Development of Imine Reductases and Reductive Aminases for Chiral Amine Synthesis

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Abstract

Novel biocatalysts for the enantioselective reduction of imines and reductive amination of a broad range of carbonyl compounds have been developed. Unlike other imine reductases (IREDs), the IRED from Amycolaptosis orientalis (AoIRED) features an aprotic “catalytic” residue Asn171 and as such became an interesting candidate for detailed mechanistic, specificity and stereoselectivity studies. AoIRED has been shown to be an efficient catalyst for the enantioselective reduction of imines and iminium ions to yield the corresponding chiral amines in high conversions and good to excellent enantioselectivity. The enzyme exhibits unusual stereoselective properties, displaying a selectivity switch for structurally similar substrates and in certain cases for the same substrate depending on the age of the enzyme. Mutagenesis studies have highlighted important residues that may play key roles in the substrate specificity and stereoselectivity of the enzyme.The reductive aminase from Aspergillus oryzae (AspRedAm) is a multifunctional catalyst that efficiently catalyses i) the reductive coupling of carbonyl compounds and amine nucleophiles, ii) the enantioselective reduction of prochiral cyclic and preformed imines or iii) the oxidative deamination of amines towards kinetic resolution of racemic amines. Detailed kinetic studies have led to the construction of a kinetic model/mechanism and based on structure guided investigation of conserved active site residues, a putative catalytic mechanism has been proposed. It has also been possible to engineer wild-type AspRedAm for improved stereoselectivity as well as to invert the enzyme's enantioselectivity towards a range of substrates. Using AspRedAm as a catalyst, efficient systems have been developed that allow the kinetic resolution of several racemic amines.This thesis has been organised into separate chapters each addressing a specific theme. Chapter 1 gives an overview of recent advances in the field of amine biocatalysis with emphasis on biocatalytic imine reduction and reductive amination; it also outlines the objectives of this project. Chapter 2 describes methods and materials used in these studies while Chapters 3-7 present and discuss results from different projects that constitute the work in this thesis. Initial discovery and characterisation studies of IREDs are described in Chapter 3. Chapter 4 describes detailed characterisation of AoIRED with particular emphasis on stereoselectivity and synthetic applicability while Chapter 5 presents and discusses results from the study of the reductive aminase (AspRedAm) from Aspergillus oryzae. Chapters 6 and 7 respectively describe the engineering of AoIRED and AspRedAm, and the application of AspRedAm in kinetic resolution of racemic amines. The results from these chapters have been summarised and discussed in Chapter 8 and recommendations for future directions in this field have been offered.

Keyword(s)

Imine reductases, reductive aminases, biocatalysis, biotransformation; protein engineering, catalysis, kinetic resolution

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