Synthesis and characterization of anaerobic degradation biomarkers of n-alkanes via hydroxylation/carboxylation pathways

Article abstract of DOI:10.1255/ejms.1402

Metabolite profiling is a powerful method in research on anaerobic biodegradation of hydrocarbons. Hydroxylation and carboxylation are proposed pathways in anaerobic degradation but very little direct evidence is available about metabolites and signature biomarkers. 2-Acetylalkanoic acid is a potential signature metabolite because of its unique and specific structure among possible intermediates. A procedure for the synthesis of four homologues with various carbon chain lengths was proposed and the characteristics of 2-acetyl-alkanoic acid esters were investigated using four derivatization processes, namely methyl, ethyl, n-butyl and trimethylsilyl esterification. Four intermediate fragments observed were at m/z 73 + 14n, 87 + 14n, 102 + 14n (n = 1, 2 and 4 for methyl, ethyl and n-butyl ester, respectively) and [M - 42]+ for three of the derivatization methods. For silylation, characteristic ions were observed at m/z 73, 117, [M - 42]⁺ and [M - 55]⁺ . These are basic and significant data for the future identification of potential intermediates of the hydroxylation and carboxylation pathways in hydrocarbon degradation.

Full text of DOI:10.1255/ejms.1402

J. Zhou et al., Eur. J. Mass Spectrom. 22, 31–37 (2016)
31
Received: 12 November 2015
■
Revised: 22 December 2015 ■ Accepted: 23 December 2015 ■ Publication: 23 December 2015
EUROPEAN
JOURNAL
OF
MASS
SPECTROMETRY
Synthesis and characterization of anaerobic
degradation biomarkers of n-alkanes via
hydroxylation/carboxylation pathways
Jing Zhou,a Xin-Yu Bian,a Lei Zhou,a Serge Maurice Mbadinga,a,c Shi-Zhong Yang,a Jin-Feng Liu,a Ji-Dong Gub and
Bo-Zhong Mua,c
*
aState Key Laboratory of Bioreactor Engineering and Institute of Applied Chemistry, East China University of Science and Technology,
Shanghai 200237, PR China. E-mail: bzmu@ecust.edu.cn
bSchool of Biological Sciences, University of Hong Kong, Pokfulam Road, Hong Kong SAR, PR China
cShanghai Collaborative Innovation Center for Biomanufacturing Technology, Shanghai 200237, PR China
Metabolite profiling is a powerful method in research on anaerobic biodegradation of hydrocarbons. Hydroxylation and carboxylation
are proposed pathways in anaerobic degradation but very little direct evidence is available about metabolites and signature biomarkers.
2-Acetylalkanoic acid is a potential signature metabolite because of its unique and specific structure among possible intermediates. A
procedure for the synthesis of four homologues with various carbon chain lengths was proposed and the characteristics of 2-acetyl–
alkanoic acid esters were investigated using four derivatization processes, namely methyl, ethyl, n-butyl and trimethylsilyl esterifica-
tion. Four intermediate fragments observed were at m/z 73 + 14n, 87 + 14n, 102 + 14n (n = 1, 2 and 4 for methyl, ethyl and n-butyl ester,
respectively) and [M − 42]+ for three of the derivatization methods. For silylation, characteristic ions were observed at m/z 73, 117,
[M − 42]+ and [M − 55]+. These are basic and significant data for the future identification of potential intermediates of the hydroxylation
and carboxylation pathways in hydrocarbon degradation.
Keywords: hydroxylation/carboxylation, potential biomarkers, 2-acetylalkanoic acid, synthesis of biomarkers, mass spectral characteristics
Introduction
It has been established since the 1990s that anaerobic degra- is an exception.2,17 This strain was elucidated for n-alkane
dation of alkanes plays an important role in nature.1,2 To
date, metabolite profiling has elucidated at least four kinds
of initial activation steps in anaerobic alkane degradation.3,4
A group of anaerobic microorganisms capable of degrading
n-alkanes under nitrate-reducing,5 sulfidogenic6–8 and
methanogenic conditions9–12 has been reported.13 Increasing
evidence supports that fumarate addition plays a dominant
activation by another mechanism compared to most other
alkane-degrading strains, which showed an initial pathway
of fumarate.2,17 A putative biochemical pathway of degrada-
tion for alkanes, namely carboxylation, was proposed by So
et al. based on 13CO2-labeling experiments.18 Strain Hxd3
would metabolize an alkane to form fatty acids via carboxyl–
−
ation with inorganic carbon (HCO3 /CO2), most likely at C-3,
role in the anaerobic activation of alkanes by those micro– with subsequent removal of two sub-terminal carbon atoms
organisms.14–16 However, isolated sulfate-reducing strain Hxd3, from the alkane chain.18,19 Callaghan et al.20 characterized
a sulfate reducer closely related to the genus Desulfococcus, an alkane-degrading and nitrate-reducing consortium, and
ISSN: 1469-0667
doi: 10.1255/ejms.1402
© IM Publications LLP 2016
All rights reserved

