Synthesis and evaluation of a radioiodinated trisaccharide derivative as a synthetic substrate for a sensitive N-acetylglucosaminyltransferase V radioassay

• Published in: Bioorganic & medicinal chemistry 2011-07, Vol.19 (14), p.4312-4321
• Main Authors: Mukai, Takahiro, Hagimori, Masayori, Arimitsu, Kenji, Katoh, Takahiro, Ukon, Misa, Kajimoto, Tetsuya, Kimura, Hiroyuki, Magata, Yasuhiro, Miyoshi, Eiji, Taniguchi, Naoyuki, Node, Manabu, Saji, Hideo
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biocatalysis; Biological and medical sciences; Carbohydrate Conformation; chemistry; Chromatography, High Pressure Liquid; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Glycosylation; glycosyltransferases; hydrogen peroxide; Male; metastasis; N-acetylglucosaminyltransferase V; N-Acetylglucosaminyltransferases - analysis; N-Acetylglucosaminyltransferases - metabolism; oxidants; quantitative analysis; Radioassay; Radiolabeled substrate; radiolabeling; Radioligand Assay; Rats; Rats, Wistar; Stereoisomerism; Transferases; tributyltin; Trisaccharides - chemical synthesis; Trisaccharides - chemistry
• ISSN: 0968-0896; 1464-3391
• DOI: 10.1016/j.bmc.2011.05.047

N-acetylglucosaminyltransferase V (GnT-V) is one of the most relevant glycosyltransferases to tumor invasion and metastasis. Based on previous findings of molecular recognition between GnT-V and synthetic substrates, we designed and synthesized a p-iodophenyl-derivatized trisaccharide, 2-(4-iodophenyl)ethyl 6-O-[2-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-α-d-mannopyranosyl]-β-d-glucopyranoside (IPGMG, 1) and its radiolabeled form, [125I]IPGMG ([125I]1), for use in assays of GnT-V activity in vitro. The tributyltin derivative, 2-[4-(n-tributylstannyl)phenyl]ethyl 6-O-[2-O-(3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-β-d-glucopyranosyl)-3,4,6-tri-O-acetyl-α-d-mannopyranosyl]-2,3,4-tri-O-acetyl-β-d-glucopyranoside (21), was synthesized as a precursor for the preparation of [125I]1. The iododestannylation of 21 using hydrogen peroxide as an oxidant followed by deacetylation yielded [125I]1. When [125I]1 was incubated in GnT-V-expressing cells with a UDP-GlcNAc donor, the production of β1–6GlcNAc-bearing IPGMG (IPGGMG, 2) was confirmed by radio-HPLC. In kinetic analysis, 1 was found to be a good substrate with a Km of 23.7μM and a Vmax of 159pmol/h. μg protein. [125I]1 would therefore be a useful synthetic substrate for the quantitative determination of GnT-V activity.