Streptopyridines, volatile pyridine alkaloids produced by Streptomyces sp. FORM5

Article abstract of DOI:10.3762/bjoc.10.146

Streptomyces sp. FORM5 is a bacterium that is known to produce the antibiotic streptazolin and related compounds. We investigated the strain for the production of volatiles using the CLSA (closed-loop stripping analysis) method. Liquid and agar plate cultures revealed the formation of new 2-alkylpyridines (streptopyridines), structurally closely related to the already known 2-pentadienylpiperidines. The structures of the streptopyridines A to E were confirmed by total synthesis. The analysis of the liquid phase by solvent extraction or extraction with an Oasis adsorbent showed that streptazolin and 2-pentadienylpiperidine are the major compounds, while the streptopyridines are only minor components. In the gas phase, only the streptopyridines could be detected. Therefore, an orthogonal set of analysis is needed to assess the metabolic profile of bacteria, because volatile compounds are obviously overlooked by traditional analytical methods. The streptopyridines are strain specific volatiles that are accompanied by a broad range of headspace constituents that occur in many actinomycetes. Volatiles might be of ecological importance for the producing organism, and, as biosynthetic intermediates or shunt products, they can be useful as indicators of antibiotic production in a bacterium.

Full text of DOI:10.3762/bjoc.10.146

Streptopyridines, volatile pyridine alkaloids produced
by Streptomyces sp. FORM5
Ulrike Groenhagen, Michael Maczka, Jeroen S. Dickschat
and Stefan Schulz*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2014, 10, 1421–1432.
Institut für Organische Chemie, Technische Universität Braunschweig,
Hagenring 30, 38106 Braunschweig, Germany
doi:10.3762/bjoc.10.146
Received: 07 February 2014
Accepted: 28 May 2014
Published: 24 June 2014
Email:
Stefan Schulz* - stefan.schulz@tu-braunschweig.de
* Corresponding author
This article is part of the Thematic Series "Natural products in synthesis
and biosynthesis".
Keywords:
headspace analysis; natural products; polyketide biosynthesis;
pyridine derivatives; streptazolin; volatile compounds
Associate Editor: A. Kirschning
© 2014 Groenhagen et al; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
Streptomyces sp. FORM5 is a bacterium that is known to produce the antibiotic streptazolin and related compounds. We investi-
gated the strain for the production of volatiles using the CLSA (closed-loop stripping analysis) method. Liquid and agar plate
cultures revealed the formation of new 2-alkylpyridines (streptopyridines), structurally closely related to the already known
2-pentadienylpiperidines. The structures of the streptopyridines A to E were confirmed by total synthesis. The analysis of the liquid
phase by solvent extraction or extraction with an Oasis adsorbent showed that streptazolin and 2-pentadienylpiperidine are the
major compounds, while the streptopyridines are only minor components. In the gas phase, only the streptopyridines could be
detected. Therefore, an orthogonal set of analysis is needed to assess the metabolic profile of bacteria, because volatile compounds
are obviously overlooked by traditional analytical methods. The streptopyridines are strain specific volatiles that are accompanied
by a broad range of headspace constituents that occur in many actinomycetes. Volatiles might be of ecological importance for the
producing organism, and, as biosynthetic intermediates or shunt products, they can be useful as indicators of antibiotic production
in a bacterium.
Introduction
Actinomycetes are excellent producers of diverse and bioactive products as antibiotics, cytotoxic compounds, immunosuppres-
secondary metabolites. These metabolites belong to many sants etc. In addition, actinomycetes are also able to produce
different structural classes including polyketides, nonribosomal and release a wide variety of volatile compounds with bouquets
peptides, terpenoids, alkaloids, lipids and others. Such com- composed of up to 100 different compounds [1-6]. Major
pounds became a major source of biologically active natural volatile classes comprise aliphatic compounds derived from
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fatty acid metabolism, terpenes, aromatic compounds, sulfur
compounds, and pyrazines [1]. Apart from pyrazines, indole,
and a few strain specific compounds [2] such as methyl pyrrole-
2-carboxylate, emitted by Stackebrandtia nassauensis, or
2-acetylpyrrole from Saccharopolyspora erythraea, volatile
alkaloids are rarely produced by actinomycetes.
We became interested in the strain Streptomyces sp. FORM5 to
elucidate whether volatile formation is linked to the production
of other, usually less volatile secondary metabolites and
whether different compounds can be detected by headspace
analysis [7] compared to commonly used solvent extraction or
adsorption/extraction procedures. Strain FORM5 has been
reported to produce the tetrahydrocyclopenta[b]pyridine deriva-
tives streptazones B1 (1), B2 (2), C (3), the 4-pyridone deriva-
tive streptazone D (4) with a pentadienyl side chain, and strep-
tazolin (5) (Figure 1) [8]. Streptazolin is produced by several
Figure 1: Alkaloids produced by Streptomyces strain FORM5.
streptomycetes [8-12], and is formed biosynthetically by a analysis new secondary metabolites can be found that eluded
polyketide mechanism [13]. The respective polyketide synthase earlier analysis. The separate analysis of headspace and liquid
gene cluster has not been identified yet.
phase is complementary and in combination allows better evalu-
ation of the metabolic potential of an investigated microor-
The streptazones are relatively small compounds suggesting ganism.
that they may be volatile enough to find them in the headspace
above bacterial cultures, although the presence of hydrogen Results and Discussion
bond donor and acceptor sites hints to good solubility in the The volatiles released by agar plate cultures of strain Strepto-
aqueous phase.
