Luminol anchors improve the electrochemical-tyrosine-click labelling of proteins. Issue 46 (17th November 2021)

• Record Type: Journal Article
• Authors: Depienne, Sébastien; Alvarez-Dorta, Dimitri; Croyal, Mikael; Temgoua, Ranil C. T.; Charlier, Cathy; Deniaud, David; Mével, Mathieu; Boujtita, Mohammed; Gouin, Sébastien G.
• Is Part Of: Chemical science. Volume 12:Issue 46(2021)
• Page Start: 15374
• Page End: 15381
• Publication Date: 2021-11-17
• Subjects: Chemistry -- Periodicals; 540.5
• DOI: 10.1039/d1sc04809k
• Languages: English
• ISSNs: 2041-6520

Abstract

Abstract : We assessed the relative efficiencies of tyrosine anchors in the electrochemical conjugation of peptides and proteins. Luminol derivatives showed faster reaction kinetics, complete tyrosine-chemoselectivity, and possible double modification. 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.