Characterization of the Transition-State Structures and Mechanisms for the Isomerization and Cleavage Reactions of Uridine 3‘-<i>m</i>-Nitrobenzyl Phosphate

Abstract

The transition-state structures and mechanisms of the isomerization to the 2‘-isomer and cleavage reactions of uridine 3‘-m-nitrobenzyl phosphate to m-nitrobenzyl alcohol and a 2‘,3‘-cyclic UMP at 86 °C and at pH 2.5, 5.5, and 10.5 have been characterized through kinetic isotope effects. The 18O primary isotope effect of 1.0019 ± 0.0007 and the secondary isotope effect of 0.9904 observed for the cleavage reaction at pH 2.5 are consistent with a neutral phosphorane-like transition-state structure. The cleavage and isomerization reactions at pH 2.5 proceed through a neutral phosphorane intermediate. The 18kbridge and 18knonbridge of unity measured for the pH-independent isomerization reaction at neutral pH support a stepwise mechanism with a monoanionic phosphorane intermediate. The primary and secondary isotope effects of 1.009 ± 0.001 and of 0.9986 ± 0.0004 observed for the pH-independent cleavage reaction are consistent with either a stepwise mechanism through a monoanionic phosphorane intermediate or with an ANDN reaction with a transition state resembling a monoanionic phosphorane intermediate. The absolute requirement of a water-mediated proton transfer for the formation of a phosphorane intermediate is proven by the absence of the isomerization reaction in anhydrous tert-butyl alcohol. The primary isotope effect of 1.0272 ± 0.0001 for the cleavage reaction at pH 10.5 is consistent with a concerted reaction through a transition state in which the leaving group departs with almost a full negative charge.