Xanalteric Acids I and II and Related Phenolic Compounds from an Endophytic Alternaria sp.
Isolated from the Mangrove Plant Sonneratia alba
Julia Kjer,†Victor Wray,‡RuAngelie Edrada-Ebel,§Rainer Ebel,⊥Alexander Pretsch,|Wenhan Lin,*,3and Peter Proksch*,†
Institut fu ¨r Pharmazeutische Biologie und Biotechnologie, Heinrich-Heine-UniVersita ¨t Du ¨sseldorf, UniVersita ¨tsstrasse 1, Geb. 26.23, 40225
Du ¨sseldorf, Germany, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany, Strathclyde Institute of
Pharmacy and Biomedical Science, UniVersity of Strathclyde, The John Arbuthnott Building, 27 Taylor Street, Glasgow G4 0NR, U. K.,
Department of Chemistry, UniVersity of Aberdeen, Meston Building, Meston Walk, AB24 3UE, Aberdeen, U.K., SeaLife Pharma GmbH,
Technopark 1, 3430 Tulln, Austria, and National Research Laboratories of Natural and Biomimetic Drugs, Peking UniVersity, Health Science
Center, 100083 Beijing, People’s Republic of China
ReceiVed July 11, 2009
Two new 10-oxo-10H-phenaleno[1,2,3-de]chromene-2-carboxylic acids, xanalteric acids I (1) and II (2), and 11 known
secondary metabolites were obtained from extracts of the endophytic fungus Alternaria sp., isolated from the mangrove
plant Sonneratia alba collected in China. The metabolites were confirmed to be of fungal origin, and the structures of
the new natural products were unambiguously elucidated on the basis of extensive one- and two-dimensional NMR
spectroscopic studies and mass spectrometric analysis. The two new compounds 1 and 2 exhibited weak antibiotic
activity against multidrug-resistant Staphylococcus aureus. Altenusin (3) displayed broad antimicrobial activity against
several additional multidrug-resistant bacterial and fungal strains.
Terrestrial fungi are known as rich sources of biologically active
secondary metabolites that are indispensable for medicinal and
agricultural applications. However, due to the frequent rediscovery
of previously described metabolites of fungi from traditionally
investigated habitats, the attention of natural product researchers
has been increasingly attracted to fungi from less common sources
and ecological niches such as fungal endophytes.1-3An endophytic
fungus spends a part or all of its whole life cycle inside living tissues
of a host plant and establishes a relationship with its host that may
range from symbiotic to slightly pathogenic. The fungus obtains
nutrients and protection from the host, whereas it is generally
assumed that its metabolites may enhance the host’s growth and
competitiveness.3,4In continuation of our search for new bioactive
natural products from fungal endophytes,5-8we isolated an
Alternaria strain from the leaves of Sonneratia alba, a mangrove
tree mainly used for the production of timber, but also as a
traditional medicine against injuries of the skin or intestinal
parasites.9In their natural habitat, mangroves are prone to numerous
stress factors such as changing tides and salinity as well as abundant
(pathogenic) microorganisms and insects, which demand a special
fitness of the plant.10,11Therefore they epitomize an ecological
niche and potential habitat of specific microorganisms. Alternaria
is a cosmopolitan fungal genus, and several species are known as
plant pathogens. Although several endophytic Alternaria strains
have been investigated before,5isolates from particular habitats
frequently yield novel natural products. This prompted us to
investigate the compounds produced by the mangrove-derived
Alternaria sp. We isolated two new secondary metabolites (1 and
2) as well as several known polyketides and report on the structure
elucidation of the new natural products. Their fungal origin was
confirmed, and this is the first report of fungal metabolites featuring
a 10-oxo-10H-phenaleno[1,2,3-de]chromene skeleton hitherto only
reported from bacteria.
Extracts of Alternaria sp., when grown on solid rice medium,
yielded two new carboxylic acids, xanalteric acids I (1) and II (2),
together with the known fungal metabolites alternarienonic acid,
altenusin (3), altenuene, 4′-epialtenuene, alternariol, altertoxin I (4),
2,5-dimethyl-7-hydroxychromone, and alternarian acid. When the
fungus was grown in liquid medium, compounds 1, 2, 3, altenuene,
alternariol, and 4 were likewise detected. Additionally, the known
compounds alternariol-5-O-methyl ether and the perylene deriva-
tives alterperylenol (5) and stemphyperylenol (6) were also obtained.
