Griseorhodins D-F, neuroactive intermediates and end products of post-PKS tailoring modification in griseorhodin biosynthesis

Article abstract of DOI:10.1021/np500155d

The griseorhodins belong to a family of extensively modified aromatic polyketides that exhibit activities such as inhibition of HIV reverse transcriptase and human telomerase. The vast structural diversity of this group of polyketides is largely introduced by enzymatic oxidations, which can significantly influence the bioactivity profile. Four new compounds, griseorhodins D-F, were isolated from a griseorhodin producer, Streptomyces sp. CN48+, based upon their enhancement of calcium uptake in a mouse dorsal root ganglion primary cell culture assay. Two of these compounds, griseorhodins D1 and D2, were shown to be identical to the major, previously uncharacterized products of a grhM mutant in an earlier griseorhodin biosynthesis study. Their structures enabled the establishment of a more complete hypothesis for the biosynthesis of griseorhodins and related compounds. The other two compounds, griseorhodins E and F, represent new products of post-polyketide synthase tailoring in griseorhodin biosynthesis and showed significant binding activity in a human dopamine active transporter assay.

Full text of DOI:10.1021/np500155d

Article
pubs.acs.org/jnp
Griseorhodins D−F, Neuroactive Intermediates and End Products of
Post-PKS Tailoring Modification in Griseorhodin Biosynthesis
Zhenjian Lin,† Malcolm M. Zachariah,† Lenny Marett,†,§ Ronald W. Hughen,§ Russell W. Teichert,⊥
Gisela P. Concepcion,‡ Margo G. Haygood,∥ Baldomero M. Olivera,⊥ Alan R. Light,§
and Eric W. Schmidt*,†,⊥
†Department of Medicinal Chemistry, L.S. Skaggs Pharmacy Institute, University of Utah, Salt Lake City, Utah 84112, United States
‡Marine Science Institute, University of the Philippines, Diliman, Quezon City 1101, Philippines
§Department of Anesthesiology, University of Utah, Salt Lake City, Utah 84112, United States
⊥Department of Biology, University of Utah, Salt Lake City, Utah 84112, United States
∥Department of Environmental and Biomolecular Systems, OGI School of Science & Engineering, Oregon Health & Science
University, Beaverton, Oregon 97006, United States
S
* Supporting Information
ABSTRACT: The griseorhodins belong to a family of extensively modified
aromatic polyketides that exhibit activities such as inhibition of HIV reverse
transcriptase and human telomerase. The vast structural diversity of this
group of polyketides is largely introduced by enzymatic oxidations, which can
significantly influence the bioactivity profile. Four new compounds,
griseorhodins D−F, were isolated from a griseorhodin producer, Streptomyces
sp. CN48+, based upon their enhancement of calcium uptake in a mouse
dorsal root ganglion primary cell culture assay. Two of these compounds,
griseorhodins D1 and D2, were shown to be identical to the major,
previously uncharacterized products of a grhM mutant in an earlier
griseorhodin biosynthesis study. Their structures enabled the establishment
of a more complete hypothesis for the biosynthesis of griseorhodins and
related compounds. The other two compounds, griseorhodins E and F,
represent new products of post-polyketide synthase tailoring in griseorhodin
biosynthesis and showed significant binding activity in a human dopamine active transporter assay.
riseorhodins belong to a large family of bioactive,
Garomatic polyketides that are synthesized in actino-
mycetes by type II polyketide synthase (PKS) proteins.1,2 The
closest relatives of griseorhodin originate as pentangular
polyketides that undergo reduction in the C-19/20 region
(Figure 1A). Within this group (Figure 1), griseorhodins,
fredericamycins, and benastatins have been extensively studied.3
These compounds almost certainly originate in very similar
polyketide products that are reduced in the C-19/20 region, but
that differ in chain length because they originate with starter
units of different lengths.1 After the polyketide assembly and C-
19/20 region reduction steps, the compounds are tailored by
modifying enzymes, of which oxidases are especially prevalent.
