The analytical and insecticidal assays with the extract of the herbaceous perennial plant, Phryma leptostachya L., have been conducted, including the isolation, structure elucidation and total syntheses of the active plant components, as well as derivation of congeners and analysis of structural requirements for the biological activity. So far, novel insecticidal sesquilignans such as HAEDOXAN A, C-acetate, D, F-acetate, G-acetate, and H-acetate, of which content totally below 5% of the extract, were revealed to be responsible for the peculiar flaccid paralysis followed by the insect death. In association with piperonyl butoxide, haedoxan A showed very high insecticidal activity against housefly as comparable with deltamethrin. The synthetic studies clarified the activity is tightly linked with the absolute configuration (1S, 2R, 5R, 6S, 2"R, 3"R) among the 32 stereoisomers. The molecule could take a conformation as the opened 15-crown-5, suggesting the ability to chelate with metal ions such as Ca^<+2>, Mg^<+2>, and Na^<+1> in the nervous system of insects. The discussions will be focused on the total synthesis and absolute configuration of (+)-haedoxan D as well as the structure-activity relationship concerning with about 50 congeners of haedoxan obtained by the isolation and syntheses. The previous assignment of the absolute configuration of (1S, 2S, 5R, 6S)-(+)-phrymarolin I and (1S, 2S, 5R, 6S,2"R,3"R)-(+)-haedoxan D, depending on the large acetylation shift of 2-H in their molecules, have been revised to (1S, 2R, 5R, 6S) and (1S, 2R, 5R, 6S, 2"R, 3"R), respectively, based on their NOESY spectra.
Four new flavonoids linderatin (1), linderatone (2), methyllinderatone (3), and isolinderatone (4) were isolated from the leaves of L. umbellata var. lancea, L. umbellata, and L. umbellata var. membranacea, and their structures were established by chemical and spectroscopic means. The structure of linderatin (1) was determined by direct comparison with the product which was synthesized by condensation of 2',4',6'-trihydroxydihydrochalcone (6) and piperitol (18). Hydrogenolysis of linderatone (2) with Raney Ni gave linderatin (1) and methylation of linderatone (2) with CH_2N_2 afforded methyllinderatone (3). Therefore, the structures of linderatone and methyllinderatone were represented by the formula 2 and 3, respectively. Finally, isolinderatone was transformed into the dihydrochalcone derivative (15) which was identical with the product derived from rubranine (16) with known stereostructure. Thus, the structure of isolinderatone was established to be 4. These new compounds discussed above were very curious products in which a flavonoid unit is linked with a cyclic monoterpene.
The crude drug 'Brazil ginseng' has been used medicinally in Brazil, chiefly as a tonic, an aphrodisiac and as antidiabetic.1 In the course of our investigation of the constituents of 'Brazil ginseng', we found that at least two kinds of plants, Pfaffia paniculata and P. iresinoides (Amaranthaceae) were used for the preparation of the crude drug. From the former plant, pfaffic acid(1), the hexacyclic nortriterpene and its glycosides possessing cytotoxicity were isolated. While from the latter, a large amount of ecdysterone(2) was isolated together with three new ecdysteroid glycosides and novel yellow pigments, iresinoside A(5) and B(7). These results stimulate our interests in investigating another kinds of plants of this family, P. glomerata, P. pulverulenta and P. tuberosa. All of the plants, except P. paniculata contained 2 as the major component. From P. glomerata and P. pulverulenta, rubrosterone(8)9 regarded as ecdysteroid metabolite was isolated. Four new pfaffane-type nortriterpenes(9-12) were isolated from P. pulverulenta together with 1 and β-D-glucuronopyranosyl pfaffate(13). The structures of the new compounds were elucidated by chemical and extensive spectral analyses. ^<13>C NMR data(Table 3) were fully assigned by using 2D NMR techniques(^1H-^1H COSY, ^<13>C-^1H COSY, long-range ^<13>C-^1H COSY and INADEQUATE).
About 200 alkaloids have been detected from Neotropical poison frogs of the family Dendrobatidae, some of which are used as precious tools for the neuro-pharmacological studies. Recently new cis-decahydroquinoline and 5,8-di-substituted indolizidine alkaloids were isolated from D. auratus and determined their stereochemistry through 1D-HOHAHA nmr studies. A representative cis-decahydroquinoline alkaloid 195A (5) was served as a model for subsequent conformational anlysis of cis-243(1) C_<17>H_<25>N and cis-219(2) C_<15>H <25>N. The conformational analysis through the 1D-HOHAHA studies indicated anti-coprostane(5β)type cis-juncture (Fig.1) and 2-equatorial propyl side chain (Fig.2) which had been established by x-ray crystallo-graphic analysis. For cis-243(1) the results of the 1D-HOHAHA indicated that the conformation of 8aH was axial on the β-ring [coprostane(5β)type cis-juncture](Fig.3).2-Equatorial and 8-axial conformations of the side-chains(Fig.3) were also suggested. Noe between 2H and 8'H, 12'H and 8aH supported the structure (1). In the case of the 5-methyl-8-substituted indolizidine 235B (6), rigorous confirmation of the equatorial 8-methyl group was achieved through 1D-HOHAHA studies. The new sub-classes of indolizidine and decahydroquinoline alkaloids are now presented their structure.
Trichosporins are a linear peptide mixture produced by Trichoderma polysporum whose structures are closely similar to each other. They inhibits the growth of Lentinus edodes. These peptides belong to peptaibols in which the N-terminal is protected by an acetyl group and the C-terminal by a phenylalaninol. Recently, we have isolated trichosporins[TS]-B-IIIb(1), c(2), IVb(3), c(4), d(5) and VIa(6), b(7) and established their primary structures by positive ion FAB-MS spectrometry, respectively. TS-B-IVb,d and VIb are the first examples of isovaline containing peptides isolated from T. polysporum. The connectivities of amino acid residues are established by means of COSY and NOESY NMR techniques. In addition, uncoupling activities of these peptides on rat liver mitochondria were examined.
