面包酵母催化不对称合成4-氯-(R)-3-羟基丁酸乙酯

以4 氯乙酰乙酸乙酯为原料,以面包酵母为手性生物催化剂,选择性合成光学活性4 氯 (R) 3 羟基丁酸乙酯。经IR、GC MS、1HNMR和旋光度测定,表明所得产品的结构与预期的结构一致。分别考察了面包酵母用量、葡萄糖浓度、底物投入量、pH值、反应时间以及反应温度等因素对产品比旋光度的影响。结果表明,4 氯乙酰乙酸乙酯不对称生物还原反应的适宜条件为:面包酵母6 0 0g/L、葡萄糖2 0g/L、反应温度34℃、底物加量16mL/L、反应时间4 8h、pH为5 ,产品的比旋光度为[α]2 0D = 13 9°。     

固定化细胞有机相催化不对称还原β-羰基酯

将酵母细胞用海藻酸钙包埋后用于有机相催化不对称还原4-氯乙酰乙酸乙酯制备光学活性的4-氯-3-羟基丁酸乙酯,从中筛选得到具有较高立体选择性和还原能力的菌株假丝酵母SW0401,将此菌株的细胞固定化细胞作为研究对象,系统考察了固定化条件、固定化细胞大小、反应溶剂、初始底物浓度、辅助底物、固定化细胞热处理和抑制剂对还原反应的影响。结果表明,上述因素对反应的摩尔转化率和产物（S）-CHBE光学纯度有显著影响。固定化时所用缓冲液的pH值为7．0时和固定化细胞颗粒平均直径为2.5mm较合适,以正己烷为反应介质时反应的摩尔转化率和产物光学纯度最优,初始底物浓度以54.7mmol／L为宜,辅助底物以1-己醇为佳。对固定化细胞的热处理和添加抑制剂烯丙醇均能够明显改善产物的光学纯度,但对提高摩尔转化率有负面影响。     

生物催化不对称还原制备氟代手性醇

由于氟原子的特殊性质，化合物中引入氟原子可显著改变其物理化学性质。因此，氟原子在药物中的应用越来越广。此外，80%药物分子结构属于手性分子。其中，氟代手性醇常见于手性药物结构中，该类结构的合成方法研究具有重要的意义。不对称还原含氟酮是合成此结构的常见方法。与化学还原方法相比，生物催化还原具有对映选择性强、产率高和易于分离纯化等优点。生物催化，特别是酶催化还原含氟酮类化合物成为手性药物合成领域的研究热点。本文从纯化酶催化和全细胞催化两个方面，综述了近年来含氟酮生物催化还原合成氟代手性醇的研究进展，并分析总结了氟代对酮生物催化还原的影响，最后对生物催化还原法未来的发展进行了展望。     

不对称生物还原制备手性药物

近年来手性药物的发展非常迅速,手性合成药物的出现不仅提高了药效,而且有利于克服现行消旋体药物在治疗上的副作用。本文介绍了用生物还原法制备高光学纯度手性醇前体的一些方法。     

还原酶催化羰基不对称还原的应用进展

羰基不对称还原作为合成手性醇的重要方法,已成为近年来有机合成的研究热点。与传统化学法相比,利用还原酶催化前手性羰基化合物的不对称还原具有显著优势。介绍了还原酶的来源与形式,对完整细胞还原酶与游离还原酶在手性药物不对称合成中的应用进行了简要综述。     

羰基生物还原法合成手性醇的研究进展

手性醇是合成许多光学活性药物、农用化学品及其他精细化学品的关键手性砌块。羰基生物还原法理论上可实现100%转化率,且反应条件温和,对环境十分友好,被普遍认为是生产手性醇的绿色、高效途径。综述了近年来利用生物信息学、高通量筛选和蛋白质工程的发展对新型、高效生物催化剂开发的影响,特别是利用相关技术手段开发羰基还原酶的进展。     

不对称还原2-辛酮的菌株筛选和反应条件的研究

从本实验室保藏菌株中筛选出一株酒明串珠菌Oenococcus oeni CECT 4730,能不对称还原2-辛酮得到R-2-辛醇。对反应条件的研究表明：Tris-硼酸（TBE）是最佳缓冲液,葡萄糖是最佳辅助底物,壬烷是最佳有机相,当采用300 mmol/L的TBE缓冲液、300 mmol/L的葡萄糖、1：1（体积比）的两相比、150 g/L的湿菌体质量浓度下,可以实现3%的2-辛酮转化为R-2-辛醇（转化率〉99%）,并且e.e.值为97%。     

高分子膜载体固定化酵母催化2-辛酮不对称还原

以戊二醛交联尼龙6膜载体固定化面包酵母DX213,采用固定化酵母细胞催化2-辛酮不对称还原得到(R)-2-辛醇。系统考察了有机溶剂、反应时间、pH、底物、辅助底物和热处理等因素对反应的产率和光学选择性的影响。结果表明,上述因素对酵母细胞催化不对称合成(R)-2-辛醇反应均有显著影响。二氯甲烷为该反应最适有机溶剂,在固定化细胞57 g/L(50℃预热50 min),水相与有机溶剂相体积比4/1,pH 7.0,初始2-辛酮浓度为60 mmoL/L(分别在反应0,10,17 h等分添加),蔗糖5.7 g/L和28℃条件下反应48 h,(R)-2-辛醇的产率和e.e.值分别达到89.3%和96.8%。     

利用微生物重组技术促进羰基不对称还原研究进展

手性醇是许多手性药物合成的关键手性砌块,利用微生物细胞催化相应前手性羰基化合物不对称还原,是合成手性醇的重要方法之一。但应用野生微生物催化时,反应的时空产率、立体选择性较低。详细介绍了利用微生物重组技术以促进前手性羰基化合物不对称还原反应合成手性醇的国内外研究进展。从酶的种类、表达系统以及辅酶再生系统3个方面对重组细胞催化反应体系的构建进行了概述。同时按照反应底物的类型,对重组微生物在催化不同类型羰基化合物不对称还原合成手性醇中的应用分别进行了归纳和介绍。     

疏水离子液体中生物不对称还原制备手性醇

针对近平滑假丝酵母全细胞不对称还原2-羟基苯乙酮制备光学纯(R)-苯基乙二醇反应中底物的质量浓度、产量及质量平衡低的问题,运用多相萃取生物转化的原理,比较不同非水介质对不对称还原反应效率的影响,构建具有良好生物相容性和高质量平衡的水/疏水离子液体1-丁基-3-乙基咪唑六氟磷酸盐([BEIM]PF6)双相反应体系。考察该体系下辅助底物种类、辅助底物用量、底物质量浓度、催化剂用量、离子液体比例、p H和反应温度对生物催化反应的影响,通过正交试验设计和响应面法优化不对称还原2-羟基苯乙酮的反应条件,在最优反应条件下,产物质量浓度、产率和质量平衡得率分别达到15.35 g/L、76.8%和84.3%,产物的对映消旋值(e.e.值)大于99.9%。     

Asymmetric reduction of prochiral ketones to chiral alcohols catalyzed by plants tissue

As an important organic compound, chiral alcohols are the key chiral building blocks to many single enantiomer pharmaceuticals. Asymmetric reduction of the corresponding prochiral ketones to produce the chiral alcohols by biocatalysis is one of the most promising routes. Asymmetric reduction of different kinds of non-natural prochiral ketones catalyzed by various plants tissue was studied in this work. Acetophenone, 4'-chloroacetophenone and ethyl 4-chloroacetoacetate were chosen as the model substrates for simple ketone, halogen-containing aromatic ketone and beta-ketoesters, respectively. Apple (Malus pumila), carrot (Daucus carota), cucumber (Cucumis sativus), onion (Allium cepa), potato (Soanum tuberosum), radish (Raphanus sativus) and sweet potato (Ipomoea batatas) were chosen as the biocatalysts. It was found that these kinds of prochiral ketoness could be reduced by these plants tissue with high enantioselectivity. Both R- and S-form configuration chiral alcohols could be obtained. The e.e. and chemical yield could reach about 98 and 80% respectively for acetophenone and 4'-chloroacetophenone reduction reaction with favorable plant tissue. And the e.e. and yield for ethyl 4-chloroacetoacetate reduction reaction was about 91 and 45% respectively.     

Alternative synthetic route to chiral N-substituted camphor-derived beta-amino alcohols

An alternative route from (1R)-(+)-camphor to chiral N-substituted camphor-derived beta-amino alcohol (4b-e) consists of four steps with a total yield of 28%. N-Alkylation of camphor-derived beta-amino alcohol (4a) involves condensation and hydride reduction in one pot without isolation of intermediates. Condensation of 4a with aldehydes or ketones generates a mixture of 1,3-oxazolidines (6) and imino-alcohols (7), which are reduced to 4b-e by NaBH(4).     

Biotransformations of organosilicon compounds: enantioselective reduction of acyl silanes by means of baker's yeast

The enantioselective reduction of acyl silanes has been performed employing baker's yeast (BY) in fermenting conditions: a series of substrates of different structure was investigated, showing that the reactivity as well as the level of enentioselectivity depends on the steric bulk of the substituents on the acyl silane. The products α-hydroxy silanes were obtained with chemical and optical yields over 90% in the most favourable cases.     

