Azobenzene-caged sulforhodamine dyes: a novel class of `turn-on' reactive probes for hypoxic tumor cell imaging

New sulforhodamine-based fluorescent `turn-on' probes have been developed for the direct imaging of cellular hypoxia. Rapid access to this novel class of water-soluble `azobenzene-caged' fluorophores was made possible through an easily-implementable azo-coupling reaction between a fluorescent primary arylamine derived from a sulforhodamine 101 scaffold (named SR101-NaphtNH(2)) and a tertiary aniline whose N-substituents are neutral, cationic, or zwitterionic. The detection mechanism is based on the bioreductive cleavage of the azo bond that restores strong far-red fluorescence (emission maximum at 625 nm) by regenerating the original sulforhodamine SR101-NaphtNH(2). This valuable fluorogenic response was obtained for the three `smart' probes studied in this work, as shown by an in vitro assay using rat liver microsomes placed under aerobic and then under hypoxic conditions. Most importantly, the probe namely SR101-NaphtNH(2)-Hyp-diMe was successfully applied for imaging the hypoxic status of tumor cells (A549 cells).