Salacinol and kotalanol are new class of potent glycosidase inhibitors, isolated by presenters from Ayruvedic traditional medicine Salacia reticulata, having the unique zwitter-ionic structure comprising of 1-deoxy-4-thio-D-arabinofranosyl cation and the sulfate anion in the alditol side chain. Elucidation of the stereostructure of kotalanol, which has long been unknown and very recently approved by Pinto and co-workers by the synthesis, by the independent manner involving the degradation of natural kotalanol is presented. In the detradation of 2, characteristic deprotective cyclization of heptitols to anhydroheptitols was found to occur to a large extent. Structural elucidation of salalprinol, one of the sulfonium analogs recently isolated from the same species, by the synthesis is also presented. Revisions of the structures of new constituents from Salacia species, neosalacinol and 13-membered cyclic sulfoxide, recently reported as constituents responsible for the α-glucosidase inhibitory activity by Minami and Osaki and co-workers, respectively, are presented. In relation to this study, synthetic route of de-O-sufonated salacinol, which was proved as potent as 1, has been developed. Finally, conditions for the quantitative analysis of 1, 2, and their de-O-sulfonates (3 and 4) by LC-MS for the qualitative evaluation of Salacia extracts is discussed.
The marine animal commonly known as the "firefoms" is dangerous for humans because if they are carelessly handled with bare hands, serious dermatitis results. However the toxic substance of these animals has remained unknown in a long while. We isolated as a novel amphipathic substance, named complanine, from an amphinomid polycheates, Eurythoe complanata. The structure of complanine was spectroscopically revealed to be a trimethylammonium with an unsaturated carbon chain. The total synthesis of (-)-complanine was also accomplished by a synthon approach, by using a chiral malic acid as a starting material. The stereochemistry of this molecule was determined to have an R configuration. Syntheses of the related compounds, neocomplanines, were also achieved. From a biological point of view, complanine enhanced PKC activity in combination with TPA in the presence of Ca^<2+> in vitro; this result suggest that it could bind PKC at the same site as phosphatidylserine. This may explain the molecular mechanism behind its inflammation-inducing activity.
Tabernaemontana sphaerocarpa Blume and T. elegans Staph. are a member of the Apocynaceae family that occur in Indonesia, and are found mostly in Java. Traditionally, the leaves have been used as a laxative, the flowers as a cardiotonic agent, and the latex for removing warts. Tabernaemontana species so far have been shown to produce various skeletal alkaloids, including iboga-type alkaloids such as ibogamine, aspidosperma-type alkaloids such as taberhanine, and vobasinyl-ibogan bisindole alkaloids such as conodiparine A. In our search for structually and biogenetically interesting alkaloids from tropical plants found in Indonesia, novel tetrakis-monoterpene indole alkaloid, alasmontamine A, consisting of bis-vobtusine type skeleton, and two new bis-monoterpene indole alkaloids, biscarpamontamine A, having an aspidosperma-iboga type skeleton, and biscarpamontamine B, based on aspidosperma-aspidosperma type skeleton, were isolated from Tabernaemontana elegans and T. sphaerocarpa, respectively, together with vobtusine, vobtusine lactone, 3-hydroxyvobtusine, ibogamine, voacangine, 3-hydroxyvoacangine, and vobasine. The structures of alasmontamine A and biscarpamontamines A and B were determined by 1D & 2D-NMR data (^1H-^1H COSY, HSQC, HMBC, NOESY, TOCSY) and CD spectra. Five bis-monoterpene indole alkaloids isolated in this research was evaluated for cytotoxicity against five human cancer cell lines, HL60, RPMI8226, NCI-H226, HCT116, and MCF7. As a result of analysis of cytotoxicity mechanism against HL60 cells, vobtusine was found to induce apoptosis via the caspasse-dependent pathway.
Club moss (Lycopodiaceae) is known to be a rich source of Lycopodium alkaloids possessing unique heterocyclic ring systems such as C_<16>N, C_<16>N_2, and C_<27>N_3, which have attracted great interest from biogenetic, synthetic, and biological points of view. In our continuing efforts to find new Lycopodium alkaloids, lycopladine H (1), lyconadins C-E (2-4), lycoplaidines F-G (5-6), and complanadine E (7) were isolated from Lycopodium domplanatum. The structures for 1-7 were elucidated on the basis of spectral data. Lycopladine H (1) is an unprecedented C_<16>N-type Lycopodium alkaloid possessing a novel fused-tetracyclic ring system consisting of an azocane ring fused to a [2,2,2,]-bicyclooctane ring and 3-piperidinone ring, Lyconadin C (2) is a new tetracyclic Lycopodium alkaloid consisting of cyclohexane, cycloheptane, pyperidine, and pyridone rings. Lyconadin C (2) might be a biosynthetic precursor of lyconadin A. Lyconadins D (3) and E (4) are the first fastigiatine-type alkaloids isolated from Lycopodium complanatum. Lycopladine F (5) is a rare C_<16>N_2-type Lycopodium alkaloid possessing an amino acid residue (C_4N), while lycopladine G (6) is a 17-keto and 20-methylester form of lycopladine F (5). Complanadine E (7) is a new dimeric Lycopodium alkaloid consisting of two C_<16>N_2-type skeletons, which corresponds to 1,2,3,4,5,N-1-hexahydro complanadine A.
NMR techniques are important to determine the chemical structures of natural products. Since the structures of natural products are rich in varieties, the corresponding NMR spectral data give us important structural information. We started to collect NMR spectral data at spring 2002 to build a database named by "CH-NMR-NP". The data bave been collected from research paters mainly from 2000 to the present. The Journals are selected to be nine (Chem. Pharm. Bull., J. Antibiotics, J. Nat. Prod., Phytochemistry(Phytochemstry Lett.), Tetrahedron, Tetrahedron Lett., J. Org. Chem. Eur., J. Org. Chem., Magn, Reson. Chem.), and if necessary, we collect the data from other Jounals. At present (July 2009) total number of NMR spectra of natural products is 15,500. An item of the "CH-NMR-NP" includes ^<13>C and ^1H NMR data, name (trivial name or chemical name), molecular formula, molecular weight, origin (natural resource), solvent, shift references, ^1H measuring frequency, reference (top author's name and Journal information) and comments. One of the important criteria to adopt the data is that all the ^<13>C shift values are described in a pater and look reasonable to the chemical strucre. We do not take a compound without any description on ^1HNMR data. If the structures are very close with each other, we adopt one of them. The database is not comprehensive. The "search" function is available for sub-structure, name, molecular weight, ^<13>C and ^1H chemical shifts and so on. As the display system the chemical structure with numbering is shown where the numbers correspond to the spectral assignment. All the carbons on a chemical structure are given numeric numbers except for equivalent positons. The ^<13>C and ^1H shifts are shown with the spectral assignments. If the precise description was made for ^1H NMR incluging the coupling constants, we included the information. The ^<13>C spectral pattern is created from the shift values and the number of equivalent carbons. Each peak is shown with the assignment by the number on the chemical structure and can be classified by C, CH, CH_2 and CH_3. At present, a trial version of CD-ROM of CH-NMR-NP is available which has functions of search compounds and display the spectral data.
For the quantification of natural products, chromatographic methods such as GC-FID, GC-MS, HPLC and LC-MS are widely used. The reliability of analysis depends on the purities of the target compounds used as reference materials. However, it is very difficult to obtain pure compounds and determine the purities that are traceable to the International System of Units (SI), because most of the target compounds can be only obtained from natural source. For all of these reasons, the present chromatographic methods result in degrading the reliability of analysis data at present. To improve the reliability of the analytical data in the field of natural products chemistry, we are developing quantitative nuclear magnetic resonance (qNMR) as one of simple absolute quantitation methods that is able to determine the purity or content with SI-traceability. qNMR is based on the fact that the signal intensities of a given NMR resonance are directly proportional to the molar amount of that nucleus in the sample. If appropriate instrument setting has been made, any NMR use can apply NMR spectroscopy to quantitative analysis of any compound. To build up SI-traceable qNMR analysis and boost the convenience, we contrived that 2-Dimethyl-2-silapentane-5-sulfonate-d_6 sodium salt (DSS-d_6) and hexamethyldisilane (HMD) were set as qNMR reference materials after correction of the concentrations by using certified reference materials (CRMs) such as dietyl phthalate (DEP) and potassium hydrogen phthalate (PHP). Thus qNMR can calculate the purity or content with SI-traceability from the ratio of the signal intensities of target compound to qNMR reference materials. qNMR analysis needs no reference compound but alst ti is rapid and simple with overall analysis time of only 10-20 min. In this study, we show two applications using qNMR analysis; the abslute quantification of carminic acid in the reagents and cochineal dyes, the direct quantification of rutin and quercetin in buckwheat without separation step. Our approach tus represents an absolute quantitation method with SI-traceability that will be readily utilized to analysis and quality control natural products.
In our ongoing efforts to isolate novel marine cyanobacterial metabolites with antitumor activity, we found Bisebromoamide (1) and Biselyngbyaside (2). We report here the structure determination and preliminary biologicaal characerization Lyngbya sp. was collected at Okinawa Prefecture. A crude organic extract of this material was subjected to bioassay-guided fractionation by solvent partition, ODS-HPLC to afford Bisebromoamide (1) as a colorless oil. The molecular formula of 1 was determined to be C_<51>H_<72>BrN_7O_8S on the basis of HR-ESIMS and NMR data. The gross structure of 1 was established on the basis of spectroscopic data. To determmine the absolute configuration of the eight chiral centers, we conducted chiral HPLC analysis of the acid hydrolysis. These analysis identified 4-MePro and 2-Opp as (2S, 4S) and (2S). Therefore, the absolute stereostructure of Bisebromoamide (1) was determined to be as shown in Figure 1. Bisebromoamide (1) exhibited potent cytotoxicity against HeLa S_3 cells with an IC_<50> value of 0.04μg/mL. Bisebromoamide (1) was evaluated against a disease-oriented panel composed of 39 human cancer cell lines, the average GI_<50> value of 0.1μg/mL. We reported relative stereostructure of 2 last year, determination of he absolute stereostructure of 2 was described here. The absolute stereochemistries of C3, C17, and the 3-O-methylglucoside moiety in 2 were determined by the modified Mosher's method and synthetic means. These results established that the absolute stereochemistries of C3 and C17 were 3S and 17R. Futhermore, tribromobenzoylation of sugar afforded methyl 2,4,6-tri-O-(4-bromobenzoyl)-3-O-methyl-α-D-glucose, which was identified by ~1H NMR data and CD data, it was prepared from D-glucose. The absolute stereochemistries of C7 and C10 were determined by enantioselective synthesis of the degradation product from natural 2, 7S and 10S, respectively. Based on these findings, the complete stereostructure of Biselyngbyaside (2) was determined as shown in Figure 5. Biselyngbyaside (2) exhibits broad-spectrum cytotoxicity in a human tumor cell line panel, and 2 likely inhibits cancer cell proliferation through a novel mechanism.
Scince modified Mosher's method was reorted an 1991, many reports have been pubished on the NMR methods that enable the elucidation of the absolute configuration of chiral alchols, amines, carboxylic acids, diols, and sulfoxides. Most of these reports showed us that the derivatives of α-methoxy-α-(trifluoromethyl)phenylacetic acid (MTPA) are powerful tools to deteremine the absolute configuration and widly apllicable to various kind of compounds. On the other hand, our group has developed alternative method to deteremined absolute configuration by using of axially chiral reagents like binaphthalene derivatives, and the methodlogy to elucidate the stereochemistry of chiral alcohols, cyclic chiral alkens, and α-exo-methylene-γ-lactone by using NOEs have been developed. In this synposium, we will present the new methodlogy for the determinations of absolute stereochemisties of diols and β-chiral primary alcohols with axially chiral reagent: MBC (1) and MBCA (2), which derivatize them to collesponding acetals and esters, respectively. NOE correlations of the derivatives revealed their configuration, and it means that the absolute configurations of the diols and β-chiral primary alcohols are determined, because the stereochemistry of the reagents were known. In addition, we propose determinations of the absolute configuration of α-chiral and β-chiral primary amines with 1.
The leaves of soybeans (Glycine max L.) suddenly senesce (wither) over the field even in midsummer. Although this senescence is believed to be induced so as to deliver enough nutrients to the soybean seeds, what causes it remains unknown. The senescence inducing activity (SIA) of pod extracts reached its maximum(49%) on July 31 when soybean began to senesce. This suggests that the SIA of pod extracts triggers the process of monocarpic senescence. However, the amoung of abscisic acid, senescence inducing substance, did not aler signnificantly over this period. Moreover, the detected concentration could not explain the SIA on July 31. This difference suggests the presence of other substances in the crud extracgts besides abscisic acid. It was revealed that jasmonic acid and 12-hydroxyjasmonic acid isolated from soybean pods extracts accelerate the senescence induced by abscisic aced. Quantification analyses employing GCMS showed us that the amount of 12-hydroxyjasmonic acid in pods synchronizes with the commencement of senescence. The maximum amount reached 10.2μmol/kg, which would be sufficient to enhance the activity of abscisic acid. On the other hand, the amount of jasmonic acid was almost constant (around 0.8μmol per kg of pod) during this period. These results have led to the conclusion that 12-hydroxyjasmonic acid plays the role of a synergist for abscisic acid in soybean senescence.
Highly efficient screening method of naturally occurring products that bind to a specific target protein was demonstrated by using hVDR-magnetic beads. The native ligand 1α,25(OH)_2VD_3 was selectively bound by hVDR magnetic beads when present in a mixture of natural compounds. Furthermore, this method was proven to be applicable to the identification of natural products, which interact with a specific protein immoblized on the beads, from an extract of a natural resource. Two new natural compounds were isolated by this method. This approach will be helpful for the discovery of novel, neturally occurring products that bind to specific target proteins. A high-throughput screening system for small molecule-inhibitors of the basic helix-loop-helix (bHLH) transcriptional repressor factor Hes1 was construcrted. Successful demerization of Hes1 immobilized on a microplate and fluorophore (Cy3)-labeled Hes1 was confirmed. Using this system, several natural products were identified as the first Hes1 dimer inhibitors. Of these, two compounds which were isolated from myxomycetes (true slime molds) inhibited Hes1 from N box-dependent suppression of the gene expression in C3H10T1/2 cells.
In prokaryotes, ubiquinone and menaquinone(MK) are obligatory components of the electron-transfer pathway. Escherichia coli, which is a facultative anaerobe, utilizes ubiquinone under aerobic conditions, but uses MK 8 when it is grown anaerobically. By contrast, many Gram-positive aerobes such as Bacillus subtilis contain only MKs. MK biosynthesis is therefore essential for the survival of these strains. MK is biosynthesized from chorismate by seven enzymes (MenA-MenH) in E. Coli. However, a bioinformatic analysis of whole genome sequences has suggested that some microorganisms, including pathogenic species such as Helicobacter pylori and Campylobacter jejuni, do not have orthologs of the men genes, even though they synteesize MK. Here, we investigated this novel pathway in a nonpathogenic Streptomyces coelicolor A3(2) grown on [U-^<13>C]glucose was quite different from that of E. coli. We searched for candidate genes participating in the pathway by in silico screening, and the involmement of these genes in the pathway was confirmed by gene-disruption experiments. We also used mutagenesis to isolate mutants that required menaquinone for their growth, and used these mutants as hosts for shotgun cloning experiments. Metabolites that accumulated in the culture broth of the mutants were isolated and their structures were determined. Taking these results together, we deduced the outline of the alternative pathway, which formed chorismate in a similar manner to the known pathway, but then branched to follow a completely different pathway. Since humans and some useful intestinal bacteria utilize the different pathway, this new pathway would be an attractive target for the development of chemotherapeutics.
Neomycins belong to a family of clinically important 2-deoxystreptamine (2DOS) containing aminoglycoside antibiotics including kanamycin, gentamicin, tobtamycin, and butirosin. In 2005, biosynthetic genes for neomycin were identified from the producer strain Streptomyces fradiae and the functions of those gene products have been extensively investigated with recombinant proteins. Among seven conserved enzymes encoded in the neomycin, butirosin, kanamycin, gentamicin, and tobramycin biosynthetic gene clusters, NeoC, NeoS and NeoE were already reorted to be responsible for the construction of 2DOS from D-glucose 6-phosphate (G6P). In the present study, a putative glycosyltransferase NeoM was characterized as UDP-GlcNAc:2DOS GlcNAc transferase. Comparative analysis of aminoglycoside biosynthetic genes revealed that four conserved ribostamycin-related enzymes couls be responsible for the ribosylation of neamine. As a result of enzymatic analysis with recombinant proteins derived from the butirosin producer Bacillus circulans, we verified that BtrL (NeoL homolog) catalyzes phosphoribosylation of ribostamycin, and BtrP (NeoP homolog) catalyzes following dephospholylation giving ribostamycin. Two conserved neomycin-related enzymes seemed to be involved in the last glycosylation in neomycin biosynthesis. One of the enzymes NeoF was then found to catalyze the GlcNAc transfer reation onto ribostamysin. The NeoF reaction product was further deacetylated by repetitive functional N-Ac-paromamine deacetylase NeoD, followed by dehydrogenation (NeoQ) and transamination (NeoB) leading to neomycin C. In addtion to previous results from our and British groups, all biosynthetic enzymes to synthesize neomycin C from G6P have been finally characterized.
Hybrid class of natural products such as meroterpenoids offer an unique opportunity to engineer their biosynthesis to provide novel structures due to their emergence from both polyketide and terpenoid pathways. Pyripyropene A, a potent adyl-CoA: cholesterol acyltransferase inhibitor by Aspergillus fumigatus, is one of such examples and is truly promising leads for cholesterol lowering drug. The biosynthesis of pyripyropene A involves a fungal iterative type I polyketide synthase (PKS), a prenyltransferase (PT) and a terpene cyclase as key enzymes in the pathway. In order to identify the entire biosynthetic genes necessary for producing pyripyropene A, we have searched a publicly available genomic DNA database of A. fumigatus Af293 for a gene cluster that contains both PKS and PT genes. As a result, we were able to identify a gene cluster that spans 22 kb with 9 genes, indluding genes for PKS, PT, CoA ligase, FAD-dependent monooxygenase (FMO), cytochrome P450 and acetyltransferase, all corresponds to pyripyropene A biosynthesis except for a lack of gene supposed to be involved in a cyclization of terpenoid moiety. The PKS of this cluster enceded by pyr2 possesses ketosynthase (KS), acyltransferase (AT) and acyl carrier protein (ACP) domains but no other domains, which is consistent with a simple reaction involving condensaton and release of a triketide intermediate to produce pyrone, 2, without further modification such as reduction or methylation. The presence of a CoA ligase gene pyr1 is consistent with the formation of unusual CoA substrae, 1, as the first step of its biosynthesis. In order to characterize each gene product, we have undertaken a reconstitution approach by co-expressing them in heterologous fungal host. Two plasmids were employed for co-expression of each gene in the cluster: pTAex3R harboring argB marker and pPTR with pyrithiamine resistant marker. All of the co-expressed genes were placed under the amylase promoter, and these plasmids were introduction into arginine auxotrophic mutant A. oryzae M-2-3 strain by protoplast-PEG method to give transformant and selected for arginige auxotrophy and antibiotic resistance. In these works, hte following points were identified. 1) Pyr1 and Pyr2 are novel enzymes that activate and incorporate nicotinic acid into polyketide system, respectively to produce the pyrone 2. 2) UbiA like prenyltransferase (Pyr6) in the cluster catalyzed prenylation of 2 to 3. 3) Pyr4 is a novel terpene cyclase enzyme involved in cyclization of terpenoid moiet of 4 to yield 5. 4) The basic carbon skeleton of pyripyropene A was reconstitured in heterologous fungal expression system. The functional characterization of the pyripyropene A biosynthetic gene cluster from A. fumigatus led to the discovery of a novel terpene cyclase unprecedented with abnormal sequence character. Identification of homologous genes in other meroterpenoid or indole-diterpene biosynthetic gene clusters suggests that this novel terpene cyclase is involved in the biosynthesis of various terpenoid bearing secondary metabolites produced by fungi.
Natural products display impressive activities against a wide range of targets, including viruses, microbes and tumors. However, their clinical use is hampered frequently by their scarcity and undesirable toxicity. Not only can engineering Escherichia coli for plasmid-based pharmacophore biosynthesis offer alternative means of simple and easily-scalable production of valuable yet hard-to-obtain compounds, but also carries a potential for providing a straightforward and efficient means of preparing natural product analogs. The quinomycin family of nonribosomal peptides, including echinomycin, triostin A and SW-163s, are important secondary metabolites imparting antibiotic antitumor activity via DNA bisintercalation. Previously we have shown the production of echinomycin and triostin A in E. coli using our convenient and modular plasmid system to introduce these heterologous biosynthetic pathways into E. coli. However, we have yet to develop a novel biosynthetic pathway capable of producing bioactive unnatural natural products in E. coli. Here we present successful engineering of the E. coli echinomycin biosynthetic pathway for the production of a synthetic analog TANDEM by site-specific mutagenesis of an echinomycin biosynthetic gene. Furthermore, we report an identification of a new gene cluster responsible for the biosynthesis of SW-163s that involves previously unknown biosynthesis of (+)-(1S, 2S)-norcoronamic acid and generation of aliphatic side chains of various sizes via iterative methylation of an unactivated carbon center. Substituting an echinomycin biosynthetic gene with a gene from the newly identified SW-163 biosynthetic gene cluster, we were able to rationally re-engineer the plasmid-based echinomycin biosynthetic pathway for the production of a novel bioactive compound in E. coli.
Plant type III polyketide synthases (PKSs) exhibit unusually broad, promiscuous substrate specificities; the structurally simple homodimeric proteins accept a variety of nonphysiological substrates, including aromatic and aliphatic CoA thioesters, to produce an array of chemically and structurally divergent unnatural polyketides. Here we present most recent examples including enzymatic formation of (i) tricyclic and tetracyclic unnatural novel alkaloidal scaffolds by PKS1 from Huperzia serrata, and (ii) a C_<19> hexzketide stilbene and a C_<21> heptaketide chalcone by octaketide synthase (OKS) from Aloe arborescens. Manipulation of the enzyme reactions by combination of the precursor directed biosynthesis and sructure-guided engineering of the enzyme would thus lead to further production of unnatural novel polyketide and alkaloid scaffolds.
Polyether natural products such as monensin, salinomycin, and brevetoxin are one of the biosynthetic pathway of the polyether skeletons, which is a structural feature of these natural products, was proposed as follows; epoxidation of linear polyene, produced by polyketide synthase (PKS), followed by the cyclization of the generated polyepoxide to afford polyether skeleton (known as Cane-Celmer-Westley model). After this proposal, several groups attempted to prove this hypothesis, however, a detailed mechanism to the enzymatic polyether ring formation is still unconfirmed. Recently, we identified the whole biosynthetic gene cluster of lasalocid A, which is one of the most important ionophore antibiotics produced by Streptomyces lasaliensis. Among this cluster, lsd19, which showed significant homology to the putative epoxide hydrolase genes such as monBI and monBII involved in monensin biosynthesis, was assumed to be responsible for the construction of the polyether skeleton. Actually, in vitro analysis of Lsd19 clearly showed that Lsd19 catalyzes polyether ring formation from bisepoxyprelasalocid to lasalocid A. This is the first direxct experimental evidence of Cane-Celmer-Westley model. In thid presentation, details of the functional analysis of Lsd19 will be discussed.
Peridinin is known as a representative auxiliary light harvesting pigment for photosynthesis in the sea. In the peridin-chloropyll a-protein (PCP) complex, exceptionally high (>95%) energy transfer efficiencies from peridinin to Chl a has been reported. This energy transfer efficiency is thought to be related to the unique structure of peridinin, which possesses allene and ylidenbutenolide functions and the unusual C37 carbon skeleton referred to as a 'nor-carotenoid' in contrast to the typical C40 system of carotenoids. There are, however, no studies on the relation between the structural features of peridinin and its super ability for the energy transfer. We then focused on the subjects of why peridinin possessesa unique allene group and an irregular C37 skeleton and how these functions play a role in the exceptionally high energy transfer. In order to understand the questions, we have achieved the syntheses of six different peridinin derivatives as a series modifying the allene bond and the length of the π-electron conjugated chain. We then measured their ultrafast time-resolved optical absorption and Stark spectra. The ultrafast time resolved optical absorption spectra showed that the lifetime of the ICT state converged to a value of 10±1 ps in methanol for all peridinin derivatives regardless of the extent of π-electron conjugation. These data strongly support the notion that the S_1 and ICT states behave independently. Meanwhile, the Stark spectra of peridinin and these derivatives showed that the |Δμ| value of peridinin was largest among six compounds, although the number of the conjugated double bond of D-1 and C39-peridinin is larger than that of peridinin. This result apparently shows that the allene bond and C37 skeleton of peridinin effectively contribute to production of the large dipole moment in the molecule, which would result in the high energy transfer efficiencies to Chl a. This would be at least a partial answer to the question of why peridinin possesses the unique structure.
Iron is an essential element for plant growth. Gramineous plants have generally developed a distinct strategy to efficiently acquire insoluble iron, which is characterized by the synthesis and secretion of an iron-chelating substance, phytosiderophore (PS) such as mugineic acid (MA), and by a specific uptake system for iron(III)-MAs complexes. In this study, we have identified a gene specifically encoding an iron(III)-MAS transporter (HvYS1) in barley. This gene as well as the encoded protein is specifically expressed in ghe epidermal cells of the roots and gene expression is greatly enhanced under iron-deficient conditions. The localization and substrate specificity of HvYS1 indicate that it is a specific transporter in barley roots. In contrast, ZmYS1, reported as iron-MAs transporter from maize, possesses broad substrate specificity despite a high homology with HvYS1. By assessing the transport activity of a series of HvYS1-ZmYS1 chimeras, we revealed that the outer membrane loop between the sixth and seenth transmembrane regions is essential for the substrate specificity. Circular dichroism spectra revealed that a synthetic peptide corresponding to the loop of HvYS1 forms an α-helix in solution, whereas that of ZmYS1 is flexible. We disclosed that the structural difference at this particular loop determines the substrate specificity of the HvYS1 transporter. We also achieved an efficient short-step synthesis of MA and 2'-deoxymugineic acid (DMA). Our new synthetic method enabled a sufficient supply of these compounds that are essential for further biological studies.
Trehalose-6,6'-dimycolate (1, TDM, used to be so called cord factor) is a glycolopid distributed in the cell wall of Mycobacterium tuberculosis, and shows significant anti-tumor activity based on an immunoadjuvant activity. However, TDM was not yet applied for practical use due to its significant toxicity. Datta and Takayama reported the purification of related trahalose diester, trehalose-6,6'-dicorynomycolate (TDCM, 2) from Corynebacterium diphtheria in 1993. We have reported the synthesis of four diastereomeric TDCMs, and showed that the synthetic (2R,3R,2'R,3'R)-TDCM (2) is identical with natural TDCM. We have also demonstrated that 2 is a potnnt anti-tumor substance containing inhibitory activity of experimental lung metastasis. Furthermore, we found that TDCM composed of shorter fatty acid chain is not lethal for mice. Therefore, we have continued tha synthetic study to deelop more active and less toxic analogues based on 2 as the seed compound. We prepared more than 100 of TDCM analogues such as 3a-d, 4 and structurally simple (achiral fatty acid derivatives) 5a-i and 6a-g. Derivative 6b induced release of cytokines such as MIP-2 and IFN-γ more potent than TDCM, and activated phagocytosis and supeoxide production in macrophages. We named this new immunostimulating agent 6b as vizantine. To analyze antitumor activity of vizantine, we investigated prophylactic and therapeutic effect on metastases of breast cancer FM3A cells. When the 6b was administered i.p. into mice 24h before the cancer inoculation into abdominal cavity, metastases of FM3A cells were completely inhibited. Administration of 6b after cancer inoculation also significantly inhibited the metastasis. Furthermore, i.p. administration of 6b to mice 24h before the injection of Clostridium perfringens resulted in 80% survival of mice. This is the first report of the adjuvant-induced protection of mice against C. perfringens infection. Vizantive was also effective for the infecction with P. aeruginosa that causes the opportunistic infectious disease. Therefore, we would like to emphasize vizantine (6b) as an eitirely new drug effective for cancer and infectous diseases based on the intensive immunostimulating activity.
Bacterial cell wall peptidoglycan (PGN) is a potent immunopotentiater and an adjuvant for antibody production. We have recently revealed that intracellular proteins, Nod1 and Nod2, are innate immune receptors of PGN, which recognize diaminopimelic acid (DAP) containing peptide components and muramyldipeptide (MDP) respectively, using synthesized PGN partial structures. We have also revealed that DAP-type PGN containing bacteria release Nod1 ligands to the environments. Genetic studies have shown that polymorphsms in human NOD1 gene significantly associated with susceptibility to several diseases including allergic deseases such as asthma. However, the function and the immunostimulating mechanism of Nod1 have not yet been well understood. We thus analyzed the PGN fragments released to the environment, and synthesized DAP-type PGN fragments including tracheal cytotoxin (TCT). Based on the preparation strategy of the β(1→4) glycans using appropriately protected GlcNTroc and MurNTroc (or MurNTroc(anh)), and a newly developed synthetic method of DAP, we achieved the first chemical synthesis of TCT and a repeating unit of DAP-type PGN fragment. We also synthesized a series of the partial structures, and observed human-Nod1 stimulation activities. The results demonstrated that the glycan partof the ligands enhance the Nod1 activation. We also prepared a library of γ-D-glutamyl-DAP(iE-DAP) derivatives and found N-myristoyl-iE-DAP as a stronger Nod1 activator. The compound enabled us to investigate Nod1 function in vivo.
Mycobacterial arabinan has been found as a common constituent of both arabinogalactan (AG) and liporrabinomannan (LAM), which are attracting particular attention among furanoside-containing glycans. Here, we wish to report the convergent synthesis of docosasaccharide arabinan motif 1 from mycobacterial cell wall (Fig. 1), which features the stereoselective construction of 1,2-cis-β-arabinofuranoside linkages and the fragment coupling using thioglycoside converted from 1,2-acetonide moiety. The retro-synthetic disconnections of target docosasaccharide 1 have been made at linear sequences to be separated to two identical heptasaccharide donors (Araf_7) and an octasaccharide diol acceptor (Araf_8)(Fig. 2). Araf_3 and Araf_5 intermediates were synthesized from 5 (Sch. 1, 2). Two β-linked Araf residues in Araf_7 were stereoselectively introduced by using our TIPDS-[2-4] and NAP-[6,7] protected donors (2, 3 and 4) to Araf_5 diol acceptor 19 (Sch. 3). Introduction of thioglycoside leaving group from 1,2-acetonide moiety has been carrid out through acidic hydrolysis of acetonide followed by treatment under (TolS)_2-Bu_3P conditions and subsequent acetylation of 2-OH group to gibe Araf_5 and Afaf_7 glycosyl donors (25 and 26)(Sch. 4). Araf_8 fragment has been synthesized from Araf_5 donor 25 and Araf_3 acceptor 10 (Sch. 5). Glycosylation between Araf_7 donor 26 and Araf_8 diol acceptor 28 under NIS-AgOTf conditons gave Araf_<22> derivative in high yield, which was assigned by Mass and NMR techniques, effectively. Conventional deprotections of 29 afforded 1 (Sch. 6).
Telomestatin, isolated from Streptomyces anulatus 3533-SV4, is a potent specific telomerase inhibitor. The unique macrocyclic structure of telomestatin consists of the macrocyclic linkage of two methyloxazoles, five oxazoles and one thiazoline ring. Because of the unique chemical structure along with its unusual and potent biological activity, telomestatin is an attractive synthetic target and a candidate for the design of analogues relevant to anti-cancer drug development. The synthesis of telomestatin derivatives and their biological evaluation are presented. Initially, we achieved the total synthesis of (S)-telomestatin and evaluated its biological activity by telomeric repeat amplification protocol. It was found that (S)-telomestatin is more potent than the natural product, (R)-telomestatin. Toward the further diversity-oriented synthesis of telomestatin, we examined palladium-catalzed cross-coupling reacton of 12 that is the macrocyclic key intermediate containing 5-bromooxazole. We found that the cross-coupling r ea ctions such as Suzuki-Miyaura reactoin of 12 with several aryl boronic acids proceeded under mild conditions leading to the corresponding telomestatin derivatives in moderate yields.
Overman rearrangement has been widely used in the total synthesis since a carbon-nitrogen bond can be formed in stereoselective manner through chirality transfer of an enantiopure allylic alcohol. In order to render Overman rearrangement more practical, we developed two new versions of thie reaction; (a) the orthoamide Overman rearrangement (5→6), (b) the cascade Overman rearrangement (7→8). The orthoamide Overman rearrangement could allow us to have a single rearranged product from allylic diol 4 without any protection and deprotection sequence of Cl hydroxy group. On the other hand, the cascade Overman rearrangement of diimidate 7 could establish diamino functional groups in a single operation. We report herein the total syntheses of broussonetines F (1) and L (2) utilizing the orthoamide Overman rearrangement, and (-)-agelastatin A (3) based on the cascade Overman rearrangement. 1. Total synthesis of broussonetines F and L The broussonetines are idolated from the branches of the Asian paper mulberry tree Broussonetia kazinoki, and show striking glycosidase inhibitory activities. The Overman rearrangement of orthoamide 12, which drived from L-tartaric acid, introduce an amino group, giving 11 stereoselectively. This compound was successfully transformed into pyrrolidine 9 in 12 steps. The toal synthesis of broussonetine F (1) was accomplished through the Suzuki-Miyaura cross-coupling between 9 and 10. 2. Total synthesis of (-)-agelastatin A (-)-Agelastatin A (3) is an alkaloid isolated from the deep water marin sponge Agelas dendromorpha. The vicinal diamino moiety was efficiently installed by the cascade Overman rearrangement of diimidate 21, derived from D-tartaric acid. Subsequent Mislow-Evans rearrangement (20→19), followed by the ring-closing metathesis reaction provided 18. We have successfully achieved the total synthesis of 3 through the intramolecular aza-Michael addition of 30, followed by deprotection.
Haplophytine (1) is the major dimeric indole alkaloid isolated from the Mexican plant Haplophyton cimicidum by Snyder in 1952. After two decades, Cava and Yates revealed the structure of 1 by X-ray crystallography. Compound 1 is composed of two segments, which are connected by forming a quaternary carbon center. The left-hand segment has a bicyclic-[3.3.1] structure including a bridged ketone and a cyclic aminal functionalities. The right-hand segment possesses a hexacyclic aspidosperma skeleton. While its unique structure has attracted considerable intersts from chemists, this compound has not yet been synthesized due to its complex structure. Recently, we have achieved the first total synthesis of 1 through our highly convergent route. Preparation of tricyclic ketone 3 was commenced with construction of the quaternary carbon center by the d'Angelo's asymmetric Michael reaction using ethyl thioacrylate. Tha Michael adduct was then conerted to the 11-membered secondary amine derivative via palladium mediated coupling of the thiolester moiety with organozinc reagent, and 11-membered ring formation by N-alkylation with Ns amide. The desired tricyclic ketone 3 was obtained by highly stereoselective intramolecular Mannich reaction of the 11-membered cyclic secondary amine derivative 14. The left-hand segment was synthesized via Friedel-Crafts alkylaton to construct the quaternary carbon center at the 4α position, and oxidative skeletal rearrangement of 1,2-diaminoethene derivative 23 to f orm the characteristic structure of the left segment. The union of these highly elaborated synthetic intermediates was carried out by Fischer indole synthesis to give the nanocyclic indolenine compound 28 and undesired indole compound 29. After extensive optimization, we found that careful control of the reaction temperature and the apropriate choice of acid and solvent were essential to obtain the desired indolenine 26 preferentially. Finally, indolenine 26 was converted to (+)-haplophytine (1) by a five-step sequence including reductive N-methylation and oxidative lactone formation.
Physalins, which are steroidal constituents isolated from Physalis plants, contain a unique 13,14-seco-16,24-cycloergostane skeleton. The structures of physalins A (1) and B (2) were first determined in 1969, and so far, many kinds of physalins have been identified. Physalins are characterized by a highly oxygen-functionalized, complex, fused and bridged ring system, and type B physalins, such as physalin B (2), are composed of eight rings, including an intramolecular acetal ring (H-ring, C(14)-O-CH_2(27)-C(25)). Physalins show antitumor activity in vitro and in vivo. Furthermore, other unique activities of 2 have recently been reported, includin inhibition of hedgehog/GLI-mediated transcription, inhibition of PMA-induced NF-кB activation, anti-inflammatory and immunomodulatory activity, and inhibition of the ubiquitin-proteasome pathway. This time, we focused on the synthesis of characteristic DFGH-ring system of type B physalin 3 to elucidate the biological activity of this moiety and to develop the synthetic methodology for total synthesis. In this symposium, we report the first synthesis of the type B physalin DFGH-ring system. During the course of the synthesis, efficient methods for the synthesis of intermediates 16 and 25 by utilizing 2,3-Wittig rearrangement and Baeyer-Villiger oxidation were developed. Moreover, we succeeded in the construction of the DFGH-ring system through a four-step domino reaction, including an unusual oxy-Michael addition, in one pot under simple basic conditions. Examination of H-ring formation via two different approaches revealed that the oxy-Michael strategy (path b) to construct the H-ring of physalins is superior to the acetal formation strategy (path a).
(+)-Spiculoic acid A (1) and (+)-zyggomphic acid (6) are novel secondary metabolites of polyketide origin, which were isolated from the marine sponges Plakortis angulospiculatus or P. zyggompha by Andersen et al. or by Amade et al., respectively. These natural products showed in vitro cytotoxicity against the human breast cancers MCF-7 cell of MDA-MB-231 cells. Recently, we have accomplished the first total syntheses of 1 using a highly stereoselective intramolecular Diels-Alder (IMDA) reaction as a key step. The total synthesis of 1 began with the known enantiomerically homogeneous 7. Key steps to an IMDA substrate 16 include 1) asymmetric crotylation of 8 using a Brown's (E)-crotylborane, 2) (E)-selective Wittig olefination for preparing vinyl bromide 11, and 3) Suzuki-Miyaura coupling for preparing the substraste 16. The IMDA reaction of 16 proceeded under thermal conditions to provide an endo-adduct 17 as a result of complete π-facial selection. The adduct 17 was efficiently transformed into 1 via a Horner-Wadsworth-Emmons reaction for installing a styryl moiety. On the other hand, asymmetric crotylation of 8 using a Roush's (Z)-crotyl boronate provided 21. The IMDA reaction of substrate 26, derived from 21, provided an exo-adduct 27 with complete π-facial selectivity. The cycloadduct 27 was eventually transformed into (+)-2,5,6-tri-epi-spiculoic acid A (30). Finally, total synthesis of 6 was accomplished starting from intermadiate 10, which was subjected to a Wittig reacton providing 31. The Suzuki-Miyaura coupling of vinyl boronate 32 derived from 31 with (E,E)-dienyl iodide 34 provided a triene 35. The IMDA reaction of 35 efficiently provided an endo-adduct 36. Installation of a styryl unit into the side chain in 36 eventually led to a more functionalized natural product 6.
Chronic hepatitis C continuea to be the most important cause of chronic liver disease, potentially resulting in cirrhosis, hepatocellular carcinoma, and the need for liver transplantation. The current standard of care for HCV infection is treatment with pegylated interferon alpha in combination with Ribavirin; however, this regimen has limited efficacy and side effects. Newer anti-HCV drugs in development target several viral enzymes such as NS3/4A protease and RNA polymerase but drugs targeting viral enzymes may result in the development of viral resistance. Our interest has been in identifying a novel HCV replication inhibitor that manipulates host cell factor. Using a HCV replicon cell culture sysrem, we identified NA255 from a natural screening source as a novel HCV replication inhibitor. To further expand the possibility of NA255 as a new anti-HCV therapy, we initiated a chemical modification program by using total synthesis. We developed a method of concise enantioselective synthesis of NA255 derivatives based on the method reported by Hatakeyama in 1998. We utilized Sharpless AE and its regioselective epoxide ring opening reaction as the key steps, followed by simultaneous oxidation of two primary alcohols (scheme 1). We have synthesized over 300 NA255 derivatives and established the SAR. Optimization of NA255 using the medicinal chemistry approach resulted in teh identification of NA808 as a candidate for development. We also established an efficient and robust synthetic route for NA255 derivatives toward scale-up synthesis using an enantioselective aldol reaction. Evans auxiliary, (S)-4-Benzyl-oxazolidin-2-one, provided the undesired relative configuration (anti/syn=7:1, enolate face selection=10:1). The reversal of relative configuration was achieved by the employment of 4-i-Pr-5,5-diphenyloxzolidinthione. It is unusual that relative stereochemistry is controlled by substituents on position 5 of the oxazolidine-2-thione. The addition of lithium chloride was essential to achieve both high conversion and stereoselectivity (without the additive, typically 50% of the conversion with a varied ratio of 10-4:1 for the enolate face selection was observed). Interstingly, the structure of a protecting group with the primary alcohol of the ketoester did not have an effect on the stereoselectivity. In this presentation, this novel enantioselective aldol reaction will be reported in datail, as well as the synthetic scheme of the NA255 derivatives utilizing this reaction as a key step.
A great deal of attention has been paid both in the scientific literature and teh general media to the high potential risk of a worldwide spread of avian H5N1 influenza virus, the death reate of which is over 50%. Indeed, shoud this virus acquire the ablility to become capable of spreading easily and directly from human to human it could very possibly cause a disastrous pandeic. (-)-Oseltamivir phosphate (Tamiflu), a neuraminidase inhibitor used in the treatment of both type A and type B human influenza, is one of the most promising therapeutics, and many nations have plans to stock a significant amount of this compound in case of a possible influenza outbreak. An efficient, enantioselective total synthesis of (-)-oseltamivir (1 via separated three one pot operation has been accomplished, demonstrating the power of asymmetric reactions catalyzed by organocatalysts, in particular diphenylprolinol silyl ether. The present synthesis has several noteworthy features: 1) A highly functionalized chiral cyclohexane framework of the correct configuration is synthesized in the first one-pot operation, which consists of a succession of reactions, including a diphenylprolinol silyl ether-mediated asymmetric Michael reaction, a domino Michael reaction/Horner-Wardsworth-Emmons reaction combined with retro-aldol and retro-Michael reactions, a thiol-Michael reaction, and a base catalyzed isomerization. 2) Three reactions occur in the third one-pot operaton: a Curtius rearrangement, the reduction of a nitro group to an amine, and a retro-Michael reaction of the thiol. 3) The Curtius rearrangement proceeds at room temperature without heating, decreasing the potential hazards. 4) The domino reaction consisting of a Curtius rearrangement and amide formation is a direct method for the synthesis of 14. This synthesis requires nine reactions, a toal of three separate one-pot operations, and one purificaton by column chromatography. The total yield of (-)-oseltamivir from nitroalkene 4 is 57%. All the reagents are inexpensive. The metal-based reagents employed in the present total synthesis contain either alkali-metal ions (Na, K, and Cs) or nontoxic Zn. No special care is needed to exclude water or air. Thus, the present procedure is suitable for large-scale preparation.
Kendomycin (1), an ansa-type compoung having a unique quinone methide portion connected to a highly-substituted tetrahydropyran, was isolated from Streptomyces species. 1 possesses a potent activities as an endothelin receptor antagonist and an antiosteoporotic compoung as well as antibacterial and cytotoxic activities. Herein, we report the total synthesis of kendomycin (1) featuring the intramolecular Dotz(Doetz) reaction. We anticipated that the intramolecular Dotz reaction of Fischer chromium carbene complex 2 would afford oxametacyclophane 3 (Scheme 1). Actually, the intramolecular Dotz reaction using the simple substrate 6 produced the desired oxametzcyclophane 7 with exclusive regioselectivity (Scheme 2). Further, more complicated substrates 19 and 22 were applied to the reaction as model studies of kendomycin (1) synthesis. The intramolecular Dotz reaction of 19, containing all substituents in the tetrahydropyran portion, proceeded with high regioselectivity to give the oxametacyclophane 20 (Scheme 3). Subsequent cyclization of the product was diastereoselectively performed, affording 21. Also in the case of 22 having the foothold for the construction of the p-quinone methide portion, the intramolecular Dotz reaction proceedee with high regioselectivity to give the oxzmetacyclophane 23 (Scheme 4). The p-quinone 27 was synthesized from 23 in seven steps containing IBX oxidation of phenol. Ring closure and tautomerization was easily performed by applying 27 on the silica gel TLC to give the p-quinone methide 28. Based on the above results, we challenged the total synthesis of kendomycin (1)(Scheme 5-8). The Suzuki coupling of the left half segment 36, synthesized from 29, and the right half segment 40, derived from the ent-29, gave 41 in excellent yield. Diastereoselective tetrahydropyran-ring formation and the protecting group manipulation of 41 afforded the precursor of the chromium carbene complex. The resulting 48 was converted to the carbene complex 49 and it was subjected to the intramolecular Dotz reaction to give the desired oxametacyclophane 50 in good yield. After oxdative cleavage of terminal olefin followed by the Claisen rearrangement, the resulting 52 was oxidized with IBX and applied on silica gel, giving p-quinone methide 54. After deprotection of TBS group as the last step, we accomplished the total synthesis of kendomycin (1).
(-)-Zampanolide (-)-1 was isolated from Okinawa sponge (Faciospongia rimosa) by Higa and Tanaka in 1996. Five years later, Riccio and co-workers isolated (+)-dactylolide (+)-2 possessing antipodal macrolactone ring of (-)-1 from Dactylospongia sp. collected in Vanuatsu island. The enantiomeric relationship vis-a-vis the common macrecyclic domains of (-)-1 and (+)-2, is a rare occurrence in marine natural products. This observaton aptly suggests that if dactylolides were either a direct biosynthetic precursor to zampanolide, or perhaps a degradaton product thereof, one should expect to either find both (-)-1 and (-)-2 in Fasciospongia rimosa or (+)-1 together with (+)-2 in Dactylospongia sp.; however, neither was the case. (-)-Zampanolide (-)-1 is a potent cytotoxic agent and possesses a unique molecular architecture comprised of a highly unsaturated macrolactone ring, a bridged 2,6-cis-tetrahydropyran unit and an exocyclic N-acylhemiaminal group with the pendant N-(2Z,4E)-hexadienoylamide (23). In this pater, we report a concise total synthesis of (-)-1 and (-)-2. The key synthetic transformations involve i) the Kita-Trost macrolactonization, ii) a short seco-acid formation by a mind HWE reaction, iii) consecutive Sonogashira/Kumada-Tamao-Corriu couplings, iv) stereo selective cis-THP ring formation. A biological evaluation of (-)-1 and its diastereomer 24 revealed an importance of the stereochemistry of the hemiaminal moiety. The analysis of the extract obtained from the Okinawa sponge by LC-MS turned up no evidence of (-)-dactlolide as a distinct entity in the sponge extract. Therefore, we conclude that (+)-dactylolide is indeed a naturally occurring entity, and currently, we hypothesized that the macrolactone ring may from after formation of N-acylhamiaminal in the sponge.
Saxitoxin (STX) (2) and its analogues known as causative agents of paralytic shellfish poisoning, so called PSP, are potent neurotoxins produced by harmful dinoflagellates. This fatal intoxication is attributed to STXs' potent affinity against the voltage gated sodium channels (NaChs), thus the toxins strongly block the influx of sodium ion and inhibit the depolarization process of neuronal cells. We have recently accomplished total synthesis of (-) and (+)-doSTX (ent-2 and 2) and (+)-STX (1) by the use of 1,3-dipolar cycloaddition reaction and unique IBX oxidation reaction. In this paper, we described the NaCh inhibitory activity of novel synthetic STX derivatives 19-22. We also succeeded in developing the new synthetic methodology for constructing the cyclic guanidine skeleton under the extremely mild conditions, which successfully allow us to the total synthesis of (+)-dcSTX (3) and (+)-GTX3 (7) from "protected" saxitoxinol 34.
Manzamine A (1), a β-carboline alkaloid, was isolated from a marine sponge in Okinawa. It has drawn a great deal of attention because of its attractive biological activities, including angicancer, antimicrobial and antimalarial. Its complex and unique structure including the pentacyclic diamine core skeleton has attracted many synthetic chemists and a number of synthetic investigations have been reported to date. Inspired by its challenging structure, we initiated our synthetic studies on manzamine A several years ago. Our synthetic studies commenced with synthesis of vinylogous ester 8, which was prepared from commercially available alcohol 9 in 9 steps. It was easil converted to the diene 7 and we focused on Diels-Alder reaction with butenolide 6. The instability of the diene 7 was the major problem in this reaction but when the reaction was conducted in reflexing toluene in the presence of sodium acetate and molecular asieves 3A, the Diels-Alder adduct 5 was obtained in excellent yield. Then the adduct 5 was converted to macrocyclization precursor 15 in 8 steps. Macrocyclization was acomplished by intramoluecular Mitsunobu reaction of nosyl amide though the desired product 3 has a surprisingly rigid structure. Next quaternary stereogenic center was constructed with complete control of the stereochemistry owing to the steric effect of the macrocycle in our key intermediate 3. After epoxidation of enone 17, the pruduct 18 was put under dehydrating condition and the resultant allyl cyanate underwent [3,3]-sigmatropic rearrangement to provide the isocyanate 19. Then the isocyanate was converted to the amine in the absence of water and the resultant imine 20 was stereoselectively reduced. Following acylation of the amine provided the amide 22. Then construction of the ring E was attempted by ring-closing metathesis reaction. With Grubbs' 2^<nd> generation catalyst 26 or Hoveyda-Grubbs' 2^<nd> generation catalyst 27, the desired compound 23 was obtained. After several investigations, Grubbs' 2^<nd> generation catalyst 26 gave a better result. Then nethyl ester and amide was reduced by alane without damaging the nosyl group and the following Dess-artin oxidation provided the aldehyde 28. The ring A was constructed by reductive amination and further functional group elaboration led to tatal synthesis of ircinal A (2). A new and efficient conversion of ircinal A (2) to manzamine A (1) is now under investigaton.
Cladiellin terpenoids, which are widely found from soft corals, have attracted the attention of synthetic organic chemists because of their unique structure involving a ten-menbered carbocycle with a transannular oxygen bridge. Polyanthellin A (1), isolated from Briareum steckei in 1989 by Bowden, exhibits the antimalarial activity. Litophynin F (2) was isolated from Litophyton sp. in 1991 by Ochi. Toward the total synthesis of these natural products, a new method for constructing the Cladiellin skeleton was developed on the basis of the [6+4] cycloaddition reaction of 4 having a 11-oxabicyclo[6.2.1]undecane skeleton which is a common substructure in Cladiellin terpenoids. With a view to inducing a decomplexation reaction, cobalt complex 4 was treated with I_2 and Cu(OAc)_2 in acetic acid. Although the product 5 was a 11-oxabicyclo[5.3.1]undecane derivative arising from a novel rearrangement reaction, it was found to be a useful intermediate of the total synthesis. Thus, the six-membered ring moiety was constructed via a conjugate addition reaction followed by and intramolecular cyclization reaction. After installation of the isopropyl group by a conjugate addition reaction, the cladiellin skeleton of the natural product was reconstructed. Finally, the total synthesis of Polyanthellin A (1) was accomplished through functional group manipulation involving oxymercuration reaction of alcohol 22. Litophynin F (2) was also synthesized from the common intermediate 22 by using the selective allylic oxidation reaction via π-allylpalladium complex.
Fomitellic acids, isolated from the mycelium of a basidiomycete, Fomitella fraxinea, are specific inhibitors of DNA polymerase α and β. It was also reported that inhibition of DNA polymerase activity by fomitellic acids induced neurite outgrowth in PC-12 cells. Structurally, fomitellic acids belong to the triterpenoid family of compounds, which are characterized by a highly oxygenated steroidal AB ring moiety. Herein, we report a total synthesis of fomitellic acid B (2) based on a radical cascade cyclization of epoxypolyene. Our synthetic approach began with the vinylogous Mukaiyama aldol reaction between the enal 11 and the vinylketene silyl N,O-acetal 12. After protection of the secondary alcohol in aldol adduct 13, the chiral auxiliary was removed to give allylic alcohol 14 with 95% ee. Sharpless asymmetric epoxidation of 14 gavd epoxyalcohol 15, which was converted into a key intermediate 17 in 3 step. The vinyl iodide 27, stereoselectively prepared from (-)-Wieland-Miescher ketone, was treated with t-BuLi, and the resulting vinyl lithium was then reacted with the aldehyde 17. Subsequent acetylation of the secondary alcohol provided the cyclization precursor 28a. The trans-decaline skeleton was stereoselectively constructed by means of titanium(III)-mediated radical cascade cyclization of epoxypolyene 28a. The cyclization product 29 was found to possess the desired stereochemistry and to be formed via a trans-fused chair/boat-like transition state. After protection of the secondary alcohol in 29, treatment of the resulting benzoate ester with HCl provided diol 31, which was converted into carboxylic acid 33 in 4 sreps. Allylic oxidation at C7 position with NaClO_2, followed by construction of the side chain moiety afforded enone 35. Finally, deprotection of acetyl and benzoyl groups with NaOH completed the synthesis of fomitellic acid B (2).
Maduropeptin, an extremely potent antitumor agent, was isolated from the groth filtrate of Actinomadura madurae. It was identified as a 1:1 complex of an acidic carrier apoprotein (32kDa) and 9-membered ring enediyne chromophore, which showed selective DNA strand scission such as 5'-TCTT/3'-AGAA and 5'-TCTC/3'-AGAG. The chromophore (1), isolated as a methanol adduct, possesses a labile 9-membered diyne core, a macrolactam ansa0bridge and an aminosugar moieties. The protected aglycon 4 had been synthesized as reported on 49^<th> symposium on the chemistry of natural products. Here we report the total synthesis of the (-)-maduropeptin chromophore and its structure revision. The glycosyl donor 3 was synthesized from known lactone 5, which was derived from L-serine according to Nicolaou's procedure, in two steps. Glydocylation of the C9 tertiary hydroxyl group of 2, which was derived from 4 in 4 steps, in CH_2Cl_2 using TMSOTf as a Lewis acid proceeded smoothly to afford axial glycoside 9 in 40% yield. Removal of two benzoyl groups and all TES groups of 9 led to completion of the total synthesis of 1. However, the ^1H and ^<13>C NMR spectra of synthetic 1 were found to differ from those of the natural product. In consequence, the proposed structure of the natural chromophore of maduropeptin was revised as structure 1', which possesses the antipodal madurosamine moiety. Stereoselective glycosylation using enantiomeric sugar moiety ent-3, which was derived from D-serine, was conducted under the similar conditions (30% yield based on 50% recovery of 2). Deprotection of all protecting groups of 10 afforded the desired chromophore 1'. The ^1H and ^<13>C NMR spectra, as well as HRMS and NOE correlations, were in good agreement to those of the natural product. Thus, the first total synthesis and structure revision of (-)-maduropeptin chromophore were accomplished.
Grandisine alkaloids 1-7 isolated from the leaved of Elaeocarpus grandis display affinity for human δ-opioid receptor. Although compounds 1-7 are attractive target molecules for total synthesis because of their structural diversity and pharmacological activity, only grandisine A (1) has been synthesized so far. Hence, we undertook synthesis of other grandisine alkalodis via a flexible route applicable to various grandisine analogues as candidate selective δ-opioid receptor agonists. We p resent here the first total synthesis of grandisine B (2), D (5) and F (4) from the common key intermediate 9, which was prepared by a highly stereoselective Bronsted acid-mediated Morita-Baylis-Hillman(MBH) reaction via N-acyl iminium ion. MBH ring closure reaction of aldehyde 11, obtained from (S)-malic acid, using the combination of TfOH and Me_2S in acetonitrile afforded the desired indolizidine trans-15 with high stereoselectivity. Indolizidine trans-15 was converted into the key intermediate 9 by way of several steps including boron-aldol reaction with (S)-5-methylcyclohexenone (23). α,β-Unsaturated ketone of 9 was protected as the thiophenol adduct 25, followed by converted into the corresponding thioamide 26 by treatment with Lawesson's reagent. Thioamide 26 was exposed to Meerwein's salt, followd by treated with NaBH_3CN to afford 5. Fortunately, grandisine D (5), on treatment with ammonia solution, underwent intermolecular 1,4-addition of ammonia followed by intramolecular imine formation to afford 2 as a sole product. After several experiments, we were delighted to fund that lactam 9 was a good starting material for 4. Treatment of ketone 9 with ammonia solution caused intermolecular 1,4-addition of ammonia follwed by ring closure of enolate to provide tetracyclic compound 32 as a single isomer. The tetracyclic structure 32 was converted into the Boc derevative 33, followed by two-step reduction of amide moiety to give amine 34. Finally, the total synthesis of 4 was accomplished by removal of the Boc group with TFA.
Gambierol (1) is one of the characteristic polycyclic ethers, which was isolated from ciguatera causative dinoflagellate Gambierdiscus toxicus. It shows strong neurotoxicity and the syptoms resemble those caused by ciguatoxins. Its structure has a ladder-shaped trans-fused octacyclic ring system, four axial methyl groups at fused ring positions, and a triene side chain. Because of the intersting biological activity andn characteristic architecture, many synthetic efforts have been devoted, culminationg three total syntheses and one f ormal total synthesis. Herein we report the total synthesis of gambierol (1). The salient features of the route include: 1) direct carbon-carbon bond formation on an oxirane ring and the subsequent sulfonyl-assisted 6-endo cyclization, 2) a ring-expansion approach to seven-menbered ether rings, and 3) radical cyclization. Our synthesis commenced with the coupling reaction between griflate 8 and epoxy sulfone 16 to provide 17, which was sequentially treated with MgBr_2 and DBU to afford the D ring 18. After Wacker oxidation of 19, the resulting methyl ketone was transformed into keto acrylate 20. Samarium iodide-mediated ketyl radical cyclization of 20 furnished the CD ring 21, which was converted to the BCD ring 15 via an acid-catalyzed 6-endo cyclization of epoxy olefin 23. Stereselective epoxidation of 15 with VO(acac)_2-TBHP followed by the reaction with dithiane 2 and hydrolysis of the dithioketal group provided hydroxy ketone 26. Reductive etherification with Et_3SiH and SnCl_4 followed by manipulation of the protecting groups furnished the ABCD ring triflate 29. For the construction of the EF ring, triflate 29 was treated with the anion derived from epoxy sulfone 5, and the subsequent 6-endo cyclization afforded ketone 31. Treatment of the ketone with TMSCHN_2 followed by reduction yielded the ABCDE ring alcohol 32. The alcohol was converted into keto acrylate 38, which was then subjected to ketyl radical cyclization with SmI_2 to afford the ABCDEF ring ester 39. The ester was transformed into triflate 42 by a nine-step sequence. Construction of the G and H rings was carried out by the doupling reacton of the oxiranyl anion derived from 6 with triflate 42 to provide heptacyclic ketone 44 after 6-endo cyclization. Iteration of the coupling of 6 with triflate 45 followed by cyclization furnished octacyclic ketone 46. Ring expansion reaction of 46 and stereoselective methylation of 47 afforded the ABCDEFGH ring alcohol 48. Conversion of the alcohol to (Z)-vinyl iodide 49 was achieved by TRAP oxidation followed by Wittig reaction. Removal of the benzyl groups with DDQ in dichloroethane and the TES group with TsOH privided triol 50. Finally, Stille coupling of 50 with vinyl stannane 7 afforded gambierol (1).
