Wells-Dawson phosphotungstates as mushroom tyrosinase inhibitors: a speciation study.

In order to elucidate the active polyoxotungstate (POT) species that inhibit fungal polyphenol oxidase (AbPPO4) in sodium citrate buffer at pH 6.8, four Wells-Dawson phosphotungstates [α/β-PWO] (intact form), [α-PWO] (monolacunary), [PWO] (trilacunary) and [HPWO] (hexalacunary) were investigated. The speciation of the POT solutions under the dopachrome assay (50 mM Na-citrate buffer, pH 6.8; L-3,4-dihydroxyphenylalanine as a substrate) conditions were determined by W-NMR, P-NMR spectroscopy and mass spectrometry. The intact Wells-Dawson POT [α/β-PWO] shows partial (~ 69%) disintegration into the monolacunary [α-PWO] anion with moderate activity (K = 9.7 mM). The monolacunary [α-PWO] retains its structural integrity and exhibits the strongest inhibition of AbPPO4 (K = 6.5 mM). The trilacunary POT [PWO] rearranges to the more stable monolacunary [α-PWO] (~ 62%) accompanied by release of free phosphates and shows the weakest inhibition (K = 13.6 mM). The hexalacunary anion [HPWO] undergoes time-dependent hydrolysis resulting in a mixture of [HPWO], [PWO], [PWO(HO)] and [α-PWO] which together leads to comparable inhibitory activity (K = 7.5 mM) after 48 h. For the solutions of [α/β-PWO], [α-PWO] and [PWO] the inhibitory activity is correlated to the degree of their rearrangement to [α-PWO]. The rearrangement of hexalacunary [HPWO] into at least four POTs with a negligible amount of monolacunary anion interferes with the correlation of activity to the degree of their rearrangement to [α-PWO]. The good inhibitory effect of the Wells-Dawson [α-PWO] anion is explained by the low charge density of its protonated forms H[α-PWO] (x = 3 or 4) at pH 6.8.