An improved method for the synthesis of 1-monoolein

Article abstract of DOI:10.1016/j.molcatb.2013.08.004

Monoacylglycerols (MAGs) are precursors for the synthesis of many active lipids and an important amphiphilic emulsifiers which are widely used in food, pharmaceutical, and cosmetic industries. In this study, we reported an improved method for the synthesis of 1-monoolein using 1,2-acetonide glycerol as starting reactant. Firstly, commercial oleic acid was purified using our previous method and then 1,2-acetonide-3-oleoylglycerol was synthesized by the esterification of 1,2-acetonide glycerol with purified oleic acid using Novozym 435 lipase as catalyst. Finally, the cleavage of unpurified 1,2-acetonide-3-oleoylglycerol in methanol was conducted to obtain 1-monoolein. The effects of reaction system, addition amount of solvent, lipase load, reaction temperature and time on 1,2-acetonide-3-oleoylglycerol content in the crude reaction mixture were investigated. Under the optimal conditions, 94.6% 1,2-acetonide-3-oleoylglycerol in crude reaction mixture was obtained. 1-Monoolein was synthesized further by cleaving unpurified 1,2-acetonide-3-oleoylglycerol in methanol at room temperature with Amberlyst-15 resin as catalyst. The cleavage reaction resulted in the formation of 76.5% 1-monoolein and 96.2% 1-monoolein was obtained at 72.8% yield after repeated recrystallization in hexane to remove nonpolar impurities and water washing to remove glycerol. The main novelties for the synthesis of 1-monoolein are the use of Novozym 435 lipase instead of chemical catalysts used in previous studies to catalyze the esterification of 1,2-acetnode glycerol with free fatty acids and scalable crystallization method used instead of column chromatography to purify 1-monoolein.

Full text of DOI:10.1016/j.molcatb.2013.08.004

Journal of Molecular Catalysis B: Enzymatic 97 (2013) 130–136
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
An improved method for the synthesis of 1-monoolein
Xiaosan Wanga,b,∗, Qingzhe Jina, Tong Wangb, Jianhua Huanga, Xingguo Wanga,∗∗
a State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122,
PR China
b Department of Food Science and Human Nutrition, Iowa State University, 2312 Food Science Building, Ames, IA 50011, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Monoacylglycerols (MAGs) are precursors for the synthesis of many active lipids and an important
amphiphilic emulsifiers which are widely used in food, pharmaceutical, and cosmetic industries. In this
study, we reported an improved method for the synthesis of 1-monoolein using 1,2-acetonide glycerol
as starting reactant. Firstly, commercial oleic acid was purified using our previous method and then 1,2-
acetonide-3-oleoylglycerol was synthesized by the esterification of 1,2-acetonide glycerol with purified
oleic acid using Novozym 435 lipase as catalyst. Finally, the cleavage of unpurified 1,2-acetonide-3-
oleoylglycerol in methanol was conducted to obtain 1-monoolein. The effects of reaction system, addition
amount of solvent, lipase load, reaction temperature and time on 1,2-acetonide-3-oleoylglycerol content
in the crude reaction mixture were investigated. Under the optimal conditions, 94.6% 1,2-acetonide-3-
oleoylglycerol in crude reaction mixture was obtained. 1-Monoolein was synthesized further by cleaving
unpurified 1,2-acetonide-3-oleoylglycerol in methanol at room temperature with Amberlyst-15 resin as
catalyst. The cleavage reaction resulted in the formation of 76.5% 1-monoolein and 96.2% 1-monoolein
was obtained at 72.8% yield after repeated recrystallization in hexane to remove nonpolar impurities and
water washing to remove glycerol. The main novelties for the synthesis of 1-monoolein are the use of
Novozym 435 lipase instead of chemical catalysts used in previous studies to catalyze the esterification
of 1,2-acetnode glycerol with free fatty acids and scalable crystallization method used instead of column
chromatography to purify 1-monoolein.
