The scent of bacteria: Headspace analysis for the discovery of natural products

Article abstract of DOI:10.1021/np300468h

Volatile compounds released by 50 bacterial strains, 45 of them actinobacteria in addition to three chloroflexi and two myxobacteria, have been collected by use of a closed-loop stripping apparatus, and the obtained headspace extracts have been analyzed by GC-MS. Excluding terpenes that have recently been published elsewhere, 254 compounds from all kinds of compound classes have been identified. For unambiguous compound identification several reference compounds have been synthesized. Among the detected volatiles 12 new natural products have been found, in addition to mellein, which was released by Saccharopolyspora erythraea. The iterative PKS for this compound has recently been identified by in vitro experiments, but mellein production in S. erythraea has never been reported before. These examples demonstrate that headspace analysis is an important tool for the discovery of natural products that may be overlooked using conventional techniques. The method is also useful for feeding experiments with isotopically labeled precursors and was applied to investigate the biosynthesis of the unusual nitrogen compound 1-nitro-2-methylpropane, which arises from valine. Furthermore, several streptomycetes emitted compounds that were previously recognized as insect pheromones, thus questioning if bacterial symbionts are involved in insect communication.

Full text of DOI:10.1021/np300468h

Article
pubs.acs.org/jnp
The Scent of Bacteria: Headspace Analysis for the Discovery of
Natural Products
Christian A. Citron, Patrick Rabe, and Jeroen S. Dickschat*
Institute of Organic Chemistry, Technical University of Braunschweig, Hagenring 30, 38106 Braunschweig, Germany
S
* Supporting Information
ABSTRACT: Volatile compounds released by 50 bacterial
strains, 45 of them actinobacteria in addition to three
chloroflexi and two myxobacteria, have been collected by use
of a closed-loop stripping apparatus, and the obtained
headspace extracts have been analyzed by GC-MS. Excluding
terpenes that have recently been published elsewhere, 254
compounds from all kinds of compound classes have been
identified. For unambiguous compound identification several
reference compounds have been synthesized. Among the
detected volatiles 12 new natural products have been found, in
addition to mellein, which was released by Saccharopolyspora
erythraea. The iterative PKS for this compound has recently
been identified by in vitro experiments, but mellein production
in S. erythraea has never been reported before. These examples demonstrate that headspace analysis is an important tool for the
discovery of natural products that may be overlooked using conventional techniques. The method is also useful for feeding
experiments with isotopically labeled precursors and was applied to investigate the biosynthesis of the unusual nitrogen
compound 1-nitro-2-methylpropane, which arises from valine. Furthermore, several streptomycetes emitted compounds that
were previously recognized as insect pheromones, thus questioning if bacterial symbionts are involved in insect communication.
acteria, especially actinomycetes, are prolific producers of
various secondary metabolites including polyketides,
technique.1,2 After a collection time of about one day the
adsorbed volatiles were eluted with dichloromethane, and the
extracts were analyzed by GC-MS. Each microorganism was
investigated in at least four replicates to test for reproducibility.
The species included in this study are summarized in Table 1 of
the Supporting Information (SI), the identified compounds are
listed in Table 2 of the SI, and one representative chromato-
gram of each investigated species is shown in Figure 1 of the SI.
Compound identification was performed by the following
approaches: (a) comparison of the mass spectrum to electronic
mass spectral libraries,15 (b) comparison of the retention index
to tabulated literature data,15 (c) comparison with a
commercially available authentic sample, (d) chemical synthesis
of a reference compound.
A total of 254 volatile compounds from various classes were
identified. Some of these compounds were almost ubiquitously
present, whereas others occurred in only one or a few species.
Each compound class will be discussed separately in the
following sections. A detailed description of the terpenoids that
make up the largest and struturally most diverse class of
volatiles from actinomycetes with more than 150 different
compounds identified in 35 strains was recently published
elsewhere.3
B
nonribosomal peptides, terpenoids, alkaloids, and lipids. The
traditional method of screening for new natural products starts
with a fermentation followed by culture extraction and
compound purification. During concentration steps volatile
secondary metabolites are easily lost and, therefore, have
frequently been overlooked.
An approach for the discovery of volatile secondary
metabolites requires specialized headspace techniques such as
the closed-loop stripping apparatus (CLSA).1,2 Using this
method we have recently identified a large number of terpenes
emitted by bacteria, most of them actinomycetes, known to
encode terpene cyclases.3 Only a few other reports exist on
volatiles from actinomycetes,4−13 and a general overview
summarizing the accumulated knowledge about bacterial
volatiles was published in 2007.14 Following the survey on
terpenoid bacterial volatiles,3 here we report on the volatiles
from other compound classes. A discussion of the significance
of the analytical results with respect to the potential of finding
new natural products or known natural products from
previously unrecognized sources is given.
RESULTS AND DISCUSSION
■
The volatiles released by 50 bacterial strains, mainly actino-
mycetes together with three chloroflexi and two myxobacteria,
were collected on charcoal using the CLSA headspace
Received: July 4, 2012
© XXXX American Chemical Society and
American Society of Pharmacognosy
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Figure 1. Hydrocarbons.