The latter compounds were missing when the fungus was cultivated
on rice medium. All known compounds were identified by their
spectroscopic characteristics involving one- and two-dimensional
NMR spectroscopy and mass spectrometry as well as comparison
with literature data.5,12-15
Compound 1, obtained as a red powder, had the molecular
formula C20H12O7as determined by HRESIMS (m/z 365.0656 [M
+ H]+), corresponding to eight double-bond equivalents. Taken
together with the UV/vis spectrum, which exhibits maxima at 413
and 514 nm, these data suggested that 1 featured a complex aromatic
ring system, which is in accordance with three sets of ortho-coupled
* Corresponding authors. (P.P.) Tel: ++49-211-81-14163. Fax: ++49-
211-81-11923. E-mail: email@example.com. (W.L.) Tel: ++86-
10-82806188. Fax: ++86-10-82802724. E-mail: firstname.lastname@example.org.
†Heinrich-Heine Universita ¨t, Du ¨sseldorf.
‡Helmholtz Centre for Infection Research, Braunschweig.
§University of Strathclyde, Glasgow.
⊥University of Aberdeen.
|SeaLife Pharma, Tulln.
J. Nat. Prod. XXXX, xxx, 000
10.1021/np900417g CCC: $40.75
XXXX American Chemical Society and American Society of Pharmacognosy
1H and13C NMR, HMBC, and NOESY Spectroscopic Data for Xanalteric Acids I (1) and II (2)
xanalteric acid I (1)
xanalteric acid II (2)
6.17, d (3.0)
5.93, d (2.3)
3b, 12a, 12b,
A: 4.22, d (17.3)
B: 3.66, d (17.3)
A: 3.88, d (18.3)
B: 3.71, d (18.0)
3b, 2, 12a, 12b
2, 3b, 9b, 12a,12b, COOH
5.06, d (3.0)
1, 3a, 12b, COOH-2
7.46, d (9.1)
7.35, d (7.4)
3a, 4, 6a
7.43, d (8.8)
7.42, d (8.9)
3a, 4, 6a
8.24, d (8.8)
8.16, d (9.3)
3a, 3b, 4, 6a, 6b
8.24, d (9.1)
8.24, d (8.8)
3b, 4, 6b, 12b
9.00, d (9.1)
9.04, d (7.5)
6a, 6b, 9, 9a,
8.98, d (9.5)
9.07, d (8.6)
6a, 9, 9b
7.38, d (9.1)
7.35, d (7.4)
6b, 9, 9a, 9b
7.38, d (9.1)
7.37, d (9.2)
6b, 9, 9a
6.94, d (10.0)
6.94, d (9.4)
9, 9a, 9b, 12a
6.92, d (9.8)
6.89, d (9.8)
8.74, d (10.1)
8.62, d (7.3)
6b, 9a, 9b,10,
8.53, d (10.0)
8.62, d (9.8)
9b, 10, 12a, 12b
3b, 12a, 12b,
aMeasured in MeOH-d4.bmeasured in DMSO-d6at 500 MHz.