myces sp. FORM5 were collected by CLSA for one day on a
charcoal filter and eluted with dichloromethane. The extract was
In our study the volatile bouquet of the actinomycete Strepto- then analyzed using GC–MS. More than 40 different com-
myces sp. FORM5 was investigated and several new 2-alky- pounds were identified in the headspace extract (Table 1,
lated pyridines were identified using the closed-loop stripping Figure 2, and Figure S1 in Supporting Information File 1).
analysis (CLSA) [7] headspace technique followed by GC–MS Nevertheless, the major compounds (compounds 8, 9, 11, and
analysis and synthesis of the target compounds for structure 12 in Figure 2) attracted our interest, because they were
verification. Agar plate cultures and liquid cultures were unknown, thus giving room for the discovery of new volatile
investigated and the liquid phase analyzed for the presence of secondary metabolites. High resolution GC–MS revealed a
secondary metabolites. The results showed that by headspace molecular composition of C10H11N (found 145.09136, calcd
Figure 2: Part of the total ion chromatogram of the headspace extract of Streptomyces sp. FORM5 with the structures of 2-propylpyridine (6),
2-pentylpyridine (7), (E)-2-(pent-3-en-1-yl)pyridine (8), 2-((1Z,3E)-penta-1,3-dien-1-yl)pyridine (9), 2-((1Z,3Z)-penta-1,3-dien-1-yl)pyridine (10),
2-((1E,3Z)-penta-1,3-dien-1-yl)pyridine (11), 2-((1E,3E)-penta-1,3-dien-1-yl)pyridine (12).
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145.08910) for the major compound 12 and C10H13N (found erentially (1E)-configured products. After conversion of 17 into
147.10428, calcd 147.10475) for the minor component 8. The the respective Wittig salt 18 and reaction with 2-pyridinecarb-
mass spectrum (Figure 3) of the major compound pointed to the aldehyde (19) a mixture of four diastereoisomers of 2-(1,3-
presence of an extended aromatic and conjugated π-system and pentadienyl)pyridine was formed (Figure 4), all showing similar
the formation of a stable [M − 1]+ ion. The methyl loss to form mass spectra. These four diastereomers proved to be identical to
the base peak [M − 15]+ indicated the presence of a methyl the natural compounds 9 to 12 by comparison of mass spectra
group in the compound. Careful analysis of the extract revealed and GC retention. The major compound, 2-((1E,3E)-penta-1,3-
that a small peak (7) eluted slightly earlier than compound 8. dienyl)pyridine (12), and the 1Z,3E-isomer 9 could be isolated
The mass spectrum of compound 7 matched that of in almost pure form, but a large amount of material was lost
2-pentylpyridine, present in public databases [14]. From these during the purification process (17% yield for compound 12 and
data we concluded that the unknown compound 12 might be a 3% yield for compound 9 after purification). The large coupling
2-(pentadienyl)pyridine with conjugated side chain and that the constants 3J around 15 Hz between H-1 and H-2 (15.8 Hz) as
compounds 9–12 might be diastereomers. Compound 8 should well as H-3 and H-4 (15.1 Hz) in the side chain indicated the
carry a pentenyl side chain, while compound 6 showed a mass E-configuration of both double bonds for compound 12. The
spectrum identical to the known one of 2-propylpyridine.
mass spectrum and retention time proved to be identical to com-
pound 12, the major compound of the natural extract. We
The target compounds were then synthesized to prove the struc- propose the name streptopyridine A for this new natural com-
tural proposal. Pentadienylpyridines 9–12 were synthesized by pound. The minor product isolated pure under these conditions
Wittig reaction using different conditions (Scheme 1). A was 2-((1Z,3E)-penta-1,3-dienyl)pyridine (9, streptopyridine B),
Wittig–Schlosser reaction [15] starting from a commercially indicated by the coupling constants 3J1,2 = 11.8 Hz and 3J3,4 =
available 5:1 E/Z-mixture of 1-bromobut-2-ene (17) led to pref- 15.1 Hz.
Figure 3: Mass spectra of a) (E)-2-(pent-3-en-1-yl)pyridine (streptopyridine E, 8), b) (1Z,3E)-penta-1,3-dien-1-yl)pyridine (streptopyridine A, 9),
c) 2-((1E,3E)-penta-1,3-dien-1-yl)pyridine (streptopyridine B, 12).
Scheme 1: Synthesis of streptopyridines A to E (8–12) and the 2-alkylpyridines 6 and 7.
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Beilstein J. Org. Chem. 2014, 10, 1421–1432.
To delineate the stereochemistry of the other two isomers 10 fried chicken [18], or 2-propylpyridine (6), present in sesame
and 11, the conditions of the Wittig reaction were changed to seed oil [19], but are not known from bacteria. Natural prod-
favor Z-configured products by using NaHMDS as base. Again, ucts of bacteria containing a pyridine ring are rare. As an
all four diastereomers were formed (Figure 4). Together with example, 1-(2-pyridinyl)ethanone was identified as a volatile of
knowledge of the structures of 9 and 12, the product ratio in the Enterobacter agglomerans [20]. Highly substituted pyridine
Z-selective reaction indicates that compound 10 (streptopyri- derivatives can be found in bacterial thiopeptide antibiotics
dine C) is 2-((1Z,3Z)-penta-1,3-dienyl)pyridine, while com- [21].
pound 11 is 2-((1E,3Z)-penta-1,3-dienyl)pyridine (streptopyri-
dine D): The formation of only small amounts of 11 indicates The streptopyridines of Streptomyces sp. FORM5 are struc-
that this compound is 1E,3Z-configured, because it is disfa- turally related to known secondary metabolites by Strepto-
vored by the 1Z-selective reaction conditions and the minor myces. The piperidine derivatives 2-((1E,3E)-1,3-penta-
amounts of the Z-configured isomer in the diastereomeric mix- dienyl)piperidine (20) and 2-((1E,3E)-1,3-pentadienyl)-
ture of Wittig salts. Conclusively, 10 must be the last possible piperidin-4-ol (SS20846A, 21, Figure 5) have been reported
1Z,3Z-stereoisomer. Both compounds 10 and 11 could not be from other streptomycetes [8,22,23]. They constitute hydro-
isolated in pure form, but their mass spectra and retention times genated analogs of the streptopyridines and occur together with
were identical to those of the natural products.
streptazolin (5).
Figure 4: Total ion chromatograms of the product mixtures of isomers
9–12 synthesized under E-selective (a) and Z-selective Wittig reaction
conditions (b).
Figure 5: Structures of piperidine derivatives 20–26.
The mass spectrum of compound 8 indicated that the side chain
contained only one double bond, the position of which had to be
determined. A 1-pentenyl side chain seemed unlikely because
of the high abundance of the m/z 93 ion in the mass spectrum of
8, which we assumed is a McLafferty ion that is commonly
observed in 2-alkylpyridines [16]. Basing on the structure of
streptopyridine A, a 3-pentenyl side chain seemed to be most
likely. Coupling of (E)-3-pentenylmagnesium bromide with
2-chloropyridine under Fürstner conditions with iron(III) acetyl-
acetonate as catalyst [17] yielded (E)-2-(pent-3-enyl)pyridine
(8) that proved to be identical to the natural compound, now
called streptopyridine E. The mass spectra of 2-(pent-1-
enyl)pyridine and 2-(pent-2-enyl)pyridine, compounds synthe-
sized for comparison, are shown in Supporting Information
File 1 and differ from that of 8. Finally, pyridines 6 and 7 were
synthesized also by Fürstner cross-coupling and proved to be
identical to the natural products.
We then tested whether the streptopyridines can also be
detected under conventional cultivation and isolation pro-
cedures. Therefore, strain FORM5 was grown as liquid culture.
Headspace analysis revealed the production of the streptopy-
ridines also under these conditions. The liquid cultures were
either extracted with ethyl acetate (E-extract) or filtered over
Oasis adsorption material. The latter was than extracted with
ethyl acetate (O-extract). These extracts were analyzed by
GC–MS. While the E-extract did not show the presence of any
streptopyridine, they were present as trace components in the
O-extract. Both extracts showed two major constituents, one
with a molecular mass of 151 u (C10H17N, HRMS found
151.13656, calcd 151.13605), and the other with a molecular
mass of 207 u (C11H13NO3, HRMS found 207.09056, calcd
207.08949). Although the compounds were not isolated, the
mass spectral data and the previous reports of this class of com-
pounds from Streptomyces led us to conclude that these com-
2-Alkylpyridines have been reported earlier as aroma compo- pounds are indeed streptazolin (5, molecular mass 207) and
nents, like 2-butylpyridine or 2-pentylpyridine (7) identified in 2-(penta-1,3-dien-1-yl)piperidine (20, molecular mass 151,
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mass spectra and high resolution data see Supporting Informa- mass spectrum see Supporting Information File 1). Its oxi-
tion File 1). Both compounds are accompanied by diastereo- dation product is streptazone D (4) in which the ring double
mers exhibiting the same mass spectrum but different retention bond moves into conjugation with the carbonyl group by
times. Minor components of the extracts could be tentatively imine–enamine tautomerisation. Finally compound 23 is
assigned by their mass spectra to be the hydroxypiperidine 21, produced, a derivative of piperidine 21 with an additional
occurring again as a pair of diastereomers, and streptazone D hydroxy group with unknown location in the ring. Compounds
(4). The mass spectrum of 4 matches the published data [8]. 22–25 (Figure 5) as well as the streptazolin isomer have not
Streptazone B1/B2 and/or C elute as broad peak from the GC been reported from nature before.
column; a discrimination basing on the mass spectrum is not
possible.
We then tested whether the streptopyridines are biosyntheti-
cally produced via a polyketide sequence similar to that
Several additional N-containing compounds occur in the reported for streptazolin, involving a pentaketide precursor [13],
extracts, some of them can be tentatively identified basing on or whether a specific pyridine precursor, e.g., pipecolic acid,
their mass spectra. Two compounds exhibiting a small molec- with chain elongation was used. Feeding experiments with
ular ion at m/z 153 and a dominating base peak at m/z 84 are 13C2-sodium acetate showed incorporation of up to five acetate
likely 2-(3-pentenyl)piperidines (22, mass spectrum see units by GC–MS, indicated by a mass shift of 10 amu for the
Supporting Information File 1). The ion m/z 84 is typical for molecular ion, from m/z 145 to m/z 155 (Figure 6a and 6b).
