Monomyristin and Monopalmitin Derivatives: Synthesis and Evaluation as Potential Antibacterial and Antifungal Agents

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.


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 [1].Staphylococcus aureus (S. aureus), one of the gram-positive bacteria, causes skin infections [2], while Candida albicans (C.albicans) gives rise to nosocomial and superficial infections [3].Many researchers have been putting effort into developing an effective and efficient antibacterial and antifungal agent from natural product isolation [4][5][6].He et al. (2017) reported polyketide derivatives from Emericella sp.TJ29 and evaluated their antibacterial activity [7].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 [8].Because of this, researchers prefer to develop synthetic compounds as antibacterial and antifungal agents [9][10][11][12].
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 [15,16].In our previous work, the 1-monolaurin 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 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).

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 1 H-and 13 C-NMR spectra of 1-monomyristin are shown in Figures S1 and S2, respectively.Both 1 H-and 13 C-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 13 C-NMR spectra also show 3 peaks at 63.35, 65.16 and 70.28 for the inequivalent carbon atoms in the glycerol backbone.

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 FTIR, GC-MS, and 1 H-and 13 C-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 1 H-and 13 C-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 1 H-and 13 C-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 1 H-and 13 C-NMR spectra of 2-monomyristin are shown in Figures S3 and S4, respectively.Both 1 H-and 13 C-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 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 1 H-and 13 C-NMR analysis.The absence of the broad peak at 3200-3400 cm −1 on tripalmitin FTIR spectrum shows that all the hydroxyl

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 FTIR, GC-MS, and 1 H-and 13 C-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 1 H-and 13 C-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 1 H-and 13 C-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 1 H-and 13 C-NMR spectra of 2-monomyristin are shown in Figures S3 and S4, respectively.Both 1 H-and 13 C-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 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 1 H-and 13 C-NMR analysis.The absence of the broad peak at 3200-3400 cm −1 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 1 H-and 13 C-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 1 H-and 13 C-NMR.The 1 H-and 13 C-NMR spectra of 2-monopalmitin are shown in Figures S5 and S6, respectively.

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.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.