A novel carbazolyl GFP chromophore analogue: synthesis strategy and acidic pH-activatable lysosomal probe for tracing endogenous viscosity changes

Abstract

Intracellular viscosity changes of the internal microenvironment lead to many diseases, including cancer, inflammation, and neurodegenerative diseases. A novel, acidic pH-activatable carbazolyl GFP chromophore analogue, Lys-CzFP, was designed for tracing lysosomal viscosity changes. The developed synthesis is an efficient, novel, one-pot procedure. Lys-CzFP serves as a fluorescent molecular rotor and the internal carbazole and benzazole moieties of Lys-CzFP serve as rotators, which can rotate around the single C–C bond in the π-conjugated bridge. Lys-CzFP showed a long emission wavelength at 560 nm and a large Stokes shift of 78 nm. The fluorescence intensity of Lys-CzFP exhibited a significant enhancement with increasing viscosity. The fluorescence intensity increased by 98-fold within 5 s from 1.0 cP to 1412 cP, while the fluorescence quantum yield (ϕ_F) increased from 0.003 to 0.253 and the fluorescence lifetime increased from 0.25 ns to 1.12 ns, respectively. Furthermore, interference experiments indicated that Lys-CzFP could particularly respond to viscosity. Additionally, Lys-CzFP had excellent biocompatibility, low cytotoxicity, and lysosomal localization. Finally, Lys-CzFP was successfully applied to tracing endogenous viscosity changes in living cells.