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• W2068505823 abstract "Biosynthesis of the prostaglandin endoperoxide by the cyclooxygenase (COX) enzymes is accompanied by formation of a small amount of 11R-hydroxyeicosatetraenoic acid (HETE), 15R-HETE, and 15S-HETE as by-products. Acetylation of COX-2 by aspirin abrogates prostaglandin synthesis and triggers formation of 15R-HETE as the sole product of oxygenation of arachidonic acid. Here, we investigated the formation of by-products of the transformation of 5S-HETE by native COX-2 and by aspirin-acetylated COX-2 using HPLC-ultraviolet, GC-MS, and LC-MS analysis. 5S,15S- dihydroxy (di)HETE, 5S,15R-diHETE, and 5S,11R-diHETE were identified as by-products of native COX-2, in addition to the previously described di-endoperoxide (5S,15S-dihydroxy-9S,11R,8S,12S-diperoxy-6E,13E-eicosadienoic acid) as the major oxygenation product. 5S,15R-diHETE was the only product formed by aspirin-acetylated COX-2. Both 5,15-diHETE and 5,11-diHETE were detected in CT26 mouse colon carcinoma cells as well as in lipopolysaccharide-activated RAW264.7 cells incubated with 5S-HETE, and their formation was attenuated in the presence of the COX-2 specific inhibitor, NS-398. Aspirin-treated CT26 cells gave 5,15-diHETE as the most prominent product formed from 5S-HETE. 5S,15S-diHETE has been described as a product of the cross-over of 5-lipoxygenase (5-LOX) and 15-LOX activities in elicited rat mononuclear cells and human leukocytes, and our studies implicate cross-over of the 5-LOX and COX-2 pathways as an additional biosynthetic route. Biosynthesis of the prostaglandin endoperoxide by the cyclooxygenase (COX) enzymes is accompanied by formation of a small amount of 11R-hydroxyeicosatetraenoic acid (HETE), 15R-HETE, and 15S-HETE as by-products. Acetylation of COX-2 by aspirin abrogates prostaglandin synthesis and triggers formation of 15R-HETE as the sole product of oxygenation of arachidonic acid. Here, we investigated the formation of by-products of the transformation of 5S-HETE by native COX-2 and by aspirin-acetylated COX-2 using HPLC-ultraviolet, GC-MS, and LC-MS analysis. 5S,15S- dihydroxy (di)HETE, 5S,15R-diHETE, and 5S,11R-diHETE were identified as by-products of native COX-2, in addition to the previously described di-endoperoxide (5S,15S-dihydroxy-9S,11R,8S,12S-diperoxy-6E,13E-eicosadienoic acid) as the major oxygenation product. 5S,15R-diHETE was the only product formed by aspirin-acetylated COX-2. Both 5,15-diHETE and 5,11-diHETE were detected in CT26 mouse colon carcinoma cells as well as in lipopolysaccharide-activated RAW264.7 cells incubated with 5S-HETE, and their formation was attenuated in the presence of the COX-2 specific inhibitor, NS-398. Aspirin-treated CT26 cells gave 5,15-diHETE as the most prominent product formed from 5S-HETE. 5S,15S-diHETE has been described as a product of the cross-over of 5-lipoxygenase (5-LOX) and 15-LOX activities in elicited rat mononuclear cells and human leukocytes, and our studies implicate cross-over of the 5-LOX and COX-2 pathways as an additional biosynthetic route. Oxygenation of arachidonic acid by either of the two cyclooxygenase (COX) isozymes yields the prostaglandin endoperoxide PGH2 as the major product and the mono-hydroxylated 11-hydroxyeicosatetraenoic acid (HETE) and 15-HETE as by-products of about 2–5% abundance (1Hamberg M. Samuelsson B. Oxygenation of unsaturated fatty acids by the vesicular gland of sheep.J. Biol. Chem. 1967; 242: 5344-5354Abstract Full Text PDF PubMed Google Scholar). 