Polyketide glycosides from bionectria ochroleuca inhibit candida albicans biofilm formation

Article abstract of DOI:10.1021/np500531j

One of the challenges presented by Candida infections is that many of the isolates encountered in the clinic produce biofilms, which can decrease these pathogens' susceptibilities to standard-of-care antibiotic therapies. Inhibitors of fungal biofilm formation offer a potential solution to counteracting some of the problems associated with Candida infections. A screening campaign utilizing samples from our fungal extract library revealed that a Bionectria ochroleuca isolate cultured on Cheerios breakfast cereal produced metabolites that blocked the in vitro formation of Candida albicans biofilms. A scale-up culture of the fungus was undertaken using mycobags (also known as mushroom bags or spawn bags), which afforded four known [TMC-151s C-F (1-4)] and three new [bionectriols B-D (5-7)] polyketide glycosides. All seven metabolites exhibited potent biofilm inhibition against C. albicans SC5314, as well as exerted synergistic antifungal activities in combination with amphotericin B. In this report, we describe the structure determination of the new metabolites, as well as compare the secondary metabolome profiles of fungi grown in flasks and mycobags. These studies demonstrate that mycobags offer a useful alternative to flask-based cultures for the preparative production of fungal secondary metabolites.

Full text of DOI:10.1021/np500531j

Article
pubs.acs.org/jnp
Polyketide Glycosides from Bionectria ochroleuca Inhibit Candida
albicans Biofilm Formation
Bin Wang,†,‡,§ Jianlan You,†,‡,§ Jarrod B. King,†,‡ Shengxin Cai,†,‡ Elizabeth Park,†,‡ Douglas R. Powell,‡
and Robert H. Cichewicz*,†,‡
‡
†Natural Product Discovery Group, Institute for Natural Products Applications and Research Technologies, and Department of
Chemistry & Biochemistry, Stephenson Life Science Research Center, University of Oklahoma, 101 Stephenson Parkway, Norman,
Oklahoma 73019, United States
S
* Supporting Information
ABSTRACT: One of the challenges presented by Candida
infections is that many of the isolates encountered in the clinic
produce biofilms, which can decrease these pathogens’
susceptibilities to standard-of-care antibiotic therapies. Inhib-
itors of fungal biofilm formation offer a potential solution to
counteracting some of the problems associated with Candida infections. A screening campaign utilizing samples from our fungal
extract library revealed that a Bionectria ochroleuca isolate cultured on Cheerios breakfast cereal produced metabolites that
blocked the in vitro formation of Candida albicans biofilms. A scale-up culture of the fungus was undertaken using mycobags (also
known as mushroom bags or spawn bags), which afforded four known [TMC-151s C−F (1−4)] and three new [bionectriols B−
D (5−7)] polyketide glycosides. All seven metabolites exhibited potent biofilm inhibition against C. albicans SC5314, as well as
exerted synergistic antifungal activities in combination with amphotericin B. In this report, we describe the structure
determination of the new metabolites, as well as compare the secondary metabolome profiles of fungi grown in flasks and
mycobags. These studies demonstrate that mycobags offer a useful alternative to flask-based cultures for the preparative
production of fungal secondary metabolites.
fermentation medium. A series of several early successes
using this method9,10 has spurred our interests in extending its
application to include a broader range of fungal isolates.
Employing this solid-phase culture method, a small-scale
extract of Bionectria ochroleuca was identified that inhibited C.
albicans biofilm formation. Assay-guided purification yielded
seven bioactive polyketide glycosides including three new
metabolites, bionectriols B−D (5−7). Compounds 5−7 exhibit
structural similarities to other compounds in this metabolite
family, which include TMC-151s,11,12 bionectriol A,13 rosli-
pin,14,15 and cladionol.16 In this paper, we report the
purification of several known and new natural products bearing
an O-linked glycosylated polyketide scaffold, as well as
demonstrate that these compounds inhibit C. albicans biofilm
formation. As an additional element of our investigation, we
explored the application of mycobags (commonly referred to as
mushroom bags or spawn bags) as a convenient high-surface-
area container for the cultivation of fungi. Our studies
demonstrate that mycobags are effective tools for the scale-up
preparation of fungi, yielding profiles that are similar to those
obtained for solid-phase fungal cultures grown in traditional
Erlenmeyer flasks.
