KR20040054842A - Method for preparing an (R)- or (S)- form of N-(2,6-dimethyl phenyl) alanine and a counter enantiomeric form of N-(2,6-dimethyl phenyl) alanine ester thereto using enzyme - Google Patents

Method for preparing an (R)- or (S)- form of N-(2,6-dimethyl phenyl) alanine and a counter enantiomeric form of N-(2,6-dimethyl phenyl) alanine ester thereto using enzyme Download PDF

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KR20040054842A
KR20040054842A KR1020020081152A KR20020081152A KR20040054842A KR 20040054842 A KR20040054842 A KR 20040054842A KR 1020020081152 A KR1020020081152 A KR 1020020081152A KR 20020081152 A KR20020081152 A KR 20020081152A KR 20040054842 A KR20040054842 A KR 20040054842A
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alanine
dimethylphenyl
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lipase
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박오진
이상후
박태윤
이상현
정원교
서병우
최증순
조군호
정재호
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주식회사 엘지생명과학
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Abstract

PURPOSE: A method for preparing (R)- or (S)-N-(2,6-dimethyl phenyl) alanine and stereoisomers of corresponding ester thereto using enzyme are provided, thereby cheaply and simply conducting the optical resolution of racemic (R), (S)-N-(2,6-dimethyl phenyl) alanine ester to prepare (R)- or (S)-N-(2,6-dimethyl phenyl) alanine having antimicrobial activity, and ester thereto in higher yield. CONSTITUTION: The method for preparing (R)- or (S)-N-(2,6-dimethyl phenyl) alanine and stereoisomers of corresponding ester thereto using enzyme comprises the steps of: (A) reacting racemic (R), (S)-N-(2,6-dimethyl phenyl) alanine ester of formula (1) with enzyme which specifically hydrolyzes (R)- or (S)- form of optical isomers of racemic (R), (S)-N-(2,6-dimethyl phenyl) alanine ester to prepare (R)- or (S)-N-(2,6-dimethyl phenyl) alanine; and (B) separating the (R)- or (S)-N-(2,6-dimethyl phenyl) alanine from unreacted corresponding (R)- or (S)-N-(2,6-dimethyl phenyl) alanine ester, wherein R is selected from optionally substituted C1-18 linear or branched chain alkyl or alkenyl, optionally substituted C3-6 cycloalkyl, optionally substituted aryl alkyl, and optionally substituted heteroaryl alkyl.

Description

효소를 이용한 (Ｒ)- 또는 (Ｓ)-Ｎ-(2,6-디메틸페닐)알라닌과 그것의 대응 에스테르 화합물의 입체이성질체의 제조방법 {Method for preparing an (R)- or (S)- form of N-(2,6-dimethyl phenyl) alanine and a counter enantiomeric form of N-(2,6-dimethyl phenyl) alanine ester thereto using enzyme}Method for preparing stereoisomers of (R)-or (S) -N- (2,6-dimethylphenyl) alanine and its corresponding ester compound using enzymes {Method for preparing an (R)-or (S)-form of N- (2,6-dimethyl phenyl) alanine and a counter enantiomeric form of N- (2,6-dimethyl phenyl) alanine ester with using enzyme}

본 발명은 효소를 이용하여 입체특이적으로 (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌((R),(S)-N-(2,6-dimethyl phenyl) alanine)과 그것의 대응 에스테르 화합물을 제조하는 방법에 관한 것이다. 더욱 상세하게는, 라세미(racemic) (R),(S)- N-(2,6-디메틸페닐) 알라닌 에스테르 중 어느 한 종류((R)-광학이성질체 또는 (S)-광학이성질체)에 대해 입체특이적 가수분해성을 갖는 효소를 상기 라세미 혼합물과 반응시켜 (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌을 광학분할하여 제조하거나, 또는 미반응물로부터 유기용매를 사용하여 (S)- 또는 (R)-N-(2,6-디메틸페닐) 알라닌 에스테르 화합물을 추출함으로써 이들을 광학분할하여 제조한다. 또는, 효소에 의한 가수분해 반응으로부터 제조된 특정한 알라닌을 에스테르화 반응시켜 광학분할된 (S)- 또는 (R)-N-(2,6-디메틸페닐) 알라닌 에스테르를 제조한다.The present invention is stereospecifically (R)-or (S) -N- (2,6-dimethylphenyl) alanine ((R), (S) -N- (2,6-dimethyl phenyl)) using an enzyme alanine) and a corresponding ester compound thereof. More specifically, to racemic (R), (S) -N- (2,6-dimethylphenyl) alanine esters of any one type ((R) -optical isomer or (S) -optical isomer) Or (S) -N- (2,6-dimethylphenyl) alanine by optically reacting an enzyme having stereospecific hydrolytic activity with the racemic mixture, or from an unreacted organic solvent. They are prepared by optical division by extracting the (S)-or (R) -N- (2,6-dimethylphenyl) alanine ester compound using Alternatively, a specific alanine prepared from the hydrolysis reaction by an enzyme is esterified to prepare an optically divided (S)-or (R) -N- (2,6-dimethylphenyl) alanine ester.

라세미 (R),(S)-N-(2,6-디메틸페닐) 알라닌과 이의 에스테르 화합물은 항균활성(antifungal activity)을 가지는 Metalaxyl, Benalaxyl, Furalaxyl 등의 합성을 위한 중간체로 알려져 있다. 이들 화합물의 항균활성은 대체로 (R)-이성질체에 기인한다.Racemic (R), (S) -N- (2,6-dimethylphenyl) alanine and its ester compounds are known as intermediates for the synthesis of Metalaxyl, Benalaxyl, Furalaxyl and the like having antifungal activity. The antimicrobial activity of these compounds is largely due to the (R) -isomer.

Metalaxyl의 (R)-광학이성질체인 Metalaxyl-M의 합성은 키랄(chiral) 금속 촉매의 존재하에서 에나미드(enamide)의 수소첨가반응에 의하여 합성됨이 보고되어있다 (Pesticide Science, Vol. 54, 1998, pp 302-304). 이 반응은 고가의 금속촉매를 사용하며 고온과 고압의 반응조건하에서 광학순도(% enantiomeric excess, %ee) 95.6%ee로 Metalaxyl-M을 제조한다.Synthesis of Metalaxyl-M, the (R) -isomer of Metalaxyl, has been reported to be synthesized by the hydrogenation of enamide in the presence of a chiral metal catalyst ( Pesticide Science , Vol. 54, 1998). , pp 302-304). This reaction uses an expensive metal catalyst to produce Metalaxyl-M with 95.6% ee of optical purity (% enantiomeric excess,% ee) under high temperature and high pressure reaction conditions.

광학활성물질 (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌을 제조하는 방법 중의 하나로는, 라세미 혼합물에 광학활성 분할제(resolving agent)로서 광학적으로 순수한 아민(예를 들어, (R)- 또는 (S)-phenylethylamine)을 첨가하여 분리하는 광학분할기술(optical resolution)이 알려져 있다 (미국특허 제4,919,709호). 그러나, 상기 광학활성 분할제는 가격이 비싸고 공정이 복잡하다는 단점을 가지고 있다.One method for preparing the optically active substance (R)-or (S) -N- (2,6-dimethylphenyl) alanine is an optically pure amine (e.g., an optically active resolving agent) in the racemic mixture. For example, optical resolution for separating by adding (R)-or (S) -phenylethylamine) is known (US Pat. No. 4,919,709). However, the optically active dividing agent has the disadvantage of being expensive and complicated in process.

(R)-N-(2,6-디메틸페닐) 알라닌 메틸 에스테르를 합성하기 위한 하나의 방법으로서, 메틸 (S)-2-(히드록시)-프로파노에이트를 출발물질로 하여 이의 술포네이트 유도체를 제조하고, 염기 존재하에서 2,6-디메틸 아닐린과 반응시키는 방법이 보고되어있다 (WO00/76960). 그러나, 이 반응을 통해서는, 높은 광학순도의 (R)-N-(2,6-디메틸페닐) 알라닌 메틸 에스테르를 얻기 어려울 뿐만 아니라 (95%ee 이하), 고온과 유기용매를 사용한다는 문제점도 있다. 상기 (R)-N-(2,6-디메틸페닐)알라닌 메틸 에스테르는 chiral 형태의 benalaxyl, furalaxyl에도 적용가능한데, Metalaxyl-M의 경우 요구되는 광학순도는 95%ee 이상인 것이 바람직하다.As a method for synthesizing (R) -N- (2,6-dimethylphenyl) alanine methyl ester, a sulfonate derivative thereof having methyl (S) -2- (hydroxy) -propanoate as a starting material Is prepared and reacted with 2,6-dimethyl aniline in the presence of a base (WO00 / 76960). However, through this reaction, it is not only difficult to obtain (R) -N- (2,6-dimethylphenyl) alanine methyl ester of high optical purity (95% ee or less), but also a problem of using a high temperature and an organic solvent. have. The (R) -N- (2,6-dimethylphenyl) alanine methyl ester is also applicable to beralaxyl and furalaxyl in the chiral form. In the case of Metalaxyl-M, the optical purity required is 95% ee or more.

한편, 특정 라세미 혼합물을 광학분할하기 위하여 에스터라아제(esterase), 리파아제(lipase), 프로테아제(protease) 등의 효소를 이용하여 선택적으로 거울상 이성질체를 가수분해시키는 방법들이 알려져 왔다. 예를 들어, 라세미 메틸-2-클로로프로피오네이트(Methyl-2-chloropropionate)를Candida rugosa유래의 리파아제를 이용하여 가수분해하는 방법이 보고되어있다 (Biotechnology & Bioengineering 30, 1987, pp 995-999). 또한, 정제된Candida rugosa유래의 리파아제를 이용하여 (R)-2-(4-히드록시페녹시) 프로피온산를 합성하는 것이 보고되었다 (WO90/15146).On the other hand, methods for selectively hydrolyzing enantiomers using enzymes such as esterase, lipase, protease and the like to optically divide a specific racemic mixture have been known. For example, a method of hydrolyzing racemic methyl-2-chloropropionate using lipase from Candida rugosa has been reported (Biotechnology & Bioengineering 30, 1987, pp 995-999 ). It has also been reported to synthesize (R) -2- (4-hydroxyphenoxy) propionic acid using purified lipase from Candida rugosa (WO90 / 15146).

