Baeyer-Villiger单加氧酶的蛋白质改造及其催化氧化反应研究新进展

Baeyer-Villiger单加氧酶(BVMO)作为一种重要的生物催化剂,可以催化各种有机酮/醛化合物的Baeyer-Villiger氧化反应,以及一些含硫、硒、硼等杂原子底物的氧化反应.Baeyer-Villiger单加氧酶催化的氧化反应具有选择性高、反应条件温和、高效等优点,已成为不可或缺的有机合成工具,被广泛应用于各种手性化合物的合成中.近年来利用生物信息学分析和基因挖掘技术,从众多微生物中找到了多种新型的BVMO;另外利用蛋白质工程技术对已知的野生型BVMO进行改造,从而扩大底物范围,提高热稳定性和反应活性,改善酶的立体、区域和化学选择性.这些都进一步扩大了Baeyer-Villiger单加氧酶催化氧化反应的应用范围.以不同底物结构的Baeyer Villiger氧化反应为主线,综述了近5年来国内外对野生型以及蛋白质工程改造的BVMO催化氧化反应研究的新进展.     

分子氧氧化法合成ε-己内酯的研究进展

内酯化合物在医药、高分子等领域都有重要的应用.其中ε-己内酯是生物可降解材料聚己内酯(poly(ε-caprolactone))的单体以及聚酯的改性剂,目前年产量近10万吨,未来前景广阔.本文主要介绍了本课题组在分子氧氧化法合成内酯化合物特别是ε-己内酯方面所做的工作,以及国内外关于内酯化合物合成的研究进展以及展望.本课题组在传统的Baeyer-Villiger氧化环酮化合物制备内酯产物的研究基础上加以改进,分别从环己酮、K/A油(环己酮和环己醇的混合物)、环己烷出发,以氧气为氧化剂,一步反应得到ε-己内酯,较传统Baeyer-Villiger氧化法更加简单、高效、绿色环保,具有潜在的工业发展前景.     

Baeyer-Villiger氧化反应的不同催化体系

Baeyer-Villiger氧化反应能控制产物的立体化学结构,在有机合成中对功能基转化和环扩张有重要的意义,因此氧化所得的产物可以广泛应用于合成许多天然产物和药物中间体以及一些高分子材料的单体等,是目前有机化学研究的热点之一。随着对该反应研究的深入,其催化剂的类型也在不断地增多,包括均相催化剂、非均相催化剂、生物催化剂。均相催化剂选择性和转化率虽高,但不及非均相催化剂重复利用率高。生物催化剂绿色环保,是未来研究的重点之一。本文从均相催化、非均相催化和生物催化三个方面对Baeyer-Villiger氧化反应相关的研究新进展进行了阐述,重点介绍了不同催化体系下催化剂与反应底物之间的作用,总结了有关催化反应的机理,并对Baeyer-Villiger氧化反应的发展进行了展望。     

SBA-15负载硅钨酸催化环己酮Baeyer-Villiger氧化

采用浸渍法制备了H4O40SiW12/SBA-15催化剂,通过红外光谱、X射线衍射、N2吸附-脱附和透射电镜等方法对催化剂进行了表征.结果表明,H4O40SiW12能均匀分散于SBA-15的孔道中,催化剂仍保持载体的介孔结构和硅钨酸的Keggin型结构.将催化剂用于环己酮的Baeyer-Villiger氧化反应中,考察...     

Wells-Dawson型杂多酸的制备及其在Baeyer-Villiger氧化反应中的应用

合成了Wells-Dawson结构的磷钨酸,用红外光谱、紫外光谱和X射线粉末衍射等方法对其结构进行了表征。将所制备杂多酸应用于催化环酮类化合物的Baeyer-Villiger氧化反应中,探讨了催化剂用量、H2O2的用量、反应温度、体系溶剂和反应时间对催化剂性能的影响。结果发现,所得催化剂对环酮类底物的催化氧化反应表现出良好的催化性能。在2-金刚烷酮的催化氧化中,以30%H2O2为氧化剂,在1,2-二氯乙烷溶剂中于80℃反应8 h后,2-金刚烷酮的转化率和产物2-金刚烷内酯的选择性都达到了99%以上。同时,催化剂具有一定的重复使用性。     

