Second Generation Artificial Hydrogenases Based on the Biotin‐Avidin Technology: Improving Activity, Stability and Selectivity by Introduction of Enantiopure Amino Acid Spacers

We report on our efforts to create efficient artificial metalloenzymes for the enantioselective hydrogenation of N ‐protected dehydroamino acids using either avidin or streptavidin as host proteins. Introduction of chiral amino acid spacers – phenylalanine or proline – between the biotin anchor and the flexible aminodiphosphine moiety 1 , combined with saturation mutagenesis at position S112X of streptavidin, affords second generation artificial hydrogenases displaying improved organic solvent tolerance, reaction rates (3‐fold) and ( S )‐selectivities (up to 95 % ee for N ‐acetamidoalanine and N ‐acetamidophenylalanine). It is shown that these artificial metalloenzymes follow Michaelis–Menten kinetics with an increased affinity for the substrate and a higher k cat than the protein‐free catalyst (compare k cat 3.06 min −1 and K M 7.38 mM for [Rh(COD) Biot‐1 ] + with k cat 12.30 min −1 and K M 4.36 mM for [Rh(COD) Biot‐( R )‐Pro‐1 ] + ⊂ WT Sav). Finally, we present a straightforward protocol using Biotin‐Sepharose to immobilize artificial metalloenzymes (>92 % ee for N ‐acetamidoalanine and N ‐acetamidophenylalanine using [Rh(COD) Biot‐( R )‐Pro‐1 ] + ⊂ Sav S112W).