Photometric Characterization of the Reductive Amination Scope of the Imine Reductases from Streptomyces tsukubaensis and Streptomyces ipomoeae

Abstract

Imine reductases (IREDs) have emerged as promising enzymes for the asymmetric synthesis of secondary and tertiary amines starting from carbonyl substrates. Screening the substrate specificity of the reductive amination reaction is usually performed by time-consuming GC analytics. We found two highly active IREDs in our enzyme collection, IR-20 from Streptomyces tsukubaensis and IR-Sip from Streptomyces ipomoeae, that allowed a comprehensive substrate screening with a photometric NADPH assay. We screened 39 carbonyl substrates combined with 17 amines as nucleophiles. Activity data from 663 combinations provided a clear picture about substrate specificity and capabilities in the reductive amination of these enzymes. Besides aliphatic aldehydes, the IREDs accepted various cyclic (C -C ) and acyclic ketones, preferentially with methylamine. IR-Sip also accepted a range of primary and secondary amines as nucleophiles. In biocatalytic reactions, IR-Sip converted (R)-3-methylcyclohexanone with dimethylamine or pyrrolidine with high diastereoselectivity (>94-96 % de). The nucleophile acceptor spectrum depended on the carbonyl substrate employed. The conversion of well-accepted substrates could also be detected if crude lysates were employed as the enzyme source.

Synthesis of β‐Chiral Amines by Dynamic Kinetic Resolution of α‐Branched Aldehydes Applying Imine Reductases

“…Enzymatic approaches represent a promising alternative: Amine transaminases give the primary β‐chiral amine in a DKR directly, as demonstrated in the synthesis of niraparib or pregabalin . Additionally, IREDs and reductive aminases have been explored for the one‐step asymmetric synthesis of secondary or tertiary amines from ketones . Until now, these reductive aminations have been investigated only to prepare α‐chiral amines.…”

“…In previous works we and others demonstrated that simple achiral aldehydes are converted by IREDs and reductive aminases (RedAms; this subgroup of IREDs catalyze imine formation directly in the active site rather than using the pre‐formed imine out of the reaction solution and are hence especially efficient in reductive amination because they require only stoichiometric amounts or a small excess of the amine nucleophile substrates). Employing the IRED from Streptomyces ipomoeae , aliphatic aldehydes are converted with similar efficiency or outperform the reaction with the model substrate cyclohexanone with some amine nucleophiles such as pyrrolidine.…”

“…[9] Additionally, IREDs and reductive aminases have been explored for the one-step asymmetric synthesis of secondary [10] or tertiary amines from ketones. [11] Until now, these reductive aminations have been investigated only to prepare α-chiral amines. Interestingly, there are a few examples from natural biosynthesis pathways demonstrating that IREDs also mediate imine reductions with β-selectivity: plants produce the cyclic β-chiral amine metabolites festuclavine, [12] tetrahydroalstonine, [13] and vitrosamine, [14] which are obtained by reducing an intramolecularly formed imine.Because a variety of pharmaceuticals represent acyclic βchiral amines (Scheme 1), we wanted to explore the potential of IREDs for intermolecular reductive amination by dynamic kinetic resolution of α-branched aldehydes (Scheme 2).…”

“…A highly enantioselective enzyme is required for a successful dynamic kinetic resolution. In addition, the reaction needs to be carried out under conditions that allow the remaining enantiomer to racemize, e. g. via keto-enol tautomerism of the non-consumed aldehyde enantiomer.In previous works we [11] and others [10a,15] demonstrated that simple achiral aldehydes are converted by IREDs and reductive aminases [10a] (RedAms; this subgroup of IREDs catalyze imine formation directly in the active site rather than using the pre-[a] P. Matzel, S. Wenske, Dr. M. Höhne…”

Stereoselective Biotransformations of Cyclic Imines in Recombinant Cells of Synechocystis sp. PCC 6803

“…Since then, IREDs have rapidly assumed an important role as highly stereoselective catalysts for amine synthesis. [18][19][20][21][22][23][24] IREDs strictly require NADPH and are preferentially used in cellular systems, which makes them very suitable for application in photosynthesis-driven whole-cell biotransformations.…”