Enantioselective, intermolecular benzylic C-H amination catalysed by an engineered iron-haem enzyme

Abstract

C-H bonds are ubiquitous structural units of organic molecules. Although 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-haem cofactor.

Figure 1. Intermolecular C–H amination, a simplifying transformation for chiral amine synthesis

Intermolecular C–H amination enables direct and convergent functionalization in which a simple alkane and a nitrogen atom source are brought together in a single step. In principle, any C–H bond in the substrate is a potential site of functionalization.

Figure 2. Proposed mechanism of cytochrome P411-catalyzed intermolecular C–H amination

Reaction of the aminating reagent, tosyl azide, with the ferrous porphyrin (1) generates an enzyme-bound iron nitrenoid intermediate (2). This nitrenoid then inserts into a C–H bond in the alkane, delivering a benzylic amine product. The nitrogen atoms in a plane represent the enzyme’s heme cofactor. Ts = 4-toluenesulfonyl; Ser = serine.

Figure 3. Evolution of a cytochrome P411 catalyst for enantioselective C–H amination on increasingly challenging substrates

Directed evolution, via sequential rounds of site-saturation mutagenesis and screening, improved both the conversion and enantioselectivity of P411-catalyzed C–H amination. The initial variant P-4 shows significant activity only on the electronically-activated 4-ethylanisole (3); evolved variants display activity on inherently less activated substrates. Reactions were performed using whole E. coli cells overexpressing the P411 variant, resuspended to OD₆₀₀ = 30, with 5 mM alkane and 5 mM tosyl azide, under anaerobic conditions. Results are the average of experiments performed with duplicate cell cultures, each used to perform duplicate chemical reactions (four reactions total). Bars represent yield; numbers above bars represent enantiomeric excess (ee); both are color-coded to match the substrate (blue = 4-ethylanisole; red = 4-ethyltoluene; purple = ethylbenzene). Error bars correspond to one standard deviation. P-4 gives predominantly the S enantiomer in the amination of 4-ethylanisole (3); all other variant/substrate combinations give predominantly the R enantiomer.

Figure 4. Kinetic isotope effect and enzyme structural studies

a, The kinetic isotope effect in enzymatic C–H amination was determined from independent in vitro rate experiments. b, Active site view of the P-4 A82L A78V F263L crystal structure, showing the heme in white and the iron atom in orange. Key active site residues are labeled and shown as sticks in blue. Residue S400 ligates the iron center; mutations at positions 78, 82, 263, and 267 enhance C–H amination activity and/or selectivity. All beneficial mutations identified in this study lie in the P411 active site on the distal face of the heme.