Thiol-mediated DNA alkylation by the novel antitumor antibiotic leinamycin
Thiol-mediated DNA alkylation by the novel antitumor antibiotic leinamycin
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DOI：
10.1021/ja960892w
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发表时间：
1996-07-17
影响因子：
15
通讯作者：
Saitoh, Y
中科院分区：
文献类型：
--
作者：
Asai, A;Hara, M;Saitoh, Y
莱纳霉素 (1) 是从链霉菌属的培养液中分离出来的。 1989年，通过光谱分析、1X射线晶体学、4和化学合成阐明了1-3及其结构。 5 这种抗生素含有一种不寻常的 1, 3-dioxo-1, 2-dithiolane 部分，该部分通过螺键与 18 元内酰胺相连，似乎是一类新的天然产物。 1在一些小鼠肿瘤模型中表现出显着的抗肿瘤活性。 2 我们之前报道过，1 在体外存在硫醇辅助因子的情况下会诱导 DNA 单链断裂。我们在这里报告了 1 诱导的硫醇激活和 DNA 裂解的详细化学反应。将 1.5 当量的 2-巯基乙醇 (2-ME) 添加到 1 的 MeOH/10 mM 磷酸盐缓冲液 (pH 7) (1/9, v/v) 溶液中，导致快速转化为主要产物和几种次要产物。其他硫醇，包括乙硫醇、二硫苏糖醇、半胱氨酸和谷胱甘肽，可提供大约等量的 2。我们使用反相 HPLC 从反应混合物中分离出 2（产率 70%），但由于 2 在 DMSO 中不稳定，因此无法对 2 进行全面表征。用K2CO3和碘甲烷处理2得到稳定的甲酯2a（68%，产率）。通过 1D 和 2D-NMR 实验对 2a 进行表征，建立了意想不到的结构，其中缺少 1, 3-二氧代-1, 2-二硫戊环部分和 6, 7-烯烃，并观察到新的 3, 7-硫醚键和 6-羟基。在富含 MeOH 的溶剂 MeOH/0.5 M 磷酸盐缓冲液（pH 7）（99/1，v/v）中用 2-ME 处理 1，得到作为主要产物的甲醇加合物 2b6。 1与小牛胸腺DNA在2-ME(药物/DNAbp/2ME)1/20/1.5)存在下反应后，通过乙醇沉淀纯化DNA。 DNA 显示出与发色团复合物的紫外光谱特征。尽管在 4℃ 下稳定，但在 37℃ 时观察到发色团从 DNA 中逐渐释放。释放的速率和效率随着温度的进一步升高而增加。在制备规模上，我们从来那霉素处理的小牛胸腺 DNA 中分离出释放的发色团 3（1 的产率为 75%）。在 13C NMR 谱中，3 的所有共振都与 2 的共振相当，并且发现了 5 个额外的共振，表明 2 中添加了嘌呤残基。在 1H-NMR 谱中，所有共振也与 2 的共振相当。在 7.77 ppm (1H) 处发现了一个额外的不可交换共振。唯一能产生不可交换共振的核碱基是鸟嘌呤。鸟嘌呤 H-8 和 6-CH3 之间的 NOE 观察结果与鸟嘌呤 N-7 对 C-6 碳的烷基化一致。这些光谱数据表明3是莱那霉素-N7鸟嘌呤加合物。这得到了钠盐 C27H31O7N7S2Na 的分子式的支持：HRFABMS (M+ Na)+ m/z 652.1600，计算值 652.1624。 1 不与鸟苷核苷酸单体或单链 DNA 发生反应，表明 DNA 的烷基化可能归因于该分子与双链 DNA 的独特相互作用。为了确定链切割的特异性，我们检查了 1 与从质粒 pBR322 获得的 5' 端标记 DNA 片段 (89bp) 的反应。我们的初步结果表明，1 在硫醇存在的情况下会诱导 DNA 的非选择性链断裂。 3 然而，用热哌啶处理这些裂解产物会产生以鸟嘌呤特异性裂解形式迁移的片段。最近的报告 7-9 表明，对 N7 鸟嘌呤或 N3 腺嘌呤修饰的 DNA 进行热处理会产生一些产物……
Leinamycin (1) was isolated from the culture broth of a Streptomyces sp. in 1989, 1-3 and its structure was elucidated by spectroscopic analysis, 1 X-ray crystallography, 4 and chemical synthesis. 5 This antibiotic contains an unusual 1, 3-dioxo-1, 2-dithiolane moiety, which is connected to the 18-membered lactam through a spiro linkage, and appeared to be a new class of natural product. 1 exhibited significant antitumor activity in some murine tumor models. 2 We previously reported that 1 induces single-strand scission of DNA in the presence of thiol cofactors in vitro. We report here the detailed chemistry of thiol-activation and DNA-cleavage induced by 1. Addition of 1.5 equiv of 2-mercaptoethanol (2-ME) to a solution of 1 in MeOH/10 mM phosphate buffer (pH 7)(1/9, v/v) resulted in a rapid conversion to a major product with several minor products. Other thiols including ethanethiol, dithiothreitol, cysteine, and glutathione afforded approximately