Received 27 February 2013
Received in revised form 8 April 2013
Accepted 6 August 2013
Available online 20 August 2013
Keywords:
Esterification
Enzymatic synthesis
1-Monoolein
Novozym 435 lipase
Purification
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
example, monolaurin, monomyristin, monolinolein, and monoli-
nolenin have shown the antimicrobial activities [8]. Monoolein has
Monoacylglycerols (MAGs) are nonionic surfactants and emul-
sifiers with hydrophilic and hydrophobic parts in the molecules.
They can be widely used due to excellent emulsifying, stabilizing,
conditioning and plasticizing properties [1,2]. It has been reported
that approximately 200,000–250,000 metric tons of emulsifiers are
produced each year worldwide, of which MAGs account for approx-
imately 75% of the total [3,4]. Secondly, MAGs can be used for the
synthesis of many types of lipids including structured triacylglyce-
rols (TAGs) and diacylglycerols (DAGs), phospholipids, glycolipids
and lipoproteins [5,6]. Thirdly, MAGs from omega-3 polyunsatu-
rated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA) may have beneficial effects in human
health [7]. In addition, certain MAGs have unique applications. For
shown strong antioxidation and anti-atherosclerotic properties in
animal experiments [9,10]. Thus, there has been great interest in
the synthesis of monoolein.
Generally, MAGs were synthesized either by chemical and enzy-
matic glycerolysis of native oils (or commercial product) [2,11,12],
enzymatic esterification of glycerol with free fatty acids [13–16],
chemical esterification of 1,2-acetonide glycerol with free fatty
acids and then the cleavage of 1,2-acetonide-3-fatty acyl glycerol
in methanol to produce MAGs [17–19], or other chemical methods
[20]. In addition, MAGs can also be synthesized either by enzymati-
cally irreversible esterification of glycerol with saturated fatty acid
vinyl esters [21,22] or enzymatically irreversible esterification of
1,2-acetnoide glycerol with saturated fatty acid vinyl esters and
then the cleavage to produce MAGs [23].
Many methods are available for the synthesis of MAGs, but pure
monoacid MAGs synthesis by chemical and enzymatic glycerolysis
of native oil is impossible since these TAGs contain many types of
fatty acids, while pure TAGs are much expensive for the studies.
One step enzymatic esterification of glycerol with free fatty acid
also results in the formation of many types of glycerides in the reac-
tion mixture containing free fatty acids, MAGs, DAGs and TAGs and
∗
Corresponding author at: State Key Laboratory of Food Science and Technol-
ogy, School of Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi,
Jiangsu 214122, PR China. Tel.: +86 510 85876799; fax: +86 510 85876799.
∗∗
Corresponding author. Tel.: +86 510 85876799; fax: +86 510 85876799.
E-mail addresses: wxg1002@qq.com (X. Wang),
wxstongxue@163.com (X. Wang).
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.08.004

X. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 130–136
131
Table 1
their isomers. The monoolein content in the crude reaction mix-
ture is about 50% [8]. The main impurity was DAGs, which can not
be fully removed by crystallization. Column chromatography is an
effective method to remove DAGs, but this method is not a readily
scalable purification method. Thus, we used 1,2-acetonide glycerol
as starting material to synthesize monoolein because of no DAGs
and TAGs formed during esterification and cleavage reactions.
Chemical esterification of 1,2-acetonide glycerol with free
fatty acids and then the cleavage of acetonide group to produce
pure MAGs is popular and was a very effective process. How-
ever, chemical catalysts such as 4-dimethylaminopyridine (DMAP),
pyridine and N-ethyl-Nꢀ-(3-dimethylaminopropyl)-carbodiimide
hydrochloride (EDCI), are highly toxic. EDCI is more expensive
($396/25 g from Sigma–Aldrich) than Novozym 435, and this ester-
ification reaction needs a large amount of EDCI. Therefore, the
synthesis of pure MAGs on a large scale by this method is nei-
ther economical nor environmentally sound. In addition, a very
small amount of chemical catalysts in the monoolein product will
significantly affect the animal experiment results and monoolein
synthesized by the use of toxic catalysts is not allowed to be used
as material to conduct human study. Thus, this method has its dis-
advantages.