라세미 혼합물의 분할에 효소를 이용하는 것은 매우 효과적일 수는 있지만, 어떠한 라세미체에 대해 효소 분할이 가능한지를 확인하는 것과, 특정 라세미체의 분할에 어떠한 효소가 특별히 효과적인지를 확인하는 것은 매우 어려운 일이다. 예를 들어, 미국특허 제5,928,933호는 라세미 4-옥소-1,2-피롤리딘디카르복실산 디알킬 에스테르의 광분할을 위하여 프로테아제, 리파아제 및 에스테라아제 중에서 선택된 44 가지 효소에 대해 반응 특이성을 실험하였고, 그 중 한 종류의 효소만이 95%의 광학순도를 나타냄을 보이고 있다. 이와 같이, 사용하는 효소의 종류 및 기질의 화학적 구조 등에 의해 이성질체의 선택성 및 광학순도(%ee)가 달라지므로 지속적인 연구를 통해 기질에 적합한 조합을 찾아내는 것이 무엇보다도 중요하다,Using enzymes for the cleavage of racemic mixtures can be very effective, but it is very difficult to determine which racemates can be cleaved and which enzymes are particularly effective for cleavage of specific racemates. It's work. For example, US Pat. No. 5,928,933 discloses reaction specificity for 44 enzymes selected from proteases, lipases and esterases for light splitting of racemic 4-oxo-1,2-pyrrolidinedicarboxylic acid dialkyl esters. The experiment showed that only one type of enzyme showed 95% optical purity. As such, the selectivity and optical purity (isee) of the isomers vary according to the type of enzyme used and the chemical structure of the substrate, so it is important to find a suitable combination for the substrate through continuous research.

한편, 본 발명에서 광학분할의 대상으로 삼고있는 라세미체인 (R),(S)-N-(2,6-디메틸페닐) 알라닌 에스테르에 대하여, 그것의 광학분할에 효소를 이용한 예는 아직까지 보고되어있지 않다. 기존의 효소를 이용한 광학분할은 주로 prop류의 제초제의 중간물질인 aryloxypropionic acid의 합성, -profen류의 항염증제의 중간물질인 arylpropionic acid의 합성에만 국한되었고, 본 발명에서 다루고자 하는 (R),(S)-N-(2,6-디메틸페닐) 알라닌 메틸 에스테르의 광학분할에 효소를 사용한 예가 없다.On the other hand, for the racemate (R), (S) -N- (2,6-dimethylphenyl) alanine ester which is the object of optical division in the present invention, the example of using an enzyme in its optical division is still It is not reported. Optical splitting using existing enzymes was mainly limited to the synthesis of aryloxypropionic acid, an intermediate of prop herbicides, and the synthesis of arylpropionic acid, an intermediate of anti-inflammatory drugs of -profen, and (R), ( There is no example of using an enzyme for the optical separation of S) -N- (2,6-dimethylphenyl) alanine methyl ester.

이에 본 발명자들은, 광범위한 연구를 수행한 결과, 라세미 (R),(S)-N-(2,6-디메틸페닐) 알라닌 에스테르를, 그것의 특정 광학이성질체(R형 또는 S형 광학이성질체)에 대해 가수분해 특이성을 갖는 효소와 상온, 상압에서 반응시킬 때, 광학 순도가 매우 우수한(> 96-99%ee) (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌과, 그것의 대응 (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌 에스테르를 제조할 수 있음을 발견하고, 본 발명에 이르게 되었다.The present inventors have conducted extensive research and found that racemic (R), (S) -N- (2,6-dimethylphenyl) alanine esters can be converted to specific optical isomers thereof (type R or type S optical isomers). (R)-or (S) -N- (2,6-dimethylphenyl) alanine with excellent optical purity (> 96-99% ee) when reacted with an enzyme having hydrolysis specificity at room temperature and atmospheric pressure And the corresponding (R)-or (S) -N- (2,6-dimethylphenyl) alanine ester can be produced, and the present invention has been attained.

따라서, 본 발명의 목적은 효소에 의한 광학분할에 의해 높은 광학순도를 갖는 (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌과, 그것의 대응 (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌 에스테르를 경제적으로 제조할 수 있는 방법을 제공하는데 있다.Accordingly, it is an object of the present invention to provide (R)-or (S) -N- (2,6-dimethylphenyl) alanine having high optical purity and its corresponding (R)-or ( The present invention provides a method for economically preparing S) -N- (2,6-dimethylphenyl) alanine ester.

우선, 본 발명의 (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌의 제조방법은,First, the method for producing (R)-or (S) -N- (2,6-dimethylphenyl) alanine of the present invention,

(A) 하기 화학식 1의 라세미 (R),(S)-N-(2,6-디메틸페닐) 알라닌 에스테르를, 그것의 특정 광학이성질체(R형 또는 S형 광학이성질체)에 대해 가수분해 특이성을 가지는 반응 유효량의 효소와 반응시켜, (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌을 생성하는 단계; 및(A) Hydrolysis specificity of racemic (R), (S) -N- (2,6-dimethylphenyl) alanine ester of formula (1) with respect to its specific optical isomer (R type or S type optical isomer) Reacting with an effective amount of an enzyme having to produce (R)-or (S) -N- (2,6-dimethylphenyl) alanine; And

(B) 상기 반응물 중의 (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌을, 미반응 대응 (S)- 또는 (R)-N-(2,6-디메틸페닐) 알라닌 에스테르로부터 분리하는 단계를 포함하는 것으로 구성되어있다.(B) unreacted corresponding (S)-or (R) -N- (2,6-dimethylphenyl) to (R)-or (S) -N- (2,6-dimethylphenyl) alanine in the reaction product; And a step of separating from an alanine ester.

상기 식에서, R 은 치환되거나 또는 치환되지않은 탄소수 1-18의 선형 내지 가지형 알킬 또는 알케닐, 치환되거나 또는 치환되지않은 탄소 3-6의 시클로알킬, 치환되거나 또는 치환되지않은 아릴 알킬, 및 치환되거나 또는 치환되지않은 헤테로아릴 알킬로 이루어진 군에서 선택된다.Wherein R is substituted or unsubstituted linear to branched alkyl or alkenyl having 1 to 18 carbon atoms, substituted or unsubstituted cycloalkyl having 3 to 6 carbon atoms, substituted or unsubstituted aryl alkyl, and substituted Or unsubstituted heteroaryl alkyl.

상기 R 의 구체적인 예로는, 메틸, 에틸, n-프로필, 이소프로필, n-부틸, 2-부틸, 2-펜틸, 3-메틸-1-부틸, 2-에틸-1-헥실, 2-클로로에틸, 2-브로모에틸, 3-클로로프로필, 3-브로모프로필, 2,2-디클로로에틸, 1-클로로-2-프로필, 올레일, 시클로헥실, 1-시클로프로필메틸, 알릴, 페닐, 벤질, 프로파아르길, 2-페녹시-1-에틸, 2,4-디클로로벤질, 메톡시에틸, 에톡시에틸, 1-티오에톡시에틸 등을 들 수 있다.Specific examples of R include methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-butyl, 2-pentyl, 3-methyl-1-butyl, 2-ethyl-1-hexyl, 2-chloroethyl , 2-bromoethyl, 3-chloropropyl, 3-bromopropyl, 2,2-dichloroethyl, 1-chloro-2-propyl, oleyl, cyclohexyl, 1-cyclopropylmethyl, allyl, phenyl, benzyl , Propargyl, 2-phenoxy-1-ethyl, 2,4-dichlorobenzyl, methoxyethyl, ethoxyethyl, 1-thioethoxyethyl and the like.

다음으로, 본 발명의 (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌 에스테르의 제조방법은,Next, the method for producing (R)-or (S) -N- (2,6-dimethylphenyl) alanine ester of the present invention,

(A') 상기 화학식 1의 라세미 (R),(S)-N-(2,6-디메틸페닐) 알라닌 에스테르를, 그것의 특정 광학이성질체(R형 또는 S형 광학이성질체)에 대해 가수분해 특이성을 가지는 반응 유효량의 효소와 반응시켜, (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌을 생성하는 단계; 및,(A ') Hydrolysis of the racemic (R), (S) -N- (2,6-dimethylphenyl) alanine ester of the formula (1) with respect to its specific optical isomer (R type or S type optical isomer) Reacting with an effective amount of an enzyme having a specificity to produce (R)-or (S) -N- (2,6-dimethylphenyl) alanine; And,

(B') 상기 반응물 중의 미반응 대응 (S)- 또는 (R)-N-(2,6-디메틸페닐) 알라닌 에스테르를, 생성된 (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌으로부터 분리하는 단계를 포함하는 것으로 구성되어있다.(B ') The unreacted corresponding (S)-or (R) -N- (2,6-dimethylphenyl) alanine ester in the reaction product is produced by (R)-or (S) -N- (2,6 -Dimethylphenyl) alanine.

상기 단계(A') 및 (B')는 앞서 설명한 단계(A) 및 (B)와 사실상 동일하다. 경우에 따라서는, 상기 단계(B') 대신에 하기 단계(C')를 포함하거나 또는 단계(B') 이후에 하기 단계(C')를 더 포함할 수 있다.The steps (A ') and (B') are substantially the same as the steps (A) and (B) described above. In some cases, the step C 'may be included instead of the step B' or the step C 'may be further included after the step B'.

(C') 상기 (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌을 알코올(R-OH; 여기서의 R 은 화학식 1에서와 동일함)과 반응시켜, (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌 에스테르를 합성하는 단계.(C ′) reacting (R)-or (S) -N- (2,6-dimethylphenyl) alanine with an alcohol (R-OH; wherein R is the same as in Formula 1), wherein (R) Or synthesizing (S) -N- (2,6-dimethylphenyl) alanine ester.

이하에서는, 본 발명을 더욱 구체적으로 설명한다.Hereinafter, the present invention will be described in more detail.

광학분할을 행하는 대상인 상기 라세미 (R),(S)-N-(2,6-디메틸페닐) 알라닌 에스테르의 제조방법은 당업계에 공지되어 있는바, 그러한 하나의 예로는, 2-브롬 프로피온산(2-bromopropionic acid)과 2,6-디메틸아닐린(dimethylaniline)의 반응을 들 수 있다. 그러나, N-(2,6-디메틸페닐) 알라닌 에스테르의 라세미 혼합물(이하, 라세미 에스테르 혼합물로 약칭하기도 함)은, R형 광학이성질체와 S형 광학이성질체 그자체의 물리화학적 특성 차이가 적으므로, 일반적인 분리 방법들로는 분리하기 매우 어렵다.The method for preparing the racemic (R), (S) -N- (2,6-dimethylphenyl) alanine ester, which is an object of optical division, is known in the art, and one example thereof is 2-bromine propionic acid. (2-bromopropionic acid) and 2,6-dimethylaniline (dimethylaniline) reaction. However, racemic mixtures of N- (2,6-dimethylphenyl) alanine esters (hereinafter also abbreviated as racemic ester mixtures) have little difference in the physicochemical properties of the R- and S-type optical isomers themselves. Therefore, it is very difficult to separate by common separation methods.

