A number of marine cyclic bromo ethers were isolated from red algae, especially from Laurencia nipponica, whose components were precisely and continuously investigated by Irie. Kurosawa, and their co-workers since late 1960s. These bromo ethers are characterized by the fact that they have terminal enyne or bromo allene moieties, medium-sized cyclic ether skeletons, and a straight carbon chain with (6R,7R)- or (6S,7S)-configurations. It must be emphasized that each population of this single species metabolites completely-different major bromo ethers depending on growth locality in the case of Oshoro Ray. We have proved that a typical mono-cyclic bromo ether, deacetyl-laurencin (2) with (6R,7R)-configurations arises biosynthetically from its precursor (2E,6R,7R)-laurediol (11) by using lactoperoxidase (LPO) and bromopproxidase (BPO) and that 2 is successively converted to laurefucin (3) and laureoxanyne (4) in the same enzymatic reaction conditions.8) In order to prove the biosynthesis of bromo ethers having (6S,7S)-configurations, prelaureatin (13) was proposed to be a key intermediate of laureatin (5). isolaureatin (6), and laurallene (7). Actually the intermediate 13 was converted by LPO (or crude BPO extracted from the alga) into 5, 6, and 7 in the presence of hydrogen peroxide and bromide ion. Then, (3Z,6S,7S) laurediol (12) was subjected to the same enzymatic reaction using LPO to afford prclaureatin (13) in 3% yield. Thus, we tried to isolate the proposed intermediate 13 from the neutral extracts (31g) of the alga (wet weight 15kg) and really found 2mg of the compound, which clarified the whole biosynthetic pathway of the (6S,7S)-group bromo ethers. The results mentioned above indicate that bromoperoxidase in the alga cyclizes laurediols (11 and 12) into two regio-isomers 2 and 13, respectively, without substrate selectivity and that this single enzyme catalizes the multi-steps of bromo etherification reactions in vivo.

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Bialaphos (BA) is a tripeptide produced by Streptomyces hygroscopicus SF1293 and is characterized by the presence of a unique C-P-C bond. Through biosynthetic studies on BA we revealed the involvement of three enzymes catalyzing C-P bond formation. Two of them, phosphoenolpyruvate phosphomutase and carboxyphosphonoenolpyruvate phosphonomutase, have been purified and characterized. They catalyze reactions of a similar type, i.e., the intramolecular rearrangement of phosphate esters to form a C-P bond. On the other hand, the remaining enzyme catalyzes P-methylation of a phosphinic acid. The reaction mechanisms of these enzymes have been revealed in detail as summarized in Fig. 1.

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Biosynthetic studies of dynemicin A (DNM-A) were carried out on the basis of the incorporation of singly and doubly ^<13>C-labeled acetates, Imethyl-^<13>CImethionine and I^<15>N, 2-^<13>CIglycine by cultures of Micromonospora chersina N956-1. The incorporation results show that the compound is bio-synthesized from two heptaketide chains to compose the bicyclic enediyne core and the anthraquinone moiety, respectively, both of which are derived from seven head to tail coupled acetate units, the carboxyl group derived from one carbon of an acetate unit and the O-methyl group derived from methionine. Intact incorporation of glycine was not observed but a carbon was incorporated into the O-methyl group. The related C_<15> enediyne ring skeleton in the esperamicin/calicheamicin class of antibiotics may be similarly derived as the enediyne core of DNM-A. DNM-A rearranged smoothly to dynemicin H, on treatment with aqueous sodium hydrosulfite in dioxane. The same reaction in d_8-dioxane gave DNM-H deuterated specifically at 24- and 27-positions indicating the generation of biradical species at these positions. Dynemicins O, P and Q which have a bridging phenylene group, a vicinal diol and an oxo group in place of the respective 1,5-diyn-3-ene bridge, the epoxide and the carboxyl group in DNM-A. They also showed rather high activities against bacteria and tumor cells.

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(+)-Geodin (7), an anti-fungal antibiotic produced by Aspergillus terreus, is a seco-anthraquinone derived from octaketide anthraquinone emodin (2) as shown in Fig. 1. Asterric acid (9), a main metabolite of Penicillium frequentans, is biosynthesized in a similar pathway as (+)-geodin (7) except a chlorination step. Enzymes involved in these biosyntheses were studied to clarify and compare their molecular properties. Emodinanthrone oxygenase (EAO) catalyzes the fixation of molecular oxygen at C-10 position of emodinanthrone (1) without any requirement of external electron donor. EAO from both A. terreus and P. frequentans were membrane bound proteins and solubilized by Triton X-100. The solubilized enzymes were purified by column chromatographies as shown in Table I and II. The visible spectra of both enzymes showed no apparent absorption such as flavin or heme. The enzyme activity was inhibited by o-phenanthroline in the presence of 2-mercaptoethanol. These facts suggested the presence of non-heme ferric iron in the active center of EAO like lipoxygenase. Hence, the reaction mechanism of EAO was proposed as shown in Fig. 3. Emodin O-methyltransferase from both A. terreus and P. frequentans, desmethylsulochrin O-methyltransferase from P. frequentans were purified to apparent homogeneity. Sulochrin oxidase (SO), a phenol oxidative coupling enzyme of P. frequentans was also purified and revealed to be copper protein like dihydrogeodin oxidase (DHGO) from A. terreus. However, SO and DHGO showed different properties, such as effects of zinc ion and copper chelating inhibitors.

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In a continuing study in search of new bioactive models in crude drugs, we have been working in these years on the chemical constituents of leguminous plants and it has gradually become clear that triterpene oligoglycosides and some isoflavonoids obtained from Puerariae Flos, Desmodii Herba and Abri Herba exhibit protecting effect against hepatic injury. Now, we have investigated the ingredients of Astragali Semen, the seeds of Astragalus complanatus R. BR., and isolated six oleanene-type triterpene oligoglycosides (1-6), four sesquiterpene derivatives (7-10), eighteen flavonoid glycosides (11-28) and two sesquiterpene-flavonol complexes (29 and 30). Sesquiterpene-flavonol complexes (29 and 30) were characterized as unique ester complexes of rhamnocitrin glycoside (17) and dihydrophaseic acid (8). The pattern of the constituents in this plant was different from the usual leguminous one with respect to including sesquiterpene derivatives relating to abscisic acid, and sesquiterpene-flavonoid complexes.

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Beautiful flower color are mostly composed of anthocyanins. In 1919 Shibata and collaborates proposed the metal-complex theory for blue color development of flower petals. In 1958 Hayashi et al isolated a metallo-anthocyanin, commelinin, from blue petals of Commelina communis (tsuyukusa in Japanese). We determined that commelinin is composed with six molecules each of an anthocyanin, malonylawobanin (M) and a flavone, flavocommelin (F), and two atoms of magnesium, and the gross structure was [M_6F_6Mg_2]^<6-> by electrophoresis, metal analysis by ICP, and molecular weight (10,000) obtained by ultracentrifugation. In commelinin counterclockwise self-association of MM and FF was suggested by negative Cotton effect on CD. By ^1H NMR M are observed to be copigmented with F by cross arrangement. The metal chelated position was determined to be 3' and 4' o-dihydroxy group of B-ring of M by complexation experiment of commelinin-like complex using various anthocyanins. On basis of above-mentioned results we succeeded in X-ray crystallographic analysis of Cd-commelinin (trigonal, P321, a=b=31.256A, c=33.676A, R factor 0.132 at 1.0A resolution), which has the same space group of natural commelinin, and the complete structure of commelinin was determined (Fig. 8). Two cadmium atoms were located on three fold axis at 5.1A distance each other and the two M molecules, which chelates another metals each other, are self-associated. FF are inserted between MMs. Totally all the components are arranged as cyclo-MMFFMMFFMMFF-around the metals. These associations are derived from hydrophobic interaction between aromatic rings and the hydrophilic interaction of glucoses, which cover the outer part of the molecule. Commelinin is a stoichiometorical supra molecule.

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Some of Vitaceaeous plants have been used as anti-inflammatory in the treatment of liver diseases. In our continuous effort to discover anti-hepatotoxic constituents from Oriental medicinal plants, we observed that methanol extract of Vitis coignetiae exhibited strong inhibition against cytotoxicity induced by several hepatotoxic drugs using primary cultured rat hepatocytes. Further, ethyl acetate solubles of the methanol extract were also anti-hepatotoxic in the assay. Fractionation of the solubles led to the isolation of a new stilbene oxidative dimer, ε-viniferin diol (1), and two new stilbene oxidative tetramers, vitisins A (3) and B (8). Vitisins A (3) and B (8) were isolated as mixtures with their stereoisomers,4 and 9, respectively. Their structures were elucidated on the basis of extensive spectroscopic analyses, especially by the combination of 2D NMR methods such as HMBC experiment, as well as some reactions. Evaluation of the oligostilbenes isolated from Vitaceaeous plants was caried out. Thus, treatment of mice with ε-viniferin (2) has been found to strongly inhibit the increase of GPT value after carbon tetrachloride injection. While vitisin A (3) and ampelopsin C (11) produced severe damage in the mice liver. These observations were confirmed by histological investigation.

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Oligomeric hydrolyzable tannins having a valoneoyl group as linking unit of monomeric constituents have been classified into three types, based on the location of hexahy-droxydiphenoyl (HHDP) moiety of valoneoyl group on the glucose residue [HHDP part at O-4/O-6 (Type-A), O-2/O-3 (Type-B) and O-3/O-6 (Type-C) of glucose core]. The orientation of valoneoyl group in several dimers, representing those of each type, were determined by ^1H-^<13>C long-range COSY. A convenient and generally applicable method, for assigning the orientation of valoneoyl group in each type of oligomers, was established based on the difference of the chemical shifts of valoneoyl proton signals of the above-mentioned dimers and known oligomers of related structures. The behaviors of the oligomers upon partial hydrolysis at the valoneoyl moiety, under various conditions, were shown to be different depending on the location of valoneoyl group on the glucose core. The optimal condition was determined for the ether cleavage in the valoneoyl group and other related structural units, which is frequently observed upon partial hydrolysis of oligomers in hot water. This is applicable to chemical correlations of the oligomers with their monomeric constituents.

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Tuber forming substances from plants and microorgansms were surveyed using a bioassay consisting single-node stem segment culture. From leaves of Jerusalem artichoke (Helianthus tuberosus. L), two cyclopentanoid fatty acids, jasmonic acid(2) and methyl tuberonic acid glucoside(3), and six polyacetylene compounds(4-9) were isolated and the chemical structures were determinded. From the fungi, Lasiodiplodia theobromae IFO 31059, a cyclohexene epoxide(10) and mellein(11) were isolated. Cyclopentanoid fatty acids(2,3) have revealed tuber forming activity at more than 10^<-5>M and cyclohexene epoxide(10) at 10^<-4>M. Polyacetylene compounds(4-9) and mellein(11) have displayed weak activity.

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In the course of our study on neurotrophic substances in natural products, four novel dimeric isocuparene-type sesquiterpenes, mastigophorenes A (1), B (2), C (6), and D (7) together with a new 3,4-dihydroxylated isocuparene 3 were isolated from the liverwort Mastigophora diclados collected in Borneo. These new dimeric compounds belong to unique class of natural products made up of two molecules of (-)-herbertenediol (4), a co-metabolite in the title plant via different types of carbon-carbon bond. The structures of mastigophorenes A (1) and B (2) were elucidated on the basis of the extensive spectroscopic analyses and their absolute configurations with respect to biphenyl bond axis of 1 and 2 were assigned as S and R on the basis of the CD spectra, respectively Mastigophorenes C (6) was clarified to have an unsymmetrical dimer linked between the C-15 and C-3 positions of 4, whereas another symmetric dimer, mastigophorene D (7) was verified to form a carbon-carbon bond at the C-15 positions of two molecules of 4 by extensive analyses of spectroscopic data. Plausible biosynthetic pathway of these dimers initiated by one electron oxidation of (-)-herbertenediol (4) is also proposed. Compounds 1, 2, and 7 exhibited interesting neurotrophic properties in neuronal cell culture system of fetal rat cerebral hemisphere. Namely, they could enahnce not only neuritic sprouting but also choline acetyltransferase (ChAT) activity at 10^<-5>〜10^<-7> M.

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During the investigation on antitumor compounds from natural sources, the MeOH extract of the Brazilian plant "Favela", Cnidoscolus phyllacanthus (MART.) PAX et K. HOFFM. (Euphorbiaceae), was found to show a cytotoxic activity against P-388 lymphocytic leukemia in cell culture. Bioactivity-guided fractionation has led to the isolation of cytotoxic compounds, faveline methyl ether(1), faveline(2), deoxofaveline(3), and neofavelanone(8), as well as 12-hydroxyfaveline methyl ethers(4, 5), and 12-oxofaveline methyl ether(6) as related compounds. Two novel tetracyclic compounds, faveline(7) and neofavelanone(8) belong to unique class of fused tetracyclic system containing cyclopropane or cyclobutene ring. The structures of those compounds have been elucidated from spectroscopic data.

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Recetly, much attention has been given to the subject of differentiation inducers as new types of anti-tumor substances. We studied the cell differentiation inducers of A. paniculata Nees (Acanthaceae). The methanol extract of the whole plant showed the differentiation inducing activity on mouse myeloid leukemia (M1) cells. From ethyl acetate soluble fraction of the extract, thirteen ent-labdan tyep diterpenoids containing six new compounds and three new dimers of diterpenoids were isolated. The structures of the diterpenoids were determined on the basis of spectral methods, such as ^1H-NMR, ^<13>C-NMR, H-H COSY, C-H COSY. Their structures were illustrated in Fig 1 and Fig 2. We tested the cell diferentiation inducing activity of these diterpenoids on the M1 cells. The inducibility of phagocytosis of the M1 cells was observed for the marker of the activity. As a result, aglycons of diterpenoids except for AP-6 showed the strong phagocytosis and growth inhibitory activity, whereas diterpenoid glucosides except for AP-13 did not show such activities. The dimers of diterpenoids also showed the strong phagocytosis. In order to investigate the structure-activity relationship, several kinds of derivatives were derived from these natural diterpenoids and tested the inducibility of phagocytosis.

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During our investigations on bioactive substances from Okinawan marine organisms, we have isolated six new antineoplastic isomalabaricane-type triterpenes, named stelliferins A-F (1〜6), from Okinawan marine sponge Jaspis stellifera. The molecular formula C_<32>H_<48>O_4 of 1 was established by HREIMS. The UV spectrum of 1 indicated the presence of a trienone chromophore. The structure of 1 was deduced by a combination of ^1H-^1H COSY, HOHAHA, HMQC, and HMBC experiments as well as chemical means. The stereostructure of 1 was elucidated by NOESY spectra and modified Mosher's method. Structures of stelliferins B-F (2〜6) were determined from comparison of the spectroscopic data with those of 1. Stelliferins A-F (1〜6) exhibited potent antineoplastic activities against murine leukemia L1210 cells (IC_<50> 0.57〜2.1μg/mL) and human epidermoid carcinoma KB cells (IC_<50> 1.4〜6.0μg/mL) in vitro.

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CD Spectra of acetates of chiral cyclic and acyclic aliphatic alcohols exhibit the Cotton effects near 215nm, and their signs are dependent on the absolute configuration of the adjacent carbinyl carbons. Thus the esters with R configuration show a positive Cotton effect, while that of S structure exhibit a negative peak (Table 1). Although the parent alcohols are almost transparent, their contributions are subtracted from the acetate curves. The above phenomena may be explained by the asymmetric interactions between the chiral center and the ester group. The sign of the Cotton effectmay be predicted by the models 9 and 10. Analogusly, methoxyglyoxalates exhibit the peaks at 247nm with the same sign as those of corresponding acetates (Table 1). Further, the difference spectra of the respective pairs disclosed a pair of maxima at 247 and 214nm (see Fig. 3), indicating the presence of excitone coupling between the vicinal ester groups. Practical usefulness of this investigation is demonstrated by the determination of absolute configuration of a side chain hydroxyl group of 11, isolated from Aconitum carmiehaeli leaves. The difference CD spectrum of the acetates 12.A and 13.A, derived from 11, exhibited a negative Cotton effect due solely to the contribution of 24-acetoxyl group. The configuration at this center was thus assigned to be S.

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Part I. Lobatriene (1) has been obtained from the Okinawan soft coral of Sinularia species. The attempts to determine the stereochemical relationship of the substituents on its A and B rings by means of NMR spectroscopy have been found to be fruitless, because several unexpected NOE's appear between the protons on both the rings in the NOESY spectrum possible due to free rotation of the rings around the single bond connecting them. Therefore, chemical transformations of 1 have been carried out to produce the glycol (5) and the secondary alcohol (8). The application of the CD method in the presence of Eu(fod)_3 as well as the modified Mosher's method has resulted in the elucidation of the absolute configuration of C-17. Use of the modified Mosher's method1 to 1 for 8 has led to S configuration of C-4. These findings have revealed the absolute configurations of all the chiral centers of lobatriene as shown in structure 1. Part II. Isoclavukerin A (1) has been isolated from the Okinawan soft coral of Clavularia species. The relative stereochemistry of its substituents has been established by comparison of its spectral properties with those of clavukerin A (2), the structure including absolute configuration of which has been firmly established by Kitagawa's group. Finally, the absolute configuration of isoclavukerin A has been determine by application of the modified Mosher's method to the alcohol (4a) formed by autoxidaiton of 1, although the benzoate chirality method15 happened to be inapplicable to its benzoate (5) because of antipararell nature of the benzoate group and the olefinic group of 5.

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To determine the absolute configuration and conformation of chiral acyclic 1,3- and 1,2-glycols, the CD exciton chirality method has been applied to various acyclic 1,3- and 1,2-bis(p-bromobenzoates). Acyclic anti-1,3-dibenzoates exhibit typical exciton split CD Cotton effects, the sign of which agrees with the sign of the screw sense between two benzoate chromophores in the conformation expanded in a zigzag form. For example, the CD spectrum of anti-(2S,4S)-8 exhibited bisignate Cotton effects of positive chirality: λ_<ext>253.5nm, Δε+22.1 and λ_<ext>237.0nm, Δε-12.3, A=+34.4. On the other hand, syn-dibenzoates showed a weak single Cotton effects irrespective of the asymmetric structure: e.g., syn-(2R,4S)-15, λ_<ext>240.5nm, Δε+4.1. Other 1,3-dibenzoates composed of primary and secondary alcoholic benzoates exhibit exciton split CD Cotton effects of half intensity in comparison with those of anti-1,3-dibenzoates: e.g., (2S)-1, λ_<ext>252.6nm, Δε+13.9 and λ_<ext>235.9nm, Δε-3.9, A=+17.8. These results provided the CD method for determination of the absolute stereochemistry of acyclic 1,3-glycols. The same method could be applied to acyclic 1,2-glycol dibenzoates. In the case of syn-(2S,3S)-21, CD spectrum showed positive first and negative second Cotton effects indicating positive exciton chirality in the stable conformation of 21: λ_<ext>252.8nm, Δε+11.8 and λ_<ext>238.2nm, Δε-8.1, A=+19.9. These results are consistent with the conclusion of the conformational analysis that the conformer C with a positive exciton chirality in Figure 5 is the most stable. The CD method obtained here was next applied to acyclic 1,2,3-triols. Dibenzoate syn-(2S,3S)-28 derived from triol (-)-26 exhibited typical exciton Cotton effects of positive chirality, λ_<ext>254.1nm, Δε+9.9 and λ_<ext>238.5nm, Δε-9.6, A=+19.5, while anti-(2S,3R)-31 showed single Cotton effect reflecting meso-structure of dibenzoate part. To determine the absolute configuration of meso compound 31, it was converted to dibenzoate 33 composed of terminal primary and secondary benzoates. The CD spectrum of 33 exhibited exciton Cotton effects which enabled us to determine the absolute configuration as indicated: λ_<ext>253.0nm, Δε-18.4 and λ_<ext>237.0nm, Δε+8.0, A=-26.4. It was thus clarified that the CD exciton chirality method is also applicable to acyclic polyol systems as in the case of cyclic compounds.

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In tea (Camellia sinensis) plants, caffeine is synthesized from xanthosine, 7-methylxanthosine, 7-methylxanthine and theobromine, and S-adenosylmethionine is utilized as the actual source of the methyl groups. In addition to the pathway leading to the formation of xanthosine, which is the first methylated purine compound for caffeine biosynthesis from AMP, IMP and XMP, the formation from guanine nucleotides is proposed, based on available evidence for the catabolism of guanine nucleotides in plants, as: GMP→guanosine→xanthosine. There are seasonal variations in the caffeine content and biosynthetic capacity for caffeine synthesis in tea leaves. The results indicated that the biosynthesis of caffeine occurs in young leaves during the early stages of shoot development (April to June) and that one of the most important limiting factors for the synthesis of caffeine is the activity of various N-methyltransferases. The metabolism of [8-^<14>C]adenine in isolated stamens and petals from the flower buds of four different species of Camellia revealed that the biosynthetic pathway to caffeine is operative in the stamens and petals of tea flowers, but the reaction from theobromine to caffeine is defficient in C. irrawadiensis, while the entire biosynthetic pathway for purine alkaloids is not functional in flowers of C. japonica and C. sasanqua, as it is in leaves of these Camellia plants.

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The technology of plant cell suspension cultures to generate useful secondary metabolites was emploied for two medicinal plants originally producing the indole alkaloids. I. Biotransformation of Ajmaline in Plant Cell Cultures of Rauwolfia Serpentina Benth. From the methanol extracts of the plant cell suspension cultures of R. serpentina, which have been cultivated in the alkaloid-production medium after feeding of ajmaline (1), three new indole alkaloids, raumacline (2), N_b-methylraumacline (3), and 19(S)-hydroxy-N_b-methylraumacline (4), were isolated. These structures first elucidated by spectroscopic analysis were unambiguously determined by the chemical synthesis from ajmaline (1). These new compounds are the first examples of the macroline-type indole alkaloids having the trans relationship between C15 and C16 positions. II. Isolation of Novel Indole Alkaloids from Cell Cultures of Aspidosperma Quebracho Blanco Schlecht. From the cell suspension cultures of A. quebracho blanco, two novel monoterpenoid indole alkaloids, aspidochibine (19) and 3-oxo-14,15-dehydrorhazinilam (21), were isolated, though the production amounts of them were very low at this stage. The structure elucidation and the stereochemical analysis were made by mainly 2D NMR technique. Aspidochibine (19), which has a characteristic ten-membered lactone, is a completely new structural class of the quebrachamine series.

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During the course of our screening program for new antitumor antibiotics, we isolated an antibiotic, designated AL081, from the culture broth of an actinomycete identified as Streptomyces gannmycicus. Recently, AL081 was found to be identical with viridenomycin, an antifungal antibiotic, by direct comparison. The structure of viridenomycin, however, has not been clarified because of its instability in various solvents. The molecular formula of viridenomycin was established as C_<34>H_<37>NO_6 by FD-MS (m/z 556, MH^+) and elemental analysis (C 73.58, H6.56, N 2.42, O 17.44; calcd. C 73.49, H 6.71, N 2.52, O 17.28). The structure of viridenomycin was elucidated by using a variety of two-dimensional NMR techniques as shown in Fig. 1. The structure of viridenomycin is a novel 24-membered macrocyclic polyene lactam antibiotic. Viridenomycin prolonged the survival periods of mice bearing P388 leukemia (ILS 23.0%) and B16 melanoma cells (ILS 37.0%).

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Two bufodienolide alkaloids were isolated from skin extract of Indonesian toad (Bufo melanostictus) collected at Solok in West Sumatera. Both major alkaloid A and minor alkaloid B were shown to have molecular weight 624 through SIMS mass spectroscopy. ^<13>C-Nmr spectra indicated their complexed components including more than sixteen methylene groups. From their components HOHAHA (TOCSY) HC-COSY studies constructed six J_<HH>-coupling sequential blocks including suberic acid and 1,4-Butanediamine (Fig. 2, Fig. 3). Combination process of the blocks by long-range HC-COSY confirmed existances of ester linkage of suberic acid on the 3-hydroxyl group of hellebrigenine (4) and of amide linkage between suberic acid and 1,4-butanediamine. NOE study revealed that amino terminal of the side chain binded the 19-carbon of helle-brigenine through (E)-imine linkage to build up a 20-membered ring. ^1H-Nmr analysis suggested configuration of 3-acyloxy substituent as axial for alkaloid A and as equatorial for alkaloid B. As NOE indicated common cis A/B-ring juncture (5-axial-Hydroxyl-groups on A-ring), some strain would be expected on the macro ring in case of alkaloid B (Fig. 4). ^<13>C-Nmr assignments of the alkaloids are shown in Table 1. It is notable for chemoecology that skin extract from B. melano-stictus collected at Bogor on West Jawa mainly contained alkaloid B expected to have strain on its macroring and that any trace of alkaloid A unstrained was not detected on HPLC.

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The marine sponge Theonella sp., from which we isolated cyclotheonamides, contained orange pigments associated with cytotoxicity. The isolation and structure elucidation of two metabolites are the subject of this paper. The BuOH phase of the initial solvent partitioning scheme was successively purified by Sephadex LH2O and reverse-phase chromatographies to yield aurantosides A and B (1 and 2, respectively). Aurantoside A had a molecular formula of C_<36>H_<46>N_2O <15>Cl_2 as revealed by the FABMS, NMR spectra, and elemental analysis. Its gross structure was determined by the interpretation of 2D NMR spectra to be a tetramic acid glycoside: one side chain was a dichlorinated conjugated heptaene, while the other was a methylenecarboxyamide; xylopyranose, arabinopyranose, and a 5-deoxypentofuranose, whose hydroxyl group on C2 was methylated, were linked via nitrogen to the pyrrolidone ring, in order. Aurantoside B gave almost superimposable NMR spectra to that of 1, except for the absence of O-methyl group. Detailed analysis of spectral data indicated that the 2-O-methyl group of the 5-deoxypentose unit of aurantoside A was replaced by a hydroxyl group in aurantoside B.

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We have synthesized various types of ^<13>C, ^<18>O, ^<15>N and ^2H labeled δ-amino levulinic acid (ALA) and utilize these compounds in the study of biosynthesis of corrinoids and porphyrinoids. The biosynthesis of porphyrins were studied in Arthrobacter hyalinus by using stable isotope tracer techniques and hight-field NMR spectroscopy. [2-^<13>C]-isopropanol, [1-^<13>C]-glutamate and ^<13>C ALA were incorporated in uro'gen III. ^<15>N-ALA, ^<15>N-riboflavine and ^<15>N-potassium cyanide incorporated V.B_<12> showed ^<15>N signals on ^<15>N-NMR. These results supported that nitrogens of corrin ring were derived from ALA. Also, nucleoside and cyano group were derived from riboflavine and potassium cyanide. To clarify the origins of amide-nitrogen of V.B_<12> [1-^<13>C]-ALA and L-[amide-^<15>N] glutamine were administered to P.shermanii. The ^<13>C-NMR spectrum of the V.B_<12> subsequently isolated showed distinct ^<13>C-^<15>N coupling and isotope shift at six amide carbons. However, the C-57 amide carbon showed neither coupling, nor shift. Thus, it was concluded that the nitrogensof 6 amides of the side chain were derived from glutamine. Distinct isotop-shifted peaks were observed in the ^<13>C-NMR spectrum of uroporphyrin III octamethylester isolated after incorporation of [1-^<13>C, 1, 1, 4-^<18>O_3]-ALA. This indicates that undilution of ^<18>O occurred at the acethyl chain of A ring of uro'gen III. This result supports the assumption that the lactone formation of the A ring contraction, as proposed by Eschenmoser. Further more feeding experiment of ^<13>C-labeld ALA in 50% D_2O medium and that of ^<13>C, ^2H-doubly labeled ALA into bacteriochlorophyll showed α, β isotope-shifted peak atring carbons. In the ^<13>C-{^1H}{^2H} NMR spectrum of bacteriochlorophyll, the numbers of deuterium atoms directorly attached to ^<13>C carbons were observed as shifted signals.

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In our screening program for cholecystokinin (CCK) binding inhibitors of CCK type B (CCKB) receptors from microbial origin, we isolated a novel CCKB receptors antagonist, Ro 09-1468 from the culture broth of Streptomyces sp. NR0489. The molecular formula of Ro 09-1468 was determined to be C_<31> H_<38>O_8S, on the basis of HRFAB-MS, ^1H and ^<13>C NMR spectral data. The structure of Ro 09-1468 was determined to be 1 (Fig. 1), a macrocyclic compound with α-acyltetronic acid and tetrahydro-thiophene moieties, based on two-dimensional NMR techniques. Ro 09-1468 specifically inhibited CCK8 (the carboxy terminal octapeptide of CCK) binding to receptors (CCKB) in rat cerebral cortex membranes with an IC_<50> of 3.2nM.

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Biosynthesis of okadaic acid, a potent inhibitor of protein phosphatase 2A, and prorocentrolide, a macrolide having an unprecedented 26-membered carbocyclic ring and a partially hydrated isoquinoline in the molecule, was investigated by feeding experiments. First, the 13C NMR signals of okadaic acid were assigned by analyses of 2D NMR spectra. Feeding experimente were carried out by adding (1-13C), (2-13C), or [1,2-13C] sodium acetate to cultures of Prorocentrum lima. 13C NMR spectra (JEOL, 400MHz, CDC13/CD3OD 2:1) of labeled compounds showed 15 folds enhancement of signal intensities of labeled carbons. Out of 44 carbons of okadaic acid 39 were labeled, but carbons at the two termini could not be labeled with acetic acid. Out of 55 carbons of prorocentrolide 49 carbons were labeled. Three carbons consisting the isoquinoline ring could not be labeled. Two carbons were labeled with both [1-13C] and [2-13C] acetates. The labeling patterns suggested a type of mixed polyketides, involving building blocks formed via malonate, succinate, and 3-hydroxy1-3-methylglutarate.

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In the course of our continuing research aimed at the isolation of biological active compounds from mushrooms, we found nine new compounds in an edible mushroom Hericium erinaceum. Fresh fruiting bodies of H. erinaceum were extracted with acetone, and the extract was concentrated and fractionated by solvent partitions (CHCl_3 then ethyl acetate). Repeated column chromatography (SiO_2) followed by HPLC and/or recrystallization of the CHCl_3 extract gave eight new benzene-derivatives hericenones (3-5, 7-9) and a novel fatty acid(10). Hericenone A(1), B(2) and the fatty acid(10) were cytotoxic against HeLa cells; minimum concentration giving complete death of the cells for 1 was 100μg/ml, for 2 was 6.3μg/ml, and for 10 was 100μg/ml, respectively. Hericenones C(3)-E(5) and F(7)-H(9) were not toxic toward the cells, but exhibited stimulating activity of nerve growth factor (NGF)-synthesis in vitro; in the presence of 3, 4 and 5 at 33μg/ml, mouse astroglial cells secreted 10.8±0.8, 23.5±1.0 and 13.9±2.1pg/ml NGF into the culture medium, respectively. 7-9 exhibited similar level of the activity. 3-5 and 7-9 are first active compounds isolated from other than animals. On the other hand, 1 and 2 did not have the stimulating activity. 3-5 and 7-9 have also inhibitory activity of prostaglandin E_2 production in vitro.

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It has been well known that the guard cells of green leaves of Allium cepa (onion) showed an intrinsic green fluorescence under UV irradiation. However, the causal principal has not been characterized until now. Here, we report isolation and structure determination of the fluorescing compounds from the epidermal strips of green onion leaves. Two new flavonol glycosides were isolated from the aqueous methanol extract by using polyamide column, Sephadex LH-20 column and reverse phase HPLC as summarized in Scheme 1. These structures were determined to be kaempeferol -3-O-sophoroside-7-O-glucuronide 1 and quercetin-3-O-sophoroside-7-O-glucuronide 2 by ^1H and ^<13>C NMR (Table 1), UV (Figure 2), FAB Mass, and hydrolytic reactions (enzymic and acidic). From their fluorescence properties (Figure 3), 1 and 2 is seemed to be responsible for major fluorescing compounds in guard cells of Allium cepa. Particularly, the spectrum of 2 which is the most abundant flavonoid in the epidermis is almost identical with that of intact guard cell.

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Our ^1H-, ^<13>C-NMR and Mass spectral studies of isoprenoid substituted phenols have revealed that the structures of isoprenoid substituted flavonoids are easily determined with the following methods. 1) The chemical shifts of hydrogen-bonded hydroxyl groups at the C-5 position of 6-prenylated flavonoids appeared more downfield (0.25-0.29ppm, in acetone-d_6) than those of the corresponding 6-nonsubstituted flavonoids, while those of the hydroxyl groups of 8-prenylated flavonoids appeared more upfield (0.04-0.1ppm). 2) The EI-Mass spectra of 8-prenylated flavones, isoflavones and flavonols showed the intense fragment ions corresponding to M-15, M-55 and M-68. On the other hand, 6-prenylated flavones, isoflavones and flavonols showed the intense M-43 and M-55 ions. It was also found that the presense of relativity between the chemical shifts of the hydrogen-bonded hydroxy groups and ratios of intensity of the fragment ions ([M-43]/[M-55] of 6-prenylated flavonoids. 3) The chemical shifts of the methylene carbons of the prenyl groups of prenylated phenols were depend on the substituents located at the adjacent positions. The prenyl groups could be classified into six types according to the natures of atoms at the ortho-positions (hydrogen, oxygen or carbon). From the chemical shift of the methylene carbon, which kinds of groups located at the ortho-positions of the prenyl group can be suggested. Furthermore, using the above methods some wrong structures of isoprenoid substututed flavonoids were found. The structures of 8-prenyleriodictyol, 6-prenyleriodictyol, albanins D, E, glepidotin A, broussoflvonols C, D were revised to 1, 2, 9, 10, 11, 25 and 26, respectively, and the revised structures were confirmed with synthetic or spectroscopic methods.

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Furaquinocins A (1), B (2), C (3), D (4), E (5), F (8), G (7) and H (8) were isolated from the culture broth of Streptomyces sp. KO-3988 and their structures have been determined on the basis of their spectroscopical and chemical properties. Furaquinocins possess a unique structure consisting of naphtho[1,2b]furan-6,9-dione. The biosynthetic pathway to furaquinocins was investigated by means of incorporation experiments with [1-^<13>C]-acetate, [1,2-^<13>C_2]-acetate, and [methy-^<13>C]-ι-methionine. Furaquinocins are derived from a pentaketide, two mevalonates, and two C_1 units from ι-methionine. It may be worth noting that the carbon for CH_3-8 does not arise from propionate and the concurrent attachment of an isoprenoid side chain and a C_1 unit from methionine onto the polyketide backbone is also interesting. Although a few isoprenoid antibiotics produced by actinomycetes have been reported, furaquinocins may be the first example involving a carbon-carbon bond between an inside position of the isoprenoid chain (C-3) and the polyketide nucleus (C-3a). Biogenetical relationships of furaquinocin group were also discussed. These antibiotics showed cytocidal activities against HeLa S3 and B16 melanoma cells in vitro. Neither substance possessed antimicrobial activities against Gram-positive and Gram-negative bacteria, fungi or yeast at a concentarion of 1,000μg/ml.

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In Japan, a red natural coloring material, which is produced by Monascus anka, has been used as a food additive. During the course of selection of a high yield strain, one mutant strain, M. anka U-1, was found to produce new yellow pigments designated as Y-1 and Y-2. Before using this coloring material as a new food additive more information is needed. Therefore, we attempted the isolation and structural elucidation of the pigments. Physico-chemical properities of Y-1 and Y-2, except for NMR data are shown in Table 1. In addition to the FAB-MS data ^1H-NMR analyses revealed the structural similarity of Y-2 and Y-1 except for the longer side chain (C_7H_<11>) on Y-2. Since NMR studies including CH COSY (Table 2) could not afford further correlations than the subset of the partial structures of Y-1 (Fig. 2), 2D-INADEQUATE experiments were employed to complete the carbon skeleton (Fig. 3). On the basis of the spectroscopic studies the two possible structures of Y-1 were proposed (Fig. 4). Although the structures of Y-1 involved a novel skeleton, which has never been reported, an oxidative cleavage and a successive rearrangement of rubropunctatin, which is normally one of the major pigment in Monascus family, yields the structure of Y-1 (Chart 1). To confirm the structure of Y-1 and the above biosynthetic hypothesis, incorporation experiments of [1-^<13>C] sodium acetate (AcONa) and [1,2-^<13>C_2 AcONa into Y-1 were carried out. The incorporation ratios of carbons in both experiments were almost the same (1.1-1.4%). In the incorporation experiments of [1-^<13>C] AcONa the expected carbons (C, E, I, M, P, Q, R, S, T, U) were enriched. The 2D-INADEQUATE analyses of Y-1 enriched by [1,2-^<13>C_2] AcONa is consistent with the cleavage between carbons J and R and the rearrangement involving carbons J and I. The simultaneous labellings of the adjacent acetate units were observed except for the adjacent units L-S and Q-N (Fig. 5,6). The ratio calculated was about 50%. The lack of C-C coupling between both acetate units suggests that two polyketide chains are biosynthesized independently.

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In the course of our investigation of antitumor microbial metabolites which interact with DNA, we isolated 1, 3-5 and 7 from the fermentation broth of Streptomyces. These compounds showed antibacterial activity, antitumor activity in vivo, and caused single strand breakes in supercoiled plasmid DNA in vitro. Their physico-chemical data gave us the following information. The molecular formurae are C_<25>H_<18>O_9 for 1, C_<27>H_<24>O_9 for 5 and C_<29>H_<26>O_<11> for 7. All of them have fused aromatic rings and epoxids. 1 has 5, 8-dihydroxy naphthoquinone moiety in the chromophore. 1 and 3-5 have carboxylic acid groups. Their whole structures were determined based on analysis of the spectroscopic data, mainly of 2D NMR (HMQC, HMBC etc.). In order to improve instability and poor solubility, 1 was converted into the metyl ester(2) and analysed. 3 and 4 had the same fused ring chromophore as that of 5, then the difference among them were in the sidechain moieties, which were confirmed by comparison of the NMR spectra. Degradation product of 5 in DMSO was determined to be the decarboxylate (6). 1, 3-5 and 7 possesses the same anthra-γ-pyrone skeleton as the pluramycin-group antitumor antibiotics. However, 1 and 3-5 have a carboxyl group on C-13, no C-glycosylated sugar on the D ring, and 7 has no aminosugar on the D ring but a deoxyketose on C-9. So, 1, 3-5 and 7 are distinctly different from them.

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Some Carex spp. (Cyperaceae) contain a considerable amount of oligostilbenes (oligomers of 3,5,4'-trihydroxystilbene). In this paper, we report the structural determination of the oligo-stilbenes found in C. pumila Thunb. Methanol extracts of the subterranean parts of C. pumila were fractionated by column and thin layer chromatography to afford five oligostilbenes, kobophenol B (1), miyabenol A (2) and hopeaphenol (3) as tetramers, miyabenol C (4) as a trimer and ε-viniferin (5) as a dimer. All isolates showed an antibacterial activity against Staphylococcus aureus. Compound 1 showed a molecular formula of C_<56>H_<40>O_<12> which corresponded to a tetrastilbene. Permethylation of 1 yielded an octamethyl ether (7). The plain structure was elucidated principally with the aid of 2D NMR (HH- and CH-COSY, and COLOC). The EIMS fragmentation pattern of 7 well supported this structure. The stereochemistry of 1 was determined as (7aR,8aR,10aR,12aS,7bR,8bS,7cR,8cS,7dR,8dR) by the 2D NOESY results and the biogenetic relationship with 5. The stereochemistry of 2 was also determined as (7aR,8aR,7bS,8bS,7cR,8cR) by taking the NOESY results of a related tetrastilbene, miyabenol B (6) into account. Compound 3 has an interesting C_2 symmetric structure. The spatial relationship between two equivalent units was elucidated by a 2D coupled HMQC-ROESY technique.

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Recently more than ten hybrid strains have been produced by means of cell fusion technique using two different strains IFO 6200 and 4692, and many interesting metabolites have been obtained from the mycelia of the hybrid strains ME 0005, KO 0011, KO 0031, and KO 0052. We wish to report the isolation and structure determination of ten new curvlarin-type compounds, six new sesterterpene-type compounds, two new pyrones with long side chains and a new indol-type alkaloid. These structures have been elucidated by means of spectroscopic analyses, some chemical evidence, X-ray cystallographic analyses and/or molecular mechanics calculation. ^<13>C-NMR analysis of the curvlarin-type metabolites obtained by feeding experiments with sodium [1,2-^<13>C_2] acetate showed that the curvlarin-type metabolites are produced from eight molecules of acetic acid as same as curvlarin and dehydrocurvularin. In these metabolites, furthermore, the carbon atom (C_<12>)bearing the oxygen function is originally derived from the methyl group of acetate molecule, clealy indicating that they are not formed in such a β-oxidation manner as seen in dehydrocurvularin, but directly formed by enzymatic oxygenation from such a less oxidized precursor as curvularin. Especially, compound 27 has been found to have the high potent antifeedant activity against Plutella xylostella.

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In search of new bioactive metabolites from symbiotic marine microorganisms, we have isolated two peptides from a fish-possesing fungus and three macrolides from dinoflagellates which all showed antineoplastic activity. Fellutamides A (1) and B (2), antineoplastic acylpeptides, have been isolated from the cultured fungus Penicillium fellutanum Biourge which was isolated from the stomach of the fish Apogon endekataenia Bleeker. Amphidinolide F (3) is a new antineoplastic 25-membered macrolide isolated from the cultured dinoflagellate Amphidinium sp. which was a symbiont of the Okinawan flatworm Amphiscolops magniviridis. Amphidinolides G (6) and H (7), new 27- and 26- membered macrocyclic lactones with extremely potent antineoplastic activity, have been obtained from the cultured dinoflagellate Amphidinium sp. which was a symbiont of the Okinawan flatworm Amphiscolops breviviridis. The structures of these bioactive metabolites have been elucidated from spectroscopic data including 2D NMR and chemical means.

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Interest in microalgae as new sources of structurally-novel and biologically-active compounds has increased in recent years. In the course of our screening program of bioactive substances from microalgae, we found that a marine cyanobacterium Oscillatoria sp. isolated from Gokashowan-Bay, Mie Prefecture, showed significant activity in the echinoderm egg assay. The reddish blue-green alga was cultured in 1000L plastic tanks. Methanol and methanol/dichloromethane (1:1) extracts of the algal wet cells were partitioned between water and diethyl ether. The lipophilic layer was subjected to silica gel and ODS column chromatography followed by reversed phase HPLC to give an inhibitor of development of fertilized echinoderm eggs (0.0005%yield). It was named Oscillariolide (1). Its molecular formula was deduced as C_<41>H_<69>O_<11>Br from ^<13>C and ^1H NMR and HRFABMS. This compound was suggested to have 7 hydroxy groups from ^1H NMR spectra and formation of heptaacetate (2). The extensive analyses of ^1H-^1H COSY, HOHAHA, HMQC and HMBC spectra revealed the structure of Oscillariolide. Oscillariolide inhibited the cell division of fertilized starfish eggs at a concentration of 0.5μg/ml.

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The organic extract of the sea hare Aplysia kurodai collected in Mie Prefecture, which showed remarkable cytotoxicity, was subjected to bioassay-guided fractionation by repeated chromatography, resulting in the isolation of an active component named aplyronine (1). Aplyronine (1) was found to exhibit the potent in vivo antitumor activities against various murine tumors. The gross structure of 1 was deduced by means of 2D NMR spectroscopy (^1H-^1H and ^<13>C-^1H COSY, HMBC) and was confirmed on the basis of the detailed NMR spectral analysis of two amino acid derivatives (5 and 6) and four fragments (8a, 8b, 9, and 10) obtained by chemical degradation of 1. As part of a study on the absolute stereostructure of aplyronine (1), an enantioselective synthesis of the fragment 10 was carried out, disclosing the absolute stereochemistry of eight chiral centers (C23-C26 and C29-C32) in 1 as shown in 10c.

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Marine dinoflagellates are a rich source of novel polyether compounds, some of which are chemically and pharmacologically significant, e.g., okadaic acid, maitotoxin and brevetoxins. Many of them also inhibited the growth of fungus. Therefore, we initiated screening of cultured dinoflagellates for antifungals in order to find lead compounds of pharmaceutical importance. During the screening, we found potent activity in the extracts of Gambierdiscus toxicus. The active principles named gambieric acids (GA's) were unprecedentedly potent. In terms of the minimum inhibitory dose against Aspergillus niger, 3 was 2000 time more potent than amphotericin-B. Their structures were elucidated on the basis of NMR data and found to belong to brevetoxin-type polyethers (GA-A 1, GA-B 2, GA-C 3 and GA-D 4) consisting of continuously fused 9 ether rings (7/6/6/7/9/6/6/6/6) with an additional five-membered ring in a side chain. The structures are, however, very unique in bearing a terminal carboxylic acid, and methylgultarate hemiesters in the other terminus.

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Pamamycin-607 (1)(MW 607), which has been isolated from Streptomyces alboniger, induces aerial mycelium-differentiation from substrate mycelium in an aerial mycelium-less mutant of the same strain. Our previous study showed the presence of 6 pamamycin homologues ranging in MW from 593 to 663, and found that the homologues with smaller MW were higher than the larger ones in the aerial mycelium-inducing activity. We describe here the structure elucidation of four new homologues, pamamycin-635A(2), 635B(3), 649A(4) and 649B(5), preparation of desdimethylaminopamamycin-607, and the structure-activity relationship of pamamycins. The structures of pamamycins were elucidated from diol fragments obtained by their LiAlH_4 reduction, the structures of which were analyzed by GC-MS method being compared with those of the known diols, S and L, obtained from pamamycin-607 in a similar manner. Relative stereochernistries of pamamycins were determined by NOE difference spectroscopy, chemical shifts and J_<HH> values of their CF_3COOD salts. The absolute stereochemistry of pamamycin-607 was determined by derivatizing it to a furfuryl derivative(11), which coincided in the optical rotation with that of an authentic compound derived from nonactin antibiotic. The absolute stereochemistry of pamamycin-635A was elucidated by using the smaller LiAlH_4 reduction product, S_1. The secondary alcohol group at 8' of S_1 was converted to (S)- and (R)-MTPA esters, to which the advanced Mosher's method was applied, and the absolute stereochemistry at 8' of S_1, hence, that of pamamycin-635A was determined as S and 2, respectively. Pamamycin-635B showed aerial mycelium-inducing activity at 10μg/disc, which was ca. 1/3 that of pamamycin-607. In contrast, pamamycin-635A and the mixture of pamamycin-649A and 649B were not active. Characteristic structural feature to distinguish the active homologues from the inactive ones is that the active homologues have CH_3-group at C-9(R_2), whereas the inactive ones have C_2H_5. Desdimethylpamamycin-607 prepared by Hoffman degradation lost aerial mycelium-inducing activity. Thus, the dimethylamino-group in the side chain is necessary for the activity.

