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KR100657204B1 - The method of making optically active 3-hydroxy-?-butyrolactone by enzymatic method - Google Patents

The method of making optically active 3-hydroxy-?-butyrolactone by enzymatic method Download PDF

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KR100657204B1
KR100657204B1 KR1020040057426A KR20040057426A KR100657204B1 KR 100657204 B1 KR100657204 B1 KR 100657204B1 KR 1020040057426 A KR1020040057426 A KR 1020040057426A KR 20040057426 A KR20040057426 A KR 20040057426A KR 100657204 B1 KR100657204 B1 KR 100657204B1
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butyrolactone
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황순욱
유혜연
정선호
이태임
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Abstract

본 발명은 하기 [반응식 1]에서 일반식 (1)로 표시되는 라세믹 4-클로로-3-히드록시부틸산 알킬 에스테르(4-chloro-3-hydroxybutyric acid alkyl ester)로부터 효소적 방법에 의해 일반식 (3)으로 표시되는 광학활성 3-히드록시-감마-부티로락톤(3-hydroxy-γ-butyrolactone)의 제조방법에 관한 것이다. 좀 더 상세하게는 수용액상 또는 수용액을 포함하는 용매상에서 반응물인 라세믹 4-클로로-3-히드록시부틸산 알킬 에스테르를 리파제 또는 리파제 생산능을 갖는 미생물을 이용하여 입체 선택적으로 가수분해 반응하여 광학활성 3-히드록시-감마-부티로락톤을 제조하는 방법에 관한 것이다. The present invention is generalized by enzymatic method from racemic 4-chloro-3-hydroxybutyric acid alkyl ester represented by the general formula (1) in the following [Scheme 1]. It relates to a method for producing optically active 3-hydroxy-gamma-butyrolactone represented by formula (3). More specifically, the racemic 4-chloro-3-hydroxybutyl acid alkyl ester, which is a reactant, in an aqueous solution or a solvent containing the aqueous solution is subjected to stereoselective hydrolysis reaction using a microorganism having a lipase or lipase producing ability to optically react. A method for preparing active 3-hydroxy-gamma-butyrolactone.

본 발명의 방법은 기존에 보고된 방법들에 비해 공정이 쉽고, 분리 및 회수가 간편하여 실제 공정에 유용할 것으로 판단된다.Compared to the previously reported methods, the method of the present invention is easy to process, and easy to separate and recover.

[반응식 1]Scheme 1

Figure 112006019834397-pat00001
Figure 112006019834397-pat00001

( R = CnH2n+1(n=1~8) )(R = C n H 2n + 1 (n = 1 ~ 8))

메틸 4-클로로-3-히드록시부티레이트, 에틸 4-클로로-3-히드록시부티레이트, 부틸 4-클로로-3-히드록시부티레이트, 3-히드록시-감마-부티로락톤, 리파제, 가수분해반응, 광학활성Methyl 4-chloro-3-hydroxybutyrate, ethyl 4-chloro-3-hydroxybutyrate, butyl 4-chloro-3-hydroxybutyrate, 3-hydroxy-gamma-butyrolactone, lipase, hydrolysis reaction, Optical activity

Description

효소적 방법에 의한 광학활성 3-히드록시-감마-부티로락톤의 제조 방법 {The method of making optically active 3-hydroxy-γ-butyrolactone by enzymatic method}The method of making optically active 3-hydroxy-γ-butyrolactone by enzymatic method}

본 발명은 라세믹 4-클로로-3-히드록시부틸산 알킬 에스테르(4-chloro-3-hydroxybutyric acid alkyl ester)로부터 효소적 방법에 의해 광학활성 3-히드록시-감마-부티로락톤(3-hydroxy-γ-butyrolactone)을 제조하는 방법에 관한 것이다. 좀 더 상세하게 수용액상에서 리파제 또는 리파제 생산능을 갖는 미생물을 이용하여 [반응식 1]에서 일반식 (1)로 표시되는 라세믹 4-클로로-3-히드록시부틸산 알킬 에스테르를 입체 선택적으로 가수분해 반응하여 일반식 (3)으로 표시되는 광학활성 3-히드록시-감마-부티로락톤을 제조하는 방법에 관한 것이다.

