Isolation and Structure Elucidation of JBIR-157, a Skeletally Novel Aromatic Polyketide Produced by the Heterologous Expression of a Cryptic Gene Cluster

Abstract

Heterologous expression of natural compound biosynthetic gene clusters (BGCs) is a robust approach for not only revealing the biosynthetic mechanisms leading to the compounds, but also for discovering new products from uncharacterized BGCs. We established a heterologous expression technique applicable to huge biosynthetic gene clusters for generating large molecular secondary metabolites such as type-I polyketides. As an example, we targeted concanamycin BGC from Streptomyces neyagawaensis IFO13477 (the cluster size of 99 kbp), and obtained a bacterial artificial chromosome (BAC) clone with an insert size of 211 kbp that contains the entire concanamycin BGC. Interestingly, heterologous expression for this BAC clone resulted in two additional aromatic polyketides, ent-gephyromycin, and a new compound designated as JBIR-157, together with the expected concanamycin. Bioinformatic and biochemical analyses revealed that a cryptic biosynthetic gene cluster in this BAC clone was responsible for the production of these type-II polyketide synthases (PKS) compounds. Here, we describe the production, isolation, and structure elucidation of JBIR-157, determined primarily by a series of NMR spectral analyses.

Introduction

Chemists have long sought naturally occurring secondary metabolites with structural diversity and bioactivity.¹⁾ Natural products and their derivatives currently account for around 40% of clinical drugs.²⁾ Microorganisms can produce many classes of secondary metabolites, such as polyketides, peptides, alkaloids, terpenoids, and saccharides. Recent advances in genome sequencing revealed that a Streptomyces genus, one of the most abundant sources of natural products, can carry more than 30 potential biosynthetic gene clusters.^3,4) However, despite efforts to isolate and characterize secondary metabolites, most biosynthetic gene clusters are transcriptionally inactive under laboratory cultivation conditions,⁵⁾ indicating that many natural products remain undiscovered. We decided that heterologous expression technology is the most promising strategy for utilizing cryptic biosynthetic genes to produce compounds. Several heterologous expression experiments have previously been investigated, leading to a small number of reports regarding large molecules, such as macrolides biosynthesized by type I polyketide synthases (PKS). The limited success of previous studies is due to the large size of the gene cluster, prompting us to develop bacterial artificial chromosome (BAC) technology, which can clone up to 300 kb of DNA fragments. Our technique has enabled the heterologous production of mediomycin, neomediomycin,⁶⁾ JBIR-156,⁷⁾ quinolidomicin,⁸⁾ and desertomycin,⁹⁾ whose biosynthetic gene clusters are 161, 183, 137, 213, and 127 kbp in length, respectively. Subsequently, we attempted to produce concanamycin(s) by using our heterologous expression technique for a large-molecular-weight natural product. Heterologous expression of the BAC clone pKU503ccn harbouring concanamycin BGC derived from S. neyagawaensis IFO13477 resulted in the production of concanamycin.^10,11) We further revealed that the same transformant produces two other aromatic polyketides, ent-gephyromycin (1) and a novel compound designated as JBIR-157 (2, Fig. 1), when grown in a medium different from that used to produce concanamycin. Herein, we report the fermentation, isolation, structure elucidation, and proposed biosynthetic pathway of ent-gephyromycin (1) and JBIR-157 (2).

Fig. 1. Structures of (a) ent-Gephyromycin (1) and (b) JBIR-157 (2)

Results and Discussion

Fermentation, Isolation, and Structure Elucidation

Streptomyces avermitilis SUKA32/SAP1::pKU503ccn (carrying both the concanamycin cluster and ent-gephyromycin/JBIR-157 clusters)¹¹⁾ was inoculated into 50-mL tubes containing 15 mL of seed medium consisting of 0.5% glucose, 1.5% soybean meal, and 0.5% yeast extract (adjusted to pH 7.5 before sterilization). The test tubes were shaken on a reciprocal shaker (320 rpm) at 27 °C for 2 d. A 2.5 mL aliquot of the vegetative culture was used to inoculate a baffled 500-mL flask containing 100 mL of production medium comprising 6.0% glucose, 0.2% ammonium sulfate, 0.05% dipotassium hydrogenphosphate, 0.2% sodium chloride, 0.5% calcium carbonate, 0.2% yeast extract, 0.01% MgSO₄·7H₂O, 0.005% FeSO₄·7H₂O, 0.005% ZnSO₄·7H₂O, and 0.005% MnSO₄·7H₂O in 1 L of deionized water adjusted to pH 7.2. Fermentation was carried out on a rotary shaker at 27 °C for 5 d at 180 rpm.

The fermentation broth (1 L) was centrifuged and the supernatant was extracted with EtOAc (300 mL×3). The organic phase was dried over Na₂SO₄, then evaporated to dryness. The residue (111.0 mg) was loaded onto a medium pressure liquid chromatography (MPLC) silica gel column and eluted with a n-hexane–EtOAc stepwise solvent system (0 and 25% EtOAc) and a chloroform–MeOH stepwise solvent system (0, 1, 2, 5, 10, and 30% MeOH). The 5% MeOH eluate was evaporated in vacuo, then the residue (17.9 mg) was fractionated by preparative HPLC using 54% aqueous MeOH containing 0.1% formic acid to afford compound 1 (2.1 mg). Similarly, the residue of the 10% MeOH eluate (17.0 mg) was purified under the same HPLC conditions to yield compound 2 (2.9 mg).

Compound 1 was obtained as a pale yellow oil and high resolution (HR)-electrospray ionization (ESI)MS analysis showed it to have the molecular formula C₁₉H₁₈O₈. The planar structure of 1 was determined by ¹H- and ¹³C-NMR, a series of two dimensional (2D) NMR analyses, double quantum filtered correlation spectroscopy (COSY) (DQF-COSY), gradient-enhanced heteronuclear single quantum coherence with adiabatic pulses (HSQCAD), and gradient-selected heteronuclear multiple bond correlation using adiabatic pulses (HMBCAD) (Table 1, Fig. 1(a)) to establish that 1 is ent-gephyromycin by comparison of its NMR data and specific rotation ([a]²⁴_D=+67 (c 0.2, MeOH) with literature values.^11–13)

Table 1. ¹H- and ¹³C-NMR Data for JBIR-157 (2)

Position	δC	δΗ (multiplicity, J in Hz)
1	179.3	—
2	86.6	—
3	48.3	2.56 (d, 9.7)
		1.99 (dd, 9.7, 2.8)
4	85.3	—
5	49.1	2.26 (dd, 14.2, 2.7)
		1.91 (d, 14.0)
6	78.6	—
7	35.6	2.00 (m)
		1.39 (ddd, 13.8, 10.5, 5.6)
8	18.3	2.75 (m)
		2.56 (m)
9	122.7	—
10	191.0	—
11	116.5	—
12	161.8	—
13	125.7	7.16 (d, 8.3)
14	134.7	7.47 (t, 7.7)
15	119.4	7.51 (d, 7.0)
16	132.4	—
17	184.5	—
18	164.7	—
19	19.8	1.51 (s)

NMR data were measured at 600 MHz for ¹H and 150 MHz for ¹³C with the residual solvent peak as the internal standard (δ_H 3.31, δ_C 49.0 ppm).

Compound 2 was obtained as a pale red oil, and HR-ESIMS analysis indicated the same molecular formula as ent-gephyromycin (1) and urdamycin X, a urdamycin analogue produced by Streptomyces fradiae with mutations of both urdQ and urdR.¹⁴⁾ However, the NMR spectra of 2 did not match those of either compound.

Structure determination of 2 was based on ¹H, ¹³C-NMR, a series of 2D-NMR analyses, and an Incredible Natural Abundance Double Quantum Transfer Experiment (INADEQUATE). The tabulated ¹H- and ¹³C-NMR data deduced by the HSQC spectrum are shown in Table 1. The data show signals for a methyl group [δ_C 19.8 (C-19)], four methylenes [δ_C 49.1 (C-5), δ_C 48.3 (C-3), δ_C 35.6 (C-7), and δ_C 18.3 (C-8)], three aromatic methines [δ_C 134.7 (C-14), δ_C 125.7 (C-13), and δ_C 119.4 (C-15)], three oxygenated quaternary carbons [δ_C 85.2 (C-2), δ_C 83.8 (C-4), and δ_C 77.2 (C-6)], three quaternary aromatic carbons [δ_C 132.4 (C-16), δ_C 122.7 (C-9), and δ_C 116.5 (C-11)], two oxygenated quaternary aromatic carbons [δ_C 164.7 (C-18) and δ_C 161.8 (C-12)], one ester carbonyl carbon [δ_C 179.3 (C-1)], and two ketone carbons [δ_C 191.0 (C-10) and δ_C 184.5 (C-17)].

The proton sequence from aromatic proton H-13 (δ_H 7.16) to H-15 (δ_H 7.51) through H-14 (δ_H 7.47) suggests the presence of a 1,2,3-trisubstituted benzene ring. HMBC correlations were obtained from aromatic proton H-13 to a quinone carbonyl carbon C-10 (δ_C 191.0), aromatic carbons C-11, C-15 and C-12, from aromatic proton H-14 to C-12 and C-16, from H-15 to C-11, C-13 and quinone carbonyl carbon C-17 (δ_C 184.5) at the peri-position, from methylene protons H₂-8 (δ_H 2.75, 2.56) to C-9, C-10, and C-18. These data together with the ¹³C chemical shift values for C-12 (δ_C 161.8) and C-18 (δ_C 164.7) supported the 3,8-dihydroxy-10-methyl-1,4-naphthoquinone moiety, as shown in Fig. 2(a).

Fig. 2. Structure Determination for JBIR-157 (2)

(a) Correlations in DQF-COSY and key correlations in HMBCAD spectra of 2. (b) Correlations in INADEQUATE spectra of 2. Structures of (c) 18-O-methyl JBIR-157 (2-Me) and (d) 2,12,18-O-triacetyl JBIR-157 (2-Ac). (e) ROESY correlations of 2. (f) Structures used for molecular modeling. 2a has the same relative configuration as JBIR-157 (2).

The remaining substructure was determined by INADEQUATE spectroscopy, a powerful NMR technique that provides direct evidence of carbon connections. It is difficult to determine these connections unambiguously using only ¹H − ¹H correlations and ¹H − ¹³C long-range couplings. The carbon sequences from methylene carbon C-7 to carbonyl carbon C-1 through oxygenated quaternary carbon C-6 (δ_C 77.2), methylene carbon C-5, oxygenated quaternary carbon C-4 (δ_C 83.8) coupled to methyl group C-19, methylene carbon C-3, and oxygenated quaternary carbon C-2 (δ_C 85.2) coupled to methyl group C-6, established the tri-oxygenate cyclopentane moiety, as shown in Fig. 2(b). The connectivity of the naphthoquinone moiety and this substructure was verified by the spin coupling between methylene protons H₂-7 (δ_H 1.39, 2.00) and H₂-8.

Evidence of a naphthoquinone moiety, an ester carbonyl carbon, and cyclopentane ring requiring ten degrees of unsaturation led to 2 being inferred to be a lactone ring system. Methylation and acetylation of 2 provided the predicted ring substructure, yielding 18-O-methylated (2-Me) and 2,12,18-O-triacetylated (2-Ac) as products, respectively (Figs. 2(c), (d), Supplementary Figs. S12, S13). This finding, together with the carbonyl stretching absorption band in IR spectra (1770 cm⁻¹), suggests the presence of γ-lactone and not β-lactone. The planar structure of 2 was determined as shown in Fig. 1(b), and contains a 1,4-naphthoquinone moiety and a 2-oxabicyclo[2.2.1]heptan-3-one scaffold.

