Shahed Hussain

Asymmetric Reduction of Cyclic Imines Catalyzed by a Whole‐Cell Biocatalyst Containing an (S)‐Imine Reductase

In view of the importance of chiral amines as building blocks for biologically active pharmaceutical drugs, considerable effort has been devoted to the development of asymmetric catalytic methods for their preparation. Notable advances have been made by using transition-metal catalysis, organocatalysis, and artificial transfer hydrogenases. However, biocatalytic approaches have emerged as being especially important, including those based upon transaminases, monoamine oxidases, and recently engineered NADH-dependent l-amino acid dehydrogenases (NADH is the reduced form of NAD = nicotinamide adenine dinucleotide). These methods are complementary in terms of the required substrates (amine, ketone) and also the amine that they generate (primary, secondary, or tertiary). In some cases, these biocatalytic processes have been successfully demonstrated at an industrial scale for the manufacture of recently launched drugs. However, there are no general biocatalytic strategies available that are based upon asymmetric reduction of imines as a route to enantiomerically pure amines. Imine reductases (IREDs) catalyze the reduction of imines to amines by utilizing NADH or NADPH as a cofactor (Scheme 1, NADPH is the reduced form of NADP = nicotinamide adenine dinucleotide phosphate).

An (R)‐Imine Reductase Biocatalyst for the Asymmetric Reduction of Cyclic Imines

Although the range of biocatalysts available for the synthesis of enantiomerically pure chiral amines continues to expand, few existing methods provide access to secondary amines. To address this shortcoming, we have over‐expressed the gene for an (R)‐imine reductase [(R)‐IRED] from Streptomyces sp. GF3587 in Escherichia coli to create a recombinant whole‐cell biocatalyst for the asymmetric reduction of prochiral imines. The (R)‐IRED was screened against a panel of cyclic imines and two iminium ions and was shown to possess high catalytic activity and enantioselectivity. Preparative‐scale synthesis of the alkaloid (R)‐coniine (90 % yield; 99 % ee) from the imine precursor was performed on a gram‐scale. A homology model of the enzyme active site, based on the structure of a closely related (R)‐IRED from Streptomyces kanamyceticus, was constructed and used to identify potential amino acids as targets for mutagenesis.

A reductive aminase from Aspergillus oryzae

Reductive amination is one of the most important methods for the synthesis of chiral amines. Here we report the discovery of an NADP(H)-dependent reductive aminase from Aspergillus oryzae (AspRedAm, Uniprot code Q2TW47) that can catalyse the reductive coupling of a broad set of carbonyl compounds with a variety of primary and secondary amines with up to >98% conversion and with up to >98% enantiomeric excess. In cases where both carbonyl and amine show high reactivity, it is possible to employ a 1:1 ratio of the substrates, forming amine products with up to 94% conversion. Steady-state kinetic studies establish that the enzyme is capable of catalysing imine formation as well as reduction. Crystal structures of AspRedAm in complex with NADP(H) and also with both NADP(H) and the pharmaceutical ingredient (R)-rasagiline are reported. We also demonstrate preparative scale reductive aminations with wild-type and Q240A variant biocatalysts displaying total turnover numbers of up to 32,000 and space time yields up to 3.73 g l−1 d−1. An enzyme (AspRedAm) capable of coupling carbonyls with a variety of amines in a reductive amination has now been discovered. Kinetic studies revealed that the enzyme catalysed both the imine formation step, as well as the reduction step. Structure and mutagenesis studies have highlighted essential catalytic residues and preparative scale examples have demonstrated total turnover numbers of up to 32,000.

Structure, Activity and Stereoselectivity of NADPH-Dependent Oxidoreductases Catalysing the S-Selective Reduction of the Imine Substrate 2-Methylpyrroline.

Oxidoreductases from Streptomyces sp. GF3546 [3546‐IRED], Bacillus cereus BAG3X2 (BcIRED) and Nocardiopsis halophila (NhIRED) each reduce prochiral 2‐methylpyrroline (2MPN) to (S)‐2‐methylpyrrolidine with >95 % ee and also a number of other imine substrates with good selectivity. Structures of BcIRED and NhIRED have helped to identify conserved active site residues within this subgroup of imine reductases that have S selectivity towards 2MPN, including a tyrosine residue that has a possible role in catalysis and superimposes with an aspartate in related enzymes that display R selectivity towards the same substrate. Mutation of this tyrosine residue—Tyr169—in 3546‐IRED to Phe resulted in a mutant of negligible activity. The data together provide structural evidence for the location and significance of the Tyr residue in this group of imine reductases, and permit a comparison of the active sites of enzymes that reduce 2MPN with either R or S selectivity.

Substrate promiscuity of cytochrome P450 RhF

Cytochrome P450 RhF displays a high degree of substrate promiscuity, mediating a range of O-dealkylations, aromatic hydroxylations, epoxidations and asymmetric sulfoxidations. The self-sufficient nature of this CYP coupled with its ability to catalyse the oxidation of a wide range of functional groups highlights this enzyme as an excellent starting template for directed evolution and promising alternate to P450 BM3.

Combined Imine Reductase and Amine Oxidase Catalyzed Deracemization of Nitrogen Heterocycles

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.