Lipase-Catalyzed Regioselective Synthesis of Dextrin Esters

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Lipase-Catalyzed Regioselective Synthesis of Dextrin Esters HAKYONG LEE, Satoshi Kimura, and Tadahisa Iwata Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01374 • Publication Date (Web): 06 Nov 2018 Downloaded from http://pubs.acs.org on November 8, 2018

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Lipase-Catalyzed Regioselective Synthesis of Dextrin Esters Hak Yong Lee1, Satoshi Kimura1,2, and Tadahisa Iwata1* 1Science

of Polymeric Materials, Department of Biomaterial Sciences, Graduate School of

Agricultural and Life Sciences, The University of Tokyo, Japan 2Department

of Plant & Environmental New Resources, College of Life Sciences, Kyung Hee

University, Republic of Korea

KEYWORDS polysaccharide, dextrin ester, lipase enzyme, enzymatic esterification, regioselective substitution.

*Address correspondence to Tadahisa Iwata, Science of Polymeric Materials, Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan. Tel: (+81)3-5841-5266. Fax: (+81)3-5841-1304. E-mail: [email protected]

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ABSTRACT

Four lipase enzymes were investigated as catalysts in the synthesis of regioselectively mono-substituted dextrin esters from dextrin and vinyl acetate. An immobilized lipase enzyme (Lipozyme TL IM) exhibited the highest activity. This enzyme showed regioselective substitution of the dextrin at the primary hydroxyl group (C6 position) under optimal conditions (60°C for 24 hours, using a 1:3 molar ratio of glucose unit/vinyl acetate and 2.5 U/mL enzyme dosage in an organic solvent). To compare the reactivity of other vinyl esters, mono-substituted dextrin esters (degrees of substitution [DS] ≈ 1) with varying side-chain lengths (C2-12) were synthesized. With increasing side-chain length, the initial catalytic activity of the lipase enzyme decreased, resulting in lower DS values. However, the final DS values of the mono-substituted dextrin esters with longer side-chains were higher than those of the shorter chain analogues, because of an increase in affinity between the substrate and acyl donor.


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1. Introduction Dextrin is a polysaccharide produced by the partial hydrolysis of starch or glycogen. Its structure consists of an α(1→4) linked main chain with α(1→4,6) linked branches. Depending on the degree of hydrolysis, dextrin is classified as amylodextrin, erythro-dextrin, achro-dextrin or malto-dextrin, and each type exhibits different properties.1 A general characteristic of dextrin is that it dissolves well in polar solvents, such as water. This allows for its use in the adhesive, paint, cosmetic and biomedical industries.2,

3

However, because neat dextrin does not exhibit

thermoplasticity or hydrophobicity – a consequence of strong hydrogen bonding – its use in other applications is limited. The limited properties of dextrin can be modified by substitution reactions of the hydroxyl groups, which result in the insertion of functional molecular chains. The conditions of these substitution reactions, such as the components, positions and chain lengths of the substituents, have a significant effect on the final properties of the dextrin.4, 5 Esterification is one of the most popular modification reactions used to obtain thermoplastic and hydrophobic polysaccharides.6, 7 This reaction can be catalyzed by chemical reagents or enzymes. Chemical esterification is suitable for the production of polysaccharides with a high degree of substitution (DS), because both primary and secondary hydroxyl groups are substituted in a non-selective reaction.8,

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Previously, we synthesized fully-substituted polysaccharide esters using chemical esterification, and reported that the thermoplasticity and hydrophobicity of the polysaccharide were significantly increased.10,

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However, this reaction has a

limitation when regioselectively-substituted amphiphilic polysaccharide esters are


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desired. Although regioselectively-substituted polysaccharide esters can be obtained through chemical esterification using sequential protection and deprotection reactions, these additional steps can support unwanted side reactions, resulting in an expensive and uneconomic purification.12, 13 An alternative route for the synthesis of polysaccharide esters is enzymatic esterification. This route is not only environmentally benign; it is also highly regioselective, allowing for the synthesis of polysaccharide esters with controlled structures and functionalities.14, 15 Enzymes are generally efficient catalysts for the hydrolysis of esters, but in the absence of water (i.e., in organic solvents) they catalyze the reverse reaction. Most enzymes are easily denatured and inactivated in organic solvents, and therefore many studies on enzymatic esterification have been reported for the synthesis of monosaccharides, oligosaccharides and polysaccharide derivatives. These studies indicate a high DS for the enzymatic synthesis of carbohydrate derivatives, but the reactions exhibit a lack of regioselectively, due to the specific conditions used to amplify the enzyme’s catalytic activity (such as substrate pretreatment, microwave treatment and the addition of ionic liquids).14, 15 For a successful regioselective substitution reaction, it is important to choose an enzyme with excellent activity in organic solvent. Several reports have classified enzymes by their degree of solvent tolerance and have explored the industrial applications of solvent-tolerant enzymes.16 Lipases have gained attention by many industries, because of their general ease of handling, broad substrate tolerance, high stability towards temperatures and solvents, and convenient commercial availability.17 Furthermore, lipases are able to esterify the primary hydroxyl group


