The mechanisms of base-catalyzed 5-hydrogen exchange and 5-hydroxymethylation of 1-substituted uracils have been examined to provide insight into the mechanism of the analogous reactions catalyzed by thymidylate synthetase. Kinetic studies of 5-H exchange demonstrate that the reaction requires attack of a nucleophile at the 6 position of the heterocycle to form carbanionic intermediates, as in classical addition reactions of α,β-unsaturated carbonyl compounds. The intermediate then accepts a proton from water to form the corresponding carbon acid; reversal of these steps results in 5-H exchange. Proton transfers at the 5 position of the saturated pyrimidine intermediates have been found to be susceptible to general acid-base catalysis and may be rate determining. The exchange reaction and 5-hydroxymethylation are greatly facilitated by intramolecular catalysis involving addition of a nucleophile attached to the 1 substituent to the 6 position of the heterocycle; in fact, the rate of intramolecular catalyzed exchange of one of the reactive ionic species of 2′,3′-O-isopropylideneuridine is comparable to that of the enzyme-catalyzed reaction. Depending upon the efficacy of the intramolecular catalyst the rate-determining step of the exchange may be either nucleophilic attack at the 6 position of the heterocycle or proton transfer reactions at the 5 position. From these results, a minimal mechanism for the thymidylate synthetase catalyzed 5-H exchange of 2′-deoxyuridylate and its condensation with 5,10-methylenetetrahydrofolic acid is proposed.
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