Toxin production of the marine dinoflagellate Prorocentrum lima was first confirmed in the course of the survey of ciguatera causative organisms. The alga produces two groups of polyethers, one of which comprises okadaic acid and its congeners. In this paper we report the structures of new toxic constituents of the other group, prorocentrolide 1 and its congener 2 from P. lima. P. lima was isolated at Sesoko Island, Okinawa in 1985 and cultured in seawater enriched with an ES-1 nutrient mixture. After culturing at 25℃ for 35 days, the cells were collected by filtration, and extracted with aceton and Me0H. The extract was fractionated into Et_2O, BuOH and water soluble fractions. Okadaic acid and its derivatives were found in the Et_2O layer, while prorocentrolide was obtained in the BuOH layer. The toxin was then purified through columns of silica gel, Toyopearl HW-40, and ODS (MeCN-0.05N HOAc, 1:9-3:7, linear gradient). At the final stage of purification, 1 was separated from 2. The toxin was obtained as a colorless amorphous solid; [α]23/D 136.5°(c 0.147, MeOH); UVmax: 235nm (ε=13600, MeOH); IR (KBr): 3400, 1715, 1670, 1640, 1200, 1060cm^<-1>; FABMS, m/z 980 (M+H)^+; LD_<99> (mice, i.p.), 0.4mg/kg. The toxin induced paralysis in a mouse upon intraperitoneal injection and killed it within 25min. The connectivities of the protons were elucidated mainly by homo and hetero COSY. The linkages around the quaternary carbons (C1/C2, C21/C22, C21/C46 C45/46 and C46/C47) were established by COLOC, HMBC and long range C-H COSY sequences (Fig.2). The position of ether rings were determined on the basis of NOE's (Fig.4) and of coupling constants of oxymethines on each the ring. Prorocentrolide seems to be unique in bearing a hexahydroisoquinoline and a 26-membered aliphatic ring that seem to be the largest among natural products reported so far.
In the course of our search for bioactive metabolites in Japanese marine invertebrates, we encountered a sponge of the genus Mycale collected in Gokasho Bay, Mie, which showed a marked antifungal activity. From the sponge three antifungal substances, named mycalamides A, B and C, have been obtained by the bioassayguided isolation work. Mycalamides are not only antifungal but also cytotoxic against B-16 melanoma with IC_<50> 0.5-1.0ng/mL. The structure elucidation was done by spectral analyses including extensive 500MHz NMR studies as well as by comparison of the spectral data with those of kabiramides, ulapualides and halichondramide. It was disclosed that mycalamides are novel macrolides embracing a trisoxazole unit and a sidechain with an N-methyl formamide terminus, which are closely related to kabiramides and ulapualides. The present study may, provide an evidence tnat the nudibranch macrolides originate from marine sponges which nudibranchs prey on.
Fungal metabolites inducing abnormal hyphal morphology of a phytopathogenic fungus, ex. Cochliobolus lunatus, were surveyed. From this screening, the following metabolites were identified as the active inducer of the hyphal morphology, i.e., lunatoic acid A (1), (-)-mitorubrinic acid (3), dehydroaltenusin (4), sydonol (5), terreic acid (6), mycorrhizin A (9), chloromycorrhizin A (10), pyrenolide A (11). One of the common molecular characteristics of these metabolites is high reactivities to nucleophiles such as SH-compounds. Since the adducts lost the activities, this high reactivity of molecules could relate the activities inducing abnormal hyphal morphology. From this studies a new metabolite, pyrenolide I (12), was isolated from the fungus, Pyrenophora teres, which produces pyrenolide A. The plain structure of pyrenolide I was established by spectroscopic method. Pyrenolide I showed inhibition of the cell (HL-60) (IC_<50> 4ug/ml). Another new metabolites having the activity of inducing abnormal hyphal morphology were isolated from an unidentified fungus. The structure of the one was established as 13 by spectroscopic and chemical methods.
Two new phenolic glycosides, named KS-501(1) and KS-502(2), are inhibitors of calmoduline-dependent cyclic nucleotide phosphodiesterase (IC_<50>=0.99μg/ml and 2.8μg/ml, respectively) isolated from the culture broth of Sporothrix sp. The physico-chemical properties of 1 and 2 are summarized in Table 1. The ^1H and ^<13>C NMR spectra of 1 showed the presence of tri- and tetra-substituted benzene rings, two n-heptyl side chains, two hydoxy groups, one ester carbonyl group and one furanose-type sugar moiety (Table 2). Additional one carboxy group was present on the aromatic ring of 2. Methanolysis of 1 furnished an aglycone (3) and methyl D-galactopyranoside (4). Further methanolysis of 3 provided 3-heptyl resolcinol (6) and methyl 2,4-dihydroxy-6-heptyl benzoate (7), confirming the structure of 3. The location of the sugar moiety in 1 was determined to be at C-2' by the LSPD experiment of 1(Fig. 2) and the NOE experiment of methylate (8) (Fig. 3). The structure of 2 is different from that of 1 with respect to the aglycone moiety. The aglycone (9) of 2 was composed of two molecules of 7. The position of ester and glycosidic linkage was confirmed by the NOE experiment of methylate (10) shown in Fig. 4. The structure of 9 was also confirmed by methanolysis of 10 to furnish two methylate 11 and 12 (Fig. 5).
In the continuation of the previous studies, isolation and structure elucidations of the gangliosides of Acanthaster planci were conducted with the object of searching for the biologically active compound. A water-soluble lipid fraction, which was obtained by fractionation with organic solvents of the chloroform-methanol extract of A. planci, was separated with reversed phase followed by normal phase column chromatography to give two molecular species of ganglioside(AG-2 and AG-3). On thebase of chemical and spectral evidences, AG-2 and AG-3 were characterized as penta- and hexaglycoside of ceramide consisted of heterogeneous phytosphingisine and 2-hydroxy fatty acid as shown in Fig.3 and 5. By means of preparative separation using reversed phase HPLC, five major compounds acanthaganglioside A(1)〜E(5) were isolated in the pure state from AG-2 and AG-3, and they were asigned as shown in Fig.3 and 5. The islation of these pure gangliosides from starfishes is the first one ever reported. Moreover, these sugar moieties is unique in that neuraminic acid is combined with 4-hydroxy group of galactopyranose unit.
As one of our continuous synthetic studies on the spiro cyclic natural products, we describe the synthetic studies on halochamigrane type sesquiterpenes, (±)-nidifocene(1) and (±)- laurencial(2). 1, Synthetic studies on (±)-nidifocene(1) 1-1, Total synthesis of the bromochloro derivatives 3a and 3b The bromochloro derivatives 3a and 3b were regio- and stereo-selectively synthesized via the olefine 7 as a column key intermediate. The regio- and stereoselective introduction of "BrCl" to the double bond of the olefine 7 was achieved via the brocohydrins 17a and 18. 1-2, Synthetic studies on (±)-nidifocene(1) Dehalonidifocene 27 was synthesized starting fro^ the spirodienone 19 by the use of the methodology developped above. 2, Total synthesis of (±)-laurencial(2) and (±)-3-epi-laurencial(29) (±)-Laurencial(2) and (±)-3-epi-laurencial(29) were synthesized via the tricyclic ketone 30 starting from the olefine 32. The construction of the tricyclic skelton of 30 was achieved by the bromonium ion induced cyclization of ketoacetonide 35.
Natural hatching stimuli glycinoeclepins A(1), B(2), and C(3), isolated from dried kidney bean roots, have been confirmed synthetically to have a potent biological activity for the second stage of soybean cyst nematode (Heterodera glycines). Characteristic structures of glycinoeclepins having a common methylene moiety at C19 and various side chains suggest that a cycloartane-type triterpene would be one of the presumable biosynthetic precursors. Accordingly, we chose cycloastragalol(8), isolated from Chinese drug Tragacantha, as a starting material for the biogenetic-type synthesis of glycinoeclepin A analogues. As a model experiment, we could construct the A-ring fragment (7-oxabicyclo[2.2.1]heptane system) of the title target in a quantitative yield starting with a cycloartane(5). As a next stage, cycloastragalol(8) was converted into diene(24) involving the functionalization for the B-ring cleavage as well as formation of the A-ring moiety. Compound 24 was then transformed into the desired product(29), which was formed by a hydride shift from C17 to C16 followed by a methyl migration from C13 to C17 in a backbone rearranged manner. The second methyl migration to C13 from C14 is now under investigation. The structure and biological activity-relationships of the synthetic intermediates would be also discussed briefly.
Synthesis of the alkaloids, 5-hydroxy-6-methoxyonychine (5) (isoursuline) and 6-hydroxy-5-methoxyonychine (6) (ursuline), was accomplished by an application of a synthetic method for constructing cycloalkenopyridines from oxime O-crotyl ethers and the synthesis revealed that the structure of the azafluorenone from Oxandra xylopioides should be revised to 6-hydroxy-5-methoxyonychine from 5-hydroxy-6-methoxyonychine. Furthermore, the synthesis of kinaba-line (7) will be described. During the course of the synthesis of onychines, the chemical shift differences of the signals of C_7-H and C_1, C_7, and C_<7a> on ^1H-and ^<13>C-NMR were available to the stereostructure determination of E- and Z-isomers of oxime O-methyl and O-allyl ethers of indan-1-ones.
Chemical synthesis of myo-inositol phosphates which are involved as metabolites in the intracellular signal transduction system have been accomplished during these two years by several groups. We have continuously made efforts to explore practical syntheses of them. As a result, D-3,6-di-O-benzy1-4,5-di-O-(dibenzyl phosphoryl)-myo-inositol 4 have now been found to be a versatile synthetic intermediate which may be converted conveniently into various inositol phosphates. We wish to report here preparation of optically active 4 and its utility for synthesis of inositol 1,2-cyclic-4,5- 1, 1,4,5- 2, and 2,4,5-trisphosphate 3. There is no report on chemical synthesis of the cyclic derivative 1 which was suggested to have a role as a second messenger. Racemic diol d14 was readily prepared starting from myoinositol and practically resolved by the use of a chiral column (Chiralcel OD). For the first time, optically active 4 was treated with phosphorodichloridate 12 to give quantitatively 1,2-cyclic phosphate 13 which was then deprotected at once by hydrogenolysis to afford 1. Difference in reactivity of equatorially and axially disposed 1,2-cis hydroxyl groups of 4 enabled selective introduction of the phosphoryl function at either position by way of 1-triethylsilyl intermediate 14 leading to Ins(1,4,5)P_3 2 or Ins(2,4,5)P_3 3. In a preliminary experiments aiming at preparation of tritiumlabeled inositol phosphates, LiBH_4-reduction of the ketone 19 derived from 14 by oxidation (DMSO-Ac_2O) was found to proceed stereo-selectively giving 14 as a sole stereoisomer.
Asymmetric synthesis of some lactonic pheromones has been investigated through combination of enzymatic and chemical procedures. Our strategy for biotransformation of organic substrates is the use of immobilized biocatalysts in aqueous solutions and in water-organic solvent two-phase systems. For the synthesis of two enantiomers of phoracantholide I (1), diethyl 3-oxoglutarate was regioselectively alkylated and subsequently hydrolyzed with decarboxylation to give a keto acid. The keto acid was then subjected to an enzymatic reduction with immobilized baker's yeast entrapped in gels of carrageenan to produce the corresponding chiral hydroxy acid. The chiral hydroxy acid was converted to both (R)- and (S)-1 by known chemical procedures. The utility of the present methodology of combined enzymatic and chemical system is also demonstrated by the preparation of each enantiomer of 5-hexadecanolide (2) and 4-dodecanolide (3). As a result of the reuse of the baker's yeast immobilized in carrageenan beads, it has become clear that immobilized baker's yeast shows outstanding reproducibility for optical purities of the product.
Teurilene (1) is a marine triterpene isolated from the red alga Laurencia obtusa. The molecule of this compound is characterized by beautiful arrangement of eight asymmetric carbons in Cs symmetry, and it arouses special interest in its synthesis and conformational properties. We would like to report the first total synthesis of teurilene (1) and a short step synthesis by stereocontrolled double cyclization of a C_<30>-suqualene derivative 12. In the course of the studies on total synthesis of thyrsiferols (2) we found a "rule" of V^<5+> oxidation-cyclization of bishomoallyl alcohol system, i.e. a 5-substituted 4-en-1-ol 4 gave cis-2,5,-disubstituted tetrahydrofuran 6 through syn-epoxide 5, while 4-substituted 4-en-1-ol 7 gave trans-2,5,5-trisubstituted tetrahydrofuran 2 through anti-epoxide 8. This rule was extremely valuable to assemble stetreoselectively 2,4-substituted tetrahydrofuran moieties and usefulness was successfully demonstrated by application to the syntheses.
As one of the synthetic studies on complex carbohydrates, the formal synthesis of D-erythro-C_<18>-sphingosine (2), an essenstial constituent of sphingolipids, from methyl 2,3-O-isopropylidene-D-glycerate(1) are described. First, we studied the Horner-Wittig reaction of phosphonate 4, which was synthesized from dimethyl methylphosphonate and 1, and found the use of caesium carbonate as base in isopropyl alcohol effective to promote this reaction and to give (E)-olefin selectively. In this manner intermediate 6 was obtained in 85% yield. Next, we tried the stereoselective reduction of ketone 6 in tetrahydrofuran using several hydrides and found L-Selectride gave the desired syn-glycerol derivative(7a) in 90% d.e. stereoselectively. Finally, the deprotection of isopropylidene group of 7a and the following benzylidenation in general manner gave the benzylidene glycerol derivative (12), from which the synthesis of 2 have been reported. Thus, the formal synthesis of 2 was completed. Further the phosphonate 4 described herein is expected to be an efficient intermediate of other poly-functional natural products.
