Discovery, characterization, and synthetic potential of two novel bacterial aryl-alcohol oxidases.

The search for novel synthetic tools to prepare industrial chemicals in a safer and greener manner is a continuing challenge in synthetic chemistry. In this manuscript, we report the discovery, characterization, and synthetic potential of two novel aryl-alcohol oxidases from bacteria which are able to oxidize a variety of aliphatic and aromatic alcohols with efficiencies up to 4970 min mM. Both enzymes have shown a reasonable thermostability (thermal melting temperature values of 50.

L-Cysteine-Catalysed Hydration of Activated Alkynes.

Hydration reactions consist of the introduction of a molecule of water into a chemical compound and are particularly useful to transform alkynes into carbonyls, which are strategic intermediates in the synthesis of a plethora of compounds. Herein we demonstrate that L-cysteine can catalyse the hydration of activated alkynes in a very effective and fully regioselective manner to access important building blocks in synthetic chemistry such as β-ketosulfones, amides and esters, in aqueous media. The mild reaction conditions facilitated the integration with enzyme catalysis to access chiral β-hydroxy sulfones from the corresponding alkynes in a one-pot cascade process in good yields and excellent enantiomeric ratios.

One-Pot Chemoenzymatic Cascade for the Enantioselective C(1)-Allylation of Tetrahydroisoquinolines.

Herein, we report a one-pot, chemoenzymatic process for the synthesis of enantioenriched C(1)-allylated tetrahydroisoquinolines. This transformation couples a monoamine oxidase (MAO-N)-catalyzed oxidation with a metal catalyzed allylboration, followed by a biocatalytic deracemization to afford allylic amine derivatives in both high yields and good to high enantiomeric excess. The cascade is operationally simple, with all components added at the start of the reaction and can be used to generate key building blocks for further elaboration.

Stereoselective Three-Step One-Pot Cascade Combining Amino- and Biocatalysis to Access Chiral γ-Nitro Alcohols.

The combination of small-molecule catalysis and enzyme catalysis represents an underexploited area of research with huge potential in asymmetric synthetic chemistry due to both compatibility of reaction conditions and complementary reactivity. Herein, we describe the telescopic synthesis of chiral nitro alcohols starting from commercially available benzaldehyde derivatives through the one-pot three-step chemoenzymatic cascade combination of a Wittig reaction, chiral-thiourea-catalysed asymmetric conjugate addition, and ketoreductase-mediated reduction to access the corresponding target compounds in moderate to excellent overall isolated yields (36-80 %) and high diastereomeric and enantiomeric ratios (up to >97 : 3). This represents the first example of the combination of an organocatalysed asymmetric conjugate addition via iminium ion activation and a bioreduction step catalysed by ketoreductases.

New Trends and Future Opportunities in the Enzymatic Formation of C-C, C-N, and C-O bonds.

Organic chemistry provides society with fundamental products we use daily. Concerns about the impact that the chemical industry has over the environment is propelling major changes in the way we manufacture chemicals. Biocatalysis offers an alternative to other synthetic approaches as it employs enzymes, Nature's catalysts, to carry out chemical transformations.

Asymmetric synthesis of primary amines catalyzed by thermotolerant fungal reductive aminases.

Chiral primary amines are important intermediates in the synthesis of pharmaceutical compounds. Fungal reductive aminases (RedAms) are NADPH-dependent dehydrogenases that catalyse reductive amination of a range of ketones with short-chain primary amines supplied in an equimolar ratio to give corresponding secondary amines. Herein we describe structural and biochemical characterisation as well as synthetic applications of two RedAms from spp.

Screening and characterization of a diverse panel of metagenomic imine reductases for biocatalytic reductive amination.

Finding faster and simpler ways to screen protein sequence space to enable the identification of new biocatalysts for asymmetric synthesis remains both a challenge and a rate-limiting step in enzyme discovery. Biocatalytic strategies for the synthesis of chiral amines are increasingly attractive and include enzymatic asymmetric reductive amination, which offers an efficient route to many of these high-value compounds. Here we report the discovery of over 300 new imine reductases and the production of a large (384 enzymes) and sequence-diverse panel of imine reductases available for screening.

Engineered formate dehydrogenase from Chaetomium thermophilum, a promising enzymatic solution for biotechnical CO fixation.

