Leguminous plants close their leaves in the evening, as if to sleep, and open them early in the morning according to circadian rhythm controlled by biological clock. Our studies on the circadian leaf-movement, called nyctinasty, led to discovery the leaf-opening and leaf-closing substances that control nyctinasty. We revealed that some receptor for the leaf-opening subustance of Cassia mimosoides L., potassiumu lespedezate (1, bioactivity: 1×10^<-6> M), is localized in motor cells by using fluorescent probe (2), and then we synthesized 2 type photoaffinity probes based on 1 to identify the receptors. One is the probe 3 (bioactivity: 5×10^5 M) induced photoaffinity group and biotin unit into 6'-position of the glycon moiety, which remained high bioactivity because of bulky photoaffinity unit away from active aglycon moiety. The others are 4 (bioactivity: 8×10^<-5> M) and 5 (bioactivity: 1×10^<-4> M) induced photoaffinity unit into 2'-position of the glycon moiety, which are expected high labeling yield because of adjacent photoaffinity group to aglycon moiety. Then we collected large amount of motor cells by cutting sections of plant pulvini containing motor cells under the stereoscopic microscope. Successive homogenization and centrifugations gave the membrane fraction. After being added 4 and irradiated UV-lamp (365 nm), the membrane fraction was analyzed by SDS-PAGE, transferred to PVDF membrane and detected by chemiluminescence. In the result of it, we detected specific bands at 210, 180 kDa which disappeared under the condition of presence of 10000-fold molar excess non-labeled leaf-opening substance. Next, we examined the localization of these specific bands of proteins. The photolabeling experiment was carried out for the membrane fraction prepared from cutting sections of plant leave, but these bands were not found.
Two ^<13>C-labeled amphotericin Bs (AmBs) were prepared by biosynthetic methods. We attained the high level incorporation of [u-^<13>C_6]glucose into AmB resulting uniformly ^<13>C labeled [u-^<13>C]AmB (ca. 50% enriched). The site-selective ^<13>C labeled [tri-^<13>C]AmB was prepared by feeding with sodium [3-^<13>C]propionate feeding (ca. 15% enriched). The ^<13>C-^<13>C correlation spectrum of [u-^<13>C]AmB/dilauroylphosphatidylcholine (DLPC) allowed us to assign all ^<13>C signals on AmB in membrane. The ^<13>C-^<31>P REDOR experiments for dimyristoylphosphatidylcholine (DMPC) membrane using [tri-^<13>C]AmB showed the prominent dephasing effects between the phosphate group in PC and C41 carboxyl carbon in the polar head of AmB. In addition, C39/C40 methyl carbons also gave rise to the significant reduction of their ^<13>C NMR peak, implying that the both terminal parts of AmB reside close to the surface of DMPC membrane. Conversely, the same REDOR experiments with use of distearoylphosphatidylcholine (DSPC) showed no dephasing for the C39/C40 methyl signals while a marked reduction of the C41 carbonyl was again observed. These observations should be most reasonably accounted for by the notion that AmB can span across the DMPC membrane with a single-length interaction but cannot across the DSPC membrane due to its greater thickness. These results may provide the first direct spectroscopic evidence for the formation of a single-length channel across biomembrane, which was previously suggested by channel current recording experiments.
Mycophenolic acid (MPA) (1), known as an inhibitor of inosine monophosphate dehydrogenase (IMPDH) was found to inhibit the differentiation of 3T3-L1 pre-adipocytes1) into mature adipocytes. Inhibition of IMPDH could be abrogated by the addition of guanosine or guanosine derivatives in the case of 1 as an inducer of differentiation in erythroleukemia cell lines such as HL-60 or K562 cells.5),6),7) However, the inhibitory activity of 1 did not affect by the addition of guanosine or any other derivatives, XMP, GMP and GTP. This finding suggested that 1 inhibits 3T3-L1 cell differentiation via the molecular target except IMPDH. To confirm this possibility, we performed the structure-activity relationship study synthesizing various analogs of MPA (Fig. 1). The SAR study suggested also that the target molecule of 1 is not IMPDH8) but a different specific factor. We therefore focused our attention on the issue if MPA inhibits the function of peroxisome proliferatior-activated receptor gamma (PPARγ), one of the key nuclear receptor on adipocyte differentiation. Mycophenolic acid (1) was found to inhibit the function of PPARγ activated by pioglitazon under the reporter assay conditions (Fig. 2). The 1:1 specific interaction between MPA and immobilized PPARγ was detected based on surface plasmon resonance (SPR) as shown in Fig. 3. The results indicated that MPA is at least a partial antagonist of PPARγ. The alcohol analog of MPA (6) was found to be an inhibitor having a higher affinity toward PPARγ. We next focused our attention on design and synthesis of HDAC (histone deacetylase) inhibitor. Hydroxamic acid analog of MPA, namely MPAHA (13) was successfully found to be an inhibitor of HDAC, whereas 1 did not show inhibition of HDAC (Fig. 4). In conclusion, IMPDH is not the only molecular target8),9) toward MPA (1), and 1 could be an attractive leading compound to develop useful bioprobes or medicines having molecular targets except IMPDH.
Conventional and novel protein kinase C (PKC) isozymes are activated by binding of tumor promoters to their C1 domains (C1A, C1B). Recent investigations revealed that novel PKC isozymes (δ, ε, η) are involved in tumor promotion in vivo and their C1B domains play dominant roles on their activation by tumor promoters. Design of new agents with high selectivity for novel PKC C1B domains is indispensable to elucidate the precise mechanism of tumor promotion. 1-Hexyl-indolactam-V (1) was selected as a lead compound for such agents since 1 showed a binding preference for the C1B domains of novel PKC isozymes. Compound 1 existed as two stable conformers due to the cis-trans isomerization of the lactam amide function. Conversion of the indole ring of 1 to the indoline ring achieved the conformational fixation of 1. Highly improved selectivity for novel PKC C1B domains was observed in the trans-amide analogue (3), while the binding selectivity of the cis-amide analogue (2) was almost similar to that of 1. The docking simulation revealed that the binding mode of 3 with the PKCδ C1B domain is quite different from those of 1 and 2. Detailed analysis of the CH/π interaction using a PKCδ C1B mutant, in which Pro-11 was substituted for 4,4-difluoro-Pro, indicated that the CH/π interaction between the hydrogen atom at position 4 of Pro-11 and the benzene ring of 3 is critical for the binding of 3 to the PKCδ C1B domain. Based on these results, the conformationally analogous compound of 3 containing an indole ring (4), which would form effectively the CH/π interaction with Pro-11 of the PKCδ C1B domain, was designed. Compound 4 showed significant binding affinity and selectivity for the C1B domains of all novel PKC isozymes, especially for PKCδ and θ. Since PKCδ has a tumor suppresser role, 4 might be an antitumor agent as well as an analytical agent of tumor promotion.
Histone deacetylases (HDACs) and histone acetyltransferases (HATs) regulate the acetylation of histones and are involved in the transcriptional activity of certain genes. HDAC inhibitors, such as tricostatin A and trapoxin A, are known to cause cell cycle arrest, differentiation or apoptosis of tumor cells, and a p53-independent induction of the cyclin dependent kinase inhibitor, p21. In addition to the effects on tumor, antiangiogenic effects by HDAC inhibitors are reported, recently. Therefore, HDAC inhibitors are expected as potential agents for the treatment of cancer. In our screening for HDAC inhibitors from Japanese marine invertebrates, we encountered the marine sponge Mycale izuensis. collected in the Amakusa Islands whose aqueous extract was highly active. Bioassay-guided fractionation led to the isolation of five active compounds, azumamides A-E. The molecular formula of azumamide A (1) was determined to be C_<27>H_<39>N_5O_5 on the basis of HR-FABMS and NMR data. Interpretation of 2D NMR spectra indicated that the structure of 1 was composed of Ala, Val, and Phe, as well as an unprecedented novel amino acid, 3-amino-2-methyl-5-nonenedioic acid 9-amide (Amnaa). The absolute stereochemistry of Ala, Val, and Phe was determined by Marfey analysis to be all D. Stereochemistry of Amnaa was determined as 2S, 3R by comparing ^1H NMR chemical shifts of the MTPA amide derivative with those of the model compounds. The structures of azumamides B-E (2-5) were also determined following the same procedure as in the case of 1. Azumamides A-E (1-5) inhibited HDAC with IC_50 values ranging 23-617 ng/mL. Furthermore, azumamide A showed inhibitory activity against HDAC expressed in K562 cells as well as cytostatic effects on WiDr (human colon cancer cell) and K562 (human leukemia cell) cells with IC_50 values of 5.8 and 4.5 mM, respectively.
Lipid droplets accumulation in macrophages is one of the central stages of foam cells formation leading to the development of atherosclerosis in the arterial wall. Inhibition of lipid droplets accumulation in macrophages may control atherosclerogenesis. Beauveriolide III (1), isolated from the culture broth of Beauveria sp. FO-6979, is a 13 membered cyclic depsipeptide and leads to reduction of the number and size of lipid droplets in macrophages without any cytotoxic effect on macrophages. Its in vivo efficacy was shown in LDL receptor knockout mice, reducing the atherogenic lesion in aortas and hearts. Although 1 was expected as a potential lead compound for the treatment and the prevention of atherosclerosis, its structure-activity relationships have not been clearly elucidated. Herein we report an efficient route to synthesize a library of beauveriolide analogues using solid support and their biological evaluation Our synthetic strategy involves solid phase assembly of linear depsipeptide followed by solution phase macrolactamization as shown in Scheme 4. Loading of Fmoc alanine A onto 2-chlorotrityl chloride resin 23, condensation with Fmoc phenylanine B followed by coupling with ester unit C using PyBrop gave 25. Deprotection and cleavage from the resin under acid condition released linear depsipeptide 22 in 93% purity and 98% crude yield. Finally, the cyclization of 22 with EDCI and i-Pr_2NEt under high dilution condition was attained to provide beauveriolide III (1) in 51% yield. Based on this strategy, a library synthesis of beauveriolide analogues composing building blocks A, B and C was achieved by split & pool method using AccuTag^<TM> System yielding 202 member beauveriolide analogues. Inhibitory activity of the beauveriolide analogues against CE synthesis by macrophages was evaluated (Figure 2). Linear depsipeptides including NBV113 were found to spoil activity. It appears that the ring construction plays a crucial role in inhibitory of CE synthesis. The S configuration of 3-hydroxy ester moiety is important to maintain the activity. It is interesting to note that NBV248 having 4-chlorophenylalanine is 5 times as potent as beauveriolide III (1).
Tuberculosis (TB) is still the greatest single infectious cause of mortality in the world. Moreover, the spread of RIV promotes the increase in the number of tuberculosis patients. However, efficient anti-TB drugs with new mechanism of action have not been developed for last over thirty years; only 5 agents have been used as first-line anti-TB drugs. Isoniazid and ethambutol showing specific inhibition against mycobacterial growth are known to inhibit the cell wall biosynthesis. From the whole genome sequence of Mycobacterium tuberculosis, a number of enzymes are involved in the cell wall biosynthesis, which is a promising target for development of new anti-TB drugs. We have screened for antimycobacterial agents from microbial metabolites having a specific narrow-range spectrum against mycobacteria. As a part of this program, the culture broth of Rhodococcus sp. K01-B0171 isolated from the inside of the soil aggregates was selected (Fig. 2). As a result, unique cyclic peptides designated lariatins A and B were purified from supernatant of the 4 day-old culture broth of this strain by HP-20, ODS and LH-20 column chromatography and reversed-phase HPLC. The structures were elucidated by spectral analysis and advanced protein chemical methods. These peptides consist of 18 and 20 L-amino acid residues with an internal linkage between the α-amino group of ^1Gly and the γ-carboxyl group of ^8Glu. To our knowledge, the compound having the linkage is only reported for microcin J25, whose structure was recently revised from the head-to-tail cyclic structure. The three-dimensional structure of lariatin A is estimated 'lasso' structure from NMR data; the tail segment (W9-P18) passes through the ring segment (G1-E8). Lariatins A and B showed strong inhibition only against M. smegmatis among 14 microorganisms tested on the paper disk assay. The MIC values of lariatins A and B were 3.13 and 6.25 μg/mL for M. smegmstis Takeo, respectively. Lariatin A inhibited the growth of M. tuberculosis (Table 3).
Batzelladines A-E, members of a novel class of guanidine alkaloids containing a tricyclic guanidine subunit, were isolated from Caribbean sponge by scientists at SmithKline Beecham in 1995. Later in 1997, four other metabolites, termed batzelladines F-I, were also obtained from the similar source. Batzelladines A (1), B and D (2) were reported to inhibit the binding of the HIV glycoprotein gp120 to the human CD4 receptor on T cells, while batzelladines F-I induce the dissociation of the protein kinase p56^<lck> from CD4. The unique structures of these guanidine alkaloids and their important biological activities resulting from modulation of protein-protein interaction have inspired considerable synthetic attention. We describe the enantioselective total synthesis of batzelladine A (1) and D (2). Our synthesis features 1) stereoselective construction of the cyclic guanidine system via successive 1,3-dipolar cycloaddition reactions and subsequent cyclizations, 2) one-step formation of the α,β-unsaturated aldehyde 25 from the primary alcohol 19 with TPAP-NMO, providing an efficient route to the lefthand bicyclic guanidine alcohol 18 and 3) direct esterification of the bicyclic carboxylic acid 17 with the guanidine alcohol 18 to construct the whole carbon skeleton of batzelladine alkaloids. The synthetic route we have developed is very straightforward and should be flexible enough to prepare variety of batzelladines and their derivatives. To elucidate the binding inhibition mechanism of batzelladine alkaloids, affinity gel-assisted HPLC analyses were examined. As a result, batzelladines were realized to bind to CD4 selectively.
