KR20040063264A - Method for preparing optically active (R)-2-chloromandelic acid and its ester derivatives using Baker's Yeast - Google Patents
Method for preparing optically active (R)-2-chloromandelic acid and its ester derivatives using Baker's Yeast Download PDFInfo
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Abstract
Description
본 발명은 효모를 이용한 광학활성 (R)-2-클로로만델릭산 ((R)-2-chloromandelic acid) 및 그 에스테르의 제조방법에 관한 것으로, 좀 더 상세하게는 알킬 2-클로로-벤조일포르메이트(Alkyl 2-Chloro- benzoylformate)를 버퍼(buffer) 용액에 용해시킨 후, 빵을 만들 때 사용하는 효모(Baker's yeast)를 작용시켜 케톤(ketone)기를 (R)-폼의 알코올로 비대칭 환원(asymmetric reduction)시키는 생물학적 방법에 의한 광학활성 (R)-2-클로로만델릭산 및 그 에스테르의 제조방법에 관한 것이다.The present invention relates to a method for producing optically active (R) -2-chloromandelic acid ((R) -2-chloromandelic acid) and its ester using yeast, and more specifically, alkyl 2-chloro-benzoylform After dissolving the mate (Alkyl 2-Chloro-benzoylformate) in a buffer solution, the yeast (Baker's yeast) used to make bread is acted to asymmetrically reduce the ketone group to an alcohol of (R) -form. The present invention relates to a method for producing optically active (R) -2-chloromandelic acid and esters thereof by asymmetric reduction.
상기 (R)-2-클로로만델릭산 및 그 에스테르 화합물은 혈전방지제로 사용되는 플라빅스(Plavix)의 합성에 필요한 중간체로 사용되고 있다. 플라빅스(Plavix)는 1998년 사노피(Sanofi)사에 의해 출시된 혈전방지제로서 화학명은 (S)-클로피도그렐 바이설파이트((S)-Clopidogrel bisulfate)이며, 하기 화학식 1로 표시된다.The (R) -2-chloromandelic acid and ester compounds thereof are used as intermediates for the synthesis of Flavix, which is used as an antithrombotic agent. Flavix is an antithrombotic agent released by Sanofi in 1998 and its chemical name is (S) -clopidogrel bisulfate, and is represented by the following Chemical Formula 1.
1989년 사노피(Sanofi)사가 발표한 (S)-클로피도그렐 바이설파이트의 제조방법(미국특허 제4,847,265호)을 보면, 사노피사는 라세믹체의 2-클로로만델릭산(2-chloromandelic acid)에 테트라하이드로시에노[3,2-c]피리딘 (tetrahydrothieno[3,2-c]pyridine)을 도입한 후에 캄포설포닉산(camphorsulfonic acid)을 사용한 화학분할(chemical resolution)을 통하여 광학활성의 (S)-클로피도그렐 바이설파이트를 제조하였다. 이 방법의 경우 반응의 후단에서 화학분할을 수행함으로써 수율 및 경제성이 떨어지는 단점이 있다.According to the manufacturing method of (S) -clopidogrel bisulfite (US Patent No. 4,847,265) published by Sanofi in 1989, Sanofi was treated with tetrahydro in 2-chloromandelic acid of racemic body. After the introduction of tetrahydrothieno [3,2-c] pyridine, the chemical activity of camphorsulfonic acid was used to determine the optical activity of (S)-. Clopidogrel bisulfite was prepared. This method has a disadvantage in that yield and economical efficiency are reduced by performing chemical division at the rear end of the reaction.
