Mycenaaurin A, an antibacterial polyene pigment from the fruiting bodies of mycena aurantiomarginata

Article abstract of DOI:10.1021/np100155z

A new polyene pigment, mycenaaurin A (1), was isolated from fruiting bodies of Mycena aurantiomarginata. Mycenaaurin A consists of a tridecaketide that is flanked by two amino acid moieties. These are likely to be derived biosynthetically from S-adenosylmethionine. The tridecaketide itself contains an α-pyrone, a conjugated hexaene, and an isolated alkenyl moiety. The structure of the new pigment was established by 2D NMR spectroscopic methods and APCIMS. The absolute configuration of the four stereogenic centers was determined by degradation of 1 by ozonolysis and GC-MS comparison of the resulting fragments, after appropriate derivatization, with authentic synthetic samples. Mycenaaurin A (1) might act as a constitutive defense compound, since it exhibits antibacterial activity against the Gram-positive bacterium Bacillus pumilus.

Full text of DOI:10.1021/np100155z

1350
J. Nat. Prod. 2010, 73, 1350–1354
Mycenaaurin A, an Antibacterial Polyene Pigment from the Fruiting Bodies of Mycena
aurantiomarginata
Robert J. R. Jaeger and Peter Spiteller*
Institut fu¨r Organische Chemie und Biochemie, Albert-Ludwigs-UniVersita¨t Freiburg, Albertstrasse 21, D-79104 Freiburg, Germany
ReceiVed March 9, 2010
A new polyene pigment, mycenaaurin A (1), was isolated from fruiting bodies of Mycena aurantiomarginata. Mycenaaurin
A consists of a tridecaketide that is flanked by two amino acid moieties. These are likely to be derived biosynthetically
from S-adenosylmethionine. The tridecaketide itself contains an R-pyrone, a conjugated hexaene, and an isolated alkenyl
moiety. The structure of the new pigment was established by 2D NMR spectroscopic methods and APCIMS. The
absolute configuration of the four stereogenic centers was determined by degradation of 1 by ozonolysis and GC-MS
comparison of the resulting fragments, after appropriate derivatization, with authentic synthetic samples. Mycenaaurin
A (1) might act as a constitutive defense compound, since it exhibits antibacterial activity against the Gram-positive
bacterium Bacillus pumilus.
The fruiting bodies of the basidiomycete Mycena aurantiomar-
ginata (Fr.) Que´l. (German name: Feuriger Helmling) are small
mushrooms that often occur in coniferous forests. They are
characterized by a bell-shaped cap of 1 to 2 cm diameter, by a thin
stipe of 3 to 10 cm length, and by the strikingly orange color of
their gills.1 Recently, we determined the structures of several fungal
pigments from red Mycena species. A number of novel red and
blue pyrroloquinoline alkaloids were present in the red latex of
Mycena sanguinolenta2 and the red latex of Mycena haematopus,3
as well as in the pinkish fruiting bodies of Mycena rosea.4 So far,
pyrroloquinoline alkaloids have been found almost exclusively in
marine organisms;5 hence the isolation of a number of pyrrolo-
quinoline alkaloids in Mycena species was surprising. Consequently,
we became interested not only in the chemistry of red Mycena
species but also in that of the orange-colored species M. aurantio-
marginata. Metabolic profiling of the fruiting bodies of M.
aurantiomarginata by HPLC-UV indicated that the compound
responsible for the orange color was likely to be a new polyene. In
this paper we describe the isolation and the structure elucidation
of the coloring principle of the fruiting bodies of M. aurantio-
marginata, which we have named mycenaaurin A (1).
spectrum, recorded in DMSO-d6, exhibited 36 resonances between
δC 19.2 and 173.8, which were attributed to seven quaternary
carbons and 19 CH, eight CH2, and two CH3 groups with the help
1
of the HSQC spectrum (Table 1). The H NMR spectrum showed
the resonances of 13 protons that were assigned to a polyene system
on account of their chemical shift values (δ 5.80-6.92, Table 1).