The major structural differences between the mature natural
products are introduced during these tailoring steps.4 For
example, the early intermediates en route to griseorhodin
undergo a series of steps culminating in oxidative C−C bond
cleavage and rearrangement to yield the mature metabolites.5
By contrast, benastatins undergo an early methylation event,
and the final products contain the same core carbon skeleton as
found in the post-PKS intermediate.6 Fredericamycin under-
goes a tailoring event leading to a carbocyclic moiety.7 Finally,
these mature products undergo a further series of degradation
reactions in the producing bacteria to yield a large number of
known products.6,7
In the polyketide group that exhibits reduction in the C-19/
20 region, the first post-PKS intermediate is thought to be an
unstable monomer that readily undergoes radical reactions.5,7
1H NMR spectra have been obtained for these intermediates in
fredericamycin and griseorhodin biosynthesis, but the com-
pounds have not been completely characterized.5,7 In
benastatin biosynthesis,6 methyl groups are added at C-8 to
this unstable intermediate, leading to the stable benastatin
product. When the C-methyltransferase was knocked out,
instead of obtaining a monomeric precursor, a mixture of
unstable dimers was obtained, which readily broke down in the
presence of air. These dimers were characterized by NMR,
revealing the probable intermediate in benastatin biosynthesis
(Figure 1B).6
Received: February 17, 2014
Published: May 1, 2014
© 2014 American Chemical Society and
American Society of Pharmacognosy
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Figure 1. Structural diversity in the griseorhodin and its close biosynthetic relatives. (A) Postulated early steps in griseorhodin and fredericamycin
1
biosynthesis. The structures of the intermediates in brackets are supported by H NMR spectra and MS data. Here, we show that for the
griseorhodin case the spectra represent those of a dimer. (B) Benastatin biosynthesis. A methyltransferase knockout led to accumulation of an
unstable dimer, while methylation affords the natural product. The dimer in the griseorhodin pathway is similar to that of benastatin.
Chart 1
We have been studying metabolites from bacterial associates
of marine mollusks.8 One of these strains, Streptomyces sp.
CN48+, provided an extract that was bioactive in a neuroassay.
The active components were identified as a series of novel
compounds, which included both dimers of griseorhodin
precursors and griseorhodin degradation products. The
precursor dimers 1a and 1b were shown to be identical to
the earliest known intermediate in griseorhodin biosynthesis,
which was previously uncharacterized.5 Like the early
benastatin intermediate,6 it exists as a series of unstable dimers,
which may be the true substrates of early biosynthetic tailoring
enzymes. These early precursor dimers and novel degradation
products 3 and 4 provide information that aids in under-
standing the griseorhodin biosynthetic pathway.
RESULTS AND DISCUSSION
■
Extracts of strain CN48+ were strongly active in the mouse
dorsal root ganglion (DRG) assay, directly stimulating Ca2+
influx. In an assay-guided procedure, the active extract was
subjected to C18 flash chromatography, followed by HPLC, to
yield three neuroactive compounds, griseorhodins D−F (1, 3,
and 4), which were responsible for the activity of the extract.
The molecular formula C52H38O16 was assigned to each of
the two constitutional isomers (1a and 1b) on the basis of
HRESIMS analysis. 1H, 13C, HSQC, HMBC, COSY, and
NOESY NMR spectra were used in the structure determi-
1
nation. The H NMR spectrum of 1a showed signals of 17
protons, including three exchangeable hydroxyl protons, four
singlet aromatic protons, a singlet methine proton, six
methylene protons, and three methyl protons (Table 1). In
1
CD3CN, the H NMR spectrum of 1a had several unusually
broad peaks, which complicated the structural analysis. These
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Table 1. NMR Data of Compound 1a and 1b in CD3CN-d3
1a
1b
δH (J in Hz)
no.
δC
δH (J in Hz)
δC
a
1
ND
ND
2
108.2 C
161.1 C
120.7 C
150.1 C
120.9 CH
139.2 C
57.7 CH
145.4 C
109.7 CH
164.2 CH
102.7 CH
165.2 C
111.3 C
192.0 C
116.4 C
158.1 C
125.1 C
108.0 C
ND
3
4
121.1 C
ND
5
6
7.42 brs
4.37 s
ND
7.28 brs
4.46 s
7
8
57.5 CH
ND
9
10
11
12
13
14
15
16
17
18
19
6.00 brs
6.22 s
109.2 CH
ND
6.35 brs
6.15 s
102.6 CH
165.3 C
112.4 C
ND
115.1 C
158.6 C
125.2 C
Figure 2. Key NOESY, HMBC, and COSY correlations for
compounds 1a, 1b, 3, and 4.
20.2 CH2 2.76 m;
2.33 m
20.4 CH2 2.83 m;
2.50 m
between H-8 and C-8 in both 1a and 1b indicated that the
compounds were dimeric and fused at C-8.