pportunistic Candida infections pose a significant health
Oburden to immunocompromised patients, as well as
healthy adult women.1 According to the Centers for Disease
Control (CDC), approximately 7% of infants, 31% those
suffering from AIDS, and 20% of cancer patients undergoing
chemotherapy develop oral candidiasis.1 Even more common is
vulvovaginal candidiasis, which is estimated to occur one or
more times in 75% of women who are otherwise healthy.1 A
major obstacle to effectively treating these infections is that
many clinical Candida isolates form biofilms that afford
resistance against a variety of antifungal drugs including
amphotericin B.2−4 Therefore, inhibitors and/or agents that
disrupt fungal biofilms could provide an effective means for
helping control Candida infections. Since 2011, our group has
engaged in a screening effort to detect metabolites that inhibit
the formation of Candida albicans biofilms. This has led to
several discoveries including waikialoid A,5 mutanobactins,6 and
shearinines.7
A consistent challenge associated with the preparation of
secondary metabolites from fungi is that under many laboratory
culture conditions fungi produce restricted sets of natural
products that represent only a fraction of their overall
biosynthetic potential. Informal observations suggest that in
many cases the switch from broth-based to solid-phase
fermentation conditions may lead to the generation of more
chemically diverse secondary metabolite profiles.5,8 Recently,
our group introduced an unconventional solid-phase cultivation
approach employing Cheerios breakfast cereal as the
Received: June 30, 2014
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Chart 1
Figure 1. Effects of different culture vessels on the secondary metabolome of B. ochroleuca grown on a Cheerios-based medium. The B. ochroleuca
cultures were grown in 1 L Erlenmeyer flasks (A) or mycobags (B). The resulting secondary metabolome profiles (PDA detection at 200−400 nm)
from the flask-derived (C) and mycobag-derived (D) cultures reveal relatively modest changes in their respective metabolite composition.
cases, the fungi exhibited remarkably similar metabolite profiles,
indicating that mycobags are a suitable alternative to flask-based
fermentation. Significantly, the culturable surface area of one
mycobag (1855 cm2) is equivalent to that of approximately 18
× 1 L Erlenmeyer flasks. Considering that mycobags are
relatively inexpensive (∼0.50−0.60 USD/bag with one hydro-
phobic gas exchange patch), occupy limited vertical space
(mycobags can be incubated in a relatively flattened position),
and require no cleaning (mycobags are disposed of after
autoclaving), their use for solid-phase fungal cultures presents
many advantages.
RESULTS AND DISCUSSION
■
Fungal isolate BVK-SMA-2 was obtained from a soil sample
collected in Buffalo Valley, Oklahoma. A BLAST analysis of its
ITS sequence data demonstrated that the isolate was 99%
identical to B. ochroleuca. For the purpose of comparing its
growth and metabolite profiles using two solid-phase culturing
techniques, the B. ochroleuca was inoculated onto Cheerios
breakfast cereal in (a) 50 × 1 L Erlenmeyer flasks and (b) 3
mycobags (each 53 cm × 35 cm). The amounts of Cheerios
and inoculum introduced into the mycobags and flasks were
scaled relative to one another in order to ensure the ratios of
inoculum to culturable surface area in the two types of vessels
were approximately equivalent. Both sets of cultures were
incubated at 25 °C under identical lighting for 4 weeks. The
secondary metabolites from both sets of B. ochroleuca cultures
were extracted with EtOAc for chemical analysis and biological
testing.
Extracts were dissolved in MeOH−H2O (9:1), and the
supernatants were subject to LC-PDA-ESIMS analysis. The
resulting metabolite profiles for both sets of B. ochroleuca
cultures were judged to be similar (Figure 1). As an extension
of this observation, the secondary metabolite profiles for several
