The cycloSal approach has been used in the past for the synthesis of a range of phosphorylated bioconjugates. In those reports, cycloSal nucleotides were allowed to react with different phosphate nucleophiles. With glycopyranosyl phosphates as nucleophiles, diphosphate-linked sugar nucleotides were formed. Here, cycloSal-nucleotides were used to prepare monophosphate-linked sugar nucleotides successfully in high anomeric purity and high chemical yield. The method was successfully used for the synthesis of three nucleotide glycopyranoses as model compounds. The method was then applied to the syntheses of CMP-N-acetyl-neuraminic acids (CMP-Neu5NAc) and of four derivatives with different modifications at their amino functions (N-propanoyl, N-butanoyl, N-pentanoyl and N-cyclopropylcarbonyl). The compounds were used for initial enzymatic studies with a bacterial polysialyltransferase (polyST). Surprisingly, the enzyme showed marked differences in terms of utilisation of the four derivatives. The N-propanoyl, N-butanoyl, and N-pentanoyl derivatives were efficiently used in a first transfer with a fluorescently labelled trisialo-acceptor. However, elongation of the resulting tetrasialo-acceptors worsened progressively with the size of the N-acyl chain. The N-pentanoyl derivative allowed a single transfer, leading to a capped tetramer. The N-cyclopropylcarbonyl derivative was not transferred.