Identification and synthesis of volatiles released by the myxobacterium Chondromyces crocatus

Article abstract of DOI:10.1016/j.tet.2004.03.005

Cultures of the myxobacterium Chondromyces crocatus on agar plates were analysed by closed-loop-stripping analysis or solid phase micro extraction. The odour profiles consist mainly of pyrazines, sesquiterpenoids and some aromatic compounds, summing up to more than 50 components. Several new pyrazines as 2-(1-hydroxy-1-methylethyl)-3-methoxypyrazine (9), 2-(1-hydroxy-1-methylpropyl)- 3-methoxypyrazine (10), and 2-(1-hydroxy-2-methylpropyl)-3-methoxypyrazine (11) were identified besides several known pyrazines. A major pyrazine occurring in most samples was 2,5-bis-(1-methylethyl)pyrazine (3). While the well known sesquiterpenoid geosmin (1) was present in low amounts, the related compound (1(10)E,5E)-germacradien-11-ol (21) was identified in most samples in larger quantities. Other prominent sesquiterpenoids not reported before from microorganisms were (6S,10S)-6,10-dimethylbicyclo[4.4.0]dec-1-en-3-one (16), which was accompanied by smaller amounts of several derivatives. The biosynthesis of these compounds is discussed in relation to the recently proposed biosynthetic pathways to 1 and 21.

Full text of DOI:10.1016/j.tet.2004.03.005

Tetrahedron 60 (2004) 3863–3872
Identification and synthesis of volatiles released by the
myxobacterium Chondromyces crocatus
a,*
a
Stefan Schulz, Jens Fuhlendorff and Hans Reichenbach
b
a
Institut f u¨ r Organische Chemie, Technische Universit a¨ t Braunschweig, Hagenring 30, D-38106 Braunschweig, Germany
Gesellschaft f u¨ r Biotechnologische Forschung, Abteilung Naturstoffbiologie, Mascheroder Weg 1, D-38124 Braunschweig, Germany
b
Received 18 November 2003; revised 9 February 2004; accepted 2 March 2004
Abstract—Cultures of the myxobacterium Chondromyces crocatus on agar plates were analysed by closed-loop-stripping analysis or solid
phase micro extraction. The odour profiles consist mainly of pyrazines, sesquiterpenoids and some aromatic compounds, summing up to
more than 50 components. Several new pyrazines as 2-(1-hydroxy-1-methylethyl)-3-methoxypyrazine (9), 2-(1-hydroxy-1-methylpropyl)-3-
methoxypyrazine (10), and 2-(1-hydroxy-2-methylpropyl)-3-methoxypyrazine (11) were identified besides several known pyrazines. A major
pyrazine occurring in most samples was 2,5-bis-(1-methylethyl)pyrazine (3). While the well known sesquiterpenoid geosmin (1) was present
in low amounts, the related compound (1(10)E,5E)-germacradien-11-ol (21) was identified in most samples in larger quantities. Other
prominent sesquiterpenoids not reported before from microorganisms were (6S,10S)-6,10-dimethylbicyclo[4.4.0]dec-1-en-3-one (16), which
was accompanied by smaller amounts of several derivatives. The biosynthesis of these compounds is discussed in relation to the recently
proposed biosynthetic pathways to 1 and 21.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Myxobacteria are a unique group of bacteria, characterised
by the formation of multicellular fruiting bodies, the most
complex behaviour so far observed in any procaryote. They
are also known to produce a wide variety of secondary
metabolites which often show high pharmacological or
1
Scheme 1.
fungicidal activity, e.g., the epothilones and myxalamides.
In contrast, the analysis of volatile compounds from
myxobacteria has received relatively little attention. The
widespread bacterial product geosmin (1) has been
2
. Results
2
identified in Nannocystis exedens (Scheme 1), and is
For the analyses of the volatiles, C. crocatus was grown on
agar plates on different media. Cultures between 15 and 24
days old were then sampled by headspace methods and the
obtained volatiles analysed by GC–MS. Two different
methods were used for the headspace analysis. Agar plates
were introduced in a custom made sample holder of a
present in many other species, detectable by its
characteristic odour. Stigmolone (2) has been isolated
from fruiting Stigmatella aurantiara and is believed to be
the pheromone responsible for the attraction of the cells in
3
,4
the fruiting body formation process of this species. In an
attempt to further characterise the metabolic spectrum of the
myxobacteria, we started to investigate the volatiles
released from cultures of several myxobacteria. The results
obtained from C. crocatus are presented here. Cultures of
this organism produce a very characteristic odour unlike that
of any other myxobacterium, which allows to recognise the
bacterium simply by smelling.
5
closed-loop-stripping apparatus (CLSA). Air was
circulated for 6–8 h over the culture, and the volatiles
trapped by a charcoal filter were extracted with 20 ml of
CH Cl (see Fig. 1). This procedure allowed storage of the
solution for later handling. In an alternative procedure, a
small hole was drilled into the Petri dish just prior to
analysis, a solid-phase micro-extraction (SPME) syringe
was introduced through this hole, and the SPME fibre
exposed to the volatiles. After 30 min, the SPME syringe
was retracted and introduced into a GC–MS system, where
the trapped volatiles were directly analysed by desorption
2
2
Keywords: Myxobacteria; Ketones; Pyrazines; Degraded sesquiterpenes;
Sesquiterpenes; CLSA analysis.
