Deconins A-E: Cuparenic and mevalonic or propionic acid conjugates from the basidiomycete Deconica sp. 471

Article abstract of DOI:10.1021/np5010104

Bioassay-guided fractionation of antibacterial extracts from cultures of a basidiomycete from Northern Thailand, which represents a new species of the genus Deconica, yielded the terpenoid deconin A (1), whose structure was elucidated by spectral methods (NMR, HRMS) as a cuparenic/mevalonic acid conjugate. The absolute configuration of 1 was determined after saponification and comparison of specific rotations of the resulting cuparenic acid and mevalonolactone with authentic standards and literature data. Six minor congeners (2-7) were isolated and identified, and their antimicrobial and cytotoxic effects are reported. Compounds 1-4 are the first natural products featuring an unmodified mevalonic acid residue as a building block.

Full text of DOI:10.1021/np5010104

Note
pubs.acs.org/jnp
Deconins A−E: Cuparenic and Mevalonic or Propionic Acid
Conjugates from the Basidiomycete Deconica sp. 471
Frank Surup,†,‡ Benjarong Thongbai,§ Eric Kuhnert,†,‡ Enge Sudarman,†,‡ Kevin D. Hyde,§
and Marc Stadler*,†,‡
†Department of Microbial Drugs, Helmholtz Centre for Infection Research GmbH, Inhoffenstraße 7, 38124 Braunschweig, Germany
‡German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig, 38124 Braunschweig, Germany
§School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand
S
* Supporting Information
ABSTRACT: Bioassay-guided fractionation of antibacterial
extracts from cultures of a basidiomycete from Northern
Thailand, which represents a new species of the genus
Deconica, yielded the terpenoid deconin A (1), whose structure
was elucidated by spectral methods (NMR, HRMS) as a
cuparenic/mevalonic acid conjugate. The absolute configu-
ration of 1 was determined after saponification and
comparison of specific rotations of the resulting cuparenic
acid and mevalonolactone with authentic standards and
literature data. Six minor congeners (2−7) were isolated and
identified, and their antimicrobial and cytotoxic effects are
reported. Compounds 1−4 are the first natural products featuring an unmodified mevalonic acid residue as a building block.
asidiomycetes have been found to produce a broad range
of structurally diverse antibiotics. A fair example is the
molecular formula C21H30O5, implying 7 degrees of unsatura-
tion. The 1H NMR spectrum exhibited signals of two aromatic
methines with dual intensity and six methylenes and four
singlet methyl signals. The 13C and DEPT spectra indicated 21
carbons, being two carbonyls, two aromatic methines (dual
intensity), two aromatic quaternary carbons, six methylenes,
four methyls, and three quaternary sp3 hybridized carbon
atoms. 1H,1H COSY and 1H,13C HMBC spectra indicated
mevalonic acid and 4-(1,2,2-trimethylcyclopentyl)benzoic acid
moieties (Figure 1). An HMBC correlation from H2-5′ to C-1
explained the deep field shift of H2-5′ and established the
chemical structure. To reveal the absolute stereochemistry of
compound 1, the metabolite was saponified with sodium
hydroxide, and the free cuparenic acid (6) was isolated
subsequently by preparative HPLC. Its negative optical rotation
indicated an S configuration opposite to its optical antipode
(R)-(+)-cuparenic acid known from Cupressales.7 In this study,
the correlation of the absolute R configuration of cuparenic acid
with a positive specific rotation had been demonstrated by total
synthesis starting from (+)-cuparene. Furthermore, mevalono-
lactone, which spontaneously formed at pH 3,8 was identified as
R by GC/MS comparison with authentic standards.9 Therefore,
an 8S,3′R configuration was concluded for compound 1.
In addition to 1, a series of structurally related oxidized
metabolites that differed in geometry and substitution pattern
was isolated from crude extracts of multiple fermentations.
