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Scytoscalarol (1), a new antimicrobial sesterterpene bearing a guanidine group, was isolated from the cultured cyanobacterium Scytonema sp. (UTEX 1163) by bioassay-guided fractionation. The chemical structure was determined by spectroscopic analysis including MS, 1D and 2D NMR. Scytoscalarol (1) showed antimicrobial activities against Bacillus anthracis, Staphylococcus aureus, Escherichia coli, Candida albicans and Mycobacterium tuberculosis with MIC value in the range from 2 to 110 μM.
Cyanobacteria are a promising but underexplored source for biologically active secondary metabolites.1,2 Numerous biologically active natural products have been reported from cyanobacteria, inclusive of alkaloids, polyketides, and linear as well as cyclic peptides.1-3 However, terpenes are not commonly found in cyanobacteria, and there are only a few reports of cyanobacterial terpenes.4-7 In our search for antimicrobial compounds from cyanobacteria, a guanidine-bearing sesterterpene, scytoscalarol (1) was isolated from the cultured terrestrial cyanobacterium Scytonema sp. (UTEX 1163). Scytoscalarol (1) has a scalarane skeleton, a class of sesterterpenes commonly found in various species of marine sponges of the order Dictyoceratida.8 Several scalarane sesterterpenes have been reported to exhibit significant cytotoxicity in human cancer cell lines9 and antimicrobial activity against various microorganisms.10 Scytoscalarol (1) is the first sesterterpene reported from cyanobacteria and the first guanidine-containing sesterterpene from a natural source. Herein, we reported the isolation, structural elucidation and biological activity of this compound.
Scytonema sp. was initially acquired from the Culture Collection of Algae at the University of Texas at Austin (UTEX 1163). In our screening, the crude extract of Scytonema sp. (UTEX 1163) was found to possess antibacterial activity against Bacillus anthracis (MIC 40 μg/mL) and was mass cultured in the laboratory for chemical investigation. The antibacterial MeOH:CH2Cl2 extract was fractionated using a diaion HP20SS resin column and Scytoscalarol (1) was obtained as a precipitate from fraction 3. The structure was determined by interpretation of MS, 1D and 2D NMR data.
Scytoscalarol (1) was obtained as a white amorphous solid. The molecular formula was determined by HRMS as C26H45N3O2 (m/z 416.3651 [M+H]+) and implied six degrees of unsaturation. Analysis of 1H, 13C, COSY and HSQC NMR spectra suggested the presence of four isolated CHCH2CH2 spin systems (C-1 to C-3, C-5 to C-7, C-9-C-11-C-12, and C-14 to C-16) and one CHCH2 (C-18-C-25) spin system (Figure 1). Two proton resonances at δH 4.94 and 4.48 were assigned to an exomethylene moiety (C-24, δC 107.6). The 1H NMR spectrum also contained resonances for five isolated singlet methyl groups at δH 0.82, 0.85, 0.91, 0.88 and 0.77.
The 13C NMR spectrum contained 26 carbon signals, three of which appeared above 100 ppm. Two of these were assigned to the exomethylene moiety (C-17 δC 147.8 and C-24 δC 107.6). The quaternary carbon at δC 158.6 ppm was attributed to a guanidine group based on the chemical shift 11 as well as consideration of the molecular formula. Of the six degrees of unsaturation required by the molecular formula, two were accounted for by the exomethylene moiety and the guanidine group. Hence scytoscalarol (1) was deduced to be tetracyclic.
The planar structure of 1 was determined by analysis of HMBC data (Table 1 and Figure 1). The isolated CHCH2CH2 spin system from C-1 to C-3 was connected to the C-5 to C-7 spin system via a gem-dimethyl group at C-4 based on the correlations from both H3-19 and H3-20 to C-3 and C-5. HMBC correlations from H3-21 to C-10, C-1, and C-5, as well as from H-5 to C-1 connected C-1 and C-5 to C 10 to form a six membered ring (A). HMBC correlations from H3-21 to C-9 also connected the C-9-C-11-C-12 spin system to C-10. HMBC correlations from H3-22 to C-8, C-9 and C-7 connected both C-7 and C-9 to C-8 and formed a second six membered ring (B). In addition, a correlation from H3-22 to C-14 as well as from H-7ax to C-14 indicated that the C-14 to C-16 spin system was also connected to C-8. HMBC correlations from H3-23 to C-13, C-12 and C-14 completed ring C. Correlations from both H3-23 and H-12ax to C-18 placed C-18-C-25 spin system adjacent to C-13. The final ring (D) was deduced by HMBC correlations from H2-24 to C-16, C-17 and C-18, thus connecting C-18 and C-16 via C-17 of the exomethylene group and establishing a tetracylic scalarene-type sesterterpene skeleton. The chemical shift of C-1 (δc 81.0) combined with one oxygen atom required by the molecular formula, placed a hydroxy group at C-1. This was confirmed by a HMBC correlation from OH proton (δH 4.04, observed in DMSO-d6) to C-1. The guanidine moiety at C-25 was confirmed by a HMBC correlation from H2-25 to the guanidine carbon C-2′, as well as the COSY correlation between NH-1′ and H2-25.
The relative configuration was determined by analysis of the 2D NOESY spectrum (Figure 2 and Table 1). NOESY correlations observed between H-11ax and H3-21, H3-22 and H3-23 as well as between H3-23 and H2-25 indicated all these groups to be in the β plane. This led to the determination of the relative configuration as 8S*, 10S* and 13S*. Correlations between H-9 and both H-5 and H-14 indicated H-5, H-9 and H-14 all to be in the α plane. NOESY correlations between H-5 and H-1, as well as between H-14 and H-18 indicated H-1 and H-18 to be in the α plane as well and determined the relative configuration as 1R*, 5S*, 9S*, 14S* and 18S*. The rings A, B, C and D were all-trans and the relative configuration of scytosclarol was determined as 1R*, 5S*, 8S*, 9S*, 10S*, 13S*, 14S* and 18S* as shown above.