*
Corresponding author. Tel.: þ49-0531-391-7353; fax: þ49-0531-391-5272;
5
e-mail address: stefan.schulz@tu-bs.de
from the fibre (see Fig. 1).
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.03.005

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S. Schulz et al. / Tetrahedron 60 (2004) 3863–3872
A second group of pyrazines consists of methoxypyrazines.
The well known 2-methoxy-3-(1-methylethyl)pyrazine,
-methoxy-3-(2-methylpropyl)pyrazine, and 2-methoxy-3-
1-methylpropyl)pyrazine were identified in varying
2
(
amounts in the samples, but the most prominent member
of this compound class was always 2-methyl-3-methoxy-
pyrazine.
Three unknown compounds 9, 10, and 11 showed related
mass spectra with low intensity ions below m/z¼100, typical
for pyrazines, and a base peak at m/z¼153 or 139 (see
Fig. 2). The shift of 16 amu compared to both the molecular
ion and the m/z¼137 ion of 2-methoxy-3-(1-methylethyl)-
pyrazine in compound 9 indicated the presence of an
additional oxygen atom outside the aromatic nucleus.
Furthermore, the additional ion at m/z¼59 amu can be
þ
attributed to a [C H OH] fragment. The compound was
3
6
easily silylated with N-trimethylsilyltrifluoroacetamide
MSTFA), consistent with the presence of a hydroxy
(
function. We concluded, that the structure of 9 was 2-(1-
hydroxy-1-methylethyl)-3-methoxypyrazine. This assign-
ment was confirmed by synthesis starting from methoxy-
pyrazine (8), which can be selectively deprotonated at C-3
with lithium tetramethylpiperidide. Reaction with acetone
furnished the desired compound, which proved to be
identical with natural 9.
Figure 1. Methods used for volatile collection. The CLSA method
furnished storable solutions, while the SPME procedure was used for direct
analysis.
9
Compounds were identified by GC–MS analysis and
comparison of retention times with synthetic reference
samples. Unknown compounds were synthesised to confirm
the results. Control experiments were performed with
sterilised agar plates without bacteria to exclude compounds
emanating from the agar or the Petri dishes.
In a similar manner, compound 10 was identified as 2-(1-
hydroxy-1-methylpropyl)-3-methoxypyrazine and 11 as
2-(1-hydroxy-2-methylpropyl)-3-methoxypyrazine. Both
compounds were also synthesised as shown in Scheme 3
and exhibited identical mass spectra and retention times as
the natural compounds. To further strengthen our identi-
fication, a positional isomer of 10 was synthesised. Starting
Our results indicate that two major compound classes,
pyrazines and sesquiterpenoids, are formed by the bacteria.
Smaller amounts of aromatic compounds were also found.
The identified compounds are listed in Table 1.
1
0
from 2,6-dichloropyrazine (12), transformation into 2,6-
diiodopyrazine (13) with NaI, followed by treatment with
1
1
Both alkylpyrazines and alkylmethoxypyrazines are pro-
duced by C. crocatus. In several samples a major pyrazine
with a molecular ion of m/z¼164 was present. The spectrum
was very similar to that of 2,5-bis-(1-methylethyl)pyrazine
sodium methoxide according to Turck et al. furnished
2-iodo-6-methoxypyrazine (14). After transmetallation with
1
2
tert-butyllithium according to Street et al., reaction with
butanone yielded 6-(1-hydroxy-1-methylpropyl)-3-
methoxypyrazine (15). Its mass spectrum and retention
time showed slight differences to the natural compound 10,
thus confirming our initial assignment of the natural
compound (Scheme 3).
6
7
published recently by Zilkowski et al. and Beck et al. To
confirm the identification, the different regioisomers of bis-
(
1-methylethyl)pyrazine were synthesised. The reaction of
8
isopropylmagnesium bromide with pyrazine afforded
1-methylethyl)pyrazine, also present in the extracts. This
(
compound was reacted again with isopropylmagnesium
bromide to furnish a 76:20:4 mixture of the 2,6, 2,5, and 2,3-
bis-(1-methylethyl)pyrazines, which could be assigned by
their NMR spectra. The major compound produced by
C. crocatus proved to be identical to 2,5-bis-(1-methyl-
ethyl)pyrazine (3), while 2,6-bis-(1-methylethyl)pyrazine
Sesquiterpenoids represent the second major group of
compounds. The well known characteristic odorous com-
pound geosmin (1) was present in the samples in varying
amounts, sometimes not detectable by GC–MS, but by the
human nose. A major constituent in most analyses was
(1(10)E,5E)-germacradien-11-ol (21), which was identified
by comparison with a synthetic sample.
(4) was a minor component in the samples (Scheme 2).
Comparison with a synthetic sample also confirmed the
presence of 2,5-bis-(2-methylpropyl)pyrazine (7) and other
alkylmethylpyrazines (see Table 1). Two further compounds
exhibited a molecular ion of m/z¼162, 2 amu less than the
molecular ion of 3 and 4. The spectra showed a base peak at
M215 (m/z¼147), and an intense M228 ion at (m/z¼134),
typical for isopropyl branched pyrazines. Furthermore, an
intense ion at M21 occurs, often observed in vinylpyrazines.