B
drug retapamulin, which was approved in 2007, but actually had
been developed from pleuromutilin, a metabolite of Clitopilus
scyphoides that had already been known for several decades.1
Basidiomycetes are still underexploited compared to bacteria
and ascomycetes,2 and they constitute promising sources for
the discovery of new antibiotics, which are urgently needed. We
are presently focusing on antibiotics from Asian tropical
basidiomycetes, which have been poorly investigated for both
their secondary metabolite production and their taxonomy.3,4
Deconica sp. 471 was isolated from a fruiting body collected
at Mt. Doi Mae Salong in Chiang Rai Province, Northern
Thailand. Morphological studies and sequencing of the
nucleotide sequences of 18S rDNA, internal transcribed spacers
(ITS), and large subunit rRNA gene (LSU) indicated that
strain 471 represents a new species in the genus Deconica.5 This
genus was established in 2009 to accommodate basidiomycetes
of the family Strophariaceae that were formerly placed in the
genus Psilocybe but do not produce hallucinogenic alkaloids of
the psilocybin type and constitutes a yet widely unknown sister
group of Psilocybe sensu stricto.6 The crude extracts from
submerged cultures of Deconica sp. 471 showed antimicrobial
activities against Gram-positive bacteria, i.e., Bacillus subtilis and
Staphylococcus aureus. Bioactivity-guided fractionation by RP-
HPLC, using B. subtilis as the indicator organism, led to the
isolation of the novel antimicrobial metabolite 1, which
constitutes the active principle.
Deconin A (1) (Chart 1) showed a molecular ion cluster [M
Received: December 15, 2014
+ H]+ at m/z 363.2166 in the HRESIMS, which provided the
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/np5010104
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Journal of Natural Products
Note
cisoidal arrangement between the C-12 hydroxy group and the
H3-15 methyl. Thus, 4 was established as the (R)-12-hydroxy
derivative of 1.
Chart 1. Metabolites Isolated from Deconica sp. 471
Deconin E (5), which was also obtained as a colorless oil,
had a molecular formula of C19H26O6 as determined by
HRESIMS. Despite the absence of two carbon atoms, the NMR
data pointed to a structural similarity to 4. In addition to the
shift of the hydroxy group to C-11, indicated by COSY
correlations between H2-10, H-11, and H2-12, the key
difference was the replacement of the mevalonic acid moiety
by a 2,3-dihydroxy-2-methylpropionic acid unit. The stereo-
chemistry of the sesquiterpenoid moiety was assigned from
ROESY correlations: While H-11 correlated to Hb-10 and Hb-
12, ROESY correlations were observed from H3-15 to H3-14
and Ha-12. Therefore, H3-15 and the C-11 hydroxy group are
cisoidal and an 11R configuration was concluded. The C-2′
configuration remained unassigned.
The molecular formula C15H20O2 of metabolite 6 was
1
provided by HRESIMS. H and 13C NMR resembled those of
the cyclic part of deconin A (1); COSY and HMBC
correlations identified cuparenic acid. The negative optical
rotation indicated an S configuration and established the
structure of 6 as ent-cuparenic acid.7
The molecular formula C15H18O4 of 7 specified two
additional oxygen atoms and an additional double-bond
equivalent compared to 6.16 The 13C NMR spectrum indicated
the replacement of two methylene groups by a ketone and an
oxymethine carbon. HMBC correlations from H3-13/H3-14 to
oxymethine C-10 and H-10/H2-12 to ketone C-11 identified 7
as 10-hydroxy-11-oxo-cuparenic acid. Finally, ROESY correla-
tions between H-11 and H3-13 and between H3-14 and H3-15
indicated a 10R configuration. Mosher’s (MTPA) esters were
synthesized for validation of the absolute configuration; the
observed positive ΔδSR chemical shift differences for H3-13 and
H3-14 on one hand and the negative ones for Ha-12 and Hb-12
on the other confirm the 8R,10R configuration.
Cuparene-type sesquiterpenes (see Figure S2 in the
Supporting Information for comparison) are widely distributed
through all divisions of life, since they have been isolated from
higher plants,7 liverworts,10 basidiomycete fungi,11 and the sea
hare Aplysia dactyloma.12 Interestingly, from a biosynthetic and
evolutionary point of view, some, like cuparene, exist as both
enantiomers in nature, which is an uncommon phenomenon.13
Whereas cuparenes from higher plants are regularly R, their S
counterparts have been isolated from fungi and liverworts.14
Consequently the S-chiralities of deconin and the ent-cuparenic
acid derivatives are consistent. However, deconins A−E (1−5)
are not simple sesquiterpenoids, but in 1−4 the sesquiterpene
structure is extended by mevalonic acid to form the common
scaffold. Although mevalonic acid is the precursor of all
terpenes biosynthesized via the HMG-CoA reductase pathway,
it has to the best of our knowledge never been observed as a
substituent of another terpene skeleton. Deconins A−D (1−4)
share this unprecedented structural entity.
Figure 1. Key 1H,1H COSY (bold lines) and 1H,13C HMBC (arrows)
correlations of deconin A (1).
Deconin B (2) was isolated as a colorless oil. Its molecular
formula, C21H28O6, was deduced from a prominent pseudo-
molecular ion cluster in its HRESIMS spectrum, indicating the
formal substitution of two hydrogen atoms by an oxygen atom.
1
The main difference in the 13C and H,13C HSQC NMR
spectra of 2 compared to 1 was the replacement of a methylene
by a ketone group (δC 219.6). 1H,13C HMBC correlations from
H2-10 and H2-12 located the ketone at C-11, characterizing 2 as
the 11-oxo derivative of 1.
The molecular formula C21H30O6 of deconin C (3) was
obtained by HRESIMS. The major difference in the NMR data
of 3 compared to 2 was the presence of two additional
methylenes (δH 2.42, 2.34) and the integral of only one proton
for the olefinic protons H-3 and H-4. HMBC correlations from
H2-6 to C-4, C-5, and C-8 and from H2-7 to C-1, C-2, and C-3
indicated the aromatic ring was reduced to a cyclohexadiene
system. Therefore, 3 was identified as the 6,7-dihydro-11-oxo
derivative of 1.
Deconin D (4), a colorless oil, was identified as another
C21H30O6 isomer by HRESIMS. The location of the new
hydroxy group was found at C-12 (δC 75.8) by COSY
correlations from H2-11 to H-12 and HMBC correlations from
H2-10, H2-11, and H3-15 to C-12. ROESY correlations between
H-12 and H3-13 and between H3-14 and H3-15 indicated a
The biological activity of metabolites 1−7 was assessed with
a broad test panel of bacteria and fungi (see Table S1,
Supporting Information). Deconin A (1) had weak activity
(MIC = 67 μg/mL) against Bacillus subtilis, Staphylococcus
aureus, and Mucor hiemalis. Cuparenic acid (6) had weak
activity (MIC = 67 μg/mL) against M. hiemalis. Both deconins
D (4) and E (5) showed weak activity (MIC = 67 μg/mL)
against S. aureus. Furthermore, deconin D (4) also exhibited a
moderate cytotoxic effect (IC50 = 7.4 μM) in a proliferation
B
DOI: 10.1021/np5010104
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Journal of Natural Products
Note
(4.8 mg) at tR = 32−32.5 min, 6 (0.4 mg) at tR = 41−41.5 min, and 7
(1.4 mg) at tR = 23.5−24 min.
assay against the mouse fibroblast cell line L-929. Since these
activities were in the same range as the crude extract;
cumulative, synergistic effects of the deconins cannot be
excluded.
Deconin A (1): colorless oil; [α]25 −22.4 (c 0.2, MeOH); UV
D
(MeOH) λmax (log ε) 244 (4.4) nm; IR (KBr) 3431, 2967, 2875, 1718,
1
1610, 1461, 1385, 1279, 1191, 1115, 1018, 776, 712 cm−1; H NMR
see Table 1; 13C NMR see Table 1; HRESIMS m/z 363.2166 ([M +
H]+, calcd for C21H31O5, 361.2166).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
determined with a PerkinElmer 241 polarimeter; UV spectra were
recorded with a Shimadzu UV−vis spectrophotometer UV-2450.
NMR spectra were recorded with a Bruker 500 MHz Avance III
spectrometer with a BBFO(plus) SmartProbe (1H 500 MHz, 13C 125
MHz) and a Bruker 700 MHz Avance III spectrometer with a 5 mm
TCI cryoprobe (1H 700 MHz, 13C 175 MHz). HRESIMS mass spectra
were obtained with an Agilent 1200 series HPLC-UV system
combined with an ESI-TOF-MS (Maxis, Bruker) [column 2.1 × 50
mm, 1.7 μm, C18 Acquity UPLC BEH (Waters), solvent A: H2O +
0.1% formic acid; solvent B: AcCN + 0.1% formic acid, gradient: 5% B
for 0.5 min increasing to 100% B in 19.5 min, maintaining 100% B for
5 min, RF = 0.6 mL min−1, UV detection 200−600 nm].
