separatedby approximately 3.4Å(the van der Waals thickness of a phenyl ring) to accommodate the lig-
and. This base pair separation can only occur if there is rotation about torsional bonds in the phosphodi-
ester backbone, such that unwinding around the helical axis occurs.
Intercalation of multiple ligands into a stretch of short rod-like DNA and concomitant helix unwinding
leads to an overall lengthening of the duplex. This change in DNA length is reflected as a change in
hydrodynamic character, and hence the intrinsic viscosityandsedimentation coefficient of the DNA
change upon intercalation. The extent of helix unwinding produced by intercalation is dependent on the
nature of the bound drug, especially its geometry and physico–chemical properties. The amount of helix
unwinding can be determined experimentally by use of covalently closed circular DNA, with reference
to a standard value of 26° per bound ethidium bromide drug. For canonical B-DNA, the normal backbone
twist angle is 36°. Binding of ethidium reduces this twist angle to 10° and hence the DNA unwinds by 26°.
Binding of proflavin or other related acridines causes unwinding of DNA by about 17°, whereas binding
of anthracycline antibiotics results in only 11° unwinding of DNA. The effects of localised unwinding at
the intercalation site are propagated along the DNA helix and result in structural perturbations over long
distances from the bound drug. The helical twist angle for the two base pairs immediately surrounding the
bound drug may be little changed from 36°. In such cases, there is usually a significant helical unwinding
at adjacent base pairs. Hence the unwinding angle is the sum of cumulative changes in helical twist across
all affected base pairs.
The process of base-pair separationthat occurs during intercalation also produces a number of other
changes in backbone conformation and base-pair and base-step geometry. In the X-ray-derived molecular
structure of ethidium intercalated between a G C dinucleotide (Figure 9.5), ethidium is stacked with its
long axis parallel to the long axis of the adjacent base pair. The exocyclic amino groups point towards the
diester oxygen atoms of the DNA phosphate groups and provide additional electrostatic and hydrogen-bond
stabilization of the complex. The base pairs are slightly kinked, since the out-of-plane phenyl group limits
full intercalation of the cationic phenanthridinium ring (Figure 9.5a). The phenyl and ethyl substituents of
ethidium lie in the minor groove of the complex.
Various molecular events contribute to the overall free energy observed for intercalation reactions. Some
of these are enthalpic in nature and others entropic. One of the most important energetic driving forces for
drug binding is the favourable hydrophobic transfer of the drug from bulk solvent into the DNA binding
site. The non-polar ligand is removed from the aqueous environment and this disrupts the hydration layer
around the ligand, resulting in an entropically favourable release of site-specific water molecules. The DNA
binding site must undergo an energetically unfavourable conformational transition to form a cavity into which
the drug intercalates. Whilst this deformation is unfavourable from a point-of-view of configuration, it has
additional features that give rise to a favourable entropy change. Localized unwinding of the helix at the inter-
calationsite results in an increase in the distance between adjacent phosphates on the backbone. This gives
rise to a reduction of localized charge density and hence the release of condensed counter-ions (Section
9.3.1). Additional counter-ions are released during the process of intercalation of the cationic ligand owing
to the polyelectrolyte effect (Section 9.3).
Ligand intercalation is also associated with favourable enthalpic contributions to free energy arising
from the formation of noncovalent interactions between the drug and base pairs. These noncovalent inter-
actions involve several different forces, such as the hydrophobic effect, reduction of coulombic repulsion,
van der Waals interactions, -stacking, and hydrogen bonding. Quantitative apportionment of the overall
bindingfree energy for intercalators and groove binders16–18is discussed later (Sections 9.6.4 and 9.7.5).
9.6.2 The Anthracycline Antibiotic Daunomycin
The anthracycline antibiotics, daunomycin(daunorubicin) and adriamycin(doxorubicin) as well
as some derivatives have been frontline anticancer drugs for many years. Daunomycin is perhaps the
best-studied intercalating drug to date with over 20 high-resolution structures known and numerous stud-
ies on the kinetics and thermodynamics of its interaction with DNA.^19
350 Chapter 9