Most Streptomyces strains are equipped with only the MEP (2-C-methyl-D-erythritol 4-phosphate) pathway for the formation of IPP (isopentenyl diphosphate). In addition to this pathway. some Streptomyces strains use the tnevalonate pathway to produce secondary metabolites. Streptomyces sp. KO-3988 was the first example to have "two" distinct mevalonate gene clusters. Our investigations had shown that the organism possessed "two" mevalonate pathway gene clusters with one cluster being responsible for production of antitumor agents. the furaquinocins. Subsequent gene analysis of the second mevalonate pathway gene cluster showed that it was flanked by the GGDP (geranylgeranyl diphosphate) synthase gene at its upstream region. Since GGDP is the precursor of diterpenes, the strain was assumed to produce diterpene compounds that had never been isolated from this strain. We thus attempted to isolate diterpenes from fermentation broth of this strain and succeeded in purifying five novel diterpenes named oxaloterpins A (1), B (2), C (3), D (4) and E (5) together with a previously-described compound. viguiepinone (6). Their structures were established based on extensive NMR and mass spectral analyses. The absolute configurations of oxaloterpins were determined by the modified Mosher's method.

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Fragment ions obtained by MS/MS technique was quite effective for the amino acid sequencing for linear peptides. However, in the case of cyclic peptides, fragmentation pattern was complicated because the cleavages occurred randomly and fragment ions were generated as a_n, b_n, c_n, x_n, y_n and z_n series ions, so we have never obtained sufficient sequence information. In order to overcome this problem we have made attempt to apply the ion trap LC/MS with MS^n technique and characterized the fragment ions obtained from microcystins, anabaenopeptins and aeruginopeptins as the cyclic peptides. In the case of anabaenopeptins, MS^2 analysis did not provide enough sequence information of the cyclic structure and the MS^3 analysis was applied to the sequencing of the constituent amino acids. The diagnostic fragment ions were obtained by the MS^3 analysis and these ions were quite effective to obtain the sequence information of constituent amino acid. Meanwhile, MS^2 analysis was enough to obtain the sequence information of microcystins and aeruginopeptins. In any case, resulting fragment ions obtained from the cyclic structure were formed by way of the two-bond fission mechanism of the precursor ion, in which an initial fission of the cyclic structure to a linearized one and subsequent fission(s) at the peptide bonds are included. Moreover, their fragmentation showed the similar pattern among the structure related compounds, indicating that the cleavages occurred at definite peptide bonds. In addition, the resulting fragment ions are generated as b_n series ions and the mass difference facilitates the amino acid sequencing. Consequently, ion trap LC/MS with MS^n technique provide informative sequence information and the resulting fragment ions are reproducible among the structure related compounds and reliable for the sequencing of constituent amino acid of the cyclic structure. Finally, we would like to propose a structural analysis method for cyclic peptide based on ion trap LC/MS technology, in which advanced Marfey's method and MS^n techniques with two-bond fission mechanism are included.

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Daphniphyllum alkaloids are a structually diverse group of natural products with unique fused-heterocyclic skeletons, which are elaborated by trees of the genus Daphniphyllum (Daphniphyllaceae). These unusual ring systems have been challenging targets for total synthesis as well as biosynthetic studies. We have isolated novel types of Daphniphyllum alkaloids from Daphniphyllum macropodum, D. humile, D. teijsmanni, and D. glaucescense. In our continuing search for structually unique and biogenetically interesting new alkaloids, calyciphyllines C-G (1-5) have been isolated from D. calycinum, and their structures and relative stereochemistry were elucidated on the basis of spectroscopic data. Calyciphylline C (1) is a novel Daphniphyllum alkaloid with an unprecedented fused-hexacyclic skeleton having an azetidine ring. Calyciphyllines D (2) and F (4) are novel alkaloids containing a unique fused-pentacyclic skeleton with a 8-azatricyclo[4.2.1.0.^<4,8>]nonane ring system. Calyciphylline E (3) is a new alkaloid with a fused-heptacyclic ring system, while calyciphylline G (5) is a novel alkaloid possessing an unprecedented fused-hexacyclic skeleton containg 5-azatricyclo[6.2.1.0]undecane ring system. Effects of these alkaloids on neurotrophic factor biosynthesis in 1321N1 human astrocytoma cells were examined by a semiquantitative RT-PCR method to find that the mRNA expressions for NGF were enhanced.

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Iron acquisition is a vital process for bacteria to survive, since indispensable proteins such as chytochromes require iron to function properly. In aqueous solution, iron exists as two charged ionic form Fe(II) or three charged one Fe(III). These ferric ions are constantly insufficient in natural environments because the solubility of ferric ion is as low as 10-18M in water of biological pH, while optimum growth of one bacterial cell needs 1um of iron. In order to utilize such a low concentration of ferric ion in natural habitats, bacteria excrete low molecular compounds called siderophores, which have the ability to chelate ferric ion. Streptomycetes are gram-positive bacteria which are important as a source of bioactive compounds. In the soil where Streptomycetes dwell, it is competitive to acquire enough amount of iron to grow. Recently new kind of the peptide siderophore, coelichelin, was found in Streptomyces coelicolor by the combination of genome-mining and chemical analysis. This finding prompted us to explore the diversity of the siderophore among streptomycetes and accomplish the screening in search for new siderophore. On the course of the screening, we found new peptide siderophore named antichelin. By several separation steps, the compound was isolated with high purity. The structure of antichelin was determined by analyses of NMR and TOF-MS spectral data. As a result, antichelin was turned out to be the related compound to known siderophores such as foroxymithine and antichelin. Previously, we found that the inoculation of foroxymithine caused rapid aerial formation to S. coelicolor. There is the possibility that this kind of siderophore may function as a signal for deficiency of iron. We are interested in the biological role of this class of siderophores for the cell of streptomycetes besides iron uptake, and the further investigation to pursue this possibility will be continued.

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Blepharismins (BPs) are toxic pigments of a negatively phototactic ciliate Blepharisma japonicum. This pigment was first reported in 1905, and was extensively studied by Giese. The chemical structure of BP(s) was elucidated in 1997 independently by Song, and Naoki. with analyzing HMQC and HMBC NMR spectra of BP-C or tetramethoxy derivatives of BPs. BPs (1), which consist as a mixture of five congeners, are structurally related to naturally occurring polycyclic phenanthroperylene quinones such as hypericin, a photodynamic toxin of Hypericum, and stentorin isolated from another negatively phototactic ciliate Stentor coeruleus. BPs are shown to be converted to stentorin via oxyblepharismines (OxyBPs) by UV-irradiation, whose structures were determined by Spitzner. Lensi studied OxyBP-choromoprotein association, and helical properties of OxyBP with or without the chromoprotein. The function of BPs has not been fully elucidated, but three functions have been clarified: light perception, defense against predators or UV irradiation. BPs are highly ring-condensed compound with a lot of substituted sp^2 carbons, therefore assignment of the ^<13>C NMR signals has not been completely achieved in the previous reports. Herein we like to report full assignment of the ^<13>C NMR signals of BP-C (1c) using samples obtained by feeding of ^<13>C-labeled sodium acetates to the cells of B. japonicum in the culture media.

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Stilbenoids represented by resveratrol have drawn much attention from the chemical community due to their roles in food and beverage, and because of their diverse biological activities. These compounds, either in glycosylated or non-glycosylated form, are typically found as oligomers in limited plant families such as Dipterocarpaceae, Vitaceae, Cyperaceae, and Gnetaceae. The rich structural variation and multifunctional bioactivity make stilbenoid oligomers interesting targets for detailed phytochemical investigations. Dipterocarpaceaeous plants are well-known to contain resveratrol oligomers, and their occurrences in Vatica, Vateria, Shorea, and Hopea genera have been disclosed in our previous works. The genus Upuna, which belongs to the tribe Dipterocarpeae in the largest subfamily Dipterocarpoideae, comprises monotypic species and is rare species endemic to Indonesia. Although structural elucidations of resveratrol oligomers have been made in some of the above-mentioned related genera, no examination of Upuna has been reported yet. In our current phytochemical studies of Dipterocarpaceae, the chemical constituents of U. borneensis were examined, and 17 new resveratrol oligomers were isolated, together with 18 known resveratrol derivatives. The structural elucidation of them, and their NMR characteristics due to steric hindrance are presented. In our bioactive screening experiment for above-mentioned compounds including their derivatives, we examined in vitro cytotoxicity on a panel of human tumor cell lines. Among the isolates, vaticanol C exhibited growth suppression and induction of apoptosis via the loss of mitochondrial membrane potential and consequent caspases activation. The detailed mechanisms are not clearly understood. We decided to attempt to gain further insight into the mechanism underlying vaticanol C induced apoptosis in HL-60 cells. Treatment of HL-60 cells with vaticanol C was found to cause a marked decrease in the level or phosphorylated extracellular signal-regulated kinase (ERK) concurrent with inhibited phosphorylation or its upstream kinase mitogen-activates protein kinase kinase (MEK). Moreover, exposure to vaticanol C led to a significant reduction in the level or phosphorylated Akt. Thus, vaticanol C induced inbibition of both ERK and Akt phospborylation, resulting in reduced phosphorylation of Bad. These results suggest that vaticanol C might induce apoptosis via a mechanism involving activation of Bad through disruption of pro-survival signaling pathways.

