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The potential of L-fuculose-1-phosphate aldolase (FucA) as a catalyst for the asymmetric aldol addition of dihydroxyacetone phosphate (DHAP) to N-protected amino aldehydes has been investigated. First, the reaction was studied in both emulsion systems and conventional dimethylformamide (DMF)/H2O (1:4 v/v) mixtures. At 100 mM DHAP, compared with the reactions in the DMF/H2O (1:4) mixture, the use of emulsion systems led to two- to three-fold improvements in the conversions of the FucA-catalyzed reactions. The N-protected aminopolyols thus obtained were converted to iminocyclitols by reductive amination with Pd/C. This reaction was highly diastereoselective with the exception of the reaction of the aldol adduct formed from (S)-N-Cbz-alaninal, which gave a 55:45 mixture of both epimers. From the stereochemical analysis of the resulting iminocyclitols, it was concluded that the stereoselectivity of the FucA-catalyzed reaction depended upon the structure of the N-Cbz-amino aldehyde acceptor. Whereas the enzymatic aldol reaction with both enantiomers of N-Cbz-alaninal exclusively gave the expected 3R,4R configuration, the stereochemistry at the C-4 position of the major aldol adducts produced in the reactions with N-Cbz-glycinal and N-Cbz-3-aminopropanal was inverted to the 3R,4S configuration. The study of the FucA-catalyzed addition of DHAP to phenylacetaldehyde and benzyloxyacetaldehyde revealed that the 4R product was kinetically favored, but rapidly disappeared in favor of the 4S diastereoisomer. Computational models were generated for the situations before and after CC bond formation in the active site of FucA. Moreover, the lowest-energy conformations of each pair of the resulting epimeric adducts were determined. The data show that the products with a 3R,4S configuration were thermodynamically more stable and, therefore, the major products formed, in agreement with the experimental results.

Water-in-oil (W/O) highly concentrated emulsions (gel emulsions) of water/C14E4/aliphatic hydrocarbon systems were investigated as reaction media for the aldolic condensation of dihydroxyacetone phosphate (DHAP) with acceptor aldehydes such as phenylacetaldehyde (1) and benzyloxyacetaldehyde (2), catalyzed by d-fructose-1,6-bisphosphate aldolase from rabbit muscle (RAMA). Prior to any enzymatic reaction, both the formation and stability of the W/O gel emulsions in the presence of reactants were assessed. It was found that the aldehydes improved greatly the kinetic stability of W/O gel emulsions at 25 °C by decreasing the hydrophile−lipophile balance temperature (THLB) of the water/C14E4/aliphatic hydrocarbon systems. Interestingly, the stability of RAMA in W/O gel emulsions was improved by 7- and 25-fold compared to that in aqueous medium or conventional dimethylformamide/water 1/4 v/v mixture, respectively. It was found that the equilibrium yields and enzymatic activity depended on both the aldehyde partitioning between the continuous and dispersed phases and the water−oil interfacial tension. The highest enzymatic activities were achieved in W/O gel emulsion systems with the lowest water−oil interfacial tension. The equilibrium yield depended on the water−oil interfacial tension for the hydrophobic phenylacetaldehyde, and on the partition coefficient for the hydrophilic benzyloxyacetaldehyde. Optimum equilibrium product yields (65−70%) were achieved at either the lowest water−oil interfacial tension or partition coefficient values.

The potential of dihydroxyacetone phosphate (DHAP)-dependent aldolases to catalyze stereoselective aldol additions is, in many instances, limited by the solubility of the acceptor aldehyde in aqueous/co-solvent mixtures. Herein, we demonstrate the efficiency of emulsion systems as reaction media for the class I fructose-1,6-bisphosphate aldolase (RAMA) and class II recombinant rhamnulose-1-phosphate aldolase from E. coli (RhuA)-catalyzed aldol addition between DHAP and N-benzyloxycarbonyl (N-Cbz) aminoaldehydes. The use of emulsions improved the RAMA-catalyzed aldol conversions by three to tenfold relative to those in conventional DMF/water mixtures. RhuA was more reactive than RAMA towards the N-Cbz aminoaldehydes regardless of the reaction medium. With (S)- or (R)-Cbz-alaninal, RAMA exhibited preference for the R enantiomer, while RhuA had no enantiomeric discrimination. The linear N-Cbz aminopolyols thus obtained were submitted to catalytic intramolecular reductive amination to afford the corresponding iminocyclitols. This reaction was diastereoselective in all cases examined; the face selectivity was controlled by the stereochemistry of the newly formed hydroxyl group originating from the aldehyde. Characterization of the resulting iminocyclitols allowed the assessment of the diastereoselectivity of the enzymatic aldol reactions with respect to the N-protected aminoaldehyde. RAMA formed single diastereoisomers from N-Cbz-glycinal and from both enantiomers of N-Cbz-alaninal, while 14 % of the epimeric product was observed from N-Cbz-3-aminopropanal. Diastereoselectivity from RhuA was lower than that observed from RAMA. Interestingly, a single diastereoisomer was formed from (S)-Cbz-alaninal, whereas only a 34 % diastereomeric excess was observed from its enantiomer (i.e., (R)-Cbz-alaninal).