other fungi grown under both types of solid-phase culture
conditions were also compared (Figure S1). In the majority of
After comparing the B. ochroleuca secondary metabolomes
generated under both sets of conditions, the extracts were
pooled, and the bioactive compounds were purified using
normal-phase, size-exclusion, and reversed-phase chromatog-
raphy methods to yield four known O-linked glycosylated
polyketides, TMC-151s C−F (1−4), and three new metabo-
lites, named bionectriols B−D (5−7). Metabolites 1−4 were
1
identified by comparisons of their MS data, H and 13C NMR
spectra, and optical rotation values to published values.11
Compound 5 was obtained as a colorless, amorphous solid
that yielded an adduct ion with m/z 625.3934 [M + Na]+ under
HRESIMS conditions, which was determined to represent the
molecular formula C32H58O10Na (calcd 625.3928, Δ 0.96
ppm). The 1H, 13C, and HSQC NMR data (Table 1) suggested
B
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Table 1. NMR Data for Compounds 5−7 (δ in ppm, methanol-d4)
Article
a
5
6
7
carbon
δC, type
δH (m, J in Hz)
δC, type
δH (m, J in Hz)
δC, type
δH (m, J in Hz)
1
179.9, qC
44.7, CH
81.8, CH
136.2, qC
135.1, CH
37.1, CH
84.3, CH
134.7, qC
134.8, CH
36.2, CH
87.3, CH
34.2, CH
43.9, CH2
28.8, CH
46.0, CH2
32.9, CH
29.9, CH2
11.5, CH3
15.0, CH3
10.8, CH3
17.8, CH3
11.3, CH3
18.6, CH3
15.5, CH3
21.3, CH3
20.7, CH3
177.8, qC
44.7, CH
82.1, CH
135.9, qC
135.3, CH
36.6, CH
84.3, CH
137.2, qC
134.3, CH
36.9, CH
84.6, CH
134.7, qC
135.0, CH
36.1, CH
87.4, CH
34.3, CH
43.9, CH2
28.8, CH
46.1, CH2
32.9, CH
29.9, CH2
11.5, CH3
14.7, CH3
10.7, CH3
17.7, CH3
11.4, CH3
17.8, CH3
11.1, CH3
18.7, CH3
15.5, CH3
21.2, CH3
20.7, CH3
102.7, CH
72.4, CH
75.5, CH
68.9, CH
75.7, CH
65.6, CH2
181.5, qC
45.2, CH
82.0, CH
136.8, qC
134.1, CH
36.7, CH
83.9, CH
137.5, qC
133.6, CH
37.1, CH
84.4, CH
134.9, qC
134.7, CH
36.2, CH
87.1, CH
34.2, CH
43.9, CH2
28.8, CH
46.1, CH2
32.9, CH
29.9, CH2
11.6, CH3
15.2, CH3
10.9, CH3
17.8, CH3
11.5, CH3
17.8, CH3
11.3, CH3
18.6, CH3
15.5, CH3
21.3, CH3
20.7, CH3
102.5, CH
72.7, CH
75.7, CH
68.4, CH
78.3, CH
62.9, CH2
2
2.54 (m)
2.68 (m)
2.49 (m)
3
4.05 (d, 9.7)
4.08 (d, 10.0)
4.02 (d, 9.5)
4
5
5.33 (d, 9.1)
2.62 (m)
5.36 (d, 9.2)
2.65 (m)
5.34 (d, 9.6)
2.65 (m)
6
7
3.71 (d, 9.1)
3.72 (d, 9.0)
3.72 (d, 8.6)
8
9
5.54 (d, 9.5)
2.74 (m)
5.29 (d, 9.3)
2.61 (m)
5.30 (d, 8.9)
2.61 (m)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
1′
2′
3′
4′
5′
6′
3.48 (dd, 6.3, 3.5)
1.86 (m)
3.64 (m)
3.68 (d, 9.1)
0.99, 1.40 (m, m)
1.62 (m)
5.52 (d, 9.5)
2.73 (m)
5.55 (d, 9.2)
2.74 (m)
0.91, 1.25 (m, m)
1.44 (m)
3.43 (dd, 6.4, 3.2)
1.85 (m)
3.49 (dd, 6.4, 3.5)
1.85 (m)
1.08, 1.41 (m, m)
0.89 (d, 6.3)
0.97 (d, 7.2)
1.64 (br s)
0.99, 1.40 (m, m)
1.62 (m)
0.98, 1.39 (m, m)
1.62 (m)
0.92, 1.25 (m, m)
1.45 (m)
0.90, 1.26 (m, m)
1.45 (m)
0.78 (d, 6.8)
1.64 (br s)
1.10, 1.42 (m, m)
0.88 (m)
1.08, 1.38 (m, m)
0.87 (m)
0.99 (d, 6.9)
0.95 (d, 6.8)
0.89 (d, 6.3)
0.89 (d, 6.3)
0.98 (d, 7.0)
1.63 (br s)
0.96 (d, 6.8)
1.64 (br s)
0.79 (d, 6.8)
1.67 (br s)
0.80 (d, 6.8)
1.67 (br s)
0.75 (d, 6.7)
1.64 (br s)
0.77 (d, 6.8)
1.64 (br s)
0.97 (d, 7.2)
0.94 (d, 7.1)
0.89 (d, 6.2)
0.89 (d, 6.2)
4.46 (br s)
0.98 (d, 6.9)
0.94 (d, 6.5)
0.89 (d, 6.3)
0.89 (d, 6.3)
4.49 (br s)
102.6, CH
72.7, CH
75.7, CH
68.5, CH
78.3, CH
62.9, CH2
4.47 (br s)
3.89 (d, 2.8)
3.91 (d, 3.0)
3.39 (dd, 9.6, 3.1)
3.51 (dd, 9.6, 9.6)
3.36 (m)
3.89 (d, 2.8)
3.37 (dd, 9.5, 3.1)
3.58 (dd, 9.6, 9.5)
3.15 (m)
3.37 (dd, 9.5, 3.2)
3.58 (dd, 9.5, 9.4)
3.15 (m)
3.75 (dd, 11.6, 5.2)
3.87 (dd, 11.1, 2.1)
4.26 (dd, 11.8, 7.2)
4.41 (dd, 11.9, 1.7)
3.77 (dd, 11.6, 5.2)
3.87 (dd, 11.0, 1.9)
7′
8′
1″
172.8, qC
21.0, CH3
68.0, CH2
2.09 (s)
4.20 (dd, 11.5, 6.1)
4.47 (dd, 11.3, 2.4)
3.91 (m)
2″
3″
4″
5″
6″
70.4, CH
71.1, CH
71.0, CH
72.9, CH
65.2, CH2
3.82 (m)
3.80 (m)
3.69 (m)
3.64, 3.82 (m, m)
a
Assignments based on H, 13C, and HSQC NMR (13C 100/1H 400 MHz) experiments at room temperature.