We therefore conclude that these compounds are 2-(1-
methylethenyl)-5-(1-methylethyl)pyrazine (5) and 2-(1-
methylethenyl)-6-(1-methylethyl)pyrazine (6).
Furthermore, a major component 16 with a molecular ion at
m/z¼178 occurred. Its mass spectrum (Fig. 2) was similar,
but not identical, to several spectra of dimethyl-
bicyclo[4.4.0]decenones and methyloctahydrobenzocyclo-
heptanones present in the Wiley and NIST databases.
Therefore this compound was believed to represent a
degradation product of 21, related to 1, namely 6,10-
dimethylbicyclo[4.4.0]dec-1-en-3-one (16). To prove our
assignment, 16 was synthesised by Robinson annellation of
2,6-dimethylcyclohexanone and butenone according to

S. Schulz et al. / Tetrahedron 60 (2004) 3863–3872
Table 1. Compounds identified in Chondromyces crocatus headspace
3865
No
Compound
Sample
CLSA
SPME
Cm c5
y
Cm c5
p
Cm c2
y
Cm c2
p
1
2
3
4
5
6
7
8
9
2-(Methoxymethyl)furan
Anisol
2,5-Dimethylpyrazine
2-Methoxy-3-methylpyrazine
(1-Methylethyl)pyrazine
Benzyl alcohol
2-Methyl-6-(1-methylethyl)pyrazine
3-Methoxy-2,6-dimethylpyrazine
3-Methoxy-2,5-dimethylpyrazine
2-Methyl-5-(1-methylethyl)pyrazine
1-Phenylethanol
x
Trace
xx
x
Trace
xx
x
x
xxx
xx
xx
x
x
x
x
xx
x
x
x
x
x
x
x
x
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
x
Trace
x
x
x
x
2-Methoxy-3-(1-methylethyl)pyrazine
Nonanal
2-Phenylethanol
Dimethyl-(1-methylethyl)pyrazine
2,6-Bis(1-methylethyl)pyrazine (4)
2-Methoxy-3-(1-methylpropyl)pyrazine
1,4-Dimethoxybenzol
2-Methoxy-3-(2-methylpropyl)pyrazine
2,5-Bis(1-methylethyl)pyrazine (3)
Methyl salicylate
2-(1-Hydroxy-1-methylethyl)-3-methoxypyrazine (9)
2-(1-Methylethenyl)-6-(1-methylethyl)pyrazine (6)
Benzothiazol
2-(1-Methylethenyl)-5-(1-methylethyl)pyrazine (5)
endo-Bornyl acetate
2-(1-Hydroxy-1-methylpropyl)-3-methoxypyrazine (10)
2-Aminoacetophenone
Bicycloelemene
2-(1-Hydroxy-2-methylpropyl)-3-methoxypyrazine (11)
Methyl anthranilate
2,5-Bis(2-methylpropyl)pyrazine
a-Elemene
Geosmin (1)
x
x
x
Trace
Trace
x
x
x
x
x
x
x
x
xx
x
x
xx
Trace
Trace
Trace
x
xx
x
x
x
x
x
x
Trace
xx
Trace
xx
Trace
Trace
x
x
xx
x
x
xx
xxx
xxx
x
xx
x
x
xx
x
x
x
x
x
Trace
Trace
x
x
x
xx
x
x
xx
x
x
Trace
Trace
x
xx
Trace
x
xxx
x
Trace
Trace
Trace
x
x
x
x
Methyl 2-methoxybenzoate
b-Ylangene
b-Copaene
x
Trace
Trace
xx
x
x
iso-Germacrene D
6,10-Dimethylbicyclo[4.4.0]decan-3-ol (18)
Eremophilene
6,10-Dimethylbicyclo[4.4.0]decan-3-ol (18)
x
Trace
x
Trace
x
x
x
x
xx
p
p
p
(1R ,6R ,10R )-6,10-Dimethylbicyclo[4.4.0]decan-3-one (17)
(1S ,6R ,10R )-6,10-Dimethylbicyclo[4.4.0]decan-3-one
x
x
p
p
p
x
(2)-Germacrene D
a-Muurolene
Zonarene
xxx
xx
xxx
x
x
1,4-Cadinadiene
p
p
p
p
(6R ,10S )-6,10-Dimethylbicyclo[4.4.0]dec-1-en-3-one (6S ,10R -16)
(6S,10S)-6,10-Dimethylbicyclo[4.4.0]dec-1-en-3-one (6S,10S-16)
2-Benzyl-3-methoxypyrazine
Trace
xxx
x
x
xxx
x
x
xxx
xxx
Trace
x
xx
Cubenol
1(10)E,5E-Germacradien-11-ol (21)
xx
xxx
xx
xxx
x
xx
x
xxx
xxx, xx, x, trace: relative proportion of component in an extract. SPME: sample analysed by SPME; CLSA: sample analysed by CLSA; Cm c2 and Cm c5:
strain names; y: yeast medium; p: peptone medium. Compounds found in blank runs are not shown.