따라서, 상기 라세미 에스테르 혼합물 중에서, R형 광학이성질체[(R)-N-(2,6-디메틸페닐) 알라닌 에스테르] 및 S형 광학이성질체[(S)-N-(2,6-디메틸페닐) 알라닌 에스테르] 중의 어느 하나에 대해 가수분해 특이성을 가진 효소(이하, 특이적 가수분해 효소로 약칭하기도 함)를 사용하는 본 발명은, 상기 라세미 에스테르 혼합물의 광학분할을 위한 종래의 합성 또는 분리방법들과 비교하여, 간단하고 저렴한 방법에 의해 특정한 광학이성질체를 제조한다는 점에서, 당해 분야에서 혁신적인 장점을 가진다.Thus, in the racemic ester mixture, the R type optical isomer [(R) -N- (2,6-dimethylphenyl) alanine ester] and the S type optical isomer [(S) -N- (2,6-dimethylphenyl The present invention using an enzyme having a hydrolysis specificity (hereinafter also abbreviated as a specific hydrolase) for any one of the above-described alanine esters) is a conventional synthesis or separation for optical separation of the racemic ester mixture. Compared with the methods, there is an innovative advantage in the art in that specific optical isomers are prepared by simple and inexpensive methods.

(R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌의 제조방법에 관한 상기 단계(A)는, 상기 라세미 에스테르 혼합물을 수용액, 또는 소량의 유기용매를 포함하는 유기용매-수용액 혼합액에 녹인 뒤, 온도와 pH를 일정하게 유지한 상태에서 특이적 가수분해 효소와 반응시킨다.The step (A) of the method for preparing (R)-or (S) -N- (2,6-dimethylphenyl) alanine may include the racemic ester mixture in an aqueous solution or an organic solvent comprising a small amount of an organic solvent. Dissolve in aqueous solution and react with specific hydrolase while keeping temperature and pH constant.

따라서, 상기 특이적 가수분해 효소가 R형 광학이성질체에 대해서만 특이적으로 가수분해 능력을 가진 경우에는, 단계(A)의 반응에 의해 R형 광학이성질체만이 가수분해되어 (R)-N-(2,6-디메틸페닐) 알라닌이 생성되고, S형 광학이성질체, 즉, (S)-N-(2,6-디메틸페닐) 알라닌 에스테르는 미반응상태로 남게 된다. 반대로, 상기 특이적 가수분해 효소가 S형 이성질체에 대해서만 가수분해 능력을 가진 경우에는, 단계(A)의 반응에 의해 S형 광학이성질체만이 가수분해되어 (S)-N-(2,6-디메틸페닐) 알라닌이 생성되고, R형 광학이성질체, 즉, (R)-N-(2,6-디메틸페닐) 알라닌 에스테르는 미반응 상태로 남게 된다.Therefore, in the case where the specific hydrolase has a specific hydrolytic ability only with respect to the R-type optical isomer, only the R-type optical isomer is hydrolyzed by the reaction of step (A), and thus (R) -N- ( 2,6-dimethylphenyl) alanine is produced, and the S-type optical isomer, ie, (S) -N- (2,6-dimethylphenyl) alanine ester, remains unreacted. Conversely, in the case where the specific hydrolase has a hydrolytic ability only for the S isomer, only the S-type optical isomer is hydrolyzed by the reaction of Step (A) to (S) -N- (2,6- Dimethylphenyl) alanine is produced, and the R-type optical isomer, ie, (R) -N- (2,6-dimethylphenyl) alanine ester, remains unreacted.

반응계가 수용액만으로 이루어진 경우에는, 상기 단계(B)에서 유기용매를 사용하여 미반응 에스테르 화합물(R형 또는 S형 광학이성질체)을 추출할 수 있다. 반면에, 반응계가 유기용매-수용액 혼합액으로 이루어진 경우에는, 미반응 에스테르 화합물이 물에는 용해되지않지만 유기용매에 용해되므로, 유기용매층을 분리함으로써 이를 손쉽게 분리할 수 있다.When the reaction system consists of only an aqueous solution, the unreacted ester compound (R-type or S-type optical isomer) may be extracted using the organic solvent in the step (B). On the other hand, when the reaction system is composed of an organic solvent-aqueous liquid mixture, the unreacted ester compound is not dissolved in water but is dissolved in an organic solvent, and thus it can be easily separated by separating the organic solvent layer.

한편, 수용액에 포함되어있는 (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌은 수용액을 산성화한(acidify) 뒤 유기용매를 이용하여 추출하고, 그로부터 다시 유기용매를 제거함으로써, 광학적으로 순수한 (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌을 얻을 수 있게 된다. 실험결과, 이들의 (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌의 광학 순도는 매우 높은 것으로 확인되었다 (> 96-99%ee).Meanwhile, (R)-or (S) -N- (2,6-dimethylphenyl) alanine contained in the aqueous solution is acidified with an aqueous solution, extracted using an organic solvent, and the organic solvent is removed therefrom. This makes it possible to obtain optically pure (R)-or (S) -N- (2,6-dimethylphenyl) alanine. As a result, the optical purity of these (R)-or (S) -N- (2,6-dimethylphenyl) alanine was found to be very high (> 96-99% ee).

또한, 미반응 대응 화합물로서 분리되는 (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌 에스테르나, 또는 그것의 제조방법과 관련하여 상기 단계(C')에서 (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌의 에스테르화 반응에서 제조된 (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌 에스테르 역시 높은 광학 순도를 가지는 것으로 확인되었다 (> 96-99%ee).In addition, (R)-or (S) -N- (2,6-dimethylphenyl) alanine ester, which is isolated as an unreacted corresponding compound, or (R) in the above step (C ') in connection with a preparation method thereof. Or (R)-or (S) -N- (2,6-dimethylphenyl) alanine esters prepared in the esterification reaction of (S) -N- (2,6-dimethylphenyl) alanine also have high optical purity. It was confirmed to have (> 96-99% ee).

상기 화학식 1에서의 R 이, 알릴(allyl), 2-클로로에틸(2-chloroethyl), 메톡시에틸(methoxyethyl), 또는 에톡시에틸(ethoxyethyl)인 경우에는, 반응시간이 짧고, 더욱 높은 광학순도가 얻어질 수 있으므로, 더욱 바람직하다.When R in Formula 1 is allyl, 2-chloroethyl, methoxyethyl, or ethoxyethyl, the reaction time is short, and the optical purity is higher. Is more preferable since can be obtained.

상기 특이적 가수분해 효소는, R형 광학이성질체나 S형 광학이성질체의 어느 하나를 특이적으로 가수분해하는 능력을 가지고 있다면 특별히 제한되는 것은 아니지만, 미생물, 동물 또는 식물 유래의 리파아제(lipase), 프로테아제(protease) 또는 에스터라아제(esterase) 중에 선택된 것이 바람직하다.The specific hydrolase is not particularly limited as long as it has the ability to specifically hydrolyze either the R-type or the S-type isomer, but it is a lipase or a protease derived from a microorganism, an animal, or a plant. It is preferred that it is selected from protease or esterase.

상기 효소 중에서 반응의 전환율과 광학순도가 높은 리파아제가 더욱 바람직하다. 그 중에서도 Pseudomonas 유래 리파아제 AK, Toyobo Immobilized lipase, Liopprotein lipase, Burkholderia 유래의 리파아제 PS, AH, Alcaligenes 유래의 리파아제 QLM, Candida 유래 리파아제 OF 등에서 선택된 하나 또는 둘 이상인 것이 enatioselectivity와 속도면에서 특히 바람직하다.Among the enzymes, lipases having high conversion and high optical purity are more preferable. Among them, one or more selected from Pseudomonas-derived lipase AK, Toyobo Immobilized lipase, Liopprotein lipase, Burkholderia-derived lipase PS, AH, Alcaligenes-derived lipase QLM, Candida-derived lipase OF, and the like are particularly preferable in terms of enatioselectivity and speed.

사용되는 효소의 종류에 따라 R형과 S형 광학이성질체에 대한 선택성이 결정되며, 사용형태는 분말 또는 수용액일 수 있다. 경우에 따라서는, 재사용을 위해 담체에 고정화한 것일 수도 있다. 효소의 고정화 방법은 고분자 담체나 세라이트(celite)와 같은 무기담체에 효소를 부착하는 등 다양한 예가 당업계에 공지되어있으므로, 이에 대한 자세한 설명은 본 명세서에서 생략한다.Depending on the type of enzyme used, the selectivity for the R-type and S-type optical isomers is determined, and the use form may be a powder or an aqueous solution. In some cases, it may be immobilized on a carrier for reuse. Enzyme immobilization method is known in the art, such as attaching the enzyme to an inorganic carrier such as a polymer carrier or celite (celite), so a detailed description thereof will be omitted herein.

효소의 사용량은, 반응온도, pH, 수용액의 량, 반응시간 등 여러 반응요소들에 의해 결정되므로 유효 사용량은 가변적이지만, 기질(라세미 에스테르 혼합물)의 중량을 기준으로 대략적으로 0.1-100 중량%인 것이 바람직하며, 너무 적은 경우에는 반응시간이 길어지는 문제점이 있고, 너무 많은 경우에는 효소의 과량 사용이라는 경제적 측면과 분리 등의 반응공정적 측면에서 바람직하지 않다.Since the amount of enzyme is determined by various reaction factors such as reaction temperature, pH, amount of aqueous solution, reaction time, and the like, the effective amount varies, but is about 0.1-100% by weight based on the weight of the substrate (racemic ester mixture). In the case of too small, there is a problem in that the reaction time is long, and in the case of too much, it is not preferable in terms of economics such as excessive use of enzyme and in terms of reaction process such as separation.

반응조건은 특별히 제한되는 것은 아니지만, 효소반응의 최적화를 위하여 0-60℃, pH 3-12가 바람직하다. 더욱 바람직한 반응온도는 30-50℃이다. 앞서의 설명과 같이, 단계(A)의 반응은 수용액내에서 진행될 수 있으며, 기질의 용해도를 증가시키고 생성물의 효소활성 저해를 줄이기 위하여 소량의 유기용매를 포함하는 유기용매-수용액 혼합액의 사용도 가능하다.The reaction conditions are not particularly limited, but 0-60 ° C. and pH 3-12 are preferred for the optimization of the enzyme reaction. More preferred reaction temperature is 30-50 ° C. As described above, the reaction of step (A) can be carried out in an aqueous solution, and it is also possible to use an organic solvent-aqueous solution containing a small amount of organic solvent in order to increase the solubility of the substrate and reduce the inhibition of enzymatic activity of the product. Do.

