Oxidation of aminophenols by 4a-hydroperoxy-5-ethyllumiflavin anion. Flavoenzyme hydroxylase mechanism

Article abstract of DOI:10.1021/ja00163a037

Product and kinetic studies have been carried out (absolute t-BuOH solvent, O₂-free N₂ atmosphere, 30°C) for the oxidation of a number of aminophenolate anions (o-, m-, and p-amino, 2-amino-5-methyl, 3-methyl-4-amino, p-N,N-dimethylamino) and aminonaphtholate anions (1-amino-2-naphtholate, 4-amino-1-naphtholate, 2-amino-3-naphtholate) by the hydroperoxy anion of 5-ethyl-3-methyl-4a,5-dihydro-4a-hydroperoxylumiflavin (4a-FlEt-OO^-). All but m-aminophenolate and 2-amino-3-naphtholate underwent oxidation in the stopped-flow time range. Under the pseudo-first-order conditions of [phenolate] or [naphtholate] >> [4a-FlEt-OO^-] the pseudo-first-order rate constant (k(obsd)) exhibited saturation on increase of [phenolate] or [naphtholate]. The maximum value of k(obsd) at saturation was independent of the structure of the oxidizable substrate, showing that 4a-FlEt-OO^- is endothermically converted to a species (X) that is trapped by phenolate or naphtholate anions. The rate constant for 4a-FlEt-OO^- → X (~0.3 s^-1) is, within experimental error, identical with that previously observed for the dioxygen-transfer reaction from 4a-FlEt-OO^- to a number of di-tert-butylphenolate and 3-methylindolate anions. p-Aminophenolate anion yields p-benzoquinone while p-(N,N-dimethylamino)phenolate provides both p-benzoquinone and (N,N-dimethylamino)-p-benzoquinone. Migration of the N,N-dimethylamino substituent in the formation of (N,N-dimethylamino)-p-benzoquinone is proposed to occur via a (N,N-dimethylamino)aziridine cation intermediate. Oxidation of o-aminophenolate and 5-methyl-2-aminophenolate provides the corresponding phenoxizanones. The stoichiometry of these oxidations are in accord with 4a-FlEt-OO^- + aminophenolate → FlEt^- + o-quinone imine followed by condensation of o-quinone imine with remaining aminophenolate and a second oxidation by 4a-FlEt-OO^- to yield dihydrophenoxazinone. Dihydrophenoxazinone is then proposed to proceed to phenoxizanone by oxidation with HOO^-, which is a product of the initial oxidations with 4a-FlEt-OO^-. The mechanism of the flavoenzyme mixed function hydroxylation of phenolate anions is discussed in terms of the relative stereochemical disposition of enzyme bound flavin cofactor 4a-hydroperoxide and phenolate substrate - deduced from the X-ray coordinates of p-hydroxybenzoate hydroxylase with flavin and 3,4-dihydroxybenzoate bond at the active site.