The phenomenon of plant seedlings bending toward light, to optimize the exposure of their photosynthetic organs, is called "phototropism", while the bending of the organs of a plant toward or away from the earth is called "gravitropism". We have hitherto reported evidence that the differential flank growth that causes phototoropic curvature is regulated by a local gradient induced at the site of illumination, of blue light-induced growth inhibitors interfering with the action of evenly distributed auxin (Bruinsma-Hasegawa theory). To date, different plant species turn out to produce different and very specific growth inhibitors as candidates for phototropism-regulating and gravitropism-regulating substances. In this study, we report a role of inhibitors in the phototropism and gravitropism of higher plants. We have already isolated two photo-induced growth inhibitory substances related to phototropism, 4-MTBI and Raphanusanin from radish (Raphanus sativus) hypocotyls and DIMBOA and MBOA from maize (Zea mays L) coleoptiles. To understand the mechanism of growth inhibition induced by these phototropism-regulating substances, physiological and genetic analyses have been performed. We found that phototropic stimulation induces H_2O_2 accumulation via the up-regulation of phototropism-regulating substances. We also found that phototropism-regulating substances contribute to their growth inhibitory activity by promoting H_2O_2 synthesis and subsequent lignification, which could increase the stiffness at the primary cell-wall level. These results suggest a close association between an oxidative burst and phototropic curvature. A growth inhibitor, 3,6'-Disinapoylsucrose, of which content in etiolated radish hypocotyls was increased under 500 g gravity, but decreased under stimulated microgravity was isolated and determined by 2D NMR and MS. The distribution of endogenous 3,6'-Disinapoylsucrose between the peripheral cell layers of the upper and lower flank of radish hypocotyls during gravitropic curvature was analysed using a physicochemical assay. These results suggest that gravitropic stimulation suppresses the growth rate of upper side of radish hypocotyls by increasing the content of 3,6'-Disinapoylsucrose in the peripheral cell layers of the upper halves, causing a gravitropic response.
The determination of absolute configuration of chiral molecules is an essential but difficult step in natural product chemistry. Chiroptical spectroscopy is the sole technique that can nonempirically determine molecular chirality without need of crystallization. One of the most widely used is the exciton chirality method in electronic circular dichroism (ECD), developed by Harada and Nakanishi, but the requirement for two or more appropriate UV-vis chromophores restricts its applicability. Meanwhile, vibrational circular dichroism (VCD) spectroscopy using theoretical calculation has been a recent trend for its reliability and convenience; however, this approach has been hampered by the low sensitivity of vibrational absorption and the computational demand. In exploration of a more universal, sensitive method, we envisioned the potential of an exciton coupling approach in VCD. To test its feasibility, we synthesized various mono- and biscarbonyl chiral compounds and examined their VCD spectra. All the biscarbonyl compounds exhibited a bisignate VCD coupling whose intensity was 〜20 times higher than that of monocarbonyl compounds as a result of an interaction of two carbonyl groups. Furthermore, the signs of these couplets are consistent with the absolute twist of the two C=O bonds, which proved the reliability of "the VCD exciton chirality method" for determination of absolute configuration. Not only can the VCD exciton chirality method be used as conveniently as the ECD exciton chirality method, but also it can analyze molecules that are outside of the coverage of ECD and other spectroscopic techniques. In this study, we demonstrated the applicability of the VCD approach to various types of molecules such as spirobiscarbonyl compounds, α-amidelactam, α-acyloxyketone, and α-hydroxyketone. Furthermore, we recently revealed that this method can elucidate the chirality of diacylglycerolipids, major components of cell membrane. There have been few methods to analyze the absolute configuration of glycerolipids, but our current approach should provide new insight into evolution, and also clarify the detailed relationship between their chirality and biological functions. Last, the VCD exciton approach can be used as a signal intensifier. In some cases, less than 20 μg of analyte was sufficient to observe a VCD couplet, by which one can determine its absolute configuration. This method should find various usages in future, e.g., analysis of minuscule molecules with or without using theoretical calculation or time-resolved VCD measurement.
Natural products are an important source of novel therapeutics with enormous structural diversity exceeding our imagination. Especially, Type I polyketide synthetase (PKS) produces highly diverse structures with variation of chain length, cyclization pattern, heteroatom incorporation etc. Recently, we discovered two classes of biosynthetically unique polyketides, alchivemycin (1 and 2) and laetimycin (3) from Streptomyces. Their structures, biosynthetic and stereochemical studies are herein described. Alchivemycins A (1) and B (2) are unique polycyclic metabolites produced by Streptomyces. Their absolute configurations were assigned by chiral anisotropy method and CD analysis. These compounds contain a biosynthetically unprecedented ring system, tetrahydrooxazine. Incorporation experiments with ^<13>C labeled precursors revealed that this unusual heterocyclic ring is derived from acetates and N-hydroxyglycine and assembled by Dieckmann-type intramolecular cyclization. 14-Membered macrolide laetimycin (3) was obtained from another strain of Streptomyces. To confirm its absolute configuration, dichlorolaetimycin (6) was prepared from 3 and compared with chlorotonil A (4) which has the same carbon backbone with 3. Surprisingly, 6 and 4 showed inversed CD curves, and opposite optical rotation values (6: [α]_D^<25> = +140; 4: [α]_D^<25> = -160). We thus concluded that the absolute configurations of all seven chiral centers in 3 are opposite to those of 4.
The coral reef is very important as primary producer and as places for an ecosystem at the tropical and the subtropical zone. To keep and protect corals, we have studied chemical substances which influent on their ecosystems. We have been interested in biologically intriguing phenomena that several kind of marine organisms overgrow live corals. Thus, it was expected that they may produce some toxic compounds against corals. Red algae Laurencia papillosa and Cyanobacteria Lyngbya spp. that overgrew hermatypic corals were collected at Okinawa. The extracts of these algae were purified using chromatography to give new compounds papillamide (1), maedin (2), lyngbyacyclamides A (4) and B (5). The structures were determined by the combinations of NMR and MS spectroscopic analyses, degradations and synthetic studies. The biological activities of 4 and 5 are quite interesting, since they showed significant cytotoxicity toward B16 cells but no toxicity toward brine shrimp. Macrodiolide luminaolide (11) was isolated from the crustose coralline algae Hydrolithon reinboldii as a novel metamorphosis-enhancing compound for scleractinian coral larvae. The planar structure was already reported, and relative stereo chemistries were described this report. The stereo chemistries of DHP ring of C7-C11 and zigzag carbon chain parts of C3-C7, C17-C22 and C26-C28 were determined by ROESY spectra and ^3J_<HH> coupling constants. Remaining part of C11-C17 was elucidated by JBCA method.
In the course of our chemical and biological combination screening programs, JBIR-129 (1) and JBIR-139 (2), which consist of a highly oxygenated 34-membered macrolactone ring and deoxysugar moieties, were isolated from the fermentation broth of Streptomyces sp. RK74. The planar structures of 1 and 2 were established based on the analyses of 1D and 2D NMR, ESI-TOF-MS, IR and UV spectra. The relative configurations of sugar units were determined by analyzing vicinal ^1H-^1H coupling constants and steric information. The relative configuration of aglycone was elucidated by using J-based configuration analysis. Both 1 and 2 showed potent cytotoxic activities against human ovarian adenocarcinoma SKOV-3 cells with the IC_<50> values of 0.3 and 0.4 μM, respectively.
