A novel type of geosmin biosynthesis in myxobacteria

Article abstract of DOI:10.1021/jo050449g

The biosynthesis of geosmin (1) and (1(10)E,5E)-germacradien-11-ol (2), two volatile terpenoid compounds emitted by the myxobacteria Myxococcus xanthus and Stigmatella aurantiaca, was investigated in feeding experiments with different labeled precursors.

Full text of DOI:10.1021/jo050449g

A Novel Type of Geosmin Biosynthesis in Myxobacteria
Jeroen S. Dickschat,† Helge B. Bode,‡ Taifo Mahmud,§ Rolf Mu¨ller,‡ and Stefan Schulz*,†
Institut fu¨r Organische Chemie, Technische Universita¨t Braunschweig, 38106 Braunschweig, Germany,
Institut fu¨r Pharmazeutische Biotechnologie, Universita¨t des Saarlandes, 66123 Saarbru¨cken, Germany,
and Oregon State University, Corvallis, Oregon 97331
stefan.schulz@tu-bs.de
Received March 8, 2005
The biosynthesis of geosmin (1) and (1(10)E,5E)-germacradien-11-ol (2), two volatile terpenoid
compounds emitted by the myxobacteria Myxococcus xanthus and Stigmatella aurantiaca, was
investigated in feeding experiments with different labeled precursors. In these experiments, the
volatiles released by the cell cultures grown on agar plates were collected with a closed-loop stripping
apparatus (CLSA) and analyzed by GC-MS. [2H10]Leucine and [4,4,4,5,5,5-2H6]dimethylacrylate
were fed to wild-type strains and bkd mutant strains, which are impaired in the degradation of
leucine to isovaleryl-CoA. [2H10]Leucine was incorporated into 1 and 2 only by the wild-type strains
via the biosynthetic pathway that involves leucine degradation and branching into the mevalonate
pathway. Dimethylacrylyl-CoA (DMA-CoA) is an intermediate in the leucine degradation and in
the recently discovered pathway from HMG-CoA to isovaleryl-CoA. The corresponding free acid,
[4,4,4,5,5,5-2H6]dimethylacrylic acid, was incorporated into 1 and 2 only by the mutants impaired
in leucine degradation. [4,4,6,6,6-2H5]Mevalonic acid lactone (12) was synthesized and fed to M.
xanthus and S. aurantiaca wild-type strains and a double mutant strain of M. xanthus. This strain
does not degrade leucine and is impaired in the reduction of 3-hydroxy-3-methylglutaryl-CoA to
mevalonic acid. The mass spectral analysis of labeled 1 and 2 obtained in these feeding experiments
led to a biosynthetic scheme to 1 with intermediate 2. This pathway differs from that observed in
the liverwort Fossombronia pusilla and thus suggests microbial geosmin biosynthesis following a
route different from that in liverworts. Our results are supported by a 1,2-hydride shift of the
tertiary hydrogen atom at C-4a into the ring opposite to that in F. pusilla.
Introduction
because it can be accumulated by fish and other foodstuff
to produce an off-flavor and resists conventional water
treatment.9 Several biological functions such as growth
inhibition have been described, but this seems to be the
case in high concentration only.9 Geosmin is known as a
signal to the glass eel Anguilla anguilla. The eels are
attracted to the compound in a low salinity environment,
which leads to the speculation that this combination acts
as a landmass marker.10 More importantly, humans avoid
water inhabited by toxic cyanobacteria that release 1.11
Recently, we have investigated the profile of volatile
compounds emitted by the myxobacterium Myxococcus
xanthus (strain DK1622) by use of a modified closed-loop
stripping apparatus (CLSA).12 Several terpenoid com-
pounds are produced by this strain. (-)-Geosmin (1) is
Geosmin (1) is a degraded sesquiterpene with low odor
threshold that is produced by several microorganisms and
responsible for the characteristic odor of freshly plowed
earth.1 It was first isolated from the actinomycete Strep-
tomyces griseus2 and subsequently also found in fungi,3
cyanobacteria,4 plants,5 mosses,6 protozoans,7 and in-
sects.8 The occurrence of 1 is of considerable importance
† Technische Universita¨t Braunschweig.
‡ Universita¨t des Saarlandes.
§ Oregon State University.
(1) Buttery, R.; Garibaldi, J. J. Agric. Food Chem. 1976, 24, 1246-
1247.
(2) (a) Gerber, N. N.; Lechevalier, H. A. Appl. Microbiol. 1965, 13,
935-938. (b) Gerber, N. N. Tetrahedron Lett. 1968, 25, 2971-2974.
(3) Bjurman, J.; Kristensson, J. Microbios 1992, 72, 47-54.
(4) Ju¨ttner, F. Water Sci. Technol. 1995, 31, 69-78.
(5) Spiteller, D.; Jux, A.; Piel, J.; Boland, W. Phytochemistry 2002,
61, 827-834.
(8) Omura, H.; Kuwahara, Y.; Tanabe, T. J. Chem. Ecol. 2002, 28,
2601-2612.
(9) Watson, S. B. Phycologia 2003, 42, 332-350.
(10) Tosi, L.; Sola, C. Ethology 1993, 95, 177-185.
(11) Jardine, C.; Gibson, N.; Hrudey, S. Water Sci. Technol. 1999,
40, 91-98.
(6) Spo¨rle, J.; Becker, H.; Allen, N. S.; Gupta, M. P. Z. Naturforsch.
1991, 46c, 183-188.
(7) Hayes, S. J.; Hayes, K. P.; Robinson, B. S. J. Protozool. 1991,
38, 44-47.
