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Gutingimycin A Highly Complex Metabolite from a Marine Streptomycete.

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Marine Natural Products
Gutingimycin: A Highly Complex Metabolite
from a Marine Streptomycete**
Rajendra P. Maskey, Madhumati Sevvana, Isabel Usn,
Elisabeth Helmke, and Hartmut Laatsch*
In our search for novel bioactive secondary metabolites of
marine streptomycetes, the ethyl acetate extract of the isolate
B8652 drew our attention by its high antibiotic activity against
Staphylococcus aureus, Escherichia coli, Bacillus subtilis, and
Streptomyces viridochromogenes (T 57). The new 2,3-dihydro-1,4-anthraquinone parimycin[1] and the yellow trioxacarcines A–F,[2] which exhibit intensive green fluorescence in
solution, were mainly responsible for this activity.
For the isolation of a further, highly polar trioxacarcin
derivative, the culture filtrate had to be concentrated and
lyophilized, and the residue was further extracted with
methanol. In this way, a 50-L fermentation yielded 120 mg
of a novel natural product with the trioxacarcin skeleton,
however, with higher complexity, which we have named
gutingimycin (1 c), after Gutingi, the old name of G3ttingen.
for this group of compounds. The (+)- and ()-ESI mass
spectra exhibit signals at m/z = 1050 ([M+Na]+) and 1028
([M+H]+), and at m/z = 1063 ([M+Cl]) and 1026 ([MH]),
respectively, which indicate a molecular weight of 1027. Highresolution mass spectrometry suggest the molecular formula
to be either C49H57NO23 or C47H57N5O21. The 13C NMR, H,H
COSY, HMQC, and HMBC spectra as well as the coupling
constants in the 1H NMR spectrum confirm that the sugar
residues I and II are the same those as in trioxacarcin B (1 b)
(Figure 1). The aglycon (Figure 2) is identical to that of 1 b as
Figure 1. HMBC couplings (H!C) of the sugar residues in gutingimycin (1 c).
Figure 2. HMBC couplings (H!C) in the aglycon of gutingimycin
(1 c).
Like the other trioxacarcines, gutingimycin (1 c) is yellow
in substance, fluoresces green in solution upon irradiation
with 366-nm light, and delivers an 1H NMR spectrum typical
[*] Dr. R. P. Maskey, Prof. Dr. H. Laatsch
Institut f%r Organische und Biomolekulare Chemie
Georg-August-Universit+t G,ttingen
Tammannstrasse 2, 37077 G,ttingen (Germany)
Fax: (+ 49) 551-399-660
M. Sevvana, Dr. I. Us>n
Institut f%r Anorganische Chemie
Georg-August-Universit+t G,ttingen (Germany)
Dr. E. Helmke
Alfred-Wegener-Institut f%r Polar- und Meeresforschung
Bremerhaven (Germany)
[**] Marine Bacteria, Part 26. This work was supported by the
Bundesministerium f%r Bildung und Forschung (BMBF 03F0346A).
R.P.M. thanks the Deutschen Akademischen Austauschdienst
(DAAD) for a Ph.D. scholarship. Part 25: R. P. Maskey, E. Helmke,
H. Laatsch, J. Antibiot. 2003, 56, 942–949.
Angew. Chem. Int. Ed. 2004, 43, 1281 –1281
well, except for the residue at C17. The HMBC couplings of
the anomeric protons at d = 5.74 (1’’’H), 5.52 (1’’H), and
5.36 ppm (4H) with the carbon signals at d = 103.3 (C13), 72.2
(C4), and 100.8 ppm (C1’’), respectively, allow the connection
of the sugars with the aglycon, yielding the partial structure 1 c
with the mass 877 (C42H53O20) and a still-unknown residue R3.
This initial assignment was further confirmed by comparison
of the 13C NMR data with those of trioxacarcin B (1 b).
In the 1D and 2D NMR spectra of gutingimycin,
altogether nine acidic protons were detected. Three of them
can be assigned unambiguously to the aglycon and two to the
sugars. The third OH group of the sugar I showed couplings
neither in the H,H COSY nor in the HMBC spectrum. From
the remaining signals at d = 8.86, 7.55, 6.64, and 6.10 ppm,
three must belong therefore to the residue R3 in 1 c.
In the 1H NMR spectrum a CH singlet at d = 8.23 ppm is
observed, which is not present in the spectrum of 1 b.
