Antioxidants were widely used for food protection, and some of them are also for the defense of biological molecules against oxidative damage. However, synthetic antioxidants are suspected to be carcinogenic and toxic, and use of the natural antioxidants is desired. In the course of our investigation on natural antioxidants, we focused on the oil cake. Oil plants should have antioxidants in their seeds to prevent their oil from oxidative damage, and the antioxidants from oil cakes would develop their utilities and should improve ecological problems in oil industry. Here we report isolation and identification of antioxidants (1-11) from safflower and cotton oil cake. The dried safflower oil cake was extracted with hot MeOH. After the partition of the extract, the EtOAc-soluble fraction was separated by repeated SiO_2 column chromatography and HPLC to yield the seven serotonin derivatives, 1 (322mg), 2 (371mg), 3 (12.4mg), 4 (18.1mg), 5 (4.0mg) 6 (396mg) and 7 (184mg), respectively. The dried cotton oil cake was extracted with MeOH. The EtOAC-soluble was separated by the similar method as isolation of 1-7 to yield compounds 8 (5.0mg), 9 (5.7mg), 10 (6,3mg) and 11 (7.0mg). Structures of 1-11 was established mainly by detailed analysis of the various NMR spectra. Their antioxidative activity of 1-11 were determined by the ferric thiocyanate method and the DPPH radical scavenging activity assay. In these tests, compounds 1-5, 9, 10 and 11 were stronger antioxidative activity than the commonly used natural antioxidant (α-tocopherol) and comparable to synthetic antioxidant (BHA). Thus these compounds were supposed to play an important role in prevention of oil oxidation in the plants and could become food additive.

抄録全体を表示

Planar structures of the five yellow coloring matters of safflower (Carthamus tinctorius L.) have recently been elucidated. In our continuing search for these compounds, the absolute structures of these five coloring matters(2〜6) were investigated. The compounds 2〜6 have a common asymmetric carbon at C* position in their molecule and the compounds 4 and 5 have another asymmetric carbon at C** position (scheme 1). Each of these coloring matters gave no single crystals for X-ray crystallographic analysis. A cid hydrolysis and subsequent methylation of 4 and 5 yielded the chalcone type derivatives 8a(+66°) and 8b(-66°) as a viscous oil, respectively. On the other hand, two enantiomers, 8R(+61°) and 8S(-75°), were synthesized by the method described in scheme 2. The spectral data of 8a and 8b were completely identical with those of synthetic 8R and 8S, respectively. This result confirmed that the absolute configuration of C** were R in 4 and Sin 5. The X-ray crystallographic analysis of heptaacetate of 4 showed that the absolute configuration of 4 at C* position is S, but the heptaacetate of 5 gave no single crystal. So, two model compounds 17a(C*=S) and 17b(C*=R) were synthesized by the method described in scheme 3. The absolute configuration of C* of 5 was elucidated as S by the comparison of its CD spectrum with those of 4 and synthetic 17a and 17b. The absolute configurations of C* of the other yellow coloring matters 2, 3 and 6 were also elucidated as S by the investigation of their CD spectra.

抄録全体を表示

DNA topoisomerase II can relax supercoiled DNA and resolve knotted or catenated DNA rings. Therefore, functional significance of the enzyme seems to be involved in proliferative processes such as DNA replication, chromosome condensation, and chromosome segregation. By above mentioned reason, topoisomerase II inhibitors are noted as cellular targets of anticancer and antimicrobial agents. In the course of our screening program of topoisomerase II inhibitors, we isolated many inhibitory polyphenols (sixty-two compounds) together with five new compounds from the plants belonging to the Guttiterae, the Leguminosae, the Cyperaceae, the Cupressaceae, and the Berberidaceae, respectively. These structures were characterized by a combination of various NMR technique including COSY, HMBC and NOESY, and chemical evidences. From kobresia neparensis, a stilbene tetramer (2) and two stilbene trimers (1 and 3) were isolated as new resveratrol oligomer, in addition two new lignans, 4 from thujopsis dolabrata and 5 from mahonia japonica. After inhibitory screening of isolated polyphenols against topoisomerase II, sixteen compounds were found to show significant inhibitory activity. Particularly, a xanthone 6 and five stilbene oligomers 1, 2, 3, 8, 9, 10, 11, and 13 exhibited potent inhibitory activity at less than 1μg/ml concentration (MIC).

抄録全体を表示

In the course of our search for new neurotrophic factor-like substance and superoxide anion scavenger in natural product, we have studied chemical constituents in the wood of Garcinia subelliptica belonging to Guttiferae. Phytochemical data indicate that Garcinia species are rich in xanthones with various biological activities, and also the methanol extract of G. subelliptica exhibits antioxidant activity. Extensive studies on chemical components in the EtOAc soluble portion of title plant resulted in the isolation of fourteen new xanthones 1〜14, two new benzophenones 15 and 16, and six new phloroglucin derivatives 23〜28 named subellinone and garsubellins A〜E with a tetrahydrofuran ring in addition to the known xanthones. Their structures have been elucidated mainly on the basis of srectroscopic data and confirmed by converting them into the corresponding known compounds. In this paper, the structure of garsubellin A (24) are solely discussed in detail. All the compounds isolated at present study were tested for their antioxidant properties using three in vitro assay systems, viz., antilipidperoxidation activity in rat brain homogenates, free radical scavenging activity of α,α-diphenyl-β-picrylhydrazyl radical and superoxide anion scavenging activity in the xanthine-xanthine oxidase system. The results are summarized in Table 2. In particular, 1,4,5-trihydroxyxanthone (10) and symphoxanthone (21) could scavenge approximately 80% of superoxide anion at 5μgml_<-1>. Assay results imply that antioxidative xanthones should have a catechol part or a 1,4-hydroquinone moiety as a common structural unit. Further, garciniaxanthone B (2) was found to enhance not only neurite outgrowth but also choline acetyltransferase activity (ChAT) at 10μM in primary culture of rat cerebral hemisphere 10 days after seeding, whereas garsubellin A (24) could increase ChAT activity by 54% at 10μM in comparison with a control culture containing 0.5% EtOH in culture of P 10 rat septal neurons.

抄録全体を表示

Glycosphingolipids (GSLs) are contained in the cell membranes, and have recently been implicated in many physiological functions. Gangliosides, sialic acid-containing GSLs, are especially enriched in brains and nervous tissues, and are involved in the regulation of many cellular events. Recently, a number of GSLs have been obtained from the marine invertebrates such as echinoderms, poriferans, and mollusks. We have also been researching the biologically active GSLs from the echinoderms for elucidating the structure-function relationships of GSLs, and for developing the novel medicinal resources. In this time, we conducted the isolation and characterization of biologically active GSLs from the sea cucumber Holothuria pervicax, and could obtain three cerebroside molecular species, HPC-1, HPC-2, HPC-3 from less polar fraction of the CHCl_3/MeOH extract of the whole bodies of H. pervicax, and three ganglioside molecular species, HPG-1, HPG-3, HPG-8 from the water-soluble lipid fraction. The structure of the GSLs were characterized on the basis of spectral (^1H-NMR, ^<13>C-NMR, ^1H-^1H COSY. HSQC, and positive ion FAB mass) and chemical (methanolysis and methylation analysis) evidences. Namely, HPC-1, HPC-2, and HPC-3 are sphingosine-type cerebroside, sphingosine-type cerebroside with 2-hydroxy fatty acid, and phytosphingosine-type cerebroside composed of 2-hydroxy fatty acid, respectively. On the other hand, HPG-1, HPG-3, and HPG-8 are ganglioside molecular species derived from the phytosphingosine-type cerebroside HPC-3. The sugar moieties of them are unique in respect of that the sialic acid directly bind to glucose of cerebroside, and that HPG-1 and HPG-3 possess tandem-type disialosyl oligosaccharide moieties. The gangliosides HPG-1, HPG-3, and HPG-8 shows neuritogenic activities toward the rat pheochromocytoma cell line PC12 cell at 10μg/ml in vitro.

抄録全体を表示

For the determination of 3D molecular structure and absolute configuration of polysaccharide compounds, an X-ray crystallography method is the most powerful technique. However, the method can not be used if a compound does not crystallize. A general technique for determination of the stereo structure and stereochemistry is the use of chemical reaction to decompose into small fragments for relating with the known compounds. If both methods can not work, or to determine stereo structures in solution, the NMR data and stereo structure calculation by computer can be used effectively. Its appearance some years ago, that is an epoch-making method of analyzing the 3D molecular structure of natural organic compounds in solution, can be used as an important method. This calculation algorithm can be classified into the two categories. The first is the method to use the Descartes coordinates system for describing 3D positions of atoms in a molecule, is generally used. The second is the DADAS (Distance Analysis in Dihedral Angle Space) method, using the dihedral angle coordinates system. This is used in the MolSkop as the DADAS90 program for calculations of molecular structures, using the distance constraints from the NMR data. It is simply rotating the defined chemical bond as the variable, with fixed the bond lengths, bond angles. The DADAS90 can determine stable stereo molecular structures of polysaccharides from the aspects of conformers. The new technique, used by computer, with the MolSkop system and the sugar structure library newly developed, has been applied for steric positioning analysis of oligosaccharide.

抄録全体を表示

Kibdelosporangium chichiharuensis was isolated from soil in Chichiharu, China. During screening of antimicrobial activity against Bacillus subtilis, five 14-membered macrolides (1)〜(5) were found from the cultured broth of Kibdelosporangium chichiharuensis. The macrolides (1)〜(5) were purified from broth filtrate by Diaion HP-20, silica gel chromatography and HPLC. The planar structure of (1) was determined by two-dimensional NMR techniques and CI-MS. The planar structure of (2)〜(5) were determined by comparison of NMR and MS spectral data of (1). As a result, (1) and (3)〜(5) were found to be newly isolated 14-membered macrolides. The planar structure of (2) was identified with 23672 RP that was reported by Arnoux et al. Pharmacological evaluation revealed that (1) possessed higher antimicrobial activities than erythromycin A in vivo, although in vitro activities were lower. Thus (1) is a good lead compound for investigation of macrolide for further potent antibiotics.

抄録全体を表示

In the course of screening for new antitumor secondary microbial metabolites, we have isolated four novel triene-ansamycins, cytotrienins A (1), B (2), C (3), and D (4), which possess a unique 1-amino-1-cyclopropane carboxylic acid moiety, from the fermentation broth of Streptomyces sp. isolated from a soil sample through a separation procedure guided by the apoptosis-inducing activity in vitro. Both 1 and 2 exhibited a potent apoptosis-inducing activity on HL-60 cells (a human promyelocytic leukemia cell line). Molecular formulas of 1, 2, 3, and 4 were determined to be C_<37>H_<48>N_2O_8, C_<37>H_<50>N_2O_8, C_<37>H_<46>N_2O_8, and C_<37>H_<48>N_2O_8 from HR-FABMS spectral data, respectively. Structures of 1, 2, 3, and 4 were elucidated on the basis of spectroscopic data, especially by detailed analyses of their _^1H and ^<13>C NMR spectra with aid of 2D NMR spectroscopy including FG-DQFCOSY, FG-HMQC, and FG-HMBC techniques as shown in Fig. 1 and Fig. 3. This is the first example in a series of triene-ansamycin antibiotics, demonstrating that the compounds involve a 1-amino-1-cyclopropane carboxylic acid unit, such as cytotrienins A(1)〜D(4). Cytotrienins are clearly different from the triene-ansamycin antibiotics possessing D-alanine such as mycotrienins at C-11. Compounds 1〜4 were considered to be a series of related biogenetic analogs of mycotrienins. It is interest in attempting to study the biosynthesis of 1〜4, especially for the 1-amino-1-cyclopropane carboxylic acid moiety. Both 1 and 2 induced apoptosis to HL-60 cells with ED_<50> values of 0.005μg/mL. The HL-60 cells treated with 0.01μg/mL of 1 for 16 hours contained the condensed chromatin and fragmentated nuclei as visualized by staining with Hoechst Dye 33258 (Fig. 4: A〜D). When HL-60 cells were treated with 0.01μ/mL of 1, agarose gel electrophoresis of DNA revealed a "ladder" pattern as shown in Fig. 4(E), indicating preferential DNA degradation at the internucleosomal cleavage of genomic DNA. These data suggest that cell death induced by 1 resulted a programmed cellular respone, apoptosis. Further studies on the biological studies of cytotrienins are in progress.

抄録全体を表示

The characteristic structural feature of polyenemacrolide antibiotics includes a hydrophobic conjugated polyene part and a hydrophilic 1,3-polyol part. The stereochemical determination of such a polyol chain is a difficult task. There are two factors underlying the difficulties; these compounds exhibit poor crystalline properties for X-ray analysis and give a complex overlapping NMR signals which prevent assignment of signals. To date, there are several strategies for the stereochemical determination of these compounds. We now report an efficient stereochemical determination of RK-397 (1) based on combined use of two useful methods; acetonide-^<13>C-NMR analysis developed by Rychnovsky and lactone-^1H-NMR analysis developed by us. RK-397 (1) was converted into tetraacetonide 7. The ^<13>C-NMR spectrum of 7 showed three syn acetonides and one anti acetonide. The relative configurations at C13, 15 and C25, 27 were assigned as syn by analysis of 2D-NMR spectrum of 7. Acetylation of 1 followed by ozonolysis, reduction and acetylation provided 8 and 9. The absolute stereochemistry of 8 was determined as 30S, 31S by converting into diol 10. Decaacetate 9 was converted into acetoxy lactone 15 and unsaturated lactone 16. The ^1H-NMR analysis of both compounds showed 23,25-anti and 21,23-syn relationships. CD spectrum of 16 showed positive Cotton effect, which indicates an R configuration at C23. The ^<13>C-NMR spectrum of tetraacetonide 18, prepared from 16, showed two syn acetonides and one anti acetonide. To determine the relative configuration at C19, 21, lactone 16 was converted into acetoxy lactone 21. The ^1H-NMR analysis of 21 showed 19, 21-anti. configuration. Thus, all chiral centers of RK-397 (1) were assigned efficiently by combined use of acetonide-^<13>C-NMR analysis and lactone-^1H-NMR analysis. Interestingly, the configurations at C19 and C21 of 1, corresponding to 14-desmethyl mycoticin A, are different from those of mycoticin A.

抄録全体を表示

In the course of our screening program for novel antibiotics, we have isolated epoxyquinomicins A (1), B (2), C (3) and D (4) from the culture broth of Amycolatopsis sp. MK299-95F4. 1 and 2 showed weak antimicrobial activity against gram positive bacteria and cytotoxicity at the concentration of 2〜18μg/ml. 3 and 4 showed almost no antimicrobial activity and no cytotoxicity at the concentration of 100μg/ml. On the other hand, these antibiotics exhibited the improvement of collagen induced arthritis in vivo. The fermentation was carried out for 4 days with a medium consisting of glycerol, dextrin, Bacto-soytone, yeast extract and some inorganic salts. Butyl acetate extract of broth supernatant (1.8L) was concentrated, then purified by silica gel column chromatography to give pure 1 (18mg), 2 (19mg), 3 (44mg) and 4 (77mg). The structure of 1 was examined first. The molecular formula C_<14>H_<10>NO_6Cl was determined by HRFAB-MS. The structure of 1 was deduced by various NMR spectral analyses including ^1H-NMR, ^<13>C-NMR, ^1H-^1H COSY, HMQC and HMBC. X-Ray crystallography of 1 supported this structure in addition to absolute stereochemistry. Thus, 1 was determined to be (5R,6S)-2-(3-chloro-2-hydroxybenzoylamino)-5-hydroxymethyl-5,6-epoxy-2-cyclohexene-1,4-dione. 2 was elucidated to be dechlorinated derivative of 1 by comparison of the spectral data of 2 with those of 1. The differences in the molecular formula, ^1H- and ^<13>C-NMR spectra between 1 and 4 suggested that 4 possessed a hydroxyl group at C-1 position instead of keto group in 1. The trans orientation between C-1 hydroxy group and epoxy ring was determined by X-ray analysis. As the same manner, 3 was determined to be dechlorinated derivative of 4. Epoxyquinomicins have unique structures with highly oxidating. So we investigated the total synthesis of these compounds. 9 was prepared from commercially available 3-hydroxy-4-nitrobenzaldehyde in four steps (67% yield). During the reduction of nitro group in 9, salicyloyl group was migrated to give desired amide concomitant with the deprotection of phenolic acetate to afford 10. After deprotection of primary acetyl group, 11 was selectively oxidized with Fremy's salt to give a quinone 12. After epoxidation, the total synthesis of (±)-epoxyquinomicin B was achieved efficiently in 8 steps from 5 (11% total yield). Enantiomeric synthesis is now further investigating.

抄録全体を表示

Norzoanthamine (1), is a zoanthamine-type alkaloid isolated from the colonial zoanthid Zoanthus sp. Norzoanthamines inhibit IL-6 production. Furthermore, norzoanthamine hydrochloride, which suppresses the decrease in bone weight and strength in ovariectomized mice, could be a good candidate for an osteoporotic drug. Although the relative configuration of norzoanthamine was established based on an X-ray crystallographic analysis, the absolute conformation of norzoanthamines, including zoanthamines which have been reported previously, remains unclear. To investigate their biogenesis and mechanism of biologocal action, we conducted chemical and spectroscopic studies to determine the absolute configuration of norzoanthamines. We report here the absolute stereochemistry, a proposed biogenesis and bioactivities of norzoanthamines.

抄録全体を表示

In the course of our continuous search for soft coral metabolites, we have isolated four new butenolidic briaran diterpenes (4-7) from a Briareum species collected in the sea near Satsuma Peninsula. Their broad structural features and relative positions of substituents were deduced from high-field 2D-n.m.r experiments (1H, 1H- and 1H, 13C-COSY, HMBC, and COLOC). The relative configurations for the briaranes were mainly based on NOE data (N.O.e. difference spectroscopy and NOESY experiments). However, because of the flexibility of the ten-membered ring of the briaran the stereochemistries at C3 and C4 remained uncertain. Thus, conformational analysis by molecular mechanics (MM) calculations using MacroModel package were carried out to confirm the stereochemistries at C3 and C4 and also to determine the most stable conformations of the compounds in solution. The coupling constants for the MM-calculated conformers agreed well with the experimentally observed values.

抄録全体を表示

Cyclic heptapeptide hepatotoxins, microcystins, are major toxins of the toxic cyanobacterial waterblooms. Poisonous drinking water by microcystins is one of the most serious problems in the world. However, in natural environment, it is known that a part of microcystins are converted to non-toxic geometric isomers by sun light. During investigations of photo-detoxification of microcystins, we found two new photoreaction products. In this paper, we described the isolation and the structure elucidation of these photoreaction products. microcystin-LR (1) in aqueous solution was irradiated UV for 30min (0.234 joule) at 0℃. After the irradiation, the photoreaction products were isolated with reverse-phased HPLC using 60% methanol in 50mM phosphate buffer (pH 3.0). In this condition, three products were detected. The slowest-eluted product was identified as [6-(Z)-Adda^5]microcystin-LR (2). The faster-eluted product (3) was obtained as colorless solid. The molecular formula of 3 was the same as that of microcystin-LR. However, The UV spectrum of 3 was quite different from that of microcystin-LR. On the ^1H-NMR spectrum, aromatic ring was disappeared. The extensive NMR analyses of COSY, HMBC, and ROESY spectra revealed the structure of the tricyclo-Adda (3-amino-5-4',6'-dimethyl-3'-methoxy-tricyclo[5,4,0,0^<1',5'>]-undeca-8',10'-dien-6'-yl-2-methyl-4-(E)-pentenoic acid) moiety (Fig. 2.). Another amino acid units were not changed. From these results, the structure of 3 was determined as [tricyclo-Adda^5]microcystin-LR. The peak which was slightly slower than microcystin-LR with HPLC analysis, was isolated. The UV spectra of the compound (4) was resembled with that of microcystin-LR. From the HRFABMS spectrum, the molecular formula of 4 was the same as that of microcystin-LR. The extensive analyses of the NMR spectra elucidated the structure of 4 as [4-(Z)-Adda^5]microcystin-LR.

抄録全体を表示

The screening assay method for antimitotic substances, which detect the characteristic deformation (curling effect) on mycelia germinated from conidia of Pyricularia oryzae P-2b, has been applied to cultured broth of filamentous fungi isolated from tropical marine environments. A new antimitotic compound which showed the characteristic curling effect on mycelia, has been obtained from two strains of filamentous fungi, TUF 95F47 and TUF 95F143, isolated from submerged (-3m) mangrove branches in coral reef of Pohnpei. TUF 95F47 was identified as Phomopsis sp., and TUF 95F143, resembled F47, is under identification. Each fungus was cultured in Potato Dextrose medium (half nutrients, 50% sea water) at 20℃ for 3 weeks, and the broth was adjusted pH to 6 with 2% HCl, added acetone and extracted with ethyl acetate. The organic extract was separated by silica gel column chromatography (CHCl_3-H_2O=1: 1) and HPLC (ODS, CH_3CN-H_2O=1: 1) to give the bioactive compound named phomopsidin. The structure was assigned based on the ^1H and ^<13>C NMR, ^1H-^1H COSY, HMQC, HMBC and NOESY data to be a cis-decaline derivative possessing a conjugated diene carboxylic acid side chain. The compound would be biosynthesized via biological Diels-Alder reaction. Phomopsidin inhibited the assembly of microtubule proteins, purified from porcine brain, at IC_<50> of 5.7μM.

抄録全体を表示

A new NMR methodology was developed for configurational assignments in acyclic structures of natural products. Unlike a conventional method based on NOE data, this methodology is applicable to stereochemical analysis even in the case multiple conformers are present since it is based on the dihedral angle dependency of long-range carbon-proton coupling constants (^<2,3>J_<C,H>)(Fig. 1). In this methodology, the combination of the coupling constants which are categorized to Large, Medium, or Small allows one to determine the relative stereochemistry among adjacent asymmetric centers or asymmetric centers separated by a methylene (Figs. 2, 3, and 4). The combined use of hetero half-filtered TOCSY (HETLOC) and phase-sensitive HMBC (PS-HMBC) experiment was shown to be effective for measuring ^<2'3>J_<C,H> values of complicated natural products (Figs. 6 and 7). Amphidinol 3 (AM3, 1), which was isolated from the dinoflagellate Amphidinium klebsii, was subjected to this methodology. To facilitate the measurements of ^<2'3>J_<C,H> of AM3, a ^<13>C-enriched sample was prepared by culturing the dinoflagellates in a medium containing 0.01% NaH^<13>CO_3. The cells obtained from 200L of the medium yielded 8mg of AM3, which was enriched with ^<13>C at ca. 25%. With this sample, 2D INEPT-INADEQUATE, which directly provides the information on C-C connectivity, was measured to confirm the proposed planar structure of AM3 and to assign the ^<13>C chemical signals unambiguously. Then HETLOC, PS-HMBC, and their variants were measured, and the coupling constants thus obtained, partly coupled with NOE data, led to the stereochemical assignments for the C20-C27 and C32-C51 portions of AM3 as shown in Fig. 5.

