Characterization of the transition-state structures and mechanisms for the isomerization and cleavage reactions of uridine 3′-m-nitrobenzyl phosphate

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 degreesC and at pH 2.5, 5.5, and 10.5 have been characterized through kinetic isotope effects. The O-18 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 DH 2.5 proceed through a neutral phosphorane intermediate, The (18)k(bridge) and (18)k(nonbridge) 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 A(N)D(N) 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.