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 [alpha/beta-(P2W18O62)-W-V-O-VI](6-) (intact form), [alpha(2)-(P2W17O61)-W-V-O-VI](10-) (monolacunary), [(P2W15O56)-W-V-O-VI](12-) (trilacunary) and [(H2P2W12O48)-W-V-O-VI](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 W-183-NMR, P-31-NMR spectroscopy and mass spectrometry. The intact Wells-Dawson POT [alpha/beta-(P2W18O62)-W-V-O-VI](6-) shows partial (similar to 69%) disintegration into the monolacunary [alpha(2)-(P2W17O61)-W-V-O-VI](10-) anion with moderate activity (K-i = 9.7 mM). The monolacunary [alpha(2)-(P2W17O61)-W-V-O-VI](10-) retains its structural integrity and exhibits the strongest inhibition of AbPPO4 (K-i = 6.5 mM). The trilacunary POT [(P2W15O56)-W-V-O-VI](12-) rearranges to the more stable monolacunary [alpha(2)-(P2W17O61)-W-V-O-VI](10-) (similar to 62%) accompanied by release of free phosphates and shows the weakest inhibition (K-i = 13.6 mM). The hexalacunary anion [(H2P2W12O48)-W-V-O-VI](12-) undergoes time-dependent hydrolysis resulting in a mixture of [(H2P2W12O48)-W-V-O-VI](12-), [(P8W48O184)-W-V-O-VI](40-), [(P2W19O69)-W-V-O-VI(H2O)](14-) and [alpha(2)-(P2W17O61)-W-V-O-VI](10-) which together leads to comparable inhibitory activity (K-i = 7.5 mM) after 48 h. For the solutions of [alpha/beta-(P2W18O62)-W-V-O-VI](6-), [alpha(2)-(P2W17O61)-W-V-O-VI](10-) and [(P2W15O56)-W-V-O-VI](12-) the inhibitory activity is correlated to the degree of their rearrangement to [alpha(2)-(P2W17O61)-W-V-O-VI](10-). The rearrangement of hexalacunary [(H2P2W12O48)-W-V-O-VI](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 [alpha(2)-(P2W17O61)-W-V-O-VI](10-). The good inhibitory effect of the Wells-Dawson [alpha(2)-(P2W17O61)-W-V-O-VI](10-) anion is explained by the low charge density of its protonated forms H-x[alpha(2)-(P2W17O61)-W-V-O-VI]((10-x)-) (x = 3 or 4) at pH 6.8.