Chemoenzymatic synthesis of unsymmetric N-glycans for high resolution glycome analyses and glycan arrays

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Glycans occurring in various glycoconjuates are essential components of cells. The glycan part of biomolecules influences their properties and thus the biological processes they are involved in. However, the effects of carbohydrates within biological systems are still poorly understood. This is mainly attributed to limited access to pure glycans and glycoconjugates from natural sources. Additionally, the structure elucidation of natural glycans is hampered by the presence of numerous isomeric structures in combination with limited amounts of sample. In most cases these shortcomings can only be overcome by synthesis.For glycan-based interaction studies additional requirements need to be met. Ideally, glycosylated biomolecules should be investigated using the natural entity for establishing structure-activity relationships. However, in most cases this is not feasible. Limitations arise through the additional synthetic effort and the loss of precious material in macroscopic assays. As an alternative, glycan microarrays were developed. This technology minimizes the consumption of glycans and allows to conduct many experiments in parallel. The composition of many of the currently used N-glycan arrays is not systematic and furthermore the purity of the printed compounds is frequently not disclosed. There is a lack of unsymmetrically substituted N-glycans, which comprise the majority of naturally occurring N-glycans but are quite difficult to obtain. We have developed a chemoenzymatic method to diversify selectively benzylated N-glycans thus providing access to complete libraries of unsymmetrically galactosylated multiantennary N-glycans. These N-glycans are readily derivatized at their azido group and will serve to complement an existing microarray with symmetrical N-glycans. By providing complete sets of hitherto unavailable reference compounds we intend to improve the methods for N-glycan analysis in glycomics allowing for precise structure assignment. The project aims at:1) developing methods for the enzymatic diversification of complex N-glycans.2) the synthesis of galactosylated reference libraries of unsymmetric N-glycans with complete substitution patterns based on benzylated N-glycan azides.3) establishing an extended N-glycan array for the detailed elucidation of lectin specificities.4) the synthesis of labelled reference libraries of unsymmetric N-glycans for use in glycomics and glycoprotein profiling.5) developing methods for the synthesis of reference libraries of unsymmetric N-glycans bearing core modifications.