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Versie 7.Indd UvA-DARE (Digital Academic Repository) Biocatalytic synthesis of α-chiral amines Selective immobilization of enzymes and their application in batch and continuous flow Böhmer, W. Publication date 2019 Document Version Final published version License Other Link to publication Citation for published version (APA): Böhmer, W. (2019). Biocatalytic synthesis of α-chiral amines: Selective immobilization of enzymes and their application in batch and continuous flow. General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:10 Oct 2021 Biocatalytic synthesis of α-chiral amines: selective immobilization of enzymes and their application in batch and continuous ow of α-chiral and continuous Biocatalytic synthesis of enzymes in batch amines: selective immobilization and their application Wesley Böhmer Wesley 2019 2019 - University of Amsterdam Wesley Böhmer omslag_2.indd 1 12-11-2019 16:04:20 Biocatalytic synthesis of α-chiral amines: selective immobilization of enzymes and their application in batch and continuous flow Wesley Böhmer 2019 © 2019, W. Böhmer, Amsterdam This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 638271, BioSusAmin) Printed by Amsterdam University Press Biocatalytic synthesis of α-chiral amines: selective immobilization of enzymes and their application in batch and continuous flow ACADEMISCH PROEFSCHRIFT ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus prof. dr. ir. K.I.J. Maex ten overstaan van een door het College voor Promoties ingestelde commissie, in het openbaar te verdedigen in de Aula der Universiteit op donderdag 19 december 2019, te 15.00 uur door Wesley Böhmer geboren te Alkmaar Promotiecommissie: Promotor: dr. F. Mutti Universiteit van Amsterdam prof. dr. J. H. van Maarseveen Universiteit van Amsterdam Overige leden: prof. dr. W. Kroutil Universität Graz prof. dr. R. Wever Universiteit van Amsterdam prof. dr. B. de Bruin Universiteit van Amsterdam dr. N. R. Shiju Universiteit van Amsterdam dr. T. Noël Technische Universiteit Eindhoven Faculteit der Natuurwetenschappen, Wiskunde en Informatica Table of contents Summary...................................................................................................................................................................8 List of abbreviations............................................................................................................................................9 Chapter 1 Enzyme immobilization facilitating biocatalytic routes for the synthesis of enantiomerically pure α-chiral amines.............................................10 1.1 Chemical and biocatalytic syntheses of chiral α-amines...................................11 1.2 Enzyme immobilization..................................................................................................15 1.2.1 Advantages of using enzyme immobilization.......................................................15 1.2.2 Immobilization techniques...........................................................................................16 1.2.3 Support materials.............................................................................................................18 1.2.4 Partition and diffusion phenomena...........................................................................21 1.3 Immobilization of enzymes for the production of α-chiral amines in batch and flow reactors.............................................................................24 1.4 Thesis outline.....................................................................................................................25 1.5 References...........................................................................................................................26 Chapter 2 Reductive amination of carbonyl compounds by amine dehydrogenases.................................................................................................................31 2.1 Introduction........................................................................................................................32 2.2 Optimization of the reaction conditions.................................................................34 2.3 Influence of the temperature and time studies....................................................36 2.4 Substrate scope of the reductive amination using AmDHs.............................37 2.5 Representative biocatalytic reductive amination in preparative scale.......................................................................................................................................46 2.6 Conclusion...........................................................................................................................47 2.7 Experimental section.......................................................................................................49 2.7.1 General information........................................................................................................49 2.7.2 Optimized procedure for the biocatalytic reductive amination on analytical scale......................................................................................49 2.7.3 Preparative biocatalytic reductive amination for the synthesis of (R)-4b....................................................................................................................................49 2.7.4 Procedure for enzymatic synthesis of chiral reference amines using ωTAs.........................................................................................................................49 2.7.5 Chemical synthesis of substrates...............................................................................50 2.7.6 Analytics...............................................................................................................................52 2.8 References...........................................................................................................................53 Chapter 3 Co-immobilized dehydrogenases applied for the asymmetric hydrogen-borrowing bio-amination of alcohols...........................................................................................................................57 3.1 Introduction........................................................................................................................58 3.2 Results & discussion.......................................................................................................62 3.2.1 Expression and purification of dehydrogenases..................................................62 3.2.2 Catalytic activity of purified alcohol dehydrogenases.......................................62 3.2.3 Co-immobilization of dehydrogenases...................................................................64 3.2.4 Optimizations for the bio-amination with co-immobilized dehydrogenases................................................................................................................64 3.2.5 Application of co-immobilized dehydrogenases in the amination of alcohols...........................................................................................................................70 3.2.6 Elaboration on limitations in recycling of the co-immobilized ADH-AmDH cascade.......................................................................................................70 3.2.7 Extending the reactivity of the ADH-AmDH cascade.........................................72 3.3 Conclusion...........................................................................................................................74 3.4 Experimental section.......................................................................................................76 3.4.1 General information........................................................................................................76 3.4.2 Expression and purification of dehydrogenases..................................................76 3.4.3 Activity testing of ADHs.................................................................................................76 3.4.4 General procedure for EziG metal-ion affinity co-immobilization of dehydrogenase............................................................................................................77 3.4.5 General procedure for analytical scale reactions with co-immobilized dehydrogenases...............................................................................77 3.4.6 Preparative scale amination reaction with co-immobilized dehydrogenases................................................................................................................78
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