Highly efficient synthesis of chiral alcohols with a novel NADH-dependent reductase from Streptomyces coelicolor

An NADH-dependent reductase (ScCR) from Streptomyces coelicolor was discovered by genome mining for carbonyl reductases. ScCR was overexpressed in Escherichia coli BL21, purified to homogeneity and its catalytic properties were studied. This enzyme catalyzed the asymmetric reduction of a broad range of prochiral ketones including aryl ketones, α- and β-ketoesters, with high activity and excellent enantioselectivity (>99% ee) towards β-ketoesters. Among them, ethyl 4-chloro-3-oxobutanoate (COBE) was efficiently converted to ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE), an important pharmaceutical intermediate, in water/toluene biphasic system. As much as 600 g/L (3.6 M) of COBE was asymmetrically reduced within 22 h using 2-propanol as a co-substrate for NADH regeneration, resulting in a yield of 93%, an enantioselectivity of >99% ee, and a total turnover number (TTN) of 12,100. These results indicate the potential of ScCR for the industrial production of valuable chiral alcohols.     

Integration of enzyme, strain and reaction engineering to overcome limitations of baker's yeast in the asymmetric reduction of alpha-keto esters

We report on the development of a whole-cell biocatalytic system based on the popular host Saccharomyces cerevisiae that shows programmable performance and good atom economy in the reduction of alpha-keto ester substrates. The NADPH-dependent yeast reductase background was suppressed through the combined effects of overexpression of a biosynthetic NADH-active reductase (xylose reductase from Candida tenuis) to the highest possible level and the use of anaerobic reaction conditions in the presence of an ethanol co-substrate where mainly NADH is recycled. The presented multi-level engineering approach leads to significant improvements in product optical purity along with increases in the efficiency of alpha-keto ester reduction and co-substrate yield (molar ratio of formed alpha-hydroxy ester to consumed ethanol). The corresponding alpha-hydroxy esters were obtained in useful yields (>50%) with purities of > or =99.4% enantiomeric excess. The obtained co-substrate yield reached values of greater than 1.0 with acetate as the only by-product formed.     

Enantiofacial selective reduction of 2-allyl-2-carboethoxy-cyclopentanone mediated by baker's yeast

Enantioselective reduction of 2-allyl-2-carboethoxy-cyclopentanone (2) was accomplished in high enantiomeric excess ( > 99%), using baker's yeast in the presence of CuO, to obtain the (+)-2-allyl-2-carboethoxy-cyclopentanol derivative (6). This methodology also provides an entry to corresponding β-keto ester (-)-(2), representing an important strategy to prepare chiral functionalized 2-oxabicyclic[3.3.0]octane and 2-oxabicyclic[4.4.0]nonane derivatives, useful synthons to access new bioactive compounds. © 1996 Wiley-Liss, Inc.     

Influence of the ethanol and glucose supply rate on the rate and enantioselectivity of 3-oxo ester reduction by baker's yeast

Baker's-yeast-mediated reductions of ketones hold great potential for the industrial production of enantiopure alcohols. In this article we describe the stoichiometry and kinetics of asymmetric ketone reduction by cell suspensions of bakers' yeast (Saccharomyces cerevisiae). A system for quantitative analysis of 3-oxo ester reduction was developed and allowed construction of full mass and redox balances as well as determination of the influence of different process parameters on aerobic ketone reduction. The nature of the electron donor (ethanol or glucose) and its specific consumption rate by the biomass (0-1 mol.kg dw(-1).h(-1)) affected the overall stoichiometry and rate of the process and the final enantiomeric excess of the product. Excess glucose as the electron donor, i.e. a very high consumption rate of glucose, resulted in a high rate of alcoholic fermentation, oxygen consumption, and biomass formation and therefore causing low efficiency of glucose utilization. Controlled supply of the electron donor at the highest rates applied prevented alcoholic fermentation but still resulted in biomass formation and a high oxygen requirement, while low rates resulted in a more efficient use of the electron donor. Low supply rates of ethanol resulted in biomass decrease while low supply rates of glucose provided the most efficient strategy for electron donor provision and yielded a high enantiomeric excess of ethyl (S)-3-hydroxybutanoate. In contrast to batchwise conversions with excess glucose as the electron donor, this strategy prevented by-product formation and biomass increase, and resulted in a low oxygen requirement.     

Asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (<Emphasis Type="Italic">R</Emphasis>)-4-chloro-3-hydroxybutanoate with two co-existing,recombinant<Emphasis Type="Italic"> Escherichia coli</Emphasis> strains

Two recombinant strains, E. coli M15 (pQE30-alr0307) and E. coli M15 (pQE30-gdh0310), which were constructed to express, respectively, an NADPH-dependent aldehyde reductase gene and a glucose dehydrogenase gene, were mixed in an appropriate ratio and used for the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (R)-4-chloro-3-hydroxybutanoate. The former strain acted as catalyst and the latter functioned in NADPH regeneration. The biotransformation was completed effectively without any addition of glucose dehydrogenase or NADP⁺/NADPH. An optical purity of 99% (ee) was obtained and the product yield reached 90.5% from 28.5 mM substrate. Revisions requested 27 July 2004/23 September 2004; Revisions received 21 September 2004/29 November 2004