The histone acetyltransferase (HAT) activity of p300 is essential for androgen receptor (AR) function. Androgen-independent prostate cancer cells require AR-mediated transcriptional activation for their growth. These observations indicate that p300 HAT is a promising target to overcome such hormone-resistant cancer cells. We sought p300 HAT inhibitors among microbial metabolites. To find new p300 HAT inhibitors, our libraries consisting of 19,320 culture broths of microbes were screened. The one of the hit samples, produced by culturing the fungus Penicillium sp. NF13650, was purified. Consequently, two new compounds, NK13650A and NK13650B, were successfully isolated. Structural analysis demonstrated that these compounds have unique chemical structures; they are pseudopeptides containing citric acid. The five components comprising NK13650A were linearly connected in the order aspartate, citrate, homoserine, dehydro-DOPA, and arginine through two amide bonds, an ether bond, and a diketopiperazine ring. And NK13650B was determined to be similar structure to NK13650A with the exception of the lack of an aspartate moiety. Absolute configurations of each amino acid were determined by an advanced version of Marfey's method. Consequently, all chiral centers originating from the amino acids in NK13650A, aspartate, homoserine and arginine, were confirmed to have an S configuration. The citric acid had different substituents at C-1 and C-5 carboxyl groups, and the C-3 quaternary carbon accordingly became a chiral center. To determine this absolute configuration, we developed a useful biosynthesis-based method that involves feeding of a ^<13>C-labeled biosynthetic precursor of citrate. Citrate is biosynthesized from acetyl-CoA and oxaloacetic acid by citrate synthase under strict stereochemical control, and the carboxylic acid of acetyl-CoA is arranged as the pro-S carboxylic acid of citrate. Therefore, if biosynthetic precursor of acetyl-CoA, labeled by a stable isotope ^<13>C, could be incorporated into NK13650A, the stereochemistry could be readily deter-mined by ^<13>C NMR. Conclusively, ^<13>C NMR spectrum resulting from feeding of [2,3-^<13>C_2]pyruvate reveal the C-3 quaternary carbon in the citrate was determined to have an S configuration. NK13650s selectively inhibited the activity of p300 HAT but not that of Tip60 HAT. NK13650s showed inhibitory activity against agonist-induced AR transcriptional activation, and NK13650A treatment inhibited hormone-dependent and -independent growth of prostate cancer cells; it had no inhibitory activity against cancer cells which don't require functions of nuclear receptor. These results strongly suggested that NK13650s are promising lead compounds for novel anti-prostate cancer drugs.
The ubiquitin-proteasome system controls a wide range of cellular events including cell cycle progression, and defects associated with this system result in various diseases including cancer and neurodegenerative disorders. Thus, the ubiquitin-proteasome system is emerging as a significant target in anticancer therapies. In addition, nowadays, inhibitors targeting the ubiquitin system including E1, E2 and E3 enzymes, the delivery system, and deubiquitinating enzymes are also candidates for anticancer drugs. In the continuing search for ubiquitin-activating enzyme (E1) inhibitors, hyrtioreticulins A-E (1-5) were isolated from the marine sponge Hyrtios reticulatus, along with a known alkaloid, hyrtioerectine B (6). Structural elucidation on the basis of spectral data showed that 1, 2, and 5 are new tetrahydro-13-carboline alkaloids, while 3 and 4 are new azepinoindole-type alkaloids. Possibly, 1-6 would be biosynthesized by the Pictet-Spengler reaction with L-tryptophan and L-alanine. While trans/cis ratios differ greatly among the three pairs of isolates, 0.67 (1/2), 0.53 (3/4), and 0.041 (5/6), thermodynamically unstable cis isomers are dominant for these pairs. Hyrtioreticulins A and B (1 and 2) inhibited El enzyme with IC50 values of 0.75 and 11 μg/mL, respectively, measured by their inhibitory abilities against the formation of an E1-ubiquitin intermediate. So far, only five E1 inhibitors, panapophenanthrine, himeic acid A (7), largazole, and hyrtioreticulins A and B (1 and 2), have been isolated from natural sources and, among them, 1 is the most potent El inhibitor.
During our studies on search for bioactive natural products from various sources, we have investigated bioactive metabolites of actinomycete strains collected several places in Chiba area, and isolated new bioactive aromatic heterocyclic natural products, including izumiphenazines A-D (1-4) from the cultured broth of Streptomyces sp. IFM 11204 isolated soil sample of Izumi-forest in Chiba city, izuminosides A-C (9-11) from Streptomyces sp. IFM 11260 collected also in Izumi-forest in Chiba city, new pyranonaphthoquinones (12-15) yorophenazine (16), and yoropyrazone (17) from Streptomyces sp. IFM 11307 collected from Yoro-valley, and katorazone (18) from Streptomyces sp. IFM 11299 collected from Katori city. The structures of new compounds were elucidated on the basis of spectral data, and their TRAIL resistance overcoming activity was evaluated to reveal that compound 12 at 0.1 μM proved to be the most active in this series, with 33% decrease in cell viability in the presence of TRAIL (100 ng/mL).
Plipastatin A1 is a cyclic depsilipopeptide which was first isolated by Umezawa et al in 1986 from Bacillus cereus BMG3O2-fF67 as a phospholipase A2 inhibitor. In the same issue of the journal, Jung et al independently reported fengycin from Bacillus subtilis strain F-29-3 as an antibiotic lipopeptide. Although the final structure was not disclosed in that report, they reported the identical amino acids and the fatty acid as to those from plipastatin A1 as the acid hydrolysates. The structure of fengycin was revealed in 1999 by Budzikiewicz that it is a diastereomer of plipastatin A1 (named fengycin IX) possessing D-Tyr4 and L-Tyr10 residues, while plipastatin A1 has the reversed permutation, L-Tyr4 with D-Tyrl 0. Due to the promising antibiotic activity of this substance and empirical safeness of the producer, fengycin is expected to be a novel biocontrol to produce more than 220 scientific papers and counting. Some of those papers dealt with the biogenesis of fengycins to disclose NRP (non-ribosormal-peptide) synthase clusters. Those involved sequences suggesting racemases. However, positions of the expected racemases accorded with plipastatin A1 in spite of fengycin producers. The enzyme cluster which rationally explains Budzikiewicz's fengycin IX has not so far been reported. These facts have brought structural confusion between these molecules. Although many papers followed Budzikiewicz's structure, considerable number of recent reports mentioned that these are identical compounds supposedly in order to avoid the contradiction between the structure and the biogenesis. However, there was no experimental evidence. The confusion has become more serious, because at least five other structures exist as fengycin IX. The present studies experimentally proved that plipastatin A1 is the K^+ salt whereas fengycin IX is the free form or the TFA salts. Although these molecules gave considerably different ^1H NMR spectra, the quite similar ^1H NMR spectrum to that of fengycin IX was changed to provide nicely accorded spectrum to that of plipastatin A1 when the sample was converted into the K^+ salt. Our structural studies led a conclusion that the structures of these compounds should be settled into that of plipastatin Al by Umezawa.
Malaria caused by parasites of the geuns Plasmodium is one of the leading infectious diseases in many tropical and some of temperate regions. During our studies on new lead agents against malaria from medicinal plants, cassiarin F (1), a novel hybrid alkaloid consisting of cassiarin A (2) and a biphenyl unit with an acetonyl moiety, have been isolated from the flowers of Cassia siamea (Leguminosae), which have been widely used in traditional medicine, particularly for the treatment of periodic fever and malaria. Furthermore, from the leaves of C. spectabilis (Leguminosae), (-)-iso-6-spectaline (3), (-)-iso-6-cassine (4), (-)-cassine (5), and (+)-spectaline (6) have been isolated. The structures of cassiarin F (1), (-)-iso-6-spectaline (3), (-)-iso-6-cassine (4), (-)-cassine (5), and (+)-spectaline (6) were determined by 1D & 2D-NMR data (^1H-^1H COSY, HSQC, HMBC, and ROESY) and optical rotation value. The biaryl moiety of 1 was considered to be racemic from its small optical rotation value and CD spectrum, in which no Cotton effect was observed. Cassiarin F (1) might be biosynthetically produced by a transformation of chrobisiamone A. A total synthesis of the unique tetracyclic cassiarin F (1) was achieved by employing the Suzuki coupling constructing biaryl unit, nucleophilic aromatic substitution, and Houben-Hoesch type ring construction as key steps. Alkaloids (1, 3, and 4) isolated in this research were evaluated for antiplasmodial activity against Plasmodium falciparum 3D7 strain in vitro and showed modest antiplasmodial activity.
Ionophore polyethers, a structurally unique group of natural polyketides, has a polycyclic ether skeleton with multiple stereocenters. Structural diversity of these natural products is derived from combination of the number and size of ether rings. In 1983, Cane, Celmer, and Westley proposed a unified biosynthetic model (CCW model) for polyether construction in which epoxidation of the linear polyene intermediate in a stereoselective manner followed by regioselective cascade cyclization provided these polyethers. After the pioneering work by Leadlay and co-workers on identification of monensin biosynthetic gene cluster, a series of gene inactivation experiments established the involvement of polyene and polyepoxide intermediates in polyether construction. Thus, the CCW model consisting of epoxidation and epoxide-opening reaction was firmly established. Recently, the second step of polyether formation has been studied extensively by our group. We found that recombinant epoxide hydrolase Lsd19 catalyzes energetically disfavored conversion of the chemically synthesized putative precursor, bisepoxide of prelasalocid, to lasalocid in a highly regioselective manner and that this epoxide hydrolase shows promiscuous substrate specificity. Here we present most recent results including (i) functional analysis of flavin dependent monooxygenase Lsd18, a putative epoxidase in lasalocid biosynthesis, (ii) structural analysis of Lsdl9, and (iii) i enzymatic epoxide opening cascades catalyzed by a pair of epoxide hydrolase, MonBI and MonBII, involved in monensin biosynthesis.
