9.7.5.1 Structures of Hoechst 33258–DNA Complexes. Hoechst 33258–DNA structures are
extremely well studied. NMR analysis has revealed a number of locations and conformations of the drug in
5 -AATT sites. The phenolic ring is able to undergo rapid ring-flips, which involve 180° rotations about the
phenol–benzimidazole bond, even though the phenolic ring is tightly accommodated between the walls of
the minor groove. Dynamic breathing motions must occur in the DNA that results in transient widening of
the minor groove to allow the ring to flip. The piperazine ring also adopts several different conformations.
These two rings at the termini of the molecule are generally more mobile than the bis-benzimidazole core.
In one Hoechst 33258–A2T2 X-ray crystal structure, the ligand is located in the central 5 -AATT tract
in a similar manner to netropsin. Hydrogen bonds are observed from the benzimidazole amide groups to
O-2 atoms on thymine and N-3 atoms on adenine. Hoechst 33258 is isohelical with the curvature of the
DNA minor groove and numerous molecular contacts are made with the walls of the groove. The phenolic
ring of the ligand is twisted 8° with respect to the adjacent benzimidazole ring and the two benzimidazole
rings are twisted 32° with respect to each other. The piperazine ring is only slightly puckered and is almost
Reversible Small Molecule–Nucleic Acid Interactions 367
+ β Py Py Py ImIm Py Py PyFigure 9.16 (a) The Dervan Pairing Rules, which describe how different combinations of imidazole (Im), pyrrole
(Py) and hydroxypyrrole (Hp) rings placed opposite each other can be used to distinguish all four types
of base pairs. (b) and (c) These pairing rules can be applied to target region of DNA with the sequence
5 -AGTACT (dimethylaminopropylamine residue)