Synthesis and photophysics of a new family of fluorescent 9-alkyl-substituted xanthenones.

9-Alkyl xanthenones with different aliphatic pendant groups have been easily prepared by means of nucleophilic addition of the corresponding Grignard derivative to a tert-butyldimethylsilyl ether (TBDMS)-protected 3,6-dihydroxy- xanthenone. The photophysical behavior of the new dyes has been explored by using absorption, steady-state-, and time-resolved fluorescence measurements. We determined the equilibrium constants, visible spectral characteristics, fluorescence quantum yield, and decay times. Remarkably, they retain similar fluorescent properties of fluorescein including the characteristic phosphate-mediated excited-state proton-transfer (ESPT) reaction. 6-Hydroxy-9-isopropyl3H- xanthen-3-one (5) was investigated in living cells; it presented a good permeability and efficient accumulation inside the cytosol. For the first time, we reported that the requirement of an aryl group at C-9 is no longer needed and new fluorescent sensors can be therefore easily developed.