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.
Bioinspired Thiophosphorodichloridate Reagents for Chemoselective Histidine Bioconjugation
