Identification of Advanced Reaction Products Originating from the

Apr 15, 2009 - Initial 4-Oxo-2-nonenal-cysteine Michael Adducts ... 4-Oxo-2-nonenal (ONE), an aldehyde originating from the peroxidation of ω6 ...
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Chem. Res. Toxicol. 2009, 22, 957–964

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Identification of Advanced Reaction Products Originating from the Initial 4-Oxo-2-nonenal-cysteine Michael Adducts Yuuki Shimozu, Takahiro Shibata, Makoto Ojika, and Koji Uchida* Graduate School of Bioagricultural Sciences, Nagoya UniVersity, Nagoya 464-8601, Japan ReceiVed February 11, 2009

4-Oxo-2-nonenal (ONE), an aldehyde originating from the peroxidation of ω6 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 °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-acetyl5-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. Introduction Lipid peroxidation in tissue and in tissue fractions represents a degradative process, which is the consequence of the production and the propagation of free radical reactions primarily involving membrane polyunsaturated fatty acids (PUFAs), and has been implicated in the pathogenesis of numerous diseases including atherosclerosis, diabetes, cancer, and rheumatoid arthritis, as well as in drug-associated toxicity, postischemic reoxygenation injury, and aging (1). The peroxidative breakdown of PUFAs has also been implicated in the pathogenesis of many types of liver injury and, especially, hepatic damage induced by several toxic substances. The lipid peroxidation leads to the formation of a broad array of different products with diverse and powerful biological activities. Among them are a variety of different aldehydes (2). The primary products of lipid peroxidation, lipid hydroperoxides, can undergo carbon-carbon bond cleavage via alkoxyl radicals in the presence of transition metals, giving rise to the formation of short-chain aldehydes. These reactive aldehydic intermediates readily form covalent adducts with cellular macromolecules, including proteins, leading to the disruption of important cellular functions. The important agents that give rise to the modification of protein may be represented by R,β-unsaturated aldehydic intermediates, such as 2-alkenals, 4-hydroxy-2-alkenals, and 4-oxo-2-alkenals (3, 4). * To whom correspondence should be addressed. E-mail: uchidak@ agr.nagoya-u.ac.jp.

One of the most reactive aldehydes originating from the peroxidation of ω6-PUFAs may be 4-oxo-2-nonenal (ONE)1 (5, 6). West et al. (7) compared the apoptotic responses induced by a series of toxic R,β-unsaturated aldehydes, including ONE, in a human colorectal cancer cell line and demonstrated that the apoptotic response induced by ONE and other related aldehydes involves the activation of caspases, proteolysis of downstream caspase targets, and nucleosomal DNA fragmentation. Our recent study showed that the p53 signaling pathway is involved in neuronal cell death induced by ONE (8). More strikingly, the ONE-induced activation of the p53 pathway was found to mediate the gene expression of Fas, suggesting the involvement of the Fas/FasL signaling pathway, which is known to be one of the downstream mediators in the p53-dependent apoptotic pathway. It has been reported that ONE reacts with biological molecules, such as protein and DNA, at rates significantly greater than other lipid peroxidation-derived aldehydes (5, 9-13). Upon reaction with proteins, ONE selectively modifies the nucleophilic side chains of lysine, histidine, cysteine, and arginine (14). The predominant initial reaction appears to involve the Michael addition to the central ONE double bond, more at C3 than at C2, to give substituted 4-oxononanals. These adducts are relatively unstable and could be further converted to stable 1 Abbreviations: BSA, bovine serum albumin; COSY, correlation spectroscopy; DNPH, 2,4-dinitrophenylhydrazine; ESI, electrospray ionization; HMBC, 1H-detected heteronuclear multiple bond correlation; HMQC, 1 H-detected heteronuclear multiple quantum correlation; ONE, 4-oxo-2nonenal; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; TMS, tetramethylsilane.

10.1021/tx900059k CCC: $40.75  2009 American Chemical Society Published on Web 04/15/2009

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products, which include dihydrofuran, dihydropyrrole, and isomeric 4-ketoamide derivatives originating from the reaction of ONE with lysine (15) and a substituted imidazole derivative with arginine (16). ONE also forms furan derivatives upon its reaction with cysteine and histidine derivatives (17, 18). 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 remains to be established. In the present study, to gain further structural insight into sulfhydryl modification by the lipid peroxidation product, we carried out a comprehensive study on the identification of ONEN-acetyl-L-cysteine adducts and identified several advanced reaction products that originated from the initial Michael adducts.

