An Improved method for synthesis of N-stearoyl and N-palmitoylethanolamine

Article abstract of DOI:10.1007/s11746-012-2017-y

Certain N-acylethanolamines interact with cannabinoid receptors and have anorexic and neuroprotective effects. Traditional methods for the synthesis of N-acylethanolamines use fatty acid chlorides, fatty acid methyl esters, free fatty acids and triacylgly

Full text of DOI:10.1007/s11746-012-2017-y

J Am Oil Chem Soc
DOI 10.1007/s11746-012-2017-y
ORIGINAL PAPER
An Improved Method for Synthesis of N-stearoyl
and N-palmitoylethanolamine
Xiaosan Wang
•
Tong Wang Xingguo Wang
•
Received: 2 August 2011 / Revised: 7 December 2011 / Accepted: 27 December 2011
Ó AOCS 2012
Abstract Certain N-acylethanolamines interact with
cannabinoid receptors and have anorexic and neuropro-
tective effects. Traditional methods for the synthesis of
N-acylethanolamines use fatty acid chlorides, fatty acid
methyl esters, free fatty acids and triacylglycerols as acyl
donors to react with ethanolamine. In the present study, we
investigated the feasibility of using fatty acid vinyl esters
as the acyl donor to synthesize N-stearoyl and N-palmi-
toylethanolamine. Theoretically, the use of fatty acid vinyl
esters should lead to an irreversible reaction because the
volatile acetaldehyde by-product is easily removed. Four
reaction conditions, i.e. catalyst concentration, substrate
ratio, temperature, and time were evaluated. The reaction
performed at mild temperatures and with an excess amount
of ethanolamine which acted as both reactant and solvent
resulted in the formation of high purity N-stearoyl and
N-palmitoylethanolamine. When 20 mmol ethanolamine
was reacted with 1 mmol vinyl stearate at 80 °C for 1 h
with 1% sodium methoxide as catalyst, N-stearoyletha-
nolamine with 96% purity was obtained after the removal
of excess ethanolamine without further purification, while
N-palmitoylethanolamine with 98% purity was obtained by
reacting with the same substrate ratio at 60 °C for 1.5 h
with 3% catalyst. Complete conversion of vinyl stearate
and palmitate was achieved.
Keywords Acyl donor Á Amidation Á Fatty acid vinyl
esters Á N-Acylethanolamines Á Optimization Á Synthesis
Introduction
N-Acylethanolamines from fatty acids are an important
class of alkanolamides that function as nonionic surfac-
tants and have a wide range of applications in the lubri-
cants, surfactants and detergents, cosmetics, and other
industries [1–3]. Certain N-acylethanolamines are lipid
mediators in animals and plants [4–6]. It has been
reported that N-acylethanolamines of different chain
length and structure exhibit a variety of biological activ-
ities. N-Palmitoylethanolamine has anti-inflammatory
activity, and attenuates pain sensation [5, 7]. They have
also been shown to reduce allergic reaction, inhibit mast
cell degranulation [8] and exert neuroprotective effects in
rats and mice [9]. These actions were accompanied by
changes in nitric oxide production [10] and the expression
of pro-inflammatory proteins [11]. N-Oleoylethanolamine
exerts anorexigenic effects by binding to the nuclear
receptor in the periphery tissues, leading to body fat loss
[12, 13]. N-Stearoylethanolamine shows pro-apoptotic
and anorexic effects [14]. They could affect cell signaling
and elicit biological effects potentially through targets
other than cannabinoid receptors, such as exerting anor-
exic effects in mice via down-regulation of a liver enzyme
expression [6], and having anti-inflammatory activity by a
passive IgE-induced cutaneous anaphylaxis [15]. There-
fore, these are an interesting class of compounds for
animal and human applications.
