Identification of the Peroxidation Products of 13-Hydroxy-Î³-linolenate

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Articles Identification of the Peroxidation Products of 13-Hydroxy-γ-linolenate and 15-Hydroxyarachidonate: Mechanistic Studies on the Formation of Leukotriene-like Diols† Christopher L. Rector,‡,§ Donald F. Stec,‡ Alan R. Brash,| and Ned A. Porter*,‡,§ Department of Chemistry and Center in Molecular Toxicology, Vanderbilt UniVersity, NashVille, Tennessee 37235, and Department of Pharmacology, Vanderbilt UniVersity Medical Center, NashVille, Tennessee 37232 ReceiVed April 17, 2007

Monohydroxy-γ-linolenates and arachidonates were oxidized in the presence of R-tocopherol and free radical initiators at 37 °C. The dihydroxylinolenate products were analyzed and identified by use of a combination of liquid chromatography, mass spectrometry, and NMR techniques. A mechanism for the formation of the dihydroxylinolenates is proposed based on product analysis of oxidations using varied concentrations of R-tocopherol. The mechanism for monohydroxyarachidonate oxidation is the same as that of monohydroxylinolenates. However, arachidonate diol analysis is more complicated because of the formation of additional regioisomers that are a result of the parent arachidonate possessing multiple bisallylic hydrogens. Introduction Free radical chain oxidation of lipids, often referred to as lipid peroxidation, is of interest due to the various roles it plays in processes ranging from modulating enzyme activity to providing biologically active compounds (1–5). In addition, lipid peroxidation has been implicated in a variety of pathologies such as atherosclerosis, cancer, and various neurodegenerative disorders like Alzheimer’s and Parkinson’s diseases (6–8). The mechanism of autoxidation of fatty acids and esters has been extensively studied and reviewed (9–12). The various types of products, as well as the proposed mechanisms for their formation, have been areas of focus in previous research. It is known that variations in hydrogen atom donor concentration can lead to differences in product formation from the autoxidation of fatty acids and esters. In the absence of a hydrogen atom donor, the intermediate peroxyl radicals, for example, 3, can undergo a multitude of processes such as rearrangements, fragmentations, or cyclizations to form new classes of compounds such as isoprostanes, 4, or other cyclic peroxide products (Scheme 1) (10). In the presence of hydrogen atom donors, the first products formed by autoxidation of polyunsaturated fatty acids are monohydroperoxides; that is, 12-hydroperoxyeicosatetraenoic acid (12-HPETE,1 5) is one of the six regioisomers formed from the oxidation of arachidonic acid. The mechanism for the autoxidation of methyl linoleate has been extensively studied and has served as a model for other fatty acids and esters (9, 10, 13). From initial studies on lipid † This contribution honors Dr. Larry Marnett’s long commitment to scientific excellence and personal integrity. * To whom correspondence should be addressed. Tel: 615-343-4371. Fax: 615-343-5478. E-mail: [email protected]. ‡ Department of Chemistry, Vanderbilt University. § Center in Molecular Toxicology, Vanderbilt University. | Vanderbilt University Medical Center.

peroxidation, it was shown that good hydrogen atom donors trap kinetic cis,trans-hydroperoxides (6 and 7) as the major products (Scheme 2). Even though the possibility of forming a nonconjugated bisallylic hydroperoxide at C-11 of linoleic acid (8) was hypothesized, this product was not observed and isolated until recently (14). This new product from linoleate, isolated in oxidations carried out in the presence of high concentrations of good antioxidants, has led to the discovery of new hydroperoxides from other fatty acids and has opened new mechanistic possibilities for lipid peroxidation in the presence of excellent hydrogen atom donors. A recent study has shown that abstraction of bisallylic hydrogen atoms from a monohydroperoxide or alcohol (5) occurs approximately six times faster than abstraction of bisallylic hydrogens to form pentadienyl radicals from parent fatty acids or esters such as 1 (367 M-1 s-1 vs 62 m-1 s-1) (15). Thus, one concludes that hydrogen atoms in hydroperoxides such as those at C-7 in 12-HPETE (5) undergo hydrogen 1 Abbreviations: 12-HPETE, 12-hydroperoxyeicosatetraenoic acid; LTA4, leukotriene A4; LTB4, leukotriene B4; R-TOH, R-tocopherol; 13S-HOTrE, 13S-hydroxyoctadeca-6Z,9Z,11E-trienoic acid; 15S-HETE, 15S-hydroxyeicosa-5Z,8Z,11Z,13E-tetraenoic acid; MeOAMVN, 2,2′-azobis(4-methoxy2,4-dimethyl valeronitrile); 13S-HPOTrE, 13S-hydroperoxyoctadeca6Z,9Z,11E-trienoic acid; 15S-HPETE, 15S-hydroperoxyeicosa-5Z,8Z,11Z,13Etetraenoic acid; PPh3, triphenylphosphine; DIEA, diisopropylethylamine; PFB 13S-HOTrE, 2,3,4,5,6-pentafluorobenzyl 13S-hydroxyoctadeca6Z,9Z,11E-trienoate; PFB 15S-HETE, 2,3,4,5,6-pentafluorobenzyl 15Shydroxyeicosa-5Z,8Z,11Z,13E-tetraenoate; LC-Ag+-CIS-MS, liquid chromatography silver coordination ion spray mass spectrometry; PFB (OOH, 13S-OH) OTrE, 2,3,4,5,6-pentafluorobenzyl hydroperoxy, 13S-hydroxyoctadeca-6Z,9Z,11E-trienoate; PFB (OOH, 15S-OH) ETE, 2,3,4,5,6-pentafluorobenzyl hydroperoxy, 15S-hydroxyeicosa-5Z,8Z,11Z,13E-tetraenoate; LCEC-APCI-MS, liquid chromatography electron capture atmospheric pressure chemical ionization mass spectrometry; diHOTrE, dihydroxylinolenate; diHETE, dihydroxyarachidonate; CID, collision-induced dissociation; NMBHA, N-methyl benzohydroxamic acid; SPE, solid-phase extraction; SRM, selective reaction monitoring; SIM, selective ion monitoring; ECCD, exciton-coupled circular dichroism.