Materials and Methods General Experimental Procedures. The 1H NMR spectra (400 or 600 MHz) and 13C NMR spectra (100 or 150 MHz) were recorded at 27 °C on an AMX 400 (400 MHz) or an AMX 600 (600 MHz) spectrometer (Bruker, Rheinstetten, Germany). In all cases, tetramethylsilane (TMS) or the solvent peak served as the internal standard for reporting chemical shifts, which are expressed as parts per million downfield from TMS (δ scale). The 1H-1H COSY (correlation spectroscopy), HMQC (1H-detected heteronuclear multiple quantum correlation), and HMBC (1H-detected heteronuclear multiple bond correlation) experiments were carried out using an AMX 400 (400 MHz) or an AMX 600 (600 MHz) instrument. Liquid chromatography/mass spectrometry (LC/MS) was conducted using a Platform II (VG Biotech) in an electrospray ionization positive (ESP+) mode. The high-resolution mass spectral data were obtained using a Mariner Biospectrometry Workstation (Applied Biosystems, Foster City, CA) in the positive electrospray ionization (ESI) mode. High-performance liquid chromatography (HPLC) was conducted using a JASCO system (JASCO, Tokyo). Preparation of ONE. ONE was synthesized from 2-pentylfuran according to the procedure of Sun et al. (19). N-Bromosuccinimide (1.9 g) and pyridine (10 mL) were added to 2-pentylfuran (2.3 g) in THF/acetone/water (5:4:2) on an ice bath. The reaction mixture was stirred for 1 h at this temperature and then kept at ambient temperature. After 2 h, the mixture was extracted with CH2Cl2 (3 × 50 mL). The combined CH2Cl2 layer was dried (Na2SO4) and concentrated in vacuo, and the residue was subjected to silica gel chromatography using hexanes-EtOAc (3/1, v/v) as the eluent to give 556.7 mg (24% yield) of ONE. Reaction of N-Acetyl-L-cysteine with ONE. The reaction mixture containing 10 mM N-acetyl-L-cysteine was incubated with 10 mM ONE in 0.1 M sodium phosphate buffer (pH 7.4) or citrate buffer (pH 4.0). After incubation for 24 h at 37 °C, the reaction mixtures were analyzed by a reverse-phase HPLC on a Cadenza CD-C18 column (4.6 mm id. × 250 mm, Imtakt, Kyoto, Japan) eluted with a linear gradient of water containing 0.1% TFA (solvent A)-acetonitrile (solvent B) (time ) 0 min, 10% B; 40-50 min, 100% B) at a flow rate of 0.5 mL/min. The elution profiles were monitored by absorbance at 240 nm. 2,4-Dinitrophenylhydrazine (DNPH) Derivatization of the Initial ONE-Cysteine Michael Adduct. An aliquot of 10 mM ONE-cysteine adduct (I) was treated with an equal volume of 5 mM DNPH in 2 N HCl. After the DNPH mixture was allowed to stand at room temperature for 30 min in the dark, the reaction mixture was extracted with CHCl3. The CHCl3 layer was concentrated in vacuo, and the residue was analyzed by a reverse-phase HPLC on a Cadenza CD-C18 column (4.6 mm id. × 250 mm, Imtakt, Kyoto, Japan) eluted with a linear gradient of water containing 0.1% TFA (solvent A)-acetonitrile (solvent B) (time ) 0 min, 10% B; 40-50 min, 100% B) at a flow rate of 0.5 mL/ min. The elution profiles were monitored by absorbance at 240 nm. Modification of Protein by the Initial ONE-Cysteine Michael Adduct. Bovine serum albumin (BSA) (1 mg/mL) was incubated with the ONE-cysteine adduct (I) (0, 0.2, 0.6, and 2.0 mM) in 0.1