1
the presence of nine methyl, three methylene, six methine, one
oxygenated methylene, eight oxygenated methine, and four
olefinic carbons. The HMBC spectrum displayed a correlation
from H3-19 to C-1, indicating the presence of an ester carbonyl
carbon that had not been detected in the 13C spectrum. The
other features of the 13C spectrum of 5 were similar to those for
1, except that the sugar alcohol moiety and one propenyl
subunit (equivalent to C-2, C-3, and C-21 in 1) were missing
(Figure S5). The carbon chemical shifts at δC 62.9, 68.5, 72.7,
75.7, 78.3, and 102.6 denoted the presence of a hexopyranose.
An HMBC correlation from H-1′ to C-11 indicated that the
hexopyranose moiety was attached to C-11. Additional HMBC
correlations that were critical for verifying the planar structure
of 5 are illustrated in Figure 2. On the basis of 2D ROESY
correlations between H3-20 and H-6, as well as H3-22 and H-10
(Figure 2), both the C-4−C-5 and C-8−C-9 olefins were
determined to be E-configured.
C
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Another new metabolite was purified from the fungal crude
extract; however, its HRESIMS, 1D and 2D NMR (Figures
S21−27), and optical rotation data were identical to those of
the base-hydrolysis product 7. Compound 7 was subjected to
acid hydrolysis in 1 M methanolic HCl at 70 °C and extracted
with EtOAc. The water layer was neutralized and dried under
vacuum to yield 9, which was determined to be methyl-α-
mannopyranoside based upon comparisons of its 13C NMR
spectrum (δ 102.7, 72.0, 72.5, 68.4, 74.4, 62.7, and 55.2, Figure
S32) with published data.18 A comparison of the optical
rotation value for 9 ([α]D25 +60 [c 0.09, H2O]) with authentic
methyl-α-D-mannopyranoside ([α]D25 +80 [c 10, H2O])
suggested that 9 is D-configured. During the course of these
studies, colorless, needle-like crystals of 7 were fortuitously
obtained from a concentrated methanol solution of the
metabolite. This provided an opportunity to use X-ray
diffraction analysis to verify the planar structure of 7, as well
as assign its absolute configuration (Figure S31). Results from
the X-ray experiment revealed the absolute configuration of
2R,3S,6S,7S,10S,11S,14S,15R,16S,18S,20S.
Compounds 3, 4, and 6 produced modest inhibition of C.
albicans cell growth with IC50 values of 36.3 11.3, 41.0 5.6,
and 24.1 4.5 μM (30.8 9.6, 33.6 4.6, and 21.8 4.1 μg/
mL), respectively. The biofilm-inhibiting positive control ETYA
(5,8,11,14-eicosatetraynoic acid) and all of the other purified
metabolites were inactive in this assay at concentrations of up
to 100 μM. However, all the compounds exhibited biofilm
inhibition properties with IC50 values ranging from 1.1 to 5.0
μM (0.9 to 3.0 μg/mL), and most of them displayed increased
inhibition relative to ETYA (Table 2, Figure 4). The potencies
Figure 2. Key HMBC and ROESY correlations for 5.
The relative configuration of the glycan moiety was
ascertained based on gated decoupling and ROESY NMR
experiments. In the gated decoupling experiment (Figure S6),
the coupling constant between the anomeric carbon and
hydrogen atoms was determined to be 1JC‑1′,H‑1′ = 154.5 (<160),
indicating a β-orientation.17 ROESY correlations between H-1′
and H-2′, H-1′ and H-3′, H-1′ and H-5′, H-2′ and H-3′, H-2′
and H-5′, and H-3′ and H-5′ were observed (Figure 2), which
were consistent with a mannose moiety. Additional compar-
isons between our carbon chemical shift data (C-1′ to C-6′)
and those published for similar compounds11 further
corroborated this assignment. Further efforts to address the
absolute configuration of the presumably polyketide portion of
the metabolite proved unsuccessful; thus the absolute
configuration of the aglycan component of 5 was not
determined.
Table 2. Inhibition of C. albicans SC5314 Biofilm Formation
and Growth with Compounds 1−7
Compound 6 was obtained as a colorless, amorphous solid.