Objectives: Formate dehydrogenases (FDHs) are NAD(P)H-dependent enzymes that catalyse the reversible oxidation of formate to CO. The main goal was to use directed evolution to obtain variants of the FDH from Chaetomium thermophilum (CtFDH) with enhanced reduction activity in the conversion of CO into formic acid.

Results: Four libraries were constructed targeting five residues in the active site.

One-Pot Biocatalytic Cascade Reduction of Cyclic Enimines for the Preparation of Diastereomerically Enriched -Heterocycles.

Ene-reductases (EREDs) catalyze the reduction of electron-deficient C═C bonds. Herein, we report the first example of ERED-catalyzed net reduction of C═C bonds of enimines (α,β-unsaturated imines). Preliminary studies suggest their hydrolyzed ring-open ω-amino enones are the likely substrates for this step.

Biocatalytic N-Alkylation of Amines Using Either Primary Alcohols or Carboxylic Acids via Reductive Aminase Cascades.

The alkylation of amines with either alcohols or carboxylic acids represents a mild and safe alternative to the use of genotoxic alkyl halides and sulfonate esters. Here we report two complementary one-pot systems in which the reductive aminase (RedAm) from Aspergillus oryzae is combined with either (i) a 1° alcohol/alcohol oxidase (AO) or (ii) carboxylic acid/carboxylic acid reductase (CAR) to affect N-alkylation reactions. The application of both approaches has been exemplified with respect to substrate scope and also preparative scale synthesis.

Imine Reductases, Reductive Aminases, and Amine Oxidases for the Synthesis of Chiral Amines: Discovery, Characterization, and Synthetic Applications.

Synthesis of the chiral amine moiety is a key challenge for synthetic organic chemistry due to its prevalence in many biologically active molecules. Imine reductase and amine oxidase enzymes have enabled the biocatalytic synthesis of a host of chiral amine compounds. In this chapter, procedures for the synthesis of chiral amines using imine reductases (IREDs), the recently discovered IRED homologues reductive aminases, and amine oxidases (AOs) are described.

NAD(P)H-Dependent Dehydrogenases for the Asymmetric Reductive Amination of Ketones: Structure, Mechanism, Evolution and Application.

Asymmetric reductive aminations are some of the most important reactions in the preparation of active pharmaceuticals, as chiral amines feature in many of the world's most important drugs. Although many enzymes have been applied to the synthesis of chiral amines, the development of reductive amination reactions that use enzymes is attractive, as it would permit the one-step transformation of readily available prochiral ketones into chiral amines of high optical purity. However, as most natural "reductive aminase" activities operate on keto acids, and many are able to use only ammonia as the amine donor, there is considerable scope for the engineering of natural enzymes for the reductive amination of ketones, and also for the preparation of secondary amines using alkylamines as donors.

Discovery of a new metal and NAD-dependent formate dehydrogenase from Clostridium ljungdahlii.

Over the next decades, with the growing concern of rising atmospheric carbon dioxide (CO) levels, the importance of investigating new approaches for its reduction becomes crucial. Reclamation of CO for conversion into biofuels represents an alternative and attractive production method that has been studied in recent years, now with enzymatic methods gaining more attention. Formate dehydrogenases (FDHs) are NAD(P)H-dependent oxidoreductases that catalyze the conversion of formate into CO and have been extensively used for cofactor recycling in chemoenzymatic processes.

A biocatalytic cascade for the amination of unfunctionalised cycloalkanes.

Here we describe a one-pot, three-enzyme, cascade involving a cytochrome P450 monooxygenase, an alcohol dehydrogenase and a reductive aminase for the synthesis of secondary amines from cycloalkanes. Amine product concentrations of up to 19.6 mM were achieved.

Biocatalytic Routes to Enantiomerically Enriched Dibenz[c,e]azepines.

Biocatalytic retrosynthetic analysis of dibenz[c,e]azepines has highlighted the use of imine reductase (IRED) and ω-transaminase (ω-TA) biocatalysts to establish the key stereocentres of these molecules. Several enantiocomplementary IREDs were identified for the synthesis of (R)- and (S)-5-methyl-6,7-dihydro-5H-dibenz[c,e]azepine with excellent enantioselectivity, by reduction of the parent imines. Crystallographic evidence suggests that IREDs may be able to bind one conformer of the imine substrate such that, upon reduction, the major product conformer is generated directly.

A reductive aminase from Aspergillus oryzae.