Shimozu et al. M sodium phosphate buffer (pH 7.4) for 24 h at 37 °C. SDSpolyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli (20). The protein was stained with Coomassie Blue. Identification of Adducts I and II. ONE (50 mM) was added to a solution of N-acetyl-L-cysteine (50 mM) in 0.1 M sodium phosphate buffer (pH 7.4). After incubation for 1 h at 37 °C, the mixture was subjected to preparative HPLC on a Develosil ODSHG-5 column (8.0 mm id. × 250 mm, Nomura Chemicals, Aichi, Japan), using 30% aqueous CH3CN containing 0.1% TFA to isolate I (61.8%, based on ONE) and II (21.5%, based on ONE). 2-(Acetylamino)-3-(2-oxo-1-(2-oxoethyl)heptylsulfanyl)propionic Acid (I). 1H NMR (CDCl3, 600 MHz): δ 9.71 (2H, d, J ) 4.8 Hz, H-1), 4.80 (1H, dd, J ) 5.4, 12.6 Hz, H-2′a), 4.78 (1H, dd, J ) 5.4, 13.2 Hz, H-2′b), 3.81 (1H, t, J ) 6.0 Hz, H-3a), 3.80 (1H, t, J ) 6.0 Hz, H-3a), 3.26 (2H, dd, J ) 9.0, 18.6 Hz, H-2), 3.11 (1H, dd, J ) 4.8, 13.2 Hz, H-1′b), 3.00 (2H, m, H-1′a), 2.89 (1H, dd, J ) 6.0, 13.2 Hz, H-1′b), 2.85 (2H, dt, J ) 4.8, 18.6 Hz, H-2), 2.80-2.74 (2H, m, H-5), 2.66-2.61 (2H, m, H-5), 2.10 (6H, s, H-5′), 1.67-1.57 (4H, m, H-6), 1.37-1.27 (8H, m, H-7, H-8), 0.91 (6H, t, J ) 7.2 Hz, H-9). 13C NMR (CDCl3, 150 MHz): δ 205.7 (2C, C-4b), 199.5 (C-1b), 199.4 (C-1a), 172.5 (2C, C-4′), 171.6 (2C, C-3′), 52.4 (2C, C-2′), 45.5 (2C, C-3), 45.1 (C-2a), 44.9 (C2b), 40.3 (C-5b), 40.2 (C-5a), 31.9 (2C, C-1′), 31.5 (2C, C-7), 23.8 (2C, C-6), 23.1 (2C, C-5′), 22.6 (2C, C-8), 14.1 (2C, C-9). 2-(Acetylamino)-3-(1-formyl-3-oxooctyl)heptylsulfanyl)propionic Acid (II). 1H NMR (CDCl3, 600 MHz): δ 9.43 (1H, d, J ) 1.8 Hz, H-1), 9.42 (1H, d, J ) 1.2 Hz, H-1), 4.87-4.84 (1H, m, H-2′), 4.79 (1H, dd, J ) 6.0, 12.0 Hz, H-2′), 3.78-3.74 (2H, m, H-2), 3.07-3.01 (6H, m, H-3, H-1′), 2.76 (2H, dd, J ) 6.0, 18.0 Hz, H-3), 2.50-2.43 (4H, m, H-5), 2.14 (3H, s, H-5′), 2.11 (3H, s, H-5), 1.63-1.57 (4H, m, H-6), 1.34-1.26 (8H, m, H-7, H-8), 0.90 (6H, t, J ) 7.2 Hz, H-9). 13C NMR (CDCl3, 150 MHz): δ 208.3 (C-4), 208.1 (C-4), 193.4 (C-1), 193.3 (C-1), 172.2 (2C, C-4′), 171.9 (C-3′), 171.6 (C-3′), 52.6 (2C, C-2′), 48.5 (2C, C-2), 43.2 (C-5), 43.1 (C-5), 41.7 (C-3), 41.4 (C-3), 32.9 (C-1′), 31.5 (C-1′), 31.4 (2C, C-7), 23.6 (2C, C-6), 23.1 (2C, C-5′), 22.6 (2C, C-8), 14.0 (2C, C-9). Identification of Adduct III. ONE (50 mM) was added to a solution of N-acetyl-L-cysteine (50 mM) in 0.1 M sodium phosphate buffer (pH 7.4). After incubation for 24 h at 37 °C, the mixture was subjected to preparative HPLC on a Develosil ODS-HG-5 column (8.0 mm id. × 250 mm, Nomura Chemicals) using a gradient of 10-60% aqueous CH3CN containing 0.1% TFA to isolate III (0.63%, based on ONE). 4-Acetyl-5-hydroxyl-6-(2-oxoheptyl)thiomorpholine-3-carboxylic Acid (III). 1H NMR (CDCl3, 600 MHz): δ 5.48 (1H, dd, J ) 3.0, 4.8 Hz, H-2′), 5.44 (1H, d, J ) 2.4 Hz, H-1), 3.46 (1H, ddd, J ) 2.4, 4.8, 9.0 Hz, H-2), 3.55 (1H, dd, J ) 4.8, 13.8 Hz, H-1′), 3.16 (1H, dd, J ) 3.0, 13.8 Hz, H-1′), 2.93 (1H, dd, J ) 9.0, 18.0 Hz, H-3), 2.60 (1H, dd, J ) 4.8, 18.0 Hz, H-3), 2.44 (2H, t, J ) 7.2 Hz, H-5), 1.62-1.56 (2H, m, H-6), 1.35-1.25 (4H, m, H-7, H-8), 0.90 (3H, t, J ) 7.2 Hz, H-9). 13C NMR (CDCl3, 150 MHz): δ 208.5 (C-4), 175.5 (C-3′), 174.5 (C4′), 76.1 (C-1), 51.7 (C-2′), 43.5 (C-3), 43.5 (C-5), 42.7 (C-2), 31.5 (C-7), 30.2 (C-1′), 23.6 (C-6), 22.6 (C-8), 21.6 (C-5′), 14.1 (C-9). Identification of Adducts IVa, IVb, and V. ONE (10 mM) was added to a solution of N-acetyl-L-cysteine (10 mM) in 0.1 M sodium phosphate buffer (pH 7.4). After incubation for 3 days at 37 °C, the mixture was subjected to preparative HPLC on a Develosil ODS-HG-5 column (8.0 mm id. × 250 mm, Nomura Chemicals) using a gradient of 10-60% aqueous CH3CN containing 0.1% TFA. A secondary purification was conducted using 25% aqueous CH3CN containing 0.1% TFA to give IVa (3.7%, based on ONE), IVb (2.6%, based on ONE), and a 1:1 mixture of the diastereomers of V (5.1%, based on ONE). 2-(Acetylamino)-3-[(3-butyl-4-oxocyclopent-2-en-1-yl)sulfanyl]propionic Acid (IVa). 1H NMR (CDCl3, 600 MHz): δ 7.16 (1H, m, H-1), 4.82 (1H, m, H-2′), 4.01 (1H, m, H-2), 3.15 (1H, dd, J ) 4.8, 13.8 Hz, H-1′), 3.00 (1H, dd, J ) 5.4, 13.8 Hz, H-1′),

4-Oxo-2-nonenal-cysteine Michael Adducts

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Figure 1. HPLC profiles of ONE-cysteine adducts monitored at UV 240 nm. N-Acetyl-L-cysteine (10 mM) was incubated with an equimolar concentration of ONE (10 mM) in 0.1 M phosphate buffer (pH 7.4) at 37 °C.

