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

Abstract

Latent 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.

Valkyrie Probes: A Novel Class of Enzyme‐Activatable Photosensitizers based on Sulfur‐ and Seleno‐Rosamines with Pyridinium Unit**

“…For the determination of absorption and fluorescence features of these novel xanthene derivatives, we have selected two distinct aqueous buffers (phosphate buffered saline (PBS, 10 mM phosphate+2.7 mM KCl and 137 mM NaCl), pH 7.3 as simulated physiological conditions, and Tris.HCl buffer pH 8.0 well suited for in vitro enzyme assays with ALP [25,30] ), and DMSO. The permanent mono‐ or dicationic charge makes those dyes and probes a remarkable solubility in water and in polar solvents, especially under neutral pH conditions.…”

“…As demonstrated in our contributions devoted to analytical validations of different classes of enzyme-responsive fluorogen-ic platforms, [30,36] it is of prime importance to combine conventional fluorescence-based assays with complementary RP-HPLCfluorescence and RP-HPLC-MS analyses, to confirm the sensing mechanism of such probes. This is also the only way for assessing both efficacy and selectivity of the studied enzymetriggered cascade reaction (conversion rate and possible formation of side-products).…”

Revisiting the Chemistry and Photophysics of 3‐(N‐Methylpyridinium‐4‐yl)Coumarins for Designing “Covalent‐Assembly” and “Molecular Disassembly” Fluorescent Probes**

“…Conversely, positive results obtained with the ALP‐responsive “covalent‐assembly” fluorescent probe 17 open interesting perspectives. Indeed, the use of latent C ‐nucleophiles related to 2‐( N ‐methylpyridinium‐4‐yl)acetonitrile 5 in the Knoevenagel condensation step offers the unique opportunity to achieve facile late‐stage functionalization of analyte‐responsive caged precursors of coumarins [50] and pyronins [51] simply by changing the substituent used for pyridine N ‐quaternarization. This will enable to tune their physico‐chemical properties (especially water solubility), targeting ability, pharmacokinetics ( e. g ., introduction of a PEG linker) or to confer them release capabilities of a molecule of interest ( e. g ., a drug or a second reporter).…”