Diffusion-ordered NMR spectroscopy (DOSY) is a versatile and powerful NMR technique and a noninvasive analytical method for mixture analysis that does not require prior physical separation of the analysis. In our search for new metabolites from natural resources, DOSY was applied for constituent analysis of crude bromopyrrole fractions separated from an Okinawan marine sponge Agelas sp. (SS-1056) so that two new bromopyrrole alkaloids, agesamides A (1) and B (2), have been isolated. The structures and relative stereochemistry of 1 and 2 were elucidated fromspectroscopic data. Furthermore, two new bromopyrrole alkaloids, nagelamides J (3) and K (4), have been isolated from another Okinawan marine sponge Agelas sp. (SS-1077), and the structures and relative stereochemistries were elucidated from spectroscopic data. Nagelamide J (3) is the first bromopyrrole alkaloid possessing a cyclopentane ring fused to an amino imidazole ring and nagelamide K (4) is a unique dimeric bromopyrrole alkaloid containing an ester linkage. Nagelamide J (3) exhibited antimicrobial activity against Staphylococcus aureus (MIC, 8.35μg/ml) and antifungal activity against Cryptococcus neoformans (MIC, 16.7μg/ml), while nagelamide K (4) exhibited antimicrobial activity against Micrococcus luteus (MIC, 16.7μg/ml).

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Marine dinoflagellates are a rich source of various biologically and physiologically active secondary metabolites. Among them, large polyol and polyether compounds composed of a long carbon backbone that is highly functionalized by oxygen are some of the most unique and unusual compounds. The true origin of secondary metabolites isolated from marine invertebrates such as sponges, ascidians, nudibranchs, has been suggested to be mostly microorganisms, which are accumulated in the host animals through a symbiotic relationship or a food chain. However, the true physiological functions or roles of polyol compounds in the ecosystem or symbiotic relationship have rarely been clarified. Based on its structural, biological, and conformational diversity and uniqueness, various potential abilities of such polyol compounds are considered, i.e. chemical communication with host animals, defense materials, or nutrient sources. To establish their roles in symbiotic organisms, we have investigated polyol compounds from symbiotic dinoflagellates. Recently, we isolated two novel long carbon-chain polyol compounds, symbiodinolide (1) from Symbiodinium sp., and durinskiol A (2) from the cultured symbiotic dinoflagellate Durinskia sp. Their partial relative stereostructures were elucidated based on 2D-NMR and MS/MS analysis and degraded reactions. Symbiodinolide (1), a 62-membered novel polyol macrolide with a molecular weight of 2,859 mu, exhibited a potent voltage-dependent N-type calcium ion channel-opening activity at 7 nM. Durinskiol A (2) possessed a 6,5,6-bis-spiroacetal ring, two terminal olefins, two sugar units, and six- and seven-membered ether rings. It caused a short body length, abnormal pigment pattern, and pericardiac and yolk-sac edema in zebrafish. We describe here the structure and biological activity of these polyol compounds.

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Pentaketide chromone synthase (PCS) from Aloe arborescens is a novel plant-specific type III polyketide synthase (PKS) that produces 5,7-dihydroxy-2-methylchromone from five molecules of malonyl-CoA. On the basis of the crystal structures of wild-type and M207G mutant PCS, F80A/Y82A/M207G triple mutant was constructed and shown to produce an unnatural novel nonaketide naphthopyrone by sequential condensations of nine molecules of malonyl-CoAs. This is the first demonstration of the formation of a nonaketide by the structurally simple type III PKS. A homology model predicted that the active-site cavity volume of the triple mutant is increased to four times of that of the wild-type PCS.

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Alternaria solani, a causal fungus of potato early blight disease, produces phytotoxic reduced-type polyketides such as solanapyrones and alternaric acid. In the course of our study on fungal polyketide synthases (PKSs), solanapyrone biosynthetic gene cluster was identified in A. solani. Four new PKS gene fragments were amplified from A. solani genomic DNA. Sequences of entire PKS genes and surrounding regions were determined by PCR-based gene walking method. One of the gene clusters contained a gene for a typical iterative type I PKS with C-methyltransferase domain which is necessary for the prosolanapyrone synthesis and five genes for O-methyltransferase, dehydrogenase, transcription factor, FAD-dependent oxidase, and cytochrome P450. These genes were considered to be just enough for solanapyrone biosynthesis, and thus named soil 〜6. The Soil PKS was expressed in Aspergilhts oryzae and its product was identified to be a solanapyrone precursor. desmethylprosolanapyrone I (5). Expression of Sol5 in A. oryzae confirmed its solanapyrone synthase activity to convert prosolanapyrone II (7) to solanapyrone A (1) by oxidation and Diels-Alder cyclization. The purified enzyme from the recombinant Pichia pastoris showed the same stereo-specificity in conversion of (7) to (1) as the native solanapyrone synthase does.

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2-Deoxystreptamine (DOS) is a common aglycon of many important aminoglycoside antibiotics such as neomycin and butirosin. The biosynthesis of DOS starts from carbocyclization of D-glucose-6-phosphate by 2-deoxy-scyllo-inosose synthase (DOIS), and the subsequent transamination provides 2-deoxy-scyllo-inosamine (DOIA). DOIA is then oxidized and transaminated to form DOS. So far, DOIS and the aminotransferase have been enzymatically and genetically clarified. An enzyme for DOIA oxidation was the last enzyme to be characterized in the DOS biosynthesis. A cell-free extract of Streptomyces fradiae, a neomycin producer, was reported to have an NAD dependent dehydrogenase activity toward DOIA. In the neomycin biosynthetic gene cluster, only one NAD dependent dehydrogenase gene, neoA, has been found, and thus the gene product NeoA was supposed to be involved in the DOIA oxidation. In fact, the recombinant NeoA was found to oxidize DOIA to yield amino-DOI. On the other hand, a gene disruption study of a functionally uncharacterized btrN gene encoding a presumed radical SAM (S-adenosyl-L-methionine) protein suggested involvement of BtrN in the DOIA oxidation. The btrN gene was then heterologously expressed in E. coli and recombinant BtrN was used for enzymatic assay. Anaerobic purification and in vitro reconstitution of the [4Fe-4S] cluster resulted in the formation of active BtrN, which catalyzed the oxidation of DOIA to amino-DOI in the presence of SAM. Further kinetic studies and isotope tracing using [3-^2H]DOIA as a substrate showed that BtrN catalyzes the oxidation reaction through a radical mechanism. This type of enzyme entries to a new family of alcohol dehydrogenase using organic radical derived from SAM. The present study clearly demonstrated that two distinct dehydrogenases, NeoA and BtrN, catalyzing the same reaction in completely different mechanism in the DOS biosynthesis, are operative in S. fradiae and B. circulans, respectively.

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Various structures are found in peptides that was isolated as natural products. For example, N-termini of natural peptides are often modified by fatty acid groups. Furthermore, many of them involve various special structures (e.g. D-amino acids, N-methyl amino acids) as building blocks. On the other hand, the products prepared from in vitro translation system are limited to peptides that consist of limited amino acids. Here, we would like to present the method for ribosomal synthesis of peptide having a wide variety of special structures in the sequence. In vitro translation system can effectively generate peptide pool, whose sequence is encoded by mRNA sequence, just by preparing random DNA sequence. Therefore, the approach for synthesizing bioactive peptides by using in vitro translation system can be powerful tool to construct large and high-quality peptide libraries. To establish this method, we used both the Flexizyme system and the PURE translation system. First, unusual amino acids were charged onto tRNA with the Flexizyme system, which is RNA catalyst facilitating tRNA acylation. Subsequently the translation with the PURE system, which is lacking certain amino acids, was carried out in the presence of the tRNA involving unusual amino acids. As a result, the various structures, which includes N-acyl groups, D-amino acids, and α,β-unsaturated structures, were introduced into the peptide products of in vitro translation system.