Tyrosine specific kinases involving cell signal transduction pathway play a crucial role in cell proliferation or differentiation, and are important target molecules for cancer chemotherapy. We found that Microbispora rosea subsp. hibaria isolated form soil sample produced v-src tyrosin kinase inhibitor, and the active principles were named hibarimicins. Hibarimicins were extracted from fermented broth of the producing microorganism, and purified by Diaion HP20, ODS-silica gel column chromatography, ODS- TLC and HPLC. Hibarimicins were determined to be glycosides of octacyclic aglycons by spectroscopic analyses. Combination of three kinds of aglycon (R, P, Y) and four kinds of deoxy sugars gives more than ten species of hibarimicins. Asymmetric carbons were located in both ends of the aglycon and the carbohydrate moiety, and their absolute configurations were determined by improved Mosher's method. Absolute configuration of axial chirality at C2-C2' bond was determined to be Sa for hibarimicin A〜K by circular dichroism exciton chirality method. Feeding experiments with labeled acetate indicated that hibarimicin aglycon was biosynthesized through polyketide pathway, dimerization and glycosylation. Hibarimicins selectively inhibited tyrosin kinase in multiple protein kinase assay system using v-src transformed NIH3T3 cells.
In the course of the studies investigating key substances in the mycoparasitism between Lambertella and Monilinia, we isolated lambertellols A (1) and B (2), novel naphthoquinones carrying a spiro-butenolide in the molecules. The structures of them were elucidated by detailed NMR studies involving NOE experiments. Chemical derivatization into dibenzoate 6 and the following CD spectral measurement as well as conformational search deduced the absolute stereochemistry of 2 as (3R,4S). The absolute stereochemistry of 1 was estimated to be (3R,4S) by mechanistic analysis for the isomerization between 1 and 2. Labeling experiments disclosed their biosynthetic pathways. We achieved to introduce the labeled acetates as much as 40% incorporation rate by reducing the time for the cultivation, which realized to provide 2D-INADEQUATE spectra of both 1 and 2 in good quality employing sub-milligram of the samples. This high incorporation also made us allow isolating labile lambertellol C (12) by taking advantage of characteristic signal pattern of molecular ion peaks due to existence of many isotopomers in LC-MS. Our studies revealed that L. sp. 1346 produces lambertellols very much, while L. corni-maris makes quite small amount of them under simple cultivation conditions in PS and PSA media. Interestingly, addition of the culture broth of Monilinia remarkably accelerated the production of lambertellols. Our biological investigations revealed that both 1 and 2 inhibit hyphal germination of Monilinia fructicola, one of the hosts of L. corni-maris. These studies suggested that lambertellols can be substances responsible to the mycoparasitism and lambertellols play offensively at the stage invading into the host.
Ulva and Enteromorpha are two well-known, green marine algal genera. It has been well known that these green macroalgae lose their natural morphology during short-term cultivation under aseptic conditions, or during long-term cultivation in nutrient-added seawater. These phenomena led to the belief that "undefined morphogenetic factors" that were indispensable to the foliaceous morphology of macroalgae existed throughout the oceans. We have previously reported that many bacteria belonging to the Cytophaga-Flavobacterium-Bacteroides group have differentiation-inducing activity against germ-free Monostroma oxyspermum. Moreover, culture supernatants of these bacteria also showed inducing activity in the normal germination of germ-free spores of some Ulva and Enteromorpha plants. We report here for the first time a novel and an intensive algal differentiation inducer named "Thallusin" that was purified from an epiphytic marine bacterium YM2-23 isolated from a green alga. The structure of Thallusin was determined by NMR, MS and X ray diffraction analysis of its derivatives. Thallusin was thought to be a new essential vitamin-like compound of algae and showed extremely strong activity to induce the normal germination of Ulva and Enteromorpha under the germ-free conditions.
Marine organisms produce various molecules with remarkable physiological activities. In particular, huge polyol and polyether compounds, such as palytoxin, and halichondrin, are some of the most attractive molecules in natural products chemistry. These compounds are composed of a long carbon backbone functionalized by oxygen atom, and have been called "super-carbon-chain compounds". The symbiotic marine dinoflagellate Symbiodinium sp., which is a type of zooxanthellae, is found in a wide range of marine invertebrates. In our continuing search for biologically active compounds, we have isolated symbiodinolide (1), a novel super-carbon-chain compound, and an amphoteric iminium metabolite, symbioimine (2) from the dinoflagellate culture, isolated from the marine acoel flatworm Amphiscolops sp. The molecular weight of symbiodinolide (1) was deduced to be 2,859 by ESI-TOF MS. 2D-NMR (COSY, HOHAHA, HSQC, HMBC) analysis of seven partial structures including 99 carbon atoms suggested that 1 was a congener of zooxanthellatoxins (ZTXs), vasoconstrictive marine macrolide. Comparison of the NMR data between 1 and ZTXs allowed to show planar structure of 1. Structure of symbioimine (C_<19>H_<23>NO_6S) was deduced by spectroscopic analysis and X-ray crystallographic analysis. This compound has a characteristic 6,6,6-tricyclic iminium ring structure and an aryl sulfate moiety. Symbioimine (2) inhibited the differentiation of RAW264 cells into osteoclasts (EC_50=44μM), whereas its cell viability was not effected. Thus, 2 is an antiresorptive drug candidate for the prevention and treatment of osteoporosis in postmenopausal women. Studies on the isolation, structure elucidation, and biological activities of these compounds will be presented.
Arabidopsis thaliana that is well known as a model plant by taking advantage of its molecular biological background, while bioactive substances of A. thaliana have been hardly reported except for some examples of plant hormones. So, we demonstrated search for bioactive substances from A. thaliana. As a result, four new glycolipids, named arabidopsides A (1), B (2), C (3), and D (4), were isolated from the aerial parts of this plant, together with a monogalacotsyl diacylglyceride (5). The structures of 1〜4 were elucidated by HRESIMS, 2D NMR, enzymatic reaction, and chemical means. Arabidopsides A〜D (1〜4) were rare monogalactosyl and digalactosyl diacylglycerides containing 12-oxophytodienoic acid (OPDA) and/or dinor-oxophytodienoic acid (dn-OPDA). OPDA and dn-OPDA are biosynthetic precursors of jasmonic acid (JA), so it suggests that arabidopsides A〜D (1〜4) are related to JA biosynthesis pathway. Compound 1 exhibited inhibitory effect on the growth of the root of cress (Lepidium sativum) at 5×10^<-5>M. On the other hand, compound 5 induced flower bud formation of A. thaliana exposed to long day condition for only 1 day, which the decrease of a HPLC peak corresponded with compound 5 was detected in the extract of flower buds-forming A. thaliana comparing with that of non-flower buds-forming ones. These results suggest that compound 5 as the precursor or the substrate of flower bud-forming substance(s) plays important roles in the flower bud formation in A. thaliana.