活性炭负载磷钨酸催化合成环己酮缩乙二醇

研究了活性炭负载磷钨酸催化合成环己酮缩乙二醇的反应 ,考察了催化剂负载量、反应时间、酮醇物质的量比、反应温度等因素对缩酮反应的影响 .结果表明 ,活性炭负载磷钨酸是合成缩酮的良好催化剂 ,在优化条件下 ,缩酮产率达 94.8% ,催化剂可重复使用     

负载锑的水滑石催化环己酮Baeyer-Villiger氧化制ε-己内酯

 通过浸渍法制备了负载Sb的镁铝水滑石类化合物Sb/HT, 用X射线衍射(XRD)和热重(TG)等技术对催化剂的结构进行了表征,研究了其对环己酮Baeyer-Villiger(BV)氧化制ε-己内酯的催化性能,考察了催化剂制备条件和反应条件对催化活性的影响以及催化剂的再生性能. 结果表明,在乙腈作为溶剂的体系中,负载Sb的镁铝水滑石对环己酮氧化制ε-己内酯具有较高的催化活性. 在水滑石前驱体中镁/铝摩尔比为3, Sb含量为1.5%和焙烧温度为450 ℃时制备的Sb/HT催化剂的催化活性最高,在10 ml乙腈和140 mmol 30%H2O2中于70 ℃反应4 h后,环己酮的转化率达42%,ε-己内酯选择性达94%. 催化剂可多次再生重复使用,催化活性稳定.     

甲基酮香料的仿生合成新方法研究

四氢叶酸辅酶在生物体内的作用是转移不同氧化态的一碳单元, 当一碳单元处于甲酸氧化态时, 活性部位是具有五元环状结构的咪唑啉环. 模拟四氢叶酸辅酶转移一碳单元的反应, 以苯并咪唑甲基碘盐作为四氢叶酸辅酶甲酸氧化态模型, 以格利雅试剂甲基碘化镁作为接收一碳单元的亲核试剂, 将甲酸氧化态的一碳单元转移给甲基碘化镁, 成功合成了三种重要的甲基酮香料甲基己基酮、甲基壬基酮和甲基十一烷基酮, 其结构用元素分析, 1H NMR, IR和MS等方法进行了表征, 并对反应机理和反应条件进行了讨论, 为甲基酮香料提供了一种仿生合成新方法.     

[Co（p—Me—PPA）2（H2O）2]Cl的合成及其催化环己烯氧化反应的研究

环己烯的选择性氧化是一个较难的课题,原冈是环己烯存在两个活性部位易工发生氧化反应：一是烯两基位的氢易被氧化生成环己烯醇和环己烯酮的产物：二是双键易被氧化成环氧化物、环己酮或Ｃ—ＣＸＸ键断裂生成难成酸等。因此人们一直在努力寻找具有高活性,高选择性的催化剂用以催化环己烯的氧化反应［’＊’］。本文合成的Ｋ叶卜＊卜呷Ａ从比０）。厂１,在０。作氧源下ｍ于催化环己烯氧化反应时呈现出较好的转化率和环己烯酮选择性。１【Ｃｏ（Ｐ－Ｍｅ－ＰＰＡ）。（Ｈ。０）。」Ｃ！的合成采ｍ２一毗咙甲酸和ｐ一甲基本胺作原料合成了【…     

(+)-菊花烯酮的Baeyer-Villiger反应

Baeyer-Villiger反应是酮在过氧酸氧化下形成酯的反应,是有机反应中合成酯的主要方法,这一反应可应用在环酮扩环形成含氧杂环内酯的反应上．由于反应是立体控制的,已广泛应用于天然产物,例如：抗生素、类固醇和信息素中间体的合成,某些用其它方法难以合成的羟基酸可由内酯水解得到。     

Access to optically pure 4- and 5-substituted lactones: a case of chemical-biocatalytical cooperation

