A novel strategy to design latent ratiometric fluorescent pH probes based on self-assembled SNARF derivativesElectronic supplementary information (ESI) available: Chemical structure of SNARF derivatives, detail of synthesis, spectra data and cell image. See DOI: 10.1039/c3ra43928cDedicated to Professor Seiji Shinkai on the occasion of his 70th birthday

In this study, we describe a rational design strategy to develop a latent ratiometric fluorescent pH probe from self-assembled seminaphthorhodafluor (SNARF) derivatives in aqueous conditions. Analysis of the characteristics of SNARF derivatives with protected phenolic groups allowed classification of the threshold between the assembled and monomeric states according to the Hansch-Fujita hydrophobic parameters for a substituent inserted as a SNARF-OH phenolic-protecting group. The esterase-activated latent ratiometric fluorescent pH probe SNARF-OBn(OAc) was characterized in the application of tuneable small-molecule aggregation and disaggregation. Before esterase treatment, SNARF-OBn(OAc) formed self-assembled clusters, emitting no fluorescence. The significant fluorescence was observed after esterase treatment because the self-assembled clusters of SNARF-OBn(OAc) diffuse as SNARF-OH monomers, demonstrating the intracellular application of SNARF-OBn(OAc) as a latent ratiometric fluorescent pH probe for floating cells. Furthermore, to directly visualize the state of self-assembled SNARF derivatives for investigating the mechanism of their cellular uptake, SNARF-Dan was rationally designed and applied for cellular imaging. Through the cellular application of SNARF-Dan, it was suggested that the self-assembled SNARF derivatives were introduced into the cell via macropinocytosis. We describe a rational design strategy to develop a latent ratiometric fluorescent pH probe from self-assembled SNARF derivatives in aqueous conditions.