Experimental design for optimization of esterification between 1,2-acetonide glyc-
erol and oleic acid.a
Level
X1
X2 (mL)
X3 (wt%)
X4 (◦C)
X5 (h)
1
2
3
4
5
Solvent-free
Dichloromethane
Hexane
0.5
1.0
1.5
2.0
5
8
12
15
20
40
50
60
70
80
2
4
6
8
10
Methanol
X1 = reaction system, X2 = solvent amount, X3 = Novozym 435 lipase load,
X4 = reaction temperature, X5 = reaction time.
a
Differences among means were compared at P = 0.05 level.
2.2. Purification of oleic acid
Commercial oleic acid was purified based on our previously
optimized procedure and conditions with appropriate modifica-
tion [24]. In brief, the purification processes included two steps:
removal of stearic acid and then linoleic acid. The commercial
oleic acid product of 100 mL was mixed with 250 mL chloroform
at −27 ◦C for 12 h to remove stearic acid by discarding the stearic
acid crystals and collecting the liquid phase by vacuum filtration in
a cold room (about 5 ◦C). Chloroform was removed under reduced
pressure to obtain purified oleic acid. In the second step, The
low-temperature crystallization was conducted by mixing 100 mL
purified oleic acid with 400 mL methanol at −25 ◦C for 4 h to remove
linoleic acid by collecting the oleic acid crystals and discarding liq-
uid phase by vacuum filtration in a cold room. Methanol was evapo-
rated under reduced pressure to obtain the final oleic acid product.
Finally, enzymatically irreversible esterification of unsaturated
fatty acid vinyl esters with glycerol or 1,2-acetonide glycerol is not
feasible for the synthesis of 1-monoolein because commercial vinyl
oleate is hard to get.
In the present study, we improved the method for the synthesis
of MAGs by esterification of 1,2-acetonide glycerol with free fatty
acids. The enzymatic method was used instead of chemical method
using DMAP and EDCI as catalysts to synthesize 1-monoolein [18].
Firstly, commercial oleic acid was purified based on our previous
method and then 1,2-acetonide-3-oleoylglycerol was synthesized
by the enzymatic esterification of 1,2-acetnode glycerol with puri-
fied oleic acid. The unpurified 1,2-acetonide-3-oleoylglcyerol was
cleaved in methanol with Amberlyst-15 resin as catalyst to produce
1-monoolein. The effect of reaction system, addition amount of sol-
vent, lipase load, reaction temperature and time were investigated
to maximize 1,2-acetonide-3-oleoylglycerol content in the crude
reaction mixture.
2.3. Optimization of enzymatic esterification of 1,2-acetonide
glycerol with oleic acid
The design and reaction route for the optimization experiments
are outlined in Table 1 and Fig. 1. The effects of reaction system,
addition amount of solvent, lipase load, reaction temperature and
time on 1,2-acetonide-3-oleoylglycerol content in crude reaction
mixture were investigated. The reaction conditions were optimized
for each factor at a time, where other factors were fixed at a constant
level. After one of factors was optimized, the optimal value of this
factor was used for next factors optimization. All reactions were
run in duplicate and data were expressed as means SD.
2. Materials and methods
2.1. Materials
2.3.1. Effect of reaction system
The reaction was conducted with agitation at 50 ◦C for 2 h by
reacting 1 mmol oleic acid with 1.2 mmol 1,2-acetonide glycerol
with 15% (w/w, relative to total reactants) Novozym 435 lipase as
catalyst. The esterification reaction was performed in solvent-free
or solvent system (1 mL dichloromethane, hexane or methanol) to
investigate the effect of reaction system. At the end of the reac-
tion, lipase was removed by filtration and solvent was evaporated
under reduced pressure. The crude reaction product containing 1,2-
acetonide-3-oleoylglycerol was diluted with hexane and injected
to GC directly. The content of 1,2-acetonide-3-oleoylglycerol was
Most of chemicals including 1-monoolein (>99%), a mixture
of diolein (85% 1,3-diolein and 15% 1,2-diolein), Amberlyst-15
resin, glycerol, 1,2-acetonide glycerol, linoleic acid (>99%) and
oleic acid (89.6%) was purchased from Sigma–Aldrich Chemical
Co. (Shanghai, China). Immobilized lipase from Candida antarctica
(Novozym 435) was provided by Novozymes (Beijing, China). This is
an lipase immobilized on a macroscopic acrylate and has a declared
activity of 10,000 PLU (propyl laurate unit)/g. All organic solvents
used have >99% purity.
