Monomyristin and monopalmitin derivatives: Synthesis and evaluation as potential antibacterial and antifungal agents

Article abstract of DOI:10.3390/molecules23123141

In the present work, monoacylglycerol derivatives, i.e., 1-monomyristin, 2-monomyristin, and 2-monopalmitin were successfully prepared from commercially available myristic acid and palmitic acid. The 1-monomyristin compound was prepared through a transesterification reaction between ethyl myristate and 1,2-O-isopropylidene glycerol, which was obtained from the protection of glycerol with acetone, then followed by deprotection using Amberlyst-15. On the other hand, 2-monoacylglycerol derivatives were prepared through enzymatic hydrolysis of triglycerides in the presence of Thermomyces lanuginosa lipase enzymes. The synthesized products were analyzed using fourier transform infrared (FTIR) spectrophotometer, gas or liquid chromatography-mass spectrometer (GC-MS or LC-MS), and proton and carbon nuclear magnetic resonance (¹H- and¹³C-NMR) spectrometers. It was found that monomyristin showed high antibacterial and antifungal activities, while 2-monopalmitin did not show any activity at all. The 1-monomyristin compound showed higher antibacterial activity against Staphylococcus aureus and Aggregatibacter actinomycetemcomitans and also higher antifungal activity against Candida albicans compared to the positive control. Meanwhile, 2-monomyristin showed high antibacterial activity against Escherichia coli. The effect of the acyl position and carbon chains towards antibacterial and antifungal activities was discussed.

Full text of DOI:10.3390/molecules23123141

molecules
Article
Monomyristin and Monopalmitin Derivatives:
Synthesis and Evaluation as Potential Antibacterial
and Antifungal Agents
Jumina 1,*, Asma Nurmala 1, Anggit Fitria 1, Deni Pranowo 1, Eti Nurwening Sholikhah 2,
Yehezkiel Steven Kurniawan 1 and Bambang Kuswandi 3
1
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada,
Sekip Utara, Yogyakarta 55281, Indonesia; asma_nurmala@yahoo.co.id (A.N.); anggitfitria@gmail.com (A.F.);
maspranowo@ugm.ac.id (D.P.); kurniawan94steven@gmail.com (Y.S.K.)
Department of Pharmacology and Therapy, Faculty of Medicine, Universitas Gadjah Mada, Sekip Utara,
2
Yogyakarta 55281, Indonesia; etinurweningsholikhah@ugm.ac.id
Faculty of Pharmacy, University of Jember, Jember 68121, Indonesia; b_kuswandi.farmasi@unej.ac.id
3
*
Correspondence: jumina@ugm.ac.id; Tel.: +62-2745-45188
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 8 November 2018; Accepted: 26 November 2018; Published: 29 November 2018
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: In the present work, monoacylglycerol derivatives, i.e., 1-monomyristin, 2-monomyristin,
and 2-monopalmitin were successfully prepared from commercially available myristic acid
and palmitic acid. The 1-monomyristin compound was prepared through a transesterification
reaction between ethyl myristate and 1,2-O-isopropylidene glycerol, which was obtained from
the protection of glycerol with acetone, then followed by deprotection using Amberlyst-15.
On the other hand, 2-monoacylglycerol derivatives were prepared through enzymatic hydrolysis
of triglycerides in the presence of Thermomyces lanuginosa lipase enzymes. The synthesized
products were analyzed using fourier transform infrared (FTIR) spectrophotometer, gas or liquid
chromatography-mass spectrometer (GC-MS or LC-MS), and proton and carbon nuclear magnetic
resonance (1H- and 13C-NMR) spectrometers. It was found that monomyristin showed high
antibacterial and antifungal activities, while 2-monopalmitin did not show any activity at all.
The 1-monomyristin compound showed higher antibacterial activity against Staphylococcus aureus
and Aggregatibacter actinomycetemcomitans and also higher antifungal activity against Candida albicans
compared to the positive control. Meanwhile, 2-monomyristin showed high antibacterial activity
against Escherichia coli. The effect of the acyl position and carbon chains towards antibacterial and
antifungal activities was discussed.
