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Birnbaumin A and B Two Unusual 1-Hydroxyindole Pigments from the УFlower Pot ParasolФ Leucocoprinus birnbaumii.

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Angewandte
Chemie
Structure Elucidation
Birnbaumin A and B: Two Unusual
1-Hydroxyindole Pigments from the “Flower Pot
Parasol” Leucocoprinus birnbaumii**
Andrea Bartsch, Monika Bross, Peter Spiteller,
Michael Spiteller, and Wolfgang Steglich*
Dedicated to Professor Timm Anke
on the occasion of his 60th birthday
Occasionally the Yellow Parasol or Flower Pot Parasol
(Leucocoprinus birnbaumii (Corda) Singer) appears in flowerpots and greenhouses, where it attracts attention because of
its intense yellow color and delicate shape. It originated in the
tropics and was distributed worldwide with plants and potting
soil.[1] Until now, the chemical constituents of the fungus have
remained unknown. Herein, we report the unusual structures
of its yellow pigments, which we have named birnbaumin A
and B.
To isolate the pigments, the fruit bodies were extracted
carefully with methanol. Subsequent separation of the extract
by preparative reversed-phase HPLC yielded birnbaumins A
and B, together with l-tryptophan. Although the two pigments have similar UV/Vis spectra, the major compound,
birnbaumin B, exhibits a bathochromic shift of the absorption
maximum from 322 (for birnbaumin A) to 356 nm (both in
MeOH). Birnbaumin A shows a [M+H]+ peak in the ESI
mass spectrum at m/z 361. High-resolution measurements
revealed the molecular formula C16H20N6O4. According to
ESI MS, birnbaumin B differs from birnbaumin A by an
additional oxygen atom.[2] ESI MS/MS spectra of the pigments display a fragmentation of the molecules into two
characteristic parts. A fragment with a peak at m/z 200
(C7H14N5O2) is common to both compounds, whereas the
other fragment shows a peak at m/z 160 (C9H6NO2) in the
spectrum of birnbaumin A and at m/z 176 (C9H6NO3) in that
of birnbaumin B. The high content of Na+ and K+ ions
detected by atom absorption spectroscopy of the samples
[*] Dr. A. Bartsch, Dr. M. Bross, Dr. P. Spiteller, Prof. Dr. W. Steglich
Department Chemie
Ludwig-Maximilians-Universtt Mnchen
Butenandtstrasse 5–13, Haus F, 81377 Mnchen (Germany)
Fax: (+ 49) 89-2180-77756
E-mail: wos@cup.uni-muenchen.de
Prof. Dr. M. Spiteller
Institut fr Umweltforschung
Universitt Dortmund
Otto-Hahn-Strasse 6, 44227 Dortmund (Germany)
[**] This work was supported financially by the Deutsche Forschungsgemeinschaft (SFB 369). We are indebted to Till Hgele (Botanischer Garten Mnchen), Claudia Dubler, Tina Hbscher, Dr. Franz
von Nussbaum, and many others for helping to collect the fungi and
to Dr. Andrew J. Hall for linguistic help.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2005, 44, 2957 –2959
suggests that the birnbaumins have the ability to complex
metal ions.
Further insight into the structure of the birnbaumins was
provided by their NMR spectra. The 1H NMR spectrum
([D6]DMSO; DMSO = dimethyl sulfoxide) of birnbaumin A
shows signals corresponding to a 1,2-substituted benzene ring,
a single hydrogen atom on an aromatic ring (at dH =
7.98 ppm), and a NHCH2CH2CH2CH2NH unit. Additionally, at low field there is a sharp singlet at dH = 11.95 ppm and
a broad signal at dH = 12.32 ppm, which we assign to OH
protons. The 13C NMR spectrum contains 16 signals, which
correspond to four methylene and five aromatic methine
groups, as well as seven quaternary carbon atoms (signals at
dC = 109.8, 121.4, 134.1, 151.4, 156.7, 161.6, and 183.8 ppm).
The HMBC spectra of birnbaumin A enabled the deduction
of the partial structure shown in Scheme 1, whereby an
Scheme 1. Partial structures of birnbaumins A (R = H) and B (R = OH)
with the key ions observed in the ESI MS/MS spectra.
a fragmentation between the two carbonyl groups would
explain the generation of the key ions with m/z 160 and 200 in
the mass spectra. According to the NMR spectra and HMBC
experiments, birnbaumin B contains an additional hydroxy
group on the indole ring system at the 7-position, which is in
agreement with the production of the fragment ions with
m/z 176 and 200.
To confirm the substitution pattern, birnbaumin B was
reduced with zinc in glacial acetic acid. The reductive loss of
the N-hydroxy group causes a large shift of the 2-H signal in
the 1H NMR spectrum ([D6]DMSO) from dH = 7.73 to
8.59 ppm; a coupling of 2.9 Hz with the adjacent NH hydrogen atom of the indole (dH = 12.40 ppm) also appears. Moreover, the unknown end group, C2H4N3O, loses its oxygen
atom in the course of the reduction and is transformed by
addition of two hydrogen atoms into C2H6N3. This transformation excludes the possibility of an amidinourea[3] or
carbamoylguanidine end group. The excellent agreement of
the aromatic signals of the reduction product with those of the
synthetic comparison compound 1 confirms the location of
the hydroxy group at the 7-position in birnbaumin B
(Scheme 2).[4]
To elucidate the structure of the end group, the birnbaumins A and B were permethylated at 0 8C with diazomethane/
ether in methanol to yield a dimethyl ether and a trimethyl
ether, respectively. The reaction involved methylation of the
hydroxy group on the nitrogen atom of the indole (dH = 12.32
and 12.06 ppm, respectively) and the hydrogen-bonded OH
group (dH = 11.95 and 11.89 ppm, respectively), as well as of
the phenol functionality in birnbaumin B. The O-methyl
groups could be assigned by analysis of the NOESY spectra;
DOI: 10.1002/anie.200500082
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2957
Communications
lation of the OH hydrogen atom to the amidoxime carbon
atom. Furthermore, the 1H,15N HMBC correlations for
birnbaumin B are in agreement with the proposed structures
(see Supporting Information).
We propose the aminal structure 5 for the reduction
product, which was obtained in the form of its trifluoroacetate
salt. Signals in its NMR spectra at dH = 5.27 and dC = 58.0 ppm
indicate the presence of a methine group. The aminal group is
apparently stabilized by a hydrogen bond to the adjacent
amidinium functionality.
Scheme 2. Comparison of the NMR spectroscopic data (dH (above)
and dC values (below, when two values are given) of reduced
birnbaumin B (top) with those of the synthetic reference compound 1
(spectra recorded in [D6]DMSO).[4]
this analysis also confirms the substitution pattern of the
birnbaumins (Scheme 3). The correlation in the HMBC
spectrum of the signal at dH = 3.88 ppm for the methoxy
The birnbaumins are characterized by several unusual
structural features. For example, to the best of our knowledge,
simple N-hydroxyoxamidines were unknown until now, and 1hydroxyindoles,[5] in contrast to their more stable O-methyl
derivatives,[6] occur only rarely in nature. Similarly, only a few
natural 7-hydroxyindoles have been described to date,
including the coscinamides, isolated from marine sponges.[7]
These indolylglyoxylic acid amides are structurally similar to
birnbaumins A and B. We propose a pathway for the
biosynthesis of the birnbaumins which starts from l-tryptophan, citrulline, glycine, and nitrite (see Supporting Information). Experiments to verify this hypothesis are underway.