Fig. 1. Reaction routes of enzymatic synthesis of 1-monoolein.

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X. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 130–136
based on the area ratio and excess 1,2-acetonide glycerol came out
together with solvent.
was evaporated under reduced pressure. Finally, the purified prod-
uct was determined by GC after derivatization as described in the
following section.
2.3.2. Effect of addition amount of solvent
The reaction was carried out in CH2Cl2 with agitation at 50 ◦C for
2 h by reacting 1 mmol oleic acid with 1.2 mmol 1,2-acetonide glyc-
erol with 15% Novozym 435 lipase as catalyst. The addition amount
of solvent was varied from 0.5 to 2.0 mL to investigate the effect of
addition amount of solvent. The product was analyzed by GC as
described above.
2.7. Quantitative analysis of synthesized products
2.7.1. Quantification of 1,2-acetonide-3-oleoylglycerol
1,2-Acetonide-3-oleoylglycerol was determined by GC without
derivatization. Since the 1, 2-acetonide glycerol came out together
with the solvent, purity of 1,2-acetonide-3-oleoylglycerol was cal-
culated based on the area ratio of 1,2-acetonide-3-oleoylglycerol
and total sample peaks except 1,2-acetonide glycerol.
2.3.3. Effect of lipase load
The reaction was carried out in 0.5 mL CH2Cl2 with agitation
at 50 ◦C for 2 h by reacting 1 mmol oleic acid with 1.2 mmol 1,2-
acetonide glycerol with Novozym 435 lipase (5 to 20%) as catalyst.
The product was analyzed by GC as described above.
2.7.2. Quantification of 1-monoolein
The anhydrous reaction product of 1-monoolein was placed
into a 2 mL glass vial for producing its ether derivative for GC
quantification. Pyridine (0.5 mL) was added followed by hexame-
thyldisilazane (0.15 mL) and trimethylchlorosilane (0.05 mL). The
mixture was shaken for 15–30 s and allowed to stand for 10 min to
allow the upper phase turn clear. The purity of 1-monoolein was
calculated according to the peak area ratio. The peak at 10.5 min
was attributed to the silylation reagent based on retention times
determined by control injections as previously reported [25].
Monoolein derivative and 1, 2-acetonide-3-oleoylglycerol
were identified and quantified by GC-14B gas chromatography
(Shimadzu, Tokyo, Japan) equipped with a flame ionization detec-
tor (FID) using a 30 m × 0.25 mm × 0.25 ␮m (length × I.D × film
thickness) fused-silica capillary column PEG-20000. The oven tem-
perature was programmed from 150 to 310 ◦C at a rate of 10 ◦C/min,
and then held at 310 ◦C for 15 min. Injector and detector tempera-
tures were set at 320 ◦C.
2.3.4. Effect of reaction temperature
The reaction was carried out in 0.5 mL CH2Cl2 with agitation at a
certain temperature (40–80 ◦C) for 2 h by reacting 1 mmol oleic acid
with 1.2 mmol 1,2-acetonide glycerol with 8% Novozym 435 lipase
as catalyst. The product was analyzed by GC as described above.
2.3.5. Effect of reaction time
The reaction was carried out in 0.5 mL CH2Cl2 with agitation at
60 ◦C by reacting 1 mmol oleic acid with 1.2 mmol 1,2-acetonide
glycerol with 8% Novozym 435 lipase as catalyst. The reaction time
was varied from 2 to 10 h to investigate the effect of reaction time.
The product was analyzed by GC as described.
