Abstract
The biosynthesis of orotic acid is briefly reviewed. This investigation is concerned with elucidating the stereo-chemical requirements of dihydro-orotic dehydrogenases the enzyme involved in the final stage of the biosynthesiss, with respect to the configuration of 4,5-dihydro-orotic acid(I). The unambiguous synthesis of cis- and trans-4,5-dihydro-5-methylorotic acids (II and III), by cyclisation of the hitherto unknown N-ethoxycarbonyl-threo- and erythro-methylasparagines, is described. Threo- and erythro-methylaspartic acids were prepared, and conversion of them into N-ethoxycarbonyl-threo- and erythro-methylasparagines by various routes was studied. The reduction, both chemical and catalytic, of orotic acid and of 5-methylorotic acid-was also studied, cis-4,5-dihydro-5-methylorotic acid being obtained from the latter. The physical properties of the threo and erythro series of derivatives of beta-methylaspartic acid have been compares, also those of the cis- and trans-4,5-dihydro-5-methylorotic acids, with a view to correlating the properties with configuration. Possible methods for the synthesis of 5-fluoro-4,5-dihydro-orotic acid are reviewed. Preliminary experiments on the synthesis of 5-bromo-4,5-dlhydro-orotic acid were carried out. Enzymic studies were carried out with L-, and DL-4,5- dihydro-orotic acid and with cis- and trans-4,5-dihydro-5-methyl-orotic acids as substrates. Ehrlich ascites cells were used as a source of the enzyme dihydro-orotic dehydrogenase in the first instance. The oxidation of the "dihydro-orotic acids" to the "orotic acids" was measured spectrophotometrically. The cis-isomer of 5-methyldihydro-orotic acid was apparently metabolised to some extent, but the enzyme preparation was not sufficiently pure to furnish unambiguous assay results. Subsequent experiments were performed with an enzyme preparation obtained from Zymobacterium oroticum. This enzyme preparation proved to be more satisfactory and was active towards trans-4,5-dihydro-5-methylorotic acid but not towards the cis-isomer. The results for the oxidation of the substrates, and for the reduction of orotic acid with or without 5-methylorotic acid present do not entirely accord with published observations. It is, however, concluded that the enzyme-catalysed dehydrogenation proceeds by a trans-elimination mechanism.