Wells–Dawson phosphotungstates as mushroom tyrosinase inhibitors: a speciation study

In order to elucidate the active polyoxotungstate (POT) species that inhibit fungal polyphenol oxidase ( Ab PPO4) 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 Ab PPO4 ( 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.