2.4. Synthesis of 1,2-acetonide-3-oleoylglycerol on a 200 mmol
scale
2.8. Qualitative analysis of synthesized products
When the optimal reaction conditions were established, the
optimal conditions were used to synthesize 1,2-acetonide-3-
oleoylglyceorol on a 200 mmol scale. The reaction was carried out in
100 mL CH2Cl2 with agitation at 60 ◦C for 8 h by reacting 200 mmol
oleic acid with 240 mmol 1,2-acetonide glycerol with 8% Novozym
435 lipase as catalyst. The product was analyzed by GC as described
above at the end of reaction.
Standards including 1-monoolein and a mixture of diolein (85%
1,3-diolein and 15% 1,2-diolein) were used to identify the peaks.
Partial acyglycerol isomers such as 1-monoolein and 2-monoolein,
and 1,2-diolein and 1,3-diolein were separated very well by GC
based on our experiences and previous studies [22].
1H NMR qualitative analysis of 1-monoolein was done in CDCl3
as solvent using tetramethyl silane (TMS) as internal standard
with a Bruker NMR spectrometer (Avance III 400 MHz, Switzerland)
operating at 400 MHz.
2.5. Synthesis of 1-monoolein by the cleavage of
1,2-acetonide-3-oleoylglycerol on a 200 mmol scale
3. Results and discussion
The cleavage of 1,2-acetonide-3-oleoylglycerol was carried out
based on the previous method [18]. 1,2-Acetonide-3-oleoylglycerol
(200 mmol) was mixed with 6 g Amberlyst-15 in 400 mL methanol
at room temperature for 24 h. At the end of reaction, Amberlyst-
15 was removed by filtration and methanol was removed under
reduced pressure, and then the sample was analyzed by GC after
derivatization as described in the following section.
3.1. Purification of oleic acid
The commercial oleic acid product contained 89.6% oleic acid,
4.2% stearic acid, and 6.2% linoleic acid. Stearic and linoleic acids
were removed by two-step purification. The separation of oleic
acid from other free fatty acids was based on their solubility dif-
ference in solvent. The solubility of oleic acid at −30 ◦C is 23.3 g
oleic acid/100 g chloroform, while the solubility of stearic acid at
−10 ◦C is 0.08 g stearic acid/100 g chloroform. Thus, when commer-
cial oleic acid was mixed with chloroform at −27 ◦C, stearic acid
crystallized from the solution and was separated from oleic acid.
Oleic acid content was increased from 89.6 to 93.3 0.4% in this
purification step. Stearic acid was fully removed and the product
contained 6.7 0.2% linoleic acid as impurity. In the second step,
purified oleic acid was mixed with methanol because the solubility
of oleic acid at −20 ◦C is 4.02 g oleic acid/100 g methanol, compared
to 233 g linoleic acid/100 g methanol. Thus, when purified oleic acid
was mixed with methanol at −25 ◦C, oleic acid crystallized from
methanol, whereas linoleic acid was still dissolved in methanol.
2.6. Purification of 1-monoolein from the cleavage product of
1,2-acetonide-3-oleoylglycerol
The reaction product was purified by two steps. Firstly, the reac-
tion product was purified by repeated recrystallizaiton in hexane
at −30 ◦C for 5 h to remove unreacted oleic acid, 1,2-acetonide
glycerol and methyl oleate formed by methanolysis. The crystal
containing 1-monoolein was collected and liquid phase was dis-
carded. Glycerol was then separated from monoolein after the
product was mixed with CH2Cl2 and water. Monoolein was purified
by collecting the lower layer containing 1-monoolein and discard-
ing aqueous phase containing glycerol. After purification, solvent

X. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 130–136
133
Fig. 4. The effect of lipase load on 1,2-acetonide-3-oleoylglycerol content in crude
reaction mixture.
Fig. 2. The effect of reaction system on 1,2-acetonide-3-oleoylglycerol conent in
crude reaction mixture. Reaction conditions: 1 mL solvent or solvent-free, 1:1.2
molar ratio of oleic acid to 1,2-acetonide glycerol, 15% (w/w, relative to total reac-
tants) Novozym 435 lipase and 50 ◦C for 2 h.
Reaction conditions: 1:1.2 molar ratio of oleic acid to 1,2-acetonide glycerol, 0.5 mL
dichloromethane and 50 ◦C for 2 h.
