Chaunopyran A: Co-Cultivation of Marine Mollusk-Derived Fungi Activates a Rare Class of 2-Alkenyl-Tetrahydropyran

Article abstract of DOI:10.1021/acs.jnatprod.7b00144

Co-cultivation of Chaunopycnis sp. (CMB-MF028) and Trichoderma hamatum (CMB-MF030), fungal strains co-isolated from the inner tissue of an intertidal rock platform mollusc (Siphonaria sp), resulted in transcriptional activation of a rare class of 2-alkenyl-tetrahydropyran, chaunopyran A (7), and biotransformation and deactivation of the antifungal pyridoxatin (1), to methyl-pyridoxatin (8). This study illustrates the complexity of offensive and counter-offensive molecular defenses encountered during fungal co-cultivation, and the opportunities for activating new, otherwise transcriptionally silent secondary metabolites.

Full text of DOI:10.1021/acs.jnatprod.7b00144

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Article
pubs.acs.org/jnp
Chaunopyran A: Co-Cultivation of Marine Mollusk-Derived Fungi
Activates a Rare Class of 2‑Alkenyl-Tetrahydropyran
Zhuo Shang, Angela A. Salim, and Robert J. Capon*
Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD 4072,
Australia
S
* Supporting Information
ABSTRACT: Co-cultivation of Chaunopycnis sp. (CMB-MF028) and
Trichoderma hamatum (CMB-MF030), fungal strains co-isolated from the
inner tissue of an intertidal rock platform mollusc (Siphonaria sp), resulted
in transcriptional activation of a rare class of 2-alkenyl-tetrahydropyran,
chaunopyran A (7), and biotransformation and deactivation of the
antifungal pyridoxatin (1), to methyl-pyridoxatin (8). This study illustrates
the complexity of offensive and counter-offensive molecular defenses
encountered during fungal co-cultivation, and the opportunities for
activating new, otherwise transcriptionally silent secondary metabolites.
he search for bioactive microbial secondary metabolites
Thas inspired basic and applied science since early in the
last century. Notwithstanding a history replete with remarkable
successes, traditional biodiscovery is increasingly constrained by
excessive rediscovery of known metabolites. That the latter is
indicative of technical challenges rather than a depleted
resource is evidenced by modern microbial genomics, which
has catalogued many thousands of biosynthetic gene clusters
(BGCs) encoding an array of unexplored secondary metabo-
lites. That this biosynthetic potential has eluded past efforts at
exploration is for the most part explained by the observation
that many are transcriptionally silent under standard culture
conditions, rendering their gene products (i.e., secondary
metabolites) inaccessible. To overcome this impasse requires
new cultivation strategies capable of routine and cost-effective
activation of silent BGCs. One promising line of inquiry builds
on the ecology-inspired hypothesis that silent BGCs are
transcriptionally regulated by environmental stimuli, in
particular, chemical cues released by other microbes. For
example, a chemical cue released by one microbe may elicit an
“on-demand” activation of one or more silent BGCs in an
opposing strain, leading to the production of defensive
secondary metabolite(s). Indeed, these activated defensive
metabolites can themselves be chemical cues that trigger a
retaliatory transcriptional activation. Inspired by recent
successes in co-cultivation-mediated microbial biodiscovery,1−5
we set out to investigate the possible benefits and practicalities
of this approach.
Figure 1. CMB-MF028 metabolites 1a/b and 2−6, and CMB-MF030
co-cultivation products 7 and 8a/b.
speculated that the CMB-MF028 genome may encode silent
BGCs under the regulatory control of chemical cues produced
by co-isolated fungi. To test this hypothesis, extracts prepared
from 30 day ISP2 agar plate co-cultivations of CMB-MF028
with each of five co-isolated fungal strains were subjected to
HPLC-DAD-MS analysis (Figure S1). Significantly, only co-
cultivation with Trichoderma hamatum (CMB-MF030) co-
incided with activation of new chemistry (i.e., 7 and 8a/b)
(Figure 2). Figure 2A reveals a co-cultivation agar plate with
separate growth zones for (i) CMB-MF028 and (ii) CMB-
In an earlier report we described the polyketide pyridinones
1−2 and tetramic acids 3−6 from a Chaunopycnis sp. (CMB-
MF028) isolated from the inner tissue of a pulmonate false
limpet Siphonaria sp, collected from intertidal rock platforms
near Brisbane, Australia (Figure 1).6 Based on growing interest
in co-cultivation as a method for accessing new metabolites, we
Received: February 17, 2017
© XXXX American Chemical Society and
American Society of Pharmacognosy
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Figure 2. Agar plate co-cultivation of CMB-MF028 and CMB-MF030. (A) Image of the co-culture agar plate, and (B) HPLC-DAD (210 nm)
chromatograms of co-culture sampling of (i) CMB-MF028, (ii) CMB-MF030, and (iii) growth inhibitory interface, and of monocultures of (iv)
CMB-MF028 and (v) CMB-MF030.
1
Table 1. H (600 MHz) and 13C (150 MHz) NMR Data for
MF030, and (iii) a growth inhibitory interface. Figure 2B
features windows (Rt 9−11.5 min) from analytical HPLC-DAD
(210 nm) chromatograms of analytes prepared from co-
cultivation (i−iii) and monocultures (iv−v) of CMB-MF028
and CMB-MF030. In these analyses the CMB-MF030
monoculture (v) reveals no secondary metabolites, whereas
the CMB-MF028 monoculture (iv) reveals the known
metabolites 1a/b (equilibrating atropisomers) and 3. By
contrast, in the co-cultivation study, (i) exhibits a chromato-
gram featuring 1a/b and 3 at levels comparable to that of the
CMB-MF028 monoculture (iv); (ii) exhibits low levels of 3,
and the new compounds 8a/b and 7; and (iii) reveals 1a/b, 3,
8a/b and 7. UPLC-DAD (254 nm) and HPLC-ESIMS with
single ion extraction monitoring analysis could not detect 8a/b,
and could only detect extremely low levels of 7 in CMB-MF028
monoculture, a situation that was not changed by altering
cultivation conditions or media (Figure S2). Significantly,
multiple replicate ISP2 agar plate and ISP2 broth co-
cultivations of CMB-MF028 and CMB-MF030 consistently
produced 7 and 8a/b in isolable yields (Figure S3).
A time course investigation of ISP2 broth co-cultivations
revealed production of 7 and 8a/b at day 1, peaking on day 5,
with sustained levels of production across all 8 days of the study
(Figures S4). CMB-MF028 was also incubated in the presence
of sterile dead mycelia, mycelia acetone extract, and EtOAc and
butanol extracts of ISP-2 broth cultivations of CMB-MF030, as
well as an EtOAc extract of a co-culture of CMB-MF028 with
CMB-MF030; however, none of these conditions increased
production of 7 or 8a/b (Figure S5). To investigate this
phenomenon further, an EtOAc extract obtained from a scaled
up broth co-cultivation (3 L) of CMB-MF028 and CMB-
MF030 was subjected to solvent trituration, followed by normal
and reversed-phase chromatography to yield 7 and 8a/b.
HRESI(+)MS analysis of 7 returned a sodium adduct ion
indicative of a molecular formula (C14H22O3, Δmmu + 0.7)
requiring four double bond equivalents (DBE). Analysis of the
1D NMR (DMSO-d6) data for 7 (Table 1) revealed resonances
attributed to a 3,4-dihydroxytetrahydropyran (C-2 to C-6)
bearing a C-2 n-1,3,5-nonatrienyl side chain (C-7 to C-15),
accounting for all four DBE. Diagnostic 2D NMR (DMSO-d6)
Chaunopyran A (7) in DMSO-d6
position
δC, type
δH, mult. (J in Hz)
COSY
3, 7
HMBC
2
3
79.8, CH
75.9, CH
3.46, dd (9.0, 5.6)
3, 4, 7, 8
2.81, ddd (9.0, 9.0,
5.4)
2, 4, 3-OH 2, 4, 7
4
72.1, CH
3.45, overlap
3, 5β, 4-OH
−
5α
34.2, CH2 1.77, ddd (12.2, 5.0, 5β, 6β
3, 4
1.5)
5β
6α
1.42, m
4, 5α, 6α,
6β
4, 6
2, 4
