Synthesis of a phosphoramidite coupling unit of the pyrimidine (6-4) pyrimidone photoproduct and its incorporation into oligodeoxynucleotides
Synthesis of a phosphoramidite coupling unit of the pyrimidine (6-4) pyrimidone photoproduct and its incorporation into oligodeoxynucleotides
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DOI：
10.1021/ja9603158
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发表时间：
1996-08-14
影响因子：
15
通讯作者：
Ohtsuka, E
中科院分区：
文献类型：
--
作者：
Iwai, S;Shimizu, M;Ohtsuka, E
紫外线(UV)会导致DNA损伤，从而诱导突变、细胞转化和细胞死亡。在相邻的嘧啶位置，形成两种主要的光产物，即顺式环丁烷嘧啶二聚体和嘧啶(6-4)嘧啶酮光产物。1诱导3‘胸腺嘧啶向胞嘧啶或胞嘧啶向胸腺嘧啶转变的(6-4)光产物2的致突变性比顺式二聚体强得多。3(6-4)光产物在DNA修复研究中也很重要，因为有人认为这种损伤是通过核苷酸切除修复途径在人类细胞中移除的。4如前所述，在这些研究中，在单个位置含有受损碱基的寡核苷酸非常有用。5 Smith和Taylor报道了一种在单个位置含有(6-4)光产物的寡核苷酸的制备，其中一个六聚体用紫外线照射，然后用高效液相色谱法进行纯化。6虽然该六聚体通过连接被延长到49聚体，用于蛋白质结合7和酶反应的分析，8该程序在链长、序列和产量上受到限制。解决这个问题的一种方法是使用二核苷酸构建块，就像为顺式胸腺嘧啶二聚体开发的那样。9在本通信中，我们合成了胸苷(3‘-5’)胸腺嘧啶(6-4)光产物的亚磷酰胺偶联单元，并将其掺入8-聚体和30-聚寡聚脱氧核苷酸中，设计了(6-4)光产物(6)的亚胺偶联单元，以便在DNA合成器上得到普遍应用。4，4‘-二甲氧基三苯基和2-氰乙基分别用于保护5’-羟基和核苷磷酸。最初，计划保护由光产物形成产生的羟基，但发现该羟基的反应性极低，如下所述。选择了在顺式胸腺嘧啶二聚体偶联单元的制备中成功应用的乙酰丙基10作为3‘-羟基的瞬时保护基团。
Ultraviolet (UV) light causes lesions in DNA, which induce mutations, cellular transformation, and cell death. At adjacent pyrimidine sites, two major types of photoproducts, namely the cis-syn cyclobutane pyrimidine dimer and the pyrimidine (6-4) pyrimidone photoproduct, are formed. 1 It appears that the (6-4) photoproduct, which induces 3′ thymine-to-cytosine or cytosine-to-thymine transitions, 2 is much more mutagenic than the cis-syn dimer. 3 The (6-4) photoproduct is also important in studies of DNA repair, because it has been suggested that this lesion is removed in human cells via the nucleotide excision repair pathway. 4 For these studies, oligonucleotides containing the damaged base at a single site are of great use, as demonstrated previously. 5 Smith and Taylor reported the preparation of an oligonucleotide containing the (6-4) photoproduct at a single site, in which a hexamer was irradiated with UV and then purified by HPLC. 6 Although this hexamer was elongated by ligation to a 49-mer, which was used for analyses of protein binding7 and enzyme reactions, 8 this procedure suffers from limitations in chain length, sequence, and yield. One way to solve this problem is to use a dinucleotide building block, as developed for the cis-syn thymine dimer. 9 In this communication, we describe the synthesis of a phosphoramidite coupling unit of the (6-4) photoproduct of thymidylyl (3′-5′) thymidine and its incorporation into 8-mer and 30-mer oligodeoxynucleotides.The phosphoramidite coupling unit of the (6-4) photoproduct (6) was designed in order that it would be used generally on DNA synthesizers. The 4, 4′-dimethoxytrityl and 2-cyanoethyl groups were used for the protection of the 5′-hydroxyl group and the internucleoside phosphate, respectively. At the beginning, protection of the hydroxyl group generated by photoproduct formation was planned, but it was found that the reactivity of this hydroxyl was extremely low, as described below. A levulinyl group, which was used successfully in the preparation of the coupling unit of the cis-syn thymine dimer, 10 was chosen for the transient protection of the 3′-hydroxyl group.