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.