Enzymatic Asymmetric Synthesis of Enantiomerically Pure Aliphatic, Aromatic and Arylaliphatic Amines with (R)‐Selective Amine Transaminases

Abstract

Seven (R)-selective amine transaminases (R-ATAs) recently discovered by an in silico-based approach in sequence databases were produced recombinantly in Escherichia coli and subjected to partial purification by ammonium sulfate precipitation. A range of additives and various buffers were investigated to identify best conditions to ensure good storage stability and stable activity during biocatalysis. All enzymes show pH optima between pH 7.5-9. These R-ATAs were then applied in the asymmetric synthesis of twelve aliphatic, aromatic and arylaliphatic (R)-amines starting from the corresponding prochiral ketones using a lactate dehydrogenase/glucose dehydrogenase system to shift the equilibrium. For all ketones, at least one enzyme was found that allows complete conversion to the corresponding chiral amine having excellent optical purities > 99% ee. Variations in substrate profiles are also discussed based on the phylogenetic relationships between the seven R-ATAs. Thus, we have identified a versatile toolbox of (R)-amine transaminases showing remarkable properties for application in biocatalysis.

Features and technical applications of ω-transaminases

“…Using this approach, 17 new 5-TAs showing excellent (R)-stereoselectivity were discovered. Recently, the same group demonstrated asymmetric synthesis of 12 (R)-amines using the new (R)-selective 5-TAs (Schätzle et al 2011). Crucial factors to 5-TA process design Biocatalytic process development often suffers from complicated enzyme properties that do not match industrial requirements.…”

“…The first example of the asymmetric synthesis was studied by coupling a lactate dehydrogenase reaction to the 5-TA reaction to convert pyruvate to lactate (Shin and Kim 1999a). Because dehydrogenase requires an external cofactor NAD(P)H, coupling the cofactor recycling system using an additional dehydrogenase with the 5-TA reaction was later demonstrated (Fuchs et al 2010;Mutti et al 2011;Koszelewski et al 2008b;Schätzle et al 2011). As an alternative to the reduction of pyruvate into lactate, decarboxylation of pyruvate by pyruvate decarboxylase was studied (Hohne et al 2008).…”

Kinetic resolution of amines by (R)-selective omega-transaminase from Mycobacterium vanbaalenii

“…(R)-Enantiomer can be obtained from racemic mixture through a kinetic resolution with (S)-selective v-TA. (S)-Enantiomer can also be produced from the corresponding prochiral ketone through the asymmetric synthesis with (S)-selective v-TA [1]. Although asymmetric synthesis approach is more favored as it can theoretically generate 100% yield, in contrast to the 50% yield produced by the resolution approach, it often suffers from poor conversion due to unfavorable thermodynamic equilibrium and severe product inhibition [4,14].…”

“…Recently, various (R)-selective vTAs were identified with the aid of computational method [22]. Based on the current advance, the asymmetric synthesis of (R)-amine from corresponding ketone by using (R)-selective v-TAs were extensively studied [1,7,8,10]. In previous study, we successfully developed a one-pot one-step deracemization method with (S)-selective v-TA from Polaromonas sp.…”

“…Optically active amines play an important role in agrochemical, chemical and pharmaceutical industry [1]. Therefore, the development of a procedure for the production of chiral amines in an efficient and cost effective way is imperative.…”