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d-Fructose 6-phosphate aldolase (FSA) catalyzes the asymmetric cross-aldol addition of phenylacetaldehyde and hydroxyacetone. We conducted structure-guided saturation mutagenesis of noncatalytic active-site residues to produce new FSA variants, with the goal of widening the substrate scope of the wild-type enzyme toward a range of para- and meta-substituted arylated aldehydes. After a single generation of mutagenesis and selection, enzymes with diverse substrate selectivity scopes were identified. The kinetic parameters and stereoselectivities for a subset of enzyme/substrate combinations were determined for the reactions in both the aldol addition and cleavage reaction directions. The achieved collection of new aldolase enzymes provides new tools for controlled asymmetric synthesis of substituted aldols.

Methods for the synthesis of phenylacetaldehydes (oxidation, one-carbon chain extension) were compared by using the synthesis of 4-methoxyphenylacetaldehyde as a model example. Oxidations of 4-methoxyphenylethanol with activated DMSO (Swern oxidation) or manganese dioxide gave unsatisfactory results; whereas oxidation with 2-iodoxybenzoic acid (IBX) produced 4-methoxyphenylacetaldehyde in reasonable (75%) yield. However, Wittig-type one-carbon chain extension with methoxymethylene-triphenylphosphine followed by hydro­lysis gave an excellent (81% overall) yield of 4-methoxyphenylacetaldehyde from 4-methoxybenzaldehyde (a cheap starting material). This approach was subsequently used to synthesise a set of 10 substituted phenylacetaldehydes in good to excellent yields.