Towards a Photo-Driven Artificial Hydrogenase Using the Biotin-Streptavidin Technology Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Sascha Georg Keller aus Deutschland Basel, 2019 Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel edoc.unibas.ch Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Thomas R. Ward Prof. Dr. Oliver S. Wenger Basel, den 12. Dezember 2017 Prof. Dr. Martin Spiess Dekan (…) water will one day be employed as a fuel, that hydrogen and oxygen that constitute it, used singly or together, will furnish an inexhaustible source of heat and light, of an intensity of which coal is not capable. Someday the coal rooms of steamers and the tenders of locomotives will, instead of coal, be stored with these two condensed gases, which will burn in the furnaces with enormous caloric power (…). I believe, that when the deposits of coal are exhausted, we shall heat and warm ourselves with water. (…) Water will be the coal of the future (…). Jules Verne (1828 – 1905), “L’Ile Mystérieuse”1874 Für meine Familie Abstract It is estimated that the world’s population will reach 11 billion by 2100 and thus the energy need will increase. Already today this is a delicate topic since the use of finite energy sources as coal and oil are depleting. Furthermore, the CO2 released is negatively effecting the climate. Artificial photosynthesis to produce hydrogen as a clean fuel is one possibility that caught much attention. The reaction product of hydrogen and oxygen is solely water. Many small molecule catalysts have already been reported that produce hydrogen, but they are not as active as natural hydrogenases. Natural enzymes have a highly evolved and sophisticated coordination sphere around the catalytic center, as well as hydrogen, proton and electron channels. These features are hardly added by synthetic modification of a ligand of a small molecule catalyst. Incorporation of those catalysts into a protein scaffold would add a second coordination sphere that mimics natural enzymes. In this context, this thesis explores the biocompatibility of some molecular catalysts that produce hydrogen from formic acid for future in vivo or protein incorporation applications. Here, we could show that certain complexes show good turnovers and high recyclability rates, as well as oxygen tolerance under bio-compatible reaction conditions. To address the matter of electron transfer in proteins and their surface we explored the electron transfer properties of a dyad of an electron donating triarylamine and a bio-conjugated ruthenium photosensitizer. Different distances of the dyad were tested and the best system was further improved. Not only was an electron acceptor also bio-conjugated to the proteins N-terminus, but also a negative patch of three negatively charged amino acids was introduced in close proximity to the I photosensitizer. This negative patch increased the local concentration of the electron acceptor, even when not tethered to the protein. We could show that the protein can successively be bio-conjugated in three different ways: i) biotin-binding, ii) nucleophilic substitution on a cysteine residue and iii) N- terminal modification. The compatibility of these biorthogonal bio-conjugation procedures may open a new possibility of assembling catalytic systems on a protein surface. The thesis further discusses the use of the biotin-streptavidin technology to assemble an artificial hydrogenase to perform hydrogen evolution. A small molecule pentapyridin ligated Co catalyst was incorporated into different mutants of streptavidin and photocatalytic hydrogen evolution was measured. It could be shown that a lysine that was incorporated via mutagenesis has a beneficial influence on turnover numbers and rates. We also found that it decreased the initial lag- phase, often seen in small molecule hydrogen evolution catalysts, significantly. These findings support the idea that basic residues in close proximity to a hydrogen reducing or oxidizing catalyst have a positive impact, giving insights into its mechanism. The fact that the biotin-streptavidin provides a catalyst incorporated into a protein binding pocket also enables to exclude a heterolytic hydrogen evolution mechanism often proposed, since the Co-centers are too far away to react with each other, at least in our system. We envision that these findings will help to develop artificial hydrogenases that are at least as active as natural hydrogenases. II Self-Citations Self-Citations During the course of my PhD at the University of Basel, four manuscripts were published. The preprints and the corresponding supporting information of these four manuscripts are integrated in this thesis. Chapter 3 This chapter is an adapted version of „Evaluation of Formate Dehydrogenase Activity of Three-Legged Pianostool Complexes in Dilute Aqueous Solution“, Sascha G. Keller, Mark R. Ringenberg, Daniel Häussinger and Thomas R. Ward, Eur. J. Inorg. Chem. 2014, 34, 5860–5864. Chapter 4 This chapter is an adapted version of „Light-Driven Electron Injection from a Biotinylated Triarylamine 2+ Donor to [Ru(diimine)3] -Labeled Streptavidin“, Sascha G. Keller, Andrea Pannwitz, Fabian Schwizer, Juliane Klehr, Oliver S. Wenger and Thomas R. Ward, Org. Biomol. Chem. 2016, 14, 7197-7201. Chapter 5 This chapter is an adapted version of „Streptavidin as a Scaffold for Light-Induced Long-Lived Charge Separation“, Sascha G. Keller, Andrea Pannwitz, Hendrik Mallin, Oliver S. Wenger and Thomas R. Ward, Chem. Eur. J. 2017, 23, 18019-18024. III Chapter 6 This chapter is an adapted version of „Photo‐Driven Hydrogen Evolution by an Artificial Hydrogenase Utilizing the Biotin‐Streptavidin Technology“, Sascha G. Keller, Benjamin Probst, Tillmann Heinisch, Roger Alberto and Thomas R. Ward, Helv. Chim. Acta. 2018, 101, e1800036. IV Table of Content ABSTRACT ........................................................................................................................................ I SELF-CITATIONS ............................................................................................................................. III TABLE OF CONTENT ....................................................................................................................... V ABBREVIATIONS ......................................................................................................................... VIII 1. GENERAL INTRODUCTION ....................................................................................................... 1 2 THEORETICAL BACKGROUND .................................................................................................. 5 2.1 HYDROGEN ............................................................................................................................... 5 2.1.1 WHAT IS HYDROGEN? ................................................................................................................ 5 2.1.2 WHERE CAN HYDROGEN BE FOUND? ............................................................................................. 6 2.1.3 HOW CAN HYDROGEN BE PRODUCED? ........................................................................................... 6 2.2 NATURAL PHOTOSYNTHESIS ........................................................................................................ 7 2.3 ARTIFICIAL PHOTOSYNTHESIS ....................................................................................................... 9 2.3.1 PHOTOCATALYTIC WATER SPLITTING .............................................................................................. 9 2.3.2 PHOTOCATALYTIC WATER OXIDATION .......................................................................................... 10 2.3.3 PHOTOCATALYTIC WATER REDUCTION ......................................................................................... 10 2.3.4 PHOTOSENSITIZERS .................................................................................................................. 11 2.4 NATURAL HYDROGENASES ........................................................................................................ 14 2.5 BIOINSPIRED HYDROGENASE MODEL SYSTEMS ............................................................................. 17 2.6 TOWARDS ARTIFICIAL HYDROGENASE ENZYMES ............................................................................. 20 2.6.1 ARTIFICIAL METALLOENZYMES ................................................................................................... 20 2.6.2 BIOTIN-STREPTAVIDIN TECHNOLOGY ........................................................................................... 21 2.6.3 ARTIFICIAL HYDROGENASE MIMICS ............................................................................................. 23 2.7 AIM OF THIS THESIS .................................................................................................................. 29 3 EVALUATION OF FORMATE DEHYDROGENASE ACTIVITY OF THREE-LEGGED PIANOSTOOL COMPLEXES IN DILUTE AQUEOUS SOLUTION ................................................................................ 30 3.1 ABSTRACT .............................................................................................................................. 31 3.2 INTRODUCTION ......................................................................................................................