Solvent Dependence of Enzymatic Enantioselectivity in Ionic Liquids

Enantioselective synthesis is a key process in modern chemistry, and is particularly important in the fields of pharmaceuticals, foods, and pesticides, since the different enantiomers or diastereomers of a molecule often have different biological activity. Therefore, the enantiose‐ lectivity is the most valuable feature of enzymes from the standpoint of their application as practical catalysts. In nonaqueous reaction system the enantioselectivity of enzymes has been markedly dependent upon organic solvents [1-5]. On the other hand, enzymatic reactions in hydrophilic solvents have the advantage of the solubility of a variety of substrates, including amino acid derivatives, which are poorly soluble in hydrophobic solvents [5]. However, when a hydrophilic solvent is used as a reaction medium, the enzyme molecule directly contacts with the solvent, and thereby its activity and enantioselectivity are strongly influenced by the nature of solvents [1, 2]. Moreover, as the enzyme is insoluble in nonaqueous media, which are 100% organic solvent media or aqueous solutions containing high amount of organic solvents, and is suspended, the reactivity of enzymes tends to be strongly influenced by the dispersion state of enzymes.


Introduction
Enantioselective synthesis is a key process in modern chemistry, and is particularly important in the fields of pharmaceuticals, foods, and pesticides, since the different enantiomers or diastereomers of a molecule often have different biological activity. Therefore, the enantioselectivity is the most valuable feature of enzymes from the standpoint of their application as practical catalysts. In nonaqueous reaction system the enantioselectivity of enzymes has been markedly dependent upon organic solvents [1][2][3][4][5]. On the other hand, enzymatic reactions in hydrophilic solvents have the advantage of the solubility of a variety of substrates, including amino acid derivatives, which are poorly soluble in hydrophobic solvents [5]. However, when a hydrophilic solvent is used as a reaction medium, the enzyme molecule directly contacts with the solvent, and thereby its activity and enantioselectivity are strongly influenced by the nature of solvents [1,2]. Moreover, as the enzyme is insoluble in nonaqueous media, which are 100% organic solvent media or aqueous solutions containing high amount of organic solvents, and is suspended, the reactivity of enzymes tends to be strongly influenced by the dispersion state of enzymes.
Ionic solvent that is liquid at room temperature has attracted increasing attention as innovative nonaqueous media for the chemical processes because of the lack of vapor pressure, the thermal stability, the high polarity, and so on [6]. Chemical and physical properties of ionic liquids can be changed by the appropriate modification of organic cations and anions, which are constituents of ionic liquids. Biotransformation in ionic liquids has extensively been studied [6,7]. We have so far reported that protease-catalyzed esterification of amino acid and peptide synthesis are highly enhanced in ionic liquids, compared to organic solvents, and the thermal stability of proteins is markedly improved [8,9].
In the chapter, the solvent effect of ionic liquids on the enantioselectivity of enzymes is mainly discussed on the basis of enzyme kinetics [10].

