Some aplysiatoxin-related compounds have been isolated from an Okinawan cyanobacterium Okeania hirsuta. The structure of a natural product isolated as 30-methyloscillatoxin D (1a) from this cyanobacterium in our previous report was re-investigated and revised to be a 7-epimer of 30-methyloscillatoxin D (4a) using precise NMR analysis. The synthesis of O-Me-7-epi-30-methyloscillatoxin D (4b) led to confirmation of the stereochemistry of 4a. This is the first report of a 7-epi-type aplysiatoxin-related compound from a natural source.
Moore et al, in 1985, isolated 30-methyloscillatoxin D (1a) for the first time along with debromoaplysiatoxin (3) from a mixture of marine cyanobacteria Schizothrix calcicola and Oscillatoria nigroviridis.1 In July 2010, an outbreak of the marine cyanobacterium Okeania hirsuta (formerly reported as Moorea producens, see Supplemental Material) occurred in Okinawa, Japan. The sample used in this study was collected at that time and at that site. In 2019, we reported the re-isolation of 30-methyloscillatoxin D (1a) from this cyanobacterium.2 Recently Nishikawa et al reported the total synthesis of 30-methyloscillatoxin D (1a) and O-Me-30-methyloscillatoxin D (1b).3,4 Comparison of the 1H NMR data of the synthesized 1a3 with our reported 1a2 showed a significant difference. Thus, the structure of the compound which was formerly reported by us as 1a2 was re-investigated and revised to be 7-epi-30-methyloscillatoxin D (4a). Finally, the structure of 4a was confirmed by comparison of 1H and 13C NMR data of the natural compound 4a and synthesized compound 4b. This is the first report of the isolation of a 7-epi-type aplysiatoxin-related compound from a natural source. In this study, we also isolated 1a itself and 17-bromo-30-methyl-oscillatoxin D (2) from the same cyanobacterium sample.
Results and Discussion
7-Epi-30-methyloscillatoxin D (4a) was isolated as a white solid ([α]D17 + 31.4 (c 0.7, MeOH)). The UV maxima observed at 199 nm (ε = 13376), 215 nm (ε = 6259), and 275 nm (ε = 2021) indicated the presence of an aromatic group. To determine the molecular formula of C32H44O8, high-resolution electrospray ionization (ESI) mass spectrometric analysis was used (Supplemental Figure S1) ([M + H]+ at m/z 557.3064, calcd. for C32H45O8, 557.3109), and the results agreed with those of 1a. However, the proton chemical shifts from H-2 to H-12 of this compound differed from 1a (Table 1). The 1H NMR (Supplemental Figure S2), 13C NMR (Supplemental Figure S3), and HSQC (Supplemental Figure S4) spectra of 4a showed seven methyl groups (two singlets, four doublets, and a methoxy), four methylenes, four aliphatic methines, four oxygenated methines, two olefinic methines, four aromatic protons, and seven quaternary carbons (one aliphatic, one oxygenated, two aromatics in a phenol moiety, two esters, and a ketone). The proton connectivities of H-4 (H3-26) to H2-5, H-10 to H-15, H-17 to H-19, and H2-28 to H3-31 were assigned using 1H-1H COSY (Supplemental Figure S5). The proton signals at H-8 and H-9 measured in acetone-d6 had the same chemical shifts and were observed as a singlet peak due to second-order coupling. The partial structures were assembled using HMBC (Supplemental Figure S6) correlations from H-2 to C-1, C-3 and C-7, H3-26 to C-3, H3-24 to C-7 and C-8, H3-25 to C-7, and H-15 to C-16. The position of γ-lactone was deduced from the proton chemical shift at H-29 (δH 5.41). The planar structure of 4a was the same as that of 1a, suggesting that 4a is a stereoisomer of 1a.