Hydrocarbons. The hydrocarbons identified in this study
include unbranched and methyl-branched alkanes, alkenes, and
cycloalkanes (Figure 1). The methyl-branched compounds 3-
methylnonane (1), 3-methyldecane (2), 2,6-dimethylundecane
(6), and 3,7-dimethyldecane (7) were released by Chitinophaga
pinensis, while 3-methyldodecane (3), 2-methylundecane (4),
and 2-methyltridecane (5) were found in Streptomyces
griseoflavus and tridec-1-ene (8) was identified as the main
compound from Streptomyces auratus. This olefin has not been
described previously as a volatile from a particular bacterium,
but its shorter homologue undec-1-ene is well known from
Pseudomonas.16 The cyclic compounds propylcyclohexane (9),
pentylcyclohexane (10), and trans-decalin (11) were produced
by C. pinensis.
compounds, whereas compound 24 may require one α-
oxidation step for its biosynthesis.
Cyclohept-4-enol (28) was emitted by Streptomyces sviceus,
while 2,3-butanediol (29) was found in several strains. This diol
was previously described as a volatile from Bacillus and shown
to promote growth and induce systemic resistance in
Arabidopsis thaliana.17,18 Several unsaturated methyl-branched
alcohols (30−33) were detected. The compounds 3-methylbut-
3-enol (30) and 3-methylbut-2-enol (31) represent the formal
hydrolysis products of the terpene precursors isopentenyl
diphosphate and dimethylallyl diphosphate, respectively, but
alternative biosynthetic origins, e.g., from leucine, may also be
possible. Octan-3-ol (34) and oct-1-en-3-ol (35) are known as
fungal volatiles comprising the typical fungal smell.19 The
isomer (E)-oct-2-en-1-ol (36) and its higher homologues 37
and 38 were also occasionally found.
Alcohols. Primary alcohols predominated, but a few
secondary alcohols, one cycloalkanol, and one diol were also
found (Figure 2). Several species emitted unbranched alcohols
Aldehydes and Ketones. Several aldehydes and ketones
were identified, in addition to hydroxyketones, diketones, and a
few cyclic ketones (Figure 3). The aldehydes ranging from
hexanal (39) to tetradecanal (47) were widespread, with nearly
the complete series present in C. pinensis and Herpetosiphon
aurantiacus. The α,β-unsaturated analogues 48−50, prenal
(51), and the bis-unsaturated (E,E)-nona-2,4-dienal (52)
occurred less often. Furthermore, methyl ketones (53−62)
and ethyl ketones (63−66) were identified that can arise via
fatty acid biosynthesis from β-keto acids or, in the case of
incorporation of a terminal methylmalonyl-CoA unit (CoA =
coenzyme A), from α-methyl-β-keto acids, respectively, by
decarboxylation.2,20 Interestingly, the compound 3-ethylpentan-
2-one (61) derived from an internal ethylmalonyl-CoA
elongation unit was emitted by Streptomyces bottropensis. We
have recently also identified a series of blastmycinone
derivatives with internally ethyl branched O-acyl moieties,4
thus reflecting the biosynthetic potential of this particular
microorganism.
Figure 2. Alcohols.
In K. setae the typical fungal metabolites19 octan-3-one (64)
and oct-1-en-3-one (67) occur together with the related
alcohols 34 and 37. In addition, a variety of α-hydroxyketones
(68−75) together with the diones 76 and 77 were found. The
hydroxyketones were widespread and are known as precursors
of alkylated pyrazines in Corynebacterium glutamicum.5 Among
cyclic ketones cyclopentanone and α,β-unsaturated cyclo-
pentenone derivatives 78−82 were identified. The trisubsti-
tuted 2,3,5-trimethylcyclopent-2-enone (82) is released in large
quantities by Streptomyces coelicolor and is a new natural
product. The structurally related 2,3-dimethyl-5-methylenecy-
clopent-2-enone has previously been reported from S. coelicolor
as a decarboxylation product of methylenomycin C.21 Similarly,
82 can also be regarded as a shunt product of methylenomycin
ranging from hexanol (12) to decanol (16). The complete
series was found in Streptomyces parvulus, while particularly
large amounts of 12 were released by Kitasatospora setae and
Streptomyces avermitilis. Furthermore, various ω-1 methyl-
branched alcohols (17−22) and a series of odd ω-2 methyl-
branched alcohols (23−27) were identified. This pattern is
indicative of their formation from the corresponding branched
amino acids valine, leucine, and isoleucine by transamination
and oxidative decarboxylation, fatty acid chain elongation of the
thus obtained starter units, and final reduction to the alcohols.
