Isolation of 6-Deoxytetrodotoxin from the Pufferfish, Takifugu pardalis, and a Comparison of the Effects of the C-6 and C-11 Hydroxy Groups of Tetrodotoxin on Its Activity

Abstract

Identification of new tetrodotoxin (TTX, 1) analogues would be significant in the elucidation of its biosynthetic pathway and a study of its structure-activity relationships. In this study, a new TTX analogue, 6-deoxyTTX (2), was isolated from the ovary of the pufferfish, Takifugu pardalis, and the structure was determined using spectroscopic methods. Compound 2 was also identified in other marine animals, Nassarius snail and blue-ringed octopuses, using LC-MS. Furthermore, we investigated the voltage-gated sodium channel blocking activity of 2 by examination of the inhibitory activities to cytotoxicity induced by ouabain and veratridine in mouse neuroblastoma cells (Neuro-2a). The activities were then compared with those of 1, 11-deoxyTTX (3), and 6,11-dideoxyTTX (4). The EC50 value for 2 was estimated to be 6.5±2.2 nM, approximately 3-fold larger than that of 1 (2.1±0.6 nM) and approximately 20-fold smaller than that of 3. These results suggested that contribution of the C-6 hydroxy group to the activity is less than that of the C-11 hydroxy group.

Study of the matrix effects of tetrodotoxin and its content in cooked seafood by liquid chromatography with triple quadrupole mass spectrometry

“…6). The structure assignment of these analogs can be verified as well by the relative eluting sequence of Amide column as reported by Yotsu-Yamashita et al [27], Rodriguez et al [31], and Kudo et al [50]. Two peaks were found for the transitions m/z 290→162 and m/z 290→272.…”

“…Kudo et al applied the single transition of [M+H] + →162 to screen and 1 H NMR and 13 C NMR to identify 8-epi-5,6,11-trideoxyTTX, 4,9-anhydro-8-epi-5,6,11-trideoxyTTX, 1-hydroxy-8-epi-5,6,11-trideoxyTTX, and 1-hydroxy-4,4a-anhydro-8-epi-5,6,11-trideoxyTTX from the newt, Cynops ensicauda popei [16]. Kudo et al also used NMR spectroscopy and high-resolution MS to identify 6-deoxyTTX from pufferfish [50]. Although seven analogs were found besides TTX in cooked seafood according to the screening method mentioned in this study, the concentrations were relatively lower.…”

“…It may be biologically concentrated up the food chain in specific animals that consume TTX-containing bacteria [20]. The LD 50 of TTX in mammals was reported to be 2-10 g/kg following intravenous administration and 10-14 g/kg after subcutaneous administration [21]. Many cases of poisoning have been reported following the consumption of foods containing TTX, such as nassarius or fugu [9,20].…”

Differential binding of tetrodotoxin and its derivatives to voltage‐sensitive sodium channel subtypes (Nav1.1 to Nav1.7)

“…Most importantly, 5-deoxyTTX (7), 5,6, lack the 10,7-hemilactal scaffold (Figure 1), which appears to be necessary to achieve high affinity for VSSCs. The significance of the hydroxy functionality of 4-OH, 6-OH, 8-OH and 11-OH in TTX (1) for achieving high affinity to VSSCs was reported by Yotsu-Yamashita et al (Choudhary et al, 2003;Yotsu-Yamashita et al, 2003;Kudo et al, 2014;Saruhashi et al, 2016) and proposed in modelling studies (Lipkind and Fozzard, 1994;Choudhary et al, 2003;Tikhonov and Zhorov, 2005). In addition, the present study investigated the blocking activity of VSSCs by 8-deoxyTTX (3), a synthetic analogue of TTX (1) that has not yet been identified from natural sources, in whole-cell patch-clamp recordings and emphasizes the importance of hydroxyl functionality in the high-affinity binding of TTX (1).…”

“…Here, tetrodotoxin (TTX, 1, Figure 1), saxitoxin (STX) and μ-conotoxin block the sodium current by occluding the ion permeation pathway (Narahashi et al, 1964;1967;Ohizumi et al, 1986). The molecular determinants of the high affinity binding of TTX (1) to VSSCs have been investigated using various TTX analogues in binding assays with rat brain synaptosomes (Yotsu-Yamashita et al, 1999), or cell-based assays using the neuroblastoma cell line Neuro 2A (YotsuYamashita et al, 2003;Kudo et al, 2014;Saruhashi et al, 2016). However, rat brain synaptosomes and mouse neuroblastoma Neuro 2A cells express multiple subtypes of VSSCs (Lou et al, 2005).…”