Table 1. NMR Data (1H 700 MHz, 13C 175 MHz) of
Deconin A (1) in Methanol-d4
pos
δC, type
δH, m (J in Hz)
HMBC
1
168.2, C
128.7, C
129.8, CH
128.3, CH
154.7, C
52.1, C
2
3/7
4/6
5
7.97, d (8.6)
7.53, d (8.6)
1, 5, 3/7
2, 4/6, 8
8
9
45.5, C
10
40.8, CH2
1.79, m
8, 9, 11, 12, 13, 14
8, 9, 11, 12, 13, 14
8, 9, 10, 12
Fermentation and Bioactivity-Guided Isolation of Deconin A
(1). The mycelium from a cultivation in 1 L of YMG medium was
separated from the culture broth by centrifugation (10 min at 1000g)
and filtration. The wet mycelium was extracted twice with acetone
(350 mL) in an ultrasonic bath at 30 °C for 30 min; the combined
extracts were concentrated in vacuo at 40 °C. The remaining aqueous
residue was diluted to 200 mL with water and extracted twice with 200
mL of ethyl acetate. The combined organic phases were dried over
sodium sulfate and again evaporated in vacuo (40 °C) to dryness. The
crude extract (ca. 50 mg) was dissolved in methanol. The crude extract
was adsorbed through an RP-18 cartridge, eluted with methanol, and
used for preparative HPLC as described below. The crude mycelial
extract was fractionated by preparative RP-HPLC (column: 250 × 21
mm, VP Nucleodur C18 Gravity 7 μm, gradient: 40% to 100%
acetonitrile with 0.5% acetic acid in 30 min, 100% for 15 min; flow
rate: 15 mL/min). Fractions at tR = 12−13 min were bioactive and
contained 1.3 mg of 1.
1.62, m
11
12
20.6, CH2
37.9, CH2
1.89, m
2.62, dt (12.8, 9.1)
1.79, m
5, 8, 11, 15
5, 8, 9, 15
13
14
15
1′
26.8, CH3
24.7, CH3
24.7, CH3
175.8, C
0.60, s
8, 9, 10, 14
1.15, s
8, 9, 10, 13
1.35, s
5, 8, 9, 12
2′
3′
4′
47.1, CH2
71.0, C
2.56, s
1′, 3′, 4′, 6′
2′, 3′, 5′, 6′
40.9, CH2
2.16, dt (14.5, 6.7)
2.11, dt (14.5, 6.7)
4.51, t (6.7)
5′
6′
62.5, CH2
27.6, CH3
3′, 4′, 1
2′, 3′, 4′
1.41, s
Fermentation and Isolation of Metabolites 2−7. Five small
agar disks (ca. 1 cm diameter) with well-grown mycelia (10−12 days
old) from agar plates (YM 6.3 medium) were used to inoculate 10
Erlenmeyer flasks (500 mL) containing 200 mL of YM 6.3 medium.
The submerged cultures were incubated on a rotary shaker at 25 °C
and 140 rpm. Samples were taken to determine the production of 1 by
HPLC-MS during the fermentation; production started after 14 days
and reached its maximum at 18 days. After the sugar was used up, the
wet mycelium (ca. 68 g) was obtained as described above. The crude
extract was filtered with methanol through an RP solid-phase cartridge
(Strata-X 33 mm, Polymeric Reversed Phase; Phenomenex, Aschaffen-
burg, Germany) to yield 95 mg of crude product. The latter was
divided into three portions and each subjected to preparative HPLC
(PLC 2020, Gilson, Middleton, WI, USA). A VP Nucleodur C18 ec
column (250 × 21 mm, 5 μm; Macherey-Nagel) was used as stationary
phase. The mobile phase was composed of deionized water (Milli-Q,
Millipore, Schwalbach, Germany) as solvent A and acetonitrile as
solvent B. A flow rate of 15 mL min−1 was used for the following
gradient: 10−50% solvent B in 30 min, 50−100% in 15 min, afterward
isocratic conditions at 100% for 10 min. UV detection was carried out
at 210 and 340 nm, and fractions were collected and combined
according to the observed peaks. 2 (3.6 mg) was obtained at a
retention time (tR) = 32−32.5 min, 5 (0.8 mg) at tR = 27.5−28 min,
and a mixture of 2 and 4 (3.2 mg) at tR = 32.5−33.5 min. From this
mixture a final step of silica gel HPLC (column 250 × 21 mm,
Nucleosil 100 7 μm, gradient from dichloromethane to dichloro-
methane/methanol (90:10) in 60 min, flow 20 mL/min) provided 1.2
mg of 2 (tR = 9−12 min) and 1.2 mg of 4 (tR 12−16 min).
