Artificial phosphate transferases and hydrogen tranferases based on Biotin-Streptavidin technology

Enzymes have been the subject of numerous studies and have been extensively used to catalyse difficult chemical reactions. By extension, artificial metalloenzymes that bridge the gap between organometallic catalysis and enzymatic catalysis are attracting increasing attention in the scientific community due to their optimization potential by chemical and genetic methods. The active site of such artificial systems are based on the versatility of organometallic complexes, whereas the molecular scaffold, that provides the enantioselectivity as in natural enzymes, generates the second coordination sphere. The first part of this thesis presents new artificial metallohydrolases incorporating Ce(IV) as the active catalyst. The interest in this new class is motivated by the possibility to create new highly selective artificial nucleases for novel biomedical applications. The use of chiral substrates mimicking DNA or RNA coupled with colorimetric assay techniques allows developing high throughput screening methods to identify novel active and selective artificial metalloenzymes. The knowledge acquired by the creation of novel artificial hydrolases allows transposing the technology to a new class of artificial metalloenzymes such transfer
hydrogenases. The second part of this thesis presents the incorporation of catalytically active biotinylated complexes within streptavidin, creating artificial metalloenzymes for the transfer hydrogenation of prochiral ketones. The optimization of the second coordination sphere, based on a crystal structure of the active hybrid catalyst and successive rounds of saturation mutagenesis at selected positions termed “designed evolution”, afforded highly active and selective catalysts for the reduction of challenging ketone substrates. The well-defined active site of the hybrid catalysts can be redesigned for the reduction of novel substrates such as imines to create the first example of an artificial imine reductase.