(692b) Hydroclassified Combinatorial Saturation Mutagenesis for Evolving Stereoselectivity and Cosubstrate Specificity of a Diaryl Alcohol Dehydrogenase

Optically active diaryl alcohols is an important class of chemical chiral compounds which could be applied in the synthesis of pharmaceuticals, agrochemicals and natural products, such as antihistamine, hydragogue, and antitussive drugs. Among them, chiral (4-chlorophenyl)-(pyridine-2-yl)-methanol (CPMA) is a vital intermediate for anti-allergic drug Betahistine and could be synthesized by asymmetric reduction of its corresponding (4-chlorophenyl)-(pyridine-2-yl)-ketone (CPMK). Due to the strong steric hindrance of the diaryl groups, very few alcohol dehydrogenases have been reported with desirable activity and enantioselectivity. Previously, a diaryl alcohol dehydrogenase from Kluyveromyces polyspora (KpADH) was identified by genome hunting, however with a moderate enantioselectivity of 81%. Inspired by the smallest amino acid alphabets strategy used in the saturation mutagenesis of an epoxide hydrolase^[1], we proposed a hydroclassified combinatorial saturation mutagenesis (HCSM) strategy for construction of smart libraries of KpADH with reduced library size and hydroclassified amino acids. Additionally, a highly sensitive carbonyl group-dependent colorimetric method was developed using 2,4-dinitrophenylhydrazine (DNPH) for high-throughput screening of smart libraries of KpADH toward various ketones including diaromatic ketones, aliphatic ketones/diketones, and aromatic ketones^[2]. Hot spots in the substrate-binding pocket were predicted based on molecular docking analysis. After site-directed and combinatorial mutagenesis, and the enantioselectivity of KpADH was enhanced from 81% to 99.7%ee (R). Additionally, the stereoselectivity of KpADH was inverted to 97.8%ee (S) by reshaping substrate binding pocket based on hydrogen bonds networks. Consequently, our study provides carbonyl reductases with promising applications in the preparation of optically active diaryl alcohols.

[1] Sun, Z.; Lonsdale, R; Kong X. D.; Xu J. H.; Zhou J.; Reetz M. T. Angew. Chem. Int. Ed. 2015, 12, 12410-12415.

[2] Zhou, J.; Xu, G.; Han R.; Dong, J.; Zhang W.; Zhang, R.; Ni, Y. Catal. Sci. Technol. 2016, 6, 6320-6327.