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 tyrosina se. While the catalytic mechanism of phenol oxidation by tyrosinase ha s been well studied, little work has been done to determine the influe nce 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-pr opylphenols and catechols. We have previously demonstrated that tyrosi nase initially oxidizes hydroxychavicol (4-allylcatechol) to an o-quin one (3,5-cyclohexadien-1,2-dione) which because of the relatively acid ic protons in the benzyl position, readily isomerizes to the tautomeri c p-quinone methide (4-allylidene-2,5-cyclohexadien-1-one, QM) (Bolton et al., 1994). We have confirmed through GSH trapping studies that ox idation of 4-allylphenol by tyrosinase yields the same o-quinone GSH c onjugates as hydroxychavicol. In contrast, the presence of additional ortho substituents dramatically alters the mechanism of tyrosinase-cat alyzed oxidation of 4-alkylphenols. For example, eugenol (4-allyl-2-me thoxyphenol), which possesses 1 ortho-methoxy substituents, is not oxi dized to a o-quinone or a QM. However, when both ortho positions are s ubstituted with methoxy groups, direct tyrosinase-catalyzed QM formati on is observed. The identity of the 4-allyl-2,6-dimethoxyphenol-QM was confirmed by CDCl3 extraction of the post incubate and characterizati on 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 spectrop hotometrically or by monitoring increases in the GSH trapped conjugate s by HPLC. The data showed that the tyrosinase-catalyzed rates of oxid ation decreased in the following order; hydroxychavicol > 4-allylpheno l, 4-allyl-6-methoxycatechol > 4-allyl-2,6-dimethoxyphenol much greate r 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 i s also a dramatic substituent effect on the rate of tyrosinase oxidati on. (C) 1997 Elsevier Science Ireland Ltd.