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Sterols are important as structural components of plasma membranes and precursors of steroidal hormones in both animals and plants. The differences in the biosynthesis of sterols between plants and animals are believed to begin at the step of cyclization of oxidosqualene, which is cyclized to lanosterol in animals and to cycloartenol in plants. Last year, three laboratories independently identified lanosterol synthase genes from dicotyledonous plant species including Arabidopsis, Panax, and Lotus. As for Arabidopsis lanosterol synthase (LAS1), in planta function was demonstrated through overexpression of LAS1 in transgenic plant cells. From these results, it was elucidated that plants have two routes of the steroidal backbone synthesis. It is important to understand whether biosynthetic pathway of phytosterols via lanosterol generally exists or not. To answer this question, we designed a tracer experiment using [6-^<13>C^2H_3]mevalonate. By the elucidation of deuterium on C-19 behavior of phytosterol, it is possible to clarify that phytosterol is biosynthesized via either cycloartenol or lanosterol; two deuterium retained and three deuterium retained phytosterol are biosynthesized via cycloartenol and lanosterol, respectively. The number of deuterium can be analyzed by ^<13>C-NMR; carbon spectrum connected a deuterium will be theoretically shifted to approximately 0.3ppm up-field by isotope effect. According to this stratagy, [6-^<13>C^2H_3]rnevalonate was synthesized and fed to the seedlings of wild type and LAS1 overexpression plant of Arabidopsis. To enhance the incorporation ratio of the labeled mevalonate, lovastatin (inhibitor of HMG-CoA reductase) was applied at the same time. After incubation, extracted sitosterols were analyzed by ^1H, ^2H-double decoupled ^<13>C-NMR. Although main peak was two deuterium-retained peak, three deuteriums retained peak was observed as minor peak. The peak intensity of sitosterol in LAS1 overexpression was approximately three times as strong as that in WT. This is the first direct evidence that the biosynthetic pathway of phytosterol via lanosterol existed in plant cells.

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The interdisciplinary field of systems biology has evolved rapidly in recent years. A prerequisite for deriving the benefits of such a systems approach is a reliable and well validated bioanalytical platform across complementary measurement modalities, especially transcriptomics, proteomics, and metabolomics. Of these, metabolome analysis is more difficult than genome or proteome analysis because of the diverse chemical and physical properties of metabolites. Nuclear magnetic resonance (NMR) techniques can provide information about the global pool of metabolites, including both soluble low-molecular-weight compounds and insoluble macromolecules. We have been developing new methodologies for metabolomics that combine stable isotope labeling and multidimensional heteronuclear NMR analysis. At this conference, we focus on the methodologies for measuring metabolites. To show the validity of our method, we also describe how we can obtain information on metabolic networks in ^<13>C-labeled Arabidopsis thaliana. The problem of spectral overlap in NMR spectroscopy encountered in NMR-based metabolomics can be alleviated by increasing the spectral dimensionality. However, a large amount of macromolecules, such as proteins, increases the line width and decreases the efficiency of magnetization transfer by spin-spin relaxation resulting in a decrease in detectable correlation signals. In order to establish extraction methodologies that are applicable to a wide range of organisms, we evaluated a series of NMR solvents using the distribution of the full-width at half-maximum and the number of signals observed in ^1H-^<13>C heteronuclear single quantum coherence (HSQC) spectra using ^<13>C-labeled plants, bacteria, and animal samples. In addition, we optimized the concentration of HEPES-d_<18> buffer to keep the pH and chemical shifts constant, and to avoid decreasing the sensitivity of the cryogenic probe. As a result, we chose CD_3OD/HEPES-d_<18> and hexafluoroacetone trideuterate (HFA・3D_2O)/HEPES-d_<18> buffer as extraction solvents. Detailed assignments of the metabolites will be presented. Furthermore, we examined the insoluble fractions, which are required to more fully appreciate all of the functions of metabolites and metabolic networks in whole cells, using high-resolution magic angle spinning (HR-MAS) analysis. Concerning metabolic network analysis, we investigated how to obtain information about biochemical reactions, C-C bond formation, and the cleavage of the major metabolites, such as free amino acids, in crude extracts based on the analysis of the ^<13>C-^<13>C coupling pattern in two-dimensional (2-D)-NMR spectra. For example, the combination of different extraction solvents allows one to distinguish complicated ^<13>C-^<13>C fine couplings at the Cα position of amino acids. As another approach, the f1-f3 projection of the HCACO spectrum also helps in the analysis of ^<13>C-^<13>C connectivities. Using these new methods, we present an example that involves monitoring the incorporation profile of [^<13>C_6]glucose into A. thaliana and its metabolic dynamics, which change in a time-dependent manner with atmospheric ^<12>CO_2 assimilation.

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The filamentous fungus, Lambertella sp. 1346 (L. sp. 1346) and Lambertella corni-maris are mycoparasites on Monilinia fructigena. We have isolated lambertellols A (1) and B (2) from L. sp. 1346 as a candidate of the substances related to the mycoparasitism. The other fungus L. corni-maris produces only trace amount of 1 and 2, however this fungus was found to produce them in a substantial amounts under acidic conditions. Our investigations revealed that the host M. fructigena changed its surroundings into acidic conditions, which suggests that the acidic conditions acted as kairomones that stimulated the production of 1 and 2. Since lambertellols A (1) and B (2) are labile to isomerize each other and to gradually decompose into lambertellin (3) even in methanol, the active species have not been figured out. In order to dissolve this question, we synthesized enantiomeric pairs of 1 and 2 as well as some other stable analogues.

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We have designed and synthesized a novel NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ). DHMEQ is synthesized from dihydroxyaniline through several steps. Optically active DHMEQ can be prepared by a chiral colomun (1). DHMEQ inhibited animal models of rheumatoid arthritis, cancer cachexia, and renal inflammation, and it also suppressed the growth of prostate carcinoma, thyroid carcinoma, breast carcinoma, pancreatic carcinoma, multiple myeloma, and adult T-cell leukemia in nude or SCID mice without any toxicity (2). However, its molecular target has not been elucidated. (-)-DHMEQ strongly inhibited the activation of NF-κB in EMSA. Although DHMEQ was shown to strongly inhibit nuclear translocation of NF-κB in Jurkat and COS-1 cells (3) and in ATL cells (4), we found that (-)-DHMEQ rather weakly inhibited the nuclear translocation in NSCLC A549 cells. Then, we looked into the mechanism of inhibition in A549 cells, and found that (-)-DHMEQ inhibited the binding of NF-κB to κB DNA. It did not inhibit the binding of Oct-1. Next we prepared recombinant p65 including the DNA binding region and NLS. In the reconstitution system with recombinant p65 and κB DNA, the mobility shift was clearly observed, that was inhibited by (-)-DHMEQ. Physical binding of (-)-DHMEQ to p65 was analyzed by surface plasmon resonance. (-)-DHMEQ was added to the fixed p65 plate, and we could observe the interaction between them. In MALDI-TOF MS analysis, addition of (-)-DHMEQ shifted the MW peak of recombinant p65. On the other hand, an inactive analogue of DHMEQ did not shift the peak. Thus it is likely that (-)-DHMEQ directly binds to p65.

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Aggregation of the 42-mer amyloid β (Aβ42) plays a central role in the pathogenesis of Alzheimer's disease. Our recent research on the systematic replacement of Aβ42 with proline suggested that the formation of a turn structure at Glu-22 and Asp-23 could be essential to the potent aggregative ability and neurotoxicity of Aβ42. We verified the existence of this turn structure in the minor conformer of wild-type Aβ42 and E22K-Aβ42 (Italian mutation), by solid-state NMR using dipolar assisted rotational resonance (DARR). In E22K-Aβ42, the ionic interaction between Lys-22 and Asp-23 might promote the turn formation at this site. In order to identify the toxic conformation of Aβ42, we synthesized Aβ42-lactam(22K-23E) as a conformationally restricted analogue of the minor conformer, whose side chains of Lys-22 and Glu-23 are linked with an amide bond. Aβ42-lactam(22K-23E) showed much stronger aggregative ability and neurotoxicity than E22K-Aβ42. This implies that the minor conformer with a turn at Glu-22 and Asp-23 of Aβ42 should be considered as a toxic form. Neurotoxicity of Aβ42 is closely related to the radicalization at both Tyr-10 and Met-35. Our previous study reminds us that the S-oxidized radical cation at position 35, the ultimate toxic radical species, would be produced effectively through oxidation by the phenoxy radical at position 10 in the toxic conformer. Electron spin resonance (ESR) spectrometry using spin-labeling with MTSSL revealed that these residues are close to each other in Aβ42. This finding clearly accounts for the reason why the toxic conformer is more pathogenic than the physiological one.