20
21
22
23
24
25
26
31.2 CH2 2.74 m
30.7 CH2 2.73 m
Chemical degradation experiments were sufficient to
elucidate the remaining connectivities in 1a and 1b.
Compounds 1a/b were not stable when exposed to air,
especially in DMSO solutions. Both 1a and 1b were slowly
converted to a red compound, 2, which could be purified by
HPLC. NMR experiments showed that 2 was identical to the
previously reported metabolite KS-619-1.9 In fact, the only
major difference of the NMR data between 1a/b and 2 was the
presence of a methine residue (δH 4.37 and δC 57.7 for 1a; δH
4.46 and δC 57.5 for 1b) in place of a quinone carbonyl group.
Therefore, the core structure of compounds 1a/b was
determined. The core structure of 1a/b presented half the
number of atoms in the molecular formula of 1a/b (C26H19O8
vs C52H38O16), further reinforcing that 1a and 1b are
homodimers.
Mass spectrometric data provided further evidence that 1a/b
were dimers of the reduced form of 2 (Figure 3). In particular,
the ion at m/z 458 suggested a radical ion fragment [M/2 −
H]−. Another ion at m/z 414, which is equivalent to a loss of 44
Da in comparison to an ion at m/z 458, suggested loss of CO2,
while an ion at m/z 372 was assigned as loss of CH2CO. The
fragmentation pattern of 1a/b was identical to that of 2, but
with a mass shift of 16 Da resulting from the additional oxygen.
In hydroxylactones such as benastatin F3 and precursors of
fredericamycin,7 an arylacetone group readily undergoes
tautomerization to form the hydroxylactone. In analogy, we
proposed that the H-16 and H-18 might be broadened by
tautomerism. Following the benastatin F precedent, we treated
compound 2 with a drop of 28% ammonium hydroxide,
revealing the presence of the “missing” methylene and methyl
protons and confirming the identity of the side chain.
Unfortunately, ammonium hydroxide led to immediate
decomposition of 1a/b.
ND
120.4 CH
ND
120.2 CH
6.75 s
6.74 s
140.1 C
ND
ND
ND
ND
3.27 brs
3.23 brs
ND
28.5 CH3 1.76 s
11.87 s
28.2 CH3 1.75 s
17-OH
12.10 s
13-OH
11.94 s
11.80 brs
12.3 brs
1-COOH
12.16 brs
a
ND: not detected.
broad signals were attributed to isomers that were interconvert-
ing on the NMR time-scale rather than to exchangeable
protons. Two broad singlet proton signals at δH 6.00 and 7.42,
which have no HSQC correlation, were assigned as aromatic
methine protons because they could not be exchanged with
extensive D2O treatment. Similarly, a broad signal for two
protons at δH 3.27 with a 35 Hz peak width at half-height also
was not exchangeable in D2O and showed a NOESY
correlation with a methyl signal at δH 1.76, indicating that
the signal corresponded to methylene protons.
We were able to observe the HMBC correlations (Figure 2)
for every sharp proton signal (one methine proton, two
aromatic singlet protons, two exchangeable phenol hydroxyl
protons, and one methylene proton): from H-8 to six aromatic
sp2-hybridized carbons (C-7, δC 139.2; C-9, δC145.4; C-6, δC
120.9; C-16, δC 116.4; C-10, δC 109.7; C-14, δC 111.3), from
H-10 to C-13, C-14, and C-10, from H-22 to C-20, C-4, and C-
2, from 17-OH to C-17, C-18, and C-16, from 13-OH to C-13,
C-14, and C-12, and from H-19 to C-5. Together with a COSY
correlation between H-20 and H-19 and NOESY correlations
between H-8/H-10 and H-8/H-6, these HMBC signals
indicated a polyphenol substructure. Furthermore, the chemical
shifts of the two phenol hydroxyl protons (δH 11.94 and 11.87)
indicated the presence of a conjugated ketone, which was
further confirmed by a quaternary carbon observed at 192.0
ppm in the 13C NMR spectrum. Finally, an HMBC correlation
1a/b are similar to products obtained in mutants of the
benastatin pathway,6 with the differences being the length and
oxidation state of the side chain at C-3 and the saturation state
of the C5−C6 bond. As found with the benastatin derivatives,
NMR experiments were not sufficient to differentiate between
possible stereoisomers at C8−C8′. This problem was
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Figure 3. ESI−MS fragmentation of compounds 1a/b and 2.
