Spiro Bicyclic Guanidino Compounds from Pufferfish: Possible Biosynthetic Intermediates of Tetrodotoxin in Marine Environments

Abstract Tetrodotoxin (TTX, 1 ) is a potent neurotoxin that is widely found in both terrestrial and marine animals; however, the biosynthetic pathway and genes for TTX have not yet been elucidated. Previously, we proposed that TTX originated from a monoterpene; this hypothesis was based on the structures of cyclic guanidino compounds that are commonly found in toxic newts. However, these compounds have not been detected in marine organisms. Instead, a series of deoxy analogues of TTX were found in toxic marine animals; thus, we further screened for TTX‐related compounds in marine animals. Herein, we report seven novel spiro bicyclic guanidino compounds 2 – 8 that were isolated from the pufferfish Tetraodon biocellatus . In compounds 2 – 5 and 7 – 8 , a six‐membered cyclic guanidino amide is spiro‐fused with 2,4‐dimethyl cyclohexane, whereas in compound 6 , the same cyclic guanidino amide is spiro‐fused with 2,3,5‐trimethylcyclopentane. Compounds 2 – 5 and 7 – 8 have the same carbon skeleton and relative configuration as TTX. Thus, we proposed that compounds 2 – 8 are biosynthetic intermediates of TTX in marine environments. TTX could be biosynthetically derived from compound 7 via intermediates 2 – 5 through several oxidations, amide hydrolysis, and formation of the hemiaminal and lactone found in 5,6,11‐trideoxyTTX, the major TTX analogue, whereas compounds 6 and 8 might be shunt products. LC‐MS analysis confirmed the wide distribution of compounds 2 , 3 , or both in TTX‐containing marine animals, namely pufferfish, crab, octopus, and flatworm, but compounds 2 – 8 were not detected in newts.