High-yield synthesis of a [3H]ethylenediamine ditetrodotoxin derivative

3H]Tetrodotoxin ( 3H]TTX) and a 3H]ethylenediamine derivative of TTX are the most widely used ligands for the study of the Na channel. The former ligand presents a low specific radioactivity (1 Ci/mmol) while the latter is highly labeled (30 Ci/mmol). However, its two-step synthesis, i.e., mild oxidation followed by coupling of 3H]ethylenediamine, has been described with a low overall yield of 1.7%. In this work, more favorable experimental conditions are defined for the limiting reaction, i.e., the oxidation step, using 14C]testosterone as a model molecule. Applied to the oxidation of tetrodotoxin, this procedure produces yield values of 30-50%, as determined by high-performance liquid chromatography. Moreover, two oxidized TTX molecules appear to be covalently linked to 3H]ethylenediamine, yielding a new labeled tetrodotoxin derivative with a specific radioactivity of 45 Ci/mmol and a dissociation constant of 0.6 nM for electroplax membranes.