Figure 112006019834397-pat00002

( R = CnH2n+1(n=1~8) )The present invention relates to optically active 3-hydroxy-gamma-butyrolactone by enzymatic method from racemic 4-chloro-3-hydroxybutyric acid alkyl ester. hydroxy-γ-butyrolactone). In more detail, using a microorganism having a lipase or lipase-producing ability in an aqueous solution, stereoselective hydrolysis of the racemic 4-chloro-3-hydroxybutyl acid alkyl ester represented by the general formula (1) in [Scheme 1] Reaction relates to a method for producing optically active 3-hydroxy-gamma-butyrolactone represented by the general formula (3).
Figure 112006019834397-pat00002

(R = C n H 2n + 1 (n = 1 ~ 8))

상기에서 언급한 광학활성 3-히드록시-감마-부티로락톤은 L-카르니틴(L-carnitine), 감마-아미노-베타-히드록시부틸산 (r-amino-beta-hydroxybutyric acid), 고지혈증 치료제, 에이즈 치료제 등의 의약품 중간체나 농약 중간체로서 활용범위가 넓다. 또한 본 발명에 의한 제조방법은 기존의 방법에 비해 간편하고, 반응 후 분리 및 회수가 쉽기 때문에 실제 공정에 유리하게 이용될 수 있다.The above-mentioned optically active 3-hydroxy-gamma-butyrolactone is L-carnitine, gamma-amino-beta-hydroxybutyric acid, therapeutic agents for hyperlipidemia, Wide range of applications as pharmaceutical intermediates and pesticide intermediates such as AIDS treatments. In addition, the production method according to the present invention can be advantageously used in the actual process because it is simpler than the existing method, and easy to separate and recover after the reaction.

현재까지 보고된 (S)-3-히드록시-감마-부티로락톤을 제조하는 기술은 다음과 같다.The technology for producing (S) -3-hydroxy-gamma-butyrolactone reported to date is as follows.

Yuasa등 (Liebigs Ann. IRecueil. 1997, 1877-1879)은 에틸 4-클로로-3-옥소부타노에이트(ethyl 4-chloro-3-oxobutanoate)를 Ru-(R)-p-tolyl-BINAP촉매를 이용하여 94%ee 광학순도를 갖는 에틸 (S)-4-클로로-3-히드록시부타노에이트(ethyl 4-chloro-3-hydroxybutanoate)를 합성하고 여기에 염산을 처리하여 (S)-3-히드록시-감마-부티로락톤을 제조하였다. 이때 수율은 83%이었고, 광학순도는 유지가 되었다. 하지만 상기의 공정은 (S)-에틸 4-클로로-3-히드록시부타노에이트의 광학순도에 따라 (S)-3-히드록시-감마-부티로락톤의 품질이 결정되어지며 실제 공정에 적용하기는 어렵다.Yuasa et al. (Liebigs Ann. IRecueil. 1997, 1877-1879) used ethyl 4-chloro-3-oxobutanoate as a Ru- (R) -p-tolyl-BINAP catalyst. Was synthesized ethyl (S) -4-chloro-3-hydroxybutanoate having 94% ee optical purity using hydrochloric acid and treated with (S) -3- Hydroxy-gamma-butyrolactone was prepared. At this time, the yield was 83%, the optical purity was maintained. However, in the above process, the quality of (S) -3-hydroxy-gamma-butyrolactone is determined by the optical purity of (S) -ethyl 4-chloro-3-hydroxybutanoate and is applied to the actual process. It's hard to do.

Suzuki등(Enzyme Microbiology and Technology, 1999, 24. 13-20)은 탈염소화능을 갖는 미생물을 이용하여 라세믹 에틸 4-클로로-3-히드록시부티레이트로 부터 99.8%ee의 광학순도를 갖는 (R)-에틸 4-클로로-3-히드록시부티레이트와 92.4%ee의 광학순도를 갖는 (S)-3-히드록시-감마-부티로락톤을 제조하였다. Suzuki et al. (Enzyme Microbiology and Technology, 1999, 24. 13-20) have an optical purity of 99.8% ee from racemic ethyl 4-chloro-3-hydroxybutyrate using a microorganism with dechlorination (R). ) -Ethyl 4-chloro-3-hydroxybutyrate and (S) -3-hydroxy-gamma-butyrolactone having an optical purity of 92.4% ee were prepared.