11-HETE is exclusively of the 11R configuration, similar in configuration to the first oxygenation of arachidonic acid to the 11R-peroxyl radical that will form the 9,11- endoperoxide of PGH2 (2Hawkins D.J. Brash A.R. Eggs of the sea urchin Strongylocentrotus purpuratus, contain a prominent (11R) and (12R) lipoxygenase activity.J. Biol. Chem. 1987; 262: 7629-7634Abstract Full Text PDF PubMed Google Scholar, 3Baer A.N. Costello P.B. Green F.A. Stereospecificity of the hydroxyeicosatetraenoic and hydroxyoctadecadienoic acids produced by cultured bovine endothelial cells.Biochim. Biophys. Acta. 1991; 1085: 45-52Crossref PubMed Scopus (26) Google Scholar). 15-HETE is formed as a mixture of the 15S- and 15R-enantiomers, in contrast to the configuration of C15 in PGH2, which is strictly S (4Schneider C. Boeglin W.E. Prusakiewicz J.J. Rowlinson S.W. Marnett L.J. Samel N. Brash A.R. Control of prostaglandin stereochemistry at the 15-carbon by cyclooxygenases-1 and 2. A critical role for serine 530 and valine 349.J. Biol. Chem. 2002; 277: 478-485Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The HETE by-products are thought to arise from a slightly different alignment of substrate in the active site compared with when PGH2 is formed (5Thuresson E.D. Lakkides K.M. Smith W.L. Different catalytically competent arrangements of arachidonic acid within the cyclooxygenase active site of prostaglandin endoperoxide H synthase-1 lead to the formation of different oxygenated products.J. Biol. Chem. 2000; 275: 8501-8507Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), rather than resulting from incomplete or otherwise faulty catalysis. Neither 15- hydroperoxy-eicosatetraenoic acid (HPETE) nor 11-HPETE can serve as substrates for formation of PGH2 in the cyclooxygenase reaction as demonstrated for the COX-1 enzyme (6Hamberg M. Samuelsson B. On the mechanism of biosynthesis of prostaglandins E1 and F1α.J. Biol. Chem. 1967; 242: 5336-5343Abstract Full Text PDF PubMed Google Scholar, 7Porter N.A. Wolf R.A. Pagels W.R. Marnett L.J. A test for the intermediacy of 11-hydroperoxyeicosa-5,8,12,14- tetraenoic acid [11-HPETE] in prostaglandin biosynthesis.Biochem. Biophys. Res. Commun. 1980; 92: 349-355Crossref PubMed Scopus (19) Google Scholar). It has not been established whether formation of the HETE by-products follows a particular biological rationale.Acetylation by aspirin (acetylsalicylic acid) of a serine residue in the oxygenase active site channel of both COX isozymes has discrete effects on the catalytic activities of the two enzymes (8DeWitt D.L. el-Harith E.A. Kraemer S.A. Andrews M.J. Yao E.F. Armstrong R.L. Smith W.L. The aspirin and heme-binding sites of ovine and murine prostaglandin endoperoxide synthases.J. Biol. Chem. 1990; 265: 5192-5198Abstract Full Text PDF PubMed Google Scholar, 9Lecomte M. Laneuville O. Ji C. DeWitt D.L. Smith W.L. Acetylation of human prostaglandin endoperoxide synthase-2 (cyclooxygenase-2) by aspirin.J. Biol. Chem. 1994; 269: 13207-13215Abstract Full Text PDF PubMed Google Scholar). Whereas COX-1 loses all oxygenase activity following treatment with aspirin, acetylated COX-2 gains a novel catalytic activity and forms 15R-HETE as the sole product (10Holtzman M.J. Turk J. Shornick L.P. Identification of a pharmacologically distinct prostaglandin H synthase in cultured epithelial cells.J. Biol. Chem. 1992; 267: 21438-21445Abstract Full Text PDF PubMed Google Scholar, 11Mancini J.A. O’Neill G.P. Bayly C. Vickers P.J. Mutation of serine-516 in human prostaglandin G/H synthase-2 to methionine or aspirin acetylation of this residue stimulates 15-R-HETE synthesis.FEBS Lett. 1994; 342: 33-37Crossref PubMed Scopus (84) Google Scholar).The major product formed by oxygenation of the 5-lipoxygenase product, 5S-HETE, with COX-2 is a bicyclic di-endoperoxide with structural similarities to the arachidonic acid derived PGH2 (12Schneider C. Boeglin W.E. Yin H. Stec D.F. Voehler M. Convergent