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In order to clarify the chemical mechanisms of catechin oxidation during black tea production, in vitro enzymatic oxidation of epigallocatechin 3-O-gallate was examined in detail, and production mechanism of theacitrin C, an unstable black tea pigment, was confirmed. In addition, a new catechin dimer produced by decomposition of theacitrins was isolated from commercial black tea. The results suggested that the theacitrins are precursor of minor black tea polyphenols. Furthermore, it was demonstrated that the C-3 hydroxyl group of epigallocatechin plays an important role in the production of theacitrins and proepitheaflagallin. Enzymatic oxidation of epigallocatechin 3-O-acetate yielded three products, the composition of which was different from those obtained by oxidation of epigallocatechin. The difference was caused by formation of the hemiacetal structures between the free C-3 hydroxyl group and carbonyl groups generated at the B-rings. On enzymatic oxidation of epigallocatechin, we succeeded to isolate an unstable dimeric product having bicyclo[3.2.1]octane skeleton. The bicyclooctane structure was stabilized by hemiacetal formation between its carbonyl group and the C-3 hydroxyl group. Based on the HPLC analysis of the reaction mixture, the product was presumed to be a precursor of proepitheaflagallin. On the other hand, when the C-3 hydroxyl group was acylated, the bicyclo[3.2.1]octane precursor was decomposed to give theacitrins. The results indicated that the production and decomposition of bicyclo[3.2.1]octane-type intermediates are key reactions in the catechin oxidation.

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The South American medicinal plant called Pau ferro is a tall tree belonging to the leguminous genus Caesalpinia. Little chemical constituents other than gallic acid and ellagic acid in this plant have been reported so far. The acetone extracts of this bark exhibited the remarkable inhibitory activity on DNA topoisomerase II, and induced potent apoptosis in human leukemia cell line HL-60. In search of the bioactive substances, a variety of flavonoids including novel chalcone oligomers (1-5), prenyl flavone (7) and known compounds (amentoflavone (6), karanjachromene (8), 3',5'-dimethoxy-[2",3": 7,8]-furano flavone (9), desmethoxy kanugin (10)) were found from the bark extracts. In this paper, we report their compounds concerning the chemical structures and the biological activities. The molecular formulas of 1-5 and 7 were determined by HR-FABMS and HR-EIMS. Their planar structures were elucidated by the MS fragmentations and by using various NMR techniques (^1H, ^<13>C,^1H^1H-COSY, HMQC, and HMBC). The relative configurations of fran ring in 1-5 were deduced to be trans positions from NOESY. The stereochemistry around cyclobutane ring of 5 was presumed from the NOE difference spectra. Interestingly, 5 was a first example of chalcone trimer having cyclobutane structure. The chemical structures of all known compounds were identified by the comparison with published spectroscopic data. The isolated compounds were mainly evaluated for three bioactivities: inhibitory activity on DNA topoisomerase II, apoptosis induction in human leukemia cell line HL-60, and antidiabetic efficacy. 5 and 6 exhibited the inhibitory activities on topo II with IC_<50>-value of 2.1 and 8.3μM, respectively. 5 also revealed apoptosis in HL-60 with IC_<50> = 5.2μM. 8 showed cytotoxicity reaction of IC_<50> = 8.2μM. PPARα or γ activation potency as to antidiabetic efficacy was recognized on 9.

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In Japan, many kinds of dietary supplements containing Phellinus linteus ('meshimakobu' in Japanese) are in the markets. These products were prepared from either natural fruit bodies, cultivated fruit bodies, cultured mycelia or the mixture of them, but all of them are used for same purposes. We supposed that there must be differences in the chemical constituents among them and these differences may lead to variation in the biological activities. We compared the HPLC profiles of the MeOH extracts prepared from authentic P. linteus natural fruit body, cultured mycelia and cultivated fruit bodies. Their HPLC chromatograms were different from each other. The HPLC profiles of the extracts from the fruit bodies cultivated on the two different host species were remarkably similar. We isolated compounds corresponding to peaks characteristic to the natural fruit body extract and determined their structures as the novel fused aromatic compounds, meshimakobnol A (1) and B (2) by spectroscopic methods including the detection of longer-range C-H coupling constants (^<4,5>J_<C-H>) An additional new compound, phellifuropyranone A (3) was also isolated together with known compounds, phelligridin E (4), G (5) and I (6), from natural fruit body. The characteristic compounds in cultivated fruit bodies were isolated and identified as known compounds, hypholomine B (7) and inoscavin A (8). Next, we measured the content of meshimakobnol A (1) in authentic P. linteus natural fruit body, commercially available natural and cultivated fruit bodies and cultured myceria. The content of 1 in the authentic P. linteus natural fruit body was 0.08% and 0.06 to 0.14% in the commercially available products from natural fruit body origin. In contrast, the cultivated fruit body also contained 1 but only in very small amount, 0.021%. 1 could not be detected in cultured mycelia. These results meshimakobnol A (1) could become one of indicators for discriminating between the natural and those from cultivated fruit bodies or cultured mycelia. Meshimakobnol A (1) showed proliferation inhibiting activity against the mouse melanoma cells and human lung cancer cells in vitro.

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Peroxisome proliferators-activated receptor-γ (PPAR-γ), which belongs to the nuclear receptor superfamily, is expressed at high levels in the adipose tissue and functions as a master regulator of adipocyte differentiation. We have evaluated extracts of 70 spices and herbs for their PPAR-γ ligand-binding activity and found that the nonaqueous fractions of Glycyrrhiza uralensis F. and G glabra L. had higher activity than other materials tested. Previously, several phenolic compounds with PPAR-γ ligand-binding activity were isolated from the extract of G uralensis and glycyrin, one of the main PPAR-γ ligands of G uralensis, significantly decreased blood glucose levels of the genetically diabetic KK-A^y mice. In this study, we have isolated a total of 30 phenolic compounds, including 7 new ones (3, 4, 7, 14, 25, 27, 29), from the EtOH extract of G glabra using a PPAR-γ ligand binding activity-guided fractionation method. Among the isolated compounds, echinatin (1), 3,4,2',4'-tetrahydroxy-3',5-diprenylchalcone (4), (3R)-2',3',7-trihydroxy-4'-methoxyisoflavan (10), kanzonol X (11), glabridin (12), 2',4'-dihydroxy-5'-formyl-6",6"-dimethyl pyrano[2",3": 7,8] isoflavane (14), shinpterocarpin (20), licoagrocarpin (22), shinflavanone (23), (2R, 3R)-3,4',7-trihydroxy-3'-prenylflavone (25), and 4',7-dihydroxy-3',8-diprenylflavone (26) showed significantly PPAR-γ ligand-binding activity and were concluded to be responsible for the activity of the EtOH extract of G glabra. The enrich fraction of the hydrophobic phenolic compounds from G glabra, which was prepared by further extracting G glabra EtOH extract with medium-chain triglycerides, suppressed an increase in blood glucose level in obese diabetic KK-A^y mice. This result indicates that G glabra hydrophobic phenolics have hypoglycemic effect, possibly mediated via activation of PPAR-γ.

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During the course of screening for antioxidants from microorganism metabolites our research group so far isolated several interesting 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging compounds. Recently we found a Streptomyces sp. USF-6280 strain isolated from the soil sample collected in Shizuoka-city, Japan, produced new 2-pyrone compounds which had a DPPH radical-scavenging activity. The cultivation of Streptomyces sp. USF-6280 was done at 30℃ for four days in the following medium: glucose 1%, polypeptone 0.2%, meat extract 0.1%, yeast extract 0.1%, adjusted at pH 7.2. The culture filtrate was extracted with ethyl acetate (EtOAc) at pH 3, and the extract was applied to silica gel column chromatography and Sephadex LH-20 column chromatography. Finaly, Compound 1, Compound 2 and Compound 3 were isolated by ODS-HPLC. The structures of these compounds were determined by the analysis of the FABMS, 1D-NMR and 2D-NMR spectra. Compound 1 and Compound 2 were novel 2-pyrone compounds, and these were designated surugapyrone A and surugapyrone B, respectively. Compound 3 was identified as germicidin. Biosynthesis of surugapyrone A (1) was studied by feeding experiments with a variety of ^<13>C-labeled compounds. The results of feeding experiments indicated that surugapyrone A was biosynthesized via a polylketide pathway of isobutyrate (a starter unit), acetate, and propionate.

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α-Ketol of octadecadienoic acid (KODA, Figure 1) has been suggested to play a role in the photoperiod-regulated flowering in Pharbitis nil. The level of KODA in cotyledons is temporarily controlled during short-day conditions. In our previous study, we have investigated this issue by studying the metabolism of exogenously-applied KODA in P nil seedlings by the spectroscopic methods and identified 4 major metabolites (D18, D16, D14, K16) as described in Figure 2. However, the stereochemistry of reduction of KODA into D18 has not been examined. In the present study, we carried out elucidating the absolute configuration of D18 by the measuring the long-range coupling constant using J-resolved 2D-NMR experiments. Furthermore, to obtain insight in the structural requirements of FN1 for flower inducing activity, we synthesized the analogues of FN1 and evaluated their activity.

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Fire corals (Millepora spp.) which live in coral reefs are ones of those venomous organisms. They are known to cause severe pain and inflammatory effects to human by accidental contacts in tropical and sub-tropical areas. Although some previous studies showed that fire corals' nematocysts contain a complex of bioactive polypeptides, precise characterization of toxins have not yet been investigated. We present here the isolation and partial chemical characterization of three new bioactive polypeptides from the fire corals. Nematocysts are the responsible organs for stinging cases. Nematocysts exist on the surface part of the fire corals with the symbionts (zooxanthelae). Thus, the isolation of nematocysts was crucial for obtaining crude venom without impurities originating from the other tissues, especially from algal symbionts. The side fraction containing zooxanthelae showed a fluorescent color of the pink by ultraviolet irradiation. Fluorescent protein (ca. 15kDa) was isolated using molecular exclusion HPLC. Crude extract from the isolated nematocysts was purified by using anion exchange HPLC and molecular exclusion HPLC. Bioactivity of each separated fraction was checked with hemolytic activity and cytotoxicity tests. Finally, a hemolytic toxin (ca. 100kDa) and cytotoxin (ca. 20kDa) were successfully isolated in their active forms. The 100kDa toxin possessed potent hemolytic activity (EC_<50>, 36ng/ml) toward 0.8% suspension of sheep red blood cells. The cytotoxic polypeptide showed potent cytotoxicity (EC_<50>, 79ng/ml) toward L1210 mouse leukemia cells. The primary structure of the cytotoxin deduced from the corresponding cDNA sequence was determined. It was revealed that the cytotoxin was a new member of the agglutinin family. This is the first characterization of proteinaceous toxin from Millepora spp. Precise structure elucidation of hemolytic toxin and fluorescent protein is now in progress.

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Angiogenesis is the process of generating new capillary blood vessels. Tumor growth and metastasis are highly dependent on angiogenesis. Therefore, specific inhibitors of angiogenesis are expected as promising antitumor agents. In the course of our study of bioactive substances from marine organisms, we focused on a search for anti-angiogenic substances and have isolated novel steroidal alkaloids named cortistatins A-H consisting of abeo-9(10-19)-androstane skeletons, from the Indonesian marine sponge Corticium simplex. Further detailed examination of the extract of the same marine sponge led us to isolate three novel active derivatives having isoquinoline unit in the side chain, named cortistatins J (9), K (10), and L (11). These compounds showed cytostatic anti-proliferative activity against human umbilical vein endothelial cells (HUVECs) with the 60 to 1100-fold selective index, compared with normal human cells and several tumor cell lines. Structure-activity relationship study revealed that the isoquinoline unit played crucial role for the anti-angiogenic property of cortistatins. Considering the information of the SAR study, we designed some simplified analogs (12 and 20) having A-ring unit and isoquinoline unit. The analogs 12 and 20 were synthesized efficiently from readily available materials. We also executed some biological studies of cortistatin A (1) including proteome analysis of HUVECs treated with 1, in order to elucidate action-mechanism of cortistatins.

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A growing number of marine fungi have been reported to produce novel and potentially life-saving bioactive secondary metabolites. Herein, we report chemical investigation of the metabolites obtained from the extracts of some sorts of marine fungi culture filtrate. Mycosphaerella molleriana was cultured in 1/2 PD 50% sea water for 19 days under 20℃. We succeeded in isolation of a new compound (1) from the EtOAc extract. The absolute configuration of 1 was elucidated from the measurement of CD spectrum of its benzoated. Criptosphaeria eunomia var. eunomia was cultured in 1/2 PD 50% sea water for 4 weeks under 20℃. A new compound (2) from the EtOAc extract as well as two known compounds (3,4) were obtained. These compounds were recrystallized from CHCl_3 to give solvated crystals and their absolute configurations were finely determined by X-ray. We previously reported three antibacterial chlorine-containing components of A. ostianus strain 01F313 that had been isolated from an unidentified marine sponge collected at Pohnpei. Expecting that bromine-containing compounds might be obtained when a medium composed of a bromide solution in place of seawater was used, we cultivated the same strain in a bromine-modified 1/2 PD medium. Although we were unable to isolate the brominated compounds, we found that the metabolites were considerably different from those obtained from the strain cultured in the seawater medium, and succeeded in isolating six new compounds (5,6,7,10,11 and 12) as well as two known compounds 8 and 9. We elucidated the structures of the new compounds and the absolute configurations of the obtained compounds by the application of the modified Mosher's method.

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Marine dinoflagellates Amphidinium species are known as producers of unique secondary metabolites. In our continuing investigation for new anticancer drug leads from the dinoflagellate Amphidinium sp., we have resulted in the discovery of a new Amphidinium strain producing unknown cytotoxic macrolides, and the isolation of novel cytotoxic macrolides from this strain. From 263 Amphidinium strains isolated from benthic or free-swimming sources collected off South-East Islands. Japan, the HYA024 strain with the ability producing novel cytotoxic macrolides were discovered by using the genomics and metabolomics analyses. The algal cells obtained from the culture were extracted with MeOH/toluene. The toluene-soluble fractions of the extract were subjected to SiO_2 gel, C_<18>, and then NH_2-SiO_2 columns. Macrolide-containing fractions were separated by C_<18> HPLC to afford iriomoteolides-1a (1) and 1b (2). Structure elucidations of 1 and 2 were carried out using detailed analyses of 2D NMR spectra. The relative and absolute stereochemistries were assigned by the combination of conformational analyses using NMR data and modified Mosher's method of 1. On the other hand, the structure of 2 was elucidated on the basis of the spectral data and the conversion reaction from 1 to 2. Iriomoteolides-1a (1) and 1b (2) are 20-membered macrolides having a novel carbon skeleton. Iriomoteolide-1a (1) exhibited potent cytotoxicity against human B lymphocyte DG-75 cells (IC_<50>: 0.002μg/mL), while the IC_<50> value (0.9μg/mL) of 2 against DG-75 cells is ca. 500 times less potent than that of 1. This result suggests that the presence of the 6-membered hemiacetal ring and/or the exomethylene unit in 1 is important for the potent cytotoxicity for 1.

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Ascidians (tunicates, Ascidiacea) are important bioresources for bioactive secondary metabolites. The characteristic feature of compounds obtained from ascidians is nitrogenous structures. Many interesting alkaloids have been isolated from ascidians and showed various biological activities. We have reported seven new polysulfur aromatic alkaloids, lissoclibadins 1-7, together with four known compounds from an Indonesian ascidian Lissoclinum cf. badium (Polyclinidae) collected in the coral reef at North Sulawesi. These compounds were dopamine derivatives possessing heterocyclic rings with sulfur atoms. Because of the unique structures and efficient bioactivities, we continued the isolation of new lissoclibadin derivatives from this ascidian, and seven new compounds, named lissoclibadins 8 (1), 9 (2), 10 (3), 11 (4), 12 (5), 13 (6), and 14 (7), were isolated. The structures of new compounds were assigned on the basis of their spectroscopic data. These compounds were unique polysulfur dopamine derivatives with tetramic (1), trimeric (2), dimeric (3, 4, and 5), and monomeric (6 and 7) structures. Compounds 1, 2, 6, and 7 showed the growth inhibitory activity against the murine leukemia cell line L1210 at IC_<50> values of 2.00 (2.54), 0.38 (0.30), 2.20 (0.70), and 1.80 (0.64)μM (μg/mL), respectively. Compound 3 was also cytotoxic to several cancer cell lines in vitro as potent as the activities of Iissoclibadins 1 and 2. Compounds 4 and 5 reduced the cell proliferation of L1210 to 60% of a control at 20.0μM (9μg/mL).

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Root tumor of melon was first found in 1982 in Japan. This diseese is characerized by roots developing many galls, and growth-retardation and wing in the above-ground parts of plants, The pathogen of this diseese is a Streptomyces, which is thought to be unrelated to previousty descrbed plant-pathogenic Streptomyces species. About 10-20% spores of the pathogen, Streptomyces sp. B-9-1, were ungerminated spores, A heat-shock treatment increased the colony forming rate to 110-115% of the untreated contral, and it was more effective in SDS solution. These stirmutative effects suggested that the spores contained a factor that inhibited their own germination. Studies of self-inhibitors are of interest in understanding the regulatory mechanisms of spore germination, and will lead to the development of new techniques that prevent or manage diseases. We isolated a spore germination inhibitor from the liquid-cultured material of Streptomyoces sp. B-9-1, the causal organism of root tumors in melon, and idernffied it as anthranrific acid. However, anthranfic acid showed an IC_<50> at ca 50μg/ml, and its content in spores was very few, indicating that its contribution to the inhibition of spore germination was smal. Reinvestigation of germination inhibitor led us to find a new inhibitor in the spore extract of another strain CB-1-1 (yield, 0.25mg from 3,500 Petridishes). The inhibitor had a characteristic UV spectrum (λ_<max> 263, 318nm), The molecular formula of the inhibitor was determined to be C_7_H_7N_3O_3 by high resolution LC-ESHVIS. In-source CID-MS analysis of the inhibitor and reaction product of the inhibitor with CH_2N_2 led to the fact that the inhibitor contained carboxyl group and acetyl group. The possible compounds, 2-amino-β-axopyrazinepropanoic acid and 3-acetylaminopyrazine-2-carboxylic acid, were selected based on these structural characteristics and UV spectrum specutated by computational chemistry. The possible compounds were synthesized, and were compared with the inhibitor by LC-MS analysis and the inhibitor was determined to be 3-acetylaminopyrazine-2-carboxylic acid. Moreover, we confimed that the inhibitor was produced by other Streptomyces spp., S. coeticolor A3(2) and S. griseus NBRC3237, and we consider the possibility that the inhibitor is a common regulator of spore germination in Streptomyces.

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Wasps of the family Pompilidae are well known for their unique oviposition behavior. Female wasps land on spiders and sting to paralyze their prey instantaneously. The paralyzed spider is then carried or dragged to a nest which, in some cases, the female wasps build from scratch or create by substantially modifying an existing cavity. The female then lays an egg on the paralyzed spider and the larva devours the prey. This interesting function of solitary wasp venom has attracted a great deal of interest. The solitary spider wasp Cyphononyx dorsalis is well known to hunt spiders: it uses its stinger to paralyze its prey to feed its larva. This wasp venom was fractionated by bioassay-guided chromatography. Cation-exchange chromatography indicated that the pI value of the active principle was >6.5. 2D-PAGE analysis of the active fraction obtained by gel permeation chromatography showed three major spots of proteins. Two that appeared at pI of >6.5 were analyzed by in-gel digestion and protein sequencing. Three proteins were identified: an arginine kinase-like protein that was highly homologous to that of honeybee. an elastase like-protein that was homologous to that of fire ant, and an unknown protein that was not homologous to any protein in the database. Recombinant proteins expressed in E. coli were purified and used for bioassay. The recombinant arginine kinase-like protein exhibited paralytic activity against spiders with the same characteristic symptoms as the crude venom.

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Chiral derivatizing agents (CDAs) are frequently used for the NMR determination of the absolute configurations of chiral secondary alcohols and chiral primary amines. In the determination, the validity of the assignments of the absolute configurations strongly depends on the actual conformations of the chiral derivatives in solutions. Here we propose a direct method for verification of the preferred conformation of CFTA esters (CFTA = α-cyano-α-fluoro-p-tolylacetic acid) in solution by observing the C=O stretching absorption in IR spectra. Each IR spectrum of the CFTA ester exhibited two C=O stretching absorption bands conceivably due to two conformers. In all the esters, the absorptions of higher wavenumbers were stronger than that of lower ones. By the ab initio calculations, two stable conformers, the sp and the ap were found for each diastereomeric ester. The calculated wavenumbers of C=O stretching for the sp and the ap conformers well agreed with the higher and the lower wavenumbers of the absorptions experimentally observed, respectively. These results indicate that the predominance of the sp conformers in solutions which was previously deduced from ^1H chemical shift differences of the diastereomeric CFTA esters. Thus, the verification of the preferred conformation in solution is possible by simple IR measurement and this must greatly increase the reliability of the CFTA method.

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Osteoporosis is caused by an imbalance between bone resorption and bone formation, which results in bone loss and fractures after mineral flux. Osteoclast-like multinucleated cells can be differentiated in vitro from co-cultures of mouse bone marrow cells and osteoblastic cells by treatment with osteotropic factors, 1α,25-dihydroxyvitamin D3 (1α,25(OH)_2D_3) and prostaglandin E2 (PGE2). During screening for osteoclast-formation suppressing effects of the extracts of various mushrooms by using the assay, we found very strong activity in the extract of the mushroom Agrocybe chaxingu. Therefore, an attempt was made to isolate the active principles from mushroom and to determine their structures. Powder of the dried fruiting bodies of Agrocybe chaxingu was extracted with CH_2Cl_2, EtOAc and then EtOH. The CH_2Cl_2-soluble fraction only showed the suppressing activity. After repeated chromatography of the fraction, compounds 1 and 2 were purified as the active principles. Osteoclast differentiation was estimated by TRAP-(+) multinucleated cell formation. The addition of compound 1and 2 (3.1μg/ml, 6.8mM) reduced the number of TRAP-(+) multinucleated cells to 66% and 0%, respectively.

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Innate immunity is the first line of defense against infectious microorganisms. The basic mechanisms of pathogen recognition and response activation are evolutionarily conserved. In mammals, the innate immunity has an instructive role in the adaptive immune response specific to antigen by inducing co-stimulatory molecules and cytokines, indicating that a concerted and interactive innate and adaptive immune reaction is key for the regulated immune response. Therefore, from a pharmaceutical point of view, innate immunity is a good target for the development of immune regulators to suppress unwanted immune responses, such as septic shock, inflammatory diseases and autoimmunity, and to stimulate protective immune responses to some of the disease that largely elude the immune system, such as infectious diseases and cancer. For pharmaceutical screening, we established an in vitro culture based on the innate immune response of Drosophila, which is equivalent to that of mammals. The expression of a reporter gene, Dpt-lac Z, reflected the LPS-dependent activation of innate immunity. Search for natural substances regulating innate immunity by the systems led us the isolation of 17 compounds including a cyclopentanediol derivative, TP-1 (1), and a ceramide derivative, TP-76 (2), as innate immune suppressors. Furthermore, we synthesized some derivatives of TP-1 to evaluate structure-activity relationship of these compounds. Methylamide, phenylamide and methylsulfoneamide analogs exhibited innate immune suppressive activities 15 times more potent than that of TP-1 (1).

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During the spring or summer a circle or are of stimulated grass or of toadstools may appear in lawns and other grassy places. These are turf abnormalitines called fairy rings, "Fairy ring diseases" are caused by about sixty kinds of mushroom-forming fungi. Among fairy-ring forming mushrooms, Lepista sordida was investigated as a source of plant-growth regulators The culture broths of L. sordida were filtered The filtrates were partitioned between hexane and water, then ethyl acetate and water. The aqueous layer was dried under reduced pressure, and the resulting residue was extracted with ethanol. Effect of the obtamed fractions on turf growth was examined. The bioassay-guided separation of both ethyl acetate soluble part and ethanol soluble part gave compounds 1 (2-azahypoxanthine) and 2 (1H-umdazole-4-carboxamide), respectively Root growth of bentgrass seedlings exposed to 10μM of 1 was accelerated(320% of control) Compound 2 also promoted the root elongation(134% of control). Shoot and root of bentgrass (Agrostis stolomfera) were successively extracted with hexane, ethyl acetate, ethanol, and then water Since the hexane soluble part of the root showed significant inhibition against growth of L. sordida, it was repeatedly chromatographed on the basis of the result of the bioassay. As a result, compound 3 was purified This compound inhibited the growth of the fungus The symbiotic relation between L sordida and turfgrass is now being elucidated.

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We have previously cloned a DNA fragment that contained four ORFs and was confirmed to participate in viguiepinol {3-hydroxypimara-9(11),15- diene} biosynthesis by a heterologous expression experiment, from Streptomyces sp. strain KO-3988. Of the four ORFs, ORF2 and ORF4 were confirmed to encode an ent-CDP synthase and a GGDP synthase, respectively, by experiments using recombinant enzymes. In this study, ORF3, that did not show similarities with any other known proteins was expressed in E. coli and used for functional analysis. The purified ORF3 product clearly converted ent-CDP into PMD. Since ORF2 and ORF3 are the first examples of enzymes with these biosynthetic functions from prokaryotes, enzymatic properties of both enzymes were investigated. ORF2 is likely to be a dimer and requires a divalent cation such as Mg^<2+> and Zn^<2+> for its activity. The optimum pH and temperature were 5.5 and 35℃.The Km value was calculated to be 13.7±1.0μM for GGDP and the kcat value was 3.3 x 10^<-2>/sec. ORF3 is likely to be a monomer and also requires a divalent cation. The optimum pH and temperature were 7.0 and 30℃. The Km value for ent-CDP was estimated to be 2.6±0.2μM and the kcat value was 1.4 × 10^<-3>/sec.

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Terpenoids are widely distributed in nature, including hormones that regulate life cycles of mammals, insects and plants. Preparation of useful labeled-compounds makes powerful tools for studying comprehensive analysis and biosynthesis of terpenoids. Here, we synthesized fully ^<13>C-labeled prenyl diphosphates using enzyme cocktails which contain recombinant enzymes responsible for biosynthesis of farnesyl diphosphate (FDP) and geranylgeranyl diphosphate (GGDP) from mevalonate. Six cDNAs encoding mevalonate kinase (MVK), phosphomevalonate kinase (PMVK), diphosphomevalonate decarboxylase (DPMVD), isopentenyl diphosphate isomerase (IPI), FDP synthase (FPS) and GGDP synthase (GGPS) were cloned from Neurospora crassa and expressed in E. coli to produce recombinant enzymes as His-tagged proteins. These enzymes were purified by Ni-NTA affinity chromatography and combined with a reaction buffer containing cofactors and the substrate [U-^<13>C_6]mevalonate (97% ^<13>C-label). The ^<13>C-labeled mevalonate was prepared by secretion into the fermentation broth of Saccharomycopsis fibuligera, which was incubated with a [U-^<13>C_6]glucosecontaining medium. The enzyme cocktail containing five enzymes (MVK, PMVK, DPMVD, IPI and FPS) produced ^<13>C-labeled FDP from^<13>C-labeled mevalonate without dilution by natural abundance. Furthermore, uniformly ^<13>C-labeled GGDP was successful ly synthesized in the presence of GGPS into the above enzyme cocktai The ^<13>C-labeled FDP was analyzed by GC-MS, ion-pair HPLC-ESI-MS and two-dimensional ^<13>C-NMR (C-C COSY). The NMR signals were obtained from only 1.3mg of the sample and easily assigned on the basis of the cross-peaks by C-C COSY. We are synthesizing ^<13>C-labeled mevalonate and prenyl diphosphates from 99.9% ^<13>C-labeled acetate in vitro.

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Diterpenes are one of biologically important pharmaceutical sources from natural origin. In higher plant (angiosperm), tricyclic and tetracyclic diterpenes are biosynthesized successively by two-step cyclization from geranylgeranyl diphosphate (GGDP) via ent-copalyl diphosphate (CDP). Each cyclization was catalyzed by two distinct diterpene cyclases (DTCs). copalyl diphosphate synthase (CPS, type-B DTC) and ent-kaurene synthase (KS. type-A DTC). Diterpene cyclases are classified into two groups, (type A and B) on the basis of cyclization mechanisms and aspartate-rich motifs. Plant CPS belongs to type B cyclases and second aspartate of DXDD motif in CPS acts as the proton donor to initiate the cyclization of GGDP. On the other hand, type A DTC has DDXXD motif and initiates the cyclyzation of CDP by an elimination of the diphosphate group and rearranges the carbon skeleton of CDP to form tricyclic and tetracyclic diterpene skeleton. Ent-kaurene is tetracyclic diterpene. and kaurane-type diterpenes exhibit various interesting biological activities. Ent-kaurene is also key intermediate in gibberellin biosynthesis, and is synthesized sequentially by type B and type A DTCs (CPS and KS) in angiosperm. Therefore. the biosynthetic genes of ent-kaurene in angiosperm have been extensively studied, and the biosynthetic pathway is thought to be conserved among plant kingdom. These suggest that the ent-kaurene synthetic genes in lower land plant would he appropriate candidates to discuss the evolution of DTCs in land plants. We have identified ent-kaurene synthetic gene, PpCPS/KS, from the moss Physcomitrella patens and have analyzed PpCPS/KS function by in vitro cell-free protein expression system. Heterologously expressed PpCPS/KS enzyme directly converted GGDP to ent-kaurene skeleton, indicating that moss PpCPS/KS is a bifunctional DTC, as well as fungus and gymnosperm DTCs. In addition, other DTCs have not found by whole genome seach in P. Patens. The liverwort Jungermannia species have been reported to produce a various bioactive kaurane diterepens. We have found that the cultured cell of J. subulata produced ent-kaurane diterepens same as wild plant, suggesting ent-kaurene synthetic gene is expressed even in the cultured cell. We cloned diterpene synthase cDNA (JsDTC1) from J. subulata cultured cells. JsDTC1 encode polypeptide of 886 amino acids and showed high similarity to PpCPS/KS. The two DXDD and DDXXD motifs were conserved.in JsDTC1, suggesting JsDTC1would be a bifunctional DTC. The functional analysis of JsDTC1 demonstrated that JsDTC1 converted GGDP to cyclic diterpenes. The identification of JsDTC1 products are now under investigation.

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In this study, we present the biosynthesis of firefly luciferin, focusing on its chirality, which has remained ambiguous since its chemical structure was first reported in 1961. We show that the firefly contains a considerable amount of L-luciferin, an enantiomer of active D-luciferin, and that the enantiomer is enzymatically inverted to D-luciferin. Furthermore, the chirality of cysteine, the putative biosynthetic precursor of D-luciferin, consists of L-cysteine. These facts suggest that firefly luciferin is synthesized in vivo from L-cysteine and the biosynthetic intermediate is L-luciferin.

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Echinomycin (1), triostin A (2) and thiocoraline are the best known quinoxaline/quinoline antibiotics and contain twin aromatic rings linked to a cross-bridged cyclic octapeptide dilactone core. This class of antibiotics exerts potent antitumor and antiviral activities by binding to DNA via the twin aromatic rings. Recently, we have identified the entire gene cluster encoding nonribosomal peptide synthetases (NRPSs) responsible for the formation of echinomycin and achieved total biosynthesis of 1 in Escherichia cob. The C-terminal thioesterase (TE) domain from the echinomycin NRPS is surmised to catalyze the dimerization of a tetrapeptide to an octapeptide and its subsequent macrolactonization leading to the C_2-symmtetic dilactone skeleton. In this paper, we report chemo-enzymatic synthesis of triostin A and its analogs using the excised TE domain (Ecm-TE) from the echinomycin NRPS. Firstly, a tetrapeptide thioester substrate which mimics its natural acyl-S-enzyme substrate was synthesized and subjected to the enzymatic transformations. The excised Ecm-TE domain was shown to promote in vitro dimerization-cyclization, and subsequent formation of the disulfide bridge allowed us the chemo-enzymatic synthesis of triostin A (2). We then investigated the Ecm-TE catalyzed macrolactonization of octapeptidyl-S-NAC substrates. Although the desired macrolactones was obtained, Ecm-TE competitively hydrolyzed the products to form the significant amounts of the undesired carboxylic acids. To prevent the hydrolysis of cyclized products, we explored the enzymatic lactonization in the presence of DNA as a trapping reagent for the cyclized products. The addition of DNA successfully suppressed the undesired hydrolysis and remarkably improved the yield of cyclized products. Furthermore, DNA-intercalating activities of the resulting cyclic peptides were evaluated.

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The "sorbicillinoids" and "bisorbicillinoids", defined as the term used to designate groups composed of monomeric and dimeric sorbicillin-related natural products, have been attractive in their diverse frameworks and interesting biological activities. In the course of our screening program for antioxidants from microorganisms, we isolated 12 sorbicillinoids and bisorbicillinoids from the fermentation broth of a Trichoderma sp. USF-2690 strain. In our previous study, we established that a functional quinol, sorbicillinol (2), was an important starting point for bisorbicillinoids biosynthesis. However, the biosynthetic mechanism of sorbicillinoids formed via the polyketides pathway and the biosynthetic relationship among sorbicillinoids had been remained obscure. Our experiments for incorporation of [1-^<13>C]-labeled sodium acetate ([1-^<13>C]-SA) gave [1-^<13>C]SA-sorbicillin ([1-^<13>C]-SA-1), [1-^<13>C]-SA-2, [1-^<13>C]-SA-oxosorbicillinol ([1-^<13>C]-SA-3), and [1-^<13>C]-SA-bisvertinolone ([1-^<13>C]-SA-4). Each [1-^<13>C]-SA labeled compound was utilized as a precursor for production of sorbicillinoids (1, 2, and 3) and a bisorbicillinoid (4). The results of incorporation study of partially labeled sorbicillinoids satisfied the proof that one-way biosynthetic routes from 2 to 1 and from 2 to 3 existed. Further investigation making use of HPLC analysis with DAD gave a new sorbicillinoids member 5-epihydroxyvertinolide (5), a postulated end product from the biogenesis. The incorporation patterns of [1-^<13>C]- and [1,2-^<13>C2]-SA demonstrated a biosynthetic route for 5 involving a breakage reaction of carbon-carbon bond. The observation hit on a renewed idea for the sorbicillinoid biosynthesis via an "intermediate B".

