Kandenols A–E, Eudesmenes from an Endophytic Streptomyces sp. of

Note pubs.acs.org/jnp

Kandenols A−E, Eudesmenes from an Endophytic Streptomyces sp. of the Mangrove Tree Kandelia candel Ling Ding,† Armin Maier,‡ Heinz-Herbert Fiebig,‡ Wen-Han Lin,§ Gundela Peschel,† and Christian Hertweck*,†,⊥ †

Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Beutenbergstraße 11a, 07745 Jena, Germany ‡ Oncotest GmbH, Am Flughafen 12-14, 79108 Freiburg, Germany § State Key Laboratory of Natural and Biomimetic Drugs, Peking University, 100191 Beijing, People's Republic of China ⊥ Chair of Natural Product Chemistry, Friedrich Schiller University, 07737 Jena, Germany S Supporting Information *

ABSTRACT: Five novel eudesmene-type sesquiterpenes, kandenols A−E (1−5), have been isolated from Streptomyces sp. HKI0595 derived from the mangrove plant Kandelia candel. Their structures were established through NMR and mass spectrometry, and absolute configurations were established by the Mosher method and comparison of CD spectra with αrotunol and β-rotunol. The kandenols are reminiscent of various plant-derived eudesmenes, yet kandenols B and C are unusual because of their hydroperoxide moieties. Kandenol E is the first bacterial agarofuran, which belongs to an important group of antibiotics. Whereas the kandenols display no cytotoxicity against 12 human cell lines, weak to moderate antimicrobial activities were detected against Bacillus subtilis ATCC 6633 and Mycobacterium vaccae IMET 10670. recently identified the first example of bacterial indolosesquiterpenes,10,11 diverse ansa-macrolides named divergolides,12 and new germicidins that incorporate an unprecedented isobutylmalonyl extender unit.13 Here we report the discovery and characterization of five new bacterial terpene metabolites that are reminiscent of plant-like metabolites belonging to the families of eudesmenes and agarofurans. Streptomyces sp. HKI0595 was isolated as an endophyte from the stem of the mangrove plant Kandelia candel. HPLC-MS of culture extracts revealed a complex metabolic profile comprising several compounds according to preliminary deconvolution using the HKI Natural Product Database. The culture filtrate of a 200 L fermentation of Streptomyces sp. HKI0595 was passed through an XAD-161 M resin column, and the eluted mixture was subjected to sequential flash chromatography on silica gel, purification on Sephadex LH-20, and final purification by preparative reverse-phase HPLC. In this way, five new sesquiterpenes (Figure 1), named kandenol A (2.4 mg), kandenol B, (1.8 mg), kandenol C (4.3 mg), kandenol D (4.2 mg), and kandenol E (3.0 mg), were isolated. Kandenol A (1) has a molecular formula of C15H24O3 by HRESIMS. The 1H NMR spectrum indicated the presence of an olefinic proton (δ 5.83, H-3), one oxygenated methine (δ 4.23, H-8), and four methyl groups (δ 1.90, 1.31, 1.26, 1.05);

T

erpenoids, with over 55 000 isolated so far, represent the largest and most diverse class of natural products. They play crucial ecological roles, such as biological messengers as attractants, deterrents, antifeedants, and phytoalexins.1 Clinically, various terpenoids are used as therapeutics, such as the well-known anticancer drug diterpenoid paclitaxel and antimalarial sesquiterpene artemisinin.2,3 However, most terpenoids are eukaryotic secondary metabolites. Apart from geosmin, the terpene product responsible for the characteristic earthy smell of actinomycetales,4,5 the vast resource of bacterial terpene metabolites has been neglected until recently.6 With the advent of bacterial genomics, it has become more and more obvious that mevalonate- and non-mevalonate terpenoid pathways are ubiquitously encoded in Actinomycetes. 7 However, since bacterial terpene cyclases show substantial differences in overall primary amino acid sequences,8 when mining for the corresponding genes, it is often challenging to predict the exact terpene structures. Nonetheless, the genome mining results encourage the search for new bacterial terpenes,9 not only in the sequenced strains but also in wild-type isolates. However, due to the low yields, difficult detection, and complications in the separation process, most terpenes have been overlooked. Taking advantage of large-scale fermentation (>200 L), modern screening methods (HPLC/UV/HRMS), and advanced separation techniques, we were able to determine natural products produced in extremely low yields. For example, in the context of mangrove endophytes we have © XXXX American Chemical Society and American Society of Pharmacognosy

