Research Article pubs.acs.org/journal/ascecg
Basic Amino Acids as Green Catalysts for Isomerization of Glucose to Fructose in Water Qiang Yang, Matthew Sherbahn, and Troy Runge* Department of Biological Systems Engineering, University of WisconsinMadison, 460 Henry Mall, Madison, Wisconsin 53706, United States S Supporting Information *
ABSTRACT: Fructose is not only an important food and beverage ingredients, but also a renewable resource for production of 5-hydroxymethylfurfural. This study investigated nontoxic basic amino acids (arginine, lysine, and histidine) as isomerization catalysts to isomerize glucose to fructose in water. The results showed that arginine was the most effective isomerization catalyst with a 31% maximum fructose yield with 76% selectivity achieved under the investigated reaction conditions. A mechanistic study verified that the isomerization reaction catalyzed by arginine proceeded through an enediol intermediate, which formed after the deprotonation at C-2 position of acyclic glucose. KEYWORDS: Isomerization, Amino acids, Arginine, Lysine, Glucose, Fructose
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disturbances in vision and systemic health effects.16−20 Since most of these reported acid and base catalysts have some toxicity, they would not be considered for food applications and would not follow green chemical principles for biorenewable chemical production. Therefore, it is desirable to explore nontoxic isomerization catalysts. Amino acids contain basic amine and acidic carboxylic acid functional groups. All amino acids are amphoteric, but the side chains can make them weak bases. There are three basic amino acids: arginine, lysine, and histidine. Arginine is a conditionally essential amino acid, while lysine and histidine are essential amino acids. They are widely found in a variety of foods such as milk and wheat, and can be also produced from fermentation of glucose and fructose.21,22 Being part of our food system, the basic amino acids are not toxic, unlike the reported organic amines and other chemical catalysts. Therefore, for this study, we tested the basic amino acids as green catalysts for the isomerization of glucose to fructose in water.
INTRODUCTION High-fructose corn syrup (HFCS), a mixture of glucose, fructose, and minor amounts of oligosaccharides, has been widely used in foods and carbonated beverages, as a flavor enhancer, sweetener, and color developer.1 Fructose is also a renewable resource for production of 5-hydroxymethylfurfural (HMF), which is a versatile platform chemical for production of chemicals and liquid fuels.2 Although fructose is industrially important for both food and biorenewables, it is not found in great abundance compared to other carbohydrates and is typically produced via isomerization of glucose, which is widely abundant, and low-cost. Fructose is typically industrially produced by immobilized glucose isomerases.3 However, the enzymatic isomerization process has several drawbacks, including high-cost, longer reaction time, need for buffers, and irreversible deactivation. By contrast, chemical catalysts have wider operating temperature, longer lifetime, and better resistance to impurities. Chromium chloride and aluminum chloride can achieve approximately 20− 26% fructose yields.4,5 Chromium chloride has relatively lowtoxicity,6 while aluminum chloride is highly toxic and causes neurological conditions.7,8 Sn-β zeolite, a heterogeneous Lewis acid catalyst, can yield approximately 31% fructose from glucase.9,10 However, metal can be leached into the product solution during the isomerization reaction, which can cause health concerns if used as a food ingredient and catalytic issues if used for chemicals or fuels.11,12 Although organic Brønsted bases can achieve 10−36% fructose yields with 40−73% selectivity, they are highly toxic as they can also create strong odors.12−14 For example, tertiary amines can cause graphic © XXXX American Chemical Society
