In DNA, alkylation at N7-dG facilitates the formation of apurinic/apyrimidinic (AP) sites, which equilibrate between deoxyribose α and β anomers via a transient aldehyde intermediate. Mitoxantrone (MTX), an anthracenedione anti-cancer agent, may conjugate with AP sites to form a reversible Schiff base, which may be isolated following chemical reduction. We characterized reduced AP site conjugates formed by the reaction of the MTX secondary amine by NMR and mass spectrometry. We examined thermodynamic and structural changes induced by these conjugates in 5`-d(C¹A²G³A⁴C⁵T⁶X⁷A⁸G⁹A¹⁰C¹¹T¹²)-3`,5`-d (A¹³G¹⁴T¹⁵C¹⁶T¹⁷C¹⁸A¹⁹G²⁰T²¹C²²T²³G²⁴ )-3`, X = MTX-AP site conjugate. Thermal melting temperature (Tm) studies revealed that these thermodynamically stabilized DNA molecules containing an AP site, but decreased Tm by 5.5 °C compared to a native duplex. NMR revealed 1H deshielding at C18, complementary to the MTX-AP conjugate, along with 1H deshielding in the modified strand at neighboring nucleotides T6, A8, G9, and A10. Increased 1H shielding was observed at T15 C16, T17, the A19 H1` and H2` deoxyribose protons, the 3`-neighboring nucleotide to C18, and G20 H2` deoxyribose proton. At the nucleotides surrounding MTX, the magnitudes of changes in chemical shifts of H2` deoxyribose protons were greater than those of H2`` deoxyribose protons, suggesting the greater impact of the MTX anthracenedione ring on the deoxyribose H2` as compared to the H2`` protons. 1H NOE data revealed two cross-peaks for the MTX H6/H7 protons with the methyl groups of T6 and T17, suggesting their positioning in the major groove. Chemical shift changes were seen for base pairs A8: T17, G9: C16, and A10: T15 (immediately 3` from MTX) compared to only one base pair immediately 5` to MTX, T6:A19. 1H NOE data at different temperatures suggested two conformations for MTX-AP site conjugates, in which the anthracenedione ring may be either outside or intercalating into the DNA. In both, the non-conjugated 2-[(2-hydroxy ethyl)amino]ethyl amino side chain of MTX extended towards base pairs A8:T17, G9:C16, and A8:T17. For the AP-site-bound sidechain, the 2-hydroxy ethyl moiety interacted with both T6 and A8. This study yields a deeper thermodynamic and structural understanding of MTX conjugation to AP sites in DNA.

Carmelo Rizzo

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