64.9, CH2 3.79, ddd (11.6, 5.0, 5β, 6β
1.5)
6β
7
3.45, overlap
5α, 5β, 6α
2, 8
2, 4
131.7, CH
130.6, CH
130.5, CH
132.5, CH
130.6, CH
134.7, CH
5.77, dd (15.4, 5.6)
2, 8, 9
2, 10
8
6.21, dd (15.4, 10.6) 7, 9
6.14, dd (15.1, 10.6) 8, 10
9
7, 8, 10, 11
8, 12
10
11
12
6.18, overlap
9, 11
6.08, dd (15.1, 10.3) 10, 12
9, 13
5.71, dt (15.1, 7.0)
11, 13
12, 14
10, 11, 13,
14
13
34.3, CH2 2.05, dt (7.0, 7.0)
11, 12, 14,
15
14
22.0, CH2 1.37, tq (7.0, 7.3)
13.6, CH3 0.87, t (7.3)
13, 15
12, 13, 15
13, 14
15
14
3
3-OH
4-OH
−
−
4.94, d (5.4)
4.82, d (4.6)
2, 3, 4
4
3, 4, 5
HMBC and COSY correlations (Figure 3) supported the
assigned planar structure, while the magnitudes of J7,8 (15.4
Hz), J9,10 (15.1 Hz), and J11,12 (15.1 Hz) confirmed an all-E
configuration about the triene moiety. Axial configurations for
H-2, H-3, and H-4 were evident from J2,3 (9.0 Hz) and J3,4 (9.0
Hz), and were confirmed by ROESY correlations between H-3
and 4-OH, and H-2 and 3-OH (Figure 3). Thus, the structure
and relative configuration for chaunopyran A (7) were assigned
as indicated.
To support the proposed structure for 7, and to address the
issue of absolute configuration, the model tetrahydropyran 7c
was synthesized using the methodology outlined in Scheme 1.
Adapting a published procedure,7 a sample of L-rhamnose was
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3
Figure 3. Diagnostic NMR (DMSO-d6) data and correlations for chaunopyran A (7) (* Values in parentheses are J coupling constants).
Scheme 1. Synthesis of Model Tetrahydropyran 7c (i) Ac2O, HBr/AcOH, Zn/CuSO4 (71%); (ii) H2, Pd/C, (92%); (iii)
NH4OH, MeOH, (21%)
1
Table 2. H (600 MHz) and 13C (150 MHz) NMR Data for Methyl-Pyridoxatin (8a/b) in DMSO-d6
8 (major atropisomer)
8 (minor atropisomer)
a
a
position
δC, type
δH mult. (J in Hz)
δC, type
δH, mult. (J in Hz)
1
157.5, C
113.2, C
45.8, CH
41.7, CH
41.7, CH2
−
−
159.8, C
113.7, C
45.7, CH
42.8, CH
42.0, CH2
−
−
2
3
2.33, dd (10.5, 10.5)
2.57, dd (11.0, 11.0)
4
2.99, m
2.77, m
5a
1.66, ma
1.68, ma
5b
0.82, mb
0.84, mb
6
31.4, CH
44.2, CH2
1.53, mc
31.5, CH
44.4, CH2
1.53, mc
7a
1.68, md
1.67, md
7b
0.66, me
0.68, me
8
31.4, CH
143.6, CH
112.3, CH2
2.29, m
32.4, CH
143.4, CH
112.4, CH2
2.13, m
9
5.49, mf
5.46, mf
10a
4.73, ddd (17.2, 2.4, 0.8)g
4.62, dd (10.3, 2.4)h
0.63, d (6.6)i
0.89, d (6.6)j
−
4.74, d (17.2, 2.4, 0.8)g
4.63, dd (10.3, 2.4)h
0.64, d (6.6)i
0.88, d (6.6)j
−
10b
11
20.6, CH3
22.8, CH3
162.4, C
20.4, CH3
22.7, CH3
161.4, C
12
1′
2′
3′
133.0, CH
97.9, CH
63.9, CH3
7.58, d (7.8)
5.83, d (7.8)
3.82, s
132.7, CH
98.7, CH
63.8, CH3
7.55, d (7.8)
5.80, d (7.8)
3.81, s
N-OCH3
a
Resonances with the same superscript letter (a-j) across atropisomers are overlapping.
