Directed evolution of an alcohol dehydrogenase for the desymmetric reduction of 2,2-disubstituted cyclopenta-1,3-diones by enzymatic hydrogen transfer

Abstract

Chiral 2,2-disubstituted 3-hydroxycycloketones are highly desired intermediates for the construction of complex molecules with multiple chiral centres. Structure-guided directed evolution of the alcohol dehydrogenase TbADH from Thermoanaerobacter brockii was performed to enhance the enzyme activity toward ethyl secodione (1a) in an attempt to use isopropanol for substrate-coupling cofactor regeneration. Modulating the amino acid residues in the substrate tunnel and active centre resulted in mutants Tb2 (N114G/L294P), Tb3 (N114G/M285L/L294P) and Tb4 (W110V/N114G/M285L/L294P) with improved activity toward 1a, but decreased activity for isopropanol. Mutant Tb2 catalyzed the reduction of 1a to give (13R,17S)-ethyl secol with 94% yield and >99.5% ee and de, using isopropanol for NADPH regeneration. However, the substrate-coupling NADPH regeneration was not effective for variants Tb3 and Tb4 due to their low activity toward isopropanol. Mutants Tb2 and Tb3 showed high activity and stereoselectivity toward a series of 2-methyl-2-benzyl-cyclopenta-1,3-diones, giving the corresponding (2R,3S)-ketols with up to 99% ee and up to 99% de. Using Tb3 as the catalyst, (2R,3S)-2-cyanoethyl-2-methyl-3-hydroxycycloketone was prepared with >99% ee, 92% de and 90% yield.