X. Wang Á T. Wang (&)
Department of Food Science and Human Nutrition,
Iowa State University, 2312 Food Science Building,
Ames, IA 50011-1061, USA
e-mail: tongwang@iastate.edu
X. Wang Á X. Wang
State Key Laboratory of Food Science and Technology,
School of Food Science and Technology, Jiangnan University,
No. 1800 Lihu Road Wuxi, 214122 Jiangsu,
People’s Republic of China
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J Am Oil Chem Soc
N-Acylethanolamines from fatty acids theoretically can
be formed from a fatty acyl donor and ethanolamine. Four
basic methods have been reported for synthesizing
N-acylethanolamines according to the differences in the
acyl donor. In general, they are synthesized by reacting
fatty acid chlorides [16, 17], fatty acid methyl esters
Materials and Methods
Materials
Most chemicals including N-acylethanolamines standards
were purchased from the Sigma Aldrich Chemical Com-
pany (St. Louis, MO, USA). Vinyl stearate and palmitate
were purchased from Tokyo Chemical Industry (Tokyo,
Japan). Ethanolamine ([99%) was purchased from Fisher
Scientific (Fair Lawn, NJ, USA). Candida antarctica lipase
(Novozym 435) was provided by Novozymes America
(Blair, NE, USA). This is an immobilized lipase and has a
declared activity of 10,000 PLU (propyl laurate unit)/g.
[
18, 19] and triacylglycerols [20] with alkanolamine, or by
a direct reaction between free fatty acids and alkanolamine
in the presence of catalysts at low temperatures or without
any catalyst at high temperatures [1]. Yield and purity
improvement was usually achieved by removal of water,
methanol, glycerol or hydrochloric acid. However, only
60–90% N-acylethanolamines were produced from free
fatty acids, fatty acid methyl esters and triacylglycerols at
temperatures above 100 °C, usually at 180 °C for 6–12 h
21]. It has been reported that about 99% N-acylethanol-
Synthesis of N-Stearoylethanolamine Using Vinyl
Stearate Based on Procedures Already Reported
for Other Acyl Donors
[
amine was obtained if two moles of fatty acid and one mole
of ethanolamine were first reacted at 180 °C to give the
N,O-bis-acylethanolamine, which was then transesterified
with another mole of ethanolamine to form the N-acyl-
ethanolamine [19]. Nevertheless, the reaction conducted at
high temperature resulted in the formation of products with
dark color which influences the product’s quality. The
addition of deodorizers and antioxidants has been sug-
gested to improve the quality of the final products [21]. The
main problems for synthesis of N-acylethanolamines are
the low conversion of reactants and the esterification
reaction occurring in the reaction, which result in low yield
of N-acylethanolamines.
Amidation of Ethanolamine with Vinyl Stearate
at 105 °C in a Solvent-Free System
The procedure of Farris [18] was followed in which a fatty
acid methyl ester was used as the acyl donor. Ethanolamine
(1.1 mmol) was placed in a 10-mL round bottom flask and
then vinyl stearate (1 mmol) was added followed by 0.5%
sodium methoxide (w/w, relative to total reactants). The
mixture was agitated at 105 °C. Samples were withdrawn
after 1 and 12 h of reaction.
Purity of commercial alkanolamides for surfactant pur-
poses was about 80% [22], but with the increasing knowledge
and interest in N-acylethanolamines as lipid mediators in
animals, plants, or humans, high purity N-acylethanolamines
are needed to validate their biological functions in cellular and
animal systems. Currently, this type of study is limited to
small animal experiments due to the lack of access to these
compounds in a desired purity and quantity. For example, the
prices of N-stearoyl and N-palmitoylethanolamines from the
Sigma Chemical Company are $82/5 mg and $60.8/10 mg.
Thus, it is necessary to establish a simple, effective and eco-
nomical synthesis method to support studies examining the
biological and nutritional properties of N-acylethanolamines
in large animal and human subjects.
Amidation of Ethanolamine with Vinyl Stearate
at 45 °C in Hexane
This experiment was carried out based on the procedure of
using a free fatty acid as acyl donor [23]. Ethanolamine
(1 mmol) in hexane (5 mL) was mixed with vinyl stearate
(1 mmol) in a 10-mL round bottom flask in the presence of
Novozym 435 lipase at 10, 20, 30, 40 and 65% (relative to
total reactants) level. The reaction was conducted with
agitation at 45 °C for 5 and 20 h before hexane was
removed under reduced pressure. The enzyme was filtered
out so it could be reused if needed. The experiment was
also conducted under similar conditions except that sodium
methoxide was used at 1, 2 and 3% levels, and the reaction
was at ambient temperature (25 °C) for 14 h.
