Cyclic dipeptides (CDPs, diketopiperazines) and their derivatives are widely distributed in nature as secondary metabolites. Although some of dehydro-CDPs are known as cell cycle inhibitors, their effective syntheses have not been reported. We found that Streptomyces albulus KO23, an albonoursin-producing actinomycete, had a biosynthetic pathway from cyclo (Leu-Phe) to albonoursin, cyclo (ΔLeu-ΔPhe) by the fed-batch culture and the resting-cell experiments. And this enzyme activity was found to be effectively extracted in the cell-free extract of this actinomycete. This is the first report for the dehydrogenation of amino acid residues at α,β-positions in CDPs. Furthermore, this enzyme system enables us to synthesize several didehydro- and tetradehydro-CDPs from the corresponding CDPs. Among dehydro-CDPs prepared, the tetradehydro-CDPs exhibited cytotoxicity, while the didehydro-CDPs had no activity, indicating that dehydrogenation at α,β-positions of both amino acid residues in CDPs is required for cytotoxicity. Based on the above results, we speculated that a tetradehydro-CDP prepared from a didehydro-CDP exhibiting cytotoxicity might be a potent cytotoxic compound. Dehydrophenylahistin synthesized by this enzyme system from (-)-phenylahistin, which was recently reported to be a new cell cycle inhibitor, exhibited 1000 times higher inhibitory activity toward the first cleavage of sea urchin embryo than (-)-phenylahistin, and thus, would be a promising lead compound for antitumor agents.
Isoprenoids are chemically diverse in nature, ubiquitous in living organisms, and crucial in biological processes. The biosynthesis of such isoprenoids proceeds through mevalonate and nonmevalonate pathways depending upon organisms and cellular organella, isopentenyl diphosphate (IPP) being a key intermediate in both cases. Metabolic engineering and control of these pathways should thus provide new opportunity to study intriguing chemistry and biochemistry involved and to develop selective chemotherapeutic agents and isoprenoid-related materials. We describe a new practical approach to the preparation of highly- and multiply deuterated isoprenoids, zeaxanthin and diterpene anitibiotic terpentecin being as examples, and its potential for analyzing the biosynthetic mechanism of isoprenoids, based on the metabolic engineering of microorganisms. Obviously, deuterium-labeled compounds are invaluable in biochemical, bioorganic as well as physicochemical research. Metabolically engineered E. coli DK223 (pTMV20, pACCAR25ΔcrtX) produces zeaxanthin under the presence of mevalonate. Fully deuterated mevalonolactone-d_9 (MVL-d_9), which had been synthesized, was supplemented to the culture of the above triply-engineered E. coli, and the biosynthesized zeaxanthin was extracted and purified by repeated chromatography. All the zeaxanthin formed was proved to be derived only from the supplemented MVL-d_9. This was the first example of such highly and multiply deuterated zeaxanthin, and clearly demonstrated significant potential of the present approach for the preparation of various isotope-labeled isoprenoids. Additional example of this approach was also demonstrated in the mechanistic study of cyclization reaction in the biosynthesis of diterpene anitibiotic terpentecin. Straightforward stereochemical analysis of isoprenoid biosynthesis was demonstrated by one-shot labeling of MVL-d_9 and ^1H NMR spectroscopy. Precise analysis of the simplified proton spectra of highly deuterated isoprenoids, especially under the deuterium decoupled conditions, appeared to be beneficial for mechanistic enzymology, particularly, for the key transformation involving proton attack and proton quench as observed in the terpene cyclase reactions.
Enzymatic synthesis of diterpene, aphidicolin (ACL), a specific inhibitor of DNA polymerase α has been studied. cDNA encoding aphidicolin-16β-ol synthase (ACS) has been cloned and overexpressed in Escherichia coli. Partially purified enzyme converted geranylgeranyl diphosphate to tetracyclic diterpene, aphidicolan-16β-ol. This enzyme also converted syn-copalyl diphosphate (syn-CDP) to aphidicolan-16β-ol, supporting intermediacy of syn-CDP. The mycelial extract of an ACL producer, Phoma betae afforded a number of diterpene hydrocarbons. Their structures suggested that these are originated from the corresponding cation intermediates. Based on this observation, a cyclization mechanism of the reaction catalyzed by ACS was proposed.
1. The physiological function of chalcone synthase and stilbene synthase are the formation of naringenin chalcone and resveratrol, respectively. The enzymes use 4-coumaroyl-CoA, performs three condensations with malonyl-CoA, and folds the resulting tetraketide into a new aromatic ring system. We investigated the capacity to build novel and unusual polyketides from alternative substrates. Three types of products were obtained: (i) complete reaction (chlcone-or stilbene-type), (ii) three condensations without formation of aromatic ring (CTAL-type pyrone derailment), (iii) two condensations (BNY-type pyrone derailment). For both enzymes, all product types were obtained from 4-fluorocinnamoyl-CoA and analogs in which the coumaroyl moiety was replaced by furan or thiophene. The results show that minor modifications can be used to direct the enzyme reaction to form a variety of different and new products. Manipulation of the biosynthesis of polyketides by synthetic analogs could lead to development of a chemical library of pharmaceutically interesting novel polyketides. 2. Benzalacetone synthase (BSA) is a novel plant-specific polyketide synthase that catalyzes a one step decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA. A cDNA encoding BAS was for the first time cloned and sequenced from Rheum palmatum, a medicinal plant rich in pharmaceutically important phenylbutanone glucosides. The cDNA encoded a 42 kDa protein that shares 60-75% amino acid sequence identity with other members of the CHS-superfamily enzymes. Interestingly, R. palmatum BAS lacks the active-site Phe215 residue (numbering in CHS) which has been proposed to help orient substrates and intermediates during the sequential condensation of 4-coumaroyl-CoA with malonyl-CoA in CHS.
Biphenyl dioxigenase is known as an initial key enzyme to degrade environmental pollutants, which are derived from coal and petroleum (mainly polycyclic aromas). The genes coding for this enzyme are ordinarily composed of four gene components (bphA1A2A3A4). The bphA1 genes encoding the large (α) subunit of this enzyme was modified by DNA shuffling between the corresponding genes of Pseudomonas pseudoalcaligenes KF707 and Burkholderia cepacia LB400. Among them, we found a gene [bphA1 (2072)] that codes for a large subunit with extremely broad substrate specificity. The biphenyl dioxygenase genes including this modified bphA1 (modified bphA1: bphA2A3A4) were introduced into bacteria Escherichia coli and Streptomyces lividans on vectors pUC118 and pIJ6021, respectively. Using these recombinant bacteria, we succeeded in converting many heterocyclic aromatic compounds to the corresponding 1,2-dihydrodiol and/or monohydroxy derivatives with high efficacy. Even the aromatic compounds containing primary amine were able to be converted by protecting the amine as phthalic imide. Probably in many cases, the above biological technique is a simpler and effective method compared with the case synthesized by organic chemistry. Furthermore, it would be possible to complements the methods of combinatorial chemistry through extending the applicable category of the biotechnological method shown in this article. In the near future, we would like to aim at the establishment of the biological technologies that we call "BioCombiChem" (Biology-based Combinatorial Chemistry).
Streptomyces avermitlis has a life cycle with complex morphological differentiation and the ability to produce many secondary metabolites, one of which, avermectin, is commercially important in human and veterinary medicine. The major interest of the genus Streptomyces is the diversity of the production of secondary metabolites as an industrial microorganism. We have determined ca. 99% of the genome of S. avermitilis ATCC31267. The genome is composed of about 8.7 million base pairs in linear chromosome, that is the largest bacterial genome sequences, and provides insights into intrinsic diversity of the secondary metabolite production in Streptomyces. Twenty-five kinds of secondary metabolite gene clusters were found in the genome of S. avermitlis. Total length of these clusters occupies about 6.4% of the genome. A locus of pte gene cluster contained genes for five type-I modular polyketide synthases (PKSs) and postpolyketide modification enzymes. These PKSs consisted of 13 modules carrying 57 catalytic domains. In consideration of organization of domains in each module, five PKSs would catalyze to make 26-membered macrocyclic lactone with five conjugated double bonds that is a pentaene compound.
The inhibitors of glycosidases have attracted increasing research interest as chemotherapeutic agents and as useful molecular probes to understand the function of glycoproteins and the catalytic mechanism of glycosidases. The inhibitors of glycosidases have generally been designed to mimic the charge and/or the shape of the oxocarbenium ion-like transition-state or intermediate, but the incorporation of a nitrogen positive center into a sugar pyranose ring requires relatively long reaction sequence and often disrupts the structure of the sugar pyranose ring to affect the selectivity of inhibitors. We designed novel potent and selective glycosidase inhibitors, β-glycosylamines 1, 2 and 3, by incorporating a positive charge outside the sugar pyranose ring near C-1 so that the whole structure of the sugar moiety is maintained. The β-glycosylamidines 1-3 were readily synthesized without using protecting groups from the corresponding β-glycosylamine by reaction with the thioimidate in dry pyridine. Since the thioimidates are easily accessible from amides and nitriles, this synthetic route is a general method to accesss a variety of β-glucosylamidines with various glycon and aglycon structures. The β-glycosylamidines 1-3 strongly inhibited the corresponding β-glycosidase with Ki as low as 0.1 μM with substantial selectivity according to the glycon- and α-,β-specificities of glycosidases. Thus, β-Glu-amidine 1 selectively and competitively inhibited a β-glucosidase from A. niger with a Ki of less than 0.1 μM, but did not inhibit the α-glucosidases, α- and β-galactosidases, and β-xylosidase. Similarly, β-Gal-amidine 2 and β-Xyl-amidine 3 inhibited the corresponding β-glycosidase most potently, but showed little activity against α-glycosidases. We also synthesized and evaluated several β-glucosylamidine derivatives 1a-g with various aralkyl groups in the aglycon moiety. Most of them served as potent inhibitors of β-glucosidases with K_i of μM range, but substantial selectivity was observed among β-glucosidases such as mold and almond enzymes, according to the structure of the aglycon moiety. In some cases where a hydrophilic aglycon was involved, the selectivity reached as high as 10^6. The selective and easily accessible nature of β-glycosylamidines have employed to prepare an adsorbent for affinity chromatography of glucosidases by one-pot reaction of β-glucosylamine, iminothiolane-HCl and an affinity matrix with maleimide groups. A crude enzyme extract of tea leaves was passed through the column, and one of the β-glucosidase was selectively and quantitatively adsorbed onto the column. The adsorbed enzyme was eluted by glucose to give an almost pure β-glucosidase which showed a single band electrophoretically.
Solid phase synthesis is now widely recognised as the edge technology for rapid and efficient oligosaccharide construction. However, it has several disadvantages which should be overcome, i) the reduced reactivity of substrates, ii) the difficulty of real-time reaction monitoring, iii) limitations on ability of scale-up reactions, iv) purification of the desired compounds. We choose the PEG (Ave M. W. 550) as a polymer support. It gives homogeneous conditions in reaction mixture, so the reactivity of substrate bound to PEG does not diminished. The scale up of the reaction is possible because the low molecular weight of PEG. Due to its high polarity of PEG, purification of PEG bound sugar was quite simple using silica gel column chromatography. Using the nitro group introduced linker, which is quite stable under typical glycosylation reactions, the oligosaccharide was synthesised on PEG. The monitoring of the glycosylation reaction was performed by MALDI-TOF MAS based on the characteristic signal pattern which derives from normal distribution of PEG chain length. The reaction monitoring of deprotection of chloroacetyl group was performed by colorimetric assay by use of (p-nitrobenzyl)pyridine. Chloroacetyl group was selectively reacts with (p-nitrobenzyl) pyridine to give red color under basic conditions. The reaction was semi-quantified by use of NIH Image software. "Catch and release strategy" for the purification of polymer supported oligosaccharide was developed. Solid phase bound cysteine captures the glycosylated product having the chloroacetyl group. Cleavage reaction of Fmoc group releases the sugar via intramolcular cyclization process into the solution phase. By repetition of glycosyltion/capture/release cycle, the poly(lactosamine) was synthesized on polymer support.
Glycosidases are enzymes that are involved in several important biological processes such as digestion, biosynthesis of glycoproteins and catabolism of glycoconjugates. Glycosidase inhibitors have a potential to produce a number of beneficial therapeutic effects and are of biotechnological relevance. Thus carbohydrate heteroanalogues are potential inhibitors of carbohydrate-processing enzymes, which are useful tools in the study of N-linked glycoprotein biosynthesis, in that they can act as substrate analogues. Spohr and Spiro previously reported that the azadisaccharide 1-deoxy-3-O-(α-D-glucopyranosyl)-mannojirimycin (1) effectively inhibited glycoprotein-processing hydrolase endo-α-D-mannosidase (IC_<50>=1.7×10^<-6>M) in vitro, which was the enzyme responsible for the cleavage of α-D-Glc-(1→3)-D-Man from the GlcMan_9GlcNAc_2 oligosaccharide present inmmature N-linked glycoproteins. However, this disaccharide can be cleaved by intracellular glucosidases, therefore has a very short half-life. Then we developed the disaccharides 5-thio-glucosyl-α(1→3)-DMJ(2), 5-thio-glucosyl-α(1→3)-5-thio-mannoside (3), and 5-thio-glucosyl-α(1→3)-α-mannoside (4), the glycosyl unit in 1 was replaced with 5-thioglucose, for more metabolically-stable inhibitors for endo-α-D-mannosidase. Furthermore, we planned to synthesize new disaccharide (5), deoxymannojirimycin linked to pseudosugar such as validamine, which has amino-linkage in the molecule. The validamine, a glycosyl doner to construct the amino linkage of the disaccharide, is known as an excellent α-glucosidase inhibitor. Our designed analogues can be expected as a potent endo-α-D-mannosidase inhibitor in account of the resistance to hydrolysis in the metabolic system as well as the different inhibitory mechanism.
Linderol A (4), which has the potent inhibitory activity on melanin biosynthesis of cultured B-16 melanoma cells, was isolated from the fresh bark of Lindera umbellata (Lauraceae) (Fig. 2). The first total synthesis of (±)-linderol A (4) was achieved via 20 steps in 7.64% overall yield from 4,6-dimethoxysalicylaldehyde (5). 4,6-Dimethoxysalicylaldehyde (5) was converted to the coumarin (6) by Knoevenagel reaction. The coumarin derivative (6), which had an electron-withdrawing group at the 3-position, was treated with dimethylsulfoxonium methylide (2.2 eq) to afford the tricyclic 2-substituted cyclopenta[b]benzofuran-3-ol derivative (7) (Fig. 1). The benzofuran (7) was converted to the α,β-unsaturated ketoester (11), which was treated with isopropylmagnesium bromide in the presence of CuI and BF_3-etherate to afford stereoselectively the desired enolester 8 as a single product (Scheme 2, Table 1). Decarboxylation of 8 and one-carbon enlargement of the cyclopentanone ring gave the enol ester (13). Decarboxylation of 13 via 4 steps followed by Wittig olefination of the obtained ketone (15) afforded the exo-olefin (16) (Scheme 3). After the stereoselective cis-1,2-dihydroxylation with MC OsO_4, Friedel-Crafts reaction of the corresponding cyclic carbonate (21) was carried out by treatment with acetic anhydride/Sc(OTf)_3 to give regioselectively the 4-acetyl compound (22). Selective demethylation of 22 with BBr_3 afforded the phenol (23), and then alkaline hydrolysis of the cyclic carbonate function in 23 gave the diol (24). The ditosylate 25 was reduced by NaBH_3CN to convert the-CH_2OTs portion at the 6-position to the α-methyl group. Finally, treatment of 26 with benzaldehyde in the presence of tert-BuOK followed by alkaline hydrolysis gave crystalline (±)-4 (Scheme 6).
Trinervitane (1) is a unique tricyclic diterpene isolated as defence substance of termite soldiers by G. D. Prestwich. Herein we describe the first synthesis of 2,3-dihydroxytrinervitanes (1, 2α and 2β OH). Construction of trinervitane skeleton Bicyclic allyl chloride 9, prepared from 7,16-secotrinervitane (8), was treated with AgClO_4 at -20℃ to provide the dihydroxy trinervitane 10 in 68% yield. On the other hand, kempane diol 13 was formed in 50% yield by the same reagent at +20℃ The cyclization of diacetyl-derivertive 14 to 15 with AgClO_4 proceeded in high yield when carried out in the presence of pyridine. Conformational analysis The conformation of 17 and its plausible cyclization mode was discussed with the help of physical evidence including X-ray crystallographic analysis. The evidence indicates that the bisect conformation of the allyl chloride 17 changes to the bent conformation at allyl cation intermediate generated by the action of AgClO_4. This change is crucial for the facilitation of the five-membered ring formation, leading to the trinervitane skeletons. Total synthesis of (±)-2,3-dihydroxytrinervitanes Regio- and face-selective hydrogenation of triene 20 was achieved by PtO_2 in CH_2Cl_2 to give 21 with the 6: 1 ratio at C-12 position (Scheme 3). Isomerization of 23 to the conjugated enone 24 with DBU in refluxing toluene proceeded in high yield. The ring-opening of the epoxide 25 was carried out by treatment with TMSCl at -10℃ to give allyl alcohol 26, which was transformed to an easily separable 1: 1 mixture of 2,3-dihydroxytrinervitanes, 1a and 1b. Synthetic study of optically active secotrinervitane Aiming at the asymmetric synthesis of trinervitane type diterpenes, a synthetic route to the dihydrosecotrinervitane 37 was explored starting from (R)-citronellol (29) (Scheme 4). Coupling reaction of allylester 30 and Grignard reagent 31 gave dihydrogeranylgeraniol 32, which was converted into epoxy alcohol 36. The cyclization of 36 to 37 is unsuccessful at present.
Six novel drimane sesquiterpenoids, mniopetals A-F (1-6), were isolated from the fermentation broth of Mniopetalum sp. 87256 and their relative configurations were determined by Steglich and co-workers in 1994. These natural products inhibit RNA-directed DNA polymerases (reverse transcriptases) of some RNA viruses, such as human immunodeficiency virus (HIV)-1. The structural characteristics of mniopetals are (1) a tricyclic framework including a trans-fused octahydronaphthalene skeleton, (2) five or six contiguous stereogenic centers including an angular asymmetric quaternary carbon, and (3) a variety of oxygen functionalities such as a γ-hydroxy-γ-lactone ring (C ring). Thereby, to establish their unsettled absolute stereochemistries, we have been engaged in enantiospecific synthetic studies of mniopetal E and F, the former is the prototype of mniopetals A-D. In the latter, the hydroxy group at C-2 in mniopetals A-E is lacking. Recentry, we have accomplished the total syntheses of mniopetal E (5) and mniopetal F (6). The key reaction for our total synthesis of mniopetal E was the intramolecular Diels-Alder reaction of triene 21, which was prepared from known epoxide 15 as depicted in Sheme 3. Heating 21 in a toluene solution at 180℃ provided the desired 22 as the major product. This stereochemical outcome is explainable by secondary orbital interaction and 1,3-diaxial repulsion in their transition states as shown in Figure 2. The transformation of the cycloadduct 22 into mniopetal E was achieved in 6 steps via lactone 30 (Scheme 4). The ^1H NMR spectrum of the synthetic 5 was well matched with that of the reported data for natural 5. Netx, epoxide 15 was selected again as the starting material for the total synthesis of mniopetal F. From 15, the analogous reaction sequence used for mniopetal E synthesis afforded triene 33 (Scheme 5). The effect of hydroxy-protecting group at C-1 on stereoselectivity in the Diels-Alder reaction was investigated (Table 1). The reactions of silyl ethers 35-39 provided the desired cycloadducts A predominantly. The stereochemical outcome is explainable in consideration of the stereoelectronic effect of trialkylsilyloxy group, which overcomes the 1,3-diaxial repulsion as shown in TS-A (Figure 3). The total synthesis of mniopetal F was achieved from the dimethylisopropylsilyl ether 45 (Scheme 6). Deprotection of the silyl ether and acetal exchange enabled the separation of diastereomers 51 and 52. The total synthesis was achieved from 51 in the same way as described in Scheme 4. The spectral data of the synthetic 6 was also well matched with those of the reported data for natural 6. The optical rotations of the synthetic 5 and that of 6 led the conclusion that the absolute stereochemistries of these natural products are those as depicted.
Conformational analysis of the side chain of 1α,25-dihydroxyvitamin D_3 [1,25-(OH)_2D_3, 1] and its 20-epimer 2 revealed that the spatial region occupied by the side chain of these vitamin Ds can be divided to four areas. We designed and synthesized four diastereomers 3-6 at C(20) and C(22) of 22-methyl-1,25-(OH)_2D_3 whose side-chain mobility is restricted in one of these four areas. We tested biological activities of these four analogs. These studies allowed us to propose the relationship between the spatial region of the vitamin D side-chain and the activity, an active space group concept. This concept has been generally accepted as explaining the three-dimensional structure and activity relationship of almost all known vitamin D analogs. We constructed the three-dimensional structure of the ligand binding domain (LBD) of vitamin D receptor (VDR) based on the crystal structure of retinoic acid receptor by the homology modeling technique. We docked 1,25-(OH)_2D_3 1 as a ligand into the constructed VDR-LBD. Three residues forming the hydrogen bonds with the functionally important 1α- and 25-hydroxyl groups of 1 were identified and confirmed by the mutational analysis: the 1α-hydroxyl group is forming pincer type hydrogen bonds with S237 and R274 and the 25-hydroxyl group is interacting with H397. By the computational docking studies based on the mutational analysis of the VDR, we obtained the docking models of the VDR with the functionally and structurally interesting ligands. From these studies we suggested key structural factors to bestow the augmented activities on 20-epi-vitamin Ds.
1α, 25-Dihydroxyvitamin D_3 (1α, 25-(OH)_2D_3, calcitriol), the biologically active metabolite of vitamin D_3, is recognized as a steroid hormone. In order to separate these activities, especially cell differentiation and proliferation activity from calcemic activity, and to enhance the specific activities, a number of analogues have been synthesized by many laboratories. In recent years, it has been proven that applications of solid phase chemistry, capable of generating combinatorial natural small molecule libraries, to drug discovery process is extremely effective in terms of searching a wide range of analogues rapidly. Herein, we describe two solid-phase synthesis of the vitamin D_3 system for a three-component library (i.e. the A-ring, the CD-ring, and the side chain) in only four steps including two nucleophilic additions. One is for 11-hydroxy analogues, and the other is for versatile analogues. In the latter strategy, the sulfonate-linked CD-ring 28 was initially immobilized on PS-DES resin to give solid-supported CD-ring 32 (Scheme 4). Similarly, solid-supported CD-ring 32 was prepared by attachment of the CD-ring 25 to the chlorosulfonate resin 33. The vitamin D_3 system was synthesized by Homer-Wadsworth-Emmons reaction of the A-ring phosphine oxide to a solid-supported CD-ring, followed by simultaneous introduction of the side chain and cleavage from resin with a Cu^1-catalyzed Grignard reagent. Parallel synthesis of the vitamin D_3 analogues was accomplished by a split & pool method utilizing radiofrequency encoded combinatorial (REC) chemistry, and a manual parallel synthesizer for side chain diversification and deprotection. In addition, we wish to report an efficient synthesis of various A-ring moieties by means of the Pd(0)-catalyzed intramolecular Mizoroki-Heck reaction.
Ingenol, isolated from the genus Euphorbia, has been of great interest as a synthetic target because of its unusual structure involving an "inside-outside" bridged BC ring coupled with a broad spectrum of biological activities. To date, four strategically distinct approaches have been disclosed, however, the natural product itself has yet to succumb to total synthesis. On the other hand, we had reported an efficient method for constructing an ingenane skeleton via "tandem cyclization-rearrangement strategy", while stereoselective introduction of the oxygen functionalities on the AB ring moiety remained as the final problem in ingenol synthesis. We designed a new synthetic route involving key intermediate 15 which has two oxygen functionalities on both of the A and B rings. Stereoselective synthesis of trans-decalinol derivative 6 was achieved through a chelation-controlled aldol reaction of enone 4 and an intramolecular alkylation reaction of ester 5. Under the influence of Lewis acid 10, cobalt complex 9 underwent a cyclization reaction to yield cyclic cobalt complex 11, which was subjected to Birch reduction followed by cyclopropanation to give allyl alcohol 13. Transformation of the tetracyclic carbon framework into an ingenane skeleton was achieved via stereoselective epoxidation of 13 followed by treatment with trimethylaluminum. The C1 hydroxyl group of the key intermediate 15 was effectively used as a flag for installation of the C19 methyl group and the C1-C2 double bond. Under the influence of a guanidine base, ketone 18 yielded conjugated diene 19 via isomerization of the C1-C2 double bond. Treatment of diene 20 with an excess amount of osmium tetroxide gave polyol 21, containing all of the stereogenic centers of ingenol. Functionalization of the A ring was accomplished via selective protection of the C3, C4, and C5 hydroxyl groups and a dehydration reaction. Finally, the allyl alcohol moiety of the B ring was constructed through a Horner-Emmons reaction and a [2,3]-rearrangement reaction of an allyl selenoxide compound, and the first total synthesis of ingenol was completed.
Sex pheromones have been identified from female moths of more than 500 species. About 75% of them are composed of unsaturated C_<10>-C_<18> straight chain compounds with a functional group at the terminal position (type I). Another 15% of the pheromones consist of unbranched C_<17>-C_<23> (3Z,6Z,9Z)-trienes, (6Z,9Z)-dienes, and their monoepoxy derivatives (type II), which lack a terminal functional group. The type II pheromones have been identified from species in highly evolved families, such as Geometridae, Noctuidae, Arctiidae, and Lymantriidae. In spite of the great number of species in the above families, the structural diversity known to date for the pheromones in type II is limited. It is expected that some females produced further modified compounds, and recently we successfully identified novel pheromone components from two lymantrid species. From virgin females of the tussock moth, Orgyia postica, single EAG-active component was found in a pheromone extract. This compound named posticlure possesses a trans-epoxy ring and was identified as (6Z,9Z,11S,12S)-11,12-epoxyhenicosa-6,9-diene by means of GC-MS, ^1H NMR and chiral HPLC analyses, and further chemical derivation followed by the GC-MS analysis. In a field test with the pheromone synthesized stereoselectively, the male moths were specifically attracted to the (11S,12S)-isomer but not to the antipode. Based on the information of a diepoxy pheromone produced by Leucoma salicis, we synthesized all stereoiosmers of diepoxyalkenes derived from (3Z,6Z,9Z)-trienes with a C_<18>, C_<19>, or C_<21> straight chain by a MCPBA oxidation of optically active epoxyalkadienes. While the positional isomers showed similar Rts on a capillary GC, detailed inspection of their mass spectra indicated the following diagnostic ions for determining the chemical structures; m/z 128, 167, M-87, and M-85 for (Z)-cis-3,4-cis-6,7-diepoxy-9-enes, m/z 111, M-125, and M-69 for (Z)-cis-6,7-cis-9,10-diepoxy-3-enes, and m/z M-125 and M-139 for (Z)-cis-3,4-cis-9,10-diepoxy-6-enes. Mass chromatographic analysis monitoring these fragment ions revealed the existence of new pheromonal compounds, (3R,4S,6S,7R,9Z)-3,4-6,7-diepoxyhenicos-9-ene and its (3S,4R,6S,7R)-isomer, in an extract from virgin females of another tussock moth, Perina nuda. These two diepoxy compounds effectively enhanced attractiveness of the major component, (3Z,6S,7R,9Z)-6,7-eopxyhenicosa-3,9-diene, in a field.
Amphidinolide C (1) is a unique 25-membered macrolide isolated from a marine dinoflagellate Amphidinium sp. (strain Y-5). The gross structure of 1 has been elucidated by 2D-NMR data, whereas the stereochemistry remains undefined. During our search for bioactive metabolites from marine dinoflagellates, relatively large amounts of 1 have been recently isolated from two strains (Y-56 and Y-71) of the genus Amphidinium, which allowed us to investigate the stereochemistry of 1 Absolute configurations at C-12, C-13, C-20, C-23, C-24, and C-29 of amphidinolide C (1) were assigned by combination of NMR analyses including J-based configuration analysis and Mosher's method, while absolute configurations at C-7 and C-8 of 1 were assigned by application of Mosher's method for erythro glycol and comparison of ^1H-NMR data of natural specimen with those of synthetic C-1-C-10 segments. Absolute configurations at C-3, C-4, C-6, and C-16 of 1 were elucidated by comparison of NMR data of degradation products of 1 with synthetic C-1-C-7 and C-16-C-18 segments. On the other hand, five new 19-membered macrolides amphidinolides T1-T5 (2-6) have been isolated from the Y-56 and Y-71 strains of the genus Amphdinium. The gross structures were elucidated by 2D-NMR analyses. Absolute configurations at C-2, C-13, C-14, and C-18 of amphidinolide T1 (2) were assigned on the basis of NMR data of the MTPA esters of 2 and those of degradation products of 2, while absolute configurations at C-7, C-8, and C-10 of 2 were elucidated by comparison of ^1H-NMR data of C-1-C-12 segment with those of synthetic model compounds for tetrahydrofuran portion. The absolute stereochemistry of amphidinolides T2-T5 (3-6) were also elucidated by similar methods. The stereostructure of 2 was also confirmed by a single crystal X-ray diffraction analysis.
Most blue flower color development is due to a metalloanthocyanin, a stoichiometric self-assembled supramolecular pigment consisting of six molecules of anthocyanin, six molecules of flavone and two metal atoms. On formation of metalloanthocyanin, very fine and strict molecular recognition, including chiral molecular recognition, occurs. The manner of stacking of the aromatic chromophores in metalloanthocyanin is chiral, and these phenomena must arise from a chirality of sugars attached to the anthocyanin and flavone. In the present report, chiral molecular recognition on formation of a metalloanthocyanin, protodelphin (1), a blue petal pigment of Salvia patens is described. The composition of 1 was determined to be six malonylawobanins (3), six apigenin diglucosides (2a) and two Mg^<2+>, and the structure was very similar to that of commelinin. We have developed a reliable glycosylation method for less reactive phenols in flavonoids utilizing new Lewis Acid-Base-Promoted-Glycosylation with the peracetylglucosyl fluoride, and succeeded in preparing natural and unnatural apigenin 7,4'-di-O-β-glucosides, substituted with D- and/or L-glucose. Using these synthetic apigenin derivatives reconstruction experiment was done. Protodelphin-like metalloanthocyanin was produced from only apigenin glycosides which contain D-glucose at the 4' position, indicating that the D-glucosyl residue at the 4'-OH is indispensable for formation of a metal complex pigment. The three flavone molecules in 1 associate to form a M (minus) helical structure just like a propeller with three blades. They are bound at the pivot point by a strong hydrogen bond network among hydroxyl groups of the 4'-O-β-D-glucopyranosides. The two sets of M-helical flavone associates fit closely into the vacant space formed from the metal-complex of six molecules of 3 and two ions of Mg^<2+>. In conclusion, malonylawobanin (3) chooses only the D-chirality of the 4'-O-glucosyl residue in apigenin 7,4'-diglucosides to form the metalloanthocyanin, protodelphin. This restrict chiral and structural recognition realizes the entire self-assembly of metalloanthocyanin and is responsible for the beautiful blue-flower color.
Angiogenesis is defined as the generation and growth of new blood vessels from pre-existing vessels. Angiogenic factors including basic Fibroblast Growth Factor (bFGF) and Vascular Endothelial Growth Factor (VEGF) stimulate endothelial cell proliferation. Sprouting endothelial cell break down the extracellular matrix, and then migrate to cancer cells, finally lumen is formed. Because tumor growth is depended on a blood supply (oxygen and nutrients), angiogenesis inhibitors may potentially be used as new therapeutic agents for cancer treatment. In connection with our studies on the isolation of angiogenesis inhibitor for development of new anti-cancer agents, we have investigated the anti-angiogenic activity of 200 medicinal plants and 120 marine invertebrates. Among them, the MeOH extractives from Saussurea lappa, Caesalpinia sappan, and Anemarrhena asphodeloides from medicinal plants, and the EtOH extractives from the bryozoan Dakaria subovoidea and the sponge Haliclona olivacea from marine invertebrates showed inhibitory effect on BAECs proliferation. Further investigation has led to the isolation of eight anti-angiogenic compounds (1-8). In the present studies, we have found that costunolide (3) and cis-hinokiresinol (4) selectively inhibit bFGF or VEGF-induced endothelial cells proliferation and migration. From these results, we hypothesized that costunolide and cis-hinokiresinol would inhibit angiogenesis by blockade of angiogenic factors signaling pathway. To be anticipated, costunolide and cis-hinokiresinol inhibited the VEGF KDR/Flk-1 tyrosine phosphorylation. Moreover, cis-hinokiresinol reduced the vessel growth induced by VEGF in mouse corneal neovascularization model. Compounds 5-8 isolated from marine invertebrates inhibited BAECs proliferation. Among them, compounds 6 and 8 inhibited bFGF-induced BAECs mitogenesis more selectively in a dose dependent manner.
Gastrulation is one of the fundamental events in embryogenesis, and is the first process closely related to cellular differentiation. During our search for inhibitors of starfish (Asterina pectinifera) embryonic development, we found that the n-BuOH extract of a new actinomycete species belonging to the genus Micromonospora inhibited gastrulation of A. pectinifera embryos. Bioassay-guided chromatographic fractionation led to the isolation of new bioactive macrolides designated micromonospolides A (1), B (2) and C (3). Spectroscopic data showed that micromonospolides A (1), B (2) and C (3) contain a 16-membered lactone ring as that of bafilomycins. When 8-hours-old embryos at the early blastula stage of A. pectinifera were cultured in the presence of 1-3, the progression of embryonic development was arrested at the late blastula stage just prior to gastrulation at minimum inhibitory concentrations of 0.010, 0.011 and 1.6 μg/mL, respectively. Micromonospolide B (2) showed nearly the same inhibitory activity as that of 1, indicating that the presence of an N-(3-hydroxy-2-cyclopentenone-2-yl)-fumarylester monoamide functionality at C-21 in 1 does not affect the activity. Micromonospolide C (3) was 100-fold less active than 1 or 2, indicating that the opening of the tetrahydropyran ring (C-19 to C-23) and the subsequent formation of the α,β-unsaturated ketone functionality resulted in the decrease in potency of the inhibitory activity. On the other hand, the minimum inhibitory concentration of bafilomycin A_1, which has an isopropyl group attached to a tetrahydropyran ring, was 0.10 μg/mL corresponding to one-tenth of the activity of 1. The potent inhibitory activity of 1 and 2 against gastrulation of the starfish embryo seems to be due to the replacement of the isopropyl group in bafilomycin A_1 by the (E,E)-1,3-pentadienyl group.
During cerebral ischemia and subsequent reperfusion injury, neuronal degeneration is caused by the excitatory amino acid, L-glutamic acid, which acts as a neurotransmitter in the major part of brain. The ionotropic L-glutamate receptors, which are considered to play an important role in neuronal cell death, are mainly classified into two types; NMDA (N-methyl-D-aspartic acid) and AMPA/KA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid/kainate) receptors. Since the AMPA/KA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) was reported to protect neuronal cells from ischemia injury even administered after an ischemic attack, AMPA/KA receptor antagonists are expected to treat ischemia-reperfusion injury such as stroke. In the course of our screening for substances that protect chick primary telencephalic neurons from kainate toxicity, we have isolated a potent kainate-toxicity suppressor designated as kaitocephalin from Eupenicillium sheariiPF1191. The structure of kaitocephalin (C_<18>H_<21>N_3O_9Cl_2) was elucidated on the basis of spectral analyses including 2D NMR (HMQC, DQF-COSY, ^1H-^<13>C HMBC and ^1H-^<15>N HMBC) to consist of a pyrrolidine moiety with tricarboxylic acids and a dichlorohydroxybenzoate substructure as shown in Fig. 1. The absolute structure of kaitocephalin was also determined by NMR studies after series of chemical modifications to be 2S3S4R7R9S. Kaitocephalin protected rat hippocampal neurons from kainate and AMPA toxicity with similar potency to those of known AMPA/KA antagonists without showing cytotoxic effect. Although, kaitocephalin protected rat hippocampal neurons from kainate and AMPA toxicity, it failed to block the Ca^<2+> influx elicited by kainate (Kaitocephalin effectively blocked Ca^<2+> influx induced by AMPA). This result is incompatible with the hypothesis of Ca^<2+> induced neurotoxicity. Thus, kaitocephalin is expected to be a useful tool to reveal the role and cytotoxic action mechanism of kainate receptor.
L-Glutamate acts as the principal excitatory neurotransmitter in the mammalian central nervous systems. The extracellular glutamate concentrations are tightly controlled by a transport system that limits the activation of receptors during signaling and that maintains below excitotoxic level. Non-transportable blockers have been required as indispensable tools for the investigation of transporters. We have been developing novel blockers by two approaches. One is the screening from conformationally constrained glutamate analogs and the other is the synthesis of THA (threo-β-hydroxyaspartates) derivatives. We found CCG-III and CMP-III (MPDC) are substrates while CPG-II is a blocker for EAAT1. Superimposition studies using CMP-III (MPDC) as a template provided a putative pharmacophre, in which the distal carbaxylate is close to α-amino group. Although the functional groups of CPG-II were able to fit with the proposed model, the bulky five-membered ring moiety seemed to overhang the pharmacophore. We guess such a bulky substituent plays an important role in blocker activity. On the other hand, both the L- and D-THA are excellent substrates of EAAT1-4 and blockers for EAAT5. We considered that introduction of a bulky substituent on the β-hydroxy group would be useful to develop blockers. Among the synthesized THA derivatives, L-TBOA (L-threo-β-benzyloxyaspartate) is, so far, the most potent blocker for all EAAT subtypes (EAAT1-5). The functional groups of the folded form of glutamate are well superimposed with those of the extended form of aspartate. If L-TBOA takes the extended conformation, its benzyl group was well overlapped with the bulky substituents of dihydrokainate and CPG-II. Next, we synthesized L-TBOA analogs with functional groups suitable for linking to an affinity column such as amino and biotinyl groups. Although para-substituted analogs lost the activity, meta-substituted analogs inhibited [^<14>C]Glu uptake with almost comparable potency as TBOA.
Kaitocephalin (1), a novel AMPA receptor antagonist, was isolated from Eupenicillium shearii PF1191 by Seto, Shin-ya and co-workers, and its absolute configuration was shown to be 1. We were interested in this unique structure and its potent biological activities, and have been investigating stereoselective synthesis of kaitocephalin and its analogs to determine the absolute configuration of 1 and to clarify the structure-bioactivity relationships. A synthesis of compound 1b was accomplished employing a stereoselective C-C bond formation reaction under sonication condition with zinc and CuI (6b+7→8b) as a key step (Scheme 1). Unfortunately, ^1H-NMR spectral data of 1b was not identical with those of natural compound. Then, 9-epi-1b was synthesized in the same manner (Scheme 2), but this compound was not identical either. It was assumed that the stereochemistry of natural kaitocephalin have been assigned incorrectly. So, ^1H-NMR spectra of model compounds 14a-d (14c has not been synthesized yet), particularly, chemical shifts and coupling constants of H-2 and H-3 were compared. As shown in Fig. 1, 14d showed the most similar pattern to that of natural kaitocephalin. We therefore assumed that the correct stereostructure of natural kaitocephalin should be 1d and a synthesis of 1d was carried out in the same manner as shown in Scheme 4. The ^1H-NMR spectrum of compound 1d was identical with natural one. At present, we are confirming the stereochemistries of the intermediates and optimizing the each reaction condition. Thus, we developed the efficient route to the synthesis of kaitocephalin skeleton and tentatively concluded the correct relative configuration of natural kaitocephalin to be 2R^*, 3S^*, 4R^*, 7R^*, 9S^*. Antagonistic activity of 1b, 9-epi-1b, 14a, 14b, 14d and 15 to the kainate toxicity on rat hippocampal neurons are shown in Table 1. Compounds 1b and 9-epi-1b showed weaker activities than the natural compound. On the other hand, analogs 14a, b, d and 15 showed no inhibition activities.
Interleukin 6 (IL-6) is a multifunctional cytokine that acts on immune response, hematopoiesis and the nervous system. Thus, IL-6 plays one of the central regulatory roles in host defense mechanisms. However, it has been demonstrated that IL-6 produced by tumor cells causes cancer cachexia, and also stimulates the proliferation of tumor cells in an autocrine/paracrine manner. Therefore, it may be possible that the inhibition of IL-6 activity relieves cancer cachexia and suppress the growth of IL-6 dependent tumor cells. In the course of a search for inhibitors of IL-6 activity obtained from microoragnisms, madindoline A (MDL-A) and B (MDL-B) were discovered from the fermentation broth of Streptomyces nitrosporeus K93-0711. We previously described the determination of the complete absolute stereochemistries of MDL-A and B and the first total syntheses of these materials. We describe here more efficient total synthesis (second generation) via stereoselective acylation of ester by coordination with 3a-hydroxyfuroindoline moiety as the chiral ligand, and the intramolecular acylation of allylsilane. When we also prepared [^3H]-madindoline A, we clarified that [^3H]-madindoline A binds to gp-130, selectively. Moreover, madindoline A inhibited the differentiation of osteoblast cells.
A versatile chiral building block 1 containing a bicyclo[3.2.1]octane framework has been prepared by employing either chemical or enzymatic resolution method. Thus, racemic alcohol (±)-11 used for chemical resolution and racemic acetate (±)-12 used for enzymatic resolution were first prepared from norbornadiene 5 via racemic enone (±)-1. On the asymmetric hydrogen transfer reaction using a chiral diamine-Ru^<II> complex (Ru^<II>-(IS,2S)-TsDPEN), (±)-11 furnished optically enriched (+)-1 (73%ee) and enantiopure alcohol (-)-11, while (±)-12 furnished enantiopure alcohol (+)-11 and enantiopure acetate (-)-12 under hydrolysis conditions in a buffer solution in the presence of Lipase PS. Enantiopure chiral building block 1 could be obtained in both enantiomeric forms from the resolution products thus obtained by employing conventional procedure. Owing to its biased framework, 1 exhibits inherent convex-face selectivity to give rise to a variety of aldol products 2 containing either a cyclohexanone moiety or cyclopentanone moiety by diastereoselective modification of its enone functionality and the secondary oxygen functionality. The aldols 2 carrying a cyclohexanone afford 3,4-substituted cyclohexanones, while the aldols 2 carrying a cyclopentanone afford 2,3-disubstituted cyclopentanones 4 by retro-aldol reaction. On the basis of the inherent stereochemical nature of the chiral building block 1 and the retro-aldol reactivity, diastereocontrolled synthesis of some natural products, 1α,25-dihydroxyvitarnin D3 calcitriol, an antitumor sesquiterpene (+)-vernolepin, a potential indole alkaloid intermediate yohimbone 39, an antibiotic diterpene (+)-ferruginol, and an analgesic alkaloid (-)-morphine, has been investigated. (1) Synthesis of calcitriol-Although the intended convergent synthesis of calcitriol using both enantiomers of the chiral building block 1 has not been accomplished yet, the construction CD-ring moiety 13 starting from (-)-1 has been completed and very close intermediate 20 of the A-ring moiety starting from (+)-1 has been attained. (2) Synthesis of vernolepin-A close intermediate 33 constituting a formal synthesis of (+)-vernolepin has been attained starting from (-)-1 by employing an acid-catalyzed retro-aldol reaction as the key step. (3) Synthesis of yohimbone-Yohimbone 39 has been accomplished in efficient manner involving one-step conversion of 36 into 38 through an acid-catalyzed tandem retro-aldol and intramolecular Pictet-Spengler reaction. (4) Synthesis of ferruginol-A novel and efficient route to (+)-ferruginol has been established using (+)-1 as starting material by discovering a novel tandem acid-catalyzed retro-aldol and oxonium ion-mediated hydrophenanthrene formation reaction. (5) Synthesis of morphine-Based on the tandem rertro-aldol and oxonium ion-mediated hydrophenanthren formation reaction discovered as the key step, a novel and efficient route to (-)-morphine has been developed starting from (-)-1.
We have developed the general methodology for the asymmetric allylation of the tricyclic N-acyl-N,O-acetals incorporating (R)- or (S)-2-(1-aminoethyl)phenol [(R)- or (S)-10] as a chiral auxiliary, leading to the chiral (R)- or (S)-allylated piperidones and pyrrolidones. This methodology was successfully applied to alkaloid synthesis. The chiral tricyclic N-acyl-N,O-acetal 14a prepared from δ-ketodecanoic acid 6 and the chiral aminophenol (R)-10 was subjected to nucleophilic allylation with TiCl_4-allyltrimethylsilane to provide the (6R)-allylated piperidone 16 in high stereoselectivity (16: 1). The stereochemical outcome of this reaction can be rationalized on the basis of conformational control involving minimization of the allylic strain in the iminium intermediate. Subsequent four-step transformation of the chiral 6,6-dialkylpiperidone 16 thus obtained led to the first asymmetric synthesis of the coccinellid alkaloid (-)-(R)-adalinine (1), establishing the absolute configuration of natural (-)-adalinine to be R. The TiCl_4-induced allylation of the tricyclic N,O-acetal 21 installing the chiral auxiliary (S)-1O proceeded in high yield with retentive selectivity accounted for by a conformational model similar to that described above, yielding the (5S)-allylated pyrrolidone 22a. After conversion to the aldehyde 25, five-carbon chain elongation via the Horner-Emmons reaction with the phosphonate 26 led to the 5-(3-propanoyl-2-propenyl)pyrrolidone 28. Four-step conversion of 28 provided the Dendrobatid alkaloid (-)-indolizidine 167B (2). By applying a similar sequence, (-)-indolizidine 209D (3) was obtained from 25 via eight-carbon chain extension. Finally, the first total synthesis of marine alkaloid (-)-stellettamide B (4) has been achieved by a sequence based on amide coupling of the chiral 1-(aminomethyl)indolizidine fragment 33, prepared by TiCl_4-mediated asymmetric allylation of the tricyclic N-acyl-N,O-acetal 37 incorporating (R)-10, with the chiral trienoic acid fragment ent-34. This synthesis led to revision of the published relative stereochemistry of the natural product and established its absolute stereochemistry to be 1S,4S,8aR,6"R.
The natural products containing α,α-disubstituted α-amino acid structures attracted significant attention because of their interesting biological activities such as antifungal, immunosuppressive, enzyme inhibitory and neurotrophic factor activities. We report here a synthetic protocol for such natural products using rearrangement of allylic trichloroacetimidates (Overman rearrangement) derived from aldohexoses, and total syntheses of myriocin (1) from D-mannose and sphingofungin E (2) from D-glucose. Myriocin (1) is an antimicrobial and immunosuppressive agent isolated from Myriococcum, Myceria, and Isaria. The amino acid precursor 10 was prepared in 7: 1 stereoselectivity by using Overman rearrangement of the (E)-allylimidate derived from D-mannose. Ozonolysis of 10 followed by oxidation and esterification afforded diastereomeric pure ester 13, which was converted to (E)-allylbromide 16 in 5 steps. The sulfone 17, an aliphatic part of myriocin, was lithiated with nBuLi and reacted with 16 to afford the coupling product 18. Saponification and subsequent Birch reduction gave crude carboxylic acid, whose protecting groups were removed to afford the known lactone 20. Final saponification furnished the total synthesis of myriocin. Sphingofungin E (2) isolated from Paecilomyces, is a novel antifungal agent, and known as a strong inhibitor of serinepalmitoyl transferase. Stereoselective synthesis of sphingofungin E with the similar synthetic procedures to those employed for the synthesis of myriocin was explored. Overman rearrangement of (Z)-allylimidate prepared from D-glucose in 8 steps, showed moderate stereoselectivity, and gave rearranged product 23a, whose structure was determined by X-ray analysis of 24, as the major product,. Ozonolysis of 23a followed by reduction gave carbinol 25, which was converted into allylbromide 27 stereoselectively. Coupling reaction of 27 with sulfone 17 afforded the backbone of sphingofungin E 28. Treatment of 28 with lithium naphthalide followed by oxidation gave carboxylic acid 29. Acid hydrolysis of 29, followed by saponification completed the total synthesis of sphingofungin E.
Vinblastine (1), isolated from Catharanthus roseus, has been widely used in cancer chemotherapy, and a variety of derivatives have been derived from vinblastine to search for more efficient drugs. In addition, its complex structure has attracted a great deal of attension of many synthetic chemists, and several total syntheses have been reported, which, however, employed natural vindoline (2) as starting material. For the effective preparation of a range of derivatives, total synthesis of the whole vinblastine is essential. Herein, a total synthesis of (+)-vinblasitne, including (-)-vindoline, is reported. We planned to prepare the two indole parts, which constitute vinblastine, and couple these indole parts near the last stage of the synthesis. The preparation of the bottom part, vindoline (2), which we have already reported, has been improved for a large-scale preparation. The requisite indole fragment was synthesized utilizing radical cyclization of thioanilide 7, followed by Mannich reaction of malonate to give acrylate derivative 9. The preparation of 2,4-dinitrobenzensulfonamide 10 was carried out via enzymatic resolution of the cyanohydrin intermediate. The upper part of vinblastine was prepared as indoloazacycloundecane 42. The stereoselective introduction of the tertially alcohol was performed by a combination of 1,3-dipolar cycloaddition of nitrile oxide and Baeyer-Villiger oxidation (29 to 33). The indole nucleus was formed utilizing radical cyclization of thioamide 36. Construction of the 11-membered ring was successfully achieved by macrocyclization of nosyl amide 40. The coupling of 42 with (-)-vindoline (2) occurred with the desired stereochemistry to afford 45 as a sole isomer and further manipulations afforded (+)-vinblastine (1), whose spectroscopic data were identical to those of the natural sample.
Polytheonamides are potent cytotoxic metabolites isolated from the marine sponge Theonella swinhoei. The ^1H and ^<13>C NMR spectra indicated polytheonamide B to be a polypeptide with unusual residues. Due to the blocking of the N-terminal amino group and the presence of many unusual residues, the methodology of structure elucidation for proteins was not applicable. The acid hydrolysate of polytheonamide B was analyzed by amino acid analysis and 2D NMR which disclosed most of the component amino acid residues. With this knowledge, interpretation of 2D NMR data was effected leading to the sequence of the 48 reside peptide. The structure of the 44th residue was elucidated on the basis of the elemental analysis and spectral data. Polytheonamide A gave the same oxidation product as polytheonamide B indicating that they are isomeric at the sulfoxide in the 44th residue.
In our continuing study on polyphenolic constituents of Melastomataceous plants, we have reported the isolation and structure determination of ellagitannin oligomers such as nobotanin K (1). A chromatographic survey of the tannins in this plant family revealed that Tibouchina multiflora and Monochaetum multiflorum, endemic to Colombia, are rich in ellagitannin oligomers. We report herein the structure elucidation of 16 new ellagitannins and polyphenols isolated from leaves of the above two plants. The aqueous acetone homogenates of dried leaves of T. multiflora and M. multiflorum were concentrated and extracted successively with Et_2O, EtOAc and n-BuOH, respectively. Each extract was submitted to column chromatography over Diaion HP-20, Toyopearl HW-40, MCI GEL CHP-20P and/or preparative HPLC to yield two new ellagitannins [nobotanins O and P (2)] along with 15 known tannins and related polyphenols from the former, and 41 ellagitannins and related polyphenols including 14 new compounds [benzyl 6'-O-galloyl-β-D-glucopyranoside, 4-O-(6'-O-galloyl-β-D-glucopyranosyl)-cis-p-coumaric acid, 6'-O-galloylprunasin, monochaetin (3), nobotanins Q(4), R, S(5), T(6), U and V, and melastoflorins A(7), B(8), C(9) and D(10)] from the latter. Sixteen new compounds including four ellagitannin tetramers (2, 4-6) and four pentamers (7-10) were characterized by spectroscopic (^1H-^1H COSY, TOCSY, ^1H J-resolved, DEPT, NOESY, HMQC and/or HMBC) and chemical methods. Although approximately 200 oligomeric ellagitannins have been found in nature to date, most of them were dimers, and the number of trimers-tetramers was limited. Melastoflorins A (7), B(8), C(9) and D(10) are the first examples of pentameric hydrolyzable tannins composed of different monomeric units (casuarictin, pterocaryanin C, and their derivatives) with C1 glucopyranose cores. The present study should prompt a further search for this class of higher oligomers in nature.
A chemical structure of TIME-EA4 protein which was isolated from the diapausing eggs of the silkworm, Bombyx mori, as a key to the termination of embryonic diapause, was investigated in detail. The ESI(electrospray ionization)-Q-TOF(a tandem quadrupole/orthogonal-acceleration time-of-flight) mass spectrometer combined with the appropriately-adjusted nano-HPLC system was utilized to determine the glycosylation site and the glycan structure. LC-MS analysis of EA4 and deglycosylated EA4 indicated that the carbohydrate moiety of EA4 has the mass of 730 Da. Then, EA4 was digested with trypsin and chymotrypsin to identify the glycosylated peptide. The peptide fragment from Gly21 to Phe25 was found to carry the carbohydrate moiety. LC-MS/MS analysis of this peptide fragment revealed the sequence of the attached oligosaccharide (-HexNAc-HexNAc-Hex-Hex) and the glycosylation site (Asn22) at the same time. Utilizing the nano-LC-ESI-Q-TOF-MS, MS/MS, the copper binding sites in TIME-EA4 were also characterized. EA4 was treated with hydrogen peroxide, intended to specifically oxidize histidine residues coordinated to the copper ion as a mass spectrometric probe. The oxidized EA4 was then fragmented with trypsin and α-chymotrypsin. Separation of the peptide mixture with the nano-HPLC and the on-line ESI-Q-TOF MS analysis revealed only one peptide fragment was oxidized to a significant extent. Further analysis of the oxidized peptide fragment with nano-LC-ESI-Q-TOF-MS/MS disclosed that His50 and His52 were specifically oxidized. Therefore, these two histidine residues were identified to be coordinated to the redox-active copper ion.
Toxins in venom grants of wasps, spiders, or scorpions have attracted much attention because of their intriguing biological activities as neurotoxins. Studies for isolation and structure determination of the venom components are extensive. These toxins are composed of complex composites including peptides, arylethanolamines, and acylpolyamines. In a venom grant, only a small amount of toxins is available in the level of pico to nano mole. Structure determination of these components requires a highly sensitive analytical method. Our approach to this subject is the use of various types of MS. Herein, we wish to report a new methodology for the structure analysis of peptide by MS/MS based on the use of squaric acid as a charge-remote fragmentation (CRF) inducing functional group. Model peptides containing a squaryl group (Sq) in the N-termini were synthesized by condensation of a Sq amino acid or a Sq acid diester with a tetrapeptide. FAB/CID/MS of the Sq group-containing peptides exhibited clear CRF patterns. These are the first observation that the Sq group induced CRF. The successful application of the present method to bradykinin and angiotensin-I in a small scale (10 nmol) are also disclosed.
Cyanobacteria (blue-green algae) are photoautotrophic prokaryotes which include a large variety of species with varied morphological, physiological, and biochemical properties. It is well known that some kinds of cyanobacteria produce large amounts of exopolysaccharides. Although the quantity and biochemical properties are diverse depending on the species, these polysaccharides are thought to play important roles in desiccation tolerance, UV tolerance, nutrient uptake, and interactions with other organisms in the establishment of symbiosis. Nostoc commune, a nitrogen-fixing filamentous cyanobacterium, also produces copious amounts of polysaccharide around the cells and has remarkable desiccation tolerance. Despite conspicuous features of polysaccharide of Nostoc commune, little is known about its chemical structure due largely to its structural complexity. In general, polysaccharides of cyanobacteria are more complex both in their sugar composition and in their linkage types than those of other bacteria or macroalgae, and at this time only a few structures of cyanobacterial polysaccharides have been proposed. In this study, we investigated the chemical structure of water-soluble polysaccharide from desiccation-tolerant cyanobacterium Nostoc commune. The water-soluble polysaccharide (glycan) was extracted and purified from field-grown Nostoc commune. Sugar compositional analysis revealed that this polysaccharide contained D-glucose, D-galactose, D-xylose, and D-glucuronic acid in the molecular ratio of 8: 4: 4: 1 together with unknown uronic acids and trace amount of ribose. One of the unknown uronic acid was identified as 2,3-O-(1-carboxyethylidene)-D-glucuronic acid by NMR analysis of the methanolysate of the polysaccharide. Partial hydrolysis of the polysaccharide by acid (0.2 N TFA, 90℃, 5h) or by crude enzyme which was obtained from culture brose of Paenibacillus sp. afforded 5 oligosaccharide fragments. After pyridylamination, these oligosaccharides were isolated by reverse phase column chromatography, and structures were elucidated by 2D NMR. By comparison with the structures of these oligosaccharides, we concluded that the polysaccharide had a 5-membered repeating unit viz.→4)-β-D-Glcp-(1→4)-β-D-Xylp-(1→4)-[β-D-GlcAp-(1→6)]-β-D-Glcp-(1→4)-β-D-Galp-(1→, and residues in the repeating unit were partially substituted by 3-O-(1-carboxyethyl)-D-glucuronic acid or 2,3-O-(1-carboxyethylidene)-D-glucuronic acid in the GlcA moiety and by 2-O-Me-Man in the branched Glc moiety as shown in Figure 5.
Stereochemistry is an essential attribute for accurate NMR chemical shift prediction. We have developed a new computer system CAST/CNMR for ^<13>C-NMR chemical shift prediction considering stereochemistry, efficiently, for structure analyses of natural products. Prediction by CAST/CNMR is based on a structure-observed ^<13>C-NMR chemical shift database where structural information including stereochemistry are systematically and canonically described by the CAST (CAnonical-representation of STereochemistry) coding method, which was developed for this purpose. CAST code is based on dihedral angles that are uniquely defined by a molecular tree structure. A model database was constructed by using data of terpenoids, polyketides, and polyethers for the applications. Synthetic compounds are also used for construction of the database. The prediction is performed by matching with the database considering structures from an atom to be predicted to arbitrary level specified by the user. In the present study, structural environments including relative stereochemistry to γ-level are mainly considered. Size of rings was also considered in some applications for more highly accurate prediction. Predictions of ^<13>C-chemical shifts by CAST/CNMR were examined for some terpenoids such as arisugacin F, 17-deoxyaphidicolin, 20-hydroxyecdysone, deoxoscalarin-3-one (1), and 12-epi-deoxoscalarin-3-one (2). Highly accurate ^<13>C-NMR chemical shifts were predicted for them by considering stereochemistry. Some miss-assignments in literatures were also found during the examinations. CAST/CNMR gave good results to assign the ^<13>C-NMR chemical shift for hemibrevetoxin B (3).
(-)-Neplanocin A is a carbonucleoside isolated from Ampullariella regularis in 1981, and have a S-adenosylhomocystein hydrolase inhibitory activity. Until today, several examples of the total synthesis were reported. However, there is no precedent of the total synthesis using intra-molecular aldol reaction for ring construction of neplanocin A. During the course of our studies on development of asymmetric reaction, we have already developed a lithium thiolate-catalyzed stereoselective Michael-aldol tandem reaction of α,β-unsaturated esters with aldehydes. We describe here a development of lithium thiolate-initiated Michael-aldol tandem cyclization reaction as an extension of intermolecular tandem reaction, and a total synthesis of (-)-neplanocin A by using the present cyclization as a key reaction for construction of five-membered ring. The reaction of ω-oxo-α,β-unsaturated ester 6 with 0.2 equiv of lithium thiophenolate (PhSLi) in the presence of 2 equiv of phenyl trimethylsilyl sulfide (PhSTMS) in THF at 0℃ for 2h gave cyclization products 7a and 7b in 61% and 18% yields, respectively. The presence of PhSTMS, which traps lithium alkoxide as a stable TMS ether, was essential for both high yield and stereoselectivity. High yield and stereoselectivity was also obtained by using lithium benzylthiolate (BnSLi) as a nucleophile which has higher nucleophilicity than that of PhSLi. The reaction of 6 with 1.2 equiv of BnSLi in THF at -20℃ proceeded within 1h to give cyclization product 11 in 95% yield as a sole product. We applied this cyclization reaction to total synthesis of (-)-neplanocin A. TBS-protected chiral ω-oxo-α,β-unsaturated ester 23, which was derived from D-mannitol, was treated with 1.2 equiv of BnSLi in THF at-20℃ for 0.5 h to give three separable cyclization products 24a, 24b and 25 in 34%, 28% and 11% yields, respectively. After conversions of protective groups, the benzylsulfanyl part of 29 was removed by oxidation to sulfoxide and subsequent thermal elimination. Through the functional group transformations of 30, we have achieved a total synthesis of (-)-neolanocin A.
Peridinin (1), which was isolated from fish-water dinoflagellates causing red tide, has been known as an auxiliary light harvesting pigment for photosynthesis in the sea. The structure of this carotenoid was determined by Jensen's group in 1980. This molecular is highly oxidized unique C37 nor-carotenoid containing allene and ylidenebutenolide functions in the conjugated main polyene chain, in addition to the functionalized cyclohexane rings at both ends of the molecular. The first synthesis of peridinin was reported by Ito and co-workers in 1993. In that synthesis, however, the control of stereochemistry and therefore the yields were left out of consideration. For the stereocontrolled synthesis of peridinin, the questions are the following: (1) stereocontrolled construction of the all-trans-conjugated olefin chain containing the (Z)-γ-ylidenebutenolide moiety, and of the asymmetric allene function, and(2)stereocontrolled construction of the oxygen functions at the terminal cyclohexane ring. We resolved these questions and overcomed problems by effectively utilizing our own or new synthetic methods, and achieved the highly efficient total synthesis of peridinin by controlling the stereochemistry of six asymmetric carbons and six of seven geometries of the double bonds in this particular molecular. In this conference, we disclose in detail our synthetic efforts on the total synthesis of peridinin. The highly stereoselective introduction of the oxygen functions at a cyclohexane terminal was actually realized with the Sharpless asymmetric epoxidation by finding the strictly precise reaction conditions, and the key intermediate, (-)-epoxyaldehyde E was prepared in high purity. The allene half-segment A of peridinin was synthesized from the compound E by a transformation into the acetylene derivative 4, followed by the Sonogashira coupling with the conjugated vinyl iodide 5, and then S_N2' hydride reduction. The ylidenebutenolide segment Y was efficiently synthesized from the same compound E by a coupling of our own furan-Wittig reagent W followed by ^1O_2 oxygenation, acetylene formation, and then novel and effective three-step one-pot formation of the (Z)-ylidenebutenolide moiety. Finally, the both of segment A and Y was successfully coupled by utilizing the modified Julia-Kocienski olefination. The 15Z-peridinin thus obtained stereoselectively, gradually isomerized into all-trans-peridinin at room temperature. Thus, we achieved the stereocontrolled efficient synthesis of peridinin. The knowledge obtained from our present synthesis is believed to essencially contribute to the polyfunctional carotenoid synthesis.
In the course of our screening for NADH-fumarate reductase (NFRD) inhibitors, nafuredin (1), which is potentially a selective antiparasitic agent, was isolated from the fermentation broth of a fungal strain, Aspergillus niger FT-0554. Nafuredin (1) inhibited NFRD of Ascaris suum with an IC_<50> value of 12 nM without cytotoxicity and exerted anthelmintic activity against Haemonchus contortus in in vivo trials with sheep. These useful biological activities of 1 attracted our attention and prompted us to undertake the total synthetic study. In the beginning, we elucidated the absolute configuration of 1 as (2R,3S,4S,5R,6E,8E,10R,12E,14E,16S)-3,4-epoxy-2-hydroxy-4,10,12,16-tetramethyl-6,8,12,14-octadecatetraeno-5-lactone by degradation and synthetic studies (Schemes 1, 2, 3, and Fig. 1). Next, we embarked on the total synthetic study and envisioned a convergent approach towards 1 via a stereoselective one-pot Julia olefination between the sulfone 17 and aldehyde 18 followed by appropriate functional group elaboration of the resulting 16 (Scheme 4). The sulfone 17 and aldehyde 18 were derived efficiently from D-glucose and (S)-(-)-2-methyl-1-butanol 15, respectively (Schemes 5 and 6). The one-pot Julia olefination between 17 and 18 could be effected to provide the desired (6E,8E,12E,14E)-alcohol 31 in 79% yield as a single isomer (Scheme 7). Finally, 31 was converted to nafuredin (1) via formation of epoxide and lactone. Synthetic nafuredin (1) was identical with natural (1) in all respects ([α]D, ^1H- and ^<13>C-NMR, IR, FAB-MS and inhibitory activity against NFRD). We have achieved the first total synthesis of nafuredin. Investigation of the structure-activity relationship and biological studies of 1 are currently in progress.
Trachyspic acid was isolated from the culture broth of Talaromyces trachyspermus SANK 12191 after screening for low molecular weight substances that exhibit heparanase inhibitory activity. This new metabolite possesses a unique spiroketal structure consisting of the 2-nonyl-3-furanone and the tetrahydrofuran having citric acid unit. However, even the relative configuration of this compound has not been determined yet. We describe the first synthesis of (±-trachyspic acid, thereby establishing its relative configuration to be 3R^*4S^*6S^*. Aldol reaction of tert-butyl 4-(p-methoxybenzyl)oxy-2-oxo-butyrate (9) and tert-butyl 4-pentenoate (10) gave (3R^*,4S^*)-11a and (3R^*,4S^*)-11b in a ratio of 3: 2. Upon silylation and oxidative cleavage of the olefinic double bond, 11a and 11b were converted to key aldehydes 12a and 12b, respectively. The crucial Ni(II)/Cr(II)-mediated reaction of 12a with triflate 16, prepared from 3-(1,3-dioxolan-2-yl)dodecan-2-one (15), turned out to occur successfully to produce adduct 17a in 84% yield. Similarly, Ni(II)/Cr(II)-mediated reaction of 12b with 16 afforded 17b in 59% yield. Upon Dess-Martin oxidation, desilylation, and HClO_4-treatment, 17a and 17b gave the corresponding spiroketal 19a and 19b in very good yields. After acetylation of 19a and 19b, the corresponding acetates were each converted to ketone 22a and 22b by a three-step sequence involving removal of the p-methoxybenzyl protecting group, oxidation of the primary alcohol, and ozonolysis of the exomethylene group. Finally, exposure of 22a and 22b to TFA furnished the corresponding tricarboxylic acid 23a and 23b, respectively. At this stage, we found that 23b was identical with trachyspic acid by ^1H NMR comparison. The trimethyl ester of 23b also exhibited spectral properties in accord with those reported for the trimethyl ester of natural trachyspic acid.
Recently biologically active and structurally unique triterpene polyethers, which are thought to be biogenetically squalene-derived natural products, have been isolated from both marine and terrestrial plants (Figure 1). Among the polyethers, our synthetic targets in this symposium are cytotoxic 14-deacetyl eurylene (3), eurylene (5), and longilene peroxide (4), isolated from the wood of Eurycoma longifolia by Itokawa et al. (Table 1). Although the stereostructures and conformations of 3-5 have been elucidated by X-ray crystallographic analysis and spectroscopic methods, the absolute configuration of 4 had not hitherto been determined. Furthermore, total syntheses of 3 and 4 with potent cytotoxic activities have never been accomplished. Here we report the efficient and stereoselective total synthesis of (+)-14-deacetyl eurylene (3) and (+)-eurylene (5), featuring monool- and diol-differentiated chemoselective oxidative cyclizations promoted by rhenium(VII) and chromium(VI) oxo species, respectively. We also report the first enantioselective total synthesis of (-)-longilene peroxide (4) through the concept of two-directional synthesis utilizing its molecular symmetry and the determination of its absolute configuration. Our retrosynthetic analysis of 3 and 5 is illustrated in Scheme 1. These compounds have 8 asymmetric carbon centers and the trans and cis tetrahydrofuran (THF) rings. The most important key events for the total synthesis are the stereoselective construction of these THF rings and the differentiation of the 14-hydroxy group. To solve these problems, we focused on the hydroxy-directed syn oxidative cyclization of acyclic bishomoallylic alcohols promoted by rhenium(VII) and chromium(VI) oxides (Scheme 2). The treatment of triol 6 with an excess of (CF_3CO_2)ReO_3・2CH_3CN and TFAA in CH_2Cl_2/CH_3CN (9/1) at-40℃ for 1.5 hr diastereoselectively gave the expected trans THF product 16 in 84% yield (Scheme 3). On the other hand, 16 was treated with a stoichiometric amount of PCC in CH_2Cl_2 at rt for 30 min to produce (+)-14-deacetyl eurylene (3) with complete cis diastereoselectivity. Finally, selective acetylation of the 14-hydroxy group in 3 afforded another objective (+)-eurylene (5). The retrosynthetic analysis of 4 is depicted in Scheme 4. It was envisaged that the hydro-peroxy group can be introduced by oxidizing the terminal double bonds in the C_2 symmetric triTHF ether 23 with singlet oxygen. The two 2,2,5-trisubstituted THF rings in 23 will be constructed in a two-directional manner through Shi's asymmetric epoxidation of bishomoallylic alcohol 19 followed by epoxide-opening reactions. The monoTHF ether 19 will be, in turn, derived from the diepoxide 17 by extending both side chains with the C_<10> unit 18, still in the two-directional mode. According to the synthetic plan, the first asymmetric total synthesis of (-)-longilene peroxide (4) has been achieved starting from the optically active C_2 symmetric diepoxide 17 (Scheme 5). Thus, the unknown absolute configuration of longilene peroxide has been determined by this synthesis as shown in the structural formula 4.
Recently, much attention has been paid to several shellfish toxins. Pectenotoxins are a family of cyclic polyether macrolide toxins, in which pectenotoxin 2 was isolated by not only from the digestive glands of poisonous scallop (Patinopecten yessoensis) and also from an identified dinoflagellate (Dinophysis fortii) as the causative organism. Although this compound possesses an unusual and complex molecular structure and shows significant diarrhetic intoxication as well as actin depolymerizing activity, there has been no report on completion of the total synthesis. On the other hand, gymnodimine, isolated from New Zealand oysters (Tiostrea chilensis) and the dinoflagellate (Gymnodinium cf. mikimotoi), is characteristic structurally and toxically. Pinnatoxins have been isolated from the Okinawan bivalve (Pinna muricata). Specially, pinnatoxin A was first isolated as the major toxic compound and the absolute structure was clarified by the first total synthesis of ent-pinnatoxin A by the Kishi group. We have continued studies aiming to the total syntheses of these shellfish toxins, the detail of which will be described. Pectenotoxin 2 was disconnected to the left side (1) and the right side compounds (2), which were planned to construct the main 34-membered lactone system via their Julia-type coupling and the following ring closing metathesis in the 31 and 30 positions. We have succeeded in the efficient synthesis of both the components (1 and 2). Concerning the synthesis of gymnodimine and pinnatoxin A, we have developed the new exo-Diels-Alder reactions by applying Ellman's and Evans' reagents starting from the N-protected α-methylene lactams (19 and 22) as the dienophiles, respectively. When 19 was reacted with the diene (20) in the presence of (-)-siam ligand, CuCl_2, and AgSbF_6, the cyclo-addition occurred to give 21 in 30% isolated yield with 95: 5 diastereo-selectivity. Further reaction using 27 with 19 is now under way in our laboratory. The optically pure methylene lactam 22 was treated with the diene 23 as a model to afford the adduct 24 in 42% isolated yield with 93: 7 diastereo-selectivity. Finally, in order to construct the common B,C,D,E,F ring system of pinnatoxins, the tetra-ketone 28 was prepared and reacted with HF-Py. The extensive solvent effect was observed; when MeCN was used as a solvent, the desired construction proceeded smoothly to provide 29 in high yield. On the basis of these results, we are now investigating toward the total syntheses of these target compounds.
A convergent synthesis of polycyclic ethers has been achieved by the intramolecular allylation of α-acetoxy ethers and subsequent ring-closing metathesis. The carboxylic acid 2 and alcohol 3 were connected by DCC coupling to give the ester 4 in 90% yield. After deprotection of the silyloxy group, the alcohol 5 was converted to the allylic stannane 8 via the mixed acetal 7 in good yield. The ester 8 was then subjected to the Rychnovsky protocol to give the α-acetoxy ether 9 as a mixture of diastereoisomers. The cyclization precursors 10-15 were prepared in a similar manner, and the results of the cyclization are summarized in Table 1. Treatment of 9 with 4 equiv of BF_3・OEt_2 gave a 70: 30 mixture of the cyclized products 16 and 17 in 79% yield (entry 1). Similarly, the cyclization of the substrates 10-15 proceeded stereoselectively to give the desired products in good yields (entries 2-7). We next examined the ring-closing metathesis of the products 18, 20, 22, 24, 25, and 27 (Table 2). Treatment of 18 with Grubbs catalyst 29 gave the tetracyclic ether 30 in 91% yield (entry 1). Similarly, the reactions of 20, 22, and 24 proceeded smoothly to afford the corresponding polycyclic ethers 31-33 in good yields (entries 2-4). Although the reactions of 25 and 27 with 29 were unsatisfactory, the use of the more active catalyst 34 provided the desired pentacyclic ethers 35 and 36 in high yield (entries 5 and 6). Based on these results, we have started the convergent synthesis of gambierol 1 and have synthesized the ABC and FGH ring segments, 48 and 60, from 2-deoxy-D-robose as shown in Schemes 2 and 3, respectively. Further studies toward the total synthesis of gambierol are now in progress in our laboratories.
Ciguatoxin (CTX1B) and its congeners, naturally occurring polycyclic ethers originating from marine unicellular alga, are the principal toxins associated with ciguatera fish poisoning. These compounds are extremely potent neurotoxins that bind to voltage-sensitive Na^+-channels (VSSC) and inhibit depolarization to allow Na^+ influx to continue. The structural complexity and exceptionally potent neurotoxicity, as well as their scarcity in natural sources, have attracted the interest of synthetic community. Practical synthetic access to ciguatoxins and their designed unnatural analogues are required for further studies aimed at fully defining the structural basis for their high-affinity binding to VSSC and/or its activation. We have recently developed a highly convergent synthetic route to the fused polycyclic ether structure based on B-alkyl Suzuki coupling reaction. Herein we describe our studies toward the total synthesis of 51-hydroxyCTX3C (1), a representative ciguatoxin congener with comparable toxicity to CTX1B. Our convergent synthetic plan for 1 involves the separate construction of two advanced intermediates representing the ABCD (2) and FGHIJKLM (3) ring fragments followed by their union via Horner-Wittig coupling and hydroxy thioketal cyclization. The ABCD ring fragment 2 was stereoselectively prepared via hydroboration-Suzuki coupling of the B and D rings (4 and 5, respectively) followed by closure of the C ring, and construction of the A ring by ring-closing metathesis reaction. The synthesis of the FGHIJKLM ring fragment 41, a potential precursor to 3, featured palladium-catalyzed carbonylation of lactone-derived enol phosphate to construct the nine-membered F ring and a two-stage B-alkyl Suzuki coupling of the FG (6), I (7) and KLM (8b) ring fragments. The present synthesis demonstrated the general applicability of our strategy based on B-alkyl Suzuki coupling to the convergent synthesis of polycyclic ether system. Progress toward the completion of the total synthesis of 1 through coupling of 2 and 3 is underway.
Ciguatoxins are the toxic principles of ciguatera, a widespread seafood poisoning of dinoflagellate origin. The structures consist of 13 trans-fused cyclic ethers including eight- and nine-membered oxacycles. Here, we will present our achievement of the first total synthesis of ciguatoxin CTX3C (1) using ring-closing metathesis (RCM) as the key transformation. The ABCDE fragment 2 was synthesized by an alkylative coupling between the AB ring 4 and the E ring 5, followed by construction of the CD ring portion using RCM. The synthesis of the HIJKLM ring fragment 3 was started from the I ring 6 and the LM ring 7, which were coupled by esterification to afford the dithioacetal 17. Intramolecular carbonyl olefination of 17 using a low-valent titanium reagent produced the cyclic enol ether 16 reproducibly, the product 16 was then converted to the IJKLM fragment 19. Furthermore, the H ring moiety was successfully constructed in 19 in a similar manner to our previously established route, utilizing an acid catalyzed vinylepoxide-alcohol cyclization. In order to establish a viable coupling strategy of the fragments, the EFGH ring fragment 36 was chosen as a model compound. The desired mixed acetal 32 was obtained by the reaction of the seven-membered acetal 31 with TMSSPh in the presence of TMSOTf in 87% yield. Subsequently, the RCM reaction of 35 successfully provided the EFGH ring fragment 36. Next, the developed coupling method was applied to the total synthesis. Condensation between the ABCDE ring fragment 2 and the HIJKLM ring fragment 3 afforded the seven-membered acetal 37. Finally, deprotection of the tris-benzyl CTX3C (41) under Birch conditions gave synthetic 1, identical in all respect with the natural product. We have thus realized the first total synthesis of CTX3C (1) using a powerful, highly convergent, and efficient strategy.