The HRESIMS data for 6 provided an adduct ion [M + Na]+ at
m/z 929.5474, which represented the molecular formula
a
IC50 for biofilm inhibition,
μM (μg/mL)
IC50 for growth inhibition,
C46H82O17Na (calcd 929.5450, Δ 2.58 ppm). The H, 13C,
1
compound
μM (μg/mL)
and HSQC NMR spectra (Table 1) suggested the presence of
12 methyl, three methylene, seven methine, three oxygenated
methylene, 13 oxygenated methine, six olefinic, and two ester
carbonyl carbons. The 13C NMR spectrum of 6 was similar to
that of 3 with the major exceptions being the presence of
additional methyl (δC 14.7), methine (δC 44.7), and oxygenated
methine (δC 82.1) groups (Figures S13 and S14). HMBC
correlation data were employed to deduce the planar structure
of 6, which revealed that the new carbons were located adjacent
to the C-1 ester. A ROESY experiment was performed revealing
correlations between H3-24 and H-6, H3-26 and H-10, and H3-
28 and H-14, thus verifying that the olefins were E-configured.
In addition, correlations between H-1′ and H-2′, H-1′ and H-
3′, H-1′ and H-5′, H-2′ and H-3′, H-2′ and H-5′, and H-3′ and
H-5′ were detected, indicating that the glycan was a mannose.
1
1.1 0.7 (0.9 0.6)
1.5 0.1 (1.2 0.1)
1.5 0.1 (1.3 0.1)
1.2 0.5 (1.0 0.4)
5.0 2.4 (3.0 1.4)
1.5 0.2 (1.4 0.2)
2.4 0.0 (1.7 0.0)
3.4 0.0 (1.2 0.0)
>100 (81)
2
>100 (78)
3
36.3 11.3 (30.8 9.6)
41.0 5.6 (33.6 4.6)
>100 (60)
4
5
6
24.1 4.5 (21.8 4.1)
>100 (70)
7
ETYA
>100 (30)
a
IC50 values are expressed as the concentrations corresponding to 50%
of the maximum biofilm formation.
of the B. ochroleuca-derived biofilm inhibitors are similar to
waikialoid A (IC50 value of 1.4 μM),5 yet are 2 to 5 times more
potent than the mutanobactins and shearinines.6,7 All of the
compounds were tested for their abilities to synergistically
enhance the activity of amphotericin B against C. albicans. FICI
1
A coupling constant of JC‑1′,H‑1′ = 154.1 was observed for the
anomeric C−H bond supporting a β-orientation.
Further efforts to substantiate the absolute configurations of
mannose and mannitol in 6 were pursued using a modification
of Kohno’s approach.11 Compound 6 was hydrolyzed in 0.4 M
NaOH, the reaction mixture was neutralized, and products
were partitioned against EtOAc to yield 7. The water layer was
dried under vacuum and reacted with Ac2O in anhydrous
pyridine, and the products were extracted with EtOAc to yield
8, hexa-O-acetylmannitol (Figures S29, S30). A comparison of
the optical rotation values for 8 ([α]2D5 +24 [c 0.13, CHCl3])
and authentic hexa-O-acetyl-D-mannitol ([α]D25 +26 [c 0.693,
CHCl3]) suggested 8 was D-configured.
Figure 3. Key HMBC and ROESY correlations for 6.
D
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rDNA gene was sequenced at the Laboratory for Genomics and
Bioinformatics, OU Health Sciences Center. Sequence data for the
isolate (GenBank accession KM000126) was compared by BLAST
analysis to sequences publicly available through the NCBI database.
For the 1 L Erlenmeyer flask cultures, an 80 mL volume of Cheerios
breakfast cereal was added to each flask, and the flasks were autoclaved
using a solid-cycle at 121 °C for 30 min. After the flasks were cooled to
room temperature, a 48 mL aliquot of autoclaved 0.3% sucrose with
0.005% chloramphenicol was pipetted into the flasks to rehydrate the
cereal. One ∼5 mm × 5 mm chunk of the fungal isolate grown on an
agar plate was added to each flask, and the cultures were incubated for
4 weeks.
When mycobags were used as the fermentation vessels, Cheerios
breakfast cereal was preautoclaved using a solid-cycle at 121 °C for 30
min. After cooling, a 500 mL volume of Cheerios was added to each
bag, the openings of the bags were folded over once, the folds were
sealed with clamps, and bags were autoclaved using a liquid-cycle at
121 °C for 30 min. Eighteen ∼5 mm × 5 mm chunks of fungal culture
grown on an agar plate were prepared and mixed with the cereal. The
cereal was then rehydrated with 850 mL of an autoclaved solution
consisting of 0.3% sucrose with 0.005% chloramphenicol. The bags
were placed flat to facilitate the formation of a monolayer of Cheerios
across the bottom of the bag, and the cultures were incubated for 4
weeks.
