Revisiting the Chemistry and Photophysics of 3-(N-Methylpyridinium-4-yl)Coumarins for Designing "Covalent-Assembly" and "Molecular Disassembly" Fluorescent Probes

The constant need for high-performance aniline- or phenol-based fluorophores suitable for the construction of activity-based fluorescent probes, led us to study both synthesis and photophysics of C3-N-methylpyridinium-4-yl substituted 7-(dialkylamino)/7-hydroxycoumarins. Indeed, in the field of photoactive organic molecules, the positively charged N-alkylpyridinium-4-yl groups are often used as acceptor units to dramatically impact spectral features through promoting intramolecular charge transfer (ICT) processes. They are also known as effective water-solubilizing and mitochondria targeting moieties. The poor fluorescence efficiency of cationic 7-hydroxycoumarin derivatives in aqueous physiological conditions was highlighted and rationalized by the predominance of a neutral quinonoid form in such buffer medium. The ability of the excited singlet state (S1) of this neutral species to undergo intersystem crossing (ISC) to triplet state (T1) was partly supported by phosphorescence measurements of singlet oxygen. We also took advantage of green-emissive properties of 7-(diethylamino)-3-(N-methylpyridinium-4-yl)coumarin to successfully design and validate a novel small-molecule fluorescent probe for the detection of alkaline phosphatase (ALP), based on the "covalent-assembly" principle. A practical use of ortho-formylated 7-hydroxy-3-(N-methylpyridinium-4-yl)coumarin was next considered with the synthesis of a Fe(III)-salen complex whose the potential as a "molecular disassembly" probe for fluorogenic sensing of pyrophosphate (PPi) anion was assessed.