Mycenarubins A and B, red pyrroloquinoline alkaloids from the mushroom Mycena rosea

Article abstract of DOI:10.1002/ejoc.200600826

Two previously unknown red alkaloid pigments, mycenarubin A (1), and a dimer thereof, mycenarubin B (6), have been isolated from fruiting bodies of Mycena rosea and the structures of these pyrroloquinoline alkaloids have been established by 2D NMR and ESI

Full text of DOI:10.1002/ejoc.200600826

FULL PAPER
DOI: 10.1002/ejoc.200600826
Mycenarubins A and B, Red Pyrroloquinoline Alkaloids from the Mushroom
Mycena rosea
Silke Peters[a] and Peter Spiteller*[a]
Dedicated to Professor Gerhard Spiteller on the occasion of his 75th birthday
Keywords: Alkaloids / Configuration determination / Fungi / Natural products / Pyrroloquinolines
Two previously unknown red alkaloid pigments, mycenaru- pound. The fungal pigments are structurally related to the
bin A (1), and a dimer thereof, mycenarubin B (6), have been
isolated from fruiting bodies of Mycena rosea and the struc-
tures of these pyrroloquinoline alkaloids have been estab-
lished by 2D NMR and ESI-MS methods. Their absolute
configurations were determined by comparison of their CD
damirones, isolated from a marine sponge, and may be bio-
synthetically derived from tryptophan and S-adenosylme-
thionine.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
spectra with the CD spectrum of a synthetic model com- Germany, 2007)
ent only in the fruiting bodies. Here we describe the isola-
Introduction
tion and the structural elucidation of these red pyrroloquin-
oline alkaloids, which we have named mycenarubin A (1)
and mycenarubin B (6).
Mycelial cultures of fungi have for decades been proven
to be valuable sources of bioactive compounds, such as
penicillin[1] or the antifungal strobilurins.[2] Today, countless
mycelial cultures have already been extensively screened for
novel bioactive secondary metabolites. In addition to their
mycelia, higher fungi also form short-lived fruiting bodies,
which have been less well investigated, despite the fact that
they often contain completely different secondary metabo-
lites[3] in order, for instance, to protect themselves against
parasites and predators by means of the presence of toxic
or repellent compounds.[4]
Results and Discussion
The mycenarubins were extracted from frozen fruiting
bodies of M. rosea with methanol at 25 °C and purified by
HPLC on a preparative RP-18 column, with 100 g of the
frozen fruiting bodies yielding 17 mg mycenarubin A (1)
and 1 mg mycenarubin B (6).
Mycena rosea (Bull.) Gramberg (German name: Rosa
Rettichhelmling), a relatively small mushroom, belongs
among the less well investigated mushrooms, despite being
widespread in beech forests in Europe.[5] So far, no second-
ary metabolites apart from volatiles such as hexanal and
hexan-2-one[6] have been reported either from its fruiting
bodies or from its mycelium. The fruiting bodies of Mycena
rosea are rarely attacked by predators, an indication that
they may contain repellent compounds, and this stimulated
us to investigate its fruiting bodies and mycelial cultures for
secondary metabolites. By HPLC-UV comparison of the
metabolite pattern of the fruiting bodies with that of the
mycelial cultures, we identified two new red pigments pres-
The UV/Vis spectrum of 1 exhibited absorption maxima
at 244, 358 and 539 nm, while in the HR-ESI-MS, 1 showed
an [M + H]+ ion at m/z 290.11339, consistently with the
molecular formula C14H15N3O4. The presence of 14 car-
bons in the 13C NMR confirmed the molecular formula,
1
while the H NMR spectrum exhibited 11 non-exchange-
able protons. According to HSQC correlations, the signals
in the 13C NMR were attributable to four CH2 groups, three
CH groups and seven quaternary carbon atoms. In the
COSY spectrum, the protons of the CH2 group at δH
1.96 ppm (δC = 26.65 ppm) showed couplings both to the
protons of the CH2 group at δH = 3.01 ppm (δC
=
=
38.46 ppm) and to the diastereotopically split protons of
the CH2 group at δH = 3.65 and 3.22 ppm (δC = 49.91 ppm),
indicating the presence of a chain of three consecutive CH2
groups. The CH2 chain must be flanked by two hetero-
atoms, because there are no 3JC,H correlations from the pro-
tons of the central CH2 group at δH = 1.96 ppm in the
HMBC, and the chemical shifts of the flanking CH2 groups
[a] Institut für Organische Chemie und Biochemie II der Techni-
schen Universität München,
Lichtenbergstraße 4, 85747 Garching, Germany
Fax: +49-89-289-13210
E-mail: peter.spiteller@ch.tum.de
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
Eur. J. Org. Chem. 2007, 1571–1576
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1571

S. Peters, P. Spiteller
FULL PAPER
at δC = 49.91 ppm and δC = 38.46 ppm indicate that these HMBC, although only the couplings to the carbons at δC
3
heteroatoms are both nitrogen atoms. In the HMBC, the = 126.24 and 172.60 ppm are strong JC,H couplings. The
protons of the CH2 group at δH = 3.65 and 3.22 ppm (δC = CH group at δH = 5.26 ppm is located relatively far away
49.91 ppm) exhibit couplings not only to the two other car- from the other aromatic CH group, since they only share
bons of the ϾN–(CH2)3–NϽ unit at δC = 26.65 and the HMBC correlation to the carbon at δC = 126.24 ppm.
