A straightforward method for the simultaneous preparation of radiolabeled l-dihydroörotic and N-carbamyl-l-aspartic acids

A straightforward method is presented for the simultaneous enzymatic preparation of radiolabeled l-dihydroörotic and N-carbamyl-l-aspartic acids. [carboxyl-¹⁴C]Orotic acid is incubated in the presence of excess NADH to drive the reaction catalyzed by l-dihydroörotate dehydrogenase in the reverse direction, forming l-[¹⁴C]dihydroörotic acid. The contamination of the commercial reagent enzyme, l-dihydroörotate dehydrogenase from Zymobacterium oroticum, with l-dihydroörotase yields N-carbamyl-l-[¹⁴C]aspartate as a second product of the reaction. The products are purified by ion-exchange chromatography using ammonium bicarbonate buffer and desalted by repeated lyophilization. Over 95% of the starting radioactivity is recovered in the two products which, in a typical preparation, were present in a ratio of 4:6, l-[¹⁴C]dihydroörotic acid: N-carbamyl-l-[¹⁴C]aspartic acid. The identities of the reaction products are established by chromatographic, electrophoretic, and enzymic means.