Metabolites of the Anaerobic Degradation of n-Hexane by Denitrifying Betaproteobacterium Strain HxN1

Article abstract of DOI:10.1002/cbic.201900375

The constitutions of seven metabolites formed during anaerobic degradation of n-hexane by the denitrifying betaproteobacterium strain HxN1 were elucidated by comparison of their GC and MS data with those of synthetic reference standards. The synthesis of 4-methyloctanoic acid derivatives was accomplished by the conversion of 2-methylhexanoyl chloride with Meldrum's acid. The β-oxoester was reduced with NaBH₄, the hydroxy group was eliminated, and the double bond was displaced to yield the methyl esters of 4-methyl-3-oxooctanoate, 3-hydroxy-4-methyloctanoate, (E)-4-methyl-2-octenoate, and (E)- and (Z)-4-methyl-3-octenoate. The methyl esters of 2-methyl-3-oxohexanoate and 3-hydroxy-2-methylhexanoate were similarly prepared from butanoyl chloride and Meldrum's acid. However, methyl (E)-2-methyl-2-hexenoate was prepared by Horner–Wadsworth–Emmons reaction, followed by isomerization to methyl (E)-2-methyl-3-hexenoate. This investigation, with the exception of 4-methyl-3-oxooctanoate, which was not detectable in the cultures, completes the unambiguous identification of all intermediates of the anaerobic biodegradation of n-hexane to 2-methyl-3-oxohexanoyl coenzyme A (CoA), which is then thiolytically cleaved to butanoyl-CoA and propionyl-CoA; these two metabolites are further transformed according to established pathways.

Full text of DOI:10.1002/cbic.201900375

Accepted Article
Title: Identification of Metabolites of the Anaerobic Degradation of n-
Hexane by Denitrifying Betaproteobacterium Strain HxN1
Authors: Jens Christoffers, Julian Küppers, Nico Mitschke, Simone
Heyen, Ralf Rabus, and Heinz Wilkes
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10.1002/cbic.201900375
ChemBioChem
Identification of Metabolites of the Anaerobic Degradation of n-
Hexane by Denitrifying Betaproteobacterium Strain HxN1
Julian Küppers,[+,a] Nico Mitschke,[+,b] Simone Heyen,[b] Ralf Rabus,[b] Heinz Wilkes,[b]
Jens Christoffers*[a]
[a]J. Küppers,[+] Prof. Dr. J. Christoffers
Institut für Chemie, Carl von Ossietzky Universität Oldenburg, 26111 Oldenburg
(Germany)
E-Mail: jens.christoffers@uol.de
Homepage: https://uol.de/oc-christoffers/
[b]N. Mitschke,[+] S. Heyen, Prof. Dr. R. Rabus, Prof. Dr. H. Wilkes
Institut für Chemie und Biologie des Meeres (ICBM), Carl von Ossietzky Universität
Oldenburg, 26111 Oldenburg (Germany)
[+]These authors contributed equally to this work.
Abstract: The constitutions of seven metabolites formed during anaerobic
degradation of n-hexane by the denitrifying betaproteobacterium strain HxN1 were
elucidated by comparison of gas chromatographic and mass spectrometric data with
synthetic reference standards. Synthesis of 4-methyloctanoic acid derivatives was
accomplished by conversion of 2-methylhexanoyl chloride with Meldrum’s acid. The
β-oxoester was reduced with NaBH4, the hydroxy group eliminated and the double
bond displaced to yield the methyl esters of 4-methyl-3-oxooctanoate, 3-hydroxy-4-
methyloctanoate, (E)-4-methyl-2-octenoate and (E)- and (Z)-4-methyl-3-octenoate,
respectively. The methyl esters of 2-methyl-3-oxohexanoate and 3-hydroxy-2-
methylhexanoate were similarly prepared from butanoyl chloride and Meldrum’s acid.
However, methyl (E)-2-methyl-2-hexenoate was prepared by HWE-reaction, followed
by isomerization to methyl (E)-2-methyl-3-hexenoate. This investigation, with the
exception of 4-methyl-3-oxooctanoate which was not detectable in the cultures,
completes the unambiguous identification of all intermediates of anaerobic
biodegradation of n-hexane to 2-methyl-3-oxohexanoyl-CoA which is then thiolytically
cleaved to butanoyl-CoA and propionyl-CoA the two latter being further transformed
1
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10.1002/cbic.201900375
ChemBioChem
according to established pathways.
Key Topic: Anaerobic Biodegradation
Graphical Abstract: Metabolites formed during anaerobic degradation of n-hexane
by the denitrifying betaproteobacterium strain HxN1 were identified by comparison
with newly synthesized reference standards using GC-MS.
Anaerobic Biodegradation: X = SCoA
Me
Me
Me
Me
Me
Me
O
OH
O
X
O
SCoA
O
Me
Me
Me
Me
Me
Me
O
O
O
X
SCoA
X
X
Chemical Synthesis: X = OMe
Keywords: alkanes, anaerobic degradation, "Aromatoleum" sp. HxN1, C‒H-bond
activation, oxoester
Introduction
The key challenge in the biodegradation of saturated hydrocarbons is C‒H-bond acti-
vation. The initial activation step requires the cleavage of a C‒H-bond, being associa-
ted with a high energy barrier, which has to be overcome. In oxic environments (pre-
sence of O2) this is accomplished by the well-studied oxygenase enzymes employing
O2-derived highly reactive oxygen species.[1] Investigations on the biodegradation of
hydrocarbons under anoxic conditions (absence of O2) led to the discovery of a large
diversity of novel microorganisms and biochemical transformations (for overviews
see[2]).
The betaproteobacterium strain HxN1, which affiliates with the newly described ge-
nus Aromatoleum,[3] was originally isolated from ditch sediments in Bremen (Ger-
many) and has been shown to completely oxidize n-hexane (1) to CO2 under strictly
anoxic conditions coupled to denitrification.[4] Based on metabolite and EPR studies,
anaerobic degradation of n-hexane (1) by strain HxN1 has been proposed to proceed
via an 1-methylpentyl radical, which adds to fumarate yielding (1-methylpentyl)succi-
nate (MPS, 2, Scheme 1, step a) in a reaction catalyzed by a glycyl radical enzyme.[5]
2
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ChemBioChem
Subsequent investigations with stereoisomers of (2,5-2H2)hexane revealed inversion
of configuration at C-2 of n-hexane (1) during formation of dicarboxylic acid 2. Based
on this evidence it has been suggested that C‒H-bond cleavage and C‒C-bond for-
mation may proceed in a concerted manner which thus would avoid a highly reactive
1-methylpentyl radical as a free intermediate.[6] Further degradation of MPS (2) was
inferred from metabolite analysis to proceed by the following reaction sequence
(Scheme 1):[7] Thioesterification to (1-methylpentyl)succinyl-CoA (3, step b) is follo-
wed by a vitamin B12-dependent mutase-catalyzed rearrangement of the C-skeleton
in the succinyl-CoA moiety by an 1,2-acyl shift (step c) forming (2-methylhexyl)malo-
nyl-CoA (4) to then furnish 4-methyloctanoyl-CoA (5a) by decarboxylation (step d).