1
Table 2. H and 13C NMR Data of Compounds 3 and 4 in
previously solved in the benastatin case using molecular
modeling,6 which showed that one compound was a meso
(R,S) dimer, existing as readily HPLC-separable rotamers.
However, those rotamers interconverted over time. A second
compound was racemic (R,R, S,S, or a combination of both)
and existed as a single rotamer, which closely coeluted with one
of the meso rotamers. The chemical shift differences between
these proposed isomers were minor, and none exhibited
different chemical shifts across the dimer interface. Here, the
two isolated compounds were well separated by HPLC, and
they slowly interconverted in the NMR tube. This
interconversion was slower than for the benastatin relatives
and was co-incident with monomer (2) formation. Nonethe-
less, this similarity makes it likely that 1a/1b represent the meso
(R,S) dimer. A third constitutional isomer closely coeluted with
1b, but was present in minor amounts and could not be
isolated. On the basis of similarity to the benastatin case, this
may represent the rac isomer.
CD3CN-d3
3
4
no.
δC
δH (J in Hz)
δC
δH (J in Hz)
1
167.6 C
104.8 C
148.8 C
142.8 C
137.9 C
116.7 CH
129.9 C
104.6 CH
152.4 C
19.1 CH3
26.8 CH2
33.2 CH2
173.9 C
167.6 C
104.8 C
148.9 C
142.2 C
137.6 C
117.5 CH
129.9 C
104.9 CH
152.2 C
19.9 CH3
28.8 CH2
33.8 CH2
173.1 C
52.4 CH3
2
3
4
5
6
6.89 s
6.45 s
6.87 s
6.45 s
7
8
9
10
11
12
13
2.28 s
2.27 s
3.07 t (7.6)
2.71 t (7.6)
3.05 t (7.6)
2.72 t (7.6)
13-OMe
3.66 s
Griseorhodin E (3) was assigned a molecular formula of
C13H12O6 on the basis of ESIMS analysis and 1D NMR data
(Table 2). The 1H NMR spectrum of 3 showed the presence of
two singlet aromatic protons at δH 6.89 and 6.45, two
methylene groups at δH 3.07 and 2.17, and a methyl group at
δH 2.28. The 13C NMR spectrum of 3 showed the presence of
13 signals, including two carbonyls, eight sp2 carbons, and three
sp3 carbons. The ratio of proton number to carbon number
suggested a highly substituted aromatic structure in 3. The
HMBC correlations from H-11 to C-4, C-5, C-6 and from H-6
to C-2, C-3, C-4 suggested a pentasubstituted phenyl ring. The
chemical shifts of C-2 and C-3 indicated a 2,3-dihydroxy
substitution. The 1H−1H COSY correlation between H-11 and
H-12, along with the HMBC correlation from H-12 to the
carbonyl carbon (C-13), suggested the presence of a 2-
carboxyethyl group at C-5. The HMBC correlations from H-10
to C-9, C-8 and from H-8 to C-6, C-7, C-9, along with a long-
distance correlation from H-6 to C-1, suggested a benzoenol
lactone substructure in 3. Together, these data were consistent
with the structure assigned to 3. The NMR spectra (Table 2) of
griseorhodin F (4) were very similar to those of 3. The only
difference was an extra methoxyl group observed in the NMR
spectra of 4. The HMBC spectrum showed the proton of the
methoxyl had a correlation to the carbonyl carbon C-1,
indicating 4 is a methyl ester of 3.
The precursor of 1a/b is similar to a precursor in the
benastatin pathway, which was proposed to be short-lived and
to break down via reactive benzyl radicals.6 By analogy, it
seemed possible that 1a/b were dimerized precursors in the
griseorhodin pathway. Indeed, griseorhodins were also obtained
from the culture broth of strain CN48+. To test this hypothesis,
we sequenced the genome of CN48+. The resulting draft
genome (11.5 Mbp) was assembled into 169 contigs (N50 =
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Scheme 1. Proposed Pathway for the Biosynthesis of 3 and 4
642 kbp). BLAST searching for aromatic type II polyketide
synthase homologues in the genome revealed only a single
significant hit (GenBank accession number KJ610894), which
was nearly identical to the previously reported griseorhodin
biosynthetic gene cluster2 (GenBank accession number
AF509565; identical gene content, syntenic, and 99% DNA
sequence identical). Thus, genomic data strongly implicated the
griseorhodin biosynthetic gene cluster as being responsible for
griseorhodin D−F biosynthesis, since no other potential type II
pathway was present in the genome.
the dimeric forms could be the actual in vivo substrates of the
early tailoring enzymes.