oxygenation of arachidonic acid by 5-lipoxygenase and cyclooxygenase-2.J. Am. Chem. Soc. 2006; 128: 720-721Crossref PubMed Scopus (26) Google Scholar). The most significant difference between the two endoperoxides is that the typical cyclopentyl ring of PGH2, comprised of carbons 8 through 12, is extended to a seven-membered ring by insertion of a peroxide bridge from C8 to C12 in the 5-HETE derived di-endoperoxide. In addition, the di-endoperoxide contains two hydroxy groups, one at carbon 5 stemming from the 5S-HETE substrate and the other at C15, equivalent to the 15-hydroxy in the prostaglandins. The relative and absolute stereochemistries of carbons 9, 11, and 15 are the same in PGH2 and the di-endoperoxide, i.e., 9S, 11R, and 15S (13Griesser M. Boeglin W.E. Suzuki T. Schneider C. Convergence of the 5-LOX and COX-2 pathways. Heme-catalyzed cleavage of the 5S-HETE-derived di-endoperoxide into aldehyde fragments.J. Lipid Res. 2009; 50: 2455-2462Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar).Here, we report the structural identification and absolute configuration of two by-products of the COX-2 reaction with 5S-HETE. In addition, we analyzed the reaction of acetylated recombinant COX-2 with 5S-HETE. Finally, formation of diHETEs from exogenous 5S-HETE was confirmed to be dependent on COX-2 in two mouse cell lines, RAW264.7 and CT26.EXPERIMENTAL PROCEDURESMaterialsArachidonic acid was purchased from NuChek Prep, Inc. (Elysian, MN), lipopolysaccharide (LPS) (serotype 0111:B4) was from Calbiochem, and RAW264.7 and CT26 cells were obtained from ATCC (Manassas, VA). 5S-HETE was prepared by chemical synthesis from arachidonic acid as described (13Griesser M. Boeglin W.E. Suzuki T. Schneider C. Convergence of the 5-LOX and COX-2 pathways. Heme-catalyzed cleavage of the 5S-HETE-derived di-endoperoxide into aldehyde fragments.J. Lipid Res. 2009; 50: 2455-2462Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). 15R-HETE and 11R-HETE were prepared through vitamin E-controlled autoxidation of arachidonic acid methyl ester and purified by consecutive RP-, straight phase-, and chiral phase HPLC [Chiralpak AD (14Schneider C. Boeglin W.E. Brash A.R. Enantiomeric separation of hydroxy-eicosanoids by chiral column chromatography: effect of the alcohol modifier.Anal. Biochem. 2000; 287: 186-189Crossref PubMed Scopus (47) Google Scholar)], and a final step of mild hydrolysis of the methyl ester using KOH.Cell cultureRAW264.7 cells were cultured in DMEM and grown at 37°C in an atmosphere of 5% CO2. Cells of passages 5 and 6 only were used. Cells were stimulated by treatment with 100 ng/ml LPS and 10 units/ml of IFN-γ for 6 h to induce expression of COX-2. CT26 cells were cultured in RPMI 1640 medium. 5S-HETE, 5 μg dissolved in 1 μl of ethanol, was added to ∼70% confluent cells in 100 mm dishes, and after 10 min at 37°C, the culture medium was removed, acidified to pH 4, and extracted using a 30 mg Waters HLB cartridge. Products were eluted from the cartridge with methanol, evaporated, and dissolved in 50 μl of LC-MS solvent A. CT26 and RAW264.7 control cells were not treated with LPS. In some experiments, CT26 and activated RAW264.7 cells were treated with 2 mM aspirin, respectively (from a 40 mM stock solution in DMSO), or with 10 μM NS-398 (from a 10 mM stock solution in ethanol) 30 min prior to incubation with 5S-HETE.Reaction of recombinant COX-2 with 5S-HETEThe reaction of 5S-HETE (120 μg total; containing 300,000 cpm of [1-14C]5S-HETE) with recombinant human COX-2 was performed in four separate 2 ml reactions with 30 μg substrate each as described (12Schneider C. Boeglin W.E. Yin H. Stec D.F. Voehler M. Convergent oxygenation of arachidonic acid by 5-lipoxygenase and