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The development of efficient and sustainable methods for the synthesis of nitrogen heterocycles is an important goal for the chemical industry. In particular, substituted chiral piperidines are prominent targets due to their prevalence in medicinally relevant compounds and their precursors. A potential biocatalytic approach to the synthesis of this privileged scaffold would be the asymmetric dearomatization of readily assembled activated pyridines. However, nature is yet to yield a suitable biocatalyst specifically for this reaction. Here, by combining chemical synthesis and biocatalysis, we present a general chemo-enzymatic approach for the asymmetric dearomatization of activated pyridines for the preparation of substituted piperidines with precise stereochemistry. The key step involves a stereoselective one-pot amine oxidase/ene imine reductase cascade to convert N-substituted tetrahydropyridines to stereo-defined 3- and 3,4-substituted piperidines. This chemo-enzymatic approach has proved useful for key transformations in the syntheses of antipsychotic drugs Preclamol and OSU-6162, as well as for the preparation of two important intermediates in synthetic routes of the ovarian cancer monotherapeutic Niraparib.

The unique ability of the 'electrochemical leaf' (e-Leaf) to drive and control nanoconfined enzyme cascades bidirectionally, while directly monitoring their rate in real-time as electrical current, is exploited to achieve deracemisation and stereoinversion of secondary alcohols using a single electrode in one pot. Two alcohol dehydrogenase enzymes with opposing enantioselectivities, from

The importance of energized nanoconfinement for facilitating the study and execution of enzyme cascades that feature multiple exchangeable cofactors is demonstrated by experiments with carboxylic acid reductase (CAR), an enzyme that requires both NADPH and ATP during a single catalytic cycle. Conversion of cinnamic acid to cinnamaldehyde by a package of four enzymes loaded into and trapped in the random nanopores of an indium tin oxide (ITO) electrode is driven and monitored through the simultaneous delivery of electrical and chemical energy. The electrical energy is transduced by ferredoxin NADP+ reductase, which undergoes rapid, direct electron exchange with ITO and regenerates NADP(H). The chemical energy provided by phosphoenolpyruvate, a fuel contained in the bulk solution, is cotransduced by adenylate kinase and pyruvate kinase, which efficiently convert the AMP product back into ATP that is required for the next cycle. The use of the two-kinase system allows the recycling process to be dissected to evaluate the separate roles of AMP removal and ATP supply during presteady-state and steady-state catalysis.

The enantioselective oxidation of 2° alcohols to ketones is an important reaction in synthetic chemistry, especially if it can be achieved using O

Covering: 2015 up to July 2021The market for cosmetics is consumer driven and the desire for green, sustainable and natural ingredients is increasing. The use of isolated enzymes and whole-cell organisms to synthesise these products is congruent with these values, especially when combined with the use of renewable, recyclable or waste feedstocks. The literature of biocatalysis for the synthesis of ingredients in cosmetics in the past five years is herein reviewed.

The development of efficient and sustainable methods for the synthesis of nitrogen heterocycles is an important goal for the chemical industry. In particular, substituted chiral piperidines are prominent targets due to their prevalence in medicinally relevant compounds and their precursors. A potential biocatalytic approach to the synthesis of this privileged scaffold would be the asymmetric dearomatization of readily assembled activated pyridines. However, nature has yet to yield a suitable biocatalyst specifically for this reaction. Here, by combining chemical synthesis and biocatalysis, we present a general chemo-enzymatic approach for the asymmetric dearomatization of activated pyridines for the preparation of substituted piperidines with precise stereochemistry. The key step involves a stereoselective one-pot amine oxidase/ene imine reductase cascade to convert N-substituted tetrahydropyridines to stereo-defined 3- and 3,4-substituted piperidines. This chemo-enzymatic approach has proved useful for key transformations in the syntheses of the antipsychotic drugs Preclamol and OSU-6162, as well as for the preparation of two important intermediates in synthetic routes of the ovarian cancer monotherapeutic Niraparib.

Chiral amine diastereomers are ubiquitous in pharmaceuticals and agrochemicals

The ability to drive and observe rapid enzyme catalysis in both directions is an important and natural consequence of immobilizing cascade components within electrode nanopores and coupling reactions to the fast, quasi-reversible NADP+/NADPH electrochemistry mediated by bound ferredoxin-NADP+ reductase. This approach has been exploited to investigate and control the redox interconversions between a ketone and secondary alcohol enantiomers catalyzed by enantioselective alcohol dehydrogenase variants. An anticipated advantage of the bidirectionality, in allowing a single cascade to be cycled back and forth while exploiting kinetic selectivity, was the ability to achieve a simple one-electrode de-racemizer; significantly, this was overturned because the nanoconfined enzyme system strongly directs racemization, the thermodynamic outcome. By modifying the concept, it was easily demonstrated that efficient one-pot de-racemizers can be achieved by using two electrodes in sequence, each containing an alcohol dehydrogenase that is enantioselective for one of the half-cycles.

