There is intense interest in late-stage catalytic C-H bond functionalization as an integral part of synthesis. Effective catalysts must have a broad substrate range and tolerate diverse functional groups. Drug molecules provide a good test of these attributes of a catalyst. A library of P450BM3 mutants developed from four base mutants with high activity for hydrocarbon oxidation produced human metabolites of a panel of drugs that included neutral (chlorzoxazone, testosterone), cationic (amitriptyline, lidocaine) and anionic (diclofenac, naproxen) compounds. No single mutant was active for all the tested drugs but multiple variants in the library showed high activity with each compound. The high conversions enabled full product characterization that led to the discovery of the new P450 reaction type of oxidative decarboxylation of an α-hydroxy carboxylic acid and the formation a protected imine from an amine, offering a novel route to α-functionalization of amines. The substrate range and varied product profiles suggest that this library of enzymes is a good basis for developing late-stage C-H activation catalysts.

Cytochromes P450 have been recently identified as a promising class of biocatalysts for mediating C–H aminations via nitrene transfer, a valuable transformation for forging new C–N bonds. The catalytic efficiency of P450s in these non-native transformations is however significantly inferior to that exhibited by these enzymes in their native monooxygenase function. Using a mechanism-guided strategy, we report here the rational design of a series of P450BM3-based variants with dramatically enhanced C–H amination activity acquired through disruption of the native proton relay network and other highly conserved structural elements within this class of enzymes. This approach further guided the identification of XplA and BezE, two “atypical” natural P450s implicated in the degradation of a man-made explosive and in benzastatins biosynthesis, respectively, as very efficient C–H aminases. Both XplA and BezE could be engineered to further improve their C–H amination reactivity, which demonstrates their evolvability for abiological reactions. These engineered and natural P450 catalysts can promote the intramolecular C–H amination of arylsulfonyl azides with over 10 000–14 000 catalytic turnovers, ranking among the most efficient nitrene transfer biocatalysts reported to date. Mechanistic and structure–reactivity studies provide insights into the origin of the C–H amination reactivity enhancement and highlight the divergent structural requirements inherent to supporting C–H amination versus C–H monooxygenation reactivity within this class of enzymes. Overall, this work provides new promising scaffolds for the development of nitrene transferases and demonstrates the value of mechanism-driven rational design as a strategy for improving the catalytic efficiency of metalloenzymes in the context of abiological transformations.

We report the development of engineered myoglobin biocatalysts for executing asymmetric intramolecular cyclopropanations resulting in cyclopropane-fused γ-lactones, which are key motifs found in many bioactive molecules. Using this strategy, a broad range of allyl diazoacetate substrates were efficiently cyclized in high yields with up to 99% enantiomeric excess. Upon remodeling of the active site via protein engineering, myoglobin variants with stereodivergent selectivity were also obtained. In combination with whole-cell transformations, these biocatalysts enabled the gram-scale assembly of a key intermediate useful for the synthesis of the insecticide permethrin and other natural products. The enzymatically produced cyclopropyl-γ-lactones can be further elaborated to furnish a variety of enantiopure trisubstituted cyclopropanes. This work introduces a first example of biocatalytic intramolecular cyclopropanation and provides an attractive strategy for the stereodivergent preparation of fused cyclopropyl-γ-lactones of high value for medicinal chemistry and the synthesis of natural products.

We report the development of an iron-based biocatalytic strategy for the asymmetric synthesis of fused cyclopropane-γ-lactams, which are key structural motifs found in synthetic drugs and bioactive natural products. Using a combination of mutational landscape and iterative sitesaturation mutagenesis, sperm whale myoglobin was evolved into a biocatalyst capable of promoting the cyclization of a diverse range of allyl diazoacetamide substrates into the corresponding bicyclic lactams in high yields and with high enantioselectivity (up to 99% ee).These biocatalytic transformations can be performed in whole cells and could be leveraged to enable the efficient (chemo)enzymatic construction of chiral cyclopropane-γ-lactams as well as βcyclopropyl amines and cyclopropane-fused pyrrolidines, as valuable building blocks and synthons for medicinal chemistry and natural product synthesis.

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