Chemical properties and biological activities of three highly purified major tetrodotoxin analogues

Tetrodotoxin (TTX), the pufferfish toxin, has the potential to cause fatal food poisoning because of its potent voltage-gated sodium channel (Na_v) blocking activity. 4-epiTTX, 11-norTTX-6(S)-ol, and 11-oxoTTX are the major TTX analogues found in marine animals; thus, their chemical properties and biological activities should be determined. In this study, these three TTX analogues were purified to a high level (purity >97%) from pufferfish and newts. The ratios of the hemilactal to the 10,7-lactone forms were determined using ¹H NMR, and ¹³C NMR data were also obtained. 4-epiTTX underwent considerable transformation to TTX in physiological conditions. The Na_v blocking activity of these analogues was evaluated by whole-cell patch-clamp recording using a human Na_v1.2, colorimetric cell-based assay and mouse bioassay. The toxicity equivalency factors (TEFs setting TTX 1) of the three analogues were estimated; 4-epiTTX (0.06), 11-norTTX-6(S)-ol (0.02), and 11-oxoTTX (1.2) using patch-clamp recording, and 11-norTTX-6(S)-ol (0.50) and 11-oxoTTX (0.42) using the mouse bioassay. These data confirmed the low Na_v blocking activity of 4-epiTTX and high activity of 11-oxoTTX.