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.

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.

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.

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.

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.

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].

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.

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.

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.

Biochemical characterization of NADH:FMN oxidoreductase HcbA3 from Nocardioides sp. PD653 in catalyzing aerobic HCB dechlorination.

Nocardioides sp. PD653 genes hcbA1, hcbA2, and hcbA3 encode enzymes that catalyze the oxidative dehalogenation of hexachlorobenzene (HCB), which is one of the most recalcitrant persistent organic pollutants (POPs). In this study, HcbA1, HcbA2, and HcbA3 were heterologously expressed and characterized. Among the flavin species tested, HcbA3 showed the highest affinity for FMN with a K d value of 0.75±0.17 µM. Kinetic assays revealed that HcbA3 followed a ping-pong bi-bi mechanism for the reduction of flavins. The K m for NADH and FMN was 51.66±11.58 µM and 4.43±0.69 µM, respectively. For both NADH and FMN, the V max and k cat were 2.21±0.86 µM and 66.74±5.91 sec-1, respectively. We also successfully reconstituted the oxidative dehalogenase reaction in vitro, which consisted of HcbA1, HcbA3, FMN, and NADH, suggesting that HcbA3 may be the partner reductase component for HcbA1 in Nocardioides sp. PD653.

Synthetic studies of biologically active natural products contributing to pesticide development.

Natural product research, including total synthesis, is becoming increasingly important for the discovery of pesticide seeds and leads. Synthetic studies of biologically active compounds such as antibiotics (enacyloxins, polynactin, pamamycins, spirofungin A and B, glutarimides and antimycins), phytopathogenic toxins (pyricuol, pyriculariol, tabtoxinine-ß-lactam, gigantenone, phomenone and phaseolinone), marine derived products (pteroenone, ß-D-Asp-Gly, didemniselinolipid B, cortistatin A, sanctolide A and gizzerosine), POPs (dieldrin, endosulfan, HCB), plant hormones (abscisic acid and jasmonic acid), insect pheromones (endo-brevicomin etc.), especially using a variety of biotransformation are described.

Dissipation, dehalogenation, and denitration of chloroaromatic compounds by Nocardioides sp. strain PD653: Characterization of the substrate specificity.

The substrate range of Nocardioides sp. strain PD653, capable of mineralizing hexachlorobenzene, was investigated based on the dissipation of substrates and the liberation of halogen ions. Strain PD653 dehalogenated 10 out of 18 halophenol congeners; however, it could dehalogenate only hexachlorobenzene out of seven halobenzene congeners tested. Moreover, dehalogenation activities were shown for chloronitrobenzenes, along with an increase in the number of substituted chlorine atoms except for 2,3,4,5-tetrachloro-1-nitrobenzene. These results suggested that this strain might be applicable to remediate soil contaminated with these persistent chloroaromatic compounds.

Mechanism-based tuning of insect 3,4-dihydroxyphenylacetaldehyde synthase for synthetic bioproduction of benzylisoquinoline alkaloids.

Previous studies have utilized monoamine oxidase (MAO) and L-3,4-dihydroxyphenylalanine decarboxylase (DDC) for microbe-based production of tetrahydropapaveroline (THP), a benzylisoquinoline alkaloid (BIA) precursor to opioid analgesics. In the current study, a phylogenetically distinct Bombyx mori 3,4-dihydroxyphenylacetaldehyde synthase (DHPAAS) is identified to bypass MAO and DDC for direct production of 3,4-dihydroxyphenylacetaldehyde (DHPAA) from L-3,4-dihydroxyphenylalanine (L-DOPA). Structure-based enzyme engineering of DHPAAS results in bifunctional switching between aldehyde synthase and decarboxylase activities. Output of dopamine and DHPAA products is fine-tuned by engineered DHPAAS variants with Phe79Tyr, Tyr80Phe and Asn192His catalytic substitutions. Balance of dopamine and DHPAA products enables improved THP biosynthesis via a symmetrical pathway in Escherichia coli. Rationally engineered insect DHPAAS produces (R,S)-THP in a single enzyme system directly from L-DOPA both in vitro and in vivo, at higher yields than that of the wild-type enzyme. However, DHPAAS-mediated downstream BIA production requires further improvement.

Author Correction: Mechanism-based tuning of insect 3,4-dihydroxyphenylacetaldehyde synthase for synthetic bioproduction of benzylisoquinoline alkaloids.

In the original version of this Article, the abbreviation of 3,4-dihydroxyphenylacetaldehyde synthase presented in the first paragraph of the Discussion section was given incorrectly as DYPAA. The correct abbreviation for this enzyme is DHPAAS. This error has been corrected in both the PDF and HTML versions of the Article.

Chemo-enzymatic synthesis of p-nitrophenyl ß-D-galactofuranosyl disaccharides from Aspergillus sp. fungal-type galactomannan.

ß-d-Galactofuranose (Galf) is a component of polysaccharides and glycoconjugates. There are few reports about the involvement of galactofuranosyltransferases and galactofuranosidases (Galf-ases) in the synthesis and degradation of galactofuranose-containing glycans. The cell walls of filamentous fungi in the genus Aspergillus include galactofuranose-containing polysaccharides and glycoconjugates, such as O-glycans, N-glycans, and fungal-type galactomannan, which are important for cell wall integrity. In this study, we investigated the synthesis of p-nitrophenyl ß-d-galactofuranoside and its disaccharides by chemo-enzymatic methods including use of galactosidase. The key step was selective removal of the concomitant pyranoside by enzymatic hydrolysis to purify p-nitrophenyl ß-d-galactofuranoside, a promising substrate for ß-d-galactofuranosidase from Streptomyces species.

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.

Synthesis of Sulfo-Sialic Acid Analogues: Potent Neuraminidase Inhibitors in Regards to Anomeric Functionality.

The design, synthesis and application of N-acetylneuraminic acid-derived compounds bearing anomeric sulfo functional groups are described. These novel compounds, which we refer to as sulfo-sialic acid analogues, include 2-decarboxy-2-deoxy-2-sulfo-N-acetylneuraminic acid and its 4-deoxy-3,4-dehydrogenated pseudoglycal. While 2-decarboxy-2-deoxy-2-sulfo-N-acetylneuraminic acid contains no further modifications of the 2-deoxy-pyranose ring, it is still a more potent inhibitor of avian-origin H5N1 neuraminidase (NA) and drug-resistant His275Tyr NA as compared to the oxocarbenium ion transition state analogue 2,3-dehydro-2-deoxy-N-acetylneuraminic acid. The sulfo-sialic acid analogues described in this report are also more potent inhibitors of influenza NA (up to 40-fold) and bacterial NA (up to 8.5-fold) relative to the corresponding anomeric phosphonic acids. These results confirm that this novel anomeric sulfo modification offers great potential to improve the potency of next-generation NA inhibitors including covalent inhibitors.

Product Selectivity of Esterification of L-Aspartic Acid and L-Glutamic Acid Using Chlorotrimethylsilane.

TMSCI works as an acid catalyst precursor for selective esterification of L-aspartic and L-glutamic acids in the presence of primary, secondary and tertiary alcohols. Although excess TMSCI was required for the completion of esterification, the resulting alkyl TMS ether could be azeotropically removed by simple evaporation with alcohol.

Study on the metabolites of isoalantolactone in vivo and in vitro by ultra performance liquid chromatography combined with Triple TOF mass spectrometry.

Isoalantolactone (IAL) is an active sesquiterpene naturally present in many vegetables, condiments and medicinal plants. In this study, an efficient strategy was developed for the identification of metabolites following the in vivo metabolism and in vitro biotransformation of IAL with rat intestinal bacteria utilizing ultra performance liquid chromatography combined with Triple TOF mass spectrometry (UPLC-Triple TOF-MS/MS). As a result, 46 metabolites including 34 novel sulfur-containing products were identified. The results demonstrated that IAL could undergo general metabolic reactions, including oxidation, hydration, hydrogenation, demethylation, cysteine conjugation and N-acetylcysteine conjugation, but the most noticeable characteristic of IAL metabolism was the H2S addition to the double bond between C-11 and C-13 and subsequent reactions to produce a series of novel sulfur-containing dimers. This is the first study of IAL metabolism in vivo, whose results provide novel and useful data for a better understanding of the safety and efficacy of IAL.