Enzymatic assembly of carbon-carbon bonds via iron-catalysed sp3 C-H functionalization

Abstract

Although abundant in organic molecules, carbon-hydrogen (C-H) bonds are typically considered unreactive and unavailable for chemical manipulation. Recent advances in C-H functionalization technology have begun to transform this logic, while emphasizing the importance of and challenges associated with selective alkylation at a sp³ carbon^1,2. Here we describe iron-based catalysts for the enantio-, regio- and chemoselective intermolecular alkylation of sp³ C-H bonds through carbene C-H insertion. The catalysts, derived from a cytochrome P450 enzyme in which the native cysteine axial ligand has been substituted for serine (cytochrome P411), are fully genetically encoded and produced in bacteria, where they can be tuned by directed evolution for activity and selectivity. That these proteins activate iron, the most abundant transition metal, to perform this chemistry provides a desirable alternative to noble-metal catalysts, which have dominated the field of C-H functionalization^1,2. The laboratory-evolved enzymes functionalize diverse substrates containing benzylic, allylic or α-amino C-H bonds with high turnover and excellent selectivity. Furthermore, they have enabled the development of concise routes to several natural products. The use of the native iron-haem cofactor of these enzymes to mediate sp³ C-H alkylation suggests that diverse haem proteins could serve as potential catalysts for this abiological transformation, and will facilitate the development of new enzymatic C-H functionalization reactions for applications in chemistry and synthetic biology.

Figure 1 |. Enzymatic C–H functionalization systems.

a, Methylation catalysed by cobalamin-dependent radical SAM enzymes, as illustrated by Fom3 in fosfomycin biosynthesis. b, Oxygenation catalysed by cytochrome P450 monooxygenase (top) and envisioned alkylation reaction achieved under haem protein catalysis (bottom). Structural illustrations are adapted from Protein Data Bank (PDB) ID code 5UL4 (radical SAM enzyme) and PDB 2IJ2 (cytochrome P450_BM3). Ad, adenosyl; Cys, cysteine; R, organic group; X, amino acid.

Figure 2 |. Haem protein-catalysed sp³ C–H alkylation.

a, Select subset of haem proteins tested for promiscuous C–H alkylation activity. Structural illustrations are of representative superfamily members with the haem cofactor shown as red sticks: cytochrome P450_BM3 (PDB 2IJ2), sperm whale myoglobin (PDB 1A6K), and Rma cytochrome c (PDB 3CP5). TTN, total turnover number; n. d., not detected; WT, wild type; Mb, sperm whale myoglobin, HGG, Hell’s Gate globin; cyt c, cytochrome c; Hth, Hydrogenobacter thermophilus. b, Directed evolution of a cytochrome P411 for enantioselective C–H alkylation (reaction shown in (a)). Bars represent average TTNs from reactions performed in quadruplicate; each TTN data point is shown as a grey dot. Enantioselectivity results are represented by green diamonds. Unless otherwise indicated, reaction conditions were haem protein in E. coli whole cells (OD₆₀₀ = 30, (a)) or in clarified E. coli lysate (b), 10 mM substrate 1a, 10 mM ethyl diazoacetate, 5 vol% EtOH in M9-N buffer at room temperature under anaerobic conditions for 18 hours. Reactions performed with lysate contain 1 mM Na₂S₂O₄. TTN is defined as the amount of indicated product divided by total haem protein as measured by the hemochrome assay. See Supplementary Information for the complete list of haem proteins tested and detailed experimental procedures.

Figure 3 |. Substrate scope for benzylic C–H alkylation with P411-CHF.

a, Experiments were performed using E. coli expressing cytochrome P411-CHF (OD₆₀₀ = 30) with 10 mM substrate 1a–1l and 10 mM ethyl diazoacetate at room temperature (RT) under anaerobic conditions for 18 hours; each reported TTN is the average of quadruplicate reactions. See Supplementary Fig. S12 for the full list of alkane substrates. ^†Si–H insertion product 3h’ is also observed (Supplementary Fig. S7). b, Reaction selectivity for carbene C–H insertion or cyclopropanation can be controlled by the protein scaffold. Experiments were performed as in (a) using the indicated P411 variant. ^ξd.r. is given as cis : trans; e.r. was not determined.

Figure 4 |. Application of P411 enzymes for sp³ C–H alkylation.

a, Allylic and propargylic C–H alkylation. Unless otherwise indicated, experiments were performed using E. coli expressing cytochrome P411-CHF with 10 mM substrate 4a–4e and 10 mM ethyl diazoacetate; each reported TTN is the average of quadruplicate reactions. ^#TTN was calculated based on isolated yield from a reaction performed at 0.25 mmol scale. ^†Cyclopropene product was also observed (Supplementary Fig. S8). *Hydrogenation, followed by hydrolysis. b, Enzymatic alkylation of substrates containing α-amino C–H bonds. Unless otherwise indicated, experiments were performed at 0.5 mmol scale using E. coli expressing cytochrome P411-CHF with substrates 7a–7f and ethyl diazoacetate; TTNs were calculated based on isolated yields of products shown. ^ξIsolated in 9 : 1 r.r. for 8f : 8f’. eReduction, halogen exchange, and Suzuki-Miyaura cross-coupling. c, Enzymatic C–H alkylation with alternative diazo reagents. Unless otherwise indicated, reactions were performed at 0.5 mmol scale using E. coli expressing cytochrome P411-CHF with coupling partner 1a or 7a and diazo compounds 9a–9d; TTNs were calculated based on isolated yields of products shown. ^⏊Variant P411-IY T327I was used. See Supplementary Information for the complete list substrates (Fig. S12 and Fig. S13), information about enzyme variants, and full experimental details.