脂肪酶催化拆分N取代苯基α氨基丙酸的研究
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摘要
手性N取代苯基α氨基丙酸是一类重要的手性合成中间体, 特别是在农药领域, 是很多手性农药的关键中间体。目前对于旋光纯N取代苯基α氨基丙酸的合成，主要采用化学拆分方法，但化学拆分方法存在很多缺点。
本论文选择脂肪酶作为催化剂，通过立体选择性酯水解反应成功的拆分了N取代苯基α氨基丙酸，确定了最佳拆分条件，研究了酶的固定化修饰和不同类型添加剂对酶催化活性和立体选择性的影响，从分子水平上解释了酶催化立体选择性水解机制，并对光学纯N取代苯基α氨基丙酸进行了应用基础方面的研究。
一．N取代苯基α氨基丙酸的毛细管电泳分离
采用高效毛细管区带电泳法，以环糊精作为手性选择剂，对外消旋模型化合物2-[(2-甲基-6-乙基)苯基氨基]丙酸(NEMPA)的两个对映体进行了手性分离，比较了环糊精种类、环糊精浓度、电解质溶液pH值、温度和电场强度对分离的影响。实验结果表明，本文采用DM-β-CD作为手性选择剂，不需要处理毛细管柱，就可以使对映体得到较好分离，经筛选确定的最佳实验条件为：DM-β-CD浓度为40 mmol/L，三乙胺/乙酸缓冲溶液浓度为100 mmol/L，溶液pH =5.5，反向电场强度为340 V/cm，电泳温度 20℃，样品浓度50 mg/L，紫外检测器200 nm，对映体达基线分离，建立了利用毛细管电泳检测NEMPA对映体过量值的新方法，该方法也适用于其它的N取代苯基α氨基丙酸对映体过
量值的检测。
二．脂肪酶催化拆分N取代苯基α氨基丙酸的研究
以NEMPA作为模型化合物，研究了在温和条件下，通过脂肪酶的立体选择性酯水解反应来制备光学活性N取代苯基α氨基丙酸的可行性。首先从不同来源的脂肪酶中，筛选出三种具有反应优势的脂肪酶：分别为南极假丝酵母脂肪酶（CAL-B）、假单胞菌脂肪酶（PSL）和枯草杆菌脂肪酶（BSL2）。
进一步研究了脂肪酶的微环境对其催化拆分的影响，并对三种脂肪酶进行比较。研究结果表明，微环境对所选择的三种脂肪酶的催化活活性和立体选择性影响在某些方面具有相似性，主要表现在以下方面：反应温度较低时，脂肪酶的活性和立体选择性较高，温度高时，则有所下降；三种脂肪酶在偏碱性条件下，酶活力和立体选择性均有不同程度下降，但反应速度有所提高；PSL 和BSL2催化活性和立体选择性和烷基酯的链长和类型密切相关；从酶催化拆分的动力学曲线可以看出，我们选取的三种脂肪酶均没有表现出界面激活和底物抑制现象，完全受动力学控制。
三．改善脂肪酶立体选择性及催化活性的研究
分别研究了体系中添加的有机溶剂、表面活性剂、大环多胺和离子液体对脂肪酶CAL-B和BSL2催化活性和立体选择性的影响，并分别对这四种添加剂的不同影响结果给出合理的解释。
有机溶剂对CAL-B和BSL2的催化活性和立体选择性的影响，存在很大程度的相似性，反应体系中加入有机溶剂后，两种酶的催化活性均有不同程度的降低，可以提高立体选择性的有机溶剂（分别为乙醚和异丙醚）都属于醚类。
所选择的三种表面活性剂中，Tween-80对CAL-B和BSL2的立体选择性均有提高的作用，但对酶的催化活性影响不同，它可以提高CAL-B的活性，而降低BSL2的催化活性。
带有亲酯性长链的大环多胺可以显著提高酶的催化活性和立体选择性，尽管带有亲酯性长链的大环多胺可以促进更多的底物分子与酶活性部位相互作用，但链长达一定程度后，可能由于大环多胺较长的亲酯链阻碍了酶对底物分子空间上的识别，立体选择性降低。
和纯水相体系相比，在反应体系中加入离子液体后，提高了CAL-B的立体选择性(E=44.7), 大约是纯水体系的4倍(E=10.6), 低于在乙醚-水两相体系中的立体选择性（E=117.8）。但离子液体可以解决传统有机溶剂的乳化问题, 同时产物溶于水相, 与存在于离子液体相的酯分开, 有利于产品的回收，还可以循环利用。
四．脂肪酶固定化的研究
从四种经过不同修饰的中孔分子筛中筛选出只经过煅烧的分子筛SBA-15作为脂肪酶PSL的固定化载体，最佳固定化条件为：缓冲液离子强度为25mmol/L，pH 8.0，酶和分子筛的比例为1：15，温度为4℃，固定化时间为2小时。偶联率为69.2%, 活力回收为59.9%。结果显示，脂肪酶经固定化后具有较好的活性（固定化酶的比活高于游离酶1.2倍）和稳定性（可重复使用8次以上）。在反应体系中添加15%乙醚和40mg/mlCTAB后，固定化酶活性可分别提高到1.1倍和1.32倍，固定化酶在有机溶剂中的稳定性高于游离酶，在表面活性剂中稳定性略低于游离酶。初步确定了固定化机制是酶分子首先进入分子筛SBA-15的孔洞中，然后和分子筛的内表面的活性基相互作用。
五．脂肪酶催化拆分N取代苯基α氨基丙酸的理论研究