Keywords: monomyristin; monopalmitin; synthesis; antibacterial; antifungal
1. Introduction
Harmful microorganisms cause serious problems in industrial food products. One of the
gram-negative bacteria, Escherichia coli (E. coli), has been reported for serious food poisoning
and diarrhea [
infections [ ], while Candida albicans (C. albicans) gives rise to nosocomial and superficial infections [
Many researchers have been putting effort into developing an effective and efficient antibacterial and
antifungal agent from natural product isolation [ ]. He et al. (2017) reported polyketide derivatives
from Emericella sp. TJ29 and evaluated their antibacterial activity [ ]. However, isolation from natural
resources has several disadvantages, such as time-consuming process, low-isolation yield, high price,
limited resources, and disturbance of food and environmental sustainability [ ]. Because of this,
researchers prefer to develop synthetic compounds as antibacterial and antifungal agents [9–12].
1]. Staphylococcus aureus (S. aureus), one of the gram-positive bacteria, causes skin
2
3].
4–6
7
8
Molecules 2018, 23, 3141; doi:10.3390/molecules23123141
www.mdpi.com/journal/molecules

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Fatty acids and monoacylglycerol derivatives are widely developed as antibacterial agents [13].
Monoacylglycerol can be prepared through selective glycerolysis by using an inorganic base catalyst
under a nitrogen atmosphere [14]. Lauric acid and monocaprin have been investigated as intravaginal
microbicides to correct sexually transmitted diseases [15
compound was prepared in a simple and efficient method from lauric acid and glycerol in the presence
of p-toluenesulfonic acid as the catalyst [17 18]. Chinatangkul et al. (2018) reported that monolaurin
,16]. In our previous work, the 1-monolaurin
,
compounds can be loaded onto shellac nanofibers and used as an antimicrobial agent [19]. Even though
monolaurin derivatives have been well investigated, only few reports are found for the synthesis and
evaluation of other monoacylglycerol derivatives, such as monomyristin and monopalmitin.
In this work, we prepared other derivatives of monoacylglycerol, i.e., 1-monomyristin,
2-monomyristin, and 2-monopalmitin, from myristic acid and palmitic acid. The 1-monomyristin
derivative was synthesized through esterification of myristic acid with ethanol and further reacted
with 1,2-O-isopropylidene glycerol, then followed by hydrolysis using Amberlyst-15 as a solid acid
catalyst. The 2-monoacylglycerol derivatives were initially produced via esterification of glycerol
with ethyl myristate to form triglyceride, followed by selective hydrolysis of the triglyceride using
Thermomyces lanuginosa lipase enzymes (TLIM).
2. Results and Discussion
2.1. Synthesis of 1-Monomyristin
The synthesis of the 1-monomyristin compound consisted of four steps, i.e., synthesis of
1,2-O-isopropylidene glycerol and ethyl myristate separately, synthesis of isopropylidene glycerol
myristate, and synthesis of 1-monomyristin (Figure 1a). Selective monosubstitution of glycerol on
position 1 can be obtained after the protection of the two hydroxyl groups of glycerol by using
acetone. In this work, glycerol was reacted with acetone in the presence of p-toluenesulfonic
acid (pTSA) as an acid catalyst to obtain a ketal derivative of glycerol, i.e., 1,2-O-isopropylidene
glycerol. The 1,2-O-isopropylidene glycerol was then transesterified with ethyl myristate in basic
conditions to obtain isopropylidene glycerol myristate. Finally, the isopropylidene glycerol myristate
was deprotected using Amberlyst-15. The chemical structure of the intermediates was confirmed
from the FTIR and GC-MS analysis. The 1-monomyristin product was successfully synthesized in
this work in a quantitative yield (100%) by a simple stirring method with small amount (0.04 g)
of Amberlyst-15 as heterogeneous catalyst. The FTIR spectra of 1-monomyristin show a broad
peak at 3456 cm−1 and a strong peak at 1735 cm−1 due to the presence of hydroxyl and carbonyl
groups, respectively. The purity of the product was confirmed from LC chromatograms and the
mass spectrum show [M + tH]+ at m/z = 303. The 1H- and 13C-NMR spectra of 1-monomyristin are
shown in Figures S1 and S2, respectively. Both 1H- and 13C-NMR spectra confirmed the formation
of 1-monomyristin product. The presence of the hydroxyl groups of the 1-monomyristin product is
shown as singlet peak at 2.18 ppm, while the protons of the glycerol are observed at chemical shifts
more than 3.50 ppm because the carbons were directly bonded to oxygen atoms. The 13C-NMR spectra
also show 3 peaks at 63.35, 65.16 and 70.28 for the inequivalent carbon atoms in the glycerol backbone.

