Biosynthesis of the isoprenyl units of the Diels-Alder type adducts in Morus alba callus cultures has been examined by comparing their biosynthesis with that of β-sitosterol (3) produced in the same callus cultures. [1-^<13>C]- and [2-^<13>C]Acetates administered to Morus alba cell cultures were incorporated intact into the isoprenyl unit of 3. [2-^<13>C]Mevalonolactone was also incorporated intact into the expected positions of 3. In the case of the Diels-Alder type adduct chalcomoracin (1), the acetate incorporated into the isoprenyl unit was not the intact acetate administered, but resulted from reorganization from the original acetate through the TCA cycle. Furthermore, [2-^<13>C]mevalonolactone was not incorporated into the isoprenyl unit of 1. These different ways of the incorporation of acetate and mevalonate into the isoprenyl units of 1 and 3 suggest that the mevalonate biosynthesis in 1 and 3 may proceed through a different time schedule. In an analogous experiment with [2-^<13>C]-L-leucine, the incorporation of the ^<13>C-labeled carbon into the isoprenyl units was not observed on either 1 or 3, but the ^<13>C-labeling was incorporated into the polyketide-derived aromatic moieties of 1 as in the case of [1-^<13>C]acetate. This result indicates that the administered L-leucine was metabolized in the cell cultures to [1-^<13>C]acetyl CoA, which then participated in the polyketide synthesis. Reexamination of the biosynthesis of the isoprenyl units of 1 by the pulse administration of [2-^<13>C]acetate revealed that the randomization of the ^<13>C-labeling between cis- and trans-methyls has taken place presumably due to the isomerization of the prenyl group to the diene form for the Diels-Alder type reaction.
From Rosa rugosa leaves, three major carotane sesquiterpenes were isolated and their structures elucidated. Rugosal A (1) isolated as the major antifungal substance of the leaf extracts together with its corresponding acid rugosic acid A (2) and its precursor carota-1,4-dienaldehyde (3) constitute the highest propotion of the leaf extracts. Compound (3) was converted into (1) through autoxidation reaction process with two O_2 molecules. As a result of quantitative analyses carried out, dynamic changes in their concentration was observed at different growth period and also variations when the plant was subjected to various stresses. Young leaves were found to contain high concentration of (3), while (1) and (2) were pooled in the mature leaves. When leaves were placed in the dark or when leaves were detached, the concentration of (3) was drastically reduced. Compound (1) and (2) were found only in the exudates of mushroom-shaped trichomes, while (3) was involved chloroplast grains, suggesting these compounds function as defensive agents. Although neither soybean-lipoxygenase nor crude protein fractions of the leaves exhibited peroxidation activity, (1) is considered to be converted biochemically because unregulated radical reaction around chloroplast bodies may be lethal. The study of the physiological functions of these compounds warrant further investigation.
Deoxygenation reaction involved in the biosynthesis of fungal melanin was investigated by using synthesis, theoretical chemistry and enzymology. A biomimetic synthesis of scytalone (1), a simple derivative of tetralone, was reinvestigated and it was revealed that Scyatalone (1) is formed from 1,3,6,8-tetrahydroxynaphthalene (2; 1,3,6,8-THN) by sodium borohydride only in the presence of sodium methoxide. The reactive species of this reaction was clarified as a non-synmmetrical keto-tautomer, THN4H by NMR spectroscopy. The structure of the most stable ionic species of 1,3,6,8-THN (2) in CH_3ONa-CH_3OH was studied by the semi-empirical (AM1) and ab initio (Gaussian 86; 3-21+G//3-21G) molecular orbital calculations. It was suggested that the most stable species was a non-synmmetical keto-tautomeric trianion, which is in good agreement with the observations by NMR spectroscopy. Furthermore, transition state structures and stabilities were simulated for the hydride attack to the carbonyl carbons at C-1 and C-3 of THN4H. The results suggested that C-3 carbon was more favorable for the reduction, which coincided with the experimental fact. The enzymatic activities which reduce 1,3,6,8-THN and 1,3,8-trihydroxynaphthalene (1,3,8-THN) to scytalone (1) and vermelone (3) respectively, were detected in a cell free extract from shake cultures of Phialophora lagerbergii. The both reductive activities were observed in the same enzyme fractions in the partial purification using ammonium sulfate precipitation and DEAE cellulose column chromatography. Further purification and characterization of the enzyme are now in progress.
Virginiae butanolide A (VB-A) is one of the virginiamycin inducing factors isolated from the culture broth of Streptomyces virginiae, and has a unique 2,3-disubstituted butanolide skeleton which is common in other signal molecules in Streptomyces. The biosynthesis of VB-A in S. antibioticus, which was found as a high producer of VB-A, was studied by feeding experiments with labeled precursors. ^<13>C NMR and CI-MS analysis of the VB-A dibenzoate samples obtained by feeding experiments with sodium [1-^<13>C]- and [2-^<13>C]acetate, sodium [1-^<13>C]isovalerate and [1,3-^<13>C]glycerol showed that the VB-A molecule originated from two moles of acetic acid and each mole of isovaleric acid and glycerol. Further feeding experiments with ^<13>C-double labeled N-acetylcysteamine thioester of β-ketoacid (1) and glycerol-d_5 indicated that a probable biosynthetic pathway of VB-A is a coupling between a β-ketoacid derivative and a C_3 unit from glycerol, such as dihydroxyacetone or its derivatives.
The proposed mechanism of P-450 inhibitors possessing heteroaromatics suggests that these chemicals might block a wide range of oxidation steps in biosynthesis of secondary metabolites. This possibility was examined for the following three natural products. 1. Chaetoglobosin A (1): Treatment of Chaetomium subaffine with metyrapone caused the accumulation of three plausible intermediates, named as prochaetoglobosin I (2), II (3) and III (4). Their structures including relative configuration were determined by spectroscopic methods. On the basis of incorporation experiment with [1-^<13>C, ^<18>O_2]sodium acetate, origin of oxygen atoms at C-1 and C-23 of 1 was established. These data allow us to propose the biosynthetic pathway of 1. 2. Aphidicolin (5): Late biosynthetic stage of tetrahydroxyditerpene, aphidicolin (5) was investigated. The possible precursors, 3, 17, 18-trideoxyaphidicolin (6) and 3-deoxyaphidicolin (7) were found in the culture, in which inhibitor was added. The structures were identified by comparison of the authentic samples from either natural or synthetic.The conversion of ^<14>C-labeled 6 to 5 and to 7 was achieved under the conditions with and without inhibitor. 3. Nigericin (10): The oxidation sequence of 10 was examined by the inhibitor experiment. Identification of grisorixin (11) from the medium treated with inhibitor suggests that 30-hydroxylation occurred at final step.
Soft corals have proved to be a rich source of unusual diterpenoid structures. The first discovered example was xenicin, which possesses a novel bicyclic diterpene skeleton. Since then, many other xenicins and xenolides have been isolated from different Xenia species, as well as other soft corals and gorgonians. Recently, we have investigated the chemical constituents of Xenia florida and Xenia sp., two soft corals collected from the Kagoshima area. Many new xenolide-related compounds have been characterized, using high-field NMR, MS, IR and UV spectroscopy. Of particular interest are a series of compounds which contain a new tricyclic structure. Details of the isolation and characterization of these and the other metabolites will be described.
In the course of our investigation of the biologically active metabolites of marine invertebrates, we found that the methanol extracts of a gorgonian octocoral of the family Acanthogorgiidae collected at Sukumo Bay, Kochi Prefecture, showed marked lethalty to brine shrimp, Artemia salina L., and insect growth inhibitory activity against silkworm, Bombyx mori L. Bioassay-guided fractionation of the extract led to isolation of five 9,10-secosterols 1-5 and two diterpenoids 6 and 7, which are responsible to the observed activity, together with three furan derivatives 8-10. Their structures have been characterized by extensive 2D NMR studies, including scrutiny of J-resolved spectra, EIMS analysis, and chemical methjods. Biological activities of isolated compounds against brine shrimp and silkworm were evaluated. Secosteroids 1-5 were significantly toxic to brine shrimp and their LD_<50>-values ranged from 1.0 to around 10ppm. Diterpenoids 6 and 7 were active to both of tested animals but furan derivatives 8-10 were inactive to both of them.
We focused our research interests into surveying new antioxidants among the extracts of marine organisms. Among the organic solvent extracts of viscera of marine fish and bivalves, the extract of the short-necked clam, Ruditapes philippinarum, showed very low POV in the conventional antioxidant test. From the extract, new chlorophyll a related compounds, chlorophyllone a (1), C_<33>H_<32>N_4O_3, and chlorophyllonelactone a (2), C_<33>H_<32>N_4O_4 and an unknown compound (3) as well as pyropheophorbide a (4) were isolated as main antioxidative compounds. We were interested in the origin of these compounds and surveyed them in the extracts of the other bivalves, the scallop Patinopecten yessoensis and the oyster Crassostrea sp., to isolate 2, 3, 4 and 10-epichlorophyllone a (5) and 1, respectively. Spectroscopic analysis allowed us to assign the interesting structures with additional ring VI to these new chlorophyll a related compounds. This suggests the presence of another new degradation pathway of chlorophyll a. From the extract of attached diatoms mixture mainly containing Fragilaria oceanica, Nitzschia closterium, etc. cultured for culturing juvenile abalone, 1 was isolated, suggesting that these new chlorophyll a related compounds are not artifact formed in the bivalves' viscera, but produced by microalgae. The origin is now under investigation.
In the course of our screening program of Japanese marine invertebrates for potential biomedicals, a marine sponge of the genus Theonella inhibited various proteinases, particularly thrombin. We have isolated two active substances, named cyclotheonamides A and B, which proved to be novel cyclic peptides. Cyclotheonamide A was isolated from the 1987 collection by means of solvent partitioning, gel filtration, and three successive reverse-phase HPLC's, whereas cyclotheonamide B was isolated from the 1989 collection. ^1H and ^<13>C NMR spectra indicated that cyclotheonamide A contained Pro, Phe, 2,3-diaminopropionic acid (Dpr), and two unusual amino acid residues, 3 and 4. Amino acid 3 was an analog of Arg with a ketone group between the carboxyl and methane carbons. Amino acid 4 was a Tyr analog with an ethelene unit between the carboxyl and methine carbons. α-Amino group of Dpr was formylated. The sequence of the five residues was deduced by a combination of HMBC and NOESY experiments. Thus, cyclotheonamide A was cyclo[Phe-4-isoD(CHO)pr-Pro-3]. Cyclotheonamide B has an acetamide instead of formylamide in cyclotheonamide A. The stereochemistry of the amino acids except 3 was deduced by chiral GC analysis after degradation. Cyclotheonamide A inhibited thrombin with IC_<50> 0.076 ug/mL.
Conformational analysis of antitumor cyclic hexapeptide, RA series obtained from Rubia akane and R. cordifolia was studied by spectroscopic (NMR, CD, X-ray analysis) and computational chemical methods (molecular dynamics and molecular mechanics calculation). A conbination of different homo- and heteronuclear two-dimensional NMR techniques at 500MHz have enabled us to assign completely the ^1H and ^<13>C signals of the two conformers A and B of RA-VII in CDCl_3. The structures of three conformers (A, B and C) in DMSO-d_6 solution was also derived from 2D-NMR techniques, temperature dependences of NH protons and NOE experiments. Distances deduced from the NMR measurements were used for refinements by restrained molecular dynamics calculations using AMBER program. These conformational analysis found that these conformers were caused by geometrical isomerization and main populated conformer A exhibits a typical type II β-turn structure, which is similar to the crystal structure analyzed by X-ray. N-methyl derivative of RA-VII (RA-VII-NMe) exhibited the more weakly populated conformer A in solution may be responsible for its reduced biological activity in comparison to RA-VII. Further, the presence of highly strained 14-menbered ring was necessitated to maintain the typical type II β-turn structure appeared in conformer A and was considered to play an important part to show an antitumor activity.
In the course of our investigation of the chemical constituents having with the biological activities of the fungus, four new polyacetylene triglycerides (1-4) and three new dibenzocarbazoles (5-7) were isolated from the myxomycetes Lycogala epidendrum and their structures characterized by spectroscopic methods and chemical transformation.
Aureobasidins, new antifungal antibiotics, were isolated from the culture medium of Aureobasidium pullulans No. R106. We isolated 18 of these antibiotics and named them aureobasidins A〜R (1-18). All aureobasidins were cyclic depsipeptides consisting of eight of α-amino acid units and one hydroxy acid unit. The molecular formula of 1 was found to be as C_<50>H_<92>N_8O_<11> by elemental analysis and HRFAB-MS. The amino acids isolated from the acid hydrolysate of 1 were β-hydroxy-N-methylvaline, N-methylvaline, proline, alloisoleucine, N-methylphenylalanine, leucine, and phenylalanine. The hydroxy acid was 2-hydroxy-3-methylpentanoic acid. Mild alkaline hydrolysis of 1 gave 1a and 1b. The acid hydrolysate of 1a gave sarcosine instead of the β-hydroxy-N-methylvaline in the hydrolysate of 1. The sequence of the hydroxy acid and amino acids of 1a, which was a linear chain peptide, was found by NMR and FAB-MS. Compound 1b was found to have 2,3-didehydro-N-methylvaline replacing the β-hydroxy-N-methylvaline in 1. From those results, the structure of 1 was identified. The structures of 2〜18 were also examined.
