Pharmaceuticals and Surfactants from Alga-Derived Feedstock: Amidation of Fatty Acids and Their Derivatives with Amino Alcohols

Article abstract of DOI:10.1002/cssc.201500526

Amidation of renewable feedstocks, such as fatty acids, esters, and Chlorella alga based biodiesel, was demonstrated with zeolites and mesoporous materials as catalysts and ethanolamine, alaninol, and leucinol. The last two can be derived from amino acids present in alga. The main products were fatty alkanol amides and the corresponding ester amines, as confirmed by NMR and IR spectroscopy. Thermal amidation of technical-grade oleic acid and stearic acid at 180°C with ethanolamine were non-negligible; both gave 61% conversion. In the amidation of stearic acid with ethanolamine, the conversion over H-Beta-150 was 80% after 3 h, whereas only 63% conversion was achieved for oleic acid; this shows that a microporous catalyst is not suitable for this acid and exhibits a wrinkled conformation. The highest selectivity to stearoyl ethanolamide of 92% was achieved with mildly acidic H-MCM-41 at 70% conversion in 3 h at 180°C. Highly acidic catalysts favored the formation of the ester amine, whereas the amide was obtained with a catalyst that exhibited an optimum acidity. The conversion levels achieved with different fatty acids in the range C12-C18 were similar; this shows that the fatty acid length does not affect the amidation rate. The amidation of methyl palmitate and biodiesel gave low conversions over an acidic catalyst, which suggested that the reaction mechanism in the amidation of esters was different. Pores versus acidity: The structures and properties of zeolites and mesoporous materials are investigated as catalysts for the amidation of renewable feedstocks, such as fatty acids, esters, and Chlorella alga based biodiesel, with ethanolamine, alaninol, and leucinol as nitrogen sources.

Full text of DOI:10.1002/cssc.201500526

DOI: 10.1002/cssc.201500526
Full Papers
Pharmaceuticals and Surfactants from Alga-Derived
Feedstock: Amidation of Fatty Acids and Their Derivatives
with Amino Alcohols
Anastasia Tkacheva,[a] Inkar Dosmagambetova,[a] Yann Chapellier,[a] Päivi Mäki-Arvela,[a]
Imane Hachemi,[a] Risto Savela,[a] Reko Leino,[a] Carolina Viegas,[b] Narendra Kumar,[a]
Kari Eränen,[a] Jarl Hemming,[a] Annika Smeds,[a] and Dmitry Yu. Murzin*[a]
Amidation of renewable feedstocks, such as fatty acids, esters,
and Chlorella alga based biodiesel, was demonstrated with
zeolites and mesoporous materials as catalysts and ethanol-
amine, alaninol, and leucinol. The last two can be derived from
amino acids present in alga. The main products were fatty al-
kanol amides and the corresponding ester amines, as con-
firmed by NMR and IR spectroscopy. Thermal amidation of
technical-grade oleic acid and stearic acid at 1808C with etha-
nolamine were non-negligible; both gave 61% conversion. In
the amidation of stearic acid with ethanolamine, the conver-
sion over H-Beta-150 was 80% after 3 h, whereas only 63%
conversion was achieved for oleic acid; this shows that a micro-
porous catalyst is not suitable for this acid and exhibits a wrin-
kled conformation. The highest selectivity to stearoyl ethanola-
mide of 92% was achieved with mildly acidic H-MCM-41 at
70% conversion in 3 h at 1808C. Highly acidic catalysts favored
the formation of the ester amine, whereas the amide was ob-
tained with a catalyst that exhibited an optimum acidity. The
conversion levels achieved with different fatty acids in the
range C12–C18 were similar; this shows that the fatty acid
length does not affect the amidation rate. The amidation of
methyl palmitate and biodiesel gave low conversions over an
acidic catalyst, which suggested that the reaction mechanism
in the amidation of esters was different.
Introduction
Algae are rich in lipids, proteins, and sugars, and thus, have at-
tracted attention as a potential source of biofuels and chemi-
cals. The utilization of lipids for the production of fuels has
been intensively investigated.[1] On the other hand, the use of
proteins that are abundant in, for example, green alga Chlorella
sp. as a feedstock, for the production of chemicals, has been
scarcely studied.[1] The main components in proteins are amino
acids, which can be catalytically hydrogenated to give the cor-
responding amino alcohols.[2] The latter react with acids to
form alkylamides, which are used in various applications, such
as surfactants[3] and pharmaceuticals.[4–15] Paltmitoyl ethanol
amide (PAE) was already recognized as an anti-inflammatory
agent five decades ago.[4] Later on, PAE was used in several
pharmaceuticals, as summarized in Refs. [5,6]. Various N-acyla-
mides as a nitrogen source, in addition to ethanolamine, in-
cluding leucinol and alaninol, have been synthesized by
a chemical method and used for the treatment of obesity and
related disorders,[7] as well as for treating depression.[8] Apart
from various diseases in which PAE has been reported as a po-
tential drug, such as psoriasis,[9] angina,[10] migraine,[11] Alzheim-
er’s disease,[12] Parkinson’s disease,[13] and cancer,[14] N-acyla-
mides have also been used as surfactants and in cosmetics.[3,15]
Lauryl ethanolamide is of particular interest, since it is not
toxic nor mutagenic.[15] In addition, stearyl ethanolamide,
oleoyl ethanolamide, and palmityl ethanolamide are used as
foam boosters.[16]
Fatty acid alkanol amides occur in nature in blue mussels[17]
and in the seeds of legumes.[18] For example, the extraction of
N-acylethanolamides from the flesh of the green-lipped
mussel, Perna canaliculus, is performed by using chloroform
and methanol as solvents,[17] which are not environmentally
benign. Fatty acid alkanol amides are conventionally synthe-
sized by using activated acids, that is, fatty acid chlorides[19,20]
as raw materials. The chlorides can easily react with, for exam-
ple, aminocarboxylic acids in the presence of sodium hydrox-
ide, resulting in N-fatty acyl amino acid sodium salts, which are
active surfactants. Furthermore, several methods to produce
fatty acid amides by using homogeneous catalysts and amines
have been reported and recently reviewed by Lundberg
et al.[19] Thermal amidation of free fatty acids with amino alco-
hols was also demonstrated by Patil et al.[21] In their work, the
yield of amide was 74% after 10 h in the absence of any sol-
vent at 1408C.
[a] A. Tkacheva, I. Dosmagambetova, Y. Chapellier, Dr. P. Mäki-Arvela,
I. Hachemi, Dr. R. Savela, Prof. Dr. R. Leino, Dr. N. Kumar, Dr. K. Eränen,
J. Hemming, Dr. A. Smeds, Prof. Dr. D. Yu. Murzin
Johan Gadolin Process Chemistry Centre
Šbo Akademi University, 20500 Turku (Finland)
E-mail: dmurzin@abo.fi
[b] C. Viegas
Federal University of Rio de Janeiro
Department of Chemical Engineering, School of Chemistry
Rio de Janeiro (Brazil)
Supporting Information for this article is available on the WWW under
http://dx.doi.org/10.1002/cssc.201500526.
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Figure 1. a) The reaction scheme for the production of oleoyl ethanol amide (3) from oleic acid (1) and ethanol amide (2), as well as N,O-dioleoyl ethanola-
mine (5) and water (4). Structures of b) alaninol and c) leucinol.
