Formation of a beta-pyrimidine nucleoside by a free pyrimidine base and ribose in a plausible prebiotic reaction.

Glycosylation of pyrimidine bases to give nucleosides under plausible prebiotic conditions has not been achieved, and this represents a serious challenge to the “RNA world” and to many of its proposed precursors.1 Three decades ago, Orgel and co-workers demonstrated that adenine and hypoxanthine form glycosidic bonds with D-ribose when dried and heated together, although adenine gives mainly the product of attack at the NH2 group.2 As much as 20% of the nucleoside was reported from hypoxanthine (BRSM).2 Unfortunately, neither cytosine nor uracil gives rise to nucleosides under these conditions, nor does guanine, a fact attributed to its low solubility.1 Some researchers have taken to exploring completely different pathways to prebiotic pyrimidine nucleoside formation, including the construction of the cytosine base on a sugar phosphate.3 However, suggestions that the original bases may have differed from the canonical AUCG leave open the possibility that some of these bases might form glycosides under prebiotic conditions.4 Indeed, urazole, a five-membered ring derived from hydrazine, readily forms glycosides.3c In search of alternative pyrimidine bases, we have explored the condensation of ribose with 2-pyrimidinone (Figure 1) by both experiment and calculation. Drying and heating of 2-pyrimidinone with ribose using the reaction conditions described by Orgel2 produces zebularine in 12% yield. Zebularine formation was confirmed by mass spectrometry (m/z ) 227 Da), HPLC mobility (Figure 2, peak 2), UV spectrophotometry, and NMR analysis of HPLC-purified reaction products (Supporting Information (SI)). To the best of our knowledge, this work represents the first successful synthesis of a pyrimidine nucleoside from a free base and an unactivated sugar in a plausible prebiotic reaction. Additional products were also observed, including two assigned as the â-pyranosyl (coelutes with zebularine) and R-furanosyl (peak 1) nucleosides (Figure 2, SI). The R-pyranosyl anomer may be present in low yield, but was not detected. Thus, the total yield of pyrimidine nucleosides formed in the reaction is even greater than that reported here for zebularine. As seen in Figure 2, the relative ratios of the products differ dependent upon the reaction conditions, yet typically favor â-anomers, as previously observed for ribosides of urazole.3c Nucleoside formation by purines and ribose under drying conditions is an acid-catalyzed reaction that can also be promoted by divalent metal ion salts at neutral pH.2 In the absence of Mg2+, 2-pyrimidinone yields zebularine when dried and heated with ribose from a slightly acidic solution of pH 2.1 (Figure 2). The actual pH at reaction is not known, but is likely to be less than 1 because of the drying conditions. Buffering the sample pH to 6.3 reduces zebularine production to an unobservable level, although other reaction products are still observed (Figure 2). Thus, our results indicate that the zebularine formation is similar to the purine glycosylation reaction in that it is both acid-catalyzed and is promoted by Mg2+ near neutral pH. It is of interest to compare the nucleoside yields from 2-pyrimidinone, adenine, and hypoxanthine. The relative yields of â-furanosyl nucleosides produced in three different reaction conditions were determined by HPLC analysis. The results presented in Table 1 demonstrate that 2-pyrimidinone, surprisingly, forms â-nucleosides in greater yields than both adenine and hypoxanthine. Uracil did not form nucleosides in detectable yields under any of the conditions tested. In addition, we conducted nucleoside degradation studies using the same reaction conditions that were used for the nucleoside syntheses. Transition state theory predicts † Georgia Institute of Technology. ‡ Jackson State University. § University of California. Figure 1. Chemical structures of pyrimidine bases and zebularine, the â-furanosyl ribonucleoside of 2-pyrimidinone.