treated with Ac2O and catalytic HBr/acetic acid to effect
peracetylation, with further addition of HBr/AcOH generating
the intermediate anomeric bromide, which on workup in Zn/
CuSO4 returned the acetylated glycal 7a. Subsequent hydro-
genation and deacetylation yielded 7b and 7c, respectively. The
1D NMR (DMSO-d6) data for 7c compared very well with the
natural product 7 (Figures S16 and S17), confirming the
proposed structure, including relative configuration. Notwith-
standing different C-2 side chains, as the [α]D for 7 (−25, c
0.05, MeOH) was opposite in sign to 7c (+11, c 0.15, MeOH),
we tentatively propose a 2R,3S,4R absolute configuration for
chaunopyran A (7) (Figure 1).
Chaunopyran A (7) belongs to a rare class of fungal 2-
alkenyl-tetrahydropyran, selected examples of which (glycinyl
esters) are potent inhibitors of the cytochrome P450 lanosterol
C-14 demethylase, and as such exhibit broad-spectrum
antifungal activity. The first members of this structure class,
reported in 1991 by Merck, Sharpe, and Dohme researchers
from Penicillium restrictum, were the antifungal glycinyl esters
restricticin and N,N-dimethylrestricticin, and the alcohol
restrictinol.8 In 1992, Roche researchers reported five new
congeners (Ro 09−1543, Ro 09−1545, Ro 09−1547, Ro 09−
1549, and Ro 09−1544) from Penicillium sp. NR6564 and
Aspergillus sclerotiorum Huber,9 while Bristol-Myers Squibb
researchers reported the glycinyl esters lanomycin and
glucolanomycin, and the alcohol lanomycinol from Pycnidio-
phora dispersa.10 Our discovery of 7 represents the first natural
product addition to this structure class in over 30 years.
Consistent with established structure activity relationship
studies, as 7 did not possess a glycinyl ester moiety it did not
exhibit fungicidal or fungistatic activity (against either our assay
strain Candida albicans (ATCC 90028), or the co-cultivation
strain Trichoderma hamatum (CMB-MF030)). As co-culture
activation of 7 would seem to suggest a defensive response, we
were initially perplexed to discover that 7 lacked antifungal
activity. Based on prior literature, glycinyl ester analogues of 7
could be expected to have exceptionally potent antifungal
properties, and as such may only be produced under co-
cultivation conditions at very low concentrations. Unfortu-
nately, all efforts to detect putative glycinyl ester of 7 proved
unsuccessful. At this point it is worth noting that, despite a
good DAD response, 7 is only weakly responsive to ESIMS
analysis. The failure to detect low levels of putative glycinyl
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esters may be attributed to their instability during MS
ionization.
HRESI(+)MS analysis of 8a/b returned a sodium adduct ion
indicative of a molecular formula (C16H23NO3, Δmmu −0.5)
suggestive of a methylated analogue of pyridoxatin (1a/b).
Comparison of 1D and 2D NMR (DMSO-d6) data for 8a/b
(Table 2 and Figure 4) with 1a/b confirmed this hypothesis,
1a/b to 8a/b. Supportive of this hypothesis, CMB-MF030
cultures treated with an extract of CMB-MF028 rich in 1a/b
effect a quantitative biotransformation of 1a/b to 8a/b (Figure
5). Given its relationship to 1a/b, we attribute a 3R,4S,6R,8S
absolute configuration to methyl-pyridoxatin (8a/b) (Figure 1).
In summary, our observations document complementary
chemical-ecology responses encountered during co-cultivation
of CMB-MF028 and CMB-MF030 (Figure 2). During co-
cultivation, CMB-MF028 deploys a constitutively produced
antifungal agent 1a/b that we propose has the potential to
inhibit the growth of CMB-MF030. Confronted by this
challenge, CMB-MF030 biotransforms and deactivates 1a/b
to the nonantifungal 8a/b. As CMB-MF028 continues to
produce 1a/b, and CMB-MF030 continues to biotransform 1a/
b to 8a/b, the relative kinetics of these processes determines
the equilibrium for this encounteron an agar plate, co-
cultivation is evidenced by a growth inhibition interface
between these cultures (Figure 2). We speculate that, in an
effort to alter the balance of the battle in its favor, and in
response to an as yet unknown co-cultivation cue, CMB-
MF028 activates a silent PKS to produce chaunopyran A (7).
