Reaching New Biocatalytic Reactivity Using Continuous Flow Reactors

The use of flow reactors in biocatalysis has increased significantly in recent years. Chemists have begun to design flow systems that even allow new biocatalytic reactions to take place. This concept article will focus on the design of flow systems that have allowed enzymes to go beyond their limits in batch. The case is made for moving towards fully continuous systems. With flow chemistry increasingly seen as an enabling technology for automated synthesis, and with advancements in AI-assisted enzyme design, there is a real possibility to fully automate the development and implementation of a continuous biocatalytic processes. This will lead to significantly improved enzyme processes for synthesis.     

E factors, green chemistry and catalysis: an odyssey

The development of green chemistry is traced from the introduction of the concepts of atom economy (atom utilisation) and E factors in the early 1990s. The important role of catalysis in reducing or eliminating waste is emphasised and illustrated with examples from heterogeneous catalytic oxidations with hydrogen peroxide, homogeneous catalytic oxidations and carbonylations and organocatalytic oxidations with stable N-oxy radicals. Catalytic reactions in non-conventional media, e.g. aqueous biphasic, supercritical carbon dioxide and ionic liquids, are presented. Biotransformations involving non-natural reactions of enzymes, e.g. ester ammoniolysis, and the rational design of semi-synthetic enzymes, such as vanadate phytase, are discussed. The optimisation of enzyme properties using in vitro evolution and improvement of their operational stability by immobilisation as cross-linked enzyme aggregates (CLEA) are presented. The ultimate in green chemistry is the integration of catalytic steps into a one-pot, catalytic cascade process. An example of a chemoenzymatic synthesis of an enantiomerically pure amino acid in water and a trienzymatic cascade process using a triple-decker oxynitrilase/nitrilase/amidase CLEA are discussed. Finally, catalytic conversions of renewable raw materials are examined and the biocatalytic aerobic oxidation of starch to carboxy starch is presented as an example of green chemistry in optima forma i.e. a biocompatible product from a renewable raw material using a biocatalytic air oxidation.     

离子液体的制备及其在酶催化反应中的应用

离子液体，尤其是非水溶性离子液体可以作为一种溶剂或酶的载体用于非水相酶促反应中，也可以用于双相体系中的酶促反应。本文概括性介绍了常见离子液体的制备，总结和讨论了离子液体中酶的活性、稳定性、反应选择性以及各类酶在离子液体中的催化反应行为。离子液体的物性及其与酶的相容性对酶本身及酶促反应都有很大的影响。在非水相酶促反应中，离子液体的极性作用不遵从通常用来判别大多数有机物溶剂行为的规则，比如lgP规则。     

Asymmetric catalysis as a method for the synthesis of chiral organophosphorus compounds

This review discusses methods for the metallo-, organo- and biocatalytic asymmetric synthesis of chiral organophosphorus compounds with many applications in stereoselective synthesis with references to updated literature reports as well as the author’s original research. Asymmetric catalytic hydrogenation and reduction with chiral organometallic complexes, together with actively used asymmetric organocatalytic versions of various reactions enable us to synthesize chiral organophosphonates and phosphinates with high enantioselectivity and purity. Asymmetric catalysis is also an effective tool to realize some classic reactions of phosphorus chemistry in a stereospecific manner.     

The Oxidation of Thiols by Flavoprotein Oxidases: a Biocatalytic Route to Reactive Thiocarbonyls

Flavoprotein oxidases are a diverse class of biocatalysts, most of which catalyze the oxidation of C? O, C? N, or C? C bonds. Flavoprotein oxidases that are known to catalyze the oxidation of C? S bonds are rare, being limited to enzymes that catalyze the oxidative cleavage of thioethers. Herein, we report that various flavoprotein oxidases, previously thought to solely act on alcohols, also catalyze the oxidation of thiols to thiocarbonyls. These results highlight the versatility of enzymatic catalysis and provide a potential biocatalytic route to reactive thiocarbonyl compounds, which have a variety of applications in synthetic organic chemistry.     

Extending the biocatalytic scope of regiocomplementary flavin-dependent halogenase enzymes

Flavin-dependent halogenases are potentially valuable biocatalysts for the regioselective halogenation of aromatic compounds. These enzymes, utilising benign inorganic halides, offer potential advantages over traditional non-enzymatic halogenation chemistry that often lacks regiocontrol and requires deleterious reagents. Here we extend the biocatalytic repertoire of the tryptophan halogenases, demonstrating how these enzymes can halogenate a range of alternative aryl substrates. Using structure guided mutagenesis we also show that it is possible to alter the regioselectivity as well as increase the activity of the halogenases with non-native substrates including anthranilic acid; an important intermediate in the synthesis and biosynthesis of pharmaceuticals and other valuable products.     