抄録全体を表示

DNA topoisomerase I (topo I) is a nuclear enzyme which controls the topological state of DNA and is recognized as a target for anticancer drugs. In the course of our study on new topo I inhibitors of marine origin, several active lipids were isolated from an Australian sponge Amphimedon sp. and the Japanese Bryozoa Watersipora cucullata. From the hexane soluble portion of Amphimedon sp. C_<27>-C_<30> long chain fatty acids 1-8 were isolated. Among them amphimic acids A(1)-C(3) are unusual fatty acids that contain a cyclopropylidene moiety. The structures of these fatty acids were determined by spectroscopic methods (NMR, IR, HRFABMS, linked scan MS/MS etc.) and GC-MS analysis of ozonolysis products. The enantioselective synthesis of 1 was carried out to determine the absolute configuration. On the other hand, from the butanol soluble portion of W. cucullata novel ceramide 1-sulfates 22 and 23 were isolated as potent topo I inhibitors. The gross structures were determined by spectroscopic analysis, and their absolute stereochemistry was elucidated from the optical data ([α]_D, CD) of degradation products.

抄録全体を表示

We have already reported that phenylglycine methyl ester (PGME) is a convenient tool to determine the absolute configuration of the chiral tertiary carboxylic acids. This new method was analogous to "Modified Mosher's Method": (R) and (S)-PGME are condensed with the chiral tertiary carboxylic acids, the NMR signals of the protons of both diastereomers are assigned, and Δδ value of each proton is calculated by the equation Δδ=δ(S)-diastereomer δ(R)-diastereomer. The protons with +Δδ and -Δδ are placed on the right and left sides of the model [B] (see text), respectively, which indicate the correct absolute configuration of the carboxylic acids. In the present study, the PGME method was extended to the chiral quaternary carboxylic acids that possess an oxy-function such as hydroxy, alkoxy, and acyloxy groups at the α-position of the carboxyl group. The linear compounds examined in this study are (i) α-hydroxycarboxylic acids [1-hydroxy-1-methylbutanoic acid and its homologues and 1-ethyl-1-hydroxypentanoic acid], (ii) α-methoxycarboxylic acids [1-methoxy-1-methylbutanoic acid and its homologues], and (iii) a-acetoxycarboxylic acids [1-acetoxy-1-methylbutanoic acid and its homologues]. The + and-Δδ values obtained from the (R) and (S)-PGME amides [Δδ=δ(S)-diastereomer-δ(R)-diastereomer] are systematically arranged to show the correct absolute configurations without exception. The present method was verified by applying it to the cyclic compounds, chlorogenic acid, (1S)-(-)-camphanic acid (1R, 3R, 4R, 5R)-(-)-quinic acid, sclareol, and (S)-Trolox^[○!R], the absolute configurations of which are known. The configurations indicated by the PGME method were all identical with the known ones.

抄録全体を表示

We reported here that the structures including absolute stereochemistry of W493 A (1) and B (2) were elucidated by a combination of spectral analysis and enantioselective synthesis of 3-hydroxy-4-methyltetradecanoic acid (3). In the course of a screening for antifungal substances by the bioassay observing hyphal swelling of C. Miyabeanus, 1 and 2 were isolated from the culture broth of Fusarium sp. by CHCl_3 extraction and various chromatographies. The molecular formula of 1 and 2 were determined as C_<45>H_<74>O_<11>N_7 and C_<44>H_<72>O_<11>N_7, respectively from HRFAB-MS. The IR, ^1H, and ^<13>C NMR spectra were suggested the presence of a peptide structure, and their planar structures containing 3 as a component were determined by amino acid analysis and DQFCOSY, HMQC, HMBC experiments. Consequently, the structure of 2 was identified to be a cycloheptadepsipeptide, cyclo(3-D-allo-Thr-L-Ala-D-Ala-L-Gln-D-Tyr-L-Ile). W493 A was identified with the structure of 1 that was replaced the isoluesine residue in 2 with valine (Fig. 1). The NMR assignments for 1 and 2 were shown in Table 1 and 2. The absolute configuration of each amino acid residue was determined by chiral HPLC. The sequence of the two succesive alanines was determined on the analysis of a partial acid hydrolysate of 2 by LC-ESI-MS and chiral HPLC (Fig. 2, 3). To obtain four isomers of 3, we employed Sharpless asymmetric epoxidation. Tridec-2-yn-1-ol (6), which was prepared from 4 and 5, was converted to the epoxy alcohol 8 through partial hydrogenation and asymmetric epoxidation, 8 was successively submitted to epoxy cleavage with Me_3Al, tosylation, substitution of cyanide ion, and hydrolysis of the nitrile to give 3a. On the other hand, for inversion of the C-3 configuration, 1,2-diol 9 was converted to epoxide 13 through a three-step reaction sequence. The epoxide 13 was successively submitted to epoxy cleavage of cyanide ion and hydrolysis of the nitrile to give 3b (Scheme 1). According to essentialy the same procedure, 3c and 3d were synthesized from the enantiomer of 8. The five HMTAs (3) from hydrolysis of 2 and synthesis were derivatized to Mosher esters using 2NMA, and their NMR spectra were compaired one another. Since the spectra of the natural and 3b were identical, the absolute configuration of HMTA in 2 was determined to be 3S and 4R. The structures of 1 and 2 were reported only in 1985 as the components of the mixture, and also the stereochemistry of 3 containing in these compounds had not determined yet. These results led to the conclusion that we determined the total structure of 2, containing (3S,4R)-3-hydroxy-4-methyltetradecanoic acid (3b), which was shown in Fig. 1.

