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 (Org. Biomol. Chem., 2017, 15, 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 ad hoc 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. In vitro 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 (e.g., in vivo molecular imaging, image-guided drug delivery and theranostics) for which they will be used.