Natural products produced by microorganism have been contributing to our life. Especially in a medicinal field, various bioactive natural products play important roles. However, even now more variety of compounds has been strongly demanded for establishment of the tailor-made medicine and for problem of drug resistance. The recent remarkable advances in genetic technology allow us to estimate the putative function of enzymes. In this research, a methodology of search for new natural compounds based on gene information was developed. Streptomyces halstedii HC34 is a producer of polyketide antitumor antibiotic vicenistatin. During the analysis of vicenistatin biosynthetic genes, the polyketide synthase (PKS) genes, which are different from vicenistatin, were cloned from S. halstedii. This strongly suggested that S. halstedii produced a new polyketide, which led us to start the isolation study. The inspection of the fermentation broth of S. halstedii resulted in isolating new polyketide compounds, halstoctacosanolides A and B. By several NMR experiments, those structures were determined to have a 28-membered macrolactone. The deduced structures are identical with the partial structure predicted by domain pattern of the cloned PKSs. Further, the whole halstoctacosanolide biosynthetic gene cluster (his cluster), which had remained unidentified part, was clarified. The complete hls cluster spanning 100kbp include nine genes, and these genes encode seven PKSs (hlsA-G) and two cytochrome P450 monooxygenases (hlsH, 1). The module structures of these PKS genes well reflect the chemical structure of halstoctacosanolides. Then, the gene disruptions of each P450 gene were carried out. The prepared hlsH gene mutant lost the productivity of halstoctacosanolide A, and the hlsI gene mutant did not produce halstoctacosanolides A and B. These results clearly indicated that HlsH and HlsI were responsible for the oxidation at C-4 and C-32, respectively. Furthermore, a new derivative, halstoctacosanolide C, was successfully isolated from the hlsI mutant and the structure was determined as 32-deoxyhalstoctacosanolide B.
A growing number of functionally divergent type III polyketide synthases (PKSs), the chalcone synthase (CHS) superfamily enzymes, have been cloned and characterized, which include recently obtained pentaketide chromone synthase (PCS) and octaketide synthase (OKS) from aloe (Aloe arborescens). Recombinant PCS expressed in E. coli catalyzed successive condensations of malonyl-CoA to produce a pentaketide, 5,7-dihydroxy-2-methylchromone, while recombinant OKS yielded octaketides, SEK4 and SEK4b, the longest polyketides produced by the structurally simple type III PKS. PCS and OKS share 92% amino acid sequence identity, and maintain the conserved Cys-His-Asn catalytic triad. The most characteristic feature is that the CHS active site residue 197 (numbering in Medicago sativa CHS) is uniquely replaced with Met in PCS and Gly in OKS, respectively. Site-directed mutagenesis revealed that the chemically inert single residue 197 lining the active-site cavity determines the polyketide chain length and the product specificity depending on the size of the side chain.
Polyether antibiotics exemplified as lasalocid and monensin contain multiple asymmetric centers. While their biosynthetic mechanism with exquisite stereo-control has attracted great interests, the configurations of the double bonds in a putative linear polyketide intermediate, as well as the modes of oxidative cyclization, have remained unidentified. Due to structual complexity, it is difficult to clalify the biosynthetic mechanism for monensin. We therefore focused lasalocid having more simple structure. Identification of entire lasalocid biosynthetic gene cluster has been accomplished. This cluster contains polyketide synthase gene and genes for epoxidation and hydrolysis presumably responsible for polyether formation. To identify the putative intermediate, we designed deuterium-labeled prelasalocid possessing (E, E) configuration of the trisubstituted olefins. The olefins of right segment 9 installing a deuterium label were constructed utilizing a palladium- and zirconium-mediated conversion of terminal acetylenes. Assembly of 9 with the central segment 10 based on the B-alkyl Suzuki-Miyaura reaction led to 11. After incorporation of the CD_3 group at C12, the resulting 14 was connected with the left segment 16 by an anti-aldol reaction. Based on the highly convergent strategy, the prelasalocid has been synthesized in a stereo-controlled manner.
Based upon the genomic database of Mycobacterium tuberculosis H37Rv, the gene Rv3377c,which is a putative terpene cyclase, was amplified by PCR and ligated into BamH I/Hind III site of pET 22b(+), and expressed in E. coli. BL21(DE3). The expressed Rv3377c gene product is almost insoluble (inclusion body), but coexpression of Rv3377c and chaperon in E. coli afforded the gene product as an active form. The functionally expressed gene product had an enzyme activity only for geranylgeranyl diphosphate (GGPP), but inert to geranyl-pp (GPP), farnesyl-pp (FPP), squalene and oxidosqulaene. The structural analysis of the enzymatic product revealed that this enzyme encodes the diterpene cyclase for producing the halimane diterpene skeleton. We named tuberculosinol 1 for the enzyme product. The detailed investigation on the gene product indicated that the actual product by the Rv3377c enzyme was tuberculosinol diphosphate 2. Cyclic diterpene skeletons are usually constructed after removing the PP functional group. After the trials to find the dephosphorylating enzyme, it has turned out that the flanking gene Rv3378c is responsible for the dephosphorylation reaction of 2, yielding the hydroxylated product (isotuberculosinol 3) and 1 resulting from a water attack. Both of the Rv3377c and Rv3378c have been found only the pathogenic species, but not in the non-pathogenic one. Recently, Russell and coworkers reported that the survival of Mycobacterium tuberculosis in the macrophage was attenuated by the gene disruption Thus, the two genes may be closely related to the pathogenicity. We evaluated the effect of 1 on phagocytic activity against zymosan, a yeast cell wall component that produces inflammation, using human macrophage-like U937 cells. Fluorescence microscopic analysis revealed that the phagocytosis of FITC-labeled zymosan by U937 cells treated with 10μM of 1 was reduced by about 50% compared to the cells without treatment. We discuss the detailed enzyme characterization including the kinetic data and substrate specificities. The substrate specificity of the Rv3378c enzyme was remarkably broad as well as cyc2, which was found in Streptomyces griseolosporeus. The Rv3378c and cyc2 accepted copalyl-PP (CDP), ent-CDP and syn-CDP as substrate, leading to the successful construction of a variety of diterpene skeletons.
DNA-templated organic synthesis (DTS) has recently emerged as a general approach to translate DNA templates into DNA-linked synthetic small molecules. DNA templates not only direct chemistries to construct small molecules but also serve as amplifiable bar codes used to identify the chemical structure of the small molecules. Therefore, through DTS, synthetic small molecules can now be subjected to in vitro selection methods for discovery of novel functional molecules. We are interested in exploring the potential of a selection-based approach to the discovery of functional small molecules. In order to discover protein binding small molecules, we have developed a selection method suitable for small-molecule products of DTS. The method involves an affinity pull-down assay of the DNA-linked ligands using a target protein immobilized on beads, followed by PCR amplification and sequencing of DNA templates encoding the bound ligands to identify chemical structures of small molecules that bound to proteins. We have found that our selection method can provide up to a 10^4-fold enrichment of the active ligand in a single step at a very minute scale (<1 pmol of total library compounds and a protein target). Encouraged by these results, we are currently in the process of evaluating the protein binding properties of a library of 1000-membered DNA-linked small molecules derived from an N-acyloxazolidine core structure. In principle, this integrated discovery system based on DNA-encoded small molecule libraries and in vitro selection should be useful for the discovery of novel synthetic small molecules with any desired protein binding functions. Unique properties of DTS inspired us to pursue a new approach to reaction discovery. Specific juxtaposition of two DNA-linked substrates by DNA duplex formation among others in a single highly dilute solution would enable rapid and simultaneous examination of their bond-forming reactivity. The use of DNA microarray technology as a novel high-throughput DNA sequence identification method for in vitro selection led us to discover an efficient carbon-carbon bond forming reaction between a terminal alkyne and alkene in the presence of a palladium salt. Although this approach is only applicable to DNA compatible aqueous reaction conditions, it should be a powerful methodology for discovering unknown reactions between different types of substrates under a vast range of reaction conditions that are yet to be explored.
RK-682 (1), which was isolated from Streptomyces sp. 88-682, inhibits various enzymes such as phospholipase A_2, HIV-1 protease, heparanase, some dual-specificity phosphatases (DSP), and a protein tyrosine phosphatase (PTP), CD45. We already reported the structure-activity relationship studies of RK-682 derivatives at both enzyme and cell levels. Their cell level activity was, however, found to be weaker compared to the enzyme level activity indicating their poor membrane permeability. The 3-acyltetronic acid structure of RK-682, which exists as its anionic form under physiological conditions, is critical for the inhibition of phosphatases and proposed to act as phosphate mimic. But the observed low enzyme selectivity and poor membrane permeability also seem to be caused by this highly acidic structure. To create a more selective phosphatase inhibitor that can be effectively incorporated into cells, electronically neutral RK-682 derivatives were designed and synthesized. Among them, the RK-682 enamide (RE) derivatives were found to inhibit dual-specificity protein phosphatases, VHR and Cdc25A, but not PTP1B or heparanase. Finally, we found that some derivatives also remarkably inhibited proliferation of human leukemia HL-60 cells.
Fusicoccin (FC) and cotylenin (CN), fusicoccane diterpene glycosides, have been known to exhibit strong plant hormone-like activities through activation of H^+-ATPase in plant cells. Recently, the mode of action has been clarified at molecular level. FC forms a stable ternary complex with membrane-boundary H^+-ATPase and cytoplasmic 14-3-3 protein, and maintains the activated stage of the H^+-ATPase. Since 14-3-3 proteins are ubiquitously expressed in all eukaryotic cells, it has been anticipated that FC/CN might affect on animal cells as well. In fact, we found that CN induces functional and morphological differentiation of human myeloid leukemia cells (HL-60). However, FC was less efficient in differentiation of HL-60 in spite of the fact that FC and CN have identical activities on plant tissues. There are several differences in chemical structures of FC and CN such as i) +/-3-OH; ii)+/- 12-OH; iii) complex sugar in CN; iv)+ /- epoxide on sugar moiety. We have carried out SAR study using natural FC/CN congeners and several semi-synthetic FC derivatives. Eventually, we have succeeded in clarifying that the absence of 12-OH is essential for DIA; it is striking that the only one hydroxyl group crucially affects on DIA. The epoxide on the sugar is not essential but strengthens the activity. Considering the mode of action of FC on plant tissues, it is plausible that DIA of CN is also related to 14-3-3 protein function. So we then carried out preliminary experiments to prove modulation ability of CN on 14-3-3: phospho-peptide complexes. Isothermal titration calorimetry (ITC) revealed that CN can stabilize the complex of 14-3-3 protein and its target peptide having a …RSX_1pSX_2P… motif, only when X_2 is Val.
The interaction between actin and aplyronine A, a potent antitumor and actin-depolymerizing substance of marine origin, was investigated by photoaffinity labeling experiments and X-ray structure analysis of actin complex with aplyronine A. Photoaffinity probes consisting of a side-chain portion of aplyronine A as a ligand, a diazirine moiety as a photoaffinity group, and a fluorophor as a detecting group were synthesized. Photolabeling experiments between actin and the probe were carried out. Actin was successfully photolabeled by the fluorescent probe and visualized clearly. The present results provide the first chemical evidence for the direct interaction between actin and the side-chain portion of aplyronine A. X-ray structure analysis of actin-aplyronine A complex was carried out. The analysis showed that Aplyronine A bound to a hydrophobic cleft between subdomains 1 and 3 of actin. The structural feature is that the trimethylserine moiety protrudes toward the bulk solvent region.
Paralytic shellfish poisoning (PSP) toxins such as Saxitoxin (STX) cause human fatalities by blocking Na^+ conductance in nerve fibers. Bivalve molluscs, the primary vectors of PSP in humans, show marked inter-species variation in their capacity to accumulate PSP toxins. Here we demonstrate that there is also inter-population variation in PSP resistance within a species. Softshell clam Mya arenaria were collected from two areas. One of them is subject to recurrent annual toxic blooms of Alexandrium spp. (PSP area), and the other has never suffered PSP up to date (PSP-free area). The clams from PSP area were far more tolerant to PSP toxins than those from PSP-free area, accumulated higher amount of toxicity, and survived. The amino acid sequences of voltage-dependent Na^+ channels in the pore regions, the binding site of STX, turned out to be different by only one amino acid between PSP toxin-sensitive and resistant clams. Patch clamp recording proved the substitution made the channel about 1,500-fold less sensitive to STX. This study establishes a molecular basis for intraspecific variation in toxin resistance to PSP.
Most benthic marine invertebrates have a planktonic larval stage in their life cycle, and then larvae settle and metamorphosis into adult form. In Cnidarians, metamorphosis is naturally triggered by external cues and not by the elevation of an internal hormone level as in amphibians or insects. Various species of crustose coralline red algae (CCA) and bacterial biofilms have been shown to be the primary sources of external inducers of metamorphosis for coral larvae. We have focused on the relationship between CCA and coral larvae mediated by their metabolites. In our observational study, a fragment of coral rubble with CCA induced larval metamorphosis of the scleractinian coral Pseudosiderastrea tayamai, however coral larvae didn't settle on directly surface of the CCA. These results suggested that coral rubble with CCA have two types of chemical signals i.e., defense chemical and natural inducer. We have established a simple bioassay using P. tayamai larvae for such biological phenomena. The methanol extract of coral rubble with CCA showed toxic activity against the coral larvae. Bioassay-guided isolation of methanol extract afforded two new brominated toxic compounds, corallinafuran (1, 10^<-6>M) and corallinaether (2, 10^<-7>M). In contrast, the natural inducer and compounds that enhanced its effect in larval metamorphosis were isolated from methanol extracts of same coral rubble with CCA. A bromotyrosine derivative, 11-deoxyfistularin-3 (3, 10^<-7>M) isolated from the CCA, induced the metamorphosis of P. tayamai larvae (22.2±22.2%). In the presence of fucoxanthinol (4, 10^<-9>M) and fucoxanthin (5, 10^<-9>M), the percentage of metamorphosis induced by 3 was further enhanced to 88.9±13.6 and 94.4±13.6%, respectively. Both carotenoids are also found in the coral rubble with CCA. These results suggest that bromotyrosine derivative and carotenoids have a synergistic effect in the metamorphosis of P. tayamai larvae. This effect may provide a higher selectivity for recruitment than a single-component natural inducer for recognizing suitable substrata for larval metamorphosis. Thus, the effect might offer a survival advantage for benthic marine invertebrates.
Determination of absolute configuration has often required chemical derivatization. Vibrational circular dichroism (VCD), a recently developed chiroptical spectroscopy, measures differential absorption of left vs. right circularly polarized infrared light caused by molecular vibrational transition. VCD is becoming one of the most powerful and convenient technique for chiral studies, due to the applicability to all organic compounds without any derivatization in solution state with the aid of reliable theoretical calculation. We have applied this method to natural products. In this paper, we discuss VCD assignment of the absolute configurations, which are not easily determined by ordinary methods. Absolute stereochemistries of brominated marine sesquiterpenes from Laurencia 1, 2 and 4 were studied by VCD. Comprehensive conformation search followed by DFT calculation was performed to calculate VCD spectra. The observed VCD spectra showed good agreement with the calculated ones, by which compounds 1 and 2 were assigned as 7S,10S. Peroxidic diastereomers 4a and 4b were separated by chiral HPLC and their VCD spectra were measured. By comparison of experimental and theoretical VCD, their absolute stereochemistries were determined as 1R,4R,7S,10S and 1S,4S,7S,10S, respectively. This is the first case to apply VCD to determination of absolute configuration of the chiral peroxy compounds. Stereochemical studies of cruciferous phytoalexins 5-9 were also conducted by means of a nonempirical as well as an empirical manner. In this study, VCD was demonstrated to be effective also for tertiary alcohols and spiro-compounds. Stereochemistries on a glycerol moiety of lipids and glycolipids were also examined by VCD. By focusing on C=O stretching VCD, an empirical rule to distinguish the chirality of glycerol sn-2 carbon has been established.
Marine organic metabolites that are composed of a long carbon backbone functionalized by oxygen atoms have been called "super-carbon-chain compounds". To proceed further functional studies of these compounds, we first revealed the whole shape of these molecules in the water solution state, by using palytoxin (PTX). PTX is a marine natural product with a molecular weight of 2680. PTX shows potent toxicity against mice and interacts with Na/K ATPase. Since the acetamide of PTX (NAcPTX) shows reduced pharmacological activity, elucidation of the structures of both PTX and NAcPTX in solution would be interesting from a biological perspective. We solved these structures by using the small angle X-ray scattering (SAXS) technique with an intense synchrotron X-ray source. PTX was found to exist as a dimer, while NAcPTX existed as a monomer. We simulated low-resolution models for PTX and NAcPTX from the SAXS profiles by using a chain-like ensemble of dummy residues modeling methods. The overall shape of NAcPTX in water was horseshoe-like in water, while PTX was prolonged oblate. Interestingly, single-site acetylation at the N-terminus of PTX greatly affect the association of PTX molecules.
Marine sponges are known to be a prolific source of secondary metabolites with antitumor property. As part of our program to discover novel antitumor leads from natural sources, we discovered a group of novel cytotoxic macrolides from an aqueous extract of the Papua New Guinea marine sponge Cinachyrella enigmatica. In this session, we will present complete structure determination of enigmazole A, one of the most abundant and structurally the simplest members of this class of sponge metabolites. Spectroscopic analyses based on NMR and MS experiments revealed that 1 is a novel 16-membered macrolide of mixed polyketide/peptide origin and is characterized by a phosphate ester and a uniquely substituted oxazole as a side chain moiety. The complete stereochemical assignment of all of its 8 chiral centers was achieved through extensive chemical derivatization/degradation studies including N,N'-dimethyl diamidation of the phosphate group, anisotropic shift analysis after derivatization with PGME or MTPA, and non-enzymatic dephosphorylation accompanied by subsequent hydrolysis of the macrolide ring and δ-lactonization, and by development of a novel chiral analysis procedure for volatile ketones. Compound 1 was cytotoxic against IC-2 mouse mast cell progenitor cells with IC_<50> values of 0.3μg/mL. Studies on the structure of other congeners as well as on the mode of action of these unique macrolides are currently under way.
Fish and shellfish diseases are the most serious concerns for aquaculture today. Large amounts of antibiotics are used to control these diseases which may pose environmental and hygienic problems. In our search for environmentally-friendly drugs for aquaculture from Japanese invertebrates, we found that the extract of an unidentified marine tunicate collected in southern Japan exhibited activity against the bacterium Vibrio anguillarum, a causative agent of the Vibriosis in fish. Bioassay-guided fractionation afforded two novel serinolipid derivatives named shishididemniols A (1) and B (2). The structure of shishididemniol A (1) was elucidated by interpretation of spectral data, and the application of the modified Mosher method to 1 and its suitable degradation products. Shishididemniol B (2) was the chlorohydrin of 1, which was demonstrated by chemical interconversion. Compounds 1 and 2 exhibited antibacterial activity against fish pathogenic bacterium Vibrio anguillarum.
Angiogenesis is the process of generating new capillary blood vessels. Tumor growth and metastasis are highly dependent on angiogenesis. Therefore, specific inhibitors of angiogenesis are expected as promising antitumor agents. In the course of our study of bioactive substances from marine organisms, we focused on a search for anti-angiogenic substances and have isolated four novel steroidal alkaloids named cortistatins A (1), B (2), C (3), and D (4) consisting of abeo-9(10-19)-androstane and isoquinoline skeletons, from the Indonesian marine sponge Corticium simplex. The absolute stereostructures of 1-4 were elucidated by detailed 2D-NMR, CD, and X-ray crystallographic analyses. We have further isolated the abeo-9(10-19)-androstane-type alkaloids named cortistatins E (5), F (6), G (7) and H (8) having N-methyl piperidine or pyridine moiety in the side chain from the same marine sponge. Compounds 1, 2, 3, and 4 inhibited proliferation of human umbilical vein endothelial cells (HUVECs) with high selectivity. Among four substances, 1 showed the strongest anti-proliferative activity (IC_<50>=0.0018μM) against HUVECs, in which selective index was more than 3000-fold in comparison with normal fibroblast or several tumor cells, while 5, 6, 7, and 8 didn't show selective anti-proliferative activity. The anti-angiogenic property of 1 was examined by the migration assay using a chemotactic chamber and the Matrigel tubular formation assay. Compound 1 inhibited migration and tubular formation of HUVECs induced by VEGF or bFGF at 2-200nM concentrations, dose dependently. To elucidate the action mechanism of 1, we examined the influence of 1 on ERK pathway which was activated by growth factor signal. Compound 1 didn't inhibit the ERK phosphorylation at 10nM-1μM concentrations.
Streptomycetes are gram-positive soil bacteria which have complex life cycle similar to that of fungi. Besides, whole genome sequences of several strains were completed recently. On the basis of these backgrounds, S. coelicolor is a model organism for the study of morphological changing system. When it is cultured on an agar medium, it makes substrate mycelium. After 2-3 days, the generation of aerial hyphae starts from substrate mycelium into the air. Finally, spores are produced from aerial hyphae through metamorphose. It was known that the signal-cascade system via several signal molecules involved in the aerial hyphae formation of S. coelicolor. The final product of the cascade was known as SapB (Spore Associate Protein B) which induces aerial hyphae directly. We recently found that SapB was a lantibiotic-like peptide derived from the product of the developmental gene ramS. The structure was deduced as 1 by N-terminus sequencing after reduction and tandem TOF-mass. SapB lacked of the antimicrobial activity despite the fact that it had a lantibiotic-like structure. This was the first example of a morphogenetic role for an antibiotic molecule. To figure out the diversity of lantibiotic like peptide in streptomycetes, we isolated another peptide SapT from S. tendae. The structure of SapT was elucidated as 2 by analyzing the data of NMR and TOF-MS spectra. Although SapT has different structure from that of SapB, it showed a similar inducing activity of aerial formation to S. coelicolor. Surprisingly, hydrophobin, an aerial hyphae inducing protein of fungi, also induced aerial formation of S. coelicolor, although hydrophobin is a glycosylated protein which does not have any structure similarity to SapT and SapB. It was indicated by Wosten et al. that hydrophobin had amphiphatic structure and cause surfactant effect on surface of agar plate. Lowering surface tension by the surfactant activity helped to escape substrate mycelium into the air. We applied 3D molecular modeling method to SapB and SapT. The result indicated that the peptides had amphipatic structures and induced aerial hyphae in the same manner with hydrophobin.
Sarcophytoxide (1) is a cembranoid isolated from soft coral, Sarcophyton glaucum. It is worth noting that this compound is contained in the organism in quite a large amount; 13g of crystalline 1 is deposited by keeping the hexane extract of the dry soft coral (240g) in a refrigerator overnight. Sarcophytoxide (1) has only weak biological activities. Its structure is characterized by an epoxide, a dihydrofuran, three olefin bonds, and a fourteen-membered ring. We intended to convert it chemically to compounds that have strong pharmaceutical activities. As the result, we succeed in the chemical transformation to an aromatic compound 12 that exhibits cytotoxic activity against A549 cell. 1 was treated with 0.5 equiv of TMSOTf or TfOH in benzene at room temperature for 20 minute. An aromatic compound 12 was obtained in 15% yield. The absolute configuration was determined by modified Mosher's method after a reduction to secondary alcohol 15. At the same time, we discovered unusual epoxide-ketone rearrangement. A ketone obtained as a major product by the rearrangement of sarcophytoxide is contradictory to that expected from the ordinary epoxide-ketone rearrangement. We suggest a new reaction mechanism of the epoxide-ketone rearrangement.
Interleukin-6 (IL-6) is a multifunctional cytokine exhibiting important roles in host defense, acute phase reaction, immune responses, nerve cell function, and hematopoiesis. Elevated serum IL-6 levels have been observed in a number of pathological conditions including bacterial and viral infections, trauma, autoimmune diseases, inflammations, and malignancies. Now we found that IL-6 level enhancement compound during our synthetic study of trehalose-6,6'-dimycolate (1, TDM, used to be so called cord factor) analogues. TDM is a glycolipid distributed in the cell wall of Mycobacterium tuberculosis, and shows significant anti-tumor activity based on a immunoadjuvant activity. However due to its significant toxicity, TDM was not yet applied for practical use. In 1993, Datta and Takayama reported the purification of trehalose-6,6'-dicorynomycolate (2c, TDCM) from Corynebacterium spp. We have reported the synthesis of four diastereomeric TDCMs based on a strategy of Katsuki-Sharpless epoxidation and then Noyori's BINAP-Ru catalyzed asymmetric reduction of a racemic β-ketolactone. Therefore, we showed that the synthetic (2R,3R,2'R,3'R)-TDCM (2c, hereafter abbreviate to RRRR-TDCM-C_<14>) is identical with natural TDCM, and also demonstrated that 2c and SSSS-TDCM-C_<14> (3c) showed significant anti-tumor activity as well as inhibitory activity of experimental lung metastasis based on the immunoadjuvant activity. Furthermore, we found that the significant lethal toxicity in mice by TDM (1) was no more observed on the shorter analogue TDCMs. Therefore, we have elucidated that the 2,3-anti stereochemistry (RR or SS) on fatty acid residue is promising for biological activities. The chain length of the fatty acid residue should be also important for the biological activity, and thus, we designed general synthetic procedure for trehalose diesters with 2,3-anti stereochemistry and a series of chain length by using Noyori's asymmetric reduction of β-ketoesters and following anti-selective alkylation according to Frater to give β-hydroxy alcohols as the key steps. And we prepared trehalose diesters (TDCM) 2a-d, 3a-d, and 4a-d as well as monoesters (TMCM) 5a-d and 6a-d. Immunological activities of TDCMs and TMCMs were evaluated by determining IL-6 level enhancement on mice serum, and found that RRRR-TDCM-C_<14> (2c) and RRSS-TDCM-C_<14> (4c) show significant IL-6 level enhancement activities.
NKT cell is a potent producer of immunoregulatory cytokines. T cell receptor of NKT cell recognizes CD1d protein-α-galactosylceramide complex, and activated NKT cell can produce the both Th1 (immunostimulant) and Th2 (immunosuppressant)-types cytokines. KRN7000 (2), developed by Kirin Brewery Co. Ltd., is a derivative of natural α-galactosylceramide, agerasphins (main component is agelasphin-9b, 1). KRN7000 is a strong stimulant to NKT cells. However, it induces both Th1 and Th2-type cytokines production of NKT cell by single stimulation. The analogue OCH (3), possessing shorter alkyl chains as developed by Yamamura et al., induces predominant production of interleukin (IL)-4, a key cytokine of Th2 type, over Th1 type cytokines. The total amount of IL-4, produced by NKT cells by stimulation with OCH, however, was less than that in the case of 2. For improvement of this problem, we developed two kinds of new galactosylceramides. One of them is those possessing a ring structure on the sphingosine chain. This analogue was synthesized from the known epoxide 6. The selective reductive-epoxide opening reaction to 6 afforded alcohol 7 or 12. The four-membered ring part was constructed from alcohol 7, whereas alcohol 12 was converted to the five-membered ring part. The resulting products were converted to the corresponding galactosylceramides respectively. Another kind of the analogues (21) were those possessing sulfonamide chain instead of the acyl chain. Those analogues could be synthesized easily from the commercially available phytosphingosine (17). Both analogues could make complexes with CD1d, and induce NKT cells to produce Th1 type cytokine predominantly.
Sphingolipids such as sphingomyelin, ceramide, sphingosine, and sphingosine 1-phosphate are known as secondary lipid messengers in mammalian cells and cell membranes, and a great deal of attention has been devoted to studies of the biological processes regulated by sphingolipids. Among them, sphingomyelin has been known as a major component to form raft domain, which has been proposed as the particular organism in a mammalian cell membrane to efficiently transmit various biological signals. Then, efficient synthetic method for these sphingolipids and providing various kinds of their analogues are strongly desired. Now, we established the widely applicable synthetic method for these attractive compounds, and successfully synthesized various kinds of sphingolipids and their analogues including fluorescence and photoaffinity labeled derivatives by utilizing an olefin cross metathesis reaction. This method was very effective to construct their backbone skeleton possessing various functional groups at the terminal. Meanwhile, our method is also very successful to provide the substrate analogues, which possess CH_2, S, NH groups instead of an oxygen atom of the phospholic acid ester in the native sphingolipids, respectively. By utilizing this method, we synthesized many kinds of sphingolipids such as sphingosine, ceramide, sphingomyelin, fluorescence labeled sphingosine 1-phosphate analogues 15, 16, and sphingomyelin analogues 17, 18, 19. Next, we actually utilized our synthesized tool molecules to investigate the behavior of sphongosine 1-phosphate (S1-P) in cell. S1-P is known as an extracellular mediator participating in cell differentiation, cell growth and so on. Directing toward elucidation of the sphingosine 1-phosphate behavior in cell, we examined the effectiveness of fluorescence labeled sphingosine 1-phosphate 14, 15, and 16 in Chinese Hamster Ovary (CHO) cell, respectively. As a result, fortunately, all analogues were recognized as a ligand by sphingosine 1-phosphate receptor. Each analogue, however, played different behavior in cell. Analogue 14 was metabolized to corresponding sphingosine by cellular phosphatases, while analogues 15 and 16 were not metabolized. Furthermore, the analogue 15 activated MAPK and showed no activation after addition of PTx in cell. On the other hand, the analogue 16 did not show this behavior. Thus, they have strongly been expected as effective tool molecules toward elucidation of sphingosine 1-phosphate behavior in cell.
Glycosyl donors bearing azido functionality at C-2 position have been used for α-glycosylation reactions since 30 years ago. However, its preparation is not easy and the stereoselectivities in glycosylation reactions are not always high. To overcome these difficulties, we develop the novel glycosyl donors having N-benzyl-2,3-oxazolidinone for 1,2-cis selective glycosylation of 2-amino sugars. We also demonstrate its application to the synthesis of bioactive oligosaccharide. Glycosylation reactions using N-benzyloxazolidinone carrying glycosyl donors were highly α-selective. Advantages of the donors include facile preparation in gram-scale and easy deprotection of N-benzyl-oxazolidinone moiety. A one-pot oligosaccharide synthesis involving two 1,2-cis glycosidic linkages was also demonstrated using the novel glycosyl donors. To demonstrate the utility of this donor, we synthesized the anti-Helicobacter pylori oligosaccharide. The target oligosaccharide was terminal α-GlcNAc residues having core 2 type O-linked oligosaccharide. The terminal α-GlcNAc is suggested to be essential for the bioactivity. We synthesized the oligosaccharide from non-reducing terminal α-GlcNAc moieties to the reducing end, because the synthetic intermediates can be submitted to the biological assay in the aim of structure-activity relationships study.
Oligo-α(2,8) and α(2,9) sialic acids 1 and 2 are located at the non-reducing end of glycoconjugates on the cell surface, and play a central role in cell surface recognition phenomena. However, the poor availability of the pure sialo-containing glycoconjugates from the natural sources makes it difficult to elucidate their biological activity. Thus, an effective methodology for the synthesis of oligosialic acids is required. However, oligosialic acids represent difficult and challenging synthetic targets. Herein, we report on the synthesis of α(2,8) and α(2,9) tetrasialic acids 24 and 32 by direct glycosidation strategy. The first approach for the synthesis of oligosialic acids involves coupling N-Troc sialyl donor 3 and acceptor 4. The building blocks were amenable to the synthesis of an α(2,8) disialic acid 5 and, however, did not enable an access to the synthesis of an α(2,8) trisialic acid 7. Next we designed the sialyl donors and acceptors containing a 5-N,4-O-carbonyl protecting group. Glycosylation of the 5-N,4-O-carbonyl protected sialyl acceptors 15a and 15d containing two hydroxyl groups at C7, 8 and C8, 9 positions with the sialyl donor 14 containing the cyclic protecting group provided α(2,8) and α(2,9) disialic acids 16a and 16d in excellent yields and selectivity, respectively. The cyclic protecting group enabled α-selective glycosidation without acetonitrile effects and was effective for improving reactivity of the C8 hydroxyl group toward glycosylation. Using the sialyl building blocks, we succeeded in the synthesis of tetra-α(2,8) and α(2,9) sialic acids 24 and 32 in high yield and selectivity.
Corilagin (1) is a ^1C_4/B-ellagitannin, isolated from Caesalpina coriaria by Schmidt in 1951. The ^1C_4/B-ellagitannin is a subclass of ellagitannins whose hexahydroxydiphenoyl (HHDP) group(s) bridge the non-adjacent hydroxy groups of the glucose, and thus the ring conformation of the glucose part is restricted for the ^1C_4 or skew boat form. The ^1C_4/B-ellagitannins have not been synthesized, despite the identification of numerous such compounds. Compound 1 has an (R)-HHDP bridge between the 3- and 6-oxygens, and a β-galloyl ester at the anomeric position. Wide range of biological activities have been reported on the compound such as potentiation of β-lactam antibiotics against MRSA. In this paper, we report a synthesis of corilagin that is the first total synthesis of the ^1C_4/B-ellagitannin. The 3,6-O-HHDP bridge was constructed by the following sequence: (1) opening of a pyranose ring, (2) coupling of galloyl parts on the 3- and 6-oxygens, and (3) reproduction of the pyranose ring. First, the total synthesis of 1 was achieved by combining our synthetic route and Feldman's coupling method for the HHDP part. However, the feature of the Feldman's method that is the production of a mixture of regioisomers made the isolation of the synthetic intermediates difficult in each step, and as the result, HPLC purification was needed for the final separation of 1. We thus developed a new preparative method for the HHDP group using symmetrical galloyl moieties applying the Brussee's coupling. With this new method, the synthesis of 1 was improved; every synthetic intermediate became a single isomer.
Oligosaccharide chains on the protein play important roles for several biological events as a posttranslational modification, therefore synthesis of glycoprotein and glycopeptide having intact and homogeneous oligosaccharide chains have been paid an attention. In order to investigate function of oligosaccharide on protein, we have examined to establish synthetic method of glycopeptide having homogeneous oligosaccharide and then to synthesize glycoprotein. For the synthesis of protein, native chemical ligation has been used as a powerful coupling method of two peptide fragments. The ligation occurs between the C-terminal thioester in the peptide and Cysteine at the N-terminal of another peptide to afford native amide bond formation. A convenient synthetic method of glycopeptide-thioester is, therefore, essential for the synthesis of glycoprotein by native chemical ligation. Although several preparation methods of peptide-thioester have been reported, a convenient preparation method of sialylglycopeptide-thioester has not been established yet. We have examined to establish a concise synthetic method of glycopeptide thioester having intact complex type dibranched disialyloligosaccharide. The synthetic strategy employs a coupling reaction between benzylthiol and free carboxylic acid at the C-terminal of glycopeptide in which peptide side chain are protected. After construction of glycopeptide on the HMPB-PEGA resin through Fmoc-strategy, protected glycopeptide was released by use of acetic acid/trifluoroethanol. Then we examined a coupling reaction between C-terminal carboxylic acid and benzylthiol under -20 degree in DMF by use of PyBOP/DIPEA. This condition is the best to form thioester and to avoid racemization. Finally, protecting groups of glycopeptide-thioester were removed by 95 TFA to afford glycopeptide-thioester having intact and homogeneous complex type disialyloligosaccharide in good yields. In addition, we have examined the synthesis of glycoprotein, Chemokine (MCP-3) by use of this technique. These glycoprotein consists of 76 amino acids and have asparagines linked oligosaccharide. Our synthetic strategy for glycoproteins uses coupling of peptide-segment strategy and then these will be coupled by repetitive native chemical ligation. This strategy afforded desired glycoprotein having a homogeneous oligosaccharide first time.
Enteropathogenic Escherichia coil (EPEC) belong to a family of related bacterial pathogens, including enterohemorrhagic E. coli (EHEC) O157: H7 and other human and animal diarrheagenic pathogens that form attaching and effacing lesions on host epithelial surfaces. Intimate attachment requires the type III-mediated secretion of bacterial proteins, several of which are translocated directly into the host cell. Therefore, it may be possible that the interruption of type III-mediated secretion relieves infectious diarrhea without elimination of microbial pathogens. In the course of a search for inhibitors of the type III-mediated secretion system in microorganisms, Guadinomine C_1 (1), C_2 (2) and K01-0509 B (3) were discovered from the fermentation broth of Streptmyces sp. K01-0509. Due to the significant activity and the unique structure of 1 and 2, we undertook their total synthesis. We achieved the asymmetric total synthesis and determination of absolute configuration of Guadinomine C_2 (56), involving asymmetric syntheses of 3'-epi-Guadinomine C_2(49) and K01-0509 B (13) and simple piperazinone compounds (24, 30, 32). The total synthesis of 3, a plausible biosynthetic intermediate of 1 and 2, features Sharpless asymmetric epoxidation and the stereocontrolled introduction of the cyclicguanidine with asymmetric nitroaldol reaction, followed by iodine-mediated redox cyclization. These reactions allowed the cyclic guanidine part and the adjacent hydroxy group to be assembled effectively. The syntheses of model compounds including the piperazinone and peptide unit of 1 and 2 feature Evans aldol reaction, regioselective azidolysis, S_N2 cyclization and condensation. By using these successive reactions, we established the synthetic route to the piperazinone moiety and elucidated its stereochemistry. Finally, we completed the efficient synthesis of Guadinomine C_2 and its epimer through the enantioselective preparation of diol, entailing asymmetric α-aminooxylation and diastereoselective nucleophilic addition. Piperazinone and peptide units plus the cyclic guanidine part were built up as well as model compounds and K01-0509 B. The synthetic strategy enabled us to complete the first asymmetric total synthesis and determination of absolute configuration of Guadinomine C_2.
In 1961, siomycins were isolated from the culture broth of Streptomyces sioyaensis by the Shionogi group. The characteristic structure of this thiostrepton family of peptide antibiotics is the bicyclic structure containing a dehydropiperidine, a dihydroquinoline, four thiazoles, a thiazoline, dehydroamino acids, and a dihydroxyisoleucine. These antibiotics show high activities against Gram-positive bacteria, mycobacteria, and human malaria parasite. Also these antibiotics show immunosuppressive properties. Siomycin A was selected as a synthetic target and divided into the five segments, segments A, B, C, D, and E. The coupling of these five segments and the two cyclization reactions finished the total synthesis of siomycin A. The segment A (the dehydropiperidine portion) was synthesized featuring the coupling between the azomethine ylide and the enantiopure sulfinimine, the subsequent stereoselective reduction of the 6-membered imine, and the regioselective dehydrogenation of the piperidin ring. The segment B (the pentapeptide portion), containing the dihydroxyisoleucine, thiazoline, and dehydroamino acid, was synthesized featuring the β-lactone opening by phenylselenylation, the vinylzinc addition to the chiral sulfinimine, and the Wipf oxazoline-thiazoline conversion method. The segment C (the dihydroquioline portion) was synthesized featuring the modified Reissert-Henze reaction, the homolytic heteroaromatic substitution reaction, the one-pot olefination via the new Matsumura-Boekelheide rearrangement using trifluoromethanesulfonic anhydride and triethylamine, the Katsuki asymmetric epoxidation, the stereoselective addition reaction controlled by the stereocenter of the pen-position, and the regioselective opening of the epoxide function with the L-valine derivertive using catalytic Yb(OTf)_3. The segments D and E (the dehydropeptide portions) was synthesized from two phenylselenoalanines. The consecutive coupling of the segments A, C, and D followed by cyclization between the segments A and D afforded the monocyclic core portion (A-C-D). Finally the total synthesis of siomycin A was achieved by the coupling of this portion and the segment B, followed by the one-pot regioselective cyclization of the resulting coupling product and amidation of the segment E onto the cyclization product.
