Synthesis of Trifluoromethylated Purine Ribonucleotides and Their Evaluation as 19F NMR Probes.

Protected guanosine and adenosine ribonucleosides and guanine nucleotides are readily functionalized with CF3 substituents within the nucleobase. Protected guanosine is trifluoromethylated at the C8 position under radical-generating conditions in up to 95% yield and guanosine 5'-oligophosphates in up to 35% yield. In the case of adenosine, the selectivity of trifluoromethylation depends heavily on the functional group protection strategy and leads to a set of CF3-modified nucleosides with different substitution patterns (C8, C2, or both) in up to 37% yield. Further transformations based on phosphorimidazolide chemistry afford various CF3-substituted mono- and dinucleoside oligophosphates in good yields. The utility of the trifluoromethylated nucleotides as probes for 19F NMR-based real-time enzymatic reaction monitoring is demonstrated with three different human nucleotide hydrolases (Fhit, DcpS, and cNIIIB). Substrate and product(s) resonances were sufficiently separated to enable effective tracking of each enzymatic activity of interest.

Analysis of mononucleotides by tandem mass spectrometry: investigation of fragmentation pathways for phosphate- and ribose-modified nucleotide analogues.

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.