Arylidene Meldrum's Acid: A Versatile Structural Motif for the Design of Enzyme‐Responsive “Covalent‐Assembly” Fluorescent Probes with Tailor‐Made Properties**

Abstract

AbstractLatent cyclic carbon‐centered nucleophiles (latent C‐nucleophiles) are recently proving their value in the field of reaction‐based fluorescent probes, far beyond their primary utility in organic synthesis. They are typically used to introduce a Michael acceptor moiety acting as a recognition/reaction site for analyte to be detected or as a kinetic promoter of fluorogenic cascade reactions triggered by a reactive species. C‐nucleophiles bearing a further reactive handle offer an additional opportunity for tuning the physicochemical/targeting properties or providing drug‐releasing capabilities to these probes, through the covalent attachment of ad hoc chemical moiety. In order to implement such strategy to fluorogenic/chromogenic enzyme substrates based on the “covalent‐assembly” principle, we have explored the potential of some functionalized derivatives of barbituric acid, piperidine‐2,4‐dione and Meldrum's acid. Our investigations based on the rational design and analytical validations of enzyme‐responsive caged precursors of fluorescent pyronin dyes and 7‐(diethylamino)coumarin‐3‐carboxylic acid, led to identify a versatile candidate suitable for this late‐stage structural optimization approach. This Meldrum's acid derivative enables to either enhance water solubility or achieve the reversible conjugation of a targeting ligand, while promoting in situ formation of fluorophore upon enzymatic activation. This study opens the way to novel multifunctional fluorescence imaging probes and optically modulated small conjugate‐based theranostics.