Investigation of the fundamental microscopic processes occurring in organic reactions is essential for optimising both organocatalysts and synthetic strategies. In this study, single-molecule fluorescence microscopy was employed to study the Diels-Alder reaction catalysed by a first-generation MacMillan catalyst, providing direct insights into its kinetic dynamics. This reaction proceeds via a series of reversible processes under equilibrium conditions (S -><- IM1 -><- IM2 -> P, IM1 and IM2: N,O-acetal and iminium ion intermediates, respectively). The individual reaction trajectories of single molecules were directly observed in real-time, and the kinetic transitions between the different states were quantitatively analysed using a hidden Markov model, thereby enabling precise determination of the kinetic rate constants and transition probabilities at the single-molecule level. In particular, the unique structural features of the MacMillan catalyst were probed to reveal how specific interactions stabilise the reaction intermediates and influence their kinetic behaviours. These findings highlight the importance of single-molecule fluorescence microscopy in understanding the fundamental mechanisms of organic reactions and guiding the rational design of more effective catalysts.
