title: Efficient synthesis of glycopeptide-α-thioesters with a high-mannose type oligosaccharide by means of tert-Boc-solid phase peptide synthesis.
abstract: High-mannose type oligosaccharides consist of nine mannose and two N-acetylglucosamine residues (Man(9)GlcNAc(2):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 H(2)N-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.
authors.affiliation: Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Makimura
authors.forename: Tatsuto
authors.initials: T
authors.lastname: Kiuchi
authors.forename: Masayuki
authors.initials: M
authors.lastname: Izumi
authors.forename: Simone
authors.initials: S
authors.lastname: Dedola
authors.forename: Yukishige
authors.initials: Y
authors.lastname: Ito
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kajihara
mesh_terms: D000818:Animals; D001216:Asparagine / Q000737:chemistry; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D004530:Egg Yolk / Q000737:chemistry; D004789:Enzyme Activation; D004952:Esters / Q000737:chemistry / Q000302:isolation & purification; D005449:Fluorenes / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification*; D009682:Magnetic Resonance Spectroscopy; D008351:Mannans / Q000737:chemistry / Q000302:isolation & purification; D008358:Mannose / Q000737:chemistry*; D008361:Mannosidases / Q000737:chemistry; D015394:Molecular Structure; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification; D010447:Peptide Hydrolases / Q000737:chemistry; D017510:Protein Folding; D059748:Proteolysis; D060327:Solid-Phase Synthesis Techniques / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: C496789:9-fluorenylmethoxycarbonyl; D004952:Esters; D005449:Fluorenes; D006023:Glycoproteins; D008351:Mannans; D009844:Oligosaccharides; C475461:mannosyl(9)-N-acetylglucosamine2; D001216:Asparagine; D008361:Mannosidases; D010447:Peptide Hydrolases; D008358:Mannose