Brevenal (1), a new family of marine polycyclic ether, was isolated from the Florida red tide dinoflagellate Karenia brevis by Baden and co-workers in 2004. This compound inhibits the binding of tritiated dihydrobrebetoxin-B to the voltage-sensitive sodium channels in a concentration dependent manner and acts as a nontoxic brevetoxin antagonist. Moreover, a significiant improvement of tracheal mucus velocity was observed in an animal model asthma. As well as the novel biological activities, the unique structural features have attracted attention of synthetic chemists. In 2006, the first total synthesis and structure revision of 1 were reported by Sasaki and co-workers. In this paper, we wish to report eht recent results and our efforts on the total synthesis of brevenal (1). The ABC ring segment 3 was prepared from the known compound 7 the B-alkyl Suzuki-Miyahara coupling of the phosphate 11 and the alkylborate from the iodine 5. Direct methylation of the O,S-acetal 16 was performed by using Me_2Zn/Zn(OTf)_2 in highly stereoselective manner. The ABC ring fragment obtained and the known alchol 4 were connected by our own synthetic strategy including the intramolecular allylation of α-acetoxy ether followed by ring-closing metathesis to furnish the pentacyclic ether 2. The right hand diene was introduced by the Nicolaoua's protocol. Construction of the left hand side chain, highly substituted dienyl moiety, was archieved by the modified Horner-Wadsworth-Emmons reaction with the phosphonate 33, newly developed, to afford the dienyl ether 34 as the sole product. A seris of functional transformation and deprotections of 34 furnished brevenal (1). The synthetic 1 exhiibited physical and spectroscopic data identical to those reported previously.
In 1996, auripyrones A and B were isolated from the sea hare Dolabella auricularia (Aplysiidae) by Yamada and co-workers. Auripyrones A and B exhibited cytotoxicity against HeLa S_3 cells with an IC_<50> values of 0.26 and 0.48μg/mL, respectively. The main structural features of auripyrones are a γ-pyrone ring and a spiroacetal moiety. We planned the synthesis of auripyrones A and B by using diastereoselective aldol-type reaction with 2,6-diethyl-3,5-dimethyl-4-pyrone as a key step and have developed an efficient aldol-type reacton with 2,6-diethyl-3,5-dimethyl-4-pyrone. This key reaction has the benefit of straightforward access even to complex molecules and the construction of two stereogenic centers at once. Furthermore, we have achieved the total synthesis of auripyrone A by using this aldo-type reaction.
(+)-Irciniastatin A (1) and irciniastatin B (2) were isolated in 2004 from the marine sponges Ircinia ramose and shown to exhibit extremely potent cytotoxic activity (1.0-0.1ng/mL) against several human cancer cell lines. In the same year, psymberin (1') was isolated from an undescribed sponge, Psammocinia sp. ad the extremely toxic principle and the close structural assignment lead the conclusion that psymberin was identical with irciniastatin A. The promising theraputic potential coupled with the limited availability of these natural products has made contributions of synthetic community significant to play crucial roles in full strucutural elucidation as well as SAR studies for further investigations. With our intention to conduct chamical biology studies based on irciniastatins/psymberin, we planned a total synthesis of irciniastatin A that would enable acquision of all the diastereomers. Herein, we report a total synthesis of irsiniastatin A/psymberin. The total synthesis began with the construction of three segments; left 3, central 4, and right segment 7, featuring efficient regio-, and diastereoselective ring opening reaction of chiral epoxy alcohols that were synthesized via Sharpless AE. The central segment 23 and left segment 44 thus obtained were connected in a highly diastereocontrolled manner by the aldol reaction to afford 45 with the aid of inherent substrate control. The subsequent diastereoselective reduction of the aldol moiety, δ-lactone formation, Curtius rearrangement gave carbamate 48. The crucial merger of the left-cantral segment 48 with right segment 49 was attained with the judicious combination of the protecting groups and activating method. Finally, the total synthesis of (+)-irciniastatin A/psymberin was achieved by global deprotection of all protecting groups.
During the course of our project directed towards the total synthesis of natural products with allelopathic activity, we intended to synthesize the biogenetically related helianane-type terpenoids, heliespirones A (1), C (2) and heliannuol E (3) based on the intramolecular Hosomi-Sakurai type reaction. Reaction of the substrates (5) possessing a benzoquinone and an allysilane moieties with TBSOTf in isobutyronitrile at -10℃ provided the unexpected rearranged products (11) with good yield and diastereoselectivity. This type of transformation has never been reported so far. Employing the rearranged product (11a) the first enantioselective total syntheses of heliespirones A (1) and C (2) were successfully completed. In addition, an alternative enantioselective total synthesis of heliannuol E (3) was also accoplished starting from 11a. It was demonstrated that our newly developed Lewis acid mediated rearrangement can be applied to the total synthesis of helianane-type sesquiterpenoids with allelopathic activity.
The dirhodium(II) complex-catalyzed tandem cyclic carbonyl ylide formation-1,3-dipolar cycloaddition reaction sequence represents one of the most efficient methods for the rapid assembly of complex oxapolycyclic systems. Consequently, the development of a catalytic enantioselective version of this sequence has become a challenging objective. In this context, we previously reported that the tendem formation of keto- or ester-carbonyl ylide from α-diazoketone and intermolecular 1,3-dipolar cycloaddition under the influence of Rh_2(S-BPTV)_4 or Rh_2(S-PTTL)_4, respectively, give cycloadducts with up to 93% ee. Very recently, we also reported high levels of enantioselection (up to 99% ee) for the intermolecular cycloaddition of 2-diazo-3,5-diketoesters-derive dcarbonyl ylides with arylacetylene, alkoxyacetylene and styrene dipolarophiles using Rh_2(S-TCPTTL)_4. In order to demonstrate the synthetic potential of this methodology, we addressed catalytic asymmetric syntheses of oxabicyclic natural products. The 1,3-dipolar cycloaddition of cyclic carbonyl ylide dereved from α-diazoketone 11 with 2 equiv of aromatic aldehyde 12d using 1 mol % of Rh_2(S-BPTV)_4 in benzotrifluoride proceeded smoothly to afford exo-cycloadduct 10d in 67% yield with 87% ee. Peterson olefination of enantiomerically pure 10d followed by DIBAL-H reduction and chlorination afforded the allyl chloride Z-17. S_N2'-Substitution of Z-17 with MeMgI in the presence of CuCN・2LiCl at -40℃ gave the desired product 19 as a major product. Deprotection of silyl protecting group provided psoracorylifol C (7). The 1,3-dipoar cycloaddition of carbonyl ylide derived from α-diazo-β-ketoester 22 with 3 equiv of phenylacetylene 23d using 1 mol % of Rh_2(S-TCPTTL)_4 afforded the 8-oxabicyclo[3.2.1]octan-2-one 24d in 73% yield with 95% ee. Catalytic hydrogenation of the double bond provided 25 in 93% yield with perfect endo diastereoselectivity. Silylation of enantiomerically poure 25 followed by enol triflate formation and reductive elimination of the trifluoromethanesulfonate group furnished alkene 28. Reduction of the ester group in 28 and subsequent silylation gave 29. Allylic oxidation of 29 with SeO_2 followed by oxidation with MnO_2 and subsequent deprotection of two TBDPS groups completed the asymmetric synthesis of 8.
YW3699 (1) was isolated from the culture of Codinaea simplex by Wand et al. It is a sesterterpenod having 5-8-6-5 membered rings consecutively and possesses inhibitory activity against glycosylphosphatidylinositol (GPI) synthesis. We have planned he synthesis of 1 using ring closing metathesis (RCM) to give an eight-menbered ring of 1. Although compounds 4a and 4b successfully afforded 5a and 5b, respectively, these compounds did not have the desired stereochemistry. According to a new synthetic plan (Scheme 2.), compound 12a was prepared by using the Banford-Stevens-Shapiro reaction of a hydrazone of 2-allycyclopentanone with aldehyde 9 and epoxidation. Among four diastereoisomers (12a〜12d), only 12a cyclized giving 13a in 80% yield. The structure of 13a was confirmed by an X-ray crystallographic analysis (Scheme 3.). In designing a synthetic route toward chiral YW3699 (1), we selected commercially available (-)-carvone (16) as a starting material. (-)-Isocarvone (17), prepared from 16 by a known route, was acylated, and reduced to diol 27. The stereoselective installation of a C2 unit at the C-12 quaternary center was carrid out via Claisen-type [3,3]-sigmatropic rearrangement. Iodolactonization and reductive removal of the iodine atom afforded lactone 34. The ring D will be prepared by aldol reaction, and the ring A might be attached by the Shpiro reaction as before giving a precursor of the RCM reaction. An eight-membered ring would be constructed by the RCM reaction. Futher studies toward chiral YW3699 (1) along these lines are in progress.