In the present study, we investigated an alternative
method for the preparation of high purity N-stearoyl and
N-palmitoylethanolamines. Vinyl stearate was used as the
acyl donor to study the reaction conditions and the effects
of catalyst type, solvent and temperature on the purity of
N-stearoylethanolamine. Finally, a solvent-free system and
sodium methoxide catalyst were chosen to synthesize
N-stearoyl and N-palmitoylethanolamines and a large
excess ethanolamine was used as both the reactant and
solvent for the synthesis.
Amidation of Ethanolamine with Vinyl Stearate
at 50 °C in a Solvent-Free System
The procedure of Kolancilar [24], in which laurel oil was
used as the acyl donor, was followed. Vinyl stearate
(1 mmol), ethanolamine (10 mmol), and sodium methox-
ide (2%, relative to total reactants) were placed in a 10-mL
round bottom flask and the mixture was agitated at 50 °C
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J Am Oil Chem Soc
for 2 and 3 h. The reaction mixture was then mixed with
reaction temperature and time. For catalyst concentration
optimization, ethanolamine (20 mmol) and vinyl stearate
or palmitate (1 mmol) were mixed at 60 °C for 1 h with
different concentrations of sodium methoxide (0.5–4%
for the synthesis of N-stearoylethanolamine, 1–4% for
N-palmitoylethanolamine). For optimizing the reactant
ratio, different quantities of ethanolamine (5–25 mmol)
were reacted with 1 mmol fatty acid vinyl ester at 60 °C
for 1 h. Sodium methoxide at 1% was used to optimize the
molar ratio of ethanolamine to vinyl stearate, while 3%
sodium methoxide was used to optimize the molar ratio of
ethanolamine to vinyl palmitate since these catalyst con-
centrations were confirmed to be the optimum values for
the two reactions. After 20:1 molar ratio was confirmed as
the optimal ratio for the synthesis, ethanolamine (20 mmol)
and vinyl stearate or palmitate (1 mmol) were mixed at
different temperatures (40–80 °C) for 1 h with 1 or 3%
sodium methoxide to optimize the reaction temperature.
Finally, ethanolamine (20 mmol) and vinyl stearate
5
mL distilled water at 6 °C for 1 h and the process was
repeated three times to remove excess ethanolamine.
After all the reaction conditions as described above were
examined, a set of conditions was identified to optimize the
synthesis of N-stearoyl and N-palmitoylethanolamines.
Optimization for Amidation Reaction at Mild
Temperature in a Solvent-Free System
The design for the optimization experiments is outlined
in Tables 1, 2 and Fig. 1. The yields of N-stearoyl and
N-palmitoylethanolamines were studied as a function of one
of the four following variables while the other three vari-
ables were held constant: sodium methoxide concentration,
molar ratio of ethanolamine to fatty acid vinyl ester,
Table 1 Experimental design for optimization of amidation between
vinyl stearate and ethanolamine
Level
X
1
(%, relative
to total substrates)
X
ratio)
2
(molar
X
3
(°C)
4
X (h)
Ethanolamine (20 mmol)
and vinyl stearate (1 mmol)
1
2
3
4
5
0.5
1.0
2.0
3.0
4.0
5:1
10:1
15:1
20:1
25:1
40
50
60
70
80
0.5
1.0
2.0
3.0
4.0
Reaction at 80 °C
for 1h with 1%
CH ONa as catalyst
3
X
1
, sodium methoxide, reactions conducted by reacting 20 mmol
ethanolamine with 1 mmol vinyl stearate at 60 °C for 1 h; X , molar
ratio of ethanolamine to vinyl stearate, reactions conducted at 60 °C
for 1 h with 1% sodium methoxide; X , temperature, reactions con-
ducted by reacting 20 mmol ethanolamine with 1 mmol vinyl stearate
for 1 h with 1% sodium methoxide; X , time, reactions conducted by
reacting with 20 mmol ethanolamine with 1 mmol vinyl stearate at
0 °C with 1% sodium methoxide
2
Mixture products including
3
N-stearoylethanolamine
4
Removal of excess
ethanolamine by
water washing or
recrystallization
8
Table 2 Experimental design for optimization of amidation between
vinyl palmitate and ethanolamine
Level
X
1
