The fractions containing product were combined and lyophilized to give the N-glycan cluster (15) (quantitative yield). enzymatic reaction at the two GlcNAc residues could be fulfilled to afford novel hybrid clusters. Lectin microarray studies revealed unusual properties FLJ14936 in glyco-epitope expression by this panel of structurally well-defined synthetic N-glycans. These new compounds are likely Zinc Protoporphyrin valuable for functional glycomics studies to unveil new functions of both glycans and carbohydrate-binding proteins. Keywords:chemoenzymatic synthesis, N-glycans, clusters, transglycosylation, lectin microarray == Introduction == N-linked glycosylation is a predominant covalent modification of proteins in eukaryotes. It is well documented that N-glycans of glycoproteins are involved in many important biological events including protein folding, ER-associate protein degradation, cell differentiation, cell adhesion, host-pathogen interaction, cancer metastasis, and autoimmunity1. Glycoproteins are often characterized by their Zinc Protoporphyrin structural micro-heterogeneity in terms of the components of the attached glycans. It becomes clear that distinct N-glycans can confer significantly different effects on the structure and function of a given glycoprotein. This was exemplified by recent discoveries that the attachment of subtly different N-glycans at the conserved N-glycosylation site of the Fc domain could result in dramatically different impacts on the ADCC function of monoclonal antibodies and on the anti-inflammatory activity of intravenous immunoglobulin (IVIG)2. On the other hand, not only does the fine structure of the glycans provide the basis for molecular recognition, but the way of their presentation, e.g., the monovalent vs. multivalent format, is also of paramount importance in governing the specificity and strength in carbohydrate-protein interactions3. Recent advances in glycan and glycan-binding protein microarray technology have offered exciting new opportunities to unveil the mysteries of glycans in various cellular processes and disease states4. Nevertheless, functional glycomics studies are still limited by the availability of structurally well-defined N-glycans and related glycoconjugates, which are difficult to obtain in homogeneous forms from natural source5. We and others have previously demonstrated that a class of endo–N-acetylglucosaminidases (ENGases), including the Endo-A fromArthrobactor protophormiaeand the Endo-M fromMucor hiemali, were able to transfer an oligosaccharideen blocfrom either natural N-glycans or synthetic sugar oxazolines to a GlcNAc-containing moiety to form a new -1,4-glycosidic linkage in a regio- and stereo-specific manner, leading to the synthesis of complex oligosaccharides, N-glycopeptides and N- glycoproteins6. Endo-A is specific for high-mannose or hybrid type N-glycans and has been applied for the synthesis of high-mannose type oligosaccharides and N-glycopeptides7, whereas Endo-M is able to work on three major types (high-mannose, hybrid, and complex type) of N-glycans and has been particularly useful Zinc Protoporphyrin for synthesizing complex type N-glycopeptides8. In particular, the recent findings that synthetic oligosaccharide oxazolines (the mimics of the oxazolinium ion intermediate of the enzymatic reaction) could be used for Endo-A catalyzed transglycosylation have significantly expanded the scope of the chemoenzymatic method for glycopeptide and glycoprotein synthesis911. It was found that the highly activated sugar oxazolines corresponding to the truncated or modified N-glycans could serve as substrates for the Endo-A catalyzed transglycosylation but the ground-state products formed were refractory to enzymatic hydrolysis due to the slight structural modification. Moreover, the discovery of several ENGase-based glycosynthases, including EndoM-N175A; EndoA-N171A, and EndoA-E173Q that could promote transglycosylation with sugar oxazolines of natural N-glycans but lack the ability to hydrolyze the product, has enabled the synthesis of homogeneous glycoproteins carrying full-size natural N-glycans1214. Subsequent studies indicate that Endo-A and Endo-M could accommodate diverse structures in the aglycon portions of GlcNAc- or Glc-tagged acceptors for transglycosylation, permitting the introduction of N-glycans into a wide range of natural products, unnatural peptides, and even polysaccharides15,16. The relaxed substrate specificity of Endo-A and Endo-M, together with the powerful transglycosylation potential of the glycosynthase mutants, prompted us to examine the possibility to glue multiple N-glycans to the complex-type GlcNAc2Man3GlcNAc2-Asn core through tandem enzymatic transglycosylation. We report in this paper the chemoenzymatic synthesis of a class of novel N-glycan clusters containing multiple N-glycan cores, in which all monosaccharide.