Investigating Interaction Dynamics of an Enantioselective Peptide‐Catalyzed Acylation Reaction

Modern nuclear magnetic resonance (NMR) methods like carbon relaxation dispersion in the rotating frame (13C‐R1ρ) and proton chemical exchange saturation transfer (1H‐CEST) are key methods to investigate molecular recognition in biomacromolecules and to detect molecular motions on the µs to s timescale, revealing transient conformational states. Changes in kinetics can be linked to binding, folding, or catalytic events. Here, we investigated whether these methods allow detection of changes in the dynamics of a small, highly selective peptide catalyst during recognition of its enantiomeric substrates. The flexible tetrapeptide Boc‐l‐(π‐Me)‐His‐AGly‐l‐Cha‐l‐Phe‐OMe, used for the monoacetylation of cycloalkane‐diols, is probed at natural abundance using 13C‐R1ρ and 1H‐CEST. Indeed, we detected differences in dynamics of the peptide upon interaction with the diol. Importantly, these differ depending on the enantiomer of the substrate used. These enantiospecific influences of the substrates on the dynamics of the peptide are rationalized using computational techniques. We find that even though one enantiomer reacts faster, as confirmed by reaction monitoring, the other is more tightly bound in DCM (as confirmed by 1H‐saturation transfer difference (STD) measurements). These findings provide insights into the recognition of the substrates and explain the selectivity differences observed between the solvents toluene and DCM.