Pelianthinarubins A and B, Red Pyrroloquinoline Alkaloids from the Fruiting Bodies of the Mushroom Mycena pelianthina

Article abstract of DOI:10.1021/acs.jnatprod.5b00942

Pelianthinarubin A (1) and pelianthinarubin B (2), two previously unknown pyrroloquinoline alkaloids, have been isolated from fruiting bodies of Mycena pelianthina. The structures of these alkaloids have been deduced from their HR-(+)-ESIMS and 2D NMR data. The absolute configurations of the pelianthinarubins A (1) and B (2) were assigned by analysis of the NOE correlations and coupling constants and by comparison of the CD spectra of 1 and 2 and of hercynine obtained by degradation of 1 with suitable compounds of known absolute configuration. The pelianthinarubins A (1) and B (2), which contain an S-hercynine moiety, differ considerably from the known pyrroloquinoline alkaloids from marine organisms and other Mycena species, such as the mycenarubins, the haematopodins, and the sanguinones.

Full text of DOI:10.1021/acs.jnatprod.5b00942

Article
pubs.acs.org/jnp
Pelianthinarubins A and B, Red Pyrroloquinoline Alkaloids from the
Fruiting Bodies of the Mushroom Mycena pelianthina
Anna Pulte, Silke Wagner, Herbert Kogler, and Peter Spiteller*
̈
̈
Institut fur Organische und Analytische Chemie, Universitat Bremen, Leobener Straße NW2C, D-28359 Bremen, Germany
*
S Supporting Information
ABSTRACT: Pelianthinarubin A (1) and pelianthinarubin B
2), two previously unknown pyrroloquinoline alkaloids, have
(
been isolated from fruiting bodies of Mycena pelianthina. The
structures of these alkaloids have been deduced from their HR-
(
+)-ESIMS and 2D NMR data. The absolute configurations of
the pelianthinarubins A (1) and B (2) were assigned by
analysis of the NOE correlations and coupling constants and
by comparison of the CD spectra of 1 and 2 and of hercynine
obtained by degradation of 1 with suitable compounds of
known absolute configuration. The pelianthinarubins A (1)
and B (2), which contain an S-hercynine moiety, differ
considerably from the known pyrroloquinoline alkaloids from marine organisms and other Mycena species, such as the
mycenarubins, the haematopodins, and the sanguinones.
Mycena pelianthina (Fr.) Quel
́
., the black-edged bonnet
the initial and an RP-8 column for the final purification step. A
100 g amount of frozen fruiting bodies yielded 1.7 mg of
pelianthinarubin A (1), 0.5 mg of pelianthinarubin B (2), 2.5
mg of mycenarubin A (3), and 12.1 mg of mycenarubin D (4).
The UV/vis spectra of the two red pelianthinarubins A (1)
and B (2) are almost identical. Pelianthinarubin A (1) shows
four absorption maxima at λ 243, 281, 358, and 517 nm, while 2
exhibits absorption maxima at λ 243, 285, 355, and 513 nm.
The UV/vis spectra of 1 and 2 are closely related to the UV/vis
(
German name: Schwarzgezahnelter Rettichhelmling), is a
̈
relatively small mushroom possessing a purplish-colored cap
and a typical radish-like odor. It is widely distributed in
hardwood or mixed hardwood−conifer forests in Europe and
Northern America. Its common name is derived from its
dark-edged gills. So far there are no reports on the chemical
structures of the pigments of M. pelianthina. A comparative
metabolic profiling of fruiting bodies of M. pelianthina by
1
,2
3
4
3
HPLC-UV with those of Mycena rosea, Mycena sanguinolenta,
spectra of mycenarubin A (3), suggesting the presence of a
5
and Mycena haematopus revealed the presence of mycenarubin
A (3) and mycenarubin D (4) together with previously
unknown pyrroloquinoline alkaloids. In this article we describe
the isolation and structural elucidation of two new red
pyrroloquinoline alkaloids, which we have named pelianthina-
rubins A (1) and B (2) and the identification of 3 and 4 in M.
pelianthina.
pyrroloquinoline core structure.
The HR-(+)-ESIMS spectrum of pelianthinarubin A (1)
+
exhibits an [M] ion at m/z 468.1879, corresponding to the
+
molecular formula C H N O (calculated: m/z 468.1878).
23
26
5
6
The odd number of nitrogen atoms in combination with an
even molecular mass indicates that 1 is a permanent cationic
compound. When compared with 3, pelianthinarubin A (1)
contains an additional C H N O fragment.
