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

Abstract

Nitrogen mustard alkylating agents are important cancer drugs. Much interest has been 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 electrospray ionization and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry to study the structure of the DNA complexes of two minor groove binding polybenzamide mustards, alkamin and alkamini; the former is a bis-half-mustard in which reactive groups are disposed at each end of the ligand, and the latter is its monofunctional analog. Alkamin is potently cytotoxic and active in experimental mouse tumor models, whereas alkamini is not. We have studied their interaction with the DNA dodecamer d(CGCGAATTCGCG) 2 , designated A2T2, and we 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 hydrolyzed but, in addition, forms a variety of interstrand cross-links between adenines A5/A6 and A5Ј/A6Ј, an interstrand cross-link between G4 and A6Ј, and an intrastrand cross-link between G4 and A6. We conclude that the marked cytotoxicity of alkamin and its experimental antitumor activity could be the consequence of its ability to cross-link cellular DNA at AT tract sequences.

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

“…Thus, for example, CA m G-tracts (cf. A6/T6) would appear to offer novel sites for interstrand cross-linking as well as the expected sites for intrastrand cross-links, and in fact, the only sequences that would not permit interstrand cross-linking would be GA m G. Taken together, this work and that of Abdul Majid et al (13) illustrate that positioning monofunctional mustard groups on either end of a polybenzamide MGB ligand is an excellent strategy for enhancing the formation of interstrand cross-links at all manner of AT-tracts, including most in which the adenines are all in one strand. The importance of cross-links to biological activity is made clear in the potent cytotoxic and experimental antitumor activity of alkamin, properties that the monofunctional alkamini lacks (10,11).…”

“…However, a tandem MS/MS ESI measurement with SS A6-L as the parent ion (Figure S12a of the Supporting Information) unequivocally shows reaction with G10, and the full spectrum supports the above assignments. Figure S12a of the Supporting Information also reveals that fragmentation in the tandem experiment is more resistant to collision-induced decay for this DNA complex than found previously for A2T2 (13) and A3T3 adducts, so much so that the dominant ions in the spectrum are the intact complex and the deadenylated DNA. The resistance of the latter to fragmentation results in only a limited number of peaks that usefully define the binding sites.…”

“…Base Alkylation. The data presented here and in Abdul Majid et al (13) enable a direct comparison of the modes of binding of alkamin and alkamini to the range of AT-tracts represented by A2T2, A3T3, and A6/T6 DNAs, to which end Figures 3 and 4 also include the binding data for A2T2. In A2T2, alkamini alkylates G4, A5, and A6; in A3T3, it binds to A4, A5, A6, and G10; and in A6/T6, it reacts with A4-A9 and with G10 and G22 (Figure 3).…”

“…In contrast, alkamini is 35-fold less cytotoxic and lacks in vivo activity, pointing to the importance of alkamin cross-links as a determinant of its biological activity (10,11). Recently, we have used matrix-assisted laser desorption ioniza- tion (MALDI) and electrospray ionization (ESI) mass spectrometry to characterize the binding of these agents to the oligonucleotide d(CGCGAATTCGCG) 2 , A2T2, and have found that alkamin forms a variety of monofunctional adducts and interstrand and intrastrand cross-links (13).…”