Tuning the pKa of two-photon bis-chromophoric probes for ratiometric fluorescence imaging of acidic pH in lysosomes

Lysosomes are organelles containing many hydrolytic enzymes responsible for degrading macromolecules. Abnormal lysosomal pH changes are known to associate with dysfunction of cells linking to various diseases such as cancer and neurodegenerative disorders. Thus, it is of paramount importance to monitor lysosomal pH changes in order to investigate the pathological conditions. We report herein two novel, highly sensitive and fast responsive bis-chromophoric ratiometric two-photon fluorescent probes with different emission wavelengths, namely VP and VL for acidic pH sensing in live cells. Importantly, by adopting bis-chromophoric approach, the VP and VL probes bearing pyridyl and quinolyl as acid sensing sites exhibit pK(a) values of 4.62 and 5.26, respectively, which are ideal for quantitative analysis of lysosomal pH changes in live cells. These two biocompatible probes are not only highly lysosomal targeting, sensitive towards pH change with distinct emission color shifting but also highly two-photon active in cells with excellent photostability and reversibility. These probes were successfully applied to ratiometrically track and image pH fluctuation in lysosomes of HeLa cells by one- and two-photon excited fluorescence microscopy. For the first time, we have demonstrated here that the bis-chromophoric strategy is a useful tool to effectively modify and tune the pK(a) of a fluorescent probe.