Synthesis of several 2-substituted 3-(p-hydroxyphenyl)-1-propenes and their characterization as mechanism-based inhibitors of dopamine β-hydroxylase

Three substrate analogs of dopamine β-hydroxylase, viz. 2-X-3-(p-hydroxyphenyl)-1-propenes (where X = Br, Cl, H), have been synthesized, and all behave as substrates requiring O₂ and ascorbate for the enzyme-catalyzed hydroxylation reaction. The products have been characterized by mass spectrometry as the respective 2-X-3-hydroxy-3-(p-hydroxyphenyl)-1-propenes. The relative k(cat) values for these compounds at pH 5.5, 0.25 mM O₂ are 49 min^-1 (2-H), 8.6 min^-1 (2-Cl), and 7.0 min^-1 (2-Br). All three compounds have the characteristics of mechanism-based inhibitors of dopamine β-hydroxylase since incubation of enzyme with these compounds under turnover conditions leads to a time-dependent loss of activity. The k(inact) values at pH 5.5, 0.25 mM O₂ are 0.08, 0.20, and 0.51 min^-1, respectively, for the 2-Br-, 2-Cl-, and 2-H-substituted analogs. No reactivation was observed after exhaustive dialysis of enzyme inactivated by 2-Br-3-(p-hydroxyphenyl)-1-propene, suggesting irreversible inactivation of dopamine β-hydroxylase.