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。     

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。     

羰基还原酶及其催化不对称合成大基团手性羟基类化合物的研究进展

手性羟基化合物以其独特的光、热和化学性质广泛应用于医药、农药、精细化工、功能材料等行业.立体专一性羰基还原酶能够直接针对关键手性位点催化不对称还原潜手性底物获得目的手性产物.基于羰基还原酶的底物多样性,具有不同化学结构和功能的醇类、酯类、氨基酸、环氧化合物等重要手性中间体能够通过不对称还原途径实现单一光学活性对映体的高效制备.然而,针对具有应用价值的含有大基团、结构复杂的潜手性羰基化合物,已知的羰基还原酶通常催化活性较低.本文综述了生物催化不对称氧化还原反应的特点和规律及其关键立体选择性羰基还原酶的性质和结构特征,并在此基础上,重点针对大基团手性羟基化合物的不对称合成,总结了羰基还原酶及其催化系统开发和应用的研究进展,并进一步提出解决该关键问题的主要发展策略.     

不对称还原前手性芳香酮微生物的筛选及反应特性

含芳香基手性醇是许多手性药物合成的关键手性砌块,生物催化不对称还原前手性酮是合成该类醇的重要方法之一.以4'-氯-苯乙酮为模型底物,从土壤中筛选得到一株能高效催化前手性芳香酮不对称还原合成相应手性醇的菌株,鉴定表明该菌株为白地霉( Geotrichum candid ).进一步考察了其催化4'-氯-苯乙酮不对称还原的反应特性,发现还原4'-氯-苯乙酮的产物主要为 S-4'-氯苯乙醇.在合适的反应条件下,其产率达到35%,对映选择性高于97%.     

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。     

NAD(P)H依赖型氧化还原酶不对称还原胺化制备手性胺的研究进展

不对称还原胺化反应是制备医药中间体手性胺结构单元的重要反应。目前已有许多不同种类的酶被应用于合成手性胺,其中NAD(P)H依赖型氧化还原酶催化的还原胺化反应最为引人注目,因为其能够一步将潜手性酮化合物完全转化为光学纯的手性胺化合物。文中以亚胺还原酶、氨基酸脱氢酶、冠瘿碱脱氢酶和还原性酮胺化酶为例,从NAD(P)H依赖型氧化还原酶的结构特征、作用机理、分子改造及催化应用等方面,综述了其在不对称还原胺化合成手性胺领域的研究进展。     

一种来源于毕赤酵母的高对映选择性羰基还原酶的性质及底物谱分析

手性醇是一类非常重要的化合物,羰基还原酶催化酮的不对称还原生成对应的手性醇.从毕赤酵母Pichia pastoris GS115基因组数据中找到一个潜在的NADPH依赖的羰基还原酶,研究毕赤酵母 P.pastoris GS115中的羰基还原酶.根据其核酸序列设计引物,从P.pastoris GS115基因组中扩增到目的基因ppcr,大肠杆菌BL21 (DE3)中表达,Ni-NTA纯化,对酶的性质和底物谱进行了研究.PPCR的最适反应温度为35℃,最适反应pH为6.0,低于45℃时有很好的稳定性.对3-甲基-2-羰基丁酸乙酯的Km和kcat分别为9.48 mmol/L和0.12 s-1. PPCR表现出广泛的底物谱和很高的对映选择性,对醛、α-酮酯、芳香族β-酮酯及芳香族酮都表现出了很好的活性,在测定的底物中,除极少数底物外,ee值均达到97％以上.因此,PPCR具有较好的应用前景.     

