Identification of Advanced Reaction Products Originating from the Initial 4-Oxo-2-nonenal-cysteine Michael Adducts

Abstract

4-Oxo-2-nonenal (ONE), an aldehyde originating from the peroxidation of omega6 polyunsaturated fatty acids, preferentially reacts with the cysteine residues of protein. Despite the fact that there has been significant recent interest in the protein reactivity and biological activity of ONE, the structural basis of the ONE-cysteine adducts remain to be established. In the present study, to gain a structural insight into the sulfhydryl modification by ONE, we characterized reaction products that originated from the initial ONE-cysteine Michael adducts. N-Acetyl-L-cysteine (10 mM) was incubated with an equimolar concentration of ONE in 0.1 M phosphate buffer (pH 7.4) at 37 degrees C. Within 1 h of incubation, the reaction of N-acetyl-L-cysteine with ONE resulted in the formation of two (C-2 and C-3) Michael addition products possessing a carbonyl functionality. Subsequent incubation of the reaction mixture resulted in their disappearance and concomitant formation of advanced reaction products, including a minor product III and major products IVa, IVb, and V. Product III was identified to be a thiomorpholine derivative, 4-acetyl-5-hydroxyl-6-(2-oxoheptyl)thiomorpholine-3-carboxylic acid, which might have originated from the C-2 Michael addition product. The major products were identified to be the novel 2-cyclopentenone derivatives, that is, 2-(acetylamino)-3-[(3-butyl-4-oxocyclopent-2-en-1-yl)sulfanyl]propionic acid (IVa and its isomer IVb) and 2-(acetylamino)-3-[(4-butyl-5-oxocyclopent-3-en-1-yl)sulfanyl]propionic acid (V), which might be generated through the base-catalyzed cyclization of the C-2 and C-3 Michael addition products, respectively. The furan derivative, which has been reported as the end product of the Michael adducts, was found to be formed only under acidic conditions. Thus, this study identified the novel ONE-cysteine adducts, including the most prominent 2-cyclopentenone derivatives, that originated from the initial Michael adducts.

Identification of a Lipid Peroxidation Product as the Source of Oxidation-specific Epitopes Recognized by Anti-DNA Autoantibodies*

“…ONE also forms furan derivatives upon its reaction with the cysteine and histidine derivatives (43,44). In our recent study, we carried out a comprehensive study on the identification of the ONE-N-acetyl-L-cysteine adducts and identified several advanced reaction products that originated from the initial Michael adducts (45). We tried to elucidate the structure of the autoantigenic epitopes recognized by the MRL-lpr-derived mAb DSO and identified the ONE-cysteine adduct as a candidate epitope.…”

Effect of lipid oxidation products on the formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in model systems

“…4-Hydroxy-2-nonenal was synthesized according to the procedure of Gardner, Bartelt, and Weisleder (1992). 4-Oxo-2-nonenal was prepared from 2-pentylfuran according to Shimozu, Shibata, Ojika, and Uchida (2009). 4-Oxo-2-hexenal was prepared analogously to 4-oxo-2-nonenal from 2-ethylfuran.…”

Lipid Peroxidation Modification of Protein Generates Nϵ-(4-Oxononanoyl)lysine as a Pro-inflammatory Ligand

“…Preparation of ONE-ONE was synthesized by the oxidative opening of the 2-pentylfuran ring as described previously (22). Briefly, N-bromosuccinimide (Wako Pure Chemicals, Japan) and pyridine were sequentially added to 2-pentylfuran in THF/ acetone/water (5:4:2) at ice bath temperature.…”