According to the 13C NMR spectrum, the heteroatom-con-
DOI: 10.1002/anie.200352312
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
nected C atoms with signals at d = 157.7, 153.4, and
151.7 ppm, a CH group at d = 140.4 ppm, and a C atom at
d = 108.3 ppm must be attributed to the partial structure R3.
In the HMBC spectrum, the aromatic proton signal at d =
8.23 ppm couples to the carbon signals of the residue R3 at d =
157.7, 153.4, and 108.3 ppm.
Formally, gutingimycin (1 c) is formed from trioxacarcin A
(1 a) by the attack of a nucleophile, which the mass difference
indicates to have a molecular weight of 151. Among the
compounds isolated from microorganisms with this mass and
the same number of C atoms and acidic protons, only guanine,
isoguanine, and akalone are plausible candidates.[3] As
HMBC correlations connecting the residue R3 with the
trioxacarcin skeleton or determining its connectivity were
missing, further confirmation of the structure was not
The remaining questions were finally solved by a crystal
structure analysis: Gutingimycin (1 c) crystallizes in the
monoclinic space group P21 with one molecule of the
metabolite and four molecules of water in the asymmetric
unit[4] (Figure 3). It consists of the trioxacarcin A substructure
Figure 3. X-ray crystal structure of gutingimycin (1 c) as a stereoplot.
(1 a), whose epoxide ring has been opened, however, through
the nucleophilic attack of N7 of the guanine base at atom C17.
The guanine substructure is oriented nearly parallel to the
planar condensed ring system of 1 c with a distance of
approximately 3.2 A between the two planes. This conformation is stabilized by an intramolecular hydrogen bond
between 2’’-OH and the hydrogen atom at N6’. Based on
the previously reported absolute configuration of the sugars
l-trioxacarcinose A and B,[5] we assigned the absolute configuration of the trioxacarcines as well as that of 1 c.
A compound structurally related to gutingimycin (1 c) was
obtained by Saito et al.[6] from the reaction of kapurimycin A3
(2) with DNA as well as with synthetic oligonucleotides
(Scheme 1). At 0 8C the ionic intermediate 3 was produced,
which on heating at 90 8C decomposed to form 4. The latter
had not been obtained previously as a natural product.
Trioxacarcin A (1 a) forms a highly polar, stable complex with
DNA in the same manner, from which at 100 8C 1 c is easily
liberated. It seems plausible, therefore, that gutingimycin (1 c)
is not formed from smaller precursors, but by the attack of 1 a
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Scheme 1. In the reaction of kapurimycin A3 (2) with DNA as well as
with synthetic oligonucleotides the ionic intermediate 3 is produced,
which on heating at 90 8C decomposes to form 4, a compound related
to gutingimycin.
on the cellular DNA in analogy to the attack of 2. In this way
alkylation at N7 occurs, the aminal bond of the nucleotide
breaks, and with strand cleavage, the extremely water-soluble
1 c is formed.
According to the structure, gutingimycin
(1 c) should not exhibit alkylating properties,
which coincides with the fact that 1 c (IC70 =
3.4 mg mL1) is much less cytotoxic than 1 a
(IC70 = < 0.3 mg mL1). The MIC values of
trioxacarcin A (1 a) for Bacillus subtilis, Streptomyces viridochromogenes (T 57), Staphylococcus aureus, and Escherichia coli are in the
range of 0.15–2.5 mg mL1 in comparison to
gutingimycin (1 c) which has > 20 mg mL1.
The highly cytotoxic actinomycin D intercalates in a similar way into the DNA at
neighboring guanine–cytosine base pairs,[7]
however, does not lead to strand breakage.
The numerous alkylating agents used in the
cancer therapy, like the N- or S-lost derivatives,
also attack the guanine residues preferably,
although without intercalating. It is now of great interest to
determine which intermediates in this reaction may occur and
if structurally related antibiotics, for example, the indomycinones, react in a similar manner. Details of the interaction of
1 a and 1 c with DNA are currently under investigation.
Experimental Section
Materials, methods, and test procedures have been described
previously.[8] The marine streptomycete isolate B8652 was incubated
in a 50-L fermenter under our standard conditions[1] with malt extract/
yeast extract/glucose for 3 d at 28 8C and extracted with ethyl acetate
(extract A). The aqueous phase was concentrated under vacuum
(40 8C water-bath temperature) to approximately 3 L and lyophilized.
Extraction of the residue with methanol gave extract B.