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Figure 1. The synthesis scheme of (a) 1-monoacylglycerol and (b) 2-monoacylglycerol derivatives.
2.2. Synthesis of 2-Monomyristin and 2-Monopalmitin
The synthesis of 2-monomyristin was carried out through trimyristin synthesis and followed by
enzymatic hydrolysis with TLIM as catalyst (Figure 1b). Trimyristin was prepared by an esterification
reaction between glycerol and myristic acid under acidic conditions. The excess of myristic acid
was removed in basic conditions because sodium myristate is soluble in water. The product
remained in the organic phase. The chemical structure of trimyristin product was confirmed by
1
FTIR, GC-MS, and H- and 13C-NMR analysis. The absence of the broad peak at 3200–3400 cm−1
on FTIR spectra shows that all the hydroxyl groups of the glycerol were completely esterified with
myristic acid. The purity of the product was confirmed from GC chromatograms while the mass
spectrum corresponds to the trimyristin fragmentation. Both 1H- and 13C-NMR spectra confirmed
the absence of hydroxyl proton and the presence of carbon of C=O ester at 173.54 ppm, respectively.
Afterwards, the myristate esters at the edge of the trimyristin were selectively hydrolyzed in the
presence of TLIM as the catalyst. The 2-monomyristin compound was purified through preparative thin
layer chromatography (PTLC) and its chemical structure was confirmed by FTIR, GC-MS, and 1H- and
13C-NMR analysis. A broad peak appeared due to the presence of hydroxyl groups of 2-monomyristin.
The purity of the product was confirmed from LC chromatograms and the mass spectrum corresponds
to the 2-monomyristin fragmentation. The 1H- and 13C-NMR spectra of 2-monomyristin are shown
1
in Figures S3 and S4, respectively. Both H- and 13C-NMR spectra confirmed the formation of
2-monomyristin product.
The synthesis process of 2-monopalmitin is similar to the synthesis of 2-monomyristin (Figure 1b).
Tripalmitin was prepared from an esterification reaction and then hydrolyzed in the presence of TLIM

Molecules 2018, 23, 3141
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as the catalyst to obtain 2-monopalmitin as the final product. The chemical structure of both tripalmitin
and 2-monopalmitin was confirmed by FTIR, GC-MS, and 1H- and 13C-NMR analysis. The absence of
−1
the broad peak at 3200–3400 cm on tripalmitin FTIR spectrum shows that all the hydroxyl groups of
the glycerol were completely esterified with palmitic acid. The purity of the tripalmitin product was
confirmed from the presence of a single peak from the GC chromatogram, and its mass spectrum is in
agreement with the expected fragmentation of tripalmitin compound. Both 1H- and 13C-NMR spectra
confirmed the absence of hydroxyl proton and the presence of the C=O ester, respectively. On the
other side, a broad peak appeared due to the presence of the hydroxyl groups of 2-monopalmitin.
The purity of the product was confirmed from LC chromatograms, and the structure of the product
was further confirmed by the mass spectra and 1H- and 13C-NMR. The 1H- and 13C-NMR spectra of
2-monopalmitin are shown in Figures S5 and S6, respectively.
2.3. Antibacterial and Antifungal Assays of Products
The antibacterial and antifungal activities of each synthesized product were evaluated against
E. coli, S. aureus, and C. albicans. The result of the biological assay is shown in Table 1. Bold values
were made when the inhibition zone of the sample is larger than that of the positive control. It was
found that 0.25% 2-monomyristin exhibited higher antibacterial activity than the positive control
(1.00% 4-isopropyl-3-methylphenol) against E. coli. It was also found that 0.50% 1-monomyristin
and 0.50% 2-monomyristin showed higher antibacterial activity compared to the positive control
against S. aureus. However, only 1-monomyristin showed antifungal activity against C. albicans.