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(C6-OH) of glucose, which allows for the efficient synthesis of regioselectivelysubstituted polysaccharides without the need for protection and deprotection reactions. Extensive research has been carried out on the modification of polysaccharides using lipases. Three groups of researchers (Hero J. Heeres et al., Adel Sayari et al. and Rintu Banerjee et al.) have reported starch derivatives with high DS values of approximately 2.8.18-20 However, these esterification reactions are non-regioselective, requiring either ionic liquid or microwave treatment. Although the regioselective, lipase-catalyzed preparation of starch derivatives has been reported by Richard A. Gross et al., their results showed DS values in the 0.4–0.8 range, which are insufficient to demonstrate high reactivity.21 In this study, we focus on substitution reactions of polysaccharides that operate with complete regioselectively. Branched dextrin, with two types of glycosidic bonds, is a suitable substrate for enzymatic esterification, because it has good solubility in organic solvent (a consequence of the extra degrees of freedom provided by rotation at the C6 position). Using dextrin as a substrate, four commercial lipase enzymes were investigated in the regioselective synthesis of dextrin esters, and the parameters that affect the enzyme activity were identified. The regioselectively-substituted dextrin esters were then characterized in detail to determine their structure.

2. Experimental 2.1. Materials


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Hyper-branched dextrin was prepared from waxy corn starch by partial amylase digestion (Mw = 1,070,000 g/mol). The used enzymes were four commercial lipase enzymes, which are Lipozyme CALB, Novozyme 435, Lipozyme TL 100L and Lipozyme TL IM. All lipase enzymes were supplied by Novozymes (Bagsvaerd, Denmark) and their characteristic in aqueous system are shown in Table 1. Dextrin and liquid type enzymes were treated by freeze drying before reaction to remove the water. Vinyl acetate, propionate, hexanoate and laurate were purchased from Wako Pure Chemicals (Tokyo, Japan). All other chemicals were used without further purification.

2.2. Optimization of enzyme activity Table 1 shows the activity of each enzyme, but the enzyme in the organic solvent system act quite differently unlike in the aqueous system. Therefore, the activity of four enzymes between dextrin and vinyl acetate were identified in organic solvent by varying influence factors to find optimal condition. The standard reaction were carried out at 60 °C for 12 hours in super dehydrated dimethyl sulfoxide (DMSO, 10 ml) with freeze-dried dextrin (2.5 mmol) and vinyl acetate (7.5 mmol) and enzyme (liquid type;10 ml or immobilized type; 0.1 g). And then, the influence parameters such as enzyme type, solvent system, reaction temperature, molar ratio of glucose unit to vinyl acetate, dextrin concentration, enzyme dosage and reaction time were investigated. The activity of enzyme was determined based on the DS value,


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calculated by the integration ratio of the acyl protons to the C1 proton of glucose in 1H-NMR

spectrum.

𝐃𝐒 =

𝐏𝐞𝐚𝐤 𝐚𝐫𝐞𝐚 [𝑪𝑯𝟑]/𝟑 𝐏𝐞𝐚𝐤 𝐚𝐫𝐞𝐚 [𝑯𝟏 𝐨𝐟 𝐠𝐥𝐮𝐜𝐨𝐬𝐞]

2.3. Enzymatic synthesis of regioselectively mono-substituted dextrin esters Regioselectively mono-substituted dextrin esters (mono-DexEs) were synthesized according to the following procedure using lipase enzymes as a biocatalyst (Scheme 1). The homogeneous mixtures were prepared in super dehydrated DMSO (10 ml) by mixing freeze-dried dextrin (2.5 mmol) and vinyl ester (7.5 mmol) and Lipozyme TL IM (0.1 g; 2.5 U/ml). The mixtures were kept at 60 °C with increasing reaction time up to 120 hours in air-bath shaker with 200 rpm. At this time, Lipozyme TL IM was replaced with new one every 2 days. After the reaction, the mixtures were cooled to room temperature, and enzyme was filtered by nylon mesh (50 μm of pore size). The filtrate solution were precipitated with ethanol followed by centrifugation (8000 rpm, 5 min). The precipitate were washed with water and ethanol to remove the residual enzyme, solvent, unreacted reagents, and byproduct. Lastly, the obtained lipase-catalyzed dextrin esters were dried in vacuum at 100 °C for 24 h.

