Substituent‐Dependent Spirolactone Stabilization in Tetramethylamino Rhodamines: From Non‐Emissive to pH‐Responsive Fluorogenic Probes

Herein, we present the synthesis of stimuli‐responsive rhodamine dyes by functionalizing tetramethylamino rhodamine at the 6‐position of the lactone moiety with different aliphatic and aromatic amines, resulting in shifting of the equilibrium toward the spirolactone form. Fluorescence studies showed that at neutral pH, the functionalization stabilized the dyes in a non‐emissive spirolactone state. However, acid‐induced ring opening displayed a turn‐on emission for aliphatic amine‐substituted derivatives, while aromatic amine‐substituted molecules remained non‐emissive which differs from the behaviour commonly observed for rhodamine systems. The influence of substitution on the spirolactone–zwitterion equilibrium as well as photophysical properties and pH regulations was explained with the help of single‐crystal X‐ray diffraction and density functional theory. The pH‐regulated emission of one compound was demonstrated to image intracellular acidic environments, signifying the potential of these molecules as a tracker for the alteration of pH inside cells, commonly observed in diseased cells.