The application of heterogeneous catalysts makes the pro-
cess more environmentally friendly, and it would be desirable
to decrease the amount of waste produced, especially com-
pared with stoichiometric chemical synthesis. The aim is thus
to decrease the E factor, which is defined as one kilogram of
waste produced per kilogram of product.[22] Catalytic transfor-
mations of free fatty acids or their esters are of practical inter-
est, since these production methods are more environmentally
benign compared with conventional ones. To the best of our
knowledge, there are only a few publications in the literature
on catalytic amidation of fatty acids to give K-alkyl amides
(Figure 1).[23,24] Fatty acid amides can also be prepared in the
presence of enzymatic[25–27] or solid acid catalysts.[23,24] The en-
zymatic synthesis of amides is typically performed at 50–
708C,[25] whereas zeolites need a higher temperature (120–
1808C).[25] Musteata et al.[23] described direct amidation of oleic
acid with ethanolamine at 1208C in octane as a solvent by
using ZSM-5 and UL-MFI with a mesoporous structure as cata-
lysts to give, after 4 h reaction, amine and ester by-products
yields of 35 and 26%, respectively. Furthermore, the in situ
amidation of algae, giving 95% yield of fatty acid alkyl amides
during 90 min at 1208C with sulfuric acid as a catalyst, was re-
ported by Mudiyanselage et al.[24] Alternatively, a two-step
method was applied, in which fatty acid methyl ester (FAME)
was prepared in the first step by using Amberlyst-15 as a cata-
lyst at 908C for 6 h. Subsequent phase separation and amida-
tion of FAME at 1208C for 2 h with sodium methoxide as a cata-
lyst resulted in more than 95% yield of fatty acid alkyl
amides.[24]
As catalysts, two different types of zeolites, H-Beta and ZSM-
5, were investigated, with varying SiO2/Al2O3 ratios, H-Beta-22,
H-Beta-150, H-Beta-300, and H-ZSM-5-23, together with meso-
porous materials H-MCM-41 and H-MCM-36. In zeolites, the
number denotes the silica to alumina ratio used. Catalyst selec-
tion was based on the acidity and varying pore sizes, that is,
medium pore size zeolite H-ZSM-5, large pore zeolite H-Beta,
as well as mesoporous materials were investigated in the ami-
dation of fatty acids. ZSM-5 zeolite belongs to the pentasil
group of high-silica zeolites and has three-dimensional pore
systems, with 10-membered rings, straight channels with di-
mensions of 0.52”0.57 nm, and sinusoidal channels with di-
mensions of 0.53”0.56 nm. The ZSM-5 zeolite has a channel
intersection of 0.9 nm.[28,29] The structure of Beta zeolite has
three-dimensional pore systems, with 12-membered rings, c-di-
rection channels with dimensions of 0.76”0.64 nm, and two
12-ring channels perpendicular to the c direction with pores of
0.76”0.64 and 0.55”0.55 nm.[28–30] MCM-22 is composed of 10-
membered rings containing cavities of 0.71 nm.[31] Pillared H-
MCM-36 was prepared from MCM-22, according to a procedure
reported in the literature.[32] The cavity sizes of H-MCM-41 and
H-MCM-36 are 2.0–3.0[33] and 2.5 nm,[34] respectively. One im-
portant structural difference in these zeolites is the smaller
pore size of ZSM-5 (0.57 nm) compared with that of H-Beta
(0.76 nm). In particular, in the synthesis of large organic prod-
ucts, pores that are too small can retard the reaction. A com-
parative experiment with lipase as a catalyst was also per-
formed to compare the product formed qualitatively. In the
case of FAMEs, basic CeO2 was also used as a catalyst. In addi-
tion, in some cases, different reactor operations, initial reactant
concentrations, and reaction temperatures were applied.
A comparison of the batch operation mode with reactive distil-
lation was made to study the effect of water, since water as
a reaction product can inhibit the reaction, and thus, limit con-
version of the fatty acid. Preliminary experiments were per-
formed by using the batch operation. Purging mode, involving
opening of the reactor for a short time periodically to evapo-
rate hexane and part of the formed water, was also applied. Al-
ternatively, the reactive distillation method was used to shift
the equilibrium towards the amide. To the best of our knowl-
Herein, we aim to demonstrate the catalytic amidation of
fatty acids and esters with amino alcohols, in particular, FAME
prepared by in situ transesterification of Chlorella alga with sul-
furic acid as a catalyst. Chlorella alga, in addition to containing
lipids, is also rich in proteins, carbohydrates, and sugars.[1] To
improve the utilization of different fractions in Chlorella alga,
one method could be to utilize its lipids and amino acids for
the synthesis of fatty acid alkyl amides. The following feed-
stocks were used: stearic, oleic, linoleic, palmitic, and lauric
acids; methyl palmitate; and Chlorella-derived FAME by using
ethanolamine, alaninol, or leucinol as nitrogen sources.
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edge, direct amidation of fatty acids with alaninol as a nitrogen
source or amidation of Chlorella-based FAME with zeolites as
catalysts have not been reported previously.
(416 mmolgcat.À1) and the smallest amount was determined for
H-Beta-300 (54 mmolgcat.À1) zeolite catalyst.
The variation in the SiO2/Al2O3 ratios of H-Beta zeolite cata-
lysts, H-Beta-22, H-Beta-150, and H-Beta-300, exhibited differ-
ences in the Brønsted and Lewis acid sites (Table 1). The H-
Beta-150 zeolite catalyst exhibited the largest amount of
Brønsted acid sites (217 mmolgcat.À1).[39] The largest amount
Results and Discussion
Catalyst characterization results
of Lewis acid sites was obtained for H-Beta-150 zeolite
À1
catalyst (125 mmolgcat.
(61 mmolgcat.À1).
)
followed by H-MCM-36-P22-28
Four different zeolites, together with mesoporous MCM-41 and
MCM-36, were used as catalysts in the amidation of fatty acids
and their esters. In addition, lipase was used for qualitative
comparison. The structures of two different types of zeolites,
Beta and ZSM-5, were confirmed by XRD.[35–39] The XRD pattern
of pillared Na-MCM-36-28 resembled that of Na-MCM-22.[38]
Analogously, the XRD pattern for MCM-41 was similar to that
reported in Ref. [40]. Characterization results for the zeolites,
including nitrogen and pyridine adsorption methods, are pre-
sented in Table 1.
Characterization of the morphology of Na-MCM-41, Na-
MCM-36, H-Beta-300, H-Beta-150, H-Beta-22, and H-ZSM-5-23
was carried out by using SEM. The Na-MCM-36 mesoporous
material exhibited platelet-shaped crystals (Figure 2).
Characterization of amino acids in fresh Chlorella alga
Chlorella alga contains large amounts of crude protein. Amino
acid analysis[42] revealed that the main amino acids in Chlorella
were glutamic acid (11.8%), aspartic acid (9.0%), leucine
(8.2%), arginine (8.2%), alanine (8.2%), lysine (7.6%), and gly-
cine (6.6%). Hydrogenated aminoalcohol counterparts, namely,
ethanolamine from glycine, ala-
The specific surface areas of H-Beta-22, H-Beta-150, and H-
Beta-300 zeolites were similar (Table 1). However, the H-Beta
zeolite catalysts exhibited higher surface areas than the
medium pore H-ZSM-5-23 zeolite catalyst (443 m2 gÀ1). The H-
ninol from alanine, and leucinol
Table 1. Catalyst characterization results from nitrogen adsorption and pyridine adsorption/desorption. Pyri-
dine was desorbed at 250, 350, and 4508C; these temperatures denote the sum of weak, medium, and strongly
bound sites; the sum of medium and strongly bound acid sites; and the sum of strongly bound acid sites, re-
spectively.
from leucine, were selected for
amidation experiments.
À1
À1
Catalyst
Specific surface area Brønsted acid sites [molgcat.
]
Lewis acid sites [molgcat.
]
Ref.
Results from the amidation ex-
periments
À1
[m2 gcat.
]
2508C
3508C
4508C
2508C
3508C
4508C
H-MCM-41
944
513
305
686
546
443
26
87
54
167
217
416
11
70
49
143
178
395
3
44
23
72
102
284
40
61
28
20
32
9
12
6
4
[38]
[32]
[35]
[37]
[39]
[35]
H-MCM-36-P22-28
H-Beta-300
H-Beta-22
H-Beta-150
H-ZSM-5-23
Several different screening ex-
periments were performed for
the amidation of fatty acids,
their esters, and algae-based bi-
odiesel (see Tables 2–6, below).
The first task was to confirm
and identify the reaction prod-
ucts, followed by investigation
33
125
36
26
63
8
6
30
3
MCM-41 mesoporous catalyst exhibited a higher surface area
than that of the H-MCM-36 mesoporous catalyst. The explana-
tion for the higher surface area of H-MCM-41 than that of H-
MCM-36 can be attributed to structural differences and the
presence of micropores in H-MCM-41. The specific surface area
À1 [41]
of CeO2 was 136 m2 gcat.
.
The acidity of the zeolites determined by pyridine desorp-
tion, which showed that the Brønsted acidity of H-ZSM-5 was
2.3-fold that of H-Beta-150 and the concentration of Brønsted
acid sites for H-Beta-22 was also quite high, whereas H-Beta-
300 exhibited a very low concentration of Brønsted acid sites.
The difference in the amounts of Lewis acid sites in the four
zeolites was much lower. The highest Lewis acid site concen-
tration of only 61 mmolgcat.À1 was observed for H-MCM-36-28.
Determination of the acid sites for H-MCM-41, H-MCM-36, H-
Beta-300, H-Beta-22, H-Beta-150, and H-ZSM-5-23 zeolite cata-
lysts revealed the presence of both Brønsted and Lewis acid
sites. The largest amount of Brønsted acid sites among the
zeolite
catalysts
was
determined
for
H-ZSM-5-23
Figure 2. SEM image of Na-MCM-36.
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of the reaction conditions and reactor operation mode, togeth- reaction rate, different reactor operation modes were tested
er with varying the reaction conditions, catalysts, and feed.
together with analysis of water in the final product. The inten-
tion was to determine whether the presence of water had an
effect on conversion.
Reaction products in the amidation of stearic acid with etha-
nolamine
In the amidation of stearic and oleic acid with ethanolamine
over H-Beta-22 by reactive distillation, periodic purging of the
reactor seven times as well as a closed batch-reactor operation
were applied to study the effect of water on conversion and
product distribution. The comparative results for these experi-
ments are shown in Table 2. During reactive distillation, more
than 90% conversion of stearic acid was achieved in 3 h with
90% selectivity towards the
In the amidation of stearic acid with ethanolamine in the pres-
ence of H-Beta-22 at 1808C, the conversion of stearic acid after
3 h was 69% (Table 2; in purging mode, when periodically re-
moving a total of 1.4 g of the reaction mixture from the reac-
tor). The two main reaction products eluted from a short GC
main desired product, 2-steara-
Table 2. Amidation of stearic acid with H-Beta-22 at 1808C by using either batch operation or purging mode.
The initial concentration of fatty acid was 0.72m with an equimolar amount of ethanolamine. The catalyst
amount was 0.5 g.
midoethanol. Part of the solid
product, however, sublimated
inside the column and thus this
operation mode was not further
pursued. When periodic purging
mode of the reactor with H-
Beta-22 was applied, a slightly
lower conversion, 69%, com-
pared with the case of reactive
distillation was obtained. On the
other hand, in a batch reactor,
the conversion was only 56%. In
Reactor operation
mode
Conversion
[mol%]
Selectivity [mol%]
Water content
[a]
amide
ester amide
other products
[molLÀ1
] ([%])
batch
purging
purging[c]
56
69
74
78
85
50
20
13
4
2
2
46
0.035–0.0004 (9%)[b]
0.110–0.0011 (14%)[b]
n.d.
[a] Determined by KF titration. [b] From the theoretical amount of water. [c] Butylene oxide was added as
a water trap.
column at 14.6 and 22.3 min, respectively, with the corre- the final product achieved from batch-mode reactor operation,
sponding selectivities of 85 and 13%, respectively. The final the amount of water determined by Karl Fischer titration corre-
product mixture after purification, according to the procedure sponded to only 9% of the theoretical amount of water
reported by Fernandez-Perez and Otero,[25] contained 2-steara- formed, according to calculations based on conversion
midoethanol. The other product was 2-stearamidoethyl stea- (Table 2), whereas 3.1-fold more water was present in the prod-
rate. The current results are in agreement with the analogous uct obtained in the purging-mode experiment. This result
products formed by lipase-catalyzed amidation of acids with shows that a large part of water has already evaporated from
ethanolamine,[25] for which the structures of the products were the product in both cases and that even a slightly higher
also confirmed by IR and NMR spectroscopy. On the other amount of water in the latter did not decrease the conversion.
hand, lipase-catalyzed amidation of oleic acid with ethanol- When an equimolar amount of a chemical water trap, 1,2-buty-
amine only resulted in the formation of amide, even if pres- leneoxide, was also applied in a separate experiment, the con-
ence of aminoester was also speculated.[26] In their work, the version level increased substantially to 74%. The selectivity,
only analytical method used was GC with a long capillary however, towards amide was lower, at only 50%; this was
column. The same column was also tested in our work; howev- probably due to a consecutive reaction of the formed butane-
er, according to our results, 2-stearamidoethyl stearate was not diol with fatty acids. These results clearly show that the pres-
eluted from the long capillary column, which could undermine ence of water retards the reversible amidation reaction and re-
the results reported by Wang et al.[26] Zeolite-catalyzed amida- moval of water increases the conversion, analogously to the re-
tion of oleic acid was investigated by Musteata et al.[23] and sults reported by Tufvesson et al.,[46] at the same time, decreas-
gave the fatty acid amide and amino ester as the main prod- ing the consecutive formation of ester amide.
ucts, according to HPLC results without any further confirma-
tion. It was also stated by Tufvesson et al.[46] that, if the ester
Comparison of thermal and catalytic amidation of oleic acid
amine formed, it would spontaneously react further to give
the corresponding alkylamide, and further to give the N,O- Thermal amidation of oleic acid at 1808C resulted in 61% con-
diacyl derivative of ethanolamine; thus indirectly confirming version in 3 h (Table 3 and Figure 3). This conversion is relative-
that the main products should be the fatty acid amide and ly high, but can be explained by the fact that oleic acid itself
N,O-diacyl derivative of ethanolamine.
