Influence of quinone methide reactivity on the alkylation of thiol and amino groups in proteins: studies utilizing amino acid and peptide models

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Abstract

Quinone methides (QMs) are electrophiles formed in several biological processes including direct oxidations of 4-alkylphenols by cytochromes P450. These species may be responsible for the adverse effects of certain phenolic compounds through protein alkylation, but little information is available concerning specific targets or the resulting mechanisms of cell injury. The present goal was to determine the most likely sites of adduct formation among competing protein nucleophiles utilizing QMs of varying electrophilicity. Reactions of poorly reactive, moderately reactive, and highly reactive QMs, 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone (BHT-QM), 6-tert-butyl-2-(2′-hydroxy-1′,1′-dimethylethyl)-4-methylene-2,5-cyclohexadienone (BHTOH-QM), and 2-tert-butyl-6-methyl-4-methylene-2,5-cyclohexadienone (BDMP-QM), respectively, were investigated in aqueous solutions with nucleophilic amino acids. Each QM rapidly formed a thioether derivative of cysteine with little or no competition from the addition of water (hydration). The α-amino groups were the primary sites of alkylation for all other amino acids examined including lysine, histidine, tyrosine, and serine, and the pseudo-first order rates were 5 to 8-fold greater than the rates of hydration. Alkylation of the side chain nitrogens of lysine and histidine occurred at about one-fourth the rate of hydration for BDMP-QM, but no reaction was detectable for BHT-QM and no reactions occurred between QMs and amino acid hydroxyl groups. The results indicate that, based on chemical reactivity, peptide alkylation should occur in the order cysteine thiol>N-terminal amino>Nε-lysine=NIm-histidine, with side chain modifications occurring only with the more electrophilic QMs. Reactions of QMs with the tripeptide Gly-His-Lys confirmed the results with amino acids as Nα-glycine alkylation predominated, but side chain adducts also formed with BHTOH-QM and BDMP-QM. Human hemoglobin was treated with QMs, hydrolyzed, and assayed by HPLC-thermospray mass spectrometry. This work revealed that Nε-lysine was the main alkylation site, emphasizing the importance of factors, in addition to chemical reactivity, which influence protein modification by electrophiles.

Introduction

Phenolic compounds with appropriate alkyl substituents in the 4-position can be oxidized by cytochromes P450 or peroxidases to p-quinone methides, electrophiles capable of undergoing non-enzymatic Michael additions with cellular nucleophiles as shown in Fig. 11, 2. These species exhibit a wide range of reactivities which are influenced by substituents at the exocyclic methylene carbon as well as by substituents on the cyclohexadienone ring 3, 4, 5. For example, 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone (BHT-QM), a quinone methide derived from the antioxidant butylated hydroxytoluene (BHT), reacts relatively slowly with nucleophiles due to shielding of the oxo group by bulky hydrophobic substituents 6, 7. Low reactivity in this case is explained by the lack of hydrogen bonding interactions with water, effectively reducing positive charge density at the site of nucleophile attack. Substituting one bulky tert-butyl substituent by a methyl group, as in 2-tert-butyl-6-methyl-4-methylene-2,5-cyclohexadienone (BDMP-QM) (Fig. 1), markedly increases the reactivity of the quinone methide due to the enhanced hydrogen bonding interactions between the oxo group and solvent [6]. The reactivity of 6-tert-butyl-2-(2′-hydroxy-1′,1′-dimethylethyl)-4-methylene-2,5-cyclohexadienone (BHTOH-QM) is intermediate between that of BHT-QM and BDMP-QM as intramolecular hydrogen bonding partly compensates for the hindered access of water to the oxo group [7].

The formation of quinone methides in vitro has been linked to cytotoxicity in studies with hepatocytes 6, 8, liver slices 9, 10, bronchiolar Clara cells [11]and keratinocytes from mice or rats [12]. Data from in vivo studies implicate quinone methides in hepatotoxicity [13], pulmonary toxicity [14], carcinogenicity [15], and tumor promotion 16, 17. Adducts resulting from the reactions of these electrophiles with glutathione, proteins, and DNA have been determined following exposures to the corresponding phenolic precursors both in vitro and in vivo 9, 10, 11, 12, 18, 19. Quinone methides also inhibit enzymes irreversibly through covalent modifications of critical residues 20, 21. There is abundant evidence, therefore, that the generation of quinone methides results in adduct formation with important biochemical and toxicological consequences.

The principal targets for attack by quinone methides and quinoid species in general are cysteine-containing compounds including glutathione and proteins with accessible thiol residues 1, 18, 22, 23, 24. Soft (i.e. delocalized and polarizable) electrophiles including quinoids, α, β-unsaturated aldehydes, and other Michael acceptors readily combine with soft nucleophiles such as thiols and react more slowly with hard nucleophiles such as amino and hydroxyl groups 1, 4, 25. The relative rates for addition of water to BHT-QM, BHTOH-QM and BDMP-QM are 1:8:65 at 25°C [6]demonstrating substantial increases in reactivity toward a hard nucleophile with increasing stabilization of the ionic resonance form (Fig. 1). It is known that BDMP-QM alkylates proteins at nucleophilic sites other than thiols, as adducts were produced with horse heart myoglobin that does not contain cysteine [26], and amino groups of DNA bases are alkylated by BHTOH-QM [19]. The goal of the current project was to probe potential targets for covalent modifications of proteins by quinone methides of varying reactivities utilizing amino acids and a peptide model to assess chemical reactivities at various nucleophilic sites. The work was extended to a model protein in which lysine residues were found to be the principal sites of adduct formation. These data will facilitate further studies of quinone methide-protein binding, a necessary step in elucidating mechanisms by which these electrophiles injure cells.

Section snippets

Chemicals

All chemicals were purchased from Aldrich (Milwaukee, WI) or Sigma (St. Louis, MO) unless otherwise indicated. 2-tert-Butyl-4,6-dimethylphenol was obtained from TCI America (Portland, OR) and 6-tert-butyl-2-(2′-hydroxy-1′,1′-dimethylethyl)-4-methylphenol was synthesized as described [27]. The quinone methides were prepared by adding 5 g of lead dioxide to 200 mg of each phenol dissolved in 200 ml of pentane and stirring for 30 min at 25°C [6]. This mixture was filtered, 200 ml of dry

Results

Reactions of quinone methides with amino acids were investigated under pseudo-first order conditions with excess amino acid at pH 7.4. The order of reactivity (Table 1) agrees with previous results, i.e. BHT-QM<BHTOH-QM<BDMP-QM, and all three reacted most rapidly with cysteine yielding the thioether adduct shown in Fig. 2 as the only product in each case. With other amino acids, water addition to quinone methides (hydration) competed with adduct formation and generated the corresponding benzyl

Discussion

The reactivities of quinone methides are influenced by both intermolecular and intramolecular interactions affecting the relative contributions of the resonance forms shown in Fig. 11, 2, 3, 4, 5, 6, 7. In aqueous media at pH 7.4 the low reactivity of BHT-QM with various nucleophiles indicates predominance of the cyclohexadienone structure. Rates of adduct formation with BDMP-QM were substantially greater, 100-fold in the case of Nα-alkylation due to a larger contribution of the charged

Acknowledgements

This research was supported by National Institutes of Health grants ES06216 and CA41248.

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