Monoethyl ester of dipicolinic acid from bacterial spores.

The discovery and certain aspects of the biosynthesis of dipicolinic acid (pyridine-2,6-dicarboxylic acid) in exudates of germinated spores and in intact spores of aerobic bacilli have been recently reported (Powell, 1953; Perry and Foster, 1955). In the latter paper it was noted that maximum recovery of dipicolinic acid from intact spores necessitated an initial treatment with hot dilute inorganic acid, implying, as one posibility, that the dipicolinic acid exists in the spore in a bound inorganic or organic combination, or both. In view of the fact that calcium dipicolinate was isolated from exudates of germinated spores of Bacillus megaterium (Powell, 1953), and in view of the report that spores of aerobic bacilli are uniquely enriched in calcium (Curran et al., 1943), the possibility that the natural form of dipicolinic acid in intact spores is exclusively or mainly calcium dipicolinate had to be considered. Quantitative analyses were therefore made of the content of calcium and of dipicolinic acid in the same batches of spores of BaciUus cereus var. mycoides. Spores obtained from a growth medium containing yeast extract and spores obtained endotrophically contained, respectively, only 0.14 and 0.021 milliequivalents of calcium per milliequivalent of dipicolinic acid (Perry and Foster, 1955a). Hence, the bulk, if not all, of the dipicolinic acid of spores obtained under these conditions does not eist as the calcium salt, nor, in all probability, as the salt of any other inorganic cation. In the course of examining paper chromatograms of ether extracts of hydrolyzed spores according to the procedure previously described for identification and quantitative estimation of 1 This work was supported in part by the Microbiology Branch of the Office of Naval Research, by the Division of Biology and Medicine of the Atomic Energy Commission, and by a grant-in-aid from the American Cancer Society upon recommendation of the Committee on Growth of the National Research Council. dipicolinic acid (Perry and Foster, 1955), a second, relatively minor, ultraviolet absorbing spot was observed in certain extracts. It proved to be a combined form of dipicolinic acid. This paper describes the purification and isolation of that material in crystalline form. Its identification as the monoethyl ester of dipicolinic acid, and confirmation by chemical synthesis are also described.