title: Influence of quinone methide reactivity on the alkylation of thiol and amino groups in proteins: studies utilizing amino acid and peptide models.
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'-hydroxyl-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 alpha-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 > N epsilon-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 N alpha-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 N epsilon-lysine was the main alkylation site, emphasizing the importance of factors, in addition to chemical reactivity, which influence protein modification by electrophiles.
authors.affiliation: Department of Medicinal Chemistry and Pharmacognosy (M/C 781), College of Pharmacy, University of Illinois at Chicago 60612-7231, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Bolton
authors.forename: S B
authors.initials: SB
authors.lastname: Turnipseed
authors.forename: J A
authors.initials: JA
authors.lastname: Thompson
mesh_terms: D000478:Alkylation; D000588:Amines / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism*; D002084:Butylated Hydroxytoluene / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D045563:Indolequinones; D007211:Indoles / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008956:Models, Chemical; D010455:Peptides / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011809:Quinones / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000378:metabolism*
chemical_list: D000588:Amines; D000596:Amino Acids; C110180:BDMP-QM; C110172:BHTOH-QM; D045563:Indolequinones; D007211:Indoles; D010455:Peptides; D011506:Proteins; D011809:Quinones; D013438:Sulfhydryl Compounds; C068040:quinone methide; D002084:Butylated Hydroxytoluene; C024709:2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone