Transition metals have been playing very important roles in modern synthetic organic chemistry. We succeeded in the first asymmetric total syntheses of (+)-crinamine (1), (-)-haemanthidine (2) and (+)-pretazettine (3) in crinine-type alkaloids, using palladium-catalyzed asymmetric amination, and intramolecular carbonyl-ene reaction as the key steps. Palladium-catalyzed asymmetric amination of 12a with 8 gave (S)-15 with 84% ee in 87% yield. On the other hand, 13a afforded (S)-16 with only 60% ee under the same reaction conditions. The ees of 15 and 16 were slightly increased when the reactions were carried out at 0 ℃, although the starting materials were recovered. However, in the case of enol carbonate 12c and 13c, the reactions proceeded smoothly even at -20℃ because the deprotonation from 9 was accelerated due to the strong basity of counter anion in π-allyl palladium complex II, and 15 with 92% ee, and 16 with 74% ee were obtained. As expected, when a toluene solution of 18 was heated at 230-240℃, the desired intramolecular carbonyl-ene reaction proceeded, and hexahydroindole 20 was obtained in 30-40% yield (conversion yield: 46-60%) as a sole product. On the other hand, SnCl_4-catalyzed (10 mol %) carbonyl-ene reaction of 18 gave an unusual rearrangement product 19a in 89% yield. To synthesize (+)-1, recrystalyzation of 16 with 74% ee gave an optically pure (-)-16 from the mother liquor. Deacetalyzation of 16 following intramolecular carbonyl-ene reaction gave 21. Allylic oxidation of 21 with SeO_2 gave alcohol 22 as a sole product, which was followed by treatment with Ms_2O and then MeOH gave 23 as a major product. Detosylation of 23 followed by methylenation and then deacetylation proceeded smoothly to give (+)-1. Subsequentry, treatment of 25 with HC(OMe)_3 in the presence of Montmorillonite K-10 gave 24 in high yield. Detosylation of 24 followed by formylation gave compound 26, which was treated with POCl_3, followed by deacetylation, gave (-)-2. Using a known method, (+)-3 was synthesized from (-)-2. The [α]_D value, melting point and spectral data of synthetic 2 and 3 agreed with those reported in the literature.

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Asymmetric dihydroxylation of a series of 2-alkenylfurans afforded the corresponding 1,2-glycols in good to excellent optical yields. On oxidative ring-expansion followed by acid-catalyzed cyclization, the glycols obtained furnished the dioxabicyclo[3.2.1]octane products carrying an enone, a masked formyl and a 1,2-glycol functionalities in their molecules. Owing to their biased bicyclic framework, the reactions around the enone functionality occurred diastereoselectively from the convex face of the molecules which allowed enantio- and diastereo-controlled construction of a variety of natural products having tertiary and quaternary stereogenic centers. In the present paper we demonstrated the general method for the synthesis of the key chiral building blocks and their exploitation for the construction of four insect pheromons having the same framework such as (-)-β-multistriatin, (+)-exo-brevicomin, (-)-exo-isobrevicomin, and both enantiomers of frontalin, both enantiomers of levoglucosenones, five of eight possible hexoses, a cyclitol conduritol F, an insect pheromone serricornin, and a penultimate intermediate of three aromatic bisabolane sesquiterpenes such as (+)-α-curcumene, (+)-nuciferol, and (+)-nuciferal. Two novel nickel-mediated reactions were also discovered during the present investigation: the chemospecific de-O- and de-N-allylation with diisobutylaluminum hydride in the presence of a catalytic amount of NiCl_2-dppp and the coupling reaction of two equivalents of an allyl ether and one equivalent of trimethylaluminum in the presence of a catalytic amount of NiCl_2-dppp.

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Allene-1,3-dicarboxylates are useful for the Michael addition and [4+2] or [2+2] cycloaddition. The recognized methodology for their synthesis has, however, not been satisfactory in terms of the yield and the reaction-steps required. Focusing on the excellent dehydrating ability of 2-chloro-1,3-dimethylimidazolinium chloride (DMC), we exploited a novel efficient synthetic method for allene-1,3-dicarboxylates (2) from acetone-1,3-dicarboxylates (1) in one step. A typical reaction of dimethyl acetone-1,3-dicarboxylate (1a) with DMC (1.2 eq.) in dichloromethane at room temperature for 1 h in the presence of triethylamine (3 eq.) afforded dimethyl allene-1,3-dicarboxylate (2a) in 90 % yield. Optically active di-L-menthyl (R)-allene-1,3-dicarboxylate (2f-R) has been prepared by Kanematsu et al. through optical resolution. In order to improve this synthetic method, we examined the epimerization of di-(-)-L-menthyl allene-1,3-dicarboxylates (2f-R and S) with the assistance of triethylamine. As a result, we exploited a new crystallization-induced asymmetric transformation for the synthesis of di-L-menthyl (R)-allene-1,3-dicarboxylate: crystallization from a diastereomeric mixture (R: S=4: 5) of di-(-)-L-menthyl allene-1,3-dicarboxylates (2f) in pentane with 0.05 equivalent of triethylamine at low temperature (-20℃) gave 2f-R in 90 % yield (>98 %de). The former process opened a new expeditious route to a 1-azabicyclo[3.3.0]octane skeleton of pyrrolizidine alkaloids from dimethyl acetone-1,3-dicarboxylate and bis(2-chloroethyl)amine via a Michael addition and a novel tandem cyclization. The latter process was applied to a formal asymmetric synthesis of (-)-epibatidine utilizing the highly diastereoselective Diels-Alder reaction of 2f-R with N-Boc-pyrrole as a key reaction.

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Recently, cytotoxic triterpene polyethers, which are thought to be biogenetically squalene-derived natural products, have been isolated from both marine and terrestrial plants (Figure 1). Among them, our synthetic targets are teurilene (3) isolated from the red alga Laurencia obtuse and the wood of Eurycoma longifolia and glabrescol (5) extracted from the branches and wood of Spathelia glabrescens (Rutaceae). These unique polyethers 3 and 5 become achiral meso molecules due to Cs symmetry in spite of possessing 8 and 10 asymmetric centers, respectively, within the molecules. In this symposium, we report the efficient total synthesis of meso polyether teurilene (3) through the highly effective combination of the concept of two-directional synthesis and the rapidly progressive rhenium(VII) chemistry. In model experiments, syn oxidative cyclizations of bishomoallylic tert-alcohols 8-13 promoted by rhenium(VII) oxide have been accomplished with excellent diastereoselectivities (Tables 2 and 3). The critical trans or cis selectivity between the 2- and 5-positions of the tetrahydrofuran (THF) ring in the products has been observed, depending on the substrates employed. The steric discrimination between the two substituents R_S and R_L at the carbinol center in the alkoxyrhenium intermediate appears to be responsible for the high trans selectivity (Figure 2). On the other hand, the intramolecular coordination of the THF ring neighboring the hydroxyl group to rhenium appears to be important for high cis selectivity (Figure 3). Finally, the oxidative cyclizations of bishomoallylic diol 6 with an excess of (CF_3CO_2)ReO_3・2CH_3CN and TFAA in CH_2Cl_2-CH_3CN mixed solvent system successfully proceeded in a two-directional manner to give the desired teurilene (3) with the complete trans-syn diastereoselectivity (Scheme 2 and Figure 4).

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Recent development of asymmetric metal catalysis has realized highly enantioface-selective oxidation of carbon-carbon double bonds. On the other hand, enantiotopic selective C-H bond oxidation has so far been met with limited success: moderate to good enantioselectivity has been achieved only in the oxidation of activated benzylic and allylic C-H bonds. However, we have demonstrated that (salen)manganese(III) complexes are excellent catalysts not only for asymmetric epoxidation of simple olefins but also for enantioselective hydroxylation of prochiral benzylic carbon. As an extension of enantiotopic selective C-H oxidation, we examined Mn-salen catalyzed oxidative asymmetric desymmetrization of meso-heterocycles. This class of compounds are readily available in bulk and the resulting oxidation products are useful building blocks in organic synthesis. At first we examined asymmetric desymmetrization of meso-cyclic ethers in the presence of various Mn-salen complexes and found that high enantioselectivity (up to 90% ee) was realized in the reactions of meso-tetrahydrofuran derivatives, when complex 3 was used as a catalyst. The resulting lactols are useful building blocks. As their application, we synthesized (-)-alloyohimbane (13) from lactol 7 only in seven steps in a straight forward manner. We next examined asymmetric desymmetrization of meso-pyrrolidine derivatives. Moderate to good enantioselectivity was also observed, though the selectivity depended on the N-protecting group. Enantioselectivity up to 82% ee was obtained in the reaction of pyrrolidine derivative N-protected with phenylacetyl group. It is noteworthy that the oxidation of C-H bond occurred chemoselectively. For example, acetonide is tolerable to the present conditions. In conclusion, we were able to disclose that salen-catalyzed enantiotopic selective C-H oxidation is of high synthetic use.

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We are studying gibberellin (GA) biosynthesis of the fungus Phaeosphaeria sp. L487 that has GA 3β-hydroxylation step similar to that in plants. (1) Elucidation of late stages of plant-like GA biosynthetic pathway in Phaeosphaeria sp. L487 We identified GA_1, GA_4, GA_9, GA_<12>, GA_<15>, GA_<20>, GA_<24> and GA_<25> from extracts of Phaeosphaeria's culture filtrate. GA_1 was accumulated in the culture broth at 3 weeks after inoculation (GA_1> 100 mg litre^<-1>). To deremine late stages of the GA biosynthetic pathway in Phaeosphaeria, feeding studies using isotope-labeled GA intermediates were carried out. The fungus metabolized GA_<12>-aldehyde to GA_1 via GA_<12> → GA_<15> → GA_<24>→GA_9→GA_4 and GA_<20>. The 3β-hydroxylation, which is the activation step of endogenous GAs in plants, occurred at steps on GA_9 and GA_<20> to produce GA_4 and GA_1, respectively. (2) Molecular cloning, characterization and identification of catalytic domains of ent-kaurene synthase from Phaeosphaeria sp. L487 ent-Kaurene is the first cyclic intermediate of GA biosynthesis and is synthesized from geranylgeranyl diphosphate (GGDP) via copalyl diphosphate (CDP) in both fungi and plants. In order to clone a cDNA encoding ent-kaurene synthase (KS) from the fungus Phaeosphaeria sp. L487, degenerate primers based on consensus motifs of plant copalyl diphosphate synthase (CPS) and other diterpene cyclases were used to amplify a cDNA fragment encoding the fungal CPS homologue. A full-length cDNA was obtained from the fungal cDNA library and has an open reading frame encoding 946 amino acids with 106-kDa. A fusion protein produced by expression of the cDNA in E. coli catalyzed the two-step cyclization from GGDP to ent-kaurene. Thus, it revealed that ent-kaurene synthase from the fungus is a bifunctional diterpene cyclase. To determine active sites of the enzyme, site-directed mutagenesis was carried out in the aspartatelglutamate-rich motifs, which are thought to be involved in CPS/KS activities. Mutation of Asp132 to Ala132 decreased CPS activity. Mutation of Asp320 to Ala320 had no CPS activity, whereas it increased KS activity. In contrast, mutation of Asp656 to Ala656 had no KS activity. The catalytic domains for two enzyme activities, CPS and KS, on the polypeptide were identified by functional analysis of truncated proteins prepared from deletion clones from the cDNA. A 63-Kda protein translated from the N-terminus of wild type (amino acids 1-568, CTP-2) showed CPS activity, whereas a 59-KDa protein (amino acids 1-530, CTP-1) did not have CPS activity. In contrast, a 66-KDa protein lacking the N-terminus of wild type (359-946 amino acid residues, NTP-3) showed KS activity. A 101-KDa and a 63-KDa protein truncated 44 and 381 amino acid residues from the N-terminus of wild type, respectively, did not have any functions for CPS and KS activity. In conclusion, the fungal ent-kaurene synthase has separate CPS and KS domains fused tandem for the two cyclization reactions.

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Farnesyl diphosphate (FPP) synthase[2.5.1.10] catalyzes the condensation of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate(DMAPP) and with geranyl diphophate (GPP) to produce E,E-FPP as a final product and does not catalyze a condensation beyond FPP. Recently, it was observed that in Bacillus stearothermophilus FPP synthase, a replacement of tyrosine with histidine at 81 position which is located on the fifth amino acid before aspartate-rich motif, caused the mutated FPP synthas to catalyze geranylgeranyl diphosphaye (C_<20>) synthesis. Then we constructed the mutated FPP synthase, in which tyrosine-81 was substituted with glycine and studied the substrate specificity of this enzyme using the substrate analogs (1-28) as depicted in Fig 1. The results showed that the reactivities of the all analogs with the mutant enzyme (Y81G) were higuer than those of the wild enzyme as shown in Table 1 and 2. In the case of analogs having hydrocarbon as side chain (3-9), the reactivities were higher than those of the wild enzyme. The number of IPP-incoporation also was bigger than that of the wild enzyme. These results were same to those of the natural substrates as reported before. It has been reported that the substrate specificity of FPP synthase of Bacillus stearothermophilus is more stringent than that of FPP synthase of pig liver. Namely, the wild enzyme can hardly accept the analogs having oxygen atom in their chain. On the other hand, FPP synthase of pig liver can accept them. The mutant enzyme can also accept these analogs (14-19) as substrate better than the wild enzyme as shown in Table 1. This observation showed that the substrate specificity of the mutant enzyme is changed and becomes similar to that of FPP synthase of pig liver. And this fact suggest that this mutant enzume may be used for organic synthesis better than the wild enzyme because of accepting analogs having hetero atom such as oxygen atom. The analogs (20-21), which have no methyl group at 3-position of DMAPP analog, were tested to show that these analogs can not be accepted by both of the wild and the mutant enzyme. This finding was also found in the reaction of FPP synthase of mammmalian FPP synthase. This fact strongly indicates that the methyl group at 3-position of GPP is very crucial for the substrate recognition of.FPP synthase including the mutant enzyme.

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Archaea (archaebacteria) including methanogens, extreme halophiles, extreme thermophiles, and thermoacidophiles are distinct from prokaryotes and eukaryotes. The most characteristic feature of archaea is found in the chemical structure of its membrane core lipid, the biosynthesis of which has been studied with new approaches. A synthetic method of (RS)-mevalonolactone-d_9 was developed starting from deuterated dimethoxyphenylacetone and trimethyl phosphonoacetate. The 7-steps synthesis yielded mevalonolactone-d_9 in 43% overall yield. Worth to note is that the deuterium sources were easily available deuterium oxide and lithium aluminum deuteride. Synthesized mevalonolactone-d_9 was applied to the biosynthetic studies of the core membrane lipid, 2,3-di-O-phytanyl-sn-glycerol, of two halophilic archaea, Haloarcula japonica and Halobacterium halobium. Mevalonolactone-d_9 was highly incorporated into the phytanyl chains of the archaeal lipid, and the total enrichment was approximated to be as high as 70-90%. Mevalonate pathway appears to be clearly responsible for the biosynthesis of the phytanyl chains of the core lipid in the archaeal membrane. Further, since fully deuterated mevalonolactone was highly incorporated to the phytanyl group, we were able to trace the fate of incoming protium atoms during lipid biosynthesis by ^1H NMR spectroscopy. Saturation of the geranylgeranyl group to the phytanyl group was shown to take place through the addition of hydrogen in a syn-manner by analyzing the behavior of protium on the heavily deuterated archaeal lipid.

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Extracts of trees have been used as drugs, insecticides, perfumes, and dyes since a long time ago. But those were recently used a little for development of chemicals having potent effects. More recently, from a safe point of view in those chemicals, extracted compornents of trees were focused again because of variety and mild effect on organism. Hinokitiol is a compornent which have been studied best in practice. Purified oil of Thujopsis dolabrara var. hondae Makino (Aomori hiba) and Chamaecyparis taiwanesis Masamune et Suzuki (Taiwan hiba) contain about 1 wt% of Hinokitiol, which inhibits the growth of a wide variety of bacterial species even in a few thousand-fold dilution condition. Extraction of hiba oil gives a large amount of aqueous layer as a by-product, That aqueous layer contains a very small amount of Hinokitiol, but has never been tried to collect and purify. Now we established industrial technology in production of Hinokitiol with low cost and high purity, from that aqueous layer. Crude oil was recovered with an absorptive resin from aqueous layer which was obtained by steam distillation of Ma chips, and purified to give pure Hinokitiol effectively.

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Hiba oil is obtained from Thujopsis dorabrata SIEBOLD et ZUCCARINI var. hondae MAKINO. Hiba oil consists of two parts of Acidic oil and Neutral oil. Hinokitiol is contained in the Acidic oil. Hinokitiol is utilized for Industrial use due to its excellent biocidal efficacy. Meanwhile, the Neutral oil which constitutes 92% of Hiba oil has not been utilized. Research has been started to use the Neutral oil for wood preservatives, which leads the discovery of termiticidal efficacy for the Neutral oil as known for Hiba oil. It was found out that main active ingredient as temiticides is thujopsene which is the major component of the Neutral oil. Cedrol which is the known termiticide contained in Sciadopitys verticillata S. et Z. is also found in the Neutral oil but is not lilely to be the main component in the Neutral oil. Research is still under way.

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Lignans are widely distributed secondary metabolites, which consist of two phenylpropane units, linked by 8,8' bonds. Recently, 78-kilodalton protein named as 'dirigent protein' has been isolated from Forsythia suspensa, which effects stereoselective bimolecular phenoxy radical coupling in the presence of an oxidase and one electron oxidant On the other hand, lignan as unusual naphthalene derivative, 1-(3,4-dihydroxyphenyl)-6,7-dihydroxy-1,2-dihydro-2,3-naphthalenedicarboxylic acid (DDDN, 1) has been isolated from gametophytes of Pellia epiphylla. Further oxygenated lignan, jamesopyrone (2) has been isolated from Jamesoniella autunvidis. HPLC analyses of two lignans, 1 and 2 have been demonstrated on 53 species of Bryophytes. The amounts of lignans in liverworts were larger than those in mosses. Chiral HPLC analyses of lignans in twelve liverworts clarified their enantiomerical deviations. They were considered to be biosynthesized from two caffeic molecules, a common phenolic unit in liverworts. It suggested that the stereochemical mechanisms of lignan biosynthesis in liverworts were different from that in vascular plant having a great diversity. Thus we had an interest for the biosynthesis of lignans in liverworts. The formations of the lignans 1, 2, and scapaniapyrone (3) in liverwort, J.autumnalis were investigated. Feeding experiment of [8-^2H] caffeic acid (CA, 9) on axenic cultures of J. autumnalis has shown that 2 and 3 were derived from the coupling of two intact caffeic molecules. Furthermore, (+)-1 was enantioselectively formed from 9 in cell free extracts of liverwort J. autumnalis. In the bimolecular coupling model system, we found the formation of furofuran-type lignan 2,6-exo-bis (3,4-dihydroxyphenyl)-3,7-dioxabicyclo [3.3.0] octane-4,8-dione (10) from 9 with NaIO_4. NMR analyses of oxidation of 9 with NaIO_4 in MeOH-d_4 showed the formation of 3,4-dioxocinnamic acid (o-quinone, 11). It was further confirmed by the reaction of 11 in MeOH with 1,2-phenylenediamine to produced phenazine derivative. The first step of the bimolecular coupling was thought as the oxidation of 9 to highly reactive 11 which has both electrophilic and oxidative properties. ^1H-^1H COSY analyses of 10-Mes formed by couplings of ^<13>C- or non- labeled 9 with 11 revealed that 11 as an intermediate of the formation of 10 from 9.