This mechanism yields the complete series of ω-1 methyl-
branched alcohols, but only the odd ω-2 methyl-branched
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Figure 3. Aldehydes and ketones.
biosynthesis. Cyclohexanone (83) and cyclohept-4-enone (84)
were also occasionally found. Compound 84 is related to the
alcohol 28, and both compounds may originate via the
phenylacetate catabolon as previously suggested for tropone.22
Neither the alcohol 28 nor the accompanying ketone 84 has
been reported from any organism before.
compounds 110−112. The identification of the methyl-
branched compounds was based on the retention indices of
these compounds, which were 40 units lower than for the
unbranched isomers that were commercially available. This
difference of retention indices is evident for a methyl branch in
the ω-1 position, which can further be rationalized by the
biosynthesis of these compounds from valine- and leucine-
derived starter units, respectively.8 Finally, the acetate esters
113−115 were identified. In most strains only one or a few
esters were found, whereas a potpourri of several esters
occurred in Actinosynnema mirum, Stackebrandtia nassauensis,
and Streptomyces violaceusniger. While most esters were present
only in traces, 87 was emitted as one of the main compounds
by S. auratus, as was its isomer 90 by Streptomyces lividans. The
volatiles 93 and 109 from A. mirum and 97 from S. nassauensis
are new natural products.
Lactones. Lactones are well known cell-to-cell signaling
molecules in Streptomyces, and as such, they control various
processes including sporulation and antibiotic production.25,26
The A-factor from Streptomyces griseus was discovered in the
1960s by Khokhlov and is a butanolide with a 2-acyl side chain
and a 3-hydroxymethyl function (Figure 5).27 The structurally
related factor I from Streptomyces viridochromogenes and
virginiae butanolide A (VB-A) from Streptomyces virginiae
exhibit a hydroxylated 2-alkyl group with different stereo-
chemistry at the hydroxy function.28,29 The factor NFX-4 from
Carboxylic Acids and Esters. Two previous studies on the
volatiles released by Micromonospora aurantiaca and Chitino-
phaga have revealed that bacteria can produce a large diversity
of volatile fatty acids and fatty acid methyl esters.8,23 During the
course of this study the carboxylic acids 3-methylbutyric acid
(85) and 2-methylbutyric acid (86) were identified in C.
pinensis and H. aurantiacus, whereas various methyl esters such
as the branched compounds 87−97 were found within the
whole set of strains (Figure 4). Unsaturated esters included the
compounds 98−102 and methyl 2,4-dimethylhexa-2,4-dienoate
(103), which has previously been described from the fungus
Fusarium fujikuroi.24 The unsaturated esters 98 and 99 likely
originate from leucine and isoleucine by transamination,
oxidative decarboxylation, FAD-dependent introduction of the
olefinic double bond, and methylation of the corresponding
free acid. In addition, the α- and β-hydroxy esters 104−109
occurred. Compounds 105, 107, and 108 also exhibit the
leucine- and isoleucine-derived carbon backbones, respectively,
and can be regarded as alternative precursors for 98 and 99.
Ethyl esters occurred less often and were represented by the
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Figure 4. Carboxylic acids and esters.
Streptomyces antibioticus is a butanolide substituted with a 2-
alkyl group and, in contrast to the other butanolides, has a 3-
hydroxy function and a 4-methyl group.30 This substitution
pattern is also found in the blastmycinones, a large class of
butanolides produced by Streptomyces ambofaciens and various
other streptomycetes known as degradation products of the
fungicidal antimycins.4,31 Gram-negative bacteria use a wide
range of species-specific N-acylhomoserine lactones, such as N-
(3-oxohexanoyl)-L-homoserine lactone from Vibrio fisheri, for
this purpose.32,33 Although these molecules are biosynthetically
different from the signaling factors in streptomycetes, the
Figure 5. Lactone signaling compounds from bacteria.
Figure 6. Lactones.
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Figure 7. Aromatic compounds.
structural resemblence between these two compound classes is
intriguing.
Interestingly, neither in the present nor in our previous study11
were the leucine-derived congeners found. Finally, 2H-pyran-2-
one (140) occurred in Thermomonospora curvata. Some strains
included in this study released only one or a few lactones.
Particularly rich in lactones were the headspace extracts from
Saccharopolyspora erythraea, T. curvata, and the streptomycetes
S. albus, S. ambofaciens, S. citreus, S. clavuligerus, S. griseus, and S.
odorifer. Since several butanolides are known to act as signaling
compounds in streptomycetes, a similar role of the lactones
identified here cannot be ruled out.
Aromatic Compounds. Aromatic compounds made up by
far the largest class of volatiles. A series of aromatic
hydrocarbons (141−148) was found (Figure 7). Particularly
interesting is the occurrence of propylbenzene (143) together
with its saturated counterpart propylcyclohexane (9) in C.
pinensis. Compound 143 may either be biosynthetically derived
from 9 or, more likely, be its biosynthetic precursor, because
the biosynthesis of both compounds from phenylalanine is the
easiest explanation. However, pentylbenzene, which would
similarly be the precursor of (or derived from) pentylcyclohex-
ane (10), was not detected. Mesitylene (145) has previously
been described as a natural product that originates in the light-
Volatile butenolides have previously been reported from
streptomycetes by Gerber.9 The headspace extracts of various
actinomycetes included in this study also contained a large
variety of butanolides and butenolides, while strains from other
taxa did not release any lactones, suggesting that the substance
class is characteristic for actinomycetes. Besides the unsub-
stituted pentan-4-olide (117) and pent-2-en-4-olide (125) and
a series of 4-alkylated lactones, a few butanolides and
butenolides with alkyl substitutents in the 2-position or with
two alkyl substitutents were found (Figure 6). The compound
4-methylhex-5-en-4-olide (128) is also known as lavender
lactone and contains a vinyl substituent. The α,β-unsaturated
butenolides 134 and 136, the corresponding β,γ-unsaturated
butenolides 135 and 137, and the pentanolides 138 and 139
have previously been reported from a marine streptomycete.11
During the course of this study these lactones were detected in
various streptomycetes (S. albus, S. citreus, S. ghanaensis, S.
griseus, and S. parvulus). A retrobiosynthetic analysis of these
compounds suggests that three are derived from valine (134,
135, 138) and three originate from isoleucine (136, 137, 139).
