Chiral methyl mandelates are useful synthons in organic transformation and pharmaceutical synthesis.Green synthesis of these valuable compounds by direct C–H activating oxidative hydroxylation has attracted keen inte...Chiral methyl mandelates are useful synthons in organic transformation and pharmaceutical synthesis.Green synthesis of these valuable compounds by direct C–H activating oxidative hydroxylation has attracted keen interest.Described herein is achieving the stereoselective and efficient bio-hydroxylation of methyl 2-phenylacetates to the chiral methyl mandelates by directed evolution of the cytochrome P450DA hydroxylase.In the present study,a new colorimetric high-throughput screening assay was successfully developed based on a dualenzyme cascade for the engineering of the P450DA's hydroxylation activity.Several beneficial variants with enhanced bio-hydroxylation activity were created by combining random mutagenesis and site-saturated/directed mutagenesis strategies.Whole-cell bio-hydroxylation of various methyl 2-phenylacetates using the best septupletmutant P450DA-11 yielded the corresponding chiral methyl mandelates in up to 92%isolated yields and>99%ee.展开更多
基金supported by the National Natural Science Foundation of China(Nos.21961048 and 32271537)Science and Technology Department of Zunyi(Nos.ZSKRPT-2020-5,ZSKH-2018-3 and ZSKRPT-2021-5).
文摘Chiral methyl mandelates are useful synthons in organic transformation and pharmaceutical synthesis.Green synthesis of these valuable compounds by direct C–H activating oxidative hydroxylation has attracted keen interest.Described herein is achieving the stereoselective and efficient bio-hydroxylation of methyl 2-phenylacetates to the chiral methyl mandelates by directed evolution of the cytochrome P450DA hydroxylase.In the present study,a new colorimetric high-throughput screening assay was successfully developed based on a dualenzyme cascade for the engineering of the P450DA's hydroxylation activity.Several beneficial variants with enhanced bio-hydroxylation activity were created by combining random mutagenesis and site-saturated/directed mutagenesis strategies.Whole-cell bio-hydroxylation of various methyl 2-phenylacetates using the best septupletmutant P450DA-11 yielded the corresponding chiral methyl mandelates in up to 92%isolated yields and>99%ee.
