Medium-chain (E)-prenyl diphosphate synthases are of special interest because of their heteromeric structures in contrast to other classes of prenyltransferases that are tightly coupled homodimers. Heptaprenyl diphosphate (HepPP) synthase of Bacillus subtilis is composed of two dissociable subunits, component I and component II, which are encoded by two cistrons in a novel gene cluster of gerC operon. This enzyme essentially requires the coexistence of both subunits for its catalysis. Expression vector systems for the two structural genes, gerC1 and gerC3, were constructed separately, and the two components were overproduced in Escherichia coli cells. After purification, their dynamic interactions in forming a catalytically active complex were investigated by gel filtration and immunoblotting analyses. When a mixture of the two components that had been preincubated in the presence of Mg^<2+> and farnesyl diphosphate (FPP) was subjected to Superdex 200 gel filtration, a significant elution peak appeared in a region earlier than those observed when they were chromatographed individually. This fraction contained both components I (GerC1) and -II (GerC3), and it corresponded to a molecular mass that is in accord with the sum of the values of the two components. Cross-linking studies indicate that the two essential subunits, FPP, and Mg^<2+> form a ternary complex, which seems to represent a catalytically active state of the HepPP synthase. On the other hand, no complex was formed in the presence of isopentenyl diphosphate or inorganic pyrophosphate and Mg^<2+>. A photoaffinity analogue of FPP was shown to preferentially label the GerC1 subunit, suggesting that GerC1 possesses a specific affinity for the allylic substrate. Furthermore, the photoaffinity labeling of GerC1 significantly increased in the presence of GerC3. The mechanism of catalysis of this unique heteromeric enzyme is understood by assuming that association and dissociation of the two subunits facilitate turnover of catalysis for the synthesis of amphipathic product from soluble substrates.
Preferential labeling of the FPP-portion of chloroplastidic terpenoids incorporating isotopically labeled mevalonate were observed in cultured cells of liverworts and hornworts. Chloroplasts were isolated from cultured cells of liverworts, Heteroscyphus planus and incubated with ^3H-FPP to explain a reason for the non-equivalent labeling. ^3H-FPP was effectively taken into chloroplasts and converted to GGPP, while mevalonate was much poor precursor for the formation og GGPP. Thus uptake of FPP and its rapid conversion to GGPP is the reason for the non-equivalent labeling. It is also shown that a diverse source of supply (mevalonate pathway, 1-deoxy-D-xylulose pathway and reutilization of CO_2 from the carboxy carbon during acetate assimilation) is provided for the biosynthesis of the phytyl side-chain and carotenoids. A labeling pattern of striatol (3 in Fig.3) incorporating [2-^<13>C]- and [4,4-^2H_2]-mevalonate in cultured cells of Ptycanthus striatus indicated a proton addition to C-10 of the C10/C11 double bond and a concerted series of 1,2-migrations of hydrogen and methyl, followed by a specific elimination of a proton (not deuterium atom) at C-10 to form 3. The most uncommon tricyclic sesquiterpene, kelsoene (6 in Fig,4) was proven to be biosynthesized via a germacradienyl cation (4) and alloaromandendranyl cation (5). A labeling pattern of β-barbatene verified a 1,4-hydride shift and a double 1,2-methy migration in its formation in H. planus.
Enzymatic Cyclization of squalene and oxidosqualene lead to sterols and triterpenes in bacteria, fungi, plants, and animals. The cyclases for these reactions catalyze formation and stabilization of polycyclic carbocations and direct the enzyme-specific, templated formation of new carbon-carbon bonds in regio- and stereochemically defined manner. Studies of these enzymes are now progressing rapidly and promise to reveal the intimate three-dimensional structural details of the enzyme-catalyzed processes. In particular, the development of mechanism-based irreversible inhibitors, photoactivatable inhibitors, and numerous substrate analogs have helped to unravel the stepwise events occurring in the catalytic sites of these enzymes by covalent modification of specific amino acid residues. Here we report affinity labeling of vertebrate oxidosqualene: lanosterol cyclase and bacterial squalene: hopene cyclase by three active-site probes developed in our laboratory; (i) [^3H(3S)29-methylidene-2,3-oxidosqualene (29-MOS), (ii) [^3H]18-thia-2,3-oxidosqualene, and (iii) [^3H]Ro 48-8071. The mechanism-based irreversible inhibitors and the photoactivatable active-site targeted inhibitor have afforded important insights into the cyclization mechanism. The next generation of results will place the mechanistic understanding into a structural context, and the roles for specific active site residues will be elucidated through peptide mapping and crystallographic studies.
The biosynthetic pathway of avermectin has all but been elucidated. The proposed pathway is based on evidence obtained from several studies, including incorporation of labeled precursors into the avermectins and identification of key intermediates produced by biosynthetically blocked mutants or wild type-producing strains. The results of the incorporation of ^<13>C-labeled precursors and analysis of the resulting ^<13>C-labeled avermectin indicate that the avermectin aglycons are derived by head-to-tail condensation of various acyl groups which is similar to the biosynthesis of the other polyketides. The polyketide synthases use the appropriate CoA ester as a primer and add acetate units from malonyl-CoA and propionate units from methylmalonyl-CoA to assemble the polyketides. Avermectin aglycon is formed by addition of the starter unit (2-methylbutyrate or isobutyrate), of 12 acyl condensations in the order P-A-A-A-A-P-P-A-P-A-P-A (P, propionyl; A, acetyl). Within the 95 kbp gene cluster for avermectin biosynthesis, the central 65 kbp segment was found to be required for aglycon biosynthesis by phenotypic analysis of strains containing deletion or insertion mutations in this region. A sequence analysis of the central 65 kbp segment within the gene cluster for avermectin biosynthesis indicated that this segment encodes avermectin polyketide synthases. The avermectin polyketide synthase genes are organized into two converging blocks of ORFs. From the results of sequencing analysis, a feature of the two regions, aveAI and aveAII, is that they encode four kinds of large multifunctional polypeptides contains domains possessing putative fatty acid synthase-like activities. The PKS genes for avermectin biosynthesis are organized into two convergently transcribed clusters of six modules. The avermectin PKS proteins appear to contain two, three, or four synthase units (SU). PKS-1 with two SUs and PKS-2 with four SUs contain modules 1 and 2, and 3-6, respectively, whereas PKS-3 and -4 with three SUs contain modules 7-9, and modules 10-12, respectively. The 12 SUs correspond to the 12 cycles required for synthesis of the initial aglycon.
Polyketides are one of the largest and most important group of natural products. In spite of their structural diversity, the initial reactions of polyketide biosynthesis follow a common scheme that condensation of acyl primer with malonate units to form β-polyketomethylene intermediates and their cyclizations catalyzed by so called polyketide synthases (PKS). Although molecular genetic analysis of bacterial polyketide biosynthesis genes have been extensively carried out, only several PKS genes have been cloned from eukaryotic filamentous fungi, which are another rich source of polyketides, especially, aromatic compounds. All fungal PKS genes so far cloned code for iterative multifunctional type I PKS polypeptides. Though, little has been known how fungal PKSs control their reactions, that is, how to regulate the chain length of β-polyketomethylene intermediates and their cyclizations. Thus, we carried out functional analysis of Aspergillus nidulans wA gene which was assumed to code for PKS involved in conidial spore pigment biosynthesis. The wA gene was expressed in a heterologous fungus Aspergillus oryzae using fungal expression plasmid pTAex3. By starch induction, the A. oryzae transformant produced compounds of which structures were identified to be citreoisocoumarin (1) and its derivatives. In the course of these experiments, we found one base missing in the reported nucleotide sequence of wA gene that causes a frame shift in its C-terminus region. As a result, it was found that isocoumarins were produced by WA polypeptide with truncated C-terminus. The intact WA expression plasmid reconstructed was introduced into A. oryzae. The transformant produced a novel naphthopyrone compound YWA1 (4) by starch induction in place of isocoumarins. Both isocoumarins and naphthopyrone YWA1 (4) are heptaketides, but their cyclization patterns are different. That is, the former lack the second aromatic ring cyclization, in which the truncated C-terminus of WA polypeptide was suggested to be involved. By C-terminus modifications of WA polypeptide including further truncations and site-directed mutagenesis, C-terminus region, containing Ser^<1967> and His^<2129>, was identified to function as Claisen type cyclase. One of the characteristic feature of the WA polypeptide is that it contains two ACP motifs. Site-directed mutagenesis on Ser^<1682> and Ser^<1804> which are presumed phosphopantetheine modification sites revealed that both of two ACPs function independently in WA polyketide synthase reaction.
