A new aromatic pair, 2-hydroxy-6-methoxybenzamide/1-methylpyrrole at the terminal position of hairpin polyamides has been designed for distinguishing T . A from A . T base pairs and both from G . C/C . G in the minor groove of DNA. Four eight-ring hairpin polyamides with benzamide (Bz), 2-hydroxybenzamide (Hb-l), 2-hydroxy-6-methylbenzamide (Hb-2), and 2-hydroxy-6-methoxybenzamide (Hb-3) at the N-terminal position were synthesized. The equilibrium association constants (K-a) were determined at four DNA sites which differ at a single common position, 5'-TNTACA-3' (N = T, A, G, C). Quantitative DNase I footprint titration experiments reveal that (Hb-3)PyPyPy-(R)(H)2(N)gamma-ImPyPyPy-beta-Dp (4) bound the sequences 5'-TTTACA-3' and 5'-TATACA-3' with high affinity; K-a = 2.6 x 10(10) M-1 and K-a = 8.4 x 10(9) M-1, respectively, a 3-fold specificity for T vs A was found. Importantly, the sequences 5'-TGTACA-3' and 5'-TCTACA-3' are bound with 50 and 200 times lower affinity, revealing an overall specificity of Hb-3/Py of T > A >> G > C. These results expand the repertoire of sequences targetable by hairpin polyamides.