cyclooxygenase-2.J. Am. Chem. Soc. 2006; 128: 720-721Crossref PubMed Scopus (26) Google Scholar). The products were extracted using a Waters HLB cartridge and analyzed by RP-HPLC using a Waters Symmetry C18 5-μm column (4.6 × 250 mm) eluted with a gradient of acetonitrile/water/acetic acid programmed from 20/80/0.01 (by vol) to 70/30/0.01 (by vol) within 20 min at 1 ml/min flow rate. The elution profile was monitored using an Agilent 1200 diode array detector coupled on-line to a Packard Radiomatic A100 Flo-one radioactive detector. The by-products eluting at 19.8 and 20.5 min retention time were collected, extracted from HPLC solvent, and stored in methanol at −20°C until further analysis.Reaction of aspirin-acetylated COX-2 with 5S-HETERecombinant human COX-2 (0.5 μM final concentration) was diluted in 1 ml of 100 mM Tris-HCl buffer pH 8.0 and treated with 2 mM aspirin in a 37°C water bath for 30 min (15Rowlinson S.W. Crews B.C. Goodwin D.C. Schneider C. Gierse J.K. Marnett L.J. Spatial requirements for 15-(R)-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid synthesis within the cyclooxygenase active site of murine COX-2. Why acetylated COX-1 does not synthesize 15-(R)-HETE.J. Biol. Chem. 2000; 275: 6586-6591Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). A control reaction incubated with arachidonic acid and analyzed by LC-MS before and after treatment showed >95% inhibition of PG formation. The buffer was supplemented with hematin (1 μM) and phenol (500 μM), and 30 μg of 5S-HETE were added. After 5 min at room temperature, 25 μl of methanol were added, the mixture was acidified to pH 4 with glacial acetic acid, and loaded onto a preconditioned Waters Oasis HLB cartridge. After washing with water, the products were eluted with methanol. The 5,15-diHETE product was isolated using RP-HPLC as described above for the native enzyme.Synthesis and isolation of diHETE reference compounds5S,15S-diHETE was synthesized by reaction of 5S-HETE with the LOX-1 isozyme from soybean seeds (16Brash A.R. Song W-C. Detection, assay, and isolation of allene oxide synthase.Methods Enzymol. 1996; 272: 250-259Crossref PubMed Google Scholar). To 3 ml of 100 mM K2HPO4 buffer pH 10 were added 100 μg of 5S-HETE and 1 μl of soybean lipoxygenase solution (∼25,000 units; Sigma). 5R,15S-diHETE was synthesized using 5R-HETE (100 μg) as substrate and 3 μl of soybean lipoxygenase solution. After 1 min reaction time, the solution was acidified (pH 4) and extracted with methylene chloride. The organic extract was evaporated, dissolved in methanol, and treated with 200 μg of triphenylphosphine (TPP) for 15 min at room temperature. 5S,15S-DiHETE and 5R,15S-diHETE were isolated by RP-HPLC using a Waters Symmetry C18 column (4.6 × 250 mm) eluted with a solvent of methanol/water/acetic acid (80/20/0.01, by vol) at 1 ml/min flow rate and UV detection at 235 nm.5S,15R-DiHETE was synthesized by reaction of 15R-HETE with recombinant human 5-LOX. For the enzymatic transformation, a pellet of Sf9 insect cells expressing 5-LOX (∼300 μl) was sonicated and transferred to 1 ml of PBS containing 2 mM CaCl2 and 1 mM ATP. 15R-HETE (50 μg) was added and the reaction was allowed to proceed for 15 min at room temperature. The reaction was terminated by the addition of 250 μl of methanol and 10 mg of NaBH4. After 15 min at room temperature, the pH was adjusted to 3 using 1 N HCl, and the products were extracted using methylene chloride. 