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Tuber development of potato plants (Solanum tuberosum L.) was controlled by environmental factors, mainly by photoperiods. Short days stimulate this process, but long days have opposite effect against this process. This means potato plants distinguish the day length. But it is still unknown the relationships between day length and potato tuber inducing compounds, jasmonic acid (JA), 12-hydroxyjasmonic acid (tuberonic acid, TA), and tuberonic acid glucoside (TAG), since there is no qualitative and quantitative analysis method for these compounds. The synthesis of deuterium labeled TAG, TA, JA, (1R, 2R) 3-ox-2-(2'-pentenyl)-cyclopentanebutanoic acid (OPC 4: 0), (1R 2R) 3-oxo-2-(2'-pentenyl)-cyclopentanehexanoic acid (OPC 6: 0), and (1R, 2R) 3-oxo-2-(2'-pentenyl)-cyclopentaneoctanoic acid (OPC 8: 0) were accomplished. The qualitative and quantitative analysis for the endogenous jasmonides was carried out using these compounds as internal standards and monitoring by liquid chromatography/mass spectrometry (LC-SIM). This methodology was applied to estimate the endogenous jasmonides, TAG, TA, JA, OPC 4: 0, OPC 6: 0, and OPC 8: 0, in potato plant. The contents of TAG, TA, JA in the leaflets of potato (Solanum tuberosum L. cv. Irish Cobbler) which were harvested in July, Hokkaido, were estimated to be 7-5μg/1g leaflets, 170-100 ng/1g leaflets and 20-10ng/1g leaflets, respectively, and those of OPC 4: 0, OPC 6: 0, OPC 8: 0 to be below ng level/1g leaflets. The contents of TAG of the leaflets of potato which grown under long and short day conditions were analyzed, respectively, by means of this developed methodology. The contents of TAG were higher in the leaflets grown under long days than in those grown in short days. It is suggested that the short day condition might stimulate the transportation of jasmonids from leaflets to the other parts. The bio-synthetic pathway for TA and TAG was established in the first time because the deuterium leveled JA and OP 8: 0 were converted to TAG in the cultures on single-node segments of potato stem in vitro.

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Cryptomeria japonica and Chamaecyparis obtusa Endl. commonly has utilized wooden houses in japan. These has high durability which were means antifungi and termite-resistance. The termiticidal substances of C. japonica are not reported yet. Therefore, the termiticidal substances of C. japonica were examined. The heartwood of C. japonica was shown highly the termiticidal activities by termite test. It was found that termiticidal activity of the ethanol extractives from the heartwood of C. japonica was transfer into neutral fraction. The neutral fraction was chromatographed on a silica-gel column using chlorform, chlorform/methanol, methanol, 8 fractions were obtained. The termiticidal activity of fraction 4 had remarkable. Other fractions did not shown termiticidal activity. The fraction 4 was rechromatographed on a silica-gel column using hexane, hexane/acetone methanol, 6 fractions was obtained. The termiticidal activity of fraction 4-2 had remarkable. The fractionation of the fraction 4-2 by HPLC was conducted using a,μBONDASPHERE C-18 column(1.9×15cm), detection was carried out by 326nm, and obtained 4 fractions. The fraction 4-2-2 had highly termiticidal activity. Other fractions did not shown termiticidal activity. The fraction 4-2-2 was further separated to same HPLC, and obtained three compounds. These compounds had highly termiticidal activity. Finally, the termiticidal compounds from the heartwood of C. japonica were identified compound 1 (β-eudesmol), compound 2 (sandaracopimarinol) and compound 3 (16-phyllocladanol) respectively, through comparision of spectrum data of ^1H-NMR, ^<13>C-NMR, FAB-MS.

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During the course of our search for functional molecules in edible fungi, we have reported the characterization and structural determination of the sphingolipids in Hypsizigus marmoreus (Bunashimeji, a mushroom) by B/E constant linked scan fast atom bombardment (FAB) mass spectrometry, that is, the fatty acid composition, the location of double bonds in the long-chain base (C19-sphingadienine), or the presence of phosphodihexose. In addition to these compounds, we also found many polyisoprenepolyols in a hot water-ethanol extract of an edible mushroom. In the polyisoprenepolyols, gymnopilins were discovered independently by two groups almost at the same time in 1983 (Nozoe et al. and Aoyagi et al.) from Gymnopilus spectabilis (o-waraitake, an hallucinogenic mushroom), as a bitter principle of the mushroom. However, gymnoprenol which has a free terminal hydroxy group instead of half-ester of 3-hydroxy-3-methylglutaric acid, has no bitter taste. Recently, Nozoe and Shirahama reported that these polyisoprenepolyols have an antitumor activity and also excitatory activity in central neurons. In the polyisoprenepolyols isolated from Hypsizigus marmoreus (Bunashimeji), we found new polyisoprenepolyols, which seem to be the precursor of gymnopilins. This compound has a terminal olefinic carbon group instead of a free terminal hydroxy group in gymnoprenol, and we named as hypsiziprenol. In the past, the structures of these polyisoprenepolyols were determined by chemical degradation studies, which require large amounts of sample. Here we describe, how information on the isoprenoid sequence and the number of hydroxy groups present can conveniently be derived by mass spectrometric methods that require only minute amounts of sample.

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Thelophoraceous brown fungus Sarcodon scabrosus (Fr.) Karst. which grows in coniferous and deciduous woods of Europe and Japan has been recognized as an inedible mushroom because of its strongly bitter taste which may derive from bitter terpenoids. During our studies on biologically active constituents of Japanese mushrooms (Basidiomycetes), we found a new diterpenoids which showed strong inductive activity of the nerve growth factor (NGF) synthesis in 1321N1 human astrocytoma cells, from S. scabrosus. The fruit body (1.4 kg) of S. scabrosus was extracted with MeOH and then the extract was partitioned between n-hexane-EtOAc and water. The n-hexane-EtOAc extract was subjected repeatedly to silica gel chromatography and reversed-phase HPLC on ODS to give new cyathane diterpenoids, scabronine A (1), G (3) and J (4), and episcabronine A (2). The stereostructures for those compounds (1-4) were elucidated on the bases of the spectroscopic analysis of natural products and their derivatives. Scabronine A (1) and scabronine G methyl ester (7) showed potent inductive activity of the NGF synthesis in 1321N1 human astrocytoma cells. Namely, 1 released 746 pg/mL of NGF at 100 μM from 1321N1 cells under basal release of 147 pg/mL of NGF during two days. Furthermore, reverse transcription polymerase chain reaction (RT-PCR) revealed that 1 increased NGF mRNA by 1.99-fold in 1321 N1 cells. Some NGF inducers such as catecholamines, benzoquinones, hericenones, fellutamides, kansuinin A, pyrroloquinoline quinones and erinacines have been hitherto reported. A strong NGF inducer with low molecular size such as scabronine A (1) or scabronine G methyl ester (7) will be a useful drug for serious neuronal diseases such as Alzheimer's disease. Scabronines will also be helpful to clarify the mechanism of NGF synthesis and secretion.

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Taxol^[○!R], as one of the most important anti-cancer drugs currently on the market for ovarian and breast cancer, has generated world wide interest in various areas, and extensive chemical studies have been carried out. In China, there distributed 5 kinds of yew tree: Taxus chinensis Rehd, T. yunnanesis Cheng et L. K. Fu, T. cuspidata Sieb et Zucc, T. wallichiana Zucc, T. mairei Cheng et L. K. Fu, all of them have been extensively studied in recent 15 years. T. mairei is an evergreen tall tree and mainly distributed in Jiangxi, Fujian and Taiwan Provinces, southeast of China. Previous studies have resulted in the isolation of more than 20 new taxanes from this plant, most of them have the normal taxane skeletons with 6/8/6 membered ring system. In search of new precursors fit for semisynthesis of taxol analogous, we re-examined the needles and the bark of this plant growing in a different area from those examined by other groups. Methanol extraction of the needles and bark of Taxus mairei, after treated with activated charcoal and concentration, was subjected to normal phase column chromatography, each fractions were repeatedly chromatographed on silica gel column and preparative TLC, finally afforded fourteen novel compounds, their structure were characterized belong to 11(15→1)abeotaxane, 2(3→20)abeotaxane, bicyclic taxoid diterpenes, respectively, on the basis of 1D and 2D NMR spectra including ^1H, ^<13>C NMR, ^1H-^1H COSY, DEPT, HMBC, HMQC, NOESY and ROESY techniques. Most of these novel taxane diterpenes posses different skeletons from usual taxoids consisting of a 6/8/6-membered ring system, of them, 3, 4 and 7 are rare example of taxoids involving a 6/12-membered bicyclic rings, of which the skeleton has been more recently reported from yew tree. 3,7-dien-9-one bicyclic taxanes (5, 6) with the verticillene skeletons have never been isolated from yew trees. 2(3→20)Abeotaxanes (10-13) were isolated for the first time from Taxus mairei. 14 was the second example of 11(15→1)abeotaxane with epoxidic ring at C-4(20) among more than 250 natural taxanes; compound 8 was a rare example of 6/8/6-membered ring system with a 14β-OH. In the bark, taxol and several taxoids bearing a xylose unit at C-7 were isolated as the major component. The bark of T. mairei is a suitable new resource for the production of taxol. In addition to taxoids, lignans, ecdysteroids, abietanes, 9(10→20)abeoabietanes, vomifoliol and its derivatives were also isolated from the bark of T. mairei.

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A simple method for the isolation of taxol and other taxane diterpens from the needles of Japanese yew, Taxus cuspidata sieb. et Zucc. was developed. The ethyl acetate extracts of the needles were washed with acid and base to remove basic and phenolic compounds. The separation of taxol and its analogs from the neutral extracts by the combination of silica gel fresh chromatography and HPLC was achieved easily and in good yields. The taxol (22) and cephalomannine (24) were obtained in 0.004 % and 0.003 % yields respectively based on fresh needles. 13 new taxane diterpen alkaloids (taxine NA-1-NA-11 (1-11), 2(3->20)abeotaxine NA-1 (12) and NA-2 (13)) and 8 new taxanes (taxinine NN-1, 2, 5, and NN-6 (14-17), 11(15->1)abeotaxinine NN-1 (18), 2(3->20)abeotaxinine NN-1 (19), NN-2 (20), 3,13-cyclotaxinine NN-1 (21)) were isolated from the crude alkaloid fraction by the combination of alumina column chromatography and reversed phase HPLC(ODS) eluted with a mixture of ammonium acetate buffer (pH4.7)-MeOH-MeCN. Taxol and its analogs were isolated from the callus culture of Taxus cuspidata, together with taxuynnanine C (31) and its analogs (32〜35) in high yields (0.49 %). The callus culture was developed with young stem tissue of the yew grown at Aobayama of Sendai in 1997 and maintained in Gamborg B5 medium containing 0.5mg/L NAA at 27℃ in darkness. After growth for 40-50 days, biomass was scraped from solid medium and freeze-dried. The dry callus calture was extracted with hexane, ethyl acetate, and MeOH successively. These extracts were separated by the combination of silica gel fresh chromatography and HPLC. Taxol (22), 7-epitaxol (23), and taxuyunnanineA (28) were isolated from the extracts of EtOAc and MeOH in 0.014%, 0.0015 %, and 0.009 % yields, respectively. Taxuynnanine C (31) and its analogs (32), (33), (34), and (35) were obtained from these extracts in 0.135 %, 0.014 %, 0.009 %, 0.275 %, and 0.053 % yields, respectively.

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The triterpene sapogenins from the Cactaceae mainly Lemaireocereus were first studied by Djerassi et al. In this study, the MeOH extracts of 71 cactaceous plants belonging to 52 genera were screened for the presence of saponins and 26 plants were found to contain the saponins. We examined precisely for 13 plants and isolated 37 triterpene sapogenins including 17 new compounds (Table 1 and Figs. 1-5). Cactaceous plants were extracted with CHCl_3, and then repeatedly with MeOH. The MeOH extract was hydrolyzed with 3.5% HCl at 110℃ for 2.5 h. The CHCl_3-soluble fraction was subjected to column chromatography on silica gel and furthermore purified by HPLC over silica gel, eluted with CHCl_3-MeOH or n-hexane-acetone, resulting in the isolation of triterpenes. The structures of triterpenes were determined by two-dimentional NMR techniques and single-crystal X-ray diffraction studies. Machaerocerus eruca Br. & R. contained 13 triterpens including 5 new compounds (Table 1). Four of 5 new compounds were named machaerogenin (1), having hydroxymethyl at C-30 and formed 5-membered lactone between C-21 and C-28, 21-ketobetulinic acid (2), 16β-hydroxybetulinic acid (3), and 22β-hydroxystellatogenin (4). The last new one was determined to be 3β,19α-dihydroxygermanican-28-oic acid and named machaeroceric acid (5). The stereochemistry of H-18 for germanicanes is α-configuration in contrast with oleanane. All germanicanes known to data are characterized by a Δ^<18,19> olefinic linkage in their molecules, but machaeroceric acid (5) has no such bond. Trichocereus bridgesii Br. & R. contained 2 new compounds named bridgesigenin A (14) and B (15). Trichocereus pachanoi Br. & R. contained 2 new compounds, which have new skeleton named pachanan, named pachanol A (16) and B (17), together with 14 and 15. The methyl group at C-27 of the pachanan skeleton was combined at 15-C. Moreover, pachanol C(18) and bridgesigenin C (19), new compounds, were isolated from an enzymatic hydrolyzate of MeOH ext. of T. pachanoi. Isolatocereus dumortieri Backbg. Contained a new compound named pachanol D (20) together with dumortierigenin (21). The structure of 20 was determined by the single-crystal X-ray diffraction. Twenty triterpenes from cactaceous plants were tested for antinociceptive effects (Table 2) and inhibitiory effects of TPA-induced ^<32>Pi-incorporation into phospholipids of HeLa cells.

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C_<11> and C_<13>-norisoprenoid glucosides were obtained from Vieginia Tabacco about twenty years ago, but their chemical stereostructure have not been reported so far. In this study, the absoute structures of eight glucosides (3〜5,8,9,11), as well as several C_<13>-norisoprenoid (5a〜8a) have been determined by use of C-13 NMR substitution effects of alkanes, C-13 NMR glucosidation shifts rule, and reaction of allylcation. Furthermore, two glucosides of (3S,5R,6R,7E,9R)-kiwiionol from plant of Actinidiaceae, kiwiionoside (1) (its absolute structure has been determined using X-ray crystal analysis and other methods), actiriidioionoside (2) havebeen found as the precursors. The absolute structures of eight glucosides are clarified as follow; (6S,9R)-roseoside (3), (6R,9R)-3oxo-α-ionol β-D-glucopyranoside (4), (3S,5R,6S,9R)-5,6-epoxy-actinidioionoside (5), (3S,5R)-(-)-loliolide 3-O-β-D-glucopyranoside (8), (3S)-3-hydroxy-β-damascone 3-O-β-D-glucopyranoside (9), three glucosidic isomers of (3S, 9R)-megastigma-5-en-7-yne-3,9-diol (11a-c), and the C_<13>-norisoprenoid from volatile fraction as follow; (3S,5R,6S,9R)-5,6-epoxykiwiionol (5a), (3S,5R,6R,9R)-3,6-epoxy-7-megastigmene-5,9-diol (6a), and so on.

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Four new cardenolide glycosides were isolated from the seeds of Corchorus olitorius L. (Japanese name "Moroheiya") with four known digitoxigenin glycosides (coroloside, glucoevatromonoside, deglucocoroloside,evatromonoside) and four known strophanthidin glycosides (erysimoside, olitoriside, corchoroside A and helveticoside). On the basis of chemical and spectroscopic evidence, their structures were established as cannogenol 3-O-β-D-glucopyranosyl-(1→4)-O-β-D-boivinopyranoside, periplogenin 3-O-β-D-glucopyranosyl-(1→4)-O-β-D-digitoxopyranoside, digitoxigenin 3-O-β-D-glucopyranosyl-(1→6)-O-β-D-glucopyranosyl-(1→4)-O-β-D-digitoxopyranoside and canarigenin 3-O-β-D-glucopyranosyl-(1→4)-O-β-D-altromethylopyranose (or canarigenin 3-O-β-D-glucopyranosyl-(1→4)-O-β-D-allomethylopyranose). The acute oral toxicity of isolated cardiac glycosides was tested in male ddY mice (6 weeks of age). The LD_<50>, values of a mixture of erysimoside and olitoriside (6/4), and a mixture of coroloside and glucoevatromonoside (1/1) were>500mg/kg. Further toxicity testing could not be carried out because the amounts of the cardiac glycosides isolated were insufficient. HPLC analyses revealed that dark grayish green seeds contained more cardiac glycosides than dark grayish yellow seeds. Also, it suggested that the former contained SP diglycosides rather than SP monoglycosides and DG glycosides. Aerial part of C. olitorius has been used as a vegetable and sources of health tea and health food products in Japan. Therefore, analytical method of cardiac glycosides in these samples was developed. Methanol extracts of the samples were hydrolyzed in methanol-0.3mol/L HCl(2: 1) for 3h at 70℃ (hydrolysis of SP glycosides) or 6 days at room temperature (hydrolysis of DG glycosides). The hydrolysate was subjected to a Bond Elut Silica cartridge to clean up and the eluate was applied to HPLC. The HPLC analyses revealed that the edible samples did not contain SP and DG glycosides.

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Amamistatins A (1) and B (2), novel bioactive lipopeptides, have been isolated from an actinomycete. Amamistatins A (1) and B (2) inhibited mast cell growth at 20ng/mL under the existence of SCF (stem cell factor). Since mast cells proliferate, differentiate, and are activated in allergic inflammatory tissues, they play a key role for allergic responses. A potent and selective inhibitor of mast cell growth could be a candidate for antiinflammatory drugs. Molecular formulas of 1 and 2 were determined to be C_<37>H_<55>N_5O_<11> and C_<36>H_<53>N_5O_<10> from high resolution FABMS spectra. The IR spectrum showed absorption bands at 1740, 1660, 1500 cm^<-1> that were assigned to ester, amide, and nitro groups. FeCl_3 test of 1 indicates the presence of enol structure. The 2D NMR (phase sensitive DQF COSY, HOHAHA, HSQC, HMBC) spectra suggested four partial structures (two lysine, 2,2-dimethyl-3-hydroxy acid, and trisubstituted benzene ring). The HMBC correlations shown in Figure 1 disclosed connectivties of partial structures. Since further evidence for the connectivities of the partial structures could not be obtained from NMR data, the degradation reactions of 1 were carried out. Amamistatin A (1) has two N-OH groups. Considering the molecular formula of 1, C26 must be bonded to the C30 and C30 is connected to C29. Thus, the gross structure of amamistatins A and B are shown as 1 and 2, respectively. The stereochemical assignment of amamistatins is in progress.

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The advanced Marfey's method including DL-FDLA derivatization is non-empirical method for determination of the absolute configuration of primary aminno compounds including amino acids using LC/MS (Fig. 1(A)). In this method, their absolute configuration can then be determined based on their elution order under reversed phase conditions. We have proposed a separation mechanism for the resolution of both resulting diastereomers, which includes a stable and fixed conformation assisted by the intramolecular hydrogen bondings between the nitro groups and α-amino groups of the target compound and the leucinamide moiety. This conformation can be readily confirmed by UV spectral analysis and an NOE experiment. If this fixed conformation can also be formed in the case of (R, S)-FDPEA, it is expected to be available as a chiral anisotropic reagent for the determination of absolute configuration of primary amino compounds including amino acids (Fig. 1 (B)). We tried to confirm the absolute configuration of several primary amino compounds and C-3 of Ahda in microginin from cyanobacteria by (R, S)-FDPEA method (Figs. 3 and 4). Through these experiments, we established another non-empirical method for the determination of the absolute configuration of a primary amino compound using NMR spectroscopy (Fig. 2), which has the following advantages over conventional methods: (1) Derivatization with (R, S)-FDPEA is much easier than with a conventional reagent. (2) The conformation of the resulting FDPEA derivative is definitely fixed by the characteristic hydrogen bonding and arrangement of related functional groups, so that the Δδ values determined by (R, S)-FDPEA method are larger than those obtained by conventional method. (3) The fixed conformation can be easily confirmed using UV and NOE difference spectra. In addition, the obtained results can also support the fact that a primary amino compound derivatized with FDLA or FDPEA has the definite conformation shown in Fig. 1.

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We reported the structures of six pyrrolidine alkaloids, broussonetine A-F, as glycosidase inhibitors from Broussonetia kazinoki SIEB. (Moraceae) in 37th symposium (Tokushima). In our continuing works, we obtained six new pyrrolidine alkaloids called broussonetines G (1), H (2), I (3), J (4), K (5) and L (6) from the same tree. The present report deals with the isolation, structural elucidation and inhibitory activity to some glycosidases. Broussonetines G-L were obtained from hot water extracts by ion exchange chromatography [Amberlite CG-50 (H^+ form), DOWEX 50W X4 (pretreated with 0.2M HCOONH_4 buffer)], SiO2 chromatography and reverse phase HPLC. The planar structures of 1-6 were determined by spectroscopic analysis, including two dementional NMR techniques. The relative stereostructure was elucidated based on NOESY spectrum. The absolute stereostructure of 3 and 4 were confirmed by the application of benzoate rule to the 3, 4-dihydroxyl pyrrolidine ring and a new version of Mosher's method to the C-3". Further studies of the absolute configulations of 1, 2, 5, 6 are now in progress. Compounds 1, 2, 5, 6 inhibited β-glucosidase, β-galactosidase, β-mannosidase. Broussonetines E and F, having a hydroxy group on C-1', inhibited α-glucosidase, but 1, 2, 5, 6 did not inhibit α-glucosidase, in spite of the presense of the same group.

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The chinese traditional drug Ch'an Su is the preparation from the skin secretions of the local toads such as Bufo bufo gargarizanz cantor or Bufo melanostrictus Schneider. Although the steroidal bufadienolides with an α-pyrone ring at C-17 position were the major biologically and pharmaceutically active components, the water-soluble basic components such as catecholamines and indolealkylamines were also responsible for the activity. Until now, the twelve indole alkylamines: serotonine, bufotenine, bufotenidine, bufoviridine, bufothionine, O-methylbufotenine, dehydrobufotenin, bufoviridine-N-sulphate, O-methylbufotenine-N-sulphate, 5-hydroxy-N-methyl-tryptamine, 5-methyltrypt-amine and 5-methyl-N-methyltrypt-amine have been isolated from many kinds of toad. However, in Ch'an Su, the presence of bufotenine, bufotenidine, bufothionine and dehydrobufotenine were irregularly reported. A consideration of the above facts and an interest in the water-soluble components of Ch'an Su have led to the present investigation. Here we describe the isolation and structural elucidation of two novel indole alkaloids, bufobutanoic acid 2 and bufopyramide 3, in addition to the isolation of known bufotenine 1 (Figure. 1). Bufobutanoic acid 2 and bufopyramide 3 inhibited for the murine P388 lymphocytic leukemia cells at IC_<50> valuses of 22μg/ml and 7.6μg/mi, respectively. On the other hand, the two known bufadienolides, bufarenogin 4 and ψ-bufarenogin 5 were isolated from the same source by the above chromatography (Figure. 1). In the NMR spectral analysis, the solution state conformations of two toad poison bufadienolides, bufarenogin 4 and ψ-bufarenogin 5, which are the epimers at C-12, were analyzed by using NOE experiments and molecular dynamics simulation study. Interestingly, the C-ring of bufarenogin 4 with 12β-hydroxy function was shown to take a chair form, whereas that of ψ-bufarenogin 5 with 12α-hydroxy function, a boat form. Thus, we have discussed about conformational preference of two bufadienolides, 4 and 5.

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During the course of our search for microbial metabolites which exhibited bioactivity against insects, we isolated insecticidal okaramines A (16), B (17), D (19), E (20) and F (21) from fermentation products of okara (the water insoluble residue of whole soybean) with Penicillium simplicissimum ATCC 90288. We also obtained okaramine C (18) from Penicillium simplicissimum AHU 8402. In the continuing search for okaramine congeners to investigate biosynthesis of okaramines and structure-activity relationships. Okaramines H (2) and I (3) have been isolated from Aspergillus aculeatus KF-428. Furthermore, okaramines G (1), J (4)〜P (10) and related compounds 11〜15 have been isolated as minor metabolites of ATCC 90288. Their structures were established by NMR and other spectroscopic studies. The absolute stereochemistry of the hydrogen at C-2 of 11 and 13 was determined to be L-form by the chiral HPLC analysis of their acid hydrolysate. The LD_<50> values of 16, 17 and 18 were 8, 0.2, 8μg/g diet, respectively. 4 and 5 had no activity. These facts suggested that an azetidine ring in 17 and an isoprene side chain at N-8 in 16 played an important role in expressing the activity. However 1 showed 1/5 of the activity of 18, on other hand, 8 and 9 had no activity. From the NOESY experiments of 1, 8, 9 and 18, it is possible to consider that the conformational change of azocinoindole moiety influences insecticidal activity. We proposed a biogenetic pathway of okaramine congeners and related compounds on the basis of the structures.

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In continuing our phytochemical studies of Citrus plants, we have isolated many coumarins, acridone alkaloids, flavonoids, and other compounds including many new compounds. Especially, dimeric coumarins, dimeric acridone alkaloids and acridone-coumarin dimers are diagnostic constituents of this genus. Among the new compounds, we report here the structure elucidations of 25 new acridone alkaloid-coumarin dimers from the roots of five Citrus plants. Acridone-coumarin dimers could be classified into three groups. The first group is "acrimarine" constructed by various acridone alkaloids and suberosin (1) as coumarin moiety [acrimarine-A (2), -B (3), -C (4), -D (5), -E (6), -F (7), -G (8), -H (9), -K (10), -M (11) from the roots of Funadoko, and acrimarine-I (12), -J (13), -N (14) from the roots Yalaha]. The second group is "neoacrimarine" constructed by various acridone alkaloids and various coumarins except for suberosin (1) [neoacrimarine-A (15), -B (16), -E (17), -F (18) from the roots Yalaha, neoacrimarine-C (19), -D (20) from the roots Hassaku, and neoacrimarine-G (21), -H (22), -I (23), -J (24), -K (25) from the roots Marsh grapefruit]. The third group is "dioxinoacrimarine" which has a 1,4-benzodioxane ring connecting the acridone and coumarin moieties [dioxinoacrimarine-A (26) from the roots Yalaha]. The structure of these dimeric compounds were elucidated by spectroscopic methods including 2D-NMR particulaly HMBC spectrum.

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A simple procedure for assigning the absolute configuration of two secondary hydroxyl groups located at the C-3 and C-n positions of 1,3,5,…,n-polyols (n=odd number) is presented. The most preferred conformation of benzoylated acyclic 1,3-polyols in solution is considered to adopt the fully extended zigzag carbon-carbon skeleton, and the following general rule has been widely accepted: the exciton coupling CD curves derived from benzoylated 1,3-polyols consist simply of two characteristic shapes, either a distinctive negative or positive couplet if the polyol contains odd number of 1,3-anti-moieties or a very weak CD if an even number of 1,3-anti and/or 1,3-syn moieties are present. Based on these general rules, we analyzed the CD spectra of four isomers of 1,3,5-triol 5, four isomers of 1,3,5,7-tetrol 8, eight isomers of 1,3,5,7,9-pentol 9, and eight isomers of 1,3,5,7,9,11-hexol 10 and the following predictable general trend was observed. 1) A positive CD Cotton effect is correlated to the α-configuration at n-position of 1,3,5,…,n-polyols (n=odd number), while a negative CD to β-configuration at n-position, when the structure of the polyol is depicted as Fig.1. 2) The amplitude (A value) of the Cotton effect is diagnostic for the relative stereochemistry between C-3 and C-n positions: all 3,n-anti isomers gave rise to a stronger coupling with |A|>14, whereas for the 3,n-syn isomers, weaker couplings were observed and the values are |A|<12. The general trend observed provides a unique and simple method for directly determining the absolute configurations of the C-3 and C-n positions of acyclic 1,3-polyols containing a primary hydroxyl group.

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Slime molds have attracted attention as phylogenetically interesting organisms that have characteristics for both plants and animals in thire unique life cycle. Amoebae, appeared by germination of spores, consume bacteria by phagocytosis and multiply by division. Starvation causes the amoebae to aggregate with signal of cyclic AMP, and it differentiates into two types of cells, stalk cells and spore cells, to form fruit bodies. Slime molds are classified into two classes, Myxomycetae and Acrasiomycetae. The latter is called cellular slime molds, and used as a model of embryology and differentiation in biology because its cultivation method has been established. But, as for thire metabolites, only DIF-1, a differentiation factor of slime molds, and a few compounds have been reported. In this study, we focused on the secondary metabolites of slime molds, and isolated seven novel compounds from Dictyosterium sp. Dibenzofuran compounds D-1 (1) and D-2 (2) were isolated from D. medium. D-3 (3) and D-4 (4), bearing α-pyron moiety, and phosphatidyl lipids D-5 (5) and D-6 (6)with unique fatty acid were isolated from D. discoideum. Moreover, norditerpenoid D-7 (7) was obtained from D. purpreum. These structures were elucidated by the spectroscopic and chemical methods. The absolute configurations of D-3 (3) and D-4 (4) were determined by the comparison of their CD spectra with that of a known compound.

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In our screening project on immunomodulatory constituents of fungi, six new immunosuppressive α-pyrones named multiforisins A-F have so far been isolated from Gelasinospora multiforis. Successively, it was found that the crude extracts of Gelasinospora heterospora and G. santi-florii showed immunosuppressive activity. Among the four constituents 1-4 isolated from Gelasinospora heterospora, 1-3 exhibited immunosuppressive activity. The spectral data including two dimensional ^1H- and ^<13>C-NMR and the chemical correlation to multiforisins A(5) and B(6), indicated that 3, 1 and 4, which were also isolated from G. multiforis together with 5 this time, were elucidated to be new homologues of the multiforisins and named multiforisins G, H, and I, respectively (Chart 1). Compound 2, which were also isolated from G. santi-florii this time, was deduced to be identical with sordarial from Sordaria macrospora. The absolute configurations at positions 3' and 4' in 2, which had been not yet decided, were finally determined to be R and S, respectively (Chart 2). Among the six constituents 2 and 11-15 isolated from G. santi-florii, 12, 2 and 11 exhibited immunosuppressive activity. The main active principle 12 and an inactive one 13 were identical with seco-anthraquinone-type compounds, nidulalins A and B, from Emericella nidulans var lata, respectively. Compounds 15 and 11 were deduced to be new compounds related to 12, and 14 was found to be identical with 1-hydroxy-3-methylxanthone from Anixiella micropertusa, as shown in Chart 3. To our knowledge, this is the first time that sordarial and nidulalin A have been isolated as immunosuppressive constituents from natural source. The immunosuppressive activities (IC_<50> values) of 1-6, 11-16 and the three compounds as positive control in Table 1 suggested that the presence of ?CH_2OH at position 3 or 5 in the multiforisins and that of the unique A-ring moiety in 12 were very important for the appearance of the immunosuppressive activities of the multiforisins and 12, respectively. Investigation on the mode of action of 12 suggested that the immunosuppressive effect of 12 was not due to the ability to inhibit IL-2 production.

