C–H bonds are ubiquitous structural units of organic molecules; while these bonds are generally considered to be chemically inert, the recent emergence of methods for C–H functionalization promises to transform the way synthetic chemistry is performed. The intermolecular amination of C–H bonds represents a particularly desirable and challenging transformation for which no efficient, highly selective, and renewable catalysts exist. Here we report the directed evolution of an iron-containing enzymatic catalyst, based on a cytochrome P450 monooxygenase, for the highly enantioselective, intermolecular amination of benzylic C–H bonds. The biocatalyst is capable of up to 1,300 turnovers, exhibits excellent enantioselectivities, and provides access to valuable benzylic amines. Iron complexes are generally poor catalysts for C–H amination: in this catalyst, the enzyme’s protein framework confers activity on an otherwise unreactive iron-heme cofactor.
One of the greatest challenges in
protein design is creating new
enzymes, something evolution does all the time, starting from existing
ones. Borrowing from nature’s evolutionary strategy, we have
engineered a bacterial cytochrome P450 to catalyze highly enantioselective
intermolecular aziridination, a synthetically useful reaction that
has no natural biological counterpart. The new enzyme is fully genetically
encoded, functions in vitro or in whole cells, and
can be optimized rapidly to exhibit high enantioselectivity (up to
99% ee) and productivity (up to 1,000 catalytic turnovers) for intermolecular
aziridination, demonstrated here with tosyl azide and substituted
styrenes. This new aziridination activity highlights the remarkable
ability of a natural enzyme to adapt and take on new functions. Once
discovered in an evolvable enzyme, this non-natural activity was improved
and its selectivity tuned through an evolutionary process of accumulating
beneficial mutations.
Small carbocycles are structurally rigid and possess high intrinsic energy due to their significant ring strain. These unique features lead to broad applications, but also create challenges for their construction. We report the discovery and engineering of hemeproteins that catalyze the formation of chiral bicyclobutanes, one of the most strained four-membered systems, via successive carbene addition to unsaturated carbon–carbon bonds. Enzymes that produce cyclopropenes, putative intermediates to the bicyclobutanes, were also identified. These genetically-encoded proteins are readily optimized by directed evolution, function in Escherichia coli, and act on structurally diverse substrates with high efficiency and selectivity, providing an effective route to many chiral strained structures. This biotransformation is easily performed on preparative scale and the resulting strained carbocycles can be derivatized, opening myriad potential applications.
The introduction of fluoroalkyl groups into organic compounds can significantly alter pharmacological characteristics. One enabling but underexplored approach for the installation of fluoroalkyl groups is selective C(sp 3)-H functionalization due to the ubiquity of C-H bonds in organic molecules. We have engineered heme enzymes Experimental details, and spectral data for all new compounds. (PDF) X-ray crystallographic data for 3a (CIF) X-ray crystallographic data for 5e (CIF)
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