Church K M, Wurdeman R L, Zhang Y, Chen F X, Gold B
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68198-6805.
Biochemistry. 1990 Jul 24;29(29):6827-38. doi: 10.1021/bi00481a011.
The synthesis and characterization of a series of compounds that contain an N-alkyl-N-nitrosourea functionality linked to DNA minor groove binding bi- and tripeptides (lexitropsins or information-reading peptides) based on methylpyrrole-2-carboxamide subunits are described. The lexitropsins (lex) synthesized have either a 3-(dimethylamino)propyl or propyl substituent on the carboxyl terminus. The preferred DNA affinity binding sequences of these compounds were footprinted in 32P-end-labeled restriction fragments with methidiumpropyl-EDTA.Fe(II), and in common with other structural analogues, e.g., distamycin and netropsin, these nitrosoureas recognize A-T-rich runs. The affinity binding of the compound with the dimethylamino terminus, which is ionized at near-neutral pH, appeared stronger than that observed for the neutral dipeptide. The sequence specificity for DNA alkylation by (2-chloroethyl)nitrosourea-lex dipeptides (Cl-ENU-lex), with neutral and charged carboxyl termini, using 32P-end-labeled restriction fragments, was determined by the conversion of the adducted sites into single-strand breaks by sequential heating at neutral pH and exposure to base. The DNA cleavage sites were visualized by polyacrylamide gel electrophoresis and autoradiography. The alkylation of DNA by Cl-ENU-lex was compared to that by N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (CCNU), which has no DNA affinity binding properties. While all the Cl-ENU compounds generate DNA breaks as a consequence of the formation of N7-alkyl-guanine, the Cl-ENU-lex compounds induced, in a time- and dose-dependent fashion, intense DNA cleavage bands at adenine, cytosine, and thymine residues associated with affinity binding sites. These non-G cleavages induced by Cl-ENU-lex were inhibited by the coaddition of distamycin at concentrations that did not affect G alkylation break sites. CCNU, even at much higher concentrations, does not generate any similar detectable lesions at non-G sites. Therefore, linking the Cl-ENU moiety to minor groove binders is a viable strategy to qualitatively and quantitatively control the delivery and release of the ultimate DNA alkylating agent in a sequence-dependent fashion.
描述了一系列化合物的合成与表征，这些化合物包含与基于甲基吡咯 -2- 甲酰胺亚基的DNA小沟结合二肽和三肽（lexitropsins或信息读取肽）相连的N-烷基-N-亚硝基脲官能团。合成的lexitropsins（lex）在羧基末端具有3-（二甲基氨基）丙基或丙基取代基。这些化合物的优选DNA亲和结合序列通过用甲基丙基 -EDTA.Fe(II) 对32P末端标记的限制片段进行足迹分析确定，并且与其他结构类似物（例如，偏端霉素和纺锤菌素）一样，这些亚硝基脲识别富含A-T的序列。在接近中性pH下离子化的具有二甲基氨基末端的化合物的亲和结合似乎比中性二肽观察到的更强。使用32P末端标记的限制片段，通过在中性pH下顺序加热并暴露于碱将加合位点转化为单链断裂，确定了具有中性和带电荷羧基末端的（2-氯乙基）亚硝基脲-lex二肽（Cl-ENU-lex）对DNA烷基化的序列特异性。通过聚丙烯酰胺凝胶电泳和放射自显影观察DNA切割位点。将Cl-ENU-lex对DNA的烷基化与不具有DNA亲和结合特性的N-（2-氯乙基）-N'-环己基-N-亚硝基脲（CCNU）的烷基化进行比较。虽然所有Cl-ENU化合物由于形成N7-烷基鸟嘌呤而产生DNA断裂，但Cl-ENU-lex化合物以时间和剂量依赖性方式在与亲和结合位点相关的腺嘌呤、胞嘧啶和胸腺嘧啶残基处诱导强烈的DNA切割带。在不影响G烷基化断裂位点的浓度下共添加偏端霉素可抑制Cl-ENU-lex诱导的这些非G切割。即使在高得多的浓度下，CCNU在非G位点也不会产生任何类似的可检测损伤。因此，将Cl-ENU部分与小沟结合剂连接是一种可行的策略，可按序列依赖性方式定性和定量地控制最终DNA烷基化剂的递送和释放。