Scheme 3. Important NOE and HMBC correlations for birnbaumin B
trimethyl ether (2; spectra recorded in [D6]DMSO).
group positioned at the end of the side chain with the signal
for an unsaturated carbon atom at dC = 151.5 ppm is especially important, since it is consistent only with structure 2
(Scheme 3). In accordance with this deduction, the signal at
dH = 3.10 ppm for one of the terminal methylene groups
shows a correlation with the carbon-atom signal at dC =
156.9 ppm. Analogous correlations are also observed in the
spectra of birnbaumin A dimethyl ether, thus allowing the
assignment of structures 3 and 4 to birnbaumins A and B. The
hydrogen bond in the terminal N-hydroxyamidine residue
explains unambiguously the characteristic sharp singlet in the
1
H NMR spectra of the birnbaumins and the HMBC corre-
2958
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Experimental Section
Isolation of the pigments: The fresh, deep-frozen, or lyophilized fungi
were extracted on a shaker with nitrogen-saturated MeOH under the
exclusion of light until complete decolorization was observed. The
solvent was then evaporated under vacuum. The residue was
dissolved in MeOH (HPLC-grade), prepurified on an RP-18 cartridge, and then separated by preparative HPLC (column: nucleosil
100 C-18, 7 mm, 16 250 mm (Macherey & Nagel); solvent A:
CH3CN/H2O (1:9)+0.25 % trifluoroacetic acid (TFA), solvent B:
CH3CN; gradient: 100 % A linear in 35 min to 50 % A/50 % B; flow
rate: 5.00 mL min 1; detection: UV). The purity of the compounds
was checked by analytical HPLC (column: nucleosil 100 C-18, 5 mm,
4 250 mm (Knauer); solvent: as above; gradient: 100 % A linear in
25 min to 50 % A/50 % B; flow rate: 1.0 mL min 1). Retention times:
tR(4) = 11.2 min; tR(l-tryptophan) = 12.3 min; tR(3) = 15.5 min. The
pigments were stored under a nitrogen atmosphere at 20 8C. From a
42-g batch of fresh fruit bodies, 59 mg of birnbaumin A (0.14 %),
228 mg of birnbaumin B (0.54 %), and 58 mg of l-tryptophan
(0.14 %) were isolated. Yields can differ depending on the age and
the condition of the fungi.
3: yellow pigment; TLC: Rf = 0.84 (RP-18, MeOH+2 drops of
TFA); UV/Vis (MeOH): lmax (lg e) = 212 (4.42), 250 (4.03), 322 nm
(3.96); IR (KBr): ñ = 3403 s, 3205 m, 2945 m, 2863 m, 1665 s, 1625 m,
1575 m, 1537 m, 1508 m, 1483 m, 1452 m, 1438 m, 1371 m, 1340 m,
1324 m, 1250 m, 1203 s, 1139 m, 1066 m, 1009 m, 890 m, 842 m, 801 m,
751 m, 722 m, 674 m, 600 m, 426 cm 1 m; 1H NMR (600 MHz,
CD3OD, reference: d = 3.35 ppm, 25 8C): d = 1.68 (br m, 4 H, 12-H,
www.angewandte.org
Angew. Chem. Int. Ed. 2005, 44, 2957 –2959
Angewandte
Chemie
13-H), 3.26 (t, J = 6.1 Hz, 2 H, 11-H), 3.39 (t, J = 6.1 Hz, 14-H,
partially hidden by MeOH signal), 7.34 (dd, J = 7.8, 7.5 Hz, 1 H, 5-H),
7.40 (dd, J = 8.0, 7.5 Hz, 1 H, 6-H), 7.56 (d, J = 8.0 Hz, 1 H, 7-H), 7.92
(s, 1 H, 2-H), 8.25 ppm (d, J = 7.8 Hz, 1 H, 4-H); 1H NMR (600 MHz,
[D6]DMSO, reference: d = 2.49 ppm, 25 8C): d = 1.48 (br m, 4 H, 12-H,
13-H), 3.10 (pseudo q, J = 5.6 Hz, 2 H, 14-H), 3.17 (pseudo q, J =
5.6 Hz, 2 H, 11-H), 7.27 (dd, J = 7.5, 7.4 Hz, 1 H, 5-H), 7.33 (dd, J =
7.8, 7.4 Hz, 1 H, 6-H), 7.52 (d, J = 7.8 Hz, 1 H, 7-H), 7.57 (br s, 1 H,
N15-H), 7.99 (s, 1 H, 2-H), 8.10 (d, J = 7.5 Hz, 1 H, 4-H), 8.28 (br t, J =
5.6 Hz, 1 H, N10-H), 11.95 (s, 1 H, 17-NOH), 12.32 ppm (br s, 1 H, 1OH); 13C NMR (151 MHz, CD3OD, reference: d = 49.0 ppm, 25 8C):
d = 27.1 (C12), 27.7 (C13), 39.5 (C11), 42.1 (C14), 110.4 (C7), 111.8
(C3), 122.5 (C4), 123.5 (C3a), 124.5 (C5), 125.1 (C6), 135.6 (C2), 136.2
(C7a), 152.2 (C17), 158.6 (C16), 164.3 (C9), 185.9 ppm (C8);
13
C NMR (151 MHz, [D6]DMSO, reference: d = 39.5 ppm, 25 8C):
d = 26.0 (C12), 26.2 (C13), 38.1 (C11), 40.4 (C14), 109.6 (C7), 109.8
(C3), 120.8 (C4), 121.4 (C3a), 122.9 (C5), 123.6 (C6), 133.6 (C2), 134.1
(C7a), 151.4 (C17), 156.7 (C16), 161.6 (C9), 183.8 ppm (C8); MS
(FAB+): m/z (%): 361 (100) [M+H]+; HRMS (FAB+): m/z: 361.1576
[M + H]+, calcd for C16H21N6O4 : 361.1624; HR MS/MS (ESI+,
30 eV): m/z: 361, 200 [C7H14N5O2]+, 184 [C7H14N5O]+, 160
[C9H6NO2]+, 157 [C6H13N4O]+.
4: yellow pigment; TLC: Rf = 0.88 (RP-18, MeOH+2 drops of
TFA), Rf = 0.25 (silica gel 60 F254, nBuOH/H2O/EtOH/AcOH
4:1:1:1); UV/Vis (MeOH): lmax (lg e) = 214 (4.74), 248 (4.34), 267
(sh, 4.19), 356 nm (4.16); IR (KBr): ñ = 3415 m, 3210 m, 2930 m,
2856 m, 1666 s, 1628 m, 1538 m, 1493 m, 1436 m, 1374 m, 1332 m,
1279 m, 1144 m, 1086 w, 1045 w, 986 w, 865 m, 837 m, 799 m, 723 m,
673 cm 1 m; 1H NMR (600 MHz, CD3OD, reference: d = 3.35 ppm,
25 8C): d = 1.66 (br m, 4 H, 12-H, 13-H), 3.23 (br t, J = 6.5 Hz, 2 H, 11H), 3.37 (t, J = 6.6 Hz, 14-H, (partly hidden by MeOH signal), 6.73 (d,
J = 7.9 Hz, 1 H, 6-H), 7.10 (dd, J = 7.9, 7.9 Hz, 1 H, 5-H), 7.74 (d, J =
7.9 Hz, 1 H, 4-H), 7.78 ppm (s, 1 H, 2-H); 1H NMR (600 MHz,
[D6]DMSO, reference: d = 2.49 ppm, 25 8C): d = 1.47 (br m, 4 H, 12H, 13-H), 3.10 (pseudo q, J = 5.7 Hz, 2 H, 14-H), 3.16 (pseudo q, J =
6.2 Hz, 2 H, 11-H), 6.67 (d, J = 7.5 Hz, 1 H, 6-H), 7.01 (dd, J = 7.8,
7.5 Hz, 1 H, 5-H), 7.53 (br t, J = 5.7 Hz, 1 H, N15-H), 7.55 (d, J =
7.8 Hz, 1 H, 4-H), 7.73 (s, 1 H, 2-H), 8.25 (t, J = 6.2 Hz, 1 H, N10-H),
9.83 (s, 1 H, 7-OH), 11.89 (s, 1 H, 17-NOH), 12.06 ppm (s, 1 H, 1-OH);
13
C NMR (151 MHz, CD3OD, reference: d = 49.0 ppm, 25 8C): d =