Preseparation of extract A on silica gel gave a polar, yellow,
gutingimycin-containing fraction with green fluorescence under UV
light. Further separation of the fraction by preparative thin-layer
chromatography (4 plates 20 F 20 cm, CHCl3/15 % MeOH/0.1 %
AcOH) delivered a crude product with Rf = 0.21 (CHCl3/10 %
MeOH) and intensive green fluorescence at 366 nm, which yielded
Angew. Chem. Int. Ed. 2004, 43, 1281 –1281
against F2 by least squares. All non-hydrogen atoms were refined
anisotropically. The hydrogen atoms were placed in geometrically
ideal positions and refined with a riding model, in which the
methyl groups can rotate on their local axes. CCDC-213711
contains the supplementary crystallographic data for this paper.
These data can be obtained free of charge via (or from the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK;
fax: (+ 44) 1223-336-033; or
K. Shirahata, T. Iida, N. Hirayama, Tennen Yuki Kagobutsu
Toronkai Koen Yoshishu 1981, 24, 199 – 206.
a) K. L. Chan, H. Sugiyama, I. Saito, M. Hara, Phytochemistry
1995, 40, 1373 – 1374; b) K. L. Chan, H. Sugiyama, I. Saito,
Tetrahedron Lett. 1991, 32, 7719 – 7722.
U. Hollstein, Chem. Rev. 1974, 74, 625 – 652.
R. P. Maskey, R. N. Asolkar, E. Kapaun, I. Wagner-D3bler, H.
Laatsch, J. Antibiot. 2002, 55, 643 – 649.
I. UsPn, G. M. Sheldrik, Curr. Opin. Struct. Biol. 1999, 9, 643 – 648.
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
60 mg yellow gutingimycin (1 c) after twofold chromatography on
Sephadex LH-20 (4 F 100 cm, CHCl3/40 % MeOH). Purification of
extract B in the similar way delivered an additional 60 mg of 1 c.
H NMR (CDCl3, 500 MHz): d = 13.88 (s, H/D exchangeable, 1 H,
9-OH), 8.86 (br s, H/D exchangeable, 1 H, NH), 8.23 (s, 1 H, 8’H), 7.55
(br s, H/D exchangeable, 1 H, NH), 7.51 (s, 1 H, 5H), 6.64 (br s, H/D
exchangeable, 1 H, NH), 6.52 (br s, H/D exchangeable, 1 H, 2OH),
6.10 (br s, H/D exchangeable, 1 H, OH), 5.74 (d, 3J = 3.2 Hz, 1 H,
1’’’H), 5.52 (d, 3J = 4.2 Hz, 1 H, 1’’H), 5.42 (s, H/D exchangeable, 1 H,
16OH), 5.36 (t, 3J = 2.4 Hz, 1 H, 4H), 5.28 (d, 3J = 4.0 Hz, 1 H, 12H),
5.13 (1 H, 4’’H), 5.12 (1 H, 11H), 5.11 (s, 1 H, 16H), 5.05 (d, 2J =
15.1 Hz, 1 H, 17HA), 5.02 (q, 3J = 6.3 Hz, 1 H, 5’’’H), 4.93 (q, 3J =
6.6 Hz, 1 H, 5’’H), 4.77 (t, 3J = 3.2 Hz, 1 H, 2H), 4.73 (d, 3J = 4.1 Hz,
H/D exchangeable, 1 H, 3’’OH), 4.37 (d, 2J = 15.9 Hz, 1 H, 17HB), 4.01
(s, H/D exchangeable, 1 H, 4’’OH), 3.97 (s, 3 H, 10OCH3), 3.75 (m,
1 H, 3’’’H), 3.67 (s, 3 H, 16OCH3), 3.55 (s, 3 H, 16OCH3), 2.99 (td, 3J =
3.9 Hz, 2J = 13.3 Hz, 1 H, 3HA), 2.64 (s, 3 H, 6CH3), 2.51 (s, 3 H,
4’’’COCH3), 2.30 (td, 3J = 3.4 Hz, 2J = 14.2 Hz, 1 H, 2’’’HA), 2.24 (s, 3 H,
4’’OCOCH3), 2.23 (m, 1 H, 2’’HA), 2.00 (td, 2J = 13.2 Hz, 3J = 13.2,
2.7 Hz, 1 H, 3HB), 1.85 (d, 2J = 14.8 Hz, 1 H, 2’’HB), 1.78 (d, 2J =
13.6 Hz, 1 H, 2’’’HB), 1.25 (d, 3J = 6.7 Hz, 3 H, 5’’CH3), 1.18 (s, 3 H,