Antioxidative substances may prevent a variety of diseases caused by active oxygen species. In the course of our screening program for antioxidants of microbial origin, we have isolated three new active substances, named benthocyanins A, B and C, from the cultured broth of an actinomycete identified as Streptomyces prunicolor. The molecular formulas of benthocyanins A, B and C were determined as C_<31>H_<28>N_2O_4, C_<31>H_<28>N_2O_4 and C_<31>H_<29>N_3O_3, respectively, by high resolution FAB-MS. The structure of benthocyanin A was elucidated by using a variety of two-dimensional NMR techniques and confirmed by X-ray crystallographic analysis as shown in Fig. 1. The structures of benthocyanins B and C were also determined as shown in Fig. 1 on the basis of spectral comparison with benthocyanin A and NMR analysis. The structures of the benthocyanins are characterized by a new modified phenazine skelton with geranyl and phenyl residues. Benthocyanins A, B and C inhibited lipid-peroxidation in rat liver microsomes with IC_<50> values of 0.60 μg/ml, 0.07 μg/ml and 0.67 μg/ml, respectively.
The cell wall of plant pathogenic fungi Phytophthora is mainly composed of highly branched β-(1,3)glucan. We realized that this typical cell wall composition should be useful to investigate the inhibitor of β-(1.3)glucan synthesis. The culture filtrates derived from fungi were screened with the objective of detecting any morphological change that might be occurring in hyphae. On the basis of our screening, four strains of fungi, Hemicarpenteles acanthosporus, Hemicarpenteles paradoxus, Bipolaris sp. and Acremonium strictum, were found to produce active compounds which induced highly branched and noded hyphae to P. capsici The active principles isolated from the culture filtrates of H. acanthosporus, H. paradoxus and Bipolaris sp. were identified as (+)-isoepoxydon(2)(minimum dose activity inducing morphological change 100 μg/disc), brefeldin A(2)(50 μg/disc) and ophiobolin A(4)(10 μg/disc) respectively. Accompanied with brefeldin A, 7-oxobrefeldin A(3) was also isolated, however, it only showed growth inhibitory activity. (+)-Isoepoxydon and ophiobolin A showed equivqlent activity of ID50 at a concentration of 50 μg/ml and 25 μg/ml respectively with the regard to inhibition test using UDP-glucose and β-(1,3)glucan synthase prepared from baker's yeast. A yellow coloured compound was isolated from the culture filtrate of A. strictum and its molecular formula was determined by HR-FABMS as C28H32O9. This compound was revealed to be identical to bisvertinolone(6), however, we revised the structure as (5). Using NOE experiment and CD spectrum, we determined the absolute stereochemistry of this compouned as (7). This compound showed strong activity in vivo(10 μg/disc), however, it showed weak inhibitory activity in vitro(400 μg/ml). Therefore, we are now investigating the inhibitory activity to β-(1,6)glucan synthesis.
A phytopathogenic bacterium, Pseudomonas syringae pv. syringae attacks a wide range of hosts including agriculturally important crops. It has been known that the bacteria produced two types of phytotoxins, syringomycin and syringotoxin, depending on the hosts: syringomycins were produced by isolates from many hosts eg. stone fruit, maize and so on, and syringotoxins were obtained by isolates from citrus trees. Those were non-specific toxins and also showed antimicrobial activity. Their structures has been uncertain for about two decades since the first report in 1968. We studied the phytotoxins produced by two strains isolated recently in Japan. First we cultured an isolate from lilac blight (SY 12 strain) and found new toxins termed syringostatins (1). The structures of main components, syringostatins A (1a) and B (1b), were determined by the FAB mass spectra and extensive NMR works using a 600 MHz NMR spectrometer. Furthermore, syringomycins were isolated from the culture broth of a sugar cane isolate (SC1 strain). The structure of syringomycin (2) was also established in a similar way. The semi-purified syringotoxins (3) were kindly provided by Prof. DeVay of Univ. California, Davis. Those were finally purified with HPLC and the main component, syringotoxin B (3b), proved to be an analog of syringostatin A. Those three toxins belong to a family of cyclic lipodepsipeptides. The stereochemistry of the amino acids was determined by the Marfey's method.
Cyanobacteria of Microcystis species have been promptly studied from the environmental, toxicological, biological, and chemical point of views because they are responsible for water blooms frequently producing potent hepatotoxins. During our works on the toxins, we found that an HPLC fraction contained a peptide, which exhibited tyrosinase inhibitory activity. This report describes elucidation of its marvelous structure. The methanol extract of an axenical clonal strain of M. viridis was separated by HPLC to afford a colorless solid, microviridin. Microviridin (1) produced 14 amino acids on hydrolysis. The GCMS analysis using a chiral column indicated that all the amino acids are in an L-form. The structure of 1 was elucidated by spectroscopic analyses including 2D NMR and chemical reactions. Microviridin possesses the novel tricyclic structure containing two ester linkages, which is the first example of tricyclic depsipeptide from natural resources.
In search of new bioactive substances from marine organisms, we have investigated the chemical constituent of the Okinawan marine sponge Theonella sp. and isolated a potent cytotoxic dimeric macrolide, swinholide A (3). The absolute stereostructure of 3, having a dimeric dilactone structure with a 44-membered ring, has been determined on the basis of its chemical behavior and an X-ray crystallographic analysis of its derivative (12). Following the characterization of 3, three new congeneric dimeric macrolides, named swinholide B (14), swinholide C (15), and isoswinholide A (18), have been isolated. The absolute stereo-structures of these dimeric macrolides (14, 15, and 18) together with misakinolide A (19), which was previously isolated from another Okinawan marine sponge of Theonella sp., have been elucidated. These dimeric macrolides (3, 14, and 15) exhibited potent cytotoxic activities for KB cell lines (IC_<50> 0.04, 0.04, and 0.05 μg/ml, respectively) while 18 showed a weaker cytotoxicity (IC_<50> 1.1μg/ml). These results suggest that the size and conformation of the lactone-ring are essential for exhibiting cytotoxic activities.
During our studies on bioactive metabolites from Okinawan marine organisms, we have isolated three bromopyrrole alkaloids, ageliferin (1), bromoageliferin (2), and dibromoageliferin (3), as potent actomyosin ATPase activators from the Okinawan marine sponge Agelas sp. Theonelladins A〜D (4〜7) are novel pyridine alkaloids with powerful Ca-releasing activity in sarcoplasmic reticulum from the Okinawan marine sponge Theonella swinhoei. From another Okinawan marine sponge Niphates sp. have been obtained niphatesines A〜D (8〜11), new antineoplastic pyridine alkaloids. Rigidin (12), a novel alkaloid with calmodulin antagonistic activity, has been isolated from the Okinawan marine tunicate Eudistoma cf. rigida. A new antiinflammatory diterpenoid, brianolide (13), has been obtained from the Okinawan gorgonian Briareum sp. The stereostructure of 13 was established by a single crystal X-ray analysis. Amphidinolide E (14), a novel antileukemic 19-membered macrolide has been isolated from the cultured symbiotic dinoflagellate Amphidinium sp. The structures of these bioactive metabolites have been elucidated from spectroscopic data including 2D NMR and chemical means.
Screening of seven species of marine dinoflagellates for antifungal substances led to discovery of highly potent gambieric acid in Gambierdiscus toxicus and amphidinol in Amphidinium klebsii. Amphidinol, which was three times more potent than amphotericin-B, was purified from cultured cells mainly by gel pearmeation chromatography. A part of the culture (20 L) was done with added NaH^<13>co_3. About 4% enrichment of ^<13>C was attained. UV and IR spectra, respectively, indicated the presence of a conjugated trien and a sulfate ester in the molecule. The latter moiety was further confimed by determination of SO_4^<2-> after solvolysis. FAB-MS indicated the molecular weight to be 1488 (C_<73>H_<125>O_<27>SNa). The major part of the structure was deduced by a combined use of 2D-NMR, such as ^1H-^1H COSY, ^1H-^1H HOHAHA, PS-NOESY and PS C-H COSY. Proton signals on a linear hydrocarbon chain with six double bonds (C54-C69) and a long carbon chain with three hydorxyls (C6-C17) were unassignable due to heavy overlapping and had to be solved by long range ^<13>C-^1H COSY and ^<13>C-^1H HOHAHA. With the ^<13>C-enriched preparation, measurements were successful with only 3 mg. As the ^<13>C signals in the repeating moieties were too congested to be correlated with ^1H signals, these ^<13>C observing methods were more informative to establish these parts of the sturcutre than were ^1H observing methods (HMQC, HMBC). Detailed analyses of ^<13>C-^1H 2D-NMR data thus enabled us to deduce the whole structure of amphidinol as shown in Fig. 3.
Melithasterols A-D (1-4), isolated from a gorgoinan coral Melithaea ocracea of Okinawa, were shown to be cholestane-, 24-methyl-22-dehydrocholestane-, 22-dehydrocholestane-, and 24-methylenecholestane-derivatives. respectively, having a common 3β, 7α -dihydroxy-5α, 6α-epoxy-Δ^8 steroid nucleus. This was confirmed by direct comparison with the authentic sample prepared by lead tetra-acetate oxydation of cholest-6-ene- 3β, 5α, 8α-triol 3-monoacetate. Twelve new polyhydroxysterols were isolated from an Alcyonium sp. (7,8,9), and Sclerophytum sp. (14,16,19b,20,21,22a,23,26,28) soft corals collected off the coasts of the Andaman and Nicobar Islands in the Indian Ocean. All the Sclerophytum sp. soft corals contained 17a or its 25-deacetyl derivative. Compounds 7 to 9 were identified as 24-methylenecholest-5-ene- 3β, 16β-diol-3-0-α-L fucoside (7) and its 7β-(8) and 7α-hydroxy (9) derivatives. Compound 14 was shown to be 24S-24-methylcholest-5-ene-3β,25ξ,26-triol and was correlated to the known compound 15 by 5α, 6β-glycolation. Compound 16 was shown to be 5β, 6α-isomer of 17b, correlating to 17b by PCC oxidation followed by dehydration. Compound 19b was identified with 25-deacetyl derivative of the known compound lobosterol (19a) by hydrolysis of authentic lobosterol. Compounds 20 and 21 were shown to be 7-dehydro-(20) and 22E-dehydro-(21) derivative of 17a and 17b, respectively, by comparisons of their spectral data with reference compounds. Compounds 22a and 23 were 24S-methylcholestane-3β, 5α 25-triol-6-one 25-monoacetate (22a) and its 25-deacetoxyl derivative (23). Partial PCC oxidation of 17a afforded 22a. Compounds 26 (andamansterol) and 28 (nicobarsterol) were shown to be gorgost-5-ene-3β, 9α. 11α, 21-tetrol and novel secosteroid, 11,21-cyclo-B-homo-11-oxa-9, 11-secoergostane-3β, 6α, 12ξ-triol-9-one, respectively, by spectral analyses (H-HCOSY, HMQC, HMBC).
On a course of chemotaxonomical study of Polypodiaceous ferns, we have investigated triterpenoid components of three Microsorium species, M. brachylepis (Baker) Nakaike (collected in Okinawa), M. normale Ching and M. punctatum Copel. (both in Taiwan). More than 50 triterpenoids were detected from the three species along with new triterpenoids described below. Triterpenoid acids: From M. brachylepis and M. normale, fern-7-, fern-8-, fern-9(11)-en-28-oic acid (1-3) and adian-5-en-28-oic acid (4) were isolated as their methyl esters (1a-4a). These compounds are the first example of C-28 oxygenated migrated hopane triterpenoids. Compound 1-4 were not detected from M. punctatum. Cycloartanoids: Cycloartanoid fatty acid ester fraction of M. normale was subjected to HPLC [C_<18>, 5μ, CH_3CN-CHCl_3 (7:3)]. 31-norcyclomargenyl palmitate (5b) and linoleate (5c), and 30,31-bisnorcyclomargenyl palmitate (6b) and linoleate (6c) were isolated. Their structures were established by comparisons of spectral data with related compounds. Free alcohol (5) and its acetate (5a) were also isolated from M. normale and M. punctatum. C_<31>-Triterpenoid hydrocarbons:: C_<31>-Triterpenoid hydrocarbons: 21-methyl-bacchara-12,22(29)-diene (7) and 21-methyldammara-18(28),22(29)-diene (8) were isolated only from the type species of this genus, M. punctatum. The structure of 7 was established by HMBC 2D NMR spectoscopy. The structure of 8 was also established by spectral data. The isolation of C_<31>-triterpenoid hydrocarbon from a natural source is the first example. The occurrence of C_<31> triterpenoid hydrocarbons only in M. punctatum and the absence of 1-4 in M. punctatum is of chemotaxonomic interest.
Three new pentacyclic triterpenes named hancokinol (1), hancolupenone (2) and hancolupenol (3) have been isolated from cynanchum hancokianum, which is distributed Inner Mongolia and is known as a chinese folk medicine possessing antitumor activities. These structure have been elucidated by means of spectroscopic and X-ray analyses. Assignments of all protons and carbons have been made by DEPT, ^1H-^1H, ^<13>C-^1H COSY experiments Stereochemistry have been determined by NOE experiments. These structure and stereochemistry were coincided with the result of X-ray analyses. Absolute structures were determined by CD spectra comparing with the data of related compounds. Hancokinol (1) has a new triterpenoid skelton and is the first example of triterpenoid having a B/C cis ring juncture. Hancolupenone (2) is also a new pentacyclic triterpene derived from lupane-type skelton. Hancolupenol was deduced to be 3-dihydrohancolupenone by chemical transformation from (2) to (3).
Quassinoids, bitter principles of Simaroubaceae plants have been extensively investigated from the interest in structure determination and their useful biological activities. Particulary, the recent studies of this family have received renewed attention because of antimalarial and antitumor activities shown by some quassinoids. In previously, we have reported the isolation and determination of ten novel des-4-methylated picrasane type quassinoids, named javanicins A-J and quassinoids glucosides, named javanicinosides A-C, from the barks, leaves and woods of Indonesian Picrasma javanica B1. further investigation led to the isolation of nine new same type quassinoids, named javanicins K, L, N, O, P, Q, R, S and T along with known quassinoids, quassin, neoquassin, nigakilactones B, F and G, isolated from the leaves and woods of the same plant. In conclusion, about 150 quassinoids have so far been isolated from Simaroubaceae plants. Although the number and the positions of methyl groups are the same on their basic skeletons and all quassinoids so far known have a methyl group at C-4, these javanicins and javanicinosides lack the methyl group at C-4. Most of the numerous quassinoids known have the C 20 basic skeleton, our study is the first report on the isolation and structure determination of the des-4-methylated quassinoids.
ACT-toxins I and II are host-specific toxins produced by Alternaria alternata tangerine pathotype causing citrus brow n spot disease in USA. Very interestingly, both toxins are toxic to Japanese pear. Their chemical structures were determined to be 1 and 2 containing the same trienoic acid moietywith AK and AF-toxins produced by A. alternata Japanese pear pathotype and strawberry pathotype, respectively. The structures of ACT-toxins are very similar with those of AK and AF-toxins but also with those of ACR-toxins, produced by A. alternata rough lemon pathotype. Furthermore, outline of biosynthetic pathway of ACT-toxins were determined by isolation and structure determination of the related compounds (9, 10 and 12) from culture filtrates of the ACT-toxin producer.
In our screening program for phospholipase C (PLC) inhibitors from microbial origin, we isolated a novel PLC inhibitor, Ro 09-1450 from the culture broth of Penicillium vinaceum Gilman & Abbott NR6815. Ro 09-1450 was purified by EtOAc extraction, precipitation and recrystallization from EtOAc. The molecular formula of Ro 09-1450 was determined to be C_<28>H_<16>O_<14> by elemental analysis, NMR and mass spectrometry. The structure of Ro 09-1450 was determined to be 1 by 2D-NMR spectroscopy (INADEQUATE, HMBC and COLOC) and chemical derivatization. Ro 09-1450 showed selective inhibitory activity against PLCs from rat brain, murine colon 26 adenocarcinoma and murine fibroblastsNTH3T3 with IC_<50>'s of 5.4μM, 9.3μM and 44μM respectively.
Our study on neurotrophic substances in the metanol extract of a medicinal plant, Magnolia obovata THUNB. led to the isolation of a number of novel sesquiterpene-neolignans named eudesobovatols A (1) and B (2), eudeshonokiols A (3) and B (4), clovanemagnolol (5), and caryolanemagnolol (6). These new compounds belong to unique class of natural products made up of sesquiterpene and biphenyl-type neolignan via an ether bond. The structures of eudesobovatols A (1) and B (2), eudeshonokiols A (3) and B (4) were elucidated on the basis of the extensive spectroscopic analyses and confirmed by identifying (+)-γ-eudesmol as terpene units, and obovatol and honokiol corresponding to respective aromatic units produced on treatment of CF_3COOH. Clovanemagnolol (5) was clarified to have the structure consisted of tricyclic sesquiterpene clovanediol and magnolol linked through an ether bond by 2D-NMR, in particular, HMBC and MS spectra and its tentative structure was established by a biomimetic synthesis starting from caryophyllene β-oxide and magnolol, whereas caryolanemagnolol was assigned as the structure of 2 based on a combination of various 2D-NMR methods and is most likely to be biosynthesized from caryophyllene α-oxide and magnolol. Compound 1, 5, and 6 exhibited interesting neurotrophic properties on neuronal cell culture system of fetal rat cerebral hemisphere, which not only accelerate neurite sprouting but also enhance choline acetyltransferase activity at 10^<-5>〜10^<-7>M.
The molecular weight and the structure of thermally labile and non-volatile organic compounds can be analyzed by fast atom bombardment mass spectrometry (FAB-MS). However, the FAB spectra display variuos features according to the structure of sample and a liquid matrix used. In this report, a few fundamental problems observed in the FAB spectra of phenolic compounds, isolated from the root bark of the mulberry tree and related plants, are solved. First problem, i) why the FAB spectra give the fragment peaks coinciding with the corresponding EI and/or CI spectra, is solved with a tandem MS (MS/MS), and it is proved that the M^<+・> and (M+H)^+ ion produced under FAB conditions results in an EI-like and a CI-like fragmentation, respectively. Second problem, ii) why the FAB spectra give a number of fragment peaks, despite the FAB is a soft ionization method, is solved by presenting a method which evaluates the internal energy of the M^<+・> ion produced under FAB conditions. Using the method, it is revealed that the energy of the M^<+・> ion sometimes corresponds to that of the M^<+・> ion produced at the electron impact energy of 70eV or above. Third problem; iii) why the FAB spectra of the complex natural products such as glycosides and oligosaccharides frequently give the intense peaks of (M+C)^+ (C=NH_4, Na, K,..) ions, is discussed. This phenomenon can be explained by assuming the formation of a coodination complex M…C^+ in the matrix solution. The assumption is supported from the ^<13>C-NMR spectral data of sucrose (5).
In the course of our isoflavonoid research in Leguminosae, an unprecedented amino-substituted isoflavone was isolated from the root bark of Piscidia erythrina L. The root bark of P. erythrina collected in Mexico was extracted with methanol and the extracts were fractionated by Si gel column chromatography. A series of preparative TLC made it possible to isolate piscerythramine (1). The reaction of 1 with Ehrlich's reagent to yield a yellow pigment on Si gel thin layer plates suggested the presence of an amino group in the molecule. The FD-MS spectrum showed the molecular ion at m/z 451, and EI-HR-MS (M^+ 451.1997) revealed the molecular formula to be C_<26>H_<29>NO_6. ^1H-NMR, UV and EI-MS indicated 1 to be a 5,7-dihydroxylated isoflavone with two prenyl groups on the B-ring. The structure was established as 4'-amino-5,7,3'-trihydroxy-5'-methoxy-2',6'-bis(3,3-dimethylallyl)isoflavone from the results of the extensive NOE experiments and of methylation reaction. This is the first report on naturally occurring amino-flavonoids. In this connection, we revised the structure of a diprenyl isoflavone reported as 6'-prenylpiscerythrone (5,7,2',4'-tetra-hydroxy-5'-methoxy-3',6'-bis(3,3-dimethylallyl)isoflavone), which was one of the major isoflavone found in P. erythrina, to be 5,7,3',4'-tetrahydroxy-5'-methoxy-2',6'-bis(3,3-dimethylallyl)-isoflavone.
In addition to previously reported dimeric hydrolyzable tannins, woodfordins A, B, C and oenothein B, three new trimeric and a tetrameric hydrolyzable tannins (woodfordins D, E, oenothein A, and woodfordin F) have been isolated from an Indonesian crude drug called "Sidowaya" [dried flowers of Woodfordia fruticosa (Lythraceae)]. Oenothein A was also isolated from Oenothera biennis (Onagraceae). They were found to have macrocyclic structures, like oenothein B (1) and woodfordin C (2). Their structures, 4, 14, 5, and 15, having a woodfordinoyl group as one of the structural units connecting monomers, were established based on the spectroscopic analyses and chemical degradation. The host-mediated anti-tumor activity of various types of oligomeric hydrolyzable tannins was also examined. Among the screened oligomeric hydrolyzable tannins, which can be structurally classified into four types, only the oligomers composed of tellimagrandin I (12) and its galloyl analog, such as oenothein B and woodfordin C, exhibited potent activity.
Formerly, We discovered that the crude extract of an Ascomycete, Neosartorya fischeri, caused severe tremor and peritonitis in mice and proved that the tremorgenic principles of the fungus were identical with the neurotropic mycotoxins, fumitremorgins A and B from a Fungus imperfecti, Aspergillus fumigatus. Very recently, we have isolated from N. fischeri a new fatally toxic metabolite tentatively named NFA which causes peritonitis in mice. NFA (1), pale yellow powder, C_<23>H_<29>NO_7, [α]D^<26>-65.0°, is an acidic compound. Physico-chemical properties and spectral data including detailed ^1H- and ^<13>C-NMR data of 1 and its derivatives, 2,3,4,5 and 6 have suggested that 1 is composed of four moieties, a, b, c and d. Construction of the structure of 1 from the four moieties has been attained on the basis of HMBC- and COLOC-NMR data. The structure of NFA has been proposed to be 1. Comparison of the^H- and ^<13>C-NMR data of 1 with those of some known fungal metabolites, 7, 8 and 9, which have the common 2(1H)-pyridone skeleton, has supported that 1 is a new member of the fungal metabolites containing 1,4-dihydroxy-2(1H)-pyridone moiety. Biogenesis of 1 has been also proposed in the similar manner to the biosynthesis of 9.
A chromatographically unseparable mixture of two C_<22>-terpenoid hydrocarbons, juniperene (1) and isojuniperene (2), has been found to occur in the leaf wax of Juniperus chinensis L. cv. 'Kaizuka'. Their structures were determined by analyzing the mixture on the basis of NMR spectroscopy including 2D-INADEQUATE method and by degradating it into a racemic dicarboxylic acid 10. Furthermore, the syntheses of (-)-1 and (-)-2 from natural (-)-elemol demonstrated that the absolute stereostructures of juniperene and isojuniperene should be represented by (-)-1 and (+)-2, respectively. Although the proposed structures does not conform to the "isoprene rule", the compounds may be regarded as abnormal terpenoids which were biosynthesized by an enzymatic non-concerted [4+2]-cycloaddition reaction between pre-isogeijerene and β-myrcene and subsequent stereoselective Cope rearrangement, or by the stereospecific cycloaddition reaction between (±)-isogeijerene and β-myrcene, as shown in Fig. 5.
We have reported on a convenient method, an advanced Mosher method, to elucidate the absolute configurations of secondary alcohols. This method was based on the analyses of the difference of the chemical shifts (^1H) between the (+) and (-)-MTPA esters of the alcohols. This method has been successfully applied to elucidation of the absolute configurations of marine diterpenes 1, 6-9. However, in the case of a marine triterpene, sipholenol-A (10a), this method could not be applied. The main reason of this anomaly is that the MTPA group occupies the sterically crowded axial position. This problem was surmounted by conversion of 10a into episipholenol-A (11a), in which the hydroxy group exists in sterically freer equatorial position. Application of this method to 11a led to elucidation of its absolute configuration as well as that of 10a shown in the respective structures. This result agreed with that determined by the X-ray crystallography of the (-)-MTPA ester of 10a. The influence of the steric hinderence on the conformation of the MTPA moiety, which is essential to the advanced Mosher method was studied by applying this method to the model compounds, 4, 12-14, the absolute configurations of which are known.
A strain of Basidiomycetes sp. has been to produce glycosidic antibiotics glykenin (GK), which exhibit inhibitory activity against gram-positive bacteria. GK complex are composed of two major components (GK-III and IV) and five minor components (GK-I, II, V〜VII) on the silica gel TLC. Alkaline hydrolysis of GK complex gave deacetyl derivative DG-A〜C, which are basic structures of GK. DG-A〜C are composed of three unusual hydroxylated C_<26> long-chain fatty acids (1a〜c) as aglycones and trisaccharide, respectively. The location of the trisaccharide chain was determined by permethylation, methanolysis and PCC oxidation of aglycone 1a〜c The HPLC analyses of peracetyl phenacyl esters of GK-II〜IV and VI, showed three peaks which were coincided with those of the peracetyl phenacyl esters of DG-A〜C. Major components GK-III and IV, separated by repeated silica gel chromatography, have molecular weight 970 that was indicated the presence of two acetyl groups on the structures of DG-A〜C. The positions of two acetyl groups in GK-III and IV were determined by the fragmentation analysis of negative SIMS spectra and by DQF ^1H-COSY, Relayed COSY and 2-Relayed COSY techniques and their structures were determined as shown in Fig. 5.