Extraction and Compound Purification. The cultures were
homogenized and extracted with EtOAc. The EtOAc extract (53.8 g)
was subjected to silica gel flash chromatography with elution carried
out using a step gradient of hexane−EtOAc (100:0, 50:50, 0:100) and
EtOAc−MeOH (50:50, 0:100) to yield five fractions. Bioassay analysis
of the resulting fractions revealed all of the biofilm inhibition activity
was retained in fraction 4 (Fr.4). Accordingly, Fr.4 (EtOAc−MeOH,
50:50, 4.6 g) was subjected to Sephadex LH-20 column chromatog-
raphy and eluted with MeOH−DCM (1:1), providing 16 subfractions.
Recombination of the 16 fractions based on their TLC profiles yielded
four subfractions (Fr.4-1−4-4). Fr.4-2 (0.9 g) was the only fraction
exhibiting biofilm inhibition. Further purification of its components
using C18 HPLC (250 mm × 20.2 mm, 5 μm) with a MeOH−H2O
gradient (from 80:20 to 100:0), as well as isocratic C18 HPLC (250
mm × 10 mm, 5 μm) with MeCN−H2O containing 0.1% HCOOH
(55:45, 72:28, or 78:22), yielded four known [TMC-151 C−F (1−4)]
and three new polyketide glycosides [5 (3 mg), 6 (12 mg), and 7 (4
mg)].
Figure 4. Effects of compounds 6 and 7 (2.5 μM) on C. albicans
SC5314 biofilm formation. The C. albicans cultures were prepared in
RMPI 1640-MOPS medium and treated with compounds 6 (A), 7
(B), or DMSO vehicle (C) before incubating at 37 °C for 48 h. Panels
A−C illustrate representative fluorescent images taken from triplicate
cultures.
(fractional inhibitory concentration index) values for all seven
compounds were found to be <0.5, indicating synergistic effects
with amphotericin B. All of the compounds were found to
enhance the potency of amphotericin B up to 4-fold (Table 3).
EXPERIMENTAL SECTION
■
General Experimental Procedures. UV data were recorded on a
Hewlett-Packard 8452A diode array spectrophotometer. Optical
rotation measurements were made on an AUTOPOL III automatic
polarimeter. NMR spectra were obtained on a Varian Unity Inova
1
spectrometer (400 MHz for H and 100 MHz for 13C) with a broad
band probe at 20 0.5 °C using methanol-d4 (Cambridge Isotope
Laboratories, Inc.) as the solvent. HRESIMS spectra were collected on
an Agilent 6538 ultra high definition Accurate-Mass Q-TOF system.
The LC-MS data were acquired on a Shimadzu UFLC coupled to a
quadrupole mass spectrometer using a Phenomenex Kinetex C18
column (3.0 mm × 75 mm, 2.6 μm particle size) and MeCN−H2O
(with 0.1% HCOOH) gradient (1:9 to 0:10 in 12 min) followed by a
100% MeCN wash. Column chromatography was conducted using
silica gel 60 (40−63 μm particle size) and Sephadex LH-20. TLC silica
gel 60 F254 plates from EMD Chemicals Inc. were used for TLC.
Separations were also carried out using a Shimadzu HPLC system
equipped with LC-6AD solvent delivery pumps coupled to an SPD-
10AV UV−vis detector or a Waters system equipped with a 1525
binary HPLC pump coupled to a 2998 PDAD detector. Both systems
utilized Phenomenex C18 columns (21.2 × 250 mm or 10 × 250 mm,
5 μm particle size) for preparative runs. X-ray diffraction data for
compound 7 were collected using a diffractometer with a Bruker APEX
ccd area detector and graphite-monochromated Mo Kα radiation (λ =
0.710 73 Å). All solvents were of ACS grade or better.
Base Hydrolysis of 6. A 1 mL aliquot of 0.4 M NaOH was added
to 6 (10 mg), and the mixture was stirred at room temperature for 2 h.
After the reaction mixture was neutralized with 1 M HCl, 1 mL of
water was added and the mixture was partitioned with EtOAc (5 × 3
mL). The pooled EtOAc layers were reduced under vacuum to yield 7
(7.4 mg). The water layer was dried under vacuum and acetylated as
described in the following section.
