 제1471838호에는 d-N-벤조일-트랜스-2-아미노사이클로헥산 카르복실산(d-N-benzoyl-trans-2-aminocyclohexane carboxylic acid)과 l-N-벤조일-트랜스-2-아미노사이클로헥산 카르복실산(l-N-benzoyl-trans-2-aminocyclohexane carboxylic acid)을 이용하여 라세믹 2-아미노-1-부탄올로부터 각각의 광학활성 이성질체인 (S)-2-아미노-1-부탄올과 (R)-2-아미노-1-부탄올을 제조하는 방법이 기재되어 있다. 그러나 이 방법 역시 약 10여 단계에 이르는 복잡한 재결정 과정을 반복해야 하므로 효율성이 떨어지며, 그 결과 만들어지는 (S)-2-아미노-1-부탄올 및 (R)-2-아미노-1-부탄올의 광학순도가 92∼94%ee로서 광학순도가 높지 않은 단점이 있다.Nippon Soda's U.S. Patent Publication No. 1471838 discloses dN-benzoyl-trans-2-aminocyclohexane carboxylic acid and lN-benzoyl-trans-2- (S) -2-amino-1-butanol, which is an optically active isomer from racemic 2-amino-1-butanol, with aminocyclohexane carboxylic acid (lN-benzoyl-trans-2-aminocyclohexane carboxylic acid); A method for preparing (R) -2-amino-1-butanol is described. However, this method also requires less than about 10 complex recrystallization steps, resulting in inefficiency, resulting in optical (S) -2-amino-1-butanol and (R) -2-amino-1-butanol. There is a disadvantage that the optical purity is not high as 92-94% ee.

한편, 상기 공정들과는 다르게, 리파제 효소를 사용하여 라세믹 화합물을 분리하는 공정이 개발되었다. 프랜칼란시(Francalanci) 및 쎄스티(Cesti) 등은 라세믹 2-아미노-1-부탄올로부터 라세믹 2-아미노-N-에톡시카보닐-1-부탄올 (2-amino-N-ethoxycarbonyl-1-butanol)을 합성한 후, 돼지 췌장 유래의 리파제 (Porcine pancreatic lipase)를 이용하여 에틸 아세테이트(ethyl acetate)와 반응시켜 (S)-2-아미노-N-에톡시카보닐-1-부탄올 및 (R)-2-아미노-N-에톡시카보닐-1-부탄올 아세테이트를 합성한 후, 이로부터 (S)-2-아미노-1-부탄올 및 (R)-2-아미노- 1-부탄올을 제조하였다. (S)-2-아미노-1-부탄올의 광학순도는 98.4%ee, (R)-2-아미노-1-부탄올의 광학순도는 60%ee를 나타내었다. 이때, 효소의 기질을 라세믹 2-아미노-N-벤질옥시카보닐-1-부탄올로 변경하여 반응한 결과 생성된 (R)-2-아미노-1-부탄올의 광학순도는 90.4%ee를 나타내었다(유럽 공개특허 제0222561호, J. Org. Chem.,52(23), pp.5079-82,1987).On the other hand, unlike the above processes, a process for separating racemic compounds using lipase enzymes has been developed. Francalanci and Cesti et al. Describe racemic 2-amino-N-ethoxycarbonyl-1-butanol from racemic 2-amino-1-butanol (2-amino-N-ethoxycarbonyl-1). -butanol) and then reacted with ethyl acetate using Porcine pancreatic lipase (S) -2-amino-N-ethoxycarbonyl-1-butanol and ( R) -2-amino-N-ethoxycarbonyl-1-butanol acetate was synthesized and then (S) -2-amino-1-butanol and (R) -2-amino-1-butanol were prepared therefrom It was. The optical purity of (S) -2-amino-1-butanol was 98.4% ee, and the optical purity of (R) -2-amino-1-butanol was 60% ee. At this time, the optical purity of the (R) -2-amino-1-butanol produced as a result of the reaction of the enzyme substrate to racemic 2-amino-N-benzyloxycarbonyl-1-butanol was 90.4% ee. (Ep. 0222561, J. Org. Chem., 52 (23), pp. 5079-82,1987).

