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

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), N-methylphenylurazole (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 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 offer the interesting possibility for the double tagging of solvent-exposed Y

Click-electrochemistry for the rapid labeling of virus, bacteria and cell surfaces

The remodeling of microorganism surfaces with biomolecules is a powerful tool to study the role of membrane receptors in chemical biology and to develop drug delivery systems in gene therapy using viral vectors and cell-based therapies. Methods for direct covalent ligation of these surfaces remain poorly reported, and mostly based on metabolic engineering for bacteria and cells functionalization. In the latter case, a tagged precursor must first be enzymatically metabolized and delivered to the outer cell membrane to become available for chemo-selective labeling. While effective, a faster method avoiding the bio-incorporation step would be highly complementary. This would also need to be compatible with organisms showing poor levels of precursor assimilation or lacking the metabolic function. Here, we used N-methylluminol (NML), a fully tyrosine-selective protein anchoring group after one-electron oxidation, to label the surface of viruses, living bacteria and cells. The functionalization was performed electrochemically and in situ by applying a 750 mV vs Ag/AgCl electric potential to aqueous buffered solutions of tagged NML containing the viruses, bacteria or cells. The electro-coupling was performed with NML anchors bearing a bioorthogonal azide, biotin, or carbohydrate (mannose and N-acetyl galactosamine) handles. The broad applicability of the click-electrochemistry method was explored on recombinant adeno-associated viruses (rAAV2), E. coli (Gram-) and S. epidermis (Gram+) bacterial strains, and HEK293 and HeLa eukaryotic cell lines. Surface electro-conjugation was achieved in minutes to yield functionalized rAAV2 that conserved both structural integrity and infectivity properties, and living bacteria and cell lines that were still alive and able to divide. As NML activation immediately stops if there is no current, the method offers reproducible temporal control on the degree of surface functionalization. Thus, click-electrochemistry should significantly expand the scope of bioconjugation methods

Novel chemical tyrosine functionalization of adeno-associated virus improves gene transfer efficiency in liver and retina

Decades of biological and clinical research have led to important advances in recombinant adeno-associated viruses rAAV-based gene therapy gene therapy. However, several challenges must be overcome to fully exploit the potential of rAAV vectors. Innovative approaches to modify viral genome and capsid elements have been used to overcome issues such as unwanted immune responses and off-targeting. While often successful, genetic modification of capsids can drastically reduce vector yield and often fails to produce vectors with properties that translate across species. Here, we describe a chemical bioconjugation strategy to modify tyrosine residues on AAV capsids using specific ligands, thereby circumventing the need to genetically engineer the capsid sequence. Aromatic electrophilic substitution of the phenol ring of tyrosine residues on AAV capsids improved the in vivo transduction efficiency of rAAV2 vectors in both liver and retinal targets. This tyrosine bioconjugation strategy represents an innovative technology for the engineering of rAAV vectors for human gene therapy