효소반응을 위한 기질의 농도 역시 여러 반응요소들에 의해 변화될 수 있으며, 효소반응의 최적화를 위해서는 500 mM - 1 M이 바람직하지만, 더 높은 농도로 사용될 수도 있다. 단계(A) 및 (A')의 가수분해반응에 있어서, 반응초기에는 에스테르 화합물이 물에 잘 녹지 않지만 물에 용해된 에스테르 화합물과 효소의 접촉에 의해 반응이 진행된다. 효소반응의 속도를 증가시키고 효소의 활성을 유지하기 위하여, 최소량의 물과 주로 유기용매로 이루어진 반응액에서도 가능하다.The concentration of the substrate for the enzymatic reaction can also be varied by several reaction factors, and 500 mM-1 M is preferred for the optimization of the enzymatic reaction, but higher concentrations can be used. In the hydrolysis reaction of steps (A) and (A '), the reaction proceeds by the contact of an ester compound and an enzyme dissolved in water, although the ester compound is not easily dissolved in water at the beginning of the reaction. In order to increase the rate of enzymatic reaction and maintain the activity of the enzyme, it is possible even in a reaction solution consisting of a minimum amount of water and an organic solvent.

효소반응의 상기 용매로서, 아세톤, 아세토나이트릴, 알코올 등과 같은 친수성 용매와, 이소프로필에테르, tert-부틸 메틸 에테르(tert-butyl methyl ether: TBME), 클로로포름, 디클로로메탄, 사염화탄소, 헥산, 톨루엔 등과 같은 소수성 용매가 사용될 수 있고, 경우에 따라서는 이들의 이상(two-phase) 반응용매도 사용될 수 있다.As the solvent for the enzymatic reaction, a hydrophilic solvent such as acetone, acetonitrile, alcohol and the like, isopropyl ether, tert-butyl methyl ether (TBME), chloroform, dichloromethane, carbon tetrachloride, hexane, toluene and the like The same hydrophobic solvent may be used, and in some cases, these two-phase reaction solvents may also be used.

효소반응의 최적 조건은 앞서 설명한 내용뿐만 아니라 기타 다양한 반응요소들도 함께 고려하여 적절히 설정될 수 있다.Optimum conditions of the enzyme reaction may be appropriately set in consideration of various other reaction factors as well as the above description.

상기 단계(B) 또는 (B')에서의 추출에 사용되는 유기용매로는 에틸아세테이트, 이소프로필에테르, tert-butyl methyl ether(TBME), 클로로포름, 디클로로메탄, 사염화탄소, 헥산, 톨루엔 등으로부터 선택된 하나 또는 이들의 혼합물이 바람직하다.The organic solvent used for extraction in step (B) or (B ') is one selected from ethyl acetate, isopropyl ether, tert-butyl methyl ether (TBME), chloroform, dichloromethane, carbon tetrachloride, hexane, toluene and the like. Or mixtures thereof.

상기 단계(C')에서의 에스테르 반응은 카르복실기를 가진 (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌과 알코올의 반응으로서, 최종적으로 소망하는 (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌 에스테르를 제조할 수 있는 반응조건이라면 특별히 제한되지 않으며, 이러한 에스테르 반응은 당업계에 공지되어있으므로, 이에 대한 설명은 본 명세서에서 생략한다.The ester reaction in step (C ′) is a reaction of (R)-or (S) -N- (2,6-dimethylphenyl) alanine with a carboxyl group with alcohol, and finally the desired (R)-or ( The reaction conditions for preparing S) -N- (2,6-dimethylphenyl) alanine ester are not particularly limited, and such ester reactions are well known in the art, and thus description thereof is omitted herein.

이하, 다수의 실시예들을 참조하여 본 발명의 구체적인 내용을 예시하지만, 본 발명의 범주가 그것에 의해 한정되는 것은 아니다.Hereinafter, the specific details of the present invention will be illustrated with reference to a number of embodiments, but the scope of the present invention is not limited thereto.

[실시예 1] 라세미 (R),(S)-N-(2,6-디메틸페닐) 알라닌 메틸 에스테르를 가수분해하는 효소의 선별 (스크리닝) Example 1 Screening of enzymes that hydrolyze racemic (R), (S) -N- (2,6-dimethylphenyl) alanine methyl esters (screening)

450 microL (0.1 M, pH 7)의 인산 칼륨(potassium phosphate) 완충용액에, 기질용액 50 microL(라세미 (R),(S)-N-(2,6-디메틸페닐) 알라닌 메틸 에스테르 50 microL를 500 uL의 아세토니트릴에 녹인 용액)을 혼합하고, 25 ㎎의 효소분말(액체인 경우 20 microL)을 추가한 후, 30C에서 각각 1 일 및 2 일간 반응시켰다. 반응의 전환율 분석은 HPLC를 이용하여 C18 역상 컬럼(reverse phase column)에서 아세토니트릴:물(70:30, 0.1% trifluoroacetic acid)을 이동상으로 하여, UV파장 254 nm에서 분석하였다. 실험에 사용된 효소의 종류와 전환율의 결과를 하기 표 1에 나타낸다.50 microL of substrate solution (racemic (R), (S) -N- (2,6-dimethylphenyl) alanine methyl ester) 50 microL in 450 microL (0.1 M, pH 7) of potassium phosphate buffer The solution was dissolved in 500 uL of acetonitrile), and 25 mg of enzyme powder (20 microL in the case of liquid) was added, followed by reaction at 30C for 1 day and 2 days, respectively. The conversion analysis of the reaction was carried out using acetonitrile: water (70:30, 0.1% trifluoroacetic acid) as a mobile phase in a C18 reverse phase column using HPLC using a UV wavelength of 254 nm. The results of the type and conversion rate of the enzyme used in the experiment are shown in Table 1 below.

EnzymeEnzyme sourcesource conv.(%)conv. (%) 1day1day 2day2day OptimaseOptimase Bacillus licheniformisBacillus licheniformis 6161 8383 AlcalaseAlcalase Bacillus licheniformisBacillus licheniformis 6060 8383 Lipase PSLipase PS Burkholderia cepaciaBurkholderia cepacia 5050 5656 Novozym 435Novozym 435 Candida antarcticaCandida antarctica 100100 -- Lipase OFLipase OF Candida rugosaCandida rugosa 4747 5757 Lipase PLLipase PL Alcaligenessp. Alcaligenes sp. 3232 4949 Lipase PS-DLipase PS-D Burkholderia cepaciaBurkholderia cepacia 5353 6161 Lipase PS-CLipase PS-C Burkholderia cepaciaBurkholderia cepacia 5656 6060 Lipase ALLipase AL Achrobactersp. Achrobacter sp. 1919 2323 Lipase QLMLipase QLM Alcaligenessp. Alcaligenes sp. 6060 7676 Proleather FG-FProleather FG-F Bacillus subtilisBacillus subtilis 5656 7979 Acylase AmanoAcylase amano Aspergillus melleusAspergillus melleus 5151 6565 Protease PSProtease PS Bacillus sp.Bacillus sp. 100100 -- Immobililized lipase (Toyobo)Immobililized lipase (Toyobo) Pseudomonassp. Pseudomonas sp. 4545 5050 Lipoprotein lipase (Toyobo)Lipoprotein lipase (Toyobo) Pseudomonassp. Pseudomonas sp. 6161 6969 Lipase AHLipase AH Burkholderia cepaciaBurkholderia cepacia 5656 6262 PLE-AL amanoPLE-AL amano Porcine liverPorcine liver 100100 -- Alcalase 0.6LAlcalase 0.6L Bacillus licheniformisBacillus licheniformis 3535 4848 Alcalase 2.5LAlcalase 2.5L Bacillus licheniformisBacillus licheniformis 6565 8484 Novozym 525LNovozym 525L Candida antarcticaCandida antarctica 100100 -- ChiroCLEC-CABChiroCLEC-CAB Candida antarcticaCandida antarctica 100100 --

상기 표 1에서, 일정한 시간 경과 후의 반응 전환율이 50% 전후인 효소는 R형 광학이성질체나 S형 광학이성질체에 대해 반응의 광학이성적 선택성이 높을 가능성이 있는 효소를 의미한다고 할 수 있다. 그러한 점에서, 각종 리파아제는 상기 라세미체에 대한 광학이성적 선택성이 높은 후보군임을 예측할 수 있다.In Table 1, an enzyme having a reaction conversion rate of about 50% after a predetermined time elapses may refer to an enzyme having a high optical isoselectivity of the reaction with respect to the R-type or S-type isomer. In this regard, it can be predicted that various lipases are candidate groups having high optical isoselectivity for the racemate.

[실시예 2]Example 2

(R)-N-(2,6-디메틸페닐) 알라닌 메틸 에스테르가 풍부한 경우(R:S = 90:10)와 (S)-N-(2,6-디메틸페닐) 알라닌 메틸 에스테르가 풍부한 경우(R:S = 4:96)를 제조하여 실시예 1과 같은 방법으로 반응시켜 전환율을 측정하였다. 사용된 효소의 종류와 전환율의 결과를 하기 표 2에 나타낸다.Rich in (R) -N- (2,6-dimethylphenyl) alanine methyl ester (R: S = 90: 10) and When (S) -N- (2,6-dimethylphenyl) alanine methyl ester was enriched (R: S = 4: 96), a conversion rate was measured by reacting in the same manner as in Example 1. The results of the type of enzyme used and the conversion rate are shown in Table 2 below.

EnzymeEnzyme sourcesource conv.(%)conv. (%) (R)-ester(R) -ester (S)-ester(S) -ester Lipase AKLipase AK Pseudomonas fluorescensPseudomonas fluorescens 7979 1717 Lipase AHLipase AH Burkholderia cepaciaBurkholderia cepacia 4747 1212 Lipase PSLipase PS Burkholderia cepaciaBurkholderia cepacia 8686 1717 Lipase QLMLipase QLM Alcaligenes sp.Alcaligenes sp. 9696 1919

상기 표 2의 결과에서, 특히 리파아제 AK, PS, QLM 등이 상기 라세미체의 R형 광학이성질체에 대해 높은 광학이성적 선택성을 가지고 있음을 알 수 있다.In the results of Table 2, in particular, it can be seen that lipase AK, PS, QLM and the like have high optical isoselectivity with respect to the R-type optical isomer of the racemate.

