Cycle Polyamide Motif for Recognition of the Minor Groove of DNA

Motifs for covalent linkage of side-by-side complexes of pyrrole−imidazole (Py−Im) polyamides in the DNA minor groove provide for small molecules that specifically recognize predetermined sequences with subnanomolar affinity. Polyamide subunits linked by a turn-specific γ-aminobutyric acid (γ) residue form hairpin polyamide structures. Selective amino-substitution of the prochiral α-position of the γ-turn residue relocates the cationic charge from the hairpin C terminus. Here we report the synthesis of pyrrole resin as well as a solid-phase strategy for the preparation of cycle polyamides. The DNA binding properties of two eight-ring cycle polyamides were analyzed on a DNA restriction fragment containing six base pair match and mismatch binding sites. Quantitative footprint titrations demonstrate that a cycle polyamide of sequence composition cyclo-(γ-ImPyPyPy-(R)H2Nγ-ImPyPyPy-) binds a 5‘-AGTACT-3‘ site with an equilibrium association constant K a = 7.6 × 1010 M-1, a 3600-fold enhancement relative to the unlinked homodimer (ImPyPyPy-β-Dp)2·5‘-AGTACT-3‘, and an 8-fold enhancement relative to hairpin analogue ImPyPyPy-(R)H2Nγ-ImPyPyPy-C3−OH·5‘-AGTACT-3‘. Replacement of a single nitrogen atom with a C−H (Im→Py) regulates affinity and specificity of the cycle polyamide by 2 orders of magnitude. The results presented here suggest that addition of a chiral γ-turn combined with placement of a second γ-turn within the hairpin structure provides a cycle polyamide motif with favorable DNA binding properties.