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induced polyene splicing of the polyketide neoaureothin to
orinocin.34
Besides these hydrocarbons various oxygenated aromatic
compounds were found. Phenol (149) was emitted only by S.
violaceusniger, whereas benzyl alcohol (150) occurred in several
strains. Its formate ester 151 was only present in Streptomyces
Spalangia endius.44 Both methyl esters 179 and 180 are also
known as constituents of the defensive secretions from the
beetle Chrysopeplus expolitus.45 The occasional co-occurrence of
mellein and methyl 6-methylsalicylate or of methyl 6-
methylsalicylate and methyl 6-ethylsalicylate in one species
supports their common biosynthesis. Results from feeding
experiments with isotopically labeled acetate were in agreement
with a polyketide origin of 181 and the free carboxylic acid of
179, 6-methylsalicylic acid.46 Recently the iterative polyketide
synthase (PKS) for mellein in S. erythraea encoded by
SACE5532 (or pks8) has been identified by in vitro
experiments with the recombinant enzyme,47 but so far mellein
production by S. erythraea has never been reported. Therefore,
our observations confirm that mellein is indeed produced by S.
erythraea. The high sensitivity of our analytical method
furthermore enables the detection of potential side products
of the mellein PKS. The biosynthesis of mellein, 179, and 180
by one and the same PKS would be explainable by slight
modifications of a common mechanism: the incorporation of
one acetyl-CoA starter and three malonyl-CoA extension units
leads to 179, 180 is made using a propionyl-CoA starter unit
and three malonyl-CoA units, and 181 requires an acetyl-CoA
starter and four malonyl-CoA elongation units, but formation
of the aromatic ring is mechanistically the same for all three
compounds. A gene knockout experiment is currently under-
way to investigate whether production of 179 and 180 also
depends on the mellein PKS.
sp. Tu 6071, and benzyl acetate (152) was released by S.
̈
erythraea and Streptomyces sp. Tu 6071. Benzaldehyde (153),
̈
acetophenone (154), and methyl benzoate (155) were also
found in various species, with 153 and 154 being the main
constituents of the headspace extracts from C. pinensis and S.
nassauensis. One of the most widespread bacterial volatiles is 2-
phenylethanol (156),14 which was identified in 42 of the 50
strains investigated in this study and has been previously
reported from several actinomycetes,6,8,11−13 myxobacte-
ria,2,33,35 and enterobacteria.36,37 In contrast, its derivatives
such as the methyl ether 157 and the esters 158−161 occurred
less frequently. The biosynthesis of 156 can be rationalized
from phenylalanine by transamination to phenylpyruvate and
subsequent decarboxylation to phenylacetaldehyde (164),
which is found in S. erythraea and S. ambofaciens. A final
reduction of 164 yields 156. Oxidation of 164 may yield
phenylacetic acid as precursor of the esters 162 and 163, which
were both identified in a few strains. Phenylacetone (165),
present in headspace extracts of S. erythraea and both strains of
Streptomyces filamentosus, may be generated by a two-carbon
elongation of phenylacetyl-CoA with malonyl-CoA and
subsequent decarboxylation of the free β-keto acid. A similar
reaction sequence with methylmalonyl-CoA furnishes 1-
phenylbutan-2-one (166), which is released by S. nassauensis.
The related compounds 4-phenylbutan-2-one (167) and 1-
phenylpentan-3-one (168) were detected in Rubrobacter
xylanophilus. Their formation can be rationalized from
phenylpropanoyl-CoA by elongation with either malonyl-CoA
or methylmalonyl-CoA, respectively, and decarboxylation of the
resulting free β-keto acid. The ketone 167 is known from the
myxobacterium Chondromyces crocatus,35 while 168 has not
been reported from bacteria before. Compounds with a rather
unusual oxidation pattern were 2-hydroxy-1-phenylethanone
(169) from T. curvata, 1-phenylpropane-1,2-dione (170) from
S. erythraea, and 1-phenylethyl isobutanoate (171) from S.
ambofaciens.
Furans. Furans were detected in several of the investigated
strains. Furfuryl formate (187) was detected only in S.
clavuligerus, whereas 2-acetylfuran (188) is one of the most
widespread compounds of this class, which was emitted by 20
of the 50 strains and is the main volatile from Haliangium
ochraceum (Figure 8). The compound 2-hexanoylfuran (189)
Some strains produced derivatives of salicylic acid that can
arise from the shikimate pathway intermediate chorismic acid.
