The enzymatic cyclisations of acyclic squalene 1 and 2,3-oxidosqualene (OS) to form polycyclic triterpenes have fascinated chemists and biochemists for over half a century. X-ray crystallographic analysis of prokaryotic squalene-hopene cyclase (SHC) was reported in 1997. More recently, three-dimensional structure of human lanosterol synthase has successfully been elucidated. SHC catalyses the conversion of 1 into the pentacyclic triterpenes of hopene 2 and hopanol 3, the ratio being ca. 5: 1, respectively. This polycyclization cascade is attained by single enzyme. 1 is folded in all pre-chair conformation inside the reaction cavity and cyclized in regio- and stereospecific manner through a series of carbocationic intermediates, leading to the formation of new five C-C bonds and nine chiral centers. 1. Critical role of steric bulk size of amino acid residues for the folding conformation of squalene. Several examples are presented in this symposium. A306V mutant gave the tetracyclic products that are constructed by the chair-chair-chair-boat conformation. The Y420W and L607W afforded a novel enzymic product, neoachillapentaene, which is produced via the constrained boat form. These single amino acid substituted mutants enforced 1 into a boat structure, despite all pre-chair conformation being adopted by the wild-type SHC. These findings indicate that the folding conformation is directed by the steric bulk size of the active site residues. 2. Eukaryotic cyclases (OSCs) accept only (3S)-2,3-oxidosqualene (OS) as the substrate, but are inert to the (3R)-OS. The original substrate of SHC is 1, but both (3S)- and (3R)-OSs also can be accepted to afford 3β-hydroxy- and 3α-hydroxyhopenes, respectively. Comparison of amino acid alignment between OSCs and SHCs indicates that Gly600 is conserved in all SHCs, but this residue is lacking in all the OSCs. The G600-deletion mutant was constructed and incubated with 1 and the (3R,S)-racemic OSs. This mutant accepted only (3S)-OS, but were inert to 1 and (3R)-OS, indicating that the substrate specificity of prokaryotic cyclase was successfully altered to that of eukaryotic cyclases. 3. The site-specific incorporation experiments of unnatural amino acids into SHC were planned to validate the concept of cation/π interaction for the polycyclisation cascade of squalene. The targeted positions of SHC were mutated into amber codon (UAG). The site-specific in vivo incorporation of O-methoxy- and p-aminophenylalanines into positions 305 and 605 was successfully achieved according to Schultz's protocol. By using the system of cell-free protein synthesis, fluorinated phenylalanines were site-specifically incorporated into their positions. The specific activities of the SHCs into that fluorophenylalanines were site-specifically incorporated were decreased in inverse proportion to the number of substituted fluorine atoms, clearly indicating that cation/π interaction operated during the polycyclisation cascade.
Nonribosomal peptides (NRPs) comprise many pharmacologically important natural products that display a wide range of interesting biological activities, such as antibiotic (vancomycin), antitumorgenic (bleomycin A2) and immunosuppressive (cyclosporin A) activities. These compounds are produced by various organisms but often with poor yields. Our interest in studying the NRP biosynthesis stems from the potential for engineering the biosynthetic pathways for efficient, versatile production of NRPs and their rationally redesigned analogs. Echinomycin is a NRP secondary metabolite from Streptomyces lasaliensis that belongs to the large family of quinoxaline antibiotic/anticancer natural products. The members of this class of compounds have bicyclic aromatic quinoxaline chromophores attached to the C_2 symmetric cyclic peptide core structure. Isolation and sequencing of the entire Ecm biosynthetic gene cluster has been accomplished. Eight genes (ecm2-4, 8, 11-14) are predicted to be involved in the biosynthesis of quinoxaline-2-carboxylic acid (QC), while five genes (ecm1, 6, 7, 17, 18) are thought to be responsible for the peptide backbone formation and modifications. The aryl carrier protein (ArCP) required for the incorporation of QC into echinomycin is absent from this cluster. Instead, fatty acid synthase acyl carrier protein (FabC) appears to substitute for the echinomycin ArCP, as previously reported for the related triostin A biosynthetic pathway. Subsequently, the echinomycin biosynthetic genes (ecm1-4, 6-8, 11-14, 16-18 and fabC) and sfp, whose translational product is required for phosphopantetheinylation of heterologous acyl carrier proteins in E. coli, were introduced to E. coli to generate a strain capable of echinomycin biosynthesis from simple carbon and nitrogen sources. This is the first report of biosynthesis of biological active form of heterologous NRPs in E. coli. Because of the fast growth rate and ease of maintenance and manipulations of E. coli, this NRP production scheme should serve as foundation toward establishing a general biosynthetic system for economic and flexible production of peptide natural products and their analogs.
Protein kinase C (PKC) is a family of enzymes which play important roles in intracellular signal transduction. We designed a series of novel PKC ligands having an isobenzofuranone template, based on the proposed interaction of DAG (1,2-diacyl-sn-glycerol), a physiological PKC ligand, with the PKCδC1b ligand-binding domain. Since interaction of the hydrophobic alkyl chain of the isobenzofuranone derivatives with lipid membrane is expected to be critical for the PKC activation, we were interested in the synthesis of the isobenzofuranone derivatives having the hydrophobic alkyl chain at various positions of the benzene ring. All four regioisomers were synthesized, and their activities as PKC ligands were evaluated. The isobenzofuranone derivatives having a 7- or 6-alkyl group bound to PKCαC1b domain strongly, on the other hand, 5- or 4-substituted derivative showed lower affinity toward PKCαC1b domain. Effects of these synthetic ligands on the PKCα phosphorylation activity were also examined. While the 7- or 6-substituted derivative was found to be a strong activator of PKCα, 5-substituted derivative activated PKCα only weakly. Surprisingly, 4-substitued derivative showed no activation of PKCα even at high concentration at which significant binding to PKCα was observed. These results suggest that position of the hydrophobic chain is critical for the PKC activation. 7-Substituted isobenzofuranone derivatives which have straight alkyl chain or bulky alkyl group were also synthesized. The domain selectivity of synthetic ligands toward the C1a and C1b domain were evaluated using overlay plot method. The compound which have bulky alkyl group at the both acyl and phenolic substituent showed the strongest binding affinity toward the C1b domain. In contrast, the compounds which have straight alkyl chain at the acyl position might preferably bind toward C1a domain, because significant PKCα activation was observed at the low concentration at which binding toward the C1b domain was not observed. All of these compounds showed the synergistic effect with TPA. The compound which have acetyl group at acyl position showed moderate PKCα binding affinity. Surprisingly, at high concentration it showed inhibition of PKCα activity induced with 1μM TPA.
With their imposing molecular structure, the ladder-shaped polyethers pose considerable challenge to synthetic chemists. Because the linear construction of the ether rings is virtually impossible due to the large and complex structure, the development of an efficient methodology for coupling the fragments, which is suitable for use in the advanced stages of synthesis, has been particularly important for the total synthesis. In this presentation, we will report the new convergent total synthesis of 51-hydroxyCTX3C (2), which has one of the most complex structures among the natural polycyclic ethers. Recently, we have developed a direct method to form O,S-acetal 5 from a secondary alcohol 3 and a halosulfide 4. The O,S-acetal of 6, synthesized from 5, was used as radical donor to cyclize the seven-memberd ether ring of 7, which was further transformed to fused ether structure 8 through the C-C bond formation (method A). Alternatively, the enol ether of 9 that was prepared from 5 was utilized as a radical acceptor to form six-memberd ether ring 10, from which 8 was synthesized via the reductive etherification (method B). While the method B was successfully applied to the CD-ring region of 2, the method A enabled the construction of the central FG-ring system after the final coupling between the left (ABCDE) and right (HIJKLM) ring system. By using these free radical approaches, the first total synthesis of 51-hydroxyCTX3C (2) was accomplished in a concise and efficient fashion.
Lucilactaene (1), a cell-cycle inhibitor in p53-Transfected cancer, is a synthetically challenging molecule because of its rare hexahydro-3a-hydroxy-5-oxo-2H-furo [3,2-b]pyrrol-6-yl ring system and its substituted and conjugated E,E,E,E,E pentaene moiety, which is unstable to acid, base, and light. Lucilactaene (1), which readily undergoes racemization, has been synthesized for the first time in optically pure form via a biomimetic pathway. The conditions under which racemization occurs were elucidated during this total synthesis. The careful isolation of lucilactaene (1) from both the broth and the mycelia under neutral, nonracemizing conditions demonstrated that the isolable natural product is in fact itself racemic. This total synthesis, which enabled verification of the absolute configuration of lucilactaene (1), has several noteworthy features: All the reactions from NG-391 (2) are mild enough not to affect the labile E,E,E,E,E pentaene moiety; ether formation from 2 to 25 and 28 and the intramolecular Michael reaction from 26 to 27 or from 30 to 31 are both highly stereoselective; the reductive removal of the epoxide with Sml_2 without effecting demethoxylation, andthe deprotection of a hemiaminal under neutral, oxidative conditions via vinyl ether 33 by using a newly developed phenylselenylethyl protecting group, are also useful transformations.
Aerobic oxidation of alcohols, especially chemo- and enantioselective ones, is a current topic, because alcohol oxidation is of tremendous importance in organic synthesis. Therefore, many methodologies for oxidations using dioxygen as the stoichiometric oxidant have been developed, but most of the oxidations need forced reaction conditions or addition of some mediators to perform the oxidations in a catalytic manner. On the other hand, alcohol oxidizing enzymes like Galactose Oxidase (GOase) catalyze the oxidation at a bodily temperature. Recent studies have disclosed that GOase-catalyzed oxidation proceeds via single electron transfer (SET) and intramolecular hydrogen atom transfer (Scheme 1). We recently found that (ON)Ru(salen) complexes catalyzed aerobic oxidation of alcohols at ambient temperature under irradiation of visible light without any additives. In this study, we disclosed that suitable (ON)Ru(salen) complexes catalyzed chemoselective oxidation of primary alcohols in the presence of secondary alcohols or regioselective oxidation of terminal hydroxy group of polyols (Scheme 2). Furthermore, it was found that desymmetrization of meso-diols could be effected with high selectivity by using well-designed (ON)Ru(salen) complexes (Figure 1). In the course of the studies, it was also found that the stereochemistry and reaction rate were affected by the apical ligand of ruthenium complexes (Table 1). To understand this apical ligand effect, we examined kinetics and kinetic isotope effect (KIE) in the aerobic oxidations using 4 and 5 (Scheme 2 and Table 2). The rate raw for the oxidation with 4 was rate=k_<Cl>[alcohol][cat.][O_2], while that for the oxidation with 5 was rate=k_<OH>[cat.][O_2]^<0.33-0.42>. KIE studies also indicated that ligand exchange step contributes to rate determining step (RDS) for the oxidation with 4, while hydrogen atom transfer step contributes to RDS for the oxidation with 5. Based on these results, we proposed that the mechanism of the present oxidation includes SET, hydrogen atom transfer and ligand exchange (Scheme 3).^<3c> It is noteworthy that the oxidation of mono-ols with (ON)Ru(salen) complexes involves intramolecular hydrogen atom transfer via a phenoxy radical intermediate (Scheme 3, A and B) in accord with the oxidation catalyzed by Goase, while the oxidation of diols proceeds via alkoxy radical intermediate (Scheme 3, C and D). Chemoselective oxidation of primary alcohols with complex 1 or 2 was also demonstrated to proceed via an intramolecular hydrogen atom transfer.^<5b> In conclusion, we achieved enantio- and chemoselective aerobic oxidation of meso-diols and primary alcohols using (ON)Ru(salen) complexes, respectively, and demonstrated that its mechanism is intrinsically similar to that of the Goase-catalyzed oxidation, though SET needs visible light irradiation.
Fostriecin is a potent antitumor agent, and it is suggested that its activity is attributed to the inhibition of the mitotic entry checkpoint through its highly selective serine/threonine phosphatase 2A (PP2A) inhibition. Because of its unique mechanism of tumor suppression, fostriecin is an ideal lead compound for antitumor drugs. We would like to present here the synthesis of fostriecin and 8-epi-fostriecin, evaluation of their biological activity, and conformational analysis by theoretical calculation to understand the difference of their biological behavior. It is four catalytic asymmetric reactions that feature our synthesis of fostriecin and 8-epi-fostriecin; cyanosilylation of ketone, allylation, direct aldol reaction and Noyori reduction. Especially two reactions developed in our laboratory played important roles. The cyanosilylation constructed the tetrasubstituted chiral center at C8. Both enantiomers could be obtained just by switching the center metals of the catalyst. Direct catalytic asymmetric aldol reaction using acetylene ketone as donor is a versatile reaction which can introduce synthetically useful alkyne moiety. In our synthesis the product alkynone was converted into cis-vinyl iodide, the substrate for the subsequent Stille coupling. By synthesizing 8-epi-fostriecin, we could show here the effectiveness of the strategy, constructing all of the stereocenters by catalytic asymmetric reactions. Just switching the enantioselectivity of catalysts makes it possible to synthesize stereoisomers of a natural product. Biological assay and conformational analysis of fostriecin and 8-epi-fostriecin are now proceeding. The assay is about the inhibitory activity against PP1 and PP2A. Because both of the methyl group and hydroxyl group at C8 are thought to be important for the biological activity, the assay's results of the two compounds might be very much different from each other. And conformational analysis was performed using NMR technique and theoretical calculations. The former revealed the environmental difference around C8 stereocenter and phosphate moiety. Conformation search by MM calculation implicated that coordination of hydroxyl group at C8 to the sodium cation of the phosphate might affect the environmental difference and therefore the biological activity. Now ab initio calculation is being conducted to optimize the geometry of the stable comformers.
Recently, highly oxidized and structurally unique triterpene polyethers, which are thought to be biogenetically squalene-derived natural products (oxasqualenoids), have been isolated from both marine and terrestrial lives. Among them are intricatetraol (1) isolated from the red alga Laurencia intricata in 1993 and enshuol (2) isolated from the red alga Laurencia omaezakiana Masuda sp. in 1995 by Suzuki et al. A crude fraction including intricatetraol (1) as a major component exhibits cytotoxic activity against P388 with IC_<50> of 12.5μg/mL. There have also been many other types of oxasqualenoids; however, it is often difficult to determine their stereostructures even by the current highly advanced spectroscopic methods, especially in acyclic systems including quaternary carbon centers. In such cases, it is effective to predict and synthesize the possible stereoisomers. Although the plane structures and partial stereochemistries of 1 and 2 were also elucidated by NMR methods as shown in 1 and 2, determination of the entire stereochemistries of compounds 1 and 2 has not been reached. In this symposium, we report that the total assignment of the incomplete stereostructures of unique bromine-containing (+)-intricatetraol (1) and (+)-enshuol (2) to the structural formulas 3 and 4, respectively, has been achieved through their first asymmetric total syntheses featuring biogenetic-like regioselective ether ring formations to secure the stereochemical pathway (Schemes 2 and 3).
Dysiherbaine (DH, 1) and its congener neodysiherbaine A (2), isolated from the Micronesian marine sponge, Dysidea herbacea, are novel excitatory amino acids with potent convulsant activity. DH activates non-NMDA type glutamate receptors [AMPA and kainic acid (KA) receptors] with considerable preference over KA receptors. Moreover, it has been shown that DH binds to the GluR5 and GluR6 KA receptor subunits with high affinity. Due to these intriguing pharmacological properties to KA receptors, DH and its designed analogues are anticipated to serve as useful tools for understanding the structure and functions of glutamate receptors in the central nervous system. In order to reveal the detailed structure-activity relationship profiles of DH, we undertook a diverted synthesis of structural analogues of DH. In this paper, we report the synthesis of simplified analogue 3,8,9-epi-neodysiherbaine A (4a), 8-epi-neodysiherbaine A (4b), 8-deoxyneodysiherbaine A (4c) and 9-deoxyneodysiherbaine A (4d) from a common intermediate 5. The synthesis of 5 started with C-glycosylation of allylsilane 9 with diacetyl-L-arabinal (10), which led to C-glycoside 8 as the sole product. Chemo- and stereoselective dihydroxylation using (DHQD)_2AQN and subsequent epoxidation delivered epoxy alcohol 7, which underwent epoxide opening/5-exo ring-closure during chromatography on silica gel, leading to bicyclic ether core 12. Stereoselective construction of the amino acid chain was efficiently realized by DuPHOS-mediated asymmetric hydrogenation of enamido ester 17 to generate key intermediate 5 via 18. Global deprotection of 5 by acid hydrolysis furnished analogue 4a. Selective deprotection of the acetonide group of 5 was achieved by using DDQ to afford diol 19, which was further converted to other analogues (3 and 4b-d) via cyclic sulfate 20 and thiocarbonate 24. The toxicity of analogues 4a-d were tested on mice. Intracerebral injection of analogue 4a did not induce any convulsant behavior even at higher dose (20μg/mouse), whereas that of 4b induced a sleeper effect. Analogues 4c and 4d were found to show convulsant activity although the potency was much weaker than that of the natural DH (1). In the radioligand binding assay using rat synaptic membrane preparation, 4b displaced [^3H]KA and [^3H]AMPA with IC_<50> values of 24.1±6.8μM and 9.7±2.3μM, respectively. In contrast, analogue 4a did not displace these radioligand from receptors even at 100μM. In addition, detailed pharmacological studies revealed that simplified analogue 3 is a selective antagonist for GluR5 KA receptors. Further neurophysiological studies of analogues are underway and will be described.
Dysiherbaine (1) and neodysiherbaine A (2) were isolated from a Micronesian sponge Dysidea herbacea by Sakai and co-workers and found to be selective agonists of AMPA-KA type glutamate receptors in the central nervous system. Their intriguing molecular architectures and biological activity have generated a great deal of interest as synthetic targets and the total synthesis has already been achieved by Snider et al., Sasaki et al., Chamberlin et al., and our group. However, efficient stereoselective construction of the C4 quaternary center remains unsolved in these syntheses. In this symposium, we describe a total synthesis of neodysiherbaine A in which its all stereocenters including the C4 quaternary center were constructed in completely stereocontrolled manner. Our synthesis proceeded through the following six major transformations; (i) conversion of triacetoxy D-glucal 12 into alkenyl iodide 19 via diastereoselective dihydroxylation, Kotsuki's acetylene coupling, and regio- and stereoselective reductive iodination, (ii) palladium catalyzed cross-coupling reaction of organozinc reagent 20 with alkenyl iodide 19, (iii) diastereoselective epoxidation of 22 under Katsuki-Sharpless asymmetric epoxidation conditions, (iv) acid-catalyzed cyclization 26 with inversion of the stereochemistry of the C4 quaternary center, (v) oxidation of the resulting 1,2-glycol moiety to the carboxylic acid, (vi) acidic hydrolysis giving neodysiherbaine A.
Azadirachtin (1) is a C-seco-limonoid isolated from the neem tree Azadirachta indica A. Juss (Meliaceae) and possesses potent antifeedant and growth inhibitory activities. Its complicated structure as well as its activities fascinate synthetic chemists and many synthetic efforts have been made, but the total synthesis of Azadirachtin has not been reported yet. Now we achieved a synthesis of model compound 2 which was fully functionalized at the dacalin moiety using an intramolecular Diels-Alder reaction and a radical cyclization as key reactions. In order to construct A-ring and the bridged ether, the intramolecular Diels-Alder reactions of 12 and 15 were examined, but the desired endo adducts 13 and 16 were obtained in low selectivity. So we devised the Diels-Alder reaction-decarboxylation-Claisen rearrangement strategy to use the undesired exo adduct. The intramolecular Diels-Alder reaction of 37 gave a cyclization-decarboxylation product 38 which was converted into 39 through Claisen rearrangement. In this process one carbon atom which was lost by decarboxylation could be recovered. The correct stereochemistries at C-3 and C-4 positions of Azadirachtin was introduced by oxidative cleavage of hydroxycyclohexenone ring followed by ozonolysis and aldol reaction (40→43). B-ring formation was then examined and radical cyclization (48→2) was found to proceed smoothly with controlling the newly-introduced stereochemistries. In summary, model compound 2, which had a fully functionalized decalin unit of Azadirachtin, was synthesized efficiently in short steps (19 steps). Other possible modes of radical cyclization such as 50→52 and 51→52 toward the total synthesis of Azadirachtin are now under investigations.
Solanoeclepin A (1), isolated in 1986 from a large quantity of potatoes as the most active hatching agent of potato cyst nematodes, possesses a unique heptacyclic structure containing all ring sizes from three to seven carbocycles with various oxygen functional groups. Toward the total synthesis of 1, we designed a key intramolecular cyclization reaction of epoxy nitrile 5 to construct the highly strained bicyclo[2.1.1]hexane skeleton. Initially, we developed a novel cyclopentene annulation method for the synthesis of fused carbocycles in a large scale. Thus, 3-methyl-2-cyclohexen-1-one was subjected to a conjugate addition reaction with the carbanions generated from 4-methoxy-3-butenenitrile to give rise to enol acetate 12 after acetylation of the enolate intermediate. Treatment of acetate 12 with hydrochloric acid effected an intramolecular cyclization reaction giving rise to bicyclic enone 6 in good yield. Acetate rac-14 derived from the annulation product underwent optical resolution using a lipase to afford (+)-14 in the optically pure form. Synthesis of the cyclization precursor 5 from enone 6 was quite difficult, because of the trans-fused bicyclo[4.3.0]nonane skeleton with quaternary carbon atoms at both the angular positions. The difficulty was overcome by using a Lewis acid mediated rearrangement reaction of epoxy alcohol 21, which was derived from acetate rac-14 in five steps. The key construction of the core structure of 1 was accomplished by treating epoxide 5 with LDA, by which highly strained tricyclic compound 4 was obtained in high yield. The stereostructure of 4 containing the crucial tricyclo[22.214.171.124^<1.6>]decane skeleton was unambiguously confirmed by a X-ray crystallographic analysis of its p-bromobenzoate 25. we also established an efficient synthetic route to introduce a cyclopropane carboxylic acid side chain. A model study for constructing the left-hand substructure of 1 was examined on the basis of an intramolecular Diels-Alder reaction. Under the influence of dimethylaluminum chloride, furan derivative 37 was converted into the desired tetracyclic compound 38 in moderate yield. Further studies toward the total synthesis of Solanoeclepin A (1) are now progress in our laboratory.
Altemicidin (1) was the first 6-azaindene monoterpene alkaloid isolated as a metabolite of microorganisms by Takeuchi and coworkers in 1989. In addition to its potent acaricidal activity, Altemicidin has been shown to strongly inhibit the growth of tumor cells. The first total synthesis of Altemicidin was achieved by A. S. Kende et al in 1995. The unique β-hydroxyl α-disubstituted-α-amino acid structure was an attractive feature of this molecule and prompted synthetic studies in our laboratories. Our synthetic strategy was based on the idea that key intermediate 2, with Altemicidin's azabicyclo[4.3.0.]skelton, could be constructed from amide 4 via lactam 3 by the ring expansion. In turn amide 4 could be synthesized from β-ketoester 5 wherein the tetrasubstituted carbon and the neighboring secondary alcohol could be stereoselectively installed early on in the synthesis(Scheme 1). The synthesis initiated from the treatment of α-diazo-β-ketoester 6 with rhodium acetate to afforded bicyclo[3.3.0] β-ketoester 7 in excellent yield(Scheme 2). The bicyclo[3.3.0] framework provided excellent stereochemistry for the hydroxymethylation and carbonyl reduction to furnish diol 8. Amide 9 was quickly obtained after hydroxy group protections and an ester to amide transformation. Amide 9 was converted into lactam 11 by oxidative cleavage of double bond and subsequent hemiaminal reduction. Then the activation of lactam by nosyl group and opening of lactam followed by cyclization gave cyclic enamide 13. Cyclic enamide 13 which has Altemicidin's azabicyclo[4.3.0]skelton was successfully synthesized in 13 steps from bicyclic β-ketoester 7. After the introduction of the formyl group by Vilsmeier type reaction and the removal of nosyl group, the methylation smoothly occurred(Scheme 3). Deprotections followed by Curtius rearrangement gave oxazolidinone 16. Alcohol 17, oxazolidinone opening product, was obtained from the Boc imide under hydrolysis conditions. Like this, we succeeded in the introduction of the nitrogen into the tetrasubstituted carbon and formylation after the construction of the basic skelton. Final transformations toward the natural product are currently under investigation. Importantly, the synthesis of optically active Altemicidin has also been examined. In our laboratories, it was found out that the combination of chiral rhodium catalyst and chiral auxiliary derived from lactic acid gives high diastereoselectivity for C-H insertion reactions(Scheme 4). Applying this method to α-diazo β-ketoester 6, the C-H insertion reaction proceeded to give β-ketoester 21 with good diastereoselectivity. The determination of the absolute configulation and the application to total syntesis of Altemicidin is now under investigation.
Tetrodotoxin (TTX) is a toxic principle of puffer fish poisoning. TTX is a useful biological tool because of its specificity for blocking voltage-dependent sodium channels. 5,6,11-Trideoxy TTX (1), one of the congeners of TTX, has also been isolated from ovaries of puffers, Fugu poecilonotus, by Yasumoto et al. in 1995 and received significant interests in view of biosynthetic relationship with TTX. Synthetic studies of TTX and its congeners have been extensive due to their biological activities as well as their molecular complexity consisting of a quaternary carbon center attached to an amino group, concomitant polyol system, a cyclic guanidine containing an aminal, and an ortho acid for TTX or a δ-lactone for some of the congeners. In this report, we wish to describe the first total synthesis of 5,6,11-trideoxy TTX (1) and its 4-epimer 2. The synthesis was commenced with the known triol 7, prepared from (-)-quinic acid in 4 steps, which was converted to Strecker precursor 6 in 6 steps. 6 was subjected to an asymmetric transferring Strecker synthesis for the diastereoselective construction of a quaternary carbon center involving an amino group at C8a. Installation of a hydroxy group to C7 was accomplished by the Mislow-Evans rearrangement to give 20. Initial hydrogenation of 20 for the introduction of β-methyl group at C6 and subsequent pyrrolidine ring opening via lactam 24 gave alcohol 25. The hydroxyethyl group of 25 was transformed to a vinyl group in 3 steps to give 26. Inversion of the hydroxy group at C7 was performed by oxidation to a diketone followed by diastereoselective reduction to give cis-diol 4, This was converted to aldehyde 28, which, upon treatment with TMSCN in the presence of Et_3N, gave a cyanohydrin as an inseparable mixture of diastereomers. The mixture was guanidylated to give 29. Ozonolysis of the desired (9S)-29 followed by exposure to 20% TFA afforded a mixture of 5,6,11-trideoxyTTX (1), and its 4-epimer 2 and anhydro derivative 31. Thus, the total syntheses of 1 and 2 were accomplished.
Esterifications, sulfonylations, amide formations, and silylations are well recognized as frequently used reactions for the natural product synthesis and the process chemistry. As an "endless software development", we introduce herein our recent studies in this area. (i) Esterification, thioesterification, and amide formation using novel condensation agents. We introduce the titled condensations between 1: 1 mixture of carboxylic acids and alcohols, thiols, or amines using Me_2NSO_2Cl / Me_2NR and p-TsCl / N-methylimidazole reagents. These methods are currently utilized in the process chemistry and would be applied to the natural product synthesis. (ii) Pyridine-free sulfonylation methods for alcohols utilizing sterically uncrowded tertiary amine catalysts. As a promising candidates for the replacement of conventional TsCl / pyridine-method (low reactivity, requirement of ca. 10 eqiuv of Py, and prone to the undesirable side chlorides formation), we developed efficient pyridine-free improved methods, which utilized Me_3N・HCl / Et_3N and Me_2N(CH_2)_nNMe_2 as a key protocol. These methods are currently utilized in the natural product synthesis and an industrial scale production of fine chemicals. (iii) Catalytic methods for the preparation of silyl ethers and enol silyl ethers. The presence of catalytic TBAF (0.02 equiv) significantly promoted the silylation of alcohols using a variety of available R_3Si-N, R_3Si-H, and (R_3Si)_2 under mild conditions. A novel mild and powerful silylations agent, Si-BEZAs (silyl benzamides) with pyridinium triflate catalyst is also introduced. An efficient method for the preparation of enol silyl ethers using a novel agent, silazanes with NaH or DBU catalyst is also described, wherein TMS and TBDMS groups were smoothly introduced into ketones and aldehydes under mild conditions.
Nearly a half-century ago, Blomquist predicted that trans-cycloalkene should have inherent chirality. Since then, medium-sized cycloalkenes with stable planar chirality have aroused much theoretical and synthetic interest. On the other hand, their amine congener with sole planar chirality has not appeared so far, to the best of our knowledge. Here we wish to report the discovery of the remarkably stable planar chirality in a simple cyclic amine and amide 7 and their utility for asymmetric synthesis. Recently, we have reported the finding of planar chirality in nine-membered cyclic ether 1. Based on this result, we envisioned that the amine analogues should also have planar chirality and so they would be valuable chiral molecules. To realize this chemistry, we chose N-tosyl amide 7a as a target molecule, and it was successfully synthesized from neryl acetate in four steps: allylic oxidation with SeO_2, Mitsunobu reaction with methyl-N-(p-toluenesulfonyl) carbamate, base-promoted deprotection, and intramolecular Mitsunobu reaction. Analytical and semi-preparative scale HPLC using a chiral column successfully separated both enantiomers of 7a, which separation is ascertained by CD analysis of both fractions. It should be noted that the enantiomeric purity of isolated enantiopure 7a (crystal) is not significantly changed at ambient temperature for a week at least. Enantioenriched 7 thus obatained is valuable as a novel type of chiral building block. For example, hydroboration of enantio-enriched (S)-7a (>98% ee) with 9-BBN provides the stereomerically pure alcohol (3S,4R)-9 in 92% yield. The transannular reaction also proceeds in a stereospecific manner, in which a Pd(II)-catalyzed Cope rearrangement of (R)-7a (>98% ee) afforded pyrrolidine (3R,4S)-10 (>98% dr, >98% ee) in 87% yield. These results clearly show that the planar chiral cyclic amine is a valuable source for optically active heterocyclic compounds.
Although there are a number of examples of 6π-azaelectrocyclization reactions, studies on the reactivity of 1-azatrienes toward azaelectrocyclization are limited, and applications of this reaction to natural product synthesis are also limited. Recently, we found that (E)-3-carbonyltrienal compound A irre-versibly reacted with the specific lysine residue of phospholipase A_2 (PLA_2) to produce 1,2-dihydropyridine via facile 6π-azaelectrocyclization leading to inactivation of the enzyme (Figure 1). Moreover, we found that the presence of both the C4-carbonyl group and the C6-alkenyl group in 1-azatrienes significantly contributed to the acceleration of the azaelectrocyclization (Figure 2). Next, we successfully realized the asymmetric 6π-azaelectrocyclization by using a chiral cis-aminoindanol (-)-a, which play not only as a chiral auxiliary but also as a nitrogen source (Figure 3). We also found 7-substituted aminoindanol (-)-b was more effective in the reaction with more general aldehydes 2 and 3 (Table 1). The aminoalcohol (-)-b made possible to control the direction of the π orbital rotation in concerted disroratory pericyclization of the azatrienes (Figure 4). In order to achieve further efficient synthesis of poly-substituted piperidine alkaloids, we investigated one-pot asymmetric 6π-aza-electrocyclization. Heating a mixture of vinyl stannane, vinyl iodide, and cis-aminoindanol derivative (-)-b in the presence of Pd (0) catalyst successfully provided the expected compound 3b in good yield and high stereoselectivity. This one-pot asymmetric 6π-azaelectrocyclization protocol can be applied into wide range of compounds (Figure 6). We also established stereocontrol synthesis of chiral 2,4,6-substituted piperidine derivatives (Scheme 1, 2). We actually applied these methods to the formal asymmetric synthesis of indole alkaloid, 20-epiureine and to the asymmetric synthesis of indolizidine alkaloid, dendroprimine (Scheme 3, 4).
In organic synthesis, a plenty of human efforts is necessary to supply synthetic intermediates. The time and energy in simply repeating processes in syntheses can be reduced utilizing automated synthesis apparatus. It is expected that laboratory automation can realize highly efficient, reproducible and safe synthesis of organic compounds. We report here the total synthesis of (±)-baccatin III (2) utilizing automated synthesizers. Initially, the synthetic routes of the A-ring 15 and C-ring 19 from geraniol (11) were established. The all synthetic steps of the A and C-rings were successfully applied to the automated synthesizer by optimizing the reaction conditions adequate to the synthesizer. We also developed the Ti(III)-catalyzed radical cyclization of epoxy-alkene and it was found to be effective as the stoichiometric system previously reported. The synthetic route of the B-ring cyclization precursor 27 from A-ring 15 and C-ring 19 was established. All reactions were accomplished utilizing a high performance solution phase automated synthesizer that we had recently developed. The crucial 8-membered ring formation was effectively assisted by micro-wave irradiation. It was found that the intramolecular alkylation of 27 was accelerated in the presence of excess amount of LiN(TMS)_2. The reaction period was dramatically decreased (10h→15min). Regio- and stereo-selective allylic oxidation and dihydroxylation of exo-alkene provided 30. Oxetane formation without deconjugation of Δ-11 to exo-alkene was crucial. Treatment of 31 with i-Pr_2NEt in HMPA provided the desired 32. After oxidation at 9, 13-positions of 32, we accomplished the total synthesis of (±)-baccatin III (2).
Mycalamide A was originally isolated from a New Zealand marine sponge in 1988. Mycalanide A exhibits immunosuppressive activity by suppressing T-cell more effectively than FK506. In addition, mycalamide A possesses a quite unique structure bearing a tetrahydropyran ring and a trioxadecalin ring system bridged by an N-acyl aminal bond, therefore mycalamide A has attracted much attention of synthetic organic chemists. Despite the availability of many synthetic routes for mycalamide A, there still exists a need to develop strategies more efficient than those currently in existence. As a result of our ongoing studies on natural product synthesis, we became interested in developing a novel synthetic route for the synthesis of mycalamide family. (I) Development of a novel δ-lactone synthesis In order to prepare the left segment 7 of mycalamide A, a unique one-pot nucleophilic substitution-lactonization process was created using cyclopropane derivative B and a phenyl selenide. This technique was applied to the corresponding cyclopropane alcohol, derived from 5, providing δ-lactone 6 in good yield. (II) Development of a unique catalytic system in cross-aldol reactions For stereoselective construction of the right segment 12 of mycalamide A, a novel catalytic system, Yb(OTf)_3-TMSCl, was devised using a cyclohexanone and a ketene silyl acetal as a model case. The desired aldol products 9 was obtained as a single stereoisomer employing the above methodology. (III) Total synthesis of mycalamide A A total synthesis of mycalamide A has been achieved by coupling left segment 7 and right segment 12 followed by functional group manipulations.
Tubelactomicin A, B, D, and E (1-4) are novel macrolide antibiotics, isolated from the culture bloth of Nocardia sp. These antibiotics show potent and specific antibacterial activities against acid-fast bacteria including drug-registant strains. Tubelactomicins are tricyclic macrolides, consisting of a trans-fused octahydronaphthalene substructure (the lower-half part) with six contiguous stereogenic centers, and a highly functionalized 16-membered lactone (the upper-half part). Interested in these unique structures and biological activities, we involved in studies on the total synthesis of tubelactomicins, which have been completed recently. Each tubelactomicin is expected to be synthesized in a convergent manner by an assembly of the 16-membered lactone unit 5 or 6 and an octahydronaphthalene unit 7, 8 or 9, respectively. The syntheses of the upper-half segments 5 and 6 are as follows. Methyl (R)-lactate was converted into a hexanal derivative 10. Compound 10 was then subjected to a Baylis-Hillman reaction or a Wittig reaction to introduce the two kinds of trisubstituted olefin parts. The resulting compounds 11 and 15 were transformed into the upper-half segments 5 and 6, respectively, by standard functional group manipulations, including a syn-stereoselective Evans aldol reaction. The lower-half segments 7-9 were synthesized from diethyl (R)-malate. As a key step for the construction of the highly functionalized trans-fused octahydronaphthalene, an intramolecular Diels-Alder (IMDA) approach was adopted. The substrates for the IMDA reaction were synthesized by a six-step sequence, including Horner-Emmons reaction of aldehyde 29 with phosphonates 24 or 27. In all cases, the IMDA reactions provided an endo-cycloadduct with excellent π-facial selectivity and good endo-selectivity. The lower-half segments 7-9 were synthesized from the endo-cycloadducts 36-endo or 37-endo. The assemblies of the upper-half segments 5 or 6 and the lower-half segments 7, 8 or 9 were achieved by a sequence of Stille coupling and macrolactonization. Finally, thus obtained macrolides 43-46 were converted into (+)-tubelactomicin A, B, D, and E (1-4).
In 1997, Tanahashi et al. isolated four phenolics from the cultured lichen mycobiont of Graphis scripta var. pulverulenta, which were called graphislactones A-D. Since two of them, graphislactones C and D, were found to exhibit anti-tumor activity against the human bladder cancer cell, our interest has focused on the total synthesis of the graphislactones. In this report, we describe their synthesis through a Pd-mediated biaryl coupling reaction of phenyl benzoate derivatives as the key step. For their structural features, the graphislactones A-C have highly oxygenated 6H-dibenzo[b,d]pyran-6-one skeletons. To obtain these compounds, we envisioned phenyl benzoate derivatives as good precursors. These esters would be prepared by a simple esterification between the corresponding phenols and benzoic acids furnishing the required functionalities on each aromatic ring. Initially, as the precursors of the six-membered ring lactones 15, 20, and 22, the phenyl benzoate derivatives 14, 19, and 21 were prepared respectively. The Pd-mediated biaryl coupling reaction was successful to afford the desired products, which were transformed into graphislactones through the deprotection process. On the other hand, graphislactone D has a different ring system from the above graphislactone A-C. We thought that the core skeleton, 5H-dibenzo[c,e]oxepin-7-one, would be synthesized by the reconstruction of the lactone ring from the 6H-dibenzo[b,d]pyran-6-one. Thus, the lactone 25 was envisioned as a key intermediate for graphislactone D. After transformation into 25 by a similar route to graphislactone C, the treatment of the resulting 25 with an excess amount of K_2CO_3 in MeOH was effective for the direct formation of the seven-membered ring lactone 27. Final deprotection of the benzyl group was also successful, and the synthesis of graphislactone D was accomplished. The synthesis of ulocladol, which is significantly related to graphislactone D, will be also discussed.
Stachyflin (1) was isolated from the culture broth of Stachbotrys sp. RE-7260 by Shionogi's group in 1997 and reported to exhibit potent antivairal activity against influenza A/WSN/33 (H1N1) virus in vitro with an IC_<50> value of 0.003μM with a novel mechanism of action. The structure of 1 involves a unique pentacyclic ring system with five asymmetric carbon centers, in which an etheral oxygen is joined at the cis-fused decaline juncture. This attractive biological activity and its intriguing structure prompted us to embark on a project directed towards the total synthesis of stachyflin (1). Our synthetic strategy for stachyfline (1) is shown in Scheme 1, which features the domino epoxide-opening/carbocation rearrangement/cyclization reaction (4→5) as the key step to elaborate its unique benzo[d]xanthene skeleton. The synthesis commenced with Birch reductive alkylation between the known Wieland-Miescher ketone derivative (+)-2 and the benzyl bromide 3, synthesized from 3,5-dihydroxybenzoic acid, to give a mixture of the expected coupling products 15 and 16 in moderate yield. After several steps involving stereoselective hydrogenation, the acid catalyzed isomerization of exocyclic olefin to the corresponding endocyclic olefin, and epoxidation reaction, the precursor 4 for the key reaction was obtained. Next, the key domino epoxide-opening/carbocation rearrangement/cyclization reaction of epoxide 4 was explored. After several experiments, we found that the expected domino reaction proceeded by exposure of 4 to BF_3・Et_2O and trifluoroacetic acid (TFA) in dichloromethane, which led to the formation of the desired cyclized product 5 in reasonable yield (41%). Further modification of 5 including deprotection of 3,4-dimethoxybenzyl (3,4-DMB) and methyl groups would accomplish the total synthesis of optically active stachyflin (1).
Arabinofuranoside is found in the plant and mycobacterium as arabinan, whose 5-membered ring structure is absolutely different from 6-membered pyranose ring in human oligosaccharide. Its biosynthesis is the one of the new target for anti-mycobacterial agents. β-Arabinofuranoside is located at the non-reducing terminal in mycobacterial cell wall arabinan 1 (Figure 1), even though mainly α-linkage of arabinofuranosides existed in these arabinans. Because β-arabinofuranosides has a 1,2-cis stereochemistry, the synthesis of β-arabinoluranosidic linkage is troublesome compared to that of α-isomer. Here, we wish to report 1) the effect of protections of glycosyl donors on the stereoselectivity of arabinofuranosylation and 2) the synthesis of the terminal hepta-arabinofuranoside of mycobacteral arabinan 1. 1) Development of stereoselective arabinofuranosylation First of all, we examined the β-selective glycosylation of perbenzyl-d_7 protected arabinofuranosyl donor using our novel methodology for high-throughput screenings (HTS) of O-glycosylation conditions (Figure 1). However β-selectivity could not be improved from 〜110 screenings examined on the effect of solvent and temperature (Scheme 1). So, we synthesized several donors with various non-participating protections possibly existing in different shape. Both α- and β-stereoselective arabinofuranosylations were achieved only by changing of the protection of donor (Table 1, 2). The combibation of 3,5-tetraisopropyldisiloxanylidene protected arabinofuranosyl donor (2i, 2j) with the matched acceptor 3 gave β-isomer predominantly (Table 3). 2) Synthesis of branched heptaarabinofuranoside 1 of mycohacterial cell wall arabinan For the synthesis of 1, we designed the matched pentaarabinofuranoside acceptor 11, which was prepared from methyl α-D-arabinofuranoside through the double α-arabinofuranosylation of trisaccharide 9. Double β-arabinofuranosylation of 9 with the donor 2i was achieved stereoselectively to give heptaarabinofuranoside derivative 12, which was deprotected as usual to give the target branched oligoarabinofuranoside 1 (Scheme 2).
During our search for bioactive natural products from tropical plants, we investigated the chemical constituents of Curcuma parviflora Wall. (Zingiberaceae) collected in north-east Thailand. This plant is a perennial herb widely distributed over a forest area of the northern part of Thailand, and is used as an ornamental plant, and is edible, and also it has been said to be used for detoxification of scorpion bites in certain areas. Extensive investigation of extracts of the underground part of this plant led to the isolation of cytotoxic sesquiterpene-dimers, parviflorenes A-I (1-9), and their structures were elucidated by spectroscopic studies including X-ray crystal analylsis. Parviflorene A (1) and compounds 2,4,6, and 7 possess an unprecedented unsymmetrical bis-cadinane skeleton, while compound 3 is a dimer of cadinane and iso-cadinane, and compound 5 possesses another novel carbon framework consisting of two cadinanes with different bond-connection. Studies on determination of absolute stereochemistry of parviflorenes A (1), B (2), D (4), F (6), and G (7) were also described here. These new compounds showed cytotoxicity against P388 murine leukemia cells and other tumor cell lines. Parviflorenes A (1) and F (6) were cytotoxic against all tested tumor cell lines in the human cancer cell line panel assay, and DNA microarray and real time PCR studies revealed that parviflorene F (6) enhanced the gene expression of TRAIL-R2 by 4.9 times at the concentreation of 8μg/mL.
Structure analyses of underivatized neutral oligosaccharides are systematically performed by UV matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI TOF MS) and UV-MALDI ion-trap time-of-flight mass spectrometry (ion-trap/TOF MS) acquired in negative-ion mode. Interestingly their fragmentation significantly differ each other. In postsource decay (PSD) in UV-MALDI TOF MS, cross-ring cleavage at the reducing terminal predominates. On the other hand, glycosyl bond cleavage (C-type fragmentation) takes place preferentially in collision induced dissociation (CID) in UV-MALDI ion-trap/TOF MS. The cross-ring cleavage in PSD similar to that in in-source decay occurs via a non-ergodic path characteristic of the UV-MALDI process itself. During ion-trap/TOF MS experiments the deprotonated molecular ions survive during several tens of milliseconds after CID event because the high internal energy chlorinated precursor ions are cooled by collisional cooling in the ion trap. The results obtained suggest that: the PSD from the chlorinated precursor ion in UV-MALDI TOF MS might proceed as a two-step reaction; in the first, a high internal energy deprotonated molecular ion is generated as a reaction intermediate during the flight in the drift tube, and in the second, the rapid decomposition from the deprotonated molecular ion takes place.
Amphezonol A (1), a novel polyhydroxyl compound, was isolated from a marine dinoflagellate Amphidinium sp. (Y-72), and the structure was elucidated on the basis of 2D NMR data including HSQC-TOCSY and INADEQUATE spectra (920MHz) using the ^<13>C-enriched sample. Amphezonol A (1), C_<62>H_<114>O_<24>, was the first member of a new class of polyketide natural products consisting of a C_<60>-linear aliphatic chain with twenty-one hydroxyl groups, and two tetrahydropyran and a tetrahydrofuran rings. Cloning of polyketide synthase (PKS) gene from a dinoflagellate Amphidinium sp. was attempted and PKS gene fragments were obtained by PCR amplification from degenerated primer sets designed on the basis of the conserved amino acid sequences in known type I PKSs. The PCR analysis, using primer sets designed from these PKS gene fragments, revealed that these DNA sequences exist only in the dinoflagellates producing polyketides.
Axinelloside A was isolated from the lipophilic extract of the Japanease marine sponge Axinella infundibula collected off Shikinejima Island as a strong human telomerase inhibitor (IC_<50> 2.0μg/mL). The ^1H and ^<13>C NMR spectra suggested axinelloside A to be a highly sulfated lipopolysaccharide. The sugar composition was determined by GC and GC/MS analyses of the alditol acetate derivatives of the acid hydrolysates, whereas fatty acids were identified by FABMS and NMR data of both alkaline and acid hydrolysates. Together with these results, interpretation of 2D NMR data of axinelloside A could determine not only the sequence of sugars but also the position of fatty acids (Figure 3). The deuterium isotope shift experiment indicated that the 19 oxygen-linked carbons were sulfated. MS analyses confirmed the complete structure of axinelloside A including the glycosyl sequence. Axinelloside A consists of twelve sugars, e.g., a scyllo-inositol, a D-arabinose, five D-galactoses, and five L-fucoses, together with a (R)-3-hydroxyoctadecanoic acid, three (E)-2-hexadecenoic acids, and 19 sulfates (Figure 7). The molecular formula of axinelloside is C_<137>H_<219>O_<117>S_<19>Na_<19> as the 19 sodium salt.
Based on 1,2,3-triols 1a〜d, 1,2,3,4-tetraols 2a〜h, and 1,2,3,4,5-pentaols 3a〜p, NMR databases with four types of profile-descriptors (^<13>C-, ^1H-, and ^1H(OH)-chemical shifts and vicinal spin-coupling constants) for contiguous polyols are reported. To assess systematically the relative values of these databases, a case study has been conducted on heptaols 4a〜d, through which the γ-and δ-effects have been recognized to refine the ^<13>C and ^1H chemical shift profile predicted via an application of the concept of self-contained nature. The magnitudes of γ- and δ-effects depend on a specific stereochemical arrangement of the functional groups present in both the inside and outside of a self-contained box and are significant only for the stereoisomers belonging to a specific sub-group. With the exception of the stereochemical arrangement of functional groups belonging to a specific sub-group, the γ- and δ-effects can, at the first order of approximation, be ignored for the stereochemical analysis of unknown compounds. For the stereoisomers belonging to a specific sub-group, it is necessary to refine, with incorporation of the γ- and δ-effects, the profile predicted at the first order of approximation. With use of heptaols 4a〜d, the values of ^3J_<H,H> profiles have been assessed. Three methods using profiles of ^3J_<H,H> constant with three contiguous configurations, profiles consisting of three contiguous ^3J_<H,H> constants, and profiles consisting of two contiguous ^3J_<H,H> constants, have been developed. A stereochemical assignment of heptaols 4a〜d based on these three ^3J_<H,H> profiles are conducted very simply and clearly. The approach by using the logics with Universal NMR Databases (^<13>C-δ, ^1H-δ, ^3J_<H,H>) developed for a case of contiguous polyols should be applicable for other structural cluster in (acyclic) natural products as well.
GRP78 acts as a molecular chaperone in endoplasmic reticulum (ER) by associating transiently with incipient proteins as they traverse the ER and aiding in their folding and transport. Furthermore, the GRP78 protein is also induced under various stress condition such as glucose starvation, inhibition of protein glycosylation by tunicamycin, perturbation of ER function and protein movement by brefeldin A, and suppression of ER-calcium-ATPase pump by thapsigargin. The enhancement of ER stress response (also known as the unfolded protein response) takes part in the resistant mechanism against chemotherapy and hypoxic stress in solid tumor. The ER stress response causes an increase in gene expression of a number of ER chaperones such as GRP78 and GRP94. Thus, substances that directly down-regulate grp78 transcription are expected to be useful drugs for the treatment of solid tumor. In the course of our screening for inhibitors of luciferase expression, which is regulated under the control of GRP78 promoter, by the treatment of tunicamycin, we isolated a novel compound designated as prunustatin A from Streptomyces violaceoniger 4521-SVS3 as a down-regulator of the grp78 gene. The structure of prunustain A (C_<34>H_<40>N_2O_<12>) was elucidated on the basis of spectral analyses including 2D NMR (HMQC, DQF-COSY, HMBC). Prunustatin A consisted of a 3-formylamino-2-hydroxybezoic acid moiety as a chromophore and 15-membered macrocycles composed of an L-threonine, an L-lactatic acid, a 2R,3R-4-hydroxy-2,2-dimethyl-3-oxo-5-phenylpentanoic acid and a 2S,3S-2-hydroxy-3-methylpentanoic acid moieties. In the evaluation system we employed, prunustatin A reduced this reporter gene expression at the IC_<50> value of 1.9nM. Prunustatin A also completely inhibited the induction of endogenous GRP78 protein induced by 2-deoxyglucose at the concentration of 100nM. Prunustatin A, glucose starvation and 2-DG treatment alone did not induce cell death in cancer cells. However, prunustatin A specifically elicited global cell death when combined with 2-deoxyglucose. Thus, it is expected that prunustatin A would be a promising cancer chemotherapeutic agent against solid tumor.
In 1972, cymbidine A (1) was isolated from the 80% ethanol extract of the orchidaceous plant Cymbidium goeringii as a hypotensive and diuretic principle. In those days, compound 1 was suggested to contain several amino acids and amino sugars, but further structure elucidation was difficult because of its structural complexity. Our recent investigation on cymbindine A (1) resulted in the elucidation of the whole structure. The molecular formula of cymbidine A (|α|_D^<22>-28.4°) was found to be C_<39>H_<63>N_7O_<20> by HRESIMS and ^<13>C NMR data (Table 1). Based on the amino acid analysis of the hydrolysate of 1 (Figure 2), valine (Val), glutamic acid (Glu), alanine (Ala) and an unusual amino acid were detected. The HMQC revealed the direct correlations between ^<13>C and ^1H signals (Table 1). The analysis of ^1H-^1H COSY and/or TOCSY indicated the partial structures a〜g (Figure 3) in 1. The partial structures were connected by HMBC correlations to give the plane structure of 1 (Figure 3). The absolute configurations of the three amino acid residues in 1 were determined to be L-Val, D-Glu and D-Ala by the advanced Marfey method. The unusual amino acid was found to be 2,6-diaminopimelic acid (A_2pm) whose meso stereochemistry was assigned by comparison of HPLC retention time with those of DD-, LL- and meso-A_2pm. The relative configurations of two aminosugars were elucidated by the analysis of ^1H-^1H spin coupling constants as well as NOESY spectral data (Figure 4), to exhibit that these aminosugars were N-acetylglucosamine and 1,6-anhydro-N-acetyl muramic acid.
It is well known that the anti-apoptotic protein Bcl-2 and its homologue Bcl-X_L are overexpressed in many human cancers and play a crucial role in cancer progression. Therefore, the functional blockade of Bcl-2/Bcl-X_L will be a promise for novel anticancer therapeutics by way of improving tumorigenesis. In the course of screening for an inhibitor of Bcl-X_L, anti-apoptotic function using human SCLC Ms-1 cells that overexpress Bcl-X_L, we found Incednine (1) in the fermentation broth of the strain Streptomyces sp. ML694-90F3. 1 was isolated as a HCl salt by using the Centrifugal liquid-liquid Partition Chromatography, effectively. The planar structure of 1, C_<42>H_<63>N_3O_8 ([M+H]^+, m/z 738.4680, Δ-1.3mmu), was elucidated on the basis of the various NMR spectra data including TOCSY experiments (Fig. 1). The relative stereochemistry of 1 was determined by the analysis of NOE experiments and coupling constants (Fig. 2). The conformation of the aglycon was confirmed by Discovery III-computations. The absolute stereochemistry of 1 was established by modified Mosher's method and X-ray crystallographic analysis (Fig. 3, 4). Incednine has a unique skeletal structure, "enol-ether amide" in the 24-membered macrolactum core, with an attachment of a disaccharide. Bcl-X_L-overexpressing Ms-1 cells displayed resistance to several anti-tumor agents, however, anti-tumor agents-induced cell death was observed only when cells were treated with 1. Overexpression of Bcl-X_L inhibited cell death in Bax-overexpressing HEK293T cells by forming a complex with Bcl-X_L and Bax, whereas it failed to inhibit cell death in the presence of 1 without affecting the formation of Bcl-X_L and Bax complex. The inhibitory mechanism of anti-apoptotic Bcl-X_L by 1 is now under investigation.
The ComX pheromone is a posttranslationally modified oligopeptide that stimulates natural competence, controlled by quorum sensing in Bacillus subtilis. The ComX pheromone possesses a tryptophan residue, which is modified by isoprenylation, but the precise structure is not known. To elucidate the precise nature of the ComX modification, we determined the molecular formula of ComX_<RO-E-2> pheromone from the strain RO-E-2, and assumed the modification might be a simple substitution of a tryptophanyl proton by a geranyl group. Then we synthesized the ComX_<RO-E-2> peptides containing 6 kinds of geranylated tryptophans. Although each synthetic peptide had similar hydrophobicity to the natural pheromone, but none had biological activity. Using a previously reported Escherichia coli expression system, we optimized the culture and purification conditions. We isolated the ComX_<RO-E-2> pheromone, observed NMR spectra, and determined the structure for the tryptophan residue in ComX_<RO-E-2> pheromone. Furthermore, we carried out a conformational search, and proposed its absolute stereochemistry. To confirm the stereochemistry, we synthesized the corresponding ComX_<RO-E-2> peptide and other diastereomers. The proposal ComX_<RO-E-2> peptide only showed an identical ^1H NMR spectrum to that of the natural pheromone, and a very similar biological activity. We have thus succeeded in synthesizing the ComX_<RO-E-2> pheromone and determining the absolute structure of the pheromone. The novel posttranslational modification of the ComX_<RO-E-2> pheromone, showing that addition of a geranyl group to a tryptophan residue results in the formation of a unique ring structure.
It has been known that some aquatic organisms change their morphology to defend themselves in response to kairomones released from their predators, although very few kairomones are chemically identified. Scenedesmus, unicellular fresh-water phytoplankton, forms colonies in the presence of its grazer, Daphnia. Hessen and van Donk (1993) discovered the involvement of a chemical substance released from the Daphnia in stimulation of colonies. The addition of filtered medium from a Daphnia magna culture to unicellular Scenedesmus subspicatus achieved within a few days morphological change into 2-, 4-, and 8-colonies, while the controls remained unicellular. Their report has aroused the interest of many scientists to attempt to identify the kairomone, but the chemical substance that triggers this behavior has not been identified. Here we report the identification of the Daphnia kairomones as aliphatic sulfates that cause the morphological change in a unicellular green alga Scenedesmus gutwinskii var. heterospina (NIES-802) at 0.1-1000ng/ml concentrations. The kairomones were synthesized to rule out the possibility that the isolated substances are inactive and they might still be contaminated with a minute amount of 'super active compound'.
Marine microorganisms are now recognized as a promising source for the development of new pharmaceuticals. We have been collecting "new" bacteria from the marine environment, mainly from marine invertebrates. ("New" means that the homology of the 16S rDNA sequence of the collected bacterium is less than 98% by a BLAST search.) In the course of screening for antitumor substances from "new" bacteria, we found the cyclic peptide compound, mechercharmycin A (1), and its linear congener, mechercharmycin B (2), from the new marine-derived Firmicutes, Thermoactinomyces sp. YM3-251. We also found urukthapelmycin A (3) from Thermoactinomyces sp. YM11-542, which was closely related to the mechercharmycins-producing strain. Mechercharmycin A (1) is a cyclic peptide bearing four oxazoles, a thiazole and the peptide moiety. The connection of the peptide moiety was easily determined as an order of dehydroalanine, valine and isoleucine by the analyses of 2D NMR data. The remaining structure, which is presumed to have contained four consecutive oxazoles and a thiazole, could not be fully determined by the NMR studies, because the lack of any correlation signals between the oxazole rings by HMBC analyses. Consequently, the structure of 1 was determined by an X-ray crystallographic analysis. The absolute configuration of 1 was determined from X-ray anomalous dispersion of the S atom. Mechercharmycin B (2) is a linear congener of 1 which has four oxazoles and the same peptide moiety as that of 1. The connections of the oxazole-2-carboxylic acid methyl ester and of the oxazole-4-carboxylic acid amide could not determined by the NMR studies, but instead by a fragment analysis of the LC-MS/MS data, enabling the complete structure of 2 to be determined. The absolute stereochemistry of 2 was determined by the Marfey's method and by a chiral HPLC analysis. The structure of urukthapelmycin A (3) was determined from an analysis of the NMR data and by a comparison of the spectral data with 1. The absolute stereochemistry of 3 was determined by the same procedure as that used for 2. The antitumor activities of 1 and 3 were relatively strong, the respective IC_<50> values for A549 (human lung cancer) cells being 4.0×10^<-8>M and 1.2×10^<-8>M, under our assay conditions, while 2 did not exhibit any inhibitory activity toward these cells even at 4×10^<-6>M. The cyclic structure of 1 and 3 must have been essential for its strong antitumor activity. Further investigations of the antitumor potential of 1 and 3 are in progress.
Beautiful flower colors are mostly due to anthocyanins and the mechanism of color development, especially of the blue color, has been attracting much interests. The petal color of blue morning glory, Ipomoea tricolor cv. Heavenly Blue, changes from purplish red in the bud to blue in the fully open flower. The same pigment, heavenly blue anthocyanin (HBA), is responsible for both colors. Therefore, the color change might be estimated by pH change of the vacuoles. By direct measurement of vacuolar pH using a pH-sensitive microelectrode, this color change was clarified to be caused by an unusual increase in vacuolar pH from 6.6 to 7.7 in the colored adaxial and abaxial cells. To clarify the mechanism underlying the alkalization of epidermal vacuoles in the open petals we focused on vacuolar H^+-ATPase (V-ATPase), H^+-pyrophosphatase (V-PPase) and an isoform of Na^+/H^+ exchanger (NHX1). We isolated red and blue protoplasts from the petals in buds and fully open flower, respectively, and purified vacuolar membranes. The membranes contained V-ATPase, V-PPase and NHX1, which were immunochemically detected, with relatively high transport activity. Na^+/H^+ exchanger could be detected only in the vacuolar membranes prepared from flower petals and its protein level was the highest in the colored petal epidermis of the open flower. These results suggest that the increase of vacuolar pH in the petals during flower-opening is due to active transport of Na^+ and/or K^+ from cytosol into vacuoles through a sodium- or potassium-driven Na^+(K^+)/H^+ exchanger NXH1 and that V-PPase and V-ATPase may prevent the over-alkalization. This systematic ion transport maintains the weakly alkaline vacuolar pH, producing the sky-blue petals.
Although there are many membrane-active organic compounds including ionophores and channel-forming molecules, the general way to define the conformations of the membrane-bound compounds is not yet established. To address this problem we employed SDS micelles and fast-tumbling bicelles, both of which allow us to measure high-resolution liquid NMR of bound molecules due to their fast reorientation in suspension. We have applied these method to the conformation analyses of amphidinol 3 (AM3) and salinomycin. Salinomycin is a representative ionophore antibiotic. Conformations of salinomycin determined in SDS micelle and bicelle were found to be different. This is possibly attributable to the morphological difference of micelle and bicelle; the former lacks bilayer portion and has high curvature, while the latter is thought to have bilayer structures. Then we investigated the structure of AM3 in membraneous environments. AM3 is a unique dinoflagellate metabolite bearing potent membrane-permeabilizing activity. We previously showed that AM3 forms ion-channel with the inner diameter of 2.0-2.9nm. NMR-constrained modeling experiments in micelle have revealed for the fist time that amphidinols generally take a hairpin configuration, which plausibly account for their potent antifungal and other membrane permeabilizing activities. Conformational study of AM3 in bicelle is now in progress, and the results will be presented.
Nyctinastic movement of leguminous plants is controlled by the glucosylated leaf-movement factor, whose concentration changes throughout the day, regulated by β-glucosidase. Therefore, in order to understand the controlling mechanism of the movement by circadian clock, purification of this key enzymeis essential. Many isozymes of β-glucosidase are known to exist in plant body. To purify the key enzyme from this mixture, affinity chromatography is a powerful tool. We have tried two affinity gel 4 and 5, which is an usual tool used for purification of β-glucosidase. But they were both not effective, from the luck of aglycon part or the low inhibition constant. According to this experiment, we concluded that affinity ligand should be an analog of the leaf-movement factor, and at the same time it should be a strong inhibitor. Gluconoamidine is known to show strong inhibition toward β-glucosidase. Thus, we have planed to synthesize 7, an analog of leaf-movement factor with gluconoamidine part, instead of glucose as the glucoside part. Several methods are known for the synthesis of gluconoamidine compound using thionolactam(6) and amine compound. But there are several restriction. 1) Benzyl protected sugar derivative are necessary 2) Needs of a reactive amine group. And also, in some cases the epimerization at C2 of the sugar moiety occurs. To overcome these restrictions, we have developed a novel method for the synthesis of gluconoamidine compound. And by employing this method we have synthesized 7. Compound 7 showed strong and selective inhibition toward commercially available β-glucosidase. Encouraged by this result we synthesized an affinity gel 16, using 7 as a ligand. Purification of the crude enzyme, extracted from Lespedeza cuneata, by affinity gel 16 was effective. We are now working on further purification of the key enzyme.