Incednine is a 24 membered macrolactam glycoside isolated from Streptomyces sp. ML694-90F3 as an inhibitor of anti-apoptotic function of Bcl-2/Bcl-xL oncoproteins. In the present study, to understand the biosynthesis of the unique amino acid starter unit of macrolactam aglycon (incednam), a series of incorporation study and enzymatic analyses of the biosynthetic enzymes encoded in the corresponding gene cluster were conducted. Feeding experiments with [1-^<13>C_1] acetate, [1,2-^<13>C_2] acetate, [1-^<13>C_1] propionate, and [^<13>C_3]glycerol revealed that incednam is constructed with five malonyl-CoA, four methylmalonyl-CoA and one methoxymalonyl-CoA (ACP) as the extender units of polyketide synthases (PKSs). Interestingly, one intact acetate was also incorporated into the C1-C2 position of 3-aminobutyrate moiety, suggesting that the starter unit would be derived from L-glutamic acid rather than L-lysine and acetoacetyl-CoA. In fact, intact incorporation of L-[^<13>C_5,^<15>N_1]glutamic acid was observed. Further, to elucidate the missing biosynthetic link between L-glutamic acid and the starter unit, possible deuterium labeled amino acids were synthesized and fed to the producer strain. As a result, the free form of 3-[3-^2H]aminobutyric acid and β-[2,2,4,4-^2H_4]glutamic acid were efficiently incorporated to the starter unit, indicating that these amino acids are direct precursors of the starter unit of incednam. These results also implied that the unprecedented β-decarboxylation of β-glutamic acid to give 3-aminobutyric acid would be involved in the starter unit biosynthesis. As the next, to fish out the expected unique biosynthetic gene cluster of incednine, we used unique deoxysugar biosynthetic genes and methoxymalonyl-ACP biosynthetic gene as probes. Consequently, the incednine biosynthetic gene (idn) cluster, which is localized to a 138 kb contiguous DNA from Streptomyces sp. ML694-90F3, was identified. The idn gene cluster contains six modular type I PKS genes (idnPl-P6), deoxysugar biosynthetic genes (idnS1-S9), possible starter biosynthetic genes (idnLM1-LM7) and several modification enzymatic genes. To understand the unique starter unit biosynthesis, we attempted to characterize several recombinant enzymes, which were expressed in heterologous hosts. One of the most striking results was that IdnLM3 was found to catalyze β-decarboxylation of β-glutamic acid to give 3-aminobutyric acid. In the presentation, we will discuss the unique starter biosynthetic pathway based on the characterized enzymatic functions of the incednine biosynthetic enzymes.
Calyculin A is the major cytotoxic compound isolated from the Japanese marine sponge Discodermia calyx. Its potent cytotoxicity is attributed to specific inhibition of protein phosphatases 1 and 2A, which are ubiquitous in eukaryotic cells and essential for the major post-translational modifications of proteins. Therefore, calyculin A serves as a useful biochemical tool to evaluate intracellular signal transductions. The structure of calyculin A featuring a 5,6-spiroketal, a phosphate group, a dimethylamino group, a conjugated oxazole and a tetraene could be biosynthesized via a hybrid pathway comprised of PKS and NRPS. The β-branch unit found in the tetraene moiety suggested that the biosynthetic pathway should include trans-AT PKS. It was therefore possible to directly correlate KS phylogeny with structural moieties found in a polyketide without having to analyze other domains in a module. These predictive rules should assist significantly in the cloning of trans-AT PKS gene clusters. Taking advantage of this rule, we started cloning the biosynthetic gene cluster of calyculin A from metagenomic DNA of D. calyx. The PCR with several known degenerate primers for ketosynthase, afforded KS amplicons belonging to trans-AT PKS. There were a few trans-AT KS amplicons showing similarities to bryostatin and bacillaene PKS, which could be anticipated to be common clades present in calyculin PKS. Therefore, after construction of a fosmid library of D. calyx, screening based on the trans-AT amplicons was conducted to obtain positive clones. Consequently, 12 clones covering >150 kbp were obtained and subjected to sequence analysis by Illumina Genome Analyzer II. The entire sequence disclosed that NRPS codes, KS clades, KR selectivity and MT location of respective modules were in good agreement with the putative biosynthetic pathway of calyculin A. With the putative biosynthetic gene cluster of calyculin A in hand, we are now trying to identify the bacteria responsible for calyculin A production.
The article describes the detailed mechanism of the formation of the spiro-carbon moiety in an anti-mitotic fungal nonribosomal peptide (NRP), spirotryprostatin A, mediated by a key enzyme, FAD-dependent monooxygenase (FMO). The presence of spiro-carbon moieties is prevalent among natural products, yet the detailed mechanism of their biosynthesis is poorly characterized. Those spiro-centers remain to be a significant challenge to synthetic chemistry as well. Thus, we sought to investigate how a sterically constrained chiral spiro-carbon could be synthesized enzymatically. We focused on spirotryprostatins because of their useful biological activity and complex chemical structure involving a spiro-carbon moiety. Since similar structural motif is also found in other indole alkaloid natural products, we felt that our findings here will have a broad impact on our understanding of the mechanism of complex natural product biosynthesis. What is frequently the major obstacle in conducting detailed mechanistic studies of enzymes involved in complex natural product biosynthesis is the lack of adequate supply of substrates and reference materials. This was true for studying the spiro-carbon formation in spirotryprostatins. To resolve this problem, we successfully carried out metabolic engineering of yeast for heterologous production of tryprostatins (substrates for spirotryprostatins formation) and other compounds involved in the biosynthesis of tryprostatins and spirotryprostatins using our engineered strain of Saccharomyces cerevisiae, SCKW5. Using the compounds obtained from yeast, we characterized the activity of an FMO, Afua_12060, involved in the fumiquinazoline biosynthesis and confirmed that Afua_12060 was responsible for an poxidation-driven semipinacol-type rearrangement of tryprostatin A that set up the compound for the ensuing formation of spirotryprostatin A. Comparison of our finding with the proposed biosynthetic mechanism of related fumiquinazoline and notoamide natural products revealed that FMO-catalyzed epoxidation of unactivated indole plays a key role in the formation of various complex chemical structures (namely spiro-carbon and imidazoindolone ring systems). However, what was most intriguing about our finding was the discovery of an unusual crosstalk between two biosynthetic pathways (fumiquinazoline and fumitremorgin biosynthetic pathways) that resulted in the formation of new compounds with interesting chemical features (spirotryprostatins).
Vialinin A (1) is a novel compound isolated from the dry fruiting bodies of an edible Chinese mushroom, Thelephora vialis, and exhibits an extremely potent inhibitory activity against tumor necrosis factor (TNF)-α release from RBL-2H3 cells (IC_<50> = 0.09 nM). TNF-α is a proinflammatory cytokine, and dysregulated TNF-α release is implicated in many diseases such as rheumatoid arthritis (RA). Therefore, a new type of inhibitor against TNF-α release would be a promising candidate for therapeutic agents for RA. We designed and synthesized an advanced vialinin A analog, 5',6'-dimethyl-1,1':4',1"-terpheny1-2',3',4,4"-tetraol (DMT, 2) with a comparable inhibitory activity (IC_<50> = 0.02 nM) to that of 1. To identify the target biomolecule of 1 and 2, fluorescent derivatives and a biotinylated one of 2 were prepared through click chemistry. The fluorescent derivatives defined a pharmacophore in DMT through their intracellular behaviors and their TNF-α release inhibitory activities. A protein of approximately 100 kDa was obtained using the biotinylated DMT (biotin-DMT, 17), and the protein was identified as a deubiquitinating enzyme (DUB) by peptide mass fingerprinting analysis. Vialinin A (1) and DMT (2) were found to inhibit the enzymatic activities of the recombinant human DUB. RNAi experiments using siRNA-based knockdown of the DUB suggested a close correlation between lack of the DUB and TNF-α release inhibition.