중간체로서 광학활성의 (R)-2-클로로만델릭산을 얻는 방법으로는 화학분할, 비대칭 촉매(asymmetric catalysis) 또는 효소분할(enzymatic resolution) 등이 있다. 2001년 노다 히로부미(NODA hirobumi) 등이 발표한 일본 특허공개 제2001-072644호에서는 (R)-N-벤질-1-페닐에틸아민((R)-N- benzyl-1-phenylethylamine)을 분할제(resolving agent)로 사용하여 라세믹 2-클로로만델릭산의 부분입체이성질체 염(diastereomeric salt)을 만들고 이를 재결정 과정을 통하여 부분입체이성질체 순도(diastereomeric excess)로 99.0%의 순수한 염(salt)으로 만들었다. 이때의 수율은 90.4%를 나타내었다. 이렇게 만들어진 부분입체이성질체 염을 산성화를 통하여 (R)-2-클로로만델릭산으로 만들었는데 이때 광학순도는 100% e.e. 였으며, 수율은 90%를 나타내었다. 그러나 이 방법은 이론상 수율이 50%을 넘지 못하고 재결정 과정을 필요로 하는 단점이 존재한다.Methods for obtaining optically active (R) -2-chloromandelic acid as an intermediate include chemical cleavage, asymmetric catalysis or enzymatic resolution. In the presentation, such as the 2001 Noda Hirobumi (NODA hirobumi) JP-A No. 2001-072644 (R) - N - benzyl-1-phenylethylamine ((R) - N - benzyl -1-phenylethylamine) dividing the was used as a resolving agent to produce diastereomeric salts of racemic 2-chloromandelic acid, which were recrystallized to 99.0% pure salts with diastereomeric excess. . Yield at this time was 90.4%. The diastereomeric salts thus prepared were made into (R) -2-chloromandelic acid by acidification, with optical purity of 100% ee and yield of 90%. However, this method has the disadvantage that theoretical yield does not exceed 50% and requires recrystallization process.
2002년 노쓰 마이클(NORTH MICHAEL) 등은 국제공개특허 WO 02-10095호에서 키랄 리간드(Chiral ligand)를 가진 촉매를 이용하여 광학활성의 (R)-2-클로로만델릭산을 합성하는 방법을 발표하였다. 이 방법은 살렌(salen) 계열의 키랄 리간드와 Ti를 사용하여 2-클로로벤즈알데하이드의 시안산화(cyanation)와 아세틸화(acetylation)을 이용한다. 88.6%의 수율로 얻어진 (R)-O-아세틸-시아노히드린((R)-O-acetyl-cyanohydrin)은 88.3%의 e.e.를 가지는데, 이의 가수분해를 통하여 (R)-2-클로로만델릭산을 제조한다. 이렇게 얻어진 (R)-2-클로로만델릭산를 시드(seed)로 사용하는 재결정 과정을 통하여 99% e.e.의 (R)-2-클로로만델릭산을 얻는다. 이 방법은 화학분할방법에 비하여 이론 수율이 높지만, 비대칭 반응을 수행하는 반응온도가 -42℃를 유지해야 하고 재결정 과정을 여러 번 반복해야 하는 단점이 있다.In 2002, North Michael et al. Published a method for synthesizing optically active (R) -2-chloromandelic acid using a catalyst having a chiral ligand in WO 02-10095. It was. This method uses cyanation and acetylation of 2-chlorobenzaldehyde using salen-type chiral ligands and Ti. Was obtained in a yield of 88.6% (R) - O - acetyl - cyanohydrin ((R) - O -acetyl- cyanohydrin) is I have the ee of 88.3%, through the hydrolysis thereof, (R) -2- chloro-only Prepare delic acid. The recrystallization process using (R) -2-chloromandelic acid thus obtained as a seed (seed) to obtain 99% ee (R) -2-chloromandelic acid. This method has a higher theoretical yield than the chemical splitting method, but has a disadvantage in that the reaction temperature for asymmetric reaction must be maintained at -42 ° C and the recrystallization process must be repeated several times.
시아노히드린은 효소 반응에서도 이용이 되는데, 2001년 디버사(Diversa)(Co.)는 유럽공개특허 제1242589호와 국제공개특허 WO 01-48175호에서 니트릴라제(nitrilase)를 사용한 시아노히드린의 가수분해와 연속적인 라세믹화(racemization)를 통하여 98% e.e.로 (R)-2-클로로만델릭산을 제조하는 방법을 발표하였다. 이때 사용한 니트릴라제는 디버사의 직접진화(Direct evolution) 기술을 사용하여 만든 효소로서, 이 방법 역시 화학분할방법에 비하여 수율이 높지만, 새로 만든 효소의 대량생산 등 해결해야 하는 과제가 많아 당장 상업화하기에는 어려운 단점이 있다.Cyanohydrin is also used in enzymatic reactions. In 2001, Diva (Co.) used cyanohydrin using nitrileases in European Patent No. 1252589 and WO 01-48175. A method for preparing (R) -2-chloromandelic acid at 98% ee was disclosed through hydrolysis and continuous racemization of. The nitrile used at this time is an enzyme made using Diver's Direct evolution technology. This method also has higher yield than the chemical splitting method, but there are many problems to be solved such as mass production of newly produced enzyme. It has a hard disadvantage.
