Synthesis of Macrolactone Core of ent-Formosalide A via Regioselective Ether Cyclization.

Formosalide A is a cytotoxic macrolide isolated from the dinoflagellate Prorocentrum sp, whose structure is characterized by functionalized 5- and 6-membered ether rings embedded in the macrolactone and an all cis-tetraene side chain. Here, we report the synthesis of the macrolactone core of ent-formosalide A. Our approach is highlighted by the Au-mediated 6-exo-dig cyclization for the synthesis of the 6-membered ether ring, which proceeded in a highly regioselective manner. Control experiments demonstrated that the acyclic protecting group of the C9,C10-diol was crucial for the desired 6-exo-dig cyclization. Theoretical studies were performed focusing on structural component analysis, which suggested that the C8-C9-C10-C11 dihedral angle induced by the protecting group controlled the regioselectivity. An additional 6 steps including Shiina macrolactone formation from the 6-membered ether ring completed the synthesis of the macrolactone core of ent-formosalide A.

Formal Total Synthesis of Batrachotoxin Enabled by Radical and Weix Coupling Reactions.

Batrachotoxin (1), originally isolated from a Columbian poison-dart frog, is a steroidal alkaloid. Its 6/6/6/5-membered carbocycle (ABCD-ring) contains two double bonds, one nitrogen, and five oxygen functionalities. We developed a radical-based convergent strategy and realized the total synthesis of 1 in 28 steps. The AB-ring and D-ring fragments were efficiently synthesized and linked by exploiting a powerful Et3B/O2-mediated radical coupling reaction. Vinyl triflate and vinyl bromide were then utilized for a Pd/Ni-promoted Weix coupling reaction to cyclize the C-ring. A hydroxy group of the C-ring was stereoselectively installed by a decarboxylative hydroxylation reaction to prepare an advanced intermediate of our previous total synthesis of 1.

Initial Stage of Syringyl Lignin Formation from Sinapyl Alcohol.

Elucidating the detailed structure and formation mechanism of lignin, especially understudied syringyl (S) lignin, advances our knowledge of lignocellulosic biomass. To examine the early stages of S-lignin formation from sinapyl alcohol (SA), the FMR (flow microreactor) method and the Zutropf (gradual addition of SA) method with limited amounts of H2O2 were employed for the peroxidase-catalyzed dehydrogenative polymerization of SA. Only ß-ß dimers and not ß-O-4 dimers were obtained as initial dimerization products. Six new oligoligognols up to pentamers with ß-ß and ß-O-4 structures were identified. The erythro isomer was preferentially formed over the threo isomer in the ß-O-4 structures, similar to that found in naturally occurring S-rich hardwood lignin. Although minor substructures, the α-oxidized ß-ß and ß-O-4 structures and spirodienone (ß-1) structure identified in this study demonstrate the characteristic features of S-rich lignin. Based on the identified products, the initial formation mechanism of S-lignin from SA was proposed.

Computational Study Focusing on a Comprehensive Conformational Analysis of Transition States for Aza-Spiro Ring Formations with N-Alkoxyamides.

An accurate understanding of conformations in transition states is a critical piece in the theoretical analysis of complex molecular reactions. In this study, we investigated conformationally diverse transition states during intramolecular aza-spiro ring formation with an allylsilane moiety and N-alkoxy iminium ion, a key reaction in the synthesis of fasicularin by Sato and Chida et al., and identified the origins of stereoselectivity of the cyclization. A large number of conformational isomers with forming C-C bonds were comprehensively analyzed using Cremer-Pople puckering parameters. It was found that the conformations of the transition states had different puckering preferences depending on the reactant's double-bond geometry and the product's stereochemical configuration. Furthermore, an asymmetric aza-spiro cyclization with a tolyl group as a chiral auxiliary was investigated, showing that conformational anchoring by both a CH-O hydrogen bond and the CH-π interaction was critical for the asymmetric induction.

Isolation and structure determination of allopteridic acids A-C and allokutzmicin from an unexplored actinomycete of the genus Allokutzneria.

Two classes of new polyketides, allopteridic acids A-C (1-3) and allokutzmicin (4), were isolated from the culture extract of an actinomycete of the genus Allokutzneria. The structures of 1-4 were elucidated through the interpretation of NMR and MS analytical data. Compounds 1-3 possess the same carbon skeleton with pteridic acids but their monocyclic core structures are distinct from the spiro-bicyclic acetal structures of pteridic acids. Compound 4 is a linear polyketide of an unprecedented class, featured by a guanidino-terminus and an epoxide modification. Compounds 1-3 promoted the root elongation of germinated lettuce seeds by ca. 10-40% at 1~10 µM whereas 4 retarded the seed growth. Compound 4 exhibited weak antimicrobial activity against Candida albicans with MIC 25 µg mL-1.

Species-specific secondary metabolism by actinomycetes of the genus Phytohabitans and discovery of new pyranonaphthoquinones and isatin derivatives.

To further exploit secondary metabolic potential of a minor actinomycete genus Phytohabitans within the family Micromonosporaceae, metabolite profiling by HPLC-UV analysis, combined with 16S rDNA sequence-based phylotyping were attempted on seven Phytohabitans strains available at the public culture collection. The strains were grouped into three clades and each exhibited unique and distinct metabolite profiles, which were highly conserved among strains within the same clade. These results were consistent with previous observations on two other actinomycetes genera, reconfirming species-specificity of secondary metabolite production, which were conventionally thought to be strain-specific. A strain RD003215, belonging to the P. suffuscus clade, produced multiple metabolites, some of which were presumed to be naphthoquinones. Liquid fermentation followed by chromatographic separation of the broth extract led to the discovery of three new pyranonaphthoquinones, designated habipyranoquinones A-C (1-3), and one new isatin derivative, (R)-N-methyl-3-hydroxy-5,6-dimethoxyoxindole (4), along with three known synthetic compounds, 6,8-dihydroxydehydro-α-lapachone (5), N-methyl-5,6-dimethoxyisatin (6), and 5,6-dimethoxyisatin (7). Structures of 1-4 were unequivocally elucidated by NMR, MS, and CD spectral analysis, with assistance of density functional theory-based NMR chemical shift prediction and ECD spectral calculation. Compound 2 displayed antibacterial activity against Kocuria rhizophila and Staphylococcus aureus with MIC 50 µg/mL and cytotoxicity against P388 murine leukemia cells with an IC50 value of 34 µM. Compounds 1 and 4 also showed cytotoxicity against P388 cells with IC50 values of 29 and 14 µM, respectively.

Krasilnikolides A and B and Detalosylkrasilnikolide A, Cytotoxic 20-Membered Macrolides from the Genus Krasilnikovia: Assignment of Anomeric Configuration by J-Based Configuration Analysis.

A chemical investigation of strain RD003821, belonging to the underexplored actinomycetes genus Krasilnikovia, led to the discovery of three novel polyketides: two 20-membered glycomacrolides, krasilnikolides A (1) and B (2), and an aglycone of 1, detalosylkrasilnikolide A (3). A major challenge in the structure elucidation of 1 was to determine the anomeric configuration of the α-l-6-deoxytalose (6dTal) unit, which was achieved by J-based configuration analysis (JBCA) that incorporated anomeric carbon- and proton-specific two-bond 13C-1H spin-spin coupling constants as diagnostic parameters. The updated criteria for the conformation/configuration assignment facilitated discrimination of three out of four stereochemical variants at the anomeric and the adjacent C2 positions, which expanded the scope of the JBCA method to determination of the anomeric configuration of aldohexopyranoses. Compounds 1 and 2 are the first macrolides decorated by 6dTal. Compounds 1-3 exhibited cytotoxicity against P388 murine leukemia cells with IC50 values of 14, 8.4, and 3.9 µM, respectively. In addition, 1-3 were antibacterial against the Gram-positive bacterium Kocuria rhizophila with MIC values of 25, 50, and 100 µg/mL. 1 was inhibitory against Staphylococcus aureus with an MIC of 50 µg/mL.

Collective Synthesis of Aplysiatoxin/Oscillatoxin Analogues by a Bioinspired Strategy.

Interest in the marine cyanobacteria natural products aplysiatoxin (ATX) and oscillatoxin (OTX) has been renewed recently due to the discovery of many new analogues, some exhibiting intriguing biological activities. We sought to develop a collective synthesis of these natural products, hypothesizing that ATX could serve as a common biosynthetic precursor. Herein, we reveal that the core structure of ATX has unique multiple reactivities giving access to the distinct ring structures of five of the analogues, depending upon the specific conditions used. Based on these findings, syntheses of the O-Me derivative of five analogues neo-deBr-ATX-B, OTX-H, OTX-D, neo-deBr-ATX-H, and OTX-I were achieved from the main fragment of ATX as a common intermediate in a few steps. These synthetic studies also led us to revise the relative configuration in the elucidated structures of neo-deBr-ATX-B and OTX-H, and obtain unnatural 8- and 12-membered lactones from the same intermediate.

Bisprenyl naphthoquinone and chlorinated calcimycin congener bearing thiazole ring from an actinomycete of the genus Phytohabitans.

A bisprenyl naphthoquinone, phytohabinone (1), and a calcimycin congener with unusual modifications, phytohabimicin (2), were isolated from the culture extract of Phytohabitans sp. RD003013. The structures of 1 and 2 were determined by NMR and MS analyses, and the absolute configuration of 2 was established by using electronic circular dichroism (ECD) calculation. The prenylation pattern of 1 was unprecedented among the known prenylated naphthoquinones. Compound 2 represents a spiroacetal core of polyketide origin substituted with a thiazole carboxylic acid and a dichrolopyrrole moiety, which is an unprecedented modification pattern in the known calcimycin family natural products. Remarkably, 2 showed moderate antimicrobial activity against a Gram-negative bacterium Ralstonia solanacearum while calcimycin was inactive. Additionally, 2 inhibits the migration of EC17 cancer cells at noncytotoxic concentrations.

Cyclic enaminones and a 4-quinazolinone from an unidentified actinomycete of the family Micromonosporaceae.

Four novel cyclic enaminones, designated RD4123A-D (1-4), and a new 4-quinazolinone metabolite, RD4123E (5), were isolated from the culture extract of an unidentified actinomycete strain RD004123, which belongs to the family Micromonosporaceae. Structures of 1-5 were determined by spectroscopic analyses using NMR, MS, and electronic circular dichroism (ECD), combined with quantum chemical calculations of ECD and NMR chemical shifts and biosynthetic consideration. Compounds 1-5 showed weak to modest cytotoxicity against murine leukemia P388 cells, while being inactive against bacteria and fungi.

Stereocontrolled Synthesis of C20S-C26 and C20R-C26 Fragments of Amphidinolide L.

Amphidinolide L is a cytotoxic macrolide isolated from marine symbiotic dinoflagellates of the genus Amphidinium. While its planar structure and the absolute stereochemistry of the C21-C26 part have been determined, six stereocenters have remained unassigned. Aiming at structure determination, we have developed a synthetic route to the C20S-C26 and C20R-C26 fragments via the Li-mediated stereocontrolled aldol reaction. Two aldehydes, 16 with the C22-hydroxy group and 19 with the C22-TES ether, were synthesized from lactone 4. The aldol reactions using the Li-enolate of 4-methyl-2-pentanone in THF provided the C20S-C26 fragment 20 from 16 and a 1:3.5 mixture of the C20-C26 fragment 22 favoring the C20R-isomer. Mechanistic studies based on an extensive search of transition states in explicit solvents indicated that the C20S-isomer would be generated via a tri-solvated transition state, while the C20R-isomer would be formed via a di-solvated transition state. The calculation emphasizes the importance of the coordination network as a higher-order complex composed of solvent molecules, aldehyde, enolate, and Li atoms in the reaction of 16 to minimize steric interactions but maximize the stabilizing effect by the coordination of solvents. The presence of the rotationally free aldehyde in the reaction of 19 results in moderate diastereoselectivity.

Catellatolactams A-C, Plant Growth-Promoting Ansamacrolactams from a Rare Actinomycete of the Genus Catellatospora.

Catellatolactams A-C (1-3), three novel ansamacrolactams, were isolated from the culture extract of an underexplored rare actinomycete of the genus Catellatospora. Spectroscopic and spectrometric analyses by NMR and MS elucidated the structure of 1 to be a lactamized pentaketide presumably extended on a 3-amino-5-hydroxybenzoic acid starter unit. Compounds 2 and 3 further received epoxidation and intramolecular cross-linking to incorporate a 2-indolinone unit, with a 3-amino-5-hydroxybenzoic acid pendant on 3. The absolute configurations of 2 and 3 were unequivocally established to both be 2S,6R,7R by comparison of the experimental NMR chemical shifts and ECD spectra with those predicted by DFT-based quantum chemical calculation. While 1-3 showed no appreciable antimicrobial activity or cytotoxicity, root elongation of germinated lettuce seeds was promoted by 2 and 3 at 1-10 µM.

Marinoquinolones and Marinobactoic Acid: Antimicrobial and Cytotoxic ortho-Dialkylbenzene-Class Metabolites Produced by a Marine Obligate Gammaproteobacterium of the Genus Marinobacterium.

Chemical investigation of the culture extract of a marine obligate proteobacterium, Marinobacterium sp. C17-8, isolated from scleractinian coral Euphyllia sp., led to the discovery of three new o-dialkylbenzene-class metabolites, designated marinoquinolones A (1) and B (2) and marinobactoic acid (3). Spectroscopic analysis using MS and NMR revealed the structures of 1 and 2 to be 4-quinolones with an o-dialkylbenzene-containing side chain at C3 and 3 to be a fatty acid bearing an o-dialkylbenzene substructure. The 4-quinolone form of 1 and 2 was unequivocally determined by comparison of the 1H, 13C, and 15N chemical shifts of 1 with those predicted for 2-methyl-4-quinolone A and its tautomer 2-methyl-4-quinolinol B by quantum chemical calculation. Compound 1 was proven to be racemic by X-ray crystallographic analysis and chiral-phase HPLC analysis of its chemical degradation product. Compounds 1-3 exhibited antimicrobial activity against bacteria and filamentous fungi at MIC of 6.3-50 µg/mL. In addition, all compounds showed cytotoxicity against P388 murine leukemia cells at micromolar ranges.

RCM approach to the CDE-tricyclic structure of nakiterpiosin.

A synthetic approach to the CDE-tricyclic structure of nakiterpiosin, a marine-derived antimitotic C-nor-D-homosteroid, is reported. The trans-disubstituted indanone was synthesized from a commercially available carboxylic acid in 9 steps, and the ring closing metathesis of the indanone constructed the trans-fused 5/6-membered ring system. The present approach enables the concise synthesis of the functionalized CDE-tricyclic structure, which will serve as a synthetic intermediate toward nakiterpiosin.

Kumemicinones A-G, Cytotoxic Angucyclinones from a Deep Sea-Derived Actinomycete of the Genus Actinomadura.

Chemical investigation of the fermentation products of a deep sea water-derived actinomycete, Actinomadura sp. KD439, identified seven new angucyclinones, designated as kumemicinones A-G (1-7), together with the known SF2315B and miaosporone E. NMR and MS spectroscopic analyses, combined with X-ray crystallography and quantum chemical calculations of NMR chemical shifts and electronic circular dichroism (ECD) spectra, uncovered the structures of new angucyclinones as regioisomers of SF2315B at the allyl alcohol unit (1 and 2), an epoxy ring-opened γ-hydroxy enone isomer (3), a B/C-ring-rearranged product (4), or dimers with a new mode of bridging (5-7), adding new structural variation to this antibiotic group. The absolute configuration of SF2315B was also determined by comparison of ECD spectra with those of 1 and 2. All the angucyclinones exhibited cytotoxicity against P388 murine leukemia cells, with IC50 values ranging from 1.8 to 53 µM.

Effect of pH on the Dehydrogenative Polymerization of Monolignols by Laccases from Trametes versicolor and Rhus vernicifera.

Dehydrogenative polymerization of coniferyl alcohol (CA) and sinapyl alcohol (SA) was conducted using commercial laccases, fungal laccase from Trametes versicolor (LacT) and plant laccase from Rhus vernicifera (LacR), at pH 4-7 to investigate how the enzymatic polymerization of monolignols differs between these two laccase systems. The enzyme activity of LacT was the highest at pH 4, whereas that of LacR was the highest at pH 7. A dehydrogenation polymer (DHP) was obtained only from CA in both laccase systems, although the consumption rate of SA was higher than that of CA. 1H-13C HSQC NMR analysis showed that DHPs obtained using LacT and LacR contained lignin substructures, including ß-O-4, ß-O-4/α-O-4, ß-ß, and ß-5 structures. At pH 4.5, the ß-O-4 structure was preferentially formed over the ß-O-4/α-O-4 structure, whereas at pH 6.5, the ß-O-4/α-O-4 structure was preferred. The pH of the reaction solution was more vital to affect the chemical structure of DHP than the origin of laccases.

Nomimicins B-D, new tetronate-class polyketides from a marine-derived actinomycete of the genus Actinomadura.

Three new tetronate-class polyketides, nomimicins B, C, and D, along with nomimicin, hereafter named nomimicin A, were isolated from the culture extract of Actinomadura sp. AKA43 collected from floating particles in the deep-sea water of Sagami Bay, Japan. The structures of nomimicins B, C, and D were elucidated through the interpretation of NMR and MS analytical data, and the absolute configuration was determined by combination of NOESY/ROESY and ECD analyses. Nomimicins B, C, and D showed antimicrobial activity against Gram-positive bacteria, Kocuria rhizophila and Bacillus subtilis, with MIC values in the range of 6.5 to 12.5 µg/mL. Nomimicins B and C also displayed cytotoxicity against P388 murine leukemia cells with IC50 values of 33 and 89 µM, respectively.

Structure Determination, Biosynthetic Origin, and Total Synthesis of Akazaoxime, an Enteromycin-Class Metabolite from a Marine-Derived Actinomycete of the Genus Micromonospora.

A new enteromycin-class antibiotic, akazaoxime (1), possessing an aldoxime functionality in place of O-methyl nitronic acid, was isolated from the cultured extract of a marine-derived actinomycete of the genus Micromonospora, along with known A-76356 (2). The structure of 1, including the absolute stereochemistry of three chiral centers, was established by comprehensive analysis of nuclear magnetic resonance (NMR) and mass spectrometry data coupled with magnetic anisotropy analysis of its phenylglycine methyl ester derivatives. The stereochemistry of 2, not determined previously, was proven to be the same as that of 1 on the basis of the similarity of their NMR and specific rotation data. Precursor feeding experiments using 13C-labeled compounds elucidated that the carbon skeletons of 1 and 2 are constructed from propionate (methylmalonate), leucine, and glycine. Establishment of the concise and flexible synthetic route to 1 enabled us to implement biological evaluation of 1 and its unnatural analogues, demonstrating weak to moderate antimicrobial activities of 1 against Gram-positive Kocuria rhizophila [minimum inhibitory concentration (MIC) of 50 µg/mL] and those of synthetic analogues against a plant pathogen Glomerella cingulata (MIC of 50 µg/mL) and a human pathogen Trichophyton rubrum (MIC of 25-50 µg/mL).

TMKS8A, an antibacterial and cytotoxic chlorinated α-lapachone, from a sea slug-derived actinomycete of the genus Streptomyces.

TMKS8A (1), a new chlorinated α-lapachone derivative, along with five known related metabolites, A80915 C (2), SF2415B1 (3), chlorinated dihydroquinone 3 (4), SF2415B3 (5), and A80915 C (6), were identified from the culture extract of Streptomyces sp. TMKS8, which was isolated from a sea slug, Paromoionchis tumidus. The structure of 1 was determined by the analysis of NMR and MS spectral data, assisted by NMR chemical shift prediction using DFT-based calculation. The absolute configuration was determined to be R by comparison of experimental and calculated ECD spectra. Compound 1 displayed antimicrobial activity against Gram-positive bacteria with MIC values ranging from 6.25 to 12.5 µg ml-1 and cytotoxicity against murine leukemia P388 cells with IC50 9.8 µM.

Cyclopeptides from the Mushroom Pathogen Fungus Cladobotryum varium.

Three new cyclopeptides with serial Phe residues were identified with the aid of HPLC-DAD analysis, from the culture broth of Cladobotryum varium, a fungal pathogen causing mushroom cobweb disease. Cladoamides A (1) and B (2) have two consecutive N-methylphenylalanine units in the destruxin class cyclic depsipentapeptide framework, while cladoamide C (3) has a three consecutive Phe motif in a cyclopentapeptide structure. Of these three cyclopeptides, 1 showed potent autophagy-inducing activity at 10 µg/mL, comparable to a positive control, rapamycin. For the determination of the absolute configurations of the Ile residues in 1 and 3, new conditions for separating Ile and allo-Ile, using a pentafluorophenyl-bonded solid phase and methanolic solvent, were established within the analytical scheme of the advanced Marfey's method, thus offering a convenient alternative to the C3 Marfey's method, which requires elution with a three-solvent mixture. The sequence of two d-Phe and one l-Phe in 3 was determined through NMR chemical shift prediction by DFT-based calculations and chemical synthesis, which demonstrated the significance of noncovalent interactions in the accurate calculation of stable conformers for peptides with multiple aromatic rings.