Angew. Chem. Int. Ed. http://doi.org/dc7g (2019)
A biocatalytic asymmetric strategy for the sustainable and step-economic synthesis of these compounds was lacking. Now, Poelarends and colleagues report that EDDS lyase can convert fumaric acid to optically pure N-(3,3-dimethylbutyl)-l-aspartic acid and N-[3-(3-hydroxy-4-methoxyphenyl)propyl]-l-aspartic acid, which can potentially be subsequently converted to neotame and advantame, respectively, by a peptidase-catalysed amide-bond-coupling reaction (pictured, bottom). First, the authors showed that wild-type EDDS lyase allows for the synthesis of N-(3,3-dimethylbutyl)-l-aspartic acid and N-[3-(3-hydroxy-4-methoxyphenyl)propyl]-l-aspartic acid under excess of the amine substrate with high conversion and excellent enantioselectivity, but reaction times were long (7 days). Therefore, to enhance the hydroamination activity of EDDS lyase, protein engineering — based on the structure of EDDS lyase in complex with its natural substrate (S,S)-ethylenediamine-N,N′-disuccinic acid ((S,S)-EDDS) — was applied. Two residues (Asp290 and Tyr320) that were assumed to position the amine substrate in the active site were chosen for mutation. The mutant EDDS(D290M/Y320M) showed a 1,140-fold increase in activity over the wild-type enzyme. To rationalize the effect of the mutations, crystal structures of this enzyme variant were solved and the neotame precursor was docked into the wild-type and mutant crystal structure. Favourable apolar–apolar contacts of the mutated residues with the 3,3-dimethylbutyl moiety lead to a stronger binding and presumably a more productive binding of the amine compound.
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