Chiral cyclopropane rings are key pharmacophores in pharmaceuticals and bioactive natural products, making libraries of these building blocks a valuable resource for drug discovery and development campaigns. Here, we report the development of a chemoenzymatic strategy for the stereoselective assembly and structural diversification of cyclopropyl ketones, a highly versatile yet underexploited class of functionalized cyclopropanes. An engineered variant of sperm whale myoglobin is shown to enable the highly diastereo-and enantioselective construction of these molecules via olefin cyclopropanation in the presence of a diazoketone carbene donor reagent. This biocatalyst offers a remarkably broad substrate scope, catalyzing this reaction with high stereoselectivity across a variety of vinylarene substrates as well as a range of different α-aryl and α-alkyl diazoketone derivatives. Chemical transformation of these enzymatic products enables further diversification of these molecules to yield a collection of structurally diverse cyclopropane-containing scaffolds in enantiopure form, including core motifs found in drugs and natural products as well as novel structures. This work illustrates the power of combining abiological biocatalysis with chemoenzymatic synthesis for generating collections of optically active scaffolds of high value for medicinal chemistry and drug discovery.
The biocatalytic toolbox has recently been expanded to include enzyme-catalyzed carbene transfer reactions not occurring in Nature. Herein, we report the development of a biocatalytic strategy for the synthesis of enantioenriched α-trifluoromethyl amines through an asymmetric N–H carbene insertion reaction catalyzed by engineered variants of cytochrome c 552 from Hydrogenobacter thermophilus . Using a combination of protein and substrate engineering, this metalloprotein scaffold was redesigned to enable the synthesis of chiral α-trifluoromethyl amino esters with up to >99% yield and 95:5 er using benzyl 2-diazotrifluoropropanoate as the carbene donor. When the diazo reagent was varied, the enantioselectivity of the enzyme could be inverted to produce the opposite enantiomers of these products with up to 99.5:0.5 er. This methodology is applicable to a broad range of aryl amine substrates, and it can be leveraged to obtain chemoenzymatic access to enantioenriched β-trifluoromethyl-β-amino alcohols and halides. Computational analyses provide insights into the interplay of protein- and reagent-mediated control on the enantioselectivity of this reaction. This work introduces the first example of a biocatalytic N–H carbenoid insertion with an acceptor–acceptor carbene donor, and it offers a biocatalytic solution for the enantioselective synthesis of α-trifluoromethylated amines as valuable synthons for medicinal chemistry and the synthesis of bioactive molecules.
Hemoproteins have recently emerged as a promising class of biological catalysts for promoting carbene transfer reactions not found in nature. Despite this progress, our mechanistic understanding of the interplay between productive and unproductive pathways in these reactions is limited. Using a combination of spectroscopic, structural, and computational methods, we have investigated the mechanism of a myoglobin-catalyzed cyclopropanation reaction with diazoketones. Our studies shed light into the nature and kinetics of key catalytic steps in this reaction, including formation of an early heme-bound diazo complex intermediate, the rate-determining nature of carbene formation, and the mechanism of the cyclopropanation step. Importantly, our studies reveal the existence of a complex mechanistic manifold behind this hemoprotein-catalyzed cyclopropanation, wherein the cyclopropanation pathway competes with alternative pathways, including formation of an N-bound carbene adduct of the protein heme cofactor, which was isolated and characterized by X-ray crystallography, UV-Vis, and Mössbauer spectroscopy. This species is able to regenerate the active biocatalyst, thus constituting a non-productive, yet non-destructive detour from the main catalytic cycle. These findings improve our understanding of biocatalytic cyclopropanations and the ensuing mechanistic picture is expected to offer a blueprint for both the mechanistic analysis of other hemoprotein-catalyzed carbene transfer reactions and the design and engineering of carbene transferases.
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