10.1021/jo050449g CCC: $30.25 © 2005 American Chemical Society
Published on Web 05/26/2005
5174
J. Org. Chem. 2005, 70, 5174-5182

Geosmin Biosynthesis in Myxobacteria
TABLE 1. Incorporation Rates in Feeding Experiments
one of the major compounds and is responsible for the
earthy smell of the cultures. It is also produced by other
myxobacteria like Nannocystis exedens.13 Myxobacteria,
in general, are known for the production of several
secondary metabolites that often show interesting bio-
logical activities14 and biosyntheses.15,16 Previously un-
known pathways for the biosynthesis of iso-fatty acids
and its precursor isovaleryl-CoA have been identified.17-19
However, volatile secondary metabolites were only in-
vestigated recently.12 Other terpenoids released by M.
xanthus are (1(10)E,5E)-germacradien-11-ol (2), often
produced in large amounts, and the trace component (-)-
germacrene D (3). Biosynthetic studies can be carried out
by feeding labeled precursors to agar plate cultures and
subsequent analysis with the CLSA technique.12 This
method was used in the present study to investigate the
biosynthesis of terpenes in M. xanthus.
experimenta
1b
2b
Myxococcus xanthus
DK1622 + [2H10]leucine
JD300 + [2H10]leucine
DK1622 + [2H6]DMAA
JD300 + [2H6]DMAA
DK1622 + 12
6
0
8
0
0
0
49
44
46
Stigmatella aurantiaca
DW 4/3-1 + [2H10]leucine
DW 4/3-1 bkd- + [2H10]leucine
DW 4/3-1 + [2H6]DMAA
DW 4/3-1 bkd- + [2H6]DMAA
DW 4/3-1 + 12
3
0
4
0
0
0
15
22
19
25
a Feeding experiments with Myxococcus xanthus (wild-type
strain DK1622 and bkd mutant strain JD300) and Stigmatella
aurantiaca (wild-type strain DW 4/3-1 and bkd mutant strain)
supplying different labeled precursors. b Incorporation rates in %
into volatiles 1 and 2. The rates are given as ratio of labeled to
unlabeled isoprene units used for the formation of the sesquiter-
penoids. This ratio was calculated from the ion chromatograms of
molecular ions of the different isotopomers.
contained up to six deuterium atoms, accompanied by
minor amounts of isotopomers with five deuterium atoms.
The same pattern is found for 2 by an increase of the
molecular ion from m/z ) 222 to 228 and 227 (compare
Figure 1f and g). Leucine can enter the mevalonate
pathway by the leucine-dependent isoprenoid pathway,
giving rise to dimethylallyl pyrophosphate (DMAPP) and
isopentenyl pyrophosphate (IPP),18,20 the universal build-
ing blocks in the terpene biosynthesis. Herein, [2H10]-
leucine is degraded by transamination and subsequent
oxidative decarboxylation to [2H9]isovaleryl-CoA (IV-
CoA), and then further to [2H7]dimethylacrylyl-CoA
(DMA-CoA) as outlined in Scheme 1. This pathway is
continued by the carboxylation to [2H6]-3-methylgluta-
conyl-CoA (MG-CoA) and the addition of water to result
in [2H6]-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), a
central intermediate in the mevalonate pathway to
terpenes.21 Reduction furnishes [2H6]mevalonic acid (MVA),
and several phosphorylation and decarboxylation steps
lead to [2H6]IPP and [2H6]DMAPP. The pro-R proton at
C-2 of IPP is removed in the isomerization process from
IPP to DMAPP. The stereochemistry at C-2 of labeled
IPP and therefore the number of deuterium atoms
retained in DMAPP is thus determined by the stereo-
chemical course of the MG-CoA hydration. This step is
catalyzed by the methylglutaconyl-CoA hydratase and
proceeds by the syn-addition of water from the Si-side.22
The enzyme is part of the crotonase superfamily, an
enzyme superfamily catalyzing biotransformations in
which enolate anion intermediates derived from acyl-CoA
substrates need to be stabilized.23 A transformation
similar to the hydration of MG-CoA is the addition of
water to enoyl-CoA intermediates in the fatty acid
â-oxidation. The enoyl-CoA hydratase (E.C. 4.2.1.17) is
involved in the stereospecific hydration of R,â-unsatur-
ated acyl-CoA thioesters that proceeds also via the syn-
Results and Discussion
During investigations on the biosynthesis of 9-methyl-
decan-3-one and (S)-9-methyldecan-3-ol,12 the two main
components in the headspace profile of M. xanthus,
labeled [2H10]leucine was fed to the wild-type strain
DK1622 and to a mutant strain JD300. The latter strain
is impaired in the degradation of leucine to isovaleryl-
CoA due to a mutation in the bkd gene (branched-chain
keto acid dehydrogenase). Headspace extracts were
sampled from agar plate cultures by CLSA and analyzed
by GC-MS.12 Unexpectedly, the incorporation of [2H10]-
leucine into 1 and 2 by the wild-type strain was observed,
while the mutant strain showed no incorporation (for
incorporation rates, see Table 1). Identical feeding ex-
periments were carried out with the myxobacterium
Stigmatella aurantiaca (wild-type strain DW 4/3-1 and
bkd mutant strain) whose results matched those obtained
with M. xanthus.
The retention time of a deuterated compound is always
shortened as compared to that of its unlabeled counter-
part.12 Thus, pure mass spectra of labeled compounds
arising from deuterated precursors can be obtained, even
in the presence of unlabeled material. The incorporation
of [2H10]leucine into 1 is indicated by a shift of the
molecular ion from m/z ) 182 to 188 and 187, respectively
(compare Figure 1a and b). Conclusively, labeled 1
(12) Dickschat, J. S.; Wenzel, S. C.; Bode, H. B.; Mu¨ller, R.; Schulz,
S. ChemBioChem 2004, 5, 778-787.
(13) Trowitzsch, W.; Witte, L.; Reichenbach, H. FEMS Microbiol.
Lett. 1981, 12, 257-260.
(14) Kopp, M.; Irschik, H.; Gross, F.; Perlova, O.; Sandmann, A.;
Gerth, K.; Mu¨ller, R. J. Biotechnol. 2004, 107, 29-40.
(15) Sandmann, A.; Sasse, F.; Mu¨ller, R. Chem. Biol. 2004, 11,
1071-1079.