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In the course of a screening program to discover new anti-ulcer agents from Actinomycetes, we isolated a novel angucycline series compound P371A1 (1) and A2 (2) from Streptomyces sp. P371, together with other congeners P371B1 and P371B2. The molecular formula of P371A1 (1) was established to be C_<48>H_<66>N_2O_<20> based on the HRFAB-MS and ^<13>C NMR spectra. It was demonstrated through the analyses of the 1D- and 2D- NMR spectra and MS/MS spectra, together with some derivatizations that 1 is a novel angucycline series compound consisting of the angucycline unit (X_1-X_2) and the four sugar units (X_3, X_4, X_5 and X_6) as shown in Fig 2. P371A2 (2) was elucidated to be the C-4 acetoxy derivative of 1 through comparison of their ^1H- and ^<13>C- NMR and NOESY spectra as well as of their 5% Methanolic HCl degradation products. The absolute structure of the C-glycoside unit (X_3) in 1 and 2 was disclosed by applying the modified Mosher method to (S)-(-)- and (R)-(+)-MTPA esters (7 and 8) of the aglycone 6. P371A1 (1) showed a significant suppresion (percentage inhibition: ca. 61%) against pentagastrin-stimulated acid secretion, when 10mg/kg was given intraperitoneally to urethane anesthetized rats 30 min prior to the onset of pentagastrin infusion (i.v. 60μg/kg/hr). In constrast, 1 did not have any effect on acid secretory response induced by either histamine or carbachol in the experiments carried out under the same condition. Interestingly, intraperitoneal administration of 1 to unanesthetized rats 30 min prior to an oral dosage of HCl/ethanol (60% in 150mM HCl) or a subcutaneous administration of indomethacin (25mg/kg) prevented significantly and dose-dependently the lesion formation in gastric mucosa. The percentage inhibition at 1, 3 and 10mg/kg were 52.6, 81.6 and 83.6%, respectively, in the HCl/ethanol-induced lesion model and were 57.0, 64.9 and 72.8%, respectively, in the indomethacin-induced lesion model. In the binding assay, 1 was not bound to the CCK_B/gastrin receptor. Thus, 1 was presumed to hinder the gastrin from stimulating a histamine release from enterochromaffin-like (ECL) cells. Since the possibility is not ruled out that metabolites of 1 acted as a CCK_B/gastrin receptor antagonist, a further study is neccesarry to establish the definite mechanism.

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An R-type lipopolysaccharide (LPS) from Erwinia carotovora FERM P-7576 was studied after strong alkaline degradation and mild acid hydrolysis. The resulting products were analyzed by fast atom bombardment MS, one- and two-dimensional ^1H and ^<13>C NMR spectroscopy, dephosphorylation and methylation analysis. With the structure of lipid A and the core moiety, the complete LPS structure was established and confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS of the native LPS. 1. Introduction A rough mutant E. carotovora FERM P-7576 excreted LPS spontaneously into the cultural medium. Structural studies of LPS are important for better understanding its role in the bacterium-plant interaction and specific phase behavior of LPS-containing membranes. 2. Materials and Methods Extracellularly produced LPS was isolated and purified from the cell free culture solution. LPS was acid hydrolyzed to lipid A and core oligosaccharide in 0.1M H_2SO_4 at 100℃ for 20 min. The lipidA and oligosaccharide was characterized by component analysis, methylation analysis, mass and NMR spectrometry. The ionization matrix employed for the positive ion FAB-MS measurement of the lipid A was an admixture of glycerol, thioglycerol, and meta-nitrobenzyl alcohol (1: 1: 1, v/v). The core oligosaccharide was measured in glycerol and thioglycerol mixture (1: 1). MALDI-MS of the native LPS was performed in the linear TOF mode. The LPS sample was dispersed in aqueous triethylamine, treated with a cation-exchange resin and admixed with 2,4,6-trihydroxy-acetophenone for ionization matrix. 3. Results Component and methylation analysis of the oligosaccharide indicated terminal galactose, phosphate linked heptose, 5-linked Kdo, etc. ^1H-NMR indicated eight anomeric sugar protons, other peaks were assigned by two dimensional methods. Positive ion FAB-MS of the lipid A yielded informative ions appeared by cleavage of glycosidic linkage of diglucosamine backbone and further loss of acyl moiety (Fig. 1). The negative ion MALDI-TOF mass spectrum of the native LPS yielded molecular ions and fragment ions that reflected the heterogeneity of LPS with respect to the core region and to the number of the secondary O-linked fatty acid residues within lipid A (Fig. 2). Fragment ions resulted from cleavage of lipid A and showed the heterogeneity with respect to the degree of acylation of lipid A and to the number of sugar residues in the core. By these data the complete structure of the E. carotovora rough-type LPS could be proposed as illustrated in Fig. 3.

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To obtain the useful information on the structure of minute amounts of natural glycosphingolipids, we have been developing the mass spectrometrical technique. We have reported the usefulness of the CAD-MS/MS of [M+Na]^+ ions obtained in the (+)-FABMS to the structure elucidation of ceramide, cerebroside, and ceramide-lactoside, possessing α-hydroxy fatty acid in their ceramide part. On going our previous study, we attempted to apply this method to gangliosides from echinodermata by using gangliosides which have already been characterized in our laboratory. When (+)-FABMS of acanthaganglioside C (1) was measured, [M+Na]^+ ion was observed. However, in the FABMS/MS of [M+Na]^+ ion, the characteristic fragment ion originated in the fission of the amide bond was not observed (Fig. 3). As we have proposed, the fission of the amide bond would not occur without the formation of the stable five-membered chelate ring (Fig. 1). In order to reduce the effect of carboxyl group which must be disturbed the formation of the chelate ring, 1 was treated with DMSO-CH_3I to give methyl ester derivative acanthaganglioside C-ME (2). When (+)-FABMS/MS of 2 was measured, the highly intense and characteristic fragment ion, arisen from the fission of the amide bond of ceramide part, was observed (Fig. 4). In the FABMS/MS of AG-2 ME, methyl ester derivative of acanthaganglioside molecular species, each [M+Na]^+ ions provided the characteristic fragment ions with highly intense, too (Fig. 5). Furthermore, in the case of the disialoganglioside molecular species (LLG-3), too, this method could be applied (Fig. 7). The method must be very useful for the determination of ceramide structure of gangliosides possessing α-hydroxy fatty acyl moiety in their ceramide part. In addition, these methyl ester derivatives were useful for their NMR analysis. Usually, underivatized gangliosides from echinodermata give low resolutive NMR spectra. When their methyl ester derivatives were measured, the high resolutive spectra were obtained in the ^1H- and ^<13>C-NMR (Fig. 8).

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Recently blue-green algae (cyanobacteria) attract much attention because of their secondary metabolites with potent biological activities and unusual structures. Blue-green algae have also been checked for their metabolism influenced by symbiotic or parasitic microorganisms. For example, lichens, symbiotic organisms consisting of algae and fungi, produce peculiar secondary metabolites called lichen substances. Nostoc commune is a blue-green alga which often grows on wet ground surface in jelly clumps consisting of polysaccharides. Our preliminary experiment showed that many microorganisms including bacteria, fungi, and actinomycetes were residing in the jelly clumps of field grown Nostoc commune. On the other hand the methanol extract of this alga exhibited antifungal activity against 7 fungi (Aspergillus cardidus, Cladosporium herbarum, Botrytis fabae, Glomerella cingulata, Sclerotium rolfsii, Rhizoctoniasolani, Cercospora beticala) suggesting that the antimicrobial substance(s) may control microbial flora of the jelly clumps. As the first step to clarify the relationship between the alga and co-residing microorganisms, we isolated one of the antimicrobial principles-named nostofungicidine (1) from field-grown algal sample. The structure of nostofungicidine was elucidated mainly by chemical degradation and by extensive NMR measurements including DQF-COSY, HOHAHA, HMBC, and ROES Y techniques. Nostofungicidine was a novel lipopeptide containing a long chain beta amino acid, 3-amino-6-hydroxy stearic acid (Ahs) in its structure. Nostofungicidine was not found in the axenic culture of this alga and in the individual cultures of each tested microbial strain isolated from jelly clumps. On the other hand, one of the co-residing strain (tentatively identified as an actinomycete) produced a known antibiotic, echinomycin. Echinomycin had no antialgal effect toward axenic Nostoc commune at the concentration of 100 micro-g/ml. These results suggest that co-residing microorganisms may play an important role in the accumulation of microbial flora controlling substances not only through production of antimicrobial compound(s) but also through stimulation of the algal secondary metabolism.

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Xanthomonas sp. strain SB-K88 that has originally been isolated from a fibrous root surface of a sugar beet endangered to infection with Polymyxa betae has been found to deppress sugar beet damping-off disease, and the active substance named xanthobaccin A has already been isolated from culture filtrate of the bacterium as a structurally unknown antibiotic that drastically calmed damping-off disease in a sugar beet seedling pot assay. We did structural elucidation of xanthobaccin A by spectroscopic analyses and chemical derivatization, including its relative configuration. Xanthobaccin A (1), having a chromophore to show its unique UV and fluorescence λmax, was formulated in FD- and FAB-HR-MS to be C_<29>H_<38>N_2O_6. In the ^<13>C-NMR including some 2D-NMR (HMQC, HMBC and DEPT-HMQC) spectra, all of the carbons of 1 were characterized to have a unique 5,5,6-tricyclic carbon skeleton consisting of two C12-acetogenine units and also an α,β-hydroxyornithine part. These two partial structures were combined via an amidebond and a tetramic acid-like cyclization to form the macrocyclic lactam molecule. The acetogenine class hydrophobic part involving the 5,5,6-tricyclic structure was converted to the dicarboxylic acid dimethyl ester derivative (2) by acidic methanolysis of 1, and its structure was also proven by the NMR analyses. Consequently, structure 1 was elucidated to be a new macrocyclic lactam antibiotic having a new carbon skeleton. Compound 1 was closely related with an antibiotic, ikarugamycin (3), isolated from Streptomyces phaeochrmogenes var. ikaruganensis in their planar structures and biogenesis; therefore compound 1 is also considered to have a wide physiologically active spectrum against several organisms as does 3.

