Received for publication August 23, 2006.
Revised January 17, 2007.
Accepted for publication January 18, 2007.

Structure of the d(CGCGAATTCGCG)2 Complex of the Minor Grove Binding Alkylating Agent Alkamin Studied by Mass Spectrometry

Abstract

Nitrogen mustard alkylating agents are important cancer drugs. Much interest has focused on redirecting their covalent adducts from the N7 atoms of guanine in the major groove of DNA to the N3 atoms of adenine in the minor groove by attaching mustard groups to AT-selective minor groove binding ligands. Here we describe the use of ESI and MALDI-TOF mass spectrometry to study the structure of the DNA complexes of two minor groove binding polybenzamide mustards, alkamin and alkamini, the former being a bis-half-mustard in which reactive groups are disposed at each end of the ligand, and the latter its monofunctional analogue. Alkamin is potently cytotoxic and active in experimental mouse tumour models, whereas alkamini is not. We have studied their interaction with the DNA dodecamer d(CGCGAATTCGCG)2, designated A2T2, and provide a detailed analysis of the observed DNA-ligand adduct ions and their fragmentation products. We find that alkamini alkylates A2T2 at guanine G4, and adenines A5 and A6 in a manner consistent with covalent attack on purine N3 atoms from the minor groove of the AT-tract. Alkamin also forms monofunctional adducts at G4 and both adenines, in which the second mustard arm is hydrolysed, but, in addition, forms a variety of inter-strand crosslinks between adenines A5/A6 and A5'/A6', an inter-strand crosslink between G4 and A6', and an intra-strand crosslink between G4 and A6. We conclude that the marked cytotoxicity of alkamin and its experimental antitumour activity could be the consequence of its ability to crosslink cellular DNA at AT-tract sequences.

Key words: Structure determinations, Structure-activity relationships and modeling, Mass Spectroscopy, Receptor binding studies, DNA damage and repair, Transcription targets