Journal of Natural Products, XXXX, Vol. xxx, No. xxNotes
aromatic protons (δH7.46/8.24, 9.00/7.38, and 6.94/8.74; H-5/H-
6, H-7/H-8, and H-11/H-12, respectively; Table 1) that were
observed in the1H NMR spectrum. Two vicinal coupling protons
at δH6.17 and 5.06 (H-1/H-2) form a fourth spin system, with their
coupling constant of 3.0 Hz suggesting that at least one of them
must have a pseudoequatorial orientation. When the
spectrum was measured in DMSO-d6, four hydroxy protons (δH
3.3, 5.75, 8.73, and 15.12; OH-1, OH-4, COOH, and OH-9) were
detected. The striking downfield shift of OH-9 is typical of a strong
hydrogen bond, which is in accordance with the downfield shift of
C-10 (δC 191.0) observed in the13C NMR spectrum, which is
indicative of an aromatic carbonyl group. The COSY spectrum
revealed the correlation between OH-1 (overlapping with water
peak) and H-1 (δH5.93). HMBC data allowed the arrangement of
the different spin systems to be deduced. The quaternary carbons
C-9a (δC 112.9) and C-9b (δC 126.4) show correlations to the
aromatic doublets H-8 (δH7.38) and H-11 (δH6.94), and H-7 (δH
9.00) and H-12 (δH8.74), respectively. Moreover, H-7 and H-5
(δH7.46) show correlations with C-6a (δC126.9), whereas H-8
and H-6 (δH8.24) correlate with C-6b (δC122.9). Protons H-6 and
H-1 both exhibit correlations to C-3b. The correlations of H-11
and H-1 to C-12a and of H-12 and H-2 to C-12b, respectively,
provide the remaining connections of the five ring core structure
of 1. NOESY correlations that were observed between the aromatic
protons H-6 and H-7, and H-12 with H-1, confirm this result. The
presence of the oxygen in the heterocyclic ring system is cor-
roborated by the downfield shifts of the aromatic carbons C-3a (δC
140.6) and C-2 (δC81.7). The presence of a carboxyl group at C-2
is indicated by the downfield shift of the carbon (δC174.7), which
correlates with H-1 as well as with H-2. ESIMS/MS experiments
supported this structural feature by the observed facile decarboxy-
lation of 1 in the negative as well as in the positive ionization mode.
Hence, 1 was found to be the new natural product 1,4,9-trihydroxy-
lic acid, for which we propose the name xanalteric acid I.
Compound 2, also obtained as a red powder, displayed physical
characteristics comparable to those of 1. The HRESIMS again
exhibited a peak at m/z 365.0656 [M + H]+, and in the MS/MS
experiment a facile decarboxylation of the compound was observed.
1H NMR and HMBC spectra revealed very similar signals for the
three spin systems of the aromatic protons H-5/H-6, H-7/H-8, and
H-11/H-12 as found for 1, whereas the fourth spin system attracted
our attention due to its upfield shift (compared to 1, Table 1) and
the large coupling constant of 17.3 Hz, indicating the presence of
geminal protons. This observation was confirmed by a HMQC
experiment, which displayed correlations of protons H-1A and H-1B
to C-1 at δC34.8. Accordingly, 2 (2,4,9-trihydroxy-10-oxo-2,10-
dihydro-1H-phenaleno[1,2,3-de]chromene-2-carboxylic acid) is an
isomer of 1, for which we propose the name xanalteric acid II.
This is the first report of fungal substances featuring a 10H-
phenaleno[1,2,3-de]chromene skeleton. A structurally similar com-
pound, xanosporic acid (8), was previously isolated as a degradation
product of the fungal phototoxic compound cercosporin (7), which
had been added to the fermentation broth of Xanthomonas campes-
tris pv zinniae.16-18Cercosporin (7) was catabolized by the
bacteria, thereby yielding the corresponding acid (8), which was
extracted from the fermentation broth and identified after lac-
toniszation (structure of lactone not shown) (Figure 1). Viewed
against this background it appeared possible that xanalteric acids I
(1) and II (2) could have originated in a similar way (Figure 2),
especially as the isolated metabolites 4-6 seem to support the
presence of the putative precursors. Potentially endosymbiotic
bacteria that reside in fungal hosts could be involved, as recently
reported for the fungus Rhizopus sp. inhabited by Burkholderia
sp.19,20This possibility of a bacterial origin of xanalteric acids I
and II could, however, be discarded on the basis of two experiments:
When the well-known antibiotics penicillin G and chloramphenicol
were added to the fermentation broth of Alternaria sp., production
of xanalteric acids by the fungus still proceeded. When a PCR
experiment involving total DNA of the fungus and the eubacterial
primers 27f and 149r was performed, no bacterial DNA could be
identified following gel electrophoresis.
Extracts of Alternaria sp. obtained by fermentation on rice or in
liquid growth media exhibited strong cytotoxicity against L5178Y
cells at a concentration of 10 µg/mL. Recent investigations of our
group had already indicated the remarkable cytotoxic effects of 3,
alternariol, and alternariol-5-O-methyl ether, whereas only marginal
or no cytotoxic activity had been found for alternarienonic acid,
altenuene, 4′-epialtenuene, and 2,5-dimethyl-7-hydroxychromone.5
As 5 and 6 were obtained in minute amounts that prevented in
Vitro testing, only 1 and 2 together with the known alternarian acid
were investigated for their cytotoxicity against L5178Y cells using
the MTT assay. However, all three compounds exhibited only
marginal activity (Table 2).
