Acetate-Derived Metabolites from the Brown Alga Lobophora

Article pubs.acs.org/jnp

Acetate-Derived Metabolites from the Brown Alga Lobophora variegata Adrián Gutiérrez-Cepeda,†,‡ José J. Fernández,*,† Manuel Norte,† Sofia Montalvaõ ,§ Paï vi Tammela,§ and María L. Souto*,† †

Institute for Bio-Organic Chemistry “Antonio González”, Center for Biomedical Research of the Canary Islands (CIBICAN), Department of Organic Chemistry, University of La Laguna, 38206 La Laguna, Tenerife, Spain § Centre for Drug Research, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, FI-00014 Helsinki, Finland S Supporting Information *

ABSTRACT: Seven new nonadecaketides (1−7), lobophorols A−C, lobophopyranones A and B, and lobophorones A and B, along with the first naturally occurring related metabolites (8−10), were isolated from specimens of Lobophora variegata collected from the Canary Islands. Their structures were determined by extensive spectroscopic methods. In addition, an insight into the biosynthesis of these compounds on the basis of the involvement of type III polyketide synthases is proposed. Lobophorol A (1) showed significant antibacterial activity against Staphylococcus aureus.

T

he brown alga Lobophora variegata J.V Lamoroux (Dictyotaceae) has been widely studied for its ecological role in nature, such as in the biosorption of heavy metal ions from aqueous solutions.1 Although there are comparatively few phytochemical studies on this alga, prior research has led to a number of structurally interesting natural products2 exhibiting significant biological activities including antibacterial (phenolic lipids),3 antiparasitic, antioxidant, antiprotozoal (sulfoquinovosyldiacylglycerols),4 HIV reverse transcriptase inhibition (polysaccharides),5 anticoagulant and anti-inflammatory properties (sulfated galactofucans),6 sub-micromolar antifungal activity, cytotoxicity to a variety of cancer cell lines,7 and actin filament disruption8 (macrolide: lobophoride). L. variegata is the most common species of algal beds in the Canary Islands. The Lobophora communities suffer remarkably low levels of microbial infection7 and appear to act as a buffer against sea urchin domination trends.9 In the course of exploring interactions between these macroscopic alga and sea urchins, we initiated a phytochemical study of extracts from L. variegata with the aim of searching for bioactive metabolites. Herein, we report the isolation and structure elucidation of 10 new polyketides (1−10). Although their structures are simple, several of these metabolites present unique structural features in natural products. Evaluation of the antibacterial activities of 1−3, 5−7, and 10 against Enterococcus faecalis, Escherichia coli, and Staphylococcus aureus is also reported.

Final purification by normal-phase HPLC gave compounds 1− 10. Lobophorol A (1) was isolated as optically active colorless crystals. HREIMS data revealed a molecular formula of C19H32O3 (m/z 308.2353 [M]+). Analysis of the 13C and HSQC NMR spectra of 1 showed the presence of two methyl, 12 methylene, one methine, and four nonprotonated carbons (Table 1). Among them, two carbonyls (δC 204.0 and 195.4) and two olefinic carbons (δC 159.7 and 139.6) were evident. The remaining one degree of unsaturation indicated that the structure contained one ring. The 1H NMR spectrum showed signals attributable to an oxymethine (δH 4.30 dddd, J = 4.1, 4.3, 7.4, and 8.9 Hz, H-6), three deshielded methylenes (δH 2.75 dd, J = 4.1, 16.2 Hz/2.52 dd, J = 8.9, 16.2 Hz, H2-5; 2.70 dd, J = 4.3, 17.9 Hz/2.48 dd, J = 7.4, 17.9 Hz, H2-7; 2.22 m, H29), two methyls (δH 2.34 s/0.88 t, J = 7.1 Hz), and partially overlapped aliphatic methylenes between δH 1.49 and 1.25 (Supporting Information, Figure S1A). From the molecular formula and 1H NMR integration data, 1 was shown to possess a saturated C11 side chain. Analysis of the COSY and HSQC spectra revealed the presence of one spin system comprising C5→C-7 linked through HMBC correlations to one of the carbonyl carbons at δC 195.4 (C-4) and to the olefinic carbon at δC 159.7 (C-8) (Figure S1B and C). In addition, the correlations observed in the HMBC spectrum from H2-5, the protons of the methyl ketone (H3-1, δH 2.34), and one of the aliphatic methylenes (H2-9) to the carbon at δC 139.6 (C-3) completed the structure. Attempts to elucidate the absolute configuration of C-6 using Mosher’s ester methodology10 either

