An epoxide hydrolase from Vigna radiata (VrEH2) affords partial enantioconvergence (84% ee) in the enzymatic hydrolysis of racemic p-nitrostyrene oxide (pNSO), mainly due to insufficient regioselectivity for the (S)-enantiomer (r S = α S /β S = 7.3). To improve the (S)-pNSO regioselectivity, a small but smart library of VrEH2 mutants was constructed by substituting each of four key residues lining the substrate binding site with a simplified amino acid alphabet of Val, Asn, Phe, and Trp. Among the mutants, M263N attacked almost exclusively at Cα in the (S)-epoxide ring with satisfactory regioselectivity (r S = 99.0), without compromising the original high regioselectivity for the (R)-epoxide (r R = 99.0), resulting in near-perfect enantioconvergence (>99% analytical yield, 98% ee). Structural and conformational analysis showed that the introduced Asn263 formed additional hydrogen bonds with the nitro group in substrate, causing a shift in the substrate binding pose. This shift increased the difference in attacking distances between Cα and Cβ, leading to an improved regiopreference toward (S)-pNSO and affording near-perfect enantioconvergence.

Practical “green” manufacture of d-tagatose through a bi-enzymatic coupled system from galactitol refined from the waste xylose mother liquor is presented.

Baeyer-Villiger monooxygenases (BVMOs) are versatile biocatalysts in organic synthesis that can generate esters or lactones by inserting a single oxygen atom adjacent to a carbonyl moiety. The regioselectivity of BVMOs is essential in determining the ratio of two regioisomers for converting asymmetric ketones. Herein, we report a novel BVMO from Pseudomonas aeruginosa (PaBVMO); this has been exploited for the direct synthesis of medium-chain α,ω-dicarboxylic acids through a Baeyer-Villiger oxidation-hydrolysis cascade. PaBVMO displayed the highest abnormal regioselectivity toward a variety of long-chain aliphatic keto acids (C -C ) to date, affording dicarboxylic monoesters with a ratio of up to 95 %. Upon chemical hydrolysis, α,ω-dicarboxylic acids and fatty alcohols are readily obtained without further treatment; this significantly reduces the synthetic steps of α,ω-dicarboxylic acids from renewable oils and fats.

The chiral vicinal C–O/C–N bifunctional groups generated from enzymatic hydrocyanation represents a useful methodology. However, construction of the pharmacophore of β2-adrenoreceptor agonists with this method remains a great challenge because of complete racemization of the benzylic alcohol during deprotection of the acetal groups. In this study, structure-guided redesign of a hydroxynitrile lyase originating from Prunus communis (PcHNL5) enables a highly enantioselective hydrocyanation of rigid benzo-ketal aldehyde which was proved to be resistant against racemization during the deprotection step, with dramatically improved productivity (>95% conversion vs <1%). X-ray structure analysis and kinetic study revealed the side chain of L331 tunes the substrate adaptability of bulky and rigid benzo-ketal aldehyde, thereby facilitating the formation of a series of valuable unnatural cyanohydrins in high enantiopurities and good yields. Furthermore, the HNL variant L331A was successfully applied for a gram-scale chemo-enzymatic synthesis of (R)-salmeterol, a long-term β2-adrenoreceptor agonist, in an optically pure form (>99% ee) with an overall yield of 54%, which is the highest value reported.