10.1021/tx700120r CCC: $37.00  2007 American Chemical Society Published on Web 10/02/2007

Formation of Linolenate and Arachidonate Diols

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Scheme 1. Autoxidation of Fatty Acids and Routes to Secondary Products

Scheme 2. Kinetic Hydroperoxides from Autoxidation of Methyl Linoleate

atom transfer to peroxyl radicals more rapidly than abstraction of the bisallylic C-10 hydrogen in the parent arachidonic acid (1). Therefore, peroxidation products derived from fatty acid precursors having three or more olefinic units are more reactive towards free radical oxidation than the parent lipid. While the extent of peroxidation of these lipid precursors in vivo may not be extensive, the first-formed products are themselves potential substrates for subsequent oxidation. The simplest secondary oxidation products from 5 would come from the addition of molecular oxygen to the newly formed heptatrienyl radical to form dihydroperoxides (10–13) after trapping of the peroxyl radical by a hydrogen atom donor (Scheme 3). This report focuses on the formation of the corresponding diols and hydroxyhydroperoxides produced by oxidation of monohydroxylinolenates and monohydroxyarachidonates. Dihydroperoxides of polyunsaturated fatty acids have been previously prepared from a variety of enzymatic and nonenzymatic techniques. In addition to the use of singlet oxygen, autoxidations in the presence of Fe+2/ascorbic acid or other hydrogen atom donors have been a common nonenzymatic method for forming dihydroperoxides (16–19). The enzymatic formation of dihydroperoxides or their corresponding diols has received more attention. Earlier studies have shown that high concentrations of soybean lipoxygenase can form dihydroperoxides from arachidonic acid (20). Subsequent studies using leukocytes, which are known to produce various lipoxygenases, showed the formation of diols from linolenates and arachidonates (21). These diols are part of a biologically active class of compounds known as leukotrienes, which have potent effects ranging from bronchoconstriction to stimulation of leukocyte function (22). Leukotrienes were first observed from their formation in rabbit leukocytes by the later identified 5-lipoxygenase enzyme (21). Additional studies also later indicated that various types of leukotrienes are formed via the unstable epoxide intermediate leukotriene A4 (LTA4, 14) (21). A 15-series of leukotrienes derived from 15-lipoxygenases in porcine and human leukocytes were identified and determined to be structurally similar to the initially reported 5-series leukotrienes (23, 24). In mechanistic

studies using 18O2 and H218O, several of the epimeric leukotriene products contained labeled oxygen at both the C-8 and the C-15 positions derived solely from molecular 18O2. These results indicated that either a free radical-induced or an enzymatic incorporation of molecular oxygen was responsible for several of the diols rather than the hydrolysis of an unstable epoxy intermediate, such as LTA4 forming leukotriene B4 (LTB4, 15) (25). We describe here the autoxidation of selected linolenate and arachidonate hydroxyl fatty esters in the presence and absence of a good hydrogen atom donor, R-tocopherol. In addition to previously reported diols, several novel compounds from the reduction of bisallylic hydroperoxides are formed from the oxidation of γ-linolenates and arachidonates in the presence of high concentrations of R-tocopherol (R-TOH) (19, 23, 25–27). The majority of this work focuses on the oxidation of the esters of 13S-hydroxyoctadeca-6Z,9Z,11E-trienoic acid (13S-HOTrE) since arachidonate precursors such as 15-hydroxyeicosa5Z,8Z,11Z,13E-tetraenoic acid (15-HETE) give a more complex product mixture that complicates analysis. By varying concentrations of R-TOH, changes in product ratios were observed that allow a mechanism for product formation to be proposed. 2,3,4,5,6-Pentafluorobenzyl esters were chosen for these experiments to facilitate the use of highly sensitive atmospheric pressure chemical ionization–mass spectrometry (APCI-MS) for the analysis of low concentrations of some oxidation products.

Materials and Methods Materials. Polyunsaturated fatty acids were purchased from NuChek Prep (Elysian, MN). 2,2′-Azobis(4-methoxy-2,4 dimethyl valeronitrile) (MeOAMVN) was purchased from Wako Chemicals USA, Inc. (Richmond, VA). HPLC hexanes were purchased from Burdick and Jackson (Muskegon, MI). All other reagents were purchased from Aldrich (Milwaukee, WI) or Acros Organics (Morris Plains, NJ). Instruments. Analytical HPLC was performed using a Waters 600E pump with a Waters 717plus Autosampler or a Waters 2690 Separations Module HPLC. Both were equipped with a Waters 996 photodiode array detector (PDA). The autosamplers and PDAs were controlled by Millennium chromatography software (Waters, Milford, MA). Semipreparative HPLC was performed on a Waters 600E pump with a Waters 2487 dual wavelength detector. Mass spectrometric analysis was performed using a Finnigan TSQ-7000 triple-quadrupole mass spectrometer equipped with a standard API-1 electrospray ionization source fitted with a 100 µm deactivated fused Si capillary for coordination ion spray (CIS)-MS experiments. Electron capture (EC)-APCI experiments used the same TSQ-7000 with an APCI source attached. Circular dichroism experiments were performed using an Aviv model 215 circular dichroism (CD) spectrophotometer. NMR spectra were collected using either a 300 or a 600 MHz NMR with Bruker software.

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Scheme 3. Dihydroperoxides from Autoxidation of Monohydroperoxy Fatty Acids