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Staurosporine and rebeccamycin produced by actinomycetes are members of the family of indolocarbazole alkaloids that are expected to be the lead compounds of antitumor drugs, because they are strong inhibitors of Protein Kinase C or DNA topoisomerases, which play important role for cell cycling. Staurosporine biosynthetic gene cluster cloned from Streptomyces sp. TP-A0274 consists of 15 orfs spanning 22 kb, and rebeccamycin biosynthetic gene cluster cloned from Lechevarieria aerocolonigenes ATCC39243 consists of 10 orfs spanning 16 kb. Gene disruption and bioconversion experiments revealed the indolocarbazole biosynthetic pathway. First, indole-3-pyruvic acid, which is derived from tryptophan, is coupled by StaD, which is a over 500kDa large heme protein complex, to yield chromopyrrolic acid (CPA). Next, this dicarboxylic bisindole compound, CPA is transformed into the indolocarbazole skeleton via intramolecular C-C bond formation and oxidative decarboxylation catalyzed by cytochrome P450 StaP (StaP, CYP245A1). In this study, we also report X-ray crystal structures of CPA-bound and -free forms of StaP. Based on the crystal structure of StaP-CPA complex, we propose that C-C bond formation occurs via an indole cation radical intermediate. Our crystallographic study shows the first crystal structures of enzymes involved in formation of the indolocarbazole core and provides valuable insights into the process of indolocarbazole biosynthesis, combinatorial biosynthesis of indolocarbazoles, and the diversity of cytochrome P450 chemistry.

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Tetrodotoxin (TTX) and saxitoxin (STX) bind to a single site in the outer pore of the voltage-gated sodium channels (Na_vs), formed by the amino-acid residues in the outer-pore loops (p-loops) located between the S5 and S6 segments of each of the homologous domain (I-IV) of the a-subunit. Since puffer fish and newts accumulate TTX at high concentration in their tissues, they are thought to have special defense systems against their own TTX. We previously obtained a cDNA encoding Na_v from Fugu pardalis skeletal muscle (fMNa1=fNav1.4a). In fNav1.4a protein, the aromatic amino acid in p-loop region of Domain I in TTX-sensitive Nays was replaced by Asn. Also, Kaneko et al. reported that similar mutation was found in Na_v of retinal neuron of the newt, Cynops pyrrhogaster. In this study, we confirmed that these mutations are responsible to TTX-resistance of puffer fish and newts by evaluation of IC_<50>-TTX values of the corresponding mutants of rNav1.2a transiently expressed in HEK293 cells by electrophysiological study.

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Firefly D-luciferin, the specific substrate for the bioluminescence reaction, has the same chirality, with a stereogenic centre, as unnatural D-cysteine. The enantiomer L-luciferin has the same chirality as natural L-cysteine, and behaves as a competitive luciferase inhibitor, as do dehydroluciferin, fatty acids. Firefly luciferase was able to convert L-luciferin into luciferyl-CoA even under ordinary aerobic luciferin-luciferase reaction conditions. D-Luciferin inhibited the luciferyl-CoA synthetase activity of L-luciferin, whereas L-luciferin retards the bioluminescence reaction of D-luciferin, meaning that both enzyme activities are prevented by the enantiomer of its own substrate. It is anticipated that luciferyl-CoA was easily epimerized at neutral pH. Therefore L-luciferin can be converted into D-luciferin by firefly luciferase, an esterase, ATP, Mg^<2+>, and CoA. Because L-luciferin was converted luciferyl-CoA by firefly luciferase, and then epimerized luciferyl-CoA was hydrolysis by esterase to yield D- and L-luciferin. Whereas the regenerated L-luciferin would be rapidly re-converted to luciferyl-CoA, D-luciferin could be used for the light-producing reaction. We have identified a novel route of D-luciferin formation from L-luciferin in vitro that allows the construction of a new firefly bioluminescence system using L-luciferin.

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The sepals of Hydrangea macrophylla show various colors from red through purple to blue and famous for its easy color change. However, any colored sepals consisted of the same components; one anthocyanin, delphinidin 3-glucoside (1) and three co-pigments, 5-O-caffeoylquinic acid (neochlorogenic acid, 2), 5-O-p-coumaroylquinic acid (3) and 3-O-caffeoylquinic acid (chlorogenic acid, 4). For blue color development, Al^<3+> has been clarified to be essential and we obtained stable blue solution by mixing 1, 2 and Al^<3+>. However the mechanism of flower color variation and the chemical structure of the blue pigment are still obscure. To clarify the color variation of hydrangea, we established "Single Cell Analysis Method". After recording the absorption spectrum of a cell, the vacuolar pH of the cell was measured by using a microelectrode. By micro-HPLC technique, the composition of 1-4 and Al^<3+> was determined. Combining the results, we could reproduce the cell color from blue to red by mixing the components in various pH aq. solutions.

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A new antifungal antibiotic, fusarielin A (FSA), was obtained from a culture of a Fusarium sp. K432. FSA induced curling on mycelia germinated from conidia of Pyricularia oryzae P-2b. This biological feature was utilized as a screening assay for compounds that interfere with microtubule function, such as rhizoxin, griseofulvin and nocodazole. So we have speculated that this feature was caused by the inhibition of the assembly of microtubule protein. But FSA had no inhibitory activity to the assembly or disassembly of microtubule protein prepared from porcine brain. Recently, it was reported that ICM0301A, whose structure is similar to FSA, inhibited the growth of a human umbilical vein endothelial cell (HUVEC) and showed significant anti-angiogenic activity. Based on the results, we expected that FSA would show the inhibitory activity to angiogenesis. In fact, FSA showed anti-angiogenic activity. Our purpose of the,research is to reveal the target molecule of FSA and the molecular mechanism of anti-angiogenic activity elicited by FSA.

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Ladder-shaped polyether (LSP) compounds, such as brevetoxins and ciguatoxins, are thought to interact with a common motif generally occurring in membrane proteins. In this study, to examine the above hypothesis, we assessed the interactions between polycyclic ethers and various proteins using surface plasmon resonance (SPR), SDS-PAGE and NMR experiments. SPR experiments showed that membrane proteins containing transmembrane α-helices had significant affinity for yessotoxin (YTX) and a desulfated derivative (dsYTX) with the dissociation constants of about 10-100μM, whereas water-soluble proteins and a membrane protein forming a β-sheet showed relatively weak affinity for YTX and dsYTX. These results suggest that LSPs recognize membrane proteins, particularly transmembrane α-helix. Then, we examined the influence of molecular length of LSP on the affinity using SPR and SDS-PAGE. In order to specifically observe the effects of molecular length, artificial mono, tetra, hepta and deca cyclic ether (ALPs 1, 4, 7 and 10a) were synthesized and used in the experiments. The results thus obtained revealed that the affinity to transmembrane α-helix increases with increasing molecular length. In addition, decacyclic ALP10b that has four hydroxyl groups on both molecular termini was found to precipitate some membrane proteins, which may be due to specific intermolecular recognitions unique to ALP10b.

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Most marine natural products are thought to be involved in defensive systems of their host organisms, and to be produced not by hosts but by microorganisms. However, few studies could experimentally prove this system. Even today, there are many natural products whose biological functions, the origins, and biosynthesis still remain enigmatic. In this study, we show a biological function of a marine alkaloid norzoanthamine in its host organism, the colonial zoanthid Zoanthus sp., a possible symbiotic relationship between Zoanthus sp. and a fungus through norzoanthamine, and the collagenase-inhibitory effect of norzoanthamine. This Zoanthus sp. contains the alkaloid at more than 0.19% of wet weight in its epidermal tissue as shown by quantitative determination with a HPLC system and its molecular ion peak mapping on slices of the animal with MALDI-TOF MS. At this level of concentration, norzoanthamine is shown to suppress degradation of proteins in general, indicating that its biological function in Zoanthus sp. could be to protect the epidermal tissue from external stress. As a matter of, equally interest, a presumable symbiotic fungus identified as Aspergillus fumigatus was isolated from Zoanthus sp. Production of norzoanthamine by the fungus was indicated by MS/MS analysis on the extract from the cultured fungus. These results imply a symbiotic relationship between Zoanthus sp. and the fungus involving norzoanthamine. As another finding, we confirmed the collagenase-inhibitory effect of norzoanthamine was shown using the Sirius Red method, which could be related to its biological function. This effect may be involved in the mechanism of its reported in vivo bioactivity as a candidate of a drug against osteoporosis.

