Improved synthesis of monopalmitin on a large scale by two enzymatic methods

Article abstract of DOI:10.1007/s11746-013-2291-3

Monoacylglycerols (MAG) are precursors for the synthesis of symmetrical and unsymmetrical triacylglycerols (TAG). In the present study, we improved two methods for synthesizing MAG. One method involved the enzymatic transesterification of vinyl palmitate with glycerol catalyzed by Novozym 435 lipase, and the other method was an enzymatic esterification of 1,2-acetonide glycerol with palmitic acid catalyzed by Novozym 435 lipase and then the cleavage of 1,2-acetonide-3-palmitoyl glycerol in methanol catalyzed by Amberlyst-15 to produce monopalmitin. Pure monopalmitin was obtained after repeated crystallization. The main novelties of this study are twofold: Novozym 435 proved to be very effective in catalyzing the transesterification between vinyl palmitate and glycerol without absorbing glycerol onto silica gel; and the enzyme catalyzed reaction between 1,2-acetonide glycerol and palmitic acid was simpler and safer than the typical method of using 4-dimethyl aminopyridine and N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride as catalysts. Our methods for the synthesis of monopalmitin are much simpler and environmentally friendlier than the reported methods, and they are economical and scalable to larger quantity production.

Full text of DOI:10.1007/s11746-013-2291-3

J Am Oil Chem Soc
DOI 10.1007/s11746-013-2291-3
ORIGINAL PAPER
Improved Synthesis of Monopalmitin on a Large
Scale by Two Enzymatic Methods
Xiaosan Wang
Tong Wang
•
Xingguo Wang
•
Qingzhe Jin
•
Received: 9 September 2012 / Revised: 27 March 2013 / Accepted: 14 June 2013
Ó AOCS 2013
Abstract Monoacylglycerols (MAG) are precursors for
the synthesis of symmetrical and unsymmetrical triacyl-
glycerols (TAG). In the present study, we improved two
methods for synthesizing MAG. One method involved the
enzymatic transesterification of vinyl palmitate with glyc-
erol catalyzed by Novozym 435 lipase, and the other
method was an enzymatic esterification of 1,2-acetonide
glycerol with palmitic acid catalyzed by Novozym 435
lipase and then the cleavage of 1,2-acetonide-3-palmitoyl
glycerol in methanol catalyzed by Amberlyst-15 to produce
monopalmitin. Pure monopalmitin was obtained after
repeated crystallization. The main novelties of this study
are twofold: Novozym 435 proved to be very effective in
catalyzing the transesterification between vinyl palmitate
and glycerol without absorbing glycerol onto silica gel; and
the enzyme catalyzed reaction between 1,2-acetonide
glycerol and palmitic acid was simpler and safer than the
typical method of using 4-dimethyl aminopyridine and
Keywords Lipid synthesis ꢀ Novozym 435 lipase ꢀ
Monopalmitin ꢀ Structured lipids ꢀ Transesterification
Introduction
Monoacylglycerols (MAG) are important amphiphilic
emulsifiers and are widely used in food, pharmaceutical,
and cosmetic industries [1]. It has been reported that
approximately 200,000–250,000 metric tons of emulsifiers
are produced each year worldwide, of which MAG account
for approximately 75 % of the total [2, 3]. Secondly, MAG
are used as precursors for the synthesis of unsymmetrical
[4] and symmetrical triacylglycerols (TAG) [5, 6] that can
be used to study structure and function relationships.
Thirdly, MAG containing omega-3 polyunsaturated fatty
acids such as eicosapentaenoic acid (EPA) and docosa-
hexaenoic acid (DHA) may have beneficial effects in
humans with cardiovascular disorders [7]. In addition,
certain MAG have been tested for unique applications. For
example, monolaurin may be useful in the prevention and
treatment of bacterial infection [8] or used as a general
micro biocide [9]. Monopentadecanoyl glycerol is used as a
hair care additive [7], and 2-arachidonoyl glycerol has been
suggested to act as an endogenous agonist at the brain
cannabinoid receptor [10].
0
N-ethyl-N -(3-dimethylaminopropyl)-carbodiimide hydro-
chloride as catalysts. Our methods for the synthesis of
monopalmitin are much simpler and environmentally
friendlier than the reported methods, and they are eco-
nomical and scalable to larger quantity production.