The β-oxidation sequence of the latter via 4-methyl-2-octenoyl-CoA (6a, step e) and
3-hydroxy-4-methyloctanoyl-CoA (7a, step f) leads to 4-methyl-3-oxooctanoyl-CoA
(8a, step g), which undergoes thiolytic cleavage (step h) forming acetyl-CoA (9) and
2-methylhexanoyl-CoA (10a). A second round of β-oxidation starting with 2-methyl-
hexanoyl-CoA (10a) would involve sequential formation of 2-methyl-2-hexenoyl-CoA
(11a, step i), 3-hydroxy-2-methylhexanoyl-CoA (12a, step j) and 2-methyl-3-oxohex-
anoyl-CoA (13a, step k), with the latter being thiolytically cleaved into butanoyl-CoA
(16) and propionyl-CoA (17). A third round of β-oxidation would then transform C4-
compound 16 into two acetyl-CoA (9). While the three acetyl-CoA (9) formed are ter-
minally oxidized to CO2 in the TCA cycle, the propionyl-CoA (17) could be converted
to fumarate by the methylmalonyl-CoA pathway formally utilizing one equivalent of
CO2, and thus recycling the co-substrate of the initial n-hexane activation reaction.
The aim of the present study was to complete our metabolite-based understanding of
the anaerobic degradation of n-hexane (1) downstream of MPS (2), by unambi-
guously elucidating the constitutions of intermediates 6a, 7a, and 11a–15a which to
date have only tentatively been assigned based on mass spectrometric data. This
task was accomplished by chemical synthesis of the respective compounds (as their
methyl esters 6b, 7b, and 11b–15b) and their application as reference standards for
unambiguous metabolite identification by GC-MS. Compounds 14a and 15a with iso-
merized C‒C-double bond are actually not included in the metabolic pathway shown
in Scheme 1, anyhow, the respective methyl esters 14b and 15b are present in the
methylated culture extract of strain HxN1.
3
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ChemBioChem
CO2
COSCoA
CO2
O
(a)
(b)
(c)
CO2
Me
Me
Me
Me
Me
Me
Me
Me
SCoA
SCoA
O2C
Me
(MPS)
Me
Me
Me CO2
+
CO2
1
2
3
4
(d)
 CO2
O
O
OH
O
O
O
(g)
(e)
(f)
SCoA
Me
Me
SCoA
SCoA
Me
Me
Me
Me
Me
Me
8a
7a
6a
5a
(h)  AcSCoA (9)
O
O
OH
O
O
O
(j)
(k)
(i)
SCoA
SCoA
SCoA
SCoA
Me
Me
Me
Me
10a
11a
12a
13a
(l)
O
O
O
O
+
Me
SCoA
Me
SCoA
Me
SCoA
SCoA
17
Me
14a
Me
15a
Me
16
Scheme 1. Proposed pathway for the anaerobic degradation of n-hexane (1) by strain HxN1, for corresponding transformations (a)
to (l) see text.
4
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ChemBioChem
Results and Discussion
Organic Synthesis. The synthesis of the 4-methyloctanoic acid series started from
commercially available 2-methylhexanoic acid 18. The latter was first converted to its
acid chloride, which was isolated and purified by distillation (Scheme 2).[8] The con-
version with Meldrum's acid and subsequent solvolysis with methanol under standard
conditions (with pyridine and catalytic amounts of DMAP)[9] furnished however com-
plex reaction mixtures containing only small amounts of β-oxoester 8b.[10] After some
experimentation, the use of an overstoichiometric amount of DMAP (1.8 equiv.) tur-
ned out to furnish compound 8b in preparatively useful quantities (70% yield). Treat-
ment of compound 8b with NaBH4 gave both diastereoisomers of the β-hydroxyester
7b[11] (78% yield) without any stereoselectivity (dr 53:47). The isomers were not se-
parated, but its mixture submitted to elimination via the methanesulfonate to furnish
α,β-unsaturated ester 6b[12] in 77% yield and exclusively as the (E)-isomer. The C‒C-
double bond was shifted to furnish the β,γ-unsaturated isomer 15b[13] by a two-step
strategy originally introduced by Orsini et al.[14] The first step is the allylic bromination
with NBS–AIBN, yielding intermediate product 19 (79%), which was then submitted to
reduction with Zn–AcOH with displacement of the C‒C-double bond. Compound 15b
(63%) was obtained as a mixture of diastereoisomers (E/Z 2:1), which were not sepa-
rated. The configuration was assigned by the following NOE-experiments: Irradiation
of the 4-CH3 group at 1.62 ppm lead to almost no NOE effect at the olefinic signal at
5.31 ppm; thus, this signal belongs to the (E)-isomer. If the 4-CH3 group at 1.73 ppm
is irradiated, a pronounced NOE of the olefinic signal is observed, hence, this is the
(Z)-isomer.
5
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ChemBioChem
O
(a)
CO2Me
CO2H
Me
Me
Me
Me
18
8b, 70%
(b)
OH
CO2Me
(c)
(e)
Me
Me
CO2Me
Me
Me
Me
Me
6b, 77%
7b, 78% ( 53:47)
dr
(d)
CO2Me
CO2Me
Me Br
Me
, 79%
, 63% ( / 2:1)
15b E Z
19
Scheme 2. Synthesis of members of the 4-methyloctanoic acid series. Reagents and
conditions: (a) 1. 4.0 equiv. SOCl2, 76°C, 3 h; 2. distillation; 3. 0.9 equiv. Meldrum's
acid, 1.8 equiv. DMAP, CH2Cl2, 23°C, 15 h; 4. MeOH, 65°C, 24 h; (b) 1.2 equiv.
NaBH4, MeOH, 23°C, 17 h; (c) 1.4 equiv. MeSO2Cl, 9.0 equiv. NEt3, CH2Cl2, 23°C,
28 h; (d) 1.0 equiv. NBS, 6 mol% AIBN, CHCl3, 75°C, 19 h; (e) 8.2 equiv. Zn, AcOH,
23°C, 1 h.
The synthesis of the 2-methylhexanoic acid series also started with the acylation of
Meldrum's acid,[9] in this case with butanoyl chloride (20) furnishing oxoester 21[9,15] in
moderate yield (38%); anyhow, pyridine and only catalytic amounts of DMAP could
be applied here (Scheme 3). The methylation with MeI occurred with K2CO3 in ace-
tone with surprisingly high selectivity towards the mono-alkylated product 13b[16]
(55%). Reduction with NaBH4 gave the alcohol 12b[17] (57%) as an inseparable mix-
ture of two diastereoisomers (dr 3:2). Upon attempts of elimination using the same
protocol as for compound 6b (Scheme 2) the α,β-unsaturated compound 11b (see
Scheme 4) was obtained, however, together with unspecified impurities, which could
not be separated by column chromatography. For this reason, compound 11b was
accessed as outlined below.
6
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ChemBioChem
O
(a)
(c)
CO2Me
COCl
Me
Me
Me
20
21, 38%
(b)
OH
O
CO2Me
CO2Me
Me
Me
12b, 57% ( 3:2)
Me
13b, 55%
dr
Scheme 3. Synthesis of members of the 2-methylhexanoic acid series. Reagents and
conditions: (a) 1. 0.9 equiv. Meldrum's acid, 0.2 equiv. DMAP, 1.8 equiv. pyridine,
CH2Cl2, 23°C, 19 h; 2. MeOH, 65°C, 4 h; (b) 1.5 equiv. MeI, 1.0 equiv. K2CO3, ace-
tone, 23°C, 3.5 h; (c) 1.2 equiv. NaBH4, MeOH, 23°C, 3 h.
