Alpha,beta-Dehydro-3,4-dihydroxyphenylalanine Derivatives: Rate and Mechanism of Formation

The amino acid L-3,4-dihydroxyphenylalanine (DOPA), when present in the primary sequence of proteins, does not form melanin upon oxidation to the quinone, since its amine moiety participates in a peptide bond and cannot undergo internal cyclization. Instead, peptidyl DOPA quinone is available for other reactions. We have investigated the oxidation chemistry of a low molecular weight peptidyl DOPA analog, N-acetylDOPA ethyl ester (NAcDEE), and have shown that a major product of oxidation is an unsaturated DOPA derivative, N-acetyl-alpha,beta-dehydroDOPA ethyl ester (NAc delta DEE) (see companion paper, Rzepecki et al., Arch. Biochem. Biophys. (1991) 285, 17-26). In the present study, we have explored kinetic and mechanistic features of the conversion of NAcDEE to NAc delta DEE and found that the reaction requires: (i) oxidation of NAcDEE to the quinone, (ii) the presence of a Lewis base as a catalyst (phosphate anion was the best of those tried in the pH range 6.0-8.0), and (iii) prevention of competing reactions such as Michael additions. Conversion efficiencies in the presence of Lewis bases ranged between 12 and 19% at pH 8.0 and 35 and 90% at pH 6.0. At least two separate reaction mechanisms appeared necessary to explain the kinetic data: (i) a pseudo-first-order mechanism at pH 6.0 and above, and (ii) an additional second-order mechanism at higher pH which involved both NAcDEE catechol and quinone. The apparent pseudo-first-order rate constants increased with pH from 2.36 X 10(-4) s-1 at pH 6.0 to about 30 X 10(-4) s-1 at pH 8.0 in 0.1 M sodium phosphate. Tautomerization of DOPA quinone to dehydroDOPA may thus be a factor in the sclerotization of natural structures incorporating DOPA containing proteins.