(16) Wenzel, S. C.; Kunze, B.; Ho¨fle, G.; Silakowski, B.; Scharfe, M.;
Blo¨cker, H.; Mu¨ller, R. ChemBioChem 2005, 6, 375-385.
(17) Bode, H. B.; Dickschat, J. S.; Kroppenstedt, R. M.; Schulz, S.;
Mu¨ller, R. J. Am. Chem. Soc. 2005, 127, 532-533.
(18) Mahmud, T.; Bode, H. B.; Silakowski, B.; Kroppenstedt, R. M.;
Xu, M.; Nordhoff, S.; Ho¨fle, G.; Mu¨ller, R. J. Biol. Chem. 2002, 277,
32768-32774.
(20) Bode, H. B.; Zeggel, B.; Silakowski, B.; Wenzel, S. C.; Reichen-
bach, H.; Mu¨ller, R. Mol. Microbiol. 2003, 47, 471-481.
(21) Lynen, F. Angew. Chem. 1965, 77, 929-944.
(22) Messner, B.; Eggerer, H.; Cornforth, J.; Mallaby, R. Eur. J.
Biochem. 1975, 53, 255-264.
(23) Holden, H. M.; Benning, M. M.; Haller, T.; Gerlt, J. A. Acc.
Chem. Res. 2001, 34, 145-157.
(19) Mahmud, T.; Wenzel, S. C.; Wan, E.; Wen, K. W.; Bode, H. B.;
Gaitatzis, N.; Mu¨ller, R. ChemBioChem 2005, 6, 322-330.
J. Org. Chem, Vol. 70, No. 13, 2005 5175

Dickschat et al.
FIGURE 1. Mass spectra of terpenoid compounds present in the headspace extracts of Myxococcus xanthus and Stigmatella
aurantiaca. Mass spectra of 1 (a), [2H6]-1 after feeding of [2H10]leucine (b), [2H5]-1 after feeding of [2H6]DMAA and incorporation
of one labeled IPP unit (c), [2H10]-1 after feeding of [2H6]DMAA and incorporation of two labeled IPP units (d), 1g after feeding of
12 (e), 2 (f), [2H6]-2 after feeding of [2H10]leucine (g), [2H5]-2 after feeding of [2H6]DMAA (h), and 2g after feeding of 12 (i).
5176 J. Org. Chem., Vol. 70, No. 13, 2005

Geosmin Biosynthesis in Myxobacteria
SCHEME 1. Biosynthesis of the Terpene Building
Blocks [2H6]IPP and [2H6]DMAPP from
[2H10]Leucinea
SCHEME 2. Biosynthesis of Terpenoids via FPP
from DMAPP and IPPa
a Asterisks indicate fully deuterated carbons.
addition of water.24 Furthermore, in recent investigations
on Pseudomonas putida, an operon encoding the leucine
catabolic pathway was identified.25 The enzyme methyl-
glutaconyl-CoA hydratase (E.C. 4.2.1.18) is encoded by
one gene of this operon. Because of the presence of the
same catalytic residues as found in other enzymes of this
superfamily, it is supposed to catalyze the syn-addition
of water. Therefore, the 1-fold deuterated carbon in [2H6]-
HMG-CoA (C-2) generated on the leucine pathway is
suggested to be S-configured. This stereochemistry is
retained in the next steps to [2H6]IPP. The following
reversible isomerization to DMAPP is catalyzed by type-I
isomerases in animals, plants, and fungi, whereas ar-
chaea and many eubacteria use a structurally unrelated
type-II isomerase.26 The isomerization process is associ-
ated with the loss of the pro-R proton for both types of
isomerases.26 Thus, the isomerization of (S)-[2H6]IPP
furnishes [2H6]DMAPP. Hypothetically, (R)-[2H6]IPP would
lead to [2H5]DMAPP.
The universal sesquiterpene precursor farnesyl pyro-
phosphate (FPP, 4) is formed from one unit of DMAPP
and two units of IPP. This process is associated with the
loss of the pro-R proton at C-2 of IPP (Scheme 2).27 The
statistical incorporation of the [2H10]leucine-derived iso-
prene units [2H6]DMAPP and (S)-[2H6]IPP leads to the
formation of three isotopomers of [2H6]FPP (4a, 4b, and
4c). In contrast, R-configured [2H6]IPP would yield three
isotopomers of [2H5]FPP. Therefore, the observed forma-
tion of [2H6]-1 (1b and 1c) and [2H6]-2 (2a, 2b, and 2c),
which is indicated by the molecular ions at m/z ) 188
and 228, is only explainable by the formation of (S)-[2H6]-
IPP. The occurrence of six deuteriums in labeled 1
indicates by inversion of the arguments that syn-addition
of water from the Si-side to MG-CoA must take place in
the leucine-dependent isoprenoid pathway of the myxo-
bacteria. The generation of [2H5]-1 and [2H5]-2 that are
found in low amounts can be explained by the re-
isomerization of [2H6]DMAPP to (S)-[2H5]IPP and back
a Labeling patterns in the sesquiterpene precursor FPP, 1, and
2 after feeding of [2H10]leucine and incorporation of one labeled
isoprene unit. Completely deuterium-labeled carbons are indicated
by asterisks.
to [2H5]DMAPP. In this process, the terminal (E)-methyl
group can either lose an H or D, thus giving access to
(S)-[2H5]IPP.
The isopropylidene group of [2H6]DMAPP incorporated
into 4a is lost during the biosynthesis of 1 as outlined
below, forming 1a. This compound coelutes with un-
labeled 1 because of its low deuterium content and is not
detectable due to the low incorporation rates.
The molecular ions and diagnostic fragment ions
expected in the mass spectra of labeled 1 and 2 after
feeding of different deuterium-labeled precursors are
summarized in Figure 2. The mass spectrum of labeled
[2H6]-2 shows fragment ions at m/z ) 59 and 64, respec-
tively, in a ratio of about 2:1 after feeding of [2H10]leucine
(Figure 1g). These fragment ions result from R-cleavage
next to the alcohol function, giving m/z ) 64 for 2a. This
ion points to a content of five deuterium atoms according
to the composition [C3H2D5O]+ (Figure 2a). Thus, the
isopropylidene group in labeled 4a must contain up to
five deuteriums as is expected for the degradation of
[2H10]leucine to [2H6]DMAPP via the leucine-dependent
isoprenoid biosynthesis.
