Puupehenol, a Potent Antioxidant Antimicrobial Meroterpenoid from a

Feb 10, 2015 - DKI College of Pharmacy, University of Hawaii at Hilo, Hilo, Hawaii 96720, United States .... RSC Advances 2015 5 (106), 87730-87738 ...
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Puupehenol, a Potent Antioxidant Antimicrobial Meroterpenoid from a Hawaiian Deep-Water Dactylospongia sp. Sponge Kehau Hagiwara,† Jaaziel E. Garcia Hernandez,† Mary Kay Harper,‡ Anthony Carroll,§ Cherie A. Motti,⊥ Jonathan Awaya,∥ Hoang-Yen Nguyen,∥ and Anthony D. Wright*,† †

DKI College of Pharmacy, University of Hawaii at Hilo, Hilo, Hawaii 96720, United States Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112, United States § School of Environment, Griffith University, Gold Coast Campus, Brisbane, QLD 4222, Australia ⊥ Australian Institute of Marine Science, PMB no. 3, Townsville MC, Townsville, QLD 4810, Australia ∥ Department of Biology, University of Hawaii at Hilo, Hilo, Hawaii 96720, United States ‡

S Supporting Information *

ABSTRACT: From the organic extract of a deep-water Hawaiian sponge Dactylospongia sp., a new potent antioxidant and antimicrobial meroterpenoid, puupehenol (1), was isolated. The structure of 1 was determined using spectroscopic techniques (1H and 13C NMR, MS, IR, UV, [α]D). The known compound puupehenone (2) was also isolated and suggested as a probable artifact of the isolation procedures. Complete unambiguous 1 H and 13C NMR data are provided for compounds 1 and 2. Bioassays performed with 1 and 2 showed them both to be very effective antioxidants and to have antimicrobial properties.

I

these data revealed the extract of sample D0008, Dactylospongia sp., had interesting antioxidant properties and 1H NMR data that showed the presence of aromatic and terpenoid moieties. On the basis of these observations the sample was selected for further investigation. The CH3OH extract of the sponge was fractionated by RP-HPLC to yield two meroterpenoids, the new metabolite puupehenol (1) and the known compound puupehenone (2).20 The molecular formula of 1 was determined to be C21H30O4 by accurate mass measurement. The IR spectrum of 1 contained a broad absorbance signal centered on 3250 cm−1 for an OH functionality. From the 1H and 13C NMR data of 1 (Table 1) 20 carbon and 27 proton resonances were observed, indicating one carbon resonance to be degenerate and the three protons not observed to be part of three OH groups. The 13C NMR data contained resonances consistent with the presence of one aromatic ring that was highly functionalized with oxygen-containing functionalities (C-14, C-15, and C-17), as well as two sp3-carbons bearing oxygen (C-8, 76.8 ppm and C11, 76.0 ppm). The absence of any other sp2-carbons or carbonyl absorbances in the IR spectrum of 1 revealed the fourth oxygen atom was present as an ether and the molecule to be tetracyclic. The 1H and 13C NMR data of 1 showed the aromatic moiety to be tetrasubstituted and the two aromatic protons to have a 1, 4 (para) relationship. Cross-peaks in the gHMBC NMR spectrum of 1 between H-11 and C-12 and C17, and between H-9 and C-12, were the only obvious correlations to aromatic-ring carbons. These correlations also

t is widely acknowledged that the marine environment provides a plethora of natural products without structural precedent anywhere else in the natural world.1,2 Of the 22 000 natural products isolated from marine organisms over the past 30 years,3 many have proven potential as therapeutics1,2,4 and are used by the agricultural, cosmeceutical, food supplement, and chemical industries.5,6 The most probable reason for this chemical biodiversity is the compounds are typically biosynthesized or sequestered by sessile or extremely slowly moving marine life forms found in a wide range of marine environments. This lack of mobility renders such organisms vulnerable to attack by predators, and as a result, organisms such as algae, sea grasses, sponges, tunicates, soft corals, gorgonians, and nudibranchs have evolved highly specialized chemical defenses. Sponges belonging to the class Demospongiae are excellent examples of this, members being renowned for their ability to produce many and varied biologically active secondary metabolites.7−9 Sponges of the family Thorectidae seem particularly well adapted to their environments and are well known for their ability to produce compounds such as pelorol,10 ilimaquinone,11,12 a variety of quinones,9 hydroquinones,13 benzoxazoles,14 γ-lactones,9,13 and sesterterpene lactones,15 all of which probably have specialized ecological roles as well as their potential utility in one of the aforementioned chemically related industries. In the current investigation a number of samples collected by an ROV operating in the Au’au Channel off the Hawaiian island of Maui were examined. A small piece (2 cm3) of each sample was extracted with CH3OH and assayed for its antioxidant16,17 and antimicrobial18 activities. A standardized 1H NMR spectrum was also recorded for each extract.19 Examination of © XXXX American Chemical Society and American Society of Pharmacognosy