Deconin B (2): colorless oil; [α]25 −3.9 (c 0.4, MeOH); UV
D
(MeOH) λmax (log ε) 240 (4.4) nm; IR (KBr) 3445, 2970, 1718, 1610,
1
1460, 1407, 1281, 1191, 1116, 1017, 858, 777, 713 cm−1; H NMR
(CD3OD, 500 MHz) δ 7.97 (2 H, d, J = 8.4 Hz, H-3, H-7), 7.53 (2 H,
d, J = 8.4 Hz, H-4, H-6), 4.49 (2 H, t, J = 6.7 Hz, H2-5′), 3.31 (1 H, d,
J = 18.0 Hz, Ha-12), 2.57 (2 H, d, J = 1.8 Hz, H2-2′), 2.44 (1 H, d, J =
18.9 Hz, Ha-10), 2.35 (1 H, d, J = 18.0 Hz, Hb-12), 2.23 (1 H, d, J =
18.9 Hz, Hb-10), 2.10 (2 H, m, H2-4′), 1.46 (3 H, s, H3-15), 1.38 (3 H,
s, H3-6′), 1.28 (3 H, s, H3-14), 0.73 (3 H, s, H3-13); 13C NMR
(CD3OD, 125 MHz) δ 219.6 (C-11), 174.9 (C-1′), 167.9 (C-1), 151.4
(C-5), 130.3 (C-3, C-7), 129.6 (C-2), 128.2 (C-4, C-6), 71.0 (C-3′),
62.6 (C-5′), 53.2 (C-10), 51.4 (C-12), 50.7 (C-8), 46.8 (C-2′), 42.9
(C-9), 40.8 (C-4′), 27.6 (C-6′), 26.7 (C-13), 24.8 (C-15), 24.2 (C-
14); HRESIMS m/z 377.1967 ([M + H]+, calcd for C21H29O6,
377.1959).
Deconin C (3): colorless oil; [α]25 −45 (c 0.1, MeOH); UV
D
(MeOH) λmax (log ε) 238 (4.4), 301 (4.1) nm; IR (KBr) 3440, 2963,
1726, 1610, 1574, 1461, 1405, 1384, 1280, 1224, 1123, 1117, 1017,
858, 777, 712 cm−1; 1H NMR (CD3OD, 500 MHz) δ 7.03 (1 H, dt, J
= 6.1 Hz, 1.3 Hz, H-3), 5.98 (1 H, brd, J = 6.1 Hz, H-4), 4.32 (2 H, t, J
= 6.7 Hz, H2-5′), 2.95 (1 H, d, J = 18.0 Hz, Ha-12), 2.52 (2 H, d, J =
1.5 Hz, H2-2′), 2.43 (1 H, m, H2-7), 2.37 (1 H, m, H2-6), 2.34 (1 H,
m, Ha-10), 2.18 (1 H, m, Hb-10), 2.13 (1 H, d, J = 18.0 Hz, Hb-12),
2.00 (2 H, m, H2-4′), 1.34 (3 H, s, H3-6′), 1.24 (6 H, s, H3-14, H3-15),
1.04 (3 H, s, H3-13); 13C NMR (CD3OD, 125 MHz) δ 219.6 (C-11),
174.9 (C-1′), 168.8 (C-1), 152.4 (C-10), 135.3 (C-3), 127.3 (C-2),
120.5 (C-4), 71.0 (C-3′), 62.5 (C-5′), 53.5 (C-10), 51.8 (C-12), 51.4
(C-8), 46.7 (C-2′), 42.3 (C-9), 40.7 (C-4′), 27.6 (C-6′), 26.9 (C-6),
26.6 (C-13), 25.0 (C-14) 22.9 (C-15), 22.6 (C-7); HRESIMS m/z
379.2119 ([M + H]+, calcd for C21H31O6, 379.2115).