또한, 프랜칼란시(Francalanci) 및 쎄스티(Cesti) 등은 라세믹 2-아미노-N-에톡시카보닐-1-부탄올 아세테이트를 역시 돼지 췌장 유래의 리파제를 이용하여 선택적으로 가수분해하여 (S)-2-아미노-1-부탄올과 (R)-2-아미노-1-부탄올을 제조하는 방법도 발표하였는데, 이때 (S)-2-아미노-1-부탄올의 광학순도는 99.2%ee, (R)-2-아미노-1-부탄올의 광학순도는 75%ee를 나타내었다(유럽 공개특허 제0239122호, J. Org. Chem.,52(23), pp.5079-82,1987).In addition, Francalanci and Cesti et al. Selectively hydrolyze racemic 2-amino-N-ethoxycarbonyl-1-butanol acetate using a lipase derived from porcine pancreas (S A method of preparing 2-amino-1-butanol and (R) -2-amino-1-butanol was also disclosed, wherein the optical purity of (S) -2-amino-1-butanol was 99.2% ee, ( The optical purity of R) -2-amino-1-butanol showed 75% ee (European Patent No. 0239122, J. Org. Chem., 52 (23), pp. 5079-82,1987).

그 외, 일본의 치소(Chisso)사는 아미노아실라제(aminoacylase) 보유 미생물을 이용한 (S)-2-아미노-1-부탄올의 제조방법을 개발하였고(일본 공개특허 제58-198296호), 베비나카티(Bevinakatti) 등은 리파제(Porcine pancreatic lipase)를 이용하여 라세믹 N,O-디아세틸-2-아미노-1-부탄올(N,O-diacetyl-2-amino-1-butanol)을 트랜스에스테르화 반응시켜 광학활성 2-아미노-1-부탄올을 제조하는 방법을 개발하였으며(Tetrahedron: Asymmetry, 1(9), 583-6, 1990), 고터(Gotor) 등은 리파제(Porcine pancreatic lipase)를 이용하여 라세믹 2-아미노-1-부탄올을 아실화 반응시켜 광학활성 2-아미노-1-부탄올을 제조하는 방법을 개발하였다(J. Chem. Soc., Perkin Trans. 1, 21, 2885-9, 1992). In addition, Chisso of Japan has developed a method for producing (S) -2-amino-1-butanol using aminoacylase-containing microorganisms (Japanese Patent Laid-Open No. 58-198296), Bevinakatti et al. Transesterified racemic N, O-diacetyl-2-amino-1-butanol using phosphase pancreatic lipase. The reaction method was developed to produce optically active 2-amino-1-butanol (Tetrahedron: Asymmetry, 1 (9), 583-6, 1990), and Gotor was prepared using lipase (Porcine pancreatic lipase). A method of preparing optically active 2-amino-1-butanol by acylating racemic 2-amino-1-butanol (J. Chem. Soc., Perkin Trans. 1, 21, 2885-9, 1992 ).                         

상기에 언급된 방법들은 모두 결핵치료제인 에탐부톨의 중간 원료인 (S)-2-아미노-1-부탄올을 제조하는 방법들이며, (R)-2-아미노-1-부탄올을 고순도로 제조하는 방법은 발표된 바가 없다. 위에서 언급한 기술 중 니폰 소다(Nippon Soda)사의 기술과 프랜칼란시(Francalanci)사 등이 발표한 기술(유럽 공개특허 제0222561호)에서 (R)-2-아미노-1-부탄올은 90% 이상의 광학순도로 얻어질 수 있지만, 실제 의약 중간체로 사용하기에는 광학순도가 너무 낮은 단점이 있다.The above-mentioned methods are all methods for preparing (S) -2-amino-1-butanol, which is an intermediate raw material of etabutol, a tuberculosis treatment agent, and for preparing (R) -2-amino-1-butanol with high purity. No announcement was made. Among the above-mentioned technologies, (R) -2-amino-1-butanol is more than 90% in Nippon Soda and the technology published by Francalanci (European Patent No. 0222561). Although it can be obtained with optical purity, there is a disadvantage that the optical purity is too low for use as an actual pharmaceutical intermediate.