(% by
substrate)
X
ratio)
2
(molar
X
3
(°C)
4
X (h)
Pure
N-stearoylethanolamine
1
2
3
4
5
6
1.0
2.0
2.5
3.0
3.5
4.0
5:1
10:1
15:1
20:1
25:1
40
50
60
70
80
0.5
1.0
1.5
2.0
2.5
3.0
Preparation of
derivatives with
silylation reagent
X
1
, sodium methoxide, reactions conducted by reacting 20 mmol
ethanolamine with 1 mmol vinyl palmitate at 60 °C for 1 h; X , molar
ratio of ethanolamine to vinyl palmitate, reactions conducted at 60 °C
for 1 h with 3% sodium methoxide; X , temperature, reactions con-
ducted by reacting 20 mmol ethanolamine with 1 mmol vinyl stearate
for 1 h with 3% sodium methoxide; X , time, reactions conducted by
reacting with 20 mmol ethanolamine with 1 mmol vinyl stearate at
0 °C with 1% sodium methoxide
2
GC analysis
3
4
Fig. 1 A general flow chart for the synthesis of N-stearoyl-
ethanolamine
6
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J Am Oil Chem Soc
(
1 mmol) were mixed at 80 °C with 1% sodium methoxide
products on a 20 9 20 cm silica plate (250-lm thickness),
with the standards applied in separate lanes to further
validate the purity of the water-washed final lipid products.
to optimize the reaction time since 80 °C was chosen as the
optimum. The same ratio of reactants was mixed at 60 °C
with 3% sodium methoxide to optimize the reaction time
for the N-palmitoylethanolamine synthesis. For all the
products, excess ethanolamine was removed from the
product by addition of distilled water (5 mL) and crystal-
lization of the amide at 6 °C for 1 h. The N-acylethanol-
amines were washed three more times with water. All
optimization experiments were conducted in duplicate.
NMR Analysis for Structure Confirmation
1
H-NMR qualitative analysis of N-stearoyl and N-palmi-
toylethanolamine products was done by using a Varian
MR-400 Spectrometer (Foster City, CA, USA) with CDCl3
as solvent and TMS as the internal standard (chemical shift
of 0 ppm).
Procedure for Preparing of N-Stearoyl
and N-Palmitoylethanolamines Derivatives
for GC Analysis
Results and Discussion
The anhydrous reaction mixture containing amide (about
Selection of Reaction Parameters According
to the Reported Conditions
5
mg) was placed in a 2-mL glass vial and treated with
pyridine (0.5 mL), hexamethyldisilazane (0.15 mL) and
trimethylchlorosilane (0.05 mL). The mixture was shaken
for 15–30 s and then allowed to stand for 1 h or stored in a
freezer (0 °C) overnight during which time the upper layer
became clear [25]. N-Stearoyl and N-palmitoylethanolam-
ines standards were used as external standards and the
peaks were identified according to the GC retention time.
The purity of the N-acylethanolamines was calculated
based on their peak areas relative to the total peak area of a
particular sample.
The reaction conditions which were used for the synthesis
of N-acylethanolamines in previous studies that used
other acyl donors were tested in this study, and the purity
of N-acylethanolamines in the reaction mixture was
chosen as a parameter to study the reaction conditions.
Since N-stearoylethanolamine has a high melting point
(about 95 °C), amidation reaction between vinyl stearate
and ethanolamine should be conducted at [95 °C in a
solvent-free system or at lower temperature in the pres-
ence of solvent and catalyst, such as a lipase or sodium
methoxide.
Samples were identified and quantified by an HP 5890
Series II capillary GC (Hewlett-Packard, PA) equipped
with
0 m 9 0.25 mm 9 0.25 lm (length 9 ID 9 film thick-
ness) fused silica bonded phase capillary column SP-1
Supelco, Bellefonte, PA). The carrier gas (helium) flow
a
flame ionization detector (FID) using
a
When the amidation reaction was carried out at 105 °C
in the solvent-free system, 67.9 and 73.0% N-stearoyleth-
anolamine was obtained at 1 and 12 h, respectively. The
reaction after 12 h did not result in a dramatic increase in
N-stearoylethanolamine content compared to that at 1 h.
