Biochimica et Biophysica Acta, 664 (1981) 219-225 219 Elsevier/North-Holland BiomedicalPress BBA 79259 SYNTHESIS AND PROPERTIES OF NEW PHOTOACTIVABLE DERIVATIVES OF TETRODOTOXIN M. BALERNA *, A. LOMBET,R. CHICHEPORTICHE, G. ROMEY and M. LAZDUNSKI Centre de Biochimie du CNRS, Facultd des Sciences, Parc Valrose, 06034 Nice Cedex (France) (Received December 8th, 1980) Key words: Tetrodotoxin derivative; Neurotoxin synthesis; Photoactivation The Pfitzner-Moffatt oxidation procedure has been used to prepare two new photoactivable derivatives of tetro- dotoxin that have been synthesized with high specific radioactivities (17.5 Ci/mmol and 30 Ci/mmol). They specifically bind to axonal membranes with affinities of 5.2-14.2 nM. They dissociate from their membrane com- plex with half-lives of 10.8 and 20 min. In the dark, these compounds give a reversible block of the sodium chan- nels. After ultraviolet irradiation, they induce an irreversible blockade of the nerve channels. Introduction Many neurotoxins are known to specifically alter the functioning of the voltage-dependent sodium channel. They can be separated in four main groups based on their action on at least four different types of receptors [1]: (i) tetrodotoxin and saxitoxin which block Na ÷ entry through the Na ÷ channel with- out altering the gating mechanism of the channel [2,3]; (ii) batrachotoxin, veratridine, aconitine and grayanotoxin which alter both the activation and the inactivation of the Na ÷ channel [4-7]; (iii) pyre- throids which inhibit sodium inactivation [8,9] and (iv) polypeptide toxins like scorpion and sea anemone toxins [10,11] which also inhibit the inactivation process. Tetrodotoxin is still the most widely used toxin in the analysis of the properties of the fast Na ÷ channel. The receptor of this toxin has been biochem- ically identified in a variety of preparations of excit- able membranes [3,12] and even partially purified [13-151. Highly radioactive tetrodotoxin derivatives recently obtained by synthesis [16] have been found *Present address: Unity of Gynaecology-Endocrinology, Hospital 'La Carit~', CH-6600 Locarno, Switzerland. Abbreviations: NaCNBH3, sodium cyanoborohydride; DCCD, dicyclohexylcarbodiimide; Me2SO, dimethyl sub foxide; FNAB, 1-flu oro-2-nitro-4-azidobenzene; NAP, 2-nitro-4 -azidophenyl. to associate to the tetrodotoxin-binding component and to exhibit biological properties similar to those of tetrodotoxin itself. Photoactivable derivatives of tetrodotoxin have also been synthesized; in the dark they give a reversible block of the sodium channel and this block becomes irreversible after ultraviolet irradiation [17,18]. The purpose of this paper is to describe the synthesis and the properties of new photoaffinity derivatives of tetrodotoxin which we find more useful than those which have been previ- ously described. Materials and Methods Chemicals. Tetrodotoxin citrate-free was obtained from Sankyo Chemicals Co. 4-fluoro-3-nitroaniline, NaCNBHa, DCCD and crystallized H3PO4 (99% purity) were purchased from Fluka AG. Dried Me2SO, 2,4-dinitrophenylhydrazine, ethylenediamine, Silica Gel GF2s4 and cellulose plates were from Merck. All solvents were of the best analytical grade and they were dried in the presence of preactivated 3 A molecular sieves. [3H]Lysine (20 Ci/mmol), [3H]ethylenediamine (30 Ci/mmol)were obtained from the Commissariat ~ l'Energie Atomique, France. Synthesis. FNAB was prepared from 4-fluoro-3- nitroaniline according to Fleet et al. [19]. After recrystallization the product was checked for their purity by infra red and ultraviolet spectroscopy and stored at -15°C in the dark. 0 005-2736/81/0000-0000/$02.50 © Elsevier/North-Holland BiomedicalPress