1
3
Ziegler and Hwang. The resulting 1:1 mixture of the cis
configuration of the naturally occurring compound using
GC–MS with chiral phases. The racemate is well separated
on a chiral cyclodextrin phase. As can be seen in Figure 5,
only the 6S,10S-enantiomer occurs naturally. It exhibits the
same absolute configuration at C-6 and C-10 as natural
p
p
p
p
and trans-diastereomers 6R ,10S -16 and 6R ,10R -16
was transformed into the thermodynamically more
stable trans-isomer by treatment with base. This isomer
proved to be identical with natural major component 16.
The cis-isomer occurs also naturally, but only in trace
amounts.
1
5
geosmin produced by several microorganisms.
p
Hydrogenation of (6R ,10R )-16 with Pd/C furnished
p
p
p
p
We then synthesised an enantiomerically enriched sample
of 6S,10S-16 (60% ee) according to the procedure of
Revial which was used to determine the absolute
preferentially the cis-fused (1R ,6R ,10R )-6,10-dimethyl-
bicyclo[4.4.0]decan-3-one 17, but also minor amounts of
1
4
p
p
the trans-fused (1R ,6S ,10S )-diastereomer, which
p
16

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S. Schulz et al. / Tetrahedron 60 (2004) 3863–3872
spectrum. One of the two protons on C-2 show two large
coupling constants which can only be explained by its axial
position and an antiperiplanar arrangement to the vicinal
proton at C-10. This conformation is opposite to the one
1
7
recently observed in the related 10-nor derivative of 17.
The reduction of the mixture of 17 with LiAlH gave four
4
6,10-dimethylbicyclo[4.4.0]decan-3-ols (18). At least two
of these alcohols were also present in trace amounts in some
samples, but the relative configuration of them was not
determined because of extensive peak overlapping in GC
and difficulties in separating the synthetic mixture.
Considerable differences especially in the relative amount
of the compounds produced were found between different
experiments. In one experiment the same agar plate was
investigated by both the SPME and the CLSA technique
(see Table 1 and Fig. 3). The SPME technique seems to be
very good for sampling of less volatile material, which can
be seen by the total absence of compounds 1–12 in Table 1.
The technique is very sensitive for hydrocarbon sesquiter-
penes, which are the peaks of highest abundance in the gas
chromatogram. Oxygenated compounds are less well
captured, as can be seen by the total absence of 10 and
the reduced amount of 16. In contrast, the CLSA method
disfavoured hydrocarbon sesquiterpenes and seems to give a
more total view of the compounds produced by C. crocatus.
Scheme 2.
were both present as minor components in the samples. The
relative configuration of the major synthetic product
p
p
p
(
1
1R ,6R ,10R )-17 was established by comparison of its
3
C NMR data with the published ones of all four
1
6
diastereomers of 17. Its preferred conformation, depicted
1
in Scheme 4, was deduced by analysis of the H NMR
Figure 2. Mass spectra of 2-(1-hydroxy-1-methylethyl)-3-methoxypyrazine (9), 2-(1-hydroxy-1-methylpropyl)-3-methoxypyrazine (10), 2-(1-hydroxy-2-
methylpropyl)-3-methoxypyrazine (11), (6R ,10R )-6,10-dimethylbicyclo[4.4.0]dec-1-en-3-one (6R,10R-16), (6R ,10S )-6,10-dimethylbicyclo[4.4.0]dec-
p
p
p
p
p
-en-3-one (6R,10S-16), and (1R ,6R ,10R )-6,10-dimethylbicyclo[4.4.0]decan-3-one (17).
p
p
1

S. Schulz et al. / Tetrahedron 60 (2004) 3863–3872
3867
The two different C. crocatus strains Cm c2 and Cm c5 also
showed some differences in their profile of the volatiles (see
Table 1 and Fig. 4).
The amount of the compounds identified varied consider-
ably between the two different culture media used.
Sesquiterpenoids were present predominantly on the
peptone medium, while they were markedly reduced on
the yeast medium. In contrast, the pyrazines were always
present as major components on the yeast medium, while
different cultures on peptone furnished different results
ranging from low to major amounts. Furthermore, consider-
able quantitative variability occurs between different
experiments performed with the same strain.
3. Discussion
Major components of the headspace of most C. crocatus
samples were the pyrazines 2-methoxy-3-methylpyrazine
and 9 as well as the sesquiterpenoids 16, 21, and
occasionally 1. Both mono- and dialkylpyrazines as well
as alkylmethoxypyrazines occur. Pyrazines are important
aroma compounds and have been found abundantly in
1
8
several foodstuff. Furthermore, several pyrazines are
7
,19
known to be produced by microorganisms,
but so far
they have not been reported from myxobacteria. The 2,5-
bis-(1-methylethyl)pyrazine (3) has been previously
identified as an attractant of Carpophilus beetles to
oranges upon which the beetles fed. It is believed to be of
Scheme 3. Synthesis of pyrazines. Reagents and conditions: (a) LiTMP,
acetone; (b) LiTMP, butanone; (c) LiTMP, isobutanal; (d) sulfolane,
p-TsOH, NaI, 15-crown-5, 4 h, 150 8C; (e) NaOMe, MeOH; (f) 280 8C,
t-BuLi, butanone, then to rt.
