Latent Fluorophores Based on a Self-Immolative Linker Strategy and Suitable for Protease Sensing

The self-immolative spacer <i>para</i>-aminobenzyl alcohol (PABA) was used as a key component in the design of new protease-sensitive fluorogenic probes whose parent phenol-based fluorophore is released through an enzyme-initiated domino reaction. First, the conjugation of the phenylacetyl moiety to 7-hydroxycoumarin (umbelliferone) and 7-hydroxy-9<i>H</i>-(9,9-dimethylacridin-2-one) (DAO) by means of the heterobifunctional PABA linker has led to pro-fluorophores <b>6a</b> and <b>6d</b> whose enzyme activation by penicillin amidase was demonstrated. The second part of this study was devoted to the extension of this latent fluorophore strategy to the caspase-3 protease, a key mediator of apoptosis in mammalian cells. Fluorogenic caspase-3 substrates <b>11</b> and <b>13</b> derived from umbelliferone and DAO, respectively, were prepared. It was demonstrated that pro-fluorophore <b>11</b> is a sensitive fluorimetric reagent for the detection of this cysteine protease. Furthermore, <i>in vitro</i> assays with fluorogenic probe <b>13</b> showed a deleterious effect of biological thiols on fluorescence of the released acridinone fluorophore DAO that, to our knowledge, had not been reported until now.