groups suggests that these compounds should bind in the minor groove. However, solution studies, (NMR,
LD and DNA unwinding) have established unequivocally that these ligands are intercalators.
It is true to say that DNA base pairs can have significant intrinsic propeller twist, so there is no reason
why unfused aromatic ring compounds should not intercalate and enhance the base pair interactions
responsible for propeller twist. Yet, why should 4,6-diphenylpyridine (Figure 9.18) intercalate whilst sim-
ilar compounds such as Hoechst 33258 bind in the minor groove?
For ligands of this nature, the only reasonable potential binding modes are intercalation or minor groove
binding at AT-rich sites. This is because the major groove is too wide to form sufficient favourable contacts
while G C minor grooves present a steric block viathe guanine amino group. The choice of whether the lig-
and intercalates or binds at A T minor grooves is probably in large part determined by the presence or absence
of functional groups capable of participating in hydrogen bonds, notably ligand amides. We have already
seen that appropriately placed NH groups on the ligand can donate hydrogen bonds to N-3 atoms of ade-
nine and O-2 atoms of thymine in forming strong minor groove complexes. Examination of the structure
of 4,6-diphenylpyridine shows that it has no reasonable hydrogen bond donors, either in the rings or in the
connecting bonds, and so there is no strong minor groove complex. However, the aromatic rings in the ligand
can form good stacking interactions with the base pairs and hence it forms a strong intercalation complex.
The ligand 4 ,6-diamidino-2-phenylindole(DAPI, Figure 9.18) is an unfused aromatic diamidine that
can select between intercalation and groove binding. Many biophysical studies have shown that DAPI is a
minor groove binder at A T sequences and DNA footprinting has identified a binding site size of about three
base pairs, in accord with its molecular length. DAPI also has some structural similarity to the unfused
intercalators discussed above and solution studies provided the surprising result that it can bind to G C
sequences by intercalation.
These findings can be rationalized in terms of DNA structure. Both A T and G C sequences form good
intercalation sites; however, only A T base pairs are favourable minor groove binding sites for unfused
aromatic cations like DAPI, netropsin, Hoechst 33258, etc. Therefore DAPI selects the appropriate binding
mode depending on sequence, it intercalates at G C sites but binds in the minor groove at A T sites.
Intercalation of DAPI at G C sites has a binding constant similar to other intercalators such as proflavin. When
it binds at A T minor grooves, it spans more base pairs and makes more specific contacts and hence it binds
with higher affinity in the minor groove.
Reversible Small Molecule–Nucleic Acid Interactions 371
Figure 9.18 Structures of DNA binding ligands SN 6999, chloroquine, DAPI and 4,6-diphenylpyrimidine