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EXPERIMENTAL SECTION
Materials. Arginine, lysine, histidine, 4-guanidinobutyric acid, 3guanidinopropionic acid, guanidineacetic acid, triethylamine, morpholine, piperazine, ethylenediamine, piperidine, pyrrolidine, 1,5,7triazabicyclo [4.4.0] dec-5-ene, tetramethylguanidine, D,L-glyceraldehyde, D-fructose, D-glucose, and D-deuterated glucose (glucose-2-D) were bought from Sigma-Aldrich. All chemicals were used as received. Received: March 23, 2016 Revised: April 22, 2016
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DOI: 10.1021/acssuschemeng.6b00587 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering
Figure 1. Isomerization of glucose in water by 5 mol % (a), 8 mol % (b), and 10 mol % (c) arginine. Reaction conditions: 10 wt % glucose, 5 mol % or 8 mol % or 10 mol % arginine relative to glucose, 2−20 min, 1 mL H2O, 80−120 °C. Characterization and Analysis Method. 1H, 13C, and 1H−13C HSQC (heteronuclear single-quantum correlation) NMR (nuclear magnetic resonance) spectra were collected with a Bruker Biospin AVANCE 500 MHz NMR spectrometer (Bruker Corporation). Liquid chromatography−mass spectrometry (LC−MS) analysis was conducted on a Shimadzu LCMS-2020 (Shimadzu) system using the dual ion source method for ionization. Glucose, mannose, and fructose were analyzed on an Agilent 1220 Infinity high-performance liquid chromatography (HPLC, Agilent Technologies) system equipped with
Isomerization of Glucose to Fructose. Isomerization experiments of glucose (10%, wt/wt) aqueous solutions with basic amino acids were carried out in 6 mL thick-walled glass reactors at different temperatures (80−130 °C). The isomerization experiment was allowed to proceed for specific times (2−20 min), and then was stopped by rapidly cooling the reactor in an ice bath. Small aliquots of the filtered and diluted reaction media were taken for carbohydrate (fructose, glucose, and mannose) analysis by high-performance liquid chromatography. B
DOI: 10.1021/acssuschemeng.6b00587 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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ACS Sustainable Chemistry & Engineering
Table 1. Effects of Arginine Dosage and Reaction Temperature on Rate Constant and Calculated Apparent Activation Energy for Glucose Isomerization by Argininea Ea (kJ mol−1)
dosage (%)
temp (°C)
10−4 × κ (s−1)
YFru (%)
SFru (%)
YMan (%)
carbon balance (%)
10−3 × TOF (molFru mol−1Arg s−1)
44 ± 1.3
5
120 110 100 90 80 120 110 100 90 80 120 110 100 90 80
4.0 3.0 2.0 1.2 0.9 6.1 4.4 3.1 2.0 1.3 6.4 4.8 3.8 2.2 1.5
26 21 17 10 5 30 24 21 13 6 31 27 21 17 14
78 90 81 85 74 78 72 89 80 58 76 81 86 95 94
0.66 0.54 0.31 0.21 0.05 0.76 0.61 0.42 0.31 0.11 0.81 0.72 0.53 0.46 0.35
93.33 98.21 96.32 98.44 98.29 92.30 91.28 97.82 97.06 95.76 91.02 94.39 97.11 99.56 99.46
5.78 4.67 3.78 2.22 1.11 4.17 3.33 2.92 1.81 0.83 3.44 3.00 2.33 1.89 1.56
8
10
Reaction conditions: 10 wt % glucose, 5 mol % or 8 mol % or 10 mol % arginine relative to glucose, 1 mL H2O, 2−20 min, 80−120 °C. Carbon balance is defined as the ratio of moles of carbon in products (fructose and mannose) and unreacted glucose to the mole of carbon in the initial glucose.
a
an Agilent Hi-Plex H analytical column (7.7 mm × 300 mm), a BIORAD guard column, and a refractive index detector (RID). Yield of fructose (%) and selectivity for conversion to fructose (%) were calculated as the mole percentages of the initial glucose and reacted glucose, respectively. Organic acids (acetic, formic, glycolic, and lactic acid) were determined using high-performance ion chromatography (HPIC, ICS-3000, Dionex, Sunnyvale, CA), equipped with a UV−vis detector and Superlcogel C-610H analytic (30 cm × 7.8 mm) and guard (5 cm × 4.6 mm) columns.
