Synthesis and mass spectra of rearrangement bio-signature metabolites of anaerobic alkane degradation via fumarate addition

Article abstract of DOI:10.1016/j.ab.2020.113746

Metabolite profiling in anaerobic alkane biodegradation plays an important role in revealing activation mechanisms. Apart from alkylsuccinates, which are considered to be the usual biomarkers via fumarate addition, the downstream metabolites of C-skeleton rearrangement can also be regarded as biomarkers. However, it is difficult to detect intermediate metabolites in both environmental samples and enrichment cultures, resulting in lacking direct evidence to prove the occurrence of fumarate addition pathway. In this work, a synthetic method of rearrangement metabolites was established. Four compounds, namely, propylmalonic acid, 2-(2-methylbutyl)malonic acid, 2-(2-methylpentyl)malonic acid and 2-(2-methyloctyl)malonic acid, were synthesized and determined by four derivatization approaches. Besides, their mass spectra were obtained. Four characteristic ions were observed at m/z 133 + 14n, 160 + 28n, 173 + 28n and [M - (45 + 14n)]⁺ (n = 0 and 2 for ethyl and n-butyl esters, respectively). For methyl esterification, mass spectral features were m/z 132, 145 and [M - 31]⁺, while for silylation, fragments were m/z 73, 147, 217, 248, 261 and [M - 15]⁺. These data provide basis on identification of potential rearrangement metabolites in anaerobic alkane biodegradation via fumarate addition.

Full text of DOI:10.1016/j.ab.2020.113746

AnalyticalBiochemistry600(2020)113746
Contents lists available at ScienceDirect
Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
Synthesis and mass spectra of rearrangement bio-signature metabolites of
anaerobic alkane degradation via fumarate addition
Jing Chena,d, Lei Zhoua,d, Yi-Fan Liua,d, Zhao-Wei Houb, Wei Lib, Serge Maurice Mbadingaa,d
Jing Zhoua,d, Tao Yanga,d, Jin-Feng Liua,d, Shi-Zhong Yanga,d, Xiao-Lin Wub,∗∗, Ji-Dong Guc,
Bo-Zhong Mua,d,∗
,
a State Key Laboratory of Bioreactor Engineering and School of Chemistry and Molecular Engineering, East China University of Science and Technology, 130 Meilong Road,
Shanghai, 200237, PR China
b Research Institute of Daqing Oilfield Company Limited, PetroChina, Daqing, Heilongjiang, 163712, PR China
c School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong Special Administrative Region, PR China
d Engineering Research Center of Microbial Enhanced Oil Recovery, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Alkylmalonic acids
Rearrangement bio-signature metabolites
Fumarate addition
Mass spectral characteristics
Synthesis of biomarkers
Metabolite profiling in anaerobic alkane biodegradation plays an important role in revealing activation me-
chanisms. Apart from alkylsuccinates, which are considered to be the usual biomarkers via fumarate addition,
the downstream metabolites of C-skeleton rearrangement can also be regarded as biomarkers. However, it is
difficult to detect intermediate metabolites in both environmental samples and enrichment cultures, resulting in
lacking direct evidence to prove the occurrence of fumarate addition pathway. In this work, a synthetic method
of rearrangement metabolites was established. Four compounds, namely, propylmalonic acid, 2-(2-methylbutyl)
malonic acid, 2-(2-methylpentyl)malonic acid and 2-(2-methyloctyl)malonic acid, were synthesized and de-
termined by four derivatization approaches. Besides, their mass spectra were obtained. Four characteristic ions
were observed at m/z 133 + 14n, 160 + 28n, 173 + 28n and [M - (45 + 14n)]+ (n = 0 and 2 for ethyl and n-
butyl esters, respectively). For methyl esterification, mass spectral features were m/z 132, 145 and [M - 31]+
,
while for silylation, fragments were m/z 73, 147, 217, 248, 261 and [M - 15]+. These data provide basis on
identification of potential rearrangement metabolites in anaerobic alkane biodegradation via fumarate addition.
