Mechanistic variation in the glycosyltransfer of N-acetylneuraminic acid

N-acetylneuraminic acid is an acidic nine-carbon amino ketose typically found at the non-reducing terminus of glycoproteins and glycolipids. The presence of a carboxylate group adjacent to the anomeric center suggest that this sugar could have transition states with highly stabilized oxocarbenium ion character during transfer reactions at the anomeric carbon. Kinetic isotope effect (KIE) experiments were used to probe the transition state for solvolysis of UMP-NeuAc, sialyltransferase-catalyzed transfer of UMP-NeuAc to N-acetyl-lactosamine, trans-sialidase catalyzed transfer of alfa(2--3) Neu-Lac or alfa(2--3) Neu-Gal, and acid catalyzed hydrolysis of alfa(2--3) Neu-Lac. The two key positions of isotope substitution in the N-acetyl neuraminic acid residue were the C3’ position, di-substituted with deuterium, and the C2’ position, substituted with either carbon-13 or carbon-14. The solvolysis reaction had a beta–2H KIE of 1.28 and a primary 14C KIE of 1.03. The sialyltransferase-catalyzed reaction had a b-2H KIE of 1.22 and a 14C KIE of 1.03. Trans-sialidase had a b–2H KIE of 1.05 and a primary 13C KIE of 1.03, equivalent to a 14C KIE of 1.06. Solvolysis of the trans-sialidase substrate gave a beta-2H KIE of 1.06, and a primary 13C KIE of 1.015. The results indicate a very late transition state for solvolysis of CMP-NeuAc, without nucleophilic participation. The sialyltransferase transition state is similar, but with less charge development. Trans-sialidase has a transition state with diminished charge development and considerable nucleophilic character, which leads to a covalent intermediate. The glycosyltransfer of N-acetylneuraminic acid glycosides is not limited to the classical dissociative mechanism.