Alex Junino

Direct observation of the reaction of the quinone-methide from 5,6-dihydroxyindole with the nucleophilic azide ion

Investigation of the oxidation of melanagenic intermediates and, in particular, the ultimate oxidative polymerisation of 5,6-dihydroxyindole (1) is important for a comprehensive understanding of the biosynthesis of melanin. Oxidation of 1 leads to the formation of its corresponding quinone methide, a reactive intermediate. We report a novel approach using a fast reaction technique to produce the quinone methide of 1in situ and study its chemistry. In aqueous solution at pH 9, the quinone methide has a lifetime of < 0.1 s due to interaction with the solvent. As an example of the chemistry of the quinone methide of 1, it was demonstrated that it interacts with the nucleophilic azide ion. From comparison with the corresponding reactions of the quinone-methide from 5-methoxy-6-hydroxyindole (2), it is inferred that the indoloquinone of 1 is involved in the pathways to melanin formation. The importance of the involvement of the quinone methide of 1 in the pathway to melanin formation is in contrast to the pathway involving dimer products arising from 1.

Characterisation of the intermediates produced upon one-electron oxidation of 4-, 5-, 6- and 7-hydroxyindoles by the azide radical

One-electron oxidation of a series of monohydroxylated indoles (HI) by the azide radical in the pH range 5–9 has been studied using the technique of pulse radiolysis with spectrophotometric detection. One-electron oxidation of 4-, 5-, 6- and 7-hydroxyindoles results in the formation of the corresponding indoloxyl radicals, the optical absorption spectra of which are independent of pH (5–9). It is confirmed, using the N(1)-methyl substituted analogue of 6-HI, that deprotonation of the resulting radical cation of the hydroxyindoles occurs preferentially from the hydroxy group to yield the corresponding indoloxyl radical. Such deprotonation would be consistent with the resulting indoloxyl radical having a low pKa.With the exception of 4-HI, the indoloxyl radicals decay bimolecularly in the dose/pulse range of 1–30 Gy to yield semi-permenent products (2k= 2–4 × 109 dm3 mol–1 s–1). With 4-HI, the decay of the indoloxyl radical changes from second-order to first-order kinetics on lowering the dose/pulse. At 1 Gy/pulse, the first-order kinetics are dependent upon the concentration of 4-HI. The second-order rate constant for reaction of the indoloxyl radical with 4-HI was determined to be 4.8 × 107 dm3 mol–1 s–1. The decay of these semi-permanent products from the indoloxyl radicals is first order and depends upon the concentration of azide. The second-order rate constants determined for this reaction depend markedly on the hydroxyindole used (k= 159–821 dm3 mol–1 s–1). The semi-permanent product arising from the bimolecular decay of the indoloxyl radicals is discussed in terms of the formation of reactive quinone-methides and/or -imines.

Reactions of indolic radicals produced upon one-electron oxidation of 5,6-dihydroxyindole and its N(1)-methylated analogue

The reactions of indole semiquinone radicals produced following one-electron oxidation of 5,6-dihydroxyindole (DHI) and its N-methyl-substituted analogue (MeDHI) have been studied using pulse radiolysis with spectrophotometric detection in the pH range 5–10 using different dose/pulse values (1–20 Gy/pulse). Using a dose/pulse of 18.5 Gy the semiquinone radicals of DHI and MeDHI decay predominantly by second order kinetics. The second order rate constants for disappearance of the semiquinone radicals are dependent upon the pH. Values of rate constants for decay of the semiquinone radical of DHI (pKa= 6.8) at pH 5.5 and 9.1 are 3.8 × 109 and 1.0 × 108 dm3 mol–1 s–1 respectively at room temperature. In contrast, at a lower dose/pulse of ≤2 Gy, the semiquinone radical decays predominantly by first order kinetics which are dependent upon the concentration of the indole when pH pKa. of the semiquinone radical. The second order rate constants for interaction of the semiquinone radical of DHI and MeDHI with DHI and MeDHI at pH 8.8 were determined to be 1.6 × 106 and 2.3 × 106 dm3 mol–1 s–1 respectively. The decay of the semiquinone radical results in the formation of a semi-permanent product(s) with a lifetime of ca. 10 ms. These products are discussed in terms of the formation of a reactive quinone-methide and/or -imine.

One-electron oxidation of 5,6-dihydroxy-2,3-dihydroindole: the influence of Zn2+

One electron oxidation of 5,6-dihydroxy-2,3-dihydroindole (DDI) and its N-ethyl analogue by the azide radical, N3˙, at pH 5 and 9 was studied using pulse radiolysis to further the understanding of chemical pathways leading to melanin formation. One electron oxidation of DDI yields the benzosemiquinone radical with a pKa value of 5.3. With the N-ethyl analogue, the corresponding benzosemiquinone radical is formed, consistent with one of the hydroxy groups being the preferred site for deprotonation of the initially formed radical cation. The benzosemiquinone radicals of DDI disproportionate to yield a stable dopachrome-like product. In the presence of Zn2+ at pH 5.0, the benzosemiquinone radicals of DDI react to form, by an alternative route, a Zn ion complex of the o-semiquinone radical with a rate constant of 3.0 × 106 dm3 mol–1 s–1. This Zn ion complex decays by second order kinetics to yield a Zn2+–quinone complex which has a lifetime of 3–4 ms. Auto-oxidation of DDI is suggested to lead to the formation of a dopachrome-like intermediate. From these studies, it is concluded that Zn2+ significantly influences the reactions involving the semiquinone radical of DDI and these alternative reaction pathways may help to clarify the initial biochemical stage of the free-radical pathway(s) leading to melanin formation.