Abstract
Synthetic nucleotide and nucleic acid analogues are useful research tools and modern therapeutics. Hence, methods for the rapid and unambiguous identification of mononucleotides derived from organic syntheses or biological materials are of broad interest. Here, we analysed over 150 mononucleotides (mostly nucleoside 5′-mono-, 5′-di-, and 5′-triphosphates) and their structurally related nucleobase-, phosphate-, and ribose-modified analogues by electrospray tandem mass spectrometry (ESI/MS/MS), identifying characteristic fragmentation ions that may be helpful in structure determination. While positive-ion mode yielded fragments derived mainly from nucleobases, negative-ion mode provided insight into the structures of phosphoryl and phosphoribosyl moieties, enabling the determination of structural features such as the number of phosphate groups and the presence of ribose or phosphate substitutions. Based on these data, we proposed fragmentation pathways that were confirmed by experiments with [18O]-isotopologues. We demonstrated the utility of ESI(−)/MS/MS in the analysis of structurally related compounds by analysing isomeric and isobaric nucleotides and applying ESI(−)/MS/MS to rapid identification of nucleotide synthesis products. We formulated general rules regarding nucleotide structure–fragmentation pattern relationships and indicating characteristic fragmentation ions for the interpretation of ESI(−)/MS/MS spectra of nucleotides and their analogues. The ESI(−)/MS/MS spectra of all nucleotides are available in an on-line database, msTide, at www.msTide-db.com.
Highlights
Synthetic nucleotide and nucleic acid analogues are useful research tools and modern therapeutics
The unmodified nucleotides were purchased from commercial sources, the nucleotide analogues were synthesized previously and taken from our ‘in-house’ collection, and the18O-labelled isotopologues were synthesized for the purpose of this work using standard nucleotide chemistry and H218O, as described in the Methods and shown in Supplementary Fig. S2
electrospray ionization (ESI)(+)/MS/MS spectra of nucleotides contain fragmentation ions derived from the nucleobase only, with ions derived from ribose or phosphate not observed or observed at very low intensities
Summary
Synthetic nucleotide and nucleic acid analogues are useful research tools and modern therapeutics. Little is known regarding the general rules governing the fragmentation of nucleoside oligophosphates and, in particular, their phosphate- and ribose-modified analogues, as previous reports have usually focused on a single molecule type or narrow subgroup of molecules[14, 27, 28] To fill this gap, we took advantage of our ‘in house’ library of nucleotide analogues to perform a broad and in-depth study on the fragmentation of nucleotides and their synthetic analogues by tandem mass spectrometry. The results of our study should be relevant to those working on the biological aspects of the structure and function of modified nucleotides as well as in synthetic nucleotide chemistry
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