따라서, 상기와 같은 이유로 간단한 공정을 이용하여 경제적으로 우수하며, 광학순도가 높은 광학활성의 (R)-2-클로로만델릭산 또는 그 에스테르 유도체를 생산할 수 있는 공정의 개발이 필요하다.Therefore, it is necessary to develop a process that can produce economically superior (R) -2-chloromandelic acid or ester derivatives thereof having excellent optical purity and high optical purity by using a simple process for the same reason.
이에 본 발명자들은 위에서와 같은 문제점을 해결하면서 경제적인 제조공정을 개발하고자 출발물질로 알킬 2-클로로-벤조일포르메이트(alkyl 2-chloro-benzoylformate)를 사용하고, 값싸고 쉽게 구할 수 있는 효모를 이용한 α-케토(keto) 에스테르의 비대칭 환원반응이라는 간단한 제조 공정으로 (R)-2-클로로만델릭산 및 그 에스테르 화합물을 제조하는 방법을 개발하였으며, 본 발명은 이에 기초하여 완성되었다. 상기 비대칭 환원반응은 이론 수율이 100%로서 효소 또는 화학 분할(이론 수율 50%)에 비해 월등히 높은 생산성을 획득할 수 있고, 또한 효모(Baker's Yeast)는 쉽게 구할 수 있을 뿐만 아니라 가격이 매우 싸기 때문에 기존 방법에 비해 경제적인 제조 공정의 개발이 가능하다.In order to solve the problems described above, the present inventors use alkyl 2-chloro-benzoylformate as a starting material to develop an economical manufacturing process, and use cheap and easily obtained yeast. A method for preparing (R) -2-chloromandelic acid and its ester compound has been developed by a simple manufacturing process called asymmetric reduction of α-keto ester, and the present invention has been completed based on this. Since the asymmetric reduction reaction yields a theoretical yield of 100%, it is possible to obtain significantly higher productivity compared to enzyme or chemical splitting (50% of theoretical yield), and because yeast (Baker's Yeast) is not only easily available but also very cheap. It is possible to develop an economical manufacturing process compared to the existing method.
따라서, 본 발명의 목적은 효모라는 미생물을 이용하여 제조공정이 비교적 간단하고, 친환경적 공정이면서 시간당 생산 효율이 증가되어 경제적인 알킬 2-클로로-벤조일포르메이트로부터 광학활성의 (R)-2-클로로만델릭산 및 그 에스테르 화합물을 제조하는 방법을 제공하는데 있다.Accordingly, an object of the present invention is to use an optical microorganism (R) -2-chloro from an alkyl 2-chloro-benzoylformate, which is a relatively simple manufacturing process using a microorganism called yeast, which is an environmentally friendly process and an hourly production efficiency is increased. It is to provide a method for producing mandelic acid and ester compounds thereof.
상기 목적을 달성하기 위한 본 발명의 제조 방법은 인산 버퍼 용액 존재 하에서, 알킬 2-클로로-벤조일포르메이트를 효모로 선택적으로 환원 반응시키는 것으로 구성된다.The production method of the present invention for achieving the above object consists in selectively reducing the alkyl 2-chloro-benzoylformate to yeast in the presence of a phosphate buffer solution.
이하 본 발명을 좀 더 구체적으로 살펴보면 다음과 같다.Looking at the present invention in more detail as follows.
전술한 바와 같이, 본 발명에서는 효모에 의한 비대칭 환원반응의 기질로 알킬 2-클로로-벤조일포르메이트를 사용하였는데, 이 화합물은 라세믹 2-클로로만델릭산을 HCl 존재하에서 용매로 사용하는 다양한 알코올과의 반응으로 라세믹 알킬 2-클로로만델레이트로 만들고, 이를 NaOCl과 염화벤질트리에틸암모늄과 같은 상전이 촉매를 이용한 산화반응을 통하여 제조된다.As described above, in the present invention, alkyl 2-chloro-benzoylformate was used as a substrate for asymmetric reduction reaction by yeast, and this compound used various alcohols using racemic 2-chloromandelic acid as a solvent in the presence of HCl. It is prepared by racemic alkyl 2-chloromandelate, which is prepared by oxidation using a phase transfer catalyst such as NaOCl and benzyltriethylammonium chloride.
