dibenzofurans and pseudodepsidones from the lichen
TRANSCRIPT
1
Dibenzofurans and Pseudodepsidones from the Lichen
Stereocaulon paschale Collected in Northern Quebec
Claudia Carpentier,† Emerson Ferreira Queiroz, ‡ Laurence Marcourt,‡ Jean-Luc Wolfender, ‡
Jabrane Azelmat, § Daniel Grenier, § Stéphane Boudreau, Normand Voyer*,†
† Département de Chimie and PROTEO, Université Laval, 1045 avenue de la Médecine, Québec
G1V 0A6, Canada
‡ School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Rue
Michel-Servet, CH-1211 Geneva 4, Switzerland
§ Groupe de Recherche en Écologie Buccale, Faculté de Médecine Dentaire, Université Laval,
2420 rue de la Terrasse, Québec G1V 0A6, Canada
Centre d’études nordiques, Département de Biologie, Université Laval, 1045 avenue de la
Médecine, Québec G1V 0A6, Canada
2
ABSTRACT. Chemical investigation of the methanol extract of the lichen Stereocaulon
paschale collected in Nunavik, Canada, led to the isolation and identification of two new
dibenzofurans (1 and 3) and eleven known lichen metabolites. The structures of the new
compounds were established by analysis of 1D and 2D NMR spectroscopic and high-resolution
mass spectrometric data. Herein, the first isolation of ascomatic acid dibenzofuran derivatives (1-
3) from a whole lichen organism is reported. In addition, some of the isolated metabolites
showed antibacterial activity against the oral pathogens Porphyromonas gingivalis and
Streptococcus mutans.
3
Over the last few decades, the expansion of shrub species in lichen-dominated ecosystems has
had a negative impact on lichen abundance. In Nunavik (subarctic Québec), this expansion is
mainly associated with the densification of dwarf birch (Betula glandulosa Michx.) stands.1,2
This species has seen its radial growth increase rapidly with the warming trend observed since
the 1990s.3 As a result, lichens such as Stereocaulon paschale (L.) Hoffm. (Stereocaulaceae)
appear to be under threat in a large portion of the subarctic region. Hence, it is important to carry
out phytochemical investigations on lichens of northern Quebec prior to their decline.
The genus Stereocaulon contains about 130 species; from the 40 species that have been
studied phytochemically,4 only 75 metabolites have been identified.5 For example, only a few
metabolites have been isolated from S. paschale previously, including ethyl haematommate,
methyl-β-orsellinate, haematommate, lobaric acid, and heteroglycan.6-8 Moreover, several
interesting biological activities were reported for metabolites isolated from S. alpinum (anti-
inflammatory,9 antioxidant,10 antibacterial,10 tyrosinase protein phosphatase 1B inhibition,11,12 5-
lipoxygenase inhibition,13 human tumor cell line cytotoxicity 12), S. halei (antioxidant4), S.
evolutum (antiviral – HCV5,14), and S. sasakii (antimitotic15).
The methanol extract was chosen in this first thorough phytochemical study of the lichen
Stereocaulon paschale as more material was available than the other extracts produced. Medium-
pressure liquid chromatography (MPLC-UV) fractionation of the methanol crude extract led to
the isolation and identification of two new dibenzofurans, 1 and 3, and eleven known lichen
metabolites. In this contribution, the antimicrobial properties of the isolated compounds against
the major pathogens, Streptococcus mutans, Porphyromonas gingivalis, and Candida albicans,
are also reported.
4
The methanol crude extract was analyzed by HPLC-PDA and HPLC-TOF-HRMS to identify
compounds that have already been isolated from the genus Stereocaulon.5,15-17 This dereplication
procedure provided an unambiguous assignment of the molecular formula of compounds 4-13
(Figure S76 and Table S1, Supporting Information). Comparison of these data with those
previously reported for compounds isolated from species of the genus Stereocaulon enabled the
dereplication of methyl-β-orsellinate (4), methyl-haematommate (5) and lobaric acid (13) as
metabolites frequently reported in Stereocaulon species. Similarly, compound 6 was identified as
sakisacaulon A from Stereocaulon sasakii, and compounds 7-11 as diphenyl ethers described in
S. azoreum and S. alpinum. Compounds (1-3) exhibited molecular formulas that could not be
associated with secondary metabolites previously identified in Stereocaulon lichens and were
isolated for de novo structure determination (Figure S76 and Table S1, Supporting Information).
