Chemoenzymatic synthesis of a novel glycopeptide using a microbial endoglycosidase.
The chemoenzymatic synthesis of a glycopeptide by chemical synthesis of N-acetylglucosaminyl peptide and enzymatic transfer of an oligosaccharide is described. We synthesized glycosylated Peptide T which blocks infection of human T cells by human immunodeficiency virus. The first step of the chemoenzymatic method is the solid-phase chemical synthesis of N-acetylglucosaminyl Peptide T (Ala-Ser-Thr-Thr-Thr-Asn(GlcNAc)-Tyr-Thr) with an N-acetylglucosamine moiety bound to the asparaginyl residue by a solid-phase method. This product was prepared in high yield by the dimethylphosphinothioic mixed anhydride method without protecting the hydroxyl functions of the sugar moiety using Fmoc-N-acetylglucosaminyl asparagine instead of Fmoc-asparagine. The second step was transglycosylation of complex type oligosaccharide to N-acetylglucosaminyl Peptide T by a microbial endoglycosidase. The endo-beta-N-acetylglucosaminidase of Mucor hiemalis transfer the oligosaccharide of human transferrin glycopeptide to N-acetylglucosaminyl Peptide T. The transglycosylation product was confirmed to be the glycosylated Peptide T with a sialo biantennary complex type oligosaccharide by mass spectrometry. The glycosylated Peptide T was highly stable against proteolysis in comparison to native Peptide T and N-acetylglucosaminyl Peptide T.
Yamamoto
K
K
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan. yamamotk@kais.kyoto_u.ac.jp
Fujimori
K
K
Haneda
K
K
Mizuno
M
M
Inazu
T
T
Kumagai
H
H
D000118:Acetylglucosaminidase / Q000378:metabolism; D000998:Antiviral Agents / Q000138:chemical synthesis; D002240:Carbohydrate Sequence; D006020:Glycopeptides / Q000138:chemical synthesis*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006031:Glycosylation; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009089:Mucor / Q000201:enzymology; D015717:Peptide T / Q000031:analogs & derivatives*; D011402:Pronase / Q000378:metabolism
D000998:Antiviral Agents; D006020:Glycopeptides; D015717:Peptide T; D006026:Glycoside Hydrolases; D000118:Acetylglucosaminidase; D011402:Pronase