따라서, 상기와 같은 이유로 간단한 공정을 이용하여 경제적으로 우수하며, 98% 이상의 광학순도가 높은 광학활성 (R)-2-아미노-1-부탄올을 생산할 수 있는 공정의 개발이 필요하다. Therefore, there is a need for the development of a process that is economically superior using a simple process and can produce optically active (R) -2-amino-1-butanol having a high optical purity of 98% or more.

이에, 본 발명자들은 적합한 효소 또는 미생물로 라세믹 2-아미노-1-부탄올의 에스테르 화합물을 처리하면, 높은 광학순도의 (R)-2-아미노-1-부탄올을 제조할 수 있음에 착안하여 본 발명을 완성하였다. Accordingly, the present inventors pay attention to the fact that by treating the ester compound of racemic 2-amino-1-butanol with a suitable enzyme or microorganism, it is possible to prepare (R) -2-amino-1-butanol of high optical purity. The invention has been completed.

따라서, 본 발명의 목적은 가수분해 효소를 이용하여 라세믹 2-아미노-1-부탄올의 에스테르 화합물로부터 광학활성 (R)-2-아미노-1-부탄올을 제조하는 방법을 제공하는데 있다.It is therefore an object of the present invention to provide a method for producing optically active (R) -2-amino-1-butanol from ester compounds of racemic 2-amino-1-butanol using a hydrolase.

상기 목적을 달성하기 위한 본 발명의 제조방법은 pH 6∼9의 버퍼용액 존재하에서, 미생물 유래의 가수분해 효소를 이용하여 라세믹 2-아미노-1-부탄올의 에스테르 화합물을 선택적으로 가수분해시키는 것으로 구성된다.The production method of the present invention for achieving the above object is to selectively hydrolyze the ester compound of racemic 2-amino-1-butanol in the presence of a buffer solution of pH 6-9 using a microbial hydrolase It is composed.

이하 본 발명을 좀 더 구체적으로 살펴보면 다음과 같다.Looking at the present invention in more detail as follows.

전술한 바와 같이, 본 발명에서는 기질인 라세믹 N,O-디아세틸-2-아미노-1-부탄올(N,O-diacetyl-2-amino-1-butanol)을 pH 6∼9의 버퍼용액에 적절히 용해시킨 후, 효소와 기질의 비율을 1 : 10 ∼ 10 : 1로 하여 리조퍼스(Rhizopus) 속 또는 캔디다(Candida) 속 미생물 유래의 가수분해 효소를 별도의 처리 공정 없이 첨가한 다음, 상기 반응 혼합물을 20∼40℃의 온도에서 100∼300rpm으로 교반시켜 (S)-이성질체만을 선택적으로 가수분해시킴으로서 광학활성을 갖는 (R)-2-아미노-1-부탄올을 제조한다.As described above, in the present invention, racemic N, O-diacetyl-2-amino-1-butanol (N, O-diacetyl-2-amino-1-butanol) as a substrate is added to a buffer solution of pH 6-9. After appropriate dissolution, the ratio of enzyme and substrate is 1: 10 to 10: 1, and hydrolysis enzymes derived from Rhizopus genus or Candida genus microorganisms are added without a separate treatment step. The mixture is stirred at 100 to 300 rpm at a temperature of 20 to 40 ° C. to selectively hydrolyze only the (S) -isomer to produce (R) -2-amino-1-butanol having optical activity.

본 발명의 반응 공정은 하기 반응식 1과 같다.The reaction process of the present invention is shown in Scheme 1 below.

Figure 112002037817732-pat00001
Figure 112002037817732-pat00001

본 발명에 사용되는 가수분해 효소는 리조퍼스(Rhizopus) 속 또는 캔디다 (Candida) 속 미생물로부터 얻어지며, 상기 효소는 분말이나 액상 또는 담체로 고정화된 것 또는 효소를 함유하는 생물세포 및 고정화된 생물세포이다.The hydrolase used in the present invention is obtained from microorganisms of genus Rhizopus or Candida, which enzymes are immobilized in powder, liquid or carrier, or biological cells containing enzymes and immobilized biological cells. to be.