equal amounts of 2. We isolated 2 from the reaction mixture with reverse-phase HPLC (70%, yield), but the full characterization of 2 failed due to its instability in DMSO. Treatment of 2 with K2CO3 and iodomethane afforded a stable methyl ester 2a (68%, yield). Characterization of 2a by 1D and 2D-NMR experiments established an unexpected structure, in which the 1, 3-dioxo-1, 2-dithiolane moiety and the 6, 7-olefin were missing and a new 3, 7-sulfide linkage and 6-hydroxyl group were observed. Treatment of 1 with 2-ME in a MeOH-rich solvent, MeOH/0.5 M phosphate buffer (pH 7)(99/1, v/v), afforded the methanol adduct 2b6 as the main product. After reaction of 1 with calf thymus DNA in the presence of 2-ME (drug/DNAbp/2ME) 1/20/1.5), the DNA was purified by ethanol precipitation. The DNA showed a UV spectrum characteristic of the complex with a chromophore. Although it was stable at 4 C, gradual release of the chromophore from the DNA was observed at 37 C. The rate and efficiency of the release increased with further an increase in the temperature. On a preparative scale, we isolated the released chromophore 3 from the leinamycin-treated calf thymus DNA (75% yield from 1). In the 13C NMR spectrum of 3 all of the resonances were comparable to those of 2, and five additional resonances were found, suggesting the addition of a purine residue to 2. In the 1H-NMR spectrum, all of the resonances were also comparable to those of 2. One additional nonexchangeable resonance at 7.77 ppm (1H) was found. The only nucleobase that gives one nonexchangeable resonance is guanine. Observation of the NOE between the guanine H-8 and the 6-CH3 is consistent with alkylation of the C-6 carbon by the N-7 of guanine. These spectroscopic data revealed that 3 is a leinamycin-N7 guanine adduct. This was supported by the molecular formula that was established as the sodium salt C27H31O7N7S2Na: HRFABMS (M+ Na)+ m/z 652.1600, calcd 652.1624. 1 did not react with guanosine nucleotide monomer or single stranded DNA, suggesting that the alkylation of DNA could be attributable to the unique interaction of this molecule with double stranded DNA. To determine the specificity of the strand cleavage, we examined the reaction of 1 with a 5′-end-labeled DNA fragment (89bp) obtained from plasmid pBR322. Our preliminary results indicated that 1 induces nonselective strand scission of DNA in the presence of thiol. 3 However, treatment of these cleaved products with hot piperidine resulted in the production of fragments that migrated as guanine-specific cleavage. Recent reports7-9 have shown that thermal treatment of the DNA that is modified on N7 guanine or N3 adenine produce some products …