Oxidative metabolism might also lead to formation of 3 and
4, which we propose to be breakdown products of
griseorhodins. In this model, the spiroketal moiety of
griseorhodins would be subject to further enzymatic carbon−
carbon bond cleavage events, leading to formation of the
degradation products (Scheme 1). It is noteworthy that
compounds 1, 3, and 4 are major products of strain CN48+
under many different culture conditions, where they are
produced in nearly the equivalent amounts to griseorhodins
themselves. By contrast, it appears that these compounds are
not major constituents of previously reported griseorhodin
producers.5 It is unclear what causes these differences, but here
the availability of an additional strain led to the isolation and
structure elucidation of abundant intermediates that might
otherwise be difficult to obtain. For example, in the case of
fredericamycin oxidation, isolation of early products required
genetic manipulation.7
It seemed possible that the different product ratios in strain
CN48+ might be due to differences in oxygen tension or
oxidation state in the medium. Therefore, we manipulated the
culture conditions either by adding H2O2 or by limiting O2.
While peroxide increased the production of every compound,
oxygen limitation decreased production of all compounds.
Therefore, oxidation did not directly impact the production of
dimers versus mature griseorhodin A formation. In different
media conditions, CN48+ produced mainly the dimers 1a/b
when extra sodium was left out of the medium. When
supplemented with NaCl, mainly mature griseorhodins A and
75 were produced (Figure S8). It is well known that marine and
other bacterial strains are salt responsive in terms of their
chemical profiles.12
Compounds 1, 3, and 4 were identified because of their
activity in the DRG assay. This phenotypic assay uses a primary
culture of DRG neurons, which contains >12 individual cell
types that respond differently to neuroactive agents.13−15 The
different responses are caused largely by different combinations
of receptor and ion channel subtypes in these different cell
types, which reflect their phenotype in nature. DRG neurons
are the primary sensory neurons, and their individual functions
reflect sensations such as touch, pain, heat, cold, and itch.
Therefore, in a single assay, multiple, physiologically relevant
phenotypes are simultaneously assayed. The assay is performed
using a Ca2+-responsive dye, Fura-2. In the DRG assay, 3 and 4
increased the Ca2+ concentration in over 80% of the DRG cells
at 20 μg/mL. By contrast, 1a and 1b did not directly depolarize
cells, but instead potentiated the response to KCl. When 1a and
1b were administered at 10 μg/mL, subsequent addition of 25
mM KCl led to an increase in the Ca2+ influx in comparison to
controls in the same well.
Yunt et al.5a previously knocked out the genes potentially
responsible for grisheorhodin biosynthesis in Streptomyces sp.
JP95, which like CN48+ was isolated from a marine
invertebrate. When the gene grhM was deleted, two constitu-
tional isomer intermediates with a measured molecular formula
of C26H20O8 (m/z 461.1240, nearly half of the molecular
weight of 1a/b) were obtained, while all downstream
griseorhodin metabolites were abolished. Like 1a/b, the
products of the grhM mutant were unstable and difficult to
work with. Initially, the grhM mutant products were postulated
to be identical to the monomeric form of 1a/b. However, in
our examination of 1a/b, the identical ion (m/z 461.1) was also
found in many mass spectrometry conditions, which was
assigned as the major fragment of 1a/b. Furthermore,
examination of the HPLC data from Yunt et al.5b showed
that the two major isomer products of grhM mutant had a very
1
similar elution profile to 1a/b. Their H NMR spectra were
almost identical to a spectrum of 1a/b. Therefore, it seemed
likely that deletion of grhM leads to accumulation of 1a/b. To
test this hypothesis, we extracted the compounds from the
grhM mutant, which was kindly provided by Dr. Jorn Piel. Both
̈
HPLC-ESIMS and NMR experiments confirmed these
compounds are identical. Therefore, 1a/1b are the major
products from the grhM mutant.