Further insight into the biosynthesis of 1 was possible
by the following observations: The chromatographic peak
of [2H6]-1 obtained after feeding of [2H10]leucine originates
from the two isotopomers 4b and 4c (Scheme 2), thus
containing the two isotopomers 1b and 1c. This is
indicated by a shift of the highly diagnostic base peak of
natural 1 from m/z ) 112 to m/z ) 113 and 117,
respectively, in the expected 1:1 ratio (Figures 1b and
2a). Other diagnostic ions found at m/z ) 125 and 126 in
unlabeled 1 move to m/z ) 130 and 131. These major ions
arise from a rearrangement process according to Scheme
3 and can be used to localize the deuterium atoms.5 The
formation of 1b (Figure 2a) results in five deuterium
(24) Wu, W.-J.; Feng, Y.; He, X.; Hofstein, H. A.; Raleigh, D. P.;
Tonge, P. J. J. Am. Chem. Soc. 2000, 122, 3987-3994.
(25) Wong, B. J.; Gerlt, J. A. Biochemistry 2004, 43, 4646-4654.
(26) Laupitz, R.; Gra¨wert, T.; Rieder, C.; Zepeck, F.; Bacher, A.;
Arigoni, D.; Rohdich, F.; Eisenreich, W. Chem. Biodiversity 2004, 1,
1367-1376.
(27) Dewick, P. M. Medicinal Natural Products; Wiley: Chichester,
2002.
J. Org. Chem, Vol. 70, No. 13, 2005 5177

Dickschat et al.
SCHEME 3. MS Fragmentation Pathways to the
Diagnostic Fragment Ions of 1
SCHEME 4. Biosynthesis of 1 in the Myxobacteria
M. xanthus and S. aurantiaca
of 1 in S. coelicolor is lost after the deletion of the
N-terminal domain, but not after the removal of the
C-terminal domain.29 Conclusively, Cane and Watt pos-
tulated 2 to be an intermediate in the biosynthesis of 1.
A geosmin-overproducing strain of Streptomyces citreus
also emits considerable amounts of 2, thus suggesting
this compound to be the sesquiterpene precursor of 1.30
Pollak and Berger postulated a route for the biosynthesis
of 1 relying on volatile side products they identified in
cultures of S. citreus.30 Based on the mass spectral data
obtained after feeding of [2H10]leucine, we proposed a
biosynthetic pathway to 1 with intermediate 2 similar
in its early steps to the biosynthetic scheme postulated
by Pollak and Berger (Scheme 4). Herein, FPP is cyclized
to hedycaryol (5) and further isomerized to 2. Protonation
initiates the formation of the bicyclic carbon skeleton. The
isopropyl moiety is then removed by the loss of acetone.
Protonation of the double bond facilitates the attack of
water in combination with a 1,2-hydride shift from the
tertiary bridgehead carbon into the right ring. The
formation of 1 with the correct relative configuration is
in coherence with this mechanism. The hydride shift
leading to isotopomer 1c from 4c results in the formation
of an ion at m/z ) 113.
FIGURE 2. Labeling patterns in 1 and 2 after feeding of [2H10]-
leucine and incorporation of one labeled isoprene unit (a).
Labeling patterns in 1 and 2 after feeding of [2H6]DMAA and
incorporation of one labeled isoprene unit (b). Labeling pat-
terns in 1 and 2 after application of 12 and incorporation of
three labeled isoprene units (c). Expected molecular ions and
diagnostic fragment ions are shown. Asterisks indicate fully
deuterated carbons.
atoms in fragment I (m/z ) 117) and four deuterium
atoms in fragment II (m/z ) 130). One deuterium is
present in fragment I (m/z ) 113) and five deuterium
atoms in fragment II (m/z ) 131) of isotopomer 1c. These
data are not in agreement with the biosynthetic path-
ways to 1 suggested by Boland et al. for Streptomyces5
and Cane and Watt for Streptomyces coelicolor.28
The biosynthesis of 1 is controversially discussed in
the literature. Recently, the gene encoding the enzyme
responsible for the production of 1 and 2 in Streptomyces
coelicolor was identified.28,29 This protein has two do-
mains: The N-terminal domain is used for the formation
of 2,28 while the C-terminal domain is not involved in the
biosynthesis of either 1 or 2 and shows no sesquiterpene
synthase activity in vitro. Furthermore, the production
The last step in the pathway postulated by Boland et
al. (Scheme 5a) is characterized by a hydrogen shift of
the same hydrogen, but into the left ring of 1.5 Thus, the
(28) Cane, D. E.; Watt, R. M. Proc. Natl. Acad. Sci. U.S.A. 2003,
100, 1547-1551.
(29) Gust, B.; Challis, G. L.; Fowler, K.; Kieser, T.; Chater, K. F.
Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 1541-1546.
(30) Pollak, F. C.; Berger, R. G. Appl. Environ. Microbiol. 1996, 62,
1295-1299.
5178 J. Org. Chem., Vol. 70, No. 13, 2005

Geosmin Biosynthesis in Myxobacteria
SCHEME 5. Biosynthesis of 1 in the Liverwort
Fossombronia pusilla (a) and Two Suggested
Pathways for the Biosynthesis of 1 in Streptomyces
sp. (b)
cyclization leads to a bicyclic cation that is suggested to
be an intermediate in both pathways. The methine
hydrogen attached to the tertiary bridgehead carbon is
lost in the following steps of both pathways under
discussion. Consequently, both pathways would require
the diagnostic fragment ion at m/z ) 112 after the
incorporation of 4c. It can therefore be ruled out that
either of them is used by M. xanthus.
