The odor of a nontoxic tetrodotoxin analog, 5,6,11-trideoxytetrodotoxin, is detected by specific olfactory sensory neurons of the green spotted puffers.

Toxic puffers accumulate tetrodotoxin (TTX), a well-known neurotoxin, by feeding on TTX-bearing organisms and using it to defend themselves from predators. Our previous studies have demonstrated that toxic puffers are attracted to 5,6,11-trideoxytetrodotoxin (TDT), a nontoxic TTX analog that is simultaneously accumulated with TTX in toxic puffers and their prey. In addition, activity labeling using immunohistochemistry targeting neuronal activity marker suggests that TDT activates crypt olfactory sensory neurons (OSN) of the green spotted puffer. However, it remains to be determined whether individual crypt OSNs can physiologically respond to TDT. By employing electroporation to express GCaMP6s in OSNs, we successfully identified a distinct group of oval OSNs that exhibited a specific calcium response when exposed to TDT in green spotted puffers. These oval OSNs showed no response to amino acids (AAs), which serve as food odor cues for teleosts. Furthermore, oval morphology and surface positioning of TDT-sensitive OSNs in the olfactory epithelium closely resemble that of crypt OSNs. These findings suggest that TDT is specifically detected by crypt OSNs in green spotted puffer. The TDT odor may act as a chemoattractant for finding conspecific toxic puffers and for feeding TTX-bearing organisms for effective toxification.

Isolation of Paraphaeoketones: A Plausible Biosynthetic Explanation for Paraphaeolactones via a Benzilic Acid Rearrangement Rather than a Favorskii Rearrangement.

Paraphaeoketones A-C (1-3) were isolated from the culture broth of Paraphaeosphaeria sp. KT4192. Their structures and relative configurations were determined using spectroscopic analysis and verified through density functional theory (DFT)-based chemical shift calculations. The absolute configurations of these compounds were determined by comparing the experimental electronic circular dichroism (ECD) spectra with those based on DFT calculations. We also propose a plausible biosynthetic route to 1-3. While our prior studies on the isolation and structural elucidation of paraphaeolactones (e.g., 4) led us to suggest a Favorskii rearrangement for their biosynthesis, the isolation of 2 prompted the proposal of an alternative biosynthesis for 4, featuring a benzilic acid rearrangement of 2. Moreover, an in vitro conversion of 2 into 4 was achieved successfully, suggesting that a biosynthetic pathway for paraphaeolactones involving a benzilic acid rearrangement is more plausible than the previously presumed Favorskii rearrangement pathway. Arguments based on DFT calculations for these pathways are also described.

Total Synthesis of 19-Nordigitoxigenin, An Antiaroside Y Aglycon.

The first total synthesis of 19-nordigitoxigenin, an aglycon of antiroside Y, has been achieved. The key steps of our synthesis are (i) construction of the 19-norsteroid ring system via a Mizoroki-Heck reaction between a bromoanisole corresponding to the A-ring and cyclic alkene incorporating the CD-rings, followed by a Friedel-Crafts-type cyclodehydration, and (ii) incorporation of the butenolide moiety at C17 via a silyl-tethered radical cyclization and subsequent ozone oxidation.

The Asymmetric Total Synthesis and Configuration Confirmation of Aplysiaenal and Nhatrangin A, Truncated Derivatives of Aplysiatoxin and Oscillatoxin.

Asymmetric total syntheses of aplysiaenal (1) and nhatrangin A (2), truncated derivatives of the aplysiatoxin/oscillatoxin family of marine natural products, from configurationally defined intermediates are described. NMR spectra of our synthesized nhatrangin A did not match with either those obtained from authentic samples of the natural product or material obtained via two other total syntheses, but were similar to that obtained from a sample obtained in a third total synthesis. By independently synthesizing the fragments used in its total syntheses, we were able to confirm the configuration of nhatrangin A and clarified that the discrepancy in the spectroscopic data is due to salt formation of the carboxylic acid moiety.

