Enzymatic nitration of the oleic acid derivatives: Synthesis, isolation and characterization

Article abstract of DOI:10.1016/j.molcatb.2013.10.001

Unsaturated fatty acids are nitrated endogenously to produce nitrated lipids. Recent studies have shown that these nitrated lipids have high chemical reactivity and profound biological implications. We report an efficient, enzymatic synthesis of nitrated derivatives of the oleic acid. The methyl oleate could react with NO and horseradish peroxidase (HRP)/H₂O ₂/NO based nitrating systems to give various nitration products which could be isolated by silica gel column and TLC fractionation, respectively. As reacting directly with NO, the obtained products contain (E)-methyl 9-nitrooctadec-9-enoate (1), (E)-methyl 10-nitrooctadec-9-enoate (2), (E)-methyl 9-nitrooctadec-10-enoate (3) and (E)-methyl 10-nitrooctadec-8-enoate (4), characterized by extensive IR, NMR and GC-MS analysis. Whereas the products of the reaction between the methyl oleate and NO with the presence of HRP/H ₂O₂ were mainly composed of (E)-methyl 9-nitrooctadec-9-enoate and (E)-methyl 10-nitrooctadec-9-enoate. The improving selectivity of the products is attributed to the HRP catalysis system.

Full text of DOI:10.1016/j.molcatb.2013.10.001

Journal of Molecular Catalysis B: Enzymatic 98 (2013) 87–91
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Enzymatic nitration of the oleic acid derivatives: Synthesis, isolation
and characterization
Jing Lia, Jing Tiana,∗, Xu Feib,∗∗, Xiuying Wangb, Longquan Xub, Yi Wanga
a School of Biological Engineering, Dalian Polytechnic University, Dalian 116034, PR China
b Modern Education Technical Department, Dalian Polytechnic University, Dalian 116034, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Unsaturated fatty acids are nitrated endogenously to produce nitrated lipids. Recent studies have shown
that these nitrated lipids have high chemical reactivity and profound biological implications. We report
an efficient, enzymatic synthesis of nitrated derivatives of the oleic acid. The methyl oleate could react
with NO and horseradish peroxidase (HRP)/H2O2/NO based nitrating systems to give various nitration
products which could be isolated by silica gel column and TLC fractionation, respectively. As reacting
directly with NO, the obtained products contain (E)-methyl 9-nitrooctadec-9-enoate (1), (E)-methyl
10-nitrooctadec-9-enoate (2), (E)-methyl 9-nitrooctadec-10-enoate (3) and (E)-methyl 10-nitrooctadec-
8-enoate (4), characterized by extensive IR, NMR and GC–MS analysis. Whereas the products of the
reaction between the methyl oleate and NO with the presence of HRP/H2O2 were mainly composed of
(E)-methyl 9-nitrooctadec-9-enoate and (E)-methyl 10-nitrooctadec-9-enoate. The improving selectivity
of the products is attributed to the HRP catalysis system.
Received 13 August 2013
Received in revised form
18 September 2013
Accepted 2 October 2013
Available online 11 October 2013
Keywords:
Methyl oleate
Unsaturated fatty acid
Enzymatic nitration
Horseradish peroxidase
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
have shown that NO reacts with unsaturated fatty acids and esters
at high concentrations and in the presence of oxygen to get nitra-
Nitrated derivatives of unsaturated fatty acids represent an
increasingly important area at the crossroads of biology and
medicine. Several recent studies have indicated that these nitrated
derivatives of unsaturated fatty acids have significant chemical
reactivity and profound biological activities. Bioactive nitroalkene
derivatives of oleic acid have been identified in plasma, red cells,
and urine of healthy humans [1–3]. Under physiological condi-
tions, they can elevate production of nitric oxide (NO), a powerful
biological effector, which could regulate vascular relaxation [4–7].
Furthermore, the nitration products of unsaturated fatty acid have
the potential to function as biomarkers or mediators of processes.
