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Pestalone, a New Antibiotic Produced by a Marine Fungus in Response to
Bacterial Challenge
Mercedes Cueto,†Paul R. Jensen,†Chris Kauffman,†William Fenical,*,† Emil Lobkovsky,‡and Jon Clardy⊥,‡
Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California at San Diego,
La Jolla, California 92093-0204, and Department of Chemistry and Chemical Biology, Cornell University,
Ithaca, New York 14850
Received May 30, 2001
The isolation and structure determination of a new chlorinated benzophenone antibiotic, pestalone (1),
is described. The new compound was produced by a cultured marine fungus only when a unicellular
marine bacterium, strain CNJ-328, was co-cultured in the fungal fermentation. The fungus, isolated from
the surface of the brown alga Rosenvingea sp. collected in the Bahamas Islands, was identified as an
undescribed member of the genus Pestalotia. The structure of 1, initially assigned with only modest
confidence by combined spectral and chemical data, was confirmed by single-crystal X-ray analysis.
Pestalone (1) exhibits moderate in vitro cytotoxicity in the National Cancer Institute’s 60 human tumor
cell line screen (mean GI50 )6.0 µM). More importantly, pestalone shows potent antibiotic activity against
methicillin-resistant Staphylococcus aureus (MIC )37 ng/mL) and vancomycin-resistant Enterococcus
faecium (MIC )78 ng/mL), indicating that pestalone should be evaluated in advanced models of infectious
disease.
The development of resistance toward current antibiotics
continues to be a significant problem in the treatment of
infectious disease,1-4and therefore the discovery and
development of new antibiotics is evolving as a high priority
in biomedical research. As part of a continuing program
to evaluate the drug potential of marine-derived microor-
ganisms5and to develop new methods to enhance second-
ary metabolite production, we have begun exploring the
use of mixed fermentation for the discovery of antibiotics
that show efficacy against drug-resistant pathogens. In this
report, we describe the isolation and characterization of
pestalone (1), a new benzophenone antibiotic that exhibits
potent antibacterial activity against drug-resistant bacte-
ria. This antibiotic was obtained from the mixed fermenta-
tion of a marine deuteromycete (strain CNL-365, Pestalotia
sp.) and an unidentified, antibiotic-resistant marine bac-
terium. It was not detected when either strain was cultured
individually.
Mixed fermentation has been used in the food industry6
and to enhance enzyme production;7however, it is not clear
that this method has been used extensively by the phar-
maceutical industry for secondary metabolite discovery.
Given that antibiotics may be produced in nature to provide
a competitive advantage,8it is possible that the pathways
responsible for the biosynthesis of certain compounds are
regulated by factors elicited by one microbe and detected
by another. This proposal is supported by evidence that
antibiotic production can be induced in response to micro-
bial antagonism9and that the production of specific
secondary metabolites can be increased up to 400-fold when
strains are grown in the presence of an antagonist.10 The
isolation of pestalone (1) demonstrates that mixed fermen-
tation can lead to the discovery of the new antibiotics and
suggests that this method may have utility for drug
discovery.
Pestalone (1) was isolated as yellow crystals which
showed molecular ions at m/z438/440/442 by LREIMS,
which demonstrated the characteristic isotope molecular
ions for two chlorine atoms in the molecule. The molecular
formula was established as C21H20O6Cl2by HRFABMS
[M +H]+m/z439.0731: calcd for C21H20O635Cl2439.0715
(11 degrees of unsaturation). The infrared spectrum of 1
showed typical absorptions for an aromatic aldehyde (1635
cm-1), a ketone (1620 cm-1), and hydroxyl groups (3225
cm-1). The UV spectrum, which showed broad absorptions
at 340 nm, confirmed the presence of an aromatic ring with
an extended chromophore.
The 1H NMR spectrum of 1(Table 1) showed the
presence of two olefinic methyl groups (δ1.34, 3H, s; 1.48,
3H, s); one aromatic methyl group (δ2.50, 3H, s); a methoxy
group (δ3.30, 3H, s); three exchangeable phenolic protons
* To whom correspondence should be addressed. Tel: (858) 534-2133.
Fax: (858) 558-3702. E-mail: wfenical@ucsd.edu.
⊥To whom correspondence regarding X-ray analysis should be addressed.
Tel: (607) 255-7583. Fax: (607) 255-1253. E-mail: JCC12@cornell.edu.
†Scripps Institution of Oceanography.
‡Cornell University.
1444 J. Nat. Prod. 2001, 64, 1444-1446
10.1021/np0102713 CCC: $20.00 © 2001 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/19/2001
(δ10.1, 1H, s; 11.2, 1H, s; 13.2, 1H, s); and an aldehyde
proton (δ9.80, 1H, s). The 1H NMR COSY spectrum showed
that two methylene protons (δ3.10, 2H, bd, J)6.9 Hz)
were coupled to an olefinic proton at δ4.98 (1H, t, J)6.9
Hz). The olefinic proton showed further allylic coupling to
two methyl groups at δ1.34 and 1.48, thus indicating the
presence of an isopentene functional group.