32
Anaerobic Degradation Biomarkers of n-Alkanes
Figure 1. (A) Possible metabolic mechanisms initiated by carboxylation or hydroxylation. The common intermediates of these two
pathways are highlighted in bold. (B) The pathway of anaerobic ethylbenzene degradation mechanism through an initial hydroxylation
catalyzed by EBDH.
obtained evidence for a degradation mechanism analogous to
the proposed carboxylation pathway under nitrate-reducing
conditions.
advance the detection of 2-acetylalkanoic acids in environ-
mental and enrichment cultural samples.
However, recent omics-based analysis indicates that the
genome of Hxd3 contains genes that encode an ethylbenzene
dehydrogenase-like enzyme, which catalyzes the anaerobic
hydroxylation of ethylbenzene [Figure 1(A)].3,21,22 The new
Experimental
Chemical synthesis
hypothetical activation strategy suggested an initial sub- All chemical reagents used in the syntheses were commer-
terminal hydroxylation of the alkanes by an EBDH-like enzyme, cially available, of analytical grade and highest purity.
followed by oxidation to a ketone and its subsequent carboxy- n-Nonanol, ethyl acetoacetate, Na, ethanol, hexane, ethyl
lation at C-3.3,21 The latter process would be consistent with
acetate, HBr, H2SO4, Na2SO4, NaHCO3 and HCl were of analyt-
ical grade. N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)
was purchased from Sigma-Aldrich (Shanghai Trading Co.
Ltd, Shanghai, China). Where necessary, solvents were either
de-watered using standard techniques or purchased as anhy-
drous form. In the following, 2-acetylundecanoic acid is used
previous studies [Figure 1(B)].14,19
For metabolite analysis, deuterated long-chain fatty acids
were detected while the proposed typical intermediates such
as 2-ethylalkanoic acid and 2-acetylalkanoic acid were not
identified in previous studies.18,19 Notably, 2-acetylalkanoic
acid, as a common intermediate, occurs in two similar path- as an example to describe the synthetic route presented in
ways, namely hydroxylation and carboxylation.
Figure 2.
Alkylsuccinate is a biomarker of fumarate addition. We
had synthesized five biomarkers, specifically 2-ethylsuccinic
acid, 2-(1-methyloctyl)succinic acid, 2-(1-methylpentadecyl)
succinic acid, 2-cyclohexylsuccinic acid and 2-benzylsuccinic
Preparation of bromononane. H2SO4 (0.035 mol, 5.04 g)
was added dropwise into a mixture of n-nonanol (0.035mol,
5.04 g) and HBr (0.0385 mol, 6.63 g) at 0 °C. The reaction
was heated to reflux for 1h at 120°C. After cooling to room
acid, and subsequently obtained their mass spectra char- temperature, steam distillation was used to purify the target
acteristics in previous work which was concerned with the products. NaHCO3 was added to adjust the pH to >7. The oil
investigation of fumarate addition pathway in oilfield produc- phase was separated and the water phase was extracted with
tion water samples.23,24 However, the corresponding mass
spectrometry information of 2-acetylalkanoic acids is still not
available in official libraries. In the research work reported
here, we established a method for the chemical synthesis of
hexane three times successively. Then, the organic phase was
collected and dried with Na2SO4. The solvent was removed and
the yield was approximately 90.0%.
Preparation of ethyl 2-acetylundecanoic acid ester. Sodium
2-acetylalkanoic acids and obtained the characteristic frag- ethoxide was produced by Na (0.035mol, 0.80g) and ethanol
ments of them by GC-MS using four derivatization approaches, (10 mL) at 0 °C.23 Ethyl acetoacetate was added into the
namely methyl, ethyl, n-butyl and trimethylsilyl (TMS) esteri- sodium ethoxide/ethanol solution dropwise and stirred for
fication. Our results provide the lacking data and greatly
30min. Then, bromononane was added into the mixture. After