1. Introduction
gene analysis [14–16]. Functional genes can be relatively easy to ac-
quire by molecular techniques, but intermediate metabolites are hard to
Hydrocarbons, the critical components of crude oil, were reported
to be biodegraded anaerobically under sulfate-reducing, nitrate-redu-
cing and methanogenic conditions [1–4]. Research on biochemical
mechanisms involved in the anaerobic biodegradation of hydrocarbons
deepens our understandings on how the microorganisms activate al-
kanes in complex anoxic subsurface environments [5–7]. Based on pure
cultures and enrichment cultures from laboratory studies, several initial
activation pathways of alkane degradation were proposed, such as fu-
marate addition, hydroxylation/carboxylation and alkyl-CoM pathways
[8–12]. Typically, pathway exploration of anaerobic hydrocarbon de-
gradation is mainly divided into two main means. One is through meta-
omics approaches, but there still exist some limitations [13]. The other
is a combination of key-metabolite detection and related functional
detect, which eventually causes a lack of strong evidence to confirm the
presence of activation mechanisms. Therefore, investigations on bio-
signature metabolites contribute to the exploration of degradation
pathways.
In most cases, alkanes are anaerobically activated via addition to
fumarate, resulting in the generation of 1-methylalkyl succinic acids,
which is followed by the C-skeleton rearrangement reaction to form 2-
(2-methylalkyl)malonic acids and, subsequently, oxidization to fatty
acids and further conversion to methane and carbon dioxide [17–19].
Alkylsuccinates are commonly recognized as biomarkers indicating the
presence of fumarate addition [17,19]. In previous studies, alkylsucci-
nates with different carbon chain lengths were synthesized, and the
mass spectral characteristics of these derivatives were also obtained and
∗ Corresponding author. State Key Laboratory of Bioreactor Engineering and School of Chemistry and Molecular Engineering, East China University of Science and
Technology, 130 Meilong Road, Shanghai, 200237, PR China.
∗∗ Corresponding author.
E-mail addresses: wuxldq@petrochina.com.cn (X.-L. Wu), bzmu@ecust.edu.cn (B.-Z. Mu).
https://doi.org/10.1016/j.ab.2020.113746
Received 17 March 2020; Received in revised form 12 April 2020; Accepted 15 April 2020
Availableonline22April2020
0003-2697/©2020ElsevierInc.Allrightsreserved.

J. Chen, et al.
AnalyticalBiochemistry600(2020)113746
summarized [20–22], which provides substantial convenience for the
identification of fumarate addition to alkanes occurred in environ-
mental samples and enrichment cultures [15,23]. But due to the short
duration and low concentration of alkylsuccinates, and the relatively
low sensitivity of the instruments [4,18], the knowledge on the fuma-
rate addition pathway of in situ petroleum reservoirs and enrichment
cultures is not enough [17,24]. Besides, little was known about the
detection of downstream metabolites, especially C-skeleton rearrange-
ment metabolites. Since the biological source of rearrangement product
alkylmalonate is unique, it can also be considered as an indicator for the
fumarate addition pathway. However, a lack of standard substances and
insufficient commercial sources of rearrangement bio-signature meta-
bolites lead to unavailable mass spectrometry information in official
libraries. Thus, a general synthesis approach and mass spectral features
of these bio-signature metabolites with different derivatization methods
are necessary.
In the current study, a synthetic method for 2-(2-methylalkyl)
malonic acids was established. Four synthesized compounds that differ
in terms of carbon chain lengths were treated via four different deri-
vatization methods (namely methyl, ethyl, n-butyl and trimethylsilyl
esterifications) to acquire the informative fragments of various mole-
cular masses by gas chromatography-mass spectrometer (GC-MS). The
obtained mass spectral characteristics provide fundamental basis on
identification of alkylmalonates as an alternative to be indicative of
intermediates in anaerobic alkane degradation via the fumarate addi-
tion pathway.
rotary evaporation, and distilled water was added to the flask.