2-alkylpiperidines [24] and cannot be formed in piperidine 20 Because of the dilution with unlabelled acetate the 13C10
because of the adjacent double bond. The 3-pentenyl side chain isotopomer is of low abundance (Figure 6c). Nevertheless, the
is also supported by a small ion series at m/z 94 and 108 and dose dependent incorporation of the acetate units clearly
present in streptopyridine E (8) and streptenols A (28) and B, showed the presence of this isotopomer. Feeding of 2 mM
biosynthetic precursors of this compound family (see below). sodium 13C2-acetate to agar plate cultures showed double
Another compound, only present in the O-extract exhibits a incorporation compared to 1 mM sodium 13C2-acetate
molecular mass of 149 u, 2 units less than the major compound (Figure 6d). The results hint to a biosynthesis of the streptopy-
20. The mass spectrum (see Supporting Information File 1) ridines via the polyketide pathway.
resembles that of 20. The additional double bond resides most
likely at the nitrogen, arriving at the 1-piperideine structure 24. The proposed biosynthesis of the streptopyridines is shown in
A similar structure has the antibiotic nigrifactin (26), a hexake- Scheme 2. The streptopyridines might be as well as the strepta-
tide produced by S. nigrifaciens, differing only in the length of zones, streptenols (27) and piperidinols (20, 21) [8,9] precur-
the side chain [25]. Piperideine 24 lacks a N–H and might get sors or side products in the biosynthesis of streptazolin (5) [8].
lost during work-up of the E-extract, contrary to the other The biosynthesis of 5 has been investigated earlier [13]. Mayer
alkylpiperidines. It can be detected as trace component in the and Thiericke proposed a pentaketide precursor 34 that is trans-
headspace extract as well (see Table 1). As with piperidines 20 formed into the amine 33. This amine is N-carboxylated,
and 21, a hydroxypiperideine 25 seems to be present (Figure 5, cyclized, and further processed to form 5 [13].
Figure 6: a) Mass spectrum of streptopyridine A (12), b) mass spectrum of 12 after feeding of 2 mM 13C2-sodium acetate, c) single ion chro-
matogram of 151, 153, 155 m/z after feeding with 2 mM 13C2-sodium acetate; d) single ion chromatogram of the 153 m/z molecular ion after feeding
with 2 mM 13C2-acetate, 1 mM sodium 13C2-acetate, and a non fed culture as comparison. The ions at m/z 151, 153, and 155 indicate the incorpor-
ation of three, four, or five 13C2-acetate units.
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Scheme 2: Proposed biosynthesis of the streptopyridines. PKS: polyketide synthase; red: reduction; ta: transamination; ox: oxidation; elim: elimina-
tion.
The streptopyridines might similarly be formed from the luteogriseus FH-S 1307 [9], S. fimbriatus [26], S. cirratus [27],
pentaketide 29, showing the reduction status occurring in the and Streptomyces sp. HS-HY-045 [28]. This might indicate that
previously isolated streptenols A and C (27) [9]. Reduction and the double bond hydrogenation occurs already during the
transamination leads to the amine 30 that cyclizes to the pentaketide biosynthesis in these cases. Similarly, additional
piperideine 25. Elimination of water leads to the dihydropyri- hydrogenation leads to the saturated alkylpyridine 7 and a
dine 31 that is oxidized to the major streptopyridine 12 possibly tetraketide to propylpyridine (6).
by spontaneous autoxidation in air. Isomerization of the double
bonds during this pathway may lead to the isomers 9–11. In the bouquet of the headspace extract of Streptomyces strain
Reduction of 25 would lead to piperidine SS 20846A (21) FORM5 several other compounds besides the pyridines 6–12
[9,23]. Oxidation of intermediate 25 can also form streptazone were identified (Figure 7, Table 1). The most abundant of these
D (4) by oxidation which would concomitantly shift the double were dimethyl disulfide (35), accompanied by other sulfur
bond into conjugation to the carbonyl group. Reductive removal components as dimethyl trisulfide (36), dimethyl tetrasulfide
of the hydroxy group, e.g., via 31 followed by double bond (37), and S-methyl methanethiosulfonate (50). The hydroxyke-
reduction may lead to piperideine 24, a precursor of the major tones acetoin (40), and longer variants 38, 39, 41, and 42 occur
liquid phase component 22. Alternatively, this compound might often in bacteria and are precursors of alkylated pyrazines [29]
originate from the reduced PKS precursor 32 that is trans- like 2,5-dimethylpyrazine (44). Aldehydes, ketones, and
formed via 33 into 24. Piperidine 22 is then obtained via imine aromatic compounds commonly found as volatiles from
reduction as described. On the other hand, this pathway may bacteria were present in trace amounts: 5-hepten-2-one (43),
also be the entry into the streptopyridine formation by oxi- 2-acetylfuran (45), 3-octanone (48), nonanal (54), decanal (55),
dation of 24 to, e.g., 31, followed by final oxidation.
1-phenyl-2-propanone (57), 1-phenyl-1,2-propandione (59),
benzaldehyde (46), methyl benzoate (51), 2-phenylethanol (56),
A 3-pentenyl side chain as found in streptopyridine 8 and other 1-phenyl-2-propanol (58), ethyl benzoate (52), and methyl
derivatives occurs also in streptenol A (28), isolated from S. 2-phenylacetate (53) [2]. Of special interest is 5-hepten-2-one
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Beilstein J. Org. Chem. 2014, 10, 1421–1432.
Figure 7: Compounds detected in the headspace of Streptomyces sp. FORM5.
Table 1: Volatile compounds identified in the headspace extract of Streptomyces strain FORM5. The amounts of the compounds are given as 0–2%
(x), 2–8% (xx), >8% (xxx) relative to the largest peak area in the total ion chromatogram. The identification of the compound based on comparison of
mass spectrum to a data base spectrum (ms), comparison of retention index to a published retention index on the same or similar GC fused silica
capillary column (ri), and/or based on comparison to a synthetic or commercially available reference compound (std).
GCa
compoundb
I (exp.)
I (lit.)c
Ident.