acts also as an acid catalyst. The reproducibility test showed
that experimental error was less than 2% (Figure 3). Selectivity
towards the corresponding amide was 91%. In addition, small
amounts of the N,O-diacyl derivative of the ester amine (ester
amide) were formed. Thermal amidation of oleic acid was dem-
Amidation with different reactor operation modes and the
addition of a chemical water trap
The amidation of fatty acids is a reversible reaction, in which onstrated previously by Patil et al.,[21] who used a minor molar
the formation of water can retard the reaction. To enhance the excess of oleic acid relative to ethanolamine (0.166:0.1) at
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Amidation of different feedstock
Table 3. Amidation of technical-grade oleic acid with and without cata-
lyst at 180 and 1208C.[a]
Amidation of several fatty acids, namely, stearic, linoleic, pal-
mitic, and lauric acid, was performed with ethanolamine as a ni-
trogen source (Table 4). Alaninol and leucinol were also used
for amidation of technical-grade oleic acid (Table 5) as well as
FAMEs, for example, methylpalmitate and biodiesel derived
from Chlorella alga (Table 5).
Catalyst
T
Conversion
Selectivity [mol%]
amide ester amide other products
[8C] [mol%]
no catalyst 180
H-Beta-22
H-Beta-22
61
64
22
91
90
>99
9
6
0
4
0
180
120
traces
[a] The initial concentration of fatty acid was 0.72m and an equimolar
amount of ethanolamine was used. The amount of catalyst used was
0.5 g.
A comparison of the conversion levels in the amidation of
different substrates with H-Beta-150 as a catalyst showed that
these conversion levels were very close to each other (Table 4),
except for technical-grade linoleic acid, which exhibited
a slightly lower conversion. Preliminary results, based on a very
limited set of data, reveal that the chain length does not have
a large effect on the amidation rate. Technical-grade linoleic
acid, however, can contain some impurities; thus retarding the
reaction rate. Selectivity towards the desired amide was typi-
cally about 87% and the other main product was the corre-
sponding ester amine.
Amidation of methylpalmitate was restricted to only 17%
conversion after 3 h of reaction over H-Beta-22 (Table 6). An in
situ hydrolysis–amidation experiment was also performed to
study the feasibility of this two-step approach. Due to the
presence of solid methyl palmitate, together with an equimolar
amount of ethanolamine and water, it was not, however, tech-
nically feasible to rapidly hydrolyze methylpalmitate during the
experiment. In the acid-catalyzed amidation of fatty acids, it
has been confirmed that the intermediate is an ammonium
carboxylate salt.[48,49] During amidation of an ester, methanol is
formed as a byproduct instead of water, and thus, the reaction
Figure 3. Reproducibility test for the thermal amidation of technical-grade
oleic acid with an equimolar amount of ethanolamine at 1808C. The initial
concentration of oleic acid was 0.72m and the solvent was anhydrous
hexane.
Table 4. Amidation of different fatty acids with ethanolamine by using H-Beta-150 as
a catalyst at 1808C.[a]
1408C to give 74% yield of the amide in addition to
6.5% ester amide after 10 h. The initial concentration
of oleic acid was 0.58m in the study by Patil et al.[21]
These results are not directly comparative with those
reported herein due to the use of technical-grade
oleic acid as a reactant and hexane as a solvent in
the current case, along with different reaction tem-
peratures, times, and different initial concentrations
of reactants. Nevertheless, qualitatively there is
agreement between this work and that reported in
the literature. It could also be expected that the reac-
tion would be faster at a higher temperature, since
water is ionized at high temperature and the pKw
Fatty acid
Conversion
[mol%]
Selectivity [mol%]
amide
ester amide
other products
stearic acid
80
61
63
73
73
85
89
95
99
94
6
9
3
0
3
9
2
2
1
3
technical-grade linoleic acid
technical-grade oleic acid
palmitic acid
lauric acid
[a] The initial concentration of fatty acid was 0.72m and an equimolar amount of etha-
nolamine was used. The amount of catalyst used was 0.5 g.
value for water decreases with increasing temperature; the
pKw is about 11.44 at 1758C.[47] Water thus partially acts as
a Brønsted acid catalyst. The conversion of oleic acid was only
22% after 3 h at 1208C over H-Beta-22 as a catalyst. The main
product was the amide with more than 99% selectivity, where-
as no ester amide was detected. This result is different from
that achieved by Musteata et al.,[23] who reported that an ester
amine was also formed in addition to the amine over ZSM-5-
20 as a catalyst in octane as a solvent.
Table 5. Amidation of methyl palmitate with different catalysts at
1808C.[a]
Catalyst
Conversion
[mol%]
Selectivity [mol%]
ester amide other products
amide
H-Beta-22
H-Beta-22 (H2O)
CeO2
biodiesel[b]
17
8
20
28
>99
>99
>99
44
0
0
0
0
0
0
0
54
[a] The initial concentration of fatty acid was 0.72m and an equimolar
amount of ethanolamine was used. The amount of catalyst used was
0.5 g. [b] Biodiesel with an initial concentration of 0.23m, 96 mg of cata-
lyst, and a reaction time of 6 h.
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Low conversion of about 49% within 3 h was achieved in
the amidation of technical-grade oleic acid with alaninol. The
corresponding thermal amidation experiment was not, howev-
er, performed to analyze the catalytic effect of H-Beta-150. The
main products were N-(2-hydroxy-1-methylethyl)-9Z-octadec-
amide (Figure S3 in the Supporting Information) and 9Z-octa-
decanoic acid-2-{[(9Z)-1-oxo-9-octadecenyl]amino}propyl ester
(Figure S4 in the Supporting Information). The formation of the
Table 6. Amidation of technical-grade oleic acid with different aminoal-
cohols by using H-Beta-150 as a catalyst at 1808C, unless otherwise
stated.
Aminoalcohol
Conversion
[mol%]
Selectivity [mol%]
amide
ester amide
other products
ethanolamine
alaninol
leucinol
63
49
59
95
92
93
3
4
2
2
4
5
1
amide was confirmed by H NMR spectroscopy; data matched
[a] The initial concentration of fatty acid was 0.72m and an equimolar
amount of ethanolamine was used. The amount of catalyst used was
0.5 g.
that reported in the literature.[54] The ester amide, 9Z-octadeca-
noic acid-2-{[(9Z)-1-oxo-9-octadecenyl]amino}propyl ester is
a colorless oil and the NMR spectroscopy results match those
previously published.[55]
mechanism of amidation is not the same as that for the amida-
tion of fatty acids. In addition, methanol is less acidic than
water at 1808C; thus making the reaction mixture less acidic
than in the case of fatty acid amidation.
Amidation of technical-grade oleic acid with leucinol result-
ed in 59% conversion in 3 h (Table 6) and the formation of two
main products, which were identified as N-(1-hydroxymethyl-3-
methylbutyl)-9Z-ocatadecamide (Figure S5 in the Supporting
Information), as confirmed by 1H and 13C NMR spectroscopy,
and the corresponding ester amide (Figure S6 in the Support-
ing Information), as confirmed by NMR spectroscopy data. The
COSY, HSQC, and HMBC plots of stearoyl leucinolamide and
the ester amide are shown in Figures S7 and S8 in the Support-
ing Information, respectively. It can be generally stated that, in
small-ampoule experiments with lipase and pure oleic acid in
hexane at reflux, the main products were the same as those
obtained with zeolites.
