Incorporation of the flexible amino acid beta-alanine (beta) into hairpin polyamides composed of N-methylpyrrole (Py) and N-methylimidazole (Im) amino acids is required for binding to DNA sequences longer than seven base pairs with high affinity and sequence selectivity. Pairing the alpha-substituted-beta-amino acids (S)-isoserine ((S)Is), (R)-isoserine ((R)Is), beta-aminoalanine (Aa), and alpha-fluoro-beta-alanine (Fb) side-by-side with beta in hairpin polyamides alters DNA binding affinity and selectivity relative to the parent polyamide containing a beta/beta pairing. Quantitative DNase I footprinting titration studies on a restriction fragment containing the sequences 5'-TGC (N) under bar CTA-3' ((N) under bar = A, T, G, and C) show that the polyamide ImPy(S)IsImPy-gamma-PyPy beta ImPy-beta-Dp ((S)Is/beta pairing) binds to (N) under bar = T (K-a = 4.5 x 10(9) M-1) in preference to (N) under bar = A (K-a = 6.2 x 10(8) M-1). This result stands in contrast to the essentially degenerate binding of the parent ImPy beta ImPy-gamma-PyPy beta ImPy-beta-Dp (beta/beta pairing) to (N) under bar = T and (N) under bar = A, and to the slight preference of ImPy beta ImPy-gamma-PyPy(S)IsImPy-beta-Dp (beta/(S)Is pairing) to (N) under bar = A over (N) under bar = T. Additionally, this study reveals that incorporation of (R)Is, Aa, and Fb into polyamides significantly reduces binding affinity. Therefore, DNA binding in the minor groove is sensitive to the stereochemistry, steric bulk, and electronics of the substituent at the alpha-position of beta-amino acids in hairpin polyamides containing beta/beta pairs.