Transition metal catalysts within protein scaffolds−three case studies on the development and engineering of artificial metalloenzymes

Abstract

Transition metal catalysis provides versatile methods for the preparation of synthetically useful compounds. Some of these transformations can also be realized by enzymatic catalysis at much higher activity and selectivity. The research field of artificial metalloenzymes (ArMs) lies at the interface of traditional transition metal catalysis, organocatalysis and enzymatic catalysis. In this line of research, new-to-nature transition metal complexes are incorporated into suitable protein scaffolds to explore the reactivity and selectivity of the resulting constructs, thereby greatly expanding the repertoire of enzymatic catalysts. To match the high efficiency and selectivity displayed by enzymatic catalysts, ArMs need to be optimized via effective engineering methods such as directed evolution. Within the scope of this thesis, complexes of the transition metal elements Ru, Rh, and Ir were combined with mutants of human carbonic anhydrase II (hCA II), and streptavidin from Steptomyces avidinii, respectively. Specifically, three types of ArMs were developed : i) an artificial metathesase based on human carbonic anhydrase II mutants, ii) a dirhodium carbenoid transferase and iii) artificial transfer hydrogenase, the latter two based on streptavidin (Sav) mutants as the protein scaffold. The first subsection of chapter 1 provides a general introduction on ArMs, including anchoring and engineering strategies and presents their synthetic applications. A particular focus is set on the achievements of ArMs in the past two years. The further subsections of chapter 1 detail important background information for the constructs under investigation. Chapter 2 describes the incorporation of arylsulfonamide anchored Hoveyda-Grubbs Ru-catalysts into human carbonic anhydrase II to create an ArM for ring-closing metathesis. The binding affinity of a novel Ru-cofactor for hCA II scaffold was determined. The catalytic activity of the ArMs was evaluated with different olefin substrates under optimized conditions. The best ArM/substrate pair was employed in test reactions under pH-neutral conditions at a low catalyst concentration (10 uM). Such studies are an important prerequisite for the application of ArMs in vivo. In chapter 3, the development of a dirhodium based artificial carbenoid transferase for intermolecular cyclopropanation and C-H insertion reactions is outlined. ArMs which combine either bidentate or monodentate dirhodium carboxylate complexes with streptavidin were compared in terms of their carbene transfer activity. Docking simulations of the biotinylated dirhodium catalysts within streptavidin predicted the position of the rhodium center in respect to the biotin-binding vestibule. Biocompatibility of the dirhodium complex was tested by performing the dirhodium ArM catalyzed cyclopropanation reaction in the presence of whole E. coli cells. Chapter 4 summarizes a study on the directed evolution of an iridium-based artificial transfer hydrogenase. The starting point was a computed structure of a chimeric streptavidin scaffold containing an inserted loop partially shielding the active site, which was predicted by Rosetta design. Site-directed mutagenesis at four residues was performed to build up a mutant library for screening. A self-immolative substrate which releases a fluorescent product upon reduction was developed and used in the evolution process to increase the screening throughput. Around 1000 clones were screened with the hits sequenced. The promising mutants were purified and their performance was investigated for imine reduction in vitro.