Halogenated volatiles from the fungus Geniculosporium and the actinomycete Streptomyces chartreusis

Article abstract of DOI:10.3762/bjoc.9.311

Two unidentified chlorinated volatiles X and Y were detected in headspace extracts of the fungus Geniculosporium. Their mass spectra pointed to the structures of a chlorodimethoxybenzene for X and a dichlorodimethoxybenzene for Y. The mass spectra of some constitutional isomers for X and Y were included in our databases and proved to be very similar, thus preventing a full structural assignment. For unambiguous structure elucidation all possible constitutional isomers for X and Y were obtained by synthesis or from commercial suppliers. Comparison of mass spectra and GC retention times rigorously established the structures of the two chlorinated volatiles. Chlorinated volatiles are not very widespread, but brominated or even iodinated volatiles are even more rare. Surprisingly, headspace extracts from Streptomyces chartreusis contained methyl 2-iodobenzoate, a new natural product that adds to the small family of iodinated natural products.

Full text of DOI:10.3762/bjoc.9.311

Halogenated volatiles from the fungus
Geniculosporium and the actinomycete
Streptomyces chartreusis
Tao Wang, Patrick Rabe, Christian A. Citron and Jeroen S. Dickschat*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2013, 9, 2767–2777.
Institut für Organische Chemie, TU Braunschweig, Hagenring 30,
38106 Braunschweig, Germany
doi:10.3762/bjoc.9.311
Received: 29 August 2013
Accepted: 07 November 2013
Published: 03 December 2013
Email:
Jeroen S. Dickschat* - j.dickschat@tu-bs.de
* Corresponding author
This article is part of the Thematic Series "Natural products in synthesis
and biosynthesis".
Keywords:
constitutional isomerism; GC-MS; natural products; organohalogen
compounds; volatiles
Associate Editor: S. Bräse
© 2013 Wang et al; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
Two unidentified chlorinated volatiles X and Y were detected in headspace extracts of the fungus Geniculosporium. Their mass
spectra pointed to the structures of a chlorodimethoxybenzene for X and a dichlorodimethoxybenzene for Y. The mass spectra of
some constitutional isomers for X and Y were included in our databases and proved to be very similar, thus preventing a full struc-
tural assignment. For unambiguous structure elucidation all possible constitutional isomers for X and Y were obtained by synthesis
or from commercial suppliers. Comparison of mass spectra and GC retention times rigorously established the structures of the two
chlorinated volatiles. Chlorinated volatiles are not very widespread, but brominated or even iodinated volatiles are even more rare.
Surprisingly, headspace extracts from Streptomyces chartreusis contained methyl 2-iodobenzoate, a new natural product that adds
to the small family of iodinated natural products.
Introduction
Today several thousands of halogenated natural products are tions can strongly differ from those found in marine ecosys-
known, most of them being produced by marine organisms [1]. tems, but the overall trend is similar, and therefore chlorine and
The majority of halogenated natural products are chlorinated or bromine are also the most widespread halogens in natural prod-
brominated, while iodinated and particularly fluorinated com- ucts from terrestrial organisms.
pounds are significantly less abundant (Table 1) [2]. Reasons
for this distribution are (I) the different concentrations of the Many biologically active halogenated secondary metabolites are
halogenides in sea water and (II) the differences in their redox known from actinomycetes including vancomycin [4], chloram-
potentials [3]. In terrestrial habitats local halogenide concentra- phenicol [5], rebeccamycin [6], marinopyrrols [7,8], armeni-
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Results and Discussion
Table 1: Approximate number of known halogenated natural products
and redox potential and natural abundance of the halogenides in sea
water.
Chlorinated volatiles from Geniculosporium
The volatiles emitted by agar plate cultures of the fungus
Geniculosporium sp. 9910 were trapped on charcoal filters by
using a closed-loop stripping apparatus (CLSA) [20]. After one
day of collection a solvent extract of the trapped material was
analysed by GC–MS. The analytical process was performed
three times to demonstrate reproducibility of the results, and a
representative chromatogram is shown in Figure 1. Besides
the main compound 1 and some structurally related volatiles 2
and 3 that were identified by delinating a structural proposal
from the mass spectra followed by synthesis of reference com-
pounds [21], two additional volatiles X and Y were observed in
all investigated samples. Their mass spectra are shown in
Halogenide
Natural
Redox
Sea water
productsa
potential [V]b [mg L−1]b
F−
Cl−
Br−
I−
30
+2.87
+1.36
+1.07
+0.54
1.4
2200
1900
100
18100
68
0.06
aEstimated number of known natural products in 2002 according to
reference [2]. bRedox potentials and concentrations in sea water as
given in reference [3].