In connection with our stereochemical study of sporaviridin, a polyol macrolide antibiotic, we succeeded in developing a convergent and general protocol for syn-1,3-polyol synthesis, which is extendable to higher homologues of this series by repetition. Our synthesis started with the coupling reaction of the chiral building block (9) and the epoxide (10) to give the hydroxy ketone (12) after dethioacetalization. The highly syn-diastereoselective reduction of 12 was achieved using LiAlH_4-LiI, giving a syn-1,3-diol derivative (13). The LiAlH_4-LiI reduction is a useful method for preparing syn-1,3-diols from β-alkoxy and β-alkoxy-β'-hydroxy ketones. The selectivity of this reduction is syn:anti =95:5 (see Table I and II). A second coupling of 17 and 9, followed by dethioacetalization, syn-selective reduction of 19 with LiAlH_4-LiI, and deprotection of 20 gave all-syn-heptol derivative (22). The success of the present convergent synthesis is particulary owed to the developments of a new chiral building block (9) and a highly syn-selective LiAlH_4-LiI reduction method. Applications of this new method were demonstrated in the synthses of the attractant (27) of Ambrosia beetle and (-)- and (+)-tarchonanthuslactone (32), which contain syn-1,3-polyol subunits.
We have established ragioselective synthesis of [^<13>CJALA labeled at 1,2,3,4, and 5 positions (as the important precursors for the biosynthesis of corrinoid and porphyrinoid) by a simple, and complementar method, staring from ^<13>C sodium acetate. We also complete the whole assignment of complicated ^1H-NMR signals of Vitamin B_<12'> by the incorporation of labeled precursors and the 2D-NMR measurements. We isolated and partly purified the enzymes which transform ALA to PBG and Uro'gen I and could observe their time-pass changing by NMR. We have been interested in the origin of hetero atom of vitamin B_<12>. We revealed the origin of proton of corrin ring. The 3, 87, 13, 18, and 19 positioned protons are derived from water in the culture. We also made clear that the amide oxygens of vitamin B_<12> are derived from ALA without exchanging, except that of acetyl group of A ring, which result supports the hypothesis of lactone-intermediate suggested by Eschenmoser. We studied the origin of nitrogen, and could observe the ^<15>N-NMR signals of the nitrogens of pyrrole, nucleotide, and cyano group of vitamin B_<12>. We found that ALA was biosynthesized from glutamate, and glycine is metabolized to methionine, which was transformed into the 10b methyl ester of chlorophyll-a in Euglena gracilis, from a number of feeding experiments. We also confirmed ALA was incorporated into bacteriochlorophyll-a in Rhodo. spheroides.
Optically active Diels-Alder type addcuts have been isolated from the mulberry tree (Moraceae). They have unique structures which are regarded as the adducts of prenylchalcones with dehydroprenylphenols and of two dehydroprenylphenols. The biosynthetic study of the Diels-Alder type adducts was carried out using Morus alba callus cultures. Callus cultures induced from the seedlings and the leaves were subjected to selection, giving rise to cell strains having a high pigments productivity. From the callus cultures, Diels-Alder type adducts, chalcomoracin (1), kuwanons J (2), Q (3), R (4), V (5) and mulberrofuran E (6), were obtained. The biosynthetic pathway of these Diels-Alder type adducts was studied by the administration experiments of [1-^<13>C] and [2-^<13>C] acetates into the compounds. The feeding experiments with the ^<13>C labeled acetates showed high incorporation of ^<13>C atoms into chalcomoracin (1) and kuwanon J (2) (Fig. 3-a,b and 4-a,b). The ^<13>C labeled positions of carbon skeletons in these compounds indicated that both compounds consist of two isoprenylated cinnamoylpolyketide precursors (Fig. 6 and 7) and that the carbon skeleton is not retrotype. Furthermore, it was confirmed that the 2-arylbenzofuran skeleton of 1 was synthesized through the condensation (C-3〜C-8 ring closure) of cinnamoylpolyketide and then decarboxylation (Fig. 5). These results suggest that the all Diels-Alder type adducts from the mulberry are synthesized from two isoprenylated cinnamoylpolyketide precursors.
The concept of tuber forming stimulus of potato has been suggested by grafting experiments. Recently the occurrence of the substances has been evidenced by our experiment using a bioassay of a potato single-node stem segment culture. The object of this study was to isolate and determine the chemical structure of the substance from potato leaves by using of above-mentioned bioassay. The ethanol extracts of potato leaves (100kg) was separated by means of the subsequent liquid chromatographies and HPLCs. Purification processes resulted in 2.7mg of the active compound (1) which was able to induce tuberization in vitro even in lower concentration of 0.01 ppm. The FD mass spectrum showed the molecular ion peak at m/z 388 and that of the acetate at m/z 556. These data with that of GLC suggested that the active compound (1) is a monoglucoside. The PMR spectrum (δ 4.26, d, J=8.0Hz) and CMR spectrum (δ 104.4) suggested the presence of β-D-glucose moiety. EI-HR MS of the acetate (2) showed the molecular formula, C_<26>H_<36>O_<13>. The ^1H-NMR COSY experiment revealed the partial structures, I and II. These data suggested that the active compound (1) was similar to jasmonic acid (3). The chemical structure of the active compound (1) was determined to be 3-oxo-2- (5-β-D-glucopyranosyloxy-2-cis-pentenyl)-cyclopentane-1-acetic acid. A (cis)-double bond and a carboxyl group were essential for the activity. The active compound (1) and/or similar compound(s)(jasmonic acid?) were presented in all kinds of plants.
Dammarane-type triterpene glycosides, named actinostemmoside A, B, C, D, G and H, baccharane-type triterpene glycosides, actinostemmoside E and F, and oleanane-type triterpene glycosides, lobatoside A, B, C, D, E, F, G and H were isolated from the herb of Actinostemma lobatum MAXIM. (Cucurbitaceae). Their structures were elucidated on the basis of the spectral and chemical evidences as shown in the text. The structure of actinostemmoside F was elucidated mainly on the basis of two dimensional-incredible natural abundance double quantum transfer experiment (2D-INADEQUATE) spectrum. Among dammarane-type actinostemmosides, D is the glycoside of the first naturally occurring dammarane having the (20R)-configuration, and actinostemmosides E and F are the second baccharane-type triterpene glycosides isolated from the natural source. Lobatoside B, C, D, E, F and G are cyclic bisdesmosides similar to tubeimoside I isolated from the tuber of Bolbostemma paniculatum (MAXIM.) FRANQUET. (Cucurbitaceae), and this is the second instance of the isolation of cyclic bisdesmoside from the plant kingdom.
