Abstract
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.
Highlights
Carbonyl groups are not typically found in proteins, so when introduced, they create additional reactivity that has proven widely useful.[1]
During the course of our investigation into other difluoroalkyl radicals with proteins, we have discovered the sufficient reactivity of HCF2· toward native residues when generated under mild, reductive conditions and a reactivity that is coupled through their inherent chemoselectivity with a Reimer-Tiemann hydrolytic manifold in heteroaromatic residue (X = Trp > His, Figure 2c) adducts.[17,18]
The resulting, nonproteinogenic residue C-formyl-Trp (Cfw) bears a minimally sized reactive group at C2 that we show here proves useful for selective, electrophilic carbonyl-mediated modification in proteins as well acting as an enhanced fluorophore
Summary
Carbonyl groups are not typically found in proteins, so when introduced, they create additional reactivity that has proven widely useful.[1]. The resulting, nonproteinogenic residue C-formyl-Trp (Cfw) bears a minimally sized reactive group at C2 that we show here proves useful for selective, electrophilic carbonyl-mediated modification in proteins as well acting as an enhanced fluorophore. Borodeuteride-mediated detection on model peptides revealed an expected mass shift in >95% of the peaks corresponding to both singly and doubly formylated species (Figure 4c) This was complemented by LCMS/MS fragmentation analyses that revealed Trp residues in KFWAWH as the main site of difluoromethyl radical addition, along with a minor population localized on His or Phe (Supplementary Figure S9). To assess the selectivity of the reaction, we performed tryptic digestion and analyzed resulting peptides via LC-MS/MS This revealed desired difluoromethylated (hetero)aromatic products and some nonspecific N-formylation (corresponding to mass of apparent hydrolysis and as had been seen in peptide models (see above)) (Supplementary Figure S13). 6b) similar to observations made on proteins in vitro (see above)
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