Solanapyrone synthase has been partially purified from a phytopathogenic fungus Alternaria solani. On the basis of the chromatographic behavior, it is speculated that single enzyme catalyzes both oxidation of 6 and the subsequent Diels-Alder reaction of 7. The enzyme is monomeric and showed native molecular weight 40 kD and pl 4.25. The substrate specificity of this enzyme was also examined with various substrate analogs. Enzymatic Diels-Alder reaction of 6 in semi-preparative scale afforded (-)-1 with high enantioselectivity and with good exo-selectivity which is difficult to attain by chemical methods. In addition, the crude enzyme from A. solani has been able to perform a kinetic resolution of (±)-3.
Okadaic acid (1) is a polyether compound produced by the marine dinoflagellate Prorocentrum lima. Although its biosynthesis attracts considerable attention since the carbon skeleton has been shown to be synthesized via an unusual route, a limited amount of information is available for the formation of its ether rings. In this study, P. lima was cultured in the presence of N_2 /^<18>O_2 (4: 1), 1mM [^<18>O_2] AcONa and 17% H_2^<18>O, respectively, and a few microgram of 1 isolated from the extract of each cultured cells was subjected to collision-induced dissociation tandem mass spectrometry (CID-MS/MS) to elucidate the ^<18>O-incorporation pattern. The extensive determination of ^<18>O/^<16>O ratios for each product ion bearing the different numbers of incorporated ^<18>O atoms resulted in the complete assignment of the labeled positions with the accurate isotope ratios. These incorporation patterns from ^<18>O_2 and [^<18>O_2] acetate suggest that cyclization of a β-epoxide intermediate at C22-C23 yields ether rings C, D, and E, and the terminal carboxylic acid is formed by Baeyer-Villiger oxidation. A labeling pattern shown by the H_2^<18>O experiments implies that oxygen atoms of water are incorporated via hydration of carbonyl groups, since labeled oxygen functionalities are mostly derived from carbonyl precursors. However, the great difference in the incorporation ratios were observed among them; a hydroxy group at C24 was labeled at 19% while the incorporation in the ether oxygen of ring E was less than 1%. These facts suggest that oxygen incorporation from H_2^<18>O may provide information as to when the reduction of carbonyl groups to hydroxyls takes place in the course of the polyketide biosynthesis; i.e., which enzymes, polyketide synthases (PKSs) or post-PKS enzymes, are implicated in the reaction.
3-Isopropylmalate dehydrogenase (IPMDH, EC 220.127.116.11) catalyzes an oxidation-decarboxylation reaction of (2R, 3S)-3-isopropylmalate (IPM) into 2-oxoisocaproate with the aid of NAD^+ in the penultimate step of the biosynthetic pathway of an essential amino acid L-leucine. We have been involved in the mechanistic and molecular recognition studies on IPMDH derived from the extremely thermophilic bacteria Thermus thermophlus HB8. The finding that 2-O-methyl-3-isopropylmalate (1) is an uncompetitive inhibitor of IPMDH, prompted to design conformationally restricted substrate analogs, in which the hydroxy oxygen is intramolecularly bound to an isopropyl carbon to form a ring structure. The oxirane 2 was the most inhibitory among those synthesized. IPMDH appeared to recognize preferentially the anticonformation of the butanedioic acid structure. (2R, 3S)-3-vinylmalic acid (VM) was designed, as a mechanism-based inhibitor. The synthesis of VM was pursued in 6 steps from diethyl (R)-malate. Besides its weak activity as a substrate, VM appeared to be a competitive and mechanism-based inhibitor (K_i values 25 nM) for IPMDH as deduced from the time-dependent and kinetic analyses. By GC-MS and ^1H-NMR analysis of the enzyme reaction of VM, 2-oxo-3-pentenoate (7) was detected as the product. Furthermore, we synthesized deuterium-labeled VM (VM-d_3) and monitored the enzyme reaction by ^2H-NMR spectroscopy. While the signals of VM-d_3 (at δ_D 5.1 and 5.8 ppm) disappeared by consumption of VM-d_3, the signals which derived from 7 emerged at δ_D 2.0 and 7.1 ppm. These results suggest that the transient covalent bond, which was formed between IPMDH and 7, inhibits the IPMDH reaction. Arg94Lys mutant of IPMDH was prepared by site-directed mutagenesis. The mutant enzyme had the same Km value as the native enzyme. But its catalytic efficiency lowered 10^<-2> times as compared with wild-type IPMDH. Arg94 of IPMDH appeared to stabilize the transition state of the IPMDH reaction and participate in the catalytic efficiency.
Owing to its high proliferation and its ability of secondary metabolite production, hairy roots induced by infection of Agrobacterium rhizogenes to plants are expected to be a valuable culture system to produce useful secondary metabolites of plants. Hairy roots were induced by inoculation with A. rhizogenes on sterile seedlings of Hyoscyamus niger. Hyoscyamine (1) as the main tropan alkaloid was isolated in high yield, together with scopolamine (2), 7β-hydroxyhyo-scyamine (3) and 6β-hydroxyhyoscyamine (4) from the hairy roots of H. niger. The highest content (2.7% dry weight) of 1 was observed in MS liquid medium. In the course of our study on the alkaloid production, it was shown that the hairy roots of H. niger gave phytoalexins, lubimin (5) and solavetivone (6), by treatment with Cu^<2+>. The hairy roots of H. albus also gave 1 and 2 by treatment with Cu^<2+>. The hairy roots of H. albus gave seven phytoalexins, 5-11, by treatment with methyl jasmonate (JAMe). The Structures of four new phytoalexins, 8-11, were determined to be (3R,4S,5R,9R)-3-hydroxy-9-tigloyloxysolavetivone, (3R,4S,5R,7S,9R)-3-hydroxy-9-(3-methyl-2-butenoyloxy)solavetivone, (3R,4S,5R,7S,9R)-3-hydroxy-9-isobutanoyloxysolavetivone and (3R,4S,5R,7S,9R)-3,9-dihydroxysolavetivone by mean of spectral methods. The induction pattern of phytoalexins by treatment with JAMe was different with those treated with Cu^<2+>. Co-treatment of the hairy roots with JAMe and Cu^<2+> gave only 2 in several time higher yield than the treatment with JAMe.
Monoepoxy derivatives of (Z,Z,Z)-3,6,9-triunsaturated hydrocarbons have been identified as sex pheromone components of female moths which distribute in foreign countries and belong to Geometridae, one of the biggest family in Lepidoptera. In order to obtain some information about pheromones of Japanese species, the C_<18>-C_<23> trienes were systematically synthesized from linolenic acid and converted into a mixture of three racemic monoepoxides, 3,4-, 6,7- and 9,10-epoxydienes. After separation by MPLC and structure determination by 2D NMR, each positional isomer was offered to random screening tests and specific male attraction of ca. 20 species was newly observed. Besides this finding, their GC-MS data were utilized to identify a pheromonal C_<19>-3,4-epoxydiene secreted by an adult female of the Japanese giant looper, Ascotis selenaria cretacea, a serious defoliator of tea gardens. Chiral HPLC is helpful not only to determine the absolute configuration of a natural pheromone but also to prepare optically pure enantiomers for a biological assay. Using Chiralpak AS and AD columns operated under a normal phase condition and a Chiralcel OJR column operated under a reversed phase condition, enantiomers of each epoxydienes were successfully resolved, and then stereochemistry of the separated enantiomers was determined by a modified Mosher's method after methanolysis of the epoxy ring. Analysis of the natural pheromone of A. s. cretacea showed that the female produced the racemic mixture, while the males interestingly attracted to the pure (3R,4S)-isomer more strongly than the racemic mixture. Furthermore, epoxidation in the pheromone gland of A. s. cretacea was examined in vivo. GC-MS analysis of the gland extracts treated with a deuterated C_<19>-triene and other unsaturated hydrocarbons revealed that a monooxygenase of the pheromone biosynthesis regiospecifically attacked only the (Z)-double bond at 3-position but substrate specificity of this enzyme was rather low.
In the rocky sublittoral zones of Japanese waters, macroalgal communities are seen to flourish where it provides a luxuriant environment for the marine inhabitants. However, in recent years, these macroalgal communities are declining and being replaced by crustose coralline red algae. This phenomenon is called "Isoyake" and has been seen to spread particularly along the southwest coast of Hokkaido. In "Isoyake" areas dense population of sea urchins and coralline flats similar to the sea desert are observed. Rise in seawater temperature, excessive grazing by sea urchins, sloughing of epidermal tissue of crustose algae and decrease in organic and inorganic nutrients have been suggested as the primary factors involved in causing and maintaining this "Isoyake" phenomenon. In addition to these factors, we supposed that secondary metabolites of coralline red algae also play important roles in causing and maintaining Isoyake phenomenon. Therefore, our objectives are to clarify following two problems on the basis of allelochemicals. 1) Why do Laminaria communities recruit in "Isoyake areas" in the year when seawater temperatures are low? 2) Why do sea urchins gather numerously in "Isoyake areas"? In order to prove these questions, we investigated suppression of Laminaria's growth, attraction of sea urchin larvae, inducement of larval settlement and metamorphosis of sea urchins, and the feeding stimulant of sea urchins. As bialgal cultures of an articulated coralline red alga Corallina pilulifera and Laminaria sporelings were performed under various temperature, it became obvious that both the Laminaria spp.'s gametophytes maturation and normal sporophyte formation were suppressed at higher temperatures. Present findings also indicate that crustose coralline red alga Lithophyllum yessoense and C. pilulifera not only induced larval settlement and metamorphosis of the sea urchins, Strongylocentrotus intermedius and S. nudus, but also attracted swimming larvae of these sea urchins. The benthic diatoms isolated from the surfaces of L. yessoense also displayed similar activities. Bioassay-guided separation of C. pilulifera extract afforded several glyceroglycolipids which induce larval settlement and metamorphosis of sea urchins. In addition, these glycolipids attracted the swimming larvae and some of them were shown to be feeding stimulants for adult sea urchins. Similar glycolipids were also found to be present in L. yessoense. Consequently, it is suggested that glycolipids play important roles in maintaining Isoyake phenomenon.
