Reaction of Oxidized Polysialic Acid and a Diaminooxy Linker: Characterization and Process Optimization Using Nuclear Magnetic Resonance Spectroscopy

Native polysialic acid (natPSA) is a high-molecular-weight glycan composed of repeat units of α-(2 → 8) linked N-acetylneuraminic acid (Neu5Ac). Mild periodate oxidation of PSA selectively targets the end sialic acid ring containing three adjacent alcohols generating a putative aldehyde, which can be used, after attachment of a linker molecule, for terminal attachment of PSA to protein. Previously, we showed that the oxidized PSA (oxoPSA) contained a hemiacetal at the oxidation site and can react with a linker containing an aminooxy group in a conjugation reaction to form a stable oxime linkage. Thus, reagents containing an aminooxy group may be prepared for conjugation of PSA to the carbohydrate moiety of therapeutic proteins, thereby increasing their half-life. These aminooxy–PSA reagents can selectively react with aldehyde groups generated by mild NaIO4 oxidation of glycans on the surface of the target protein. To comprehend the conjugation, unoxidized tetrasialic acid and Neu5Ac were reacted in model ...