The relative configuration of 2 was determined as shown in Fig. 2(e) by rotating frame nuclear Overhauser effect spectroscopy (ROESY) correlations between H-3a/H-5a and H-5b/H-7, and we calculated the theoretical ¹³C-NMR chemical shift values of two possible stereoisomers, 2a and 2b, using density functional theory (DFT) calculations (Fig. 2(f)), the calculated values corresponding to 2a are in good agreement with the experimental value (Supplementary Table S1). Given the predicted biosynthetic pathway (Fig. 3), the absolute configuration of 2 is likely to be as shown in Fig. 1(b). The C-4 position of 2, corresponding to the C-3 position of ent-gephyromycin, was predicted to be in the R configuration. This determination was supported by the comparison of the experimental electronic circular dichroism (ECD) spectrum with the calculated (Supplementary Fig. S10).

Fig. 3. Proposed Biosynthetic Pathway for ent-Gephyromycin and JBIR-157 (2)

All intermediate structures and assignments of enzymes are hypothetical.

Prediction of the Biosynthetic Route

We predicted the biosynthetic route for 1 and 2 after branching from the hypothetical intermediate 4 (Fig. 3). Compound 4 would be a substrate for Baeyer–Villiger monooxygenase (BVMO) type FAD-dependent monooxygenase to give epoxide 5 or hemiacetal 6, possible precursors for 1 or 2, respectively. The C-3 hydroxyl could attack C-12a of 5 to form the bridge structure of 1. The hemiacetal ring of 6 could open to give diketone 7, which might undergo benzylic acid rearrangement-like ring shrinking reaction to yield cyclopentane carboxylic acid 8. Otherwise, 6 could directly convert to 8 via a Favorskii-type rearrangement using the hemiacetal oxygen as an electron sink. The egp cluster encodes two putative BVMOs, EgpO4 and the N-terminal domain of EgpO5. EgpI show 65% amino acid identity to the alpha/beta hydrolase family of thioesterase.¹¹⁾ In the egp cluster, since there is no other enzyme besides EgpI that activates carboxylic acids, we speculate that this enzyme is involved in the formation of the lactone ring. We will further validate these hypotheses by future biochemical analysis.

Biological Activities of ent-Gephyromycin, JBIR-157, and Its Derivatives

Gephyromycin C was reported to induce apoptosis against prostate cancer PC3 cells through the inhibition of heat shock protein Hsp90.¹⁵⁾ We investigated biological activities of isolated compounds and derivatives of JBIR-157. We examined the cytotoxic activities of 1, 2, 2-Me, and 2-Ac against human ovarian adenocarcinoma SKOV-3, malignant pleural mesothelioma Meso-1, and T lymphoma Jurkat cells. As the results, JBIR-157 and its derivatives showed moderate cytotoxic activities. The magnitude of their activities was in order 2-Me, 2-Ac and 2. 2-Me exhibited cytotoxicity against SKOV-3, Meso-1, and Jurkat cells with IC₅₀ values of 6.02, 6.01, and 3.51 µM, respectively (Supplementary Table S3). While, the IC₅₀ values of etoposide against SKOV-3 and Meso-1 were 3.98 and 3.89 µM, respectively. These data suggested that the cytotoxic activities of 2-Me and etoposide against SKOV-3 and Meso-1 were almost identical.

We also examined antimicrobial and antioxidative activities of these compounds. Only 2-Me showed quite slight antimicrobial activity against Staphylococcus aureus with the IC₅₀ value of 40.2 µM (Supplementary Table S4). Other all the compounds did not show any significant antimicrobial (Supplementary Table S4) and antioxidant activities (data not shown). Interestingly, ent-gephyromycin and JBIR-157 did not show significant activities, but the derivatives were more potent, suggesting that the 18-OH substituted new skeletal substructure may become a useful pharmacophore.