Two resonances (δH 5.37 and 5.41) were attributed to an isolated
alkenyl group. Two methyl signals (δH 0.99 and 1.82) were present
in the aliphatic region, as well as the resonances of several CH
1
and CH2 groups that appeared in the H NMR spectrum between
δH 1.33 and 4.23. The resonance of an exchangeable amide proton
(δH 8.05) was detectable only when DMSO-d6 rather than CD3OD
was used as solvent. Correlations in the COSY and HSQC spectra
allowed the assignment of five spin systems of 1 (Table 1). The
polyene system and the isolated alkenyl group were part of a
(CHdCH)6-CH2-CH-(CH2)2-(CHdCH)-CH2-CH-CH3 fragment. In
addition, 1 contained two (CH2)2-CH fragments, an isolated CH
group, and an isolated CH3 group. One (CH2)2-CH fragment was
part of a homoserine residue on account of the characteristic
chemical shift values of H-2 ′′ (δH 4.23), H2-4′′ (δH 3.38), C-2′′
(δC 49.1), and C-4′′ (δC 63.8) (Table 1 and Figure 1). HMBC
correlations from H-2′′ (δH 4.23) and H-3′′ (δH 1.69/1.86) to the
quaternary carbon atom C-1′′ (δC 173.8) allowed assignment to the
carboxy group of the homoserine residue. HMBC correlations from
H-2′′ (δH 4.23) and H3-2′′′ (δH 1.82) to the quaternary carbon C-1′′′
(δC 169.2) revealed that the homoserine moiety was N-acetylated.
An HMBC correlation from H2-4′′ (δH 3.38) to C-25 (δC 74.6)
indicated that the N-acetyl homoserine residue was attached to the
central (CHdCH)6-CH2-CH-(CH2)2-(CHdCH)-CH2-CH-CH3 frag-
ment via an ether bond between C-4′′ and C-25. HMBC correlations
from the protons H-6, H-7, H-4, H2-3′, and H2-4′ allowed the
assignment of the positions of the carbons C-1 to C-5. The chemical
shift values of these carbons are typical for a γ-hydroxy-R-pyrone
moiety.6 HMBC correlations from H-6 and H-7 of the central
fragment to the quaternary carbon C-5, from H-6 to C-4, and from
H-4 to C-6 indicated the connection of the R-pyrone moiety to the
polyene residue via C-5 and C-6. The second (CH2)2-CH fragment
was also part of an amino acid residue, as indicated by its chemical
shift values and typical HMBC correlations from H-2′ to C-1.
HMBC correlations from H2-3′ to C-2 and from H2-4′ (δH 2.51) to
the quaternary carbon atom C-1 (δC 164.2) and to C-2 and C-3
suggested this amino acid moiety was connected to the R-pyrone
via C-4′ and C-2. All double bonds of 1 had trans-configuration,
Results and Discussion
Mycenaaurin A (1) was extracted from frozen fruiting bodies of
M. aurantiomarginata with methanol. The resulting crude extract
was filtered, concentrated, and fractionated on a preparative RP-
18 column. A water-methanol gradient was used for HPLC, and
the compound was detected by its UV/vis absorption at λ 420 nm.
The yield of 1 from 10 g of the frozen fruiting bodies was 3.5 mg.
Mycenaaurin A exhibited absorption maxima at λ 400, 417, and
436 nm in the UV/vis spectrum, which are characteristic for polyene
pigments. The molecular formula C36H48N2O10 was deduced from
the HRAPCIMS, revealing that 1 contained 14 degrees of unsat-
uration. In accordance with the molecular formula, the 13C NMR
3
as revealed by the large JHH coupling constants of 14-15 Hz of
* To whom correspondence should be addressed. Tel: +49-761-203-
6043. E-mail: peter.spiteller@ocbc.uni-freiburg.de.
the polyene protons and of the protons of the isolated alkenyl group.
10.1021/np100155z  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/09/2010

Antibacterial Polyene Pigment from Mycena
Journal of Natural Products, 2010, Vol. 73, No. 8 1351
1
Table 1. 13C and H NMR Spectroscopic Data (600 MHz, DMSO-d6, 300 K) of Mycenaaurin A (1)
δH, mult.
(J in Hz)
position
δC, mult.
