Deeper Insight into the Protease-Mediated Formation of Pyronin Dyes and Possible Implications in the Design of Fluorogenic Probes for Bioimaging and Theranostic Prodrugs

Abstract

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.

Synthetic Routes to Novel Fluorogenic Pyronins and Silicon Analogs with Far-Red Spectral Properties and Enhanced Aqueous Stability

“…However, the availability of Sipyronin dyes 4 and 5 has enabled us to perform some photophysical and stability studies in simulated physiological conditions. The latter ones clearly support the relevance of molecular amino group [34][35][36], these novel (Si-)pyronin derivatives will be very useful as references to validate "covalent-assembly" fluorescent probes based on in situ formation of (Si-)pyronin triggered by the analyte to be detected [38][39][40][41][42]. Finally, tunable reactivity of their meso-position towards different nucleophiles can be used to design reversible reaction-based fluorescent probes particularly well suited for real-time imaging of redox status changes in vivo [73][74][75].…”

“…1) [40,43]. A one-step synthesis through a conventional acid-catalyzed condensation reaction (vide supra) provides after tedious and time-consuming purification procedures these fluorophores in poor to modest yields even if our group has recently proposed a less straightforward but more effective synthetic approach towards these fluorophores [42].…”

Synthetic routes to novel fluorogenic pyronins and silicon analogs with far-red spectral properties and enhanced aqueous stability

“…As a natural extension of our work, this versatile methodology could also be applied to facile their use as fluorogenic dyes in "smart" probes based on protection-deprotection of a primary amino group [34][35][36], these novel (Si-)pyronin derivatives will be very useful as references to validate "covalent-assembly" fluorescent probes based on in situ formation of (Si-)pyronin triggered by the analyte to be detected [38][39][40][41][42]. Finally, tunable reactivity of their meso-position towards different nucleophiles can be used to design reversible reaction-based fluorescent probes particularly well suited for real-time imaging of redox status changes in vivo [73][74][75].…”

“…Among the different fluorescent probe design strategies commonly used for selective detection of (bio)analytes through chemical reactions, those based either on (1) protection-deprotection of an amino group (i.e., aniline-based pro-fluorophores) [34][35][36] or (2) "covalent-assembly" principle [37] are readily implementable to pyronin scaffolds. Some recent examples of the literature illustrate this trend and highlight benefits in terms of sensing performances, that might accrue form it [38][39][40][41][42] (Fig. 1).…”

“…1) [40,43]. A one-step synthesis through a conventional acid-catalyzed condensation reaction (vide supra) provides after tedious and time-consuming purification procedures these fluorophores in poor to modest yields even if our group has recently proposed a less straightforward but more effective synthetic approach towards these fluorophores [42]. Fig.…”