Hydrochloric acid was added to adjust the pH to < 1. The obtained
intermediate product was extracted three times with ethyl acetate. The
combined oil layers were dried over anhydrous sodium sulfate and
evaporated [21]. In this step, the brominated product replaced the
active hydrogen from diethyl methylmalonate. The yield was approxi-
mately 68.5%.
2.2.3. Preparation of ethyl 2-methylalkylcarboxylate
This step was conducted under Krapcho decarboxylation condition
[26]. The above product, namely, diethyl 2-alkyl-2-methylmalonate
(0.035 mol), was added to a 100-mL flask and dissolved with 20 mL
DMSO. LiCl (0.07 mol, 2.96 g) and distilled water (0.07 mol, 1.26 g)
were added into the mixture, which was transferred to an oil bath at
150 °C and heated to reflux for 10 h. Following cooling to room tem-
perature, distilled water was added, and extraction was conducted with
ethyl acetate three times. The organic phase was combined, and the
solvent was dried over anhydrous Na2SO4 and evaporated. The yield
was approximately 60%.
2.2.4. Preparation of 2-methylalkyl alcohol
LiAlH4 powder (0.035 mol, 1.33 g) was added into a dry 100-mL
flask. In an ice water bath, 20 mL of the prepared re-distilled anhydrous
THF was added dropwise with a constant pressure liquid funnel and
stirred until the LiAlH4 powder was completely dissolved. The drying
tube was used throughout the process to prevent moisture in the air
from entering the reaction system. The intermediate product, namely,
ethyl 2-methylalkylcarboxylate, that was synthesized in the previous
step was dissolved in 10 mL of re-distilled THF. Then, the mixture
above was added dropwise to a solution of LiAlH4 in THF by adjusting a
constant pressure liquid funnel with a drying tube. The mixture was
stirred vigorously at room temperature for 6 h until the whole reaction
mixture turned into a gray turbid liquid [27].
The following procedure was conducted to quench the reaction.
When quenching of the reaction, a constant-pressure liquid funnel was
used to strictly control the rate of addition. When adding water at the
beginning, a drop was added and stirred for a specified duration prior to
continuing. The solution gradually changed from gray to white turbid.
At the same time, precipitation could also occur in the reaction, and if
the stirrer is unable to stir, a volume of anhydrous THF could be added
to dissolve the precipitation. Water was added until no hydrogen was
released from the solution. At this time, a large amount of white pre-
cipitation appeared in the flask, and dilute hydrochloric acid was added
dropwise under an ice water bath. The mixture was stirred vigorously
until the precipitation dissolved; then, the solution was clarified. The
obtained clarification liquid was extracted with ethyl acetate three
times. Then, the organic phase was combined and the solvent was re-
moved from via the addition of anhydrous sodium sulfate and eva-
poration. The yield was approximately 43%.
2. Experimental
2.1. Materials
All reagents that were used (HBr, H2SO4, HCl, NaOH, Na, LiAlH4, n-
hexane, Na2SO4, n-hexyl alcohol, n-propanol, ethyl acetate, ethanol,
methanol, LiCl, DMSO, THF, NaHCO3, diethyl methylmalonate and
petroleum ether) in the chemical synthesis were of analytical grade and
of the highest purity and purchased from Shanghai Lingfeng Chemical
Reagent Co. Ltd (Shanghai, China). Sodium sulfate and ethanol were
pretreated to remove water prior to use or were purchased in anhydrous
form. N, O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) was pur-
chased from Sigma-Aldrich (Shanghai Trading Co. Ltd, Shanghai,
China).
2.2. Synthesis of 2-(2-methylalkyl)malonic acid
2.2.1. Preparation of 1-bromoalkane
Alkylalcohol (0.035 mol) and HBr (0.039 mol) were mixed into a
100-mL flask. At 0 °C, H2SO4 was cautiously added, dropwise, into the
mixture above and stirred intensely [25]. After returning to room
temperature, the mixture was transferred to an oil bath and heated to
reflux at 120 °C for 1 h. After cooling to room temperature, steam
distillation was conducted to initially purify the target product.
NaHCO3 was added into the mixture to adjust the pH to > 7. The
mixture was extracted with n-hexane three times and organic phase was
obtained. Then, anhydrous Na2SO4 was used to dry the solvent. The
yield was approximately 90%.
