D-Glucose-Fatty Acid Ester Synthesis with Or Without a Biocatalyst in The

D-Glucose-fatty Acid Ester Synthesis with or without a Biocatalyst in the Same Organic Media Serap Çetinkaya1＊ , Ali Fazıl Yenidünya1, Faika Başoğlu2, and Kamuran Saraç3 1 Department of Molecular Biology and Genetics, Science Faculty, Sivas Cumhuriyet University, TURKEY 2 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Europen University, Lefke, Mersin, TURKEY 3 Department of Chemistry, Faculty of Arts and Science, Bitlis Eren University, Bitlis, TURKEY

Abstract: Esterification of D-glucose with oleic- and palmitic acids were carried out in the absence and presence of a biocatalyst, Candida antarctica lipase. The reaction medium was a mixture of dimethyl sulphoxide and tert-butanol (1:4, v/v). The reaction products were analysed by FTIR, 1H-NMR and 13C- NMR, HSQC, and by ESI-MS. Results indicated that the ester products formed were 6-O-glucose oleate and 6-O-glucose palmitate both in the absence and in the presence of the biocatalyst, with yields above 90%.

Key words: enzymatic synthesis, glucose ester, non-enzymatic synthesis, oleic acid, palmitic acid

1 Introduction experiments. In these reports information on the esterifica- Sugar fatty acid esters（SFAEs）are generally known as tion in control experiments, without using a biocatalyst, biodegradable, nonirritant, nontoxic, nonionic surfactants. has been insufficient2, 3, 5）. This article reported for the first The length of the hydrophobic fatty acid tail and the size of time that glucose could also form ester bonds with various the hydrophilic sugar head render them to possess a wide fatty acids in the absence of a biocatalyst in an organic hydrophilic-lipophilic balance（HLB）values, ranging from 1 medium, dimethyl sulphoxide and tert-butanol（1:4, v/v）at to 16, and enable them to be used as emulsifying agents in 55℃. This reaction medium has often been used for the cosmetics and food. Their antimicrobial and antitumoral enzymatic synthesis of carbohydrate-fatty acid esters2, 4）. activities have also been exploited by the pharmaceutical Both in the presence and in the absence of Candida ant- industry. Chemical or enzymatic methods have been em- arctica lipase esterification yield was above 90％ after 24 h ployed in their synthesis1）. Differing number of acyl group incubation, and stereospecificity of the reaction was also often contributes to the physical properties, such as to the the same. These findings therefore rendered the role of the features of the micelles formed or to the degree of foaming, enzyme reaction contestable in esterification. The results of the esters. were confirmed by FTIR（Fourier Transform Infrared Spec- The initial aim was to synthesize a compound which troscopy）, 1H-NMR and 13C-NMR（Nuclear Magnetic Reso- might mimic the structure and function of the biomolecule nance Spectroscopy）, HSQC（Heteronuclear Single sphingosine. After realising that the reaction media were Quantum Coherence Spectroscopy）, and by ESI-MS enough for the esterification, our interest turn to the syn- （Electro Spray Ionization-Mass Spectrometry）. thesis of sugar-fatty acid esters. Fructose and ribose were also esterified with various fatty acids using the same reaction media. As this pentose sugar molecule has been chosen for the subunit of nucleic acids, its esters with fatty 2 Experimental acids would promise a biosurfactant molecule with novel 2.1 Reagents biological and biotechnological properties. Experimenta- Dimethyl sulphoxide（DMSO; ≥ 99.5％）, D-Glucose, and tion was designed according to the information available in oleic acid（≥ 99％）were obtained from Merck, and tert-bu- the literature2, 3）. Immobilized Candida antarctica lipase tanol（≥ 99.5％）, lauric- and palmitic acids, and immobi- has been used as the biocatalyst2, 4, 5）and the mixtures of di- lized lipase B of Candida antarctica were purchased from methyl sulphoxide: tert-butanol2, 4）or 2-methyl-2-butanol3, 6） Sigma-Aldrich. Molecular sieves were bought from Alfa-Ae- as the organic reaction media in many of the esterification sar.