9
11
2
2
1
The H NMR spectrum, recorded at 283 K in D O, exhibits
2
2
3 nonexchangeable protons in total. Consequently, 1 contains
three exchangeable protons. The HSQC spectrum shows
signals accounting for three equivalent CH , four CH , and
3
2
six CH groups. Thus, 1 must contain 10 carbon atoms with no
hydrogens attached. The COSY spectrum in combination with
1
the H NMR spectrum reveals the presence of an X-Me group,
3
two CH CH fragments, a CH CH CH fragment, and three
2
2
2
aromatic CH singlets. In the COSY spectrum, two of the
aromatic CH groups exhibit weak couplings to each other,
RESULTS AND DISCUSSION
■
while the third CH group at δ 6.94 ppm is isolated. In the
H
The pelianthinarubins A (1) and B (2) as well as the
mycenarubins A (3) and D (4) were extracted from frozen
fruiting bodies of M. pelianthina with MeOH at 25 °C. The
alkaloids were purified by HPLC, using an RP-18ec column for
Received: October 21, 2015
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.5b00942
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 1. NMR Spectroscopic Data of Pelianthinarubin A (1) (600 MHz, D O, 283 K), Pelianthinarubin B (2) (700 MHz, D O,
2
2
2
83 K), and Mycenarubin A (3) (900 MHz, D O, 300 K)
2
3
pelianthinarubin A (1)
pelianthinarubin B (2)
mycenarubin A (3)
a
a
no.
2
δC
δH (mult., J in Hz)
6.94 (s)
δC
δH (mult., J in Hz)
δC
δH
6.95
128.6, CH
118.4, qC
26.7, CH2
128.7, CH
118.6, qC
26.7, CH2
7.04 (s)
3.24 (m)
4.23 (m)
128.7, CH
118.8, qC
26.8, CH2
2
3
a
b
3.17 (m)
3.20 (Heq)
.15 (Hax)
3
4
5
6
7
8
8
8
9
1
68.6, CH
156.6, qC
101.0, qC
178.1, qC
173.5, qC
127.0, qC
126.0, qC
178.8, qC
46.3, CH2
4.15 (m)
68.7, CH
156.9, qC
101.3, qC
178.3, qC
173.7, qC
127.2, qC
126.2, qC
179.0, qC
46.4, CH2
68.4, CH
159.0, qC
95.2, CH
182.1, qC
173.9, qC
126.8, qC
127.5, qC
179.3, qC
51.2, CH2
4.25
a
5.36
a
b
0
1
3.32 (dm, 14.2, H )
3.40 (dm, 14.1, H )
3.75 (Hb)
3.32 (Ha)
2.06
eq
eq
b
b
3
.18 (ddm, 16.0, 14.2, Hax)
3.29 (m, Hax)
1
31.3, CH2
2.27 (ddm, 16.0, 13.6, Hax)
.13 (dm, 13.6, Heq)
31.5, CH2
2.35 (ddm, 14.7, 13.9, Hax)
2.21 (dm, 13.9, Heq)
5.30 (m)
28.0, CH2
39.8, CH2
2
1
1
2
3
2
49.5, CH
173.8, qC
81.1, CH
28.5, CH2
5.18 (m)
49.5, CH
172.3, qC
83.8, CH
68.8, CH
3.11
′
′
′
3.80 (dm, 11.2)
4.05 (d, 10.3)
5.19 (d, 10.3)
3.04 (m)
b
3
.12 (dd, 13.4, 11.2)
4
6
8
9
′
′
′
′
136.5, qC
140.6, qC
c
c
139.8, CH
119.9 CH
7.70 (s)
7.12 (s)
3.15 (s)
140.3, CH
120.1, CH
56.1, 3 × CH3
7.79 (s)
c
c
7.34 (s)
54.5, 3 × CH3
3.34 (s)
a
6
13
7
NMR chemical shifts were adjusted to HDO δ = 4.75 at 300 K, and C values have been recalibrated using IUPAC recommendations, which
b
c
changed the chemical shifts by 1.3 ppm low field. The chemical shift value was obtained from the HSQC spectrum. Weak correlations are present
in the COSY spectrum between H-6′ and H-8′. Chemical shifts have been checked using N. Haider, W. Robien; http.://nmrpredict.orc.univie.ac.at/
c13robot/robot.php.