There are literature reports describing that esters have also
been transformed into amides in the presence of homo-[50] and
heterogeneous base catalysts.[51] For example, as a heterogene-
ous catalyst, CaO was applied in the amidation of soybean
oil.[51] It is, however, known that CaO is prone to leaching at
high temperature in the presence of fatty acids.[52] In the case
of leaching, fatty acids can be saponified; thus leading to ho-
mogeneous catalysis. When water is present in the case of
sodium methoxide as a catalyst, fatty acids can be also saponi-
fied.[51] To avoid such saponification and eventual involvement
of homogeneous catalysis, herein CeO2 was used as a solid
base catalyst. Its performance was not, however, much better
than that of H-Beta-22 (Table 5).
The abovementioned results achieved with H-Beta-150
showed that the rate of acylation of these three aminoalcohols
decreased in the following order: ethanolamine>leucinol>
alaninol. It was anticipated that acylation of ethanolamine with
oleic acid would proceed faster than that of the other aminoal-
cohols due to its smaller size. An interesting result showing
that leucinol is acylated faster than alaninol, which is not
straightforward to explain. The methyl group close the amino
group in alaninol probably results in greater steric hindrance
than that of the methylpropyl group (Figure S9 in the Support-
ing Information). In this case, the amino group has more free
space and is able to adsorb on the acid sites. Selectivities to
the desired product amide were above 80% in all cases.
Amidation of Chlorella-based purified biodiesel was per-
formed over H-Beta-22 catalyst at 1808C by using the batch re-
actor mode. Due to a limited availability of alga-derived biodie-
sel, a lower initial FAME concentration of 0.23m was used; this
was 32% of the concentrations applied for the amidation of
other feedstocks. Conversion of biodiesel was only 28% after
6 h of reaction; this was probably due to a low initial concen-
tration of the substrate as well as presence of catalyst poisons
in the feedstock, such as sulfur (46 mgkgÀ1, as determined by
inductively coupled plasma (ICP)). The main products in the
amidation of biodiesel were amides of unsaturated C18 esters.
A comparative study was made for the amidation of techni-
cal-grade oleic acid by using alaninol and leucinol as nitrogen
sources (Table 6) with H-Beta-150 as a catalyst. In the case of
ethanolamine, the catalyst applied was H-Beta-22, which exhib-
ited slightly fewer Brønsted acid sites than H-Beta-150.
Amidation of stearic acid with ethanolamine by using differ-
ent zeolites and mesoporous materials as catalysts
The performance of different types of zeolite catalysts, namely,
three H-Beta zeolites with different SiO2/Al2O3 ratios and
a highly acidic medium pore zeolite H-ZSM-5, was compared
for the amidation of stearic acid with ethanolamine after 3 h of
reaction time (Table 7). In addition, two mesoporous materials,
MCM-36 and MCM-41, were tested. The catalytic results were
compared with the thermal amidation of stearic acid with
ethanolamine under the same conditions to give 61% conver-
sion in 3 h. The highest conversion of stearic acid was achieved
with relatively acidic H-Beta-150 (Table 7): 80% after 3 h, which
shows that this catalyst is very efficient in the amidation of
stearic acid. An interesting result is found for MCM-41 and
MCM-36, namely, these catalysts are quite active, especially H-
MCM-41, although it exhibits the lowest amount of acid sites
Amidation of technical-grade oleic acid with ethanolamine
resulted in 63% conversion after 3 h (Table 6). Comparing this
result with the thermal amidation of technical-grade oleic acid
with ethanolamine (Table 3), it can be observed that H-Beta-
150 exhibits only a minor catalytic effect; this is probably due
to its wrinkled conformation with a torsion angle of 1208.[53]
Thus it can be concluded that, in the case of the amidation of
oleic acid with ethanolamine, the pore size of Beta zeolite is
not adequate and a more suitable catalyst, for example, a mes-
oporous material, should be found for the amidation of oleic
acid.
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Analogously to mesoporous materials,
a similar
Table 7. Amidation of stearic acid with ethanolamine by using different catalysts at
1808C.[a]
trend was observed for microporous zeolites H-
MCM-22 and H-Beta-150, which showed that three-
fold more ester amide formed over the strongly
acidic catalyst H-Beta-22 than that over H-Beta-150.
In the case of H-ZSM-5, the formation of ester amide
is probably partially inhibited by its smaller pore size
than that of H-Beta as well as a lower conversion.
These single-point analyses give an indication that
mesoporous materials and large-pore zeolites with
an optimum acidity are potential catalysts for the
amidation of saturated fatty acids. If the catalyst
acidity was too high, both catalyst deactivation and
a decrease in the selectivity towards the amide were
observed.
Catalyst
Conversion
[mol%]
Selectivity [mol%]
amide ester amide other products ester amide/amide
Ratio
no catalyst
H-MCM-41
H-MCM-36
H-Beta-300
H-Beta-300[b]
H-Beta-22
H-Beta-150
H-ZSM-5
61
70
66
38
42
69
80
67
80
91.5
79
95
98
83
85
88
13
3
14
0
0
15
6
7
5.5
7
5
2
2
9
13
0.08
0.06
0.20
0
0
0.18
0.06
0.10
9
[a] The initial concentration of fatty acid was 0.72m and an equimolar amount of etha-
nolamine was used. The amount of catalyst used was 0.5 g. [b] Double the amount of
catalyst was used.
Conclusions
(Table 1). Thus, it can be concluded that, not only the amount
of Brønsted acid sites, but also the pore size, are decisive for
determining the activity of zeolites and mesoporous materials
in the amidation of acids. The difference in conversion was
minor for H-MCM-41, H-MCM-36, and H-Beta-22. Very low con-
version was obtained in the case of H-Beta-300 for the amida-
tion of stearic acid, even lower than that recorded in the ther-
mal amidation of stearic oleic acid (Table 7), which shows that
H-Beta-300 did not act as a catalyst. An additional experiment
with a twofold amount of H-Beta-300, namely, 1 g, was per-
formed to further study its catalytic effect. The results revealed
that with 1 g of H-Beta-300 the conversion was only slightly
higher than that with 0.5 g of catalyst (Table 7). This unexpect-
ed result can be interpreted by the fact that the acid sites in
H-Beta-300 are probably blocked with strongly adsorbed stea-
ric acid, leading to a decreased conversion level. This result
can be affected by two factors, namely, the larger pore size
and lower acidity of H-Beta-300 relative to that of H-ZSM-5.
Relatively low conversion, 67%, was also achieved with H-
ZSM-5, which exhibited the smallest pores of all studied cata-
lysts. On the other hand, this catalyst was very acidic, leading
to more profound deactivation with organic coke. The conver-
sion of stearic acid was 21% higher with H-ZSM-5 than that
with H-Beta-300. The results indicated that even very mildly
acidic mesoporous materials were able to catalyze the amida-
tion of fatty acids, whereas large-pore zeolite H-Beta-300,
which possessed mild acidity, was not active.