Lipid rafts (Fig. 1) are a membrane microdomain comprised of sphingomyelin (SM) and cholesterol (Chol). Although lipid rafts are assumed to be implicated in diverse biological events, the molecular mechanism of their formation remains largely unknown because the elusive nature due to rapid association and dissociation equilibrium of lipid molecules. Thus, we attempted to elucidate the molecular basis of raft formation and dynamics by NMR measurements of model membranes consisting of isotope-labeled lipids. ^2H NMR experiments were first carried out by using regioselectively deuterated SMs (Fig. 2A) to analyze the dynamics of alkyl chains of SM. The broad band spectra indicated that the order of middle parts of both alkyl chains of SM is higher in the presence of Chol (Fig. 3) than that in the absence of Chol. Next, to deduce the structure basis of raft formation, particularly the conformation of SM, we carried out the solution and solid state NMR experiments. The results indicated that the orientation of the amide plane of SM is hardly changed between the presence and absence of Chol, whereas Chol significantly enhances the order of the amide part of SM. In addition, it was implied that SM changes the orientation of the phosphate group upon interaction with Chol (Fig. 5). Finally, intermolecular REDOR experiments were carried out to deduce the interaction between SMs in the rafts, indicating that intermolecular hydrogen bonds between SMs are efficiently formed at the amide part in the presence of Chol (Fig. 6).
Aggregation of the 42-residue amyloid β-protein (Aβ42) is a potential target in the therapeutics of Alzheimer's disease (AD). Although there are accumulated reports on flavonoids that prevent the aggregation of Aβ42, the inhibitory mechanism remains fully unanswered. Our group recently reported that the extracts of Silybum marianum seeds alleviated the aggregation of Aβ and cognitive impairment using a transgenic mouse model of AD. Here we identified in the extracts taxifolin, a catechol-type flavonoid, which suppressed Aβ42 aggregation. Structure-activity relationships of O-methyl taxifolins showed the significance of 3',4'-hydroxyl groups on B-ring in the preventive activity. Sodium periodate, an oxidant, accelerated the prevention of Aβ42 aggregation, whereas no inhibition was found under the anaerobic condition. These inhibitory effects were in good agreement with the formation of o-quinone moiety in taxifolin. Since the presence of taxifolin-Aβ42 adduct was detected by mass spectrometry, Aβ42 mutants substituted at the basic amino acid residues (Arg5, Lys16, Lys28) with n-leucine were prepared to identify the reaction site. Taxifolin did not suppress the aggregation of Aβ42 mutants at Lys16, but significantly inhibited that of the mutant at Arg5. These findings suggest the specific inhibitory mechanism of Aβ42 aggregation, in which the formation of o-quinone structure in flavonoids could be involved in the suppression of Aβ42 aggregation by targeting Lys16.
Lipid rafts, which are sphingomyelin (SM) and cholesterol enriched microdomain in plasma membrane, have attracted a great deal of attention in recent years. These domains play a key role in biological behaviors such as signal transduction in plasma membrane. SM is well known as an essential lipid for constructing microdomains, however, lipid rafts are still unclear including the detail mechanism of the domain construction. To understand the physical properties of the SM bilayers and SM/cholesterol domains, we designed several SM analogs such as ceramide phosphoethanol analogs having smaller head group than natural SM, N- or O-methylated analogs for understanding the role of hydrogen bonds in SM, and ceramide phosphoserine replacing the choline moiety in SM with L-serine to evaluate the ceramide skeleton. The syntheses of these analogs were achieved by the development of our previously established method for the sphingolipid synthesis. In order to compare the physical properties of SM analogs with those of natural SM and glycerophospholipids, gel-to-fluid-phase transition temperature (Tm) of each SM analog bilayers and the thermo stability of the SM analog/cholesterol domains were measured by DPH anisotropy and fluorescence quenching experiment with CTL and 7SLPC, respectively. As a result, we obtained the following valuable information: (1) the head group size affected membrane properties and cholesterol interactions, and the larger size of the head group such as choline increased the sterol affinity, (2) the secondary hydroxyl group was important for the interaction with cholesterol, (3) preventing the intra- and intermolecular hydrogen bonding in SM affected gel-phase destabilization, and (4) ceramide skeleton was important to form microdomains.
Exploring efficient strategies for identifying the natural products and their derivatives that selectively regulate protein/protein interactions is one of the most actively studied topics in chemical biology. Especially, the selective activation and/or inhibition of specific phosphorylation-mediated protein/protein interactions within a signaling pathway may direct a specific biological response on the cellular level. In pursuit of such molecules, we developed a Template-Assisted Transcription Synthesis. The approach is based on our findings that (i) the bis-lysine structure such as 1 interacts with the phosphorylated compounds, and (ii) the benzyl-protected histidine significantly accelerates the peptide-based Huisgen cycloaddition, so that the structure 1 can efficiently be modified by the peptide library to strongly and selectively bind to the target phosphorylated protein in a template-assisted fashion. As a model case, we applied to preparing the Grb2/SH2 domain mimicry. The template-assisted transcription synthesis between the bis-lysine 1 and the acetylene-peptide library 2 was performed in the presence of the cyclic phosphopeptide 3 (known ligand for Grb2/SH2 domain) as the template. The clicked peptides 4a, b, and c as the template-assisted products, exhibited the micromolar-level dissociation constants to the phosphopeptide 3. Conformational restriction of the initial hit structure 4a by the solid-supported ring-closing metathesis gave the cyclic peptide 9, further enhancing the interaction with the peptide 3, i.e., Kd of 590 nM. One of the clicked product 4a, the peptidyl mimic of the Grb2-SH2 domain, was found to be internalized into A431 cancer cells, where the EGFR-induced cellular signalling is operative. It selectively bound to the phosphorylated Shc protein, one of the Grb2-SH2 interacting signaling proteins, induced A431-selective apoptosis and tumor growth inhibition. The clicked peptide 4a even exhibited the A431-selective inhibition of tumor growth without any toxic effects and inflammatory responses in the preliminary animal experiments. The results described in this symposium demonstrate the tailor-made synthesis of artificial receptors using a Template-Assisted Transcription Synthesis that is clearly distinct from previous methods. This approach may efficiently and rapidly provide small-molecular regulators that selectively control a specific cell signaling pathway or protein/protein interactions.
Natural Killer T (NKT) cells have been identified as a novel lymphocyte lineage, and they recognize lipid antigens presented by CD1d protein with their T cell receptors. KRN7000 (1, Fig. 1) developed by Kirin Brewery Co. is known as one of the external stimulant to NKT cells, and has phytosphingosine (11, Fig. 3) as its sphingoid base part. In nature, as the C_<18>-sphingoid bases, sphingosine (10), sphinganine (12), and 6-hydroxysphingosine (13) exist abundantly, and 11 is a minor sphingosine base in mammals. We synthesized the analogs 18, 20, and 21 of KRN7000 (1), possessing natural sphingoid base 10, 12, and 13, respectively, and investigated their immunostimulatory activity. Among 1, 18, 20, and 21, sphingosine-type analog (18, RCAI-161) showed the most potent immunostimuratory activity, and induced ca. two times larger amount of IFN-γ production than 1 did in mice in vivo (iv). In 2011, Brenner and co-workers reported the internal natural lipid ligand of NKT cells to be β-GluCer (9, Fig. 3). According to their report, 9 was accumulated during infection, and activates NKT cells in mice and humans. We synthesized 13-glucosylceramide (26) from the properly protected glucose donor (22) and the ceramide (15) as shown in Figure 3. The chemical purity of 26 was determined as >99.9% by HPLC analysis. Synthesized 26 induced almost no IFN-γ production with very small production of IL-4. In addition, we synthesized sphingosine-type β-galactosylceramide (19), and found 19 to induce very small IFN-γ production, while a small amount of IL-4 secretion was observed.
Ynamides have been widely recognized as a versatile synthetic unit in recent organic synthesis. Here we report new regio- and stereoselective transformations of ynamide and their applications including natural product synthesis. 1) Synthesis of γ-Siloxyenamides by Ni-Catalyzed Three-Component Coupling of Ynamide, Aldehyde and Silane: The coupling reaction proceeded in the presence of a nickel-IMes catalyst to give the corresponding γ-silyloxyenamide derivative 5 in a highly stereoselective manner. The γ-siloxyenamide could be converted into β-amino acid derivative 7 via Ireland-Claisen rearrangement. 2) Ruthenium-Catalyzed Regioselective Coupling of Ynamide and Ethylene Leading to 2-Amino-1,3-Diene: Ruthenium-catalyzed hydrovinylation-type cross coupling of ynamides and ethylene proceed via ruthenacyclopentene to give 2-amino-1,3-diene derivatives 11 and 12 in a highly regioselective manner. The 2-amino-1,3-diene derivatives reacted with various dienophiles or singlet oxygen to give a cyclic enamide derivative 13 or 14. 3) Total Synthesis of (-)-Herbindoles A, B, and C: The total syntheses of (-)-herbindoles A, B and C as naturally occurring forms were accomplished for the first time through transition metal-catalyzed intramolecular [2+2+2] cyclization between dialkynylynamide 19. This strategy provided a highly efficient synthetic route to all three herbindoles from the identical indoline derivative 20 as a common intermediate.
