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Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9057
Published, JLR Papers in Press, September 8, 2005. DOI 10.1194/jlr.C500015-JLR200
1 To whom correspondence should be addressed. e-mail: igor.butovich{at}utsouthwestern.edu
ABSTRACT
Potato tuber lipoxygenase was shown to convert 17(S)-hydro(pero)xydocasahexaenoic acid in 10,17(S)-dihydro(pero)xydocosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid [10,17(S)-diHDHA] which was formed apparently through a double lipoxygenation mechanism. No traces of 10,17(S)-dihydro(pero)xydocosahexa-4Z,7Z,11E,13E,15Z,19Z-enoic acid were found among the reaction products.
It is very likely that a described earlier "neuroprotectin D1" [or "10,17(S)docosatriene"], a novel and potent anti-inflammatory compound derived from docosahexaenoic acid, was, in fact, 10,17(S)-dihydroxydocosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid formed through a double lipoxygenation mechanism instead of a previously thought epoxidation/isomerization mechanism.
Abbreviations: COSY, correlation spectroscopy; DHA, docosahexaenoic acid (
-3 C22:6); LOX, lipoxygenase; ptLOX, potato tuber lipoxygenase; sLOX, soybean lipoxygenase; 10,17(S)-diHDHA, 10,17(S)-dihydroxydocosahexaenoic acid; 17(S)-HDHA, 17(S)-hydroxydocosahexaenoic acid; 17(S)-HPDHA, 17(S)-hydroperoxydocosahexaenoic acid
Supplementary key words 10,17(S)-docosatriene • 10,17-dihydroxydocosahexaenoic acid • lipoxygenase • double lipoxygenation • nuclear magnetic resonance • cis,trans-geometry
Recently, several papers were published that described a novel class of potent anti-inflammatory lipid compounds formed from docosahexaenoic acid (-3 C22:6; DHA) by various cells and isolated enzymes (1–6). One of the derivatives, 10,17(S)-dihydroxydocosahexaenoic acid (10, 17-diHDHA), was postulated to be formed by lipoxygenase (LOX) with unidentified specificity (2–4) and/or in a LOX-like reaction through an epoxidation/isomerization of 17(S)-hydroperoxydocosahexaenoic acid (5, 6). The compound was termed 10,17(S)-docosatriene (and neuroprotectin D1) and was proposed to be 10,17(S)-dihydroxydocosahexa-4Z,7Z,11E,13E,15Z,19Z-enoic acid. Along with the various compounds formed in vivo, the authors of those papers mentioned that the same derivatives of DHA were produced in a "one-pot" reaction catalyzed by soybean and potato tuber LOXs (sLOX and ptLOX, respectively) (5, 7).
Considering that this potent bioregulatory compound is formed in vivo in extremely low quantities (1–7) and that it is not available commercially, it was important to develop and describe an effective method of 10,17(S)-diHDHA synthesis.
Recently, my colleagues and I published the results of our study of ptLOX-catalyzed transformations of DHA, 17(S)-hydroperoxydocosahexaenoic acid [17(S)-HPDHA], and 17(S)-hydroxydocosahexaenoic acid [17(S)-HDHA] (8). The ptLOX-catalyzed oxidation of DHA unexpectedly led to two novel oxylipins, 10(S)-HDHA and 10(S), 20-diHDHA, that were not described before, whereas in the reactions with 17(S)-HPDHA or 17(S)-HDHA, another pair of products, 7,17(S)-diH(P)DHA and 10,17(S)-diH(P)DHA, were formed, apparently through a double lipoxygenation mechanism. The epoxidation/isomerization mechanism proposed in the earlier papers (1–7) was ruled out, as ptLOX did not need the hydroperoxy group at C17 of the substrate to form 10,17(S)-diH(P)DHA, which would have been a prerequisite for epoxidation to occur. Instead, the enzyme swiftly transformed 17(S)-HDHA to 10,17(S)-diH(P)DHA. In keeping with the double lipoxygenation mechanism, the expected geometry of the conjugated triene fragment in 10,17(S)-diH (P)DHA was proposed to be 11E,13Z,15E (8), which obviously differed from the one postulated by Serhan and colleagues (1–5, 7) and Bazan and colleagues (6). At that time, we had no direct information on the geometrical features of 10,17(S)-diH(P)DHA formed by ptLOX. No new information on the structure of neuroprotectin D1 or 10,17(S)-docosatriene has been published since then, either.