Effect of reaction medium on α-chymotrypsin-catalyzed esterification
As a model enzyme, bovine pancreas α-chymotrypsin has been employed as shown in Fig. 1, since it has been well investigated regarding its structure, properties, and functions [11]. α-Chymotrypsin belongs to the S1 family that is one of the most predominant families of serine protease. Peptide and synthetic ester substrates are hydrolyzed in an aqueous solution by serine protease. The S1 family contains a catalytic triad system consisting of aspartate, histidine, and serine that work together to control the nucleophilicity of the serine residue during catalysis [12]. The serine proteases are widely distributed in nature, where they perform a variety of different functions. The binding site for a polypeptide substrate consists of a series of subsites across the surface of the enzyme, as seen in Fig. 2 [12]. In the figure, P and S are the substrate residues and the subsites, respectively. Except at the primary binding site S 1 for the side chains of the aromatic substrates of α-chymotrypsin, there is no obvious, well-defined cleft or groove for substrate binding. The subsites run along the surface of the protein. The binding pocket for the aromatic side chains of the specific substrates of α-chymotrypsin is a well-defined slit in the enzyme 1 to 1.2 nm deep and 0.35 to 0.4 by 0.55 to 0.65 nm in cross section. This gives a very snug fit, since an aromatic ring is about 0.6 nm wide and 0.35 nm thick. The binding pocket in αchymotrypsin may be described as a hydrophobic pocket, since it is lined with the nonpolar side chains of amino acids. It provides a suitable environment for the binding of the nonpolar or hydrophobic side chains of the substrates. In nonaqueous media, α-chymotrypsin can function as a synthetic catalyst for esterfication, transesterification, and peptide synthesis, and α-chymotrypsin-catalyzed esterification of Nacetyl-tryptophan (Ac-Trp-OH) with ethanol (EtOH) is proceeded as shown in Fig. 3. The esterification obeys ping-pong kinetics [12,13]. The enzyme and substrate first associate to form a noncovalent enzyme-substrate complex (E⋅Ac-Trp-OH) held together by physical forces of attraction. This is followed by the attack of the hydroxyl of serine (Ser-195), which is one of amino acid residues in a catalytic triad system, on the substrate to give acyl enzyme intermediate (Ac-Trp-E) releasing water. The acyl enzyme intermediate and ethanol associate to form an acyl enzyme-ethanol complex (Ac-Trp-E⋅EtOH). This is followed by the nucleophilic attack of ethanol on the carbonyl group in Ac-Trp-E to give N-acetyl-tryptophan ethyl ester (Ac-Trp-OEt). It is assumed that the esterification proceeds through the steady state approximation. The rate of N-acetyl-tryptophan (Ac-Trp-OH) is given as where E and E⋅Ac-Trp-OH are α-chymotrypsin and enzyme-substrate complex, respectively. Similarly, the rates of E, enzyme-substrate complex (E⋅Ac-Trp-OH), acyl enzyme intermediate (Ac-Trp-E), acyl enzyme-ethanol complex (Ac-Trp-E⋅EtOH), H 2 O, and N-acetyl-tryptophan ethyl ester (Ac-Trp-OEt) are as follows: Since all enzyme present is either free or complexes, where [E] 0 is the total concentration of enzyme in the system. Assuming the steady state approximation on the rate of E⋅Ac-Trp-OH, from Eq. 3 Assuming the steady state approximation on the rate of Ac-Trp-E⋅EtOH, from Eq. 5 where K s2 is the dissociation constant of Ac-Trp-E⋅EtOH. From Eq. 13 From Eq. 8 Concerning the equation obtained from Eqs. 10, 11, 14, and 15, (k 2 +k 4 )[EtOH] is much greater than k 2 K S2 because of excess [EtOH]. Consequently, The reaction rate where Thus, the reaction rate v corresponds upon Michaelis-Menten type model.

Effect of solvent on activity and enantioselectivity of α-chymotrypsin
The performances of enzymes such as activity and specificity in nonaqueous media markedly depend upon the nature of solvents [1,2]. In order to assess the effect of reaction media on kinetic parameters of α-chymotrypsin, the reaction rates in ionic liquids and organic solvents containing 5% (v/v) water were measured at different concentrations of substrates at 25 °C. Figure 4 shows the structures of solvents used in this study. In the figure, 1-ethyl-3-methylimidazolium tetrafluoroborate, 1-ethyl-3-methylimidazolium bis(fluorosulfonyl)imide, 1butyl-3-methylimidazolium hexafluorophosphate,  [13].
Thus, the reaction rate v corresponds upon Michaelis-Menten type model.

