Synthesis of Rings DEF of Solanoeclepin A.

An improved synthesis of rings DEF of solanoeclepin A has been achieved from ent-Hajos Parrish ketone. A key tricyclo[5.3.2.01,6]decene intermediate having an additional vinyl group as a precursor of a hydroxyl functionality was synthesized, in which the key steps included (i) a [2,3]-Wittig rearrangement to provide trans-hydroindene with C11(R)-configuration, (ii) the introduction of a vinyl group as a masked OH at C6, (iii) an oxymercurative aldol to synthesize the tricyclo[5.3.2.01,6]decene moiety, (iv) an oxidative C-C bond cleavage to yield an aldehyde and an unsaturated methyl ketone, and (v) a radical cyclization for the cyclobutane ring formation to provide the tricyclo[5.2.1.01,6]decene compound.

Photochemically Induced Aryl Azide Rearrangement: Solution NMR Spectroscopic Identification of the Rearrangement Product.

Photolysis of ethyl 3-azido-4,6-difluorobenzoate at room temperature in the presence of oxygen results in the regioselective formation of ethyl 5,7-difluoro-4-azaspiro[2.4]hepta-1,4,6-triene-1-carboxylate, presumably via the corresponding ketenimine intermediate which undergoes a photochemical four-electron electrocyclization followed by a rearrangement. The photorearrangement product was identified by multinuclear solution NMR spectroscopic techniques supported by DFT calculations.

Multicomponent Strategy for the Synthesis of Prostaglandin E2 Methyl Ester under Anion Relay Chelation Control.

Starting with four components, the enantioselective synthesis of prostaglandin E2 methyl ester has been achieved through a highly stereoselective heteroatom-directed conjugate addition reaction and cyclopentanone ring cyclization as the key steps. This asymmetric strategy includes (i) an asymmetric Reformatsky reaction; (ii) conjugate addition of a chiral vinyllithium reagent; (iii) cyclization to form a sulfonylated cyclopentanone in one-pot; followed by (iv) allylation of the side chain. Four carbon-carbon bond-forming processes and three stereogenic centers were established, with the steps from (ii) to (iii) being achieved in a one-pot process.

Stereochemical Course of Wittig Rearrangements of Dihydropyran Allyl Propargyl Ethers.

[2,3]-Wittig rearrangements of sugar-derived dihydropyran allyl propargyl ethers located at the 2- or 4-position have been studied as useful means for extending the carbon chains of the 4- or 2-position with chirality transfer. The stereochemical course of these reactions depends on the following factors: (1) deprotonation of pro-R or pro-S-H, (2) equilibration of the lithiated stereogenic carbanion, (3) conformational inversion during the rearrangement, and (4) concerted [2,3]- or [1,2]-Wittig rearrangement. In some cases, a stepwise mechanism that involves the allyl-C-O bond cleavage is shared as the first step by both the [2,3]- and [1,2]-Wittig rearrangements. The stereochemical courses of the rearrangements are compared among the lithiated reactants to determine the reaction pathways. These mechanisms in the polyoxygenated dihydropyran ring system were further supported by DFT calculations.

Asymmetric synthesis of 3-azide-4-fluoro-l-phenylalanine.

The asymmetric synthesis of N-Fmoc-protected 3-azide-4-fluoro-l-phenylalanine as a photoactive phenylalanine analog has been achieved by Schöllkopf's alkylation.

Total synthesis of chiriquitoxin, an analogue of tetrodotoxin isolated from the skin of a dart frog.

The first total synthesis of chiriquitoxin, the most structurally complex analogue of tetrodotoxin isolated from a Costa Rican dart frog, has been accomplished from a newly designed intermediate for a variety of tetrodotoxin derivatives. The synthesis includes the third total synthesis of tetrodotoxin in this laboratory, and its intermediate was transformed into chiriquitoxin by a stereocontrolled aldol reaction with a D-camphor-derived lactone for installation of the unique side chain, and a new deprotection of methylthiomethyl (MTM) ether by using a Pummerer rearrangement.

Molecular mechanism of Symplectoteuthis bioluminescence--part 4: chromophore exchange and oxidation of the cysteine residue.

Symplectin is one of the few photoproteins, which forms covalent bonds with the dehydro-coelenterazine (DCL) at the binding sites and the active site. This binding takes place through the SH's of the cysteine residues via conjugate addition reaction. This photoprotein contains the chromophore molecules at the binding cites first, and then moves to the active cite Cys-390 for the luminescence. The current study focuses on these dynamic aspects of the chromophore using the natural photoprotein by analyzing the fluorescence changing of the DCL chromophores analogs with 8-(4'-methoxyphenyl)- or 8-(2'-naphthyl)-group and 2-(2',4'-difluorophenyl)-group. Exchanges of these chromophores were monitored the fluorescence at slightly acidic media and also from the luminescence function observed at the optimum pH 7.8. The non-fluorescent naphthyl analogs was even proven to make the covalent bond formation at pH 6.0 and evidently to obtain the corresponding luminescent product amide by liquid chromatographic detection from the spent solutions.

Synthesis of tetrodotoxin, a classic but still fascinating natural product.

Tetrodotoxin, a toxic principle of puffer fish intoxication, is one of the most famous marine natural products due to its densely functionalized structure and potent toxicity. Despite its small molecular size (MW 319 g mol⁻¹), tetrodotoxin has long been well known as a formidable molecule in natural product synthesis. We have devoted more than twenty years to developing synthetic strategies for this molecule, resulting in the preparation of a variety of analogues of tetrodotoxin for biological experiments. This account describes a brief history of tetrodotoxin research and an overview of our synthetic efforts toward tetrodotoxin with the underlying logic and strategy.

An improved synthesis of (-)-5,11-dideoxytetrodotoxin.

We describe an improved synthesis of (-)-5,11-dideoxytetrodotoxin from an enone, which was used for synthesis of tetrodotoxin and its analogues in this laboratory. One of the major modifications was to establish a two-step guanidinylation of trichloroacetamide of a highly functionalized intermediate, which allowed us to prepare (15)N(2)-labeled 5,11-dideoxytetrodotoxin for biosynthetic investigations.

First identification of 5,11-dideoxytetrodotoxin in marine animals, and characterization of major fragment ions of tetrodotoxin and its analogs by high resolution ESI-MS/MS.

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.

A relaxin-like peptide purified from radial nerves induces oocyte maturation and ovulation in the starfish, Asterina pectinifera.

Gonad-stimulating substance (GSS) of starfish is the only known invertebrate peptide hormone responsible for final gamete maturation, rendering it functionally analogous to the vertebrate luteinizing hormone (LH). Here, we purified GSS of starfish, Asterina pectinifera, from radial nerves and determined its amino acid sequence. The purified GSS was a heterodimer composed of 2 different peptides, A and B chains, with disulfide cross-linkages. Based on its cysteine motif, starfish GSS was classified as a member of the insulin/insulin-like growth factor (IGF)/relaxin superfamily. The cDNA of GSS encodes a preprohormone sequence with a C peptide between the A and B chains. Phylogenetic analyses revealed that starfish GSS was a relaxin-like peptide. Chemically synthesized GSS induced not only oocyte maturation and ovulation in isolated ovarian fragments, but also unique spawning behavior, followed by release of gametes shortly after the injection. Importantly, the action of the synthetic GSS on oocyte maturation and ovulation was mediated through the production of cAMP by isolated ovarian follicle cells, thereby producing the maturation-inducing hormone of this species, 1-methyladenine. In situ hybridization showed the transcription of GSS to occur in the periphery of radial nerves at the side of tube feet. Together, the structure, sequence, and mode of signal transduction strongly suggest that GSS is closely related to the vertebrate relaxin.

Synthesis of glycocinnasperimicin D.

The first total synthesis of amino sugar antibiotic glycocinnasperimicin D (1) has been achieved by a convergent, three-component coupling strategy. The key steps involve the Heck-Mizoroki reaction by using the iodophenyl glycoside 50 and acryl amide 32 to furnish the right core structure of 1, and the construction of the urea glycoside employing the reaction of glycosyl isocyanate 8 with amino sugar 9. Glycosyl isocyanate 8 was prepared by the oxidation of isonitrile 10, which displayed excellent reactivity in the coupling event. Synthetic roadblocks, encountered during this synthetic effort, have led to the development of the alpha-selective, Lewis acid catalyzed phenyl glycosylation process with 2-amino-hexopyranose and a procedure for acetonide deprotection without affecting the silyl ethers.

Selective protein modification by the hydroperoxide intermediate in a photoprotein, aequorin.

During the course of protein modification program, we employed a recombinant aequorin, the apo-protein reconstituted with coelenterazine, and found out that the photolytic hyperperoxide modified three -S-SCH2CHOHCHOHCH2SH groups to -S-SCH2CHOHCH=CH-S=O)H or -S-SCH2CHOHCH=CH-S(=O)OH of terminal DTT connected to cysteine residues of the C145, C152 and C180, which turned out to locate near the chromophore.

Oxidatively induced Cu for Mn exchange in protein phosphatase 1gamma: a new method for active site analysis.

Protein phosphatase 1gamma, a serine/threonine phosphatase, is a metalloprotein that coordinates two Mn(2+) in the active site when expressed in Escherichia coli in a buffer containing MnCl(2). Herein, we report on the oxidatively induced copper for manganese exchange in protein phosphatase 1gamma, thus enabling firm confirmation of the four histidine (His) amino acid residues (His66, His125, His173, and His248) involved in metal coordination. By exchanging manganese with copper the oxidation yields for the peptides increased dramatically, thus simplifying detection of the oxidized peptides and analysis of the oxidation sites within the oxidized peptides. We also found that when copper was added during the oxidation process a new metal coordination center was formed at cysteine 39, 105, 140, and 155.

Molecular heterogeneity of TIME-EA4, a timer protein in silkworm diapause eggs.

TIME-EA4 is an ATPase that measures time intervals as a diapause-duration clock found in diapause eggs of the silkworm, Bombyx mori. In the current studies, we report the molecular heterogeneity of TIME-EA4 protein regarding not only amino acid L62V, but also the numbers and linkage patterns of the sugar chain attached to the Asn(22) residue. These sugar chain structures were determined in a pico-molar amount of the protein by combining the methods of chemical modification (Smith degradation) and nano-HPLC-electrospray ionization-quadrupole-time of fight-mass spectrometry (ESI-Q-TOF-MS) and -MS/MS. The Japanese and bi-voltine Thai silkworm strains were compared to show the heterogeneity represented by four kinds of molecular species. Judicious choice of the combination methods led us to find the first example of a linkage-position difference in the glycosidic bonds even in sugar moieties of the same molecular weight; thus, the Man(1-6)Man(1-4)GlcNAc(1-4)GlcNAc structure in the C108 pure strain and Man(1-3)Man(1-4)GlcNAc(1-4)GlcNAc structure in the Kinshu-Showa hybrid. A total of five kinds of molecular heterogeneity was determined, including the amino acids in TIME-EA4 protein. This paper describes the details for determining the sugar chain linkage in TIME-EA4 from the diapause eggs of various silkworm strains.

Prevention of copper-induced calcium influx and cell death by prion-derived peptide in suspension-cultured tobacco cells.

