Electrochemically promoted tyrosine-click-chemistry for protein labelling – E-CLICK

New series of phenylurazole and luminol derivatives were synthesized to study their electrochemical behaviors to identify the most promising electro-active targets for the electro-oxidation/tyrosine-click tandem reaction.

Our results show that the N-methylated anchors of phenylurazole and luminol are more reactive, to the point of doubling the change in phenol when an excess of reagent is present. Three peptides were chosen to further studies and then were compared in terms of the selectivity and kinetic of azido-armed derivatives with regard to protein modification. More complex targets, such as bovine albumin, the enzymes mannosidase and glucose oxidase, and the therapeutic antibody Trastuzumab, were effectively conjugated electrochemically and by a fluorescein probe thanks to the two-step strategy 1) electro-bio-conjugation with the azide derivative of luminol, 2) click reactions with cyclooctyne-fluorescein.

We have designed an elegant method for labelling tyrosine sites of protein, the resulting method is selective for the phenol site of tyrosine. The resulting method is simple and allows the rapid development of a series bio-conjugated proteins that could have a strong impact in the therapeutic field. We are currently investigating these different possibilities. The optimized electro-bio-conjugation methodology of tyrosine is being applied to more complex systems of biological/medical interest, with new functional synthetic derivatives of luminol.

- Luminol anchors improve the electrochemical-tyrosine-click labelling of proteins. Depienne, S.; Alvarez-Dorta, D.; Croyal, M.; Temgoua, R.C.T.; Charlier, C.; Deniaud, D. ; Mével, M. ; Boujtita, M. ; Gouin, S.G. Chem. Sci., 2021, 12, 15374-15381.

- Click-electrochemistry for the rapid labeling of virus, bacteria and cell surfaces. Depienne, S.; Bouzelha, M.; Courtois, E.; Pavageau, K.; Lalys, P-A.; Marchand, M.; Alvarez-Dorta, D.; Nedellec, S.; Marin-Fernandez, L.; Grandjean, C.; Boujtita, M.; Deniaud, D.; Mével, M.; Gouin,S. 2023. chemrxiv.org/engage/chemrxiv/article-details/6401f35737e01856dc159a87. Nat. Commun, 2023, 14, 5122.

-Structural insights into a cooperative switch between one and two FimH bacterial adhesins binding pauci- and high-mannose type N-glycan receptors. E-M Krammer, C Bridot, S Serna, B Echeverria, S Semwal, B Roubinet, K van Noort, R Wilbers, G Bourenkov, J de Ruyck, L Landemarre, NC Reichardt, J Bouckaert*. www.jbc.org/article/S0021-9258(23)00269-7/fulltext, J. Biol. Chem. 2023, 299(5), 104627;

-Demande de brevet: Chemically-modified adeno-associated viruses. Gouin, S.G.; Deniaud, D.; Mével, M.; Depienne, D.; Alvarez-Dorta, D.; Bouzelha, M. Priority date 12/01/2023, EP23305042.6.

- Demande de brevet: Methods for cell surface remodelling. Gouin, S.G.; Deniaud, D.; Mével, M.; Depienne, D.; Alvarez-Dorta, D. ; Bouzelha, M., Priority date 12/01/2023, EP23305040.0.

Bioorthogonal ligation methods are extensively explored for the development of protein conjugates, which are of considerable importance in the therapeutic field and in biotechnology industries. Popular applications include i) the conjugation of toxins, imaging agents, or radiopharmaceuticals to monoclonal antibodies in cancer therapies, ii) the design of glycoconjugate vaccines where microbial oligosaccharides are coupled to a protein carrier, iii) the pegylation of therapeutic proteins to improve their serum half-lifes and therapeutic index. Direct protein modifications are generally performed onto amino group of the abundant lysine amino-acid with N-hydroxysuccinimide-activated esters, sulfonyl chlorides or iso(thio)cyanates. Alternatively, the relatively rare cysteine residues can also be modified for single-site functionalization through disulfide exchange and Michael addition with maleimides. In comparison, the remaining 18 amino-acid have been much less exploited. One of the most promising recent alternatives, is the click-like reactions specifically targeting the tyrosine (Y) residues. Chemical oxidation of phenyl-urazole anchors like 4-phenyl-3H-1,2,4-triazole-3,5(4H)-dione (PTAD) react with phenol side chain of Y through an ene-like reaction. The method proved relatively chemoselective for Y, allowing the specific modification of peptides and proteins. Nevertheless, the method suffers from serious drawbacks such as (i) the use of an oxidizing chemical to generate the highly reactive PTAD species, (ii) the rapid PTAD decomposition in the presence of water (iii) the use of a specific buffer to scavenge the formation of an isocyanate side-product from rapid PTAD decomposition, resulting in the unwanted modification of lysines. This cross-reactivity limit the scope of the tyrosine-click reaction and the high PTAD reactivity prevents the site-specific targeting of a selected Y.
The electrochemically promoted tyrosine-click-chemistry for protein labelling (e-CLICK) project will considerably broaden the scope of the click-Y by providing the reactive PTAD specie on demand, in different buffers, and without the needs of an oxidizing chemical. This ambitious goal will be reached by using a more promising strategy in terms of chemo-selectivity and protein modification. We propose here to develop the first electrochemically driven probes for Y-specific protein conjugations. The application of an appropriate electrochemical potential will allow us to activate the PTAD dormant specie in situ, on demand, without oxidizing the sensitive amino-acids from the protein or the ligands linked to the PTAD. Our preliminary results prove the feasibility on biologically relevant peptides and proteins. High coupling yields were obtained with a complete Y-selectivity and without compromising protein functionality.
The possibility to activate a dormant urazole specie in situ will also offer the unique opportunity for site-specific Y electro-labeling (ss-e-click). In the currently described click-Y approaches, the most accessible Y are labeled preferentially but the distribution of the anchored urazoles at the protein surface is broad and aspecific. We strongly believe that ss-e-click will allow the selective labelling of Y residues at the entrance of protein binding sites. To reach this ambitious goal, we plan to attach the urazole anchor to a specific ligand of the targeted protein through an appropriate flexible linker. Y selectivity should be achieved after competitive binding followed by electrooxidation.
We forsee that the e-CLICK and ss-e-CLICK methodology will nicely complement the arsenal of current biorthogonal ligations, allowing site-specificity in the design of covalent conjugate inhibitors, molecular probes for protein target identification, or the design of therapeutically relevant bio-conjugates.