Even though tetrodotoxin (TTX) is a widespread toxin in marine and terrestrial organisms, very little is known about the biosynthetic pathway used to produce it. By describing chemical structures of natural analogs of TTX, we can start to identify some of the precursors that might be important for TTX biosynthesis. In the present study, an analog of TTX, 5,11-dideoxyTTX, was identified for the first time in natural sources, the ovary of the pufferfish and the pharynx of a flatworm (planocerid sp. 1), by comparison with totally synthesized (−)-5,11-dideoxyTTX, using high resolution ESI-LC-MS. Based on the presence of 5,11-dideoxyTTX together with a series of known deoxy analogs, 5,6,11-trideoxyTTX, 6,11-dideoxyTTX, 11-deoxyTTX, and 5-deoxyTTX, in these animals, we predicted two routes of stepwise oxidation pathways in the late stages of biosynthesis of TTX. Furthermore, high resolution masses of the major fragment ions of TTX, 6,11-dideoxyTTX, and 5,6,11-trideoxyTTX were also measured, and their molecular formulas and structures were predicted to compare them with each other. Although both TTX and 5,6,11-trideoxyTTX give major fragment ions that are very close, m/z 162.0660 and 162.1020, respectively, they are distinguishable and predicted to be different molecular formulas. These data will be useful for identification of TTXs using high resolution LC-MS/MS.

The isolation and characterization of rabbit and human cDNAs revealed a new low density lipoprotein receptor (LDLR)-related protein (LRP) designated as LRP5. Human LRP5 cDNA encodes a 1, 616-amino acid type I membrane-like protein with three ligand binding repeats in its extracellular region. LDLR-deficient cells transduced by recombinant adenovirus containing human LRP5 exhibited increased binding of apolipoprotein E (apoE)-enriched beta-migrating very low density lipoprotein. Northern blotting and in situ hybridization revealed a high level of LRP5 expression in hepatocytes and the adrenal gland cortex. In LDLR-deficient Watanabe heritable hyperlipidemic rabbits, LRP5 mRNA was increased in the liver and accumulated in cholesterol-laden foam cells of atherosclerotic lesions.

Identification of new tetrodotoxin (TTX) analogs from TTX-possessing animals might provide insight into its biosynthesis and metabolism. In this study, four new analogs, 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, were isolated from the newt, Cynops ensicauda popei, and their structures were determined using spectroscopic methods. These are the first 8-epi-type analogs of TTX that have been found in a natural source. Furthermore, we examined the composition of the TTX analogs in this newt and in the ovary of the puffer fish, Fugu poecilonotus, using LC/MS. The results indicate that TTX and 11-deoxyTTX were present in both sources. However, 6-epiTTX and 8-epi-type analogs were detected only in the newt, while 5,6,11-trideoxyTTX was a specific and major analog in the puffer fish. Such considerable differences among analog compositions might reflect differences in the biosynthesis or metabolism of TTX between these animals.

Here, we describe the synthesis of the genetically predicted biosynthetic intermediates of the neurotoxin saxitoxin (STX) (1), 2, 6 and 7, and identification of 2 and 6 in toxin-producing microorganisms. This is the first chemical evidence supporting the genetically predicted biosynthetic route toward 1.

BACKGROUND AND PURPOSEThe development of subtype-selective ligands to inhibit voltage-sensitive sodium channels (VSSCs) has been attempted with the aim of developing therapeutic compounds. Tetrodotoxin (TTX) is a toxin from pufferfish that strongly inhibits VSSCs. Many TTX analogues have been identified from marine and terrestrial sources, although their specificity for particular VSSC subtypes has not been investigated. Herein, we describe the binding of 11 TTX analogues to human VSSC subtypes Na v 1.1-Na v 1.7. EXPERIMENTAL APPROACHEach VSSC subtype was transiently expressed in HEK293T cells. The inhibitory effects of TTX analogues on each subtype were assessed using whole-cell patch-clamp recordings.

The biosynthesis of tetrodotoxin (TTX), a potent neurotoxin consisting of a 2,4-dioxaadamantane skeleton and a guanidine moiety, is an unsolved problem in natural product chemistry. Recently, the first C5-C10 directly bonded TTX analogue, 4,9-anhydro-10-hemiketal-5-deoxyTTX, was obtained from toxic newts and its carbon skeleton suggested a possible monoterpene origin. On the basis of this hypothesis, screening of predicted biosynthetic intermediates of TTX was performed using two MS-guided methods. Herein, five novel cyclic guanidine compounds from toxic newts are reported which commonly contain a cis-fused bicyclic structure including a six-membered cyclic guanidine. These structures could be biosynthetically derived from geranyl guanidine through oxidation, cyclization, and/or isomerization steps. LC-MS analysis confirmed the widespread distribution of the five novel compounds in toxic newt species. These results support the hypothesis that TTX is derived from a monoterpene.

Recently, liquid chromatography-mass spectrometry (LC-MS) has been recognized as a useful method to analyze tetrodotoxin (TTX), the primary toxin for puffer fish poisoning. TTX usually exists with its chemically interchangeable analogs, such as 4-epiTTX and 4, 9-anhydroTTX. TTX and 4-epiTTX have the same molecular weight and the same fragmentation pattern by tandem mass spectrometry (MS-MS). In this study, we optimized conditions for hydrophilic interaction liquid chromatography (HILC)-MS-MS to quantitate TTX, 4-epiTTX, 4,9-anhydroTTX, and 5,6,11-trideoxyTTX with good separation. The relationships between the applied amounts of each TTX analog to the HILC-MS-MS instrument and the peak areas showed good linearity in the ranges examined. The lower limits of detection (signal-to-noise ratio = 3) were 64 pg on-column for TTX, 128 pg for both 4-epiTTX and 4,9-anhydroTTX, and 180 pg for 5,6,11-trideoxyTTX using an API 2000 mass spectrometer.

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.