Luminol Anchors Improve the Electrochemical-Tyrosine-Click Labelling of Proteins

Abstract

New methodologies for the chemo-selective modifications of peptides and native proteins are of great importance in chemical biology and for the developm ent of therapeutic conjugates. Less abundant and uncharged amino-acid residues are interesting targets to form less heterogeneous conjugates and preserve biological functions. Phenylurazole (PhUr), Nmethylphenylurazole (NMePhUr) and N-methylluminol (NMeLum) derivatives were described as tyrosine (Y) anchors after chemical or enzymatic oxydations. Recently, we developed the first electrochemical Y-bioconjugation method coined eYclick to activate PhUr in biocompatible media. In this work, we assessed the limitations, benefits and relative efficiencies of eY-click conjugations performed with a set of PhUr, NMePhUr and NMeLum derivatives. Results evidenced a high efficiency of NMeLum that showed a complete Y-chemoselectivity on polypeptides and biologically relevant proteins after soft electrochemical activation. Side reactions on nucleophilic or heteroaromatic amino-acids such as lysine or tryptophan were never observed during mass spectrometry analysis. Myoglobine, bovine serum albumin, a plant mannosidase, glucose oxidase and the therapeutically relevant antibody trastuzumab were efficiently labelled with a fluorescent probe in a twostep approach combining eY-click and strain-promoted azide-alkyne cyclization (SPAAC). The proteins conserved their structural integrity as observed by circular dichroism and the trastuzumab conjugate showed a similar binding affinity for the natural HER2 ligand as shown by bio-layer interferometry. Compared to our previously described protocol with PhUr, eY-click with NMeLum species showed faster reaction kinetics, higher (complete) Y-chemoselectivity and reactivity, and offer the interesting possibility for the double tagging of solvent-exposed Y.

Electrochemistry for the Chemoselective Modification of Peptides and Proteins

“…Improved reaction efficiencies for both of the second-generation reagents (N-methyl luminol and 1-methyl-4-phenylurazole) were achieved compared to the original phenylurazolean observation which was further corroborated by Gouin and co-workers in a 2021 study. 99 However, Nakamura and co-workers observed significant dual modification of angiotensin II on the phenolic side chain of the tyrosine residue using N-methyl luminol. Intriguingly, modification of a larger protein, the HER2 antibody trastuzumab, proceeded smoothly for phenylurazole and Nmethyl luminol derivatives, but poorly for the 1-methyl-4phenylurazole reagent; a rationale for this experimental observation was not discussed.…”

Thermally Triggered Triazolinedione–Tyrosine Bioconjugation with Improved Chemo- and Site-Selectivity

“…When chemo-selective electrochemical oxidation of the urazole (versus its cargo and versus the protein) can be achieved, this "e-Y-clicking" constitutes a remarkable improvement of the method. 45,55 Due to our long-standing interest in TAD chemistry, 52,56−59 we hypothesized that the use of dynamic TAD-indole adducts would be a good alternative to generate reactive TADs in situ for bioconjugation purposes (Scheme 1c). TADs and indoles react swiftly at room temperature in a click-like fashion, but can be reversed at elevated temperatures (120 °C).…”