Nitrated derivatives of unsaturated fatty acids have emerged
during the past decade. Yet, the biological and toxicological prop-
erties of nitrated fatty acid derivatives have remained largely
unexplored, also because of the lack of convenient synthetic
approaches. In fact, it is very difficult to get a single nitration prod-
uct of unsaturated fatty acids which will be isolated in pure form
and structurally characterized [8]. For example, some recent works
tion products. But it is a challenge to separate the nitration products
[9,10].
At the same time, some experimental schemes of enzymatic
synthesis were put forward [1,11–13]. As we all know, compared
to chemical synthesis, enzymatic synthesis has presented many
advantages, such as high specificity, high efficiency, better environ-
mental property as well as better adjustability. Several peroxidases
were tested as nitration catalysts, such as horseradish peroxidase,
soybean peroxidase and lactoperoxidase [14–16]. For example, HRP
could catalyze the nitration of tryptophan derivatives in the pres-
ence of H2O2 and NaNO2 [17].
In this paper, we report the synthesis, structural characteriza-
tion of nitrated methyl oleate. The methyl oleate reacts with NO
and HRP/H2O2/NO based nitrating systems to give different nitra-
tion products. Under biological catalysis conditions, there are few
by-products. The products 1 and 2 have been described as only
products of nitration of methyl oleate.
2. Experimental
∗
2.1. Materials and general methods
Corresponding author at: School of Biological Engineering, Dalian Polytechnic
University, 1# Qinggongyuan Road, Dalian 116034,
PR China. Tel.: +86 411 86324485.
Corresponding author at: Modern Education Technical Department, Dalian Poly-
technic University, 1# Qinggongyuan Road, Dalian 116034,
PR China. Tel.: +86 411 86323691 201; fax: +86 411 86322038.
E-mail addresses: tianjing@dlpu.edu.cn (J. Tian), feixu@dlpu.edu.cn (X. Fei).
Methyl oleate (90%) was obtained from Dalian Medical Con-
naught Biotechnology Co., Ltd. Horseradish peroxidase (HRP,
250 U/mg) was from Beijing Biodee Biotechnology. Sodium nitrite
(99%) was purchased from Amethyst Chemicals, hydrogen peroxide
∗∗
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.10.001

88
J. Li et al. / Journal of Molecular Catalysis B: Enzymatic 98 (2013) 87–91
136.5 ( NO2C CH–), 3/4: 1H NMR (400 MHz, CDCl3) ı (ppm)
5.81 ( NO2CH CH CH ,m, 1H), 5.63 ( NO2CH CH CH , dd,
1H), 4.85 ( NO2CH CH CH , q, 1H). 13C NMR (100 MHz, CDCl3)
ı (ppm) 138.8 ( NO2CH CH CH ), 124.8 ( NO2CH CH CH ),
90.25 ( NO2CH CH CH ). GC–MS: EI-MS m/z 341 (M+), m/z 263
([M–HNO2–OCH3]+).
(water solution, 30%). All other chemicals including cyclohexane,
hexane, ethyl acetate, chloroform, alcohol, acetic acid and anhy-
drous sodium sulfate were analytical reagent grade quality, and
used without further purification. NO was prepared through the
reaction of NaNO2 (3 g) in water (20 mL) with 3% sulfuric acid
(60 mL).
IR spectra were taken on a Spectrum One-B infrared spectropho-
tometer (Perkin-Elmer, USA). Nuclear magnetic resonance (NMR)
spectra were measured on a Bruker AVANCE NMR spectrometer
at a resonance frequency of 400 MHz for 1H, 100 MHz for 13C. The
chemical shifts of 1H, 13C NMR spectra were referenced to TMS
at 0 ppm, deuterated chloroform at 76.0 ppm, respectively. For all
isolated compounds, 1H and 13C NMR resonances due to –OCH3,
COOCH3, C-2 (CH2), C-3/C-4/C-5/C-6/C-7/C-12/C-13/C-14C/-15/C-
16/C-17 (CH2) and C-18 (CH3) groups appear in the 1H/13C NMR
spectra at ı (ppm) 3.67 (s, 3H)/51.7, 174.3, 2.30 (t, 2H)/34.2, 1.6–1.2
(m, 22H)/32.3–22.7, 0.89 (t, 3H)/14.3 in the order.
Preparative TLC and analytical HPTLC were performed on F254
silica gel plates (1 and 0.25 mm, respectively). 10% H2SO4 was used
for product detection on TLC plates. The column chromatography
was performed on silica gel (300–400 mesh). Purity of isolated
compounds was determined by 1H NMR analysis.
2.5. Reaction of methyl oleate with HRP/H2O2/NO
A solution of methyl oleate (592 mg, 2.0 mmol) in cyclohex-
ane (20 mL) was added to 0.1 M phosphate buffer (20 mL, pH 5.6)
with HRP (250 U/mg, 8 mg) and kept at 30 ◦C under vigorous stir-
ring. After bubbling nitrogen for 30 min, the NO gas (produced as
described above) was slowly bubbled into the above mixture. Sub-
sequently, the equal volume (6.7 mmol each) of hydrogen peroxide
(30%) was added per 30 min. After 8 h, the reaction mixture was
allowed to stand for separation of layers in a separatory funnel
and the organic layer was preserved, dried over anhydrous sodium
sulfate, and evaporated to dryness under reduced pressure. Then
the mixture was analyzed by FT-IR spectrophotometer. Nitro com-
pounds were discovered, IR (KBr, cm−1): 3004 (
1629 (C C), 1555 (NO2), 847 (C NO2).
C H), 1743 (C O),
2.2. GC–MS analysis
2.6. Isolation of (E)-methyl 9-nitrooctadec-9-enoate (1),
(E)-methyl 10-nitrooctadec-9-enoate (2)
GC–MS was carried out on a GC instrument coupled with a
quadrupole mass spectrometer(Agilent, USA. Helium was the car-
rier gas with a 1.19 mL/min flow rate. The following analytical
conditions were used: 30 m DB-5MS capillary column (0.25 mm i.d.,
0.25 ␮m d.f.). The separation program was initiated at 60 ◦C and
kept for 2 min. Then column temperature was ramped to 280 ◦C
at the rate of 10 ◦C/min. The sample injection volume was 1 ␮L
and injector temperature was 180 ◦C with split ratio 10:1. The ion
source was set to 230 ◦C. Ionization voltage was 70 eV, and detector
voltage was 1.3 kV, the solvent delay was set to 5 min. The MS scan
range was 33–400 (m/z) with a scan interval of 0.5 s.
The products from step 2.5 were fractionated by preparative
TLC (cyclohexane/ethyl acetate/acetic acid 60:7:6 (v/v), eluant B)
and high-performance TLC plates (cyclohexane/chloroform/ethyl
acetate/alcohol/acetic 24:2:1:1:2 (v/v), eluant C) to give fraction
Iꢀ (a mixture of 1 and 2, 24 mg, 98% purity). Iꢀ: 1H NMR (400 MHz,
CDCl3) ı (ppm) 7.06 (–NO2C CH, q, 1H), 2.55 ( CNO2 CH2 , t, 2H),
2.22 ( CH2CH CNO2 , q, 2H), 13C NMR (100 MHz, CDCl3) ı (ppm)
152.1 (–NO2C CH ), 136.5 (–NO2C CH ). GC–MS: EI-MS m/z 341
(M+), m/z 263 ([M HNO2 OCH3]+).
2.3. Reaction of methyl oleate with NO (other nitric oxides):
general procedure
3. Results and discussion
3.1. Reaction of methyl oleate with NO: isolation and
characterization of nitration products
Nitrogen was bubbled for 30 min into a vigorously stirred solu-
tion of methyl oleate (2.072 g, 7 mmol) in cyclohexane (20 mL) to
remove the oxygen. Then NO gas (produced as described above)
was slowly bubbled for about 18 h into the methyl oleate solution.
