Interaction of flavones and their bromoacetyl derivatives with NAD(P)H:quinone acceptor oxidoreductase

Flavones are a new type of inhibitor of NAD(P)H:quinone acceptor oxidoreductase (DT-diaphorase, EC 1.6.99.2). To further characterize the flavone binding site, three bromoacetyl derivatives of flavones, i.e., 7-bromoacetylflavone, 5-hydroxyl-7-bromoacetylflavone, and 7,8-dibromoacetylflavone, have been synthesized. These compounds have been found to be potent inhibitors that inactivate the rat quinone reductase in a time-dependent manner, suggesting that they can be used as affinity labels for the enzyme. Among the three bromoacetyl derivatives, 7,8-dibromoacetylflavone is the most potent inhibitor; however, its labeling of the quinone reductase is the least stable, so that the enzyme regains activity after a short incubation. In contrast, the inactivation of the quinone reductase by 5-hydroxyl-7-bromoacetylflavone is stable. Accordingly, this flavone derivative is the most suitable compound for labeling the flavone binding site of the enzyme. Electrospray mass spectrometry has been applied to demonstrate that 5-hydroxyl-7-bromoacetylflavone labels this enzyme in a stoichiometric manner.