한편, 천종필등(특10-0310935, 미국특허 제6,122,122호)은 아밀로펙틴을 효소와 반응시켜 올리고당을 제조하고 이를 염기성 음이온 교환수지 및 산화제와 반응시켜 (S)-3,4-디히드록시-부틸산을 얻고, 이를 탈착하여 에스테르화 반응과 고리화 반응을 통하여 광학순도가 높게 유지되는 (S)-베타-히드록시-감마-부티로락톤을 제조하는 방법을 개발하였다. 상기의 방법 이외에도 녹말이나 테트리고(말토올리고 당)를 원료물질로 사용하여 보다 높은 수율로 제조하는 방법(미국특허 제6,124,122)이 알려져 있으나 이러한 방법들은 다당의 분자들을 분해하여 제조하는 방법이기 때문에 분해 과정 중에 원하지 않는 여러가지 화합물이 생성되며 이때 생성되는 화합물들의 대부분은 목적 화합물과의 구조적 유사성으로 인해 물리적 성질이 비슷하여 정제나 제거가 매우 어려워 순도가 떨어지는 단점이 있다.On the other hand, Chun Jong Pil et al. (Japanese Patent No. 10-0310935, U.S. Patent No. 6,122,122) prepare oligosaccharides by reacting amylopectin with enzymes and reacting them with basic anion exchange resins and oxidizing agents (S) -3,4-dihydroxy-butyl. An acid was obtained, and a method of preparing (S) -beta-hydroxy-gamma-butyrolactone in which the optical purity was maintained through the esterification reaction and the cyclization reaction was developed. In addition to the above method, a method of producing a higher yield using starch or tetrigo (maltooligosaccharide) as a raw material (US Pat. No. 6,124,122) is known, but these methods are decomposed because they are prepared by decomposing polysaccharide molecules. Various undesired compounds are produced during the process, and most of the resulting compounds have similar physical properties due to structural similarities to the target compound, which is very difficult to purify or remove.

또한 곽병성 등(특2003-0004902)은 치환된 사과산(malic acid) 유도체를 수소화하여 생성되는 디-히드록시-부틸산 메틸 에스테르를 산촉매 존재하에서 고리화 반응시켜 (S)-베타-히드록시-감마-부티로락톤을 제조하였다. 그러나 이러한 방법은 고온 고압 반응으로 정해진 압력과 온도 범위가 벗어날 경우 생성물의 광학순도와 수율이 낮아지고 촉매의 비활성화 속도가 증가 되는 단점이 있다. In addition, Kwak, Byung-Sung et al. (Sec. 2003-0004902) cyclized a di-hydroxy-butyl acid methyl ester produced by hydrogenating a substituted malic acid derivative in the presence of an acid catalyst (S) -beta-hydroxy-gamma. Butyrolactone was prepared. However, this method has a disadvantage in that the optical purity and yield of the product is lowered and the deactivation rate of the catalyst is increased when the pressure and the temperature range are out of the high temperature and high pressure reaction.

이호성 등(특2003-0065192)은 고순도의 (S)-에피클로로하이드린(epichlorohydrin)으로 부터 (S)-4-클로로-3-히드록시부티로니트릴(4-chloro-3-hydroxybutyronitrile)을 합성하고 이것을 원료물질로 하여 (S)-3-히드록시-감마-부티로락톤을 제조하였다. 그러나 상기의 제조방법은 최초의 원료물질인 (S)-에피클로로하이드린의 가격이 비싸기 때문에 제조원가가 높은 단점이 있다.Lee Ho-Sung et al. (Sec. 2003-0065192) synthesized (S) -4-chloro-3-hydroxybutyronitrile from high purity (S) -epichlorohydrin. (S) -3-hydroxy-gamma-butyrolactone was prepared as a starting material. However, the manufacturing method has a disadvantage in that the manufacturing cost is high because the price of the first raw material (S)-epichlorohydrin is expensive.

