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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.

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.