After the second step purification, oleic acid was increased from
93.3 0.4 to 97.8 0.5% at 76.6 2.5% overall yield.
The most common solvent used in the low temperature crystal-
lization for the purification of polyunsaturated fatty acids (PUFAs)
is acetone [26,27]. However, using acetone as solvent is hard to
fully remove saturated fatty acids. In this study, chloroform and
methanol were chose as solvents in low temperature crystalliza-
tion process and stearic acid was fully removed after purification.
Low temperature crystallization method for the purification of oleic
acid is scalable.
3.2. Enzymatic synthesis of 1,2-acetonide-3-oleoylglycerol
The optimization results are outlined in Figs. 2–6. The
main impurities in the crude reaction mixture were unreacted
oleic acid, excess 1,2-acetonide glycerol and 1,2-acetonide-3-
linoleoylglycerol formed from the esterification of 1,2-acetonide
glycerol with linoleic acid, which was from impure oleic acid prod-
uct. In this study, reaction system, addition amount of solvent,
lipase load, reaction temperature and time were optimized to
maximize the 1,2-acetonide-3-oleoylglycerol content in the crude
reaction product.
Fig. 5. The effect of reaction temperature on 1,2-acetonide-3-oleoylglycerol content
in crude reaction mixture. Reaction conditions: 1:1.2 molar ratio of oleic acid to
1,2-acetonide glycerol, 0.5 mL dichloromethane, 8% Novozym 435 lipase, and for
2 h.
3.2.1. The effect of reaction system
The solvent-free and solvent systems were first investigated.
Reactions conducted in dichloromethane and hexane resulted in
a higher 1,2-acetonide-3-oleoylglycerol content in crude reaction
Fig. 3. The effect of addition amount of dichloromethane on 1,2-acetonide-3-
oleoylglycerol content in crude reaction mixture. Reaction conditions: 1:1.2 molar
ratio of oleic acid to 1,2-acetonide glycerol, 15% Novozym 435 lipase and 50 ◦C for
2 h.
Fig. 6. The effect of reaction time on 1,2-acetonide-3-oleoylglycerol content in
crude reaction mixture. Reaction conditions: 1:1.2 molar ratio of oleic acid to 1,2-
acetonide glycerol, 0.5 mL dichloromethane, 8% Novozym 435 lipase, and 60 ◦C for
2 h.

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X. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 130–136
product compared to those conducted in solvent-free system and
methanol (Fig. 2). The reactants interaction may be lower in the
solvent-free system compared to solvent system and thus leaded
to a lower esterification rate, while reaction conducted in methanol
resulted in the loss of lipase activity by striping away the water
that is associated with the lipase [28] because existence of water
around the lipase is essential for the maintenance of enzyme activ-
ity. Nonpolar solvents can maintain the lipase activity and increase
the interaction of 1,2-acetonide glycerol with oleic acid. Thus,
dichloromethane was selected for the further experiments.
3.2.2. The effect of addition amound of solvent
Addition amount of solvent was optimized because solvent
amount affects the solubility of reactants, product and lipase con-
centration. A low dichloromethane amount results in low product
solubility, whereas a high dichloromethane amount dilutes the
enzyme and the reactants and may affect the reaction negatively.
The results showed that 1,2-acetonide-3-oleoylglycerol content in
the crude reaction product decreased with the increasing addi-
tion amount of solvent probably because of the decrease in the
dilution of reactants and lipase concentration (Fig. 3). The dilu-
tion of reactants reduces their interaction in reaction mixture and
may slow the reaction rate. The maximum value was observed
at 0.5 mL addition amount of dichloromethane and 1,2-acetonide-
oleoylglycerol content was (89.7 3.4)% at this condition. Thus,
0.5 mL dichloromethane was selected for the next experiments.
Fig. 7. GC chromatogram of synthetic 1,2-acetonide-3-oleoylglycerol by the enzy-
matic esterification.