The methanol extract (2.5 g) was fractionated by medium-pressure liquid chromatography
(MPLC) to isolate the potentially novel compounds 1-3. To ensure similar selectivity for a
precise separation prediction, an efficient scale-up from HPLC to MPLC was performed by
geometric transfer of the analytical HPLC conditions to preparative MPLC using
chromatographic calculations.18 This fractionation also led to the isolation of the other
constituents (4-13) that were identified as compounds previously described in the genus
Stereocaulon by the dereplication procedure used (Figure S76 and Table S1, Supporting
Information). Compounds 4, 5, and 13 were isolated directly from the MPLC fractionation
without any further purification. Methyl β-orsellinate (4), methyl haematommate (5) and lobaric
acid (13) were confirmed by 1D and 2D NMR, and HRMS.11,19 It has been reported that
monoaryl compounds 4 and 5 can be artifacts from the methanolysis of the depside atranorin.20
However, no trace of the depside atranorin was detected by HPLC-PDA-MS in the hexanes,
5
dichloromethane and methanol extracts. In addition, no trace of atranorin was observed by
HPLC-PDA-MS in a freshly prepared crude acetone extract. Therefore, compounds 4 and 5 are
not artifacts from methanolysis.
Further purification was achieved by semi-preparative HPLC and led to the isolation at the
milligram scale of ten compounds (1-3 and 6-12). The additional spectroscopic data obtained by
1D and 2D NMR confirmed the conclusions made by dereplication of compounds 6-12:
sakisacaulon A (6),15 methyl lobarin (7),10 esterified lobarin (8),17 methyl sakisacaulon (9),5,17
esterified sakisacaulon (10),17 anhydro sakisacaulon A (11),5,17 norlobaric acid (12)5. In addition,
the investigation showed that 2 corresponded to isostrepsilic acid, a dibenzofuran previously
isolated from mycobiont culture of Usnea orientalis.20 Diphenyl ethers (6-11) are closely related
to lobaric acid (13), the major compound of the crude extract, and norlobaric acid (12) is the
non-methylated derivative of 13. The co-occurrence in the lichen S. paschale of
pseudodepsidones 6-11 and the depsidone lobaric acid (13) suggests that they may be produced
by the cleavage of the depsidone linkage of lobaric acid (Figure S81, Supporting Information). 4
Compound 1 was isolated as a white-gray solid and the HRESIMS showed a molecular ion at
m/z 243.0662 [M-H]- (calcd for C14H11O4-, 243.0663), corresponding to C14H12O4. The 1H NMR
and HSQC spectra revealed the presence of a methyl group (δH/δC 2.83/21.8, H-1'), a
hydroxymethyl group (δH/δC 5.03/63.9, H-9'), and four aromatic protons (δH/δC 6.88/111.6,
6.81/96.7, 6.71/95.0 and 6.58/113.4 for H-8, H-6, H-4 and H-2, respectively). The coupling
constant values between H-2 and H-4 (J = 2.0 Hz) and between H-6 and H-8 (J = 2.2 Hz)
indicated that these four aromatic protons belong to two aromatic rings (A for H-2 and H-4 and
B for H-6 and H-8) and that they are in a meta position to each other. The HMBCs correlations
from the hydroxymethyl (H-9') to C-8, C-9 (C 134.7) and C-9a (C 114.8) allowed this
6
methylene to be located on ring B in an ortho position to H-8, whereas the methyl group H-1'
was placed on ring A in an ortho position to H-2 due to its correlations with C-1 (C 132.4), C-2
and C-9b (C 115.7). The remaining carbons were assigned to oxygenated sp2 quaternary carbons
as indicated by their 13C NMR chemical shifts (C 157.9, 157.8, 155.7 and 155.6 for C-5a, C-4a,
C-3 and C-7, respectively).21-23 On the basis of the molecular formula (C14H12O4), a dibenzofuran
structure was proposed. The NOESY correlations from the hydroxymethyl to the methyl H-1'
and H-8 and from the methyl to H-2 confirmed the structure of 1 as a new dibenzofuran, 9-
(hydroxymethyl)-1-methyldibenzofuran-3,7-diol. The UV spectrum of 1 (218, 239, 256 and 309
nm) was consistent with a dibenzofuran structure. Similar UV spectra were obtained for
norascomatic acid (or hypostrepsilic acid), hypostrepsilalic acid and strepsilin, three
dibenzofurans previously isolated from cultured mycobiont of Evernia esorediosa, S. japonium
and S. evolutum.5,21,22 The 1H NMR and 13C NMR spectra of compound 1 showed close
similarities to analogous data for isostrepsilic acid (2) (Table 1).20 When compared with
isostrepsilic acid (2), the 1H NMR spectrum of 1 displayed an additional aromatic proton (H-2),
which replaced the carboxylic acid group.
