Alkaloids from the mushroom pseudobaeospora pyrifera, pyriferines A-C

Article abstract of DOI:10.1021/np800365f

Three novel alkaloids (1-3), named pyriferines A-C, were isolated from fruiting bodies of Pseudobaeospora pyrifera. They possess an unusual eight-membered N/O-acetal ring, derived from L-glutamic acid, that is connected to an enolized 1,3-diketo moiety. The structures were determined by spectroscopic methods, and the absolute configuration of the glutamic acid moiety was established using GC-MS after Mosher-type derivatization.

Full text of DOI:10.1021/np800365f

1620
J. Nat. Prod. 2008, 71, 1620–1622
Alkaloids from the Mushroom Pseudobaeospora pyrifera, Pyriferines A-C
Dang Ngoc Quang,†,§ Peter Spiteller,‡ Andrea Porzel,† Ju¨rgen Schmidt,† Torsten Geissler,† Norbert Arnold,† and
Ludger Wessjohann*,†
Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle/Saale, Germany, Institut fu¨r
Organische Chemie and Biochemie II, Technische UniVersita¨t Mu¨nchen, Lichtenbergstrasse 4, D-85747 Garching, Germany, and Faculty of
Chemistry, Hanoi UniVersity of Education, 136 XuanThuy Road, CauGiay, Hanoi, Vietnam
ReceiVed June 19, 2008
Three novel alkaloids (1-3), named pyriferines A-C, were isolated from fruiting bodies of Pseudobaeospora pyrifera.
They possess an unusual eight-membered N/O-acetal ring, derived from L-glutamic acid, that is connected to an enolized
1,3-diketo moiety. The structures were determined by spectroscopic methods, and the absolute configuration of the
glutamic acid moiety was established using GC-MS after Mosher-type derivatization.
The fungal genus Pseudobaeospora (Tricholomataceae) is very
rare and only described for Europe and North America. Thirteen
species are known, but their chemical constituents are unknown.1
The genus was first established by Singer2,3 and recently generically
circumscribed by Bas.1,4 The species Pseudobaeospora pyrifera
Bas & L. Krieglst. (Agaricales) investigated herein was described
in 1998.5 P. pyrifera is characterized by a 0.5-2.5 cm wide cap
and a 1-3.5 cm long and 0.3-0.5 cm broad stipe. Cap and stem
are pruinose sordid pink to pruinose vinaceous; the lamellae are
rather dark reddish to violaceous pink colored. This little species
grows on the ground in more or less moist areas (Pruno-Fraxinetum
Figure 1. Important 1H-1H COSY (bold lines), HMBC, and
NOESY correlations of pyriferine A (1).
woods with Alnus, Prunus padus, Fraxinus). P. pyrifera is
characterized by a greenish-blue to brownish-green reaction of the
pileipellis with KOH solution. In this work, we describe the isolation
and structural characterization of three novel unusual alkaloids that
possess unprecedented skeletons (1-3).
C16H24O7N2. According to the DEPT spectrum, one conjugated
ketone (δC 201.3), one enol carbon (δC 183.1), two carbonyl groups,
and other signals as described in the Experimental Section were
present. The 1H-1H COSY spectrum of 1 (Figure 1) provided
evidence for partial structures CH2-CH2-CH-O and CH-
CH2-CH2-. The proton connected to the oxygen-bearing carbon
(δH 4.03) had HMBC correlations with C-2, C-4, C-5, and 3-OMe;
therefore, the signal should belong to H-3. The methyl group at δH
1.56 showed HMBC correlations with C-1, C-2, C-5, C-6, and C-7;
thus, it should be located at C-6. The second methyl group is
attached to C-8 due to its HMBC correlations with C-7 and C-8.
Since C-6 (δC 68.2) was observed at relatively low field, it is likely
connected to an oxygen atom. The presence of a glutamic acid
moiety was also revealed by HMBC correlations between H-13/
C-11, C-12, and C-14 and between H-11/C-10 and C-12 (Figure
1) and by comparison with data in a previous publication.6 The
C-14 carbonyl group (δC 173.7) coupled to H-12 and H-13 in the
HMBC spectrum and together with the low-field shift of C-8
indicated that C-14 is connected to C-8 via an oxygen atom.