The resulting solvent was evaporated to give the yellow transparent
liquid products and they were analyzed by FT-IR spectrophotome-
ter. Nitro compounds were discovered: IR (KBr, cm−1) 1743 (C O),
1645 (C C), 1555 ( NO2), 847 (C NO2).
We investigated nitration products of methyl oleate (methyl
(Z)-octadec-9-oleate) to gain some information on the nitration
reactions of unsaturated fatty acid derivative. The reaction of
methyl oleate with NO afforded crude nitration products. After
careful silica gel column chromatography and preparative TLC frac-
tionation, the main products (fraction I) were eventually obtained
and readily identified as the nitroalkenes 1/2 and the nitroallyl
derivatives 3/4 (Rf = 0.41–0.57 eluant A). By 1H NMR analysis, an
approximate formation ratio of (1 + 2)/(3 + 4) was estimated to be
1:2. Through repeated TLC on silica gel, the fraction I was separated
to the fraction II and III. However, they were still characterized as
a mixture, which contained regioisomers of 1/2 (14.3% yield) or
3/4 (26.9% yield) separately. As their polarity has a striking sim-
ilarity, attempts to isolate these regioisomers were thwarted by
the unfavorable chromatographic behavior. The fraction II and III
were exhibited in the NMR spectra. The nitration products 1–4 were
proved fairly stable and could be preserved for at least 6 months
under the storage conditions (anhydrous conditions, 5 ◦C) (Fig. 1).
FT-IR analysis: As shown in Fig. 2(a), the characteristic absorp-
tions of ester group appeared around 1743 cm−1 (C O) and the
region of 1200–1300 cm−1 (C–O), and there was no adsorption of
double bond (C C) at about 1640 cm−1 due to symmetrical struc-
ture for methyl oleate. The spectrum of the crude nitration products
Fig. 2(b) shows the absorption peak of the double bond (C C)
2.4. Isolation of (E)-methyl 9-nitrooctadec-9-enoate (1),
(E)-methyl 10-nitrooctadec-9-enoate (2), (E)-methyl
9-nitrooctadec-10-enoate (3) and (E)-methyl
10-nitrooctadec-8-enoate (4)
The reaction was carried out as described above and the
obtained liquid products were fractionated by column chro-
matography (3.6 cm × 30 cm) using hexane/ethyl acetate/acetic
acid (12:1:1 to 11:1.5:1.5 v/v gradient mixtures) to afford fraction
I (a mixture of 1, 2, 3, and 4) (Rf = 0.38–0.50, cyclohexane/ethyl
acetate/acetic acid 60:9:8 (v/v), eluantA). And then, the fraction
II and III were obtained through repeated TLC on silica gel. How-
ever, they were still characterized as a mixture, which contained
regioisomers of 1/2 or 3/4. The products exhibited in the NMR spec-
tra. 1/2: 1H NMR (400 MHz, CDCl3) ı (ppm) 7.06 ( NO2C CH
,
q, 1H), 2.55 ( CNO2 CH2 , t, 2H), 2.22 ( CH2CH CNO2–, q,
2H). 13C NMR (100 MHz, CDCl3) ı (ppm) 152.1 ( NO2C CH–),

J. Li et al. / Journal of Molecular Catalysis B: Enzymatic 98 (2013) 87–91
89
Fig. 1. The main nitrated products from reaction of methyl oleate with NO 1
(E)-methyl 9-nitrooctadec-9-enoate, 2 (E)-methyl 10-nitrooctadec-9-enoate, 3 (E)-
methyl 9-nitrooctadec-10-enoate, 4 (E)-methyl 10-nitrooctadec-8-enoate.
at 1645 cm−1 due to the NO2 group which changed symmetry
of structure. At the same time, the absorbance of aliphatic nitro
group appeared at 1555 and 847 cm−1 (Fig. 2(b)). Furthermore, the
absorptionpeak of the carbonhydrogenbond on the olefin (C
C H)
at 3004 cm−1 (Fig. 2(a)) got weak significantly after substitution
reaction of –NO2 group (Fig. 2(b)).