Effect of Solvent on Activity and Enantioselectivity of α-Chymotrypsin
The performances of enzymes such as activity and specificity in nonaqueous media markedly depend upon the nature of solvents [1,2]. In order to assess the effect of reaction media on kinetic parameters of α-chymotrypsin, the reaction rates in ionic liquids and organic solvents containing 5% (v/v) water were measured at different concentrations of substrates at 25 o C. Figure 4 shows the structures of solvents used in this study. In the figure, 1-ethyl-3-methylimidazolium tetrafluoroborate, 1-ethyl-3-methylimidazolium bis(fluorosulfonyl)imide,   Figure 5 shows the k cat in ionic liquids and THF at 5% (v/v) water and 25 °C. The k cat is known as the turnover number or molecular activity as it represents the maximum number of substrate molecules that the enzyme can turn over to product in a set time. The k cat, L for L-enantiomer was much greater than k cat, D for D-enantiomer in ionic liquids and THF. α-Chymotrypsin absolutely favors L-enantiomer in aqueous solutions. Consequently, the structural specificity of native enzyme remained to some extent in ionic liquids and THF. The k cat was strongly dependent upon the kind of solvents. The k cat, L for L-enantiomer in [ 4 ] can act as a thermal stabilizer for proteins in aqueous solutions [14]. The k cat is first-order rate constant that refers to the properties and reactions of the enzyme-substrate, enzyme-intermediate, and enzyme-product complexes. It is suggested that the solvent dependence of k cat is due to the conformational changes in the enzyme. As a result, the k cat, L / k cat, D in [C 2 mim][BF 4 ] was the greatest, while that in [C 4 mim][PF 6 ] was the smallest, as shown in Fig. 6.    Fig. 8.      the specificity for competing substrates [12]. Consequently, the (k cat /K M ) L / (k cat /K M ) D corresponds upon the enantioselectivity.    The k cat /K M is referred to as the specificity constant, and determines the specificity for competing substrates [12]. Consequently, the (k cat /K M ) L / (k cat /K M ) D corresponds upon the enantioselectivity. Figure 10 reveals that the enantioselectivity of α-chymotrypsin in ionic liquids can be forced to span a 325-fold range simply by switching from one solvent to another under otherwise identical conditions. Thus, D-enantiomer can sufficiently be employed as a substrate of αchymotrypsin in ionic liquids, although α-chymotrypsin catalyzes biotransformation of Lenantiomer in nature.
8 Figure 9 shows the k cat /K M in ionic liquids and THF at 5% (v/v) water and 25 o C. The (k cat /K M ) L for L-enantiome the specificity for competing substrates [12]. Consequently, the (k cat /K M ) L / (k cat /K M ) D corresponds upon the

Effect of Water Content on Activity and Enantioselectivity of α-Chymotrypsin
A common thread in all studies of enzymes in anhydrous solvents is that the amount of water associated with the enzyme is a k determinant of the properties (e.g. activity, stability, and specificity) that the enzyme exhibits [1]. Moreover, water can act as substrate in reactions using hydrolytic enzymes. In order to assess the effect of water content on kinetic parameters

Effect of water content on activity and enantioselectivity of αchymotrypsin
A common thread in all studies of enzymes in anhydrous solvents is that the amount of water associated with the enzyme is a key determinant of the properties (e.g. activity, stability, and specificity) that the enzyme exhibits [1]. Moreover, water can act as a substrate in reactions using hydrolytic enzymes. In order to assess the effect of water content on kinetic parameters of α-chymotrypsin, the reaction rate in ionic liquids and organic solvents containing 1% (v/v) water was measured at different concentrations of substrates at 25 o C. Figure 11 shows the k cat in ionic liquids and THF at 1% (v/v) water and 25 o C. The k cat depended upon the kind of solvents. The k cat, L for L-enantiomer was much greater than k cat, D for Denantiomer in ionic liquids and THF, similar to the case at 5% (v/v) water. at 1% (v/v) water exhibited 18-fold decrease, compared to that at 5% (v/v) water. When a certain amount of water is added into the nonaqueous enzymatic reaction system, some water is bound to the enzyme, and thereby has a large influence on the enzyme performance, while the other amount of water is dissolved in the solvent [1]. Water associated with the enzyme activates the enzyme by increasing the internal flexibility of the enzyme molecule, since water acts as a plasticizer to increase the flexibility [15]. On the other hand, the k cat is the kinetic parameter that refers to the properties and reactions of the enzyme-substrate, enzymeintermediate, and enzyme-product complexes due to the conformational changes in the enzyme. Accordingly, the water content markedly affected the conformation of enzymes in                 The optimum water content for the enzyme performance is due to the balance between kinetic rigidity and thermodynamic stability of enzyme structures, and is called essential water [16]. The kinetic rigidity is relaxed by increasing water content, while native enzyme structure gradually changes through thermodynamic stability. For instance, the activity increases with an increase in the flexibility of rigid enzyme in ionic liquids, and it decreases with an increase in disturbance of enzyme structure [9].     [FSI] decreased with increasing water content. The optimum water content for the enzyme performance is due to the balance between kinetic rigidity and thermodynamic stability of enzyme structures, and is called essential water [16]. The kinetic rigidity is relaxed by increasing water content, while native enzyme structure gradually changes through thermodynamic stability. For instance, the activity increases with an increase in the flexibility of rigid enzyme in ionic liquids, and it decreases with an increase in disturbance of enzyme structure [9].

