Isomerization of ribose to ribulose using basic amino acids as a catalyst

Abstract

In a batch reactor, 0.01 mol/L arginine, lysine, or histidine, which are natural basic amino acids, was used as an environmentally friendly, “green”, catalyst to isomerize 0.2 mol/L ribose to the corresponding ketose, ribulose, at 110 °C. The changes over time in the conversion of ribose, the yield of ribulose, pH, and the absorbance of the reaction mixture at 280 and 420 nm were measured. The yield of ribulose was highest (ca. 8.5 %) when arginine was used as a catalyst, followed by lysine. Ribulose was also produced with histidine, but the yield was very low (ca. 1.5 %). On the other hand, the coloration, which was evaluated by the absorbance of the reaction mixture at 280 and 420 nm, was highest when lysine was used, followed by arginine. Therefore, arginine was the most suitable green catalyst for isomerizing ribose to ribulose among the three basic amino acids tested.

Introduction

The isomerization and epimerization of reducing sugars by Lobry de Bruyn-Alberda van Ekenstein (LBAE) transformation via an enediol intermediate in alkaline solutions, such as sodium hydroxide or potassium hydroxide, has long been understood (Sowden and Schaffer, 1952). This transformation is also used to convert common saccharides, which are abundant in nature, to rare ones, which are generally called rare sugars because of their low abundance in nature, the physiological and physical functions of which have been attracting attention in recent years (Beadle et al., 1992; Dendene et al., 1994; Choudhary et al., 2011). Rare sugars have relatively more ketoses than aldoses. The production of rare sugars by isomerization of aldoses to their corresponding ketoses using metal catalysts or enzymes has been investigated, but challenges remain in terms of cost and reaction time (Shukla et al., 1985; Shen et al., 2012; Gounder and Davis, 2013). We have reported that isomerization and epimerization of sugars can proceed in water (Usuki et al., 2007), aqueous alcohol (Gao et al., 2015a; Gao et al., 2015b), or phosphate buffer (Onishi et al., 2020; Onishi et al., 2022) under their subcritical fluid conditions. The yields in isomerization of aldoses to their corresponding ketoses in phosphate buffer under subcritical conditions were lower, in the order pentoses < hexoses < disaccharides (Onishi et al., 2022).

Recently, isomerization of glucose to fructose using basic amino acids, including arginine, lysine, and histidine, as catalysts (referred to as “green” catalysts because they are relatively safe and environmentally friendly) has been investigated, with arginine reported to catalyze isomerization most efficiently (Kim and Lee, 2008; Yang et al., 2016). However, in the isomerization of sugars using basic amino acids as catalysts, coloration due to the Maillard reaction is inevitable (Kim and Lee, 2008; Lamberts et al., 2008). We investigated the isomerization of galactose to tagatose using arginine and found that the highest yield was comparable to that obtained from the common alkaline-catalyzed reaction (Milasing et al., 2023).

Here, we aimed to compare the catalytic ability and degree of coloration of three basic amino acids, namely arginine, lysine, and histidine, in the isomerization of ribose to ribulose, ribose being one of the pentoses with a low isomerization yield in phosphate buffer under subcritical conditions, and ribulose being used as a starting material for the synthesis of pharmaceuticals and chemicals (Hricovíniová et al., 2000).

Materials and Methods

Materials d(-)-Ribose (purity: > 99 %), d(-)-ribulose (< 95 %), l(+)-arginine (< 98 %), l(+)-lysine (> 95 %), lhistidine (> 98 %), glycerol (guaranteed reagent), acetonitrile (HPLC grade), and distilled water (HPLC grade) were purchased from FUJIFILM Wako Pure Chemical Corp., Osaka, Japan.

Isomerization of ribose to ribulose d-Ribose and a basic amino acid were dissolved in milli-Q water at concentrations of 0.2 and 0.01 mol/L, respectively. That is, the molar ratio of an amino acid to ribose was fixed to be 0.05 mol/mol-ribose. The initial pH for a mixture with arginine, lysine, and histidine was 9.87, 9.48, and 7.36, respectively. The substrate solution of 2.5 mL was placed in a 3-mL vial. The vial was fitted with an airtight lid, and no solution leaked out until at least 120 °C. Eight vials were placed in a heating bath (EL-02 bath, Major Science Co., Taoyuan, Taiwan) containing 80 % (w/w) glycerol heated to 110 °C to initiate the isomerization. A type-K thermocouple was inserted through a hole in the lid of a vial and placed in the center of a vial containing the same volume (2.5 mL) of water as the substrate solution. The hole was sealed with adhesive to prevent leakage of the liquid inside. The temperature of the water was measured at appropriate time intervals using a thermometer (Thermometer AD-5605H, A & D, Tokyo, Japan). At appropriate time intervals, one vial was removed from the bath and immediately placed in ice water to stop the reaction. Experiments were performed in triplicate for each amino acid.