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Albizzia plants close their leaves in the evening, as if to sleep, and open them in the morning according to the circadian rhythum. Potassium β-D-glucopyranosyl-12-hydroxyjasmonate was isolated as leaf-closing factor (LCF) of Albizzia saman. We developed molecular probes consisting of modified LCF in order to identify its mode of action. However, it is often discussed whether molecular probes containing a larger and less polar functional unit such as fluorescence dye can bind to genuine binding protein of a natural product in living organisms. So, we need appropriate negative control. And an enantiomer of a biologically active natural product can be used as an ideal control because all physical propaties except for optical rotation and affinity to binding proteins are identical between a pair of enantiomers. Thus, enantio-differential approach that compares the results using a pair of probes that was prepared from a pair of enantiomers would be an ideal method in the bioorganic study using molecular probes. We synthesized probe 11 and biologically inactive enantiomer-type probe 12. And carried out fluorescence labeling study using 11 and 12. The results was clearly shown that the motor cells contain some specific binding protein that can differentiate the stereochemistry of the ligands. Similarly, we synthesized enantio pair-type photoaffinity-labeling probes 20 and 21, and used them for photoaffinity labeling of receptor for LCF. By using protoplast of motor cell, we found 35kDa membrane protein strictly recognizes the stereochemistry of LCF, and it is highly likely that the protein is the specific receptor for LCF.

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Regulation of gene expression by transcription factors touches many aspects of eukaryotic biology, and its systematic, external control by organic molecules represents a challenge in chemistry. Our group has discovered by screening chemical libraries a unique small molecule that modulates gene transcription. The synthetic molecule, named "adamanolol," represents the first small molecule that modulates gene transcription by targeting transcription factor-coactivator interaction. We synthesized adamanolol and its derivatives and obtained structure-activity relationship, which enabled the design of the second-generation compound named "wrenchnolol." Wrenchnolol mimics an alpha-helical activation domain of transcription factor ESX: it may serve as a small-molecule activation module when coupled with a DNA binding molecule. Our group has in fact succeeded in designing a completely organic, synthetic transcription factor that activates transcription in vitro. The results demonstrate that it is possible to generate a transcription factor out of completely organic components. Nevertheless, the first small molecule transcription factor, STF1, had limited cell permeability and was not suited for cell culture or animal studies. We have now synthesized the second generation of small molecule transcription factors with better physical properties. We also developed a series of wrenchnolol analogs that may be more potent in activating transcription. Continued efforts may lead to the design of small-molecule transcription factors whose activities are comparable to those of naturally occurring transcription factors. As another application of wrenchnolol, we synthesized a biotinylated version of wrenchnolol, and examined its ability to activate the muscle cell differentiation in the presence of a fusion protein of streptavidin and the DNA-binding domain of MyoD, a master regulator of myogenesis in human cells. Although the differention rates were not optimized yet, the biotinylated wrenchnolol clearly enhanced the myogenesis in cell culture. The results suggest that wrenchnolol is capable of activating the transcription of endogenous genes once it is recruited to their promoters.

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Amphotericin B (AmB, 1) is a polyene antibiotic that has been used for treatment of systemic fungal infections. It is generally accepted that an ion-permeable channel formed across bilayer membranes is responsible for the pharmacological activity of AmB. A "barrel-stave" model has been most frequently discussed as the channel complex. Although many investigations about the channel structure have been carried out, details of the structures are not fully understood. To investigate the ion-channel structure, we applied solid-state NMR for evaluating the interaction between AmBs under membrane environments. First we prepared <13>^C-, <19>^F-, and 2^H-labeled AmBs [tri-^<13>C]AmB(4), derivatives 5 and 6, respectively) by biosynthetic or chemical synthetic methods. Then these labeled AmBs were mixed with DMPC and subjected to solid-state NMR measurements. The ^<13>C{<19>^F}REDOR spectra of DMPC membranes containing the ^<19>F-AmB and ^<13>C-AmB showed that the interatomic interactions were significantly weak in ergosterol-containing membrane as compared with those in sterol-free membrane. These results suggest that ergosterol enhances the mobility of AmB assemblies or changes intermolecular distances in AmB aggregates. To examine the mobility of AmB assemblies, solid-state 2^H-NMR was measured using 2^H-labeled AmB. The results showed ergosterol significantly increases mobility of AmB, while AmB is almost immobilized in sterol-free membrane. These results suggest that in sterol-free membranes AmBs are aggregated to undergo phase separation and in ergosterol-PC membranes AmBs form small assemblies, which may correspond to ion-permeable channels.

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Prion diseases, also known as transmissible spongiform encephalopathies, are believed to be caused by accumulation of an abnormal isoform of the prion protein (PrP^<Sc>) in the central nervous system. The proposed replicative cycle of prion is relatively simple. It encompasses misfolding of a single protein, the cellular prion protein (PrP^C) into PrP^<Sc>. The problem is that there is no significant effective medicine for these diseases. Therefore we planned to search for new effective compounds from natural resources using competitive ELISA. In consequence, pheophytin a (Phe-a), which has high-affinity to PrP^C was found from a medicinal plant, Conandron ramondiodes, and a brown algae Ecklonia kurome. Further investigation clarified that Phe-a inhibits the formation of PrP^<Sc> in prion-infected neuroblastoma cells (ScN2a and F3). This finding suggest that Phe-a might be useful for the prevention of prion diseases, but a mechanism to inhibit the prion replication is still unknown. Then we investigated the interactive analysis of prion and Phe-a using some spectroscopic methods(CD and NMR spectra). As a result, it revealed that one mole of Phe-a binds to the second α-helix of PrP^C 121-231, and stabilizes its conformation.

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The hedgehog (Hh)/GLI signaling pathway has been implicated not only in a variety of developmental processes in wide range of organisms, but also in the formation and development of different tumors including skin, brain, prostate, upper gastrointestinal tract, pancreas and lung. Targeting Hh/GLI signaling has been expected as an effective cancer therapeutic strategy. To find the specific inhibitors of Hh/GLI signaling pathway from natural resources, a cell-based screening assay system targeting transcriptional activator GLI1, which constitutes the final step in the Hh signaling pathway, was constructed. A pGL4-Luc reporter vector inserted with 12×GLI binding sites was stably transfected into HaCaT cell line expressing GLI1 under tetracycline repressor control. A suitable protocol for the screening was then established. The screening of our natural products library with the above constructed system identified six active compounds; staurosporinone (1), 6-hydroxystaurosporinone (2), arcyriaflavin C (3), 5,6-dihydroxyarcyriaflavin A (4), zerumbone (5), and zerumbone epoxide (6). Their IC_<50> values of GLI1 transcriptional inhibitory activity were 1.8, 3.6, 11.3, 6.9, 3.0 and 55μM, respectively. The next screening of our natural plants extracts library gave 15 hit plants. Repeated chromatography separations of the MeOH extracts of Physalis minima and Zizyphus cambodiana gave active compounds. P minima gave two compounds; physalin F (10) and physalin B (11), and Z. combodiana a compound; colubrinic acid (13). Their IC_<50> values were 0.66, 0.62 and 29μM, respectively. It was reported that glil, ptchl, bcl2 are the Hh/GLI-mediated transcriptional targets. The influences of these inhibitors (1, 5, 10 and 11) on these protein expressions were investigated with western blotting analysis. These inhibitors decreased the expression of GLI1 and PTCH1, which are related Hh signaling pathway. Moreover, BCL2, which is related with apoptosis, was also decreased.

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Papuamides A (1) and B (2) are novel cyclic depsipeptides isolated from the sponges collected at Papua New Guinea by Boyd and co-workers in 1999. They are known to inhibit the infection of human T-lymphoblastoid cells by HIV-1_<RF> in vitro. In addition to their novel biological activities, they have intriguing structures containing (4Z,6E)-2,3-dihydroxy-2,6,8-trimethyldecanoic acid (Dhtda) and 7 unusual amino acid residues. The stereochemistry of papuamides remains to be determined because of the uncertainty regarding the 3-OMeTyr and Dhtda parts. Recently we have clarified the absolute stereochemistry of 3-OMeTyr as (2R,3R). In this symposium we disclose our efforts on the structural determination of Dhtda and synthetic studies of papuamide B Dhtda-Gly part was synthesized by using stereoselective addition of alkynyl lithium generated from dibromoalkene (4) in situ to aldehyde (5) as a key step. Other diastereomers were synthesized according to the same procedure. The synthesis of (2R,3R)-3-OHLeu (22) and (2R,3R)-3-OMeTyr (29) were achieved by the use of asymmetric hydrogenation via dynamic kinetic resolution catalyzed by the chiral ruthenium or iridium complex originally developed by us. Other unusual amino acid residues, (2S,3S,4R)-DiMeGln (37) and (2S,3R)-Dab (42), were synthesized by diastereoselective Michael addition to chiral bicyclic lactam (30) and proline catalyzed asymmetric hydrazination of aldehyde (38) as a key step, respectively. The macrolactamization of heptapeptide (48) was achieved by FDPP as a coupling reagent. The consecutive coupling of cyclodepsipeptide (49), DiMeGln (37), Dab (42), Thr, and (2R,3S,8R)-Dhtda-Gly unit (16) using DEPBT followed by deprotection afforded (2R,3S,8R)-Dhtda-papuamide B (2). The synthesis of other isomers derived from Dhtda with different stereochemistry has been actively underway.

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Saxitoxin (STX) (1) and its derivatives are known to be neurotoxins that block the voltage-gated sodium channels, which are critical for depolarization and conduction in most excitable cells. STX (1) also binds to other receptor proteins, such as calcium and potassium channels, and appears to be of considerable biological importance. In this paper, we described an enantioselective total synthesis of (-)-decarbamoyloxysaxitoxin (doSTX) (7), which has the same skeleton as STX and its natural analogues. Our synthesis of doSTX features direct oxidation with o-iodoxybenzoic acid (IBX) at the α-position of the β-amino-substituted ketone, which was synthesized based upon the 1,3-dipolar cycloaddition reaction between methyl crotonate (13) and the chiral nitrone 12. Oxidation of the ketone derived from alcohol 22 at C4 selectively took place with IBX (1.1 equiv.) in DMSO, and gave the aminal 23 in 51% yield. Treatment of 23 with sodium borohydride at 0℃ diastereoselectively gave the alcohol, whose Boc group was deprotected with TFA, and a Cbz-protected guanidine group was installed to give 26. The structure of 26 was confirmed unequivocally by X-ray crystallographic analysis. The synthesis of 7 was completed with the following three steps from 26. Deprotection of the four Cbz groups with hydrogen in the presence of Pd(OH)_2, followed by treatment with TFA, gave the decarbamoyloxysaxitoxinol 27 in 60% overall yield. Finally, oxidation of the alcohol with dimethylsulfoxide and diisopropylcarbodiimide afforded (-)-doSTX (7) in 60% yield. All the spectroscopic data of the synthetic material were consistent with the reported data for the natural product. The optical rotation value of 7 was -23.3°(c 0.7, MeOH).

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A number of polymeric Nb-methyltryptamine-derived alkaloids that link together two to eight pyrrolidinoindoline units have been isolated from rubiaceous plants. Some of them have a variety of biological activities involving potent opioid agonistic activity. Recently, we have investigated the alkaloidal constituents in Psychotria rostrata native to Malaysian rainforest and have found three novel tryptamine-derived alkaloids, named pscyhotrimine (1). psychotetramine (2) and psychopentamine (3). To establish their structures as well as to develop the efficient synthetic route, we embarked on the total synthesis of these structurally unique alkaloids. 1. Total synthesis of new trimeric alkaloid, psychotrimine All the hitherto known tryptamine-related polymeric alkaloids are composed of pyrrolidinoindoline units and link together at the C3a-C3a' and C3a-C7' positions. Characteristically, psychotrimine (1) is the first example of this class of alkaloid that contains the tryptamine unit in the molecule as well as possesses the linkage at C3a-Na (indole) and C7-Na (indole) positions. We planned a synthetic strategy that employs the copper mediated intramolecular and intermolecular amination of halobenzenes as key steps. Starting from 2-bromobenzaldehyde and indoline, we prepared dimeric compound (10) via intramolecular amination of amidine (8). Onto the C7 position of indoline core in 12, an additional tryptamine unit was installed by intermolecular amination to furnish the total synthesis of (±)-1. Here, we have proven the structure of psychotrimine. 2. Synthetic study of new tetrameric alkaloid, psychotetramine Psychotetramine (2) is a new tetrameric tryptamine-derived alkaloid containing the known dimeric alkaloid, chimonanthine, in the lower segment and one tryptamine unit in the upper part. To reveal the structure inferred by spectroscopic analyses, we planned the convergent strategy that involved the copper mediated intermolecular amination between iodide (12) and the stereoisomers of chimonanthine, the later of which have been synthesized by oxidative coupling of tryptamine derivatives with hypervalent iodine (III) reagent.

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Serotobenine (1) was isolated from the seed of safflower by Sato and co-worker in 1985 and the structurally-related compound decursivine (3) was isolated by Fong et al. in 2002 Both of these compounds 1 and 3 are characterized by the fused polycyclic ring systems including indole, dihydrobenzofuran and 8-membered lactam rings. Although biosynthesis of both compounds would be similar, 1 existed as a racemic form and 3 was composed of an optically active form. The difference of stereochemistry also prompted us to investigate the synthetic study of these compounds. Herein, we report the total synthesis of optically active serotobenine (1). Recently we found that an intramolecular C-H insertion reaction by the combination of Davies Rh catalyse and the chiral auxiliary provided an optically active trans-2,3-dihydro-3-benzofuran derivatives efficiently. We envisioned that this method would be applicable for the synthesis of the optically active dihydrobenzofuran ring of 1. The indole derivative 9, readily synthesized by Leimgruber-Batcho's protocol was converted to 4-allylindole derivative 10 by regioselective Claisen rearrangement (Scheme 4). As shown in Scheme 5, conversion to the diazoester 15 from 10 was achieved by diazotransfer reaction. Upon treatment of 15 with achiral rhodium catalyst Rh_2(OAc)_4, the C-H insertion reaction was proceeded smoothly to afforded dihydrobenzofuran 16 in high diastereoselectivity. After the transformation to the activated ester 18, construction of 8-membered lactam ring was accomplished by after reduction of azide group. Finally, deprotection of Ts group and Bn group was carried out in stepwise manner and total synthesis of (-)-serotobenine (1) has been accomplished. We are currently investigating the stereochemical behavior of 1, according to our racemization hypothesis, as shown in Scheme I.

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Amathaspiramides A-F are a class of marine alkaloids isolated from a New Zealand collection of the bryozoan Amathia wilsoni. Since these alkaloids were available in a minute quantities from the bryozoan, only preliminary pharmacological evaluation of these alkaloids has been reported, i.e., amathaspiramide E exhibited a potent antiviral activity and both A and E showed moderate antifungal and cytotoxic activities. The structures of amathaspiramides are characterized by their unique spirobicyclic framework containing three contiguous chiral centers and a dibrominated aromatic ring. We have been reported that the enolate-Claisen rearrangement of an α-acyloxy-α-alkenylsilane gave the rearranged product with a complete transfer of the original chirality. Based on these results, we planned the total synthesis of amathaspiramide F (1) using the enolate-Claisen rearrangement of the α-acyloxy-α-alkenylsilane with a Z-olefin geometry 4 (route A) or 5 (route B) as the key step: the reaction would produce 8 or 9 possessing the requisite chiral centers at CS and C9 (erythro relationship) together with a vinylsilane group convertible to the hemiaminal moiety of 1. The enolate-Claisen rearrangement of 4 (route A) having a proline as the acyloxy group was found to give undesired threo isomer 8 as the major product. On the other hand, the acyclic route (route B) underwent stereoselective rearrangement to give desired erythro isomer 9 (threo/erythro = 1: 7). The rearranged product 9 was converted to the spirobicyclic hemiaminal 12 via the pyrrolidine 10. The completion of the total synthesis of 1 was accomplished by the following sequence of reactions: (1) dibromination of the phenol group with n-Bu_4NBrCl_2 (2) methylation of the phenol with MeI, and (3) removal of the TFA group with LiBH_4.

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Claisen rearrangement is a powerful reaction that generates carbon-carbon bonds from carbon-oxygen bonds with chirality transfer. However, applications of this reaction to the generation of a quaternary carbon in the cyclohexane ring system possessing a sterically hindered ortho-substituted phenyl group, and reports on its cascade-version are limited. In this paper, we report the total synthesis of (+)-galanthamine (1) and formal total synthesis of (-)-morphine (2) starting from carbohydrates in which the key stereoselective generation of the quaternary carbons was achieved by the Claisen rearrangement. In the synthesis of an Amaryllidaceae alkaloid galanthamine (1), it was found 2-nitrophenol-catalyzed Johnson-Claisen rearrangement of cyclohexenol 5 possessing o-substituted phenyl group, prepared from D-glucose using Ferrier's carbocyclization, afforded the rearranged product 3 in high yield. The dibenzofuran skeleton was effectively constructed by the Br^+ mediated cyclization. After introduction of an endo olefin, the Pictet-Spengler type cyclization, followed by reduction of the amide function completed the chiral and non-biomimetic synthesis of 1. In the morphine (2) synthesis, D-glucal was converted into cyclohexene diol 12 possessing a cathecol moiety using the Ferrier's carbocyclization and Suzuki-Miyaura coupling. The cascade Claisen rearrangement of 12 in the presence of 2-nitrophenol successfully provided the double-rearranged product 11 in moderate yield. The epoxide-mediated dibenzofuran formation, followed by intramolecular Friedel-Crafts reaction and Birch reduction provided (-)-dihydroisocodeine (23), representing the formal synthesis of 2.

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Salinosporamide A, a potent proteasome inhibitor isolated from actinomycete bacteria Salinospora tropica, has attracted much attention in the chemical and biological communities due to its intriguing molecular architecture as well as characteristic biological properties. We envisaged Conia-ene type reaction of a 2-(but-3-ynamido)malonate for the construction of its γ-lactam core. Although there was no precedent for the synthesis of γ-lactams by Conia-ene reaction, we found that Ni(acac)_2-Yb(OTf)_3 and In(OTf)_3 promoted cyclization of 12 to 13 in good yields, respectively. Moreover, we also found that SiO2 effected this conversion in excellent yield although the reaction was rather slow. It should be stressed that each of these conditions did not cause any substantial racemization as well as isomerization of the exo olefin of 13. Lactam 13, thus prepared, was stereoselectively converted to Corey's intermediate 21 via selenoacetalization giving 17, discrimination of the two esters giving 19, cyclohexynylation giving 20, and deprotection. Following the Corey's procedure, we achieved a total synthesis of salinosporamide A. Neooxazolomycin, a potent antitumor antibiotic isolated from Streptomyces strains, was also synthesized by a convergent strategy that features a highly stereoselective approach involving the above-mentioned Conia-ene type cyclization, dihydroxylation accompanied by lactonization, and Nozaki-Hiyama-Kishi reaction to construct right hand segment 29 and an improved assembly of left hand segment 30. In the key Conia-ene type cyclization of 26 to 27, Ni(acac)_2-Yb(OTf)_3 and In(OTf)_3 turned out to effect this conversion in complete E-selectivity, while SiO_2 did not cause this cyclization at all.

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(-)-Conophylline (1), a bisindole alkaloid constituted of highly oxygenated vincadifformine-tabersonine moieties, was isolated from Tabernaemontana divaricata in 1992. It was shown to possess a potent inhibitory activity of ras function. The interesting biological activities coupled with the unique structural features have attracted our attention as a synthetic target. Herein, we report a convergent total synthesis of (-)-conophylline (1) utilizing a regio- and stereoselective coupling of the upper unit 5 and the lower unit 4 as a key reaction. The synthesis of the lower unit 4 is shown in Schemes 2-5. The Key indole intermediate 13 was synthesized through our tin-mediated indole formation from isocyanide 11, which was readily prepared from a commercially available benzoate 7 in a 11-step sequence including monoprotection of phenolic hydroxyl groups, nitration, Wittig reaction, and conversion to isocyanide. The 2-stannylindole, obtained by the radical cyclization was converted to 2-iodoindole 12 by immediate treatment with iodine and reduction of the ester group. Finally, installation of the methyl acrylate moiety and manipulation of protective groups provided the key indole unit 13. The alcohol 13 thus obtained was coupled with 14 by Mitsunobu reaction to give the desired protected secondary amine 15. The crucial construction of aspidosperma skelton was executed by sequential reactions including deprotection of Boc group, cleavage of DNs, and cyclization under heating to furnish compound 18. Dehydration of 18 gave tabersonine derivative 19, which was then converted to the lower unit 4 by protection of nitrogen with Troc group followed by stereoselective epoxidation according to Szantay's procedure. On the other hand, the upper unit 5 was readily synthesized from 21, which was a key intermediate in our (-)-vindoline synthesis, according to the above synthetic route to 4. With both the upper and the lower units in hand, we next focused on the coupling of these units by Polonovski-Potier reaction. We found that upon treatment of the N-oxide 23 with TFAA, generation of iminium salt 24 and subsequent nucleophilic addition of the upper unit 5 occurred regio- and stereoselectively to give the desired coupling compound 25 as a single isomer. The characteristic dihydrobenzofuran moiety was constructed efficiently via the cleavage of the allyl group followed by intramolecular 5-exo ring closing reaction. Finally, the mesyl and Troc groups were removed with LDA to complete a total synthesis of (-)-conophylline (1). In the same manner, we have completed a total synthesis of (-)-conophyllidine (2) as well. As an alternative route to access the key intermediate 21, we have developed a 9-step sequence starting from natural (-)-vindoline (29) in 27% overall yield. We believe that these synthetic pathways should be amenable to the synthesis of a wide variety of conophylline analogs.

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Due to the novel hexahydropyrrolo[3,2-c]quinoline nucleus as well as their potential use in medicinal chemistry, martinelline (1) and martinellic acid (2), isolated from the root bark of the tropical plant Martinella iquitosensis, have spurred intense efforts to synthesize them. Although there have been reported several total syntheses, the issue on the elucidation of the absolute configurations of 1 and 2 is still the subject of serious arguments. In addition, there have been reported few methodologies that allow facile construction of the tricyclic core in an enantiocontrolled manner. Herein, we describe the first asymmetric total synthesis of (-)-martinelline (1) and the second total synthesis of (-)-martinellic acid (2) based on the highly efficient Lewis base-promoted intramolecular Mukaiyama-Mannich/hemiaminalization reaction using imine 18. Unexpectedly, a comparison of their specific rotations suggested that the reported natural Martinella alkaloids are nearly racemic or mixtures of two enantiomers. To determine which of the two enantiomers confers biological activities, both enantiomers were also synthesized. Evaluation of the GPCR antagonist activity toward H_1-hiatamine receptors on human astrocytoma 1321N1 cells revealed that only (-)-(3aS,4S,9bS)-martinelline (1) exhibits GPCR antagonist activity.

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Spirocyclic scaffolds are embedded in many biologically active natural compounds, including candidates for medicines, perfumes and agricultural chemicals. Therefore, a development of efficient synthetic methods directed at those structures is strongly demanded. To this end, we have recently developed synthetic methodologies for two different spirocyclic frameworks based on Claisen rearrangement; (1) multifunctionalized spiro[4.5]decanes through Claisen rearrangement of bicyclic 2-(alkenyl)dihydropyrans, and (2) spirocyclic oxindoles through a sequential intramolecular Ullmann coupling (IUC) and Claisen rearrangement of 2-iodoindoles bearing allylic alcohol moiety. We have developed the Claisen rearrangement protocol by which bicyclic 2-(alkenyl)dihydropyrans bearing a functional group at the C4 position can be transformed to the corresponding spiro[4.5]decanes in good-to-excellent yields with excellent stereoselectivities. We applied this method to a concise total synthesis of (-)-Gleenol (3), from hindered dihydropyran 9 bearing all requisite functional groups with proper stereochemistry. The most striking features of this synthesis are that remarkable solvent effects in the key Claisen rearrangement has been observed, and that the rearrangement of 9 led to the fully functionalized spiro[4.5]decane 10 in a single step. Related Claisen rearrangement in (alkenyl)pyranoindole systems can be also achieved. Thus, a one-pot IUC/Claisen rearrangement sequence from 2-iodoindoles 14 was found to provide spirocyclic oxindoles 16 in good yields with excellent stereoselectivities. We applied this sequence to the asymmetric synthesis of pyrrolidinoindoline alkaloids, (-)-debromoflustramine B (22). Enantiomerically pure 2-lodoindole 17 was subjected to the IUC/Claisen rearrangement conditions to furnish spirocyclic oxindole (+)-16 in good yield with perfect asymmetric transmission. Further transformations including oxidative cleavage of the olefin in (+)-16 provided the pyrrolo[2,3-b]indoline 20. Finally, the total synthesis of 22 was accomplished via a two-step sequence.

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TAN-1085 (1) is an antibiotic produced by Streptomyces sp. S-11106. This compound has attracted considerable synthetic interest not only from its important biological activities, e.g., angiogenesis and aromatase inhibitory activitities, but also from its unique structural features composed of a benzo[a]anthraquinone chromophore with a vicinal diol at the B -ring, one of which is glycosylated with a rhodinose. We recently completed the first total synthesis of this compound, clarifying unknown the relative and absolute stereochemistries, and therefore the remaining problem was the asymmetric synthesis of the aglycon portion. Herein, we report a promising method to construct the axially chiral biaryl structure and asymmetric total synthesis of target molecule. The key point of our strategy was that a new class of axially chiral compounds, "axially chiral styrenes" was used as a chiral building blocks. It is the styrene derivatives whose rotation around the C-C bond that connects aryl and ethenyl moieties is restricted by having large subsutituents on their skeleton. In planning the effective access to the target structure, we envisaged a consecutive chiral transfer approach starting from the axially chial styrene. Through a series of model experiments, we were able to design chiral styryl sulfoxide 25. After connection with a benzocyclobutenone 3 followed by pericyclic ring enlargement to the biaryl 5, the "styryl tereochemistry" was cleanly transferred into the axial stereochemistry, which in turn was transferred into the central stereochemistries of the vicinal diol via pinacol cyclization. The overall process provided the targeted aglycon 6 in highly enantio-enriched form, and introduction of the sugar moiety 7 accomplished the asymmetric total synthesis of 1.

