Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Minnamide A, a Linear Lipopeptide from the Marine Cyanobacterium Okeania hirsuta Shimpei Sumimoto,† Masayuki Kobayashi,† Rio Sato,† Seiichi Shinomiya,† Arihiro Iwasaki,† Shoichiro Suda,‡ Toshiaki Teruya,§ Toshiyasu Inuzuka,|| Osamu Ohno,⊥ and Kiyotake Suenaga*,† †
Department of Chemistry, Keio University, 3-14-1, Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan Department of Chemistry, Biology and Marine Science, Faculty of Science, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan § Faculty of Education, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan || Life Science Research Center, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan ⊥ Department of Chemistry and Life Science, Kogakuin University, 2665-1 Nakano, Hachioji, Tokyo 192-0015, Japan
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‡
S Supporting Information *
ABSTRACT: Minnamide A is a lipopeptide with a unique repeating structure consisting of hydroxy and proposed β-branched methyl groups. The absolute configuration of minnamide A was determined by a combination of chemical degradation, chiral HPLC analyses, and synthetic methods. Minnamide A showed growth-inhibitory activity toward HeLa cells with an IC50 value of 0.17 μM and rapidly induced cell death at a concentration of 2 μM. Minnamide A induced the copper-mediated accumulation of reactive oxygen species.
M
collected cyanobacterium was extracted with MeOH. The extract was filtered, concentrated, and partitioned between EtOAc and water. The EtOAc layer was further partitioned between 90% aqueous MeOH and hexane. The 90% aqueous MeOH layer was subjected to fractionation guided by growthinhibitory activity against HeLa cells. Fractionation was performed by reversed-phase open column chromatography (ODS silica gel, MeOH−H2O) and reversed-phase HPLC (Cosmosil 5C18 MS-II, MeCN-H 2 O; Cosmosil 5PE-MS, MeCN-H2O) to give 188.2 mg of 1. The molecular formula of 1 was determined to be C74H132N10O18 by HRESIMS (m/z 1471.9594, calcd for C74H132N10O18Na [M + Na]+ 1471.9619). NMR data of 1 are summarized in Table S1. The 1H, 13C, COSY, HMQC, and HMBC spectra of 1 suggest the presence of 10 carbonyl groups (δC 176−170), nine methines corresponding to the α-positions of amino acid derivatives (δH/δC 5.64/56.7, 5.58/59.0, 5.41/ 51.8, 5.28/61.4, 5.28/49.1, 5.27/48.9, 5.21/56.0, 5.15/62.4), four N-methyl groups (δH/δC 3.34/30.9, 3.33/31.1, 3.32/30.7, 3.25/31.1), one O-methyl group (δH/δC 3.69/55.2), three oxymethylenes (δH/δC 4.24 and 4.10/62.9, 4.22/62.9, 3.88/ 62.8), four oxymethines (δH/δC 4.57/66.8, 4.19/66.4, 4.12/ 66.4, 3.98/68.4), two primary methyl groups (δH /δC 0.92/14.6, 0.85/23.2), and 12 secondary methyl groups (δH /δC 1.14/19.6,
any natural products display interesting biological activities, which depend on their various structures. Marine cyanobacteria are good sources of compounds with therapeutic potential and useful tools for biological research.1 Cyanobacteria produce diverse lipopeptides, many of which are products of nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) hybrid pathways.2,3 Recently, genomic analysis has shown that gene shuffling combined with specific tailoring enzymes of NRPS/PKS genes contributes to the uniqueness of secondary metabolites in cyanobacteria.4 Here, we report the isolation, structure elucidation, and preliminary biological characterization of minnamide A (1). 1 possesses a unique repeating structure consisting of hydroxy and proposed β-branched methyl groups in a fatty acid moiety. The β-branched methyl groups of polyketides are biosynthesized by aldol reactions of an enolate of acetyl CoA to β-ketothioester and subsequent dehydration, decarboxylation, and double bond reduction.5,6 Some NRPS/PKS natural products or polyketides possessing proposed β-branched methyl groups have been isolated from cyanobacteria, such as apratoxin A,6 janadolide,7 phormidolide,8 and oscillariolide.9 However, 1 is the first natural product that has been shown to possess a structure consisting of hydroxy and proposed β-branched methyl groups. The marine cyanobacterium Okeania hirsuta (1.75 kg, wet weight) was collected at Minna Island, Okinawa. Collected cyanobacterium was identified by phylogenetic and morphological analyses (Supporting Information (SI) S8−S11). The © XXXX American Chemical Society
Received: January 11, 2019
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DOI: 10.1021/acs.orglett.9b00135 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
Scheme 1. Partial Hydrolysis of 1 and Preparation of MTPA Esters
1.12/19.6, 1.08/20.7, 1.05/20.1, 1.00/19.7, 0.98/18.6, 0.97/ 22.1, 0.94/22.0, 0.85/23.2, 0.81/14.9, 0.80/19.0, 0.78/23.3). Intensive interpretation of the COSY, TOCSY, HMQC, and HMBC spectra revealed the presence of one N-methylglutamine (N-Me-Gln), two N-methylvalines (N-Me-Val), two serines (Ser), two leucines (Leu), one N-methylisoleucine (N-Me-Ile), one 2-amino-3-(4-methoxyphenyl)propan-1-ol (Amp), and one 3,7,11,15-tetrahydroxy-5,9,13-trimethyloctadecanoic acid (fatty acid). The sequences of these partial structures in 1 were determined on the basis of HMBC and ROESY data (Figure 1, Table S1). Eight HMBC correlations [NH (Amp)/C1 (Gln),
HPLC analyses (SI S15). Therefore, Ser1 and Leu1 were eventually identified as the L-form and D-form, respectively. Thus, the absolute configurations of all amino acids and Amp were determined as shown in 1. The absolute configuration of the four hydroxy groups in the fatty acid of 1 were determined by a modified Mosher’s method.11 1 was partially hydrolyzed and then subjected to methyl esterification of the resultant carboxylic acid to give methyl ester 2 and cyclic ether 3, probably formed from dehydration followed by oxy-Michael addition (Scheme 1). The 2 was reacted with 0.4 molar equiv of (R)- and (S)-α-methoxyα-trifluoromethylphenylacetic acid (MTPA) chlorides. Esterification occurred nonselectively, and careful HPLC purification gave eight mono-MTPA esters 4a−7b (Scheme 1).12 A consistent distribution of positive and negative Δδ values around C3, C7, C11, and C15 allowed us to assign a configuration of 3S,7R,11R,15R (see the SI). To determine the absolute configuration at C5 in the fatty acid of 1, the stereochemistry of 3 was elucidated based on the analyses of NOESY and coupling constants, as shown in Figure 2. The NOESY correlations (H-3/H-19, H-19/H-6b, and H-4a/
Figure 1. Gross structure of minnamide A (1) based on 2D NMR correlations.
N-Me (N-Me-Gln)/C1 (N-Me-Val1), N-Me (N-Me-Val1)/C1 (Ser1), NH (Ser1)/C1 (N-Me-Val2), N-Me (N-Me-Val2)/C1 (Leu1), NH (Leu1)/C1 (Ser2), NH (Ser2)/C1, N-Me (N-MeIle)/C1 (Leu2), and NH (Leu2)/C1 (fatty acid)] and three ROESY correlations [NH (Amp)/H-2 (N-Me-Gln), N-Me (NMe-Val2)/H-2 (Leu2) and NH (Ser2)/H-2 (N-Me-Ile)] revealed the gross structure of 1, as shown in Figure 1. To distinguish between N-Me-Ile and N-Me-allo-Ile, acid hydrolysate of 1 was compared with authentic standards using reversed-phase HPLC (Cosmosil PBr) (SI S13). This result showed the presence of N-Me-allo-Ile residue. Absolute configurations of the Amp moiety and amino acids were determined by chiral-phase HPLC analyses or Marfey’s analyses10 of the acid hydrolysate of 1. These results showed the presence of (S)-Amp, N-Me-L-Gln, N-Me-L-Val, D/L-Ser, D/ L -Leu and N-Me- D -allo-Ile residues (SI S14−S17). To distinguish the D/L-Ser and D/L-Leu residues, 1 was hydrolyzed under two different mild acid conditions, and we obtained two partial hydrolysates S32 with one Ser (SI S11) and 2 with one Leu (Scheme 1). Compound S32 was completely hydrolyzed, and obtained Ser2 was determined to be D-form by Marfey’s analyses10 (SI S16). Compound 2 was completely hydrolyzed, and obtained Leu2 was determined to be L-form by chiral-phase
Figure 2. Configuration and conformation for 3 established on the basis of selected 3JH,H and NOESY correlations.
H-7) were observed. These data indicated that three protons (H-3, H-19, and H-6b) and two protons (H-4a and H-7) were oriented in the same face of the tetrahydropyran ring. The orientation between H-5 and H-6a was determined to be synperiplanar based on the coupling constants (JH5−H6a = 11.0 Hz, JH4a‑H5 = 3.0 Hz) and NOESY correlations (H-4a and H-7). B
DOI: 10.1021/acs.orglett.9b00135 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters These data revealed that the relative stereochemistry of 3 was determined as shown in Figure 2. We estimated that 3 was produced by dehydration of C3-OH followed by oxy-Michael addition from the 2. Based on the absolute configuration of C7 described above, we established the absolute configuration of C5 in the fatty acid of 1 to be S. To determine the configuration of C13 in the fatty acid of 1, we prepared another cyclic ether 9. 2 was transformed into mesylate 8, which was spontaneously cyclized to give 9 (Scheme 2). The stereochemistry of the six-membered ring was
Scheme 3. Preparation of MOM Ether Derived from 2 and Synthesis of (9R)-10 and (9S)-10
Scheme 2. Preparation of Cyclic Ether 9
elucidated based on the analysis of NOESY and coupling constants, as shown in Figure 3. The large coupling constants
Figure 4. Induction of cell death by 2 μM of 1 in HeLa cells. (A−E) Visualization of HeLa cell viability over time with minnamide A. (F) Phase contrast microscopy of cell death induced by minnamide A.
Figure 3. Configuration and conformation for 9 established on the basis of selected 3JH,H and NOESY spectra.
(JH11−H12b = 12.1 Hz, JH12b‑H13 = 12.1 Hz, JH13−H14b = 12.3 Hz) and NOESY correlations (H-11/H-13 and H-12b/H-14b) indicated that H-11, H-12b, H-13, and H-14b were in the axial positions. These results showed that the relative stereochemistry of 9 was as shown in Figure 3. On the basis of the absolute configuration of C11 described above, we established the absolute configuration of C13 in the fatty acid of 1 to be S. To clarify the absolute configuration of C9, we prepared MOM ether 10 from 2 and synthesized its two possible stereoisomers, (9R)-10 and (9S)-10 (Scheme 3; for details see the SI). The 1H NMR spectrum of synthetic compound (9S)-10 was identical to that of 10 derived from 2. Thus, the complete absolute stereostructure of 1 was established as shown in 1. Minnamide A inhibited the growth of HeLa cells with an IC50 value of 0.17 μM. In addition, we observed that cell death was rapidly induced by treatment with highly concentrated 1 (Figure 4A−E). The dead cells were morphologically similar to necrosis (Figure 4F), and the massive production of small surface evaginations (bubbles) was observed by phase contrast microscopy.13 Next, we evaluated the effects of various potential inhibitors on minnamide A-induced cell death (Figure 5). Cell death was prevented by antioxidants (α-tocopherol, coenzyme
Figure 5. Viability of HeLa cells preincubated with inhibitors and treated with 2 μM of minnamide A for 4 h.
Q10, N-acetyl-L-cysteine, thiourea), copper chelators (bathocuproinedisulfonic acid and ammonium tetrathiomolybdate), an iron chelator (deferoxamine), and nonspecific metal chelators (EDTA, CDTA, and DMSA). These results suggest that reactive oxygen species (ROS) and some metal ions are essential for minnamide A-induced cell death. To assess the specificity of metal ions for minnamide Ainduced cell death, we evaluated the effects of various metal ions for minnamide A-induced cell death (Figure S4). Co-treatment C
DOI: 10.1021/acs.orglett.9b00135 Org. Lett. XXXX, XXX, XXX−XXX
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with copper and manganese significantly potentiated minnamide A-induced cell death, whereas iron, zinc, chromium, and lead showed weak potentiation. In contrast, minnamide Ainduced cell death was not potentiated by co-treatment with magnesium, cobalt, or selenium. As there is a well-known relationship between metal ions and lipid ROS production for regulated cell death,14,15 we quantified lipid ROS production using the fluorescent probe C11-BODIPY (Figure 6). Treatment with 1 resulted in a dose-dependent
Letter
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
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 Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (16H03285 and 17H05450). We thank Kaneka Corporation for their gift of (S)-Roche ester.
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Figure 6. Lipid ROS assessed in HeLa cells treated with minnamide A in the presence or absence of inhibitors.
increase in lipid ROS production. This increase in ROS was prevented by α-tocopherol and bathocuproinedisulfonic acid, an antioxidant and a copper chelator, respectively. These results indicate that copper ions are involved upstream of lipid ROS accumulation. Recently, various types of regulated cell death have been defined on a genetic, biochemical, pharmacological, and functional basis. Ferroptosis is characterized by the irondependent accumulation of lethal lipid ROS.14 The relationship between iron and lipid ROS accumulation in ferroptosis is similar to the relationship between copper and lipid ROS accumulation in minnamide A-induced cell death. Minnamide A-induced cell death may involve copperdependent lipid ROS accumulation. Elevated copper levels have been observed in some cancer cells,16 and metal-induced oxidative stress is involved in some neurodegenerative diseases.17−19 In conclusion, minnamide A was isolated from the marine cyanobacterium O. hirsuta. 1 is the first discovered natural product that contains a fatty acid with a repeating structure consisting of hydroxy and proposed β-branched methyl groups. In addition, 1 showed growth-inhibitory activity in HeLa cells with an IC50 value of 0.17 μM and induced necrosis-like regulated cell death. Minnamide A induced the copper-mediated accumulation of ROS.
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REFERENCES
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(13) Rello, S.; Stockert, J. C.; Moreno, V.; Gámez, A.; Pacheco, M.; Juarranz, A.; Cañete, M.; Villanueva, A. Apoptosis 2005, 10, 201−208. (14) Dixon, S. J.; Lemberg, K. M.; Lamprecht, M. R.; Skouta, R.; Zaitsev, E. M.; Gleason, C. E.; Patel, D. N.; Bauer, A. J.; Cantley, A. M.; Yang, W. S.; Morrison, B.; Stockwell, B. R. Cell 2012, 149, 1060−1072. (15) Maher, P. Free Radical Biol. Med. 2018, 115, 92−104. (16) Gupte, A.; Mumper, R. J. Cancer Treat. Rev. 2009, 35, 32−46. (17) Ahuja, A.; Dev, K.; Tanwar, R. S.; Selwal, K. K.; Tyagi, P. K. J. Trace Elem. Med. Biol. 2015, 29, 11−23. (18) Cheignon, C.; Jones, M.; Atrián-Blasco, E.; Kieffer, I.; Faller, P.; Collin, F.; Hureau, C. Chem. Sci. 2017, 8, 5107−5118. (19) Lan, A. P.; Chen, J.; Chai, Z. F.; Hu, Y. BioMetals 2016, 29, 665− 678.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b00135. HPLC chromatograms for determination of absolute configurations; phylogenetic tree for cyanobacterial identification; synthesis of (9R)-10 and (9S)-10; detailed experimental procedures (PDF) NMR spectra for all new compounds (PDF) D
DOI: 10.1021/acs.orglett.9b00135 Org. Lett. XXXX, XXX, XXX−XXX