Received: October 10, 2014

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correlations to C-3, C-4, and C-5; that CH3-21 was connected to C-10, which in turn formed direct C−C bonds with C-1, C5, and C-9; and that C-20 bonded directly with C-8, which in turn formed direct C−C bonds with C-7, C-9, and one of the four oxygen atoms within 1. From the gCOSY NMR spectrum, direct C−C bonds were deduced between C-1 and C-2 and between C-2 and C-3, completing the A ring, between C-5 and C-6, and C-6 and C-7, completing the B ring, and between C-9 and C-11. With this part of the analysis complete all except one ring within 1, the position of the three OH groups and the ether was complete. The presence of an NOE interaction between H-11 and H-13 (Table 1) and the fact that H-9 and H11 have zero coupling means these protons are at approximately 90° to each other. This not only confirmed the C-11/C-12 bond but also established the ether linkage, and hence the final ring closure, between C-8 and C-17, as well as the location of the three hydroxy functions at C-11, C-14, and C-15. With the planar structure of 1 established, its relative configuration required resolution. From the gNOESY NMR data of 1 (Table 1) observed interactions between H3-19 and H3-21 showed them to be on the same side of the molecule and the A and B rings to be trans-fused. Further, NOES between H9 and H3-20 and between H-5 and H-9 showed them to be on the same face of 1, and rings B and C to be cis-fused. Finally, the zero coupling between H-9 and H-11 meant OH-11 had to be on the same face of 1 as H-9; thus the relative configuration of the molecule is as shown in 1. For 1, the trivial name of puupehenol is proposed. The known compound 2,20 puupehenone, was also isolated and fully characterized by NMR (Table 1 and Supporting

showed C-11 to bond with C-12, a deduction supported by the NOE interaction between H-11 and H-13. The deduction of the C-11/C-12 bond also confirmed the oxygen substitution at the other three carbons in the aromatic ring, C-14, C-15, and C-17. Once all protons and carbons were assigned their respective resonances from the results of a gHSQC NMR experiment, it was possible to discern the remaining three rings and the relative configuration of 1. From the gHMBC NMR measurement (Table 1) it was evident CH3-18 and CH3-19 constituted a gem-dimethyl group based on their proton

Table 1. 1H and 13C NMR Data (1H 400 MHz, 13C 100 MHz) of Puupehenol (1) in CD3OD and of Puupehenone (2) in CDCl3 puupehenol (1) carbon

13

C ppm

1

41.9, CH2

2

20.3, CH2

3

43.9, CH2

4 5 6 7

35.2, 57.2, 20.4, 42.9,

C CH CH2 CH2

8 9 10 11 12 13 14 15 16 17 18 19 20 21

76.8, 55.8, 39.0, 76.0, 116.2, 118.9, 141.1, 148.4, 105.6, 149.6, 35.2, 23.3, 28.8, 15.9,

C CH C CH C CH C C CH C CH3 CH3 CH3 CH3

1

H (ppm) 1.19, 2.02, 1.50, 1.62, 1.28, 1.44,

m m m m m m

1.05, 1.63, 1.66, 2.54,

m m m m

puupehenone (2) gNOESYa 11

1.59, s

11, 20

4.11, s

13, 9, 1eq, 21

6.72, s

11

6.22, s 0.96, 0.87, 1.21, 0.68,

s s s s

21 9 11, 19

δ 13Ca

δ 13C

41.5, CH2

39.2, CH2

18.1, CH2

18.1, CH2

41.9, CH2

41.6, CH2

33.2, 53.6, 18.1, 39.1,

C CH CH2 CH2

33.3, 53.8, 18.4, 40.0,

C CH CH2 CH2

78.6, 54.7, 39.8, 140.2, 129.0, 105.0, 147.2, 181.8, 105.9, 162.5, 33.6, 21.8, 27.9, 15.0,

C CH C CH C CH C C CH C CH3 CH3 CH3 CH3

78.9, 54.8, 40.7, 140.7, 129.2, 105.2, 147.5, 182.1, 106.0, 162.8, 33.7, 21.9, 28.0, 15.0,