To answer the question of whether the geometry of 10,17(S)-diHDHA was of 11E,13Z,15E-type or 11E,13E, 15Z-type, the product was synthesized exactly as described previously (8). Briefly, an sLOX-generated 17(S)-HDHA was treated with ptLOX, and the resulting product was reduced with sodium borohydride to form 10,17(S)-diHDHA. The compound was purified by normal-phase HPLC on a 4.6 x 300 mm Waters µBondapak silica gel column in a hexane-i-propanol-acetic acid mobile phase (95:5:0.1, v/v/v; 1 ml/min flow rate) at 30°C on a Waters Alliance 2695 HPLC system with a Waters 2996 diode-array detector operating in scan mode (200–350 nm) (Fig. 1A). The purity of the compound was rechecked by analytical normal-phase HPLC on a silica gel column under the same conditions. The product showed an ultraviolet spectrum of a conjugated triene, with characteristic maxima at 260.5, 269.8, and 280.5 nm (Fig. 1B). The molecular weight of the product was determined by electrospray ionization mass spectrometry on an LCQ Deca XP Max MSn ion trap mass spectrometer (Thermo Electron Corp.). The product was dissolved in methanol and directly infused with a syringe pump at a flow rate of 2 µl/min. A prominent parent peak with m/z ratio of 359.15 (M–H+, consistent with the molecular formula C22H32O4; Fig. 1C) and a chloride adduct (m/z 395.5, M+Cl–) were observed in the negative ion zoom scan mode, whereas in the positive ion mode, the most prominent peak had an m/z value of 383.56 (M+Na+). Mass spectrometric analysis of the parent ion with m/z 359.15 produced a daughter ion with m/z 341 (M–H+–H2O), whereas fragmentation of the latter gave an ion with m/z 323 (M–H+–2H2O).
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10, which is indicative of cis-geometry of the vinyl group, whereas J11,12 and J15,16 were 15, clear evidence of the trans-nature of the C11-C12 and C15-C16 bonds (Scheme 1)
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of 6.15–6.24 ppm that is seen, for example, in all-trans isomers of 9(S)- and 13(S)-hydroxylinoleyl alcohols (9), 9(S)- and 13(S)-hydroxymonolinoleoyl glycerols (10), 5,6-dihydroxyeicosapenta-7E,9E,11Z,14Z,17Z-enoic acid (11), 5-oxo-eicosatetra-7E,9E,11Z,14Z-enoic acid (12), 14,15-dihydroxyeicosatetra-5Z,8Z,10E,12E-enoic acid (13), and 5,12(S)-dihydroxyeicosatetra-6E,8E,10E-enoic acid (14). Generally, a proton system similar to the 11E,13E,15Z conjugated triene would have produced at least six distinctive groups of signals of olefinic protons (13), whereas ptLOX-synthesized 10,17(S)-diHDHA gave only four (Figs. 2, 3), which is consistent with the highly symmetrical structure of 10, 17(S)-dihydroxydocosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid (Scheme 1).
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4.165) of our compound produced cross peaks with trans-olefinic protons ( 5.73, C11 and C16) but not with cis-olefinic protons ( 5.97, C13 and C14). This observation suggests an 11E,13Z,15E arrangement of the product's triene fragment as well, as both the resonances would have been seen in its 11E,13E,15Z or 11Z, 13E,15E derivatives. The last observed resonance ( 6.72) belongs to protons at C12 and C15. Interestingly, the same compound was obtained through sLOX-only catalyzed oxidation of 1) DHA, 2) 17(S)-HPDHA, and 3) 17(S)-HDHA (unpublished data). No traces of the 11E,13E,15Z isomer were found among the products of any of these reactions, either.
Therefore, under the tested conditions potato LOX produced only 11E, 13Z, 15E isomer of 10,17(S)-dihydroxydocosahexaenoic acid that was apparently formed through the double lipoxygenation mechanism. These findings need to be considered when making this and similar compounds involves plant LOXs (5, 7, 15) as the compounds made in vitro may differ from the compounds biosynthesized in living cells.
ACKNOWLEDGMENTS
This work was supported by National Institutes of Health Grant 5P30AR041941 (to I.B.). The author expresses his sincere gratitude to Carl Bachmann (Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas) for his excellent help in conducting the NMR experiments.
Manuscript received June 30, 2005 and in revised form July 28, 2005 and in re-revised form August 19, 2005.
-3-derived local mediators in anti-inflammation and resolution. Pharmacol. Ther. 105: 7–21.[CrossRef][Medline]
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