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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Ypaoamides B and C, Linear Lipopeptides from an Okeania sp. Marine Cyanobacterium Kosuke Sueyoshi,† Miki Yamada,† Aki Yamano,† Kaori Ozaki,† Shimpei Sumimoto,‡ Arihiro Iwasaki,‡ Kiyotake Suenaga,‡ and Toshiaki Teruya*,† †
Faculty of Education, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan Department of Chemistry, Keio University, 3-14-1, Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan
‡
S Supporting Information *
ABSTRACT: Two new pyrrolinone-containing lipopeptides, ypaoamides B (1) and C (2), were isolated from an Okeania sp. marine cyanobacterium collected in Okinawa. Their structures were determined by spectroscopic analysis and Marfey’s analysis of acid hydrolysates. Ypaoamides B (1) and C (2) stimulated glucose uptake in cultured rat L6 myotubes. In particular, ypaoamide B (1) showed potent activity and activated AMPactivated protein kinase.
D
iabetes mellitus (DM) is a serious chronic disease and a major public health problem worldwide. The number of adults living with DM has almost quadrupled since 1980, reaching 422 million in 2014.1 Type 2 diabetes mellitus (T2DM), which is characterized by insulin resistance with relatively low insulin secretion, accounts for over 90% of DM cases worldwide.2 T2DM is also known to be associated with a number of health complications, such as retinopathy, neuropathy, and cardiovascular disease.3 Therefore, the development of therapeutic agents for T2DM is required. Marine natural products are a prolific source of lead compounds in drug discovery.4,5 In particular, marine cyanobacteria are known to produce various secondary metabolites with unique structures and biological activities.6,7 For example, biselyngbyaside, a macrolide glycoside isolated from a Lyngbya sp., exhibited broad-spectrum cytotoxicity in a human tumor cell line panel8 and inhibited osteoclastogenesis.9 Yoshinone A, isolated from a Leptolyngbya sp., is composed of a γ-pyrone and a linear side chain and inhibited the differentiation of 3T3-L1 cells into adipocytes without cytotoxicity.10 In our search for novel biologically active substances, we recently isolated ypaoamides B (1) and C (2), which stimulate glucose uptake in cultured L6 myotubes. Herein, we report the isolation, structure determination, and biological activities of compounds 1 and 2. An Okeania sp. marine cyanobacterium was collected from the coast of Irijima, Okinawa, and extracted with MeOH. The concentrated extract was partitioned between EtOAc and H2O, and the EtOAc layer was further partitioned between n-hexane and 90% aqueous MeOH. The material obtained from the aqueous MeOH layer was subjected to fractionation using ODS silica gel column chromatography (MeOH−H2O) and reversed-phase HPLC to isolate ypaoamides B (1) and C (2). Ypaoamide B (1) was obtained as a colorless oil, and its molecular formula was determined to be C23H38N2O4 by HRESIMS. The NMR data for 1 are summarized in Table 1. The 1H NMR © XXXX American Chemical Society and American Society of Pharmacognosy
spectrum of 1 indicated the presence of a tert-butyl group (δH 0.87), an O-methyl group (δH 3.74), and olefinic protons (δH 6.10, 6.75, and 7.58). A detailed analysis of the 2D NMR spectra allowed the assignment of two substructures, A and B (Figure 1a). COSY correlations between H2-2″/H2-3″, H2-3″/H2-4″, and H2-4″/H2-5″ and HMBC correlations between H3-7″/C-5″ and C-6″ led to the assignment of partial structure C-2″ to C-7″. Additionally, a COSY correlation between H2-4′/H2-5′ and HMBC correlations between H-2′/C-3′, H2-4′/C-3′, and H3-6′/C-3′ led to the assignment of partial structure C-2′ to C-5′. HMBC correlations between H2-2″/C-1″ and H2-5′/C-1″ connected these two partial structures through an amide carbonyl group C-1″, giving substructure A. The double-bond configuration at C-2′/C-3′ was determined to be E by a NOESY correlation between H-2′/H3-6′. The partial structure C-1 to C-8 was assigned from sequential COSY correlations from H-2/H3-8 (Figure 1a) and an HMBC correlation between H-2/C-1. The chemical shifts of H-4 (δH 4.89) and C-4 (δC 62.7) indicated the presence of a nitrogen atom at C-4. Received: January 29, 2018