The 13C NMR spectrum showed signals at δ202.4 and
192.0, which were assigned to ketone and aldehyde car-
bonyls. Three methyl groups resonating at δ17.6, 19.2, and
25.8; one methoxy carbon at δ61.6; one methylene at δ
26.1; two olefinic carbons at δ103.2 and 123.0; and 12
quaternary aromatic carbon signals were also observed.
NMR experiments, including 2D COSY, HMQC, and HMBC,
confirmed the presence of an isoprene fragment and linked
this group to an aromatic ring with a phenolic carbon on
the adjacent carbon (C-5). The chemical shift of the
aromatic proton at C-4, and HMBC correlations of this
proton to C-6 and C-2, established the presence of a
pentasubstituted aromatic ring (C-1 to C-6) with the
isopentenyl functionality at C-6. Establishing the substitu-
tion pattern of the second aromatic ring, which involved
assigning the positions of a methyl group, two chlorine
atoms, a methoxy group, and a phenolic hydroxyl group,
was not accomplished with confidence. Although the sub-
stitution pattern could be assigned by analysis of carbon
chemical shift data, the lack of substantive HMBC cor-
relations made conclusive assignments impossible.
To confidently assign the substituent pattern on the
second ring (C-7 to C-12) of pestalone (1), an X-ray
diffraction experiment was undertaken which solved the
structure in an unambiguous manner. The X-ray perspec-
tive drawing of the structure of pestalone is shown in
Figure 1. Two intramolecular H-bonds exist between
O(1)-H and O(6) of 2.62 Å and between O(5)-H and O(3) of
2.52 Å, and an intermolecular H-bond appears between
O(2)-H and O(6) of 2.78 Å; symmetry transformations used
to generate equivalent atoms: #1 -x+2,y-1/2,-z+3/2.
These results explain the low chemical shifts of the three
phenolic protons. The planes formed by the two rings were
almost perpendicular in the crystal, indicating the re-
stricted rotation of the C-1 to C-13 and C-7 to C-13 bonds.
That pestalone did not adopt a fully planar structure was
also shown in the 13C NMR spectrum. The ketone signal
at δ204.4 indicated that there was little conjugation
between the ketone and the two rings. However, the UV
absorption at 340 nm and the planar relationship between
the ketone and chlorine-containing ring (resulting from the
H-bond between the ketone and the OH at C-12) did
indicate extended conjugation through the ketone should
be observed.
Although the structure of pestalone is relatively rare and
unrelated to most other natural products, the C-20 des-
methyl analogue 2has been reported, but only in a patent,
as a product of a fungal strain of the genus Chrysosporium.
Compound 2is reported to inhibit the enzyme testosterone-
5R-reductase.11
Pestalone was not produced in control experiments in
which the fungus and bacterium were cultured individu-
ally, suggesting that the production of pestalone is induced
by bacterial competition. Neither the organic extract nor
the cell-free broth of the bacterium were found to induce
antibiotic production by the fungus, suggesting that regu-
lation may not be chemically mediated. Interestingly, the
production of pestalone could be induced, although in very
low yield, in a pure culture of the fungus CNL-365 by the
addition of ethanol (1% v/v) after the fermentation had
proceeded for 24 h. This result clearly demonstrates that
pestalone is a product of fungal biosynthesis in response
to an external trigger. It is not produced by a mixed
biosynthesis in which the fungus transforms a bacterial
metabolite.
Pestalone (1) was found to exhibit moderate in vitro
cytotoxicity in the National Cancer Institute’s human
tumor cell line screen (mean GI50 )6.0 µM). More
importantly, pestalone showed potent antibacterial acti-
vity against methicillin-resistant Staphylococcus aureus
Table 1. 1H and 13C NMR Data for Pestalone (1) in Acetone-d6
C no. 13C NMRa1H NMRbHMBC correlations
1 146.9
2 113.3
3 163.9 11.20 (s, OH) 103.2, 113.3, 163.9
4 103.2 6.60 (s) 26.1, 113.3, 117.4, 146.9,
163.9, 192.0
5 163.6 10.10 (s, OH) 103.2, 117.4, 163.6
6 117.4
7 117.9
8 157.1
9 119.4
10 145.5
11 119.8
12 158.4 13.20 (s, OH) 117.9, 119.8, 158.4, 202.4
13 202.4
14 192.0 9.80 (s) 103.2, 113.3, 146.9, 163.9
15 26.1 3.10 (d, 6.9) 117.4, 123.0, 132.1, 146.9,
163.6
16 123.0 4.98 (t, 6.9) 17.6, 25.8
17 132.1
18 17.6 1.34 (s) 25.8, 123.0, 132.1
19 25.8 1.48 (s) 17.6, 123.0, 132.1
20 61.9 3.30 (s) 157.1
21 19.2 2.50 (s) 119.4, 119.8, 145.9, 157.1,
158.4, 202.4
aRecorded at 100 MHz. Assignments are by DEPT, HMQC, and
HMBC methods. bRecorded at 300 MHz. Assignments are by
COSY, HMQC, and HMBC methods.