HSQC spectrum this aromatic proton is directly connected
chemical shifts are similar to those of C-2a, C-8a, and C-8b of 3
(Table 1). Likewise, the CH CH fragment with δ 3.17 (δ
C
1
3
with the carbon resonance at δ 128.6 ppm; this C chemical
C
2
H
shift resembles that of C-2 in compound 3 (Table 1).
Moreover, the proton at δH 6.94 ppm exhibits HMBC
correlations to three aromatic carbons (Figure 2), whose
26.7) and 4.15 (δ 65.5) ppm in 1 exhibits similar chemical
C
shift values compared to those of the CH CH fragment
2
consisting of C-3 and C-4 of mycenarubin A (3). The HMBC
correlations of H-3 and H-4 of 1 (Figure 2) resemble those of
the corresponding protons in 3. Therefore, δ 156.6 ppm was
C
assigned to C-5a, δ 46.3 ppm to C-10, and δ 178.8 ppm to C-
C
C
9
, indicating that 1 contains the same pyrroloquinoline core
structure as mycenarubin A (3). Instead of a CH CH CH
2
2
2
moiety as in 3, pelianthinarubin A (1) contains a CH CH CH
2
2
fragment. According to the HMBC correlation from H-4 to C-
1
0, this residue (δ 46.3 (C-10); δ 31.3 (C-11); δ 49.5 (C-
C C C
1
2)) is attached via the CH group (C-10) to the nitrogen N-5
2
(
Figure 2). Consequently, C-12 is attached to C-11 and two
additional substituents. Its chemical shift (δC 49.5 ppm) is
approximately 10 ppm downfield-shifted when compared with
C-12 of 3, indicating that the two other substituents of C-12 are
a nitrogen, as evident in comparison with 3, and a quaternary
carbon. By comparing the carbon chemical shifts of 1 with 3, δC
Figure 1. Fragments A and B of pelianthinarubin A (1).
1
01.0 ppm in 1 was assigned to C-6, δ 178.1 ppm to C-7, and
C
δC 173.5 ppm to C-8. In contrast to C-6 in 3, C-6 in 1 has no
hydrogen attached to it, indicating that pelianthinarubin A (1)
carries one additional substituent at C-6. Due to the presence of
a characteristic fragment ion at m/z 271.0711 in the HR-
(+)-ESIMS/MS, the CH CH CH residue is connected via C-
Figure 2. Pelianthinarubin A (1) with selected HMBC correlations
2
2
(
red arrows). Additional HMBC correlations displayed by pelianthi-
12 to the carbon at C-6, thus forming an additional ring, when
compared to mycenarubin A (3). Consequently, pelianthinar-
narubin B (2) are shown in blue.
B
DOI: 10.1021/acs.jnatprod.5b00942
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Journal of Natural Products
Article
ubin A (1) consists of partial structure A and a fragment B
The absolute configuration of pelianthinarubin A (1) with its
three stereocenters (at positions 4, 12, and 2′) was established
by CD spectroscopy, NOE measurements, and analysis of the
(Figure 1) attached at C-12.
According to the HR-(+)-ESIMS/MS data, fragment B is
1
1
equivalent to C H N O . By analysis of the H NMR and the
coupling constants in the H NMR data. In order to determine
9
15
3
2
COSY spectrum the hydrogens of fragment B are assigned to
the stereochemistry at position 2′, the hercynine moiety of
pelianthinarubin A (1) was cleaved off under mild acidic
conditions and the resulting hercynine (5) was purified via
HPLC. Then, the CD spectrum of hercynine derived from the
natural product 1 was recorded and compared to the CD
the three chemically equivalent CH groups (δ 3.15, δ 54.5
3
H
C
ppm), a CH CH fragment (δ 3.04 and 3.12, δ 28.5; δ 3.80,
2
H
C
H
δ 81.1 ppm), two different aromatic CH groups (δ 7.12, δ
C
H
C
1
19.9 and δH 7.70, δC 139.8 ppm), and one exchangeable
proton. The chemical shifts of the CH groups (δ 3.15, δ
C
8
spectra of freshly synthesized R- and S-hercynine, revealing the
3
H
5
4.5 ppm) and an HMBC correlation from the H-9′ protons to
stereocenter at position 2′ to be S configured (Figure 3). To
assign the absolute configuration at position 4, the CD
spectrum of pelianthinarubin A (1) was compared with that
of mycenarubin A (3), which is known to be S configured at C-
4. Since the CD spectra of 1 and 3 exhibit nearly the same
Cotton effects, the absolute configuration of C-4 of
C-9′ (Figure 2) indicate the presence of a trimethylammonium
moiety, explaining the cationic nature of 1. This residue is
attached to the CH of the CH CH fragment on account of a
2
3
3
JCH correlation from H-9′ to C-2′ (δ 81.1 ppm). H-2′ (δ
C
H
3
.80 ppm) shows a characteristic HMBC correlation to the
carbon atom C-1′ (δ 173.8 ppm), which is, due to its chemical
shift, part of a carboxyl moiety. Likewise, the diastereotopic
protons H-3′ (δ 3.04 and 3.12, δC 28.5 ppm) show a
correlation to C-1′. Moreover, HMBC correlations from H-3′
to the carbon C-4′ (δ 136.5 ppm) and to C-8′ (δ 7.12, δ
pelianthinarubin A (1) is also S (Figure 3).
C
The absolute configuration of the stereocenter at C-12 was
deduced indirectly from the spatial orientation of H-4 and H-12
to each other. The orientation was assigned by analysis of the
H
NOEs and coupling constants of the protons H-4, H-10 , H-
ax
C
H
C
1
0 , H-11 , H-11 , H-12, H-6′, and H-8′. The coupling
1
19.9 ppm) indicate that C-3′ is connected via C-4′ to an
eq
ax
eq
1
constants in the H NMR spectrum of 1 are not well-resolved,
aromatic moiety (Figure 2). Within the COSY spectrum H-8′
but coupling constants larger than 10 Hz are visible. H-12 does
displays a weak coupling to H-6′ (δ 7.70, δ 139.8 ppm).
H
C
not exhibit any large coupling constants, while H-11 displays
Since all but two nitrogen atoms are now assigned, C-6′, C-8′,
and C-4′ must be part of an imidazole residue. Hence, fragment
B is hercynine (5), a common natural product.
ax
two and H-11eq and H-10eq one large coupling constant. For
pelianthinarubin A (1) H-10ax is overlapping with other
resonances. However, the phase-sensitive COSY recorded for
pelianthinarubin B (2), which has the same core structure as
pelianthinarubin A (1), delivers more information. The cross-
peak of H-10ax to H-11ax exhibits one large active coupling
constant of at least 10 Hz, indicating a stable and preferred
conformation of this six-membered ring with bis-axially
orientated protons (pseudochair; see Figure 4). H-12 is
equatorially oriented because this resonance is lacking large
coupling constants and is correlated via NOEs to both protons
at C-11 (δ 2.13, 2.27 ppm). The proton H-11 at δ 2.27 ppm
In the ROESY data correlations are observed between H-12
(
1
7
δ 5.18 ppm) and H-6′ (δ 7.70 ppm) as well as between H-
H
H
H
H
2 and H-8′ (δ 7.12 ppm), which suggest a bond between N-
H
exhibits two large coupling constants, while the proton H-11 at
δH 2.13 ppm exhibits only one large coupling constant.
Consequently, the proton H-11 at δH 2.27 ppm is axially
oriented, and the proton H-11 at δH 2.13 ppm is in the
′ and C-12, leading to the constitution formula of
pelianthinarubin A (1) (Figure 4).
equatorial position. The NOE correlation between H-11 at δH
ax
2
.27 ppm and H-10 at δ 3.32 ppm and the absence of a NOE
eq H
between H-11 and H-10 at δ 3.18 ppm support the axial/
ax
ax
H
equatorial assignment based on the coupling constants. This
assignment is in agreement with the NOE correlations between
H-11 and both protons at H-10. Moreover, H-4 is located in
eq
the axial position; this is supported by the presence of a strong
NOE correlation between H-4 and H-10 and the absence of
ax
eq
an NOE correlation between H-4 and H-10 (Figure 4), thus
ax
ax
Figure 3. CD spectra in H O of 1, 2, and 3 as well as S-hercynine and
hercynine derived from 1.
2
leading to the 4S, 12R, 2′S configuration for 1.
The HR-(+)-ESIMS spectrum of pelianthinarubin B (2)
+
shows an [M] ion at m/z 484.1827 corresponding to the
+
molecular formula C H N O (calculated: m/z 484.1827),
23
26
5
7
differing from that of 1 only by the presence of one additional
1
oxygen atom. Comparing the H NMR spectra of 1 and 2,
recorded in D O, the spectrum of 2 shows only 22
2
nonexchangeable protonsone less than the spectrum of
1
and a higher chemical shift value for H-3′ with an integral
of 1 instead of 2 (Table 1). Hence, pelianthinarubin B (2)
contains an additional OH group at C-3′ in comparison with 1.
