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Bromocatechol conjugates from a Chinese marine red alga, Symphyocladia latiuscula
Phytochemistry 158 (2019) 20–25
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Bromocatechol conjugates from a Chinese marine red alga, Symphyocladia latiuscula
T
Xiuli Xua, Haijin Yanga,b, Zeinab G. Khalilc, Liyuan Yina, Xue Xiaoc, Angela A. Salimc, Fuhang Songb,∗∗, Robert J. Caponc,∗ a
School of Ocean Sciences, China University of Geosciences, Beijing, 100083, PR China CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, PR China c Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, 4072, Australia b
A R T I C LE I N FO
A B S T R A C T
Keywords: Symphyocladia latiuscula (Rhodomelaceae) Bromocatechol Bromophenol Red alga Natural products Quinone methide
This study describes an investigation into polybromocatechol conjugates isolated from a marine red alga, Symphyocladia latiuscula (Harvey) Yamada, collected from coastal waters off Qingdao, China. We report on the isolation and characterisation of eight undescribed aconitic acid conjugates, symphyocladins R-X, including a likely solvolysis artifact of symphyocladin S, and an undescribed furanoyl conjugate, symphyocladin Y. Structure elucidation was achieved by detailed spectroscopic analysis. A plausible biosynthetic pathway linking all these co-metabolites through a cascade of quinone methide additions is proposed.
1. Introduction Symphyocladia latiuscula (Harvey) Yamada (Rhodomelaceae) is a marine red alga primarily distributed along the coasts of Korea, Japan and northern China. In 1980, Japanese samples of S. latiuscula were first reported to produce disodium 2,3,6-tribromo-5-hydroxybenzyl 1′,4disulfate (Kurata and Amiya, 1980a) and bis(2,3,6-tribromo-4,5-dihydrobenzyl) ether (Kurata and Amiya, 1980b), with subsequent accounts of additional bromophenols from Korean (Choi et al., 2000) and Chinese (Duan et al., 2007; Liu et al., 2011; Wang et al., 2005; Xu et al., 2012a, 2009; 2012b, 2014; 2013a, 2013b) collections. Bromophenols reported from this algae have been reported to exhibit antibacterial (Kurata and Amiya, 1980b), antifungal (Xu et al., 2012), antiviral (Park et al., 2005), anticancer (Lee et al., 2007), free radical scavenging (Choi et al., 2000), aldose reductase inhibitory (Wang et al., 2005) and Taq DNA polymerase inhibitory (Jin et al., 2008) activities. This report describes our efforts to extend the discovery and characterisation of S. latiuscula chemical diversity, to include nine undescribed polybromocatechol conjugates (1–9). 2. Results and discussion An EtOH extract of a Chinese collection of S. latiuscula was subjected to solvent/solvent partitioning with EtOAc/H2O to generate an EtOAc
∗
soluble extract, followed by fractionation and HPLC-MS profiling to pre-concentrate and prioritize materials rich in polybrominated metabolites. Subsequent purification by reversed phase HPLC yielded nine metabolites that were identified by detailed spectroscopic analysis (Tables 1–4 and Supporting Information) as undescribed polybromophenol-aconitic acid conjugates, symphyocladins R–X (1–2, 4–8), a likely solvolysis artifact 3 of symphyocladin S, and an undescribed polybromophenol–furanoyl conjugate, symphyocladin Y (9). The structure elucidation of 1–9 is summarized below (see Fig. 1). HRESI(+)MS measurements suggested that 1 (C20H12Br6O10, Δmmu +0.7), 2 (C21H14Br6O10, Δmmu −1.1) and 3 (C23H18Br6O10, Δmmu −0.9) were homologues of the known metabolites, symphyocladin H (10) and I (11) (Xu et al., 2017), incorporating an additional tribromocatechol moiety. For example, comparison of the NMR (methanol-d4) data for symphyocladin R (1) (Figs. S2–3, Tables 1 and 4 & Table S1) with 11 revealed a common carbon skeleton featuring additional substitution at C-4 by a tribromocatechol moiety (C-1″ to C-7″). The regiochemistry of the latter was evidenced by diagnostic HMBC correlations, and COSY correlations from H-4 to H2-7″ (Fig. 2). A less sterically constrained E Δ2,3 configuration was assigned by consideration of the 13C NMR chemical shift for C-1 in 1 (δC 170.0) compared to 10 (Z Δ2,3 δC 166.5) and 11 (E Δ2,3 δC 170.3), and by the absence of a ROESY correlation between H-4 and H2-7'. Comparison of the NMR (methanol-d4) data for symphyocladin S (2) (Figs. S5–6, Tables 1 and 4
Corresponding author.; Corresponding author. E-mail addresses: [email protected] (F. Song), [email protected] (R.J. Capon).
∗∗
https://doi.org/10.1016/j.phytochem.2018.10.026 Received 8 February 2018; Received in revised form 10 October 2018; Accepted 26 October 2018 0031-9422/ © 2018 Elsevier Ltd. All rights reserved.
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124.5). The regiochemistry of the latter was apparent from chemical shift differences about H2-7'/C-7′ and H2-7″/C-7″ in 4 vs 5, and diagnostic HMBC correlations in 5 from H-2′ to C-7′, and from H2-7′ to C-2' (Fig. 5). Comparison of the NMR (methanol-d4) data for symphyocladin V (6) (Fig. S17, Tables 2 and 4 & Table S6) with 4 confirmed a common scaffold incorporating a H-2″ aromatic methane (Fig. 3, HMBC correlations from H-2″ to C-7″, and from H2-7″ to C-2″), with the only significant differences being attributed to conversion of the C-5 CO2Me in 4 to a C-5 CO2H in 6 (δC 175.6). HRESI(+)MS measurements suggested that 7 (C19H12Br6O8, Δmmu 0.0) was a decarboxy analogue of 1, while 8 (C21H15Br5O10, Δmmu −0.4) was isomeric with 4 and 5. By contrast, 9 (C12H7Br3O4, Δmmu −0.3) was deemed to be a simpler substituted tribromocatechol. Comparison of the NMR (acetone-d6) data for symphyocladin W (7) (Figs. S19–20, Tables 3 and 4 & Table S7) with 1 revealed differences readily attributed to loss of the C-1 CO2H moiety, as evidenced by the appearance of a H-2 olefinic methine (δH 5.52) exhibiting COSY correlations to a diastereotopic H2-7′, and HMBC correlations to C-3 and C4 (Fig. 4). A Z Δ2,3 configuration was assigned to 7 on the basis of ROESY correlations between H-2 and both of H-4 and H2-7″ (Fig. 4). Analysis of the NMR (acetone-d6) data for symphyocladin X (8) (Figs. S22–23, Tables 3 and 4 & Table S8) revealed a C-2 bromocatechol substituted C-1 to C-6 substructure in common with 11, including a C-5 CO2Me moiety and a Z Δ2,3 configuration. The former was confirmed by diagnostic HMBC correlations to C-5, while the latter was evident from ROESY correlations between H2-7′ to H2-4 (Fig. 4). Unlike all other symphyocladins, 8 incorporates a dibromocatechol residue (C-1′ to C7′) bearing a C-2′ to C-7″ fusion to a tribromocatechol residue (C-1″ to C-7″). The regiochemistry of this fusion was confirmed by ROESY correlations between H2-7″ and both H2-7′ and H2-4 (Fig. 4). Analysis of the NMR (acetone-d6) data for symphyocladin Y (9) (Figs. S25–26, Tables 3 and 4 & Table S9) revealed resonances attributed to a tribromocatechol residue (C-1′ to C-7′), in common with many other symphyocladins, fused via C-7′ to a 2-furanoyl (C-1 to C-5). The 2furanoyl substitution pattern was assigned by comparing experimental 13 