Chem. Res. Toxicol. 2008, 21, 895–903
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Intermolecular Peroxyl Radical Reactions during Autoxidation of Hydroxy and Hydroperoxy Arachidonic Acids Generate a Novel Series of Epoxidized Products Claus Schneider,†,§ William E. Boeglin,†,§ Huiyong Yin,†,‡,§ Ned A. Porter,‡,§ and Alan R. Brash*,†,§ Departments of Pharmacology and Chemistry, Vanderbilt UniVersity, and the Vanderbilt Institute of Chemical Biology, Vanderbilt UniVersity, NashVille, Tennessee 37232 ReceiVed October 1, 2007
We report on the identification of novel epoxide products formed during the autoxidative transformation of 15S-hydroxy- and 15S-hydroperoxy-eicosatetra-5Z,8Z,11Z,13E-enoic acids (15S-HETE and 15SHPETE). These epoxides account for about 20–30% of the polar compounds detected during the early stages of autoxidation. Their common structural features are retention of the original 15S-hydroxy or 15S-hydroperoxy moiety with epoxidation of the 11Z or 13E double bonds in the conjugated diene of the starting material. Four main epoxyalcohol isomers were characterized from the hydroxy fatty acid 15SHETE, comprising two pairs of diastereomers with either an 11,12-trans or 13,14-trans epoxide functionality. Four main epoxyhydroperoxides identified from 15S-HPETE comprised two pairs with cis or trans epoxide configuration at the 11,12 position. To account for these transformations, we propose a mechanism involving peroxyl radical dependent dimerization or oligomerization of the fatty acid hydroxy or hydroperoxy derivatives into covalent intermediates resulting in intermolecular transfer of oxygen from the peroxyl radical to the epoxide group. Autoxidation of [18O2]-15S-HPETE carrying an O-18 labeled hydroperoxide showed that the 11,12-cis epoxy oxygen of the epoxy-hydroperoxide product was enriched in the labeled oxygen, providing evidence that in part it was derived directly from the starting hydroperoxide and not from molecular oxygen. Thus, intermediate dimerization and possibly oligomerization of fatty acid peroxyl radicals provides a mechanism of epoxidation of fatty acid derivatives during lipid peroxidation and a potential route to other products including aldehydes formed via carbon chain cleavage. Introduction Fatty acid hydroperoxides are among the first covalently complete products in the autoxidation reactions of polyunsaturated lipids with molecular oxygen. These unstable hydroperoxides are, in turn, degraded into a complex mixture of derivatives during the propagation phase of autoxidation. The ensuing reactions include cleavage of the fatty acid carbon chains, producing a series of reactive aldehydes, among the most prominent and best characterized of which are the 4-hydroxyalkenals, with 4-hydroxy-nonenal (4-HNE1) being the prototypical product (1–5). These aldehydes have been investigated extensively for bioactivities, and as a result are implicated in numerous sequelae of oxidative stress (6–9). The studies reported here are a part of efforts to elucidate the mechanism(s) of autoxidative transformation of ω6 hydroxy and hydroperoxy fatty acids into the R,β-unsaturated aldehydes 4-HNE and 4-HPNE, respectively. Previous studies showed that the configuration of the ω6 hydroxy and ω6 hydroperoxy group of the starting material is retained in the aldehydic cleavage products * Corresponding author. Alan R. Brash, Department of Pharmacology, Vanderbilt University Medical School, 23rd Ave. S. at Pierce, Nashville, TN 37232-6602. Tel: 615-343-4495. Fax: 615-322-4707. E-mail:
[email protected]. † Department of Pharmacology. ‡ Department of Chemistry. § Vanderbilt Institute of Chemical Biology. 1 Abbreviations: CID, collision-induced dissociation; DBU, 1,8diazabicyclo[5.4.0]undec-7-ene; ESI, electrospray ionization; HETE, hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; 4-HNE, 4-hydroxy-2E-nonenal; 4-HPNE, 4-hydroperoxy-2E-nonenal.
(10, 11). This finding led to the proposal of a mechanism for aldehyde formation via Hock-cleavage of a suitable dihydroperoxide intermediate (11). Subsequent autoxidation studies using the chemically synthesized linoleic acid dihydroperoxides largely disproved this pathway as a route to carbon chain cleavage and aldehyde synthesis during lipid peroxidation (12). In the course of autoxidation experiments using 15S-hydroxyand 15S-hydroperoxy-eicosatetraenoates (15S-HETE and 15SHPETE, respectively) as model substrates, we noted the formation of a group of products with mobility on HPLC between that of the starting compounds and the more polar fractions containing HNE. Members of the group were characterized by having no distinct chromophore, exhibiting only end absorbance in the UV. We hypothesized that a structural analysis of these products could possibly provide some new insights on the pathways leading from fatty acid hydro(pero)xides to aldehydes by carbon chain cleavage. Here, we report the isolation and structural characterization of these products as epoxyhydro(pero)xy derivatives of arachidonic acid. The products are formed in a mechanism involving dimerization or oligomerization of fatty acid peroxyl derivatives. Subsequent breakdown of the peroxide bridge forming the dimer or oligomer can give rise to the epoxyhydro(pero)xy derivatives and potentially also to aldehydic cleavage products.
