Separately, racemic sodium fluoro(phenyl)pyruvate (FPP, 2e, R = Ph) was prepared by the established glycidic ester route. Formaldehyde (FA) was used initially as a model acceptor to simplify the products to one nascent stereocenter. Next, a collection of HpcH mutants with size reduction of varying degrees at the Trp-19, Phe-170, and Leu-212 residues were prepared in hopes of steric alleviation towards bulky fluoro-donors (Table , Figure ). Assays were conducted by incubation of substrates with 0.1 mol% enzyme and Mg 2+containing HEPES pH 7.5 buffer, followed by H2O2 decarboxylation of aldol adducts and analysis of resulting α-fluoroacids by F NMR (Figure , Table -S4). In the reactions of FP (1) or FKB (2a) with FA, the best conversions (>50%) were observed with wild-type enzyme (wt-HpcH), showing that mutations decreased the intrinsic enzyme activity and were detrimental with respect to sterically non-challenging donors. However, in the case of FKV (2b), wt-HpcH was ineffective (<2%), while single mutations at Phe-170 or Leu-212 gave increased conversion (6-12%). Thus, steric relief becomes important upon introducing a C-3 ethyl group. Furthermore, the necessary incubation time to collect reliable screening data increased corresponding to the C-3 substituent size (4 h for FP, 16 h for FKB, 48 h for larger substrates). We also observed that the consumption of total FKB never exceeded 50%, and showed with a separate experiment that the racemic donor undergoes kinetic resolution (Figure ). The bulkiest donors FKMV (2c) and FKPB (2d) were not tolerated by any enzyme variant even in forcing conditions with glycolaldehyde, an extremely good acceptor. Lastly, FPP (2e) which bears a C-3 aryl group was unstable in water and reacted nonenzymatically with FA. These results show that HpcH or its point mutants can utilize extended ß-fluoro-αketoacids such as FKB and FKV to produce alkyl-branched fluorine chiral centers.