The synthesis of MAG for emulsifier purposes is usually
conducted by glycerolysis of native fats and oils with lipase
as catalyst [11, 12] or at high temperature and pressure.
This reaction usually results in the formation of a mixture
of lipids including many types of MAG and diacylglycerols
X. Wang ꢀ Xingguo Wang ꢀ Q. Jin
State Key Laboratory of Food Science and Technology, School
of Food Science and Technology, Jiangnan University, No. 1800
Lihu Road, Wuxi, Jiangsu 214122, People’s Republic of China
Xingguo Wang ꢀ T. Wang (&)
(
DAG) which is undesirable for the further synthesis of
Department of Food Science and Human Nutrition,
Iowa State University, 2312 Food Science Building,
Ames, IA 50011-1061, USA
structured lipids. MAG can also be obtained by direct
esterification between free fatty acids and glycerol with
lipase as catalyst, but generally, the yield and purity of
e-mail: tongwang@iastate.edu
1
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J Am Oil Chem Soc
MAG are low [13, 14]. In contrast, chemical methods are
believed to be more effective for the synthesis of pure
MAG compared to enzymatic methods [15, 16]. However,
these reactions involve multiple protective and deprotec-
tive processes and toxic pyridine is usually used as catalyst.
Recently, a pure MAG is synthesized by esterifying 1,2-
palmitate (98 %) was purchased from Tokyo Chemical
Industry (Tokyo, Japan). Immobilized lipase from Candida
antarctica (Novozym 435) was provided by Novozymes
(Blair, NE, USA). All organic solvents used had [99 %
purity.
acetonide glycerol with free fatty acid in the presence of
0
Optimization of the Enzymatic Synthesis
4
-dimethylaminopyridine (DMAP) and N-ethyl-N -(3-
of Monopalmitin Using Glycerol and Vinyl Palmitate
dimethylaminopropyl)-carbodiimide hydrochloride (EDCI)
as catalysts [17, 18]. DMAP is a highly toxic compound
and EDCI is very expensive ($396/25 g from Sigma-
Aldrich), and this esterification reaction needs a large
amount of EDCI. Therefore, the synthesis of pure MAG on
a large scale by this method is neither economical nor
environmentally sound. In another method, pure MAG
were obtained by conducting irreversible transesterification
between fatty acid vinyl esters and glycerol that was
absorbed on silica gel in the presence of Lipozyme RM IM
The design and reaction routes for the optimization
experiments are outlined in Table 1 and Fig. 1. The effects
of solvent type, the amount of lipase and solvent, and
reaction time on monopalmitin content were investigated.
The reaction conditions were optimized for each factor at a
time, while other factors were fixed at a constant level. All
reactions were run in duplicate and data are expressed as
means ± standard deviations (SD).
The effect of solvent type Optimization for solvent type
was carried out by reacting 5 mmol glycerol with 1 mmol
vinyl palmitate in 2 mL dichloromethane, methyl tert-butyl
ether, methanol, or hexane with 10 % (w/w, relative to
total reactants) Novozym 435 as catalyst. The reaction was
conducted with agitation at ambient temperature for 6 h.
The resulting mixture was heated at 60 °C for 6 min to
completely dissolve the product and then the lipase was
filtered off and the solvent was evaporated under reduced
pressure. After the removal of glycerol by washing with
water, the anhydrous reaction product was derivatized and
quantified by GC as described in the following section. All
reaction mixtures were treated by the same method before
GC analysis unless otherwise stated.
[
19, 20]. In general, the lipase can be used ten times
without significant loss of enzyme activity [21, 22].
However, absorption of glycerol on silica gel led to non-
recyclable lipase in this case. Although fatty acid vinyl
esters are relatively expensive and some unsaturated fatty
acid vinyl esters are commercially unavailable, the unique
irreversibility of the transesterification suggests a complete
reaction.
In the present study, we report two improved methods
for the synthesis of pure MAG on a large scale. Relatively
pure monopalmitin can be obtained either by the esterifi-
cation of 1,2-acetonide glycerol with palmitic acid with
Novozym 435 as catalyst and then the cleavage of 1,2-
acetonide-3-palmitoyl glycerol in methanol with Amber-
lyst-15 as catalyst, or by the transesterification between
vinyl palmitate and glycerol without the absorption of
glycerol on silica gel and with Novozym 435 as catalyst.
