Second, although CGP 40215A lacks the curvature usually seen with DNA minor-groove binders, DNA interaction studies indicate a high binding affinity with AT-rich sequences. First, it has been found to have biological activity against a number of parasitic microorganisms ( 14, 15). 1) ( 13) has four unique properties distinguishing it from other DNA binding agents.

It is interesting, however, that there has been no detailed study of a binding-linked protonation of a minor-groove binding agent. This acidic environment presumably plays a critical role in protonation of ligands with basic groups that have pK values in an appropriate range. Experimental probing of the minor groove with carboxylic groups in mixed sequences has confirmed the calculated results ( 12). For nucleic acid systems, theoretical treatments have led to the proposal that the DNA minor-groove environment is more acidic than its surroundings ( 11). Consequently, the thermal stability of these structures or complexes is highly pH-dependent, and their binding affinities vary with proton activity. A number of DNA intercalators also have proton linkages to binding interactions ( 9, 10). A typical example for small molecule-nucleic acid systems is the protonation of aminoglycosides on interaction with the RNA major groove ( 5– 8). With proteins, for example, a number of trypsin/thrombin inhibitors have been found to be protonated upon binding ( 3), and the protonation of cytosine-rich DNA sequences is observed upon folding into an i-motif ( 4). Proton uptake or release is often found in biological systems where protonation or deprotonation processes occur upon interactions or folding ( 1, 2). Energetic contributions from different factors were also estimated for the ligand/DNA complex. The surface plasmon resonance binding studies indicate that the charge density per phosphate in DNA hairpins is smaller than that in polymers. Biosensor and calorimetric experiments indicate that the binding affinities vary with pH values and salt concentrations due to protonation and electrostatic interactions. Solvent accessible surface area calculations suggest that surface burial accounts for about one-half of the observed intrinsic negative heat capacity change. The observed binding enthalpy increases as a function of temperature indicating a negative heat capacity change that is typical for DNA minor-groove binders. The exothermic enthalpy of complex formation varies with different pH conditions.

The two methods established a proton-uptake profile as a function of pH. Calorimetric titrations in different buffers and pH conditions support the proton-linkage process and are in a good agreement with spectroscopic titrations. Spectroscopic studies indicate an increase of 2.7 pK a units in the linking group when the compound binds to an A/T minor-groove site. Both amidines are positively charged under experimental conditions, but the linking group for the two phenyl amidines has a pK a of 6.3 that is susceptible to a protonation process. The energetics for binding of a diphenyl diamidine antitrypanosomal agent CGP 40215A to DNA have been studied by spectroscopy, isothermal titration calorimetry, and surface plasmon resonance biosensor methods.

Groove agent 2.0