이렇게 제조된 알킬 2-클로로-벤조일포르메이트를 pH 6∼8의 인산 버퍼 용액에 넣고 효모를 별도의 처리 공정 없이 첨가한 다음, 상기 반응 혼합물을 20∼37℃의 온도에서 100∼300rpm으로 교반시켜 α-케토 그룹(keto group)을 선택적으로 환원 반응시킴으로서 광학활성을 갖는 알킬 (R)-2-클로로만델레이트를 제조한다.The alkyl 2-chloro-benzoylformate thus prepared was added to a phosphate buffer solution at pH 6-8, and yeast was added without a separate treatment step, and the reaction mixture was stirred at 100-300 rpm at a temperature of 20-37 ° C. By selectively reducing the α-keto group, an alkyl (R) -2-chloromandelate having optical activity is prepared.
본 발명의 비대칭 환원반응 공정은 하기 반응식 1과 같다.The asymmetric reduction process of the present invention is shown in Scheme 1 below.
여기서, R은 저급 알킬기, 바람직하게는 메틸기, 에틸기 또는 부틸기, 좀더 바람직하게는 메틸기이다.Here, R is a lower alkyl group, preferably a methyl group, an ethyl group or a butyl group, more preferably a methyl group.
본 발명에 사용되는 바람직한 효모는 사카로미케스 세레비제(Saccharomyces cerevisiae) Type II로서 직접 배양하여 사용할 수도 있으며, 시그마사와 같은 시약회사 또는 많은 효모 생산 회사에서 제품으로 나와 있으므로 구매하여 사용할 수 있다.Preferred yeasts used in the present invention may be directly cultured as Saccharomyces cerevisiae Type II, and may be purchased and used as a product from a reagent company such as Sigma or many yeast production companies.
본 발명에 있어서, 효소 반응 온도는 20∼37℃가 바람직하며, 반응 온도가 낮아질수록 반응 속도가 떨어지고 반응온도가 높아질수록 생성물의 광학순도가 떨어져 최적온도는 30℃ 정도인 것으로 나타났다. 또한 효소 반응시의 pH는 pH 6∼8의 범위가 적정한 것으로 나타났는데, pH가 6.0 미만일 경우에는 반응이 일어나지 않았고, pH가 8.0을 초과하면 광학순도가 떨어지는 문제점이 있으며, 최적 pH는 7.0인 것으로 나타났다.In the present invention, the enzyme reaction temperature is preferably 20 ~ 37 ℃, the lower the reaction temperature, the lower the reaction rate, the higher the reaction temperature, the lower the optical purity of the product was found to be about 30 ℃. In addition, the pH of the enzyme reaction was found to be in the proper range of pH 6 ~ 8, when the pH is less than 6.0, the reaction did not occur, when the pH exceeds 8.0 there is a problem that the optical purity falls, the optimum pH is 7.0 appear.
또한, 상기 효모와 기질의 비율은 무게비로 1 : 0.1∼10인 것이 바람직한데, 상기 기질의 양이 상기 범위를 벗어나면 반응 효율이 떨어지는 경향이 있다.In addition, the ratio of the yeast and the substrate is preferably 1: 0.1 to 10 by weight ratio, but when the amount of the substrate is out of the range, the reaction efficiency tends to be inferior.
본 발명에 따라 제조된 광학활성의 알킬 2-클로로만델레이트는 다이셀(Daicel) 사의 키랄 칼럼인 키랄셀(Chiralcel) OD-H 칼럼을 써서 HPLC에서 R-폼과 S-폼을 쉽게 분석할 수 있다. 이때 HPLC로 분석하기 위해서는 n-헥산과 이소프로필 알코올을 97 : 3의 중량 비율로 함유하고 있는 이동상을 0.5ml/분의 속도로 흘려주고 흡광도를 254nm로 하여 분석하면 되며, 각 생성물의 머무름 시간 (retention time)은 하기 표 1과 같다.The optically active alkyl 2-chloromandelate prepared according to the present invention can be easily analyzed for R-form and S-form in HPLC using Chiralcel OD-H column, which is a chiral column of Daicel. Can be. In this case, for analysis by HPLC, a mobile phase containing n-hexane and isopropyl alcohol in a weight ratio of 97: 3 was flowed at a rate of 0.5 ml / min, and the absorbance was set at 254 nm, and the retention time of each product ( retention time) is shown in Table 1 below.