[실시예 3]라세미 (R),(S)-N-(2,6-디메틸페닐) 알라닌 알릴 에스테르는 바, 와 라세미 (R),(S)-N-(2,6-디메틸페닐) 알라닌 2-클로로에틸 에스테르 광학분할을 위한 효소선별 Example 3 Racemic (R), (S) -N- (2,6-dimethylphenyl) Alanine Allyl Esters are obtained from bar and racemic (R), (S) -N- (2,6-dimethyl Phenyl) Enzyme Screening for Alanine 2-Chloroethyl Ester Optical Fractionation

효소분말 2 ㎎을 0.1 M 인산 칼슘 버퍼(pH 7.0) 1 mL에 현탁시킨 후, 라세미 (R),(S)-N-(2,6-디메틸페닐) 알라닌 알릴 에스테르 20 ㎎을 투입한 뒤, 혼합물을 35℃에서 200 rpm으로 세이킹(shaking)하였다. 각각 3시간 및 24시간 후에 20 l를 견본추출(samping)하여 헥산:이소프로필 알코올의 혼합액(hexane:isopropyl alcohol =95:5)에 녹인 후, 5% Na2CO3를 사용하여 pH 10으로 조절하였다. 유기용매층에는 에스테르 화합물이 존재하였으며, 물층은 1N HCl을 사용하여 pH 3-4로 조절한 후, 에틸 아세테이트로 추출하였다. 층분리 후 유기용매를 감압증류에 의해 제거하고 헥산:이소프로필 알코올의 혼합액(95:5)에 녹인후, 선택도를 분석하였다.2 mg of enzyme powder was suspended in 1 mL of 0.1 M calcium phosphate buffer (pH 7.0), followed by 20 mg of racemic (R), (S) -N- (2,6-dimethylphenyl) alanine allyl ester. The mixture was shaken at 200 ° C. at 35 ° C. After 3 hours and 24 hours, 20 l of sample was sampled and dissolved in a mixture of hexane: isopropyl alcohol (hexane: isopropyl alcohol = 95: 5), and then adjusted to pH 10 using 5% Na 2 CO 3 . It was. An ester compound was present in the organic solvent layer, and the water layer was adjusted to pH 3-4 using 1N HCl, and then extracted with ethyl acetate. After separation of the layers, the organic solvent was removed by distillation under reduced pressure, dissolved in a mixed solution of hexane: isopropyl alcohol (95: 5), and the selectivity was analyzed.

상기 라세미 (R),(S)-N-(2,6-디메틸페닐) 알라닌 알릴 에스테르 대신에 라세미 (R),(S)-N-(2,6-디메틸페닐) 알라닌 2-클로로에틸 에스테르를 사용하였다는 점을 제외하고, 상기 동일한 방법으로 실험을 반복하였다.Racemic (R), (S) -N- (2,6-dimethylphenyl) alanine 2-chloro instead of racemic (R), (S) -N- (2,6-dimethylphenyl) alanine allyl ester The experiment was repeated in the same manner except that ethyl ester was used.

분석조건은 다음과 같다.The analysis conditions are as follows.

*HPLC 분석조건 * HPLC analysis condition

chiracel OD column, 30℃, 0.8mL, 254nmchiracel OD column, 30 ° C, 0.8mL, 254nm

acid 분석 - Hexane:Isopropyl acohol:Trifluoroacetic acid = 95: 5:0.1Acid analysis-Hexane: Isopropyl acohol: Trifluoroacetic acid = 95: 5: 0.1

ester 분석 - Hexane:Isopropyl acohol = 100:1ester analysis-Hexane: Isopropyl acohol = 100: 1

실험 결과를 하기 표 3에 나타낸다.The experimental results are shown in Table 3 below.

EnzymeEnzyme allyl esterallyl ester 2-chloroethyl ester2-chloroethyl ester conv.(%)conv. (%) %eep(R)% ee p (R) conv.(%)conv. (%) %eep(R)% ee p (R) 3hr3hr 24hr24hr 3hr3hr 24hr24hr 3hr3hr 24hr24hr 3hr3hr 24hr24hr Lipase AKLipase AK 33.533.5 50.350.3 99.499.4 98.898.8 49.249.2 -- 97.897.8 -- Lipase GFLipase GF -- 23.123.1 -- 100100 -- 14.014.0 -- 98.298.2 Lipase AGLipase AG -- 16.716.7 -- 5050 -- -- not selectivenot selective Lipase AHLipase AH 20.620.6 50.250.2 100100 99.299.2 49.849.8 -- 98.698.6 -- Novozym 435Novozym 435 not selectivenot selective not selectivenot selective Esterase CCEEsterase CCE -- 66.766.7 -- 44 not selectivenot selective Esterase PPEEsterase PPE -- 2.32.3 -- 85.485.4 -- 4.14.1 -- 91.891.8 Lipase TLLipase TL 14.414.4 53.253.2 94.894.8 80.880.8 53.553.5 -- 86.386.3 -- Lipase ULLipase UL -- 2.02.0 -- 100100 4.14.1 6.26.2 9393 2222 Lipase ALLipase AL -- 3.83.8 -- 100100 6.26.2 25.125.1 91.491.4 95.695.6 Lipase QLMLipase QLM 41.441.4 49.749.7 99.099.0 97.097.0 -- 60.860.8 -- 6262 Immobilizedlipase (Toyobo)Immobilizedlipase (Toyobo) 7.77.7 47.747.7 95.895.8 96.696.6 22.322.3 52.752.7 90.490.4 7070 Lipase BG*Lipase BG * -- -- -- -- -- 46.1(17hr)46.1 (17hr) -- 91.5(17hr)91.5 (17hr)

Conv. = ees/(ees+ eep)Conv. = ee s / (ee s + ee p )

Conv.는 전환율을 의미하며 eep와 ees는 생성물과 반응물의 광학순도를 나타내주는 인자이다. 다음과 같은 식으로 나타낼 수 있다.Conv. Is the conversion rate and ee p and ee s are factors that indicate the optical purity of products and reactants. It can be expressed as follows.

eep= [(R)-N-(2,6-디메틸페닐) 알라닌 - (S)-N-(2,6-디메틸페닐) 알라닌]/ [(R)-N-(2,6-디메틸페닐) 알라닌 + (S)-N-(2,6-디메틸페닐) 알라닌]ee p = [(R) -N- (2,6-dimethylphenyl) alanine-(S) -N- (2,6-dimethylphenyl) alanine] / [(R) -N- (2,6-dimethyl Phenyl) alanine + (S) -N- (2,6-dimethylphenyl) alanine]

ees= [(R)-N-(2,6-디메틸페닐) 알라닌 에스터 - (S)-N-(2,6-디메틸페닐) 알라닌 에스터]/ [(R)-N-(2,6-디메틸페닐) 알라닌 에스터 + (S)-N-(2,6-디메틸페닐) 알라닌 에스터]ee s = [(R) -N- (2,6-dimethylphenyl) alanine ester-(S) -N- (2,6-dimethylphenyl) alanine ester] / [(R) -N- (2,6 -Dimethylphenyl) alanine ester + (S) -N- (2,6-dimethylphenyl) alanine ester]

상기 표 3에서 볼 수 있는 바와 같이, 특정 리파아제는 상기 라세미체의 알릴 에스테르와 2-클로로에틸 에스테르 모두에 있어서 50% 전후의 높은 전환율과 광학이성적 반응 선택성을 가지며, 에스테르의 종류에 따라 최적 반응시간은 다름을 알 수 있다.As can be seen in Table 3, certain lipases have high conversion and optical isomeric reaction selectivity around 50% for both the allyl ester and 2-chloroethyl ester of the racemate, and the optimum reaction according to the type of ester. Time is different.

[실시예 4]Lipase OF에 의한 (R),(S)-N-(2,6-디메틸페닐) 알라닌 2-클로로에틸 에스테르의 광학분할 Example 4 Optical Fractionation of (R), (S) -N- (2,6-dimethylphenyl) alanine 2-chloroethyl ester by Lipase OF

20 ㎎의 라세미 (R),(S)-N-(2,6-디메틸페닐) 알라닌 2-클로로에틸 에스테르를 0.1 M 인산 칼륨 버퍼(pH 7.0) 1 mL에 넣고, Lipase OF를 2 ㎎ 첨가한 뒤, 35℃에서 200 rpm으로 세이킹하면서 반응을 수행하였다. 반응의 분석은 실시예 3과 같이 수행하였다. 17 시간 반응 후 41.4% 전환율에서 (S)-N-(2,6-디메틸페닐) 알라닌을 70.8 %ee로 얻어졌다. Candida rugosa 유래의 Lipase OF는 여러가지의 isozyme이 혼합된 형태로서 그 중의 특정한 isozyme의 경우 광학순도를 더 높일 수 있을 것이다.20 mg of racemic (R), (S) -N- (2,6-dimethylphenyl) alanine 2-chloroethyl ester was added to 1 mL of 0.1 M potassium phosphate buffer (pH 7.0) and 2 mg of Lipase OF was added. After that, the reaction was performed while shaking at 200 rpm at 35 ° C. Analysis of the reaction was carried out as in Example 3. (S) -N- (2,6-dimethylphenyl) alanine was obtained as 70.8% ee at 41.4% conversion after 17 hours reaction. Lipase OF, derived from Candida rugosa, is a mixture of several isozymes, which may enhance optical purity for certain isozymes.

[실시예 5]에스테르의 종류에 따른 효소 반응성 Example 5 Enzyme Reactivity According to Ester Type

다양한 라세미 (R),(S)-N-(2,6-디메틸페닐) 알라닌 에스테르 20 ㎎을 0.1 M 인산 칼륨 버퍼(pH 7.0) 1 mL에 녹인 뒤, Lipase PS를 2 ㎎ 첨가하여, 실시예 3과 같은 방법으로 실험을 행하고 분석을 실시하였다. 그 결과를 하기 표 4에 나타낸다.20 mg of various racemic (R), (S) -N- (2,6-dimethylphenyl) alanine esters were dissolved in 1 mL of 0.1 M potassium phosphate buffer (pH 7.0), followed by addition of 2 mg of Lipase PS. The experiment was conducted in the same manner as in Example 3, and the analysis was performed. The results are shown in Table 4 below.