2.91 (1H, dd, J ) 6.0, 19.2 Hz, H-3), 2.41 (1H, dd, J ) 1.8, 19.2 Hz, H-3), 2.20 (2H, t, J ) 7.8 Hz, H-6), 2.10 (3H, s, H-5′), 1.47 (2H, m, H-7), 1.34 (2H, m, H-8), 0.92 (3H, t, J ) 7.2 Hz, H-9). 13 C NMR (CDCl3, 150 MHz): δ 207.3 (C-4), 172.4 (C-3′), 171.2 (C-4′), 155.8 (C-1), 148.2 (C-5), 52.4 (C-2′), 43.6 (C-3), 42.1 (C2), 32.3 (C-1′), 29.9 (C-7), 24.5 (C-6), 23.2 (C-5′), 22.6 (C-8), 14.0 (C-9). 2-(Acetylamino)-3-[(3-butyl-4-oxocyclopent-2-en-1-yl)sulfanyl]propionic Acid (IVb). 1H NMR (CDCl3, 600 MHz): δ 7.17 (1H, m, H-1), 4.84 (1H, m, H-2′), 4.01 (1H, m, H-2), 3.19 (1H, dd, J ) 4.8, 13.8 Hz, H-1′), 3.08 (1H, dd, J ) 5.4, 13.8 Hz, H-1′), 2.92 (1H, dd, J ) 6.6, 19.2 Hz, H-3), 2.41 (1H, dd, J ) 1.8, 19.2 Hz, H-3), 2.20 (2H, t, J ) 7.8 Hz, H-6), 2.10 (3H, s, H-5′), 1.47 (2H, m, H-7), 1.34 (2H, m, H-8), 0.92 (3H, t, J ) 7.2 Hz, H-9). 13 C NMR (CDCl3, 150 MHz): δ 207.5 (C-4), 172.3 (C-3′), 171.2 (C-4′), 155.9 (C-1), 148.1 (C-5), 52.3 (C-2′), 43.9 (C-3), 42.5 (C2), 33.2 (C-1′), 29.8 (C-7), 24.5 (C-6), 23.2 (C-5′), 22.6 (C-8), 14.0 (C-9).

2-(Acetylamino)-3-[(4-butyl-5-oxocyclopent-3-en-1-yl)sulfanyl]propionic Acid (V). 1H NMR (CDCl3, 600 MHz): δ 7.32 (1H, m, H-1a), 7.29 (1H, m, H-1b), 4.76 (1H, m, H-2′b), 4.66 (1H, dd, J ) 6.6, 12.6 Hz, H-2′a), 3.54 (1H, dd, J ) 2.4, 7.2 Hz, H-3a), 3.47 (1H, dd, J ) 5.4, 14.4 Hz, H-1′b), 3.42 (1H, dd, J ) 2.4, 7.2 Hz, H-3b), 3.17 (1H, dd, J ) 5.4, 14.4 Hz, H-1′a), 3.14 (1H, m, H-2a), 3.11 (1H, m, H-2b), 3.11 (1H, dd, J ) 6.6, 14.4 Hz, H-1′a), 2.98 (1H, dd, J ) 4.2, 14.4 Hz, H-1′b), 2.45 (1H, br d, J ) 2.4 Hz, H-2a), 2.42 (1H, br d, J ) 2.4 Hz, H-2b), 2.21 (4H, t, J ) 7.2 Hz, H-6), 2.16 (3H, s, H-5′b), 2.12 (3H, s, H-5′a), 1.48 (4H, m, H-7), 1.35 (4H, m, H-8), 0.92 (6H, t, J ) 7.2 Hz, H-9). 13C NMR (CDCl3, 150 MHz): δ 209.4 (C-4a), 209.0 (C-4b), 173.2 (C-4b′), 172.2 (C4′a), 172.0 (C-3′a), 171.9 (C-3′b), 156.9 (C-1a), 156.4 (C-1b), 145.5 (C-5b), 145.3 (C-5a), 54.2 (C-2′b), 53.2 (C-2′a), 45.9 (C-3b), 45.7 (C-3a), 35.9 (C-2a), 35.5 (C-2b), 33.5 (C-1′b), 33.1 (C-1′a), 29.8 (2C, C-7), 24.9 (2C, C-6), 23.0 (C-5′a), 22.9 (C-5′b), 22.6 (2C, C-8), 14.0 (2C, C-9). Identification of Adducts VI and VII. ONE (50 mM) was added to a solution of N-acetyl-L-cysteine (50 mM) in citrate buffer (pH 4.0). After incubation for 24 h at 37 °C, the mixture was subjected to preparative HPLC on a Develosil ODS-HG-5 column (8.0 mm id. × 250 mm, Nomura Chemicals) using 40% aqueous CH3CN containing 0.1% TFA to give VI (0.63%, based on ONE) along with the known adduct, 2-(acetylamino)-3-(2-pentylfuran-3-ylsulfanyl)propionic acid (VII) (6.0%, based on ONE). 4-Acetyl-6-(2-oxoheptyl)-3,4-dihydro-2H-1,4-thiazine-3-carboxylic Acid (VI). 1H NMR (CDCl3, 400 MHz): δ 6.73 (1H, s, H-1), 5.78 (1H, t, J ) 3.6 Hz, H-2′), 3.43 (1H, dd, J ) 3.6, 13.2 Hz, H-1′), 3.20 (2H, d, J ) 6.4 Hz, H-3), 3.07 (1H, dd, J ) 3.6, 13.2 Hz, H-1′), 2.50 (2H, t, J ) 7.2 Hz, H-5), 2.27 (3H, s, H-5′), 1.63-1.55 (2H, m, H-6), 1.35-1.23 (4H, m, H-7, H-8), 0.90 (3H, t, J ) 7.2 Hz, H-9). 13C NMR (CDCl3, 100 MHz): δ 207.7 (C-4), 171.9 (C-3′), 169.1 (C-4′), 120.6 (C-1), 108.4 (C-2), 50.7 (C-2′), 48.8 (C-3), 42.2 (C-5), 31.5 (C-7), 27.3 (C-1′), 23.5 (C-6), 22.6 (C-8), 21.7 (C-5′), 14.1 (C-9). 2-(Acetylamino)-3-(2-pentylfuran-3-ylsulfanyl)propionic Acid (VII). 1H NMR (CDCl3, 400 MHz): δ 6.46 (1H, d, J ) 3.2 Hz, H-1), 5.99 (1H, t, J ) 3.2 Hz, H-2), 4.82 (1H, dd, J ) 4.8, 12.4 Hz, H-2′), 3.23 (2H, d, J ) 4.8 Hz, H-1′), 2.61 (2H, t, J ) 3.6 Hz, H-5), 2.04 (3H, s, H-5′), 1.64 (2H, m, H-6), 1.35-1.32 (4H, m, H-7, H-8), 0.91 (3H, t, J ) 7.2 Hz, H-9). 13C NMR (CDCl3, 100 MHz): δ 173.3 (C-3′), 171.1 (C-4′), 160.8 (C-4), 141.4 (C-3), 119.6 (C-1), 107.3 (C-2), 52.7 (C-2′), 37.6 (C-1′), 31.5 (C-7), 28.6 (C5), 27.7 (C-6), 23.1 (C-5′), 22.5 (C-8), 14.2 (C-9).

