Sequence specific DNA cleavage by conjugates of benzotriazoles and minor groove binders
Sequence specific DNA cleavage by conjugates of benzotriazoles and minor groove binders
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DOI：
10.1021/ja971042z
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发表时间：
1997-08-13
影响因子：
15
通讯作者：
Wender, PA
中科院分区：
文献类型：
--
作者：
Touami, SM;Poon, CC;Wender, PA
小分子不可逆地修饰核酸的能力引起了人们的极大兴趣，因为许多医学上有用的天然产品的生物活性来自于它们与DNA的选择性相互作用。1此外，以特定序列方式裂解核酸的化合物有可能用作获取结构和遗传信息的试剂。虽然DNA裂解剂的复杂性从羟基自由基到限制性内切酶，但许多研究都集中在设计新的化合物上，这些化合物可以被触发来产生有效和选择性的裂解剂。2最近，我们描述了一类新的DNA裂解剂的开发，称为苯并三氮唑光核酸酶。3这些分子被设计用来产生反应性苯基，被证明在光化学激活时能够有效和选择性地裂解DNA。虽然目前正在研究切割的实际机制，但很明显，这些新化合物提供了几个优点。值得注意的是，它们很容易获得，可以很容易地修改以结合一系列功能，并且在被光触发之前保持不活动状态。在这里，我们描述了苯并三氮唑光核酸酶和DNA小槽结合剂的新型结合物的合成和研究，这些结合剂被发现具有更高的切割效率和独特的切割选择性。我们的苯并三氮唑光核酸酶的DNA切割效率和选择性有望通过共价连接到特定的DNA识别元件而得到提高。这样的修饰有望增加DNA附近裂解亚基的局部浓度，也可以用于选择特定的DNA序列。虽然有几种潜在的合适的DNA结合化合物，但我们最初选择将我们的三氮唑定位在DNA的小凹槽中，方法是将它们连接到具有良好特性的netropsin类结合剂上。4 Netropsin和地塞霉素是双和三吡咯，它们通过静电相互作用、氢键和van der Waals接触与DNA的细沟紧密结合。5虽然这些天然产物优先结合四个和五个碱基对(BP)AT区，但最近报道了几种允许靶向多个位点的修饰。6此外，在固体载体上制备此类化合物的新方法为进一步通过杂交核酸酶灵活地控制序列特异性提供了基础。制备了7个化合物5-7，它们含有苯并三氮唑型的可光激活的DNA裂解单元，以验证我们最初的假设。由于已经证明增加N-甲基吡咯单元的数量会增加这些分子的结合亲和力，5a决定系统地探索一个、两个和三个吡咯单元对切割效率和选择性的影响。低聚(N-甲基吡咯甲酰胺)结合部分(1、2和3)按照标准的文献方法分别经过四步、六步和八步合成。8由苯并三唑-5-羧酸制备的活化酯4在这些合成中被用作多功能偶联伙伴，顺利地以六到十步合成了化合物5、6和7(方案1)。重要的是，这些分子都吸收300 nm以上的光，允许在DNA存在的情况下选择性地进行光激发。9通过监测环状超螺旋DNA(形式I)向环状松弛(形式II)和线状(形式III)DNA的转化，初步检测了5、6和7的DNA切割能力。杂化化合物(3-90μM)在…中用焦平面滤光灯照射
The ability of small molecules to irreversibly modify nucleic acids has generated considerable interest given that many medicinally useful natural products derive their biological activity from their selective interaction with DNA. 1 In addition, compounds which cleave nucleic acids in a sequence specific manner are potentially useful as reagents for accessing structural and genetic information. While DNA cleaving agents range in complexity from hydroxyl radicals to restriction enzymes, much research has focused on the design of novel compounds which can be triggered to generate potent and selective cleavage agents. 2 Recently, we described the development of a new class of DNA cleaving agents termed “benzotriazole photonucleases”. 3 These molecules, designed to generate a reactive phenyl radical, were shown to efficiently and selectively cleave DNA upon photochemical activation. While the actual mechanism of cleavage is presently under investigation, it is clear that these novel compounds offer several advantages. Notably, they are readily available, can be easily modified to incorporate a range of functionality, and remain inactive until triggered by light. Herein we describe the synthesis and study of novel conjugates of benzotriazole photonucleases and DNA minor groove binders, agents which have been found to exhibit enhanced cleavage efficiency and unique cleavage selectivity. The DNA cleavage efficiency and selectivity of our benzotriazole photonucleases were expected to be enhanced by covalent attachment to specific DNA recognition elements. Such a modification would be expected to increase the local concentration of the cleaving subunit in proximity to the DNA, and could also be used to select for specific DNA sequences. While there are several potentially suitable DNA binding compounds, we initially chose to position our triazoles in the minor groove of DNA by attaching them to well-characterized netropsin-like binders. 4 Netropsin and distamycin are di-and tripyrroles which tightly bind to the minor groove of DNA through a combination of electrostatic interactions, hydrogen bonds, and van der Waals contacts. 5 While these natural products preferentially bind four and five base pair (bp) AT tracts, several modifications have recently been reported which allow for the targeting of multiple sites. 6 In addition, new methods for the preparation of such compounds on a solid support provide the basis for further flexible control of sequence specificity by hybrid nucleases. 7 Compounds 5-7, which contain a photoactivatable DNA cleaving unit of the benzotriazole type, were prepared to test our initial hypothesis. Since it has been shown that increasing the number of N-methylpyrrole units increases the binding affinity of these molecules, 5a it was decided to systematically explore the effect of one, two, and three pyrrole units on cleavage efficiency and selectivity. The oligo (N-methylpyrrolecarboxamide) binding moieties (1, 2, and 3) were synthesized in four, six, and eight steps, respectively, according to standard literature procedures. 8 The activated ester 4, prepared from benzotriazole-5-carboxylic acid, was used as a versatile coupling partner in these syntheses, which proceeded smoothly to afford compounds 5, 6, and 7 in six to ten steps overall (Scheme 1). Importantly, these molecules all absorb light above 300 nm, allowing for selective photoexcitation in the presence of DNA. 9The DNA cleavage ability of 5, 6, and 7 was initially tested by monitoring the conversion of circular supercoiled DNA (form I) to circular relaxed (form II) and linear (form III) DNA. Hybrid compounds (3-90 μM) were irradiated with Pyrexfiltered light in the …