Engineering the Donor Selectivity of D‐Fructose‐6‐Phosphate Aldolase for Biocatalytic Asymmetric Cross‐Aldol Additions of Glycolaldehyde

Abstract

D-Fructose-6-phosphate aldolase (FSA) is a unique catalyst for asymmetric cross-aldol additions of glycolaldehyde. A combination of a structure-guided approach of saturation mutagenesis, site-directed mutagenesis, and computational modeling was applied to construct a set of FSA variants that improved the catalytic efficiency towards glycolaldehyde dimerization up to 1800-fold. A combination of mutations in positions L107, A129, and A165 provided a toolbox of FSA variants that expand the synthetic possibilities towards the preparation of aldose-like carbohydrate compounds. The new FSA variants were applied as highly efficient catalysts for cross-aldol additions of glycolaldehyde to N-carbobenzyloxyaminoaldehydes to furnish between 80-98 % aldol adduct under optimized reaction conditions. Donor competition experiments showed high selectivity for glycolaldehyde relative to dihydroxyacetone or hydroxyacetone. These results demonstrate the exceptional malleability of the active site in FSA, which can be remodeled to accept a wide spectrum of donor and acceptor substrates with high efficiency and selectivity.

Asymmetric assembly of aldose carbohydrates from formaldehyde and glycolaldehyde by tandem biocatalytic aldol reactions

“…Notably, inversion of the stereochemistry at C3 (that defined during the approach of the acceptor aldehyde to the FSA-donor intermediate) in the first addition of 1a would afford L-glucose, a non-caloric sweetener of complex preparation by alternative procedures 32 . Such C3 inversion appeared feasible, because the stereochemical approach of the aldehyde to the aldolase-donor complex is often compromised when dealing with non-natural substrates 26,33 and it has been successfully inverted by engineering several aldolases 34 . In a preliminary exploration, we observed that dimerization of 1a with FSA A165G provided an ∼4:1 mixture of D-threose (2a) and L-erythrose (4a), as inferred from chromatographic and NMR analysis (Supplementary Table 28 and Supplementary Fig.…”

“…28a-c). Attempts to engineer the most active FSA variants 26 to form L-erythrose (for example, library FSA L107Y/A129G/A165G/S166X ) were unsuccessful. In a second step, a set of FSA variants was assayed for selective aldol addition of 1a to L-erythrose (4a) (Supplementary Table 28).…”

“…In a, the two reactive carbon atoms are shown in a pre-reactive disposition at a distance of 3.3 Å and the D-threose carbonyl oxygen is forming a hydrogen bond with an essential water molecule, which is also hydrogen-bonded to residues Q59, T109 and Y131. It is postulated that this water molecule mediates proton transfer from the phenol group of residue Y131 to the acceptor aldehyde group 26 , once the C-C bond is formed, to generate the corresponding iminium cation (b). Subsequent nucleophilic attack of the water on the iminium-activated carbon atom, promoted by proton abstraction by the Y131 phenolate, would yield the hemiaminal precursor of D-idose (c).…”

“…Subsequent nucleophilic attack of the water on the iminium-activated carbon atom, promoted by proton abstraction by the Y131 phenolate, would yield the hemiaminal precursor of D-idose (c). All the structures shown were modelled and energy minimized using a previously described protocol 26 . The Schrödinger Suite 2014-3 was used to carry out all calculations 31 .…”

“…The substrate specificity of FSA has been tailored in our laboratory by a structure-guided programme of active-site engineering to expand its scope of application 25,26 . Mutations at positions L107 and particularly A129 were found to be crucial for its donor activity and selectivity, whereas the A165G mutation enhances the reactivity of poorly reactive aldehydes [25][26][27] . These studies have provided a toolbox of FSA variants with different synthetic capabilities as a function of the acceptor and donor substrates.…”