Synthesis and Characterization of Photoswitchable Covalent Ligands for the β 2 ‐Adrenoceptor

Abstract The β 2 ‐adrenergic receptor (β 2 AR) is a critical target for the treatment of airway diseases such as asthma or chronic obstructive pulmonary disease (COPD). To better understand its mechanism of action and study its dynamics, photoswitchable ligands provide a distinct advantage due to their ability to be activated with high spatiotemporal control. In this study, we developed a series of molecular tools featuring different combinations of pharmacophores, covalent warheads, and linker lengths. These compounds were characterized using highly specific assay protocols to evaluate their covalent binding capabilities, with the goal of identifying optimal covalently‐bound photoswitches. Among these, the covalently‐bound photoswitchable receptor agonist 6 exhibited a significant functional switch. To gain deeper insight into the binding thermodynamics and kinetics of this molecular tool, as well as the molecular determinants involved, we performed metadynamics (MetaD) simulations and analyzed their results using a Markov State Model (MSM) obtained by applying the Maximum Caliber (MaxCal) principle to the MetaD‐derived free energies. These studies suggest that photoswitching of compound 6 occurs within the binding pocket of the β 2 AR. Consequently, compound 6 holds promise as a valuable tool for investigating β 2 AR activation kinetics and dynamics with high accuracy, thereby facilitating high‐resolution biophysical studies.