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Maduropeptin, an extremely potent antitumor agent, was isolated from the groth filtrate of Acti nomadura madurae. It was identified as a 1: 1 complex of an acidic carrier apoprotein (32kDa) and 9-membered ring enediyne chromophore. The structure of intact chromophore has not been elucidated because it is too labile for isolation, and instead methanol adduct (1) was isolated from the methanol extract and was characterized except for the absolute stereochemistry. The chromophore (1) possesses a labile 9-membered diyne core, a macrolactam ansa-bridge and an aminosugar. We developed a new reductive olefination method from diol derivatives using SmI_2, and succeeded in synthesizing the aglycon (5). Installation of the 9-membered diyne ring is one of the most important steps in the synthesis of maduropeptin chromophore. We developed a new cyclization method using LiN(SiMe_2Ph)_2 and CeCl_3, which was superior to the previous combination, i.e., LiHMDS/CeCl_3. TBS group of cyclized 7 was removed and the alcohol (16) was converted to the azide. The TBDPS group was also removed and the alcohol (17) was oxidized to the corresponding carboxylic acid, which was converted to the pentafluorophenyl ester. Then, macrolactam (19) was formed spontaneously after Staudinger reaction. X-ray crystallographic analysis of 19 proved that the two triple bonds are greatly bent. To complete the synthesis of the aglycon (5), p-trifluoromethyl benzoate was chosen as the diol derivative. The corresponding dibenzoate (20) was exposed to the reduction conditions using SmI_2, so that the desired 4,13-Z-olefin (21) was solely produced. This regioselectivity would be controlled by the repulsion between the ansabridge and the C13-p-trifluoromethyl benzoate. Protected aglycon (22) was a mixture of two atropisomers, which were in equilibrium at room temperature. Finally, all protecting groups were removed and aglycon (5) was synthesized for the first time.

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An extreme thermophile, Thermus thermophilus HB8 is a microorganism living in a severe hot environment. Recently, three kinds of glycolipid have been extracted from the cell membrane of the microorganism, and these structures have been determined. All these glycolipids showed immunostimulating activities. In order to examine the detailed biological activities and to search the immunostimulatory mechanism, we synthesized the most unique glycolipids, phosphoglycolipid 1a, by applying newly developed α-selective glycosylation. The synthetic scheme is shown in Scheme 1. First, we examined the α-selective glycosylation of 2-azide sugar 2 and the glyceric acid unit 3. We used the 2-azide sugar, 3,4,6-tri-O-benzyloxy-2-azide-D-glucopyranosides, without the neighboring group effect at 2-position, as the form of 3-nitro-2-pyridyl glycoside. The 3-nitro-2-pyridyl glycoside is easily activated with a catalytic amount of Lewis acid, and we were able to obtain the α-glycoside 4 as the major compound. The α-glycoside was then provided to the phosphorylation with phosphoroamidite 6a to construct the skeleton. Final deprotection with reductive conditions successfully afforded the desired compound 1a. We also synthesized the diastereomer 1b, which has the different configuration at the diacyl glycerol moiety. Only 1a showed the immunostimulatory activity, suggesting the existence of a receptor that recognizes diacyl glycerol part.

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It has been revealed that certain prokaryotes, such as Campylobacter jejuni, have asparagine (Asn)-linked glycoproteins [1]. However, the structures of their glycans are distinct from those of eukaryotic origin. They consist of a bacillosamine (Bac) residue linked to Asn, α-1,4-GalNAc repeat, and branching β-Glc residue, namely GalNAc-α(1,4)-GalNAc-α(1,4)-[Glc-β(1,3)-]GalNAc-α(1,4)-GalNAc-α(1,4)-GalNAc-α(1,3)-Bac [2]. Our achievements toward the synthesis of the novel N-linked glycan have been reported, including 1) development of PFP strategy [4] for the stereoselective synthesis of α(1,4)-GalNAc repeating motifs using 4-O-pentafluoropropionyl (PFP) protected donors [5], 2) construction of the branched hexasaccharide Glc_1GalNAc_5 (7) [5], 3) syntheses of Bac (10) and N-Asn-linked Bac (11) [6] using our efficient methodology for N-glycosyl Asn [7]. Here, we describe the completion of stereoselective synthesis of a N-glycan derived from C. jejuni, a heptasaccharide, GlcGalNAc_5Bac (1) [8]. The synthesis started from Bac acceptor (9), which was consecutively glycosylated with 4-O-PFP protected donors (4, 6) to give the target heptasaccharide GlcGalNAc_5Bac (1). For the substrate of C. jejuni oligosaccharyl transferase, we also completed the synthesis of undecaprenol (39) starting from trans,trans-farnesol and nerol.

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Ciguatoxin (1) is a principal toxin of ciguatera which is one of the most wide spread seafood poisonings. Our concept for the total synthesis toward Ciguatoxin (1) is based on acetylene dicobalthexacarbonyl complex-mediated ether-cyclizations. The reaction proceeds via a dicobalthexacarbonyl complex-stabilized cationic intermediate, which called Nicholas effect, subsequently undergoes endo cyclization to give a syn product thermodynamically. We have explored various decomplexation methods of the endo-acetylene cobalt complex. The retrosynthetic analysis toward ciguatoxin is revealed that the A, F, and G ring would be cyclized at the latest stage after the coupling between acetylene of left segment 11 and aldehyde of right segment 12. The right segment would be divided further three subsegments (16, 17 and 18). Synthesis of left segment 11 was started from 19. The methylketone moiety of 19 was protected as 2-naphtylmethyl ether. The modification of E-ring was proceeded by following steps: epoxidation of vinylsilane, Peterson reaction, ring opening of epoxysilane, inversion of hydroxyl group to afford 11. Subsegment 17 was prepared through heteroconjugate addition reaction of excess amount of lithium acetylide and vinyl sulfone 23. Reduction of the desilylated product 26 using SmI_2 gave subsegment 17. After coupling between 17 and 18, the coupling product was cyclized via acetylene cobalt complex followed by dihydroxylation and spiroketalization to give JKLM-ring fragment. Sonogashira coupling of vinyltriflate 31 with acetylene 16 followed by I-ring cyclization to cyclic cobalt complex 33. Intramolecular 1.4-addition of enone afforded H-ring cyclized product 35. The cyclic ketone 35 was transformed into endo-cyclic olefin 36 and subsequent reduction with Crabtree catalyst to give 37 as a single isomer. Segment coupling between 11 and 12 followed by F-ring cyclization to 39. The cyclic cobalt complex 39 was converted into corresponding ketone via ligand exchange with dppm. The hydroxyketone was cyclized by reductive etherification to give BCDEFGHIJKLM-ring 41 in moderate yield. Construction of A-ring to 1 is now underway.

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During this decade, we have been engaged in synthetic and structural studies on highly oxidized and structurally diverse cytotoxic triterpene polyethers, which are thought to be biogenetically squalene-derived natural products (oxasqualenoids), isolated form both marine and terrestrial organisms. In particular, we have been interested in the biogenesis of polyTHF rings-containing oxasqualenoids which might be synthesized in a single step by the sequential oxacyclization from acyclic precursors squalene polyepoxides. In this symposium, we report that a novel oxasqualenoid omaezakianol (1) was isolated from the red alga Laurencia omaezakiana Masuda sp. by Suzuki et al. in 1994, and the structure including the absolute configuration has recently been found to be shown by a structural formula 6 through spectroscopic methods (Figure 1), the derivatization experiments (Scheme 1), and the asymmetric total synthesis (Scheme 2). We also report that the total synthesis of neighboring tri- and tetraTHF rings-containing teurilene (7) (Scheme 3) and (+)-omaezalcianol (6) (Scheme 4), respectively, has been accomplished on the basis of the biogenetic hypothesis (Scheme 1).