应用生物法还原羰基化合物

还原羰基化合物是生成手性化合物的一种常见方法,利用生物催化反应的特异性,应用生物法进行这一反应具有广阔的应用前景,其中包括采用酶、微生物、值物细胞等作为催化剂还原羰基化合物的反应。该文介绍了有关这一反应的研究现状。     

生物催化立体选择性氧化还原中存在问题及其发展策略

以立体选择性氧化还原酶或其全细胞催化的不对称氧化还原反应已经成为转化光学活性手性醇及其他手性化合物的有效手段。然而,生物催化氧化还原反应体系存在着催化活性与专一性、反应体系与催化稳定性等生物催化剂所固有的局限性问题,而且,生物氧化还原反应必需辅酶及其再生问题也是限制该转化途径产业化应用的一个重要因素。围绕上述生物催化立体选择性氧化还原中存在的关键问题,现代分子生物技术及反应工程的不断突破和发展为改善生物催化立体选择性氧化还原在催化剂本身和反应工程方面的局限性提供了有效的发展策略,为其进一步大规模产业应用提供了发展基础。     

手性技术与生物催化

简要介绍了手性,手性技术与生物催化的基本概念。手性,是指一个有机分子具有不对称性,形成两种空间排布方式不同的对映异构体。手性技术即生产手性化合物的技术,手性化合物的制备方法主要有手性源、外消旋体拆分、不对称合成等几种。生物催化,即利用酶或微生物等生物材料催化进行某种化学反应,被认为是手性化合物生产取得突破的关健技术。文章还介绍了生物催化外消旋体拆分、生物催化不对称合成等几种生产手性化合物的应用实例。     

Novel bioreduction system for the production of chiral alcohols

Chiral alcohols are useful intermediates for many pharmaceuticals and chemicals. Enzymatic asymmetric reduction of prochiral carbonyl compounds is a promising method for producing chiral alcohols. There have been many attempts to construct bioreduction systems for the industrial production of chiral alcohols. This review focuses on the establishment of a novel bioreduction system using an Escherichia coli transformant co-expressing genes for carbonyl reductase and cofactor-regeneration enzyme. This bioreduction system could be useful as an all-purpose catalyst for asymmetric reduction reactions.     

Production of chiral alcohols from prochiral ketones by microalgal photo-biocatalytic asymmetric reduction reaction

Microalgal photo-biocatalysis is a green technique for asymmetric synthesis. Asymmetric reduction of nonnatural prochiral ketones to produce chiral alcohols by microalgal photo-biocatalysis was studied in this work. Acetophenone (ACP) and ethyl acetoacetate (EAA) were chosen as model substrates for aromatic ketones and β-ketoesters, respectively. Two prokaryotic cyanophyta and two eukaryotic chlorophyta were selected as photo-biocatalysts. The results proved that nonnatural prochiral ketones can be reduced by microalgal photo-biocatalysis with high enantioselectivity. Illumination is indispensable to the photo-biocatalysis. For aromatic ketone, cyanophyta are eligible biocatalysts. For ACP asymmetric reduction reaction, about 45% yield and 97% e.e. can be achieved by the photo-biocatalysis reaction with Spirulina platensis as biocatalyst. On the contrary, chlorophyta are efficient biocatalysts for β-ketoester asymmetric reduction reaction among the four tested algae. For EAA asymmetric reduction reaction, about 70% yield and 90% e.e. can be achieved with Scenedesmus obliquus as biocatalyst. The microalgae used in this study outperformed other characterized biocatalysts such as microbial and plant cells.     

Highly Enantioselective Production of Chiral Secondary Alcohols with Candida zeylanoides as a New Whole Cell Biocatalyst

The increasing demand for biocatalysts in synthesizing enantiomerically pure chiral alcohols results from the outstanding characteristics of biocatalysts in reaction, economic, and ecological issues. Herein, fifteen yeast strains belonging to three food originated yeast species Candida zeylanoides, Pichia fermentans, and Saccharomyces uvarum were tested for their capability for asymmetric reduction of acetophenone to 1‐phenylethanol as biocatalysts. Of these strains, C. zeylanoides P1 showed an effective asymmetric reduction ability. Under optimized conditions, substituted acetophenones were converted to corresponding optically active secondary alcohols in up to 99% enantiomeric excess and at high yields. The preparative scale asymmetric bioreduction of 4‐nitroacetophenone ( 1m ) by C. zeylanoides P1 gave (S)‐1‐(4‐nitrophenyl)ethanol ( 2m ) with 89% yield and > 99% enantiomeric excess. Compound 2m has been obtained in an enantiomerically pure and inexpensive form. Additionally, these results indicate that C. zeylanoides P1 is a promising biocatalyst for the synthesis of chiral alcohols in industry.     