Salicylaldehyde (172) was present in H. aurantiacus and T.
curvata, o-hydroxyacetophenone (173) was found in R.
xylanophilus, and methyl salicylate (174) was produced by
five strains. Several further compounds identified in this study
occurred only in one or very few strains. These are methyl p-
toluate (175), 4-methoxyacetophenone (176), p-cresol (177),
p-methylanisol (178), methyl 6-methylsalicylate (179), methyl
6-ethylsalicylate (180), mellein (181), veratrole (182), 1,2,3-
trimethoxybenzene (183), eugenol (184), diphenylmethanol
(185), and benzophenone (187).
Particularly interesting is the common occurrence of mellein
and the two structurally related methyl esters 179 and 180 in S.
erythraea. Mellein was first recognized in the fungus Aspergillus
melleus in 1950 by Burton.38 In ants mellein and methyl 6-
methylsalicylate function as alarm or trail pheromones, and in
some species both metabolites occur as mixtures.39−42 In
addition, mellein is known from the giant danaine butterfly Idea
leuconoe, where it acts as a male sex pheromone,43 and methyl
6-methylsalicylate is a female sex pheromone from the wasp
Figure 8. Furans.
was released by S. bottropensis, 2-pentylfuran (190) was
identified in S. avermitilis, and the ketone 1-(2-furyl)butan-3-
one (191) occurred in S. filamentosus. A series of methyl
furancarboxylates was found including methyl 2-furoate (192),
which was produced by several strains, whereas its isomer
methyl 3-furoate (194) was limited to S. griseoflavus. Methyl 5-
methyl-2-furoate (193) was detected only in Streptomyces
cattleya and S. griseoflavus, and dimethyl furan-2,4-dioate (195)
was present in the extracts from S. griseoflavus and S. parvulus.
The compounds 192 and 195 have also been reported from
other streptomycetes,13 but compound identification was based
only on their mass spectra and not on comparison to authentic
samples. Since the mass spectra of isomers such as 192 and
194, or 195 and the 2,3-, 2,5-, and 3,4-disubstituted analogues,
are very similar, compound identification in this earlier report
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remained tentative. Therefore, for unambiguous compound
identification all possible regioisomers of these furan derivatives
were obtained by synthesis (cf. below) or from commercial
suppliers. The furanone 196 and the corresponding methylated
derivative 197 were identified in S. clavuligerus. The furan 193
has not been reported as a natural product before.
Streptomyces pristinaespiralis. This compound may arise by
chlorination of 198 and represents a new natural product.
Further dialkyl (poly)sulfides were 2,4-dithiapentane (205)
from S. bottropensis and S. griseoflavus and methyl methyl-
thiomethyl disulfide (206).
Cyclic polysulfides included 1,2,4-trithiolane (207) from S.
filamentosus and tetrathiolane (208), pentathiane (209), and
hexathiepan (210) from S. bottropensis. Several sulfur hetero-
cycles including 207 are known from Shiitake mushrooms
(Lentinus edodes),48 while 208 and 209 have not been reported
from natural sources before. Cyclooctasulfur (211) was also
present in a few headspace extracts.
Sulfur Compounds. A structurally diverse array of sulfur
volatiles was identified (Figure 9), with the headspace extracts
Several methyl alkanethioates (212−215) were occasionally
found. The two volatiles methyl 3-(methylsulfanyl)prop-2-
enoate (216) emitted by S. nassauensis and S. griseoflavus and 3-
(methylsulfanyl)propionitrile (217) released by S. clavuligerus
are probably methionine-derived. The compound 218 from S.
bottropensis was unambiguously identified as 2-
(methylsulfanyl)aniline by comparison to all three possible
regioisomers and is a new natural product. This metabolite has
been discussed as a degradation product of benzothiazole
(220),49 which was, however, not emitted by S. bottropensis, but
only by a few other strains. The thiazole derivative
acetylthiazole (219) also occurred in some bacterial bouquets.
The new natural product thiophene-2-carboxylate (221) is the
sulfur analogue of the furan derivative 192 and was found in S.
nassauensis. The compound 2-methyltetrahydrothiophen-3-one
(222) is derived from homocysteine and pyruvate50 and was
detected in Saccharopolyspora spinosa.
Figure 9. Sulfur compounds.
from R. xylanophilus and S. bottropensis being particularly rich in
these compounds. The most widespread sulfur compounds
were the dimethyl polysulfides ranging from dimethyl disulfide
(198) to dimethyl pentasulfide (201). The oxidation product of
198, S-methyl methanethiosulfonate (202), was also present in
various samples, while the formal oxidation product of dimethyl
sulfide, dimethyl sulfoxide (203), was detected only in S.
auratus. The chlorinated sulfur volatile dichloromethyl methyl
disulfide (204) was tentatively identified from its mass
spectrum and was found in R. xylanophilus, S. griseus, and
Nitrogen Compounds. Several alkylated pyrazines (223−
235) were identified (Figure 10). Some of these pyrazines such
as 2,5-dimethylpyrazine (226) were particularly widespread,
whereas others occurred in only one or a few samples, e.g., 2-
ethyl-3,5-dimethylpyrazine (233) from Streptomyces sp. Tu
̈
6071. The biosynthesis of alkylated pyrazines has recently been
investigated in our laboratories and proceeds via acetoin and its
derivatives.5 For this reason acetoins (cf. the discussion of
aldehydes and ketones above) and pyrazines frequently co-
Figure 10. Nitrogen compounds.
