Covalent attachment of small molecules to proteins has been a target of numerous synthetic endeavors because this process covers nonimmunogenetic molecules into immonogenetic materials. The application of this concept to carbohydrates provided the foundation to generate anti-oligosaccharide antibodies. Active ester linkers such as the readily available DSG, MBS and the synthesized maleimide bifunctional linkers have become very useful in the area of bioconjugate chemistry. These linkers allow the conjugation of amino group containing molecules such as peptides having amino acid residues and protein. This allows the construction of various moieties including protein-conjugate haptens, immobilized antibodies or enzymes, immuno-conjugates or -toxins, and immunodiagnostic agents. Here, we describe a new approach to biocojugation utilizing the active ester linker containing acid chloride 1 and 2 that may take place the acylation. Although the active ester method has been studied in carbohydrate field, this linker has not previously been used for bioconjugation saccharides. The active ester linkers 1 and 2 are obtained from the carboxyl dichloride and N-hydroxyphthalimide in one step. N-hydroxyphthalimide gives 1 and 2 (34 and 44%) by succinyl chloride and glutaryl chloride in the presence of pyridine. We introduced active ester linkers into a modified glucose by two kinds of path. One is of using DCC to introduce active ester linker into 6-OH of modified glucose by two steps. Another is of using acid chloride linker containing acid chloride into 4,6-OH modified glucose with pyridine by one step. Furthermore, we tried to introduce the linker into cellulose by one step.

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Hemibrevetoxin B (1), isolated from the red tide dinoflagellate Gymnodinium breve by Shimizu group, is a trans-fused tetracyclic ether. To date, many research groups reported total synthesis of 1. Notably, most of them adopted a linear strategy, though a convergent strategy would have comparable efficiency with linear one even in the synthesis of such small polyether. In this context, we planned to synthesize 1 through a convergent strategy that would allow the large-scale preparation. During our studies, Holton group reported first convergent total synthesis of 1 very recently, where the ABCD ring part was constructed in 15 steps after connection of the cyclic A-ring part with the acyclic C12-C21 (BCD-ring) segment. Here, we disclose alternate successful convergent construction of the ABCD ring system 2 of 1 from the A- and D-ring segments (5 and 6, respectively). Previously, we developed an effective convergent method for the synthesis of trans-fused tetracyclic ethers adopting coupling reaction of an acyl anion equivalent with an aldehyde followed by reductive cyclization reactions. Therefore, dimethyldithioacetal mono-S-oxide 5 and aldehyde 6 were selected as A- and D-ring segments, respectively, in our synthetic plan for 1. The segment 5 was prepared in 14 steps from 2,4,6-tri-O-acetyl-D-glucal 7 in 14% total yield. The aldehyde 6 was synthesized in 19 steps from γ-butyrolactone 14 in 11% total yield. Coupling reaction of deprotonated 5 with 6 followed by simultaneous removal of TBS and dithioacetal groups afforded ketodiol 4, which was subjected to reductive cyclization, oxidation, and deprotection to give 3. Formation of cyclic S,O-acetal 34 from 3 followed by oxidation with mCPBA and in situ treatment with AlMe_3 produced tetracyclic 2. Thus, the ABCD-ring part 2 was successfully constructed in 7 steps from 6 in 12% total yield.

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Heliannuols A-K are the sesquiterpenes isolated from the Spanish cultivated sunflower Helianthus annuus L. cv. SH-222. They exhibited bioactivities as phytotoxic allelochemicals and are potential sources of new type herbicides with new modes of action, which may be less harmful than herbicides presently used in agriculture. The enantioselective synthesis of heliannuol E has already been reported by Shishido, although its bioactivity has uncovered. In addition to such a background, the chroman framework of this molecule would be an attractive synthetic target of the anodic oxidation-ring expansion protocol developed by our group. Electrolysis might be one of powerful tools for organic synthesis, because electron transfer reactions can be conducted under high cost-performance and safety conditions, without toxic reagents. We present our investigation process. Thus, anodic oxidation of halogenated phenol derivatives(1, 5) provided the corresponding spiro compounds(2, 6), which on treatment with a Lewis acid gave the chroman derivatives (3, 4, 7, 8), By employing this key reactions, (8R,10S)- and (8R,10R)-heliannuol E. have been synthesized. Comparison of their spectral data and optical rotations revealed that the natural (-)-heliannuol E has the latter absolute configuration. In addition application of our synthetic methodology to other heliannuols, allolopathogenic assessment of heliannuols and related compounds would be presented.

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Pentenocins A (1) and B (2) were isolated by Omura et al. in 1999. The planar structures of 1 and 2 were determined by NMR spectral analysis, however, the relative and absolute configurations of these cyclopentenones were not elucidated. We report here the syntheses of the four possible racemic pentenocins B 3〜6 and the first synthesis of (+)-pentenocin B, and the determination of the relative and absolute configurations of natural pentenocin B. We synthesized the four possible racemic pentenocins B 3〜6 to determine the relative configuration of pentenocin B using racemic ketodicyclopentadiene (KDP) 8 as the common starting material. 8 was converted to the 1,4-diols 10a and 10b as an 86:14 mixture of diastereomers. Although the configurations of the diastereomers 10a and 10b were not determined, the major alcohol of 10a was transformed to the ketone 13a. The retro Diels-Alder reaction of 13a was performed to the cyclopentenone 14a. Finally, 14a was treated with 90% TFA to give (±)-pentenocin B 3 or 4. The minor alcohol 10b was transformed to (±)-pentenocin B 4 or 3 by the same procedures as those for the major alcohol 10a to (±)-pentenocin B 3 or 4. To synthesize (±)-pentenocin B 5 or 6, the mixture of the 1,4-diols 10a and 10b was converted to the α,β-unsaturated ketones 18a and 18b via a Wharton rearrangement. 18a was then converted to (±)-pentenocin B 5. The Z-isomer 18b was transformed to (±)-pentenocin B 6 by the same procedures as those for 18a to 5. The NMR spectrum of synthetic pentenocin B 3 or 4 from the major alcohol 10a was in agreement with those of natural pentenocin B from the comparison of NMR spectra between reported natural pentenocin B and synthetic (±)-pentenocins B 3〜6. Having secured the diastereoselective route to pentenocin B, we then conducted the enantioselective synthesis to determine the absolute stereochemistry of natural pentenocin B. (-)-KDP 8 was transformed to (+)-pentenocin B 3 as shown in scheme 4 with the same procedures as described for the synthesis of the racemic pentenocin B 3 or 4. The (R)-stereochemistry of the C-6 secondary alcohol in (+)-pentenocin B 3 was determined using the modified Mosher method and the X-ray analysis. We then developed new synthetic routes to (+)-pentenocin B 3 from (-)-KDP 8 as shown in the scheme 5 to improve the synthetic yield. In conclusion, we have realized the first enantiocontrolled synthesis of (+)-pentenocin B and determined its absolute stereochemistry to be 4S,5R,6R as compared with the value of optical rotation with natural pentenocin B.

