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.