The kinetic parameters of the first and second oxygenation of arachidonic acid by soybean lipoxygenase-1 were determined and found to be for the first step at pH 10.0, Km (arachidonic acid) = 8.5 +/- 0.5 microM; kcat = 225 +/- 7 s-1 and for the second step at pH 8.7 Km (15-HPETE) = 440 +/- microM; kcat = 25 +/- 1 s-1. In the second oxygenation for which 15-Ls-hydroperoxy 5-cis, 8-cis, 11-cis 13-trans-eicosatetraenoic acid is a substrate, two isomeric dihydroperoxy fatty acids are formed. After separation of the corresponding dihydroxy esters by high-performance liquid chromatography, they were identified by mass-spectrometry, 1H- and 13C-NMR spectroscopy as 8-DS, 15-LS-dihydroperoxy 5-cis, 9-trans, 11-cis, 13-trans-eicosatetraenoic acid and 5-DS, 15-LS-dihydroperoxy 6-trans, 8-cis, 11-cis, 13-trans-eicosatetraenoic acid. Independent evidence for the absolute configurations was obtained by capillary gas-liquid chromatography of diastereomeric R-(-)-2-butyl esters of the acetylated 2-hydroxy carboxylic acids produced by oxidative ozonolysis of the acetylated dihydroxy fatty acids. It is concluded that soybean lipoxygenase-1 produces hydroperoxides with predominantly the S-configuration irrespective of the position in the fatty acid which is oxygenated.

No abstract

A method is presented for determination of the enantiomeric composition of hydroxyperoxides formed by enzymic oxygenation of unsaturated fatty acids. After reduction of the hydroperoxy group with NaBH4, and esterification, the positional isomers of the resulting hydroxy compounds are separated by high performance liquid chromatography. The latter are subsequently subjected to a chiral derivatization to form diastereomeric alpha-methoxy-alpha-trifluoromethylphenylacetate esters. Determination of the diastereomeric composition by a NMR shift experiment furnishes the enantiomeric composition of the parent hydroperoxides. The method has been applied to the hydroperoxides formed by incubation of linoleic acid by corn germ or soybean lipoxygenase. Our results indicate that under the conditions used the hydroperoxides are mainly enantiospecifically formed.

The absolute configurations of a number of unsaturated hydroperoxy fatty acids obtained by lipoxygenase catalysis were investigated by capillary gas-liquid chromatography after proper derivatization. To this end the hydroperoxy groups were reduced and the resulting hydroxyl groups acetylated. Oxidative ozonolysis of the acetylated methyl esters yielded acetylated 2-hydroxycarboxylic acids, which were converted into R-(--)-2-butyl esters and then reacetylated. The ratio of the resulting diastereomers, which reflects the optical purity of the chiral centers in the parent hydroperoxy fatty acids, was determined by capillary gas-liquid chromatography. Application of this simple method to a number of mono- and dihydroperoxy fatty acids obtained by incubation with soybean lipoxygenase-1 or -2, or by corn-germ lipoxygenase yields enantiometric compositions which are in good agreement with results obtained by other methods.

The insertion of phenyl isocyanide into ethanol under the influence of base and Cu(PhNC),BF, is investigated mechanistically. In pre-equilibria the BFF anion is exchanged for eOC,H,. By intramolecular nucleophilic attack of eOC,H, on coordinated isocyanide an [(ethoxy)-(phenylimino)methyl]copper intermediate is formed. The substituent effect supports this conclusion. In a rapid reaction with ethanol the intermediate decomposes and the insertion product, ethyl Nphenylformimidate, is formed. It reacts slowly to give N,N-diphenylformamidine, which is precipitated at the end of the reaction as a copper complex.