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A series of nicotinamide-containing compounds based on the structure of a triazine dye, C.I. Reactive Blue 2, which is known to interact at the coenzyme-binding sites of several NAD(P)(H)-dependent dehydrogenases,1,2 were designed, synthesized, and characterized. The preparation of these compounds is described. Reduction of the coenzyme mimics with sodium borohydride led to an increase in absorption at 356 nm, analogous to the behavior of the natural coenzyme, NAD+.3 When incubated with horse liver alcohol dehydrogenase and ethanol at 25°C and pH 9.0, one of the mimics, Blue N-3, was converted into a new compound with an increased absorption at 356 nm and an Rf value on thin-layer chromatography identical to that of the reduced form produced by treatment with sodium borohydride. The oxidized and reduced forms of Blue N-3 could be separated by reverse-phase ion pair high-performance liquid chromatography. This technique could be used to measure the extent of Blue N-3 reduction: Approximately 90 turnovers were calculated for each enzyme active site over a 48-h period. Gas chromatography analysis suggested that ethanol was simultaneously converted to acetaldehyde. Blue N-3 represents the first example of a new generation of potentially inexpensive, stable, and active biomimetic redox coenzymes. |
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