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DOI: 10.1039/A702943H (Paper) Reference Section for: J. Chem. Soc., Perkin Trans. 2, 1998, 149-158

1H NMR investigations on the solution structure of the oligonucleotide 5′-d(ACCT5GATGT)-3′/5′-d(ACATCA5GGT)-3′ and its interaction with tallimustine

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Enzio Ragg, Stefania Mazzini, Rita Bortolini, Nicola Mongelli and Roberto D’Alessio

Abstract

The conformation of the DNA duplex 5′-d(ACCT5GATGT)-3′/5′-(ACATCA5GGT)-3′ has been studied by means of 2D 1H NMR and restrained molecular mechanics and dynamics calculations. This sequence contains the tract ‘T4GA’, which has previously been proved to be the recognition sequence of tallimustine, a distamycin-A analogue, containing a nitrogen mustard moiety and with interesting anti-leukemia properties. The tridecamer has been found to possess a B-type DNA conformation with local variations in the helical parameters which may be related to the sequence. Guanine in position 9, a residue supposed to be a key point for tallimustine recognition, is at the end of a homopyrimidinic tract where the propeller twist is found to decrease down to 9° at the flanking residue A10. Analysis of the minor groove geometry has revealed an increase of the width up to G9 and a return to average values at the level of residues A10–C18. The reversible interaction of this oligonucleotide with tallimustine has also been investigated by NMR and UV spectroscopy. The association constant Ka is 2 × 107 dm3 mol–1. The off-rate constant is 1.5 ± 0.5 s–1 as measured by NOESY-exchange experiments performed at R = [drug]/[DNA] = 0.5. A multiple mode of binding of tallimustine with the oligomer has been detected by analysis of 1H NMR spectra; however, the chemical shift analysis and several inter-molecular NOE interactions observed at low drug∶DNA ratios have provided sufficient structural information to define the major orientation of the drug in the minor groove. The positively charged amidinic moiety is situated at the beginning of the AT-rich region and the phenyl group resides close to the T8∶A19 base pair. The specificity of interaction of tallimustine depends on a favourable steric interaction at the T8G9A10 tract, as a result of the widening of the minor groove allowing accommodation of the phenyl moiety in the floor of the groove. Under the conditions applied in the NMR studies, negligible alkylation has been detected by HPLC measurements. However, after nine days of incubation at T = 37 °C several peaks corresponding to alkylation products are detected giving further indications of a multiple mode of binding of tallimustine with the oligonucleotide.

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