J. Zhou et al., Eur. J. Mass Spectrom. 22, 31–37 (2016)
33
Figure 2. Synthesis of 2-acetylundecanoic acid.
1
further stirring for 1h, the mixture was refluxed for 6h at 80°C.
Ethanol was removed firstly by rotary evaporation. Deionized
water and HCl were added into the product until the pH was
Ethyl 2-acetyldodecanoic acid ester. H NMR (400 MHz,
CDCl3, TMS; d, ppm): 0.86 (t, J = 6.8Hz, 3H), 1.14–1.39 (m, 17H),
1.83 (m, J = 17.5, 10.1Hz, 2H), 2.22 (s, 3H), 3.43 (t, J = 7.4Hz,
<1. The oil phase was separated and the water phase was 1H), 4.18 (q, J = 7.1Hz, 2H).
extracted with ethyl acetate three times successively. Then,
the organic phase was collected and dried by passing through
Na2SO4. The solvent was removed and the yield was approxi- Four homologues with various carbon chain lengths were
Derivatization
mately 68.5%.
derivatized to methyl, ethyl, n-butyl and trimethylsilyl esters,
Preparation of 2-acetylundecanoic acid. NaOH (0.035mol, respectively. Noteworthy, the mass spectra of ethyl ester
1.40 g) and H2O (20 mL) were added to ethyl 2-acetylun– products were obtained directly after synthesis. Briefly, the
decanoate (0.035mol, 8.96g), and the mixture was refluxed for
4h at 100°C. After cooling to room temperature, hexane was
used three times successively to extract the non-dissolved
organic substances. Then, the water phase was acidified with
HCl, and extracted three times with ethyl acetate and dried
by passing through Na2SO4. The solvent was removed and the
yield was about 92.5%.
methods were as follows. A solution of 10% H2SO4–CH3OH/
H2SO4–butanol was added into each of the four compounds
and heated at 90°C for 1h for methyl/butyl derivatizations.26
For silylation, BSTFA and acetonitrile were rapidly mixed
with each of the compounds and heated at 60°C for 30min.27
Details of specific methods of derivatization were published
previously by Bian et al.23
2-Acetylpentanoic, 2-acetylhexanoic and 2-acetyldeca-
noic acids were also synthesized according to the method
described above. Notably, the steps of Part B (Figure 2) should
be kept water free. Thin-layer chromatography was used to
GC-MS analyses were carried out using an Agilent 7890A
GC instrument equipped with an HP-5MS capillary column
(30m × 0.25mm × 0.25µm) and mass detector (MSD 5975). The
injection port temperature was 280°C and the oven tempera-
monitor the progress of the whole reaction. The 2-acetylalka- ture was initially held at 60°C for 2min, and then increased
noic acids were purified via column chromatography (petro- at 10 °C min−1 to 280 °C, held for 30 min. The MS detector
leum ether–ethyl acetate, 8:1). The four compounds were
derivatized to ethyl esters for GC-MS analysis via a method
described elsewhere.7,23,25
acquired data in the scan mode, from 30 to 1000 mass units.
Results and discussion
NMR data
1
Ethyl 2-acetylpentanoic acid ester. H NMR (400 MHz, CDCl3, Alkanes can be degraded anaerobically via a number of
TMS; d, ppm): 0.86 (t, J = 6.7Hz, 3H), 1.26–1.31 (m, 5H), 1.81 (m, specific biochemical mechanisms, such as fumarate addition,
J = 14.8, 7.4Hz, 2H), 2.06 (s, 3H), 3.39 (t, J = 7.3Hz, 1H), 4.15 (q, carboxylation, hydroxylation etc. These proposed biochem-
J = 7.1Hz, 2H).
ical pathways can be observed in either enrichment cultures
Ethyl 2-acetylhexanoic acid ester. 1H NMR (400 MHz, CDCl3, or pure cultures under laboratory conditions. However, it
TMS; d, ppm): 0.86 (t, J = 6.7Hz, 3H), 1.26–1.32 (m, 7H), 1.81 (m, is difficult to identify them in hydrocarbon-rich subsurface
J = 14.8, 7.4Hz, 2H), 2.06 (s, 3H), 3.39 (t, J = 7.3Hz, 1H), 4.16 (q, environments, including oil reservoirs and contaminated envi-
J = 7.1Hz, 2H).
ronments. An efficient method to investigate the anaerobic
1
Ethyl 2-acetylundecanoic acid ester. H NMR (400 MHz, degradation mechanisms in these environments is the direct
CDCl3, TMS; d, ppm): 0.87 (t, J = 6.8Hz, 3H), 1.17–1.37 (m, 15H), detection and confirmation of the key degradation metab-
1.83 (m, J = 14.8, 7.4Hz, 2H), 2.22 (s, 3H), 3.42 (t, J = 7.4Hz, 1H), olites produced from these biochemical degradation path-
4.19 (q, J = 7.1Hz, 2H).
ways. For carboxylation and hydroxylation mechanisms, the

