Allosteric Regulation of Photophysics and Binding in Oxazine‐macrocycle Complexes at Single‐molecule Resolution

Dye-macrocycle complexes have become invaluable tools for various applications. The mechanisms of complex formation and photophysical changes can be complex and difficult to characterize. This is due to limited information available from bulk experiments on heterogeneity, complex stoichiometry, kinetic rates, and a distinction of photophysical effects caused by complex formation or collisional processes. Recent advances in the use of single-molecule spectroscopy have begun to bridge this gap. Here, we investigate the interactions between the oxazine fluorophore ATTO655 and two macrocyclic hosts, cucurbit[8]uril (CB8) and p-sulfonatocalix[4]arene (sCX4). Although ATTO655-CB8 shows the classical picture of an inclusion complex, the interactions of ATTO655 with sCX4 are largely unprecedented. ATTO655 forms dim exclusion complexes via external binding to the aromatic moeities of sCX4. Here, the upper portal and hydrophobic cavity of sCX4 remain accessible for known guests such as the neurotransmitter choline. In this ternary complex, choline reduces the binding affinity and prevents the formation of photoinduced dark states observed in single-molecule traces, which presents a form of allosteric regulation of binding and photophysics. Our study is a showcase for the power of single-molecule spectroscopy to unravel fundamental mechanisms of host-guest systems, which can facilitate their use in advanced bioimaging and sensing applications.