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

The biosynthesis of tetrodotoxin (TTX, ), 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 and . Mass spectrometry (MS)-guided screening identified two new -hydroxy TTX analogues in newts: 1-hydroxy-8-TTX () and 1-hydroxy-8-5,11-dideoxyTTX (, previously reported as 1-hydroxy-5,11-dideoxyTTX). We prepared a new analogue, 8--5,11-dideoxyTTX (), from via -OH reduction and confirmed the presence of in using hydrophilic interaction liquid chromatography (HILIC)-LCMS. The presence of 8--type TTX analogues in both and supports a branched biosynthetic pathway of terrestrial TTX, which produces 6- and 8-epimers. In addition, new bicyclic guanidinium compounds Tgr-238 () and Tgr-240 () were identified as putative shunt products of our proposed TTX biosynthesis pathway. A structural analysis of Cep-228A (), another bicyclic compound, was performed using NMR. Based on the structures of - and their analogues, we propose a model of the shunt and metabolic pathways of the terrestrial TTX biosynthesis.