Mebamamides A and B, Cyclic Lipopeptides Isolated from the Green

Article pubs.acs.org/jnp

Mebamamides A and B, Cyclic Lipopeptides Isolated from the Green Alga Derbesia marina Arihiro Iwasaki,† Osamu Ohno,† Shinpei Sumimoto,† Teruhiko Matsubara,‡ Satoshi Shimada,§ Toshinori Sato,‡ and Kiyotake Suenaga*,† †

Department of Chemistry, Keio University, 3-14-1 Hiyoshi, Kohoku-ku Yokohama, Kanagawa 223-8522, Japan Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Kohoku-ku,, Yokohama, Kanagawa 223-8522, Japan § Department of Biology, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo, 112-0012, Japan ‡

S Supporting Information *

ABSTRACT: Mebamamides A and B, new lipopeptides with four D-amino acid residues and a 3,8-dihydroxy-9-methyldecanoic acid residue, were isolated from the green alga Derbesia marina. Their gross structures were elucidated by spectroscopic and ESIITMS analyses. The absolute configurations except for the two leucines were revealed based on chiral-phase HPLC analyses of the acid hydrolysate and a modified Mosher’s method. A distinction between D-Leu and L-Leu in the sequence was established by the application of a dansyl−Edman method to the partial acid hydrolysate. Mebamamide A did not exhibit any growth inhibitory activity against HeLa and HL60 cells at 10 μM, and mebamamide B did not exhibit any growth inhibitory activity against those cells at 100 μM. Additionally, it was suggested that mebamamide B induced the differentiation of HL60 cells into macrophagelike cells at 100 μM.

M

arine organisms have been considered to be good sources of novel bioactive substances.1 Marine green algae, in particular, produce several important compounds. For instance, kahalalide F isolated from the green alga Bryopsis sp. has attracted attention due to its antitumor activity.2,3 Against this background, several efforts to discover bioactive substances from marine green algae, including Derbesia marina (Lyngbya) Solier (Derbesiaceae, Bryopsidales), have been reported.4,5 In our continuing search for new bioactive substances from marine organisms,6−10 we investigated the constituents of the green alga Derbesia marina and isolated two lipopeptides, mebamamides A (1) and B (2).

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chromatography (ODS silica gel, MeOH−H2O) and repeated reversed-phase HPLC to give mebamamide A (1, 15.6 mg) and B (2, 11.0 mg). Mebamamide A (1) was obtained as a colorless oil. The NMR data for 1 are summarized in Table 1. The molecular formula of 1 was found to be C59H93N9O15 by ESIMS. The 1H and 13C NMR data suggested that 1 was a peptidic compound,

RESULTS AND DISCUSSION

The green alga Derbesia marina (700 g, wet weight) was collected at Mebama, Mie, Japan, and extracted with MeOH. The extract was filtered, concentrated, and partitioned between EtOAc and H2O. The EtOAc-soluble material was further partitioned between 90% aqueous MeOH and hexane. The material obtained from the aqueous MeOH portion was subjected to fractionation with reversed-phase column © XXXX American Chemical Society and American Society of Pharmacognosy

Received: February 19, 2015

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DOI: 10.1021/acs.jnatprod.5b00168 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 1. NMR Data for Mebamamide A (1) in CD3OD position

δCa, type

δHb (J in Hz)

Val 1 2 3 4 5

171.6, C 59.4, CH 31.8, CH 19.0, CH3 19.2, CH3

1 2 3a 3b 4 5/6

173.4, C 52.8, CH 41.0, CH2

4.22, 2.12, 0.92, 0.91,

d (7.5) m m m

4.39, 1.67, 1.51, 1.60, 0.98,

m m m m m

173.3,c C 60.9, CH 28.1, CH2

1 2 3a 3b 4 5/9 6/8 7

172.7, C 54.5, CH 38.7, CH2 137.9, 130.6, 129.7, 128.1,

1 2 3 4

170.4, C 57.6, CH 71.2, CH 18.1, CH3

1 2

175.6, C 51.0, CH

25.7, CH2 48.2, CH2

4.34, 2.05, 1.60, 1.95, 1.61, 3.66, 2.85,

dd (7.8, 3.1) m m m m m ddd (16.4, 8.9, 1.5)

1.46, d (7.4)

1 2 3a 3b 4a 4b 5a 5b

173.6, C 61.8, CH 30.3, CH2 26.1, CH2 48.7, CH2

4.47, 2.18, 2.05, 2.18, 1.96, 3.84, 3.65,

m m m m m m m

174.1,c C 51.5, CH 41.2, CH2

4.71, 1.66, 1.53, 1.66, 0.88,

m m m m m

25.7, CH 18.88, CH3

Ser 1 2 3a 3b fatty acid 1 2a 2b 3 4 5 6 7 8 9 10 11 acetyl group 1 2

4.77, dd (8.9, 7.1) 3.07, dd (13.6, 8.9) 2.99, dd (13.6, 7.1) 7.20, m 7.27, m 7.22, m

Thr 4.56, d (1.6) 5.62, dq (6.6, 1.6) 1.23, d (6.6)

Ala

a

17.8, CH3

1 2 3a 3b 4 5/6

Phe

C CH CH CH

3

Leu2

Pro1 1 2 3a 3b 4a 4b 5a 5b

δHb (J in Hz)

Pro2

Leu1

26.5, CH3 22.9, CH3

δCa, type

position

172.4, C 56.8, CH 63.1, CH2

174.7, C 44.9, CH2

4.50, m 3.81, m 3.80, m

70.2, CH 38.4, CH2 26.6,d CH2 26.5,d CH2 32.3, CH2 79.8, CH 32.7, CH 21.8, CH3 23.7, CH3

2.37, 2.36, 3.92, 1.50, 1.48, 1.34, 1.54, 4.73, 1.81, 0.90, 0.89,

m m m m m m m m m m m

173.0, C 21.0, CH3

2.04, s

4.44, q (7.4)

Measured at 100 MHz. bMeasured at 400 MHz. cThese carbon signals are interchangeable. dThese carbon signals are interchangeable.

Figure 1. Partial structures based on 2D NMR analyses of 1.

with 10 deshielded methine protons (δH 4−5) and 11 carbonyl carbons (δC 170−180). Further analyses of COSY, HMBC, HMQC, and NOESY data revealed that 1 was composed of a fatty acid residue, 8-acetoxy-3-hydroxy-9-methyldecanoic acid, and nine amino acid residues, including serine, two leucines,

two prolines, alanine, threonine, phenylalanine, and valine. On the basis of two NOE correlations, H-5 of Pro1/H-2 of Phe and H-5 of Pro2/H-2 of Leu2, and three HMBC correlations, H-2 of Phe/C-1 of Thr, H-2 of Thr/C-1 of Ala and H-3 of Thr/C-1 of Val, the following partial sequences were clarified: Leu2-Pro2 B



Article

Scheme 1. Preparation of the Acyclic Derivative 3

Figure 2. Fragmentation pattern of 3 in ESI-ITMS.

of 1 was subjected to the dansyl−Edman method.12 Treatment of 1 with 9 M HCl in 1,4-dioxane at room temperature (rt) for 14 h afforded partial acid hydrolysis product 5. Edman degradation of 5 gave peptide 6, and dansylation of 6 afforded dansylated peptide 7. Complete acid hydrolysis of 7 afforded the dansylated leucine and the leucine, respectively (Scheme 3). Chiral-phase HPLC analyses of these compounds revealed that the leucine in the side chain of 2 was the L-form and the other was the D-form, respectively. With regard to the fatty acid moiety, the absolute configurations of the stereogenic centers at C-3 and C-8 of 2 were determined to be 3R and 8S on the basis of the modified Mosher’s method (see Supporting Information, p S19).13 Because mebamamide B (2) has the same configuration as 1 as demonstrated above, the absolute configurations of mebamamides were clarified as shown in 1 and 2. To evaluate the growth inhibitory activities of mebamamides (1 and 2) against cancer cells, MTT assays were performed with HeLa and HL60 cells. The cells were treated in 96-well plates with various concentrations of the compounds (0.1−100 μM) for 72 h. The data from these assays revealed that 1 is inactive against those cells at 10 μM (IC50 against HeLa cells, 48 μM; IC50 against HL60 cells, 19 μM), whereas 2 did not exhibit any growth inhibitory activity against those cells at 100 μM. In addition, 2 did not show any cytotoxicity toward HL60 cells on the basis of a trypan blue dye exclusion assay (see Supporting Information, p S31). However, 2 significantly induced the adhesion of HL60 cells (Figure 3A). On the basis of the observation of cells stained with May−Giemsa solution, the morphological features of the adherent cells treated with 2 were similar to those of cells treated with 12-O-tetradecanoyl-

and Ala-Thr(-Val)-Phe-Pro1 (Figure 1, Supporting Information, Table S1). Further sequence determination based on 2D NMR was not possible because of severe overlap of the chemical shifts of the carbonyl carbons. To establish the gross structure of 1, the acyclic derivative 3 was prepared by the elimination reaction of 1 under basic conditions (Scheme 1). A fragmentation study of 3 by negative electrospray ionization ion trap mass spectrometry (ESI-ITMS) revealed that the complete sequence of 1 was fatty acid-Ser-Leu2-Pro2Ala-Thr-Phe-Pro1-Leu1-Val (Figure 2). Mebamamide B (2) was obtained as a colorless oil. The NMR data for 2 are summarized in Table 2. The molecular formula of 2 was found to be C57H91N9O14 by ESIMS, which represents 42 mass units less than that of 1. Additionally, the 1H and 13C NMR features of 2 were analogous to those of 1 except for the disappearance of an acetyl group. Acetylation of 1 and 2 gave the triacetate 4 (Scheme 2), respectively, and the 1H NMR spectra and the specific rotations of both triacetates matched each other. Therefore, the structure of 2 including its absolute configuration was determined to be a deacetylated analogue of 1. The absolute configurations of the stereogenic centers except for the two leucine residues and the fatty acid moiety were determined by Marfey’s method11 and chiral-phase HPLC analyses of the acid hydrolysates of 1. These analyses established the configurations of valine, two prolines, phenylalanine, threonine, alanine, and serine to be L, L, D, L, D, and D, respectively. The analyses described above clarified that the two leucine residues in 1 were composed of L and D forms. To distinguish them from one another, the partial acid hydrolysate C



