J. Lipid Res.
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Originally published In Press as doi:10.1194/jlr.D500042-JLR200 on January 3, 2006

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Journal of Lipid Research, Vol. 47, 854-863, April 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

Methods

A one-step method of 10,17-dihydro(pero)xydocosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid synthesis by soybean lipoxygenase

Igor A. Butovich1

Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9057

Published, JLR Papers in Press, January 3, 2006.

1 To whom correspondence should be addressed. e-mail: igor.butovich{at}utsouthwestern.edu

A product of lipoxygenase (LOX) oxidation of docosahexaenoic acid (DHA), 10,17-dihydro(pero)xydocosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid [10,17(S)-diH(P)DHA] was obtained through various reaction pathways that involved DHA, 17(S)-hydro(pero)xydocosahexa-4Z,7Z,11Z,13Z,15E,19Z-enoic acid [17(S)-H(P)DHA], soybean lipoxygenase (sLOX), and potato tuber lipoxygenase (ptLOX) in various combinations. The structure of the product was confirmed by HPLC, ultraviolet (UV) light spectrometry, GC-MS, tandem MS, and NMR spectroscopy. It has been found that 10,17(S)-diH(P)DHA formed by sLOX through direct oxidation of either DHA or 17(S)-H(P)DHA was apparently identical to the product of ptLOX oxidation of the latter. The sLOX- and ptLOX-derived samples of 10,17-diHDHAs coeluted under the conditions of normal, reverse, and chiral phase HPLC analyses, displayed identical UV absorption spectra with maxima at 260, 270, and 280 nm, and had similar one-dimensional and two-dimensional proton NMR spectra. Analysis of their NMR spectra led to the conclusion that 10,17-diHDHA formed by sLOX had solely 11E,13Z,15E configuration of the conjugated triene fragment, which was identical to the previously published structure of its ptLOX-derived counterpart. Based on the cis,trans geometry of the reaction products, the conclusion is made that in the tested conditions sLOX catalyzed direct double dioxygenation of DHA. Compared with the previously described two-enzyme method that involved sLOX and ptLOX, the current simplified one-enzyme procedure uses only sLOX as the catalyst of both dioxygenation steps.

Supplementary key words docosahexaenoic acid • 10,17-dihydroxydocosahexaenoic acid • 10,17(S)-docosatriene • neuroprotectin D1 • nuclear magnetic resonance • mass spectrometry • protectin D1

Abbreviations: BSTFA, bis(trimethylsilyl)trifluoroacetamide; C12E10, monododecyl ether of decaoxyethylene glycol; 1D, one-dimensional; 2D, two-dimensional; DHA, docosahexaenoic acid ({omega}-3 C22:6); 10,17-diH(P)DHA, 10,17-dihydro(pero)xydocosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid; EI, electron-impact; ESI, electrospray ionization; 1H, 1H-DQCOSY, double quantum correlation spectroscopy; LOX, lipoxygenase; neuroprotectin D1 and 10,17(S)-docosatriene, 10,17(S)-dihydroxydocosahexaenoic acid with postulated 4Z,7Z,11E,13E,15Z,19Z geometry of the double bonds; NP HPLC, normal phase high performance liquid chromatography; ptLOX, potato tuber lipoxygenase; 17(S)-H(P)DHA, 17(S)-hydro(pero)xydocosahexa-4Z,7Z,11Z,13Z,15E,19Z-enoic acid; sLOX, soybean lipoxygenase; UV, ultraviolet


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