*Article* **Chemical Synthesis of a Functional Fluorescent-Tagged** α**-Bungarotoxin**

**Oliver Brun 1,2,3, Claude Zoukimian 4, Barbara Oliveira-Mendes 1, Jérôme Montnach 1, Benjamin Lauzier 1, Michel Ronjat 1, Rémy Béroud 4, Frédéric Lesage 3,5, Didier Boturyn <sup>6</sup> and Michel De Waard 1,4,7,\***


**Abstract:** α-bungarotoxin is a large, 74 amino acid toxin containing five disulphide bridges, initially identified in the venom of *Bungarus multicinctus* snake. Like most large toxins, chemical synthesis of α-bungarotoxin is challenging, explaining why all previous reports use purified or recombinant α-bungarotoxin. However, only chemical synthesis allows easy insertion of non-natural amino acids or new chemical functionalities. Herein, we describe a procedure for the chemical synthesis of a fluorescent-tagged α-bungarotoxin. The full-length peptide was designed to include an alkyne function at the amino-terminus through the addition of a pentynoic acid linker. Chemical synthesis of α-bungarotoxin requires hydrazide-based coupling of three peptide fragments in successive steps. After completion of the oxidative folding, an azide-modified Cy5 fluorophore was coupled by click chemistry onto the toxin. Next, we determined the efficacy of the fluorescent-tagged α-bungarotoxin to block acetylcholine (ACh)-mediated currents in response to muscle nicotinic receptor activation in TE671 cells. Using automated patch-clamp recordings, we demonstrate that fluorescent synthetic α-bungarotoxin has the expected nanomolar affinity for the nicotinic receptor. The blocking effect of fluorescent α-bungarotoxin could be displaced by incubation with a 20-mer peptide mimicking the α-bungarotoxin binding site. In addition, TE671 cells could be labelled with fluorescent toxin, as witnessed by confocal microscopy, and this labelling was partially displaced by the 20-mer competitive peptide. We thus demonstrate that synthetic fluorescent-tagged α-bungarotoxin preserves excellent properties for binding onto muscle nicotinic receptors.

**Keywords:** toxins; peptide chemistry; native chemical ligation; α-bungarotoxin; click chemistry; automated patch-clamp; fluorescent peptide; TE671 cells; nicotinic acetylcholine receptor

**Key Contribution:** The first report of the chemical synthesis of a fluorescent α-bungarotoxin that leads to a bioactive peptide.