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In the biosynthesis of isoprenoid compounds construction of the fundamental prenyl chain backbone of short chain prenyl compounds is catalyzed by several kinds of prenyltransferases or prenyl chain elongating enzymes, which catalyze the sequential condensation of isopentenyl diphosphate (IPP) with an allylic prenyl diphosphate such as dimethylallyl diphosphate (DMAPP, C_5) or geranyl diphosphate (GPP,C_<10>). To develop convenient biotransformation systems for the syntheses of such biologically active isoprenoid compounds with short prenyl chain backbone, we fed IPP and the allylic diphosphates (DMAPP or GPP) directly to tissue-cultured cells of pumpkin, Cucurbita maxima. After long term incubations followed by alkaline phosphatase treatment, we detected some of the biocatalytic reaction products such as geraniol (GOH), farnesol (FOH), geranylgeraniol (GGOH), and geranylfarnesol (GFOH) in the cultured mediums fed with DMAPP and IPP, or with GPP and IPP. Furthermore, from the cultured medium fed with GOH and isopentenol (IOH), we obtained GGOH along with small amounts of FOH and GFOH. These results suggest that diphosphorylation of these short chain prenyl alcohols occurred in the cultured cells, followed by the prenyl chain elongation by the action of some prenyltransferases.

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The azuki bean weevil, Callosobruchus chinensis L. and the cowpea weevil, C. maculatus, are serious cosmopolitan pests of stored products such as cowpea, azuki or other pulses. The structures of the copulation release pheromones were proposed to be as 1 and 2. However, it was impossible to determine the absolute configurations because of the scarcity of the materials and the incorporation of other acids. In order to determine the absolute configurations of the natural products, we re-isolated the natural pheromones and synthesized optically active 1 and 2 via Evans asymmetric alkylation as a key step. The natural products and the synthetic samples were converted into the corresponding Ohrui-Akasaka esters, that were subjected to the 2D-HPLC analyses. The natural products were confirmed to be stereochemically impure. The stereoisomer constitution of 1 was determined to be R: S=3.3: 1 and that of 2 was determined to be (2R,6S): (2S,6R): (2S,6S): (2R,6R)=43: 38: 18: trace. (R)-1 was reported to be almost a half as active as (S)-1. This means that the female C. chinensis L. produces the biologically weaker enantiomer as a major isomer. On the other hand, there was an inclination for the more contained isomer of 2 in the natural product, to be more active.

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Produced by marine bacterium, thallusin is an algal morphogenesis inducer that is indispensable for the foliaceous morphology of macroalgae. Thallusin, which was isolated in 2005 after chasing for more than 50 years, showed foliaceous morphology-inducing activity at the exceptionally low concentration of 1ag/mL at the first stage. The structure 1 was established via a spectral study as well as via the single crystal X-ray diffraction study of a thallusin derivative. In 2006, Snider and co-workers achieved the total synthesis of compound 1 from sclareol oxide 2. Because compound 1 did not show the foliaceous morphogenesis-inducing activity, the absolute structure of thallusin was determined to be the antipodal 3. Thallusin can be obtained in very limited amounts from cultivation. Thus, in the present study, the total synthesis of (±)-thallusin and analogues were undertaken to allow a detailed examination of thallusin's biological activity. The key steps of the synthesis is a Hg(OTf)_2・PhNMe_2-induced olefin cyclization of homofarnesyl acetate 6 leading to a bicyclic compound 7, and Suzuki coupling of enol triflate 25 with a pyridylboronic acid derivative 26. Hg(OTf)_2 also acted as a catalyst to isomerize the double bond into the more thermodynamically stable isomer when treated in toluene. Synthetic (±)-thallusin as well as an analogue showed morphogenesis activity. On the other hand, we have developed a variety of Hg(OTf)_2-catalyzed reactions base upon the significant π-philicity of this reagent. For example, the reaction of arylalkyne 33 with 0.1mol % of Hg(OTf)_2・3TMU affords 34 in 99% yield. Protonation of vinyl mercuric intermediate 35 with in situ formed TfOH leading to cation 36 is the key step of the catalytic syntem. Now we introduce oxygen functionality nearby as the protonation site and succeeded the Hg(OTf)_2-catalyzed olefin cyclization. Reaction of 37 with 0.2mol % of Hg(OTf)_2 afforded 38 in 90% yield, and the reaction of Hg(OTf)_2 with 1mol % of Hg(OTf)_2 afforded 42 in 84% yield.

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We have recently reported a novel Rh(I)-catalyzed cascade reaction by combination of a hydroacylation of 4,6-dienal and a cycloisomerization of the resultant triene, giving a bicyclo[5.3.0]decenone derivative (Scheme 1). By the use of the cascade reaction, the synthesis of (±)-epiglobulol has been also accomplished. Herein, we report the construction of polycyclic compounds using the Rh(I)-catalyzed cascade reaction with the aim of the synthesis of Guanacastepenes (Scheme 2). Treatment of syn-3 with 10 mol% of [Rh(dppe)]ClO4 in dichloroethane at 65 ℃ for 6 h gave the desired tricyclic compound syn-4 in 34% yield along with syn-S, anti-4, and anti-5 in 8%, 7%, and 23% yields, respectively (Scheme 3). It was thought that bicyclic compound syn-5 was formed through reductive elimination from 6 (Scheme 4). On the other hand, anti-4, and anti-5 should be produced from anti-3, which would be formed from syn-3 via epimerization of a-proton of the aldehyde moiety syn-3. In the cyclization of anti-3 under the same conditions, bicyclic compound anti-5 was produced as the major product in 32% yield and the desired tricyclic compound anti-4 was produced in only 9% yield. These results indicate that the stereochemistry of cyclohexane ring in 3 affects on the reaction course. In order to prevent the isomerization of syn-3 to anti-3, the reactions of syn-3 were examined under the various conditions (Table 1). It was found that the cyclization of syn-3 in the presence of MS 4A gave the cyclized product syn-4 in 61% yield as the major product, while anti-4 and anti-5 derived from anti-3 were not obtained.

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The gibberellins are devided into two groups, the larger of which is C_<19> gibberellins [gibberellic acid: GA3; a typical representative], with most of the remaining have 20 carbons. The latter possesses entgibberellane framework. GA_<112> belongs to C_<20> gibberellins, and GA_<12> is believed to be a common intermediate in the biosynthesis of all gibberellins. Since the question of how to establish practical routes for the construction of C_<20> gibberellins was open, we made a start in the study of a stereoselective synthesis of GA_<112> employing the palladium-catalyzed cycloalkenylation developed by us. As a result, a highly stereoselective total synthesis of chiral GA_<112> was achieved by a combination of palladium-catalyzed cycloalkenylation and reverse electron demand intramolecular Diels-Alder reaction. The most important aspect of the present synthesis is that all stereoisomers produced were used, and most of the reaction yields are good. The present synthesis. would provide some perspectives both on the determination of tentative new structures and on the exploration of their bioactivities.

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Previously, we isolated valtrate (2) as an inhibitor for nuclear export of NES (nuclear export signal) possessing proteins such as Rev and MEK from the medicinal plant, Valerianae Radix. Furthermore, this principle was shown to exhibit anti-HIV and selective cytotoxic activity against MEK activated tumor cells, respectively. Because of acyl groups instable in serum, valtrate (2) was presumed to show little efficacy in vivo. In addition, preparation for a deacylated derivative of 2 was revealed to be difficult due to a labile hemi-vinyl acetal moiety. In this context, establishment of total synthetic protocol of 2 or its analog with similar biological activity seems essential to exploration for medicinal leads with in vivo potency. With the aid of molecular orbital calculation by PM3, we designed 5,6-dihydroanalog (3) as an isostere of 2. By use of asymmetric Diels-Alder reaction and intramolecular acetal cyclization of 17 as key steps, a methyl-vinyl acetal 18 with iridoid skeleton was prepared. Successive construction of exo-olefin, 1-O-valely1 portion, and an acetoxymethyl function from 18 provided an allyl alcohol 29 by a combination of moderate chemical conversions. Stereoselective epoxidation of exo-olefin in 29 followed by Mitsunobu reaction furnished the desired 5,6-dihydroanalog (3). Since 5,6-dihydroanalog (3) exhibited nearly the same biological activity as valtrate (2), the analogs of 3 with different acyl groups were also synthesized to clarify their steric effect. As a result of synthesis of the three analogs (3a-3c) according to the synthetic route of 3 and evaluation for biological activity, 1-O-acetylanalog (3b) was shown to display more potent activity than valtrate (2).