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RESULTS AND DISCUSSION The marine alga L. variegata was collected by scuba divers at El Médano (Tenerife, Canary Islands). The CH2Cl2/MeOH (1:1, v/v) extract was subjected to purification using a series of chromatoghaphic steps over Sephadex LH-20 and silica gel. © XXXX American Chemical Society and American Society of Pharmacognosy

Received: May 8, 2015

A

DOI: 10.1021/acs.jnatprod.5b00415 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

methyl ketone unit, and a hydroxy group ortho to it, as well as 10 methylenes and a triplet methyl resonance, which were part of an undecyl aliphatic chain. These data enabled the structure of lobophorol B to be assigned as 2.14 HREIMS analysis of 3 revealed a molecular formula of C19H30O3 with five degrees of unsaturation. The NMR spectra of 3 were very similar to those of 2 (Table 1). The structure of 3, however, possessed an additional hydroxy group. This OH group was placed at C-6, which from its chemical shift (δC 160.3) was clearly an oxygenated aromatic carbon atom. This was evidenced by HMBC correlations from H-5 to C-4, C-6, and C-7. Compound 3 was named lobophorol C, and its structure is closely related to 2,4-dihydroxy-6-alkylacetophenones isolated from the plant Knema glomerata15 and the bacterium Azobacter vinelandii,16 only differing in the alkyl chain lengths. Lobophopyranone A (4), a yellow oil, was shown by HREIMS to have a C19H34O2 molecular formula, indicating three degrees of unsaturation. 13C NMR analysis revealed all 19 resonances including one carbonyl and two olefinic carbons, accounting for two degrees of unsaturation. Hence, compound 4 possesses a single ring. IR and UV spectroscopy showed bands attributed to pyrone (1685 and 1613 cm−1; λmax 264 nm) and ether functions (1126 cm−1). The 1H and 13C NMR signals [H3-1 (δH 1.99 s), H-3 (δH 5.30 s), H2-5 (δH 2.40 dd, J = 12.6, 16.6 Hz/2.35 ddd, J = 0.8, 4.6, 16.6 Hz), and H-6 (δH 4.35 dddd, J = 4.6, 5.1, 7.4, 12.6 Hz); δC 193.1 (C), 174.4 (C), 104.7 (CH), 79.3 (CH), 40.8 (CH2), and 21.1 (CH3)] (Tables 2 and 3) suggested that 4 was a 2,3-dihydro-γ-pyranone, disubstituted by a vinylic methyl group and a long aliphatic chain having 13 carbons, which was further confirmed by 2D NMR data analysis (Figure S3). In addition, the orientation of the side chain was deduced to be equatorial because of the diaxial relationship (J = 12.6 Hz) between H-5ax at δH 2.40 and H-6. The absolute configuration of lobophopyranone A (4) was established through comparison with related (+) and (−) synthetic dihydropyrones.17,18 Compound 4 showed a negative [α]D; thus, an S configuration could be suggested for this metabolite. Compound 5 was obtained as a white, amorphous solid with [α]25D +33 (c 0.20, CHCl3). The spectroscopic data of lobophopyranone B (5) (Tables 2 and 3) were extremely similar to those of 4. However, the HREIMS signal at m/z 310.2512 indicated that 5 had the molecular formula C19H34O3, and the IR absorption at 3421 cm−1 supported the presence of an additional hydroxy group. Furthermore, a characteristic fragment for loss of water was observed at m/z 293 [MH − H2O]+, as well as the presence of a 1H NMR signal at δH 3.84 (m) assigned to an oxygenated methine at C-8 instead of the methylene group that is in 4. Correlations observed in the COSY and HMBC spectra supported the proposed structure (Figure S4). This alcohol moiety was estimated to be in the syn configuration, as evidenced by 3JH,H coupling constants.19,20 For instance, the coupling constants between H-6 and H2-7 were 5.2 and 7.3 Hz. The absolute configuration of 5 was therefore proposed to be 6S and 8S. Lobophorone A (6) was shown by HREIMS to have a molecular formula of C19H32O2, consistent with a molecular ion peak at m/z 292.2411. Its IR spectrum indicated the presence of an α,β-conjugated ketone functionality (1658 cm−1). Analysis of the 13C and HSQC spectra of 6 revealed one carbonyl, four olefinic resonances, one oxygenated methine, 11 methylenes, and two methyls (Table 3). Moreover, three deshielded vinyl protons at δH 7.50 (br dd, J = 2.7, 10.1 Hz),