＊Correspondence to: Serap Çetinkaya, Department of Molecular Biology and Genetics, Science Faculty, Sivas Cumhuriyet University, TURKEY E-mail: [email protected] Accepted March 29, 2020 (received for review March 21, 2020) Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online http://www.jstage.jst.go.jp/browse/jos/ http://mc.manusriptcentral.com/jjocs

737 S. Çetinkaya, A. F. Yenidünya, F. Başoğlu et al.

2.2 Synthesis of glucose esters （Agilent 1260）was used for assessing the conversion per- Esterification reactions with immobilized lipase B of centage of sugar monomers into sugar-fatty acid ester Candida antarctica（S）and control reactions（C）, in the using an Agilent ZORBAX Carbohydrate Analysis Column. absence of a catalyst, were performed under the same con- FTIR（Perkin Elmer 400）, Nuclear Magnetic Resonance（1H- ditions（Fig. 1）. The esterification procedure described by NMR, Varian UNITY INOVA 500 MHz; 13C-NMR Bruker Deng and Zimmermann2）was followed with minor modifica- spectrometer, 125 MHz）, and Mass Spectrometry（Thermo tions: final reaction volume（25 mL）included 20％ DMSO Finnigan LCQ LC-MS/MS Spectrometer）were used for the （v/v）, 80％ tert-butanol（v/v）, 0.2 M D-glucose, and 0.2 M structural analysis of the ester products. oleic acid or 0.6 M palmitic acid. The enzymatic reactions included 140 mg immobilised Candida antarctica lipase B. Water produced during esterification was removed by in- cluding 2 g molecular sieve into the reaction mixture. Es- 3 Results terification was performed for 24 h in a shaker incubator at 3.1 6-O-glucose oleate（10S） 200 rpm at 55℃. Samples were then centrifuged at 5,000 The spectra obtained indicated that glucose existed as rpm and supernatants were transferred into fresh tubes α- and β-anomers in the ester products. and stored at －20℃. FTIR（ATR, cm－1）: 3383（O-H）, 1718（ester C＝O）, 1187 （C-O）, 2881, 2968（aliphatic C-H）. 1 2.3 Thin-Layer Chromatography H-NMR（500 Mhz, DMSO-d6/TMS）δ 0,86（m, 3H, CH3）, For the qualitative analysis of ester products thin layer 1,14（s, 12H, oleic acid H-5,7,12,15,16,17）, 1,23（s, 8H, oleic chromatography was employed. At the bottom of the chro- acid H-4, 6, 13, 14）, 1,47（t, 2H, J＝6,83 Hz, oleic acid H-3）, matography sheets（silica gel 60, Merck）a margin（1 cm） 1,97（q, 4H, J＝ 6,84 Hz, oleic acid H-8, 11）, 2,15（t, 2H, J＝ was left. 7,32 Hz, oleic acid H-2）, 2,40（s, H, glucose H-6）, 2,49-2,50 Small aliquots of the esterification reaction, 2 μL, were （m, 4H, glucose H-2,3,4,5）, 3,40（s, 2H, glucose H-7）, 4,19 loaded on top of the sheet. The samples were resolved （s, 4H, OH）, 5,30（t, 2H, J＝ 4,88 Hz, oleic acid H-9,10）. 13 using a mixture of chloroform（70 mL）, methanol（20 mL）, C-NMR（125 MHz, DMSO-d6/TMS）δ 9,05, 14,36（CH3）, acetic acid（8 mL）, and water（2mL）. After drying, the 22,43, 22,54, 22,81（oleic acid C17）, 24,95, 25,64（oleic acid banding patterns were visualized with p-anisaldehyde/ C3）, 27,01, 27,85（oleic acid C8 and C11）, 28,95, 29,02,

H2SO4 or with orcinol for 10 min at 100℃, and their Rf 29,04, 29,08, 29,15, 29,29, 29,54（oleic acid C4, 5, 6, 13, 14, values were determined2）. 15）, 31,35, 31,50, 31,70（oleic acid C7 and C12）, 31,89 （oleic acid C16）, 34,13, 36,09, 36,37（oleic acid C2）, 61,66 2.4 Structural analyses of the ester products （open chain/cyclic glucose C6）, 67,18, 67,38, 67,57（open High Performance Liquid Chromatography, FTIR, carbon chain glucose C3）, 73,52（cyclic glucose C3）, 72,39, 72,81 and proton NMRs were performed at Research Laboratory （cyclic glucose C2/ open chain glucose C5）, 70,72（open Centre, Erciyes University, Kayseri, Turkey. Mass Spec- chain glucose C4）, 70,72, 77,19（cyclic glucose C4）, 73,52 trometry analyses were performed at Research Laboratory （cyclic glucose C3）, 71,03, 75,25（cyclic glucose C5）, 92,66

Centre, Istanbul University, Istanbul, Turkey. HPLC （cyclic glucose C1）, 174,77（CH2COO）.