采用半经验量子化学方法，优化了R型和S型2-[(2-甲基-6-乙基)苯基氨基]丙酸甲酯(NEMPAME)分子的空间构型，得到了稳定的几何构型；以脂肪酶PSL 、CAL-B活性中心的晶体结构为基础，与优化的NEMPAME分子几何构型构建了NEMPAME与酶反应的四面体过渡态，在酶的活性腔中优化底物分子的过渡态，获得的最稳定构型存在差异，反应在生成热上。在PSL与NEMPAME
作用时，R构型的过渡态要比S构型的过渡态稳定，说明PSL对NEMPAME具有手性选择性，催化R构型酯的水解
Enantiopure N-substituted phenyl α-alanines are important intermediates for the chiral synthesis, especially in the field of chiral pesticides. The most commonly used methodology for the preparation of enantiopure N-substituted phenyl α-alanine is chemical resolution, but the method of chemistry have some disadvantages.
The main purpose of this research is enantioselective hydrolysis of N-substituted phenyl α-alanine by using lipase as the catalyst. Optimum reaction conditions are achieved. The influence on lipase activity and enantioselectivity by immobilization of lipase and addition of different additives to the reaction system is studied. The mechanism of the enantioselective lipase-catalyzed hydrolysis is explained based on the molecular level. The application of enantiopure N-substituted phenyl α-alanine is also performed in the chemical synthesis.
1. Separation of N-substituted phenyl α-alanine by Capillary Electrophoresis
Capillary electrophoresis was firstly used for the chiral separation of N- (2-ethyl-6-methyl-Phenyl)-alanine(NEMPA) in this paper, using cyclodextrin as chiral additive. The effects of cyclodextrin type, variation of cyclodextrin concentration, background electrolyte pH, temperature and field strength were
investigated. Optimum separation was achieved for NEMPA in 100 mmol/L triethylamine/acetic acid buffer, pH= 5.5, by using 40 mmol/L DM-β-CD as a buffer additive. The analysis was performed with applied voltage at 340V/cm, the temperature at 20℃ , sample concentration (50mg/L) and the absorbance was recorded at 200 nm. A new method of using capillary electrophoresis for the detection of enantiomeric excess of NEMPA has been established. The method is also applied to determin the enantiomeric excess of other N-substituted phenyl α-alanine.
