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-H-3], [1-C-14-N-acetyl], [2-C-14], [1-C-14], and [9-H-3;3,3'-H-2] in yields of 78%, 86%, 76%, 85%, and 35%, respectively. The beta-dideuterium kinetic isotope effect for solvolysis at pH 5.0, 37 degrees C, was 1.276 +/- 0.008; the primary C-14 isotope effect at C2, the anomeric carbon, was 1.030 +/- 0.004; and an unusually large secondary C-14 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 alpha- and beta-methyl glycosides of NeuAc, and small amounts of the elimination product 2,3-dehydro-N-acetyl neuraminic acid. The very large beta-H-2 KIE, small primary C-14 KIE, and the large secondary C-14 KIE at the carboxylate carbon are consistent with a very late oxacarbenium 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.