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.

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.

Hypervalent iodine-mediated gem-difluorination of vinyl halides enabled by exclusive 1,2-halo migration

β-Difluorinated alkyl halides are of significant value in the modular synthesis of gem-difluorinated molecules.An exclusive 1,2-halo migratory gem-difluorination of vinyl halides with in situ-generated PhIF_(2)·HF is described.This protocol provides a general and practical approach towards a wide variety ofβ-difluorinated alkyl bromides.Bothα-andβ-bromoalkyl alkenes are suitable substrates,leading to two distinct types of products.The extension of this protocol to vinyl chloride and iodide are also feasible.The synthetic versatility of this method has been highlighted by the late-stage modification of complex small molecules and further transformations of theβ-difluorinated alkyl halides to valuable CF_(2)-containing compounds.