Novozym 435 has proven to be an effective catalyst for
these two reactions. The reactions are scalable since simple
crystallization steps can be used to purify the products. The
reactions are also environmentally benign since lipase was
used as catalyst instead of hazardous chemicals. The fea-
sible method of monopalmitin synthesis allows us to fur-
ther synthesize kilogram quantities of structured lipids for
the ultimate nutritional studies.
The effect of solvent amount Optimization for solvent
amount was conducted by reacting 5 mmol glycerol with
Table 1 Experimental design for the optimization of the transeste-
rification between vinyl palmitate and glycerol
Level
X
1
X
2
(mL)
X
3
(%)
4
X (h)
a
1
2
3
4
5
Dichloromethane
MTBE
0
1
2
3
4
5
2
10
15
20
–
6
10
14
24
Methanol
Hexane
–
All reactions were conducted at ambient temperature for 6 h by
reacting 5 mmol glycerol with 1 mmol vinyl palmitate in 1 mL
MTBE with 10 % Novozym 435 as catalyst unless otherwise stated
Materials and Methods
1
X = solvent type, conducted at ambient temperature for 6 h by
Most of the chemicals including dipalmitin ([99 %, a
mixture of 1,2-dipalmitin and 1,3-dipalmitin), monopalm-
itin ([99 %), palmitic acid ([98 %), glycerol ([99 %),
reacting 5 mmol glycerol with 1 mmol vinyl palmitate in 2 mL sol-
vent with 10 % Novozym 435 (w/w, relative to total reactants) as
catalyst. X = solvent volume, X = lipase load, X = reaction time
2
3
4
1
,2-acetonide glycerol ([98 %), Amberlyst-15, and EDCI
[99 %) were purchased from Sigma-Aldrich Chemical
Co. (St. Louis, MO, USA) except the following: vinyl
MTBE methyl tert-butyl ether
a
When solvent-free system was selected, the reaction was conducted
at 30 °C in order to keep the reactants liquid
(
1
23

J Am Oil Chem Soc
Fig. 1 Enzymatic synthesis of
monopalmitin. rt room
O
OH
OH
temperature, MTBE methyl tert-
butyl ether. a and b are two
different enzymatic methods
optimized
O
O
14
Novozym 435
MTBE, rt
+
OH
O
14
+
CH CHO
3
(a)
(b)
OH
OH
O
O
O
O
OH
14
Novozym 435
Amberlyst-15
O
+
14
O
OH
o
CH Cl , 55 C
CH OH, rt
2
2
O
3
O
O
H
OH
O
14
1
mmol vinyl palmitate in 0, 1, 2, 3, and 4 mL methyl tert-
to obtain pure monopalmitin. The purification methods of
monopalmitin are outlined in Fig. 2a. After the removal of
excess glycerol by washing with water, the crystals were
dissolved in methanol at 60 °C and placed at ambient
temperature for 1 h to crystallize the unreacted vinyl pal-
mitate and DAG side reaction products. The resulting
crystals were filtered off by vacuum filtration, and the fil-
trate was evaporated and the residue was redissolved in
hexane. The solution was then placed at ambient temper-
ature for about 40 min to recrystallize monopalmitin. The
yield and purity of monopalmitin were calculated based on
the weight of the initial material and final product after
purification.
butyl ether with 10 % Novozym 435 as catalyst with agi-
tation at ambient temperature for 6 h. The lipase was fil-
tered off after 2 mL methyl tert-butyl ether was added into
solvent-free system to dissolve the product at 60 °C.
Methyl tert-butyl ether was evaporated under reduced
pressure to give a white crystalline product.
The effect of lipase load Optimization for lipase quantity
was conducted with agitation at ambient temperature for
6
h by reacting 5 mmol glycerol with 1 mmol vinyl pal-
mitate in 1 mL methyl tert-butyl ether with 5, 10, 15, and
0 % Novozym 435 (relative to total reactants). The lipase
2
was filtered off after the product was dissolved in methyl
tert-butyl ether at 60 °C and methyl tert-butyl ether was
evaporated under reduced pressure to give a white crys-
talline product.