Table 1. 1H and 13C NMR Assignments of Adducts I and II position

adduct I

1

9.71 (2H, d, J ) 4.8 Hz)

2

3.26 2.85 3.80 3.81

3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′

(2H, (2H, (1H, (1H,

dd, J ) 9.0, 18.6 Hz) dt, J ) 4.8, 18.6 Hz) t, J ) 6.0 Hz) t, J ) 6.0 Hz)

2.80-2.74 2.66-2.61 1.67-1.57 1.37-1.27 0.91 3.00 3.11 2.89 4.80 4.78

(6H, (2H, (1H, (1H, (1H, (1H,

adduct II

(2H, (2H, (4H, (8H,

m) m) m) m)

t, J ) 7.2 Hz) m) dd, J ) 4.8, 13.2 dd, J ) 6.0, 13.2 dd, J ) 5.4, 12.6 dd, J ) 5.4, 13.2

2.10 (6H, s)

9.43 (1H, d, J ) 1.8 Hz) 9.42 (1H, d, J ) 1.2 Hz) 3.78-3.74 (2H, m) 3.07-3.01 (2H, m) 2.76 (2H, dd, J ) 6.0, 18.0 Hz) 2.50-2.43 (4H, m) 1.63-1.57 (4H, m) 1.34-1.26 (8H, m) 0.90 (6H, t, J ) 7.2 Hz) 3.07-3.01 (4H, m)

Hz) Hz) Hz) Hz)

4.87-4.84 (1H, m) 4.79 (1H, dd, J ) 6.0, 12.0 Hz) 2.14 (3H, s) 2.11 (3H, s)

adduct I

adduct II

199.4, 199.5

193.3, 193.4

45.1, 44.9

48.5 (2C)

45.5 (2C)

41.4, 41.7

205.7 (2C) 40.2, 40.3

208.1, 208.3 43.1, 43.2

23.8 (2C) 31.5 (2C) 22.6 (2C) 14.1 (2C) 31.9 (2C)

23.6 (2C) 31.4 (2C) 22.6 (2C) 14.0 (2C) 31.5, 32.9

52.4 (2C)

52.6 (2C)

171.6 (2C) 172.5 (2C) 23.1 (2C)

171.6, 171.9 172.2 (2C) 23.1 (2C)

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Figure 2. Carbonyl functionality of the initial ONE-cysteine Michael addition products. (A) HPLC chromatograms of authentic and DNPH-treated ONE-cysteine Michael adduct. Chromatogram: a, authentic adduct I; b, DNPH-treated adduct I. (B) SDS-PAGE analysis of BSA treated with the initial ONE-cysteine adduct. BSA (1 mg/mL) was incubated with the ONE-cysteine adduct (I) (0, 0.2, 0.6, and 2.0 mM) in 0.1 M sodium phosphate buffer (pH 7.4) for 24 h at 37 °C.

Table 2. 1H and 13C NMR Assignments of Adduct III position 1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′

δH (ppm) 5.44 3.46 2.93 2.60

(1H, (1H, (1H, (1H,

d, J ) 2.4 Hz) ddd, J ) 2.4, 4.8, 9.0 Hz) dd, J ) 9.0, 18.0 Hz) dd, J ) 4.8, 18.0 Hz)

2.44 (2H, t, J ) 7.2 Hz) 1.62-1.56 (2H, m) 1.35-1.25 (4H, m) 0.90 3.55 3.16 5.48

(3H, (1H, (1H, (1H,

t, J ) 7.2 Hz) dd, J ) 4.8, 13.8 Hz) dd, J ) 3.0, 13.8 Hz) dd, J ) 3.0, 4.8 Hz)

2.32 (3H, s)

δC (ppm) 76.1 42.7 43.5 208.5 43.5 23.6 31.5 22.6 14.1 30.2 51.7 175.5 174.5 21.6

Results and Discussion Reaction of N-Acetyl-L-cysteine with ONE. N-Acetyl-Lcysteine (10 mM) was incubated with an equimolar concentration of ONE (10 mM) in 0.1 M phosphate buffer (pH 7.4). After incubation at 37 °C for times ranging from 1 to 72 h, the reaction mixture was analyzed by a reverse-phase HPLC monitored at 240 nm. As shown in Figure 1, several peaks corresponding to the ONE-N-acetyl-L-cysteine adducts were detected. Within the first hour, adducts I and II were detected as the major products. Subsequent incubation resulted in the disappearance of these products and concomitant formation of four products, III, IVa, IVb, and V. By 24 h, IVa, IVb, and V were the major products. After 72 h of incubation, the yields of IVa, IVb, and V were more prominent, whereas both I and II were undetectable. In addition, IVa, IVb, and V were detected as the major products

Figure 3. Key HMBC correlations of III.

