Effects of the N-terminal acylamido group of imidazole- and pyrrole-containing polyamides on DNA sequence specificity and binding affinity.

The N-terminal formamido group on imidazole- and pyrrole-containing polyamides causes stacked polyamides to bind in the minor groove of DNA in the staggered motif, and it also increases the binding affinity compared to those of non-formamido compounds. To further investigate the role of the N-terminal acylamido in affecting sequence specificity and binding affinity, six polyamide analogues containing the core triheterocyclic structure IPI were designed and synthesized, and the acylamido moiety reported herein includes the following: formamido (f-IPI, 1), acetamido (Ac-IPI, 2), trifluoroacetamido (Tf-IPI, 3), N-methylureido (Mu-IPI, 4), N-methylpyrrole-2-carboxamido (PIPI, 5), and the (13)C-labeled formamido-IPI compound ((13)C-f-IPI, 6). In addition, two nonacylated IPI compounds were also synthesized and examined, namely, the amino-containing (NH(2)-IPI, 7) and non-formamido (nf-IPI, 8) compounds. The binding characteristics of compounds 1-8 were investigated using methods of molecular biology and biochemistry, which included biophysical techniques, such as DNA melts, circular dichroism, isothermal titration calorimety, and surface plasmon resonance and DNase I footprinting. With the exception of nf-IPI and NH(2)-IPI, all other compounds preferentially interacted with the cognate sequence, 5'-ACGCGT-3'. The biophysical results suggest that all six compounds bind within the minor groove at their cognate DNA sequence as stacked, staggered, antiparallel dimers. The order from highest to lowest binding affinities is as follows: f-IPI > P-IPI > Ac-IPI > Mu-IPI > Tf-IPI >> NH(2) and nf-IPI. Hence, having an acylamido moiety at the N-terminus is important for the binding of polyamides to DNA in a stacked and staggered motif. According to footprinting analysis, P-IPI (5), Ac-IPI (2), Mu-IPI (4), and Tf-IPI (3) exhibited some enhancement in sequence preference for their cognate 5'-ACGCGT-3' over f-IPI (1). NMR analysis of the [(13)C]f-IPI-CGCGnmr complex showed a slight downfield shift in the formamido (13)C signal indicating that the moiety remained intact. The trend in binding affinity suggests that steric factors play a role, in which small and planar aromatic acylamido units such as f, Ac, and P are preferred. Polar groups, such as in Mu-IPI (4) and Tf-IPI (3), afforded negative effects on binding affinity, compared to that of Ac-IPI (2).