C NMR chemical shifts with values calculated for alternate 2-furanoyl and 3-furanoyl configurations. The 2-furanoyl configuration was further confirmed by the observation of a single ROESY correlation between H-3 and H2-7' (Fig. 4), where the alternate 3-furanoyl configuration would be expected to exhibit correlations from H2-7′ to two furan methines. While the 6-OEt moiety observed in 3 is likely due to ethanolysis, induced during extraction and handling with EtOH, the absence of a comparable 6-OMe homologue supports the proposition that methanolysis artifacts were not produced, and that the 5-OMe substituted cometabolites 3–5 and 8 are natural products. Although 1–7 share a common sp3 chiral C-4 carbon, as all fail to exhibit measureable optical rotations, we conclude they are racemic. This assessment was consistent with the lack of a measureable CD spectra for 3 and 5 (Figs. S28–29). The racemic character of 1–7 could reflect a lack of stereoselectivity during biosynthesis (Fig. 5), or an inherent C-4 chemical reactivity and associated ease of epimerization. Structural similarities across 1–8 suggest a highly conserved biosynthesis. Building on this observation, we propose a plausible biosynthetic pathway linking 1–8 (Fig. 5). In this proposal, esters derived from E-aconitic acid form adducts with quinone methides generated from either 2,3,6-tribromo-4,5-dihydroxybenzyl alcohol or 3,6-tribromo-4,5-dihydroxybenzyl alcohol. These adducts can then undergo either; (i) addition to a quinone methide to generate 1–6 (highlighted in red in Fig. 4); or (ii) a C-1 decarboxylation mediated addition to a quinone methide to generate 7 (highlighted in blue in Fig. 4); or (iii) a 1,3-hydride shift double bond migration followed by addition to a quinone methide to generate 8 (highlighted in green in Fig. 4). Although 1–7 incorporate a single chiral sp3 center, as they do not exhibit measurable optical rotations, they are presumed to be racemic. The absence of double bond migrations during isolation and handling
Table 1 1 H NMR data (600 MHz) for compounds 1–3. position
1a δH, mult (J in Hz)
2a δH, mult (J in Hz)
3b δH, mult (J in Hz)
4 5-OCH3 6-OCH2CH3 6-OCH2CH3 7′a 7′b 7″a 7″b
3.92, dd (10.8, 3.6)
3.94, dd (10.8, 3.0) 3.69, s
4.79, d (18.0) 4.51, d (18.0) 3.82, dd (14.4, 3.6) 3.70, dd (14.4, 10.8)
4.80, d (18.0) 4.48, d (18.0) 3.83, dd (13.8, 3.0) 3.67, dd (13.8, 10.8)
4.05, 3.67, 4.31, 1.35, 4.86, 4.51, 3.90, 3.67,
a b
dd (10.8, 3.6) s q (7.2) t (7.2) d (18.0) d (18.0) dd (14.4, 3.6) dd (14.4, 10.8)
Methanol-d4. Acetone-d6.
Table 2 1 H NMR data (600 MHz) for compounds 4–6 in methanol-d4. position
4 δH, mult (J in Hz)
5 δH, mult (J in Hz)
6 δH, mult (J in Hz)
4 5-OCH3 2′ 7′a 7′b 2″ 7″a 7″b
3.99, dd (9.6, 5.4) 3.67, s
4.56, 3.75, 6.89, 4.30, 3.73,
3.95, m
4.50, 4.44, 6.88, 3.51, 2.99,
d (16.8) d (16.8) s dd (13.8, 5.4) dd (13.8, 9.6)
dd (10.2, 3.0) s s d (16.2) d (16.2)
3.85, dd (14.4, 3.0) 3.72, dd (14.4, 10.2)
4.49, 4.41, 6.93, 3.42, 3.04,
d (16.8) d (16.8) s m dd (13.8, 8.4)
Table 3 1 H NMR data (600 MHz) for compounds 7–9 in acetone-d6. position
7 δH, mult (J in Hz)
2 3 4 5 5-OCH3 7′a 7′b 7″a 7″b
5.52, dd (6.0, 4.8) 3.57, dd (9.6, 4.8)
4.62, 4.08, 3.71, 3.70,
dd (18.4, 6.0) dd (18.4, 4.8) m m
8 δH, mult (J in Hz)
9 δH, mult (J in Hz)
3.00, s
7.48, dd (3.6, 0.6) 6.73, dd (3.6, 1.2) 7.90, dd (1.2, 0.6)
3.60, s 4.02, d (2.5)
4.77, s
4.73, d (2.5)
& Table S2) with 1 revealed the only significant difference being the presence of a C-5 CO2Me moiety (δH 3.69), as evidenced by diagnostic HMBC correlations from H-4 and the CO2Me, to C-5. Likewise, comparison of the NMR (methanol-d4) data for 3 (Figs. S8–9, Tables 1 and 4 & Table S3) with 1 revealed the only significant difference being the presence of C-5 CO2Me (δH 3.67) and C-6 CO2Et (δH 4.31, q, OCH2CH3 and 1.35, t, OCH2CH3) moieties, confirmed by HMBC correlations to C5 and C-6 respectively (Fig. 2). The presence of C-6 CO2Et suggested that 3 is a likely ethanolysis artifact of symphyocladin S (2). HRESI(+)MS measurements confirmed 4 (C21H15Br5O10, Δmmu −0.9) and 5 (C21H15Br5O10, Δmmu −0.3) as monodebromo, and 6 (C20H13Br5O10, Δmmu +0.5) as a monodebromo hydrolysed analogue of 2. Comparison of the NMR (methanol-d4) data for symphyocladin T (4) (Figs. S11–12, Tables 2 and 4 & Table S4) with 2 confirmed a common scaffold incorporating an E Δ2,3 configuration, as evidenced by the 13C NMR chemical shift for C-1 (δC 170.0). The C-5 CO2Me regiochemistry was confirmed by HMBC correlations from H-4 and CO2Me to C-5. The only significant difference in the NMR data for 4 compared to 2 was attributed to the presence of a H-2″ aromatic methine (δH 6.88, s; δC 126.5), the regiochemistry of which was determined by diagnostic HMBC correlations from H-2″ to C-7″, and H2-7″ to C-2″ (Fig. 3). Comparison of the NMR (methanol-d4) data for symphyocladin U (5) (Figs. S14–15, Tables 2 and 4 & Table S5) with 4 confirmed a common scaffold incorporating an E Δ2,3 configuration (C1 δC 170.7), and a C-5 CO2Me, with the only significant differences being attributed to an alternate H-2′ aromatic methine (δH 6.89, s; δC 21
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Table 4 13 C NMR data (150 MHz) for compounds 1 - 9. position
1a δC, type
2a δC, type
3b δC, type
4a δC, type
5a δC, type
6a δC, type
7b δC, type
8b δC, type
9b δC, type
1 2 3 4 5 6 5-OCH3 6-OCH2CH3 6-OCH2CH3 1′ 2′ 3′ 4′ 5′ 6′ 7′ 1″ 2″ 3″ 4″ 5″ 6″ 7″
170.0, C 148.7, C 129.4, C 48.7, CH 176.0, C 170.0, C
169.9, C 149.1, C 129.1, C 48.8, CH 174.5, C 169.8, C 52.8, CH3
170.0, C 144.4, C 131.3, C 48.9, CH 174.2, C 170.1, C 52.7, CH3
170.7, C 144.6, C 135.3, C 47.9, CH 174.3, C 169.9, C 52.9, CH3
170.0, C 143.5, C 131.7, C 49.1, CH 175.6, C nd
145.7, C 130.4, C 51.1, CH 173.2, C 167.4, C
165.3, C 135.8, C 142.0, C 29.3, CH 168.3, C 165.8, C 52.8, CH3
183.9, 153.1, 118.3, 113.3, 148.0,
130.8, C 119.4, C 114.6, C 145.0, C 144.8, C 115.3, C 42.1, CH2 131.8, C 118.8, C 114.6, C 144.7, C 144.3, C 114.6, C 39.4, CH2
130.7, C 119.4, C 114.6, C 145.1, C 144.8, C 115.3, C 42.0, CH2 131.5, C 118.7, C 114.0, C 144.8, C 144.3, C 114.6, C 39.3, CH2
167.4, C 147.7, C 128.3, C 47.8, CH 172.4, C 166.7, C 52.4, CH3 61.6, CH2 14.5, CH3 130.5, C 119.0, C 113.9, C 144.2, C 143.9, C 114.7, C 41.4, CH2 131.1, C 118.4, C 113.4, C 143.9, C 143.5, C 114.0, C 39.2, CH2
130.3, C 119.2, C 114.2, C 145.4, C 145.1, C 115.1, C 41.4, CH2 131.8, C 126.5, C 109.9, C 143.7, C 144. 5, C 113.4, C 37.2, CH2
131.4, C 124.5, C 110.0, C 143.4, C 145.3, C 112.9, C 38.5, CH2 131.3, C 118.8, C 114.6, C 145.1, C 144.9, C 114.6, C 39.6, CH2
130.2, C 118.7, C nd nd nd 114.8, C 41.1, CH2 nd 126.1, C 109.6, C 143.4, C nd 113.0, C 36.8, C
132.8, C 117.4, C 113.7, C 144.1, C 144.0, C 113.2, C 40.6, CH2 131.9, C 118.3, C 114.0, C 144.0, C 143.9, C 114.0, C 39.3, CH2
131.8, C 125.7, C 116.7, C 144.1, C 144.0, C 115.1, C 32.4, CH2 132.6, C 118.7, C 114.4, C 143.4, C 144.3, C 114.6, C 43.6, CH2
128.4, C 118.4, C 113.5, C 144.5, C 143.9, C 114.1, C 48.8, CH2
a b
C C CH CH CH
Methanol-d4. Acetone-d6, nd – not detected.