抄録全体を表示

In the continuous studies of protease inhibitors of microalgae, we have found that the cyanobacterium Microcystis aeruginosa is a new and rich source of unique biologically active compounds. Here we report the isolation and structure elucidation of novel biologically active peptides of M. aeruginosa. The 80% methanol extract of lyophilized alga (M. aeruginosa NIES-299) was subjected to solvent partition, ODS column chromatography followed by ODS HPLC to obtain microginin-type peptides 1-4. Their structures were determined by FABMS and 2D NMR spectra. The absolute stereochemistries of usual and N-Me amino acids were determined to be all L-form by the HPLC analysis of the Marfey derivatives. The absolute stereochemistry of their β-amino acids was deduced by a combination of spectral and chemical studies. Microginin-type peptides 8-10 were also isolated from the cyanobacterium M. aeruginosa (NIES-90, 478). 1-4 and 10 inhibited leucine aminopeptidase, and 10 also inhibited angiotensin-converting enzyme. The 80% methanol extract of lyophilized alga (M. aeruginosa NIES-88, 90, 478) was subjected to solvent partition, ODS column chromatography followed by ODS HPLC to obtain micropeptin-type peptides 13-21. Their structures were determined by FABMS and 2D NMR spectra. The absolute stereochemistries of usual and N-Me amino acids were determined to be all L-form by the HPLC analysis of the Marfey derivatives or the chiral GC analysis of N-trifluoroacetyl isopopyl ester derivatives. The absolute stereochemistries of the glyceric acid 3-O-sulfate (disulfate) in 13-15 were determined to be all D-form by chrial GC analysis of O-heptafluorobutyryl isopropyl ester derivatives. The absolute stereochemistries of Ahp were deduced as follows. PCC oxidation of 13-15 followed by hydrolysis with HCl afforded Glu, which was proved to be L-form from the HPLC analysis of the Marfey derivatives. The reduction of 16-21 with NaBH_4 followed by hydrolysis with HCl afforded pentahomoserine and Pro, which were proved to be L-form from the HPLC analysis of the Marfey derivatives. The relative stereochemistries of Ahp of 13 and 16 were decided as Fig. 1 and 2 by NOESY correlations. Therefore, the stereochemistries of Ahp were decided to be (3S, 6R)-3-amino-6-hydroxy-2-piperidone. 13-15 inhibited plasmin, and 16, 18-21 inhibited chymotrypsin. The 80% methanol extract of lyophilized alga (M. aeruginosa NIES-88) was subjected to solvent partition, ODS column chromatography followed by ODS HPLC to obtain kawaguchipeptin A (22) and B (23). Their structures were determined by FABMS and 2D NMR spectra. The absolute stereochemistries of usual amino acids were determined to be all L-form besides Leu in 22 by the HPLC analysis of the Marfey derivatives or the chiral GC analysis of N-trifluoroacetyl isopopyl ester derivatives. 22 and 23 inhibited the growth of the gram positive bacterium Staphylococcus aureus.

抄録全体を表示