Efficient synthesis of glycopeptide-α-thioesters with a high-mannose type oligosaccharide by means of tert-Boc-solid phase peptide synthesis

High-mannose type oligosaccharides consist of nine mannose and two N-acetylglucosamine residues (Man9GlcNAc2:M9) and play an important role in protein folding processes in the endoplasmic reticulum. A highly efficient preparation method of this asparaginyl-M9-oligosaccharide from hen egg yolk was established by a two-step proteolysis with commercially available proteases and subsequent purification using high performance liquid chromatography (HPLC). To avoid the hydrolysis of the desired M9-oligosaccharide during the proteolysis steps, several commercially available proteases were screened for their contamination with mannosidases. The α-amino group of the resultant H2N-Asn-(M9-oligosaccharide)-OH was protected with 9-fluorenylmethyloxycarbonyl (Fmoc) group for convenient separation by HPLC. The structure of Fmoc-Asn-(M9-oligosaccharide)-OH thus obtained was confirmed by ESI-MS spectrometry and several NMR experiments. Using this Fmoc-Asn-(M9-oligosaccharide)-OH, the synthesis of the M9-glycopeptide-α-thioester was demonstrated by means of tert-Boc-solid phase peptide synthesis. These tert-Boc conditions afforded the M9-glycopeptide-α-thioester in moderate yield.

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► Fmoc-Asn-(Man9GlcNAc2:M9)-OH was prepared from hen egg yolk.
► Isolated yield of Fmoc-Asn-(Man9GlcNAc2:M9)-OH was optimized by use of several proteases.
► The structure of Fmoc-Asn-(Man9GlcNAc2:M9)-OH isolated was analyzed by NMR.
► Boc solid phase synthesis of M9-glycopeptide-α-thioester was demonstrated.