O6-3-[125I]iodobenzyl-2′-deoxyguanosine ([125I]IBdG): synthesis and evaluation of its usefulness as an agent for quantification of alkylguanine-DNA alkyltransferase (AGT) Original Research Article

The development of O6-(3-[125I]iodobenzyl)-2′-deoxyguanosine ([125I]IBdG), the glycosylated analogue of the O6-3-iodobenzylguanine (IBG), as an agent for the in vivo mapping of the DNA repair protein alkylguanine-DNA alkyltransferase (AGT) is described. Synthesis of its tin precursor, O6-3-trimethylstannylbenzyl-2′-deoxyguanosine (TBdG) was achieved in four steps from deoxyguanosine. Radioiodination of TBdG in a single step gave [125I]IBdG in 70–85% isolated radiochemical yield. [125I]IBdG bound specifically to pure AGT with an IC50 of 7.1 μM. From paired-label assays, [125I]IBdG showed a 2- to 3-fold higher cellular uptake than [131I]IBG in DAOY medulloblastoma, TE-671 rhabdomyosarcoma, SK-Mel-28 melanoma, and HT-29 colon carcinoma human cell lines. Uptake of both labeled compounds in these cell lines decreased with increasing concentrations of unlabeled O6-benzylguanine (BG) when BG was present in the medium during incubation with the labeled compounds. Compared to BG, unlabeled IBdG diminished the uptake of [125I]IBdG and [131I]IBG in DAOY cells more efficiently (IC50 < 1 μM vs >10 μM for BG). There was no significant change in cell-bound activity of [125I]IBdG and [131I]IBG when BG was removed from the incubation medium before incubating cells with the tracers, suggesting that only a very small portion of radioactivity taken up by the cells is AGT bound. This was corroborated by gel-electrophoresis performed on extracts from cells treated with varying amounts of BG and then incubated with [125I]IBdG in the presence of BG. No radiolabeled AGT band was discernable by phosphor-imaging, signifying low cellular AGT binding of the radiotracer. In contrast, when cell extracts were prepared from BG pre-treated cells and aliquots were incubated with [125I]IBdG subsequently, the intensity of radiolabeled AGT band decreased linearly as a function of BG concentration. This suggests that the low level of [125I]IBdG that binds to AGT does so in a concentration dependent manner. These data suggest that IBdG is transported across the cell membrane to a higher degree than IBG. However, to be a practical tracer for quantifying cellular AGT, considerable localization of such derivatives need to occur within the cell nucleus where AGT is present predominantly.