5S,15R-DiHETE was purified by RP-HPLC as described above for the 5S,15S-diastereomer.5S,11R-DiHETE was synthesized by reaction of 5S-HETE with the recombinant linoleic acid 9R-LOX from Anabaena sp. PCC7120 expressed in Escherichia coli, a gift from Alan R. Brash at Vanderbilt University (17Zheng Y. Boeglin W.E. Schneider C. Brash A.R. A 49-kDa mini-lipoxygenase from Anabaena sp. PCC 7120 retains catalytically complete functionality.J. Biol. Chem. 2008; 283: 5138-5147Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The substrate, 5S-HETE (100 μg), was added to 3 ml of 100 mM Tris-HCl pH 7.5 containing 150 mM NaCl and 0.01% CHAPS. The reaction was initiated by addition of 1 μl of the purified Anabaena 9R-LOX. After 3 min reaction time, the solution was acidified to pH 4 using 1 N HCl, and the products were extracted using a 30 mg Waters HLB cartridge and eluted with methanol. After evaporation of the solvent, the residue was dissolved in 100 μl of methanol, and the products were reduced with 150 μg TPP at room temperature for 15 min. 5S,11R-DiHETE was isolated using RP-HPLC conditions as described above for 5S,15S-diHETE. HPLC-purified 5S,11R-diHETE was dissolved in CDCl3 for NMR analysis using a Bruker AV-II 600 MHz spectrometer equipped with a cryoprobe. Chemical shifts are reported relative to the signal for residual CHCl3 at δ 7.25 ppm.A mixture of 5,11-diHETE diastereomers was synthesized by autoxidation of racemic 11-HETE. Three 200 μg aliquots of 11-HETE were evaporated in small plastic tubes and placed in an oven at 37°C. After 2 h, the samples were dissolved in 50 μl of methanol, treated with triphenylphosphine (TPP), and analyzed using RP-HPLC. The diastereomers eluted as a single peak and purification was performed as described for 5S,15S-diHETE.SP-HPLC analysis of diHETEsThe 5,15-diHETE diastereomers were resolved using an Agilent Zorbax RX-SIL 5-μm column (4.6 × 250 mm) eluted with hexane/isopropanol/acetic acid (95/5/0.1, by vol) at 1 ml/min flow rate. The 5,11-diHETEs were analyzed using the same HPLC conditions after conversion to the methyl ester derivatives with diazomethane. Eluting peaks were monitored using an Agilent 1200 series diode array detector.CD spectroscopyAliquots of ∼20 μg each of 5S-HETE, 15S-HETE, 15R-HETE, 11S-HETE, 11R-HETE, and the enzymatically synthesized standards of 5S,15S-diHETE, 5R,15S-diHETE, and 5S,11R-diHETE were treated with ethereal diazomethane for 30 s, evaporated, and dissolved in 50 μl of dry acetonitrile. To the solution was added 1 μl of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and a few grains each of 4-dimethylaminopyridine (DMAP) and 2-naphthoylchloride. The reaction was carried out at room temperature overnight, the solvent was evaporated, and the residue was dissolved in methylene chloride and washed with water twice. Purification of the methyl ester, 2-naphthoyl derivatives of the HETEs and diHETEs was achieved by RP-HPLC using a Waters Symmetry C18 column (4.6 × 250 mm) eluted with a solvent of methanol/water/acetic acid (95/5/0.01, by vol) at 1 ml/min flow rate and UV detection at 235 nm. Samples were extracted from HPLC solvent using methylene chloride and dissolved in acetonitrile to an optical density (OD) of 0.75 absorbance unit (AU) for HETE derivatives and OD 1.5 AU for diHETE derivatives (when possible), respectively. Circular dichroism (CD) spectra were recorded using an Aviv Model 215 CD spectrometer at room temperature in a 1 cm pathlength cuvette scanning from 350–200 nm. The 1H NMR spectrum (600 MHz) of the 2-naphthoate derivatized 5S,11R-diHETE methyl ester was recorded in CD3CN, δ 1.93 ppm.GC-MS and LC-MS analysisFor GC-MS analysis, 5,15-diHETE and 5,11-diHETE formed by reaction of COX-2 with 5S-HETE were purified using RP- and SP-HPLC and methylated using ethereal diazomethane. Hydrogenation was performed in 100 μl of ethanol in the presence of palladium/carbon and bubbling with hydrogen gas for 5 min. Trimethylsilyl ethers were prepared using bis(trimethylsilyl)trifluoroacetamide at room temperature for 1 h. The