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 pK(a) value of FAM to adapt well to pH measurement under diverse pH conditions. With increasing length of polyarginine, the pK(a) value of FAM was tuned from 6.20 +/- 0.06 to 5.17 +/- 0.05. The key mechanism for pK(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 pK(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 pK(a) values for organelle-specific pH measurement.