Daphniphyllum alkaloids with unique heterocyclic ring systems are of current interest from a biogenetic point of view or as challenging targets for total synthesis. Recently, many Daphniphyllum alkaloids indicating the unique biogenetic path have been isolated. In our project on a search for biogenetic intermediates of Daphniphyllum alkaloids, a series of daphnicyclidins, daphmanidins, daphniglaucins, and calyciphyllines were isolated from the leaves, stems, and fruits of Daphniphyllum humile, D. teijsmanni, D. glaucescense, and D. calycinnum, together with a series of daphnezomines. The structures of these new alkaloids were elucidated on the basis of extensive 2D NMR techniques, X-ray analysis, CD spectra, and chemical derivatization. Daphnicyclidins A - H (1-8), J (9), and K (10) are novel types of alkaloids consisting of fused hexa- or penta-cyclic ring system, respectively. The biogenetic origin of daphnicyclidins seems to be yuzurimine-type alkaloids such as yuzurimine A and macrodaphniphyllamine with an appropriate leaving group at C-4. From biogenetic points of view, cleavage of C-1 - N-1 bond of daphniglaucin A (13) will give the skeleton of daphniglaucin D (16). Daphmanidins A (11) and B (12) might be generated from a common imine intermediate A. On the other hand, the biogenetic origin of daphniglaucin C (15) and calyciphylline B (18) seems to be an imine intermediate C, which might be produced through fragmentation reaction of the secodaphniphylline-type skeleton (B) derived from an imine intermediate A. Biogenetically, cleavage of C-6 - C-7 bond of an intermediate D will give the skeleton of daphniglaucin C (15). Calyciphylline B (18) might be generated from attack of the carboxyl group to C-5 of the intermediate C and cleavage of C-4 - C-5 and C-8 - C-9 bond followed by C-7 - C-9 bond formation. There are currently more than sixty Daphniphyllum alkaloids. This phytochemical developments in the structures of the new alkaloid types, such as the daphnezomines, the daphnicyclidins, the daphmanidins, the daphniglaucins, and the calyciphyllines will bring increasing structural variation to this alkaloid group. These unusual ring systems have attracted great interest as challenging targets for total synthesis or biosynthetic studies. Diversity of the ring skeletons in Daphniphyllum alkaloids might be related to the diversity of the biosynthetic pathways and the relevant enzymes. Widespread efforts for understanding the properties of these complex and fascinating alkaloids will result in further developments in this field.
Recently, our interests focus on the synthesis and the bio-screening of the natural-originated compounds in enantio-pure form on the basis of the new developed synthetic methods in this group. In this presentation, the synthesis and evaluation of the biologically active compounds are illustrated by the on-going subjects. For example, in light of our recent discovery of Ni(0) catalyzed homo-coupling and cross-coupling reactions, together with consulting the therapeutic information of Chinese traditional medicine from plants, the screenings of the compounds obtained by the above mentioned reactions have been undertaken. The preliminary results showed that the phthalide-type compounds showed good neuroprotection activities, and the coumarinyl derivatives exhibited excellent anti-fungal effects. The second topic is on the GABA neurotransmitter. The GABA reuptake is effected by selective transporter called GAT-1. The reduction of GABAergic neuronal activity plays an important role in a number of neurological disorders, including epilepsy, auxiety and pain. Several designed compounds bearing with (R)-nipecotic acid moiety were synthesized and found very active in inhibiting GABA reuptake showing the possible usage for the analgesic chemotherapeutic treatment.
The development of carbon-carbon (C-C) bond formation allows chemists to build various molecules with excellent yield and high selectivity under mild conditions. However no one has accomplished a novel C-C bond formation to the desired site of proteins without losses of their tertiary structures and innate properties. In this contribution, we have succeeded in the first site-specific introduction of a new functional group into the Ras protein using Mizoroki-Heck reaction. We firstly incorporated, 4-iodophenylalanine, a chemoselective tag to Mizoroki-Heck reaction into the nonsense codon of c-Ha-ras by using in vitro protein synthesis. Site-specific incorporation was determined by both LC/MS and LC/MS/MS analyses. Secondly, we optimized reaction conditions for the post-translational modification of proteins under physiological conditions. Glu-4-iodoPhe-Thr tripeptide as a model of Ras carrying 4-iodophenylalanine (Iphe-Ras) was used for this optimization experiments. The coupling reaction has been completely done at 5℃ within 93 hour. In addition, Ras was not decomposed under the same conditions, and still retained its binding activity against the Ras-binding domain of Raf-1 (Raf-1-RBD). Then, Iphe-Ras was subjected to Mizoroki-Heck reaction with vinylated biotin at 5℃ for 50 hour. The covalent attachment of biotin to Ras was detected with Western blotting and the undisturbed activity of Ras after biotinylation was demonstrated with in vitro binding assays. This is the first report that a new C-C bond has been introduced into the target sites of proteins. It is reported that small peptides are chemoselectively modified in aqueous media by using organometallic catalysts. However, full preparation is not committed for preventing potential degradation and retention of fragile tertiary structures when proteins are used as a substrate in organometallic reactions. We have shown that Mizoroki-Heck reaction is benign enough to maintain activities of proteins. Our developed methodology enables us to take more advantage of intelligent materials or devices mimicking subtle biological processes near future.
Plants produce isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) as the common C_5-isoprenoid units through the plastidic methylerythritol phosphate (MEP) pathway and the cytosolic mevalonate (MVA) pathway. To assess which IPP/DMAPP pathways contribute for the biosynthesis of gibberellins (GAs), metabolites from each isoprenoid pathway were selectively labeled with <13>^C in Arabidopsis seedlings. Efficient <13>^C-labeling was achieved by blocking the endogenous pathway genetically or chemically during the feed of a <13>^C-labeled precursor specific to the MEP or MVA pathways. GC-MS analysis demonstrated that GAs are predominantly synthesized through the MEP pathway but also the MVA pathway partially contributed to GA biosynthesis in plants. Using our in vivo isoprenoid labeling system, the early stage of cytokinin (CK) biosynthesis in plants was next studied. LC-MS analysis demonstrated that the prenyl group of trans-zeatin (tZ) and isopentenyladenine mainly originates from the MEP pathway, whereas that of cis-zeatin is primarily biosynthesized through the MVA pathway. Plastid localization of four adenosine phosphates-isopentenyltransferases in Arabidopsis cells supported the idea that the MEP pathway primarily provides DMAPP to trans-zeatin and isopentenyladenine. To distinguish origins of the prenyl group of tZ-type CKs in TMR (Agrobacterial isopentenyltransferase)-introduced plants, we have prepared [3,4-<18>^O_2]1-deoxy-D-xylulose, which is incorporated into the MEP pathway to give [1-<18>^O]1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (a hydroxylated derivative of DMAPP and a possible direct precursor of prenyl group of tZ riboside monophosphate). We will present new findings from labeling experiments in Arabidopsis seedlings and Catharanthus cell cultures using this <18>^O-labeled precursor.
Alternaria solani, a causal fungus of potato early blight disease, produces phytotoxic reduced-type polyketides such as solanapyrones and alternaric acid. In the course of our study on fungal polyketide synthases (PKSs), cloning and functional analysis of reduced-type PKS genes from A. solani were carried out. Three PKS gene fragments were amplified from A. solani genomic DNA by polymerase chain reaction and their entire gene sequences were determined. These PKS genes, named pksN, pksF, and pksK, encode typical iterative type I reduced-type PKSs with KR, ER, and DH domains in addition to KS, AT, and ACP domains. A methyltransferase domain was found in the PKSN. These PKS genes were subcloned into a fungal expression vector pTAex3R to construct expression plasmids, pTA-pksN, pTA-pksF, and pTA-pksK. A. oryzae transformants with these plasmids were cultured in induction medium and their products were analyzed. The PKSN product isolated from pTA-pksN transformant was identified to be a new decaketide pyrone with eight methyl groups derived from methionine, named alternapyrone. The pTA-pksF transformant produced two yellow pigments, AS405 and AS423. The structure of AS405 was determined to be a C_<23> polyene compound by physico-chemical analysis. Although the structure of AS423 has not been identified yet, AS423 is considered to be formed from the C_<22> intermediate. Structure determination of AS423 is now underway.
The biosynthesis of clinically important aminocyclitol antibiotics, particularly containing 2-deoxystreptamine (DOS), has been studied. Recently, we identified the butirosin biosynthetic gene cluster from Bacillus circulans, however, most of the ORFs could not been functionally identified by means of simple homology search. We envisioned, therefore, that related biosynthetic gene clusters of similar DOS-containing aminoglycoside antibitotics may give significant information as to the crucial biosynthetic genes for the core structure of the antibiotic. Thus, the neomycin biosynthetic gene cluster has been tackled and identified accordingly from Streptotnyces fradiae. As a result, most of the btr genes were found in the neomycin gene cluster, which appears to suggest that those genes are responsible for the biosynthesis of the core ribostamycin in both antibiotics. Our attention was next turned to the enzymatic reaction by each biosynthetic gene product, particularly for the core structure biosynthesis. As a result of the btrD gene disruption, BtrD was suggested to be involved in the preparation of glycosyl donor prior to the glycosylation of DOS. BtrD was expressed in E. coli and was found to be a novel nucleotidylyltransferase, which synthesizes UDP-glucosamine from UTP and GlcN-1-P, although no significant homology was found between BtrD and the well known dTDP-glucose synthase. BtrS was shown to be a dual functional aminotransferase in the DOS biosynthesis by gene disruption studies. Precise mechanistic and stereochemical studies of BtrS reaction showed that BtrS can distinguish prochirality of DOS, where the chirality of DOI was not strictly distinguished. These results suggested that the α-positions of the inosose carbonyl are not the determining element of recognition, but rather a plausible hydrogen-bonding at one of the β-positions of inosose carbonyl should be significant.
In 1983, we developed an olefin cyclization agent, mercuric triflate, Hg(OTf)_2, and offered the first experimental evidence that the olefin cyclization takes place by a stepwise mechanism via conformationally flexible cationic intermediates by trapping intermediates 8, 9, and 10 to give tert-alcohols 5, 6, and 7, respectively, during the cyclization of geranylgeranyl acetate (4) in the presence of water. Now we have focused on the C-ring problem of sterol biosynthesis that includes rearrangement from the more stable tert-cation 15 to a less stable six-membered ring secondary cation 16. We have achieved a model study using diol 18 and investigated the fate of the corresponding tert-cation 19 generated by the reaction with Lewis acids. The cation generated with BF_3・Et_2O, SnCl_4, Sc(OTf)_3, FeCl_3, TiF_4 and CF_3SO_3H underwent a hydride shift to generate tert-cation 20 and afforded spiro-cyclic 21 as the sole product. On the other hand, TiCl_4 selectively induced rearrangement into the six-membered ring secondary cation 22 and afforded 23 and related six-membered ring products. CONFLEX-MOPAC calculation elucidated the existence of two conformers 24 and 25, for the naked cation of 19. Parallel (the five-membered ring and the cationic plane) conformer 24 is favorable for the hydride shift to form 20 and the perpendicular 25 should lead to C-C bond migration to form 22. The ab initio calculation of the conformation of cations in the presence of the counteranion showed that [TiCl_4,OH] significantly destabilizes parallel cation 24 and should lead to six-membered ring secondary cation 22. On the other hand, counteranions [TiF_4OH] and [BF_3OH] destabilize six-membered ring secondary cation 22, and a hydride shift into 20 should be the dominant pass. We surmise that the control of cation conformation (parallel or perpendicular) by the carboxylates of glutamic and aspartic acids to act as counteranion in an enzymatic cavity is a distinct possibility. Therefore, this result is the first example that overcame the big Markovnikov wall experimentally and theoretically at least to our knowledge. We have also investigated Hg(OTf)_2-catalyzed hydration of alkyne affording methyl ketone 36, 38, and 40, Hg(OTf)_2-catalyzed hydroxylative enyne cyclization to give 47, and Hg(OTf)_2-(TMU)_3 catalyzed arylyne cyclization leading to 50 and 51 with highly efficient catalytic turnover. Protodemercuration by in-situ generated TfOH of vinyl mercuric intermediates 42, 46, and 54 should be the key step that regenerates the catalyst Hg(OTf)_2.
1. 1-Methylidenesqualene (1-MS) and 25-MS were converted to 30-methylidene-hop-22(29)-ene by recombinant squalene: hopene cyclase from Alicyclobacillus acidocaldarius. It was remarkable that both analogues afforded the same product; the hopanyl intermediate cation, stabilized by the methylidene residue enabled a rotation of the isobutenyl group at C-21 prior to the final proton elimination. Further, the enzyme also converted 26-MS and 27-MS to novel pentacyclic C_<31> polyprenoids; a dammarene derivative with a 220.127.116.11+6 ring system and 26-methylidene-hop-22(29)-ene, respectively. The broad substrate specificity of the enzyme provided important information on the structure and function of squalene: hopene cyclase. 2. Recombinant oxidosqualene: β-amyrin cyclase from Pisum sativum converted 22,23-dihydro-2,3-oxidosqualene into a 4: 1 mixture of euph-7-en-3β-ol and bacchar-12-en-3β-ol. This is the first demonstration of the enzymatic formation of the baccharene skeleton with a six-membered D-ring. In the absence of the terminal double bond, the proton initiated cyclization first generated the tetracyclic dammarenyl cation, followed by a backbone rearrangement with loss of H-7α leading to the formation of euph-7-en-3β-ol, while D-ring expansion to the baccharenyl cation and subsequent 1,2-hydride shifts with H-12α elimination yielded bacchar-12-en-3β-ol. The recombinant enzyme also converted 24,30-bisnor-2,3-oxidosqualene into a 3: 1: 0.2 mixture of 29,30-bisnor-β-amyrin, 29,30-bisnorgermanicol, and 29,30-bisnor-δ-amyrin. Furthermore, incubation with [23-<13>^C]- and [23,23-2^H]-labeled isotopomers established that the cyclization reaction proceeded through formation of a thermodynamically favored bisnoroleanyl secondary cation with a less-strained six-membered E-ring.
Oligosaccharides on cell surface play important roles in many biological processes. Most biologically active oligosaccharides are rare, and are difficult to be purified. Additionally, their structural diversity based upon stereo- and regio-isomers makes it difficult to not only determine their structures, but also to accomplish its chemical synthesis in comparison with those of oligopeptides and oligonucleotides. We have investigated the one-pot glycosylation based on the chemoselective activation of glycosyl donors attached with different leaving groups with appropriate activators. Herein we report the high-speed synthesis of biologically active oligosaccharides (dimeric Lewis X and sialo-containing glycosyl amino acids) by one-pot glycosylation. In the first topic, we descried the synthesis of a protected dimeric Lewis X epitope by sequential one-pot glycosylation and an application to the automated synthesis of a protected dimeric Lewis X library. We designed sequential one-pot four-step glycosylations to provide octasaccharide 12 from five different building blocks 2 and 4-7. One-pot glycosylation using 2-5 provided the glycosyl fluoride 3 in good yield. The following one-pot glycosylation involving glycosylation of 2 with the glycosyl fluoride 3 and coupling with additional two building blocks 4 and 5 afforded a protected octasaccharide 12 in excellent yield. The synthesis of 12 member hepta-to di-saccharides 13 and 14 was accomplished by the one-pot glycosylation utilizing an solution-phase automated synthesizer. In the next topic, we report one-pot glycosylation involving sialylation to provide sialo-containing glycosyl amino acids 19 and 20. We first examined glycosidation of thiosialoside varying the protecting group at the C5 amino group. N-Troc thiosialoside 21 was found to be effective for sialylation. Using the sialyl donor 21, linear and branched type one-pot glycosylations were accomplished to provide linear and branched trisaccharides 27 and 31 in excellent yields with good stereoselectivity at the anomeric position.
The pradimicin-benanomicin class antibiotics constitute a new class of natural products with a benzo[a]naphthacenequinone, amino acid and disaccharide moieties. They show anti-fungal and anti-HIV activities, which are related to the selective binding abilities to mannose-rich oligosaccharides, e.g., the mannane surface of fungi or gp 120 of HIV. The unique structure as well as the significant bioactivities les us to undertake the synthesis of this class of natural product. We have previously reported the stereoselective synthesis of the pradimicinone, the common aglycon of this class of natural products, by using the intramolecular pinacol coupling of the biaryl dialdehyde. A remaining problem is the regioselective introduction of the sugar to the pseudo-C_2 symmetric diol structure of the aglycon. Herein, we have completed total synthesis of pradimicin C. This synthesis features two key points; 1) Diastereoselective ring opening reaction of biaryl lactone 14 was conducted by employing optically active amino alcohol derived from D-valine as a nucleophile, gaving the axially chiral compound 15 in good yield with a high stereoselectivity, and 2) stereoselective semi-pinacol cyclization of acetal-aldehyde 18 by using Sml_2 with Lewis acid and water afforded the requisite tetracyclic 19, of which the vicinal diol moiety was suitably discriminated ready for introduction of disaccharide. Metallocene-promoted glycosylation of 20, derived from 19, effected to give glycoside 22, and further transformations involving the construction of E-ring skeleton and removal of all protecting groups led us to complete the total synthesis of 2.
Tetrodotoxin (TTX, 1), isolated as a toxic principle of puffer fish poisoning, is a potent neurotoxin as a specific voltage-gated Na^+ ion channel blocker. The structure was elucidated by Hirata-Goto, Tsuda and Woodward independently in 1964 to be a highly functionalized cage molecule which has 8 contiguous chiral center, cyclic guanidinium with hemiaminal and dioxa-adamantan structure. The remarkable biological activity and the structural complexity made it an attractive target for total synthesis. However only a total synthesis of racemic TTX was reported by Kishi-Goto in 1972 until the first asymmetric total synthesis of TTX in our laboratory in 2002. Now we wish to report here an alternative and efficient total synthesis of (-)-TTX starting from 4, an intermediate for our total synthesis of 11-deoxyTTX (2). This synthetic strategy was based on total syntheses of deoxy TTX analogs in our laboratory. Installation of hydroxyl group at allylic position of 4 with SeO_2 was followed by epoxidation and ozonolysis to give an epoxy aldehyde 10. Diastereoselective addition of magnesium acetylide to 10 and protecting group manipulation gave acetylene 12. Oxidative cleavage of the acetylene to carboxylic acid via α-keto acid and subsequent intramolecular epoxide opening gave a hydroxy lactone 16. After transformation of 1,2-diol protected as acetonide into dimethyl acetal, selective removal of acetates at C-9 and 10 hydroxyl groups gave diol 21, which was treated with TBSOTf to give an ortho ether 22. Deprotection of trichloroacetamide with DIBAL-H and following guanidinylation with di-Boc isothiourea gave a fully functionalized guanidine 24. Finally, global deprotection under acidic condition provided tetrodotoxin (1).
Merrilactone A (1) was isolated from pericarps of Illicium merrillianum. Because of its neurotrophic activity, 1 is expected to possess therapeutic potential to treat neurodegeneration associated with Alzheimer's and Parkinson's diseases. Apart from the biological aspects, the highly oxygenated and caged pentacyclic skeleton of 1 serves interesting synthetic challenges. In this paper, we report the efficient total synthesis of (±)-1. The construction of cis-bicyclo[3.3.0]octane framework embedded within 1 was envisioned to involve the desymmetrization of meso-diketone 3 through an intramolecular aldol reaction. Eight-membered meso-diketone 3 was efficiently prepared through pairwise symmetrical functionalizations from commercially available 2 in nine steps. In the crucial intramolecular aldol reaction, the reaction conditions and the protecting groups of 3 were found to significantly affect the diastereoselectivity. When the dichlorobenzyl (DCBn) protected 3d was treated with base 13, the desired isomer 4d was obtained as an exclusive isomer. Thus, this single reaction remarkably established the relative stereochemistry of four chiral centers (C4, C5, C6, C9). In regard to the total synthesis of 1 from 4d, the proper orchestration of the functional group manipulation was the most critical issue. The key transformations included (i) radical cyclization to set the highly congested C9 quaternary center (18→19), (ii) stereoselective reduction of C7-ketone utilizing the rationally designed enol ether (24→25), (iii) regioselective oxidation of tetraol (26→27), and (iv) acid-treatment of α-epoxide 29, which gave rise to the targeted merrilactone A (1).
Sordarin, isolated in 1971 as a metabolite of Sordaria araneosa, is a potent and selective inhibitor of fungal protein synthesis. The aglycon of sordarin, sordaricin (1), has a unique tetracyclic diterpene core containing trans-perhydroindene and a strained bicyclo[2.2.1]heptene frameworks with successive chiral centers. Sordaricin (1) is proposed to be biosynthesized through an intramolecular [4+2] cycloaddition. There have been two reports on the syntheses of sordaricin (1), both of which employed the intramolecular [4+2] cycloaddition as a key step. We planed to synthesize (±)-sordaricin (1) by developing a new synthetic strategy to construct the strained norbornene system. The bicyclic[5.3.0]decan-3-one 5 bearing four successive chiral centers [C(9), C(10), C(13), C(5)] could be synthesized stereoselectively from cyclopropanol 9 by the oxidative radical cyclization with Mn(pic)_3 via β-keto radical intermediate. Stereoselective and regioselective introduction of β-ketoester unit at C(3) of bicyclic ketone 10 derived from 9 and the successive Knoevenagel cyclization worked well to afford tricyclic compound 4, which was transformed to 3 in good yields. The construction of the strained bicyclo[2.2.1]heptan-2-one 2 was successfully achieved from tricyclic compound 3. When 3 was exposed to a catalytic amount of Pd(PPh_3)_4 and NaH, the desired intramolecular allylation (the Tsuji-Trost reaction) proceeded to furnish 2 in 92% yield. The structure of 2, which contains all of the stereogenic centers of sordaricin (1), was secured by X-ray crystallographic analysis. The ketone 2 was converted into 24 by the transformation to enol triflate 23 and the successive treatment with higher order cuprate derived from (2-Th)Cu(CN)Li and isopropylmagnesium bromide in the presence of HMPA. Dialdehyde 26 was derived from 24 by two-steps sequences: (i) conversion to bis(phenylboronic ester) 25 by the oxidation with OsO_4-PhB(OH)_2 and (ii) oxidative cleavage of the 25 with NalO_4. The resulting dialdehyde of 26 was reduced with NaBH_4 to the corresponding diol, and the selective protection of the less hindered C(19)-hydroxy group with TBS was subsequently carried out to afford 27. Oxidation of the C(17)-hydroxy group of 27 to aldehyde, followed by desilylation with TsOH, provided sordaricin ethyl ester 28. Finally, deethylation of ester 28 proceeded smoothly with propanethiolate to give (±)-sordaricin (1).
Strychnine (1), a well-known poison first isolated in 1818, has generated considerable attention among synthetic chemists due mainly to its architecturally complex structural features including the unique heptacyclic framework as well as the six contiguous chiral centers. After the first total synthesis reported by Woodward in 1954, strychnine remains popular as a target for demonstrating new reactions and novel synthetic strategies. In this paper we report a concise stereocontrolled total synthesis of (-)-strychnine (1), wherein efficient synthetic methodologies developed in our laboratories played crucial roles. Notable features of our synthesis include: (1) Palladium-mediated coupling of the sterically demanding indolylmalonate 11 and the cyclic vinyl epoxide 12 provided a rapid access to the 2,3-disubstituted indole derivative 10, which has all the requisite carbon atoms for the construction of Wieland-Gumlich aldehyde (6), the known precursor of strychnine; (2) The ring-closing double N-alkylation of 2-nitrobenzenesulfonamide under Mitsunobu conditions was quite effective, providing the nine-membered cyclic secondary amine derivative 18 in 95% yield; (3) A critical geometrical control of the trisubstituted olefin was secured by incorporation of the double bond into cyclohexenone; (4) Removal of Ns group from the multifunctional substrate 21 could be executed under mild conditions, which was followed by facile transannular cyclization to afford pentacyclic strychnos skeleton 23. We consider that our synthetic strategy provides not only a unique and effective solution for the construction of strychnine, but also versatile and facile access to variety of polycyclic indole alkaloids.
Brevetoxin B (1), a potent neurotoxin was isolated from the red tide organism Gymnodinium breve Davis in 1981. The unique structural features and biological activity of this molecule have attracted the attention of synthetic chemists. We have already reported the efficient method for the convergent synthesis of polycyclic ethers via the intramolecular allylation and subsequent ring-closing metathesis. Herein we report the convergent synthesis of the polycyclic framework of 1 based on our own methodology. The reaction of the bicyclic compound 12, prepared from the known compound 9, with the allylic silane 13 in the presence of TMSOTf gave the desired product 14, stereoselectively. The JK ring segment 6 was synthesized in 11 steps including one-carbon elongation from 14. Palladium-catalyzed coupling of the ketene acetal triflate 22, prepared from the known compound 18, and the organozinc reagent 23 gave the enol ether 24, which was converted to the BC ring segment 7. The connection of 7 and the FG ring segment 8 followed by several transformations afforded the allylic stannane 29. The partial reduction of 29 with DIBALH followed by trapping of the resulting aluminum hemiacetal with acetic anhydride provided the acetoxy ether 30. However, the yield was very low. Alternatively, the chlorosulfide 31, prepared from 26, was treated with the alcohol 8 in the presence of AgOTf to provide the O,S-acetal 32. Cyclization of 33 using AgOTf as a Lewis acid gave the desired product 34, stereoselectively. The construction of the E ring and the A ring via ring-closing metathesis afforded the ABCDEFG ring segment 5. The alcohol 5 and the carboxylic acid 6 were connected successfully by Yamaguchi conditions to give the ester 41. The construction of the polycyclic framework of 1 was performed via the intramolecular allylation and subsequent ring-closing metathesis to yield 46. Completion of the total synthesis of 1 is currently under way.
Gymnocin-A (1) is a polycyclic ether toxin, isolated from the notorious red-tide forming dinoflagellate Karenia mikimotoi. The toxin molecule displays in vitro cytotoxicity against P388 lymphocytic leukemia cells (EC_<50>=1.3μg/mL). Structurally, gymnocin-A is characterized by 14 contiguous and saturated ether rings and a 2-methyl-2-butenal side chain. The number of contiguous ether rings is the largest among the polycyclic ethers hitherto known. Herein, we describe a convergent and efficient total synthesis of gymnocin-A by using our developed B-alkyl Suzuki-Miyaura coupling-based methodology. We planned to assemble the polycyclic ether backbone of 1 by a particularly challenging Suzuki-Miyaura cross-coupling between the ABCD and FGHIJKLMN fragments (2 and 3, respectively). The symmetry elements of fragment 3 allowed further division into two fragments, the GHI and KLMN (4 and 5, respectively) rings, both of which would be derived from a common precursor 6. Common precursor 6 was synthesized by using B-alkyl Suzuki-Miyaura coupling-based methodology, then converted into the GHI (4) and KLMN (5) ring fragments. Hydroboration of 4 followed by Suzuki-Miyaura reaction with 5 provided cross-coupled product 20 in good yield. Ring-closure of the J ring by radical reduction of fused thioketal 22 and formation of the F ring generated the FGHIJKLMN ring fragment 2. Synthesis of the ABCD ring fragment 3 started with tricyclic ether 7, which was elaborated to α,β-epoxy ketone 26. Selenium-mediated reduction of 26 installed the C10 hydroxyl group, and the tetrahydrofuran ring A was constructed by radical cyclization of β-alkoxyacrylate 28 to give ester 29. A further eight-step sequence was required to complete the synthesis of 3. Hydroboration of 3 with 9-BBN and cross-coupling with enol triflate 2 in the presence of aqueous Cs_2CO_3 and Pd(PPh_3)_4 in DMF at room temperature proceeded smoothly to furnish the desired cross-coupled product 31 in excellent yield (81%). The C17 hydroxyl group was installed by stereoselective OsO_4 oxidation of the silyl enol ether, derived from ketone 32. The E ring was formed by radical reduction of fused thioketal 35 to generate tetradecacyclic polyether skeleton 36. Finally, incorporation of a side chain completed the first total synthesis of gymnocin-A (1). The synthetic gymnocin-A was identical to the natural sample by 1^H and <13>^C NMR and MS spectra, thus confirming the structure of gymnocin-A. Moreover, cytotoxicity of synthetic gymnocin-A was comparable to that of natural sample.
Stevastelins are novel cyclic depsipeptides isolated from a culture broth of penicillium, and reported to show a potent immunosuppressive activity. Herein we describe the total synthesis and structure confirmation of stevastelins B (1) and B3 (3), and the synthesis and structure revision of stevastelin C3 (2). For a synthesis of the fatty acid precursor 13 with four contiguous chiral centers in stevastelins, we chose L-quebrachitol (5), an optically active cyclitol obtained in large quantities from the serum of the rubber tree, as the starting material. Stereo- and regioselective introduction of two methyl groups into 5 gave ketone 6, which was converted into 13 by regioselective Baeyer-Villiger reaction, followed by introduction of an alkyl group. Stepwise introduction of the peptide moiety into 13 gave an amino carboxylic acid, whose macrolactamization under the Shioiri's conditions successfully gave macrocycle 19. Removal of the O-benzyl group in 19, followed by selective O-acetylation furnished stevastelin B (1). For a synthesis of stevastelins C3 (3) and B3 (4), cyclization precursor 21 was prepared from 13. Macrolactamization of the derived amino carboxylic acid by Shioiri's procedure effectively constructed the 13-membered cyclic structure. Deprotection of the O-benzyl group gave the proposed structure of stevastelin C3 (3). Selective O-acetylation furnished stevastelin B3 (4). Although synthetic stevastelin B3 (4) was identical with the natural product, synthetic stevastelin C3 (3) was not, implying that the proposed structure of stevastelin C3 (3) might be incorrect. Spectral analysis and degradation study of natural stevastelin C3 suggested thate the natural product should be a 5-deoxy derivative of the proposed structure. To confirm the structure of the natural product, 5-deoxy diol (22) was synthesized from 13. Introduction of the peptide, followed by macrolactamization and deprotection gave macrocycle (24), whose spectral data were found to be fully identical with those of natural stevastelin C3. On the basis of this synthesis, it was revealed that the structure of stevastelin C3 is not 3 but should be revised to 24.
Lasonolide A (1) was isolated from an extract of the shallow water Caribbean marine sponge, Forcepia sp. by McConnell. This compound was discovered to inhibit the in vitro proliferation of A-549 human lung carcinoma cells as well as cell adhesion in a newly developed whole cell assay that detects signal transduction agents. Because of the intriguing structural features, for example the 20 membered polyene macrolide and the characteristic quaternary stereogenic center at C22, notable biological profiles and limited availability, lasonolide A represents an attractive target for total synthesis. Several synthetic attempts have been reported and to date two total syntheses have been communicated by Korean groups. In particular, Lee and co-workers revised the proposed structure and established the absolute configurations as shown in Figure 1. We report here the enantioselective total synthesis of the natural enantiomer (+)-lasonolide A employing a convergent strategy. Namely, the diastereoselectively synthesized three segments, the C1-C17, the C 18-C25 and the C26-C35 segments, were connected by sequential cross metathsis reaction, Yamaguchi's macrolactonization and Wittig reaction.
Based on the fact that some of the bioactive materials isolated marine animals have been produced by bacteria, we have focused our attention on new antitumour materials from microorganisms inhabiting the marine environment. As part of our ongoing seach for new antitumour metabolites produced by microorganisms from marine organisms, peribysins A (1)- I (9) have been isolated from a strain of Periconia byssoides OUPS-N 133 originally separated from sea hare Aplysia kurodai. The relative stereostructures of 1- 9 have been elucidated on the basis of spectroscopic analyses using 1D and 2D NMR techniques and some chemical transformations. Compounds 1-9 were examined together with haerbimycin A as standard sample in the cell adhesion assay using human-leukaemia HL-60 cells and human-umbilical-vein endothelial cells (HUVEC). As a result, all of these compounds inhibited the adhesion of HL-60 cell to HUVEC more potently than herbimycin A. Among them, 1 and 4 exhibited especially potent inhibitory activities, and their activities were 127 to 380 times as potent as herbimycin A.
Marine polycyclic ether toxins, represented by brevetoxins and ciguatoxins, have attracted intense attention due to their unique structures and extremely potent biological activities. One of the common structural features among these polycyclic ethers is trans-fused medium-sized ether rings, which are speculated to play important roles in exhibiting their biological activities based on their conformational flexibility. Here we surveyed detailed conformational properties of such ether rings by NMR analysis of synthetic models and force field calculation. In order to show validity of conformational analysis using partial structure models of natural polycyclic ethers, brevetoxin B (G)HI ring model 7a was first synthesized. 7a reproduced NMR chemical shifts of the corresponding part of brevetoxin B. This indicates that 7a reproduces conformation of the corresponding part in the natural product. In addition, yessotoxin (F)GH ring model 10a and its C26 epimer 10b were synthesized. Only 10a reproduced NMR chemical shifts of the corresponding part of yessotoxin. Thus stereochemistry of C26 of yessotoxin was confirmed, simultaneously showing validity of partial structure models again. Next, brevetoxin A (F')GH ring model 13 was synthesized and signal broadening of its <13>^C NMR spectrum was observed due to a slow conformational change in the NMR time scale. Two sets of <13>^C NMR signals derived from two conformers appeared at -90℃. Analyses of <13>^C NMR chemical shifts at both 20℃ and -90℃ and proton-proton coupling constants showed that the eight-membered G ring of 13 is a conformational mixture of 'crown' and 'boat-chair' in the ratio of 83: 17 at 20℃ (energy difference is ca. 3.9 kJ/mol). Dynamic NMR study on 13 revealed that activation energy of the conformational change is ca. 45 kJ/mol. However 13 did not reproduce NMR chemical shifts of the corresponding part of natural brevetoxin A, indicating that 13 differs in conformation from G ring of brevetoxin A. So brevetoxin A FGH ring model 17 was redesigned and synthesized. 17 reproduced NMR chemical shifts of the corresponding part of brevetoxin A and major conformation of G ring of 17 is boat-chair, which was predicted by chemical shift comparison between 13 and brevetoxin A. In 2000, Isobe at al. reported synthesis and conformational analysis of ciguatoxin HIJ ring model 18. Its <13>^C NMR spectrum was reported to contain 14 peaks in spite of its symmetrical planar structure, explained to be due to slower conformational change of two asymmetric 'boat-chair' enantiomers than NMR time scale. We resynthesized this model 18 via a different route, reveling that their synthesized model requires to be reinvestigated. In addition, synthesis and detailed stereochemical studies on a yessotoxin DEF ring model, a gambierol DEF ring model, their desmethyl model, and a gambieric acid BC ring model will be presented.
The proanthocyanidins are known as condensed tannins and/or oligomeric flavonoids, in which flavan-3-ol units are linked together by carbon-carbon bonds from the 4-position of one unit to the 8-position of the next unit. Many biological activities, powerful antioxidant activity, free-radical-scavenging activity and an anti-tumor-promoting effect, have been reported for flavonoids and their investigation is now increasingly important. Proanthocyanidins have been obtained from many kinds of plants, but because they are usually obtained as a complex mixture of structurally related compounds, it is very difficult to isolate them in a pure form. Consequently, we planned to develop efficient synthetic methods leading to procyanidins with a high level of purity and stereoselectivity for biological assay. We previously developed that TMSOTf-catalyzed intermolecular- and intramolecular-condensation and described their inhibitory activity of the Maillard reaction. We report here the simple efficient method to synthesize 3-substituted procyanidin oligomers constituted with (+)-catechin and (-)-epicatechin using TMSOTf-catalyzed intermolecular condensation. And we also report the stereoselection of 3,4-cis and 3,4-trans catechin and catechin condensation under intramolecular coupling method. The inhibitory activity of the Maillard reaction, antioxidant activity, DPPH radical scavenging activity, DNA polymerase inhibitory activity and DNA metabolic enzymes inhibitory activity were tested using synthesized procyanidin oligomers including 3-substituted oligomers. From the results of these activity tests, it became apparent that the oligomer length and the presence of substituted groups at the C-3 position in the proanthocyanidin oligomer were very important for biological activities, however; the antioxidant activity and DPPH scavenging activity of these compounds were not parallel to the other inhibitory activities. Further work is in progress to clarify the relationships between the structure of proanthocyanidins and their bioactivities.