reagents were evaporated and the samples were dissolved in hexane. GC-MS analysis was carried out in the EI mode (70 eV) using a ThermoFinnigan DSQ mass spectrometer equipped with a 5 m SPB-1 column (0.1 mm i.d., film thickness 0.25 μm) and a temperature program from 100°C, hold 2 min, and then increased to 260°C at 20°C/min.LC-MS was performed using a ThermoFinnigan Quantum Access instrument equipped with an electrospray interface and operated in the negative ion mode. User modified parameters of sheath and auxiliary gas pressures, temperature, and voltage settings were optimized using direct infusion of a solution of PGD2. A Waters Symmetry Shield C18 3.5 μm-column (2.1 × 150 mm) was eluted with a linear gradient of acetonitrile/water, 10 mM NH4OAc (5/95, by vol; solvent A) to acetonitrile/water, 10 mM NH4OAc (95/5, by vol) at a flow rate of 0.2 ml/min within 10 min. Negative ion collision-induced dissociation (CID) mass spectra of the standards of PGD2, 5S-HETE, 5S,15S-diHETE, and 5S,11R-diHETE were obtained. The fragmentation patterns were used to establish ion transitions for analyses in the selected reaction monitoring (SRM) mode. The following transitions were monitored: for PGD2 and PGE2: m/z 351 → 271; 5-HETE: m/z 319 → 115; 5,15-diHETE: m/z 335 → 201; and 5,11-diHETE: m/z 335 → 183. Relative levels of prostaglandins and diHETEs between treatments were calculated using peak areas of the signals in the SRM chromatograms.RESULTSReaction of native and acetylated COX-2 with 5S-HETERP-HPLC analysis of the transformation of [1-14C]5S-HETE by recombinant COX-2 shows one main product that was identified previously as a highly oxygenated di-endoperoxide (12Schneider C. Boeglin W.E. Yin H. Stec D.F. Voehler M. Convergent oxygenation of arachidonic acid by 5-lipoxygenase and cyclooxygenase-2.J. Am. Chem. Soc. 2006; 128: 720-721Crossref PubMed Scopus (26) Google Scholar), in addition to two minor, less polar peaks designated I and II representing by-products of the reaction (Fig. 1A). When COX-2 was treated with aspirin prior to incubation with 5S-HETE, one major product (III) was formed with retention time similar to peak I in the untreated enzyme (Fig. 1B). Both I and III had a characteristic UV spectrum with a λmax at 243 nm that was readily identified as 5,15-diHETE (Fig. 1C) (18Maas R.L. Turk J. Oates J.A. Brash A.R. Formation of a novel dihydroxy acid from arachidonic acid by lipoxygenase-catalyzed double oxygenation in rat mononuclear cells and human leukocytes.J. Biol. Chem. 1982; 257: 7056-7067Abstract Full Text PDF PubMed Google Scholar). The UV spectrum of peak II had a maximum at 238 nm with shoulders around 228 nm and 247 nm (Fig. 1C). The retention time and UV spectrum of II implicated that the product also contained two hydroxy groups and conjugated diene moieties.The products I and II were isolated using RP-HPLC and further purified as the methyl ester derivatives using SP-HPLC. GC-MS analysis in the EI mode (70 eV) of the hydrogenated, TMS-ether derivatives confirmed the identification of the first peak I as 5,15-diHETE. Characteristic α-cleavage fragments were found at m/z 203 (55% relative intensity) and m/z 311 [after loss of O-trimethylsilyl (OTMS); 9%] for the 5-hydroxy, and at m/z 173 and 341 (after loss of OTMS) (56% and 7%, respectively) for the 15-hydroxy group; the base peak was at m/z 73. Peak III from the aspirin-acetylated COX-2 reaction was identified as 5,15-diHETE based on identical UV spectra and retention times on RP-HPLC, and in addition to subsequent experimental evidence as described below.Product II gave a very weak [M+] (m/z 502) and [M-CH3+] (m/z 487) ion, with characteristic α-cleavage fragments at m/z 203 (42%) and m/z 311 (after loss of OTMS; 