Tuning the pKa of Carboxyfluorescein with Arginine-Rich Cell-Penetrating Peptides for Intracellular pH Imaging

5-Carboxylfluorescein (FAM) is a conventional pH-responsive fluorophore widely used in fluorescence labeling and imaging. Because of its nonfluorescent structure under acidic conditions, FAM has long been limited to pH determination in a neutral–basic environment. Here, we modified the optical properties of FAM with cationic arginine-rich cell-penetrating peptides (CPPs), tuning the p K a value of FAM to adapt well to pH measurement under diverse pH conditions. With increasing length of polyarginine, the p K a value of FAM was tuned from 6.20 ± 0.06 to 5.17 ± 0.05. The key mechanism for p K a variations was attributed to intramolecular electrostatic attraction and the positive charge of cationic CPPs tend to stabilize the fluorescent dianionic form of FAM. Apart from tunable p K a, arginine-rich CPPs also improved the water solubility, membrane permeability, and organelle-specific localization of FAM. Two conjugated probes FAM-R 12 and FAM-(F x r) 3 were selected to monitor intracellular pH fluctuations. Compared to FAM-(F x r) 3, highly positively charged FAM-R 12 was more effective in lower pH condition and realized targeted visualization of lysosomal pH changes. The arginine-rich CPP-based strategy offers a promising approach to obtain optimized fluorescent pH probes with adjustable p K a values for organelle-specific pH measurement.