The reaction at 105 °C resulted in the formation of prod-
ucts with a dark color, which influences product quality.
Therefore, the reaction in a solvent system at a lower
temperature was then tested.
3
(
rate was 32.3 mL/min, and the split ratio was seven. The
oven temperature was programmed from 140 to 300 °C at a
rate of 10 °C/min, and then held at 300 °C for 5 min.
Injector and detector temperatures were set at 300 °C.
TLC Chromatography of N-Stearoyl
and N-Palmitoylethanolamine Products
Results for the synthesis of N-stearoylethanolamine in
the solvent system are presented in Table 3. Although the
previous researchers used these reaction conditions to
synthesize N-acylethanolamine, the desired product was
not formed in this study under low concentrations of lipase
when vinyl stearate was used as the acyl donor. Instead, a
A
developing solvent of
A
(toluene:ethyl ace-
tate:ether:acetic acid = 80:10:10:0.2, v/v) and B (100%
methanol) in 80:15 ratio (v/v) were used to separate the
Table 3 Synthesis of
N-stearoylethanolamine in a
solvent system with lipase as
catalyst
Time of reaction (h)
5
20
Lipase (% relative to total substrate) 10
20
0
30
0
40
0
65
0
10
20 30
40
0
65
0
Vinyl stearate (%)
85.4
73.6
0
0
O-stearoylethanolamine (%)
N-stearoylethanolamine (%)
7.2 100 100 100 17.4 5.3
72.3 10.9
100 100 100 26.5
61.6
0
0
0
0
0
0
0
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J Am Oil Chem Soc
side reaction product, O-stearoylethanolamine was pro-
duced in high concentration. When the enzyme addition
was increased to 65%, there was 72.3% N-stearoyletha-
nolamine produced after 5 h. Further addition of enzyme is
not feasible for the experiment. When the enzyme was
substituted with sodium methoxide, no N-stearoylethanol-
amine was produced after 14 h with the catalyst concen-
tration at 1, 2 and 3% (w/w) level (based on total reactants).
Therefore, a reaction using excess ethanolamine as solvent
to dissolve N-stearoylethanolamine was tested in the next
experiment.
When excess ethanolamine (109) was used to act as
both reactant and the solvent to dissolve the N-stearoyl-
ethanolamine product, 92.9% N-stearoylethanolamine was
produced with 2% sodium methoxide at 50 °C after 2 h.
This experiment proved that the N-stearoylethanolamine
can be synthesized by using excess ethanolamine as a
solvent rather than using hexane probably because hexane
diluted the catalyst and the reactants.
From all above preliminary experiments using the
unique vinyl stearate as the acyl donor and various con-
ditions as reported in the literature, we identified the
reaction system and variables to examine in our single
factor optimization experiments.
Optimization of Reaction Conditions for the Synthesis
of N-Stearoyl and N-Palmitoylethanolamine
with Sodium Methoxide Catalyst
Fig. 2 Effect of sodium methoxide concentration on the content of
N-stearoyl and N-palmitoylethanolamine in the final amidation
product
The effects of sodium methoxide concentration, molar ratio
of ethanolamine to fatty acid vinyl ester, temperature, and
time on the amidation reaction were investigated. The
results are shown in Figs. 2, 3, 4, and 5. Data are expressed
as means ± SD.
products at 60 °C. The reason for the slight reduction in the
content of N-acylethanolamine at a molar ratio above 20:1
is unknown. The 20:1 molar ratio was selected as optimal
reaction condition for further reaction optimization.
For sodium methoxide concentration optimization
(
Fig. 2), the content of N-stearoylethanolamine formed
with 1% catalyst was high but it tended to decrease slightly
when the sodium methoxide was increased from 2–4%. An
explanation for the changes is that the increase in the
catalyst concentration may result in the formation of
O-stearoylethanolamine produced by the esterification
reaction (Fig. 6). Spontaneous acyl migration of O-stearoy-
lethanolamine will happen, and thus the reaction typically
proceeds towards amidation [26]. In contrast, 3% sodium
methoxide was optimal for the synthesis of N-palmitoy-
lethanolamine. In general, the amidation reaction is
dominant.
