The designed peptides pyridine-2-carboxamidonetropsin (2-PyN) and 1-methylimidazole-2-carboxamidonetropsin (2-ImN) are crescent-shaped analogs of the natural products netropsin and distamycin A. 2-PyN and 2-ImN bind the 5′-TGTCA-3′ sequence as antiparallel side-by-side dimers in the minor groove of DNA. The binding affinities of 2-PyN and 2-ImN to four different 5-bp sites on DNA were determined by quantitative MPE·Fe(II) footprint titration and compared with the tripeptide D from distamycin. The binding affinities of D to the sites 5′-TTTTT-3′ and 5′-TGTCA-3′ are 2.6 × 107 and <1 × 105 M−1, respectively (pH 7.0, 100 mM NaCl). 2-PyN binds these sites with similar affinities, 2.3 × 105 and 2.7 × 105 M−1, respectively. The affinities of 2-ImN to the same two sites are <5 × 104 and 1.4 × 105 M−1, respectively. Substitution of an N-methylpyrrole-2-carboxamide of the distamycin tripeptide by 1-methylimidazole-2-carboxamide has changed the specificities for the two binding sites by a factor of 103. The data for 2-PyN and 2-ImN binding the 5′-TGTCA-3′ site are best fit by a cooperative binding curve consistent with 2:1 peptide-DNA complexes.
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