Engineered Modular Heterocyclic-Diamidines for Sequence-Specific Recognition of Mixed AT/GC Base Pairs at the DNA Minor Groove

This report describes a breakthrough in a project to design minor groove binders to recognize any sequence of DNA. A key goal is to invent synthetic chemistry for compound preparation to recognize an adjacent GG sequence that has been difficult to target. After trying several unsuccessful compound designs, an N-alkyl-benzodiimidazole structure was selected to provide two H-bond acceptors for the adjacent GG-NH groups. Flanking thiophenes provide a preorganized structure with strong affinity, DB2831, and the structure is terminated by phenyl-amidines. The binding experimental results for DB2831 with a target AAAGGTTT sequence were successful and include a high ΔTm, biosensor SPR with a KD of 4 nM, a similar KD from fluorescence titrations and supporting competition mass spectrometry. MD analysis of DB2831 bound to an AAAGGTTT site reveals that the two unprotonated N of the benzodiimidazole group form strong H-bonds (based on distance) with the two central G-NH while the central -CH of the benzodiimidazole is close to the -C=O of a C base. These three interactions account for the strong preference of DB2831 for a -GG- sequence. Surprisingly, a complex with one dynamic, interfacial water is favored with 75% occupancy.