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Quartromicins A1 (1), A2 (2), and A3 (3) are the antibiotics isolated from Amycolatopsis orientalis. These compounds have been isolated by use of Diaion HP-20, SiO_2 and ODS column chromatographies as water-soluble amorphouses. The structure of quartromicin A3 (3) has been elucidated by combination of spectroscopic analyses and chemical reactions. These works have revealed its unique structural features with a symmetric dimeric structure that possesses a large ring composed of only C-C linkages and bears four tetronic acid moieties. To verify the structure, compounds 6-9 have been prepared and their spectroscopic properties compared with those of 3. Good agreement in the spectral data has been observed between them. The structures of quartromicins A1 (1) and A2 (2) have been determined by the analogous spectroscopic analyses and chemical correlations with 3. Quartromicin A3 exists as a salt. The results of the ICP analyses have shown the presence of Na^+, K^+, Ca^<2+>, the proportion of the cations varying lot to lot. The NMR properties of 3 in the presence of several metal cations have been studied. There have been found very few macrocyclic antibiotics, the carbon frameworks of which are composed of only C-C linkages thus far. Quartromicins A1-A3 are the largest members of this category.

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In our laboratory, an antifungal antibiotic, tautomycin was isolated from a culture of Streptomyces spiroverticillatus. Besides antifungal activity, tautomycin induced a morphological change (bleb formation) of human leukemia cells K-562. Tautomycin is a specific inhibitor of protein phosphatase 1 and 2A. On the basis of chemical degradation and spectroscopic evidence, we determined structure (1) for tautomycin. The absolute configuration at C-3' in p-bromobenzoate of 2b was determined by the exciton chirality method for determining absolute configurations of acyclic allylic alcohols. The absolute configurations S/R for C-18 and C-24 in 3 were determined by Mosher's method. Degradation product (3) from 1 was converted into isopropyliden derivertive (6). The coupling constants between protons 18/19/20 indicated that the 1,3-dioxane ring adopts a chair conformation and that the relative configurations at C-18 and C-19 in 1 are S/S (R/R). The absolute configuration at C-19 in 1 is thus S. To determine relative and absolute configurations at C-22 and C-23 isopropyliden derivertive 7 was prepared by stereoselective reduction of 1 and isopropylidene formation. The twist-boat conformations of both 1,3-dioxane rings lead to absolute configurations R/S for C-22 and C-23. Degradation product (8) was prepared from 5 through Baeyer-Villiger oxidation as a key step. The absolute configuration S for C-3 was established by Mosher's method. Treatment of 5a with p-TsOH/Ac_2O/AcOH afforded 9. The acetate was converted into the alcohol by methanolysis and the absolute configuration S for C-6 was determined by Trost's method. Biosynthetic origin of 1 was revealed by feeding experiments of ^<13>C and ^<14>C labeled precursors. The right half of 1 is synthesized by a polyketide pathway which starts with isobutyric acid followed by introduction of a glycolate, and then five acetate and five propionate units. The terminal methyl carbon was derived from [2-^<13>C]acetate which could be metabolized to β-ketocarboxylic acid, and then the terminal methyl ketone was formed by decarboxylation. The left half of the antibiotic is synthesized from propionate and a C-5 unit. The latter may be formed by a unusual assembly of three acetate units with loss of one carboxyl carbon.

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During our studies on bioactive substances from Okinawan marine organisms, we have now isolated five novel cyclic peptides from the extracts of the Okinawan marine sponges of the genus Theonella. Konbamide (1) is a unique hexapeptide containing the unknown amino acid. 2-bromo-5-hydroxy-tryptophan (BhTrp), and possessing an ureido linkage. Keramamide A (2) consists of six amino acid residues, one of which was unknown N-methyl-6-chloro-5-hydroxytryptophan (MeCht), and also contains an unusual ureido bond like 1. Three novel peptides, keramamides B-D (3-5), are structurally unique with the unusual amino acid, BhTrp, and three modified amino acid residues, α,β-unsaturated amide group, an oxazole ring, and α-keto-β-amino acid moiety. The structures of 1-5 were fully established on the basis of extensive spectroscopic analyses including several types of 2D NMR as well as FAB MS/MS experiments. The absolute stereostructures of 1-5 were revealed by the chiral GC and HPLC analyses. Konbamide (1) showed calmodulin antagonistic activity (IC_<50> 10^<-4>M). while keramamide A (2) inhibited sarcoplasmic reticulum Ca^<2+>-ATPase (IC_<50> 3×10^<-4>M). Keramamides B-D (5×10^<-8>M) completely inhibited the superoxide-generation response of the human neutrophils elicited with an fMLP.

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B. zeicola is the pathogen of leaf spot of corn. Their pathogenic races of the fungus have been differentiated according to the symptoms expressed on different corn genotype. Race 1 produces host-specific HC-toxin (Table 1). B. zeicola race 3 also exhibits high pathogenicity to rice plants by artificial inoculation. The spore-germination fluids of race 3 showed susceptibility-inducing activity on rice and corn leaves to nonpathogenic fungus. In 1990 Nishimura et al. isolated BZR-toxins as susceptibility-inducing factor produced by B. zeicola race 3. The factor consist of 4 components which are called B1, B2, B3, and B4. While each component showed little toxicity, their combinations exhibited potent phytotoxicity and susceptibility-inducing activity on rice plants (Fig. 1). We undertook the structural study of BZR-toxins to make clear the interesting phenomenon chemically. The ^1H-NMR spectra of each component revealed the presence of N-methyl-α-aminoacids residue in their structure. Their molecular weight were determined by the SIMS spectra (B1: 967, B2: 964, B3: 907). And the IR spectra showed adsorption based on amide C=O groups, ester C=O groups, amide NH groups and OH groups. Some hydrolyzed products of each component gave positive ninhydrin tests. Analysis of these results and the structures of partially hydrolysed products of each component (Scheme 1, 2, 3) elcidated the plane structures of the components as being cyclic depsipeptides shown in Fig. 3.

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Substance P is the member of the family of structurally related peptides known as tachykinins and is widely distributed in the body. Several studies suggest that tachykinins are involved in numerous physiolosical activities. In the course of our screening program for tachykinin antagonist, WS9326A was isolated as a Substance P receptor antagonist from Streptomyces violaceoniger No.9326. The structure of WS9326A was elucidated as (1), by spectroscopic analyses and chemical reactions, and finally established by the success of total synthesis. WS9326A has the following remarkable structural features: (i) to posses the new ΔMeTyr unit, (ii) the geometry of its α,β dehydro amino acid is (E)(usually (Z)-configuration), (iii) to have the novel cinnamic acid derivative as acyl group. So far WS9326A has been found to antagonize the tracheal constrictions produced by exogenously added substance P(NK_1) and neurokinin A(NK_2), with IC_<50> values of 9.7×10^<-6>M and 3.5×10^<-6>M, respectively.

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Trichoderma spp. are active as mycoparasites. Therefore, they can serve as potential biocontrol agents. Various antibiotic peptides, peptaibols, have been isolated from Trichoderma spp. Their structures are characterized as follows; 1) both N-terminal and C-terminal amino acids are protected, 2) a high proportion of abnormal amino acids, α-aminoisobutyric acid (Aib) and isovaline (Iva) are incorporated, and 3) a labile Aib-Pro peptide bond is present. New peptaibols, trichorozin (TZ)-I〜IV from T. harzianum, trichodecenin (TD)-I and -II, and trichorovins (TVs) from T. viride have been isolated from their conidia, respectively. The primary structures of the former two peptide groups were clearly established by FABMS, FABMS/MS and NMR. TZ-I〜IV are composed of eleven amino acid residues. The N-terminals are all protected by an acetyl group and the C-terminal amino acids are linked with a valinol (TZ-I, -II) or a leucinol (TZ-III, -IV). On the other hand, TD-I and -II are composed of seven amino acid residues. The N-terminals are protected by a cis-4-decenoyl group and the C-terminal amino acids are linked with a leucinol. In addition, TD-I and -II contain no Aib-Pro peptide bond. Thus, TD-I and -II were found to belong to a new type of peptaibols. TZ-I〜IV and TD-II were synthesized to confirm their structures. TVs having the molecular weights ca. 1200 showed similar FABMS spectra to those of TZs. However, TVs were hardly separatable. Thus, we tried to determine their amino acid sequences by FABMS/MS in the state of a mixture.

