Hoshinoamides A and B, Acyclic Lipopeptides from the Marine

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Hoshinoamides A and B, Acyclic Lipopeptides from the Marine Cyanobacterium Caldora penicillata Arihiro Iwasaki,† Takato Tadenuma,† Shimpe Sumimoto,† Ikuma Shiota,† Teruhiko Matsubara,‡ Yumiko Saito-Nakano,§ Tomoyoshi Nozaki,∥ Toshinori Sato,‡ and Kiyotake Suenaga*,†

J. Nat. Prod. Downloaded from pubs.acs.org by UNIV OF TEXAS AT EL PASO on 11/02/18. For personal use only.

†

Department of Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan ‡ Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan § Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan ∥ Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan S Supporting Information *

ABSTRACT: Hoshinoamides A (1) and B (2), new acyclic lipopeptides, were isolated from the marine cyanobacterium Caldora penicillata. Their structures were elucidated by spectroscopic analyses and degradation reactions. Hoshinoamides A (1) and B (2) did not exhibit any cytotoxicity against HeLa cells at 10 μM, but inhibited the in vitro growth of the malarial parasite Plasmodium falciparum (IC50 = 0.52 and 1.0 μM, respectively).

M

arine cyanobacteria are rich resources of secondary metabolites possessing unusual structures and intriguing biological activities.1 Despite their utility, the taxonomic classification of marine cyanobacteria has been neglected, and some new genera, such as Moorea and Okeania, have been proposed in recent years.2,3 Against this background, in 2015, Engene et al. established the new genus Caldora and clarified that they produce various natural products, such as dolastatin 10, symplostatin 1, and largazole.4 Since this report, several natural products, including caldoramide and caldorin, have been discovered from this genus, and its ability to produce a variety of secondary metabolites has been demonstrated.5,6 Here we describe new lipopeptides, hoshinoamides A (1) and B (2), isolated from the marine cyanobacterium Caldora penicillata. The hoshinoamides possess an unusual long-chain amino acid moiety and a hydroxyphenylbutanoic acid moiety. Although 1 and 2 did not exhibit any cytotoxicity against HeLa cells, hoshinoamides (1 and 2) showed moderate antimalarial activities.

aqueous MeOH and hexane. The material obtained from the aqueous MeOH portion was subjected to fractionation by reversed-phase column chromatography (ODS silica gel, MeOH−H2O) and repeated reversed-phase HPLC to give hoshinoamide A (1, 2.6 mg). Meanwhile, a second sample of the same cyanobacterium (1600 g, wet weight) was collected at the same location in October 2016. A similar separation operation as described above afforded hoshinoamide B (2, 7.1 mg).

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RESULTS AND DISCUSSION The marine cyanobacterium Caldora penicillata (1600 g, wet weight) was collected at Hoshino, Okinawa, Japan, in June 2014, and extracted with MeOH. The extract was filtered, concentrated, and partitioned between EtOAc and H2O. The EtOAc-soluble material was further partitioned between 90% © XXXX American Chemical Society and American Society of Pharmacognosy

Received: August 1, 2018

A

DOI: 10.1021/acs.jnatprod.8b00643 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 1. NMR Data for Hoshinoamide A in CD3OD residue Pro-O-Me

N-Me-Phe

Val

Gln

N-Me-Val

Ile

N-Me-Leu

6-aminohexanoic acid

4-(4-hydroxyphenyl)-butanoic acid

position 1 2 3 4a 4b 5a 5b 6a 6b 7 8 9a 9b 10 11/15 12/14 13 16 17 18 19 20 21 22 23 24a 24b 25a 25b 26 27 28 29 30 31 32 33 34 35 36a 36b 37 38 39 40 41a 41b 42 43 44 45 46 47 48 49 50 51 52 53 54 55

δc, typeb 52.7, CH3 173.8, C 60.8, CH 29.7, CH2 26.2, CH2 48.2, CH2 170.5, CH 57.2, CH 35.7, CH2 138.4, C 130.7, CH 129.4, CH 127.7, CH 31.5, CH3 173.14, Ccd 55.6, CHc 31.5, CH 18.1, CH3 19.9, CH3 173.06, Ccd 54.3, CH 26.0, CH2 29.9, CH2 177.4, C 171.9, C 64.1, CH 27.7, CH 20.3, CH3 19.5, CH3 32.6, CH3 174.5, C 55.5, CHc 38.4, CH 25.1, CH2 11.6, CH3 16.5, CH3 172.9, C 55.6, CHc 37.8, CH2 26.7, CH 23.8, CH3 22.1, CH3 31.8, CH3 176.5, C 34.6, CH2 25.9, CH2 27.7, CH2 30.2, CH2 40.2, CH2 176.0, C 36.6, CH2 29.2, CH2 35.5, CH2