C CH C CH C CH C C CH C CH3 CH3 CH3 CH3

δ 1H (J in Hz) 1.57, m 2.17, m 1.43, m 1.21, m 1.41, m 0.94, 1.54, 1.16, 1.67,

m m m m

2.04, d (7.0) 6.65, brd (7.0) 6.20, s

5.85, d (1.1) 0.90, 0.83, 1.21, 0.80,

s s s s

a

Values from Ravi, B. N.; Perzanowski, H. P.; Ross, R. A.; Erdman, T. R.; Scheuer, P. J.; Finer, J.; Clardy, J. Pure Appl. Chem. 1979, 51, 1893−1900, were unassigned by these authors. B

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Information Table S1). It is noteworthy that the specific rotation of our isolate was +59 in CH3OH (c 0.1) and +98 in CHCl3 (c 0.5) compared to +315 previously reported in CCl4 (c 1.64).20 These findings indicate the optical activity of 2 probably has a dependence on solvent and/or concentration. In an attempt to resolve the absolute configuration of 1, and by implication 2, attempts were made to make Mosher’s esters21,22 and p-bromobenzoate (pbb) derivatives, the latter for CD and/ or X-ray studies.23,24 Unfortunately, all attempts to make any esters at C-11 were unsuccessful. After closer inspection of the structures of 1 and 2 it was proposed that 2 was perhaps an artifact of the isolation, being formed from 1 via an acid-catalyzed dehydration elimination. The mechanism of this process would probably be driven by protonation of OH-11. Indeed, addition of 1 to slightly acidic CDCl3 in an NMR tube with slight heating (30 °C) resulted in quantitative conversion to 2 over 1 h. This finding raises the question of the original discovery of puupehenone (2)20 as a natural product. On the basis of the original isolation methods used, preparative silica TLC and CHCl3,20 it is possible that 1 or similar alcohols present in the sponge or sponge extract could have been quantitatively converted to 2. Over time it was also noted that 1 converts to 2 and 4 in the presence of CH3OH (Supporting Information). Further to this, addition of 2 to CH3OH at room temperature resulted in the formation of two methoxylated minor products, 3 and 4, as determined by subsequent NMR and MS analyses (Supporting Information Tables S2 and S3), pointing toward the formation of 2 from 1 being partially reversible. The molecular formula of 3 was established as C22H32O4 by accurate mass measurement. The NMR data for 3 showed many similarities to those for 1, with one obvious difference: the presence of a resonance in 3 associated with a methoxy at C-11 (13C: 56.3 ppm; 1H: δ 3.33, s) and a corresponding gHMBC correlation to C-11 (13C: 73.3 ppm). These data established the planar structure of 3, as 11-O-methylpuupehenol, as shown. Proton−proton coupling information and 13C NMR data comparisons made with 1 showed 3 to have the same relative configuration as that shown for 1. Accurate mass measurement of 4 showed it to have the molecular formula C42H62O6. Surprisingly, its 13C NMR spectrum contained only 21 resonances and showed many similarities to that for 3, including the presence of a resonance in 4 associated with a methoxy at C-11 (13C: 54.5 ppm; 1H: δ 3.29, s) and a corresponding gHMBC correlation to C-11 (13C: 71.4 ppm). Further analysis of the gHMBC data of 4 showed C-13 in 3 (13C: 116.8 ppm; 1H: δ 6.61, s) was absent, and in its stead was a quaternary carbon with a resonance at 122.0 ppm. This latter observation was consistent with 4 being a symmetrical dimer of 3 linked via C-13. As the proton−proton coupling information, 13C NMR data comparisons, and NOE data obtained for 4 were similar to those of 3, it was concluded that the two monomeric units had the same relative configuration as that shown for 1 and 3. Complete and unambiguous 1H and 13C NMR data are provided for 1 and 2 (Table 1) and 3 and 4 (Supporting Information). Bioassays to assess the antioxidant16,17 and antimicrobial18 activities of compounds 1 and 2 found them both to have pronounced antioxidant properties (Table 2) in the applied assay and also to be relatively active toward the Gram-positive bacteria Staphylococcus aureus and Bacillus cereus (Table 3).