2.4. Chemical synthesis of fully-substituted dextrin esters


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To compare the properties of substitution degree and position, fully-substituted dextrin esters (fully-DexEs) and random-substituted dextrin esters (random-DexEs) were synthesized by chemical esterification according to the procedure described in our previous report.10 Briefly, a heterogeneous mixture of trifluoroacetic anhydride (50 ml) and carboxylic acid (50 ml) had been stirred at 50 ℃ for 10 min. And freeze-dried dextrin (1 g) was immediately added into mixture and kept for 2 hours. After cooling to room temperature, the mixture was poured into methanol, and then precipitate was filtered. To remove the unreacted reagents and byproduct, reprecipitation was conducted in three time and was completely dried in vacuo.

2.5. Peracylation for confirmation of substitution position The first method to identify the substitution position of mono-DexEs was directly performed by two-dimensional NMR analysis. Another method was to indirectly compare the structures of mono-DexEs and fully-DexEs, but the following procedure was additionally required because of the solubility differences in common solvents; The mono-DexEs obtained through enzymatic synthesis were converted to regioselectivly per-substituted dextrin esters (regio-DexEs) by following a stepwise chemical synthesis. The substitution position of mono-DexEs was indirectly confirmed by comparison to regio-DexEs and fully-DexEs.

2.6. Methods


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2.6.1. Nuclear magnetic resonance (NMR) spectroscopy Chemical structure of were measured by 1H-NMR,

13C-NMR

and two-dimensional

NMR (JNM-A500, 500 MHz, JEOL Ltd. Japan) with dimethyl sulfoxide-d6 as a solvent, where solution concentration was set to 20 mg/ml. Chemical shifts (δ in ppm) were referenced to the resonance of tetramethylsilane (TMS; δ = 0) for dimethyl sulfoxide-d6. 2.6.2. Fourier-transform infrared (FT-IR) spectroscopy FT-IR spectra of dextrin and dextrin esters were acquired using Nicolet 6700 spectrometer (Thermo Scientific Corp. USA) using KBr disk technique. For FT-IR measurement, the samples were mixed with anhydrous KBr and then compressed into thin disk-shaped pellets. The spectra were obtained with a resolution of 2 cm−1 and a wavenumber range of 400−4000 cm−1. 2.6.3. Thermogravimetric analysis (TGA) Thermal decomposition behaviors were investigated using a Thermo plus TG8120 system (Rigaku Corp., Japan). 5 mg of each sample was put in platinum pan and heated at heating rate of 10 °C/min from 40 °C to 600 °C under nitrogen gas.

3. Results and discussion 3.1. Optimization of enzyme activity 3.1.1. Effect of reaction temperature on enzyme activity


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An important property of enzymes is that they exhibit substrate specificity under the conditions required for activity. Initially, the effect of reaction temperature was studied, because enzyme activity is sensitive to temperature. The activity of four enzymes in the catalyzed reaction between dextrin and vinyl acetate was determined at various temperatures, ranging from 40 °C to 80 °C, after 12 hours. Figure 1 shows the DS values of the dextrin acetate (DexAc) for each type of enzyme. Overall, the catalytic activity of all enzymes increased with increasing temperature. However, two of the four enzymes, Lipozyme CALB and Lipozyme TL IM, displayed high catalytic activity. Lipozyme CALB is a non-immobilized enzyme extracted from Thermomyces lanuginose, and Lipozyme TL IM is an immobilized enzyme extracted from Candida antartica B. Although both enzymes exhibited high catalytic activity, the reaction conditions were only optimized for Lipozyme TL IM in the following experiments. This is because the immobilized enzyme has superior thermal stability and reusability, which is commercially advantageous.22 The DS values of DexAc produced by the Lipozyme TL IM-catalyzed reaction at 40 °C, 50 °C, 60 °C, 70 °C and 80 °C were 0.61, 0.76, 0.90, 0.90 and 0.94, respectively. The DS values gradually increase until 60 °C, and stay relatively constant, at around 0.90, beyond this temperature. This indicates that a reaction temperature of 60 °C is sufficient for imparting acetate groups to the dextrin regioselectively.

