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 pairi ng rules. Equilibrium association constants for four eight-ring hairpins co ntaining all four pairings of Hp and Py at a single common position (ImImXP y-gamma-ImYPyPy-beta-Dp, where X/Y is Py/Py, Py/Hp, Hp/Py, and Hp/Hp) were determined at four DNA sites, 5'-TGGTCA-3', 5'-TGGACA-3', 5'-TGGACA-3', and 5'-TGGGCA-3', to study the relative binding affinities of the 16 possible complexes. The protected 3-hydroxypyrrole amino acid building block, 3-meth oxypyrrole, is prepared on a 50 g scale, and the solid-phase synthesis of h ydroxypyrrole-imidazole-pyrrole polyamides is described. Quantitative DNase I footprint titrations demonstrate that a Py/Py pair is partially degenera te 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 mo re tightly than when placed opposite A.T, G.C, and C.G base pairs. The Py/H p pair selectively binds A.T with 11-fold higher affinity over T.A and with greater than or equal to 30-fold selectivity relative to G.C and C.G. An H p/Hp pairing is disfavored opposite all four base pairs, potentially limiti ng 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.