Benchtop NMR-Based\nIn-Line Analysis of Diastereoselective\nEnzymatic α‑Amino Acid Synthesis: Quantification and\nValidation

This study investigates the application of a commercial\nlow-field\nbenchtop NMR for real-time monitoring of enzymatically catalyzed reactions,\nfocusing on the diastereoselectivity of the threonine aldolase-catalyzed\nstereoselective aldol reaction between glycine and benzaldehyde. Despite\nthe signal overlap inherent in the weak electromagnetic field of the\nbenchtop NMR system, a complemental hard modeling (CHM) approach effectively\ndifferentiates between diastereomers, enabling the determination of\nenzymatic diastereoselectivity and the transition from kinetic to\nthermodynamic control. In particular, the achievement of thermodynamic\nequilibrium in the enzymatic aldol reaction is observed for the first\ntime using in-line methods, occurring at 30% benzaldehyde conversion\nafter 2 h. In-line NMR analysis reveals a diastereomeric excess of\n37:63 (<i>erythro</i>/<i>threo</i>), which closely\naligns with off-line measurements via GC and HPLC (36:64). This determination\nof diastereomers using CHM enhances the efficiency of in-line monitoring\nin enzymatic reactions, promising significant advancements in pharmaceutical\nprocess development. Overall, the study underscores the utility of\nbenchtop NMR systems for in-line analysis of enzymatic reactions,\noffering insights into reaction mechanisms, selectivity, and equilibrium\ndynamics, thereby facilitating more efficient process optimization\nin the area of fine chemicals.