Furthermore, only one NH signal was observed when the HMBC
spectrum was recorded in DMSO-d6. The signal at δ 9.22 showed
correlations with C-7, C-8, and C-10; thus it was assigned as NH-
9. The other end of the glutamic acid moiety must be attached to
N-9 to generate an eight-membered-ring partial structure in 1.
The relative configuration within the hexene ring of 1 was
established by creation of a three-dimensional model and by its
correlations to NOESY data (Figure 1). First of all, H-3 showed a
NOE correlation with 3-OMe and appeared as a dd (δH 4.03, J )
4.4, 4.4); thus H-3 is equatorial. Accordingly 3-OCH3 must be axial.
H-3 coupled strongly with H-4e (δH 2.13) and weakly with H-4a
(δH 1.90). Furthermore, H-4e (δH 2.13) coupled with H-5e (δH 1.68).
Finally, 6-Me was determined to be axial and located on the
opposite face relative to 3-OCH3 due to the NOE correlations
between 6-Me and H-4a, H-5e (δH 1.68). Since, there was no
correlation observed between 8-Me and other protons, the relative
Compound 1 was obtained as a colorless oil. Its positive ion
ESIMS gave an [M + Na]+ ion at m/z 379.1467, corresponding to
the molecular formula C16H24O7N2Na, which was revealed by ESI-
FTICR-MS, indicating the presence of six double-bond equivalents.
Analysis of its IR spectrum suggested that it contained carbonyl
groups (1681 cm-1) and a double bond (1615 cm-1). The 1H NMR
spectrum of 1 indicated the presence of two methyl, four methylene,
two methine, and one methoxy group. Its 13C NMR exhibited signals
for 16 carbon atoms, in agreement with the molecular formula
* Corresponding author. Tel: +49 (0) 345 5582 1301. Fax: +49 (0) 5582
1309. E-mail: wessjohann@ipb-halle.de.
† Leibniz Institute of Plant Biochemistry.
‡ Technische Universita¨t Mu¨nchen.
§ Hanoi University of Education.
Dedicated to Dr. habil. Siegfried Huneck on the occasion of his 80th
birthday.
10.1021/np800365f CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/05/2008

Notes
Journal of Natural Products, 2008, Vol. 71, No. 9 1621
configuration of 8-CH3 remains unclear. In addition, LC-ESIMS/
MS spectra in both positive and negative modes were recorded.
Detailed analyses of all major fragments are shown (see S4 in the
Supporting Information). From the above discussion and spectro-
scopic evidence, compound 1 was determined to be 7-amino-2-
(2,6-dihydroxy-3-methoxy-6-methylcyclohex-1-enecarbonyl)-2-
methyl[1,3]oxazocane-4,8-dione, and it was named pyriferine A.
synthesis can eventually provide a final proof of the absolute
configuration of these stereocenters in the pyriferines.
This is the first report on chemical constituents of Pseudobaeospo-
ra sp., and the pyriferines A-C (1-3) possess unprecedented
carbon skeletons. The interconversion of compounds 1-3 is
presumably by biosynthetic conversions starting from 1. A dehydra-
tion reaction could form compound 2. Compound 3 could then be
obtained from compound 2 by oxidation (see S5 in the Supporting
Information). Labeling experiments are needed to clarify whether
pyriferines A-C (1-3) originate from shikimic acid and either the
two amino acids alanine and glutamic acid or from pyruvate and
glutamine. Although most biosynthetic steps are usually enzyme-
catalyzed, for the last step(s) nonenzymatic conversions cannot be
excluded and such reactions are not rare in fungi, e.g., by oxidative
activation of defense compounds in wounded tissue.
