Synthesis of multiantennary complex type N-glycans by use of modular building blocks.

A modular set of oligosaccharide building blocks was developed for the synthesis of multiantennary N-glycans of the complex type, which are commonly found on glycoproteins. The donor building blocks were laid out for the elongation of a core trisaccharide acceptor (beta-mannosyl chitobiose) conveniently protected with a single benzylidene moiety at the beta-mannoside. Through two consecutive regio- and stereoselective couplings the donors gave N-glycans with three to five antennae in high yields. Due to the consistent protection group pattern of the donors the deprotection of the final products can be performed by using a general reaction sequence.

A double regio- and stereoselective glycosylation strategy for the synthesis of N-glycans.

A building block approach for biantennary N-linked oligosaccharides from glycoproteins (N-glycans) has been developed. Starting from a core trisaccharide (beta-mannosyl chitobiose) containing a benzylidene-protected beta-mannoside, the attachment of the disaccharide building blocks for the antennae can be performed in a double regio- and stereoselective manner. A short synthesis of a GlcNPhtbeta1,2Man donor was developed. The benzylidene acetal moiety, as a minimal protection of the beta-mannoside, allows selective alpha-glycosylation at OH-3 of the 2,3-diol with GlcNbeta1,2Man trichloroacetimidate donors. Subsequent debenzylidenation leads to a 4,6-diol, which can be selectively extended at OH-6. Overreaction at OH-4 was generally low when phthalimido-protected donors were used. This general strategy represents a modular synthesis of N-glycans and their glycoconjugates.

Substitutions in the N-glycan core as regulators of biorecognition: the case of core-fucose and bisecting GlcNAc moieties.

Core fucosylation and the bisecting N-acetylglucosamine residue are prominent natural substitutions of the N-glycan core. To address the issue of whether these two substituents can modulate ligand properties of complex-type biantennary N-glycans, we performed chemoenzymatic synthesis of the respective galactosylated and alpha2,3/6-sialylated N-glycans. Neoglycoproteins were then produced to determine these glycans' reactivities with sugar receptors in solid-phase assays and with tumor cells in vitro as well as their in vivo biodistribution profiles in mice. Slight protein-type-dependent changes were noted in lectin binding, including adhesion/growth-regulatory galectins as study objects, when the data were related to properties of N-glycans without or with only one core substituent. Monitoring binding in vitro revealed cell-type-dependent changes. They delimited the ligand activity of this glycan type from that of chains with un- and monosubstituted cores. A markedly prolonged serum half-life was conferred to the neoglycoprotein by the galactose-terminated N-glycan, which together with increased organ retention of all three neoglycoproteins underscores the conspicuous relevance for glycoengineering of pharmaproteins. The predominant presentation of the two branches in the disubstituted N-glycan as extended (alpha1,3-antenna) and backfolded (alpha1,6-antenna) forms, revealed by molecular dynamics simulations, can underlie the measured characteristics. These results obtained by a combined strategy further support the concept of viewing N-glycan core substitutions as non-random additions which exert a modulatory role on ligand properties. Moreover, our data inspire us to devise new, non-natural modifications to realize the full potential of glycoengineering for diagnostic and therapeutic purposes.