HPLC-ESI-MS/MS Research on DNA Interstrand Cross-Links Formed by 1,3-Bis-(2-Chloroethyl)-1-Nitrosourea

摘要

Bifunctional alkylating agent 1,3-Bis-(2-chloroethyl)-1-nitrosourea (BCNU) is a significant anticancer drug in the clinical treatment of human malignancies. Several lines of evidences indicated that the cytotoxic activity of BCNU was related to their ability of inducing DNA interstrand cross-links (ICLs). In the present work, the formation of the ICLs from the treatment of calf thymus DNA with BCNU was studied with HPLC-ESI-MS/MS analysis and QM/MM computations. Double strand calf thymus DNA was treated with BCNU and digested enzymatically down to the nucleoside level. The hydrolysates were analyzed by HPLC separation followed by electrospray ionization tandem mass spectrometric conformation. The main ICL product, 1-[N³-deoxycytidyl]-1-[N¹-deoxyguanosyl]-ethane, was characterized by selected reaction monitoring (SRM) mode with ion transitions of mlz 521rarrm/z 405 ([M+H⁺-dR]⁺) and mlz 521rarrm/z 289 ([M+H⁺-2dR]⁺). QM/MM computations were carried out with ONIOM method to investigate molecular geometric structure of the DNA double helixes containing various cross-links. A stable structure of the dC(N³)-CH₂CH₂-dG(N¹) ICL was obtained, which was consistent with the result of HPLC-ESI-MS/MS analysis.