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-P(V)(2)W(VI)(18)O(62)](6−) (intact form), [α(2)-P(V)(2)W(VI)(17)O(61)](10−) (monolacunary), [P(V)(2)W(VI)(15)O(56)](12−) (trilacunary) and [H(2)P(V)(2)W(VI)(12)O(48)](12−) (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 (183)W-NMR, (31)P-NMR spectroscopy and mass spectrometry. The intact Wells–Dawson POT [α/β-P(V)(2)W(VI)(18)O(62)](6−) shows partial (~ 69%) disintegration into the monolacunary [α(2)-P(V)(2)W(VI)(17)O(61)](10−) anion with moderate activity (K(i) = 9.7 mM). The monolacunary [α(2)-P(V)(2)W(VI)(17)O(61)](10−) retains its structural integrity and exhibits the strongest inhibition of AbPPO4 (K(i) = 6.5 mM). The trilacunary POT [P(V)(2)W(VI)(15)O(56)](12−) rearranges to the more stable monolacunary [α(2)-P(V)(2)W(VI)(17)O(61)](10−) (~ 62%) accompanied by release of free phosphates and shows the weakest inhibition (K(i) = 13.6 mM). The hexalacunary anion [H(2)P(V)(2)W(VI)(12)O(48)](12−) undergoes time-dependent hydrolysis resulting in a mixture of [H(2)P(V)(2)W(VI)(12)O(48)](12−), [P(V)(8)W(VI)(48)O(184)](40−), [P(V)(2)W(VI)(19)O(69)(H(2)O)](14−) and [α(2)-P(V)(2)W(VI)(17)O(61)](10−) which together leads to comparable inhibitory activity (K(i) = 7.5 mM) after 48 h. For the solutions of [α/β-P(V)(2)W(VI)(18)O(62)](6−), [α(2)-P(V)(2)W(VI)(17)O(61)](10−) and [P(V)(2)W(VI)(15)O(56)](12−) the inhibitory activity is correlated to the degree of their rearrangement to [α(2)-P(V)(2)W(VI)(17)O(61)](10−). The rearrangement of hexalacunary [H(2)P(V)(2)W(VI)(12)O(48)](12−) 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(2)-P(V)(2)W(VI)(17)O(61)](10−). The good inhibitory effect of the Wells–Dawson [α(2)-P(V)(2)W(VI)(17)O(61)](10−) anion is explained by the low charge density of its protonated forms H(x)[α(2)-P(V)(2)W(VI)(17)O(61)]((10−x)−) (x = 3 or 4) at pH 6.8.