Fig. 1. S. latiuscula compounds 1–9.
3. Conclusions
suggests this racemic character is a function of initial achiral adduct addition. The biosynthesis of 9 most likely proceeds via a similar adduct formation, but the origin of the furanoyl nucleophile remains unclear. The proposed biosynthetic relationship informs a possible biomimetic synthesis of 1–9. All compounds did not exhibit growth inhibitory activity (i.e. IC50 > 30 μM) against a panel of Gram-positive (Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633) and Gram-negative bacteria (Escherichia coli ATCC 11775, Pseudomonas aeruginosa ATCC 10145) or a fungus (Candida albicans ATCC 10231).
We report a series of undescribed polybromocatechol conjugates 1–9 isolated from a marine red alga, S. latiuscula, collected from coastal waters off Qingdao, China. These compounds feature new carbon skeletons and very likely share a common putative biosynthetic quinone methide precursor. These metabolites demonstrate the power of convergent biosynthesis, where simple biosynthetic precursors can assemble a multitude of structurally related natural products. Knowledge of the chemistry of S. latiuscula has the potential to inspire future biomimetic syntheses, opening up access to new chemical space. 22
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Fig. 2. Diagnostic 2D NMR (methanol-d4) correlations for symphyocladins R–S (1–2), solvolysis artifact 3, and structures for symphyocladins H (10) and I (11).
4. Experimental
performed using an Agilent 1100 series separations module equipped with Agilent 1100 series diode array and/or multiple wavelength detectors and Agilent 1100 series fraction collector, controlled using ChemStation Rev.B02.01 and Purify version A.1.2 software. All solvents used for chromatographic separation were purchased from Sigma Australia. All solvents were HPLC grade.
4.1. General experimental procedures Specific rotations ([α]D) were measured on Anton Paar MCP 200 Modular Circular Polarimeter (Austria) in a 100 × 2 mm cell at 22 °C. The CD spectra were recorded on Chirascan (Applied Photophysics, UK). NMR spectra were obtained on a Bruker Avance DRX600 or DRX500 spectrometers, in the solvents indicated and referenced to residual 1H and 13C signals in deuterated solvents. Electrospray ionization mass spectra (ESIMS) were acquired using an Agilent 1100 Series separations module equipped with an Agilent 1100 Series LC/MSD mass detector in both positive and negative ion modes. High-resolution ESIMS measurements were obtained on (i) a Bruker micrOTOF mass spectrometer by direct infusion in MeCN at 3 mL/min using sodium formate clusters as an internal calibrant, or (ii) an Agilent 6520 mass spectrometer equipped with an Agilent 1200 series separation module, with elution through an Agilent Eclipse XDB-C8 column (5 μm, 4.6 × 150 mm, 1 mL/min gradient elution from 10% MeCN/H2O to 100% MeCN with 0.05% isocratic formic acid, over 15 min). HPLC was
4.2. Plant material Symphyocladia latiuscula (Harvey) Yamada (Rhodomelaceae) was collected in May 2004, on the coast of Taipingjiao, Qingdao, Shandong Province, China. The specimen identification was verified by Dr. KuiShuang Shao (Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China). A voucher specimen (No. 2004X16) was deposited at the Herbarium of the Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China. 4.3. Extraction and isolation The air-dried red alga S. latiuscula (4.3 kg) was extracted with 95%
Fig. 3. Diagnostic 2D NMR (methanol-d4) correlations for symphyocladins T–V (4–6). 23
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EtOH/H2O (20 L × 3) at room temperature (72 h). After the solvent was removed under reduced pressure at < 40 °C, a dark residue (610 g) was obtained. The residue was partitioned between EtOAc (5 L) and H2O (5 L), and the EtOAc-soluble partition (320 g) was chromatographed over silica gel, eluting with a gradient of 0–100% Me2CO/petroleum ether to yield 85 fractions (A1–A85) (Scheme S1). Fraction A49 (845 mg) was further fractionated over Sephadex LH20 using CHCl3–MeOH (2:1) (5 mL/min) to afford 22 fractions (A49.1–A49.22). Fraction A49.12 (586.4 mg) was subjected to HPLC purification (Zorbax SB-C8, 5 μm, 250 × 9.4 mm column, 3.0 mL/min, gradient elution from 10 to 100% MeCN/H2O over 15 min, with isocratic 0.01% TFA modifier) to yield 7 (4.9 mg, tR 8.35 min, 0.001%). Fraction A54 (9486 mg) was further fractionated over Sephadex LH20 using CHCl3–MeOH (2:1) (5 mL/min) to afford 21 fractions (A54.1–A54.21). Fraction A54.8 (1054 mg) was further subjected to SPE purification (Bond elute C18, 5 g), eluted with a stepwise 10% gradient from 100% H2O to 100% MeOH to afford 11 fractions (A54.8.1–A54.8.11). Fraction A54.8.7 (283.8 mg) was further subjected to HPLC fractionation (Zorbax SB-C18, 5 μm, 250 × 9.4 mm column, 3.0 mL/min, gradient elution from 50 to 60% MeCN/H2O over 12 min, with isocratic 0.01% TFA modifier) to yield 3 (14.2 mg, tR 11.83 min, 0.004%) and 9 (4.9 mg, tR 8.53 min, 0.001%) Fraction A54.9 (968 mg) was subjected to SPE purification (Bond elute C18, 5 g), eluted with a stepwise 10% gradient from 100% H2O to 100% MeOH to afford 11 fractions (A54.9.1–A54.9.11). Fraction A.54.9.7 (217.2 mg) was further subjected to HPLC fractionation (Zorbax SB-C18, 5 μm, 250 × 9.4 mm column, 3.0 mL/min, gradient elution from 50 to 70% MeCN/H2O over
Fig. 4. Diagnostic 2D NMR (acetone-d6) correlations for symphyocladins W–Y (7–9).
Fig. 5. A plausible biosynthetic relationship linking 1–8. 24
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15 min, with isocratic 0.01% TFA modifier) to yield 8 (8.2 mg, tR 9.0 min, 0.002%). Fraction A54.14 (128.4 mg) was further subjected to HPLC fractionation (Zorbax Eclipse XDB-C8, 5 μm, 250 × 9.4 mm column, 3.0 mL/min, gradient elution from 35% MeCN/H2O over 17 min, with isocratic 0.01% TFA modifier) to yield 1 (1.3 mg, tR 8.23 min, 0.0004%), 2 (2.1 mg, tR 15.94 min, 0.0006%), 4 (1.8 mg, tR 12.40 min, 0.0004%), 5 (2.4 mg, tR 13.73 min, 0.0006%), and 6 (1.2 mg, tR 6.53 min, 0.0002%). [% yields were determined on a massto-mass basis against the EtOAc crude extract].