NMR analysis: The products 1/2 (fraction II) were exhibited in
the 1H NMR spectrum as most salient features at ı 7.06 (C-9 or C-
10, alkene proton, appearing on one NMR spectrometer at 400 MHz
as a quartet, in actuality a superimposed pair of triplets, typical of
protons on E-nitroalkene functionalities) ı 2.55 (C-8 or C-11, allylic
methylene neighboring nitro group; nitroalkene more electron
withdrawing than carbonyl), and ı 2.22 (C-8 or C-11, allylic methy-
lene opposite nitro group, inductive effect of electron-withdrawing
group makes absorption peak move to higher frequencies). For
the products 3/4 (fraction III), the characteristic chemical shifts
of–CH CH CHNO2– moiety was readily apparent from the sets of
signals in the 1H NMR spectrum (Fig. 3) at ı 5.81, ı 5.63, ı 4.85 and
in 13C NMR spectrum at ı 124.8, ı 138.8, ı 90.25 in that order. The
NMR data was summarized in Table 1.
Fig. 3. 1H NMR spectra of the four main nitrated products.
oleate, of four main products which were identified as the nitrated
products 1, 2, 3 and 4 by the analysis of their mass spectra. A
weak, barely detectable molecular ion peak at m/z 341 was noted
in the EI–MS spectrum along with a fragmentation peak at m/z 263,
reflecting the loss of HNO2 and OCH3.
GC–MS analysis: The crude products were purified by silica gel
chromatographic fractionation. Then the obtained sample (fraction
I) from separation was analyzed by GC–MS, which provided a reli-
able means to monitor the generation of nitrated methyl oleate.
The GC–MS profile revealed the presence, besides unreacted methyl
3.2. Reaction of methyl oleate with HRP/H2O2/NO: isolation and
characterization of nitration products
Treatment of methyl oleate with HRP, H2O2 and NO yielded
nitration products with a high-performance thin layer chro-
matography (HPTLC) (Rf = 0.58–0.65 eluant C) to eliminate lots of
unreacted methyl oleate. Additionally, the HPTLC fraction Iꢀ con-
tains two regioisomers, which were formed in small amounts
(3.5% yield) and identified as (E)-methyl 9-nitrooctadec-9-enoate
1, and (E)-methyl 10-nitrooctadec-9-enoate 2. The FT–IR spectrum
of the crude nitration products exhibited the absorption peak at
Table 1
Selected NMR data of 9-nitro and 10-nitrooleate. ıH (ppm)(mult), ıC (ppm).
9- and 10-nitrooleate
ıH (ppm) (mult)
ıC (ppm)
CH2CH CNO2
2.22 (q)
7.06 (q)
–
2.55 (t)
5.81 (m)
5.63 (dd)
4.85 (q)
29.7
136.5
152.1
27.9
124.8
138.8
90.25
CH2-CH CNO2
CH CNO2 CH2
CH CNO2 CH2
CH CH CH NO2
CH CH CH NO2
CH CH CH NO2
Fig. 2. FT-IR spectra of (a) methyl oleate and (b) nitrated methyl oleate from the
reaction with NO.

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J. Li et al. / Journal of Molecular Catalysis B: Enzymatic 98 (2013) 87–91
Fig. 4. FT-IR spectra of (a) methyl oleate and (b) nitrated methyl oleate from the
reaction with HRP/H2O2/NO.
Fig. 6. 13C NMR spectrum of the pure main nitrated products.
the product 1/2 of the experiment carried out on methyl oleate with
NO.
1629 cm−1 (Fig. 4(b)) of the double bond C C, as well as at 1555
and 847 cm−1 indicating aliphatic nitro groups. At the same time,
the absorption peak at 3004 cm−1 (Fig. 4(a)) of the carbon hydrogen
GC–MS Analysis: GC–MS data of the main products identified in
fractions Iꢀ were given in Fig. 7, along with a plausible interpretation
of the origin of the main fragmentation peaks. Detectable molec-
ular ion peaks were obtained for mono-nitroderivatives 1 and 2.