HWE-reaction of phosphorylpropionate 22[18] with butanal gave α,β-unsaturated ester
11b[19] as mixture of diastereoisomers (61%, E/Z 4:1, Scheme 4), which could be se-
parated by column chromatography. The (E)-selectivity of the HWE-reaction in the
formation of α-methyl-α,β-unsaturated esters has been investigated before in the lite-
rature.[20] Furthermore, the NMR spectra of both diastereoisomers of ester 11b were
previously reported.[19] The (E)-isomer was submitted to two-step double bond dis-
placement as performed above to furnish the β,γ-unsaturated isomer 14b[21] (55%,
only trans-configurated) via the allylic bromide 23 (93%).
O
CO2Me
Me
(EtO)2P
CO2Me
(a)
Me
Me
22
11b, 61% ( / 4:1)
E Z
(b)
(c)
CO2Me
CO2Me
Me
Me
Me
14b, 55%
Br Me
23, 93%
Scheme 4. Synthesis of unsaturated derivatives of 2-methylhexanoic acid. Reagents
and conditions: (a) 1.0 equiv. butanal, 1.1 equiv. KOtBu, THF, 23°C, 1 h; (b) 1.0
equiv. NBS, 6 mol% AIBN, CCl4, 80°C, 19 h; (c) 2.0 equiv. Zn, AcOH, 0°C, 1 h, then
23°C, 1 h.
7
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Identification of Metabolites. Methylated culture extracts of strain HxN1 after an-
aerobic growth with n-hexane were analyzed by gas chromatography-mass spectro-
metry (GC-MS). Accordingly, all reference standards needed for unambiguous iden-
tification of metabolites have been synthesized as the respective methyl esters (see
above). Target structures of the so far unidentified metabolites were proposed based
on their mass spectrometric fragmentation patterns. Conclusive identification of the
detected metabolites was accomplished by GC co-injection experiments and compa-
rison of mass spectra (Figures 1 and 2, Figures S1–S16 in the Supporting Informa-
tion). Oxoester 8b could not be detected which may indicate that the steady-state
concentration of transient metabolite 8a is below detection limit in growing cultures of
strain HxN1. The underlying β-thiolase reaction is actually known to be far on the
side of the oxoester cleavage products.[22] Interestingly, the two diastereoisomers of
methyl 3-hydroxy-2-methylhexanoate (12b) were present in the culture extracts of
strain HxN1 in a similar proportion as furnished by the synthetic procedure. The two
diastereoisomers of hydroxyester 7b were not separable under the GC conditions
applied.
The comparison of synthetic esters 14b and 15b with the methylated extract from cul-
tures of strain HxN1 by GC-MS have doubtlessly confirmed, that these compounds
with isomerized β,γ-C‒C-double bonds are present in the extract. It might be assu-
med, however, that compounds have been formed artificially during heat deactivation
and acid treatment of the culture broth, e.g. by isomerization of compounds 6a or 11a
or by elimination of alcohols 7a or 12a, respectively. For this reason, we have perfor-
med the following control experiments: The β-hydroxyesters 7b or 12b as well as the
free carboxylic acid corresponding to ester 6b were treated under respective condi-
tions (85°C at pH 1.5, hydrochloric acid). These mixtures were then analyzed by GC-
MS and 1H NMR, and in none of the cases, compounds with β,γ-double bonds could
be detected. Therefore we conclude, that compounds 14a and 15a are native meta-
bolites of strain HxN1, though their possible roles remain unclear at this point.
8
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Figure 1. Gas chromatographic separation of isomers of methyl 4-methyloctenoates
[6b and (E)-, (Z)-15b] and co-injections with methylated culture extracts of strain
HxN1 after anaerobic growth with n-hexane (1). Depicted are the total ion chromato-
grams. The metabolite methyl 4-methyloctanoate (5b) had been identified and struc-
turally elucidated before.[7] The relative abundance of component 5b is used as an in-
ternal reference to assess the increase of peaks upon co-injection. (A) Methylated
culture extract of strain HxN1; (B) synthetic standard methyl (Z)- and (E)-4-methyl-3-
octenoate (15b) (* = impurity); (C) co-injection of extract and standard 15b; (D)
synthetic standard methyl (E)-4-methyl-2-octenoate (6b); (E) co-injection of extract
and standard 6b.
9
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ChemBioChem
Figure 2. Mass spectra of compounds eluting at 21.42 (A), 22.07 (B) and 22.15 (C) min from methylated culture extracts of HxN1 (cf.
Figure 1A) compared with the mass spectra of synthetic standards (Z)-15b (D), (E)-15b (E) and 6b (F).
10
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Conclusion
The betaproteobacterium strain HxN1 degrades n-hexane (1) under anoxic condi-
tions into three equivalents of acetyl-CoA (9), which are further oxidized to CO2 in the
TCA cycle. A metabolic pathway has been proposed earlier to proceed from (2-me-
thylhexyl)malonyl-CoA (4) through two rounds of β-oxidation via 4-methyloctanoate
derivatives 5a–8a and 2-methylhexanoate derivatives 10a–13a (Scheme 1). Extracts
of a strain HxN1 culture anaerobically grown with n-hexane (1) were actually submit-
ted to thioester hydrolysis with hydrochloric acid and methylation with diazomethane
to furnish the respective methyl esters 5b–13b for analysis by GC-MS. While the
structures of compounds 5b and 10b, the methyl esters related to thioesters 5a and
10a, have been elucidated earlier, the constitutions of methylated metabolites 6b–
13b were so far proposed based on mass spectrometric data. Furthermore, two β,γ-
unsaturated congeners 14b and 15b were also proposed to be present in the culture
extracts, although their role in the metabolic pathway remains so far unclear.
In this study, we have prepared synthetic compounds 6b–8b and 11b–15b and com-
pared them with the constituents of the methylated culture extract by gas chromato-
graphy including co-injection experiments and mass spectrometry. This enabled us to
unequivocally establish the molecular identities of seven metabolites which so far
were only tentatively identified. The only exception was the methyl ester of 4-methyl-
3-oxooctanoate (8b) which could not be detected in the extract, presumably because
the steady-state concentration of this transient metabolite is below the detection limit.
In both, the 4-methyloctanoate (6b–8b and 15b) and the 2-methylhexanoate (10a–
14a) series, the organic synthesis started with the acylation of Meldrum’s acid with
the appropriate acid chlorides to furnish the β-oxoesters 8b and 21; the latter was α-
methylated to give target compound 13b. The β-oxoesters 8b and 13b were submit-
ted to reduction with NaBH4 to give the respective β-hydroxyesters 7b and 12b (both
as mixtures of two racemic diastereoisomers). While compound 7b could be elimina-
ted to compound 6b after activation of the hydroxy group as methyl sulfonate, this
transformation was rather sluggish in the 2-methylhexanoate series. Therefore, we
have prepared compound 11b by HWE reaction of butanal with phosphorylpropio-
nate 22.
11
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Isomerization of the α,β-double bonds in compounds 6b and 11b was accomplished
in two steps: allylic bromination with NBS–AIBN furnished the γ-bromo congeners 19
and 23. The C–C-double bond was then shifted to the β,γ-positon by reduction with
Zn in AcOH to give compounds 14b and 15b.
The synthetic routes to obtain the reference standards, in particular for those of 4-
methyloctanoate series, to a certain extent mimic the reversed pathway of n-hexane
degradation in strain HxN1. This study furthers our metabolite-based understanding
of the anaerobic degradation of n-hexane (1) by "Aromatoleum" sp. HxN1. Since this
biodegradation pathway is archetypical for n-alkanes of a very broad chain length
range and diverse anaerobic microorganisms including nitrate-, sulfate-, and arsen-
ate-reducing bacteria and even a sulfate-reducing archaeon, the present findings will
serve as valuable reference for pathway-oriented studies with pure cultures as well
as environmental samples (e.g. from hydrocarbon-containing soils and sediments). It
can be envisaged that the synthetic procedures described may also provide access
to homologues with shorter and longer carbon chains and thus enable comprehen-
sive metabolite-based investigations of anaerobic biodegradation of n-alkanes in
laboratory-based as well as field studies.
Experimental Section
General. Preparative column chromatography was carried out using Merck SiO2 (35–
70 µm, type 60 A) with hexanes (mixture of isomers, bp. 64–71°C), tert-butylmethyl-
ether (MTBE), and CH2Cl2 as eluents. TLC was performed on aluminum plates
1
coated with SiO2 F254. H and 13C NMR spectra were recorded on Bruker Avance
DRX 500 and 300 instruments. Multiplicities of carbon signals were determined with
DEPT experiments. HRMS spectra of products were obtained with Waters Q-TOF
Premier (ESI) or Thermo Scientific DFS (EI) spectrometers. IR spectra were recorded
on a Bruker Tensor 27 spectrometer equipped with a diamond ATR unit. All starting
materials were commercially available.
2-Methylhexanoyl chloride. A mixture of 2-methylhexanoic acid (18) (8.7 mL, 8.0 g,
61 mmol, 1.0 equiv.) and SOCl2 (17.8 mL, 29.2 g, 246 mmol, 4.0 equiv.) was heated
to reflux for 3 h (gas evolution). Subsequently, the mixture was submitted to vacuum
distillation through a 10 cm-Vigreux column yielding 2-methylhexanoyl chloride (8.51
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1
g, 57.3 mmol, 93%) at 53°C (19 mbar) as a colorless liquid. H-NMR (500 MHz,
CDCl3): δ = 0.91 (t, J = 6.9 Hz, 3H), 1.28 (d, J = 6.9 Hz, 3H), 1.31–1.36 (m, 4H),
13
1.48–1.57 (m, 1H), 1.76–1.85 (m, 1H), 2.86 (sex, J = 6.9 Hz, 1H) ppm. C{1H}-NMR
(75 MHz, CDCl3): δ = 13.8 (CH3), 16.9 (CH3), 22.4 (CH2), 28.8 (CH2), 33.1 (CH2),
51.4 (CH), 177.8 (C) ppm. IR (ATR): nu(tilde) 2960 (s), 2935 (s), 2863 (m), 1790 (vs),
1459 (s), 1381 (m), 1144 (m), 934 (vs), 892 (m), 861 (s), 801 (w), 733 (w), 705 (s),
680 (m), 646 (w) cm–1. C7H13ClO (148.63).
Methyl 4-Methyl-3-oxooctanoate (8b). DMAP (5.86 g, 48.0 mmol, 1.8 equiv.) was
added to a solution of Meldrum's acid (3.46 g, 24.0 mmol, 0.9 equiv.) in CH2Cl2. After
stirring the mixture for 15 min at ambient temperature, 2-methylhexanoyl chloride (4.1
mL, 3.9 g, 26 mmol, 1.0 equiv, prepared as given above) was dropwise added over a
period of 15 min. After stirring the mixture for further 15 h at ambient temperature,
hydrochloric acid (2 mol L–1, 100 mL) was added and the resulting suspension vigo-
rously stirred for 5 min. The layers were separated and the organic layer was washed
with water (100 mL). Both combined aqueous layers were extracted with CH2Cl2 (2 x
75 mL, 1 x 50 mL). All four organic layers were combined, dried (MgSO4) and evapo-
rated after filtration. The residue was dissolved in MeOH (100 mL) and the solution
heated to reflux for 1 d. After evaporation, the residue was submitted to chromato-
graphy (SiO2, hexanes/MTBE 6:1, Rf = 0.38) to furnish the title compound 8b (3.13 g,
16.8 mmol, 70%) as a colorless liquid. According to 1H NMR, the compound exists as
two tautomers (keto/enol 85:15). 1H-NMR (500 MHz, CDCl3), keto tautomer: δ = 0.90
(t, J = 7.0 Hz, 3H), 1.11 (d, J = 7.0 Hz, 3H), 1.23–1.42 (m, 5H), 1.64–1.72 (m, 1H),
2.62 (sex, J = 6.9 Hz, 1H), 3.49 (s, 2H), 3.74 (s, 3H) ppm; enol tautomer: δ = 0.89 (t,
J = 7.0 Hz, 3H), 1.13 (d, J = 7.0 Hz, 3H), 1.23–1.42 (m, 5H), 1.55–1.64 (m, 1H), 2.22
(sex, J = 6.9 Hz, 1H), 3.73 (s, 3H), 4.98 (s, 1H), 12.03 (s, 1H) ppm. 13C{1H}-NMR
(125 MHz, CDCl3), keto tautomer: δ = 14.1 (CH3), 16.1 (CH3), 22.8 (CH2), 29.4 (CH2),
32.5 (CH2), 46.8 (CH), 47.6 (CH2), 52.4 (CH3), 167.9 (C), 206.6 (C) ppm; signals for
the enol tautomer could not be identified with certainty. IR (ATR): nu(tilde) 2957 (s),
2933 (s), 2875 (m), 2861 (m), 1749 (vs), 1714 (vs), 1652 (s), 1626 (s), 1451 (s), 1437
(s), 1404 (m), 1377 (m), 1311 (s), 1232 (vs), 1155 (s), 1003 (s), 840 (m), 805 (m) cm–
1. MS (EI, 70 eV): m/z (%) 186 (0.5) [M+], 155 (1), 143 (9), 130 (100), 101 (47), 98
(86), 85 (65), 74 (26), 69 (68), 59 (36), 57 (54). HRMS (ESI): calcd. 193.1416 (for
+
C10H18LiO3 ), found 193.1409 [M + Li+]. C10H18O3 (186.25).
