Artificial transfer hydrogenases based on biotin-streptavidin technology

The importance of metalloenzymes in nature is reflected by their involvement in many fundamental processes (e.g. photosynthesis, respiration, nitrogen fixation). The creation of artificial metalloenzymes for chemical and biochemical applications is an intriguing and potentially highly rewarding area of research. As a starting point, a catalytically active transition metal complex or catalyst precursor needs to be incorporated into a host protein thereby generating a hybrid, which exhibits attractive features of biocatalyst and chemocatalyst. Exploiting the biotin (strept)avidin technology for the creation of artificial metalloenzymes, is a convenient means, to ensure the cofactor localisation thanks to the high affinity of biotin for streptavidin. Synthetic cofactor and protein host can be separately modified by chemical- and genetic means, respectively and subsequently combined. The topic of this thesis is to create artificial transfer hydrogenases relying on this technology and to study the resulting constructs. With the ultimate goal of implementing efficient directed evolution protocols for the optimization of artificial metalloenzymes and for their application in vivo, the interaction between the active catalyst and the biological environment needs to be evaluated. Mutual inhibition between the synthetic catalyst and enzymes (other than Sav) was identified as one potential problem. After reviewing the main organometallic-based methods for the non-enzymatic regeneration of NADH, a solution for the frequently observed inhibition between the organometallic NADH regeneration system and the NADH dependent enzyme, namely the compartmentalization of the synthetic cofactor in Sav, will be discussed. The incorporation of the active organometallic catalyst [Cp*Ir(biot-p-L)Cl] into streptavidin, led to an active ATHase (Artificial Transfer Hydrogenase), utilized for NADH regeneration, which was subsequently successfully coupled in a cascade biocatalysis reaction with HbpA (a NADH and FADH2 dependent monooxygenase), for the selective hydroxylation of 2-hydroxybiphenyl to 2,3-hydroxybiphenyl. Next, the stereoselectivity of the ATHase mediated-NAD+ reduction with deuterated formate as a deuteride source was investigated resulting in up to 90% de.
Finally chemical variants of IrCp*/Sav- or RhCp*/Sav-based transfer hydrogenases were studied. In order to rapidly generate chemical diversity, a new approach for the creation of biotinylated complexes is presented. Tethering the biotin anchor to the Cp* moiety of the organometallic complex, thereby leaving three coordination sites vacant, enabled fast screening of libraries of bidentate ligands, which led to the identification α-amino amides as promising ligands for the asymmetric reduction of cyclic imines.