Genetic Encoding of <i>para</i>-Pentafluorosulfanyl Phenylalanine: A Highly Hydrophobic and Strongly Electronegative Group for Stable Protein Interactions

Abstract

SF₅Phe, <i>para</i>-pentafluorosulfanyl phenylalanine, is an unnatural amino acid with extreme physicochemical properties, which is stable in physiological conditions. Here we present newly developed aminoacyl-tRNA synthetases that enable genetic encoding of SF₅Phe for site-specific incorporation into proteins in high yields. Owing to the SF₅ moiety's dichotomy of strong polarity and high hydrophobicity, the unnatural amino acid forms specific and strong interactions in proteins. The potential of SF₅Phe in protein research is illustrated by (i) increasing the binding affinity of a consensus pentapeptide motif toward the β subunit of <i>Escherichia coli</i> DNA polymerase III holoenzyme by mutation of a phenylalanine to a SF₅Phe residue, (ii) site-specifically adhering β-cyclodextrin to the surface of ubiquitin, and (iii) selective detection of ¹⁹F-¹⁹F nuclear Overhauser effects in the <i>Escherichia coli</i> peptidyl-prolyl <i>cis</i>/<i>trans</i>-isomerase B following mutation of two phenylalanine residues in the core of the protein to SF₅Phe. With increasing use of the SF₅ moiety in pharmaceutical chemistry, this general method of functionalizing proteins with SF₅ groups opens unique opportunities for structural biology and <i>in vivo</i> studies.