Formation of Mono- and Bis-Michael Adducts by the Reaction of Nucleophilic Amino Acids with Hydroxymethylvinyl Ketone, a Reactive Metabolite of 1,3-Butadiene

Abstract

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 of HMVK (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, 37°C resulted in 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 (HSQC). 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. Formation of adduct 2 was dependent upon the presence of both adduct 1 and HMVK whereas formation of adduct 3 was dependent upon 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 2 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 autooxidation 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.

Characterization of HKI-272 Covalent Binding to Human Serum Albumin

“…However, there are reports that Michael addition can take place between the -amine of lysine and the functional group of ␣,␤-unsaturated keto under physiologic pH and temperature if some electrophilic groups are introduced. For example, Barshteyn and Elfarra (2009) recently observed that a lysine adduct with hydroxymethylvinyl ketone was formed when N␣-acetyl lysine was incubated with the compound at 37°C, pH 7.4. We can propose that the intramolecular hydrogen bond between the hydroxyl oxygen and the hydrogen of the ␣-or ␤-carbon of the carbonyl group could contribute to enhancement of electrophilicity of the ␤-carbon, leading to Michael addition at physiologic pH and temperature.…”

“…We can propose that the intramolecular hydrogen bond between the hydroxyl oxygen and the hydrogen of the ␣-or ␤-carbon of the carbonyl group could contribute to enhancement of electrophilicity of the ␤-carbon, leading to Michael addition at physiologic pH and temperature. In addition, on incubation of hydroxymethylvinyl ketone with hemoglobin followed by tryptic digestion, the compound was found to be covalently bound to several nucleophilic AA residues, including cysteine and lysine residues in hemoglobin (Barshteyn and Elfarra, 2009 …”

Mass Spectral Analyses of Hydroxymethylvinyl Ketone-Hemoglobin Adducts Formed after in Vivo Exposure of Sprague−Dawley Rats to 3-Butene-1,2-diol

“…Characterization of the cyclic diadduct 2 after incubation of HMVK with NAL (14) led us to investigate the formation of such diadducts on amino groups of Hb in vivo (Figure 2) after rats were treated with 25 or 200 mg/kg BDD. Upon trypsin digestion, multiple modified peptides containing HMVK cyclic diadduct 2 were detected by LC/ESI/MS in rats treated with 25 and 200 mg/kg BDD, respectively (Table 2A).…”

“…HMVK-GSH conjugate may undergo a spontaneous or a GSH-transferase-catalyzed retro-Michael reaction as demonstrated previously with the GSH conjugates of trans-4-phenyl-3-buten-2-one and 4-hydroxy-2-nonenal (25, 26). Interestingly, the kinetics of HMVK disappearance and HMVK adduct formation in the presence of nucleophilic amino acids suggest the reaction of HMVK with amino groups could be more reversible than the reaction with N-acetyl-L-cysteine since HMVK was not completely depleted and its levels did not change in between 30 min and 6 h after incubation with excess valinamide (14). …”

“…We have also characterized two cyclic HMVK-NAL diadducts consisting of two HMVK moieties that formed as a result of a bis-Michael addition reaction (14) (Figure 2). We proposed that cyclic diadduct 1 formed via enolization of one of the HMVK moieties followed by cyclization to form an octameric ring which further undergoes loss of water and autooxidation resulting in cyclic diadduct 2 (Figure 2).…”