Residue-Selective Protein C-Formylation via Sequential Difluoroalkylation-Hydrolysis

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.

Stereoretentive Post-Translational Protein Editing

“…The former provides a reactive acetophenone carbonyl-containing moiety with the consequent potential for further application in diverse bioconjugation. 63 The latter allows chemical generation of L-homoallylglycine (Hag), an archetypal ‘tag’ sidechain for thiyl-ene ligations 64 The ability to introduce this chemically now complements its typical introduction via sense-codon reassignment.…”

Late-stage difluoromethylation: concepts, developments and perspective

“…98 In 2021, Davis and co-workers demonstrated the site-selective difluoromethylation of tryptophan (Trp) residues in proteins (Scheme 20B). 99 A range of proteins undergoing difluoromethylation at the C2 position of Trp, include annexin A5, lysozyme, cationic trypsin and lactalbumin. Once installed, the CF 2 H spontaneously hydrolysed resulting in net C-H formylation.…”

“…Seminal work in this direction has recently been reported by Davis, Gouverneur and co-workers, who developed site-selective photocatalytic hydrodifluoromethylation and difluoromethylation of dehydroalanine and tryptophan residues in proteins, respectively. 99,134…”

Visible Light‐Mediated Metal‐Free Chlorodifluoromethylation of Arenes and Heteroarenes by a Hypervalent Iodine EDA Complex

“…[8][9][10][11] Recent efforts towards aryl difluoromethylation have centred on direct radical transformations, enabling more facile late-stage functionalization of drug-like molecules. [12][13][14][15] Photochemical generation of alkyl radicals of this type has emerged as an important reaction motif. [16] In particular, electron donor-acceptor (EDA) complexes, comprised of the molecular aggregation of an electron rich donor with an electron deficient acceptor, have revolutionized a wide array of visible light mediated synthetic transformations.…”

“…As a result, many transition metal mediated procedures have been developed to furnish difluoromethylated (hetero)arenes via cross coupling, but most of these methods require prefunctionalized substrates and expensive metals [8–11] . Recent efforts towards aryl difluoromethylation have centred on direct radical transformations, enabling more facile late‐stage functionalization of drug‐like molecules [12–15] . Photochemical generation of alkyl radicals of this type has emerged as an important reaction motif [16] .…”