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Novel Approaches for Using Dehydrogenases and Ene-Reductases for Organic Synthesis

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Novel Approaches for Using Dehydrogenases and Ene-Reductases for Organic Synthesis Novel approaches for using dehydrogenases and ene-reductases for organic synthesis Serena GARGIULO Novel approaches for using dehydrogenases and ene-reductases for organic synthesis Proefschrift ter verkrijging van de graad van doctor aan de Technische Universiteit Delft, op gezag van de Rector Magnificus prof. ir. K.C.A.M. Luyben, voorzitter van het College voor Promoties, in het openbaar te verdedigen op dinsdag 24 februari 2015 om 10.00 uur door Serena GARGIULO Master of Science in Molecular and Industrial Biotechnology van Università degli Studi di Napoli “Federico II”, Italië, geboren te Napels, Italië Dit proefschrift is goedgekeurd door de promotor: Prof Dr. I.W.C.E. Arends Copromotor Dr.F.Hollmann Samenstelling promotiecommissie: Rector Magnificus, voorzitter Prof Dr. I. W. C. E. Arends Technische Universiteit Delft, promotor Dr. F. Hollmann Technische Universiteit Delft, supervisor Prof. Dr. S. De Vries Technische Universiteit Delft Prof. Dr. R. Piccoli Università degli Studi di Napoli “Federico II” Prof. Dr. W. van Berkel Wageningen Universiteit Prof. Dr. G. Grogan University of York Dr. Stephan Luetz Novartis Pharma Prof. Dr. E. J. R. Sudholter Technische Universiteit Delft, reservelid ISBN 978-94-6108-919-9 The research reported in this thesis was supported by the Marie Curie Initial Training Network BIOTRAINS, financed by the European Union through the 7th Framework People Programme (grant agreement number 238531) Ai miei Angeli, in terra e in cielo, a chi ha creduto in me Table of contents Chapter 1 Introduction……………………………………………………………………………1 Chapter 2 A photoenzymatic system for alcohol oxidation……………………………………..29 Chapter 3 A biocatalytic redox isomerization…………………………………………………...47 Chapter 4 Synthetic nicotinamide cofactors for biocatalytic reduction of activated C=C bonds…………………………………………………………………………...67 Chapter 5 Structure of the alcohol dehydrogenase from Thermus sp . ATN1……………..…….87 Chapter 6 Heterologous expression and characterisation of the Ene-reductases from Deinococcus radiodurans and Ralstonia metallidurans …………..………………..105 Summary……………………………………………………………………………......125 Samenvatting……………………………………………………………………………127 Acknowledgments………………………………………………………………………130 Curriculum Vitae………………………………………………………………………..136 List of publications……………………………………………………………………...137 Chapter 1: Introduction 1 Introduction 1.1 Enzymes as smart redox catalysts Redox reactions comprise a wide variety of essential synthetic transformations. Therein the increase/decrease in the oxidation state of one species is accompanied by the decrease/increase in the oxidation state of another species, denoted as the oxidant/reductant. For selective redox transformations in the liquid phase a broad repertoire of stoichiometric reagents are part of the organic toolbox for already many decades 1. However their use is commonly not in line with the 12 principles of green chemistry 2. Catalysts that can facilitate the use of green oxidising and reducing agents play a major role in a transition to more sustainable technologies. With regard to the oxidation case, catalytic methods only recently started to replace the traditional use of stoichiometric oxidants (MnO 2, Jones reagent, Dess-Martin periodinane, etc.), ultimately allowing for O 2 or H 2O2 to be used as oxidants. Seminal examples include the tetra-n-propylammonium perruthenate (TPAP) catalyst utilising N- methylmorpholine N-oxide (NMNO) or O 2 as oxidant for the conversion of primary alcohols to carboxylic acids; the Fenton’s reagent employing H 2O2 for the degradation of phenolic compounds in waste water; the Al(i-PrO 3) (Oppenauer reagent) utilising acetone for the selective oxidation of secondary alcohols to ketones and the 2,2,6,6-tetramethyl-1- piperidine-N-oxyl (TEMPO) and its derivatives which have been exploited for alcohol 3 oxidation either in combination with sodium hypochlorite or O2 . Reduction catalysts are relatively well embedded and usually consist of Noble metals on surfaces (Pd, Pt, Ru, Raney-Ni) that can activate H2 as the reductant. In case of chiral reduction catalysis, commonly chiral ligands are employed that, in combination with metals such as Rh Ru, or Pd and Rh, lead to high enantioselectivities and selective transformations 4, 5. In general, thus most research efforts have been devoted to the development of transition metal-based catalysts. Very often these are robust catalysts, stable in organic media over a considerably broad range of temperature and pressure. However, main points of concern about their preparation and use are the need for toxic solvents or additives, the combined use of O 2 with flammable solvents and the constant depletion