Article

Table 2. NMR Data for Mebamamide B (2) in CD3OD position

δCa, type

δHb (J in Hz)

Val 1 2 3 4 5

171.6, C 59.4, CH 31.8, CH 18.9, CH3 19.2, CH3

1 2 3a 3b 4 5/6

173.4, C 52.8, CH 41.2,c CH2

1 2 3a 3b 4a 4b 5a 5b

173.3,d C 60.9, CH 28.1, CH2

4.22, 2.12, 0.94, 0.90,

d (7.4) m m m

4.39, 1.67, 1.51, 1.59, 0.94,

m m m m m

25.72,e CH2 48.2, CH2

dd (7.9, 3.1) m m m m m ddd (16.2, 7.6, 1.8)

172.7, C 54.5, CH 38.8, CH2 137.9, 130.6, 129.6, 128.1,

1 2 3 4

170.4, C 57.6, CH 71.2, CH 18.2, CH3

1

175.6, C

C CH CH CH

51.0, CH 17.8, CH3

4.44, q (7.2) 1.46, d (7.2)

1 2 3a 3b 4a 4b 5a 5b

173.6, C 61.8, CH 30.3, CH2

1 2 3a 3b 4 5/6

174.1,d C 51.5, CH 41.0,e CH2

26.1, CH2 48.7, CH2

4.46, 2.18, 2.05, 2.19, 1.97, 3.85, 3.65,

m m m m m m m

4.71, 1.65, 1.51, 1.66, 0.89,

m m m m m

25.69,e CH 19.4, CH3

Ser 1 2 3a 3b fatty acid 1 2a 2b 3 4 5 6 7a 7b 8 9 10 11

Phe 1 2 3a 3b 4 5/9 6/8 7

2 3

Leu2

Pro1 4.34, 2.07, 1.59, 1.95, 1.61, 3.66, 2.84,

δHb (J in Hz)

Pro2

Leu1

26.1, CH 22.9, CH3

δCa, type

position

4.77, dd (8.4, 7.1) 3.06, dd (13.3, 8.4) 2.99, dd (13.3, 7.1) 7.21, m 7.27, m 7.22, m

Thr 4.55, d (1.5) 5.62, dq (6.8, 1.5) 1.23, d (6.8)

Ala

172.4, C 56.8, CH 63.1, CH2

174.7, C 44.9, CH2 70.3, CH 38.6, CH2 26.7,f CH2 27.2,f CH2 35.0, CH2 77.3, 34.8, 21.8, 23.6,

CH CH CH3 CH3

4.50, m 3.81, m 3.80, m

2.38, 2.37, 3.93, 1.52, 1.54, 1.35, 1.51, 1.35, 3.28, 1.64, 0.90, 0.90,

m m m m m m m m m m m m

a

Measured at 100 MHz. bMeasured at 400 MHz. cThese carbon signals are interchangeable. dThese carbon signals are interchangeable. eThese carbon signals are interchangeable. fThese carbon signals are interchangeable.

Scheme 2. Acetylation of 1 and 2

macrophage-like cells. Meanwhile, 1 did not induce the adhesion of HL60 cells (see Supporting Information, p S31), and it was indicated that 1 did not exhibit differentiationinducing activity. Mebamamides A (1) and B (2) were isolated from the green alga Derbesia marina. The planar structures of 1 and 2 were

phorbol 13-acetate (TPA), which induces the differentiation of HL60 cells into macrophages14 (Figure 3B). Additionally, 2induced adherent cells exhibited phagocytotic activity, similar to TPA-treated cells, on the basis of an assay with FITCconjugated latex beads (Figure 3C). These results suggested that 2 induced the differentiation of HL60 cells into D



Article

Scheme 3. Distinction between Two Leucine Residues by the Dansyl−Edman Method

any growth inhibitory activity against HeLa and HL60 cells at 10 μM, while 2 did not show any cytotoxicity at up to 100 μM. Additionally, it was suggested that 2 induced the differentiation of HL60 cells into macrophage-like cells at 100 μM but that 1 did not at all. These results indicated that the presence of a hydroxy group at C-8 of the fatty acid moiety of 2 might be essential for its differentiation-inducing activities. A detailed investigation of the structure−activity relationship of 1 and 2 is in progress.