Analysis The concentrations of ribose remaining and ribulose produced in the reaction mixture were determined using high-performance liquid chromatography (HPLC). The equipment comprised a separation column (Cosmosil^® Sugar-D; 4.6 mm I.D. × 250 mm, Nacalai Tesque, Kyoto, Japan), a guard column (Cosmosil^® Sugar-D; 4.6 mm I.D. × 10 mm, Nacalai), an LC-10ADvp pump (Shimadzu Corp., Kyoto), and a refractive index detector (Shodex RI-101, Showa Denko, Tokyo). The sample volume injected was 5 µL, and the mobile phase used was 88 % (v/v) acetonitrile at a flow rate of 1.0 mL/min. The elution profiles of ribose and ribulose were recorded using a Chromatopac recorder (C-R8A, Shimadzu). It was confirmed that ribulose was the product of the isomerization of ribose by comparing the elution time of authentic ribulose and the product during HPLC.

As ribose is a reducing sugar, it is colored by the Maillard reaction with amino acids (Nakamura et al., 2006). The Maillard reaction produces substances that absorb not only visible light but also UV light (Murata, 2021); therefore, the absorbance of the reaction mixture at both 280 and 420 nm was measured using a spectrophotometer (UV-1280, Shimadzu).

The pH of the reaction mixture was measured at room temperature using a pH meter (LAQUAtwin-pH-22, HORIBA Advanced Techno, Kyoto).

Results and Discussion

Isomerization using arginine, lysine, and histidine Figs. 1(a), (b), and (c) show the changes over time of the temperature of the reaction mixture, the fraction of remaining ribose, the yield of ribulose, pH, and the absorbance at 280 and 420 nm when arginine, lysine, and histidine, respectively, were used as catalysts. Delidovich et al. (2018) reported that isomerization of ribose using NaH₂PO₄ and Na₂HPO₄ as a catalyst resulted in ribulose as the major product, with small quantities of xylulose, arabinose, and lyxose. However, no products other than ribulose were detected in the HPLC chromatograms for any of the amino acids because of very small quantities of the other pentoses. Ribose dissolved in water with no amino acid was used as a control, and Fig. 1(d) shows the changes when the control was treated under similar conditions. In water with no basic amino acids, the pH decreased below 6 immediately after the reaction was initiated. No ribulose formation in water would be ascribed to the low pH since the LBAE transformation does not occur in such pH range (Sowden and Schaffer, 1952).

Fig. 1.

Changes in temperature (dashed line), fraction of remaining ribose (C_S/C_S0; □), yield of ribulose (C_P/C_S0; ◯), pH (◇), absorbance at 280 nm (A₂₈₀, ▽), and absorbance at 420 nm (A₄₂₀, △) during the isomerization of ribose to ribulose with (a) arginine, (b) lysine, and (c) histidine at ca. 110°C. Figure 1(d) represents the changes for the treatment of ribose dissolved in water. The concentrations of ribose and each amino acid were 0.2 and 0.01 mol/L, respectively. Symbols and bars represent mean ± standard deviation (n = 3). Most of the bars are behind the symbols. The solid curves were drawn empirically.

As the isomerization was performed using a batch reactor, it took 5 to 10 min to reach the set temperature, after which the temperature was maintained at 110 ± 1.5 °C. Although some slight isomerization was observed during the period when the temperature was increasing, the decrease in ribose and the formation of the corresponding ketose, ribulose, became more pronounced once the temperature was more than 90 °C. The yield of ribulose depended on the type of amino acid used, although the yield was almost constant for each amino acid at reaction times longer than 15 min. Among the three basic amino acids, the highest yield of ribulose, ca. 8.5 %, was obtained when arginine was used as a catalyst. Even after the yield of ribulose became constant, the concentration of ribose and the absorbance of the reaction mixture at 280 and 420 nm continued to decrease and increase, respectively. This tendency was more pronounced when lysine was used as a catalyst, which trended to promote the Maillard reaction.

The yield of ribulose was higher for arginine (12.48) > lysine (10.54) ≫ histidine (9.33). The figures in parentheses indicate the dissociation constant (pK) of the amino group of the side chain of each amino acid. The amino acids with the stronger basicity were more catalytic for the isomerization of ribose to ribulose. The apparent equilibrium constants K_app = C_P/C_S, where C_S and C_P represent the concentrations of ribose and ribulose, respectively, at 15 min of reaction time, when the yield of ribulose was almost constant, were 0.107. 0.068, and 0.010 for arginine, lysine, and histidine, respectively. Since the equilibrium constant should be independent of the type of catalyst used, the difference in the apparent equilibrium constants may have been due to the Maillard reaction or the caramelization of sugars that proceeded in parallel with isomerization.