NMR (150 MHz, acetone-d6), see Table 4; HRESIMS m/z 450.7819 [M H]- (calcd for C12H678Br3O4, 450.7822). Acknowledgments This work was supported in part by the National Key Research and Development Program of China (2018YFC0311002, 2017YFD0201203, 2017YFC1601300), the Chinese Government Fundamental Research Funds for the Central Universities, and the University of Queensland, Institute for Molecular Bioscience.
4.3.1. Symphyocladin R (1) Light brown solid; [α]D25 0 (c 0.01, MeOH); 1H NMR (600 MHz, methanol-d4), see Table 1; 13C NMR (150 MHz, methanol-d4), see Table 4; HRESIMS m/z 914.5365 [M + Na]+ (calcd for C20H1278Br381Br3O10Na, 914.5362).
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.phytochem.2018.10.026. References
4.3.2. Symphyocladin S (2) Light brown solid; [α]D25 0 (c 0.01, MeOH); 1H NMR (600 MHz, methanol-d4), see Table 1; 13C NMR (150 MHz, methanol-d4), see Table 4; HRESIMS m/z 906.5688 [M + H]+ (calcd for C21H1578Br381Br3O10, 906.5699).
Choi, J.S., Park, H.J., Jung, H.A., Chung, H.Y., Jung, J.H., Choi, W.C., 2000. A cyclohexanonyl bromophenol from the red alga Symphyocladia latiuscula. J. Nat. Prod. 63, 1705–1706. https://doi.org/10.1021/np0002278. Duan, X.-J., Li, X.-M., Wang, B.-G., 2007. Highly brominated mono- and bis-phenols from the marine red alga Symphyocladia latiuscula with radical-scavenging activity. J. Nat. Prod. 70, 1210–1213. https://doi.org/10.1021/np070061b. Jin, H.J., Oh, M.Y., Jin, D.H., Hong, Y.K., 2008. Identification of a Taq DNA polymerase inhibitor from the red seaweed Symphyocladia latiuscula. J. Environ. Biol. 29, 475–478. Kurata, K., Amiya, T., 1980a. Disodium 2,3,6-tribromo-5-hydroxybenzyl 1′, 4-disulfate, a new bromophenol from the red alga, Symphyocladia latiuscula. Chem. Lett. 9, 279–280. https://doi.org/10.1246/cl.1980.279. Kurata, K., Amiya, T., 1980b. Bis(2,3,6-tribromo-4,5-dihydroxybenzyl) ether from the red alga, Symphyocladia latiuscula. Phytochemistry 19, 141–142. https://doi.org/10. 1016/0031-9422(80)85032-1. Lee, J.H., Park, S.E., Hossain, M.A., Kim, M.Y., Kim, M.N., Chung, H.Y., Choi, J.S., Yoo, Y.H., Kim, N.D., 2007. 2,3,6-tribromo-4,5-dihydroxybenzyl methyl ether induces growth inhibition and apoptosis in MCF-7 human breast cancer cells. Arch. Pharm. Res. 30, 1132–1137. Liu, X., Li, X., Gao, L., Cui, C., Li, C., Li, J., Wang, B., 2011. Extraction and PTP1B inhibitory activity of bromophenols from the marine red alga Symphyocladia latiuscula. Chin. J. Oceanol. Limnol. 29, 686–690. https://doi.org/10.1007/s00343-0110136-1. Park, H.J., Kurokawa, M., Shiraki, K., Nakamura, N., Choi, J.S., Hattori, M., 2005. Antiviral activity of the marine alga Symphyocladia latiuscula against herpes simplex virus (HSV-1) in vitro and its therapeutic efficacy against HSV-1 infection in mice. Biol. Pharm. Bull. 28, 2258–2262. Wang, W., Okada, Y., Shi, H., Wang, Y., Okuyama, T., 2005. Structures and aldose reductase inhibitory effects of bromophenols from the red alga Symphyocladia latiuscula. J. Nat. Prod. 68, 620–622. https://doi.org/10.1021/np040199j. Xu, X., Piggott, A.M., Yin, L., Capon, R.J., Song, F., 2012a. Symphyocladins A-G: bromophenol adducts from a Chinese marine red alga, Symphyocladia latiuscula. Tet. Lett. 53, 2103–2106. https://doi.org/10.1016/j.tetlet.2012.02.044. Xu, X., Song, F., Fan, X., Fang, N., Shi, J., 2009. A novel bromophenol from marine red alga Symphyocladia latiuscula. Chem. Nat. Compd. 45, 811–813. https://doi.org/10. 1007/s10600-010-9501-0. Xu, X., Yang, H., Khalil, Z.G., Yin, L., Xiao, X., Neupane, P., Bernhardt, P.V., Salim, A.A., Song, F., Capon, R.J., 2017. Chemical diversity from a Chinese marine red alga, Symphyocladia latiuscula. Mar. Drugs 15, 374. https://doi.org/10.3390/md15120374. Xu, X., Yin, L., Fang, N., Fan, X., Song, F., 2012b. Bromophenol coupled with diketopiperazine from marine red alga Symphyocladia latiuscula. Chem. Nat. Compd. 48, 622–624. https://doi.org/10.1007/s10600-012-0327-9. Xu, X., Yin, L., Gao, J., Gao, L., Song, F., 2014. Antifungal bromophenols from marine red alga Symphyocladia latiuscula. Chem. Biodivers. 11, 807–811. https://doi.org/10. 1002/cbdv.201300239. Xu, X., Yin, L., Gao, L., Gao, J., Chen, J., Li, J., Song, F., 2013a. Two new bromophenols with radical scavenging activity from marine red alga Symphyocladia latiuscula. Mar. Drugs 11, 842–847. https://doi.org/10.3390/md11030842. Xu, X., Yin, L., Wang, Y., Wang, S., Song, F., 2013b. A new bromobenzyl methyl sulphoxide from marine red alga Symphyocladia latiuscula. Nat. Prod. Res. 27, 723–726. https://doi.org/10.1080/14786419.2012.695362.
4.3.3. Likely solvolysis artifact (3) Light brown solid; [α]D25 0 (c 0.01, MeOH); 1H NMR (600 MHz, acetone-d6), see Table 1; 13C NMR (150 MHz, acetone-d6), see Table 4; HRESIMS m/z 934.6003 [M + H]+ (calcd for C23H1978Br381Br3O10, 934.6012). 4.3.4. Symphyocladin T (4) Light brown solid; [α]D25 0 (c 0.01, MeOH); 1H NMR (600 MHz, methanol-d4), see Table 2, 13C NMR (150 MHz, methanol-d4), see Table 4; HRESIMS m/z 826.6605 [M + H]+ (calcd for C21H1678Br281Br3O10, 826.6614). 4.3.5. Symphyocladin U (5) Light brown solid; [α]D25 0 (c 0.01, MeOH); 1H NMR (600 MHz, methanol-d4), see Table 2; 13C NMR (150 MHz, methanol-d4), see Table 4; HRESIMS m/z 826.6611 [M + H]+ (calcd for C21H1678Br281Br3O10, 826.6614). 4.3.6. Symphyocladin V (6) Light brown solid; [α]D25 0 (c 0.01, MeOH); 1H NMR (600 MHz, methanol-d4), see Table 2; 13C NMR (150 MHz, methanol-d4), see Table 4; HRESIMS m/z 812.6462 [M + H]+ (calcd for C20H1478Br281Br3O10, 812.6457). 4.3.7. Symphyocladin W (7) Light brown solid; [α]D25 0 (c 0.01, MeOH); 1H NMR (600 MHz, acetone-d6), see Table 3; 13C NMR (150 MHz, acetone-d6), see Table 4; HRESIMS m/z 848.5644 [M + H]+ (calcd for C19H1378Br381Br3O8, 848.5644). 4.3.8. Symphyocladin X (8) Light brown solid; 1H NMR (600 MHz, acetone-d6), see Table 3; 13C NMR (150 MHz, acetone-d6), see Table 4; HRESIMS m/z 826.6610 [M + H]+ (calcd for C21H1678Br281Br3O10, 826.6614). 4.3.9. Symphyocladin Y (9) Light brown solid; 1H NMR (600 MHz, acetone-d6), see Table 3; 13C
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Bromocatechol conjugates from a Chinese marine red alga, Symphyocladia latiuscula
T
Xiuli Xua, Haijin Yanga,b, Zeinab G. Khalilc, Liyuan Yina, Xue Xiaoc, Angela A. Salimc, Fuhang Songb,∗∗, Robert J. Caponc,∗ a
School of Ocean Sciences, China University of Geosciences, Beijing, 100083, PR China CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, PR China c Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, 4072, Australia b
A R T I C LE I N FO
A B S T R A C T
Keywords: Symphyocladia latiuscula (Rhodomelaceae) Bromocatechol Bromophenol Red alga Natural products Quinone methide
This study describes an investigation into polybromocatechol conjugates isolated from a marine red alga, Symphyocladia latiuscula (Harvey) Yamada, collected from coastal waters off Qingdao, China. We report on the isolation and characterisation of eight undescribed aconitic acid conjugates, symphyocladins R-X, including a likely solvolysis artifact of symphyocladin S, and an undescribed furanoyl conjugate, symphyocladin Y. Structure elucidation was achieved by detailed spectroscopic analysis. A plausible biosynthetic pathway linking all these co-metabolites through a cascade of quinone methide additions is proposed.