Chaunopyran A (7) is the first new example of a rare class of
fungal 2-alkenyl-tetrahydropyrans to be reported in over 30
years. Prior studies on the structure class determined that
glycinyl esters were exceptionally potent antifungals, whereas
simple alcohols such as 7 were not antifungal. Whether CMB-
MF028 is stimulated by co-culture with CMB-MF030 to
produce glycinyl esters of 7 at levels below detection, or
whether the BGC is corrupted and lacks the necessary NRPS
module for incorporating the glycinyl residue, remains an
intriguing, but unresolved question.
Figure 4. Diagnostic 2D NMR (DMSO-d6) correlations for methyl-
pyridoxatin (8a/b).
with the only significant difference being the appearance of
resonances for an N-OCH3 moiety (major atropisomer δH 3.82;
δC 63.9; minor atropisomer δH 3.81; δC 63.8)as previously
documented for 1a/b, the NMR and HPLC data for 8a/b
revealed major and minor atropisomers (5:3).
Importantly, whereas 1a/b is a potent siderophore (logKapp
34 binding affinity for Fe(III))6 and is known to exhibit
antifungal properties dependent on retaining an unmodified N-
OH moiety,11 the N-OMe analogue 8a/b in our hands exhibits
no Fe(III) binding affinity, nor is it cytotoxic to mammalian or
fungal cells. As 1a/b is constitutively produced by CMB-
MF028,6 we speculate that under co-cultivation condition
CMB-MF030 counters this antifungal agent by biotransforming
Figure 5. HPLC-DAD (210 nm) chromatograms of CMB-MF030 biotransformation of 1a/b to 8a/b and single ion extraction (SIE) with m/z 264
and m/z 278. (a) Monoculture of CMB-MF030; (b) monoculture of CMB-MF028; CMB-MF030 cultivated in the presence of EtOAc extract of
CMB-MF028 on (c) day 0 and (d) day 7. Peaks for metabolites are annotated and highlighted.
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culture plate of CMB-MF028 and CMB-MF030 was incubated at 26.5
°C for 5 days, after which it was sliced into three parts, as shown in
Figure 2A. Each part was extracted with EtOAc, and concentrated in
vacuo to yield extracts which were resuspended in MeOH (5 mg/mL)
and analyzed by HPLC-DAD-MS (Zorbax SB-C8 column, 150 × 4.6
mm, 5 μm, 1 mL/min gradient elution from 90% H2O/MeCN to
100% MeCN over 15 min with isocratic 0.05% formic acid modifier).
Production, Isolation, and Characterization of 7 and 8a/b. A
scaled-up (3 L) ISP-2 broth of co-cultivation of Chaunopycnis sp.
(CMB-MF028) and T. hamatum (CMB-MF030) was incubated at 180
rpm/min at 26.5 °C for 5 days. An EtOAc extract (501.0 mg) was
generated and sequentially triturated to afford hexane (0.7 mg),
CH2Cl2 (202.5 mg), and MeOH (301.0 mg) soluble fractions. HPLC-
DAD analysis localized the induced metabolites in the CH2Cl2 and
MeOH fractions, which were combined and subjected to reversed-
phase (C18) SPE and preparative HPLC fractionation (Luna C18
column, 250 × 21.2 mm, 10 μm, 20 mL/min gradient elution from
85% to 10% H2O/MeCN over 20 min) to afford chaunopyran A (7)
(tR = 12.7 min; 1.5 mg, 0.3% of EtOAc extract) and the crude methyl-
pyridoxatin (8a/b) (tR = 13.4−14.4 min; 6.0 mg) contaminated with a
small amount of pyridoxatin (1a/b). The latter fraction was further
purified by normal phase column chromatography (SiO2; petroleum
ether:acetone 5:1−2:1) to afford pure methyl-pyridoxatin (8a/b) (5.5
mg, 1.1% of EtOAc extract).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Specific optical rotations
([α]D) were measured on a JASCO P-1010 polarimeter in a 100 × 2
mm cell at room temperature. UV−visible spectra were obtained on a
Varian Cary 50 UV−visible spectrophotometer with 1 cm quartz cells.
Nuclear magnetic resonance (NMR) spectra were acquired on a
Bruker Avance 600 MHz spectrometer with either a 5 mm PASEL
1H/D-13C Z-Gradient probe or 5 mm CPTCI 1H/19F-13C/15N/
DZ-Gradient cryoprobe, controlled by TopSpin 2.1 software. In all
cases spectra were acquired at 25 °C (unless otherwise specified) in
solvents as specified in the text, with referencing to residual 1H or 13
C
signals in the deuterated solvents. Electrospray ionization mass
spectrometry (ESIMS) experiments were carried out on an Agilent
1100 series LC/MSD (quadrupole) instrument in both positive and
negative modes. High-resolution ESIMS spectra were obtained on a
Bruker micrOTOF mass spectrometer by direct injection in MeCN at
3 μL/min using sodium formate clusters as an internal calibrant. High-
performance liquid chromatography-diode array-mass spectrometry
(HPLC-DAD-MS) data were acquired on an Agilent 1100 series
separation module equipped with an Agilent 1100 series HPLC/MSD
mass detector and diode array multiple wavelength detector.
Semipreparative and preparative HPLCs were performed using Agilent
1100 series HPLC instruments with corresponding detectors, fraction
collectors, and software inclusively. Ultrahigh-performance liquid
chromatograph-diode array (UPLC-DAD) data were obtained on an
Agilent 1290 infinity UPLC system.
Fungal Collection, Isolation, and Taxonomy. Both of the fungi
Chaunopycnis sp. (CMB-MF028) and Trichoderma hamatum (CMB-
MF030) were isolated in 2012 from the inner tissue of a marine
pulmonate false limpet Siphonaria sp. collected at the rocky intertidal
zone of Moora Park, Shorncliffe, Queensland. The fresh Siphonaria
sample was transported in a sterile tube (50 mL) on ice to the
laboratory, where it was rinsed in sterile natural seawater for 1 min and
subjected to surface sterilization in 70% EtOH (v/v) for 30 s after
which it was washed with sterile natural seawater to remove traces of
EtOH. Subsequently, the sample was dissected under aseptic
conditions and the inner tissue placed on PYG agar plates (comprising
2% glucose, 1% peptone, 0.5% yeast extract, 0.02% chloramphenicol,
1.7% sea salt, and 1.5% agar). The plates were sealed and incubated at
26.5 °C for 3−4 weeks. Pure cultures of fungi CMB-MF028 and CMB-
MF030 were obtained by single-colony serial transfer on agar plates
and then cryopreserved at −80 °C in 15% aqueous glycerol. Fungus
CMB-MF028 formed circular white colonies with fan-shaped wrinkles
and no spores on peptone yeast glucose (PYG) agar and ISP-2 agar.
Fungus CMB-MF030 grew quickly with white floccus aerial hyphae
and yellow-green spores after 10 days cultivation on PYG agar. The
BLAST search showed the amplified ITS sequence for CMB-MF028
(GenBank accession no. KP881722) has 98% homology with other
members of the genus Chaunopycnis sp., and ITS sequence for CMB-
MF030 (GenBank KU593648) has 100% homology with other
members of the species T. hamatum.
Chaunopyran A (7). Pale yellow oil; [α]D22 − 25 (c 0.05, MeOH);
UV−vis (MeOH) λmax (log ε) 260 (3.71), 269 (3.77), 279 (3.69) nm;
1D and 2D NMR (600 MHz, DMSO-d6) data, Table 1 and Figures S6
and S7; ESI(+)MS m/z 221 [M − H2O + H]+, 203 [M − 2H2O +
H]+; HRESIMS m/z 261.1454 [M + Na]+ (calcd for C14H22O3Na,
261.1461).
22
Methyl-Pyridoxatin (8a/b). White powder; [α]D − 15 (c 0.12,
MeOH); UV−vis (MeOH) λmax (log ε) 285 (3.84) nm; 1D and 2D
NMR (600 MHz, DMSO-d6) data, Table 2 and Figures S8 and S9;
ESI(+)MS m/z 278 [M + H]+, ESI(−)MS m/z 276 [M − H]−;
HRESIMS m/z 300.1575 [M + Na]+ (calcd for C16H23NO3Na,
300.1570).