The roots and stems of Salacia reticulata WIGHT ("Kotala himbutu" in Singhalase, Celastraceae) have been extensively used as a specific remedy for diabetes in Ayurvedic system in Indian traditional medicine. As a continuing part of our screening for antidiabetogenic principles of natural medicine and medicinal foods, we have found that the water-soluble fractions from the roots and stems of S. reticulata strongly inhibited the increase of serum glucose levels after the administration of sucrose or maltose, but not glucose, in rats. Furthermore, the fractions inhibited rat intestinal maltase and sucrase in vitro, although the extract even at high dose did not have any effect on experimental hyperglycemia induced by injection of alloxan in mice. On the other hand, the lipophilic fraction showed inhibitory activity for rat lens aldose reductase and, as the active components, new triterpene kotalagenin 16-acetate (5) was isolated together with several diterpenes and triterpenes. Through bioassay-guided separation, two potent α-glucosidase inhibitors called salacinol (1, 0.0079%) and kotalanol (4, 0.0002%) have been isolated from the water-soluble fraction together with many sugars and glycosides. The absolute stereostructure of salacinol was determined on the basis of chemical and physicochemical evidence, which included the alkaline degradation to 1-deoxy-4-thio-D-arabinofuranose (2) and the X-ray crystallographic analysis. The molecular conformation showed the unique spiro-like configuration of the inner salt comprised of 1-deoxy-4-thio-D-arabinofuranosyl sulfonium cation and 1-deoxy-D-erythrosyl-3-sulfate anion. The structure of kotalanol was also elucidated in a similar manner as that of 1 to be the inner salt comprised of 1-deoxy-4-thio-D-arabinofuranosyl sulfonium cation and 1-deoxyheptosyl-3-sulfate anion. Salacinol (1) and kotalanol (4) were found to exhibit the competitive inhibition for the intestinal α-glucosidase of rat. Their inhibitory activities against sucrase and maltase were nearly equal to those of a commercial α-glucosidase inhibitor acarbose, whereas their activities against isomaltase were much more potent than that of acarbose. 1-Deoxy-4-thio-D-arabinofuranose (2) lacked the activity (IC_<50>>400 μg/ml) and its methyl sulfonium iodide (3) showed weak activity (sucrase: IC_<50> 129 μg/ml; maltase: IC_<50>>400μg/ml). This evidence revealed that the spiro-like inner salt structure of 1 and 4 was essential for the potent α-glucosidase inhibitory activity. Furthermore, 1 more strongly inhibited the increase of serum glucose levels in sucrose-loaded rats than acarbose.
Mitragyna speciosa Korth. (Rubiaceae) is a tropical plant indigenous to Thailand and the Malay Peninsula. The leaves of this plant were traditionally used as a stimulant like coca or as a substitute for opium. But the use of this plant is now illegal in these countries because of its narcotism. With the object to clarify pharmacological profiles of this plant species as well as to find new lead-compounds from this medicinal resources, the following studies focusing on the constituents in the leaves of M. speciosa native to Malaysia were carried out. I: Isolation and Structure Elucidation of New Indole Alkaloids from the Leaves of M. speciosa in Malaysia. From the methanol extract of the mature leaves of M. speciosa in Malaysia, six 9-methoxy-Corynanthe-type indole alkaloids, i.e., mitragynine, speciogynine, speciociliatine, paynantheine, mitragynaline, and 7α-hydroxy-7H-mitragynine were isolated. In addition, three new indole alkaloids, 3,4,5,6-tetradehydromitragynine, mitralactonal, and mitrasulgynine carrying a sulfonate function, were isolated and their structures were elucidated by spectroscopic analysis and/or chemical transformation. From the young leaves of this plant, two new alkaloids, named mitralactonine and 9-methoxymitralactonine, were isolated together with six known indole alkaloids. The unique structure of mitralactonine was finally established by total synthesis. Determination of the absolute configuration at the C20 position by means of chiral total synthesis will be presented. II: Studies on the Asymmetric Total Synthesis of a Minor Mitragyna Alkaloid, Speciogynine. First asymmetric total synthesis of speciogynine, which is a minor constituents in M. speciosa and a diastereoisomer of the major component, mitragynine, at the C20 position, was studied. Our basic approach to speciogynine features the utilization of asymmetric desymmetrization of the prochiral cyclic imide to construct the fundamental core structure of Corynanthe-type alkaloids in chiral form. Based on this strategy, the key intermediate, 21-oxo-20-deethyl-speciogynine, having the absolute configuration of the natural alkaloid could be synthesized. The final stages of the total synthesis is currently being investigated. III: Synthesis of the Mitragynine Derivatives, Structure-Activity Relationship, and the Action Mechanisms of the Antinociceptive Activity of the Mitragyna Alkaloids. A potent opioid agonistic property of mitragynine, a major constituent of this plant, and mitragynine pseudoindoxyl, a oxidative derivative of mitragynine, was clarified in vitro experiments. Mitragynine pseudoindoxyl, whose agonistic potency is 20-40 fold greater than that of morphine, acts on mu- and delta-opioid subtype receptors. The essential structural feature for revealing the activity was elucidated by study of the structure-activity relationship using natural and synthetic derivatives.
Recently, we have developed a highly sensitive fluorescent derivatization reagent, 2-(anthracene-2,3-dicarboximido)ethyl trifluoromethanesulfonate (AE-OTf), for carboxylic acids. The reagent made it possible to detect fatty acids at sub-fmol levels. The ^1H-NMR studies on some AE-O-esters indicate that these compounds take the bent conformation as shown in Fig.1. Since some proton signals of the hydrocarbon chain were shifted to higher field, those protons would be located over the anthracene ring. Therefore, it can be expected that the chiral carboxylic acids having chiral centers at a remote position from their carboxyl groups should be descriminated by labeling with chiral AE-OTf analogues. Here, we report the preparation of chiral AE-OTf analogues 1-7 (Fig.3) and the results of the discrimination of their diastereomeric derivatives of some branched fatty acids 8-28 (Fig.4) on a reversed phase HPLC and ^1H-NMR. The branched fatty acid derivatives were separeated on an ODS column eluted with acetonitrile/ methanol/water or acetonitrile/methanol/n-hexane below 0O℃. In general, lower the column temperature is, better separation was obtained. Among seven reagents tested, 1-(2,3-anthracenedic arboximido)-2-propanol (2) and trans-2-(2,3-anthracenedicarboximido)-1-cyclohexa-nol (3) showed excellent separation ability. All chiral branched acids except 7-methylnonanoic acid (13) was separated into two peaks by labelling with trans-2-(2,3-anthracenedicarboximido)-1-cyclohexa nol (3), while 13 could be separated by labelling with 1-(2,3-anthracenedicarboximido)-2-propanol (2). These results allow us to determine the absolute comfiguration of chiral branched acids which have chiral centers remote from the carboxyl group up to 11 bond distances at fmol levels by derivatizing with these reagents. With respected to elution order of (S) and (R) isomers, there was a rule that (S) isomers chiral center was located at an even number position were eluted faster than (R) isomer when labelled with (S) or (S,S) configurated reagents, while (R) isomer were eluted fasterwith those having at odd number position. The branched methyl groups of 2-(2,3-anthracenedicarboximido)-1-propanol (1) derivatives gave different chemical shifts on 1H-NMR measured in CDCl_3. This meant that it was also possible to discriminate their chirality on 1H-NMR. It was also possible to discriminate theenantiomers of 12-methylpentadecanoic acid, which would be possible to bederived from a ceramide isolated from a marine plankton.
The CD exciton chirality method has been extensively applied to various natural products and chiral synthetic compounds to determine their absolute configurations in a nonempirical manner. However, application of the CD exciton chirality method has been limited to chiral compounds with two or more functional groups, because exciton CD Cotton effects are observable only for the interaction between two or more chromophores. This has been a weak point of the CD exciton chirality method, and therefore it has never been applied to mono-functional chiral compounds. We report here a new strategy for determining the absolute configuration of mono-fucntional compounds by the CD exciton chirality method. Di(1-naphthyl)acetic acid 2, an achiral CD auxiliary with two naphthalene chromophores was designed, because the ^1B_b transition of naphthalene chromophore located around 230 nm is the most ideal for observing exciton CD Cotton effects. This CD auxiliary was condensed with various chiral mono-alcohols. For example, ester 6 prepared from di(1-naphthyl)acetic acid 2 and (S)-(-)-α-methylbenzyl alcohol 6a exhibits intense exciton split CD Cotton effects of positive exciton chirality around 225 nm indicating that the long axes of two naphthalene chromophores constitute a clockwise screw sense: CD (EtOH) λ_<ext> 229.6 nm Δε +81.1) and 219.8 (-44.9), A = +126.0. To correlate the positive exciton chirality with the absolute configuration of 6a, the conformational analysis of 6 was carried out by using the CONFLEX-MM3 program. It was revealed that the most stable conformer of 6 had a positive exciton chirality in agreement with the observed CD spectra. It was also found that the population of the conformer with positive exciton chirality exceeds that of conformers with negative chirality: positive/negative 70: 30. The absolute configuration of mono-alcohol 6a was thus determined by the new strategy of the CD exciton chirality method. The absolute configurations of other chiral mono-alcohols including steroids were similarly determined by this new strategy of the CD exciton method.
The genera, Nephila Nephilengys, etc., belonging to the Araneidae family spiders store various acylpolyamines in their venom glands which paralyze insects by blocking to nerve-muscle signal transduction of glutaminergic synapses. Because of structurally related compounds and complexity of venom constituents, it was difficult to separate and identify all of those compounds. Tandem mass spectrometer equipped with Liquid MALDI ionization source allowed to identify all of those constituents without extensive sample clean up procedures. Detailed structures of those acylpolyamines were determined by the observation of high energy collision induced dissociation (CID) and charge remote fragmentation method. It was able to classify acylpolyamines obtained Araneidae family spiders into type-A to F among those Type-C and E have isomeric structures. Genus Nephilengys exclusively store type-C acylpolyamines, on the other hand, genus Nephila stores type-E compounds which might suggest the applicability of chemotaxonomy.
In the course of our screening for specific apoptosis inducers in transformed cells, we isolated a novel 20-membered macrolide, apoptolidin (1), from Nocardiopsis sp. 1 induced apoptotic cell death in rat glia cells transformed with the adenovirus E1A oncogene (IC_<50> 11ng/ml) but not in normal glia cells or normal fibroblasts (IC_<50>>100μg/ml). The molecular formula of 1 was determined to be C_<58>H_<96>O_<21> from high-resolution FAB-MS. COSY and HMBC experiments generated a polyketide chain (2) and three hexoses (3〜5) as the partial structures. Methanolysis of 1 followed by hydrolysis yielded D-oleandrose (5), L-olivomycose (4), and a novel sugar, 6-deoxy-4-O-methyl-L-glucose (3). ^1-^<13>C long-range correlations constructed a 20-membered macrolide ring, a 6-membered hemiketal ring and three glycosidic linkages. High-field carbon shifts for the allylic methyls and a large vicinal coupling constant indicated all (E) configurations for the five olefinic bonds. A clue to the absolute stereochemistry of the macrolide ring was found between the 6-deoxy-4-O-methyl-L-glucose moiety and the vicinal ring methines (C-8 and C-9). NOEs including the most important one observed between 3-H and 17-H identified the absolute configurations of all the asymmetrical carbons. The stereochemistry of 1 thus obtained was confirmed by distance analysis in dihedral angle space (DADAS) using a JEOL MolSkop system.
We have demonstrated that several minor components which amount to 2-3 wt % of the lipoteichoic acid (LTA) fraction from Enterococcus hirae ATCC 9790 possess cytokine-inducing activity, whereas the major component of over 90 wt % has no activity. The chemical structure of the major component coincides with the structure proposed for LTA by Fischer. In the present paper, we report structural analysis of a biologically active minor component in the LTA fraction and an improved separation procedure for active glycoconjugates from bacterial cells. The active minor component, designated OS-4L, from the LTA fraction was subjected to hydrolysis with aqueous HF to give a polysaccharide, low molecular weight hydrophilic products, and lipophilic products. The polysaccharide was composed mainly of highly branching mannan as determined by NMR and MS analyses. The structure was close to that of a yeast mannan, which, however, showed no cytokine-inducing activity. The low molecular weight products consisted of phosphate and glycerol, suggesting the presence of a glycerophosphate structure in OS-4L. The lipophilic products was not fully characterized because of its limited amount. By employing a more efficient separation procedure, in which active fractions were directly separated from a crude extract by anion exchange chromatography, several new active components different from above OS-4L were isolated from the same bacteria. On HF hydrolysis, the major new active components gave lipophilic products similar to those from above OS-4L, suggesting the importance of these lipophilic parts for the biological activity.
Marine microalgae produce various types of compounds, including nitrogenous neurotoxins, polyether sea food toxins, sulfonium compounds of dimethyl sulfide precursors and antineoplastic macrolides. Among them, dinoflagellate metabolites such as ciguatoxin and maitotoxin are unique and spectacular in terms of their complex structures, potent activities and intricate biogenesis. Symbiotic dinoflagellates so-called zooxanthellae are distributed over a wide range of marine invertebrates and were thought to produce bioactive metabolites which were isolated from marine invertebrates containing zooxanthella inside of the body. However, most trials failed to establish the production of bioactive substances by symbiotic organisms because of difficulties of their culture and adjustment of the culture conditions for the desired metabolite production. In studies on the biogenesis of vasocontrictive macrolides, zooxanthellatoxins isolated from a symbiotic dinoflagellate Symbiodinium sp., we have investigated metabolites of the dinoflagellate cultured under different conditions. Four new compounds were isolated from 70% EtOH extract of the cells cultured in an f/2 medium. Two betaines (zooxanthellabetaine-A and -B) were obtained from a neutral fraction of n-BuOH soluble portion and the structure of zooxanthellabetaine-A was determined as 4-(4-hydroxybenzoyloxy)-3-(trimethylammonio)-butyrate. The EtOAc soluble portion afforded a new C-30 alkaloid, zooxanthellamine, and a new ceramide, symbioramide-C16. The structural similarity of zooxanthellamine to zoanthid alkaloids, zoanthamines, suggested an algal origin of these zoanthamines. Zooxanthellamine might be derived biogenetically from a polyketide chain presumably started from a glycine unit, like other marine toxins such as zooxanthellatoxin and palytoxin.
Marine microorganisms are potentially prolific sources of highly bioactive secondary metabolites that might represent useful leads in the development of new pharmaceutical agents. As part of our ongoing search for new antitumour metabolites produced by microorganisms from marine organisms, we have previously isolated gymnastatins A (1), B, C, D (2), E (3) - H (4) from a strain of Gymnascella dankaliensis originally separated from the marine animal Halichondria japonica, and their relative stereostructures have already been reported. Further investigation for metabolites of this fungal strain has now led to the isolation of nine additional new compounds, gymnastatins I(5)-M(9) and gymnasterones A(10)-D(13). The stereostructures of these metabolites have been elucidated by spectroscopic analyses. Furthermore, the absolute stereo-structures for compounds 1-4 were determined by spectroscopic analyses, some chemical transformations and X-ray structure analysis. The total syntheses of gymnastatins H (4) and I (5) were also achieved. Among the compounds tested this time, compounds 5 - 7 and 11 - 13 exhibited significant cytotoxicity against cultured P388 cells.
During the course of our investigation in search for new bioactive substances from marine organisms, we isolated an extremely potent cytotoxic polyketide designated callystatin A (1) from the marine sponge Callyspongia truncata and elucidated the absolute stereostructure of 1 by both physicochemical and synthetic means. The scarcity of natural supply prompted us to engage in total synthesis of 1 for further biological evaluation and confirmation of the absolute stereostructure. Consequently, the first total synthesis of callystatin A (1) was achieved by use of a trans-selective Wittig olefination utilizing allylic tributylphosphorus ylide and asymmetric Evans aldol condensation as the key reactions. On the other hand, an anti-tumor antibiotic, leptomycin B (23), which has a fairly similar chemical structure to callystatin A (1), was shown to induce arrest of cell cycle at G1 and G2 phase. Additionally, it was very recently found to inhibit nuclear export signal (NES)-mediated transport of Rev and U snRNA protein through specific binding chromosome maintenance region 1 (CRM1) protein. In spite of its attractive biological activities, no study on the stereostructure of 23 as well as those of several congeners has been reported. Thus, we have established the absolute stereostructure of leptomycin B as 23 having 4S, 5R, 10R, 16R, 18S, 19R, and 20S configurations by asymmetric total synthesis.