The utilization of proteins and lipids in alga as renewable feed-
stocks for the synthesis of pharmaceuticals and surfactants is
of great interest. The amidation of fatty acids and aminoalco-
hols over zeolites and mesoporous materials as catalysts has
scarcely been investigated, to date. Aminoalcohols can be de-
rived from the catalytic hydrogenation of amino acids, which
are abundant in, for example, Chlorella alga. The purified bio-
diesel synthesized by in situ transesterification of Chlorella alga
with sulfuric acid and methanol at 608C was converted into
the corresponding amides over zeolitic catalysts by using etha-
nolamine as a nitrogen source. In addition, several fatty acids
and aminoalcohols were transformed into amides over zeolites
and mesoporous materials as catalysts at 120–1808C.
The main products in the amidation of fatty acids were fatty
alkanolamides and ester amines, as confirmed by NMR and IR
spectroscopy. Typically, a high selectivity, above 75%, to the
desired amide was achieved over active, large-pore zeolites
and mesoporous catalysts. The product distribution was analo-
gous to that obtained with the enzyme lipase as a catalyst in
hexane at reflux.
Two types of materials, H-Beta and H-ZSM-5, which repre-
sented large and medium pore size zeolites, respectively, were
selected as amidation catalysts. The SiO2 to Al2O3 ratio of H-
Beta was also varied to study the effect of catalyst acidity. In
addition to zeolites, mesoporous materials MCM-41 and MCM-
36 were used as catalysts. The catalysts were characterized by
different methods, including XRD, SEM, nitrogen physisorption,
and FTIR spectroscopy of pyridine adsorption.
Typically amidation of fatty acids is very selective towards
the desired fatty alkanol amide (Table 7). In the case of active
catalysts, selectivities towards the amide of more than 73%
were achieved. The highest selectivity of 87% towards the
amide at a conversion of 76% was obtained with the most
active catalyst, H-Beta-150. The second main product was the
corresponding ester amide, although very small amounts of
other, unknown products were obtained. When comparing the
product distribution obtained over different catalysts after 3 h
at 1808C, the ratio between the molar yields of ester amide to
amide showed that more ester amide was formed with meso-
porous H-MCM-36, which also contained 14.7-fold more strong
Brønsted acid sites compared with its counterpart H-MCM-41.
The amidation results revealed that higher conversion in the
reaction between stearic acid and ethanolamine over H-Beta-
22 was achieved. When the reactor was purged 7 times during
3 h of reaction, a total of 1.4 g of the reaction mixture was re-
leased. This mode of operation was required to remove water,
since the reaction was limited by thermodynamics in classical
batch operation.
Thermal amidation of stearic and oleic acid with ethanola-
mine resulted in 61% conversion at 1808C in 3 h. For oleic
acid, this conversion level was only slightly lower than that
achieved with H-Beta-150, and it was concluded that the cata-
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cations. Analogously to Na-MCM-22, Na-MCM-41 was transformed
lytic effect of H-Beta-150 was very minor for the amidation of
oleic acid, which had a wrinkled conformation. On the other
hand, the catalytic effect of H-Beta-150 was much higher in
the amidation of linear stearic acid, giving 80% conversion in
3 h.
into the ammonium form and calcined at 5008C.[37]
For comparison, an immobilized enzyme catalyst, acrylic resin
based lipase from Candida antarctica (Sigma) was used. In addition,
commercially available CeO2 (CeIV oxide, <25 nm, Aldrich) was
used as a catalyst.
Amidation of methyl palmitate and biodiesel from Chlorella
alga gave quite low conversion levels. This result could be ex-
plained by the fact that the reaction intermediates were differ-
ent in the case of fatty acid esters than those with fatty acids,
and thus, the reaction was not catalyzed by acid sites as easily
as for the fatty acids. A base-catalyzed amidation of methyl
palmitate with CeO2 gave, however, only 20% conversion after
3 h. One reason for the low yield of amide in biodiesel amida-
tion was the presence of small amounts of sulfur.
Catalyst characterization methods
Nitrogen adsorption measurements were performed with a Sorpto-
matic 1900 instrument. Prior to measurements, catalysts were out-
gassed at 1508C for 3 h. The specific surface area was calculated
by using the Dubinin method.[59]
The crystal structure was investigated by XRD by using a Philips
X’Pert Pro MPD instrument and monochromated CuKa radiation at
40 kV/50 mA. The X-ray beam was collimated with a 0.258 diver-
gence slit and a fixed 20 mm mask. The measured diffractograms
were analyzed with Philips X’Pert HighScore and MAUD pro-
grams.[60]
The effect of different catalysts was investigated in the ami-
dation of stearic acid with ethanolamine. The highest conver-
sion in the amidation of stearic acid with ethanolamine was
achieved with H-Beta-150, which exhibited quite a high con-
centration of Brønsted acid sites. With the most acidic catalyst,
H-ZSM-5, the conversion was only 67% after 3 h due to the
small pore sizes of the zeolite. Mildly acidic mesoporous mate-
rials H-MCM-41 and H-MCM-36 both exhibited nearly the same
conversion levels as that of relatively acidic, large-pore zeolite
H-Beta-22. Thus, it can be concluded that both acid site con-
centration and pore structure determine the amidation rate of
fatty acids. Fatty acid amide formation was favored over ester
amide formation with an optimum acidity of the catalyst. If the
catalyst acidity was too high, more ester amide was formed.
Future studies are devoted to kinetic analysis and detailed in-
vestigation of the reaction network.
SEM analysis was performed with a LEO Gemini 1530 microscope
with a Thermo Scientific UltraDry Silicon Detector (SDD) equipped
with secondary and backscattered electron detectors and with an
In-Lens detector.
The catalyst morphology was studied by TEM (JEM 1400 plus with
acceleration voltage of 120 kV and resolution of 0.98 nm) with
a Quemsa II MPix bottom-mounted digital camera.
The amount of Brønsted and Lewis acid sites in the catalysts was
measured with pyridine (Sigma–Aldrich, >99.5%) adsorption/de-
sorption with an ATI Mattson FTIR spectrometer. The catalyst was
pressed into small tablets. The molar extinction coefficients were
those reported by Emeis[61] for quantification of Brønsted (bands at
n˜ =1545 cmÀ1) and Lewis (bands at n˜ =1455 cmÀ1) acid sites.
Experimental Section
Feedstock
Raw materials
Different feedstocks, namely, stearic acid (Sigma Aldrich, 95%),
oleic acid (Sigma Aldrich, >99%), technical-grade oleic acid (Fluka,
70%), technical-grade linoleic acid (Aldrich, 60–74% linoleic acid,
oleic acid), palmitic acid (Sigma, >99%), methyl palmitate (Sigma
Aldrich, >97%), and lauric acid (FCC, >98%), were tested. Three
amino alcohols, ethanolamine (Sigma Aldrich, >98%), dl-alaninol
(Aldrich, 98%), and (S)-(+)-leucinol (Aldrich, 96%), were used as
the nitrogen source. In addition, biodiesel was used, which was
prepared from Chlorella alga by in situ transesterification at 608C
for 4 h with methanol and 20 wt% sulfuric acid as a catalyst.
According to the supplier, Chlorella alga (Fuqing King, Drarmsa
Spirulina Co., Ltd., P.R. China) contained 57.3% protein, 17.3 wt%
carbohydrates, 2.3 wt% chlorophyll, 7% moisture, and ash.
