Late-stage functionalisation of alkyne-modified phospha-xanthene dyes: lysosomal imaging using an off-on-off type of pH probe.

Near-infrared (NIR) fluorescent molecules are of great importance for the visualisation of biological processes. Among the most promising dye scaffolds for this purpose are P[double bond, length as m-dash]O-substituted phospha-xanthene (POX) dyes, which show NIR emission with high photostability. Their practical utility for and imaging has recently been demonstrated. Although classical modification methods have been used to produce POX-based fluorescent probes, it is still a challenge to introduce additional functional groups to control the localisation of the probe in cells. Herein, we report on the development of POXs that bear a 4-ethynylphenyl group on the phosphorus atom. These dyes can subsequently be functionalised with azide-tagged biomolecules a late-stage Cu-catalysed azide/alkyne cycloaddition (CuAAC) reaction, thus achieving target-selective labelling. To demonstrate the practical utility of the functionalised POXs, we designed a sophisticated NIR probe that exhibits a bell-shaped off-on-off pH-response and is able to assess the degree of endosomal maturation.