Formate Oxidase (FOx) from Aspergillus oryzae: One Catalyst Enables Diverse H2O2‐Dependent Biocatalytic Oxidation Reactions

An increasing number of biocatalytic oxidation reactions rely on H₂O₂ as a clean oxidant. The poor robustness of most enzymes towards H₂O₂, however, necessitates more efficient systems for in situ H₂O₂ generation. In analogy to the well‐known formate dehydrogenase to promote NADH‐dependent reactions, we here propose employing formate oxidase (FOx) to promote H₂O₂‐dependent enzymatic oxidation reactions. Even under non‐optimised conditions, high turnover numbers for coupled FOx/peroxygenase catalysis were achieved.     

Employing modular polyketide synthase ketoreductases as biocatalysts in the preparative chemoenzymatic syntheses of diketide chiral building blocks

Chiral building blocks are valuable intermediates in the syntheses of natural products and pharmaceuticals. A scalable chemoenzymatic route to chiral diketides has been developed that includes the general synthesis of α-substituted, β-ketoacyl N-acetylcysteamine thioesters followed by a biocatalytic cycle in which a glucose-fueled NADPH-regeneration system drives reductions catalyzed by isolated modular polyketide synthase (PKS) ketoreductases (KRs). To identify KRs that operate as active, stereospecific biocatalysts, 11 isolated KRs were incubated with 5 diketides and their products were analyzed by chiral chromatography. KRs that naturally reduce small polyketide intermediates were the most active and stereospecific toward the panel of diketides. Several biocatalytic reactions were scaled up to yield more than 100?mg of product. These syntheses demonstrate the ability of PKS enzymes to economically and greenly generate diverse chiral building blocks on a preparative scale.     

Engineered Biocatalytic Synthesis of β-N-Substituted-α-Amino Acids

Non-canonical amino acids (ncAAs) are useful synthons for the development of new medicines, materials, and probes for bioactivity. Recently, enzyme engineering has been leveraged to produce a suite of highly active enzymes for the synthesis of β-substituted amino acids. However, there are few examples of biocatalytic N-substitution reactions to make α,β-diamino acids. In this study, we used directed evolution to engineer the β-subunit of tryptophan synthase, TrpB, for improved activity with diverse amine nucleophiles. Mechanistic analysis shows that high yields are hindered by product re-entry into the catalytic cycle and subsequent decomposition. Additional equivalents of l -serine can inhibit product reentry through kinetic competition, facilitating preparative scale synthesis. We show β-substitution with a dozen aryl amine nucleophiles, including demonstration on a gram scale. These transformations yield an underexplored class of amino acids that can serve as unique building blocks for chemical biology and medicinal chemistry.     

Modern trends in biocatalytic synthesis of chiral compounds

Characteristic features and modern trends in the biocatalytic synthesis of chiral compounds have been discussed. New processes of biocatalytic synthesis use at least two enzymes. Whole cells of recombinant strains are utilized as biocatalysts, and the complete set of target enzymes can be expressed in a single cell.     

3D-Printed Phenacrylate Decarboxylase Flow Reactors for the Chemoenzymatic Synthesis of 4-Hydroxystilbene

Continuous flow systems for chemical synthesis are becoming a major focus in organic chemistry and there is a growing interest in the integration of biocatalysts due to their high regio- and stereoselectivity. Methods established for 3D bioprinting enable the fast and simple production of agarose-based modules for biocatalytic reactors if thermally stable enzymes are available. We report here on the characterization of four different cofactor-free phenacrylate decarboxylase enzymes suitable for the production of 4-vinylphenol and test their applicability for the encapsulation and direct 3D printing of disk-shaped agarose-based modules that can be used for compartmentalized flow microreactors. Using the most active and stable phenacrylate decarboxylase from Enterobacter spec. in a setup with four parallel reactors and a subsequent palladium(II) acetate-catalysed Heck reaction, 4-hydroxystilbene was synthesized from p-coumaric acid with a total yield of 14.7 % on a milligram scale. We believe that, due to the convenient direct immobilization of any thermostable enzyme and straightforward tuning of the reaction sequence by stacking of modules with different catalytic activities, this simple process will facilitate the establishment and use of cascade reactions and will therefore be of great advantage for many research approaches.     

Research activities in the field of enzyme engineering in the framework of the russian state scientific program “novel methods in bioengineering”

The article describes the research activities in the field of enzyme engineering in Russia. The discussion is focused on fundamental studies of biocatalytic processes that expand utilization of enzymes, biocatalytic synthesis of organic products from renewable raw mate rials, enzymes for hydrolysis of cellulose and lignocellulose materials, immobilized cells, new enzyme-based drugs, enzymes in fine organic synthesis, bioanalytic devices, biosensors, and biofuels.     

Stereoselective biotransformations using fungi as biocatalysts

The development of novel biocatalytic methods is a continuously growing area of chemistry, microbiology, and genetic engineering due to the fact that biocatalysts are selective, easy-to-handle, and environmentally friendly. A wide range of reactions are catalyzed by microorganisms. Fungi can be considered as a promising source of new biocatalysts, mainly for chiral reactions. Chemo-, regio-, and stereoselective processes are very important in the synthesis of many chemical, pharmaceutical, and agrochemical intermediates; active pharmaceuticals; and food ingredients. This report reviews stereoselective reactions mediated by fungi, such as stereoselective hydroxylation, sulfoxidation, epoxidation, Baeyer–Villiger oxidation, deracemization, and stereo- and enantioselective reduction of ketones, published between 2000 and 2007.     