Compound 3 was isolated as a white solid. The HRESIMS showed a deprotonated molecular
ion at m/z 301.0362 [M-H]- (calcd for C15H9O7-, 301.0364), consistent with a molecular formula
of C15H10O7. The NMR data of 3 were very similar to those of isostrepsilic acid except that
signals for a hydroxymethyl unit were missing.20 The HMBC correlations from H-8 (H/C
7.01/110.8) to C-7 (C 156.4), C-6 (C 99.8), C-9a (C 113.5) and particularly C-9' (C 171.6)
supported the carboxylic acid function being positioned at C-9. All these data indicated that 3 is
a new dibenzofuran, 3,7-dihydroxy-1-methyldibenzofuran-2,9-dicarboxylic acid.
7
In this contribution, the phytochemical investigation of S. paschale has led to the first
isolation of ascomatic acid dibenzofurans derivatives (1-3) from a whole lichen organism.20-24
Indeed, isostrepsilic acid (2), norsacomatic acid (or hypostrepsilic acid), and hypostrepsilalic
acid have been isolated up to now only from mycobiont cultures of Usnea orientalis, Evernia
esorediosa, and Stereocaulon japonicum.20-22,24 Miyagawa et al. have suggested that the
dibenzofuran isostrepsilic acid is an intermediate compound in the oxidation pathway from
norascomatic acid to hypostrepsilalic acid.21 Based on these results, compounds 1, 3 and
isostrepsilic acid (2) appear to be derivatives of norsacomatic acid (or hypostrepsilic acid), and
might be produced by final modification including decarboxylation and oxidation reactions.
Isostrepsilic acid (2) could be produced by the oxidation of a methyl group of norsacomatic acid
to form a hydroxymethyl. Sequential steps of oxidation of this hydroxymethyl would lead to an
aldehyde (hypostrepsilalic acid) and finally to a carboxylic acid (3) (Scheme 1). Compound 1
could be formed from the decarboxylation of isostrepsilic acid (2). Structural similarities
between compounds 1, 3, and isostrepsilic acid (2) were also indicative of this close biosynthetic
relationship (Scheme 1).
Miyagawa et al. also suggested that the osmotic stress due to the culture conditions, could
enhance the formation of ascomatic-type dibenzofurans.20-22 The fact that dibenzofurans 1-3
occur naturally in S. paschale suggests that the extreme growing conditions in the Nunavik
region enhance the lichen’s ability to produce new secondary metabolites. HPLC-PDA-MS
analysis performed on a freshly prepared acetone extract demonstrated the presence of
dibenzofurans 1-3, indicating that these compounds are indeed occurring naturally in the lichen
S. paschale.
8
Interestingly, Bhattari et al. have reported that lobastin, a pseudodepsidone isolated from S.
alpinum, and lobaric acid were active against the Gram-positive bacteria, B. subtilis and S.
aureus.10 Based on those results, the antimicrobial activity of the isolated compounds from the
methanol extract were investigated against a fungus, Candida albicans and two bacteria:
Porphyromonas gingivalis and Streptococcus mutans, to identify new antimicrobial agents.
