Isolation of Paraphaeoketones: A Plausible Biosynthetic Explanation for Paraphaeolactones via a Benzilic Acid Rearrangement Rather than a Favorskii Rearrangement.

Paraphaeoketones A-C (1-3) were isolated from the culture broth of Paraphaeosphaeria sp. KT4192. Their structures and relative configurations were determined using spectroscopic analysis and verified through density functional theory (DFT)-based chemical shift calculations. The absolute configurations of these compounds were determined by comparing the experimental electronic circular dichroism (ECD) spectra with those based on DFT calculations. We also propose a plausible biosynthetic route to 1-3. While our prior studies on the isolation and structural elucidation of paraphaeolactones (e.g., 4) led us to suggest a Favorskii rearrangement for their biosynthesis, the isolation of 2 prompted the proposal of an alternative biosynthesis for 4, featuring a benzilic acid rearrangement of 2. Moreover, an in vitro conversion of 2 into 4 was achieved successfully, suggesting that a biosynthetic pathway for paraphaeolactones involving a benzilic acid rearrangement is more plausible than the previously presumed Favorskii rearrangement pathway. Arguments based on DFT calculations for these pathways are also described.

Highly Functionalized Spirobisnaphthalenes from Roussoella sp. KT4147.

Highly functionalized spirobisnaphthalenes, preussomerins N (1) and O (2), and simpler compounds, such as 2,3-α-epoxypalmarumycin CP18 (3), 3α-hydroxy-CJ-12,372 (4), and 16 known structurally related congeners, were isolated from a culture broth of Roussoella sp. KT4147. Structural analysis revealed that 1 was a dimer of preussomerin G (6), connected by a nitrogen atom, and 2 was a derivative of 6 with a macommelin substructure. Preussomerin N (1) was considered to be biosynthetically derived via the Michael-type 1,4-addition of ammonia to 6, followed by another Michael addition to another molecule of 6. Contrarily, 2 was suggested to be derived through an endo-Diels-Alder cycloaddition between a diene derived from the (E)-enol form of macommelinal via an ene-reaction and dienophile 6. Compounds 1 and 2 exhibited potent cytotoxicity against COLO-201 human colorectal cancer cells.

Integrasone Derivatives Isolated from Lepteutypa sp. KT4162 and Their Anti-HIV-1 Integrase Activity.

Five integrasone derivatives, integrasone C (1), isointegrasone C (2), integrasone D1 (3), integrasone D2 (4), and integrasone E (5), were isolated from the culture broth of Lepteutypa sp. KT4162. Neither conventional NMR analyses nor DFT (density functional theory)-based computationally assisted chemical shift discussions were sufficient to elucidate the relative configuration of the 1,4-epoxydiol moiety. A combined analysis using the calculated nJCH values and HMBC spectra was helpful to establish the relative configuration. The absolute configurations of 1-5 were determined using DFT-based ECD (electronic circular dichroism) spectral analysis. Biological assays of these compounds revealed that 2 potently inhibits HIV-1 integrase without cytotoxicity.

HIV-1 Integrase Inhibitor, Dihydroobionin B, and the Investigation of Its Extraordinary Specific Rotation.

Dihydroobionin B (1), a chiral congener of known obionin B, was isolated from Pseudocoleophoma sp. KT4119, a freshwater fungus collected from a submerged wood block in Kochi Prefecture, Japan, in 2020. The planar structure of 1 was characterized by mass and NMR spectral analysis and confirmed by density functional theory (DFT)-based chemical shift calculations. Its absolute structure was determined by electronic circular dichroism spectral analysis. Notably, 1 exhibited an extraordinarily large specific rotation [[α]20D +1080 (c 0.056, CHCl3)], which was verified by DFT-based specific rotation calculations. However, these calculations indicated that the sign of the specific rotation based on static analysis was insufficient to determine the absolute configuration in this case. Furthermore, Pseudocoleophoma KT4119 produced coleophomapyrones A (2) and B (3) and coleophomaldehyde A (4). While this is the first report of 2 isolated from a natural source, it has also been prepared previously using a synthetic approach. Compound 1 potently inhibited HIV type 1 integrase (IC50 = 0.44 µM) without significant cytotoxicity. Finally, docking experiments were conducted to propose a plausible mechanism for the behavior of 1.

Pseudo-Enantiomeric Paraphaeolactones and Their Biosynthetic Derivatives from Paraphaeosphaeria sp. KT4192: A Proposition of a Favorskii Rearrangement for Their Biosynthesis.

Paraphaeolactones A1, A2, B1, and B2 (1-4, respectively), known arthropsadiol D (5), massariphenone (6) and its positional isomer 7, and massarilactones E (8) and G (9) were isolated from the culture broth of Paraphaeosphaeria sp. KT4192. Although the structural resemblance between 1 and 2 implies that these comprised a diastereomeric pair at the C-2 stereogenic center, electronic circular dichroism (ECD) spectral analyses revealed that they were pseudo-enantiomers possessing the common (2R)-configuration. Paraphaeolactones B1 and B2 (3 and 4) were the derivatives of 2, which equipped the 3-(1-hydroxy-2-oxopropyl)-4-methylcatechol moiety via an acetal bond at C-10. The relative configurations of their acetal carbons were elucidated by NOE experiments, and those of C-8' were deduced independently by ECD spectral analysis. The present study disclosed that 1-5, 8, and 9 contain a methylcyclohexene substructure with the same absolute configuration. This prompted us to reinvestigate the absolute configurations of known structurally related fungal metabolites, allowing us to conclude that the methylcyclohexene moieties of these natural products have the same absolute configuration despite the variety of configurations of other stereogenic centers. The plausible biosynthetic routes for 1-9 are discussed on the basis of the above conclusion. We propose a Favorskii rearrangement as the key transformation for biosyntheses of 1-4.

Structural Determination, Total Synthesis, and Biological Activity of Iezoside, a Highly Potent Ca2+-ATPase Inhibitor from the Marine Cyanobacterium Leptochromothrix valpauliae.

Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is a membrane protein on the endoplasmic reticulum (ER) that transports Ca2+ from the cytosol into the ER. As its function is associated with various biological phenomena, SERCA has been recognized as a promising druggable target. Here, we report the second-strongest SERCA-inhibitory compound known to date, which we isolated from the marine cyanobacterium Leptochromothrix valpauliae and named iezoside (1). The structure of iezoside (1) is fundamentally different from that of any other SERCA inhibitor, and its potency is the strongest among marine natural products (Ki 7.1 nM). In this article, we report our comprehensive analysis of iezoside (1), which covers its isolation, structural characterization supported by density functional theory (DFT) calculations and statistical analysis, total synthesis, and clarification of the mode of action of its potent antiproliferative activity (IC50 6.7 ± 0.4 nM against HeLa cells).

Cyclohelminthol CPs: Scope and Limitations of Density Functional Theory-Based Structural Elucidation of Natural Products.

The effectiveness and limitations of density functional theory (DFT) calculations in the structural determination of complexed and conformationally flexible natural products were demonstrated using the cyclohelminthols CP-1 (1) CP-2 (2), CP-3 (3), and CP-4 (4) newly isolated from Helminthosporium velutinum yone96. Prior to DFT calculations, the structures were tentatively assigned using conventional spectroscopic analyses. The structures were verified with reference to DFT-derived 13C and 1H NMR chemical shifts, 3JHH and nJCH values, and electronic circular dichroism (ECD) spectra. The 13C chemical shift calculations were very effective for verifying the ring-structure moieties but less effective for verifying the geometry of the side chain in which the juncture asymmetric carbon (C-16) was apart from the ring-structure moiety. However, 1H chemical shift calculations compensated for the imperfection of the latter. ECD spectral calculations were used to determine the absolute configurations. Calculations for virtual simple model molecules enabled us to evaluate the reliability of the ECD spectral calculation and derive the chiral torsion responsible for the characteristic Cotton effects.

Isolation and structural elucidation of novel fusicoccan dehydroxypericonicin A from Roussoella sp.

A potent antifungal fusicoccane dehydroxypericonicin A (1) was isolated from Roussoella sp. along with known allantofuranone (2). The relative structure of 1 was fully elucidated by NMR spectroscopic manner. The suggested relative structure was confirmed by density functional theory (DFT)-based 13C NMR chemical shift calculations. The absolute configuration was investigated by a spectral comparison of the experimental electronic circular dichroism spectrum with that based on the DFT calculations.

Semipinacol rearrangement of a bicyclo[7.2.0]undecane framework into a bicyclo[6.3.0]undecane skeleton: a model study on the biosynthesis of seiridiasteriscane A.

Seiridiasteriscane A is an asteriscane-type sesquiterpenoid bearing a trans-fused bicyclo[6.3.0]undecane skeleton. Although its biosynthesis has been proposed to involve a semipinacol rearrangement of a putative intermediary acetate bearing a bicyclo[7.2.0]undecane ring system (presumably derived from coisolated pestalotiopsin M) followed by epimerization of the resulting cis-fused seiridiasteriscane B, such a type of semipinacol rearrangement has never been reported so far. Our model study revealed that a 1-hydroxybicyclo[7.2.0]undecan-2-yl acetate underwent a smooth and stereospecific semipinacol rearrangement with the assistance of Et2AlCl to give the corresponding bicyclo[6.3.0]undecane-9-one. In addition, the resulting cis-fused 5,8-bicyclic ketone was partially epimerized to the corresponding trans-fused ketone by prolonged adsorption onto a silica gel plate. These results may support a recently proposed biosynthetic pathway of seiridiasteriscane A.

Cyclohumulanoid Sesquiterpenes from the Culture Broth of the Basidiomycetous Fungus Daedaleopsis tricolor.

A series of cyclohumulanoids, i.e., tricocerapicanols A-C (1a-1c), tricoprotoilludenes A (2a) and B (3), tricosterpurol (4), and tricoilludins A-C (5-7) were isolated along with known violascensol (2b) and omphadiol (8) from the culture broth of Daedaleopsis tricolor, an inedible but not toxic mushroom. The structures were fully elucidated on the basis of NMR spectroscopic analysis, and the suggested relative structures were confirmed via density functional theory (DFT)-based chemical shift calculations involving a DP4 probability analysis. In the present study, the 1H chemical shifts were more informative than the 13C chemical shifts to distinguish the diastereomers at C-11. The absolute configurations of 1-5 were determined by comparing the experimental and calculated electronic circular dichroism (ECD) spectra. For 6 and 7, the same chirality was assigned according to their biosynthetic similarities with the other compounds. The successful assignment of some Cotton effects was achieved by utilizing DFT calculations using simple model compounds. The plausible biosynthesis of 1-7 was also discussed on the basis of the structural commonality and general cyclohumulanoid biosynthesis. Compounds 2a and 5 were found to simultaneously induce hyphal swelling and branching at 5.0 µg/mL against a test fungus Cochliobolus miyabeanus.

Biochemistry-Guided Prediction of the Absolute Configuration of Fungal Reduced Polyketides.

Highly reducing polyketide synthases (HR-PKSs) produce structurally diverse polyketides (PKs). The PK diversity is constructed by a variety of factors, including the ß-keto processing, chain length, methylation pattern, and relative and absolute configurations of the substituents. We examined the stereochemical course of the PK processing for the synthesis of polyhydroxy PKs such as phialotides, phomenoic acid, and ACR-toxin. Heterologous expression of a HR-PKS gene, a trans-acting enoylreductase gene, and a truncated non-ribosomal peptide synthetase gene resulted in the formation of a linear PK with multiple stereogenic centers. The absolute configurations of the stereogenic centers were determined by chemical degradation followed by comparison of the degradation products with synthetic standards. A stereochemical rule was proposed to explain the absolute configurations of other reduced PKs and highlights an error in the absolute configurations of a reported structure. The present work demonstrates that focused functional analysis of functionally related HR-PKSs leads to a better understanding of the stereochemical course.

Biosynthetic Machinery of 6-Hydroxymellein Derivatives Leading to Cyclohelminthols and Palmaenones.

Oxygenated cyclopentene systems are unique structural motifs found in fungal polyketides such as terrein, cyclohelminthols, and palmaenones. Here we report the identification of the biosynthetic gene clusters for cyclohelminthols and palmaenones and the functional characterization of the polyketide synthases and halogenases involved in the construction of 6-hydroxymellein derivatives. Heterologous expression in Aspergillus oryzae demonstrated that 6-hydroxymellein is a common biosynthetic intermediate and that chlorination occurs in the early stages of its products' biosynthesis. This was further confirmed by in vitro enzymatic reactions conducted in the presence of recombinant proteins. Plausible means of biogenesis of fungal polyketides from 6-hydroxymellein derivatives, additionally supported by the reported labeling patterns of terrein and structurally related fungal polyketides, are also discussed. This study sets the stage for elucidation of the biosynthetic machinery of fungal polyketides of this type.

DFT calculation-assisted stereo-structural assignment of arundifungin.

Arundifungin (1) has been reported as a potent antifungal agent against Candida and Aspergillus spp; however, only its planar structure has been disclosed. This paper describes the assignment of the relative and absolute configuration of 1, which includes (a) determination of the relative configuration of the ABCD polycyclic ring moiety on the basis of detailed nuclear magnetic resonance (NMR) analysis, followed by the confirmation with density functional theory (DFT)-based 13 C NMR chemical shift calculations, (b) determination of the absolute configuration of the ABCD polycyclic ring moiety by observing a positive Cotton effect at 217 nm because of the C-8/C-9 tetrasubstituted double bond and its reproduction using DFT calculations, (c) determination of the configurational relationship between C-17 and C-20 by a combination of nuclear Overhauser effect (NOE) analysis and DFT-based conformational analysis, and (d) determination of the relative and absolute configuration of the C-24 and C-25 asymmetric centers on the acyclic side chain by a combination of chemical derivatization including modified Mosher's method and DFT-based conformational analysis, followed by electronic circular dichroism (ECD) spectral reproduction. Present study also discovered 26-deoxyarundifungin (2) of which relative structure was readily elucidated by 1 H and 13 C spectral comparison with those of 1. Since 2 exhibits slightly weaker antifungal activity against Cochliobolus miyabeanus than 1, the hydroxy group at C-26 moderately contributes to the activity.

Structure-activity relationships of trichothecenes against COLO201 cells and Cochliobolus miyabeanus: The role of 12-epoxide and macrocyclic moieties.

The novel trichothecene 12-deoxytrichodermin (3) was isolated from the fungus Trichoderma sp. 1212-03, and included with other known natural trichothecenes in a structure-activity relationship investigation against a human colon cancer cell line (COLO201) and filamentous fungus Cochliobolus miyabeanus. This revealed that the 12-epoxide functionality is critical for the cytotoxicity of simple trichothecenes trichodermin (4) and deoxynivalenol (2), while not critical for the cytotoxicity of roridin J (6) and epiisororidin E (8). In contrast, 12-epoxide is essential for the antifungal activity.

Identification of neomacrophorins isolated from Trichoderma sp. 1212-03 as proteasome inhibitors.

Neomacrophorins I-III (1-3) and X have previously been isolated from Trichoderma sp. 1212-03. Their mode of action against cancer cells and the mechanism of biosynthesis of the characteristic [4.4.3] propellane framework in neomacrophorin X have not been reported. The isolation and characterization of neomacrophorins IV (4), V (5), and VI (6) is reported. Epoxyquinones 1, 4, and 6 potently induced apoptotic cell death in human acute promyelocytic leukemia HL60 cells, while epoxysemiquinols 2, 3, and 5 showed weak activity. This indicates that the epoxyquinone moiety is crucial for apoptosis-inducing activities of neomacrophorins. We also found that neomacrophorins inhibit proteasome in vitro, and 1, 4, and 6 induced significant accumulation of ubiquitinated proteins in HL60 cells. These activities were completely suppressed by a nucleophile, N-acetyl-l-cysteine (NAC). The analysis of reaction mechanisms using LC-MS suggested that C2' and C7' of neomacrophorins could be Michael acceptors in the reaction with NAC methyl ester (NACM). These findings indicated that the electrophilic properties of neomacrophorins are responsible for both their potent biological effects and the biosynthesis of unique [4.4.3] propellane framework in neomacrophorin X.

Isolation of Peribysins O, P, and Q from Periconia macrospinosa KT3863 and Configurational Reinvestigation of Peribysin E Diacetate from Periconia byssoides OUPS-N133.

Peribysins O (1), P (3), and Q (4) were isolated from Periconia macrospinosa KT3863. The relative configuration of the 6,7-epoxide of 1 was elucidated by performing quantitative NOE experiments. The structure of 2, which is a tautomer of 1 present in CDCl3 solutions in 5% abundance, was also fully characterized by NMR analysis. Their absolute configurations were independently determined by the modified Mosher's method (for 1 and 3), the electronic circular dichroism (ECD) exciton coupling theory after conversion into dibenzoate 9 (for 3), and theoretical ECD calculations (for 1, 3, and 4). The obtained relative structures 1, 3, and 4 were verified by calculating their 13C chemical shifts using density functional theory (DFT). Although the established (4 S)-enantiomer for 1-4 is in accordance with that of other peribysins isolated from the related fungus Periconia byssoides OUPS-N133, Danishefsky's total synthesis of peribysin E (5) led to the subsequent revision of the (2 R,4 S,5 R,6 S,7 S,8 R,10 S)-enantiomer to the (2 S,4 R,5 S,6 R,7 R,8 S,10 R)-enantiomer. This discordance led us to reinvestigate the configuration using time-dependent DFT-based ECD spectral calculations, which supported the original (4 S)-enantiomer.

Efficient Protocol for Accurately Calculating 13C Chemical Shifts of Conformationally Flexible Natural Products: Scope, Assessment, and Limitations.

An efficient protocol for calculating 13C NMR chemical shifts for natural products with multiple degrees of conformational freedom is described. This involves a multistep procedure starting from molecular mechanics and ending with a large basis set density functional model to obtain accurate Boltzmann conformer weights, followed by empirically corrected density functional NMR calculations for the individual conformers. The accuracy of the protocol (average rms <4 ppm) was determined by application to ∼925 diverse natural products, the structures of which have been confirmed either by X-ray crystallography or independent synthesis. The protocol was then applied to ∼ 2275 natural products, the structures of which were elucidated mainly by NMR and MS data. Five to ten percent of the latter compounds exhibited rms errors significantly in excess of 4 ppm, suggesting possible structural or signal assignment errors. Both data sets are available from an online browser ( NMR.wavefun.com ). The procedure can be and has been fully automated and is practical using present-generation personal computers, requiring a few hours or days depending on the size of the molecule and number of accessible conformers.

Biosynthetic study of conidiation-inducing factor conidiogenone: heterologous production and cyclization mechanism of a key bifunctional diterpene synthase.

Conidiogenone, a diterpene with a unique structure, is known to induce the conidiation of Penicillium cyclopium. The biosynthetic pathway of (-)-conidiogenone has been fully elucidated by the heterologous expression of biosynthetic genes in Aspergillus oryzae and by in vitro enzyme assay with 13C-labeled substrates. After construction of deoxyconidiogenol by the action of bifunctional terpene synthase, one cytochrome P450 catalyzes two rounds of oxidation to furnish conidiogenone. Notably, similar biosynthetic genes are conserved among more than 10 Penicillium sp., suggesting that conidiogenone is a common conidiation inducer in this genus. The cyclization mechanism catalyzed by terpene synthase, which involves successive 1,2-alkyl shifts, was fully elucidated using 13C-labeled geranylgeranyl pyrophosphate (GGPP) as substrate. During the structural analysis of deoxyconidiogenol, we observed broadening of some of the 13C signals measured at room temperature, which has not been observed with other structurally related compounds. Careful examination using techniques including 13C NMR studies at -80 °C, conformational analysis and prediction of the 13C chemical shifts using density functional theory gave insights into this intriguing phenomenon.

Cyclohelminthols Y1-Y4 Metabolites Possessing Two Spirocyclopropanes in Their Structure.

Cyclohelminthols Y1-Y4 (1-4) were isolated from the culture broth of Helminthosporium velutinum yone96. These compounds are diastereomers to each other featuring 3-azabicyclo[3.1.0]hexane-6-spirocyclopentane linked with a cyclopentanespirocyclopropane framework. Their planar structures were established via the comparison of their spectra with the simpler analogue cyclohelminthol X as well as analysis of their HMBC spectra. Although the proton-deficient core frameworks of 1-4 prevented us from obtaining configurational information via conventional NMR analysis, their total structures involving the relative and absolute configurations were established using density functional theory (DFT)-based molecular modeling calculations. The present study demonstrates the effectiveness of the comparison between the theoretical and experimental δ13C values for stereochemical analysis by focusing on the carbons that show relatively large δ13C deviations among the isomers. The G-ring of these molecules most likely originates from the cyclopropanation of the C6C7 double bond with the carbene equivalent 6 derived from cyclohelminthol IV (7), which was isolated from the same producer fungus. Preliminary biological experiments revealed the potent cytotoxicity of the (6 S)-isomers against COLO201 cells, whereas the (6 R)-isomers exhibited weak activity. The antifungal assay with Cochiobolus miyabeanus showed a slightly different profile.

Cyclohelminthol X, a Hexa-Substituted Spirocyclopropane from Helminthosporium velutinum yone96: Structural Elucidation, Electronic Circular Dichroism Analysis, and Biological Properties.

Helminthosporium velutinum yone96 produces cyclohelminthol X (1), a unique hexa-substituted spirocyclopropane. Although its molecular formula and NMR spectral data resemble those of AD0157, being isolated from marine fungus Paraconiothyrium sp. HL-78-gCHSP3-B005, our detailed analyses disclosed a totally different structure. Chemical shift calculations and electronic circular dichroism spectral calculations were quite helpful to establish the structure, when those were performed based on density functional theory. The carbon framework of cyclohelminthols I-IV is found at the C1-C8 propenylcyclopentene substructure of 1. Thus, 1 is assumed to be biosynthesized by cyclopropanation between an oxidized form of cyclohelminthol IV and a succinic anhydride derivative 4. Cytotoxicity for two cancer cell lines and proteasome inhibition efficiency are measured.