Dicarbonyl reduction by single enzyme for the preparation of chiral diols

Chiral diols are a group of key building blocks useful for preparing a variety of important chiral chemicals. While the preparation of optically pure diols is generally not an easy task in synthetic organic chemistry, three classes of enzymes, namely dicarbonyl reductase, dioxygenase and epoxide hydrolase, display remarkable ability to stereoselectively introduce two hydroxyl groups in a single-step enzymatic conversion. In this tutorial review, we pay special attention to dicarbonyl reductases that directly produce chiral diols through the bio-reduction of two carbonyl groups. The dicarbonyl reductases include diketoreductase, α-acetoxy ketone reductase and sepiapterin reductase. We present these exceptional enzymes in the context of source and properties, structure and catalytic mechanism as well as biocatalytic application. In addition to the broad substrate specificity, the excellent stereoselectivity and high catalytic efficiency of these enzymes have positioned them as valuable biocatalysts. With more sophisticated understanding of the structure-function relationship, the practical utilities of these enzymes associated with their interesting chemistry will be considerably appreciated over time. Moreover, rational redesign and molecular evolution of these unusual biocatalysts will truly enable their broader applications in the synthesis of chiral diols in the future.     

生物催化不对称合成beta-羟基酸衍生物

手性β-羟基酸及其衍生物是应用化工和有机合成的关键中间体, 生物催化的不对称合成方法以其绿色环保, 简洁高效及高立体选择性已然成为一个新兴的研究热点。本文较系统的总结了生物催化的β-羟基酸及其衍生物不对称合成研究工作,重点介绍了脂肪酶,腈代谢酶及还原酶在合成手性β-羟基酸衍生物中的应用。最后,对今后生物催化不对称合成β-羟基酸的发展方向做一展望。     

Nitroxides: applications in synthesis and in polymer chemistry

This Review describes the application of nitroxides to synthesis and polymer chemistry. The synthesis and physical properties of nitroxides are discussed first. The largest section focuses on their application as stoichiometric and catalytic oxidants in organic synthesis. The oxidation of alcohols and carbanions, as well as oxidative C-C bond-forming reactions are presented along with other typical oxidative transformations. A section is also dedicated to the extensive use of nitroxides as trapping reagents for C-centered radicals in radical chemistry. Alkoxyamines derived from nitroxides are shown to be highly useful precursors of C-centered radicals in synthesis and also in polymer chemistry. The last section discusses the basics of nitroxide-mediated radical polymerization (NMP) and also highlights new developments in the synthesis of complex polymer architectures.     

Microbial and Enzymatic Processes for the Production of Biologically and Chemically Useful Compounds [New Synthetic Methods (69)]

In recent years, the most significant development in the field of synthetic organic chemistry has been the application of biological systems to chemical reactions. Reactions catalyzed by enzymes and enzyme systems display far greater specificities than more conventional organic reactions. Biological and/or enzymatic syntheses and transformations, that is, “microbial transformations,” have great potential. Some of these reactions have already been shown to have useful applications in the fields of synthetic organic chemistry and biotechnology. This article reviews the current status of the rapidly developing field of microbial transformation, the methodology, available technological procedures, and fields of application being described especially in relation to conventional organic synthesis methods.     

Design and evolution of enzymes for non-natural chemistry

Enzymes are used in biocatalytic processes for the efficient and sustainable production of pharmaceuticals, fragrances, fine chemicals, and other products. Most bioprocesses exploit chemistry found in nature, but we are now entering a realm of biocatalysis that goes well beyond. Enzymes have been engineered to catalyze reactions previously only accessible with synthetic catalysts. Because they can be tuned by directed evolution, many of these new biocatalysts have been shown to perform abiological reactions with high activity and selectivity. We discuss recent examples, showcase catalyst improvements achieved using directed evolution, and comment on some current and future implications of non-natural enzyme evolution for sustainable chemical synthesis.     

Production of Hydroxy Acids: Selective Double Oxidation of Diols by Flavoprotein Alcohol Oxidase

Flavoprotein oxidases can catalyze oxidations of alcohols and amines by merely using molecular oxygen as the oxidant, making this class of enzymes appealing for biocatalysis. The FAD‐containing (FAD=flavin adenine dinucleotide) alcohol oxidase from P. chrysosporium facilitated double and triple oxidations for a range of aliphatic diols. Interestingly, depending on the diol substrate, these reactions result in formation of either lactones or hydroxy acids. For example, diethylene glycol could be selectively and fully converted into 2‐(2‐hydroxyethoxy)acetic acid. Such a facile cofactor‐independent biocatalytic route towards hydroxy acids opens up new avenues for the preparation of polyester building blocks.     

离子液体及其在生物催化中的应用

综述了在离子液体中酶及完整细胞的生物催化反应的研究状况, 初步总结了影响离子液体中酶催化反应的因素, 离子液体作为绿色反应介质在生物转化工业中将有广阔的应用前景.