Pelianthinarubin B (2) carries one additional stereocenter at
C-3′. In order to deduce its stereochemistry, the spatial
Figure 4. Pelianthinarubin A (1) with selected ROESY correlations.
C
DOI: 10.1021/acs.jnatprod.5b00942
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Journal of Natural Products
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orientation of H-2′ and H-3′ to each other was analyzed by
first on a C18 ec column (Nucleodur, 100 Å, 5 μm, 21 × 250 mm,
Machery-Nagel) with the following gradient: Within 45 min linear
from 100% H O (+ 0.01% HOAc) to MeOH−H O (75:25), then
ROESY experiments and determination of the coupling
1
constants in the H NMR. Since H-2′ (δ 4.05 ppm) and H-
2
2
H
within 5 min linear to 100% MeOH; flow rate 12 mL/min; UV
3
′ (δ 5.19 ppm) are lacking an NOE to each other but display
H
3
detection at 360 nm. A second separation step was performed on a C
8
a large coupling constant J(H-2′, H-3′) of 10.3 Hz, these
column (Nucleosil, 100 Å, 8 × 250 mm) with the following gradient:
Starting from MeOH−H O (5:95) linear to MeOH−H O (75:25)
protons are antiperiplanar to each other. The nine protons of
2
2
the NMe group and H-8′ of the imidazole ring both show
3
within 45 min, then to 100% MeOH within 5 min (flow rate 4 mL/
min; UV detection at 360 nm). Hercynine samples were purified using
a C18 ec column (Nucleodur, 100 Å, 5 μm, 21 × 250 mm (Machery-
NOEs to H-2′ and H-3′ but not to each other. Therefore, these
two residues are located opposite to each other (see Figure 5).
Nagel)) and the following isocratic separation conditions: 100% H O
2
(
+ 0.2% HOAc); flow rate 12 mL/min; UV detection at 220 nm; UV:
Thermo Scientific Genesys 10S UV/vis spectrometer operated by the
software VISIONlite; CD: J-715 spectropolarimeter (Jasco); NMR:
1
13
Bruker Avance DRX-600 ( H at 600.22, C at 150.91 MHz), Bruker
1
Avance II spectrometer equipped with a TXI cryo probe ( H at
7
00.20, 13C at 176.08 MHz), Bruker Avance III spectrometer equipped
1
13
1
with a TCI cryo probe ( H at 500.30, C at 125.81 MHz). H
chemical shifts were calibrated using the chemical shift of the HDO
solvent signal corrected for the well-known temperature dependency
6
13
of water (δ (HDO) = 4.94 ppm at 283 K). The C chemical shifts
were calibrated using the IUPAC recommendations using the
H
7
conversion factor of 0.251 449 53 for calculating the absolute
frequency of 13C (0.0 ppm). LC-ESIMS spectra were obtained on
an LCQ DecaXP Plus ESIMS spectrometer (Thermo Fisher Scientific
Inc., USA). The spectrometer was operated in positive mode (0.625
Figure 5. Newman projections of the hydroxyhercynine moiety.
−1
spectrum s ; mass range 50−1000). Nitrogen was used as sheath gas
80 arbitrary units) and helium served as collision gas. The
spectrometer was equipped with a Hewlett-Packard HPLC system
Series 1100) consisting of degasser, pump, DAD detector, and
(
Since the stereocenter at C-2′ is S configured, C-3′ also has to
be S configured. This is in agreement with the stereochemistry
of β-hydroxyergothioneine, which is closely related to β-
hydroxyhercynine, exibiting similar chemical shifts and coupling
(
autosampler (injection volume 5 μL). The separations were performed
with a C18 ec column (Nucleodur, 100 Å, 5 μm, 3 × 250 mm,
Machery-Nagel) using the following gradient program: Within the first
45 min linear from 100% H O (+ 0.01% HOAc) to MeOH−H O
9
constants in the proton NMR.