For selecting the best temperature, the reaction tem-
perature was varied from 40 to 80 °C. Results from Fig. 4a
show that the increase in temperature was beneficial to the
reaction. This is probably because of the increase in the
rate of both amidation reaction and acyl migration, which
resulted in more ester being converted to amide. The
reaction at 80 °C resulted in the formation of 95.4%
N-stearoylethanolamine. The color of the product became
dark if the temperature was increased further. Therefore,
80 °C was selected as the optimal temperature for
the synthesis of N-stearoylethanolamine. However, the
response of N-palmitoylethanolamine to temperature was
different from that of the N-stearoylethanolamine, in that
N-palmitoylethanolamine yield did not change as sharply
with the changes in temperature. The content of N-palmi-
toylethanolamine seemed to have reached a maximum at
60 °C (Fig. 4b). The different optimal temperatures for
Responses of N-stearoyl and N-palmitoylethanolamine
to the molar ratio of ethanolamine to vinyl ester were
similar. The content of N-acylethanolamines increased
gradually with the increase in molar ratio from 5:1 to 20:1
(
Fig. 3). Reactions with 5:1 and 10:1 ratio were incomplete
because it was difficult to completely dissolve the final
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J Am Oil Chem Soc
Fig. 3 Effect of molar ratio of ethanolamine to vinyl ester on the
content of N-stearoyl and N-palmitoylethanolamine in the final
amidation product
Fig. 4 Effect of reaction temperature on the content of N-stearoyl
and N-palmitoylethanolamine in the final amidation product
these two N-acylethanolamines may be due to the differ-
ence in the sodium methoxide concentration used.
identified above. There was 95.3% N-stearoylethanolamine
and 96.5% N-palmitoylethanolamine in the final product
after washing with water.
The content of N-stearoylethanolamine after 1 h of the
reaction was significantly higher than other time points
Thin layer chromatography purity confirmation of the
N-acylethanolamine was done to show that there were no
other lipids that may not have been eluted from the GC
column. The Rf values of N-stearoyl and N-palmitoyleth-
anolamine were about 0.49–0.48, and the Rf values of the
corresponding standards were all about 0.48. There was no
other major lipid impurity except some very faint bands of
residual fatty acyl vinyl esters or O-acylethanolamine.
Structural confirmation of N-acylethanolamine was done
(
Fig. 5a). This may be due to the side reactions if the
reaction time was prolonged. In contrast, the reaction time
did not seem to be a significant factor for the synthesis of
N-palmitoylethanolamine. Therefore, 1 and 1.5 h were
considered as the optimal times for the synthesis of
N-stearoyl and N-palmitoylethanolamine.
After all the factors were examined, 1% sodium meth-
oxide, 20:1 molar ratio of ethanolamine to vinyl stearate,
1
8
0 °C and 1 h were chosen for the synthesis of N-stea-
by using H NMR. Nuclear magnetic resonance analysis
roylethanolamine, compared to 3%, 20:1, 60 °C and 1.5 h,
respectively, for the synthesis of N-palmitoylethanolamine.
The purity of N-stearoyl and N-palmitoylethanolamine was
gave the expected amide peak (d 5.9, 1H, –CH
CONH–) and we did not see any NMR peak of –COO
CH CH NH (d 4.2–4.4, 2H) and –COOCH CH NH
(d 1.1–1.5, 2H).
2 2
CH
2
2
2
2
2
2
9
5.7 ± 0.6 and 98.0 ± 0.5%, respectively, under these
optimal conditions.