2. Lipase-catalyzed resolution of N-substituted phenyl α-alanine
Using (R, S)–NEMPA as a model compound, we herein have attempted to develop a stereoselectively hydrolytic process for studying the feasibility of resolution of the racemic N-substituted phenyl α-alanine via enzyme. The use of lipase as chiral catalyst to prepare enantiopure N-substituted phenyl α-alanine from the corresponding racemic esters has not been widely studied. The study focused on the identification of suitable lipases for enantiomeric resolution of (R, S)-NEMPA. Six kind of lipases were screened out for the enantioselective hydrolysis of racemic NEMPAME to yield chiral NEMPA. The screened lipase are CAL-B, PSL and BSL2.
We further studied the influence of microenvironment on the lipase-catalyzed resolution and made comparison among the three lipases. The results suggest that the effects of the microenvironment on the activity and enantioselectivity of the three lipases are similar. The higher selectivity and activity of lipase could be achieved by operating at lower temperatures. In contrast, at higher temperatures, the reaction occurred with low enantiopreference. The E value and activity of the three lipases decreased in the pH range of alkalinity. The activity and enantioselectivity of BSL2 and PSL were influenced by the size of alkyl group of
the ester moiety. According to the kinetic curve, the three lipases did not show the phenomena of interfacial activation and substrate inhibition under these assay conditions and the resolution of the lipase-catalyzed was controlled by kinetics entirely.
3. Improve the activity and enantioselectivity of lipase
Effects of the organic solvent、detergent、macrocyclic polyamine and ionic liquid on the activity and enantioselectivity of CAL-B and BSL2 were studied in the reaction system, and the reasonable explainations for the effects were given.
The effect of organic solvent on the activity and enantioselectivity of CAL-B and BSL2 is similar. Addition of certain organic solvent to the reaction mixture results in the improvement of enantioselectivity of lipases, whereas the activity of lipases are deteriorated. Diisopropyl ether and diethyl ether which are both belonged to the ether can drast
本论文选择脂肪酶作为催化剂，通过立体选择性酯水解反应成功的拆分了N取代苯基α氨基丙酸，确定了最佳拆分条件，研究了酶的固定化修饰和不同类型添加剂对酶催化活性和立体选择性的影响，从分子水平上解释了酶催化立体选择性水解机制，并对光学纯N取代苯基α氨基丙酸进行了应用基础方面的研究。