Additionally, all compounds except 2,5-dimethyl-7-hydroxy-
chromone were tested for their antibiotic activity against multire-
sistant bacterial and fungal strains (E. coli, Klebsiella pneumoniae,
Enterococcus faecium, Enterococcus cloacae, Staphylococcus au-
reus, Streptococcus pneumonia, Pseudomonas aeruginosa, Acine-
tobacter baumanii, Candida albicans, Candida krusei, Aspergillus
faecalis, and Aspergillus fumigatus). In these studies xanalteric acids
I (1) and II (2) showed weak antibacterial activity against
Staphylococcus aureus with MIC values of 250 -125 µg/mL.
Altenusin (3) exhibited broad antimicrobial activity against several
resistant pathogens with MIC values of 31.25-125 µg/mL (Table
3), whereas all other compounds lacked antibiotic properties toward
the tested bacteria and fungi.
General Experimental Procedures. Optical rotations were deter-
mined on a Perkin-Elmer-241 MC polarimeter. IR data were recorded
on a Nicolet 4800 FTIR-FMIR spectrometer using a diamond as ATR
crystal.1H,13C, and two-dimensional NMR spectra were recorded on
Figure 1. Structures of cercosporin (7) and xanosporic acid (8).
Figure 2. Possible precursor of xanalteric acid I (1).
2. Cytotoxicity Assay for
L5178Y growth in %
(at 10 µg/mL)
Alternaria rice EtOAc
Alternaria liquid EtOAc
xanalteric acid I (1)
xanalteric acid II (2)
NotesJournal of Natural Products, XXXX, Vol. xxx, No. xx C
Bruker ARX 500 or AVANCE DMX 600 NMR spectrometers. ESIMS
was conducted on a Finnigan LCQ Deca mass spectrometer, and
HRESIMS spectra were obtained on a FTHRMS-Orbitrap (Thermo-
Finnigan) mass spectrometer. HPLC analysis was performed using a
HPLC (Dionex P580) system coupled to a photodiode array detector
(UVD340S). Routine detection was at 235, 254, 280, and 340 nm. The
separation column (125 × 4 mm, L × i.d.) was prefilled with Eurospher-
10 C18 (Knauer, Germany), and the following gradient was used
(MeOH, 0.02% H3PO4 in H2O): 0 min, 10% MeOH; 5 min, 10%
MeOH; 35 min, 100% MeOH; 45 min, 100% MeOH. Solvents were
distilled before use, and spectral grade solvents were used for
spectroscopic measurements. TLC was performed on TLC plates
precoated with silica Si 60 F254(Merck, Germany). The compounds
were detected and fractions monitored by their UV absorbance at 254
and 366 nm and by spraying the plates with anisaldehyde reagent.
Fungal Material. The fungus Alternaria sp. was isolated from fresh
healthy leaves of Sonneratia alba J.E. Smith (family Sonneratiaceae,
order Myrtales). The plant material was collected in Dong Zhai Gang
Mangrove Garden on Hainan Island, China, in October 2005. A voucher
specimen (code no. 6) was deposited at one of the authors’ laboratory
(P.P.). Following surface sterilization of the leaves with 70% EtOH
for 2 min the samples were air-dried under a laminar air flow. To
distinguish the remaining epiphytic fungi from endophytic fungi, an
imprint of the leaf surface on biomalt agar was performed. Small tissue
samples from inside the leaves were cut aseptically and pressed onto
agar plates containing an antibiotic to suppress bacterial growth
(composition of isolation medium: 15 g/L malt extract, 15 g/L agar,
and 0.2 g/L chloramphenicol in distilled water, pH 7.4-7.8, adjusted
with 10% NaOH or 36.5% HCl). After incubation at room temperature,
the fungal strain under investigation was found to grow exclusively
out of the leaf tissue, but not on the agar plates taken from the imprint
of the leaf surface. From the growing cultures pure strains of Alternaria
sp. were isolated by repeated reinoculation on malt agar plates.
