The results are reported of mass-spectrometric studies of the nucleobases adenine 1h (1, R Ï H), guanine 2h, and cytosine 3h. The protonated nucleobases are generated by electrospray ionization of adenosine 1r (1, R Ï ribose), guanosine 2r, and deoxycytidine 3d (3, R Ï deoxyribose) and their fragmentations were studied with tandem mass spectrometry. In contrast to previous EI-MS studies of the nucleobases, NH 3 elimination does present a major path for the fragmentations of the ions [1h Ï© H] Ï© , [2h Ï© H] Ï© , and [3h Ï© H] Ï© . The ion [2h Ï© H ÏȘ NH 3 ] Ï© also was generated from the acyclic precursor 5-cyanoamino-4-oxomethylenedihydroimidazole 13h and from the thioether derivative 14h of 2h (NH 2 replaced by MeS). The analyses of the modes of initial fragmentation is supported by density functional theoretical studies. Conjugate acids 15-55 were studied to determine site preferences for the protonations of 1h, 2h, 3h, 13h, and 14h. The proton affinity of the amino group hardly ever is the substrate's best protonation site, and possible mechanisms for NH 3 elimination are discussed in which the amino group serves as the dissociative protonation site. The results provide semi-direct experimental evidence for the existence of the pyrimidine ring-opened cations that we had proposed on the basis of theoretical studies as intermediates in nitrosative nucleobase deamination. [1,2]. This chemistry has been studied extensively because of the dietary and environmental exposure of humans to these substances [3][4][5]. Toxicological studies of deamination became more significant when it was recognized that endogenous nitric oxide [6,7] causes nitrosation [8,9], and that this process is accelerated by chronic inflammatory diseases [10,11]. It has been known for a long time that deamination of adenine 1, guanine 2, and cytosine 3 (Scheme 1) results in the formation of hypoxanthine, xanthine, and uracil, respectively, and these products are thought to result from DNA base diazonium ions 4-6, respectively, by direct nucleophilic dediazoniation. The discovery of oxanine formation [12][13][14] in the nitrosative deamination of guanine challenged the generality and completeness of this mechanism. Theoretical studies revealed that unimolecular dediazoniation of guaninediazonium ion 5 is accompanied or immediately followed by pyrimidine ringopening [15,16] and that cytosine-catalysis promotes the process [17,18]. The resulting 5-cyanoimino-4-oxomethylene-4,5-dihydroimidazole is a highly reactive intermediate and undergoes acid-catalyzed 1,4-addition via cyano-N or imino-N protonated 5-cyanoimino-4-oxomethylene-4,5-dihydroimidazoles, 9 and 10, respectively [19].Labeling studies support this reaction mechanism for oxanine formation [20]. Moreover, we synthesized 5-cyanoamino-4-imidazolecarboxamide and studied its cyclization chemistry [21] and its proficiency for cross-link formation [22]. The unimolecular dediazoniation of the diazonium ions of adenine and cytosine can proceed without ring-opening but the cations 7 and 11 formed in this...