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^V₂W^VI₁₈O₆₂]^6- (intact form), [α₂-P^V₂W^VI₁₇O₆₁]^10- (monolacunary), [P^V₂W^VI₁₅O₅₆]^12- (trilacunary) and [H₂P^V₂W^VI₁₂O₄₈]^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 ¹⁸³W-NMR, ³¹P-NMR spectroscopy and mass spectrometry. The intact Wells-Dawson POT [α/β-P^V₂W^VI₁₈O₆₂]^6- shows partial (~ 69%) disintegration into the monolacunary [α₂-P^V₂W^VI₁₇O₆₁]^10- anion with moderate activity (K_i = 9.7 mM). The monolacunary [α₂-P^V₂W^VI₁₇O₆₁]^10- retains its structural integrity and exhibits the strongest inhibition of AbPPO4 (K_i = 6.5 mM). The trilacunary POT [P^V₂W^VI₁₅O₅₆]^12- rearranges to the more stable monolacunary [α₂-P^V₂W^VI₁₇O₆₁]^10- (~ 62%) accompanied by release of free phosphates and shows the weakest inhibition (K_i = 13.6 mM). The hexalacunary anion [H₂P^V₂W^VI₁₂O₄₈]^12- undergoes time-dependent hydrolysis resulting in a mixture of [H₂P^V₂W^VI₁₂O₄₈]^12-, [P^V₈W^VI₄₈O₁₈₄]^40-, [P^V₂W^VI₁₉O₆₉(H₂O)]^14- and [α₂-P^V₂W^VI₁₇O₆₁]^10- which together leads to comparable inhibitory activity (K_i = 7.5 mM) after 48 h. For the solutions of [α/β-P^V₂W^VI₁₈O₆₂]^6-, [α₂-P^V₂W^VI₁₇O₆₁]^10- and [P^V₂W^VI₁₅O₅₆]^12- the inhibitory activity is correlated to the degree of their rearrangement to [α₂-P^V₂W^VI₁₇O₆₁]^10-. The rearrangement of hexalacunary [H₂P^V₂W^VI₁₂O₄₈]^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 [α₂-P^V₂W^VI₁₇O₆₁]^10-. The good inhibitory effect of the Wells-Dawson [α₂-P^V₂W^VI₁₇O₆₁]^10- anion is explained by the low charge density of its protonated forms H_x[α₂-P^V₂W^VI₁₇O₆₁]^(10-x)- (x = 3 or 4) at pH 6.8.