27.0 (C12), 27.6 (C13), 39.5 (C11), 42.0 (C14), 110.6 (C6), 111.6 (C3),
113.8 (C4), 125.2 (C7a), 125.3 (C5), 126.2 (C3a), 135.7 (C2), 145.2
(C7), 152.1 (C17), 158.5 (C16), 164.2 (C9), 185.6 ppm (C8); 13C NMR
(151 MHz, [D6]DMSO, reference: d = 39.5 ppm, 25 8C): d = 26.2
(C12), 26.4 (C13), 38.3 (C11), 40.6 (C14), 109.3 (C6), 109.8 (C3),
111.8 (C4), 123.7 (C7a), 124.0 (C5), 124.5 (C3a), 134.1 (C2), 144.3
(C7), 151.8 (C17), 156.9 (C16), 161.9 (C9), 183.7 ppm (C8); 15N NMR
(60.8 MHz, [D6]DMSO, reference: benzamide, dN = 73.6): d = 47.0
(C16=NH), 55.1 (N15), 83.8 (N10), 161.0 (N1), 344.5 ppm (C17=N
OH); MS (ESI+): m/z (%): 377 (100) [M+H]+; HRMS (ESI+): m/z:
377.1594 [M+H]+, calcd for C16H21N6O5 : 377.1573; HR MS/MS
(ESI+,
30 eV): m/z: 359.1464 [C16H19N6O4]+, 200.1140
[C7H14N5O2]+, 184.1190 [C7H14N5O]+, 176.0339 [C9H6NO3]+,
157.1081 [C6H13N4O]+.
[3] The amidinourea end group is present in different metabolites
derived from red algae: a) gigartinine: K. Ito, Y. Hashimoto,
Nature 1966, 211, 417; M. V. Laycock, J. S. Craigie, Can. J.
Biochem. 1977, 55, 27 – 30; b) gongrine: K. Ito, Y. Hashimoto,
Agric. Biol. Chem. 1965, 29, 832 – 835; c) nicaeensin: R. Chillemi,
R. Morrone, A. Patti, M. Piattelli, S. Sciuto, J. Nat. Prod. 1990, 53,
1220 – 1224.
[4] In Scheme 2, the compounds are shown in their nonprotonated
forms. They are present, however, in the form of their trifluoroacetate salts after HPLC purification.
[5] a) For arcyroxepin A, see: W. Steglich, B. Steffan, T. Eizenhfer,
B. Fugmann, R. Herrmann, J. D. Klamann, Ciba Found. Symp.
1990, 154, 56 – 65; b) for nocathiacins, see: J. E. Leet, W. Li, H. A.
Ax, J. A. Matson, S. Huang, R. Huang, J. L. Cantone, D. Drexler,
R. A. Dalterio, K. S. Lam, J. Antibiot. 2003, 56, 232 – 242; T.
Sasaki, T. Otani, H. Matsumoto, N. Unemi, M. Hamada, T.
Takeuchi, M. Hori, J. Antibiot. 1998, 51, 715 – 721.
[6] M. Somei, Adv. Heterocycl. Chem. 2002, 82, 101 – 155.
[7] H. R. Bokesch, L. K. Pannell, T. C. McKee, M. R. Boyd, Tetrahedron Lett. 2000, 41, 6305 – 6308; see also: N. Lindquist, W. Fenical,
Tetrahedron Lett. 1990, 31, 2521 – 2524.
Received: January 10, 2005
Published online: April 7, 2005
.
Keywords: alkaloids · dyes/pigments · natural products ·
N-hydroxyoxamidines · structure elucidation
[1] R. Watling, Fungi, The Natural History Museum, London, 2004.
[2] In the ESI MS/MS spectra concomitant ions were observed in
very low intensity and were assigned to the corresponding Ninddeoxybirnbaumins.
Angew. Chem. Int. Ed. 2005, 44, 2957 –2959
www.angewandte.org
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2959
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