38.46 ppm, but also to the carbon of the CH group at Since mycenarubin A is a highly unsaturated compound
67.05 ppm and a quaternary carbon at 157.71 ppm, while with nine double bond equivalents, and also in view of the
the protons of the other flanking CH2 group at δH
=
unusual shielded nature of the aromatic CH group at δH =
3.01 ppm (δC = 38.46 ppm) show only couplings to the two 5.26 ppm, with a carbon shift of δC = 93.94 ppm, this group
other carbons of the ϾN–(CH2)3–NϽ unit. Therefore, the should be placed adjacent to the deshielded carbon at δC =
latter CH2 group is connected to an NH2 group, while the 157.71 ppm, which is directly connected to the carbon at δC
nitrogen connected to the other CH2 of the side chain at δC = 126.24 ppm (Figure 2).
= 49.91 ppm belongs to a tertiary amine.
The second spin system present in the COSY spectrum
reveals the presence of a CH2–CH fragment consisting of
the CH2 group with two diastereotopically split protons at
δH = 3.05 and 3.10 ppm (δC = 25.52 ppm) and the CH
group at δH = 4.15 ppm (δC = 67.05 ppm). The CH2–CH
unit is connected to the tertiary amine nitrogen through the
3
CH group at δC = 67.05 ppm, since there are JC,H coup-
lings in the HMBC from the proton of the CH group at δH
= 4.15 ppm (δC = 67.05 ppm) to the carbon of the side
chain at δC = 49.91 ppm and vice versa from the protons of
the side chain at δH = 3.65 and 3.22 ppm to the carbon
Figure 2. Substructure B of mycenarubin A (1) with selected NOE
and HMBC correlations.
In the partial structure deduced so far, three quaternary
carbons, two oxygen atoms, one nitrogen atom and one ex-
changeable proton are left to complete the structure of my-
cenarubin. The two carbons at δC = 172.60 and 180.79 ppm
are attributable by their chemical shifts to C=O groups in
a quinone moiety. The exchangeable proton therefore has
to be located at the nitrogen and the quaternary carbon at
125.49 ppm in the bridge position between the NH group,
the C=O group at δC = 172.60 ppm and the quaternary car-
bon at 126.24 ppm. This assignment unambiguously gives
the pyrroloquinoline alkaloid structure 1 for mycenaru-
bin A (Figure 3). The relative configurations of the dia-
stereotopic protons were assigned by NOE measurements
(Figure 3).
3
of the CH2–CH moiety at δC = 67.05 ppm. A strong JC,H
coupling from the diastereotopic protons at δH = 3.05 and
2
3.10 ppm and a weaker JC,H coupling from the proton of
the CH2–CH unit at δH = 4.15 ppm to a quaternary proton
at δC = 177.95 ppm indicates that a carboxylic group is di-
rectly attached to the CH group. All protons of the CH2–
CH unit show couplings to a quaternary carbon at δC
=
117.46 ppm. The CH2–CH unit is therefore connected to an
aromatic moiety through the CH2 group. These results are
consistent with substructure A (Figure 1).
Figure 1. Substructure A of mycenarubin A (1) with selected
COSY and HMBC correlations.
The aromatic CH group at δH = 6.85 ppm (δC
=
127.44 ppm) shows HMBC correlations to the quaternary
carbons at δC = 117.46, 125.49 and 126.24 ppm. In the
ROESY, a NOE can be detected between the proton at δH
= 6.85 ppm and the proton at δH = 3.10 ppm, so the CH
group at δH = 6.85 ppm should be located adjacent to the
Figure 3. Mycenarubin A (1) with selected NOEs for the assign-
ment of the diastereotopic protons.
Mycenarubin A shares its pyrroloquinoline core struc-
quaternary carbon at δ = 117.46 ppm. The quaternary car- ture with haematopodin,[7] the only other pyrroloquinoline
bon at δC = 126.24 ppm should also be located adjacent to alkaloid so far isolated from fungi. The chemical shift val-
the carbon at 117.46 ppm, since the protons at δH = 3.05, ues of the core structure of haematopodin are very similar
3.10 and at 6.85 ppm all exhibit 3JC,H couplings to this car- to those of 1. Like 1, haematopodin possesses a stereocentre
bon (Figure 2). The CH group at δH = 5.26 ppm (δC
=
at C-4, but a cyclic O-alkyl moiety is present instead of the
93.94 ppm) exhibits JC,H couplings to the quaternary car- carboxylic acid. Since the two compounds have similar CD
bons at δC = 117.46, 126.24, 172.60 and 180.79 ppm in the spectra, a preliminary deduction can be made that the ste-
1572
www.eurjoc.org
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 1571–1576

Pyrroloquinoline Alkaloids from Mycena rosea
FULL PAPER
reochemistry of mycenarubin A (1) is (S), from the known
stereochemistry of haematopodin. This assignment is in
agreement with the hypothetical biosynthesis of mycenaru-
bin A from -tryptophan and S-adenosylmethionine.