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
메틸 2-클로로만델레이트의 제조Preparation of Methyl 2-chloromandelate
2-클로로만델릭산(33g, 0.18mol)을 메탄올 100㎖에 녹인 후, 진한 염산 2㎖를 첨가한다. 반응물을 12시간 동안 교반하며 80℃로 가열한다. 반응물을 상온으로 식힌 후, CH2Cl2를 사용하여 추출한다. 추출액을 NaHCO3포화용액으로 씻은 후, MgSO4로 수분을 제거한다. 용매 CH2Cl2를 제거하고, 증류를 통하여 메틸 2-클로로만델레이트(32g, 0.16mol)를 얻었다.2-chloromandelic acid (33 g, 0.18 mol) is dissolved in 100 ml of methanol, and then 2 ml of concentrated hydrochloric acid is added. The reaction is heated to 80 ° C. with stirring for 12 h. The reaction is cooled to room temperature and extracted using CH 2 Cl 2 . The extract is washed with saturated NaHCO 3 solution and then water is removed with MgSO 4 . The solvent CH 2 Cl 2 was removed and methyl 2-chloromandelate (32 g, 0.16 mol) was obtained through distillation.
실시예 2Example 2
메틸 2-클로로-벤조일포르메이트의 제조Preparation of Methyl 2-chloro-benzoylformate
상기 실시예 1에서 얻은 메틸 2-클로로만델레이트(32g, 0.16mol)를 EtOAc 50㎖에 녹이고, NaOCl(13%, 81㎖, 0.22mol) 수용액을 가한다. 여기에 상전이 촉매로 염화벤질트리에틸암모늄(0.7g, 3.2mmol)를 첨가하고, 상온에서 6시간동안 교반하여 반응한다. EtOAc를 사용하여 유기층을 추출한 후, MgSO4로 수분을 제거한다. 용매를 제거하고, 증류를 통하여 메틸 2-클로로-벤조일포르메이트 (29g, 0.14mol)를 얻었다.Methyl 2-chloromandelate (32 g, 0.16 mol) obtained in Example 1 was dissolved in 50 mL of EtOAc, and aqueous NaOCl (13%, 81 mL, 0.22 mol) solution was added. Benzyltriethylammonium chloride (0.7 g, 3.2 mmol) was added thereto as a phase transfer catalyst, and the reaction was stirred at room temperature for 6 hours. After extracting the organic layer using EtOAc, water is removed with MgSO 4 . The solvent was removed and distillation gave methyl 2-chloro-benzoylformate (29 g, 0.14 mol).
실시예 3Example 3
효모를 이용한 메틸 2-클로로-벤조일포르메이트의 비대칭 환원반응Asymmetric Reduction of Methyl 2-Chloro-benzoyl Formate Using Yeast
100mM 인산용액 (pH 7.0) 150㎖에 메틸 2-클로로-벤조일포르메이트 1.5g, 효모(사카로미케스 세레비제(Saccharomyces cerevisiae) Type II 7.5g, 포도당 4.5g을 넣고 30℃에서 반응을 수행하였다. 반응중의 pH는 20% 수산화나트륨 용액을 이용하여 6.5∼7.5 사이로 유지되도록 하였다. 일정 시간마다 반응 샘플을 채취하여 반응 진행 상황을 분석하였다. 4시간 반응 후 GC 상에서 분석된 환원반응의 전환율은 99.9%를 기록하였고, 이때 LC상에서 광학순도를 측정한 결과, 생성된 생성물인 메틸 (R)-2-클로로만델레이트의 광학순도는 98.5% e.e. 였다.In 150 ml of 100 mM phosphoric acid solution (pH 7.0), 1.5 g of methyl 2-chloro-benzoylformate, 7.5 g of yeast ( Saccharomyces cerevisiae Type II, 4.5 g of glucose) were added, and the reaction was performed at 30 ° C. During the reaction, the pH was maintained between 6.5 and 7.5 using 20% sodium hydroxide solution, and reaction samples were taken at regular intervals to analyze the progress of the reaction. % Was recorded and the optical purity of the resulting product, methyl (R) -2-chloromandelate, was 98.5% ee.
