Chem. Res. Toxicol. 1993,6,413-416
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Polyunsaturated Fatty Acid Alkoxyl Radicals Exist as Carbon-Centered Epoxyallylic Radicals: A Key Step in Hydroperoxide-AmplifiedLipid Peroxidation Allan L. Wilcoxl and Lawrence J. M a r n e t t * A. B. Hancock, Jr., Memorial Laboratory of Cancer Research, Center in Molecular Toxicology, Department of Biochemistry, Vanderbilt Uniuersity School of Medicine, Nashuille, Tennessee 37232-0146 Received March 10,1993
13-Hydroperoxyoctadeca-9,11,15-trienoic acid wm reacted with a catalytic amount of 5,10,15,20tetraphenyl-21H,23H-porphyrin iron(II1) chloride in dichloromethane containing 2,4,6-tri-tertbutylphenol. The principal products were identified as 13-oxooctadeca-9,11,15-trienoic acid, 13-oxotrideca-9,11-dienoicacid, and a series of isomeric epoxyaryl ethers [9-(2,4,6-tri-tertbutylphenoxy)-12,13-epoxyoctadec-l0-enoic acids and ll-(2,4,6-tri-tert-butylphenoxy)-12,13epoxyoctadec-9-enoic acids]. The epoxyaryl ethers are coupling products of 2,4,6-tri-tertbutylphenoxyl radical and an epoxyallylic radical formed by cyclization of an intermediate alkoxyl radical. The high yield of epoxyaryl ethers relative to 13-oxotrideca-9,11-dienoicacid suggests the equilibrium between alkoxyl radical and epoxyallylic radical lies predominantly toward epoxyallylic radical. This cyclization appears to be a key step in the amplification of lipid peroxidation by polyunsaturated fatty acid hydroperoxides. Introduction Polyunsaturated fatty acid hydroperoxidesare reduced by a variety of metal complexes and metalloproteins to alkoxyl radicals (1-9). Alkoxyl radicals are believed to be responsible for some,of the deleterious effects of lipid peroxidation because they are organic analogs of the highly reactive hydroxyl radical (10). Work from several laboratories has established that the chemical fate of alkoxyl radicals depends on the functional groups in the vicinity of the radical center. For example, 1 undergoes extensive cyclization to an epoxyallylic radical, whereas 2 undergoes predominantly 8-scission to aldehyde and octenyl radical (Figure 1) (11-14). In both cases, the driving force is provided by production of an allylic carbon-centered radical. An intriguing question regards the extent to which cyclization competes with 8-scission in an alkoxyl radical with both options such as 3 (Figure 1). Radicals analogous to 3 are intermediates in the metal-catalyzed decomposition of 8-, 9-, 11-,and 12-hydroperoxy derivatives of arachidonic acid, which are expected to have widespread occurrence in peroxidized phospholipid membranes (15). To explore the fate of radical 3,13-hydroperoxyoctadeca9,11,15-trienoic acid, (4) was reacted with a catalytic amount of 5,10,15,20-tetraphenyl-21H,23H-porphyrin iron(111)chloride (TPP-Fe3+)2 in dichloromethane. TPP-Fe3+ reduces organic hydroperoxidesby one electron to alkoxyl radicals and is oxidized to a ferryl-oxo complex (5). 2,4,6Tri-tert-butylphenol (TBPH) was added to support catalytic reduction and to minimize secondary reactions such as hydrogen abstraction from the hydroperoxide by the
* To whom correspondence should be addressed.
'Present address: Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, 825 NE 13th St., Oklahoma City, OK 73104. *Abbreviations: 5,10,15,20-tetraphenyl-2lZf,23H-porphyrin iron(II1) TBPH; correlated specchloride, WP-FeS+;2,4,6-tri-tert-butylphenol, troscopy, COSY.
3
Figure 1. Alkoxyl radicals (1 and 2) that undergo cyclization and @-scissionto allylic radicals. Radical 3 can undergo both @-scissionand cyclization to allylic radicals.
ferryl-oxo complex (16-18). Inclusion of TBPH led to the production of a series of novel epoxyaryl ethers which were the major class of reaction products. The extent of formation of these products indicates that cyclization occurs preferentially to @-scissionand that polyunsaturated fatty acid alkoxyl radicals exist predominantly as carbon-centered epoxyallylic radicals.