Relationship between the yield of ribulose and pH during isomerization Fig. 2 shows the relationship between the pH of the reaction mixture and the yield of ribulose. In the phosphate buffer system under subcritical conditions, isomerization of sugars by LBAE transformation did not proceed when the pH of the reaction mixture (measured at room temperature) fell below 6.3 (Onishi et al., 2022). When arginine and lysine were used as catalysts, the yields became constant at a pH of approximately 7.8 (Fig. 2), which was higher than 6.3, and isomerization did not proceed further. This suggests that the reaction may have almost reached apparent equilibrium at this point. When histidine was used as a catalyst, isomerization did not reach apparent equilibrium within 30 min, due to its weak catalytic activity; also, because the pH was more than 6.3, the yield of ribulose gradually increased with the decreasing pH with reaction time.

Fig. 2.

Relationship between the yield of ribulose (C_P/C_S0) and the pH of the reaction mixture treated with (◯) arginine, (□) lysine, or (△) histidine at ca. 110°C. The concentrations of ribose and each amino acid were 0.2 and 0.01 mol/L, respectively. Symbols and bars represent mean ± standard deviation (n = 3). Solid curves were drawn empirically.

Selectivity in isomerization of ribose to ribulose Fig. 3 shows the change in selectivity of isomerization of ribose to ribulose with arginine, lysine, and histidine. For each amino acid, at the beginning of the reaction when the fraction of consumed ribose, i.e., the conversion of ribose, was low, the relationship between the yield of ribulose and the conversion of ribose could be approximated by a straight line passing through the origin, and from the slope of the straight line, the selectivity was estimated to be 0.70, 0.56, and 0.21 for arginine, lysine, and histidine, respectively. In the cases of both arginine and lysine, the apparent selectivity decreased because the ribulose concentration did not increase even as the conversion of ribose increased. The selectivity for lysine, which is more prone to the Maillard reaction, was lower than that for arginine. Therefore, of the three basic amino acids tested, arginine was the most suitable for isomerization of ribose to ribulose. This result was similar to those obtained for the isomerization of galactose to tagatose (Milasing et al., 2023) and glucose to fructose (Yang et al., 2016).

Fig. 3.

Relationship between the yield of ribulose (C_P/C_S0) and the fraction of consumed ribose (conversion of ribose) (1–C_S/C_S0) during the isomerization of ribose to ribulose with (◯) arginine, (□) lysine, or (△) histidine at ca. 110°C. The concentrations of ribose and each amino acid were 0.2 and 0.01 mol/L, respectively. Symbols and bars represent mean ± standard deviation (n = 3). The dashed line indicates that the selectivity in the isomerization of ribose to ribulose is one.

Color development during isomerization As shown in Fig. 1, the degree of coloration (absorbance at 280 and 420 nm) of the reaction mixture varied according to the type of amino acid used as catalyst. Clearly, lysine resulted in a greater degree of coloration than arginine and histidine. However, as shown in Fig. 4, the relationship between absorbance at 280 nm and that at 420 nm during the isomerization of ribose to ribulose was represented by a straight line passing through the origin regardless of the type of amino acid. This linear relationship would be an apparent phenomenon, since the compounds formed in the Maillard reaction and in the caramelization and decomposition of sugars are different (Murata, 2021; Onishi et al., 2022).

Fig. 4.

Relationship between absorbance at 280 nm (A₂₈₀) and that at 420 nm (A₄₂₀) of the reaction mixture during the isomerization of ribose to ribulose with (◯) arginine, (□) lysine, or (△) histidine at ca. 110°C. The concentrations of ribose and each amino acid were 0.2 and 0.01 mol/L, respectively. Symbols and bars represent mean ± standard deviation (n = 3). The solid line was drawn empirically.

Conclusions

The catalytic activity of three basic amino acids, arginine, lysine, and histidine, for the isomerization of ribose to ribulose was compared; arginine exhibited the greatest catalytic activity, followed by lysine. The coloration of the reaction mixture was most pronounced when lysine was used as a catalyst. Therefore, among three basic amino acids tested, arginine was most suitable for the isomerization of ribose to ribulose.

Acknowledgements This work was partially supported by the Invitational Fellowships for Research in Japan (Short-term program) of the Japan Society for the promotion of Science (awarded to P.K.). The authors thank Mr. H. Gohki, Mr. K. Nakamura, and Mr. R. Fukuzono for their technical assistance.

Conflict of interest There are no conflicts of interest to declare.

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