Received: June 1, 2012

A

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oxygenated carbon signal (δ 75.4, C-5) appeared in the 13C NMR spectrum. The location of this quaternary carbon at C-5 was established by HMBC data, where H-1, H-3, H-9, Me-14, and Me-15 show correlations to C-5 (Figure 2). The structure of 2 was finally established as 7-epi-5,8,11-trihydroxy-3eudesmen-2-one and named kandenol B. Selective NOESY experiments confirmed that both 1 and 2 share the same relative configuration, since irradiation at Me-15 caused no signal enhancement on H-8. HRESIMS and 13C NMR data of 3 revealed a molecular formula of C15H24O5, indicating the same number of doublebond equivalents, but one additional oxygen atom compared to kandenol B. The 13C NMR spectrum revealed three oxygenated carbon signals (δ 86.4, 75.4, 69.5), and the unusual downfield signal (δ 86.4, C-5) suggested the presence of a hydroperoxy substituent. The location of the hydroperoxyl group at C-5 was established by the HMBC correlations (Figure 2) between H-1, H-6, Me-14, and Me-15 and C-5. 3 was deduced to be 7-epi-5hydroperoxy-8,11-dihydroxy-3-eudesmen-2-one and named kandenol C. Kandenol D (4) has a molecular formula of C15H24O4, suggesting the same number of double-bond equivalents as in 3, but lacking one oxygen atom. By comparison of the 1HNMR spectra between 3 and 4, we noted that the oxygenated proton signal for H-8 was missing in 4. In the 13CNMR spectrum, two signals of oxygenated carbons were observed, resonating at δ 87.0 (C-5) and δ 72.7 (C-11). HMBC correlations (Figure 2) between H-1, H-3, H-6, Me-14, and Me-15 and C-5 confirmed that the position of the hydroperoxy group, which remained the same as in kandenol C. 4 was confirmed as 7-epi-5hydroperoxy-11-hydroxy-3-eudesmen-2-one, named kandenol D. The presence of the hydroperoxide group in both 3 and 4 was also supported by results from the MS/MS experiment (Supporting Information). Fragments corresponding to a loss of H2O followed by a loss of H2O2 were observed for both compounds. For kandenol E (5), the molecular formula of C15H22O3 was deduced from HRESIMS and 13C NMR data, which indicated an additional double-bond equivalent compared to 2. The 1H NMR spectrum exhibited a pattern similar to that for 2, indicating the presence of one olefinic proton (δ 5.90, H-3), one oxygenated methine (δ 4.20, H-8), and four methyl groups (δ 1.98, 1.36, 1.32, 1.20). Formation of an epoxy ring between C-5 and C-11 was proposed based on the number of oxygen atoms and double-bond equivalents. This was further supported by 13C NMR data, which showed signals from two oxygenated carbons (δ 84.7, C-5; δ 81.1, C-11), and all observed HMBC correlations (Figure 2) confirmed the proposed structure of 5 (7-epi-5,11-epoxy-8-hydroxy-3-eudesmen-2-one), which was named kandenol E. Kandenol E (5), which has a relatively rigid carbon skeleton, was chosen for a circular dichroism study. In the CD spectrum of 5 we observed a diagnostic positive Cotton effect at 328 nm (see Supporting Information), which is due to an n−π* excitation. According to the helicity rule,14 the absolute configurations of 5 was proposed to be 5R, 7S, 8S, 10R. Comparison of the CD spectra (Supporting Information) of αrotunol and β-rotunol (Figure 3), which were isolated from the Chinese medicinal plant Cyperus rorundus,15 confirmed that 5 and α-rotunol share the same overall configuration. Furthermore, Mosher’s method16 was applied to elucidate the absolute configuration of 1. Through analysis of the 1H