다이셀(Daicel)사에서 발표한 일본특허(공개번호2002-204699)의 경우 미생물을 이용하여 광학활성 에틸 4-클로로-3-히드록시-부티레이트로 부터 광학활성 3-히드록시-감마-부티로락톤을 제조하였다. 이 때 광학순도는 93.9%ee 이상이었으나 반응물의 농도가 낮아서 실제 공정에 적용하기는 힘들다. 또한 이들은 라세믹 에틸 4-클로로-3-히드록시-부티레이트로부터 광학활성 3-히드록시-감마-부티로락톤을 제조하였으나 반응물의 농도 및 광학순도가 매우 낮았다. 또한 미생물을 이용하여 에틸 4-클로로-3-히드록시-부티레이트로 부터 광학활성 3-히드록시-감마-부티로락톤을 제조하는데 있어서 반응 메카니즘을 정확하게 설명하지 못하고 있다.Japanese Patent Publication No. 2002-204699 published by Daicel Co., Ltd. discloses optically active 3-hydroxy-gamma-butyro from optically active ethyl 4-chloro-3-hydroxy-butyrate using microorganisms. Lactones were prepared. At this time, the optical purity was more than 93.9% ee but the concentration of the reactants is low, so it is difficult to apply to the actual process. They also prepared optically active 3-hydroxy-gamma-butyrolactone from racemic ethyl 4-chloro-3-hydroxy-butyrate, but the concentration and optical purity of the reactants were very low. In addition, the reaction mechanism in preparing optically active 3-hydroxy-gamma-butyrolactone from ethyl 4-chloro-3-hydroxy-butyrate using microorganisms is not accurately described.

본 발명에 의한 제조방법은 리파제 또는 리파제 생산능을 갖는 미생물을 이용하여 라세믹 4-클로로-3-히드록시부틸산 알킬 에스테르를 가수분해 반응하여 광학활성 3-히드록시-감마-부티로락톤을 제조하는 방법으로 현재까지 보고된 바가 없는 새로운 공정이다. The production method according to the present invention hydrolyzes racemic 4-chloro-3-hydroxybutyl acid alkyl esters using microorganisms having lipase or lipase-producing ability to produce optically active 3-hydroxy-gamma-butyrolactone. It is a new process that has not been reported to date as a manufacturing method.

이에 본 발명자들은 일반식 (1)로 표시되는 라세믹 4-클로로-3-히드록시부틸산 알킬 에스테르를 반응함에 있어서 리파제 또는 리파제 생산능을 갖는 미생물을 사용하여 가수분해 반응으로 광학활성 3-히드록시-감마-부티로락톤을 제조하고자 하였다. The inventors of the present invention have optically active 3-hydrides in a hydrolysis reaction using microorganisms having lipase or lipase production ability in reacting racemic 4-chloro-3-hydroxybutyl acid alkyl ester represented by the general formula (1). Roxy-gamma-butyrolactone was prepared.

본 발명에 의한 방법은 기존의 방법과는 달리 가수분해 반응에 의한 방법으로, 공정이 매우 간단하고 높은 광학순도를 갖는 3-히드록시-감마-부티로락톤을 제조할 수 있다. 따라서, 본 발명의 목적은 효소 또는 미생물을 사용하여 일반식 (1)로 표시되는 에스테르 화합물로부터 광학활성 3-히드록시-감마-부티로락톤을 제조하는 공정을 제공하는데 있다. Unlike the conventional method, the method according to the present invention is a method by hydrolysis reaction, and thus, 3-hydroxy-gamma-butyrolactone having a very simple process and high optical purity can be prepared. It is therefore an object of the present invention to provide a process for producing optically active 3-hydroxy-gamma-butyrolactone from an ester compound represented by the general formula (1) using an enzyme or a microorganism.

상기 목적을 달성하기 위한 본 발명의 제조 방법은 수용액상 또는 수용액을 포함하는 용매상에서 라세믹 에스테르 화합물을 리파제 또는 리파제 생산능을 갖는 미생물을 촉매로 사용하여 입체선택적으로 가수분해 반응시키는 것으로 이루어진다.The production method of the present invention for achieving the above object consists of stereoselective hydrolysis reaction using a racemic ester compound in the aqueous solution phase or a solvent containing an aqueous solution using a microorganism having a lipase or lipase production capacity as a catalyst.