상기 리조퍼스(Rhizopus) 속 미생물로는 리조퍼스 오리재(Rhizopus oryzae) 가 있으며, 캔디다(Candida) 속 미생물로는 캔디다 루고사(Candida rugosa)가 바람직하다.The microorganism of the genus Rizopus (Rhizopus) is the Rhizopus oryzae , Candida (Candida) is a Candida rugosa (Candida rugosa ) is preferred.

상기 가수분해 효소는 상업적으로 판매되는 것을 사용하거나 제조하여 사용할 수 있다. 상업적으로 판매되는 효소는 예를 들어, 아마노(Amano)사의 Ppeptidase R(프로테아제)과 유로파(Europa)사의 Europa Esterase 2(Cr/F5)가 있으나, 이에 한정되는 것은 아니다. The hydrolase may be used commercially available or manufactured. Commercially available enzymes include, but are not limited to, Ppeptidase R (protease) from Amano and Europa Esterase 2 (Cr / F5) from Europa.

본 발명에 있어서, 효소 반응 온도는 선택된 효소의 활성온도에 따라 변화되므로 특별히 제한 받지는 않으나, 바람직한 반응 온도는 20∼40℃가 적당하며, 상기 온도가 20℃ 미만이면 반응 속도가 너무 늦어지는 단점이 있고, 40℃를 초과하면 광학순도가 떨어지는 문제가 있다. 또한, 효소 반응시의 pH는 pH 6∼9의 범위가 적당하며, 상기 범위를 벗어날 경우 반응 속도 및 광학순도가 떨어지는 문제점이 있고, 가수분해 반응시 효소와 기질의 비율은 1 : 10 ∼ 10 : 1이 적당하며, 상기 효소와 기질의 비율이 상기 범위를 벗어나면 반응 속도가 느려지거나 사용되는 효소의 양이 너무 많아 경제성이 떨어지는 문제점이 있다. In the present invention, the enzyme reaction temperature is not particularly limited because it is changed depending on the activity temperature of the selected enzyme, the preferred reaction temperature is 20 to 40 ℃ suitable, if the temperature is less than 20 ℃ reaction rate is too slow There exists a problem that optical purity falls when it exceeds 40 degreeC. In addition, the pH of the enzyme reaction is in the range of pH 6 ~ 9 is appropriate, if there is a problem that the reaction rate and optical purity is lowered, the ratio of enzyme and substrate during the hydrolysis reaction is 1: 10 ~ 10: 1 is appropriate, and the ratio of the enzyme and the substrate is out of the range, there is a problem that the reaction rate is slow or the amount of the enzyme used is too large and economic efficiency is low.

본 발명에 따라 제조된 광학 활성의 N,O-디아세틸-2-아미노-1-부탄올의 분석 조건 및 방법은 기체크로마토그래피를 이용하여 아래와 같이 분석할 수 있다.Analysis conditions and methods of optically active N, O-diacetyl-2-amino-1-butanol prepared according to the present invention can be analyzed using gas chromatography as follows.

