Fluorogenic Substrates Based on Fluorinated Umbelliferones for Continuous Assays of Phosphatases and β-Galactosidases

Abstract

Fluorogenic substrates based on 4-methylumbelliferone (4-MU) have been widely used for the detection of phosphatase and glycosidase activities. One disadvantage of these substrates, however, is that maximum fluorescence of the reaction product requires an alkaline pH, since 4-MU has a pKa ≈ 8. In an initial screening of five phosphatase substrates based on fluorinated derivatives of 4-MU, all with pKa values lower than that of 4-MU, we found that one substrate, 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), was much improved for the detection of acid phosphatase activity. When measured at the preferred acid phosphatase reaction pH (5.0), DiFMUP yielded fluorescence signals that were more than 10-fold higher than those 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 β-galactosidase substrate was also prepared based on 6,8-difluoro-4-methylumbelliferone. This substrate, 6,8-difluoro-4-methylumbelliferyl β-d-galactopyranoside (DiFMUG), was found to be considerably more sensitive than the commonly used substrate 4-methylumbelliferyl β-d-galactopyranoside (MUG), for the detection of β-galactosidase activity at pH 7. DiFMUP and DiFMUG should have great utility for the continuous assay of phosphatase and β-galactosidase activity, respectively, at neutral and acid pH.