failed or led to ring aromatization. Thus, only a tentative proposal for the absolute configuration of metabolite 1 as 6S can be suggested. This assignment is based on the reports in the literature where side chain modifications in model synthetic compounds produced a change in the magnitude but not the sign of the specific rotation.11−13 Lobophorol B (2) was isolated as a light yellow oil. HREIMS analysis of 2 gave a molecular ion at m/z 290.2234 consistent with a molecular formula of C19H30O2 and five degrees of unsaturation. The 1H NMR and COSY spectra showed signals attributable to three adjacent aromatic protons at δH 7.28 (dd, J = 7.8 and 8.1 Hz), 6.80 (dd, J = 1.1 and 8.1 Hz), and 6.75 (dd, J = 1.1 and 7.8 Hz), indicating together with 13C NMR and HMBC data the presence of a trisubstituted phenyl moiety (Table 1, Figure S2). Furthermore, similar to the previously described compound 1, the molecule was found to contain a B



Article

Table 1. 1H and 13C NMR Data (600 MHz, CDCl3) for Compounds 1−3 lobophorol A (1) position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 a

δC, type 31.9, 204.0, 139.6, 195.4, 46.5,

CH3 C C C CH2

65.9, CH 38.5, CH2 159.7, 35.4, 27.9, 29.6, 29.4, 29.6, 29.7, 29.4, 29.4, 31.6, 22.7, 14.1,

C CH2 CH2 CH2 CH2 CH2a CH2a CH2a CH2a CH2 CH2 CH3

lobophorol B (2)

δH, mult. (J in Hz) 2.34, s

b 2.75, dd (4.1, 16.2) a 2.52, dd (8.9, 16.2) 4.30, dddd (4.1, 4.3, 7.4, 8.9) b 2.70, dd (4.3, 17.9) a 2.48, dd (7.4, 17.9) 2.22, 1.49, 1.31, 1.29, 1.28, 1.28, 1.28, 1.28, 1.25, 1.28, 0.88,

m m m m m m m m m m t (7.1)

δC, type 32.4, 206.3, 122.1, 161.0, 116.0,

δH, mult. (J in Hz)

CH3 C C C CH

2.67, s

lobophorol C (3) δC, type

6.80, dd (1.1, 8.1)

25.1, 207.1, 116.1, 166.2, 101.8,

134.2, CH 122.0, CH

7.28, dd (7.8, 8.1) 6.75, dd (1.1, 7.8)

160.3, C 111.7, CH

144.2, 35.8, 32.5, 29.7, 29.3, 29.6, 29.6, 29.4, 29.5, 31.9, 22.7, 14.1,

2.85, 1.59, 1.37, 1.28, 1.28, 1.28, 1.28, 1.28, 1.26, 1.29, 0.88,

142.1, 44.1, 25.1, 29.4, 29.5, 29.5, 29.6, 29.5, 29.5, 31.9, 22.7, 14.1,


dd (7.8, 8.0) m m m m m m m m m t (7.1)


CH3 C C C CH

6.25, br s

6.22, br s


2.56, s 1.28, 1.27, 1.27, 1.27, 1.27, 1.25, 1.28, 0.88,

m m m m m m m t (7.1)

Carbons may be interchanged.

Table 2. 1H NMR Data (600 MHz, CDCl3) for Compounds 4−7 lobophopyranone A (4)

lobophopyranone B (5)

lobophorone A (6)

lobophorone B (7)

position



1 3 5

2.00, s 5.34, s (ax) 2.46, dd (12.1, 16.7) (eq) 2.44, br dd (5.4, 16.7) 4.63, dddd (5.2, 5.4, 7.3, 12.1) 1.99, ddd (7.3, 8.1, 14.6) 1.79, ddd (3.3, 5.2, 14.6) 3.84, m

2.15, s 5.65, s 7.50, br dd (2.7, 10.1)

2.40, s 5.07, s 6.02, br dd (2.3, 9.7)

6.40, 2.29, 2.23, 3.97,

br ddd (2.7, 6.2, 10.1) ddd (3.9, 6.2, 18.2) dddd (2.7, 2.7, 10.9, 18.2) dddd (3.9, 4.6, 6.6, 10.9)

6.30, 2.32, 2.25, 4.05,

ddd (2.4, 6.3, 9.7) ddd (3.8, 6.3, 18.2) dddd (2.3, 2.4, 10.7, 18.2) dddd (3.8, 4.4, 7.2, 10.7)

9

1.99, s 5.30, s (ax) 2.40, dd (12.6, 16.6) (eq) 2.35, ddd (0.8, 4.6, 16.6) 4.35, dddd (4.6, 5.1, 7.4, 12.6) 1.79, m 1.64, m 1.45, m 1.38, m 1.33, m