Compound 2 was also purified as a colorless oil. Its molecular
formula, C16H22O6N2 (ESI-FTICR-MS), corresponded to a dehy-
drated compound 1. Its NMR spectroscopic data resembled those
of 1, except for the presence of one exo-methylene group with
signals at δ 4.89 and 5.62 in its 1H NMR spectrum. The
exo-methylene was placed at C-6 due to HMBC correlations
between 6-CH2 and C-1, C-5, C-6. The relative position of H-3
was established as equatorial since it appeared as a dd (J ) 5.4,
5.4 Hz); therefore 3-OCH3 is axial. Consequently, pyriferine B was
determined to be 7-amino-2-(2-hydroxy-3-methoxy-6-methylenecy-
clohex-1-enecarbonyl)-2-methyl[1,3]oxazocane-4,8-dione.
Preliminary tests9,10 show that pyriferines A and B (1, 2) inhibit
acetylcholinesterase, an important medicinal target, e.g., in Alzhe-
imer’s disease, to a minor extent (20% and 7% at 100 µM,
respectively).
Pyriferine C (3) exhibited an [M + Na]+ ion at m/z 359.1214 in
the ESI-FTICR-MS, corresponding to the molecular formula
C16H20O6N2Na, two hydrogen atoms less than compound 2. The
spectroscopic data were very similar to those of 2, except for signals
typical for an aromatic ring. The locations of substituents on the
aromatic ring were determined by 2D NMR spectra. The 3-OMe
group coupled to the aromatic C-3 in the HMBC spectrum. Two
proton signals (δ 7.13 and 6.78) appeared as doublets with a large
coupling constant (J ) 7.8 Hz), correlating in the HMBC to C-2,
C-3, C-6 and C-1, C-3, respectively; thus they were assigned as
H-4 and H-5. In addition, the methyl group at δ 2.47 is located at
C-6 on the basis of HMBC correlations between 6-Me and C-1,
C-5, C-6. Their positioning was supported by a NOESY spectrum
in which correlations between H-4 and 3-OCH3, H-5 and between
H-5 and H-4, 6-Me were also detected. To confirm the proposed
structure, compound 3 was treated with N-methyl-N-trimethylsi-
lyltrifluoroacetamide to give the silylation product (3a, see the
Supporting Information), which was analyzed by GC-EIMS. The
EI mass spectrum exhibited the molecular peak at m/z ) 624,
indicating the presence of four TMS groups in 3a.
Experimental Section
General Experimental Procedures. Column chromatography was
carried out on Sephadex LH-20 (Amersham Pharmacia Biotech).
Preparative HPLC was performed with a Varian ProStar 218 system
and an ODS C-18 column (5 µm, 150 × 20 mm i.d., YMC). Optical
rotations were measured on a JASCO DIP-1000 polarimeter with MeOH
as solvent. UV spectra were obtained on a Jasco V-560 spectropho-
tometer in MeOH. IR spectra were measured on a Thermo Nicolet 5700
FT-IR spectrometer, on an ATR crystal (diamond). The H and 13C
1
NMR spectra were recorded on a Varian Unity Inova 500 spectrometer
and Bruker AMX-600 using CD3OD as solvent. Chemical shifts are
given relative to TMS as internal standard (1H) and 49.0 ppm from
CD3OD as standard (13C). High-resolution ESI mass spectra were
recorded on a Bruker Apex III Fourier transform ion cyclotron
resonance (FTICR) mass spectrometer. LC-ESIMS/MS were recorded
on a Finnigan MAT TSQ Quantum Ultra AM system. The GC-EIMS
measurements of the trimethylsilyl derivatives of 3 were performed
on a Voyager/Trace GC 2000 (Thermo Quest CE Instruments) under
the following conditions: 70 eV EI, source temperature 200 °C, column
DB-5MS (J&W, 30 m × 0.25 mm, 0.25 µm film thickness), injection
temperature 250 °C, interface temperature 300 °C, carrier gas He, flow
rate 1.0 mL/min, constant flow mode, splitless injection, column
temperature program 60 °C for 1 min, then raised to 300 °C at a rate
of 10 °C min-1 and then held at 300 °C for 15 min. The GC-EIMS
measurement for 4 was analyzed by a Fisons Instruments GC 8000
series and MD 800 equipped with the same column used to analyze
3a. Temperature program: starting from 100 to 160 °C at 30 °C/min,
then 160 to 200 °C with 1 °C/min, finally 200 to 270 °C at 30 °C/min.
Mass selective detector: m/z 202, 240, 248, and 332.
To establish the absolute configuration of the glutamic acid
moiety, compound 3 was hydrolyzed to give glutamic acid followed
by methylation using diazomethane7 and esterification with (S)-R-
methoxy-R-trifluoromethylphenylacetylchloride [(S)-MTPA-Cl] to
form compound 4. Compound 4 was analyzed by GC-MS using a
mass selective detector8 and compared to 5 and 6 as standards,
which were derived from D- and L-glutamic acid, respectively. From
the GC chromatograms (see the Supporting Information for
preparation and structures of compounds 4, 5, and 6), it was
established that the glutamic acid moiety of compound 3 is
S-configured (L-glutamic acid). Accordingly, the glutamic acid
moieties of 1 and 2 are assumed to have L-configurations by
analogy. Therefore, pyriferine C (3) is 7S-amino-2-(2-hydroxy-3-
methoxy-6-methylbenzoyl)-2-methyl[1,3]oxazocane-4,8-dione (please
note that the IUPAC 7S-carbon corresponds to the stereocenter at
C-13).
Fungus Material. Fruiting bodies of Pseudobaeospora pyrifera were
collected in Germany, state of Bavaria, in Lower Franconia near
Kitzingen (Klosterforst) at the loco-type (leg. and det. L. Krieglsteiner).
A voucher specimen is deposited in REG.
Extraction and Isolation. Dried basidiocarps of P. pyrifera (3.08
g) were extracted two times with MeOH (100 mL) in an ultrasonic
bath for 45 min. The methanolic extract was concentrated in Vacuo to
obtain a red-brown residue (400 mg), which was subjected to Sephadex
LH-20 column chromatography using CHCl3/MeOH (1:1) as eluent.
Further purification was carried out by repeated preparative HPLC on
an ODS C-18 column, using H2O (A) and CH3CN (B) as a solvent
system (linear gradient: 0-20 min, 3% to 25% B; 20 to 23 min: 25%
to 100% B; isocratic flow of B to 27 min; linear gradient: 27-30 min,
0 to 97% A), flow rate 20 mL/min to afford 1 (2.7 mg, 4.4 min), 2 (1.6
mg, 16.2 min), and 3 (4.8 mg, 22.6 min).
Unfortunately, the flexible eight-membered ring does not allow
an unequivocal stereochemical correlation of the defined stereo-
center at C-13 to the center at C-8, and thus the absolute
stereochemistry at C-8 and in the six-membered ring in relation to
C-13 is not defined. Neither extensive molecular modeling calcula-
tions nor NMR studies provided any hint at a preferred orientation.
Thus, it cannot be excluded that the cis-related O-substituents at
C-3 and C-6 may both have opposite absolute configuration. P.