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Bacillus polymyxa L-1129 produced a new peptide antibiotic complex, named LI-Fs, composed of more than ten components. In this reports we will describes the isolation, structure elucidation and biological activity of each components, LI-F03a, LI-F03b, LI-F04a, Li-F04b, LI-F05a, LI-F05b, LI-F06a, LI-F06b, LI-F07a, LIF07b, LIF08a and LIF08b. These antibiotics were active against gram positive bacteria, mycobacteria and wide range of fungi and yeast, but not for gram negative bacteria. The antibiotic complex LI-Fs (400mg) was isolated from 10 liters fermentation broth using several kind of column chromatography. HPLC analysis showed that the complex was composed of at least 12 components. The components were isolated by HPLC, and the structures of each components were determined by 1D and 2D NMR and MS spectra coupled with amino acid analyses to be hexadepsipeptide containing 15-guanidino-3-hydroxypentadecanoic acid as side chain. The structure of LI-F05a was identified to be a cyclohexadepsipeptide [L-thr(1)-D-val(2)-L-Ile(3)-D-allo Thr(4)-D-Asn(5)-D-Ala(6)] and LI-F05b was identified as an isomer of LI-F05a that was replaced the Asn residue in LI-F05b with Gln, similar pairs were also found in other components, (LI-F06a-LI-F06b, LI-F07a-LI-F07b, LI-F08a-LI-F08b) (Fig. 4). Four amino acids; L-Thr(1); D-Ala(6); D-Asn(5) or D-Gln(5); D-allo Thr(4) and one side chain were commonly found in the LI-Fs antibiotics. Thus, these moieties seems to be fundamental for biological activity of LI-Fs antibiotics.

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During our search for new manzamine-related alkaloids, we have isolated nakadomarin A (1), a novel furan-containing hexacyclic alkaloid consisting of an unprecedented 8/5/5/5/15/6 ring system, and ma'eganedin A (2), a new tetrahydro-β-carboline alkaloid with a methylene carbon bridge between N-2 and N-27, from an Okinawan marine sponge Amphimedon sp. EtOAc-soluble fraction of the sponge Amphimedon sp. collected off Kerama Islands, Okinawa, was purified by silca gel and alumina column chromatographies to afford nakadomarin A (1, 1.8x10^<-3>%, wet weight) and ma'eganedin A (2, 9x10^<-4>%) together with several known manzamine alkaloids. The structures containing relative stereochemistry were elucidated on the basis of mainly 2D NMR data. Absolute configurations of ma'eganedin A (2) were deduced as 1R, 10R, 11S, 12R, 24S, 25R, and 26R from the CD Cotton curve. Since nakadomarin A (1) and ircinal A possess the common structures, nakadomarin A (1) may be generated from ircinal A through retro-Mannich, Mannich, and dehydroxylation processes. On the other hand, ma'eganedin A (2) may be biogenetically derived from ircinal B.

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During our continuing search for structurally unique secondary metabolites from marine dinoflagellates, we previously isolated a series of cytotoxic macrolides, amphidinolides, from dinoflagellates Amphidinium sp. Recently we investigated another strain of Amphidinium sp. (strain number Y-52), which was isolated from the inside cells of the Okinawan marine acoel flatworm Pseudaphanostoma luteocoloris, and isolated two new polyhydroxyl compounds, luteophanols B (1) and C (2), both consisting of a C_<64>-linear aliphatic chain. The structure elucidation of 1 and 2 were elucidated on the basis of chemical degradation and newly developed 2D NMR experiments, E-HSQC and E-HSQC-TOCSY. E-HSQC and E-HSQC-TOCSY are very useful techniques for structure elucidations of complex natural products possessing many aliphatic methine and methylene carbons such as luteophanols B (1) and C (2). CH_2-selected E-HSQC-TOCSY^6 experiments seem to be suitable to assign very closely resonated carbon signals, since this method affords the high resolution in F_1 axis by limiting the F_1 spectral width, in which sp^2 methylene carbons are resonated.

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Multidrug resistance (MDR) in tumor cells has been recognized as one of major obstacles to successful cancer chemotherapy. Overexpression of membrane glycoprotein (e.g. P-glycoprotein) has been observed in MDR tumor cell lines and P-glycoprotein is believed to function as energy-dependent efflux pump. In the course of our study of bioactive substances from marine organisms, we focused on a search for reversing substances of MDR in tumor cells and isolated novel polyhydroxylated sterol acetate named agosterols from a marine sponge of Spongia sp. An AcOEt soluble portion of acetone extract of the titled frozen sponge (collected in July at Ago Bay, Mie Prefecture) showed strong growth inhibition at 10μg/ml concentration against P-glycoprotein overexpressing MDR tumor cells (KB-C2) in the presence of 0.1μg/ml of colchicine, while it exhibited little cytotoxicity against parental KB-3-1 cells at 10μg/ml. This fraction was subjected to bioassay-guided separation (growth inhibition assay against KB-C2 in the presence of colchicine) to afford the active component named agosterol A (1) together with the related sterols named agosterols B, C, D2, and A4. The detailed ^1H- and ^<13>C-NMR analysis of agosterol A (1) clarified the planar structure and the absolute stereostructure of 1 was elaborated on the bases of ROESY analysis, modified Mosher's method, and CD analysis of 22-ketoderivative. Chemical structures of the other related agosterols were also determined by detailed NMR analysis. Agosterol A (1) completely reversed the resistance to colchicine in KB-C2 cells at 3μg/ml and also the resistance to vincristine in KB-CV60 cells, which overexpress multidrug resistance-associated protein (MRP), at 1μg/ml. So far, there are few agents which reverse MDR caused by overexpression of MRP. Agosterol A (1) may be a pharmaceutical candidate for reversing MDR and also may be useful for detailed explication of the molecular mechanism of MRP.

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In the course of screening for novel cell cycle inhibitors, WS9885B(1), C_<24>H_<32>O_5, was isolated from the fermentation broth of Streptomyces sp. No9885. During the NMR measurements, the decomposition of 30% of 1 took place. This instability and ^1H signal overlap made the structure determination difficult. The sample recovered was acetylated to give WB9885B-Ac-1(3) and -2(2). The molecular formula of 2 was established as C_<28>H_<36>O_7, indicating 2 was diacetate of 1. An unprecedented hexacyclic structure was elucidated, but the instability of 2 hindered further structural analysis. In contrast to 1 and 2, 3 was stable in solution. The molecular formula of 3 was determined to be C_<28>H_<36>O_8, one more oxygen than that of 2. The planar structure of 3 was determined by H-H, C-H COSY and HMBC data. The relative configuration of 3 was assumed by a combination of NOE's and vicinal coupling constants as shown in Fig. 4. Finally, X-ray crystallographic analysis of 3 confirmed the structure. In CD_2Cl_2 solution, 2 decomposed gradually and ^1H signals newly generated were superimposable with those of 3. The transformation of 2 to 3 can be explained by epoxidation of the strained double bond. This finding established the structure of 2 and also the structure of 1. The absolute stereochemistry of 1 is now under investigation. WS9885B showed broad antitumor activities in vitro (Table 2) as strong as Taxol and exhibited no antimicrobial activities in vitro (Table 3). WS9885B arrested the cell cycle of HT29 at G2/M phase and promoted assemblies of tublins in vitro. These results showed that WS9885B may have a microtubule-stabilizing activity like Taxol. It is intriguing to note that the stable epoxide 3 is devoid of the antitumor activities.

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We have been studying structure and function of eukaryotic DNA polymerases, which are divided into at least 6 classes designated as α, β, γ, δ, ε and ζ. In the process of our investigations, the need for an inhibitor of each of the DNA polymerases has arisen. The roles of the DNA polymerases in vivo are still mostly obscure, and for the elucidation of the precise roles of each DNA polymerase, the use of the appropriate inhibitors would be quite useful. We therefore have established an assay method to detect DNA polymerase inhibitors, and we have used it to screen the extracts of many organisms for the inhibitors. In the screening, an important aspect was the type of natural product as a source of inhibitors. Not only several fungi, mushrooms and higher plants were found to produce such inhibitors, but also some algae were indicated to produce them. We found inhibitors from a basidiomycete, Fomitella fraxinea, well-known fatty acids and novel triterpenoids (fomitellic acids) (Fig. 1). We also found from a basidiomycete, Ganoderma lucidum, an ergosterol peroxide (Fig. 2), incisterols (Fig. 3) and two cerebrosides which were called the fruiting body-inducing substances (Fig. 4). Sulfolipids (Sulfoquinovosyl diacylglycerol, SQDG) (Fig. 5), which is the strongest DNA polymerase inhibitors, were found in a fern, Athyrium niponicum, and sea alga, Gigartina tenella. The fern compounds were isolated and identified as previously identified sulfolipid compounds from a cyanobacteria which are AIDS-antiviral agents.

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In the continuous studies of enzyme inhibitors from cyanobacteria, we have found that Nostoc minutum (NIES-26) and N. linckia (NIES-25, 28) were a rich source of novel enzyme inhibitors. Here we report the isolation and structure elucidation of novel enzyme inhibitors form N. minutum and N. linckia. N. minutum (NIES-26) and N. linckia (NIES-25, 28) were obtained from the NIES-collection (Microbiol Culture Collection, the National Institute for Environmental Studies, Japan) and cultured in 10L glass bottles containing CB medium. The 80% methanol extracts of lyophilized cyanobacteria were subjected to solvent partitions, ODS column chromatography followed by ODS HPLC to yield nostopeptin-type peptides (1-10). Their structures were determined by FABMS and 2D NMR spectra. The absolute stereochemistries of usual and N-Me amino acids were determined to be all L-form by the HPLC analysis of the acid hydrolyzates derivatized with Marfey's reagent. 1, 2, 5, 6, and 8 inhibited elastase and chymotrypsin but 3, 4, and 7 had no elastase and chymotrypsin inhibitory activities. The 80% methanol extracts of lyophilized cyanobacteria [N. minutum (NIES-26) and N. linckia (NIES-25, 28)] were subjected to solvent partitions, ODS column chromatography followed by ODS HPLC to yield miutumamides A-D (12-15). Their structures were determined by FABMS and 2D NMR spectra. The absolute stereochemistries of usual amino acids were determined to be all L-form by the HPLC analysis of the acid hydrolyzates derivatized with Marfey's reagent. The absolute stereochemistries of Amopa and Hmp remains to be defined. 12-15 inhibited tyrosinase with the IC_<50> of 30μg/mL.

