Harnessing Cyclohexadiene Chemistry for Chemical Modification of Unprotected Peptides

Because of its chemical inertness, phenylalanine is one of the proteinogenic amino acids that does not have many chemical labeling methods for, particularly in unprotected peptide substrates. Inspired by a recently reported mechanochemical Birch reduction method, we tested a reduction-based approach as well as inverse electron-demand Diels-Alder reactions to develop a phenylalanine-targeting bioconjugation method using 1,4-cyclohexadiene as a key chemical species. Chemoselective reduction of the phenyl to cyclohexadienyl group was achieved for dipeptides and tripeptides that do not contain tryptophan residues. Through a computation- and experiment-guided approach, we also demonstrated the possibility of 1,4-cyclohexadiene-based Diels-Alder reactions on an unprotected peptide using a series of monosubstituted tetrazine reagents.