Resolution of Identity in Gas-Phase Dissociations of Mono- and Diprotonated DNA Trinucleotide Codons by ¹⁵N-Labeling and Computational Structure Analysis

Abstract

Dissociations of DNA trinucleotide codons as gas-phase singly and doubly protonated ions were studied by tandem mass spectrometry using 15N-labeling to resolve identity in the nucleobase loss and backbone cleavages. The monocations showed different distributions of nucleobase loss from the 5′-, middle, and 3′-positions depending on the nucleobase, favoring cytosine over guanine, adenine, and thymine in an ensemble-averaged 62:27:11:<1 ratio. The distribution for the loss of the 5′-, middle, and 3′-nucleobase was 49:18:33, favoring the 5′-nucleobase, but also depending on its nature. The formation of sequence w 2 + ions was unambiguously established for all codon mono- and dications. Structures of low-Gibbs-energy protomers and conformers of dAAA+, dGGG+, dCCC+, dTTT+, dACA+, and dATC+ were established by Born–Oppenheimer molecular dynamics and density functional theory calculations. Monocations containing guanine favored classical structures protonated at guanine N7. Structures containing adenine and cytosine produced classical nucleobase-protonated isomers as well as zwitterions in which two protonated bases were combined with a phosphate anion. Protonation at thymine was disfavored. Low threshold energies for nucleobase loss allowed extensive proton migration to occur prior to dissociation. Loss of the nucleobase from monocations was assisted by neighboring group participation in nucleophilic addition or proton abstraction, as well as allosteric proton migrations remote from the reaction center. The optimized structures of diprotonated isomers for dAAA2+ and dACA2+ revealed combinations of classical and zwitterionic structures. The threshold and transition-state energies for nucleobase-ion loss from dications were low, resulting in facile dissociations involving cytosine, guanine, and adenine.

The DNA Radical Code. Resolution of Identity in Dissociations of Trinucleotide Codon Cation Radicals in the Gas Phase

“…It may be noted that protonation of thymine in trinucleotides has been found to be energetically disfavored, as judged by (TTT+H) + , in which low-energy structures were protonated at the phosphate ester groups instead in thymine. 48 This is expected to have a large effect on the structures of the (TXT+2H) 2+ precursor dications and (TXT+2H) +• cation radicals. Ion Structures and Energetics.…”

“…Standard backbone cleavages in trinucleotides by phosphate ester elimination can result in the formation of d 2 + ions retaining the 5′- and middle nucleobases or w 2 + ions retaining the 3′- and middle nucleobases. ,,− With hydrogen-rich cation radicals containing an additional hydrogen atom in the radical-carrying nucleobase, one also observes backbone dissociations that are associated with hydrogen atom transfer, forming odd-electron ( w 2 +H) +• and even-electron ( w 2 +2H) + and ( d 2 +2H) + ions. The main types of dissociation by nucleobase loss, phosphate ester backbone cleavage, and cross-ring fragmentations for 60 codon trinucleotide cation radicals are summarized in the master table (Table ).…”

“…Custom-made trinucleotides (95% pure) were purchased from Integrated DNA Technologies, Inc. (Coralville, IA), as reported previously .…”

“…Electrospray ionization was performed in acetonitrile–water–acetic acid solutions at 50:50:1 component ratios and 10–20 μM concentration of the unlabeled trinucleotides and DBCE. The 15 N-labeled trinucleotides that were only available in nmol quantities were dissolved in 1 mL of the solvent mixture, and the solutions were electrosprayed as described previously . Mass-selected doubly charged ions were subjected to ion–ion reactions with fluoranthene anion radicals (ETD-MS 2 , 150 ms), and the cation radicals formed were selected for collision-induced dissociation (CID-MS 3 ).…”

“…Our interest was in identifying the main dissociation types of these cation radicals and their relationship to the trinucleotide sequence and electronic structure. Previous comprehensive studies of trinucleotide mono- and dications , elucidated the tendency for position and base-specific nucleobase loss and phosphate backbone cleavage occurring between the 5′- and middle position, unambiguously forming w 2 + fragment ions. We now wish to show that dissociations of trinucleotide cation radicals display a much richer chemistry regarding backbone cleavage, proton and hydrogen atom transfer reactions, and 5′- and 3′-deoxyribose cross-ring cleavages.…”