Chemoenzymatic synthesis of hydrophobic glycoprotein: synthesis of saposin C carrying complex-type carbohydrate.
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.
Department of Applied Biochemistry, Tokai University, 4-1-1 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan. hojo@keyaki.cc.u-tokai.ac.jp
Hironobu
H
Hojo
Hiromasa
H
Tanaka
Masashi
M
Hagiwara
Yuya
Y
Asahina
Akiharu
A
Ueki
Hidekazu
H
Katayama
Yuko
Y
Nakahara
Azusa
A
Yoneshige
Junko
J
Matsuda
Yukishige
Y
Ito
Yoshiaki
Y
Nakahara
D000118:Acetylglucosaminidase / Q000737:chemistry* / Q000378:metabolism*; D002241:Carbohydrates / Q000737:chemistry*; D002352:Carrier Proteins; D006020:Glycopeptides / Q000737:chemistry* / Q000378:metabolism; D006023:Glycoproteins / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D057927:Hydrophobic and Hydrophilic Interactions; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D049231:Saposins / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism
D002241:Carbohydrates; D002352:Carrier Proteins; D006020:Glycopeptides; D006023:Glycoproteins; D049231:Saposins; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase