Published: March 10, 2011 r2011 American Chemical Society 3127 dx.doi.org/10.1021/bi102028a | Biochemistry 2011, 50, 3127–3136 ARTICLE pubs.acs.org/biochemistry Hx, a Novel Fluorescent, Minor Groove and Sequence Specific Recognition Element: Design, Synthesis, and DNA Binding Properties of p-Anisylbenzimidazole-imidazole/pyrrole-Containing Polyamides Sameer Chavda, † Yang Liu, ‡ Balaji Babu, † Ryan Davis, † Alan Sielaff, † Jennifer Ruprich, † Laura Westrate, † Christopher Tronrud, † Amanda Ferguson, † Andrew Franks, † Samuel Tzou, † Chandler Adkins, # Toni Rice, † Hilary Mackay, † Jerome Kluza, § Sharjeel A Tahir, § Shicai Lin, § Konstantinos Kiakos, § Chrystal D. Bruce, # W. David Wilson, ‡ John A. Hartley, § and Moses Lee* ,† † Division of Natural and Applied Sciences and Department of Chemistry, Hope College, Holland, Michigan 49423, United States ‡ Department of Chemistry, Georgia State University, Atlanta, Georgia 30302, United States § Cancer Research UK Drug-DNA Interactions Research Group, UCL Cancer Institute, Paul O’Gorman Building, 72 Huntley Street, London WC1E 6BT, UK # Department of Chemistry, Erskine College, Due West, South Carolina 29639, United States b S Supporting Information P yrrole (P) and imidazole (I)-containing polyamide analogues of the naturally occurring distamycin A (1) bind in the minor groove of DNA in a stacked, antiparallel 2:1 (ligand/DNA) motif. 1À3 Early studies have led to the establishment of a set of pairing rules for DNA base pair recognition; a P/P stacking recognizes an A/T or T/A base pair, an I/P stacking binds to a G/C base pair, while a P/I stacking recognizes a C/G base pair. In addition, a stacked I/I pairing targets a T/G mismatch. 4À6 As a result of the high degree of sequence specificity that these molecules exhibit, their potential use as regulators of gene expression has been widely explored. 7 However, attempts to inhibit the transcription of genes that code for a disease in cell lines have been met with moderate success which presumably points toward poor cellular uptake of some of these polyamides and their limited ability to achieve nuclear localization. 8,9 Some success in this area has been achieved through the attachment of Received: December 21, 2010 Revised: March 9, 2011 ABSTRACT: With the aim of incorporating a recognition element that acts as a fluorescent probe upon binding to DNA, three novel pyrrole (P) and imidazole (I)-containing polyamides were synthesized. The compounds contain a p-anisylbenzimida- zolecarboxamido (Hx) moiety attached to a PP, IP, or PI unit, giving compounds HxPP (2), HxIP (3), and HxPI (4), respec- tively. These fluorescent hybrids were tested against their complementary nonfluorescent, non-formamido tetraamide counterparts, namely, PPPP (5), PPIP (6), and PPPI (7) (cognate sequences 5 0 -AAATTT-3 0 ,5 0 -ATCGAT-3 0 , and 5 0 - ACATGT-3 0 , respectively). The binding affinities for both series of polyamides for their cognate and noncognate sequences were ascertained by surface plasmon resonance (SPR) studies, which revealed that the Hx-containing polyamides gave binding con- stants in the 10 6 M À1 range while little binding was observed for the noncognates. The binding data were further compared to the corresponding and previously reported formamido-triamides f-PPP (8), f-PIP (9), and f-PPI (10). DNase I footprinting studies provided additional evidence that the Hx moiety behaved similarly to two consecutive pyrroles (PP found in 5À7), which also behaved like a formamido-pyrrole (f-P) unit found in distamycin and many formamido-triamides, including 8À10. The biophysical characterization of polyamides 2À7 on their binding to the abovementioned DNA sequences was determined using thermal melts (ΔT M ), circular dichroism (CD), and isothermal titration calorimetry (ITC) studies. Density functional calculations (B3LYP) provided a theoretical framework that explains the similarity between PP and Hx on the basis of molecular electrostatic surfaces and dipole moments. Furthermore, emission studies on polyamides 2 and 3 showed that upon excitation at 322 nm binding to their respective cognate sequences resulted in an increase in fluorescence at 370 nm. These low molecular weight polyamides show promise for use as probes for monitoring DNA recognition processes in cells.