In order to verify the (S) configuration of 1, (S)-4-car-
boxydamirone C (5), lacking only the side chain of 1, was
synthesized from the known 6,7-bis(benzyloxy)indole 2.[8]
The indole derivative 2 was condensed with serine in glacial
acetic acid and acetic anhydride to provide the racemic N-
acetyl derivative 3,[9] which was stereoselectively deacety-
lated with acylase I to afford 6,7-bis(benzyloxy)--trypto-
phan (4).[9] The benzyl groups of 4 were removed by cata-
lytic hydrogenolysis[7a] and the resulting compound was oxi-
datively cyclised (air) to yield 5 (Scheme 1).[7a]
Figure 4. Mycenarubin B (6) with chemical shift values and se-
lected NOEs and HMBC couplings.
The CD spectrum of mycenarubin B (6) is similar to that
of mycenarubin A (1), so the stereochemistries of the two
quaternary carbons carrying the carboxylic groups should
also be (S).
The mycenarubins are structurally related – apart from
to the already mentioned red fungi pigment haematopo-
din[5] – to a large number of marine alkaloids,[10] such as
Scheme 1. Stereoselective synthesis of (S)-4-carboxydamirone C the batzellines,[11] damirones,[12] isobatzellines,[13] makalu-
vamines[14] and discorhabdins,[15] all of them isolated from
(5).
marine sponges. In contrast, wakayin, the first alkaloid of
Since the CD spectra of 1 and 5 are nearly identical, the this class, was isolated from a Fijian ascidian.[16] Moreover,
configuration for mycenarubin A is (S). In addition, the makaluvamin A has been isolated not only from a marine
carbon and proton NMR spectroscopic data for 5 confirm sponge, but also from a culture of the myxomycete Didym-
the proposed structure of 1.
ium bahiense, a terrestrial organism.[17] The presence of the
The minor alkaloid 6 exhibited a UV spectrum that re- mycenarubins A and B and of haematopodin in Mycena
sembled that of mycenarubin A, with absorption maxima species confirms that pyrroloquinoline alkaloids are not re-
at 245, 354 and 539 nm, so 6 also possesses a pyrroloquino- stricted to marine sources but seem also to be common in
line core structure. The molecular formula C28H28N6O7, de- some terrestrial organisms.
duced from the [M + H]+ ion at m/z 561.20789 in the HR-
Among the pyrroloquinoline alkaloids, compounds con-
ESI-MS, indicates that 6 originates from mycenarubin A by taining a para-iminoquinone moiety, such as the isobatzel-
dimerisation of two molecules of 1 and loss of one molecule lines, often exhibit wide ranging bioactivities;[10] the isobat-
1
of water. The dimeric structure of 6 is supported by its H zellines, for instance, are cytotoxic towards P388 murine
NMR and 13C NMR spectra, since these spectra both con- leukaemia cells and active against Candida albicans.[13] The
sist of two similar signal sets. In order to elucidate how the makaluvamines, which also contain para iminoquinone sys-
two monomers are connected to each other, first the proton tems, typically exhibit cytotoxicity towards tumour cell
and then the carbon NMR spectroscopic data for the two lines[10] through inhibition of the function of mammalian
monomers were assigned analogously with those for mycen- topoisomerase II,[14] but only mild activity against Bacillus
3
arubin A. In the HMBC, two significant JC,H couplings subtilis and other standard test organisms.[18] The batzel-
between the diastereotopic protons at C-12Ј and the carbon lines and damirones, however, are characterised by ortho
at C-7 allowed structure 6, in which two units of 1 are con- quinone core structures and are much less active than the
nected to each other through an imine moiety, to be de- isobatzellines;[10] only the isobatzellines, and not the batzel-
duced for mycenarubin B (Figure 4).
lines, for instance, are cytotoxic and moderately antifun-
Eur. J. Org. Chem. 2007, 1571–1576
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1573

S. Peters, P. Spiteller
FULL PAPER
gal.[13] Likewise, the ortho-quinoid mycenarubin A (1) did
not show antimicrobial activity against bacteria such as
Escherichia coli, Bacillus brevis or B. subtilis, and fungi such
as Cladosporium cucumerinum.
Unlike the previously known pyrroloquinoline alkaloids,
the mycenarubins possess carboxylic groups at C-4. The
presence of the carboxylic group supports the hypothesis
that pyrroloquinoline and pyrroloiminoquinone alkaloids
are in general biosynthetically derived from -tryptophan.
Mycenarubin B (6) is the first example of the occurrence of
a dimeric pyrroloquinoline alkaloid in nature.
HPLC Profiling: For comparison of the metabolite pattern of the
fruiting bodies with that of the mycelial cultures, water and meth-
anol extracts both of the fruiting bodies and of the mycelial cul-
tures were injected into the HPLC. At 360 nm, only the extracts of
the fruiting bodies showed significant absorptions.
Isolation Procedure: The frozen fruiting bodies (20 g) were crushed
and extracted with MeOH (2ϫ50 mL) at 25 °C for 1 min and the
red extract was then concentrated in vacuo at 40 °C. The resulting
residue was dissolved in H2O (5 mL), prepurified with an RP-18
cartridge and separated by preparative HPLC (UV detection at
360 nm). Mycenarubin A (1) was obtained as a red solid; 100 g of
frozen fruiting bodies were required to obtain 17 mg of 1 and 1 mg
of 6.
