In situ formation of pyronin dyes for fluorescence protease sensing

We report a reaction-based strategy for the fluorogenic detection of protease activity. Based on the ``covalent-assembly'' probe design principle recently put forward by the Yang group for detection of Sarin related threats (J. Am. Chem. Soc., 2014, 136, 6594-6597), we have designed two unusual nonfluorescent caged precursors (mixed bis-aryl ethers) which are readily converted into a fluorescent unsymmetrical pyronin dye through a domino cyclisation-aromatisation reaction triggered by penicillin G acylase (PGA) or leucine aminopeptidase (LAP). Fluorescence-based in vitro assays and HPLCfluorescence/- MS analyses support the claimed activation mechanism whose the further implementation to ``smart'' imaging agents for the study of protease function in vivo is expected.