6
microbial origin. To the best of our knowledge, the
Figure 3. Gas chromatograms of volatiles collected from the same culture by SPME (A) or CLSA (B) methods. 25 m BPX-5, 60 8C, 5 min isothermal, then
with 5 8C/min to 300 8C.

3868
S. Schulz et al. / Tetrahedron 60 (2004) 3863–3872
Figure 4. Gas chromatograms of volatiles collected from the same strain (Cm c5) of Chondromyces crocatus cultured on peptone (A) or yeast (B) medium. 25
m BPX-5, 60 8C, 5 min isothermal, then with 5 8C/min to 300 8C.
hydroxyalkyl-methoxypyrazines 9, 10, and 11 have not been
reported before from nature.
related to 1. It has been patented. The derivatives 17 and 18
have so far not been reported as natural products.
While geosmin (1) is widespread in microorganisms, the
germacradienol 21 has been isolated before only from
Recently the gene responsible for the production of 1 and 21
2
4,25
in Streptomyces coelicor has been identified.
The
2
0
Streptomyces coelicor, where it occurs together with 1,
2
protein it codes for has two domains. While one domain is
needed for production of 1, the other one is required for the
production of 21. It has been proposed by Crane and Watt
1
and from the liverwort Dumortiera hirsuta. The trans-
bicyclodecanone 16 has been previously identified in the
2
2,23
24
plant Vetiver zizanioides,
and is structurally closely
that 21 is an intermediate in the biosynthesis of 1, while
Boland and co-workers independently postulated the
isomer 1(10),4-germacradien-11-ol (hedycaryol) (20) as
Figure 5. Gas chromatographic separation of 16. A: racemate; B: synthetic
S,10S-16; C: natural sample (Cm c2); D: coinjection racemate and natural
6
sample; X: 21. 15 m Hydrodex-6-TBDMS programmed as follows: 3 min
isothermal at 110 8C, then heated with 1 8C/min to 140 8C.
Scheme 4.

S. Schulz et al. / Tetrahedron 60 (2004) 3863–3872
3869
Scheme 5. Proposed biosynthetic pathway to the sesquiterpenoids 1, 16, 17, 18, and 21. Compounds identified in C. crocatus are shown in boxes.
1
5
precursor. The latter hypothesis was supported by feeding
studies with labelled precursors with a Streptomyces strain
and MS analysis of the products. It is presented in Scheme 5
as path a. The labelling patterns observed by Boland et al.
are not consistent with the proposed pathway by Crane and
4. Conclusion
In essence we have shown that efficient analysis of the
volatiles produced by myxobacteria can be performed by
analysis of the headspace of simple agar plate cultures.
Several new pyrazines and sesquiterpenoids could thus be
found and the complex odour bouquet of C. crocatus with
more than 50 components delineated.
2
4
Watt. It seems likely that 21 is formed by the independent
pathway c in Scheme 5 via isomerisation.
The formation of the degraded sesquiterpenoids 16, 17 and
18 can be explained by a modification of the geosmin
pathway. According to Boland, the geosmin biosynthesis
proceeds via the proposed intermediate 22. After hydro-
genation of the C-9–C-10 double bond to the intermediate
5. Experimental
†
2
1
3, cyclisation furnishes the cation 24, which finally forms
after addition of water. If the hydrogenation step is
5.1. General remarks
1
13
omitted and 22 directly cyclised via path b, the homoallylic
cation 25 is formed. This cation can be attacked by water to
form the alcohol 26 as depicted in Scheme 5. In both the
biosynthesis of 1 and 26 a 1,2-H shift occurs, which is
completed by attack of water on C-10 in the case of 1, and
on C-2 in the case of 26. The latter compound can then be
transformed into the derivatives 16, 17, and 18 by oxidation
and reduction.
H and C NMR spectra were obtained with Bruker AC-
200 and AMX-400 instruments. For NMR experiments,
CDCl was used if not mentioned otherwise; the internal
standard was tetramethylsilane. GC–MS investigations
were carried out with a Hewlett–Packard model 5973
mass selective detector connected to a Hewlett–Packard
model 6890 gas chromatograph. Analytical GLC analyses
were performed with a CE instruments GC 8000 gas
chromatograph equipped with a flame ionisation detector
and split/splitless injection. An apolar BPX-5 (SGE)
3
capillary column was used with H as the carrier gas. All
2
†
reactions were carried out under an inert atmosphere of N2
For convenience, the numbering scheme used is based on the numbering
of the germacrane skeleton.
in oven-dried glassware. Dry solvents: dry toluene was

3870
S. Schulz et al. / Tetrahedron 60 (2004) 3863–3872
distilled from Na, CH Cl from CaH , THF from K and Na.
2
0 8C then cooled to 270 8C. A solution of methoxypyrazine
(0.27 ml, 2.7 mmol) in diethyl ether (2 ml) was added and
the mixture stirred for 30 min. Then the corresponding
ketone or aldehyde was added dropwise and the mixture
stirred overnight, during which time it was allowed to warm
to room temperature. Then 10 ml water was added, the
mixture extracted twice with CH Cl , the organic phase
dried with MgSO and the solvent removed. The residue
4
was purified by column chromatography (silica, petrol
ether/diethyl ether 5:1 containing 0.5% triethylamine).