Furthermore, the sesquiterpene 3 is emitted by M.
xanthus as a trace component, but no incorporation of
[2H10]leucine is observed because of the low amounts
present in the extract. In a recent study on Streptomyces
coelicolor, the cyclization of FPP to 2 and 3 is shown to
be catalyzed by the same enzyme.35 An alternative
pathway to 2 via nerolidyl pyrophosphate and a common
cationic intermediate in the biosynthesis of 2 and 3
(helminthogermacradienyl cation) is presented. This
pathway is also consistent with the data obtained after
feeding [2H10]leucine.
In a subsequent experiment, [4,4,4,5,5,5-2H6]dimeth-
ylacrylate ([2H6]DMAA), one of the proposed intermedi-
ates on the catabolic route from leucine to HMG-CoA,18,19
was fed to the wild-type strains and the bkd mutant
strains of M. xanthus and S. aurantiaca. While the wild-
type strains did not incorporate [2H6]DMAA into the
isoprenoids, the mutant strains readily incorporated this
labeled intermediate into 1 and 2 (Figure 1c, d, and h).
One feeding experiment (feeding of [2H6]DMAA to the
mutant strain JD300 of M. xanthus) did not yield 2, and
therefore no incorporation into this compound was ob-
served. The incorporation rates for [2H6]DMAA in the
case of the wild-type strains seem to be low. Accordingly,
the incorporation is beyond the limits of detection in the
GC-MS system. In contrast, the mutant strains impaired
in the production of dimethylacrylyl-CoA from leucine
show significant incorporation (Table 1). The incorpora-
tion of one and two labeled isoprene units into 1 was
indicated by a shift of the molecular ion from m/z ) 182
to 187 and 192, respectively. Thus each [4,4,4,5,5,5-2H6]-
DMAA is responsible for the occurrence of five deuterium
atoms in 1. The incorporation of one unit of [2H6]DMAA
leads to three FPPs with different labeling patterns
similar to 4a, 4b, and 4c, only missing the olefinic
deuterium. While the incorporation of [2H5]FPP leads to
the complete loss of deuterium in 1 (similar to 4a),
labeling in the other two respective isoprene units affords
the two different isotopomers 1e and 1f (Figure 2b),
which cannot be separated by GC. The incorporation of
[2H6]DMAA into the second isoprene unit of FPP (similar
to 4b) results in a base peak shift from m/z ) 112 to 117,
while the incorporation into the third isoprene unit of
FPP (similar to 4c) gives rise to a fragment with m/z )
112 (Figure 1c). Furthermore, the incorporation of two
units [2H6]DMAA into 1 is accompanied by a base peak
at m/z ) 117 (Figure 1d). In the case of 2, the incorpora-
tion of only one unit [2H6]DMAA is observed furnishing
the coeluting isotopomers 2d, 2e, and 2f (Figure 1h,
Figure 2b). This is indicated by a common molecular ion
at m/z ) 227 and fragment ions at m/z ) 59 and 64,
respectively, in the expected ratio of about 2:1.
incorporation of 4c following this pathway would neces-
sarily lead to the detection of the diagnostic fragment
ion at m/z ) 112. Boland et al. used different labeled
precursors as [5,5-2H2]DOX ([5,5-2H2]deoxyxylulose),
[4,4,6,6,6-2H5]MVA, and [2,2-2H2]MVA, respectively. [5,5-
2H2]DOX was used in investigations on Streptomyces sp.
(sesquiterpenes can arise in streptomycetes through the
deoxyxylulose phosphate pathway as well as the meva-
lonate pathway depending on the growth phase31,32),
whereas [4,4,6,6,6-2H5]MVA and [2,2-2H2]MVA were used
in investigations on the liverwort Fossombronia pusilla
(sesquiterpenes are formed in this species via the meva-
lonate pathway only33,34). While the results of feeding
experiments with F. pusilla employing [4,4,6,6,6-2H5]-
MVA clearly indicated the hydrogen shift into the left
ring of 1 and therefore gave evidence for the pathway
outlined in Scheme 5a, the feeding of [5,5-2H2]DOX to
Streptomyces sp. led to an ambiguous result. The H-2 in
FPP is not labeled, and thus the fate of this diagnostic
hydrogen atom cannot be followed. Both pathways shown
in Schemes 4 and 5a are in accordance with the frag-
mentation pattern of labeled 1 obtained by Boland et al.
after feeding of [5,5-2H2]DOX to Streptomyces sp. It
should be noticed, however, that different organisms
might use different pathways for the biosynthesis of the
same compound.
Two alternative pathways suggested by Cane and
Watt28 are presented in Scheme 5b (pathways a and b).
Both comprise the formation of 2 by cyclization of FPP
to 5 and subsequent isomerization. The proton-mediated
(31) Seto, H.; Watanabe, H.; Furihata, K. Tetrahedron Lett. 1996,
37, 7979-7982.
Deuterium-labeled mevalonic acid lactone [4,4,6,6,6-
2H5]MVA (12) was synthesized to further investigate the
(32) Seto, H.; Orihara, N.; Furihata, K. Tetrahedron Lett. 1998, 39,
9497-9500.
(33) Warmers, U.; Ko¨nig, W. A. Phytochemistry 2000, 53, 645-650.
(34) Thiel, R.; Adam, K. P. Phytochemistry 2002, 59, 269-274.
(35) He, X.; Cane, D. E. J. Am. Chem. Soc. 2004, 126, 2678-2679.
J. Org. Chem, Vol. 70, No. 13, 2005 5179

Dickschat et al.