Acyclic monoterpene primary alcohol: NADP^+ oxidoreductase, the key enzyme in the biosynthesis of monoterpene alcohols and iridoids in plants, is unstable and has been only poorly characterized. However we have established the conditions to stabilize the enzyme from Rauwolfia serpentina cells, followed by purification of it to homogeneity. It is a monomer with a molecular weight of about 44,000 and contains zinc ions. Various branched - chain allylic primary alcohols such as nerol (6), geraniol (7) and 10-hydroxygeraniol (1) were good substrates, but ethanol was inert. Menthol (16), cyclic monoterpene secondary alcohol, was also inert as a substrate, but inhibited the enzyme competitively. The enzyme exclusively requires NADP^+ or NADPH as the cofactor. Steady - state kinetic studies showed that the dehydrogenation proceeds by an ordered Bi - Bi mechanism. NADP^+ binds the enzyme first and then NADPH is the second product released from it. Neral, 10-hydroxygeranial (2) and 10-oxogeraniol (3) were enzymatically reduced in the presence of stereospecifically deuterium - labelled NADPH's. Capillary gas chromatography - mass spectrometric analysis of the products showed that the enzyme exclusively transfers the pro-R hydrogen at C-4 of NADPH.
We have studied on the constituents and the stereochemical aspects of lupin alkaloids in differentiated plants and undifferentiated tissue culture of Thermopsis species in Leguminous plants. A new alkaloid, (+)-lupanine N-oxide (14), have been isolated from T. lupinoides with nine known alkaloids (11-15, 17, 18, 21-23) (scheme 2). Two new alkaloids, (-)-O-acetylbabtifoline (19) and (-)-tetrahydrocytisine (24), have been isolated from T. chinensis with nine known alkaloids (11,12,16,17,18,20-23). The structure of new alkaloids were determined by spectroscopic methods and by chemical transformations. We have also studied on the production of alkaloids with callus culture of T. lupinoides and Sophora flavescens. The differentiated plants of T. lupinoides and T. chinensis accumulated (+)-lupanine (11)(7S:9S:11S) series alkaloids together with the antipodal (-)-anagyrine (17)(7R:9R:11R) series. It is interesting from the biosynthetic point of view that the sparteine-type alkaloids (11-14) and the α-pyridone-type alkaloids (16-23) have opposite absolute configuration with each other. However, (+)-lupanine (11)(6R:7S:9S:11S) was the only detectable alkaloid in the green callus of T. lupinoides. In the green callus of S. flavescens, matrine was the alkaloid mainly. The multiple shoots of S. flavescens additionally produced not only matrine but also 5,6-dehydrolupanine (16) and anagyrine (17). These results suggest that the biosynthesis of (+)-lupanine (11) and matrine, both of which are assumed to be the early intermediates in biosynthesis of lupin alkaloids, are related to greening of the tissues in Thermopsis and Sophora (scheme 1).
We have recently isolated and characterized several phytoalexins from cruciferous vegetables: Chinese cabbage, cabbage, and Japanese radish. These phytoalexins(1〜13) are synthesized de novo under stress conditions and structurally unique in possessing an indole or oxindole nucleus linked to a sulfur-containing moiety. Structural features suggest close biosyntheic relationships between these phytoalexins and indole glucosinolates(15〜17) which are known as biologically-active secondary metabolites in cruciferous plants. Time-course studies of cruciferous phytoalexins in UV-irradiated turnip root tissue suggested that brassinin(1) would be transformed into spirobrassinin(6) via cyclobrassinin(4). Feeding experiments with D-labeled brassinin(1) indicated that both cyclobrassinin(4) and spirobrassinin(6) were derived from brassinin(1). However, no incorporation of D-labeled cyclobrassinin(4) or dioxibrassinin(5) into spirobrassinin(6) was observed. Feeding experiments with D-labeled amino acids showed that brassinin(1) was biosynthesized from tryptophan and methionine. Feeding experiment with DL-tryptophan-α-^<13>C revealed the increase of ^<13>C content of the imino carbon atom in 6, suggesting that molecular rearrangement, such as Lossen type, would be involved in a biosynthetic pathway to brassinin(1).
Stereochemistry and mechanism in the chain elongation of E-prenyl chain in the biosynthesis of geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) were investigated. (i) A chain-length-specific GPP synthase was detected in leaves of Pelargonium roseum; GPP synthase was separated from FPP synthase by hydrophobic chromatography on Butyl-Toyopearl. This separation demonstrates that there are two specific synthase, GPP synthase and FPP synthase, in higher plant. Biosynthesis of GPP with the GPP synthase was found to involve the addition of dimethylallyl diphosphate to the double bond of isopentenyl diphosphate (IPP) from its si-si face and then the syn elimination of the C(2)-H of IPP. (ii) Evidence for the generation of geranyl cation intermediate during the enzymatic condensation of GPP to IPP with the FPP synthase from pumpkin was given by monitoring the enzymatic reaction by means of ^<13>C NMR measurements. This is direct evidence to approve the generation of carbocation intermediate during the enzymatic prenylation, and supports the involvement of the "stepwise" mechanism in the prenylation.
A pathogenic fungus of apple trees. Alternaria alternata (AKI-3 strain) produced two cyclic peptides alternariolide 1 (AM-Toxin I) and tentoxin 2. 1 is known as a host-specific toxin which caused brown necrotic spots on trees of susceptible cultivar. 2 is also known a potent phytotoxin against several plant including apple trees. Now the fungi were grown with ^<14>C-labelled amino acids (U-L-Ser, U-L-Ala, U-L-Phe, U-Gly) in Richard's medium for 2 weeks at 28℃. Both the peptides 1 and 2 were isolated and purified by silica gel TLC. Incorporation of each amino acid into 1 (6.5×10^<-5>〜1.9×10^<-3>μCi/μmole) and 2 (7.9×10^<-5>〜2.8×10^<-3>μCi/μmole) was confirmed. From the radioactivities of components separated by paper chromatography of the hydrolysis of 1, metabolic precursors of dehydroalanine and an aromatic amino acid moieties in 1 may be considered to be serine and phenylalanine, respectively.
In early studies on the seed germination inhibitors from a Hibiscus, we have isolated some new fatty acid methyl esters. In continuation of our studies, we isolated three cyclopropene compounds, methyl sterculate (1), malvalate (2) and 2-hydroxysterculate (7), as the active constituents along with two new compounds, 5 and 6. As malvalate (2) may be derived from sterculate (1) by a biological α-oxidation mechanism, 2-hydroxysterculate (7) would be an intermediate. The absolute configulation of the asymmetric carbon atom (C-2) of the compound 7 was determined as R by using a dibenzoate chilarity methode on the 1,2-diol derivative. We also found out a new ring opening oxidation of these cyclopropenoids by air to give a pair of conjugated enones, 3-A and 3-B, 4-A and 4-B, and 8-A and 8-B. From this air oxidation reaction and ozonolysis of the cyclopropenoids, we proposed a biogenetic pathway on the compounds isolated from Hibiscus rosa-sinensis.