Figure 1. Chemical structures of compounds 1−5.

various other signals appeared in the aliphatic region. The 13C NMR spectrum exhibited 15 carbon signals, including signals for one carbonyl (δ 198.8, C-2), two olefinic carbons (δ 161.7, C-4; δ 126.7, C-3), two from oxygenated sp3 carbons (δ 74.8, C-11; δ 68.7, C-8), and 10 other aliphatic carbons. COSY correlations easily established a partial structure for 1 (Figure 2). An α,β-unsaturated ketone was inferred from the carbon data and confirmed by HMBC correlations (Figure 2).

Figure 2. COSY and key HMBC correlations for compounds 1−5.

A eudesmene-type skeleton was proposed on the basis of the HMBC correlations between H-1 and H-9 and C-15; H-5 and C-4 and C-10; and Me-12, Me-13 and C-7 and C-11 (Figure 2). The relative configuration was established by 2D NOESY experiments, revealing NOE effects between H-5 and H-8, and H-8 and H-12 (13). On the basis of these data, 1 was deduced to be 7-epi-8,11-dihydroxy-3-eudesmen-2-one and named kandenol A. According to the HRESIMS data, compound 2 has a molecular formula of C15H24O4 and thus differs from 1 by one additional oxygen atom. The 1H NMR spectrum of 2 displayed a similar pattern to 1, showing signals for one olefinic proton (δ 5.80, H-3), one oxygenated methine (δ 4.04, H-8), and four methyl groups (δ 1.98, 1.26, 1.23, 1.01). One additional B

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As to the biological context of the kandenols, it is remarkable that an endophytic bacterium produces plant-like metabolites. The observation that endophytes have the capability of biosynthesizing compounds with the same skeletons as their host plants29 raises the question whether pathways have coevolved and/or the corresponding genes were transferred horizontally between plants and endophytic microbes. Irrespective of the origin, the metabolites generated in the symbiosis provide a pool of compounds that could contribute to survival, e.g., by repelling herbivores and defeating plant pathogens. Likewise, the kandenols could serve as defensive substances for the bacterial producer and the host. To test this hypothesis, all kandenols were assayed for cytotoxic and antimicrobial activities. In an MIC test using three bacterial test strains (Table 2), kandenols were found to be active against the growth of Bacillus subtilis and Mycobacterium vaccae, albeit with only moderate activity. Kandenols showed no cytotoxicity against 12 human cell tumor lines, a prerequisite for developing kandenols into selective antimicrobial agents or other therapeutics.

Figure 3. Structurally related sesquiterpenes from plants and bacteria: α-Rotunol and β-rotunol from the Chinese medicinal plant Cyperus rorundus, selina-4(14),7(11)-diene-8,9-diol from a marine Streptomyces sp., 1,6,11-eudesmanetriol and 11-eudesmene-1,6-diol from a Drymaria diandra endophytic Streptomyces sp.

NMR, COSY, and NOESY spectra of both MTPA derivatives we were able to determine the absolute configuration of 1 as 5S, 7S, 8S, 10R (Figure 4). On the basis of biogenetic considerations, all kandenols are proposed to share the same skeleton. Thus, we could deduce the absolute configuration for all other kandenols.

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EXPERIMENTAL SECTION

General Experimental Procedures. NMR spectra were recorded on a Bruker Avance III 500 and a Bruker Avance III 600. IR spectra were recorded on a Bruker FT-IR (IFS 55) spectrometer. UV spectra were recorded on a Cary 1 Bio UV−visible spectrophotometer (Variant). Optical rotation was recorded on a Propol digital automatic polarimeter (Dr. Wolfgang Kernchen GmbH, Seelze, Germany), and CD spectra were recorded on a J-810-150s spectropolarimeter (JASCO, Groß Umstadt, Germany). ESIMS data were obtained on a triple quadrupole mass spectrometer (Quattro; VG Biotech, Cheshire, UK). HRESIMS were recorded on a Finnigan TSQ Quantum Ultra AM Thermo Electron. Open column chromatography was performed on silica gel 60 (Merck, 0.04−0.063 mm, 230−400 mesh ASTM) and Sephadex LH-20 (Pharmacia). TLC analysis was performed on silica gel plates (Sil G/UV254, 0.20 mm, Macherey-Nagel). Preparative HPLC was performed on a Waters HPLC system using a Nucleosil 100-5 C18 column (5 μm, 250 × 16 mm) with a UV detector. Strain Isolation and Taxonomic Classification. The stems of K. candel were collected in Xiamen, Fujian Province, People’s Republic of China, in June 2002 and authenticated by Prof. Peng Lin, Xia Men University, People’s Republic of China. A voucher sample of the plant is deposited in the National Research Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing, China (Mangrove 200208082). HKI0595 was isolated from the stems of the plant. It was deposited in the strain collection of the HKI, Jena, Germany. The nucleotide sequence of 16S DNA was sequenced and deposited under the accession number JQ937274 in GenBank at www.ncbi.nlm.nih.gov. Fermentation and Isolation. Liquid medium 79 (dextrose 10 g, bacto peptone 10 g, casamino acids 1 g, yeast extract 2 g, NaCl 6 g, H2O 1 L) (2 × 100 mL/flask) was inoculated with a suspension of mycelium and spores (about 1 × 1 cm) of Streptomyces sp. HKI0595 grown on agar slants or agar plates. After incubation for 48 h on a rotary shaker at 28 °C, the culture was transferred to 3200 mL of medium 27 (glucose 20 g, soybean powder 20 g, NaCl 5 g, CaCO3 3 g, H2O 1 L, eight 1 L-scale Erlenmeyer flasks with 400 mL of medium 27 each) and incubated at 28 °C under shaking conditions for 48 h to yield prefermentation culture, which was poured into a 300 L-scale fermenter filled with 200 L of medium 27 and fermented for 5 days. The fermentation broth of Streptomyces sp. HKI0595 was separated into culture filtrate and mycelia by filtration. The culture filtrate was extracted with ethyl acetate and evaporated to dryness. Separation of the culture filtrate (15 g) by flash silica gel chromatography (column 50 × 3 cm, CH2Cl2−CH3OH (0−50%) gradient, yielded fractions 1− 10. By RP-C18 column chromatography (MeOH−H2O as gradient), fraction 3 was fractionated into 13 subfractions. One of the subfractions containing 4 was fractionated by HPLC (RP-C18,

Figure 4. Elucidation of the absolute configuration of 1 using the modified Mosher method; Δδ values (ΔS − ΔR) obtained for (R)and (S)-MTPA esters of 1.

The structures of the kandenols are intriguing because they belong to the family of eudesmanes (and selinanes) that are secondary metabolites from a variety of plants, such as Inula japonica,17 Blumea balsamifera,18 and Nectandra cissif lora.19 Related bacterial natural products are extremely scarce. The only known examples are the recently described selina4(14),7(11)-diene-8,9-diol from a marine Streptomyces sp.20 and 1,6,11-eudesmanetriol and 11-eudesmene-1,6-diol from an endophytic Streptomyces sp. of Drymaria diandra21 (Figure 3). Together with these compounds, the kandenols represent the first selinane/eudesmane sesquiterpenes from bacteria. Kandenol E possesses a tricyclic backbone that is characteristic of agarofurans, an important group of antibiotics isolated from plants, especially the family Celastraceae.22 Agarofurans display a range of biologically activities; they have been shown to be cytotoxic,23 antibacterial,24 and anti-inflammatory.25 It should be noted that kandenol E represents the first agarofuran isolated from a bacterium. Furthermore, kandenols C and D possess hydroperoxide moieties, which are relatively rare in nature. Notably, peroxide groups may be essential for the biological activities in various drugs, such as antimalarial and antihelminthic terpenoids.26,27 Only a few natural hydroperoxyl-substituted terpenes have been reported, such as monoterpene hydroperoxides from Chenopodium ambrosioides27 and limonene hydroperoxides that are formed after oil gland injury in lemon fruits.28 Given the presence of the reactive hydroperoxide function, it is surprising that both kandenols C and D are stable at room temperature. C