Novel acyclic diterpene glycosides, named Capsiansides, were obtained from the part of the polar ingredients in the fresh fruits of Capsicum plants, C.annuum L. var. fasciculatum Irish, C.annuum L. var. conoides Bailey, and C.annuum L. var. grossum Their structures were disclosed mainly by the spectroscopic means (^1H-^1H, ^1H-^<13>C and ^1H-^<13>C long range COSY and NOE NMR experiments). They are classified into two groups, the glycosides (Capsiansides I-V) of the geranyl-linalool derivatives and the ones (Capsiansides A-E) having two acyclic diterpene moieties in the respective molecule. These acyclic diterpenes occur rarely in nature. Capsiansides C and D of them were shown to have inhibitory activity for angiotensin converting enzyme. We have plans to perform various pharmacological tests and to practice cyclization with regard to these compounds by the aid of catalytic acid or enzyme.
From the results of various screening tests, cytotoxicity, Sarcoma 180A, antihistamine and anti-bariumchloride, Zingiberaceous plants were assumed to be containing some kinds of active components. First of all, Curcuwa xanthorrhiza,Indonesian plant, was studied chemically and 8 kinds of bisabolane type sesquiterpenes were isolated. Two sesquiterpenes of then were observed to have strong activity against Sarcoma 180A. Alpinia spp. are widely distributed in Japan and in other tropical Countries. A. japonica was found to contain many kinds of eudesnane, agarofuran, erenophilane type sesquiterpenes. A peroxide, hanalpinol(8), obtained from the sane plant was converted into furopelargone B(13). Moreover, some kitinds of peroxide sesquiterpenes were also isolated from the sane plant, so molecular orbital calculation using MNDO method was adopted to assume the biosynthetical pathway of these peroxides. YakuchinoneA and B were isolated from A. oxyphylla as pungent principles. The former was noticed to be active for the inhibition of prostaglandin biosynthesis. Further, A. internedia, A. officinarun, A. speciosa and A. katsunadai were studied chemically and many components were isolated. Finally,Chemotaxonomical consideration was applied to Alpinia species.
The Dictyotaceae brown algae produce various types of diterpenes. Chromatographic separation of the methanol extract of the seaweeds, Dictyota dichotoma and Pachydictyon coriaceum, afforded five new diterpenes, 3, 5, 6, 8, and 9. The structures of these compounds were elucidated by spectroscopic analyses. The absolute configuration of dictyotin A (3) was established by the CD spectrum of the benzoate derivative, and that of dictyotin B (5) was also determined by the chemical correlation with dilophol (10). Dictyotins have a same carbon skeleton as biflora-4,10(19),15-triene (14), which was isolated from the frontalgland secretion of termite soldiers, but the absolute configuration of dictyotins is opposite to that of 14.
Among Japanese gastropods, the abalone Haliotis discus hannai Ino, one of the herbivorous turban shell, is known to feed preferentially on the brown algae of the family Laminariaceae, such as Eisenia bicyclis ("Arame"), Undaria pinnatifida ("Wakame"), and Laminaria ("Konbu") species.1) The feeding attractants2) and the feeding stimulants3) for young abalone H. discus Reeve have been studied. By contrast, these herbivorous abalones can scarcely be seen in the community of the brown algae Dilophus okamurai ("Fukurin-amiji") and Ecklonia stolonifera ("Tsuru-arame"), thus suggesting that these brown algae contain feeding-deterrent substance against the abalone. A bioassay method4) has been used to examine effects to the settlement and the metamorphosis of the swimming larvae (veliger) of the abalone H. discus hannai. Furthermore, the cellulose plate method3b) has been used to examine feeding deterrents of the young abalone H. discus hannai. From the neutral extract of Dilophus okamurai eight active compounds,1-8 have been isolated. On the other hand, two compounds 10 and 11, the former of which showed a very week activity, have been isolated from the neutral extract of Ecklonia stolonifera.
It is now well established that most marine invertebrates have evolved chemical defense mechanisms that employ biologically active secondary metabolites. In our preliminary screening for biological activities of marine invertebrates, the crude extracts of a Litophyton sp. (Nephtheidae, Alcyonacea, Octocorallia) showed an insect growth inhibitory activity against the silkworm, Bombyx mori L. Monitoring the fractionations of the methanol extract by artificial diet feeding bioassay against silkworm larvae led to the isolation of eleven diterpenoids 1-11, which are responsible to the observed activity, together with two lipid metabolites 12 and 13. Their structures have been characterized by extensive 2D NMR studies, chemical methods, and molecular mechanics calculations. The absolute configuration of these diterpenoids was determined by the CD spectrum of the p-bromobenzoate 15 based on the exciton chirality method of allylic alcohol benzoate. The effective doses (ED_<50>) for 50% growth inhibition of the diterpenoids tested against silkworm larvae ranged from 1.8 to around 100 ppm.
Sarcophytol A (1a) is a simple monohydroxycembratetraene which we previously isolated from Sarcophyton qlaucum, a ubiquitous soft coral found in the coral reefs of Indo-Pacific coastal waters. Recently, Fujiki and co-workers found that 1a and its derivative sarcophytol B (2a) efficiently inhibit the activity of the powerful tumor-promoter teleocidin in a two-stage carcinogenesis experiment on the mouse dorsal skin. In view of this unique activity of 1a and 2a, the lipid extract of S. glaucum was re-investigated and was found to contain ten new cembranoids, sarcophytol M (5), H (11a), O (15), I (17), G (22a), K (23), P (24a), Q (25), N (26a), and J (28a). The structures of these new compounds were elucidated by means of their spectroscopic data, and most of them were confirmed by chemical correlation. The major component 1a was found to give several secondary products on standing at room temperature. The major products were the dihydrofuran derivative 37 and the novel [9.3.0] bicyclic system 32, the latter being known to occur in a soft coral Cespitularia sp. Compounds 2a, 20 and and 22a were found to give similar bicyclic system, in low yields, indicating this reaction to be characteristic of 14-hydroxy-1,3-diunsaturated cembrane system. Their formation process, involving the initial formation of 3,4-epoxy derivative followed by transannular cyclization,is disscussed.