4%) indicating a 5-hydroxy group, and at m/z 229 (38% relative intensity) and m/z 285 (after loss of OTMS) (5%) indicative of a 11-hydroxy group. The LC-ESI mass spectrum confirmed the molecular weight as 336 and also gave a major fragment at m/z 183 and a minor fragment at m/z 115, compatible with two hydroxyls at carbons 5 and 11 (Fig. 1D). Based on UV, GC-MS, and LC-MS analyses, product II was identified as 5,11-diHETE.1H NMR and H,H COSY data for product II were recorded using a chromatographically and spectroscopically (UV, LC-MS/MS) identical standard of 5S,11R-diHETE that was prepared as described below. The 1H NMR spectrum showed eight signals in the double bond region that appeared as a pair of two similar motives of four protons each comprised of the two conjugated cis,trans-dienes (H7: δ 6.57 ppm, dd, J = 15.1 Hz/11.0 Hz; H8: δ 6.13, dd, J = 11.0; H6: δ 5.70, dd, J = 14.9 Hz/6.3 Hz, H9: δ 5.55, m; and H13: δ 6.51, dd, J = 14.9 Hz/11.4 Hz; H14: 5.96, dd, J = 11.0 Hz; H12: δ 5.67, m; H15: δ 5.46, m). Two protons attached to carbons bearing a hydroxyl group were located at 4.25 ppm (H11: δ 4.25, dt, J = 6.3 Hz/6.1 Hz) and 4.17 ppm (H5: δ 4.17, dt, J = 6.2 Hz/6.0Hz). H4 was detected as a cross-peak from H5 in the H,H-COSY spectrum at 1.57 ppm, H3 was a multiplet (1.70 ppm) and was coupled to the triplet signal of H2 at 2.34 ppm (J = 7.4 Hz). Both protons of H10 were detected as a multiplet at 2.47 ppm, and H16 was a dt signal at 2.17 ppm (J = 7.6 Hz/7.2 Hz).The configuration of C-15 in the 5,15-diHETE products (I and III) and of C-11 in the 5,11-diHETE (II) was established by coelution with corresponding diHETE diastereomers of known configuration. The configuration of the 5-hydroxy group in all diHETE products was expected to be unchanged from the starting substrate, 5S-HETE.Synthesis of standards of diastereomeric diHETEsTable 1 gives an overview of the diHETE standards prepared as reference compounds. Authentic 5S,15S-diHETE was prepared by reaction of soybean LOX-1 with 5S-HETE. Synthesis of 5S,15R-diHETE by reaction of 15R-HETE with the recombinant human 5-LOX gave only a minor yield of product, albeit it was sufficient to determine the retention times on RP- and SP-HPLC. In addition, the enantiomer 5R,15S-diHETE was prepared by reaction of 5R-HETE with the lipoxygenase from soybean seeds. 5S,15R-diHETE and 5R,15S-diHETE have indistinguishable retention times on RP- and SP-HPLC.TABLE 1Overview of the standards of 5,15-diHETEs and 5,11-diHETEs; their method of synthesis and HPLC retention timesRetention Time (min)diHETEMethod of PreparationRP-HPLCgWaters Symmetry C18 column (250 x 4.6 mm) eluted with methanol/water/acetic acid 80/20/0.01 at 1 ml/min flow rate.SP-HPLChAgilent Zorbax RX-SIL column (250 x 4.6 mm) eluted with hexane/isopropanol/acetic acid 95/5/0.1 at 1 ml/min flow rate. Retention times for the 5,11-diHETEs are for the methyl ester derivatives.5S,15S- (I)aThe roman numerals in parentheses refer to the numbering of the peaks in Fig. 1A, B.5S-HETE + soybean LOX6.812.35S,15R-bdiHETEs with the same superscript letter are enantiomers. (I, III)15R-HETE + hum. 5-LOXeThis reaction gave a very low yield.6.812.85R,15S-bdiHETEs with the same superscript letter are enantiomers.5R-HETE + soybean LOX6.812.85S,11R-cdiHETEs with the same superscript letter are enantiomers. (II)5S-HETE + Anabaena LOX / 11R,S-HETE autoxidation7.318.35R,11S-cdiHETEs with the same superscript letter are enantiomers.11R,S-HETE autoxidation7.318.35S,11S-ddiHETEs with the same superscript letter are enantiomers.11R,S-HETE autoxidation7.317.85R,11R-ddiHETEs with the same superscript letter are enantiomers.11R,S-HETE autoxidationfAlthough 5R-HETE was readily converted by the Anabaena LOX formation of 5R,11R-diHETE