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2.23, m 4.20, m 1.96, 1.53, AB (15.0)

1.32, 1.36, 1.98, 1.20,

51.5, CH 67.9, CH 41.7, CH2

40.4, 81.1, 30.3, 22.7, 19.7, 24.8,

C C CH3 CH3 CH3 CH3 s s d (1.4) s 1.16, 1.15, 2.16, 1.01, C C CH3 CH3 CH3 CH3 41.6, 72.7, 27.1, 26.9, 20.5, 23.6,

43.1, CH 22.8, CH2 36.1, CH2

s s d (1.4) s

2.49, 2.22, AB (11.7)

2.45, td (11.4, 2.6); 1.34, signal overlap 1.75, signal overlap 1.72, 1.37, m 1.89, ddd (13.6, 13.6, 4.0); 1.30, signal overlap

5.90, m

198.9, C 129.0, CH 155.8, C 84.7, C 27.4, CH2 5.75, m

201.4, C 126.4, CH 168.8, C 87.0, C 31.8, CH2

s s d (1.3) s 1.23, 1.26, 1.98, 1.01, 36.8, 74.8, 29.4, 23.9, 21.8, 22.5, 10 11 12 13 14 15

C C CH3 CH3 CH3 CH3

47.1, CH 68.7, CH 46.6, CH2 7 8 9

1.90, m 47.2, CH 4.23, m 68.n5, CH 2.02, dd (13.6, 6.9); 1.48, dd 43.4, CH2 (13.6, 8.5) 41.8, C 75.0, C 1.26, s 30.0, CH3 1.31, s 23.8, CH3 1.90, d (1.3) 18.9, CH3 1.05, s 22.7, CH3

1.87, dd (14.2, 3.7); 1.33, dd (13.8, 13.5) 1.93, m 4.04, ddd (10.9, 10.9, 4.6) 1.80, dd (13.4, 11.3); 1.62, dd (13.4, 4.6)

198.8, C 126.7, CH 161.7, C 41.0, CH 24.4, CH2 2 3 4 5 6

2.61, m 1.73, 1.63, m

compd

B. subtilis

M. vaccae

VRE

1 2 3 4 5 cip MeOH

50 50 >100 50 50 100

12.5 12.5 12.5 25 12.5 0.1 >100

>100 >100 >100 >100 >100 0.78 >100

MeOH−H2O as gradient) to afford pure 4 (4.2 mg). Using a Sephadex LH-20 column, fraction 6 was separated into four fractions, A−D. After separation of fraction A by RP-C18 column chromatography (MeOH−H2O), six subfractions (6a−6f) were obtained. Further separation of the subfractions by HPLC (RP-C18, MeOH−H2O) yielded the following substances: 1 (2.4 mg), 2 (1.8 mg), 3 (4.3 mg), and 5 (3.0 mg). Antimicrobial Test. An MIC test was carried out to test compounds 1−5 against three strains: Bacillus subtilis ATCC 6633, Mycobacterium vaccae IMET 10670, and Enterococcus faecalis 1528 R10. The assay was done by a standard broth microdilution method.30 Cytotoxicity Assay. A modified propidium iodide assay was used to determine the cytotoxic activities of compounds 1−5 against 12 cell lines derived from solid human tumors. The test procedure has been described elsewhere.31 Cell lines tested were derived from patient tumors engrafted as a subcutaneously growing tumor in NMRI nu/nu mice or obtained from the American Type Culture Collection, Rockville, MD, USA, National Cancer Institute, Bethesda, MD, USA, or Deutsche Sammlung von Mikroorganismen and Zellkulturen, Braunschweig, Germany. Inhibitory concentrations are provided as 50% inhibition of cell growth (absolute IC50, determined by two-pointcurve-fit after plotting compound concentration versus fluorescence intensity). Physicochemical Data of New Compounds. Kandenol A (1): colorless solid; [α]23 D 7.3 (c 0.91, MeOH); CD (c 0.20, MeOH) Δε241 2.45; UV (MeOH) λmax (log ε) 242 (3.50) nm; IR (film) νmax 3363, 2958, 2837, 1659, 1462, 1378, 1261, 1022, 901, 797, 723, 674, 661, 631 cm−1; ESIMS m/z [M + H]+ 253.1, [M + Na]+ 275.1, [2 M + Na]+ 527.2, [M − H]− 251.1, [2 M − H]− 503.3; HRESIMS m/z 253.1785 [M + H]+ (calcd for C15H25O3 253.1803); NMR data, see Table 1. Kandenol B (2): colorless solid; [α]23 D −36.2 (c 1.36, MeOH); CD (c 0.70, MeOH) Δε222 −3.77, Δε263 3.98; UV (MeOH) λmax (log ε) 237 (3.74) nm; IR (film) νmax 3351, 2963, 2930, 2855, 1652, 1440, 1413, 1375, 1279, 1227, 1161, 1120, 1073, 1056, 1042. 1013, 958, 903, 862, 769, 723, 691, 681, 674, 661, 645, 629 cm−1; ESIMS m/z [M + H]+ 269.1, [2 M + H]+ 537.2, [M + Na]+ 291.0, [2 M + Na]+ 559.2, [3 M + Na]+ 827.4; HRESIMS m/z 291.1540 [M + Na]+ (calcd for C15H24O4Na 291.1572); NMR data, see Table 1. Kandenol C (3): colorless solid; [α]23 D −16.5 (c 3.22, MeOH); CD (c 0.80, MeOH) Δε214 −5.42, Δε266 7.32; UV (MeOH) λmax (log ε) 235 (3.99) nm; IR (film) νmax 3263, 2971, 2936, 1651, 1443, 1378, 1280, 1227, 1158, 1053, 1015, 953, 899, 673, 630 cm−1; ESIMS m/z [M + Na]+ 307.0, [2 M + Na]+ 591.2, [3 M + Na]+ 875.4; HRESIMS m/z 307.1540 [M + Na]+ (calcd for C15H24O5Na 307.1521); NMR data, see Table 1. Kandenol D (4): colorless solid; [α]23 D −12.4 (c 2.36, MeOH); CD (c 1.10, MeOH) Δε212 −1.79, Δε265 9.98; UV (MeOH) λmax (log ε) 236 (3.92) nm; IR (film) νmax 3202, 2969, 2927, 2531, 1658, 1444, 1409, 1376, 1342, 1314, 1286, 1263, 1229, 1211, 1124, 1036, 975, 954, 928, 879, 804, 756, 668 cm−1; ESIMS m/z [M + H]+ 269.2, [2 M + H]+ 537.3, [M + Na]+ 291.2, [2 M + Na]+ 559.3, [3 M + Na]+ 827.4;

200.3, C 127.0, CH 5.75, m 167.7, C 86.4, C 31.9, CH2 2.43, dd (14.4, 3.4); 1.34, dd (14.4, 14.4) 48.3, CH 1.94, m 69.5, CH 4.04, ddd (10.9, 10.9, 4.5) 45.3, CH2 1.90, dd (13.1, 11.2); 1.52, dd (13.2, 4.6) 43.5, C 75.4, C 29.2, CH3 1.23, s 24.3, CH3 1.22, s 20.6, CH3 2.15, d (1.3) 23.4, CH3 1.02, s 5.80, m

197.3, C 126.1, CH 164.9, C 75.4, C 35.1, CH2

δC, type 1

5.83, m

δH (J in Hz) 5

2.69, dd (15.3, 0.7); 1.95, d (15.3)

δC, type 5

49.3, CH2

δH (J in Hz) 4

2.88, d (17.0); 1.84, dd (17.0, 1.3)

δC, type 4

49.0, CH2 2.85, d (16.9); 2.00, dd (16.9, 1.3)

δH (J in Hz) 3 δC, type 3

50.0, CH2 2.65, d (17.0); 2.16, dd (17.0, 1.2)

δH (J in Hz) 2 δC, type 2

47.9, CH2

test strain

a B. subtilis: Bacillus subtilis ATCC 6633; M. vaccae: Mycobacterium vaccae IMET 10670; VRE: vancomycin-resistant Enterococcus faecalis 1528 R10; cip: ciprofloxacin.