GrhM is similar to FdmM from fredericamycin biosynthesis.7
FdmM was proposed to be an oxidase that performs the first
post-PKS aromatic hydroxylation in the pathway.10 Similar
proteins are encoded in many different type II PKS pathways,
where they may perform very similar functions.11 In the
griseorhodin pathway, GrhM appears to oxidize the C-14
position in 1a/b. Subsequently, other positions in the
polyketide are oxidized and modified by a series of tailoring
enzymes, leading ultimately to formation of mature griseo-
rhodins. Several other early intermediates in the pathway could
also not be readily characterized.5 We speculate that some of
these compounds might also be dimeric forms. For example,
the grhM mutant product was initially identified as possibly
being the monomer by MS, but our analysis shows that the
dimeric form readily provides ions corresponding to the
monomer due to fragmentation. Under many conditions, this
ion dominates the spectrum. This might also be true of some of
the other intermediates in the series,7 possibly indicating that
To determine the potential molecular targets and potencies
underlying these observed phenotypes, we further screened 1−
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Griseorhodin F (4): pale yellow solid; UV (MeOH) λmax 233, 260,
4 at the National Institute of Mental Health’s Psychoactive
Drug Screening Program (PDSP). A primary assay was
performed binding 46 targets at a final concentration of 10
μM of each compound. Initial hits were obtained in radioligand
displacement assays. For targets where significant binding
activity was detected, secondary binding assays were performed,
and Ki values were calculated using radioligand displacement
with test compound concentrations from 1 to 10 000 nM. Only
compound 3 exhibited activity below the 10 μM threshold, and
only in a human dopamine active transporter (DAT) assay,
with a Ki of 557 nM. Compound 4 did not show any binding
activity below 10 μM. We could not find any report of DAT in
DRG cells, making it unlikely that this target was responsible
for the activity in the phenotypic assay.
1
362 nm; H and 13C NMR (see Table 2); ESIMS m/z 265.1 [M +
H]+.
Chemical Modification of Griseorhodin D. Compound 2 was
synthesized by dissolving 1a (2 mg) in DMSO-d6 (500 μL) and
incubating the mixture for 10 days at −20 °C. Compound 2 (55%
yield) was purified by HPLC using 65% MeCN in H2O with 0.05%
TFA.
DRG Assay. DRG cells were obtained from mice, plated in medium
with additives, and loaded with Fura-2 AM (Molecular Probes).13
Experiments were performed as previously described.8e
Receptor Affinity Screen. Assays for the following receptors were
performed by the PDSP/NIMH: (1) muscarinic receptors: M1, M2,
M3, M4, M5; (2) serotonin receptors: 5ht1a, 5ht1b, 5ht1d, 5ht1e,
5ht2a, 5ht2b, 5ht2c, 5ht3, 5ht4, 5ht5a, 5ht6, 5ht7; (3) GABA
receptors: BZP (rat brain site), GABA A, GABA B; (4) histaminergic
receptors: H1, H2, H3, H4; (5) dopamine receptors: D1, D2, D3, D4,
D5; (6) transporters: NET, SERT, DAT; (7) opiate receptors: DOR,
KOR, MOR; (8) adrenergic receptors: Alpha1A, Alpha1B, Alpha1D,
Alpha2A, Alpha2B, Alpha2C, Beta1, Beta2, Beta3; (9) others: Sigma 1,
Sigma 2, Ca+ channel. Detailed online protocols are available for all
assays at the PDSP/NIMH Web site (http://pdsp.med.unc.edu/).
EXPERIMENTAL SECTION
■
General Experimental Procedures. UV spectra were obtained
using a PerkinElmer Lambda2 UV/vis spectrometer. NMR data were
collected using either a Varian INOVA 500 (1H 500 MHz, 13C 125
MHz) NMR spectrometer with a 3 mm Nalorac MDBG probe or a
Varian INOVA 600 (1H 600 MHz, 13C 150 MHz) NMR spectrometer
equipped with a 5 mm 1H[13C,15N] triple resonance cold probe with a
z-axis gradient, utilizing residual solvent signals for referencing. High-
resolution mass spectra (HRMS) were obtained using a Bruker
(Billerica, MA, USA) APEXII FTICR mass spectrometer equipped
with an actively shielded 9.4 T superconducting magnet (Magnex
Scientific Ltd., UK), an external Bruker APOLLO ESI source, and a
Synrad 50W CO2 CW laser.