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RESULTS AND DISCUSSION Glucose Isomerization. Previous studies have demonstrated that isomerization performance of amine catalysts were primarily dependent upon their basicity, which was measured by the pKa value of its conjugated acid.13−15 Arginine with a guanidinium group was the most basic amino acid, and its αamine and side amine have characteristic pKa values of about 9.04 and 12.48, respectively (Scheme S1). The isomerization of glucose by arginine was kinetically investigated through changing reaction time (2−20 min), reaction temperature (80−120 °C), and dosage (5−10 mol % relative to glucose). The results were presented in Figure 1 and Table 1. The results show that higher fructose yields and selectivity were obtained when the reactions were carried out under higher temperature for longer time with higher dosage of arginine. Specifically, a 31% maximum fructose yield with 76% selectivity was achieved by the 10 mol % arginine (relative to glucose) when the reaction was carried out at 120 °C for 15 min. For comparison, several organic amines were also investigated under the same reaction conditions. The results presented in Figure 2 show that arginine can achieve a similar level of fructose yield to triethylamine (32%), piperidine (30%), piperazine (27%), ethylenediamine (26%), and pyrrolidine (28%). However, due to the relatively higher fructose yield, arginine achieved a better glucose-to-fructose selectivity than the organic amines studied (76% vs 45−62%). Similar to arginine, 1,5,7-triazabicyclo [4.4.0] dec-5-ene (TBD, pKa = 21 in tetrahydrofuran) has a bicyclic guanidine, and tetramethylguanidine (TMG, pKa = 13.6) also contains guanidine. The TBD and TMG can achieve 32% fructose yields. However, arginine showed better fructose selectivity than the TBD and
Figure 2. Comparison of arginine and lysine with other organic amines: Arg, arginine; Lys, lysine; TEA, triethylamine; Mor, morpholine; Pip, piperazine; Eth, ethylenediamine; Pipe, piperidine; Pyr, pyrrolidine; TBD, 1,5,7-triazabicyclo [4.4.0] dec-5-ene; TMG, tetramethylguanidine. Reaction conditions: 10 wt % glucose, 10 mol % catalyst relative to glucose, 15 min, 1 mL H2O, 120 °C.
TMG. It was hypothesized that the presence of carboxylic acid group acts as a pH buffer and was responsible for the improved fructose selectivity. Because the carboxylic acid is a pretty good source of protons, a proton transfer may occur from one site to the other in arginine. As a result, the presence of carboxylic acid makes the guanidinium group (pKa = 12.48) in arginine less basic than the guanidine groups (pKa = 13.6) in TBD and TMG. Therefore, arginine with a similar fructose yield and a lower glucose conversion showed better fructose selectivity. Arginine contains the guanidinium, primary amine, secondary amine, imine, and α-amine functional groups, which all play important roles on the basicity of arginine. Specifically, the primary and secondary amines and imine in the guanidinium side chain have the abilities to delocalize protons, and the αamine can shield the guanidinium side chain from the carboxylic acid group. The combined effect of these functional C
DOI: 10.1021/acssuschemeng.6b00587 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering
Figure 3. Isomerization of glucose by lysine. Reaction conditions: 10 wt % glucose, 20 mol % lysine relative to glucose, 2−20 min, 1 mL H2O, 90− 130 °C.
Table 2. Effects of Lysine Dosage and Reaction Temperature on Rate Constant and Calculated Apparent Activation Energy for Glucose Isomerization by Lysinea Ea (kJ mol−1)
dosage (%)
temp (°C)
10−4 × κ (s−1)
YFru (%)
SFru (%)
YMan (%)
carbon balance (%)
10−3 × TOF (molFru mol−1Lys s−1)
30.9
5 10 20
120 120 130 120 110 100 90 120
1.7 2.1 8.2 5.9 4.7 3.7 2.9 10.8
9 12 20 15 19 14 11 19
92 79 46 48 68 75 69 34
0.23 0.26 0.45 0.31 0.25 0.19 0.12 0.41
99.45 97.07 76.97 84.06 91.31 95.52 95.18 63.53
2.00 1.33 1.11 0.83 1.06 0.78 0.61 0.53
40
Reaction conditions: 10 wt % glucose, 5 mol %, 10 mol %, 20 mol %, or 40 mol % lysine relative to glucose, 2−20 min, 1 mL H2O, 90−130 °C. Carbon balance is defined as the ratio of moles of carbon in products (fructose and mannose) and unreacted glucose to the mole of carbon in the initial glucose.