Until some years ago pyrroloquinoline alkaloids have been
considered to be typical marine alkaloids, since nearly all known
11
2
2
(75:25), within the next 5 min linear to 100% MeOH, flow rate 0.66
mL/min. HR-ESIMS spectra were obtained on an LTQ Orbitrap
ESIMS spectrometer (Thermo Fisher Scientific Inc., USA). The
10
representatives, for instance the batzellines, damirones,
1
2
13
14
isobatzellines, makaluvamines, and discorhabdins, were
isolated from marine sources. However, from 2006 to 2008 our
group was able to isolate a considerable number of new
pyrroloquinoline alkaloids from fruiting bodies of terrestrial
−1
spectrometer was operated in the positive mode (1 spectrum s ; mass
range 50−1000) with a nominal mass resolving power of 60 000 at
m/z 400 with a scan rate of 1 Hz with automatic gain control to
provide high-accuracy mass measurements within 2 ppm deviation
using polydimethylcyclosiloxane ([(CH ) SiO] , m/z 445.120025) as
3
5
Mycena species, such as M. rosea, M. haematopus, and
M. sanguinolenta.
3
2
6
4
internal lock mass. Nitrogen was used as sheath gas (6 arbitrary units),
In contrast to the pyrroloquinoline alkaloids isolated from
and helium served as collision gas.
marine sources, the pyrroloquinoline alkaloids from Mycena
Mushrooms. Fruiting bodies of M. pelianthina (Fr.) Quel
det. P. Spiteller) were collected in September and October of 2004 to
014 in beech forests near Muhltal or Wellenburg in Bavaria,
́
. (leg. et
3
−5
species often feature a carboxylic group at C-4.
The
2
̈
presence of this carboxylic group fits well to the hypothetical
biosynthesis of 1 and 2 from L-tryptophan, S-adenosylmethio-
nine, and histidine.
Germany, and immediately frozen and stored at −35 °C after their
collection. Voucher samples of M. pelianthina are deposited at the
Institut fu
Germany.
̈
̈
r Organische und Analytische Chemie, Universitat Bremen,
Pyrroloquinoline alkaloids have attracted considerable
interest, since pyrroloquinoline alkaloids containing a para-
iminoquinone moiety often exhibit wide-ranging bioactivities.
For instance, the discorhabdins and isobatzellines display strong
Biological Tests. For plate diffusion assays 0.1 mg (0.21 μmol) or
.5 mg (1.07 μmol) of 1 (respectively 0.1 mg (0.21 μmol) or 0.5 mg
0
(1.03 μmol) of 2) was dissolved in distilled water and dropped onto
paper discs (⦶ 6 mm, thickness 0.5 mm), which then were dried
under sterile conditions. The dry paper discs were placed on agar
plates inoculated with the test organism (Escherichia coli, Bacillus brevis,
Bacillus subtilis, and Cladosporium cucumerinum). The plates were
incubated at 37 °C for 24 h (bacteria) and at 18 °C for 48 h (C.
cucumerinum). In order to test herbicidal acitivity 1 mg (2.13 μmol) of
1
2,15
activity toward a large panel of tumor cell lines.
Pelianthinarubins A (1) and B (2) were not active against
Escherichia coli, Bacillus brevis, Bacillus subtilis, or Cladosporium
cucumerinum. Although 1 and 2 possess no activity toward the
springtail Folsomia candida or herbicidal activity toward garden
cress (Lepidium sativum), they might play an ecological role in
1
(respectively 0.5 mg (1.03 μmol) of 2) was dissolved in 1 mL of
16,17
the chemical defense
of M. pelianthina.
distilled and sterilized water, and the solution was used to drench a
cotton ball (with a weight of approximately 0.4 g) within a sterile glass
container with a lid. Then, 10 Lepidium sativum seeds were placed on
the cotton ball, and their growth at 25 °C was monitored for the next 7
days in comparison to 10 L. sativum seeds growing under the same
conditions but on a cotton ball drenchend only in water. After 7 days
the weight and length of the seedlings were measured. For activity
tests against springtails (Collembola) 0.5 mg (1.07 μmol) of 1
(respectively 0.3 mg (0.62 μmol) of 2) was dissolved in 250 μL of
EXPERIMENTAL SECTION
General Experimental Procedures. Evaporation of the organic
solvents was performed under reduced pressure using a rotary
■
evaporator or in the case of H O by freezing the samples and
2
lyophilizing them. Preparative HPLC was performed on Waters 590EF
pumps equipped with an automated gradient controller 680 and a
Knauer UV/vis detector. Extracts from M. pelianthina were separated
D
DOI: 10.1021/acs.jnatprod.5b00942
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
distilled and sterilized water, and this solution was transferred in a
sterile glass container with a lid. Then, seven specimens of Folsomia
candida were placed in this container, also. After 5 days at 25 °C their
survival rate was compared to seven specimens being in a container
with only water.
Extraction and Isolation. Frozen fruiting bodies (100 g) were
extracted with 500 mL of MeOH for 10 min at 200 rpm and 25 °C.