They also gave a poorly discernible molecular ion peak at m/z 341,
together with peak (at m/z 263) due to subsequent losses of HNO2
and OCH3. These results indicate that we have synthesized nitrated
methyl oleate successfully under the system of HRP/H2O2/NO.
bonds on the olefin (C
reaction (Fig. 4(b)).
C H) in methyl oleate was weakened after
NMR Analysis: The structure of synthetic nitro-methyl oleate (a
1:1 mixture of C-9- and C-10-regioisomers) was confirmed by 1H
and 13C NMR spectra The products displayed virtually identical lH
NMR spectrum (Fig. 5) featuring a characteristic set of resonances
around ı 7.06 (q) (C-9 or C-10, alkene proton, appearing on one
NMR spectrometer at 400 MHz as a quartet, in actuality a super-
imposed pair of triplets), ı 2.55 (t) (C-8 or C-11, allylic methylene
neighboring nitro group), and ı 2.22 (q) (C-8 or C-11, allylic methy-
lene opposite nitro group). 13C NMR spectrum (Fig. 6) indicated
that synthetic nitro-methyl oleate was a mixture of two regioiso-
mers, with carbon peaks appearing as doublets. The equal height of
the doublets illuminated a 1:1 ratio of the regioisomers. The peaks
appearing at 152.1 and 136.5 ppm are the carbons ␣ and ␤ to the
alkenyl nitro group, respectively. The results were in accord with
3.3. Mechanistic issues
In order to synthesize nitrated unsaturated fatty acids or unsatu-
rated fatty acid derivative, different experimental approaches have
been used, including incubation of fatty acids with acidified nitrite
(NO2−) and oxidation with peroxynitrite (ONOO−) or other NOx,
nitrogen dioxide (·NO2) [6,8,18–20]. However, enzyme catalysis
mechanisms of lipid nitration remain unknown.
It has been reported that ·NO2 could react with unsatu-
rated lipids and lipid radicals leading to nitroalkenes, nitroallyl
derivatives and nitrohydroxy derivatives [8,21]. The mechanism
of unsaturated fatty acid nitration by ·NO2 involves an attack
to the double bond yielding a nitroalkyl radical which, at low
oxygen concentrations, combines with other ·NO2 molecules
to form nitro intermediates. By losing nitrous acid (HNO2) or
Fig. 5. 1H NMR spectrum of the pure main nitrated products.
Fig. 7. The mass spectrum of the nitrated products.

J. Li et al. / Journal of Molecular Catalysis B: Enzymatic 98 (2013) 87–91
91
hydrolysis, nitroalkenes or nitrohydroxy derivatives can be formed
Appendix A. Supplementary data
respectively. In addition, another mechanism is that electrophilic
subtitution at the double bond by NO2+ yields nitroalkenes. [20,22].
In this article, the two nitration reaction systems give various
nitration products. The products 1–4 from the NO system can be
envisaged as originating from two independent pathways of evolu-
tion of the primary ␣-nitroalkyl radical intermediates, namely free
radical combination with another molecule of •NO2 and ␤ H-atom
abstraction. As a result, the products 1/2 can be obtained via the loss
of HNO2 from labile 1, 2-nitronitrite intermediates and the prod-
ucts 3/4 containing the 1-nitro-2-alkenyl moiety can be obtained
via ␤-hydrogen atom abstraction from the nitroalkyl radicals. The
mechanism was supported by recent literature evidence [23,24].
However, no trace of the nitration products 3/4 could be
observed in HRP/H2O2/NO system, thus ruling out their involve-
ment in the formation of 3/4. On the basis of available evidence it is
not possible to draw any substantiated mechanism for the products
formation for the system of HRP/H2O2/NO. But obviously enzyme
catalysis improves selectivity and is a very effective method to
promote generation of nitroalkene products.
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.molcatb.
2013.10.001.
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Acknowledgments
This work was supported by National Natural Science Founda-
tion of China (No. 21204007), Science and Technology Research
Project of the Education Department of Liaoning province
(L2011080), Natural Science Foundation of Liaoning Province of
China (2013020167).
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