Palom Y, Lipman R, Musser S M, Tomasz M
Department of Chemistry, Hunter College, City University of New York, New York 10021, USA.
Chem Res Toxicol. 1998 Mar;11(3):203-10. doi: 10.1021/tx970205u.
A minor N6-deoxyadenosine adduct of mitomycin C (MC) was isolated from synthetic oligonucleotides and calf thymus DNA, representing the first adduct of MC and a DNA base other than guanine. The structure of the adduct (8) was elucidated using submilligram quantities of total available material. UV difference spectroscopy, circular dichroism, and electrospray mass spectroscopy as well as chemical transformations were utilized in deriving the structure of 8. A series of synthetic oligonucleotides was designed to probe the specificities of the alkylation of adenine by MC. The nature and frequency of the oligonucleotide-MC adducts formed under conditions of reductive activation of MC were determined by their enzymatic digestion to the nucleoside level followed by quantitative analysis of the products by HPLC. The analyses indicated the following: (i) (A)n sequence is favored over (AT)n for adduct formation; (ii) the alkylation favors the duplex structure; (iii) at adenine sites only monofunctional alkylation occurs; (iv) the adenine-to-alkylation frequency in the model oligonucleotides was 0.3-0.6 relative to guanine alkylation at the 5'-ApG sequence but only 0.02-0.1 relative to guanine alkylation at 5'-CpG. The 5'-phosphodiester linkage of the MC-adenine adduct is resistant to snake venom diesterase. The overall ratio of adenine to guanine alkylation in calf thymus DNA was 0.03, indicating that 8 is a minor MC-DNA adduct relative to MC-DNA adducts at guanine residues in the present experimental residues in the present experimental system. However, the HPLC elution time of 8 coincides with that of a major, unknown MC adduct detected previously in mouse mammary tumor cells treated with radiolabeled MC [Bizanek, R., Chowdary, D., Arai, H., Kasai, M., Hughes, C. S., Sartorelli, A. C., Rockwell, S., and Tomasz, M. (1993) Cancer Res. 53, 5127-5134]. Thus, 8 may be identical or closely related to this major adduct formed in vivo. This possibility can now be tested by further comparison.
从合成寡核苷酸和小牛胸腺DNA中分离出了丝裂霉素C（MC）的一种次要的N6-脱氧腺苷加合物，这是MC与除鸟嘌呤以外的DNA碱基形成的首个加合物。利用亚毫克量的全部可用材料阐明了该加合物（8）的结构。在推导8的结构过程中，采用了紫外差示光谱法、圆二色性、电喷雾质谱法以及化学转化方法。设计了一系列合成寡核苷酸来探究MC对腺嘌呤的烷基化特异性。在MC的还原活化条件下形成的寡核苷酸-MC加合物的性质和频率，通过将其酶解至核苷水平，然后用HPLC对产物进行定量分析来确定。分析结果表明：（i）对于加合物形成，（A）n序列比（AT）n更有利；（ii）烷基化有利于双链结构；（iii）在腺嘌呤位点仅发生单功能烷基化；（iv）在模型寡核苷酸中，相对于5'-ApG序列处鸟嘌呤的烷基化，腺嘌呤的烷基化频率为0.3 - 0.6，但相对于5'-CpG处鸟嘌呤的烷基化，仅为0.02 - 0.1。MC-腺嘌呤加合物的5'-磷酸二酯键对蛇毒磷酸二酯酶具有抗性。小牛胸腺DNA中腺嘌呤与鸟嘌呤烷基化的总体比例为0.03，这表明在本实验系统的当前实验残基中，相对于鸟嘌呤残基处的MC-DNA加合物，8是一种次要的MC-DNA加合物。然而，8的HPLC洗脱时间与先前在用放射性标记的MC处理的小鼠乳腺肿瘤细胞中检测到的一种主要的、未知的MC加合物的洗脱时间一致[比赞内克，R.，乔达里，D.，新井，H.，笠井，M.，休斯，C. S.，萨托雷利，A. C.，罗克韦尔，S.，和托马兹，M.（1993年）《癌症研究》53，5127 - 5134]。因此，8可能与体内形成的这种主要加合物相同或密切相关。现在可以通过进一步比较来检验这种可能性。