Identification of Fungal Cultures. The fungus (strain no. JCM9.2)
was identified using a molecular biological protocol by DNA amplifica-
tion and sequencing of the ITS region as described previously.21The
strain was identified as Alternaria sp.; however, due to the lack of
similar sequences in GenBank, identification of the strain to the species
level was not possible. A voucher strain is kept at the one of the authors’
laboratory (P.P.). The sequence data have been submitted to and
deposited at GenBank (accession no. FJ465171).
Isolation of bacterial 16S rDNA genes was achieved using a
modification of the protocol mentioned above. PCR was performed
with the primer pair 27f (AGAGTTTGATCCTGGCTCAG) and 149r
(GGTTACCTTGTTACGACTT)22using the following amplification
program: 1: 94 °C for 10 min, 2: 94 °C for 2 min, 3: 65 °C for 1.30
min (steps 2-4 were repeated 35 times), 4: 65 °C for 10 min, 5: 4 °C
until workup. Escherichia coli and Bacillus subtilis were used as
Cultivation. Mass growth of the fungus for the isolation and
identification of new metabolites was carried out in Erlenmeyer flasks
(1 L each). The fungus was grown on rice solid medium (to 100 g
commercially available rice was added 110 mL of distilled water, and
the mixture was kept overnight prior to autoclaving, 20 flasks) or in
liquid Wickerham medium (3 g yeast extract, 3 g malt extract, 5 g
peptone, 10 g glucose, distilled water added up to 1000 mL, pH
7.2-7.4, adjusted with 10% NaOH or 36.5% HCl, liquid medium/flask;
300 mL per flask, 20 flasks) at room temperature under static conditions
and daylight for 45 or 28 days, respectively.
To suppress the potential growth of bacteria, chloramphenicol at a
concentration of 200 mg/L or penicillin G at a concentration of 500
mg/L was added in a second experiment to the liquid Wickerham
medium mentioned above.
Extraction and Isolation. The rice culture was extracted with
EtOAc. The extract obtained was dried and partitioned between
n-hexane and 90% MeOH. The 90% MeOH-soluble material (4.6 g)
was then fractionated by vacuum-liquid chromatography (VLC) over
silica gel using a step gradient method of elution employing CH2Cl2
and MeOH as solvent systems. Fraction 3 (80% CH2Cl2) was chro-
matographed over silica gel using CH2Cl2and MeOH (90:10, v/v) as
eluent mixture. Alternarienonic acid (30.5 mg) was obtained from the
combined fractions 5 and 6 following purification over a Sephadex
LH-20 column with MeOH as eluent. Fractions 3 and 4 were combined
and rechromatographed via normal-phase VLC using a step gradient
of CH2Cl2 and MeOH as mobile phase. Fraction 3 from the silica
column yielded altenusin (3, 18.8 mg), and fraction 5 was further
purified over a RP-18 column and H2O/MeOH (7:3, v/v) to give
altenuene (3.0 mg; [R]20D-62 (c 0.02, MeOH)), 4′-epialtenuene (2.2
mg; [R]20D -140 (c 0.02, MeOH)), and 2,5-dimethyl-7-hydroxy-
chromone (1.1 mg) after semipreparative HPLC over a RP-18 column
with MeOH/H2O (25:75, v/v) as eluent. Alternariol (36.1 mg) and
altertoxin I (4, 4.3 mg) were obtained from fraction 7 and final
purification by semipreparative HPLC over a RP-18 column with
MeOH/H2O (35:65, v/v) as eluent. VLC fractions 5 to 7 were combined
and purified over a Sephadex LH 20 column with MeOH as mobile
phase, followed by semipreparative HPLC over a RP-18 column with
a mixture of MeOH/H2O as eluent to yield alternarian acid (5.7 mg).
The new compounds xanalteric acid I (1, 4.5 mg) and xanalteric acid
II (2, 5.7 mg) were obtained from the combined VLC fractions 5 to 7
after final purification by semipreparative HPLC over a RP-18 column
with a gradient of MeOH/H2O as eluent.
