Biocatalytic stereoselective synthesis of methyl mandelates by engineering a cytochrome P450 hydroxylase

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.

Microbial synthesis of anti-tumor agent oxysophoridine through one step by filamentous fungus

Sophoridine and oxysophoridine are alkaloids extracted from Chinese traditional medicinewith significant antitumor activities. Computer-aided design and preliminary structure-activity relationship studiesshowed that the biological activity of sophoridine can be improved after oxidation (oxysophoridine) whileits toxicity significantly decreased. A new method to prepare oxysophoridine from natural product Sophoridineby biotransformation was established for the first time except for traditional extraction from plants and chemicalsynthesis. Positive biocatalytic reaction was detected by TLC. Structure of conversion product was elucidatedbased on NMR spectroscopy. Optimizations of reactions including substrate concentration, pH, conversion timeand inoculation quantity were complemented which analyzed by LC-MS. Results showed that filamentous fungusXylariales. sp F005 (CCTCC M2014660) has the capacity of synthetizing oxysophoridine from sophoridinein the selected 37 strains. The yield of oxysophoridine reached highest at conditions pH 6.0, the third day ofconversion time, 5.0 mg substrate and 5% inoculums respectively. The method for preparing oxysophoridinecatalyzed by enzymes with green and sustainable synthetic processes was developed.

Transition-metal catalyzed asymmetric reactions under continuous flow from 2015 to early 2020

During last two decades,transition-metal catalyzed asymmetric reactions under continuous flow system have attracted a widespread attention.With its prominent advantages including higher safety and efficiency,precise control,better heat/mass transfer,easier scale-up and better sustainability,it has impressed both the academia and related industries.However,comparing with the prosperous development of transition-metal catalyzed asymmetric reactions in batch,utilization of this type of chemistry under continuous flow is still at an early stage.To inspire more potential industrial application and further studies of this chemistry in flow,this review sum-marized the recent advances of transition-metal catalyzed asymmetric reactions under continuous flow.Moreover,we further discussed the encountered challenges including reactor engineering,catalyst design,catalyst deacti-vation,pressure drop,clogging,side reactions etc.,as well as their corresponding solutions and achievements in this mini-review.