1. Introduction Symphyocladia latiuscula (Harvey) Yamada (Rhodomelaceae) is a marine red alga primarily distributed along the coasts of Korea, Japan and northern China. In 1980, Japanese samples of S. latiuscula were first reported to produce disodium 2,3,6-tribromo-5-hydroxybenzyl 1′,4disulfate (Kurata and Amiya, 1980a) and bis(2,3,6-tribromo-4,5-dihydrobenzyl) ether (Kurata and Amiya, 1980b), with subsequent accounts of additional bromophenols from Korean (Choi et al., 2000) and Chinese (Duan et al., 2007; Liu et al., 2011; Wang et al., 2005; Xu et al., 2012a, 2009; 2012b, 2014; 2013a, 2013b) collections. Bromophenols reported from this algae have been reported to exhibit antibacterial (Kurata and Amiya, 1980b), antifungal (Xu et al., 2012), antiviral (Park et al., 2005), anticancer (Lee et al., 2007), free radical scavenging (Choi et al., 2000), aldose reductase inhibitory (Wang et al., 2005) and Taq DNA polymerase inhibitory (Jin et al., 2008) activities. This report describes our efforts to extend the discovery and characterisation of S. latiuscula chemical diversity, to include nine undescribed polybromocatechol conjugates (1–9). 2. Results and discussion An EtOH extract of a Chinese collection of S. latiuscula was subjected to solvent/solvent partitioning with EtOAc/H2O to generate an EtOAc
∗
soluble extract, followed by fractionation and HPLC-MS profiling to pre-concentrate and prioritize materials rich in polybrominated metabolites. Subsequent purification by reversed phase HPLC yielded nine metabolites that were identified by detailed spectroscopic analysis (Tables 1–4 and Supporting Information) as undescribed polybromophenol-aconitic acid conjugates, symphyocladins R–X (1–2, 4–8), a likely solvolysis artifact 3 of symphyocladin S, and an undescribed polybromophenol–furanoyl conjugate, symphyocladin Y (9). The structure elucidation of 1–9 is summarized below (see Fig. 1). HRESI(+)MS measurements suggested that 1 (C20H12Br6O10, Δmmu +0.7), 2 (C21H14Br6O10, Δmmu −1.1) and 3 (C23H18Br6O10, Δmmu −0.9) were homologues of the known metabolites, symphyocladin H (10) and I (11) (Xu et al., 2017), incorporating an additional tribromocatechol moiety. For example, comparison of the NMR (methanol-d4) data for symphyocladin R (1) (Figs. S2–3, Tables 1 and 4 & Table S1) with 11 revealed a common carbon skeleton featuring additional substitution at C-4 by a tribromocatechol moiety (C-1″ to C-7″). The regiochemistry of the latter was evidenced by diagnostic HMBC correlations, and COSY correlations from H-4 to H2-7″ (Fig. 2). A less sterically constrained E Δ2,3 configuration was assigned by consideration of the 13C NMR chemical shift for C-1 in 1 (δC 170.0) compared to 10 (Z Δ2,3 δC 166.5) and 11 (E Δ2,3 δC 170.3), and by the absence of a ROESY correlation between H-4 and H2-7'. Comparison of the NMR (methanol-d4) data for symphyocladin S (2) (Figs. S5–6, Tables 1 and 4
Corresponding author.; Corresponding author. E-mail addresses: [email protected] (F. Song), [email protected] (R.J. Capon).
∗∗
https://doi.org/10.1016/j.phytochem.2018.10.026 Received 8 February 2018; Received in revised form 10 October 2018; Accepted 26 October 2018 0031-9422/ © 2018 Elsevier Ltd. All rights reserved.
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124.5). The regiochemistry of the latter was apparent from chemical shift differences about H2-7'/C-7′ and H2-7″/C-7″ in 4 vs 5, and diagnostic HMBC correlations in 5 from H-2′ to C-7′, and from H2-7′ to C-2' (Fig. 5). Comparison of the NMR (methanol-d4) data for symphyocladin V (6) (Fig. S17, Tables 2 and 4 & Table S6) with 4 confirmed a common scaffold incorporating a H-2″ aromatic methane (Fig. 3, HMBC correlations from H-2″ to C-7″, and from H2-7″ to C-2″), with the only significant differences being attributed to conversion of the C-5 CO2Me in 4 to a C-5 CO2H in 6 (δC 175.6). HRESI(+)MS measurements suggested that 7 (C19H12Br6O8, Δmmu 0.0) was a decarboxy analogue of 1, while 8 (C21H15Br5O10, Δmmu −0.4) was isomeric with 4 and 5. By contrast, 9 (C12H7Br3O4, Δmmu −0.3) was deemed to be a simpler substituted tribromocatechol. Comparison of the NMR (acetone-d6) data for symphyocladin W (7) (Figs. S19–20, Tables 3 and 4 & Table S7) with 1 revealed differences readily attributed to loss of the C-1 CO2H moiety, as evidenced by the appearance of a H-2 olefinic methine (δH 5.52) exhibiting COSY correlations to a diastereotopic H2-7′, and HMBC correlations to C-3 and C4 (Fig. 4). A Z Δ2,3 configuration was assigned to 7 on the basis of ROESY correlations between H-2 and both of H-4 and H2-7″ (Fig. 4). Analysis of the NMR (acetone-d6) data for symphyocladin X (8) (Figs. S22–23, Tables 3 and 4 & Table S8) revealed a C-2 bromocatechol substituted C-1 to C-6 substructure in common with 11, including a C-5 CO2Me moiety and a Z Δ2,3 configuration. The former was confirmed by diagnostic HMBC correlations to C-5, while the latter was evident from ROESY correlations between H2-7′ to H2-4 (Fig. 4). Unlike all other symphyocladins, 8 incorporates a dibromocatechol residue (C-1′ to C7′) bearing a C-2′ to C-7″ fusion to a tribromocatechol residue (C-1″ to C-7″). The regiochemistry of this fusion was confirmed by ROESY correlations between H2-7″ and both H2-7′ and H2-4 (Fig. 4). Analysis of the NMR (acetone-d6) data for symphyocladin Y (9) (Figs. S25–26, Tables 3 and 4 & Table S9) revealed resonances attributed to a tribromocatechol residue (C-1′ to C-7′), in common with many other symphyocladins, fused via C-7′ to a 2-furanoyl (C-1 to C-5). The 2furanoyl substitution pattern was assigned by comparing experimental 13 C NMR chemical shifts with values calculated for alternate 2-furanoyl and 3-furanoyl configurations. The 2-furanoyl configuration was further confirmed by the observation of a single ROESY correlation between H-3 and H2-7' (Fig. 4), where the alternate 3-furanoyl configuration would be expected to exhibit correlations from H2-7′ to two furan methines. While the 6-OEt moiety observed in 3 is likely due to ethanolysis, induced during extraction and handling with EtOH, the absence of a comparable 6-OMe homologue supports the proposition that methanolysis artifacts were not produced, and that the 5-OMe substituted cometabolites 3–5 and 8 are natural products. Although 1–7 share a common sp3 chiral C-4 carbon, as