Chemical Synthesis of 7c. L-Rhamnose monohydrate (1.0 g)
suspended in Ac2O (3.5 g, 6.2 mol equiv) was treated at room
temperature (rt) with 22% HBr/AcOH (0.1 g). After stirring for 1 h,
an additional aliquot of 22% HBr/AcOH (10.5 g, 5.3 mol equiv) was
introduced and the mixture stirred overnight, after which anhydrous
NaOAc (2.5 g) was added to neutralize the excess HBr, and the
resulting solution treated with Zn (10.0 g) and CuSO4 (0.16 g
suspended in H2O (10 mL) and AcOH (5 mL) containing sodium
acetate (7.5 g). The resulting mixture was stirred vigorously at rt for 3
h, after which it was partitioned between H2O (100 mL) and EtOAc
(100 mL). The EtOAc solubles were washed successively with H2O
(100 mL), saturated aqueous NaHCO3 (100 mL), and saturated brine
(100 mL), after which they were concentrated in vacuo to yield a
colorless oil which was purified by column chromatography (SiO2;
petroleum ether: EtOAc 30:1−15:1) to afford the acetylated glycal 7a
(310.7 mg, 31%). Subsequently, 7a was dissolved in MeOH (10 mL)
and hydrogenated in the presence of Pd/C (31 mg) for 1.5 h to yield
7b (286.6 mg, 92%) as colorless oil. A sample of 7b in MeOH (5 mL)
was treated with K2CO3 (100 mg) and stirred for 15 min to yield, after
standard solvent extraction, a colorless oil, which was purified by
column chromatography (SiO2, increasing polarity from
CH2Cl2:MeOH (40:1) to CH2Cl2:MeOH(20:1) to afford 7c (58.9
mg, 21%).
Analytical Cultivation and Chemical Profiling of Co-Culture
in ISP-2 Broth. The co-culture of CMB-MF028 and CMB-MF030,
inoculated from their respective seed culture (2 mL for each culture),
was cultivated at 180 rpm for 5 days in a Schott flask (250 mL)
containing ISP-2 broth (0.4% glucose, 0.4% yeast extract, and 1% malt
extract in 50 mL distilled H2O). After cultivation, the broth was
extracted with EtOAc (50 mL) and the organic phase concentrated in
vacuo to yield an organic extract (7.6 mg). A solution of the extract
prepared in MeOH (5 mg/mL) was subjected to HPLC-DAD-MS
analysis (Zorbax SB-C8 column, 150 × 4.6 mm, 5 μm, 1 mL/min
gradient elution from 90% H2O/MeCN to 100% MeCN over 15 min
with isocratic 0.05% formic acid modifier). The EtOAc extract for the
monoculture control of CMB-MF028 and CMB-MF030 was also
analyzed using the same HPLC method.
7a. Colorless oil; 1H NMR (CDCl3) δH 4.11 (dq, J = 8.3, 6.6 Hz, H-
2), 5.03 (dd, J = 8.3, 6.2 Hz, H-3), 5.34 (ddd, J = 6.2, 3.0, 1.2 Hz, H-4),
4.78 (dd, J = 6.2, 3.0 Hz, H-5), 6.43 (dd, J = 6.2, 1.2 Hz, H-6), 1.31 (d,
J = 6.6 Hz, H-7), 2.09 (s, 3-OAc), 2.04 (s, 4-OAc); 13C NMR (CDCl3)
δC 72.7 (C-2), 72.0 (C-3), 68.5 (C-4), 99.0 (C-5), 146.2 (C-6), 16.8
(C-7), 21.1 (3−OCOCH3), 21.3 (4−OCOCH3), 170.1 (3-OCOCH3),
170.9 (4-OCOCH3); HRESIMS m/z 237.0744 [M + Na]+ (calcd for
C10H14O5Na, 237.0739).
Analytical Cultivation and Chemical Profiling of Co-Culture
on ISP-2 Agar. Fungus CMB-MF028 was inoculated with a single
colony on the left side (2 cm from the edge) of an ISP-2 agar plate.