13
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Methyl 3-Hydroxy-4-methyloctanoate (7b). At 0°C (ice–water bath), NaBH4 (62 mg,
1.6 mmol, 1.2 equiv.) was added to a solution of compound 8b (254 mg, 1.36 mmol,
1.0 equiv.) in MeOH (5 mL). After stirring the mixture for 17 h at ambient temperature,
AcOH (1 mol L–1, 10 mL) was added. It was extracted with MTBE (3 x 15 mL) and the
organic layers were combined, dried (MgSO4) and evaporated after filtration. The
residue was submitted to chromatography (SiO2, hexanes/MTBE 1:1, Rf = 0.40) to
furnish the title compound 7b (200 mg, 1.06 mmol, 78%) as a colorless liquid. Accor-
1
1
ding to H NMR, the compound exists as two diastereoisomers (dr 53:47). H-NMR
(500 MHz, CDCl3), major isomer: δ = 0.89 (t, J = 6.6 Hz, 3H), 0.91 (d, J = 6.7 Hz,
3H), 1.08–1.41 (m, 5H), 1.42–1.55 (m, 1H), 1.55–1.64 (m, 1H), 2.47 (dd, J = 16.3 Hz,
J = 9.7 Hz, 1H), 2.49 (dd, J = 16.3 Hz, J = 2.6 Hz, 1H), 2.83 (d, J = 3.8 Hz, 1H), 3.71
(s, 3H), 3.87 (dddd, J = 9.7 Hz, J = 6.4 Hz, J = 3.8 Hz, J = 2.6 Hz, 1H) ppm; minor
isomer: δ = 0.89 (t, J = 6.6 Hz, 3H), 0.91 (d, J = 6.7 Hz, 3H), 1.08–1.41 (m, 5H),
1.42–1.55 (m, 2H), 2.45–2.48 (m, 2H), 2.68 (d, J = 3.7 Hz, 1H), 3.71 (s, 3H), 3.91–
13
3.96 (m, 1H) ppm. C{1H}-NMR (125 MHz, CDCl3), major isomer: δ = 14.2 (CH3),
15.0 (CH3), 23.09 (CH2), 29.5 (CH2), 32.1 (CH2), 37.8 (CH2), 38.3 (CH), 51.9 (CH3),
72.0 (CH), 174.1 (C) ppm; minor isomer: δ = 14.2 (CH3), 14.4 (CH3), 23.07 (CH2),
29.6 (CH2), 32.6 (CH2), 38.2 (CH), 38.8 (CH2), 51.9 (CH3), 71.4 (CH), 174.0 (C) ppm.
IR (ATR): nu(tilde) 3480 (s, br), 2957 (s), 2928 (s), 2873 (m), 2860 (m), 1725 (vs),
1460 (m), 1438 (m), 1379 (w), 1338 (w), 1338 (w), 1277 (m), 1194 (m), 1170 (vs),
1048 (s), 1013 (m), 989 (s) cm–1. MS (EI, 70 eV): m/z (%) 170 (0.5), 139 (4), 128 (4),
+
103 (100), 74 (12), 71 (42). HRMS (ESI): calcd. 195.1572 (for C10H20LiO3 ), found
195.1570 [M + Li+]. C10H20O3 (188.26).
Methyl (E)-4-Methyl-2-octenoate (6b). At 0°C (ice–water bath), MeSO2Cl (626 mg,
5.47 mmol, 1.4 equiv.) and NEt3 (3.56 g, 35.0 mmol, 9.0 equiv.) were added to a so-
lution of compound 7b (735 mg, 3.90 mmol, 1.0 equiv.) in CH2Cl2 (20 mL). After stir-
ring the mixture for 28 h at ambient temperature, saturated aqueous NH4Cl solution
(40 mL) was added. The mixture was extracted with CH2Cl2 (3 x 40 mL) and the or-
ganic layers were combined, dried (MgSO4) and evaporated after filtration. The resi-
due was submitted to chromatography (SiO2, hexanes/MTBE 1:1, Rf = 0.62) to fur-
1
nish the title compound 6b (513 mg, 3.01 mmol, 77%) as a colorless liquid. H-NMR
(500 MHz, CDCl3): δ = 0.88 (t, J = 7.0 Hz, 3H), 1.04 (d, J = 6.7 Hz, 3H), 1.21–1.41
14
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(m, 6H), 2.29 (qtdd, J = 7.9 Hz, J = 6.8 Hz, J = 6.4 Hz, J = 1.2 Hz, 1H), 3.73 (s, 3H),
5.77 (dd, J = 15.7 Hz, J = 1.2 Hz, 1H), 6.87 (dd, J = 15.7 Hz, J = 7.9 Hz, 1H) ppm.
13C{1H}-NMR (125 MHz, CDCl3): δ = 14.2 (CH3), 19.6 (CH3), 22.9 (CH2), 29.5 (CH2),
35.9 (CH2), 36.7 (CH), 51.5 (CH3), 119.3 (CH), 155.3 (CH), 167.5 (C) ppm. IR (ATR):
nu(tilde) 2958 (s), 2929 (s), 2873 (m), 2859 (m), 1724 (vs), 1656 (s), 1458 (m), 1435
(s), 1379 (w), 1352 (m), 1310 (m), 1268 (s), 1213 (m), 1175 (s), 1151 (m), 1138 (m),
1036 (w), 1012 (w), 983 (m), 941 (w), 916 (w), 861 (w), 725 (w), 715 (w), 646 (w) cm–
1. MS (EI, 70 eV): m/z (%) 170 (1.5) [M+], 155 (2), 141 (5), 139 (29), 128 (56), 127
+
(54), 96 (100). HRMS (ESI): calcd. 177.1467 (for C10H18LiO2 ), found 177.1465 [M +
Li+]. C10H18O2 (170.25).
Methyl (E)-4-Bromo-4-methyl-2-octenoate (19). NBS (0.53 g, 3.0 mmol, 1.0 equiv.)
and AIBN (30 mg, 0.18 mmol, 6 mol%) were added to a solution of compound 6b
(0.51 g, 3.0 mmol, 1.0 equiv.) in CHCl3 (5 mL). After stirring the mixture for further 19
h at 75°C, it was cooled (ice–water bath) and filtered, the residue rinsed with CH2Cl2
(5 mL) and the filtrate was washed with water (3 x 20 mL). The organic layer was
dried (MgSO4) and evaporated after filtration. The residue was submitted to Kugel-
rohr distillation (175°C, 0.9 mbar) to furnish the title compound 19 (0.59 g, 2.4 mmol,
1
79%) as a colorless liquid. H-NMR (300 MHz, CDCl3): δ = 0.92 (t, J = 7.0 Hz, 3H),
1.30–1.46 (m, 4H), 1.87 (s, 3H), 1.91–2.03 (m, 2H), 3.77 (s, 3H), 5.91 (d, J = 15.7
Hz, 1H), 7.09 (d, J = 15.7 Hz, 1H) ppm. 13C{1H}-NMR (75 MHz, CDCl3): δ = 13.9
(CH3), 22.6 (CH2), 28.1 (CH2), 29.4 (CH3), 45.0 (CH2), 51.8 (CH3), 65.4 (C), 118.5
(CH), 151.8 (CH), 166.7 (C) ppm. IR (ATR): nu(tilde) 2956 (m), 2938 (m), 2920 (w),
2877 (m), 1727 (vs), 1653 (w), 1437 (s), 1382 (w), 1313 (m), 1283 (s), 1199 (m),1177
(m), 1158 (m), 1045 (m), 1015 (m), 985 (w), 868 (w), 723 (w), 634 (w), 621 (w) cm–1.
+
HRMS (ESI): calcd. 249.0485 (for C10H18BrO2 ), found 249.0484 [M + H+].
C10H17BrO2 (249.15).