Table 2. Results of Ferric Reducing Antioxidant Power (FRAP)a Assay Conducted with 1 and 2 compoundb

average FRAPa,c

SDa,c

CH3OH vitamin Cd 1 2

4.7 870 2500 2600

0.5 130 29 17

Units are μM. bUnless otherwise stated concentrations were 1 mg/ mL. cResults are based on measurements made in triplicate. d Concentration was 0.1 mg/mL. a

Table 3. Antibacterial Activities of 1 and 2a organism Staphylococcus aureus Bacillus cereus Escherichia coli Pseudomonas aeruginosa a

zone of inhibition (mm), compound, and conc (μg/disc) 4, 1, 10; 4, 2, 10; 31, ampicillin, 10; 0, CH3OH, 10 3, 1, 10; 3, 2, 10; 9, ampicillin, 10; 0, CH3OH, 10 all compounds inactive at 10 μg/disc; 20, ampicillin, 10; 0, CH3OH, 10 all compounds and controls inactive at 10 μg/disc

Activities were determined employing a disc diffusion assay.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotation data were collected employing a Rudolph Research Analytical Autopol IV Automatic polarimeter. IR spectra were measured using a Thermo Scientific Nicolet iS10 FTIR spectrophotometer fitted with a Smart iTR. UV spectra were recorded with a Shimadzu UV-1800 UV−vis spectrophotometer. NMR spectra for compounds 1 and 2 were measured on a Bruker Avance DRX 400 MHz NMR spectrometer; spectra for 3 and 4 were measured on a Bruker Avance 600 MHz NMR spectrometer equipped with a cryoprobe. All NMR spectra were referenced to NMR solvent signals as follows (δ 7.26 and 77.0 ppm for CDCl3, and δ 3.34 and 49.9 ppm for CD3OD). FT-ICR-HRESIMS measurements were performed on an unmodified Bruker BioAPEX 47e mass spectrometer equipped with an Analytica of Branford model 103426 electrospray ionization (ESI) source in both positive and negative mode. Direct infusion of the sample (∼0.2 mg/mL in CH3OH) was carried out using a Cole Palmer 74900 syringe pump at a rate of 150 μL/h. The instrument was calibrated using a methanolic solution of CF3COONa (0.1 mg/mL in CH3OH). RP (C18-Silica) HPLC was undertaken employing a Shimadzu Prominence HPLC system consisting of a degasser, two pumps, autosampler, dual wavelength detector, and LC Solution software for data analysis, coupled to a 250 × 21.2 mm, 5 μm Ultra II C18 column (Restek, Bellefonte, PA, USA). Sponge Material. The sponge Dactylospongia sp. was collected on April 3, 2009, at a depth of 130 m by remote-operated submersible (ROV) from a sandy, silty barren bottom in the Au’au Channel (20°46.303′ N, 156°40.245′ W) off the Hawaiian island of Maui. The lobate sponge (ca. 150 cm3; see image in Support Information) was yellow upon collection; in spirits the sponge is dark brown, fibrous, and only slightly compressible. The skeleton is a dense reticulation of fibers that are not clearly differentiated as primary or secondary fibers. The fibers are slightly laminated in cross section, uncored, and occasionally pithed, and frequently become fasciculate as they ascend to the sponge surface and form conules. Tertiary fibers are scattered among the network and commonly concentrated below the surface. This sponge is comparable to the two described species of the genus, but as those have been reported from predominantly shallow water Indo-Pacific reef habitats, we denote this sponge as Dactylospongia sp. A voucher specimen (accession number D0008) is stored at the Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah, USA. Extraction and Isolation. Freeze-dried sponge material (7.12 g) was exhaustively extracted with CH3OH to yield 1.67 g of extract. The C