SCHEME 6. Synthesis of 12a
into 2 can be observed (Figure 4b). The incorporation
rates were higher in the experiment with M. xanthus
than with S. aurantiaca (Table 1). The mass spectrum
of 1g (Figure 1e) showed a molecular ion at m/z ) 191,
and the diagnostic fragment ions shifted from m/z ) 112
to 117 and m/z ) 126 to 133. These data further
corroborate the suggested biosynthetic pathway to 1
(Scheme 4), whereas the pathway described by Boland
et al. (Scheme 5a) would require m/z ) 116 and m/z )
134 due to a 1,2-hydride shift into the left instead of the
right ring of 1. The pathways suggested by Cane and
Watt (Scheme 5b) would require the detection of m/z )
116 and m/z ) 133, because the respective deuterium
atom is lost in both pathways. The mass spectrum of 2g
(Figure 1i) showed a molecular ion at m/z ) 234 and a
base peak at m/z ) 62, indicating the presence of three
deuterium atoms in the isopropyl group (Figure 2c).
In conclusion, it was shown that the leucine-dependent
isoprenoid biosynthesis can also operate for the produc-
tion of sesquiterpenes.19 In myxobacteria, this novel
pathway seems to be sort of a back-up system, because
wild-type strains only poorly incorporate leucine. The
biosynthetic pathway to 1 was clarified by feeding small
amounts of labeled leucine, DMAA, and MVA to M.
xanthus and S. aurantiaca that were cultivated on agar
plates. Leucine is transformed via the leucine-dependent
isoprenoid biosynthetic pathway to HMG-CoA, further
supported by the incorporation of the intermediate
DMAA. Cyclization and isomerization of FPP leads to 2.
The data are consistent with, but do not prove the
intermediacy of 2 in the formation of 1. The subsequent
steps, cyclization to the bicyclic system, loss of acetone,
and proton-mediated addition of water in combination
with a 1,2-hydride shift, are in accordance with the
fragmentation pattern observed after feeding of the
precursors. In particular, the biosynthetic pathway to 1
is different from that described for the liverwort Fossom-
bronia pusilla. Obviously, two independent pathways to
1 exist in nature. Whether the pathway described here
also operates in Streptomyces remains to be determined
(as well as its dependence on the MVA or DOX path-
ways), because the data in the literature do not allow a
discrimination.5
a (a) NaH, THF, DMF, BnBr, 58%; (b) PCC, CH2Cl2, 78%; (c)
D3CMgI, Et2O, 90%; (d) PCC, CH2Cl2, 93%; (e) K2CO3, D2O, 94%;
(f) Rieke-Zn, ethyl bromoacetate, THF, 79%; (g) H2, Pd/C, Et2O;
(h) p-TsOH, CH2Cl2, 85% over two steps.
biosynthetic pathway to 1 (Scheme 6). One of the alcohol
groups of propane-1,3-diol (6) was protected as benzyl
ether to give 7. Oxidation with PCC provided the alde-
hyde 8 that was treated with the Grignard reagent of
[2H3]methyl iodide. PCC oxidation furnished 10a. The
required additional deuterium labeling was introduced
into 10a, resulting in 10b by H/D exchange. While strong
bases (NaOMe in MeOD) led immediately to the elimina-
tion of benzyl alcohol, K2CO3 in D2O required prolonged
reaction times (7 d), but resulted in the formation of 10b
in high yield (94%). NaOD in D2O caused a fast H/D
exchange (2 h), but some elimination occurred. A Refor-
matsky reaction36 with Rieke zink37,38 and ethyl bromo-
acetate then furnished 11. Cleavage of the benzyl ether
moiety with Pd/C in a hydrogen atmosphere gave 13 and
12 arising by spontaneous cyclization. Another portion
of 12 was obtained from 13 by cyclization with p-
toluenesulfonic acid. This procedure allows the conve-
nient synthesis of 12 in a gram scale.
In subsequent experiments, 12 was fed to M. xanthus
and S. aurantiaca wild-type strains and a double mutant
strain of M. xanthus (DK5624). The latter is impaired in
the transformation of HMG-CoA to MVA in addition to
the bkd negative genotype39 and does not produce 1 and
2 (compare Figure 3a and 3b). Total ion chromatograms
obtained after the application of 12 are depicted in Figure
3c (DK1622) and d (DK5624). The wild-type strain used
12 and natural MVA for the production of 1 and 2 with
different degrees of labeling. In contrast, the mutant
strain only emitted 1g and 2g derived from 12 with the
maximum deuterium content of 9 or 12 deuterium atoms,
respectively. The isotopomers of 1 and 2 with different
deuterium content obtained from the wild-type strains
were separable by gas chromatography, but the isoto-
pomers with identical deuterium content coeluted. This
is shown for 1 by the ion chromatograms for the expected
molecular ions above m/z ) 182 (Figure 4a). Similarly,
the incorporation of one to three labeled isoprene units
Experimental Section
Strains, Culture Conditions, Feeding Experiments.
Myxococcus xanthus (strains DK162240 and the bkd mutant
strain JD30041) and Stigmatella aurantiaca (strain DW4/3-1
with the corresponding bkd mutant18) have been described
previously. Cultivation and feeding experiments using these
strains were performed as described.12 Labeled compounds
were dissolved in MeOH and spread on top of freshly prepared
agar plates with 40 µg/mL kanamycin if required (for DK5624)
to reach a final concentration of 1 mM. Plates were inoculated
with 500 µL of a log-phase liquid culture of the required strain
and incubated 1-3 d until significant growth was visible.
Sampling. Volatile organic compounds emitted by cell
cultures of Myxococcus xanthus and Stigmatella aurantiaca
were collected using the CLSA technique as described previ-
ously.12
Preparation of [1,1,1-2H3]-4-Benzyloxy-2-butanol (9).
The synthesis of the known compounds42 7 and 8 is described
(36) Rathke, M. W. Org. React. 1975, 22, 423-460.
(37) Rieke, R. D.; Uhm, S. J.; Hudnall, P. M. J. Chem. Soc., Chem.
Commun. 1973, 267-268.
(38) Rieke, R. D.; Tzu-Jung Li, P.; Burns, T. P.; Uhm, S. T. J. Org.
Chem. 1981, 46, 4324-4326.
(40) Kaiser, D. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 5952-5956.
(41) Toal, D. R.; Clifton, S. W.; Roe, B. A.; Downard, J. Mol.