2
2
All other chemicals were commercially available (Fluka,
Aldrich) and used without further treatment, if not stated
otherwise. All reactions were monitored by thin layer
chromatography (TLC) carried out on Macherey-Nagel
Polygram SIL G/UV254 silica plates visualised with heat
gun treatment with 10% molybdato phosphoric acid in
ethanol. Column chromatography was performed with
Merck silica gel 60 (70–200 mesh). All new compounds
2
2
1
were determined to be .95% pure by HPLC, GLC, or H
NMR spectroscopy. Identification of known compounds
was performed by comparison of mass spectra with those in
the databases Wiley7, NIST 2.0, and MassFinder 2.3
5.4.1. 2-(1-Hydroxy-1-methylethyl)-3-methoxypyrazine
(9). Using the general procedure with abs. acetone
(0.3 ml, 4.1 mmol) gave a colourless oil in 23% (105 mg)
yield. Odour: strong herbaceous and root like smell.
2
6
(
Essential Oils), taking their retention indices into account
when no reference sample was available. Identification of
known sesquiterpenes was performed by comparing mass
2
7
1
spectra and retention times with critical evaluated data.
H NMR (400 MHz, C D ) d 1.80 (s, 6H, 2CH ), 3.64 (s,
6
6
3
Chiral GC was performed using a 15 m Hydrodex-6-
TBDMS column (Macherey and Nagel) with a constant flow
of 1 ml/min He installed into the GC–MS system to allow
peak determination. The oven was programmed as follows:
3H, OCH ), 5.52 (s, 1H, OH), 7.65 (d, J¼2.7 Hz, 1H, ArH),
3
1
3
7.70 (d, 1H, ArH); C NMR (100 MHz, C D ) d 28.1 (2C,
6
6
0
0
C-2 ), 53.1 (H CO), 71.3 (C-1 ), 134.1 (C-6), 139.6 (C-5),
3
150.8 (C-2), 157.5 (C-3); HR-EI-MS calculated for
C H N O 168.0899. Found 168.0903.
3 min isothermal at 110 8C, then heated with 1 8C/min to
40 8C.
8
12 2 2
1
5
.4.2. 2-(1-Hydroxy-1-methylpropyl)-3-methoxypyra-
5
.2. Microorganisms and culture conditions
zine (10). Using the general procedure with butanone
gave a colourless oil. Yield 43%. Odour: herbaceous, root
like.
Strains Cm c2 and Cm c5 of C. crocatus were isolated at the
GBF in 1982 and 1985 from soil samples with decaying
plant material collected on the island of Madeira, Portugal
and near Igua c¸ u, Brazil, respectively. While Cm c2 requires
a bacterial symbiont for growth, Cm c5 is a pure strain. The
organisms were cultivated either on VY/2-(yeast-) agar
1
0
H NMR (400 MHz, CDCl ) d 0.67 (t, 3H, H-3 ), 1.55 (s,
3
0
0
3H, C-1 –CH ), 1.87 (dq, J
3
(dq, J2
0 0
¼7.4 Hz, 1H, H-2b ), 2.10
2b ,3
0
0
0
¼7.4 Hz, J
0 0
¼14.8 Hz, 1H, H-2a ), 4.01 (s, 3H,
a ,3
2a ,2b
OCH ), 5.27 (s, 1H, HO), 8.05 (d, J¼2.8 Hz, 1H, ArH), 8.06
3
1
3
(
bakers’yeast, 0.5%; CaCl ·2H O, 0.1%; cyanocobalamin,
2
(d, J¼2.8 Hz, 1H, ArH); C NMR (100 MHz, CDCl ) d 8.1
2
3
0
0
0
0
0
.5 mg/l; agar, 1.5%; pH 7.2, autoclaved) or on CY-
(C-3 ), 26.1 (C-1 –CH ), 32.3 (C-2 ), 53.5 (H CO), 73.6
3
3
(
0
peptone-) agar (peptone from casein, tryptically digested,
.3%; yeast extract, 0.1%; CaCl ·2H O, 0.1%; agar, 1.5%;
pH 7.2, autoclaved). Cultures in 8 cm Petri dishes were
(C-1 ), 133.8 (C-6), 139.5 (C-5), 149.3 (C-2), 157.4 (C-3);
HR-EI-MS calculated for C H N O 182.1055. Found
182.1064.
2
2
9 14 2 2
incubated at 30 8C the dark.
5
.4.3. 2-(1-Hydroxy-2-methylpropyl)-3-methoxypyra-
5
.3. Headspace analysis
zine (11). Similar to the synthesis of 9, using methyl-
propanal instead of acetone. Yield 32%. Odour: vetiver root,
potato peels.
The sample vessel of a commercial CLSA system
Brechb u¨ hler) was replaced by a custom made glass
(
1
chamber. The chamber consisted of two parts connected
by a planschliff; a standard agar plate fits in the lower part,
while the upper part developed conical into a NS 29 female
joint, in which the CLSA adapter fits. Before every
headspace sampling from bacteria, the apparatus was
thoroughly cleaned with CH Cl and dried in an oven.
