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Directed Evolution of an Amine Oxidase for the Preparative Deracemisation of Cyclic Secondary Amines

Enantiomerically pure primary and secondary amines are widely used as chiral auxiliaries and resolving agents and are also valuable intermediates for the synthesis of pharmaceuticals and agrochemicals. Although enantiomerically pure amines are traditionally prepared by classical resolution of the corresponding racemate, alternative approaches have been developed based upon i) asymmetric reduction of imines, ii) hydroamination of alkenes and iii) lipase-catalysed kinetic resolution of racemic amines. However, secondary amines, many of which have pronounced biological activity, are poor substrates for lipases compared to the corresponding primary amines, with only a few documented examples in the literature. Hence the use of lipase resolution does not offer a general route to this class of chiral molecule. Moreover, to date it has generally not been possible to achieve the in situ racemisation of amines to effect a dynamic kinetic resolution process due to the relatively harsh conditions required to racemise amines. Against this backdrop, we sought to extend our chemo-enzymatic deracemisation method to encompass chiral secondary amines. Based upon our earlier work with a-amino acids, we recently reported the deracemisation of a-methylbenzyl amine (a-MBA, 1) in a one-pot procedure by the combined use of an enantioselective amine oxidase and ammonia borane as the reducing agent (Scheme 1). In order to identify an enzyme with appropriate activity and enantioselectivity towards a-methylbenzylamine, the amine oxidase from Aspergillus niger (MAO-N) was subjected to directed evolution, with (S)-1 as the probe substrate, by random mutagenesis and selection employing a high-throughput agarplate-based colorimetric screen. This approach led to the identification of an important amino acid substitution (Asn336Ser) that resulted in a variant enzyme possessing significantly enhanced activity (ca. 50-fold) and greater enantioselectivity towards 1 than the wild-type enzyme. Subsequently, we showed that this variant was also characterised by broad substrate specificity, being able to oxidize a wide range of chiral primary amines with high enantioselectivity. However, although this variant showed some activity towards chiral secondary amines (relative activity of 1-methyltetrahydroisoquinoline (MTQ, 2) 15 % of a-MBA), the rates of oxidation were too low to permit efficient preparative deracemisation reactions. Our goal therefore was to evolve a “secondary amine oxidase” for preparative-scale deracemisation reactions to complement the existing “primary amine oxidase”. The MAO-N gene used as the starting point for further directed evolution contained four amino acid substitutions compared to the wild-type. In addition to the Asn336Ser mutation, which is important for catalytic activity/enantioselectivity, mutations were Arg259Lys and Arg260Lys (improved expression) and Met348Lys (improved activity). This gene was subjected to random mutagenesis, by using the E. coli XL1-Red mutator strain (mutation frequency ca. 1–2 base changes per gene), followed by transformation and screening of the library (ca. 20 000 clones) against (R/S)-2 as the substrate, as previously described. A number of clones (ca. 10) showed greater activity than the parent, with one in particular appearing to be significantly more active. Purification of this variant amine oxidase showed that it possessed a kcat value about 5.5-fold higher than the parent towards (S)-2 (Table 1) and also a higher KM