3’’CH3), 1.08 ppm (d, 3J = 6.6 Hz, 3 H, 5’’’CH3); 13C NMR (CDCl3,
125.7 MHz): d = 210.9 (4’’’COCH3), 207.6 (C1), 173.2 (4’’OCOCH3),
162.9 (C9), 157.7 (C6’), 153.0 (C4’), 152.7 (C8), 151.7 (C2’), 144.6
(C10), 142.4 (C6), 140.4 (C8’), 135.7 (C10a), 128.4 (C4a), 116.8 (C5),
114.5 (C8a), 113.1 (C7), 108.8 (C9a), 108.3 (C5’), 108.2 (C15), 103.3
(C13), 100.8 (C1’’), 99.5 (C16), 92.9 (C1’’’), 84.4 (C14), 78.5 (C4’’’),
75.8 (C4’’), 72.2 (C4), 71.6 (C12), 69.5 (C3’’’), 69.0 (C11), 68.2 (C2),
67.2 (C3’’), 63.7 (C5’’’), 62.7 (10OCH3), 62.2 (C5’’), 55.4 (16OCH3),
56.2 (16OCH3), 46.2 (C17), 37.8 (C2’’), 37.0 (C3), 32.9 (C2’’’), 27.8
(4’’’COCH3), 27.2 (3’’CH3), 21.2 (4’’OCOCH3), 20.2 (6CH3), 16.8
(5’’CH3), 14.6 ppm (5’’’CH3); (+)-ESI-MS: m/z = 1050 [M+Na), 1028
[M+H]; ()-ESI-MS: m/z = 1063 [M+Cl], 1027 [MH); HRMS
calcd for C47H57N5O21: 1027.354604, found: 1027.3257; UV/Vis
(MeOH): lmax (lg e) = 269 (5.53), 399 (4.96) nm; IR (KBr): ñ = 3438,
2938, 2362, 1695, 1626, 1445, 1385, 1225, 1089, 1000, 871, 822, 755, 672,
554 cm1; [a]20
D = 56.5 (c = 584 mg mL , CHCl3).
Received: July 4, 2003 [Z52312]
Keywords: antibiotics · glycosides · gutingimycin ·
natural products · structure determination
[1] R. P. Maskey, E. Helmke, H. H. Fiebig, H. Laatsch, J. Antibiot.
2002, 55, 1031 – 1035.
[2] a) R. P. Maskey, E. Helmke, O. Kayser, H. H. Fiebig, H. Laatsch,
J. Antibiot., submitted; b) F. Tomita, T. Tamaoki, M. Morimoto,
K. Fujimoto, J. Antibiot. 1981, 34, 1519 – 1524; c) T. Tamaoki, K.
Shirahata, T. Iida, F. Tomita, J. Antibiot. 1981, 34, 1525 – 1530.
[3] H. Izumida, K. Adachi, M. Nishijima, M. Endo, W. Miki, J. Mar.
Biotechnol. 1995, 2, 115 – 118.
[4] Crystallographic data for 1 c: C47H57O21N5 + 4 H2O, Mr = 1100.04,
monoclinic, space group P21, a = 17.736(3), b = 7.091(2), c =
19.816(3) A, g = 90.008, V = 2510.3(9) A3, Z = 2, 1calc = 1.379,
F(000) = 1164, l = 1.54178 A, T = 133 K, crystal dimensions 0.3 F
0.1 F 0.05 mm3, 2 2q 118, of 18 834 measured data, 3897 were
independant (Rint = 0.015 Friedel mates merged). Refinement:
737 parameters, 729 restraints, R1(%) = FoFc/Fo = 4.41 for
3357 Fo > 4s(Fo) and 5.67 for 3897 (all data), wR2 = [w(F2oF2c/
w(F2o)2]1/2 = 0.1100 (all data), min/max residual electron density
0.24/0.23 e A3. Data for 2 c were collected on a three-circle
diffractometer with a CCD detector and f scans on a flash-frozen
crystal in an oil drop in a loop. The data was integrated with the
program SAINT. All data were corrected semiempirically for
systemic errors such as absorption. The structure was solved by
using the dual-space recycling method (SHELXD[9]) and refined
Angew. Chem. Int. Ed. 2004, 43, 1281 –1283
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gutingimycin, complex, metabolico, streptomyces, highly, marina
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