一．N取代苯基α氨基丙酸的毛细管电泳分离
采用高效毛细管区带电泳法，以环糊精作为手性选择剂，对外消旋模型化合物2-[(2-甲基-6-乙基)苯基氨基]丙酸(NEMPA)的两个对映体进行了手性分离，比较了环糊精种类、环糊精浓度、电解质溶液pH值、温度和电场强度对分离的影响。实验结果表明，本文采用DM-β-CD作为手性选择剂，不需要处理毛细管柱，就可以使对映体得到较好分离，经筛选确定的最佳实验条件为：DM-β-CD浓度为40 mmol/L，三乙胺/乙酸缓冲溶液浓度为100 mmol/L，溶液pH =5.5，反向电场强度为340 V/cm，电泳温度 20℃，样品浓度50 mg/L，紫外检测器200 nm，对映体达基线分离，建立了利用毛细管电泳检测NEMPA对映体过量值的新方法，该方法也适用于其它的N取代苯基α氨基丙酸对映体过
量值的检测。
二．脂肪酶催化拆分N取代苯基α氨基丙酸的研究
以NEMPA作为模型化合物，研究了在温和条件下，通过脂肪酶的立体选择性酯水解反应来制备光学活性N取代苯基α氨基丙酸的可行性。首先从不同来源的脂肪酶中，筛选出三种具有反应优势的脂肪酶：分别为南极假丝酵母脂肪酶（CAL-B）、假单胞菌脂肪酶（PSL）和枯草杆菌脂肪酶（BSL2）。
进一步研究了脂肪酶的微环境对其催化拆分的影响，并对三种脂肪酶进行比较。研究结果表明，微环境对所选择的三种脂肪酶的催化活活性和立体选择性影响在某些方面具有相似性，主要表现在以下方面：反应温度较低时，脂肪酶的活性和立体选择性较高，温度高时，则有所下降；三种脂肪酶在偏碱性条件下，酶活力和立体选择性均有不同程度下降，但反应速度有所提高；PSL 和BSL2催化活性和立体选择性和烷基酯的链长和类型密切相关；从酶催化拆分的动力学曲线可以看出，我们选取的三种脂肪酶均没有表现出界面激活和底物抑制现象，完全受动力学控制。
三．改善脂肪酶立体选择性及催化活性的研究
分别研究了体系中添加的有机溶剂、表面活性剂、大环多胺和离子液体对脂肪酶CAL-B和BSL2催化活性和立体选择性的影响，并分别对这四种添加剂的不同影响结果给出合理的解释。
有机溶剂对CAL-B和BSL2的催化活性和立体选择性的影响，存在很大程度的相似性，反应体系中加入有机溶剂后，两种酶的催化活性均有不同程度的降低，可以提高立体选择性的有机溶剂（分别为乙醚和异丙醚）都属于醚类。
所选择的三种表面活性剂中，Tween-80对CAL-B和BSL2的立体选择性均有提高的作用，但对酶的催化活性影响不同，它可以提高CAL-B的活性，而降低BSL2的催化活性。
带有亲酯性长链的大环多胺可以显著提高酶的催化活性和立体选择性，尽管带有亲酯性长链的大环多胺可以促进更多的底物分子与酶活性部位相互作用，但链长达一定程度后，可能由于大环多胺较长的亲酯链阻碍了酶对底物分子空间上的识别，立体选择性降低。
和纯水相体系相比，在反应体系中加入离子液体后，提高了CAL-B的立体选择性(E=44.7), 大约是纯水体系的4倍(E=10.6), 低于在乙醚-水两相体系中的立体选择性（E=117.8）。但离子液体可以解决传统有机溶剂的乳化问题, 同时产物溶于水相, 与存在于离子液体相的酯分开, 有利于产品的回收，还可以循环利用。
四．脂肪酶固定化的研究
从四种经过不同修饰的中孔分子筛中筛选出只经过煅烧的分子筛SBA-15作为脂肪酶PSL的固定化载体，最佳固定化条件为：缓冲液离子强度为25mmol/L，pH 8.0，酶和分子筛的比例为1：15，温度为4℃，固定化时间为2小时。偶联率为69.2%, 活力回收为59.9%。结果显示，脂肪酶经固定化后具有较好的活性（固定化酶的比活高于游离酶1.2倍）和稳定性（可重复使用8次以上）。在反应体系中添加15%乙醚和40mg/mlCTAB后，固定化酶活性可分别提高到1.1倍和1.32倍，固定化酶在有机溶剂中的稳定性高于游离酶，在表面活性剂中稳定性略低于游离酶。初步确定了固定化机制是酶分子首先进入分子筛SBA-15的孔洞中，然后和分子筛的内表面的活性基相互作用。
五．脂肪酶催化拆分N取代苯基α氨基丙酸的理论研究
采用半经验量子化学方法，优化了R型和S型2-[(2-甲基-6-乙基)苯基氨基]丙酸甲酯(NEMPAME)分子的空间构型，得到了稳定的几何构型；以脂肪酶PSL 、CAL-B活性中心的晶体结构为基础，与优化的NEMPAME分子几何构型构建了NEMPAME与酶反应的四面体过渡态，在酶的活性腔中优化底物分子的过渡态，获得的最稳定构型存在差异，反应在生成热上。在PSL与NEMPAME
作用时，R构型的过渡态要比S构型的过渡态稳定，说明PSL对NEMPAME具有手性选择性，催化R构型酯的水解
Enantiopure N-substituted phenyl α-alanines are important intermediates for the chiral synthesis, especially in the field of chiral pesticides. The most commonly used methodology for the preparation of enantiopure N-substituted phenyl α-alanine is chemical resolution, but the method of chemistry have some disadvantages.
