Model studies of topaquinone-dependent amine oxidases. 2. Characterization of reaction intermediates and mechanism

Article abstract of DOI:10.1021/ja00139a003

The reaction of 2-hydroxy-5-tert-butyl-1,4-benzoquinone (1a) and benzylamine in acetonitrile was studied under anaerobic conditions. Addition of benzylamine to the quinone 1a solution generates the anionic form of the quinone (λ(max) at 492 nm), followed by the formation of the product Schiff base 11 with λ(max) at 368 nm and the aminoresorcinol 13 with λ(max) at 304 nm. The rapid dissociation of the 2-hydroxyl proton was confirmed by the isolation of the amine salt 5a in the reaction of tert-butylamine and 1a. The substrate Schiff base 6a was not spectrally detected due to its lower extinction coefficient and rapid conversion to the product Schiff base 11. However, when α-methylbenzylamine was employed as a substrate, the formation of the substrate Schiff base 7 was detected by ¹H NMR and UV-vis spectroscopy. Cyclohexylamine, n-propylamine, and ammonia also gave the substrate Schiff bases 8, 9, and 10, respectively. Both the steric bulk and the acidity of the C₁ proton of the substrate are found to be factors controlling the further reaction (C₁ proton abstraction). Detailed structural analysis of the substrate Schiff base was performed on 8 by 2D NMR spectroscopy, showing that 8 is in its amine salt form and has undergone nucleotophilic addition at C₁, the carbonyl carbon next to the 2-hydroxyl group. UV-vis spectroscopy supports the view that 8 is not a solvent-separated ion pair (λ(max) at 454 nm) but an intimate ion pair (λ(max) 352 nm) in CH₃CN. The latter λ(max) value is very similar to λ(max) observed for the Schiff base complex seen in bovine serum amine oxidase and different from a Schiff base complex with 4-methoxy-5-tert-butyl-1,2-benzoquinone 14. The product Schiff base 11 was prepared by the reaction of the hydrochloride salt of the aminoresorcinol 13 and benzaldehyde. It has an ε value 10 times larger than that of the substrate Schiff base (7, 8, or 9) at 368 nm. Treatment of 11 with benzylamine yielded the aminoresorcinol 13 and the product, N-benzylidenebenzylamine (PhCH = NCH₂Ph). Comparison of these results to catalytic properties of the copper amine oxidases provides support for an aminotransferase mechanism from a Schiff base of topa in a localized p-quinone form.