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

Abstract

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.