Directed evolution of nonheme iron enzymes to access abiological radical-relay C(sp3)-H azidation

Abstract

We report the reprogramming of nonheme iron enzymes to catalyze an abiological C(sp³)‒H azidation reaction through iron-catalyzed radical relay. This biocatalytic transformation uses amidyl radicals as hydrogen atom abstractors and Fe(III)‒N₃ intermediates as radical trapping agents. We established a high-throughput screening platform based on click chemistry for rapid evolution of the catalytic performance of identified enzymes. The final optimized variants deliver a range of azidation products with up to 10,600 total turnovers and 93% enantiomeric excess. Given the prevalence of radical relay reactions in organic synthesis and the diversity of nonheme iron enzymes, we envision that this discovery will stimulate future development of metalloenzyme catalysts for synthetically useful transformations unexplored by natural evolution.

Figure 1.

(A) Radical relay C—H functionalization involves an initial hydrogen atom transfer (HAT) mediated by a het2roatom-centered radical (X•) followed by the trapping of the carbon-centered radical with a redox-active metal complex. (B) Mechanism employed by natural non-heme iron enzymes for C(sp³)—H halogenation/azidation. (C) Integration of radical relay chemistry into non-heme iron enzymes enables unnatural C—H functionalization reactions.

Figure 2.

(A) Protein residues selected for mutagenesis (pink: loop residues surounded the active site (N191, F216, Q255, F359), green: residues on the C—terminal α-helix (K361, L367, N363), blue: residues on the β barrel of the C-terminal domain (V189, S230, P243, N245, Q269, Q334, F336, R353) (PDB: 1T47). (B) A high-throughput screening platform for detection of enzymatic azidation products. (C) Representative variants identified during the directed evolution of Sav HppD. Experiments were performed at analytical scale using suspensions of E. coli expressing Sav HppD variants (OD₆₀₀ = 10), 10 mM substrate 1NF, 25 mM NaN₃, 2.5 mM Fe²⁺ in KPi buffer (pH 7.4) at room temperature under anaerobic conditions for 24 hours (Table S2).

Figure 3.

(A) Substrate scope of Sav HppD Az1 and Sav HppD Az2. Experiments were performed at analytical scale using suspensions of E. coli expressing Sav HppD variants in KPi buffer (pH 7.4) at room temperature under anaerobic conditions for 24 hours (detailed conditions see Table S3). The absolute configuration of enzymatically synthesized azidation product 1 was determined to be S via X-ray crystallography. The absolute configurations of all other azidation products were inferred by analogy. (B) Preparative scale synthesis and absolute configuration determination. (C) One-pot chemoenzymatic synthesis by in situ derivatization of enzymatic azidation products via CuAAC. Detailed conditions see section IX of the SI.

Figure 4.

(A) Left: Mössbauer spectrum of Sav HppD Az1•Fe(II) complex (top, black) and the spectroscopic changes upon azide addition (bottom, black). The upward and the downward absorption peaks represent the disappeared and the appeared spectral components after the addition of azide. The colored solid lines represent spectral simulations (see SI for detailed discussion); Right: EPR spectrum of Sav HppD Az1•Fe(II)•N₃ complex after incubation with 18NF for 60 min (black) and the spectral simulation (red). (B) Left: Optical absorption spectra of Sav HppD Az1•Fe(II)•N₃ complex with 18NF (black) and after incubation with 18NF for 60 min (red). The inset shows the reaction scheme; Right: The time dependent change the 505 nm feature. (C) Active site arrangement of Az2 variant with 1NF substrate bound in a near-attack conformation for N—F activation characterized from MD simulations (see SI for details, Fig. S13). (D) Reaction mechanism obtained from DFT calculations employing a truncated active-site model build from MD simulations (see Fig. S18 for details) (energies in kcal/mol, distances in Å, and angles in deg.).