Traditionally, synthesis of N-acylethanolamines has
been performed with free fatty acids, fatty acid methyl
esters, fatty acid chlorides and triacylglycerols. To the best
of our knowledge, this was the first time a fatty acid vinyl
For a scaled-up synthesis, ethanolamine (600 mmol)
was reacted with vinyl stearate or palmitate (30 mmol)
with agitation under the optimal reaction conditions
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J Am Oil Chem Soc
product was O-acylethanolamine, resulting from an ester-
ification reaction between ethanolamine and the fatty acid
vinyl ester. However, spontaneous acyl migration would
happen if a large amount of O-acylethanolamine were to be
produced [21]. Since the acyl migration step was too fast to
be monitored, the reaction appeared to proceed via direct
amidation when the conditions were suitable [21, 26]. In
our system, the formation of N,O-bis-acylethanolamine
was not favored due to the large excess of ethanolamine
and it was not seen. Therefore, the impurities in the final
product might have included ethanolamine (if not com-
pletely washed away by water), fatty acid vinyl ester and
O-acylethanolamine. Under the optimal reaction condi-
tions, almost all the fatty acid vinyl ester was converted as
verified by GC, while ethanolamine was removed by
washing with water. Thus, the main impurity was O-acy-
lethanolamine, as shown in the GC chromatogram
(Fig. 7b). During the process of product purification, the
non-polar acyl vinyl esters, N-acylethanolamines and
O-acylethanolamine could not be removed by washing and
thus no loss of these compounds could occur. Therefore,
the purity or percentage of N-acylethanolamines in the final
lipid extract as determined by GC could be used as an
indicator to monitor the amidation reaction, as we have
done in this report.
For GC product characterization, the peak at
11.1–11.2 min was attributed to the silylation reagent
Fig. 5 Effect of reaction time on the content of N-stearoyl and
N-palmitoylethanolamine in the final amidation product
based on retention times determined by control injections.
In addition, the area of N-acylethanolamines increased
proportionally to the amount of sample used, whereas the
area of the peak at 11.1–11.2 min remained almost con-
stant even after the sample size was increased from 3 to
12 mg. This observation was also reported by another
researcher [29]. Peaks that had common retention times as
the silylating reagent peak were not included for the purity
calculation.
ester has been used as the acyl donor to synthesize
N-acylethanolamines. The possible reaction routes for
acylation of ethanolamine with vinyl stearate are presented
in Fig. 6. Three reactions might have occurred in the system.
The amidation reaction was dominant and resulted in the
formation of N-acylethanolamines and ethenol which tau-
tomerized to a non-nucleophilic acetaldehyde immediately
As reported in the literature, products with a N-acyle-
thanolamine content ranging from 60 to 90% were
obtained when free fatty acids [1, 23], fatty acid methyl
[27, 28]. Acetaldehyde (boiling point of 20.8 °C) could be
evaporated at ambient temperature. The main side reaction
Fig. 6 Possible reactions
between ethanolamine and vinyl
stearate
Main reaction
:
O
O
CH ONa
3
CH (CH ) C-O-CH=CH2
+
+
NH -CH -CH -OH
CH (CH ) C-NH-(CH ) -OH
+
[CH =CH-OH]
3
2 16
2
2
2
3
2 16
2 2
2
N
-stearoylethanolamine
CH CHO
Side reactions
:
3
O
O
CH ONa
3
CH (CH ) C-O-CH=CH2
NH -CH -CH -OH
CH (CH ) C-O-(CH ) -NH2 + CH CHO
3 2 16 2 2 3
3
2 16
2
2
2
O-stearoylethanolamine
O
O
O
CH ONa
3
2
CH (CH ) C-O-CH=CH2
+
NH -CH -CH -OH
CH (CH ) C-O-(CH ) -NH-C-(CH ) CH3 + 2CH CHO
3 2 16 2 2 2 16 3
3
2 16
2
2
2
N, O-bis-stearoylethanolamine
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J Am Oil Chem Soc
optimal conditions because none of these reactants were
detected by GC.
(
a)
The unique property of our reaction is its irreversibility.