실시예 4Example 4
(R)-2-클로로만델릭산의 제조Preparation of (R) -2-chloromandelic acid
NaOH (0.6g, 0.015 mol)을 20㎖ 물에 녹이고, 상기 실시예 3에서 얻은 광학순도 98.5% e.e.의 메틸 2-클로로만델레이트(2.2g, 0.011 mol)을 첨가한다. 상온에서 6시간 동안 교반하여 반응한 후, 산성조건에서 EtOAc로 추출한다. 용매를 제거하고 얻은 생성물은 톨루엔/헥산에서 재결정하여 순수한 (R)-2-클로로만델릭산(1.5g, 0.008mol, e.e.>99%)를 얻었다. ([a]d= -131.94, c=3, H2O, 30.6℃).NaOH (0.6 g, 0.015 mol) is dissolved in 20 ml water and methyl 2-chloromandelate (2.2 g, 0.011 mol) of 98.5% ee optical purity obtained in Example 3 is added. After stirring for 6 hours at room temperature, the mixture was extracted with EtOAc under acidic conditions. The solvent was removed and the product obtained was recrystallized from toluene / hexane to give pure (R) -2-chloromandelic acid (1.5 g, 0.008 mol, ee> 99%). ([a] d = -131.94, c = 3, H 2 O, 30.6 ° C).
실시예 5Example 5
비대칭 환원반응에 미치는 온도의 영향Effect of temperature on asymmetric reduction
100mM의 인산용액(pH 7.0) 150㎖에 메틸 2-클로로-벤조일포르메이트를 1.5g, 사카로미케스 세레비제(Saccharomyces cerevisiae) Type II 7.5g, 포도당 4.5g을 넣은 뒤 온도를 20, 30 및 37℃로 변화시켜 반응하였다. 반응중의 pH는 20% 수산화나트륨 용액을 이용하여 6.5∼7.5 사이로 유지되도록 하였다. 일정 시간마다 반응 샘플을 채취하여 반응 진행 상황을 분석하였다. 반응 온도에 따른 반응 결과는 하기 표 2와 같다. 반응 결과 37℃의 경우 반응 속도는 빨라졌으나 생성물의 광학순도가 낮아졌으며, 20℃의 경우에는 반응 속도가 30℃의 경우보다 떨어져 최적온도는 30℃ 정도인 것으로 나타났다.150 g of 100 mM phosphoric acid solution (pH 7.0) was added 1.5 g of methyl 2-chloro-benzoylformate, 7.5 g of Saccharomyces cerevisiae Type II and 4.5 g of glucose, followed by temperature 20, 30 and 37. The reaction was carried out by changing to ℃. The pH during the reaction was kept between 6.5 and 7.5 using 20% sodium hydroxide solution. Reaction samples were taken at regular intervals to analyze the reaction progress. The reaction results according to the reaction temperature are shown in Table 2 below. As a result of the reaction, the reaction rate was increased at 37 ° C., but the optical purity of the product was lowered.
그러나 상기 반응에서 광학순도가 떨어지는 경우에도, 실시예 4와 같은 조건으로 가수분해 및 재결정 반응을 수행하면 광학순도 99% e.e. 이상의 (R)-2-클로로만델릭산을 제조할 수 있었다.However, even when the optical purity is poor in the reaction, when the hydrolysis and recrystallization reaction is carried out under the same conditions as in Example 4, the optical purity is 99% e.e. The above (R) -2-chloromandelic acid could be manufactured.
실시예 6Example 6
비대칭 환원반응에 미치는 pH 의 영향Effect of pH on Asymmetric Reduction
실시예 5의 반응 조건에서 반응 온도는 30℃로 유지하고 기타 조건은 같은 상태에서 반응 pH를 pH 6 미만, pH 6.5∼7.5 사이 유지, 및 pH 8을 초과하는 조건으로 pH의 영향을 측정하였다. 그 결과 pH 6 미만에서는 반응이 일어나지 않았으며, pH 8을 초과하는 경우는 전환율은 99% 이상 유지하였으나 생성물(product)의 광학순도가 약 93.5% e.e.로서 pH 6.5∼7.5 사이를 유지한 경우에 비해 떨어졌다. 따라서 반응의 최적 pH는 7.0인 것으로 나타났다.In the reaction conditions of Example 5, the reaction temperature was maintained at 30 ° C., and the other conditions were the same, and the influence of pH was measured under conditions such that the reaction pH was maintained between pH 6, pH 6.5 to 7.5, and pH 8 and above. As a result, the reaction did not occur below pH 6, and the conversion rate was maintained at 99% or more when the pH was exceeded 8, but the optical purity of the product was about 93.5% ee, compared to the case where the pH was maintained between 6.5 and 7.5. fell. Thus, the optimum pH of the reaction was found to be 7.0.
