Degree Name

Doctor of Philosophy


Department of Chemistry


The binding of the antitumour antibiotics pyrindamycin A (duocarmycin C2) and pyrindamycin B (duocarmycin C1) to self-complementary oligonucleotides has been studied using electrospray ionisation mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS). The pyrindamycins bind via non-covalent interactions in the minor groove of DNA with subsequent alkylation of the N3 of adenine (particularly within 5'-AAA and 5'- TTA sequences). The relative stability of adducts with different oligonucleotides can be inferred from the ESI mass spectra. M S / M S spectra of alkylated adducts enable facile identification of the site of alkylation. W e show here that the site(s) of alkylation by the pyrindamycins are strongly influenced by the location of the preferred high affinity binding sites within these short oligonucleotides. Alkylation of guanine by pyrindamycin A is shown to occur only upon binding to single-stranded oligonucleotides, providing further confirmation of the importance of the initial non-covalent interaction in conferring sequence selectivity. These data clearly demonstrate value of using ESI-MS/MS to prescreen ligand-oligonucleotide complexes prior to performing more detailed structural studies.

In the second part of this work, the binding of nogalamycin to unusual DNA structures was explored by nuclear magnetic resonance (NMR) spectroscopy. The first of these was an oligonucleotide 5'-d(GCGAAGCACGAAGT)-3', which was designed to form a double hairpin or loop sequence. T w o further oligonucleotides, 5'-d(GTGCGAAGCTAC)-3' and 5'-d(GCTACGAAGTGC)-3' were designed to incorporate a mispaired thymine, which may then form a bulge. The folding of the hairpins was evidenced by anomalous chemical shift values for the sugar H4', H5' and H5" protons (characteristic of the adenine involved in the loop) and the presence of imino protons (protected from exchange by hydrogen bonding interactions in the stem).

Nogalamycin formed 1:1 complexes with each of the oligonucleotides with the ligand bound at the TpG step. Upon ligand binding, changes in chemical shift values for the DNA resonances were observed, with the largest differences noted for the nucleotides in the intercalation site. Nogalamycin bound to 5'-d(GCGAAGCACGAAGT)-3' at the nicked site and stabilised the double hairpin structure through co-axial stacking interactions. The intercalation of nogalamycin to 5'-d(GTGCGAAGCTAC)-3' gave rise to an equilibrium in solution between a frame-shifted conformation and bulged conformation. The binding of nogalamycin to 5'-d(GCTACGAAGTGC)-3' stabilised the bulge positioned at T3. Finally, ESI-MS and melting temperature data indicate that the relative stability of the complexes formed with nogalamycin is in the order: 5'-d(GCGAAGCACGAAGT)-3'-Ng > 5'- d(GCTACGAAGTGC)-3'-Ng > 5'-d(GTGCGAAGCTAC)-3'-Ng.



Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.