2.2.5. Preparation of 1-bromo-2-methylalkane
This synthetic method was the same as that for the preparation 1-
bromoalkane in step 2.2.1. The yield was approximately 82%.
2.2.6. Preparation of diethyl 2-(2-methylalkyl)malonate
In this step, the 1-bromo-2-methylalkane that was obtained in the
above step was reacted with diethyl malonate. The method was as
described in step 2.2.2, and the yield was approximately 60%.
2.2.2. Preparation of diethyl 2-alkyl-2-methylmalonate
To a 100-mL flask equipped with a drying tube, 10 mL absolute
ethanol was added. Na (0.035 mol, 0.80 g) was added in an ice water
bath, and the solution was stirred untill Na was completely dissolved.
The reaction apparatus was transferred to room temperature. Diethyl
methylmalonate was added dropwise, which was followed by stirring
for 30 min. Then, the prepared 1-bromoalkane was added into the
above mixed system, stirred for 1 h, and placed in an oil bath at 80 °C
for 6 h. The anhydrosity of the entire reaction was maintained. After the
reaction cooled to room temperature, the ethanol was removed via
2.2.7. Preparation of 2-(2-methylalkyl)malonic acid
2-(2-Methylalkyl)malonic acid was acquired via an ester hydrolysis
reaction. The product that was acquired in the above step, namely,
diethyl 2-(2-methylalkyl)malonate, was added with NaOH (0.07 mol,
2.80 g) and H2O (20 mL) into a 100-mL flask and heated to reflux at
100 °C for 4 h. After cooling to room temperature, the mixture was
extracted with n-hexane 3 times to remove undissolved organic matter
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J. Chen, et al.
AnalyticalBiochemistry600(2020)113746
[20]. Then, the aqueous phase was collected and acidified with hy-
drochloric acid to pH < 1 and extracted with ethyl acetate three times.
Finally, the oil phase was combined and dried over anhydrous sodium
sulfate and evaporated. The yield was approximately 90%.
The final product, namely, 2-(2-methylalkyl)malonic acid, was
purified via column chromatography (petroleum ether: ethyl
acetate = 15:1, v/v).
Diethyl 2-(2-methylbutyl)malonate. 1H NMR (400 MHz,
CDCl3, TMS; δ, ppm) δ = 4.24-4.17 (m, 4H), 3.47-3.41 (m, 1H), 2.01-
1.93 (m, 1H), 1.72-1.65 (m, 1H), 1.42-1.34 (m, 2H), 1.31-1.20 (m, 6H),
1.23-1.17 (m, 1H), 0.91-0.86 (m, 6H).
Diethyl 2-(2-methylpentyl)malonate. 1H NMR (400 MHz, CDCl3,
TMS; δ, ppm): δ = 4.19-4.10 (m, 4H), 3.39-3.33 (m, 1H), 1.91-1.83 (m,
1H), 1.64-1.54 (m, 1H), 1.43-1.26 (m, 2H), 1.22-1.17 (m, 8H), 1.12-
0.99 (m, 1H), 0.87-0.75 (m, 6H).
Diethyl 2-(2-methyloctyl)malonate. 1H NMR (400 MHz, CDCl3,
TMS; δ, ppm): δ = 4.21-4.16 (m, 4H), 3.45-3.41 (m, 1H), 1.98-1.91 (m,
1H), 1.70-1.63 (m, 1H), 1.42-1.38 (m, 1H), 1.28-1.25 (m, 16H), 0.89-
0.86 (m, 6H).
2.3. NMR analysis
Diethyl 2-(2-methylalkyl)malonates were analyzed via 1H NMR. The
spectra were recorded on a Bruker AM 400-MHz spectrometer, and
tetramethylsilane (TMS) was used as an internal standard.
3.2. Mass spectrometry characterization of methyl, ethyl and n-butyl
esterification products
2.4. Derivatization
The mass spectral characteristics are presented in Fig. 3 and Figs.