Scheme 4. Formation of Leukotriene B4 from Arachidonic Acid

Synthesis of 2,3,4,5,6-Pentafluorobenzyl (PFB) Hydroxy Polyunsaturated Fatty Esters. The stereospecific 13S-hydroperoxyocta-6Z,9Z,11E-trienoic acid (13S-HPOTrE) and 15S-hydroperoxyeicosa-5Z,8Z,11Z,13E-tetraenoic acid (15S-HPETE) were synthesized by using a previously reported procedure that was slightly modified (28). The free acid (500 mg) was placed in a pressure reaction vessel (Andrews Glass Co., Vineland, NJ) with 20 mL of 0.1 M sodium borate buffer (pH 9) and 1 mL of ethanol. This mixture was cooled for 5 min in an ice bath and followed by the addition of 40 mg of soybean lipoxygenase type 1B (Sigma, St. Louis, MO). The reaction vessel was sealed and pressurized with 40 psi O2. The reaction was rapidly stirred for 3 h. The status of the reaction was checked by TLC (4:6 hexanes:diethyl ether). If incomplete, 10 mg of additional lipoxygenase was added, the vessel was repressurized, and the reaction was continued in an ice bath. The arachidonic acid was completely reacted after 5 h; however, the γ-linolenic acid was never completely converted. To the mixture was added a 1 M HCl solution until the milky mixture had a pH of 3. After adding 50 mL of brine, the unreacted fatty acids and hydroperoxides were extracted (3 × 50 mL) with CH2Cl2. The collected organics were backwashed with 50 mL of H2O, dried with MgSO4, and concentrated under reduced pressure. The residual yellow oil was dissolved in 10 mL of dry Et2O. After the oil was cooled in an ice bath, 0.4 g of triphenylphosphine (PPh3) was slowly added. After this reacted for 30 min, the solid precipitate was removed using filtration, and the solvent was removed. The yellow oil was dissolved in 8 mL of dry acetonitrile. To this was added 0.75 mL of diisopropylethylamine (DIEA), followed by 0.75 mL of PFB bromide. This was stirred at 40 °C under N2 for 30 min. To the mixture was added 50 mL of H2O and 0.5 mL of 1 M HCl. The mixture was extracted (3 × 50 mL) with hexanes. The organics were backwashed with 50 mL of H2O, dried with MgSO4, and concentrated under reduced pressure. The products were purified by column chromatography with silica (hexanes:EtOAc ) 9:1) to give the stereospecific PFB hydroxy fatty esters [average ≈ 400 mg (≈50% yield) over three steps]. 2,3,4,5,6-Pentafluorobenzyl 13S-Hydroxyocta-6Z,9Z,11Etrienoate (PFB 13S-HOTrE). 1H NMR (300 MHz, CDCl3): δ ) 0.87 (t, 3H, J ) 6.6 Hz), 1.23–1.67 (12H), 2.06 (q, 2H, J ) 7.2 Hz), 2.32 (t, 2H, J ) 7.4 Hz), 2.89 (t, 2H, J ) 6.5 Hz), 4.15 (q, 1H, J ) 6.3 Hz), 5.17 (s, 2H), 5.32–5.40 (3H), 5.68 (dd, 1H, J )

6.8, 15.2 Hz), 5.96 (t, 1H, J ) 10.9 Hz), 6.49 (dd, 1H, J ) 11.1, 15.2 Hz). 13C NMR (75 MHz, CDCl3): δ ) 14.0, 22.6, 24.4, 25.1, 26.1, 26.8, 28.9, 31.8, 33.8, 37.3, 53.3, 72.8, 125.3, 127.8, 128.3, 129.8, 130.3, 136.6, 173.0. 19F (282 MHz, CDCl3; standard, C6F6): δ ) -140.4, -150.9, -160.0. HRMS: calcd for C25H31F5O3Li (M + Li)+, 481.2353; found, 481.2352. 2,3,4,5,6-Pentafluorobenzyl 15S-Hydroxyeicosa-5Z,8Z,11Z,13Etetraenoate (PFB 15S-HETE). 1H NMR (300 MHz, CDCl3): δ ) 0.87 (t, 3H, J ) 6.7 Hz), 1.23–1.73 (10H), 2.08 (q, 2H, J ) 7.1 Hz), 2.32 (t, 2H, J ) 7.4 Hz), 2.76 (t, 2H, J ) 5.6 Hz), 2.93 (t, 2H, J ) 5.8 Hz), 4.15 (q, 1H, J ) 6.0 Hz), 5.17 (s, 2H), 5.30–5.41 (5H), 5.68 (dd, 1H, J ) 6.7, 15.2 Hz), 5.97 (t, 1H, J ) 11.1 Hz), 6.49 (dd, 1H, J ) 11.1, 15.0 Hz). 13C NMR (75 MHz, CDCl3): δ ) 14.4, 23.0, 25.0, 25.5, 26.0, 26.5, 26.8, 32.2, 33.6, 37.7, 53.6, 73.2, 125.6, 128.0, 128.4, 128.8, 129.1, 129.3, 130.5, 137.1, 173.3. 19 F (282 MHz, CDCl3; standard, C6F6): δ ) -140.3, -150.9, -159.9. HRMS: calcd for C27H33F5O3Li (M + Li)+, 507.2510; found, 507.2503. Autoxidation of PFB Fatty Esters. A solution of 0.1 M PFB polyunsaturated ester, 0–1.8 M R-TOH, and 0.05 M MeOAMVN in dry benzene was oxidized at 37 °C under air for 4 h. The reactions were quenched with excess BHT in hexanes. For dihydroxy compounds, the reaction mixture was reduced with excess P(OCH3)3 in hexanes. The samples were prepped for analysis using Varian BondElut silica SPE cartridges that were conditioned with hexanes. After the samples were loaded, excess R-TOH and unoxidized PFB ester were removed using hexanes:EtOAc (85: 15), and the oxidation products were rinsed from the solid-phase extraction (SPE) cartridge with EtOAc. HPLC Analysis Conditions. Reversed phase (RP)-HPLC analysis of the oxidation mixtures was performed using a Supelco Discovery C-18 column (4.6 mm × 25 cm). For the PFB 13SHOTrE oxidations, a gradient of MeOH:H2O (70:30f90:10) between 5 and 45 min in each run at 1 mL/min was used for the separations. The PFB 15S-HETE oxidation products were analyzed the same way using a MeOH:H2O gradient (80:20f90:10). Normal phase (NP)-HPLC analyses were performed using a Beckman Ultrasphere 5 µm silica column (4.6 mm × 25 cm) with hexanes/ i PrOH (97.5:2.5) for the mobile phase at 1 mL/min. The products were detected at 235 and 268 nm.



Scheme 5. Kinetic Products from Autoxidation of PFB 13 S-HOTrE (16)