수펠코사의 키랄 칼럼인 CP-Chirasil-Dex CB 칼럼(길이 25m, 내경 0.25mm)을 100℃에서 5분간 가열하고, 5℃/분의 속도로 200℃까지 증가시킨 후, 200℃에서 15분간 정지시킨다. 담체(carrier)로 헬륨기체를 1.4㎖/분의 속도로 흘리고, 250℃에서 FID(Flame Ionization Detector)를 사용하여 검출하면, (S)-N,O-디아세틸-2-아미노-1-부탄올은 19.3분, (R)-N,O-디아세틸-2-아미노-1-부탄올은 19.5분에서 검출된다. 가수분해 반응의 결과 생성된 N-아세틸-2-아미노-1-부탄올의 경우 20.1분에서 검출되며, 이 경우 (S)-이성질체와 (R)-이성질체가 구별되지는 않는다.The CP-Chirasil-Dex CB column (length 25m, inner diameter 0.25mm), a Sufelco company chiral column, was heated at 100 ° C for 5 minutes, increased to 200 ° C at a rate of 5 ° C / min, and then stopped at 200 ° C for 15 minutes Let's do it. When helium gas was flowed at a rate of 1.4 ml / min as a carrier and detected using a Flame Ionization Detector (FID) at 250 ° C., (S) -N, O-diacetyl-2-amino-1-butanol Silver is detected at 19.3 minutes and (R) -N, O-diacetyl-2-amino-1-butanol at 19.5 minutes. N-acetyl-2-amino-1-butanol produced as a result of the hydrolysis reaction is detected at 20.1 minutes, in which case the (S) -isomer and the (R) -isomer are not distinguished.

이하 실시 예를 통하여 본 발명을 좀 더 구체적으로 설명하지만 하기 예에 본 발명이 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

실시예 1 Example 1

50g의 2-아미노-1-부탄올을 5ℓ의 디클로로메탄에 녹인 혼합물에 114g의 트리메틸아민 및 115g의 무수초산을 차례로 넣어준 후 상온에서 교반하였다. 반응이 끝나면 물을 넣고 추출하고, 유기층에 MgSO4를 넣어 상기 물을 제거한 다음 여과하였다. 여과액을 증류시킨 후 87.2g의 흰색 고체인 라세믹 N,O-디아세틸-2-아미노-1-부탄올을(수율 92%)을 회수하였다. To a mixture of 50 g of 2-amino-1-butanol dissolved in 5 L of dichloromethane were added 114 g of trimethylamine and 115 g of acetic anhydride in order, followed by stirring at room temperature. After the reaction, water was added and extracted, MgSO 4 was added to the organic layer to remove the water and filtered. After distilling the filtrate, 87.2 g of a white solid, racemic N, O-diacetyl-2-amino-1-butanol (yield 92%) was recovered.

실시예 2Example 2

100mM 인산용액(pH 7.0) 5㎖에 실시예 1에서 얻은 라세믹 N,O-디아세틸-2-아미노-1-부탄올 0.1g을 넣고 pH를 7.0으로 조정한 다음, 미생물 또는 동물 유래의 가수분해 효소 0.5g을 넣고 30℃에서 반응을 수행하였다. 반응액 0.5㎖를 취해 1㎖의 에틸 아세테이트로 반응물을 추출해 위에서 언급한 분석방법으로 기체크로마토그래피에서 분석한 결과 하기 표 1과 같은 결과를 얻었다. 5 ml of 100 mM phosphoric acid solution (pH 7.0) was added 0.1 g of racemic N, O-diacetyl-2-amino-1-butanol obtained in Example 1, and the pH was adjusted to 7.0. 0.5 g of enzyme was added and the reaction was carried out at 30 ° C. 0.5 ml of the reaction solution was extracted with 1 ml of ethyl acetate and analyzed by gas chromatography using the aforementioned analytical method.

효소enzyme 미생물microbe 광학순도(%ee)Optical purity (% ee) N,O-디아세틸-2-아미노 -1-부탄올의 절대배열Absolute configuration of N, O-diacetyl-2-amino-1-butanol 비 고Remarks 프로테아제Protease Rhizopusoryzae Rhizopus oryzae 100100 SS Aspergillusniger Aspergillus niger 00 100% 가수분해100% hydrolysis Aspergillusoryzae Aspergillus oryzae 00 100% 가수분해100% hydrolysis 리파아제Lipase Pseudomonascepacia Pseudomonas cepacia 00 100% 가수분해100% hydrolysis Candida antarctica Candida a ntarctica 00 100% 가수분해100% hydrolysis Alcaligenes sp.Alcaligenes sp. 00 100% 가수분해100% hydrolysis 에스테라제Esterase Candidarugosa Candida rugosa 91.291.2 SS