1H and 13C NMR Spectroscopic Data (in Acetone-d6) for 7-epi-30-Methyloscillatoxin D (4a), O
-Me-7-epi-30-Methylocsillatoxin D (4b), and 30-Methyloscillatoxin D (1a) (δ, ppm; J, Hz).
No
δH (J in Hz)
δC
4a(1) (600 MHz)
4b (400 MHz)
1a (600 MHz)
4a(1) (150 MHz)
4b (100 MHz)
1a (150 MHz)
1
166.7
166.7
169.2
2
3.96 s
3.96, s
4.01 s
62.6
62.7
65.2
3
205.3
205.3
205.7
4
2.65 m
2.65, m
2.81 m
41.6
41.6
41.4
5a
1.54 dd (13.1, 6.5)
1.51, dd (13.0, 6.5)
1.37 m
45.9
46.0
44.0
5b
1.76 m
1.74, t (13.0)
1.72 m
6
41.5
41.5
41.3
7
84.3
84.3
84.7
8
5.81 s
5.81, s
5.77 d (10.5)
137.4(2)
137.4(3)
134.6
9
5.81 s
5.81, s
5.51 dd (3.0, 10.8)
125.1(2)
125.1(3)
126.9
10
2.36 m
2.31-2.42, m
2.15 m
30.5
30.4
31.0
11
3.28 dd (9.5, 1.7)
3.27, dd (9.5, 1.5)
3.15 m
81.1
81.2
79.0
12
1.64 m
1.58–1.67, m
1.72 m
35.5
35.6
34.3
13a
1.36 m
1.36, m
1.39 m
31.8
31.8
31.6
13b
1.36 m
1.36, m
1.48 m
14a
1.61 m
1.58–1.67, m
1.55 m
37.4
37.5
36.8
14b
1.78 m
1.79, m
1.72 m
15
4.02 dd (6.6, 6.6)
4.08, t (6.5)
4.02 dd (6.6, 6.6)
85.2
85.2
84.7
16
145.6
145.6
145.6
17
6.74 m
6.80–6.90, m
6.79 br.d (7.4)
118.9
120.0
118.8
18
7.15 dd (7.5, 7.8)
7.25, t (8.0)
7.17 dd (7.4, 7.8)
130.2
130.3
130.1
19
6.73 m
6.80–6.90, m
6.74 br.d (8.4)
115.3
113.7
115.1
20
158.6
161.0
158.4
21
6.78 dd (1.9, 1.9)
6.80–6.90, m
6.83 m
114.3
113.1
114.3
22
0.86 d (6.9)
0.86, d (7.0)
0.89 d (7.2)
13.7
13.7
13.3
23
0.84 d (7.3)
0.84, d (7.0)
0.88 d (7.2)
17.2
17.3
16.9
24
0.95 s
0.93, s
0.92 s
27.2
27.3
25.1
25
1.32 s
1.32, s
1.31 s
24.7
24.8
22.6
26
0.93 d (6.4)
0.92, d (6.5)
0.99 d (6.2)
14.9
14.8
14.5
27
175.1
175.1
174.8
28a
2.38 d (18.1)
2.38. brd (18.0)
2.53 d (17.4)
37.6
37.6
37.4
28b
3.02 dd (18.0, 5.9)
3.01, dd (18.0, 6.0)
3.02 dd (18.0, 5.9)
29
5.41 m
5.41, dd (5.0, 4.5)
5.37 m
72.0
72.1
73.6
30
4.75 m
4.75, qd (6.5, 4.0)
4.80 m
79.4
79.5
79.4
31
1.27 d (6.5)
1.27, d (6.5)
1.41 d (6.4)
14.9
15.0
14.9
32
3.15 s
3.16, s
3.15 s
56.7
56.8
56.6
20-OH
8.22 s
8.25
(1) By re-assignment in this study, the chemical shifts of 4a were slightly revised from formerly reported values (4a had been incorrectly assigned as 30-methyloscillatoxin D at that time).2
(2), (3) 13C chemical shifts were interchangeable.
The stereostructure around C-7 in 4a was deduced from the rotating frame nuclear Overhauser effect spectroscopy (ROESY) experiments. The rotating-frame Overhauser effect (ROE) correlations of H-2/H-4, H-2/H3-25, and H-4/H3-25 indicated that H-2 (δH 3.96), H-4 (δH 2.65), and H3-25 (δH 1.32) were oriented in the β position. The ROE correlations, H-8 (δH 5.81)/H-2 and H-8/H3-25, show that C-7 has an R-configuration (Figure 1). Therefore, 4a was deduced to be a C-7 epimer of 1a. The large coupling constant (9.5 Hz) between H-10 (δH 2.36) and H-11 (δH 3.28) and the ROE correlation H-11/H3-23 (δH 0.84) indicates the anti-position of H-10/H-11. The observed small coupling constant (1.7 Hz) of H-11/H-12 (δH 1.64) was typical for oscillatoxins, probable due to the gauche conformation of H-11/H-12. Therefore, the configuration at C-12 was suggested to be S. The configurations of C-15, C-29, and C-30 were deduced from the proton chemical shifts and coupling constants. Therefore, the configuration, except for C-7 in 4a, was the same as that of 1a. These observations elucidate the structure of 4a (Figure 2).
Rotating-frame Overhauser effect (ROE) correlations of compound 4a.
Structures of compounds 1a to 6.
The stereochemical assignment (Table 1) was confirmed by comparison of 1H and 13C NMR data of 4a and 4b. Compound 4b was synthesized from a stereochemically-defined intermediate 7 (Figure 3),4 which was previously obtained as a byproduct in the synthesis of O-Me-30-methyloscillatoxin D (1b).4 Transesterification of 7 with lactone 85 was carried out in the presence of DMAP under toluene reflux conditions to provide 4b in 27% yield. The 1H and 13C NMR spectra around C-7 of the natural compound 4a are in good agreement with those of the synthetic compound 4b, except for the aromatic moiety (Table 1).
Synthesis of 4b from 7.
The isolation of 1a and 4a from the same sample (Supplemental Figures S11 and S12) confirms that 4a was not an artifact produced during isolation. In our previous report, we mistakenly described the cytotoxicity of 4a as 90% at the tested dose (10 µg/mL).2 However, after re-examination, the cytotoxicity of 4a was corrected to 25% inhibition at the tested dose (10 µg/mL). Unfortunately, we could not examine the cytotoxicity of the newly isolated 1a because of its instability. Isolated 1a and 2 were rapidly converted to oscillatoxin F (5)6 and 16-bromooscillatoxin F (6) (see Supplemental Material) within a month under dry, dark, and refrigerated conditions. Compound 6 has not been obtained as a natural product, so far. Interestingly, 4a was stable for over two years under the same conditions.
Previously, the cyanobacterium sample used in this study had been identified as Moorea producens by morphological observations using microscopy.7 However, this cyanobacterium was found in this study to be Okeania hirsuta by re-identification using gene analysis methods (see Supplemental Material). This O hirsuta sample strain (20100713-a) is a chemically rich species. Over 40 compounds have been isolated from this O hirsuta sample, of which more than 20 were new compounds.2,7–12
Conclusion
In this study, 30-methyloscillatoxin D (1a), 17-bromo-30-methyloscillatoxin D (2), and 7-epi-30-methylocsillatoxin D (4a) were isolated and identified from the cyanobacterium O hirsuta sample strain (20100713-a). The synthesis of O-Me-7-epi-30-methyloscillatoxin D (4b) confirmed the stereochemistry of 7-epi-30-methylocsillatoxin D (4a). Compound 4a is the first report of a 7-epi-type aplysiatoxin-related compound from a natural source.
Experimental
General
Optical rotations were measured using either a JASCO P-2100 or a JASCO DIP-370 (JASCO Co., Tokyo) using a 10 mm length cell. Infrared spectra (IR) were recorded on a JASCO FT/IR-4100 (JASCO Co., Tokyo). NMR spectra were recorded in acetone-d6 using either a Bruker AVANCE III 600 spectrometer or a Bruker AVANCE-400 (400 MHz; Bruker Co., Billerica) spectrometer. UV spectra were measured using a HITACHI U-3000 spectrometer (Hitachi High-Tech Science Co., Tokyo). HPLC was performed using a Hitachi Chromaster HPLC System (Hitachi High-Tech Science Co., Tokyo). HR-ESI-MS spectral data were determined using either a Bruker micrOTOF QII (Bruker Co., Bremen) mass spectrometer or an Agilent 6220 Accurate-Mass TOF (Agilent Technologies, Santa Clara). Bioassay results were recorded on a Model 550 microplate reader (Bio-Rad, California).
Biological Material
Samples of the marine cyanobacterium Okeania hirsuta (20100713-a) were collected from Kuba Beach, Nakagusuku, Okinawa, Japan in July 2010. O hirsuta was a dominant cyanobacterial species in the sample. The sample also contained some unidentified diatoms. The cyanobacterium was identified by 16S rRNA sequence analysis and morphological and chemotaxonomic observations (see Supplemental Material). This biological material had previously been incorrectly identified as Moorea producens by morphological observations using microscopy.2,7–12
Extraction and Isolation
Extraction and purification of the frozen O hirsuta sample (wet weight: 9.7 kg) was performed in the same manner as described in a previous paper.2 The EtOAc layer was evaporated to dryness. The EtOAc layer was fractionated using an open glass column measuring 40 × 400 mm and packed with ODS resin (Cosmosil 75C18-OPN, Nacalai Tesque Inc., Kyoto) with stepwise elution with 50%, 70%, 90%, and 100% methanol. The 70% methanol eluate was then purified via HPLC using a reversed-phase column (Cosmosil 5C18-AR-II, 10 × 250 mm, Nakalai Tesque Inc., Kyoto). 7-Epi-30-methyloscillatoxin D (4a, 0.9 mg) had been already isolated and incorrectly identified as 30-methyloscillatoxin D in a former paper.2 Finally, 30-methyloscillatoxin D (1a, 1.7 mg, see Supplemental Material), and 17-bromo-30-methyloscillatoxin D (2, 1.3 mg, see Supplemental Material) were also isolated.
Synthesis of O-Me-7-epi-30-Methyloscillatoxin D (4b)
To a solution of spiro ether 7 (14.0 mg, 28.8 μmol) and lactone 8 (14.4 mg, 0.115 mmol) in toluene (1.5 mL) was added DMAP (3.4 mg, 28 μmol). Then, the solution was refluxed for 2 h and diluted with saturated NH4Cl solution (10 mL) after cooling to room temperature. The mixture was extracted with EtOAc (10 mL × 3). The combined organic extracts were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by preparative TLC (EtOAc/hexane = 1/10 to 1/1) to afford 4b (4.5 mg, 27%) as a colorless oil. [α]D23 + 97 (c 0.23, CHCl3). IR (film): νmax (cm−1) 2965, 2933, 1786, 1767, 1716, 1487, 1457, 1261, 1164, 1120, and 1055. HR-MS (ESI, positive): calcd. For C33H46NaO8 [M + Na]+, 593.3085; Found 593.3078. 1H and 13C NMR chemical shifts of 4b are shown in Table 1.
Biological Test
Cytotoxicity assays against mouse L1210 leukemia cells were carried out for 4a. Bioactive assays were performed using the XTT colorimetric reaction method, as previously reported.13
Supplemental Material
sj-docx-1-npx-10.1177_1934578X231173799 - Supplemental material for 7-Epi-30-Methyloscillatoxin D From an Okinawan Cyanobacterium Okeania hirsuta
Supplemental material, sj-docx-1-npx-10.1177_1934578X231173799 for 7-Epi-30-Methyloscillatoxin D From an Okinawan Cyanobacterium Okeania hirsuta by Nao Kanda, Bo-Tao Zhang, Akihisa Shinjo, Mitsunobu Kamiya, Hiroshi Nagai, Hajime Uchida, Yusuke Araki and
Toshio Nishikawa, Masayuki Satake in Natural Product Communications
Footnotes
Acknowledgments
This study was supported by JSPS KAKENHI (grant numbers 22K05817 for H.N., 17H06406, and 19H02896 for N.T.). The authors would like to thank Ms. Haruka Shimada (Tokyo University of Marine Science and Technology) for her support during the sample identification experiments. We would like to thank Editage () for English language editing.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Ethical Approval
Not applicable, because this article does not contain any studies with human or animal subjects.
Funding
The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the JSPS KAKENHI (grant number 22K05817 for H.N., 17H06406 and 19H02896 for N.T.).
Informed Consent
Not applicable.
Statement of Human and Animal Rights
Not applicable.
ORCID iD
Hiroshi Nagai
Supplemental Material
Supplemental material for this article is available online.
References
1.
Entzeroth M, Blackman AJ, Mynderse JS, Moore RE. Structures and stereochemistries of oscillatoxin B, 31-noroscillatoxin B, oscillatoxin D, and 30-methyloscillatoxin D. J Org Chem. 1985;50(8):1255‐1259.
2.
NagaiHWatanabeMSatoS, et al.New aplysiatoxin derivatives from the Okinawan cyanobacterium Moorea producens. Tetrahedron. 2019;75(17):2486‐2494.
3.
ArakiYHanakiYKitaM, et al.Total synthesis and biological evaluation of oscillatoxins D, E, and F. Biosci Biotech Biochem. 2021;85(6):1371‐1382.
4.
NokuraYArakiYNakazakiANishikawaT. Synthetic route to oscillatoxin D and its analogues. Org Lett. 2017;19(21):5992‐5995.
5.
HarckenCBrücknerR. Elucidation of the stereostructure of the Annonaceous acetogenin (+)-montecristin through total synthesis. New J Chem. 2001;25(1):40‐54.
6.
TangYHWuJFanTT, et al.Chemical and biological study of aplysiatoxin derivatives showing inhibition of potassium channel Kv1. 5. RSC Adv. 2019;9(14):7594‐7600.
7.
JiangWBuYKawaguchiM, et al.Five new indole derivatives from the cyanobacterium Moorea producens. Phytochem Lett. 2017;22(3):163‐166.
8.
NagaiHSatoSIidaK, et al.Oscillatoxin I: a new aplysiatoxin derivative, from a marine cyanobacterium. Toxins (Basel). 2019;11(6):366.
9.
KawaguchiMSatakeMZhangBT, et al.Neo-aplysiatoxin A isolated from Okinawan cyanobacterium Moorea producens. Molecules. 2020;25(3):457.
10.
MurakamiAHayashiJIIgawaK, et al.Natural dolapyrrolidone: isolation and absolute stereochemistry of a substructure of bioactive peptides. Chirality. 2020;32(9):1152‐1159.
11.
IguchiKSatakeMNishioY, et al.Debromooscillatoxins G and I from the cyanobacterium Moorea producens. Heterocycles. 2021;102(7):1287‐1293.
12.
SatakeMIguchiKWatanabeRUchidaHNagaiH. Aplysiadione and aplysiaenal: truncated biosynthetic intermediates of aplysiatoxins from a cyanobacterium. Results Chem. 2021; 3:100206.
13.
KawabataTLindsayDJKitamuraM, et al.Evaluation of the bioactivities of water-soluble extracts from twelve deep-sea jellyfish species. Fish Sci. 2013;79(3):487‐494.
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) for English language editing.