Macrocycle Conformational Flexibility as a Key to Enantioselective Recognition

The differentiation and assignment of absolute configurations of enantiomers in mixtures remain a significant challenge for both experimental and computational methods. In recent years, diffusion-NMR (DOSY) experiments using chiral resolving agents have emerged as a promising approach for enantiodiscrimination. While many resolving agents demonstrate strong enantioselective capabilities, limitations remain in reliably assigning absolute configurations and elucidating the molecular basis of chiral recognition. In this study, we combine NMR experiments and computational approaches to investigate the differentiation of mandelic Acid (MA), α-methylbenzylamine, methyl mandelate, Mosher's acid, 2-butanol, and camphor enantiomers by a chiral macrocycle (MAC) assembled from N,N'-bis-(6-acylamino-2-pyridinyl)-isophthalamide units and BINOL derivatives. NMR measurements reveal a stereopreference of MAC for the (S)-enantiomer, as evidenced by its lower diffusion coefficient compared to the (R)-enantiomer. Our computational modeling indicates that this enantioselectivity arises when the MAC adopts a closed conformation, rather than the expected open form. Temperature-dependent ¹H and selective ROESY measurements further support an equilibrium between open and closed MAC conformers in solution, as also suggested by extensive conformational searches. Our combined experimental and computational approach demonstrates that only the closed MAC-enantiomer complexation simultaneously accounts for the observed diffusion-NMR and shielding differences and can rationalize the observed enantiodifferentiation. The results show that the macrocyclic receptor exhibits conformational flexibility that must be considered in chiral recognition events. Therefore, these insights advance our understanding of enantioselective interactions and provide a framework for the future design of chiral resolving agents for differentiation by diffusion-NMR and computational methods.