Acetylation of Mannitol. The solid product from the water layer
was dissolved in 800 μL of anhydrous pyridine with 200 μL of acetic
anhydride. The mixture was stirred at room temperature for 2 h. When
the reaction was completed, 5 mL of EtOAc was added and the
Fungal Isolate Procurement and Culture Conditions. A soil
sample was collected from beneath a granite monument on the
grounds of one of the author’s (J.B.K.) family property in Buffalo
Valley, Oklahoma. The sample was processed using the method
described by Du et al.,9 and 51 fungal isolates were purified. Isolate
BVK-SMA-2 was obtained on a starch-milk agar plate. The large-
ribosomal-subunit internal transcribed spacer 1 (ITS1) region of the
Table 3. In Vitro Interaction between Amphotericin B and Compounds 1−7 Using the Checkerboard Method
amphotericin B [A, μM (μg/mL)]
compound [B, μM (μg/mL)]
a
single
b
compound
MIC
MICcombination
FICA
MICsingle
MICcombination
FICB
FICI
c
1
2
3
4
5
6
7
5 (4.6)
5 (4.6)
5 (4.6)
5 (4.6)
5 (4.6)
5 (4.6)
5 (4.6)
1.25 (1.15)
1.25 (1.15)
1.25 (1.15)
1.25 (1.15)
1.25 (1.15)
1.25 (1.15)
1.25 (1.15)
0.25
0.25
0.25
0.25
0.25
0.25
0.25
>100 (81)
>100 (78)
50 (42)
5 (4)
<0.05
<0.01
0.03
<0.30 S
<0.26 S
0.28 S
1.25 (1)
1.25 (1.1)
1.25 (1.1)
5 (3)
50 (41)
0.03
0.28 S
>100 (60)
50 (45)
<0.05
0.03
<0.30 S
0.28 S
1.25 (1.1)
5 (3.5)
>100 (70)
<0.05
<0.30 S
a
b
The MIC was defined as the lowest concentration causing prominent growth reduction (≥80% reduction in the metabolic activity). FICI = FICA +
c
FICB = (MICA combination/MICA single) + (MICB combination/MICB single). S, synergistic interaction when FICI ≤ 0.5.
E
dx.doi.org/10.1021/np500531j | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
solution was washed repeatedly with H2O (5 × 2 mL). The EtOAc
layer was reduced under vacuum to yield 8 (4.3 mg).
For determining the inhibition of biofilm formation, the medium
was removed by aspiration from cells that were actively growing in
microtiter plates, and the wells were washed twice with sterile PBS to
remove nonadherent cells. Fresh medium (100 μL RPMI-1640 plus
MOPS) was then added back to each well. Biofilm formation was
measured using the XTT assay. All experiments were performed in
triplicate on three separate occasions. The 50% inhibitory concen-
tration values (IC50) for biofilm formation inhibition were calculated
using GraphPad Prism 5. The effects of compounds on biofilm
formation were confirmed by fluorescence microscopy (Operetta,
PerkinElmer). After washing with PBS, yeasts were stained with
Calcofluor white and held at 37 °C for 10 min.
Checkerboard Assay for Synergistic Activity. To evaluate the
synergistic effects of compound 1−7 with amphotericin B, a
checkerboard assay was used.22 C. albicans cells were seeded in 96-
well plates and treated with different concentrations of test
compounds alone or in combination with amphotericin B in RPMI-
1640 plus MOPS medium at 37 °C for 48 h. The viability of the yeast
was measured using the XTT assay, and the MIC for growth was
defined as the lowest antifungal concentrations that caused ≥80%
reduction in the metabolic activity. The interactions of test compounds
with amphotericin B were determined according to the resultant FICI
values. FICI values were calculated as follows: FICI = FICA + FICB =
(MICA combination/MICA single) + (MICB combination/MICB single). The
interpretation of the FICI data was determined as follows: ≤0.5,
synergistic effect; >0.5 but <4, indifference (no effect); and ≥4,
antagonistic effect.
Acid Methanolysis of 7. A solution of 1 M methanolic HCl (800
μL) was added to 7 (3.7 mg), and the solution was refluxed at 70 °C
for 1.5 h. After the reaction mixture was neutralized with 1 M NaOH,
1 mL of water was added, and the solution was partitioned against
EtOAc (5 × 3 mL). The water layer was evaporated under vacuum to
provide 9 (∼1.0 mg).
Single-Crystal X-ray Diffraction Analysis of 7. A colorless
prism-shaped crystal of dimensions 0.420 × 0.230 × 0.100 mm was
selected for structural analysis. The sample was cooled to 100 K. Cell
parameters were determined from a nonlinear least-squares fit of 2682
peaks in the range 2.26° < θ < 17.05°. A total of 20 189 data were
measured in the range 1.129° < θ < 19.780° using ω oscillation frames.
The data were corrected for absorption by the empirical method,
giving minimum and maximum transmission factors of 0.963 and
0.991. The data were merged to form a set of 4239 independent data
with R(int) = 0.1301 and a coverage of 50.9%. The monoclinic space
group C2 was determined by systematic absences and statistical tests
and verified by subsequent refinement. The structure was solved by
direct methods and refined by full-matrix least-squares methods on
F2.19 Positions of hydrogens bonded to carbons were initially
determined by geometry and refined by a riding model. Hydrogens
bonded to oxygen atoms were located on difference maps and were
refined with a riding model. Non-hydrogen atoms were refined with
anisotropic displacement parameters. Hydrogen atom displacement
parameters were set to 1.2 (1.5 for methyl) times the displacement
parameters of the bonded atoms. A total of 489 parameters were
refined against 210 restraints and 4239 data to give wR(F2) = 0.4094
and S = 1.142 for weights of w = 1/[σ2(F2) + (0.1400P)2 + 80.00P],
Bionectriol B (5): colorless, amorphous solid; UV (MeOH) λmax
(log ε) 206 (4.31) nm; [α]2D5 +3 (c 0.24, MeOH); H and 13C NMR
1
(see Table 1); HRESIMS m/z 625.3934 [M + Na]+ (calcd for
C32H58O10Na, 625.3928).