Levels of Tetrodotoxins in Spawning Pufferfish, Takifugu alboplumbeus.

Tetrodotoxin (TTX), also known as pufferfish toxin, is an extremely potent neurotoxin thought to be used as a biological defense compound in organisms bearing it. Although TTX was thought to function as a chemical agent for defense and anti-predation and an attractant for TTX-bearing animals including pufferfish, it has recently been demonstrated that pufferfish were also attracted to 5,6,11-trideoxyTTX, a related compound, rather than TTX alone. In this study, we attempted to estimate the roles of TTXs (TTX and 5,6,11-trideoxyTTX) in the pufferfish, Takifugu alboplumbeus, through examining the location of TTXs in various tissues of spawning pufferfish from Enoshima and Kamogawa, Japan. TTXs levels in the Kamogawa population were higher than those in the Enoshima population, and there was no significant difference in the amount of TTXs between the sexes in either population. Individual differences were greater in females than in males. However, the location of both substances in tissues differed significantly between sexes: male pufferfish accumulated most of their TTX in the skin and liver and most of their 5,6,11-trideoxyTTX in the skin, whereas females accumulated most of their TTX and 5,6,11-trideoxyTTX in the ovaries and skin.

[Epithelioid Hemangioendothelioma of the Upper Mediastinum:Report of a Case].

A 81-year-old woman was referred to our hospital for neck discomfort. Chest computed tomography (CT) showed a tumor in the upper mediastinum. Fluorodeoxyglucose-positron emission tomography (FDG-PET) showed the mild accumulation in the tumor. Percutaneous biopsy was performed and epithelioid hemangioendothelioma was suspected, and the surgical treatment was performed. The histological study showed polygonal and irregular cells with nuclear atypia in myxoma-like substrate compatible with epithelioid hemangioendothelioma.

Green spotted puffers detect a nontoxic TTX analog odor using crypt olfactory sensory neurons.

Toxic puffers accumulate their defense substance (tetrodotoxin; TTX) through the food chain. Although the previous study suggests that 5,6,11-trideoxyTTX, a nontoxic TTX analog detected simultaneously with TTX in toxic puffers or their prey, acts as an olfactory chemoattractant for grass puffers, it is unclear whether toxic puffers are commonly attracted to 5,6,11-trideoxyTTX, and which types of olfactory sensory neurons (OSNs) detect 5,6,11-trideoxyTTX. Here, we demonstrated that green spotted puffer, a phylogenetically distant species from the grass puffer, is attracted to 5,6,11-trideoxyTTX. 5,6,11-TrideoxyTTX administration made green spotted puffers stay longer at the administered site, whereas a food odor (l-Arg) made them actively swim throughout the aquarium. Attractive responses were not observed when TTX or its vehicle was administered, nor when 5,6,11-trideoxyTTX was administered to anosmic fish. Furthermore, double immunohistochemistry with activity marker and crypt OSN marker antibodies labeled oval cells with apical invagination on the olfactory epithelium surface treated with 5,6,11-trideoxyTTX. These results suggest that 5,6,11-trideoxyTTX acts as an olfactory chemoattractant detected by crypt OSNs, and attraction to 5,6,11-trideoxyTTX odor appears to be a trait shared by toxic puffers for social communication or effective toxification.

Total Syntheses of the Proposed Biosynthetic Intermediates of Tetrodotoxin Tb-210B, Tb-226, Tb-242C, and Tb-258.

The collective synthesis of the four spiro-cyclic guanidines Tb-210B, Tb-226, Tb-242C, and Tb-258, all of which have been isolated from puffer fish and are considered possible biosynthetic intermediates of tetrodotoxin, has been achieved. Our synthesis is based on the stepwise deoxygenation or hydroxylation of a common intermediate, prepared from a known oxazoline.

Collective Synthesis of Aplysiatoxin/Oscillatoxin Analogues by a Bioinspired Strategy.