The marine worm order Cephalodiscida (Hemichordata phylum) contains two tubeliving genera occurring primarily at considerable depths in the Southern hemisphere. Initial investigation of bioactive constituents in this worm class (Pterobranchia) led to our discovery of the powerful murine P388 Lymphocytic leukemia (PS system) cell growth inhibitor cephalostatin 1 (1) in the South African (Indian Ocean) Cephalodiscus gitchristi. Subsequently, we isolated and assigned structures to the related disteroidal alkaloids cephalostatins 2(2), 3(3), and 4(4). We now report that further bioassay (PS) guided study of C. gilchristi has led to the isolation of two unusual disteroidal alkaloids bearing aromatic Crings designated cephalostatins 5(5) and 6(6) that retain significant cell growth inhibition (PS ED_<50> 10^<-2>μg/ml). With the X-ray crystal structure assigned cephalostatin 1(1) as a reference point, and the completely interpreted nmr spectra for cephalostatins 1, 2, 3, and 4, it became possible using detailed analysis of high field two-dimensional nmr data and the mass spectra, to unequivocally assign structures 5 and 6 respectively to cephalostatins 5 and 6.
Twenty three diterpenoid alkaloids (1-23) have been isolated from Aconitum japonicum Thunb. and their structures were ditermined on the basis of chemical and spectral evidences. Secojesaconitine (19) has an unusual skeleton, and the structure was determined by X-ray analysis. By treatment with acetic acid and acetylation, 19 gave normal aconitine type alkaloid. Acetyloxy group at C_8 position in the aconitine type alkaloid was readily exchanged for alkoxy group in alcoholic media (16 and 17). Aconitine (4) was transformed into penduline (8) by three steps involving deoxygenation of bridgehead hydroxyl. Analgesic activity of aconitine and its related compounds was investigated by acetic acid-induced writhing method. The acute toxicity of 8-O-methyl-14-benzoylaconine was much lower and the activity was approximately same as morphine's.
Indolactam-V(1), which is a partial structure of tumor promote teleocidins, olivoretins and their derivatives, has a nine membered ring containing an amide bond, and exists in two conformational states in solution, SOFA and TWIST (1:2 in CD_3OD). The conformation of the nine membered ring in indolatams depends on the substituent at C12-position, and determines the relative positions of hetero atoms and orientations of hydrogen bonding whic'h act significant roles on the tumor promoter recepter. The conformations of the nine membered ring of teleocidins and olivoretins determined by X-ray crystallography have been reported to be an alternative of two conformers, SOFA and TWIST. In this paper we report two new conformations of the nine membered ring of indolactams, ie. (±)-indolactam-G(3) and (±)-epi-indolactam-V(2) which have slight or no activity of tumor promotion, determined by X-ray crystallography and NMR spectroscopy. Quantitative analyses of these four conformations by molecular mechanics were performed. To these compounds, we have applied molecular dynamical calculations for the purpose of analyzing the process of ring conformational changes and obtaining the structures of the unknown conformers observed in NMR spectra. We have successflly made clear the favorable path of TWIST-SOFA conversion as well as the equilibrium between several conformers for each compound.
The five pigments (1)-(5) and a colorless substance 6 were present in the dorsal skin of frogs of the nine selected species belonging to Rhacophoridae, Ranidae, Hylidae, and Bufonidae. These pigments were identified as pterin-6-carboxylic acid, xanthopterin, isoxanthopterin, erythro-biopterin, 6-hydroxymethylpterin, and guanine, respectively. Another pigment 7 was specifically present in the skin of genus Rhacophorus and was deduced to be a pteridine derivative composed of five molecules of pterin-6-carboxylic acid. The nine species were found to be divided into six chemotypes on the basis of their pigment composition. Three gangliosides were isolated from the brain tissues of bull frog (Rana gatesbeiana). The structures of these gangliosides were established to be disialosylgangliotetraosyl-ceramide (III^6NeuAcIV^3NeuAc-GgOse_4Cer), trisialosyl-gangliotetraosylceramide (III^6NeuAcIV^3(NeuAc)_2-GgOse_4Cer), and tetrasialosylgangliotetraosylceramide(III^6(NeuAc)_2IV^3(NeuAc)_2-GgOse_4Cer) by phisico-chemical method. Similarity in the dis-tribution of these gangliosides was seen among hemisph, diencephalon, and a mixed tissue of opticlobe, cerebellum, and medulla oblongata of the brain. The brain tissues of Rana nigromaculata and Rhacophorus arboreus also included the same gangliosides as those of bull frog.
"Bidara upas", the tuber of Merremia mammosa CHOIS (Convolvulaceae), is an Indonesian folk medicine which is said to be useful for treating diabetes and affection of the throat and respiratory system. As a part of our investigations of Indonesian medicinal plants, we have examined the chemical constituents of the tuber. We have isolated nine new resin-glycosides named merremosides a, b, c, d, e, f, g, h_1, and h_2, among which the structures of a, b, c, d, f, and g were reported previously where the 11R configuration of jalapinolic acid, the common aglycone of merremosides, was based on the application of Horeau's method. In order to confirm the absolute configuration of jalapinolic acid, we have synthesized jalapinolic acid (16), the 11-epimer (18), and the oligoside (21a) by use of Sharpless epoxidation, and we have found that jalapinolic acid has in fact 11S configuration. Consequently, the structures of merremosides a (1), b (2), c (3), d (4), f (5), and g (6) have been formulated as shown. Furthermore, based on the chemical and physicochemical evidence, the structures of merremosides e (19), h_1 (27), and h_2 (28) have been determined. Merremosides are macrocyclic lactone oligosides. It has been found that merremoside a (1) exhibits ion-transport activity for K^+, Na^+, and Ca^<++>ions through artificial membrane, whereas all merremosides have been shown to transport Ca^<++> ion by the human erythrocyte method. Merremosides b (2) and d (4) have been found to exhibit anti-serotonic activity.
Spores of many fungi in dense population, either in pastules or in suspension, germinate poorly or not at all. In some cases this is due to spore dormancy, but more commonly it is caused by substances present in or produced by spores which inhibit their own gemination. This active priciple is designated as spore germination self-inhibitor. Self-inhibitor from uredospores of oat rust fungus, Puccinia coronata f. sp. avenae, was identified as methyl cis-3,4-dimethoxycinnamate. The ED_<50> value of the cis-form of the inhibitor was 12.5pg/ml, whereas the trans-isomer had little or no activity. Irradiation of light induces isomerization from cis- to trans-form. For prevention of the isomerization methyl 3,4-dimethoxybenzalmalonate which has the symmetrical charactor was selected and synthesized. This compound expressed the same level of the activity of the natural ones. The self-inhibitors of Colletotrichum spiecies were also investigated. Their structures were demonstrated to be (E)- and (Z)-ethylidene-1,3-dihydroindol-2-one and (2S)-(3-indolyl)-propionic acid. These are the different compounds from gloeosporone which was isolated from the same strain of the fungus as its spore germination self-inhibitor.
