Ammonia elimination from protonated nucleobases and related synthetic substrates

Abstract

The results are reported of mass-spectrometric studies of the nucleobases adenine 1h (1, R ϭ H), guanine 2h, and cytosine 3h. The protonated nucleobases are generated by electrospray ionization of adenosine 1r (1, R ϭ ribose), guanosine 2r, and deoxycytidine 3d (3, R ϭ 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 ϩ H] ϩ , [2h ϩ H] ϩ , and [3h ϩ H] ϩ . The ion [2h ϩ H Ϫ NH 3 ] ϩ 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...

Study of forced decomposition behavior of lamivudine using LC, LC–MS/TOF and MSn

“…Relative behavior in the literature [16]. The ion of m/z 94 resulted from neutral loss of water from cytosine, which is also reported previously [17].…”

“…It is proposed that the drug was ionized to protonated cytosine (m/z 112) on replacement of oxathiol moiety by hydrogen [16]. The subsequent generation of fragments of m/z 94, 95 and 69 from protonated cytosine during MS 3 study resulted from loss of H 2 O, NH 3 and HNCO, respectively.…”

Identification of 8-methyladenosine as the modification catalyzed by the radical SAM methyltransferase Cfr that confers antibiotic resistance in bacteria

“…For both compounds, initial fragmentation of the intact nucleoside yields a single fragment ion at m/z 150 corresponding to the characteristic neutral loss of ribose (132 Da). The dominant fragmentation of nucleoside molecular ions in electrospray ionization is the CID of the N-C glycosidic bond that leads to the replacement of the sugar moiety with a hydrogen atom (Wilson and McCloskey 1975;Qian et al 2007). Following isolation and fragmentation of the nucleobase at m/z 150, a second neutral loss of 17 Da is observed corresponding to the loss of NH 3 and a minor peak at m/z 108 corresponding to the loss of cyanamide (NH 2 CN).…”

“…Following isolation and fragmentation of the nucleobase at m/z 150, a second neutral loss of 17 Da is observed corresponding to the loss of NH 3 and a minor peak at m/z 108 corresponding to the loss of cyanamide (NH 2 CN). Neutral loss of ammonia is also a characteristic of CID of nucleobases (Nelson and McCloskey 1992;Jensen et al 2007;Qian et al 2007); hence, MS 3 was insufficient to distinguish the two isobaric adenosine derivatives.…”

Identification of urinary modified nucleosides and ribosylated metabolites in humans via combined ESI-FTICR MS and ESI-IT MS analysis

“…Recently, the coupling of HPLC with mass spectrometric detection via electrospray ionization ion trap mass spectrometry (ESI-IT MS) [15,19], ESI tandem MS [20,21], and fast atom bombardment (FAB) [22,23] has been established. Sophisticated mass spectrometric setups like ESI-oa-TOF MS [24] and MALDI-TOF MS [25] have already proven to be valuable tools for the structural elucidation of RNA metabolites.…”