An artificial copper‐Michaelase featuring a genetically encoded bipyridine ligand for asymmetric additions to nitroalkenes

Artificial metalloenzymes (ArMs) are an attractive approach to achieving “new to nature” biocatalytic transformations. In this work, a novel copper‐dependent artificial Michaelase (Cu_Michaelase) comprising a genetically encoded copper‐binding ligand, i.e. (2,2‐bipyridin‐5‐yl)alanine (BpyA), was developed. For the first time, such an ArM containing a non‐canonical metal‐binding amino acid was successfully optimized through directed evolution. The evolved Cu_Michaelase was applied in the copper‐catalyzed asymmetric Michael addition of 2‐acetyl azaarenes to nitroalkenes, yielding various γ‐nitro butyric acid derivatives, which are precursors for a range of high‐value‐added pharmaceutically relevant compounds, with good yields and high enantioselectivities (up to >99% yield and 99% ee). Additionally, the evolved variant could be further used in a preparative‐scale synthesis, providing chiral products for diverse derivatizations. X‐ray crystal structure analysis confirmed the binding of Cu(II) ions to the BpyA residues and showed that, in principle, there is sufficient space for the 2‐acetyl azaarene substrates to coordinate. Kinetic studies showed that the increased catalytic efficiency of the evolved enzyme is due to improvements in apparent KM for both substrates and a notable threefold increase in apparent kcat for 2‐acetyl pyridine. This work illustrates the potential of artificial metalloenzymes exploiting non‐canonical metal‐binding ligands for new‐to‐nature biocatalysis.