EsterEster conv.(%)conv. (%) %eep(R)% ee p (R) 3hr3hr 24hr24hr 3hr3hr 24hr24hr 2-chloroethyl2-chloroethyl 48.348.3 52.652.6 97.097.0 90.290.2 2-bromoethyl2-bromoethyl 36.536.5 62.162.1 84.484.4 21.621.6 3-chloropropyl3-chloropropyl 12.212.2 48.048.0 99.299.2 99.499.4 3-bromopropyl3-bromopropyl 6.26.2 38.838.8 100100 100100 2,2-dichloroethyl2,2-dichloroethyl 35.535.5 51.851.8 98.698.6 93.293.2 1-chloro-2-propyl1-chloro-2-propyl -- 10.710.7 -- 100100 methoxyethylmethoxyethyl 39.639.6 52.152.1 99.099.0 92.092.0 ethoxyethylethoxyethyl 48.148.1 51.251.2 98.898.8 95.495.4 1-thioethoxyethyl1-thioethoxyethyl 10.010.0 34.434.4 88.088.0 96.896.8 isopropylisopropyl -- 11.211.2 -- 86.886.8 2-butyl2-butyl -- 12.712.7 -- 100100 2-pentyl2-pentyl -- 9.29.2 -- 61.461.4 3-methyl-1-butyl3-methyl-1-butyl -- 25.325.3 -- 100100 2-ethyl-1-hexyl2-ethyl-1-hexyl -- 28.428.4 -- 96.896.8 benzylbenzyl -- < 10<10 -- 96.696.6 2-phenoxy-1-ethyl2-phenoxy-1-ethyl 14.714.7 37.937.9 97.697.6 95.895.8 2,4-dichlorobenzyl2,4-dichlorobenzyl -- 4.14.1 -- 6565 oleyloleyl -- 12.012.0 -- 98.498.4 allylallyl 32.532.5 50.550.5 99.699.6 98.098.0 propargylpropargyl 42.842.8 53.553.5 96.696.6 87.087.0 methylmethyl 17.117.1 41.541.5 96.696.6 97.297.2 ethylethyl 3737 -- 98.698.6 -- n-propyln-propyl 3737 -- 97.697.6 -- n-butyln-butyl 33.933.9 -- 98.498.4 -- cyclohexylcyclohexyl 0.260.26 -- 23.223.2 -- 1-cyclopropylmethyl1-cyclopropylmethyl -- 8.88.8 -- 99.099.0 phenylphenyl -- -- 23.223.2 -- trifluoroethyltrifluoroethyl -- -- -- not selectivenot selective n-butanethioestern-butanethioester -- -- 45.045.0 --

Conv. = ees/(ees+ eep)Conv. = ee s / (ee s + ee p )

상기 표 4에서 볼 수 있는 바와 같이, 2-chloroethyl, 2-bromoethyl, allyl, methoxyethyl, ethoxyethyl ester 등이 특히 바람직하다.As can be seen in Table 4, 2-chloroethyl, 2-bromoethyl, allyl, methoxyethyl, ethoxyethyl ester and the like are particularly preferred.

[실시예 6]라세미 (R),(S)-N-(2,6-디메틸페닐) 알라닌 2-클로로에틸 에스테르의 광학분할 Example 6 Optical Fractionation of Racemic (R) and (S) -N- (2,6-dimethylphenyl) alanine 2-chloroethyl ester

라세미 (R),(S)-N-(2,6-디메틸페닐) 알라닌 2-클로로에틸 에스테르 500 ㎎을 2.5 mL 인산 칼륨 버퍼(pH 7.0)에 넣고(200 g/L scale), 효소 10 ㎎(ester : enzyme = 50:1)을 첨가한 뒤, 35℃에서 200 rpm으로 세이킹하여 반응을 행하였다. 반응의 분석은 실시예 3과 같은 방법으로 수행하였다. 그 결과를 하기 표 5에 나타낸다.500 mg of racemic (R), (S) -N- (2,6-dimethylphenyl) alanine 2-chloroethyl ester was placed in 2.5 mL potassium phosphate buffer (pH 7.0) (200 g / L scale), and enzyme 10 After addition of mg (ester: enzyme = 50: 1), the reaction was carried out by shaking at 35 rpm at 200 rpm. Analysis of the reaction was carried out in the same manner as in Example 3. The results are shown in Table 5 below.

EnzymeEnzyme SourceSource conv.(%)conv. (%) %eep(R)% ee p (R) 6hr6hr 24hr24hr 36hr36hr 6hr6hr 24hr24hr 36hr36hr Lipaes AHLipaes AH Burkholderia cepaciaBurkholderia cepacia 6.26.2 21.121.1 31.531.5 96.096.0 98.898.8 98.298.2 Lipase AKLipase AK Pseudomonas fluorescen sPseudomonas fluorescen s 16.816.8 36.836.8 45.445.4 96.896.8 98.898.8 97.297.2 Lipase PSLipase PS Burkholderia cepaciaBurkholderia cepacia 21.221.2 40.740.7 45.345.3 98.698.6 98.698.6 95.295.2 Lipase QLMLipase QLM Alcaligenes sp.Alcaligenes sp. 25.025.0 44.044.0 47.147.1 98.498.4 97.097.0 94.894.8 Lipase TLLipase TL Pseudomonas stutzeriPseudomonas stutzeri 25.925.9 41.641.6 44.844.8 94.494.4 90.490.4 88.488.4

Conv. = ees/(ees+ eep)Conv. = ee s / (ee s + ee p )

반응시간에 따라 약간의 차이는 있지만, 상기 라세미체는 표 5의 리파아제들에서 우수한 전환율과 광학순도를 나타내며, 특히, Lipase AK, PS, QLM 등은 50%에 가까운 전환율과 높은 광학순도를 보여준다.Although there is a slight difference depending on the reaction time, the racemate shows excellent conversion and optical purity in the lipases of Table 5, in particular, Lipase AK, PS, QLM, etc. show a conversion rate and high optical purity close to 50%. .

[실시예 7] 라세미 (R),(S)-N-(2,6-디메틸페닐) 알라닌 에스테르의 기질-효소 비율에 따른 광학분할 Example 7 Optical Segmentation According to Substrate-Enzyme Ratios of Racemic (R), (S) -N- (2,6-dimethylphenyl) alanine Ester

1 g의 각종 라세미 (R),(S)-N-(2,6-디메틸페닐) 알라닌 에스테르를 2.5 mL(400 g/L) 0.5 M 인산 칼륨 버퍼(pH 7)에 첨가한 뒤, Lipase PS 양을 달리하면서 기질과 효소의 비율을 변화시켜, 35℃에서 200 rpm으로 세이킹하며 반응을 행하였다. 분석은 실시예 3과 동일하게 수행하였다. 그 결과를 하기 표 6에 나타낸다.1 g of various racemic (R), (S) -N- (2,6-dimethylphenyl) alanine esters was added to 2.5 mL (400 g / L) 0.5 M potassium phosphate buffer (pH 7), followed by Lipase. The ratio of substrate and enzyme was varied while varying the amount of PS, and the reaction was carried out at 35 rpm at 200 rpm. The analysis was performed in the same manner as in Example 3. The results are shown in Table 6 below.

Lipase PSLipase PS 2-chloroethyl ester2-chloroethyl ester methoxyethyl estermethoxyethyl ester ethoxyethyl esterethoxyethyl ester amountamount conv.(%)conv. (%) %eep(R)% ee p (R) conv.(%)conv. (%) %eep(R)% ee p (R) conv.(%)conv. (%) %eep(R)% ee p (R) (ester:enzyme)(ester: enzyme) 1d.1d. 2d.2d. 1d.1d. 2d.2d. 1d.1d. 2d.2d. 1d.1d. 2d.2d. 1d.1d. 2d.2d. 1d.1d. 2d.2d. 10 ㎎ (100:1)10 mg (100: 1) 18.218.2 31.231.2 98.698.6 98.098.0 15.715.7 30.330.3 98.498.4 96.896.8 15.515.5 26.226.2 99.299.2 99.099.0 20 ㎎ (50:1)20 mg (50: 1) 29.429.4 39.339.3 98.498.4 97.697.6 28.728.7 38.138.1 98.298.2 97.697.6 22.522.5 31.131.1 99.099.0 98.498.4 50 ㎎ (25:1)50 mg (25: 1) 35.535.5 41.941.9 98.298.2 96.696.6 40.640.6 45.045.0 97.497.4 98.298.2 28.028.0 39.439.4 98.698.6 97.897.8

Conv. = ees/(ees+ eep)Conv. = ee s / (ee s + ee p )

상기 표 6에서 보는 바와 같이, 효소의 사용량이 많을수록 전환율은 높아짐을 알 수 있고, 효소의 최적 사용량은 효소의 종류, 에스테르의 종류, 전환율 및 광학순도 등 다양한 요소들을 모두 고려하여 선택하는 것이 바람직함을 알 수 있다.As shown in Table 6, it can be seen that the higher the amount of the enzyme is used, the higher the conversion rate, and the optimal amount of the enzyme is preferably selected in consideration of various factors such as the type of the enzyme, the type of the ester, the conversion rate and the optical purity. It can be seen.

[실시예 8]고정화 Lipase PS에 의한 라세미체 (R),(S)-N-(2,6-디메틸페닐) 알라닌 메틸 에스테르의 광학분할 Example 8 Optical Segmentation of Racemate (R), (S) -N- (2,6-dimethylphenyl) Alanine Methyl Ester by Immobilized Lipase PS

0.1 g Lipase PS를 0.1 M 인산 칼슘 버퍼(pH 7.0) 10 mL에 녹인 뒤, 교반하면서 상온에서 방치한 후 필터링하여 불용성 물질을 제거하였다. 용액 4.5 mL을 1 M 인산 칼륨 버퍼(pH 7.0)에 혼합한 후, Sepabeads FP-EP16 (Resindion) 30 ㎎과혼합하고 상온에서 24 시간동안 교반하였다. 그런 다음, 고정화 효소액을 필터링하였다. 이렇게 비드(beads)에 고정된 효소를 0.1 M 트리스 버퍼(Tris buffer :pH 7) 1 mL에 넣고, (R),(S)-N-(2,6-디메틸페닐) 알라닌 메틸 에스테르 10 ㎎을 첨가한 후, 35℃에서 200 rpm으로 세이킹하면서 17 시간동안 반응시켰다. 분석은 실시예 3과 같은 방법으로 수행하였다.0.1 g Lipase PS was dissolved in 10 mL of 0.1 M calcium phosphate buffer (pH 7.0), left at room temperature with stirring, and filtered to remove insoluble matters. 4.5 mL of the solution was mixed in 1 M potassium phosphate buffer (pH 7.0), then mixed with 30 mg of Sepabeads FP-EP16 (Resindion) and stirred at room temperature for 24 hours. Then, the immobilized enzyme solution was filtered. The enzyme immobilized in beads was added to 1 mL of 0.1 M Tris buffer (pH 7), and 10 mg of (R), (S) -N- (2,6-dimethylphenyl) alanine methyl ester was added. After the addition, the reaction was carried out at 35 rpm at 35 rpm for 17 hours. The analysis was performed in the same manner as in Example 3.