FORM5
ms, std
ms, std
ms, ri
acetoin (40)
n.d.d
n.d.
xx
xx
xx
x
a
b
dimethyl disulfide (35)
2-hydroxypentan-3-one (38)
3-hydroxypentan-2-one (41)
5-hepten-2-one (43)
829
822*
838
ms, ri
833
ms
c
909
x
ms
d
2-hydroxyhexan-3-one (39)
3-hydroxyhexan-2-one (42)
2,5-dimethylpyrazine (44)
2-acetylfuran (45)
910
x
ms
914
x
e
f
925
925
923
978
978
987
994
1001
1024
ms, ri, std
ms, ri, std
ms, ri, std
ms, ri, std
ms, ri, std
ms, ri, std
ms, ri, std
ms, ri, std
x
925
x
g
h
i
benzaldehyde (46)
977
x
dimethyl trisulfide (36)
1-octen-3-ol (47)
979
x
987
x
j
3-octanone (48)
994
x
k
l
2-propylpyridine (6)
1000
1023
1082
1083
1108
1116
1128
1129
1143
1147
1183
1189
1199
1205
x
cyclohept-4-enone (49)
m/z = 118, 105, 77, 51, 39
S-methyl methanethiosulphonate (50)
methyl benzoate (51)
nonanal (54)
x
n
o
x
1083
1104
1116
ms, ri
x
ms, ri, std
ms, ri, std
x
p
q
x
m/z = 106, 135, 79
x
2-phenylethanol (56)
1-phenyl-2-propanone (57)
1-phenyl-2-propanol (58)
1-phenyl-1,2-propandione (59)
methyl 2-phenylacetate (53)
2-methylisoborneol (60)
2-pentylpyridine (7)
1129
ms, ri, std
ms
x
x
ms
x
1186
1187
1201
1205
ms, ri,
ms
x
x
ms, ri, std
ms, ri, std
x
r
x
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Table 1: Volatile compounds identified in the headspace extract of Streptomyces strain FORM5. The amounts of the compounds are given as 0–2%
(x), 2–8% (xx), >8% (xxx) relative to the largest peak area in the total ion chromatogram. The identification of the compound based on comparison of
mass spectrum to a data base spectrum (ms), comparison of retention index to a published retention index on the same or similar GC fused silica
capillary column (ri), and/or based on comparison to a synthetic or commercially available reference compound (std). (continued)
s
t
(E)-2-(pent-3-enyl)pyridine (8)
m/z = 132, 93, 118, 41, 106
decanal (55)
1207
1212
1216
1219
1233
1246
1256
1283
1321
1327
1352
1362
1366
1429
1465
1499
1529
1208
1216
ms, ri, std
ms, ri, std
xxx
x
u
v
x
m/z = 118, 93, 46, 130, 52
dimethyl tetrasulfide (37)
benzothiazole (61)
x
1234
1246
1252
ms, ri, std
ms, ri, std
ms
x
x
ethyl 2-phenylacetate
x
w
m/z = 118, 147, 132, 91, 51
m/z = 79, 104, 133, 51, 117
2-((1Z,3E)-penta-1,3-dienyl)pyridine (9)
2-((1Z,3Z)-penta-1,3-dienyl)pyridine (10)
2-((1E,3Z)-penta-1,3-dienyl)pyridine (11)
2-((1E,3E)-penta-1,3-dienyl)pyridine (12)
geosmin (62)
x
x
x
y
1334
1358
1363
1368
1430
ms, ri, std
ms, ri, std
ms, ri, std
ms, ri, std
ms, ri, std
xx
xxx
xx
xxx
x
z
aa
ab
ac
ad
ae
af
m/z = 105, 120, 91, 204, 176
valencene (63)
x
1498
ms, ri, std
ms
x
m/z = 148, 163, 120
x
aCompound assignment refers to Figure S2 (Supporting Information File 1), bartefacts found in both control and inoculated samples are not listed,
ctaken from NIST Chemistry WebBook [14] or our own data base, *DB-5, A: artifact; dnot determined.
(43) that may be a shunt product of the streptopyridine E as described. This consecutive approach allows a broad
biosynthesis: It contains a 3-pentenyl chain motif adjacent to a overview on the metabolites. The E- or O-extracts can be
carbonyl group, thus resembling the structural requirements of a analyzed by HPLC–MS and GC–MS. Small, basic compounds
possible streptopyridine E biosynthesis that follows the logic as as the piperidines are released from the liquid medium and can
presented in Scheme 2. Cyclohept-4-enone (49) has recently be detected only by the extraction methods. Their basicity and/
been reported from several actinomycetes [2]. Some terpenes or their ability to interact by hydrogen bonding with water obvi-
could be identified as well. 2-Methylisoborneol (60) and ously prevent release from the water phase in substantial
geosmin (62) are commonly produced by Streptomyces and amounts. Volatile compounds like the streptopyridines are less
other actinomycetes [3,30]. In addition, the sesquiterpene soluble, have a lower basicity and can be detected as major
valencene (63) was found, but the amount of terpenes released compounds in the headspace. Both analytical methods have a
is quite low compared to other streptomycetes. Some nitrogen different analytical window and complement each other.
containing trace components could not be identified. In the E-
and O-extracts similarly other nitrogen containing trace compo- The detected volatiles fall into different groups. Most of the
nents occurred, although the small amounts excluded their iden- compounds listed in Table 1 can be regarded as volatiles
tification.
