Sequence Selectivity of 3-Hydroxypyrrole/Pyrrole Ring Pairings in the DNA Minor Groove

Hairpin polyamides containing the aromatic amino acids 3-hydroxypyrrole (Hp), pyrrole (Py), and imidazole (Im) are capable of discriminating all four Watson−Crick base pairs in the DNA minor groove according to a set of pairing rules. Equilibrium association constants for four eight-ring hairpins containing all four pairings of Hp and Py at a single common position (ImIm X Py-γ-Im Y PyPy-β-Dp, where X / Y is Py/Py, Py/Hp, Hp/Py, and Hp/Hp) were determined at four DNA sites, 5‘-TGG T CA-3‘, 5‘-TGG A CA-3‘, 5‘-TGG C CA-3‘, and 5‘-TGG G CA-3‘, to study the relative binding affinities of the 16 possible complexes. The protected 3-hydroxypyrrole amino acid building block, 3-methoxypyrrole, is prepared on a 50 g scale, and the solid-phase synthesis of hydroxypyrrole−imidazole−pyrrole polyamides is described. Quantitative DNase I footprint titrations demonstrate that a Py/Py pair is partially degenerate for A•T and T•A, but disfavors G•C and C•G base pairs by 53- and 17-fold, respectively. An Hp/Py pair placed opposite T•A binds at least 20-fold more tightly than when placed opposite A•T, G•C, and C•G base pairs. The Py/Hp pair selectively binds A•T with 11-fold higher affinity over T•A and with ≥30-fold selectivity relative to G•C and C•G. An Hp/Hp pairing is disfavored opposite all four base pairs, potentially limiting certain slipped motifs available to unlinked dimers in the minor groove. This study serves to guide the design of second-generation polyamides for DNA recognition.