Synthesis of Monopalmitin Using Glycerol Acetonide
and Palmitic Acid
The effect of reaction time Optimization for reaction
time was conducted with agitation at ambient temperature
for 2, 6, 10, 14, and 24 h by reacting 5 mmol glycerol with
Synthesis of monopalmitin by this method included two
steps: the synthesis of 1,2-acetonide-3-palmitoyl glycerol
and then the cleavage of 1,2-acetonide-3-palmitoyl glyc-
erol in methanol with Amberlyst-15 as catalyst to produce
monopalmitin. The effects of reaction temperature and
time on the content of 1,2-acetonide-3-palmitoyl glycerol
in the crude reaction mixture were investigated. These
reactions were conducted separately with agitation at 35,
1
mmol vinyl palmitate in 1 mL methyl tert-butyl ether
with 10 % Novozym 435. The lipase was filtered off after
the product was processed as above.
Enzymatic Synthesis of Monopalmitin Using Glycerol
and Vinyl Palmitate on a Large Scale
45, and 55 °C for 2, 4, 6, 8, and 10 h by reacting 11 mmol
After all factors were optimized, large scale synthesis of
monopalmitin was done using the conditions that were the
same as the optimized conditions determined on a 1-mmol
scale. Glycerol (500 mmol, 46 g) and vinyl palmitate
1,2-acetonide glycerol and 10 mmol palmitic acid in 5 mL
dichloromethane with 10 % Novozym 435 as catalyst. The
lipase was removed by filtration and the solvent was
evaporated under reduced pressure. The reaction product
was determined by GC directly without derivatization.
Cleavage of 1,2-acetonide-3-palmitoyl glycerol was
conducted with agitation at ambient temperature for 36 h
by mixing 10 mmol 1,2-acetonide-3-palmitoyl glycerol
with 15 mL pure or 95 % aqueous methanol and with 0.5 g
Amberlyst-15 as catalyst. The reaction product was deter-
mined by GC after the removal of glycerol by washing with
water and derivatization as described in the following
section. All treatments were done in duplicate.
(
100 mmol, 28.25 g) were mixed with agitation at ambient
temperature for 6 h in 100 mL methyl tert-butyl ether with
0 % Novozym 435 as catalyst. The lipase was filtered off
after the product was dissolved in methyl tert-butyl ether at
0 °C and methyl tert-butyl ether was evaporated under
1
6
reduced pressure to give a white crystalline product. This
large scale reaction was conducted in duplicate.
Purification of monopalmitin on a large scale After the
large scale synthesis, some purification steps were essential
1
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J Am Oil Chem Soc
(a)
(b)
Crude reaction
mixture
Crude reaction
mixture
Water washing
Evaporated solvent
Crystal 1
Glycerol
Crystal 1
Solvent
1
2
. Dissolved in methanol
. Recrystallized at rt
1
. Dissolved in hexane
2. Recrystallized at rt
Discarded
Discarded
Filtrate 1
Crystal 2
Discarded
Purified
monopalmitin
Filtrate 1
1
2
. Evaporated solvent
. Dissolved the crystal
in hexane
Recrystallizad at rt
Filtrate 2
3
. Recrystallized at rt
Purified
monopalmitin
Purified
monpalmitin
Filtrate 2
Discarded
Discarded
Fig. 2 Purification of monopalmitin synthesized by the two enzymatic methods corresponding to the two reactions outlined in Fig. 1
Synthesis of Monopalmitin on a Large Scale Using
Glycerol Acetonide and Palmitic Acid
monopalmitin fractions were combined and this hexane
purification method was repeated three times until no
glycerol and palmitic acid peaks were shown on GC. The
purity and yield were calculated based on the weight of the
initial material and final product after purification.
The reaction conditions for large scale synthesis of
monopalmitin were the same as the optimal conditions
established on the 10-mmol scale. Synthesis of 1,2-aceto-
nide-3-palmitoyl glycerol was conducted with agitation at
Quantitative Analysis of the Synthesized Products
5
5 °C for 10 h by reacting 606 mmol 1,2-acetonide glyc-
erol with 551 mmol palmitic acid in 250 mL dichloro-
methane using 10 % Novozym 435. After the removal of
the lipase by filtration and evaporation of the solvent under
reduced pressure, the product was used directly to conduct
the cleavage reaction without purification.
Quantification of monopalmitin The anhydrous reaction
product of monopalmitin (about 2 mg) was placed in a
2-mL glass vial for producing its ether derivative for GC
quantification. Pyridine (0.5 mL) was added followed by
hexamethyldisilazane (0.15 mL) and trimethylchlorosilane
(0.05 mL). The mixture was shaken for 15–30 s and
allowed to stand for 30 min to allow the upper phase to
become clear. The purity of monopalmitin was calculated
according to the peak area ratio. A peak at 11.1–11.2 min
was attributed to the silylation reagent based on retention
time determined by control injections as previously
reported [23].