The cyclotheonamides which were isolated from the marine sponge Theonella swinhoei collected off Hachijo-jima Island, 300 km south of Tokyo are unusual cyclic peptides containing two new amino acids, α-ketohomoarginine (k-Arg) and vinylogous tyrosine (v-Tyr). Significantly, they revealed potent inhibitory activity against serine proteases including thrombin, trypsin and plasmin. Since the cyclotheonamides had become important model compounds for serine protease inhibitors, we further examined the extract of T. swinhoei, which has resulted in isolation of five new peptides related to the cyclotheonamides: pseudocyclotheonamides A_1 (2), A_2 (3), B_2 (4), C (5), and D (6). Pseudocyclotheonamide A_1 (2) had a molecular formula of C_<36>H_<45>N_9O_8 as established by HR-FABMS, which was the same as that of cyclotheonamide A (1). Interpretation of 2D NMR spectra indicated 2 consisted of the same component amino acids whose sequence was same as that of 1. However, it was found to contain unusual piperazinone and piperidinoiminoimidazolone rings. Absolute stereochemistry of 2 was determined by combination of NMR and Marfey analysis. Pseudocyclotheonamide A_2 (3) was an epimer of 2 at the β-position of e-Tyr (ethyleneinserted Tyr) unit in the piperazinone ring. Pseudocyclotheonamide B_2 (4) has an additional methylene unit between α-nitrogen of D-Phe and β-nitrogen of Dpr. NMR data and Marfey analysis led to the same stereochemistry of 4 as 3. Pseudocyclotheonamide C (5) had a piperidinoiminoimidazolone ring but v-Tyr unit remained intact, while pseudocyclotheonamide D (6) was a linear peptide in which k-Arg residue was missing.These compounds 2-6 inhibited thrombin with IC_<50> values of 1.0, 3.0, 1.3, 0.19, 1.4, and 0.33 μM, respectively, while they inhibited trypsin with IC_<50> values of 4.5, >10, 6.2, 3.8, >10, and 6.7 μM, respectively.
Marine polyether compounds have fascinated many scientists by their unique and complex structures and specific biological activities, as exemplified by the brevetoxins, the ciguatoxins, and maitotoxin. Although elucidation of the absolute stereochemistry of these compounds is essential for studying their mode of actions, biosynthesis, and chemical synthesis, difficulties existed due to their noncrystalline natures, and extremely limited availabilities. By applying anisotropic NMR reagents and chiral HPLC regeants, we successfully elucidated the absolute configuarations of pectenotoxin-6 (PTX6), dinophysistoxin-1 (DTX1), gambierol and gambieric acid. The absolute configurations of PTX6 and DTX1 were determined by preparing amides with anisotropic NMR reagents, (R)- and (S)-phenylglycine methyl esters (PGME). The axial C6-OH group in gambierol was converted to equatorial orientation and then esterified with (R)- and (S)-MTPA. NMR spectra of these esters indicated the absolute configuration at C6 to be S. The stereostrucures at C3, C9 and C48 in gambieric acid-B were determined by NMR measurements of its PGME amides and MTPA esters and by chiral HPLC analysis of an AP-OTf ester of an oxidative degradation product.
DNA topoisomerases, mechanistically devided into two main classes types I and II, are critical enzymes that control the level of DNA supercoiling by catalyzing the passage of individual DNA strands or double strands through one another. DNA topoisomerases are involved in biological processes of DNA metabolism such as replication, transcription, recombination and chromosome segregation at mitosis. Therefore, compounds that inhibit these enzymes as the primary cellular target are of special interest since those are promising candidates for anticancer drugs. In our continuing studies towards antitumor compounds, we have isolated oleic acid (1) from the fungus Syncephalastrum spp. and six known pentacyclic triterpenoids including ursolic acid (2) from Tabebuia caraiba and Campsis radicans as DNA topoismerase II inhibitors. Acids 1 and 2 inhibited the catalytic topoismerase II activity (the conversion of catenated kDNA to minicircle monomers) in a dose-dependent manner. However, DNA cleavage assay did not afford linear DNA that generates via double-strand breaks, indicating that both acids do not stabilize the cleavable complex consisting of DNA and topoismerase II. On the other hand, inhibitory effects of 1 and 2 on topoismerase II-mediated DNA relaxation were reduced by increasing amounts of ATP in the buffer. These behaviors suggest that acids 1 and 2 competes with ATP for binding at the ATPase domain of the enzyme, though allosteric effect cannot be ruled out. Structure-activity relationship study of acids 1 and 2 was carried out since both acids have a novel carbon framework as the topoismerase II inhibitor. In the case of fatty acids, carbon chains that have at least 14 methylens were required for topoismerase II inhibition and C-C double bonds more than one reduced the activity. On the other hand, the 3β-OH of 2 was found to show a negative effect on the topoismerase II inhibition. In fact, the inhibitory activity of the deoxy form of 2 was markedly enhanced compared with that of 2. Furthermore, the inhibitory activity of both 1 and 2 disappeared completely when the carboxyl group was reduced to the cotresponding alcohol. These results indicate that the presence of the carboxyl group is essential and the lipophilicity of molecules is contributable to increase the inhibitory activity.
The role of prostacyclin (PGI_2, 1) in the central nervous system (CNS) has still been unclear because of the lack of a specific ligand for a PGI_2 receptor in CNS. In this context, we recently elaborated 15R-TIC (2) with high binding affinity and selectivity for novel IP2 receptor which is specifically expressed in CNS neurons. The R configuration of the hydroxy-bearing C(15) in 2 is fascinating, because, in general, the configuration of biological active PGs at C(15) position is known to be S. In this symposium, we describe synthesis of the TIC derivatives for structure-binding affinity relationship in addition to biological actions. In consequence, 15-deoxy-16-m-tolyl-17,18,19,20-tetranorisocarbacyclin (5) (referred to as 15-deoxy-TIC) exhibited, among others, highest binding affinity and selectivity for a IP2 receptor. 5 has been prepared based on the combination of the Witting reaction and Pd(0)-mediated coupling of an allyl carbonate and a sulfone. Thus, Witting reaction of aldehyde 7 and a phosphorane led to 8. The reduction of 8 and subsequent methoxy carbonylation gave allyl carbonate 9 which was treated with disulfone 10 in the presence of a 1: 1 mixture of Pd(0) and diphenylphosphinoethane to give 11. Reductive removal of phenyl sulfonyl groups of 11 and subsequent deprotection of THP group gave methyl ester 12. Finally, alkaline hydrolysis of 12 led to 5. 2 and 5 prevented apoptotic cell death of hippocampal neurons induced under high oxygen (50%) atmosphere. 6, isocarabacyclin, and natural PGs except for 1 did not show such a biological effect.
Glycopeptide antibiotics represented as vancomycin (1) are final defense against bacterial infection. The mechanism of this class of antibiotics inhibits gram positive bacterial cell wall biosynthesis due to bind the key peptide fragment, L-Lys-D-Ala-D-Ala with five hydrogen bonding. However recently some enterococci showed resistance against vancomycin by changing the terminal D-Ala to D-Lac and the activity was significantly decreased by approximately 3000 times by luck of one hydrogen bonding. Recent X-ray crystal structure of vancomycin showed asymmetric dimmer and the glucose-C6 hydroxyl group of one monomer faced to the binding pocket. Therefore we focused this position and hypothesized to build additional hydrogen bonding or hydrophobic effect by introducing several functional group to improve activity. First, two amines and one carboxyl group were needed to be protect with aloc and allyl groups. The protected vancomycin was treated with 2-mesitylenesulfonyl chloride and pyridine at 4℃ to give glucose C6 sulfonated intermediate selectively and displaced with KI then deprotected aloc and allyl groups with Pd catalyst. This key intermediate (3) was displaced with several nucleophiles to give glucose-C6 modified vancomycins. The best analogues were 2-thio-6-azathymine (5) which is more active against susceptible strains and 5-thio-4-amino-3-hydrazino-1, 2, 4-triazole (6, Purpald^[○!R]) which showed specific activity against resistant strains of 46 derivatives. However activities of these glucose-C6 modified vancomycin analogues against vancomycin resistant Enterococci were not enough. Introduction of N-4-(4-chlorophenyl)benzyl group on vancosamine amine greatly improved their activities. N-4-(4-chlorophenyl)benzyl glucose-C6-iminotriphenylphosphine analogue (11) was most active. Furthermore Time-Kill Studies showed modification of glucose-C6 and vancosamine amine exhibited bactericidal activity. The mode of action of these glucose-C6 modified analogues was not clear. Their binding affinity against Ac_2-t-Lys-D-Ala-D-Ala and Ac_2-L-Lys-D-Ala-D-Lac are almost identical with parent vancomycin. This imply there was the other mode of action. ES I mass spectra of them suggested their interesting physical characters. Pseudoaglycon tetramer ion peak was observed on the ESI mass spectrum of 5 which was most active against all vancomycin susceptible strains. No dimer or tetramer peak was observed in the mass spectrum of 6. It is possible that aggregation in the solution might take part in improving activity. Further study is currently in progress in our laboratory.