Conclusion

We reported herein the isolation and structure elucidation of ent-gephyromycin (1) and JBIR-157 (2). Compound 2 contains a unique 2-oxabicyclo[2.2.1]heptan-3-one moiety. We identified the new products of type II PKS by utilizing the BAC cloning technique, a high-capacity approach for capturing large DNA inserts, and by heterologous expression using a host strain that does not naturally produce secondary metabolites. Involvement of the BVMO family of enzymes in the formation of the bicyclic system of JBIR-157 was suggested by annotation analysis of the biosynthetic gene cluster. We examined the biological activities of isolated compounds, and JBIR-157 including its derivatives showed moderate cytotoxicity against SKOV-3, Meso-1, and Jurkat cells. Further biosynthetic analysis and structure–activity relationship studies on the unique bicyclic structure of JBIR -157 are currently underway.

Experimental

General

NMR spectra were generally recorded on a Varian NMR 600 NB CL spectrometer (Varian, CA, U.S.A.). INADEQUATE spectra were measured with a Bruker (Billerica, MA, U.S.A.) Avance III 800 spectrometer equipped with cryogenic TXO probe. The coupling constants (J) are given in Hertz. Measurements were carried out at room temperature. Chemical shifts (δ) are reported in ppm using the residual solvent signal as an internal standard. The data are reported as (s = singlet, d = doublet, dd = double doublet, dt = double triplet, m = multiplet or unresolved, coupling constant(s), integration). ¹³C-NMR spectra were recorded with complete ¹H decoupling. Infrared (IR) spectra were recorded on an IRSpirit FT-IR spectrometer (Shimadzu Co., Kyoto, Japan). Absorption band positions are given in wavenumber (cm⁻¹). HR-ESIMS data were recorded using a Waters XevoG2-Tof mass spectrometer (Waters, MA, U.S.A.). Optical rotations were measured on a Horiba SEPA-500 polarimeter (Horiba, Kyoto, Japan) using a sodium lamp at a wavelength of 589 nm. Normal phase MPLC was carried out using a SNAP Ultra column (Biotage Co., Ltd., Yokohama, Japan). Reversed-phase MPLC was carried out using a Purif-Pack ODS-60 column (Shoko Scientific Co., Ltd., Yokohama, Japan). Preparative RP-HPLC was conducted using a CAPCELL PAK C18 MGII column (20 i.d. × 150 mm, Shiseido Co., Ltd., Tokyo, Japan).

JBIR-157 (2)

Red oil; [a]²⁴_D −35 (c 0.50, MeOH); UV λ_max (MeOH) nm (logε): 436 (3.45), 321 (4.17), 248 (4.20); IR (ATR) cm⁻¹: 3346, 2976, 2941, 1770, 1648, 1616, 1457, 1375; ¹H-NMR (600 MHz, CD₃OD) and ¹³C-NMR (150 MHz, CD₃OD), see Table 1; ESI-MS m/z: 411.1065 (Calcd for C₂₀H₂₀O₈Na: 411.1056).

Synthesis of 18-O-Methyl JBIR-157 (2-Me)

To a solution of JBIR-157 (2) (5.0 mg, 0.013 mmol) in toluene/methanol (9/1, 0.5 mL) at room temperature was added 0.6 mol/L trimethylsilyldiazomethane hexane solution (50 µL). After stirring for 30 min, the solution was concentrated. The crude material was purified using column chromatography using 10% CHCl₃/MeOH solution as eluent to yield 2-Me as a pale yellow oil (3.9 mg, 0.010 mmol, 77%). Compound 2-Me; pale yellow oil; [a]²⁴_D −42 (c 0.38, MeOH); UV λ_max (MeOH) nm (logε): 318 (3.91), 264 (3.98); IR (ATR) cm⁻¹: 3451, 2943, 2360, 1782, 1669, 1628, 1457, 1312, 1266, 1245; ¹H-NMR (600 MHz, CD₃OD) and ¹³C-NMR (150 MHz, CD₃OD), see Table S2; ESI-MS m/z: 411.1065 (Calcd for C₂₀H₂₀O₈Na: 411.1056).

Synthesis of 2,12,18-O-Triacetyl JBIR-157 (2-Ac)

To a solution of JBIR-157 (2) (5.0 mg, 0.013 mmol) in pyridine (0.5 mL) at room temperature was added acetic anhydride (2 drops). After stirring overnight, the solution was partitioned between EtOAc and water. The organic layers were combined, dried over Na₂SO₄, filtered, and concentrated. The crude material was purified by column chromatography using 2% CHCl₃/MeOH solution as an eluent to yield 2-Ac as a pale yellow oil (2.1 mg, 0.004 mmol, 31%). Compound 2-Ac; pale yellow oil; [a]²⁴_D −50 (c 0.21, MeOH); UV λ_max (MeOH) nm (logε): 304 (3.86), 256 (3.99); IR (ATR) cm⁻¹: 3503, 2982, 2938, 2342, 1772, 1975, 1594, 1370, 1335, 1219; ¹H-NMR (600 MHz, CD₃OD) and ¹³C-NMR (150 MHz, CD₃OD), see Table S2; ESI-MS m/z: 523.1230 (Calcd for C₂₅H₂₄O₁₁Na: 523.1219).

Calculation of Theoretical EDC Spectra of 2

Conformational searches and DFT calculations were carried out on Avogadro software¹⁶⁾ and Gaussian 16 program,¹⁷⁾ respectively. 2 was submitted to conformational searches at the Molecular Mechanics (MMFF94s). The initial stable conformers with Boltzmann distributions over 1% were further optimized by DFT calculations at the B3LYP/6-31G(d) level. The stable conformers with Boltzmann distributions over 1% were subjected to TDDFT calculations at the B3LYP/6-31G + (d,p) level in the presence of acetonitrile with a polarizable continuum model. The resultant rotatory strengths of the lowest 30 excited states for 2 was converted into Gaussian-type curves with half-bands (0.2 eV) using GaussView 6 software.¹⁸⁾

Calculation of ¹³C-NMR Chemical Shift Values of 2

Calculated ¹³C-NMR data were obtained employing ωB97X-V/6-311 + G(2DF,2P)-[6-311G*]//ωB97X-D/6-31G* model based on the literature.^19–21) The calculated ¹³C-NMR chemical shift values were expected by DFT calculation. The calculations were performed with Spartan ’20.

Cytotoxicity Assays

The cytotoxic activities of 1, 2, 2-Me, and 2-Ac against human ovarian adenocarcinoma SKOV-3 cells, malignant pleural mesothelioma Meso-1 cells, and immortalized human T lymphocyte Jurkat cells were examined. SKOV-3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium supplemented with 10% fetal bovine serum, penicillin (50 U/mL), and streptomycin (50 µg/mL). Meso-1 cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum, penicillin (50 U/mL), and streptomycin (50 µg/mL). Jurkat cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum, penicillin (50 U/mL), streptomycin (50 µg/mL), and GlutaMAX. All cell lines were seeded in 384-well plates at the density of 1000 cells/well in 20 µL media and incubated at 37 °C in a humidified incubator with 5% CO₂. Samples were resolved in dimethyl sulfoxide (DMSO). After 4 h, two-fold diluted samples were added to the cell culture at the final concentration of 0.5% and incubated for 72 h. Cell viabilities were measured using CellTiter-Glo Luminescent Cell Viability Assay and EnVision Multilabel Plate Reader.

Antimicrobial Assays

Staphylococcus aureus and Micrococcus luteus were used in this study. The bacteria were precultured in LB medium until an optical density at 620 nm become 0.5. The bacteria suspension was diluted 2000-fold with LB medium and inoculated into each well of 384-well plates, followed by adding serially diluted test compounds. After incubation for 24 h at 37 °C, optical density for each plate was measured at 620 nm using EnVision Multilabel Plate Reader.

Acknowledgments

This work was supported by AMED under Grant JP19ae0101045 to KS.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials. Electronic supplementary information (ESI) available: 1D and 2D-NMR spectra for 1, 2, and its derivatives. Experimental and theoretical EDC spectrum, and calculated ¹³C-NMR chemical shifts values of 2.

References

• 1) Koehn F. E., Carter G. T., Nat. Rev. Drug Discov., 4, 206–220 (2005).
• 2) Newman D. J., Cragg G. M., J. Nat. Prod., 79, 629–661 (2016).
• 3) Doroghazi J. R., Albright J. C., Goering A. W., Ju K. S., Haines R. R., Tchalukov K. A., Labeda D. P., Kelleher N. L., Metcalf W. W., Nat. Chem. Biol., 10, 963–968 (2014).
• 4) Cimermancic P., Medema M. H., Claesen J., Kurita K., Brown L. C. W., Mavrommatis K., Pati A., Godfrey P. A., Koehrsen M., Clardy J., Birren B. W., Takano E., Sali A., Linington R. G., Fischbach M. A., Cell, 158, 412–421 (2014).
• 5) Wang B., Guo F., Dong S. H., Zhao H., Nat. Chem. Biol., 15, 111–114 (2019).
• 6) Zhang L., Hashimoto T., Qin B., Hashimoto J., Kozone I., Kawahara T., Okada M., Awakawa T., Ito T., Asakawa Y., Ueki M., Takahashi S., Osada H., Wakimoto T., Ikeda H., Shin-ya K., Abe I., Angew. Chem. Int. Ed., 56, 1740–1745 (2017).
• 7) Hashimoto T., Kozone I., Hashimoto J., Ueoka R., Kagaya N., Fujie M., Satoh N., Ikeda H., Shin-ya K., J. Antibiot., 73, 171–174 (2020).
• 8) Hashimoto T., Hashimoto J., Kozone I., Amagai K., Kawahara T., Takahashi S., Ikeda H., Shin-ya K., Org. Lett., 20, 7996–7999 (2018).
• 9) Hashimoto T., Kozone I., Hashimoto J., Suenaga H., Fujie M., Satoh N., Ikeda H., Shin-ya K., J. Antibiot., 73, 650–654 (2020).
• 10) Kinashi H., Someno K., Sakaguch K., J. Antibiot., 37, 1333–1343 (1984).
• 11) Kudo K., Nishimura T., Izumikawa M., Kozone I., Hashimoto J., Fujie M., Suenaga H., Ikeda H., Satoh N., Shin-ya K., J. Antibiot., 77, 288–298 (2024).
• 12) Bringmann G., Lang G., Maksimenka K., Hamm A., Gulder T. A., Dieter A., Bull A. T., Stach J. E., Kocher N., Muller W. E., Fiedler H. P., Phytochemistry, 66, 1366–1373 (2005).
• 13) Chang Y., Xing L., Sun C., Liang S., Liu T., Zhang X., Zhu T., Pfeifer B. A., Che Q., Zhang G., Li D., J. Nat. Prod., 83, 2749–2755 (2020).
• 14) Fedoryshyn M., Nur-e-Alam M., Zhu L., Luzhetskyy A., Rohr J., Bechthold A., J. Biotechnol., 130, 32–38 (2007).
• 15) Ding W., Ji Y., Jiang Y., Ying W., Fang Z., Gao T., Biochem. Biophys. Res. Commun., 531, 377–382 (2020).
• 16) Hanwell M. D., Curtis D. E., Lonie D. C., Vandermeersch T., Zurek E., Hutchison G. R., J. Cheminform., 4, 17 (2012).
• 17) Frisch M. J., Trucks G. W., Schlegel H. B., et al., Gaussian, Inc., Wallingford CT, 2016.
• 18) Dennington R., Keith T. A., Millam J. M., Semichem Inc., Shawnee Mission, KS, 2016.
• 19) Oikawa M., Sugeno Y., Tukada H., Takahashi Y., Takamizawa S., Irie R., Bull. Chem. Soc. Jpn., 92, 1816–1823 (2019).
• 20) Hehre W., Klunzinger P., Deppmeier B., Driessen A., Uchida N., Hashimoto M., Fukushi E., Takata Y., J. Nat. Prod., 82, 2299–2306 (2019).
• 21) Tanaka K., Manabe H., Irie R., Oikawa M., Bull. Chem. Soc. Jpn., 92, 1314–1323 (2019).