Synthesis of the A/D/E-ring Core Compounds of Maoecrystal V†.

Synthesis of the A/D/E-ring core compounds of maoecrystal V was achieved. The key Diels-Alder reactions between tricyclic α-methylene lactones and Kitahara-Danishefsky dienes afforded the spirocyclic core compounds in a regioselective and stereoselective manner.

Controlling the production of phytotoxin pyriculol in Pyricularia oryzae by aldehyde reductase.

Pyricularia oryzae is one of the most devastating plant pathogens in the world. This fungus produces several secondary metabolites including the phytotoxin pyriculols, which are classified into 2 types: aldehyde form (pyriculol and pyriculariol) and alcohol form (dihydropyriculol and dihydropyriculariol). Although interconversion between the aldehyde form and alcohol form has been predicted, and the PYC10 gene for the oxidation of alcohol form to aldehyde is known, the gene responsible for the reduction of aldehyde to alcohol form is unknown. Furthermore, previous studies have predicted that alcohol analogs are biosynthesized via aldehyde analogs. Herein, we demonstrated that an aldo/keto reductase PYC7 is responsible for the reduction of aldehyde to alcohol congeners. The results indicate that aldehyde analogs are biosynthesized via alcohol analogs, contradicting the previous prediction. The results suggest that P. oryzae controls the amount of pyriculol analogs using two oxidoreductases, PYC7 and PYC10, thereby controlling the bioactivity of the phytotoxin.

Synthesis of (12R,13S)-pyriculariol and (12R,13S)-dihydropyriculariol revealed that the rice blast fungus, Pyricularia oryzae, produces these phytotoxins as racemates.

Synthesis of assumed natural (12R,13S)-enantiomers of pyriculariol (1) and dihydropyriculariol (2), phytotoxins isolated from rice blast disease fungus, Pyricularia oryzae, was achieved using Wittig reaction or microwave-assisted Stille coupling reaction as the key step. The synthesis revealed that the natural 1 and 2 are racemates. Foliar application test on a rice leaf indicated that both the salicylaldehyde core and side chain were necessary for phytotoxic activity. The fungus is found to produce optically active phytotoxins when incubated with rotary shaker, but racemic ones when cultured using an aerated jar fermenter.

Xylomexicanins I and J: Limonoids with Unusual B/C Rings from Xylocarpus granatum.

Two tetranortriterpenoids with new skeletons, xylomexicanins I and J (1 and 2), were isolated during the investigation of chemical constituents from seeds of the Chinese mangrove, Xylocarpus granatum. Xylomexicanin I (1) is an unprecedented limonoid with bridged B- and C-rings. A biosynthesis pathway for 1 from xylomexicanin F is proposed.

Isolation of endosulfan sulfate-degrading Rhodococcus koreensis strain S1-1 from endosulfan contaminated soil and identification of a novel metabolite, endosulfan diol monosulfate.

An aerobic endosulfan sulfate-degrading bacterium, Rhodococcus koreensis strain S1-1, was isolated from soil to which endosulfan had been applied annually for more than 10 years until 2008. The strain isolated in this work reduced the concentration of endosulfan sulfate (2) from 12.25 µM to 2.11 µM during 14 d at 30 °C. Using ultra performance liquid chromatography-electrospray ionization-mass spectroscopy (UPLC-ESI-MS), a new highly water-soluble metabolite possessing six chlorine atoms was found to be endosulfan diol monosulfate (6), derived from 2 by hydrolysis of the cyclic sulfate ester ring. The structure of 6 was elucidated by chemical synthesis of the candidate derivatives and by HR-MS and UPLC-MS analyses. Therefore, it was suggested that the strain S1-1 has a new metabolic pathway of 2. In addition, 6 was expected to be less toxic among the metabolites of 1 because of its higher water-solubility.

Inhibitory effect of 13 taxane diterpenoids from Chinese yew (Taxus chinensis var. mairei) on the proliferation of HeLa cervical cancer cells.