COSY
NOESY/ROESY
HMBC
1
2
164.2, qC
99.9, qC
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
169.8, qC
104.8, CH
155.8, qC
122.9, CH
133.0, CH
131.5, CH
137.5, CH
132.5, CH
135.6, CH
132.4, CH
134.8, CH
130.9, CH
134.4, CH
132.1, CH
133.4, CH
40.9, CH2
69.2, CH
6.00, s
6, 7
2, 3, 5, 6
6.26, d (14.9)
6.92, dd (14.9, 11.6)
6.423, dd (14.1, 11.6)
6.64, dd (14.1, 11.2)
6.417, dd (16, 11.2)
6.46, dd (16, 10)
6.37, dd (16, 10)
6.39, dd (16, 10)
6.24, dd (16, 10)
6.31, dd (16, 10.8)
6.13, dd (14.4, 10.8)
5.80, d (14.4)
2.17, m
7
4, 8
4, 9
4, 5, (7), 8
5, (6), (8), C-9
6, 10
7, (10), 11
8, 12
9, 13
10, 14
11, 14
(13), 16
6, 8
7, 9
8, 10
9,11a
6,10a
7, 11
8a, 12a
9, 13a
10a, 12a
11a, 13a
10a, 14a
12a, 14
11a, 15a
13, 15
14, 16
15, 17
16, 18
12a, 16a
13a, 17
13, 17
14a, 17, 18
14, 15, (18)
15, 18, 19
16, 19
15, 16, 18
16, 17, 19
17, 19
3.45, m
1.38, m, Ha
1.33, m, Hb
2.06, m, Ha
18, 20a, 20b
19, 20b, 21a, 21b
19, 20a, 21a, 21b
20a, 20b, 21b, 22
20a, 20b, 21a, 22
21a, 21b, 23
22, 24a, 24b
23, 24b, 25
23, 24a, 25
24a, 24b, 26
25
18
17, 20, 21
18, 19, 21, 22
18, 19, 21, 22
19, 20, 22, 23
19, 20, 22, 23
20, 21, 23, 24
21, 22, 24, 25
22, 23, 25, 26
22, 23, 25, 26
23, 24, 26, 4′′
24, 25
36.5, CH2
20b, 21b, 22
20a, 21a, 22
21
28.4, CH2
20b, 21b, 22, 23
20a, 21a, 22, 23
20a, 20b, 21a, 21b, 24a, 24b, 25
21a, 21b, 24a, 24b, 25
22, 23, 24b, 25, 26
22, 23, 24a, 25, 26
22, 23, 24a, 24b, 26, 4′′
24a, 24b, 25
1.97, m, Hb
22
23
24
132.3, CH
126.1, CH
39.0, CH2
5.41, dm (13.9)
5.37, dm (13.9)
2.14, m, Ha
2.01, m, Hb
3.32, m
25
26
1′
74.6, CH
19.2, CH3
170.8, qC
52.2, CH
29.4, CH2
20.4, CH2
173.8, qC
49.1, CH
31.5, CH2
0.99, d (5.9)
2′
3.09, m
1.73, m
2.51, m
3′
(3′), 4′
(2′), 4′
2′, 3′
1′, 3′, 4′
3′
4′
1′′
2′′
3′′
2′, 4′
3′
2, 1′, 2′
1, 2, 3, 2′, 3′
4.23, m
3′′a, 3′′b
3′′a, (3′′b), 4′′, NH-2′′
2′′, 3′′b, 4′′, NH-2′′
(2′′), 3′′a, 4′′, NH-2′′
25, 2′′, 3′′a, 3′′b
1′′, 3′′, 4′′, 1′′′
1′′, 2′′, 4′′
1′′, 2′′, 4′′
25, 2′′, 3′′
1.86, m, Ha
1.69, m, Hb
3.38, m
2′′, 3′′b, 4′′
2′′, 3′′a, 4′′
3′′a, 3′′b
4′′
63.8, CH2
169.2, qC
22.4, CH3
1′′′
2′′′
1.82, s
8.05, d (7.6)
1′′′
2′′, 3′′, 1′′′
NH-2′′
2′′
2′′, 3′′a, 3′′b
a Assignment ambiguous on account of signal overlap. Weak ROESY and HMBC correlations are given in brackets.
γ-lactone. After removal of the solvents, hydroxyadipic acid-γ-
lactone was converted with diazomethane to the methyl ester 6
(Figure 2). This compound was compared by GC-MS with authentic
samples of R-6 and S-6, revealing that 1 possesses the R-
configuration at C-19. Compound S-6 was synthesized from 1-tert-
butyl 6-methyl 3-oxohexanedioate, which was reduced enzymati-
cally with baker’s yeast to (S)-1-tert-butyl 6-methyl 3-hydroxyhexane-
dioate.11,12 Subsequent acid-catalyzed transesterification of this
compound with methanol yielded S-6.
Figure 1. Selected HMBC correlations of mycenaaurin A (1).
The absolute configuration at C-25 of 1 was determined after
degradation of the homoserine ether 4. Treatment of 4 with
methanolic potassium hydroxide yielded 3-hydroxybutyric acid in
traces. Subsequent derivatization of 3-hydroxybutyric acid with
diazomethane yielded methyl 3-hydroxybutanoate (7) (Figure 2).
The GC-MS comparison of the degradation product 7 with authentic
samples of R- and S-7 showed that the degradation product was
(R)-methyl 3-hydroxybutanoate, thus revealing the R-configuration
at C-25 of 1.
