Preparative scale purification of natural glycans by closed-loop recycle HPLC

While glycoscience has become well recognized as an indispensable area in biomedical research, studies on the function of individual glycans remains a great challenge due to the lack of tools and methods. One of the greatest impediments to progress in this area is the lack of biomedically relevant complex glycans in sufficient quantity and purity for structural and functional analysis. Despite recent advances in chemoenzymatic synthesis of complex glycans, generating significant amounts of pure glycans is limited to laboratories with specialized expertise. We have previously reported the oxidative release of natural glycans (ORNG) using household bleach, which provides large quantities of biologically relevant glycans that can be a source of glycans in quantities (>mg scale) suitable for functional studies. However, the preparative scale separation of complicated glycan mixtures has not been studied due largely to the fact that gram quantities of starting glycans have not been available until now. Here we report the adoption of closed-loop, recycle HPLC to resolve closely related glycan structures, including complex glycan isomers at preparative scale (10-100 mg).