Those pathogens were chosen as they are involved in important oral infections, namely,
candidiasis, periodontal disease, and dental caries, respectively.25-27 Active compounds endowed
with a capacity to exert antimicrobial activity towards these oral pathogens have received
considerable attention as they may represent potential new therapeutic agents for the
prevention/treatment of oral infections. The MIC and MBC values measured are presented in
Table 2. None of the compounds studied was able to inhibit the growth of C. albicans at a
concentration of 80 µM. However, all pseudodepsidone-type metabolites (6-11) and lobaric acid
(13) showed moderate or weak antibacterial activities. The results suggest that the diphenyl
ethers 8 and 11, and the depsidone 13, have potential as natural antibacterial agents, and should
be investigated further.
EXPERIMENTAL SECTION
General Experimental Procedures. Analytical HPLC data were recorded on an Agilent
1260 Infinity equipped with a photodiode array detector. MPLC fractionation was performed
using a Biotage Isolera™ Prime system equipped with a SiliCycle SiliaSep ISO120 (256 x 42
mm i.d.) flash cartridge loaded with C18 as the stationary phase (40-63 µm). Semi-preparative
HPLC was carried out using a HP 1260 system with a photodiode array detector using an ACE
C18 column (5 µm, 250 x 10.0 mm i.d). The NMR spectra were recorded on an Agilent DD2 500
MHz spectrometer. Complete assignment was performed using 2D experiments (COSY, HSQC,
9
HMBC and NOESY). The chemical shifts (δ) are given in ppm with respect to the residual
solvent methanol-d4 signal (H 3,31, C 49,0 for 1H and 13C NMR, respectively), and coupling
constants (J) are given in Hz. High-resolution mass spectra (HRMS) were obtained using an
Agilent 6210 LC Time of Flight mass spectrometer with electrospray ionization (ESI) interface
in direct injection mode.
HPLC-PDA analyses were conducted on an Agilent 1260 Infinity equipped with an auto
sampler, a quaternary high-pressure mixing pump and a photodiode array detector.
Chromatographic analysis was performed with a Beckman ODS column (5 µm, 250 x 4.6 mm
i.d.) and 20 µL (10 mg/mL) of the crude extract was injected. The gradient was performed at a
flow rate of 1 mL/min with a mobile phase composed of H2O containing 0.1% TFA (A) and
MeOH (B). A linear gradient of 40% to 100% of B in 40 min followed by 100% of B for 10 min
was used to ensure a good separation. The column temperature was kept at 22.5 °C. The UV
absorbance was recorded at 254, 210, 220 and 280 nm, and the UV spectra (PDA) were recorded
between 190 and 400 nm (step, 2 nm).
HPLC-TOF-HRMS metabolites profiling of the crude extract were obtained on an Agilent
6210 LC time-of-flight mass spectrometer with an electrospray ionization (ESI) interface. The
ESI conditions were as follows: capillary voltage 4000 V, fragmentor voltage 175 V, skimmer
65 V, octopole RF peak voltage of 250 V, gas temperature of 350 °C, gas flow of 8 L/min, and
nebulizer gas pressure of 35 psi. Detection was performed in both the negative- and positive- ion
mode with a m/z range of 120-1000 and a scan time of 1 s. The mass spectrometer was calibrated
in the negative- and positive- modes using Agilent ESI-L Low Concentration Tuning Mix. The
separation was performed with a Phenomenex Ultracarb ODS column (5 µm, 150 x 4.6 mm i.d.),
and 15 µL (10 mg/mL) of the crude extract was injected. A splitter was used at the end of the
10
column to let pass only 17% of the mobile phase to the MS. The following solvent system was
used: H2O containing 0.1% formic acid (A) and MeOH (B).The constant flow rate was reduced
to 0.5 mL/min, so a shorter column was used and the separation conditions for the MS
metabolites profiling were determined by doing a geometric transfer of the HPLC-PDA
conditions. A linear gradient of 50% to 100% of B in 48 min followed by 100% of B for 12 min.
The UV absorbance was recorded at 210 and 254 nm, and the UV spectra (PDA) were recorded
between 190 and 400 nm (step, 2 nm).
