We previously revealed the structural basis of Ca2+ dependent regulation of a polyethylene terephthalate (PET)-degrading enzyme, Cut190, and proposed a unique reaction cycle in which the enzyme repeatedly binds and releases Ca2+. Here, we report crystal structures of Cut190 mutants with high thermal stability complexed with PET-like ligands that contain aromatic rings. The structural information has allowed us to perform further computational analyses using a PET-trimer bound model. Our multicanonical molecular dynamics simulations and subsequent analyses of the free energy landscapes revealed a novel intermediate form that occurs during the enzymatic reaction cycle. Furthermore, the computational analyses were used to investigate the effect of the point mutations F77L and F81L in the Ca2+-binding site, which showed that the former stabilizes the engaged and open forms to improve transition between the open and active forms, while the latter extremely increases the open form. Subsequent experiments showed that the F77L mutation increased the activity, while the F81L mutation decreased the activity. Our computational analysis has enabled us to explore the dynamics of Cut190 on a completely new level, providing key insights into how the balance between the various conformations influences the reaction cycle and ultimately how to improve the reaction cycle.

This study evaluated the ability of isolated or semisynthesized trichothecene sesquiterpenes to prevent cancer emergence and proliferation and inhibit signal transducer and activator of transcription-3 (STAT3) phosphorylation through in vitro assays. Trichothecinol A (TTC-A), which bears a hydroxy group at C3, exhibited greater cancer prevention, antiproliferation, and STAT3 phosphorylation inhibition effects than trichothecin (TTC), which lacks a hydroxy group at C3. Furthermore, trichothecinol B (TTC-B), which is a reduced derivative of TTC and has similar cytotoxic effect, showed substantially weaker chemoprotection and STAT3 phosphorylation inhibition effects than TTC. These results clearly indicate that the hydroxy group at C3 and carbonyl group at C8 are crucial for inducing both potent chemoprevention and STAT3 phosphorylation inhibition.

Based on previous studies, we synthesized a novel class of ortho- and para-naphthoquinones derivatives bearing a phenolic hydroxy or sulfonamide moiety and evaluated their in vitro antiproliferative and signal transducer and activator of transcription-3 (STAT3) phosphorylation inhibitory activities. The biological evaluations of these naphthoquinones revealed that ortho-naphthoquinones containing a phenolic hydroxyl group exhibited greater antiproliferative activity compared to compounds without a phenolic hydroxyl group. Among the synthesized para-naphthoquinones, 21, which has a condensed sulfonamide structure, showed substantially higher antiproliferative activity than that of the parent compound, and was also found to inhibit the phosphorylation of STAT3(Y705) in a dose-dependent manner. A docking simulation using AutoDock Vina suggested that 21 could directly bind to the hinge region of STAT3.

The enzymatic recycling of polyethylene terephthalate (PET) can be a promising approach to tackle the problem of plastic waste. The thermostability and activity of PET-hydrolyzing enzymes are still insufficient for practical application. Pretreatment of PET waste is needed for bio-recycling. Here, we analyzed the degradation of PET films, packages, and bottles using the newly engineered cutinase Cut190. Using gel permeation chromatography and high-performance liquid chromatography, the degradation of PET films by the Cut190 variant was shown to proceed via a repeating two-step hydrolysis process; initial endo-type scission of a surface polymer chain, followed by exo-type hydrolysis to produce mono/bis(2-hydroxyethyl) terephthalate and terephthalate from the ends of fragmented polymer molecules. Amorphous PET powders were degraded more than twofold higher than amorphous PET film with the same weight. Moreover, homogenization of post-consumer PET products, such as packages and bottles, increased their degradability, indicating the importance of surface area for the enzymatic hydrolysis of PET. In addition, it was required to maintain an alkaline pH to enable continuous enzymatic hydrolysis, by increasing the buffer concentration (HEPES, pH 9.0) depending on the level of the acidic products formed. The cationic surfactant dodecyltrimethylammonium chloride promoted PET degradation via adsorption on the PET surface and binding to the anionic surface of the Cut190 variant. The Cut190 variant also hydrolyzed polyethylene furanoate. Using the best performing Cut190 variant (L136F/Q138A/S226P/R228S/D250C-E296C/Q123H/N202H/K305del/L306del/N307del) and amorphous PET powders, more than 90 mM degradation products were obtained in 3 days and approximately 80 mM in 1 day.

In this study, based on our previous study, derivatives of naphtho[2,3-b]furan-4,9-diones were synthesized and their antimicrobial activities were evaluated. The screening of these naphthoquinones revealed that the fluorine-containing NQ008 compound exhibited potent and broad antimicrobial activities against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA), Gram-negative bacteria, and fungi. The results of the ratio of the minimum bactericidal concentration (MBC) to the minimum inhibitory concentrations (MICs) and time-kill assays suggest that the mode of action of NQ008 is bactericidal. Additionally, the results of a drug resistance study revealed that NQ008 exhibited potent antibacterial activity and may delay the development of bacteria resistance. Furthermore, NQ008 exhibited preliminary antiviral activity against the swine influenza virus and Feline calicivirus.

An enzyme, Cut190, from a thermophilic isolate, Saccharomonospora viridis AHK190 could depolymerize polyethylene terephthalate (PET). The catalytic activity and stability of Cut190 and its S226P/R228S mutant, Cut190*, are regulated by Ca2+ binding. We previously determined the crystal structures of the inactive mutant of Cut190*, Cut190*S176A, in complex with metal ions, Ca2+ and Zn2+, and substrates, monoethyl succinate and monoethyl adipate. In this study, we determined the crystal structures of another mutant of Cut190*, Cut190**, in which the three C-terminal residues of Cut190* are deleted, and the inactive mutant, Cut190**S176A, in complex with metal ions. In addition to the previously observed closed, open, and engaged forms, we determined the ejecting form, which would allow the product to irreversibly dissociate, followed by proceeding to the next cycle of reaction. These multiple forms would be stable or sub-stable states of Cut190, regulated by Ca2+ binding, and would be closely correlated with the enzyme function. Upon the deletion of the C-terminal residues, we found that the thermal stability increased while retaining the activity. The increased stability could be applied for the protein engineering of Cut190 for PET depolymerization as it requires the reaction above the glass transition temperature of PET.

The extract of Tabebuia avellanedae has been used as a folk medicine, and the various biological activities of T. avellanedae have been extensively studied. However, few studies have reported which natural products play a role in their biological effects. In this study, we evaluated representative naphthoquinones isolated from T. avellanedae and found that furanonaphthoquinones were the key structures required to exhibit STAT3 phosphorylation inhibitory activities. Our SAR analysis indicated that removal of a hydroxyl group enhanced the STAT3 phosphorylation inhibitory activity. In addition, the combined results of a mobility shift assay, SH2 domain binding assay, and docking simulation by Autodock 4.2.6 suggested that (S)-5-hydroxy-2-(1-hydroxyethyl)naphtho[2,3-b]furan-4,9-dione (1) could directly bind to the hinge region of STAT3.

A concise and efficient synthesis method for the preparation of antiproliferative ortho-naphthoquinones is described. Notably, the synthesis of ortho-furanonaphthoquinone was achieved by utilizing a regioselective oxidative conjugate addition of dimethylamine and the Sonogashira coupling/cyclization reaction as the key steps. Additionally, an improved synthesis of hydroxy-P-lapachone was established and included a regioselective prenylation by directed ortho-lithiation. In vitro antiproliferative effects of the synthesized against a panel of 39 human cancer cell lines were evaluated and the results were directly compared to those previously obtained for 1.

Endurance capacity is important for maintenance of quality of life as well as performance of endurance athletes. In order to improve endurance, intake of nutritional supplements as well as exercise training is also important. Indeed, polyphenolic extracts from plants are known to improve endurance capacity via increase of fatty acid utilization, mitochondrial biogenesis or inhibition of oxidative stress. Taheebo, the extract obtained from inner bark of Tabebuia avellanedae has been reported to have beneficial effects for treatment of inflammation, oxidative stress and obesity. Here, we investigated the effects and mechanisms of polyphenol fraction of taheebo (taheebo polyphenol; TP) on endurance capacity of mice. Single dose administration of TP significantly increased running time until exhaustion. Acute TP administration increased blood glucose and muscle glycogen levels (p < 0.05) through alteration on expression level of genes involved with glycogen metabolism and gluconeogenesis. Furthermore, TP administration decreased exercise-induced increase of protein carbonyls in skeletal muscle. These results suggest that TP administration improve endurance capacity via up-regulation of skeletal muscle glycogen levels and maintenance of blood glucose by acceleration of gluconeogenesis as well as inhibition of exercise-induced oxidative stress. Single administration of TP also increased phosphorylation of AMP-activated protein kinase (AMPK) and gene expression level of sirtuin 1 (SIRT1) but did not change the marker of mitochondrial biogenesis.

HDAC inhibitors enable histones to maintain a high degree of acetylation. The resulting looser state of chromatin DNA may increase the accessibility of DNA drug targets and consequently improve the efficiency of anticancer drugs targeting DNA, such as Topo II inhibitors. A novel class of nucleoside-SAHA derivatives has been designed and synthesized based on the synergistic antitumor effects of topoisomerase II and histone deacetylase inhibitors. Their inhibitory activities toward histone deacetylases and Topo II, and their cytotoxicities in cancer cell lines, were evaluated. Among the synthesized hybrid compounds, compound 16b showed the potent HDAC inhibitory activity at a low nanomolar level and exhibited antiproliferative activity toward cancer cell lines including MCF-7 (breast), HCT-116 (colon), and DU-145 (prostate) cancer cells at a low micromolar level. Moreover, compound 16a showed HDAC6-selectivity 20-fold over HDAC1.

[This corrects the article on p. 63 in vol. 4, PMID: 29312947.].

We developed novel nucleoside-based topoisomerase II selective inhibitors and showed that small structural units, such as catechols, are essential for DNA topoisomerase II inhibitory activity. Moreover, nucleoside analogues containing TBS and 1,3-dithian moieties had potent and selective DNA topoisomerase II inhibitory activities. In further experiments, compound 25b having a beta configuration of the thymine moiety showed relatively strong growth inhibitory activity against cancer cell lines, and was more potent against all cancer cell lines than compound 26b, which carries a thymine moiety in the alpha configuration.

Aim of study: Taheebo polyphenols (TP) are water extracts of Tabebuia spp. (Bignoniaceae), taken from the inner bark of the Tabebuia avellanedae tree, used extensively as folk medicine in Central and South America. Some anti-inflammatory drugs act by inhibiting both cyclooxygenase-2 (COX-2) and COX-1 enzymes. COX-2 syntheses prostaglandin (PG) E2, which is a species of endogenous pain-producing substance, whereas COX-1 acts as a house-keeping enzyme. Inhibiting both COX-1 and -2 simultaneously can have side effects such as gastrointestinal bleeding and renal dysfunction. Some polyphenols have been reported for its selective inhibiting activity toward COX-2 expression. Our study aimed to demonstrate the potential and mechanisms of TP as an anti-inflammation action without the side effects of COX-1 inhibition. Materials and methods: Free fatty acid-stimulated macrophage cell lines were employed to mimic macrophage behaviors during lifestyle-related diseases such as atherosclerosis and non-alcoholic steatohepatitis. Real-time polymerase chain reaction was used to detect expression of inflammatory cytokine mRNA. Griess assay was used to measure the production of nitric oxide (NO). ELISA was used to measure PG E2 production. Molecular docking was adopted to analyze the interactions between compounds from T. avellanedae and COX-2. Results: TP significantly suppressed the production of NO production, blocked the mRNA expression of iNOS, and COX-2 in both cell lines, blocked the mRNA expression of TNF-α, IL-1β, IL-6, and PGE2 in the murine cell line. However, there was no inhibitory effect on COX-1. Molecular docking result indicated that the inhibitory effects of TP on COX-2 and PGE2 could be attributed to acteoside, which is the main compound of TP that could bind to the catalytic zone of COX-2. After the interaction, catalytic ability of COX-2 is possibly inhibited, followed by which PGE2 production is attenuated. COX inhibitor screening assay showed TP as a selective inhibitor of COX-2 enzyme. Conclusion: The anti-inflammatory effects of TP can possibly regulate macrophages due to the targeted inhibition of COX-2 activity, without affecting COX-1 activity with other anti-inflammatory effects including suppression of iNOS and inflammatory cytokines. As such, TP is potentially useful in prevention and treatment of lifestyle-related disease by attenuating inflammation caused by macrophages infiltration.

Estrogen deficiency-induced obesity has a high risk of visceral fat accumulation and body weight gain. It is also associated with many adverse health conditions. Taheebo extract from Tabebuia avellanedae has been recognized as playing several biological and pharmacological roles. Therefore, we investigated whether the intake of n-BuOH extract of Taheebo shows anti-obesity effect in ovariectomized (OVX) mice. After 16 weeks of feeding, the mice administrated with 0.5% n-BuOH extract of Taheebo showed significantly decreased body weight compared with that of the control mice, and the fat mass also showed a significant decrease. In 3T3-L1 cells, supplementation with n-BuOH extract of Taheebo significantly reduced the triglyceride (TG) levels. Furthermore, bioassay-guided purification of the n-BuOH extract based on the TG levels in 3T3-L1 cells led to the isolation of compound 2 (1-dehydroxy-3,4-dihydroaucubigenin). These results suggested that the anti-obesity effect of Taheebo extract is due to its capability in preventing the accumulation of adipocyte in mice. Taheebo extract might be a promising functional food resources capable of protecting against OVX-induced obesity.

We developed facile one-pot methods for the transformation of 2-arylindoles to polyhydropyrido[1,2-a]indoles and tetracyclic quinazolinones. The copper-catalyzed oxidation of 2-arylindoles to C-acylimines followed by aza-Diels-Alder reactions or oxidative ring-expansion reactions afforded significant polycyclic heterocycles. (C) 2016 Elsevier Ltd. All rights reserved.

In this study, we describe a one-pot method to obtain a variety of 2-arylbenzoxazinones and N-benzoyl anthranilic acid by using a copper catalyst and molecular oxygen as oxidants. This protocol involves tandem cyclization and oxidative processes of 2-alkynylanilines to afford significant motifs in synthetic and medicinal chemistry with moderate yields. We also demonstrated that combining the Sonogashira coupling and the developed method realized the synthesis of 2-arylbenzoxazinones derivatives from commercially available 2-iodoanilines and terminal acetylenes. (C) 2014 Elsevier Ltd. All rights reserved.

We developed an efficient method for the transformation of indoles by utilizing a copper catalyst and molecular oxygen as the oxidant. The transformation involves a tandem oxidative process of 2-arylindoles. Our reaction afforded a variety of N-benzoyl anthranilic acids and benzoxazinones. Our investigation revealed that the choice of solvent and additives is critical in these reactions. (C) 2014 Elsevier Ltd. All rights reserved.

Ten new 3,4-seco betulinic acid (BA) derivatives were designed and synthesized. Among them, compounds 7-15 exhibited enhanced chemopreventive ability in an in vitro short-term 12-O-tetradecanoylphorbol-13-acetate (TPA) induced Epstein-Barr virus early antigen (EBV-EA) activation assay in Raji cells. Specifically, analogs with a free C-28 carboxylic acid, including 7, 8, 11, and 13, inhibited EBV-EA activation significantly. The most potent compound 8 displayed 100% inhibition at 1×10(3) mol ratio/TPA and 73.4%, 35.9%, and 8.4% inhibition at 5×10(2), 1×10(2), and 1×10 mol ratio/TPA, respectively, comparable with curcumin at high concentration and better than curcumin at low concentration. The potent chemopreventive activity of novel seco A-ring BAs (8 and 11) was further confirmed in an in vivo mouse skin carcinogenesis assay.

In continuation of our studies with chemoprevention potential of plant-derived naphthoquinone derivatives, leaf powder of the medicinal plant Lawsonia inermis L, commonly known as 'henna', was evaluated by its inhibition of the Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Lawsone (2-hydroxy- 1,4-naphthoquinone), the reddish orange pigment artifact formed during the extraction or preparation of the dye from henna leaves and believed to be the active component, was also assessed in this in vitro assay. Both showed a profound inhibition (>88%) of EBV-EA activation. In the in vivo two-stage mouse skin carcinogenesis study using UV-B radiation for initiation and TPA for tumor promotion, oral feeding of henna (0.0025%) in drinking water ad libitum decreased tumor incidence by 66% and multiplicity by 40% when compared to the positive control at 10 weeks of treatment. Similarly, in the above mouse model, orally fed lawsone (0.0025%) decreased tumor incidence by 72% and multiplicity by 50%. The tumor inhibitory trend continued throughout the 20-week test period. Similar antitumor activities were observed when henna (0.5 mg/ml) was applied topically on the back skin in the UV-B initiated, TPA promoted and peroxynitrite initiated, TPA promoted mouse skin carcinogenesis models. Topically applied lawsone (0.015 mg/ml) also exhibited similar protection against tumor formation in the 7,12-dimtehylbenz(a)anthracene induced and TPA promoted skin cancer in mice. Also, there was a delay of 1 to 2 weeks in tumor appearance in both henna and lawsone treated groups compared to control in all three test models. This study ascertains the skin cancer chemopreventive activity of henna leaf powder and lawsone when administered by either oral (through drinking water) or topical (by application on the back skin) routes. Further, it emphasizes the need for the evaluation of these henna-derived green chemopreventive candidates in combination with currently used sunscreen agents for complementary anticancer potential against UV-induced skin carcinogenesis.

Although a wide variety of cytotoxic plant extracts and phytochemicals are known to act synergistically with anticancer drug doxorubicin (D), their clinical application is hindered by safety concerns of such combination therapy. Our earlier studies showed that red beetroot (Beta vulgaris L.) extract (B), approved by Food and Drug Administration and European Union as red food color E162, reduced multi-organ tumor formations in various animal models when administered in drinking water. This led us to postulate that a long-term daily exposure to low doses of B through diet might be safe and sufficient to produce cancer chemopreventive effect in humans. Further, our recent comparative cytotoxic investigation with B and D in several human cancer cell lines indicated their potential for synergistic activity. Since B is considered safe for human use with no known toxicity, we conducted the present study to evaluate its synergistic antiproliferative activity with D against pancreatic (PaCa), breast (MCF-7) and prostate (PC-3) tumor cells of human origin. Different concentrations of B and D (0.29-290 μg/ml) and in various combinations (B:D ratio = 1:0, 1:1, 5:1, 1:5 and 0:1) were tested for cytotoxic effects against the three cancer cells. The viability of cells was assessed after 72 h incubation with various combinations of B and D using the trypan-blue staining method. The cytotoxic data were analyzed by the combination index method of Chou and Talalay to establish synergy between B and D. The results indicated that an overall positive reduction in drug concentration was achieved by D when combined with B in its cytotoxicity profile in the three human cancer cells tested. The synergistic cytotoxicity was best when the B:D ratio of 1:5 was used in PaCa cells at IC50, IC75 and IC90 dose levels and in MCF-7 cells at IC90 dose level. These results warrant further studies on the potential of red beetroot extract-doxorubicin combination in treating human cancers.

BACKGROUND: Sunscreen compounds with added benefit of skin cancer prevention have both public and commercial interests. Our earlier study using the Epstein-Barr virus early antigen in vitro assay reported on skin cancer chemoprevention potential of benzophenone sunscreens. We now report the in vivo antitumor activity of two of the benzophenone sunscreens which tested positively in the in vitro assay, octabenzone (UV-1) and dioxybenzone (UV-2), in the two-stage mouse skin carcinogenesis model using (±)-(E)-4-methyl-2-[-(E)-hydroxyamino]-5-nitro-6-methoxy-3-hexanamide (NOR-1) as inducer and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) as promoter. MATERIALS AND METHODS: Pathogen-free, female hairless mice of HOS:HR-1 strain, 15 animals per control and test groups, were used. Skin tumors were induced by a single dose of NOR-1 (390 nmol in 100 μl of acetone). One week later, TPA (1.7 nmol in 100 μl of acetone) was applied to skin twice weekly for 20 weeks as tumor a promoter. The test compounds UV-I or UV-2 were administered at 0.0025% to mice through drinking water ad libitum, starting one week prior to and stopping one week after tumor initiation. All animals were examined weekly for the development of skin papillomas. RESULTS: In both UV-1- and UV-2-treated mice, a two-week delay in tumor appearance, and significant inhibition (p<0.001) of tumor incidence (50% and 60%, respectively) and tumor burden (papilloma inhibition/mouse, 50% and 70%, respectively) were observed when compared to the positive control group. UV-2 (dihydroxy derivative) was a more potent inhibitor of skin tumor than UV-1 (monohydroxy derivative), which followed their antioxidant activity ranking. CONCLUSION: The results affirm the skin cancer chemoprevention potential of orally-ingested benzophenone sunscreens in mice and warrant studies in humans to validate synergistic protection achievable by complementation of oral and topical sunscreen usage.

The cancer chemopreventive potential of sarcophine-diol in a chemical carcinogen initiation-promotion experimental tumor model in mice was evaluated. Sarcophine-diol, when given orally, afforded significant protection in the mouse skin cancer model initiated by the topical administration of (+/-)-(E)-4-methyl-2-[ (E)-hydroxyamino]-5-nitro-6-methoxy-3-hexanamide (NOR-1) and promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA). These findings, along with our initial reports, suggest that sarcophine-diol is an effective cancer chemopreventive agent, even when administered orally and at a very low dose and thus indicating possible potential human applications in the control of malignancy.

A concise method for the synthesis of heterocycle-fused naphthoquinones such as naphtho[2,3-b]-furan-4,9-dione, 1H-benz[f]indole-4,9-dione, and naphtho[2,3-b]thiophene-4,9-dione was developed. This method employed Sonogashira coupling and tandem addition-elimination/intramolecular cyclization, and it enabled the preparation of versatile heterocycle-fused naphthoquinones from one substrate.

CONTEXT: Dimethyl dicarboxylate biphenyl (DDB) is a clinically used hepatoprotectant and has also been found to have chemopreventive activity. MATERIALS AND METHODS: Sixteen novel analogs (5-20) were designed, synthesized, and evaluated for their cancer preventive activity. The 2,2'-bismethyl ester (5-18) and ether (19, 20) DDB analogs were synthesized by insertion of various linear alkyl, short fatty acid, polar, and aromatic groups. All synthesized analogs were evaluated in an in vitro short-term 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced Epstein Barr virus early antigen (EBA-EA) activation assay. Three of the most potent compounds were also tested for inhibitory effects on skin tumor promotion in an in vivo two-stage mouse-skin carcinogenesis test using 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and TPA as a promoter. RESULTS: Compound 19 with bisprenyl ethers had the most significant cancer preventive activity (100% inhibition of activation at 1 × 10(3) mol ratio/TPA, 78.4%, 49.7%, and 10.9% inhibition at 5 × 10(2), 1 × 10(2), 1 × 10 mol ratio/TPA, respectively) in vitro. Compound 19 also exhibited a remarkable inhibitory effect on skin tumor promotion in the in vivo two-stage mouse-skin carcinogenesis test. DISCUSSION AND CONCLUSIONS: Thus, DDB analog 19 could be a valuable candidate as a cancer preventive agent or as a lead for the development of new antitumor promoter drugs.

Previous cancer chemoprevention studies from our laboratories and by other investigators have demonstrated that the extract of red beetroot (Beta vulgaris L.), the FDA approved red food color E162, can be effective in suppressing the development of multi-organ tumors in experimental animals. To further explore this finding, we have compared the cytotoxic effect of the red beetroot extract with anticancer drug, doxorubicin (adriamycin) in the androgen-independent human prostate cancer cells (PC-3) and in the well-established estrogen receptor-positive human breast cancer cells (MCF-7). This red colored anticancer antibiotic was selected for comparative cytotoxic study because its chemical structure with a planar configuration of an aromatic chromophore attached to a sugar molecule is remarkably similar to that of betanin, the beetroot extract constituent primarily responsible for its red color. Both doxorubicin and the beetroot extract exhibited a dose-dependent cytotoxic effect in the two cancer cell lines tested. Although the cytotoxicity of the beetroot extract was significantly lower when compared to doxorubicin, it continued to decrease the growth rate of the PC-3 cells (3.7% in 3 days vs. 12.5% in 7 days) when tested at the concentration of 29 µg/ml. In contrast, doxorubicin, at the same concentration level, completely inhibited the growth of the PC-3 cells in three days. Similarly, comparative studies in the normal human skin FC and liver HC cell lines showed that the beetroot extract had significantly lower cytotoxic effect than doxorubicin (8.6% vs. 100%, respectively, at 29 µg/ml concentration of each, three-day test period). The results suggest that betanin, the major betacyanin constituent, may play an important role in the cytotoxicity exhibited by the red beetroot extract. Further studies are needed to evaluate the chemopreventive potentials of the beetroot extract when used alone or in combination with doxorubicin to mitigate the toxic side-effects of the latter.

In this paper, a concise one-pot method for the construction of benzo[f]indole-4,9-dione motifs is described. These transformations proceed via a sequential palladium- and copper-catalyzed coupling reaction of 1,4-naphthoquinones with terminal acetylenes, followed by a copper-catalyzed intramolecular cyclization reaction of the resulting coupling product.

A convenient chemoenzymatic synthesis of a new class of non-natural sulfo-glycolipids - 2-O-(beta-D-sulfoquinovosyl)-monoacylglycerols (2-O-beta-D-SQMG) - derived from 2-O-(beta-D-glucopyranosyl)glycerol and carrying acyl chains of various lengths at the 1-position of the sn-glycerol moiety, was performed with the aid of a key step involving regioselective lipase-catalyzed acylation of 2-O-(6-deoxy-6-tosyl-beta-D-glucopyranosyl)-sn-glycerol (4) at its 1-position, reported here for the first time. Elaboration of the sugar moiety

A series of naphthoquinones based on the naphtho[2,3-b]furan-4,9-dione skeleton such as (-)-5-hydroxy-2-(1'-hydoxyethyl)naphtho[2,3-b]furan-4,9-dione (1) and its positional isomer, (-)-8-hydroxy-2-(1'-hydoxyethyl)naphtho[2,3-b]furan-4,9-dione (2), which are secondary metabolites found in the inner bark of Tabebuia avellanedae, were stereoselectively synthesized and their biological activities were evaluated in conjunction with those of their corresponding enantiomers. Compound 1 exhibited potent antiproliferative effect against several human tumor cell lines, but its effect against some human normal cell lines was much lower than that of mitomycin. On the other hand, its enantiomer (R)-1 was less active toward the above tumor cell lines than 1. The antiproliferative effect of 2 against all tumor cell lines was significantly reduced. These results indicated the presence of the phenolic hydroxy group at C-5 is of great important for increasing antiproliferative effect. In addition, 1 also showed higher cancer chemopreventive activity than 2, while there were no significant differences between 1 and 2 in antimicrobial activity. Both compounds displayed modest antifungal and antibacterial activity (gram-positive bacteria), whereas they were inactive against gram-negative bacteria.