Biological Tests: For plate diffusion assays, 1 (100 or 500 µg) was
dissolved in H2O and dropped onto paper discs (Ø 6 mm, thickness
0.5 mm). These discs were dried under sterile conditions and placed
on agar plates inoculated with the test organism (Bacillus brevis,
Bacillus subtilis, Escherichia coli and Cladosporium cucumerinum
CBS 117.54). The plates were incubated at 37 °C for 24 h in the
case of bacteria and at 18 °C for 48 h in the case of C. cucumeri-
num.
Experimental Section
General: Evaporation of the solvents was performed under reduced
pressure with a rotary evaporator. Preparative HPLC separations
were performed by use of two Waters 590EF pumps fitted with an
automated gradient controller 680 and a Kratos Spectroflow 783
UV/Vis detector. The samples were separated on a Luna C-18 (2)
column (5 µm, 15ϫ250 mm, Phenomenex) by use of the following
gradient program: 10 min at 100% H2O, then linear to 100%
MeOH over 30 min; flow rate: 12 mLmin–1; detection: UV at
360 nm. UV spectra were recorded on a Varian Cary 100 Bio UV/
Vis spectrometer. Optical rotation values were measured with a Ja-
sco P-1030 polarimeter and CD spectra were obtained with a Ja-
sco J-715 spectropolarimeter. NMR spectra were recorded with a
Bruker DMX 900 spectrometer fitted with a TXI cryo probe (1H
at 900.13, 13C at 226.3 MHz), with a Bruker DMX 600 spectrome-
ter fitted with a TXI cryo probe (1H at 600.13, 13C at 150.9 MHz)
and a Bruker DMX 500 spectrometer (1H at 500.11, 13C at
125.8 MHz). Chemical shifts were determined relative to the D2O
(δH = 4.65 ppm), CDCl3 (δH = 7.26, δC = 77.00 ppm) and CD3OD
(δH = 3.31, δC = 49.00 ppm) solvents as internal standards. EIMS
spectra were obtained with a ThermoElectron DSQ instrument fit-
ted with a direct insertion probe by EI at 70 eV. LC-ESIMS spectra
were recorded with a Gynkotek-HPLC fitted with a Luna C-18 (2)
column (Phenomenex, 150ϫ2 mm, 3 µm, operation temperature
40 °C, flow rate: 200 µLmin–1, gradient: 10 min isocratic at
100% H2O, then linear to 100% AcCN over 10 min) coupled with a
Finnigan TSQ 7000 fitted with a Finnigan ESI ion source interface
operating in the positive ESI mode [ionisation 4.5 kV, capillary
temperature 200 °C, mass range 50–800 amu, multiplier 1000 V
(scan modus), MS/MS: argon collision gas 2.0 mbar, sheath gas
(N2) 2.9 bar, multiplier 1400 V, collision energy automatically ro-
tated at –20, –30, –40 eV]. HRESIMS and HRESIMS/MS spectra
were obtained with a ThermoElectron LTQ-FT mass spectrometer.
Mycenarubin A (1): Red solid; m.p. 126 °C (dec.). HPLCprep: Rt =
16.4 min. [α]2D5 = +1190 (c = 0.00807, H2O). CD (H2O): λ (∆ε) =
243 (–4.2), 265 (+1.2), 275 (+1.0), 306 (+2.0), 365 (–4.7), 535 (+2.2)
1
nm. H NMR (900 MHz, D2O, 300 K): δ = 1.96 (m, 2 H, 11-H),
3.01 (m, 2 H, 12-H), 3.05 (dd, J = 16.6, 5.0 Hz, 1 H, 2-Hb), 3.10
(d, J = 16.6 Hz, 1 H, 2-Ha), 3.22 (m, 1 H, 10-Ha), 3.65 (m, 1 H,
10-Hb), 4.15 (d, J = 5.0 Hz, 1 H, 3-H), 5.26 (s, 1 H, 6-H), 6.85 (s,
1
1 H, 2-H) ppm. H NMR (600 MHz, D2O, 310 K): δ = 2.08 (m, 2
H, 11-H), 3.13 (dd, J = 16.5, 5.6 Hz, 1 H, 2-Hb), 3.15 (m, 2 H, 12-
H), 3.22 (d, J = 16.5 Hz, 1 H, 2-Ha), 3.33 (ddd, J ≈ 14 Hz, J ≈
7 Hz, J ≈ 7 Hz, 1 H, 10-Ha), 3.76 (ddd, J ≈ 14 Hz, J ≈ 7 Hz, J ≈
7 Hz, 1 H, 10-Hb), 4.25 (d, J = 5.6 Hz, 1 H, 3-H), 5.35 (s, 1 H, 6-
H), 6.94 (s, 1 H, 2-H) ppm. 13C NMR (226 MHz, D2O, 300 K): δ
= 25.52 (C-3), 26.65 (C-11), 38.46 (C-12), 49.91 (C-10), 67.05 (C-
4), 93.94 (C-6), 117.46 (C-2a), 125.49 (C-8b), 126.24 (C-8a), 127.44
(C-2), 157.71 (C-5a), 172.60 (C-8), 177.95 (CO2H), 180.79 (C-
7) ppm. UV/Vis (H2O): λmax (lgε) 244 (4.12), 358 (3.94), 539 (2.82)
nm. HR-ESIMS: m/z = 290.11339 [M + H]+ calcd. 290.11408 for
C14H16N3O4. LC-ESIMS: Rt = 18.9 min (detection: UV at λ =
360 nm) m/z = 290 [M + H]+. LC-HR-ESIMS/MS (parent ion m/z
290, 20 eV) m/z (%) = 290.11371 (40) [M + H]+, 246.12383 (100)
[M + H – CO2]+, 233 (4) [M + H – (C3H7N)], 217.09728 (12) [M
+ H – CO2 – (CH2=NH)]+, 203.08160 (32) [M + H – CO2
–
(C2H5N)], 189.06593 (24) [M + H – CO2 – (C3H7N)], 85 (10), 58
(7).
