Protein Conjugation with Triazolinediones: Switching from a General Tyrosine-Selective Labeling Method to a Highly Specific Tryptophan Bioconjugation Strategy

Abstract

Selective labeling of tyrosine residues in peptides and proteins can be achieved via a 'tyrosine-click' reaction with triazolinedione reagents (TAD). We have found that tryptophan residues are in fact often also labeled with this reagent. This off-target labeling is only observed at very low levels in protein bioconjugation but remains under the radar due to the low relative abundance of tryptophan compared to tyrosines in natural proteins, and because of the low availability and accessibility of their nucleophilic positions at the solvent-exposed protein surface. Moreover, because TAD-Trp adducts are known to be readily thermoreversible, it can be challenging to detect these physiologically stable but thermally labile modifications using several MS/MS techniques. We have found that fully solvent-exposed tryptophan side chains are kinetically favored over tyrosines under almost all conditions, and this selectivity can even be further enhanced by modifying the pH of the aqueous buffer to effect selective Trp-labeling. This new site-selective bioconjugation method does not rely on unnatural amino acids and has been demonstrated for peptides and for recombinant proteins. Thus, the TAD-Tyr click reaction can be turned into a highly site-specific labeling method for tryptophan.

Covalent Protein Inhibitors via Tyrosine Conjugation with Cyclic Imine Mannich Electrophiles

“…Lys/Cys). 32,33 Similarly, aryl diazonium salts have seen utility in Tyr conjugation, but high reactivity and poor stability limits use in a TCI. [34][35][36] In 2004, the Francis group reported a three-component Mannich reaction to selectively modify protein tyrosines and tryptophans via the Betti variant of the Mannich reaction.…”