commonly produced by various bacteria. That does not imply
that most bacteria release them, but that these volatiles often
The streptopyridines showed only weak antibacterial and cyto- occur when bacteria are analyzed. These compounds form a
static activity (R. Müller, pers. commun.), similar to that chemical structure space of volatiles released by bacteria and
described earlier by us for 2-pentylpyridine [31]. The analysis their number is limited, although this space is certainly only
of the 16S-RNA revealed strain FORM5 to be a Streptomyces. partially explored. Another group of volatile compounds
It showed 99% sequence similarity to Streptomyces forming a structure space is already known from the green part
griseosporus (R. Müller, pers. commun.).
of plants (excluding flowery parts). The plant structure space is
different from that emerging for bacteria. The bacterial struc-
The two analytical methods used in the current work are obvi- ture space includes acetoins and aromatic aldehydes, esters and
ously orthogonal to each other. They can be used on the same ketones, aliphatic compounds, sulfur compounds, but also
culture. After initial sampling of the headspace of a liquid 4-cycloheptenone that we found in several other bacteria
culture, the culture can be separated from the cells and extracted (unpublished results). The terpenes geosmin and methylisobor-
1428

Beilstein J. Org. Chem. 2014, 10, 1421–1432.
neol are typical terpenes of the actinomycetes. Various inert atmosphere (N2) in flame-dried flasks. Purification of the
sesquiterpene cyclases occur in the streptomycetes [3,30,32] synthetic products was carried out by flash chromatography
and give rise to a wide variety of sesquiterpenes, in this case using Merck silica gel 60 (70–200 mesh). Thin-layer chroma-
valencene.
tography was performed with 0.2 mm pre-coated polyester
sheets (Polygram SIL (G/UV254), Macherey-Nagel). NMR
The only strain specific compounds are the streptopyridines. spectra were obtained on either a Bruker DRX-400 (400 MHz)
They have not been reported from other strains and their or an AV III-400 (400 MHz) spectrometer and were referenced
biosynthesis seems tightly connected to the biosynthesis of against TMS (δ = 0.00 ppm) for 1H NMR and CHCl3 (δ =
streptazolin. A similar case has been reported for specific 77.16 ppm) for 13C NMR. GC–MS analysis were performed on
volatile butenolides released by streptomycetes producing the an Agilent 7890A gas chromatograph connected to an Agilent
antibiotic antimycin [33] and for other strains investigated by 6975 C inert mass detector fitted with a BPX-5 fused silica
us. The analysis of volatiles and detection of unique com- capillary column (25 m, 0.25 mm i.d., 0.25 μm film). Condi-
pounds may thus be used as a method to select microbial strains tions were as follows: inlet pressure 67 kPa, He 23.3 mL/min,
for the detection of non-volatile biologically active compounds. injection volume 1 µL, transfer line 300 °C, injector 250 °C,
Alternatively, two independent gene clusters may be respon- electron energy 70 eV. The gas chromatograph was
sible for streptazolin and streptopyridines.
programmed as follows: 5 min at 50 °C, then increasing with
5 °C/min to 320 °C. Linear retention indices were determined
The production of strain specific compounds connected to, e.g., from a homologous series of n-alkanes (C8–C32). Compounds
antibiotic biosynthesis might also have important ecological were identified by comparison of mass spectra to database
consequences. As an example, streptazolin producing strepto- spectra (Wiley 7, NIST 08 and our own created from synthe-
mycetes have been isolated from mud-dauber wasps [34]. They sized reference compounds), by comparison of the retention
have been postulated as antibiotic-producing symbionts of the index data to standards (own database and NIST Chemistry
wasps. If this is true, they should also produce specific volatiles WebBook (2013) [14]) and by synthesis of reference com-
as the streptopyridines and add to the odor of the insect. Given pounds.
the many different bacteria living on the insect, a specific odor
bouquet of the insect would arise, assembled by insect derived Organism and analysis
compounds and bacterial volatiles, finally carrying information Streptomyces strain FORM5 was isolated from a soil sample
on the individual symbiont composition. The symbiont compos- collected in Formentera (Spain) and is deposited at the Institute
ition might affect the fitness and, if it can be perceived by inter- of Organic Chemistry in Göttingen [8]. It was cultivated in
acting partners, may in the end influence the behavior of the 10 mL SM-media (20 g/L mannitol, 20g/L soy flour, 4 mL/L
insects, e.g., mate choice or aggression.
2.5 M magnesium chloride solution, 20 g/L agar only for plates)
at 28 °C for 3 days. SM-agar plates were then inoculated with
300 µL of the preculture and cultivated for 6 days at 28 °C.
Conclusion
In conclusion, the investigation of the headspace extract of Then the culture was analyzed by closed-loop stripping analysis
Streptomyces strain FORM5 revealed the occurrence of new at room temperature [7]. In this system, air is continuously
2-alkylpyridines that are structurally related to streptazolin and pumped (MB-21E, Senior Flextronics, USA) through the closed
2-pentadienylpiperidines produced by this strain. While they are system that contains an activated charcoal filter (Chromtech
the major compounds in the volatile bouquet, they occur only in GmbH, Idstein, Precision Charcoal Filter, 5 mg) and the agar
minor amounts in the liquid phase. In contrast, the major com- plate or liquid culture for 24 hours. The filter was then extracted
pounds streptazolin and 2-pentadienylpiperidine do not occur in by rinsing 3× with 15 µL dichloromethane (≥99.8%, Merck,
the headspace, thus proving the necessity to use orthogonal Germany) and the resulting headspace extract was analyzed by
analytical methods to assess the full metabolic potential of a GC–MS. The experiment was repeated at least three times. The
microorganism.
SM-medium was analyzed without inoculation as control.
Liquid cultures were analyzed similarly by the CLSA method.