Recently, a variety of novel antineoplastic compounds have been isolated from Okinawan tunicates and sponges. Pseudodistomins A(1) and B(2), potent antineoplastic piperidine alkaloids with calmodulin antagonistic activity, have been isolated from the Okinawan tunicate Pseudodistoma kanoko. Cystodytins A(7), B(8) and C(9) are novel tetracyclic aromatic alkaloids with potent antineoplastic activity and powerful Ca-releasing activity in sarcoplasmic reticulum isolated from the Okinawan tunicate Cystodytes dellechiajei. From another Okinawan tunicate Didemnum sp. has been obtained a novel pentacyclic aromatic alkaloid, ascididemin(10), with potent antileukemic activity. Cystodytins and ascididemin might be originated from symbiotic microorganisms. Prianosins A(12), B(13), C(14) and D(15), novel sulfur-containing polycyclic alkaloids with potent antineoplastic activity have been isolated from the Okinawan marine sponge Prianos melanos. A novel lanosterol-derived metabolite, penasterol(18), with potent antileukemic activity has been obtained from the Okinawan marine sponge Penares sp. This is the first isolation of a marine sterol with a 14-carboxy group potentially important within the framework of sterol biosynthesis. Metachromins A(19) and B(20) are a novel antineoplastic sesquiterpenoid quinone and a chromenol isolated from the Okinawan marine sponge HilSpospongia cf. metachromia. The structures of these tunicate and sponge metabolites have been elucidated from spectroscopic data including 2D NMR and chemical means.
The bioluminescence of dinoflagellates involves air-oxidation of luciferin (enzyme substrate) by luciferase (enzyme). On the other hand, Euphausia krills utilize highly fluorescent substance F not only as the catalyst for air-oxidation of a protein but also as the light-emitter. Fluorescent substance F exhibits chemical properties similar to those of dinoflagellate luciferin. Using alumina and ion exchange chromatography at low temperature under inert atmosphere (Sheme 1), the substance F (1) was successfully isolated from Euphausia pacifica. The structure of F was elucidated on the basis of degradation reaction summarized in Fig. 1 as well as the spectroscopic data of F (1) and oxy-F (2). The ring D part of the proposed structure, including relative stereochemistry, was unambiguously established by chemical means; ozonolysis of F, followed by CH_2N_2 treatment, yielded the expected product 7, the structure of which was determined by chemical synthesis. Dinoflagellate luciferin could be isolated from the dinoflagellate Pyrocystis lunula (Scheme 2). The structures of luciferin 8, oxidized luciferin 9 and blue compound 10 were elucidated by comparing their spectroscopic data with those of fluorescent substance F and oxy-F. Dinoflagellate luciferin and krill fluorescent substance F are apparently a member of the bile pigments. To the best of our knowledge, however, these are the first naturally occurring bile pigments, which structurally relate to chlorophylls rather than to haems. Studies on the mechanism of dinoflagellate bioluminescence is in progress.
Countercurrent chromatography (CCC) is a unique form of liquid-liquid partition chromatography that eliminates the need for a solid supporting matrix. Centrifugal devises, which comprise the majority of CCC apparatus, retain effectively the stationary phase in a helical column by gravitational or centrifugal force. Especially, high speed CCC (HSCCC) which has been extensively developed by Ito, one of the authors, is useful in the preparative-scale separation. In the present study, optimization of several parameters, selection of appropriate two-phase solvent system, stationary phase retention, revolution speed and flow rate of mobile phase, was carried out for obtaining satisfactory results. Finally HSCCC was successfully applied to the preparative-scale separation of components of sporaviridins (Fig. 1) and bacitracins (Fig. 2) under the optimized conditions. These results clearly suggest that HSCCC is promising for preparative separation of natural products and a combination of HSCCC and HPLC would greatly contribute to this field.
1)Four new hydrolyzable tannins, liquidambin (3), isorugosin A (12), isorugosin B (9) and isorugosin D (13), were isolated from the leaves of Liquidambar formosana. 2) Liquidambin (3), which could be a biogenetic precursor of C-glucosidic tannins, was isolated as an equilibrium mixture. The equilibration was found to be due to hydration of the aldehyde group at C-1 of the glucose residue in 3. 3) Isorugosin D (13), biogenetically producible from two molecules of tellimagrandin II (17), had a valoneoyl group of the orientation different from that of rugosin D (6). Isorugosins A (12) and B (9), monomeric tannins from the same plant, were isomers of rugosins A (4) and B (5) concerning the orientation of valoneoyl group. 4) Rugosins A (4), B (5) and D (6) were absent in L. formosana, and isorugosins A (12), B (9) and D (13) have not been found in Rosa rugosa and Coriaria japonica, which contain 4, 5 and 6. The C-O oxidative coupling in the formation of valoneoyl group in L. formosana, therefore will have been effected by an enzyme different from that of R. rugosa and C. japonica. 5) Cornusiin A, a major component of the fruits of Cornus officinalis, should be formulated as 19, on the basis of the chemical conversion of 19 to isorugosin B (9). Structures of camptothins A and B, which were isolated from the leaves of Camptotheca acuminata, and that of cornusiin C from the Cornus and Camptotheca species, were determined to be 20, 21 and 22, respectively.
Duocarmycin A, C_1 and C_2 produced by Streptomyces sp. are novel antitumor antibiotics which are effective against murine lymphocytic leukemia p388 and murine Sarcoma 180 in mice. In this report, we describe the structural determination of the Duocarmycins (Fig. 1). The ^1H NMR spectrum of Duocarmycin C_1 (1) revealed the presence of three aromatic protons, four methoxyl groups, one tert. methyl group, two methylene groups, one methine proton and three protons which disappeared in the presence of D_2O (Table 2). The observation of ^1H-^<13>C long range couplings through COLOC spectrum of 1 exhibited three partial structures (I, II and III) shown in Fig. 2. The attachment of the amino group between C-2 and C-9a, and of the phenolic OH group to C-9 were also established by COLOC experiment of a permethylate 4, where the long range couplings from N-methyl and O-methyl protons were observed (Fig. 3). Treatment of 1 with triethylamine in methanol at room temperature in the presence of Ag_2CO_3 produced a quinoline derivative 6, indicating the presence of nitrogen linked to C-6 and C-7a. The attachment of the chlorine to C-5 was confirmed by alkaline treatment of 4 producing a dehydrochloride 5. The partial structure III was defined by isolation of the alkaline degradation product 7. The observation of NOE between 1-NH and 9-OH confirmed the attachment of N-1 to C-9a and C-3 to C-3a in the partial structure I and II. The long range couplings between 6-H_2 and C-2'α determined by LSPD experiment established the connection of C-2'α to N-7, thus confirming the whole structure of 1 as shown in Fig. 1. The structure of Duocarmycin C_2 (2) is different from that of 1 with respect to the chlorine-containing moiety. The ^1H NMR spectrum showed that chloromethyl protons were coupled to a methine, which in tern was coupled to a nitrogen-bearing methylene. This indicated that 2 had a dihydroindole skeleton on the corresponding partial structure II of 1. The structural differences between Duocarmycin A (3) and Duocarmycin C_1 are also related to the partial structure II. The COLOC spectrum revealed the structure of 3 having dienone conjugated with the cyclopropane ring (Fig. 4). Treatment of 3 with hydrochloric acid in acetone gave 1 and 2 in the ratio ca. 1:4 (Fig. 5). This provided the evidence for the structures of Duocarmycins and meant that 1 and 2 seemed to be artifact products of 3.