Materials and Methods Chemicals. Octadeca-9,12,15-trienoicacid (linolenicacid) was purchased from Nu-Chek Prep (Elysian,MN) and [1-*4C]linolenic acid from New England Nuclear (Wilmington, DE). 13-Hydroperoxyoctadeca-9,11,15-trienoicacid (4) was prepared from [l-14C]linolenicacid (6.4X l o 7 dpm/mmol) and soybean lipoxygenase type I (Sigma Chemical, St. Louis, MO) and purified by preparative HPLC (19). Imidazole, TBPH, and TPP-FeS+were 0 1993 American Chemical Society
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5
4
Communications
6
HOO
7
1-Bu
8
Figure 2. Products identified in the reaction of 4 with TPPFe3+. Product 7 is a major product in the absence of TBPH whereas product 8 and a series of isomeric epoxyaryl ethers are the major products in the presence of TBPH. purchased from Aldrich Chemical (Milwaukee,WI). TBPH was recrystallized three times from absolute ethanol prior to use, and other chemicals were used as received from the supplier. Solvents were purchased from Baxter ScientificProducts (Stone Mountain, GA) and degassed prior to use for HPLC. Instrumentation. AnalyticalHPLC analyseswere performed using an Alltech Partisil silica 10-pm (4.5 X 250 mm) column (Alltech Associates, Deerfield, IL). Preparative HPLC separations were accomplished using a Whatman 10-pm Magnum 9 column (10 X 500 mm) (Hillsboro, OR). Eluants from columns were monitored with either a Varian 2550 variable-wavelength detector or a Hewlett-Packard 1040A diode array detector controlled with an H P 79994A Chemstation computer. Radioactive peaks were detected with either a Radiomatic Flow-One/ Beta or a Radiomatic A100 radioactive flow detector. GC-MS analyses were performed using a Supelco 6-m SPBl capillary column (Supelco, Bellefonte Park, PA) on either a HewlettPackard 5880a GC coupled to a Finnigan Incos 50 mass spectrometer or a Varian Vista 6000 GC coupled to a Nermag R10 10 mass spectrometer. UV spectra were recorded with a Hewlett-Packard 8452a spectrophotometer and processed with the aid of a Hewlett-Packard 79994A Chemstation. NMR spectra were recorded on a Bruker 300-mHz Model AM300 spectrometer in CDCl3. Reaction Conditions. Reactions were initiated by addition of TPP-FeS+ (1.4 X 1od M) to 4 (5.0 X 1W M) with and without TBPH (2.5 X M) in CHzClz at 22 OC. Reactions were analyzed by direct injection onto HPLC. Products were eluted at a flow rate of 1.5mL/min with a gradient of THF and hexane containing 0.1% acetic acid (1-3% THF over 25 min; 3-8% THF from 25 to 50 min; 8-15% THF from 50 to 55 min). To obtain enough material for NMR analysis, the reaction was scaled up and the resulting reaction mixture applied to a silica gel SepPak column and the SepPak washed with THF/ hexane with 0.1 % HOAc. The solvent was evaporated and the residue applied to the Magnum prep column. Isolated products were shown to coelute with products identified by analytical
HPLC.
Results Reaction of 4 (5 mM) with TPP-Fe3+ (0.014 mM) in CH2Cl2 for 60 min led to a complex mixture of products containing 4 % aldehyde 5 , 3 % ketone 6,22 % unreacted starting material, 28 5% isomerized hydroperoxide 7, and 43% unidentified polar products (Figure 2). The polar material may contain dimeric compounds analogous to the epoxyallylic dimers previously reported by Kaneko and Matsuo, and by Gardner et al. (7,20).The formation of 7 is most likely due to rearrangement of the 13-peroxyl
2.5
I Q
3.0
3.5
.o 4.0 B
d
€3
4.5 c
5.0
P
5.5
Figure 3. Partial two-dimensional COSY spectrum of product 8.
radical formed by H-abstraction from 4 by the ferryl-oxo complex (21) . Inclusion of TBPH (25 mM) caused a dramatic change in the product profile. Straight-phase HPLC analysis indicated that the major product zone (73%) was very nonpolar and contained multiple products. Aldehyde 5 and ketone 6 were still minor products (4% and 7 % , respectively) along with a small amount of rearranged hydroperoxide 7 (3%). The extent of conversion was greater in the presence of the phenol, as only 4% of the starting material was recovered. Rechromatography of the nonpolar product zone revealed the presence of four products with similar spectral properties. Negative ion chemical ionization m a s spectra of pentafluorobenzyl esters did not exhibit molecular ions, but contained ions at mlz 489 and 473. The ion at mlz 489 arose from the loss of tri-tert-butylphenyl from a putative molecular ion at mlz 734, and the ion at mlz 473 was due to loss of tri-tert-butylphenoxyl. lH-NMR spectra provided evidence for an epoxyaryl ether structure. A COSY spectrum (Figure 3) revealed weak coupling between the two epoxide protons at 2.86 ppm (H19 and 3.15 ppm (H12)and between H12 and a vinyl proton at 5.38 ppm (H11). HI1 was in turn coupled to a vinyl proton at 5.78 ppm (H1O),which was coupled to a methine proton a t 4.28 ppm (Hg). These connectivities are consistent with the substitution pattern of the epoxyaryl ether functional group present in 8. The remaining resonances in the NMR
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Communications
TPP-Fe3'
+
6(CH2)@2H
TPP-Fe"0H
+
C2H~
0-
OH
I
1-Bu
CBU
+
__c
CBU
otherisomers
1-Bu
Figure 4. Mechanism for the formation of epoxyaryl ethers in the reaction of 4 with TPP-FeS+in the presence TBPH.