After the collection of volatiles the liquid phase was analyzed
by two different methods. E-extract: The culture media was
Experimental
General experimental procedures
Reagents and solvents were purchased from Sigma-Aldrich centrifuged for 20 min at 4 °C and the supernatant was
Chemie GmbH (Steinheim, Germany) and Acros Organics extracted 3 times with 50 mL CH2Cl2. The extracts were dried
(Geel, Belgium) and used without further purification. Solvents with MgSO4, concentrated under reduced pressure and analyzed
were distilled before use and, if necessary, dried using standard by GC–MS. O-extract A Oasis® HLB cartridge (Waters) was
procedures. All non aqueous reactions were performed under an prewashed with 2 column volumes ethyl acetate and condi-
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Beilstein J. Org. Chem. 2014, 10, 1421–1432.
tioned with 2 column volumes water. The culture media was (m, 2H, 2× CH), 7.07–7.10 (m, 1H, CH), 7.13 (d, J = 7.8 Hz,
centrifuged for 20 min at 4 °C and the supernatant (100 mL) 1H, CH), 7.57 (td, J = 7.6, 1.9 Hz, 1H, CH), 8.51–8.53 (m, 1H,
filtered through the cartridge. The cartridge was extracted with CH); 13C NMR (CDCl3, 100 MHz) δ ppm 17.5 (CH3), 32.4
3 column volumes ethyl acetate, the eluent was dried with (CH2), 38.1 (CH2), 120.6 (CH2), 122.4 (CH2), 125.2 (CH),
MgSO4, concentrated under reduced pressure, and finally 129.9 (CH), 135.8 (CH), 148.8 (CH), 161.4 (C); EIMS m/z: 147
subjected to GC–MS analysis. For feeding experiments on the [M]+ (17), 146 (29), 133 (11), 132 (100), 119 (17), 118 (30),
biosynthesis of compound 7 the SM-media was enriched with 117 (47), 106 (25), 93 (77), 79 (17), 78 /14), 65 (18), 51 (13),
sodium 13C2-acetate (ISOTEC) in 1 mM (1.7 mg, 0.02 mmol, in 39 (19); HREIMS m/z: calcd for C10H13N, 147.1048; found,
20 mL medium) and 2 mM (3.4 mg, 0.04 mmol, in 20 mL 147.1054; GC (BPX-5) I = 1208.
medium) followed by collection of the volatiles by CLSA.
The synthesis of compounds 9 and 12 was achieved by Wittig
Synthesis of reference compounds
reaction starting with the synthesis of crotyltriphenylphosphon-
The compounds 6–8 were synthesized after the standard proce- ium bromide (18). Crotyl bromide (17, 2.23 g, 16.52 mmol) was
dure for iron-catalyzed aryl–alkyl cross-coupling described by added to a mixture of triphenylphosphine (3.67 g, 14 mmol) in
Fürstner et al [17].
THF and stirred under reflux overnight. THF was removed
under reduced pressure and the resulting solid was dissolved in
2-Propylpyridine (6): Yield (1.01 g, 8.35 mmol, 61%); Rf a mixture of CH2Cl2/MeOH (10:1). Column chromatography
(pentane/Et2O 5:1) 0.2; UV (CH2Cl2) λmax (log ε): 257 (5.77), on silica gel with CH2Cl2/MeOH (15:1, Rf = 0.25) yielded 18
262 (5.78) nm; IR (diamond) νmax: 2960, 2832, 2871, 1590, (5.12 g, 12.9 mmol, 92%) as a white solid. 1H NMR (CDCl3,
1569, 1472, 1434, 1149, 1051, 993, 751 cm−1; 1H NMR 200 MHz) δ ppm 1.35–1.42 (m, 3H, CH3), 1.58–1.65 (m, 3H,
(CDCl3, 400 MHz) δ ppm 0.97 (t, J = 7.4 Hz, 3H, CH3), 1.76 CH3), 4.54–4.69 (m, 4H, 2× CH2), 5.24–5.39 (m, 2H, 2× CH),
(sxt, J = 7.5 Hz, 2H, CH2), 2.74–2.79 (m, 2H, CH2), 7.07–7.11 5.87–6.04 (m, 2H, 2× CH), 7.65–7.92 (m, 30H, 30× CH);
(m, 1H, CH), 7.14 (dd, J = 7.8, 0.5 Hz, 1H, CH), 7.58 (tdd, J = 13C NMR (CDCl3, 50 MHz) δ ppm 18.1 (CH3), 18.2 (CH3),
7.6, 1.9, 0.9 Hz, 1H, CH), 8.52 (dt, J = 4.89, 0.94 Hz, 1H, CH); 27.2 (CH2), 28.2 (CH2), 114.6 (CH), 114.8 (CH), 117.0 (3× C),
13C NMR (CDCl3, 100 MHz) δ ppm 13.8 (CH3), 23.1 (CH2), 118.7 (3× C), 130.0 (6× CH), 130.3 (6× CH), 133.6 (6× CH),
40.4 (CH2), 120.8 (CH), 122.7 (CH), 136.1 (CH), 149.2 (CH), 133.8 (6× CH), 134.8 (3× CH), 134.9 (3× CH), 137.4 (CH),
162.3 (C); EIMS m/z: 121 [M]+ (2), 120 (9), 106 (29), 93 (100), 137.7 (CH); 31P NMR (CDCl3, 80 MHz) δ ppm 21.4 (s), 21.6
78 (12), 65 (18), 51 (15), 39 (18); HREIMS m/z: calcd for (s). At room temperature an ethereal phenyllithium solution
C8H11N, 121.0891; found, 121.0888; GC (BPX-5) I = 1001. (1.63 mL, 15.5 mmol) was added to a mixture of 18 (6.15 g,
15.5 mmol) in 30 mL dry THF and 45 mL dry diethyl ether.
