Computational mechanistic investigation of the kinetic resolution of α-methyl-phenylacetaldehyde by norcoclaurine synthase.

Norcoclaurine synthase from Thalictrum flavum (TfNCS) demonstrated high stereospecificity and yield in catalyzing the Pictet-Spengler reaction of dopamine with chiral aldehydes, achieving kinetic resolution of aldehydes. However, the mechanism and the factors contributing to the stereoselectivity remain unclear. Herein, by using quantum chemical calculations, the mechanisms of TfNCS-catalyzed reactions of dopamine with both enantiomers of α-methyl-phenylacetaldehyde are studied. The calculations reveal a mechanism mirroring the reaction of natural substrates, for which the deprotonation of the C5-H of the cyclized intermediate is rate-limiting. The calculated overall barriers are 20.1 kcal mol-1 and 21.6 kcal mol-1 for the reactions of (R)- and (S)-α-methyl-phenylacetaldehyde, respectively. The M97 and L72 residues are proposed to be the key residues contributing to the stereospecificity. The obtained detailed information is helpful for designing new variants of TfNCS with extended substrate scope, and also advancing our understanding of TfNCS reactions for potential applications.