Methyl 4-Methyl-3-octenoate (15b). Zinc powder (1.00 g, 15.3 mmol, 1.0 equiv.)
was added to a solution of compound 19 (0.46 g, 1.86 mmol, 8.2 equiv.) in AcOH (4
mL) and the solution was stirred at ambient temperature for 1 h. It was diluted with
water (20 mL), filtered and the filtrate was extracted with MTBE (3 x 20 mL). The
combined organic layers were dried (MgSO4) and evaporated after filtration. The resi-
due was submitted to chromatography (SiO2, hexanes/MTBE/CH2Cl2 100:5:1, Rf =
15
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0.43) to furnish the title compound 15b (0.20 g, 1.2 mmol, 63%) as a mixture of two
stereoisomers (E/Z = 2:1) and as a colorless oil. 1H-NMR (300 MHz, CDCl3): δ = 0.89
(t, J = 7.1 Hz, 3H), 1.22–1.44 (m, 4H), 1.62 [s, 2/3 x 3H; (E)-isomer], 1.73 [s, 1/3 x
3H; (Z)-isomer], 2.02 (t, J = 7.0 Hz, 2H), 3.05 (d, J = 7.1 Hz, 2H), 3.68 (s, 3H), 5.31
(tq, J = 7.1 Hz, J = 1.1 Hz, 1H), ppm. 13C{1H}-NMR (75 MHz, CDCl3), (E)-isomer: δ =
13.97 (CH3), 16.2 (CH3), 22.3 (CH2), 29.9 (CH2), 33.5 (CH2), 39.2 (CH), 51.7 (CH3),
115.3 (CH), 139.5 (C), 173.0 (C) ppm; (Z)-isomer: δ = 14.01 (CH3), 22.6 (CH2), 23.4
(CH3), 30.0 (CH2), 31.7 (CH2), 33.3 (CH2), 51.7 (CH3), 115.9 (CH), 139.7 (C), 173.0
(C) ppm. IR (ATR): nu(tilde) 2961 (s), 2930 (s), 2872 (m), 2859 (m), 1741 (vs), 1627
(vw), 1435 (s), 1379 (w), 1312 (m), 1261 (s), 1193 (m), 1164 (s), 1015 (m), 983 (m),
836 (w), 729 (w), 613 (w) cm–1. GC-MS (EI, 70 eV), (E)-isomer: m/z (%) 170 (19)
[M+], 138 (12), 128 (29), 127 (6), 111 (20), 109 (15), 96 (93), 85 (19), 81 (55), 74 (23),
69, (100), 68 (45), 67 (43), 65 (7), 59 (23), 55 (100); (Z)-isomer: m/z (%) 170 (19)
[M+], 138 (12), 128 (29), 127 (6), 111 (20), 109 (17), 97 (30), 96 (90), 81 (63), 74 (24),
69, (91), 67 (40), 65 (7), 59 (22), 55 (100). HRMS (ESI): calcd. 177.1461 (for
+
C10H18LiO2 ), found 177.1463 [M + Li+]. C10H18O2 (170.25).
Methyl 3-Oxohexanoate (21). DMAP (0.68 g, 5.6 mmol, 0.2 equiv.) and pyridine (4.4
g, 56 mmol, 1.8 equiv.) were added to a solution of Meldrum's acid (4.00 g, 28.0
mmol, 0.9 equiv.) in CH2Cl2 (35 mL). After stirring the mixture for 15 min at ambient
temperature, butanoyl chloride (20) (3.3 g, 31 mmol, 1.0 eq.) was added dropwise at
0°C (ice–water bath) over a period of 15 min. After stirring the mixture for further 19 h
at ambient temperature, hydrochloric acid (2 mol L–1, 100 mL) was added and the re-
sulting suspension vigorously stirred for 5 min. The layers were separated and the or-
ganic layer was washed with water (100 mL). Both combined aqueous layers were
extracted with CH2Cl2 (3 x 50 mL). All four organic layers were combined, washed
with hydrochloric acid (2 mol L–1, 2 x 50 mL) and brine (50 mL), dried (MgSO4) and
evaporated after filtration. The residue was dissolved in MeOH (35 mL) and the solu-
tion heated to reflux for 4 h. After evaporation, the residue was submitted to chroma-
tography (SiO2, hexanes/MTBE 3:1, Rf = 0.40) to furnish the title compound 21 (1.52
g, 10.5 mmol, 38%) as a colorless liquid. According to 1H NMR, the compound exists
as two tautomers (keto/enol 9:1). 1H-NMR (300 MHz, CDCl3), keto-tautomer: δ = 0.92
(t, J = 7.4 Hz, 3H), 1.63 (sex, J = 7.3 Hz, 2H), 2.51 (t, J = 7.3 Hz, 2H), 3.44 (s, 2H),
3.74 (s, 3H) ppm; enol-tautomer: δ = 0.97 (t, J = 7.5 Hz, 3H), 1.63 (sex, J = 7.3 Hz,
16
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13
2H), 2.17 (t, J = 7.5 Hz, 2H), 3.72 (s, 3H), 4.99 (s, 1H), 12.01 (s, 1H) ppm. C{1H}-
NMR (75 MHz, CDCl3), keto-tautomer: δ = 13.5 (CH3), 16.9 (CH2), 44.9 (CH2), 49.0
(CH2), 52.3 (CH3), 167.7 (C), 202.7 (C) ppm; signals for the enol tautomer could not
be identified with certainty. IR (ATR): nu(tilde) 2963 (m), 2937 (w), 2878 (w), 1744
(vs), 1714 (vs), 1630 (w), 1437 (s), 1408 (m), 1319 (s), 1259 (s), 1230 (s), 1155 (s),
1124 (m), 1072 (m), 1006 (s), 853 (w) cm–1. MS (EI, 70 eV): m/z (%) 144 (9) [M+],
129 (2), 116 (4), 101 (27), 84 (7), 71 (100), 59 (33), 57 (13). HRMS (ESI): calcd.
+
151.0941 (for C7H12LiO3 ), found 151.0943 [M + Li+]. C7H12O3 (144.17).
Methyl 2-Methyl-3-oxohexanoate (13b). Compound 21 (1.45 g, 10.1 mmol, 1.0
equiv.) was added to a solution of K2CO3 (1.39 g, 10.1 mmol, 1.0 equiv.) in acetone
(10 mL). After stirring the mixture for 30 min at ambient temperature, methyl iodide
(2.14 g, 15.1 mmol, 1.5 equiv.) was added dropwise at 0°C (ice–water bath) over a
period of 15 min. After stirring the mixture for further 3 h at ambient temperature, wa-
ter (20 mL) was added. It was extracted with MTBE (3 x 20 mL) and the organic
layers were combined, dried (MgSO4) and evaporated after filtration. The residue
was submitted to chromatography (SiO2, hexanes/MTBE 5:1, Rf = 0.37) to furnish the
1
title compound 13b (0.87 g, 5.5 mmol, 55%) as a colorless liquid. According to H
1
NMR, the compound exists as two tautomers (keto/enol 9:1). H-NMR (500 MHz,
CDCl3), keto-tautomer: δ = 0.91 (t, J = 7.4 Hz, 3H), 1.34 (d, J = 7.1 Hz, 3H), 1.62
(sex, J = 7.4 Hz, 2H), 2.47 (dt, J = 17.3 Hz, J = 7.1 Hz, 1H), 2.55 (dt, J = 17.4 Hz, J =
7.3 Hz, 1H), 3.53 (q, J = 7.2 Hz, 1H), 3.73 (s, 3H) ppm; enol-tautomer: δ = 0.96 (t, J =
7.6 Hz, 3H), 1.62 (sex, J = 7.4 Hz, 2H), 1.75 (s, 3H), 2.28 (t, J = 7.6 Hz, 2H), 3.76 (s,
3H), 12.66 (s, 1H) ppm. 13C{1H}-NMR (125 MHz, CDCl3), keto-tautomer: δ = 12.8
(CH3), 13.5 (CH3), 17.0 (CH2), 43.2 (CH2), 52.3 (CH3), 52.7 (CH), 171.1 (C), 205.8
(C) ppm; signals for the enol tautomer could not be identified with certainty. IR (ATR):
nu(tilde) 2961 (m), 2877 (w), 1745 (vs), 1714 (vs), 1455 (s), 1436 (m), 1377 (m),
1327 (m), 1248 (s), 1202 (s), 1177 (m), 1122 (m), 1072 (m), 1018 (m), 967 (w), 898
(w), 860 (m) cm–1. MS (EI, 70 eV): m/z (%) 158 (2) [M+], 129 (3), 127 (5), 115 (6), 88
+
(13), 87 (7), 83 (2), 71 (100), 59 (16). HRMS (ESI): calcd. 165.1103 (for C8H14LiO3 ),
found 165.1108 [M + Li+]. C8H14O3 (158.19).