Liquid Chromatography Silver (LC-Ag+)-CIS-MS of PFB Hydroperoxy, Hydroxy Fatty Esters. Product identification of the PFB hydroperoxy; hydroxylinolenates; 2,3,4,5,6-pentafluorobenzyl hydroperoxy, 13S-hydroxyoctadeca-6Z,9Z,11E-trienoate (PFB [OOH, 13S-OH] OTrEs); and arachidonates (2,3,4,5,6-pentafluorobenzyl hydroperoxy, 15S-hydroxyeicosa-5Z,8Z,11Z,13E-tetraenoate, PFB [OOH, 15S-OH] ETEs) were performed using conditions and experiments previously established for Ag+-CIS-MS in the identification of fatty acid methyl ester hydroperoxides (29, 30). LC separations were performed using the aforementioned RP-HPLC gradients with a narrow bore Supelco Discovery C-18 column (2.1 mm × 15 cm) with a flow rate of 200 µL/min. LC-EC-APCI-MS of PFB Dihydroxy Fatty Esters. Product identification of the PFB dihydroxylinolenates (PFB diHOTrEs) and PFB dihydroxyarachidonates (PFB diHETEs) were performed with the corona discharge needle at 5 µA, capillary temperature at 300 °C, vaporizer temperature at 475 °C, capillary voltage of -20 V, and tube lens voltage of -35 V in the mass spectrometer. Collision-induced dissociation (CID) experiments were performed with a collision energy offset of 10 V. LC separations were performed using the aforementioned RP-HPLC gradients and Discovery C-18 column (4.6 mm × 25 cm) at 1 mL/min. NMR Studies. PFB diHOTrEs were produced by oxidizing 1 g of PFB 13S-HOTrE with the above conditions in the presence of 1.8 M R-TOH. Semipreparative HPLC with a Supelco Discovery C-18 column (21.2 mm × 25 cm) using MeOH:H2O (80:20) at 20 mL/min was used to collect fractions of the diols products. After the collected fractions were concentrated and the diols were extracted with CH2Cl2, the diols were further purified using NPHPLC with hexanes:iPrOH (97.5:2.5) at 1 mL/min. All reported NMR spectra were acquired using a 14.1 T Bruker magnet equipped with a Bruker DRX spectrometer operating at proton Lamour frequency of 600.13 MHz. 1H spectra were acquired in 2.5 mm NMR tubes using a Bruker 5 mm cryogenically cooled NMR probe. Chemical shifts were referenced internally to benzene (7.1 ppm), which also served as the 2H lock solvent. Typical experimental conditions included 32K data points, an 8 ppm sweep width, a recycle delay of 1.5 s, and 32–256 scans depending on the sample concentration. The vinylic regions of the products were noted and combined with 1H,1H correlation spectroscopy (COSY) experiments to determine the double bond configurations. Stereochemical Determination of Dihydroxylinolenates. A solution of 0.2 M PFB 13 S-HOTrE, 0.3 M N-methyl benzoydroxamic acid (NMBHA), and 0.02 M MeOAMVN in dry CH3CN was oxidized for 4 h at 37 °C. The oxidation was stopped by the addition of excess butylated hydroxytoluene (BHT) and P(OCH3)3. After CH3CN was removed, unoxidized PFB 13 S-HOTrE and excess NMBHA were separated from the PFB diHOTrEs using a Varian BondElut silica SPE cartridge. The elution fractions with the diols were determined by UV absorbance at 268 nm. Individual diols were isolated using semipreparative HPLC with MeOH:H2O (80: 20) at 20 mL/min using a Supelco Discovery C-18 column (21.2 mm × 25 cm). After the solvent was removed, the hydroxyl groups were converted to naphthoate esters using a method previously used to create benzoate esters (31).

The collected diols were dissolved in 250 µL of dry CH3CN. A 25 µL amount of DBU, ≈500 µg of naphthoyl chloride, and ≈500 µg of dimethylaminopyridine were added, and the reaction mixture was stirred at room temperature overnight. After the solvent was removed, 1 mL of H2O was added to the reactions. The products were extracted with 1 mL of CH2Cl2. The dinaphthoates were then purified with RP-HPLC using MeOH:H2O (95:5) at 1 mL/min. After the fractions were extracted with CH2Cl2 and the solvent was removed, the purified fractions were redissolved in hexanes and the concentrations were adjusted so that the UV absorbance was 0.6 AU for all fractions. CD spectra were recorded with five scans between 200 and 350 nm using an Aviv spectrophotometer. The configuration of the diols was then determined from the average of the scans.

Results and Discussion Certain fatty acid diols elicit potent biological effects, for example, leukotriene B4 and various resolvins, docosatrienes, and neuroprotectins (21, 32) This has spurred an increased interest in their formation whether through enzymatic or nonenzymatic processes (21). Extensive oxidation of fatty acids or esters can lead to the formation of a number of regioisomeric and diasteromeric dihydroperoxides as well as the corresponding diols analogous to the leukotrienes. In this mechanistic study, a two-step sequence of oxidation of fatty acids was carried out to simplify the mixture of diol products formed. Thus, specific fatty ester alcohols were prepared by enzymatic methods, and subsequent free radical oxidations of these compounds were carried out in the presence and absence of an excellent hydrogen atom donor, R-TOH. A majority of the experiments carried out in this study used PFB 13S-HOTrE (16), formed by lipoxygenase-promoted oxidation of γ-linolenic acid. This compound serves as a model substrate for the corresponding arachidonate derivatives. It gives fewer diol oxidation products than arachidonate and simplifies the product analysis of secondary peroxidation. Soybean lipoxygenase is known to catalyze the stereospecific formation of hydroperoxides with S configuration at the ω-6 position of fatty acids, that is, 13S-HPOTrE and 15S-HPETE. In this report, a previously described procedure, which calls for the reaction of a 0.1 M free acid solution in 0.1 M borate buffer (pH 11):EtOH (95:5) with soybean lipoxygenase (4 mg/ mL) at 5 °C, was used to synthesize 13S-HPOTrE and 15SHPETE in quantities of several hundred milligrams for use as the oxidation substrate (28). One modification to the experimental procedure of note was extraction of the oxidation mixture with CH2Cl2 instead of Et2O, which in our hands led to a significantly greater yield of hydroperoxide. After the hydroperoxides were reduced with PPh3, 13S-HOTrE and 15S-HETE were converted to PFB esters to analyze for products by LCEC-APCI-MS, which is known to be highly sensitive for PFB esters (33).


Rector et al.

Figure 1. Chromatograms of PFB 13 S-HOTrE oxidized in the presence of 1.8 M R-TOH for 4 h at 37 °C initiated by 0.05 M MeOAMVN observed at 268 nm (black line) and 235 nm (red dashed line). The separation mobile phase was (70:30f90:10) MeOH:H2O over 40 min. (A) PFB [OOH, 13 S-OH] OTrEs and (B) PFB diHOTrEs.