After 5 days, fungus CMB-MF030 was inoculated on the right side (2
cm to the edge) of the same agar plate, by the same method. The co-
7b. Colorless oil; 1H NMR (CDCl3) δH 3.39 (dq, J = 9.4, 6.2 Hz, H-
2), 4.72 (dd, J = 9.4, 9.4 Hz, H-3), 4.92 (ddd, J = 11.5, 9.4, 5.3 Hz, H-
4), 2.06 (m, H-5a), 1.78 (tdd, J = 12.8, 11.5, 5.1 Hz, H-5b), 3.94 (ddd,
E
DOI: 10.1021/acs.jnatprod.7b00144
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Journal of Natural Products
Article
J = 11.9, 5.1, 1.7 Hz, H-6a), 3.47 (ddd, J = 11.9, 11.9, 2.1 Hz, H-6b),
1.18 (d, J = 6.2 Hz, H-7), 2.05 (s, 3-OAc), 2.02 (s, 4-OAc); 13C NMR
(CDCl3) δC 74.9 (C-2), 74.8 (C-3), 72.6 (C-4), 31.6 (C-5), 65.3 (C-
6), 18.1 (C-7), 21.1 (3-OCOCH3), 21.3 (4-OCOCH3), 170.4 (3-
OCOCH3), 170.8 (4-OCOCH3); HRESIMS m/z 239.0897 [M + Na]+
(calcd for C10H16O5Na, 239.0895).
7c. Colorless volatile oil; [α]D + 11 (c 0.15, MeOH); H NMR
(CDCl3) δH 3.21 (dq, J = 9.1, 6.1 Hz, H-2), 3.08 (dd, J = 8.9, 8.9 Hz,
H-3), 3.58 (ddd, J = 11.4, 8.9, 5.1 Hz, H-4), 1.96 (ddt, J = 12.9, 5.1, 1.9
Hz, H-5a), 1.71 (tdd, J = 12.9, 11.4, 5.0 Hz, H-5b), 3.92 (ddd, J = 11.8,
5.0, 1.6 Hz, H-6a), 3.45 (ddd, J = 12.6, 11.8, 2.1 Hz, H-6b), 1.30 (d, J
= 6.1 Hz, H-7); 13C NMR (CDCl3) δC 76.3 (C-2), 78.5 (C-3), 73.5
(C-4), 34.0 (C-5), 65.8 (C-6), 18.2 (C-7).
Biotransformation of 1a/b to 8a/b by CMB-MF030. The
EtOAc extract (50 mg, containing 1a/b as the major component) of 7
days’ cultivated fungus Chaunopycnis sp. (CMB-MF028) was added to
500 mL ISP-2 broth followed by inoculation of fungus T. hamatum
(CMB-MF030) seed culture and incubation for 7 days at 180 rpm at
26.5 °C. An aliquot (25 mL) of the 0 day and 7 days’ broth was
extracted with EtOAc respectively, followed by drying the organic
phase in vacuo to yield extracts. The extracts as well as the extracts
from 7 days’ monoculture of CMB-MF028 and CMB-MF030 under
the same cultivation condition were prepared at the concentration of 5
mg/mL and analyzed on HPLC-DAD-MS (Zorbax SB-C8 column, 150
× 4.6 mm column, 5 μm, 1 mL/min gradient elution from 90% H2O/
MeCN to 100% MeCN over 15 min, with constant 0.05% formic acid
modifier). Single ion extraction (SIE) with the protonated molecules
m/z 264 [M + H]+ for 1a/b and m/z 278 [M + H]+ for 8a/b was
performed on Agilent ChemStation software.
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.7b00144.
General experimental, fungal taxonomy, HPLC chroma-
tograms of co-cultivation study, bioassays, and NMR
spectra of 7, 7a−7c, and 8a/b (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: r.capon@uq.edu.au.
■
ORCID
Robert J. Capon: 0000-0002-8341-7754
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank B. P. Espos
́
ito (University of Sao Paulo) for helpful
̃
advice on assaying Fe(III) binding affinity and Z. G. Khalil
(UQ) for cytotoxicity assay. Z. S. acknowledges the provision of
the International Postgraduate Research Scholarships (IPRS)
and the Centennial Scholarships by the University of
Queensland. This research was funded in part by the Institute
for Molecular Bioscience, the University of Queensland and the
Australian Research Council (DP120100183).
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DOI: 10.1021/acs.jnatprod.7b00144
J. Nat. Prod. XXXX, XXX, XXX−XXX