Interest in the marine cyanobacteria natural products aplysiatoxin (ATX) and oscillatoxin (OTX) has been renewed recently due to the discovery of many new analogues, some exhibiting intriguing biological activities. We sought to develop a collective synthesis of these natural products, hypothesizing that ATX could serve as a common biosynthetic precursor. Herein, we reveal that the core structure of ATX has unique multiple reactivities giving access to the distinct ring structures of five of the analogues, depending upon the specific conditions used. Based on these findings, syntheses of the O-Me derivative of five analogues neo-deBr-ATX-B, OTX-H, OTX-D, neo-deBr-ATX-H, and OTX-I were achieved from the main fragment of ATX as a common intermediate in a few steps. These synthetic studies also led us to revise the relative configuration in the elucidated structures of neo-deBr-ATX-B and OTX-H, and obtain unnatural 8- and 12-membered lactones from the same intermediate.

Structural verification of petromyzestrosterol by total syntheses of both C14-epimers of its 3-O-methyl derivative.

The structure of petromyzestrosterol, a pheromonal steroid of the sea lamprey, was verified by total syntheses of both C14-epimers of its 3-O-methyl derivative. The key features of our synthesis involve (1) a highly stereoselective Mizoroki-Heck reaction to unite the A- and CD-ring segments and (2) Friedel-Crafts-type cyclodehydration to construct the B-ring. Petromyzestrosterol is concluded to bear an α-configured C14 hydroxy group based on a comparison of NMR data of both the synthesized C14-epimers of the 3-O-methyl derivative with those of the natural petromyzestrosterol. The downfield shifts of C9 and C12 via the γ-gauche effect in the 14ß-isomer would enable the structural elucidation of C14 in the 14-hydroxy estrogenic steroids.

[Primary Pericardial Malignant Mesothelioma:Report of a Case].

A 67-year-old woman was referred to our hospital for cough and fever. Chest computed tomography (CT) showed some masses showing slightly enhanced effect in the pericardium. FDG-PET showed the accumulation of FDG in the masses. Thoracoscopic surgical biopsy was performed to establish the diagnosis. The histological study showed proliferation of short spindle-shaped cells surrounded by lymphocyte, and the spindle cells were immunohistochemically positive for cytokeratin AE1/AE3, WT-1, D2-40, CAM5.2, intelectin-1 and negative for CEA, TTF-1, napsin A, claudin-4, calretinin, MUC4, PAX8, CD30. These findings were compatible with epithelial pericardial malignant mesothelioma.

Total Synthesis of the Cardiotonic Steroid (+)-Cannogenol.

The total synthesis of (+)-cannogenol, an aglycon common to various biologically important cardiotonic glycosides, has been achieved. Synthesis of the versatile intermediate involves Mizoroki-Heck and intramolecular Diels-Alder reactions from the enantiomerically pure CD-ring segment, newly prepared in a multidecagram scale this time. Total synthesis by the site-selective transformations of the versatile intermediate demonstrated the applicability of our synthetic approach.

Total synthesis and biological evaluation of oscillatoxins D, E, and F.

Oscillatoxins (OTXs) and aplysiatoxins are biosynthetically related polyketides produced by marine cyanobacteria. We previously developed a synthetic route to phenolic O-methyl analogs of OTX-D and 30-methyl-OTX-D during collective synthesis of these natural products. According to our synthetic strategy, we achieved total synthesis of OTX-D, 30-methyl-OTX-D, OTX-E, and OTX-F by deprotecting the O-methyl group in an earlier intermediate, and determined their biological activities. Although OTX-D and 30-methyl-OTX-D have been reported to show antileukemic activity against L1210 cell line, we found that their cytotoxicity in vitro against this cell line is relatively weak (IC50: 29-52 µm). In contrast, OTX-F demonstrated cell line-selective antiproliferative activity against DMS-114 lung cancer cells, which implies that OTXs target as yet unknown target molecules as part of this unique activity.

First Detection of Tetrodotoxins in the Cotylean Flatworm Prosthiostomum trilineatum.

