Free Radical Oxidation of (E)-Retinoic Acid by the Fenton Reagent:  Competing Epoxidation and Oxidative Breakdown Pathways and Novel Products of 5,6-Epoxyretinoic Acid Transformation

The nature of the products formed by reaction of all-trans retinoic acid (1) and its major metabolite, 5,6-epoxyretinoic acid (2), with the Fenton reagent was investigated. Oxidation of 1 in a vigorously stirred biphasic medium (0.1 M phosphate buffer, pH 7.4/ethyl acetate 5:1 v/v) with Fe²⁺/EDTA complex (2 mol equiv) and a 10-fold excess of H₂O₂ proceeded smoothly to give a very complex mixture of products. Repeated TLC fractionation of the reaction mixture after methylation allowed isolation of the main products which were identified as 2 methyl ester, (7E)-7,8-epoxyretinoic acid methyl ester (6), all-(E)-2,6-dimethyl-8-(2,6,6-trimethyl-2-cyclohexen-1-ylidene)-2,4,6-octatrienal (11), the novel (9E)-5,6,9,10-diepoxyretinoic acid methyl ester (7), and (9Z)-5,6,9,10-diepoxyretinoic acid methyl ester (8) (1:1 mixture of syn/anti isomers each), 5,6-epoxy-β-ionone (9), 5,6-epoxy-β-ionylideneacetaldehyde (10), and trace amounts of β-ionone (12), β-ionylideneacetaldehyde (13), and 4-oxoretinoic acid (3) methyl ester. When the oxidation was carried out with the substrate and the Fenton reagent at concentrations as low as 10 μM, the main detectable products were 2 methyl ester, 11, and 7/8. Under similar conditions, the epoxide 2 gave mainly products 7−10. A less efficient conversion of 1 and 2 but similar product patterns were observed with other oxidizing systems such as peroxidase/H₂O₂ and 13-hydroperoxyoctadecadienoic acid in the presence of Fe(II). Besides providing the first detailed insight into the products formed by reaction of a retinoid with the Fenton reagent, the results of this study disclose a novel nonenzymatic route from 1 to the epoxide 2 and offer an improved chemical basis to inquire into the mechanism of the antiinflammatory, antimutagenic, and cancer chemopreventive action of these retinoids.