The NOE between H-4 (δH 6.00) and H-6 (δH 6.26) established
the orientation of the pyrone moiety to the polyene chain, as shown
in Figure 1.
The absolute configuration of the four stereogenic centers (C-
2′, C-2′′, C-19, and C-25) of mycenaaurin A (1) was determined
after degradation by ozonolysis.7 Subsequent oxidation of the
degradation products with H2O2 yielded glutamic acid (2), 3-hy-
droxyadipic acid (3), and the homoserine ether 4 (Figure 2).
The absolute configuration at C-2′ was determined by conversion
of 2 to the corresponding methyl ester,8 which was reacted with
S-Mosher’s acid chloride (S-MTPA-Cl)9,10 to the amide 5 (Figure
2). The GC-MS comparison of the derivatized degradation product
5 with authentic synthetic samples of known configuration revealed
the S-configuration for C-2′ in 1.
To elucidate the absolute configuration at C-19, the degradation
products of the ozonolysis were treated with 2 N hydrochloric acid,
thus converting 3-hydroxyadipic acid (3) to hydroxyadipic acid-
To determine the absolute configuration of mycenaaurin A (1)
at C-2′′, the homoserine ether 4 present in the mixture of ozonolysis
products of 1 was hydrolyzed to homoserine, which was converted
via its methyl ester to the Mosher amide 8 (Figure 2). GC-MS
comparison of the natural product-derived 8 with synthetic samples
of known configuration revealed the S-configuration at C-2′′ of 1.
The structure of mycenaaurin A (1) is in accordance with its
hypothetical biosynthesis. The core structure consisting of C-1 to

1352 Journal of Natural Products, 2010, Vol. 73, No. 8
Jaeger and Spiteller
Figure 2. Degradation and derivatization of 1 for the determination of the absolute configuration of the stereogenic centers (DMAP:
4-dimethylaminopyridine; S-MTPA-Cl: S-Mosher’s acid chloride; Py: pyridine).
Nucleodur C-18 EC column (5 µm, 21 × 250 mm, Macherey-Nagel)
C-26 is obviously derived from a tridecaketide, starting from C-26/
using the following gradient program: 10 min 99.9% H2O/0.1% AcOH,
then within 30 min linear to 100% MeOH, then 20 min at 100% MeOH;
flow rate: 12 mL/min; detection: UV/vis at 420 nm. UV spectra were
recorded on a Varian Cary 100 Bio UV/vis spectrometer. NMR spectra
were recorded with a Bruker DMX 250 spectrometer (1H at 250.06,
13C at 62.9 MHz), with a Bruker DMX 500 spectrometer equipped
with a TXI probe (1H at 500.11, 13C at 125.8 MHz), and with a 600
MHz Bruker Avance III spectrometer equipped with a TXI cryo probe
(1H at 600.13, 13C at 150.9 MHz). Chemical shifts were determined in
δ (ppm) relative to the solvent CDCl3 (δH 7.26, δC 77.0) or DMSO-d6
(δH 2.49, δC 39.5) as internal standard. GC-MS spectra were recorded
with a Thermo Electron Trace DSQ mass spectrometer coupled with a
Thermo Electron Trace GC Ultra equipped with a PTV injector. For
sample separation, either a fused silica OPTIMA-5-Accent capillary
column (15 m × 0.25 mm, coated with a 0.25 µm layer of liquid phase,
Macherey-Nagel), a Betadex 120 capillary column (30 m × 0.25 mm,
coated with a 0.25 µm layer of liquid phase, Supelco), or a Lipodex E
capillary column (25 m × 0.25 mm, Macherey-Nagel) was used. Helium
served as carrier gas. Injection volumes were 0.2-0.5 µL of a 1-2%
(w/v) solution. N-Methyl-N-trimethylsilyltrifluoroacetamide (MSTFA)
was used for pertrimethylsilylation. Temperature programs: 1 min
isothermal at 50 °C, then 5 °C/min up to 300 °C, finally 10 min
isothermal at 300 °C (OPTIMA-5-Accent); 1 min isothermal at 50 °C,
then 5 °C/min up to 220 °C, finally 15 min isothermal at 220 °C
(Betadex 120); 1 min isothermal at 50 °C, then 5 °C/min up to 200
°C, finally 15 min isothermal at 200 °C (Lipodex E). Retention indices
Ri according to Kova´ts were determined by injection of a 0.2 µL sample
of a standard mixture of saturated straight-chain alkanes (C10-C36).20
HRAPCIMS spectra were obtained with a Thermo Scientific LTQ
Orbitrap mass spectrometer.