Oxidations of PFB 13 S-HOTrE and PFB 15 S-HETE were performed in the presence of 1.8 M R-TOH in an attempt to trap any kinetically controlled products. After sample preparations to remove unoxidized starting material and R-TOH, the hydroperoxide products or, after reduction, the diol products were analyzed by RP-HPLC. With low concentrations of R-TOH, more highly oxidized products appeared in the HPLC chromatograms, but these products were not extensively analyzed. Analysis of Hydroperoxy-Hydroxy Oxidation Products from Oxidation of PFB 13S-HOTrE. Studies to identify the secondary oxidation products of PFB 13S-HOTrE began by examination of its various hydroperoxide products. There have been few studies on the oxidation of γ-linolenates, so little is known about the secondary oxidation products from this fatty acid. The formation of 6,13-diols from γ-linolenate has been reported from oxidations catalyzed by soybean lipoxygenase and oxidations in the presence of hemoglobin (27). On the basis of chemistry established for linoleate oxidation, the expected products from the oxidation of PFB 13S-HOTrE in the presence of an excellent hydrogen atom donor are shown in Scheme 5. On the basis of this analogy, it is predicted that four different sets of product regioisomers are possible from PFB 13S-HOTrE. Oxidations of PFB 13S-HOTrE were performed in the presence of 1.8 M R-TOH over a 4 h period to trap possible kinetic products formed from oxidation of the starting substrate. After the excess R-TOH and unoxidized PFB 13S-HOTrE were removed from the samples using SPE, PFB [OOH, 13S-OH] OTrEs, such as 17–20, were analyzed using RP-HPLC (70: 30f90:10 MeOH:H2O). The notation PFB [OOH, 13S-OH] OTrE generally refers to any of the four regioisomers from the oxidation of PFB 13S-HOTrE, whereas modified notations such as PFB [6-OOH, 13S-OH] OTrE refer to specific regioisomers, that is, PFB 6-hydroperoxy, 13S-hydroxylinolenate. A number of PFB [OOH, 13S-OH] OTrEs can be seen in the representative chromatogram between 28 and 38 min (Figure 1A). The 6,13 and 12,13 regioisomers are detected at 268 nm due to the absorbance of the conjugated trienes in this region, while the conjugated dienes of the 8,13 and 10,13 isomers absorb at 235 nm. The PFB [OOH, 13S-OH] OTrE mixture was reduced with P(OCH3)3 to obtain a mixture of PFB diHOTrEs. When analyzed by RP-HPLC, these PFB diHOTrEs can be separated with better product resolution than the PFB [OOH, 13 S-OH] OTrEs using the same solvent gradient (Figure 1B). With the established HPLC conditions, LC-Ag+-CIS-MS was used to identify the PFB [OOH, 13S-OH] OTrE regioisomers. This technique allows for the identification of fatty ester

Figure 2. Ag+-CIS-MS SRM PFB 13 S-HOTrE oxidized in the presence of 1.8 M R-TOH for 4 h at 37 °C. The separation was achieved with a gradient of (70:30f90:10) MeOH:H2O between 5 and 45 min. Monitored for 107Ag+ adducts. (A) Total ion current. (B) Hock fragmentation of PFB [6-OOH, 13S-OH] OTrE. (C) Hock fragmentation of PFB [8-OOH, 13S-OH] OTrE. (D) Hock fragmentation of PFB [10OOH, 13S-OH] OTrE. (E) Hock fragmentation of PFB [12-OOH, 13SOH] OTrE.

hydroperoxides based on specific cleavages of the hydroperoxide moiety (29, 30) Selective reaction monitoring (SRM) was performed using LC-Ag+-CIS-MS, an approach that permits monitoring of the fragmentation of the parent PFB [OOH, 13SOH] OTrEs–Ag+ complex, 613 m/z, to its daughter fragments that result from cleavages of the various hydroperoxides. The PFB [6-OOH, 13S-OH], [8-OOH, 13S-OH], and [10OOH, 13S-OH] OTrE products were identified by monitoring the characteristic Hock cleavages of the hydroperoxides using SRM to follow the reactions 613 m/z f 417 m/z, 613 m/z f 443 m/z, and 613 m/z f 457 m/z, respectively (Figure 2). The PFB [12-OOH, 13S-OH] OTrE isomers do not give facile Hock fragmentations in comparison to the other regioisomers but instead undergo R-cleavage between the hydroperoxide and the hydroxy moieties at the 12- and 13-positions. The elution of the 12-OOH, 13S-OH products was determined by monitoring this R-cleavage reaction 613 m/zf 495 m/z with SRM. From the LC-Ag+-CIS-MS SRM analysis, we conclude that four distinct 8,13 stereoisomeric products are present in the product mixture as well as two 10,13 and 12,13 stereoisomers. From the SRM analysis, there appear to be only three 6,13 products. However, reduction of the hydroperoxide and analysis of the corresponding diols suggests that the separation of the [6-OOH, 13S-OH] OTrE isomers is incomplete and that there are four 6,13 stereoisomers formed. Assignment of the peaks



Scheme 6. CID Fragmentation of Dihydroxylinolenates

in the chromatogram shown in Figure 1A are as follows: peak I ) 8,13; peak II ) 8,13 and 6,13; peak III ) 6,13; peak IV ) 8,13; peak V ) 10,13; peak VI ) 8,13; peak VII ) 10,13 and 6,13; peak VIII ) 12,13; and peak IX ) 12,13. Analysis of Dihydroxy Oxidation Products from Oxidation of PFB 13-γ-HOTrE. In an effort to determine the elution order of the diHOTrEs, the PFB [OOH, 13 S-OH] OTrEs were collected and each fraction was then reduced to the corresponding PFB diHOTrEs with excess P(OCH3)3. The elution order for the PFB diHOTrE regioisomers was then tentatively determined by coinjection of the oxidation mixture with each of the diols formed from the purified fractions, Vide supra. The PFB diHOTrE regioisomers responsible for the peaks in Figure 1B were identified as follows: peak 1 ) 8,13; peak 2 ) 8,13 and 6,13; peak 3 ) 8,13; peak 4 ) 8,13 and 6,13; peak 5 ) 10,13; peak 6 ) 6,13; peak 7 ) 6,13; peak 8 ) 10,13; peak 9 ) 12,13; and peak 10 ) 12,13. EC-APCI-MS has been used in the analysis of PFB esters with GC and LC systems. The high sensitivity of the technique allows for quantitative analysis of various types of compounds (33). A mixture of PFB diHOTrEs from the oxidation of PFB 13S-HOTrE in the presence of neat R-TOH was first analyzed with LC-EC-APCI-MS using selective ion monitoring (SIM) of 309 m/z in the negative ion mode. The mass chromatogram further confirmed that the peaks observed in the chromatogram in Figure 1B are diHOTrEs. A full scan of the daughter ions from fragment 309 m/z using CID to induce fragmentation permitted identification of the regioisomers that are present in the individual peaks seen in Figure 1B. Daughter ions for the parent 309 m/z fragments indicated that each compound had losses of –H2O (-18 m/z), –CO2 (-44 m/z), –H2O + CO2 (-62 m/z), and additional combinations of H2O and CO2 loss. All of the regioisomers also undergo R-fragmentation at the 13-hydroxyl that is present in each to give a fragment of 209 m/z. The 10,13 and 12,13 diols both undergo R-cleavages at the 10 and 12 hydroxyl groups to give fragments of 153 m/z and 179 m/z, respectively (Scheme 6).The 8,13 regioisomers undergo a rearrangement to form a fragment of 195 m/z. A tentative structure for this fragment is shown in Scheme 6, but a definitive identification of the fragment would require isotope-labeling studies. Analysis of the CID from peaks tentatively identified to be 6,13 regioisomers showed a consistent 163 m/z fragment. A possible identity of this fragment is shown in Scheme 6, and a proposed mechanism for a such a fragmentation has been previously described in the negative ion mass spectrometric analysis of LTB4 (34).

