Labelling of tyrosine residues in peptides and proteins has been reported to selectively occur via a 'tyrosine-click' reaction with triazolinedione reagents (TAD). However, we here demonstrate that TAD reagents are...
Flexible in vitro translation (FIT) was used as a screening method to uncover a new methodology for peptide constraining based on the attack of a nucleophilic side-chain functionality onto an oxidized furylalanine side chain. A set of template peptides, each containing furylalanine as furan-modified amino acid and a nucleophilic residue (Cys, His, Lys, Arg, Ser, or Tyr), was produced through FIT. The translation mixtures were treated with N-bromosuccinimide (NBS) to achieve selective furan oxidation and subsequent MALDI analysis demonstrated Lys and Ser as promising residues for cyclisation. Solid-phase peptide synthesis (SPPS) was used to synthesize suitable amounts of material for further in-depth analysis and characterisation. It was found that in the case of the peptide containing lysine next to a furylalanine residue, a one-pot oxidation and reduction reaction leads to the generation of a cyclic peptide featuring a pyrrole moiety as cyclisation motif, resulting from the attack of the lysine side chain onto the oxidized furylalanine side chain. Structural evidence was provided via NMR and the generality of the methodology was explored. We hereby expand the scope of our previously developed furan-based peptide labeling and crosslinking strategy.
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.
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