Microbiol. 1995, 16, 177-189.
(39) Bode, H. B.; Kaiser, D.; Mu¨ller, R., unpublished.
5180 J. Org. Chem., Vol. 70, No. 13, 2005

Geosmin Biosynthesis in Myxobacteria
FIGURE 3. Total ion chromatograms of headspace extracts of Myxococcus xanthus. Wild-type strain DK1622 (a), a mutant strain
(DK5624) that releases no more 1 and 2 (b), DK1622 after feeding of 12 (c), and a mutant strain (DK5624) after feeding of 12 (d).
in the Supporting Information. A solution of 8 (14.8 g, 90.5
mmol, in dry diethyl ether (50 mL)) was added dropwise to an
ice-cooled solution of [2H3]methylmagnesium iodide prepared
3.60-3.72 (m, 2H), 2.91 (br s, 1H), 1.67-1.81 (m, 2H). 13C NMR
(100 MHz, CDCl3): δ ) 137.9 (C), 128.4 (2 × CH), 127.7 (CH),
127.6 (2 × CH), 73.2 (CH2), 69.0 (CH2), 67.3 (CH), 38.1 (CH2).
MS (EI) m/z (%) ) 164 (15), 147 (1), 120 (16), 107 (40), 91 (100),
79 (19), 65 (19), 59 (15), 48 (15).
2
from [2H3]methyl iodide (14.5 g, 100 mmol, 99% H) and Mg
(2.43 g, 100 mmol) in dry diethyl ether (250 mL). The reaction
mixture was stirred overnight at room temperature and then
quenched by the addition of 2 N HCl. The aqueous layer was
extracted three times with diethyl ether. The combined organic
layers were dried with MgSO4 and concentrated. The residue
was purified by column chromatography on silica gel (eluent:
pentane/diethyl ether, 1:1) to give 9 (14.9 g, 90%) as a colorless
liquid. The deuterium content was determined to be >98% by
1H NMR.
Preparation of [1,1,1-2H3]-4-Benzyloxy-2-butanone (10a).
A solution of 9 (14.9 g, 81.5 mmol) in dry CH2Cl2 (50 mL) was
added to a suspension of PCC (26.3 g, 122.3 mmol) in dry CH2-
Cl2 (300 mL). The reaction mixture was stirred overnight.
Workup and purification as described for 8 yielded 10a (13.7
g, 93%) as a colorless liquid. The deuterium content was
1
determined to be 91% by H NMR.
1
9. Rf ) 0.25; I ) 1466. 1H NMR (400 MHz, CDCl3): δ )
7.25-7.36 (m, 5H), 4.51 (s, 2H), 3.99 (dd, J ) 8.1, 2.9 Hz, 1H),
10a. Rf ) 0.25; I ) 1463. H NMR (400 MHz, CDCl3): δ )
7.25-7.36 (m, 5H), 4.50 (s, 2H), 3.73 (t, J ) 6.3 Hz, 2H), 2.70
(t, J ) 6.3 Hz, 2H). 13C NMR (100 MHz, CDCl3): δ ) 207.2
(CO), 138.0 (C), 128.3 (2 × CH), 127.7 (2 × CH), 127.6 (CH),
73.1 (CH2), 65.2 (CH2), 43.7 (CH2). MS (EI) m/z (%) ) 120 (21),
(42) Cleater, E.; Harter, J.; Ley, S. V. Heterocycles 2004, 62, 619-
634.
J. Org. Chem, Vol. 70, No. 13, 2005 5181

Dickschat et al.
added dropwise to the solution of lithium naphthalenide to give
a fine slurry of highly reactive Rieke zinc.37,38 Caution! Rieke
zinc might spontaneously ignite on air and should always be
handled in an inert gas atmosphere. Subsequently, a solution
of ethyl bromoacetate (15.2 g, 91.0 mmol) and 10b (13.2 g, 71.2
mmol) in dry THF (50 mL) was added dropwise. The Zn was
consumed; the resulting reaction mixture was stirred over-
night, and then quenched by the addition of 2 N HCl. The
aqueous layer was extracted three times with diethyl ether.
The combined extracts were dried with MgSO4. The solvents
were removed under reduced pressure. Column chromatogra-
phy on silica gel (eluent: pentane/diethyl ether, 1:1) furnished
11 (15.2 g, 79%) as a colorless oil. The deuterium content was
1
determined to be >95% by H NMR.
1
11. Rf ) 0.43; I ) 1915. H NMR (400 MHz, CDCl3): δ )
7.25-7.37 (m, 5H), 4.49 (s, 2H, CH2), 4.13 (dq, J ) 7.2, 1.8
Hz, 2H), 3.95 (br s, 1H), 3.67 (s, 2H), 2.57 (d, J ) 15.2 Hz,
1H), 2.49 (d, J ) 15.3 Hz, 1H), 1.24 (t, J ) 7.2 Hz, 3H). 13C
NMR (100 MHz, CDCl3): δ ) 172.1 (CO), 137.6 (C), 128.0 (2
× CH), 127.3 (CH), 127.3 (2 × CH), 72.9 (CH2), 70.2 (C), 66.5
(CH2), 60.1 (CH2), 45.1 (CH2), 13.8 (CH3). MS (EI) m/z (%) )
184 (1), 165 (21), 147 (12), 134 (8), 115 (5), 107 (6), 91 (100),
74 (10), 65 (11), 46 (19).
FIGURE 4. (a) Ion chromatograms showing the molecular
ions of 1 after feeding DK1622 with 12: m/z ) 182 (unlabeled
1, arising from three units MVA), m/z ) 183 ([2H1]-1, first unit
12, second and third unit MVA), m/z ) 186 ([2H4]-1, two units
MVA, second or third unit 12), m/z ) 187 ([2H5]-1, two units
12, second or third unit MVA), m/z ) 190 ([2H8]-1, first unit
MVA, second and third unit 12), and m/z ) 191 ([2H9]-1, three
units 12). The ion chromatograms at m/z ) 184, 185, 188, and
189 showed no significant peak intensities. (b) Ion chromato-
grams showing the molecular ions of 2 after feeding DK1622
with 12: m/z ) 222 (unlabeled 2, 3 × MVA), m/z ) 226 ([2H4]-
2, 2 × MVA, 1 × 12), m/z ) 230 ([2H8]-2, 1 × MVA, 2 × 12),
and m/z ) 234 ([2H12]-2, 3 × 12). The ion chromatograms at
m/z ) 223, 224, 225, 227, 228, 229, 231, 232, and 233 showed
no significant peak intensities.
