Design and characterization of pH-sensitive spirorhodamine 6G probes with aliphatic substituents.

Molecular volume representing a good observable to easily estimate pK eq for a given alkyl R-group as the lactam spirorhodamine substituent. • Spirorhodamine amides with an N-aliphatic substituent display emission quantum yields of the open form between 0.8–1.0. • pH-dependent lactam ring opening reaction displays a strong correlation with the volume of the N-aliphatic substituent. • N-aliphatic substituent has no effect on the electronic properties, such as absorption and emission spectral distribution. • Correlation with the substituent volume can be extended to a kinetic property (k (coloration)). We report synthesis and photophysical studies of spirorhodamines (SRA) with amide N-aliphatic substituents, including measurements of single-crystal X-ray structures, fluorescence emission, and quantum mechanical calculations. Variations in the equilibrium position between closed and open states, a transition that is catalysed under acidic conditions, of several SRAs sharing the same central chromophore (Rhodamine 6G) were measured. For the different substituents in the lactam position, the pH-dependent lactam ring opening reaction displays a strong correlation with the volume of the functional group. The open form shows similar absorption and emission spectrum, fluorescence efficiency and emission lifetime, regardless of the nature of the substituents. The molecular volume of the substituent is a simple and straightforward molecular design feature to control the equilibrium between the "dark" and "bright" states of the probe, without any further changes in the emission properties of the fluorescent form. These results allow rational design of pH-sensitive aliphatic SRAs fluorescent probes. [ABSTRACT FROM AUTHOR]