The Neisseria meningitidis sialic acid synthase (NeuB) catalyzes the metal-dependent condensation of N-acetylmannosamine (ManNAc) and phosphoenolpyruvate (PEP) to generate N-acetylneuraminic acid (NeuAc or sialic acid). N. meningitidis is a causative agent of meningitis and produces a capsular polysaccharide comprised of polysialic acid. This allows it to evade the immune system of the host by an act of molecular mimicry. This work describes the synthesis and characterization of the first potent inhibitor of sialic acid synthase. The inhibitor is a stable deoxy analogue of the tetrahedral intermediate presumed to form in the NeuB reaction and was synthesized as a mixture of stereoisomers at the key tetrahedral center. Inhibition studies demonstrate that one stereoisomer binds more tightly than the other and that the more potent isomer binds with micromolar affinity. An X-ray crystallographic analysis of the NeuB.inhibitor.Mn(2+) complex solved to a resolution of 1.75 A shows that the more tightly bound stereoisomer bears a (2R)-configuration. This suggests that the tetrahedral intermediate formed in the NeuB reaction also bears a (2R)-configuration. This analysis is consistent with a mechanism whereby the active site metal plays at least two roles during catalysis. First, it serves as an electrostatic catalyst and activates the aldehyde of ManNAc for attack by the alkene of PEP. Second, it serves as a source of nucleophilic water and delivers it to the si face of the oxocarbenium intermediate to generate a tetrahedral intermediate with a (2R)-configuration.