a
1.3 kJ mol−1, which was lower than those of other isomerization catalysts such as triethylamine (58 ± 8 kJ mol−1), chromium chloride (58.6−64.0 kJ mol−1), sodium hydroxide (121 kJ mol−1), aluminum chloride (110 ± 2 kJ mol−1), and Sn-β (93 ± 15 kJ mol−1).4,5,9 Lysine was assumed to play a crucial role in the isomerization by glucose isomerases.23 Relative to arginine, lysine is a weak base with pKa values of about 8.95 for the α-amine and 10.79 for the side amine. However, as shown in Figure 3 and Table 2, lysine was not as effective as arginine in isomerization of glucose to fructose. Under the same reaction conditions, lysine achieved lower fructose yields and poor selectivity than arginine, with only an approximately 20% maximum fructose yield achieved by lysine. Furthermore, increasing the lysine dosage from 5 to 40 mol % resulted in a decrease in the fructose selectivity. The estimated apparent activation energy (30.9 kJ mol−1) for lysine was lower than that (44 ± 1.3 kJ mol−1) for arginine (Figure S2 and Table 2). Therefore, compared with arginine, increasing temperature has a lessened enhancement effect on the rate constant for lysine. Histidine is an essential component of the active site of glucose isomerases.23,24 Histidine has an α-amine group (pKa = 9.17) and two acidic groups (carboxylic acid with a pKa of 1.82 and secondary amine with a pKa of 6.04 in imidazole). As a result, histidine is a very weak base, and its aqueous solution has a pH value of 7.65. Therefore, it cannot isomerize glucose to fructose
groups makes arginine a strong and effective base catalyst for the glucose isomerization. In contrast, arginine derivatives such as 4-guanidinobutyric acid, 3-guanidinopropionic acid, and guanidineacetic acid were unable to isomerize glucose (Scheme S1). Since these compounds also have the guanidinium groups, this result indicates the significance of α-amine for the basicity of arginine. If present, the α-amine and carboxylic acid would be attached to the same carbon atom, which shields the guanidinium from the carboxylic acid. Therefore, for compounds without the α-amine, the basicity of guanidinium would be significantly decreased by the carboxylic acid, which in turn would lead to lower isomerization. The isomerization performance achieved by arginine was also better than the other reported isomerization catalysts such as sodium hydroxide (typically below 10% fructose yield and very poor selectivity), chromium chloride (25.4% fructose yield and 48.6% selectivity), and aluminum chloride (26.3% fructose yield and 82.7% selectivity) (Table S1).5 Effects of temperature and arginine dosage on the rate constant were also analyzed; results are shown in Figure S1. Apparent activation energy (Ea) and turnover frequency (TOF, moles of produced fructose/mol of catalyst/time) were calculated on the basis of the kinetics data and summarized in Table 1. The results show that the TOFs increased with increasing of temperature but decreased with higher arginine dosage. Activation energy for arginine catalyzed isomerization reaction was estimated to be approximately 44 ± D
DOI: 10.1021/acssuschemeng.6b00587 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
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Figure 4. 1H NMR spectra of glucose isomerized by arginine in D2O. Reaction conditions: 10 wt % glucose, 8 mol % arginine relative to glucose, 1 mL D2O, 0−20 min, 110 °C.
Figure 5. 1H−13C HSQC NMR spectrum of isomerized glucose-2-D by arginine.
