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.

Automated summary

The investigation of four Wells-Dawson phosphotungstates revealed that the monolacunary [alpha(2)-(P2W17O61)-W-V-O-VI]10- has the strongest inhibitory effect on AbPPO4, while the trilacunary and hexalacunary species showed weaker inhibition. The rearrangement of hexalacunary species led to the formation of multiple POTs with comparable inhibitory activity. The good inhibitory effect of the monolacunary species is attributed to the low charge density of its protonated forms at pH 6.8.