3.1.2. Effect of solvent on DS value


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The selection of solvent is also an important factor in the optimization of enzymatic polysaccharide modification reactions. To obtain a high DS value, the polysaccharide should be fully dissolved and also have affinity for the enzyme. Dextrin characteristically dissolves in polar solvents. The catalytic activity of the enzyme

was

confirmed

dimethylsulfoxide

using

(DMSO),

typical

polar

solvents,

N,N-dimethylformamide

such

(DMF)

as

water,

and

N,N-

dimethylacetamide (DMAc) (Figure 2(a)). In water, the substitution reaction was not detectable, because the enzyme-catalyzed hydrolysis reaction dominated. In DMSO, DMF and DMAc, the DS values were 0.90, 0.32 and 0.23, respectively. There is a positive correlation between the solvent polarities and the DS values. Therefore, the differences in the DS values might simply be due to the differences in polarity of the solvents. The most polar solvent (DMSO) gave the highest DS value (0.90). This solvent is commonly used in experiments involving polysaccharides.23, 24

3.1.3. Effect of glucose unit/vinyl acetate molar ratio on DS value To confirm the effect of the acyl donor content on the DS value, the reaction was carried out using various molar ratios of the glucose unit to vinyl acetate (1:1, 1:2 1:3, 1:4, 1:5 and 1:6). The results are shown in Figure 2(b). As the molar ratio of vinyl acetate increases, the DS value also increases. This is because the reaction frequency between the hydroxyl group of the glucose and the ester group of vinyl acetate increases stochastically. However, there was no significant difference in the DS values at molar ratios higher than 1:3 (all conditions between 1:3 and 1:6 gave


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DS values between 0.90 and 0.94). These results indicate that a molar ratio of 1:3 is sufficient to obtain a high DS value. Previous studies on other polysaccharides have shown similar results.

3.1.4. Effect of media concentration on DS value It is well known that branched polymers, such as dextrin, have unique viscosity properties.25 Therefore, to confirm the effect of the dextrin media concentration, the catalytic activity of the enzyme was determined at various dextrin concentrations, ranging from 2 to 8 wt%. Dextrin showed good solubility in DMSO and low visible viscosity at all media concentrations. Figure 2(c) shows the DS values of DexAc produced by the lipase-catalyzed reaction versus media concentration. The DS values did not change significantly with media concentration, and the highest value obtained (0.90) was at 4 wt%. These results indicate that the most efficient media concentration for collision probability of dextrin, vinyl acetate and enzyme is 4 wt%.

3.1.5. Effect of enzyme dosage on DS value The amount of used enzyme in an enzyme-catalyzed reaction is a factor that directly influences the degree of reaction. The DS values were determined while increasing the amount of enzyme from 0.25 U/mL to 7.5 U/mL (Figure 2(d)). When a small amount of enzyme was used (between 0.25 U/mL and 1 U/mL), the DS value increased sharply with increasing enzyme dosage. Between 1 U/mL and 2.5 U/mL,


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the DS value gradually increased with increasing dosage. However, when the dosage exceeded 3.75 U/mL, the DS value began to decrease. The highest DS value (0.90) was observed at 2.5 U/mL, indicating that the enzyme is the most active at this dosage. The lower degree of reactivity with excess enzyme is thought to be due to the interruption of the movement of the enzyme to neighboring hydroxyl groups by other enzymes. It has been shown in other literature that the enzyme engages the hydroxyl groups of polysaccharide substrates in a sequential manner.25

3.1.6. Effect of reaction time on DS value To obtain fully-modified DexAc (i.e., with a DS value of 1), the reaction time was increased from 2 hours to 120 hours, and the effect of reaction time on the DS values was determined. As shown in the Figure 3, the dextrin substitution reaction occurs rapidly within the first 24 hours, giving a DS value of 1.06 at the 24-hour time point. The DS values at 48, 72 and 120 hours were 1.21, 1.20 and 1.23, respectively. These values are similar to the DS value at 48 hours, indicating that only a slight substitution reaction takes place up to the equilibrium state of the reaction. These results suggest that substitution reactions at the C2 and C3 positions are expected only after the substitution reaction takes place preferentially at the C6 hydroxyl group (i.e., regioselectively). Therefore, the time required to obtain monosubstituted dextrin acetate (mono-DexAc) (DS = 1.06) is 24 hours.


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3.2. Characterization of mono-DexAc 3.2.1. Structure properties (NMR) The structural analysis of mono-DexAc (with a DS value of 1.06) was carried out using 1H,

13C

and two-dimensional NMR. Figure 4 shows the HSQC spectrum of

mono-DexAc with the respective protons marked on each of the peaks. The hydroxyl peaks in the dextrin 1H-NMR spectrum (X-axis), showed slight changes during the enzymatic reaction; the disappearance of the peak corresponding to the hydroxyl group at the C6 position demonstrates the regioselective substitution of the dextrin. Concurrently, a strong Ac-CH3 peak is observed at 2.02 ppm, due to the addition of the acetate group. In the

13C-NMR

spectrum (Y-axis), peaks

corresponding to the Ac-CH3 (21.5 ppm) and Ac-COO- (170.7 ppm) groups of the regioselectively substituted acetate appear together, and the peaks corresponding to the glucose carbons are observed in the region of 100–60 ppm. The results of the NMR study show that the regioselective reaction proceeds successfully.

3.2.2. Confirmation of substitution position It was difficult to directly confirm the substitution positions of mono-DexAc and fully acetylated dextrin (fully-DexAc) using NMR analysis, because of the differences in solubility of the two substances in common solvents. To solve this problem, mono-DexAc was peracylated, replacing the remaining hydoxyl groups with propionate groups (regio-DexAcPr). The substitution positions of five dextrin


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esters, fully-DexAc, fully-DexPr, random-DexAcPr and regio-DexAcPr, all of which dissolved in chloroform, were identified by their carbonyl peaks in

13C-NMR

(Figure 5). The carbonyl peaks of the fully-substituted dextrin esters appear in three regions of the spectrum, but those of fully-DexPr (Pr-COO-; 174.2, 174.0 and 173.0 ppm) were found to be higher than those of fully-DexAc (Ac-COO-; 170.9, 170.6 and 179.7 ppm). All carbonyl peaks corresponding to acetate and propionate were observed in the spectrum of random-DexAcPr, which was synthesized to determine the effect of the composition ratio. The spectrum of regio-DexAcPr includes a C6 acetate peak at 170.9 ppm, in addition to the C2 and C3 propionate peaks at 173.1 ppm and 174.0 ppm, respectively. These results prove that the lipase-catalyzed reaction proceeds regioselectively, in accordance with 1H-NMR and 13C-NMR data obtained for mono-DexAc.

3.2.3. Functional group analysis (FT-IR) FT-IR analysis was performed to confirm the change in functional groups during the esterification reaction. The results are shown in Figure 6. Neat dextrin exhibited a strong and broad peak in the region of 3800–3100 cm-1. This is due to the –OH groups located at the C2, C3 and C6 positions. The peaks that appear at the regions of 3000 cm-1 and 1700–900 cm-1 correspond to C–H stretching and C–H bending, respectively (Figure 6(a)). In the case of mono-DexAc (obtained by enzymatic esterification), a strong peak appears at 1750 cm-1, which is not observed in the spectrum of neat dextrin. This peak is due to the C=O bond of acetate, which is


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present at the C6 position. In addition, the spectrum includes all of the peaks in the spectrum of neat dextrin (Figure 6(b)). Finally, Figure 6(c) shows the spectrum of fully-DexAc, which was obtained through chemical esterification. The substitution of all hydroxyl groups with acetate resulted in the disappearance of the −OH peak, while strong C=O and C–H peaks of the acetate were observed. These results are in accordance with the NMR results.

3.2.4. Thermal stability The thermal stabilities of dextrin, mono-DexAc and fully-DexAc were analyzed by thermogravimetric analysis (TGA) and the results are shown in Figure 7. The thermal stability of mono-DexAc is between that of dextrin and fully-DexAc. The decomposition temperatures at 95% weight loss (Td

95%)

of dextrin, mono-DexAc

and fully-DexAc were 290.9 °C, 310.1 °C and 342.3 °C, respectively. These results indicate that the esterification reaction enhanced the thermal stability of dextrin, as measured by Td 95%. Many previous studies on polysaccharide derivatives, including starch, curdlan, glucomannan and xylan, have shown that levoglucosan, a volatile organic compound, is produced during the pyrolysis of polysaccharides.26,

27

The

esterification of the hydroxyl groups in fully-DexAc inhibits the formation of levoglucosan, relative to mono-DexAc. mono-DexAc is less affected owing to its relatively low DS value.


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3.3. Synthesis of mono-substituted dextrin esters using the optimal conditions for Lipozyme TL IM The vinyl acyl donor plays an important role in the physicochemical properties of the final product, because it determines the length of the side-chain of the modified dextrin. The individual reactivity of each acyl donor (vinyl acetate, propionate, hexanoate and laurate) was confirmed for Lipozyme TL IM by increasing the reaction time. As shown in Table 2 and Figure 8, the lipase-catalyzed production of DexAc exhibited the fastest initial reaction rate. This is because the shorter acyl donor has less steric hindrance and a higher solvent affinity. In the case of the lipase-catalyzed production of laurate-substituted dextrin (DexLa), the reaction proceeded considerably slower than the others. This is because of the low solvent affinity the vinyl laurate (due to its high hydrophobicity). However, the DexLa produced from the lipase-catalyzed reaction showed the highest DS value (1.30) at 120 hours. This result might be because of an increase in affinity between the vinyl laurate and the mono-DexLa (i.e., the hydrophobicity of the DexLa gradually increases with the addition of laurate to the dextrin chain, resulting in an increase in affinity with vinyl laurate). All final DS values of the dextrin esters (DexEs) are higher than 1. That indicates that the lipase-catalyzed substitution reaction occurs at the C2 and C3 positions, in addition to the C6 position. However, minimal substitution takes place at the C2 and C3 positions, because the hydroxyl groups at the C6 position are substituted first. The structure analysis of mono-substituted dextrin propionate (mono-DexPr), mono-substituted dextrin hexanoate (monoDexHe) and mono-DexLa (with DS values of 1.03, 1.00 and 1.09, respectively) was


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carried out (Figure 9). The results obtained for the mono-DexEs were similar to those obtained for mono-DexAc, showing terminal methyl protons (-CH3) as well as methylene protons (-CH2-). These results demonstrate that the lipase enzyme exhibits regioselective modification of dextrin when using acyl donors other than vinyl acetate.


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4. Conclusions The activities of lipase enzymes in the regioselective synthesis of dextrin acetate were investigated. The structure of the dextrin acetate produced by the lipasecatalyzed reaction was analyzed by NMR, which confirmed the regioselective substitution. An immobilized lipase enzyme (Lipozyme TL IM) showed the most efficient reactivity. The optimal conditions for obtaining a high DS value were determined to be: a reaction temperature of 60 °C, the use of DMSO as a solvent, a 1:3 molar ratio of glucose unit/acyl donor, an enzyme dosage of 2.5 U/mL and a dextrin concentration of 4 wt%. Based on these optimal conditions, dextrin esters with varying side-chain lengths (C2-12) were obtained using the lipase-catalyzed process with increased reaction time. The initial reaction rates and final DS values of the dextrin esters were affected by the chain length of the vinyl acyl donor. As the chain length increased, the initial rate slowed (because of increased steric hindrance), but the final DS value increased (because of increased hydrophobicity). Furthermore, the substitution reaction was confirmed to occur at the C6 position of dextrin preferentially. After all the C6 positions are substituted, the reaction occurs slightly at the C2 and C3 positions. The regioselectively mono-substituted dextrin esters showed thermal stabilities between those of unmodified dextrin and the fullysubstituted dextrin esters. Overall, these results demonstrate the successful regioselective modification of dextrin using a lipase enzyme as a biocatalyst. This study contributes to the green chemistry available for the modification of polysaccharides.


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5. Acknowledgments This work was carried out as part of the “Innovative Synthesis of High-Performance Bioplastics from Polysaccharides” project supported by JST ALCA Grant Number JPMJAL1502, Japan.

References (1) Godswill, A. C. Sugar alcohols: Chemistry, production, health concerns and nutritional importance of mannitol, sorbitol, xylitol, and erythritol. International Journal of Advanced Academic Research 2017, 3, 31-66. (2) Das, D.; Pal, S. Modified biopolymer-dextrin based crosslinked hydrogels: application in controlled drug delivery. RSC Adv. 2015, 5(32), 25014-25050. (3) Tester, R. F.; Qi, X. β-limit dextrin–Properties and applications. Food hydrocolloids 2011, 25(8), 1899-1903. (4) Gonçalves, C.; Gama, F. M. Characterization of the self-assembly process of hydrophobically modified dextrin. Eur. Polym. J. 2008, 44(11), 3529-3534. (5) Carvalho, J.; Gonçalves, C.; Gil, A. M.; Gama, F. M. Production and characterization of a new dextrin based hydrogel. Eur. Polym. J. 2007, 43(7), 3050-3059. (6) Heinze, T. Esterification of polysaccharides.; Heinze, T., Liebert, T., Koschella, A.; Springer, Berlin/Heidelberg, 2006; p 232.


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(7) Iozep, A. A.; Bessonova, N. K.; Passet, B. V. Synthesis of carboxyethyl polysaccharide esters. Russ. J. Appl. Chem. 1998, 71(6), 1028-1031. (8) Chatterjee, C.; Pong, F.; Sen, A. Chemical conversion pathways for carbohydrates. Green Chem. 2015, 17(1), 40-71. (9) Cumpstey, I. Chemical modification of polysaccharides. ISRN Org. Chem. 2013. (10) Lee, H. Y.; Danjo, T.; Iwata, T. Synthesis and characterization of dextrin derivatives by heterogeneous esterification. J. Polym. Res. 2017, 24(11), 183-190. (11) Enomoto-Rogers, Y.; Iio, N.; Takemura, A.; Iwata, T. Synthesis and characterization of pullulan alkyl esters. Eur. Polym. J. 2015, 66, 470-477. (12) Chien, C. Y.; Iwata, T. Synthesis and characterization of regioselectively substituted curdlan hetero esters with different ester groups on primary and secondary hydroxyl groups. Carbohydr. Polym. 2018, 181, 200-206. (13) Fox, S. C.; Li, B.; Xu, D.; Edgar, K. J. Regioselective esterification and etherification of cellulose: a review. Biomacromolecules 2011, 12(6), 1956-1972. (14) Karaki, N.; Aljawish, A.; Humeau, C.; Muniglia, L.; Jasniewski, J. Enzymatic modification of polysaccharides: mechanisms, properties, and potential applications: a review. Enzyme Microb. Technol. 2016, 90, 1-18. (15) van den Broek, L. A.; Boeriu, C. G. Enzymatic synthesis of oligo-and polysaccharide fatty acid esters. Carbohydr. Polym. 2013, 93(1), 65-72.


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(16) Klibanov, A. M. Improving enzymes by using them in organic solvents. nature 2001, 409(6817), 241-246. (17) Kumar, A.; Dhar, K.; Kanwar, S. S.; Arora, P. K. Lipase catalysis in organic solvents: advantages and applications. Biol. Proced. Online 2016, 18(1), 2-11 (18) Junistia, L.; Sugih, A. K.; Manurung, R.; Picchioni, F.; Janssen, L. P.; Heeres, H. J. Synthesis of higher fatty acid starch esters using vinyl laurate and stearate as reactants. Starch‐Stärke 2008, 60(12), 667-675. (19) Horchani, H.; Chaâbouni, M.; Gargouri, Y.; Sayari, A. Solvent-free lipase-catalyzed synthesis of long-chain starch esters using microwave heating: Optimization by response surface methodology. Carbohydr. Polym. 2010, 79(2), 466-474. (20) Adak, S.; Banerjee, R. A green approach for starch modification: Esterification by lipase and novel imidazolium surfactant. Carbohydr. Polym. 2016, 150, 359-368. (21) Chakraborty, S.; Sahoo, B.; Teraoka, I.; Miller, L. M.; Gross, R. A. Enzyme-catalyzed regioselective modification of starch nanoparticles. Macromolecules 2005, 38(1), 61-68. (22) DiCosimo, R.; McAuliffe, J.; Poulose, A. J.; Bohlmann, G. Industrial use of immobilized enzymes. Chem. Soc. Rev. 2013, 42(15), 6437-6474. (23) Ge, J., Lu, D.; Wang, J.; Liu, Z. Lipase nanogel catalyzed transesterification in anhydrous dimethyl sulfoxide. Biomacromolecules 2009, 10(6), 1612-1618.


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(24) Pedersen, N. R.; Kristensen, J. B.; Bauw, G.; Ravoo, B. J.; Darcy, R.; Larsen, K. L.; Pedersen, L. H. Thermolysin catalyses the synthesis of cyclodextrin esters in DMSO. Tetrahedron: Asymmetry 2005, 16(3), 615-622. (25) Satoh, T.; Imai, T.; Ishihara, H.; Maeda, T.; Kitajyo, Y.; Sakai, Y.; Kakuchi, T. Synthesis, branched structure, and solution property of hyperbranched D-glucan and Dgalactan. Macromolecules 2005, 38(10), 4202-4210. (26) Ponder, G. R.; Richards, G. N. A review of some recent studies on mechanisms of pyrolysis of polysaccharides. Biomass Bioenergy 1994, 7(1-6), 1-24. (27) Ponder, G. R.; Richards, G. N.; Stevenson, T. T. Influence of linkage position and orientation in pyrolysis of polysaccharides: A study of several glucans. J. Anal. Appl. Pyrolysis 1992, 22(3), 217-229.


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List of Tables and Figures Table 1. Types and characteristics of lipase enzymes Table 2. DS values of dextrin esters for each reaction time under optimal conditions Scheme 1. Synthesis of regioselectivly mono-substituted dextrin esters using a lipase enzyme. Figure 1. DS values of dextrin acetate for each type of enzyme versus reaction temperature. Figure 2. DS values of dextrin acetate obtained by modifying various parameters: (a) the solvent, (b) the molar ratio of glucose unit/vinyl acetate (GU/VA), (c) the media concentration and (d) the enzyme dosage. Figure 3. DS values of dextrin acetate versus reaction time under optimal conditions. Figure 4. HSQC spectrum of mono-substituted dextrin acetate (DS = 1.06). Figure 5. 13C-NMR spectrum of peracylated dextrin esters. (a) fully-DexAc (DS = 3), (b) fullyDexPr (DS = 3), (c) random-DexAcPr (DS = 1.5/1.5) and (d) regio-DexAcPr (DS = 1.06/1.94). Figure 6. FT-IR spectra of dextin and dextrin esters. (a) dextrin, (b) mono-DexAc (DS = 1.06) and (c) fully-DexAc (DS = 3). Figure 7. Thermogravimetric analysis curves of dextrin and dextrin esters. (a) dextrin, (b) monoDexAc (DS = 1.06) and (c) fully-DexAc (DS = 3). Figure 8. DS values of dextrin esters versus reaction time under optimal conditions.


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Figure 9. 1H-NMR spectra of mono-substituted dextrin esters. (a) mono-DexPr (DS = 1.03), (b) mono-DexHe (DS = 1.00) and (c) mono-DexLa (DS = 1.09).


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Table 1. Types and characteristics of lipase enzymes Product name Source Type Activity Lipozyme CALB Candida antartica B Liquid lipase 5×103 U/g Novozyme 435 Candida antartica B Immobilized lipase 1×104 U/g Lipozyme TL 100L Thermomyces lanuginosa Liquid lipase 1×105 U/g Lipozyme TL IM Thermomyces lanuginosa Immobilized lipase 2.5×102 U/g * Lipase unit is the amount of enzyme activity liberates 1μmol of each specific product represented by Novozymes. For the immobilized lipase, the enzyme unit (U/g) contains the mass of substrate resin for immobilization of enzyme.


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Table 2. DS values of dextrin esters for each reaction time under optimal conditions DexEs DexAc DexPr DexHe DexLa

2h 0.29 0.17 0.09 0.02

4h 0.46 0.43 0.29 0.06

8h 0.83 0.80 0.76 0.17

12 h 0.91 0.89 0.81 0.30

24 h 1.06 1.03 1.00 0.84

48 h 1.21 1.11 1.07 1.09

72 h 1.20 1.18 1.12 1.19

120 h 1.23 1.21 1.20 1.30


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Scheme 1. Synthesis of regioselectively mono-substituted dextrin esters using a lipase enzyme.


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Figure 1. DS values of dextrin acetate for each type of enzyme versus reaction temperature.


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Figure 2. DS values of dextrin acetate obtained by modifying various parameters: (a) the solvent, (b) the molar ratio of glucose unit/vinyl acetate (GU/VA), (c) the media concentration and (d) the enzyme dosage.


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Figure 3. DS values of dextrin acetate versus reaction time under optimal conditions.


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Figure 4. HSQC spectrum of mono-substituted dextrin acetate (DS = 1.06).


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Figure 5. 13C-NMR spectrum of peracylated dextrin esters. (a) fully-DexAc (DS = 3), (b) fullyDexPr (DS = 3), (c) random-DexAcPr (DS = 1.5/1.5) and (d) regio-DexAcPr (DS = 1.06/1.94).


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Figure 6. FT-IR spectra of dextin and dextrin esters. (a) dextrin, (b) mono-DexAc (DS = 1.06) and (c) fully-DexAc (DS = 3).


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Figure 7. Thermogravimetric analysis curves of dextrin and dextrin esters. (a) dextrin, (b) monoDexAc (DS = 1.06) and (c) fully-DexAc (DS = 3).


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Figure 8. DS values of dextrin esters versus reaction time under optimal conditions.


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Figure 9. 1H-NMR spectra of mono-substituted dextrin esters. (a) mono-DexPr (DS = 1.03), (b) mono-DexHe (DS = 1.00) and (c) mono-DexLa (DS = 1.09).


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Page 39 of 39 1 2 3 4 5 6 7 8 9 10 11 O 12 13 14 CH2OH 15 O 16 17 OH 18 19 OH 20 21 22 23 24 25 26 27 28 29 30 31 32 33

Biomacromolecules

CH2OR

CH2OR O CH2OH

CH2OH O OH

Vinyl Esters

R

OH O

O

CH2OH

HO OH One-step!

OH O

OH

OH

CH O

O

OH

OH

O OH

O

n

O

CH2OR

CH2OR

OH Enzymatic CH2 Esterification

OH

Dextrin

O OH

O

OH

OH

O

OH

O

O

O

O OH OH

m


OH

n

Regioselectively-substituted

Dextrin Esters

Lipase-Catalyzed Regioselective Synthesis of Dextrin Esters

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