Transition state analysis of acid-catalyzed dAMP hydrolysis

Multiple kinetic isotope effects (KIEs) on deoxyadenosine monophosphate (dAMP) hydrolysis in 0.1 M HCl were used to determine the transition state (TS) structure and probe its intrinsic reactivity. The experimental KIEs revealed a stepwise (SN1) mechanism, with a discrete oxacarbenium ion intermediate. This is the first direct evidence for the deoxyribosyl oxacarbenium ion in solution. In 50% methanol/0.1 M HCl the products were deoxyribose 5-phosphate (dRMP) and alpha- and beta-methyl dRMP. The alpha-Me-dRMP/beta-Me-dRMP ratio was 8.5:1. Assuming that a free oxacarbenium ion is equally susceptible to nucleophilic attack on either face, this indicated that approximately 20% proceeded through a solvent-separated ion pair complex, or free oxacarbenium ion, a DN+AN mechanism, while approximately 80% of the reaction proceeded through a contact ion pair complex. The oxacarbenium ion lifetime was estimated at 10(-11)-10(-10) s. Computational transition states were found for ANDN, DN*AN, DN*AN, and DN+AN mechanisms using hybrid density functional theory calculations. After taking into account 20% of DN+AN, there was an excellent match of calculated to experimental KIEs for 80% of the reaction having a DN*AN mechanism. That is, C-N bond cleavage is reversible, with dAMP and the {oxacarbenium ion*adenine} complex in equilibrium. The first irreversible step is water attack on the oxacarbenium ion. The calculated 1'-14C KIE for a stepwise mechanism with irreversible C-N bond cleavage (DN*AN) was 1.052, in the range previously associated only with ANDN transition states, and close to the calculated ANDN value, 1.059. The 1'-14C KIE was strongly dependent on the adenine protonation state.