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extract, shown in bioassays to have antioxidant properties, was separated employing RP-HPLC (elution program: 0−30 min: 20− 100% CH3OH, 80−0% ultrapure H2O; 90 min: 100% CH3OH; 20 mL fractions collected) to yield 31 fractions. Fraction 11 contained puupehenol (1, 42.7 mg, 0.59%), and fraction 12 contained puupehenone20 (2, 31.7 mg, 0.45%). All remaining fractions contained either too little material to work with or ubiquitous substances such as lipids and sterols. Puupehenol (1): clear oil; [α]25D +68 (c 0.1, CH3OH); IR (neat film) νmax 3250, 2920, 1605, 1460 cm−1; UV (CH3OH) λmax (log ε) 298 (3.7), 225 sh (3.8), 202 (4.4) nm; CD (CH3OH) λmax (Δε) 204 (−10.5) nm; 1H (400 MHz, CD3OD) and 13C (100 MHz, CD3OD) NMR data see Table 1; FT-ICR-HRESIMS m/z (in the presence of HCl) 329.2125 [M − H2O + H]+ (calcd for C21H29O3, 329.2111); m/ z 363.1731 [M − H2O + Cl]− (calcd for C21H28O3Cl, 363.1732); m/z 343.1912 [M − 2H − H]− (calcd for dienone form C21H27O4, 343.1915). Puupehenone (2): clear oil; [α]25D +59 (c 0.1, CH3OH), [α]25D +98 (c 0.5, CHCl3) [lit. +315 (c 1.64, CCl4)20]; IR (neat film) νmax 3350, 2920, 1590, 1460 cm−1; 1H (400 MHz, CD3OD) and 13C (100 MHz, CD3OD) NMR data see Table 1 and Supporting Information Table S1; FT-ICR-HRESIMS m/z 351.1948 [M + Na]+ (calcd for C21H28O3Na, 351.1931); m/z 327.1964 [M − H]− (calcd for C21H27O3, 327.1966); m/z 363.1737 [M + Cl]− (calcd for C21H28O3Cl, 363.1732). Conversions of 1 and 2. Compounds 1 and 2 (ca. 5 mg) were dissolved in CH3OH (5 mL) and allowed to stand at room temperature overnight. 1H NMR investigation of these solutions revealed 1 to have converted to 2 and 4 (Supporting Information), and 2 to have converted to 3 and 4 (Supporting Information). 11-O-Methylpuupehenol (3): 1H (600 MHz, CDCl3) and 13C (150 MHz, CDCl3) NMR data, Supporting Information Table S2; FT-ICRHRESIMS m/z 383.2198 [M + Na]+ (calcd for C22H32O4Na, 383.2193); m/z 359.2220 [M − H]− (calcd for C 22H31O4, 359.2228), 395.1979 [M + Cl]− (calcd for C22H32O4Cl, 395.1995); dienone formation m/z 381.2034 [M − 2H + Na]+ (calcd for dienone form C22H30O4Na, 381.2036) and 739.4204 [2(M − 2H) + Na]+ (calcd for C44H60O8Na, 739.4180); dienone formation m/z 393.1842 [M − 2H − Cl]− (calcd for dienone form C22H30O4Cl, 393.1838). 11-O-Methylpuupehenol dimer (4): 1H (600 MHz, CDCl3 and DMSO-d6) and 13C (150 MHz, CDCl3 and DMSO-d6) NMR data, Supporting Information Tables S2 and S3; FT-ICR-HRESIMS m/z 739.4204 [M − 2H + Na]+ (calcd for C44H60O8Na, 739.4180). Bioassays. Ferric Reducing Antioxidant Power (FRAP) Antioxidant Assay. The FRAP assay employed was modified from the Benzie and Strain protocol.17,18 The working FRAP reagent was produced by mixing 300 mM acetate buffer (pH 3.6), 10 mM 2,4,6-tripyridyl-Striazine (TPTZ) solution, and 20 mM FeCl3·6H2O in a 10:1:1 ratio and heating to 37 °C prior to use. The 300 mM acetate buffer was prepared by mixing 3.1 g of sodium acetate trihydrate (NaOAc·3H2O) with 16 mL of glacial acetic acid and made to 1 L with doubly distilled H2O. The TPTZ solution was prepared by mixing equal volumes of 10 mM TPTZ with 40 mM HCl. For the actual assays, 150 μL of FRAP reagent was added to each well of a 96-well microtiter plate. A blank reading was then taken at 595 nm using a Bio-Rad microtiter plate reader. To each well was then added 20 μL of sample (tests were done in triplicate), incubated for 8 min at room temperature, and measured at 595 nm. Triplicate standards of known FeII concentrations were run simultaneously using concentrations between 50 and 1000 μM of FeSO4·7H2O. A standard curve was plotted and the FRAP values, in μM, for each sample were determined. Since results may vary between plates, a new standard curve was prepared for each plate. Antimicrobial Assays. The antimicrobial assays were undertaken using a disc diffusion assay as previously described.16 Employed test organisms were Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Pseudomonas aeruginosa. Test concentrations were 1.0 and 0.1 mg/ mL.

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ASSOCIATED CONTENT

S Supporting Information *

Image of the sponge while being collected by ROV. Spectra in the file are 1H and 13C NMR spectral data for compounds 1 and 2, selected 2D NMR spectra for 1−4, together with three tables of NMR data (Tables S1, S2, and S3) for compounds 2, 3, and 4, and are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +1 808 981 4521. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are indebted to J. Rooney, NOAA, Honolulu, Hawaii, USA, for provision of the sponge material investigated in this study.

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REFERENCES

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