Methyl 3-Hydroxy-2-methylhexanoate (12b). NaBH4 (0.29 g, 7.6 mmol, 1.2 equiv.)
was added to a solution of compound 13b (1.00 g, 6.32 mmol, 1.0 equiv.) in MeOH
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(20 mL). After stirring the mixture for 3 h at ambient temperature, water (20 mL) was
added. The mixture was extracted with MTBE (3 x 30 mL) and the organic layers
were combined, dried (MgSO4) and evaporated after filtration. The residue was sub-
mitted to chromatography (SiO2, hexanes/MTBE 1:1, Rf = 0.36) to furnish the title
1
compound 12b (0.58 g, 2.6 mmol, 57%) as a colorless liquid. According to H NMR,
1
the compound exists as two diastereoisomers (dr 3:2). H-NMR (500 MHz, CDCl3),
major isomer: δ = 0.93 (t, J = 7.2 Hz, 3H), 1.21 (d, J = 7.3 Hz, 3H), 1.29–1.59 (m,
4H), 2.49 (d, J = 6.9 Hz, 1H), 2.51–2.57 (m, 1H), 3.64–3.70 (m, 1H), 3.71 (s, 3H)
ppm, the signal at 2.49 ppm was identified as OH-signal by H-D-exchange (D2O); mi-
nor isomer: δ = 0.94 (t, J = 7.0 Hz, 3H), 1.18 (d, J = 7.3 Hz, 3H), 1.29–1.59 (m, 4H),
2.43 (d, J = 4.7 Hz, 1H), 2.51–2.57 (m, 1H), 3.71 (s, 3H), 3.89–3.93 (m, 1H) ppm, the
signal at 2.43 ppm was identified as OH-signal by H-D-exchange (D2O). 13C{1H}-
NMR (125 MHz, CDCl3), major isomer: δ = 10.8 (CH3), 14.2 (CH3), 18.9 (CH2), 37.1
(CH2), 45.4 (CH), 51.9 (CH3), 73.3 (CH), 176.7 (C) ppm; minor isomer: δ = 10.8
(CH3), 14.5 (CH3), 19.3 (CH2), 36.1 (CH2), 44.4 (CH), 52.0 (CH3), 71.6 (CH), 176.7
(C) ppm. IR (ATR): nu(tilde) 3457 (s, br), 2957 (s), 2874 (m), 1720 (vs), 1459 (m),
1436 (m), 1255 (m), 1197 (s), 1170 (s), 1120 (m), 1055 (m), 1026 (m), 1013 (m), 985
(m), 851 (m) cm–1. GC-MS (EI, 70 eV), major isomer: m/z (%) 145 (0.5), 129 (2), 117
(40), 111 (6), 88 (100), 85 (34), 83 (9); minor isomer: m/z (%) 145 (2), 129 (1), 117
(16), 111 (5), 88 (100), 85 (23), 83 (7). HRMS (ESI): calcd. 167.1259 (for
+
C8H16LiO3 ), found 167.1260 [M + Li+]. C8H16O3 (160.21).
Methyl (E)- and (Z)-2-Methyl-2-hexenoate (11b). At 0°C (ice–water bath), KOtBu
(6.10 g, 54.3 mmol, 1.1 equiv.) was added to a solution of methyl 2-(diethoxyphos-
phoryl)propanoate (22) (11.1 g, 49.4 mmol, 1.0 equiv.) in THF (100 mL). After stirring
the mixture for 15 min at ambient temperature, butanal (3.57 g, 49.4 mmol, 1.0
equiv.) was added at 0°C (ice–water bath). After stirring the mixture for further 1 h at
ambient temperature, saturated aqueous NH4Cl solution (40 mL) was added. The
mixture was extracted with MTBE (3 x 40 mL) and the organic layers were combined,
dried (MgSO4) and evaporated after filtration. The residue was submitted to chroma-
tography (SiO2, hexanes/MTBE/CH2Cl2 20:1:0.1) to yield the title compound (Z)-11b
(930 mg, 6.54 mmol, 13%) as the first fraction (Rf = 0.53) as a colorless liquid. Se-
condly, compound (E)-11b (3.36 g, 23.6 mmol, 48%) was eluted (Rf = 0.35), also a
colorless liquid; ratio (E/Z 3.6:1).
18
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1
(E)-Isomer: H-NMR (300 MHz, CDCl3): δ = 0.92 (t, J = 7.4 Hz, 3H), 1.45 (sex, J =
7.4 Hz, 2H), 1.81 (s, 3H), 2.13 (q, J = 7.4 Hz, 2H), 3.71 (s, 3H), 6.74 (t, J = 7.4 Hz,
1H) ppm. 13C{1H}-NMR (75 MHz, CDCl3): δ = 12.4 (CH3), 13.9 (CH3), 21.8 (CH2),
30.7 (CH2), 51.7 (CH3), 127.6 (C), 142.6 (CH), 168.8 (C) ppm. IR (ATR): nu(tilde)
2958 (m), 2931 (w), 2876 (w), 1714 (vs), 1650 (m), 1434 (m), 1280 (m), 1220 (m),
1143 (s), 1094 (m), 742 (m) cm–1. MS (EI, 70 eV): m/z (%) 142 (33) [M+], 127 (17),
111 (33), 101 (42), 95 (25), 88 (31), 83 (26), 82 (24), 81 (17), 73 (23), 69 (19), 67
+
(22), 59 (19), 55 (100). HRMS (ESI): calcd. 149.1154 (for C8H14LiO2 ), found
149.1155 [M + Li+]. C8H14O2 (142.20).
(Z)-Isomer: 1H-NMR (300 MHz, CDCl3): δ = 0.91 (t, J = 7.4 Hz, 3H), 1.41 (sex, J = 7.4
Hz, 2H), 1.88 (d, J = 1.2 Hz, 3H), 2.42 (q, J = 7.4 Hz, 2H), 3.72 (s, 3H), 5.93 (tq, J =
13
7.4 Hz, J = 1.2 Hz, 1H) ppm. C{1H}-NMR (75 MHz, CDCl3): δ = 13.8 (CH3), 20.6
(CH3), 22.6 (CH2), 31.6 (CH2), 51.1 (CH3), 126.8 (C), 143.5 (CH), 168.5 (C) ppm. IR
(ATR): nu(tilde) 2952 (m), 2930 (w), 2866 (w), 1715 (vs), 1647 (m), 1434 (m), 1274
(m), 1218 (m), 1148 (s), 1070 (m), 768 (m) cm–1. MS (EI, 70 eV): m/z (%) 142 (45),
127 (28), 111 (32), 101 (35), 95 (38), 81 (24), 67 (35), 55 (100). HRMS (ESI): calcd.