Preparation of [4,4,6,6,6-2H5]Mevalonic Acid Lactone
(12). Benzyl ether 11 (2.71 g, 10.0 mmol) was added to a
suspension of Pd/C (532 mg, 0.50 mmol, 10% Pd) in diethyl
ether (20 mL). The mixture was stirred in a H2 atmosphere at
room temperature for 2 h, filtered, and concentrated. Column
chromatography on silica gel (eluent: diethyl ether) gave 12
(0.83 g, 46%) and ethyl [2H5]mevalonate (13, 0.57 g) as colorless
liquids. For GC-MS analysis, both products were transformed
into TMS derivatives with MSTFA. Compound 13 (0.57 g, 3.15
mmol) was dissolved in dry CH2Cl2 (50 mL). A catalytic
amount of p-toluenesulfonic acid (2 mg, 0.01 mmol) was added.
The mixture was stirred for 48 h and concentrated. The residue
was purified by column chromatography on silica gel (eluent:
diethyl ether) to give another portion of 12 (315 mg, 74% from
13; total yield of 12: 1.15 g, 85%). The deuterium content was
107 (59), 91 (100), 79 (26), 65 (30), 57 (14), 51 (15), 46 (85), 39
(12).
Preparation of [1,1,1,3,3-2H5]-4-Benzyloxy-2-butanone
(10b) by H/D Exchange. A mixture of 10a (13.7 g, 75.8
mmol), K2CO3 (2.09 g, 15.2 mmol), and D2O (50 mL) was
stirred at room temperature for 7 d. The reaction mixture was
extracted three times with diethyl ether. The combined
extracts were dried (MgSO4) and concentrated. Purification
by column chromatography on silica gel (eluent: pentane/
diethyl ether, 2:1) gave 10b (13.3 g, 94%) as a colorless liquid.
The deuterium content was determined to be >95% by 1H
NMR.
1
determined to be >95% by H NMR.
1
12. Rf ) 0.10; I ) 1385. H NMR (400 MHz, CDCl3): δ )
4.60 (d, J ) 11.3 Hz, 1H), 4.34 (d, J ) 11.3 Hz, 1H), 2.71 (br
s, 1H), 2.66 (d, J ) 17.4 Hz, 1H), 2.52 (d, J ) 17.3 Hz, 1H).
13C NMR (100 MHz, CDCl3): δ ) 170.5 (CO), 68.0 (C), 65.9
(CH2), 44.6 (CH2). MS (EI) m/z (%) ) 192 (12), 162 (11), 150
(100), 133 (5), 118 (44), 103 (11), 73 (39), 59 (6), 46 (15).
1
13. Rf ) 0.25; I ) 1500. H NMR (400 MHz, CDCl3): δ )
4.21 (br s, 1H), 4.18 (q, J ) 7.1 Hz, 2H), 3.88 (dd, J ) 11.1, 2.0
Hz, 1H), 3.81 (dd, J ) 11.2, 4.1 Hz, 1H), 3.37 (br s, 1H), 2.62
(d, J ) 15.4 Hz, 1H), 2.48 (d, J ) 15.4 Hz, 1H), 1.29 (t, J ) 7.1
Hz, 3H). 13C NMR (100 MHz, CDCl3): δ ) 172.6 (CO), 71.6
(C), 60.6 (CH2), 59.1 (CH2), 45.1 (CH2), 14.0 (CH3). MS (EI)
m/z (%) ) 310 (18), 280 (6), 261 (12), 250 (7), 238 (28), 221
(10), 206 (100), 190 (20), 177 (10), 160 (25), 147 (58), 133 (13),
119 (16), 103 (42), 88 (17), 73 (59), 59 (4), 45 (5).
1
10b. Rf ) 0.25; I ) 1458. H NMR (400 MHz, CDCl3): δ )
7.25-7.36 (m, 5H), 4.51 (s, 2H), 3.72 (s, 2H). 13C NMR (100
MHz, CDCl3): δ ) 207.4 (CO), 138.0 (C), 128.4 (2 × CH), 127.7
(2 × CH), 127.6 (CH), 73.2 (CH2), 65.2 (CH2). MS (EI) m/z (%)
) 120 (12), 107 (65), 91 (96), 77 (35), 65 (27), 59 (16), 51 (13),
46 (100), 39 (11).
Preparation of Ethyl [4,4,6,6,6-2H5]-5-Benzyloxy-3-hy-
droxy-3-methylpentanoate (11). As described by Naka-
mura,43 freshly cut Li (0.66 g, 94.8 mmol) was added to a
solution of naphthalene (12.1 g, 94.8 mmol) in dry THF (50
mL). The reaction mixture immediately turned to dark green
and was stirred until the Li was totally consumed. ZnCl2 (12.9
g, 94.8 mmol) was dried at 120 °C in vacuo for 2 h and then
dissolved in dry THF (50 mL). The resulting solution was
Acknowledgment. J.S.D. thanks the Fonds der
Chemischen Industrie and the BMBF for a stipend.
Supporting Information Available: General methods;
preparation of 7 and 8; 1H and 13C NMR spectra of compounds
7-12. This material is available free of charge via the Internet
at http://pubs.acs.org.
(43) Nakamura, E. Organozinc Chemistry. In Organometallics in
Synthesis; Schlosser, M., Ed.; John Wiley & Sons: Chichester, 2002.
JO050449G
5182 J. Org. Chem., Vol. 70, No. 13, 2005