was not observed.7.317.8a The roman numerals in parentheses refer to the numbering of the peaks in Fig. 1A, B.b, c, d diHETEs with the same superscript letter are enantiomers.e This reaction gave a very low yield.f Although 5R-HETE was readily converted by the Anabaena LOX formation of 5R,11R-diHETE was not observed.g Waters Symmetry C18 column (250 x 4.6 mm) eluted with methanol/water/acetic acid 80/20/0.01 at 1 ml/min flow rate.h Agilent Zorbax RX-SIL column (250 x 4.6 mm) eluted with hexane/isopropanol/acetic acid 95/5/0.1 at 1 ml/min flow rate. Retention times for the 5,11-diHETEs are for the methyl ester derivatives. Open table in a new tab An authentic standard of 5S,11R-diHETE was prepared by reaction of 5S-HETE with the recombinant 9R-LOX from Anabaena sp PCC7120. A mixture of the 5,11-diHETE diastereomers was prepared by thin-film autoxidation of racemic 11-HETE. Initial attempts to prepare 5S,11S- and 5S,11R-diHETEs by reaction of 11S-HETE and 11R-HETE, respectively, with the recombinant human 5-LOX did not yield a significant amount of either 5,11-diHETE diastereomer. The assignment of the absolute configuration of the hydroxy groups in the diHETE standards was confirmed using CD spectroscopy (see below).Absolute configuration of 5,15-diHETEs from native and acetylated COX-2The diastereomers of 5,15-diHETE do not resolve on RP-HPLC (18Maas R.L. Turk J. Oates J.A. Brash A.R. Formation of a novel dihydroxy acid from arachidonic acid by lipoxygenase-catalyzed double oxygenation in rat mononuclear cells and human leukocytes.J. Biol. Chem. 1982; 257: 7056-7067Abstract Full Text PDF PubMed Google Scholar), but there is adequate separation on SP-HPLC to allow for secure assignment of the absolute configuration at C-15. Therefore, the 5,15-diHETEs were first isolated as a single peak using RP-HPLC and then resolved using SP-HPLC (Fig. 2). The 5,15-diHETE (peak I) isolated form the reaction of human COX-2 gave a %-ratio for 5S,15S-diHETE to 5S,15R-diHETE of 77:33, 80:20, and 75:25 in three separate experiments (Fig. 2A). The authentic standards of 5S,15S-diHETE and 5S,15R-diHETE eluted at 12.3 min and 12.8 min retention times, respectively (Fig. 2B, 2C). Peak identification was further confirmed by cochromatography with the authentic standards (Fig. 2D). Aspirin-treatment of human COX-2 resulted in a shift of the chiral distribution of 5,15-diHETE, and now the product (peak III) was 95% 5S,15R-diHETE (Fig. 3).Fig. 2Configurational analysis of 5,15-diHETE formed by the reaction of human COX-2 with 5S-HETE. Resolution of the 5,15-diHETE diastereomers was achieved using SP-HPLC as described in Experimental Procedures. A: Analysis of the 5,15-diHETE by-product from human COX-2 isolated by RP-HPLC. B, C: Elution of authentic standards of 5S,15S-diHETE and 5S,15R-diHETE, respectively. D: Cochromatography of the sample in A with the 5S,15R-diHETE standard (sample from C). All chromatograms were recorded at UV 235 nm using a diode array detector.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Configurational analysis of 5,15-diHETE formed by the reaction of acetylated human COX-2 with 5S-HETE. A: SP-HPLC analysis of 5,15-diHETE formed by acetylated human COX-2 isolated by RP-HPLC. B: Cochromatography of the sample in A with an authentic standard of 5S,15S-diHETE. The same chromatographic conditions as in Fig. 2 were used. All chromatograms were recorded at UV 235 nm using a diode array detector.View L" @default.
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• W2068505823 title "Identification and absolute configuration of dihydroxy-arachidonic acids formed by oxygenation of 5S-HETE by native and aspirin-acetylated COX-2" @default.
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