1.50, m

10

1.29, m

11 12 13 14 15 16 17 18 19

1.30, 1.28, 1.28, 1.28, 1.28, 1.28, 1.24, 1.29, 0.88,

1.42, 1.33, 1.29, 1.28, 1.28, 1.28, 1.28, 1.28, 1.26, 1.29, 0.88,

1.70, 1.56, 1.46, 1.35, 1.32, 1.28, 1.28, 1.28, 1.28, 1.28, 1.26, 1.29, 0.88,

m m m m m m m m m m m m t (7.1)

1.80, 1.63, 1.55, 1.43, 1.32, 1.27, 1.27, 1.27, 1.27, 1.27, 1.26, 1.29, 0.88,


6 7 8

m m m m m m m m t (7.1)


m m m m m m m m m m t (7.1)

6.40 (br ddd, J = 2.7, 6.2, 10.1 Hz), and 5.65 (s), an oxymethine (δH 3.97 dddd, J = 3.9, 4.6, 6.6, 10.9 Hz), an allylic methylene (δH 2.29 ddd, J = 3.9, 6.2, 18.2 Hz/2.23 dddd, J = 2.7, 2.7, 10.9, 18.2 Hz), and one methyl singlet (δH 2.15 s) revealed the structure of 6 to be bearing an unusual 2-propanone-dihydro2H-pyran-2-ylidene nucleus versus the γ-pyranones of compounds 4 and 5. This was confirmed by the analysis of the COSY and HMBC spectra of 6 (Figure S5). To complete the structural assignment of 6, we deduced the presence of one


undecyl aliphatic chain at C-8 based on 10 methylenes and a triplet methyl upfield resonance and a characteristic fragment ion observed at m/z 137.0575 [M+ − C11H23]. Based on the similarity of IR, UV, and 1H and 13C NMR of 6 and 7 (Tables 2 and 3), we could deduce that lobophorone B (7) differed from 6 only in the geometry of the C-3 olefin. This double bond was assigned to have the E configuration in 6 and the Z configuration in 7 according to the chemical shifts of H-5, H-3, and H3-1 (δH 7.50, 5.65, and 2.15 in 6 vs δH 6.02, 5.07, C



Article

Table 3. 13C NMR Data (150 MHz, CDCl3) for Compounds 4−7 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 a

4

5

6

7

δC, type

δC, type

δC, type

δC, type

21.1, 174.4, 104.7, 193.1, 40.8, 79.3, 34.4, 24.8, 29.9, 29.3, 29.4, 29.5, 29.6, 29.6, 29.6, 29.6, 31.9, 22.7, 14.1,

CH3 C CH C CH2 CH CH2 CH2 CH2 CH2 CH2 CH2a CH2a CH2a CH2a CH2a CH2 CH2 CH3

21.1, 173.7, 105.2, 192.5, 41.0, 78.5, 41.6, 69.6, 37.7, 25.4, 29.3, 29.6, 29.6, 29.6, 29.6, 29.3, 31.9, 22.7, 14.1,

CH3 C CH C CH2 CH CH2 CH CH2 CH2 CH2 CH2a CH2a CH2a CH2a CH2a CH2 CH2 CH3

31.9, 197.8, 104.4, 162.8, 121.9, 135.7, 30.2, 75.5, 35.0, 25.0, 29.5, 29.6, 29.7, 29.7, 29.7, 29.3, 32.0, 22.7, 14.1,

CH3 C CH C CH CH CH2 CH CH2 CH2 CH2 CH2a CH2a CH2a CH2a CH2a CH2 CH2 CH3

31.5, 198.4, 108.9, 159.9, 124.9, 133.7, 29.7, 76.1, 35.1, 25.3, 29.4, 29.5, 29.6, 29.6, 29.6, 29.4, 31.9, 22.7, 14.1,

Table 4. 1H NMR Data (600 MHz, CDCl3) for Compounds 8−10

CH3 C CH C CH CH CH2 CH CH2 CH2 CH2 CH2a CH2a CH2a CH2a CH2a CH2 CH2 CH3

position 1 3

lobophorone C (8)

lobophorone D (9)

lobophorone E (10 enol)

lobophorone E (10 keto)





2.05, s 5.49, s

2.24, s 3.57, br s

2.26, dd (7.3, 7.4)

2.49, dd (7.4, 7.5)

