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Seven in one blow: The efficient formation of mixed disulfides on the thiol-rich fusion protein A followed by subsequent intein cleavage gave the fragment B with all seven cysteines protected against oxidation. The native chemical ligation of B with synthetic glycopeptide thioesters provides glycoproteins.

Aktives RNase-Glycoprotein aus drei Segmenten: Das Glycoprotein-Enzym Ribonuclease C mit einem komplexen nonasaccharidischen N-Glycan wurde durch sequenzielle native chemische Ligation synthetisiert. Durch optimierte Ligations- und Isolierungsbedingungen konnte das aus 124 Aminosauren bestehende Enzym effizient aufgebaut und zuruckgefaltet werden.

Sieben auf einen Streich: Durch die effiziente Bildung von gemischten Disulfiden an dem thiolreichen Fusionsprotein A konnte nach Inteinabspaltung das Cys-Fragment B mit sieben oxidationsgeschutzten Cysteinen erhalten werden. Die native chemische Ligation von B mit synthetischen Glycopeptidthioestern ergibt Glycoproteine.

Protein-carbohydrate interactions are at the heart of a variety of essential molecular recognition events. Hevein, a model lectin related to the superantigen family, recognizes the trisaccharide core of N-glycoproteins (1). A combined approach of NMR spectroscopy and molecular modeling has permitted us to demonstrate that an Asn-linked Man(GlcNAc)(2) (2) is bound with even higher affinity than (GlcNAc)(3). The molecular recognition process entails conformational selection of only one of the possibilities existing for chitooligosaccharides. The deduced 3D structure of the hevein/2 complex permits the extension of polypeptide chains from the Asn moiety of 2, as well as glycosylation at Man O-3 and Man O-6 of the terminal sugar. Given the ubiquity of the Man(GlcNAc)(2) core in all mammalian N-glycoproteins, the basic recognition mode presented herein might be extended to a variety of systems with biomedical importance.

The Fmoc-protected heptasaccharide asparagine building block beta-GlcNAc-(1-->2)-alpha-Man-(1-->3)-[beta-GlcNAc-(1-->2)-alpha-Man-(1-->6)]beta-Man-(1-->4)-beta-GlcNAc-(1-->4)-beta-GlcNAc-(Fmoc)Asn was obtained by chemical synthesis. Two flexible strategies were developed with optimized conditions for the simultaneous debenzylation of the sugar and the amino acid part. The heptasaccharide asparagine building block is a partial structure of many glycoproteins and can be used for glycopeptide synthesis in solution and on the solid phase. In this work the heptasaccharide asparagine was elongated in solution to an Fmoc-glycopentapeptide methylester. After chemical cleavage of the Fmoc group the methylester was removed enzymatically by chymotrypsin. The use of beta-(1-->4)-galactosyltransferase and alpha-(2-->6)-sialyltransferase in the presence of alkaline phosphatase allowed the efficient transfer of four sugar units to the acceptor resulting in an undecasaccharide glycopentapeptide.

Active RNase glycoprotein from three pieces: The glycoprotein enzyme ribonuclease C, which contains a complex saccharide N-glycan, was synthesized by sequential native chemical ligation. An optimized ligation and isolation protocol allowed the efficient assembly and refolding of the 124 amino acid enzyme.

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.

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.

The achiral, nitroxyl-containing alpha-amino acid TOAC (TOAC = 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid), in combination with the chiral alpha-amino acid C(alpha)-methyl valine [(alphaMe)Val], was used to prepare short peptides (from di- to hexa-) that induced the enantioselective oxidation of racemic 1-phenylethanol to acetophenone. The best catalyst was an N(alpha)-acylated dipeptide alkylamide with the -TOAC-(alphaMe)Val- sequence folded in a stable, intramolecularly hydrogen-bonded beta-turn conformation with large, lipophilic (hydrophobic) N- and C-terminal blocking groups. We rationalized our findings by proposing models for the diastereomeric intermediates between (R)-[and (S)]-1-phenylethanol and the catalyst Fmoc-TOAC-L-(alphaMe)Val-NHiPr, based on the X-ray diffraction structure of the latter.

We have synthesized by solution methods and fully characterized two sets of terminally protected peptides based on the tricyclic Cα,α-disubstituted glycine Afc. The conformational preferences of the Afc/Gly peptides were examined by FT-IR absorption and1H NMR techniques, while those of the Afc/TOAC peptides were primarily investigated by using fluorescence spectroscopy. The X-ray diffraction structure of an Afc derivative was also analyzed. The body of solution and crystal-state experimental data conclusively confirms previous findings that the Afc residue may either adopt the fully extended (C5) or a turn conformation.