34
Anaerobic Degradation Biomarkers of n-Alkanes
data. There are certain apparent regularities in the mass
spectra of these derivatization products.
Mass spectral characteristics of methyl,
ethyl and n-butyl esterification products
Four intermediate fragments observed at m/z 73 + 14n,
87 + 14n, 102 + 14n (n = 1, 2 and 4 for methyl, ethyl and n-butyl
ester, respectively) and [M − 42]+ for the three derivatization
methods are considered as mass spectral characteristics of
2-acetylalkanoic acid esters. We take ethyl esterification as an
example to discuss the fragment ions in detail.
Figure 3. Chemical structures of the four synthetized
compounds.
The mass spectra of the four ethyl esterification compounds
are shown in Figure 4. The four most significant fragment
ions at m/z 101, 115, 130 and [M − 42]+ are supposed to be the
characteristic fragments of ethyl 2-acetylalkanoic acid. They
intermediate 2-acetyl-alkanoic acids are the key compounds
involved, but the mass spectral characteristics of these
compounds are unavailable.
Four compounds, namely 2-acetylpentanoic, 2-acetylhexa- suggest a cleavage mechanism shown in Figure 5. These
noic, 2-acetyldecanoic and 2-acetylundecanoic acids, were
synthesized in this research. Their structures are shown in
Figure 3. They were characterized by both mass spectrometry
ions corresponding to different cleavage mechanisms are
discussed in the following.
Fragment ions m/z 101, 115 and 130 (in red in Figure 4)
remained unchanged in the compounds with increasing molec-
ular weight. Ethyl esters of 2-acetylalkanoic acids undergo
EI-induced fragmentation due to McLafferty rearrangement
and g-cleavage to form m/z 130. Note that alkyl of the carbon
1
and H NMR spectroscopy. The synthesized compounds were
derivatized by four methods respectively for higher volatility of
the chemicals and enhanced detection sensitivity. The mass
spectra of them are shown in Figure 4 and the supplementary
Figure 4. Mass spectra of ethyl 2-acetylalkanoic acids. (Values in red are constant for all chemical compounds. Values in blue are
different for each compound and can reflect the length of the carbon chain.)