Eupergit C(R)(Rohm)에 대해서도 상기 동일한 방법으로 실험을 반복하였다. The experiment was repeated for Eupergit C (R) (Rohm) in the same manner .

Sepabeads FP-EP16의 경우 25% 전환율에서 98.5 %ee의 (R)-N-(2,6-디메틸페닐) 알라닌을, Eupergit(R)C의 경우 46% 전환율에서 98.4 %ee의 (R)-N-(2,6-디메틸페닐) 알라닌를 얻었다.98.5% ee (R) -N- (2,6-dimethylphenyl) alanine at 25% conversion for Sepabeads FP-EP16 and 98.4% ee (R) -at 46% conversion for Eupergit (R) C. N- (2,6-dimethylphenyl) alanine was obtained.

[실시예 9]고정화 Lipase PS에 의한 라세미 (R),(S)-N-(2,6-디메틸페닐) 알라닌 2-클로로에틸 에스테르의 광학분할 Example 9 Optical Fractionation of Racemic (R), (S) -N- (2,6-dimethylphenyl) alanine 2-chloroethyl Ester by Immobilized Lipase PS

0.1 g Lipase PS를 0.1 M 인산 나트륨 버퍼(pH 6.0) 5.0 mL에 녹인 뒤 교반하면서, 폴리아크릴레이트 수지인 XAD(R)-7HP(Rohm & Haas)의 양을 100 ㎎(resin:enzyme = 1:1), 250 ㎎, 500 ㎎, 1.0 g으로 각각 달리하여 첨가하였다. 그런 다음, 30℃에서 24 시간동안 교반한 후 필터링하여, 수지와 불용성 물질을 0.1 M 인산 나트륨 버퍼(pH 6.0)에 넣고 글루타르알데히드(glutaraldehyde)를 5 uL첨가한 후, 상온에서 3 시간동안 교반하였다. 얻어진 고정화 효소(Lipase PS 10 ㎎에 해당)를 0.5 M 인산 칼륨 버퍼(pH 8) 1 mL에 넣고, (R),(S)-N-(2,6-디메틸페닐) 알라닌 2-클로로에틸 에스테르 100 ㎎을 첨가한 후, 35C에서 200 rpm으로 세이킹하면서 16 시간동안 반응시켰다. 분석은 실시예 3과 같은 방법으로 수행하였다. 그 결과를 하기 표 7에 나타낸다.0.1 g Lipase PS was dissolved in 5.0 mL of 0.1 M sodium phosphate buffer (pH 6.0), followed by stirring, and the amount of polyacrylate resin XAD (R) -7HP (Rohm & Haas) was 100 mg (resin: enzyme = 1: 1). 1), 250 mg, 500 mg, and 1.0 g were added differently. Then, the mixture was stirred at 30 ° C. for 24 hours and then filtered. The resin and the insoluble substance were added to 0.1 M sodium phosphate buffer (pH 6.0), and 5 uL of glutaraldehyde was added thereto, followed by stirring at room temperature for 3 hours. It was. The obtained immobilized enzyme (corresponding to Lipase PS 10 mg) was added to 1 mL of 0.5 M potassium phosphate buffer (pH 8), and (R), (S) -N- (2,6-dimethylphenyl) alanine 2-chloroethyl ester After addition of 100 mg, the reaction was carried out for 16 hours while shaking at 200 rpm at 35C. The analysis was performed in the same manner as in Example 3. The results are shown in Table 7 below.

Lipase PSLipase PS XAD-7HP amountXAD-7HP amount conv.(%)conv. (%) %eep(R)% ee p (R) 100 ㎎100 mg 100 ㎎100 mg 1616 87.087.0 100 ㎎100 mg 250 ㎎250 mg 2323 93.093.0 100 ㎎100 mg 500 ㎎500 mg 4040 95.695.6 100 ㎎100 mg 1000 ㎎1000 mg 4949 96.396.3

Conv. = ees/(ees+ eep)Conv. = ee s / (ee s + ee p )

[실시예 10] 라세미 (R),(S)-N-(2,6-디메틸페닐) 알라닌 메틸 에스테르의 양산 광학분할 Example 10 Mass production optical split of racemic (R), (S) -N- (2,6-dimethylphenyl) alanine methyl ester

30 mL 증류수에 라세미 (R),(S)-N-(2,6-디메틸페닐) 알라닌 메틸 에스테르 20 mL(400 g/L 농도)를 첨가하고, 500 ㎎의 Lipase PS를 첨가한 후, 40℃에서 스터링(30%, Mettler DL70 titrator)하면서 반응을 수행하였다. 반응액의 pH를 일정하게 유지하기 위하여 1N NaOH를 자동적으로 첨가하였다(Mettler DL70 titrator). 24 시간 후에 250 ㎎의 Lipase PS를 추가로 첨가하였다. 분석은 실시예 3과 같은 방법으로 수행하였다. 40.2%의 전환율(6day)에서 96.9 %ee의 (R)-N-(2,6-디메틸페닐) 알라닌을 얻었다.To 30 mL distilled water was added 20 mL (400 g / L concentration) of racemic (R), (S) -N- (2,6-dimethylphenyl) alanine methyl ester, followed by addition of 500 mg Lipase PS. The reaction was carried out with sterling (30%, Mettler DL70 titrator) at 40 ° C. 1N NaOH was automatically added to maintain the pH of the reaction solution (Mettler DL70 titrator). After 24 hours an additional 250 mg of Lipase PS was added. The analysis was performed in the same manner as in Example 3. 96.9% ee of (R) -N- (2,6-dimethylphenyl) alanine was obtained at a conversion rate of 60.2 days of 40.2%.

[실시예 11] 효소에 의한 (R),(S)-N-(2,6-디메틸페닐) 알라닌 메틸 에스테르 광학분할과 (R)-N-(2,6-디메틸페닐) 알라닌의 분리 Example 11 By enzyme Separation of (R), (S) -N- (2,6-dimethylphenyl) alanine methyl ester optical splitting from (R) -N- (2,6-dimethylphenyl) alanine

실시예 9의 반응액을 여과하여 불용성 물질을 제거한 뒤, 여과액(filtrate)를 2.72 g의 Accurel MP1000(메탄올과 증류수로 세척한 후 사용)가 충진되어있는 유리 컬럼(glass column)에 통과시켰다. 여과액에 에틸 아세테이트를 넣고 pH 9로 조절한 후, 에틸 아세테이트층을 키랄(chiral) HPLC로 분석하여, (S)-ester:(R)-ester = 95.6:1.1(area %ratio), ester:acid = 96.7:0.87(area %ratio)를 얻었다. 또한, 수용액층을 산성화(acidify)하여 에틸 아세테이트로 추출하고 용매를 증발시킨 후, 키랄 HPLC로 분석하여, (R)-acid 97.2 %ee , ester:acid = 0.42:99.58(area % ratio)로 광학적으로 순수한 흰색의 (R)-N-(2,6-디메틸페닐) 알라닌을 정량적으로 얻었다.The reaction solution of Example 9 was filtered to remove insoluble matters, and then the filtrate was passed through a glass column filled with 2.72 g of Accurel MP1000 (used after washing with methanol and distilled water). After adding ethyl acetate to the filtrate and adjusting to pH 9, the ethyl acetate layer was analyzed by chiral HPLC, and (S) -ester: (R) -ester = 95.6: 1.1 (area% ratio), ester: acid = 96.7: 0.87 (area% ratio) was obtained. In addition, the aqueous layer was acidified, extracted with ethyl acetate, the solvent was evaporated, and analyzed by chiral HPLC. The pure white (R) -N- (2,6-dimethylphenyl) alanine was obtained quantitatively.

[실시예 12]효소에 의한 라세미 (R),(S)-N-(2,6-디메틸페닐) 알라닌 메톡시에틸 에스테르의 광학분할과 (R)-N-(2,6-디메틸페닐) 알라닌의 분리 Example 12 Optical Fractionation of Racemic (R), (S) -N- (2,6-dimethylphenyl) Alanine Methoxyethyl Ester by Enzyme and (R) -N- (2,6-dimethylphenyl Isolation of alanine

24 g의 라세미 (R),(S)-N-(2,6-디메틸페닐) 알라닌 메톡시에틸 에스테르를 0.5 M 인산 칼륨 버퍼(pH 7.0) 50 mL에 첨가하고 Lipase PS 1.0 g를 첨가한 뒤, 35℃에서 200 rpm으로 세이킹하면서 반응을 수행하였다. 그런 다음, 40% 전환율에서 반응을 중지한 후, 에틸 아세테이트를 첨가하고 수용액층의 pH를 10으로 조절한 뒤, 에틸 아세테이트층을 분리하고 물을 첨가하였다. 수용액층의 pH를 3-4로 조절하고, 에틸 아세테이트층을 MgSO4로 건조한 후 필터링하고, 에틸 아세테이트를 증발시켜, (R)-N-(2,6-디메틸페닐) 알라닌(키랄 HPLC분석, %96 ee) 5.3g 얻었다( 57.4% isolated yield).24 g of racemic (R), (S) -N- (2,6-dimethylphenyl) alanine methoxyethyl ester was added to 50 mL of 0.5 M potassium phosphate buffer (pH 7.0) and 1.0 g of Lipase PS was added, followed by 35 ° C The reaction was performed while shaking at 200 rpm. Then, after stopping the reaction at 40% conversion, ethyl acetate was added and the pH of the aqueous layer was adjusted to 10, the ethyl acetate layer was separated and water was added. The pH of the aqueous layer was adjusted to 3-4, and the ethyl acetate layer was washed with MgSO4After drying with filtered, ethyl acetate was evaporated to give 5.3 g of (R) -N- (2,6-dimethylphenyl) alanine (chiral HPLC analysis,% 96 ee) (57.4% isolated yield).