Blank runs were performed with and without sterile agar
plates to identify the compounds emanating from the CLSA
system, the agar, and the polystyrene Petri dishes. Samples
from bacteria were obtained by running the apparatus for
H NMR (400 MHz, CDCl ) d 0.72 (d, J
3
0
0
0
¼6.8 Hz, 3H,
2 ,3b
0
H-2 ), 3.88 (d, 1H, OH), 3.99 (s, 3H, OCH ), 4.76 (dd,
H-3b ), 1.08 (d, J
0
0
0
¼6.9 Hz, 3H, H-3a ), 2.16 (m, 1H,
2
,3a
3
0
1H, ArH), 8.08 (d, 1H, ArH); C NMR (100 MHz, CDCl )
J1
0
¼7.1 Hz, J
0 0
¼3.7 Hz, 1H, H-1 ), 8.03 (d, J¼2.8 Hz,
,OH
1 ,2
1
3
3
0
(C-1 ), 134.5 (C-6), 139.3 (C-5), 147.3 (C-2), 157.4 (C-3);
0
0
d 15.5 (C-3b ), 19.8 (C-3a ), 32.5 (C-2 ), 53.6 (H CO), 73.0
3
2
2
0
HR-EI-MS calculated for C H N O 182.1055. Found
182.1061.
9
14 2 2
8
with 15 ml CH Cl , and the extract stored in ampoules at
low temperature until analysis.
h. Then the 5 mg charcoal filter was extracted two times
5.4.4. 2-(1-Hydroxy-1-methylpropyl)-6-methoxypyra-
zine. Pyrazine was transformed into 12 according to a
2
2
1
0
11
published procedure. Treatment with NaI gave 13,
which was monomethoxylated by treatment with NaOMe to
1
1
5.4. General procedure for the preparation of
hydroxyalkylpyrazines (9–11)
furnish 14.
A solution of 81 mg (0.34 mmol) 14 in 5 ml diethyl ether
was added at 280 8C 0.42 ml of a 1.7 M tert-butyllithium
solution in pentane. After stirring for 30 min, 0.1 ml
(1.1 mmol) butanone was added and the mixture stirred for
A mixture of 2,2,6,6-tetramethylpiperidine (0.7 ml,
1
2
4.1 mmol), 1.6 M n-butyllithium in hexane (2.6 ml,
4.2 mmol) and diethyl ether (6 ml) was stirred for 1 h at

S. Schulz et al. / Tetrahedron 60 (2004) 3863–3872
3871
an additional 2 h at room temperature. Then 10 ml water
5.6. Synthesis of sesquiterpenoids
was added and the mixture extracted three times with
diethyl ether. The combined organic phases were dried with
Na SO , the ether removed by evaporation, and the residue
purified by chromatography (silica, diethyl ether/petroleum
ether 3:1). A colourless oil was obtained in 41% yield
5.6.1. 6,10-Dimethylbicyclo[4.4.0]dec-1-en-3-one (16).
2,6-Dimethylcyclohexanone and methylvinylketone were
2
4
1
3
reacted according of the procedure of Ziegler and Hwang
to yield a 1:1 mixture of cis- and trans-16. Treatment of this
mixture with 5% KOH in ethanol furnished a 60:1 trans/cis
(
26 mg, 0.14 mmol). Odour: potato like, earthy.
p
p
p
p
or 6R ,10R /6R ,10S -mixture of 16.
1
0
H NMR (400 MHz, CDCl ) d 0.82 (t, 3H, H-3 ), 1.55 (s,
3
0
0
H, OH), 3.99 (s, 3H, OCH ), 8.13 (s, 1H, ArH), 8.25 (s, 1H,
1
3
H, H C–C-1 ), 1.85 (q, J
3
0
0
¼7.4 Hz, 2H, H-2 ), 3.73 (s,
H NMR (400 MHz, CDCl ) d 1.07 (d, J
3
¼6.5 Hz, 3H,
2 ,3
CH3,10
1
ArH); C NMR (100 MHz, CDCl ) d 8.0 (C-3 ), 28.0
H C–C-10,), 1.24 (s, 3H, H C–C-6), 1.95–1.15 (m, 8H),
3
3
3
1
3
0
H C–C-1 ), 35.8 (C-2 ), 53.5 (H CO), 73.6 (C-1 ), 132.7 (d,
2.43–2.30 (m, 2H, H-4b, H-10), 2.49 (ddd, J4a,4b¼16.9 Hz,
3
0
0
0
(
J4a,5a¼13.2 Hz, J
¼6.0 Hz, 1H, H-4a), 5.79 (d,
3
3
4a,5b
1
3
C-Ar), 133.3 (d, C-Ar), 157.0 (q, C-Ar), 158.9 (q, C-Ar);
HR-EI-MS calculated for C H N O 182.1055. Found
J2,10¼1.5 Hz, 1H, H-2); C NMR (100 MHz, CDCl ) d
3
18.1 (CH ), 21.7 (CH ), 23.0 (CH ), 33.9 (CH ), 34.2
3
9
14
2
2
2
3
2
1
82.1070.