8% Novozym 435 lipase load, 60 ◦C reaction temperature and 8 h
reaction time. The main impurities were unreacted oleic acid,
1,2-acetonide glycerol and 1,2-acetonide-3-linoleoylglycerol since
commercially inexpensive oleic acid contains linoleic acid, which
reacted with 1,2-acetonide glycerol to produce 1,2-acetonide-3-
linoleoylglycerol. If pure oleic acid was used for the enzymatic
esterification, the 1,2-acetonide-oleoylglycerol content in the
crude reaction product will tend to be higher.
3.2.3. The effect of lipase load
The synthesis of 1,2-acetonide-3-oleoylglycerol was also con-
ducted on a 200 mmol scale. Reaction conditions for scalable
synthesis of 1,2-acetonide-3-oleoylglycerol were exactly the same
with the optimal reaction conditions identified on a small scale.
At the end of reaction, (93.4 1.3)% 1,2-acetonide-3-oleoylglycerol
was produced in the crude reaction mixture, indicating that the
enzymatic esterification between 1,2-acetonide glycerol with oleic
acid on a larger scale is feasible.
Lipase load affects the reaction rate and thus the reaction
time. High lipase load is favorable for the completion of reac-
tion during short reaction time. The effect of Novozym 435 load
is present in Fig. 4. The addition amount of Novozym 435 lipase
did not significantly affect 1,2-acetonide-3-oleoylglyceorl content
in the crude reaction mixture at the low addition amount of
dichloromethane. Novozym 435 load was saturated at a low addi-
tion amount probably due to high lipase activity and low addition
amount of solvent. The maximum 1,2-acetonide-3-oleoylglyceorl
content was observed at 20% lipase addition amount. However, in
order to reduce the operating cost, 8% Novozym 435 load was used
for the further reactions.
3.3. Synthesis of 1-monoolein
After 1,2-acetonide-3-oleoylglycerol was obtained on
a
200 mmol scale, the unpurified product was used to synthesize
1-monoolein directly without purification. 1-Monoolein was
synthesized from 1,2-acetonide-3-oleoylglycerol based on the
previous conditions [18]. When 1,2-acetonide-3-oleoylglycerol
was mixed with 6 g Amberlyst-15 in methanol at room temper-
ature for 24 h, There was 76.5% 1-monoolein produced at the
end of reaction. We also investigated the effect of cleavage time
on 1-monoolein content in crude reaction product. The results
showed that 1-monoolein content had a decreased trend with
the increasing cleavage time. This observation may result from
methanolysis of 1,2-acetonide-3-oleoylglycerol to produce impure
methyl oleate [29]. We also detected the existence of methyl oleate
in the crude reaction product and confirmed the hypothesis. The
observation agrees well with a previous study [30] even though
most researchers did not discuss this methanolysis reaction during
the cleavage reaction for the synthesis of MAGs.
The main impurities in the cleavage product were oleic acid,
methyl oleate produced from methanolysis of 1,2-acetonide-3-
oleoylglycerol, uncleaved 1,2-acetonide-3-oleoylglycerol, glycerol
originated from the cleavage of 1,2-acetonide glycerol, minor 2-
monoolein originated from acyl migration of 1-monoolein and
1-monolinolein originated from the cleavage of 1,2-acetonide-3-
linoleoylglycerol. Oleic acid, methyl oleate and 1,2-acetonide-3-
oleoylglycerol were nonpolar lipids and have a higher solubility
in hexane compared to 1-monoolein. Thus, these nonpolar lipids
can be removed by recrystallization in hexane at −30 ◦C for 5 h.
Repeated recrystallization was conducted until nonpolar lipids
3.2.4. The effect of reaction temperature
The reaction temperature also affects the reaction rate and
lipase activity. Lipase activity release needs an optimal tempera-
ture. On the one hand, reaction conducted at high temperature will
speed the completion of reaction, but the increase in reaction tem-
perature may result in the inactivation of lipase. On the other hand,
activity of lipase and reaction rate are decreased with the decrease
in temperature to a certain extent. The results for the effect of reac-
tion temperature are showed in Fig. 5. Novozym 435 activity and
reaction rate were low when the reaction was carried out at 40 ◦C.