C-25. The positions of the double bonds, the OH groups, and the
lactone ring perfectly fit this hypothesis. Both amino acid units are
probably derived from S-adenosylmethionine. Usually, the methyl
group from S-adenosylmethionine is transferred as an electrophile;
however, in the case of the biosynthesis of 1 the amino acid moiety
is apparently transferred to both the nucleophilic OH group at C-25
and to C-2, on account of the acidic nature of H-2. An electrophilic
transfer of the amino acid moiety of S-adenosylmethionine is likely
to occur not only in the case of the biosynthesis of 1 but also in
thehypotheticalbiosynthesesofthesanguinones,2ofthemycenarubins,3,4
and of aleurodisconitrile,13 which is the inactive precursor in the
wound-activated chemical defense14 of the crust fungus Aleuro-
discus amorphus.13 It is also worthy of note that mycenaaurin A
(1) is present only in fruiting bodies of M. aurantiomarginata and
does not occur in the colorless mycelial cultures of this species.
Hence, feeding experiments to prove the proposed biosynthesis
could not be performed with mycelial cultures.
A number of polyene pigments have previously been isolated
from other fungi. For instance, Boletus laetissimus and Boletus rufo-
aureus contain the boletocrocins A-G,15 while the polyene pigment
melanocrocin7 was found in Melanogaster broomeianus. Moreover,
calostomal16 is present in Calostoma cinnabarinum, Laetiporus
sulphureus contains laetiporic acids,17 and Albatrellus confluens
contains aurovertin E.18 However, in comparison to these known
fungal polyenes, the structure of mycenaaurin A (1) is more
complex.
Many polyenes are bioactive. For instance, nystatin and am-
phothericin B are used to kill human-pathogenic fungi.18 The
bioactivity of these compounds is based on the destabilization of
cell membranes, leading to an efflux of ions and small molecules
from the interior of the cells.19 When we tested 1 for antimicrobial
activity at a concentration of 0.5 µmol/disk in the agar diffusion
assay against Bacillus pumilus, there was a growth inhibition zone
of 2.5 cm in diameter. Under the same conditions the known
antibiotic nourseothricine produced an inhibition zone of 3.5 cm
in diameter. Consequently, the ecological role of mycenaaurin A
(1) could be to prevent certain bacteria from growing on the fruiting
bodies of M. aurantiomarginata.
Mushroom Material. Fruiting bodies of M. aurantiomarginata (leg.
et det. R. J. R. Jaeger and P. Spiteller) were collected between
September and December of 2004 to 2009 in coniferous forests near
Frieding, 35 km southwest of Munich. Voucher samples of M.
aurantiomarginata were deposited at the Institut fu¨r Organische Chemie
und Biochemie der Albert-Ludwigs-Universita¨t Freiburg, Germany. The
mushrooms were stored at -35 °C after collection. Mycelial cultures
of M. aurantiomarginata (CBS 494.79, Centraalbureau voor Schim-
melcultures, Amsterdam) were grown on agar plates at 18 °C for several
weeks on a medium consisting of malt extract (30 g), peptone (5 g),
agar (15 g), and H2O (1 L).
Test Organism. Bacillus pumilus (DSM 27, Deutsche Sammlung
von Mikroorganismen und Zellkulturen GmbH, Braunschweig) was
grown on agar plates at 26 °C for 24 h on a medium consisting of
peptone (10 g), NaCl (5 g), agar (15 g), and H2O (1 L).
Extraction and Isolation of 1. Frozen fruiting bodies (10 g) were
crushed after addition of MeOH (10 mL) and extracted with MeOH (2
× 100 mL) at 25 °C. Then, the combined extracts were concentrated
in vacuo at 40 °C. The resulting residue was dissolved in H2O-MeOH
Experimental Section
General Experimental Procedures. Evaporation of the solvents was
performed under reduced pressure using a rotary evaporator. Preparative
HPLC separations were performed using two Waters 590EF pumps
equipped with an automated gradient controller 680 and a Kratos
Spectroflow 783 UV/vis detector. The samples were fractionated on a

Antibacterial Polyene Pigment from Mycena
Journal of Natural Products, 2010, Vol. 73, No. 8 1353
(1:1, 5 mL), prepurified with an RP-18 cartridge, and separated on an
RP-18 column by preparative HPLC (UV/vis detection at 420 nm),
yielding 1 (3.5 mg, tR 42.0 min).
Synthetic R-6: GC-MS (Betadex 120) Ri 1688; m/z (see mass
spectrum of S-6).
