Fig. 3: Assembly and evaluation of S-Functionalized Cysteine designer enzymes in the enantioselective Friedel-Crafts alkylation reaction.

a Overview of the homo-dimeric structure of SFC_V15pAPhC (PDB: 8YRF) and enhanced view of the hydrophobic pocket showing selected positions for pAPhC catalytic residue incorporation. b Asymmetric Friedel-Crafts alkylation of enal 1a and indole 2a to form alkylation product (R)−3a by SFC_V15pAPhC and (S)−3a by LmrR_V15pAF. c Reaction yields (blue) and e.e. (yellow) values of product 3a with different SFC_pAPhC variants as well as negative controls. Reaction conditions: Enzyme varaints (5.0 mol%), 5 mM 1a and 1 mM 2a in 50 mM NaH₂PO₄, 150 mM NaCl (pH 6.5) buffer with DMF (4.0% v/v). Reactions conducted for 10 h at 4 °C and analyzed by normal-phase chiral column with e.e. and yield, unless noted otherwise. Yields are determined by HPLC analysis using a calibration curve of pure product 3a. Error bars represent ± standard deviation of the mean over 3 independent replicates. Absolute configuration of 3a assigned by comparison of order of elution on chiral normal-phase HPLC with enantioenriched reference compound and the literature^57,67. Source data are provided as a Source Data file.