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Isolation and structure determination of lysiformine from bacteria associated with marine sponge Halichondria okadai
Tetrahedron 74 (2018) 3742e3747
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Isolation and structure determination of lysiformine from bacteria associated with marine sponge Halichondria okadai Sou Kikuchi a, 1, Kayo Okada a, 1, Yuko Cho a, Shinichiro Yoshida b, Eunsang Kwon b, Mari Yotsu-Yamashita a, Keiichi Konoki a, * a b
Graduate School of Agricultural Science, Tohoku University, Aoba, Sendai 980-8572, Japan Research and Analytical Center for Giant Molecules, Graduate School of Science, Tohoku University, Aoba, Sendai 980-8578, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 March 2018 Received in revised form 16 May 2018 Accepted 18 May 2018 Available online 18 May 2018
Marine sponge Halichondria okadai is a source of numerous toxic secondary metabolites, which are considered as products of the symbiotic species associated with the sponge. In this study, we tested 720 culture conditions and purified 48 sponge-associated microbes. The ethyl acetate extract from the liquid culture of each species was tested for cytotoxicity, hemolytic activity, and brine shrimp lethal activity. Lysiformine (1) was isolated from Lysinibacillus fusiformis and its structure was determined by HRMS and NMR. © 2018 Published by Elsevier Ltd.
Keywords: Halichondria okadai Lysinibacillus fusiformis Isolation Structure determination
1. Introduction Marine sponges are filter-feeders and have various symbiotic species in their bodies.1 A recent study suggests that the symbiotic microbes occupy 40% of the whole volume of marine sponges.2 Therefore, most of the secondary metabolites isolated from marine sponges with significant biological activity are presumably produced by their symbiotic species. For example, the sponge Halichondria okadai is a rich source of toxic secondary metabolites such as okadaic acid (OA), which inhibits serine threonine protein phosphatases 1 and 2A and exhibits tumor-promoting activity,3e5 halichondrin B, which is an antitumor compound,6 and halichlorine, a potent VCAM-1 inhibitor.7 Therefore, it is useful to obtain a microbe producing each toxic secondary metabolite in view of the therapeutic applications. This concept of isolating and maintaining a culture of the symbiotic microbes is universally understood but extremely difficult to implement.8 Recent developments in DNA-sequencing technology have enabled the sequence analysis of metagenomes derived from marine sponges and isolation of the biosynthetic genes for polytheonamide B,9
* Corresponding author. E-mail address: [email protected] (K. Konoki). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.tet.2018.05.049 0040-4020/© 2018 Published by Elsevier Ltd.
calyculin B,10 and others.11 The metagenomic analysis of H. okadai was also carried out, and a new compound, halichrome A, is reported.12,13 However, genome mining is relatively inadequate for identifying the biosynthetic gene of the target compound. Consequently, we adopted the classical approach to identify the spongeassociated microbes of H. okadai in our search for novel toxic secondary metabolites. Herein, we identified 48 sponge-associated species from H. okadai by testing 720 kinds of culture conditions, which were the combination of 30 culture media, two oxygen concentrations, two temperatures, and six culture methods. From a culture of Lysinibacillus fusiformis, we isolated lysiformine (1) based on 3 independent biological assays and completed structure determination by HRMS and NMR analyses.1 2. Results and discussion 2.1. Isolation of sponge-associated microbes In total, 73 characteristic colonies appeared; however, 16S rDNA or 18S rDNA analyses showed that some of the colonies grew from the same species (Table S2). Finally, 45 species were identified in addition to three unidentified fungi. There should be an innumerable species present in the sponge, which reveals the difficulty in growing sponge-associated microorganisms.8 It should be noted
S. Kikuchi et al. / Tetrahedron 74 (2018) 3742e3747
that none of the species could be identified by metagenome screening,12,13 which is typically able to identify the abundant species present in the target specimen. Thus, the isolated microorganisms in the sponge were present in very minor amounts. From this viewpoint, the specific culture conditions readily enabled these minor species to grow, which implies that the classical approach should be performed in parallel to metagenome screening. The forty-five species belonged to seven different phyla, where fourteen species were classified as Proteobacteria, five species as Actinobacteria, twenty species as Firmicutes, two species as Bacteroidetes, one species as Deinococus-Thermus, two species as Ascomycota, and one species as Basidiomycota. The oxygen concentration requirements for the growth of these species (normal or hypoxia) were mostly identical to those described in the literature. The tryptone yeast medium (I2) led to the isolation of ten species, giving the best bacterial diversity among the different tested media. Next, nine species were isolated from the yeast starch medium (F2). The best culture method was the DPM, which resulted in the isolation of thirty-six species. We applied the ODM upon considering that the OA-producing species could be cultured in the presence of OA. Four species were grown with the ODM, but none of them were confirmed to produce OA. Furthermore, Thermoleophilum minutum,14 Brevibacillus laterosporusii,15e18 and Lysinibacillus fusiformis19,20 were the only species from which the secondary metabolites were purified. Other species of H. okadai may act as sources for novel secondary metabolites.
2.2. Purification of lysiformine (1) Each species was grown in a liquid medium and the suspension was centrifuged. The ethyl acetate extracts from the supernatant and the pellet were examined for cytotoxicity against mouse leukemia P388 cells, hemolytic activity against sheep red blood cells, and lethal activity against brine shrimp. The species that were active in any of the three assays are shown in Table S2. Among them, Brevibacillus laterosporus and Lysinibacillus fusiformis were selected for purifying the toxic substances. Three known compounds of bevibacillin,18 basiliskamide A (3),15 and loloatin B21 were isolated from B. laterosporus, whereas two known compounds, namely, 2,5-bis(3-indolylmethyl)pyrazine (2)22 and basiliskamide A (3),15 were isolated for the first time and a novel compound, lysiformine (1), was isolated from Lysinibacillus fusiformis (Fig. 1). While L. fusiformis is also known to produce tetrodotoxin,23 it was not detected in the present study (data not shown).
Fig. 1. Lysiformine (1), 2,5-bis(3-indolylmethyl)pyrazine (2),22 and basiliskamide A (3),15 isolated from Lysinibacillus fusiformis.