Much interest in the biological acitities of fullerenes have been drawn since their first discovery. As one of the first findings of biological activities, we have already reported the synthesis of water-miscible fullerene carboxylic acid and its biological activities. Thus, we found that fullerenes show the photo-induced DNA cleaving activity, cytotoxicity and enzyme inhibition. In this study, we further investigated their biological activities, including distribution analysis of dosed radio-active fullerene and interactions with DNAs. We have synthesized radio-active water-miscible fullerene carboxylic acid by [3+2] cycloaddition of trimethylene methane. When administered orally to rats, fullerene was not efficiently absorbed and was excreted primarily in the feces. When injected itravenously, it was distributed rapidly to various tissues, and most of the material was retained in the body after one week. We have expanded fullerene's photo-induced biological activities, targeting DNAs. Thus, we have designed and synthesized a novel fullerene-oligonudeotide conjugate, and examined its DNA nicking ability. When this conjugate was incubated and irradiated with oligonuculeotides with target sequence, site specific nicking of the oligonuleotide was achieved. We further continued investigating the interactions of fullerene with DNAs and found that fullerene-polyamine conjugate tightly binds to DNA in a non-specific manner, and this new fullerene gave first example of photoinduced covalent bond formation of fullerene with DNA base.
The pradimicin-benanomicin antibiotics constitute a new class of natural products with a benzo[a]naphthacenequinone core with an amino acid and a disaccharide. They show anti-fungal and anti-HIV activities, which are related to the selective binding abilities to mannose-rich oligosaccharides, e.g. the mannane surface of fungi or gp 120 of HIV. The unique structure as well as the significant biological activities led us to undertake the synthesis of this class of compounds. The most intriguing challenge among the synthetic issues is the control of the vicinal diol stereogenicities at the corner of the pentacyclic skeleton comprises, to which we explored the possible use of intramolecular pinacol coupling of bis-aldehyde. Along these lines, we found that this reaction is useful for the above issue. The SmI_2-mediated pinacol coupling of biphenyl o, o'-dialdehyde was found to proceed stereoselectivelly and gave only trans diol, which enabled us to achieve the synthesis of the trans diol structure of B ring as diastereomerically pure form. Furthermore, the reaction proved to proceed stereospecifically, thereby enabling the full transmission of the axial chirality into the two central chiralities. Thus, we studied the stereoselective and -specific pinacol coupling of bisaldehyde, providing the requisite trans-diol in enantio- and diastereomerically pure form, which served as a basic strategy for the total synthesis of the pradimicin-benanomicin class antibiotics. Total synthesis of the pradimicinone, the common aglycon of the class of natural products, has been achieved.
Radical cyclization is one of the most useful methodology for the formation of carbon-carbon bonds and has been used for the syntheses of natural products that have polycyclic system. Although regio- and stereoselectivities of radical cyclizations have been well studied, it is quite difficult to predict quantitatively the stereoselectivity of radical cyclizations. Herein, we describe about the method of stereochemical prediction for radical cyclization based on MM2 transition state models and its applications for the synthesis of steroid compounds. We planned two strategies using radical cyclization for the synthesis of steroid compounds. First approach is the synthesis of vitamin D_3 CD-ring 2 by a "radical cyclization-trapping reaction" of ω-iodoacrylonitrile derivative 5. Second approach is the synthesis of steroid BCD-ring 9, which is known as an important intermediate for the synthesis of progesterone, by a "tandem radical cyclization" of acyclic intermediate 9 or ten-membered ring compounds 12, 21. MM2 transition state model calculations suggested that the initial radical cyclization of all intermediates 5, 12 and 21 should predominantly give cyclopentylradicals 7A, 17 and 22, respectively, which have a desired trans relative stereochemistry between 13-Me and 14-H. Indeed, radical cyclizations of the intermediates 5, 12 and 21 gave the cyclized products in high yield and stereoselective syntheses of steroid intermediates 2 and 9 were accomplished. Now we examined about solid phase approach to the synthesis of steroid intermediate 9 using a tandem radical cyclization. In this session, we will discuss about detail results of calculations and experiments.
Popolohuanone E (1) isolated from the Pohnpei marine sponge Dysidea sp. along with the known arenarol (2), is a potent inhibitor of topoisomerase-II and exhibits highly selective cytotoxicity against human non-small cell lung cancer cells. The structure of 1 was revealed to have a unique 3,7,8-trihydroxydibenzofuran-1,4-dione skeleton which possesses two identical cis-fused decalin moieties the same as in 2. It has been proposed that 1 may be produced biogenetically by oxidative dimerization of 6'-hydroxyarenarol (3). Its remarkable biological properties, unique structural feature, and plausible biogenetic pathway make 1 an exceptionally intriguing and timely target for total synthesis. To date, none of the total syntheses of 1 have been reported. Herein we report a method for the synthesis of the model compounds for the tricyclic core of 1. Moreover, by employing this method as the key steps, the synthesis of the compound 29, which possesses the full carbon framework with the requisite asymmetric carbons involved in 1, was successfully achieved. Our synthetic strategy for 1 features the biogenetic-type annulation of the phenolic segment 4 with the quinone segment 5 to regioselectively construct the tricyclic core of 1 as the key step. At first, we investigated the synthesis of the model compounds 10a-10d for the tricyclic core of 1 to explore the feasibility of our synthetic strategy. The regioselective annulation of the phenols 11a-11d with the quinones 12a-12d turned out to be effected by a two-step sequence, giving rise to the requisite dibenzofuran-1,4-dione systems 10a-10d. Having completed the synthesis of the model compounds 10a-10d, we next pursued the synthesis of the cis-fused decalin segment 6 required for the synthesis of the phenolic segment 4 and the quinone segment 5, starting from the enantiomerically pure (-)-Wieland-Miescher ketone derivative 9. The sequence involves ortho ester Claisen rearrangement of the allylic alcohol 18 to construct the C-1 quatemary carbon and Ir-catalyzed hydrogenation of the exocyclic olefin 20 to control the C-2 stereochemistry. Both the phenolic segment 4 and the quinone segment 5 were efficiently synthesized through the coupling reaction of 6 with the corresponding aryllithiums 7 and 8, respectively. The crucial annulation of the phenolic segment 4 with the quinone segment 5 was accomplished by employing the reaction sequence similar to that described for the synthesis of the model compounds 10a-10d, providing the desired dibenzofuran-1,4-dione derivative 29 through the coupling product 28. Since the compound 29 is considered as a potential advanced key intermediate in our designed synthetic strategy, conversion of 29 to the target molecule 1 is in progress. We have succeeded in developing a general and efficient synthetic pathway to the 2,6-disubstituted-3,7,8-trihydroxydibenzofuran-1,4-dione derivatives representing the central tricyclic core of Popolohuanone E (1). By employing this reaction sequence as the key steps in our synthetic strategy for 1, the synthesis of the compound 29 which possesses the full carbon framework with the requisite asymmetric carbons involved in 1, was successfully achieved. Work on the total synthesis of 1 is being pursued in our laboratories.
Bryostatins, polyether macrolides isolated from the marine bryozoans Bugula neritina L. and Amathia convouta, have remarkably powerful antineoplastic activities and the potential to promote kinase C activity attracting the interest of many synthetic chemists. In particular, bryostatin 1 is now undergoing the Phase II clinical test in National Caner Institute and is recognized as a promising anticancer agent. From the biological and synthetic points of view, synthetic studies on bryostatins, particularly bryostatin 3, have been carried out. Based on the retrosynthesis of bryostatin 3 (1) (Scheme 1), we first synthesized the two segments, the top-half fragment (2) and the bottom-half fragment (3). Using Julia-Lythgoe olefination method, coupling of the two segments afforded the seco-acid derivative, which had the same functional groups as those of bryostatin 3 except for the C_<13> functionality. The following macrolactonization afforded the desired lactone (18), which presumably adopts a keto form at the C_9 position. Further synthetic study on bryostatin 3 is in progress.
A Diels-Alder (DA) reaction of 3-hydroxy-2-pyrone (1) with electron-deficient dienophile (2) was catalyzed by base and gave adducts (3 and 4) under very mild conditions. The reaction with the reactive dienophiles (2a-d) afforded corresponding bicycloadducts in nearly quantitative yield with complete stereospecificity and regioselectivity. But with the less reactive dienophiles (2e and f), the reaction only afforded the adducts in 30-0 % yields. Enantioselective DA reaction of 1 and 2a was achieved by using chiral base. By a screening of several optically pure bases it was found that application of cinchonidine as the catalyst in PhCl afforded (-)-3a in 87 % ee. The reaction of 1 with an acrylate derivatives having a chiral auxiliaries (2g and h) afforded endo-adducts (3g and h) in high diastereoselectivity (>95 %de). The resulting adducts 3g and h are practical chiral building blocks for natural product synthesis since this reaction can be applied to large scale preparation. Actually, over 10 g of 3g has been prepared with >95 %de. By using these enantioselective and diastereoselective base catalyzed DA reaction, biologically active compounds, pseudo-sugar (5b), pericosine B (10b), and a key intermediate of substance P antagonist (18) are being synthesized as shown in scheme 2, 3, and 5, respectively.