Figure 1. X-ray structure of pestalone.
Notes Journal of Natural Products, 2001, Vol. 64, No. 11 1445
(MIC )37 ng/mL) and vancomycin-resistant Enterococcus
faecium (MIC )78 ng/mL). The potency of this agent
toward drug-resistant pathogens suggests that pestalone
should be evaluated in more advanced, whole animal
models of infectious disease.
Experimental Section
Microbial Isolation and Identification. Fungal strain
CNL-365 was isolated on medium YPG (1.0% glucose, 0.5%
peptone, 0.5% yeast extract, 1.5% agar, 0.01% penicillin
G/streptomycin sulfate, 100% seawater) from a sample of the
brown alga Rosenvingea sp. collected in the Bahamas Islands
in 1996. The alga was identified according to Littler et al.12
Strain CNL-365 was identified (D. Porter, University of
Georgia) based on morphological characteristics as a deutero-
mycete belonging to the genus Pestalotia. The bacterial strain
(CNJ-328) used in the mixed fermentation was a Gram-
negative unicellular bacterium that could not be identified by
FAME (fatty acid methyl ester) analysis (Microbial ID, Inc.,
Newark, DE).
Mixed Fermentation of Pestalotia sp. Strain CNL-365
and Bacterium Strain CNJ-328. Pestalotia sp. strain CNL-
365 was cultivated in 2.8 L Fernbach flasks (20 ×1 L) while
shaking at 250 rpm at 25 °C in the seawater-based marine
nutrient medium described above. After 24 h, 10 mL of a
culture of the unicellular bacterium CNJ-328, grown in the
same medium, was added to each flask. The mixed culture
fermentation was allowed to proceed for an additional 6 days,
after which the total culture volume was extracted with 25 L
of ethyl acetate and the extract dried with anhydrous sodium
sulfate.
Purification and Properties of Pestalone (1). The dried
extract was concentrated under vacuum and, while monitoring
in vitro cytoxicity against the human colon tumor cell line
HCT-116, fractionated by reversed-phase C-18 silica flash
chromatography using gradient elution (100% H2O to 100%
MeOH). Active fractions were combined and subjected to size
exclusion chromatography (Sephadex LH-20) using isooctane-
toluene-MeOH (3:1:1) as the eluent. The final purification of
1was performed by normal-phase HPLC (Dynamax semi-
preparative column, Si gel, 60 Å, 10 mm ×250 mm) with
n-hexane-EtOAc (6:4), to yield 60.0 mg of pestalone (1).
Pestalone, isolated as yellow crystals, mp 153-155 °C, showed
the following spectral features: UV (CH2Cl2)λmax (log ) 340
(3.75), 281 (4.29), 238 (4.24) nm; IR film, (NaCl) νmax 3225,
2943, 1635, 1620, 1383, 1251, 1205 cm-1;1H and 13C NMR
data, see Table 1; EIMS m/z438/440/442 [M]+(43, 29, 6), 423/
425/427 [M -CH3]+(60, 42, 9), 407/409/411 [M -OCH3]+(59,
37, 8), 395/397/399 (100, 61, 12); HRFABMS [M +H]+m/z
439.0731 (calcd for C21H20O635Cl2, 439.0715).
X-ray Data. A single crystal (size 0.2 ×0.2 ×0.1 mm) was
selected for the diffraction experiment. The data collection was
performed at 173(2) K. The dimensions were a)8.4984(2) Å,
b)14.4130(3) Å, c)32.6934(1) Å, and β)95.31(1)°. Analysis
of the diffraction pattern showed that the crystal belonged to
the space group P21/c, with two molecules per asymmetric unit.
The refined Rfactor was 6% for data with intensities greater
the 2σ(see Supporting Information).
Acknowledgment. We thank Ms. Sara Kelly for perform-
ing the HCT-116 colon carcinoma and antibacterial bioassays.
We thank Professor David Porter (University of Georgia) for
fungal identification and the NIH, National Cancer Institute,
for financial support under Grants CA44848 and CA55060.
M.C. thanks the NATO Science Fellowship Program of Spain
for support in the form of a postdoctoral fellowship. J.C. wishes
to acknowledge financial support from the NIH, National
Cancer Institute, under Grant CA24487.
Supporting Information Available: Crystallographic data for
pestalone (1) have been deposited with the Cambridge Crystallographic
Data Centre. Copies of the data can be obtained, free of charge, on
application to the Director, CCDC, 12 Union Road, Cambridge CB2
1EZ, UK (fax: +44-(0)1223-336033 or e-mail: deposit@ccdc.cam.ac.uk).
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NP0102713
1446 Journal of Natural Products, 2001, Vol. 64, No. 11 Notes
