Fluorination has become an increasingly attractive strategy in protein engineering for both basic research and biomedical applications. Thus researchers would like to understand the consequences of fluorination to the structure, stability, and function of target proteins. Although a substantial amount of work has focused on understanding the properties of fluorinated aliphatic amino acids, much less is known about fluorinated aromatic residues. In addition, polar-π interactions, often referred to as aromatic interactions, may play a significant role in protein folding and protein-protein interactions. Fluorination of aromatic residues presents an ideal strategy for probing polar-π interactions in proteins. This Account summarizes the recent studies of the incorporation of fluorinated aromatic amino acids into proteins. Herein we discuss the effects of fluorinating aromatic residues and rationalize them in the context of polar-π interactions. The results strongly support the proposal that polar-π interactions are energetically significant to protein folding and function. For example, an edge-face interaction of a pair of phenylalanines contributes as much as -1 kcal/mol to protein stability, while cation-π interactions can be much stronger. Furthermore, this new knowledge provides guidelines for protein engineering with fluorination. Importantly, incorporating perfluorinated aromatic residues into proteins enables novel mechanisms of molecular recognition that do not exist in native proteins, such as arene-perfluoroarene stacking. Such novel mechanisms can be used for programming protein folding specificity and engineering peptide-based materials.

Makeover of an old antibiotic: The channel‐forming toxin gramicidin A (gA) has been successfully converted into agents that selectively chase after bacterial cells. These novel D‐Lys‐containing gA mutants display potent antimicrobial activity and remarkable therapeutic indexes (>1000‐fold).

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Aromatic “bonding”: Although known to exist in proteins, aromatic stacking interactions and the energetic factors important for it are not well understood. A systematic investigation of aromatic stacking interactions in a protein model system using a series of fluorinated phenylalanine analogues illustrates the importance of dipole–dipole and dipole–induced‐dipole coupling to the stability and self‐sorting properties of aromatic stacking pairs.

CH-π stacks up! Using the protein α(2) D as a model system, we estimate that a CH-π contact between cyclohexylalanine (Cha) and phenylalanine (F) contributes approximately -0.7 kcal  mol(-1) to the protein stability. The stacking F-Cha pairs are sequestered in the core of the protein, where water interference does not exist (see figure). Therefore, the observed energetic gain should represent the inherent magnitude and upper limit of the CH-π interactions.