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Deracemisation of amines using enzymes obtained by directed evolution

Reference	BB/C504751/1
Principal Investigator / Supervisor	Professor Nicholas Turner
Co-Investigators / Co-Supervisors
Institution	The University of Manchester
Department	Chemistry
Funding type	Research
Value (£)	369,974
Status	Completed
Type	Research Grant
Start date	01/09/2005
End date	31/08/2008
Duration	36 months

Abstract

The overall aim of this proposal is to develop a general method for the deracemisation of chiral amines using a combination of enantioselective amine oxidases and non-selective chemical reducing agents. Deracemisation represents a powerful new strategy for the preparation of optically pure amines which are valuable, and difficult to access, compounds used as chiral resolving agents, ligands for asymmetric synthesis and importantly intermediates in the synthesis of pharmaceuticals and agrochemicals. A key aspect of this proposal is the discovery and optimisation of various amine oxidases, suitable for deracemisation reactions, by employing directed evolution strategies. In this context we have developed a robust high-throughput colorimetric screen which is able to routinely assess libraries of up to 300,000 variant clones. This screening method can for example be used to identify variant amine oxidases possessing activity towards specific substrates of interest. Clones of interest can then be rapidly checked for enantioselectivity. We also plan to extend this screening approach to select for variants that have improved organic solvent thermostability in order to identify enzymes that are likely to perform better under the conditions required for deracemisation on a preparative scale. To date we have focussed on this identification of (S)-selective amine oxidases but now we propose to screen for (R)-selective variants such that both enantiomeric series are accessible via the deracemisation approach. In addition to generating libraries of Aspergillus niger monoamine oxidase (MAO-N) we will also examine the L-amino acid oxidase from Synechococcus sp. (L-AAO) as an alternative starting point for directed evolution. In addition to optimisation of the amine oxidases we shall also investigate the nature of the chemical reducing agent. We shall prepare polymer bound amine-borane complexes to improve the efficiency of the deracemisation reactions. We shall also examine the use of organic solvents and ionic liquids in order to increase the yield of the reactions by minimising hydrolysis of the intermediate imine. Finally, we will explore alternative methods for generating the key imine intermediate in situ using ketone and alkene precursors rather than the amine directly. We shall also examine further reaction of the imines including trapping with Grignard reagents under aqueous conditions.

Summary

unavailable

Committee	Closed Committee - Engineering & Biological Systems (EBS)
Research Topics	Industrial Biotechnology, Microbiology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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