Chemical and biological studies on 5,6-epoxyretinol, retinol, and their phosphoryl esters

Studies are reported on chemical synthesis, ultraviolet absorption spectral characteristics, and mass spectral fragmentation of 5,6-epoxyretinol and 5,6-epoxyretinylphosphate. These compounds were separated from each other and from other retinoids by a reverse phase high pressure liquid chromatographic system. A comparative study on the lability to acid of 5,6-epoxyretinylphosphate and retinylphosphate was conducted. The retroretinoid anhydroretinol is formed chemically from retinylphosphate by acid hydrolysis and biologically from retinal in cultured, spontaneously-transformed mouse fibroblasts, 3T12 cells. Similarly, acid hydrolysis of 5,6-epoxyretinylphosphate (absorption maxima 324, 310, 296 nm) in methanol yielded a low polarity retinoid with absorption maxima at 364, 346, and 330 nm, similar to the absorption spectra of retrovitamin A1 and retrovitamin A2. Mass spectral analysis was found to be in agreement with a retrostructure and permitted identification of the compound as a methoxyretrovitamin A1 methyl ether. A similar retroretinoid was formed biologically from 5,6-epoxyretinol in spontaneously-transformed mouse 3T12 cells. Thus, it appeared that these cells have the ability to convert the primary alcohols into retroretinoids, which are also formed by acid treatment of the phosphate esters. The adhesive properties of 3T12 cells were highly enhanced by culturing in the presence of 10(-6) to 10(-5) M 5,6-epoxyretinol or -retinoic acid, in analogy with the response of these cells to the parent retinoids. Moreover, in another test of biological activity, 5,6-epoxyretinylphosphate functioned as a highly active acceptor of [14C]D-mannose from GDP-[14C]mannose in a reaction catalyzed by rat liver membranes. Thus, 5,6-epoxyretinoids appear to be as active as the parent retinoids in these in vitro tests of biological activity, even though they do not replace vitamin A in its growth function in vivo.