Characterization of Dihydroxylinolenates by 1H and H,1H COSY NMR Analysis. While LC and LC-MS can be used to identify the regioisomers responsible for each peak in the chromatograms, these techniques do not allow the double bond configurations to be determined for complete product characterization. To this end, the diHOTrEs were collected by semipreparative HPLC using a combination of NP-HPLC and RP-HPLC to isolate the 12 isomers. Some of the diols are only formed if the oxidation of PFB 13-HOTrE is carried out in the presence of high concentrations of R-TOH, which of course reduces the quantities of oxidation products being formed. Nevertheless, sufficient quantities of the diHOTrEs could be obtained to permit analysis. The vinylic regions of the collected fractions were analyzed to determine the double bond configuration of the products. By using a combination of 1H and 1H,1H COSY NMR, the stereochemical configuration of the regioisomers was assigned in each of the peaks from Figure 1B. From the analysis, it was determined that six sets of epimers were responsible for the 12 product peaks. High-resolution 1H spectra were obtained that showed the distinct differences between each of the six sets of 1

Figure 3. EC-APCI-MS SRM of PFB 13S-HOTrE oxidized in the presence of 1.8 M R-TOH for 4 h at 37 °C, followed by reduction of the PFB [OOH, 13 S-OH] OTrEs to PFB diHOTrEs with excess P(OCH3)3. The separation was achieved with a gradient of (90:10f70: 30) MeOH:H2O between 5 and 45 min. (A) Total ion current. (B) Fragmentation of 6-OH, 13S-OH. (C) Fragmentation of 8-OH, 13SOH. (D) Fragmentation of 10-OH, 13S-OH. (E) Fragmentation of 12OH, 13S-OH.


Rector et al.

Figure 4. 1H NMR of the vinylic region of dihydroxylinolenates obtained from the oxidation of PFB 13S-HOTrE in the presence of 1.8 M R-TOH. NMR spectra obtained using a 600 MHz system equipped with a cryogenically cooled probe.

epimers (Figure 4). When used in combination with the COSY experiments, the identities of the peaks shown in the chromatogram of Figure 1B can be assigned. Peaks 1 and 3 were confirmed to be 8,13 diols with Z,Z,E configuration. The compounds with conjugated diene moieties isolated from peaks 2 and 4 were verified to be 8,13 diols with Z,E,E configuration. The diols with conjugated trienes in peak 2 and peak 6 are 6,13 diols with E,E,E configuration. Likewise, the conjugated trienes from peak 4 and peak 7 are 6,13 diols but have a E,Z,E double bond configuration. Peaks 5 and 8 are 10,13 diols with Z,E,E configuration. The two epimers in peaks 9 and 10 are 12,13 diols with Z,E,E configuration. Diastereomer Assignment of Oxidation Products. The configuration of the stereogenic carbon formed at the site of oxygen addition of the conjugated triene products was determined using exciton-coupled circular dichroism (ECCD). ECCD uses through-space coupling of two or more chromophores to produce a CD spectrum of a chiral molecule (35). This technique has been used in previous reports to determine the stereochemistry of lipid oxidation products from various forms of lipoxygenases (31, 35). Sample preparation for ECCD requires the conversion of hydroxy groups to an ester having a chromophore. A common approach is to use benzoate or naphthoate esters due to the large UV absorbance of these compounds. When coupled with the conjugated π-systems of the oxidized lipids, a CD spectrum can be obtained and the product stereochemistry can be determined from the Cotton effects observed. Large quantities of the PFB [6-OOH, 13S-OH] OTrEs and PFB [12-OOH, 13 S-OH] OTrEs were produced (36); the compounds were reduced to their corresponding PFB diHOTrEs and then converted to dinaphthyl esters using naphthoyl chloride. The dinaphthoates were purified by RP-HPLC, and CD spectra from 200–350 nm were then collected for each of the fractions. The concentration of each sample was manipulated such that the nominal O.D. of each was 0.6 A.U. The two 6,13 diols with the E,Z,E configuration gave the CD curves seen in Figure 5. One of the epimers showed a typical CD curve of a lipid alcohol acylnaphthoate, while the other epimer shows no evidence of chirality. This result is anticipated because the PFB 13S-HOTrE precursor gives an R,S-diHOTrE in which the two naphthoate

Figure 5. CD spectra of naphthoate ester derivatized PFB 6,13-E,Z,EdiHOTrE in hexanes. Peak 4 (black line) ) PFB 6S,13S-E,Z,EdiHOTrE. Peak 7 (red dashed line) ) PFB 6R,13S-E,Z,E-diHOTrE.

esters will experience opposite chiral environments, which cancel out in the CD spectra. In the S,S product, the two naphthoate esters will experience a similar environment and an additive effect will yield typical CD curves. By this analysis, we conclude that the 6S,13S-E,Z,E epimer elutes first using RPHPLC, while the 6R,13S-E,E,E product elutes before its 6S,13S diastereomer. Both 12,13 epimers show CD curves, because the naphtholates do not interact equally with the conjugated triene (see Supporting Information). The naphthoate at C-12 has interactions with the conjugated triene to produce a CD spectrum, but the 13S naphthoate has little or no interaction with the triene. We conclude that the 12R,13S compound elutes first followed by the 12S,13S epimer. Oxidations carried out under a variety of conditions did not provide sufficient quantities of the 8,13 or 10,13 regioisomers for CD analysis. Therefore, the stereochemical identifications of the individual 8,13 and 10,13 products were not determined. A complete summary of the identification of the diHOTrE compounds in Figure 1B is listed below, and their structures are shown in Scheme 7. Peak 1 ) 8,13S-dihydroxyocta-6Z,9Z,11E-trienoate; peak 2 ) 8,13S-dihydroxyocta-6Z,9E,11E-trienoate and 6R,13S-dihydroxyocta-7E,9E,11E-trienoate; peak 3 ) 8,13S-dihydroxyocta-