Since 1-monomyristin showed promising antibacterial and antifungal activity, 1-monomyristin was
further investigated as an antibacterial agent against Bacillus subtilis (B. subtilis) and Aggregatibacter
actinomycetemcomitans (A. actinomycetemcomitans). The antibacterial activity of 1-monomyristin against
B. subtilis and A. actinomycetemcomitans is listed in Table 2. As expected, 1-monomyristin exhibited
antibacterial activity against B. subtilis and A. actinomycetemcomitans. From these results, monomyristin
showed better antibacterial and antifungal activities compared to monopalmitin. This is probably due
to the shorter carbon chain near the monolauric acid [18]. The 1-position showed high activity for both
antibacterial and antifungal agent. It is due to the better interaction 1-monomyristin can make with
the bacterial cell wall than 2-monomyristin can.
Table 1. Antibacterial and antifungal assays of 1-monomyristin, 2-monomyristin and 2-monopalmitin
against E. coli, S. aureus, and C. albicans.
Inhibition Zone’s Average (mm)
Sample
E. coli
S. aureus
C. albicans
0.50% 1-monomyristin
1.00% 1-monomyristin
5.00% 1-monomyristin
10.0% 1-monomyristin
15.0% 1-monomyristin
0.25% 2-monomyristin
0.50% 2-monomyristin
1.00% 2-monomyristin
5.00% 2-monomyristin
10.0% 2-monomyristin
0.25% 2-monopalmitin
0.50% 2-monopalmitin
1.00% 2-monopalmitin
5.00% 2-monopalmitin
10.0% 2-monopalmitin
Positive control
1.5
1.1
4.3
-
6.0
34.0
29.5
22.0
21.0
11.0
-
10.3
5.7
5.9
9.1
18.9
23.0
20.0
13.0
13.0
5.0
-
-
-
-
-
6.6
-
-
3.5
3.6
2.4
4.1
-
-
-
-
-
-
-
-
-
-
6.8
-
-
-
-
-
12.5
-
Negative control
Negative control: 20.0% PEG 400; Positive control: 1.00% 4-isopropyl-3-methylphenol; -: No activity.

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Table 2. Antibacterial assays of 1-monomyristin against B. subtilis and A. actinomycetemcomitans.
Inhibition Zone Average (mm)
Sample
B. subtilis
A. actinomycetemcomitans
0.50% 1-monomyristin
1.00% 1-monomyristin
5.00% 1-monomyristin
10.0% 1-monomyristin
15.0% 1-monomyristin
Positive control
2.4
3.6
5.7
9.2
12.7
16.3
-
1.2
1.9
3.6
7.9
10.4
5.5
-
Negative control
Negative control: 20.0% PEG 400; Positive control: 1.00% 4-isopropyl-3-methylphenol; -: No activity.
The inhibition mechanism of 1-monomyristin against C. albicans seems to be similar to
amphotericin B compounds. The hydroxyl group in 1-monomyristin interacts with the ergosterol
on the fungi cell membrane, and therefore the function of the membrane was disrupted and cell
lysis happened [20]. In contrast to the C. albicans cell wall, which consists of the phospholipid
bilayer, ergosterol, chitin, and microfibrillar β-glucan, the bacterial cell wall is simpler. Therefore, it is
reasonable for monomyristin compounds that showed high activity to be used as antibacterial agents
against E. coli, S. aureus, B. subtilis, and A. actinomycetemcomitans. The 1-monomyristin compound
at concentrations higher than 1.00% exhibited medium antifungal activity compared with 1.00%
4-isopropyl-3-methylphenol as the positive control. These results demonstrate that 1-monomyristin is
a potential compound to be applied as an antibacterial and antifungal agent.
