TuO-12


STRUCTURAL DETERMINATION OF GLYCANS BY ELECTROSPRAY MASS SPECTROMETRY (ESI-MS)

Steven Ramsay, John MacLeod, John Redmond and Craig Freeman

Research School of Chemistry, Australian National University, Canberra, ACT 0200


Heparin and heparan sulfate are structurally related glycosaminoglycans (GAGs), part of a family of complex anionic polysaccharides. The heparin and heparan sulfate polymers play a multifunctional role in the body and are involved in many important biological activities. Recent work by Parish and co-workers has shown that heparin fragment analogues are potentially powerful anticancer drugs having both a remarkable inhibitory effect on vascularisation and an antimetastatic effect. Sequence information of these highly charged molecules is necessary therefore, to understand these interactions and will allow us to develop more potent anticancer and anti-inflammatory drugs.

A new methodology to tag oligosaccharides with fluorophores and obtain glycan sequence data with ESI-MS/MS is described. A novel reductive labelling technique has been developed using N-substituted hydroxylamines which has significant advantages over previous reductive amination labelling methods. We have optimised this reaction to give high to quantitative yields of the model anhydromannose monosaccharide derivatives and the more common monosaccharides Glc, GlcNAc and Man. Extension to complex polysaccharides such as a dextran hydrolysate and an enzymatically degraded tamarind seed xyloglucan mixture have shown that this labelling method is applicable to a broad range of reducing sugars.

Figure 1

Figure 1: Example of a Heparin oligosaccharide showing nitrous acid susceptible cleavage sites and the generation of the reactive anhydromannose moiety used for tagging.


We have successfully applied this labelling technique to an exhaustive nitrous acid degradation of bovine lung heparin (shown above), enabling the separation and assignment of tentative structures to these GAGs using reverse phase chromatography, followed by off-line ESI-MS/MS spectrometric analysis.