Cleavage of 1,2-acetonide-3-palmitoyl glycerol was
conducted with agitation at ambient temperature for 36 h
by reacting 500 mmol 1,2-acetonide-3-palmitoyl glycerol
with 750 mL 95 % aqueous methanol and 25 g Amberlyst-
1
5 as catalyst. This large scale reaction was conducted in
duplicate.
Purification of monopalmitin on a large scale The
purification method is outlined in Fig. 2b. Monopalmitin
and glycerol produced from the cleavage of excess 1,2-
acetonide glycerol were separated by adding water and
dichloromethane into the reaction mixture after the
removal of methanol. After dichloromethane was removed
under reduced pressure, the product was dissolved in
hexane at 65 °C and crystallized at ambient temperature for
about 30 min to obtain the purified monopalmitin. The
Filtrate 1 fraction was left for overnight at ambient tem-
perature to recover additional monopalmitin. The two
Quantification of 1,2-acetonide-3-palmitoyl glycerol
1,2-Acetonide-3-palmitoyl glycerol was determined by GC
without derivatization. Since 1,2-acetonide glycerol came
out together with the solvent, the purity of 1,2-acetonide-3-
palmitoyl glycerol was calculated based on the area ratio of
1,2-acetonide-3-palmitoyl glycerol and total sample peaks
except 1,2-acetonide glycerol.
GC conditions The monopalmitin derivative and 1,2-
acetonide-3-palmitoyl glycerol were identified and quan-
tified by an HP 5890 Series II capillary GC (Hewlett-
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J Am Oil Chem Soc
Packard, PA, USA) equipped with a flame ionization
Results and Discussion
detector (FID) using a 30 m 9 0.25 mm 9 0.25 lm
(
length 9 ID 9 film thickness) fused silica bonded phase
Synthesis of Monopalmitin Using Glycerol and Vinyl
Palmitate
capillary column SP-1 (Supelco, Bellefonte, PA, USA).
The carrier gas (helium) flow rate was 32.3 mL/min, and
the split ratio was seven. The oven temperature was pro-
grammed from 140 to 305 °C at a rate of 10 °C/min, and
then held at 305 °C for 15 min. Injector and detector
temperatures were set at 300 °C.
Standards including monopalmitin and dipalmitin,
which is a mixture of 1,2-dipalmitin and 1,3-dipalmitin,
were used to identify the peaks. Partial acylglycerol iso-
mers such as 1-monopalmitin and 2-monopalmitin, and
The results for the optimization of the enzymatic synthesis
of monopalmitin are presented in Fig. 3. The effects of
type and amounts of solvent, lipase load and reaction time
were investigated. The main impurities in the enzymatic
synthesis of monopalmitin were DAG and excess glycerol.
Therefore, the optimization was aimed at increasing the
monopalmitin content and decreasing the DAG content.
The solvent type affects the solubility of reactants and
enzyme activity due to the differences in solvent polarity.
When methanol, dichloromethane, methyl tert-butyl ether
and hexane were used as solvent, monopalmitin content in
the mixture after the removal of excess glycerol was 0,
1
,2-dipalmitin and 1,3-dipalmitin, were separated very well
by GC based on our study and previous reports [19, 20].
Qualitative Analysis of the Synthesized Products
7
9.8, 89.1, and 86.5 %, respectively (Fig. 3a). Methanol
1
H-NMR qualitative analysis of monopalmitin was done by
greatly decreased the catalytic activity of the lipase pos-
sibly by stripping away the water that is associated with the
lipase even though the removal of water or keeping the
water at a low level in the reaction system is beneficial to
using a Varian MR-400 spectrometer (Foster City, CA,
USA) with CDCl as solvent and TMS as the internal
3
standard (chemical shift of 0 ppm).
Fig. 3 Optimization of enzymatic synthesis of monopalmitin, illustrating the effect a solvent type, b solvent amount, c enzyme load, and
d reaction time
1
23

J Am Oil Chem Soc
drive the esterification reaction to completion. Lipases
other than phospholipases, which have been shown to
catalyze the hydrolysis of soy lecithin in water medium
However, increasing the solvent amount led to a decrease
in monopalmitin content and an increase in DAG content
possibly due to a dilution effect. Therefore, 1 mL was
selected as the optimal solvent amount for further
experiments.
