Kajino_2025_Bioorg.Med.Chem.Lett_130_130422

Rhodamines are useful fluorescent molecules for activity-based sensing. One powerful design strategy is to exploit changes in the open/closed equilibrium of rhodamine amides. In the context of amide derivatives of rhodamine B, a prototypical member of the rhodamine family, this strategy has been especially useful in the development of activity-based indicators for protons and metal ions. This is because at neutral pH, the closed form of rhodamine B amides dominates, making this otherwise bright and fluorescent dye non-fluorescent. At acidic pH, the equilibrium favors the open form. Despite a wealth of methods to trigger Lewis acid-mediated fluorogenicity of rhodamine B amides, there are far fewer ways to shift the open-close equilibrium to favor the open form at neutral pH. Here, we demonstrate that a simple substitution substantially shifts the native rhodamine B amide equilibrium to favor the open, fluorescent form at neutral pH. Rhodamine B derivatives with an N-(2'-carboxy)-phenyl substitution (an ortho anthranilic acid, RhoB-AA) show strong absorbance and emission at pH 7.2, up to -1500x greater than their unsubstituted N-phenyl derivatives (RhoB-Ph). The fluorescence of RhoB-AA at neutral pH is dependent on the free carboxylic acid. Esters of RhoB-AA are also -1500x less fluorescent than RhoB-AA and have optical properties nearly identical to the unsubstituted RhoB-Ph. Esters of RhoB-AA can be converted by esterases to the fully fluorescent RhoB-AA, demonstrating that the simple ortho anthranilic acid substitution is a powerful strategy for activity-based sensing with rhodamine amides at neutral pH.