S1, S2 and S3. Regularities were observed in the mass spectra of all the
derivatization products. Since the diethyl esterification product can be
obtained according to the designed synthetic route, it can be directly
analyzed via GC-MS. The fragment ions were examined in detail by
considering the diethyl ester as an example.
Four homologous compounds that differed in terms of carbon chain
length were derivatized to methyl, ethyl, n-butyl and trimethylsilyl
esters. The mass spectral characteristics of the diethyl ester products
could be acquired directly during chemical synthesis. Other derivati-
zations were conducted as described below.
In test tubes, 0.5 mL of 10% (v/v) H2SO4-methanol/H2SO4-butanol
solution was added into 100 mg of each product for complete mixing.
Then, the tubes were sealed and placed in an oil bath at 90 °C for
60 min for methyl/n-butyl esterifications [28]. Regarding the silylation
products, BSTFA and acetonitrile were quickly well-mixed with each of
the synthesized compounds and reacted in an oven at 60 °C for 30 min
[29].
All derivatives above were identified directly via GC-MS for mass
spectrometry characterization. All GC-MS analyses were performed on
an Agilent 6890 GC instrument equipped with an HP-5MS capillary
column (30 m × 0.25 mm × 0.25 μm) and mass detector (MSD 5975).
The experimental details regarding various methods of derivatization
and the conditions for the GC-MS program were studied previously
[20].
3.2.1. Mass spectrometry characterization of ethyl esterification products
The mass spectral characterizations of the four diethyl esterification
compounds are presented in Fig. 3. The four most significant inter-
mediate fragments, which are observed at m/z 133, 160, 173 and [M -
45]+, are speculated to be the characteristic fragment ions of diethyl 2-
(2-methylalkyl)malonate. The cleavage mechanism is proposed (Fig. 4)
for further discussion.
The strong peaks at m/z 133, 160 and 173 (in red in Fig. 3) re-
mained unchanged with changes in the relative molecular mass. It is
posited that diethyl 2-(2-methylalkyl)malonates (structure a) loses the
alkyl moiety (the R group) and forms a carbon cation of diethyl me-
thylenemalonate (structure b) at m/z 173. This fragment ion presents
the unique molecular structure of diethyl malonate from diethyl 2-(2-
methylalkyl)malonates and is indicative of the structural characteristics
of such substances. Among all of the peaks, the fragment ion m/z 160
shows the highest relative abundance in each mass spectrum. Since the
alkyl group of the carbon chain can provide a γ-H for this rearrange-
ment reaction, it is speculated that structure d undergoes EI-induced
fragmentation via McLafferty rearrangement and γ-cleavage to form
structure f at m/z 160 [30,31]. At the same time, structure g can further
proceed a McLafferty rearrangement with double hydrogen transfer,
leading to an alkenyl loss and yielding structure h, m/z 133 [31,32].
The fragment ions m/z 133, 160 and 173 are all relatively abundant in
the four mass spectra and can be used as the primary determinants of
compounds with structures of this type.
3. Results and discussion
Four 2-(2-methylalkyl)malonic acids that differed in terms of carbon
chain length, namely, propylmalonic acid, 2-(2-methylbutyl)malonic
acid, 2-(2-methylpentyl)malonic acid and 2-(2-methyloctyl)malonic
acid, were synthesized in this study (1–4, Fig. 1).
The complete synthetic route for 2-(2-methylalkyl)malonic acid is
illustrated in Fig. 2. Four synthesized compounds were derivatized via
four methods for GC-MS detection (Fig. 3, S1, S2 and S3). The diethyl
esterification compounds were identified via both mass spectrometry
(Fig. 3) and 1H NMR spectroscopy (Fig. S4).
The relative abundance of the fragment ion m/z [M - 45]+ (in blue
in Fig. 3), which corresponds to structure c, is also high in all the EI
mass spectra. As presented in Fig. 4, it is presumed that the loss of an
ethyoxyl moiety ([·OC2H5], m/z 45) from the ethyl ester group in
structure a results in the formation of fragment c [33–36]. The relative
molecular mass of the compound can be inferred from m/z [M - 45]+ to
determine the length of the alkyl chain.