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Pladienolide B (2) and D (3) are 12-membered macrolide isolated from Streptomyces platensis Mer-11107 by way of a cell-based assay that evaluated the suppression of hypoxia-induced gene expression controlled by the human VEGF promoter. They also inhibit the growth of a variety of cancer cell lines in vitro with low nanomolar IC_<50> values. COMPARE analysis indicated that the compounds have a unique mode of antitumor action. Pladienolides B and D also cause in vivo tumor regression in several human cancer xenograft models. After intensive studies, we discovered E7107 (4). Treatment of several tumor xenograft models with E7107 has led to complete remission as well as tumor shrinkage in a variety of tumor xenografts. Recently E7107 has entered Phase I clinical trial. To verify the structure of Pladienolides, and to facilitate the discovery of novel analogues with advantageous pharmaceutical profiles we have executed the first total syntheses of pladienolides B and D. In conclusion, we have achieved the first total synthesis of pladienolides B (2) and D (3), through longest linear sequences of 22 and 19 steps, respectively, in overall yields of 2.1% and 2.2 %, respectively. These syntheses confirmed the absolute stereochemistry of pladienolides B and D. Our synthetic approaches involve the first example of ring-closing metathesis for the construction of a sterically hindered aliphatic 12-membered macrolide structure and also exhibit an effective application of cross-metathesis to the synthesis of natural products. The exploitation of cross-metathesis at the culmination of our total synthesis is quite efficient and versatile because the fragments can be assembled without protecting groups to directly provide the final target in sufficient yield. We believe that this synthetic effort provides a practical route to novel pladienolide analogues that could not be obtained from natural resources.

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Reveromycin A (1) having 6,6-spiroketal core is a novel polyketide-type antibiotic with strong biological activity that makes it potentially useful for treatment of tumor, hyperpotassemia and bone disease. Spirofungins A (2) and B (3) are also polyketide-type antibiotics isolated from Streptomyces violaceusniger TO 4113 as a mixture of 2: 3 in the ratio of 4: 1 and show various antifungal activities, particularly against yeasts. The stereocontrolled total synthesis of (-)-2 and (+)-3 has been achieved with the alkyne 7, the Weinreb amide 8, and the alkenylboronatev 6 readily available from the common intermediate alkyne 5 employing Horner-Emmons reaction and Suzuki coupling reaction for the construction of the both side chains. It was determined that the absolute configuration of natural spirofungin A is 4S,5R,11R,12S,15S,18S,19R. The first synthesis proceeded with a longest linear sequence of 31 steps, affording (-)-spirofungin A and (+)-spirofungin B in 7.9% and 5.2% overall yields, respectively. The first asymmetric total synthesis of 1 was accomplished by our group using the succinylation of tertiary alcohols under high pressure as a key step. Now, we have developed a new methodology for the synthesis of the succinates of tertiary alcohols without troublesome high pressure. Namely, 3-butyn-1-ol was converted into the lactone 24 through the Sharpless asymmetric epoxidation of 32 in 14% overall yield in eleven steps. After protection of the hydroxyl groups, the dihydropyrans 29 and 30 was prepared via palladium-catalyzed coupling of the ketene acetal triflate derived from 26 and 27 and zinc derivative. Successive oxidation of 28, 29 and 30 with OsO_4 and Pb(OAc)_4 gave the succinyl derivative 31, 32 and 33. Synthesis of 1 from 33 is in progress. In connection of our studies concerning the chemical modifications and structure-activity relationships of 1, we synthesized 2,3-dihydro- 39 and 40, 4-hydroxy- 41 and 5α-hydroxy derivatives of 1. Effects on isoleusyl-tRNA synthetase activity and morphological reversionactivity on src^<1s>h-NRK cells by the 6,6-spiroketal derivatives were examined.

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Presented herein is a total synthesis of cinchona alkaloids including quinine (1) and quinidine (2). The intermediate we have designed is the piperidine 5, which was synthesized from the cyclopentenyl monoacetate 6 as disclosed in Scheme 1. Installation of the necessary two side chains on the cyclopentene ring of 6 was accomplished by Pd-catalyzed reaction with malonate anion and subsequent Claisen rearrangement with CH_2=CHOEt/Hg^<2+> to afford cyclopentene 9, which, after functional group manipulation, was submitted to oxidative cleavage at the double bond followed by construction of the piperidine ring to produce 11. Finally, Wittig reaction of the aldehyde derived from 14 with the quinoline phosphonate furnished the intermediate 5. Transformation of 5 to quinine (1) was carried out through dihydroxylation with AD-mix-β, transformation of the resulting diol 16a to epoxide 17a, and an intramolecular reaction shown in 18a (Scheme 2). Similarly, quinidine (2) was synthesized from 5. In order to replace the above method of constructing the vinyl group, allylic substitution of 19 with CH_2=CHMgBr was studied to afford 20 satisfactory (Scheme 3). In a similar way, we synthesized various analogues with i-Pr, c-Hex, and Ph groups (Scheme 4).

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We have been involved in development of asymmetric reactions using diether 2 as a chiral chelating ligand and have succeeded in asymmetric addition of organolithium reagents to α,β-unsaturated carboxylates (Scheme 1). The addition of organolithium reagent to α,β-unsaturated carboxylate gives lithium enolate intermediate, whose intermolecular trapping with another α,β-unsaturated carboxylate moiety would produce highly functionalized chiral cyclohexane building blocks, such as 4 (Scheme 2). We planned asymmetric synthesis of Amaryllidaceae alkaloids, lycorine (1) and related compounds using 4 as a key intermediate. The investigation of the key tandem asymmetric conjugate addition-cyclization reaction using chiral ligand 2 revealed that the bulky ortho-substituent, TMS group of aryllithium 6b using important to achieve the first addition step in high enantioselectivity (Scheme 3). Besides, the ethylenedioxy group of α,β-unsaturated carboxylate 3 improves diastereoselectivity in the cyclization step probably because the replacement of H with O makes 1,3-diaxial interaction more unfavorable in the transition state that gives trans-cis isomers 8 (Figure 2). With enantiomerically enriched cyclohexane 9 in hand, we started the asymmetric synthesis of lycorines (Scheme 4). Treatment of 9 with ethanolic HCl gave carboxylic acid 10, whose Curtius rearrangement gave carbamate 11 in good yield. Formation of the tetracyclic core was achieved via lactam formation and Bishler-Napieralski reaction to give ketone 14. Formal synthesis of 1-deoxylycorine was accomplished via double bond formation by IBX oxidation of silyl enolate and reduction of the resulting enone 15. Introduction of 2-hydroxy functionality to ketone 14 was achieved stereoselectively by Magnus' chemistry (Scheme5). Formation of double bond followed by reduction gave 2-epi-lycorine diacetate (23) after acetylation of the resulting 2-epi-lycorine (22).

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Over 80 unique polycyclic polyprenylated acylphloroglucinols (PPAPs) have been isolated from the family Guttiferae and related plants Fascinating chemical structures and intriguing biological activities are closely related with this class of natural products. The PPAPs, represented by hyperforin (1), feature a highly oxygenated and densely substituted bicycle[3.3.1]nonane-2,4,9-trione core with prenyl, geranyl, benzoyl, and isobutyryl side chains. Relevant to neurodegenerative diseases such as Alzhiemer's the natural product garsubellin A (2) has been shown to enhances choline acetyltransferase (ChAT) activity by 154% in P10 rat septal neurons, relative to a controls. Nemorosone (3) alternatively has displayed in vitro cytotoxicity against cancer cells over normal cell lines and possesses activity against drug resistant cancer cell lines. Clusianone (4) an anti-HIV agent displaying activity against HIV infection of C8166 human T lymphoblastoid cells additionally has promise as a potential therapeutic agent. Due to these intriguing biological activities we envisioned that the preparation of several members of this class of natural products could allow additional syntheses and provide the required materials for further pharmacological testing as well as provide access to edited structures, that may elucidate the structure activity relationships of these important natural products. The concise synthesis of garsubellin A (2) and nemorosone (3) are described. Starting from 3,5-dimethoxyphenol, the synthesis has provided garsubellin A (2) in an 18-step sequence and nemorosone (3) in a 14-step sequence. Notable transformations include dearomative allylation, diastereoselective vinylogous lactonization, iodocarbocyclization, and bridgehead functionalization reactions to construct and functionalize of the bicyclo[3.3.1] nonane carbon skeleton of the PPAP natural products garsubellin A (2) and nemorosone (3).

