Convergent synthetic routes for N-methylpyrrole (P) and N-methylimidazole (I) seco-1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benz[e]indole (CBI) conjugates with a vinyl linker were developed. New hairpin polyamide-seco-CBI conjugates, compounds 16-19, were synthesized, and their DNA sequence-specific alkylating activities were evaluated via high-resolution denaturing gel electrophoresis and high-performance liquid chromatography (HPLC) product analysis. The new synthetic route for PI conjugates with a vinyl linker consisted of the introduction of a vinylpyrrole unit (8-11) into the C terminal of a PI polyamide synthesized by (fluorenylmethoxy)carbonyl solid-phase peptide synthesis (SPPS), followed by liquid-phase coupling with seco-CBI. The yield of the conjugates was significantly improved compared with that of the method reported previously, which allows us to synthesize various substituted conjugates containing a vinyl linker. Conjugates 16-19 were designed to investigate the substituent effect of the vinyl linker, and conjugate 16S was synthesized to evaluate the reactivity between racemic and S enantiomers of the seco-CBI derivative. The results of high-resolution denaturing gel electrophoresis using 208 bp DNA fragments indicated that alkylation by compounds 16 and 17, in which the H of the vinyl linker of compound 16 was replaced with F, occurred predominantly at the A of the 5'-TTTGTCA-3' sequence at nanomolar concentrations. In clear contrast, compounds 18 and 19, which were methyl or Br derivatives of compound 16, did not exhibit any DNA alkylating activity. Moreover, HPLC product analysis using synthetic oligonucleotides demonstrated that alkylation occurred between the N3 of the adenine of the oligomer and the cyclopropane ring of 16S. Density functional calculation of substituted vinylpyrrole seco-CBI units indicated that methyl and Br substituents led to a significantly distorted geometry of the vinyl group with the pyrrole ring compared with H and F derivatives. Molecular modeling studies offered the additional information that steric hindrance reduced the DNA alkylating activity of these derivatives.