The Synthesis of Orotic Acid-C14O2H

Orotic acid-C1402H, which was required for biological studies being pursued in this laboratory, has been synthesized by the method of Nyc and Mitchell,5 starting with DL-aspartic acid-a-C1402H. The latter was prepared from ethyl malonate-C1402C2H5, as described by Galat.' This orotic acid probably contains the isotopic carbon exclusively in the carboxyl group since it evolves carbon dioxide containing most of the original radioactivity when heated to 3450 and the uracil which remains is not radioactive; this finding lends strong support to the mechanism proposed by Nyc and Mitchell (loc. cit.) for the alkaline rearrangement of 5-(carboxymethylidene)-hydantoin to orotic acid. METHOD ETHYL MALONATE-C1402C2H5 Barium carbonate-C1' (150 mg., 5 mc., Oak Ridge) was converted by the method of McCarter' into sodium cyanide-C", this was then mixed with a solution of sodium cyanide-C" (3.00 g.) in water (8 ml.). The resulting solution was used for the preparation of malonic acid, as described by Weiner.7 The reaction was conducted in a centrifuge tube (50 ml.) and the crude calcium malonate obtained was separated by centrifugation and washed twice by stirring with ice-water (10 ml.), centrifuging after each addition. The process was repeated three times with acetone, and the product, after being dried in air, was converted into malonic acid in the usual way (Weiner, loc. cit.). This crude malonic acid (4.26 g.) was treated with absolute ethanol (50 ml.) and concentrated sulfuric acid (0.5 ml.). The mixture was heated under reflux on the water bath for four hours and then evaporated in vacuo at room temperature to half its bulk. Water (30 ml.) was then added and the mixture extracted with ether (4 lots of 5 ml. each). The extract was shaken first with water (5 ml.), then with sodium bicarbonate solution (5%; 5 ml.), and then with water (5 ml.), and dried over anhydrous calcium chloride. After removing the ether by fractional distillation, using a short Vigreux column, the residue was distilled from glass wool under reduced pressure into a trap kept at-30°. A short fore-run (b.p. up to 50°/11 mm.) was discarded and the remaining distillate, nearly all of which boiled at 85-7°/11 mm., was collected as one fraction and used directly in the next stage (yield, 4.74 g.).