Proceeding towards studying the acceptor scope of the aldol reaction with ß-fluoro-α-ketoacid donors, we first optimized the individual reactions of FP (1), FKB (2a), and FKV (2b) with formaldehyde (FA, 3a) based on time and buffer pH (Table -S5). For the difficult donor FKV, the promising single mutants of HpcH were re-screened at a higher enzyme loading of (0.3 mol%), which identified HpcH F170V to be optimal. Next, the optimized conditions were used to expand the acceptor scope to a panel of 17 aldehydes (3a-3q) with a diverse spectrum of size and polarity (Figure ). Good conversions of FP were observed with every aldehyde. Furthermore, very hydrophobic aldehydes (4f, 4g, 4n) were used successfully by using a maltose-binding protein fusion (MBP-HpcH) to stabilize the enzyme. Aliphatic and polar heteroaromatic aldehydes were also well-tolerated when using FKB and FKV as donors. In most cases, reactions are not diastereoselective, because a lack of aldehyde-interacting residues leads to attack at both aldehyde faces, giving access to both configurations of the hydroxy stereocenter. All products were characterized by F NMR (Table ) and high-resolution LC-MS (Figure ).