The inhibitory effect of 13 taxanes isolated from the Chinese yew (Taxus chinensis var. mairei) on the proliferation of human cervical cancer HeLa cells were examined using an MTT assay. Four compounds having a hydrophobic cinnamate side chain showed antiproliferative activity, which may be due to increased cell permeability.

Functional and structural analysis of influenza virus neuraminidase N3 offers further insight into the mechanisms of oseltamivir resistance.

The influenza virus neuraminidase H274Y substitution is a highly prevalent amino acid substitution associated with resistance to the most heavily used influenza drug, oseltamivir. Previous structural studies suggest that the group specific 252 residue (Y252 in group 1 and T252 in group 2) might be a key factor underlying H274Y resistance. However, H274Y has only been reported in N1 subtypes, which indicates that there must be additional key residues that determine H274Y resistance. Furthermore, we found that members of NA serotype N3 also possess Y252, raising the key question as to whether or not H274Y resistance may also be possible for some group 2 NAs. Here, we demonstrate that the H274Y substitution results in mild oseltamivir resistance for N3. Comparative structural analysis of N3, N1, and their 274Y variants indicates that the interaction of residue 296 (H in N1 and nonaromatic for other serotypes) with conserved W295 is another important determinant of oseltamivir resistance.

Facile synthesis of the cyclohexane fragment of enacloxins, a series of antibiotics isolated from Frateuria sp. W-315.

An efficient and good yield synthesis of the cyclohexane moiety of enacyloxins, a series of antibiotics isolated from Frateuria sp. W-315, was achieved from d-quinic acid using a successive Barton-McCombie deoxygenation.

Xylomexicanins C and D, new mexicanolide-type limonoids from Xylocarpus granatum.

Two new limonoids, named xylomexicanins C and D, were isolated from a dichloromethane extract of the seeds of Xylocarpus granatum cultivated in Hainan, China, and their structures were elucidated on the basis of one- and two-dimensional NMR (including 1H, 13C-NMR, DEPT, 1H-1H COSY, HSQC, HMBC, and NOESY) and confirmed by high-resolution mass spectrometry. Xylomexicanin C exhibited antiproliferative activity against human breast carcinoma cells (KT).

Structure-activity relationship of 9-methylstreptimidone, a compound that induces apoptosis selectively in adult T-cell leukemia cells.

We previously reported that 9-methylstreptimidone, a piperidine compound isolated from a culture filtrate of Streptomyces, induces apoptosis selectively in adult T-cell leukemia cells. It was screened for a compound that inhibits LPS-induced NF-kappaB and NO production in mouse macrophages. However, 9-methystreptimidone is poorly obtained from the producing microorganism and difficult to synthesize. Therefore, in the present research, we studied the structure-activity relationship to look for new selective inhibitors. We found that the structure of the unsaturated hydrophobic portion of 9-methylstreptimidone was essential for the inhibition of LPS-induced NO production. Among the 9-methylstreptimidone-related compounds tested, (+/-)-4,alpha-diepi-streptovitacin A inhibited NO production in macrophage-like cells as potently as 9-methylstreptimidone and without cellular toxicity. Moreover, this compound selectively induced apoptosis in adult T-cell leukemia MT-1 cells.

Hybrid stereoisomers of a compact molecular probe based on a jasmonic acid glucoside: syntheses and biological evaluations.

12-O-ß-D-glucopyranosyl jasmonic acid (JAG) shows unique biological activities, including leaf-closing of Samanea saman. It is expected that the mode of action for such regulation is distinct from that of other jasmonates. We developed high-performance compact molecular probes (CMPs) based on JAG that can be used for the FLAG-tagging of JAG target. We synthesized four hybrid-type JAG-CMP stereoisomers (7, ent-7, 8, and ent-8), which are composed of (-)-12-OH-JA (2)/D-galactopyranoside, (-)-2/L-galactopyranoside, (+)-ent-2/d-galactopyranoside, and (+)-ent-2/L-galactopyranoside moieties, respectively, and we examined their biological features, such as the stereospecific induction of shrinkage, rate of the cellular response, and dependence on potassium channel activity. These features of the JAG-CMPs were completely consistent with those of the original JAG. These results indicate the biological equivalence of JAG and the JAG-CMPs. During the course of such biological evaluations, it was revealed that the biological activity of the CMPs is greatly dependent on the d/l-stereochemistry of a glycon moiety. To the best of our knowledge, this is the first study suggesting that the d/l-stereochemistry of the glycon moiety significantly affects the biological activity of the associated glycoside.

Relationship between the structures of taxane derivatives and their microtubule polymerization activity.

Paclitaxel (Taxol), one of the most potent anticancer drugs, is a microtubule-stabilizing compound that inhibits microtubule depolymerization within the cell. The structure of paclitaxel is composed of two key elements, a taxane ring and an N-benzoylphenylisoserine side chain at C-13. A number of natural and artificial compounds with taxane skeletons have been isolated, but almost none of their bioactivities have been evaluated. In this study, we focused on compounds having a taxane skeleton structure and examined their effects on tubulin dynamics. Although none of these compounds had an N-benzoylphenylisoserine side chain, three were found to promote tubulin assembly. On the other hand, one compound inhibited tubluin assembly in a way similar to nocodazole. These compounds exhibited novel structure-activity relationships of taxane compounds.

Antitumour activities of sesquiterpene lactones from Inula helenium and Inula japonica.

Eight sesquiterpene lactones were isolated from the roots of Inula helenium and flowers of I. japonica. Among them, isoalantolactone (3) and santamarine (6) exhibited significant growth inhibitory activities against gynecologic cancer cell lines, while others weakly inhibited the growth of the cell lines (IC50 < or = 100 microM). In addition, 3 significantly inhibited the tumour growth of S180 tumour-bearing mice. Compounds 3 and 6 were not toxic to human embryonic lung fibroblast cells in vitro. These results demonstrated that the antitumour activities are closely related to the structures of the compounds, that is, an alpha-exomethylene-gamma-lactone ring is necessary for these activities.

Synthesis and Neuraminidase Inhibitory Activity of Sialic Acid Analogues with Fluoro, Phosphono, and Sulfo Functionalities.

Methods to synthesize influenza virus inhibitors with fluoro, phosphono, and/or sulfo functional groups are described. The resulting sialic acid analogues are produced from the natural substrate N-acetylneuraminic acid as starting material. Fluorescent assay methods for inhibition of influenza neuraminidase and virus proliferation are also provided.

Influenza A Virus Neuraminidase Inhibitors.

Depending on the strain, influenza A virus causes animal, zoonotic, pandemic, or seasonal influenza with varying degrees of severity. Two surface glycoprotein spikes, hemagglutinin (HA) and neuraminidase (NA), are the most important influenza A virus antigens. NA plays an important role in the propagation of influenza virus by removing terminal sialic acid from sialyl decoy receptors and thereby facilitating the release of viruses from traps such as in mucus and on infected cells. Some NA inhibitors have become widely used drugs for treatment of influenza. However, attempts to develop effective and safe NA inhibitors that can be used for treatment of anti-NA drugs-resistant influenza viruses have continued. In this chapter, we describe the following updates on influenza A NA inhibitor development: (i) N-acetylneuraminic acid (Neu5Ac)-based derivatives, (ii) covalent NA inhibitors, (iii) sulfo-sialic acid analogs, (iv) N-acetyl-6-sulfo-ß-D-glucosaminide-based inhibitors, (v) inhibitors targeting the 150-loop of group 1 NAs, (vi) conjugation inhibitors, (vii) acylhydrazone derivatives, (viii) monoclonal antibodies, (ix) PVP-I, and (x) natural products. Finally, we provide future perspectives on the next-generation anti-NA drugs.

Synthesis of the oxazolidinone fragment of thelepamide.

Thelepamide, an unique ketide-amino acid isolated from a marine annelid worm Thelepus crispus, has a unique oxazolidinone ring derived from cysteine, glycine and valine. Rareness in nature as well as promising bioactive possibility make the oxazolidinone ring an attractive synthetic target. The hydroxy oxazolidinone fragment of thelepamide was prepared by acid-catalysed N,O-acetal formation between a ketoamide and formaldehyde. Lactone-carbonyl selective isopropyl addition to an oxazilidine-dione under Grignard conditions also forms the target compound.

Isolation of a Novel Endophytic Bacillus Strain Capable of Transforming Pentachlorophenol and Structure Determination of Pentachlorophenol Phosphate Using Single-Crystal X-ray Diffraction.

A novel approach for the remediation of upland soils contaminated with pentachlorophenol (C6HCl5O; PCP) (1), a fungicide, wood perservative, and herbicide, through the exploitation of plant-endophytic bacteria may overcome the existing issues in bioaugmentaion and phytoremidiation. In this study, we isolated the endophytic Bacillus sp. strain PCP15 and determined its metabolite of PCP (1). This strain degraded 8.03 µmol L-1 PCP (1) within 24 h and generated the novel metabolite PCP phosphate (3). The PCP15 strain showed nearly complete growth inhibition of 20 µmol L-1 PCP (1). In contrast, PCP15 showed resistance to PCP phosphate (3), indicating that the phosphorylation of PCP, which has never previously been reported in organisms, contributed to the detoxification of PCP (1) in bacterial cells. Our results show the potential for practical application of this strain in hybrid remediation of PCP (1)-contaminated soils and reveal a novel PCP (1) detoxification mechanism in organisms.

Millifolide A, a dimeric ether of degraded sesquiterpene lactones, inhibited the proliferation of human lung cancer cell line A549.

The inhibitory effect of three degraded sesquiterpene lactones, iso-seco-tanapartholide, arteludooicinolide A and millifolide A isolated from Achillea millefolium L., on anti-human lung cancer cells was examined using MTT and reporter gene assays. Millifolide A has significant inhibitory effects on the proliferation of human lung cancer cells probably through inducing cell apoptosis.

Machine learning discovery of missing links that mediate alternative branches to plant alkaloids.

Engineering the microbial production of secondary metabolites is limited by the known reactions of correctly annotated enzymes. Therefore, the machine learning discovery of specialized enzymes offers great potential to expand the range of biosynthesis pathways. Benzylisoquinoline alkaloid production is a model example of metabolic engineering with potential to revolutionize the paradigm of sustainable biomanufacturing. Existing bacterial studies utilize a norlaudanosoline pathway, whereas plants contain a more stable norcoclaurine pathway, which is exploited in yeast. However, committed aromatic precursors are still produced using microbial enzymes that remain elusive in plants, and additional downstream missing links remain hidden within highly duplicated plant gene families. In the current study, machine learning is applied to predict and select plant missing link enzymes from homologous candidate sequences. Metabolomics-based characterization of the selected sequences reveals potential aromatic acetaldehyde synthases and phenylpyruvate decarboxylases in reconstructed plant gene-only benzylisoquinoline alkaloid pathways from tyrosine. Synergistic application of the aryl acetaldehyde producing enzymes results in enhanced benzylisoquinoline alkaloid production through hybrid norcoclaurine and norlaudanosoline pathways.

Xylomexicanins K-N: limonoids from the leaves and twigs of Xylocarpus granatum.

Six new compounds, xylomexicanins K-N (1-4), granasteroid (5) and 5-methoxy-2-pentylbenzofuran-7-ol (6), along with nine known compounds were isolated from the leaves and twigs of Xylocarpus granatum. Among them, 1 was a biogenetic precursor of 1,8,9-phragmalin limonoid, and 4 represent the first example of degraded A-ring limonoid. The structures of them were elucidated on the basis of one- and two-dimensional NMR spectroscopic data (including 1H, 13C-NMR, DEPT, 1H-1H COSY, HSQC, HMBC, and NOESY) and confirmed by high-resolution mass spectrometry.[Formula: see text].