ASSOCIATED CONTENT
■
S
* Supporting Information
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Bacterial Material. Streptomyces sp. CN48+ was cultivated from
Chicoreus nobilis, obtained by professional collectors near Mactan
Island, Cebu, Philippines, as previously described.8 The strain was
cultured from dissected venom duct tissue and purified, and later the
strain was recovered from a glycerol stock and used for further
chemical analysis. The 16S gene was cloned using primers 8−27f (5′-
AGAGTTTGATCCTGGCTCAG-3′) and 1492r (5′-
TACGGYTACCTTGTTACGACTT-3′) and submitted to GenBank
(accession number KJ400005).
Fermentation and Extraction. For the first-stage seed, an agar-
grown culture of Streptomyces sp. CN48+ was inoculated into 150 mL
of ISP2 medium (0.4% yeast extract, 1% malt extract, 0.4% glucose).
After 3 days’ incubation at 22 °C with shaking, the first stage (100 mL)
was used to inoculate in a New Brunswick BioFlo110 fermenter
containing 10 L of the medium ISP2, with or without addition of 2%
NaCl, at 30 °C. After 8 days, the broth was centrifuged and the
supernatant was extracted with Diaion HP-20 resin for 4 h. The resin
was filtered through cheesecloth and washed with H2O to remove
salts. The filtered resin was eluted with MeOH to yield a primary
extract. The MeOH eluate was dried under rotavap, and the water
remaining was portioned three times against EtOAc. The organic layer
was dried to yield the crude extract.
Corresponding Author
*Tel: 801-585-5234. Fax: 801-581-7087. E-mail: ews1@utah.
edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was funded by ICBG grant U01TW008163 from
Fogarty (NIH). We thank the government of the Philippines
and the community of Mactan Island for permission to conduct
this study. Binding assay data were generously provided by the
National Institute of Mental Health’s Psychoactive Drug
Screening Program, contract no. HHSN-271-2008-00025-C
(NIMH PDSP). The NIMH PDSP is directed by B. L. Roth
MD, PhD, at the University of North Carolina at Chapel Hill
and Project Officer J. Driscol at NIMH, Bethesda, MD, USA.
REFERENCES
■
(1) Lackner, G.; Schenk, A.; Xu, Z.; Reinhardt, K.; Yunt, Z. S.; Piel, J.;
Hertweck, C. J. Am. Chem. Soc. 2007, 129, 9306−9312.
(2) Li, A.; Piel, J. Chem. Biol. 2002, 9, 1017−1026.
Purification. The extract from 2% NaCl ISP2 media culture was
separated into six fractions (Fr1−Fr6) on a C18 column using step-
gradient elution of MeOH in H2O (40%, 50%, 60%, 70%, 80%, 100%).
Fr3, eluting in 60% MeOH, was further purified by C18 HPLC using
50% MeOH in H2O with 0.05% TFA to obtain compounds 3 (1.3 mg)
and 4 (4.0 mg). The extract from NaCl-free media culture was
separated into three fractions (Fr′1−Fr′3). Fr′3, eluting in 100%
MeOH, was further purified by C18 HPLC using 87% MeCN in H2O
with 0.05% TFA to obtain compounds 1a (4.1 mg) and 1b (3.2 mg).
Griseorhodin D1 (1a): yellow solid; UV (MeOH) λmax 228, 303,
(3) Xu, Z.; Schenk, A.; Hertweck, C. J. Am. Chem. Soc. 2007, 129,
6022−6030.
(4) (a) Rix, U.; Fischer, C.; Remsing, L. L.; Rohr, J. Nat. Prod. Rep.
2002, 19, 542−580. (b) Hertweck, C.; Luzhetskyy, A.; Rebets, Y.;
Bechthold, A. Nat. Prod. Rep. 2007, 24, 162−190.
(5) (a) Yunt, Z.; Reinhardt, K.; Li, A.; Engeser, M.; Dahse, H.;
Guetschow, M.; Bruhn, T.; Bringmann, G.; Piel, J. J. Am. Chem. Soc.
2009, 131, 2297−2305. (b) Yunt, Z. Chemical Investigation of
Streptomyces albus Heterologous Expression Strains and the Biosyn-
thesis of the Aromatic Polyketide Griseorhodin A. Ph.D. Thesis,
University of Bonn, Bonn, Germany, 2012; p 272.
(6) Schenk, A.; Xu, Z.; Pfeiffer, C.; Steinbeck, C.; Hertweck, C.
Angew. Chem., Int. Ed. 2007, 46, 7035−7038.
(7) Chen, Y.; Wendt-Pienkoski, E.; Rajski, S. R.; Shen, B. J. Biol.
Chem. 2009, 284, 24735−24743.