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Recent advance of spectroscopic instrumentation such as NMR spectroscopy has greatly facilitated structural determination of natural products, so that purification and isolation has become further important in this field. A combination of mass spectrometry and chromatography is being much paid attention for simultaneous separation and structural characterization of natural products. Fast atom bombardment mass spectrometry (FABMS) has contributed for obtaining molecular weight information of polar and involatile compounds. Recently, the use of a frit probe as an interface for FABMS analysis of HPLC effluents has been established. In this method effluent from HPLC was split with a pneumatic splitter or flow splitter and the resulting smaller portion was introduced into a frit surface via a fine capillary tube, followed by FAB ionization on the frit surface as a target (Fig. 2). The method was successfully applied to structural characterization of minor components of peptide antibiotic, bacitracin (BC) and the structures of 13 components were proposed as shown in Fig. 3. We have also developed another combination technique using TLC and FABMS. In order to perform effectively TLC/FABMS, a condensation technique was devised. Namely, methanol was applied around a developed spot on a TLC plate and then the sample was condensed to the center of the spot in line with penetration of methanol (Fig. 6). The method including the condensation technique has been applied to various natural products.

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The azidophenyl derivative, (15S)-10c. has been synthesized by structural modification of isocarbacyclin methyl ester (3). First, the C(13)-C(14) double bond of 3 is selectively epoxidized by Sharpless epoxidation giving 4, whose 11- and 15-hydroxyl groups are acetylated. Epoxy ring opening of resulting 5 with CH_3COOH-H_2O and subsequent deacetylation with aqueous K_2CO_3 give 6, which is cleaved with NaIO_4 to give an aldehyde 7. Horner-Emmons reaction with 8c gives an enone 9c. Reduction of 9c with NaBH_4-CeCl_3 is followed by separation of the resulting 15-epimers and alkaline hydrolysis of the methyl ester to give (15S)-10c. This compound exhibits high affinity to the PGI_2 receptor protein(s) in mastocytoma P-815 cells with the IC_<50> value of 3nM. The tritium labeled derivative, [^3H]-(15S)-10c, synthesized by reduction of 9c with [^3H]NaBH_4-CeCl_3 followed by alkaline hydrolysis, has been used for the photoaffinity labeling experiment. Plasma membrane fraction of mastocytoma P-815 cells which is abundant in the PGI_2 receptor protein(s) is incubated with [^3H]-(15S)-10c and then irradiated with a UV lamp. The irradiated material is subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography showing a clear band around 43k. The photoreaction in presence of GTPγS decreases the intensity of this band. The addition of iloprost (11) to the incubated media completely suppresses the formation of this band. These results confirm the molecular weight of the PGI_2 receptor protein(s) in mastocytoma P-815 cells to be 43k.

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PAF (platelet activating factor, 1-O-alkyl-2(R)-acetylglyceryl-3-phosphoryl choline) causes platelet aggregation, chemotaxis and degranulation of polymorphonuclear leukocytes, smooth muscle contraction, vascular permeability, and hypotension. Recent studies have shown that PAF may be involved in many inflammatory, respiratory, and cardiovascular diseases. In our program for finding PAF antagonists from marine sources, we focused on marine fungi. We systematically screened lipophilic extracts of marine fungal isolates for inhibition of PAF-induced platelet aggregation and binding of PAF to its receptors, and found that a marine fungus Phoma sp. produced a novel PAF antagonist, phomactin A (1) and its derivatives B (2), B1 (3), B2 (4). Their structures were determined by NMR studies, chemical conversion and X-ray analysis. Phomactin A, B, B1, B2 inhibited PAF-induced platelet aggregation at IC_<50> 1.0x10^<-5>M, 1.7x10^<-5>M, 9.8x10^<-6>M, 1.6x10^<-6>M respectively, and A, B1, B2 also inhibited binding of PAF to its receptors at IC_<50> 2.3x10^<-6>M, 2.0x10^<-5>M, 5.4x10^<-6>M.

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KS-505(1) is a potent inhibitor of calmodulin dependent phosphodiesterase, elaborated by streptomyces argenteolus. Molecular formula of 1 (C_<61>H_<88>O_<17>) was determined by high resolution FAB-MS, but further analysis was unsuccessful owing to its intramolecular equilibrium. Two isomeric methyl ester (2,3) were prepared, which correspond to the tautomeric structures of 1. Detailed studies using homo- and heteronuclear two dimensional NMR spectroscopy on 2, including HMQC-HOHAHA method, exhibited the structure shown in Fig. 3. Methanolysis of 2 afforded lactone 4, which was confirmed to be D-sugar by comparison with synthetic authentic sample. Analysis of NOE data around the sugar moiety and aglycon moiety, revealed the absolute stereochemical structure of 2. 3, which is an epimeric mixture of methoxy group, was elucidated to be shown in Fig. 1 by the same manner as in 2. Finally, the position of methoxycarbonyl group which had been exist in 1, was determined by the studies of MS fragmentation of 1 and of NMR analysis of aglycons. 1 is a novel tetraterpenoid with a unique 2-O-methyl sugar, and from the biosynthetic point of view, origin of sec. methyl groups at C-1 and C-12 position is interesting and worth investigating.

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During the course of an investigation into insecticides produced by microorganisms, we got a soil isolate of Penicillium simplicissimum AK-40. This strain, when grown on okara (the insoluble residue of whole soybeans), showed strong activity to silkworms upon oral administration. We also succeeded in the isolation of novel insecticidal compounds named okaramines A (1), C_<32>H_<32>N_4O_3, and B(2), C_<33>H_<34>N_4O_5. Acetylation of 1 gave acetylokaramine A (3), whose structure was determined by an X-ray analysis. 1 is a new heptacyclic compound containing a hexahydropyrroloindole and dihydroazocinoindole, which are linked to form an additional diketopiperazine ring. The structure of 2 was elucidated by analyses of ^<13>C-, ^1H- and 2D-NMR spectra. Since we had an interest in the unique structures of okaramines, we tried to find if okaramines or similar compounds were produced by other strains. P. cimplicissimum AHU 8402 was shown to produce okaramine C (5), C_<32>H_<36>N_4O_3, together with 1 and 2. 2 was the most active among okaramines and their derivatives. 5 showed almost the same activity as 1. Despentenylokaramine A (4) had no activity. These data strongly suggest that an azetidine ring in 2 and an isoprene side chain at N^8 in 1 played an essential role in expressing the activity and that an azocine ring in 1 and 2 were unnecessary to exhibit the activity.

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Fifteen metabolites were isolated from the mycelium and culture filtrate of a strain of Aspergillus fumigatus which existed in the gastrointestinal tract of the saltwater fish Pseudolabrus japonicus. Among them, TR-2, fumitremorgin C and gliotoxin exhibited significant cytotoxicity against the cultured P-388 lymphocytic leukemia cells. Analysis of long range ^1H-^<13>C COSY and other spectral data for the five new metabolites[FQ-A(1),-B(2),-C(3),-D(4)and -E(5)], exhibiting marginal or moderate cytotoxicity, allowed assignment of their structures containing quinazolone and indoline moieties. The absolute stereostructure of 3 was determined on the basis of X-ray crystallographic analysis as well as of the production of L-(+)-alanine by its acidic hydroloysis. The stereochemistry of the other metabolites was established by deriving 1 and 5, from 3 and other chemical behavior.

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