3. Materials and Methods
3.1. Materials
Myristic acid, palmitic acid, glycerol, sulfuric acid, n-hexane, ethanol, acetone, sodium hydroxide,
p-toluenesulfonic acid (pTSA), Amberlyst-15, sodium carbonate, potassium carbonate, ethyl acetate,
chloroform, and anhydrous sodium sulfate were purchased from Merck (Darmstadt, Germany),
while TLIM was obtained from Novozymes (Bagsværd, Denmark). All chemicals were used as
received without any further purification. Thin layer chromatography (TLC) was carried out using
an aluminium plate (Merck) coated with silica gel 60 F254 (20
Sabouraud agar powder which contains 4.00% of dextrose as the microbial nutrient was purchased
from Merck.
×
20 cm). The brain-heart broth and
3.2. Equipment
Infrared spectra of the prepared compounds were recorded on a Fourier transform infrared
spectrophotometer (FT-IR, Shimadzu Prestige 21, Tokyo, Japan). The purity of the products
was evaluated from chromatograms which were obtained from either gas chromatography-mass
spectrometer (GC-MS, Shimadzu QP 2010S) or liquid chromatography-mass spectrometer (LC-MS/MS,
Acquity HPLC-SQD MassLynx v4-1 SCN 805, Waters Corporation, Milford, MA, USA). The 1H-NMR
(nuclear magnetic resonance) and 13C-NMR spectra were recorded by a JEOL JNM–ECZ500R/S1
spectrometer (JEOL Ltd., Tokyo, Japan) using tetramethylsilane as an internal standard in
deuterated chloroform.
3.3. Synthesis of 1-Monomyristin
3.3.1. Synthesis of 2,2-Dimethyl-1,3-dioxolan-4-methanol (1,2-O-isopropylidene glycerol)
Acetone (48 mL, 650 mmol) and pTSA (1.2 g) were stirred for 15 min. Chloroform (96 mL)
was added into the solution and stirred at 343 K for 30 min. Glycerol (24 mL, 330 mmol) was
then added and the mixture was heated at 393 K for 6.5 h. The lower layer was neutralized with

Molecules 2018, 23, 3141
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sodium carbonate and then dried over anhydrous sodium sulfate. The solvent was evaporated to
obtain 14.61 g of 1,2-O-isopropylidene glycerol as a colorless liquid (33.71% yield). FT-IR (cm−1):
3417 (broad, OH stretching), 2985 (C-H stretching), 1211 (C-O eter). GC: 100% 1,2-O-isopropylidene
glycerol (retention time (tR) = 8.6 min, [M − 15]+ = 117).
3.3.2. Synthesis of Ethyl Myristate
Myristic acid (12 g, 53 mmol) was esterified by using a 4% wt sulfuric acid solution in
ethanol (50 mL) and the mixture was sonicated for 4.5 h with a Branson 1210 ultrasonic cleaner
(Branson, Danbury, CT, USA). After the reaction, the solvent was evaporated. The filtrate was
diluted with ethyl acetate (40 mL) and washed with 5% wt sodium hydroxide solution until neutral.
The organic layer was dried over anhydrous sodium sulfate and the solvent was evaporated to obtain
13.25 g of ethyl myristate as a yellowish liquid (98.43% yield). FT-IR (cm−1): 2924 (C-H stretching),
1735 (C=O ester), 1458 (CH2 bending), 1180 (C-O ester). GC: 98.90% ethyl myristate (retention time
(tR) = 34.4 min, [M − 15]+ = 256).
3.3.3. Synthesis of Isopropylidene Glycerol Myristate
The 1-monomyristin compound was prepared in two-step process, i.e., synthesis of the
isopropylidene glycerol myristate followed by ketal hydrolysis using Amberlyst-15 (Sigma Aldrich,
Missouri, MO, USA). First, ethyl myristate (2.1 g, 8 mmol) was reacted with 1,2-O-isopropylidene
glycerol (4.2 g, 32 mmol) and potassium carbonate (0.31 g) at 413 K for 30 h. Diethylether (20 mL)
was used to extract the product and then neutralized with distilled water. The solvent was
evaporated to obtain 0.92 g of isopropylidene glycerol myristate as a yellowish liquid (32.12% yield).