A
DOI: 10.1021/acs.jnatprod.8b00088 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 1. NMR Spectroscopic Data for Ypaoamides B (1) and C (2) in CD3OD Yypaoamide B (1)
a
position
δC,a type
1 2 3 4 5a 5b 6 7 8 1′ 2′ 3′ 4′a 4′b 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 7″
172.3, C 126.8, CH 154.5, CH 62.7, CH 42.0, CH2 26.4, CH 22.7, CH3 24.4, CH3 166.7, C 95.6, C 176.9, C 34.0, CH2 38.5, CH2 56.7, CH3 176.2, C 37.4, CH2 28.1, CH2 25.5, CH2 45.1, CH2 31.2, C 29.9, CH3
δH, mult (J in Hz)b
ypaoamide C (2) HMBC
6.10, dd (6.0, 1.4) 7.58, dd (6.0, 1.9) 4.89, m 1.41, ddd (13.2, 9.5, 4.8) 2.05, ddd (13.2, 9.2, 3.7) 1.75, m 1.02, d (6.6) 0.93, d (6.6)
1, 3, 4 1, 2, 4 2, 3, 5, 6 3, 4, 6, 8 3, 4, 6, 7 4, 5, 7, 8 5, 6, 8 5, 6, 7
6.75, s
3′, 4′
2.94, m 3.00, m 3.43, m 3.74, s
2′, 3′, 5′ 2′, 3′, 5′ 3′, 4′, 1″ 3′
2.15, t (7.7) 1.54, m 1.27, m 1.19, m
1″, 3″, 4″ 1″, 2″, 4″, 5″ 2″, 3″, 5″, 6″ 4″, 7″
0.87, s
5″, 6″
position
δC,a type
1 2 3 4 5 1′ 2′ 3′ 4′a 4′b 5′ 6′ 7′ 8′ 1″ 2″ 3″ 4″ 5″ 6″ 7″
172.2, C 126.3, CH 155.8, CH 59.8, CH 18.3, CH3 166.5, C 95.5, CH 175.1, C 37.3, CH2 136.6, C 118.2, CH 39.6, CH2 56.8, CH3 176.4, C 37.2, CH2 28.0, CH2 25.5, CH2 45.2, CH2 31.1, C 29.9, CH3
δH, mult (J in Hz)b
HMBC
6.08, dd (6.0, 1.5) 7.42, dd (6.0, 2.0) 4.86, m 1.43, d (6.7)
1, 3, 4 1, 2, 4 2, 3, 5 3, 4
6.82, s
3′, 4′
3.62, dd (15.1, 1.2) 3.69, dd (15.1, 1.2)
2′, 3′, 5′, 6′, 7′ 2′, 3′, 5′, 6′, 7′
6.14, t (1.2) 4.04, s 3.75, s
4′, 5′, 7′ 4′, 5′, 6′, 1″ 3′
2.20, t (7.7) 1.56, m 1.28, m 1.19, m
1″, 3″, 4″ 1″, 2″, 4″, 5″ 2″, 3″, 5″, 6″ 4″, 7″
0.87, s
5″, 6″
Recorded at 125 MHz. bRecorded at 500 MHz.
indicated the presence of a doublet methyl group (δH 1.43, δC 18.3) and the absence of an isobutyl group compared with ypaoamide B (1). A detailed 2D NMR analysis revealed two substructures, A and B (Figure 1b). COSY correlations and HMBC correlations led to the assignment of partial structure C-2″ to C-7″. The methylene group at C-7′ (δH 4.04, δC 39.6) was attached to a nitrogen atom and linked to C-4′ through olefinic carbon C-5′. The chemical shifts of H-6′ (δH 6.14) and C-6′ (δC 118.2) were diagnostic of a vinyl chloride functionality,11 and the double bond between C-5′ and C-6′ was confirmed by an HMBC correlation between H-6′/C-5′. HMBC correlations from the methylene protons (H2-4′), the O-methyl group (H3-8′), and the vinyl proton (H-2′) to the olefinic carbon (C-3′) were observed, allowing partial structure C-2′ to C-7′ to be assigned. The connectivity of these two partial structures was clarified by HMBC correlations between H2-2″/C-1″ and H2-7′/C-1″. The configurations of the two double bonds at C-2′ and C-5′ were assigned as E and Z, respectively, from NOESY correlations between H-2′/H3-8′ and H2-4′/H-6′. In substructure B, the methyl-substituted pyrrolinone ring was assigned using COSY correlations, HMBC correlations, and the chemical shifts of H-4 (δH 4.86) and C-4 (δC 59.8) (Figure 1b). Although HMBC correlations between H-2′/C-1′ and H-4/C-1′ were not observed, substructures A and B should be linked through the carbonyl carbon (δC 166.5) as in compound 1, considering the molecular formula and degree of unsaturation of 2. Therefore, the gross structure of ypaoamide C (2) was determined to be as shown in Figure 1b. To determine the absolute configurations of C-4 in ypaoamide B (1) and C-4 in ypaoamide C (2), compounds 1 and 2 were subjected to ozonolysis followed by oxidative workup, acid hydrolysis, and derivatization with Marfey’s reagent.13 HPLC analysis of these products revealed the presence of L-(S)-leucine in 1 and L-(S)-alanine in 2, respectively. This completed the structural assignments of ypaoamides B (1) and C (2).
Figure 1. Gross structures of ypaoamides B (1) and C (2). (a) Key 2D NMR correlations for 1. (b) Key 2D NMR correlations for 2.
Although there was no HMBC correlation between H-4/C-1, the linkage of C-4 to C-1 through a nitrogen atom was confirmed by the chemical shifts consistent with a pyrrolinone ring.11 The sequence of substructures A and B was not determined using HMBC data, because HMBC correlations between H-2′/C-1′ and H-4/C-1′ were not observed. However, the remaining CO component and the carbonyl carbon chemical shift (δC 166.7)12 indicated the presence of an amide functionality, and substructures A and B should be connected through C-1′ considering its molecular formula and degree of unsaturation. Thus, the gross structure of ypaoamide B (1) was determined to be as shown in Figure 1a. Ypaoamide C (2) was isolated as a colorless oil and determined to have a molecular formula of C22H33ClN2O4 by HRESIMS. The NMR data for 2 are summarized in Table 1. These data B
DOI: 10.1021/acs.jnatprod.8b00088 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Structurally similar compounds ypaoamide12 and the jamaicamides11 have been reported. Ypaoamide B (1) differs only in the substituent on the pyrrolinone ring from ypaoamide. Ypaoamide C (2) has the same pyrrolinone ring terminus as seen in jamaicamides. The formation of the carbon framework of ypaoamide B (1) is predicted to proceed in a similar fashion to that of jamaicamides, and the tert-butyl group may be added from S-adenosyl methionine (SAM).14 The biological activities of ypaoamides B (1) and C (2) were evaluated using a glucose uptake assay in cultured rat L6 myotubes. Both compounds 1 and 2 showed no cytotoxicity at 10−40 μM (Figure 2a) and stimulated glucose uptake in a dose-dependent
Figure 3. Effects of ypaoamide B (1) on AMPK pathway in cultured L6 myotubes. (a) Cells were preincubated in KHH buffer without glucose for 2 h. They were then incubated in KHH buffer containing 5 mM glucose without or with 40 μM ypaomide B (1) and without or with 30 μM compound C for 8 h. Glucose uptake was measured using a Glucose C-II test kit. Values are the mean ± SD of quadruplicate determinations. (b) Cells were treated with the indicated concentrations of compound 1. The lysates were analyzed by Western blotting with anti-phosphorylated AMPK (p-AMPK) and anti-AMPK antibodies. Immunoreactive bands were quantified using Image Lab software, and ratios of p-AMPK/ AMPK are shown. The ratio of control (0 μM compound 1) was regarded as 1. Values are the mean ± SD of quadruplicate determinations.
treated with 40 μM compound 1 (Figure 3b). These results indicated that ypaoamide B (1) stimulated glucose uptake in cultured L6 myotubes via the AMPK pathway regulating cellular metabolism. In addition, we recently reported that a structurally similar compound, 6,8-di-O-acetylmalyngamide 2, also stimulated glucose uptake and activated AMPK in cultured L6 myotubes.17 In conclusion, new pyrrolinone-ring-containing lipopeptides ypaoamides B (1) and C (2) were isolated from an Okeania sp. marine cyanobacterium collected in Okinawa. The structure of the compounds was established by spectroscopic analysis and HPLC analysis of the acid hydrolysates. Both compounds stimulated glucose uptake in a dose-dependent and an insulin-independent manner in cultured L6 myotubes. Furthermore, the effect of ypaoamide B (1) on glucose uptake was by activation of the AMPK pathway. Therefore, ypaoamide B (1) may have antidiabetic properties and be a potential therapeutic candidate for the treatment of T2DM.
Figure 2. Effects of ypaoamides B (1) and C (2) on cell viability and glucose uptake in cultured L6 myotubes. (a) Cells were treated with the indicated concentrations of compounds. After incubation for 16 h, cell viability was determined based on an MTT assay. Values are the mean ± SD of quadruplicate determinations. (b) Cells were preincubated in Krebs−Henseleit−HEPES buffer (KHH buffer) without glucose for 2 h. They were then incubated in KHH buffer containing 5 mM glucose with the indicated concentrations of compounds for 16 h. Glucose uptake was measured using a Glucose C-II test kit. Values are the mean ± SD of quadruplicate determinations.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured on a JASCO P-1010 polarimeter. UV spectra were measured on a JASCO V-660 UV visible spectrometer. IR spectra were measured on a JASCO FT/IR-6100 spectrometer. All NMR spectra were recorded on a Bruker AVANCE III 500 NMR spectrometer (500 and 125 MHz for 1H and 13C NMR, respectively). Chemical shifts were reported as δ values relative to the residual solvent signals (CHD2OD: δH 3.31, δC 49.0). ESIMS data were obtained using a Waters Quattro micro API mass spectrometer; HRESIMS was performed on a Waters Micromass Q-TOF spectrometer. HPLC was carried out with a JASCO PU-2080 Plus Intelligent HPLC pump and a JASCO UV-2075 Plus Intelligent UV/vis detector. The absorbance of the assay mixture was determined using a BioTek ELx800 absorbance microplate reader. Chemicals and solvents were the best grade available and used as received from commercial sources. Compound C, an AMPK inhibitor, was purchased from SigmaAldrich. Rat L6 myoblasts were purchased from JCRB Cell Bank.
and an insulin-independent manner (Figure 2b). To clarify the involvement of AMP-activated protein kinase (AMPK), which increases insulin-independent glucose uptake in skeletal muscle,15 we examined the effect of compound C,16 a selective AMPK inhibitor, and performed Western blotting with anti-AMPK and anti-phosphorylated AMPK (p-AMPK) antibodies. Compound C significantly reduced glucose uptake stimulated by ypaoamide B (1) in cultured L6 myotubes (Figure 3a). The expression of p-AMPK (the activated form of AMPK) increased in the cells C
DOI: 10.1021/acs.jnatprod.8b00088 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Cell Viability. After the glucose uptake assay, an MTT solution (10 μL, 5 mg/mL in H2O) was added to each well, and the plate was incubated at 37 °C with 5% CO2 for 4 h. All remaining supernatant was then removed, and DMSO (50 μL) was added to each well to dissolve the resultant formazan crystal. Absorbance was measured by using a microplate reader at the wavelength of 570 nm. Western Blotting. The Western blotting was performed as previously described,17 with the following minor modifications: lysates were centrifuged at 10 000 rpm for 5 min; 40 μg/lane protein samples were loaded on the electrophoresis gel; 5% bovine serum albumin (BSA) was used instead of 80% BSA.
Collection and Gene Sequencing. Samples of an Okeania sp. marine cyanobacterium were collected by hand from the coast of Irijima, Okinawa Prefecture, Japan, in September 2010. A voucher specimen of this sample, named T1009-1, has been deposited at University of the Ryukyus. Small pieces of collected marine cyanobacteria were preserved for genetic analysis in RNAlater (Qiagen). The DNA template was prepared from a single cyanobacterial filament as described previously.18 The fragments of the 16S rRNA genes were PCR amplified using the primer set CYA19 and CYA 1371R (1 + 2 + 3).20 PCR conditions and purification of PCR products were the same as described previously.18 Sequences were determined with CYA 359F and CYA 1371R (1 + 2 + 3) primers by a commercial firm (Macrogen Japan Corp.). These sequences are available in the DDBJ/EMBL/GenBank databases under accession number LC274886. Extraction and Isolation. Approximately 1.0 kg (wet weight) of the cyanobacterial samples was extracted with MeOH (2.0 L) at room temperature for 2 weeks. The extract was filtered, and the filtrate was concentrated. The residue was partitioned between H2O (0.2 L) and EtOAc (0.2 L × 3). The material obtained from the organic layer was further partitioned between 90% aqueous MeOH (0.1 L) and n-hexane (0.1 L × 3). The aqueous MeOH fraction (0.48 g) was separated by column chromatography on ODS (5.0 g) using 40% aqueous MeOH, 60% aqueous MeOH, 80% aqueous MeOH, and MeOH. The fraction (155.7 mg) that eluted with 80% aqueous MeOH was subjected to reversedphase HPLC [Develosil ODS-HG-5 (20 × 250 mm), 80% MeOH at 5.0 mL/min, UV detection at 215 nm] to yield ypaoamides B (1, 38.3 mg, tR = 35.9 min) and C (2, 11.6 mg, tR = 28.7 min). Ypaoamide B (1): colorless oil; [α]22D +110 (c 0.62, MeOH); UV (MeOH) λmax (log ε) 268 (4.18) nm; IR (neat) 3310, 2955, 2866, 1716, 1669, 1651, 1600, 1539, 1212, 1198, 1168 cm−1; 1H NMR, 13C NMR, and HMBC data, Table 1; HRESIMS m/z 429.2701 [M + Na]+ (calcd for C23H38N2O4Na, 429.2724). Ypaoamide C (2): colorless oil; [α]22D +85 (c 0.62, MeOH); UV (MeOH) λmax (log ε) 268 (4.05) nm; IR (neat) 3313, 2953, 2864, 1717, 1668, 1652, 1599, 1334, 1298, 1199, 1164 cm−1; 1H NMR, 13C NMR, and HMBC data, Table 1; HRESIMS m/z 447.2009 [M + Na]+ (calcd for C22H33ClN2O4Na, 447.2021). Ozonolysis and Acid Hydrolysis of 1 and 2. Ozone gas was bubbled through a solution of ypaoamide B (1, 1.2 mg) in MeOH (2.0 mL) at 0 °C for 2 min. The solution was then added to a stream of nitrogen gas to remove ozone and warmed to room temperature. The solution was oxidized by addition of 30% H2O2 (3.0 mL) and stirred for 1 h. The mixture was concentrated, and the residue was dissolved in 6 M HCl (1.0 mL) and refluxed at 110 °C for 16 h. The hydrolysate was concentrated and then partitioned between H2O (3.0 mL) and EtOAc (3.0 mL). The aqueous layer was subjected to further derivatization. Using the same procedure as described above, the aqueous layer was obtained from ypaoamide C (2, 0.9 mg). HPLC Analysis of Marfey’s Derivatives of Leu and Ala. The aqueous layer containing Leu from natural 1 was dissolved in 0.5 M NaHCO3 (100 μL), and a 1% solution of Nα-(5-fluoro-2,4-dinitrophenyl)-Lalaninamide (Marfey’s reagent, 200 μL) in acetone was added followed by heating at 40 °C for 90 min. After cooling to room temperature, the reaction mixture was neutralized with 2 M HCl (25 μL) and diluted with MeOH (300 μL). Marfey’s derivatives of Ala from 2 and standard amino acids were prepared by the same procedure. These solutions were subjected to reversed-phase HPLC [Cosmosil 5C18-AR-II (4.6 × 250 mm), MeOH/20 mM NaOAc = 60:40 (solvent A) or 40:60 (solvent B), UV detection at 340 nm]. The retention times (min) of the authentic standards were as follows: L-Leu (5.6) and D-Leu (11.9) in solvent A, L-Ala (6.8) and D-Ala (15.2) in solvent B. The retention time and ESIMS product ions (m/z [M + Na]+) of the Marfey’s derivatives of Leu from compound 1 was 5.6 min (m/z 406.1), proving the configuration of Leu was L. The retention time and ESIMS product ions (m/z [M + Na]+) of the Marfey’s derivatives of Ala from compound 2 was 6.8 min (m/z 364.1), proving the configuration of Ala was L. Culture of L6Myoblasts. L6 myotubes were differentiated from myoblasts by the procedure previously described.17 Determination of Glucose Uptake. The assay was performed as previously described.17 The concentrations of compounds 1 and 2 used in this assay were 10−40 μM.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00088. 1D and 2D NMR spectra of 1 and 2 and phylogenetic tree of cyanobacteria (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Kosuke Sueyoshi: 0000-0002-7965-3236 Arihiro Iwasaki: 0000-0002-3775-5066 Kiyotake Suenaga: 0000-0001-5343-5890 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid for Young Scientists (B) (21710237) from the Ministry of Education, Culture, Sports, Science and Technology, Japan; University of the Ryukyus Strategic Research Grant.
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REFERENCES
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