New sulfoquinovosyldiacylglycerols derived from 2-O-beta-d-glucopyranosyl-sn-glycerol, carrying acyl chains of various length on the glycerol moiety, were prepared through a convenient synthetic procedure in which a sulfonate is introduced at the C-6 position of glucose by oxidation of a thioacetate in the presence of the unprotected secondary hydroxyl groups, and tested for their anti-tumor-promoting activity using a short-term in vitro assay for Epstein-Barr virus early antigen (EBV-EA) activation. Our study has allowed to ascertain the role of the 6'-sulfonate group and the need of a free hydroxyl group on the glycerol moiety in inhibiting the EBV activation promoted by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA).

The nano-sizedbeta-zeolites were synthesized first time by dry gel conversion (DGC) method without the use of sodium hydroxide. Resultant beta-zeolites had particle size of 35-80 nm with SiO2/Al2O3 ratios in the range of 25-400. They had BET surface areas in the range of 290-750 m2 x g(-1) and the external surface area of 41-283 m2 x g(-1): cumulative surface areas were in the range of 420-1050 m2 x g(-1). Beta-Zeolites modified with alkaline earth and rare earth metal species, such as Ca, La, and Ce, were synthesized by the introduction of these metal nitrates during the dry-gel preparation. To evaluate catalytic properties of these zeolites, they were applied for the isomerization/cracking of hexane and the phenol octylation. The influence of Ca, La, and Ce oxides on the catalytic properties was also examined.

A lithium ester enolate was activated by both a chiral etheral ligand and a lithium amide to form a ternary complex reagent that reacted with enoates giving the corresponding Michael addition products in reasonably high enantioselectivity of up to 97% ee. (c) 2008 Elsevier Ltd. All rights reserved.

An ethanol extract of Helichrysum maracandicum showed antiproliferative activity against cultured cells of SENCAR mouse in an in vitro assay, and activity-guided fractionation of the extract resulted in the isolation of isosalipurposide as an active substance. Naringenin chalcone, the aglycone of isosalipurposide, also showed strong antiproliferative activity. An in vivo assay of two-stage carcinogenesis on mouse skin revealed that epidermal application of isosalipurposide resulted in delayed formation of papillomas. Western blot analysis showed that the expression of p38 mitogen-activated protein kinase was suppressed by the administration of naringenin chalcone or isosalipurposide, which might be related to the anticarcinogenic activity.

Stereoselective synthesis of 1, one of biologically active naphthoquinones from a Brazilian traditional medicine Tabebuia avellanedae, was achieved by utilizing Noyori reduction as a key step. Compound 1 displayed potent cytotoxicity against several human tumor cell lines, whereas it showed lower cytotoxicity against some human normal cell lines compared with that of mitomycin. On the other hand, its enantiomer was less active toward the tumor cell lines than 1.

Rhinacanthus nasutus (L.) Kurz (Acanthaceae) is a shrub widely distributed in South China and India. In this study, the antiproliferative activity of the ethanol extract of root and aqueous extract of leaves of R. nasutus, and the supposed active moiety rhinacanthin C was assessed in vitro using the human cervical carcinoma cell line HeLa, its MDR1-overexpressing subline Hvr100-6, human prostate carcinoma PC-3 cells and human bladder carcinoma T24 cells. Rhinacanthin C was chemically synthesized and its content in the R. nasutus extracts was determined by HPLC with a photodiode array detector. The antiproliferative activity of the R. nasutus extracts was also assessed in vivo using sarcoma 180-bearing mice. It was suggested that 1) the in vitro antiproliferative activity of rhinacanthin C was comparable with or slightly weaker than that of 5-FU, 2) rhinacanthin C showed antiproliferative activity for MDR1-overexpressing Hvr100-6 cells, similarly to parent HeLa cells, 3) the in vitro antiproliferative activity of the ethanol extract of root R. nasutus was due to rhinacanthin C, whereas that of the aqueous extract of leaves of R. nasutus was due to constituents other than rhinacanthin C, and 4) both of the R. nasutus extracts showed in vivo antiproliferative activity after oral administration once daily for 14 d.

Bioassay-guided separation of extracts from the culture broth and mycelium of the fungus Trichothecium roseum, aiming at the discovery for cancer preventive agents, resulted in the isolation of three new trichothecene sesquiterpenes, trichothecinols A-C (1-3) together with three known analogues, trichothecin (4), trichodermol (5) and trichothecolone (6). Compounds 1-6 exhibited remarkably potent inhibition against Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Further compound 1 strongly inhibited TPA-induced tumor promotion on mouse skin initiated with 7,12-dimethylbenz[a]anthracene (DMBA) in two-stage carcinogenesis tests. These results suggest that compound 1 might be a valuable lead for further evaluation as a cancer preventive agent. In addition to their cancer preventive activity, compound 2 was found to show modest antifungal activity against Crypotcoccus albidus and Saccharomyces cerevisiae.

In our continuing search to discover bioactive compounds from medicinal plants, a methanolic extract of Verbascum songaricum collected in Kazakhstan was investigated. Activity-guided fractionation of the extract prepared from the plant, using a sensitive assay to monitor DNA polymerase reactions, resulted in the isolation of two known phenylethanoid glycosides, poliumoside (1) and verbascoside (2). Glycoside 1 was shown to inhibit mammalian DNApolymerases α,β,δ and ε with IC_<50> values of 42, 23, 35 and 9 μM, respectively, while those of glycoside 2 were all more than 100 μM.

The optically pure trichothecene cis-AB ring moiety (1) was synthesized starting from an optically pure butenolide (7) through the ring closing olefin metathesis for the formation of the A-ring and a Lewis acid mediated cyclization to the cis-fused tetrahedrochromane skeleton that had been converted to natural trichothecene, (+)-calonectrin.

Bioassay-guided fractionation of an extract prepared from Taraxacum officinale, aiming at the search for mammalian DNA polymerase inhibitors, led to the isolation of D-mandelonitrile-β-D-glucoside (prunasin) as the inhibitory principle.

Ointments of the skin depigmentation agent hydroquinone (HQ) have been prepared by extemporaneous nonsterile compounding in our hospital. The HQ ointments were highly effective in the treatment of various types of skin pigmentations; however, various problems have emerged including chromatic aberration of the ointments, a relatively large variability of efficacy, and mild side effects. Chromatic aberration is expected to induce non-compliance, and this may be the reason for the relatively large variability in efficacy. In this paper, the effects of various storage conditions on the chromatic aberration and HQ content of HQ ointments were evaluated, and it was suggested that the chromatic aberration was accelerated by exposure to high temperature, air and light, although these had no effect on the HQ content. In addition, various types of HQ ointments were prepared to find a formulation to minimize chromatic aberration, and it was found that the concentrations of antioxidants, Na(2)SO(3) and L(+)-ascorbic acid (AsA), seemed to be too high, and that the protective effect of AsA on chromatic aberration was mainly due to its acidifying effect.

Three new lanostane-type triterpenoids (1-3) were isolated from the bark of Abies sachalinensis along with a known compound (4). The structures of 1-4 were characterized by spectroscopic methods including NMR and MS. Compound 4 and some derivatives were tested for inhibitory effects on in vitro DNA topoisomerases I and II and found to be selective catalytic inhibitors of topoisomerase II activity with IC(50) values in the range 43-76 microM.

Ointments of the skin depigmentation agent hydroquinone (HQ) have been prepared by extemporaneous nonsterile compounding in Japan by imitating skin lightening creams commercially available in the U.S.A. and European Union. In our hospital, HQ ointments consisting of 5 or 10% HQ, 1.6% L(+)-ascorbic acid (AsA), 0.5% (w/w) Na2SO3, 10% (v/w) glycerin and hydrophilic ointment have been prepared. However, various problems have been observed including chromatic aberration of HQ ointments, relatively large variability of efficacy, and undesirable side effects although they were mild. Herein, the pharmaceutical and clinical properties of the HQ ointments were evaluated. HQ ointments were highly effective for treatment of various types of skin pigmentation. Chromatic aberration occurred during 3 months of storage, but this could be suppressed by storage at 4 degrees C. Chromatic aberration was independent of prescribed HQ content, and was not explained by alterations of HQ or p-benzoquinone (p-BQ) contents. Unexpectedly, removal of both antioxidants resulted in suppression of chromatic aberration, but an increase in p-BQ content. Acidification by removal of Na2SO3 only was further effective for the suppression of chromatic aberration, but with a decrease of p-BQ content except in the initial period. Chromatic aberration was due to water soluble material and insoluble material both formed by co-existence of HQ and p-BQ at a molecular ratio of 5:3 to 1:1. 1H-NMR analysis elucidated that the water soluble material was not HQ or p-BQ, and the insoluble material was a complex of HQ and p-BQ with non-covalent binding.

DNA topoisomerases (Topos) are enzymes that play a crucial role in DNA metabolism events such as replication, transcription, recombination, and chromosome segregation at mitosis. Thus, Topo inhibitors could be expected to have antitumor effects. Naturally occurring lupane- and oleanane-type triterpenoids isolated from the bark of Phyllanthus flexuosus were screened for human Topos I and II inhibitory activities. Olean-12-en-3beta,15alpha-diol (1), olean-12-en-3beta,15alpha,24-triol (3), lupeol (4), and betulin (6) were found to be selective catalytic inhibitors of human Topo 11 activity with IC50 values in the range of 10-39 muM.

Nine lanostane and serratane-type triterpenes including two unknown compounds were isolated from the stem bark of Pinus luchuensis. These new compounds were characterized as 3-oxolanost-9(11)-ene-24S,25-diol (1) and 29-acetoxy-3 beta -methoxyserrat-14-en-21 alpha -ol (2) on the basis of spectroscopic evidence. Some of these triterpenes were tested for the inhibitory effect on DNA topoisomerase 11 activity. Compound 1 showed a slightly less potent inhibitory activity with an IC50 value of 186 muM.

The molecular action of lithocholic acid (LCA), a selective inhibitor of mammalian DNA polymerase β (pol β), was investigated. We found that LCA could also strongly inhibit the activity of human DNA topoisomerase II (topo II). No other DNA metabolic enzymes tested were affected by LCA. Therefore, LCA should be classified as an inhibitor of both pol β and topo II. Here, we report the molecular interaction of LCA with pol β and topo II. By three-dimensional structural model analysis and by comparison with the spatial positioning of specific amino acids binding to LCA on pol β(Lys60, Leu77, and Thr79), we obtained supplementary information that allowed us to build a structural model of topo II. Modeling analysis revealed that the LCA-interaction interface in both enzymes has a pocket comprised of three amino acids in common, which binds to the LCA molecule. In topo II, the three amino acid residues were Lys720, Leu760, and Thr791. These results suggested that the LCA binding domains of pol β and topo II are three-dimensionally very similar.

As described previously, we found that new triterpenoid compounds, designated fomitellic acids A and B, which selectively inhibit the activities of mammalian DNA polymerases alpha and beta [Mizushina, Tanaka, Kitamura, Tamai, Ikeda, Takemura, Sugawara, Arai, Matsukage, Yoshida and Sakaguchi (1998) Biochem. J. 330, 1325-1332; Tanaka, Kitamura, Mizushina, Sugawara and Sakaguchi (1998) J. Nat. Prod. 61, 193-197] and that a known triterpenoid, ursolic acid, is an inhibitor of human DNA topoisomerases I and II (A. Iida, Y. Mizushina and K. Sakaguchi, unpublished work). Here we report that all of these triterpenoids are potent inhibitors of calf DNA polymerase cc, rat DNA polymerase beta and human DNA topoisomerases I and II, and show moderate inhibitory effects on plant DNA polymerase II and human immunodeficiency virus reverse transcriptase. However, these compounds did not influence the activities of prokaryotic DNA polymerases such as Escherichia coli DNA polymerase I or other DNA metabolic enzymes such as human telomerase, T7 RNA polymerase and bovine deoxyribonuclease I. These triterpenoids were not only mammalian DNA polymerase inhibitors but also inhibitors of DNA topoisomerases I and II even though the enzymic characteristics of DNA polymerases and DNA topoisomerases, including their modes of action, amino acid sequences and three-dimensional structures, differed markedly. These triterpenoids did not bind to DNA, suggesting that they act directly on these enzymes. Because the three-dimensional structures of fomitellic acids were shown by computer simulation to be very similar to that of ursolic acid, the DNA-binding sites of both enzymes, which compete for the inhibitors, might be very similar. Fomitellic acid A and ursolic acid prevented the growth of NUGC cancer cells, with LD50 values of 38 and 30 mu M respectively.

The pendant E-ring moiety of the podophyllotoxin aza-analogue 1 that is a potent inhibitor of microtubule assembly was modified in order to acquire inhibitor? activity of DNA topoisomerase II. The monophenolic analogue 2 did not exhibit human topoisomerase II inhibition, while the ortho-quinone 3 that was obtained by oxidation of 2 inhibited its catalytic activity (decatenation) in a dose-dependent manner and stimulated double strand DNA breaks in supercoiled circular plasmid DNA, resulting in the production of linear DNA. These results showed that the topoisomerase II inhibition of the ortho-quinone 3 is due to stabilization of the topoisomerase II-DNA covalent binary complex. On the other hand, the ortho-quinone 3 did not inhibit the relaxation process of supercoiled DNA by topoisomerase 1 at concentrations up to 100 mu M, nor was intercalation observed in unwinding measurements of 3. Therefore, the ortho-quinone 3 was shown to be a novel nonintercalative topoisomerase II specific inhibitor that stabilizes the cleavable complex. The present results suggest that the 4'-free hydroxyl group on the E-ring and the sugar moiety on the C-ring are not a prerequisite for topoisomerase II inhibition by podophyllotoxin derivatives.

A new ent-kaurane diterpenoid dimer, fritillebinide C (1) together with one known diterpenoid dimer fritillebinide B (2) were isolated from the bulbs of Fritillaria ebiensis G.D. Yu et G.Q. Ji. Compound 1 has been determined to be eni-3 beta-acetoxy-kauran-16 beta, 17-acetal ent-16 beta-kauran-17(S)-aldehyde (1) by means of spectral analysis and chemical evidence.

A novel ent-kaurane diterpenoid dimer, fritillebinide B (1) together with one known diterpenoid dimer fritillebinide A (2) were isolated from the bulbs of Fritillaria ebeiensis var. purpurea G.D. Yu ct P. Li. Compound 1 has been established to be ent-3 beta-acetoxy-kauran-16 beta,17-acetal ent-16 beta-kauran-17(R)-aldehyde (1) by means of spectral analysis and chemical evidence.

Three new 10-residue lipopeptaibols, trichopolyns III-V have been isolated from the fungus Trichoderma polysporum together with the known trichopolyns I and II. Structure determination has been achieved on the basis of spectroscopic and chemical evidence. These peptides have been shown to suppress the proliferation of lymphocytes in mouse allogeneic mixed lymphocyte reaction. (C) 1999 Elsevier Science Ltd. All rights reserved.

Tridentate chiral amines 7-13 mediated the asymmetric condensation reaction of lithium ester enolate 2 with benzaldehyde p-anisidine imine 3 giving the corresponding β-lactam 4 in up to 75% ee. It became apparent that coexistence of 2 and chiral lithium amides derived from 7-13 is an important factor for the enhancement of the reactivity and enantioselectivity of 2.

We examined membrane fluidity of bovine adrenal chromaffin cells and chromaffin granules using cationic trimethylammonium derivative of diphenylhexatriene (TMA-DPH) as a fluorescence probe. After adding TMA-DPH to the suspension of chromaffin cells and that of granules, it first bound to the outer layer of the plasma membrane of the dells and that of the granule membrane, then gradually penetrated the inner layer of each membrane and distributed to both leaflets of the respective membranes. Accompanying increases in the ratio of incorporated probe on the cytoplasmic side of the chromaffin cell membrane, its fluorescence anisotropy gradually decreased. However, in chromaffin granules,the fluorescence anisotropy gradually increased with increases in the ratio of incorporated probe. These findings suggest that the inner layer of the plasma membrane and outer layer of the granular membrane are more fluid than the corresponding side of each membrane, which is suitable for the fusion between both membranes. We also examined the effect of trichosporin-B VIa, a fungal ion channel forming alpha-aminoisobutyric acid-containing peptide, on the fluidity of chromaffin cells using TMA-DPH. The peptide decreased the fluorescence anisotropy and increased the fluorescence intensity in the concentration range that induced Ca2+ dependent catecholamine secretion, suggesting that a change in lipid dynamics of the lipid bilayer of the plasma membrane was induced by this peptide. (C) 1998 Elsevier Science B.V. All rights reserved.

Several kinds of naturally occurring fernane-type triterpenoids isolated from a Euphorbia genus were tested on the inhibitory effects of DNA Topoisomerases I (Topo I) and II (Topo II) activities. A-ring cleaved 3,4-seco-8 beta H-ferna-4(23),9(11)-dien-3-oic acid and its 3-hydroxyl derivative were found to be selective inhibitors of Topo II activity without the stabilization of a DNA/Topo II cleavable complex. (C) 1998 Elsevier Science Ltd. All rights reserved.

Bioassay-directed fractionation of an extract of the stem-bark of Catalpa ovata led to the isolation of three new naphthoquinones: 8-methoxydehydroiso-alpha-lapachone (1), 9-methoxy-4-oxo-alpha-lapachone (2), and (4S,4aR,10R,10aR)-4,10-dihydroxy-2,2-dimethyl-2,3,4,4 alpha,10,10 alpha-hexahydrobenzo[g] chromen-5-one (3), which is a 1,4-reductive form of 6. The known compounds 3-hydroxydehydroiso-alpha-lapachone (4), 4,9-dihydroxy-alpha-lapachone (5), 4-hydroxy-alpha-lapachone (6), and 9-methoxy-alpha-lapachone (7), and catalpalactone (8) were also isolated. Their structures were elucidated by spectral methods. These compounds all exhibited significant inhibitory activity against 1 2-O-tetradecanoylphorbol 13-acetate (TPA)-induced Epstein-Barr virus early antigen (EBV-EA) activation in Raji cells.

The external chiral ligand mediated asymmetric reaction of thiazolyllithium with aldimines was examined. The amino ether (5) gave rise to relatively high selectivity in the reaction of phenylimine, and sparteine (6) provided high selectivity in the reaction of imines bearing an alpha-methylene group. The reaction was applied to the synthesis of (S)-Doe, a component of antileukemic marine natural product, dolastatin 10.

The conjugate addition reaction of BHA alkenoates with butyl- and phenyllithiums in toluene at -78 degrees C were catalyzed by a substoichiometric amount of chiral ligands 1 and 2 to give the corresponding 3-substituted alkanoates in good to modest ees.

The chiral ligand mediated enantioselective reaction of methyllithium with furylimines was investigated in the presumed 1,4- and 1,2-addition manners. The imines bearing a phosphinoyl or sulfonyl group on the imine nitrogen atom gave rather poor enantioselectivity. On the other hand, the imines bearing a 2-substituted anisidine moiety were found to be the appropriate substrate, giving the corresponding amines with up to 91% ee.

The conjugate addition reactions of BHA alkenoates with organolithiums in toluene or toluene-hexane at -78 degrees C were mediated by the chiral ligands 1 and 2 to give the corresponding 3-substituted alkanoates in high ees and high yields. The two ligands are complementary each other, 1 is effective for phenyl-and vinyllithiums to give the adducts in 64-93% ee, while 2 is effective for butyl-and ethyllithiums to give the adducts in 91-99% ee. (C) 1997 Elsevier Science Ltd.

The reaction of N,N'-bis(4-methoxyphenyl)ethylenediimine with phenyllithium, with the mediation of a chiral ligand, provided the addition products, (1R,2R)-N,N'-bis(4-methoxyphenyl)-1,2-diphenylethanediamine of 67% ee and the meso-product, in a ratio of 41:59, The net reaction involves sequential double additions of phenyllithium. In the first addition a new chiral center is created, but with rather poor selectivity, and in the second addition kinetic discrimination takes place, giving the chiral double addition product.

Oxidation of a cytotoxic podophyllotoxin aza-analogue 2 into the quinone 3 succeeded in acquiring DNA topoisomerase II inhibitory activity at a concentration of 25 mu M due to stabilization of the cleavable complex. (C) 1997 Elsevier Science Ltd.

Trichocellins (TC) A-II and B-II, 20-residue peptaibols isolated from conidia of the fungus Trichoderma viride, have the same sequence except for the residue at position 18. Both TCs were found to form voltage-dependent ion-channels in bilayer lipid membranes (BLM) and to induce catecholamine secretion from bovine adrenal chromaffin cells through Ca2+ influx. TC-A-II (Gln18, neutral) was more effective than TC-B-II (Glu18, charged) for macroscopic current induction in BLMs and for catecholamine secretion from chromaffin cells, suggesting that Glu18 is unfavorable for the ion-channel formation in BLMs and chromaffin cell membranes. Nevertheless, single-channel recordings indicated that TC-B-II forms larger pores with longer open lifetimes than those of TC-A-II. This indicates that the negatively charged carboxyl group of Glu at position 18 stabilizes larger pores. The effects of the negative charge of Glu18 on the activities were confirmed by the use of a TC-B-II analog containing the methyl ester of Glu18.

Three new trichothecenes, trichothecinols A (1), B (2) and C (3) were isolated from the fungus Trichothecium roseum and unambiguiously characterized on the basis of spectroscopic and chemical evidence. These exhibited potent inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Copyright (C) 1996 Elsevier Science Ltd

An 11-residue peptaibol, trichorovin-XII, isolated from the fungus Trichoderma viride, was found to form voltage-dependent and cation-selective ion channels in planar lipid bilayer membranes. The channels formed were classified into two types; a short-lived channel whose conductance level was not clearly distinguished and a long-lived channel whose conductance levels were 0.51 and 1.24 nS. Trichorovin-XII is the shortest channel-forming peptide of the peptaibol family so far reported. Copyright (C) 1996 Elsevier Science Ltd

Enantioselective opening of oxide ring was achieved by the combination of a chiral ether and phenyllithium in the presence of boron trifluoride to give the corresponding alcohol in 47% ee. Copyright (C) 1996 Published by Elsevier Science Ltd

Trichosporin-B-VIa (TS-B-VIa) has a Pro(14)-kinked helical structure which is considered to be important for the formation of peptaibol-type ion-channels in lipid bilayer membranes. TS-B-VIa and its analog [Aib(14)]TS-B-VIa with Pro --> Aib substitution at position 14, resulting in a straight helical structure, were tested for ion-channel-forming activity in planar lipid bilayer membranes and for ability to induce catecholamine secretion from cultured bovine adrenal chromaffin cells. Voltage-dependent multi-channel conductance, which is characteristic of TS-B-VIa, was also observed for [Aib(14)]TS-B-VIa. In single-channel measurements, current fluctuations induced by [Aib(14)]TS-B-Vla had a shorter life-time and showed fewer substates than those induced by TS-B-VIa. Catecholamine secretion induced by these peptides at low concentrations is completely Ca2+-dependent. At high concentrations, TS-B-VIa-induced secretion was partly independent of external Ca2+, but this was not the case for the analog. The differences of behavior can be explained in terms of the differences of hydrophobicity, amphiphilicity, and magnitude of dipole moment due to the conformational changes around position 14 and the C-terninal domain caused by the Pro --> Aib substitution.

Trichosporin (TS)-B-VIa, a fungal alpha-aminoisobutyric acid (Aib)-containing peptide consisting of 19 amino acid residues and a phenylalaninol, produced both Ca-45(2+) influx into bovine adrenal chromaffin cells and catecholamine secretion from the cells. The secretion induced by TS-B-VIa at lower concentrations (2-5 mu M) was completely dependent on the external Ca2+, while that induced by TS-B-VIa at higher concentrations (10-30 mu M) was partly independent of the Ca2+, The concentration-response curves (2-5 mu M) for the TS-B-VIa-induced Ca2+ influx and secretion correlated well. The TS-B-VIa (at 5 mu M)-induced secretion was not antagonized by diltiazem, a blocker of L-type voltage sensitive Ca2+ channels. The treatment of fura-2-loaded C-6 glioma cells with TS-B-VIa (2-5 mu M) led to an increase in the intracellular free Ca2+ concentration([Ca2+](i)) in a concentration-dependent manner but the stimulatory effects of TS-B-VIa on [Ca2+](i) were only slightly observed in Ca2+-free medium, indicating that TS-B-VIa causes Ca2+ influx from the external medium into the C-6 cells. The TS-B-VIa-induced increase in [Ca2+](i) in the C-6 cells was not antagonized by diltiazem and by SK&F 96365, a novel blocker of receptor-mediated Ca2+ entry. High K+ increased neither [Ca2+](i) in the C-6 cells nor Mn2+ influx into the cells, while TS-B-VIa increased Mn2+ influx. Also in other non-excitable cells, bovine platelets, similar results were obtained. These results strongly suggest that the mechanism of Ca2+ influx by TS-B-VIa at the lower concentrations is distinct from the event of Ca2+ influx through receptor-operated or L-type voltage-sensitive Ca2+ channels in both excitable cells (the chromaffin cells) and non-excitable cells (the C-6 cells and the platelets) and that TS-B-VIa per se may form Ca2+-permeable ion channels in biological membranes. On the other hand, the peptide at the higher concentrations seems to damage cell membranes.

Replacement of the Gln(7) residue, which is well conserved among ion-channel-forming peptaibols, by an Ala in trichosporin-B-VIa (TS-B-VIa = AcUAUAUUQUIUGLUPVUUQQPheol; U = alpha-aminoisobutyric acid, Pheol = phenylalaninol) alters neither the multilevel nature of the TS-B-VIa channel nor its conductance, but abolished the rectification of the membrane current characteristic of the TS-B-VIa channel.

The mass spectral characteristics of selected peptaibols were investigated using electrospray ionization in conjunction with low-energy collision-induced dissociation (CID). Protonated and sodiated molecular species were selected as precursor ions and the resulting CID spectra are discussed with respect to fragmentation characteristics related to the structures of peptaibols. The CID spectra of peptaibols with acetylated N-terminus and with the sub-sequences of Aib-Pro were similar. The CID spectra of [M + 2Na](2+) provided structural information more than those of [M + 2H](2+) and [M + H + Na](2+). The CID of [M + 2Na](2+) can be used to deduce complete sequence information for peptaibols.

Examination of newly synthesized elongated and truncated analogues of the ion-channel-forming peptide, trichosporin-B-Vla (20 residues), for ion-channel-forming activity in lipid bilayer membranes and catecholamine secretion-inducing activity from adrenal chromaffin cells revealed that the natural compound has the optimal molecular length for both activities.

Two new labdane diterpenoids, marrubinones A (1) and B (2), were isolated from aerial parts of Marrubium astracanicum collected in Turkey. Compounds 1 and 2 were mostly characterized by two-dimensional NMR. For determination of the absolute configuration, the modified Mosher's method was applied to the 12-hydroxy forms of 1 and 2. Compounds 1 and 2 are pentacyclic diterpenoids, which differ from each other only in the stereochemistry at C-13, and they were identified as 9 alpha,13S:15,16-diepoxy-12-oxo-14-labden-19,6 beta-olide and 9 alpha,13R:15,16-diepoxy-12-oxo-14-labden-19,6 beta-olide, respectively.

A new diterpenoid, fritillebic acid (1) and four new diterpenoid dimers, fritillebins A (2) and B (3), and fritillebinides A and B, were isolated from a crude drug, bulbs of Fritillaria ebeiensis G. D. YU et G. Q. JI. The structures of compounds 1-3 were determined to be ent-3 beta-acetoxy-16 beta-kauran-17-oic acid (1), ent-16 beta-hydroxykauran-17-yl-ent-3 beta-acetoxg-16 beta-kauran-17-oate (2), and ent-3 beta-acetoxy-16 beta-hydroxykauran-17-yl ent-3 beta-acetoxy-16 beta-kauran-17-oate (3), respectively, on the basis of spectroscopic and chemical evidence.

Trichosporin (TS)-B-VIa, a peptide consisting of 19 amino acid residues and an amino alcohol, causes Ca2+-dependent secretion of catecholamines from bovine adrenal chromaffin cells. The TS-B-VIa-induced secretion was greater under alkaline conditions and at a temperature of 37 degrees C compared with that at 21 degrees or 30 degrees C. It was not observed when the peptide was eliminated from the incubation medium. These results strongly suggest that the stimulatory effect of TS-B-VIa on the secretion is reversible and dependent on the temperature and pH of the incubation medium.

The secondary structures of an ion-channel-forming icosapeptaibol, trichosporin B-VIa, and its Aib(14)-substituted derivative containing no Pro were investigated on the basis of CD and various NMR experiments in methanol, Trichosporin B-VIa has a fully helical structure with a kink stabilized by a 1<--4 hydrogen-bond between the Leu(12) CO and Val(15) NH, The helical structure is composed of 3(10)-helix in the N-terminal first turn and the C-terminal moiety following Leu(12), and a-helix in the middle region, In contrast, the Aib(14) derivative predominantly has a straight a-helical structure except for a 3(10)-helix region in the N-terminal first turn.

Trichorovins (TV)-I-XIV are new antibiotic peptides obtained from conidia of the fungus Trichoderma viride. The peptide mixture of TVs was repeatedly fractionated by preparative HPLC until individual TVs showed a single peak on their analytical HPLC chromatograms. Nevertheless, FAB-MS or NMR indicated that each of TVs-I-XIV was composed of at least two components. We attempted to elucidate their structures within the fractions by electrospray ionization (ESI)-MS, FAB-MS, FAB-MS/MS and NMR. TVs generally have molecular weights of approximately 110-1200 Da, and are characterized by an acetylated N-terminus, the presence of an aminoalcohol, e.g. leucinol, isoleucinol or valinol, at the C-terminus, and eleven residues including three alpha-aminoisobutyric acids in the molecule. Thus, it was determined that TVs belong to the class of peptaibols.

A series of peptaibols, trichosporin (TS) -Bs, induced voltage-activated conductance in lipid bilayer membranes and catecholamine secretion from bovine adrenal chromaffin cells. The order of activities is the same as that of the lipophilicity rank of TS-Bs derived from the reversed-phase HPLC capacity factors.

Trichocellins-A and -B are catecholamine-releasing peptides isolated from the fungus Trichoderma viride. Their amino acid sequences were determined by mars spectrometry and NMR spectroscopy. Trichocellins-A-l similar to VIII and -B-l and -II are each composed of 20 residues, having an N-terminal acetyl group and phenylalaninol as the C-terminal residue.

New peptaibols, trichokindins I-VII, have been isolated from the fungus Trichoderma harzianum. Their structures were characterized by spectrometric methods. Trichokindins, which are 18-residue peptides containing one to three isovaline residues, were found to induce Ca2+-dependent catecholamine secretion from bovine adrenal medullary chromaffin cells.

The effect of the trichosporin-Bs, peptide antibiotics, on the respiration of mitochondria was investigated. Trichosporin-Bs stimulated the respiratory rate of state 4 rat liver mitochondria in a dose-dependent manner. The maximum respiratory rate obtained by trichosporin-Bs was essentially the same as for 2,4-dinitrophenol and SF6847. Trichosporin-B-VIb released oligomycin-inhibited respiration of mitochondria. This means that trichosporin-Bs are uncouplers of oxidative phosphorylation. The stimulatory effect of trichosporin-B-VIb, a component of trichosporin-Bs, on mitochondrial respiration was increased by inorganic phosphate but not by other permeant anions studied. These results suggest that the stimulation of mitochondrial respiration by trichosporin-B-VIb is mediated by the same mechanism as is operating in the case of hypelcins and alamethicins. Furthermore, the relative potencies of trichosporin-Bs on the mitochondrial respiration and their relative hydrophobicities were examined. A clear relationship was observed between the uncoupling potencies of trichosporin-Bs and their relative hydrophobicities.

Three new groups of peptaibols, trichodecenins, trichorovins and trichocellins, have been isolated from conidia of the fungus, Trichoderma viride. The structures of trichodecenins-I and -II were established by positive-ion fast-atom bombardment, collision-induced dissociation mass spectrometry and two-dimensional NMR spectroscopy. Trichodecenins-I and -II have a (Z)-4-decenoyl group, six amino acid residues and a leucinol moiety in the molecules. Trichodecenin-II was synthesized by the solution-phase method.

A membrane-modifying peptide antibiotic having uncoupling activity on rat liver mitochondria, hypelcin A-III, has been synthesized by assembling five peptide fragments via the N,N'-dicyclohexylcarbodiimide method. The synthesized hypelcin A-III was identical with the natural product.

Hypelcin A is a mixture of antibiotic peptides produced by Hypocrea peltata. Hypelcin A-I, A-II, A-III, A-IV, A-V, A-VI, A-VII, A-VIII and A-IX are components of this mixture purified by reversed-phase high-performance liquid chromatography. The amino acid sequences of these peptides were determined by fast-atom bombardment mass spectrometry and fast-atom bombardment mass spectrometry/mass spectrometry with the help of NMR spectroscopy. The relative molecular masses of these peptides were all approximately 2000 and their structures were very similar.

The secondary structure of the peptaibol trichosporin B-V in methanol was investigated in detail by 600 MHz nuclear magnetic resonance spectroscopy. Its fundamental secondary structure was characterized as a helix by the circular dichroism spectrum. Inter-residual nuclear Overhauser effect patterns, 3J(NH-CalphaH) amide coupling constants, hydrogen-deuterium (H-D) exchange rates of the amide protons, and inspection of molecular models showed that the secondary structure of this peptide consists of two major alpha-helical structures because of a bent structure around a Pro residue, and that the first three amino acid residues of the N-terminal alpha-helix are arranged predominantly in a 3(10)-helical fashion.

Sequence-specific H-1- and C-13-nuclear magnetic resonance assignments of an antibiotic peptide, trichosporin B-V isolated from fungus Trichoderma polysporum, were achieved in methanol by using two-dimensional NMR techniques. The H-1 NMR spectra recorded at various concentrations (1-60 mmol dm-3) suggested that the peptide behaves predominantly as a monomer in methanol and that its conformation is not affected by its concentration. Furthermore, complete or partial H-1-resonance assignments of trichosporin B-Ia, -IIIa, -IVd and -VIb were carried out similarly. Comparison of the H-1 NMR parameters for the amide groups of trichosporin Bs suggested that their backbone conformations are very similar to each other. It is proposed that the significant differences in the biological activity of trichosporin Bs result from the lipophilicity of the individual molecules.

From the minor-components mixture of Trichosporin Bs (TS-Bs) produced by Trichoderma polysporum, ten components could be elucidated by means of linked-scan and continuous-flow fast-atom bombardment. The combination of their complementary results made the rapid structure elucidation of the minor components possible without isolation and purification. The identified compounds are antibiotic eicosapeptides and have very similar structures with relative molecular masses of approximately 2000. Four new peptide groups were found. In their structures, the first group has Gly at the amino acid position 3 in contrast to the known TS-Bs, the second one has Vxx (valine or isovaline) at position 12, and the third and the fourth ones have Ala at position 13 or 17, respectively.

Hypelcin A is a mixture of antibiotic peptides produced by Hypocrea peltata. Hypelcin A-I, A-II, A-III, A-IV, A-V, A-VI, A-VII, A-VIII and A-IX are components of this mixture purified by reversed-phase high-performance liquid chromatography. The amino acid sequences of these peptides were determined by fast-atom bombardment mass spectrometry and fast-atom bombardment mass spectrometry/mass spectrometry with the help of NMR spectroscopy. The relative molecular masses of these peptides were all ∼2000 and their structures were very similar.

From the minor-components mixture of Trichosporin Bs (TS-Bs) produced by Trichoderma polysporum, ten components could be elucidated by means of linked-scan and continuous-flow fast-atom bombardment. The combination of their complementary results made the rapid structure elucidation of the minor components possible without isolation and purification. The identified compounds are antibiotic eicosapeptides and have very similar structures with relative molecular masses of ∼2000. Four new peptide groups were found. In their structures, the first group has Gly at the amino acid position 3 in contrast to the known TS-Bs, the second one has Vxx (valine or isovaline) at position 12, and the third and the fourth ones have Ala at position 13 or 17, respectively.

A new chemotype was found in Scoparia dulcis from Taiwan, China and Thailand based on the diterpene composition. The new chemotype was characterized by the presence of scopadulciol and scopadiol. The structure of a new diterpene was determined to be 6-benzoyl-labda-8(17),13-diene-15,18-diol by spectroscopic methods.

Large quantities of a catalytically active protein have been produced in a cell free system. More than 10(9) copies of protein were produced from each DNA plasmid containing DNA(fol), the bacterial gene encoding dihydrofolate reductase (DHFR). The strategy employed, denoted gene amplification with transcription/translation (GATT), involves sequential coupling of (i) DNA amplification by the polymerase chain reaction (PCR) and (ii) in vitro RNA transcription by T7 RNA polymerase, followed by (iii) translation of the run-off transcripts in a rabbit reticulocyte system. The protein product had the expected size (18 kDa) and catalyzed the NADPH-dependent reduction of 7,8-dihydrofolic acid to 5,6,7,8-tetrahydrofolic acid as efficiently as authentic DHFR. Potential applications of the strategy include large scale production of enzymes containing synthetic amino acids and facilitation of the characterization of the function of genes encountered in genomic mapping studies.

We examined the effect of trichosporin-B-III, an alpha-aminoisobutyric acid-containing antibiotic peptide consisting of 19 amino acid residues and a phenylalaninol, on catecholamine secretion from cultured bovine adrenal chromaffin cells. Incubation of the cells with trichosporin-B-III (3-20-mu-M) caused an increase in the secretion of catecholamines. The secretion induced by trichosporin-B-III at low concentrations (3 and 5-mu-M) was completely dependent on external Ca2+, whereas that induced by higher concentrations (10 and 20-mu-M) was partly independent of Ca2+. Trichosporin-B-III at low concentration (5-mu-M) did not increase the release of lactate dehydrogenase, a marker enzyme of cytoplasm, from the cells. In contrast, the peptide at higher concentration (10-mu-M) increased the release of the enzyme. Trichosporin-B-III also caused both Ca-45(2+) influx into the cells and an increase in the intracellular free Ca2+ concentration. The increases in catecholamine secretion and Ca-45(2+) influx behaved similarly in relation to trichosporin-B-III concentration (3-10-mu-M). The time courses of the increases in secretion, Ca-45(2+) influx, and intracellular free Ca2+ concentration induced by trichosporin-B-III were also quite similar. Trichosporin-B-III-induced (at 5-mu-M) secretion was not affected by the elimination of Na+ from the incubation medium or by the addition of tetrodotoxin, a blocker of highly selective voltage-dependent Na+ channels, or hexamethonium, a blocker of nicotinic acetylcholine receptors. On the other hand, both diltiazem (2-200-mu-M) and nicardipine (1-200-mu-M), blockers of voltage-dependent Ca2+ channels, inhibited the secretion induced by trichosporin-B-III (5-mu-M) in a concentration-dependent manner. Trichosporin-B-III-induced (at 5-mu-M) secretion also was suppressed by the addition of Mn2+ (5-mu-M) to the medium. The diltiazem (20-mu-M) inhibition of trichosporin-B-III-induced (at 5-mu-M) secretion was reversed by increasing the external Ca2+ concentration. These results indicate that trichosporin-B-III causes the secretion of catecholamines from bovine adrenal chromaffin cells by two mechanisms, Ca2+ dependent and Ca2+ independent (only at high concentrations of trichosporin-B-III). Furthermore, these results strongly suggest that trichosporin-B-III, in Ca2+-dependent secretion, activates endogenous voltage-dependent Ca2+ channels, or itself forms the channels in the membranes, and induces Ca2+ influx into the cells.

Trichosporin-B-IIIb, -IIIc, -IVb, -IVc, -IVd, -VIa and -VIb are components of an antibiotic peptide mixture produced by Trichoderma polysporum. Each component was purified by reversed-phase high-performance liquid chromatography (HPLC). The amino acid sequences of these peptides, which have an unstable peptide bond, Aib-Pro, were elucidated by fast-atom bombardment mass spectrometry (FAB MS) and fast-atom bombardment mass spectrometry/mass spectrometry (FAB MS/MS) with the help of NMR spectroscopy. The molecular weights of these peptides were all ca. 2000 and the structures were very similar.

Trichoderma spp. are active as mycoparasites and, therefore, can serve as potential biocontrol agents. We found in dual culture studies on agar medium that Trichoderma harzianum killed most wood decay fungi examined. New antibiotic peptides, trichokindin (TK)-I〜X, which belong to a class of peptaibols like alamethicin and suzukacillin, were isolated from the spores of T. harzianum. The primary structure of TK-X(10) was clearly established by FABMS and FABMS/MS. The N-terminal of (10) is protected by an acetyl group and the C-terminal is linked with an isoleucinol (Iol). The peptide contains a high proportion of abnormal amino acids, α-aminoisobutyric acid (Aib) and isovaline (Iva). The peptides, trichokindin-IV〜IX (4〜9), have all about 1800 of molecular weight and have a similar structure, in which an aminoalcohol is Iol or leicinol (Lol), to TK-X. The other peptides, trichokindin-I〜III (1〜3), whose molecular weights are about 1200, are also protected by an acetyl group. Their total structure are not yet elucidated.

Trichosporins are a linear peptide mixture produced by Trichoderma polysporum whose structures are closely similar to each other. They inhibits the growth of Lentinus edodes. These peptides belong to peptaibols in which the N-terminal is protected by an acetyl group and the C-terminal by a phenylalaninol. Recently, we have isolated trichosporins[TS]-B-IIIb(1), c(2), IVb(3), c(4), d(5) and VIa(6), b(7) and established their primary structures by positive ion FAB-MS spectrometry, respectively. TS-B-IVb,d and VIb are the first examples of isovaline containing peptides isolated from T. polysporum. The connectivities of amino acid residues are established by means of COSY and NOESY NMR techniques. In addition, uncoupling activities of these peptides on rat liver mitochondria were examined.

Trichosporins are a peptide mixture isolated from culture filtrate of Trichoderma polysporum, which is antagonistic to Leutinus edodes. The pure component, trichosporin (TS)-B-V, has been obtained by preparative HPLC from Trichosporin mixture. This novel peptide has been shown to consit of one acetylated N-terminal residue, 19 amino acids and a phenylalaninol, C-terminal amino alcohol. Moreover, since this peptide contains a high proportion of α-aminoisobutyric acid (Aib) and has a moleculor weight near 2000, it belongs to the class of peptaibophols. Sequence determination (Fig. 8) was based on positive ion FAB mass spectrometry. High-field NMR data with two-demensional NMR assignment techniques were very useful in this study. Furthermore, sequences of the congeners, TS-B-I-a, -III-a, and -III-d were elucidated as shown in Fig. 8.

Trichothecium roseumの生産する抗発がんプロモーター活性ならびに発がんプロモーター活性を同時にもつ二重活性のセスキテルペンであるTTC-Aの量的確保は,菌株の選択と培養条件の工夫で達成された。また, TTC-AによるRaji細胞の多核化は, ras遺伝子の関与するシグナル伝達により制御される細胞周期の調節や細胞分化の結果生じたものと推定された。

中央アジアにおけるトルコ系民族薬物に関する第3次調査研究として、現地調査として、2003年はウズベキスタンのアラル海沿岸部からブハラにかけての地域を、2004年にはイランのカスピ海沿岸部を、また2005年には中国の新疆ウイグル自治区を調査した。また、収集した薬用植物について、天然物化学的解析を行った。 2003年の調査地のウズベキスタン西部は、アムダリア川流域の灌漑事業のために、アラル海周辺部の環境が悪化し、降雨量が少ないため砂漠化も進んでいる。この地域の薬草として目立つものはカンゾウで、分析用資料の収集をした。また、他の薬用植物数種類も収集した。この地域も含めて、ウズベキスタンでは、タビブと称される民間医が薬草の知識を伝えているが、その中身には、ロシア経由のヨーロッパのハーブの知識が多く見受けられた。2004年は、カスピ海に沿って東西に伸びるアルボルス山中に居住するトルクメンの調査をすることができたが、トルコとウズベキスタンの双方に共通するトルコ語系の呼称のものも認められ、更なる調査の必要性が明らかとなった。2005年はトルコ系のウイグル族が多い新疆省を調査した。省南部のホータンはウイグル医学が最もよく残っている地区であるが、この伝統医学はアラビア医学の系列に属するものであることが薬物とその呼称から明らかである。 この3年間には、各地で収集した薬用植物に関する天然物化学的研究や品質評価研究についても解析を進め、これまでに13報を報告している。

本研究により以下の成果を上げることが出来た。 1)リチウムアミド存在下、キラル補助基を有する酢酸エステルのリチウムエノラートのN-PMPイミンへの付加反応によって生じるジアニオンを酸化すると抗腫瘍天然物タキソールの側鎖に変換可能なα-ヒドロキシ-β-アミノエステルが高収率、高立体選択的に得られた。 2)キラル配位子制御によるアリールリチウムの共役付加を契機とするタンデム型不斉共役付加-分子内マイケル型閉環反応により得られた環化体から(-)-1-deoxylycorineを形式全合成した。 3)キラルアミドモノホスフィン配位子-ロジウム触媒を用いるラセミ5-置換及び6-置換シクロヘキセノンに対するアリールホウ素の付加反応を行うと3,5-もしくは3,6-トランスおよびシス置換シクロヘキサノンがいずれも高いエナンチオ選択性で得られた。5-トリメチルシリルシクロヘキセノンに対する付加反応を行い塩化銅で処理すると対応する5-アリールシクロヘキセノンが高収率、高エナンチオ選択的に得られた。また3,6-トランスおよびシス置換シクロヘキサノンの混合物を塩基で処理すると3,6-トランス置換シクロヘキサノンが高収率、高選択的に得られた。 4)キラルアミノアルコール配位子制御によるジンクアセチリドのニトロオレフィン、およびアルデヒドに対する不斉付加を開発した。 5)キラルオキサゾリン配位子制御による触媒的不斉分子内ヒドロアミノ化反応を開発した。 6)キラルアミドホスファン配位子制御によるジアルキル亜鉛のニトロオレフィンに対する触媒的不斉付加を開発した。 7)キラルジエーテル配位子制御によるタンデムMichael-アルキル化反応を鍵工程とする(+)-halichlorineの形式全合成を達成した。

当研究室ではこれまでにジメチル亜鉛と酸素をラジカル開始剤とする、エーテルα位水素引き抜きによる直接的新規エーテルラジカル発生法を見出している。発生したエーテルラジカルはイミンと反応し高収率で付加体を与える。本研究は我々の見いだしたエーテルラジカルの直接的発生法を機軸とする反応を開発するとともに、新たな反応設計の基盤となる活性種の化学的性質に関する新知見を得ることを目的とする。本年度は以下に示す成果を上げた。 1)ジメチル亜鉛を開始剤とするヨウ化アルキルからの一級アルキルラジカルの発生とイミンに対する付加反応を検討した。その結果、三フッ化ホウ素エチルエーテル錯体と触媒量の二価銅トリフラート存在下反応が劇的に加速することを見出し、一級アルキル付加体を高収率で得ることに成功した。本手法は二級アルキルラジカルの発生および付加反応にも適用可能である。 2)ジメチル亜鉛を開始剤とするα,β-不飽和カルボニル化合物に対するエーテル類の付加反応を検討した。α,β-不飽和N-トシルイミンをTHFおよびジメチル亜鉛の溶液に六時間かけてゆっくり滴下するとTHFの1,4-付加体が高収率で得られた。また、三フッ化ホウ素エチルエーテル錯体存在下、アルキリデンマロネートに対する付加体が高収率で得られた。 3)ジメチル亜鉛を開始剤としてキラルN-スルフィニルイミンに対する一炭素、二炭素単位エーテルの付加反応を検討した。その結果3:2〜9:1の立体選択性、および良好な収率で付加体を得ることに成功した。

中央アジアにおけるトルコ系民族薬物に関する調査研究の第2次として、(1)現地調査として、カザフスタンのタルドクルガンからウズベキスタンのナマンガンまでの天山北路に沿った地域の伝統薬と薬用植物の調査研究を行った。また、(2)収集した薬用植物について、天然物化学的解析を行った。その結果、(1)民間薬、薬用植物の証拠標本およそ400点を得、(2)3か年の研究成果の発表は論文26報となった。 (1)の多くは基原植物が同定されたが、特にバザールにて売られているものには、ヨーロッパロシアの影響が認められた。またこの地には甘草基原植物のGlycyrrhiza uralensisとG. glabraとが広範囲に分布しており、カザフスタンにはこの交配雑種とみなされるものが広く認められ、両種の種分化の点で興味ある事象である。この甘草原植物については、アルマティ〜タシケント間の各地で分析用サンプルと種子を採取した。(2)現地で薬用とされる以下のGentiana olivieri、Inula macrophylla、Ferula foetida、F. kuhistanica、F, pallida,、F. penninervis、F. sumbu、Rheum maximowiczii、Scutellaria hematochlora、Prangos tschimganica、P. pabularia、Phlomis spinidens、Dracocephalumn komarovi 13種の特徴成分と生理活性について解析し、テルペン類(セスキテルペン、ジテルペン)、クマリン、フラボノイド類、含硫化合物など、新規化合物を含む二次代謝成分100以上を単離、構造決定した。

DNAトポイソメラーゼはDNAのトポロジーを変換する酵素であり、複製・転写・組み換えといったDNAの代謝に不可欠な酵素である。トポイソメラーゼは重要な抗がん剤の主要な細胞内ターゲットのひとつであり、トポイソメラーゼの活性阻害を指標とする抗生物質や抗がん剤あるいはそれらのリード化合物の探索が可能と考えられる。今までに、天然物や合成品を含め多くのトポイソメラーゼ阻害剤が報告されているにもかかわらず、ヌクレオシンド系阻害剤についての報告は今までにされていない。 本研究では、トポイソメラーゼII(トポII)がDNAの切断と再結合に関わる一連の酵素機能の中で、ある特定のアミノ酸残基間で起こるプロトンの移動に着目し、その移動のトラップが酵素触媒作用の阻害様式の一つであるとする新規な作業仮説を基盤に据えた酵素阻害剤の設計と合成に挑戦した。プロトントラップとしてのカテコール構造の組み込みとDNAがトポIIの基質であることを考慮した結果考え出されたヌクレオシド誘導体は、デオキシリボース等価体である光学活性ブテノライドを経由する効率的なの設計と合成法の確立に到達した。 3年の研究計画期間のうち初年度に設計合成したカテコール誘導体は、ヌクレオシドとしては初めてのトポII阻害剤となり、設計にいたる発想や理論展開の正当性が示された。さらに、誘導体を合成する過程で得られた中間体についても阻害活性を調べてみると、それらは予想外に興味深い結果を示した。すなわち、阻害活性を示さなかったヌクレオシド誘導体の前駆体やその中間体がトポII阻害活性を示し、その活性は既存の阻害剤のそれを駿がした。構造活性相関から得られた知見をまとめると、(1)ヌクレオシド塩基は活性に必須ではなく、5員環ラクトンとカテコール誘導体の合目的な組合わせにより活性発現最小分子構造が形成される、(2)塩基部は酵素との親和性向上に寄与する、(3)従って、5員環ラクトン誘導体における活性の強度がそのままヌクレオシドの活性に反映される、ことが明らかとなった。一方、今回活性発現誘導に成功したヌクレオシドは、トポIには全く作用せずトポII選択的であること、また、切断複合体を形成しない作用様式を有することが明らかとなった。

α,β-不飽和カルボニル化合物に対する有機金属化合物及びヘテロ原子求核剤の共役付加反応は有機化合物の骨格形成の根幹をなす炭素-炭素結合や炭素-ヘテロ原子結合の形成法として極めて重要であり汎用されてきた。一方、アルケニルホスホナートは不飽和カルボニル化合物と同様に共役付加反応における捕捉剤の一つであるが、その反応特性の理解、その特性を生かした合成化学的利用が未だ不充分である。本研究の目的はアルケニルホスホナートに対する炭素求核剤及び窒素求核剤の反応特性の理解の深化とその特性を利用した新規合成反応の開発である。本研究により昨年までにアルケニルホスホナートのHorner-Wadswors-Emmons型反応を用いる新規アレン合成法及び分子内に二つのアルケニルホスホナート部位を有する基質を用いる分子内環化反応について報告した。 本年度は昨年度報告した環化反応を用いて酒石酸から誘導したキラル環状ビスホスフィンオキシドを中間体としてキラル環状ビスホスフィン、及びキラル環状ホスフィノカルボン酸を合成した。キラル環状ビスホスフィンオキシドをロジウム触媒による不斉水素化反応の不斉配位子として用いると、プロキラルなエナミドから高収率、高エナンチオ選択的に光学活性アミノ酸誘導体が得られた。またキラル環状ホスフィノカルボン酸をパラジウム触媒による不斉アリル化反応の不斉配位子として用いると、マロン酸エステルが良好な化学収率、高いエナンチオ選択性でアリル化されることを見いだした。

本研究で酸塩基複合体形成を基盤とする分子と分子の相互作用による活性化を基本概念として設定し、活性化と立体化学制御機能を持つ分子の設計を基盤とした触媒的不斉反応の開拓を直接の目的とする。本研究により以下の成果をあげることができた。 1.リチウムエーテルアミド存在下エステルエノラートとイミンとの反応ではβラクタムを最高96%収率74%ee、またα,β-不飽和エステルとの反応では付加体を最高67%収率92%eeで得ることに成功した。 2.触媒量のキラルアミドホスフィンとCu(OTf)_2存在下4,4-ジメチルシクロヘキセノンにジエチル亜鉛を作用させると共役エチル化体が最高64%eeで得られることが明らかになった。さらに、この銅-ジエチル亜鉛の反応系をイミンへの付加反応へ適用したところ、最高97%収率94%eeでエチル付加体を得ることに成功した。また、アミドホスフィン配位子がロジウム触媒によるアリールホウ酸のエノンへの共役付加反応に有効であり、最高99%収率96%eeで付加体が得られることを見いだした。このアミドホスフィン配位子-ロジウム錯体の構造解析研究から、キラルアミドホスフィン配位子が触媒サイクルの中、必要に応じて二座配位と単座配位を切り替える能力を有するヘミレイバル配位子であること示す知見が得られている。 3.不飽和エステルに対するリチオ化チオフェノールの高選択的付加反応を契機とするα位不斉プロトン化反応を確立し、β位には不斉点が構築されないエステルを基質にした場合でも最高92%eeの非常に高い選択性でα位を不斉プロトン化することに成功した。さらに、リチオ化チオフェノールを分子内にホルミル基を有するキラルな不飽和エステルと反応させ、良好なジアステレオ選択性にて環化体を得ることに成功した。得られた環化体から生理活性天然物(-)-Neplanocin Aの効率的合成を達成した。

本企画調査研究は、将来、「特定領域研究」への申請を視野に入れて、これまであまり科学者が手がけて来なかった未利用菌類を対象として、新規有用物質の探索ならびに持続的資源化システムの開発をめざすものであり、本年度の活動実績は以下の通りである。 1.研究打合わせ会議の開催:本企画調査研究の活動の一環として、研究打合わせ会議を平成12年9月8日学術総合センターで開催した(研究組織メンバー6名参加)。活発な意見交換の結果、将来の「特定領域研究」への申請のためには、(1)メンバー個々のさらなる研究実績の積み重ね、および(2)シンポジウムならびに総説特集の出版等による本研究分野の啓蒙活動を行うことが必須であるという結論に至った。従って本研究課題のもと企画調査研究を平成13年度も継続させることが重要であり、平成13年秋に特定領域研究(B)を申請するという計画を立てた。 2.シンポジウム開催と総説出版:上記会議の結果に基づき、(1)シンポジウム開催、(2)総説出版に関して、具体的活動を行った。(1)平成13年度日本生薬学会年会における公募シンポジウムに「未利用菌類の資源化」というタイトルで申込み、採択された。従って、平成13年9月8日(土)金沢において開催することが決定した。第2回目の同シンポジウムの開催も平成14年春に計画している。(2)中編あるいは短編の総説を各メンバーが執筆することで合意し、日本農芸化学会から出版されている「化学と生物」誌への投稿を同誌編集委員へ申込んだ。編集委員会で議論後、既に一部のメンバーへの執筆依頼が来ている。 3.セミナー活動:関東地区では、微生物(菌類)を対象とした天然物化学研究者・学生が集うセミナーとして、「菌化学セミナー」が毎年開催されており、平成12年度も10月28日に明治薬科大学で第12回目が開催され、研究代表者およびその研究室メンバーが参加・発表した。

本研究は、不完全菌Trichothecium roseumから新規に発見された発がんプロモーター活性を示すトリコテセン、トリコテシノール類やその類縁体をバイオロジカルツールとして利用し、未知なる発現機構の発見とその解明を究極の目的としている。そのために、合成ルートの効率化を高めつつ標識化や骨格変換をも可能とする光学活性体の合成法の確率がめざし研究を遂行した。 トリコテカン骨格構築の要となるシスA/B環の合成は、研究計画書に従って、D-マンニトールを出発原料とした。シスA/B環合成の鍵中間体であるスピロラクトンへの変換は、当初の計画ルートにあったアルドール縮合反応に換えて、オレフィンメタセシスによる閉環反応を組み込むことにより、立体選択性と化学収率を共に大幅に向上させることに成功した。この鍵中間体からシスA/B環への変換は、基本的には計画されたルートに従っておこなったがいくつかの改良点を加えた。この改良へ至った経緯と効率的なシスA/B環への閉環に最も寄与した点は、保護基の選択であった。すなわち、当初計画ルートにあったスピロラクトン体の水酸基の保護にトリチル基を用いることを、合成初期の段階でMOM基に変更した。これにより、スピロラクトン体の還元的開環反応により生じるジオールの保護が容易になると共に、MOM基の除去とシスA/B環への閉環反応がワンポットで可能となった。その結果、閉環における化学収率のみを比較しても、従来10%でしか得られなかったものが、今回は一挙に80%にも向上した。この閉環体を収率良く(ラセミ体として)既知のトリコテセン合成中間体に導いた。得られた化合物のNMRデータは文献値に一致したことから、ここにエナンチオ選択的なシスA/B環の合成が完了し、トリコテセン類の形式全合成に成功した。

汎用有機金属反応剤を有効な触媒的不斉反応へ組み込み、生体応答分子合成へ応用展開するためには、不斉配位子、反応系を効率化すると共に反応を一般化することが重要である。我々は本研究において、汎用有機金属反応剤を用いる触媒的不斉炭素-イオウ結合形成反応の開発に成功した。この反応に対し、反応系の効率化など反応の化学的基礎への検討を行うと共に、多種の基質との反応を検討し適用範囲の拡大を図った。その過程でチオフェノールのリチウム塩が我々が開発したキラル配位子の共存下97%に達する不斉収率でα,β-不飽和カルボニル化合物に共役付加することを見いだすに到った。この不斉収率の大幅な向上にはオルト位にアルキルもしくはシリル置換基を有するリチオ化チオフェノールを用いることにより達成された。このオルト位の置換基が嵩高いほど不斉収率が高くなった。リチオ化チオフェノールは溶液中でオリゴマーを形成しているのに対し、オルト位置換リチオ化チオフェノールはモノマーで存在しているため可溶化しており、我々が開発した3配座型不斉配位子とモノメリックな活性種を形成すると考えられる。このことが選択性向上の理由として考えられる。さらに、11種の基質で本反応の適用範囲を検討した。その結果、70-97%のいずれも高選択性を示し本反応の一般性が証明された。本反応はβラクタム、アミノ酸誘導体、脂肪酸誘導体等の不斉合成への適用が可能であり、医薬品を含めた生体応答分子の効率的な合成への応用展開が期待できる。

有機化合物の構造と反応性の相関を明確に理解することは、創薬化学の基礎となる有機合成化学の進歩にとって必須の課題である。本研究は反応に直接関わる分子の構造と選択性の発現との相互関係を、経験からばかりではなく理論的に解明することを目的とする。 ナフチルイミン、シクロヘキセニルイミンおよびアクリルイミンを基質として反応点の選択性制御の構造要因を検討した。その結果、いずれのイミンもイミン上の置換基がアルキル基だと1,4-付加、芳香族基だと1,2-付加が選択的に進行することを明らかにした。また、芳香族イミンのオルト位をイソプロピル基で置換することにより選択性を1,2-付加から1,4-付加へ制御することに成功した。分子軌道計算からオルト位をイソプロピル基で置換したイミンのLUMO係数は1,4-付加に有利である。これは共役二重結合とイミン置換基とがなす二面角はほぼ直行しており、共役系が壊れているとが原因と考えられる。さらに、このイソプロピル基が1,2-付加の反応点を遮蔽していることが1,4-付加選択性を更に高める結果になっている。このように反応の選択性決定要因を立体配座解析ならびに理論計算化学によって明確にした。即ち、付加の位置選択性は反応点のLUMO係数と立体障害により説明できることが明らかになった。 本研究により構造と立体選択性の相関が明確化されると同時に、高い選択性を与える概念的に新しい不斉反応の設計・創製が期待される。

本年度から京大薬学部に研究拠点を移したのに伴って、トポイソメラーゼ阻害活性天然物の探索研究者を共同研究者に加えることが可能となったので、一層の今後の進展が期待できる。 非天然型リグナン系化合物の構造をほんの一部修飾すると、微小管の凝集阻害に加えてDNAトポイソメラ-セ阻害活性をも示すこと、或いは、微小管凝集作用を全く失うがトポイソメラーゼ阻害活性は維持すること、など興味深い事実が明らかになってきた。 本研究では、天然物を手本とした微小管凝集阻害とトポイソメラ-セ阻害活性発現の分子構造論的基礎の確立を合成化学的に展開すると共に、より有効な抗腫瘍活性の発現を可能とする分子構造の創製を目論んだ。合成非天然型リグナン系化合物、特にポドフィロトキシン及びステガナシン系化合物のトポイソメラーゼII阻害活性発現分子の構造の特徴を、既に合成したもの及び新規合成化合物をもとに明らかにした。 1 基本構造の各部分に特徴ある化学修飾を加えた化合物を合成した。 2 また、微小管の凝集阻害活性及び細胞成長阻害活性と分子構造との相関を明らかにした。 3 次いで、凝集阻害活性とトポイソメラーゼ阻害活性を併せ持つ有効な新規化合物を化学合成的に明らかにする検討を行った。そのために、配座の固定可能な6原子直鎖状分子の設計、合成、さらに配座の検討を行った。 さらに、天然物を手本としたトポイソメラーゼ阻害活性を発現する分子構造についても例示的ではあるが、新たな展開を得た。

不完全菌Trichodermapolysporumの産生するペプタイボールと称される20残基の両親媒性ペプチド、トリコスポリンの誘導体やトリコスポンをコアとしたヘリックスバンドル(ペプチドバンドル)型複合体の合成を行い、トリコスポリンの電圧依存性イオンチャンネル形成特性とその安定化因子の明確化、またそれらの分子構造論的基礎確立への展開を目指した。天然型トリコスポリン分子の高次構造は保持されたテンプレート型2量体と4量体ならびにジスルフィド型2量体2種を含むトリコスポリン誘導体は、いずれも電位依存性のイオンチャンネルを形成した。同時に、多量体のチャンネル寿命は、単量体のそれよりも極めて長いことが明らかになった。したがって、両年度で達成された成果は、ヘリックスバンドル型複合体として制御されたトリコスポリンチャンネルの安定化に成功するにとどまらず、チャンネル形成ペプチド研究の理論的基盤であるバレルステーブモデルを実証するにまで至った。以上、本研究と同時に行ったトリコスポリン誘導体の詳細なチャンネル形成特性の結果とを合わせ、トリコスポリン分子が安定なチャンネル機能を発現するためには、 1.膜の厚さと極めて厳密に一致するペプチドのヘリックス長と 2.ペプチド-脂質-水から構成される超共役分子の形成が重要であることが結論づけられた。 また、チャンネル形成の安定化を促進する因子として、 3.ヘリックス分子の脂溶性とヘリックス構造に折れ曲がりを誘導するPro残基の重要性が明確にされた。 したがって、これらの研究成果を礎に、生体内イオンチャンネル研究や未知なる機能性材料の開発研究への展開が可能となった。 さらに興味深いことに、新たに単離したアミノ酸11残基のペプチド、トリコロビンがトリコスポリン複合体と同様に寿命の長いチャンネルを形成することを発見した。この今までに例を見ない分子挙動は、新たなチャンネル形成機構が存在する可能性を示唆している。したがって、トリコロビンを用いた今後の研究は、既存の理論をさらに発展させた分子構造論への展開が期待できる。

本研究においては、アルツハイマー型老人性痴呆症との関連が指摘されている神経栄養因子に着目し、合成品を用いる老人性痴呆症の診断用特異的免疫測定法の開発を目的として以下の研究を遂行した。研究途上において神経栄養因子の血中濃度が老人性痴呆症患者と正常人との間で有意差が認められないとの報告に接し、神経栄養因子の特異的免疫測定法の開発が老人性痴呆症の診断に一義的に展開できないことから基礎研究に重点を移した。 I.二価官能征SH-導入試薬による固相合成ペプチドの化学選択的一段階精製法および可逆的ビオチン化試薬による最終品の高効率精製法を開発した。 I.本研究においては固相合成法により各栄養因子に対応するフラグメントペプチドを合成し固相担体上でのフラグメント縮合法によりペプチド鎖を構築し二価官能性試薬を用いる化学選択的精製法により中間体及び最終品を精製する計画であった。しかしながら、神経栄養因子のようなCys含有ペプチドの精製収率が低いこと、及び固相担体上でのフラグメント縮合収率がペプチド鎖の延長に伴い著しく低下すること等により、本ストラテジーによる神経栄養因子の全合成は極めて困難であることが判明した。 II.分子内に3個のジスルフィド結合を有する神経栄養因子の最終段階でのジスルフィド形成反応への応用を目的として酸性条件化でのスルフォキシド酸化法を用いる位置選択的ジスルフィド形成法を開発した。本法は上記の理由により神経栄養因子の合成には用いられなかったが抗HIVペプチド等種々の生物活性ペプチドの合成において有用性を立証した。また本法を応用して、記憶増強作用を有するNGF合成促進因子及び数種の誘導体を合成した。 III.神経栄養因子の受容体(Trk)を介するシグナル伝達にはTrkのTyr残基のリン酸化が重要な役割を果しており、またアルツハイマー型痴呆症の発症に微小管関連蛋白質であるTau蛋白質の異常リン酸化が関連している、そこでこれらの生化学的研究に必要なリン酸化ペプチドの効率的合成法を開発した。またシグナル伝達阻害剤、およびTauリン酸化阻害剤の開発を目的として非水解性リン酸化アミノ酸の設計合成、非水解性ジペプチド等価体としての(E)-アルケン型ペプチドイソスターの立体選択的合成法を開発した。

カテコールアミンは、神経終末から分泌される神経伝達物質である。Trichoderma virideの生産する20残基の抗菌性ペプチドtrichocellin類は、神経細胞類似の副腎髄質細胞からカテコールアミンを放出させた。最初に、trichocellin類の2次構造を詳細に検討し、その構造が両親媒性を生み出す事を明らかにした。このことから、カテコールアミンの放出は、trichocellinのペプチドモノマーが細胞膜中で自己凝集することにより形成された人工的なイオンチャンネル(barrel-stave model)を経由してイオンが細胞外から細胞内に流入することにより引き起こされるという機構が推察された。電気生理学的な手法によりマクロレベルならびにシングルチャンネルレベルでのtrichocellin類により引き起こされる2分子膜のコンダクタンス値の変化を詳細に検討したところ、予想どおりペプチド分子がパラレルに4から9個凝集したイオンチャンネル(barrel-stave)を形成していることが分った。さらに、2次構造を変化させた誘導体ならびにペプチド鎖の長さの異なる誘導体を種々合成し、そのイオンチャンネル形成能を比較した。その結果、安定なイオンチャンネル形成には、20残基から構成されたプロリンによる折れ曲がりを有するヘリックス構造が重要な因子であることを明らかにした。また、イオンチャンネル形成能とカテコールアミン放出活性が一致したことから、本研究に用いた電気生理学的な手法が、カテコールアミン放出活性を有するペプチドを探索するためのプローブとなる可能性を示した。

ダイベンゾパラダイオキシン(Dと略記)フエノキサチイン(OSDと略記、Dの一方の酸素が硫黄)、チアンスレン(SSDと略記、Dの両酸素の硫黄)骨格を有する中性、酸性、塩基性物質について、それらの生物活性をin vitroならびにin vivoのレベルで検索した。【1】発癌プロモーターのTPAによるE-Bウィルス早期抗原発現を指標とする発癌プロモーション実験において、種々の中性D誘導体がいずれも発癌プロモーション抑制活性を示すことを見出した。特に2,7-ジクロロ-Dは強い活性を示し、TPAの500倍のモル比で80%以上の抑制を示す。しかも32ナノモルでもリンパ芽細胞に対する毒性は認められない。【2】酸性のD誘導体、D-1,6及び2,7-ジカルボン酸、及びそれらのメチルエステル、2,7-ジスルホン酸、2,7-ジメチル-3,8-ジスルボン酸、1,6-ジメトキシ-3,8-ジカルボン酸、及びそのメチルエステルも顕著な抗発癌促進活性を示す。D自身は殆ど活性を示さないが、1,6-ならびに2,7-ジメチル-D、1,6-ジメトキシ-3,8-ジメチル-D、1,6-ジメトキシ-D-3,8-ジアルデヒド、2,7-ジメチル-3,8-ジニトローD、塩基性の2,7-ジメチル-3,8-ジアミノ、1,6-ジメトキシ-3,8-ジエチルアミノ-Dも抗発癌促進活性をもつ。これらのうちで特に活性の顕著なのは、D-2,7-ジカルボン酸メチルエステルであり、TPAに対して100倍のモル比で発癌促進を80%以上抑制する。【3】アオツヅラフジのアルカロイドtrilobine、isotrilobineはTPAに対して100倍のモル比で発癌促進を夫々60%、及び50%抑制する。この際、リンパ芽細胞の生存率は夫々40%、60%である。【4】SSDやOSDは、TPAに対し1000倍のモル比で発癌促進を完全に抑制する。両者ともこの濃度では、リンパ芽細胞に対して毒性を示さない。

アカネ科、クワ科、ノウゼンカズラ科植物培養細胞より、それぞれ抗癌促進活性の顕著なイリドイド系モノテルペン配糖体、プレニルカルコン、及びフラノナフトキノンを高収率で得た。これらの二次代謝産物について、生成機構、in vitroならびにin vivoにおける抗発癌促進活性を調べた。【1】クチナシ果実とその培養細胞ならびにGenipa americana果実の主イリドイド成分であるgeniposideの非糖部genipinはRaji細胞のTPAによる発癌促進を強く抑制し、マウスの実験的肺癌のグリセリンによる発癌促進やDMBAをイニシエータとするTPAによる乳頭腫形成を顕著に抑制した。【2】クワ培養細胞の生産するchalcomoracin,kuwanonJなどのプレニルカルコンもTPAに対し1000倍の濃度で発癌促進を完全に阻止したが、リンパ細胞の生存率は30%であった。両者の等モル混合物は、活性はそのままでリンパ芽細胞の生存率は60%に上昇した。両物質のプレニル部分は、glucoseから解糖系を経て生成する酢酸が第一酢酸単位となり、これにtrioseから五炭糖リン酸回路を経て形成される酢酸が第2、第3の単位として結合し、メバロン酸を経て、プレニル基となることを証明した。【3】クワのフェニルプロパノイド由来部分の形成には、フェニルアラニン、チロシンのいずれもが同等の寄与をすることが^<13>C標識位置の異なる両アミノ酸の同時投与実験により証明した。【4】南米産ノウゼンカズラ科植物Tabebuia avellanedaeのカルスの生産する5-hydroxy-2-(1-hydroxyethyl)-naphtho-[2,3-b]furan-4,9-dioneや関連ナフトキノンが顕著な抗発癌促進活性を示すことを見出した。

菌類の生産する免疫抑制物質の探索を冬虫夏草菌類について行い、活性物質数種を単離しその作用機作を検討すると共に合成を計画した。 1)ISP-I並びにその類縁体の単離:ISP-Iは免疫抑制活性物質としてI.sinclairiiから、初めて単離された。いわゆる冬虫夏草菌Cordyceps sinensisの培養液について免疫抑制活性を指標として成分を検索したところISP-Iを単離同定することができた。ISP-IはMycelia sterilia菌によりI.sinclairii菌よりも効率よく生産される。その培養液から既知のISP-II、-IIIb、-Vの他に、新規活性物質として、ISP-IIIa、-Vを得た。また新たにそれらのdihidro体に相当する物質もえられた。 2)ISP-I並びに誘導体の免疫抑制活性:ISP-I及び14-deoxo-ISP-Iの動物を用いた移植実験では毒性が強く、使用にさいし安全域の狭いことが判明した。また新規天然物についてマウス同種リンパ球混合培養反応(MLR)、細胞毒性、皮膚移植について試験した。その結果はISP-Iのそれらと大きな差異は認められなかった。 3)ISP-Iの免疫抑制機作:ISP-Iはin vitroにおいて同種抗原刺激による同種細胞障害T細胞(alloCTL)の誘導をサイクロスポリン(CsA)よりも強力に抑制した。そこでin vivoにおけるISP-IのalloCTL誘導抑制について検討した。その結果ISP-IはCsAと異なりインターロイキン(IL)-2依存性のT細胞の増殖を特異的に抑制することが強く示唆された。 4)ISP-Iの合成:ISP-Iの合成の設計方針としてFragmentA〜Cに三分割した。AはD-セリン部、Bはグリコール部、Cはケトアルキル部よりなる。A部はD-バリンからSchollkopfらのcyclo(D-Val-2-amino-malonyl)bis-lactam dimethyl etherを合成した。B部はスズエノレートを活用する不斉アルドール反応により、またC部は1、8-octanediolから合成した。AとB部の結合は不斉アルキル化、A-B部とC部の結合はWittig反応により結合させISP-Iの合成を完成させることができる。

イリドイド系化合物は薬用植物成分としてに広く分布している。なかでも、抗腫瘍活性を始め種々の生理活性を有するインド-ルアルカロイドの非トリプタミン部分がイリドイド配糖体より生合成されることはよく知られている。従って、イリドイド配糖体の基本骨格であるイリダン骨格の形成機構を酵素レベルで解明することは、イリドイド関連物質の生合成研究にとって非常に重要なことである。そこで、本研究ではインド蛇木の培養細胞を酵素源として以下の研究を行い、ほぼ初期の目的を達した。 1.鎖状モノテルペン脱水素酵素。イリダン骨格を形成する閉環反応の鎖状生合成前駆物質である10ーhydroxygeraniolから10ーoxogeranialの形成にあずかる脱水素酵素を活生収率10%、87倍で、かつ単一なタンパク質として単離した。本酵素は分子量44,000のモノマ-タンパク質で、等電点pH5.4、亜鉛原子を含み、NADP(H)依存性である。Ca^<2+>,Mg^<2+>の添加は酵素反応を活性化し、チオ-ル試薬に阻害効果が認められた。本酵素反応の立体化学的過程を(4R)ー[4ー ^2H]NADPHを用いて調べた結果、proーR特異的であることが分かった。本酵素のN末端アミノ酸配列19個は、植物から単離されている他のアルコ-ル脱水素酵素との間に相同性は認められなかった。 2.イリダン骨格閉環機構。鎖状前駆体10ーoxogeranialから環状前駆体イリドジア-ルへNADPH存在下閉環反応を触媒する酵素を440倍に精製した。本酵素は分子量118,000の4量体であることが明かとなり、NADPHはNADHより2倍高い活性を示した。本酵素が還元的機構により閉環反応を触媒することは、環状モノテルペンの生合成における、geranyl pyrophosphateが関与する従来の閉環機構とは異なり新しいタイプのものである。この酵素を単離し、反応機構を解明したことは非常に意義あることである。

申請者らは冬虫夏草菌の一種Isaria sinclairiiの培養液中に強い免疫抑制活性物質の存在を認め、その活性物質ISPーIが(2S,3S,4R)ー(E)ー2ーaminoー3,4ーdihydroxyー2ーhydroxymethylー14ーoxoeicosー6ーenoicーacidの構造を持つことを明かにした。さらにISPーIとその諸誘導体につき種々の免疫抑制活性および細胞毒性を測定した。それらの結果を下記に列挙する。1)マウス同種リンパ球混合培養反応に対する抑制活性においてISPーIはCsAに比し同等以上の活性を示した。 2)ISPーIの13種の誘導体につき同活性を調べた結果、活性発現あるいは活性の上昇には、2位のアミノ基、6位の二重結合および8位から20位の炭素鎖が必要である。さらに14ーdeoxoーISPーI(ISPーIー13)がISPーI自身よりも1桁高い活性を持つことを見い出した。3)ISPーIとISPーIー13について抗羊赤血球抗体産生および細胞傷害性T細胞誘導への抑制効果を調べたところ、ISPーIはCsAよりも100倍、またISPーIー13はISPーIよりもさらに10倍強い抑制効果を示した。4)ISPーIは経口投与によるマウス同種細胞特異抗体産生に対してCsAより100倍強い抑制効果を示した。5)ISPーI誘導体のうち、ISPーIとISPーIー13は培養細胞に対する毒性が最も強いが、CsAよりも弱い。6)CsAはインタ-ロイキンー2(ILー2)などのサイトカイニンの産生を抑制するのに対し、ISPーIはILー2などによるT細胞の分裂増殖を抑制する。従って、両者は異なる作用機序により免疫抑制を発現する。その他、ISPーIを高生産するMycelia sterilia菌の菌体より新たに三種のISPーI類縁体を単離し、それらの構造を明らかにした。 このように、ISPーIおよびその同族体はCsAとは異なる作用機序により抑制効果を発現する免疫抑制剤で、今後の実際の移植実験の結果が待たれる。