pyrifera is such a rare species that the amount of 1 available was
too small to determine the absolute configuration of the C1-8 moiety
through chemical derivatization/degradation experiments. Micro-
crystallization was unsuccessful. At this point, only chemical
Pyriferine A (1): colorless oil; [R]2D0 -8.0 (MeOH, 0.08); UV λmax
nm (log ε) 269 (3.7), 234 (3.5); IR (ATR) 3359, 3252, 2925, 2852,
1681, 1615, 1538, 1448, 1258, 1139, 1021, 914 cm-1 1H NMR
;
(CD3OD, 600 MHz) δ 1.48 (3H, s, 8-Me), 1.56 (3H, s, 6-Me), 1.68
(1H, m, H-5), 1.90 (1H, m, H-4), 1.92 (1H, m, H-5), 2.05 (2H, m,
H-12), 2.13 (1H, m, H-4), 2.45 (2H, dd, J ) 7.3, 7.3 Hz, H-11), 3.42
(3H, s, 3-OMe), 3.56 (1H, dd, J ) 5.6, 5.6 Hz, H-13), 4.03 (1H, dd, J
) 4.4, 4.4 Hz, H-3); 13C NMR (CD3OD, 151 MHz) δ 22.0 (CH3, 8-Me),

1622 Journal of Natural Products, 2008, Vol. 71, No. 9
Notes
26.6 (CH2, C-4), 26.6 (CH3, 6-Me), 27.4 (CH2, C-12), 31.9 (CH2, C-11),
36.4 (CH2, C-5), 55.5 (CH, C-13), 58.2 (CH3, 3-OMe), 68.2 (C, C-6),
73.4 (CH, C-3), 91.9 (C, C-8), 117.6 (C, C-1), 173.7 (C, C-14), 174.2
(C, C-10), 183.1 (C, C-2), 201.3 (C, C-7); 1H NMR (DMSO-d6) δ 1.32
(3H, s, 8-Me), 1.37 (3H, s, 6-Me), 1.52 (1H, m, H-5), 1.70 (1H, m,
H-5), 1.75 (1H, m, H-4), 1.77 (2H, m, H-12), 2.01 (1H, m, H-4), 2.77
(2H, t, J ) 7.6 Hz, H-11), 3.12 (1H, t, J ) 6.2 Hz, H-13), 3.27 (3H,
s, 3-OMe), 3.93 (1H, t, J ) 4.1 Hz, H-3), 9.22 (1H, s, H-9); 13C NMR
(DMSO-d6) δ 21.9 (8-Me), 24.8 (C-4), 26.4 (6-Me), 26.7 (C-12), 30.8
(C-11), 35.4 (C-5), 53.3 (C-13), 56.8 (3-OMe), 65.7 (C-6), 71.3 (C-3),
90.7 (C-8), 116.4 (C-1), 169.5 (C-14), 171.3 (C-10), 179.3 (C-2), 198.2
(C-7); positive ion ESIMS 379 [M + Na]+; negative ion ESIMS 355
[M - H]-; LC-ESIMS/MS (positive) 357 (18) [M + H]+, 339 (100),
210 (82); LC-MS/MS (negative) 355 (10) [M - H]-, 337 (25), 293
(12), 276 (22), 226 (100), 208 (25), 145 (20); ESI-FTICR-MS [M +
Na]+, m/z 379.1476, calcd for C16H24O7N2Na+, 379.1481.
δ 17.0 (6-Me), 22.0 (8-Me), 27.5 (C-12), 31.9 (C-11), 55.5 (C-13),
56.8 (3-OMe), 91.6 (C-8), 120.1 (C-1), 120.5 (C-4), 123.9 (C-5), 131.2
(C-6), 145.3 (C-3), 160.9 (C-2), 174.6 (C-10, C-14), 200.7 (C-7);
positive ion ESIMS 359 [M + Na]+, negative ion ESIMS 335 [M -
H]-; LC-ESIMS/MS (positive) 337 (10) [M + H]+, 208 (40), 191 (100);
LC-ESIMS/MS (negative) 335 (13) [M - H]-, 273 (32), 206 (100),
191 (10), 128 (26); ESI-FTICR-MS [M + Na]+, m/z 359.1214, calcd
for C16H20O6N2Na+, 359.1219.
Acknowledgment. We gratefully acknowledge a postdoctoral fel-
lowship of the Alexander von Humboldt Foundation for Dr. D. N.
Quang. The authors also thank Mrs. C. Kuhnt for GC-EIMS and LC-
ESIMS/MS, Mrs. M. Su¨sse for IR and UV measurements, and Dr. W.
Brandt for calculations (all: Leibniz Institute of Plant Biochemistry,
IPB).
Pyriferine B (2): colorless oil; [R]2D0 +34.3 (MeOH, 0.06); UV λmax
nm (log ε) 297 (3.3), 219 (3.9), 204 (3.9); IR (ATR) 3209, 3041, 2927,
Supporting Information Available: Silylation of pyriferine C (3),
determination of absolute configuration of compound 3 by GC-EIMS,
fragmentation scheme of compound 1 in the LC-ESIMS/MS, and 1D
and 2D NMR spectra of pyriferines A-C (1-3). This material is
available free of charge via the Internet at http://pubs.acs.org.