δH (J in Hz)a

COSY

3.70, s 4.38, 2.23, 1.86, 1.97, 1.87, 3.50, 3.34,

dd (8.1, 6.1) m m m m m m

selected HMBC

4a, 4b 3, 4b, 5a, 5b 3, 4a, 5a, 5b 4a, 4b, 5b, 6a, 6b 4a, 4b, 5a, 6a, 6b 5a, 5b, 6b 5a, 5b, 6a

8

5.74, dd (9.0, 6.4) 3.17, m 2.90, m

9a, 9b 8, 9b 8, 9a

7.24, m 7.25, m 7.16, m 3.09c, s

12/14 11/15, 13 12, 14

4.57, 1.76, 0.65, 0.60,

d (7.2) m d (6.6) d (6.8)

19 18, 20, 21 19 19

17

4.30, 2.02, 1.93, 2.25, 1.87,

dd (8.6, 5.6) m m m m

24a, 24b 23, 24b, 25a, 25b 23, 24a, 25a, 25b 24a, 24b, 25b 24a, 24b, 25a

22

4.61, d (10.8) 2.28, m 0.98, d (6.6) 0.85, d (6.8) 3.09c, s

29 28, 30, 31 29 29

27, 32

4.78, 1.84, 1.46, 1.07, 0.86, 0.93,

d (6.2) m m m m d (6.9)

35 34, 36a, 36b, 38 35, 36b, 37 35, 36a, 37 36a, 36b 35

33

5.22, 1.75, 1.59, 1.40, 0.94, 0.89, 2.97,

dd (10.8, 5.2) m m m d (6.6) d (6.6) s

41a, 41b 40, 41b, 42 40, 41a, 42 41a, 41b, 43, 44 42 42

2.43, 1.64, 1.39, 1.53, 3.17,

t (7.2) m m m t (7.2)

48 47, 49 48, 50 49, 51 50

46

2.16, t (7.6) 1.86, m 2.52, t (7.6)

54 53, 55 54

52

B

selected NOESY

2

7, 16 10, 11, 15 10, 11, 15

6a

26 26

40, 46

52

56, 57, 61 DOI: 10.1021/acs.jnatprod.8b00643 J. Nat. Prod. XXXX, XXX, XXX−XXX


Article

Table 1. continued residue

position 56 57/61 58/60 59

δc, typeb 133.7, 130.4, 116.1, 156.6,

C CH CH C

δH (J in Hz)a 6.99, d (8.5) 6.70, d (8.5)

COSY 58/60 57/61

selected HMBC

selected NOESY

59 59

a

Measured at 400 MHz. bMeasured at 100 MHz. cThese signals are interchangeable. dDigits in the hundredths place are nonreproducible.

Figure 1. Planar structure of hoshinoamide A (1) based on 2D NMR and MSn analyses.

contiguous methylene groups was clarified based on COSY correlations. HMQC correlation revealed that the terminal methylene protons at 2.43 ppm (t, J = 7.2 Hz) were assocated with a carbon at 34.6 ppm. Meanwhile, the other terminal methylene protons at 3.17 ppm (t, J = 7.2 Hz) were located on a carbon at 40.2 ppm, and these chemical shifts suggested that it was connected to a nitrogen atom of an amide group. In addition, each terminal methylene showed an HMBC correlation to a carbonyl carbon, respectively. Based on these observations, we clarified the presence of the Aha residue. The existence of the other partial structures was established by same procedures as described above. The sequences of these fragments were determined as follows: Based on the two HMBC correlations, H-45/C-46 and H-51/C-52, the following partial sequence was clarified: HbaAha-N-Me-Leu. In addition, the NOESY correlation H-6a/H-8 revealed connectivity between Pro-O-Me and N-Me-Phe. However, overlapped NMR signals prevented us from further elucidating the sequence of 1. Therefore, we changed the NMR solvent from CD3OD to CDCl3 to obtain additional information. As a result, a new NOESY correlation between H32/H-34 was observed, indicating the connection between NMe-Val and Leu. So far, the following five partial structures were elucidated: Hba-Aha-N-Me-Leu, Leu-N-Me-Val, Gln, Val, and N-Me-Phe-Pro-O-Me. The remaining connectivity between these partial structures was clarified based on detailed analyses of the MS2 and MS3 spectra (Figures S14 and S15).

