Application of Engineered Amine Oxidases for the Synthesis of Chiral Amines A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy (PhD) in the Faculty of Engineering and Physical Sciences 2013 Diego Ghislieri School of Chemistry Contents DECLARATION ...................................................................................................................... 3 COPYRIGHT ............................................................................................................................ 4 ACKNOWLEDGEMENTS ...................................................................................................... 5 THESIS STRUCTURE ............................................................................................................. 6 ABSTRACT .............................................................................................................................. 7 INTRODUCTION .................................................................................................................... 9 THESIS OBJECTIVES .......................................................................................................... 36 ENGINEERING AN ENANTIOSELECTIVE AMINE OXIDASE FOR THE SYNTHESIS OF ALKALOIDS NATURAL PRODUCTS AND PHARMACEUTICAL BUILDING BLOCK ................................................................................................................................... 37 Supplementary information ....................................................................................... 56 MAO-N CATALYSED DERACEMISATION OF TETRAHYDRO-β-CARBOLINES ...... 95 Supplementary information ..................................................................................... 103 DERACEMISATION OF BENZYLISOQUINOLINE ALKALOIDS EMPLOYING MONOAMINE OXIDASE VARIANTS .............................................................................. 132 Supplementary information ..................................................................................... 146 ASYMMETRIC REDUCTION OF CYCLIC IMINES CATALYSED BY A WHOLE CELL BIOCATALYST PRODUCING AN (S)-IMINE REDUCTASE ......................................... 151 Supplementary information ..................................................................................... 159 SYNTHETIC CASCADES ARE ENABLED BY COMBINING BIOCATALYST WITH ARTIFICIAL METALLOENZYMES ................................................................................. 178 Supplementary information ..................................................................................... 196 CHEMO/BI-ENZYMATIC REDOX CASCADEFOR THE ENANTIOCONVERGEN TRANSFORMATION OF (±)-BENZYLISOQUINOLINES INTO (S)-BERBINES ......... 207 Supplementary information ..................................................................................... 219 TRANSAMINASES/MONOAMINE OXIDASES BIOCATALITIC CASCADE FOR THE SYNTHESIS OF CHIRAL PYRROLIDINES ..................................................................... 222 Supplementary information ..................................................................................... 226 METHODS ........................................................................................................................... 239 CONCLUSIONS AND OUTLOOK ..................................................................................... 246 Conclusions .............................................................................................................. 246 Outlook .................................................................................................................... 248 2 DECLARATION The University of Manchester PhD by published work Candidate Declaration Candidate Name: Diego Ghislieri Faculty: Engineering and Physical Sciences Thesis Title: Application of Engineered Amine Oxidases for the Synthesis of Chiral Amines Declaration to be completed by the candidate: I declare that no portion of this work referred to in this thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning. 3 COPYRIGHT The author of this thesis (including any appendices and/or schedules to this thesis) owns any copyright in it (the "Copyright")1 and he has given The University of Manchester the right to use such Copyright for any administrative, promotional, educational and/or teaching purposes. Copies of this thesis, either in full or in extracts, may be made only in accordance with the regulations of the John Rylands University Library of Manchester. Details of these regulations may be obtained from the Librarian. This page must form part of any such copies made. The ownership of any patents, designs, trademarks and any and all other intellectual property rights except for the Copyright (the "Intellectual Property Rights") and any reproductions of copyright works, for example graphs and tables ("Reproductions"), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property Rights and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property Rights and/or Reproductions. Further information on the conditions under which disclosure, publication and exploitation of this thesis, the Copyright and any Intellectual Property Rights and/or Reproductions described in it may take place is available from the Head of School of Chemistry (or the Vice-President) and the Dean of the Faculty of Engineering and Physical Sciences, for Faculty of Engineering and Physical Sciences candidates. 1This excludes material already printed in academic journals, for which the copyright belongs to said journal and publisher. Pages for which the author does not own the copyright are numbered differently from the rest of the thesis. 4 ACKNOWLEDGEMENTS First and foremost I would like to thank my supervisor, Prof Nick Turner, for giving me the opportunity to work on this interesting project in a fascinating and highly interdisciplinary environment. I am also grateful for the generous support from the Marie Curie research fellowship which made many of the collaborations possible. I am also very happy of being a part of the Turner/Flitsch group. It was great to work in such a nice atmosphere in the lab as well as in the pub afterwards. Thanks to the colleagues that collaborated on my projects (in alphabetic order) Marcos Diaz Muñoz, Dr Anthony Green, Deborah Houghton, Dr Valentin Köhler, Dr Jennifer Hopwood, Dr Friedemann Leipold, Dr Marta Pontini, Dr Joerg Schrittwieser and Dr Simon Willies. I would also like to thank Dr Mark Corbett and Dr Bas Groenendaal for their fantastic and numerous proof readings. Finally, I want to thank the Italian community of the Turner/Flitsch group, with them I have always felt at home… 5 THESIS STRUCTURE This thesis describes my work on the development of new variants of MAO-N and their application in deracemisation processes or in combination with other chemo- and biocatalysts. An introduction to the work highlights the role of amines as building blocks for drugs and the different methods to synthesise them, in particular using monoamine oxidase will be discussed in detail. The objectives of the thesis are then discussed in a separate chapter. The main body of the thesis consists of six publications which have either already been published or the manuscripts have been submitted or are ready to be submitted. The format of these papers is modified to fit in the template of the thesis, but no modifications are made to the main text. The supplementary information has been edited so that it contains only the experiments that were performed by Diego Ghislieri. References for the paper are at the end of each manuscript numbered as in the submitted version. Initially, there are four papers discussing different enzymes (MAO-N and IRED) used independently. Then, another two papers follow discussing the combination of monoamine oxidase-N with chemocatalysts (Ir-SAV system) and biocatalysts (Berberine Bridge Enzyme) in order to complete a cascade of enzymatic reactions. Another two small chapters follow the first covers the results that have not been published and the second describes the methods not discussed in the papers supplementary information. The last chapter is a conclusion and an overview of the further development of this project. 6 ABSTRACT The development of cost-effective and sustainable catalytic methods for the production of enantiomerically pure chiral amines is a key challenge facing the pharmaceutical and fine chemical industries. There is an increasing demand for broadly applicable synthetic methods which deliver the desired amine product in high yield and enantiomeric excess (e.e.). Previously we have described the development of variants of monoamine oxidase from Aspergillus niger (MAO-N) which are able to mediate the complete conversion of racemic amines to the corresponding enantiomerically pure products in a single step. In this thesis we report a panel of MAO-N variants (D5, D9 and D11) developed in our laboratory, which are able to mediate the deracemisation of primary, secondary and tertiary amines with broad structural features. In particular, we have synthesized and subjected to deracemisation a broad range of tetrahydroisoquinolines and tetrahydro-β-carbolines checking enantioselectivity and enantiopreference of our biocatalysts. A relation between lipophilicity of the substituents