SYNTHESIS AND SPECTROSCOPIC CHARACTERIZATION OF MODEL COMPOUNDS FOR THE ACTIVE-SITE COFACTOR IN COPPER AMINE OXIDASES

The synthesis and spectroscopic characterization of compounds which model different forms of the active site cofactor in copper amine oxidases have been pursued. As described, 5-(2,4,5-trihydroxybenzyl)hydantoin (1(red)H-3), its corresponding quinone (1(ox)H), and 6-amino-4-ethylresorcinol (7H-2) have been prepared. Additionally, 5-(3,4-dihydroxybenzyl) hydantoin (2(red)H-2) was synthesized for comparative purposes. Spectroscopic titrations have been employed to determine the pK(a) values of the acid-base species of the quinone (A = 1(ox)H + 1ox-; eq 3), the quinol (B = 1(red)H-3 + 1(red)H-2- + 1(red)H-2- + 1red3-; eq 2), and the aminophenol (C = 7H-3+ + 7H-2 + 7H- + 7(2-); eq 4). The quinone (1(ox)H) has a pK(a) of 4.13 +/- 0.01. The anionic species (1ox-) has a broad absorption band at around 484 nm which is characteristic of the eukaryotic copper amine oxidases. The quinol (B) and the aminophenol (C) have no absorption in this region, comparable to the reduced forms of enzymes. At pH 7.20, the two-electron redox potential of topa quinone (1ox-) is shown to be ca. 300 mV less positive than that of dopa quinone (2ox) but to be similar to that of pyrroloquinoline quinone (PQQ). The aminophenol (C' = 7H-2 + 7H- + 7(2-)) and the quinol (B) have similar acid-base properties and electrochemistry at neutral and basic conditions. The iminoquinone (9) is easily generated by oxidation of the aminophenol (C') exhibiting a blue-shifted lambda(max) which is similar to that observed in a complex of a copper amine oxidase and ammonia. (4-Nitrophenyl)hydrazine readily forms the hydrazone (azo) of 1(ox)H (3). The position of the nucleophilic addition is shown by 2D NMR experiments to be at C5, the carbonyl carbon next to the hydroxyl group.