1.58, m

a

1.33, m 1.30, m

a

1.26, 1.30, 1.27, 5.39, 5.35, 2.82, 5.33, 5.37, 2.06, 2.02, 1.28, 1.31, 0.88,

a

4

2.18, s 2.63, dd (2.4, 17.7) 2.53, dd (9.3, 17.7) 4.03, m

5

1.50, m 1.39, m

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 OH-2 OH-4

Carbons may be interchanged.

and 2.40 in 7). This contention was confirmed by the ROESY cross-peak observed between H-3 and H-5 for compound 7. It is noteworthy that these metabolites contain a structure without precedent in natural compounds. A series of three linear methyl ketones, lobophorones C−E (8−10), consisting of C15, C17, and C21 alkyl chains, respectively, were also isolated from L. variegata. Compound 8, C15H30O2, exhibited spectroscopic properties (IR absorptions: 1710 and 3329 cm−1, NMR data: δC 209.9 (C), 67.4 (CH), 30.5 (CH3) and δH 4.03 m, 2.18 s), characteristic of a methyl ketone and a secondary alcohol. The 1H and 13C NMR chemical shifts (Tables 4 and 5) were assigned using COSY, HSQC, and HMBC spectra (Figure S6). Thus, the chemical structure of 8 was determined to be 4-hydroxypentadecan-2one. The absolute configuration of dextrorotatory or levorotatory linear hydroxy ketones is known from the literature.21−23 Thus, the S configuration was assigned to lobophorone C (8). The HRESIMS data of lobophorone D (9) resulted in a molecular formula of C17H32O2. The 1H and 13C NMR spectra (Tables 4 and 5) showed characteristic resonances for an α,β conjugated methyl ketone [δH/δC, 6.85 ddd, J = 7.2, 7.6, 16.0 Hz/144.4 (CH-4), 6.15 d, J = 16.0 Hz/133.6 (CH-3), 2.27 s/ 27.0 (CH3-1), and 198.6 (C-2)] and a secondary alcohol [δH/ δC 3.78 m/70.7 (CH-6)], including a large 1H NMR coupling between the olefinic protons (3JH‑3,H‑4 = 16.0 Hz) indicating a trans configuration around the double bond. Correlations observed in the COSY and HMBC spectra (Figure S7) supported the proposed structure as being (E)-6-hydroxyheptadec-3-en-2-one. Again, the S configuration of dextrorotatory compound 9 was assigned by analogy to synthetic δ-hydroxy enones.24 Preliminary spectroscopic (IR, NMR, MS) analysis indicated a long diunsaturated alkyl chain β-diketone nature for lobophorone E (10). From the 1H and 13C NMR spectra obtained for the isolated β-diketone 10, it was apparent that there were contributions corresponding to both the enol and

a

1.41, 1.30, 1.26, 1.28,

m m m m

1.27, 1.27, 1.27, 1.27, 1.26, 1.29, 0.88,

m m m m m m t (7.1)

2.95, d (3.4)

2.27, s 6.15, d (16.0)

6.85, ddd (7.2, 7.6, 16.0) 2.44, dddd (1.3, 4.3, 7.2, 14.5) 2.34, dddd (1.0, 7.3, 7.6, 14.5) 3.78, m 1.47, 1.42, 1.29, 1.29, 1.27, 1.27, 1.27, 1.27, 1.27, 1.25, 1.29, 0.88,

m m m m m m m m m m m t (7.1)


a

a a a a

2.78, m a a a a a a a

4.21

Signal overlapped by the resonance of the major tautomer.

keto tautomeric forms (Tables 4 and 5), existing predominantly in the enol form (86%) in CDCl3 solution. As can be seen in Table 4, the C-3 hydrogens for the keto tautomer had a chemical shift of δH 3.57, whereas the C-3 hydrogen in the enol tautomer is at δH 5.49. Additionally, the C-2, C-3, and C-4 carbon atoms for both keto and enol tautomeric forms were visible at δC 201.8, 57.9, 204.8 and 191.5, 99.7, 194.3, respectively (Table 5). Olefinic hydrogen signals corresponding to the unsaturated sites contained on the alkyl chain were also observed as multiplets at δH 5.39 (m), 5.37 (m), 5.35 (m), and 5.33 (m). The EI-HRMS spectra of compound 10 provided distinctive fragmentation ions that allowed the location of the double bonds in the alkyl chain to be established (Figure S8). The Z configurations of the Δ12 and Δ15 double bonds due to the overlapping signals were proposed by final comparison with the spectroscopic data of a series of synthetic long-chain βdiketones.25 The spectroscopic data of synthesized heineicos12,15-diene-2,4-dione were in agreement with spectroscopic data of the natural product 10.25 The biosynthetic origin of these metabolites can be rationalized by the participation of a nonacetate acyl-CoA starter unit involving type III polyketide synthases (PKSs), similar to what has been described by Horinouchi and coworkers in the context of phenolic lipid biosynthesis in the nitrogen-fixing soil bacterium Azotobacter vinelandii.26,27 On D



Article

Scheme 1. Suggested Biogenesis of New Metabolitesa

Table 5. 13C NMR Data (150 MHz, CDCl3) for Compounds 8−10 8a

9

position

δC, type

δC, type

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

30.5, 209.9, 49.7, 67.4, 36.1, 25.3, 29.8, 29.5, 29.6, 29.6, 29.6, 29.3, 31.7, 22.5, 14.1,

CH3 C CH2 CH CH2 CH2 CH2 CH2 CH2b CH2b CH2b CH2b CH2 CH2 CH3

27.0, 198.6, 133.6, 144.4, 40.4, 70.7, 37.4, 25.6, 29.6, 29.6, 29.6, 29.5, 29.3, 27.0, 31.9, 22.7, 14.1,

10 enol δC, type

CH3 C CH CH CH2 CH CH2 CH2 CH2 CH2b CH2b CH2b CH2b CH2b CH2 CH2 CH3

25.0, 191.5, 99.7, 194.3, 38.2, 25.7, 29.2, 29.3, 29.8, 29.7, 27.8, 130.2, 128.0, 25.6, 127.7, 130.0, 27.7, 29.6, 31.9, 22.7, 14.1,

10 keto δC, type

CH3 C CH C CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH CH CH2 CH CH CH2 CH2 CH2 CH2 CH3

30.9, 201.8, 57.9, 204.8, 43.8,

CH3 C CH2 C CH2

c c c c c c c c c c c c c c c c

a

Abbreviations: ARSs, alkyresorcinol synthases; MCoA, malonyl coenzyme A; R, alkyl chain.

δ C determined by HSQC and HMBC. Carbons may be interchanged. cSignal overlapped by the resonance of the major tautomer. a

b

Table 6. Antibacterial Activities of Compounds Isolated from Lobophora variegata at 100 μg/mL Concentrationa

this basis, it is possible to assume that dodecanoic acid functions as a common starter unit, and several extender units of malonyl-CoA are added to generate, with the participation of a type III PKS, the corresponding polyketide intermediates. In the case of the cyclic metabolites, the chain elongation is followed by cyclization of the linear intermediate in the same active-site cavity to generate the polyketide scaffolds.28 A comparison of the classical alkylresorcinols and alkylpyrones identified in plants, fungi, or bacteria with these metabolites isolated from L. variegata highlights an unusual mode of cyclization of the initial ring leading to the respective cyclohexenes or pyrones containing two intact C2 units and their adjacent side chains (Scheme 1).29 In order to reinforce this hypothesis and the existence of this predicted type III polyketide synthase in the genome of this brown alga, an exhaustive phylogenetic study is in progress. Primary antibacterial evaluation of compounds 1−3, 5−7, and 10 at 100 μg/mL concentration against Enterococcus faecalis, Escherichia coli, and Staphylococcus aureus was carried out (Table 6). Compounds 1 and 2 inhibited the growth of S. aureus by 100% and 65%, respectively. The minimum inhibitory concentration (MIC90) of lobophorol A (1) against S. aureus was shown to be 25 μg/mL.

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compound lobophorol A (1) lobophorol B (2) lobophorol C (3) lobophopyranone B (5) lobophorone A (6) lobophorone B (7) lobophorone E (10)

Enterococcus faecalis (ATCC 29212) 23 −1.0 −9.8 23

± ± ± ±

6 8 2 2

21 ± 2 19 ± 2 −10 ± 3

Escherichia coli (ATCC 25922) 29 −3.3 −1.0 27

± ± ± ±

5 1 2 2

15 ± 14 25 ± 1 −2.4 ± 3

Staphylococcus aureus (ATCC 25923) 100 65 17 −8.5

± ± ± ±

1 2 10 2

−95 ± 2 −57 ± 49 −17 ± 7

The results are expressed as % inhibition (average ± SD, n = 3). Ciprofloxacin was used as positive control on every assay plate at minimum inhibitory concentration (MIC). The MICs against E. coli, E. faecalis, and S. aureus were 0.016, 1.0, and 0.5 μg/mL, respectively.

