To clarify a defense mechanism of liverworts against the higher plants, we examined defense reaction of the cultured cells of M. polymorpha against metabolites of higher plants, e.g. monoterpenoids. When bornyl acetate (1) was added into the cultured cells of M. polymorpha as a stress substrate, 4-[2-(3-hydroxyphenyl)ethyl]phenol (2) and 4-hydroxy-2,3-dimethyl-2-nonen-4-olide (3) were secreted from the cells as the allelochemicals. Especially, 3 induced the complete death of the cultured cells of Nicotiana tabacum. To elucidate the biosyntheses of 3, feeding experiments of [1- ^<14>C]acetic acid and [4,5-^3H] isoleucine into 3 were carried out. [1-^<14>C]Acetic acid was incorporated into 3 in 0.11 %. On the other hand, [4,5-^3H]isoleucine was incorporated into 3 in 0.016 % via angelic acid. These results suggested that 3 is biosynthesized from angelic acid and caproic acid, which were produced from isoleucine and acetic acid, respectively. Nextly, a transduction mechanism of the signal against the bornyl acetate-stimulation in the cultured cells of M. polymorpha was examined. Addition of bornyl acetate (1) to the cultured cells of M. polymorpha caused a rapid increase in the intracellular level of cAMP. Furthermore, the Sp-cAMPS, which is a cAMP agonist, activated the H_2O_2 formation and the cAMP antagonist (Rp-cAMPS) inhibited the H_2O_2 formation. These results suggest that a defense reaction against monoterpemoids exists in liverwort.
Safflower(Carthamus tinctorius L.)is well known as one of source plants of red pigments since ancient times. From the petals a red pigment, carthamin (4), several orange and yellow ones, precarthamin (3) and safflor yellow B (2) etc., and flavonol glycosides, 9 etc., were identified by us and other several groups. 3, an unstable orange yellow pigment, was confirmed to be a precursor of carthamin by us. However, the stereochemistry of the determined structural formulas and their biosynthetic path way are still unresolved. During the search study of biosynthetic intermediates into precarthamin, we now isolated a new compound, named as anhydrosafflor yellow B (1), as dark orange powder which was transformed into carthamin by standing the solution dissolved in the NMR solvent(methanol/pyridine=95/5) for a month at r.t.. It showed m/z 1045(FAB^+, [M+H]^+), λ_<max> 410nm (ε42000, Me0H), λ_<εxt>441nm (Δε+45.4) and λ_<ext>386nm (Δε-30.8). From ^1H, ^<13>C, and 2D NMR spectra(Table) the presence of two cinnamoyl, two 2,4,6-trihydroxy-4-C-glucosyl-cyclohexadienone, and a six carbon C-1 deoxyglycitol groups are confirmed. These data suggested that this compound also have a dimeric quinochalcone skeleton, such as 2 and 3, common in pigments of safflower. The δ values of the C-2 carbon of glycitol at 94.2 and the oxygen substituted sp^2 carbon at 171.3 are shifted to lower and higher field, respectively, compared with δ values of the corresponding carbon in 2. This suggested that these two carbon were binded through an ether bond and hence, a five membered ether ring was formed in 1. Since J_<H1-H2> and J_<H2-H3> in the glycitol were 8.5 and 3.4Hz respectively and it seems the glycitol was derived from D-glucose, it was determined that the juncture of the five membered ether ring was trans and the glycitol was 1-deoxy D-glucitol with a defined C-1 configuration. Because of a keto-enol tautomerism of carbonyl group to form an ether bond, two different ring closed structures are formed and furthermore two configurational isomers at C-4 in the cyclohexadienone ring should be considered. Therefore, the four structural isomers for the plane structural formula of 1 are inferred. The conformational analysis for them by a molecular mechanics simulation(Fig. 2) and the judgment for calculated structures based on the CD (plus chirality) and NOE(between glucitol and one cinnamoyl) were carried out. Consequently, a most expected structure is considered to be A shown in Fig. 2 with a extended arrangement of two cynnamoyl groups and trans configuration for the C-1 H of D-glucitol and D-glucose in the cyclohexane ring without ether bond.
Proliferation of dispersed plant cells in culture is strictly dependent on cell density, and cells in a low density culture can only grow in the presence of conditioned medium (CM). No known plant hormones have been able to substitute for CM so far. In order to quantify mitogenic activity of CM. we examined conditions for the assay system using mechanically dispersed mesophyll cells of Asparagus officinalis L., and established a highly sensitive bioassay method. By use of this system, the mitogenic activity of CM prepared from asparagus cells was characterized: it was heat stable, susceptible to pronase digestion, and resistant against glycosidase treatment. On the basis of these results, the mitogenic activity in CM was purified 10^7-fold by column chromatography, and two factors named phytosulfokine-α and β(PSK-α and PSK-β)were obtained. By amino acid sequence analysis and mass spectrometry, structures of these two factors were determined to be sulfated pentapeptide (H-Tyr(SO_3H)-Ile-Tyr(SO_3H)-Thr-Gln-OH) and sulfated tetrapeptide (H-Tyr(SO_3H)-Ile-Tyr(SO_3H)-Thr-OH). PSK-α and PSK-β were prepared by chemical synthesis and enzymatic sulfation. The synthetic peptides exhibited the same activity as the natural factors confirming the structure for PSK-α and PSK-β mentioned above. This is the first study to demonstrate the actual structure of peptidal growth factors in the plant kingdom.
Several Streptomyces species were reported to biosynthesize terpenoidal compounds via the mevalonate pathway, while the biosynthesis of pentalenolactone, a common metabolite of Streptomyces, was explained by involvement of the non-mevalonate pathway disclosed recently. These findings suggest that the genus Streptomyces utilizes either one (or both) of two different pathways for the formation of isopentenyl diphosphate (IPP) depending on species. To address this unusual issue in more detail, we have studied the biosynthesis of a primary metabolite, ubiquinone, and a secondary metabolite, naphterpin produced by Streptomyces aeriouvifer. Labeling experiments using [6-^2H_2]glucose and [U-C_6]glucose showed that most of the ubiquinone molecules was produced by the non-mevalonate pathway. On the other hand, naphterpin which was produced at later stage of the fermentation was mainly produced by the classical mevalonate pathway. In addition, we have studied the biosynthesis of an unusual tetraterpenoid, longestin (KS-505) which is produced by Streptomyces argenteolus. It turned out that the producing organism possessed only the non-mevalonate pathway which was used for the formation of longestin.
2-Deoxystreptamine 1 is a common central aglycon in the major group of clinically important aminocyclitol antibiotics. The initial step of 2-deoxystreptamine biosynthesis is the intramolecular C-C bond formation between C-1 and C-6 of D-glucose-6-phosphate 3 to form 2-deoxy-scyllo-inosose 2 by 2-deoxy-scyllo-inosose synthase. An aldol-type cyclization involving transient oxidation of C-4 with NAD^+ and subsequent elimination of inorganic phosphate has been proposed. In this study, enzyme purification was carried out from centrifuged supernatant of the disrupted Bacillus circulans cells by ammonium sulfate fractionation, followed by DEAE-, Dye-ligand-, Q-Sepharose FF-, Mono Q- (x2) and gel filtration chromatography to afford two major spieces. The molecular weight of these enzymes were estimated to be about 45,000. Cross-over experiments with doubly labeled D-[4-^2H, 3-^<18>O] glucose-6-phosphate 1 1 were performed with the partially purified enzymes. The deuterium label at C-4 of the substrate was retained at C-6 of the product without scrambling the doubly-labeled isotope, and kinetic isotope effect (k_H/k_D ≒2.4-2.7) were observed in the enzyme reaction. These results strongly suggest that "2-deoxy-scyllo-inosose synthase" is a single enzyme, which catalyzes multistep reaction involving oxidation-reduction with NAD^+ cofactor and aldol-type carbocycle formation. Further, reaction with deoxy-G-6-P was investigated to clarify whether or not the other hydroxyl groups of G-6-P are participated in the reaction. Both 2-deoxy- and 3-deoxy-G-6-P were converted to some extent into their corresponding cyclitols. This indicates that the C-2 and C-3 hydroxyl groups are involved in the substrates recognition, but are not necessary for triggering the reaction.
Molecular cloning of the gene of hexaprenyl diphosphate synthase (HexPS) of Micrococcus luteus B-P 26 was achieved, and the gene of heptaprenyl diphosphate synthase (HepPS) of Bacillus subtilis was also identified. We have already reported the cloning of the gene of HepPS of Bacillus stearothermophilus and revealed that the two essential components of this enzyme (Component I' and Component II') were encoded by the first and the third out of the three neighboring open reading frames. DNA sequence analyses of the genes encoding HexPS of M. luteus B-P 26 and HepPS of B. subtilis revealed that the locations of these genes were very analogous to that of HepPS of B. stearothermophilus. The first and the third genes of them were found to encode the two components of medium-chain prenyl diphosphate synthases (Component A and Component B of HexPS or Component I and Component II of HepPS), respectively. The deduced amino acid sequences of the larger subunits (Component B, -II and -II') have the seven highly conserved regions typical of prenyltransferases. The smaller subunits (Component A, -I and -I') have no similarities to any proteins currently registered. Enzymatic activities were screened with possible combinations between these components, and the results indicated that Component I' of HepPS of B. stearothermophilus can be replaced by Component I of B. subtilis. The resulting hybrid-type HepPS showed some properties between B. subtilis HepPS and B. stearothermophilus HepPS, i.e. two separable components and medium heat stability. Although the subunits were not exchangeable between HexPS and the two kinds of HepPSs, some conserved amino acid residues were found among them. This implies that these residues may play an important role for the expression of the catalytic activity of medium-chain prenyl diphosphate synthases with these unique two-component systems.
The cyclization of 2,3-oxidosqualene to the backbones of steroid and triterpene is one of the most important and remarkable reactions found in nature. In higher plants, the cyclization of 2,3-oxidosqualene is an important branch point between sterol and triterpene biosynthesis. To understand the enzyme reaction mechanism and the regulation of the biosynthesis of steroid and triterpene, we have been studying 2,3-oxidosqualene cyclases in plants. We have tried to purify cycloartenol synthase and β-amyrin synthase from the cell suspension cultures of Rabdosia japonica and the seedlings of Pisum sativum. All of the cyclase activities were detected in membrane bound proteins and required detergents for their optimal activities. They were successfully solubilized with Triton X-100. Each solubilizedsynthase was purified with the combination of column chromatography of hydroxylapatide, DEAE-cellulose, isoelectric focusing and gel filtration. The purified cycloartenol synthases showed a single band at 54K(Rabdosia japonica) and 56K (Pisum sativum) on SDS-PAGE, respectively, while the purified β-amyrin synthases showed a single band at 28K(Rabdosia japonica) and 35K(Pisum sativum). Dammarenediol has the different skeleton from those of cycloartenol and β-amyrin and expected to be formed in a similar manner to β-amyrin until the formation of dammarenyl cation but the reaction is terminated by the addition of water before backbone rearrangement. As there has been no report on dammarenediol synthase, we have tried to detect this enzyme in the hairy root of Panax ginseng. Panax ginseng produces a number of triterpene saponins and one of the major saponins is ginsenoside R_<b-1> whose aglycone is protopanaxadiol (12β-hydroxy-dammarenediol). The microsomal preparation from the hairy root culture of this plant was able to convert [3-^3H]-2,3-oxidosqualene to (20S)-dammarenediol. The cyclization activity was highest at relatively acidic pH, and did not require any detergent. This finding was in sharp contrast to the cycloartenol and β-amyrin synthases from Rabdosia japonica and Pisum sativum all of which absolutely required detergent for their optimal activities. Several 2,3-oxidosqualene cyclases have been cloned recently from yeast, rat, human, and plant. Several oligo DNA primers based on the highly homologous regions among the known cyclases were designed for PCR amplification of cyclase cDNA from plant. PCR performed on cDNA mixture of Pisum sativum seedling using these primers gave 289 bp product which showed 79% identity to the corresponding region of Arabidopsis thaliana cycloartenol synthase cDNA. The sequence of the full length cDNA was finally obtained with 5'- and 3' RACE methods and showed 76% identity to Arabidopsis thaliana cycloartenol synthase cDNA. The identity of this cDNA as cycloartenol synthase was confirmed by functional expression in yeast.
The whole plant of Incarvillea sinensis has been used as anti-rheumatic and analgesic medicine in China. We have examined the constituents of this crude drug in order to reveal active principle(s), and obtained a number of novel alkaloids, which were classified into two groups: monoterpene alkaloid derivatives (1-10) and spermine related compounds (12-17), whose chemical structures have been clarified by the mass measurements, various NMR techniques and X-ray analyses. In parallel with this chemical investigation, the anti-inflammatory and analgesic activities of several fractions derived from the methanolic extract have been examined by formalin test in mice. This test has revealed that incarvillateine (8) has strong analgesic and sedative activities: Compound 8 (10mg/kg, i.p.) gradually inhibited both the first (neurogenic) and second (inflammatory) phases of formalin-induced pain and reduce the spontaneous motor activity. In this experiment, it has been suggested that anti-inflammatory and analgesic activity of 8 might be induced by its sedative action. However, 8 has showed the analgesic action for this test even in the range of 1-5mg/kg, i.p., in which 8 didn't show the effect toward the spontaneous motor activity in mice. Therefore, it has become clear that 8 could be accounted for the analgesic and sedative substance, whose actions are related to influence on the central nervous system.
As products of oncogenes and tumor suppressor genes are involved in the regulation of mam-malian cell cycle and also as cancers in fact are the uncontrolled cell proliferation with deregulation of cell cycle, new cell cycle inhibitors might be good candidates for cancer chemotherapy and also be a source for providing molecular probes useful in elucidating regulatory mechanism of the cell cycle. We have therefore begun on the screening for new cell cycle inhibitors from microbial origin. During the screening, we have isolated novel diketopiperazine alkaloids, spirotryprostatins A (1), B (2), tryprostatins A (3), B (4) and cyclotryprostatins B (6), D (8), together with three new natural diketopiperazines, cyclotryprostatins A (5), C (7) and demethoxyfumitremorgin C (9), as well as several known diketopiperazines such as fumitremorgin C (10) from the fermentation broth of a fungus, Aspergillus fumigatus BM939, through a separation procedure guided by inhibitory activity on the cell cycle progression of mouse tsFT210 cells. Structures of the new natural diketopiperazines obtained were determined mainly by the use of spectroscopic methods especially by detailed analyses of their ^1H and ^<13>C NMR spectra with the aid of 2D NMR spectroscopy including PFG-HMQC and PFG-HMBC techniques. The molecules of 1 and 2 had an unique structural skeleton with a spiro ring system composed from a γ-lactam fused to a benzene ring and a five-membered hetero ring fused to a diketopiperazine moiety, which are composed from a tryptophan unit, a proline residue and an isoprenyl group. The tryptophan units have been modified by dihydrogenation at C_2/C_3 and further oxidation at C_2 positions in both 1 and 2 and modified further by dehydrogenation at C_8/C_9 positions to form an enamine ring in 2. The isolation of 1 and 2 provided the first example of a novel class of natural diketopiperazines with an unique spiro ring skeleton. The molecules of 3 and 4 are composed from a 2-isoprenyl-L-tryptophan moiety and a L-proline residue, forming a diketopiperazine unit, which are distinguished from the molecules of fumitremorgin series by the opening of the central heterocyclic ring at the C-N bond between the 18 and 10 positions. Compounds 3 and 4 provided the first example of novel diketopiperazines as a new inhibitor of the mammalian cell cycle. Compounds 1-10 inhibited the cell cycle progression of tsFT210 cells in the dose range of micromolar order and certain structure-activity relationships were also observed. The present work provided diketopiperazines 1-10 as a new group of G2/M phase inhibitors of the mammalian cell cycle. In this symposium, we will mainly represent the production, isolation, structure determination and biological activities of the new natural diketopiperazines.
Constituents of the Japanese sea hare Dolabella auricularia collected in Mie Prefecture were examined by using bioassay, and novel cytotoxic macrolide glycosides, aurisides A (1) and B (2), and a new cyclodepsipeptide aurilide (4) were isolated. Aurisides A (1) and B (2) exhibited cytotoxicities against HeLa S3 cells with IC50 values of 0.17 and 1.2μg/mL, respectively. Their gross structures were established by spectroscopic analysis including the 2D NMR technique. On the basis of the NOESY spectral analysis and the degradation experiments, their absolute stereostructures were determined to be 14-membered macrolide glycosides that contain a bromine-substituted conjugated diene structure, a cyclic hemiacetal moiety, and a 2,4-di-O-methyl-L-rhamnopyranoside part. Aurilide (4) proved to be a 26-membered cyclodepsipeptide composed of a hexadepsipeptide part and a new dihydroxy acid on the basis of the 2D NMR analysis. The stereochemistry of the hexadepsipeptide moiety was determined by the HPLC analysis of the acid hydrolysate of 4. The stereochemistry of the dihydroxy acid moiety was determined by the enantioselective synthesis of the corresponding fragment 5 obtained by degradation of aurilide (4). Of the four syntheticdiastereomers 5a, 5b, 5c, and 5d, the ^1HNMR and the CD spectra for 5d were identical to those for natural 5, establishing the absolute stereochemistry of 5. On the basis of these findings the complete stereostructure of aurilide was determined as depicted in 4.
In search of new biologically active substances from marine organisms, we have isolated four novel cytotoxic macrolides named altohyrtins A (1), B, C, and 5-desacetylaltohyrtin A (2) from the Okinawan marine sponge Hyrtios altum. These macrolides exhibited extremely potent cytotoxicities against KB cells with IC50 values of 0.01-0.3ng/ml. The absolute stereostructures of altohyrtins have been elucidated on the bases of detailed NMR analysis, application of the modified MTPA method to hexa-MTPA ester, and application of the CD exciton chirality method. In this paper, we studied the solution structure of 5-desacetylaltohyrtin A (2) on the bases of NMR analysis and restrained molecular dynamics (rMD) calculation. The 323 distance restraints, which have been obtained by analysis of NOESY spectrum, 3 dihedral angle restraints, and the 24 chiral restraints were used for calculations. RMD calculations are carried out with the protocol of the random arrays of atoms, variable force constant simulated annealing (RA-VFC SA) method. In the case of use of the chiral restraints of 24 asymmetric carbons which we have determined previously, the resulting calculated structures were converged very well to the conformation with an average value of pairwised RMSD for backbone (C-1 to C-43) of 0.50 A. Furthermore, the stereochemistry of C-14-C-16 in 2 has been verified through the calculations. Using the same restraints and calculation strategy except for the chiral restraints of C-14 - C-16, we further ran 7 other sets of rMD calculations to analyze which chirality set for C-14 - C-16 is most adequate. Among the eight sets of calculations, the calculation using the chiral restraints (14R,15S,16S) we presented previously shows the lowest values of E_<total>, RMS violation for distance restraints, RMS violation for dihedral angle restraints, and RMSD and the highest number of the accepted structures.
Marine microorganisms are potentially prolific sources of highly bioactive secondary metabolites that might represent useful leads in the development of new pharmaceutical agents. As part of our ongoing search for new antitumour metabolites produced by microorganisms from marine organisms, penochalasins A (1)-C (3) have been isolated from a Penicillium sp. originally separated from the marine alga Enteromorpha intestinalis. Furthermore, penostatins A (4)-D (7), F (8) and G (9) have been isolated from this fungal strain cultivated in a different medium from that used for production of penochalasins. The stereostructures and conformations of these metabolites have been elucidated by spectroscopic analyses and some chemical transformations. Penochalasins are a novel class of cytochalasans including a pyrrole ring and exist in different conformers arising from rotation of the C-3-C-10 and C-10-C-3' axes in CDCl_3 and pyndine-d_5. Penostatins A (4)-D (7) and G (9), and F (8) are three-cyclic terpenes including dihydropyran and including cyclooctadienone, respectively. Among these compounds, 1-6 and 8 exhibited significant cytotoxicity against cultured P388 cells.
Further examination of the tunicate extract resulted in isolation of a total of 26 ritterazines whose structures were elucidated by spectroscopic analyses. Their cytotoxicity against P388 leukemia cells and that of derivatives obtained from the most active ritterazine B led to some structure-activity relationships. Ritterazine B which was unsymmetric was the most potent cytotoxin; ritterazines A,D,E,F,G,H,I,J,K,L,M and Y were also highly cytotoxic. These compounds have the 5/6 spiro ring and 12',25'-diol functionalities in common in the western hemisphere. However ritterazines N,O,P,Q,R,S,W,X, and Z having no 5/6 spiroketal were marginally active. Therefore the 5/6 spiroketal was supposed to be a important function for the expression of cytotoxicity, because the cleavage of the 5/6 spiroketal decreased their activity. Furthermore ritterazine T which had no conventional steroidal skeleton with OH-12 in the eastern hemisphere, but the 5/6 spiro ring in the western hemisphere showed weak cytotoxicity. Therefore, the presence of a 5/6 spiro ring in one end, a 5/5 spiro ring in the other end, and the hydroxyl group on C12 are shown to be important for cytotoxic activity. However acetylation of secondary hydroxyl groups gave interesting information. Compared with oxidation, acetylation of OH-12 does not decrease cytotoxicity, but induction of acetyl groups in the western hemisphere showed weaker activity. It is possible to consider that the steric hindrance by acetyl groups in the western hemisphere influences the expression of the cytotoxicity. In the future these findings will stimulate research on the mode of action of this important class of compounds.
In our continuing search for biologically active substances from marine organisms, three novel alkaloids halichlorine (1), pinnaic acid (2) and norzoanthamine (4) were isolated. The structures of these alkaloids were elucidated mainly by detailed analysis of NMR data and MS spectral data. Halichlorine (1) was isolated from the marine sponge Halichondria okadai Kadota. This compound inhibits the induction of VCAM-1 (vascular cell adhesion molecule-1) at IC_<50> 7μg/ml. Drugs that block the induced expression of VCAM-1 may be useful for treating atherosclerosis, coronary artery diseases, angina and noncardiovascular inflammatory diseases. We report here the isolation and structural elucidation of 1. A cytosolic 85-kDa phospholipase (cPLA_2) exhibits specificity for the release of arachidonic acid from membrane phospholipids. Therefore, compounds that inhibit cPLA_2 activity have been targeted as anti-inflammatory agents. Pinnaic acid (2) isolated from the Okinawan bivalve Pinna muricata, inhibits the cPLA_2 activity at 0.2mM. Interestingly, the structure of 1 is closely related to that of 2. Therefore, each carbon atom has been tentatively numbered under biogenetic consideration of formation of the N-C23 bond. Further studies on chemistry of halichlorine, including its absolute configuration, biogenetic pathway and structure-activity relationships, are currently in progress in our laboratory. Biogenetic pathway of zoanthamine and norzoanthamines (4) exhibiting inhibitory effect of IL-6 production will be described here.
The jellyfish,Aequorea victoria, possesses a green fluorescent protein (GFP, 27kDa), which serves as the ultimate light emitter in the bioluminescence. GFP is made up of 238 amino acid residues in a single polypeptide chain and has a fluorescent chromophore emitting a green light (λ<max> = 508nm) when irradiated with an ultraviolet light. It has been supposed that the chromophore consists of an imidazolone ring, formed by a post-translational modification of the tripeptide Ser^<65>-Tyr^<66>-Gly^<67> in the primary structure. We report here the results of a spectroscopic study on a peptide fragment of GFP bearing the chromophore and on model compounds 2-4 for structural elucidation of the GFP chromophore. The mass spectroscopic analysis of the chromophore-containing peptide isolated from the protease digest of GFP establishes the chromophore formation by the dehydration-dehydrogenation mechanism in the tripeptide Ser^<65>-Tyr^<66>-Gly^<67>. The clear overlapping of the UV-vis absorption spectra of model compound 3 with those of the lysyl endopeptidase fragment of GFP indicates that the structure of the GFP chromophore consists of a 4-(4-hydroxyphenylmethylidene)imidazol-5-one ring and that the GFP chromophore has a phenolate anion structure, when GFP is in the electronically excited state. GFP lost its fluorescence completely on protease digestion. Model compound 3 also is nonfluorescent in fluid media. On the other hand, both the lysyl endopeptidase fragment and 3 became highly fluorescent in ethanol glass at 77K. These results of the fluorescent behavior of the lysyl endopeptidase fragment and 3 are explained by a competition between the photoisomerization of the exo-methylene double bond and fluorescence emission in the singlet excited state of the imidazolone chromophore. Therefore the characteristic greenish fluorescence of GFP at room temperature may be considered as being due to a restriction of the molecular motion of the chromophore within the peptide environment.
A series of taurolipids has been isolated from a fresh-water protozoan Tetrahymena thermophila, and their unique chemical features have been clarified as: taurolipid A; 2-(3-acyloxy-7,13-dihydroxyoctadecanoylamino)ethanesulfonic acid, taurolipid B (2); 2-(3-acyloxy-2,7,13-trihydroxyoctadecanoylarnino)ethanesulfonic acid, taurolipid C; 2-(3-acyloxy-2,7,12,13-tertrahydroxy-octadecanoylamino)ethanesulfonic acid. Taurolipid B exhibits growth-inhibitory activity against HL-60. Methanolysis of taurolipid B gives a tetrahydroxy compound (3), which possesses four asymmetric centers. This paper describes determination of the absolute configuration of the four hydroxy groups of 3 by using 2ATMA (1; 2-anthrylmethoxyacetic acid), a newly developed chiral anisotropic reagent. The vicinal hydroxy groups of 3 at C-2 and 3 were protected by dimethylacetalization (acetone/CuSO4/H^+) to give acetonide (4). This compound was treated with 1.0eq (S)-2ATMAIEDC/DMAP/CH_2Cl_2, giving a mixture of mono-2ATMA esters(5 and 6)together with a small amount of di-2ATMA ester (7). Figure 1 shows the ^1H-NMR spectra of 5 (a) and 6 (b), in which the protons on the same side of the anthracene ring (see conformation 1a) shift upfield remarkably. The shifted signals were easily assigned to the protons denoted in the respective spectra, and 7R, 13S configuration was determined. It should be noted that only one enantiomer of 2ATMA was necessary to elucidate the absolute configuration owing to the strong anisotropic effect of 2ATMA. To confirm the proposed absolute configuration, 4 was treated with 1.0eq. (R)-2ATMA, affording monoesters 8 and 9. TheΔδvalues (δR-δS) were calculated and they are shown in 8a and 9a, which completely verified the absolute configuration at 7 and 13 positions. The absolute configuration of the glycol at C-2 and 3 were determined by combined usage of CD and 2ATMA: By analyzing the NMR pattern of the dimethyl-1,3-dioxolane moiety of 4, the threo relationship of the two hydroxy groups was established. 4 was converted into dianisoate 12, which showed positive split Cotton effect, thus leading 2S,3R configuration. This absolute configuration was also supported by the 2ATMA method. All these experiments lead to 2S, 3R, 7R, 13S configuration of the tetrahydoxystearate.
Pinguisane-type sesquiterpenes are biosynthetically interesting since biogenesis of their structures is difficult to be simply explained with the isoprene rule. In the course of our study on pinguisane-type sesquiterpenes, we examined the liverworts Aneura pinguis and Porella grandiloba. Five new pinguisane-type sesquiterpenes, 6a-hydroxy-3-oxo-pinguis-5(10)-ene-11,6-olide, 6a-methoxy-3-oxo-pinguis-5(10)-ene-11,6-olide, 3-oxo-pinguis-5(10),6-diene-11,6-olide, 5a,6a-epoxy-3-oxo-pinguis-11,6-olide and 3-oxo-ptychanolide together with pinguisone have been isolated from an axenic culture of the liverwort, A.pinguis. Three new pinguisane-type sesquiterpenes, grandilobalide A, B and 6a-hydroxy-pinguis-4,8-dimethoxycarbony1-11,6-olide, and a new rearranged pinguisane-type, grandilobalide C, together with six known sesqui- and diterpenes have been also isolated from the liverwort P.grandiloba grown in the field. Their structures were elucidated on the basis of spectroscopic evidence and chemical correlation. A biosynthetic study on the formation of pinguisane-type sesquiterpenes was also carried out by feeding [2-^<13>C]-acetate and [6,6,6-^2H_3]-mevalonate(MVA)to the cultured gametophytes of A. pinguis. Pinguisone and its derivatives were labeled at an adequate level to determine the labeling positions by ^2H- and ^<13>C-NMR analysis. The labeling pattern indicated two-methyl migration and C-C bond cleavage of the main chain in farnesyl diphosphate in the formation of pinguisone and its derivatives. From these observations, we proposed a biosynthetic pathway of pinguisane-type sesquiterpenes. Further experiments by incorporation of [2,2-^2H_2]- and [4,5-^<13>C_2]-MVAs have been done to confirm the proposed biosynthetic pathway.
The leaves of Viburnum awabuki (Caplifoliaceae) are known to have been used as traditional fish stupefying agent and fish poison for purpose of catching fish in Okinawa islands. Kawazu reported the isolation of a piscicidal activating agent, vibsanine A, a plant growth inhibitor, vibsanine B (1), and vibsanine C (4), which belong to unprecedented 11-membered and 7-membered ring diterpenes, respectively. Hence, we have proposed the new term "Vibsanine" for this type of diterpenes. Although occurrence of intriguing diterpenoids is anticipated, further chemical study on V. awabuki has not been done since then. From viewpoint of structural interest and biological activity of vibsanine-type diterpenes we have reinvestigated chemical constituents of V. odoratissimum and V. awabuki collected in Ishigaki island and Tokushima, resulting in the isolation of seven new vibsanine-type diterpenes 2, 3, 5-9, together with three unprecedented diterpenes 10, 11 and 12 named neovibsanines A, B and D. As first of all, the stereochemistries of vibsanines B (1) and C (4) which have remained unclear have to be defined. HPLC and ^1H NMR spectrum of vibsanine B indicated that 1 is a mixture of two conformational isomers as ratio of 99: 1. The relative configurations for both conformers were elucidated on the basis of NOESY data and then were clarified to be consistent with two most stable conformations obtained by MM2 calculation. Next, upon refluxing 1 in toluene an intermolecular Cope rearrangement took place to give 7-membered ring vibsanines 4 (85.9 %), 4a (11.2 %), 4b (1.6 %) and 4c (0.2 %). This result implies that 7-membered ring vibsanines may be biosynthesized via Cope rearrangement from 11-membered ring vibsanines. The absolute configurations of vibsanines B (1) and C (4) have been unambiguously determined based on the result of X-ray crystallographic analysis of 4e. The structures of new 7-membered ring vibsanines G (5), H (6), K (7), L (8), and L hydroperoxide (9) were elucidated by spectral data and comparison of ^<13>C NMR data with those of congeners. On the other hand, the plane structures of unprecedented neovibsanines A (1), B (2) and D (3) were determined by extensive analysis of ^1H-^1H COSY, HMQC, TOCSY and HMBC and their stereochemistries were elucidated by NOESY. It is worthy of note that the neovibsanine-type compound 13 was derived via a consecutive bond cleavage and formation upon irradiation of vibsanine B (1) by high pressure Hg lamp. Finally, cytotoxicity of vibsanine-type diterpenoids were examined, in particular, to know the effect of a hydroperoxy group presented in the molecule.
The roots of Beta vulgaris L. (sugar beet, Chenopodiaceae) have been used industrially as a raw material for sugar. The fresh roots and leaves of this plant are used as a vegetable and food garnish in Japanese-style dishes. In Chinese traditional medicine, the roots of sugar beet have been known to exhibit sedative and emmenagogue-like effects. As a part of our screening to find biologically active principles contained in medicinal foodstuffs, which have been used for both medicinal and alimentative purposes, the saponin fractions from the roots and leaves of sugar beet were found to show an inhibitory effect on the elevation of plasma glucose levels in the oral glucose tolerance test in rats. By using a bioassay-guided separation, we have isolated ten new saponins named betavulgarosides I, II, III, IV, V, VI, VII, VIII, IX, and X from the roots and leaves. The absolute structures of betavulgarosides I-X with a novel acetal type acidic substituent or a dioxolant moiety were determined on the basis of physicochemical evidence using 2D NMR technique (H-H, C-H COSY, NOESY, HMBC, H-H, C-H HOHAHA) and chemical findings, which included the chemical correlation of betavulgaroside IV with known saponin momordin I. Oleanolic acid 3-O -monodesmosides such as betavulgarosides II and IV and 28-deglucosylbetavulgaroside V were found to exhibit hypoglycemic activity, while oleanolic acid 3, 28-O-bisdesmosides such as betavulgarosides I, III, and V lacked the activity. Examination of the hypoglycemic activity of oleanolic acid and related glycosides led us to presume the following structure-requirement in oleanolic acid glucuronide-saponin for the activity: the 3-O-glycoside moiety and 28-O-carboxyl group are essential to the activity. On the basis of detailed examination for the hypoglycemic activity of oleanolic acid 3-O-glucuronide, we have deduced that these betavulgarosides decrease the concentration in the blood by inhibiting absorption across the cell membranes of the digestive tract.
In our screening program for chitin synthase(CHS) inhibitor, we isolated novel nucleotide inhibitors, guanofosfocin A (1), produced by a Streptmyces sp., and guanofosfocins B (2) and C (3), produced by Trichoderma sp. Though it was very difficult to isolate the pure compound without degradation, because of its instability, we could isolate and determine their structures utilizing the radical scavenger, n-propyl gallate, during the isolation and the NMR experiments to prevent the degradation. We isolated 50mg of 1, 164mg of 2 and 15mg of 3 from each broth filtrate using a series of column chromatography. The molecular formula(C_<17>H_<25>N_5O_<17>P_2) of 1 was determined based on HRFAB-MS, ^1H-NMR^<13>C NMR and ^<31>P NMR spectra data. The characteristic UV absorption of 1 suggested the presence of purine base moiety like guanine. We detremined the structure of 1, except for the base moiety and the cyclic linkage, from the analysis of the ^1H, ^<13>C, ^<31>P NMR, HMBC, HOHAHA and selective COSY experiments. The presence of a D-mannose and 8-hydroxyguanocine substructure was comfirmed by the comparison with the authentic sample and the cyclic linkage between the two was determined by 2D NMR experiments and by comparing NMR and UV spectra of 1 with those of related compound. The structure of 2 (C_<16>H_<23>N_5O_<17>P_2)was identified as demethyl derivative of 1 with comparing UV, ^1H, ^<13>C, ^<31>P NMR and we determined the structure of 3 (C_<29>H_<43>N_9O_<26>P_2) based on the amino acid analysis, UV,^1H,^<13>C,^<31>P NMR, HMBC, pH shift experiments of ^<13>C NMR. The absolute configuration at the acetal position of 1 was suggested to be [s] by the NOESY experiments and interatomic distanse of S and R isomers calculated by molecular dynamics calculation(NEMESIS). Guanofosfosins A, B and C show potent inhibitory activity against Candida albicans CHS 2 with 0.025, 0.026 and 0.008μM, respectively.
Mycoplasmas cause infection of wide distribution throughout domestic animals, fowls, insects and plants, as well as human, and recently, the fact that mycoplasmas promote proliferation of HIV, is taken notice of people. Here we found new antimycoplasma antibiotic micacocidin (1) produced by Pseudomonas sp. Lot 57-250, and determined its chemical structure. Further, we challenged its total synthesis. Antimicrobial activity and chemical structure Micacocidin (1) shows high activity against mycoplasmas, especially Mycoplasma pneumoniae, which is causative bacteria of human pneumonia, selectively. (Table1) Micacocidin (1) is an octahedral chelated compound, contain one alkylsalicylic acid unit and three different thiazoline, thiazolidine skeletons, wherein the core is a bivalent zinc ion. (Figure 1.2) Synthesis of segment A Condensation of 10, derived from benzyl amine (7) via 6 step reactions, with protected cystein (11) gave amide (12). Treatment of 12 with phosphorus pentachloride gave thiazoline (13), though obtained achiral. After dealkylation, conversion of 13 to Weinreb amide (15) and subsequent reduction gave very unstable aldehyde (2). (Scheme 2) Synthesis of segment B After protection, N-Me-L-Cys (16) was converted to 18 by C-C bond formation reaction with CDI-method. Reduction of ketone (18) gave desired alcohol (19) stereoselectively. Configuration of 19 was substantiated by NOE examination of lactam (20) which was prepared from 19. (Scheme 3) Examination of total synthesis 19 was converted to 27 by protection of hydroxyl and hydrolysis. Condensation of 27 with 2-Me-R-CysOMe (22) gave amide (28). Cyclization and deprotection to give cysteamine (26), coupling of 26 and segment A (2) to give 29, hydrolysis and following chelation with zinc ion to complete total synthesis, are under way.(Scheme 5)
The glycopeptide antibiotic vancomycin is well known as a therapeutic drug in the treatment of methicillin resistant Staphylococcus aureus (MRSA) infection. The bactericidal activity is explained by the high affinity of this antibiotic to the terminal D-alanyl-D-alanine residue of peptidoglycan precursors in bacterial cell wall biosynthesis. Synthesis of secoaglucovancomycins (3 and 4), vancomycin analogs, has been accomplished. The biphenyl ether moiety was successfully constructed by means of thallium (III) oxidation of the corresponding halogenated tetrapeptides. Further peptide chain elongation gave the corresponding heptapeptide, and final ring closure was undertaken under the same oxidation conditions. The following deprotection and dehalogenation gave secoaglucovancomycin-1 (3) and 2 (4). The conformational analysis of these compounds is also discussed by means of ^1HNMR studies and molecular dynamics calculation (CHARMm). Secoaglucovancomycin possessed more flexible conformers than agulucovancomycin, due to the lack of the biphenyl linkage. The binding structure of these molecules with the models of bacterial cell wall is also discussed. The complexes were experimentally obtained by the co-solution of 3 or 4 with N-acetyl-D-alanyl-D-alanine. The properties estimated by ^1HNMR techniques, were different from that of aglucovancomycin. Consequently, the binding structures were also examined by molecular dynamics calculations. The synthetic aglucovancomycins were interacted by five hydrogen bondings to the model peptide, which is different from the natural case. This binding mode strongly suggested a possibility that such compounds will be effective against vancomycin resistant strains, which have terminal D-alanyl-D-lactic acid as cell wall precursor.
Calbistrin, which was independently isolated and characterized by four groups at the almost same time exhibits antifungal and NGF production enhancing activities. The absolute structures of the octahydronaphthopyranone skeleton and the conjugated tetraene dicarboxylic acid side chain, however, remained undetermined. We now report here the synthesis of these fragments to confirm their absolute configurations. The tetrahydronaphthalene moiety 8 was synthesized by intramolecular Diels-Alder reaction of silyl dienol ether 7 prepared from methyl α-D-mannopyranoside in 23 steps as shown in Scheme 2. Through conversion into the diene, introduction of a C2 unit, construction of lactol ring and oxidation was obtained the diketone 12, which was identical with naturally derived compound [IV] in all respects. Stereoselective reduction of 12 and regio selective oxidation provided the hyrdoxy ketone 14, completing the synthesis of the octahydronaphthopyranone skeleton of calbistrin. The anti (20) and syn isomers of the rasemic side chain, tetraene carboxylic acid dimethyl ester, were synthesized from allyl alcohol 15, which was prepared by the aldol reaction of trans-2-methyl-2-butenal and methyl propionate (Scheme 4). By comparison with the naturally derived diester [I], the stereochemistry of the side chain was confirmed to be anti configuration. Finally, in the similar manner, the optically active acetate 24 obtained by optical resolution with lipase, gave the anti compound 26 identical with the diester [I]. The final stage of the total synthesis of calbistrin is now in progress.
Glycoprotein oligosaccharides constitute a group of highly diverse structures. Aiming at the development of efficient synthetic methodologies for this biologically important class of molecules, our research activities have been directed to 1) stereoselective formation of β-manno glycoside and its application into convergent synthesis of asparagine-linked glycans, and 2) polymer-support synthesis of oligosaccharide. β-Manno glycosylation was achieved in a fully stereocontrolled manner, by using a P-methoxybenzyl (PMB) group as a stereocontrolling element. Thus, 2-O-PMB carrying mannosyl donors 2,8, and 9 afforded 4, 11-14 as a single stereoisomer, by treatment with DDQ in the presence of appropriate aglycon, followed by activation of the anomeric position. These products were further converted into core structure of asparagine-linked glycans 18, 19, and 21. Orthogonal glycosylation strategy previously reported by ourselves was applied into polymer-support synthesis of glycoprotein related oligosaccharides 28 and 29. Known chloride 22 was converted into polyethylene glycol-linked 24. Sequential glycosylation with 25 and 26 afforded 27, which was deprotected into 28. The existence of hydrophobic aglycon as a tag at the anomeric position enabled us to fish up the desired oligosaccharide in a simple manner by using reverse phase column. Tetrasaccharide 29 was also synthesized in a similar manner.
The preparation and use of tetrasaccharide 1 which enables a rapid and preparative scale synthesis of sialyl Lewis X (SLeX) analogs having 1-O- and 2-N-disubstituted glucosamine (GlcN) moieties is reported. Such modifications would bring a dramatic change of the physical and pharmacological properties of the SLeX analogs. Therefore, tetrasaccharide 1 is a convenient intermediate for the synthesis of various SLeX analogs, since it has convertible 2-(trimethylsilyl)ethyl (SE) glycoside and the free amino group on GlcN moiety: (a) SE glycosides can be smoothly transformed into other glycosides via the corresponding glycosyl donors such as 1-chloro sugars and (b) desirable functional groups can be introduced on the amino group by treatment with desired electrophiles such as acyl halides. The intermediate 1 was constructed from a glucosamine derivative by a highly efficient combined use of enzymatic galactosylation/sialylation and chemical fucosylation. Thus obtained 1 was converted into SLeX analogs by N-substitution followed by transformation of SE glycoside into other glycosides and deprotection. These synthesized analogs were found to inhibit cell adhesion of human leukocyte (HL-60) to recombinant soluble human E-selectin.
Ganglioside GAA-7 (1) derived from the starfish Asterias amurensis versicolor showed neuritogenic and growth-inhibitory activities towards the mouse neuroblastoma cell line (Neuro2a). On the other hand, the structure of its oligosaccharide moiety is very unique, since 1 possesses two sialic acids which directly linked to the GalNAc residue. We are conducting the reconstitution of the biologically active ganglioside 1, and now report the synthetic studies of the demethyl compound (2) of 1. The strategy for the synthesis of 2 is shown in chart 1. 1 possesses the less available N-glycolyl type sialic acid (NeuGc), therefore we prepared the glycosyl donor of NeuGc 5 from the N-acetyl type sialic acid (NeuAc) in 6 steps. For examination of its donor property, 5 was glycosylated with two lactose derivatives 8 and 11. As a result, the NeuGc-containing oligosaccharides 12 and 13 were obtained in good yields. In the preparation of the asialotrisaccharide 6, three GalNAc donors, bromide, trichloro-acetimidate and thioglycoside were examined. As a result, the thioglycoside of GalNAc 7 gave the highest chemical yield and regioselectivity. For examination of the disialylation reaction, tetra O-acetyl lactose derivative 17 was prepared and glycosylated with the NeuGc donor 5 in CH_3CN in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid. The disialylated lactose derivative 18 was obtained in 15% yield together with some monosialylated lactosides. The disialylation of the NeuGc donor 5 towards the asialooligosaccharide 6 was achieved to give 3 by one step in moderate chemical yield. Ceramide moiety was prepared from the cerebrosides derived from the starfish Acanthaster planci. The extracted cerebroside molecular species were hydrolyzed by the glycosidases (Charonia lampas), and the resultant ceramides were separated by flash chromatography to yield pure ceramide 21. The isolated ceramide 21 was easily converted to the synthetically useful ceramide 4. Glycosylation of the sialopentasaccharide 3 and the ceramide 4 thus obtained is in progress.
Maitotoxin (MTX), produced by the dinoflagellate Gambierdiscus toxicus, is the most toxic and largest non-biopolymer known to date. The elucidation of its complete structure with the absolute configuration has been regarded as one of the most exciting challenges in natural products chemistry. The gross structure and stereochemistry of the fused rings as well as directly connected ether rings were elucidated on the basis of extensive 2,3D NMR analyses and MS/MS experiments. We have recently succeeded in determination of the relative configuration along the entire polyether entity of MTX encompassing ether rings A through F. Herein we report the determination of the remaining configuration along both terminal chains and the absolute configuration of MTX. The relative configurations of the stereogenic centers in the C1-C15 acyclic terminal chain of MTX were elucidated by (1) extensive conformational analysis using ^3JH,H and ^<2,3>JC,H coupling constants obtained by a contemporary NMR technique, and (2) synthesis of model compounds 2A and 2B furnished with thus determined relative configurations, and comparison of their ^1H and ^<13>C NMR data with those for the corresponding portion of MTX, concluding that 2A represents the relative stereochemistry of the C5-C15 portion of MTX. The relative stereochemistry between C136 and C138 in the another terminal acyclic chain was unambiguously assigned on the basis of the ^3JH,H and ^<2,3>JC,H data. The stereochemical relationships among C134, C135, and C136 were also deduced from the similar NMR analysis and confirmed by the comparison of the synthesized model compound 24 with MTX in their ^1H NMR. In order to determine the absolute configurations along with the relative configuration between C 138 and C139, which was not deduced by NMR due to signal broadening, we synthesized the four stereoisomers 25a-d possible for 3,4-dimethy1-6-hepten-l-ol and compared them with a degraded fragment of a natural product by GC with chiral column to identify 25a as the natural diastereo- and optical isomer, thus establishing the 138R,139S configuration of MTX. The present results, together with the previously reported stereochemical assignments, allowed us to determine the complete absolute stereochemistry of MTX to be represented by structure 1.
The brevetoxins, represented by brevetoxin A and B, are potent neurotoxins produced by the red tide organism Gymnodium breve and constitute an important class of marine polycyclic ethers. Hemibrevetoxin B (1), whose molecular size is about half that of brevetoxins, was isolated from the cultured cells of Gymnodinium breve by Shimizu in 1989. The characteristic structural features of 1 include trans-fused 6, 6, 7, 7-tetracyclic ether rings (ABCD-ring system) having 10 chiral centers, an α-vinyl aldehyde and a (Z)-diene moieties. The unique complex structure and potent biological activity have attracted the attention of numerous synthetic organic chemists, and the total syntheses of 1 have recently been accomplished by the Nicolaou and Yamamoto groups. We have also reported several studies directed toward developing an efficient method for the synthesis of the cyclic ethers and toward the total synthesis of this class of marine polyethers. We now report the stereoselective total synthesis of hemibrevetoxin B (1), which features a novel double rearrangement-ring expansion of the bicyclic ether for the CD-ring formation, an exclusive 6-endo cyclization of the hydroxy styrylepoxide for the B-ring formation, and a direct insertion of a C-4 unit as the side chain on the A-ring. The allyl alcohol 16, prepared from geraniol, was converted into 6,6-membered bicyclic ether 22 via the Sharpless asymmetric epoxidation (AE). Upon treatment of bis(chloromethanesulfonate) 23 with Zn(OAc)2 in aq AcOH at 60-80℃, the expected double rearrangement-ring expansion took place effectively giving the 7,7,-membered bicyclic ether, which was hydrolyzed with K_2CO_3 to give 24 corresponding to the CD-ring system. The bicyclic ether 24 was converted into styrylepoxide 29, which was treated with CSA to give the desired 6-memberd ether 30 corresponding to the B-ring system. Ozonolysis of the double bond in 31 followed by reaction with allylmagnesium chloride stereoselectively produced 613-alcohol 32 along with the 6a-isomer. Ozonolysis of 32 followed by treatment with Dowex in MeOH effected acetalization of the resulting lactol and simultaneous deprotection of the acetonide, giving the diol which was oxidized with NaIO_4 to give aldehyde 33 having the ABCD-ring system. The Wittig reaction of 33 using PhSeCH_2CH_2CH=PPh_3 followed by H_2O_2 treatment gave (Z)-diene 34, which was treated with CH_2=CH(CH_2OAc)CH_2TMS in the presence of TMSOTf in MeCN to give exclusively 4β-substituted compounds 35 and 36. The TBS ether 35 was converted into the alcohol 36 by treatment with TMSOTf in MeCN. Alkaline hydrolysis of the acetate 36 gave alllyl alcohol 37, which was finally oxidized with MnO_2 in ether to give α,β-unsaturated aldehyde 1. The ^1H and ^<13>C NMR spectra of the synthetic 1 were identical with those of natural hemibrevetoxin B (1).
One of the most characteristic and interesting classes of marine toxins produced by dinoflagellates is the polycyclic ethers. Trans-fused tetrahydropyran rings are the most frequently encountered cyclic units and form a rigid backbom of this class of toxins. We have developed a novel reiterative route to trans-fused tetrahydropyran ring systems. The key features of the synthesis are alkylation of an oxiranyl anion stabilized by a sulfonyl group and a 6-endo mode of cyclization. The approach involves the following five-step process: (i) transformation of a monocyclic diol to a silyl-protected triflate by a one-pot procedure (14→4), (ii) coupling reaction of the triflate with the oxiranyl anion generated from an epoxy sulfone by deprotonation (4→15), (iii) stereo- and regioselective 6-endo mode of cyclization of the coupling product to a bicyclic ketone (15→16), (iv) stereoselective reduction of the ketone (16→17), and (v) deprotection (17→18). The resulting bicyclic compound contains useful functional group that can be explored for further elaboration of the system. Reiteration of the sequence provide predictable access to tri- and polytetrahydropyran ring systems. Application of the present method to prepare a tetrahydrofuran ring by 5-endo cyclization was demonstrated by the synthesis of (6S,7S,9R,10R)-6,9-epoxynonadec-18-ene-7,10-diol (35) isolated from the brown alga Notheia anomala.
Phomactins are novel PAF antagonists isolated from the culture filtrate of the marine fungus, Phoma sp., a parasite on the shell of the crab, Chinoecetes opilio. Their structures, contain a rare bicyclo[9.3.1]pentadecane ring system, were determined based on spectroscopic evidences, X-ray crystallography and chemical conversions. Phomactin D has strongest PAF antagonistic activity among phomactins. Thus its unique structure and biological activity prompted the authors to undertake total synthesis of phomactin D. The sequential Michael reaction of kinetic enolate of 2-cyclohexen- 1-one withα,β-unsaturated ester 2 afforded bicyclo[2.2.2]octane derivative 3 as the sole product. Ketone 3 was converted to olefin 4 in three steps: 1) NaBH_4 reduction, 2) tosylation and 3) elimination of the tosylate. Oxidative cleavage of the C(2)-C(19) double bond in 4 was successfully carried out by ozonolysis followed by NaBH_4 reduction to afford lactone 5. Compound 5 was converted to pentasubstituted cyclohexane derivative 15, having requisite four chiral centers in phomactin D, by chemical modifications. Reaction of alkenyllithium reagent, prepared from alkenyliodide 18 and ^tBuLi, with aldehyde 15 gave alcohol 19 as the sole product. Alcohol 19 was converted to allylic chloride 20. The macrocyclization of sulfone 20 was successfully carried out by treatment with KHMDS to afford 21. Removal of the phenylsulfonyl group and deprotection of benzyl and BOM ether were carried out by treatment of sodium in liq. NH_3 to afford the diol. Epoxidation of the allylic alcohol with ^tBuOOH in the presence of VO(acac)2 gave epoxide as the sole product. Finally, PDC oxidation of the primary and secondary hydroxyl groups completed the synthesis phomactin D (1).
Curacin A(1) is a novel antimitotic agent isolated from a Caribbean cyanobacterium Lyngbya majuscula, and inhibits tubulin assembly by binding to the colchicine-binding site. Since curacin A has little structural similarity to the known natural and synthetic colchicine-site ligands, it is expected to play as an unique probe for this binding site and to show interesting bioactivity. First, we proposed the absolute configurations of three chiral centers in curacin A, to be 2R, 19R and 21S, by synthesizing the four stereoisomers, (+)- and (-)- 2a, 2b as the partial structure of curacin A. Next, we developed a versatile method for the synthesis of enantiomerically pure cis-2-ethylcyclopropanecarboxylic acid, (-)-(1R, 2S)-7, a component of curacin A, and its enantiomer, (+)-(1S, 2R)-7. Double-asymmetric Simmons-Smith cyclopropanations of dienes 11 and 15 derived from diethyl L-tartrate proceeded with excellent diastereofacial selectivity (>99% de) to give dicyclopropanes 12 and 16, and they were converted to the respective isomers of 7. Finally, we achieved the total synthesis of curacin A in a highly stereo-contolled manner. The key steps were (1) an asymmetric allylation using a chiral allyltitanium reagent and a double-asymmetric Simmons-Smith cyclopropanation to introduce three chiral centers, (2) Wittig and Wittig-Horner reactions to construct the C(3-4) and C(7-10) alkenes, and (3) a direct conversion of the thiazolidine to the thiazoline. The effects of the synthesized curacin A and related compounds on microtuble assembly were also examined.
Asymmetric synthesis of (+)-coronafacic (3) acid has been completed from (R)-(+)-4-acetoxy-2-cyclopenetene-1-one 29 as a chiral source. Construction of the 1-hydrindanone frame-work was carried out via intramolecular 1, 6-conjugate addition as the key step. Asymmetric synthesis of (+)-coronamic acid (4) and its possible three stereoisomers have been completed from (R)-malic acid 19 or (S)-malic acid as chiral sources. Construction of cyclopropane frame-work was carried out via dialkylation of dibenzyl malonate with cyclic sulfate by Sharpless's method. Coupling between (+)-coronafacic acid (3) and protected coronamic acid 26, and the subsequent deprotection provided (+)-coronatine(1). This is the first asymmetric total synthesis of (+)-coronatine(1). On several biological assay systems, coronatine (1) shows a 100 to 10,000 times higher activities than that of jasmonic acid (2). Coronafacic acid (3) is more structurally similar to jasmonic acid (2) than that of coronatine (1), however, coronafacic acid (3) shows slightly weaker activities than that of jasmonic acid(2). By using of coronatine (1), coronafacic acid (3), amino acid conjugated jasmonic acids and precursors of jasmonic acid biosynthesis, the study of biological activity is in progress.
We have developed an efficient olefin cyclization agent mercury(II) triflate, Hg(OTf)_2, and applied to the syntheses of a variety of carbocyclic terpenoids. Recently we found that the Hg(OTf)_2 is formed almost instantaneously in acetonitrile as a colorless solution. When this freshly prepared Hg(OTf)_2 was treated with homogeranyl acetate (2) in acetonitrile, an organomercuric compound 3 that has a tetrasubstituted double bond was obtained selectively. Of course it corresponds to the A ring of taxol (1). Thus we designed to construct taxane skeleton from 3 according to a biomimetic entry. Although a tremendous number of synthetic studies toward taxol have been carried out, biomimetic routes have not been intensively studied. Although a reaction of homogeranyl acetate (2) with Hg(OTf)_2 in acetonitrile at-20℃ for 20min afforded organomercuric compounds 3, 4, 5, and 6 in a ratio of 78:16:3:3, a 92:8 mixture of 3 and 4 was obtained in 93% yield after 72h. Furthermore it was found that isomer due to the minor trisubstituted olefin was gradually transformed into tetrasubstituted olefin during the course of the synthesis. Thus the mixture of 3 and 4 was employed for next hydroxylation step leading to a ketone 7 after oxidation. Acetallization, hydrolysis of acetate, and Swern oxidation provided aldehyde 8. The olefinic isomer originated from 4 was completely disappeared at this stage. Alkylation to give 10 and following Red-Al reduction afforded E-olefin 11, and hydrolysis-oxidation sequence gave keto aldehyde 12. Low valent titanium promoted McMurry cyclization of the ketoaldehyde 12 in PhH-THF provided diols with bicyclo[9,3,1]-pentadecatriene skeleton 13 and 14 in 70% yield in 5:1 ratio. The stereochemistries of 13 and 14 were established by the NOESY NMR experiments of the derived carbonates 18 and 19, respectively. Since the minor diol 14 afforded single crystal, the structure was also confirmed directly.
Taxol 1 has attracted much attention because of its promising anti-cancer activities, as well as its complex molecular structure containing the unique tetracyclic skeleton. This report discloses our recent studies toward the asymmetric total synthesis of 1. In our strategies, the following transformations should be very important: 1) construction of the tricyclic carbon framework, 2) introduction of C19 methyl group on taxane skelton, 3) construction of oxetane ring. As for the point 1), we have already reported the highly efficient method for the construction of taxane skeltons, using intramolecular vinylogous aldol reaction. By applying this method, tricyclic compound 12 was easily prepared starting from optically active hydroxy aldehyde 2 (A-ring fragment) and cyclohexadiene 3 (C-ring fragment). (Eq 1) At the next stage (2) introduction of C19 methyl group), we have decided to utilize reductive cleavage of cyclopropylketone, and 20 was prepared as shown in eq 2. Although birch reduction of 20 gave the product having C19 methyl group in excellent yield, it was enol derivative 21 unexpectedly (eq 3). We are now studying about the isomerization of enol-form 21 into keto-form 22. If it is accomplished, the transformation of 22 to 8 should be very easy. We have been also investigating the transformation of synthetic intermediate 8 to taxol 1, which corresponds to the latter half of our total synthesis. The compound 8 was prepared by degradation of 10-deacetylbaccatin III (eq 5). We are currently studying about the conversion of 8 to 1 based on the Holton's procedures (eq 6).
The green flagellate Chlamydomonas eugametos and its sexuality were extensively studied by Moewus and Kuhn in 1930's to 1950's, whose work later turned out to be a scientific fraud. In 1995, two sex pheromones produced by female gametes of Chlamydomonas allensworthii were isolated and identified as lurlenic acid 1 and lurlenol 2 by Starr, Jaenicke and Marner. Here we report the first synthesis of 1 and 2. 1) First approach to lurlenic acid 1: Our first route to synthesize lurlenic acid included a palladium-catalyzed reaction as the key step, starting from commercially available geranylgeraniol, 2,3-dimethyl-p-hydroquinone and D-xylose. This synthesis gave an inseparable mixture of two compounds, lurlenic acid and its (12Z)-isomer due to the isomerization in the course of the palladium-catalyzed coupling step. This mixture was as bioactive as the natural product. 2) Second approach to lurlenic acid 1: In the second synthetic attempt, a sulfone-coupling was the key step to synthesize lurlenic acid as the only product without isomerization of the double bond at C-12. Our starting materials were farnesol, 2,3-dimethyl-p-hydroquinone and D-xylose. ^1H- and ^<13>C-NMR spectra of the synthetic lurlenic acid were identical with those of the natural product. 3) Synthesis of lurlenol 2: Lurlenol was synthesized in a similar manner. Its ^1H- and ^<13>C-NMR data were identical with those of the natural product. 4) Synthesis of compound 3: We also synthesized the compound 3 which might be the biosynthetic precursor of the two pheromones. Bioactivity of this compound is now being tested.
Efficient methodologies for regio- and stereoselective syntheses of polycyclic compounds have been developed by us using intramolecular double Michael reaction (IDM) of cyclohexenones or acyclic enones possessing α,β-unsaturated carbonyl group. We now describe a stereoselective synthesis of bicyclo[2.2.1]heptane derivatives 18, 23 and 29 from cyclopentenones 16, 17 and 28, and a total synthesis of (±)-cedranediol from 35 which was stereoselectively obtained from cyclopentenone 34 by IDM. (A) Construction of copaborneol and longiborneol frameworks IDM of cyclopentenone 16 carried out by heating with ZnCl_2-Et_3N-TMSCl, gave the tricyclic compound 18 along with the diastereoisomer 19. Treatment of 16 with TMSI-HMDS or Bu_2BOTf-HMDS stereoselectively produced 18 in a less satisfactory yield. On the other hand, reaction of 17 with TMSI or Bu_2BOTf in the presence of HMDS stereoselectively provided the corresponding seven membered compound 23 in good yields. (B) Construction of tricyclo[188.8.131.52^<1,5>]decane and stereoselective total synthesis of (±)-cedranediol. Stereocontrolled construction of the tricyclo[184.108.40.206^<1,5>]decane derivative 29 was achieved by IDM of 28 performed under three different conditions: LHMDS; ZnCl_2-Et_3N-TMSCl; TBSOTf-Et_3N. Key intermediate 35 of cedranediol was obtained stereoselectively from 34 in 86% yield using ZnCl_2-Et_3N-TMSCl. Regioselective ring expansion of 35 produced the keto ester 36, which was then converted to the diol 39 in six steps. Mono-protection of the diol 39 afforded the ether 40. Oxidation of secondary hydroxyl group of 40, followed by diastereoselective methylation and deprotection of the silyl ether furnished (±)-cedranediol.
The phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), are known to be powerful tumor promoters and activators of protein kinase C (PKC). First discovered by Nishizuka et al., PKC is a phospholipid- and calcium-dependent serine/threonine kinase, physiologically activated by 1,2-diacyl-sn-glycerol (DAG). PKC is also known to be an important target for other structurally diverse tumor promoters such as the ingenols, teleocidins, and aplysiatoxins. Structure-activity analyses of a variety of analogs of DAG and these tumor promoters have been carried out. Although many pharmacophore models have been proposed from molecular modeling, no information about the specific amino acid residues that interact with these ligands is available. Moreover it has been shown that the biological activity of 11-demethyl-13-deoxyphorbol esters, which were synthesized by our group, was not fully consistent with the pharmacophore models so far. Thus we are now interested in determing the importance of the 13-acetoxy group in phorbol ester-PKC complexes. This has led us to design a new photoaffinity probe 3, and to carry out previously unprecedented photoaffinity labeling of PKC. We have also designed a new phorbol ester-phosphatidylserine hybrid molecule 19. Because phosphatidylserins in phospho lipid membranes are known to have specific interactions with phorbol ester-PKC complexes, such a hybrid molecule can be expected to act as a specific inhibitor of PKC by preventing PKC from interacting with phospholipid membranes. The hybrid molecule was synthesized and preliminary biological activities were examined.
We have recently reported a novel stereoselective cyclization of 1,3-diene and the carbonyl group in a chain, in which the hydride nickel complex 2 plays an important role. By use of Et_3SiH as a hydride source, the catalytic cycle was established to produce 4, 9c, 10c, or 11c as a sole product. Further, we have also found that 1,3-diene serves as a ligand and affects the regio-selectivity in a side chain of the cyclized product. In the presence of 1,3-cyclohexadiene (1,3-CHD) (150mol %), the cyclized product 8, 9b, 10b, or 11b was produced predominantly. Using this cyclization, the synthesis of prostanoids was tried. The optically active substrate 13 was easily prepared via Sharpless epoxidation followed by regioselective ring opening reaction of resultant epoxide 20 with vinyl cuprate. The reaction of 13 with 2 (100mol %) in the presence of 1,3-CHD proceeded stereoselctively, affording the cyclized product 14 in 93% yield as a sole product. The cyclized product 14 was transformed into 28, which is a versatile intermediate for the synthesis of prostanoids. Further studies along this line are in progress.
Palladium(0)-catalyzed cyclization of 6-(2-alkenyl)-2,7-octadienyl acetates (7) was carried out. When the 6-substituent is an allyl group, tandem cyclization of 7a took place stereoselectively to give trans-bicyclo[3.3.0]octane 9a in 67% yield. The compounds 9b-d having (E)- or (Z)-2- alkenyl groups at 6-position underwent the cyclization leading to 12b-d, which resulted from β-elimination of the common 2-(vinylcyclopentyl)methylpalladium intermediates 8. There was found an unprecedented cis diastereoselection at C(3)-C(6) of 7a-d during the cyclization as well as trans diastereoselection at C(6)-C(7) in the particular formation of 9a. It is also worthy of mentioning that the trans-bicyclo-[3.3.0]octane 14 bearing a novel angular methyl group was formed in the Pd(0)-catalyzed tandem cyclization of 13 in 53% yield. The Pd(0)-catalyzed cyclization of 15 proceeded in acetic acid at 45℃ under a carbon monoxide atmosphere to give 16 accompanied with the meso products (yield 41%, selectivity 82%). This is resulted in the unprecedented carbopalladation of the π-allylpalladium intermediate (path a) forming the δ-alkenylpalladium A which underwent carbonylation leading to the product 16. This method was utilized to the synthesis of (±)-isoiridomyrmecin (5). The Pd(0)-catalyzed cylization of trienyl acetate 18, followed by carbonylation selectively gave the acrylic acid derivative 17, which was converted to (±)-5 in six steps. The Pd(0)-catalyzed cyclization of 24, in which the allylic acetate and the allenic moieties are tethered by two methylene carbons was examined. In this reaction, the initially formed π-allylpalladium intermediate 21 would undergo exclusively alkene insertion at the center of the allenic unit, resulting in a new π-allylpalladium complex 23, which in turn proceeded back to the product 25. In fact, shortening the tether by one carbon between the π-allylpalladium and the allenic moieties (cf. 15 and 24) has caused a drastic change of the regioselection in the cyclizations. This reaction was extended to the formation of tetracyclic diketone 6. The Pd(0)-catalyzed tandem cyclization of tetraenyl acetate 26 proceeded under carbon monoxide to give tetracyclic compound 6 in 22% yield. It is noteworthy that the six consecutive C-C bond formation involving four carbopalladation and two carbonylation insertions has taken place in one operation.
In order to establish the stereoselective synthesis of retinoid isomers, which are signal molecules of retinoid proteins such as retinochrome, rhodopsin, RAR, RXR, and etc., the reaction of β-ionone-tricarbonyliron complex 1 with carbanions was investigated. Treatment of 1, prepared from the reaction of β-ionone and dodecacarbonyltriiron, with lithiated acetonitrile afforded 2 via addition, dehydration, and migration of tricarbonyliron complex. On the contrary, the reaction between 1 and lithium enolate of ethyl acetate and subsequent dehydration by thionyl chloride provided the ethyl 9Z-β-ionylideneacetate 12 predominantly. These compounds (2 and 12) were converted to the corresponding β-ionylideneacetaldehydes (4 and 17) in excellent yields, respectively. The Emmons-Horner reaction of these compounds with C5-phosphonate followed by the sequence of decomplexation and alkaline hydrolysis gave the corresponding retinoic acids (8 and 21). The Peterson reaction of 4 with ethyl trimethylsilylacetate afforded the 11Z-isomer 23a preferentially (77%) accompanied by the 11E-isomer 22b (15%). The ester 23a was converted to the C18-ketone-tricarbonyliron complex 25 using triphenylstannyl-methyllithium. The Emmons-Homer reaction of 25 with C3-phosphonate followed by the sequence of decomplexation and DIBAL reduction led to the 11Z-retinal 28. The stereochemistry of 11 position was retained during these transformations and this is the first report for the stereselective synthesis of 11Z-retinal.
Recently, the role of prostaglandins (PGs) in brain function has attracted much attention. We succeeded in creating a stable ligand with high binding affinity and selectivity for a prostacyclin (1, PGI_2) receptor expressed in the central nervous system (CNS). The ligand, (15R)-16-m-tolyl-17,18,19,20-tetranorisocarbacyclin (3, 15R-TIC), was prepared starting from the Horner-Emmons reaction of aldehyde 4 and β-keto phosphonate 5 followed by 1,2-reduction of the enone, separation of the resulting C(15)-epimers, and hydrolysis of the methyl ester. The binding assay was conducted by the [^3H]isocarbacyclin displacement study using the rat frozen tissue sections containing the thalamus or NTS (nucleus tractus solitarius) as representative of the CNS or peripheral nervous system. Consequently, 3 exhibited high binding affinity sufficient for a receptor in the thalamus (IP_2) but showed very weak binding for a receptor in the NTS (IP_1). In contrast, cicaprost (12), a stable PGI_2 agonist belonging to an iloprost family, showed high binding affinity and selectivity for IP_1. 15S-TIC (9), the C(15)-epimer of 3, and isocarbacyclin (2) bound strongly with both of the receptors. Electrophysiological studies carried out in the CA1 region of the rat hippocampus revealed that the PGI_2 analogs have a facilitatory effect on the excitatory transmission through IP_2. The widespread expression of IP_2 in the CNS suggests that PGI_2 has important roles in neuronal activity. In order to identify IP_2, we prepared an azido-functionalized isocarbacyclin derivative 14 as a photoaffinity probe, which showed strong binding affinity for IP_2.
Streptazolin (1), originally discovered in 1981 from cultures of Streptomyces viridochromogenes, has the striking structural feature of an unusual ring system 2-azabicyclo[4.3.0]nonane (pyrindine) and an internal urethane unit both rarely distributed in nature. The isolation and characterization of this antibiotic were complicated by its tendency to undergo polymerization in concentrated form due to the presence of the conjugated diene part. We present the stereoselective synthesis of (+)-streptazolin (1) and (-)-4a,5-dihydrostreptazolin (5) by a new strategy utilizing a palladium-based approach under the nonreductive and reductive conditions, respectively, for constructing the pyrindine core skeleton bearing the Z ethylidene group. The chiral imide 21 was converted to the enyne compound 29 via N-acyliminium ion cyclization of the acetoxy lactam 25, partial reduction of 26 with DIBALH-BuLi, and formation of the dibromoolefin 28. Subsequent removal of the benzyl protecting group followed by basic treatment provided the cyclic carbamate 31, which was subjected to palladium(II)-catalyzed cyclization under the reductive conditions using polymethylhydrosiloxane as a hydride source to afford (-)-4a,5-dihydrostreptazolin (5). On the other hand, palladium-catalyzed cyclization of 31 under the nonreductive conditions led to the formation of (-)-Δ^5-streptazolin (32). Attempted isomerization of the 1,4-diene system in 32 did not resulted in any of desired streptazolin (1) with 1,3-diene system. We thus turned to the nonreductive cyclization of the bicyclic enyne compound 29. The best result in the palladium-catalyzed bicyclization of 29 was obtained when treated with 30 mol % Pd(OAc)_2 and N,N'-bis(benzylidene)ethylenediamine (BBEDA) as the ligand in benzene at reflux, thus affording a 93: 7 mixture of the 1,2,4a, 6,7,7a-hexahydro-5H-1-pyrindine (Z)-34 along with its isomerized product (Z)-35 in 84% total yield. The isomerization of the 1,4-diene in (Z)-34 to the 1,3-diene was successfully achieved by treatment with triiron dodecacarbonyl to generate the stable tricarbonyl(η^4-1,3-diene)iron complex 36, which was then converted to the glycol 37. Treatment of 37 with sodium methoxide followed by ordinary chromatography on silica gel resulted in isolation of chemically and enantiomerically pure (+)-streptazolin (1).
We found that the Lewis acid-mediated Diels-Alder reaction between cyclopentadiene and 4-tert-butoxy-2-cyclopentenone (±)-2 proceed stereoselectively in a contra-steric manner to give the endoalkoxy adduct (±)-3 in 92% yield. Based on this finding, a new and efficient preparation of optically pure ketodicyclopentadiene (KDP) (1) has been developed. Namely, racemic endo-alcohol (±)-5, generated from the contra-steric adduct (±)-3 by stereoselective reduction, afforded the optically pure acetate (-)-6 quantitatively leaving the optically pure alcohol (-)-5 in an excellent recovery when treated it with vinyl acetate in tert-butyl methyl ether in the presence of a lipase. Similarly, the racemic alcohol (±)-10 generated from racemic KDP (±)-1 gave the optically pure acetate (-)-11 and the optically pure alcohol (+)-10 in excellent yields. The contra-steric adduct (±)-3 also allowed the synthesis of the meso-diol 8 by sequential dealkylation and stereoselective reduction. The all resolved products were efficiently converted into optically pure KDP (1). Efficient preparation of optical pure KDP (1) as well as of the optically pure (β-alkoxy ketone 3 made more extensive utilization of these chiral buildings for the enantio- and diastereo-selective construction of natural products possible. Thus, new routes to the key dihydroxyaminocyclopentene segment 19 of Q nucleoside queuosine (12), the marine anthelmintic kainic acid (30), the marine triquinane sesquiterpene Δ^<9(12)>-capnellene (38), prostaglandin precursor 43, a potential precursor of the polyketide from wood-decay fungus sistodiolynne (47), and the estrogenic steroid estrone (56), have been established. Principle of the synthesis is stereoselective modification of the enone moiety of the biased tricyclic framework and thermal regeneration of the cyclopentene double bond. Based on this principle, the construction of the target molecules were successfully carried out except Δ^<9(12)>-capnellene (38) where whole carbon framework of KDP (1) was used. Newly developed procedures were based on tandem sigmatropic reaction linked to the retro-Diels-Alder step which allowed efficient preparation of prostaglandin precursor 43 and enantioconvergent construction of kainic acid (3). Another noteworthy was the stability of the β-alkoxyketone 3 under strong basic conditions which led to an enantioconvergent route to estrone (56).
The novel natural products containingα,α-disubstituted α-amino acid structures, sphingofungin E and F, myriocin, conagenin (1), and lactacystin (2), attracted significant attention because of their interesting biological activities such as neurotrophic factor, immunomodulation, immunosuppressive and enzyme inhibitory activities. We report here preparation of precursors of α,α-disubstituted α-amino acids by way of rearrangement of allylic trichloroacetimidates (Overman rearrangement) derived from carbohydrates, and total synthesis of lactacystin (2) and its analogs starting from D-glucose. The allyl alcohol derivative (4Z) prepared from diacetone-D-glucose was transformed into trichloroacetimidate (5), which was heated in toluene at 140℃ afforded rearranged products 6a and 6b (8.3:1.0) in 78% yield. It was revealed that Overman rearrangement of trichloroacetimidates derived carbohydrates proceeded generally in good yields and in moderate stereoselectivities. The rearranged product 6a was successfully converted into (S)-methylserine derivative (10), which is an amino acid part of conagenin (1). Its enantiomer (11) was also synthesized from compound 6a. Lactacystin (2), reported to show neurotrophic activity as well as inhibitory activity against proteosome, was synthesized stereoselectively using Overman rearrangement as the key transformation. Thermal rearrangement of imidate (17), followed by removal of the acetonide group provided R-19 as the major product (43% from 16). Periodate oxidation afforded aminal (20), which was transformed into γ-lactam (21). Stereoselective introduction of an isopropyl group and oxidation of vinyl function gave 28, whose coupling with N-acetyl cysteine and subsequent deprotection furnished the total synthesis of 2. The analogs of lactacystin containing hydrogen (31) and phenyl group (33) in place of the isopropyl group were prepared from compounds 21 and 24, respectively. This work revealed that the Overman rearrangement of imidates derived from carbohydrates should be an effective approach to α,α-disubstituted α-amino acids.