상기 표 1에 나타낸 바와 같이, 리조퍼스 오리재(Rhizopus oryzae) 유래의 프로테아제 및 캔디다 루고사(Candida rugosa) 유래의 에스테라제는 (S)-이성질체 만을 선택적으로 가수분해시킨 반면, 그 외의 가수분해 효소는 선택성이 없었다. 또한, 상기 표 1에 표시된 효소 외에 약 60종의 가수분해 효소를 이용하여 실험을 하였으나 대부분 선택성이 없는 것으로 나타났다.As shown in Table 1, protease from Rhizopus oryzae and esterase from Candida rugosa selectively hydrolyzed only the (S) -isomer, while other hydrolysis was performed. The enzyme was not selective. In addition, experiments were carried out using about 60 hydrolytic enzymes in addition to the enzymes shown in Table 1, but most of them were found to have no selectivity.

실시예 3Example 3

실시예 2에서 선정된 효소 중 리조퍼스 오리재(Rhizopus oryzae) 유래의 프로테아제를 이용하여 다음과 같은 반응을 수행하였다. 100mM 인산용액(pH 7.0) 400㎖에 라세믹 N,O-디아세틸-2-아미노-1-부탄올 40g(10% w/v)을 넣고 pH를 7.0으로 조정한 다음, 상기 프로테아제 40g을 넣은 후 30℃에서 반응을 수행하였다. 반응 중의 pH는 20% 수산화나트륨용액을 이용하여 6.5∼7.5로 유지시켰다. 일정 시간마다 반응 샘플을 채취하여 반응 진행 상황을 분석하였다. 47시간 반응 후 (R)-N,O-디아세틸-2-아미노-1-부탄올의 광학순도가 98.0%ee 이상 도달하였을 때, 반응을 중단시킨 후 반응액에 디클로로메탄(CH2Cl2)을 첨가하여 가수분해되지 않고 남아있는 N,O-디아세틸-2-아미노-1-부탄올을 추출하였다. 상기 추출과정을 4회 반복하여 유기 용매층을 따로 분리하고 감압증류로 디클로로메탄을 제거한 결과, 12.5g의 (R)-N,O-디아세틸-2-아미노-1-부탄올(수율 31.3%)을 회수하였다. 이 중 일부를 칼럼으로 정제한 후 (R)-N,O-디아세틸-2-아미노-1-부탄올의 광학순도를 측정한 결과 99.1%이었다. Among the enzymes selected in Example 2, the following reaction was performed using a protease derived from Rhizopus oryzae . 400 ml (10% w / v) of racemic N, O-diacetyl-2-amino-1-butanol was added to 400 ml of 100 mM phosphoric acid solution (pH 7.0), pH was adjusted to 7.0, and then 40 g of the protease was added thereto. The reaction was carried out at 30 ° C. The pH during the reaction was maintained at 6.5-7.5 using 20% sodium hydroxide solution. Reaction samples were taken at regular intervals to analyze the reaction progress. After 47 hours of reaction, when the optical purity of (R) -N, O-diacetyl-2-amino-1-butanol reached 98.0% ee or more, the reaction was stopped and dichloromethane (CH 2 Cl 2 ) was added to the reaction solution. Was added to extract N, O-diacetyl-2-amino-1-butanol which remained unhydrolyzed. The extraction process was repeated four times to separate the organic solvent layer and dichloromethane was removed by distillation under reduced pressure. As a result, 12.5 g of (R) -N, O-diacetyl-2-amino-1-butanol (yield 31.3%) Was recovered. A part of this was purified by a column, and the optical purity of (R) -N, O-diacetyl-2-amino-1-butanol was measured and found to be 99.1%.

