Fluorogenic substrates based on fluorinated umbelliferones for continuous assays of phosphatases and beta-galactosidases

Fluorogenic substrates based on 4-methylumbelliferone (4-MU) have been wide ly used for the detection of phosphatase and glycosidase activities. One di sadvantage of these substrates, however, is that maximum fluorescence of th e reaction product requires an alkaline pH, since 4-MU has a pK(a) approxim ate to 8. In an initial screening of five phosphatase substrates based on f luorinated derivatives of 4-MU, all with pK(a) values lower than that of 4- MU, we found that one substrate, 6,8-difluoro-4-methylumbelliferyl phosphat e (DiFMUP), was much improved for the detection of acid phosphatase activit y, When measured at the preferred acid phosphatase reaction pH (5.0), DiFMU P yielded fluorescence signals that were more than 16-fold higher than thos e of 4-methylumbelliferyl phosphate (MUP). DiFMUP was also superior to MUP for the detection of protein phosphatase 1 activity at pH 7 and was just as sensitive as MUP for the detection of alkaline phosphatase activity at pH 10. A beta-galactosidase substrate was also prepared based on 6,8-difluoro 4-methylumbelliferone. This substrate, 6,8-difluoro-4-methylumbelliferyl be ta-D-galactopyranoside (DiFMUG), was found to be considerably more sensitiv e than the commonly used substrate 4-methylurnbelliferyl beta-D-galactopyra noside (MUG), for the detection of beta-galactosidase activity at pH 7. DiF MUP and DiFMUG should have great utility for the continuous assay of phosph atase and beta-galactosidase activity, respectively, at neutral and acid pH . (C) 1999 Academic Press.