Scytoscalarol (1) has a scalarane skeleton and showed antimicrobial activity against Bacillus anthracis (MIC 6 μM), Staphylococcus aureus (MIC 2 μM), Escherichia coli (MIC 30 μM), Candida albicans (MIC 4 μM) and Mycobacterium tuberculosis (MIC 110 μM). Scytoscalarol was also weakly cytotoxic (IC50 135 μM) in a Vero cell assay.
This is the first report of a scalarane sesterterpene from cyanobacteria. Scalaranes represent a common class of sesterterpenes found in various species of marine sponges of the order Dictyoceratida.8,12 12-keto-scalarane derivatives have also been found in sponge-feeding Glossodoris nudibranchs. It was assumed that certain Glossodoris nudibranchs can produce these compounds by enzymatic oxidation of dietary scalaranes.13 A biosynthetic study of scalarane sesterterpenes suggested that this class of compounds might be of microbial origin.14 Several previously reported metabolites from sponges have been found to be produced by symbiotic microorganism.15 However, there are no reports of the cyanobacterium Scytonema associated with sponges of the order Dictyoceratida. More research is needed to determine if cyanobacteria are involved in the production of the scalarane-type sesterterpenoids in marine sponges.
Scytoscalarol (1) is the first guanidine-bearing sesterterpene isolated from natural sources. Many guanidine-containing polyketide, peptides and alkaloids have been reported from terrestrial, freshwater and marine microganisms and invertebrates.16 Most reports of cyanobacterial guanidine bearing compounds are from arginine-containing linear or cyclic peptides as well as alkaloids.3 Some terpenoid guanidine alkaloids have been identified from higher plants, such as the galegines from Galega officinalis17 and pterogynidine and the nitensidines from Pterogyne nitens.18,19
The optical rotation was determined on a Perkin-Elmer 241 polarimeter. IR spectra were obtained on a Jasco FTIR-410 Fourier transform infrared spectrometer. 1H and 13C NMR spectra were obtained on a Bruker Avance AVII900 MHz NMR spectrometer with 5mm ATM CPTCI Z-gradient probe, referenced to the corresponding solvent peaks. Low resolution ESI mass spectra were obtained on a ThermoFinnigan TSQuantum Triple Quadrupole Mass Spectrometer. High resolution ESI mass spectra were obtained on a Thermo Electron LTQ-FT-ICR mass spectrometer.
Scytonema sp., a terrestrial cyanobacterium, was acquired from the Culture Collection of Algae at the University of Texas at Austin (UTEX 1163). The cyanobacterium was grown in 13 L of aerated inorganic media (Z media).20 Cultures were illuminated with fluorescent lamps at 1.93 klx with an 18/6 h light/dark cycle. The temperature of culture room was maintained at 22° C. After 4 weeks, the biomass of cyanobacteria was harvested by centrifugation and freeze-dried.
The freeze-dried biomass (2.88 g) was extracted by repeated maceration with CH2Cl2:MeOH (1:1) to yield 597.6 mg of crude extract. The crude extract showed inhibitory activity against B. anthracis (MIC 40 μg/mL). A portion of crude extract (572 mg) was fractionated on a diaion HP20SS column using a gradient with increasing amount of isopropanol in water to afford 9 fractions. Fraction 3 (eluted with 40% isopropanol) was the most active fraction against both B. anthracis (MIC 2.5 μg/mL) and M. tuberculosis (MIC 10.8 μg/mL). A white amorphous powder precipitated from fraction 3 upon drying under air flow. The precipitate was purified by filtration to afford 1 (15.4 mg).
white amorphous powder; [α]D +6 (c 0.41 MeOH); IR (neat) νmax 3341, 3189, 2935, 2848, 1666, 1388, 1339, 1035 cm-1; 1H NMR and 13C (see Table 1); HRESIMS m/z 416.3651 [M+H]+ (calcd for C26H46N3O, 416.3641).
Bacillus anthracis. The fractions and compound 1 were tested with concentrations from 100 μg/mL to 48.8 ng/mL using the previously described method.21 The minimal inhibitory concentration (MIC) sample was calculated as the lowest concentration that prevents visible bacterial growth. Mycobacterium tuberculosis. The inhibitory activity of fractions and compound 1 against M. tuberculosis was performed using the Microplate Alamar blue Assay (MABA).22 Virulent H37Rv strain was used in the assay. The MIC value was determined as the lowest sample concentration effecting an inhibition of ≥90%. Other organisms. The broth microdilution MIC method was used to test the activity of 1 against bacteria23 and Candida albicans.24
The cytotoxicity of 1 was evaluated using green monkey kidney cells (Vero).25 Cell viability was measured using the CellTiter 96 aqueous nonradioactive cell proliferation assay.
We thank Dr. S. Cho, B. Wan, Y. Wang, D. Wei, B. Becker and K. Sidell from the Institute for Tuberculosis Research (ITR) at UIC for performing antibacterial, antifungal and cytotoxicity assays and Dr. A. Mesecar at UIC for B. anthracis data. We also thank Dr. C. A. Crot from the Research Resources Center (RRC) at UIC for high resolution mass spectrometry. The 900 MHz NMR spectrometer was funded by NIH P41 grant GM68944. This research was supported by NIH grant R01GM0758556.
Supporting Information Available: 1D NMR spectra in MeOD-d4 and DMSO-d6, as well as a table of NMR data acquired in DMSO-d6. This material is available free of charge via the Internet at http://pubs.acs.org.