Structural and Energetic Effects of the 2-Amino and 2'- and 3'-Hydroxy Substituents of Protonated Guanosine Nucleosides: IRMPD, ER-CID, and Theoretical Studies of Protonated Guanosine and Inosine Nucleosides.

Abstract

Inosine is a naturally occurring modified RNA nucleoside. Guanosine differs from inosine only by the 2-amino substituent of its nucleobase. The effects of the 2-amino and 2'- and 3'-hydroxy substituents on structure and glycosidic bond stability are examined via comparative studies of protonated guanosine vs inosine nucleoside analogues. Infrared multiple photon dissociation (IRMPD) action spectroscopy experiments are performed to probe structural effects, whereas energy-resolved collision-induced dissociation (ER-CID) experiments combined with survival yield analyses are performed to probe their effects on glycosidic bond stability. Density functional theory (DFT) calculations are performed to determine the stable low-energy conformations available to these systems, their relative stabilities, and infrared (IR) spectra. The structures experimentally populated are determined via comparisons of the measured IRMPD and predicted IR spectra. DFT calculations are also employed to map detailed mechanistic pathways for N-glycosidic bond cleavage of the protonated guanosine and inosine nucleosides. The influences of the 2-amino and 2'- and 3'-hydroxy substituents on glycosidic bond stability are determined from the trends in the energy-dependence of their ER-CID behavior and the activation energies predicted for glycosidic bond cleavage. The 2-amino substituent of the guanosine nucleosides has little effect on structure and is found to strengthen the glycosidic bond. The 2'- and 3'-hydroxy substituents exert a greater influence on structure via stabilizing hydrogen-bonding interactions enabled by their presence. The influences of the 2'- and 3'-hydroxy substituents on glycosidic bond stability differ. The 2'-hydroxy substituent significantly enhances glycosidic bond stability, whereas the 3'-hydroxy substituent slightly weakens glycosidic bond stability.