Another cultivation of Deconica sp. 471 in 2 L scale was processed in
the same way as described above. Parameters of the preparative RP-
HPLC separation were the same as described above, but with a flow
rate of 12 mL/min. 1 (1.0 mg) was obtained at tR = 43−43.5 min, 3
Deconin D (4): colorless oil; [α]25 −0 (c 0.0, MeOH); UV
D
(MeOH) λmax (log ε) 244 nm; 1H NMR (CD3OD, 700 MHz) δ 7.96
C
DOI: 10.1021/np5010104
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Journal of Natural Products
Note
(2 H, d, J = 8.6 Hz, H-3, H-7), 7.55 (2 H, d, J = 8.6 Hz, H-4, H-6),
5.05 (1 H, t, J = 8.8 Hz, H-12), 4.48 (2 H, t, J = 6.7 Hz, H2-5), 2.58 (2
H, brs, H2-2), 2.19 (1 H, m, Ha-11), 2.11 (2 H, m, H2-4′), 1.81 (1 H,
m, Ha-10), 1.69 (1 H, m, Hb-11), 1.49 (1 H, m, Hb-10), 1.37 (3 H, s,
H3-6′), 1.28 (3 H, s, H3-15), 1.08 (3 H, s, H3-14), 0.51 (3 H, s, H3-
13); 13C NMR (CD3OD, 175 MHz) δ 175.5 (C-1′), 168.4 (C-1),
152.4 (C-5), 130.1 (C-3, C-7), 129.0 (C-2), 128.7 (C-4, C-6), 75.8 (C-
12), 71.0 (C-3′), 62.5 (C-5′), 54.7 (C-8), 47.1 (C-2), 44.4 (C-9), 41.0
(C-4′), 37.3 (C-10), 29.8 (C-11), 28.4 (C-13), 27.7 (C-6′), 25.6 (C-
14), 17.2 (C-15); HRESIMS m/z 379.2121 ([M + H]+, calcd for
C21H31O6, 379.2115).
at 37 °C. A 60 μL amount of serial dilutions from an initial stock of 1
mg/mL in MeOH of the test compounds was added to 120 μL
aliquots of a cell suspension (50 000/mL) in 96-well microplates. After
5 days of incubation, an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay was performed, and the
absorbance measured at 590 nm using an ELISA plate reader (Victor).
The concentration, at which the growth of cells was inhibited to 50%
of the control (IC50), was obtained from the dose−response curves.
The negative control was methanol.
Absolute Configuration of 1. Deconin A (1, 0.8 mg) was
hydrolyzed with 0.1 M NaOH (500 μL) for 16 h at 25 °C. The
solution was neutralized with 1 M HCl (50 μL). Analysis by ESIMS
revealed the complete hydrolysis of 1. The mixture was fractionized by
RPHPLC; a VP Nucleodur C18 ec column (250 × 21 mm, 5 μm;
Macherey-Nagel) was used as stationary phase. The mobile phase was
composed of deionized water (Milli-Q, Millipore, Schwalbach,
Germany) as solvent A and acetonitrile as solvent B, both with
0.05% TFA. A flow rate of 12 mL min−1 was used with the gradient
from 40−100% solvent B in 30 min; ent-cuparenic acid (6, 0.3 mg)
eluted at 23.5−24 min.
