Enzymes in Carbohydrate Synthesis: N-Acetylneuraminic Acid Aldolase Catalyzed Reactions and Preparation of N-Acetyl-2-deoxy-D-neuraminic Acid Derivatives

Article abstract of DOI:10.1021/ja00227a031

This paper describes the structural characteristics of substrates accepted by N-acetylneuraminic acid (Neu5Ac) aldolase (E.C.4.1.3.3), the results from its stability studies, its use in the synthesis of Neu5Ac and 9-O-acetyl-Neu5Ac (Neu5,9Ac2), and the chemical conversion of Neu5Ac to the 2-deoxy derivatives.Values of kinetic parameters (K_m and V_max) for 14 aldoses including N-acetyl-D-mannosamine (ManNAc) and pyruvate were determined at pH 7.5 and 25 deg C in the direction of condensation.The 30-50-mmol-scale synthesis using ManNAc, excess pyruvate, and PAN-immobilized Neu5Ac aldolase provided multigram quantities of Neu5Ac (yield, 87-91 percent in solution and 67 percent in isolated products) without a significant loss of enzyme activity.The synthesis using two separate enzyme reactions, acetylation of ManNAc to 6-O-acetylManNAc catalyzed by protease N and condensation of 6-O-acetylManNAc with pyruvate catalyzed by Neu5Ac aldolase, provided Neu5,9Ac2 in 59 percent overall yield.To illustrate the utility of Neu5Ac as a synthetic starting material, a potential inhibitor of Neu5Ac-associated enzymes was prepared.Three chemical steps from Neu5Ac provided methyl 4,7,8,9-tetra-O-acetyl-N-acetyl-2-deoxy-α-neuraminic acid (2-deoxy-α-Neu4,5,7,8,9Ac5OMe) in 50 percent overall yield.Its structure was analyzed by 1H and 13C NMR spectroscopy and X-ray crystallography.