53518-15-3

Amino acid and peptide derivatives of 7-amino-4-(trifluoromethyl)coumarin are used to monitor peptidase activities. Caspase activation is measured using the fluorogenic compound N-acetyl-asp-glu-val-asp-7-Amino-4-(trifluoromethyl)coumarin (Ac-DEVD-AFC). This substrate rapidly enters cells where it is efficiently cleaved at the aspartate residue by specific caspases, yielding the fluorescent compound 7-Amino-4-(trifluoromethyl)coumarin (AFC). Following cell disruption, released 7-Amino-4-(trifluoromethyl)coumarin is separated on HPLC and detected by fluorescence. The appearance of 7-Amino-4-(trifluoromethyl)coumarin in cells is blocked by the pancaspase inhibitor benzyloxycarbonyl-val-ala-asp-fluoromethylketone, thus establishing that intracellular caspases are responsible for the cleavage. γ-glutamyl-7-amino-4-(trifluoro-methyl)coumarin is synthesized and used as a substrate for fluirimetric assay of γ-gluta-myltranspeptidase. The reaction product 7-amino-4-(trifluoromethyl)coumarin is fluorescent at neutral pH values and with excitation and emission wavelengths of 400 and 490 nm, respectively, concentration is linearly related to fluorescence over the range of 10 to 300 pmol/3 mL reaction mixture.