The solvent was evaporated, and the resulting residue dissolved in 7
Hz, H-3′a), 3.01 (1H, dd, J = 14.0, 12.4 Hz, H-3′b), 2.92 (9H, s, H-
13
9′); C NMR (151 MHz, D O, 277 K) δ 169.1 (C, C-1′), 133.3 (CH,
2
C-6′), 125.9 (C, C-4′), 116.9 (CH, C-8′), 76.0 (CH, C-2′), 51.3 (3 ×
+
CH , C-9′), 21.8 (CH , C-3′); (+)-ESIMS m/z 198 [M] .
3
2
Hercynine (5) Obtained by Degradation of Pelianthinarubin
A (1). A 2 mg amount of pelianthinarubin A (1) was dissolved in 5 mL
of H O, and 0.5 mL of concentrated AcOH was added. The solution
2
was stirred for 1 h at 50 °C. The solvent was then removed under
reduced pressure. The residue was purified by preparative HPLC,
yielding 0.5 mg of hercynine.
mL of H O. After centrifugation at 15 000 rpm and 25 °C for 5 min,
2
the supernatant was prepurified with an RP18ec cartridge by using first
H O and then H O−MeOH (50/50 v/v) as eluent to obtain two
Hercynine (5) Derived from 1: colorless solid; HPLCprep, t
= 2.8
2
2
R
fractions. The H O−MeOH fraction contained 1 and 2 and was
min; CD (H
198 [M] .
2
O) S-hercynine λ (Δε) 211 (+1.4) nm; (+)-ESIMS m/z
2
+
further purified by two subsequent preparative HPLC separations first
on RP18ec and then on an RP8 column. A 100 g amount of fruiting
bodies yielded 1.7 mg of pelianthinarubin A (1) and 0.5 mg of
pelianthinarubin B (2).
ASSOCIATED CONTENT
Supporting Information
■
*
S
Pelianthinarubin A (1): red solid; HPLCprep, t = 14.8 min (step 1),
R
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.5b00942.
1
2
2
2.5 min (step 2); LC-(+)-ESIMS t = 14.9 min; UV/vis (H O) λ
R 2 max
45, 357, 529; CD (H O) λ (Δε) 235 (−1.1), 252 (+4.2), 272 (+2.0),
2
1
95 (+2.3), 357 (−10.0), 429 (+1.6), 518 (+3.1) nm; H NMR (600
MHz, D O, 283 K, pH 9.0), see Table 1; C NMR (151 MHz, D O,
13
2
2
Selected UV/vis, CD, NMR, and mass spectra of
compounds 1, 2, and 5 (PDF)
2
H
83 K, pH 9.0), see Table 1; HR-(+)-ESIMS m/z 490.1697 (55) [M −
+
+
+
+
+
+ Na ] , 468.1879 (100) [M] (calculated for C H O N ,
23 26 6 5
+
4
68.1878), 424.1981 (78) [M − CO H] ; HR-(+)-ESIMS/MS
2
(
parent ion m/z 468.1879, 35 eV) m/z 424.1970 (28) [M −
AUTHOR INFORMATION
Corresponding Author
Tel: +49-421-218-63100. Fax: +49-421-218-63102. E-mail:
peter.spiteller@uni-bremen.de.
■
*
+
+
CO H] , 271.0711 (100) [M − hercynine] .
2
Pelianthinarubin B (2): red solid; HPLC, t = 15.5 min (step 1),
R
1
2
2
0.8 min (step 2); LC-(+)-ESIMS t = 15.4 min; UV/vis (H O) λ
R 2 max
45, 357, 527; CD (H O) λ (Δε) 234 (−1.1), 251 (+2.1), 271 (+1.3),
2
1
98 (+1.6), 355 (−6.6), 429 (+1.0), 514 (+2.2) nm; H NMR (700
Notes
13
MHz, D O, 283 K), see Table 1; C NMR (126 MHz, D O, 280 K),
see Table 1; HR-(+)-ESIMS m/z 506.1647 (66) [M − H + Na ] ,
The authors declare no competing financial interest.
2
2
+
+ +
+
+
4
4
4
84.1827 (100) [M] (calculated for C H O N , 484.1827),
23 26 7 5
ACKNOWLEDGMENTS
We are grateful to Prof. Dr. M. Spiteller, to Dr. M. Lamsho
+
■
40.1929 (62) [M − CO H] ; HR-(+)-ESIMS/MS (parent ion m/z
2
+
̈
ft,
40.1929, 35 eV) m/z 440.1919 (100) [M − CO H] , 271.0710 (100)
2
+
and to Dr. S. Zuhlke (Universitat
̈
Dortmund, Institut fur
̈
̈
[
M − hydroxyhercynine] .