A new antibiotic, phthoramycin (1; C_<40>H_<68>O_<12>) which exhibits antimicrobial activities against fungi such as the plant pathogen Phytophthora parasitica was isolated from the cultured broth of a strain of Streptomyces sp. The structure and biosynthetic origin of 1 were elucidated by the 2-D NMR spectral experiments of pentaacetylphthoramycin (2) in combination with biosynthetic means using [1-^<13>C]- and [1,2-^<13>C_2]acetate and [1-^<13>C]propionate. From the results of ^<13>C-NMR analysis of labeled compounds (2), it was revealed that the antibiotic contained nine intact acetate and six propionate units as shown in Fig. 2. The biosynthesis of okilactomycin (3), produced by a strain of Streptomyces sp., was also investigated by the feeding experiments of [1-^<13>C]- and [2-^<13>C]acetate, [1-^<13>C]propionate, [U-^<13>C_6]glucose, and L-[Me-^<13>C]methionine. The incorporation of seven intact acetate and four propionate units, a glycerol moiety from glucose, and a methyl group of methionine were observed by the ^<13>C-NMR analysis. The biosynthetic pathway may be unique as shown in Fig. 4 in light of the methyl of methionine incorporated into a methyl group of the antibiotic produced by an actinomycete.
Three dimensional structures of oligosaccharide chains largely depend on the conformational properties of the (1-6)-linkage at the branching points. In a series of our conformational studies on mono- and di-saccharides, we have applied our method of chiral deuteration at the C-6 position of D-hexose and D-pentoses to differentiate the H-6R and H-6S signals in the 1H-NMR spectroscopies. In this study, the method was successfully applied for the conformational anlyses on four branching tri-saccharides (I-IV) as follows; The Chiral deuteration at the the (1-6)-linkage moiety of I-IV, NMR (chemical shifts, coupling constants, NOE) and HSEA calculation enabled us to determine the conformational properties of the (1-6)-linkage moitey and compare the possible interactions between the 0-4 and the 0-6 residues among I-IV to show that the β-D-G1c residue at 0-4 in III and IV stabilizes the gg-conformation.
Secoiridoid glucosides of three types, oleoside (1)-, 10-hydroxyoleoside (4)- and ligustaloside (5)-type occur only in oleaceous plants. We had proposed the bisynthesis pathway of these glucosides.3) In order to examine the proposed pathway, a pair of respective stereoisomers of 8,10- epoxysecoxyloganin (6, 6a) and 8,10-epoxysecologanin (7, 7a), and their labeled derivatives were synthesized from secologanin (3) and fed to three oleaceous plants, Olea europaea, Osmanthus fragrans and Ligustrum japonicum. 8S,10-Epoxysecologanin (7) was incorporated into oleuropein (15), 10-acetoxyoleuropein (16) and ligustalosides A (13) and B (14) more efficiently than three other epoxides (6, 6a, 7a). Based on these results, we have proposed the possible biosynthetic rout of these glucosides as shown in Scheme 2. Further studies on the secoiridoids from oleaceous plants in connection with the biosynthesis resulted in the isolation of 8-epikingiside (17) from L. lucidum and five new glucosides (20, 21, 22, 23, 24) from Jasminum mesnyi.
We have found that B. cinerea, a phytopathogenic fungus, produces abscisic acid (ABA), a plant hormone. The production of ABA by B. cinerea was enhanced by irradiating blue light (450nm) on the growing colony, whereas an irradiation of near UV light (352nm) decreased the yeild severely. Effects of quality of light on the ABA production were examined with blue light-sensitive strains that were selected by the monospore culture method. Blue light was the most stimulative, red light considerably and far-red light stimulated very slightly, however, near UV and green lights showed no activity.As an energy of blue light was strengthened, the yield of ABA increased, hence, blue light seems to operate as an energy source of its production. In order to clarify what step of enzyme reaction is stimulated by blue light in the biosynthesis of ABA, HMG-CoA reductase, which catalyzed a conversion of HMG-CoA to Mevalonic acid, was measured using the mycelial homogenates. There was a close positive correlation between enzyme activity and ABA production, and as the enzyme activity was enhanced by blue light irradiation, the ABA production increased in a similar rate. Thus, HMG-CoA reductase has been shown to be a rate-limiting enzyme and stimulated by blue light irradiation in the biosynthesis of ABA in B. cinerea.
AF- and AK-toxins are host-selective toxins produced by Alternaria alternata strawberry pathotype and Japanese pear pathotype, respectively, and express different toxicities to the host-plants (Table 1). We have already succeeded in structure determinations of AF-toxins and total syntheses (Scheme 1) of AF-and AK-toxins. Here, we wish to report recent studies on the cultivation condition of AK-toxin producer, the biosynthesis of AK-toxin I (4) and the mechanism of toxinless mutation of A.alternata pathogens. Various 2'-acyl derivatives 9-11 were prepared from AF-toxin II (2) as shown in Scheme 1. All of those acyl derivatives are toxic to both Japanese pear and strawberry similarly to AF-toxin I (1) and III (3) as shown in Table 3. Those host-selectivities demonstrated our hypothesis concerning structure-activity relationships. By examination of varirous cultivation conditions of A. alternata Japanese pear pathotype, we succeeded in harvesting AK-toxin I about 10 times much than before. Since it was found that decatrienoic acid 12 is about 10 times of AK-toxin I in the culture (Figure 2), it was suggested that 12 is a precursor of AK-toxins. When 3H-labeled decatrienoic acid 12 (Scheme 3) was fed to the culture of AK-toxin producer, radioactivity was incorporated into AK-toxin I. Thus, the decatrienoic acid (12) was proved to be a biosynthetic precursor of AK-toxins (Scheme 2). Various A. alternata mutants losting their pathogenicities produce neither host-selective toxins nor their precursor decatrienoic acid 12 as shown in Table 4. This result suggests that the machanism of toxinless mutation is luck of enzyme system for the initial step in the biosynthesis of host-selective toxins.