Impact of copper on the oxidative and calcium signal transductions leading to cell death in plant cells and the effects of the copper-binding peptide derived from the human prion protein (PrP) as a novel plant-protecting agent were assessed using a cell suspension culture of transgenic tobacco (Nicotiana tabacum L., cell line BY-2) expressing the aequorin gene. Copper induces a series of biological and chemical reactions in plant cells including the oxidative burst reflecting the production of reactive oxygen species (ROS), such as hydroxyl radicals, and stimulation of calcium channel opening, allowing a transient increase in cytosolic calcium concentrations. The former was proven by the action of specific ROS scavengers blocking the calcium responses and the latter was proven by an increase in aequorin luminescence and its inhibition by specific channel blockers. Following these early events completed within 10 min, the development of copper-induced cell death was observed during additional 1 h in a dose-dependent manner. Addition of a synthetic peptide (KTNMKHMA) corresponding to the neurotoxic sequence in human PrP, prior to the addition of copper, effectively blocked both calcium influx and cell death induced by copper. Lastly, a possible mechanism of peptide action and future applications of this peptide in the protection of plant roots from metal toxicity or in favour of phytoremediation processes are discussed.

[JSPS Asian core program on cutting-edge organic chemistry in Asia].

The vision to establish this program was to establish and extend cooperative research efforts beyond the intraregional boundaries. The Japan Society for the Promotion of Science (JSPS) has taken an initiative to support an Asian Core Program, which aims to create world-class research hubs within the Asian region and foster the development of the next generation of leading researchers by establishing sustainable collaborative relations among research and educational institutions in Asian countries. Nagoya University strongly supports and is the Core University of this program with Minoru Isobe and Toshio Nishikawa serving as the coordinator. Representing their respective countries/regions, Guo-Qiang Lin and Zhu-Jun Yao (China, Shanghai), Sunggak Kim and Kwan-Soo Kim (Korea), Somsak Ruchirawat (Thailand), and Chun-Chen Liao and Biing-Jiun Uang (China, Taipei) share in the vision to enhance collaborative efforts. As coordinators they have invited many cooperative universities/institutes in their home countries/regions to start the network since 2005. Singapore (Tech-Peng Loh) has joined lately, and Hong Kong is represented by Henry Wong. All cooperating regions also agreed to support this program by acquiring matching funds for the duration of the program, that is, until March 2010. This program is jointly supported by the JSPS (Japan), the NNSFC (China, Beijing), the NSCT (China, Taipei), the KOSEF/CMDS (Korea), the NRCT/CRI (Thailand), and the IUPAC for an East Asian Network Task group project. Pauline Chiu takes the general secretary work. The initiation of the Asian Core Program and the Inauguration Conference (The 0th International Conference on Cutting-Edge Organic Chemistry in Asia; ICCEOCA-0) was held in Nagoya (2006. 3), which was followed by ICCEOCA-1 in Okinawa, Japan (2006. 10), ICCEOCA-2 in Busan, Korea (2007. 9), ICCEOCA-3 in Hangzhou, China (2008. 10). A post symposium of ICCEOCA-1 was held in Hsinchu, Taiwan (2006. 10), and a satellite symposium of ICCEOCA-2 was held in Bangkok, Thailand (2007. 11). Future international conferences will be held in Bangkok (2009. 11) and Taipei (2010).

Total synthesis of ciguatoxin.

Something fishy: Ciguatoxin (see structure) is one of the principal toxins involved in ciguatera poisoning and the target of a total synthesis involving the coupling of three segments. The key transformations in this synthesis feature acetylene-dicobalthexacarbonyl complexation.

Synthetic studies and biosynthetic speculation on marine alkaloid chartelline.

Synthetic studies and biosynthetic speculation on chartelline inspired by an unexpected reaction are described.

Protein phosphatase inhibitory activity of tautomycin photoaffinity probes evaluated at femto-molar level.

Herein we describe the further improvement of our in-house developed firefly bioluminescence assay system for the determination of inhibition of protein phosphatase (PP). The advantage with the new system is higher sensitivity as well as being time and sample efficient. The inhibition activity of tautomycin with PP1gamma was determined using the upgraded test system and Ki was found to be 4.5 nM, which compare favorably with the activity reported previously by others using different methods. The test system was then used in order to determine the activity of nine tautomycin (TTM) photoaffinity probes. One of the TTM photoaffinity probes (anti-10) was found to possess higher activity than the natural product itself with a Ki of 3.4 nM, while the remaining photoaffinity probes were found to possess Ki in the range of 8.0-213 nM.