Experimental Procedures Preparation of 15S-HPETE and 15S-HETE. 15S-HPETE and 15S-HETE were prepared as described using soybean lipoxygenase
10.1021/tx700357u CCC: $40.75 2008 American Chemical Society Published on Web 03/07/2008
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Chem. Res. Toxicol., Vol. 21, No. 4, 2008
(Sigma) (13). 15S-HETE was prepared from 15S-HPETE by reduction with sodium borohydride. Both products were isolated by RP-HPLC using a Waters Symmetry C18 column (1.9 × 30 cm) eluted with a solvent of acetonitrile/water/acetic acid (70/30/ 0.01, by vol.) at a flow rate of 12 mL/min and UV detection at 210 nm. Stock solutions of 10 or 20 mg/mL in methanol were stored at -20 °C until use. Autoxidation Reactions. For the initial analysis of product formation, 5 µg aliquots of 15S-HETE and 15S-HPETE were incubated in an open plastic tube in an oven at 37 °C for the time indicated. After the incubation time indicated, HPLC column solvent was added, and the entire reaction was injected on HPLC using a Waters Symmetry C18 column (0.46 × 25 cm) eluted with a solvent of acetonitrile/water/acetic acid (60/40/0.01, by vol.) at a flow rate of 1 mL/min and UV detection using a diode array detection (Agilent 1100). For product isolation and characterization, several 1 mg aliquots of 15S-HPETE and 15S-HETE, respectively, were evaporated from solvent and incubated as a lipid film in an open glass vial at 37 °C for 1 h. HPLC conditions were as described for the initial analyses except for UV detection at 205 nm only. The collected products were extracted into methylene chloride, evaporated, and methylated using ethereal diazomethane. All methylated products were further purified by SP-HPLC using a Whatman Partisil Si 5-µm column (0.46 × 25 cm) eluted with a solvent of hexane/isopropanol/acetic acid (100/2/0.1, by vol.) at a flow rate of 1 mL/min and UV detection at 205 nm. Preparation of Benzoyl Derivatives for CD Spectroscopy. Aliquots (20 µg each) of the methyl esters of products 5, 6, 8, and 9 were treated with 40 µg of triphenylphosphine in methanol and isolated using SP-HPLC (Whatman Partisil 5 µm, hexane/isopropanol/acetic acid 100/2/0.1, by vol.). The reduced products 5, 6, 8, and 9, and products 1, 2, and 7 were dissolved in 50 µL of acetonitrile. Benzoyl chloride and DBU, 1 µL each, and a few grains of dimethylaminopyridine were added, and the reactions were kept at room temperature overnight (14, 15). The reactions were evaporated from solvent under a stream of nitrogen, dissolved in dichloromethane, and washed with water twice. The benzoyl derivatives were isolated by RP-HPLC using a Waters Symmetry C18 5-µm column (0.46 × 25 cm) eluted with a solvent of methanol/water/acetic acid (95/5/0.01, by vol.) at a flow rate of 1 mL/min and UV detection at 235 nm (retention time ≈6.5 min). For CD spectroscopy, the derivatives were dissolved in acetonitrile to an absorbance of 0.8–1.2 AU at 228 nm. LC-MS, GC-MS, NMR, and CD Spectroscopy. For LC-ESIMS analyses, a Thermo Finnigan LC Quantum system was used. Samples dissolved in methanol/acetonitrile/water (3/2/1, by vol.) were introduced directly into the ESI interface using a syringe pump or via a Waters Symmetry C18 3-µm column (0.2 × 15 cm) eluted with a water/acetonitrile gradient containing 10 mM ammonium acetate at a flow rate of 0.2 mL/min. Samples from the experiments with O-18 labeled 15S-HPETE were resolved on the same HPLC column using isocratic elution with acetonitrile/water/acetic acid (60:40:0.01, by vol.) at a flow rate of 0.2 mL/min. The heated capillary ion lens was operated at 280 °C. Nitrogen was used as a nebulization and desolvation gas. The electrospray potential was held at 3.5 kV. Mass spectra were acquired in the negative and positive ion modes over the mass range m/z 50 to 500 