Deconin E (5): colorless oil; [α]25 +1.5 (c 0.2, MeOH); UV
D
(MeOH) λmax (log ε) 240 (4.2) nm; IR (KBr) 3415, 2961, 1721, 1610,
1
1460, 1384, 1278, 1193, 1123, 1019 cm−1; H NMR (CD3OD, 700
MHz) δ 7.95 (2 H, d, J = 8.6 Hz, H-3, H-7), 7.46 (2 H, d, J = 8.6 Hz,
H-4, H-6), 4.53 (1 H, dddd, J = 8.6 Hz, 8.0 Hz, 4.7 Hz, 2.6 Hz, H-11),
4.45 (1 H, d, J = 10.8 Hz, Ha-3), 4.38 (1 H, d, J = 10.8 Hz, Hb-3), 3.00
(1 H, dd, J = 14.0 Hz, 8.6 Hz, Ha-12), 2.00 (1 H, dd, J = 13.6 Hz, 8.0
Hz, Ha-10), 1.81 (1 H, dd, J = 13.6 Hz, 4.7 Hz, Hb-10), 1.78 (1 H, dd,
J = 14.0 Hz, 2.6 Hz, Hb-12), 1.49 (3 H, s, H3-4′), 1.47 (3 H, s, H3-15),
1.19 (3 H, s, H3-14), 0.54 (3 H, s, H3-13); 13C NMR δ, 177.2 (C-1′),
167.6 (C-1), 154.4 (C-5), 130.1 (C-3, C-7), 128.4 (C-2), 128.2 (C-4,
C-6), 74.7 (C-2′), 70.9 (C-3′), 70.5 (C-11), 52.1 (C-8), 51.5 (C-10),
48.0 (C-12), 46.0 (C-9), 27.0 (C-13), 26.1 (C-15), 24.9 (C-14), 23.0
(C-4′); HRESIMS m/z 351.1805 ([M + H]+, calcd for C19H27O6,
351.1802).
All other fractions were combined, concentrated in vacuo, and
acidified to pH 3 with formic acid. The solution (100 mL) was
extracted with CH2Cl2 (100 mL) for 16 h. The CH2Cl2 extract was
dried (Na2SO4) and analyzed by GC-MS. Retention times of
commercial R- and S-mevalonolactone were compared with the
obtained material on Chirasil-Val.
ent-Cuparenic acid (6): colorless oil; [α]25D −120 (c 0.03, MeOH);
1H NMR (CD3OD, 700 MHz) δ 7.88 (2 H, d, J = 8.2 Hz, H-3, H-7),
7.42 (2 H, d, J = 8.2 Hz, H-4, H-6), 2.58 (1 H, m, Ha-12), 1.84 (2 H,
m, H-11), 1.74 (2 H, m, Ha-10, Hb-12), 1.58 (1 H, s, Hb-10), 1.31 (3
H, s, H3-15), 1.10 (3 H, s, H3-14), 0.56 (3 H, s, H3-13); 13C NMR
(CD3OD, 175 MHz) δ, 167.9 (C-1), 154.9 (C-5), 130.0 (C-3, C-7),
128.4 (C-2), 128.3 (C-4, C-6), 52.2 (C-8), 45.5 (C-9), 40.8 (C-10),
37.9 (C-12), 26.8 (C-13), 24.7 (C-14, C15), 20.6 (C-11); HRESIMS
m/z 233.1542 ([M + H]+, calcd for C15H21O2, 233.1536).
Synthesis of (R)- and (S)-MTPA Esters of 7. For the preparation
of the (R)-MTPA ester 0.15 mg of 7 was dissolved in 600 μL of
pyridine-d5, and 7 μL of (S)-MTPA chloride was added. The mixture
was kept at 25 °C for 15 min before measurement of proton NMR
spectra: 1H NMR (pyridine-d5, 700 MHz, only relevant signals
assigned) δ 0.78 (s, H3-13), 0.97 (s, H3-14), 1.44 (s, H3-15), 2.73 (d, J
= 19.9 Hz, Hb-12), 3.32 (d, J = 19.9 Hz, Ha-12). The (S)-MTPA ester
was prepared in the same manner by the addition of 7 μL of (R)-
MTPA chloride: 1H NMR (pyridine-d5, 700 MHz, only relevant
signals assigned) δ 0.86 (s, H3-13), 1.09 (s, H3-14), 1.46 (s, H3-15),
2.69 (d, J = 19.9 Hz, Hb-12), 3.29 (d, J = 19.9 Hz, Ha-12).
