Modifying the N-terminus of polyamides: PyImPyIm has improved sequence specificity over f-ImPyIm

Abstract

Seven N-terminus modified derivatives of a previously published minor-groove binding polyamide (f-ImPyIm, 1) were synthesized and the biochemical and biophysical chemistry evaluated. These compounds were synthesized with the aim of attaining a higher level of sequence selectivity over f-ImPyIm ( 1), a previously published strong minor-groove binder. Two compounds possessing a furan or a benzofuran moiety at the N-terminus showed a footprint of 0.5 μM at the cognate ACGCGT site (determined by DNase I footprinting); however, the specificity of these compounds was not improved. In contrast, PyImPyIm ( 4) produced a footprint of 0.5 μM but showed a superior specificity using the same technique. When evaluated by thermal melting experiments and circular dichroism using ACGCGT and the non-cognate AAATTT sequence, all compounds were shown to bind in the minor-groove of DNA and stabilize the cognate sequence much better than the non-cognate (except for the non-amido-compound that did not bind either sequence, as expected). PyImPyIm ( 4) was interesting as the Δ T m for this compound was only 4 °C but the footprint was very selective. No binding was observed for this compound with a third DNA (non-cognate, ACCGGT). ITC studies on compound 4 showed exothermic binding with ACGCGT and no heat change was observed for titrating the compound to the other two DNA sequences. The heat capacity (Δ C p) of the PIPI/ACGCGT complex calculated from the hydrophobic interactions and SASA calculations was comparable to the experimental value obtained from ITC (−146 cal mol −1 K −1). SPR results provided confirmation of the sequence specificity of PyImPyIm ( 4), with a K eq value determined to be 7.1 × 10 6 M −1 for the cognate sequence and no observable binding to AAATTT and ACCGGT. Molecular dynamic simulations affirmed that PyImPyIm ( 4) binds as a dimer in an overlapped conformation, and it fits snugly in the minor-groove of the ACGCGT oligonucleotide. PyImPyIm ( 4) is an especially interesting molecule, because although the binding affinity is slightly reduced, the specificity with respect to f-ImPyIm ( 1) is significantly improved.