even with a high dosage (40 mol % relative to glucose). Other amino acids are essentially acidic. These acidic amino acids such as aspartic acid and glutamic acid cannot convert glucose to fructose, only causing byproducts. Reversibility of Glucose Isomerization. Estimated equilibrium constants for the glucose isomerization reaction
catalyzed by arginine were less than 0.6, indicating that the reaction was reversible (Table S2).3−9 The isomerization of produced fructose back to glucose is unavoidable under these conditions. In order to evaluate the reaction reversibility, the isomerization of fructose to glucose by arginine was also kinetically investigated, with the results shown in Figures S3 E
DOI: 10.1021/acssuschemeng.6b00587 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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reactant, the rate constant, glucose conversion, and fructose yield were found to be much lower than those for the unlabeled glucose, as shown in Figure S10 and Table 3. The isotopically
and S4 and Table S3. These results indicate that the apparent activation energy for the fructose isomerization (28.7 kJ mol−1) was much lower than that for the glucose isomerization (44 ± 1.3 kJ mol−1). However, the fructose isomerization reaction was much slower than the glucose isomerization reaction under the same reaction conditions (Table 1 and Table S3). The estimated equilibrium constants (0.06−0.11) in Table S4 for the isomerization of fructose to glucose were lower than those (0.07−0.49) for the isomerization of glucose to fructose (Table S2). These results support the hypothesis that glucose isomerization to fructose was favored under the reaction conditions with the arginine catalyst. Mechanistic Study. A mechanistic study was also conducted with the isomerization reaction catalyzed by arginine in deuterium oxide, with the products monitored by 1H NMR (nuclear magnetic resonance) spectroscopy with time (0−20 min). Peaks around δ = 4.0−3.8 ppm attributed to the formed fructose appeared, and their intensities increased as the reaction proceeded (Figure 4 and Table S5). Thus, hydroxide ions generated after amine reacted with water were shown to be responsible for the isomerization reaction (Scheme S2), suggesting amine shares a similar isomerization mechanism to that of sodium hydroxide.13−15 Arginine and lysine investigated in this study have amine groups and also contain carboxyl groups. Therefore, it was speculated that arginine or lysine shared similar isomerization mechanisms to other reported amines. On the basis of the proposed mechanisms, the glucose isomerization reaction starts with acyclic glucose. Therefore, any cyclic glucose first needs to be transformed to acyclic glucose, which would be achieved through breakage of the C1− O5 ether bond. However, the acyclic glucose is thermodynamically unstable. In addition to deprotonation to the enediol intermediate, it can be spontaneously isomerized to the cyclic glucose. To understand the reversibility of acyclic glucose, the effect of glyceraldehyde on the isomerization reaction was investigated, and the results were presented in Figure S6 and Table S5. It turns out that the addition of glyceraldehyde greatly suppressed the isomerization reaction. Although glyceraldehyde can likely compete with glucose for arginine to be isomerized to 1,3-dihydroxyacetone, the great suppression indirectly indicates the existence of reaction equilibrium between the acyclic glucose and cyclic glucose. The presence of glyceraldehyde was hypothetically favorable to the ringclosure reaction through changing the reaction equilibrium. It has been reported that the amine-catalyzed isomerization proceeds through an enediol intermediate formed after deprotonation at C-2 position of acyclic glucose.13−15 To verify this speculation that the deprotonation occurred at the C2 position, glucose-2-D was used as the reactant, and isomerized glucose-2-D by arginine in water was analyzed by using a 1H−13C HSQC (heteronuclear single-quantum correlation) NMR spectrometer. The isomerized glucose-2-D displays different 1H and 13C signals from glucose-2-D (Figure 5 and Figures S7, S8, and S9). Except that the unreacted glucose-2-D remains the same, there were new peaks observed around δ = 3.9−4.1 ppm (in red), which also appeared in the 1 H spectrum of fructose (Figure S8). The 1H NMR spectra for the produced fructose isomerized from glucose-2-D and unlabeled glucose show no differences (Figure S7). These results indicate that the fructose isomerized from glucose-2-D does not contain deuterium atoms and thus gained its additional proton from water. When glucose-2-D was the
Table 3. Kinetic Isotope Effects (KIEs) for Isomerization of Glucose by Arginine reactant
solvent
10−4 × κ (s−1)
KIE (κH/κD)
glucose glucose-2-D glucose
H2O H2O D2O
6.1 1.7 6.0
3.59 3.59 1.01
labeled at C-2 position induced a kinetic isotopic effect (KIE, κH/κD) of 3.59, indicating that the deprotonation of the C-2 position was the rate-limiting step in the glucose isomerization. In contrast, as a proton donor, the solvent (deuterium oxide) did not play a kinetically significant role (KIE = 1.01) (Table 3). The above NMR studies and observed kinetic isotopic effects support that arginine (or lysine) isomerizes glucose to fructose through an enediol intermediate formed after deprotonation at C-2 position (Scheme 1). Scheme 1. Proposed Reaction Pathway of Isomerization of Glucose to Fructose by Amino Acid through Enediol Intermediate (Mannose Omitted for Clarity)
Identification of Byproduct. In addition to fructose, mannose is another isomerization product of glucose (Scheme S3). However, a very low yield (typically,
