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A facile microwave assisted three-component protocol allows the synthesis of chiral aryl-1,2-mercaptoamines in water in a few minutes with high yields, bypassing the use of toxic aziridine intermediates. The chiral 1,2-mercaptoamines were then deracemized through enzymatic resolution of the racemates using monoamine oxidase (MAO-N) biocatalysts.

A library of 132 racemic chiral amines (α-substituted methylbenzylamines, benzhydrylamines, 1,2,3,4-tetrahydronaphthylamines (THNs), indanylamines, allylic and homoallylic amines, propargyl amines) was screened against the most versatile monoamine oxidase (MAO-N) variants D5, D9 and D11. MAO-N D9 exhibited the highest activity for most substrates and was applied to the deracemisation of a comprehensive set of selected primary amines. In all cases, excellent enantioselectivity was achieved (e.e. >99%) with moderate to good yields (55-80%). Conditions for the deracemisation of primary amines using a MAO-N/borane system were further optimised using THN as a template addressing substrate load, nature of the enzyme preparation, buffer systems, borane sources, and organic co-solvents., (Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.)

Objective: To clone monoamine oxidase N, that catalyses the selective oxidative deamination or deracemisation of amines into imines, in Pichia pastoris and prove the importance of choosing the proper expression system for its recombinant production., Results: Monoamine oxidase, originating from Aspergillus niger and subjected to directed evolution (MAO-N D5), was cloned and expressed in Pichia pastoris CBS7435 Mut S strain for the first time. Various transformants were screened at microscale level. The production of the clone expressing the most active enzyme was scaled-up to a 1.5 l fermenter and preparation of MAO-N D5 as a crude enzyme extract was optimised. The obstacles in the production of the enzyme in both expression systems, Escherichia coli and P. pastoris, are discussed and demonstrated., Conclusions: There was an improvement in specific productivity, which was 83 times higher in P. pastoris, clearly proving the importance of choosing the right expression host system for the specific enzymes.

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., (© 2018 Elsevier Inc. All rights reserved.)

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.

Biocatalytic approaches to the synthesis of optically pure chiral amines, starting from simple achiral building blocks, are highly desirable because such motifs are present in a wide variety of important natural products and pharmaceutical compounds. Herein, a novel one-pot ω-transaminase (TA)/monoamine oxidase (MAO-N) cascade process for the synthesis of chiral 2,5-disubstituted pyrrolidines is reported. The reactions proceeded with excellent enantio- and diastereoselectivity (>94 % ee; >98 % de) and can be performed on a preparative scale. This methodology exploits the complementary regio- and stereoselectivity displayed by both enzymes, which ensures that the stereogenic center established by the transaminase is not affected by the monoamine oxidase, and highlights the potential of this multienzyme cascade for the efficient synthesis of chiral building blocks., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The development of cost-effective and sustainable catalytic methods for the production of enantiomerically pure chiral amines is a key challenge facing the pharmaceutical and fine chemical industries. This challenge is highlighted by the estimate that 40-45% of drug candidates contain a chiral amine, fueling a demand for broadly applicable synthetic methods that deliver target structures in high yield and enantiomeric excess. Herein we describe the development and application of a "toolbox" of monoamine oxidase variants from Aspergillus niger (MAO-N) which display remarkable substrate scope and tolerance for sterically demanding motifs, including a new variant, which exhibits high activity and enantioselectivity toward substrates containing the aminodiphenylmethane (benzhydrylamine) template. By combining rational structure-guided engineering with high-throughput screening, it has been possible to expand the substrate scope of MAO-N to accommodate amine substrates containing bulky aryl substituents. These engineered MAO-N biocatalysts have been applied in deracemization reactions for the efficient asymmetric synthesis of the generic active pharmaceutical ingredients Solifenacin and Levocetirizine as well as the natural products (R)-coniine, (R)-eleagnine, and (R)-leptaflorine. We also report a novel MAO-N mediated asymmetric oxidative Pictet-Spengler approach to the synthesis of (R)-harmicine.