Scheme 7. Diols from Autoxidation of PFB 13S-HOTrE in the Presence of 1.85 M r-TOH (Peak Numbers from Figure 1B)

6Z,9Z,11E-trienoate; peak 4 ) 8,13S-dihydroxyocta-6Z,9E,11Etrienoate and 6S,13S-dihydroxyocta-7E,9Z,11E-trienoate; peak 5 ) 10,13S-dihydroxyocta-6Z,8E,11E-trienoate; peak 6 ) 6S,13S-dihydroxyocta-7E,9E,11E-trienoate; peak 7 ) 6R,13Sdihydroxyocta-7E,9Z,11E-trienoate; peak 8 ) 10,13S-dihydroxyocta-6Z,8E,11E-trienoate; peak 9 ) 12R,13S-dihydroxyocta6Z,8E,10E-trienoate; and peak 10 ) 12S,13S-dihydroxyocta6Z,8E,10E-trienoate. A comparison of UV/vis spectra from the identified PFB diHOTrEs with previously identified conjugated trienes and dienes lends additional support to the proposed structures. Diols from oxidations of arachidonic acid have been isolated and characterized by various techniques (23, 37). UV/vis absorbance analysis of conjugated trienes provides distinctive spectra with triplet-shaped absorbance curves that provide structural information based on differences in the shoulder heights. The absorbance of 8,15-diHETEs isolated from oxidations induced by soybean lipoxygenase and lipoxygenases from porcine leukocytes provide UV/vis spectra defined by tripletlike profiles with maxima of approximately 269 nm with shoulders at 260 and 280 nm. DiHETEs with a E,E,E double bond configuration displayed a slight bathochromic shift with deeper shoulders as compared to E,Z,E trienes. These 8,15-diHETE isomers correspond to the 6,13-diHOTrEs described in this work, and the UV spectra of these compounds presented in the Supporting Information display the same features as those reported for the diHETEs.

The proposed 8,13- and 10,13-diHOTrE structures contain conjugated dienes, and their spectra are also presented in the Supporting Information. These compounds are defined by a broad absorbance peak with maxima of 237 and 235 nm, respectively. This absorbance profile is similar to the previously reported spectra of 5,15-diHETE, with the exception of a slight hypsochromic shift, which further confirms the presence of conjugated dienes in these regioisomers. Proposed Mechanism for the Formation of Hydroperoxy, Hydroxylinolenates from Autoxidation of PFB 13 S-HOTrE. A series of oxidations of PFB 13S-HOTrE in the presence of varied concentrations of R-TOH provides information for the possible mechanism of formation of [OOH, 13SOH] OTrEs through autoxidation. These oxidations were performed using 0.1 M PFB 13S-HOTrE with R-TOH concentrations that varied from 0 to 1.8 M. With this wide range of antioxidant concentration, the changes in product ratios were monitored to provide information about the autoxidation mechanism. As seen in Figure 6, the range of concentrations produces distinct differences in the HPLC chromatograms of the PFB diHOTrEs. The HPLC chromatogram (Figure 6A) of products from an oxidation with no antioxidant appears distinctly different than oxidations with even small quantities of antioxidant present (Figure 6B). With no antioxidant, the 6,13-E,E,E diols (peaks A2 and A5) are major products, while the 6,13-E,Z,E products

Figure 6. Chromatograms of diols from PFB 13S-HOTrE oxidized in the presence of various concentrations of R-TOH for 4 h at 37 °C initiated by 0.05 M MeOAMVN observed at 268 (black line) and 235 nm (red dashed line). The separation mobile phase was (70:30f90:10) MeOH:H2O over 40 min. (A) 0 M R-TOH, (B) 0.05 M R-TOH, and (C) 1.8 M R-TOH.


Rector et al.

Scheme 8. Proposed Mechanism of the Autoxidation of PFB 13-HOTrE (Peak Numbers from Figure 1)

(peaks A4 and A6) are present in a relatively low abundance. The chromatography of the 12,13 diols (peaks A7–A10) is also different than that observed from those samples with antioxidant present. When no antioxidant is present, two large peaks each having a shoulder are observed in the chromatogram. The shoulders were identified as the known 12,13-Z,E,E diols (peaks A7 and A10), while the new peaks (peaks A8 and A9) were identified as 12,13 diols that have E,E,E double bond geometries. Analysis of the oxidations carried out in the presence of 0.05 M concentrations of R-TOH shows that the 12,13-E,E,Ediols, specifically peaks B8 or B9, are not present in the product mixture; the Z,E,E stereoisomers are the only 12,13 diols formed (peaks B7 and B10; Figure 6B). Both the 6,13-E,Z,E diols (peaks B4 and B6) and the 6,13-E,E,E (peaks B2 and B5) are formed at 0.05 M R-TOH as the other major products. Only at the highest concentration of R-TOH do the 8,13 and 10,13 products become significant (Figure 6C). This is shown in Figure 6C by those products that exhibit conjugated diene absorbance at 235 nm (red dashed line) but none at 268 nm due to conjugated triene. Attempts to better understand the mechanism of autoxidation lipids have been the focus of many studies (9–12), and on the basis of these earlier precendents, we propose here a mechanism for the formation of all of the products observed in this series of oxidations (Scheme 8). We suggest that the initial step in the autoxidation of PFB 13S-HOTrE is hydrogen atom abstraction from C-8, which will form a stabilized heptatrienyl radical system (15). Molecular oxygen can then add to four possible positions along the heptatrienyl radical at C-6, C-8, C-10, or C-12 to form peroxyl radicals. The crucial product-determining step is the subsequent reaction of the intermediate peroxyl radicals with a hydrogen atom donor. With a good donor present during oxidation, the four initially formed peroxyl radicals are trapped as their

respective [OOH, 13S-OH] OTrEs before the peroxyl radicals can undergo β-fragmentation and possible rearrangement, which leads to other possible products. Computational studies have predicted the rate constants for β-fragmentation of bisallylic peroxyl radicals in heptatrienyl radicals to be on the order of 1.1 × 107 s-1 for the peroxyl radical that forms the 10,13Z,E,E products and 7.4 × 107 s-1 for the peroxyl radical that leads to 8,13-Z,Z,E products (38). These rate constants for β-fragmentation are substantially higher than the 2.6 × 106 s-1 determined experimentally for the bisallylic peroxyl radical in the oxidation of methyl linoleate at 37 °C (15). Even in the presence of neat R-TOH, the bisallylic peroxyl radicals in these oxidations of PFB 13S-HOTrE still undergo β-fragmentation. The β-fragmentation of a peroxyl radical at C-10, followed by a rearrangement, leads to the formation of 8,13 products with Z,E,E and 6,13 conjugated trienes with E,E,E double bond configurations. The formation of 6,13-E,E,E and 12,13-E,E,E diols, when no R-TOH is present, comes from multiple peroxyl radical β-fragmentations and rearrangements that leads ultimately to the thermodynamically favored all-trans products. Formation of Hydroperoxy, Hydroxyarachidonates, and Dihydroxyarachidonates. Fatty esters that are more highly unsaturated can undergo oxidations similar to those described here for PFB 13S-HOTrE. As the total sites of unsaturation increase, the mechanistic complexity of the system and the possible number of products also increase, which in turn make analyses more challenging. Preliminary experiments were performed by oxidizing PFB 15S-HETE to determine if the proposed mechanism for autoxidation of PFB 13S-HOTrE can be extended to this system. Because of the presence of arachidonates in natural systems and the similarities between



