Oxidation of 4-alkylphenols and catechols by tyrosinase: Ortho-substituents alter the mechanism of quinoid formation

Numerous phenols and catechols are known to be substrates for tyrosinase. While the catalytic mechanism of phenol oxidation by tyrosinase has been well studied, little work has been done to determine the influence of substituents on the reaction. In the present investigation, we explored the effects of changing substituents at the 2 and 6 position on the mechanism of tyrosinase-catalyzed oxidation of 4-allyl and 4-propylphenols and catechols. We have previously demonstrated that tyrosinase initially oxidizes hydroxychavicol (4-allylcatechol) to an o-quinone (3,5-cyclohexadien-1,2-dione) which because of the relatively acidic protons in the benzyl position, readily isomerizes to the tautomeric p-quinone methide (4-allylidene-2,5-cyclohexadien-1-one, QM) (Bolton et al., 1994). We have confirmed through GSH trapping studies that oxidation of 4-allylphenol by tyrosinase yields the same o-quinone GSH conjugates as hydroxychavicol. In contrast, the presence of additional ortho substituents dramatically alters the mechanism of tyrosinase-catalyzed oxidation of 4-alkylphenols. For example, eugenol (4-allyl-2-methoxyphenol), which possesses 1 ortho-methoxy substituents, is not oxidized to a o-quinone or a QM. However, when both ortho positions are substituted with methoxy groups, direct tyrosinase-catalyzed QM formation is observed. The identity of the 4-allyl-2,6-dimethoxyphenol-QM was confirmed by CDCl3 extraction of the post incubate and characterization of the QM by H-1-NMR. Finally, the oxidation rates were determined by following the appearance of either the o-quinone or the QM spectrophotometrically or by monitoring increases in the GSH trapped conjugates by HPLC. The data showed that the tyrosinase-catalyzed rates of oxidation decreased in the following order; hydroxychavicol > 4-allylphenol, 4-allyl-6-methoxycatechol > 4-allyl-2,6-dimethoxyphenol much greater than eugenol (no quinoids detected). These results show that changes in the substituents ortho to the phenolic hydroxy group not only lead to an alteration in the mechanism and type of quinoid formed, there is also a dramatic substituent effect on the rate of tyrosinase oxidation. (C) 1997 Elsevier Science Ireland Ltd.