Monoamine oxidase from Aspergillus niger (MAO-N) is a flavoenzyme that catalyses the oxidative deamination of primary amines. MAO-N has been used as the starting model for a series of directed evolution experiments, resulting in mutants of improved activity and broader substrate specificity, suitable for application in the preparative deracemisation of primary, secondary and tertiary amines when used as part of a chemoenzymatic oxidation-reduction cycle. The structures of a three-point mutant (Asn336Ser/Met348Lys/Ile246Met or MAO-N-D3) and a five-point mutant (Asn336Ser/Met348Lys/Ile246Met/Thr384Asn/Asp385Ser or MAO-N-D5) have been obtained using a multiple-wavelength anomalous diffraction experiment on a selenomethionine derivative of the truncated MAO-N-D5 enzyme. MAO-N exists as a homotetramer with a large channel at its centre and shares some structural features with human MAO B (MAO-B). A hydrophobic cavity extends from the protein surface to the active site, where a non-covalently bound flavin adenine dinucleotide (FAD) sits at the base of an 'aromatic cage,' the sides of which are formed by Trp430 and Phe466. A molecule of l-proline was observed near the FAD, and this ligand superimposed well with isatin, a reversible inhibitor of MAO-B, when the structures of MAO-N proline and MAO-B-isatin were overlaid. Of the mutations that confer the ability to catalyse the oxidation of secondary amines in MAO-N-D3, Asn336Ser reduces steric bulk behind Trp430 of the aromatic cage and Ile246Met confers greater flexibility within the substrate binding site. The two additional mutations, Thr384Asn and Asp385Ser, that occur in the MAO-N-D5 variant, which is able to oxidise tertiary amines, appear to influence the active-site environment remotely through changes in tertiary structure that perturb the side chain of Phe382, again altering the steric and electronic character of the active site near FAD. The possible implications of the change in steric and electronic environment caused by relevant mutations are discussed with respect to the improved catalytic efficiency of the MAO-N variants described in the literature.

Monoamine oxidase from Aspergillus niger (MAO-N) is an FAD-dependent enzyme that catalyses the conversion of terminal amines to their corresponding aldehydes. Variants of MAO-N produced by directed evolution have been shown to possess altered substrate specificity. Crystals of two of these variants (MAO-N-3 and MAO-N-5) have been obtained; the former displays P2(1) symmetry with eight molecules per asymmetric unit and the latter has P4(1)2(1)2 or P4(3)2(1)2 symmetry and two molecules per asymmetric unit. Solution of these structures will help shed light on the molecular determinants of improved activity and high enantioselectivity towards a broad range of substrates.

A template-based mnemonic has been developed for the enzyme monoamine oxidase from Aspergillus niger and has been used to successfully identify the alkaloid (+/-)-crispine A as a target for chemo-enzymatic deracemisation yielding the biologically active (R)-enantiomer in 97% e.e.

Enantioselective oxidation of racemic O-methyl-N-hydroxycyclohexylethylamine, using a variant of monoamine oxidase N (MAO-N) from Aspergillus niger, yields unreacted (R)-enantiomer (e.e. = 99%) together with the oxime exclusively in the (E)-configuration.

Deracemization of racemic chiral tertiary amines has been achieved by combination of an enantioselective amine oxidase, obtained through directed evolution, and ammonia borane in a one-pot process.

Objective: To clone monoamine oxidase N, that catalyses the selective oxidative deamination or deracemisation of amines into imines, in Pichia pastoris and prove the importance of choosing the proper expression system for its recombinant production.Results: Monoamine oxidase, originating from Aspergillus niger and subjected to directed evolution (MAO-N D5), was cloned and expressed in Pichia pastoris CBS7435 MutS strain for the first time. Various transformants were screened at microscale level. The production of the clone expressing the most active enzyme was scaled-up to a 1.5 l fermenter and preparation of MAO-N D5 as a crude enzyme extract was optimised. The obstacles in the production of the enzyme in both expression systems, Escherichia coli and P. pastoris, are discussed and demonstrated.Conclusions: There was an improvement in specific productivity, which was 83 times higher in P. pastoris, clearly proving the importance of choosing the right expression host system for the specific enzymes. [ABSTRACT FROM AUTHOR]

