The concept of the halogen bond (or X-bond) has become recognized as contributing significantly to the specificity in recognition of a large class of halogenated compounds. The interaction is most easily understood as primarily an electrostatically driven molecular interaction, where an electropositive crown, or r-hole, serves as a Lewis acid to attract a variety of electronrich Lewis bases, in analogous fashion to a classic hydrogen bonding (H-bond) interaction. We present here a broad overview of X-bonds from the perspective of a biologist who may not be familiar with this recently rediscovered class of interactions and, consequently, may be interested in how they can be applied as a highly directional and specific component of the molecular toolbox. This overview includes a discussion for where X-bonds are found in biomolecular structures, and how their structure-energy relationships are studied experimentally and modeled computationally. In total, our understanding of these basic concepts will allow X-bonds to be incorporated into strategies for the rational design of new halogenated inhibitors against biomolecular targets or toward molecular engineering of new biological-based materials.

Halogens are important substituents of many drugs and secondary metabolites, but the structural and thermodynamic properties of their interactions are not properly treated by current molecular modeling and docking methods that assign simple isotropic point charges to atoms. Halogen bonds, for example, are becoming widely recognized as important for conferring specificity in protein-ligand complexes but, to this point, are most accurately described quantum mechanically. Thus, there is a need to develop methods to both accurately and efficiently model the energies and geometries of halogen interactions in biomolecular complexes. We present here a set of potential energy functions that, based on fundamental physical properties of halogens, properly model the anisotropic structure-energy relationships observed for halogen interactions from crystallographic and calorimetric data, and from ab initio calculations for bromine halogen bonds in a biological context. These energy functions indicate that electrostatics alone cannot account for the very short-range distances of bromine halogen bonds but require a flattening of the effective van der Waals radius that can be modeled through an angular dependence of the steric repulsion term of the standard Lennard-Jones type potential. This same function that describes the aspherical shape of the bromine is subsequently applied to model the charge distribution across the surface of the halogen, resulting in a force field that uniquely treats both the shape and electrostatic charge parameters of halogens anisotropically. Finally, the electrostatic potential was shown to have a distance dependence that is consistent with a charge-dipole rather than a simple Coulombic type interaction. The resulting force field for biological halogen bonds (ffBXB) is shown to accurately model the geometry-energy relationships of bromine interactions to both anionic and neutral oxygen acceptors and is shown to be tunable by simply scaling the electrostatic component to account for effects of varying electron-withdrawing substituents (as reflected in their Hammett constants) on the degree of polarization of the bromine. This approach has broad applications to modeling the structure-energy relationships of halogen interactions, including the rational design of inhibitors against therapeutic targets.

Interest in noncovalent interactions involving halogens, particularly halogen bonds (X-bonds), has grown dramatically in the past decade, propelled by the use of X-bonding in molecular engineering and drug design. However, it is clear that a complete analysis of the structure-energy relationship must be established in biological systems to fully exploit X-bonds for biomolecular engineering. We present here the first comprehensive experimental study to correlate geometries with their stabilizing potentials for fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) X-bonds in a biological context. For these studies, we determine the single-crystal structures of DNA Holliday junctions containing halogenated uracil bases that compete X-bonds against classic hydrogen bonds (H-bonds), estimate the enthalpic energies of the competing interactions in the crystal system through crystallographic titrations, and compare the enthalpic and entropic energies of bromine and iodine X-bonds in solution by differential scanning calorimetry. The culmination of these studies demonstrates that enthalpic stabilization of X-bonds increases with increasing polarizability from F to Cl to Br to I, which is consistent with the σ-hole theory of X-bonding. Furthermore, an increase in the X-bonding potential is seen to direct the interaction toward a more ideal geometry. However, the entropic contributions to the total free energies must also be considered to determine how each halogen potentially contributes to the overall stability of the interaction. We find that bromine has the optimal balance between enthalpic and entropic energy components, resulting in the lowest free energy for X-bonding in this DNA system. The X-bond formed by iodine is more enthalpically stable, but this comes with an entropic cost, which we attribute to crowding effects. Thus, the overall free energy of an X-bonding interaction balances the stabilizing electrostatic effects of the σ-hole against the competing effects on the local structural dynamics of the system.

Bromine halogen bonds (X-bonds) had previously been assayed using DNA junctions in which the X-bonds compete against hydrogen bonds (H-bonds) in defining the conformational form of the junction. Here, we show calorimetrically that the stabilizing effect of the X-bond in solution derives primarily from a negative enthalpy (−5 kcal/mol) but is opposed by a negative entropy (−8 cal/mol·K, equivalent to −2 kcal/mol for TΔS at room temperature), resulting in an overall stabilizing free energy of −3 kcal/mol for the X- vs H-bond. Quantum chemical energies for this X-bond are nearly identical to energies derived from the crystallographic and solution assays, confirming that the stabilizing potentials are primarily reflected in the components of the X-bond. A study in which the bromine is replaced by a methyl group (substituents that are similar in size and hydrophobicity) showed that the solvent and steric effects of burying these substituents in the tight pocket of the junction is nearly equivalent in energy to the competing H-bond. Thus, the stabilization of DNA junctions by a bromine X-bond in crystals is reflected in the enthalpic stabilization in solution and that both energies are direct measures of the X-bonding potential of the bromine in a biomolecular system.

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