FT-IR (cm−1): 3441 (broad, OH stretching), 2924 (C-H stretching), 1743 (C=O ester), 1458 (CH2 bending),
1157 (C-O ester). GC: 95.55% isopropylidene glycerol myristate (retention time (tR) = 30.0 min,
[M − 15]+ = 327).
3.3.4. Synthesis of 1-Monomyristin
The isopropylidene glycerol myristate (0.4 g, 1 mmol) was directly dissolved in ethanol (5 mL),
and the mixture was stirred at room temperature for 30 h in the presence of Amberlyst-15 (0.04 g) as the
catalyst. The mixture was filtered and evaporated to obtain 0.31 g of 1-monomyristin as the final product,
a white solid (100.00% yield). Melting point: 53.8–56.7 ◦C. FT-IR (cm−1): 3456 (broad, OH stretching),
2916 (C-H stretching), 1735 (C=O ester), 1465 (CH2 bending), 1180 (C-O ester). LC: 1-monomyristin
(retention time (tR) = 3.17 min, [M + H]+ = 303). 1H-NMR (ppm): 0.87 (t, 3H, -CH3), 1.27 (m, 20H, -CH2-),
1.63 (q, 2H, -C
11.5 Hz, -C 2-OH), 3.70 (dod, 1H, J = 6.8 and 10.8 Hz, -C
(m, 2H, -C
2-OOC-). 13C-NMR (ppm): 14.14 (-CH3), 22.70 (-
34.17 (- H2-CH2-CO), 34.42 (- H2-CO), 63.35 (-CH2-OH), 65.16 (-CH2-O-), 70.28 (-CH-OH), 174.42 (-COO-).
H
2-CH2-CO), 2.18 (s, 2H, OH), 2.35 (t, 2H, -CH2-CO), 3.60 (dod, 1H, J = 6.0 and
2-OH), 3.93 (q, 1H, C -OH), 4.13-4.31
H2-CH3), 24.92–31.93 (other -CH2-),
H
H
H
H
C
C
C
3.4. Synthesis of 2-Monomyristin
3.4.1. Synthesis of Trimyristin
Myristic acid (13.7 g, 60 mmol), glycerol (0.9 g, 10 mmol), and a 5% wt sulfuric acid solution
(0.7 mL) were reacted at 393 K for 3 h. Chloroform (96 mL) was added into the solution and
stirred at 343 K for 30 min. A 10% wt NaOH solution was added into the mixture until the pH
of the mixture reached 13. The product was extracted with n-hexane and the organic layer was
washed with distilled water until neutral. The organic phase was dried over anhydrous sodium
sulfate to obtain 9.62 g of trimyristin as a yellowish solid (54% yield). Melting point: 56.0–58.0 ◦C.
FT-IR (cm−1): 2916 (C-H stretching), 1753 (C=O), 1296 (C-O eter). GC: 100% trimyristin (retention time
(tR) = 18.9 min, [M − 227]+ = 496). 1H-NMR (ppm): 0.87 (t, 9H, -CH3), 1.27 (m, 30H, -CH2-), 1.62 (m, 6H,
-CH2-CH2-CO), 2.34 (t, 6H, OH), 2.34 (t, 6H, -CH2-CO), 4.28 (dod, 4H, J = 4.5 and 11.6 Hz, -CH2-OOC-),

Molecules 2018, 23, 3141
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5.23 (m, 1H, C
H
-OOC-). 13C-NMR (ppm): 14.30 (-CH3), 22.87 (-
C
H2-CH3), 24.86–29.85 (other -CH2-),
32.10 (-CH2-CH2-CO), 34.17 (-CH2-CO), 62.29 (-CH2-O-), 69.03 (-CH-O-), 173.54 (-COO-).
3.4.2. Synthesis of 2-Monomyristin
The trimyristin (0.75 g, 1 mmol) was directly dissolved in dry ethanol (3 mL), and the mixture was
incubated at 308 K for 24 h in the presence of TLIM (0.38 g) as the catalyst. This catalyst was reported for
its regioselectivity [21,22]. The mixture was filtered, and the filtrate was diluted with 80% vol ethanol
(20 mL). The side products were removed using n-hexane (30 mL). The aqueous phase was evaporated
and purified with PTLC with chloroform:acetone:methanol = 9.5:0.45:0.05 as the mobile phase to obtain
0.06 g of 2-myristin (18% yield) as the final product, a yellowish liquid. Melting point: 58.0–60.0 ◦C.