[
24], have been reported to show a low catalytic activity in
a polar medium [25]. In contrast, reactions conducted in a
nonpolar solvent did not result in the inactivation of the
lipase probably because the poor interaction of the lipase
with hydrophobic solvents appears to prevent the protein
from denaturation. In addition, this reaction is between two
immiscible substrates. The best solvent should be the one
capable of solubilizing the two substrates. Methanol is not
suitable in this regard. Therefore, methyl tert-butyl ether
that gave the highest monopalmitin yield was selected for
the further experiments.
Lipase is an effective biocatalyst for the transesterifi-
cation reaction and its quantity usually affects the reaction
rate. The content of monopalmitin and DAG tended to
increase and then decrease when the lipase load was
increased even though no significant differences were
observed among the different addition amounts of lipase
(Fig. 2c). With the enzyme cost taken into consideration,
10 % lipase was selected to conduct further experiments.
Finally, the reaction time was optimized. The reaction
time from 6 to 24 h did not affect the content of mono-
palmitin and DAG (Fig. 3d). The maximum value of
monopalmitin was observed at 6 h. Thus, 6 h was selected
as the optimal reaction time. Under the optimal reaction
conditions, monopalmitin content in the crude reaction
mixture on the small scale synthesis was about 94 % after
the removal of excess glycerol by washing with water. The
removal of glycerol by washing did not result in the loss of
glycerides.
The solvent amount affects the concentration of the
lipase and the solubility of the reactants. The reaction in a
solvent-free system resulted in the formation of 78.2 %
monopalmitin compared to 94.5, 92.7, 84.5, and 84.1 % in
1
, 2, 3, and 4 mL methyl tert-butyl ether, respectively
(
Fig. 3b). Glycerol did not dissolve vinyl palmitate in a
solvent-free system, which resulted in a lower content of
monopalmitin and DAG in the crude reaction mixture
compared to that in solvent systems, in which glycerol may
be partially mixed with vinyl palmitate. Thus, a solvent
system was a more effective reaction system for the syn-
thesis of monopalmitin compared to a solvent-free system.
The transesterification reaction between vinyl palmi-
tate and glycerol was also conducted on a large scale using
the optimized reaction conditions. The content of
(a)
(
b)
(c)
(
d)
(e)
Fig. 4 GC chromatograms of synthesis products on large scale.
a Enzymatic synthesis of monopalmitin before purification, b enzy-
matic synthesis of monopalmitin after purification, c enzymatic
synthesis of 1,2-acetonide-3-palmitoyl glycerol before purification,
d cleavage product of 1,2-acetonide-palmitoyl glycerol before
purification, and e cleavage product of 1,2-acetonide-3-palmitoyl
glycerol after purification
1
23

J Am Oil Chem Soc
monopalmitin in the crude reaction mixture after the
removal of excess glycerol was (84.3 ± 3.7) % (Fig. 4a).
The purity of monopalmitin reached 97–98 % with 68.5 %
yield (Fig. 4b) after purification based on the steps
described in Fig. 2a. The benefit of this purification method
was that a pure 1,3-dipalmitin ([98 %) was also obtained
in the process (data not shown). After the reaction mixture
was recrystallized in methanol at ambient temperature and
the crystals and filtrate were separated by filtration, the
crystals mainly contained 1,3-dipalmitin and vinyl palmi-
tate, while the filtrate mainly contained monopalmitin
product. Vinyl palmitate and 1,3-dipalmitin can be sepa-
rated by recrystallization in hexane. In another preliminary
study on ethanolysis of tristearin, we also found that 1,2-
distearin and 1,3-distearin can be separated by recrystalli-
zation in methanol (data not shown). This finding may lead
to a more effective method of separating DAG isomers on
analytical or commercial scales compared to column
chromatography.
a small and large scale after the removal of excess glycerol.
Novozym 435 instead of Lipozyme RM IM was used as
catalyst without the glycerol absorption onto silica gel in
methyl tert-butyl ether polar solvent. Thus, our reaction
system is much simpler and more efficient.
Synthesis of Monopalmitin Using Glycerol Acetonide
and Palmitic Acid
The GC product characterization results are outlined in
Fig. 4c–e, and the synthetic route is presented in Fig. 1b.
