Isolation and characterization of natural allene oxides: unstable intermediates in the metabolism of lipid hydroperoxides.

Abstract

Allene oxides are unstable epoxides that have been implicated as intermediates in the biotransformation of hydroperoxyicosatetraenoic acids and related hydroperoxides to ketols and cyclopentenones. Direct proof of the structure of the putative allene oxide intermediates has been hampered by their extreme instability under the conditions of their biosynthesis (t½2 15-30 sec at 0C and pH 7.4). We now report the isolation and structural elucidation of allene oxides prepared from the (13S)-hydroperoxides of linoleic and linolenic acids. The compounds were biosynthesized by using a very active enzyme preparation from flaxseed. After a 5-sec incubation at 0C, the allene oxide metabolites were extracted and purified as the methyl ester derivatives at -15TC. The structures were established by UV, CD, NMR, and oxygen-18 labeling experiments. 12,13(S)-Oxido-9Z,11-octadecadienoic acid is derived from linoleic acid, and 12,13(S)-oxido-9Z,11,15Z-octadecatrienoic acid is from linolenic acid. Analysis of the breakdown products formed on exposure to water led to identification of hydrolysis and cyclization products previously characterized as enzymic derivatives of the (13S)-hydroperoxides in flaxseed. Our results give direct proof of the structure of the allene oxide intermediates and should facilitate further investigation of the metabolism of this class of epoxide to prostaglandins, clavulones, and other stable end products. tTo whom reprint requests should be addressed. 3382The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Evidence for an Ionic Intermediate in the Transformation of Fatty Acid Hydroperoxide by a Catalase-related Allene Oxide Synthase from the Cyanobacterium Acaryochloris marina

“…The 1 H NMR spec- trum, recorded at Ϫ20°C in d-hexane (Fig. 3C), resembles that of the allene oxide formed from 13S-HPOTrE in the jasmonate biosynthetic pathway (30). There are signals from five protons in the olefinic region, notably including a doublet at H14 coupling only to H15, with no proton at C13.…”

“…The sample was then treated with ethanol (20 l) and ethereal diazomethane for 30 s at 0°C and then rapidly evaporated to dryness and stored in hexane at Ϫ80°C until further analysis. The sample was purified as described previously for an allene oxide derived from 13S-HPOTrE using a 4.5-ϫ 0.46-cm silica column operated at Ϫ15°C using a solvent system of hexane/diethyl ether (100:1, v/v) run at a flow rate of 3 ml/min (30).…”

Isolation and Characterization of Two Geometric Allene Oxide Isomers Synthesized from 9S-Hydroperoxylinoleic Acid by Cytochrome P450 CYP74C3

“…The allene oxide is then further stabilized by conversion to the methyl ester by a 60-s treatment of the hexane solution at 0°C with diazomethane. The allene oxide methyl ester can be chromatographed on HPLC in hexane-based solvents at Ϫ10°C or below (10).…”

“…The recombinant P450 enzymes were quantified using ⑀ ϭ 100,000 for the main Soret band (393 nm in CYP74A2 and 415 nm in CYP74C3). Preparation of Allene Oxides Using Flaxseed Extract (CYP74A1)-Flaxseed acetone powder (100 mg/ml) was stirred for 30 min at 0°C in 0.1 M potassium phosphate buffer, pH 6.5, containing 3 mM Zwittergent 3-14 (Calbiochem) and then centrifuged at top speed in a benchtop microcentrifuge for 5 min at room temperature, and the resulting clear solution was kept at 0°C until use (10). 9S-HPODE (4 mg) was taken to near dryness (ϳ10 l of ethanol remaining) in a 20-ml glass Schwartz vial with Teflon screw cap; 8 ml of ice-cold pentane was added and vigorously vortex-mixed, and then the vial was buried in ice.…”

“…The 18:33 allene oxide of the jasmonate pathway (in addition to being converted to a cyclopentenone by the enzyme AOC) will cyclize spontaneously in physiological buffers (typically ϳ15% cyclization, 85% hydrolysis to ␣-and ␥-ketols (12)). By contrast, the linoleic acid (C18:26) allene oxide undergoes only hydrolysis (10). By inspection, therefore, the presence of the extra 3 double bond facilitates cyclization, and study of additional fatty acid analogues supports this deduction (13).…”