FT-IR (cm−1): 3425 (broad, OH stretching), 2924 (C-H stretching), 1735 (C=O ester), 1465 (CH2 bending),
1
1180 (C-O ester). LC: 2-monomyristin (retention time (tR) = 0.33 min, [M + H]+ = 303). H-NMR (ppm):
0.85 (t, 3H, -CH3), 1.25 (m, 20H, -CH2-), 1.58 (m, 2H, -C
3.92 (m, 1H, C -OOC-), 4.19 (m, 2H, J = 6.5 and 11.5 Hz, -C
22.78 (- H2-CH3), 25.00–29.76 (other -CH2-), 32.00 (- H2-CH2-CO), 34.24 (-C
H
H
2-CH2-CO), 2.33 (t, 2H, -CH2-CO),
2-OH). 13C-NMR (ppm): 14.22 (-CH3),
H2-CO), 63.43 and 65.26
H
C
C
(-CH2-OH), 70.34 (-CH-OH), 174.63 (-COO-).
3.5. Synthesis of 2-Monopalmitin
3.5.1. Synthesis of Tripalmitin
Palmitic acid (15.4 g, 60 mmol), glycerol (0.9 g, 10 mmol), and a 5% wt sulfuric acid solution (0.7 mL)
were reacted at 393 K for 3 h. Chloroform (96 mL) was added into the solution and stirred at 343 K
for 30 min. 10% wt NaOH solution was added into the mixture until the pH of the mixture reached 13.
The product was extracted with n-hexane and the organic layer was washed with distilled water until
neutral. The organic phase was dried over anhydrous sodium sulfate to obtain 9.62 g of tripalmitin as
◦
a yellowish solid (53% yield). Melting point: 65.0–68.0 C. FT-IR (cm−1): 2916 (C-H stretching), 1705 (C=O),
1180 (C-O eter). GC: 100% tripalmitin (retention time (tR) = 21.9 min, [M
567]+ = 239). 1H-NMR (ppm):
0.87 (t, 9H, -CH3), 1.29 (m, 30H, -CH2-), 1.62 (m, 6H, -C 2-CH2-CO), 2.34 (t, 6H, OH), 2.34 (t, 6H, -CH2-CO),
4.27 (dod, 4H, J = 4.5 and 11.6 Hz, -C 2-OOC-), 5.26 (m, 1H, C
-OOC-). 13C-NMR (ppm): 14.30 (-CH3),
22.88 (- H2-CH3), 24.86–29.85 (other -CH2-), 32.10 (- H2-CH2-CO), 34.17 (- H2-CO), 62.29 (-CH2-O-),
−
H
H
H
C
C
C
69.03 (-CH-O-), 173.54 (-COO-).
3.5.2. Synthesis of 2-Monopalmitin
The tripalmitin (0.75 g, 1 mmol) was directly dissolved in dry ethanol (3 mL), and the mixture
was incubated at 308 K for 24 h in the presence of TLIM (0.38 g) as catalyst. The mixture was
filtered and the filtrate was diluted with 80%vol ethanol (20 mL). The side products were removed
by using n-hexane (30 mL). The aqueous phase was evaporated and purified with PTLC with
chloroform:acetone:methanol = 9.5:0.45:0.05 as the mobile phase to obtain 0.03 g of 2-palmitin
(8% yield) as the final product, a yellowish solid. Melting point: 67.0–70.0 ◦C. FT-IR (cm−1):
3433 (broad, OH stretching), 2916 (C-H stretching), 1735 (C=O ester), 1465 (CH bending),
2
1
1180 (C-O ester). LC: 2-monomyristin (retention time (tR) = 0.32 min, [M]+ = 330). H-NMR (ppm):
0.85 (t, 3H, -CH3), 1.25 (m, 20H, -CH2-), 1.58 (m, 2H, -C
3.92 (m, 1H, C -OOC-), 4.19 (m, 2H, J = 6.5 and 11.5 Hz, -C
22.78 (- H2-CH3), 25.00–29.78 (other -CH2-), 32.01 (- H2-CH2-CO), 34.25 (-
70.35 (-CH-OH), 174.48 (-COO-).