(-)-Lemonomycin (1) belongs to a large family of tetrahydroisoquinoline (THIQ) alkaloids, and was isolated from fermentation broth of Streptomyces candidus (LL-AP191) in 1964. In 2000, researchers at Wyeth-Ayerst discovered that 1 shows antibacterial activity against MRSA and VREF. The compound features a tetracyclic ring system including a 3,8-diazabicyclo[3.2.1]octane core, which is typical of quinocarcin alkaloids. Moreover, 1 contains a 2,6-dideoxy-4-amino sugar (lemonose) that is rare in nature. Herein, we present the enantioselective total synthesis of 1. The preparation of aglycon 18 was started from glycine derivative 8 having Oppolzer's chiral auxiliary. The synthesis of 9 was achieved via Perkin-type condensation reaction. We then focused on the construction of the bicyclo[3.2.1]octane ring system. Upon treatment of 10 with TFA, generation of the conjugated acyliminium cation and subsequent intramolecular attack of the allylsilane proceeded immediately to provide 11 with complete stereoselectivity. Stereoselective formation of B ring was achieved via thermodynamically controlled Pictet-Spengler reaction. The equilibrium presumably occurs via acid-induced ring-opening reaction to form a quinonemethide intermediate 17. Upon treatment of 18 and glycosyl fluoride 22 with TMSOTf, the desired glycosidation proceeded smoothly to give 23 with good stereoselectivity despite the presence of the tertiary amine as well as the amino nitrile functional group. Finally, further transformations of 23 eventually furnished (-)-lemonomycin (1).
Histrionicotoxin (1) and perhydrohistrionicotoxin (2), are non-competitive blockers of nicotinic acetylcholine-acceptors. The important bioactivity and the characteristic azaspirocyclic skeleton have attracted considerable interests from the synthetic community. However, there are few effective methods for enantiocontrolled construction of the azaspirocyclic ring. Herein, we report a diastereoselective construction of the core six-membered spirolactam utilizing a radical translocation-cyclization strategy and application to a total synthesis of (-)-2. In our preliminary experiments, the radical reaction using enantiomerically pure lactam 6 resulted in significant decrease of the enantiomeric purity, which indicated inversion of the sp^3 radical center generated via 1,5-hydrogen radical shift was much faster than cyclization. Based on these results, we considered a stereoselective radical reaction with chiral-transfer from other stereocenter introduced on the side chain. With this idea in mind, we examined radical reaction with diastereomeric 16 or 19 having the same chiral side chain. As we expected, the reactions of both compounds gave the same 18 albeit in low yield probably due to the undesired radical translocation to the a-position of the silyloxy group. The low yielding problem was circumvented by conducting the reaction with ketal 22 with no hydrogen. Encouraged by this successful result, we then investigated the diastereoselective radical reaction of a substrate with a chiral ketal moiety derived from a C_2-symmetrical diol. Among a variety of diols tested, we found that 2,4-pentanediol was effective for achieving high enantiomeric excess. Based on the formation of the azaspirocyclic compound 31, we accomplished a total synthesis of (-)-perhydrohistrionicotoxin (2).
Complanadine A was isolated from club moss Lycopodium complanatum by J. Kobayashi group. Its structure was characterized by a dimeric motif of lycodine-type C_<16>N_2 skeleton. So far complanadines B, D and E, which consist of the lycodine skeleton, were also reported as dimeric Lycopodium alkaloids. Interestingly complanadines A, B, D, and E induced mRNA expression level for nerve growing factor (NGF) in 1321N1 human astrocytoma cells. Because of the intriguing biological activity and complex structure, we started a synthetic program toward complanadines. The total syntheses of lycodine (5), complanadines A (1) and B (2) are described. For synthesis of lycodine, notable transformations include diastereoselective Diels-Alder and Mizoroki-Heck reactions to access a bicycle[3.3.1]nonane core in the lycodine skeleton. In the total syntheses of complanadines, C-H functionalization of pyridine N-oxide is a key feature for construction of the dimeric structure. Further synthetic studies toward natural enantiomer of complanadines as well as biological studies are now in progress, the results will be discussed in the presentation.
Gangliosides GM3, a simple glycosphingolipid, is known to inhibit cell proliferation and auto-phosphorylation of EGFR stimulated by EGF. On the other hand, plasma membrane-associated human sialidase NEU3 hydrolyzes the glycosidic linkage of sialic acid in GM3 to produce lactosylceramide. Its accumulation in cancer cells causes suppression of apoptosis. The enzymatic metabolism of GM3 by NEU3 induces the dynamic change of the structure and biological function, and it makes the evaluation for the function of GM3 more difficult. To clarify the function of GM3 and analyze the lipid microdomain containing GM3, we designed sialidase-resistant C-linked GM3 analogues, in which the oxygen atom of sialoside linkage is replaced by the carbon atom. We have already reported the synthesis of CF_2- and CH_2-linked GM3 analogues. This time, we achieved the synthesis of (R)- and (S)-CHF-linked GM3 analogues having an extra chiral center. For the stereocontrolled synthesis, we developed novel methods for selective construction of (E)- or (Z)- monofluoroexomethylene. On the basis of the inhibitory activities of these analogues for EGFR auto-phosphorylation, we concluded that the conformations regulated by exo-anomeric effect were important for the biological activity of GM3. Details of the synthesis, the biological activity, and conformational analysis of GM3 analogues will be presented.
Gangliosides are a family of glycolipids, which possess sialic acids that play important roles in biological events on cell surfaces. Ganglio-series gangliosides are composed of the tetrasaccharide, (Galβ(1,3)GalNAcβ(1,4)Galβ(1,4)Glc), which bears α(2,8) oligosialic acid units. Glycolipids that vary in number of sialic acids are known as the a-, b-, and c-series of gangliosides. Chemical synthesis of these di-/oligo-sialo-containing glycolipids and their derivatives would facilitate identification of their biological roles. However, the α(2,8) oligosialic acid unit and the compact and rigid 3,4 dibranched galactoside unit are difficult to prepare. Therefore, an effective method for the synthesis of a-, b-, and c-series of gangliosides is still needed. In this report, we describe combinatorial synthesis of ganglio-series ganglioside epitopes varying at the number of the sialic acids. The α(2,3) and α(2,8) sialylations were accomplished by use of 5N,4O-carbonyl and 7,8-O-isopropyliden, and 5N,4O-carbonyl- and 7,8-di-O-chloroacetyl-protected sialyl donors in good yields with excellent a-selectivity, respectively. The two sialyl donors enable synthesis of the di- and tri-sialylgalactosides by simple glycosylation and deprotection. We successfully prepared the ganglioside epitopes via direct construction of a compact, rigid, branched structure. We also reported on a competitive inhibiting activity of the soluble glycolipid library to binding solid-supported GD3 with Siglec-7.
Endocyclic cleavage reactions of pyranosides have been recognized as rare events in glycoscience. The pyranosides with 2,3-trans carbamate/carbonate group are easily anomerized from the β- to the α-direction via endocyclic cleavage reaction. Evidence of endocyclic reaction was shown by reduction and Friedel-Crafts reaction of acyclic cation. Significant substituent effect was observed at the carbamate nitrogen, especially and Ac group. By using the isomerization reaction via endocyclic reaction, several 1,2-cis aminosaccharides were prepared from the 1,2-trans glycosides. [chemical formula]
Regioselective manipulation of one of the multiple hydroxy groups of carbohydrates has been a fundamental challenge in organic synthesis. We have recently developed a catalytic method for regioselective acylation of glucose derivatives. This reaction directly provides the 4-O-acylated product in high regioselectivity in the presence of four free hydroxy groups including the primary one. Alternatively, five steps are required to prepare the same product in the case where the conventional protection-deprotection procedure was employed. Here we report further development of this regioselevtive protocol to the sequential protetion of glucopyranosides, the one-pot diacylation of glucose derivatives at the C(4)- and C(6)-OH, and the short total synthesis of ellagitannins. A perfectly regioselective and sequential method for the preparation of orthogonally protected glucopyranosides has been developed. An acyl group was introduced at the C(4)-OH by organocatalysis in >99% regioselectivity. TBDPS, Boc and BOM groups were sequentially introduced into the 4-O-acyl-glucopyranoside at the C(6)-OH, C(2)-OH and C(3)-OH respectively in ca. 100% regioselectivity in each steps. Each of the protective groups was readily removed to give the corresponding mono-ols with three different protective groups, which are possible intermediates for the synthesis of natural and modified oligosaccharides with structural diversity. Regioselective short total synthesis of tellimagrandin I and II have been achieved, which are polyphenolic secondary metabolites produced by dicotyledonous plants, and exhibit HCV inhibitory and DNA topoisomerase inhibitory activities. The first generation total synthesis of tellimagrandins features the one-pot regioselective diacylation of glucose derivative 11. After 4-O-selective acylation of 11 with acid anhydride 10 in the presence of organocatalyst 6, the second esterification of the 4-O-acyl-glucopyranoside at the C(6)-OH was performed with a carboxylic acid generated in situ by the first esterification step in the presence of a condensation reagent in one-pot. The second generation total synthesis of this class of natural products involves the direct chemo- and stereoselective galloylation of unprotected D-glucose. The β-selective anomeric galloylation of unprotected D-glucose followed by regioselective digalloylation gives 19 as a single stereo- and regioisomer. Compound 19 is expected to be a versatile intermediate for the short-step total synthesis of various ellagitannins and the related glycosides. The present protocol based on organocatalytic regioselective functionalization is expected to be useful toward extremely short total syntheses of carbohydrates.
The oxidation of alcohols to their corresponding carbonyl compounds is a fundamental transformation in organic chemistry. In recent years, the 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO, a nitroxyl radical)-catalyzed oxidation method has attracted increasing attention in synthetic organic chemistry because the method allows the use of various safe bulk oxidants, thereby enabling a safe and extremely efficient oxidation of alcohols with considerable operational simplicity. Another distinguishing feature of the TEMPO-catalyzed method is its capability for the selective oxidation of primary alcohols in the presence of secondary alcohols. The rationale behind such a feature is its reaction mechanism and its structure, in which four methyl groups flanking the nearby catalytic center (TEMPO^+) play key roles in preventing bulky substrates from forming the key intermediate. As a result, TEMPO is inefficient in the oxidation of structurally hindered secondary alcohols, posing a problem in the oxidation of alcohols. Such features of the TEMPO oxidation led us to envision that the use of a structurally less hindered nitroxyl radical can solve this problem and expand the scope of the oxidation of alcohols. We conceived the potential use of 2-azaadamantane N-oxyl (AZADO), a structurally less hindered class of nitroxyl radical, to address the issues. Thus, it was envisioned that the a-hydrogen flanked by the nitroxyl group of AZADO would furnish a wider catalytic center, enabling them to proceed to subsequent oxidation events. It was revealed that AZADO indeed exhibits an excellent catalytic activity for oxidation of a variety of alcohols even in which TEMPO failed to give the expected products. The synthetic use of AZADO has een exploited to develop (i) oxoammonium salt/NaClO_2 method for one-pot oxidation of primary alcohols to carboxylic acids, and (ii) oxidative rearrangement of tertiary allylic alcohos to α,β-unsaturated ketones. We also developed chirally modified AZADOs that successfully conduct a highly enantioselective oxidative kinetic resolution of racemic secondary alcohols. The versatility of the AZADO oxidation has been demonstrated in the first total synthesis of (-)-idesolide, which was isolated from the fruit of Idesia polycarpa.
Lactonamycin (1), isolated from a culture broth of Streptomyces rishiriensis, shows potent antimicrobial activities against Gram-positive bacteria including MRSA and VRE, as well as cytotoxicity against various tumor cell lines. Lactonamycin Z (2) differing solely by the sugar moiety was later isolated and is less potent against Gram-positive bacteria. Their unique hexacyclic core structure including a densely oxygenated EF-ring system has inspired synthetic efforts in several laboratories. In planning for the total synthesis of 1, we adopted the final-stage glycosylation of lactonamycinone (3), which will enable the divergent syntheses of the sugar derivatives of 1. First, we tried the construction of the model BCDEF-ring system. Ethynyltetraol 13 was synthesized via a cycloaddition reaction between quinone 9 and the diene derived from anhydride 10 followed by dihydroxylation. The EF-ring system construction from 13 was realized via a palladium-catalyzed cyclization-methoxycarbonylation, a stereoselective methanol addition, and lactonization, leading to the production of the model aglycon 15. The EF-ring turned out to be tolerant under strong acidic conditions. Based on the above results, we forwarded the total synthesis of 1 including the A-ring system. We envisioned a late-stage A-ring formation route because isoindolinones tend to be oxidized under basic conditions and have a solubility problem. As a method of A-ring formation, we applied the Bischler-Napieralski reaction to isoindolinone synthesis. The dramatic improvement of this new reaction was secured when the alkoxy moiety of the alkoxycarbonylamino function was changed from methyl (16a) to isopropyl (16c) group. Further investigation reveals a new reaction mechanism involving a phosphate intermediate A. A diene precursor 22, having an A-ring foothold, was prepared by the known phenol 18 via one carbon elongation and reductive amination. A cyloaddition reaction between the diene derived from anhydride 22 and chloroquinone 23 gave anthraquinone 24. In a similar way to the synthesis of model aglycon 15, anthraquinone 24 was converted to BCDEF intermediate 27. The challenging Bischler-Napieralski-type cyclization of 27 was succeeded, providing lactonamycinone (3) in good yield. The final glycosylation of the protected aglycon 28 with the L-rhodinose derivative 4 afforded lactonamycin (1) and its diastereomer 29.
Polycyclic polyprenylated acylphloroglucinols (PPAPs) feature complex and diverse structures, including a highly oxygenated and densely substituted bicyclo[3.3.1]nonane-2,4,9-trione core complete with prenyl or geranyl side chains, among others. The family of PPAPs consists of more than 110 members, and their number continues to increase. Interestingly, PPAPs having closely related structures show different and wide-ranging biological activities. For example, nemorosone exhibits anti-HIV and antitumor activities, hyperforin shows antidepressant and antitumor activities, and garsubellin A has anti-Alzheimer activity, while the differences in their structures lie only in the substituents. The highly stereoselective total syntheses of nemorosone and hyperforin have been accomplished via a new approach which features intramolecular cyclopropanation of α-diazo ketone (IMCP) (step I), subsequent stereoselective alkylations (step II), and regioselective ring-opening of cyclopropane (step III). Step I would be made enantioselective through the use of a chiral catalyst, and step II enable the construction of the C8 all-carbon quaternary stereogenic center for the total synthesis of hyperforin. Step III regioselectively affords the ring-opened product because the electron-donating methoxy group on the cyclopropane and the electron-withdrawing ketone cooperatively enhance the ring-opening reaction. The total syntheses of nemorosone and hyperforin include chemo-and stereoselective hydrogenation directed by the internal alkene.
Gambieric acid A (1) is a marine polycyclic ether natural product that was isolated from the ciguatera causative dinoflagellate Gambierdiscus toxicus. Despite its structural similarity to ciguatoxins and brevetoxins, 1 only weakly binds to voltage-gated sodium channels and does not exhibit lethal toxicity against mice at 1 mg/kg (ip). Instead, 1 displays extraordinary potent antifungal activity against Aspergillus niger. Our previous studies on the synthesis and NMR analysis of suitably designed model compounds have strongly suggested that the stereochemical assignment of the originally proposed structure of 1 is questionable and led us to propose that the absolute configuration of the polycyclic ether domain of 1 is opposite to that of the natural product. Here, we disclose the first total synthesis and complete stereostructure of 1. The B-ring was first synthesized based on our methodology for the synthesis of medium-sized cyclic ethers. The A-ring was next forged via stereoselective bromoetherification. Suzuki-Miyaura coupling of the A/B-ring exo-olefin with an acetate-derived enol phosphate followed by ring-closing metathesis (RCM) constructed the D-ring, and mixed-thioacetalization and one-pot oxidation/methylation methodologies were used to close the C-ring. The A/BCD- and F'GHIJ-ring fragments were coupled by means of Suzuki-Miyaura coupling. After establishment of the C25 stereogenic center by exploiting the conformational bias of the F'-ring, oxidative cleavage of the F'-ring followed by elaboration of the E-ring via stereoselective allylation of a mixed thioacetal by using glycosylation chemistry, and ensuing closure of the F-ring furnished the entire polycyclic ether backbone. Finally, the J-ring side chain was introduced by means of modified Julia-Kocienski olefination to complete the first total synthesis of gambieric acid A (1). The spectroscopic data, optical rotation value, and antifungal activity of synthetic 1 matched those of the natural product. Thus, our total synthesis confirmed the correctness of our revised structure and unambiguously established the complete stereostructure of 1.
Ophiodilactone A (1) and B (2), isolated from ophiuroid Ophiocoma scolopendrina, exhibit moderate cytotoxic activity against P388 murine leukemia cell with IC_<50> value of 5.0 and 2.2 μg/mL, respectivity. These compounds possess characteristic structures consisting of a fused γ-lactone/6-lactone skeleton with four or five contiguous stereogenic centers containg three quaternary centers and four phenyl groups. The absolute configuration of 1 was tentatively determined by its CD spectrum; however that of 2 has not been determined yet. Their unique highly substituted dilactone structures and intriguing biological activities prompt us to investigate the synthesis of ophiodilactones. Since ophiodilactone B (2) would be accessible from ophiodilactone A (1), we focused on the synthesis of 1 (Scheme 1). From a retrosynthetic perspective, we envisioned bicyclic ketone 5 as a precursor of 1. We expected that 1 would be obtained from 5 via construction of a dilactone core by Baeyer-Villiger oxidation and stereoselective introduction of benzyl and hydroxyl groups. To access 5 we selected the [2+2] cycloadditon of 6 as a key reaction. In this symposium, we will report a highly enantio- and stereoselective approach to ophiodilactone A (1). This approach involves organocatalytic asymmetric Micheal addition of dimethyl malonate to cinnamaldehyde, stereoselective formation of bicyclo [3.2.0] heptane 5e by [2+2] cycloaddition of ketene 19e, Baeyer-Villiger oxidation of cyclobutanone 20, and stereoselective introduction of benzyl and hydroxyl groups giving 26. Compound 26 was then converted to 27, a promising precursor of ophiodilactone A (1).
The pluramycins constitute a class of antitumor antibiotics with an anthrapyrone chromophore with two amino sugars attached through the C-glycoside linkages. These compounds exhibit antitumor activity by specific DNA alkylation with high sequence recognition. Considerable attention has been centered at the synthesis of these compounds, where two basic problems must be addressed, (1) bis-C-glycosylation and (2) the selective construction of anthra[1,2-b]pyrone chromophore. This presentation will deal with the total synthesis of the saptomycin B. Sc(OTf)_3-promoted C-glycosylation of tricyclic bisphenol 15 with vancosaminyl acetate 2 proceeded in a regioselective manner at C7 in high yield. Further reaction with angolosaminyl acetate 4 under the similar conditions proceeded at C5 to give bis-C-glycoside 13 possessing the two amino sugar moieties at the requisite positions. This bis-C-glycoside was derived to ynone 12 via the aldol addition to chiral, optically active ynal 14. Treatment of 12 with K_2CO_3 in Me0H cleanly promoted the pyranone formation, and the following three-step oxidation gave anthraquinone 32. We are now examining the final deprotection toward the total synthesis of saptomycin B.
In the last year, we had achieved the total synthesis of antituberculosis Hirsutellone B via direct construction of the highly strained 13-membered macrocycle utilizing Ullmann-type etherification. Based on this work, we examined an influence of the configuration at C19 position on the above mentioned 13-membered macrocyclization. As a result, the configuration of C19 position was specifically reflected to the planar chirality of MOM enol ether moiety. That is, the facial selectivity of MOM enol ether moiety on the intramolecular Ullmann reaction was completely controlled by the central chirality at C19 position. The following construction of γ-hydroxylactam moiety was also proceeded stereoselectively by reflecting the planar chirality of MOM enol ether moiety. A novel unnatural-type stereoisomer of Hirsutellone B was obtained via the hydrolysis of this moiety and subsequent isomerization to the (Z)-enol form.
Indole diterpenes are known to possess intriguing biological properties such as tremorgenic, insecticidal, mito-inhibitory, and anti-MRSA activities. Paspalinine (1) and paspalicine (2) that belong to this family of natural products were isolated from the culture broth of the ergot fungus Claviceps paspali. Paspalinine (1) bearing a hydroxy group at the C13 position causes tremors upon oral administration in cockerels, mice, and domestic animals. Their unique meroterpenoid structures as well as the interesting biological activity of 1 prompted us to embark on the total synthesis of 1 and 2. In this symposium, we report the total synthesis of 1 and the formal synthesis of 2. Synthesis of 4, the A-E ring moiety of 1 and 2, was accomplished from enantiomeric pure Wieland-Miescher ketone in 10 steps by using intramolecular Horner-Emmons reaction, stereoselective installation of the C3 angular methyl group via a cyclopropane intermediate, and Pd(II)-mediated oxidative indole ring formation as the key transformations. Conversion of 4 to heptacyclic intermediate 30 was achieved via Tsuji's palladium-catalyzed allylation and the Sharpless asymmetric dihydroxylation. Since 30 had previously been converted to 1 and 2 by Smith and co-workers, our work represents a formal synthesis of 1 and 2. We also succeeded in converting 29 (N-Boc-protected precursor of 30) into the N-Boc-protected form of paspalinine (34) by stereoselective installation of the C13 hydroxyl via a one-pot selenenylation/oxidation/[2,3]-sigmatropic rearrangement sequence. The Boc protecting group of 34 was removed to complete the total synthesis of 1 in 18 steps from Wieland-Miescher ketone.
Lepenine (1), a diterpene alkaloid isolated from aconite species, is characterized by a complex polycyclic system including bicyclo [2.2.1] and bicyclo [2.2.2] skeleton as well as 11 contiguous stereogenic centers. While a synthetic study of denudatine was reported by Wiesner and co-workers, total synthesis of the natural products that possess the carbon skeleton of lepenine has not been achieved to date. Therefore, we initiated our synthetic studies toward the total synthesis of lepenine (1). Our synthetic studies commenced with preparation of enyne 9 from commercially available o-vanillin acetate in seven steps. Enyne metathesis of 9 was performed under an atmosphere of ethylene to give diene 10 in excellent yield. Reduction of the ester of 10 afforded the primary alcohol, which was condensed with methacrylic acid. When the resultant triene 11 was heated at high dilution in the presence of sodium bicarbonate and hydroquinone, intramolecular Diels-Alder reaction proceeded smoothly to afford the desired tetracy clic lactone 12. Diels-Alder adduct 12 was converted to the Mannich reaction precursor 15 in four steps. When 15 was treated with palladium catalyst and acetic acid, removal of the Alloc group and subsequent intramolecular Mannich reaction took place via imminium ion 16, giving a complex cyclic system containing bicyclo [2.2.1] skeleton with high stereoselectivity. After removal of the benzyl group and stereoselective reduction of the ketone, transformation into orthoquinone monoketal 2 was attempted by oxidation with iodobenzene diacetate. While direct oxidation of 19 caused decomposition, oxidation of the corresponding hydrochloride salt 20 yielded orthoquinone monoketal 2. Construction of the bicyclo [2.2.2] skeleton was accomplished by Diels-Alder reaction of orthoquinone monoketal 2 and ethylene with complete stereoselectivity. Thus, we could achieve the construction of the skeleton of the natural product 1. Finally, we attempted a series of functional group manipulations toward the total synthesis of lepenine (1). After protection of the hydroxy group as a MOM ether, reductive removal of the ketal moiety was performed with samarium(II) iodide to give 22. Introduction of exomethylene unit at the a position of ketone 22 and subsequent 1,4-addition of thiophenol afforded 25. When 25 was treated with borane-THF complex at 50 ℃, the regio- and stereoselective hydroboration of the double bond and stereoselective reduction of the ketone proceeded smoothly, and diol 26 was obtained after oxidative workup and treatment with ethanolamine. Protection of the two hydroxy groups with acetic anhydride, followed by careful oxidation of the sulfur atom yielded sulfoxide 27. Sulfoxide elimination and removal of the protecting groups led to the first total synthesis of lepenine (1).
