Luminol anchors improve the electrochemical-tyrosine-click labelling of proteins

Abstract

New methods for chemo-selective modifications of peptides and native proteins are important in chemical biology and for the development of therapeutic conjugates. Less abundant and uncharged amino-acid residues are interesting targets to form less heterogeneous conjugates and preserve biological functions. Phenylurazole (PhUr), N-methylphenylurazole (NMePhUr) and N-methylluminol (NMeLum) derivatives were described as tyrosine (Y) anchors after chemical or enzymatic oxidations. Recently, we developed the first electrochemical Y-bioconjugation method coined eY-click 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 two-step 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 offers the interesting possibility of the double tagging of solvent-exposed Y.