For the extraction of natural products from the liquid culture, culture
filtrates and mycelia were collected and extracted with EtOAc or MeOH,
respectively. The MeOH portion was then taken to dryness and
partitioned between water and successively n-hexane, EtOAc, and
n-BuOH. The EtOAc crude fraction (1.5 g) was chromatographed by
VLC over silica gel using CH2Cl2/MeOH gradient elution. Alternariol-
5-O-methyl ether (6.7 mg) was obtained from the combined fractions
6, 7, and 8 (60, 50, 40% CH2Cl2) after rechromatography over Sephadex
LH20 with MeOH as eluent. The VLC fractions 3 and 4 (95 and 90%
CH2Cl2) were also combined and further purified using Sephadex LH20
and CH2Cl2/MeOH (50:50, v/v) as eluent and subsequent semiprepara-
tive HPLC over RP-18 material with a gradient of MeOH/H2O as eluent
to yield 4, 5 (1.3 mg), and 6 (1.8 mg).
Fractionation of the extracts obtained from fermentation in liquid
medium containing antibiotics was achieved by separation over Diaion
HP-20 material using a step gradient of water and methanol.
Xanalteric acid I (1): dark red powder; [R]20D -120 (c 0.03,
MeOH); UV λmax(log ?) 204.4 (4.21), 231.2 (4.23), 261.8 (4.24), 410.4
(4.06), 513.7 (3.54) nm;1H,13C NMR see Table 1; ESIMS positive
m/z 365.3 [M + H]+, 319.2 [M + H - CO2]+, 303.4 [M + H - CO2
- H2O]+, negative m/z 363.0 [M - H]-, 301.3 [M - H - CO2-
H2O]-; HRESIMS m/z 365.0656 [M + H]+(calcd for C20H13O7,
Xanalteric acid II (2): dark red powder; [R]20D +40 (c 0.019,
MeOH); UV λmax(log ?) 204.3 (4.13), 228.2 (4.22), 260.8 (4.24), 408.5
(4.08), 503.1 (3.54) nm; IR (ATR) νmax3045, 2868, 2688, 1610, 1584,
1517, 1471, 1224 cm-1;1H,13C NMR see Table 1; ESIMS positive
m/z 365.2 [M + H]+, 347.0 [M + H - H2O]+, 319.2 [M + H - CO2]+,
303.4 [M + H - CO2- H2O]+, negative m/z 363.0 [M - H]-, 301.3
[M - H - CO2- H2O]-; HRESIMS m/z 365.0656 [M + H]+(calcd
for C20H13O7, 365.0661).
Cell Proliferation Assay. Cytotoxicity was tested against the
L5178Y mouse lymphoma cell line using the microculture tetrazolium
(MTT) assay as described earlier.23,24Experiments were repeated three
times and carried out in triplicate. As negative controls, media with
0.1% (v/v) EtOH were included in all experiments.
Determination of Minimal Inhibitory Concentration (MIC). Tests
were carried out according to the EUCAST (http://www.eucast.org)
criteria in a dilution assay against the following multidrug-resistant
pathogens: Escherichia coli, Enterococcus faecium, Enterococcus
cloacae, Staphylococcus aureus, Streptococcus pneumonia, Pseudomo-
nas aeruginosa, Klebsiella pneumonia, Candida albicans, Candida
krusei, Aspergillus faecalis, and Aspergillus fumigatus. Compound
Table 3. Antimicrobial Activities for Isolated Compounds from Alternaria sp.
S. pneum.E. faecium E. cloacaeA. faecalis C. albicans
xanalteric acid I (1)
xanalteric acid II (2)
Journal of Natural Products, XXXX, Vol. xxx, No. xxNotes
concentrations were between 250 and 0.48 µg/mL. The minimal Download full-text
inhibitory concentration (MIC) of a substance was defined as the lowest
concentration at which bacterial or fungal growth was inhibited.
Acknowledgment. This project was supported by grants of the
BMBF and MOST awarded to P.P. and W.L. We are grateful to Dr.
Peters and co-workers for NMR measurements and Prof. W. E. G.
Mu ¨ller and co-workers (Johannes Gutenberg Universitaet Mainz,
Germany) for performance of the MTT assay. We also acknowledge
the Official Medicines Control Laboratory (OMCL) of the Institute for
Public Health Nordrhein-Westfalen for their support recording the IR
Supporting Information Available: Details of the HRMS spectrum
of xanalteric acid I, of the 1D and 2D1H (COSY, ROESY) and 2D
13C (HMQC, HMBC) NMR spectra of xanalteric acids I and II, and of
the 1D13C spectrum of xanalteric acid II are provided. This material
is available free of charge via the Internet at http://pubs.acs.org.
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