Four different zeolites, H-ZSM-5-23, H-Beta-22, H-Beta-150, and H-
Beta-300, in which the number denoted the silica/alumina ratio,
were used as catalysts. The sodium form of Beta-22 was prepared
in an autoclave by using a method described in the literature;[56]
the only difference was that a rotation mode was applied for 240 h
during the synthesis. After drying the zeolite at 1008C, it was con-
+
verted into the NH4 form of the Beta zeolite by calcination in an
oven at 5408C. NH4-Beta-150 and NH4-Beta-300 were purchased
from Zeolyst International and calcined at 5008C. Na-ZSM-5-23 was
purchased from Zeolyst International and ion-exchanged to trans-
form it into the protonated form. The preparation of MCM-36 start-
ed from MCM-22, which was synthesized according to a procedure
reported by Lawton et al.[57] Details of the preparation of MCM-22
are given in Ref. [38]. Thereafter, the wet cake of MCM-22 was
swelled in cetylmethylammonium chloride and tetrapropylammo-
nium hydroxide, followed by pH adjustment to 13.5 with NaOH.
The mixture was stirred for 68 h at 1008C and then at 258C for 4 h.
Subsequently, the mixture was washed with distilled water, dried
at 258C, and pillared by mixing it with tetraethyl orthosilicate
(TEOS) at 788C for 25 h. After filtration and drying at room temper-
ature, MCM-36 was hydrolyzed at pH 8 at 408C for 6 h, followed by
drying at 258C, and calcination at 5508C. The detailed procedure
was reported by Käldstrçm et al.[38] Na-MCM-41 was prepared ac-
cording to the method used by Kresge et al.[58] with a few modifi-
Amino acids in Chlorella alga were analyzed by derivatizing them
by using 6-N-aminoquinolyl hydrosuccinimidyl carbonate (AQC).
Samples containing more than 3 mg of protein were hydrolyzed
with HCl (AOAC 982.30/1982, AOAC International), which was
thereafter removed under high vacuum. Furthermore, amino acids
were dissolved in 20 mm HCl (10–20 mL) and mixed with borate
buffer (80 mL, pH 8.8) and derivatized with 10 mm ACQ (20 mL) in
acetonitrile. Separation of the derivatized amino acids was opti-
mized in a C18 column ACCQ TagTM (3.9”150 mm) with particle
sizes of 4 mm. The column and injector temperatures were 37 (flow
1 mLminÀ1) and 258C, respectively. The quantification of amino
acids was performed by comparison with a standard solution con-
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taining a mixture of free amino acids as external standards. The
free amino acids were analyzed by derivatization in post-hydrolysis
solution.
solved in acetonitrile and amide was precipitated at 08C, whereas
fatty acids remained in the solvent.
The crude purified reaction product mixtures (150 mg) from amida-
tion of stearic acid and technical-grade oleic acid, with ethanola-
mine, were coated on silica, followed by subsequent automated
flash chromatography by using RediSep Rf Gold Silica columns
(0.020–0.040 mm) with a gradient of 0–7% methanol in chloro-
form, which yielded two fractions. The amounts of product mixture
from the amidation of technical-grade oleic acid with alaninol and
leucinol were 208 and 220 mg, respectively. The NMR spectra were
recorded on a Bruker 500 MHz NMR spectrometer equipped with
The amounts of methionine and cysteine were obtained by per-
forming acid oxidation (AOAC 994.12/2000, AOAC International). To
analyze tryptophane obtained by basic hydrolysis, protein (50 mg;
AOAC 988.15/1988, AOAC International) was used. Hydrolysis oc-
curred at 110 8C for 20 h in 4.2m NaOH and 1-octanol (100 mL) in
sealed ampoules. The hydrolyzed material was resuspended in
MilliQ water (50 mL).
Chlorella alga derived biodiesel contained 77% FAME (the main
components in the FAME fraction were methyl linolenate (25.6%),
linoleate (26.2%), palmitate (32.7%), and palmitoleate (8.6)) and
7% fatty acids. The following amino alcohols were used as a nitro-
gen source: ethanolamine (Sigma Aldrich, >98%) and dl-alaninol
(Aldrich, 98%).
1
a BBO-5 mm-Zgrad probe at 258C operating at 500.13 MHz for H
and 125.76 MHz for 13C. IR spectra were recorded with a Bruker
FTIR spectrometer with a platinum single reflection diamond ATR
module.
The water content in the reaction mixture was determined by Karl
Fischer titration by using Hydranal Composite 2 (Fluka) as a titrant.
In a typical experiment, the initial concentration of the reacting
acid was 0.72m and an equimolar amount of ethanolamine in an-
hydrous hexane (100 mL; Sigma Aldrich, >99%) and catalyst
(500 mg) were used under 20 bar of argon at 1808C. The kinetic
experiments were performed in the kinetic regime by using a small
2-Stearamidoethanol
1
White solid; yield: 104 mg; Rf =0.16 (5% MeOH in CHCl3); H NMR
(CDCl3, 500.13 MHz, 258C): d=5.92 (brs, 1H), 3.73 (t, J=5.1 Hz,
2H), 3.43 (q, J=5.1 Hz, 2H), 2.20 (t, J=7.6 Hz, 2H), 1.63 (quint, J=
7.6 Hz, 2H), 1.33–1.24 (m, 28H), 0.88 ppm (t, J=7.0 Hz, 3H);
13C NMR (CDCl3, 125.76 MHz, 258C): d=174.9, 63.0, 42.8, 36.9, 32.2,
29.95, 29.93, 29.91, 29.62, 29.60, 29.5, 26.0, 22.9, 14.4 ppm (Fig-
ure S1 in the Supporting Information). IR[43] and NMR[44] spectrosco-
py data matched that reported in the literature for 2-stearamido-
ethanol (Figure S2a,b in the Supporting Information).
catalyst particle size (<63 mm) and
a high stirring speed
(1100 rpm). In batch mode, the reactor was closed for 3 h, whereas
in purging mode small amounts of reaction mixture were periodi-
cally (seven times) removed from the reactor.
Lipase-catalyzed experiments were carried out by using hexane as
a solvent, together with an equimolar amount of oleic acid and
either ethanolamine or alaninol as the nitrogen source. The reac-
tions were performed in small ampules in which the total liquid
volume was 1.4 mL. In addition to hexane, a small amount of
water was also introduced into the mixture to enhance the
enzyme activity, since the enzyme needed essential water. The
amount of lipase was 30 wt% of the fatty acid. The initial fatty acid
concentration was 0.72m in hexane at reflux at 698C and the reac-
tion mixture was stirred with a magnetic bar.
2-Stearamidoethyl stearate
White solid; yield: 38 mg; Rf =0.69 (5% MeOH in CHCl3); 1H NMR
(CDCl3, 500.13 MHz, 258C): d=5.72 (brs, 1H), 4.16 (t, J=5.4 Hz,
2H), 3.52 (q, J=5.4 Hz, 2H), 2.32 (t, J=7.6 Hz, 2H), 2.17 (t, J=
7.6 Hz, 2H), 1.64–1.59 (m, 6H), 1.32–1.22 (m, 54H), 0.88 ppm (t, J=
6.3 Hz, 6H); 13C NMR (CDCl3, 125.76 MHz, 258C): d=174.2, 173.4,
63.3, 39.0, 36.9, 34.3, 32.1, 29.85, 29.81, 29.79, 29.78, 29.66, 29.64,
29.5, 29.4, 29.3, 25.8, 25.1, 22.8, 14.3 ppm (Figure S2a,b in the Sup-
porting Information). IR[43] and NMR[45] spectroscopy data matched
that reported in the literature for 2-stearamidoethyl stearate.
