The HpcH/HpaI family of type II pyruvate aldolases (PF03328) has recently emerged as a source of useful biocatalysts. In contrast to the type I mechanism where a catalytic lysine residue must form a Schiff base with the donor, the type II mechanism requires only a divalent cation such as Mg 2+ to activate the donor as an enolate. We previously found that various homologs of this family can tolerate the aldol addition of fluoropyruvate to polar aldehydes, affording value-added products like fluorinated sugar acids with 0.01-0.1 mol% enzyme catalyst. Seeking to probe the limits of substrate scope in both donor and acceptor, we chose HpcH from Escherichia coli C for this study due to its excellent heterologous expression yield. We tested a panel of ß-fluoro-α-ketoacids bearing different C-3 substitutions to investigate as new aldolase donors (Figure , Figure ). Along with the commercially available sodium fluoropyruvate (FP, 1, R = H), we also screened ß-fluoro-α-ketobutyrate (FKB, 2a, R = Me), ß-fluoro-α-ketovalerate (FKV, 2b, R = Et), ß-fluoro-α-keto-γ-methylvalerate (FKMV, 2c, R = i-Pr), and ß-fluoro-α-keto-γ-phenylbutyrate (FKPB, 2d, R = Bn). Donors 2b-2d were prepared from the corresponding α-ketoesters through a three-step sequence of silylation with TMS-Cl, fluorination with Selectfluor reagent, and saponification with LiOH. The substrates were purified as free acids before neutralization to sodium salts for enzymatic use.