all fail to exhibit measureable optical rotations, we conclude they are racemic. This assessment was consistent with the lack of a measureable CD spectra for 3 and 5 (Figs. S28–29). The racemic character of 1–7 could reflect a lack of stereoselectivity during biosynthesis (Fig. 5), or an inherent C-4 chemical reactivity and associated ease of epimerization. Structural similarities across 1–8 suggest a highly conserved biosynthesis. Building on this observation, we propose a plausible biosynthetic pathway linking 1–8 (Fig. 5). In this proposal, esters derived from E-aconitic acid form adducts with quinone methides generated from either 2,3,6-tribromo-4,5-dihydroxybenzyl alcohol or 3,6-tribromo-4,5-dihydroxybenzyl alcohol. These adducts can then undergo either; (i) addition to a quinone methide to generate 1–6 (highlighted in red in Fig. 4); or (ii) a C-1 decarboxylation mediated addition to a quinone methide to generate 7 (highlighted in blue in Fig. 4); or (iii) a 1,3-hydride shift double bond migration followed by addition to a quinone methide to generate 8 (highlighted in green in Fig. 4). Although 1–7 incorporate a single chiral sp3 center, as they do not exhibit measurable optical rotations, they are presumed to be racemic. The absence of double bond migrations during isolation and handling
Table 1 1 H NMR data (600 MHz) for compounds 1–3. position
1a δH, mult (J in Hz)
2a δH, mult (J in Hz)
3b δH, mult (J in Hz)
4 5-OCH3 6-OCH2CH3 6-OCH2CH3 7′a 7′b 7″a 7″b
3.92, dd (10.8, 3.6)
3.94, dd (10.8, 3.0) 3.69, s
4.79, d (18.0) 4.51, d (18.0) 3.82, dd (14.4, 3.6) 3.70, dd (14.4, 10.8)
4.80, d (18.0) 4.48, d (18.0) 3.83, dd (13.8, 3.0) 3.67, dd (13.8, 10.8)
4.05, 3.67, 4.31, 1.35, 4.86, 4.51, 3.90, 3.67,
a b
dd (10.8, 3.6) s q (7.2) t (7.2) d (18.0) d (18.0) dd (14.4, 3.6) dd (14.4, 10.8)
Methanol-d4. Acetone-d6.
Table 2 1 H NMR data (600 MHz) for compounds 4–6 in methanol-d4. position
4 δH, mult (J in Hz)
5 δH, mult (J in Hz)
6 δH, mult (J in Hz)
4 5-OCH3 2′ 7′a 7′b 2″ 7″a 7″b
3.99, dd (9.6, 5.4) 3.67, s
4.56, 3.75, 6.89, 4.30, 3.73,
3.95, m
4.50, 4.44, 6.88, 3.51, 2.99,
d (16.8) d (16.8) s dd (13.8, 5.4) dd (13.8, 9.6)
dd (10.2, 3.0) s s d (16.2) d (16.2)
3.85, dd (14.4, 3.0) 3.72, dd (14.4, 10.2)
4.49, 4.41, 6.93, 3.42, 3.04,
d (16.8) d (16.8) s m dd (13.8, 8.4)
Table 3 1 H NMR data (600 MHz) for compounds 7–9 in acetone-d6. position
7 δH, mult (J in Hz)
2 3 4 5 5-OCH3 7′a 7′b 7″a 7″b
5.52, dd (6.0, 4.8) 3.57, dd (9.6, 4.8)
4.62, 4.08, 3.71, 3.70,
dd (18.4, 6.0) dd (18.4, 4.8) m m
8 δH, mult (J in Hz)
9 δH, mult (J in Hz)
3.00, s
7.48, dd (3.6, 0.6) 6.73, dd (3.6, 1.2) 7.90, dd (1.2, 0.6)
3.60, s 4.02, d (2.5)
4.77, s
4.73, d (2.5)
& Table S2) with 1 revealed the only significant difference being the presence of a C-5 CO2Me moiety (δH 3.69), as evidenced by diagnostic HMBC correlations from H-4 and the CO2Me, to C-5. Likewise, comparison of the NMR (methanol-d4) data for 3 (Figs. S8–9, Tables 1 and 4 & Table S3) with 1 revealed the only significant difference being the presence of C-5 CO2Me (δH 3.67) and C-6 CO2Et (δH 4.31, q, OCH2CH3 and 1.35, t, OCH2CH3) moieties, confirmed by HMBC correlations to C5 and C-6 respectively (Fig. 2). The presence of C-6 CO2Et suggested that 3 is a likely ethanolysis artifact of symphyocladin S (2). HRESI(+)MS measurements confirmed 4 (C21H15Br5O10, Δmmu −0.9) and 5 (C21H15Br5O10, Δmmu −0.3) as monodebromo, and 6 (C20H13Br5O10, Δmmu +0.5) as a monodebromo hydrolysed analogue of 2. Comparison of the NMR (methanol-d4) data for symphyocladin T (4) (Figs. S11–12, Tables 2 and 4 & Table S4) with 2 confirmed a common scaffold incorporating an E Δ2,3 configuration, as evidenced by the 13C NMR chemical shift for C-1 (δC 170.0). The C-5 CO2Me regiochemistry was confirmed by HMBC correlations from H-4 and CO2Me to C-5. The only significant difference in the NMR data for 4 compared to 2 was attributed to the presence of a H-2″ aromatic methine (δH 6.88, s; δC 126.5), the regiochemistry of which was determined by diagnostic HMBC correlations from H-2″ to C-7″, and H2-7″ to C-2″ (Fig. 3). Comparison of the NMR (methanol-d4) data for symphyocladin U (5) (Figs. S14–15, Tables 2 and 4 & Table S5) with 4 confirmed a common scaffold incorporating an E Δ2,3 configuration (C1 δC 170.7), and a C-5 CO2Me, with the only significant differences being attributed to an alternate H-2′ aromatic methine (δH 6.89, s; δC 21
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Table 4 13 C NMR data (150 MHz) for compounds 1 - 9. position
1a δC, type
2a δC, type
3b δC, type
4a δC, type
5a δC, type
6a δC, type
7b δC, type
8b δC, type
9b δC, type
1 2 3 4 5 6 5-OCH3 6-OCH2CH3 6-OCH2CH3 1′ 2′ 3′ 4′ 5′ 6′ 7′ 1″ 2″ 3″ 4″ 5″ 6″ 7″
170.0, C 148.7, C 129.4, C 48.7, CH 176.0, C 170.0, C
169.9, C 149.1, C 129.1, C 48.8, CH 174.5, C 169.8, C 52.8, CH3
170.0, C 144.4, C 131.3, C 48.9, CH 174.2, C 170.1, C 52.7, CH3
170.7, C 144.6, C 135.3, C 47.9, CH 174.3, C 169.9, C 52.9, CH3
170.0, C 143.5, C 131.7, C 49.1, CH 175.6, C nd
145.7, C 130.4, C 51.1, CH 173.2, C 167.4, C
165.3, C 135.8, C 142.0, C 29.3, CH 168.3, C 165.8, C 52.8, CH3
183.9, 153.1, 118.3, 113.3, 148.0,
130.8, C 119.4, C 114.6, C 145.0, C 144.8, C 115.3, C 42.1, CH2 131.8, C 118.8, C 114.6, C 144.7, C 144.3, C 114.6, C 39.4, CH2
130.7, C 119.4, C 114.6, C 145.1, C 144.8, C 115.3, C 42.0, CH2 131.5, C 118.7, C 114.0, C 144.8, C 144.3, C 114.6, C 39.3, CH2
167.4, C 147.7, C 128.3, C 47.8, CH 172.4, C 166.7, C 52.4, CH3 61.6, CH2 14.5, CH3 130.5, C 119.0, C 113.9, C 144.2, C 143.9, C 114.7, C 41.4, CH2 131.1, C 118.4, C 113.4, C 143.9, C 143.5, C 114.0, C 39.2, CH2
130.3, C 119.2, C 114.2, C 145.4, C 145.1, C 115.1, C 41.4, CH2 131.8, C 126.5, C 109.9, C 143.7, C 144. 5, C 113.4, C 37.2, CH2
131.4, C 124.5, C 110.0, C 143.4, C 145.3, C 112.9, C 38.5, CH2 131.3, C 118.8, C 114.6, C 145.1, C 144.9, C 114.6, C 39.6, CH2
130.2, C 118.7, C nd nd nd 114.8, C 41.1, CH2 nd 126.1, C 109.6, C 143.4, C nd 113.0, C 36.8, C
132.8, C 117.4, C 113.7, C 144.1, C 144.0, C 113.2, C 40.6, CH2 131.9, C 118.3, C 114.0, C 144.0, C 143.9, C 114.0, C 39.3, CH2
131.8, C 125.7, C 116.7, C 144.1, C 144.0, C 115.1, C 32.4, CH2 132.6, C 118.7, C 114.4, C 143.4, C 144.3, C 114.6, C 43.6, CH2
128.4, C 118.4, C 113.5, C 144.5, C 143.9, C 114.1, C 48.8, CH2
a b
C C CH CH CH
Methanol-d4. Acetone-d6, nd – not detected.
