Enzymatic synthesis of the core-2 sialyl Lewis X O-glycan on the tumor-associated MUC1a’ peptide

• Published in: Biochimie 2003-03, Vol.85 (3), p.275-286
• Main Authors: Gutiérrez Gallego, Ricardo, Dudziak, Gregor, Kragl, Udo, Wandrey, Christian, Kamerling, Johannis P, Vliegenthart, Johannes F.G
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Carbohydrate Sequence; Core-2 sialyl Lewis X; Enzymatic synthesis; Humans; Models, Molecular; Molecular Sequence Data; MUC1; Mucin-1 - biosynthesis; Mucin-1 - chemistry; Mucin-1 - genetics; Nuclear Magnetic Resonance, Biomolecular; O-glycopeptide; Oligosaccharides - biosynthesis; Oligosaccharides - chemistry; Oligosaccharides - genetics; Polysaccharides - biosynthesis; Polysaccharides - chemistry; Polysaccharides - genetics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/S0300-9084(03)00050-6

Starting from a tumor-associated synthetic MUC1-derived peptide MUC1a’ and using a completely enzymatic approach for the synthesis of the core-2 sialyl Lewis X glycopart, the following glycopeptide was synthesized: AHGV{Neu5Ac(α2-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)[Gal(β1-3)]GalNAc(α1-O)}TSAPDTR. First, polypeptide N-acetylgalactosaminyltransferase 3 was used to site-specifically glycosylate MUC1a’ to give MUC1a’-GalNAc. Then, in a one-pot reaction employing β-galactosidase and core-2 β6- N-acetylglucosaminyltransferase the core-2 O-glycan structure was prepared. The core-2 structure was then sequentially galactosylated, sialylated, and fucosylated by making use of β4-galactosyltransferase 1, α3-sialyltransferase 3, and α3-fucosyltransferase 3, respectively, resulting in the sialyl Lewis X glycopeptide. The overall yield of the final compound was 23% (3.2 mg, 1.4 μmol). During the synthesis three intermediate glycopeptides containing O-linked GalNAc, Gal(β1-4)GlcNAc(β1-6)[Gal(β1-3)]GalNAc, and Neu5Ac(α2-3)Gal(β1-4)GlcNAc(β1-6)[Gal(β1-3)]GalNAc, respectively, were isolated in mg quantities. All products were characterized by mass spectrometry and NMR spectroscopy.