Aromatization-Driven Hydrolysis of Pyrimidine d4Ns under Acidic Conditions: The Preferential γ-Pathway to Furfuryl...

The acid-catalyzed hydrolysis mechanism of the N-glycosidic bond in pyrimidine 2',3'-didehydro-2',3'-dideoxynucleosides (d4Ns), including d4U, d4T, d4C, and d4FC, was investigated using molecular dynamics (MD) simulations to refine microsolvated models, followed by density functional theory (DFT) calculations at the M06-2X(CPCM)/6-31+G(d) level of theory, with single-point energies refined at the M06-2X(CPCM)/6-311++G(d,p) level. Three plausible pathways─α-path (inversion at C1'), β-path (retention at C1'), and γ-path (aromatization to form a furfuryl alcohol)─were evaluated. The first stage, cleavage of the N-glycosidic bond to form an oxacarbenium ion intermediate, is the rate-determining step (RDS) for all pathways. Acidic conditions dramatically catalyze the hydrolysis, lowering the activation free energy by approximately 10 kcal mol-1 compared to neutral conditions. The γ-path exhibits a distinct kinetic and thermodynamic advantage for d4U and d4T, driven by aromatization of the furfuryl alcohol sugar, which aligns with experimental observations of γ-product formation. The 5-methyl group in d4T increases the activation barrier relative to d4U, while the 5-fluoro group in d4FC lowers the barrier by ∼2 kcal mol-1 compared to d4C, indicating an electron-withdrawing promoting effect. Heterolytic dissociation of the N-glycosidic bond is favored over homolytic dissociation in both gas and aqueous phases, consistent with the charge-separated intermediates identified. Nucleus-independent chemical shift (NICS) analyses quantify the increasing aromaticity along the γ-path, rationalizing its thermodynamic favorability. These computational insights elucidate how nucleobase protonation and substituent effects govern the stability of the glycosidic bond and the unusual formation of γ-cleavage products in acidic d4N hydrolysis.