A new type of furan-iminium cation cyclization was developed and successfully applied to the synthesis of 25, a central core of ircinal A. Compound 25 was efficiently converted to racemic ircinal A, a biogenetic and synthetic precursor of manzamine A. An asymmetric synthesis of ircinal A will also be discussed.
A concise 11-step total synthesis of (-)- and ent-(+)-vindoline (3) is detailed based on a unique tandem intramolecular [4+2]/[3+2] cycloaddition cascade of a 1,3,4-oxadiazole inspired by the natural product structure, in which three rings and four C-C bonds are formed central to the characteristic pentacyclic ring system setting all six stereocenters and introducing essentially all the functionality found in the natural product in a single step. As key elements of the scope and stereochemical features of the reaction were defined, a series of related natural products of increasing complexity were also prepared by total synthesis including both enantiomers of vindorosine (4), minovine (31), 4-desacetoxy-6,7-dihydrovindorosine (32), and 4-desacetoxyvindorosine (48) as well as N-methylaspidospermidine (52). Subsequent extensions of the approach provided both enantiomers of dihydrovindoline (47), 4-desacetoxyvindoline (49), 4-desacetoxy-6,7-dihydrovindoline (50) and 4-desacetoxy-5-desethylvindoline (51).
(+)-Yatakemycin (1), which was isolated from a culture broth of Streptomyces sp. TP-A0356, is an antitumor antibiotic that has a characteristic dienone cyclopropane ring found in duocarmycins and CC-1065. Among them, 1 has been shown to exhibit the most potent activity, and therefore has attracted a great deal of attention. The first total synthesis along with the revision of its structure and determination of the absolute configuration has recently been reported by Boger and co-workers. Herein, we describe an efficient total synthesis of 1 utilizing our copper-mediated amination for the construction of all five aryl-nitrogen bonds, allowing us to conduct a sub-gram-scale preparation of 1 in 16% overall yield over 17 steps (longest linear steps from the known starting compound 6). Synthesis of the left segment 3 commenced with dibromination of 6. Removal of the TFA group, and subsequent oxidation provided dihydroisoquinoline 7, which was readily converted to the cyclization precursor 9. The first amination reaction of 9 afforded indoline 10 with retention of the other bromo group. After conversion to the dehydroamino ester 12, the second amination was performed to provide dihydropyrroloindole 13. The Ns group and benzyl ester in 13 were then converted to Fmoc group and a methanethiol ester, respectively. Finally, an Fmoc-directed, regioselective demethylation was performed with BCl_3 to furnish the left segment 3. Our amination also proved to be highly effective for the construction of the middle segment 4. Cleavage of (S)-epichlorohydrin (18) with 2,6-dibromophenyllithium species 17 provided chlorohydrin 19, which was then converted to amination precursor 21. The crucial aryl amination took place smoothly to give tetrahydroquinoline 22. After Mizoroki-Heck reaction with a dehydroalanine derivative 23 and removal of the nosyl group, bromo group was introduced regioselectively. The second amination reaction at the sterically hindered position was achieved by using a stoichiometric amount of CuI to furnish the middle segment 4. The right-hand segment 5 was also prepared in a straightforward manner by using the aryl amination strategy. Three segments thus obtained were assembled to complete the total synthesis. After coupling of the middle segment 4 with the right segment 5, TBS ether 32 was converted into the mesylate 33. Subsequent hydrolysis provided 34, which was subjected to the condensation conditions with 3 without isolation. Two benzyl groups were then removed with BCl_3, in the presence of excess pentamethylbenzene as a scavenger of benzyl cation. Finally, spirocyclopropanation was carried out according to Boger's conditions to furnish (+)-1, which was identical in all respects to the natural product.
Oxazolomycin and neooxazolomycin are structurally closely related antibiotics isolated from Streptomyces sp. by Uemura et al. The former is the parent member of a class of polyene bicyclic antibiotics, other members being oxazolomycin B and C, 16-methyloxazolomycin, and curromycin A and B. The oxazolomycins were found to exhibit wide ranging and potent antibiotic activity, including inhibitory activity against Gram-positive bacteria, antiviral activity against vaccina, herpes simplex type I and influenza A, as well as in vivo antitumor activity. The intriguing molecular architectures and the biological activities make these compounds attractive targets for synthesis. However, the total synthesis is limited to Kende's synthesis of neooxazolomycin. We report here a novel approach to neooxazolomycin, which can be also applicable to the synthesis of oxazolomycin. Our synthesis of right hand core 22 started with methyl (S)-hydroxyisobutyrate and proceeded through three major transformations involving regio- and stereoselective iodination via intramolecular Pt-catalyzed hydrosilylation, Pd-catalyzed enolate alkenylation, and stereoselective dihydroxylation accompanied by concomitant lactonization. Nozaki-Hiyama-Kishi coupling of aldehyde 23 obtained from 22 with N-Fmoc-iododienamine 24 gave 7S-isomer 25 and 7R-isomer 26 as a 1: 1 epimeric mixture. It was gratifyingly found that Dess-Martin oxidation of this epimeric mixture followed by L-Selectride reduction of the resulting ketone produced the desired 7R-isomer 26 in excellent stereoselectivity (94% de). Removal of the silyl protecting group allowed us to obtain the Kende's intermediate 27, the synthesis of which constitutes a formal synthesis of neooxazolomycin.
Samarium(II) iodide (SmI_2) has been effective in carbon-carbon bond forming reactions and considerable attention has been focused on various intramolecular processes. During the studies on SmI_2-induced cyclization, we found a novel sequential cyclization, reductive cyclization-Dieckmann condensation-lactonization, of keto diesters I, to produce bicyclo[4.n+2.0]alkanones II (n=1, 2) bearing γ-lactone. In order to demonstrate the synthetic utility of this reaction, we choose gibberellin A_1 as a target, which is one of representative gibberellins and has a unique structural feature with eight asymmetric centers. Here we wish to describe a new route for the total synthesis of (±)-gibberellin A_1. Our synthetic strategy involves SmI_2-induced sequential reaction, IV to III, as a crucial step as shown in Scheme 2. Intermediate 17 corresponding to VI was prepared by 7 steps from enone 13, involving stereoselective aldol reaction of the lithium enolate of 13 with aldehyde 12, 1,2-addition of a propagyl group with enone 14 and introduction of methoxycarbonylmethyl group into β-position in α,β-unsaturated ketone 15. The key intermediate 26 was synthesized from 17 as shown in Scheme 3, which involves the construction of D ring using SmI_2-HPMA-induced reductive coupling, stereoselective introduction of hydroxymethyl group into α-position of ester carbonyl in 21, and formation of Z-α,β-unsaturated ester group using Still's method. Treatment of 26 with 3 equivalent of SmI_2 in THE for 10 min at room temperature gave gibbane skeleton 27 with desire stereochemistry in 67% yield (Scheme 4). 27 was easily transformed to (±)-gibberellin A_1 by 4 steps.
The nucleophilic addition of organochromium reagents to aldehydes (Nozaki-Hiyama reaction) is one of the most useful C-C bond-forming reactions, due to its high chemoselectivity and mild conditions. We have developed a new chiral ligand 1 effective for the asymmetric catalysis of Nozaki-Hiyama reactions. Various aldehydes were generally allylated or methallylated with good to excellent enantioselectivity (86-96% ee). Enantioselective synthesis of homopropargylic or allenic alcohols has also been achieved by choosing an appropriate propargyl halide (51-98% ee, 72-83% ee, respectively). Another remarkable feature of this ligand is the stability of the Cr-ligand complex, which was recovered after the reaction and recycled without diminishing the enantioselectivity and yield. We applied this powerful catalytic asymmetric reaction to the total synthesis of FR901512(7), which potently inhibits cholesterol synthesis. Since the stereochemistry of 7 has not been determined, we decided to develop the stereo-divergent synthetic route to all the stereoisomers of 7 to determine the absolute stereochemistry. The catalytic asymmetric Nozaki-Hiyama methallylation of aldehyde 12 with chiral ligand 1b gave alcohol 13 (92% ee), and the following ring-closing methathesis and highly diastereoselective hydrogenation afforded either cis-16 (94% de) or trans-16 (>99% de). Asymmetric allylation of homologated aldehyde 22 proceeded successfully to afford homoallyl alcohol 23 (91% de) again. After conversion of alcohol 23 to α,β-unsaturated lactone 24, diastereoselective epoxidation and reduction completed the asymmetric total synthesis of FR901516(8). Methanolysis and hydrolysis of 8 afforded FR901512(7) with agreement of all spectroscopic properties. Application of the developed catalytic asymmetric Nozaki-Hiyama reaction to the side-chain synthesis of calcitriol lactone will be also reported.
Recently, we have developed a new and rapid lactonization of ω-hydroxycarboxylic acids using symmetric substituted benzoic anhydrides such as 2-methyl-6-nitrobenzoic anhydride (MNBA) as a condensation reagent. This protocol could be performed using a very simple procedure and the desired lactones are obtained within a very short time under mild conditions since the reaction quickly proceeds by the promotion of a catalytic amount of basic catalysts such as DMAP or its N-oxide (DMAPO). In this presentation, we will report an effective method for the synthesis of the several cyclic compounds including natural 8-, 9-, 14- or 29-membered ring lactone moiety using the effective lactonization protocol accelerated by MNBA as part of our continuous efforts for the application of the new synthetic methodology to produce peculiarly structural lactones. I. First, erythronolide A, a 14-membered lactone ring moiety of antibiotic erythromycin A, is prepared from the corresponding seco-acids including a variety of functionalities through the mixed-anhydride method using MNBA combined with basic catalyst. Although the reaction of a substrate protected by benzylidene acetal group with free tert-hydroxyl groups affords the desired lactone in low yield, the substrate protected with MOM group on the tert-hydroxyl groups is converted to the 14-membered lactone in good yield. II. Next, an effective method for the total synthesis of 2-hydroxy-24-oxooctacosanolide, a defensive salivary secretion of the African termite Pseudacanthoterme springer, has been developed. The key lactonization to form a 29-membered ring lactone core is performed using MNBA with a catalytic amount of DMAPO. III. Octalactin A, an anti-tumor agent having an 8-membered lactone moiety, is stereoselectively prepared by means of stereoselective C-C bond forming reactions using asymmetric catalysts. The medium-sized lactone part is effectively constructed by way of the new and rapid mixed-anhydride lactonization using MNBA with a catalytic amount of DMAP or DMAPO. The use of only 5mol% of DMAP or 2mol% of DMAPO rapidly promotes formation of the medium-sized ring of the octalactins indicating the remarkable efficiency of the new lactonization protocol. IV. Finally, the total synthesis ofpseudo-2-epibotcinolide through several featured synthetic approaches is attained. First, a chiral linear precursors of the 9-membered ring compounds is stereoselectively constructed by the combination of the asymmetric aldol strategy for producing β-hydroxy ester units. The key cyclization reaction to form the 9-membered ring lactone moiety is efficiently achieved by the extremely facile and powerful mixed-anhydride method promoted by MNBA with basic promoters.
Aurisides A (1) and B (2) are macrolide glycosides isolated from Japanese sea hare Dolabella auricuralia, which exhibit cytotoxicity against HeLa S_3 cells with IC_<50> values of 0.17 and 1.2μg/mL, respectively. The main structural features of aurisides are a bromine-substituted conjugated diene, a 14-membered lactone, and a cyclic hemiacetal. We achieved the enantioselective synthesis of aurisides A and B by a convergent approach. The C1-C9 segment 4 was prepared from (R)-pantolactone using a reaction of dithiane carbanion with epoxide, stereoselective reduction of β-hydroxyketone with Me_4NBHOAc, and rhodium-catalyzed Reformatsky-type reaction as key steps. The C10-C17 segment 12 was synthesized from (R)-glycidyl trityl ether. The Nozaki-Hiyama-Kishi reaction between 4 and 12 and subsequent reactions including construction of bromine-substituted conjugated diene gave seco acid 10, which was converted into the aglycon (3) of aurisides by construction of the 14-membered lactone. The Mukaiyama glycosylation reaction of the aglycon 3 provided aurisides A (1) and B (2).
Fostriecin (1), a novel secondary metabolite of Streptomyces pulveraceus, is a selective inhibitor of protein phosphatase 2A (PP2A), and displays antitumor activity against a diverse panel of tumor cell lines and in vivo toward lymphoid leukwmias. Because of its important biological activity, fostriecin has attracted many synthetic chemists. Our synthetic strategy is to control the stereocenters using the existing stereogenic centers as much as possible. Our retrosynthetic analysis is as follows: The stereogenic center at C11 would be constructed by 1,3-asymmetric induction using C9 chiral center. The stereochemistry at C5 would be controlled by the chiral center at C8 via 1,4-asymmetric induction. A catalytic asymmetric dihydroxylation of homoallylic alcohol 8 is the only asymmetric reaction using external chiral reagent. Another noteworthy feature is the construction of a labile (Z,Z,E)-triene unit, which would be synthesized by the reduction of the more stable dieneyne at the later stage of the synthesis. In this scenario, one of the most difficult transformations would be the 1,4-chiral induction. We have developed an efficient and highly diastereoselective 1,4-asymmetric induction based on the novel methodology using cobalt-alkyne complex. Using this reaction as a key step, we have accomplished a synthesis of protected dephosphofostriecin 2, a key intermediate in Imanishi's synthesis of fostriecin in a highly stereo-selective manner.
Phoslactomycin B and fostriecin are a unique class of compounds possessing a phosphate group and, in the latte molecule, an amino group. A similarity between these two molecules suggests the same strategy will be applicable to synthesis of these molecules. Recently, we have studied synthesis of fostriecin and established a strategy using chelation-contolled addition of a vinyl anion to an α-PMB-oxy ketone and metathesis for construction of the central part and the lactone moiety of fostriecin, respectively (Org. Lett. 2002, 4, 4615). We then applied the strategy to synthesis of phoslactomycin B. Chelation controlled addition proceeded efficiently, while the lactone formation by metathesis proceeded little. Furthermore, diimine reduction of the iodo-acetylene gave a mixture of the desired product, the over-reduction product, and the recovered compound. We then studied another construction of the lactone ring through Evans aldol reaction followed by Wittig reaction and lactonization, which produced the lactone efficiently. On the other hand, Sonogashira coupling of the acetylene end with 2-cyclohexyl-1-iodoethene took place cleanly to produce the conjugated enyne efficiently. Latter, this moiety was reduced to the conjugated diene with activated Zinc. The amino group was introduced as (allyl-OCO)_2N and the phosphonate group was introduced as (allyl-O)_2P(O) group. In the last stage, these groups were removed by using Bu_3SnH and Pd catalyst to afford phoslactomycin B (Angew. Chem. Int. Ed. 2006, 45, 3320). In a similar way, deamino phoslactomycin B, a proposed intermediate in the biosynthesis of phoslactonycin B, was synthesized efficiently.
Yessotoxin (YTX, 1), which belongs to the ladder-shaped polyether family, has been isolated from the digestive glands of the scallops in association with diarrhatic shellfish poisoning. The toxin turned out later to be produced by dinoflagelate Pritocaratium sp., and further transferred and accumulated through the food chain. YTX is recently reported to be a modulator of cytosolic calcium levels of human lymphocytes and a caspase activator. Its broad spectrum of biological activities and unique arched molecular structure have therefore attracted the attention of synthetic chemists. During the course of our synthetic studies, we have developed an efficient method for the convergent synthesis of polycyclic ethers via α-cyano ethers, which was successfully applied to the synthesis of the FGHIJ-ring fragment (18) of YTX. Unification of the F-ring diol (6) and IJ-ring aldehyde (7) through acetal formation, followed by the regioselective cleavage of the acetal afforded the α-cyanoether (9). Construction of the GH ring was achieved by using ring-closing metathesis and reductive etherification. A novel method for synthesizing the ABC-ring fragment (30) of YTX has been also developed. Coupling of the A-ring triflate (19) and 2-lithiofuran (20) followed by oxidative ring expansion of the furan gave the six membered-ring enone (22). Construction of the BC ring system was achieved by means of intramolecular oxy-Michael addition and reductive etherification. Furthermore, construction of the K-ring was achieved by enyne cross metathesis reaction of alkyne (33) with β-methallylalcohol, and subsequent 6-exo cyclization of the vinylepoxide (35) with CSA. Further studies directed towards total synthesis of YTX based on our convergent strategy will be also reported.