이하 본 발명을 좀 더 상세히 설명하면 다음과 같다. 전술한 바와 같이, 본 발명은 일반식 (1)로 표시되는 에스테르 화합물에 생촉매로서 효소 또는 미생물을 첨가하여 가수분해 반응을 하여 광학활성 3-히드록시-감마-부티로락톤을 제조하는 방법에 관한 것이다.Hereinafter, the present invention will be described in more detail. As described above, the present invention is a method for producing an optically active 3-hydroxy-gamma-butyrolactone by a hydrolysis reaction by adding an enzyme or a microorganism as a biocatalyst to an ester compound represented by the general formula (1). It is about.

본 발명에 사용되는 일반식 (1)로 표시되는 라세믹 4-클로로-3-히드록시부틸산 알킬 에스테르의 R은 CnH2n+1(n=1~8) 등이 있으나 이에 한정되는 것은 아니다. 상기 일반식 (1)에서 R은 이소프로필(isopropyl), n-프로필(n-propyl), 이소부틸(isobutyl)과 벤질(benzyl) 등이 가능하다.R of the racemic 4-chloro-3-hydroxybutyl acid alkyl ester represented by the general formula (1) used in the present invention includes C n H 2n + 1 (n = 1 to 8), but is not limited thereto. no. In Formula (1), R may be isopropyl, n-propyl, isobutyl, benzyl, or the like.

본 발명에 사용되는 리파제로는 아마노사의 PS, AK, 노보자임스사의 CALB 등이며 균주로는 Candida rugosa, Rhodococcus butanica, Cunninghamella echinylata, Candida magnoliae, Burkholderia cepacia, Geotrichum candidum 등 리파제 생산능을 갖는 여러 종의 미생물이 가능하나 이에 한정되는 것은 아니다.The lipases used in the present invention include Amano's PS, AK, Novozymes' CALB, and the like. Candida rugosa , Rhodococcus butanica, Cunninghamella echinylata, Candida magnoliae, Burkholderia cepacia, Geotrichum candidum, etc. Microorganisms are possible but are not limited thereto.

본 발명에 있어서, 반응물 및 생성물들은 기체크로마토그래피(도남 인스트루먼트사, 모델 DS6200)를 이용하여 분석하였으며, 반응후 반응물을 아세트산 에틸(ethyl acetate)로 추출하여 분석하였다. In the present invention, the reactants and products were analyzed using gas chromatography (Donam Instruments, Model DS6200), and after the reaction was analyzed by extraction with ethyl acetate (ethyl acetate).

라세믹 4-클로로-3-히드록시부틸산 알킬 에스테르는 모세관(capillary) 칼럼인 G-TA(Astec 사, 30m×0.32mm)을 100℃에서 5분간 가열 후 170℃까지 분당 20℃씩 올려주었고, 170℃에서 15분을 유지하였다. 담체로는 헬륨기체를 사용하였으며 칼럼 헤드 압력을 6 psi로 유지하면서 170℃에서 FID를 사용하여 검출하였다. 이때 메틸 4-클로로-3-히드록시부티레이트는 9.31분, 에틸 4-클로로-3-히드록시부티레이트는 10.05분, 부틸 4-클로로-3-히드록시부티레이트는 12.92분, 3-히드록시-감마-부티로락톤은 18.11분에서 각각 검출되었다.The racemic 4-chloro-3-hydroxybutyl acid alkyl ester was heated in capillary column G-TA (Astec, 30m × 0.32mm) at 100 ° C. for 5 minutes and then raised to 170 ° C. by 20 ° C. per minute. 15 minutes was maintained at 170 degreeC. Helium gas was used as the carrier and was detected using FID at 170 ° C. while maintaining the column head pressure at 6 psi. In this case, methyl 4-chloro-3-hydroxybutyrate is 9.31 minutes, ethyl 4-chloro-3-hydroxybutyrate is 10.05 minutes, butyl 4-chloro-3-hydroxybutyrate is 12.92 minutes, 3-hydroxy-gamma- Butyrolactone was detected at 18.11 minutes respectively.