3.1. NMR data
Diethyl propylmalonate. 1H NMR (400 MHz, CDCl3, TMS; δ,
ppm): δ = 4.19 (q, J = 8.0 Hz, 4H), 3.33 (t, J = 7.6 Hz, 1H), 1.87 (q,
J = 7.6 Hz, 2H), 1.38-1.33 (m, 2H), 1.27 (t, J = 7.2 Hz, 6H), 0.94 (t,
J = 7.3 Hz, 3H).
Therefore, the four fragment ion peaks at m/z 133, 160, 173 and [M
- 45]+ can be regarded as the characteristic fragments that indicate the
presence of diethyl 2-(2-methylalkyl)malonates.
3.2.2. Mass spectrometry characterization of methyl esterification products
Three relatively intense peaks at m/z 132, 145 (in red) and [M -
31]+ (in blue) are visible in all the spectra of the methyl esterification
derivatives (Figure S1). Fragment ions m/z 132 and 145 are similar to
those of ethyl esters, which differ by 28 mass units, suggesting the
presence of two methylene groups, analogous to structures b and f. The
shifts of m/z are rational when methoxyl is substituted for ethoxyl.
Fig. 1. Chemical structures of the four 2-(2-methylalkyl)malonic acids.
3

J. Chen, et al.
AnalyticalBiochemistry600(2020)113746
Fig. 2. Chemical synthetic procedure for 2-(2-methylalkyl)malonic acid.
Additionally, a fragment ion m/z [M - 31]+ can be observed in all mass
spectra, indicating the elimination of a methyl group. According to
mechanism (b), the fragment at m/z 119 that corresponds to structure h
is missing from all EI mass spectra of methyl esters. However, this is
reasonable because when the methyl moiety replaces the ethyl moiety,
McLafferty rearrangement with double hydrogen transfer (steps g to h)
will not occur, resulting in the absence of the fragment at m/z 119 [31].
Therefore, the mass spectral characteristics of dimethyl 2-(2-methy-
esterification products, which coincide with mechanisms (a) and (b) in
Fig. 4. In addition, two fragment ions, namely, m/z [M - 55]+ and [M -
129]+ (in green), are observed in all mass spectra of n-butyl esters.
According to Fig. 4(c), due to the presence of γ-H on the butyl group of
fragment i, the McLafferty rearrangement with double hydrogen
transfer reaction occurs to yield fragment j at m/z [M - 55]+, and a
butenyl moiety is lost [31,37]. The butanol group of fragment j is fur-
ther removed to generate structure k at m/z [M - 129]+, which is in
agreement with previous observations [20]. Hence, the fragment ions
m/z 161, 216, 229, [M - 73]+, [M - 55]+ and [M - 129]+ are regarded
as characteristic peaks of dibutyl 2-(2-methylalkyl)malonates.
lalkyl)malonates are m/z 132, 145 and [M - 31]+
.
3.2.3. Mass spectrometry characterization of n-butyl esterification products
Likewise, the mass spectral characteristics of n-butyl esters differ by
28 or 56 mass units from those of ethyl esterification. As displayed in
Fig. S2, the mass spectral characteristics m/z 161, 216, 229 (in red) and
[M - 73]+ (in blue) can be identified in each mass spectrum of n-butyl
3.3. Mass spectrometry characterization of silylation products
As shown in Fig. S1 and S2, in contrast to alkyl esterification,
Fig. 3. Mass spectral characteristics of the synthesized diethyl 2-(2-methylalkyl)malonates as fumarate addition pathway rearrangement derivatives. The values in
red are constant for all chemical compounds. The values in blue differ among the compounds and can reflect the length of the carbon chain. (For interpretation of the
references to colour in this figure legend, the reader is referred to the Web version of this article.)
4

J. Chen, et al.
AnalyticalBiochemistry600(2020)113746
Fig. 4. Proposed cleavage of diethyl 2-(2-methylalkyl)malonate via mechanisms (a) and (b) and of dibutyl 2-(2-methylalkyl)malonate via mechanism (c).
