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.

Die Gegenwart von N-Glycanen mit einer bisecting GlcNAc-Modifikation auf Glycoproteinen hat viele Auswirkungen in der Entwicklungs- und Immunbiologie. Allerdings ist es schwierig diese N-Glycane aus der Natur oder durch Synthese zu erhalten. Für bisected N-Glycane des komplexen Typs haben wir eine flexible und allgemeine Synthesemethode entwickelt, die auf modularen TFAc-geschützten Donoren für alle Antennen basiert. Die TFAc-geschützten N-Glycane sind besonders geeignet für die späte Einführung eines bisecting GlcNAc-Rests. Damit können erstmals gleichzeitig multiantennäre N-Glycane und ihre bisected Derivate durch einen einzigen Syntheseweg erhalten werden. Dies gelingt sequentiell mit Imidatdonoren in beispiellosen Ausbeuten und sogar durch eine Eintopf-Doppelglycosylierung. Mit der neuen Methode können die seltenen bisected N-Glycane einfach erhalten werden und damit zur Aufklärung der biologischen Rollen von bisecting GlcNAc-Modifikationen bereitgestellt werden.

Human interleukin 6 (IL-6) is a potent cytokine with immunomodulatory properties. As the influence of N-glycosylation on the in vivo activities of IL-6 could not be elucidated so far, a semisynthesis of homogeneous glycoforms of IL-6 was established by sequential native chemical ligation. The four cysteines of IL-6 are convenient for ligations and require only the short synthetic glycopeptide 43–48. The Cys-peptide 49–183 could be obtained recombinantly by cleavage of a SUMO tag. The fragment 1–42 was accessible by the simultaneous cleavage of two inteins, leading to the 1–42 thioester with the native N-terminus. Ligation and refolding studies showed that the inherently labile AspPro bond 139–140 was detrimental for the sequential C- to N-terminal ligation. A reversed ligation sequence using glycopeptide hydrazides gave full-length IL-6 glycoproteins, which showed full bioactivity after efficient refolding and purification.

Human interleukin 6 (IL-6) is a potent cytokine with immunomodulatory properties. As the influence of N-glycosylation on the in vivo activities of IL-6 could not be elucidated so far, a semisynthesis of homogeneous glycoforms of IL-6 was established by sequential native chemical ligation. The four cysteines of IL-6 are convenient for ligations and require only the short synthetic glycopeptide 43–48. The Cys-peptide 49–183 could be obtained recombinantly by cleavage of a SUMO tag. The fragment 1–42 was accessible by the simultaneous cleavage of two inteins, leading to the 1–42 thioester with the native N-terminus. Ligation and refolding studies showed that the inherently labile AspPro bond 139–140 was detrimental for the sequential C- to N-terminal ligation. A reversed ligation sequence using glycopeptide hydrazides gave full-length IL-6 glycoproteins, which showed full bioactivity after efficient refolding and purification.

A modular synthesis of complex-type N-glycans containing the core fucosyl motif was optimized. The core trisaccharide building block was protected by a methoxyphenyl group for convenient core fucosylation. The trisaccharide was obtained on a large scale from the glycosylation of the corresponding chitobiosyl azide with a glucosyl donor followed by intramolecular inversion. Improved methods were established for the synthesis of the monosaccharide building blocks and for their couplings. The inversion to the β-mannoside was accompanied by previously unnoticed side-reactions resulting in the hydrolytic ring-opening of the iminocarbonate intermediate. The benzylidene-protected core trisaccharide was elongated into a biantennary N-glycan heptasaccharide by two regio- and stereoselective couplings. The final fucosylation also gave some of the β anomer, which could be removed by HPLC to give an α1,6-fucosylated N-glycan octasaccharide.

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 (β-mannosyl chitobiose) conveniently protected with a single benzylidene moiety at the β-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 building block approach for biantennary N-linked oligosaccharides from glycoproteins (N-glycans) has been developed. Starting from a core trisaccharide (β-mannosyl chitobiose) containing a benzylidene-protected β-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 GlcNPhtβ1,2Man donor was developed. The benzylidene acetal moiety, as a minimal protection of the β-mannoside, allows selective α-glycosylation at OH-3 of the 2,3-diol with GlcNβ1,2Man 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.