Fig. 1 Esterification reaction of oleic acid（a）and palmitic acid（b）with D-glucose. 738 J. Oleo Sci. 69, (7) 737-742 (2020) D-Glucose-fatty Acid Ester Synthesis

3.2 6-O-glucose oleate（10C） group were seen between 1710-1722 cm－1. The -CH asym- FTIR（ATR, cm－1）: 3364（O-H）, 1717（ester C＝O）, 1199 metric aliphatic stress vibration generally peaks at 2924- （C-O）, 2874, 2970（aliphatic C-H）. 2970 cm－1, while the symmetric aliphatic strecting vibra- 13 －1 C-NMR（125 MHz, DMSO-d6/TMS）δ 14,29（CH3）, 22,52, tions peaks at 2850-2872 cm . The palmitic acid still 24,93（oleic acid C3）, 27,00（oleic acid C8, and C11）, 28,94, present in the reaction media was indicated at 1695 cm－1. 29,02, 29,07, 29,13, 29,28, 29,47, 29,53（oleic acid 4, 5, 6, 13, 14, 15, 17）, 31,42, 31,62, 31,81（oleic acid 7,12, and 16）, 4.2 13C-NMR, 1H-NMR and HSQC 34,11（oleic acid C2）, 61,66（cyclic glucose C6/ open chain In sugar ester products, the peak indicating the carbon glucose C6）, 67,21, 67,41, 67,60（open chain glucose C3）, of functional ester group is the most important sign that 70,71（cyclic glucose C4/ open chain glucose C4）, 71,01 and the esterification reaction has taken place. The carbon 75,23（cyclic glucose C5）, 73,52（cyclic glucose C3）, 72,33 atom in the ester group of the glucose oleate（10S）was rep- and 72,79（cyclic glucose C2 / open chain glucose C5）, resented at 174.77 ppm. Although it was seen at 174.87 in 77,13 and 77,16（cyclic glucose C4）, 92,64（cyclic glucose the graph of 7C, this carbon was indicated at 174.77 again C1）, 97,31（open chain glucose C2）, 129,12, 129,98（oleic in the HSQC spectra（Fig. 2）.

acid C9 and C10）, 174,87（CH2COO）. The presence or absence of C-H interactions in HSQC 13 C-NMR（HSQC）（ 125 MHz, DMSO-d6/TMS）δ 14,36（oleic sheds light on whether the production of desired com- 7） acid CH3）, 24,95 and 25,64（oleic acid C-3）, 27,01（oleic pound is achieved . In this work, esterification was carried acid 8-11）, 31,55（oleic acid C-7, 12, 16）, 73,52（cyclic out through the interaction between the acid and alcohol glucose C-3）, 70,72 and 77,19（cyclic glucose C-4）, 92,66 groups. Thus the proof indicating that the ester product （cyclic glucose C-1）, 97,34（open chain glucose C-2）, was formed was provided by the disappearance of the 128,17 and 130,02（oleic acid C-9 and C-10）, 174,77 proton in the COOH group in oleic acid in 1H-NMR.

（CH2COO）. The carbon of COOH in acetic acid is found at 190.7 ppm and in oleic-, and palmitic acids, the carbon of the COOH 3.3 6-O-glucose palmitate（7S） group is represented between 180-182 ppm. The spectral FTIR（ATR, cm－1）: 3364（O-H）, 1722（ester C＝O）, 1196 difference between the carbon of the carboxyl group and （C-O）, 2854, 2927（aliphatic C-H）. that of the carbonyl group arises from a shielding effect 13 C-NMR（125 MHz, DMSO-d6/TMS）δ 13,74（CH3）, 22,30 caused by the carbonyl carbon. Carbonyl group in the acid （palmitic acid C15）, 24,65（palmitic acid C3）, 28,82, 28,99, can resonate. Because carbonyl of the ester group is sur- 29,14, 29,32, 30,63, 30,83 and 31,01（palmitic acid C4, 5, 6, rounded with much higher electron density its carbon 7, 8, 9, 10, 11, 12, 13）, 31,56（palmitic acid C14）, 33,94 cannot resonate and is represented by a lower peak in

（palmitic acid C2）, 36,20 and 40,14（CH2COO）, 68,08 and NMR spectra（Fig. 3）.