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EXPERIMENTAL SECTION

General Experimental Procedures. Chemicals and solvents were the best grade available and were used as received from commercial sources. Optical rotations were measured with a JASCO DIP-1000 polarimeter. IR spectra were recorded on a JASCO RT/IR-4200 instrument. All NMR spectral data were recorded on a JEOL JNMECX400 spectrometer for 1H (400 MHz) and 13C (100 MHz). 1H NMR chemical shifts (referenced to residual CHD2OD observed at δ 3.31) were assigned using a combination of data from COSY and HMQC experiments. Similarly, 13C NMR chemical shifts (referenced to CD3OD observed at δ 49.0) were assigned based on HMBC and HMQC experiments. ESI mass spectra were obtained on an LCT premier EX spectrometer (Waters). ESI-MSn spectra were obtained on an Esquire 3000 plus ion trap mass spectrometer (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). Algal Material. The green alga was morphologically classified into Derbesia marina (Lyngbya) Solier (Derbesiaceae, Bryopsidales). A voucher specimen of the alga, named 0707-07, has been deposited at Keio University. Collection, Extraction, and Isolation. The green alga was collected at Mebama, Mie Prefecture, Japan, at a depth of 0−1 m in July 2007. The collected alga (700 g) was extracted with MeOH (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 (0.2 L) and hexane (3 × 0.2 L). The aqueous MeOH fraction (340 mg) was first separated by column chromatography on ODS (5 g) eluted with 40% MeOH, 60% MeOH, 80% MeOH, and MeOH. The fraction (107 mg) eluted with 80%

Figure 3. Induction of the differentiation of HL60 cells into macrophage-like cells by 2. (A) HL60 cells were treated with 100 μM of 2. After incubation for the indicated time, the numbers of adherent/suspended cells were counted with a hemocytometer. Values are the means ± SD of quadruplicate determinations. (B) HL60 cells were incubated with 100 μM of 2 or 16 nM of 12-Otetradecanoylphorbol 13-acetate (TPA) for 72 h. Cells were then stained by the May−Giemsa staining method and observed with a microscope. (C) HL60 cells were incubated with FITC-conjugated latex beads and 100 μM of 2 or 16 nM of TPA for 48 h. Cells were then observed with a fluorescent microscope.

established by spectroscopic and MS/MS analyses of the acyclic derivative of 1. The absolute configurations except for the two leucines were revealed based on chiral-phase HPLC analyses of the acid hydrolysate and a modified Mosher’s method. The distinction between D-Leu and L-Leu in the sequence was established by application of the dansyl−Edman method to the partial acid hydrolysate. Structurally, mebamamides were revealed to contain a 3,8-dihydroxy-9-methyldecanoic acid moiety, and a high degree of D-amino acids. So far, several structurally related fatty acid residues have been reported in lipopeptides. For instance, kahalalides E, H, J, K, and Y contain a similar fatty acid moiety, 3-hydroxy-9-methyldecanoic acid.15−18 With respect to biological activities, 1 did not exhibit E



Article

MeOH was subjected to HPLC [Cosmosil 5C18-AR-II (ϕ20 mm × 250 mm); flow rate 5 mL/min; detection, UV 215 nm; solvent 79% MeOH, 0.1% TFA] to give mebamamide A (1) (15.6 mg, tR = 50.3 min) and a fraction that contained mebamamide B (2) (21.7 mg, tR = 33.9 min). The fraction containing 2 was further separated by HPLC [Cosmosil 5C18-AR-II (ϕ20 mm × 250 mm); flow rate 5 mL/min; detection, UV 215 nm; solvent 50% MeCN] to give 2 (11.0 mg, tR = 34.3 min). Mebamamide A (1). Colorless oil; [α]27D −39 (c 0.2, CH3OH); IR (film) 3308, 2960, 1733, 1653, 1539, 1456 cm−1; 1H NMR, 13C NMR, COSY, HMBC, and NOESY data, Tables 1 and Supporting Information, p S1; HRESIMS m/z 1190.6705 [M + Na]+ (calcd for C59H93N9O15Na, 1190.6689). Mebamamide B (2). Colorless oil; [α]27D −54 (c 0.2, CH3OH); IR (film) 3300, 2959, 1744, 1654, 1558, 1452 cm−1; 1H NMR, 13C NMR data, Table 2; HRESIMS m/z 1126.6766 [M + H]+ (calcd for C57H92N9O14, 1126.6764). Preparation of 3. A 0.7 M solution of NaOMe in MeOH (0.2 mL) was added to mebamamide A (1, 0.8 mg, 690 nmol), and the solution was stirred at rt for 30 min. The mixture was neutralized with an acidic ion-exchange resin (Amberlite IRC-76) and poured on a pad of the same resin. The resin was washed with MeOH. The filtrate were evaporated to dryness and purified by HPLC to afford acyclic derivative 3 (0.7 mg, 600 nmol, 87%) as a colorless oil [conditions for HPLC separation: column, Cosmosil Cholester (ϕ20 mm × 250 mm); flow rate, 5.0 mL/min; detection at 215 nm; solvent 55% MeCN, 0.1% TFA; retention time of 3, 25.5 min]; 1H NMR (400 MHz, CD3OD), δ 7.33−7.24 (m, 5H), 6.86 (q, 1H, J = 7.3 Hz), 4.72−4.76 (m, 2H), 4.58−4.46 (m, 3H), 4.41−4.34 (m, 3H), 4.28 (d, 1H, J = 5.7 Hz), 3.92−3.89 (m, 2H), 3.81 (dd, 1H, J = 11.5, 5.7 Hz), 3.77 (dd, 1H, J = 11.5, 4.9 Hz), 3.71−3.63 (m, 2H), 3.14−3.11 (m, 2H), 2.56 (m, 1H), 2.38−2.35 (m, 2H), 2.23−2.13 (m, 3H), 2.04 (s, 3H), 2.04−1.97 (m, 4H), 1.81 (m, 1H), 1.75−1.71 (m, 2H), 1.72 (d, 3H, J = 7.3 Hz), 1.67 (m, 1H) 1.60−1.41 (m, 11H), 1.44 (d, 3H, J = 7.0 Hz), 1.36−1.25 (m, 2H), 0.96−0.87 (m, 24H); HRESIMS m/z 1190.6736 [M + Na]+ (calcd for C59H93N9O15Na, 1190.6689). Preparation of 4 from 1. To a stirred solution of 1 (0.7 mg, 600 nmol) in pyridine (100 μL) were added 100 μL of acetic anhydride. The mixture was stirred at rt for 1 h and evaporated to dryness. The crude product was purified by HPLC to afford the triacetate (4, 0.7 mg, 560 nmol, 93%) as a colorless oil [conditions for HPLC separation: column, Cosmosil Cholester (ϕ20 mm × 250 mm); flow rate, 5.0 mL/min; detection at 215 nm; solvent 75% MeCN; retention time of 4, 23.3 min]; [α]31D −25 (c 0.04, MeOH); 1H NMR (400 MHz, CD3OD), δ 7.30−7.19 (m, 5H), 5.60 (dq, 1H, J = 6.3, 1.8 Hz), 5.2 (m, 1H), 4.83−4.69 (m, 5H), 4.54 (d, 1H, J = 1.5 Hz), 4.50−4.21 (m, 6H), 3.83 (m, 1H), 3.67 (m, 2H), 3.02 (dd, 1H, J = 13.3, 4.7 Hz), 2.98 (dd, 1H, J = 13.3, 9.7 Hz), 2.83 (m, 1H), 2.51 (m, 2H), 2.20− 1.84 (m, 9H), 2.12 (s, 1H), 2.05 (s, 2H), 2.04 (s, 2H), 2.02 (s, 2H), 2.00 (s, 2H), 1.80 (m, 1H), 1.69−1.44 (m, 12H), 1.45 (d, 3H, J = 7.2 Hz), 1.42−1.21 (m, 2H), 1.24 (d, 3H, J = 6.8 Hz), 0.98−0.79 (m, 24H); HRESIMS m/z 1274.6946 [M + Na] + (calcd for C63H98N9O17Na, 1274.6900). Preparation of 4 from 2. To a stirred solution of 2 (0.7 mg, 610 nmol) in pyridine (100 μL) were added 100 μL of acetic anhydride. The mixture was stirred at rt for 1 h and evaporated to dryness. The crude product was purified by HPLC to afford the triacetate (4, 0.3 mg, 240 nmol, 39%) as a colorless oil [conditions for HPLC separation: column, Cosmosil Cholester (ϕ20 mm × 250 mm); flow rate, 5.0 mL/min; detection at 215 nm; solvent 75% MeCN; retention time of 4, 23.3 min]; [α]31D −14 (c 0.02, MeOH); 1H NMR (400 MHz, CD3OD), δ 7.30−7.19 (m, 5H), 5.60 (dq, 1H, J = 6.3, 1.8 Hz), 5.2 (m, 1H), 4.83−4.69 (m, 5H), 4.54 (d, 1H, J = 1.5 Hz), 4.50−4.21 (m, 6H), 3.83 (m, 1H), 3.67 (m, 2H), 3.02 (dd, 1H, J = 13.3, 4.7 Hz), 2.98 (dd, 1H, J = 13.3, 9.7 Hz), 2.83 (m, 1H), 2.51 (m, 2H), 2.20− 1.84 (m, 9H), 2.12 (s, 1H), 2.05 (s, 2H), 2.04 (s, 2H), 2.02 (s, 2H), 2.00 (s, 2H), 1.80 (m, 1H), 1.69−1.44 (m, 12H), 1.45 (d, 3H, J = 7.2 Hz), 1.42−1.21 (m, 2H), 1.24 (d, 3H, J = 6.8 Hz), 0.98−0.79 (m, 24H); HRESIMS m/z 1252.7087 [M + H]+ (calcd for C63H98N9O17, 1252.7081).

Acid Hydrolysis, Chiral-Phase HPLC Analysis, and Marfey’s Analysis of 1. Mebamamide A (1) (0.3 mg) was treated with 9 M HCl (100 μL) for 24 h at 110 °C. The hydrolyzed product was evaporated to dryness and could be separated into each component, except for a mixture of Ala, Ser, and Thr [conditions for HPLC separation: column, Cosmosil 5C18−PAQ (ϕ4.6 mm × 250 mm); flow rate, 1.0 mL/min; detection at 215 nm; solvent H2O; retention times (min) of components, mixture of Ala, Ser, and Thr (tR = 2.9 min), Pro (tR = 3.2 min), Val (tR = 3.4 min), Leu (tR = 4.8 min), Phe (tR = 10.6 min)]. Each fraction was dissolved in H2O (50 μL) and analyzed by chiralphase HPLC, and the retention times were compared to those of authentic standards [Daicel Chiralpak (MA+) (ϕ4.6 mm × 50 mm); flow rate, 1.0 mL/min; detection at 254 nm; solvent 2.0 mM CuSO4 and 2.0 mM CuSO4−MeCN (90:10)]. With 2.0 mM CuSO4, the retention times (tR min) for authentic standards were D-Pro (2.8), LPro (5.0), D-Val (3.5), L-Val (6.2), D-Leu (8.2), and L-Leu (15.1). With 2.0 mM CuSO4−MeCN (90:10), the retention times for authentic standards were D-Phe (5.6) and L-Phe (7.1). The retention times (min) (and the respective HPLC conditions) of the amino acids in the hydrolysate were 5.0, 6.2, 8.2, 15.1 (100:0), and 5.6 (90:10), indicating the presence of L-Pro, L-Val, D-Leu, L-Leu, and D-Phe in the hydrolysate. The Ala-, Ser-, and Thr-containing fraction was dissolved in H2O (100 μL). A 1.0% 1-fluoro-2,4-dinitro-phenyl-5-L-alaninamide (Marfey’s reagent) solution in acetone (200 μL) and 50 μL of 1 M NaHCO3 were added, and the mixture was heated at 80 °C for 3 min. The solution was cooled to rt, neutralized with 1 M HCl, and evaporated to dryness. The residue was resuspended in 50 μL of MeCN−H2O (1:1), and the solution was analyzed by reversed-phase HPLC [Cosmosil 5C18-AR-II (ϕ4.6 mm × 250 mm); flow rate 1.0 mL/min; detection, UV 340 nm; solvent 0.02 M NaOAc−MeOH (45:55) and 0.02 M NaOAc−MeOH (50:50)]. With 0.02 M NaOAc− MeOH (45:55), the retention times (tR min) for authentic standards were D-Ala (24.6), D-Thr (18.9), and D-allo-Thr (10.9). With 0.02 M NaOAc−MeOH (50:50), the retention times (tR min) for authentic standards were L-Ala (13.8), L-Thr (9.3), L-allo-Thr (10.0), D-Ser (15.1), and L-Ser (10.8). The retention times (min) (and the respective HPLC conditions) of the amino acids in the hydrolysate were 24.6 (45:55), 9.3, and 15.1 (50:50), indicating the presence of DAla, L-Thr, and D-Ser in the hydrolysate (for more details, see Supporting Information, S25−S28). Preparation of 5. Mebamamide A (1) (1.0 mg, 860 nmol) was treated with 9 M HCl (100 μL) and 1,4-dioxane (100 μL) for 14 h at rt. The hydrolyzed product was evaporated to dryness and purified by HPLC to afford partial acid hydrolysate 5 (0.5 mg, 540 nmol, 63%) as a colorless oil [conditions for HPLC separation: column, Cosmosil Cholester (ϕ20 mm × 250 mm); flow rate, 5.0 mL/min; detection at 215 nm; solvent 45% MeCN, 0.1% TFA; retention time of 5, 20.9 min]; 1H NMR (400 MHz, CD3OD), δ 7.31−7.18 (m, 5H), 5.62 (dq, 1H, J = 6.7, 1.4 Hz), 4.73−4.36 (m, 5H), 4.33 (dd, 1H, J = 7.0, 2.2 Hz), 4.18 (t, 1H, J = 8.0 Hz), 4.10 (d, 1H, J = 7.3 Hz), 4.07−3.78 (m, 4H), 3.70−3.63 (m, 2H), 3.08−2.99 (m, 2H), 2.84 (m, 1H), 2.20− 1.85 (m, 7H), 1.66−1.45 (m, 11H), 1.23 (d, 3H, J = 7.3 Hz), 0.98− 0.75 (m, 18H); HRESIMS m/z 948.5168 [M + Na]+ (calcd for C46H71N9O11Na, 948.5171). Edman Degradation of 5. Partial acid hydrolysate (5) (0.25 mg) was dissolved in 200 μL of EtOH/triethylamine/H2O/phenyl isothiocyanate (7:1:1:1) and allowed to stand for 30 min at 50 °C. The reaction mixture was evaporated to dryness. The obtained phenylthiocarbamylpeptide was treated with neat TFA (200 μL) at 50 °C for 15 min. The TFA was removed in vacuo, and the residue was dissolved in 2 mL of H2O. The H2O solution was washed with hexane (1 mL, 4 times) and then extracted with EtOAc (1 mL, 4 times). The EtOAc layer was evaporated to dryness and purified by HPLC to afford the degradated peptide (6) [conditions for HPLC separation: column, Cosmosil 5C18 AR-II (ϕ20 mm × 250 mm); flow rate, 5.0 mL/min; detection at 215 nm; solvent 45% MeCN, 0.1% TFA; retention time of 6, 27.6 min]; 1H NMR (400 MHz, CD3OD), δ 7.31−7.18 (m, 5H), 5.63 (m, 1H), 4.73−4.36 (m), 4.26 (m, 1H), 4.18 F



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(dq, 1H, J = 6.8, 1.6 Hz), 5.42 (1H, m), 4.92−4.53 (m, 6H), 4.49− 4.35 (m, 3H), 4.32 (dd, 1H, J = 8.1, 3.2 Hz), 4.21 (m, 1H), 3.82 (m, 1H), 3.67 (m, 2H), 3.53 (s, 3H), 3.52 (s, 3H), 3.49 (s, 3H), 3.01 (m, 2H), 2.80 (m, 1H), 2.65 (dd, 1H, J = 15.8, 8.1 Hz), 2.45 (dd, 1H, J = 15.8, 5.6 Hz), 2.17−2.03 (m, 6H), 1.98−1.82 (m, 3H), 1.69−1.43 (m, 14H), 1.45 (d, 3H, J = 7.3 Hz), 1.22−1.10 (m, 2H), 1.23 (d, 3H, J = 6.4 Hz), 0.98−0.79 (m, 24H); HRESIMS m/z 1772.7969 [M − H]− (calcd for C87H111F9N9O20, 1772.7802). 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. HL60 cells were cultured at 37 °C with 5% CO2 in RPMI (Nissui) supplemented with 10% heat-inactivated FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin, 300 μg/mL Lglutamine, and 2.25 mg/mL NaHCO3. HeLa cells were seeded at 2 × 104 cells/well in 96-well plates (Iwaki) and cultured overnight. HL60 cells were seeded at 1 × 105 cells/well in 96-well plates. Various concentrations of compounds were then added, and cells were incubated for 72 h. Cell proliferation was measured by the MTT assay. Trypan Blue Dye Exclusion Assay. HL60 cells were seeded at 1 × 105 cells/well in 24-well plates (Iwaki, Japan) and then treated with 100 μM of 2 for 72 h. They were then stained with 0.8 mg/mL trypan blue (Sigma-Aldrich, St. Louis, MO), and the cell viability was determined by counting the number of stained (killed) cells. Determination of the Ratio between Suspended Cells and Adherent Cells. HL60 cells were seeded at 1 × 105 cells/well in 96well plates, each well containing 200 μL of medium. Then, 100 μM of 2 or 16 nM of 12-O-tetradecanoylphorbol 13-acetate (TPA) were added. After 1, 3, 6, 12, 24, 48, or 72 h, suspended cells were collected from supernatant, and adherent cells were suspended by using trypsin treatment and then collected. A number of cells were counted with the use of a hemocytometer. Morphological Observation of Cells. HL60 cells were seeded at 2 × 104 cells/well in 96-well plates, each well containing 200 μL of medium. Then 100 μM of 2 or 16 nM of TPA were added, and cells were incubated for 72 h. The cells were washed once in 200 μL of PBS and stained with May−Giemsa solution. Phagocytosis Assay. Phagocytosis was examined by fluorescent microscopy using Phagocytosis Assay Kit (Cayman Chemical) according to the manufacture’s protocol. HL60 cells were seeded at 2 × 104 cells/well in 96-well plates, each well containing 200 μL of medium. Then 20 μL of FITC-conjugated latex beads solution were added to each well. Then 100 μM of 2 or 16 nM of TPA were added, and cells were incubated for 48 h. After discarding the supernatant by careful aspiration, surface-bound fluorescence were quenched with 50 μL of trypan blue solution. Each well was analyzed by using a fluorescence microscope.

(t, 1H, J = 8.0 Hz), 4.16 (m, 1H), 3.73−3.55 (m), 3.05 (m, 1H), 2.97 (m, 1H), 2.82 (m), 2.25−2.01 (m), 1.75−1.45 (m), 1.23−1.20 (m), 1.06−0.89 (m, 18H); HRESIMS m/z 839.4988 [M + H]+ (calcd for C43H67N8O9, 839.5031). Dansylation and Total Acid Hydrolysis of the Degraded Peptide. To the degraded peptide (6) were added 0.2 mL of 10 mM dansyl chloride−acetone solution and 0.2 mL of 0.1 M NaHCO3− Na2CO3 buffer (pH 8.9). After stirring for 6 min at 70 °C, the mixture was evaporated to dryness and purified by HPLC to afford the dansylated peptide (7) [conditions for HPLC separation: column, Cosmosil 5C18 AR-II (ϕ20 mm × 250 mm); flow rate, 5.0 mL/min; detection at 215 nm; solvent 57.5% MeCN, 0.1% TFA; retention time of 7, 20.6 min]; 1H NMR (400 MHz, CD3OD), δ 8.58 (m, 1H), 8.41 (m, 1H), 8.25 (m, 1H), 7.62−7.56 (m, 2H), 7.32−7.24 (m, 5H), 5.15 (m), 4.39−4.24 (m), 4.11−4.06 (m), 2.87 (s, 6H), 1.96−1.76 (m), 1.70−1.51 (m), 1.42−1.25 (m), 0.98−0.81 (m); HRESIMS m/z 1094.5367 [M + Na]+ (calcd for C55H77N9O11SNa, 1094.5361). The dansylated peptide (7) was treated with 6 M HCl (100 μL) for 20 h at 110 °C. The hydrolyzed product was evaporated to dryness and separated into Leu and dansylated Leu, respectively [conditions for HPLC separation of Leu: column, Cosmosil 5C18−PAQ (ϕ4.6 mm × 250 mm); flow rate, 1.0 mL/min; detection at 215 nm; solvent H2O; retention time of Leu, 4.8 min]; [conditions for HPLC separation of dansylated Leu: column, Cosmosil 5C18-MS-II (ϕ4.6 mm × 250 mm); flow rate, 1.0 mL/min; detection at 215 nm; solvent 50% MeCN, 0.1% TFA; retention time of dansylated Leu, 4.0 min]. Chiral-Phase HPLC Analysis of Leucine Derived from 7. The leucine derived from 7 was dissolved in H2O (50 μL) and analyzed by chiral-phase HPLC, and the retention time was compared to those of authentic standards [Daicel Chiralpak (MA+) (ϕ4.6 mm × 50 mm); flow rate, 1.0 mL/min; detection at 254 nm; solvent 2.0 mM CuSO4]. The retention times (tR min) for authentic standards were D-Leu (8.2) and L-Leu (14.9). The retention time of the Leu derived from 7 was 8.2 min, indicating the presence of D-Leu in the cyclic peptide portion of 7. Chiral-Phase HPLC Analysis of Dansylated Leucine derived from 7. The dansylated leucine derived from 7 was dissolved in MeOH (50 μL) and analyzed by chiral-phase HPLC, and the retention time was compared to those of authentic standards [Daicel Chiralpak (IA) (ϕ4.6 mm × 250 mm); flow rate, 1.0 mL/min; detection at 254 nm; solvent 85% hexane, 15% EtOH, 0.1% TFA]. The retention times (tR min) for authentic standards were D-dansyl-Leu (7.3) and L-dansylLeu (7.5). The retention time of the dansyl-Leu derived from 7 was 7.5 min, indicating the presence of L-dansyl-Leu in 7. Synthesis of the (S)-MTPA Ester of 2 (8). To a stirred solution of 2 (0.9 mg, 800 nmol) in pyridine (100 μL) were added 3 drops of (R)-MTPACl and 4-(dimethylamino)pyridine (1.3 mg). The mixture was stirred at rt for 2 h and concentrated to give an oil, which was purified by HPLC [condition for HPLC separation: column, Cosmosil 5C18-MS-II (ϕ20 mm × 250 mm); flow rate, 5.0 mL/min; detection at 215 nm; solvent 95% MeOH; retention time, 28.0 min] to afford (S)MTPA ester 8 (1.5 mg, quant) as a white powder; 1H NMR (400 MHz, CD3OD), δ 7.56−7.37 (m, 15H), 7.29−7.18 (m, 5H), 5.61 (dq, 1H, J = 7.0, 1.9 Hz), 5.36 (1H, m), 4.92−4.53 (m, 6H), 4.49−4.35 (m, 3H), 4.32 (dd, 1H, J = 7.0, 2.4 Hz), 4.21 (m, 1H), 3.81 (m, 1H), 3.67 (m, 2H), 3.55 (s, 3H), 3.49 (s, 3H), 3.47 (s, 3H), 3.01 (m, 2H), 2.80 (m, 1H), 2.60 (dd, 1H, J = 15.5, 7.0 Hz), 2.40 (dd, 1H, J = 15.5, 6.5 Hz), 2.17−2.03 (m, 6H), 1.98−1.89 (m, 3H), 1.69−1.43 (m, 14H), 1.45 (d, 3H, J = 7.3 Hz), 1.30−1.17 (m, 2H), 1.24 (d, 3H, J = 7.3 Hz), 0.99−0.82 (m, 24H); HRESIMS m/z 1772.7933 [M − H]− (calcd for C87H111F9N9O20, 1772.7802). Synthesis of the (R)-MTPA Ester of 2 (9). To a stirred solution of 2 (0.8 mg, 710 nmol) in pyridine (100 μL) were added 3 drops of (S)-MTPACl and 4-(dimethylamino)pyridine (1.3 mg). The mixture was stirred at rt for 2 h and concentrated to give an oil, which was purified by HPLC [condition for HPLC separation: column, Cosmosil 5C18-MS-II (ϕ20 mm × 250 mm); flow rate, 5.0 mL/min; detection at 215 nm; solvent 95% MeOH; retention time, 28.0 min] to afford (R)MTPA ester 9 (1.0 mg, 560 nmol, 79%) as a white powder; 1H NMR (400 MHz, CD3OD), δ 7.55−7.37 (m, 15H), 7.29−7.18 (m, 5H), 5.60

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ASSOCIATED CONTENT

S Supporting Information *

NMR data for 1−9, HPLC charts for chiral-phase HPLC analyses, ESI-MSn spectra of 3. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone/Fax: +81-45-566-1819. E-mail: [email protected]. jp. 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 (24310160 and 20436992), the Shimadzu Science Foundation, the Novartis Foundation (Japan) for the G



Article

Promotion of Science, and the Keio Gijuku Fukuzawa Memorial Fund for the Advancement of Education and Research.

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REFERENCES

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Mebamamides A and B, Cyclic Lipopeptides Isolated from the Green

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