N-Acetyl-6,7-bis(benzyloxy)tryptophan (3): -Serine (31.9 mg,
0.304 mmol) was added to a solution of 6,7-bis(benzyloxy)indole
(50 mg, 0.152 mmol) in AcOH (400 µL) and Ac2O (100 µL). The
solution was stirred under argon for 2 h at 75 °C. After cooling to
25 °C, the mixture was diluted with diethyl ether (10 mL) and ad-
justed to pH 11 with NaOH (30%). The water layer was washed
three times with diethyl ether (10 mL) and the aqueous phase was
then adjusted to pH 3 with conc. HCl and extracted three times
with diethyl ether (10 mL). The organic phase was dried with anhy-
drous Na2SO4 and the solvent was removed. Yield: 51 mg (73%).
1H NMR (600 MHz, CDCl3, 300 K): δ = 1.94 (s, 3 H COCH3),
3.23 (dd, J = 14.9, 5.2 Hz, 1 H, 3-Ha), 3.29 (dd, J = 14.9, 4.9 Hz,
1 H, 3-Hb), 4.87 (dd, J = 5.2, 4.9 Hz, 1 H, 2-H), 5.17 (m, 2 H,
OCH2-6Ј), 5.18 (d, J = 11.1 Hz, 1 H, OCH2-7Ј-Ha), 5.20 (d, J =
11.1 Hz, 1 H, OCH2-7Ј-Hb), 6.06 (d, J = 5.8 Hz, NH), 6.81 (s, 1
H, 2Ј-H), 6.91 (d, J = 8.6 Hz, 1 H, 5Ј-H), 7.18 (d, J = 8.6 Hz, 1 H,
Mushrooms: Fruiting bodies of M. rosea (leg. et det. S. Peters and
P. Spiteller) were collected in September and October 2004, 2005
and 2006 in beech forests 20 km south of Munich (Bavaria).
Voucher samples of M. rosea are deposited at the Institut für Or-
ganische Chemie und Biochemie II der Technischen Universität
München, Germany. The mushrooms were frozen and stored at
–35 °C after collecting.
Mycelial Cultures and Cultivation Conditions: For cultivation of
M. rosea (CBS 514.79, Centraalbureau voor Schimmelcultures,
Amsterdam), 40 Petri dishes (90 mm diameter), each of them con-
taining 25 g solid MEA medium, consisting of malt extract (30 g),
peptone (5.0 g), agar (15 g) and H2O (1000 mL), were inoculated
with mycelia of M. rosea (CBS 514.79) and incubated at 20 °C.
Typically, the mycelia covered the whole agar plate within two
weeks.
1574
www.eurjoc.org
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 1571–1576

Pyrroloquinoline Alkaloids from Mycena rosea
FULL PAPER
4Ј-H), 7.29–7.32 (m, 3 H, 3ЈЈЈ-H, 4ЈЈЈ-H, 5ЈЈЈ-H), 7.32 (m, 1 H, 4ЈЈ- λmax (lgε) 242 (4.15), 344 (3.85), 516 (2.96) nm.CD (H2O): λ (∆ε)
H) 7.37 (d, J = ca. 7 Hz, 2 H, 2ЈЈЈ-H, 6ЈЈЈ-H), 7.37 (m, 2 H, 3ЈЈ-H, = 244 (–4.5), 265 (+0.7), 275 (+0.6), 304 (+1.4), 360 (–2.2), 520
5ЈЈ-H), 7.47 (d, J = 7.0 Hz, 2 H, 2ЈЈ-H, 6ЈЈ-H), 8.22 (NHCO) ppm. (+1.2) nm. HR-ESIMS: m/z = 233.05566 [M + H]+ calcd.
13C NMR (151 MHz, CD3OD, 300 K): δ = 23.05 (COCH3), 27.08 233.05568 for C11H9N2O4; LC-ESIMS: Rt = 20.7 min (detection:
(C-3), 53.33 (C-2), 72.82 (6Ј-OCH2), 75.54 (7Ј-OCH2), 109.79 (C- UV at λ = 360 nm) m/z = 233 [M + H]+. LC-ESIMS/MS (parent
3Ј), 110.55 (C-5Ј), 113.65 (C-4Ј), 122.92 (C-2Ј), 124.78 (C-3aЈ), ion m/z 233, 30 eV) m/z (%) = 233 (100) [M + H]+, 187 (88)
127.67 (C-2ЈЈ and C-6ЈЈ), 127.93 (C-4ЈЈ), 128.26 (C-4ЈЈЈ), 128.48 (C-
[M + H – HCO2H]+, 159 (10) [M + H – HCO2H – CO]+, 131 (20)
3ЈЈЈ and C-5ЈЈЈ), 128.51 (C-3ЈЈ, C-5ЈЈ, C-2ЈЈЈ and C-6ЈЈЈ), 131.23 (C- [M + H – HCO2H – CO – CO]+, 104 (22), 77 (4).