J. Zhou et al., Eur. J. Mass Spectrom. 22, 31–37 (2016)
35
Figure 5. Proposed cleavage of ethyl 2-acetylalkanoic acid by mechanisms (a) and (b), and n-butyl 2-acetylalkanoic acid (c).
chain near the carbethoxy can provide a g-H for this re– [M − 42]+ could be regarded as the characteristic fragments of
arrangement (Figure 5, mechanism a).28,29 Structure b loses a
methyl radical to form c at m/z 115. Similarly, ethyl radical is
eliminated from ion b to give fragment ion d at m/z 101. Ions
m/z 130 and 101 have strong peaks, suggesting that they are
relatively stable. Fragment ions m/z 143 and 157 are relatively
abundant in mass spectra for long-chain compounds. They
are simply generated by fracture of carbon chains.
ethyl 2-acetylalkanoic acids.
For methyl and n-butyl esterification derivatives (supple-
mentary data), except for common fragment ion m/z [M − 42]+,
the mass spectral characteristics have 14 or 28 mass units
differences from ethyl esterification. The shifts of m/z are
reasonable when methoxyl or butoxyl is substituted for
ethoxyl. In addition, fragment [M − 55]+ can be observed in
Fragment ion m/z [M − 42]+. EI mass spectra of the deriva- mass spectrum of n-butyl esterification product representing
tives presented reveal a rather intense peak for the [M − 42]+
ion (in blue in Figure 4) by elimination of a ketene [·CH2CO]
group due to McLafferty rearrangement (acetyl group can
provide a g-H for this rearrangement) (Figure 5).28 Note that
the elimination of a methyl or ethyl radical can also arise
a McLafferty rearrangement with double hydrogen transfer
mechanism according to a previous study.30
Mass spectral characteristics of silylation
The fragment ions via mechanism (a) of Figure 5 are not
based on the rearrangement, but this process was not consid- regarded as the mass characteristics for silylation derivati-
ered as characteristic fragmentation owing to either repeated
m/z value or negligible ion peaks. The molecular mass can
often be recognized by the presence of [M − 42]+ ion which
reflects indirectly the length of the carbon chain. Therefore, the
general structure of compounds can be confirmed basically in
terms of the key information. Fragment ion m/z [M − 42]+ is
constantly visible for the four derivatization methods (Figure 4
and supplementary data) since this cleavage mechanism of
2-acetylalkanoic acid esters is independent of the length of
carbon chain and also the derivatization method.
zation due to low abundance (supplementary data), except
m/z [M − 42]+. Instead, diagnostic fragmentations have been
observed in the EI mass spectra of TMS esters with m/z 73 and
117. The EI mass spectra of TMS esters show rather abundant
ions with m/z 73 suggesting the formation of trimethylsilyl
monomers. Fragment ions m/z 117, corresponding to the
formation of the Me3SiOCO+ ion by McLafferty rearrangement
and g-cleavage, also have a pronounced peak in each spec-
trum.31,32 As for m/z [M − 42]+, it has no strong correlation with
the length of the carbon chain or the derivatization method.
Also, molecular ion peaks are found in the mass spectra. Another characteristic fragment ion m/z [M − 57]+ is produced
Consequently, the fragment ions m/z 101, 115, 130 and
based on m/z [M − 42]+ by eliminating a methyl radical which

36
Anaerobic Degradation Biomarkers of n-Alkanes
Table 1. Characteristic fragment ions of 2-acetylalkanoic acid
esters.
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3-540-77587-4_64
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Derivation
Characteristic fragment ions
87, 101, 116, [M − 42]+
Methyl esterification
Ethyl esterification
n-Butyl esterification
Silylation
101, 115, 130, [M − 42]+
129, 143, 158, [M − 42]+, [M − 55]+
73, 117, [M − 42]+, [M − 57]+
belongs to TMS. Therefore, 73, 117, [M − 42]+ and [M − 57]+ are
the mass spectral characteristics of silylation.
According to the discussion above, the mass spectral charac–
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in Table 1. Each derivatization method corresponds to different
characteristic ions. Based on the information newly available,
it is possible to choose one or several of the methods for
detection of degradation intermediates to confirm carboxy-
lation and hydroxylation mechanisms by direct detection of
degradation intermediates.
Conclusions
Synthesis of chemical biomarkers in anaerobic degradation of
alkanes via hydroxylation and carboxylation mechanisms was
carried out and characteristic mass spectra were obtained.
Four homologues with various carbon chain lengths were
synthesized, and their characteristic mass spectra obtained by
GC-MS include four characteristic fragments at m/z 73 + 14n,
87 + 14n, 102 + 14n (n = 1, 2 and 4 for methyl, ethyl and n-butyl
ester) and [M − 42]+ for three of the derivatization methods,
and at m/z 73, 117, [M − 42]+ and [M − 55]+ for silylation. They
can be used to identify potential biomarkers, 2-acetylalkanoic
acids, in hydrocarbon degradation, namely carboxylation
and hydroxylation pathways. Direct detection of biomarkers
involved in carboxylation and hydroxylation pathways will
advance our knowledge of anaerobic degradation processes.
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Acknowledgements
This work was supported by the National Natural Science
Foundation of China (nos. 41530318, 41373070, 41403066) and
NSFC-RGC (41161160560).
12. D.M. Jones, I.M. Head, N.D. Gray, J.J. Adams, A.K.
Rowan, C.M. Aitken, B. Bennett, H. Huang, A. Brown,
B.F.J. Bowler, T. Oldenburg, M. Erdmann and S.R.
Larter, “Crude-oil biodegradation via methanogenesis
in subsurface petroleum reservoirs”, Nature 451, 176
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Supplementary data
Supplementary data associated with this article can be found
in the online version at http://dx.doi.org/10.1255/ejms.1402.
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