+
143.1067 (for C8H14LiO2 ), found 143.1070 [M + H+]. C8H14O2 (142.20).
Methyl (E)-4-Bromo-2-methyl-2-hexenoate (23). NBS (4.62 g, 26.0 mmol, 1.0
equiv.) and AIBN (0.26 g, 1.6 mmol, 6 mol%) were added to a solution of compound
(E)-11b (3.69 g, 26.0 mmol, 1.0 equiv.) in CCl4 (40 mL). After stirring the mixture for
further 19 h at 80°C, it was cooled (ice–water bath) and filtered, the residue rinsed
with CH2Cl2 (5 mL) and the filtrate was washed with water (3 x 20 mL). The organic
layer was dried (MgSO4) and evaporated after filtration. The residue was submitted to
chromatography (SiO2, hexanes/MTBE 7:1, Rf = 0.56) to furnish the title compound
1
23 (5.37 g, 24.3 mmol, 93%) as a colorless liquid. H-NMR (300 MHz, CDCl3): δ =
1.01 (t, J = 7.3 Hz, 3H), 1.90 (d, J = 1.4 Hz, 3H), 1.86–2.07 (m, 2H), 3.76 (s, 3H),
4.68 (dt, J = 10.7 Hz, J = 7.0 Hz, 1H), 6.81 (dq, J = 10.7 Hz, J = 1.4 Hz, 1H) ppm.
13C{1H}-NMR (75 MHz, CDCl3): δ = 12.2 (CH3), 12.6 (CH3), 32.0 (CH2), 50.3 (CH3),
52.1 (CH), 129.1 (C), 140.3 (CH), 167.9 (C) ppm. IR (ATR): nu(tilde) 2972 (m), 2956
(w), 2938 (w), 2876 (w), 1715 (vs), 1646 (m), 1435 (m), 1276 (s), 1234 (s), 1191 (m),
+
1119 (m), 748 (s) cm–1. HRMS (ESI): calcd. 242.9991 (for C8H13BrNaO2 ), found
242.9997 [M + Na+]. C8H13BrO2 (221.09).
19
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ChemBioChem
Methyl 2-Methyl-3-hexenoate (14b). Zinc powder (3.17 g, 48.5 mmol, 2.0 equiv.)
was added to a solution of compound 23 (5.37 g, 24.3 mmol, 1.0 equiv.) in AcOH
(100 mL) and the solution was stirred at 0°C for 1 h, then at ambient temperature for
1 h. It was diluted with water (50 mL), filtered and the filtrate was extracted with
MTBE (3 x 80 mL). The combined organic layers were washed successively with
hydrochloric acid (2 mol L–1, 50 mL), saturated aqueous NaHCO3 solution (150 mL),
and brine (80 mL), then dried (MgSO4), filtered and evaporated after filtration. The re-
sidue was submitted to chromatography (SiO2, hexanes/MTBE 10:1, Rf = 0.33) to fur-
1
nish the title compound 14b (1.91 g, 13.4 mmol, 55%) as a colorless liquid. H-NMR
(300 MHz, CDCl3): δ = 0.96 (t, J = 7.5 Hz, 3H), 1.22 (d, J = 7.1 Hz, 3H), 2.02 (pent, J
= 7.1 Hz, 2H), 3.09 (pent, J = 7.1 Hz, 1H), 3.66 (s, 3H), 5.46 (dd, J = 15.5 Hz, J = 7.3
Hz, 1H), 5.57 (dt, J = 15.4 Hz, J = 5.7 Hz, 1H) ppm. 13C{1H}-NMR (75 MHz, CDCl3): δ
= 13.4 (CH3), 17.5 (CH3), 25.4 (CH2), 42.7 (CH), 51.7 (CH3), 127.7 (CH), 133.8 (CH),
175.6 (C) ppm. IR (ATR): nu(tilde) 2968 (m), 2935 (w), 2876 (w), 1737 (vs), 1457 (m),
1434 (m), 1280 (m), 1249 (s), 1193 (s), 1165 (s), 1051 (m), 967 (m) cm–1. MS (EI, 70
eV): m/z (%) 142 (7) [M+], 127 (5), 88 (32), 83 (48), 67 (13), 55 (100). HRMS (ESI):
+
calcd. 149.1148 (for C8H14LiO2 ), found 143.1151 [M + Li+]. C8H14O2 (142.20).
Cultivation. The betaproteobacterium "Aromatoleum" sp. HxN1 has been subcul-
tured in our laboratory since its isolation.[4] Cultivation was carried out in defined, bi-
carbonate-buffered medium, essentially as described earlier.[5] Cultures were grown
in stopper-sealed flat glass bottles (500 mL) containing 400 mL medium under an an-
oxic atmosphere (N2/CO2 90:10, v/v). n-Hexane (1) was provided as dilution (5%, v/v)
in 2,2,4,4,6,8,8-heptamethylnonane serving as inert carrier phase. Sodium n-hexan-
oate from a sterile stock solution was added to control cultures at a final concentra-
tion of 3 mM.
Preparation of culture extracts. Extracts for metabolite analysis were obtained from
cultures of "Aromatoleum" sp. HxN1 as previously described.[5] Essentially, cultures
were inactivated by heat (85°C in a water bath for 15 min). Overlying carrier phase
(cultures with n-hexane) was removed by means of a separatory funnel, the obtained
culture broth acidified (pH 1.5 with hydrochloric acid) and thereafter extracted three
times with Et2O. Finally, the combined organic layers were dried (Na2SO4) and after
filtration, stored in teflon-sealed glass bottles until further analyses. In addition, co-in-
20
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10.1002/cbic.201900375
ChemBioChem
jection experiments for some compounds were performed using samples obtained by
solid phase extraction of cell-free supernatants of strain HxN1 grown with n-hexane
(1). Waters Oasis MAX (30 µm) was used as the solid phase. The adsorbed metabo-
lites were eluted with 5% formic acid in methanol. The identity of the metabolite pat-
tern with that of the original extracts was confirmed by GC-MS.
Derivatization and analysis of metabolites. Dried extracts were evaporated to
dryness, solubilized in CH2Cl2 and methylated using freshly prepared diazomethane
essentially as described before.[7] Methylated extracts were then analyzed by GC-MS
using a Trace GC Ultra gas chromatograph coupled to an ISQ OD mass spectrome-
ter (both Thermo Scientific). The GC was equipped with an Agilent DB5 fused silica
capillary (30 m length, 0.25 mm internal diameter, 0.25 μm film thickness). Helium
was used as carrier gas. The GC oven temperature was programmed from 40°C (2
min hold time) to 200°C at a rate of 3 K min–1 and further to 320°C (2 min hold time)
at a rate of 20 K min–1. The MS was operated in electron impact ionization mode (70
eV) at a source temperature of 220°C and a transfer line temperature of 280°C. The
mass range was 50–650 Dalton at scan cycle time of 0.2 s. The analyses of refe-
rence standards and the co-injection experiments were performed on the same
system using identical conditions.
Acknowledgement
This work was funded by the Deutsche Forschungsgemeinschaft (GRK2226). MTBE
was obtained as a generous gift from Evonik Industries, Marl, Germany. We are fur-
thermore grateful to Matthias Klat and Eric Meding for preliminary synthetic studies
and Elisabeth Jaskulska for assistance.
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