To investigate the effect of incorporation of beta-alanine in alkylating N-methylpyrrole (Py)-N-methylimidazole (Im) polyamide. seco-CBI conjugates 2-8 were synthesized by an Fmoc solid-phase method and subsequent coupling with an alkylating moiety. DNA-alkylating activities of conjugates 2-8 were evaluated by high-resolution denaturing gel electrophoresis with 202-base pair (bp) DNA fragments. Alkylation by conjugates 2 and 3, which have antiparallel pairings of beta-alanine (beta) opposite beta (beta/beta) and Py/beta, occurred mainly at the adenine (A) of the matching sequences, 5'-AGCTCC (C) under bar -3' (site 1) and 5'-AGCACC (A) under bar -3' (site 3). However, conjugate 4, with beta/Py, did not show any DNA-alkylating activities. Similarly, conjugate 5, which possessed a Py/Py pair, weakly alkylated the matching sites at micromolar concentrations. Conjugates 6 and 7. which possessed beta/beta and Py/beta pairs, respectively, alkylated at the A of the matching sequences, 5'-ACTACC (A) under bar -3' (site 2) and 5'-ACAACC (A) under bar -3' (site 4). In contrast, conjugated 8, with a Py/Py pair, showed lower activity and less alkylated DNA at sites 2 and 4 with mismatched alkylation at site I at a higher concentration than that of 6 and 7. These results demonstrate that incorporation of beta-alanine is required for the sequence-specific alkylation by seco-CBI Py-Im conjugates with a seven-base pair sequence. (C) 2007 Elsevier Ltd. All rights reserved.