Determination of the Absolute Configuration at C-25 of 1:
Degradation of Mycenaaurin A (1) to Methyl (R)-3-Hydroxybu-
tanoate (R-7). Ozone was passed through a solution of 6 mg (9 µmol)
of 1 in 5 mL of MeOH-DMSO (10:1) at -78 °C for 2 min. After
addition of three drops of 30% H2O2 the solution was allowed to warm
to room temperature and the solvents were removed. The residue was
refluxed in 2 mL 1 N methanolic KOH for 8 h; then the solvent was
removed and the residue was dissolved in H2O, acidified with aqueous
HCl, and extracted three times with EtOAc. The organic phase was
dried over Na2SO4, filtered, and concentrated in vacuo. The aqueous
phase was neutralized with aqueous NaOH, and the solvent was
evaporated. Both the organic and the aqueous phases were dissolved
in 1 mL of EtOAc and methylated for 15 min with an ethereal solution
of CH2N2. The solutions were dried over Na2SO4, filtered and
concentrated in vacuo, transferred into GC vials, and analyzed by
enantioselective GC-MS. The product (7) could be found only in traces
in the aqueous phase. Compound 7 consisted mainly of R-7, but some
S-7 was also present, which we attributed to partial racemization of
R-7 during the degradation procedure. GC-MS (Lipodex E) Ri 1237;
m/z (%) 103 (43) [M - CH3]+, 100 (5) [M - H2O]+, 87 (34), 74 (90)
[M - CH3CHO]+, 71 (44), 61 (18), 59 (18) [CO2CH3]+, 45 (48) [M -
CH2CO2CH3]+, 43 (100) [CH3CO]+.
Mycenaaurin A (1): orange solid; UV/vis (MeOH) λmax (log ε) 202
(4.51), 226 (4.55), 264 (4.24), 334 (4.48), 400 (4.95), 417 (5.03), 436
(4.94) nm; 1H NMR (see Table 1); 13C NMR (see Table 1); HRAPCIMS
m/z 669.3384 (calcd for C36H49N2O10 [M + H]+, 669.3382); HRAPCI-
MSMS (parent ion m/z 669, 35 eV) m/z (%) 651.3287 (95) [C36H47N2O9,
M + H - H2O]+, 508.2700 (100) [C30H38NO6, M + H -
HO(CH2)2CH(NHCOCH3)(CO2H)]+, 490.2592 (39) [C30H36NO5]+.
Determination of the Absolute Configuration at C-2′ of 1:
Degradation and Derivatization of 1 to (S)-Dimethyl 2-((R)-3,3,3-
Trifluoro-2-methoxy-2-phenylpropanamido)pentanedioate (2S,2′R-5).
Ozone was passed through a solution of 1.7 mg (2.6 µmol) of
mycenaaurin A (1) in 2 mL of MeOH at -78 °C for 1 min. After
addition of two drops of 30% H2O2 the solution was allowed to warm
to room temperature and the solvents were removed. The residue was
refluxed in 2 mL of MeOH and 0.2 mL of (CH3)3SiCl for 1 h. After
complete removal of the solvent, the residue was dissolved in 0.5 mL
of pyridine. Then, 3 µL (16 µmol) of (S)-3,3,3-trifluoro-2-methoxy-
2-phenylpropanoic acid chloride (S-MTPA-Cl) and catalytic amounts
of 4-dimethylaminopyridine (DMAP) were added, and the solution was
stirred under argon at 37 °C for 1 h. The solvent was removed and the
residue was dissolved in 1 mL of H2O and extracted three times with
1 mL of CHCl3. The organic phase was dried over Na2SO4, filtered,
concentrated under reduced pressure, and transferred into a GC vial.
2S,2′R-5: GC-MS Ri 2190; m/z (%) 328 (4), 202 (26) [M -
C(CF3)(OCH3)(C6H5)]+, 189 (76) [C(CF3)(OCH3)(C6H5)]+, 174 (67)
[M - (CdO)C(CF3)(OCH3)(C6H5)]+, 170 (30), 158 (8), 142 (100) [M
- C(CF3)(OCH3)(C6H5) - CO - CH3OH]+, 139 (13), 127 (14), 119
(19), 114 (20), 110 (29), 105 (45) [C7H5O]+, 98 (14), 91 (24), 82 (21),
77 (33) [C6H5]+, 69 (9) [CF3]+.
Synthetic R-7. Sodium (R)-3-hydroxybutanoate was acidified with
10 N HCl, and the free carboxylic acid was extracted with EtOAc.
The organic phase was dried over Na2SO4, filtered, concentrated in
vacuo, and methylated for 10 min with an ethereal solution of CH2N2.
The solution was dried over Na2SO4, filtered and concentrated in vacuo,
transferred into a GC vial, and analyzed by enantioselective GC-MS.