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As a C2 symmetric hydrophilic chiral ligand, a series of 2,6-bis(oxazolinyl)-pyridines (pyboxs) bearing a hydroxyalkyl group on the oxazoline ring has been synthesized from readily available amino acid derivatives. Catalytic asymmetric intermolecular cyclopropanation of electron rich terminal alkenes such as vinyl ethers and enamines with diazoesters in the presence of hydrophilic pybox ligand, pybox-hm and Ru(II) complex proceeded smoothly in protic or biphasic media to give the corresponding cyclopropanation products in 97:3 to 99:1 trans/cis ratios and 90 to 97% ee. Steric tuning of the chiral environment of pybox ligands was simply achieved by using a weak interaction between the solvent and the hydroxyl groups of the chiral ligand. The solubility of the new hydrophilic pybox and Ru(II) complexes in protic solvents is dramatically increased; hence, the efficiency of these catalysts enhanced the rate of cyclopropanation. Furthermore, the active catalyst in the water phase can be re-used several times for the cyclopropanation reaction. The hydroxymethyl derivative of pybox can provide excellent stereoselectivities for cyclopropanations of electron rich terminal alkenes, compared to the hydroxyethyl or isopropyl derivatives, in moderate yields in aqueous and protic media. We hypothesize that appropriate solvation of water or alcohols around the hydroxy group causes a more favorable chiral environment around the active site for the cyclopropanation. Work is now under way on applications for total synthesis of natural product, Belactosin A performed in aqueous media.

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Macroviracins are a new class of macrocyclic dilactones, which were isolated from the mycelium extracts of Streptmyces sp. BA-2836, and classified into eight types of congeners by the length (C_<22> or C_<24>) of the constitutive fatty acids. These compounds exhibit a powerful anti-viral activity against HSV-1 and VZV. Degradation studies of macroviracins suggested the corresponding constitutive acids to have the same relative stereochemistry. The relative and absolute configuration of macroviracins, however, remained unsolved. In this symposium, we present determination of the absolute configuration of macroviracins by synthesizing methyl ester 2a of a constitutive C_<22>-fatty acid of macroviracin A (1). Prior to the synthesis of methyl ester 2 of an acid constituted macroviracin A (1), we designed two types of glucosides (6 and 7) as the left- and right-half model of 2, respectively and synthesized them employing regioselective reduction of epoxides and stereoselctive glycosidation as key steps. Direct comparison of the ^1H-NMR data of the model compounds with those of 2 suggested that the acid 2 might have the stereochemistry of 3R, 15S, and 21R if possessing a D-glucose residue. In order to confirm the estimation, 3R,15S,21R-methyl ester 2a was synthesized through a coupling reaction of the epoxide 5a with an organocopper reagent derived from a chiral iodide 7. The spectroscopic data and specific rotation of 2a were identical with those of 2, showing macroviracin A (1) to have the structure 1a as depicted in Scheme 3. In summary, the absolute configuration of macroiviracins was unambiguously established by chemical synthesis of C_<22>-fatty acid methyl ester 2a.

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Eukaryotic cells reportedly contain at least fourteen types of DNA polymerase (pol. α,β,γ,δ,ε,ζ,η,θ,ι,κ,λ,μ,σ and Rev1). We screened for natural compounds that selectively inhibit pol. λ. The purpose was to use the compounds as tools and molecular probes to distinguish DNA polymerases and to clarify their biological and in vivo function. There have been no previous reports of inhibitors capable of distinguishing among pol. β, and λ. Petasiphenol, a bio-antimutagen isolated from a Japanese vegetable, Petasites japonicas, selectively inhibits the activities of mammalian pol. λ in vitro. The compound did not influence the activities of replicative DNA polymerases such as α,δ and ε, but also showed no effect even on the pol. β activity, the three-dimensional structure of which is thought to be highly similar to pol λ. The inhibitory effect of petasiphenol on intact pol λ including the BRCA1 C-terminus (BRCT) domain was dose-dependent, and 50% inhibition was observed at a concentration of 7.8μM. The petasiphenol-induced inhibition of the pol λ activity was non-competitive with respect to both the DNA template-primer and the dNTP substrate. Petasiphenol did not only inhibit the activity of the truncated pol λ including the pol β-like core, in which the BRCT motif was deleted in its N-terminal region. BIAcore analysis demonstrated that petasiphenol bound selectively to the N-terminal domain of pol λ, but did not bind to the C-terminal region. We found here that another phenolic compound, curcumin (diferuloylmethane), which is structurally quite similar to petasiphenol, was also a potent pol. λ inhibitor. The IC_<50> values of curcumin was 7.0μM. Based on these results, the pol λ-inhibitory mechanism of phenolic compounds such as curcumin is discussed.