Figure 4. Key HMBC correlations of IV and V.

even after 1 week of incubation (data not shown), suggesting that they represent the most prominent cysteine adducts formed by ONE. Identification of the Initial Michael Addition Products (I and II). The LC-MS analysis of adducts I and II gave the same pseudomolecular ion peaks at m/z 318 (M + H)+ (data not shown), which could be expected from the Michael addition to the central ONE double bond to give the substituted 4-oxononanals. The most likely structures may be the C-2 and C-3 Michael addition products shown below. It is also possible that adducts I and II are diastereomers of either the C-2 or the C-3 Michael addition product.

4-Oxo-2-nonenal-cysteine Michael Adducts

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Scheme 1. Proposed Mechanism for the Formation of 2-Cyclopentenone Derivatives upon Reaction of N-Acetyl-L-cysteine with ONE

To elucidate the structures, adducts I and II were subjected to NMR characterization. The assignment of the proton and carbon signals of adducts I and II was made on the basis of the proton and carbon chemical shifts, proton-proton couplings, and 1H-1H COSY, HMQC, and HMBC correlations (Table 1). These data clearly showed that both adducts I and II correspond to the Michael addition products generated through the sulfhydryl addition at C-3 and C-2 of ONE, respectively. The distinction of I over II was made on the basis of the 1H-1H COSY spectrum. The 1H-1H COSY spectrum of I showed the correlations between the aldehydic proton H-1 (9.71 ppm) with

the methylene protons H-2 (2.85 ppm), the H-2 with the methine proton H-3 (3.80 or 3.81 ppm), whereas in II, the aldehydic proton H-1 at 9.42 or 9.43 ppm would be coupled to a methine proton H-2 (3.78-3.74 ppm). Therefore, it is established that I and II are the C-3 and C-2 Michael addition adducts, respectively. Adduct I was previously reported by Sayre and coworkers (17), and our data were consistent with their results. Meanwhile, as far as we know, the isolation and identification of II has not been made until this study probably due to its instability. It was suggested that, consistent with the previous observations by Zhang et al. (17), the sulfhydryl group of N-acetyl-L-cysteine might have slightly more preference for the binding at the C-3 than at the C-2 of ONE. The 13C NMR spectrum of I analyzed in D2O showed the signal at 91.4 ppm, which was typical of a carbon atom attached to two oxygen atoms (Supporting Information, Figure S1). The signals derived from the aldehyde and ketone groups were also observed in the 1H and 13C NMR spectra. Thus, these data suggest that the initial Michael adducts may be in equilibrium with the hydrated state. Carbonyl Functionality of the Initial Michael Adducts. Because the ONE-cysteine Michael adducts (I and II) possess a free aldehyde, they are supposed to retain the carbonyl functionality. Indeed, our preliminary experiments have demonstrated that ONE is capable of introducing DNPH-reactive carbonyl groups into proteins (unpublished data). To examine whether the initial Michael adducts possess a carbonyl functionality, I was exposed to excess DNPH, and the products were analyzed by reverse-phase HPLC. As shown in Figure 2A, I indeed reacted with the carbonyl reagent and provided a new product. The LC-MS analysis of the product gave a pseudomolecular ion peaks at m/z 480, which corresponded to the reaction of one molecule of I with one molecule of DNPH and the loss of two molecules of water, consistent with a dihydropyridazine (21). The formation of these Michael adducts may, therefore, contribute to the generation of carbonyl groups in the protein exposed to ONE. In addition, when I was incubated

Table 3. 1H and 13C NMR Assignments of Adducts IVa, IVb, and V δH (ppm) position 1

adduct IVa

adduct IVb

adduct V

155.9

42.1

42.5

43.6

43.9

4

207.3

207.5

5

148.2

148.1

24.5 29.9 22.6 14.0 32.3

24.5 29.8 22.6 14.0 33.2

3

6 7 8 9 1′

2′

7.32 7.29 4.01 (1H, m) 4.01 (1H, m) 3.14 2.45 3.11 2.42 2.91 (1H, dd, J ) 6.0, 19.2 Hz) 2.92 (1H, dd, J ) 6.6, 19.2 Hz) 3.54 2.41 (1H, dd, J ) 1.8, 19.2 Hz) 2.41 (1H, dd, J ) 1.8, 19.2 Hz) 3.42

2.20 1.47 1.34 0.92 3.15 3.00

(2H, (2H, (2H, (3H, (1H, (1H,

7.17 (1H, m)

t, J ) 7.8 Hz) 2.20 m) 1.47 m) 1.34 t, J ) 7.2 Hz) 0.92 dd, J ) 4.8, 13.8 Hz) 3.19 dd, J ) 5.4, 13.8 Hz) 3.08

4.82 (1H, m)

t, J ) 7.8 Hz) 2.21 m) 1.48 m) 1.35 t, J ) 7.2 Hz) 0.92 dd, J ) 4.8, 13.8 Hz) 3.17 dd, J ) 5.4, 13.8 Hz) 3.11 3.47 2.98 4.84 (1H, m) 4.66 4.76 (2H, (2H, (2H, (3H, (1H, (1H,

(1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H,

adduct IVa adduct IVb 155.8

2

7.16 (1H, m)

δC (ppm)