Optically pure or highly enantiomerically enriched 4- and 5-substituted lactones are rather difficult to obtain. Chemical or enzymatic syntheses alone are not particularly successful. A combination of chemical catalysis and biocatalysis, however, provides a convenient route to a variety of these useful chiral compounds. In this paper we describe the synthesis of several optically pure 4- and 5-substituted lactones obtained via whole cell-catalyzed Baeyer-Villiger oxidations of highly enantiomerically enriched 3-alkyl cyclic ketones. Such chiral ketones are readily accessed by recently developed copper-catalyzed asymmetric conjugate reductions of the corresponding enones. A very high proximal regioselectivity and complete chirality transfer was obtained by employing biological Baeyer-Villiger oxidations, using recombinant E. coli strains that overexpress cyclopentanone monooxygenase (CPMO). A comparative study showed that CPMO gives superior results to those obtained with cyclohexanone monooxygenase (CHMO) catalyzed oxidations.     

Towards practical biocatalytic Baeyer-Villiger reactions: applying a thermostable enzyme in the gram-scale synthesis of optically-active lactones in a two-liquid-phase system

Baeyer-Villiger monooxygenases (BVMOs) are extremely promising catalysts useful for enantioselective oxidation reactions of ketones, but organic chemists have not used them widely due to several reasons. These include instability of the enzymes in the case of in vitro and even in vivo systems, reactant/product inhibition, problems with upscaling and the necessity of using specialized equipment. The present study shows that the thermally stable phenylacetone monooxygenase (PAMO) and recently engineered mutants can be used as a practical catalysts for enantioselective Baeyer-Villiger oxidations of several ketones on a preparative scale under in vitro conditions. For this purpose several parameters such as buffer composition, the nature of the solvent system and the co-factor regeneration system were optimized. Overall a fairly versatile and efficient catalytic system for enantioselective laboratory scale BV-oxidations of ketones was developed, which can easily be applied even by those organic chemists who are not well versed in the use of enzymes.     

Simultaneous biocatalyst production and baeyer-villiger oxidation for bioonversion of cyclohexanone by recombinant Escherichia coli expressing cyclohexanone monooxygenase

Cyclohexanone monooxygenase (CHMO) catalyzing Baeyer-Villiger oxidation converts cyclic ketones into optically pure lactones, which have been used as building blocks in organic synthesis. A recombinant Escherichia coli BL21(DE3)/pMM4 expressing CHMO originated from Acinetobacter sp. NCIB 9871 was used to produce ε-caprolactone through a simultaneous biocatalyst production and Baeyer-Villiger oxidation (SPO) process. Afed-batch process was designed to obtain high cell density for improvin production of ε-caprolactone. The fed-batch SPO process have the best results, 10.2 g/L of ε-caprolactone and 0.34 g/(L·h) of productivity, corresponding to a 10.5- and 3.4-fold enhancement compared with those of the batch SPO, respectively.     

Protein engineering of stereoselective baeyer-villiger monooxygenases

Baeyer-Villiger monooxygenases (BVMOs) have been used for decades as catalysts in stereoselective Baeyer-Villiger reactions, including oxidative kinetic resolution of racemic ketones and desymmetrization of prochiral substrates with high enantioselectivity. These complement catalytic BV processes based on chiral synthetic catalysts. However, as in any enzyme-catalyzed process, limitations exist due to the often observed narrow substrate scope and/or insufficient stereoselectivity. Recent protein engineering of BVMOs in the form of directed evolution and rational design have eliminated these traditional limitations, which is the subject of this Minireview. The main focus is on phenylacetone monooxygenase (PAMO); an unusually thermostable and robust BVMO, which has a very narrow substrate scope. Protein engineering of PAMO has provided a number of mutants that display relatively wide substrate scope, high stereoselectivity, and maintained thermostability.     

Blending Baeyer-Villiger monooxygenases: using a robust BVMO as a scaffold for creating chimeric enzymes with novel catalytic properties

The thermostable Baeyer-Villiger monooxygenase (BVMO) phenylacetone monooxygenase (PAMO) is used as a scaffold to introduce novel selectivities from other BVMOs or the metagenome by structure-inspired subdomain exchanges. This yields biocatalysts with new preferences in the oxidation of sulfides and the Baeyer-Villiger oxidation of ketones, all while maintaining most of the original thermostability.     