2
where P = [Fo + 2Fc2]/3. The final R(F) was 0.1647 for the 3058
Bionectriol C (6): colorless, amorphous solid; UV (MeOH) λmax
(log ε) 206 (4.27) nm; [α]2D5 +12 (c 0.26, MeOH); 1H and 13C NMR
(see Table 1); HRESIMS m/z 929.5474 [M + Na]+ (calcd for
C46H82O17Na, 929.5450).
observed [F > 4σ(F)] data. The largest shift/s.u. was 0.036 in the final
refinement cycle. The final difference map had maxima and minima of
0.889 and −0.427 e/Å3, respectively. The polar axis restraint was taken
from Flack and Schwarzenbach.20 The intensity data were truncated to
1.05 Å resolution because data in higher resolution shells all had ⟨I/
σ(I)⟩ < 2.0. Atoms O10′ and C6′ were disordered (the occupancies
for these atoms refined to 0.5319 and 0.4719 for the two structures
illustrated in Figure S31). Restraints on the positional parameters of
the disordered atoms and the displacement parameters of all atoms
were required. Disordered solvent was also present and was accounted
for using Babinet’s principle.21 The X-ray crystallographic data of 8
have been deposited in the Cambridge Crystallographic Data Center
under accession number CCDC 1007026. The data can be accessed
free of charge at http://www.ccdc.cam.ac.uk/.
Assays for C. albicans Growth Inhibition and Disruption of
Biofilm Formation. The C. albicans strain SC5314 was cultured in
brain heart infusion medium (BHI medium, Becton Dickinson) or
RPMI-1640 plus MOPS medium [RPMI-1640 medium (Sigma)
buffered to pH 7.0 with 0.17 M MOPS 3-(N-morpholino)-
propanesulfonic acid (Sigma)] as required. The effects of compounds
on the growth of C. albicans were tested using the method described in
the NCCLS 2008 CLSI M27-A3 guidelines. The biofilm assay was
performed as described with the following modifications. Cells of C.
albicans SC5314 were cultured in BHI medium (Becton Dickinson) at
37 °C overnight. The cells were pelleted by centrifugation, washed
with sterile PBS (phosphate-buffered saline, pH 7.4), and resuspended
in RPMI-1640 plus MOPS medium. Test compounds were prepared
in DMSO at stock concentrations of 20 mM before being serially
diluted in RPMI-1640 plus MOPS medium for testing. ETYA was used
as a positive control.7 Aliquots of yeast suspension (100 μL containing
2.5 × 103 cells mL−1) were added to the medium containing the
diluted compounds or DMSO [final concentrations did not exceed 1%
(v/v)] before being transferred to 96-well plates (Corning). After 48 h
of incubation at 37 °C, the viability of the yeast was measured using
the XTT assay. In brief, yeast cells were treated with 0.1 mg mL−1
XTT at 37 °C for 1 h. Absorbance measurements were taken at 492
nm using a microplate reader (Infinite M200). The minimum
inhibitory concentrations (MIC) were defined as the lowest antifungal
concentrations that caused ≥80% reduction in metabolic activity.
Bionectriol D (7): colorless, prism-shaped crystals; UV (MeOH)
λmax (log ε) 206 (4.29) nm; [α]2D5 +7 (c 0.12, MeOH); H and 13C
1
NMR (see Table 1); HRESIMS m/z 723.4667 [M + Na]+ (calcd for
C38H68O11Na, 723.4659).
ASSOCIATED CONTENT
■
S
* Supporting Information
1D and 2D NMR spectra and HRESIMS data for compounds
5−7 and 1D NMR spectra of 8 and 9 are available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 1-405-325-6969. Fax: 1-405-325-6111. E-mail:
rhcichewicz@ou.edu.
Author Contributions
§B. Wang and J. You contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The reported research was supported by the National Institute
of Allergy and Infectious Diseases of the National Institutes of
Health under award number R01AI085161. The X-ray
diffractometer was purchased through the National Science
Foundation (CHE-0130835). We would like to thank Dr. A.
Dongari-Bagtzoglou at the University of Connecticut Health
Center for kindly providing the C. albicans SC5314 strain used
in this study.
F
dx.doi.org/10.1021/np500531j | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
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