(C-10), 36.3 (CH ), 38.4 (CH ), 41.9 (CH ), 121.6 (C-2),
2
2
2
173.9 (C-1), 200.2 (C-3).
5
.5. General procedure for alkylation of pyrazines
5.6.2. 6,10-Dimethylbicyclo[4.4.0]decan-3-one (17). To a
solution of 6R ,10R -16 (20 mg, 0.11 mmol) in 1 ml
p
p
A Grignard solution was prepared from 0.121 g (5.0 mmol)
magnesium and 0.5 ml (5.3 mmol) 2-bromopropane in 5 ml
THF at 0 8C. After 1 h 0.2 g (2.5 mmol) of the appropriate
pyrazine in 2 ml THF were added dropwise and the solution
stirred for further 15 min. Longer reaction times resulted in
lower yields. The reaction was quenched with ammonium
chloride solution and the mixture extracted with THF. The
CH Cl was added 3 mg 5% Pd/C and the mixture stirred
2
2
under an H2 atmosphere for 30 min. The mixture was
filtered over a short plug of silica and the solvent
evaporated. The product (20 mg, 97% yield) consisted of
p
p
p
p
p
p
a 6:1 mixture of (1R ,6R ,10R )- and (1R ,6S ,10S )-17.
1
organic phases were dried with MgSO , the solvent
4
H NMR (400 MHz, CDCl ) d 0.80 (d, J
3
¼6.8 Hz, 3H,
CH3,10a
removed and the residue purified by column chromato-
graphy (petroleum ether/diethyl ether 2:1). The spectro-
scopical data are identical to those reported in the
H C–C-10), 1.04 (s, 3H, H C–C-6), 1.16–1.07 (m, 2H, H-
3
3
9, H-7e), 1.44 (m, 1H, H-9), 1.69–1.58 (m, 4H, H-1a, H-5a,
H-8a, H-8e), 1.73 (ddd, J5e,5a¼13.9 Hz, J ¼6.3 Hz, 1H,
4a,5e
1
0
literature.
H-5e), 1.87 (m, 1H, H-7a), 1.98 (m, 1H, H-10a), 2.16 (ddd,
¼2.3 Hz, 1H, H-2e), 2.20 (ddt,
2a,2e
J2e,1a¼4.8 Hz, J
4e,2e
5
.5.1. (1-Methylethyl)pyrazine. Prepared according to the
J4e,5a¼4.8 Hz, J
¼2.8 Hz, 1H, H-4e), 2.29 (t, J
¼
4e,5e
general procedure for the alkylation of pyrazines starting
from pyrazine. Yield 20% (61 mg). The spectroscopical
J2a,1a¼13.9 Hz, 1H, H-2a), 2.49 (dt, J
¼J
¼14.4 Hz,
3
4a,4e
4a,5a
1
3
1H, H-4a); C NMR (100 MHz, CDCl ) d 19.4 (CH –C-
3
1
0
data are identical to those reported in the literature.
10), 21.7 (C-8), 26.4 (CH –C-6), 27.8 (C-9), 28.9 (C-7),
3
30.1 (C-10), 33.3 (C-6), 37.1 (C-4), 37.7 (C-5), 41.3 (C-2),
47.8 (C-1), 214.0 (C-3).
5
.5.2. Bis-(1-methylethyl)pyrazine. Prepared according to
the general procedure for the alkylation of pyrazines starting
from (1-methylethyl)pyrazine in 14% yield. The reaction
time after addition of the pyrazine was reduced to 5 min. A
mixture of the 2,3-isomer (4%), the 2,5-isomer (20%), and
the 2,6-isomer (76%) was obtained.
Acknowledgements
We thank Prof. W. A. K o¨ nig, University of Hamburg, for a
gift of (1(10)E,5E)-germacradien-11-ol. We also thank Dr
R. Kaiser, Firmenich, for evaluation of the odour properties
of some of the pyrazines.
1
2
CDCl ) d 8.34 (s, 2H, ArH); C NMR (100 MHz, CDCl )
,3-Bis-(1-methylethyl)pyrazine: H NMR (400 MHz,
1
3
3
3
0
þ
d 22.12 (C-2 ); MS (70 eV) 164 (M , 81), 149 (100), 136
24), 135 (53), 134 (16), 133 (13), 121 (19), 80 (14), 52 (13),
(
4
1 (17).
References and notes
1
2
CDCl ) d 1.32 (d, J
,5-Bis-(1-methylethyl)pyrazine (3): H NMR (400 MHz,
0 0
0
ArH); C NMR (100 MHz, CDCl ) d 22.2 (C-2 ), 33.5
¼6.9 Hz, 12H, H-2 ), 8.39 (s, 2H,
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3
1 ,2
1
3
0
C-1 ), 141.8 (C-3, C-6), 159.3 (C-2, C-5); MS (70 eV) 164
3
0
(
(
þ
33 (10), 121 (17), 53 (9), 41 (10).
M , 25), 163 (12), 150 (10), 149 (100), 136 (42), 134 (16),
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3
. Plaga, W.; Stamm, I.; Schairer, H. U. Proc. Natl. Acad. Sci.
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1 ,2
0
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2
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