(4H, (4H, (4H, (6H, (1H, (1H, (1H, (1H, (1H, (1H,

m) (a) m) (b) m) (a) br d, J ) 2.4 Hz) (a) m) (b) br d, J ) 2.4 Hz) (b) dd, J ) 2.4, 7.2 Hz) (a) dd, J ) 2.4, 7.2 Hz) (b)

t, J ) 7.2 Hz) m) m) t, J ) 7.2 Hz) dd, J ) 5.4, 14.4 dd, J ) 6.6, 14.4 dd, J ) 5.4, 14.4 dd, J ) 4.2, 14.4 dd, J ) 6.6, 12.6 m) (b)

3′ 4′ 5′

2.10 (3H, s)

2.10 (3H, s)

2.12 (3H, s) (a) 2.16 (3H, s) (b)

Hz) Hz) Hz) Hz) Hz)

(a) (a) (b) (b) (a)

adduct V 156.9 (a) 156.4 (b) 35.9 (a) 35.5 (b) 45.7 (a) 45.9 (b) 209.4 (a) 209.0 (b) 145.3 (a) 145.5 (b) 24.9 (2C) 29.8 (2C) 22.6 (2C) 14.0 (2C) 33.1 (a) 33.5 (b)

52.4

52.3

172.4 171.2

172.3 171.2

23.2

23.2

53.2 (a) 54.2 (b) 172.0 (2C) 172.2 (a) 173.2 (b) 23.0 (a) 22.9 (b)

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Shimozu et al.

Scheme 2. Proposed Mechanism for the Formation of ONE-Cysteine Adducts upon Reaction of N-Acetyl-L-cysteine with ONEa

a

Solid line, under neutral conditions; dashed line, under acidic conditions.

with BSA in sodium phosphate buffer (pH 7.4) at 37 °C for 24 h, we observed a significant mobility shift in the protein bands on the SDS-PAGE analysis (Figure 2B). These data provide strong evidence that the aldehyde moiety of the initial Michael addition products has the ability to undergo secondary reactions with amino groups to form Schiff base adducts. Identification of Advanced Reaction Products Originating from the Initial Michael Adducts. The 1H and 13C NMR spectra of III showed no signals corresponding to -CHO and -NH groups (Table 2). In addition, the HMBC spectrum of III showed that the three-bond correlation of the singlet proton at 5.44 ppm (H-1) correlated with the ester carbonyl carbon at 174.5 ppm (C-4′), the R carbon of N-acetyl-L-cysteine at 51.7 ppm (C-2′), and the methylene carbon of ONE at 43.5 ppm (C-3) (Figure 3). Moreover, III has a hydroxyl group at C-1; this was supported by the chemical shift in the proton (5.44 ppm) and carbon (76.1 ppm) signals at C-1. The structure of III was thus established as 4-acetyl-5-hydroxyl-6-(2-oxoheptyl)thiomorpholine-3-carboxylic acid. Adduct III is likely to be formed from the nucleophilic addition of the secondary amine of II to the aldehyde carbon. Adducts IVa, IVb, and V were detected as the most prominent cysteine adducts formed by ONE (Figure 1). After purification, these adducts were characterized by mass spectrometry and NMR. The high-resolution ESI-MS of IVa showed a molecular ion peak at m/z 300.12573 (M + H)+ corresponding to the molecular formula of C14H22NO4S, which contained one molecule of ONE and N-acetyl-Lcysteine. The signals corresponding to the R,β-unsaturated linkage and aldehyde group of ONE were not observed in both the 1H- and the 13C NMR spectra (Table 3). The 1H-1H COSY spectrum showed the correlations between the methyl group at 0.92 ppm (H-9) with the protons at 1.34 ppm (H8), the H-8 with the protons at 1.47 ppm (H-7), and the H-7

with the protons at 2.20 ppm (H-6) but observed no correlation between the H-6 with the H-5. Moreover, the methine proton (H-1) at 7.16 ppm correlated with the methine proton at 4.01 ppm (H-2). Long-range coupling was also observed between H-1 and H-6 due to allyl coupling. The quaternary carbon signal at 148.2 ppm was assigned to C-5, because of the HMBC correlations with H-1, H-2, H-3, H-6, and H-7. In addition, the HMBC spectrum showed correlations of the carbonyl carbon at 207.3 ppm (C-4) with H-1, H-2, H-3, and H-6 (Figure 4). These data suggest the presence of a cyclopentenone structure. On the basis of the above data, IVa was identified as 2-(acetylamino)-3-[(3-butyl-4-oxocyclopent-2-en-1-yl)sulfanyl]propionic acid. On the other hand, the high-resolution ESI-MS of IVb showed the same molecular ion peak at m/z 300.12799 (M + H)+ as IVa corresponding to the molecular formula of C14H22NO4S. In addition, the NMR profiles of IVb were almost identical to those of IVa, suggesting that IVb might be a configurational isomer of IVa. The resulting complicated diastereotopic splittings as well as contributions from multiple conformers make the 1H NMR spectrum difficult to directly analyze. Therefore, we have not yet resolved their configurations. The high-resolution ESI-MS of adduct V also showed the same ion peak at m/z 300.12576 (M + H)+ as that of IVa and IVb, indicating the molecular formula of C14H22NO4S. Complete analysis of the combination of the 1H-1H COSY, HMQC, and HMBC spectra suggested that adduct V is a similar cyclopentenone derivative with the isomer ratio of about 1:1 as IVa and IVb. Distinguishable features of V from IVa and IVb are the substituted position of thioether linkage. The 1H-1H COSY spectrum showed correlations between the methine proton H-1 with the methylene protons H-2, the H-2 with the methine proton H-3. The HMBC experiment revealed long-range coupling from H-1′ to C-3, which