광학활성 (R)- 및 (S)-3-히드록시-감마-부티로락톤은 모세관(capillary) 칼럼인 G-TA(Astec 사, 30m×0.32mm)를 120℃에서 10분간 가열 후 170℃까지 분당 10℃씩 올려주었고, 170℃에서 15분을 유지하였다. 담체로는 헬륨기체를 사용하였으며 칼럼 헤드 압력을 10 psi로 유지하면서 170℃에서 FID를 사용하여 검출하였다. 이때 (R)-3-히드록시-감마-부티로락톤은 25.83분, (S)-3-히드록시-감마-부티로락톤은 26.52분에서 각각 검출되었다.Optically active (R)-and (S) -3-hydroxy-gamma-butyrolactone was heated to 170 ° C. after heating a capillary column, G-TA (Astec, 30 m × 0.32 mm) for 10 minutes at 120 ° C. The temperature was raised by 10 ° C. per minute and maintained at 170 ° C. for 15 minutes. Helium gas was used as the carrier and was detected using FID at 170 ° C. while maintaining the column head pressure at 10 psi. At this time, (R) -3-hydroxy-gamma-butyrolactone was detected at 25.83 minutes and (S) -3-hydroxy-gamma-butyrolactone at 26.52 minutes, respectively.

또한 메틸 4-클로로-3-히드록시부티레이트, 에틸 4-클로로-3-히드록시부티레이트, 부틸 4-클로로-3-히드록시부티레이트는 FT-NMR(Burker사, 모델 DPX300)로 확인을 하였으며, 각각의 분석결과는 다음과 같다.In addition, methyl 4-chloro-3-hydroxybutyrate, ethyl 4-chloro-3-hydroxybutyrate, and butyl 4-chloro-3-hydroxybutyrate were confirmed by FT-NMR (Burker, model DPX300), respectively. The analysis results of are as follows.

메틸 4-클로로-3-히드록시부티레이트 :Methyl 4-chloro-3-hydroxybutyrate:

1H-NMR (CDCl3) δ (ppm) = 2.64 (2H,d), 3.61 (2H,d), 3.72 (3H,s) 1 H-NMR (CDCl 3 ) δ (ppm) = 2.64 (2H, d), 3.61 (2H, d), 3.72 (3H, s)

4.15 (1H,br), 4.26 ~ 4.31 (1H,m) 4.15 (1H, br), 4.26-4.31 (1H, m)

에틸 4-클로로-3-히드록시부티레이트 :Ethyl 4-chloro-3-hydroxybutyrate:

1H-NMR (CDCl3) δ (ppm) = 1.28 (3H,t), 2.62 (2H,d), 3.53 (1H,br), 1 H-NMR (CDCl 3 ) δ (ppm) = 1.28 (3H, t), 2.62 (2H, d), 3.53 (1H, br),

3.60 (2H,d), 4.20 (2H,q), 4.33 (1H,m) 3.60 (2H, d), 4.20 (2H, q), 4.33 (1H, m)

n-부틸 4-클로로-3-히드록시부티레이트 :n-butyl 4-chloro-3-hydroxybutyrate:

1H-NMR (CDCl3) δ (ppm) = 0.94 (3H,t), 1.36 ~ 1.43 (2H,m), 1.59 ~1.66 1 H-NMR (CDCl 3 ) δ (ppm) = 0.94 (3H, t), 1.36 to 1.43 (2H, m), 1.59 to 1.66

(2H,m), 2.64 (2H,d), 3.45 (1H,br), 3.63 (2H,d), 4.12 (2H,t), 4.24 ~ (2H, m), 2.64 (2H, d), 3.45 (1H, br), 3.63 (2H, d), 4.12 (2H, t), 4.24-

4.29 (1H,m) 4.29 (1 H, m)

이하 실시예를 통하여 본 발명을 좀 더 구체적으로 설명하지만, 하기 예에 본 발명의 범주가 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited to the following Examples.