Thermal degradation of the flavonoid glycosides and hydrothermolysis of the triterpenoid-, steroid-, steroidal alkaloid-, cardenolide-, and flavonoid glycosides were studied. The latter cleavage reaction was applied to the structure determination of new triterpenoid glycosides and gangliosides. The useful products for structure elucidation and FD mass fragmentation analysis were obtained by means of thermal degradation of flavonoid glycosides. Hydrothermolysis of the above mentioned glycosides also afforded useful products, namely genuine aglycones, prosapogenins and oligosaccharides, which were not obtained easily by another methods. Furthermore, selective cleavage reaction of ester type glycosidic linkages using hydrothermolysis could be found out. The structures of sugar moiety of a lanostane-type triterpenoid pentaglycoside (67) isolated from the sea cucumber Pentacta australis, a gypsogenin 3,28-O-bisglycoside (69) from Gypsophila paniculata, and a ganglioside molecular species (75) from the starfish Asterias amurensis versicolor Sladen were characterized with the assistance of the effective cleavage reaction, hydrothermolysis.
The chirality of the secondary carbon atom attached to the alcohol group can be decided with high sensitivity by derivation into a 1,1'-binaphthyl-2,2'-diyl phosphate. The secondary alcohol and 1,1'-binaphthyl-2,2'-diyl phosphoryl chloride ((BNP)Cl) with the same chirality react preferentially over that with different chirality. The result is characteristically confirmed by the CD spectrum. since the binaphthol moiety causes a Davydov splitting with a very strong intensity. The chirality recognition ability of the BNP reagent is not affected by ring size, neighboring functional groups, or the configuration of the hydroxyl group, and especially exhibited with acyclic alcohols, as well as the cyclic compounds. Chirality of the asymmetric carbon atom attached to the hydroxy methylene group can be also determined with high sensitivity by derivation into the (R,S)-BNP ester. The BNP derivatives are not consistent with the sign of their CD spectra and the chirality of the asymmetric carbon atom. The inconsistent result disappears with the introduction of a verso sequence rule. The concept is that, in a bond formation reaction, the hydroxy methylene group on a reaction axis is excluded from the priority, and the three other ligands on the chiral carbon atom are put into a priority sequence and reassigned the novel chiral label (R) or (S). The sign of the (R,S)-BNP derivatives consequently accords with the verso chirality of the asymmetric carbon atom.
An efficient general method for the construction of chiral α-acetylene alcohols has been developed. The new method consists of the Katsuki-Sharpless asymmetric epoxidation of allylic alcohol substrates, conversion of the chiral epoxides into the corresponding chloro-epoxides, and brief exposure of the latter products with an excess n-butyllithium in tetrahydrofuran. The reaction leads to the formation of the corresponding terminal α-acetylene alcohols readily in excellent yields without affecting original chiral integrities introduced in the asymmetric epoxidation reaction. Employing the new chiral α-acetylene alcohol formation reaction as the key step as well as using a chiral acetylene alcohol thus generated as a common building block, some isoprenoid natural products ranging from mevalonolactone to α-tocopherol have been synthesized in an enantiocontrolled sequence of reactions. Namely, a single acetylene alcohol obtained has been sequentially elongated via a monoterpenoid citronellal, a sesquiterpenoid perhydrofarnesol, and a diterpenoid phytol by iterative application of the key reaction to complete a linear synthesis of α-tocopherol. On the other hand, the same acetylene alcohol has been separately transformed into the two known segments, chromanethanol and the perhydronor-farnesol, to complete a convergent synthesis of α-tocopherol. During these transformations, an isoprenoid biogenetic precursor mevalonolactone, olfactive monoterpenoids, linalool, cis- and trans-rose oxides, and an irregular phenolic monoterpenoid bakuchiol have been synthesized from the same acetylene alcohol in enantiocontrolled ways.
The cleavage reaction of cyclopropane ring is one of the useful method in organic synthesis. Especially the sequence of cyclopropanation-homoconjugate addition is one of the accessible methods for the synthesis of natural products possessing ring- and side-chain contiguous chiral centers. This report deals with the synthetic studies of biologically active natural products possessing such chiral centers by using the ring opening reaction of double activated cyclopropanes. Cyclopropane derivatives (3-8) have been synthesized from methyl acetoacetate by alkylation, diazo transfer followed by copper-mediated cyclopropanation. The stereochemical course of homoconjugate addition to an activated cyclopropane has been elucidated. The opening reactions proceed with inversion of absolute configuration at the apical carbon of the cyclopropane. The various adducts (16,22,25,31,44 and 46) were obtained by homoconjugate addition of nucleophiles (Gilmann reagent, Grignard reagents, cyanide anion, methanol etc.) to cyclopropane derivatives. The adducts were converted effectively into the synthetic intermediate of steroids (21 and 27), iridoid monoterpenes (30,36,37 and 38), the synthetic intermediate of thienamycin (42) and epiinvictolide (53) by routine functional group transformation.
Halimedatrial, a structurally unique marine diterpene, was reported as the chemical defence adaptation in the calcareous reef-building algae Halimeda (Udoteaceae)^1. This diterpene shows potent antimicrobial activities toward a variety of marine microorganisms and also a highly inhibitory effect on the growth of a marine bacterium and a gray fungus. The structure of halimedatrial has been elucidated by NMR analysis and chemical reactions, except the absolute configuration. Herein, we wish to describe the total synthesis of 1, (+)-halimedatrial, by an enantiospecific manner starting from (S)-4-hydroxy-2-cyclopentenone. This synthesis involves stereoselective formation of the cyclopropane ring system and construction of the diformylcyclopentene moiety as crucial steps. Formation of the cyclopropane ring system was achieved by the following reactions: i) stereoselective 1,3-dipolar addition reaction of 15, prepared from (S)-4-hydroxy-2-cyclopentenone (3) via 4,5 and 11, with diazomethane to give 16, and ii) photosensitized degradation of the pyrazoline moiety to give cyclopropane derivative 17 as a major product. Lactone 17 was then converted into 24 by elongation of the side chain in 17. Construction of the diformylcyclopentene moiety was successfully carried out by stereoselective photocycloaddition of 24 with vinylene carbonate followed by oxidative cleavage of the resulting cyclobutane ring system to give (+)-halimedatrial (1). The NMR spectrum of 1 was identical with that for natural halimedatrial, though the optical rotation of 1 observed as [α]D^<25> +73.9°(c=0.28, CHCl_3) was contrary to that of the natural one, [α]D^<25> -59°(c=0.9, CHCl3). The synthesis of the antipodal (+)-halimedatrial (1) established the absolute structure of natural halimedatrial as shown in 2.