The biological recognition of complex‐type N‐glycans is part of many key physiological and pathological events. Despite their importance, the structural characterization of these events remains unsolved. The inherent flexibility of N‐glycans hampers crystallization and the chemical equivalence of individual branches precludes their NMR characterization. By using a chemoenzymatically synthesized tetra‐antennary N‐glycan conjugated to a lanthanide binding tag, the NMR signals under paramagnetic conditions discriminated all four N‐acetyl lactosamine antennae with unprecedented resolution. The NMR data revealed the conformation of the N‐glycan and permitted for the first time the direct identification of individual branches involved in the recognition by two N‐acetyllactosamine‐binding lectins, Datura stramonium seed lectin (DSL) and Ricinus Communis agglutinin (RCA120).

The occurrence of N-glycans with a bisecting GlcNAc modification on glycoproteins has many implications in developmental and immune biology. However, these particular N-glycans are difficult to obtain either from nature or through synthesis. We have developed a flexible and general method for synthesizing bisected N-glycans of the complex type by employing modular TFAc-protected donors for all antennae. The TFAc-protected N-glycans are suitable for the late introduction of a bisecting GlcNAc. This integrated strategy permits for the first time the use of a single approach for multiantennary N-glycans as well as their bisected derivatives via imidates, with unprecedented yields even in a one-pot double glycosylation. With this new method, rare N-glycans of the bisected type can be obtained readily, thereby providing defined tools to decipher the biological roles of bisecting GlcNAc modifications.

In human serum immunoglobulin G (IgG), a rare modification of biantennary complex N-glycans lead to a β1,4-galactosylated bisecting GlcNAc branch. We found that the bisecting GlcNAc on a biantennary core-fucosylated N-glycan was enzymatically galactosylated under stringent reaction conditions. Further optimizations led to an efficient enzymatic approach to this particular modification for biantennary substrates. Notably, triand tetra-antennary complex N-glycans were not converted by bovine galactosyltransferase. An N-glycan with a galactosylated bisecting GlcNAc was linked to a lanthanide binding tag. The pseudo-contact shifts (PCS) obtained from the corresponding Dy-complex were used to calculate the conformational preferences of the rare N-glycan. Besides two extended conformations only a single folded conformation was found.

The occurrence of α1,6-linked core fucose on the N-glycans of mammalian glycoproteins is involved in tumor progression and reduces the bioactivity of antibodies in antibody-dependent cell-mediated cytotoxicity (ADCC). Since core-fucosylated N-glycans are difficult to isolate from natural sources, only chemical or enzymatic synthesis can provide the desired compounds for biological studies. A general drawback of chemical α-fucosylation is that the chemical assembly of α1,6-linked fucosides is not stereospecific. A robust and general method for the α-selective fucosylation of acceptors with primary hydroxy groups in α/β ratios exceeding 99:1 was developed. The high selectivities result from the interplay of an optimized protecting group pattern of the fucosyl donors in combination with the activation principle and the reaction conditions. Selective deprotection yielded versatile azides of all mammalian complex-type core-fucosylated N-glycans with 2-4 antennae and optional bisecting GlcNAc.

The biological recognition of complex-type Nglycans is part of many key physiological and pathological events.Despite their importance,the structural characterization of these events remains unsolved. The inherent flexibility of Nglycans hampers crystallization and the chemical equivalence of individual branches precludes their NMR characterization. By using achemoenzymatically synthesized tetra-antennary Nglycan conjugated to al anthanide binding tag,t he NMR signals under paramagnetic conditions discriminated all four N-acetyl lactosamine antennae with unprecedented resolution. The NMR data revealed the conformation of the N-glycan and permitted for the first time the direct identification of individual branches involved in the recognition by two N-acetyllactosamine-binding lectins,D atura stramonium seed lectin (DSL) and Ricinus Communis agglutinin (RCA120).N-glycans are ubiquitous in nature and functionalize glycoproteins.[1] Protein glycosylation is required for proper biological and biophysical function and often, alterations in glycosylation are related to diseases. [2] Complex glycosylation patterns containing multi-antennary N-glycans are typically found in mature glycoproteins. However,t he structural characterization of these glycans is rather challenging.U sually,N MR spectroscopy and X-ray diffraction techniques fail to provide specific answers on the structure and molecular recognition features owing to the intrinsic attributes of the glycan. Thep roperties of the glycosidic bond and especially the presence of 1-6 linkages endow al arge flexibility to the molecule.T his feature precludes crystallization or hampers the detection of enough electron density for most of the glycan part in the X-ray analysis of glycoproteins.Moreover,the standard use of the corresponding fitting programs to deduce three-dimensional structures frequently give rise to incorrect structures of the glycans.[3] Thus,a ny advance in this area is of high value. As ap romising approach, carbohydrates conjugated to lanthanide-binding tags have shown high potential toward this aim. [4,5] In its vicinity,acomplexed paramagnetic ion induces significant chemical shift changes of the NMR signals of the glycan as ar esult of dipolar interactions involving the unpaired electron of the metal. These pseudocontact shifts (PCS) depend on the distance between each proton and the metal (proportional to 1/r 3 ).[6] This methodology has first been applied to the study of small oligosaccharides (di-, tri-, and tetrasaccharides), [7][8][9][10][11] then to N-glycans.T he conformational properties of complex-type bi-antennary and high-mannosetype N-glycans were elucidated and could be resolved in each case down to the level of individual branches. [4,5] Proceeding from this experimental basis,weherein extend this concept to the level of high-degree branching,and show that it is possible to experimentally characterize the conformational behavior and recognition properties of ag alactosylated complex-type