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occur in one species. Furthermore, a few acetylpyrazines were
identified including the stem compound acetylpyrazine (236),
2-acetyl-5-methylpyrazine (237), and 2-acetyl-6-methylpyrazine
(238), which are all produced by T. curvata.
Two derivatives of anthranilic acid were released by a few
species. These were identified as 2-aminoacetophenone (239)
and methyl anthranilate (240), the latter being one of the main
components from Haliangium ochraceum. The pyrrole deriva-
tive 2-acetylpyrrole (241) was found in S. erythraea, S.
pristinaespiralis, and T. curvata, while methyl pyrrole-2-
carboxylate (242) completed the series of structurally related
five-membered heterocycles in S. nassauensis, in which the
respective furan and thiophene derivatives (194 and 221) were
also found (cf. above). The compound 242 is known as a trail
pheromone of the ant Metapone madagascaria.51 Indole (243)
was present in the extracts of three species, while tetrahy-
dropyridine (244) was a headspace constituent of T. curvata
and is known to be generated from lysine via cadaverine. It
serves as a reactive building block in the biosynthesis of various
piperidine alkaloids such as isopelletierine and anabasine from
Nicotiana.52 However, no piperidine alkaloid is known from T.
curvata that could rationalize the occurrence of 244 in this
species. The cyanides nicotinonitrile (3-cyanopyridine, 245)
and phenylacetonitrile (246) were found in T. curvata and A.
mirum, respectively. Finally, S. viridochromogenes released
isobutylaldoxime (247) and 1-nitro-2-methylpropane (248),
which are structurally and biosynthetically related. The
biosynthesis of the unusual volatiles 247 and 248 was
investigated in a feeding experiment with isotopically labeled
[2H8]valine in which a maximum incorporation of seven to
eight deuterium atoms with a ratio of ca. 1:1 into both
compounds was observed by GC-MS (Figure 2 of the SI). The
delineated biosynthetic pathway (Scheme 1) starts with the
Figure 11. Other compounds.
it was impossible to assign the structures of a distinct
diastereoisomer. The spiroketal 252 is a repellant or
antiaggregative pheromone of Paravespula vulgaris,53 while
253 is an aggregation pheromone of the beetle Pityogenes
chalcographus.54 Finally, 6-methylmaltol (254) was identified in
S. clavuligerus.
Synthesis of Reference Compounds. For unambiguous
identification several reference compounds were synthesized.
All synthetic compounds were identical to the natural products
as discussed above and listed in Table 2 of the SI.
For the synthesis of cyclohept-4-enol (28) and the
corresponding ketone 84 homoallylmagnesium bromide was
reacted with ethyl formate (255) to yield the alcohol 256
(Scheme 2). Ring-closing metathesis with Grubbs’ catalyst of
the second generation resulted in 28, while 84 was available
from 256 by a sequence of oxidation with PCC followed by
olefine metathesis.55 The β-hydroxy esters 108 and 109 were
prepared by the addition of the ester enolate of methyl acetate
to acetone or 2-methylbutanal, respectively. In contrast, the α-
hydroxy esters 105 and 107 were obtained by transformation of
the commercially available carboxylic acids into the methyl
esters using diazomethane. The lactones 126−128 were
synthesized by the addition of the required Grignard reagent
to ethyl levulinate (263),56 while the addition to the ester
function was avoided by careful control of the reaction
conditions. The adducts cyclized spontaneously to the lactones
during moderately acidic workup with ammonium chloride.
Treatment of lactone 127 with phosphoric acid and P2O5 gave
the ketone 262,57 which was methylated using LDA and MeI to
yield 82.
Scheme 1
Mellein (181) and the biosynthetically related compounds
methyl 6-methylsalicylate (179) and methyl 6-ethylsalicylate
(180) were prepared from 2,6-dihydroxybenzoic acid (263,
Scheme 3). The acid 263 was protected as acetonide 264,58
followed by conversion of the free phenol into the triflate
265.59 A Stille coupling with allyltributylstannane yielded 266,
which was deprotected with potassium hydroxide to give 6-
allylsalicylic acid (270).60,61 A metal-catalyzed cyclization62
resulted in the target compound mellein. The copper-catalyzed
methylation63 of 265 provided 267, which upon reaction with
potassium carbonate in methanol yielded in 179. Since a direct
ethylation of 265 using ethyllithium and ethyl iodide failed, the
synthesis of 180 was carried out by radical bromination63 of
267 to the benzyl bromide 268 and subsequent cross coupling
with methylmagnesium bromide using Kochi’s catalyst.64
For the identification of the dimethyl furandioate 195
released by S. griseoflavus and S. parvulus three possible
regioisomers were prepared, while the fourth regioisomer
dimethyl furan-3,4-dioate was commercially available. These
compounds show very similar mass spectra, and therefore the
natural product could be unambiguously identified only based
on its retention index in comparison to all four standards. The
pyridoxal phosphate-dependent decarboxylation of valine to
isobutylamine followed by oxidations via the imine and the
oxime 247 to the nitrite 248. The oxidation of isobutylamine to
the imine may proceed with low stereospecificity, thus resulting
in the loss of either a proton or a deuterium in this step. An
alternative pathway that cannot be distinguished on the basis of
our feeding experiment is the oxidation of isobutylamine via N-
isobutylhydroxylamine to the oxime 247.