Plant Material. The lichen Stereocaulon paschale was collected in Umiujaq, Québec,
Canada: (56° 32' 55.77'' N, 76° 32' 26.19'' O), in August 2014. The Umiujaq region is located at
the forest-tundra ecotone.28 In this region, lichens are progressively being replaced by shrub
species,29,30 a likely consequence of recent temperature warming.3 The specimens were collected
and identified by S.B. and Prof. Esther Lévesque (Centre d’études nordiques, Département des
Sciences de l’Environnement, Université du Québec à Trois-Rivières).31 The whole thallus was
air-dried and stored at room temperature for approximately two months.
Extraction and Isolation. The air-dried thallus (130.7 g) was ground in liquid nitrogen and
extracted successively under maceration and agitation at room temperature with hexanes (3 x
600 mL), dichloromethane (4 x 600 mL), and methanol (4 x 600 mL). The extracts were
concentrated under vacuum to yield, respectively, 0.39 g of a hexanes extract (0.29%), 3.22 g of
a dichloromethane extract (2.5%), and 7.01 g of a methanol extract (5.4%).
A portion of the methanol extract (2.5 g) was fractionated initially using a Biotage Isolera™
Prime system equipped with a SiliCycle SiliaSep ISO120 (256 x 42 mm i.d.) flash cartridge
loaded with C18 silica gel as the stationary phase (40-63 µm). The crude extract was mixed with
7.50 g of C18 reversed-phase silica gel (40-63 µm, carbon 17%, Siliabond C18 (17%), Silicycle)
11
and 2.25 g of Ottawa sand. To introduce the extract in the flash cartridge, the mixture was loaded
in an empty solid-load cartridge (60 mL, Silicycle) which was connected to the flash cartridge
using a plunger for a solid load cartridge (Siliasep plunger, 60 mL). The separation conditions
for the MPLC fractionation were determined by performing a geometric transfer of the HPLC-
PDA conditions.18 The solvent system used was (A) H2O containing 0.1% of TFA and (B)
MeOH. The UV absorbance was measured at 254 and 210 nm. A linear gradient from 40% to
100% in 350 min followed by 100% of B for 18 min at a constant flow rate of 10 mL/min was
performed and yielded 389 fractions. Fraction purity was monitored by HPLC-PDA. Those with
similar chromatogram were combined to afford nine fractions: (A = Fr 104-134, B= Fr 140-168,
C = Fr 199-220, D = Fr 264-273, E = Fr 273-287, F = Fr 288-292, G = Fr 303-313, H = Fr 314-
328 and I = Fr 335-365). Fractions C, D and I yielded 4 (100 mg), 5 (34 mg), and 13 (110 mg),
respectively.
Further purification was performed for fractions A, B, and E-H (Table S2, Supporting
Information). Semi-preparative HPLC was carried out using a HP 1260 system with a
photodiode array detector. The analytical tubing (i.d. 0.17 mm) of the system was replaced by
tubing with larger internal diameter (0.25 mm) to reduce the internal pressure created by the flow
rate. The final purification was performed with an Ace C18 column (5 µm, 250 x 10 mm i.d.) and
the following solvent system: (A) H2O - 0.l % TFA and (B) MeOH at a constant flow rate of
4.2 mL/min.
9-(Hydroxymethyl)-1-methyldibenzofuran-3,7-diol (1): amorphous white-gray powder;
UV (MeOH) λmax (log ɛ) 218 (3.99), 239 (3.91), 256 (3.73), 309 (3.75) nm; 1H and 13C NMR
data (CH3OH-d4), Table 1; HRESIMS m/z 243.0652 [M-H]- (calcd for C14H11O4-, 243.0663).
12
3,7-Dihydroxy-1-methyldibenzofuran-2,9-dicarboxylic acid (3): amorphous white powder;
UV (MeOH) λmax (log ɛ) 240 (3.96), 261 (3.69), 309 (3.47) nm; 1H and 13C NMR data (CH3OH-
d4), Table 1; HRESIMS m/z 301.0362 [M-H]- (calcd for C15H9O7-, 301.0364).