H
H
2-CH2-CO), 2.33 (t, 2H, -CH2-CO),
2-OH). 13C-NMR (ppm): 14.22 (-CH3),
H2-CO), 63.41 (-CH2-OH),
H
C
C
C
3.6. Antibacterial and Antifungal Assays of the Synthesized Products
The antibacterial and antifungal assays were carried out in the Microbiology Laboratory
of the Faculty of Veterinary Medicine, Universitas Gadjah Mada using the standard method as
previously described [23]. Candida albicans (C. albicans) was used as the representative of fungi,

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Staphylococcus aureus (S. aureus) and Bacillus subtilis (B. subtilis) were used as representatives
of gram-positive bacteria, and Escherichia coli (E. coli) and Aggregatibacter actinomycetemcomitans
(A. actinomycetemcomitans) were used as representatives of gram-negative bacteria.
3.6.1. Antibacterial Activity Assay of Monoacylglycerol
At first, brain heart broth (6.5 g) was dissolved in distilled water (100 mL) and sterilized using
an autoclave at 394 K under 15 psi pressure for 15 min. The solution was poured into a petri dish
and cooled down to room temperature. The selected bacteria were then introduced separately into
the prepared media. The wells for sample injection were formed by using a capillary glass, and thus
the well diameters were fixed at 6.0 mm. Each of the synthesized products was dissolved in 20.0%
polyethyleneglycol 400 (PEG 400) in distilled water as the diluent at the desired concentration (% w/v).
The sample (50 µL) was added into the wells on the culture media and incubated at 310 K for 30 h.
When the sample showed antimicrobial activity, a transparent zone (called the inhibition zone) was
formed due to the diffusion process of the sample. The antibacterial activity of the monoacylglycerol
was represented from the average diameter inhibition zone (with two replications). The 1.00%
4-isopropyl-3-methylphenol in 20.0% PEG 400 solution was used as the positive control, while the
solvent 20.0% PEG 400 was used as the negative control.
3.6.2. Antifungal Activity Assay of Monoacylglycerol
The antifungal activity assay of monoacylglycerol was carried out in a similar procedure to the
antibacterial assay, except Sabouraud agar powder, which contained 4.00% of dextrose to replace brain
heart broth, was used.
4. Conclusions
This research reported the synthesis of 1-monomyristin, 2-monomyristin and 2-monopalmitin
through simple and efficient reactions. The chemical structure of the synthesized products was
confirmed by FTIR, GC (or LC)-MS, and 1H- and 13C-NMR spectrometers. These synthesis methods
have the potential to be applied in industrial processes owing to the commercially available starting
materials, simple process, short time reaction, and high yield. From the antibacterial and antifungal
assays, the 2-monopalmitin compound did not show either antibacterial or antifungal activities.
Meanwhile, the 1-monomyristin compound exhibited higher antibacterial activity against S. aureus
and A. actinomycetemcomitans and also better antifungal activity against C. albicans compared with the
positive control. The 2-monomyristin compound performed better for antibacterial activity against
E. coli than the positive control. These results show a potential application of either 1-monomyristin or
2-monomyristin compounds as antibacterial and antifungal agents in real applications.
Supplementary Materials: The following are available online.
Author Contributions: J. conceived and designed the experiments; A.N. and A.F. performed the experiments;
D.P. and E.N.S. analyzed the data; B.K. contributed materials/analysis instrument; Y.S.K. wrote and revised the
paper; all authors read and approved the final manuscript.
Funding: This research was financially supported by the Directorate of Research and Development
KEMRISTEKDIKTI through PTUPT Grant 2017.
Acknowledgments: We thank the Microbiology Laboratory of the Faculty of Veterinary Medicine, Universitas
Gadjah Mada for their guidance on antimicrobial and antifungal assays. Asma Nurmala would like to
acknowledge DIKTI, Indonesia, for the scholarship.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).