1H NMR (250 MHz, CDCl3, 300 K) δ 4.17 (m, 1 H, H-3), 3.68 (s, 3
3
H, H-1′), 2.45 (m, 2 H, H-2), 1.20 (d, JHH ) 6.3 Hz, 3 H, H-4); 13C
NMR (63 MHz, CDCl3, 300 K) δ 173.2 (C-1), 64.2 (C-3), 51.7 (C-1′),
42.5 (C-2), 22.4 (C-4); GC-MS (Lipodex E) Ri 1239; m/z (%) 117 (2),
103 (46) [M - CH3]+, 100 (7) [M - H2O]+, 87 (35), 85 (10), 74 (100)
[M - CH3CHO]+, 71 (46), 61 (17), 59 (15) [CO2CH3]+, 45 (47) [M -
CH2CO2CH3]+, 43 (98) [CH3CO]+.
Synthetic S-7: GC-MS (Lipodex E) Ri 1214; m/z (see mass spectrum
of synthetic R-7).
Synthetic 2S,2′R-5: Synthetic 2S,2′R-5 was synthesized from
L-glutamic acid by methylation and reaction with S-MTPA-Cl as
described above: GC-MS Ri 2192; m/z (%) 328 (6), 202 (27) [M -
C(CF3)(OCH3)(C6H5)]+, 189 (44) [C(CF3)(OCH3)(C6H5)]+, 174 (66)
[M - (CdO)C(CF3)(OCH3)(C6H5)]+, 170 (19), 158 (7), 142 (100) [M
- C(CF3)(OCH3)(C6H5) - CO - CH3OH]+, 139 (6), 127 (6), 119 (10),
114 (17), 110 (28), 105 (19) [C7H5O]+, 98 (11), 91 (9), 82 (19), 77 (9)
[C6H5]+, 69 (4) [CF3]+.
Determination of the Absolute Configuration at C-2′′ of 1:
Degradation and Derivatization of 1 to (S)-Methyl 2-((R)-3,3,3-
Trifluoro-2-methoxy-2-phenylpropanamido)-4-hydroxybutanoate
(2S,2′R-8). Ozone was passed through a solution of 1.73 mg (2.59 µmol)
of 1 in 2 mL of MeOH at -78 °C for 1 min. Two drops of 30% H2O2
were added, the solution was allowed to warm to room temperature,
and the solvents were removed. The residue was refluxed in 1 mL of
concentrated HCl for 1 h. After removal of the solvent the residue was
refluxed in 2 mL of MeOH and 0.2 mL of (CH3)3SiCl for 1 h. The
solvent was removed, and the residue was dissolved in 0.5 mL of
pyridine. After addition of 3 µL (16 µmol) of S-MTPA-Cl and catalytic
amounts of DMAP the solution was stirred under argon at 37 °C for
1 h. The solvent was removed, and the residue was dissolved in 1 mL
of H2O and extracted three times with 1 mL of CHCl3. The organic
phase was dried over Na2SO4, filtered, concentrated under reduced
pressure, and transferred into a GC vial. GC-MS Ri 2056; m/z (%) 318
(7) [M - CH3O]+, 189 (84) [C(CF3)(OCH3)(C6H5)]+, 188 (35), 175
(14), 170 (22), 160 (85) [M - C(CF3)(OCH3)(C6H5)]+, 156 (34), 139
(13), 128 (59), 119 (24), 113 (35), 105 (57) [C7H5O]+, 97 (12), 91
(30), 86 (37), 77 (39) [C6H5]+, 69 (20) [CF3]+, 44 (36), 40 (100).
Synthetic 2S,2′S-8. Compound 2S,2′S-8 was synthesized from
L-homoserine by methylation and reaction with R-MTPA-Cl as
described above: GC-MS Ri 2042; m/z (%) 318 (2) [M - CH3O]+,
190 (35), 189 (100) [C(CF3)(OCH3)(C6H5)]+, 188 (25), 175 (19), 170
(80), 160 (59) [M - C(CF3)(OCH3)(C6H5)]+, 156 (20), 139 (18), 128
(77), 119 (28), 105 (68) [C7H5O]+, 100 (19), 91 (36), 86 (27), 77 (48)
[C6H5]+, 69 (13) [CF3]+.
Synthetic 2S,2′S-5. Synthetic 2S,2′S-5 was synthesized from L-
glutamic acid by methylation and reaction with R-MTPA-Cl as
described above. GC-MS Ri 2175; m/z (see mass spectrum of synthetic
2S,2′R-5).
