Acid-Catalyzed Solvolysis of CMP-N-Acetyl Neuraminate: Evidence for a Sialyl Cation with a Finite Lifetime

An investigation of the mechanism of solvolysis of CMP- N -acetyl neuraminate (CMP-NeuAc) is presented that includes synthesis of a family of isotopically labeled CMP-NeuAc's, use of new methodology for measurement of multiple kinetic isotope effects for acid-catalyzed solvolysis of CMP-NeuAc, and a stereochemical analysis of the reaction by solvolysis in aqueous methanol. The CMP-NeuAc isotopomers were synthesized with the following labels: [9- 3 H], [1- 14 C- N -acetyl], [2- 14 C], [1- 14 C], and [9- 3 H;3,3‘- 2 H] in yields of 78%, 86%, 76%, 85%, and 35%, respectively. The β-dideuterium kinetic isotope effect for solvolysis at pH 5.0, 37 °C, was 1.276 ± 0.008; the primary 14 C isotope effect at C2, the anomeric carbon, was 1.030 ± 0.004; and an unusually large secondary 14 C KIE was observed at C1, the carboxylate carbon, of 1.037 ± 0.004. Analysis of pH versus rate data and rate versus buffer concentration data establish that the solvolytic reaction is specific acid-catalyzed. Solvolysis of CMP-NeuAc at pH 5 or pH 6 in methanol/water mixtures afforded NeuAc, equal quantities of the α- and β-methyl glycosides of NeuAc, and small amounts of the elimination product 2,3-dehydro- N -acetyl neuraminic acid. The very large β- 2 H KIE, small primary 14 C KIE, and the large secondary 14 C KIE at the carboxylate carbon are consistent with a very late oxocarbenium ion-like transition state in which the carboxylate carbon is in a looser environment than in the ground state. The observation of racemization in the solvolysis reaction supports a reaction pathway that proceeds with the formation of a sialyl cation after the transition state.