4-Oxo-2-nonenal-cysteine Michael Adducts

established the thioether at the C-3 position of V (Figure 4). Allyl coupling was also observed between H-1 and H-6. Thus, V was identified as 2-(acetylamino)-3-[(4-butyl-5-oxocyclopent-3-en-1-yl)sulfanyl]propionic acid. In addition, the adduct was presumed to be nearly a 1:1 mixture of the configurational isomers, as judged by the observed peak doubling for many of the 13C signals. The yields of adducts IV (a + b) and V after 72 h of incubation were approximately 10 and 8% of the ONE, respectively. Formation of these 2-cyclopentenone adducts can be reasonably explained by the mechanism (Scheme 1) in which deprotonation occurs at the R-position (C-5) of I and II followed by cyclization to form the 2-cyclopentenone structures through an intramolecular aldol condensation. On the other hand, when ONE was incubated with N-acetylL-cysteine under acidic conditions in citrate buffer (pH 4.0), I and II were detected as the major products even after 24 h of incubation, whereas the 2-cyclopentenone derivatives were undetectable (Supporting Information, Figure S3). Instead of the 2-cyclopentenone adducts, we detected two products (VI and VII), which were not observed under neutral conditions (pH 7.4). On the basis of the NMR characterization, VII was identified to be a furan derivative, 2-(acetylamino)-3-(2-pentylfuran-3-ylsulfanyl)propionic acid (VII), which was previously identified by Zhang and Sayre (17). We have also observed that this furan derivative can be formed when ONE (10 mM) was incubated with a high concentration (100 mM) of N-acetyl-Lcysteine (data not shown). This finding and the observation that the furan type adduct (VII) originating from the C-3 Michael adduct I was formed under acidic conditions, but not under neutral conditions, suggest that the excess amount of N-acetylL-cysteine might cause the shift in pH of the reaction mixture from neutral to acidic, leading to the change in the profile of the products. With regards to VI, both the 1H and the 13C NMR spectra exhibited no signals corresponding to an R,β-unsaturated linkage. In addition, the aldehyde proton in ONE and the -NH proton in N-acetyl-L-cysteine were also not observed. The HMBC spectrum showed that the three-bond correlation of the singlet proton at 6.73 ppm (H-1) correlated with the ester carbonyl carbon at 169.05 ppm (C-4′), the R carbon of N-acetylL-cysteine at 50.65 ppm (C-2′), and the methylene carbon of ONE at 48.79 ppm (C-3). Moreover, the quaternary carbon signal at 108.43 ppm was assigned to C-2; this was supported by the HMBC correlations with H-1, H-3, and H-1′. On the basis of these data, the structure of VI was therefore established as 4-acetyl-6-(2-oxoheptyl)-3,4-dihydro-2H-1,4- thiazine-3-carboxylic acid. This adduct is likely to be generated through the dehydration of adduct III.

Conclusion Scheme 2 summarizes the mechanism for the formation of the ONE-cysteine adducts. ONE initially formed two substituted 4-oxononanals I and II as the major products. It was suggested that, due to the presence of the free aldehyde group, both adducts might possess a carbonyl functionality. They were indeed derivatized with a carbonyl reagent (Figure 2A), suggesting that the formation of these Michael adducts may contribute to the generation of carbonyl groups in the protein exposed to ONE (unpublished data). In addition, incubation of BSA with I resulted in a significant mobility shift of the protein bands on the SDS-PAGE analysis (Figure 2B). The data suggest that the initial Michael addition products may have the ability to undergo secondary reactions with amino groups to form Schiff base adducts, leading to

Chem. Res. Toxicol., Vol. 22, No. 5, 2009 963

the formation of intra- and intermolecular cross-links. We here showed that, under physiological conditions, the initial Michael adducts (I and II) could be converted to the thiomorpholine derivative (III) and 2-cyclopentenone derivatives (IVa, IVb, and V) as the ONE-cysteine adducts. Especially, the 2-cyclopentenone type adducts were the major products, representing the most prominent ONE-cysteine adducts. The formation of such cyclopentenone type adducts from the substituted 4-oxononanals was reasonable, since the 4-ketoaldehydes can be readily converted to the 2-cyclopentenone derivatives through the base-catalyzed cyclization (22). It will be important to determine to what extent the 2-cyclopentenone may constitute a marker of oxidative stress in vivo. We also confirmed the formation of the furan (VII) and dihydrothiazine (VI) derivatives upon reaction of the cysteine derivative with ONE, while they were formed under the acidic condition. These results establish the chemical precedent for interpreting the reaction of proteins with ONE generated from the peroxidation of PUFAs. The knowledge of the sulfhydryl reactivity of ONE provides an underpinning for the eventual interpretation of various types of biological activities, including apoptotic responses (7, 8), that are being observed for this important endogenously formed molecule. Furthermore, these adducts may represent the excellent immunogens that are capable of stimulating adaptive immune response. Of interest, our recent study showed that the monoclonal antibodies, showing recognition specificity toward DNA, can bind ONE-cysteine adducts (23). These findings suggest the connection between sulfhydryl modification of proteins by ONE and autoimmune response. Further studies are required to understand the biological consequences of the production of ONE under oxidative stress. Acknowledgment. This work was supported by research grants from the Ministry of Education, Culture, Sports, Science, and Technology in Japan (K.U.). Supporting Information Available: 1H and 13C NMR spectra, HPLC profiles, and 1H-1H COSY, HMQC, and HMBC spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

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