1
380 nm; H and 13C NMR (see Table 1); HRESIMS m/z 919.2241
[M + H]+ (calcd for C52H39O16, 919.2238).
Griseorhodin D2 (1b): yellow solid; UV (MeOH) λmax 228, 303,
1
380 nm; H and 13C NMR (see Table 1); HRESIMS m/z 919.2255
[M + H]+ (calcd for C52H39O16, 919.2238).
Griseorhodin E (3): pale yellow solid; UV (MeOH) λmax 233, 260,
1
362 nm; H and 13C NMR (see Table 2); ESIMS m/z 279.1 [M +
H]+.
1229
dx.doi.org/10.1021/np500155d | J. Nat. Prod. 2014, 77, 1224−1230

Journal of Natural Products
Article
(8) (a) Lin, Z.; Antemano, R. R.; Hughen, R. W.; Tianero, M. D.;
Peraud, O.; Haygood, M. G.; Concepcion, G. P.; Olivera, B. M.; Light,
A.; Schmidt, E. W. J. Nat. Prod. 2010, 73, 1922−1926. (b) Lin, Z.;
Reilly, C. A.; Antemano, R.; Hughen, R. W.; Marett, L.; Concepcion,
G. P.; Haygood, M. G.; Olivera, B. M.; Light, A.; Schmidt, E. W. J.
Med. Chem. 2011, 54, 3746−3755. (c) Lin, Z.; Flores, M.; Forteza, I.;
Henriksen, N. M.; Concepcion, G. P.; Rosenber, G.; Haygood, M. G.;
Olivera, B. M.; Light, A. R.; Cheatham, T. E.; Schmidt, E. W. J. Nat.
Prod. 2012, 75, 644−649. (d) Lin, Z.; Torres, J. P.; Ammon, M. A.;
Marett, L.; Teichert, R. W.; Reilly, C. A.; Kwan, J. C.; Hughen, R. W.;
Flores, M.; Tianero, M. D.; Peraud, O.; Cox, J. E.; Light, A. R.;
Villaraza, A. J.; Haygood, M. G.; Concepcion, G. P.; Olivera, B. M.;
Schmidt, E. W. Chem. Biol. 2013, 20, 73−81. (e) Lin, Z.; Marett, L.;
Hughen, R. W.; Flores, M.; Forteza, I.; Ammon, M. A.; Concepcion, G.
P.; Espino, S.; Olivera, B. M.; Rosenberg, G.; Haygood, M. G.; Light,
A. R.; Schmidt, E. W. Bioorg. Med. Chem. Lett. 2013, 23, 4867−4869.
(f) Lin, Z.; Koch, M.; Pond, C. D.; Mabeza, G.; Seronay, R. A.;
Concepcion, G. P.; Barrows, L. R.; Olivera, B. M.; Schmidt, E. W. J.
Antibiot. 2014, 67, 121−126.
(9) Yasuzawa, T.; Yoshida, M.; Shirahata, K.; Sano, H. J. Antibiot.
1987, 40, 1111−1114.
(10) Wendt-Pienkowski, E.; Huang, Y.; Zhang, J.; Li, B.; Jiang, H.;
Kwon, H.; Hutchinson, C. R.; Shen, B. J. Am. Chem. Soc. 2005, 127,
16442−16452.
(11) Martin, R.; Sterner, O.; Alvarez, M. A.; De Clercq, E.; Bailey, J.
E.; Minas, W. J. Antibiot. 2001, 54, 239−249.
(12) Bugni, T. S.; Ireland, C. M. Nat. Prod. Rep. 2004, 21, 143−163.
(13) Light, A. R.; Hughen, R. W.; Zhang, J.; Rainier, J.; Liu, Z.; Lee, J.
J. Neurophysiol. 2008, 100, 1184−1201.
(14) Teichert, R. W.; Raghuraman, S.; Memon, T.; Cox, J. L.;
Foulkes, T.; Rivier, J. E.; Olivera, B. M. Proc. Natl. Acad. Sci. U.S.A.
2012, 109, 12758−12763.
(15) Teichert, R. W.; Smith, N. J.; Raghuraman, S.; Yoshikami, D.;
Light, A. R.; Olivera, B. M. Proc. Natl. Acad. Sci. U.S.A. 2012, 109,
1388−1395.
1230
dx.doi.org/10.1021/np500155d | J. Nat. Prod. 2014, 77, 1224−1230