실시예 4Example 4

실시예 3에서 얻어진 (R)-N,O-디아세틸-2-아미노-1-부탄올 10g을 1N 염산용 액에 넣고 환류시켰다. 반응이 끝난 뒤 반응물에 디클로로메탄을 넣고 추출한 다음, 유기 용매층을 감압 증류하여 (R)-O-아세틸-2-아미노-1-부탄올이 얻어지면, 이를 메탄올에 녹인 후, K2CO3 13g을 넣고 교반하였다. 반응혼합물을 여과하여 K2 CO3를 제거하고 4.2g의 (R)-2-아미노-1-부탄올(수율: 82%)을 얻었다. 이를 (R)-2-아미노-1-부탄올을 폴라리미터(Polarimeter)를 이용하여 광회전도(optical rotation)를 측정한 결과 [α]D 25 = 12.38 (c=2 in ethanol)이었다. 이는 광학순도 99.1%ee에 해당하는 값이다.10 g of (R) -N, O-diacetyl-2-amino-1-butanol obtained in Example 3 was placed in a 1N hydrochloric acid solution and refluxed. After completion of the reaction, dichloromethane was added to the reaction mixture, followed by extraction. The organic solvent layer was distilled under reduced pressure to obtain (R) -O-acetyl-2-amino-1-butanol, which was dissolved in methanol and then 13 g of K 2 CO 3. Was added and stirred. The reaction mixture was filtered to remove K 2 CO 3 to obtain 4.2 g of (R) -2-amino-1-butanol (yield: 82%). The optical rotation of the (R) -2-amino-1-butanol using a polarimeter was [α] D 25 = 12.38 (c = 2 in ethanol). This value corresponds to 99.1% ee of optical purity.

실시예 5Example 5

실시예 3의 조건 중 프로테아제 대신 캔디다 루고사(Candida rugosa) 유래의 에스테라제를 사용하여 반응을 실시하였다. 100mM 인산용액(pH 7.0) 130㎖에 라세믹 N,O-디아세틸-2-아미노-1-부탄올 13g(10% w/v)을 넣고 pH를 7.0으로 조정한 다음, 상기 에스테라제 13g을 넣고 30℃에서 반응을 수행하였다. 반응 중의 pH는 20% 수산화나트륨용액을 이용하여 6.5∼7.5로 유지시켰다. 일정 시간마다 반응 샘플을 채취하여 반응 진행 상황을 분석하였다. 72시간 반응 후 (R)-N,O-디아세틸-2-아미노-1-부탄올의 광학순도가 90.5%ee에 도달하였을 때, 반응을 중단시킨 후 반응액에 디클로로메탄(CH2Cl2)을 첨가하여 가수분해되지 않고 남아있는 N,O-디아세틸-2-아미노-1-부탄올을 추출하였다. 상기 추출과정을 4회 반복하여 유기용매 층을 따로 분리하고 감압증류로 디클로로메탄을 제거한 결과, 1.6g의 (R)-N,O-디아세틸-2-아미노-1-부탄올(수율 12.3%)을 회수하였다. 광학순도 90.5%ee의 (R)-N,O-디아세틸-2- 아미노-1-부탄올 1.6g을 디이소프로필 에테르(diisopropyl ether)로 재결정한 결과, 광학순도 99.3%ee의 (R)-N,O-디아세틸-2-아미노-1-부탄올 1.24g(재결정 수율 77.3%)을 얻을 수 있었다. 여기서 얻어진 (R)-N,O-디아세틸-2-아미노-1-부탄올을 실시예 4와 같은 방법으로 처리하여 0.5g의 (R)-2-아미노-1-부탄올(수율: 80%)을 얻었다. 이 (R)-2-아미노-1-부탄올을 폴라리미터(Polarimeter)를 이용하여 광회전도(Optical rotation)를 측정한 결과 [α]D 25 = 12.4 (c=2 in ethanol)이었다. 이는 광학순도 99.2%ee에 해당하는 값이다. The reaction was carried out using an esterase derived from Candida rugosa instead of the protease under Example 3 conditions. 13 g (10% w / v) of racemic N, O-diacetyl-2-amino-1-butanol was added to 130 ml of 100 mM phosphate solution (pH 7.0), pH was adjusted to 7.0, and 13 g of esterase was added thereto. The reaction was carried out at 30 ℃. The pH during the reaction was maintained at 6.5-7.5 using 20% sodium hydroxide solution. Reaction samples were taken at regular intervals to analyze the reaction progress. When the optical purity of (R) -N, O-diacetyl-2-amino-1-butanol reached 90.5% ee after 72 hours, the reaction was stopped and dichloromethane (CH 2 Cl 2 ) was added to the reaction solution. Was added to extract N, O-diacetyl-2-amino-1-butanol which remained unhydrolyzed. The extraction process was repeated four times to separate the organic solvent layer separately, and dichloromethane was removed by distillation under reduced pressure. As a result, 1.6 g of (R) -N, O-diacetyl-2-amino-1-butanol (yield 12.3%) was obtained. Was recovered. 1.6R of (R) -N, O-diacetyl-2-amino-1-butanol having an optical purity of 90.5% ee was recrystallized with diisopropyl ether to give (R)-of an optical purity of 99.3% ee. 1.24 g of N, O-diacetyl-2-amino-1-butanol (77.3% recrystallization yield) were obtained. (R) -N, O-diacetyl-2-amino-1-butanol obtained here was treated in the same manner as in Example 4 to give 0.5 g of (R) -2-amino-1-butanol (yield: 80%) Got. The optical rotation of this (R) -2-amino-1-butanol was measured using a polarimeter. The result was [α] D 25 = 12.4 (c = 2 in ethanol). This corresponds to an optical purity of 99.2% ee.