Brevenal is a polyether natural product isolated from laboratory cultures of the Florida red tide-forming dinoflagellate, Karenia Brevis. Its gross structure, including relative stereochemistry, was disclosed based on extensive 2D NMR studies. It has been reported that brevenal competitively displaces tritiated dihydrobrevetoxin from voltage-sensitive Na^+ channels and acts as a natural brevetoxin antagonist in vivo. More importantly, brevenal improved tracheal mucus velocity in picomolar concentrations in an animal model of asthma. Thus, it may be a source of novel therapeutic agents for treatment of cystic fibrosis and other respiratory disorders such as asthma and chronic obstructive pulmonary disease (COPD). Intrigued by these biological profiles of brevenal, we embarked on the total synthesis of brevenal by means of our developed convergent methodology. Suzuki-Miyaura coupling of the AB ring enol phosphate 6 and an alkylborane derived from the DE ring exo-olefin 7 proceeded smoothly to afford coupling product 23. The C ring was then constructed by mixed-thioacetalization followed by one-pot oxidation/methylation. After construction of the left-hand (E,E)-diene side chain by means of CuTC-mediated Stille coupling, the total synthesis was completed by installing the right-hand (Z)-diene chain via Wittig reaction. However, ^1H and ^<13>C NMR spectra of synthetic 1 were not identical with those of the natural product. Detailed NMR studies suggested that the true structure of brevenal is the C26-epimer of the originally proposed structure. This notion is also supported by the postulated biosynthetic pathway of marine polyether natural products. In the event, this proved to be correct as the spectroscopic data of synthetic 2 were completely identical with those of the natural product. In addition, the optical rotation of synthetic 2 matched the value for the natural product. Thus, we succeeded in unambiguous determination of the absolute configuration of brevenal through our total synthesis endeavor.
With their imposing molecular structure, the ladder-shaped polyethers including ciguatoxins pose considerable challenge to synthetic chemists. Ciguatoxins are isolated as the causative toxins of ciguatera seafood poisoning. Previously, we reported the total synthesis of CTX3C (2) and 51-hydroxyCTX3C (3), two representative congeners of ciguatoxins. In this presentation, we report the first total synthesis of CTX1B (1), which has one of the most complex structures among ciguatoxins. The SAR study of synthetic ciguatoxins is also discussed. Recently, we have developed two methods to synthesize the polyether compounds based on forming a O,S-acetal from a secondary alcohol and a halosulfide. The O,S-acetal was used as radical donor to cyclize the seven-memberd ether ring with an acrylate. Alternatively, the enol ether that was prepared from O,S-acetal was utilized as a radical acceptor to form ether ring. By using these two free radical approaches, CTX1B (1) was assembled in a concise and efficient fashion. To elucidate the biological mechanism of ciguatoxins, we performed the SAR study of the synthetic intermediates of CTX3C (2) and 51-hydroxyCTX3C (3). The SAR study clearly indicated that the semi-rigidity of the central portion of ciguatoxins was extremely important for its potent biological activity.
Acaterin (1), isolated from the culture broth of Pseudomonas sp. A92 by Endo's group, is a γ-lactone having acyl-CoA: cholesterol acyl transferase inhibitory activity. Dehydroacaterin (2), isolated from the same strain by changing culture medium, is the immediate biosynthetic precursor of 1. In this presentation, we describe the mechanism of the conversion of 2 to 1 catalyzed by "dehydroacaterin reductase," and purification and gene cloning of this enzyme. Incubation studies of 2 in the presence of D_2O or NADPD with a cell-free extract (20,000g sup) prepared from the Pseudomonas sp. established that the hydrogen atoms from NADPH and water are incorporated into the C-5 and C-4 positions, respectively. Further, incubation studies of 2 with a partially purified enzyme in the presence of [4-pro-R-^2H]NADPH or [4-pro-S-^2H]NADPH revealed that pro-R hydrogen at C-4 of NADPH (A-specific) is used stereospecifically in this reduction. The transfer of 4-pro-R-H of NADPH was found to be proceeded via a flavine cofactor. Dehydroacaterin reductase was purified in a five-step sequence (ammonium sulfate precipitation and ion-exchange, NADPH-affinity, gel-filtration and hydroxyapatite chromatographies). Dehydroacaterin reductase gene was successfully cloned and sequenced by using N-terminal and internal amino acids sequences of the enzyme. The gene was heterologously expressed in Escherichia coli. This enzyme was shown to be a flavoprotein by UV-Vis spectrum of the recombinant protein.
The anatomical distribution of tetrodotoxin (TTX), saxitoxin (STX) and their analogs (TTXs, STXs) in three female and three male specimens of the marine puffer fish Fugu pardalis from Miyagi Prefecture, 2005, Japan, were studied. 5-DeoxyTTX, 11-deoxyTTX, and 5,6,11-trideoxyTTX were quantified by liquid chromatography/mass spectrometry (LC/MS) for the first time, and other TTXs and STXs were determined by liquid chromatography-fluorescent detection (LC-FLD). As a result, 5,6,11-tridoexyTTX was found to be the major TTX analog in all tissues tested, whereas 5-deoxyTTX and 11-deoxyTTX were minor components. Especially, in female (n=3), the ratios of 5,6,11-trideoxyTTX to total of all TTX analogs (mol/mol) in ovaries (mean±SD, 0.42±0.055) were significantly larger than those in livers (0.17±0.025)(P<0.05). In contrary, the ratios of 4,9-anhydroTTX to total of all TTX analogs in livers (0.27±0.047) were significantly larger than those in ovaries (0.073±0.040)(P<0.01). In male (n=3), all these ratios were not significantly different between livers and testis. 4-S-CysteinylTTX was detected in liver, spleen, gall, and intestine in 1-6 mol% of total of all TTX analogs, supporting our previous hypothesis that 4-S-cysteinylTTX was a metabolite of TTX. In addition, 6,11-dideoxyTTX was isolated from ovary of F. pardalis.
Since nuclear export of Rev protein is essential for HIV proliferation, we have been engaged in search for inhibitors for nuclear export of Rev to disclose new anti-HIV seeds. Consequently, 1'-acetoxychavicol acetate (ACA, 1) was isolated as an active principle by using fission yeast expressing Rev-NES-contained protein and confirmed to inhibit proliferation of HIV-1 virus. Furthermore, the mechanism of action of 1 was revealed to bind to the cystein-529 in CRM1, the receptor of nuclear export signal (NES) in Rev, by the competitive experiment in the presence of the biotinylated leptomycin B probe (2). Reactants of 1 and N-acetyl-L-cystein methyl ester established 1-acetoxy-2-ene moiety as the binding site of cystein-529 in CRM1. Analysis for structure activity relationship by synthetic analogs disclosed the hydrolysis of functional group at 4-OH in 1 to be essential for bioactivity of 1. Based on the plausible mechanism of action of 1 wih Cys-529 in CRM1, activation energy to crucial transition state by molecular orbital calculation was found to be correlated with nuclear export inhibitory activity of ACA analogs. Halogenated ACA analogs designed from this calculation showed more potent activity than 1. On the other hand, homology modeling of hCRM1 by folding recognition method was performed. Interaction energies between ACA analogs and hCRM1 were shown to be correlated with bioactivity of ACA analogs. Functional groups at 1'-C positions of ACA analogs were assumed to affect interaction energy with hCRM1 by docking study. ACA analogs possessing 1'-O-acyl or 1'-O-carbamoyl functions exhibited lower interaction energy and more potent activity than 1. Furthermore, we developed assay systems to evaluate inhibitory activity for nuclear export of Rev in HeLa cells. Synthetic ACA analogs based on these rational designs showed more potent activity than 1 in HeLa cells as well as in yeast system.