(8R,10R)-10-Hydroxy-11-oxo-cuparenic acid (7): colorless oil;
[α]25 +3 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 235 (3.8) nm;
D
IR (KBr) 3435, 2972, 1745, 1705, 1611, 1410, 1384, 1272, 1196, 1080,
cm−1; 1H NMR (CD3OD, 700 MHz) δ 7.97 (2 H, d, J = 8.6 Hz, H-3,
H-7), 7.41 (2 H, d, J = 8.2 Hz, H-4, H-6), 3.95 (1 H, d, J = 1.7 Hz, H-
10), 3.04 (1 H, dd, J = 19.8 Hz, 1.7 Hz, Ha-12), 2.47 (1 H, d, J = 19.8
Hz, Hb-12), 1.56 (3 H, s, H3-15), 1.00 (3 H, s, H3-14), 0.79 (3 H, s,
H3-13); 13C NMR (CD3OD, 175 MHz) δ 218.3 (C-11), 169.6 (C-1),
152.9 (C-5), 130.2 (C-3, C-7), 129.9 (C-2), 128.9 (C-4, C-6), 82.5 (C-
10), 48.6 (C-12), 46.3 (C-9), 45.4 (C-8), 24.5 (C-15), 23.3 (C-13),
18.1 (C-14); HRESIMS m/z 263.1282 ([M + H]+, calcd for C15H19O4,
263.1278).
Minimum Inhibitory Concentrations. MICs were determined
(Table S1) in 96-well microtiter plates in a serial dilution assay with
EBS medium (0.5% peptone [Marcor], 0.5% glucose, 0.1% meat
extract, 0.1% yeast extract, 50 mM HEPES [11.9 g/L], pH 7.0) for
bacteria and MYC medium (1.0% phytone peptone, 1.0% glucose, 50
mM HEPES [11.9 g/L], pH 7.0) for yeasts and fungi, respectively.15
First, a 20 μL stock solution of each compound at 1 mg/mL in MeOH
(2 μL of reference drugs) was pipetted into the first row (A) of the
plate, and a few minutes allowed for the solvents to evaporate.
Negative control wells were left blank. Next, 150 μL of a mixture of the
test pathogen and the culture medium in the ratio of 1:100,
respectively, was aliquoted in all the rows. To the first row was
added an additional 150 μL of the pathogen−medium mixture and
mixed by repeated pipetting, before transferring 150 μL of this mixture
to the second row. A 1:1 serial dilution was done in the subsequent
rows, and 150 μL discarded after the last row (H). Plates were
incubated on a microplate vibrating shaker (Heidolph Titramax 1000)
at 600 rpm at 30 °C for 24−48 h. The lowest concentration of the
compounds preventing visible growth of the pathogen was taken as the
MIC. The concentrations tested ranged from 66.7 to 0.1 μg/mL.
Cytotoxicity Assay. In vitro cytotoxicity (IC50) was determined
against mouse fibroblast cell line L929 cultured in EBM-2 (Lonza)
supplemented with 10% fetal bovine serum (Gibco) under 10% CO2
ASSOCIATED CONTENT
* Supporting Information
Experimental procedures, NMR and mass spectra, morpho-
logical and phylogenetic details of the producing organism.
This material is available free of charge via the Internet at
http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Tel: +49 531 6181-4240. Fax: +49 531 6181 9499. E-mail:
marc.stadler@helmholtz-hzi.de.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to P. Sysouphanthong for collecting the fruiting
body of Deconica sp., B. Balling, Y. Siebken, K. A. Ullmann, and
K. Stephan for assistance with the extraction/isolation work
during their student projects in the lab of M.S., K. I. Mohr, W.
Collisi, and B. Hinkelmann for conducting the bioassays, and C.
Kakoschke and A. Teichmann/H. Steinmetz for recording
NMR and HPLC-MS data, respectively. We thank M. Nimtz
for GC-MS measurements and R. Jansen for proofreading the
manuscript prior to submission. Financial support by the
German Academic Exchange Service (DAAD) and the Thai
Royal Golden Ph.D. Jubilee-Industry Program (RGJ) for a joint
TRF-DAAD PPP (2012−2013) academic exchange grant to
K.D.H. and M.S., and the RGJ for a personal grant to B.T. (No.
D
DOI: 10.1021/np5010104
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
Ph.D/0138/2553 in 24.S.MF/53/A.3) is gratefully acknowl-
edged. K.D.H. would like to thank the Thailand Research Fund
for a grant (BRG5580009).
REFERENCES
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