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-epiTTX (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.

中文翻译：

---

在有毒蝾螈中发现的 N-羟基型河豚毒素类似物和双环胍化合物的结构。

河豚毒素 (TTX, 1 ) 是一种广泛分布在海洋和陆地后生动物中的强效神经毒素，其生物合成仍未得到解决。一个重要的问题是识别与 TTX 生物合成途径相关的中间体和分流产物。我们通过筛选和鉴定来自Cynops ensicauda popei和Taricha granulosa 的新 TTX 相关化合物来研究 TTX 生物合成。质谱 (MS) 引导的筛选在蝾螈中鉴定了两种新的N-羟基 TTX 类似物：1-羟基-8 - epi TTX ( 2 ) 和 1-羟基-8 - epi -5,11-dideoxyTTX ( 3，之前报道为1-羟基-5,11-双脱氧TTX)。我们准备了一个新的类似物，8-Epi -5,11-dideoxyTTX ( 4 )，通过N -OH 还原从3 中提取，并使用亲水相互作用液相色谱 (HILIC)-LCMS确认了4在T. granulosa 中的存在。Cynops和Taricha中 8- epi型 TTX 类似物的存在支持陆地 TTX 的分支生物合成途径，其产生 6-和 8-差向异构体。此外，新的双环胍化合物 Tgr-238 ( 5 ) 和 Tgr-240 ( 6 ) 被鉴定为我们提出的 TTX 生物合成途径的推定分流产物。Cep-228A 的结构分析 ( 7)，另一种双环化合物，使用核磁共振进行。基于对结构的5 - 7及其类似物，我们提出了分流和地面TTX的生物合成代谢途径的典范。