a

data were obtained on a Micromass Autospec spectrometer, and HRESIMS data on a Micromass LTC Premier XE system mass spectrometer. HPLC separations were carried out with an LKB 2248 system equipped with a photodiode array detector. TLC were performed on AL Si gel Merck 60 F254 plates with visualization by spraying with phosphomolybdic acid reagent (10% in EtOH) and heating. Biological Material. Specimens of Lobophora variegata were collected by scuba diving at depths of 6−30 m at El Médano (Tenerife, Canary Islands). A voucher specimen was deposited at Departamento de Biologiá Vegetal, Botánica, Universidad de La Laguna, Tenerife (TFC Phyc 14923). Extraction and Isolation. The thawed alga was extracted three times with CH2Cl2/MeOH (1:1, v/v) at room temperature. Extracts were combined and the solvent was removed in vacuo to yield a dark green, viscous oil (15.8 g). The extract was subjected to Sephadex LH20 size exclusion chromatography eluting with CHCl3/MeOH (1:1). Selected fractions exhibiting similar TLC profiles were rechromato-

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured in CHCl3 on a PerkinElmer 241 polarimeter by using a Na lamp. Ultraviolet−visible spectra were run as MeOH solutions on a Jasco V-560. IR spectra were recorded on a Bruker IFS55 spectrometer. NMR spectra were recorded on a Bruker Avance 600 instrument equipped with a 5 mm TCI inverse detection cryoprobe operating at 600/150 MHz (1H/13C nuclei). Chemical shifts were reported in ppm referenced to solvent signals (CDCl3: δH 7.26, δC 77.0). Standard Bruker NMR pulse sequences were utilized. HREIMS E



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graphed by medium-pressure chromatography using a Lobar LiChroprep Si 60 column with n-hexane/EtOAc (7:3). Final purifications were achieved on a μ-Porasil HPLC column, 10 μm, 19 × 150 mm, using multiple rounds (eluent n-hexane/EtOAc in different ratios), yielding compounds 1 (150.0 mg), 2 (80.0 mg), 3 (30.0 mg), 4 (15.0 mg), 5 (3.0 mg), 6 (4.2 mg), 7 (3.2 mg), 8 (7.0 mg), 9 (2.9 mg), and 10 (3.0 mg). Lobophorol A (1): colorless needles; mp 62−63 °C; [α]25D +17 (c 0.23, CHCl3); UV (MeOH) λmax (log ε) 236 (3.91) nm; IR (CHCl3) νmax 3502, 2917, 2851, 1698, 1654, 1620, 1467, 1372, 1060 cm−1; 1H and 13C NMR data (CDCl3), Table 1; HREIMS m/z 308.2353 [M]+ (calcd for C19H32O3, 308.2351). Lobophorol B (2): light yellow oil; UV (MeOH) λmax (log ε) 203 (3.91), 308 (3.47) nm; IR (CHCl3) νmax 3340, 2924, 1680, 1604, 1585, 1462, 1209, 780 cm−1; 1H and 13C NMR data (CDCl3), Table 1; HREIMS m/z 290.2234 [M]+ (calcd for C19H30O2, 290.2246). Lobophorol C (3): colorless needles; mp 79−80 °C; UV (MeOH) λmax (log ε) 220 (4.05), 279 (3.69) nm; IR (CHCl3) νmax 3367, 2928, 2857, 1619, 1461, 1379, 1262, 1170, 1062, 848 cm−1; 1H and 13C NMR data (CDCl3), Table 1; HREIMS m/z 306.2203 [M]+ (calcd for C19H30O3, 306.2195). Lobophopyranone A (4): yellow oil; [α]25D −116 (c 1.14, CHCl3); UV (MeOH) λmax (log ε) 264 (3.94) nm; IR (CHCl3) νmax 2926, 2855, 1685, 1613, 1463, 1399, 1241, 1126, 1034, 809 cm−1; 1H and 13 C NMR data (CDCl3), Tables 2 and 3; HREIMS m/z 294.2521 [M]+ (calcd for C19H34O2, 294.2559). Lobophopyranone B (5): white, amorphous solid; [α]25D +33 (c 0.20, CHCl3); UV (MeOH) λmax (log ε) 263 (3.96) nm; IR (CHCl3) νmax 3421, 2923, 2852, 1651, 1604, 1401, 1341, 1126, 753 cm−1; 1H and 13C NMR data (CDCl3), Tables 2 and 3; HREIMS m/z 310.2512 [M]+ (calcd for C19H34O3, 310.2508). Lobophorone A (6): white, amorphous solid; [α]25D +20 (c 0.37, CHCl3); UV (MeOH) λmax (log ε) 276 (3.39) nm; IR (CHCl3) νmax 2924, 2854, 1658, 1623, 1564, 1463, 1262, 1041, 823 cm−1; 1H and 13 C NMR data (CDCl3), Tables 2 and 3; HREIMS m/z 292.2411 [M]+ (calcd for C19H32O2, 292.2402). Lobophorone B (7): white, amorphous solid; [α]25D +23 (c 0.29, CHCl3); UV (MeOH) λmax (log ε) 268 (3.33) nm; IR (CHCl3) νmax 2924, 2854, 1717, 1649, 1463, 1260, 1080, 1043, 723 cm−1; 1H and 13 C NMR data (CDCl3), Tables 2 and 3; HREIMS m/z 292.2369 [M]+ (calcd for C19H32O2, 292.2402). Lobophorone C (8): white, amorphous solid; [α]25D +2 (c 0.57, CHCl3); UV (MeOH) λmax (log ε) 231 (3.30) nm; IR (CHCl3) νmax 3329, 3242, 2916, 1710, 1466, 1351, 1253, 1080, 890 cm−1; 1H and 13 C NMR data (CDCl3), Tables 4 and 5; HREIMS m/z 242.2252 [M]+ (calcd for C15H30O2, 242.2246). Lobophorone D (9): white, amorphous solid; [α]25D +3 (c 0.12, CHCl3); UV (MeOH) λmax (log ε) 224 (3.98) nm; IR (CHCl3) νmax 3398, 223, 1738, 1671, 1605, 1460, 1253, 1166, 1030, 979, 805 cm−1; 1 H and 13C NMR data (CDCl3), Tables 4 and 5; HRESIMS m/z 269.2472 [M]+ (calcd for C17H33O2, 269.2481); 291.2283 [M + Na]+ (calcd for C17H33O2Na, 291.2300). Lobophorone E (10): yellow, amorphous solid; UV (MeOH) λmax (log ε) 281 (3.40) nm; IR (CHCl3) νmax 3463, 2926, 2455, 1732, 1611, 1461, 1378, 1246, 1075, 958, 722 cm−1; 1H and 13C NMR data (CDCl3), Tables 4 and 5; HREIMS m/z 320.2699 [M]+ (calcd for C21H36O2, 320.2715). Antibacterial Evaluation. Antibacterial activities against Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922), and Staphylococcus aureus (ATCC 25923) were evaluated by using a broth microdilution assay in 96-well format following the CLSI guidelines.30 Bacterial stock cultures were maintained on Mueller-Hinton agar (BD) and used for preparing overnight-grown bacterial suspension cultures in Mueller-Hinton broth (MHB, BD) for the assays. Bacterial suspension (final inoculum of 5 × 105 colony-forming units (CFU)/ mL) was mixed in the assay plate with samples diluted into MHB. Plates were incubated at 37 °C for 24 h in a shaker, and absorbance at 620 nm was measured at 0 and 24 h time-points. Results were calculated by normalizing against untreated controls and expressed as