The content of 1,2-acetonide-3-palmitoyl glycerol
increased gradually with the increase in reaction time
(Fig. 5). Increasing the reaction temperature was beneficial
to the formation of 1,2-acetonide-3-palmitoyl glycerol.
Thus, 55 °C was selected as the optimal reaction tempera-
ture. When the reaction was conducted at 55 °C for 10 h by
reacting 10 mmol palmitic acid with 11 mmol 1,2-aceto-
nide glycerol in 5 mL dichloromethane with 10 % enzyme,
1,2-acetonide-3-palmitoyl glycerol with (98 ± 0.6) %
purity was formed in the crude reaction mixture compared
to about 94 % on a large scale synthesis. We found that
further increasing the reaction time resulted in a decrease in
the 1,2-acetonide-3-palmitoyl glycerol content on the large
scale synthesis and the reason is unknown.
1
H-NMR spectroscopy of monopalmitin shows the fol-
lowing: d 0.88 (t, 3H, CH ), 1.24–1.27 (m, 24H,
3
1
2
–
2 9 CH2), 1.60–1.63 (m, 2H, O–C(O)–CH –CH ),
2 2
.32–2.36 (m, 2H, O–C(O)–CH –CH ), 3.56–3.60 (m, 2H,
2
2
CH(OH)–CH –OH), 3.90–3.94 (m, 1H, –CH –CH(OH)–
2
2
1
CH –), 4.1–4.2 (m, 2H, –CH(OH)–CH –O–C(O)–). No H
2
2
NMR peak of –CH–O–C(O)– (d 5.2–5.3, 1H) was
observed, suggesting the structure of 1- or 3-monopalmitin
rather than 2-monopalmitin.
The effect of methanol concentration on the synthesis of
monopalmitin by the cleavage of 1,2-acetonide-3-palmitoyl
glycerol in methanol was investigated. Other reaction
conditions used were obtained from the previous study
[17]. The cleavage of 1,2-acetonide-3-palmitoyl glycerol
on a small scale resulted in the formation of (84.6 ±
2.3) % monopalmitin in the crude reaction mixture when
pure methanol was used as solvent. There was
(90.8 ± 1.2) % monopalmitin formed when 95 % aqueous
methanol was used. The main side reaction was the
There are a few publications reporting the synthesis of
partial acylglycerols including MAG and DAG by the
esterification between a fatty acyl donor and excess glyc-
erol absorbed on silica gel with Lipozyme RM IM as cat-
alyst [19, 20, 26, 27]. The content of monolaurin in the
crude reaction mixture was 70 % and pure monolaurin was
obtained with 60 % yield after purification when 5:1 ratio
of glycerol and vinyl laurate was used in Berger and
Schneider’s study [19]. Waldinger and Schneider [20]
obtained pure unsaturated monoacylglycerols with
5
6–85 % yield by the transesterification between unsatu-
rated fatty acid vinyl esters with glycerol absorbed on silica
gel. Absorption of glycerol on silica gel may be essential
for the transesterification of glycerol with fatty acid vinyl
esters, methyl esters or free fatty acids with Lipozyme RM
IM to obtain a high yield because of the poor solubility of
glycerol in nonpolar organic solvents [27, 28], while polar
solvents would result in a decrease in lipase activity. Thus,
in another study, \25 % MAG was produced in the crude
reaction mixture by the transesterification of fatty acid
vinyl esters with glycerol without its absorption on silica
gel [29]. In addition, the lipase cannot be separated from
silica gel after reaction for reuse, suggesting a higher
processing cost. In our study, we found that about 94 and
Fig. 5 Effect of enzymatic synthesis condition on purity of 1,2-
acetonide-3-palmitoyl glycerol
8
4 % monopalmitin was produced in a suitable solvent on
1
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J Am Oil Chem Soc
methanolysis of 1,2-acetonide-3-palmitoyl glycerol, which
resulted in the formation of methyl palmitate (Fig. 4d).
This observation agrees well with a previous study [30]
even though most researchers did not discuss this meth-
anolysis during the cleavage reaction for the synthesis of
MAG [17, 18, 31]. The addition of water can prevent the
methanolysis reaction, but an excess amount of water was
unfavorable due to the low solubility of monopalmitin in a
highly polar solvent and possible side reaction caused by
hydrolysis of the palmitate ester. Pure monopalmitin was
obtained with 77.8 % yield after purification. In addition,
our study showed that the purification method described in
Fig. 2b was also suitable for the purification of monoolein
when -25 °C was used as the crystallization temperature.