Highly Enantioselective Production of Chiral Secondary Alcohols Using Lactobacillus paracasei BD101 as a New Whole Cell Biocatalyst and Evaluation of Their Antimicrobial Effects

Chiral secondary alcohols are valuable intermediates for many important enantiopure pharmaceuticals and biologically active molecules. In this work, we studied asymmetric reduction of aromatic ketones to produce the corresponding chiral secondary alcohols using lactic acid bacteria (LAB) as new biocatalysts. Seven LAB strains were screened for their ability to reduce acetophenones to their corresponding alcohols. Among these strains, Lactobacillus paracasei BD101 was found to be the most successful at reducing the ketones to the corresponding alcohols. The reaction conditions were further systematically optimized for this strain and high enantioselectivity (99%) and very good yields were obtained. These secondary alcohols were further tested for their antimicrobial activities against important pathogens and significant levels of antimicrobial activities were observed although these activities were altered depending on the secondary alcohols as well as their enantiomeric properties. The current methodology demonstrates a promising and alternative green approach for the synthesis of chiral secondary alcohols of biological importance in a cheap, mild, and environmentally useful process.     

Effect of chemical structure of benzofuran derivatives and reaction conditions on enantioselective properties of Aureobasidium pullulans microorganism contained in Boni Protect antifungal agent

Bioorganic asymmetric reduction of carbonyl compounds is one of the most important fundamental and practical reactions for producing chiral alcohols. The stereoselective bioreduction of prochiral ketones of benzofuran derivatives in the presence of yeast-like fungus Aureobasidium pullulans contained in the antifungal Boni Protect agent was studied. Biotransformations were carried out under moderate conditions in an aqueous and two-phase system and without multiplication of the bioreagent. Despite similar chemical structure, each of the used ketone has been reduced with varying efficiency and selectivity. One of the reasons for these results is the presence of a whole set of oxidoreductases in A. pullulans cells that are sensitive to the smallest changes in the structure of prochiral substrate. The unsymmetrical methyl ketones were biotransformed with the highest selectivity. Aureobasidium pullulans microorganism is less effective in the reduction of unsymmetrical halomethyl ketones. The presence of a heteroatom in the alkyl group significantly decreases the selectivity of the process. Finally, as a result of the preferred hydride ion transfer from the dihydropyridine ring of the cofactor to the carbonyl double bond on the re side, secondary alcohols of the S and R configuration were obtained with moderate to high enantioselectivity (55-99%).     

Recent advances in imine reductase-catalyzed reactions

Imine reductases are nicotinamide-dependent enzymes that catalyze the asymmetric reduction of various imines to the corresponding amine products. Owing to the increasing roles of chiral amines and heterocyclic compounds as intermediates for pharmaceuticals, the demand for novel selective synthesis strategies is vitally important. Recent studies have demonstrated the discovery and structural characterization of a number of stereoselective imine reductase enzymes. Here, we highlight recent progress in applying imine reductases for the formation of chiral amines and heterocycles. It particularly focuses on the utilization of imine reductases in reductive aminations of aldehydes and ketones with various amine nucleophiles, one of the most powerful reactions in the synthesis of chiral amines. Second, we report on the synthesis of saturated substituted N-heterocycles by combining them with further biocatalysts, such as carboxylic acid reductases, oxidases or transaminases. Finally, we summarize the latest applications of imine reductases in the promiscuous asymmetric hydrogenation of a highly reactive carbonyl compound and the engineering of the cofactor specificity from NADPH to NADH.     