Determination of the Absolute Configuration at C-19 of 1:
Degradation and Derivatization of 1 to Methyl 2-((R)-Tetrahydro-
5-oxofuran-2-yl)acetate (R-6). Ozone was passed through a solution
of 12 mg (18 µmol) of 1 in 5 mL of MeOH at -78 °C for 1 min. Two
drops of 30% H2O2 were added, the solution was allowed to warm to
room temperature, and the solvents were removed. The residue was
methylated for 15 min in an ethereal solution of CH2N2, and the solvent
was evaporated under reduced pressure. The residue was warmed to
50 °C in 2 N HCl for 10 min and after removal of the solvent methylated
again for 15 min in an ethereal solution of CH2N2. The reaction product
was concentrated in vacuo, transferred into a GC vial, and analyzed
by enantioselective GC-MS.
R-6: GC-MS (Betadex 120) Ri 1688; m/z (%) 140 (18) [M - H2O]+,
130 (17) [M - CO]+, 127 (19), 126 (11), 116 (11), 98 (11), 85 (100)
[M - CH2CO2CH3]+, 74 (15), 59 (13) [CO2CH3]+, 57 (13), 56 (16),
55 (19), 43 (20).
Synthetic S-6. Compound S-6 was synthesized from 1-tert-butyl
6-methyl 3-oxohexanedioate, which was reduced enzymatically with
baker’s yeast.11,12 For NMR measurements the reaction product was
purified by flash chromatography [silica gel, hexane-EtOAc (2:1)],
1
yielding an enantiomeric mixture of the lactones R- and S-6 (1:4): H
NMR (500 MHz, CDCl3, 300 K) δ 4.89 (m, 1 H, H-2), 3.71 (s, 3 H,
H-1′′), 2.82 (dd, 3JHH ) 6.6 Hz, 2JHH ) 16.3 Hz, 1 H, Ha-2′), 2.65 (dd,
3JHH ) 6.4 Hz, 2JHH ) 16.3 Hz, 1 H, Hb-2′), 2.57 (m, 2 H, H-4), 2.47
(m, 1 H, Ha-3), 1.97 (m, 1 H, Hb-3); 13C NMR (126 MHz, CDCl3, 300
K) δ 176.4 (C-5), 169.9 (C-1′), 76.2 (C-2), 52.0 (C-1′′), 39.7 (C-2′),
28.4 (C-4), 27.6 (C-3); GC-MS (Betadex 120) Ri 1685; m/z (%) 140
(18) [M - H2O]+, 130 (17) [M - CO]+, 127 (18), 126 (10), 116 (10),
108 (8), 98 (9), 85 (100) [M - CH2CO2CH3]+, 74 (13), 59 (11)
[CO2CH3]+, 56 (13), 55 (18), 54 (7), 43 (13).
Synthetic 2S,2′R-8. Compound 2S,2′R-8 was synthesized from
L-homoserine by methylation and reaction with S-MTPA-Cl as de-
scribed above: GC-MS Ri 2055; m/z (%) (see mass spectrum of 2S,2′S-
8).
Biological Tests. For agar diffusion assays, 1 (0.5 µmol) was
dissolved in MeOH (5-25 µL) and dropped onto paper discs (diameter
5 mm, thickness 0.5 mm). These discs were dried under sterile
conditions and placed on agar plates inoculated with the test organism

1354 Journal of Natural Products, 2010, Vol. 73, No. 8
Jaeger and Spiteller
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Bacillus pumilus. The plates were incubated at 26 °C for 24 h.
Nourseothricin (0.5 µmol) was used as a positive control and mannitol
(0.5 µmol) as a negative control.
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Acknowledgment. We are grateful to Prof. Dr. M. Spiteller and
Dr. M. Lamsho¨ft (Technische Universita¨t Dortmund, Institut fu¨r
Umweltforschung, Germany) for the measurement of HRAPCIMS
spectra and to S. Serdjukow for experimental assistance. Our work has
been supported by an Emmy Noether Fellowship for young investigators
of the Deutsche Forschungsgemeinschaft (SP 718/1-3) and by the Fonds
der Chemischen Industrie.
Supporting Information Available: Selected UV/vis and NMR
spectra of 1. This material is available free of charge via the Internet
at http://pubs.acs.org.
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(3) Peters, S.; Jaeger, R. J. R.; Spiteller, P. Eur. J. Org. Chem. 2008,
319–323.
(4) Peters, S.; Spiteller, P. Eur. J. Org. Chem. 2007, 1571–1576.
(5) Antunes, E. M.; Copp, B. R.; Davies-Coleman, M. T.; Samaai, T.
Nat. Prod. Rep. 2005, 22, 62–72.
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