“…To address these items and to identify the best protease-triggered reaction-based probe candidate that might combine fast/reliable fluorogenic "turn-on" response, overall stability and ability to release a second molecule of interest (in addition to pyronin AR116), we have screened a short library of Cnucleophiles whose reaction with aldehyde 2 should lead to a set of probes exhibiting their own intrinsic reactivity closely tied to the pKa value of the nucleophilic partner involved in their synthesis. To cover a broad range of C-nucleophiles that fulfills the requirements mentioned above, we have chosen to use both commercial basic organic building blocks and less conventional molecular structures: dimedone (pKa 5.2 in water) 16 , 1,3-dimethylbarbituric acid (pKa 4.7 water) 17 , Meldrum's acid (pKa 4.8 in water) 16,18 ,4, in water, see ESI † for its photophysical characterization in phosphate buffer (PB)) 19 , 1,4dimethylpyridinium iodide (pKa never determined), 1-ethyl-2,3,3-trimethylindolenium iodide (pKa never determined), edaravone (a free radical scavenger and neuroprotective agent used for therapy of amyotrophic lateral sclerosis, also known as 1-phenyl-3-methyl-5-pyrazolone, pKa 7.0 in water) 20 and NSC 645039 (4-phenyl-1,3-dihydro-2H-1,5-benzodiazepin-2one, pKa 12.7, predicted value) 21 , compared with malonitrile (pKa 11.5) 22 .…”