The increase in reaction temperature from 40 to 50 ◦C caused a
dramatic increase in 1,2-acetonide-3-oleoylglycerol content in the
crude reaction product. However, there were no significant differ-
ences observed among 50–80 ◦C. The maximum value was seen at
60 ◦C. Therefore, 60 ◦C was selected as optimal condition for the
further experiments.
3.2.5. The effect of reaction time
Finally, the reaction time was optimized. 1,2-Acetonide-3-
oleoylglycerol tended to increase with the increasing reaction
time even though no differences were observed among differ-
ent reaction times (Fig. 6). Herein, 8 h was selected as optimal
reaction time and (94.6 0.9)% 1,2-acetonide-3-oleoylglycerol was
produced in the crude reaction product (Fig. 7). The optimal reac-
tion conditions were 0.5 mL addition amoun of dichloromethane,

X. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 130–136
135
oleoylglycerol and thus a alternative route for the synthesis of pure
MAGs. In addition, the enzymatic method for the synthesis of pure
MAGs is feasible on a 200 mmol scale due to effective enzymatic
esterification and simpler recrystallization method used to purify
1-monoolein compared to column chromatography used in pre-
vious studies. This is first time to use recrystallization method to
purify 1-monoolein obtained from the cleavage of 1,2-acetonide-3-
oleoylglycerol. In addition, in contrast to previous studies [17,18],
we found that the purification of 1,2-acetonide-3-oleoylglycerol
was not essential for the synthesis of 1-monoolein. Thus, the route
reported herein is simpler. The enzymatic method for the syn-
thesis of MAGs receive little attention even though enzymatic
esterification between stearic acid and 1,2-acetonide glycerol has
been reported in a study [31], but the final product in their
study is 1,2-acetonide-stearoylglycerol rather than MAGs in our
study.
Fig. 8. GC chromatogram of synthetic 1-monoolein by the cleavage of 1,2-
acetonide-3-oleoylglycerol (after purification).
4. Conclusions
were fully removed and GC was used to monitor their contents.
Glycerol can be removed by water washing. Monolinolein and 2-
monoolein have a similar polarity and solubility with 1-monoolein
and are hard to be removed by this method (Fig. 8). Thus, the main
impurities were MAGs after recrystallization and water washing,
and (96.2 0.5)% 1-monolein was obtained at 72.8% yield after
purification.
In addition to the qualitative analysis by 1-monoolein and
diolein standards, structural confirmation of 1-monoolein was also
done by using 1H NMR. NMR analysis gave the expected H peak
from glycerol backbone and oleoyl molecular even though the
number of H gave by NMR is not exactly same with 1-monoolein
molecular due to the existence of minor impurities (Fig. 9).
In the current study, enzymatic esterification was used
instead of chemical esterification to synthesize 1,2-acetonide-
3-oleoylglycerol by esterifying 1,2-acetonide glycerol with oleic
acid using Novozym 435 as catalyst. We found that enzymatic
method was also effective for the synthesis of 1,2-acetonide-3-
Recent studies showed that monoolein had strong antioxidation
and anti-atherosclerotic properties in animal experiments (8,9).
Thus, the synthesis of pure monoolein is essential to investigate its
nutritional properties in human and animal studies. In the present
study, we improved the method of the synthesis of MAGs by using
lipase as catalyst. The improvement by replacing chemical cata-
lysts with lipase for the synthesis of monoolein is very important
because toxic chemicals are not allowed to be used in food indus-
tries. In addition, the study on nutritional functions of monoolein
in human subjects also needs a green synthetic method. Enzymatic
method for the synthesis of MAGs is not only greener compared
to chemical methods, but also very effective. In addition, we used
a simpler recrystallizaiton method instead of column chromatog-
raphy to purify monoolein. Thirdly, the route for the synthesis of
MAGs is economical due to the used of commercially inexpensive
oleic acid as starting material rather than pure oleic acid, Finally,
we simplified the procedure by using unpurified 1,2-acetonide-3-
Fig. 9. 1H NMR spectrum of synthetic 1-monoolein.

136
X. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 130–136
oleoylglycerol directly for the cleavage to produce 1-monoolein
without purification compared to previous studies.
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Acknowledgments
We thank the Chinese Scholarship Council for providing the
sponsorship to this research.
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