Hoshinoamide A (1) was obtained as a colorless oil. Hoshinoamide A (1) existed as a 4:1 mixture of rotamers in CD3OD, and the NMR data for the major rotamer of 1 are summarized in Table 1. The molecular formula of 1 was found to be C61H95N9O12 by HRESIMS. In the 1H NMR spectrum, three singlet methyl signals (δH 3.09, 3.09, and 2.97) and one relatively deshielded methyl (δH 3.70) signal indicated the presence of three N-methyl amide groups and one methyl ester moiety, respectively. In addition, five aromatic proton signals (δH 7.16−7.25) and one pair of doublet signals (δH 6.99 (2H) and 6.70 (2H)) suggested that 1 contained a monosubstituted benzene ring and a para-substituted phenol, respectively. This prediction is also supported by the observation of eight aromatic carbon signals in the 13C NMR spectrum (δC 156.6, 138.4, 133.7, 130.7 (2C), 130.4 (2C), 129.4 (2C), 127.7, and 116.1 (2C)). Furthermore, in the 13C NMR spectrum, 10 deshielded signals (δC 177.4, 176.5, 176.0, 174.5, 173.8, 173.14, 173.06, 172.9, 171.9, and 170.5) corresponding to amide or ester carbonyl carbons were detected. Next, we analyzed the COSY and HMQC spectra and clarified the connectivities of several protons and carbons as shown by bold lines in Figure 1. Then, we expanded these fragments based on the correlations in the HMBC and NOESY spectra. As a result, we revealed the presence of nine partial structures: Pro-O-Me, N-Me-Phe, Val, Gln, N-Me-Val, Ile, N-Me-Leu, 6-aminohexanoic acid (Aha), and 4-(4-hydroxyphenyl)-butanoic acid (Hba). Regarding the Aha moiety, the presence of five C



Article

Table 2. NMR Data for Hoshinoamide B in CD3OD residue Pro-O-Me

N-Me-Phe

Pro

Gln

N-Me-Val

Ile

N-Me-Leu

9-aminononanoic acid

position

δc, typeb

1 2 3 4a 4b 5a 5b 6a 6b 7 8 9a 9b 10 11/15 12/14 13 16 17 18 19a 19b 20a 20b 21a 21b 22 23 24a 24b 25 26 27 28 29 30 31 32 33 34 35 36a 36b 37 38 39 40 41a 41b 42 43 44 45 46 47 48 49 50 51 52 53

52.7, CH3 174.07, Ccd 60.8, CH 30.0, CH2 25.8, CH2 48.4, CH2 171.0, C 60.4, CH 35.7, CH2 138.7, C 130.4, CH 129.6, CH 127.7, CH 33.7, CH3 174.14, Ccd 58.8, CH 29.7, CH2 26.0, CH2 48.4, CH2 171.3, C 51.9, CH 25.8, CH2 31.9, CH2 177.7, C 171.7, C 63.8, CH 28.1, CH 20.5, CH3 19.5, CH3 32.1, CH3 174.3, Cc 55.4, CHc 38.3, CH 25.4, CH2 11.5, CH3 16.3, CH3 172.9, C 55.5, CHc 37.7, CH2 26.5, CH 23.5, CH3 21.9, CH3 31.7, CH3 176.9, C 32.5, CH2 26.03, CH2cd 25.98, CH2cd 25.93, CH2cd 26.01, CH2cd 26.03, CH2cd 30.7, CH2

δH (J in Hz)a

COSY

selected HMBC

selected NOESY

3.66, s 4.34, 2.19, 1.89, 1.92, 1.90, 3.62, 3.31,

m m m m m m m


2

5.09, dd (7.4, 7.4) 3.24, m 3.01, m

9a, 9b 8, 9b 8, 9a

7 10, 11, 15 10, 11, 15

6a, 6b

7.27, 7.29, 7.21, 3.00,

m m m m

12/14 11/15, 13 12, 14 8

18

4.79, 2.25, 1.81, 2.05, 1.98, 3.85, 3.70,

dd (8.8, 5.1) m m m m m m


4.62, 2.13, 1.99, 2.29,

m m m m

24a, 24b 23, 24b, 25 23, 24a, 25 24a, 24b

4.66, 2.28, 1.00, 0.85, 3.10,

m m d (6.4) d (6.9) s

29 28, 30, 31 29 29

4.72, 1.81, 1.49, 1.09, 0.88, 0.92,

d (6.3) m m m m d (6.8)

5.18, 1.71, 1.56, 1.39, 0.93, 0.86, 2.96, 2.41, 1.64, 1.39, 1.39, 1.39, 1.39, 1.48,

8 8

16 17 17

23 23 21a, 21b 22, 26 22, 26 26

27

28

34

35 34, 36a, 36b, 38 35, 36b, 37 35, 36a, 37 36a, 36b 35

33

32

dd (10.7, 5.3) m m m d (6.2) d (6.6) s

41a, 41b 40, 41b, 42 40, 41a, 42 41a, 41b, 43, 44 42 42

39, 46

m m m m m m m

48 47

40, 46 46

53 52, 54 D



Article

Table 2. continued residue 4-(4-hydroxyphenyl)-butanoic acid

position 54 55 56 57 58 59 60/64 61/63 62

δc, typeb 40.3, CH2 175.9, C 36.6, CH2 29.2, CH2 35.5, CH2 133.7, C 130.4, CH 116.2, CH 156.6, CH

δH (J in Hz)a

COSY

selected HMBC

3.16, t (6.9)

53

55

2.17, t (7.7) 1.85, m 2.52, t (7.7)

57 56, 58 57

55, 58

6.99, d (8.6) 6.69, d (8.6)

61/63 60/64

selected NOESY

56, 59, 60, 64

62

a

Measured at 400 MHz. bMeasured at 100 MHz. cThese signals are interchangeable. dDigits in the hundredths place are nonreproducible.

Figure 2. Gross structure of hoshinoamide B (2) based on 2D NMR and MS/MS analyses.

and δC ∼26) were typical values for those of aliphatic methylene chains, and these data also supported the presence of the Ana moiety. The absolute configurations of hoshinoamides A (1) and B (2) were determined by acid hydrolysis followed by a combination of chiral-phase HPLC analyses and advanced Marfey’s method.7 For hoshinoamide A (1), the N-Me-Phe and N-Me-Val residues were determined to be the D-forms and the other residues were the L-forms, as shown in structure 1. Meanwhile, in hoshinoamide B (2), the absolute configurations of all of the amino acid moieties except for the N-MeD-Val residue were established to be L. Next, we evaluated the biological activities of hoshinoamides A (1) and B (2) (Table 3). First, we tested their cell growth inhibitory activity by using the MTT assay. As a result, 1 and 2 did not inhibit the growth of HeLa cells at 10 μM. Next, we assessed the antimalarial activities of 1 and 2 against the asexual erythrocytic stage of the Plasmodium falciparum 3D7 clone, which is a standard reference that is sensitive to most antimalarials including chloroquine, sulfadoxine, pyrimethamine, atovaquone, and artemisinin. As shown in Table 3, hoshinoamides A (1) and B (2) exhibited antiplasmodial

As a result, the gross structure shown in Figure 1 was determined to be the unique structure that could explain the observed fragmentation patterns. Hoshinoamide B (2) was obtained as a colorless oil. Hoshinoamide B (2) also existed as a 4:1 mixture of rotamers in CD3OD, and the NMR data for the major rotamer of 2 are summarized in Table 2. The molecular formula of 2 was found to be C64H99N9O12 by HRESIMS. The 1H NMR spectrum of hoshinoamide B (2) is similar to that of 1 except for the presence of broad overlapping signals at 1.3−1.5 ppm. These signals were considered to be protons corresponding to aliphatic methylene groups; therefore, we assumed that hoshinoamide B (2) possessed a longer aliphatic chain than A (1). Detailed analyses of the 2D NMR spectra and the MS/ MS spectra revealed the gross structure of 2 as shown in Figure 2. As a result, hoshinoamide B (2) had two different residues compared to 1: a Pro residue and a 9-aminononanoic acid residue (Ana) in place of the Val residue and the 6aminohexanoic acid residue, respectively. Because of the overlapping signals, the length of the aliphatic chain in the Ana residue was determined based on the MS/MS data. The proton and carbon chemical shifts of C-49 to C-52 (δH 1.39 E



Article

Buffer for KOD-Multi & Epi-, and H2O for a total volume of 25 μL. The PCR reaction was performed as follows: initial denaturation for 2 min at 94 °C, amplification by 40 cycles of 10 s at 98 °C, 10 s at 58 °C, 1 min at 68 °C, and final elongation for 7 min at 68 °C. PCR products were analyzed on an agarose gel (1%) in TBE buffer and visualized by ethidium bromide staining. The obtained DNA was sequenced with CYA106F and 16S1541R primers. This sequence is available in the DDBJ/EMBL/Genbank databases under an accession number LC317271. The nucleotide sequence of 16S rRNA gene obtained in this study was used for phylogenetic analysis with the sequences of related cyanobacterial 16S rRNA genes.4 All sequences were aligned by SINA web service (version 1.2.11)11 with default settings. The poorly aligned positions and divergent regions were removed by Gblocks Server (version 0.91b),12 implementing the options for a less stringent selection, including the “Allow smaller final blocks,” “Allow gap positions within the final blocks,” and “Allow less strict flanking positions” options. The obtained 878 nucleotide positions have been used for phylogenetic analysis. JModeltest (version 2.1.7)13 with default settings was used to select the best model of DNA substitution for the Maximum Likelihood (ML) analysis and Bayesian analysis according to the Akaike information criterion (AIC). The ML analysis was conducted by PhyML (version 20131016),14 using the GTR+I+G model with a gamma shape parameter of 0.4770, a proportion of invariant sites of 0.5110 and nucleotide frequencies of F(A) = 0.2427, F(C) = 0.2365, F(G) = 0.3225, and F(T) = 0.1984. Bootstrap resampling was performed on 1000 replicates. The ML tree was visualized with Njplot (version 2.3).15 The Bayesian analysis was conducted by MrBayes (version 3.2.5)16 using the GTR+I+G model. The Markov chain Monte Carlo process was set at two chains, and 1 000 000 generations were conducted. Sampling frequency was assigned at every 500 generations. After analysis, the first 100 000 trees were deleted as burn-in, and the consensus tree was constructed. The Bayesian tree was visualized with FigTree (version 1.4.0, http://tree.bio.ed.ac.uk/software/figtree). As a result, the cyanobacterium formed a clade with Caldora penicillata. Therefore, the cyanobacterium was classified into Caldora penicillata. Collection, Extraction, and Isolation. The cyanobacterium, Caldora penicillata (1600 g, wet weight), was collected at Hoshino, Okinawa, Japan, in June 2014. The collected cyanobacterium was extracted with MeOH (2 × 2 L) for 1 week. The extract was filtered, and the filtrate was concentrated. The residue was partitioned between EtOAc (3 × 0.3 L) and H2O (0.3 L). The material obtained from the organic layer was partitioned between 90% aqueous MeOH (0.3 L) and hexane (3 × 0.3 L). The aqueous MeOH fraction (197 mg) was separated by column chromatography on ODS (5 g) eluted with 40% MeOH, 60% MeOH, 80% MeOH, and MeOH. The fraction (36 mg) eluted with 80% MeOH was subjected to HPLC [Cosmosil 5C18AR-II (ϕ20 × 250 mm); flow rate 5 mL/min; detection, UV 215 nm; solvent 75% MeOH] to give a fraction that contained hoshinoamide A (1) (3.3 mg, tR = 45.5 min). This fraction was further purified by HPLC [Cosmosil Cholester (ϕ20 × 250 mm); flow rate 5 mL/min; detection, UV 215 nm; solvent 75% MeOH] to give hoshinoamide A (1, 2.6 mg, tR = 44.6 min). A second collection of C. penicillata (1600 g, wet weight) was made at Hoshino, Okinawa, Japan, in October 2016. The collected cyanobacterium was extracted with MeOH (2 × 3 L) for 1 week. The extract was filtered, and the filtrate was concentrated. The residue was partitioned between EtOAc (3 × 0.3 L) and H2O (0.3 L). The material obtained from the organic layer was partitioned between 90% aqueous MeOH (0.3 L) and hexane (3 × 0.3 L). The aqueous MeOH fraction (487 mg) was separated by column chromatography on ODS (5 g) eluted with 40% MeOH, 60% MeOH, 80% MeOH, and MeOH. The fraction (126 mg) eluted with 80% MeOH was subjected to HPLC [Cosmosil 5C18AR-II (ϕ20 × 250 mm); flow rate 5 mL/min; detection, UV 215 nm; solvent 75% MeOH] to give a fraction that contained hoshinoamide B (2) (18.7 mg, tR = 47.9 min). This fraction was further purified by HPLC [Cosmosil Cholester (ϕ20 × 250 mm); flow rate 5 mL/min; detection, UV 215 nm; solvent 55% MeCN] to give hoshinoamide B (2, 7.1 mg, tR = 33.9 min).

Table 3. Growth Inhibitory Activities of hoshinoamides A (1) and B (2) IC50 values (μM)b compds

HeLa cells

Plasmodium falciparum 3D7

hoshinoamide A (1) hoshinoamide B (2) chloroquinea

14 ± 1 29 ± 7

0.52 ± 0.08 1.0 ± 0.1 0.0076 ± 0.0005

positive control. bValues are shown as the average ± SD.

a

activity with IC50 values of 0.52 and 1.0 μM, respectively. However, the IC50 value of choloroquine was significantly lower than those of hoshinoamides A (1) and B (2). In conclusion, we isolated two new acyclic lipopeptides, hoshinoamides A (1) and B (2), from the marine cyanobacterium Caldora penicillata. Their structures were elucidated by spectroscopic analyses and degradation reactions. The characteristic 4-(4-hydroxyphenyl)-butanoic acid moiety is a relatively rare motif in natural products. Except for hoshinoamides A (1) and B (2), only the stictamides, linear peptides produced by a Sticta sp. of lichen, have been reported to date as secondary metabolites that possess this motif as a partial structure.8 The other structural feature of 1 and 2 is a long chain amino acid moiety, and this motif has been discovered in some cyanobacterial natural products such as mitsoamide.9 However, to the best of our knowledge, hoshinoamide B (2) is the first natural product possessing an Ana moiety. Considering the biosynthetic pathway of the jamaicamides, this rare long chain amino acid can be biosynthesized from one β-Ala and three acetate units.10 Regarding their biological activities, hoshinoamides A (1) and B (2) showed selective toxicity against the malarial parasite P. falciparum compared with human HeLa cells. Therefore, marine Caldora sp. cyanobacteria can be considered to be prospective resources for natural products possessing intriguing structures and useful biological activities.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a JASCO DIP-1000 polarimeter. UV spectra were recorded on a JASCO V730-BIO instrument. IR spectra were recorded on a JASCO RT/IR-4200 instrument. All NMR data were recorded on a JEOL JNM-ECX400 spectrometer for 1H (400 MHz) and 13C (100 MHz). 1H NMR chemical shifts (referenced to residual CHD2OD observed at δH 3.31) were assigned using a combination of data from COSY and HMQC experiments. Similarly, 13C NMR chemical shifts (referenced to CD3OD observed at δC 49.0) were assigned based on HMBC and HMQC experiments. HRESIMS spectra were obtained on an LCT Premier XE time-of-flight (TOF) mass spectrometer (LCT premier XE, Waters). MS and MSn spectra were collected in positive mode by using an amaZon SL ion trap mass spectrometer equipped with an ESI source (Bruker Daltonics). Chromatographic analyses were performed using an HPLC system consisting of a pump (model PU-2080, JASCO) and a UV detector (model UV-2075, JASCO). All chemicals and solvents used in this study were the best grade (extra pure reagent) and available from a commercial source (Nacalai Tesque). Identification of the Marine Cyanobacterium. A cyanobacterial filament collected in October 2016 was isolated under a microscope and crushed with freezing and thawing. The 16S rRNA genes were PCR-amplified from isolated DNA using the primer set CYA106F, a cyanobacterial-specific primer, and 16S1541R, a universal primer. The PCR reaction contained DNA derived from a cyanobacterial filament, 0.5 μL of KOD-Multi & Epi- (Toyobo), 1.0 μL of each primer (0.5 μM, respectively), 12.5 μL of 2× PCR F



Article

Hoshinoamide A (1): colorless oil; [α]25D +13 (c 0.065, MeOH); UV (MeOH) λmax (log ε) 279 (3.21) nm; IR (neat) 3315, 2959, 2930, 1744, 1636, 1515, 1453, 1409, 1222 cm−1; 1H NMR, 13C NMR, COSY, HMBC, and NOESY data, Table 1; HRESIMS m/z 1168.6992 [M + Na]+ (calcd for C61H95N9O12Na, 1168.6998). Hoshinoamide B (2): colorless oil; [α]25D −77 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 279 (3.29) nm; IR (neat) 3326, 2960, 2874, 1745, 1636, 1516, 1450, 1409, 1269 cm−1; 1H NMR, 13C NMR, COSY, HMBC, and NOESY data, Table 2; HRESIMS m/z 1208.7297 [M + Na]+ (calcd for C64H99N9O12Na, 1208.7311). Determination of the Absolute Configuration of Hoshinoamide A (1). Hoshinoamide A (1, 0.3 mg) was treated with 6 M HCl (100 μL) for 21 h at 110 °C. The hydrolyzed product was evaporated to dryness and could be separated into each component. [Conditions for HPLC separation: column, Cosmosil 5C18-PAQ (ϕ4.6 × 250 mm); flow rate, 1.0 mL/min; detection at 215 nm; solvent H2O. Retention times (min) of components; Pro (3.2), Val (3.4), N-MeVal (3.6), Ile + Glu (4.4), N-Me-Leu (5.9), N-Me-Phe (12.6).] Each fraction was dissolved in H2O (50 μL) and analyzed by chiralphase HPLC, and the retention times were compared to those of authentic standards [column, DAICEL CHIRALPAK MA(+) (ϕ 4.6 × 50 mm); flow rate, 1.0 mL/min; detection at 254 nm; solvent, several conditions]. With 2 mM CuSO4 as a solvent, the retention times for authentic standards were 3.7 min (D-Pro) and 7.3 min (LPro), 3.9 min (D-Val) and 7.3 min (L-Val), 4.9 min (N-Me-D-Val) and 7.9 min (N-Me-L-Val), 7.3 min (allo-D-Ile), 9.3 min (D-Ile), 14.1 min (allo-L-Ile) and 19.3 min (L-Ile), and 10.6 min (D-Glu) and 16.9 min (L-Glu). The retention times of each amino acid from natural 1 were 7.3, 7.3, 4.9, 19.3, and 16.9 min, indicating the presence of L-Pro, LVal, N-Me-D-Val, L-Ile, and L-Glu, respectively. With 5% MeCN 2 mM CuSO4 solution as a solvent, the retention times for authentic standards were 5.8 min (N-Me-D-Leu), 8.4 min (N-Me-L-Leu), 14.9 min (N-Me-D-Phe), and 17.8 min (N-Me-L-Phe). The retention time of each amino acid from natural 1 were 8.4 and 14.9 min, indicating the presence of N-Me-L-Leu and N-Me-D-Phe, respectively. Determination of the Absolute Configuration of Hoshinoamide B (2). Hoshinoamide B (2, 0.5 mg) was treated with 6 M HCl (100 μL) for 24 h at 110 °C. The hydrolyzed product was evaporated to dryness and could be separated into each component. [Conditions for HPLC separation: column, Cosmosil 5C18-PAQ (ϕ4.6 × 250 mm); flow rate, 1.0 mL/min; detection at 215 nm; solvent H2O. Retention times (min) of components; Pro (3.3), N-Me-Val (3.7), Ile (4.6), Glu (4.7), N-Me-Leu (5.9), N-Me-Phe (13.0).] Each fraction was dissolved in H2O (50 μL) and analyzed by chiralphase HPLC, and the retention times were compared to those of authentic standards [column, DAICEL CHIRALPAK MA(+) (ϕ 4.6 × 50 mm); flow rate, 1.0 mL/min; detection at 254 nm; solvent, several conditions]. With 2 mM CuSO4 as a solvent, the retention times of each amino acid from natural 2 were 7.3, 4.9, 19.3, 16.9 min, indicating the presence of L-Pro, N-Me-D-Val, L-Ile, and L-Glu, respectively. With 5% MeCN 2 mM CuSO4 solution as a solvent, the retention time of N-Me-Leu in the hydrolysate was 8.4 min, indicating the presence of N-Me-L-Leu. With 10% MeCN 2 mM CuSO4 solution as a solvent, the retention times for authentic standards were 6.7 min (N-Me-D-Phe) and 7.6 min (N-Me-L-Phe). The retention time of NMe-Phe in the hydrolysate was 7.6 min, indicating the presence of NMe-L-Phe. Cell Growth Analysis. HeLa cells were cultured at 37 °C with 5% CO2 in DMEM (Nissui) supplemented with 10% heat-inactivated FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin, 300 μg/mL L-glutamine, and 2.25 mg/mL NaHCO3. Cells were seeded at 4 × 103 cells/well in 96-wells plates (Iwaki) and cultured overnight. Various concentrations of compounds were then added, and cells were incubated for 72 h. Cell proliferation was measured by the MTT assay. Growth-Inhibitory Assay against Malarial Parasites. The P. falciparum 3D7 line was obtained from the Malaria Research and Reference Reagent Resource Center (MR4). P. falciparum axenic culture17 and the drug sensitivity assay18 were described previously. In brief, parasites were cultivated in RPMI-1640 medium containing 5%

heat-inactivated human serum and 0.25% Albumax II (Invitrogen), 200 mM hypoxanthine (Sigma), 10 μg/mL gentamicin (Invitrogen), and human RBC (type O) at 2% hematocrit. One hundred microliter cultures at 0.3% parasitemia containing different concentrations of drugs were prepared in 96-well plates. After 72 h of cultivation in an incubator containing 5% O2, 5% CO2, and 90% N2 at 37 °C, parasite growth was monitored by measurement of the absorbance at 650 nm using a DTX880 Multimode Detector (Beckman Coulter), in the lactate dehydrogenase assay as previously described.18

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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.8b00643. NMR spectra and MS/MS spectra for hoshinoamides (1, 2); HPLC chromatograms for determination of the absolute configurations; phylogenetic tree (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Arihiro Iwasaki: 0000-0002-3775-5066 Teruhiko Matsubara: 0000-0002-8006-4324 Toshinori Sato: 0000-0002-4429-6101 Kiyotake Suenaga: 0000-0001-5343-5890 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by JSPS KAKENHI Grant Number 18K14346 and 16H03285. We thank Y. Umeki for technical assistance and the Japanese Red Cross Society for providing human RBCs and plasma. P. falciparum 3D7 line was obtained from MR4 (contributed by D. J. Carucci, MRA-102).

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REFERENCES

(1) Shah, S. A. A.; Akhter, N.; Auckloo, B. N.; Khan, I.; Lu, Y.; Wang, K.; Wu, B.; Guo, Y. W. Mar. Drugs 2017, 15, 354−383. (2) Engene, N.; Rottacker, E. C.; Kaštovský, J.; Byrum, T.; Choi, H.; Ellisman, M. H.; Komárek, J.; Gerwick, W. H. Int. J. Syst. Evol. Microbiol. 2012, 62, 1171−1178. (3) Engene, N.; Paul, V. J.; Byrum, T.; Gerwick, W. H.; Thor, A.; Ellisman, M. H. J. Phycol. 2013, 49, 1095−1106. (4) Engene, N.; Tronholm, A.; Salvador-Reyes, L. A.; Luesch, H.; Paul, V. J. J. Phycol. 2015, 51, 670−681. (5) Gunasekera, S. P.; Imperial, L.; Garst, C.; Ratnayake, R.; Dang, L. H.; Paul, V. J.; Luesch, H. J. Nat. Prod. 2016, 79, 1867−1871. (6) Shiota, I.; Iwasaki, A.; Sumimoto, S.; Tomoda, H.; Suenaga, K. Tetrahedron Lett. 2018, 59, 1261−1263. (7) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591−596. (8) Liang, Z.; Sorribas, A.; Sulzmaier, F. J.; Jiménez, J. I.; Wang, X.; Sauvage, T.; Yoshida, W. Y.; Wang, G.; Ramos, J. W.; Williams, P. G. J. Org. Chem. 2011, 76, 3635−3643. (9) Andrianasolo, E. H.; Goeger, D.; Gerwick, W. H. Pure Appl. Chem. 2007, 79, 593−602. (10) Edwards, D. J.; Marquez, B. L.; Nogle, L. M.; McPhail, K.; Goeger, D. E.; Roberts, M. A.; Gerwick, W. H. Chem. Biol. 2004, 11, 817−833. (11) Pruesse, E.; Peplies, J.; Glöckner, F. O. Bioinformatics 2012, 28, 1823−1829. (12) (a) Talavera, G.; Castresana, J. Syst. Biol. 2007, 56, 564−577. (b) Castresana. Mol. Biol. Evol. 2000, 17, 540−552. G



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(13) (a) Darriba, D.; Taboada, G. L.; Doallo, R.; Posada, D. Nat. Methods 2012, 9, 772. (b) Guindon, S.; Gascuel, O. Syst. Biol. 2003, 52, 696−704. (14) Guindon, S.; Gascuel, O. Syst. Biol. 2003, 52, 696−704. (15) Perrière, G.; Gouy, M. Biochimie 1996, 78, 364−369. (16) Ronquist, F.; Teslenko, M.; van der Mark, P.; Ayres, D. L.; Darling, A.; Höhna, S.; Larget, B.; Liu, L.; Suchard, M. A.; Huelsenbeck, J. P. Syst. Biol. 2012, 61, 539−542. (17) Alexandre, J. S. F.; Yahata, K.; Kawai, S.; Torii, M.; Kaneko, O. Parasitol. Int. 2011, 60, 313−320. (18) Gamo, F. J.; Sanz, L. M.; Vidal, J.; de Cozar, C.; Alvarez, E.; Lavandera, J. L.; Vanderwall, D. E.; Green, D. V.; Kumar, V.; Hasan, S.; Brown, J. R.; Peishoff, C. E.; Cardon, L. R.; Garcia-Bustos, J. F. Nature 2010, 465, 305−310.

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Hoshinoamides A and B, Acyclic Lipopeptides from the Marine

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