Chemoenzymatic synthesis of Neu5Ac9NAc-containing α2–3- and α2–6-linked sialosides and their use for sialidase substrate specificity studies

Abstract

O-Acetylation of sialic acid (Sia) modulates its recognition by sialic acid-binding proteins and plays an important role in biological and pathological processes. 9-O-Acetylation is the most common modification of sialic acid in human. However, study of O-acetylated sialoglycans is hampered due to the instability of O-acetyl group towards pH changes and sensitivity to esterases. Our previous studies demonstrated a chemical biology method to this problem by replacing the oxygen atom in the C9 ester group of sialic acid by a nitrogen to form an amide. Here, we synthesized a library of sixteen new 9-acetamido-9-deoxy-N-acetylneuraminic acid (Neu5Ac9NAc)-containing α2–3- and α2–6-linked sialosides with various underlying glycans using efficient one-pot three-enzyme (OP3E) sialylation systems. Neu5Ac9NAc-containing compounds with a para-nitrophenol aglycon have been used together with their 9-O-acetyl analogs in microtiter plate-based high-throughput substrate specificity studies of nine different sialidases including those from humans and bacteria. In general, similar to 9-O-acetylation, 9-N-acetyl modification of sialic acid in the substrates lowers sialic acid-cleavage activity of most sialidases. In most cases, Neu5Ac9NAc is a good analog of 9-O-acetyl sialic acid. However, exceptions do exist. For example, 9-N- and 9-O-acetyl modifications have different effects on the sialosides cleave efficiencies of a commercially available C. perfringens sialidase as well as recombinant Streptococcus pneumoniae sialidase SpNanC and Bifidobacterium infantis sialidase BiNanH2. The mechanism for the difference awaits further investigation.