2-Pentylpyridine (7): Yield (827 mg, 5.6 mmol, 56%); Rf After stirring for 20 min at rt the mixture was cooled down to
(pentane/Et2O 5:1) 0.2; UV (CH2Cl2) λmax (log ε): 257 (6.28), −78 °C and pyridine-2-carbaldehyde (19, 1.66 g, 15.5 mmol)
262 (6.29) nm; IR (diamond) νmax: 2955, 2928, 2858, 1590, was added. Afterwards the mixture was allowed to warm to
1472, 1434, 1148, 993 cm−1; 1H NMR (CDCl3, 400 MHz) δ −30 °C in 2 h and again phenyllithium solution (1.63 mL,
ppm 0.86–0.93 (m, 3H, CH3), 1.29–1.40 (m, 4H, 2× CH2), 15.5 mmol) was added. Then the mixture was cooled down
1.68–1.78 (m, 2H, CH2), 2.74–2.82 (m, 2H, CH2), 7.08 (ddd, J again to −78 °C and potassium tert-butoxide (2.61 g,
= 7.5, 4.9, 1.1 Hz, 1H, CH), 7.13 (dd, J = 7.8, 1.0 Hz, 1H, CH), 23.25 mmol) was added. Overnight the mixture was allowed to
7.53–7.60 (m, 1H, CH), 8.50–8.55 (m, 1H, CH); 13C NMR warm to rt and quenched with dest. water. The aqueous layer
(CDCl3, 100 MHz) δ ppm 13.9 (CH3), 22.5 (CH2), 29.5 (CH2), was extracted two times with diethyl ether. The combined
31.5 (CH2), 38.4 (CH2), 120.7 (CH), 122.6 (CH), 136.1 (CH), organic layers were dried with MgSO4 and the solvent was
149.1 (CH), 162.5 (C); EIMS m/z: 149 [M]+ (2), 93 (100), 120 removed under reduced pressure. Column chromatography on
(25), 106 (30), 92 (14), 78 (14), 65 (16), 51 (10), 39 (14); silica gel with pentane/diethyl ether (5:1) yielded 9 (0.36 mmol,
HREIMS m/z: calcd for C10H15N, 149.1204; found, 149.1203; 53 mg, Rf = 0.3, 3%) and 12 (2.22 mmol, 322 mg, Rf = 0.25,
GC (BPX-5) I = 1205.
14%) as yellow oils (crude yield was 62%).
2-((E)-Pent-3-en-1-yl)pyridine (8): Yield (326 mg, 2.2 mmol, 2-((1Z,3E)-Penta-1,3-dien-1-yl)pyridine (9): UV (CH2Cl2) λmax
16%); Rf (pentane/Et2O 5:1) 0.24; UV (CH2Cl2) λmax (log ε): (log ε): 271 (6.66), 300 (6.71), 308 (6.72) nm; IR (diamond)
257 (5.73), 262 (5.74) nm; IR (diamond) νmax: 3009, 2918, νmax: 3006, 2962, 2911, 1641, 1582, 1558, 1469, 1431, 1149,
2854, 1590, 1569, 1474, 1434, 1148, 1051, 966, 750 cm−1; 990, 833, 797, 741 cm−1; 1H NMR (CDCl3, 400 MHz) δ ppm
1H NMR (CDCl3, 400 MHz) δ ppm 1.62–1.64 (m, 3H, CH3), 1.85 (dd, J = 6.8, 1.7 Hz, 3H, CH3), 5.96 (dq, J = 15.2, 6.8 Hz,
2.38–2.44 (m, 2H, CH2), 2.82–2.85 (m, 2H, CH2), 5.41–5.52 1H, CH), 6.23 (d, J = 11.8 Hz, 1H, CH), 6.34 (dd, J = 11.5, 11.2
1430

Beilstein J. Org. Chem. 2014, 10, 1421–1432.
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7.9 Hz, 1H, CH), 7.40 (dd, J = 15.2, 11.0 Hz, 1H, CH), 7.61 (td,
J = 7.7, 1.9 Hz, 1H, CH), 8.60–8.62 (m, 1H, CH); 13C NMR
(CDCl3, 100 MHz) δ ppm 18.5 (CH3), 121.0 (CH), 124.1 (CH),
125.5 (CH), 128.8 (CH), 134.1 (CH), 135.2 (CH), 136.0 (CH),
149.3 (CH), 156.9 (C); EIMS m/z: 145 [M]+ (9), 144 (12), 143
(6), 142 (5), 131 (10), 130 (100), 117 (5), 103 (4), 89 (3), 78
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1429, 1145, 989, 796, 755, 740 cm−1; 1H NMR (CDCl3, 400
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15.7, 6.8 Hz, 1H, CH), 6.26 (dd, J = 15.1, 10.8 Hz, 1H, CH),
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Supporting Information
Supporting Information File 1
Total ion chromatograms of strain Streptomyces sp.
FORM5, mass spectra, 16S-RNA data, and 1H and
13C NMR spectra of the synthetic compounds.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-10-146-S1.pdf]
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Acknowledgements
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We thank Silke Wenzel and Jennifer Herrmann from the work-
group of Rolf Müller, HIPS, Saarbrücken, Germany, for
performing 16S-RNA classification and bioassays. We also
thank Axel Zeeck, Göttingen, for the strain FORM5.
doi:10.1039/c39730000075
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