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(+)-Duocarmycin SA (1), which is a potent antitumor antibiotic bears a characteristic structural features in which the tricyclic ring system containing cyclopropane are connected with the trimethoxyindole part. Due to both of the unique structure and biological activity, 1 had been attracted by many organic chemists and total syntheses had been already reported by several research groups. Herein, we will report synthetic studies of 1 by two independent routes using two different kind methods for constructing the nitrogen containing heterocycles as key steps. For the first route, the epoxide (17) which is the substrate for the ring closure reaction was synthesized from commercially available 4-amino-3-nitrophenol (14) by 5 steps in good overall yield. The reaction of 17 with BuLi in THF gave the desired 5-membered ring (18) for 82% yield as a single isomer via 5-exo selective ring closure reaction of the carbanion intermediate. Acetylation of 18, followed by regioselective nitration reaction provided 19. The substrate for Sonogashira coupling reaction was synthesized by reduction of nitro group and bromination. However, the bromide (21) was completely recovered in the all reaction conditions. At this point, as we thought that this low reactivity might be the serious steric hindrance, we turned our attention to the other synthetic route. For the second route, 2-amino-5-nitrophenol (23) was converted to the iodide (26) by benzyl ether formation, regioselective iodination, and mesylamide formation. Application of Negishiis coupling conditions to the iodide (26) with methyl propiolate afforded the unexpected indole (28) via tandem coupling and cyclization reaction. The nitro group of 28 was converted to the iodide (34) by 3 steps and it was coupled with propargyl alcohol by Sonogashira coupling reaction provided the acetylene (35). The partial reduction of the triple bond in the presence of Lindler catalyst and the resulting alcohol was subjected to intramolecular Mitsunobu reaction and deprotection gave 38 in almost perfect overall yield. The epoxidation of 38 and the reductive epoxide opening reaction provided the alcohol (40), which was converted to 41 by the reported procedure. The spectral data of 40 and 41 were identified with reported data and the formal synthesis of 1 was accomplished. Now, asymmetric total synthesis of 1 is under going in our laboratory.

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Inositol phosphates and phosphoinositides are now well known physiologically important molecules mainly as second messengers for intracellular signal transduction. As part of our efforts to establish convenient and practical syntheses of them, here we report selective phosphorylation of 3,4-diol, 2,3,6-triol and 3,4,5,6-tetrol derivatives of myo-inositol, using the phosphite-pyridinium tribromide method developed by us, and discuss also practical resolution of 1,2-cyclohexylidene-myo-inositol. Distannylene derivative transformed in situ from ketal 1 smoothly reacted with chiral acetylmandeloyl chloride to give L-3,6-dimandelate (42%) and D-products consisting of 3-mono ester (20%) as well as 3,6-ester (10%), that could be easily separated to optically pure products. In a similar manner, phosphorylation of 1 with pyrophosphate in place of the acid chloride afforded 2 in excellent yield. Diol 3 was treated with glycerol phosphite in the presence of pyridinium tribromide and 2,6-lutidine, resulting in the smooth and selective formation of 3-phosphate 4 in 88%. The product was unexpectedly deprotected at once under hydrogenolysis conditions using commercial AcOEt without purification as a solvent, giving PI(4,5)P2 with palmitoyls. Both enantiomers resolved were converted to triols 5 and 7, respectively. These triols were subjected to the same phosphorylation procedure as above, to afford exclusively 3-O-phosphorylation products 6 and 8, which were then deprotected in two-step procedure to give the same product, PI(3,5)P2. It should be noted that a pyridine-CH_2Cl_<2> (1:1) mixed solvent system was needed for the phosphorylation to take place. The role of pyridine is presumably to dissociate the aggregation state of the triols because they extraordinarily associate each other, as indicated by nmr analysis.

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Our research goal is to make possible systematic dissection of biological events by use of small molecules as biological probes (chemical genetics). To discover bioactive compounds for diverse biological targets efficiently, we constructed an integrated platform performing compound synthesis, biological screening, and data store (Figure 1). Generating a high level of skeletal diversity in library compounds is important to get as many biologically active compounds as possible for diverse targets. Here, we proposed a new strategy called as skeletal transformation to get a skeletal diversity in a library. The key of the strategy is making better use of intramolecular reactions and the ideal order of the inter- and intramolecular reactions in the library synthesis. Intramolecular reactions after conventional intermolecular library synthesis give another skeletal compound accompanied by building blocks. Consequently, the strategy is efficient to generate building blocks diversity as well as skeletal diversity. We synthesized a skeletally diverse library from a steroidal compound 3 by the skeletal transformation strategy as shown in Scheme 3. For the first and second steps usual liquid phase conditions were applicable. We found Et_2AlCl could promote the Diels-Alder reaction at the third step without cleavage of silyl linker. Simple heating of beads was enough for the last retro-Diels-Alder reaction. We screened building blocks for each step, and selected 41 nucleophiles (6 thiols; 25 secondary amines; and 10 primary amines) for the first step, 15 reagents for the second step, and 12 ynones for the third step as shown in Figure 2. Theoretically the combination of these building blocks can produce a maximum of 4275 distinct compounds. We synthesized the library encoded with chemical tags using 25650 beads (4275 compounds×6 copy) as shown in Figure 3. The purity of compounds in library part 1, 2, and 3 were 78%, 71%, and 62% respectively. The intensity of tags was enough for decoding by CAN-cleavage and GC analysis. Biological screenings of the library synthesized here is currently in progress.

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The ripe fruits of paprika (Capsicum annuum L.) is a good source of carotenoids and is used widely as a vegetable and food colorant. The red carotenoids are mainly capsanthin and capsorbin, possessing 3-hydroxy-6-oxo-κ-end group. In the course of the carotenoids studies of paprika, two new carotenoids 1 and 2 were isolated as minor components. The MeOH extract of ripe fruits of paprika (4kg) was saponified with 5% KOH/MeOH, and unsaponifiable matter was chromotographed on silica gel using an increasing percentage of acetone in hexane. The fraction eluted with acetone-hexane (1:9) was subjected to a series of HPLC on ODS with CHCl_3-acetonitrile (1:9) and on silica gel with acetone-hexane (2:8) to yield 1 (2mg) and 2 (0.5mg). The molecular formula of 1 was determined to be C_<40>H_<56>O_2 by HR FAB MS. The positive ion FAB MS/MS spectrum of the molecular ion (M^+) of 1 is shown in Fig 1a. Characteristic product ions of M-111 and M-139 which attributed to cleavage between C-5' and C-6' and between C-6' and C-7', respectively suggested the presence of 6-oxo-κ-end group in 1. The structure of 1 was determined to be 3-hydroxy-β,κ-caroten-6'-one by ^1H- and ^<13>C-NMR, COSY, TOCSY, NOESY, HSQC and HMBC data and named 3'-deoxycapsanthin. The structure of 2 was determined to be 3,4-didehydro-β,κ-caroten-6'-one by HR FAB MS, FAB MS/MS, UV-Vis and ^1H-NMR data. From the CD spectral data and biosynthetic consideration 3R, 5'R and 5'R chiralities were proposed for 1 and 2, respectively. They are the first example of carotenoids possessing 6-oxo-κ-end group.

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Trypanosoma cruzi, a parasitic protozoan, is the causative agent of Chagas' disease in Central and South America. In our search for trypanocidal compounds in medicinal plants used in Uzbekistan, we isolated new trypanocidal diterpenes from the EtOAc extract of Dracocephalum komarovi. Since komaroviquinone, a quinone-type diterpene 1, showed potent trypanocidal activity, we tested natural quinones to find out quinone-type diterpenes 2-5 from Salvia miltiorrhiza to have potent trypanocidal activity. Several quinones have been reported to have trypanocidal activity, and these compounds are thought to exert their trypanocidal action by generation of reactive oxygen species, which is catalyzed by flavo-enzymes such as trypanothione reductase, in the parasite. Recently, an enzyme, which catalyzes the two-electron reduction of 9,11-endoperoxide PGH_2 to PGF^<2α>, was isolated from T. cruzi. This enzyme (TcOYE) was found to be a member of old yellow enzyme family, which has been isolated from yeasts, plants, and bacteria but not from animals, and to catalyze one-electron reduction of trypanocidal quinones. Thus, we investigated the role of this enzyme. Under an anaerobic condition, quinone-type diterpenes 1-5 were efficiently reduced by TcOYE in the presence of NADPH as a cofactor. NADPH was almost 2-fold more effective as the cofactor than NADH. Electron spin resonance experiments showed that, under the anaerobic condition, TcOYE catalyzed one-electron reduction of 1-5 to form semiquinone radicals, and under aerobic condition, these radicals reduced molecular oxygen leading to regeneration of the quinone and formation of superoxide anion radicals, which will cause oxidative damage to cellular components resulting in the death of the parasites. Compounds 2-5, which have larger one-electron reduction potentials, were better substrate of TcOYE than 1, which has a smaller reduction potential. However, the specific activity was not completely parallel with the reduction potential, indicating the importance of the substrate specificity of TcOYE. The specificity was not also parallel with the trypanocidal activity: komaroviquinone (1), which was not the best substrate of TcOYE, showed the strongest trypanocidal activity among the tested compounds. This suggested the involvement of other flavo-enzymes in the generation of active oxygen species. An experiment to evaluate the importance of TcOYE in this process is now in progress.

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In our study on lipoxygenase inhibitors, we succeeded in designing a new assay system. Furthermore, we found four novel lipoxygenase inhibitors, tetrapetalone A (1), B (2), C (3) and D (4) by using this system. Tetrapetalone A (1), B (2), C (3) and D (4) were isolated from a culture filtrate of Streptomyces sp. USF-4727 strain. The planar structure of 1 was determined by its spectroscopic evidence and methylation with diazomethane to show the presence of a novel tetracyclic skeleton, including a nitrogen atom, and a β-rhodinosyl moiety in 1. The stereochemistry of 1 was investigated by the coupling constant in the ^1H NMR spectrum, NOE correlations and modified Mosher's method. Then, the absolute stereochemistry of all the asymmetric carbons was determined. In the similar way, we estimated the chemical structure of 2, 3 and 4. Tetrapetalone B (2) also had a tetracyclic skeleton and a β-rhodinosyl moiety as well as 1. Tetrapetalone B (2), however, had an additional acetoxy moiety at a side chain of the tetracyclic skeleton. Tetrapetalone C (3) and D (4) were revealed to be derivatives of 1 and 2, respectively, with an additional hydroxy group in their tetracyclic skeleton. Tetrapetalone A (1), B (2), C (3) and D (4) indicated the inhibitory activity against soybean lipoxygenase as well as two well-known lipoxygenase inhibitors, kojic acid and NDGA.

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Cellular localization of the marine sponge-derived excitatory amino acid dysiherbaine (1) was investigated using immunohistochemical methods. Dysiherbaine (1), isolated from a sponge Dysidea herbacea, selectively activates mammalian non-NMDA type glutamate receptors (GluRs) and induces seizure in mice. Recently 1 was found to have unusually high affinity towards GluR5 and 6, subtypes of kainite-type GluRs, and can activate these subtype receptors selectively in the heteromerically expressed receptor complex. These unusual actions of 1 to the neuronal receptors warrants its usefulness as a research tool, however, its production, biosynthesis, and roles in the natural environment are left to be investigated. Since microorganisms including a cyanobacterial, Oscillatoria spongeliae are known to be heavily associated with D. herbacea, actual producers of the secondary metabolites and their roles in the sponge and associated microorganisms are difficult problem to solve. In this study, we paved a way to approach this elusive problems by showing cellular localization of 1 using immnumohistochemical methods. We also show a presence of putative DH-binding protein of 111kD using DH-conjugated affinity chromatography and SDS-PAGE.

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The ubiquitin-proteasome pathway is the principal one for intracellular protein turnover in eukaryotic cells. This pathway is involved in many biological processes, including degradation of damaged, oxidized, or misfolded proteins. Likewise, by this pathway, processing or degradation occurs of regulatory proteins such as cyclins, cyclin-dependent kinase inhibitors (e.g., p21 and p27), tumor suppressors (e.g., p53), and 1κB, which are critical in tumor growth and inflammation. Proteasome inhibitors might be useful for the treatment of cancer and inflammatory diseases. Tyropeptins A and B, new proteasome inhibitors, were isolated from the culture broth of Kitasatospora sp. MK993-dF2. They were purified using ethyl acetate extraction, silica gel column chromatography, Sephadex LH-20 column chromatography and HPLC. The ^1H and ^<13>C NMR of tyropeptins were complicated due to the presence of an aldehyde group. Therefore, to give assignable NMR spectra, tyropeptins were converted to their alcohols by sodium borohydride. The stereochemistry of tyropeptins were determined by analysis of acid hydrolysis products from tyropeptins, and further confirmed by the total synthesis. The structures of tyropeptins A and B were found to be isovaleryl-L-tyrosyl-L-valyl-DL-tyrosinal and n-butyryl-L-tyrosyl-L-leucyl-DL-tyrosinal, respectively. Tyropeptin A inhibits the chymotrypsin-like and trypsin-like activities of 20S proteasome with IC_<50> values of 0.1μg/ml and 1.5μg/ml respectively, but did not inhibit the peptidylglutamyl-peptide hydrolyzing (PGPH) activity of 20S proteasome at a concentration of 100μg/ml. And it inhibits the intracellular proteasome activity in PC-12 cell, and causes neurite outgrowth. As expected, ubiquitinated proteins accumulated in cells treated with tyropeptin A. Hence, it appears that tyropeptin A can permeate into cells, and then inhibits the intracellular proteasome activity. To enhance the tyropeptin A potency, we constructed a structural model of tyropeptin A bound to the site responsible for the chymotrypsin-like activity of the 20S proteasome. The obtained model nicely made fit into the site. On the basis of modeling experiment, several derivatives of tyropeptin A having the bulky N-terminal moiety were designed and synthesized. The most potent compound exhibited about 20-fold enhancement in potency compared to tyropeptin A.

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We have shown that plant cells act a defense reaction against chemical stress, such as geraniol and bornyl acetate. When the cultured shoot primordia of Marchantia chamomilla and cultured cells of Glycine max were treated with 5mM geraniol, the levels of glutathione S-transferase (GST) mRNA and the GST activity reach peaks at 4 and 8h, respectively. The mRNA levels of McEREBP1 and McWRKY1 transcription factors were severely elevated to maximum at 1h, after treatment of the cultures with geraniol. These increases were specific to geraniol, not geraniol isomers, nerol and linalool. The McEREBP1 may act to a transcription factor on the GST promoter. The p20, a novel-type of trypsin inhibitor mRNA, levels were decrease by the treatment of the cells with geraniol. These results suggest that induction of GST activity and loss of protease inhibitor may participate in defence reaction by monoterpenoid treatment. We reported that when the cultured cells of Marchantia polymorpha were treated with bornyl acetate as a chemical stress agent, the cells secreted lunularin, hydrogen peroxide, peroxidase and phosphatase. A peroxidase was purified from cultured medium and a full-length cDNA clone was isolated. The purified peroxidase catalyzed the oxidative polymerization of lunurarin with hydrogen peroxide. The peroxidase mRNA level elevated to maxima at 1h after treatment with bornyl acetate. An ATPase-like phosphatase was purified from cultured medium induced to chemical stress. The phosphatase differed from normal type phosphatase secreted without chemical stress. These indicated that the secreted peroxidase and ATPase-like phosphatase reinforce cell wall to chemical stress, because the peroxidase promote oxidative coupling of lunularin and hydrogen peroxide.

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Species are shown by the history of character on individual Aconitum plants. Specifically, it is expressed by the evolutional location of three dimensional coordination consist of X-axis(Physical selection e.g. embryological morphology and ecological geography etc), Y-axis(Chemical selection e.g. molecular heredity and physiological chemistry etc) and Z-axis (Geohistorical selection e.g. pedelogical geology and absolute chronology etc). Subgenus Gymnaconitum: one yearly plants. C19-aconines generates by Wagner-Meerwein rearrangement of C20-gymnandines via 7.20-Mannich reaction of C20-atisines. This facts are the relix of metabolism in primitive Aconitum species (A. gymnandrum Maxim) Subgenus Aconitum: two yearly plants. From the existence of C19-protoaconines which was rcognized by us, theoretically and experimentally, the biosynthesis of C19-aconines was proceeded from Mannich reaction of the C19-protoaconines via the Wagner-Meerwein rearrangement of C20-atisines as the reverse course of the above Gymnaconitum. Subgenum Paraconitum: perennial plants. The biosynthesis is characterized by the oxygenation of 7,8-double bond of the C19-protoaconines.This phenomenon was also observed in the hybridization species of subgenus Aconitum. Thus further evolution of paraconitum metabolism occurres esterification with anthranilic acid derivatives or Baeyer-Villiger oxidation except methylation on 4-hydroxymethyl function of C19-lycoctonines.

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In 2000, an α-pyrone-containing natural product, TT-1, was isolated from the ethyl acetate extract of a fungi imperfecti Trichurus terrophilus culture by Fujimoto and co-workers in our laboratory. TT-1 significantly suppressed proliferation (blastogenesis) of mouse spleen lymphocytes stimulated with mitogens, concanavalin A (Con A) and lypopolysaccharide (LPS), with IC_<50> values of 0.7 and 0.5μg/mL, respectively. TT-1 was optical active and had a molecular formula of C_<25>H_<38>O_6, and its planar structure was elucidated as 1 consisting of two principal carbon-chains with an α-pyrone ring on the basis of the spectral data. Almost at the same time, Hayakawa and co-workers reported isolation of rasfonin, a new apoptosis inducer in ras-dependent cells from the fermented mycelia of Talaromyces sp. 3656-A1. The planar structure of rasfonin was identical with that of TT-1 (1). However, the absolute stereochemistry of five chiral centers of 1 remained undetermined. Here we describe the determination of absolute stereochemistry of five chiral centers of 1 as 5R, 6R, 7S, 9R, and 6'S on the basis of synthesis of partial structural units (fragments A and B) of 1 in optically active forms and comparison of their spectral and optical data with those of natural specimens. We first prepared fragment B with 6'S-configuration as shown in Scheme 1. Comparison of the ^1H NMR chemical shift data of synthetic di-(R)- and di-(S)-MTPA esters (7 and 8) with those of di-(R)- and di-(S)-MTPA esters of natural products (9 and 10) suggested that the absolute configuration of C-6' position of 1 was S. On the basis of the analysis of spectral data, it was proposed that H-5 and H-6 were cis and 1,3-dimethyl system at C-7 and C-9 was syn for the relative stereochemistry of fragment A. Two diastereomers (13 and 14) with 5-membered lactone were prepared in optically active forms, and their spectral and optical data were compared with those of a natural specimen (13) with 5-membered lactone derived from 1 to reveal that the diastereomer 13 was completely identical with natural specimen. Fragment A was therefore shown to have 5R, 6R, 7S, 9R-configurations.

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Vascular endothelial cells have essential roles in several diseases such as cancer, atherosclerosis and diabetes mellitus. In particular, adhesion of leukocyte to vascular endothelial cell is a critical step in atherosclerosis. Various adhesion molecules such as ICAM-1, VCAM-1 and E-selectin are expressed on vascular endothelial cells at sites of inflammation. These adhesion molecules are induced by inflammatory stimuli such as lipopolysaccharide (LPS), TNF-α and IL-1β, and these inductions are associated with the activation of NF-κB. In the present research, we searched naturally occurring secondary metabolites for the compounds that inhibit adhesion of LPS-stimulated human umbilical vein endothelial cells (HUVEC) and human promyelocytic cell line HL-60 cells. We also studied the biological activity and the mechanism of action of the isolated compound. In the course of our screening for inhibitors of adhesion of LPS-stimulated HUVEC and HL-60 cells, we isolated the novel compound 1785-2A from the whole culture broth of a bacterial strain. Treatment of HUVEC with LPS for 4 hours significantly increased the adhesion of HL-60 cells. Addition of the novel compound 1785-2A prior to LPS stimulation decreased HL-60 cell -HUVEC adhesion significantly at 3μg/ml. We confirmed that 1785-2A did not show cytotoxity and growth inhibition on HUVEC below 30μg/ml. 1785-2A also inhibited the cellular adhesion induced by Lipid A, the active component of LPS. However, 1785-2A did not inhibit adhesion of HL-60 cell -HUVEC when HUVEC were activated by TNF-α or IL-1β. Furthermore, 1785-2A inhibited LPS-induced expression of ICAM-1 and VCAM-1 at 3μg/ml almost completely. Thus, this novel compound 1785-2A was shown to be a new selective inhibitor of LPS signal transduction.

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Conventional and novel protein kinase C (PKC) isozymes are main targets of tumor promoters. These isozymes contain two C1 domains (C1A, C1B), both of which bind tumor promoters. Design of new ligands with high selectivity among the C1 domains of PKC isozymes is indispensable to elucidate the precise mechanism of tumor promotion. Indolactam-V (1) was selected as a lead compound for such ligands since 1 showed a binding preference for the C1B domains of novel PKC isozymes. Compound 1 exists as two stable conformers in solution at room temperature; the twist form with the cis amide and the sofa form with the trans amide. Highly improved selectivity for the C1B domains of novel PKC isozymes was observed in the sofa-restricted analogue (3), while the binding selectivity of the twist-restricted analogue (2) was similar to that of 1. This indicates that the conformation of 1 plays an important role on the binding selectivity for C1 domains of PKC isozymes. Based on this result, new conformationally-restricted analogues of 1, indolinelactam-Vs (4, 5), were synthesized. (3R)-Indolinelactam-V (4) adopted a conformation similar to the twist form of 1, while the conformation of (3S)-indolinelactam-V (5) was close to that of the sofa form. The binding selectivity of 4 and 5 for the C1B domains of novel PKCs was considerably higher than that of 1, and sofa-like 5 showed slightly higher binding selectivity than twist-like 4. These results suggest that conventional PKC isozymes (α, βI, βII, γ) strictly recognize the twist form of 1 and that the sofa-restricted analogues of 1 such as 3 and 5 would be lead compounds of new legands with high selectivity for the C1B domains of novel PKC isozymes.

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Auxin controls cell division, elongation and differentiation and, therefore through its action at the level of the cell, exerts profound effects on growth and development throughout the life of the plant. Yokonolides A (YkA) and B (YkB), spiroketal-macrolide, were isolated from Streptomyces diastatochromogenes B59 in a screen for inhibitors of β-glucoronidase expression under the control of an auxin-responsive promoter in Arabidopsis. YkB inhibit the expression of auxin-inducible genes as shown using native and synthetic auxin promoters as well as using expression profiling of 8300 Arabidopsis gene probes but does not affect expression of an abscisic acid- and a gibberellin A_3-inducible gene. The mechanism of action of YkB is to block AUX/IAA protein degradation; however, YkB is not a general proteasome inhibitor. YkB blocks auxin-dependent cell division and auxin-regulated epinastic growth mediated by auxin-binding protein 1. Gain of function mutants such as shy2-2, slr1, and axr2-1 encoding AUX/IAA transcriptional repressors and loss of function mutants encoding components of the ubiquitin-proteolytic pathway such as axr1-3 and tir1-1, which display increased AUX/IAAs protein stability, are less sensitive to YkB, although axr1 and tir1 mutants were sensitive to MG132, a general proteasome inhibitor, consistent with a site of action downstream of AXR1 and TIR. YkB-treated seedlings displayed similar phenotypes as dominant AUX/IAA mutants. Taken together, these results indicate that YkB acts to block AUX/IAA protein degradation upstream of AXR and TIR, links a shared element upstream of AUX/IAA protein stability to auxin-induced cell division/elongation and to auxin-binding protein 1, and provides a new tool to dissect auxin signal transduction.

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In the course of our studies on the bioactive constituents from Egyptian herbal medicines, we characterized the chemical structures and pharmacological activities of the constituents from several Egyptian herbal medicines, Anastatica hierochuntica, Cyperus longus, and Nigella sativa. 1. Anastatica hierochuntica: The whole plants of A. hierochuntica (Cruciferae), which is a winter annual plant of the Sahara-Arabian deserts, are prescribed in Egyptian folk medicine for fatigue and uterine haemorrhage and are used by women as a charm for child birth. Two novel skeletal benzofuranoflavanones, anastatins A (1) and B (2) and three new neolignans termed hierochins A (3), B (4), and C (5) were isolated from the methanolic extract of this herbal medicine. Their absolute stereostructures were determined on the basis of chemical and physicochemical evidence. The inhibitory effects of anastatins (1, 2) and isolated flavonoids (6-10) on D-galactosamine (D-GalN)-induced cytotoxicity in primary cultured mouse hepatocytes were examined. As a result, hepatoprotective activities of 1 (IC_<50>=20μM) and 2 (17μM) were stronger than those of other flavonoids and commercial silybin (40μM), which is well known to show potent hepatoprotective activity. In addition, we examined inhibitory effects of isolated lignans from A. hierochuntica on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated macrophages. Among them, three neolignans, 12 (IC_<50>=31μM), (+)-dehydrodiconiferyl alcohol (13, 26μM), and (+)-balanophonin (14, 23μM) inhibited NO production and induction of inducible NO synthase (iNOS) without cytotoxic effects in the MTT assay. Furthermore, in order to clarify the structural requirements of flavonoids for inhibitory activities of NO production and antigen-induced degranulation from RBL-2H3 cells, we examined inhibitory effects of 73 flavonoids on NO production and the release of β-hexosaminidase from RBL-2H3 cells and clarified several structural requirements for the activities. 2. Cyperus longus: C. longus (Cyperaceae) is widely distributed in Mediterranean, western and central Europe, tropical Africa, and western and central Asia. The whole plants of C. longus have been used as a diuretic and tonic in Egyptian traditional medicine. From the methanolic extract of this herbal medicine, a new nor-stilbene dimer, longusone A (22), three stilbene dimers, longusols A-C (23-25), and six new sesquiterpenes, cyperusols (26-31), were isolated. In addition, the constituents of C. longus were found to show DPPH radical scavenging activity and inhibitory effects on the release of β -hexosaminidase from RBL-2H3 cells and D-GalN-induced cytotoxicity in primary cultured mouse hepatocytes. 3. Nigella sativa: The seeds of N. sativa (common name "Black Cumin") have been used for foods, spices, and also prescribed in Egyptian folk medicine for asthma, flatulence, polio, kidney stones, and abdominal pain, etc. Two new dolabellane-type diterpene alkaloids 43, 44 were isolated from the methanolic extract of this herbal medicine and found to show potent reduction of triglyceride levels in primary cultured mouse hepatocytes.

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Archaea is clearly distinguished from eucarya and eubacteria by the 16S ribosomal RNA sequences, and the existence of unusual ether lipid in archaea is one of the clearest distinctions between archaea and other organisms. Furthermore, among thermophilic archaea, the genus Sulfolobus that have one of the main part of thermophilic archaea and have a quite characteristic lipid termed carditoglycerocaldarcheol, consisted from the characteristic 5-membered carbocycle compound, carditol. The biosynthesis of this unique structure of carditol, the ring closure mechanism, and the C-O bond formation between cyclopentane ring and glycerol is intriguing problem and could be examined by precise observation of the labeling experiment. Thus, we executed an investigation of the formation of the unique structure of carditol. Deuterium-labeling experiments with each [1-^2H]-, [6,6-^2H_2]- and (6S)-[6-^2H]-D-glucose were carried out. Fortunately, the labeling pattern and the degree of deuterium incorporation could be undoubtedly observed only with ^1H NMR measurement of carditoglycerocaldarcheol acetate. Deuterium was incorporated clearly at C-1 of carditol, and C-6 methylene position with stereospefically and high degree of incorporation (about 50% incorporation of deuterium) from each substrate. The deuterium from |2-^2H|-D-glucose was not incorporated to carditol. These results clarified that the carditol was biosynthesized from glucose with C-C bond formation between C-1 and C-5. The two courses are proper when efficiency of mechanism is regarded as experimental findings and Gambacorta's previous reports. (1) The one is the course that resembled biosynthesis of myo-inositol that assumes the C-4 oxidation the starting point. (2) The another one is that an enzyme does reductive coupling like a pinacol-reaction after the C-5 oxidation. Among them, the pathway (1) will be preferred from the resemblance of myo-inositol biosynthesis. Furthermore, high deuterium incorporation at C-1 of carditol suggests that activation such as oxidation at C-1 is not involved, and also may suggest that the ether formation is done after cyclization reaction.

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Marine sponges have proved to be a rich source of secondary metabolites with unusual structures as well as interesting biological activities. In the course of our continuing research on biologically active compounds from Japanese marine invertebrates, we have investigated the isolation for the cytotoxic principle from the marine sponge Chondrosia chucalla. We report here the isolation and structure elucidation of three novel cyclic depsipeptides, chondrosiamide A (1), B (2), and C (3). C. chucalla was collected by hand at depths of 10m off Hedo Point, Okinawa, Japan, in July 2000. The diethyl ether-soluble fraction of the EtOH extract showed cytotoxcity against KB cells. Bioassay guided the separation of the active fraction by Sephadex LH-20, silica gel chromatography, reversed-phase HPLC to give three active compounds, 1 (16.8mg), 2 (26.7mg), and 3 (3.9mg). The structure of chondrosiamides was determined on the basis of spectral and chemical methods. Absolute configuration of amino acid residues was determined by Marfey's analysis. Chondrosiamide A (1) and B (2) showed cytotoxicity against KB cells at the IC_<50> value of 9.7 and 2.8μM, respectively.

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The plant hormone, auxin, regulates various aspects of plant growth and development by controlling cell division, elongation, and differentiation. However, the molecular mechanism of auxin action is still unclear. Therefore, bio-probes that modulate auxin action are useful tools for study on auxin signaling. In the screening program to search for auxin signaling inhibitors by mean of transgenic Arabidopsis plant harboring early auxin-responsive promoter and reporter gene, GUS (β-glucuronidase), we found terfestatin A (trfA) as a specific inhibitor of auxin signaling from culture bloth of Streptomyces sp. F-40. The chemical structure of trfA was determined to be p-terphenyl-β-glucoside on the basis of various NMR techniques and chemical degradation. TrfA competitively inhibited the expression of early auxin-responsive Aux/IAA genes and also displayed competitive effects on auxin-induced cell division and elongation in Arabidopsis. In contrast to auxin action, trfA showed no effects on the gene expression induced by other plant hormone, such as abscisic acid, gibberellin, and cytokinin. From these results, trfA would be specific antagonist on auxin action that lead to early auxin-responsive gene expression, suggesting trfA might bind unknown auxin receptor in competitive manner.

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In the course of our studies on the function and distribution of vasoconstrictive polyhydroxy compounds zooxanthellatoxins (ZTs), we found structurally related compounds to ZTs, named zooxanthellamide A (1) and its derivatives 2, 3 from one species of the genus Symbiodinium (strain HA3-5) isolated in the free-living state from an Hawaiian tide pool. The structural determination of these compounds was performed by spectroscopic methods mainly by 2D NMR techniques. 2 and 3 were determined to be δ-lactone and macrocyclic lactone derivatives of 1, respectively. These compounds 1-3 possess a molecular weight of 2715 or 2697, which are a little smaller than those of ZTs. Although 1 is a large polyhydroxy molecule like ZTs, 1 differs from ZTs in the following points. Thus, 1 does not possess a diepoxide and an exomethylene that are present in ZTs. Amide and sulfate groups exist singly in ZTs, whereas there are a pair of both of these groups in 1. In an evaluation of vasoconstrictive activity, 3 showed a higher activity than that of a positive control ZT-A, whereas 1 and 2 did not show the activity. The presence of a macrolide structure in 3 might be important for the vasoconstrictive activity. Further studies on structural analysis of macrolide 3 are in progress.

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Since the modified Mosher's method has been reported by our group, it has become indispensable to the absolute configuration determination of natural compounds. The modified Mosher's method is based on Professor Mosher's idea that MTPA ester takes such a conformation, in which the carbinyl proton, carbonyl oxygen, and trifluoromethyl groups are located on the same plane. During the present work, some MTPA esters were obtained as crystals. Their X-ray crystallography shows that the conformations of the MTPA moieties are the same as the proposed one. Proton NMR spectra of the MTPA esters with variety of functional groups were recorded for deuterochloroform, deuterobenzene, deuteromethanol and deuteropyridine solutions. The experiments revealed that the modified Mosher's method was applicable to most of the compounds in these deutero-solvents. Interestingly, the MTPA esters of two cyclopropane compounds exhibited abnormal Δδ distribution in deuterobenzene. However, they showed normal patterns when the proton NMR spectra were recorded for their deuterochloroform, deuteromethanol, and deuteropyridine solutions.

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When the apical bud of a plant seedling is actively growing, the outgrowth of lateral buds is suppressed. This phenomenon is called "apical dominance". Apical dominance is released by excision of the apical bud or by application of auxin transport inhibitors, such as 1-naphthylphthalamic acid (NPA) and 2,3,4-triiodobenzoic acid (TIBA) to the apical bud or the internode of seedlings. However, direct application of auxin to the lateral buds of decapitated seedlings did not maintain apical dominace. We demonstrated that application of natural auxin-inhibiting substances, raphanusanin B and 6-methoxy-2-benzoxazolinone (MBOA) not only to the apical bud or the internode but also directly to the lateral buds of pea (Pisum sativum L. cv. Alaska) seedlings released apical dominance in either intact or IAA-treated, decapitated seedlings. These results suggest that antagonist(s) of the auxin-inhibiting substances, raphanusanin B or MBOA are synthesized during auxin translocation from the apical buds. We aimed to isolate and identify lateral bud growth inhibitor (s), which transport from the apical bud towards the lateral buds, from pea seedlings. The lateral bud growth inhibitor was isolated from etiolated pea seedlings and identified as indole-3-aldehyde. The indole-3-aldehyde content was significantly higher in the diffusates from explants with apical bud and IAA-treated decapitated explants, in which apical dominance is maintained, than in those from decapitated ones releasing apical dominance. When the indole-3-aldehyde was applied to the cut surface of etiolated decapitated plants or directly to the lateral buds, it inhibited outgrowth of the lateral buds. These results suggest that indole-3-aldehyde plays an important role as a lateral bud growth inhibitor in apical dominance of pea seedlings. On the other hand, flower development of the lateral buds was accelerated in Japanese pear Pyrus pyrifolia (Burm.) Nak. when vertical shoots were bent at a 45℃ angle. This acceleration was associated with notable changes in plant hormone levels. We propose that the bending of pear shoots alters hormone levels, thereby increasing the ability of the lateral buds to compete for assimilates. In the present study, we explore bioactive substance(s) involved in flower development of the lateral buds, and isolated and identified 3,5-di-O-caffeoylquinic acid from the lateral buds of bending-treatment pear shoots.

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Metabolic pathway of tetrodotoxin (TTX, 1), a potent neurotoxin in puffer fish, has not been clarified yet, for puffer fish and also for human. We found a new natural analog of TTX, 4-L-cycteineTTX (4-CysTTX, 2) from the liver of the puffer fish, Fugu pardalis, collected in spring in 2000. In this paper, structural confirmation of 2, and the reaction of TTXs with thiols are described. 2 (ca. 0.4mg) was isolated from 200g of liver of the puffer fish by sequential liquid chromatography. Based on the spectroscopic data, 2 was determined as an analog of 1 of which equatorial hydroxyl group at C4 was substituted by sulfur atom of cysteine. Configuration of cysteine was confirmed to be L by using Marfey reagent for cystine obtained from 2 by I_2 oxidation. 2 was produced by the reaction of 4,9-anhydroTTX (4) and large excess (100-500 equiv.) of cysteine in phosphate buffer (pH 7.0-8.0) at 40℃ for 90min. It was pH dependent reaction. Other thiols, glutathione (GSH), mercaptoethanol (ME), and cysteinyl glycine were similarly reacted with 4 to produce sulfur substituted compounds at C4 of 1. On the condition with much more excess of thiols (more than 20000 equiv.) and for longer reaction time (longer than 8hr), ME and GSH were also reacted with 1 to produce the same derivatives from 4.

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