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Phosphatidylinositol 3-kinase (PI 3-kinase) is a key enzyme in the signaling pathways of 3-phosphorylated polyphosphoinositides. The natural PI purified from bovine liver, which mainly contains arachidonate at the sn-2 position, is an excellent substrate of PI 3-kinase. However, synthetic distearate PI and synthetically hydrogenated natural PI were not phosphorylated by PI 3-kinase. In this study, phosphatidylinositols with saturated sn-2 fatty acids were evaluated the phosphorylation reaction with PI 3-kinase. A standard fatty acids mixture of PI [PI(C4)〜PI(C16)] at the sn-2 center was synthesized from an equimolar mixture of seven carboxylic acids (C4, C6, C8, C10, C12, C14, C16), and the PI mixture was subjected to the enzymatic reaction with PI 3-kinase. Negative ion fast atom bombardment (FAB) mass spectrometric analysis of the reaction mixture using a matrix (triethanolamine: glycerol=3: 1) showed the apparent ion peaks of monophosphorylated PI(C4), PI(C6) and PI(C8) analogs. Short sn-2 fatty acid analogs such as PI(4) and PI(C8) were proved to be excellent substrates of PI 3-kinase by means of independent phosphorylation experiments with sythetic PI. In these experiments, after PI 3-kinase reaction of the synthetic PI with [γ-^<32>P]-ATP, the resulting phosphorylated products were analyzed by TLC. The present results open a new window on the design of phosphatidylinositols with saturated sn-2 fatty acids, which are apparently more stable than natural ones.

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A new methodology using a chiral anisotropic reagent, 2NMA [methoxy-(2-naphthyl)acetic acid] has been developed for determination of absolute configurations of acyclic β- or γ-methyl substituted secondary alcohols. 2NMA is known as a powerful NMR reagent to determine the absolute configuration of a secondary hydroxy group especially on a long C-C chain compound. ^1H-NMR spectra of several model 2NMA esters derived from acyclic β- or γ-methyl substituted secondary alcohols were measured, and the Δδ values (δ_<R-ester>-δ_<S-ester>) for both syn and anti isomers were calculated. We found the significant differences of the |Δδ| values between syn and anti isomers. In the case of β-methyl substituted secondary alcohols, |Δδ| values of each proton at γ- and δ-position for syn compounds are larger than those for and compounds. While, in the case of γ-methyl substituted secondary alcohols, |Δδ| values of each proton at γ-, δ-, ε-, and ζ-position for and compounds are larger than those for syn compounds. Threshold values of |Δδ| between syn and anti have been established; in β-methyl substituted secondary alcohols, 0.35 ppm for γ-position and 0.25 ppm for δ-position, and in γ-methyl substituted secondary alcohols, 0.41, 0.23, 0.28, and 0.15 ppm for γ-, δ-, ε-, and ζ-position, respectively. That is, in the case of β-methyl substituted secondary alcohols, if these |Δδ| values at the γ- and δ-protons are larger than the corresponding threshold numbers, the relative stereochemistry should be assigned as syn. On the contrary, smaller |Δδ| values than the threshold numbers suggest the relative stereochemistry to be anti. In the case of γ-methyl substituted secondary alcohols, these |Δδ| values of protons at γ-, δ-, ε-, and ζ-positions being larger than the corresponding threshold values, the relative stereochemistry should be determined as anti. Smaller |Δδ| values than the threshold numbers show the relative stereochemistry to be syn. The absolute configuration of secondary hydroxy group is easily determined by the sign of each Δδ value as usual, thus, the absolute configurations of both methyl and hydroxyl in the acyclic β- or γ-methyl substituted secondary alcohols could be determined.

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The phase sensitive CH- and CH_2-selected 2D spectra of HSQC, HSQC-TOCSY, HSQC-relay, and HSQC-ROESY spectra were obtained by setting proper delays and ^1H-pulse angle of the editing sequence in E-HSQC followed by a suitable magnetization transfer if necessary. The magnitude mode CH_3-selected HMQC-TOCSY spectrum was obtained by applying a quadruple quantum fitter using a pulsed field gradient. These methods solved the cancellation problem caused by overlapping of the opposite phase peaks and also reached the relatively high resolution in F_1 axis by limiting the F_1 spectral width. Since in a certain total measurement time, the narrower is the F_1 spectral width which can reduce the data points in F_1 axis (t_1 increments), the larger number of transients can be accumulated, the C/H correlation methods including the insensitive magnetization transfer such as ROESY become applicable to natural products of small amount. Application of these methods for analysis of complex natural products such as oligosaccharides and a peptide in which signal overlapping is severe are also presented.

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Phytoalexins are antimicrobial stress compounds synthesized by plants after their exposure to microorganisms. During the course of our studies on stress metabolites from vegetables, we have found several new types of compounds. As an extension of the studies, medicinal plants were screened for the production of stress compounds. In the course of the study, we found that the root tissues of Glehnia littoralis (Umbelliferae) produced antifungal furanocoumarins psoralen (1), xanthotoxin (2), and bergapten (3) after stress treatment (slicing, UV-irradiation, or bacterial inoculation) followed by incubation. Although the compounds 1-3 have been reported as constituents of a crude drug Glehnia root, they were barely detectable in the fresh roots. A time-course study on the treated tissues showed greatly increasing concentrations of 1-3 after stress treatment. Next, the crude drug was prepared under several different conditions. The contents of 1-3 in the crude drug showed considerable variation depending on the method of storage and/or processing of raw material. Slow drying seemed to increase the amounts of 1-3. Similar results were obtained in other umbelliferous medicinal plants: Angelica acutiloba and A. acutiloba var. sugiyamae. Ledebouriella seseloides also gave 1-3 as stress compounds. Inoculation with Pseudomonas cichorii of root slices of Paeonia lactiflora (Paeoniaceae) resulted in the formation of hederagenin (9) as a stress compound. Hederagenin has been reported as a major constituent of callus tissue of P. lactiflora and isolated as a minor constituent of Paeoniae Radix, which is one of the most important crude drugs in traditional medicine. The present findings suggest that some reported constituents of other crude drugs could be also stress metabolites produced during processing. Furthermore, stress compounds could be used as markers to evaluate processing procedure of crude drugs.

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We examined the photolysis characterstics and antibacterial activities of hinokitiol and its organometallic compounds that were obtained to react hinokitiol with metallic ions. As for photolysis, we found that the half-life period was 2.6 hours in hinokitiol. On the other hand, the half-life period were 1.8 days in the calcium salt, 2.2 days in the sodium salt, 6.5 days in the zinc salt and 42.9 days in the copper salt, respectively. So we found that the photolysis slowed about as many as 400 times at the copper salts. This is thought to be the obstraction of the generation of the valence isomer prepared because of an optical irradiation, tjhat is, result by the oxgen, s of the hydorxyl and the ketone of hinokitiol with a metallic ions. The antibacterial activities of hinokitiol and its organometallic commpounds were claified. Minimum inhibitory concentration (MIC) of hinokitiol, the sodium salt, the potassium salt, the calcium salt against bacteria were 25〜50μg/mL, that of the magnesium salt was 200μg/mL and that of H-Cu was 20μg/mL.

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Hinoki wood (Chamaecyparis obtusa) is one of the most durable Japanese woods against the biodegradation by termite. In the former reports, we elucidated that the anti-termite activity of hinoki depends significantly on the components of wood extracts, especially α-terpinyl acetate or α-terpineol contents are the most crucial factor for its termiticidal activity. On the other hand, α-terpinyl acetate and α-terpineol are generally synthesized by rather simple method from α-pinene or β-pinene which are major components of the essential oil from sugi (Cryptomeria japonica) or akamatsu (Pinus densiflora) woods. The latter woods have little or no termiticidal activity, but their low termiticidally active extracts are assumed to be convertible into higher active ones by the simple method. This report deals with the conversion of monoterpen hydrocarbons into terpineol and the evaluation of termiticidal activities of the products. 1) Firstly, the conversion of α-pinene into α-terpineol by hydrolysis with sulfuric acid was investigated. This method is known popularly, however, it was far from the efficient method because the conversion efficiency of this reaction was about 22 mole% based on the starting material at maximum. 2) The conversions with some sulfonic acids were also investigated. Among three sulfonic acids, p-phenol-sufonic acid was the most suitable catalyst for conversion of pinenes into terpineol. The conversion efficiencies were about 47mole% for α-pinene and 59mole% for β-pinene. β-pinene is found to be converted efficiently into α-terpineol whose content is about 83w/w% based on the product. 3) The conversions with solid acids were investigated in order to omit the complicated extraction procedure. Synthetic zeolite or cationic exchange resin were used as the solid acids. Although zeolite treatment was carried out under severe condition for long time, α-terpineol was formed slightly. On the contrary, the cationic exchange resin treatment can give a good α-terpineol yield, about 95w/w% based on the product, under mild condition for short time, 4) The hydrolysis products prepared above were subjected to the termite test. All the hydrolysis products had the termiticidal activity, and the higher content of α-terpineol the product has, it shows the stronger termiticidal activity. Since the simple hydrolyses cause the efficient conversion of pinenes into α-terpineol and increase the termiticidal activity, they will be applied to the essential oil from the woods of sugi, hinoki, and akamatsu.

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We studied on the anti-bacterial effect of Hinokitiol (H) and its isomer γ-Thujaplicin (γ-T) which were purified from Aomori hiba oil against pathogenic Escherichia coli O-157, O-111 and Salmonella sp. The minimun inhibitory concentration s(MIC) of H against Vero toxin production strains of E. coli O-157 (n=3) and O-111 were 12.5μg/ml, and against other strains of E. coli (n=2) were 25μg/ml. MICs of H against Salmonella sp (n=8) were in the range of 6.3 to 25μg/ml. On the other hand, MICs of γ-T against E. coli and Salmonella were in the range of 6.3 to 25μg/ml and 12.5μg/ml, respectively. MICs of H against both species were the same concentration as other Gram negative rods. In the physiological saline containing 100μg/ml of H and approximately 10^6 of bacterial cells, 99% of E. coli type strain and 95% of Salmonella serogroup O-9 strain became extinct in half an hour. In the solution containing the same concentration of γ-T, extinction of both strains were 93% and 89%, respectively. No survived bacterial cell detected in these solution after 24 hours. In the nutrient broth containing 25μg/ml of H, the shape of both strains swelled or got longer, then certain condensation appeared at the both edges of rod cell or cells burst. In the case of 100μ/ml of H, bacterial cell did not swell but got longer and then became extinct as it was. The same was true of γ-T but these phenomena appeared later than H. These effects show that the anti-bacterial effect of H and γ-T were bactericidal but H is stronger than γ-T. The observation of the cells suggested that the bactericidal mechanism against Gram negative rods are the inhibition of cell division and denaturation of bacterial endoprotein.

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