Synthesis and study of intramolecularly-quenched fluorogenic substrates containing aminocoumarin or aminoquinolinone-type fluorophores

Abstract

The amines 7-amino-4-methylcoumarin (AMec)(1), 7-amino-4-methyl-2-quinolinone (AMeq)(2) and 3-aminocoumarin (Ac)(3) have been used as fluorophores (H–F) and the aromatic systems 2,4-dinitrophenyl (Dnp), 2,4,6-trinitrophenyl (Tnp), 2-nitrophenylsulphenyl (Nps) and 3,5-dinitrobenzoyl (DnBz) have been used as quenchers (Q) in the synthesis of twelve intramolecularly-quenched fluorogenic substrates of general structure Q–Gly–Phe–F. The study of the fluorescence properties of the substrates synthesized shows that efficient quenching of fluorescence is observed in all cases. The Dnp, Nps and Tnp groups show a higher quenching efficiency and the combination of the Dnp group with the fluorophore Ac gives the best result (99% quenching). The substrates synthesized can be used for the direct specific determination of enzymes which hydrolyse the peptide chain at any point between the interacting groups by measuring the increase in fluorescence.