at 2 s/scan in the full scan mode only. For direct liquid infusion, samples were dissolved in methanol and infused via a T-connector into the ESI interface using a syringe pump at a flow rate of 20 µL/min. The second line of the T connector supplied a solvent of acetonitrile/ water containing 10 mM ammonium acetate (90:10, by vol.) at a flow rate of 0.2 mL/min. GC-MS (EI 70 eV) analyses were performed using a Thermo Finnigan DSQ system equipped with a SPB-1 column (6 m × 0.25 mm) connected to a 5 m postcolumn. Samples were injected via split injection at 125 °C, and after 1 min, the temperature was programmed to 280 at 20 °C/min. The TMS ether derivatives were prepared by reacting the products with 10 µL of bis(trimethylsilyl)trifluoroacetamide in the presence of 1 µL of pyridine for 30
Schneider et al. min at room temperature. The reagents were evaporated under a stream of nitrogen and dissolved in hexane for GC-MS analysis. NMR spectra were recorded on a Bruker Avance DRX 400 MHz instrument using CDCl3 as solvent. Chemical shifts are reported relative to CHCl3 (δ ) 7.24 ppm). CD spectra (350–200 nm) were recorded on a Jasco J-720 spectropolarimeter using acetonitrile as a solvent. The configuration of the hydroxyl group in the products was determined from the sign of the first Cotton effects of the benzoate ester derivative. The direction of the coupling of the transition moment of the two chromophores attached to the chiral hydroxy group, i.e., the benzoate and the 13,14- or 11,12-single bond, respectively, reflects the arrangement of the substituents at the chiral center and, therefore, provides a means to determine the absolute configuration. There is no fixed relationship between the absolute (R or S) configuration of the molecule and the sign of the first Cotton effect (14, 15). (11S,12S) and (11R,12R) 11,12(trans)-Epoxy-15S-hydroxy5Z,8Z,13E-eicosatrienoic Acid (Methyl Ester) 1 and 2. First diastereomer: 1H NMR, 400 MHz, CDCl3 (δ ) 7.24 ppm), δ 5.96 (dd, J ) 6.2 Hz/15.6 Hz, 1H, H14); 5.48 (m, 1H, H8); 5.41 (m, 2H, H9, H13); 5.36 (m, 2H, H5, H6); 4.11 (m, 1H, H15); 3.65 (s, 3H, OCH3); 3.13 (dd, J ) 2.0 Hz/7.8 Hz, 1H, H12); 2.86 (dt, J ) 2.1 Hz/5.3 Hz, 1H, H11); 2.76 (dd, J ) 5.6 Hz, 2H, H7); 2.37 (m, 2H, H10a/b); 2.30 (t, J ) 7.4 Hz, 2H, H2); 2.09 (dt, J ) 6.5 Hz, 2H, H4); 1.68 (m, 2H, H3). LC-MS (ESI): m/z 373.2 [M + Na]+ (methyl ester). EI-MS (70 eV) methyl ester, TMS ether: m/z 422 (0.1%), 407 (0.2%), 351 (2.5%), 261 (7%), 241 (7%), 225 (7%), 199 (8%), 129 (42%), 73 (100%). CD (benzoyl ester, acetonitrile, 227.5 nm, ∆ ) +14.2). Second diastereomer: 1H NMR, 400 MHz, CDCl3 (δ ) 7.24 ppm), δ 5.92 (dd, J ) 6.1 Hz/15.6 Hz, 1H, H14); 5.47 (m, 1H, H8); 5.42 (m, 2H H9, H13); 5.36 (m, 2H, H5, H6); 4.11 (m, 1H; H15); 3.65 (s, 3H, OCH3); 3.13 (dd, J ) 2.1 Hz/7.7 Hz, 1H, H12); 2.86 (dt, J ) 2.1 Hz/5.3 Hz, 1H, H11); 2.76 (dd, J ) 5.7 Hz, 2H, H7); 2.38 (m, 2H, H10a/b); 2.30 (t, J ) 7.5 Hz, 2H, H2); 2.08 (dt, J ) 6.8 Hz, 2H, H4); 1.68 (m, 2H, H3). LC-MS (ESI): m/z 373.2 [M + Na]+ (methyl ester). EI-MS (70 eV) methyl ester, TMS ether: m/z 407 (0.1%), 351 (1.8%), 261 (6%), 241 (6%), 225 (6%), 199 (8%), 129 (45%), 73 (100%). CD (benzoyl ester, acetonitrile, 226.5 nm, ∆ ) +17.5). 13R,14R(trans)-Epoxy-15S-hydroxy-5Z,8Z,11Z-eicosatrienoic Acid (Methyl Ester) 3. 1H NMR, 400 MHz, CDCl3 (δ ) 7.24 ppm), δ 5.69 (dt, J ) 8.0 Hz/10.9 Hz, 1H, H11); 5.37 (m, 4H, H5, H6, H8, H9); 5.09 (dd, J ) 9.0 Hz/10.8 Hz, 1H, H12); 3.65 (s, 3H, OCH3); 3.63 (dd, J ) 2.2 Hz/8.9 Hz, 1H, H13); 3.55 (m, 1H, H15); 2.97 (m, 2H, H10a/b); 2.88 (dd, J ) 2.3 Hz/4.5 Hz, 1H, H14); 2.79 (dd, J ) 5.6 Hz, 2H, H7); 2.31 (t, J ) 7.4 Hz, 2H, H2); 2.09 (m, 2H, H4); 1.85 (d, J ) 6.4 Hz, 1H, 15-OH); 1.96 (m, 2H, H3). LC-MS (ESI): m/z 373.2 [M + Na]+ (methyl ester). EI-MS (70 eV) methyl ester, TMS ether: m/z 407 (
