Mass Spectrometry Studies of the Binding of the Minor Groove-Directed Alkylating Agent Alkamin to AT-Tract Oligonucleotides

• Published in: Chemical research in toxicology 2009-01, Vol.22 (1), p.146-157
• Main Authors: Abdul Majid, Amin M. S, Smythe, George, Denny, William A, Wakelin, Laurence P. G
• Format: Article
• Language: English
• Subjects: Adenine - chemistry; Alkylating Agents - chemistry; Alkylating Agents - toxicity; Anilides - chemistry; Anilides - toxicity; DNA Adducts - chemistry; Nitrogen Mustard Compounds - chemistry; Nitrogen Mustard Compounds - toxicity; Oligonucleotides - chemistry; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Thymine - chemistry
• ISSN: 0893-228X; 1520-5010
• DOI: 10.1021/tx800276h

Minor groove binding alkylating agents, which have potential as cancer drugs, generate cytotoxic DNA adducts that are relatively resistant to repair as a consequence of locating covalent attachment at purine N3 nitrogen atoms. Recently, we used electrospray and matrix-assisted laser desorption ionization mass spectrometry to study the binding of the minor groove-directed polybenzamide bis-half-mustard alkamin, and its monofunctional analogue alkamini, to the oligonucleotide d(CGCGAATTCGCG)2, identifying a number of inter- and intrastrand alkamin cross-links involving the GAATTC sequence [ Abdul Majid A. M. S. , Smythe G. , Denny W. A. , and Wakelin L. P. G. (2007) Mol. Pharmacol. 71, 1165−1178 ]. Here, we extend these studies to d(CGCAAATTTGCG)2, A3T3, and d(CGCAAAAAAGCG)·d(CGCTTTTTTGCG), A6/T6, in which the opportunity for both inter- and intrastrand cross-linking is enhanced. We find that both ligands alkylate all adenines in the longer AT-tracts, as well as the abutting guanines, whether they are in the same strand as the adenines or not, in a manner consistent with covalent attack on purine N3 atoms from the minor groove. Alkamin forms intrastrand cross-links involving A4 and A6 and A6 and G10 in A3T3 and all of the purines in the A6/T6 purine tract, including G10. In addition, it forms interstrand cross-links between A4, A5, A6 and A4′, A5′, A6′, between G10 and the latter adenines in A3T3, and between G22 and adenines A5 and A6 in A6/T6. The reactivity of the abutting guanines provides unexpected opportunities for both inter- and intrastrand cross-linking by alkamin, such as the interstrand cross-link in the CAAAAAAG sequence. We conclude that positioning monofunctional mustard groups on either end of a minor groove-directed polybenzamide has the capacity to enhance interstrand cross-links at all manner of AT-tracts, including most in which the adenines are all in one strand.