전술한 바와 같이, 본 발명의 방법은 가수분해 효소를 이용하여 간단한 방법으로 높은 광학순도의 광학활성 (R)-2-아미노-1-부탄올을 제조할 수 있으며, 특히 효소를 이용하여 복잡한 재결정 과정 없이 한번에 98% 이상의 높은 광학순도를 달성할 수 있기 때문에 산업적으로 매우 유용하다. As described above, the method of the present invention can produce a high optical purity optically active (R) -2-amino-1-butanol by a simple method using a hydrolase, in particular a complex recrystallization process using an enzyme It is very useful industrially because it can achieve high optical purity of more than 98% at a time.

Claims (7)

pH 6∼9의 버퍼용액 존재하에서, 리조퍼스 속(Rhizopus sp.) 미생물 또는 캔디다 속(Candida sp.) 미생물 유래의 가수분해 효소를 이용하여 라세믹 2-아미노-1-부탄올의 에스테르 화합물을 선택적으로 가수분해시키는 것을 특징으로 하는 효소적 방법에 의한 광학활성 (R)-2-아미노-1-부탄올의 제조방법.In the presence of a buffer solution of pH 6-9, ester compounds of racemic 2-amino-1-butanol are selectively selected using a hydrolytic enzyme derived from Rhizopus sp. or Candida sp. A method for producing optically active (R) -2-amino-1-butanol by enzymatic method, characterized in that hydrolysis is carried out. 제1항에 있어서, 상기 가수분해 효소는 분말, 액상 또는 담체로 고정화된 것으로서 미생물 유래의 프로테아제 또는 에스테라제인 것을 특징으로 하는 방법.The method according to claim 1, wherein the hydrolase is immobilized in powder, liquid or carrier and characterized in that it is a protease or esterase derived from a microorganism. 삭제delete 제1항에 있어서, 상기 리조퍼스 속 미생물은 리조퍼스 오리재(Rhizopus oryzae)인 것을 특징으로 하는 방법.The method of claim 1, wherein the genus of the genus of the genus is Rezopus duck (Rhizopus oryzae ). 제1항에 있어서, 상기 캔디다 속 미생물은 캔디다 루고사(Candida rugosa) 인 것을 특징으로 하는 방법.The method of claim 1, wherein the Candida genus microorganism is Candida rugosa . 제1항에 있어서, 상기 가수분해 반응의 온도는 20∼40℃인 것을 특징으로 하는 방법.The method of claim 1, wherein the temperature of the hydrolysis reaction is 20 to 40 ℃. 제1항에 있어서, 상기 가수분해 반응시 효소와 기질의 비율은 1 : 10 ∼ 10 : 1인 것을 특징으로 하는 방법.The method of claim 1, wherein the ratio of the enzyme and the substrate in the hydrolysis reaction is characterized in that 1: 10 to 10: 1.
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