Reductive amination is one of the most important methods for the synthesis of chiral amines. Here we report the discovery of an NADP(H)-dependent reductive aminase from Aspergillus oryzae (AspRedAm, Uniprot code Q2TW47) that can catalyse the reductive coupling of a broad set of carbonyl compounds with a variety of primary and secondary amines with up to >98% conversion and with up to >98% enantiomeric excess. In cases where both carbonyl and amine show high reactivity, it is possible to employ a 1:1 ratio of the substrates, forming amine products with up to 94% conversion.

Direct Alkylation of Amines with Primary and Secondary Alcohols through Biocatalytic Hydrogen Borrowing.

The reductive aminase from Aspergillus oryzae (AspRedAm) was combined with a single alcohol dehydrogenase (either metagenomic ADH-150, an ADH from Sphingobium yanoikuyae (SyADH), or a variant of the ADH from Thermoanaerobacter ethanolicus (TeSADH W110A)) in a redox-neutral cascade for the biocatalytic alkylation of amines using primary and secondary alcohols. Aliphatic and aromatic secondary amines were obtained in up to 99 % conversion, as well as chiral amines directly from the racemic alcohol precursors in up to >97 % ee, releasing water as the only byproduct.

Structural Basis for Phospholyase Activity of a Class III Transaminase Homologue.

Pyridoxal-phosphate (PLP)-dependent enzymes catalyse a remarkable diversity of chemical reactions in nature. A1RDF1 from Arthrobacter aurescens TC1 is a fold type I, PLP-dependent enzyme in the class III transaminase (TA) subgroup. Despite sharing 28 % sequence identity with its closest structural homologues, including β-alanine:pyruvate and γ-aminobutyrate:α-ketoglutarate TAs, A1RDF1 displayed no TA activity.

Highly efficient enzymatic biodiesel production promoted by particle-induced emulsification.

Background: At present, the conversion of oils to biodiesel is predominantly carried out using chemical catalysts. However, the corresponding lipase-catalysed process has important advantages, which include mild reaction conditions and the possibility of using cheap, low quality feedstocks with a high free fatty acid content. Further increases in the efficiency of the enzymatic process are desired to make it even more attractive and suitable for large-scale applications.

One-pot synthesis of enantiopure 3,4-dihydroisocoumarins through dynamic reductive kinetic resolution processes.

A straightforward chemoenzymatic synthesis of enantiopure 4-alkyl-3-methyl-3,4-dihydroisocoumarins through a ketoreductase-catalyzed one-pot dynamic reductive kinetic resolution is reported. E. coli/ADH-A cells have shown outstanding diastereo- and enantioselectivity toward the bioreduction of a series of racemic ketones, with the use of anion exchange resins or triethylamine being compatible in the same aqueous reaction medium.

Structures of a γ-aminobutyrate (GABA) transaminase from the s-triazine-degrading organism Arthrobacter aurescens TC1 in complex with PLP and with its external aldimine PLP-GABA adduct.

Two complex structures of the γ-aminobutyrate (GABA) transaminase A1R958 from Arthrobacter aurescens TC1 are presented. The first, determined to a resolution of 2.80 Å, features the internal aldimine formed by reaction between the ℇ-amino group of Lys295 and the cofactor pyridoxal phosphate (PLP); the second, determined to a resolution of 2.

Highly stereoselective chemoenzymatic synthesis of the 3H-isobenzofuran skeleton. Access to enantiopure 3-methylphthalides.

A straightforward synthesis of (S)-3-methylphthalides has been developed, with the key asymmetric step being the bioreduction of 2-acetylbenzonitriles. Enzymatic processes have been found to be highly dependent on the pH value, with acidic conditions being required to avoid undesired side reactions. Baker's yeast was found to be the best biocatalyst acting in a highly stereoselective fashion.

Asymmetric chemoenzymatic synthesis of miconazole and econazole enantiomers. The importance of chirality in their biological evaluation.

A simple and novel chemoenzymatic route has been applied for the first time in the synthesis of miconazole and econazole single enantiomers. Lipases and oxidoreductases have been tested in stereoselective processes; the best results were attained with oxidoreductases for the introduction of chirality in an adequate intermediate. The behaviors of a series of ketones and racemic alcohols in bioreductions and acetylation procedures, respectively, have been investigated; the best results were found with alcohol dehydrogenases A and T, which allowed the production of (R)-2-chloro-1-(2,4-dichlorophenyl)ethanol in enantiopure form under very mild reaction conditions.