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(-)-Nalanthalide (1), isolated from the culture of Nalanthamala sp., was found to be a novel blocker of the voltage-gated potassium channel Kv 1.3 by Merck research group. In human T cell, to block Kv1.3 channels cause to prevent membrane depolarization, which attenuates intarcellular Ca^<2+> levels for T cell activation and proliferation. Therefore, (-)-1 is expected to be a promising new lead for the immunosuppressant. A closely related diterpenoid α-pyrone (+)-sesquicillin (2), wherein the γ-pyrone ring of (-)-1 is replaced with an α-pyrone ring, was previously isolated from Acremonium sp. It was reported that (+)-2 shows a variety of biological properties such as glucocorticoid antagonist. anti-inflammatory, anticancer, and G1 phase arrest activities. We envisioned that (+)-(2) would be elaborated through conversion from the γ-pyrone ring of (-)-1 to the corresponding a-pyrone ring. And (-)-(1) would be available through a coupling of 4 with 3. The intermediate 5 would be available through the strategic [2,3]-Wittig rearrangement of 6. The synthesis began with the preparation of intermediate 6, the substrate for the key [2,3]-Wing rearrangement, starting from 8. After conversion of 8 to 6, we carried out the [2,3]-Wittig rearrangement under several reaction conditions. Finally, we found that the designed [2,3]-Wittig rearrangement of 6 proceeded smoothly and cleanly by treatment with n-butyllithium in n-hexane at-50℃→room temperature. In this reaction, the use of n-hexane as the solvent was crucial. After conversion of 5 into 4, the coupling of 4 with 3-Iithio-γ-pyrone was achieved an initial bromine/lithium exchange on 3. The coupling product was converted to the key intermediate 19. Acetylation of 19 furnished (-)-(1). Next, we attempted direct conversion of (-)-(1) to (+)-(2) under several basic conditions; however, undesired deacetylation of the C3 acetyl group in (-)-1 occurred. Consequently, conversion of the γ-pyrone ring in 19 to the α-pyrone ring under basic conditions was conducted; the desired diacetate 20 was obtained upon acetylation of the product. Finally, selective deacetylation of the α-pyrone moiety in 20 under mild basic conditions furnished (+)-(2).

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Numerous efforts have been made to produce bicyclic cyclopentanones, not only in consideration of their abundance in nature, but also their usefulness as versatile synthetic building blocks. The sequential reductive coupling-Dieckmann condensation reaction has been found quite useful as a one-step process for effectively producing oxacyclopentanecarboxylates from α,β-unsatureted esters. Electrohydrodimerization and metal-induced cyclization of cinnamate derivatives are methods generally applied for this purpose but none has been shown to produce in an intramolecular manner bicyclic oxacyclopentanecarboxylate. The authors have established two types of Sm12-induced sequential reaction to produce bicyclic cyclopentanones: 1) a sequential reductive cyclization-Dieckmann condensation of bis-α,β-unsaturated esters i with Sm-SmI_2 in the presence of catalytic amount of methanol to provide cyclopentanone ii (n=1, 2) (Scheme 2); and 2) a ring expansion reaction of 1,2-cyclobutanedicarboxylate iii with SmI_2-HMPA to provide ii (n=1 to 4) (Scheme 3), both of which involve iv and v as intermediates. The results of reactions of bis-α,β-unsaturated esters 1-4 with SmI_2 and 1,2-cyclobutanedicarboxylate 11-14 with SmI_2-HMPA are summarized in Table1 and Table 2, respectively. To show the synthetic utility of these reactions, we choose hirsutene as a synthetic target, which is typical member of triquinane sesquiterpene. And we wish to describe a new synthetic route to (±)-hirsutene, which involves SmI_2-induced sequential reactions, 3 to 6 and 23 to 25, as key steps (Scheme 5). (±)-1-Desoxyhypnophilin was also synthesized from common precursor 6 via rapid transformation of 6 into triquinane ring system 19 and reductive conversion of 19 into 21 (Scheme 4).

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Kitocephalin was isolated from Eupenicillim shearii PF1191 in 1997. This compound has the very unique framework in which three amino acids connect with C-C bonds. This compound exhibits potent inhibitory activity against neuronal cell death by the action of antagonist for NMDA and AMPA/KA receptors. Although this compound is expected to be the lead compound for development of therapentic agents against neuronal diseases, the fungus has stopped producing this compound. So, there is not the way to obtain this compound except for organic synthesis at present. These backgrounds made us develop the efficient synthetic route of kaitocephalin. Starting from known compound 1, homocoupling by treating with Grubbs 2^<nd> catalyst followed by Wacker oxidation readily provided ketone 3. Successive reductive amination and mono acylation with hydroxyl succinimide ester at primary amino group gave 4 preferentially. For the introduction of the right-hand moiety, we first tried dianion coupling, which was found to be unsuccessful. However, we tried Barbier reaction, after transforming 4 to imine 11 by treating with NBS and Et_3N, Barbier reaction proceeded to afford allylated compound 12 with desired stereoselectivity in almost quantitative yield. By exchanging the protective groups, followed by olefin-isomerization and allic oxidation, we obtained alcohol 15. Epoxidation of 15 proceeded with complete stereoselectivity and provided 16. Subsequent azidation under the Miyashita's condition gave desired product 17 along with debenzylated compound as a mixture. Rebenzylation of the mixture followed by reduction with indium and Cbz protection provided compound 18, which is an intermediate of our previous synthesis. After conducting oxidation and deprotection according to the known procedure, we obtained kaitocephalin. In summary, we achieved total synthesis of kaitocephalin in 6.5% overall yield (18 steps) from 1.

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Various structural types of 2-aminoimidazole alkaloids have been isolated from marine sources (Fig. 1). Their intriguing structures as well as diverse biological activities, some of which are of interest from the pharmaceutical points of view, make these alkaloids attractive synthetic targets. We have expected that the 2-aminoimidazol-4-carbaldehyde derivatives I (Fig. 2) would be the best starting material for these alkaloid synthesis. However, a search of the literature soon disclosed that the synthesis of I is scarcely explored. Therefore, we embarked on exploring a novel synthetic route to I. By using the reaction of t-butoxycarbonylguanidine (12a) with 3-bromo-1,1-dimethoxypro-pan-2-one (10) as a key step, we succeeded in developing a novel synthesis of I. Expeditious syntheses of oroidin (1), hymenidin (2), dispacamide (4), monobromodispacamide (5) and ageladine A (6), the representative 2-aminoimidazole alkaloids, were accomplished starting with 2-t-butoxycarbonylaminoimidazol-4-carbaldehyde (15) thus obtained. These results confirmed that 15 is very useful for synthesizing 2-aminoimidazole alkaloids. In addition, we synthesized analogs 27-35 of 6 in order to disclose its structural features required for MMP-12 inhibitory activity and to explore a novel analog that can show more excellent activity than 6. From MMP-12 inhibitory activity assay of 6 and 27-35, it appeared evident that the two bromine atoms and the three NH groups (1-NH, 14-NH, and 15-NH_2) were indispensable for 6 to exhibit excellent activity.

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The AI-77-B (1), isolated from a culture broth of Bacillus pumilus AI-77 by Shimojima and co-workers, exhibits potent gastroprotective activity without antichlorinergic, antihistaminergic, or central suppressive effects. Prompted by its interesting biological activity and unique structure, six groups have reported the total synthesis of 1. In this symposium, we report a more efficient enantioselective total synthesis of 1 and 2. Right segment 5 was synthesized from known aldehyde 11 through Cordova's asymmetric epoxidation and TFA-mediated hydroxylactonization. Benzamide derivative 24 was subjected to coupling reaction with azidoepoxide 23 prepared via Sharpless asymmetric epoxidation to furnish 25. The coupling product 25 was converted into left segment 6 via lactonizaton and reduction. Condensation between 5 and 6 followed by deprotection afforded 2. Finally, hydrolysis of its five-membered lactone moiety gave 1. Thus, an efficient enantioselective total synthesis of 1 and 2 has been accomplished in 10 steps from 11.

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We have already successeded the formal total synthesis of (±)-manzamine A (1) via key intermediate (3). For asymmetric total synthesis of (+)-manzamine A, we established effective asymmetric synthesis to key intermediate (+)-3 (Scheme 6). Catalytic allylation of 20 using only 0.15mol% [PdCl(allyl)]_2 and 0.36mol% ligand 23 gave 21 in 83% yield with 81% ee. Intramolecular Michael reaction of 28 which has β-TIPS ether proceeded with high diastereoselectivity. Therefore, optically pure (+)-3 was synthesized from ethyl 4-oxopiperidine-3-carboxylate in 12 steps and in 38% over all yield. Further investigation toward the asymmetric total synthesis of (+) manzamine A (1) from (+)-3 will be discussed.

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