Identification of Deoxy- and <i>epi</i> -Tetrodotoxin Analogues from the Newt <i>Cynops ensicauda popei</i> Suggests Stepwise Oxidation in Terrestrial Tetrodotoxin Biosynthesis

Tetrodotoxin (TTX, <b>1</b>) is a potent neurotoxin that selectively blocks voltage-gated sodium channels and occurs in various marine and terrestrial organisms, yet its biosynthetic pathway remains unresolved. In this study, five deoxy-type TTX analogues were identified from the toxic newt <i>Cynops ensicauda popei</i>. The structures of four analogues, 6-<i>epi</i>-11-deoxyTTX (<b>2</b>), 8-<i>epi</i>-6-deoxyTTX (<b>3</b>), 1-hydroxy-8-<i>epi</i>-6,11-dideoxyTTX (<b>4</b>), and 1-hydroxy-4,9-anhydro-8-<i>epi</i>-10-hemiketal-5,6,11-trideoxyTTX (<b>5</b>), were elucidated by NMR spectroscopy, and an additional natural TTX analogue, 8-<i>epi</i>-6,11-dideoxyTTX (<b>6</b>), was characterized by high-resolution LCMS/MS. The structural features of these analogues suggest that stepwise oxidation at C-6 and C-11 occurs after formation of a TTX- or 10-hemiketal-type skeleton. Although this oxidative logic parallels that proposed for marine TTX-producing organisms, the analogue profile in newts is clearly distinct from that of marine organisms and is characterized by the presence of deoxy analogues in the 6-<i>epi</i> and 8-<i>epi</i> forms. These findings indicate that, although late-stage oxidative steps in TTX biosynthesis may be conserved, structural diversification has proceeded differently in terrestrial and marine systems, reflecting divergence in their biosynthetic pathways. This study provides insight into stepwise oxidation processes potentially involved in terrestrial TTX biosynthesis. A cell-based assay showed that 6-<i>epi</i>-11-deoxyTTX (<b>2</b>) retains voltage-gated sodium channel inhibitory activity, consistent with previously reported structure-activity trends.