Dual-fluorescence pH probe for bio-labellingElectronic supplementary information (ESI) available: Synthesis and characterization of all compounds can be found. See DOI: 10.1039/c5cp05454k

Although seminaphtorhodafluor (SNARF) dyes are already widely used to measure pH in cells and at biofilms, their synthesis has low yield and results in an unspecific position of a carboxy-group. The separation of 5′- and 6′-carboxy-SNARF reveals a p K a difference of 0.15, calling into question pH measurements with the (commercially available) mixture. Here we replace the bulky external dicarboxyphenyl ring with a propionate group and evaluate the spectral properties of the new derivative. Proceeding to the ethyl-iodoacetamide, covalent linkage to cysteine protein sites is achieved efficiently as shown with a cyanobacterial phytochrome, extending the scarce application of SNARF in bio-labelling in the current literature. Application in fluorescence lifetime imaging is demonstrated both with the lifetime-based and ratiometric-yield method. Seminaphtorhodafluor (SNARF) dyes usually contain a bulky phenyl group with an unspecific position of a second carboxy group. Here a smaller SNARF derivative is characterized spectroscopically, and efficient linkage to cysteine protein sites is demonstrated.