The incorporation of fluorine can not only significantly facilitate the study of proteins but also potentially modulate their function. Though some biosynthetic methods allow global residue-replacement, post-translational fluorine incorporation would constitute a fast and efficient alternative. Here, we reveal a mild method for direct protein radical trifluoromethylation at native residues as a strategy for symmetric-multifluorine incorporation on mg scales with high recoveries. High selectivity toward tryptophan residues enhanced the utility of this direct trifluoromethylation technique allowing ready study of fluorinated protein constructs using 19F-NMR.

18 F labeling strategies for unmodified peptides with [ 18 F]fluoride require 18 F-labeled prosthetics for bioconjugation more often with cysteine thiols or lysine amines. Here we explore selective radical chemistry to target aromatic residues applying C− H 18 F-trifluoromethylation. We report a one-step route to [ 18 F]CF 3 SO 2 NH 4 from [ 18 F]fluoride and its application to direct [ 18 F]CF 3 incorporation at tryptophan or tyrosine residues using unmodified peptides as complex as recombinant human insulin. The fully automated radiosynthesis of octreotide[Trp(2-CF 2 18 F)] enables in vivo positron emission tomography imaging.Communication pubs.acs.org/JACS

The carbonyl group is now a widely useful, nonproteinogenic functional group in chemical biology, yet methods for its generation in proteins have relied upon either cotranslational incorporation of unnatural amino acids bearing carbonyls or oxidative conversion (chemical or enzymatic) of existing natural amino acids. If available, alternative strategies for directly adding the CO group through C–C bond-forming C-carbonylation, particularly at currently inaccessible amino acid sites, would provide a powerful method for adding valuable reactivity and expanding possible function in proteins. Here, following a survey of methods for HCF2· generation, we show that reductive photoredox catalysis enables mild radical-mediated difluoromethylation-hydrolysis of native protein residues as an effective method for carbonylation. Inherent selectivity of HCF2· allowed preferential modification of Trp residues. The resulting C-2-difluoromethylated Trp undergoes Reimer-Tiemann-type dehalogenation providing highly effective spontaneous hydrolytic collapse in proteins to carbonylated HC­(O)-Trp (C-formyl-Trp = CfW) residues. This new, unnatural protein residue CfW not only was found to be effective in bioconjugation, ligation, and labeling reactions but also displayed strong “red-shifting” of its absorption and fluorescent emission maxima, allowing direct use of Trp sites as UV–visualized fluorophores in proteins and even cells. In this way, this method for the effective generation of masked formyl-radical “HC­(O)·” equivalents enables first examples of C–C bond-forming carbonylation in proteins, thereby expanding the chemical reactivity and spectroscopic function that may be selectively and post-translationally “edited” into biology.

Stapled peptides with an enforced α-helical conformation have been shown to overcome major limitations in the development of short peptides targeting protein-protein interactions (PPI). While the growing arsenal of methodologies to staple peptides facilitates their preparation, stapling methodologies are not broadly embraced in synthetic library screening. Herein, we report a strategy leveraged on hybridization of short PNA-peptide conjugates wherein nucleobase driven assembly facilitates ligation of peptide fragments and constrains the peptide's conformation into an α-helix. Using native chemical ligation, we show that a mixture of peptide fragments can be combinatorially ligated and used directly in affinity selection against a target of interest. This approach was exemplified with a focused library targeting the p-53 / MDM2 interaction. One hundred peptides were obtained in a one-pot ligation reaction, selected by affinity against MDM2 immobilized on beads and the best binders were identified by mass spectrometry.