Pironetin 1 (PA-48153C) is a novel unsaturated δ-lactone derivative isolated from the fermentation broths of Streptomyces species which shows immunosuppressive and plant growth regulating activities. Several total syntheses of 1 have been reported to date, but we show here a novel approach to 1 via a convergent route using our chiral building block, (1S,5S,6R)-5-hydroxybicyclo[4.1.0]heptan-2-one 5. We dissected pironetin at C6-C7 and employed the coupling of the epoxide 16 with the dithiane 25, which would be derived from 5, as a key step. The THP ether of 5 was methylated and the following reduction gave an inseparable mixture of four diastereomers 8. After etherification, removal of THP group afforded separable isomers 9a and 9b in a ratio of 13 to 1. The desired major isomer 9a was oxidized to ketone 6. Oxidation of 6 via silyl enol ether with osmium tetroxide gave ketol 21 in quantitative yield. Reduction of cyclopropylketone moiety of 21 under Birch condition afforded a mixture of 22a and 22b. The ketol moiety of 22a and 22b was oxidatively cleaved with lead tetraacetate in benzene-methanol to give the single aldo-ester 23. Acetalization of 23 followed by reduction of ester group gave aldehyde 24. Introduction of C2-unit via Takai's protocol as E-olefin formation followed by thioacetalization afforded the dithiane 25, as C7-C14 unit of pironetin. Meanwhile, the epoxide 16 as C1-C6 unit was prepared from known epoxy alcohol 3 in 6 steps. Coupling reaction of two units, the dithiane 25 and the the epoxide 16, successfully gave the product with the whole skeleton 26 in high yield. Mercuric ion assisted hydrolysis of the thioacetal 26 gave ketol 27. Selective reduction of β-hydroxyketone 27 by Mori's method afforded and-diol 28a predominantly (91: 9). Finally, desilylation and oxidation with manganese dioxide afforded pironetin 1.
Marine carotenoids halocynthiaxanthin 1, mytiloxanthin 2 and crassostreaxanthin B3 have characteristic structures, commonly possessing a monoacetylenic end group. The cyclopentyl end group of 2 is believed to be formed in nature from the epoxide end group of 5,6-epoxy carotenoids such as 1 (Scheme 1, route a). It is also conceivable that 3 including the novel tetrasubstituted olefinic end group arises from epoxy carotenoids by opening of the C-6-oxygen bond of the oxirane ring and subsequent migration of the methyl group at the C-1 position (route b). We found that treatment of the epoxide 4a having a part structure of epoxy carotenoids with Lewis acids gave the cyclopentyl ethyl ketone 6a possessing the same configuration as 2, and the acyclic tetrasubstituted olefinic methyl ketone 5 including the partial structure of 3 (Scheme 2). It supported the proposed metabolic pathway of 5,6-epoxy carotenoids. Toward the biomimetic synthesis of 3, we examined the reaction of epoxides having several substituents at C-6 position with Lewis acids. Among these epoxides, the epoxide 23 was found to provide the tetrasubstituted compound 24 as a major product. This could be converted into the aldehyde 32 in 8 steps. This was transformed into the compound 33 through the coupling reaction with the vinyllitium 26. Then, the first total synthesis of 3 was accomplished via the double Wittig condensation of 33 with phosphonium salts 34 and 36.
A new method for synthesis of chiral cycloalkane derivatives was explored. The new method involves the formation of cycloalkane in a one-pot process using the anion of allyl phenyl sulfone and chiral epoxymesylate (Scheme 1). Total synthesis of marine eicosanoid constanolactone E and bacillariolides I-III was achieved using this new method. Constanolactone E (1), isolated from the marine red alga Constantinea simplex, is a cyclopropane-containing eicosanoid. Reaction of the lithio derivative of allyl phenyl sulfone with chiral epoxymesylate 2 gave cyclopropane 3 as a diastereomeric mixture at C-8 in a ratio of 8: 1 (Scheme 2). Cyclopropane 3 was converted to sulfone 5 via aldehyde 4. The Julia's olefination of sulfone 5 with aldehyde 6 afforded E-olefm 9. Following the selective deprotection of TBDMS ether in 9, hydroxymethyl group was converted to the methoxycarbonyl group. Deprotections of acetonide and TBDPS ether, and then lactonization completed the synthesis of constanolactone E (1). Bacillariolides I (11), II (12) and III (13), isolated from the marine diatom, Pseudo-nitzschia multiseries, are cyclopentane-containing structurally unique eicosanoids. Reaction of the lithio derivative of allyl phenyl sulfone with epoxymesylate 19, prepared from diol 14, gave cyclopentane 20 as the sole product (Scheme 3). Cyclopentane 20 was converted to lactone 21 by oxidation of terminal olefin, Jonens oxidation and removal of the phenylsulfonyl group. Deprotection of MPM ether in 21 afforded an alcohol, which was oxidized by the Swern procedure to give the aldehyde 22. Coupling reaction of the aldehyde 22 with Wittig reagent, prepared using phosphonium salt 23 afforded the (8Z,11Z,14Z,17Z)-tetraene. Finally, deprotection of MOM ether completed the synthesis of bacillariolide II (12). Bacillariolides I (11) and III (13) were synthesized from lactone 21 via epimerization of the C-7 position, respectively (Scheme 4).
We recently developed a novel acyclic stereocontrol based on the stereospecific methylation of γ,δ-epoxy acrylates with trimethylaluminum in the presence of water by which both anti and syn compounds can be highly stereoselectively synthesized from trans- and cis-γ,δ-epoxy acrylates, respectively. We report here stereospecific internal alkylation of terminal epoxides and stereospecific construction of asymmetric quaternary carbons via γ,δ-epoxy acrylates. We also report synthetic studies toward total synthesis of a marine natural product discodermolide and epothilone based on the above methodologies. Stereospecific Internal Alkylation of Terminal Epoxides Regio- and stereoselective internal alkylation of terminal epoxides has little been known. We designed such a reaction using γ,δ-epoxy acrylates with trimethylaluminum. The reaction of terminal γ,δ-epoxy acrylates 1 and 2, easily prepared from D-mannitol, with excess trimethylaluminum in the presence of water proceeded regiospecifically at the γ-position to give 3 and 4 as the sole product, respectively, with maintenance of optical integrity. Stereospecific Construction of Asymmetric Quaternary Carbons via γ-Alkyl-γ,δ-epoxy Acrylates We found that the reaction of γ-alkyl-γ,δ-epoxy acrylates with trialkylaluminum and water occurs regio- and stereo-specifically at the γ-position as well yielding an asymmetric quaternary carbon. Thus treatment of 5 and 6 with excess trimethylaluminum in the presence of water gave 7 and 8 as a single product, respectively, in which a methyl group was stereospecifically introduced at the γ-position with net inversion of configuration. Synthetic Studies on Discodermolide and Epothilone We set out synthesis of discodermolide having potent immunosuppressive activity based on the above stereospecific acyclic stereocontrol. Discodermolide was devided into three segments in which the segment B having three contiguous chiral centers and the segment C possessing five chiral centers have been highly stereoselectively synthesized as shown in Schemes 5 and 6, respectively. Stereoselective synthesis of the C1-C9 segment of epothilone having potent anticancer activity was also carried out as shown in Scheme 7 in which five asymmetric centers was highly stereoselectively constructed by repeating the above methylation reaction.
Sulfobacins A (1) and B (2), novel von Willebrand factor (vWF) receptor antagonists, have been isolated by Kamiyama et al.^1 from the culture broth of Chryseobacterium sp. (Flavobacterium sp.) NR 2993 in a soil sample collected in Iriomote Island, Okinawa Prefecture, Japan. Sulfobacin A (1) was also isolated by Kobayashi et al.^2 as flavocristamide B from Flavobacterium sp. in the marine bivalve Cristria plicata collected in Ishikari Bay, Hokkaido, Japan. Sulfobacins A (1) and B (2) inhibit the binding of vWF to the GPIb/IX receptors in a competitive manner with IC_<50>s of 0.47 and 2.2 μM, respectively.^<1a> Furthermore, sulfobacin A (1) was found to have inhibitory activity against DNA polymerase α. 2 We now wish to report the first total synthesis of sulfobacins in an effective and stereoselective manner. The left fragment 4 was obtained through the asymmetric hydrogenation of the corresponding β-keto ester 9 with a chiral Ru(II) catalyst atmospheric pressure according to the Genet's method. The right fragment 5 was constructed by the asymmetric aldol reaction using the Schiff base derived from (+)-2-hydroxy-3-pinanone ((+)-HyPN, 11). Sulfobacins were prepared from the corresponding thioacetates (17 and 24), which were constructed by coupling the left fragment 4 with the right fragment 5 by use of diethyl phosphorocyanidate (DEPC, (C_2H_5O)_2P(O)CN) as a coupling reagent The synthetic sulfobacins were identical with the natural ones in every respect (IR, ^1H NMR, ^<13>C NMR spectra, [α]_D and TLC). Thus we have completed the first total synthesis of sulfobacins.
