Isolation and Biological Activity of 9-epiTetrodotoxin and Isolation of Tb-242B, Possible Biosynthetic Shunt Products of Tetrodotoxin from Pufferfish.

Tetrodotoxin (TTX, 1) is a potent voltage-gated sodium channel blocker detected in certain marine and terrestrial organisms. We report here a new TTX analogue, 9-epiTTX (2), and a TTX-related compound, Tb-242B (4), isolated from the pufferfish Takifugu flavipterus and Dichotomyctere ocellatus, respectively. NMR analysis suggested that 2 exists as a mixture of hemilactal and 10,8-lactone forms, whereas other reported TTX analogues are commonly present as an equilibrium mixture of hemilactal and 10,7-lactone forms. Compound 2 and TTX were confirmed not to convert to each other by incubation under neutral and acidic conditions at 37 °C for 24 h. Compound 4 was identified as the 9-epimer of Tb-242A (3), previously reported as a possible biosynthetic precursor of TTX. Compound 4 was partially converted to 3 by incubation in a neutral buffer at 37 °C for 7 days, whereas 3 was not converted to 4 under this condition. Compound 2 was detected in several TTX-containing marine animals and a newt. Mice injected with 600 ng of 2 by intraperitoneal injection did not show any adverse symptoms, suggesting that the C-9 configuration in TTX is critical for its biological activity. Based on the structures, 2 and 4 were predicted to be shunt products for TTX biosynthesis.

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

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.