The amidation reaction between the free fatty acids, fatty
acid methyl esters or triacylglycerols and ethanolamine is
reversible. Yield of N-acylethanolamines might be
increased by removal of water, methanol or glycerol under
reduced pressure. However, this method was not desirable
since the ethanolamine would also be evaporated due to its
relatively low boiling point (170 °C) [21]. When a fatty
acid vinyl ester is used as the acyl donor, the amidation
reaction is irreversible because of the tautomerization of
the by-product to a non-nucleophilic acetaldehyde. Acet-
aldehyde has a boiling point of 20.8 °C, so it can be
evaporated easily without the loss of ethanolamine. Thus,
this irreversibility promoted the amidation reaction. In fact,
fatty acid vinyl esters were previously applied to the syn-
thesis of symmetrical diacylglycerol and triacylglycerol
(
b)
[
27, 30], but this was the first time they have been used for
N-acylethanolamine synthesis.
The amount of ethanolamine influenced the solubility of
products, and it perhaps acted as a catalyst [3, 24, 31].
Using ethanolamine as a solvent was economical because
this material is readily available and affordable ($50.4 per
liter from the Sigma Chemical Company).
The objective of optimization for this amidation reaction
was to find a set of reaction conditions to reduce the
esterification and promote the amidation reaction. The
series of reactions we conducted was single-factor reac-
tions. We knew that there might be interactions among
these factors or variables, and a multi-factor response
surface type of experimental design might be used to
generate some mathematical equations or prediction mod-
els. However, a recently published paper [32] discouraged
us from doing so. This work gave basic reaction informa-
tion for possible future modeling and optimization trials on
a large scale.
Fig. 7 GC chromatograms of N-palmitoyl (a) and N-stearoyletha-
nolamine (b). Peaks of fatty acid vinyl ester standards and peaks from
reaction products are superimposed for retention time comparison
esters [18] and triacylglycerols [22] were used as the acyl
donor. A high temperature and long reaction time (100 °C
for 6–12 h) in the absence of a catalyst resulted in unde-
sirable product quality. In a recent study, much milder
reaction conditions were used for the synthesis. Plastina
et al. [23] used a free fatty acid to synthesize N-acyletha-
nolamines at 40 °C for 6–15 h in hexane with lipase
Conclusion
We evaluated four reaction conditions which had been
frequently tested in earlier studies for the synthesis of
N-acylethanolamines. We showed that high temperatures
were not desirable when vinyl stearate was used as the acyl
donor as this might lead to the formation of unwanted
color. No reaction occurred when the reaction was con-
ducted at 45 °C in hexane with sodium methoxide or a low
concentration of lipase. The use of excess ethanolamine as
the solvent proved suitable for the synthesis of N-acyleth-
anolamines. The novelty of this study was that a new route
for the synthesis of N-stearoyl and N-palmitoylethanol-
amines was established, and the reaction was fast and the
(
Novozym 435). However, a high concentration of lipase
was shown to be essential for the reaction, which made the
reaction uneconomical. Their study did not show product
purity, but did report a 80% yield after preparative LC
purification. By comparison, the purity of N-stearoyl and
N-palmitoylethanolamines in the present study was
approximately 96 and 98% after the removal of excess
ethanolamine. We also showed that 100% fatty acid vinyl
ester was converted when the reaction was performed at the
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J Am Oil Chem Soc
conditions were mild. This reaction resulted in the forma-
tion of N-acylethanolamines with high purity ([ 95%) after
simply washing with water. These products could be used
in biological activity evaluations in the future.
14. Okamoto Y, Wang J, Morishita J, Ueda N (2007) Biosynthetic
pathways of the endocannabinoid anandamide. Chem Biodivers
4
:1842–1857
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naling molecules. Bioorg Med Chem Lett 20:5959–5968
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P, Piomelli D (2000) Elevated circulating levels of anandamide
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Pharmacol 408:161–168
Acknowledgments We thank Drs. Linxing Yao and Jose Gerde for
assistance with GC instrumentation from Department of Food Science
and Human Nutrition, Iowa State University. We also acknowledge
the Chinese Government for providing sponsorship for the Ph.D.
graduate student, Xiaosan Wang, who conducted this research.
17. Koutek B, Prestwich GD, Howlett AC, Chin SA, Salehani D,
Akhavan N, Deutsch DG (1994) Inhibitors of arachidonoyl eth-
anolamide hydrolysis. J Biol Chem 269:22937–22940
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8. Farris R (1979) Methyl esters in the fatty acid industry. J Am Oil
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