Candida parapsilosis: A versatile biocatalyst for organic oxidation-reduction reactions

This review highlights the importance of the biocatalyst, Candida parapsilosis for oxidation and reduction reactions of organic compounds and establishes its versatility to generate a variety of chiral synthons. Appropriately designed reactions using C. parapsilosis effect efficient catalysis of organic transformations such as deracemization, enantioselective reduction of prochiral ketones, imines, and kinetic resolution of racemic alcohols via selective oxidation. This review includes the details of these biotransformations, catalyzed by whole cells (wild type and recombinant strains), purified enzymes (oxidoreductases) and immobilized whole cells of C. parapsilosis. The review presents a bioorganic perspective as it discusses the chemo, regio and stereoselectivity of the biocatalyst along with the structure of the substrates and optical purity of the products. Fermentation scale biocatalysis using whole cells of C. parapsilosis for several biotransformations to synthesize important chiral synthons/industrial chemicals is included. A comparison of C. parapsilosis with other whole cell biocatalysts for biocatalytic deracemization and asymmetric reduction of carbonyl and imine groups in the synthesis of a variety of enantiopure products is presented which will provide a basis for the choice of a biocatalyst for a desired organic transformation. Thus, a wholesome perspective on the present status of C. parapsilosis mediated organic transformations and design of new reactions which can be considered for large scale operations is provided. Taken together, C. parapsilosis can now be considered a ‘reagent’ for the organic transformations discussed here.     

Highly enantioselective bioreduction of prochiral ketones by stem and germinated plant of Brassica oleracea variety italica

Abstract

An eco-friendly and environmentally benign asymmetric reduction of a broad range of prochiral ketones employing Brassica oleracea variety italica (stems and germinated plant) as a novel biocatalyst was developed. It was found that B. oleracea variety italica could be used effectively for enantioselective bioreduction in aqueous medium with moderate to excellent chemical yield and enantiomeric excess (ee). This process is more efficient and generates less waste than conventional chemical reagents or microorganisms. Both R- and S-configurations were obtained by these asymmetric reactions. The best ee were achieved for pyridine derivatives (92–99%). The ee in germinated plant reactions were significantly higher than those of stem reactions. The low cost and the easy availability of these biocatalysts suggest their possible use for large scale preparations of important chiral alcohols.     

Microbial formation of esters

Small aliphatic esters are important natural flavor and fragrance compounds and have numerous uses as solvents and as chemical intermediates. Besides the chemical or lipase-catalyzed formation of esters from alcohols and organic acids, small volatile esters are made by several biochemical routes in microbes. This short review will cover the biosynthesis of esters from acyl-CoA and alcohol condensation, from oxidation of hemiacetals formed from aldehydes and alcohols, and from the insertion of oxygen adjacent to the carbonyl group in a straight chain or cyclic ketone by Baeyer–Villiger monooxygenases. The physiological role of the ester-forming reactions can allow degradation of ketones for use as a carbon source and may play a role in detoxification of aldehydes or recycling cofactors. The enzymes catalyzing each of these processes have been isolated and characterized, and a number of genes encoding the proteins from various microbes have been cloned and functionally expressed. The use of these ester-forming organisms or recombinant organisms expressing the appropriate genes as biocatalysts in biotechnology to make specific esters and chiral lactones has been studied in recent years.     

Lactones 32 : New aspects of the application of Fusarium strains to production of alkylsubstituted ɛ-lactones

Wild strains Fusarium oxysporum and Fusarium avenaceum were chosen among six to the Baeyer–Villiger oxidation of alkylsubstituted cyclohexanones (1a–e) to the corresponding highly enantiomerically enriched ?-lactones. Besides the oxidation activity, the biocatalysts were also able to the reduction of carbonyl group. Only cis-methylcyclohexanols turned out to be further transformed to lactones. Biotransformations of optically enriched ketones and alcohols were also studied to give an overall view of stereoselectivity of the enzymes involved in biocatalytic pathway.