We disclose the highly diastereoselective combination of monoamine oxidase-catalyzed oxidation of meso-pyrrolidines and aza-Friedel-Crafts reactions in aqueous buffer to give valuable enantioenriched 2-substituted pyrrolidines in a formal double CH activation process. A range of secondary as well as tertiary amines were shown to be suitable substrates for the biocatalytic oxidation and subsequent addition of a variety of C-nucleophiles. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Herein we report a one-pot protocol for the deracemisation of chiral benzylic amines employing a novel monoamine oxidase-ω-transaminase cascade, allowing access to enantiopure compounds in >99 % ee. We also demonstrate that the same enzymatic cascade can be employed for the dealkylation of secondary amines with >99 % conversion. [ABSTRACT FROM AUTHOR]

Biocatalytic approaches to the synthesis of optically pure chiral amines, starting from simple achiral building blocks, are highly desirable because such motifs are present in a wide variety of important natural products and pharmaceutical compounds. Herein, a novel one-pot ω-transaminase (TA)/monoamine oxidase (MAO-N) cascade process for the synthesis of chiral 2,5-disubstituted pyrrolidines is reported. The reactions proceeded with excellent enantio- and diastereoselectivity (>94 % ee; >98 % de) and can be performed on a preparative scale. This methodology exploits the complementary regio- and stereoselectivity displayed by both enzymes, which ensures that the stereogenic center established by the transaminase is not affected by the monoamine oxidase, and highlights the potential of this multienzyme cascade for the efficient synthesis of chiral building blocks. [ABSTRACT FROM AUTHOR]

The article discusses deracemisation process of racemic chiral amines by using variants of the enzyme monoamine oxidase N from Aspergillus niger. It mentions that the deracemisation process involves a combination of an enantioselective enzymatic oxidation of the amine to form imine with a non-selective chemical reduction of the imine or iminium ion back to the racemic starting material. The reaction in this process was monitored by chiral high performance liquid chromatography.

Mirror mirror on the wallAfter several years of “reflection” on the use of S‐selective monoamine oxidase ((S)‐MAO‐N) for the deracemization of amines, an R‐selective biocatalyst has now been developed. In their Full Paper on p. 996 ff., R. S. Heath et al. describe how 6‐hydroxy‐D‐nicotine oxidase ((R)‐6‐HDNO) provides a method for preparing the mirror image S enantiomers and thereby constitutes a perfect enantiocomplementary enzyme to (S)‐MAO‐N. [ABSTRACT FROM AUTHOR]

This study focused on the production and immobilisation of the crude enzyme extract of recombinant monoamine oxidase (EC 1.4.3.4), originating from Aspergillus niger (MAO-N-D5) and expressed in Escherichia coli , in PVA gel using the LentiKats ® technique. MAO-Ns are important enzymes in the chemical industry due to their stereoselectivity and they are often used for the deracemisation of non-optically pure mixtures of amines. Biomass production, enzyme preparation, enzyme immobilisation, process parameters for the immobilised enzyme and characterisation of the enzyme are described in detail here. The biomass was prepared in laboratory bioreactors, and two different disruption techniques were compared. The activity of the enzyme was determined by biotransformation with secondary amine 3-azabicyclo [3,3,0] octane as a substrate. The crude enzyme extract showed 61.5% of the whole cell activity and the immobilised enzyme showed a wider optimum pH and temperature ranges than the free enzyme. The initial specific activity of the immobilised monoamine oxidase crude enzyme extract remained at 80% after 12 repeated biotransformations. For the first time, the full kinetic parameters of an immobilised MAO-N-D5 were obtained based on a ping-pong bi–bi reaction mechanism. The specific activity was 0.29 U g −1 (Lentikats) and the K m was 7.31 mM, which were similar in comparison to whole cell MAO-N-D5. Characterisation of immobilised MAO-N-D5 showed particular benefits in terms of activity and stability in comparison with free and whole cell MAO-N-D5; therefore, the immobilisation of this enzyme is very suitable for industrial applications. [ABSTRACT FROM AUTHOR]