Mycenarubin A (3): red solid; LC-(+)-ESIMS t = 11.1 min; m/z
Umweltforschung, Germany) for the measurement of HR-
(+)-ESIMS spectra, to J. Stelten and W. Willker for their
R
+
2
90 (100) [M + H] ; (+)-ESIMS/MS (parent ion m/z 290, CID
+
collision energy of 30%) m/z 246 (100) [M + H − CO ] , 217 (9) [M
support with NMR measurements, to the Verein fu
Munchen e. V., for mushroom material and valuable
suggestions, and to the Deutsche Forschungsgemeinschaft
̈
r Pilzkunde,
2
+
+
H − CO − (CH NH)] , 203 (17) [M + H − CO
−
2
2
2
̈
+
+
(
C H N)] , 189 (5) [M + H − CO − (C H N)] .
2
5
2
3
7
Mycenarubin D (4): red solid; LC-(+)-ESIMS t = 5.1 min; m/z
R
(
SP718/4-1) for financial support.
+
2
89 (100) [M + H] ; (+)-ESIMS/MS (parent ion m/z 289, CID
+
collision energy of 30%) m/z 245 (100) [M + H − CO ] , 202 (10)
2
+
REFERENCES
[
(
M + H − CO − (C H N)] , 188 (12) [M + H − CO
−
■
2
2
5
2
+
C H N)] .
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3
7
8
Synthesis of R- and S-Hercynine. For the synthesis of S-
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(2) Arora, D. Mushrooms Demystified; Ten Speed Press: Berkeley,
1986; p 231.
hercynine a 0.28 mL portion of a 37% aqueous CH O solution was
2
added to 350 mg (1.67 mmol) of L-histidine monohydrochloride
monohydrate in 5 mL of H O. For the synthesis of R-hercynine D-
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2
histidine monohydrochloride monohydrate was used instead of L-
histidine monohydrochloride monohydrate. After the catalyst (Pd/C)
was added, the mixture was hydrogenated at 12 bar for 50 min. The
catalyst was separated by filtration and washed with MeOH. Afterward,
the solvent was removed under reduced pressure. The colorless, thick
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7512−7515.
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2
portionwise. The solution turned milky and was removed after 10 min
by decantation. The remaining colorless oil was N,N-dimethylhistidine.
Without further purification 287 mg (1.57 mmol) of N,N-
dimethylhistidine was dissolved in 2 mL of MeOH, and with the
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addition of aqueous 25% NH OH solution the pH was adjusted to 9.
4
Then 120 μL of MeI (1.93 mmol) was added, and the reaction mixture
was heated under reflux at 60 °C for 2 h. The solvent was then
removed under reduced pressure, and the product was purified via
HPLC. A total of 119 mg (36%) of hercynine was isolated as a white
solid.
(10) Sakemi, S.; Sun, H. H.; Jefford, C. W.; Bernardinelli, G.
Tetrahedron Lett. 1989, 30, 2517−2520.
(11) Stierle, D. B.; Faulkner, D. J. J. Nat. Prod. 1991, 54, 1131−1133.
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1990, 55, 4964−4966.
Hercynine (5): colorless solid; HPLC, t = 2.8 min; CD (H O) S-
(13) Radisky, D. C.; Radisky, E. S.; Barrows, L. R.; Copp, B. R.;
Kramer, R. A.; Ireland, C. M. J. Am. Chem. Soc. 1993, 115, 1632−1638.
(14) Perry, N. B.; Blunt, J. W.; McCombs, J. D.; Munro, M. H. G. J.
Org. Chem. 1986, 51, 5476−5478.
R
2
hercynine λ (Δε) 211 (+1.4) nm, R-hercynine λ (Δε) 211 (−1.3) nm;
1
H NMR (600 MHz, D O, 277 K) δ 8.27 (1H, s, H-6′), 6.94 (1H, s,
2
H-8′), 3.58 (1H, dd, J = 12.4, 3.5 Hz, H-2′), 3.12 (1H, dd, J = 14.0, 3.5
E
DOI: 10.1021/acs.jnatprod.5b00942
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(
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(
(
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F
DOI: 10.1021/acs.jnatprod.5b00942
J. Nat. Prod. XXXX, XXX, XXX−XXX