7aЈ), 134.01 (C-7Ј), 137.44 (C-1ЈЈ), 137.55 (C-1ЈЈЈ), 146.35 (C-6Ј),
Mycenarubin B (6): Red solid; HPLCprep: Rt = 16.7 min. [α]2D5
=
170.97 (COCH3), 174.31 (C-1) ppm. EIMS: m/z (%) = 458 (1)
[M]+, 367 (2) [M – CH2Ph]+, 342 (1) [M – (NHAc)CHCO2H]+, 339
(2), 297 (2), 293 (1), 279 (1), 252 (3), 251 (3), 236 (3) [M – (NHAc)-
CHCO2H – PhCHO]+, 223 (4), 208 (2), 189 (3), 162 (2), 158 (2),
132 (6), 108 (3) [PhCH2OH]+, 107 (2), 91 (100) [PhCH2]+, 79 (4),
77 (4) [Ph]+, 65 (6), 51 (1), 43 (3).
+170 (c = 0.0061, H2O). CD (H2O): λ (∆ε) = 242 (–4.8), 264 (+2.9),
300 (+0.9), 319 (+1.0), 374 (–2.4), 432 (+0.7), 473 (+0.4), 539 (+0.9)
1
nm. H NMR (900 MHz, D2O, 300 K): δ = 2.00 (m, 2 H, 11-H),
2.05 (dm, J ≈ 16 Hz, 1 H, 11Ј-Hb), 2.23 (dddm, J ≈ 16 Hz, J ≈
13 Hz, J ≈ 13 Hz, 1 H, 11Ј-Ha), 3.03 (m, 2 H, 12-H), 3.06 (dd, J ≈
17 Hz, J ≈ 7 Hz, 1 H, 3Ј-Hb), 3.06 (dd, J ≈ 17 Hz, J ≈ 7 Hz, 1 H,
3-Hb), 3.13 (d, J ≈ 17 Hz, 1 H, 3-Ha), 3.15 (d, J ≈ 17 Hz, 1 H, 3Ј-
Ha), 3.16 (m, 1 H, 10Ј-Hb), 3.38 (ddd, J ≈ 14 Hz, J ≈ 7.5 Hz, J ≈
7.5 Hz, 1 H, 10-Ha), 3.58 (dm, J = 12.4 Hz, 1 H, 12Ј-Hb), 3.74
(ddd, J ≈ 14 Hz, J ≈ 7.5 Hz, J ≈ 7.5 Hz, 1 H, 10-Hb), 3.86 (dd, J =
12.4 Hz, J ≈ 12 Hz, 1 H, 12Ј-Ha), 3.98 (dd, J ≈ 15 Hz, J ≈ 15 Hz,
1 H, 10Ј-Ha), 4.208 (d, J ≈ 7 Hz, 1 H, 4-H), 4.213 (d, J ≈ 7 Hz, 1
H, 4Ј-H), 5.522 (s, 1 H, 6-H), 5.524 (s, 1 H, 6Ј-H), 6.77 (s, 1 H, 2Ј-
(S)-6,7-Bis(benzyloxy)tryptophan
(4):
Acylase I
(25 mg,
E.C. 3.5.1.14, Sigma: A8376) and CoCl2 (1 mg) were added to a
solution of 3 (50.0 mg, 1.09 mmol) in phosphate buffer (10 mL,
50 m, pH 7.2). The mixture was incubated at 37 °C and 100 rpm
for 3 h. The reaction was stopped by addition of MeOH (10 mL),
the solvents were removed at 40 °C in vacuo, the residue was dis-
solved in MeOH (5 mL), the precipitated enzyme was removed by
centrifugation, and the crude product was first applied onto a RP-
18 cartridge and the methanolic eluate was then purified by prepar-
ative HPLC. Yield: 13.3 mg [59% referred to the original content
of (S)-4], colourless solid; HPLCprep (gradient: 10 min at 50% H2O,
50% MeOH then linear to 100% MeOH over 20 min; flow rate:
12 mLmin–1; detection: UV at 280 nm, other conditions as de-
scribed under “General Experimental Procedures”): Rt = 20.5 min.
1H NMR (500 MHz, CD3OD, 300 K): δ = 3.10 (dd, J = 15.2,
9.4 Hz, 1 H, 3-H), 3.46 (dd, J = 15.2, 3.8 Hz, 1 H, 3-H), 3.83 (dd,
J = 9.4, 3.8 Hz, 1 H, 2-H), 5.14 (m, 2 H, OCH2-6Ј), 5.16 (m, 2 H,
OCH2-7Ј), 6.94 (d, J = 8.6 Hz, 1 H, 5Ј-H), 7.11 (s, 1 H, 2Ј-H), 7.29–
7.31 (m, 4 H, 4ЈЈ-H, 3ЈЈЈ-H, 4ЈЈЈ-H, 5ЈЈЈ-H), 7.35 (m, 2 H, 3ЈЈ-H,
5ЈЈ-H), 7.36 (d, J = 8.6 Hz, 1 H, 4Ј-H), 7.47 (d, J = 6.9 Hz, 2 H,
2ЈЈ-H, 6ЈЈ-H), 7.47 (d, J = 6.9 Hz, 2 H, 2ЈЈЈ-H, 6ЈЈЈ-H) ppm. 13C
NMR (126 MHz, CD3OD, 300 K): δ = 28.50 (C-3), 56.65 (C-2),
74.02 (6Ј-OCH2), 76.10 (7Ј-OCH2), 110.14 (C-3Ј), 111.44 (C-5Ј),
114.81 (C-4Ј), 125.21 (C-2Ј), 126.07 (C-3aЈ), 128.87 (C-4ЈЈ), 128.95
(C-2ЈЈ and C-6ЈЈ), 128.99 (C-4ЈЈЈ), 129.28 (C-3ЈЈЈ and C-5ЈЈЈ),
129.43 (C-3ЈЈ and C-5ЈЈ), 129.58 (C-2ЈЈЈ and C-6ЈЈЈ), 135.66 (C-7Ј),
139.22 (C-1ЈЈ), 139.29 (C-1ЈЈЈ), 147.67 (C-6Ј), 174.28 (C-1) ppm.