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2.3. Structure determination of lysiformine (1) MS analysis provided the monoisotopic peak of m/z 340.1459 and the molecular formula was determined as C22H17N3O (calculated m/z: 340.1444) (Fig. S1). Comparison of 1H NMR spectra measured in CD3OD (Fig. S3) and DMSO‑d6 (Fig. S10) revealed that 1 has three exchangeable protons: 3-OH, 10 -NH, and 100 -NH. From the 1 H-1H COSY spectrum (Figs. S6 and S11), two pairs of four consecutive methine protons in an aromatic region were elucidated. The spectrum showed a correlation between the amino proton 10 -NH and H-20 and another correlation between the amino proton 100 -NH and H-200 (Fig. S11). The 13C chemical shifts of the two quaternary carbons adjacent to the four consecutive methines, deduced from the HSQC (Fig. S7) and 1H-13C HMBC (Fig. S8) spectra, were 126.89 (C-3a0 ) and 137.96 (C-7a0 ) in one pair and 126.84 (C-3a00 ) and 138.52 (C-7a00 ) in the other pair (Table 1). Although 1H-13C HMBC spectra only showed correlations between 100 -NH/C-300 and 100 -NH/C-3a00 (Fig. S13), these data indicated the presence of two indole units. The high-resolution ESI-MS/MS spectra displayed a fragment peak at m/ z 223.0853, which corresponded to C14H11N2O (calculated m/z for [MþH]þ: 223.0871) (Fig. S2) and was produced by the fragmentation of an indole unit from the parent ion. This fragmentation has also been reported in the MS/MS analysis of another bisindole alkaloid, topsentin,24 and supports our structure elucidation. The degree of unsaturation in lysiformine (1) was 16. Six degrees of unsaturation were accounted for by each indole unit and four degrees of unsaturation were assigned to the remaining structural unit. The remaining protons, H-2, H-5, and H-800 , were observed as singlet signals in the 1H NMR spectra (Figs. S3 and S10). While H-2 and H-5 were observed in the aromatic region, H-800 was observed at d 4.14 and assigned to the methylene unit connected to C-800 by analyzing the HSQC spectrum (Fig. S12). Among the remaining carbons, C-3, C-4, and C-6 were quaternary, and C-3 and C-6 were observed at d 150.77 and d 152.58 (Table 1), respectively, which were downfield shifted from those of typical aromatic carbons. Because one nitrogen atom and one oxygen atom remained unassigned, the presence of a hydroxypyridine unit was postulated as the core scaffold of lysiformine (1). The connections of the two indole units and the hydroxy unit to the pyridine unit were revealed by 1H-13C HMBC correlations (Fig. 2). First, HMBC correlations from H-800 to C-200 , C-300 , and C-3a00 indicated the presence of an indolyl methyl unit (Fig. S8). The HMBC correlation from H-5 to C-30 and that from H-20 to C-4 revealed linkages between C-30 and C-4. In addition, HMBC correlations in CD3OD from H-800 to C-5/C-6 provided evidence for a connection between C-800 and C-6. The aforementioned downfield shift of C-6 in the 13C NMR spectrum suggests that this carbon could be connected to a nitrogen atom. Consequently, because of the downfield shift of C-3 in the 13C NMR spectrum as well, a hydroxy group could be connected to C-3. This explanation was supported by the HMBC correlations between the hydroxy proton of 3-OH and C-3 and C-4 when DMSO‑d6 was used as the NMR solvent (Fig. S13). Hence, lysiformine (1) isolated from L. fusiformis was identified as 2-(3-indolylmethyl)-4-(3-indolyl)-5-hydroxypyridine. 3-Hydroxypyridine, the core scaffold of lysiformine (1), is scarcely found in secondary metabolites. While Vitamin B6s and pristinamycins have 3-hydroxypyridine in their structures, agelamadin F is the only compound to have been identified from marine sponges. Similar to bisindole alkaloids, including topsentin24 and hamacanthin,25 which have been identified from marine sponges, 1 is presumably biosynthesized from two tryptophanes. However, it might not be constructed in a similar manner to the C2 symmetric compound, 2,5-bis(3-indolylmethyl)pyrazine (2).22 Therefore, further studies are necessary to elucidate the biosynthetic pathway
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S. Kikuchi et al. / Tetrahedron 74 (2018) 3742e3747
Table 1 1 H and 13C NMR spectroscopic data of 1. Position
CD3OD
DMSO‑d6 1
13
8.13 (1H, s)
136.31 148.70
H NMR
2 3 3-OH 4 5 6 10 -NH 20 30 3a0 40 50 60 70 7a0 100 -NH 200 300 3a00 400 500 600 700 7a00 800
C
9.63 (1H, s) 7.40 (1H, s) 11.38 (1H, s) 7.84 (1H, d, 3.0)
7.44 6.91 7.09 7.41
(1H, (1H, (1H, (1H,
d, 8.1) dt, 8.1, 7.5, 0.9) dt, 7.8, 7.5, 0.9) d, 7.8)
10.81 (1H, s) 7.22 (1H, d, 3.0)
7.51 6.95 7.06 7.35
(1H, (1H, (1H, (1H,
d, 7.5) dt, 7.5, 7.5, 0.6) dt, 7.5, 7.5, 0.6) d, 7.5)
4.14 (2H, s)
HMBC
1
13
C3, C4
8.02 (1H, s)
H NMR
C
HMBC
135.61 150.77
C3, C4 130.20 120.34 152.55 127.22 109.55 125.85 119.04 119.21 120.82 111.52 136.65 122.91 113.89 127.65 118.45 117.93 120.65 111.01 136.98 32.50
C3, C30
7.55 (1H, s)
C30 , C3a0 , C7a0
7.96 (1H, s)
C60 , C7a0 C3a0 , C70 C40 , C7a0 C3a0 , C50
7.26 6.84 7.07 7.36
C300 , C3a00 , C300 , C3a00 , C7a00 ,
7.15 (1H, s)
C600 , C7a00 C3a00 , C700 , C400 , C7a00 C3a00 , C500
7.48 7.01 7.13 7.40
C5, C6, C200 , C300 , C3a00
4.25 (2H, s)
(1H, (1H, (1H, (1H,
(1H, (1H, (1H, (1H,
d, 8.0) t, 8.0, 7.2) t, 8.0, 7.2) d, 8.0)
d, 7.2) t, 7.6, 7.2) t, 8.4, 7.6) d, 8.4)
134.42 123.06 152.58 129.25 110.50 126.89 120.63 121.10 122.74 112.61 137.96 124.31 113.89 128.64 119.79 119.86 122.51 112.38 138.52 33.39
C3, C30
C4, C30 , C3a0 , C7a0
C30 , C3a0 , C60 , C70 , C7a0 C3a0 , C40 , C60 , C70 , C7a0 C40 , C70 , C7a0 C3a0 , C50
C300 , C3a00 , C7a00 , C800
C300 , C3a00 , C600 , C700 , C7a00 C3a00 , C600 , C700 , C7a00 C400 , C500 , C7a00 C3a00 , C500 C5, C6, C200 , C300 , C3a00
Fig. 2. Key 2D correlations for determining the structure of 1. Key 1H-1H correlations are drawn in bold and 1H-13C correlations are indicated by arrows from 1H to 13C.
of lysiformine (1). The peak width corresponding to lysiformine (1) in HPLC analysis with H2O/MeOH as the mobile phase was significantly wider than that obtained with H2O/MeOH with formic acid as the mobile phase, whereas the former condition provided a better peak separation in the 1H NMR spectrum than the latter condition. We thus decided to purify lysiformine (1) using the formic acid-containing mobile phase and convert the protonated form to the neutral form by treating with Sep-Pak® Plus C18. However, it should be noted that the pyridinium proton was detected in the 1H NMR spectrum (Fig. S10) with the 0.23 of the integrated value when that of H-700 was defined as one proton, suggesting that the ratio of the protonated form to the neutral form was 0.23:0.77. DFT calculations depicted these characteristic structural features shown in the HPLC and NMR spectra of lysiformine (1). The dihedral angle for 5-4-30 3a0 was 51 (Fig. 3A) in the neutral form, whereas þ155 in the protonated form (Fig. 3B), suggesting that the dihedral angle of 5-43-3a0 is reflected in pH and that lysiformine (1) is axially chiral. When the scaling factors26 and contribution of the protonated form were taken into account to simulate the chemical shifts, the average differences in chemical shifts of the 1H and 13C NMR spectra from the observed chemical shifts were d 0.08 ppm and d 0.02 ppm, respectively, confirming our structure elucidation.
Fig. 3. The stabilized conformation of 1 estimated by the DFT calculations. The top and side views for (A) the neutral form and (B) the protonated form was displayed.
2.4. Cytotoxicity of lysiformine (1) The cytotoxicity was evaluated using mouse leukemia P388 cells. Due to the limited amount of the sample, however, the dose response was examined at three concentrations, 0.1 mM, 1.0 mM and 10 mM, and the rate of cell viability were 103 ± 2%, 103 ± 3%, and 50 ± 4%, respectively (mean ± standard error of the mean, n ¼ 4). Thus, the IC50 value was approximately estimated to be 10 mM.
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3. Conclusion We isolated forty-eight species of sponge-associated microorganisms from H. okadai, and purified lysiformine (1) from L. fusiformis. Other novel compounds may be found from the rest of the species and may form the basis of future therapeutic applications. 4. Experimental 4.1. General procedure MS or MS/MS spectra were measured with a micrOTOF-Q II (ESI, Q-TOF) (Bruker Daltonics Inc., Billerica, MA, USA) spectrometer connected to a Shimadzu Nexera UHPLC System (Shimadzu, Kyoto, Japan). The mass spectrometer was equipped with an ESI ion S9 source and set up in the positive ionization mode; dry gas: nitrogen 8 L/min; dry temperature: 200 C; nebulizer: 1.6 bar; capillary: 4500 V. NMR spectra were obtained on either an Agilent 600 MHz (Agilent Technologies, Inc., Santa Clara, CA, USA) or a JEOL 800 MHz (JNM-ECA800; JEOL Ltd., Akishima, Tokyo, Japan) NMR spectrometer with CDCl3 (Acros Organics, Inc.; Geel, Belgium), CD3OD (Euriso-Top, Inc.; Saint-Aubin, Orne, France), or DMSO‑d6 (Euriso-Top, Inc.; Saint-Aubin, Orne, France) as the solvent and internal standard. Spectra were referenced to residual solvent signals at dH/ C ¼ 7.26/77.0 ppm (CDCl3), dH/C ¼ 3.31/49.0 ppm (CD3OD), or dH/ C ¼ 2.5/39.6 ppm (DMSO‑d6). 4.2. Materials Mouse leukemia P388 cell line was provided by RIKEN BRC through the National Bio-Resource Project of MEXT, Japan. KODPlus, T4 ligase was purchased from Takara Bio, Inc. (Kusatsu, Shiga, Japan). PCR primers were purchased from Fasmac Co., Ltd. (Atsugi, Kanagawa, Japan). Bacto agar, bacto tryptone, and bacto yeast extract were purchased from Becton, Dickinson and Company (Franklin Lakes, New Jersey, United States). All other chemicals were purchased from Nacalai Tesque, Inc. (Nakagyo, Kyoto, Japan), Wako Pure Chemical Industries, Ltd. (Chuou, Osaka, Japan), or Sigma-Aldrich Japan, Inc. (Shinagawa, Tokyo, Japan).
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casein medium,28 (K) a cellulose medium,30 (L) two kinds of yeast extract media (L1, L2),31 and (M) a marine agar medium.32 The culture methods used were DPM, DCM,33,34 MTM,34 spongesettling method (SSM), cellulose method (CM),30 and OA diffusion method (ODM). Unless otherwise noted, the methods were identical to those described in each reference. In the classical DPM, the dissemination liquid is applied to an agar plate to start the culture. This process was modified for the ODM, where agar containing 500 nM or 1 mM OA was used to facilitate the culture OA-producing microorganisms. In the DCM, the mixture of melted agar and dissemination liquid was poured into the center of a steel washer, whose bottom was sealed with a 0.03 mm pore-size polycarbonate membrane (Avanti Polar Lipids, Inc., Alabaster, AL, USA). After the agar was solidified, the washer was covered with another 0.03 mm pore-size polycarbonate membrane to make a sandwich-like complex, i.e., a diffusion chamber. This diffusion chamber was set up on a piece of sponge specimen to start the culture. The diffusion chamber prevented the entry of contaminants but the nutrients from the sponge specimen could diffuse into the chamber, which facilitated the growth of the sponge-associated microorganisms in the environments mimicking the natural habitat. In the MTM, melted agar was poured into the center of a steel washer, whose bottom was sealed with a 0.2 mm pore-size polycarbonate membrane (Avanti Polar Lipids, Inc., Alabaster, AL, USA). After the agar solidified, the washer was covered with a 0.03 mm polycarbonate membrane to make a sandwich-like complex, i.e., a microbial trap. The microbial trap was set up on a piece of sponge specimen to start the culture. The microbial trap prevented the entry of contaminants but allowed the actinomycetes to pass through the 0.2 mm pore-size polycarbonate membrane and grow in the environments mimicking the natural habitat. In the SSM, a 2 cm3 block of marine sponge was autoclaved and mounted on an agar plate to utilize its skeleton for facilitating the culture. The dissemination liquid was poured into the autoclaved sponge specimen to start the culture. In the CM, the fresh sponge specimen was used as the source of the microorganisms instead of the dissemination liquid. An agar plate was covered with a cellulose disk, on which small pieces of H. okadai were mounted to start the culture.
4.3. Isolation of sponge-associated microbes
4.4. Analysis of 16S or 18S rDNA sequence
The sponge specimens were collected from Kanagawa prefecture, Japan.27 The dissemination liquid was prepared by immersing small pieces of the sponge specimen in EDTA-containing calcium and magnesium free artificial seawater (CMF-ASW/EDTA; 920 mM NaCl, 14.0 mM Na2SO4, 21.4 mM KCl, 4.2 mM NaHCO3, and 50.0 mM EDTA; pH 7.0) to dissociate the sponge-associated species, incubating at 4 C for 10 min, and filtering with a 77 mm-sized nylon mesh. The dissemination liquid was subjected to 720 different conditions where the temperature, culture medium, oxygen concentration, and culture method were the variables. The temperature was either 26 C or 32 C. Oxygen concentration was either normal or 5% (obtained by using AnaeroPack, an oxygen absorbent (Mitsubishi Gas Chemical, Tokyo, Japan) in a sealed box). The culture media included (A) four kinds of yeast glucose media (A1eA4),28 (B) six kinds of low-nutrient media (B1eB6), (C) two kinds of Gifu anaerobic agar media (C1, C2), (D) two kinds of glucose asparagine media (D1, D2),28 (E) three kinds of humic media (E1eE3),28 (F) two kinds of yeast starch media (F1, F2),29 (G) two kinds of glucose nitrate media (G1, G2),28 (H) two kinds of inorganic salt starch media (H1, H2),28 (I) two kinds of tryptone-yeast medium (I1, I2),28 (J) a
A single colony of isolated microbes was picked up and suspended in 10% Chelex (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The microbial DNA was extracted by incubating at 95 C for 20 min. The extracted DNA was amplified by PCR using DNA polymerase of PCR KOD Plus Neo (TOYOBO, Osaka, Japan). In the case of bacteria, the forward primer for 16S rDNA was 27f, AGAGTTTGATCMTGGCTCAG, and the reverse primer was 1522r, AAGGAGGTGATCCANCCRCA.35 The reaction mixture was subjected to PCR conditions of 94 C for 2 min, followed by 30 cycles of 98 C for 10 s, 61 C for 10 s, and 68 C for 30 s.30 In the case of fungi, the forward primer for 18s rDNA was EF4, GGAAGGGRTGTATTTATTAG, and the reverse primer was Fung5, GTAAAAGTCCTGGTTCCCC. The PCR conditions were 110 C for 2 min, followed by 40 cycles of 94 C for 1 min, 48 C for 1 min, and 72 C for 3 min.36 The amplified fragments were treated with ExoSAP-IT (Affymetrix, Inc., Santa Clara, CA, USA) or purified by Wizard SV Gel and PCR Clean-up system (Promega Corporation, Fitchburg, WI, USA) after running 1.0% agarose gel (Kanto Chemical, Tokyo, Japan) electrophoresis with Hyper Ladder I (Bioline, London, UK) as the marker. The amplified fragments were sequenced by Fasmac (Kanagawa, Japan) and referred to Blast Search to determine the species.
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4.5. Extraction from sponge-associated microbes
4.8. Hemolytic assay
Each of the 48 species was cultured in a liquid medium. The mixture was centrifuged at room temperature and 5000 g for 10 min. The supernatant was partitioned three times with an equivalent volume of EtOAc and subsequently evaporated in vacuo. The concentrate was dissolved in MeOH to a final concentration of 10 mg/mL and defined as Fraction S. The pellet was re-suspended in H2O and sonicated with VibraCell® (Sonics & Materials Inc., Newtown, CT, USA) at 0 C for 10 min. The suspension was extracted with EtOAc and the organic layer was evaporated in vacuo. The concentrate was dissolved in MeOH to a concentration of 10 mg/mL and defined as Fraction P.
Sheep red blood cells (Nippon Bio-Supp. Center, Tokyo, Japan) were centrifuged at 1000 g for 10 min at 4 C. The pellets were washed twice with ice-cold phosphate buffered saline (PBS ()). Next, the cells were diluted to be 1.0% v/v with PBS (). The cell suspension (200 mL) added to each well of a 96-well microplate was mixed with 2 mL of the sample (final concentration: 100 mg/mL MeOH). After the mixture was incubated at 37 C for 2 days, the mixture was centrifuged at 1000 g for 10 min. The supernatant was transferred to a well of another 96-well microplate, and the absorbance at 576 nm was measured on a microplate reader (BioRad Laboratories, Inc., Hercules, CA, USA). Complete hemolysis was achieved when the cells were treated with H2O. Each dose was performed in duplicate and the experiment involving multiple doses was repeated at least three times.
4.6. Purification of lysiformine (1) from Lysinibacillus fusiformis The organic layer from Fraction P of L. fusiformis was concentrated (40e45 mg) and portioned between 70% aqueous MeOH and hexane. The aqueous layer was partitioned twice with double volumes of CHCl3. The organic layer was evaporated in vacuo (30e35 mg) and subjected to silica gel chromatography (0.8 g, 60 OPN, 0.04e0.06 mm; Merck, Darmstadt, Germany) using a mobile phase of hexane (2 mL, Fractions 1e2), 4:1 hexane-EtOAc (3 mL, Fractions 3e5), 1:1 hexane-EtOAc (10 mL, Fractions 6e15), 9:1 CHCl3-MeOH (5 mL, Fractions 16e20), and 1:9 CHCl3-MeOH (5 mL, Fractions 21e25). A total of 25 fractions were analyzed by thin layer chromatography (silica gel 60, Merck, Darmstadt, Germany) using a mobile phase of 9:1 CHCl3-MeOH and subjected to two bioassays of hemolytic activity and cytotoxic activity. Fractions 21e24 (1.5 mg) were applied to the Sep-Pak® Plus C18 column (360 mg; Waters Corporation, Milford, MA, USA). Elution was performed with 10 mL of water (Fractions 1e10), 10 mL of 50% MeOH (Fractions 11e20), and 5 mL of MeOH (Fractions 21e25). Fractions 21e25 were combined and concentrated in vacuo. The residue was purified by HPLC, where a low-pressure gradient pump (PU-2089 Plus), UVeVisible detector (UV-2075 Plus), and photodiode array detector (MD2018 Plus) were controlled with JASCO LC Net II/ADC Chromatography Data Solutions (JASCO, Tokyo, Japan) to separate the samples on a reversed phase column of Cosmosil 5C18-AR-II (250 mm 10 mm i.d.; Nacalai Tesque, Kyoto, Japan). Mobile phases A and B were 50% MeOH containing 50 mM HCO2H and MeOH containing 50 mM HCO2H, respectively. The elution was performed with 100% A during the first 5 min and with 0e100% B in the linear gradient mode during the next 75 min. The flow rate was fixed at 2 mL/min, and the elution was monitored at an absorbance of 254 nm with a UV detector and within a range of 200e600 nm with a photodiode array detector. After the eluate containing compound 1 was evaporated in vacuo, the concentrate was transferred for purification over the Sep-Pak® Plus C18 column with the mobile phases of 50% MeOH and 100% MeOH. The fraction containing 1 was evaporated and subjected to HRMS and NMR analyses.
4.7. Cytotoxic assay Mouse leukemia P388 cells were maintained in an RPMI1640 medium containing 10 units/mL of penicillin, 10 mg/mL streptomycin (Thermo Fischer Scientific K.K., Yokohama, Kanagawa, Japan), and 10% v/v FBS. Aliquots of the cells (200 mL) at 5.0 104 cells/mL were mixed with the sample (100 mg/mL). After 2 days, 4 mL of WST-8 (Dojin, Kumamoto, Japan) was added to the mixture for 4 h and the cell viability was evaluated by absorbance at 450 nm using a microplate reader (model 680, Bio-Rad Laboratories, Hercules, CA).
4.9. Brine shrimp lethal assay Brine shrimps (Japan Pet Design Co., Tokyo, Japan) were hatched in artificial seawater. The next day, 30 brine shrimps were transferred to each well of a 12-well microplate. Next, 5.0 mL of the sample having a concentration of 10 mg/mL was added to each well (50 mg/mL) and the suspension was incubated at room temperature for 2 days. The lethal activity was determined by counting the number of dead brine shrimps. 4.10. Lysiformine (1) Brown amorphous powder; [a]25 D 20.450 (c 0.01, MeOH); UV (MeOH) lmax (ε0) 220 (2.61 104), 282 (6.82 103), 317 (4.43 103), 377 (1.28 103) nm; IR (film) nmax 3734, 3628, 3309, 2927, 2855, 1716, 1698, 1457, 1435, 1244, 1173, 1101, 1064, 1031, 837, 743, 699, 650, 639 cm1; 1H NMR, see Table 1; 13C NMR, see Table 1; HRESIMS m/z 340.1459 (calcd for C22H17ON3, 340.1444).
4.11. Density functional theory (DFT) calculations The initial structure drawn on GaussView6 was optimized with DFT/B3LYP/3-21G in Gaussian 16. Conformational analysis was conducted with Semi-empirical MO/PM6 by rotating dihedral angles of 5e6e800 e300 and 6e800 e300 e3a00 every 30 , which simulated total 144 conformers. The most stable conformer was subjected to the second scanning with Semi-empirical MO/PM6 by rotating dihedral angles of 3e4e30 e3a0 and 2e3eOeH every 30 and 90 , respectively, which simulated total 48 conformers. The most stable conformer was optimized with DFT/B3LYP/6-31 þ G(d,p) followed by DFT/B3LYP/6-311 þ G(2d,p) to obtain the isotropic values. The isotropic values were converted to the simulated chemical shift based on a formula, Computed chemical shift ¼ (31.883-Isotropic value)/1.0553), for calculating the chemical shift of 1H NMR and another formula, Computed chemical shift ¼ (182.113-Isotropic value)/1.0469), for calculating the chemical shift of 13C NMR. These formulae were made by considering scaling factors and enable compensation of the difference between the simulated and observed chemical shifts of NMR spectra.26 In all the calculations, DMSO was used as the solvent. The charge and the spin multiplicity was zero and one, respectively, when considering the neutral form of lysiformine, whereas one and one, respectively, when considering the protonated form of lysiformine. Acknowledgments The present study was funded by JSPS KAKENHI (Grant Numbers JP17H02199, 21603003 and 24510291). The authors thank Prof.
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Shigefumi Kuwahara and Asst. Prof. Yusuke Ogura for measuring IR spectra and optical rotations. The authors are grateful for Asst. Prof. Kaoru Yamazaki Tohoku University in creating the input files for Gaussian 16. The DFT calculations in this research were conducted with supercomputing resources at Cyberscience Center, Tohoku University. We would like to thank Editage (www.editage.jp) for English language editing. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.tet.2018.05.049. References 1. Webster NS, Taylor MW. Environ Microbiol. 2012;14:335e346. 2. Vogel G. Science. 2008;320:1028e1030. 3. Tachibana K, Scheuer PJ, Tsukitani Y, et al. J Am Chem Soc. 1981;103: 2469e2471. 4. Bialojan C, Takai A. Biochem J. 1988;256:283e290. 5. Suganuma M, Fujiki H, Suguri H, et al. Proc Natl Acad Sci USA. 1988;85: 1768e1771. 6. Hirata Y, Uemura D. Pure Appl Chem. 1986;58:701e710. 7. Kuramoto M, Tong C, Yamada K, Chiba T, Hayashi Y, Uemura D. Tetrahedron Lett. 1996;37:3867e3870. 8. Amann RI, Ludwig W, Schleifer KH. Microbiol Rev. 1995;59:143e169. 9. Freeman MF, Gurgui C, Helf MJ, et al. Science. 2012;338:387e390. 10. Wakimoto T, Egami Y, Nakashima Y, et al. Nat Chem Biol. 2014;10, 648-U193. 11. Brady SF, Simmons L, Kim JH, Schmidt EW. Nat Prod Rep. 2009;26:1488e1503.
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12. Abe T, Sahin FP, Akiyama K, et al. Biosci Biotechnol Biochem. 2012;76:633e639. 13. Abe T, Kukita A, Akiyama K, Naito T, Uemura D. Chem Lett. 2012;41:728e729. 14. Hamana K, Matsuzaki S, Niitsu M, Samejima K. FEMS Microbiol Lett. 1990;66: 35e38. 15. Barsby T, Kelly MT, Andersen RJ. J Nat Prod. 2002;65:1447e1451. 16. Krachkovskii SA, Sobol AG, Ovchinnikova TV, et al. Russ J Bioorg Chem. 2002;28: 269e273. 17. Desjardine K, Pereira A, Wright H, Matainaho T, Kelly M, Andersen RJ. J Nat Prod. 2007;70:1850e1853. 18. Yang X, Huang E, Yuan CH, Zhang LW, Yousef AE. Appl Environ Microbiol. 2016;82:2763e2772. 19. Park HB, Kim YJ, Lee JK, Lee KR, Kwon HC. Org Lett. 2012;14:5002e5005. 20. Seo MH, Kim KR, Oh DK. Appl Microbiol Biotechnol. 2013;97:8987e8995. 21. Gerard J, Haden P, Kelly MT, Andersen RJ. Tetrahedron Lett. 1996;37: 7201e7204. 22. Shaaban M, Maskey RP, Wagner-Dobler I, Laatsch H. J Nat Prod. 2002;65: 1660e1663. 23. Wang J, Fan YH, Yao ZG. Toxicon. 2010;56:640e643. 24. Shin J, Seo Y, Cho KW, Rho JR, Sim CJ. J Nat Prod. 1999;62:647e649. 25. Gunasekera SP, McCarthy PJ, Kellyborges M. J Nat Prod. 1994;57:1437e1441. 26. Lodewyk MW, Tantillo DJ. J Nat Prod. 2011;74:1339e1343. 27. Konoki K, Okada K, Kohama M, et al. Toxicon. 2015;108:38e45. 28. Miyadoh Shinji, ed. The-Society-for-Actinomycete-Japan Identification Manual of Actinomycetes. Tokyo: Business Center for Academic Society Japan; 2001. 29. Hayakawa M, Nonomura H. J Ferment Technol. 1987;65:501e509. 30. Magarvey NA, Keller JM, Bernan V, Dworkin M, Sherman DH. Appl Environ Microbiol. 2004;70:7520e7529. 31. Xi LJ, Ruan JS, Huang Y. Int J Mol Sci. 2012;13:5917e5932. 32. Zobell CE. J Mar Res. 1941;4:42e75. 33. Gavrish E, Bollmann A, Epstein S, Lewis K. J Microbiol Meth. 2008;72:257e262. 34. Lewis K, Epstein S, D'Onofrio A, Ling LL. J Antibiot. 2010;63:468e476. 35. Suzuki MT, Giovannoni SJ. Appl Environ Microbiol. 1996;62:625e630. 36. Lee YK, Kim HW, Liu CL, Lee HK. J Microbiol Meth. 2003;52:245e250.
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Isolation and structure determination of lysiformine from bacteria associated with marine sponge Halichondria okadai Sou Kikuchi a, 1, Kayo Okada a, 1, Yuko Cho a, Shinichiro Yoshida b, Eunsang Kwon b, Mari Yotsu-Yamashita a, Keiichi Konoki a, * a b
Graduate School of Agricultural Science, Tohoku University, Aoba, Sendai 980-8572, Japan Research and Analytical Center for Giant Molecules, Graduate School of Science, Tohoku University, Aoba, Sendai 980-8578, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 March 2018 Received in revised form 16 May 2018 Accepted 18 May 2018 Available online 18 May 2018
Marine sponge Halichondria okadai is a source of numerous toxic secondary metabolites, which are considered as products of the symbiotic species associated with the sponge. In this study, we tested 720 culture conditions and purified 48 sponge-associated microbes. The ethyl acetate extract from the liquid culture of each species was tested for cytotoxicity, hemolytic activity, and brine shrimp lethal activity. Lysiformine (1) was isolated from Lysinibacillus fusiformis and its structure was determined by HRMS and NMR. © 2018 Published by Elsevier Ltd.
Keywords: Halichondria okadai Lysinibacillus fusiformis Isolation Structure determination
1. Introduction Marine sponges are filter-feeders and have various symbiotic species in their bodies.1 A recent study suggests that the symbiotic microbes occupy 40% of the whole volume of marine sponges.2 Therefore, most of the secondary metabolites isolated from marine sponges with significant biological activity are presumably produced by their symbiotic species. For example, the sponge Halichondria okadai is a rich source of toxic secondary metabolites such as okadaic acid (OA), which inhibits serine threonine protein phosphatases 1 and 2A and exhibits tumor-promoting activity,3e5 halichondrin B, which is an antitumor compound,6 and halichlorine, a potent VCAM-1 inhibitor.7 Therefore, it is useful to obtain a microbe producing each toxic secondary metabolite in view of the therapeutic applications. This concept of isolating and maintaining a culture of the symbiotic microbes is universally understood but extremely difficult to implement.8 Recent developments in DNA-sequencing technology have enabled the sequence analysis of metagenomes derived from marine sponges and isolation of the biosynthetic genes for polytheonamide B,9
* Corresponding author. E-mail address: [email protected] (K. Konoki). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.tet.2018.05.049 0040-4020/© 2018 Published by Elsevier Ltd.
calyculin B,10 and others.11 The metagenomic analysis of H. okadai was also carried out, and a new compound, halichrome A, is reported.12,13 However, genome mining is relatively inadequate for identifying the biosynthetic gene of the target compound. Consequently, we adopted the classical approach to identify the spongeassociated microbes of H. okadai in our search for novel toxic secondary metabolites. Herein, we identified 48 sponge-associated species from H. okadai by testing 720 kinds of culture conditions, which were the combination of 30 culture media, two oxygen concentrations, two temperatures, and six culture methods. From a culture of Lysinibacillus fusiformis, we isolated lysiformine (1) based on 3 independent biological assays and completed structure determination by HRMS and NMR analyses.1 2. Results and discussion 2.1. Isolation of sponge-associated microbes In total, 73 characteristic colonies appeared; however, 16S rDNA or 18S rDNA analyses showed that some of the colonies grew from the same species (Table S2). Finally, 45 species were identified in addition to three unidentified fungi. There should be an innumerable species present in the sponge, which reveals the difficulty in growing sponge-associated microorganisms.8 It should be noted
S. Kikuchi et al. / Tetrahedron 74 (2018) 3742e3747
that none of the species could be identified by metagenome screening,12,13 which is typically able to identify the abundant species present in the target specimen. Thus, the isolated microorganisms in the sponge were present in very minor amounts. From this viewpoint, the specific culture conditions readily enabled these minor species to grow, which implies that the classical approach should be performed in parallel to metagenome screening. The forty-five species belonged to seven different phyla, where fourteen species were classified as Proteobacteria, five species as Actinobacteria, twenty species as Firmicutes, two species as Bacteroidetes, one species as Deinococus-Thermus, two species as Ascomycota, and one species as Basidiomycota. The oxygen concentration requirements for the growth of these species (normal or hypoxia) were mostly identical to those described in the literature. The tryptone yeast medium (I2) led to the isolation of ten species, giving the best bacterial diversity among the different tested media. Next, nine species were isolated from the yeast starch medium (F2). The best culture method was the DPM, which resulted in the isolation of thirty-six species. We applied the ODM upon considering that the OA-producing species could be cultured in the presence of OA. Four species were grown with the ODM, but none of them were confirmed to produce OA. Furthermore, Thermoleophilum minutum,14 Brevibacillus laterosporusii,15e18 and Lysinibacillus fusiformis19,20 were the only species from which the secondary metabolites were purified. Other species of H. okadai may act as sources for novel secondary metabolites.
2.2. Purification of lysiformine (1) Each species was grown in a liquid medium and the suspension was centrifuged. The ethyl acetate extracts from the supernatant and the pellet were examined for cytotoxicity against mouse leukemia P388 cells, hemolytic activity against sheep red blood cells, and lethal activity against brine shrimp. The species that were active in any of the three assays are shown in Table S2. Among them, Brevibacillus laterosporus and Lysinibacillus fusiformis were selected for purifying the toxic substances. Three known compounds of bevibacillin,18 basiliskamide A (3),15 and loloatin B21 were isolated from B. laterosporus, whereas two known compounds, namely, 2,5-bis(3-indolylmethyl)pyrazine (2)22 and basiliskamide A (3),15 were isolated for the first time and a novel compound, lysiformine (1), was isolated from Lysinibacillus fusiformis (Fig. 1). While L. fusiformis is also known to produce tetrodotoxin,23 it was not detected in the present study (data not shown).
Fig. 1. Lysiformine (1), 2,5-bis(3-indolylmethyl)pyrazine (2),22 and basiliskamide A (3),15 isolated from Lysinibacillus fusiformis.
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2.3. Structure determination of lysiformine (1) MS analysis provided the monoisotopic peak of m/z 340.1459 and the molecular formula was determined as C22H17N3O (calculated m/z: 340.1444) (Fig. S1). Comparison of 1H NMR spectra measured in CD3OD (Fig. S3) and DMSO‑d6 (Fig. S10) revealed that 1 has three exchangeable protons: 3-OH, 10 -NH, and 100 -NH. From the 1 H-1H COSY spectrum (Figs. S6 and S11), two pairs of four consecutive methine protons in an aromatic region were elucidated. The spectrum showed a correlation between the amino proton 10 -NH and H-20 and another correlation between the amino proton 100 -NH and H-200 (Fig. S11). The 13C chemical shifts of the two quaternary carbons adjacent to the four consecutive methines, deduced from the HSQC (Fig. S7) and 1H-13C HMBC (Fig. S8) spectra, were 126.89 (C-3a0 ) and 137.96 (C-7a0 ) in one pair and 126.84 (C-3a00 ) and 138.52 (C-7a00 ) in the other pair (Table 1). Although 1H-13C HMBC spectra only showed correlations between 100 -NH/C-300 and 100 -NH/C-3a00 (Fig. S13), these data indicated the presence of two indole units. The high-resolution ESI-MS/MS spectra displayed a fragment peak at m/ z 223.0853, which corresponded to C14H11N2O (calculated m/z for [MþH]þ: 223.0871) (Fig. S2) and was produced by the fragmentation of an indole unit from the parent ion. This fragmentation has also been reported in the MS/MS analysis of another bisindole alkaloid, topsentin,24 and supports our structure elucidation. The degree of unsaturation in lysiformine (1) was 16. Six degrees of unsaturation were accounted for by each indole unit and four degrees of unsaturation were assigned to the remaining structural unit. The remaining protons, H-2, H-5, and H-800 , were observed as singlet signals in the 1H NMR spectra (Figs. S3 and S10). While H-2 and H-5 were observed in the aromatic region, H-800 was observed at d 4.14 and assigned to the methylene unit connected to C-800 by analyzing the HSQC spectrum (Fig. S12). Among the remaining carbons, C-3, C-4, and C-6 were quaternary, and C-3 and C-6 were observed at d 150.77 and d 152.58 (Table 1), respectively, which were downfield shifted from those of typical aromatic carbons. Because one nitrogen atom and one oxygen atom remained unassigned, the presence of a hydroxypyridine unit was postulated as the core scaffold of lysiformine (1). The connections of the two indole units and the hydroxy unit to the pyridine unit were revealed by 1H-13C HMBC correlations (Fig. 2). First, HMBC correlations from H-800 to C-200 , C-300 , and C-3a00 indicated the presence of an indolyl methyl unit (Fig. S8). The HMBC correlation from H-5 to C-30 and that from H-20 to C-4 revealed linkages between C-30 and C-4. In addition, HMBC correlations in CD3OD from H-800 to C-5/C-6 provided evidence for a connection between C-800 and C-6. The aforementioned downfield shift of C-6 in the 13C NMR spectrum suggests that this carbon could be connected to a nitrogen atom. Consequently, because of the downfield shift of C-3 in the 13C NMR spectrum as well, a hydroxy group could be connected to C-3. This explanation was supported by the HMBC correlations between the hydroxy proton of 3-OH and C-3 and C-4 when DMSO‑d6 was used as the NMR solvent (Fig. S13). Hence, lysiformine (1) isolated from L. fusiformis was identified as 2-(3-indolylmethyl)-4-(3-indolyl)-5-hydroxypyridine. 3-Hydroxypyridine, the core scaffold of lysiformine (1), is scarcely found in secondary metabolites. While Vitamin B6s and pristinamycins have 3-hydroxypyridine in their structures, agelamadin F is the only compound to have been identified from marine sponges. Similar to bisindole alkaloids, including topsentin24 and hamacanthin,25 which have been identified from marine sponges, 1 is presumably biosynthesized from two tryptophanes. However, it might not be constructed in a similar manner to the C2 symmetric compound, 2,5-bis(3-indolylmethyl)pyrazine (2).22 Therefore, further studies are necessary to elucidate the biosynthetic pathway
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S. Kikuchi et al. / Tetrahedron 74 (2018) 3742e3747
Table 1 1 H and 13C NMR spectroscopic data of 1. Position
CD3OD
DMSO‑d6 1
13
8.13 (1H, s)
136.31 148.70
H NMR
2 3 3-OH 4 5 6 10 -NH 20 30 3a0 40 50 60 70 7a0 100 -NH 200 300 3a00 400 500 600 700 7a00 800
C
9.63 (1H, s) 7.40 (1H, s) 11.38 (1H, s) 7.84 (1H, d, 3.0)
7.44 6.91 7.09 7.41
(1H, (1H, (1H, (1H,
d, 8.1) dt, 8.1, 7.5, 0.9) dt, 7.8, 7.5, 0.9) d, 7.8)
10.81 (1H, s) 7.22 (1H, d, 3.0)
7.51 6.95 7.06 7.35
(1H, (1H, (1H, (1H,
d, 7.5) dt, 7.5, 7.5, 0.6) dt, 7.5, 7.5, 0.6) d, 7.5)
4.14 (2H, s)
HMBC
1
13
C3, C4
8.02 (1H, s)
H NMR
C
HMBC
135.61 150.77
C3, C4 130.20 120.34 152.55 127.22 109.55 125.85 119.04 119.21 120.82 111.52 136.65 122.91 113.89 127.65 118.45 117.93 120.65 111.01 136.98 32.50
C3, C30
7.55 (1H, s)
C30 , C3a0 , C7a0
7.96 (1H, s)
C60 , C7a0 C3a0 , C70 C40 , C7a0 C3a0 , C50
7.26 6.84 7.07 7.36
C300 , C3a00 , C300 , C3a00 , C7a00 ,
7.15 (1H, s)
C600 , C7a00 C3a00 , C700 , C400 , C7a00 C3a00 , C500
7.48 7.01 7.13 7.40
C5, C6, C200 , C300 , C3a00
4.25 (2H, s)
(1H, (1H, (1H, (1H,
(1H, (1H, (1H, (1H,
d, 8.0) t, 8.0, 7.2) t, 8.0, 7.2) d, 8.0)
d, 7.2) t, 7.6, 7.2) t, 8.4, 7.6) d, 8.4)
134.42 123.06 152.58 129.25 110.50 126.89 120.63 121.10 122.74 112.61 137.96 124.31 113.89 128.64 119.79 119.86 122.51 112.38 138.52 33.39
C3, C30
C4, C30 , C3a0 , C7a0
C30 , C3a0 , C60 , C70 , C7a0 C3a0 , C40 , C60 , C70 , C7a0 C40 , C70 , C7a0 C3a0 , C50
C300 , C3a00 , C7a00 , C800
C300 , C3a00 , C600 , C700 , C7a00 C3a00 , C600 , C700 , C7a00 C400 , C500 , C7a00 C3a00 , C500 C5, C6, C200 , C300 , C3a00
Fig. 2. Key 2D correlations for determining the structure of 1. Key 1H-1H correlations are drawn in bold and 1H-13C correlations are indicated by arrows from 1H to 13C.
of lysiformine (1). The peak width corresponding to lysiformine (1) in HPLC analysis with H2O/MeOH as the mobile phase was significantly wider than that obtained with H2O/MeOH with formic acid as the mobile phase, whereas the former condition provided a better peak separation in the 1H NMR spectrum than the latter condition. We thus decided to purify lysiformine (1) using the formic acid-containing mobile phase and convert the protonated form to the neutral form by treating with Sep-Pak® Plus C18. However, it should be noted that the pyridinium proton was detected in the 1H NMR spectrum (Fig. S10) with the 0.23 of the integrated value when that of H-700 was defined as one proton, suggesting that the ratio of the protonated form to the neutral form was 0.23:0.77. DFT calculations depicted these characteristic structural features shown in the HPLC and NMR spectra of lysiformine (1). The dihedral angle for 5-4-30 3a0 was 51 (Fig. 3A) in the neutral form, whereas þ155 in the protonated form (Fig. 3B), suggesting that the dihedral angle of 5-43-3a0 is reflected in pH and that lysiformine (1) is axially chiral. When the scaling factors26 and contribution of the protonated form were taken into account to simulate the chemical shifts, the average differences in chemical shifts of the 1H and 13C NMR spectra from the observed chemical shifts were d 0.08 ppm and d 0.02 ppm, respectively, confirming our structure elucidation.
Fig. 3. The stabilized conformation of 1 estimated by the DFT calculations. The top and side views for (A) the neutral form and (B) the protonated form was displayed.
2.4. Cytotoxicity of lysiformine (1) The cytotoxicity was evaluated using mouse leukemia P388 cells. Due to the limited amount of the sample, however, the dose response was examined at three concentrations, 0.1 mM, 1.0 mM and 10 mM, and the rate of cell viability were 103 ± 2%, 103 ± 3%, and 50 ± 4%, respectively (mean ± standard error of the mean, n ¼ 4). Thus, the IC50 value was approximately estimated to be 10 mM.
S. Kikuchi et al. / Tetrahedron 74 (2018) 3742e3747
3. Conclusion We isolated forty-eight species of sponge-associated microorganisms from H. okadai, and purified lysiformine (1) from L. fusiformis. Other novel compounds may be found from the rest of the species and may form the basis of future therapeutic applications. 4. Experimental 4.1. General procedure MS or MS/MS spectra were measured with a micrOTOF-Q II (ESI, Q-TOF) (Bruker Daltonics Inc., Billerica, MA, USA) spectrometer connected to a Shimadzu Nexera UHPLC System (Shimadzu, Kyoto, Japan). The mass spectrometer was equipped with an ESI ion S9 source and set up in the positive ionization mode; dry gas: nitrogen 8 L/min; dry temperature: 200 C; nebulizer: 1.6 bar; capillary: 4500 V. NMR spectra were obtained on either an Agilent 600 MHz (Agilent Technologies, Inc., Santa Clara, CA, USA) or a JEOL 800 MHz (JNM-ECA800; JEOL Ltd., Akishima, Tokyo, Japan) NMR spectrometer with CDCl3 (Acros Organics, Inc.; Geel, Belgium), CD3OD (Euriso-Top, Inc.; Saint-Aubin, Orne, France), or DMSO‑d6 (Euriso-Top, Inc.; Saint-Aubin, Orne, France) as the solvent and internal standard. Spectra were referenced to residual solvent signals at dH/ C ¼ 7.26/77.0 ppm (CDCl3), dH/C ¼ 3.31/49.0 ppm (CD3OD), or dH/ C ¼ 2.5/39.6 ppm (DMSO‑d6). 4.2. Materials Mouse leukemia P388 cell line was provided by RIKEN BRC through the National Bio-Resource Project of MEXT, Japan. KODPlus, T4 ligase was purchased from Takara Bio, Inc. (Kusatsu, Shiga, Japan). PCR primers were purchased from Fasmac Co., Ltd. (Atsugi, Kanagawa, Japan). Bacto agar, bacto tryptone, and bacto yeast extract were purchased from Becton, Dickinson and Company (Franklin Lakes, New Jersey, United States). All other chemicals were purchased from Nacalai Tesque, Inc. (Nakagyo, Kyoto, Japan), Wako Pure Chemical Industries, Ltd. (Chuou, Osaka, Japan), or Sigma-Aldrich Japan, Inc. (Shinagawa, Tokyo, Japan).
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casein medium,28 (K) a cellulose medium,30 (L) two kinds of yeast extract media (L1, L2),31 and (M) a marine agar medium.32 The culture methods used were DPM, DCM,33,34 MTM,34 spongesettling method (SSM), cellulose method (CM),30 and OA diffusion method (ODM). Unless otherwise noted, the methods were identical to those described in each reference. In the classical DPM, the dissemination liquid is applied to an agar plate to start the culture. This process was modified for the ODM, where agar containing 500 nM or 1 mM OA was used to facilitate the culture OA-producing microorganisms. In the DCM, the mixture of melted agar and dissemination liquid was poured into the center of a steel washer, whose bottom was sealed with a 0.03 mm pore-size polycarbonate membrane (Avanti Polar Lipids, Inc., Alabaster, AL, USA). After the agar was solidified, the washer was covered with another 0.03 mm pore-size polycarbonate membrane to make a sandwich-like complex, i.e., a diffusion chamber. This diffusion chamber was set up on a piece of sponge specimen to start the culture. The diffusion chamber prevented the entry of contaminants but the nutrients from the sponge specimen could diffuse into the chamber, which facilitated the growth of the sponge-associated microorganisms in the environments mimicking the natural habitat. In the MTM, melted agar was poured into the center of a steel washer, whose bottom was sealed with a 0.2 mm pore-size polycarbonate membrane (Avanti Polar Lipids, Inc., Alabaster, AL, USA). After the agar solidified, the washer was covered with a 0.03 mm polycarbonate membrane to make a sandwich-like complex, i.e., a microbial trap. The microbial trap was set up on a piece of sponge specimen to start the culture. The microbial trap prevented the entry of contaminants but allowed the actinomycetes to pass through the 0.2 mm pore-size polycarbonate membrane and grow in the environments mimicking the natural habitat. In the SSM, a 2 cm3 block of marine sponge was autoclaved and mounted on an agar plate to utilize its skeleton for facilitating the culture. The dissemination liquid was poured into the autoclaved sponge specimen to start the culture. In the CM, the fresh sponge specimen was used as the source of the microorganisms instead of the dissemination liquid. An agar plate was covered with a cellulose disk, on which small pieces of H. okadai were mounted to start the culture.
4.3. Isolation of sponge-associated microbes
4.4. Analysis of 16S or 18S rDNA sequence
The sponge specimens were collected from Kanagawa prefecture, Japan.27 The dissemination liquid was prepared by immersing small pieces of the sponge specimen in EDTA-containing calcium and magnesium free artificial seawater (CMF-ASW/EDTA; 920 mM NaCl, 14.0 mM Na2SO4, 21.4 mM KCl, 4.2 mM NaHCO3, and 50.0 mM EDTA; pH 7.0) to dissociate the sponge-associated species, incubating at 4 C for 10 min, and filtering with a 77 mm-sized nylon mesh. The dissemination liquid was subjected to 720 different conditions where the temperature, culture medium, oxygen concentration, and culture method were the variables. The temperature was either 26 C or 32 C. Oxygen concentration was either normal or 5% (obtained by using AnaeroPack, an oxygen absorbent (Mitsubishi Gas Chemical, Tokyo, Japan) in a sealed box). The culture media included (A) four kinds of yeast glucose media (A1eA4),28 (B) six kinds of low-nutrient media (B1eB6), (C) two kinds of Gifu anaerobic agar media (C1, C2), (D) two kinds of glucose asparagine media (D1, D2),28 (E) three kinds of humic media (E1eE3),28 (F) two kinds of yeast starch media (F1, F2),29 (G) two kinds of glucose nitrate media (G1, G2),28 (H) two kinds of inorganic salt starch media (H1, H2),28 (I) two kinds of tryptone-yeast medium (I1, I2),28 (J) a
A single colony of isolated microbes was picked up and suspended in 10% Chelex (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The microbial DNA was extracted by incubating at 95 C for 20 min. The extracted DNA was amplified by PCR using DNA polymerase of PCR KOD Plus Neo (TOYOBO, Osaka, Japan). In the case of bacteria, the forward primer for 16S rDNA was 27f, AGAGTTTGATCMTGGCTCAG, and the reverse primer was 1522r, AAGGAGGTGATCCANCCRCA.35 The reaction mixture was subjected to PCR conditions of 94 C for 2 min, followed by 30 cycles of 98 C for 10 s, 61 C for 10 s, and 68 C for 30 s.30 In the case of fungi, the forward primer for 18s rDNA was EF4, GGAAGGGRTGTATTTATTAG, and the reverse primer was Fung5, GTAAAAGTCCTGGTTCCCC. The PCR conditions were 110 C for 2 min, followed by 40 cycles of 94 C for 1 min, 48 C for 1 min, and 72 C for 3 min.36 The amplified fragments were treated with ExoSAP-IT (Affymetrix, Inc., Santa Clara, CA, USA) or purified by Wizard SV Gel and PCR Clean-up system (Promega Corporation, Fitchburg, WI, USA) after running 1.0% agarose gel (Kanto Chemical, Tokyo, Japan) electrophoresis with Hyper Ladder I (Bioline, London, UK) as the marker. The amplified fragments were sequenced by Fasmac (Kanagawa, Japan) and referred to Blast Search to determine the species.
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4.5. Extraction from sponge-associated microbes
4.8. Hemolytic assay
Each of the 48 species was cultured in a liquid medium. The mixture was centrifuged at room temperature and 5000 g for 10 min. The supernatant was partitioned three times with an equivalent volume of EtOAc and subsequently evaporated in vacuo. The concentrate was dissolved in MeOH to a final concentration of 10 mg/mL and defined as Fraction S. The pellet was re-suspended in H2O and sonicated with VibraCell® (Sonics & Materials Inc., Newtown, CT, USA) at 0 C for 10 min. The suspension was extracted with EtOAc and the organic layer was evaporated in vacuo. The concentrate was dissolved in MeOH to a concentration of 10 mg/mL and defined as Fraction P.
Sheep red blood cells (Nippon Bio-Supp. Center, Tokyo, Japan) were centrifuged at 1000 g for 10 min at 4 C. The pellets were washed twice with ice-cold phosphate buffered saline (PBS ()). Next, the cells were diluted to be 1.0% v/v with PBS (). The cell suspension (200 mL) added to each well of a 96-well microplate was mixed with 2 mL of the sample (final concentration: 100 mg/mL MeOH). After the mixture was incubated at 37 C for 2 days, the mixture was centrifuged at 1000 g for 10 min. The supernatant was transferred to a well of another 96-well microplate, and the absorbance at 576 nm was measured on a microplate reader (BioRad Laboratories, Inc., Hercules, CA, USA). Complete hemolysis was achieved when the cells were treated with H2O. Each dose was performed in duplicate and the experiment involving multiple doses was repeated at least three times.
4.6. Purification of lysiformine (1) from Lysinibacillus fusiformis The organic layer from Fraction P of L. fusiformis was concentrated (40e45 mg) and portioned between 70% aqueous MeOH and hexane. The aqueous layer was partitioned twice with double volumes of CHCl3. The organic layer was evaporated in vacuo (30e35 mg) and subjected to silica gel chromatography (0.8 g, 60 OPN, 0.04e0.06 mm; Merck, Darmstadt, Germany) using a mobile phase of hexane (2 mL, Fractions 1e2), 4:1 hexane-EtOAc (3 mL, Fractions 3e5), 1:1 hexane-EtOAc (10 mL, Fractions 6e15), 9:1 CHCl3-MeOH (5 mL, Fractions 16e20), and 1:9 CHCl3-MeOH (5 mL, Fractions 21e25). A total of 25 fractions were analyzed by thin layer chromatography (silica gel 60, Merck, Darmstadt, Germany) using a mobile phase of 9:1 CHCl3-MeOH and subjected to two bioassays of hemolytic activity and cytotoxic activity. Fractions 21e24 (1.5 mg) were applied to the Sep-Pak® Plus C18 column (360 mg; Waters Corporation, Milford, MA, USA). Elution was performed with 10 mL of water (Fractions 1e10), 10 mL of 50% MeOH (Fractions 11e20), and 5 mL of MeOH (Fractions 21e25). Fractions 21e25 were combined and concentrated in vacuo. The residue was purified by HPLC, where a low-pressure gradient pump (PU-2089 Plus), UVeVisible detector (UV-2075 Plus), and photodiode array detector (MD2018 Plus) were controlled with JASCO LC Net II/ADC Chromatography Data Solutions (JASCO, Tokyo, Japan) to separate the samples on a reversed phase column of Cosmosil 5C18-AR-II (250 mm 10 mm i.d.; Nacalai Tesque, Kyoto, Japan). Mobile phases A and B were 50% MeOH containing 50 mM HCO2H and MeOH containing 50 mM HCO2H, respectively. The elution was performed with 100% A during the first 5 min and with 0e100% B in the linear gradient mode during the next 75 min. The flow rate was fixed at 2 mL/min, and the elution was monitored at an absorbance of 254 nm with a UV detector and within a range of 200e600 nm with a photodiode array detector. After the eluate containing compound 1 was evaporated in vacuo, the concentrate was transferred for purification over the Sep-Pak® Plus C18 column with the mobile phases of 50% MeOH and 100% MeOH. The fraction containing 1 was evaporated and subjected to HRMS and NMR analyses.
4.7. Cytotoxic assay Mouse leukemia P388 cells were maintained in an RPMI1640 medium containing 10 units/mL of penicillin, 10 mg/mL streptomycin (Thermo Fischer Scientific K.K., Yokohama, Kanagawa, Japan), and 10% v/v FBS. Aliquots of the cells (200 mL) at 5.0 104 cells/mL were mixed with the sample (100 mg/mL). After 2 days, 4 mL of WST-8 (Dojin, Kumamoto, Japan) was added to the mixture for 4 h and the cell viability was evaluated by absorbance at 450 nm using a microplate reader (model 680, Bio-Rad Laboratories, Hercules, CA).
4.9. Brine shrimp lethal assay Brine shrimps (Japan Pet Design Co., Tokyo, Japan) were hatched in artificial seawater. The next day, 30 brine shrimps were transferred to each well of a 12-well microplate. Next, 5.0 mL of the sample having a concentration of 10 mg/mL was added to each well (50 mg/mL) and the suspension was incubated at room temperature for 2 days. The lethal activity was determined by counting the number of dead brine shrimps. 4.10. Lysiformine (1) Brown amorphous powder; [a]25 D 20.450 (c 0.01, MeOH); UV (MeOH) lmax (ε0) 220 (2.61 104), 282 (6.82 103), 317 (4.43 103), 377 (1.28 103) nm; IR (film) nmax 3734, 3628, 3309, 2927, 2855, 1716, 1698, 1457, 1435, 1244, 1173, 1101, 1064, 1031, 837, 743, 699, 650, 639 cm1; 1H NMR, see Table 1; 13C NMR, see Table 1; HRESIMS m/z 340.1459 (calcd for C22H17ON3, 340.1444).
4.11. Density functional theory (DFT) calculations The initial structure drawn on GaussView6 was optimized with DFT/B3LYP/3-21G in Gaussian 16. Conformational analysis was conducted with Semi-empirical MO/PM6 by rotating dihedral angles of 5e6e800 e300 and 6e800 e300 e3a00 every 30 , which simulated total 144 conformers. The most stable conformer was subjected to the second scanning with Semi-empirical MO/PM6 by rotating dihedral angles of 3e4e30 e3a0 and 2e3eOeH every 30 and 90 , respectively, which simulated total 48 conformers. The most stable conformer was optimized with DFT/B3LYP/6-31 þ G(d,p) followed by DFT/B3LYP/6-311 þ G(2d,p) to obtain the isotropic values. The isotropic values were converted to the simulated chemical shift based on a formula, Computed chemical shift ¼ (31.883-Isotropic value)/1.0553), for calculating the chemical shift of 1H NMR and another formula, Computed chemical shift ¼ (182.113-Isotropic value)/1.0469), for calculating the chemical shift of 13C NMR. These formulae were made by considering scaling factors and enable compensation of the difference between the simulated and observed chemical shifts of NMR spectra.26 In all the calculations, DMSO was used as the solvent. The charge and the spin multiplicity was zero and one, respectively, when considering the neutral form of lysiformine, whereas one and one, respectively, when considering the protonated form of lysiformine. Acknowledgments The present study was funded by JSPS KAKENHI (Grant Numbers JP17H02199, 21603003 and 24510291). The authors thank Prof.
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Shigefumi Kuwahara and Asst. Prof. Yusuke Ogura for measuring IR spectra and optical rotations. The authors are grateful for Asst. Prof. Kaoru Yamazaki Tohoku University in creating the input files for Gaussian 16. The DFT calculations in this research were conducted with supercomputing resources at Cyberscience Center, Tohoku University. We would like to thank Editage (www.editage.jp) for English language editing. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.tet.2018.05.049. References 1. Webster NS, Taylor MW. Environ Microbiol. 2012;14:335e346. 2. Vogel G. Science. 2008;320:1028e1030. 3. Tachibana K, Scheuer PJ, Tsukitani Y, et al. J Am Chem Soc. 1981;103: 2469e2471. 4. Bialojan C, Takai A. Biochem J. 1988;256:283e290. 5. Suganuma M, Fujiki H, Suguri H, et al. Proc Natl Acad Sci USA. 1988;85: 1768e1771. 6. Hirata Y, Uemura D. Pure Appl Chem. 1986;58:701e710. 7. Kuramoto M, Tong C, Yamada K, Chiba T, Hayashi Y, Uemura D. Tetrahedron Lett. 1996;37:3867e3870. 8. Amann RI, Ludwig W, Schleifer KH. Microbiol Rev. 1995;59:143e169. 9. Freeman MF, Gurgui C, Helf MJ, et al. Science. 2012;338:387e390. 10. Wakimoto T, Egami Y, Nakashima Y, et al. Nat Chem Biol. 2014;10, 648-U193. 11. Brady SF, Simmons L, Kim JH, Schmidt EW. Nat Prod Rep. 2009;26:1488e1503.
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