Other Compounds. A set of epoxides (249−251) were
emitted by S. spinosa (Figure 11). Most interestingly, in several
actinomycetes the spiroketals conophtorin (252) and chalcog-
ran (253) were detected. In contrast to the two diaster-
eoisomers of 252, the diastereoisomers of 253 could be
chromatographically separated, but without reference material
H
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Scheme 2
Scheme 3
2,4-regioisomer was synthesized by methylation of the
monomethyl ester 273 with diazomethane (Scheme 4). The
preparation of 273 from furan-3-carbaldehyde (271) via
carboxymethylation with methyl chloroformate to 272 and
and 194 were obtained by methylation of the corresponding
carboxylic acids.
The synthesis of 2-pentylfuran (190) was performed by
deprotonation of furan with n-BuLi followed by treatment with
oxidation was published by Sußmuth.65 The 2,5-regioisomer
̈
pentyl iodide (Scheme 5).11 The heterocyclic compounds
methyl thiophene-2-carboxylate (221) and methyl pyrrole-2-
carboxylate (242) from S. nassauensis were prepared with
diazomethane from the corresponding carboxylic acids. The
unusual nitrite 248 was obtained from isobutyl bromide (284)
by nucleophilic substitution with sodium nitrite. Finally, the
epoxides 250 and 251 were synthesized from the respective
alkenes by oxidation with m-CPBA.
275 was easily obtained from the commercially available diacid
274 by esterification with diazomethane, while the 2,3-
regioisomer (278) was synthesized from furan-2-carboxylic
acid (276) by carboxymethylation with LDA and methyl
cyanoformate to the monomethylester 277. Subsequent
esterification with diazomethane furnished 278. Comparison
of all three synthetic regioisomers and the commercially
available fourth regioisomer to the natural volatile established
its structure as dimethyl furan-2,4-dioate (195). The esters 193
I
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Article
Scheme 4
viously reported from any organism, thus demonstrating that
bacterial volatiles can be a rich source of new natural products.
Furthermore, known natural products can be identified in
previously unrecognized sources, as shown for mellein from S.
erythraea, for which the iterative PKS has recently been
reported based on in vitro experiments.47 The large structural
diversity of bacterial volatiles also reflects the fascinating
biosynthetic potential of microorganisms. The biosynthetic
capacity was highlighted by the occurrence of 1-nitro-2-
methylpropane (248) in S. viridochromogenes. Feeding experi-
ments with isotopically labeled precursors can easily be
performed with agar plate cultures using the CLSA headspace
technique and GC-MS analysis. This approach demonstrated
that the nitro compound 248 is formed from valine by
decarboxylation and successive oxidation steps via the
corresponding aldoxime. A prominent example for a similar
formation of a nitro group has been reported for the
biosynthesis of the p-nitrobenzoate starter unit for the
polyketide aureothin in Streptomyces thioluteus from p-amino-
benzoate.66
Scheme 5
In some cases bacterial volatiles were recognized as
biosynthetic shunt products of other known metabolites; for
example, 2,3,5-cyclopent-2-enone (82) found in S. coelicolor is
obviously related to the methylenomycin biosynthesis,21 and
the ethyl branched ketone 61 coincides nicely with the
occurrence of similarly ethyl branched blastmycinones in S.
bottropensis.4 Several of the other volatiles may potentially be
shunt products of biosynthetic pathways to yet unidentified
larger secondary metabolites. Another important finding of this
study is the bacterial origin of several compounds that are
known as insect pheromones including methyl 6-methylsalicy-
late (179), methyl 6-ethylsalicylate (180), mellein (181),
pyrrole-2-carboxylate (242), conophthorin (252), and chalcog-
ran (253). Together with the accumulating knowledge about
symbiotic relationships between actinomycetes and insects67
this questions the origin of these compounds in the arthropods.
Further research will be required to clarify this interesting
point. The odor marks set by many highly productive bacteria
CONCLUSIONS
■
Within the headspace extracts of 50 bacterial strains
investigated in this study, 254 compounds from various classes
were identified, excluding terpenes, which have been reported
separately.3 Although a large number of bacterial volatiles are
already known,13,14 our analyses demonstrate that new
compounds can still be discovered. In particular, the
compounds cyclohept-4-enol (28), cyclohept-4-enone (84),
2,3,5-cyclopent-2-enone (82), methyl 5-methylheptanoate
(93), methyl 3-hydroxy-4-methylhexanoate (109), methyl 4,6-
dimethyloctanoate (97), methyl 4-methylfuran-2-carboxylate
(193), dichloromethyl methyl disulfide (204), tetrathiolane
(208), pentathiane (209), 2-(methylsulfanyl)aniline (218), and
methyl thiophene-2-carboxylate (221) have not been pre-
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Article
pressure 77.1 kPa, He 23.3 mL min−1, injection volume 1.5 μL,
transfer line 300 °C, electron energy 70 eV. The operation mode was
splitless (60 s valve time), and the carrier gas was He at 1.2 mL min−1.