Flavins as organocatalysts for environmentally benign molecular transformations

Recent progress in the development of flavin-catalyzed oxidations and related reactions is described with respect to scope, limitation, and reaction mechanism. The 4a-hydroperoxyflavins, which are the most simplified model compounds of flavoenzymes, act as catalytically active species for the oxidation of organic substrates with the help of H(2)O(2) or O(2) as a mild oxidant. This principle behind the simulation of flavoenzymes led to the discovery of a variety of environmentally benign, oxidative transformations of secondary amines to nitrones, tertiary amines to N-oxides, sulfides to sulfoxides, and Baeyer-Villiger oxidations of ketones. Asymmetric oxidation of sulfides can also be performed with several chiral flavin catalysts. One of the fortunate outcomes of this study is the development of an environmentally friendly ("green") method for the "aerobic hydrogenation" of olefins, which is achieved by in situ generation of diimide with the aid of the flavin-catalyzed oxidation of hydrazine under an O(2) atmosphere.     

Microbial Baeyer-Villiger oxidation: stereopreference and substrate acceptance of cyclohexanone monooxygenase mutants prepared by directed evolution

[reaction: see text] An array of random mutants of cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 was screened against a library of structurally diverse ketones for modifications in the substrate acceptance profile and stereopreference of the enzymatic Baeyer-Villiger biooxidation. While the set of mutant biocatalysts was initially evolved for the enantiocomplementary oxidation of 4-hydroxycyclohexanone, improved and/or divergent stereoselectivities were observed for several substrates. In addition, expanded substrate acceptance to facilitate biotransformation of sterically demanding ketones was found.     

Characterization and Crystal Structure of a Robust Cyclohexanone Monooxygenase

Cyclohexanone monooxygenase (CHMO) is a promising biocatalyst for industrial reactions owing to its broad substrate spectrum and excellent regio‐, chemo‐, and enantioselectivity. However, the low stability of many Baeyer–Villiger monooxygenases is an obstacle for their exploitation in industry. Characterization and crystal structure determination of a robust CHMO from Thermocrispum municipale is reported. The enzyme efficiently converts a variety of aliphatic, aromatic, and cyclic ketones, as well as prochiral sulfides. A compact substrate‐binding cavity explains its preference for small rather than bulky substrates. Small‐scale conversions with either purified enzyme or whole cells demonstrated the remarkable properties of this newly discovered CHMO. The exceptional solvent tolerance and thermostability make the enzyme very attractive for biotechnology.     

Enhanced shape selective catalysis of mixed cyclic ketones in aerobic Baeyer-Villiger oxidation with magnetic Cu-Fe3O4 supported mesoporous silica microspheres

Various strategies have been developed to improve the conversion for the Baeyer-Villiger oxidation. However, the catalytic effects of the Baeyer-Villiger oxidation for the mixed ketones are rarely reported, though it is also important for the natural and industrial separation processes. In this report, magnetite Cu modified Fe₃O₄ supported mesoporous silica microspheres (Cu-Fe₃O₄@mSiO₂) have been successfully synthesized by two step direct hydrothermal method (DHT). Over 99% of cyclohexanone conversion was obtained with mild air oxidation and benzaldehyde as sacrificing agent over Cu-Fe₃O₄@mSiO₂. The catalytic system also shows higher conversion rates for small molecular ketones in the mixed ketone reactants, which was attributed to the enhanced mass transfer effect and Fe-Cu composite active sites in the magnetite mesoporous silica microspheres. The catalyst could be recycled for four times with similar catalytic performance, which shows enhanced shape selectivity in aerobic Baeyer-Villiger oxidations for mixed cyclic ketones.     

Regiodivergent Baeyer-Villiger oxidation of fused ketone substrates by recombinant whole-cells expressing two monooxygenases from Brevibacterium

Microbial Baeyer-Villiger oxidations of fused bicyclic ketones with a cyclobutanone structural motif were investigated using recombinant Escherichia coli cells expressing two monooxygenases from Brevibacterium. In a kinetic resolution process fused ketones were transformed to regioisomeric lactones: ‘normal’ lactones were generated by migration of the more substituted carbon atom and ‘abnormal’ lactones resulted from migration of the less substituted carbon atom. The two Baeyer-Villigerases demonstrated a significantly different stereoselectivity for the regiodivergent biotransformation.