aspirols [9], and salinosporamide [10]. Also a few volatile halo- Figure 2.
genated compounds are known, e.g., from algae as summarised
in a recent review [11]. Particularly interesting is the produc- Both volatiles were suggested to be chlorinated compounds due
tion of chloroform by termites that was estimated to account for to their characteristic isotope patterns of the molecular ions with
15% of the global chloroform emissions [12].
compound X containing one chlorine and volatile Y containing
two chlorine atoms. Furthermore, a comparison of the mass
The introduction of halogens into natural products is catalysed spectrum of X to data base spectra revealed strong similarities
by various known types of enzymes [13,14], including the to mass spectra of chlorodimethoxybenzenes, but the data base
FADH2-dependent halogenases [15], α-ketoglutarate/Fe2+- mass spectra of various constitutional isomers showed only
dependent halogenases [16], SAM-dependent halogenases marginal differences. Similarly, the mass spectrum of Y was
[17,18], and vanadium-dependent haloperoxidases [19].
very similar to data base spectra of dichlorodimethoxybenzenes,
but also in this case the mass spectra of several constitutional
Here we report on the identification of two chlorinated volatile isomers were highly similar. Therefore, the comparison of mass
compounds from the fungus Geniculosporium and a iodinated spectra was not a sufficient criterion for unambiguous com-
volatile from Streptomyces chartreusis.
pound identification, and thus the additional comparison of GC
Figure 1: Total ion chromatogram of a CLSA headspace extract from Geniculosporium.
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Figure 2: Mass spectra of A) the chlorinated volatile X and B) the chlorinated volatile Y.
retention indices of both natural products to authentic standards
of all possible structural isomers was pursued.
The six constitutional isomers of a chlorodimethoxybenzene
4a–4f as candidate structures for X are summarised in Figure 3.
Compounds 4d and 4e were commercially available, while the
remaining isomers were obtained by synthesis according to
Scheme 1. Surprisingly, no satisfying synthetic procedure for
compound 4a was available from literature, but ortho-lithiation
of veratrole (5) followed by treatment with trifluorosulfonyl
chloride and triethylamine gave acceptable yields (47%).
Conversion of 4-aminoveratrole (6) into the corresponding
diazonium salt followed by a Sandmeyer reaction resulted in 4b
(61% over two steps). The isomer 4c was efficiently prepared
Figure 3: Constitutional isomers of chlorodimethoxybenzene as candi-
date structures for X.
by double methylation of 2-chlororesorcinol (8) with potassium
carbonate and methyl iodide in acetone (88%), while 4f was
obtained by chlorination of dimethoxybenzene 9 with
benzyltrimethylammonium tetrachloroiodate in moderate yield summarised in Table 2. Mass spectra, 1H and 13C NMR spectra
(34%) [22]. All six compounds were subjected to GC–MS of all six compounds are given in Supporting Information
analysis using the same type of GC column as for the head- File 1. The structure of 4b was unambiguously assigned to the
space extract from Geniculosporium (HP5-MS) and their reten- natural product X by comparison of mass spectra and retention
tion indices together with the NMR spectroscopic data are indices (Table 2).
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Scheme 1: Synthesis of chlorodimethoxybenzenes as reference compounds for X.
Table 2: NMR spectroscopic data and GC retention indices of all constitutional isomers of chlorodimethoxybenzene in comparison to natural X.
Compound
1H NMR
13C NMR
I
4a
6.96 (m, 2H, 2 × CH)
6.81 (m, 1H, CH)
3.87 (s, 3H, OCH3)
3.86 (s, 3H, OCH3)
154.0 (Cq)
145.5 (Cq)
128.4 (Cq)
124.3 (CH)
122.0 (CH)
110.9 (CH)
60.6 (OCH3)
56.1 (OCH3)
1278
4f
6.95 (d, 1H, 4JH,H = 3.0 Hz, CH)
6.86 (d, 1H, 4JH,H = 3.0 Hz, 3JH,H = 9.0 Hz)
6.76 (dd, 1H, 3JH,H = 3.0 Hz, 4JH,H = 9.0 Hz, CH)
3.84 (s, 3H, OCH3)
153.8 (Cq)
149.4 (Cq)
123.0 (Cq)
116.1 (CH)
113.2 (CH)
112.8 (CH)
56.7 (OCH3)
55.8 (OCH3)
1292
1322
3.75 (s, 3H, OCH3)
4b
6.87 (dd, 1H, 3JH,H = 8.5 Hz, 4JH,H = 2.4 Hz, CH)
6.84 (d, 1H, 4JH,H = 2.4 Hz, CH)
6.76 (d, 1H, 3JH,H = 8.5 Hz, CH)
3.85 (s, 3H, OCH3)
149.5 (Cq)
147.8 (Cq)
125.6 (Cq)
120.2 (CH)
112.0 (CH)
111.9 (CH)
56.02 (OCH3)
55.98 (OCH3)
3.84 (s, 3H, OCH3)
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Table 2: NMR spectroscopic data and GC retention indices of all constitutional isomers of chlorodimethoxybenzene in comparison to natural X.
(continued)
4e
6.51 (d, 2H, 4JH,H = 2.2 Hz, 2 × CH)
6.34 (t, 1H, 4JH,H = 2.2 Hz, CH)
3.77 (s, 6H, 2 × OCH3)
161.1 (2 × Cq)
135.2 (Cq)
106.9 (2 × CH)
99.2 (CH)
1340
1374
55.5 (2 × OCH3)
4d
7.24 (d, 1H, 3JH,H = 8.7 Hz, CH)
6.50 (d, 1H, 4JH,H = 2.7 Hz, CH)
6.42 (dd, 1H, 3JH,H = 8.7 Hz, 4JH,H = 2.7 Hz, CH)
3.87 (s, 3H, OCH3)
159.5 (Cq)
155.6 (Cq)
130.1 (CH)
114.1 (Cq)
105.2 (CH)
100.0 (CH)
56.0 (OCH3)
55.5 (OCH3)
3.79 (s, 3H, OCH3)
4c
X
7.17 (t, 1H, 3JH,H = 8.4 Hz, CH)
6.60 (d, 2H, 3JH,H = 8.4 Hz, 2 × CH)
3.90 (s, 6H, 2 × OCH3)
156.2 (2 × Cq)
127.1 (CH)
110.6 (Cq)
104.7 (2 × CH)
56.3 (2 × OCH3)
1404
1322
For compound Y a series of eleven constitutional isomers as
shown in Figure 4 were possible. All eleven compounds were
obtained by synthesis as summarised in Scheme 2. The com-
pounds 10a, 10c, and 10e were obtained by treatment of
1,2,3,4-tetrachlorobenzene (11) with sodium methoxide in hot
HMPA. This reaction is reported to yield mainly 1,2,3-
trichloro-4-methoxybenzene and 1,2,4-trichloro-3-methoxyben-
zene by monosubstitution [23], but small amounts of disubstitu-
tion products were also formed that were rigorously purified
from the mixture by repeated column chromatography. Starting
from 1,2,3,5-tetrachlorobenzene (12) the same reaction condi-
tions also provided a mixture of mono- and disubstitution prod-
ucts, but the target compounds 10f and 10g were obtained in
better yields (29% and 17%). Derivative 10b was prepared from
3,5-dichlorocatechol (13) by methylation with potassium
carbonate and methyl iodide in high yield (80%). Accordingly,
methylation of 4,6-dichlororesorcinol (14) provided 10h in 80%
yield, and methylation of 2,3-dichlorohydroquinone (16),
prepared from benzoquinone (15) by treatment with SO2Cl2
under acidic conditions, generated 10i (13% via two steps).
Reduction of 2,6-dichlorobenzoquinone with ascorbic acid to
the corresponding hydroquinone 18 followed by methylation
yielded 48% of 10k. Chlorination of veratrole (5) with two
equivalents of benzyltrimethylammonium tetrachloroiodate
resulted in 10d (45%), while the same procedure starting from
1,4-dimethoxybenzene gave access to 10j (38%). The NMR
spectroscopic data and results of GC–MS analyses for all eleven
dichlorodimethoxybenzenes in comparison to the volatile Y are
summarised in Table 3. All mass spectra, 1H and 13C NMR
spectra are shown in Supporting Information File 1. Compari-
son of mass spectra and retention indices unequivocally estab-
lished the identity of Y and 10b (Table 3).
Figure 4: Constitutional isomers of dichlorodimethoxybenzene as
candidate structures for Y.
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Scheme 2: Synthesis of chlorodimethoxybenzenes as reference compounds for Y.
Both compounds 4b and 10b that were identified in this study chlorination from the monochlorodimethoxybenzene (4b or 4f,
as volatiles emitted by Geniculosporium have not been reported respectively), suggesting that in both organisms the sets of chlo-
as natural products before. However, the structurally related rinated compounds arise via one and the same biosynthetic
compounds 4f and 10k are known from the fungus Bjerkandera pathway. Known structurally related compounds from fungi are
adusta [24]. It is interesting to note that in both cases the di- drosophilin A (19) [25] or the trichlorinated phenols 20–22
chlorodimethoxybenzene (10b or 10k) can arise by a second (Figure 5) [26]. The biological function of 4b and 10b in
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Table 3: NMR spectroscopic data and GC retention indices of all constitutional isomers of dichlorodimethoxybenzene in comparison to natural Y.
Compound
1H NMR
13C NMR
I (HP5-MS)
10c
7.07 (s, 2H, 2 × CH)
3.92 (s, 6H, 2 × OCH3)
150.7 (2 × Cq)
127.1 (2 × Cq)
125.1 (2 × CH)
61.1 (2 × OCH3)
1339
10b
10k
6.98 (d, 1H, 4JH,H = 2.5 Hz, CH)
6.80 (d, 1H, 4JH,H = 2.5 Hz, CH)
3.86 (s, 3H, OCH3)
154.1 (Cq)
144.4 (Cq)
129.1 (Cq)
128.9 (Cq)
121.6 (CH)
111.7 (CH)
60.8 (OCH3)
56.3 (OCH3)
1426
1443
3.84 (s, 3H, OCH3)
6.84 (s, 2H, 2 × CH)
3.84 (s, 3H, OCH3)
3.76 (s, 3H, OCH3)
155.7 (Cq)
146.2 (Cq)
129.5 (2 × Cq)
114.5 (2 × CH)
60.8 (OCH3)
55.9 (OCH3)
10j
6.97 (s, 2H, 2 × CH)
3.85 (s, 6H, 2 × OCH3)
149.2 (2 × Cq)
120.9 (2 × Cq)
114.5 (2 × CH)
56.8 (2 × OCH3)
1448
1466
10a
7.15 (d, 1H, 3JH,H = 9.0 Hz, CH)
6.76 (d, 1H, 3JH,H = 9.0 Hz, CH)
3.86 (s, 3H, OCH3)
152.4 (Cq)
146.7 (Cq)
127.5 (Cq)
124.7 (CH)
124.5 (Cq)
111.0 (CH)
60.6 (OCH3)
56.2 (OCH3)
3.85 (s, 3H, OCH3)
10e
7.23 (d, 1H, 3JH,H = 9.0 Hz, CH)
6.66 (d, 1H, 3JH,H = 9.0 Hz, CH)
3.90 (s, 3H, OCH3)
155.1 (Cq)
153.2 (Cq)
127.7 (CH)
120.7 (Cq)
118.2 (Cq)
107.9 (CH)
60.6 (OCH3)
56.5 (OCH3)
1487
3.89 (s, 3H, OCH3)
10d
10h
6.90 (s, 2H, 2 × CH)
3.86 (s, 6H, 2 × OCH3)
148.3 (2 × Cq)
123.4 (2 × Cq)
112.9 (2 × CH)
56.2 (2 × OCH3)
1505
1545
7.34 (s, 1H, CH)
6.53 (s, 1H, CH)
3.90 (s, 6H, 2 × CH3)
154.5 (2 × Cq)
130.5 (CH)
114.0 (2 × Cq)
97.8 (CH)
56.5 (2 × OCH3)
10f
6.60 (s, 2H, 2 × CH)
3.89 (s, 6H, 2 × CH3)
156.4 (2 × Cq)
133.0 (Cq)
109.3 (Cq)
105.5 (2 × CH)
56.5 (2 × OCH3)
1556
1565
10g
6.62 (d, 1H, 4JH,H = 2.7 Hz, CH)
6.42 (d, 1H, 4JH,H = 2.7 Hz, CH)
3.87 (s, 3H, OCH3)
158.7 (Cq)
156.7 (Cq)
133.8 (Cq)
113.5 (Cq)
106.4 (CH)
98.6 (CH)
3.79 (s, 3H, OCH3)
56.4 (OCH3)
55.7 (OCH3)
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Table 3: NMR spectroscopic data and GC retention indices of all constitutional isomers of dichlorodimethoxybenzene in comparison to natural Y.
(continued)
10i
Y
6.81 (s, 2H, 2 × CH)
3.86 (s, 6H, 2 × OCH3)
150.3 (2 × Cq)
123.1 (2 × Cq)
110.0 (2 × CH)
56.8 (2 × OCH3)
1574
1426
Figure 5: Known natural products that are structurally related to 4b and 10b from Geniculosporium.
Geniculosporium is unknown, but the antibiotic activity of the analyses during a time period of almost two years (January
related compound drosophilin A towards bacteria is well known 2012–August 2013), using new batches of medium in all cases.
[27].
Compound 23 was repeatedly detected in all six analyses which
clearly supports the natural origin of this volatile.
A iodinated volatile from Streptomyces
chartreusis
The iodinated compound 23 has never been reported as a
During the course of our work on volatiles from various natural product. One of the few reported iodinated compounds
bacteria and fungi we rarely encountered chlorinated volatiles. is the structurally highly complex calicheamicin 24 (Figure 7)
Brominated and iodinated volatiles are even more rare. Investi- from Micromonospora echinospora [28] that also contains an
gations on the volatiles released by the actinomycete Strepto- iodinated aromatic subunit.
myces chartreusis during growth on 84 GYM medium, also
collected by use of a CLSA followed by GC–MS analysis of the Conclusion
obtained headspace extracts, led to a surprising result. The In summary, we have presented the identification of 4-chloro-
extracts contained a trace compound that was readily identified 1,2-dimethoxybenzene (4b) and 1,5-dichloro-2,3-dimethoxy-
from its mass spectrum and comparison to an authentic stan- benzene (10b) as volatile natural products released by the
dard as methyl 2-iodobenzoate (23, Figure 6A). This com- fungus Geniculosporium. Furthermore, Streptomyces
pound was observed as the principal compound in headspace chartreusis was shown to emit methyl 2-iodobenzoate (23). All
extracts during growth of S. chartreusis on 84 GYM supple- three compounds are new natural products. Future work on the
mented with 2-iodobenzoic acid suggesting that 23 arises by biological function of these metabolites and the biosynthetic
efficient methylation of the carboxylic acid (Figure 6B). pathways is now possible.
2-Iodobenzoic acid is frequently used in many chemical labora-
tories including ours, e.g., as a starting material for the prepar- Experimental
ation of the oxidising agents IBX and DMP. A contamination of Strains and culture conditions: Geniculosporium sp. 9910
a bacterial agar plate with traces of 2-iodobenzoic acid was was obtained from Barbara Schulz (Braunschweig) and grown
assumed as a possible, albeit unlikely explanation for the forma- on potato–carrot medium [29] on petri dishes and incubated for
tion of 23 in the bacterial cultures. In our laboratories all analyt- 21 d at 28 °C prior to analysis. Streptomyces chartreusis NRRL
ical work is strictly spatially separated from the other chemical 3882 (= DSM 41447) was obtained from DSMZ (Braun-
(synthetic) work. Furthermore, a contamination with 2-iodoben- schweig, Germany) and grown on 84 GYM (20.0 g rolled oats,
zoic acid as the reason for the emission of 23 by S. chartreusis boiled in 1 L of distilled water for 20 min, then oats were
was excluded by the sixfold repetition of the headspace filtered off, trace element solution (1 mL) containing 1 g L−1
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Figure 6: Total ion chromatograms of headspace extracts from S. chartreusis. A) Growth on 84 GYM showing production of 23 as a trace compound.
B) Growth on 84 GYM amended with 2-iodobenzoic acid (1 mM) results in the emission of 23 as the principal volatile compound.
Figure 7: Calicheamicin, a known iodinated compound from the actinomycete Micromonspora echinospora.
FeSO4·7H2O, 1 g L−1 MnCl4·4H2O, and 1 g L−1 ZnSO4·7H2O extracts were carried out on an Agilent 7890A connected with
was added, followed by sterilisation at 121 °C for 20 min).
an Agilent 5975C inert mass detector fitted with a HP5-MS
fused silica capillary column (30 m, 0.25 mm i. d., 0.25 μm
Headspace analyses: The volatiles released by Geniculospo- film, Agilent). GC conditions were as follows: inlet pressure
rium or Streptomyces chartreusis were trapped by use of the 77.1 kPa, He 23.3 mL min−1, injection volume 1.5 μL, transfer
CLSA (closed-loop stripping analysis) technique as described line 300 °C, electron energy 70 eV. The operation mode was
previously [30]. GC–MS analyses of the obtained headspace splitless (60 s valve time) and the carrier gas was He at
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Acknowledgements
This work was funded by the Deutsche Forschungsgemein-
schaft with an Emmy Noether fellowship (DI1536/1-3) and a
Heisenberg fellowship (DI1536/4-1) (to JSD), by the State of
Lower Saxony with a Ph. D scholarship (to TW), and by the
Beilstein Institut zur Förderung der chemischen Wissenschaften
with a Ph. D scholarship (to PR). We thank Stefan Schulz
(Braunschweig) for his support.
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