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 gamma-aminobutyric acid (gamma) residue form hairpin polyamide structures. Selective amino-substitution of the prochiral alpha-position of the gamma-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-(gamma-ImPyPyPy-(R)(H2N)gamma-ImPyPyPy-) binds a 5＇-AGTACT-3＇ site with an equilibrium association constant K-a = 7.6 x 10(10) M-1, a 3600-fold enhancement relative to the unlinked homodimer (ImPyPyPy-beta-Dp)(2).5＇AGTACT-3＇, and an 8-fold enhancement relative to hairpin analogue ImPyPyPy-(R)(H2N)gamma-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 gamma-turn combined with placement of a second gamma-turn within the hairpin structure provides a cycle polyamide motif with favorable DNA binding properties.