percent inhibition. Ciprofloxacin was used as a positive control (MIC90 values for E. faecalis, E. coli, and S. aureus were 1, 0.016, and 0.5 μg/ mL, respectively). Compounds were initially tested at a final concentration of 100 μg/mL (n = 3). Dose−response experiments were carried out for compound 1 against S. aureus using a dilution series from 100 to 1.6 μg/mL (n = 6). The MIC90 value was defined as the lowest concentration showing >90% inhibition.

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ASSOCIATED CONTENT

S Supporting Information *

These data included key 2D NMR correlations for compounds 1, 2, 4, 5, 6, 8, and 9, and key fragments of 10 in the EI mass spectrum and NMR spectra for the new compounds. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00415.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail (J. J. Fernández): [email protected]. *E-mail (M. L. Souto): [email protected]. Tel: +34 922318586. Fax: +34 922318571. Present Address ‡

Department of Chemistry, Chemistry Institute, Sciences Faculty, Autonomous University of Santo Domingo, University City, 1355 Santo Domingo, Dominican Republic.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants SAF2011-28883-C03-01 (the Spanish MINECO), EU FP7-KBBE-3-245137 (MAREX), and EU FP7-REGPOT-2012-CT2012-316137 (IMBRAIM) and by the Academy of Finland (PT, grant no. 277001). A.G.-C. acknowledges MAEC-AECID for a Doctoral Felloẃ ship. The authors thank M. Machin-Sá nchez (Departamento de Biologiá Vegetal, University of La Laguna) for the taxonomic classification and the picture of the alga.

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REFERENCES

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Acetate-Derived Metabolites from the Brown Alga Lobophora

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