2851, 1673, 1641, 1590, 1539, 1410, 1352, 1242, 1141, 1086, 874 cm-1
;
1H NMR (CD3OD, 600 MHz) δ 1.49 (3H, s, 8-Me), 1.96 (2H, m, H-4),
2.04 (2H, m, H-12), 2.35 (1H, m, H-5), 2.47 (2H, dd, J ) 7.3, 7.3 Hz,
H-11), 2.56 (1H, m, H-5), 3.45 (3H, s, 3-OMe), 3.55 (1H, dd, J ) 5.9,
5.9 Hz, H-13), 4.21 (1H, dd, J ), 5.4, 5.4 Hz, H-3), 4.89 (1H, s, 6-CH2),
5.62 (1H, s, 6-CH2); 13C NMR (CD3OD, 151 MHz) δ 22.2 (8-Me),
27.3 (C-12), 28.9 (C-5), 29.7 (C-4), 31.9 (C-11), 55.5 (C-13), 58.2
(3-OMe), 74.1 (C-3), 92.3 (C-8), 108.7 (6-CH2), 114.5 (C-1), 135.9
(C-6), 173.7 (C-14), 174.3 (C-10), 183.5 (C-2), 200.2 (C-7); positive
ion ESIMS 361 [M + Na]+, negative ion ESIMS 337 [M - H]-; LC-
ESIMS/MS (positive) 339 (10) [M + H]+, 210 (100), 192 (72); LC-
ESIMS/MS (negative) 337 (35) [M - H]-, 305 (15), 243 (15), 208
(70), 145 (100); ESI-FTICR-MS [M + Na]+, m/z 361.1370, calcd for
C16H22O6N2Na+, 361.1376.
References and Notes
(1) Bas, C. Persoonia 2003, 18, 169–199.
(2) Singer, R. Lloydia 1942, 5, 97–135.
(3) Singer, R. Mycologia 1963, 55, 115–122.
(4) Bas, C. Persoonia 2002, 18, 115–122.
(5) Bas, C.; Krieglsteiner, L. Z. Mykol. 1998, 64, 203–206.
(6) Eizenhofer, T.; Fugmann, B.; Sheldrick, W. S.; Steffan, B.; Steglich,
W. Liebigs Ann. Chem 1990, 1115, 1118.
(7) Schwetlick, K. et al. Organikum; VEB Deutscher Verlag der Wis-
senschaften: Berlin, 1977; p 672.
Pyriferine C (3): oil; [R]2D0 +12.4 (MeOH, 0.1); UV λmax nm (log
ε) 346 (3.1), 267 (3.7), 205 (4.1); IR (ATR) 3254, 3040, 2936, 2835,
1720, 1673, 1621, 1596, 1518, 1267, 1232, 1071, 1022, 816 cm-1; 1H
NMR (CD3OD, 600 MHz) δ 1.51 (3H, s, 8-Me), 2.02 (2H, m, H-12),
2.47 (3H, s, 6-Me), 2.48 (2H, dd, J ) 7.3, 7.3 Hz, H-11), 3.53 (1H,
dd, J ) 5.6, 5.6 Hz, H-13), 3.86 (3H, s, 3-OMe), 6.78 (1H, d, J ) 7.8
Hz, H-5), 7.13 (1H, d, J ) 7.8 Hz, H-4); 13C NMR (CD3OD, 151 MHz)
(8) Bringmann, G.; God, R.; Schaeffer, M. Phytochemistry 1996, 43, 1393–
1403.
(9) Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone, R. M.
Biochem. Pharmacol. 1961, 7, 88–95.
(10) Rhee, I. K.; Meent, M.; Ingkaninan, K.; Verpoorte, R. J. Chromatogr.
A 2001, 915, 217–223.
NP800365F