Triazolinedione protein modification: from an overlooked off-target effect to a tryptophan-based bioconjugation strategy.

Klaas W Decoene,An Staes,Annemieke Madder,Olivier Zwaenepoel,Kris Gevaert,Johan M Winne,Kamil Unal,Jan Gettemans

doi:10.1039/d1sc06942j

Abstract

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 actually not selective for tyrosine and that tryptophan residues are in fact also labelled with these reagents. This off-target labelling remained under the radar as it is challenging to detect these physiologically stable but thermally labile modifications with the commonly used HCD and CID MS/MS techniques. We show that selectivity of tryptophan over tyrosine can be achieved by lowering the pH of the aqueous buffer to effect selective Trp-labelling. Given the low relative abundance of tryptophan compared to tyrosine in natural proteins, this results in a new site-selective bioconjugation method that does not rely on enzymes nor unnatural amino acids and is demonstrated for peptides and recombinant proteins.

Highlights

Site-selective protein modi cation reactions are highly sought a er by researchers in both academia and industry
Given the low relative abundance of tryptophan compared to tyrosine in natural proteins, this results in a new site-selective bioconjugation method that does not rely on enzymes nor unnatural amino acids and is demonstrated for peptides and recombinant proteins
Yield optimisation experiments with tryptophan containing peptide 1e and triazolinedione reagents (TAD) reagent 2c in PBS at pH 4 demonstrated that 10 equivalents of 2c are sufficient for a conversion of over 90% (ESI Section 2.2.4†)

Summary

Introduction

Site-selective protein modi cation reactions are highly sought a er by researchers in both academia and industry. Labelling of tyrosine residues in peptides and proteins has been reported to selectively occur via a ‘tyrosineclick’ reaction with triazolinedione reagents (TAD). A novel biomimetic approach for the selective conjugation of tryptophan was developed, the original method employs UV irradiation and needs to be performed in absence of oxygen.[20] This approach was further re ned and allows for the use of visible light in presence of ambient air.[21] In 2010, Barbas and co-workers reported a click like reaction for the more abundant tyrosine (Tyr, 3.3% abundance7) using triazolinedione chemistry,[22] a er which many applications and re nements for protein conjugation followed.[23–29] Interestingly, when exploring this powerful Tyr click reaction on Trp containing peptides, we observed a high degree of off-target labelling on Trp residues, even in aqueous buffers.

Results

Conclusion