On the mechanism of formation of N‐acetyldopamine quinone methide in insect cuticle

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Abstract
The mechanism of formation of quinone methide from the sclerotizing precursor N‐acetyldopamine (NADA) was studied using three different cuticular enzyme systems viz. Sarcophaga bullata larval cuticle, Manduca sexta pharate pupae, and Periplaneta americana presclerotized adult cuticle. All three cuticular samples readily oxidized NADA. During the enzyme‐catalyzed oxidation, the majority of NADA oxidized became bound covalently to the cuticle through the side chain with the retention of o‐diphenolic function, while a minor amount was recovered as N‐acetylnorepinephrine (NANE). Cuticle treated with NADA readily released 2‐hydroxy‐3′,4′‐dihydroxyacetophenone on mild acid hydrolysis confirming the operation of quinone methide sclerotization. Attempts to demonstrate the direct formation of NADA‐quinone methide by trapping experiments with N‐acetylcysteine surprisingly yielded NADA‐quinone‐N‐acetylcysteine adduct rather than the expected NADA‐quinone methide‐N‐acetylcysteine adduct. These results are indicative of NADA oxidation to NADA‐quinone and its subsequent isomerization to NADA‐quinone methide. Accordingly, all three cuticular samples exhibited the presence of an isomerase, which catalyzed the conversion of NADA‐quinone to NADA‐quinone methide as evidenced by the formation of NANE—the water adduct of quinone methide. Thus, in association with phenoloxidase, newly discovered quinone methide isomerase seems to generate quinone methides and provide them for quinone methide sclerotization.