The main purpose of this research is enantioselective hydrolysis of N-substituted phenyl α-alanine by using lipase as the catalyst. Optimum reaction conditions are achieved. The influence on lipase activity and enantioselectivity by immobilization of lipase and addition of different additives to the reaction system is studied. The mechanism of the enantioselective lipase-catalyzed hydrolysis is explained based on the molecular level. The application of enantiopure N-substituted phenyl α-alanine is also performed in the chemical synthesis.
1. Separation of N-substituted phenyl α-alanine by Capillary Electrophoresis
Capillary electrophoresis was firstly used for the chiral separation of N- (2-ethyl-6-methyl-Phenyl)-alanine(NEMPA) in this paper, using cyclodextrin as chiral additive. The effects of cyclodextrin type, variation of cyclodextrin concentration, background electrolyte pH, temperature and field strength were
investigated. Optimum separation was achieved for NEMPA in 100 mmol/L triethylamine/acetic acid buffer, pH= 5.5, by using 40 mmol/L DM-β-CD as a buffer additive. The analysis was performed with applied voltage at 340V/cm, the temperature at 20℃ , sample concentration (50mg/L) and the absorbance was recorded at 200 nm. A new method of using capillary electrophoresis for the detection of enantiomeric excess of NEMPA has been established. The method is also applied to determin the enantiomeric excess of other N-substituted phenyl α-alanine.
2. Lipase-catalyzed resolution of N-substituted phenyl α-alanine
Using (R, S)–NEMPA as a model compound, we herein have attempted to develop a stereoselectively hydrolytic process for studying the feasibility of resolution of the racemic N-substituted phenyl α-alanine via enzyme. The use of lipase as chiral catalyst to prepare enantiopure N-substituted phenyl α-alanine from the corresponding racemic esters has not been widely studied. The study focused on the identification of suitable lipases for enantiomeric resolution of (R, S)-NEMPA. Six kind of lipases were screened out for the enantioselective hydrolysis of racemic NEMPAME to yield chiral NEMPA. The screened lipase are CAL-B, PSL and BSL2.
We further studied the influence of microenvironment on the lipase-catalyzed resolution and made comparison among the three lipases. The results suggest that the effects of the microenvironment on the activity and enantioselectivity of the three lipases are similar. The higher selectivity and activity of lipase could be achieved by operating at lower temperatures. In contrast, at higher temperatures, the reaction occurred with low enantiopreference. The E value and activity of the three lipases decreased in the pH range of alkalinity. The activity and enantioselectivity of BSL2 and PSL were influenced by the size of alkyl group of
the ester moiety. According to the kinetic curve, the three lipases did not show the phenomena of interfacial activation and substrate inhibition under these assay conditions and the resolution of the lipase-catalyzed was controlled by kinetics entirely.
3. Improve the activity and enantioselectivity of lipase
Effects of the organic solvent、detergent、macrocyclic polyamine and ionic liquid on the activity and enantioselectivity of CAL-B and BSL2 were studied in the reaction system, and the reasonable explainations for the effects were given.
The effect of organic solvent on the activity and enantioselectivity of CAL-B and BSL2 is similar. Addition of certain organic solvent to the reaction mixture results in the improvement of enantioselectivity of lipases, whereas the activity of lipases are deteriorated. Diisopropyl ether and diethyl ether which are both belonged to the ether can drast
引文
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