Enzymatic Nitrogen Insertion into Unactivated C–H Bonds
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The paper engineers cytochrome P450 enzymes, termed "nitrene transferases", that can selectively introduce nitrogen into unactivated aliphatic C-H bonds. This represents the first demonstration of enzymes that can directly aminate or amidate saturated hydrocarbons at specific positions with high stereoselectivity.
The enzymes were evolved starting from variants that showed very low initial activity towards the model substrates methylcyclohexane and ethylcyclohexane. After multiple rounds of directed evolution involving site-saturation mutagenesis and error-prone PCR, variants were obtained that could convert these substrates into chiral 1,2- or 1,3-substituted amines or amides with high site-, diastereo-, and enantioselectivity.
The engineered enzymes showed broad substrate tolerance, functionalizing diverse cycloalkanes and some linear alkanes. Mechanistic studies suggest the reactions proceed via hydrogen atom transfer to generate a substrate radical, followed by radical recombination with the nitrenoid species. Molecular dynamics simulations indicate a hydrophobic binding pocket provides favorable dispersion interactions with the hydrocarbon substrates.
Overall, this work establishes cytochrome P450 variants as promising biocatalysts for direct introduction of nitrogen functionality into unactivated carbon centers. It also provides a new logic for enzymatic C-H functionalization beyond the native oxygenation reactions, expanding the scope for biocatalytic synthesis of aminated and amidated target compounds.
https://pubs.acs.org/doi/full/10.1021/jacs.2c08285