In close quarters: Nicotinamide adenine dinucleotide phosphate (NADPH) and NADP+ are cycled rapidly between ferredoxin–NADP+ reductase (FNR) and a dehydrogenase enzyme (E2) within the 5–100 nm pores of an indium tin oxide electrode. The electrode mimics the nanoconfinement strategies of natural systems, such as the chloroplast stroma or mitochondria in living cells.In living cells, redox chains rely on nanoconfinement using tiny enclosures, such as the mitochondrial matrix or chloroplast stroma, to concentrate enzymes and limit distances that nicotinamide cofactors and other metabolites must diffuse. In a chemical analogue exploiting this principle, nicotinamide adenine dinucleotide phosphate (NADPH) and NADP+ are cycled rapidly between ferredoxin–NADP+ reductase and a second enzyme—the pairs being juxtaposed within the 5–100 nm scale pores of an indium tin oxide electrode. The resulting electrode material, denoted (FNR+E2)@ITO/support, can drive and exploit a potentially large number of enzyme-catalysed reactions.

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. These new biocatalytic methods address issues facing alternative traditional synthetic protocols such as harsh conditions, overalkylation and complicated workup procedures.

The conversion of carboxylic acids, such as acrylic acids, to amines is a transformation that remains challenging in synthetic organic chemistry. Despite the ubiquity of similar moieties in natural metabolic pathways, biocatalytic routes seem to have been overlooked for this purpose. Herein we present the conception and optimisation of a two‐enzyme system, allowing the synthesis of β‐phenylethylamine derivatives from readily‐available ring‐substituted cinnamic acids. After characterisation of both parts of the reaction in a two‐step approach, a set of conditions allowing the one‐pot biotransformation was optimised. This combination of a reversible deaminating and irreversible decarboxylating enzyme, both specific for the amino acid intermediate in tandem, represents a general method by which new strategies for the conversion of carboxylic acids to amines could be designed.

Sequence function relationships help to understand how imine reductase enzymes perform reductive amination in water., Imine reductases (IREDs) have shown great potential as catalysts for the asymmetric synthesis of industrially relevant chiral amines, but a limited understanding of sequence activity relationships makes rational engineering challenging. Here, we describe the characterization of 80 putative and 15 previously described IREDs across 10 different transformations and confirm that reductive amination catalysis is not limited to any particular subgroup or sequence motif. Furthermore, we have identified another dehydrogenase subgroup with chemoselectivity for imine reduction. Enantioselectivities were determined for the reduction of the model substrate 2-phenylpiperideine, and the effect of changing the reaction conditions was also studied for the reductive aminations of 1-indanone, acetophenone, and 4-methoxyphenylacetone. We have performed sequence-structure analysis to help explain clusters in activity across a phylogenetic tree and to inform rational engineering, which, in one case, has conferred a change in chemoselectivity that had not been previously observed.

The NADP(H)-dependent reductive aminase from Aspergillus oryzae (AspRedAm) was combined with an NADPH oxidase (NOX) to develop a redox system that recycles the co-factor. The AspRedAm-NOX system was applied initially for the kinetic resolution of a variety of racemic secondary and primary amines to yield S-configured amines with enantiomeric excess (ee) values up to 99 %. The addition of ammonia borane to this system enabled the efficient deracemization of racemic amines, including the pharmaceutical drug rasagiline and the natural product salsolidine, with conversions up to >98 % and >99 % ee Furthermore, by using the AspRedAm W210A variant it was possible to generate the opposite R enantiomers with efficiency comparable to, or even better than, the wildtype AspRedAm.

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. We also report the development of a facile high-throughput screen to interrogate their activity. Through this approach we identified imine reductase biocatalysts capable of accepting structurally demanding ketones and amines, which include the preparative synthesis of N-substituted β-amino ester derivatives via a dynamic kinetic resolution process, with excellent yields and stereochemical purities. [Figure not available: see fulltext.]

Enantioselective conversion of alcohols to ketones using air as the oxidant is achieved with high rates and efficiency using an indium tin oxide (ITO) electrode in which an alcohol dehydrogenase and a photosynthetic NADPH recycling enzyme are confined within nanopores. The massive catalytic enhancement arising from nanoconfinement is exploited in an air-driven electrochemical cell, which requires no complicating control features yet allows continuous monitoring of the reaction via the current that flows between anode (ITO: organic chemistry) and cathode (Pt: O2 from air).

Structure-guided directed evolution of choline oxidase has been carried out by using the oxidation of hexan-1-ol to hexanal as the target reaction. A six-amino-acid variant was identified with a 20-fold increased kcat compared to that of the wild-type enzyme. This variant enabled the oxidation of 10 mm hexanol to hexanal in less than 24 h with 100 % conversion. Furthermore, this variant showed a marked increase in thermostability with a corresponding increase in Tm of 20 °C. Improved solvent tolerance was demonstrated with organic solvents including ethyl acetate, heptane and cyclohexane, thereby enabling improved conversions to the aldehyde by up to 30 % above conversion for the solvent-free system. Despite the evolution of choline oxidase towards hexan-1-ol, this new variant also showed increased specific activities (by up to 100-fold) for around 50 primary aliphatic, unsaturated, branched, cyclic, benzylic and halogenated alcohols.

