Biocatalytic Asymmetric Reduction Animation Using Reductive Aminases (RedAms)

Lead Research Organisation: University of Manchester
Department Name: Chemistry

Abstract

This project aligns with the pharmaceutical and fine chemical sector. The challenge is to develop robust biocatalysts for the enantioselective reduction amination of ketones and aldehydes with amines, thereby enabling the direct one-step synthesis of biologically important target molecules. The aim of this project is to develop reductive aminase (RedAm) biocatalysts for applications in organic synthesis. Specifically we shall engineer existing wild-type RedAms such that they possess expanded substrate scope and improved stability and activity. This project falls squarely within the Industrial Biotechnology remit which is a strategic priority of BBSRC.
The reductive amination of ketones is one of the most powerful and frequently employed reactions in organic synthesis enabling a wide range of ketones to be conjugated to 1o and 2o amines. Given the fact that the products are often chiral there is an increasing desire to develop asymmetric variants of this reaction, particularly involving catalysis rather than stoichiometric use of reagents. In the field of biocatalysis there are currently two possible approaches to catalysing this reaction; (i) using engineered octopine dehydrogenases (OctDHs) and (ii) using engineered imine reductases (IREDs). The former approach employing OctDHs has been partly addressed by Codexis/Merck although to date only a patent has appeared. The alternative approach based upon IREDs has been reported although these reactions involve excessively large quantities of IRED enzyme and high ratios of amine to ketone (>20:1) in order to achieve moderate conversions. The low efficiency of IREDs for reductive amination is due to the fact that they catalyse imine reduction but not imine formation. Recently Manchester and collaborators have discovered a new sub-clade of IREDs termed reductive aminases (RedAms) which catalyse enantioselective reductive amination with high activity (kcat up to 300 min-1) and turnover number (TTN up to 200,000). Steady-state kinetics have revealed ordered binding of substrates (NADPH;ketone;amine) and importantly established that RedAms catalyse imine formation, as well as imine reduction. Through a series of ligand:protein structures Manchester have also developed a working model for the catalytic mechanism and have identified key targets for mutagenesis. The student shall aim to capitalize on these preliminary exciting results by (i) identifying novel RedAms using the prototype Aspergillus oryzae RedAm (AspRedAm) and the putitative RedAms from Pfizer as a guide sequence; (ii) characterizing and comparing the RedAms using a defined panel of amines and ketones in order to determine substrate scope together with levels of enantioselectivity and conversion; (iii) determining structures of novel RedAms, in the presence of ligands, to identify active-site amino acids for mutagenesis studies and (iv) carrying out rate-based process optimization and exploring process intensification of RedAm catalyzed reactions to determine limit the prospects for carrying out RedAm catalyzed reductive aminations on a larger scale.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M011208/1 01/10/2015 30/09/2023
1908766 Studentship BB/M011208/1 01/10/2017 30/09/2021 Thomas Thorpe