Crops grown on alkaline soils, which cover about one-third of the world lands, are prone to iron-deficiency stress because of the low solubility of iron there. To acquire the insoluble iron efficiently, graminaceous plants have developed a unique strategy characterized by the synthesis and secretion of an iron-chelator phytosiderophore and a specific uptake system of iron(III) in its complex. Mugineic acid (MA) 1 was first identified as phytosiderophore in barley and its analogues have then been isolated and identified from various graminaceous species and cultivars; they all form water-soluble 1:1 complexes with iron(III). Owing to its significant implication in plant physiology, this iron up-take system has been a subject of intensive research since its discovery 30 years ago. However, a limited supply of phytosiderophores has been a severe bottleneck particularly for the study on the transport mechanism. Therefore, it is essential to establish an ample supply source of MA 1 and/or 2'-deoxymugineic acid (DMA) 2. In addition, supply of DMA will be potentially important to solve the worldwide problem of shortage of food supply because their use may make cultivation possible even on alkaline soils which are not favorable for farming. In this presentation, 1) the investigation of potential values of DMA as a fertilizer and synthetic studies toward an ample supply of DMA, 2) the mugineic acid derivatives as molecular probes for the mechanistic elucidation, and 3) development of 1,3a,6a-triazapentalenes as a breakthrough fluorescent molecule will be discussed. 1) A practical synthesis of L-azetidine-2-carboxylic acid, an expensive starting material for DMA, was established by using asymmetric organocatalyst. The normal rice plant was able to grow even in alkaline hydroponic culture by the addition of DMA and iron(III) salt. 2) A hydroxy group in mugineic acid (MA) that was not needed for Fe(III) complexation was used for the introduction of various labeling groups through propargylation and a click reaction. Labeled MA was incorporated as a phytosiderophore into Xenopus oocytes through the HvYS1 transporter, as determined by an electrophysiological assay and fluorescence microscopy. 3) An efficient and versatile method was established for the preparation of 1,3a,6a-triazapentalenes. The 1,3a,6a-triazapentalene skeleton without an additional fused ring system was discovered to be a compact and highly fluorescent chromophore, which exhibited various interesting fluorescent properties such as a noteworthy correlation of luminescent wavelength with the Hammett σ_p value and a strongly positive solvatofluorochromism.
Neoxaline was isolated from the culture broth of Aspergillus japonicus Fg-551, together with the structurally relevant compound oxaline in 1979 by our group. Neoxaline and oxaline inhibit cell proliferation and arrest the cell cycle during M phase in Jarkat cells by the inhibition of tubulin polymerization. Neoxalines, isolated 10 families from the nature so far, are a novel class of biologically active indole alkaloid possessing unique indoline spiroaminal framework, E-dehydrohistidine and a sterically bulky reverse-prenyl group at the benzylic ring junction. Total synthesis of their biosynthetically relevant compounds have been reported by several groups. However the construction of indoline spiroaminal framework has been reported only by our group. The first asymmetric total synthesis of Neoxaline was accomplished by means of 1) stereoselective introduction of reverse-prenyl group at ring junction from 3a-trichloroacet -imidoxyfuroindoline prepared from commercially available tryptophol via aymmetric oxidative ring closure under the modified Katsuki-Sharpless epoxidation condition, 2) stereoselective construction of indoline spiroaminal by three times oxidation and cyclization twice (oxidation to nitrone, 6-memberd ring closure, oxidation to cyclic nitrone, 5-memberd ring closure) from indoline possessing two amino groups at suitable position, 3) photoisomerization of Z-dehydrohistidine to E-isomer.
Stemonamine (1) was isolated from the roots of Stemona japonica Miq. as a member of the Stemona alkaloid family. Although the racemic total syntheses of 1 or stemonamide (3) have been reported by three groups, its enantioselective synthesis has not been reported so far. In this presentation, the total synthesis of 1 using the reactions that we have developed, such as the ynolate-initiated tandem reaction and the intramolecular acylation, is described. Our synthesis commenced with condensation of the known optically active carboxylic acid 16 and the lactone 17 to give the iodide 15 in 4 steps. The intramolecular acylation of 15 using ^tBuLi afforded the 7-member ring 22 in high yield as the first key reaction. The TBS protection of 22 and the oxidative cleavage of the terminal alkene, followed by esterification, provided the methyl ester 14c. As the second key step, the tandem [2+2]cycloaddition-Dieckmann condensation using ynolate anion 6 and 14c was performed to successfully provide the enone 28c in high yield. After transformation of 28c to the diacetate 31, the acetoxy group on the C-ring was removed via the SmI_2-mediated reduction in good yield to afford the Tu's synthetic intermediate 33. At last, the total synthesis of 1 was achieved via the Tu's method. The spectroscopic data of our synthetic 1 were in good agreement with those recorded in the literature. [chemical formula]
13-Oxyingenol derivative 1 and ingenol (3) are diterpenoids isolated from the plants of Euphorbia sp. The main structural features of ingenols are a bicyclo[4.4.1]undecane skeleton with inside-outside intrabridgehead stereochemistry and a high degree of oxygenation. They and their analogues showed interesting bioactivities. The molecular complexity of ingenol derivatives, in conjunction with their potent bioactivities, has made them attractive synthetic targets. We have achieved the first total synthesis of (-)-13-oxyingenol (2) and its natural derivative 1. The efficient functionalization of the A- and B-ring parts was established by using C-2 and C-7 hydroxy groups as clues. This approach is highlighted by RCM for the construction of an inside-outside framework, regio- and stereoselective dihydroxylation for the functionalization of the A-ring, and [2,3]-sigmalropic rearrangement for the functionalization of the B-ring.
Isodehydrothyrsiferol, one of the possible structures of which was shown as 1, was isolated from the red alga Laurencia viridis by Norte et al. in 1996 (Scheme 1). The plane structure of isodehydrothyrsiferol and the relative configuration of the D-ring were determined by the NMR analysis. The stereostructure of the A,B,C-ring system was elucidated by comparing the NMR data with those of dehydrothyrsiferol (2), isolated from the red alga Laurencia pinnatifida by Martin et al. in 1984. However, the stereochemical relationship between the A,B,C- and the D-ring systems due to the intervening methylene chain and the absolute configuration have never been determined. Both of isodehydrothyrsiferol and 2 exhibit cytotoxic activity with IC_<50> = 0.01 μg/mL against murine leukemia P388. To determine the entire stereostructure of isodehydrothyrsiferol, we embarked on the total synthesis. We planned convergent strategy that united the common B,C-ring system 4 with the D-ring 5 or 6 by Suzuki-Miyaura cross coupling (Scheme 1). The common B,C-ring system 4 was constructed from the known epoxy alcohol 10^4 through two 6-exo oxacyclizations of appropriate epoxy alcohols (Scheme 2). After hydroborating D-ring 22, prepared as shown in Schemes 3 and 4, with an excess of 9-BBN, Suzuki-Miyaura cross coupling with triflate 4 in the presence of LiCl and Ph_3As^<12> afforded a coupling product 23 in 66% yield (Scheme 4). Removal of protective groups and subsequent 6-endo bromoetherification gave target molecule 1, ^1H and ^<13>C NMR spectra of which were inconsistent with those of natural isodehydrothyrsiferol. Another possible diastereomer 25 was synthesized from ent-22. ^1H and ^<13>C NMR spectra of synthetic 25 was identical to those of natural isodehydrothyrsiferol except for a sign of the optical rotation. Thus, it was found that the entire stereostructure of natural (+)-isodehydrothyrsiferol is shown as ent-25 including the absolute configuration. We have also achieved the total synthesis of dehydrothyrsiferol (2) and 15(28)-anhydrothyrsiferyl diacetate (3).
19-Hydroxy-sarmentogenin (3) is an aglycon of 19-hydroxy-sarmentogenin-3β-O-β-6-deoxyguloside (2), a cytotoxic cardenolide carbohydrate. The cardenolide family is structurally characterized by the oxygenated steroidal skeleton, which is composed of cis-fused AB- and CD-ring and β-oriented C17-butenolide. To establish a unified synthetic route to the oxygenated cardenolides, we selected 3 as the initial synthetic target. Here we report a total synthesis of 3. For efficient construction of the oxygenated steroidal structure of 3, the new convergent strategy was designed. Specifically, the functionalized AB-ring 4 and the D-ring 5 were coupled via the acetal coupling under the mild conditions, affording bromoacetal 6. The C9-11 bond of 20 was stereoselectively formed through 6-membered ring cyclization of the C11-carbon radical that was generated by treating 6 with (TMS)_3SiH and Et_3B. Then the regio- and stereoselective aldol reaction of 7 by using KN(TMS)_2 at 60 ℃ gave rise to the oxygenated steroidal structure 8a as a sole product out of the eight possible steroidal structures 8a〜h. 19-Hydroxy-sarmentgenin (3) was synthesized from 8a. Nine-step sequence from 8a, including deoxygenation of the C7-oxygen functionality and oxidative cleavage of the vinyl ether, resulted in formation of 28. The Cll-ketone of 28 was reduced to the alcohol under the Birch conditions to afford 29 as a single isomer. After the C17-ketone was converted to the vinyl iodide 30, the C17-butenolide was installed by the Cu-accelerated Stale coupling to provide 32. Stereoselective hydrogenation of the C16-17 double bond of 34 generated the desired C17 stereocenter. Finally, the global deprotection delivered the target product 3.