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. Amine oxidases have been the subject of mutagenesis approaches for improvement of substrate scope. The high-throughput screening method for determining active variants in amine oxidase libraries is illustrated. Finally, in an approach which takes inspiration from nature, many enzymes can be combined with each other in cascade reactions. The incorporation of imine reductase and monoamine oxidase biocatalysts into several cascade reactions, both in vitro and in vivo (where the approach moves toward synthetic biology), is reported.

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. Amine oxidases have been the subject of mutagenesis approaches for improvement of substrate scope. The high-throughput screening method for determining active variants in amine oxidase libraries is illustrated. Finally, in an approach which takes inspiration from nature, many enzymes can be combined with each other in cascade reactions. The incorporation of imine reductase and monoamine oxidase biocatalysts into several cascade reactions, both in vitro and in vivo (where the approach moves toward synthetic biology), is reported.

A novel amine oxidase (AO)/imine reductase (IRED) system was developed for the deracemization of racemic amines. By combining (R)-6-hydroxy-d-nicotine oxidase (6-HDNO) with an (R)-IRED, a panel of racemic 2-substituted piperidines and pyrrolidines were deracemized to yield the (S)-amines in high yields and enantiomeric excess values. Other N-heterocycles were deracemized with monoamine oxidase (MAO-N) or 6-HDNO in combination with ammonia borane, which allowed comparison of the two enzyme deracemization approaches with that involving a chemical reducing agent.

Amine oxidases are useful bio-catalysts for the synthesis of enantiomerically pure 1°, 2° and 3° chiral amines. Enzymes in this class (e.g., MAO-N from Aspergillus niger) reported previously have been shown to be highly S selective. Herein we report the development of an enantiocomplementary R-selective amine oxidase based on 6-hydroxy-D-nicotine oxidase (6-HDNO) with broadened substrate scope and high enantioselectivity. The engineered 6-HDNO enzyme has been applied to the preparative deracemisation of a range of racemic amines to yield S-configured products, for example, (S)-nicotine, in high ee.

Three complementary biocatalytic routes were examined for the synthesis of the cyclopropyl amine (1R,2S)-2, which is a key building block for the anti-thrombotic agent ticagrelor 1. By employing either a ketoreductase, amidase or lipase biocatalyst, the key building blocks for synthesis of the amine 2 were obtained in 99.9, 92.5 and 46.3 ee, respectively.

This discussion describes efforts to produce a stable, efficient electrocatalyst for four-electron O2 reduction through the direct attachment of fungal laccase, a 'blue' copper oxidase, to functionalised carbon electrode materials. Commercially available carbons, including fibrous and porous materials, offer important opportunities for achieving high conductivity over high surface areas that can be chemically functionalised. A promising approach for attaching laccase to a carbon surface is to use the diazonium coupling reaction to generate protrusive aromatic functionalities that can bind to hydrophobic residues close to the 'blue' Cu site: this site provides a fast, intramolecular electron relay into the buried trinuclear Cu active site that converts O2 rapidly and cleanly to H2O. This enhancement procedure makes possible the stable, direct electrocatalytic reduction of O2 at high potential with high efficiency in terms of turnover frequency per enzyme active site engaged with the electrode. The absence of electron-transfer mediators and simplicity of electrode system reveals the more inherent characteristics of the electrocatalytic mechanism that are masked in the waveform when a mediator is used. The study includes experiments to assess the effects of methanol and chloride ions on laccase electrocatalysis, complementing studies carried out by other groups, particularly those in which laccase is embedded in an electron-mediating gel.

This work demonstrates the first example of the immobilisation of MAO-N whole cells to produce a biocatalyst that remained suitable for repetitive use after 11 months of storage and stable up to 15 months after immobilisation. The production of Escherichia coli expressing recombinant MAO-N was scaled up to bioreactors under regulated, previously optimised conditions (10 % DO, pH 7), and the amount of biomass was almost doubled compared to flask cultivation. Subsequently, pilot immobilisation of the whole-cell biocatalyst using LentiKats® technology was performed. The amount of the immobilised biomass was optimised and the process was scaled up to a production level by immobilising 15 g of dry cell weight per litre of polyvinyl alcohol to produce 3 kg of whole-cell ready-to-use biocatalyst. The immobilised biocatalyst retained its initial activity over six consecutive biotransformations of the secondary amine model compound 3-azabicylo [3,3,0]octane, a building block of the hepatitis C drug telaprevir. Consecutive cultivation cycles in growth conditions not only increased the initial specific activity of biocatalyst produced on the industrial plant by more than 30 %, but also significantly increased the rate of the biotransformation compared to the non-propagated biocatalyst.

Attachment of substrate-like anthracene based units to the surface of pyrolytic graphite greatly enhances the adsorption of high-potential fungal laccases, 'blue' Cu enzymes that catalyse the four-electron reduction of O(2), providing a stable cathode for enzymatic biological fuel cells and electrochemical studies.