Structures of N-Hydroxy-Type Tetrodotoxin Analogues and Bicyclic Guanidinium Compounds Found in Toxic Newts

The biosynthesis of tetrodotoxin (TTX, 1 ), a potent neurotoxin widely distributed in marine and terrestrial metazoans, remains unresolved. A significant issue has been identifying intermediates and shunt products associated with the biosynthetic pathway of TTX. We investigated TTX biosynthesis by screening and identifying new TTX-related compounds from Cynops ensicauda popei and Taricha granulosa . Mass spectrometry (MS)-guided screening identified two new N -hydroxy TTX analogues in newts: 1-hydroxy-8- epi TTX ( 2 ) and 1-hydroxy-8- epi- 5,11-dideoxyTTX ( 3 , previously reported as 1-hydroxy-5,11-dideoxyTTX). We prepared a new analogue, 8- epi -5,11-dideoxyTTX ( 4 ), from 3 via N -OH reduction and confirmed the presence of 4 in T. granulosa using hydrophilic interaction liquid chromatography (HILIC)-LCMS. The presence of 8- epi -type TTX analogues in both Cynops and Taricha supports a branched biosynthetic pathway of terrestrial TTX, which produces 6- and 8-epimers. In addition, new bicyclic guanidinium compounds Tgr-238 ( 5 ) and Tgr-240 ( 6 ) were identified as putative shunt products of our proposed TTX biosynthesis pathway. A structural analysis of Cep-228A ( 7 ), another bicyclic compound, was performed using NMR. Based on the structures of 5 - 7 and their analogues, we propose a model of the shunt and metabolic pathways of the terrestrial TTX biosynthesis.