Since 1981, a number of polycyclic ethers have been isolated from marine dinoflagellates. Much attention has been paid to the synthesis of these compounds due to their unusual structures, biological activities, and the rarity in nature. Gambierol, which has a 6,6,6,6,7,6,6,7-polycyclic ether skeleton including 18 stereocenters and a triene side chain, was isolated from the cultured cells of Gambierdiscus toxicus by Yasumoto in 1993. The compound shows toxicity against mice (LD_<50> 50μg/kg), and the symptoms resemble those caused by ciguatoxins inferring the possibility that it is also implicated in ciguatera poisoning. We now report the stereocontrolled construction of the AB, E, and H ring systems of Gambierol as parts of its total synthetic study. First, synthesis of the AB ring system of gambierol was achieved from 2-deoxy-D-ribose. The key steps were the stereoselective allylation of the aldehyde 2, corresponding to the B ring, and the intramolecular hetero-Michael reaction of 5. Next, the synthesis of the E ring was accomplished from D-ribose via the intramolecular reaction of allylic stannane 24 as a key step. The undesired stereoisomer 26 formed in this reaction was converted to the desired product 33 by using DBU mediated isomerization. Finally, stereoselective synthesis of the H ring was achieved from 2-deoxy-D-ribose by using the intramolecular reaction of allylic stannane 43 as a key step. Modified Stille coupling was successfully applied for the construction of the triene side chain to give 57.
Palldium mediated cascade reaction has emerged as one of the most important methods for the preparation of a wide range of molecular frames. Here, we report two types of novel palladium mediated intramolecular cascade reactions triggered by strain release of four membered ring and their application to the natural product synthesis. 1) Palladium(0)-catalyzed cascade insertion-ring expansion reaction Palldium(0)-catalyzed cascade reaction of allenylcyclobutanols 5 and aryl halides 6 gave rise to aryl substituted cyclopentanones 7-9. The reaction pathway was presumed via the ring transformation of the π-allylpalladium intermediate in situ generated by the carbopalladation of allene. We also examined the intramolecular version of this cascade reaction. Consequently, substrates 10a,b and 12a,b, where the allene and vinyl iodide units were tethered by carbon chains, were successfully converted to the corresponding 5,7 and 5,8 ring systems 11 and 13-15. This sequential process provides a unique entry into the biologically important natural products having 5,7- and 5,8-fused ring frameworks. 2) Palldium(II)-mediated cascade ring expansion-insertion reaction The treatment of the isopropenylcyclobutanols 20a,b with palladium(II) complex afforded hydrindans 21-24 by the successive ring expansion and intramolecular insertion reactions. In this cascade reaction, diastereoselectivity of the products was found to be largely dependent on solvents employed. 3) Asymmetric synthesis of (+)-equilenin We applied the above reaction to synthesis of one of steroids, (+)-equilenin (25). In this process, novel synthetic method of chiral cyclobutanone was developed by using chiral (salen)manganese(III) catalyst 33. Thus, the aryl substituted cyclopropylidene 29 was converted to the chiral cyclobutanone 28 by asymmetric epoxidation-ring expansion reaction in the presence of 33. The isopropenylcyclobutanol 27, prepared from 28, was diastereoselectively converted to the trans-naphthohydrindan 26 using palladium(II) acetate in HMPA-THF. Asymmetric synthesis of (+)-equilenin (25) was completed by the transformation of the product 26.
(+)-Obtusenyne 1, isolated from red alga (Laurencia obtusa) by Imre et al., has some isomers from another alga as well as sea here, such as (-)-12-epi-obtusenynes 2 from sea hare (Aplysia dactylomela) by Schmitz et al. and (-)-12?,13-epi-obtusenynes 3 from red alga (Laurencia pinnatifida) by Norte et al. Their common structures involve 9-membered ether rings having halogen functional groups and alkyl side chains as geometric isomers of enynes with various stereochemistries. The absolute stereochemistries of 1 and 2 have been determined by X-ray crystallographical analyses and various chemical methods, while that including C(12) on 3 has not been defined yet. We planned to synthesize 1 and determine the stereochemistry of 3 synthetically. The direct bromination to cyclic enol ether 10 with NBS in MeOH afforded a bromo acetal 23 which was reduced to bromo ether 24 with Et_3SiH and SnCl_4. The alcohols 27 and 28, which were obtained by epoxidation of 10 with dimethyldioxirane followed by reduction with DIBAH, were stereoselectively converted to 29 and 30 with CBr_4 and Oct_3P, respectively. The (Z)- and (E)-enynes were constructed stereoselectively with Sonogashira coupling via (Z)-monobromo and (E)-monoiodo olefin, respectively, obtained by Uenishi and Takai reactions. The first total syntheses of (+)-obtusenyne, (-)-12,13-epi-obtusenynes 4, and (+)-13-epi-obtusenynes 5 were thus achieved. Furthermore, the absolute configuration of C(12) on 3 was clarified as S including its absolute whole structure.
Wortmannin (1) is a selective inhibitor of phosphoinositide 3-kinase (PI 3-kinase) and widely used as a tool for studies in intracellular signal transduction. We anticipated that its derivatives would give some interesting properties, especially subtype selectivity, so we started synthetic programs with two strategies. First we started the synthesis with hydrocortisone as a starting material and achieved its first total synthesis. However, since its long synthetic path and low overall conversion, we tried the second synthetic strategy, a catalytic asymmetric synthesis. We designed a synthetic plan shown in Scheme 3, in which two palladium-promoted reactions were planned as key steps, that is, 13→12 and 15→14. We first tried to prepare 16 in a catalytic asymmetric manner. Robinson annulation reaction using 17 and 18 in the presence of asymmetric base catalyst did not afford the optically active 16. Then we tried a Lewis acid catalyzed cyclization reaction using enol ether 23 as a substrate. 16 was obtained in reasonable chemical yield in the presence of several kinds of achiral Lewis acids such as Sc(OTf)_3. To our pleasure, it was found that the addition of iodide ion promoted the reaction. However, chiral Lewis acid catalysts reported so far could not promote the reaction. So we considered other possibilities and attempted at a kinetic resolution at intramolecular Heck cyclization step. After several efforts, we could obtain optically enriched 36 using BINAP as an asymmetric ligand in toluene. Interestingly, β-MOMO group in 35β is required for the construction of quartenary carbon center in correct configuration; α-configuration at this group mainly lead to the product, which has incorrect configuration at quartenary center formed. From 36 we explored the synthetic path toward 1 and could obtain 45, which possesses basic carbon skeleton of 1. Now we are investigating the synthesis of 1.
The antitumor antibiotic neocarzinostatin consists of an apoprotein and the chromophore 1. We describe herein a synthetic study of 1 and the 10-membered ring analog 2. An expeditious synthesis of trans-4,5-dihydroxy-2-cyclopenten-1-one 7, a key intermediate for the synthesis of 1 and 2, was achieved via an enzymatic desymmetrization of meso compounds 5 and 8. Construction of the 9-membered epoxydiyne core was accomplished by intramolecular cyclization of the aldehyde 12 with CeCl_3/LiHMDS. However, a conversion of the resultant diol 13 to the dienediyne 14 was unsuccessful. We then examined a dehydration of the secondary alcohol 19, which was successfully achieved using Tf_2O/Et_3N to give a 9-membered dienediyne 20. Glycosylation of 21 provided 23 in moderate yield, but subsequent coupling reaction of 24 with epoxydiyne fragment 25 did not proceed at all. Therefore, the coupling was conducted before glycosylation. The glycosylation of 29 with 22-β proceeded stereoselectively to give 30-α in good yield. Further synthetic study directing toward 1 is now in progress. An advanced 10-membered ring analog 39, which possesses an important functional groups in 1, such as an epoxide, naphthoate and N-methylfucosamine moieties, were synthesized. It was remarkable that 39 showed a very high DNA cleaving activity compared to the analogs which have no sugar moiety, and a different base sequence selectivity from 1. Synthesis and the DNA cleavage of 2 will be also described.
The regioselective synthesis of (+)-K252a was carried out by developing an efficient indolocarbazole synthesis for the preparation of 21. The total synthesis requires 23 steps from commercially available materials with an overall yield of 2.3% from indole-3-acetic acid. During synthetic studies on the regioselective synthesis of (+)-K252a, the regioselective synthesis of the N-protected K252a-aglycon moiety 8 was also achieved by employing the novel tin-mediated indole synthesis recently developed in our laboratories.