실시예 1Example 1

0.1 M 인산 완충용액(potassium phosphate buffer, pH 8.0) 4.5 ml이 들어있는 바이알에 라세믹 메틸 4-클로로-3-히드록시부티레이트가 10 %(v/v)가 되도록 첨가하고 리파제 CAL B 를 2 %(w/v)가 되도록 넣은 다음 30 ℃에서 반응을 수행하였다. 10분 반응 후 반응액을 아세트산 에틸로 추출하여 상기의 분석방법에 따라 분석하였다. 이 때 전환율은 21.2 % 이었으며 (S)-3-히드록시-감마-부티로락톤의 광학순도는 94.6 % e.e 이었다. To a vial containing 4.5 ml of 0.1 M potassium phosphate buffer (pH 8.0) add racemic methyl 4-chloro-3-hydroxybutyrate to 10% (v / v) and add lipase CAL B 2% (w / v) was added and the reaction was carried out at 30 ℃. After the reaction for 10 minutes, the reaction solution was extracted with ethyl acetate and analyzed according to the above analysis method. At this time, the conversion was 21.2% and the optical purity of (S) -3-hydroxy-gamma-butyrolactone was 94.6% e.e.

실시예 2-3Example 2-3

실시예 1에서 반응물로 사용된 메틸 3-히드록시부티레이트 대신 에틸 4-클로로-3-히드록시부티레이트와 부틸 4-클로로-3-히드록시부티레이트를 사용하여 반응을 수행하였으며, 각 반응의 전환율과 광학순도는 표1에 나타내었다. The reaction was carried out using ethyl 4-chloro-3-hydroxybutyrate and butyl 4-chloro-3-hydroxybutyrate instead of methyl 3-hydroxybutyrate used as the reactant in Example 1, and the conversion and optical Purity is shown in Table 1.

실시예 반응물 반응시간 (분) 전환율 (%) (S)-3-히드록시-감마-부티로락톤광학순도 (% e.e) 2 에틸 4-클로로-3-히드록시부티레이트 10 39.9 90.4 3 부틸 4-클로로-3-히드록시부티레이트 10 59.1 63.5
실시예 4-5
Example Reactant Response time (minutes) % Conversion (S) -3-hydroxy-gamma-butyrolactone optical purity (% ee) 2 Ethyl 4-chloro-3-hydroxybutyrate 10 39.9 90.4 3 Butyl 4-chloro-3-hydroxybutyrate 10 59.1 63.5
Example 4-5

삭제delete

실시예 1에서 리파제 CAL B대신 하기 표2에 명시한 리파제를 사용하여 반응을 수행하였으며, 각 반응의 전환율과 광학순도는 표2에 나타내었다In Example 1, the reaction was performed using the lipase shown in Table 2 instead of the lipase CAL B, and the conversion and optical purity of each reaction are shown in Table 2.

실시예Example 리파제 종류Lipase Type 반응시간 (시간)Response time (hours) 전환율 (%)% Conversion (R)-3-히드록시-감마-부티로락톤광학순도 (% e.e)(R) -3-hydroxy-gamma-butyrolactone optical purity (% e.e) 44 PS(아마노 사)PS (Amano company) 99 39.139.1 51.851.8 55 AK(아마노 사)AK (Amano) 2424 34.534.5 52.652.6

실시예 6-12Example 6-12

실시예 1에서 리파제 CAL B대신 하기 표2에 명시한 미생물을 사용하여 반응을 수행하였으며, 이때 기질은 5%, 미생물 균체는 20%가 되도록 하였다. 각 반응의 전환율과 광학순도는 표3에 나타내었다In Example 1, instead of the lipase CAL B, the reaction was performed using the microorganisms shown in Table 2, wherein the substrate was 5% and the microbial cell was 20%. The conversion and optical purity of each reaction are shown in Table 3.

실시예Example 미생물 microbe 반응시간 (시간)Response time (hours) 전환율 (%)% Conversion 3-히드록시-감마-부티로락톤광학순도 (% e.e)3-hydroxy-gamma-butyrolactone optical purity (% e.e) 66 Candida rugosa KTCC 7292 Candida rugosa KTCC 7292 66 13.413.4 63.4(R)63.4 (R) 77 Cunninghamella echinylata ATCC 26269 Cunninghamella echinylata ATCC 26269 4.54.5 20.420.4 45.4(S)45.4 (S) 88 Rhodococcus butanica ATCC 21197 Rhodococcus butanica ATCC 21197 4.54.5 36.836.8 85.4(S)85.4 (S) 99 Burkholderia cepacia ATCC 21808 Burkholderia cepacia ATCC 21808 3232 16.816.8 78.7(S)78.7 (S) 1010 Candida magnoliae KCCM 50046 Candida magnoliae KCCM 50046 7272 21.921.9 80.7(S)80.7 (S) 1111 Candida magnoliae KCCM 50567 Candida magnoliae KCCM 50567 100100 6.16.1 92.0(S)92.0 (S) 1212 Geotrichum candidum IFO 4597 Geotrichum candidum IFO 4597 22 8.48.4 50.7(S)50.7 (S)