68,27（cyclic glucose C-OH）, 175,66（CH2COO）. Carbon peak values of some sugar esters synthesized by Candida antarctica lipase B present in the literature 3.4 6-O-glucose palmitate（7C）（Table 1）. The ester carbon appeared to have assumed FTIR（ATR, cm－1）: 3376（O-H）, 1710（ester C＝O）, 1208 values between 173 ppm to 175 ppm3, 8）. （C-O）, 2850, 2967（aliphatic C-H）. Sugar anomers are unstable structures, and therefore 13 C-NMR（125 MHz, DMSO-d6/TMS）δ 13,69（CH3）, 22,18 the peak at 90-110 ppm of the carbon adjacent to the ring （palmitic acid C14）, 24,52（palmitic acid C2）, 28,70, 28,86, oxygen cannot always be seen by NMR4）. 29,01, 29,19, 30,96（palmitic acid C3, 4, 5, 6, 7, 8, 9, 10, 11, 12）, 30,96（palmitic acid C13）, 40,14 and 61,48（open chain 4.3 ESI-MS analysis of 6-O-glucose oleate（10S） glucose/ cyclic glucose C6）, 67,58, 67,78, 67,96（open chain In the ESI-MS spectrum（Fig. 4）the peak with the great- glucose C3）, 70,48（open chain glucose C4,5/ cyclic glucose est intensity, the Base Peak, at m/z 461.5 corresponded to C2）, 74,56（cyclic glucose C3）, 75,92（cyclic glucose C5）, the molecular ion（［ M＋NH4＋1］＋ , 100）. The peaks at m/ 76,44（cyclic glucose C4）, 92,07 and 96,60（open chain z 462.5 and m/z 459.6 represented adducts of the molecu-

glucose C2/ cyclic glucose C1）, 175,19（CH2COO）. lar ions species（［ M＋NH4＋2］＋ , 21,16）and（［ M＋NH4-1］＋, 10,90）, respectively. These findings were consistent with the expected molecular mass of 6-O-glucose oleate, 444.60 g/mol. Polar sample solutions, not containing strong 4 Discussion acidic or basic groups, can be ionized by the addition of 4.1 FTIR various salts of cations（K＋, Li＋, Na＋ etc.）to facilitate In the spectra of glucose-palmitic acid and glucose-oleic positive ion formation in the ESI analysis, while chlorine acid esters, the OH stress vibration, observed between containing salts, such as chloroform, are used to facilitate 3364-3387 cm－1, indicated the intramolecular hydrogen the formation of negative ions11）. bond. The peaks belonging to the C＝O ester functional

739 J. Oleo Sci. 69, (7) 737-742 (2020) S. Çetinkaya, A. F. Yenidünya, F. Başoğlu et al.

Fig. 2 HSQC spectrum of 6-O-Glucose oleate.

13 Fig. 3 Comparison of C-NMR spectra obtained from‘ Novo 435-catalysed’（ upper）and‘ non-catalysed’（ lower）6-O-glucose oleate（left）and 6-O-glucose palmitate.

740 J. Oleo Sci. 69, (7) 737-742 (2020) D-Glucose-fatty Acid Ester Synthesis

Table 1 Some 13C-NMR peak found for the carbon of ester carbonyl in the literature. CH COO δ13C CH COO δ13C CH δ13C Ester 2 2 3 Reference (ppm) (ppm) (ppm) 6-O-palmitoilglucose 33.7 173.0 14.1 9) 6-O-palmitoilglucopyranose 33.9 173.3 14.3 3) 5-O-palmitoil-L-(+)-arabinose 64.6 173.2 14.3 4) 6-O-glucose stearate 34.5 174.2 22.1 10) 1,6-diacyl-O-fructofuranose 35.0 175.2 14.5 8)

Fig. 4 ESI-MS spectrum of 6-O-glucose oleate.

5 Conclusion form both in the absence and in the presence of a biocata- Mechanism of non-enzymatic esterification can be briefly lyst in an organic reaction medium consisting of DMSO and summarized as follows（Fig. 5）. An esterification reaction tert-butanol or of DMSO 2-methyl-2-butanol at 55℃. The starts according to Fischer reaction. Here the proton（H＋）, reactions appeared to have reached completion after 24 h. released in the appropriate rxn environment, attacks the These reaction media and reaction conditions have been free electrons of the oxygen in the C＝O group of the acid. specifically designed for the enzymatic esterification of It terminates after the formation of a water molecule. Since sugars and fatty acids. this reaction proceeds through the SN2 mechanism, A separate manuscript was prepared for the ribose work. primary alcohols react better than secondary alcohols. Because the information on glucose-fatty acid esters, syn- Therefore, the free electrons on the oxygen of the primary thesized with a biocatalyst, has been available in literature, alcohol make an electrophilic attack on the partial positive it was decided that the glucose work should be published portion of the acid. Enzymatic esterification also occurs in first. Furthermore, these works were performed within the accordance with the synthesis mechanisms of basic organic limits of a PhD thesis. Biological activities of the ester chemistry4）. In addition, the solvents used in enzymatic products will soon be investigated in separate projects. synthesis are similar to the solvents used in this study. This study indicated that sugar-fatty acid esters could

741 J. Oleo Sci. 69, (7) 737-742 (2020) S. Çetinkaya, A. F. Yenidünya, F. Başoğlu et al.

Fig. 5 Mechanism of acid catalyzed esterification.

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