Phenanthridinium as an artificial base and charge donor in DNA
Phenanthridinium as an artificial base and charge donor in DNA
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DOI：
10.1002/anie.200353153
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发表时间：
2004-01-01
影响因子：
16.6
通讯作者：
Wagenknecht, HA
中科院分区：
文献类型：
--
作者：
Amann, N;Huber, R;Wagenknecht, HA
3，8-二氨基-5-乙基-6-苯基菲啶，又称“乙锭”，已广泛应用于核酸荧光分析中。[1]乙锭及其衍生物也是有效的杀锥虫药。[2]此外，乙锭是DNA中光诱导电荷转移过程的重要供体。[3-6]相对氧化还原电位表明，处于光激发态(Et*+)的乙锭不能氧化或还原DNA而引发空穴或电子跳跃。[7]因此，必须提供合适的电荷受体。7-去氮鸟嘌呤猝灭DNA中乙锭的发射，并已被用作空穴转移研究的受体。值得注意的是，通过烷基连接物将乙锭共价连接到5‘端的DNA双链的研究发现，DNA介导的空穴氧化转移速率与距离无关[6]，尽管跳跃模型[9]不适用于这种情况。乙锭插入DNA的定点插层对于详细研究其结合作用和电荷供体性质至关重要。我们在双链DNA的特定位置加入了乙锭的菲杂环作为人工碱基。相应的乙二胺2‘-脱氧核呋喃糖苷[10]的水解性使得有必要用连接在菲杂环N-5位上的非环连接系统来取代糖的部分(方案1)。为了合成相应的DNA构建块1，我们首先用氯甲酸烯丙酯来保护3，8-二氨基-6-苯基菲啶(2)的两个外环氨基功能。然后与1，3-二碘丙烷进行烷基化反应，得到双铝保护的菲啶衍生物3。四氢呋喃是该反应的最佳溶剂，因为起始原料3溶于四氢呋喃，而烷基化产物4不溶于四氢呋喃。因此，可以简单地通过过滤来收集4。菲啶4在典型的亲核取代条件下与DMT保护的3-氨基-1，3-丙二醇(5)相连。化合物5按文献方法合成，并带有后期自动寡核苷酸偶联所需的DMT保护基团。[11]在连接5之后，分配保护基团被三氟乙酰基交换。这一过程是必要的，因为三氟乙酰基不够稳定，不能用于菲杂环在N-5[12]的烷基化反应，但在典型的DNA加工条件下可以裂解。这种保护基团策略具有另外的优点，即烷基连接物的仲氨基功能也受到保护。用标准程序完成亚磷酰胺1的制备，并将产物用于菲修饰寡核苷酸的自动化制备。更长的耦合时间(1小时而不是标准联轴器的1.5分钟)，更高的亚磷酸盐浓度(0.2m而不是0.067 m)，以及3
3, 8-Diamino-5-ethyl-6-phenylphenanthridinium, known as “ethidium”, has been widely used in fluorescence assays with nucleic acids.[1] Ethidium and its derivatives are also potent trypanocidal drugs.[2] In addition, ethidium represents an important donor for photoinduced charge transfer processes in DNA.[3–6] Relative redox potentials indicate that ethidium in the photoexcited state (Et*+) is not able to oxidize or reduce DNA to initiate hole or electron hopping, respectively.[7] Hence, a suitable charge acceptor has to be provided. 7-Deazaguanine quenches the emission of ethidium in DNA [8] and has been applied as the acceptor in holetransfer studies.[5, 6] Remarkably, investigations of DNA duplexes with ethidium covalently attached to the 5’-end through an alkyl linker found no dependence of the rate of DNA-mediated oxidative hole transfer on the distance,[6] although the hopping model [9] cannot be applied in this case. Site-specific intercalation of ethidium into DNA is crucial for a detailed study of its binding interactions and charge donor properties. We incorporated the phenanthridinium heterocycle of ethidium as an artificial base at specific sites in duplex DNA. The hydrolytic lability of the corresponding ethidium 2’-deoxyribofuranoside [10] made it necessary to replace the sugar moiety with an acyclic linker system tethered to the N-5 position of the phenanthridinium heterocycle (Scheme 1).To synthesize the corresponding DNA building block 1, we started with the protection of the two exocyclic amino functions of 3, 8-diamino-6-phenylphenanthridine (2) by treatment with allyl chloroformate. The bisalloc-protected phenanthridine derivative 3 was then alkylated with 1, 3-diiodopropane. THF is the best solvent for this reaction because the starting material 3 is soluble in THF, whereas the alkylation product 4 is not. Hence, 4 can be collected simply by filtration. The phenanthridinium 4 was linked to DMT-protected 3-amino-1, 3-propanediol (5) under the typical conditions used for a nucleophilic substitution. Compound 5 was synthesized according to literature procedures and carries the DMT protecting group necessary for automated oligonucleotide coupling at a later stage.[11] After attachment of 5, the alloc protecting groups were exchanged by trifluoroacetyl groups. This procedure is necessary since trifluoroacetyl groups are not stable enough to be used in the alkylation of the phenanthridine heterocycle at N-5 [12] but can be cleaved under typical DNA workup conditions. This protecting-group strategy has the additional advantage that the secondary amino function of the alkyl linker is also protected. The preparation of the phosphoramidite 1 was completed by using standard procedures, and the product was used for the automated preparation of phenanthridinium-modified oligonucleotides. An extended coupling time (1 h instead of the 1.5 min used for standard couplings), a higher phosphoramidite concentration (0.2 m instead of 0.067 m), and three