54.9, CH2

δH (J in Hz) 1

Table 2. Antimicrobial Activity of Compounds 1−5 (MIC data, in μg/mL)a

1

2.37, d (15.9); 2.26, dd (15.9, 0.7)

Note

no.

Table 1. NMR Data of Compounds 1 (CDCl3), 2 (CDCl3), 3 (CD3OD), 4 (CD3OD), and 5 (CDCl3) (1H NMR, 600 MHz; 13C NMR 1, 4, 150 MHz; 2, 3, 5, 125 MHz)

Journal of Natural Products

D

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HRESIMS m/z 269.1745 [M + H]+ (calcd for C15H25O4 269.1752); NMR data, see Table 1. Kandenol E (5): colorless solid; [α]23 D 32.8 (c 1.02, MeOH); CD (c 1.01, MeOH) Δε260 −25.6, Δε328 10.4; UV (MeOH) λmax (log ε) 229 (3.85) nm; IR (film) νmax 3399, 2962, 1668, 1261, 1077, 1019, 798, 674, 631 cm−1; ESIMS m/z [M + H]+ 251.1, [M + Na]+ 273.1, [2 M + Na]+ 523.2, [2 M + H]+ 501.2; HRESIMS m/z 251.1637 [M + H]+ (calcd for C15H23O3 251.1647); NMR data, see Table 1. Preparation of (S)-MTPA Ester. (S)-MTPA ester of 1 (6): To a mixture of 1 (0.3 mg) and 4-(dimethylamino)pyridine (1.0 mg) in pyridine (400 μL) was added (R)-MTPACl (2.0 μL) at room temperature under an argon atmosphere for 12 h. The organic phase was evaporated to dryness and separated by analytical HPLC to afford the (S)-MTPA ester 6 (0.3 mg). Compound 6: ESIMS [M + H]+ 469.2, [M + Na]+ 491.1, [2 M + Na]+ 959.3; HRESIMS m/z [M + H]+ 469.2205 (calcd for C25H33F3O5, 469.2202). Characteristic 1H NMR data (CDCl3, 600 MHz): H-3 (δ 5.820), H-5 (δ 2.950), H-7 (δ 1.665), H-8 (δ 5.615), H-9 (δ 2.121, 1.697), H-12 (δ 1.320), H-13 (δ 1.455), H-14 (δ 0.791), H-15 (δ 1.871). Preparation of (R)-MTPA Ester. (R)-MTPA ester of 1 (7): according to the method to prepare the (S)-MTPA ester, the (R)MTPA ester of compound 1 was obtained as 7 (0.2 mg). Compound 7: ESIMS [M + H]+ 469.2, [M + Na]+ 491.1, [2 M + Na]+ 959.3; HRESIMS m/z [M + H]+ 469.2206 (calcd. for C25H33F3O5, 469.2202). Characteristic 1H NMR data (CDCl3, 600 MHz): H-3 (δ 5.822), H-5 (δ 2.950), H-7 (δ 1.815), H-8 (δ 5.591), H-9 (δ 2.054, 1.607), H-12 (δ 1.354), H-13 (δ 1.458), H-14 (δ 0.691), H-15 (δ 1.901).

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ASSOCIATED CONTENT

S Supporting Information *

NMR spectra of compounds 1−5. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: (+) 49-3641-5321100. Fax: (+) 49-3641-5320804. Email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Mrs. U. Valentin for screening and sample preparation, C. Heiden and M. Steinacker for fermentation and downstream processing, Mrs. H. Heinecke for HPLC analysis and preparation, Dr. F. A. Gollmick and Mrs. H. Heinecke for NMR measurements, Mrs. A. Perner for MS analyses, and Dr. M. Ramm and Mrs. C. Weigel for antimicrobial assays. We thank Mangrove Protect Association Tainan City for permission to use a Kandelia candel photograph for the TOC/abstract graphic. This project was financially supported by the BMBF (Intercommunicate).

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REFERENCES

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