Similarly, the methanolysis produced 25–30 % methyl
oleate during the cleavage of 1,2-acetonide-3-oleoyl glyc-
erol in pure methanol with Amberlyst-15 as catalyst when
synthesizing monoolein (data not shown). Therefore, a
further study should be conducted to investigate the effect
of solvent moisture content and the type of catalyst on
methanolysis of fatty acid acetonide when synthesizing
MAG using this method. In addition, further study is
necessary to use hexane instead of chloromethane to syn-
thesize 1,2-acetonide-3-palmitoyl glycerol which may be
cleaved in 95 % ethanol instead of 95 % methanol to
produce monopalmitin as food-grade MAG, because only
hexane, ethanol and acetone are allowed for use as solvents
or processing aids in the food industry.
purification is much easier and scalable compared to column
chromatography [28]. Thus, we greatly simplified the syn-
thesis of monopalmitin by modifying and improving the
reaction conditions and reducing purification cost by using a
recrystallization method.
In contrast to the transesterification between vinyl pal-
mitate and glycerol, the esterification between palmitic
acid and glycerol acetonide is reversible. In addition, the
transesterification of vinyl palmitate with glycerol can be
conducted at ambient temperature, whereas the esterifica-
tion reaction needs a higher temperature to complete.
Finally, the synthesis of monopalmitin using glycerol
acetonide needs a longer time due to the long cleavage time
in alcohol. Thus, if time and temperature are very impor-
tant considerations, the irreversible transesterification may
be a better choice than the esterification route. If synthesis
cost is a very important factor, the reversible esterification
may be used instead.
Conclusion
We improved two methods for the synthesis of mono-
palmitin on a large scale. To the best of our knowledge,
large scale synthesis of pure MAG was only conducted by
Andrews et al. who used EDCI and DMAP as catalysts [17,
36]. In this study, monopalmitin was synthesized by the
transesterification between vinyl palmitate and glycerol in
a proper solvent. The main novelty of this method is that
highly pure monopalmitin can be obtained without using
the glycerol absorption onto silica gel. We also synthesized
monopalmitin by esterifying 1,2-acetonide glycerol and
palmitic acid and then cleaving the 1,2-acetonide-3-pal-
mitoyl glycerol in 95 % methanol. This enzymatic method
is more economical due to the use of affordable free fatty
acids as the starting material, feasible due to the recrys-
tallization purification method instead of column chroma-
tography, and environmentally benign due to the use of
lipase instead of a toxic chemical catalyst.
In the past, the esterification between 1,2-acetonide
glycerol and an acyl donor was conducted by reacting 1,2-
acetonide glycerol either with fatty acid vinyl esters by lipase
catalysis [29, 32, 33] or with free fatty acids using DMAP as
catalyst [17, 18, 34], or another chemical method [31]. Even
though the esterification of 1,2-acetonide glycerol and free
fatty acids with lipase as catalyst has been reported [35], such
a method had received little attention because MAG content
in the crude reaction mixture was very low. Thus, a strong
catalyst, DMAP or a higher activity acyl donor, such as fatty
acid vinyl ester had to be used to conduct the esterification
reaction. However, fatty acid vinyl esters are more expensive
compared to free fatty acids and the esterification reaction
catalyzed by DMAP needs a large amount of expensive
EDCI. In addition, DMAP is a highly toxic chemical. Thus,
these reaction routes are not economically feasible and
environmentally friendly. In contrast, we found that Nov-
ozym 435 is an effective catalyst for the esterification
between palmitic acid and 1,2-acetonide glycerol in a
suitable solvent. Our method for the synthesis of 1,2-aceto-
nide-3-palmitoyl glycerol is much more economical and
environmentally friendly. We also found that the purification
of 1,2-acetonide-3-palmitoyl was not essential after the
reaction. The purification after the hydrolysis reaction can be
feasibly done. In general, the recrystallization method for the
Acknowledgments We thank Dr. Xueshu Li of the Department of
Chemistry, Iowa State University, for the NMR characterization of
1
-monopalmitin. We also acknowledge the Chinese Government for
providing the sponsorship for the Ph.D. graduate student, Xiaosan
Wang, who conducted this research.
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