trend of rare metals. The continuous quest for greener manufacturing routes has shed light on the high potential of enzymes as alternatives to the established procedures 6. Many reasons account for this interest towards bio-based catalysts. Enzymes, in general, have evolved 2 Chapter 1 to work under milder reaction conditions (pH, temperature, pressure) than most chemical catalysts. Furthermore they utilise water as solvent of choice, are biodegradable and can be isolated from renewable resources. Thus, even though for any given process claims of greenness should only be made after proper eco-assessment analysis, they bear the potential for more sustainable catalysis. An overview of the general characteristics of chemo- and biocatalytic systems is given in Table 1. 1. Table 1. 1: Comparison of biocatalytic and chemocatalytic strategies for oxidation and reduction reactions. Chemocatalysis Biocatalysis Organic media Aqueous media High substrate loading Moderate substrate loading Selectivity issues High chemo/stereo-selectivity Mostly robust catalysts Catalyst stability issues Oxidations Catalysts: Catalysts: Ru, Pd, Cu, Fe, Au-based; Al(i-PrO 3); 2,2,6,6- Dehydrogenases, oxidases, peroxidases tetramethyl-1-piperidine-N-oxyl (TEMPO), polymer- immobilised piperidinyl oxyl (PIPO) 7, 8. Oxidants: Oxidants: NaOCl, acetone, O 2, H 2O2 O2, H 2O2, R-OOH, acetone Reductions Catalysts: Catalysts: Rh, Pt, Ir-based, MacMillan 9 imidazolidinone; Reductases; dehydrogenases Reductants: Reductants: H2, dihydropyridine, NaBH 4 Glucose, formate, H 2 Noteworthy, the potentially lower environmental impact of most biocatalysts often goes in hand with their excellent catalytic properties: high chemo-/regio-/stereo-selectivity are some of the major advantages of enzyme catalysis. An exemplary case of regioselective enzymatic oxidation is shown in Figure 1. 1 10 . Figure 1. 1: regioselective oxidation of cholic acid by the 12 α-hydroxysteroid dehydrogenase from Clostridium group P, adapted from 10 . When studying the different classes of enzymes that catalyse redox reactions four subclasses can be distinguished: dehydrogenases, oxygenases, oxidases and peroxidases (Scheme 1. 1). Although industrially viable examples stem from all groups, the 3 Introduction dehydrogenases/reductases family represents a highly attractive target of study, as it includes a large number of enzymes well characterised and readily accessible to both scientific and industrial community. Dehydrogenases cover a wide range of substrates, and nowadays a large number of thermostable enzyme variants are available as well. Thus they provide an excellent basis for industrial processing. Scheme 1. 1: oxidoreductases subclasses, schematic mode of action (adapted from 11 ) This thesis focuses on the use of dehydrogenases/reductases (DRs), particularly alcohol dehydrogenases and ene-reductases for the development of novel approaches to the biocatalytic oxidation of alcohols and reduction of activated olefins, overall aiming at addressing the present shortcomings in the use of these enzymes and enlarging the scope of their application. 1.2 Alcohol oxidations: the need for biocatalytic alternatives Oxidation of alcohols is a fundamental reaction in organic chemistry. Popular ways to perform this kind of reaction rely on the use of heavy metal based reagents (Mn, Cr) in stoichiometric amounts or on transition metal based catalysts 12-14 . Even though these methods are by now well-developed, they often lack chemo and stereoselectivity, thus requiring complex protection and deprotection strategies in order to afford the desired product, like in the case of polyols oxidation. Moreover, high catalysts loadings and the need for significant O2 pressures in flammable solvents are some of the issues that relate to their applicability on large scale so that currently they do not appear to be the most promising route towards more sustainable processes. 4 Chapter 1 Instead, the use of oxidative enzymes bears the promise for more selective and environmental benign possibilities. 1.2.1 Alcohol dehydrogenases as biocatalytic alternative Two types of enzymes can perform oxidation of alcohols: oxidases and dehydrogenases. Alcohol oxidases are mostly flavin-dependent enzymes that catalyse the irreversible substrate oxidation by transferring reducing equivalent from the alcohol group to O 2 via the prosthetic flavin. They represent a very promising class of biocatalysts for oxidation reactions but their applicability is severely hampered by the limited number of enzymes identified and the rather narrow substrate spectrum of those that are available. Instead, hundreds of alcohol dehydrogenases have already been identified in the past which have also been reported as excellent catalysts for a wide range of applications in oxidative chemistry. In broad terms, these enzymes catalyse the interconversion
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