15]+ can be used for the identification of methyl and trimethylsilyl
Table 1
Characteristic fragment ions of diester 2-(2-methylalkyl)malonates.
esters, respectively. These results provide an alternative method for
detecting downstream biomarkers of the fumarate addition pathway in
both in situ and in the laboratory, which deepens the exploration of
anaerobic hydrocarbon degradation.
Derivation
Characteristic fragment ions
Methyl esterification
Ethyl esterification
n-Butyl esterification
Silylation
132, 145, [M - 31]+
133, 160, 173, [M - 45]+
161, 216, 229, [M - 73]+, [M - 55]+, [M - 129]+
73, 147, 217, 248, 261, [M - 15]+
CRediT authorship contribution statement
Jing Chen: Conceptualization, Methodology, Writing - original
draft. Lei Zhou: Software, Visualization, Writing - original draft. Yi-Fan
Liu: Data curation, Writing - original draft, Writing - review & editing.
Zhao-Wei Hou: Writing - original draft, Writing - review & editing. Wei
Li: Writing - original draft, Writing - review & editing. Serge Maurice
Mbadinga: Writing - original draft, Formal analysis. Jing Zhou:
Writing - original draft, Methodology. Tao Yang: Writing - original
draft, Methodology. Jin-Feng Liu: Writing - original draft, Project ad-
ministration. Shi-Zhong Yang: Writing - original draft, Project ad-
ministration. Xiao-Lin Wu: Writing - original draft, Supervision. Ji-
Dong Gu: Writing - original draft, Conceptualization, Supervision. Bo-
Zhong Mu: Writing - original draft, Project administration, Funding
acquisition, Supervision.
fragment ions at m/z 261 and 248 via mechanisms (a) and (b) in Fig. 4
are identified in each spectrum, but no ion peaks that corresponded to
the process of converting structure a to structure c via mechanism (a)
were observed. Similar to methyl esterification, the correspondence of
the missing fragment at m/z 175 to structure h is rational because
McLafferty rearrangement with double hydrogen transfer (steps g to h)
will not occur [31], whereas the four fragment ions at m/z 73, 147, 217
and [M - 15]+ (Fig. S3) are presented with relatively high abundance. It
is suggested that m/z [M - 15]+ corresponds to the loss of the methyl
group from the bis-(trimethylsilyl) esters. The fragment at m/z 217 is
presumed to be a product of the migration of the trimethylsilyl group
on the carboxyl monomer, and the ions at m/z 73 and 147 correspond
to the trimethylsilyl monomer and dimer [38–41]. Consequently, the
fragment ions at m/z 73, 147, 217, 248, 261 and [M - 15]+ can be
regarded as the mass spectral characteristics of trimethylsilyl esters.
In conclusion, the mass spectral characteristics of diester 2-(2-me-
thylalkyl)malonates are summarized in Table 1. Each of the derivati-
zation methods corresponds to a distinct sets of characteristics. Ac-
cording to the newly obtained information, it is possible to choose one
or several suitable methods for proving fumarate addition via the direct
detection of rearrangememnt bio-signature metabolites.
Declaration of competing interest
The authors declare that there is no conflict of interest.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (No. 41530318), the Shanghai International
Collaboration Program (No. 18230743300), and the Fundamental
Research Funds for the Central Universities (No. 222201817017).
4. Conclusions
Appendix A. Supplementary data
A synthetic procedure for 2-(2-methylalkyl)malonic acids as re-
arrangement bio-signature metabolites of anaerobic alkane activation
via fumarate addition was conducted. Four model products with var-
ious carbon chain lengths were derivatized via four approaches, and
their mass spectra information was determined via GC-MS. The char-
acteristic fragments were observed at m/z 133 + 14n, 160 + 28n,
173 + 28n and [M - (45 + 14n)]+ (n = 0 and 2 for ethyl and n-butyl
esters, respectively). Additionally, mass spectra features at m/z 132,
145, and [M - 31]+ and ions at m/z 73, 147, 217, 248, 261 and [M -
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ab.2020.113746.
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