title: Synthesis and evaluation of a radioiodinated trisaccharide derivative as a synthetic substrate for a sensitive N-acetylglucosaminyltransferase V radioassay.
abstract: 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, [(125)I]IPGMG ([(125)I]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 [(125)I]1. The iododestannylation of 21 using hydrogen peroxide as an oxidant followed by deacetylation yielded [(125)I]1. When [(125)I]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 K(m) of 23.7 μM and a V(max) of 159 pmol/h. μg protein. [(125)I]1 would therefore be a useful synthetic substrate for the quantitative determination of GnT-V activity.
authors.affiliation: Department of Biophysical Chemistry, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Mukai
authors.forename: Masayori
authors.initials: M
authors.lastname: Hagimori
authors.forename: Kenji
authors.initials: K
authors.lastname: Arimitsu
authors.forename: Takahiro
authors.initials: T
authors.lastname: Katoh
authors.forename: Misa
authors.initials: M
authors.lastname: Ukon
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Kajimoto
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kimura
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Magata
authors.forename: Eiji
authors.initials: E
authors.lastname: Miyoshi
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Taniguchi
authors.forename: Manabu
authors.initials: M
authors.lastname: Node
authors.forename: Hideo
authors.initials: H
authors.lastname: Saji
mesh_terms: D000818:Animals; D055162:Biocatalysis; D002236:Carbohydrate Conformation; D002851:Chromatography, High Pressure Liquid; D006031:Glycosylation; D008297:Male; D017351:N-Acetylglucosaminyltransferases / Q000032:analysis* / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D017208:Rats, Wistar; D013237:Stereoisomerism; D014312:Trisaccharides / Q000138:chemical synthesis* / Q000737:chemistry*
chemical_list: D014312:Trisaccharides; D017351:N-Acetylglucosaminyltransferases; C052254:alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase