Carbohydrate Research 414 (2015) 32e38
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Enzymatic synthesis and characterization of galactosyl monoesters Dong An, Xiaohui Zhao, Zhiwen Ye* Department of Chemical Engineering, Nanjing University of Science and Technology, Nanjing 210094, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 January 2015 Received in revised form 15 April 2015 Accepted 26 May 2015 Available online 4 June 2015
Enzyme-catalyzed synthesis of several fatty acyl-amino acid esters based on D-galactose, as well as their chemical evaluation, was performed. These novel galactosyl fatty acyl-amino acid monoesters were synthesized by utilizing lipase from lipozyme TL IM in tert-butanol with D-galactose and fatty acyl-amino acids as starting materials. The products were characterized by 1H NMR, 13C NMR and MS analysis. In addition, their primary physical properties, such as hydrophilic-lipophilic balance (HLB), critical micellar concentration (CMC), solubility in water, maximum surface excess (Gmax), and minimal surface tension (Amin) were measured. The experimental results showed that their CMC values are between 5 and 0.4 mM. The HLB values of galactosyl esters 15e17 indicate that they are useful as oil-in-water emulsions or detergents, whereas 18e22 can be employed as water-in-oil emulsifiers or wetting agents. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Surfactant Fatty acid Galactosyl ester Glycine Valine
1. Introduction Sugar esters, non-ionic surfactants, including one or more fatty acid esters chain as lipophilic moiety and hydrophilic carbohydrate moiety, are widely used in pharmaceutical formulations, food and cosmetic, often termed ‘green surfactants’.1,2 Novel galactosyl esters might be further useful in other fields like bioremediation and membrane protein extraction. They can also been used as foaming agents or emulsifiers to clean products utilizing their good detergent properties.3e5 Sugar esters are primarily synthesized by chemical methods using acid as catalyzer, which causes environmental pollution.6 The method proceeds at rigor conditions such as high temperature, high pressure and has low selectivity and plenty of outgrowths.7 In recent years, surface-active molecules of biological origin obtained by bioprocesses as well as enzymatic synthesis have been reported.8e11 Lipases are not only responsible for the hydrolysis of esters but also they can be widely used in organic synthesis. Owing to the regioselectivity and mild reaction conditions,12e16 enzymatic syntheses have potential applications on the carbohydrate research. Therefore, it is significant to select suitable hydrolases via reversed hydrolysis in order to gain the desired product without byproducts. Many lipases have been successfully used to catalyze the esterification of sugar and fatty acids, but the high cost of
lipases limits industrial use. However, galactosyl esters could be synthesized utilizing some appropriate enzyme catalysts, for instance, cheaper lipase Lipozyme TL IM.17e19 Sometimes, the enzymatic synthesis of sugar esters is restricted by the low solubility of sugars in non-polar organic solvents, in which lipases can exhibit their esterification activity.20e22 Whereas most lipases lose their esterification activity in polar solvents, in which sugars are soluble.23e25 Different strategies have been proposed to handle this problem.26,27 Some solvents are found to keep solubility of sugars and activity of lipases. For example, the uses of t-butanol have been reported in which sugars solubility and enzyme activity are given.28e30 Surface tension is one of the important parameters of liquid interfacial properties. The surface activity, CMC and adsorption capacity at the surface, which were theoretical explanation on mechanism of surfactant in the gathering process, can be determined by the surface tension data. So, the surface tension and CMC were researched firstly when a new type of surfactant was synthesized. We attempt to synthesize and study the physical properties of galactosyl fatty acyl-amino acid esters and exploit much more this kind of surfactants by enzyme catalysis in future.31e33 2. Results and discussion 2.1. Synthesis
* Corresponding author. E-mail address: [email protected] (Z. Ye). http://dx.doi.org/10.1016/j.carres.2015.05.011 0008-6215/© 2015 Elsevier Ltd. All rights reserved.
The enzymatic synthesis of galactosyl monoesters are based on the following materials: D-galactose, glycine (1), valine (2), fatty
D. An et al. / Carbohydrate Research 414 (2015) 32e38
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acyl chloride (3e6). Firstly, 7e14 were prepared from two amino acid by reaction with different fatty acyl chloride (decanoyl chloride, dodecanoyl chloride, tetradecanoyl chloride, hexadecanoyl chloride) in the presence of catalytic amounts of sodium hydroxide and purified by crystallization from methyl alcohol, respectively. Then, direct esterification of D-galactose, using fatty acyl-amino acids (7e14) and catalyst lipozyme TL IM in t-butanol, yielded two series of galactosyl esters 15e22 as shown in Scheme 1. 2.2. Structural information The 1H NMR spectra of all the final products showed that the coupling constant of H1 was about 4 Hz, indicating that the glycosidic linkage has an configuration. We conclude O6 is not esterified with the amino acid-fatty acids, because the two H6 were shown to multiple peaks with the chemical shift at d (symbol: delta) 3.10e3.14. As a consequence, the resulting ester should be linked to the anomeric carbon. The 1H and 13C NMR spectra indicated that the chemical shifts only displayed minor differences as compared to those of the corresponding starting materials. Taking compound 19 as an example, in the 1H NMR spectra, the amide proton exhibited a chemical shift at around d (symbol: delta) 8.21e8.23 as a triplet. H1 and H2, H3, H4, H5, H6 were shown chemical shift at d 5.34e5.35 as double peaks and 3.65e3.62, 3.47e3.42, 3.25e3.23, 3.22e3.19, 3.14e3.10 as multiple-peaks. The chemical shift of CH3 and CH2 in the fatty chain were mainly located from 0.83 to 2.14. In the 13C NMR spectra, the carbon of amide and carbonyl revealed significant shift at around d 173.24 and 169.53. The chemical shift of C1, C2, C3, C4, C5, C6 position on were around 95.25, 72.93, 76.71, 69.82, 78.42, 60.90.
Fig. 1. Effects of pH value on the stability of 16 at different times.
2.3. Stability study The absorbance values decreased further after 1e4 h, which means the galactosyl esters 16, 20 might be partially hydrolyzed in solution of strong acid and strong base. In contrast, they could be stable in the range of pH 5.5e8.5 solution. These experimental values indicate that these types of galactosyl fatty-acid amino-acid esters 15e22 are chemical stability in the environment of weak acid, weak base or neutrality (Fig. 1 and Fig. 2). 2.4. Optimal reaction conditions In this present study, t-butanol, which has low toxicity was served as solvent in this enzymatic reaction. The effects of various parameters on the yield were investigated in detailed. The optimum reaction conditions were gained following: 1 mmol a-Dgalactose, l.5 mmol acid, 0.49 g enzyme, 10 mL t-butanol and 0.8 g activated molecular sieve 4 Å as water absorbant after 6 h at 40 C. As can be seen, higher temperature and longer reaction time might lead to decrease the activity of enzyme and produce galactosyl diesters, resulting in diminished yield (Table 1).
Fig. 2. Effects of pH value on the stability of 20 at different times.
Lipozyme TL IM had a good catalytic activity from 25 C to 60 C in t-butanol. However, the fields of galactosyl esters 15e22 increased and decreased were happened with the reaction temperature rising. On the one hand, the field of the enzymatic reaction accelerated with the temperature rises from 25 C to 40 C. On the other hand, the irreversible change of spatial structure might
Scheme 1. Enzymatic synthesis of galactosyl esters (15e22). Reagents and conditions: a) 20% NaOH, acetone; b) D-galactose, TLL, t-butanol, 40 C.
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D. An et al. / Carbohydrate Research 414 (2015) 32e38
Table 1 Chemical yields in the enzymatic synthesis of galactosyl esters 15e22 Reagenta
T ( C)
Time (h)
Product
Yield (%)
7 7 7 7 7 8 8 9 10 11 11 12 13 14 14
25 30 35 40 45 40 40 40 40 40 40 40 40 40 40
6 6 6 6 6 7 6 6 6 6 7 6 6 8 6
15 15 15 15 15 16 16 17 18 19 19 20 21 22 22
77.3 86.4 93.1 94.5 83.1 94.2 95.8 83.2 83.1 89.6 87.2 85.3 78.5 73.2 71.7
a
Molar ratio Acyl donor(fatty acid):a-D- galactosyl¼1.5:1.
deactivate activity of lipozyme TL IM molecules at higher temperature. Thus, we selected 40 C as the optimal temperature for these reactions (Fig. 3). As can be seen in Fig. 4, the fields of galactosyl esters 15e22 increased and decreased gradually with increasing reaction time from 2 h to 8 h. The highest conversion was achieved at 6 h. It was probably connected with reversible enzymic reaction, which took place under increasing H2O by-product as the time prolonged. In addition, galactosyl diesters were further produced when the conversion of galactosyl monoesters were over a maximum value. Based on the data, the highest fields are obtained after 6 h. 2.5. Surface tension results The galactosyl esters were characterized with their HLB values, solubility in water, CMC values, Gmax, and Amin. The HLB values of galactosyl esters 15e17 slightly decreased from 14.8 to 11.2 with the chain length of fatty acid increasing. Thus, these HLB values and the water solubility from 466±11 to 219±17 mmol1 indicated that they are suitable for oil-in-water (o/w) emulsion cleansing agents or detergents. On the contrary, the HLB values of the galactosyl esters
Fig. 4. Time course of galactosyl fatty acyl-amino-acid 15e22 synthesis reaction (Reaction conditions:1 mmol D-galactose, 1.5 mmol/L fatty acyl-glycine 7e14, 0.8 g molecular sieve and 0.3 g lipozyme TL IM in 10 mL tert-butanol at 40 C and 150 rpm for 2e8 h).
18e22 between 5.5 and 9.6 are suggested their promising applications in oil-soluble hydrophobic (w/o) emulsifiers or wetting agents (Table 2). The CMC values can be determined by a plot of the surface tension versus the concentration. The plots of surface tension versus concentration (C-g) for 15e22 were demonstrated in Fig. 5 and Fig. 6. The CMC values of galactosyl fatty acyl-glycine esters 15e18 were 4.97103, 3.96103, 1.87103, and 4.8104 mol/L, and galactosyl fatty acyl-valine esters 19e22 were 4.70103, 3.74103, 1.62103 and 4.0104 mol/L, respectively. The CMC decreased as the chain length of fatty acid ester increase. It is consistent with our suggestion that the different fatty acyl-amino acid hydrophobic chain are responsible for determining the CMC values of galactosyl monoesters 15e22 in aqueous solution (Table 3). The packing densities of surfactants at the air/aqueous solution interface are also important for the interpretation of the surface activity. The maximum surface excess (Gmax), at the air/solution interface was calculated by applying the Gibbs adsorption isotherm Eq. 1. The minimum area occupied by a surfactant molecule at the air/solution interface (Amin) can be estimated from the relation (Eq. 2).34
Gmax ¼
1 dg 2:303nRT d logc
(1)
Table 2 Solubility in water and HLB value of galactosyl monoesters 15e22
Fig. 3. Effect of temperature on the field of galactosyl fatty acyl-glycine ester 15 (Reaction conditions:;1 mmol D-galactose, 1.5 mmol L1 7, 0.8 g molecular sieve and 0.3 g lipozyme TL IM in 10 mL tert-butanol at 150 rpm for 6 h).
Compounds
Solubility in water/mmol1
Measure HLB
15 16 17 18 19 20 21 22
466±11 254±10 219±17 178±14 198±11 164±14 142±6 133±10
14.3±0.5 12.4±0.4 11.0±0.2 9.1±0.5 6.9±0.4 6.5±0.4 5.9±0.7 5.1±0.4
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Table 3 The surface active parameter of 15e22 in distilled water solution at 25 C Compound
CMC (mmol/L)
gcmc (mN/m)
Gcmc (mmol/m2)
Amin (nm2)
15 16 17 18 19 20 21 22
4.97 3.96 1.87 0.48 4.70 3.74 1.62 0.40
31.44 30.52 29.13 28.09 30.84 29.75 28.68 27.82
1.01 1.00 0.99 1.16 1.02 0.97 0.95 1.02
1.64 1.66 1.67 1.43 1.63 1.71 1.75 1.63
(△Hqm). The thermodynamic parameters are estimated by the mass action model.36 △Gqm can be obtained from Eq. 3. The enthalpy of micelle formation can be obtained by using the GibbseHelmboltz equation (Eq. 4) Then the entropy of micelle formation is determined by using Eq. 5.37,38
Fig. 5. Relationship between CMC of galactosyl monoesters 15e18 and surface tension at 25.0 C.
DGqm ¼ RT ln
CMC 55:5
q DHm ¼ 2:303R
Amin
1024 ¼ NA Gmax
(2)
where c is the surfactant concentration, R is the gas constant(8.314 J mol1 K1), T is the absolute temperature, Gmax is the maximum surface excess in mmol/m2, g is the surface tension in mN/m, and (d g/d logc) is the slope in the surface tension isotherm when the concentration is near the CMC. The value of n is taken as 1 due to the fact that the galactosyl esters are conventional surfactants.35 When maximum estimated, the value of Amin is obtained using Eq. 2. NA is Avogadro's number and Amin is in nm2/molecule. 2.6. Thermodynamic analysis of micellization The micellization of galactosyl fatty-acid amino-acid esters (15e22) could be indicated by some thermodynamic parameters, such as the standard Gibbs energy change (△Gqm), the standard entropy change (△Sqm), and the standard enthalpy change
d log CMC dð1=TÞ
. q DSqm ¼ DHm DGqm T
(3)
(4)
(5)
where T is the absolute temperature, and R is the ideal gas constant (8.314 J mol1 K1). The thermodynamic parameters of galactosyl fatty acyl-amino acid esters 15e22 micellization at different temperatures were listed in Table 4. As can be seen, the CMC values decreased with the length of hydrophobic group increasing and increased as the temperature raising. More important, the galactosyl esters 15e22 micellization were spontaneous based on the negative Gibbs free energy changes. In addition, the micelle formation was exothermic process, which could be attributed to the surfactantesolvent interactions because the values of standard enthalpy change (△Hqm) were negative. The large positive △Sqm was mainly caused by the negative values of △Gqm as well as the values of T△Sqm were much bigger than △Hqm. Therefore, the spontaneous micellization of 15e22 were entropy-driven process and thermodynamic stable systems. 3. Experimental 3.1. Materials and methods NMR were recorded on a Bruker 500 MHz spectrometer. Mass spectra were performed by a Finnigan TSQ Quantum ultra AM mass spectrometer. Silica gel 200e300 mesh was used for column chromatography. CMC values were determined by a BZY-2 fully automatic surface tensiometer using the Wilhelmy Type method at 25.0 C. Lipozyme TL IM (TLL, Thermomyces lanuginosus immobilized on silica,50,000 U/g)was obtained from the SigmaeAldrich Trading Co., Ltd. (Shanghai, China). Spectrophotometer (446 nm) (Shanghai, China). Commercially available grades of organic solvents of adequate purity were used in all reactions. 3.2. General procedure for the preparation of N-acyl amino acids 7e1439
Fig. 6. Relationship between CMC of galactosyl monoesters 19e22 and surface tension at 25.0 C.
Taking compound 7 as an example, glycine 1 (4.5 g, 0.06 mol) was dissolved in the mixture of acetone and deionized water (2:1
36
D. An et al. / Carbohydrate Research 414 (2015) 32e38
Table 4 Thermodynamic parameters of galactosyl esters 15e22 at different temperature Glucosyl ester
T ( C)
CMC (mmol/L)
△Gqm (kJ/mol)
△Hqm (kJ/mol)
T△Sqm (kJ/mol)
△Sqm (kJ/mol)
15
20 25 30 35 20 25 30 35 20 25 30 35 20 25 30 35 20 25 30 35 20 25 30 35 20 25 30 35 20 25 30 35
4.5 4.97 5.07 5.25 3.55 3.96 5.04 5.15 1.27 1.87 2.08 2.13 0.40 0.48 0.62 0.68 4.33 4.70 4.91 5.07 3.55 3.74 4.02 4.37 1.43 1.62 2.09 2.12 0.23 0.40 0.43 0.47
25.20 23.08 26.96 26.57 21.77 23.64 26.73 26.66 23.53 28.32 29.58 36.87 26.19 28.87 32.11 33.85 20.17 23.22 29.83 26.59 21.17 23.79 27.19 28.13 25.77 25.86 28.78 29.71 33.21 29.32 38.59 40.05
2.89 1.79 2.84 5.38 2.35 1.73 0.41 4.08 1.31 0.93 0.61 0.78 2.88 1.79 4.73 5.44 0.45 0.67 0.72 0.97 1.23 1.77 0.79 0.56 0.72 0.77 0.64 0.68 2.97 2.88 3.24 3.72
22.31 22.01 20.24 21.19 19.42 21.91 26.32 22.58 22.22 27.39 28.97 36.09 23.31 27.08 27.38 28.41 19.72 22.55 29.11 25.62 19.94 22.02 26.40 27.57 25.05 25.09 28.14 29.03 30.24 26.44 35.35 36.33
0.076 0.074 0.067 0.069 0.066 0.074 0.080 0.074 0.081 0.092 0.096 0.117 0.080 0.091 0.090 0.094 0.067 0.076 0.096 0.083 0.068 0.074 0.087 0.090 0.085 0.084 0.093 0.094 0.103 0.089 0.117 0.118
16
17
18
19
20
21
22
by volume), 5% (wt %) NaOH aqueous solution were added dropwise into the mixture until pH was at 8.5e9.5. Then, decanoyl chloride (8.74 g, 0.04 mol) was dropwise under strongly stirring at 0e5 C. At same time, the pH was kept a range from 7.5 to 11.5 adjusted with 5% NaOH aqueous solution. Then, the mixed liquid was stirred at 0e5 C for another 2.5 h and stood at room temperature overnight. The solvent was removed by vacuum distillation, then the mixture of concentrated hydrochloric acid and water (1:1 by volume) was added to the residues until pH reached 1 to 2. White solid was gradually precipitated. The white solid was obtained by vacuum filtration and washed with eionized water until pH 7 of the filtrate, then washed with petroleum ether (15 mL) three times. The crude products were by recrystallized with methanol to obtain 7 (7.29 g, 75%). Compounds 8e14 were obtained by the same method and 71e79 % yield. 3.3. General synthesis methods of galactosyl esters 15e22 3.3.1. 1-O-(N-Decanoylglycineyl)-D-galactosyl ester (15) A solution of a-D-galactose (0.18 g, 1 mmol), N-decanoylglycine 7 (0.34 g, 1.5 mmol), lipozyme TL IM (0.49 g) and activated molecular sieve 4 Å (0.8 g) in butanol was allowed to stir at 40 C for 6 h under nitrogen. Samples from the reaction mixture were analyzed by TLC (1:1 ethyl acetate:methanol). At the end of the reaction, the mixture was extracted with 20 mL ethyl isopropanol:acetate (1:4) by stirring at rt for 15 min. Particularly, the glycosyl esters extraction was performed with 20 mL dichloromethane and the immobilized enzyme was separated from the reaction mixture by flotation allowing easy recovery. Organic solvent was removed in vacuo and the crude product was purified by column chromatography (10:1e20:1 ethyl acetate:methanol) to give white solid 15 (0.37 g, 94.5%); mp.: 100.2e101.8 C; 1H NMR (500 MHz, DMSO) d 8.22 (t, 1H, J¼6.0 Hz, NH), 5.35 (d, 1H, J¼4.0 Hz, H-1), 5.27 (d, 1H, J¼5.0 Hz,
C4eOH), 5.10 (d, 1H, J¼5.0 Hz, C2eOH), 5.01 (d, 1H, J¼5.5 Hz, C3eOH), 4.61 (t, 1H, J¼6.0 Hz, C6eOH), 3.92 (dd, 1H, J¼16.0, 6.0 Hz, aliphatic OCOCH2), 3.86 (dd, 1H, J¼16.0, 6.0 Hz, aliphatic OCOCH2), 3.65e3.62 (m, 1H, H-2), 3.47e3.42 (m, 1H, H-3), 3.25e3.23 (m, 1H, H-4), 3.22e3.19 (m, 1H, H-5), 3.14e3.10 (m, 2H, H-6), 2.12 (t, 2H, J¼7.5 Hz, aliphatic NHCOCH2C8H17), 1.50e1.48 (m, 2H, aliphatic NHCOCH2CH2C7H15), 1.24 (s, 12H, NHCOC2H4C6H12CH3), 0.87 (t, 3H, J¼7.0 Hz, aliphatic NHCOC8H16CH3). 13C NMR (125 MHz, CDCl3) d 173.24 (s, NHC]O), 169.54 (2s, C]O), 95.23 (C-1), 78.37(C-5), 76.66(C-3), 72.88(C-2), 69.79(C-4), 60.90(C-6), 35.56, 31.76, 29.52, 29.47, 29.37, 29.06, 25.67, 22.55 (each aliphatic CH2), 14.35 (CH3); ESIMS: Found, m/z 414.09; required, 414.22 [MþNa]þ. 3.3.2. 1-O-(N-Dodecanoylglycineyl)-D-galactosyl ester(16) Treatment of 8 (0.38 g, 1.5 mmol) as described for 15 gave white solid 16 (0.40 g, 95.8%); mp.: 98.3e99.7 C. 1H NMR (500 MHz, DMSO) d 8.21 (t, 1H, J¼6.0 Hz, NH), 5.35 (d, 1H, J¼4.1 Hz, H-1), 5.27 (d, 1H, J¼5.0 Hz, C4eOH), 5.10 (d, 1H, J¼5.0 Hz, C2eOH), 5.01 (d, 1H, J¼5.5 Hz, C3eOH), 4.61 (t, 1H, J¼6.0 Hz, C6eOH), 3.92 (dd, 1H, J¼16.0, 6.0 Hz, aliphatic OCOCH2), 3.86 (dd, 1H, J¼16.0, 6.0 Hz, aliphatic OCOCH2), 3.65e3.62 (m, 1H, H-2), 3.47e3.42 (m, 1H, H-3), 3.25e3.23 (m, 1H, H-4), 3.22e3.19 (m, 1H, H-5), 3.14e3.10 (m, 2H, H-6), 2.12 (t, 2H, J¼7.5 Hz, aliphatic NHCOCH2C10H21), 1.51e1.47 (m, 2H, aliphatic CH2C9H19), 1.24 (s, 16H, C8H16CH3), 0.86 (t, 3H, J¼7.0 Hz, aliphatic NHCOC10H20CH3); 13C NMR (125 MHz, DMSO) d 173.24 (s, NHC]O), 169.54 (s, C]O), 95.19 (C-1), 78.41(C-5), 76.68(C-3), 72.87(C-2), 69.80(C-4), 60.83(C-6), 35.56, 31.75, 29.51, 29.45, 29.40, 29.27, 29.03, 22.56 (each aliphatic CH2), 14.35 (CH3). ESI-MS: m/z 442.11 [MþNa]þ. 3.3.3. 1-O-(N-Myristylglycineyl)-D-galactosyl ester(17) Treatment of 9 (0.42 g, 1.5 mmol) as described for 15 gave white solid 17 (0.37 g, 83.2%); mp.: 95.8e97.4 C. 1H NMR (500 MHz,
D. An et al. / Carbohydrate Research 414 (2015) 32e38
DMSO) d 8.22 (t, 1H, J¼6.0 Hz, NH), 5.35 (d, 1H, J¼4.0 Hz, H-1), 5.27 (d, 1H, J¼5.0 Hz, C4eOH), 5.10 (d, 1H, J¼5.0 Hz, C2eOH), 5.01 (d, 1H, J¼5.5 Hz, C3eOH), 4.61 (t, 1H, J¼6.0 Hz, C6eOH), 3.92 (dd, 1H, J¼16.0, 6.0 Hz, aliphatic OCOCH2), 3.86 (dd, 1H, J¼16.0, 6.0 Hz, aliphatic OCOCH2), 3.65e3.62 (m, 1H, H-2), 3.47e3.42 (m, 1H, H-3), 3.25e3.23 (m, 1H, H-4), 3.22e3.19 (m, 1H, H-5), 3.14e3.10 (m, 2H, H-6), 2.12 (t, 2H, J¼7.5 Hz, aliphatic NHCOCH2C12H25), 1.51e1.47 (m, 2H, aliphatic NHCOCH2CH2C11H23), 1.24 (s, 20H, NHCOC2H4C10H20CH3), 0.86 (t, 3H, J¼7.0 Hz, aliphatic NHCOC12H24CH3). 13C NMR (125 MHz, CDCl3) d 173.24 (s, NHC]O), 169.54 (s, C]O), 95.25 (C-1), 78.42(C-5), 76.71(C-3), 72.93(C-2), 69.82(C-4), 60.90(C-6), 35.56(s), 31.76, 29.52, 29.47, 29.42, 29.29, 29.17, 29.06, 25.65, 22.56 (aliphatic CH2), 14.41 (q, aliphatic CH3). ESI-MS: m/z 470.15 [MþNa]þ. 3.3.4. 1-O-(N-Palmitoylglycineyl)-D-galactosyl ester (18) Treatment of 10 (0.46 g, 1.5 mmol) as described for 15 gave white solid 18 (0.39 g, 83.1%); mp.: 83.2e84.7 C; 1H NMR (500 MHz, DMSO) d 8.23 (t, 1H, J¼5.0 Hz, NH), 5.35 (d, 1H, J¼4.2 Hz, H-1), 5.27 (d, 1H, J¼5.0 Hz, C4eOH), 5.10 (d, 1H, J¼5.0 Hz, C2eOH), 5.01 (d, 1H, J¼5.5 Hz, C3eOH), 4.61 (t, 1H, J¼6.0 Hz, C6eOH), 3.93 (dd, 1H, J¼18.0, 7.0 Hz, aliphatic OCOCH2), 3.85 (dd, 1H, J¼18.0, 6.0 Hz, aliphatic OCOCH2), 3.65e3.62 (m, 1H, H-2), 3.47e3.42 (m, 1H, H-3), 3.25e3.23 (m, 1H, H-4), 3.22e3.19 (m, 1H, H-5), 3.14e3.10 (m, 2H, H-6), 2.12 (t, 2H, J¼7.5 Hz, aliphatic NHCOCH2C14H29), 1.51e1.47 (m, 2H, aliphatic NHCOCH2CH2C13H27), 1.24 (s, 24H, NHCOC2H4C12H24CH3), 0.86 (t, 3H, J¼7.0 Hz, aliphatic NHCOC14H28CH3). 13C NMR (125 MHz, CDCl3) d 173.05 (s, NHC]O), 169.30 (s, C]O), 95.00 (d, C-1), 78.16 (C-5), 76.45 (C-3), 72.68 (C-2), 69.58 (C-4), 60.66 (C-6), 35.31 (s), 31.52, 29.28, 29.24, 29.18, 29.05, 28.93, 28.81, 25.41, 22.32 (each aliphatic CH2), 14.16 (q, aliphatic CH3). ESI-MS: m/z 498.17 [MþNa]þ. 3.3.5. 1-O-(N-Decanoylvalineyl)-D-galactosyl ester (19) Treatment of 11 (0.41 g, 1.5 mmol) as described for 15 gave yellow liquid 19 (0.38 g, 89.6%); 1H NMR (500 MHz, DMSO) d 8.07 (d, 1H, J¼3.5 Hz, NH), 5.34 (d, 1H, J¼4.1 Hz, H-1), 5.22 (d, 1H, C4eOH), 5.12 (d, 1H, J¼5.0 Hz, C2eOH), 5.07 (d, 1H, J¼5.5 Hz, C3eOH), 4.66 (t, 1H, J¼6.0 Hz, C6eOH), 4.31 (dd, 1H, J¼17.0, 6.0 Hz, CH2), 4.30 (dd, 1H, J¼17.0, 6.0 Hz, CH2), 3.64e3.61 (m, 1H, H-2), 3.57e3.51 (m, 1H, H-3), 3.25e3.23 (m, 1H, H-4), 3.22e3.19 (m, 1H, H-5), 3.14e3.10 (m, 2H, H-6), 2.19e2.11 (m, 3H, CH2, CH), 1.47e1.46 (m, 2H, CH2C7H15), 1.22 (s, 12H, C6H12CH3), 1.06e1.04 (m, 9H, CH3,(CH3)2). 13C NMR (125 MHz, DMSO) d 173.90 (s, NHC]O), 171.10 (s, C]O), 95.14 (d, C-1), 76.69 (C-5), 72.80 (C-3), 69.77(C-2), 60.83(C-4), 56.60(C-6), 35.34(s), 31.65, 30.18, 29.28, 29.11, 28.91, 25.75, 19.35(CH2),18.74, 18.04 14.35 (CH3). ESI-MS: m/z 456.28 [MþNa]þ. 3.3.6. 1-O-(N-Palmitoylvalineyl)-D-galactosyl ester (20) Treatment of 12 (0.45 g, 1.5 mmol) as described for 15 gave yellow liquid 20 (0.39,85.3%); 1H NMR (500 MHz, DMSO) d 8.25 (d, 1H, J¼4.0 Hz, NH), 5.35 (d, 1H, J¼4.0 Hz, H-1), 5.20 (d, 1H, J¼5.0 Hz, C4eOH), 5.12 (d, 1H, J¼5.0 Hz, C2eOH), 5.05 (d, 1H, J¼5.5 Hz, C3eOH), 4.66 (t, 1H, J¼6.0 Hz, C6eOH), 4.30 (dd, 1H, J¼17.0, 6.5 Hz, CH2), 4.31 (dd, 1H, J¼17.0, 6.5 Hz, CH2), 3.64e3.61 (m, 1H, H-2), 3.58e3.54 (m, 1H, H-3), 3.25e3.23 (m, 1H, H-4), 3.21e3.19 (m, 1H, H-5), 3.14e3.10 (m, 2H, H-6), 2.18e2.15 (m, 3H, CH2, CH), 1.47e1.44 (m, 2H, CH2), 1.21 (s, 18H, C9H18CH3), 1.05e1.04 (m, 9H, CH3, (CH3)2). 13 C NMR (125 MHz, DMSO) d 173.90 (s, NHC]O), 171.10 (s, C]O), 95.14 (C-1), 76.69 (C-5), 72.80 (C-3), 69.77(C-2), 60.83(C-4), 56.60(C-6), 35.34(s), 31.65, 30.18, 29.28, 29.11, 28.91, 25.75, 19.35(CH2),18.74, 18.04 14.35 (CH3). ESI-MS: m/z 479.28; [MþNH4]þ.
37
3.3.7. 1-O-(N-Myristylvalineyl)-D-galactosyl ester (21) Treatment of 13 (0.49 g, 1.5 mmol) as described for 15 gave light yellow liquid 21 (0.38 g, 78.5%); 1H NMR (500 MHz, DMSO) d 8.08 (m, 1H, NH), 5.34 (d, 1H, J¼4.1 Hz, H-1), 5.22 (d, 1H, J¼5.3 Hz,C4eOH), 5.12 (d, 1H, J¼5.0 Hz, C2eOH), 5.07 (d, 1H, J¼5.5 Hz, C3eOH), 4.66 (t, 1H, J¼6.0 Hz, C6eOH), 4.31 (dd, 1H, J¼17.5, 6.5 Hz, CH2), 4.30 (dd, 1H, J¼17.5, 6.5 Hz, CH2), 3.64e3.61 (m, 1H, H-2), 3.57e3.51 (m, 1H, H-3), 3.25e3.23 (m, 1H, H-4), 3.22e3.19 (m, 1H, H-5), 3.14e3.10 (m, 2H, H-6), 2.19e2.11 (m, 3H, CH2,CH), 1.47e1.46 (m, 2H, CH2C12H24), 1.22 (s, 22H, C11H22CH3), 1.03e1.01 (m, 9H, CH3,(CH3)2). 13C NMR (125 MHz, CDCl3) d 173.90 (s, NHC] O), 171.10 (s, C]O), 95.14 (C-1), 76.69 (C-5), 72.80 (C-3), 69.77(C-2), 60.83(C-4), 56.60(C-6), 35.34(s), 31.65, 30.18, 29.28, 29.11, 28.91, 25.75, 19.35(CH2), 18.74, 18.04 14.35 (CH3). ESI-MS: m/z 512.33 [MþNa]þ. 3.3.8. 1-O-(N-Palmitoylvalineyl)-D-galactosyl ester (22) Treatment of 14 (0.53 g, 1.5 mmol) as described for 15 gave yellow liquid 22 (0.37 g, 71.7%); 1H NMR (500 MHz, CDCl3) d 8.20 (d, 1H, J¼3.5 Hz, 1H), 5.34 (d, 1H, J¼4.2 Hz, H-1), 5.22 (d, 1H, J¼5.2 Hz, C4eOH), 5.12 (d, 1H, J¼5.0 Hz, C2eOH), 5.07 (d, 1H, J¼5.5 Hz, C3eOH), 4.66 (t, 1H, J¼6.0 Hz, C6eOH), 4.31 (dd, 1H, J¼17.0, 6.5 Hz, CH2), 4.30 (dd, 1H, J¼17.0, 6.5 Hz, CH2), 3.64e3.61 (m, 1H, H-2), 3.57e3.51 (m, 1H, H-3), 3.25e3.23 (m, 1H, H-4), 3.22e3.19 (m, 1H, H-5), 3.14e3.10 (m, 2H, H-6), 2.19e2.11 (m, 3H, CH2, CH), 1.47e1.46 (m, 2H, CH2C14H29), 1.22 (s, 26H, C13H26CH3), 1.04e1.02 (m, 9H, CH3, (CH3)2). 13C NMR (125 MHz, DMSO) d 173.90 (s, NHC]O), 171.10 (s, C]O), 95.14 (C-1), 76.69 (C-5), 72.80 (C-3), 69.77(C-2), 60.83(C-4), 56.60(C-6), 35.34(s), 31.65, 30.18, 29.28, 29.11, 28.91, 25.75, 19.35(CH2),18.74, 18.04 14.35 (CH3). ESI-MS: m/z 540.36 [MþNa]þ. 3.4. Chemical stability Taking compounds 16 and 20 example, preparing 100 mL buffer solution of pH value 1.0e14.0 with hydrochloric acid and sodium hydroxide, 50 mg galactosyl ester were dissolved in buffer solution and placed for 0e8 h, respectively. The absorbance values were measured at maximum wavelength (446 nm) using spectrophotometer to ensure the chemical stability at different times. 3.5. Surface tension Surface tension measurements were measured by model BZY-2 tensiometer (Shanghai Hengping Instrument Co., Ltd., accuracy±0.1 mN m1) using the ring method (r¼0.955 cm) and utilizing constant temperature bath (DC-0506, Shanghai Hengping Instrument Co., Ltd.). The surface tension of 15e22 aqueous solutions were measured by a surface tension apparatus at 25.0 C. The glucosyl esters accuracy were prepared the solution of 1.0103 with ultrapure water. The solution of each glucosyl monoester should be diluted to concentration of 1.0107~1.0103 mol/L according to the requirement. All measurements were repeated until the values were reproducible and taken at 25.0±0.1 C.40 Acknowledgements We acknowledge support of this work by the National Science Foundation of China (Grant no. 11076017). Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.carres.2015.05.011.
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Enzymatic synthesis and characterization of galactosyl monoesters Dong An, Xiaohui Zhao, Zhiwen Ye* Department of Chemical Engineering, Nanjing University of Science and Technology, Nanjing 210094, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 January 2015 Received in revised form 15 April 2015 Accepted 26 May 2015 Available online 4 June 2015
Enzyme-catalyzed synthesis of several fatty acyl-amino acid esters based on D-galactose, as well as their chemical evaluation, was performed. These novel galactosyl fatty acyl-amino acid monoesters were synthesized by utilizing lipase from lipozyme TL IM in tert-butanol with D-galactose and fatty acyl-amino acids as starting materials. The products were characterized by 1H NMR, 13C NMR and MS analysis. In addition, their primary physical properties, such as hydrophilic-lipophilic balance (HLB), critical micellar concentration (CMC), solubility in water, maximum surface excess (Gmax), and minimal surface tension (Amin) were measured. The experimental results showed that their CMC values are between 5 and 0.4 mM. The HLB values of galactosyl esters 15e17 indicate that they are useful as oil-in-water emulsions or detergents, whereas 18e22 can be employed as water-in-oil emulsifiers or wetting agents. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Surfactant Fatty acid Galactosyl ester Glycine Valine
1. Introduction Sugar esters, non-ionic surfactants, including one or more fatty acid esters chain as lipophilic moiety and hydrophilic carbohydrate moiety, are widely used in pharmaceutical formulations, food and cosmetic, often termed ‘green surfactants’.1,2 Novel galactosyl esters might be further useful in other fields like bioremediation and membrane protein extraction. They can also been used as foaming agents or emulsifiers to clean products utilizing their good detergent properties.3e5 Sugar esters are primarily synthesized by chemical methods using acid as catalyzer, which causes environmental pollution.6 The method proceeds at rigor conditions such as high temperature, high pressure and has low selectivity and plenty of outgrowths.7 In recent years, surface-active molecules of biological origin obtained by bioprocesses as well as enzymatic synthesis have been reported.8e11 Lipases are not only responsible for the hydrolysis of esters but also they can be widely used in organic synthesis. Owing to the regioselectivity and mild reaction conditions,12e16 enzymatic syntheses have potential applications on the carbohydrate research. Therefore, it is significant to select suitable hydrolases via reversed hydrolysis in order to gain the desired product without byproducts. Many lipases have been successfully used to catalyze the esterification of sugar and fatty acids, but the high cost of
lipases limits industrial use. However, galactosyl esters could be synthesized utilizing some appropriate enzyme catalysts, for instance, cheaper lipase Lipozyme TL IM.17e19 Sometimes, the enzymatic synthesis of sugar esters is restricted by the low solubility of sugars in non-polar organic solvents, in which lipases can exhibit their esterification activity.20e22 Whereas most lipases lose their esterification activity in polar solvents, in which sugars are soluble.23e25 Different strategies have been proposed to handle this problem.26,27 Some solvents are found to keep solubility of sugars and activity of lipases. For example, the uses of t-butanol have been reported in which sugars solubility and enzyme activity are given.28e30 Surface tension is one of the important parameters of liquid interfacial properties. The surface activity, CMC and adsorption capacity at the surface, which were theoretical explanation on mechanism of surfactant in the gathering process, can be determined by the surface tension data. So, the surface tension and CMC were researched firstly when a new type of surfactant was synthesized. We attempt to synthesize and study the physical properties of galactosyl fatty acyl-amino acid esters and exploit much more this kind of surfactants by enzyme catalysis in future.31e33 2. Results and discussion 2.1. Synthesis
* Corresponding author. E-mail address: [email protected] (Z. Ye). http://dx.doi.org/10.1016/j.carres.2015.05.011 0008-6215/© 2015 Elsevier Ltd. All rights reserved.
The enzymatic synthesis of galactosyl monoesters are based on the following materials: D-galactose, glycine (1), valine (2), fatty
D. An et al. / Carbohydrate Research 414 (2015) 32e38
33
acyl chloride (3e6). Firstly, 7e14 were prepared from two amino acid by reaction with different fatty acyl chloride (decanoyl chloride, dodecanoyl chloride, tetradecanoyl chloride, hexadecanoyl chloride) in the presence of catalytic amounts of sodium hydroxide and purified by crystallization from methyl alcohol, respectively. Then, direct esterification of D-galactose, using fatty acyl-amino acids (7e14) and catalyst lipozyme TL IM in t-butanol, yielded two series of galactosyl esters 15e22 as shown in Scheme 1. 2.2. Structural information The 1H NMR spectra of all the final products showed that the coupling constant of H1 was about 4 Hz, indicating that the glycosidic linkage has an configuration. We conclude O6 is not esterified with the amino acid-fatty acids, because the two H6 were shown to multiple peaks with the chemical shift at d (symbol: delta) 3.10e3.14. As a consequence, the resulting ester should be linked to the anomeric carbon. The 1H and 13C NMR spectra indicated that the chemical shifts only displayed minor differences as compared to those of the corresponding starting materials. Taking compound 19 as an example, in the 1H NMR spectra, the amide proton exhibited a chemical shift at around d (symbol: delta) 8.21e8.23 as a triplet. H1 and H2, H3, H4, H5, H6 were shown chemical shift at d 5.34e5.35 as double peaks and 3.65e3.62, 3.47e3.42, 3.25e3.23, 3.22e3.19, 3.14e3.10 as multiple-peaks. The chemical shift of CH3 and CH2 in the fatty chain were mainly located from 0.83 to 2.14. In the 13C NMR spectra, the carbon of amide and carbonyl revealed significant shift at around d 173.24 and 169.53. The chemical shift of C1, C2, C3, C4, C5, C6 position on were around 95.25, 72.93, 76.71, 69.82, 78.42, 60.90.
Fig. 1. Effects of pH value on the stability of 16 at different times.
2.3. Stability study The absorbance values decreased further after 1e4 h, which means the galactosyl esters 16, 20 might be partially hydrolyzed in solution of strong acid and strong base. In contrast, they could be stable in the range of pH 5.5e8.5 solution. These experimental values indicate that these types of galactosyl fatty-acid amino-acid esters 15e22 are chemical stability in the environment of weak acid, weak base or neutrality (Fig. 1 and Fig. 2). 2.4. Optimal reaction conditions In this present study, t-butanol, which has low toxicity was served as solvent in this enzymatic reaction. The effects of various parameters on the yield were investigated in detailed. The optimum reaction conditions were gained following: 1 mmol a-Dgalactose, l.5 mmol acid, 0.49 g enzyme, 10 mL t-butanol and 0.8 g activated molecular sieve 4 Å as water absorbant after 6 h at 40 C. As can be seen, higher temperature and longer reaction time might lead to decrease the activity of enzyme and produce galactosyl diesters, resulting in diminished yield (Table 1).
Fig. 2. Effects of pH value on the stability of 20 at different times.
Lipozyme TL IM had a good catalytic activity from 25 C to 60 C in t-butanol. However, the fields of galactosyl esters 15e22 increased and decreased were happened with the reaction temperature rising. On the one hand, the field of the enzymatic reaction accelerated with the temperature rises from 25 C to 40 C. On the other hand, the irreversible change of spatial structure might
Scheme 1. Enzymatic synthesis of galactosyl esters (15e22). Reagents and conditions: a) 20% NaOH, acetone; b) D-galactose, TLL, t-butanol, 40 C.
34
D. An et al. / Carbohydrate Research 414 (2015) 32e38
Table 1 Chemical yields in the enzymatic synthesis of galactosyl esters 15e22 Reagenta
T ( C)
Time (h)
Product
Yield (%)
7 7 7 7 7 8 8 9 10 11 11 12 13 14 14
25 30 35 40 45 40 40 40 40 40 40 40 40 40 40
6 6 6 6 6 7 6 6 6 6 7 6 6 8 6
15 15 15 15 15 16 16 17 18 19 19 20 21 22 22
77.3 86.4 93.1 94.5 83.1 94.2 95.8 83.2 83.1 89.6 87.2 85.3 78.5 73.2 71.7
a
Molar ratio Acyl donor(fatty acid):a-D- galactosyl¼1.5:1.
deactivate activity of lipozyme TL IM molecules at higher temperature. Thus, we selected 40 C as the optimal temperature for these reactions (Fig. 3). As can be seen in Fig. 4, the fields of galactosyl esters 15e22 increased and decreased gradually with increasing reaction time from 2 h to 8 h. The highest conversion was achieved at 6 h. It was probably connected with reversible enzymic reaction, which took place under increasing H2O by-product as the time prolonged. In addition, galactosyl diesters were further produced when the conversion of galactosyl monoesters were over a maximum value. Based on the data, the highest fields are obtained after 6 h. 2.5. Surface tension results The galactosyl esters were characterized with their HLB values, solubility in water, CMC values, Gmax, and Amin. The HLB values of galactosyl esters 15e17 slightly decreased from 14.8 to 11.2 with the chain length of fatty acid increasing. Thus, these HLB values and the water solubility from 466±11 to 219±17 mmol1 indicated that they are suitable for oil-in-water (o/w) emulsion cleansing agents or detergents. On the contrary, the HLB values of the galactosyl esters
Fig. 4. Time course of galactosyl fatty acyl-amino-acid 15e22 synthesis reaction (Reaction conditions:1 mmol D-galactose, 1.5 mmol/L fatty acyl-glycine 7e14, 0.8 g molecular sieve and 0.3 g lipozyme TL IM in 10 mL tert-butanol at 40 C and 150 rpm for 2e8 h).
18e22 between 5.5 and 9.6 are suggested their promising applications in oil-soluble hydrophobic (w/o) emulsifiers or wetting agents (Table 2). The CMC values can be determined by a plot of the surface tension versus the concentration. The plots of surface tension versus concentration (C-g) for 15e22 were demonstrated in Fig. 5 and Fig. 6. The CMC values of galactosyl fatty acyl-glycine esters 15e18 were 4.97103, 3.96103, 1.87103, and 4.8104 mol/L, and galactosyl fatty acyl-valine esters 19e22 were 4.70103, 3.74103, 1.62103 and 4.0104 mol/L, respectively. The CMC decreased as the chain length of fatty acid ester increase. It is consistent with our suggestion that the different fatty acyl-amino acid hydrophobic chain are responsible for determining the CMC values of galactosyl monoesters 15e22 in aqueous solution (Table 3). The packing densities of surfactants at the air/aqueous solution interface are also important for the interpretation of the surface activity. The maximum surface excess (Gmax), at the air/solution interface was calculated by applying the Gibbs adsorption isotherm Eq. 1. The minimum area occupied by a surfactant molecule at the air/solution interface (Amin) can be estimated from the relation (Eq. 2).34
Gmax ¼
1 dg 2:303nRT d logc
(1)
Table 2 Solubility in water and HLB value of galactosyl monoesters 15e22
Fig. 3. Effect of temperature on the field of galactosyl fatty acyl-glycine ester 15 (Reaction conditions:;1 mmol D-galactose, 1.5 mmol L1 7, 0.8 g molecular sieve and 0.3 g lipozyme TL IM in 10 mL tert-butanol at 150 rpm for 6 h).
Compounds
Solubility in water/mmol1
Measure HLB
15 16 17 18 19 20 21 22
466±11 254±10 219±17 178±14 198±11 164±14 142±6 133±10
14.3±0.5 12.4±0.4 11.0±0.2 9.1±0.5 6.9±0.4 6.5±0.4 5.9±0.7 5.1±0.4
D. An et al. / Carbohydrate Research 414 (2015) 32e38
35
Table 3 The surface active parameter of 15e22 in distilled water solution at 25 C Compound
CMC (mmol/L)
gcmc (mN/m)
Gcmc (mmol/m2)
Amin (nm2)
15 16 17 18 19 20 21 22
4.97 3.96 1.87 0.48 4.70 3.74 1.62 0.40
31.44 30.52 29.13 28.09 30.84 29.75 28.68 27.82
1.01 1.00 0.99 1.16 1.02 0.97 0.95 1.02
1.64 1.66 1.67 1.43 1.63 1.71 1.75 1.63
(△Hqm). The thermodynamic parameters are estimated by the mass action model.36 △Gqm can be obtained from Eq. 3. The enthalpy of micelle formation can be obtained by using the GibbseHelmboltz equation (Eq. 4) Then the entropy of micelle formation is determined by using Eq. 5.37,38
Fig. 5. Relationship between CMC of galactosyl monoesters 15e18 and surface tension at 25.0 C.
DGqm ¼ RT ln
CMC 55:5
q DHm ¼ 2:303R
Amin
1024 ¼ NA Gmax
(2)
where c is the surfactant concentration, R is the gas constant(8.314 J mol1 K1), T is the absolute temperature, Gmax is the maximum surface excess in mmol/m2, g is the surface tension in mN/m, and (d g/d logc) is the slope in the surface tension isotherm when the concentration is near the CMC. The value of n is taken as 1 due to the fact that the galactosyl esters are conventional surfactants.35 When maximum estimated, the value of Amin is obtained using Eq. 2. NA is Avogadro's number and Amin is in nm2/molecule. 2.6. Thermodynamic analysis of micellization The micellization of galactosyl fatty-acid amino-acid esters (15e22) could be indicated by some thermodynamic parameters, such as the standard Gibbs energy change (△Gqm), the standard entropy change (△Sqm), and the standard enthalpy change
d log CMC dð1=TÞ
. q DSqm ¼ DHm DGqm T
(3)
(4)
(5)
where T is the absolute temperature, and R is the ideal gas constant (8.314 J mol1 K1). The thermodynamic parameters of galactosyl fatty acyl-amino acid esters 15e22 micellization at different temperatures were listed in Table 4. As can be seen, the CMC values decreased with the length of hydrophobic group increasing and increased as the temperature raising. More important, the galactosyl esters 15e22 micellization were spontaneous based on the negative Gibbs free energy changes. In addition, the micelle formation was exothermic process, which could be attributed to the surfactantesolvent interactions because the values of standard enthalpy change (△Hqm) were negative. The large positive △Sqm was mainly caused by the negative values of △Gqm as well as the values of T△Sqm were much bigger than △Hqm. Therefore, the spontaneous micellization of 15e22 were entropy-driven process and thermodynamic stable systems. 3. Experimental 3.1. Materials and methods NMR were recorded on a Bruker 500 MHz spectrometer. Mass spectra were performed by a Finnigan TSQ Quantum ultra AM mass spectrometer. Silica gel 200e300 mesh was used for column chromatography. CMC values were determined by a BZY-2 fully automatic surface tensiometer using the Wilhelmy Type method at 25.0 C. Lipozyme TL IM (TLL, Thermomyces lanuginosus immobilized on silica,50,000 U/g)was obtained from the SigmaeAldrich Trading Co., Ltd. (Shanghai, China). Spectrophotometer (446 nm) (Shanghai, China). Commercially available grades of organic solvents of adequate purity were used in all reactions. 3.2. General procedure for the preparation of N-acyl amino acids 7e1439
Fig. 6. Relationship between CMC of galactosyl monoesters 19e22 and surface tension at 25.0 C.
Taking compound 7 as an example, glycine 1 (4.5 g, 0.06 mol) was dissolved in the mixture of acetone and deionized water (2:1
36
D. An et al. / Carbohydrate Research 414 (2015) 32e38
Table 4 Thermodynamic parameters of galactosyl esters 15e22 at different temperature Glucosyl ester
T ( C)
CMC (mmol/L)
△Gqm (kJ/mol)
△Hqm (kJ/mol)
T△Sqm (kJ/mol)
△Sqm (kJ/mol)
15
20 25 30 35 20 25 30 35 20 25 30 35 20 25 30 35 20 25 30 35 20 25 30 35 20 25 30 35 20 25 30 35
4.5 4.97 5.07 5.25 3.55 3.96 5.04 5.15 1.27 1.87 2.08 2.13 0.40 0.48 0.62 0.68 4.33 4.70 4.91 5.07 3.55 3.74 4.02 4.37 1.43 1.62 2.09 2.12 0.23 0.40 0.43 0.47
25.20 23.08 26.96 26.57 21.77 23.64 26.73 26.66 23.53 28.32 29.58 36.87 26.19 28.87 32.11 33.85 20.17 23.22 29.83 26.59 21.17 23.79 27.19 28.13 25.77 25.86 28.78 29.71 33.21 29.32 38.59 40.05
2.89 1.79 2.84 5.38 2.35 1.73 0.41 4.08 1.31 0.93 0.61 0.78 2.88 1.79 4.73 5.44 0.45 0.67 0.72 0.97 1.23 1.77 0.79 0.56 0.72 0.77 0.64 0.68 2.97 2.88 3.24 3.72
22.31 22.01 20.24 21.19 19.42 21.91 26.32 22.58 22.22 27.39 28.97 36.09 23.31 27.08 27.38 28.41 19.72 22.55 29.11 25.62 19.94 22.02 26.40 27.57 25.05 25.09 28.14 29.03 30.24 26.44 35.35 36.33
0.076 0.074 0.067 0.069 0.066 0.074 0.080 0.074 0.081 0.092 0.096 0.117 0.080 0.091 0.090 0.094 0.067 0.076 0.096 0.083 0.068 0.074 0.087 0.090 0.085 0.084 0.093 0.094 0.103 0.089 0.117 0.118
16
17
18
19
20
21
22
by volume), 5% (wt %) NaOH aqueous solution were added dropwise into the mixture until pH was at 8.5e9.5. Then, decanoyl chloride (8.74 g, 0.04 mol) was dropwise under strongly stirring at 0e5 C. At same time, the pH was kept a range from 7.5 to 11.5 adjusted with 5% NaOH aqueous solution. Then, the mixed liquid was stirred at 0e5 C for another 2.5 h and stood at room temperature overnight. The solvent was removed by vacuum distillation, then the mixture of concentrated hydrochloric acid and water (1:1 by volume) was added to the residues until pH reached 1 to 2. White solid was gradually precipitated. The white solid was obtained by vacuum filtration and washed with eionized water until pH 7 of the filtrate, then washed with petroleum ether (15 mL) three times. The crude products were by recrystallized with methanol to obtain 7 (7.29 g, 75%). Compounds 8e14 were obtained by the same method and 71e79 % yield. 3.3. General synthesis methods of galactosyl esters 15e22 3.3.1. 1-O-(N-Decanoylglycineyl)-D-galactosyl ester (15) A solution of a-D-galactose (0.18 g, 1 mmol), N-decanoylglycine 7 (0.34 g, 1.5 mmol), lipozyme TL IM (0.49 g) and activated molecular sieve 4 Å (0.8 g) in butanol was allowed to stir at 40 C for 6 h under nitrogen. Samples from the reaction mixture were analyzed by TLC (1:1 ethyl acetate:methanol). At the end of the reaction, the mixture was extracted with 20 mL ethyl isopropanol:acetate (1:4) by stirring at rt for 15 min. Particularly, the glycosyl esters extraction was performed with 20 mL dichloromethane and the immobilized enzyme was separated from the reaction mixture by flotation allowing easy recovery. Organic solvent was removed in vacuo and the crude product was purified by column chromatography (10:1e20:1 ethyl acetate:methanol) to give white solid 15 (0.37 g, 94.5%); mp.: 100.2e101.8 C; 1H NMR (500 MHz, DMSO) d 8.22 (t, 1H, J¼6.0 Hz, NH), 5.35 (d, 1H, J¼4.0 Hz, H-1), 5.27 (d, 1H, J¼5.0 Hz,
C4eOH), 5.10 (d, 1H, J¼5.0 Hz, C2eOH), 5.01 (d, 1H, J¼5.5 Hz, C3eOH), 4.61 (t, 1H, J¼6.0 Hz, C6eOH), 3.92 (dd, 1H, J¼16.0, 6.0 Hz, aliphatic OCOCH2), 3.86 (dd, 1H, J¼16.0, 6.0 Hz, aliphatic OCOCH2), 3.65e3.62 (m, 1H, H-2), 3.47e3.42 (m, 1H, H-3), 3.25e3.23 (m, 1H, H-4), 3.22e3.19 (m, 1H, H-5), 3.14e3.10 (m, 2H, H-6), 2.12 (t, 2H, J¼7.5 Hz, aliphatic NHCOCH2C8H17), 1.50e1.48 (m, 2H, aliphatic NHCOCH2CH2C7H15), 1.24 (s, 12H, NHCOC2H4C6H12CH3), 0.87 (t, 3H, J¼7.0 Hz, aliphatic NHCOC8H16CH3). 13C NMR (125 MHz, CDCl3) d 173.24 (s, NHC]O), 169.54 (2s, C]O), 95.23 (C-1), 78.37(C-5), 76.66(C-3), 72.88(C-2), 69.79(C-4), 60.90(C-6), 35.56, 31.76, 29.52, 29.47, 29.37, 29.06, 25.67, 22.55 (each aliphatic CH2), 14.35 (CH3); ESIMS: Found, m/z 414.09; required, 414.22 [MþNa]þ. 3.3.2. 1-O-(N-Dodecanoylglycineyl)-D-galactosyl ester(16) Treatment of 8 (0.38 g, 1.5 mmol) as described for 15 gave white solid 16 (0.40 g, 95.8%); mp.: 98.3e99.7 C. 1H NMR (500 MHz, DMSO) d 8.21 (t, 1H, J¼6.0 Hz, NH), 5.35 (d, 1H, J¼4.1 Hz, H-1), 5.27 (d, 1H, J¼5.0 Hz, C4eOH), 5.10 (d, 1H, J¼5.0 Hz, C2eOH), 5.01 (d, 1H, J¼5.5 Hz, C3eOH), 4.61 (t, 1H, J¼6.0 Hz, C6eOH), 3.92 (dd, 1H, J¼16.0, 6.0 Hz, aliphatic OCOCH2), 3.86 (dd, 1H, J¼16.0, 6.0 Hz, aliphatic OCOCH2), 3.65e3.62 (m, 1H, H-2), 3.47e3.42 (m, 1H, H-3), 3.25e3.23 (m, 1H, H-4), 3.22e3.19 (m, 1H, H-5), 3.14e3.10 (m, 2H, H-6), 2.12 (t, 2H, J¼7.5 Hz, aliphatic NHCOCH2C10H21), 1.51e1.47 (m, 2H, aliphatic CH2C9H19), 1.24 (s, 16H, C8H16CH3), 0.86 (t, 3H, J¼7.0 Hz, aliphatic NHCOC10H20CH3); 13C NMR (125 MHz, DMSO) d 173.24 (s, NHC]O), 169.54 (s, C]O), 95.19 (C-1), 78.41(C-5), 76.68(C-3), 72.87(C-2), 69.80(C-4), 60.83(C-6), 35.56, 31.75, 29.51, 29.45, 29.40, 29.27, 29.03, 22.56 (each aliphatic CH2), 14.35 (CH3). ESI-MS: m/z 442.11 [MþNa]þ. 3.3.3. 1-O-(N-Myristylglycineyl)-D-galactosyl ester(17) Treatment of 9 (0.42 g, 1.5 mmol) as described for 15 gave white solid 17 (0.37 g, 83.2%); mp.: 95.8e97.4 C. 1H NMR (500 MHz,
D. An et al. / Carbohydrate Research 414 (2015) 32e38
DMSO) d 8.22 (t, 1H, J¼6.0 Hz, NH), 5.35 (d, 1H, J¼4.0 Hz, H-1), 5.27 (d, 1H, J¼5.0 Hz, C4eOH), 5.10 (d, 1H, J¼5.0 Hz, C2eOH), 5.01 (d, 1H, J¼5.5 Hz, C3eOH), 4.61 (t, 1H, J¼6.0 Hz, C6eOH), 3.92 (dd, 1H, J¼16.0, 6.0 Hz, aliphatic OCOCH2), 3.86 (dd, 1H, J¼16.0, 6.0 Hz, aliphatic OCOCH2), 3.65e3.62 (m, 1H, H-2), 3.47e3.42 (m, 1H, H-3), 3.25e3.23 (m, 1H, H-4), 3.22e3.19 (m, 1H, H-5), 3.14e3.10 (m, 2H, H-6), 2.12 (t, 2H, J¼7.5 Hz, aliphatic NHCOCH2C12H25), 1.51e1.47 (m, 2H, aliphatic NHCOCH2CH2C11H23), 1.24 (s, 20H, NHCOC2H4C10H20CH3), 0.86 (t, 3H, J¼7.0 Hz, aliphatic NHCOC12H24CH3). 13C NMR (125 MHz, CDCl3) d 173.24 (s, NHC]O), 169.54 (s, C]O), 95.25 (C-1), 78.42(C-5), 76.71(C-3), 72.93(C-2), 69.82(C-4), 60.90(C-6), 35.56(s), 31.76, 29.52, 29.47, 29.42, 29.29, 29.17, 29.06, 25.65, 22.56 (aliphatic CH2), 14.41 (q, aliphatic CH3). ESI-MS: m/z 470.15 [MþNa]þ. 3.3.4. 1-O-(N-Palmitoylglycineyl)-D-galactosyl ester (18) Treatment of 10 (0.46 g, 1.5 mmol) as described for 15 gave white solid 18 (0.39 g, 83.1%); mp.: 83.2e84.7 C; 1H NMR (500 MHz, DMSO) d 8.23 (t, 1H, J¼5.0 Hz, NH), 5.35 (d, 1H, J¼4.2 Hz, H-1), 5.27 (d, 1H, J¼5.0 Hz, C4eOH), 5.10 (d, 1H, J¼5.0 Hz, C2eOH), 5.01 (d, 1H, J¼5.5 Hz, C3eOH), 4.61 (t, 1H, J¼6.0 Hz, C6eOH), 3.93 (dd, 1H, J¼18.0, 7.0 Hz, aliphatic OCOCH2), 3.85 (dd, 1H, J¼18.0, 6.0 Hz, aliphatic OCOCH2), 3.65e3.62 (m, 1H, H-2), 3.47e3.42 (m, 1H, H-3), 3.25e3.23 (m, 1H, H-4), 3.22e3.19 (m, 1H, H-5), 3.14e3.10 (m, 2H, H-6), 2.12 (t, 2H, J¼7.5 Hz, aliphatic NHCOCH2C14H29), 1.51e1.47 (m, 2H, aliphatic NHCOCH2CH2C13H27), 1.24 (s, 24H, NHCOC2H4C12H24CH3), 0.86 (t, 3H, J¼7.0 Hz, aliphatic NHCOC14H28CH3). 13C NMR (125 MHz, CDCl3) d 173.05 (s, NHC]O), 169.30 (s, C]O), 95.00 (d, C-1), 78.16 (C-5), 76.45 (C-3), 72.68 (C-2), 69.58 (C-4), 60.66 (C-6), 35.31 (s), 31.52, 29.28, 29.24, 29.18, 29.05, 28.93, 28.81, 25.41, 22.32 (each aliphatic CH2), 14.16 (q, aliphatic CH3). ESI-MS: m/z 498.17 [MþNa]þ. 3.3.5. 1-O-(N-Decanoylvalineyl)-D-galactosyl ester (19) Treatment of 11 (0.41 g, 1.5 mmol) as described for 15 gave yellow liquid 19 (0.38 g, 89.6%); 1H NMR (500 MHz, DMSO) d 8.07 (d, 1H, J¼3.5 Hz, NH), 5.34 (d, 1H, J¼4.1 Hz, H-1), 5.22 (d, 1H, C4eOH), 5.12 (d, 1H, J¼5.0 Hz, C2eOH), 5.07 (d, 1H, J¼5.5 Hz, C3eOH), 4.66 (t, 1H, J¼6.0 Hz, C6eOH), 4.31 (dd, 1H, J¼17.0, 6.0 Hz, CH2), 4.30 (dd, 1H, J¼17.0, 6.0 Hz, CH2), 3.64e3.61 (m, 1H, H-2), 3.57e3.51 (m, 1H, H-3), 3.25e3.23 (m, 1H, H-4), 3.22e3.19 (m, 1H, H-5), 3.14e3.10 (m, 2H, H-6), 2.19e2.11 (m, 3H, CH2, CH), 1.47e1.46 (m, 2H, CH2C7H15), 1.22 (s, 12H, C6H12CH3), 1.06e1.04 (m, 9H, CH3,(CH3)2). 13C NMR (125 MHz, DMSO) d 173.90 (s, NHC]O), 171.10 (s, C]O), 95.14 (d, C-1), 76.69 (C-5), 72.80 (C-3), 69.77(C-2), 60.83(C-4), 56.60(C-6), 35.34(s), 31.65, 30.18, 29.28, 29.11, 28.91, 25.75, 19.35(CH2),18.74, 18.04 14.35 (CH3). ESI-MS: m/z 456.28 [MþNa]þ. 3.3.6. 1-O-(N-Palmitoylvalineyl)-D-galactosyl ester (20) Treatment of 12 (0.45 g, 1.5 mmol) as described for 15 gave yellow liquid 20 (0.39,85.3%); 1H NMR (500 MHz, DMSO) d 8.25 (d, 1H, J¼4.0 Hz, NH), 5.35 (d, 1H, J¼4.0 Hz, H-1), 5.20 (d, 1H, J¼5.0 Hz, C4eOH), 5.12 (d, 1H, J¼5.0 Hz, C2eOH), 5.05 (d, 1H, J¼5.5 Hz, C3eOH), 4.66 (t, 1H, J¼6.0 Hz, C6eOH), 4.30 (dd, 1H, J¼17.0, 6.5 Hz, CH2), 4.31 (dd, 1H, J¼17.0, 6.5 Hz, CH2), 3.64e3.61 (m, 1H, H-2), 3.58e3.54 (m, 1H, H-3), 3.25e3.23 (m, 1H, H-4), 3.21e3.19 (m, 1H, H-5), 3.14e3.10 (m, 2H, H-6), 2.18e2.15 (m, 3H, CH2, CH), 1.47e1.44 (m, 2H, CH2), 1.21 (s, 18H, C9H18CH3), 1.05e1.04 (m, 9H, CH3, (CH3)2). 13 C NMR (125 MHz, DMSO) d 173.90 (s, NHC]O), 171.10 (s, C]O), 95.14 (C-1), 76.69 (C-5), 72.80 (C-3), 69.77(C-2), 60.83(C-4), 56.60(C-6), 35.34(s), 31.65, 30.18, 29.28, 29.11, 28.91, 25.75, 19.35(CH2),18.74, 18.04 14.35 (CH3). ESI-MS: m/z 479.28; [MþNH4]þ.
37
3.3.7. 1-O-(N-Myristylvalineyl)-D-galactosyl ester (21) Treatment of 13 (0.49 g, 1.5 mmol) as described for 15 gave light yellow liquid 21 (0.38 g, 78.5%); 1H NMR (500 MHz, DMSO) d 8.08 (m, 1H, NH), 5.34 (d, 1H, J¼4.1 Hz, H-1), 5.22 (d, 1H, J¼5.3 Hz,C4eOH), 5.12 (d, 1H, J¼5.0 Hz, C2eOH), 5.07 (d, 1H, J¼5.5 Hz, C3eOH), 4.66 (t, 1H, J¼6.0 Hz, C6eOH), 4.31 (dd, 1H, J¼17.5, 6.5 Hz, CH2), 4.30 (dd, 1H, J¼17.5, 6.5 Hz, CH2), 3.64e3.61 (m, 1H, H-2), 3.57e3.51 (m, 1H, H-3), 3.25e3.23 (m, 1H, H-4), 3.22e3.19 (m, 1H, H-5), 3.14e3.10 (m, 2H, H-6), 2.19e2.11 (m, 3H, CH2,CH), 1.47e1.46 (m, 2H, CH2C12H24), 1.22 (s, 22H, C11H22CH3), 1.03e1.01 (m, 9H, CH3,(CH3)2). 13C NMR (125 MHz, CDCl3) d 173.90 (s, NHC] O), 171.10 (s, C]O), 95.14 (C-1), 76.69 (C-5), 72.80 (C-3), 69.77(C-2), 60.83(C-4), 56.60(C-6), 35.34(s), 31.65, 30.18, 29.28, 29.11, 28.91, 25.75, 19.35(CH2), 18.74, 18.04 14.35 (CH3). ESI-MS: m/z 512.33 [MþNa]þ. 3.3.8. 1-O-(N-Palmitoylvalineyl)-D-galactosyl ester (22) Treatment of 14 (0.53 g, 1.5 mmol) as described for 15 gave yellow liquid 22 (0.37 g, 71.7%); 1H NMR (500 MHz, CDCl3) d 8.20 (d, 1H, J¼3.5 Hz, 1H), 5.34 (d, 1H, J¼4.2 Hz, H-1), 5.22 (d, 1H, J¼5.2 Hz, C4eOH), 5.12 (d, 1H, J¼5.0 Hz, C2eOH), 5.07 (d, 1H, J¼5.5 Hz, C3eOH), 4.66 (t, 1H, J¼6.0 Hz, C6eOH), 4.31 (dd, 1H, J¼17.0, 6.5 Hz, CH2), 4.30 (dd, 1H, J¼17.0, 6.5 Hz, CH2), 3.64e3.61 (m, 1H, H-2), 3.57e3.51 (m, 1H, H-3), 3.25e3.23 (m, 1H, H-4), 3.22e3.19 (m, 1H, H-5), 3.14e3.10 (m, 2H, H-6), 2.19e2.11 (m, 3H, CH2, CH), 1.47e1.46 (m, 2H, CH2C14H29), 1.22 (s, 26H, C13H26CH3), 1.04e1.02 (m, 9H, CH3, (CH3)2). 13C NMR (125 MHz, DMSO) d 173.90 (s, NHC]O), 171.10 (s, C]O), 95.14 (C-1), 76.69 (C-5), 72.80 (C-3), 69.77(C-2), 60.83(C-4), 56.60(C-6), 35.34(s), 31.65, 30.18, 29.28, 29.11, 28.91, 25.75, 19.35(CH2),18.74, 18.04 14.35 (CH3). ESI-MS: m/z 540.36 [MþNa]þ. 3.4. Chemical stability Taking compounds 16 and 20 example, preparing 100 mL buffer solution of pH value 1.0e14.0 with hydrochloric acid and sodium hydroxide, 50 mg galactosyl ester were dissolved in buffer solution and placed for 0e8 h, respectively. The absorbance values were measured at maximum wavelength (446 nm) using spectrophotometer to ensure the chemical stability at different times. 3.5. Surface tension Surface tension measurements were measured by model BZY-2 tensiometer (Shanghai Hengping Instrument Co., Ltd., accuracy±0.1 mN m1) using the ring method (r¼0.955 cm) and utilizing constant temperature bath (DC-0506, Shanghai Hengping Instrument Co., Ltd.). The surface tension of 15e22 aqueous solutions were measured by a surface tension apparatus at 25.0 C. The glucosyl esters accuracy were prepared the solution of 1.0103 with ultrapure water. The solution of each glucosyl monoester should be diluted to concentration of 1.0107~1.0103 mol/L according to the requirement. All measurements were repeated until the values were reproducible and taken at 25.0±0.1 C.40 Acknowledgements We acknowledge support of this work by the National Science Foundation of China (Grant no. 11076017). Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.carres.2015.05.011.
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