The intrinsic basicity of the phosphate backbone exceeds that of uracil and thymine residues: protonation of the phosphate moiety is preferred over the nucleobase for pdThd and pUrd

The gas-phase conformations of the protonated forms of thymidine-5'-monophosphate and uridine-5'-monophosphate, [pdThd+H](+) and [pUrd+H](+), are investigated by infrared multiple photon dissociation (IRMPD) action spectroscopy and electronic structure calculations. The IRMPD action spectra of [pdThd+H](+) and [pUrd+H](+) are measured over the IR fingerprint and hydrogen-stretching regions using the FELIX free electron laser and an OPO/OPA laser system. Low-energy conformations of [pdThd+H](+) and [pUrd+H](+) and their relative stabilities are computed at the MP2(full)/6-311+G(2d, 2p)//B3LYP/6-311+G(d,p) and B3LYP/6-311+G(2d,2p)//B3LYP/6-311+G(d, p) levels of theory. Comparisons of the measured IRMPD action spectra and B3LYP/6-311+G(d, p) linear IR spectra computed for the low-energy conformers indicate that the dominant conformers of [pdThd+H](+) and [pUrd+H](+) populated in the experiments are protonated at the phosphate oxo oxygen atom, with a syn nucleobase orientation that is stabilized by strong P=OH+center dot center dot center dot O2 and P-OH center dot center dot center dot O4' hydrogen-bonding interactions, and C2'-endo sugar puckering. Minor abundance of conformers protonated at the O2 carbonyl of the nucleobase residue may also contribute for [pdThd+H](+), but do not appear to be important for [pUrd+H](+). Comparisons to previous IRMPD spectroscopy investigations of the protonated forms of thymidine and uridine, [dThd+H](+) and [Urd+H](+), and the deprotonated forms of pdThd and pUrd, [pdThd-H](-) and [pUrd-H](-), provide insight into the effects of the phosphate moiety and protonation on the conformational features of the nucleobase and sugar moieties. Most interestingly, the thymine and uracil nucleobases remain in their canonical forms for [pdThd+H](+) and [pUrd+H](+),unlike [dThd+H](+) and [Urd+H](+), where protonation occurs on the nucleobases and induces tautomerization of the thymine and uracil residues.