Chyan_2017_J.Org.Chem_82_4297

Fluorogenic probes are invaluable tools for spatiotemporal investigations within live cells. In common fluorogenic probes, the intrinsic fluorescence of a small-molecule fluorophore is masked by esterification until entry into a cell, where endogenous esterases catalyze the hydrolysis of the masking groups, generating fluorescence. The susceptibility of masking groups to spontaneous hydrolysis is a major limitation of these probes. Previous attempts to address this problem have incorporated auto-immolative linkers at the cost of atom economy and synthetic adversity. Here, we report on a linker-free strategy that employs adventitious electronic and steric interactions in easy-to-synthesize probes. We find that X...C horizontal lineO n-->pi* interactions and acyl group size are optimized in 2',7'-dichlorofluorescein diisobutyrate. This probe is relatively stable to spontaneous hydrolysis but is a highly reactive substrate for esterases both in vitro and in cellulo, yielding a bright, photostable fluorophore with utility in biomolecular imaging.