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Cyathane diterpenes possess an angularly fused unusual 5-6-7 tricyclic framework (cyathane skeleton (1), Scheme 1) with 1, 4-anti stereogenic quaternary carbon centers at the ring junctures. Most compounds in this large family show strong antibiotic activity, and more importantly, erinacines and scabronines exhibit significant stimulating activity in nerve growth factor (NGF) synthesis. Moreover, erinacine E, one of the most complex members in this family, was reported not only as a compound showing potent stimulating activity in NGF synthesis but also as a selective agonist of κ-opioid receptor. For the enantioselective total syntheses of cyathanes, we have developed a convergent synthetic approach using two devised chiral fragments, Fragments A and B. Fragment A was prepared via the catalytic asymmetric intramolecular cyclopropanation of the α-diazo-β-keto sulfone developed by us (Scheme 2). Fragment B was derived from the optically pure building block originally prepared by the baker's yeast-mediated asymmetric reduction of the prochiral cyclohexan-1, 3-dione (Scheme 3). We prepared enantiopure 8 via the coupling reaction of Fragment A and B, followed by ring-closure via the intramolecular aldol reaction. Further transformations from 8 via ring expansion completed the first total synthesis of (+)-allocyathin B_2 (Scheme 4). The trans-fused B, C ring juncture in cyathanes was stereoselectively constructed via the SmI_2 mediated conjugate reduction of enone 9 utilizing the β-oriented C14 hydroxy group as a proton source. Through the key steps, that is, SmI_2 mediated ring expansion, stereoselective vanadium-catalyzed epoxidation, and regioselective cleavage of the epoxide, total syntheses of (-)-erinacine I and (-)-cyathin A_3 were successfully achieved (Scheme 5). Glycosylation reactions of aglycon 18 with several xylose derivatives were extensively surveyed and, finally, we found that MeOTf mediated glycosylation of 18 with 19 stereoselectively provided the desired product to accomplish the first total syntheses of (-)-erinacine B and C (Scheme 6). Further synthetic studies on (-)-erinacine E, (-)-scabronine A, and (-)-cyanthiwigin F are now in progress, and the results obtained till the symposium will be discussed in the presentation.

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Histone deacetylases (HDACs) regulate gene expression. Although more than ten human HDACs have been determined, exact roles of them are still unclear. To investigate each role of HDACs, selective inhibitors are important. Spiruchostatin A is a potent histone deacetylase inhibitior, which is a bicyclic depsipeptide consisting of Val-statine, D-cysteine, D-alanine, and β-hydroxy acid. Toward a combinatorial library synthesis of its analogues, we have achieved a total synthesis of spiruchostatin A in both solution-phase and solid-phase. The β-hydroxy acid was prepared by asymmetric aldol reaction of the Zr-enolate of acetate derivative utilizing Seebach chiral auxiliary. After sequential coupling of Val-statine, Fmoc-D-Cys(Trt)-OH, Fmoc-D-Ala-OH, and the β-hydroxy acid, macrolactonization was achieved by Shiina's method in excellent yield. Finally, oxidative deprotection and disulfide bond formation provided spiruchostatin A. A chemical probe prepared from spiruchostatin A was utilized for elucidation of natural product/protein network analysis by a nano-flow LC-MS/MS system. It was found that 20 interacting proteins including HDAC1 and HDAC2 were fished-out. The results exhibit this method can be utilized for elucidation of not only a target protein but also interacting proteins in high speed.

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With increasing fear of a potential new influenza pandemic. the anti-influenza drug Tamiflu^[○!R]has become extremely important for protecting humans against this lethal flu. Here our third generation synthesis of Tamiflu^[○!R]is discussed. To quickly establish the synthetic route, we started our synthesis without chirality control (Figure 3.). Through the key transformations. 1) Diels-Alder reaction to construct the central carbon skeleton, 2) Curtius rearrangement of 14 to introduce the key 1,2-trans-diamide moiety, and 3) Ni-catalyzed conjugate addition of TMSCN to enone 4, we have produced Tamiflu^[○!R]successfully. We next developed the catalytic enantioselective Diels-Alder-type reaction for an asymmetric synthesis of Tamiflu^[○!R]. After intensive studies, we found that the desired cyclization proceeded in quantitative yield (endo: exo=2: 1) and 93% ee (endo) in the presence of 10mol% Ba(O^1Pr)_2. 10mol% Ligand I, and 10mol% CsF (Figure 4.). Preliminary mechanistic studies (Figure 5.) have led us to propose the mechanism shown in Figure 6. This reaction mechanism is completely different from the previously developed catalytic enantioselective Diels-Alder reactions which depend on chiral Lewis acids or Bronsted acids, and a new entry in the catalytic enantioselective Diels-Alder reaction has been developed. Thus, our third generation catalytic asymmetric synthesis of Tamiflu^[○!R]has been developed. Improvement of diastereoselectivity in the Diels-Alder-type reaction and further optimization in the late stage of the sythesis are now ongoing.

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Ligularia dictyoneura was found to be extremely diverse with respect to chemical composition, DNA sequence, as well as morphology. From 20 samples of the species, 17 eremophilane derivatives were isolated, including three new franoeremophilane derivatives, 3β-acetoxy-6β-angeloyloxyfuranoeremophilan-10β-ol (9), 1α-acetoxyfuranoeremophi lan-15, 6α-olide (11), and 6β-(2-hydroxymethylprop-2-enoyloxy)furanoeremophil-1(10)-ene (16). Seven types of chemical composition were identified in the 20 samples: one with non-furano eremophilane derivatives and the others (types 1-6) with furanoeremophilane derivatives of different oxidation levels (Table 1). Two distinct types were found for the DNA sequence of the atpB-rbcL intergenic region. The sequence of the ITSs region was also very diverse. The presence of two distinct types of the atpB-rbcL sequence and multiple ITS sequences within individuals suggests hybridization in the past. No correlation between the chemical composition and the DNA sequence indicates the presence of complex mechanism in the evolution of L. dictyoneura. Our results on inter- and intra-specific diversity in Ligularia hitherto have shown that the genus is indeed continuing its rapid evolution and suggest that production of furano-compounds confers an ecological importance.

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Four Ligularia species, L. kanaitzensis, L. vellerea, L. subspicata, and L. lamarum collected in Yunnan province of China were examined and 64 compounds were isolated. Compounds 1-15 were new. The samples of L. kanaitzensis could be divided into two groups, based on chemical composition of the root extract. Furanoeremophilane-6β,10β-diol and its derivatives were isolated from one group, while eremophilan-8-one derivatives were isolated from the other group. In contrast, intra-specific diversity was found to be small in the atpB-rbcL intergenic sequence. The samples of L. vellerea could be divided into three groups. The major components isolated from types A, B, and C were 6,15-dioxygenated furanoeremophilanes, 1,6-dioxygenated furanoeremophilanes, and 6,15-dioxygenated eremophilanolides. The difference in chemical composition is consistent with geographic distribution. Intra-specific diversity in the atpB-rbcL sequence was small. The samples of L. subspicata were grouped into two, that is, a group A producing 1-angeloyloxy derivatives and a group B producing ligularol (35) and its derivatives. Diversity was found to be present in the nucleotide sequences as well. 1,10-Dioxygenated derivatives from L. lamarum were isolated. The chemical composition was diverse. However, the DNA sequence of atpB-rbcL is currently under investigation.

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Antimitotic agents that inhibit the microtuhule formation and the mitotic arrest of eucaryotic cells, such as paclitaxel and vinblastine, are important components of current anticancer therapy. Paclitaxel is potent polymerization of purified tubulin, causing stabilization and bundling of microtuhules. The antimitotic agents have potential applications in drug development. Recently much effort has been directed to the isolation and synthesis of new antimitotic drugs that target the tubulin/microtubule system and display efficacy against drug-refractory carcinomas. During our search for bioactive compounds targeting the tubulin/microtubules from medicinal plants, we found that the extract from the fruits of Taxodium distichum and bark of Maytenus chuchuhuasca remarkably inhibit the polymerization of tubulin. Further investigation on the extract of T distichum that target tubulin resulted in the isolation of two new abietane-type diterpencs, taxodistines A and B. The structures and relative stereochemistry were elucidated on the basis of 2D NMR data. On the other hands, four known quinoid triterpenes, tingenone, 22β-hydroxytingenone, pristimerin, and celasterol have been isolated from the extract of M. chuchuhuasca which inhibit the polymerization of tubulin. Celasterol with a carboxylic acid and a diosphenol functions potently inhibited the polymerization of tubulin in a concentration-dependent manner. Taxodistine B also showed inhibition of polymerization of tubulin.

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