EIMS: m/z (%) = 416 (2), [M]+, 372 (10) [M – CO2]+, 342 (19) [M –
(NH2)CHCO2H]+, 325 (7) [M – CH2Ph]+, 308 (4), 297 (3), 289 (3),
281 (28) [M – CO2 – CH2Ph]+, 264 (10), 251 (10), 236 (30) [M –
(NH2)CHCO2H – PhCHO]+, 224 (6), 209 (5), 208 (13), 192 (3),
189 (3), 181 (6), 161 (7), 158 (3), 132 (11), 129 (12), 118 (4), 108
(28) [PhCH2OH]+, 107 (20), 106 (9) [PhCHO]+, 91 (100)
[PhCH2]+, 79 (25), 77 (19) [Ph]+, 65 (10), 51 (6).
1
H), 6.97 (s, 1 H, 2-H) ppm. H NMR (600 MHz, D2O, 298 K): δ
= 2.03 (m, 2 H, 11-H), 2.08 (dm, J ≈ 16 Hz, 1 H, 11Ј-Hb), 2.27
(dm, J ≈ 16 Hz, 1 H, 11Ј-Ha), 3.07 (dd, J ≈ 8 Hz, J ≈ 8 Hz, 2 H,
12-H), 3.09 (dd, J ≈ 16 Hz, J ≈ 7 Hz, 2 H, 3-Hb and 3Ј-Hb), 3.16
(d, J ≈ 16 Hz, 1 H, 3-Ha), 3.18 (d, J ≈ 16 Hz, 1 H, 3Ј-Ha), 3.19 (m,
1 H, 10Ј-Hb), 3.40 (ddd, J = 14.5, 7.8, 7.8 Hz, 1 H, 10-Ha), 3.61
(dm, J = 14.6 Hz, 1 H, 12Ј-Hb), 3.77 (ddd, J = 14.5, 7.8, 7.8 Hz, 1
H, 10-Hb), 3.89 (dm, J = 14.6 Hz, 1 H, 12Ј-Ha), 4.01 (dm, J ≈
15 Hz, 1 H, 10Ј-Ha), 4.24 (d, J ≈ 7 Hz, 1 H, 4-H), 4.24 (d, J ≈ 7 Hz,
1 H, 4Ј-H), 5.55 (s, 1 H, 6-H), 5.56 (s, 1 H, 6Ј-H), 6.81 (s, 1 H, 2Ј-
H), 7.00 (s, 1 H, 2-H) ppm. 13C NMR (226 MHz, D2O, 300 K): δ
= 25.23 (C-3), 25.36 (C-3Ј), 26.16 (C-11Ј), 26.80 (C-11), 38.45 (C-
12), 44.85 (C-12Ј), 50.58 (C-10), 50.60 (C-10Ј), 64.48 (C-4Ј), 67.68
(C-4), 85.47 (C-6), 96.04 (C-6Ј), 117.81 (C-2aЈ), 118.75 (C-2a),
123.61 (C-8a), 125.34 (C-8aЈ), 125.68 (C-8b), 125.70 (C-8bЈ), 127.67
(C-2Ј), 128.86 (C-2), 155.50 (C-7), 156.73 (C-5a), 158.03 (C-5aЈ),
167.59 (C-8), 172.48 (C-8Ј), 176.67 (C-9), 177.90 (C-9Ј), 180.05 (C-
7Ј) ppm. UV/Vis (H2O): λmax (lgε) 245 (4.19), 354 (3.89), 534 (3.01)
nm. HR-ESIMS: m/z = 561.20789 [M + H]+ calcd. 561.20922 for
C28H29N6O7. LC-ESIMS: Rt = 18.4 min (detection: UV at λ =
360 nm) m/z = 561 [M + H]+. LC-ESIMS/MS (parent ion m/z 561,
20 eV) m/z (%) = 517 (6) [M + H – CO2]+, 473 (100) [M + H –
CO2 – CO2]+, 455 (13), 267 (6).
Supporting Information (see also the footnote on the first page of
this article): UV/Vis, CD, NMR and mass spectra of 1, 3, 4, 5 and
6.
(S)-4-Carboxydamirone C (5): Catalytic amounts of Pd/C were
added to a solution of 4 (10.0 mg, 24.0 µmol) in MeOH (5 mL) and
the mixture was hydrogenated by application of H2 (balloon) for
1 h. After removal of the balloon, NEt3 (0.2 µL) was added and
the solution was stirred under air for 10 min at 40 °C. After that,
the red solution was filtered, the solvent was removed, and the
Acknowledgments
We are grateful to Prof. Dr. Michael Spiteller, Silke Richter, Dr.