Scheme 9. Autoxidation Products from PFB 15-HETE

15-HETE oxidation products and leukotrienes, the oxidation of 15S-HETE is perhaps of more biological relevance than 13SHOTrE. Autoxidation of PFB 15S-HETE can occur from the abstraction of a hydrogen atom from C-10 to form a heptatrienyl radical similar to that formed in the oxidation of PFB 13S-HOTrE (Scheme 9). However, the arachidonate contains additional bisallylic hydrogens at C-7 that can be abstracted to form pentadienyl radicals. The hydrogen atoms at C-7 should be abstracted approximately six times slower than those at C-10, but removal of this additional bisallylic hydrogen leads to additional products. An oxidation of PFB 15S-HETE was performed in the presence of 1.8 M R-TOH. The PFB [OOH, 15S-OH] ETEs and PFB diHETEs were analyzed with RP-HPLC in the same manner as the linolenate oxidations (Figure 7). The products are not as well-resolved in the chromatograms of the arachidonate oxidation products. Additional compounds with conjugated diene chromophores appear in the chromatograms. These diene products apparently derive from the abstraction of the C-7 hydrogen atoms, an event that can lead to three additional regioisomers formed as a pair of epimers. The PFB 15S-HETE oxidation samples were also analyzed by mass spectrometry to determine which regioisomers are formed in the peroxidation. The PFB [OOH, 15S-OH] ETEs were analyzed using Ag+-CIS-MS utilizing the same instrument conditions and RP-HPLC gradient of (80:20f90:10) MeOH: H2O gradient over 40 min (see the Supporting Information for Figure S1 and a discussion of the fragmentation patterns). From

the Hock cleavage fragmentation of these compounds, seven regioisomer epimeric pairs are detected as compared to the four sets that are formed from γ-linolenate. The elution pattern remains the same for the four regioisomers (8,15; 10,15; 12,15; and 14,15) that are analogous to the linolenates. Products from the pentadienyl radical due to hydrogen atom abstraction at C-7 (5,15; 7,15; and 9,15) are intermixed within the aforementioned compounds in the chromatogram of PFB [OOH, 15S-OH] ETEs. HPLC analysis of the PFB diHETEs showed better product resolution than was seen from the PFB [OOH, 15S-OH] ETEs. The diHETEs from the oxidation of PFB 15S-HETE were analyzed by LC-EC-APCI-MS utilizing the same conditions used in the analysis of PFB diHOTrEs. The analysis was highly sensitive for the parent carboxylate ion at 335 m/z, which results from loss of the PFB headgroup from the diHETE esters. The chromatogram peaks in Figure 7B that result from the reduction of the [OOH, 15S-OH] ETES with P(OCH3)3 were confirmed to be PFB diHETEs using SIM of 335 m/z. The diHETE regioisomers were tentatively identified using a full scan of the daughter ions derived from CID-induced fragmentation of the parent 335 m/z ions. Assignments for the peaks in Figure 7B are as follows: peaks VIIa and VIIc are assigned as 8,15-Z,E,E,E-diHETEs based on the UV spectra, in combination with the similarities between their mass spectra and those of the 6,13-E,E,E-diHOTrEs. Using similar comparisons of spectra, peaks VIIb and VIId are assigned 8,15-Z,E,Z,EdiHETEs. The remaining triene diols, peaks VIIe and VIIf, are believed to be the 14,15-Z,Z,E,E-diHETEs by analogy to the mechanism for formation of the corresponding 12,13-Z,E,E-

Figure 7. Chromatograms of PFB 15S-HETE oxidized in the presence of 1.8 M R-TOC for 4 h at 37 °C initiated by 0.05 M MeOAMVN observed at 268 (black line) and 235 nm (red dashed line). The separation mobile phase was (80:20f90:10) MeOH:H2O over 40 min. (A) PFB [OOH, 15S-OH] ETEs. (B) PFB diHETEs.


diHOTrEs. Additional characterization of the arachidonate products was not pursued at this time. However, product analyses and mechanistic studies are currently being performed on the oxidation of 15S-HETE to further relate this more biologically relevant system to the formation of leukotrienes and will be presented in future works. In conclusion, chromatography conditions were established to allow for the separation and isolation of the products formed from the oxidation of PFB 13S-HOTrE and PFB 15S-HETE. The isolated products were then characterized utilizing various analytical techniques including UV absorbance, MS, NMR, and CD to characterize the linolenate diols. With the acquired data, a mechanism is proposed for the formation of dihydroperoxides from the autoxidation of polyunsaturated fatty esters. A preliminary analysis of the arachidonate oxidations showed the autoxidations to occur by a similar mechanism. Acknowledgment. We thank Dawn Overstreet and Lisa Manier of the Vanderbilt University Mass Spectrometry Research Center for their assistance in MS measurements. We also extend thanks to Markus Voehler and the Vanderbilt University Center for Structural Biology for assistance with and use of the NMR instrumentation. Dr. Claus Schneider is thanked for his valuable conversations about ECCD, and we offer additional thanks to Professor David Wright and Dr. Scott Miller for the use and assistance with the CD spectrophotometer. N.A.P. thanks NIH (ES-13125) and the National Science Foundation (CHE-107697) for their support of this work. C.L.R. acknowledges support from the Center in Molecular Toxicology, Vanderbilt University (T32ES07028). Supporting Information Available: UV/vis spectra, NMR data, and additional CD spectra of PFB diHOTrEs. This material is available free of charge via the Internet at http://pubs.acs.org.

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Identification of the Peroxidation Products of 13-Hydroxy-Î³-linolenate

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