Improved Enzymatic Procedure for a Preparative-Scale Synthesis of Sialic Acid and KDN

Abstract

Abstract Sialic acid (N-acetylneuraminic acid) and KDN (3-deoxy-β-d-glycero-d-galacto-2-nonulosonic acid) play important roles in a variety of intercellular events. Extensive studies have been reported on the use of sialic acid aldolase as a catalyst to form the C–C bond between N-acetyl-d-mannosamine derivatives and pyruvic acid to give sialic acid and its analogs. However, the reported protocols have a significant drawback in terms of the tedious separation of the desired product from a large excess of pyruvic acid. We report here on a clean solution to this problem that relies on the utilization of pyruvate decarboxylase for catalyzing the decomposition of pyruvic acid into acetaldehyde and carbon dioxide to facilitate product isolation. Concerning our investigation to enhance the activity of this enzyme, we emphasized two points. One was a sufficient supply of cofactors for pyruvate decarboxylase; the other was an improvement of the enzyme reactor. As a result, the activity became nearly four-times as high as that previously reported. The combination of two enzymatic reactions worked well, affording sialic acid and KDN in 60% yield from N-acetyl-d-mannosamine and d-mannose, respectively. In addition, an efficient method for preparing N-acetyl-d-mannosamine by the epimerization of N-acetyl-d-glucosamine was established. Calcium hydroxide was found to be most effective for base-catalyzed epimerization.