Analysis
The following capillary column was used for quantitative analysis
of the reaction mixture: Zebron Inferno Zb-5HT, 30 m length,
0.32 mm internal diameter, and 0.25 mm film thickness. Eicosane
(Acros, 99%) was used as an internal standard. The temperature
program was as follows: 708C (5 min) heating at 58CminÀ1 to
2058C (18 min) heating at 78CminÀ1 to 3408C (10 min). Further-
more, a short GC column was used to elute heavier compounds
from the column: HP-1 column (15 m length, 0.53 mm internal di-
ameter, 0.15 mm (film thickness) by using the following tempera-
ture program: 1008C (1.5 min) heating at 128CminÀ1 to 3408C
(20 min). Injector and detector temperatures were 330 and 3408C,
respectively. The short-column method was calibrated with the
fractions isolated by TLC and the relative response factors of oleic
acid, stearic acid, the corresponding amides, and ester amides
were determined. Furthermore, N-oleoylethanolamide (Sigma Al-
drich, 98%) and palmitoylethanolamide (Sigma Aldrich) were used
for calibration of the GC method. The reaction products were iden-
tified by GC-MS. The analytical error in the GC analysis was below
1%.
N-(2-Hydroxy-1-methylethyl)-9Z-octadecamide
White wax; Rf =0.16 (5% MeOH in CHCl3); 1H NMR (CDCl3,
500.13 MHz, 258C): d=5.75 (d, J=6.1 Hz, 1H), 5.40–5.29 (m, 2H),
4.09–4.01 (m, 1H), 3.64 (dd, J=11.0 Hz 3.6 Hz, 1H), 3.52 (dd, J=
11.0 Hz, 6.1 Hz, 1H), 2.95 (brs, 1H), 2.17 (t, J=7.6 Hz, 2H), 2.03–
1.92 (m, 4H), 1.64–1.58 (m, 2H), 1.36–1.24 (m, 20H), 1.16 (d, J=
6.9 Hz, 3H), 0.87 ppm (t, J=6.9 Hz, 3H) (Figure S3 in the Support-
ing Information); 13C NMR (CDCl3, 125.76 MHz, 258C): d=174.2,
130.1, 129.8, 67.4, 47.9, 37.0, 32.0, 29.89, 29.84, 29.6, 29.45, 29.39,
29.36, 29.26, 27.34, 27.30, 25.9, 22.8, 17.2, 14.2 ppm. NMR spectros-
copy data matched that reported in the literature.[54]
9Z-Octadecanoic acid-2-{[(9Z)-1-oxo-9-octadecenyl]amino}-
propyl ester
Purification of the product was performed according to the
method described by Fernandez-Perez and Otero.[25] when the
product was dissolved in a 50/50 (vol%) mixture of chloroform and
methanol and evaporated. Thereafter, the solid product was dis-
Colorless oil; Rf =0.78 (5% MeOH in CHCl3); 1H NMR (CDCl3,
500.13 MHz, 258C): d=5.55 (brd, J=8.3 Hz, 1H) 5.41–5.29 (m, 4H),
4.31–4.24 (m, 1H), 4.11 (dd, J=11.3, 5.9 Hz, 1H), 3.99 (dd, J=11.3,
ChemSusChem 2015, 8, 2670 – 2680
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Full Papers
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4.3 Hz, 1H), 2.31 (t, J=7.5 Hz, 2H), 2.13 (t, J=7.5 Hz, 2H) 2.06–1.98
(m, 8H), 1.64-1.57 (m, 4H), 1.36–1.24 (m, 40H), 1.15 (d, J=6.9 Hz,
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258C): d=174.1, 172.7, 130.15, 130.12, 129.83, 129.81, 66.9, 44.48,
37.0, 34.3, 32.0, 29.89, 29.85, 29.82, 29.78, 29.65, 29.61, 29.50, 29.49,
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N-(1-Hydroxymethyl-3-methylbutyl)-9Z-ocatadecamide
Colorless oil; Rf =0.37 (5% MeOH in CHCl3); 1H NMR (CDCl3,
500.13 MHz, 258C): d=5.51 (d, J=7.3 Hz, 1H; NH), 5.39–5.30 (m,
2H; CH=CH), 4.06–4.01 (m, 1H; CHNHC(O)), 3.67 (dd, J=10.9,
3.0 Hz, 1H; CHaHbOH), 3.53 (dd, J=10.9, 5.7 Hz, 1H; CHaHbOH), 2.81
(brs, 1H; OH), 2.19 (t, J=7.6 Hz, 2H; CH2C(O)NH), 2.06–1.98 (m, 4H;
=CHCH2), 1.66–1.58 (m, 3H; CH2CH2CH2CO and (CH3)2CH), 1.40–1.33
(m, 22H; CH2 and (CH3)2CHCH2CHNHC(O)), 0.93 (t, J=7.0 Hz, 6H;
(CH3)2CH), 0.88 ppm (t, J=6.9 Hz, 3H; CH3CH2); 13C NMR (CDCl3,
125.76 MHz, 258C): d=174.4, 130.2, 129.9, 66.9, 50.3, 40.4, 37.0,
32.0, 29.9, 29.8, 29.7, 29.5, 29.4, 29.35, 29.28, 27.4, 27.3, 25.9, 25.1,
23.2, 22.8, 22.3, 14.3 ppm (Figure S5 in the Supporting Informa-
tion).
9Z-Octadecanoic acid-2-{[(9Z)-1-oxo-9-octadecenyl]amino}-
(4-methyl)pentyl ester
Colorless oil; Rf =0.94 (5% MeOH in CHCl3); 1H NMR (CDCl3,
500.13 MHz, 258C): d=5.40–5.30 (m, 5H; CH=CH and NH), 4.32–
4.26 (m, 1H; CHNHC(O)), 4.11 (dd, J=11.3, 5.4 Hz, 1H; CHaHbOC(O)),
4.01 (dd, J=11.3, 4.0 Hz, 1H; CHaHbOC(O)), 2.30 (t, J=7.5 Hz, 2H;
CH2C(O)O), 2.14 (t, J=7.6 Hz, 2H; CH2C(O)NH), 2.06–1.98 (m, 8H; =
CHCH2), 1.65–1.55 (m, 5H; CH2CH2CH2CO and (CH3)2CH), 1.38–1.23
(m, 42H; CH2 and (CH3)2CHCH2CHNHC(O)), 0.94–0.91 (m, 6H;
(CH3)2CH), 0.87 ppm (t, J=7.0 Hz, 6H; CH3CH2); 13C NMR (CDCl3,
125.76 MHz, 258C): d=174.0, 172.7, 130.2, 130.1, 129.9, 129.8, 66.3,
46.5, 41.0, 34.3, 32.0, 29.90, 29.85, 29.7, 29.46, 29.42, 29.37, 29.35,
29.30, 29.29, 29.27, 27.36, 27.34, 27.31, 25.9, 25.1, 25.0, 23.1, 22.8,
22.4 ppm.
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