그러나 상기 반응에서 광학순도가 떨어지는 경우에도, 실시예 4와 같은 조건으로 가수분해 및 재결정 반응을 수행하면 광학순도 99% e.e. 이상의 (R)-2-클로로만델릭산을 제조할 수 있었다.However, even when the optical purity is poor in the reaction, when the hydrolysis and recrystallization reaction is carried out under the same conditions as in Example 4, the optical purity is 99% e.e. The above (R) -2-chloromandelic acid could be manufactured.
실시예 7Example 7
비대칭 환원반응에 미치는 기질의 영향Effect of Substrate on Asymmetric Reduction
효모를 이용한 비대칭 환원반응에 미치는 기질의 영향을 알아보기 위하여 기질을 다른 에스테르로 바꾸어 실험하였다. 이때 반응 조건은 실시 예 3과 같다. 실험 결과 다른 종류의 에스테르 화합물 모두 광학 순도면에서 좋은 결과를 보여주었으며, 이중 메틸 2-클로로-벤조일포르메이트가 반응 시간 및 전환율 측면에서 가장 좋은 결과를 나타내었다. 비교실험 결과는 하기 표 3과 같다.In order to investigate the effect of the substrate on the asymmetric reduction reaction using yeast, the experiment was changed to another ester. At this time, the reaction conditions are the same as in Example 3. Experimental results showed that all other ester compounds showed good results in terms of optical purity, and methyl 2-chloro-benzoylformate showed the best results in terms of reaction time and conversion rate. Comparative experiment results are shown in Table 3 below.
전술한 바와 같이, 본 발명의 방법은 효모를 사용하여 간단한 방법으로 높은 광학순도의 광학활성 (R)-2-클로로만델릭산 및 그 에스테르 화합물을 제조할 수 있는 효과가 있다. 이 공정은 기존의 제조방법과 비교할 경우 제조공정이 비교적 간단하고 또한 가격이 매우 저렴하고 쉽게 구할 수 있는 효모를 사용함으로써, 친환경적 공정이면서 시간당 생산 효율이 증가되어 경제적이기 때문에 대량생산이 용이한 것이 장점이다. 특히 메틸 2-클로로-벤조일포르메이트를 반응 기질로 사용할 경우, 주요 혈전 치료제 의약품인 플라빅스((S)-클로피도그렐 바이설파이트)의 중간체로 사용되는 메틸 (R)-2-클로로만델레이트을 광학순도 98% e.e. 이상의 제품으로 만들 수 있어 추가의 가수분해와 재결정 과정이 필요 없는 장점이 있다.As described above, the method of the present invention has the effect of producing a high optical purity optically active (R) -2-chloromandelic acid and its ester compound by a simple method using yeast. Compared with the conventional manufacturing method, the production process is relatively simple and the price is very low, and by using yeast which can be easily obtained, it is an eco-friendly process and the production efficiency per hour is increased, so it is economical and easy for mass production. to be. In particular, when methyl 2-chloro-benzoylformate is used as a reaction substrate, optical purity of methyl (R) -2-chloromandelate, which is used as an intermediate of Plavix ((S) -clopidogrel bisulfite), a major thrombotic drug 98% ee It can be made into the above products, there is an advantage that does not require additional hydrolysis and recrystallization process.
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KR100752282B1 (en) * | 2003-10-11 | 2007-08-29 | 주식회사 이큐스팜 | Process for R or S-2-chlorostyrene oxide by using enzyme |
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KR100722155B1 (en) * | 2006-01-05 | 2007-05-28 | 엔자이텍 주식회사 | The method of making optically active 2-chloromandelic acid esters and 2-chloromandelic acids by enzymatic method |
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