Determination of Minimal Inhibitory Concentrations and Minimal Microbicidal
Concentrations. Porphyromonas gingivalis ATCC 33277, Streptococcus mutans ATCC 25175,
and Candida albicans ATCC 28366 were used. Bacteria were grown in Todd-Hewitt broth (BBL
Microbiology Systems, Cockeysville, MD, USA) supplemented with 0.001% hemin and
0.0001% vitamin K (THB-HK). Candida albicans was cultivated in Yeast Nitrogen Base (YNB;
BBL Microbiology Systems) medium supplemented with 0.5% glucose. P. gingivalis was
incubated under anaerobic conditions (N2-H2-CO2/80:10:10), while S. mutans and C. albicans
were grown aerobically, all at 37 °C. Overnight cultures of microorganisms were diluted in fresh
broth medium to obtain an optical density at 660 nm (OD660) of 0.2. Samples (100 µL) were
added to the wells of a 96-well tissue culture plate containing 100 µL of serial dilutions (80 to 5
µM) of compounds. Control wells with no substance but with substance vehicle were also
inoculated. Penicillin G (Sigma-Aldrich Canada, Oakville, ON, Canada) and Nystatin (EMD
Biosciences Inc., San Diego, CA, USA) were used as reference controls for growth inhibition of
bacteria and C. albicans, respectively. After incubation for 24 h, the concentration of compounds
that caused complete growth inhibition was recorded as the minimal inhibitory concentration
(MIC). Ten-µL samples obtained from the wells showing no visible growth were spread on solid
culture plates to determine the minimal microbicidal concentration (MBC) of the compounds.
All the above assays were run in triplicate. Penicillin G was used for bacteria and nystatin for C.
albicans as reference controls for growth inhibition (Table 2).
13
14
Scheme 1. Proposed biosynthetic relationship between compounds 1-3.
15
Table 1. 1H NMR and 13C NMR Data for Compounds (1-3) in CH3OH-d4
(1)
(3)
isostrepsilic acid (2)
position δC δH (J in Hz) HMBC
δC δH (J in Hz) HMBC
δC δH (J in Hz)
1 132.4, C
136.2, C
133.0, C
2 113.4, CH 6.58 d (2.0) 3, 4, 9b, 1'
116.7, C
117.4, C
3 155.7, C
160.2, C
157.3, C
4 95.0, CH 6.71 d (2.0) 2, 3, 4a, 9b
95.9, CH 6.89 s 2, 3, 4a, 9b
95.7, CH 6.84 s
4a 157.8, C
159.0, C
158.3, C
5a 157.9, C
158.3, C
158.4, C
6 96.7, CH 6.81 d (2.2) 5a, 7, 8, 9a
99.8, CH 7.06 d (2.3) 5a, 7, 8, 9a
96.8, CH 6.85 d (2.2)
7 155.6, C
156.4, C
156.5, C
8 111.6, CH 6.88 d (2.2) 6, 7, 9a, 9'
110.8, CH 7.01 d (2.3) 6, 7, 9a, 9'
112.6, CH 6.94 d (2.2)
9 134.7, C
129.4, C
135.4, C
9a 114.8, C
113.5, C
114.3, C
9b 115.7, C
112.5, C
115.0, C
1' 21.8, CH3 2.83 s 1, 2, 9b
21,4, CH3 2.69 s 1, 2, 9b, 2'
19.8, CH3 2.67 s
2'
173.1, COOH
9' 63.9, CH2 5.03 s 8, 9, 9a
171.6, COOH
64.0, CH2 5.02 s
Table 2. Antimicrobial Activity of the Isolated Compounds
P. gingivalis S. mutans
MICb MBCc MIC MBC
(µm) (µm) (µm) (µm)
1 - - - -
2 - - - -
3 - - - -
4 - - - -
5 - - - -
6 - - 80 80
7 80 80 80 80
8 40 40 80 -
9 80 80 80 80
10 80 80 80 -
11 20 20 10 20
13 80 80 20 80
penicillin Ga 0.29 2.3 0.15 4.7
aPositive control. bMIC, mininal inhibitory concentration.
cMB(M)C, minimal bactericidal (microbial) concentration.
- : no significant activity at 80 µM; MIC > 80 µM.
16
ASSOCIATED CONTENT
Supporting Information. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel.: + 1 418 656 3613. Fax: + 1 418 656 7916. E-mail: [email protected].
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was supported by NSERC and the FRQNT. The authors thank PROTEO, FRQNT and
NSERC for graduate scholarships. The authors are also thankful to M. Pierre Audet for his help
in NMR and MS analyses.
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