A chemical-enzymatic method for the synthesis of sialylated oligosaccharides: Synthesis of cytidine monophosphate-sialic acid analogs for use in probing the donor specificity of alpha-2,3- and alpha-2,6-sialyltransferase

Sialic acids are nine-carbon monosaccharides typically found at the termini of animal glycoconjugates and are known to be important sites for biological recognition. An effective method for the incorporation of sialic acid analogs into oligosaccharides utilizes sialyltransferase enzymes, which regio- and stereoselectively transfer sialic acid from the donor compound, cytidine monophoso- N-acetyl neuraminic acid (CMP-Neu5Ac), onto several lactose- and lactosamine-derived acceptors. Sialyltransferases have been shown to tolerate a variety of acceptor modifications, although less is known about the donor specificity due to the difficulty in synthesizing the requisite CMP-Neu5Ac sugar-donors. Consequently, a chemical synthesis of CMP-Neu5Ac analogs, which is amenable to the incorporation of derivatized sialic acids, is a focus of this work.;A key step in the synthesis of the CMP-Neu5Ac congeners was the tetrazole-promoted coupling between a sialic acid derivative and an silyl cytidine phosphoramidite. The presence or absence of electron-withdrawing substituents at C-5 and C-7 of the sialic acid was found to affect the phosphite product yields. However, quenching the promotor at low temperature with diisopropylethylamine and purifying the products by size-exclusion chromatography led to consistantly high yields (80–95%) for the coupling of each sialic acid.;Oxidation of the phosphites was accomplished with dimethyldioxirane, using a procedure developed in this laboratory, to afford the phosphates in quantitative yield. Deprotection of the silyl phosphorus protecting group was achieved by a palladium-catalyzed ester transfer reaction. The creation of the phosphate anion significantly decreased the rate of elimination in the subsequent basic hydrolysis steps. Following the removal of the hydroxyl and carboxylate protecting groups, the enzymatic substrates were provided in good yield and purity.;Five of the seven synthetic CMP-Neu5Ac derivatives were successfully transferred by the α2,3- and α2,6-sialyltransferases to lactose and lactosamine acceptors. Three naturally occurring CMP-Neu5Ac analogs modified at C-5 of sialic acid were accepted by both enzymes. Also, two unnatural congeners modified at C-5 and C-9 were accepted by the transferases. Through these reactions, a better understanding of the functional group tolerances of two sialyltransferases has been obtained.