Formation of mono- and bis-Michael adducts by the reaction of nucleophilic amino acids with hydroxymethylvinyl ketone, a reactive metabolite of 1,3-butadiene

Chem Res Toxicol. 2009 May;22(5):918-25. doi: 10.1021/tx900006b.


Previously, our laboratory has shown that hydroxymethylvinyl ketone (HMVK), a Michael acceptor oxidation product of the 1,3-butadiene metabolite, 3-butene-1,2-diol, readily reacts with hemoglobin at physiological conditions and that mass spectrometry of trypsin-digested peptides suggested adduct formation with various nucleophilic amino acids. In the present study, we characterized reactions ofHMVK (3 mM) with three model nucleophilic amino acids (6 and/or 15 mM): N-acetyl-L-cysteine (NAC),L-valinamide, and N-acetyl-L-lysine (NAL). NAC was the most reactive toward HMVK followed by L-valinamide and NAL. HMVK incubations with each amino acid at pH 7.4 and 37 degrees C resulted in the formation of a mono-Michael adduct. In addition, HMVK incubated with NAL gave rise to two additional bis-Michael adducts characterized by LC/MS, LC/MS/MS, 1H NMR, and 1H-detected heteronuclear single quantum correlation. The relative ratios of areas of NAL monoadduct (adduct 1) and diadducts (adducts 2 and 3) at 6 h were 49, 21, and 30% of total product area, respectively. The formation of adduct 2 was dependent upon the presence of both adduct 1 and HMVK, whereas the formation of adduct 3 was dependent upon the presence of adduct 2 only. Monoadducts were formed by a Michael addition reaction of one HMVK moiety with nucleophilic amino acid, whereas NAL diadducts were products of two Michael addition reactions of two HMVK moieties followed by enolization and formation of an octameric cyclic product. NAL diadduct (adduct 3) was formed by loss of a water molecule from adduct 2 followed by autoxidation of one of the hydroxy groups, yielding a diketone conjugated system. Collectively, our results provide strong evidence that HMVK can react with various nucleophilic residues and form different types of adducts, suggesting that a variety of proteins may be subjected to these modifications, which could result in loss of protein function.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acids / chemistry*
  • Butadienes / chemistry
  • Butadienes / metabolism*
  • Butanones / chemistry*
  • Chromatography, High Pressure Liquid
  • Lysine / analogs & derivatives


  • Amino Acids
  • Butadienes
  • Butanones
  • hydroxymethylvinyl ketone
  • N(alpha)-acetyllysine
  • 1,3-butadiene
  • Lysine