Fig. 1. S. latiuscula compounds 1–9.
3. Conclusions
suggests this racemic character is a function of initial achiral adduct addition. The biosynthesis of 9 most likely proceeds via a similar adduct formation, but the origin of the furanoyl nucleophile remains unclear. The proposed biosynthetic relationship informs a possible biomimetic synthesis of 1–9. All compounds did not exhibit growth inhibitory activity (i.e. IC50 > 30 μM) against a panel of Gram-positive (Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633) and Gram-negative bacteria (Escherichia coli ATCC 11775, Pseudomonas aeruginosa ATCC 10145) or a fungus (Candida albicans ATCC 10231).
We report a series of undescribed polybromocatechol conjugates 1–9 isolated from a marine red alga, S. latiuscula, collected from coastal waters off Qingdao, China. These compounds feature new carbon skeletons and very likely share a common putative biosynthetic quinone methide precursor. These metabolites demonstrate the power of convergent biosynthesis, where simple biosynthetic precursors can assemble a multitude of structurally related natural products. Knowledge of the chemistry of S. latiuscula has the potential to inspire future biomimetic syntheses, opening up access to new chemical space. 22
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Fig. 2. Diagnostic 2D NMR (methanol-d4) correlations for symphyocladins R–S (1–2), solvolysis artifact 3, and structures for symphyocladins H (10) and I (11).
4. Experimental
performed using an Agilent 1100 series separations module equipped with Agilent 1100 series diode array and/or multiple wavelength detectors and Agilent 1100 series fraction collector, controlled using ChemStation Rev.B02.01 and Purify version A.1.2 software. All solvents used for chromatographic separation were purchased from Sigma Australia. All solvents were HPLC grade.
4.1. General experimental procedures Specific rotations ([α]D) were measured on Anton Paar MCP 200 Modular Circular Polarimeter (Austria) in a 100 × 2 mm cell at 22 °C. The CD spectra were recorded on Chirascan (Applied Photophysics, UK). NMR spectra were obtained on a Bruker Avance DRX600 or DRX500 spectrometers, in the solvents indicated and referenced to residual 1H and 13C signals in deuterated solvents. Electrospray ionization mass spectra (ESIMS) were acquired using an Agilent 1100 Series separations module equipped with an Agilent 1100 Series LC/MSD mass detector in both positive and negative ion modes. High-resolution ESIMS measurements were obtained on (i) a Bruker micrOTOF mass spectrometer by direct infusion in MeCN at 3 mL/min using sodium formate clusters as an internal calibrant, or (ii) an Agilent 6520 mass spectrometer equipped with an Agilent 1200 series separation module, with elution through an Agilent Eclipse XDB-C8 column (5 μm, 4.6 × 150 mm, 1 mL/min gradient elution from 10% MeCN/H2O to 100% MeCN with 0.05% isocratic formic acid, over 15 min). HPLC was
4.2. Plant material Symphyocladia latiuscula (Harvey) Yamada (Rhodomelaceae) was collected in May 2004, on the coast of Taipingjiao, Qingdao, Shandong Province, China. The specimen identification was verified by Dr. KuiShuang Shao (Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China). A voucher specimen (No. 2004X16) was deposited at the Herbarium of the Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China. 4.3. Extraction and isolation The air-dried red alga S. latiuscula (4.3 kg) was extracted with 95%
Fig. 3. Diagnostic 2D NMR (methanol-d4) correlations for symphyocladins T–V (4–6). 23
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EtOH/H2O (20 L × 3) at room temperature (72 h). After the solvent was removed under reduced pressure at < 40 °C, a dark residue (610 g) was obtained. The residue was partitioned between EtOAc (5 L) and H2O (5 L), and the EtOAc-soluble partition (320 g) was chromatographed over silica gel, eluting with a gradient of 0–100% Me2CO/petroleum ether to yield 85 fractions (A1–A85) (Scheme S1). Fraction A49 (845 mg) was further fractionated over Sephadex LH20 using CHCl3–MeOH (2:1) (5 mL/min) to afford 22 fractions (A49.1–A49.22). Fraction A49.12 (586.4 mg) was subjected to HPLC purification (Zorbax SB-C8, 5 μm, 250 × 9.4 mm column, 3.0 mL/min, gradient elution from 10 to 100% MeCN/H2O over 15 min, with isocratic 0.01% TFA modifier) to yield 7 (4.9 mg, tR 8.35 min, 0.001%). Fraction A54 (9486 mg) was further fractionated over Sephadex LH20 using CHCl3–MeOH (2:1) (5 mL/min) to afford 21 fractions (A54.1–A54.21). Fraction A54.8 (1054 mg) was further subjected to SPE purification (Bond elute C18, 5 g), eluted with a stepwise 10% gradient from 100% H2O to 100% MeOH to afford 11 fractions (A54.8.1–A54.8.11). Fraction A54.8.7 (283.8 mg) was further subjected to HPLC fractionation (Zorbax SB-C18, 5 μm, 250 × 9.4 mm column, 3.0 mL/min, gradient elution from 50 to 60% MeCN/H2O over 12 min, with isocratic 0.01% TFA modifier) to yield 3 (14.2 mg, tR 11.83 min, 0.004%) and 9 (4.9 mg, tR 8.53 min, 0.001%) Fraction A54.9 (968 mg) was subjected to SPE purification (Bond elute C18, 5 g), eluted with a stepwise 10% gradient from 100% H2O to 100% MeOH to afford 11 fractions (A54.9.1–A54.9.11). Fraction A.54.9.7 (217.2 mg) was further subjected to HPLC fractionation (Zorbax SB-C18, 5 μm, 250 × 9.4 mm column, 3.0 mL/min, gradient elution from 50 to 70% MeCN/H2O over
Fig. 4. Diagnostic 2D NMR (acetone-d6) correlations for symphyocladins W–Y (7–9).
Fig. 5. A plausible biosynthetic relationship linking 1–8. 24
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15 min, with isocratic 0.01% TFA modifier) to yield 8 (8.2 mg, tR 9.0 min, 0.002%). Fraction A54.14 (128.4 mg) was further subjected to HPLC fractionation (Zorbax Eclipse XDB-C8, 5 μm, 250 × 9.4 mm column, 3.0 mL/min, gradient elution from 35% MeCN/H2O over 17 min, with isocratic 0.01% TFA modifier) to yield 1 (1.3 mg, tR 8.23 min, 0.0004%), 2 (2.1 mg, tR 15.94 min, 0.0006%), 4 (1.8 mg, tR 12.40 min, 0.0004%), 5 (2.4 mg, tR 13.73 min, 0.0006%), and 6 (1.2 mg, tR 6.53 min, 0.0002%). [% yields were determined on a massto-mass basis against the EtOAc crude extract].
