Complete structural elucidation of an oxidized polysialic acid drug intermediate by nuclear magnetic resonance...

Polysialic acid (PSA) is a high molecular weight glycan composed of repeat units of α(2→8) linked 5-N-acetyl-neuraminic acid. Mild periodate oxidation of PSA selectively targets the end sialic acid ring containing three adjacent alcohols generating a putative aldehyde, which can be used for terminal attachment of PSA to therapeutic proteins. The work presented here permitted complete NMR peak assignments of not only the repeat units, but also the two terminal units at each end of oxidized PSA, an intermediate, which can be used to improve drug performance. The assignments were made using a variety of NMR techniques on oligomers of sialic acid as well as oxidized PSA with molecular masses of 4 and 20 kDa. This enabled structure elucidation that showed the actual moiety formed was not the expected aldehyde or its hydrate, but is a hemiacetal between the oxidation site on the terminal sialic acid ring and the penultimate ring. The existence of a hemiacetal structure has major implications on stability, reactivity, and conjugation chemistry of oxidized PSA. The assignment process also revealed deuterium exchange of the axial hydrogen at the 3- (methylene) position of the ring, which was in agreement with the literature.