Several polyclad flatworm species are known to contain high levels of tetrodotoxin (TTX), but currently TTX-bearing flatworms seem to be restricted to specific Planocera lineages belonging to the suborder Acotylea. During our ongoing study of flatworm toxins, high concentrations of TTXs were detected for the first time in the flatworm Prosthiostomum trilineatum, suborder Cotylea, from the coastal area of Hayama, Kanagawa, Japan. Toxin levels were investigated by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS), revealing that this species contains comparable concentrations of toxins as seen in planocerid flatworms such as Planocera multitentaculata. This finding indicated that there may be other species with significant levels of TTXs. The distribution of TTXs among other flatworm species is thus of great interest.

Studies toward the Synthesis of Chartelline C.

Chartelline C, a marine alkaloid, possesses a unique core scaffold including indolenine ß-lactam and imidazole moieties linked by an unsaturated 10-membered ring. A new synthetic approach for the construction of the indolenine ß-lactam was planned, based on the inherent reactivity of chartelline A with NaOMe, triggered by bromination of bromoenamide, and proceeding through an N-acyl imine. However, the N-acyl imine intermediate was not observed. Instead, the corresponding bromoindolenine was obtained, which led to the desired indolenine ß-lactam in 92% yield.

Biomimetic Synthesis of Chaxine and its Related Compounds.

The highly oxidized natural products chaxine B and BB have been synthesized from ergosterol in eight steps according to a route inspired by their proposed biosynthesis; key steps were an oxidative cascade from a furan intermediate to an enol ester using m-chloroperbenzoic acids (MCPBA), followed by diastereoselective epoxidation and acyloxy migration. This concise synthesis resulted in the revision of the structures of chaxine B and its naturally occurring analogs and syntheses of the unnatural analogues of these natural products for biological investigations.

[Solitary Pulmonary Metastasis from Follicular Thyroid Carcinoma Resected 28 Years Earlier Coexisting with Early Lung Cancer;Report of a Case].

A 71-year-old woman was referred to our hospital for a round mass shadow in the right lower lung field in mass screening chest X-ray. Computed tomography (CT) of the chest showed a well-defined lobulating mass shadow measuring 2.2 cm in diameter in the lower lobe of the right lung and a ground glass opacity ( GGO) in the upper lobe of the left lung. She underwent video-assisted partial resection of right lower lobe of the lung. The pathological examinations indicated a pulmonary metastasis of follicular thyroid carcinoma. Three months later, video-assisted partial resection of left upper lobe of the lung was performed. Microscopically, 2 lesions of adenocarcinoma in situ were revealed.

Biosynthesis of Indole Diterpene Lolitrems: Radical-Induced Cyclization of an Epoxyalcohol Affording a Characteristic Lolitremane Skeleton.

Lolitrems are tremorgenic indole diterpenes that exhibit a unique 5/6 bicyclic system of the indole moiety. Although genetic analysis has indicated that the prenyltransferase LtmE and the cytochrome P450 LtmJ are involved in the construction of this unique structure, the detailed mechanism remains to be elucidated. Herein, we report the reconstitution of the biosynthetic pathway for lolitrems employing a recently established genome-editing technique for the expression host Aspergillus oryzae. Heterologous expression and bioconversion of the various intermediates revealed that LtmJ catalyzes multistep oxidation to furnish the lolitrem core. We also isolated the key reaction intermediate with an epoxyalcohol moiety. This observation allowed us to establish the mechanism of radical-induced cyclization, which was firmly supported by density functional theory calculations and a model experiment with a synthetic analogue.

Asymmetric Synthesis of the Aromatic Fragment of Sespendole.

Sespendole is an indole sesquiterpene alkaloid bearing two isoprenyl groups, one of which is highly oxidized. Herein, we disclose an eight-step synthesis of the aromatic fragment of sespendole in an optically pure form, starting from 4-bromo-2-fluoronitrobenzene. The key steps were a Claisen rearrangement at room temperature for introduction of the prenyl group and a coupling between the dianion generated from prenylated bromo-N-tosylanilide and a chiral epoxy aldehyde.

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.