The hydrophobic effect (or the aggregated effects that we call “the hydrophobic effect”) underlies the binding of many ligands to proteins. It involves three molecular participants: the surface of the binding pocket of the protein, the surface of the ligand, and the networks of water molecules that fill the pocket and surround the ligand. The molecular-level mechanism of the hydrophobic effect in protein-ligand binding remains a subject of substantial controversy.[1, 2] There are three primary questions of interest: i)“Do hydrophobic effects reflect the release of structured, and hence entropically unfavorable, water from hydrophobic surfaces when the ligand and surface of the binding pocket come into contact?”; ii)“Do hydrophobic effects represent the release of free-energetically unfavorable water from hydrogen-bonded networks in the binding pocket or displacement by the ligand, and the release of free-energetically unfavorable (although perhaps different) waters from the hydrophobic surface of the ligand?”; and iii)“How important in free energy are the contact interactions between the protein and the ligand in the binding pocket?”.

In a previous examination of these questions,[3] we compared the binding of a series of heteroarylsulfonamide ligands, and their “benzo-extended” analogs (Scheme 1), to human carbonic anhydrase II (HCA; EC 4.2. 1.1). In these binding events, the addition of a benzo group: i) increased the hydrophobic surface area (and the volume) of the ligand; ii) generated two new van der Waals contacts between the ligand and hydrophobic wall of HCA; but iii) did not result in a significant increase in the area of contact between the hydrophobic surfaces of the protein and ligand. The free energy of binding of the arylsulfonamide ligands increased with the additional surface area buried upon binding from the benzo-extension by− 20 cal mol 1 Å 2,[3] an amount expected for normal hydrophobic effects (− 20 to–33 cal mol