Deeper Insight into the Protease-Mediated Formation of Pyronin Dyes and Possible Implications in the Design of Fluorogenic Probes for Bioimaging and Theranostic Prodrugs
We report a rational and systematic study devoted to structural optimisation of a novel class of protease-sensitive fluorescent probes recently reported by us (<i>Org. Biomol. Chem.</i>, 2017, <b>15</b>, 2575-2584), based on the "covalent-assembly" strategy and using the targeted enzyme (penicilin G acylase as model protease) to build a fluorescent pyronin dye by triggering a biocompatible domino cyclisation-aromatisation reaction. The aim is to identify <i>ad hoc</i> probe candidate(s) that might combine fast/reliable fluorogenic "turn-on" response, full stability in complex biological media and ability to release a second molecule of interest (drug or second fluorescent reporter), for applications in disease diagnosis and therapy. We base our strategy on screening a set of active methylene compounds (C-nucleophiles) to convert the parent probe to various pyronin caged precursors bearing Michael acceptor moieties of differing reactivity. <i>In vitro</i> stability and fluorescent enzymatic assays combined to HPLC-fluorescence analyses provide data useful to define the most appropriate structural features for these fluorogenic scaffolds depending on the specifications required by the biomedical application (<i>e.g.</i>, <i>in vivo</i> molecular imaging, image-guided drug delivery and theranostics) for which they will be used.