Fig. 1: S_NAr reactions and directed evolution of an enantioselective S_NAr enzyme.

a, S_NAr reactions involve the coupling of aromatic electrophiles with diverse nucleophilic coupling partners. b, Chemical scheme showing the target S_NAr reaction between ethyl 2-cyanopropionate (1) and 2,4-dinitrochlorobenzene (2), generating product 3 containing an all-carbon quaternary carbon centre. The S_NAr enzymes developed preferentially produce the (R)-enantiomer of 3 (Supplementary Information). c, Bar chart showing reaction conversion (solid bars) and selectivity (patterned bars) achieved by S_NArase variants along the evolutionary trajectory. Reaction conditions: 1 (25 mM), 2 (2.5 mM), S_NAr variant (75 μM) in PBS pH 8.0 with 10% v/v DMSO as a co-solvent, 16 h at 30 °C. d, Michaelis–Menten kinetic analysis of S_NAr1.0 to S_NAr1.3 show a 160-fold improvement in k_obs following evolution (0.0040 ± 0.0002 min⁻¹ and 0.65 ± 0.01 min⁻¹ for S_NAr1.0 and S_NAr1.3, respectively; Extended Data Fig. 2). Assays were performed at a fixed concentration of 2 (2.5 mM) and varying concentrations of 1 (3.5–75.0 mM). e, Structure showing the six amino acid positions mutated in S_NAr1.3 mapped onto the structure of BH32.7 (PDB: 7O1D). Mutations are represented as spheres at the C_α and coloured according to their order of introduction, corresponding to the variants in Fig. 2b. Data points shown are averages of triplicate measurements, with error bars representing standard deviation. See Supplementary Data for source data.