NMR (150 MHz, acetone-d6), see Table 4; HRESIMS m/z 450.7819 [M H]- (calcd for C12H678Br3O4, 450.7822). Acknowledgments This work was supported in part by the National Key Research and Development Program of China (2018YFC0311002, 2017YFD0201203, 2017YFC1601300), the Chinese Government Fundamental Research Funds for the Central Universities, and the University of Queensland, Institute for Molecular Bioscience.
4.3.1. Symphyocladin R (1) Light brown solid; [α]D25 0 (c 0.01, MeOH); 1H NMR (600 MHz, methanol-d4), see Table 1; 13C NMR (150 MHz, methanol-d4), see Table 4; HRESIMS m/z 914.5365 [M + Na]+ (calcd for C20H1278Br381Br3O10Na, 914.5362).
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.phytochem.2018.10.026. References
4.3.2. Symphyocladin S (2) Light brown solid; [α]D25 0 (c 0.01, MeOH); 1H NMR (600 MHz, methanol-d4), see Table 1; 13C NMR (150 MHz, methanol-d4), see Table 4; HRESIMS m/z 906.5688 [M + H]+ (calcd for C21H1578Br381Br3O10, 906.5699).
Choi, J.S., Park, H.J., Jung, H.A., Chung, H.Y., Jung, J.H., Choi, W.C., 2000. A cyclohexanonyl bromophenol from the red alga Symphyocladia latiuscula. J. Nat. Prod. 63, 1705–1706. https://doi.org/10.1021/np0002278. Duan, X.-J., Li, X.-M., Wang, B.-G., 2007. Highly brominated mono- and bis-phenols from the marine red alga Symphyocladia latiuscula with radical-scavenging activity. J. Nat. Prod. 70, 1210–1213. https://doi.org/10.1021/np070061b. Jin, H.J., Oh, M.Y., Jin, D.H., Hong, Y.K., 2008. Identification of a Taq DNA polymerase inhibitor from the red seaweed Symphyocladia latiuscula. J. Environ. Biol. 29, 475–478. Kurata, K., Amiya, T., 1980a. Disodium 2,3,6-tribromo-5-hydroxybenzyl 1′, 4-disulfate, a new bromophenol from the red alga, Symphyocladia latiuscula. Chem. Lett. 9, 279–280. https://doi.org/10.1246/cl.1980.279. Kurata, K., Amiya, T., 1980b. Bis(2,3,6-tribromo-4,5-dihydroxybenzyl) ether from the red alga, Symphyocladia latiuscula. Phytochemistry 19, 141–142. https://doi.org/10. 1016/0031-9422(80)85032-1. Lee, J.H., Park, S.E., Hossain, M.A., Kim, M.Y., Kim, M.N., Chung, H.Y., Choi, J.S., Yoo, Y.H., Kim, N.D., 2007. 2,3,6-tribromo-4,5-dihydroxybenzyl methyl ether induces growth inhibition and apoptosis in MCF-7 human breast cancer cells. Arch. Pharm. Res. 30, 1132–1137. Liu, X., Li, X., Gao, L., Cui, C., Li, C., Li, J., Wang, B., 2011. Extraction and PTP1B inhibitory activity of bromophenols from the marine red alga Symphyocladia latiuscula. Chin. J. Oceanol. Limnol. 29, 686–690. https://doi.org/10.1007/s00343-0110136-1. Park, H.J., Kurokawa, M., Shiraki, K., Nakamura, N., Choi, J.S., Hattori, M., 2005. Antiviral activity of the marine alga Symphyocladia latiuscula against herpes simplex virus (HSV-1) in vitro and its therapeutic efficacy against HSV-1 infection in mice. Biol. Pharm. Bull. 28, 2258–2262. Wang, W., Okada, Y., Shi, H., Wang, Y., Okuyama, T., 2005. Structures and aldose reductase inhibitory effects of bromophenols from the red alga Symphyocladia latiuscula. J. Nat. Prod. 68, 620–622. https://doi.org/10.1021/np040199j. Xu, X., Piggott, A.M., Yin, L., Capon, R.J., Song, F., 2012a. Symphyocladins A-G: bromophenol adducts from a Chinese marine red alga, Symphyocladia latiuscula. Tet. Lett. 53, 2103–2106. https://doi.org/10.1016/j.tetlet.2012.02.044. Xu, X., Song, F., Fan, X., Fang, N., Shi, J., 2009. A novel bromophenol from marine red alga Symphyocladia latiuscula. Chem. Nat. Compd. 45, 811–813. https://doi.org/10. 1007/s10600-010-9501-0. Xu, X., Yang, H., Khalil, Z.G., Yin, L., Xiao, X., Neupane, P., Bernhardt, P.V., Salim, A.A., Song, F., Capon, R.J., 2017. Chemical diversity from a Chinese marine red alga, Symphyocladia latiuscula. Mar. Drugs 15, 374. https://doi.org/10.3390/md15120374. Xu, X., Yin, L., Fang, N., Fan, X., Song, F., 2012b. Bromophenol coupled with diketopiperazine from marine red alga Symphyocladia latiuscula. Chem. Nat. Compd. 48, 622–624. https://doi.org/10.1007/s10600-012-0327-9. Xu, X., Yin, L., Gao, J., Gao, L., Song, F., 2014. Antifungal bromophenols from marine red alga Symphyocladia latiuscula. Chem. Biodivers. 11, 807–811. https://doi.org/10. 1002/cbdv.201300239. Xu, X., Yin, L., Gao, L., Gao, J., Chen, J., Li, J., Song, F., 2013a. Two new bromophenols with radical scavenging activity from marine red alga Symphyocladia latiuscula. Mar. Drugs 11, 842–847. https://doi.org/10.3390/md11030842. Xu, X., Yin, L., Wang, Y., Wang, S., Song, F., 2013b. A new bromobenzyl methyl sulphoxide from marine red alga Symphyocladia latiuscula. Nat. Prod. Res. 27, 723–726. https://doi.org/10.1080/14786419.2012.695362.
4.3.3. Likely solvolysis artifact (3) Light brown solid; [α]D25 0 (c 0.01, MeOH); 1H NMR (600 MHz, acetone-d6), see Table 1; 13C NMR (150 MHz, acetone-d6), see Table 4; HRESIMS m/z 934.6003 [M + H]+ (calcd for C23H1978Br381Br3O10, 934.6012). 4.3.4. Symphyocladin T (4) Light brown solid; [α]D25 0 (c 0.01, MeOH); 1H NMR (600 MHz, methanol-d4), see Table 2, 13C NMR (150 MHz, methanol-d4), see Table 4; HRESIMS m/z 826.6605 [M + H]+ (calcd for C21H1678Br281Br3O10, 826.6614). 4.3.5. Symphyocladin U (5) Light brown solid; [α]D25 0 (c 0.01, MeOH); 1H NMR (600 MHz, methanol-d4), see Table 2; 13C NMR (150 MHz, methanol-d4), see Table 4; HRESIMS m/z 826.6611 [M + H]+ (calcd for C21H1678Br281Br3O10, 826.6614). 4.3.6. Symphyocladin V (6) Light brown solid; [α]D25 0 (c 0.01, MeOH); 1H NMR (600 MHz, methanol-d4), see Table 2; 13C NMR (150 MHz, methanol-d4), see Table 4; HRESIMS m/z 812.6462 [M + H]+ (calcd for C20H1478Br281Br3O10, 812.6457). 4.3.7. Symphyocladin W (7) Light brown solid; [α]D25 0 (c 0.01, MeOH); 1H NMR (600 MHz, acetone-d6), see Table 3; 13C NMR (150 MHz, acetone-d6), see Table 4; HRESIMS m/z 848.5644 [M + H]+ (calcd for C19H1378Br381Br3O8, 848.5644). 4.3.8. Symphyocladin X (8) Light brown solid; 1H NMR (600 MHz, acetone-d6), see Table 3; 13C NMR (150 MHz, acetone-d6), see Table 4; HRESIMS m/z 826.6610 [M + H]+ (calcd for C21H1678Br281Br3O10, 826.6614). 4.3.9. Symphyocladin Y (9) Light brown solid; 1H NMR (600 MHz, acetone-d6), see Table 3; 13C
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