α,β-Methylene-2′-deoxynucleoside 5′-Triphosphates as Noncleavable Substrates for DNA Polymerases: Isolation, Characterization, and Stability Studies of Novel 2′-Deoxycyclonucleosides, 3,5′-Cyclo-dG, and 2,5′-Cyclo-dT
α,β-Methylene-2′-deoxynucleoside 5′-Triphosphates as Noncleavable Substrates for DNA Polymerases: Isolation, Characterization, and Stability Studies of Novel 2′-Deoxycyclonucleosides, 3,5′-Cyclo-dG, and 2,5′-Cyclo-dT
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DOI：
10.1021/jm800692a
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发表时间：
2008-10-23
影响因子：
7.3
通讯作者：
Cho, Bongsup P.
中科院分区：
文献类型：
--
作者：
Liang, Fengting;Jain, Nidhi;Cho, Bongsup P.
我们报道了一套完整的α，β-亚甲基-2 '-dNTP（α，β-m-dNTP; N = A，C，T，G，12-15）的合成和表征，其中天然dNTP的α，β-氧键被亚甲基取代。这些核苷酸被设计为DNA聚合酶的不可裂解底物。合成需要制备2 '-脱氧核苷5'-二磷酸前体，然后进行酶促γ-磷酸化。发现所有四种合成的α，β-m-dNTP都是聚合酶β的有效抑制剂，Ki值范围为1-5 μ M。在α，β-亚甲基二磷酸的dG和dT衍生物的制备过程中，我们还分别分离出大量的3，5 ′-环-dG（16）和2，5 ′-环-dT（17）。这些新的2 '-脱氧环己糖苷通过碱催化的分子内环化（分别为N3 -> C5'和O2 -> C5'）形成。在酸性溶液中，16和17都经历糖酵解，然后完全脱嘌呤。当暴露于碱性条件时，16经历氧化脱氨基作用以产生3，5 '-cyc 1 o-2'-脱氧黄苷（19），而17仅水解为dT。
We report synthesis and characterization of a complete set of alpha,beta-methylene-2'-dNTPs (alpha,beta-m-dNTP; N = A, C, T, G, 12-15) in which the alpha,beta-oxygen linkage of natural dNTP was replaced by a methylene group. These nucleotides were designed to be noncleavable substrates for DNA polymerases. Synthesis entails preparation of 2'-deoxynucleoside 5'-diphosphate precursors, followed by an enzymatic gamma-phosphorylation. All four synthesized alpha,beta-m-dNTPs were found to be potent inhibitors of polymerase beta, with K-i values ranging 1-5 mu M. During preparation of the dG and dT derivatives of alpha,beta-methylene diphosphate, we also isolated significant amounts of 3,5'-cyclo-dG (16) and 2,5'-cyclo-dT (17), respectively. These novel 2'-deoxycyclonucleosides were formed via a base-catalyzed intramolecular cyclization (N3 -> C5' and O2 -> C5' respectively). In acidic solution, both 16 and 17 underwent glycolysis, followed by complete depurination. When exposed to alkaline conditions, 16 underwent an oxidative deamination to produce 3,5'-cyc1o-2'-deoxyxanthosine (19), whereas 17 was hydrolyzed exclusively to dT.