Transition State Analysis of Vibrio cholerae Sialidase-Catalyzed Hydrolyses of Natural Substrate Analogues

A series of isotopically labeled natural substrate analogues (phenyl 5-N-acetyl-α-d-neuraminyl-(2→3)-β-d-galactopyranosyl-(1→4)-1-thio-β-d-glucopyranoside; Neu5Acα2,3LacβSPh, and the corresponding 2→6 isomer) were prepared chemoenzymatically in order to characterize, by use of multiple kinetic isotope effect (KIE) measurements, the glycosylation transition states for Vibrio cholerae sialidase-catalyzed hydrolysis reactions. The derived KIEs for Neu5Acα2,3LacβSPh for the ring oxygen (18V/K), leaving group oxygen (18V/K), C3-S deuterium (DV/KS) and C3-R deuterium (DV/KR) are 1.029 ± 0.002, 0.983 ± 0.001, 1.034 ± 0.002, and 1.043 ± 0.002, respectively. In addition, the KIEs for Neu5Acα2,6βSPh for C3-S deuterium (DV/KS) and C3-R deuterium (DV/KR) are 1.021 ± 0.001 and 1.049 ± 0.001, respectively. The glycosylation transition state structures for both Neu5Acα2,3LacβSPh and Neu5Acα2,6LacβSPh were modeled computationally using the experimental KIE values as goodness of fit criteria. Both transition states are late with largely cleaved glycosidic bonds coupled to pyranosyl ring flattening (4H5 half-chair conformation) with little or no nucleophilic involvement of the enzymatic tyrosine residue. Notably, the transition state for the catalyzed hydrolysis of Neu5Acα2,6βSPh appears to incorporate a lesser degree of general-acid catalysis, relative to the 2,3-isomer.