Dougan H, Hobbs J B, Weitz J I, Lyster D M
TRIUMF, 4004 Wesbrook Mall, Vancouver, BC V6T 2A3, Canada.
Nucleic Acids Res. 1997 Jul 15;25(14):2897-901. doi: 10.1093/nar/25.14.2897.
Synthesis and radioiodination of a stannyl oligodeoxyribonucleotide were undertaken to evaluate a gamma ray emitting ODN ligand for thrombus imaging in vivo . Synthesis of the ODN was based on modified automatedbeta-cyanoethyl phosphoramidite chemistry with an organotin nucleoside (dU*) coupled to a thrombin binding aptamer sequence to give d(UGGTTGGTGTGGTTGG). The synthesis accommodated dU, which is destannylated by iodine or acids. Fourteen standard synthesis cycles were followed by one 'stannyl synthesis cycle', distinguished by Fmoc protection, omission of capping, oxidation by an organic peroxide and cleavage by ammonium hydroxide. The organotin nucleoside phosphoramidite {5'-[fluorenylmethoxycarbonyl]-5-(E)-[2-tri-n -butylstannylvinyl]-2'-deoxyuridine-3'-(2-cyanoethyl N,N-diisopropyl phosphoramidite)} was prepared from 5-iodo-2'-deoxyuridine. A customized mild rapid workup included deprotection with methylamine, and reverse phase HPLC with CH3CN/triethylammonium bicarbonate. Pure stannyl ODN was highly retained by reverse phase HPLC. Radioiodination of stannyl ODN (100 microg) provided 123I-labeling yields up to 97%. Five alternative oxidants were effective. High specific activity [123I]- ODN (15 000 Ci/mmol) was recovered, separated from unlabeled isomers. Excellent reverse phase HPLC resolution of ODN isomers (alternatively I, Cl, H or Br in vinyl deoxyuridine) was essential. The affinity of the iodovinyl aptamer analog (Kd = 36 nM) for human alpha-thrombin was similar to the native aptamer (Kd = 45 nM).
进行了一种甲锡烷基寡脱氧核糖核苷酸的合成和放射性碘化，以评估一种用于体内血栓成像的发射γ射线的寡脱氧核糖核苷酸配体。寡脱氧核糖核苷酸的合成基于改良的自动化β-氰基乙基亚磷酰胺化学方法，将一种有机锡核苷（dU*）与凝血酶结合适体序列偶联，得到d（UGGTTGGTGTGGTTGG）。该合成方法适用于dU，它会被碘或酸脱去锡。在进行14个标准合成循环后进行一个“甲锡烷基合成循环”，其区别在于采用芴甲氧羰基（Fmoc）保护、不进行封端、用有机过氧化物氧化以及用氢氧化铵裂解。有机锡核苷亚磷酰胺{5'-[芴甲氧羰基]-5-(E)-[2-三正丁基锡乙烯基]-2'-脱氧尿苷-3'-(2-氰基乙基N,N-二异丙基亚磷酰胺)}由5-碘-2'-脱氧尿苷制备。一种定制的温和快速后处理方法包括用甲胺脱保护，以及用乙腈/碳酸氢三乙铵进行反相高效液相色谱分析。纯甲锡烷基寡脱氧核糖核苷酸通过反相高效液相色谱法能很好地保留。甲锡烷基寡脱氧核糖核苷酸（100微克）的放射性碘化提供了高达97%的123I标记产率。五种替代氧化剂均有效。回收了高比活度的[123I]-寡脱氧核糖核苷酸（15000居里/毫摩尔），并与未标记的异构体分离。寡脱氧核糖核苷酸异构体（乙烯基脱氧尿苷中分别为I、Cl、H或Br）的出色反相高效液相色谱分辨率至关重要。碘乙烯基适体类似物对人α-凝血酶的亲和力（Kd = 36纳摩尔）与天然适体（Kd = 45纳摩尔）相似。