The GC was programmed as follows: 5 min at 50 °C increasing at 5
°C min−1 to 320 °C. Retention indices (I) were determined from a
homologous series of n-alkanes (C8−C32).
such as actinomycetes, myxobacteria, and cyanobacteria can
easily be recognized by the earthy smell due to geosmin and 2-
methylisoborneol production, but we have demonstrated that
there is still more to be discovered within the scent of bacteria.
EXPERIMENTAL SECTION
■
ASSOCIATED CONTENT
* Supporting Information
General Experimental Procedures. Chemicals were purchased
from Acros Organics (Geel, Belgium) or Sigma Aldrich Chemie
GmbH (Steinheim, Germany) and used without further purification.
All nonaqueous reactions were performed under an inert atmosphere
(N2) in flame-dried flasks. Solvents were purified by distillation and
dried according to standard methods. For all general procedures, the
relative amounts of the reagents are given as equivalents (equiv)
referring to the molar ratios of the compounds, and the relative
amounts of the solvents are given as final concentrations of the
transformed starting material (set to 1.0 equiv). Melting points were
■
S
List of investigated strains and culture conditions, medium
compositions, tabulated results of analyses, chromatograms of
headspace extracts, mass spectra obtained in feeding experi-
ments, synthetic procedures, and spectroscopic data including
1H and 13C NMR spectra of synthetic compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
measured on a Buchi 530, UV spectra were obtained using a Varian
̈
Cary 100 Bio, and IR spectra were recorded with a Bruker Tensor 27
ATR (attenuated total reflectance). NMR spectra were recorded on
Bruker DRX-400 (400 MHz) or AV III-400 (400 MHz) spectrometers
AUTHOR INFORMATION
Corresponding Author
*Tel: +49-531-391-5264. Fax: +49-531-391-5272. E-mail: j.
dickschat@tu-bs.de.
■
1
and were referenced against TMS (δ = 0.00 ppm) for H NMR and
CHCl3 (δ = 77.16 ppm) for 13C NMR. GC-MS analyses of synthetic
products were carried out with an Agilent 7890A gas chromatograph
connected to an Agilent 5975C inert mass detector fitted with a BPX-5
(25 m, 0.25 mm i.d., 0.25 μm film) and, in the case of natural products,
also on a HP-5MS fused silica capillary column (25 m, 0.25 mm i.d.,
0.25 μm film). Instrumental parameters were (1) inlet pressure, 77.1
kPa, He 23.3 mL min−1, (2) injection volume, 2 μL, (3) transfer line,
300 °C, and (4) electron energy 70 eV. The GC was programmed as
follows: 5 min at 50 °C increasing at 10 °C min−1 to 320 °C, and
operated in split mode (20:1, 60 s valve time). The carrier gas was He
at 1 mL min−1. Retention indices (I) were determined from a
homologous series of n-alkanes (C8−C32). Thin-layer chromatography
was performed with 0.2 mm precoated plastic sheets (Polygram Sil G/
UV254, Machery-Nagel). Column chromatography was carried out
using Merck silica gel 60 (70−200 mesh).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was funded by the Deutsche Forschungsgemein-
schaft DFG with an Emmy-Noether fellowship (to J.S.D.,
DI1536/1-2), by a Ph.D. scholarship of the Beilstein-Institut
zur Forderung der Chemischen Wissenschaften (to P.R.), and
̈
by a DFG grant (“Duftstoffe aus Actinomyceten”, DI1536/2-1).
We thank J. Salas (Oviedo, Spain) for S. albus J1074, W.
Wohlleben (Tubingen) for S. griseoflavus Tu 4000, B. Gust and
̈
̈
L. Heide (Tubingen) for S. lividans TK 24, H.-P. Fiedler
̈
(Tubingen) for Streptomyces sp. Tu 6071 and S. violaceusniger
Tu 4113, S. Grond (Tubingen) for S. bottropensis Dra 17, S.
parvulus Tu 64, and Streptomyces sp. Go 22/15, D. Spiteller
̈
̈
Strains, Culture Conditions, and Feeding Experiments. All
bacterial strains (Table 1 of the SI) were precultured in the
appropriate liquid medium (for medium compositions cf. SI) until
full growth (ca. 3−7 days). Subsequently, 1 mL of liquid culture was
transferred to an agar plate containing the same medium, and the
plates were allowed to dry, incubated for 2−3 days, and subjected to
CLSA headspace analysis. In the case of Roseiflexus castenholzii, which
did not grow on agar plates, the liquid cultures were directly used for
sampling of volatiles by CLSA. For each medium a control experiment
with medium alone was performed. The compounds identified in these
control experiments are not listed in Table 2. For the feeding
experiment on the biosynthesis of compound 248 by S. viridochromo-
genes SFM agar was amended with [2H8]valine (1 mM) followed by
collection of the volatiles by CLSA.
̈
̈
̈
̈
(Konstanz) for S. odorifer DSM 40347, and the U.S.
Department of Agriculture for S. cattleya NRRL 8057, S.
filamentosus NRRL 11379 and NRRL 15998, Streptomyces
scabiei 87.22, and Streptomyces xinghaiensis S187. We thank S.
Schulz (Braunschweig) for support of our group.
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