spectrum were due to the 15,16-double bond (H16, 5.54 ppm; H15, 5.3 ppm), the aryl protons (6.70 ppm), the tertbutyl group (0.90 ppm), and the aliphatic protons (0.962.48 ppm). Similar analysis of the other three nonpolar peaks established that each was an epoxyaryl ether.3 Double bond and configurational isomerization account for the different products. The presence of isomeric epoxyaryl ethers suggests they arise by coupling of the TBPH phenoxyl radical to the termini of the epoxyallylic radical derived from alkoxyl radical cyclization. The possibility they arose by phenoxyl radical-induced decompositionof the starting hydroperoxide was eliminated by a control experiment in which the TBPH phenoxyl radical was incubated with 4.4 The hydroperoxide was recovered unchanged, and no traces of epoxyaryl ethers were detectable.
Discussion Previous studies have revealed a rich chemistry of polyunsaturated fatty acid alkoxyl radicals; two facile reactions are cyclization and @-scission(7,ll-14,20). The extent to which these reactions proceed depends upon the stability of the carbon-centered radical formed. In the present series of experiments, we have explored the competition between these two reactions in an alkoxyl radical in which both processes could produce a highly stabilized allylic radical. The results indicate that cyclization is the preferred mode of reaction. Either in the absence or in the presence of TBPH, the @-scissionproduct 5 accounted for less than 5% of the products. The formation of epoxyaryl ethers in the reaction of 4 with TPP-Fe3+and TBPH is a dramatic and important result. The minimum series of reactions necessary to produce 8 and its isomers is illustrated in Figure 4. Homolytic scission of 4 by TPP-Fe3+ produces alkoxyl radical 3, which must undergo immediate cyclization to SThe structures of all these products will be the subject of a full report. 'Solutione of tri-tert-butylphenoxylradical were prepared by reaction of TBPH with 2,2-diphenylpicrylhydrazylradical (29).
the epoxyallylic radical in order to prevent @-scissionto 5. The oxidized iron derivative formed by reduction of 4 then oxidizes TBPH to a phenoxyl radical that couples to the epoxyallylic radical. The ratio of epoxyaryl ethers to aldehyde 5 is nearly 20:1, which emphasizes that the equilibrium between epoxyallylic radical and alkoxyl radical lies substantially toward epoxyallylic radical. However, these data also require that the rate of equilibration of the two radicals is slow because whenever the epoxyallylic radical opens to the alkoxyl radical, it has a significant probability of @-scissionto 5 (12-14). The low yield of 5 in the presence of TBPH indicates that the epoxyallylic radical opens to the alkoxyl radical slower than the ferryl-oxo complex oxidizesTBPH and the TBPH phenoxyl radical couples to the epoxyallylic radical. Thus, alkoxyl radical 3 exists predominantly as the carboncentered epoxyallylic radical. Gardner has previously proposed that polyunsaturated fatty acid alkoxyl radicals exist as epoxyallylic radicals because of the predominance of epoxide-containing products relative to alcohol in the reduction of linoleic acid hydroperoxide by ferrous complexes (22). One limitation of these studies was the absence of a high-yield unimolecular pathway of alkoxyl radical decomposition that could compete with cyclization. The possibility of 8scission from 3 provides such a competitive manifold. The very low yields of 8-scission products in the reaction of 3 provide strong evidence for the existence and stability of the epoxyallylic radical. Furthermore, the inability of TBPH to increase the yields of the fatty acid alcohol derived from 4 suggests that 3 does not live long enough to be reduced by a reactive phenol. These findings provide significant insights into the chemical events responsible for lipid peroxidation. Metal hydroperoxide reactions are known to amplify the extent of lipid peroxidation presumably by formation of alkoxyl free radicals (23). The alkoxyl free radicals are believed to abstract hydrogen atoms from active methylene groups of the polyunsaturated fatty acid residues of membranes, thereby initiating new radical chains. However, in studies of the metal-catalyzed decomposition of polyunsaturated
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fatty acid hydroperoxides in the presence of microsomal lipids, very low yields of the alcohols that would arise by hydrogen abstraction are obtained (22, 24, 25). This paradox can be explained by our finding that the alkoxyl radicals exist primarily as epoxyallylic radicals. Our findings also help to explain why metal-induced hydroperoxide reduction is an efficient amplifier of lipid peroxidation (26). Epoxyallylic radicals are more stable than alkoxyl radicals and couple with 02 to form peroxyl radicals (eq 1). Peroxyl radicals are the stablest of the
oxygen radicals and are highly selective for abstraction of the active methylene hydrogens of polyunsaturated fatty acids (IO,26-28). Thus, short-lived alkoxyl radicals that are the initial products of hydroperoxide reduction are converted into long-lived peroxyl radicals that are efficient initiators/propagators of lipid peroxidation.
Acknowledgment. This work was supported by a research grant (CA47479)and a training grant (ES07028) from the National Institutes of Health. NMR and mass spectrometry were supported by an NIEHS center grant (ES00267). We are grateful to Chandra Prakash for assistance in acquiring some of the mass spectra.
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