X-ray crystallographic study of DNA duplex cross-linking: simultaneous binding to two d(CGTACG)2 molecules by a bis(9-aminoacridine-4-carboxamide) derivative

Abstract

Acridine-4-carboxamides form a class of known DNA mono-intercalating agents that exhibit cytotoxic activity against tumour cell lines due to their ability to inhibit topoisomerases. Previous studies of bis-acridine derivatives have yielded equivocal results regarding the minimum length of linker necessary between the two acridine chromophores to allow bis-intercalation of duplex DNA. We report here the 1.7 A resolution X-ray crystal structure of a six-carbon-linked bis(acridine-4-carboxamide) ligand bound to d(CGTACG)2 molecules by non-covalent duplex cross-linking. The asymmetric unit consists of one DNA duplex containing an intercalated acridine-4-carboxamide chromophore at each of the two CG steps. The other half of each ligand is bound to another DNA molecule in a symmetry-related manner, with the alkyl linker threading through the minor grooves. The two crystallographically independent ligand molecules adopt distinct side chain interactions, forming hydrogen bonds to either O6 or N7 on the major groove face of guanine, in contrast to the semi-disordered state of mono-intercalators bound to the same DNA molecule. The complex described here provides the first structural evidence for the non-covalent cross-linking of DNA by a small molecule ligand and suggests a possible explanation for the inconsistent behaviour of six-carbon linked bis-acridines in previous assays of DNA bis-intercalation.

Figure 1

Molecular formula (a) and atom numbering scheme (b) of 9,9′-(1,6-hexanediyldiimino)bis{N-[2-(dimethylamino)ethyl]}acridine-4-acarboxamide, referred to as compound 1 throughout the text and designated A4C in the coordinate files. The numbering scheme is shown for only half of the ligand due to internal symmetry. The two crystallographically independent chromophores are referred to as DAC1 and DAC2, after the common name for the N-[2-(dimethylamino)ethyl]acridine-4-carboxamide monomer, DACA.

Scheme 1

Synthesis of 1: (i) Conc. H₂SO₄, 100°C, 3 h, 99% yield. (ii) (a) SOCl₂, DMF (two drops), reflux, 30 min, 85% yield; (b) N, N-dimethylethylenediamine, CH₂Cl₂, 0°C, 2 h, 67% yield. (iii) (a) Phenol, 120°C, 1 h; (b) 1,6-diaminohexane, phenol, 55°C, 24 h, 46% yield.

Figure 2

(a) Ball-and-stick representation of DNA duplex cross-linking by compound 1 in the crystal. The asymmetric unit contains a single duplex with two mono-intercalated ligands. Each molecule of compound 1 lies on an axis of 2-fold symmetry, with the other chromophore bound to a symmetry-related duplex. The DNA molecule of focus is shown in green whilst symmetry-related molecules are shown in yellow. In each case, strand B is indicated by darker shading than strand A. The ligand is shown in space filling format. (b) Schematic representation of the DNA cross-linking depicted in (a). The asymmetric unit is indicated by the shaded box and symmetry-related nucleotides are labelled with an asterisk. The major and minor grooves of the DNA are depicted as hollow and filled arrows, respectively. Alternative stereo views of Figures 2a, 4, 5, 6b and 7 are provided as online Supplementary Data.

Figure 3

Example of the final electron density map, contoured at 1 σ-level. The region shown corresponds to the intercalation site of the DAC1 chromophore, viewed from the major groove face to highlight the definition of the electron density for the side chain, which is semi-disordered in the mono-intercalator complex (15).

Figure 4

Interactions formed by the two Sr²⁺ ions in the asymmetric unit. (a) Sr1 bound in the minor groove of the DNA duplex, at the central TA step. (b) Sr2-mediating crystal packing contacts between the backbones of several DNA strands. Sr²⁺ ions are shown in magenta and water molecules in orange. Coordination bonds to the ion and hydrogen bonds (except those involved in standard Watson–Crick base pairing) are represented by grey lines.

Figure 5

DNA duplex cross-linking by DAC1 (a) and DAC2 (b). The left-hand side of each ligand is shown in a similar orientation, to highlight the similarities and differences in binding between the two independent observations.

Figure 6

Conformational analysis of the DNA. (a) Definition of DNA backbone torsion angles, using the A4(A) nucleotide (which adopts a conformation close to B-form DNA) as an example. For clarity, the ribose rings and bases are shown only as outlines. (b) Superposition of the DAC1 (green), DAC2 (yellow) and 9-amino-DACA mono-intercalator (14) (grey) binding sites.

Figure 7

The packing of the end of one DNA duplex with the opposite end of a symmetry-related duplex to form a pseudo-infinite stack. The bound ligands are shown in space filling format to illustrate that the side chain of DAC2 sterically prevents that of DAC1 from interacting with the terminal base pair of the duplex to which it is bound. The symmetry-related chromophore of each ligand has been omitted for clarity.