Two new phenolic glycosides, ilexpubsides A and B, along with four known lignan glycosides were isolated from the roots of Ilex pubescens. By spectral evidence, the structures of the new compounds were elucidated as 4-O-beta-D-[6'-O-(4''-O-beta-D-glucopyranosylvanilloyl)glucopyranosyl] vanillic acid (1) and syringinic 6'-O-beta-D-xylopyranoside (2). The known compounds were identified to be liriodendrin (3), (-)-olivil (4), tortoside A (5) and (+)-cyclo-olivil (6). All compounds were first isolated from Ilex pubescens.

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S)-N-Boc-3-hydroxypiperidine [(S)-NBHP] is a key intermediate for the synthesis of mantle cell lymphoma drug, ibrutinib. Here, KpADH, an alcohol dehydrogenase from Kluyveromyces polyspora, exhibits evolutionary potential in the asymmetric reduction of N-Boc-3-piperidone (NBPO) to (S)-NBHP. By screening key residues in substrate binding pocket of KpADH, an excellent variant Y127W was obtained with 6-fold improved activity of 119.3 U mg À 1 , 1.8-fold enhanced half-life of 147 h and strict S-stereoselectivity (> 99% ee). When catalyzed by Y127W, a complete conversion of 600 g L À 1 NBPO was achieved at a substrate to catalyst ratio (S/C) of 30 in 10 h. Based on crystal-structure of Y127W, molecular docking and dynamic simulations reveal hydrogen bonds formed between W127 and Boc group of NBPO, as well as improved structural stability mainly contribute to the increased catalytic activity and stereoselectivity of Y127W. This study offers guidance for engineering ADHs for biosynthesis of chiral heterocyclic alcohols, and provides insights into mechanisms in catalytic activity and stereoselectivity toward carbonyl-containing heterocyclic substrates.

Bioreduction of ketones with self-sufficient cofactor regeneration is green and sustainable for the synthesis of chiral secondary alcohols. In this study, a novel alcohol dehydrogenase CgADH was identified from Candida glabrata through genome hunting, exhibiting high oxidation and reduction activities. Conserved motif analysis revealed that CgADH belongs to the extended SDR subfamily. CgADH is NADP(H) dependent and displays the highest activity at pH 5.0 and 65 °C. Substrate spectrum analysis indicated that CgADH exhibited high specificity toward 2-propanol and 2,3butanediol. Rational engineering of CgADH was performed to modulate its stereoselectivity in the asymmetric reduction of bulky−bulky ketones. Two stereocomplementary mutants, C244A and V222G/C244N, were obtained with e.e. values of 99.6% (R) or 94.5% (S) toward (4-chlorophenyl)-pyridin-2-ylmethanone, respectively. Moreover, the catalytic efficiency (k cat /K M ) of C244A and V222G/C244N increased by 70-and 25-fold compared with the wild type (WT), respectively. C244A and V222G/ C244N also displayed significantly enhanced catalytic efficiency and stereoselectivity toward diaryl ketones with different substituents. Using isopropanol both as a co-substrate and as a co-solvent, a concise selfsufficient NADPH regeneration system was developed to demonstrate the advantage of CgADH. This study provides evidence of the application potential of the newly identified CgADH in the preparation of enantiopure diaryl alcohols with low byproducts and E factor.

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