Electrochemically Promoted Tyrosine-Click-Chemistry for Protein Labeling

Abstract

The development of new bio-orthogonal ligation methods for the conjugation of native proteins is of particular importance in the field of chemical biology and biotherapies. In this work, we developed a traceless electrochemical method for protein bioconjugation. The electrochemically promoted tyrosine-click (e-Y-CLICK) allowed the chemoselective Y-modification of peptides and proteins with labeled urazoles. A low potential is applied in an electrochemical cell to activate urazole anchors in situ and on demand, without affecting the electroactive amino acids from the protein. The versatility of the electrosynthetic approach was shown on biologically relevant peptides and proteins such as oxytocin, angiotensin 2, serum bovine albumin, and epratuzumab. The fully conserved enzymatic activity of a glucose oxidase observed after e-Y-CLICK further highlights the softness of the method. The e-Y-CLICK protocols were successfully performed in pure aqueous buffers, without the need for co-solvents, scavenger or oxidizing chemicals, and should therefore significantly broaden the scope of bioconjugation.

Discriminating changes in protein structure using PTAD conjugation to tyrosine

“…We confirmed by 1 H NMR the reaction with a tyrosine analogue, 3-(4-hydroxyphenyl)propionic acid (Supplemental Spectra 1). We allowed PTAD to react with the tyrosine mimic for 15 minutes and observed conversion to labeled tyrosine, consistent with previous reports (Figure 1A) 16,17 . By repeated labeling up to four times, signals for the fully converted product exceeded those for the tyrosine analogue.…”

“…The most significant changes were found for Y180 and Y475 (p = 0.009 and 0.0034, respectively, for 8 M urea; Student's ttest). Phenylisocyanate (m/z = 121) conjugations were observed for sixteen lysine residues, twice as many as previously reported 16,19 . However, this enrichment was little changed in the unfolded protein (Supplemental Figure 3C).…”

“…We assessed the products of PTAD conjugation with tyrosine to determine products our experiment designs may produce. Previous reports noted such additional reactions as conjugates with amines, second additions to tyrosine phenols, and short-lived conjugations to cysteine residues 15,16 . We confirmed by 1 H NMR the reaction with a tyrosine analogue, 3-(4-hydroxyphenyl)propionic acid (Supplemental Spectra 1).…”

“…We could also observe a double-reacted product of two PTADs on the phenolic ring, Y (2) (m/z = 700, Supplemental Figure 1A). PTAD can decompose to an isocyanate that is reactive toward amines 16,18 . The third product we observed was the phenylisocyanate product conjugated with the primary amine of tyrosine, NH (urea) (m/z = 384, Supplemental Figure 1A).…”

“…Angiotensin II is an 8 amino acid long peptide, DRVYIHPF (m/z = 1046, Figure 1B). In this reaction, we incubated PTAD in aqueous buffer containing 100 mM 2-amino-2-hydroxymethyl-propane-1,3diol (Tris) to scavenge the isocyanate through its own primary amine 16,19 . Matrix-assisted laser desorption/ionization and time of flight (MALDI-TOF) mass spectrometry was used to detect the PTAD conjugate (m/z = 1221).…”