[실시예 13](R)-N-(2,6-디메틸페닐) 알라닌의 에스테르화 반응 Example 13 Esterification Reaction of (R) -N- (2,6-dimethylphenyl) alanine

5.03 g의 (R)-N-(2,6-디메틸페닐) 알라닌(96 ee%)를 15 g의 메틸 알코올에 녹인 후, 0℃에서 티오닐 클로라이드(thionyl chloride) 3.25 g (1.05 equivalent)을 적가하였다. 2 시간동안 환류(reflux)시킨 후 GC분석한 결과, 2.5%의 (R)-N-(2,6-디메틸페닐) 알라닌가 존재함을 확인하였다. 용매를 증발시킨 후, 톨루엔 30 mL을 첨가하고 50 mL Na2CO3로 5 번 세척한 뒤, 톨루엔층을 분리하고 MgSO4로 건조하여 용매를 제거하였다. 얻어진 (R)-N-(2,6-디메틸페닐) 알라닌 메틸 에스테르(4.2g, 78% isolated yield)를 키랄 HPLC로 분석한 결과, 97.3 %ee의 광학순도를 나타내었다(chemical purity 97.2% ester determined by GC analysis).5.03 g of (R) -N- (2,6-dimethylphenyl) alanine (96 ee%) was dissolved in 15 g of methyl alcohol, followed by 3.25 g (1.05 equivalent) of thionyl chloride at 0 ° C. Added dropwise. GC analysis after reflux for 2 hours revealed that 2.5% of (R) -N- (2,6-dimethylphenyl) alanine was present. After evaporating the solvent, 30 mL of toluene was added and washed 5 times with 50 mL Na 2 CO 3 , and then the toluene layer was separated and dried over MgSO 4 to remove the solvent. The obtained (R) -N- (2,6-dimethylphenyl) alanine methyl ester (4.2 g, 78% isolated yield) was analyzed by chiral HPLC and showed an optical purity of 97.3% ee (chemical purity 97.2% ester). determined by GC analysis).

본 발명이 속하는 분야에서 통상의 지식을 가지라면, 상기 내용을 바탕으로 본 발명의 범주내에서 다양한 응용 및 변형을 행하는 것이 가능할 것이다.It will be possible to make various applications and modifications within the scope of the present invention based on the above contents, if one of ordinary skill in the art of the present invention belongs.

본 발명에 따르면, 광학이성질체들(R형 및 S형)의 물리화학적 특성의 차이가 적어서 종래의 방법으로는 광학분할이 어려운, 라세미 (R),(S)-N-(2,6-디메틸페닐) 알라닌 에스테르를 간단하고 저렴한 방법으로 광학분할하여, 항균활성을 가지는 Metalaxyl, Benalaxyl, Furalaxyl 등의 합성을 위한 중간체인 (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌과, 그것의 에스테르 화합물을 높은 광학순도와 수율로서 용이하게 제조할 수 있다.According to the present invention, the racemic (R), (S) -N- (2,6- Dimethylphenyl) alanine esters are optically divided in a simple and inexpensive way to produce (R)-or (S) -N- (2,6-dimethylphenyl), which is an intermediate for the synthesis of metalaxyl, Benalaxyl, Furalaxyl, etc. Alanine and its ester compound can be easily produced with high optical purity and yield.

Claims (12)

(A) 하기 화학식 1의 라세미 (R),(S)-N-(2,6-디메틸페닐) 알라닌 에스테르를, 그것의 특정 광학이성질체(R형 또는 S형 광학이성질체)에 대해 가수분해 특이성을 가지는 반응 유효량의 효소와 반응시켜, (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌을 생성하는 단계; 및(A) Hydrolysis specificity of racemic (R), (S) -N- (2,6-dimethylphenyl) alanine ester of formula (1) with respect to its specific optical isomer (R type or S type optical isomer) Reacting with an effective amount of an enzyme having to produce (R)-or (S) -N- (2,6-dimethylphenyl) alanine; And (B) 상기 반응물 중의 (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌을, 미반응 대응 (S)- 또는 (R)-N-(2,6-디메틸페닐) 알라닌 에스테르로부터 분리하는 단계를 포함하는 것으로 구성되어있는, 광학적으로 순수한 (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌의 제조방법.(B) unreacted corresponding (S)-or (R) -N- (2,6-dimethylphenyl) to (R)-or (S) -N- (2,6-dimethylphenyl) alanine in the reaction product; A process for preparing optically pure (R)-or (S) -N- (2,6-dimethylphenyl) alanine, comprising the step of separating from an alanine ester. (1) (One) 상기 식에서, R 은 치환되거나 또는 치환되지않은 탄소수 1-18의 선형 내지 가지형 알킬 또는 알케닐, 치환되거나 또는 치환되지않은 탄소 3-6의 시클로알킬, 치환되거나 또는 치환되지않은 아릴 알킬, 및 치환되거나 또는 치환되지않은 헤테로아릴 알킬로 이루어진 군에서 선택된다.Wherein R is substituted or unsubstituted linear to branched alkyl or alkenyl having 1 to 18 carbon atoms, substituted or unsubstituted cycloalkyl having 3 to 6 carbon atoms, substituted or unsubstituted aryl alkyl, and substituted Or unsubstituted heteroaryl alkyl. (A') 상기 화학식 1의 라세미 (R),(S)-N-(2,6-디메틸페닐) 알라닌 에스테르를, 그것의 특정 광학이성질체(R형 또는 S형 광학이성질체)에 대해 가수분해 특이성을 가지는 반응 유효량의 효소와 반응시켜, (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌을 생성하는 단계; 및,(A ') Hydrolysis of the racemic (R), (S) -N- (2,6-dimethylphenyl) alanine ester of the formula (1) with respect to its specific optical isomer (R type or S type optical isomer) Reacting with an effective amount of an enzyme having a specificity to produce (R)-or (S) -N- (2,6-dimethylphenyl) alanine; And, (B') 상기 반응물 중의 미반응 대응 (S)- 또는 (R)-N-(2,6-디메틸페닐) 알라닌 에스테르를, 생성된 (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌으로부터 분리하는 단계를 포함하는 것으로 구성되어있는, 광학적으로 순수한 (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌 에스테르의 제조방법.(B ') The unreacted corresponding (S)-or (R) -N- (2,6-dimethylphenyl) alanine ester in the reaction product is produced by (R)-or (S) -N- (2,6 A process for preparing optically pure (R)-or (S) -N- (2,6-dimethylphenyl) alanine ester, comprising the step of separating from -dimethylphenyl) alanine. 제 2 항에 있어서, 상기 단계(B') 대신에 하기 단계(C')를 포함하거나 또는 단계(B') 이후에 하기 단계(C')를 더 포함하는 제조방법.The process according to claim 2, comprising the following step (C ') instead of step (B') or further comprising the following step (C ') after step (B'). (C') 상기 (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌을 알코올(R-OH; 여기서의 R 은 화학식 1에서와 동일함)과 반응시켜, (R)- 또는 (S)-N-(2,6-디메틸페닐) 알라닌 에스테르를 합성하는 단계.(C ′) reacting (R)-or (S) -N- (2,6-dimethylphenyl) alanine with an alcohol (R-OH; wherein R is the same as in Formula 1), wherein (R) Or synthesizing (S) -N- (2,6-dimethylphenyl) alanine ester. 제 1 항에 있어서, R 이 알릴, 2-클로로에틸, 2-브로모에틸 메톡시에틸, 또는 에톡시에틸인 제조방법.The process according to claim 1, wherein R is allyl, 2-chloroethyl, 2-bromoethyl methoxyethyl, or ethoxyethyl. 제 1 항 또는 제 2 항에 있어서, 상기 효소는 미생물, 동물 또는 식물 유래의 리파아제(lipase), 프로테아제(protease) 또는 에스터라아제(esterase) 중에 선택되는 제조방법.The method according to claim 1 or 2, wherein the enzyme is selected from lipases, proteases or esterases derived from microorganisms, animals or plants. 제 5 항에 있어서, 상기 효소는 리파아제인 제조방법.The method of claim 5, wherein the enzyme is a lipase. 제 5 항에 있어서, 상기 효소는 Pseudomonas 유래 리파아제 AK, Toyobo Immobilized lipase, Liopprotein lipase, Burkholderia 유래의 리파아제 PS, AH, Alcaligenes 유래의 리파아제 QLM, 및 Candida 유래 리파아제 OF에서 선택된 하나 또는 둘 이상인 제조방법.The method according to claim 5, wherein the enzyme is one or more selected from Pseudomonas-derived lipase AK, Toyobo Immobilized lipase, Liopprotein lipase, Burkholderia-derived lipase PS, AH, Alcaligenes-derived lipase QLM, and Candida-derived lipase OF. 제 1 항 또는 제 2 항에 있어서, 상기 효소의 가수분해 반응을, 수용액, 또는 소량의 유기용매를 포함하는 유기용매-수용액 혼합액 중에서 행하는 제조방법.The production method according to claim 1 or 2, wherein the hydrolysis reaction of the enzyme is performed in an aqueous solution or an organic solvent-aqueous liquid mixture containing a small amount of an organic solvent. 제 8 항에 있어서, 상기 유기용매는, 아세톤, 아세토나이트릴, 알코올 등의 친수성 용매나, 이소프로필에테르, tert-부틸 메틸 에테르(TBME), 클로로포름, 디클로로메탄, 사염화탄소, 헥산, 톨루엔 등의 소수성 용매이거나, 또는 이들의 혼합 용매인 제조방법.The organic solvent according to claim 8, wherein the organic solvent is hydrophilic solvent such as acetone, acetonitrile, alcohol, hydrophobicity such as isopropyl ether, tert-butyl methyl ether (TBME), chloroform, dichloromethane, carbon tetrachloride, hexane, toluene and the like. A process for producing a solvent or a mixed solvent thereof. 제 1 항 또는 제 2 항에 있어서, 상기 효소의 가수분해 반응을 0-60℃ 및 pH 4-12에서 행하는 제조방법.The production method according to claim 1 or 2, wherein the hydrolysis reaction of the enzyme is performed at 0-60 ° C and pH 4-12. 제 1 항 또는 제 2 항에 있어서, 반응계가 수용액만으로 이루어진 경우에는,유기용매를 사용하여 미반응 에스테르 화합물(R형 또는 S형 광학이성질체)을 추출하고, 반응계가 유기용매-수용액 혼합액으로 이루어진 경우에는, 미반응 에스테르 화합물이 용해되어있는 유기용매층을 분리함으로써 이를 분리하는 방법.The method according to claim 1 or 2, wherein when the reaction system consists only of an aqueous solution, an organic solvent is used to extract an unreacted ester compound (R or S type optical isomer), and the reaction system consists of an organic solvent-aqueous solution mixture. The method for separating this by separating the organic solvent layer in which the unreacted ester compound is dissolved. 제 1 항 또는 제 2 항에 있어서, 상기 효소는 담체에 고정화되어있는 효소인 제조방법.The production method according to claim 1 or 2, wherein the enzyme is an enzyme immobilized on a carrier.
KR1020020081152A 2002-12-18 2002-12-18 Method for preparing an R- or S- form of N-2,6-dimethyl phenyl alanine and a counter enantiomeric form of N-2,6-dimethyl phenyl alanine ester thereto using enzyme KR100654587B1 (en)

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