상기 실시예 1-12를 통해 알 수 있는 것과 같이, 광학활성 3-히드록시-감마-부티로락톤을 제조하는 방법에 있어서 본 발명에 따른 리파제 또는 리파제 생산능을 갖는 미생물을 사용하는 가수분해 반응은 매우 간단하다. 또한 적절한 리파제 효소나 군주를 사용하면 높은 광학순도의 3-히드록시-감마-부티로락톤을 합성할 수가 있다. 본 발명의 방법은 효소 및 미생물을 이용한다는 점에서 환경친화적이고, 반응농도가 높아 실제 대량생산에 이용할 수 있으며, 고정화 효소나 고정화 균주의 경우 반복사용이 가능하여 제조 공정에서 비용을 줄일 수 있다는 장점이 있다.As can be seen through Examples 1-12, in the method for producing optically active 3-hydroxy-gamma-butyrolactone hydrolysis reaction using a microorganism having a lipase or lipase production capacity according to the present invention Is very simple. In addition, the use of suitable lipase enzymes or monarchs can be used to synthesize high optical purity of 3-hydroxy-gamma-butyrolactone. The method of the present invention is environmentally friendly in terms of the use of enzymes and microorganisms, the reaction concentration is high and can be used in actual mass production, and the use of immobilized enzymes or immobilized strains can be repeated and used to reduce costs in the manufacturing process. There is this.

Claims (3)

하기 [반응식 1]에서 일반식 (1)로 표시되는 라세믹 4-클로로-3-히드록시부틸산 알킬 에스테르를 수용액상 또는 수용액을 포함하는 용매상에서 로도코커스 부타니카(Rhodococcus butanica), 캔디다 마그놀리애(Candida magnoliae) 또는 캔디다 앤타르크티카(Candida antarctica)로부터 선택되는 미생물 유래의 리파제, 또는 로도코커스 부타니카, 캔디다 마그놀리애 또는 캔디다 앤타르크티카로부터 선택되는 리파제 생성능을 갖는 미생물을 사용하여 가수분해 반응시키는 것을 특징으로 하는 일반식 (3)으로 표시되는 광학활성 3-히드록시-감마-부티로락톤을 제조하는 방법.The racemic 4-chloro-3-hydroxybutyl acid alkyl ester represented by the general formula (1) in the following [Reaction Scheme 1] is Rhodococcus butanica and Candida magnoli in an aqueous solution phase or a solvent containing an aqueous solution. Hydrolysis using a lipase derived from a microorganism selected from Candida magnoliae or Candida antarctica , or a microorganism having a lipase producing ability selected from Rhodococcus butanica, Candida magnolia or Candida antarctica A method for producing an optically active 3-hydroxy-gamma-butyrolactone represented by the general formula (3), which is reacted. [반응식 1]Scheme 1
Figure 712006003316245-pat00004
Figure 712006003316245-pat00004
( R = CnH2n+1(n=1~4) )(R = C n H 2n + 1 (n = 1 ~ 4))
제1항에 있어서, 미생물 균주는 로도코커스 부타니카 ATCC 21197, 캔디다 마그놀리애 KCCM 50046 또는 캔디다 마그놀리애 KCCM 50567임을 특징으로 하는 방법.The microorganism strain of claim 1, wherein the microorganism strain is Rhodococcus butanica ATCC 21197, Candida Magnolia. KCCM 50046 or Candida Magnolia Method characterized in that KCCM 50567. 삭제delete
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JP2002204699A (en) 2001-01-11 2002-07-23 Daicel Chem Ind Ltd METHOD FOR PRODUCING beta-HYDROXY-gamma-BUTYROLACTONE

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JP2002204699A (en) 2001-01-11 2002-07-23 Daicel Chem Ind Ltd METHOD FOR PRODUCING beta-HYDROXY-gamma-BUTYROLACTONE

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