Marc Lamshöft and Dr. Sebastian Zülke (University of Dortmund,
Institute of Environmental Research, Germany) and to Dr. Kerstin
crude product was purified by preparative HPLC. Yield: 1.26 mg
1
(23%). Red solid. HPLCprep: Rt = 21.5 min. H NMR (600 MHz, Strupat (Thermo Electron GmbH, Bremen, Germany) for the mea-
D2O, 298 K): δ = 2.93 (dd, J = 16.1, 6.6 Hz, 1 H, 3-H), 3.05 (dd,
J = 16.1, 6.6 Hz, 1 H, 3-H), 4.25 (dd, J = 6.6, 6.6 Hz, 1 H, 4-H),
5.27 (s, 1 H, 6-H), 6.94 (s, 1 H, 2-H) ppm. 13C NMR (151 MHz,
D2O, 298 K): δ = 24.50 (C-3), 58.94 (C-4), 94.35 (C-6), 118.36 (C-
surement of LC-ESIMS and HR-ESIMS spectra and to Dr. Dieter
Spiteller (Max Planck Institute for Chemical Ecology, Jena, Ger-
many) for the measurement of optical rotation values. Our work
was generously supported by an Emmy Noether Fellowship for
2a), 125.45 (C-8b), 125.93 (C-8a), 127.43 (C-2), 159.02 (C-5a), young investigators from the Deutsche Forschungsgemeinschaft
173.74 (C-8), 178.84 (CO2H), 180.20 (C-7) ppm. UV/Vis (H2O): (DFG) (SP 718/1-1) and by the Fonds der Chemischen Industrie.
Eur. J. Org. Chem. 2007, 1571–1576
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1575

S. Peters, P. Spiteller
FULL PAPER
[9] Y. Konda-Yamada, C. Okada, K. Yoshida, Y. Umeda, S. Ar-
ima, N. Sato, T. Kai, H. Takayanagi, Y. Harigaya, Tetrahedron
2002, 58, 7851–7861.
[10] E. M. Antunes, B. R. Copp, M. T. Davies-Coleman, T. Samaai,
Nat. Prod. Rep. 2005, 22, 62–72.
[11] S. Sakemi, H. H. Sun, C. W. Jefford, G. Bernardinelli, Tetrahe-
dron Lett. 1989, 30, 2517–2520.
[12] D. B. Stierle, D. J. Faulkner, J. Nat. Prod. 1991, 54, 1131–1133.
[13] H. H. Sun, S. Sakemi, N. Burres, P. McCarthy, J. Org. Chem.
1990, 55, 4964–4966.
[1]
[2]
E. P. Abraham, Jpn. J. Antibiotics 1977, 30 (Suppl.), 1–26.
a) H. Sauter, W. Steglich, T. Anke, Angew. Chem. Int. Ed. 1999,
38, 1328–1349; b) T. Anke, G. Erkel, Non-β-lactam antibiotics,
in: The Mycota (Eds.: K. Esser, W. Bennett), vol. 10, Springer-
Verlag, Berlin, Heidelberg, 2002, pp. 93–108.
M. Stadler, O. Sterner, Phytochemistry 1998, 49, 1013–1019.
O. Sterner, R. Bergman, J. Kihlberg, B. Wickberg, J. Nat. Prod.
1985, 48, 279–288.
[3]
[4]
[5] G. Robich, Mycena dЈEuropa, Associazione Micologica Bresa-
dola, Trento, 2003, pp. 123–127.
[6] T. Talou, S. Breheret/Hulin-Bertaud, A. Gaset, Identification of
the major key flavour compounds in odorous wild mushrooms in
Frontiers of Flavour Science (Eds.: P. Schieberle, K.-H. Engel),
Deutsche Forschungsanstalt für Lebensmittelchemie, Gar-
ching, 2000, pp. 46–50.
[7] a) C. Baumann, M. Bröckelmann, B. Fugmann, B. Steffan, W.
Steglich, W. S. Sheldrick, Angew. Chem. 1993, 105, 1120–1121;
b) C. Hopmann, W. Steglich, Liebigs Ann. 1996, 7, 1117–1120.
[8] F. G. H. Lee, D. E. Dickson, J. Suzuki, A. Zirnis, A. Manian,
J. Heterocycl. Chem. 1973, 10, 649–654.
[14] D. C. Radisky, E. S. Radisky, L. R. Barrows, B. R. Copp, R. A.
Kramer, M. C. Ireland, J. Am. Chem. Soc. 1993, 115, 1632–
1638.
[15] N. B. Perry, J. W. Blunt, J. D. McCombs, M. H. G. Munro, J.
Org. Chem. 1986, 51, 5476–5478.
[16] B. R. Copp, C. M. Ireland, J. Org. Chem. 1991, 56, 4596–4597.
[17] M. Ishibashi, T. Iwasaki, S. Imai, S. Sakamoto, K. Yamaguchi,
A. Ito, J. Nat. Prod. 2001, 64, 108–110.
[18] E. W. Schmidt, M. K. Harper, D. J. Faulkner, J. Nat. Prod.
1995, 58, 1861–1867.
Received: September 21, 2006
Published Online: December 21, 2006
1576
www.eurjoc.org
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 1571–1576