Chemoenzymatic Synthesis of Hydrophobic Glycoprotein: Synthesis of Saposin C Carrying Complex-Type Carbohydrate

• Published in: Journal of organic chemistry 2012-11, Vol.77 (21), p.9437-9446
• Main Authors: Hojo, Hironobu, Tanaka, Hiromasa, Hagiwara, Masashi, Asahina, Yuya, Ueki, Akiharu, Katayama, Hidekazu, Nakahara, Yuko, Yoneshige, Azusa, Matsuda, Junko, Ito, Yukishige, Nakahara, Yoshiaki
• Format: Article
• Language: English
• Subjects: Acetylglucosaminidase - chemistry; Acetylglucosaminidase - metabolism; Bioconversions. Hemisynthesis; Biological and medical sciences; Biotechnology; Carbohydrates - chemistry; Carbohydrates with 4, 5, 6, ... C atoms, dissacharides and oligosaccharides; Carbohydrates. Nucleosides and nucleotides; Carrier Proteins; Chemistry; Exact sciences and technology; Fundamental and applied biological sciences. Psychology; Glycopeptides - chemistry; Glycopeptides - metabolism; Glycoproteins - chemical synthesis; Glycoproteins - chemistry; Glycoproteins - metabolism; Glycosylation; Hydrophobic and Hydrophilic Interactions; Lipids; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase - chemistry; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase - metabolism; Methods. Procedures. Technologies; Molecular Sequence Data; Organic chemistry; Peptides; Preparations and properties; Saposins - chemical synthesis; Saposins - chemistry; Saposins - metabolism
• ISSN: 0022-3263; 1520-6904
• DOI: 10.1021/jo3010155

The complex-type N-linked octasaccharide oxazoline having LacNAc as the nonreducing end sugar was efficiently synthesized using the benzyl-protected LacNAc, mannose, and β-mannosyl GlcNAc units as key building blocks. To achieve a highly β-selective glycosylation with the LacNAc unit, the N-trichloroacetyl group was used for the protection of the amino group in the LacNAc unit. After complete assembly of these units and deprotection, the obtained free sugar was successfully derivatized into the corresponding sugar oxazoline. On the other hand, the N-acetylglucosaminylated saposin C, a hydrophobic lipid-binding protein, was chemically synthesized by the native chemical ligation reaction. On the basis of the previous results related to the synthesis of the nonglycosylated saposin C, the O-acyl isopeptide structure was introduced to the N-terminal peptide thioester carrying GlcNAc to improve its solubility toward aqueous organic solvents. The ligation reaction efficiently proceeded with the simultaneous O- to N-acyl shift at the O-acyl isopeptide moiety. After the removal of the cysteine-protecting group and folding, saposin C carrying GlcNAc was successfully obtained. The synthetic sugar oxazoline was then transferred to this glycoprotein using the mutant of endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) (glycosynthase), and the saposin C carrying the complex-type nonasaccharide was successfully obtained.