Effect of reaction temperature on enantioselectivity of α-chymotrypsin in ionic liquids
Enzymatic reactions, as well as chemical reactions, obey the Arrhenius correlation between reaction rate constant and temperature, although the temperature range is quite limited. Accordingly, it is considered that the enantioselectivity is strongly influenced by the reaction temperature. In order to estimate the effect of reaction temperature on α-chymotrypsincatalyzed esterification in ionic liquids, the kinetic parameters in ionic liquids and THF containing 5% (v/v) water were investigated at 40 o C. Figure 17 shows the k cat in ionic liquids and THF at 5% (v/v) water and 40 o C.         As seen in Fig. 22, the enantioselectivity of α-chymotrypsin in [C 2 mim][BF 4 ] at 40 o C was lower than that at 25 o C, while that in [C 2 mim][FSI] increased with increasing the reaction temperature. On the other hand, the enantioselectivity of α-chymotrypsin in THF drastically dropped with an increase in the reaction temperature. The temperature dependence of the enantioselectivity of protease and lipase is markedly affected by organic solvents [3]. Thus, the result indicates that the temperature dependence of the enantioselectivity is controlled by changing the reaction medium from one ionic liquid to another, similar to the case of organic solvents.
α-chymotrypsin in THF drastically dropped with an increase in the reaction temperature. The temperature depe the enantioselectivity of protease and lipase is markedly affected by organic solvents [3]. Thus, the result indicat temperature dependence of the enantioselectivity is controlled by changing the reaction medium from one ioni another, similar to the case of organic solvents.

Relationship between catalytic efficiency and enantioselectivity of α-chymotrypsin in liquids
As mentioned above, the enantioselectivity is strongly influenced by a kind of ionic liquids, water content, an temperature. In order to assess the correlation between the catalytic efficiency and the enantioselectivity, the α-chymotrypsin in THF drastically dropped with an increase in the reaction temperature. The temperat the enantioselectivity of protease and lipase is markedly affected by organic solvents [3]. Thus, the resu temperature dependence of the enantioselectivity is controlled by changing the reaction medium from another, similar to the case of organic solvents.  6. Relationship between catalytic efficiency and enantioselectivity of α-chymotryp liquids As mentioned above, the enantioselectivity is strongly influenced by a kind of ionic liquids, water co Figure 22. Effect of reaction temperature on the enantioselectivity of α-chymotrypsin-catalyzed esterification in ionic liquids and THF at 5% (v/v) water.

Relationship between catalytic efficiency and enantioselectivity of αchymotrypsin in ionic liquids
As mentioned above, the enantioselectivity is strongly influenced by a kind of ionic liquids, water content, and reaction temperature. In order to assess the correlation between the catalytic efficiency and the enantioselectivity, the (k cat /K M ) L / (k cat /K M ) D was plotted against the (k cat / K M ) L , as seen in Fig. 23. One can observe an increase in the (k cat /K M ) L / (k cat /K M ) D with increasing the (k cat /K M ) L . The catalytic efficiency (k cat /K M ) L is attributable to the enzyme structure [11]. Therefore, the higher the native structure of enzymes is kept, the larger the enantioselectivity becomes.
(kcat /KM)D was plotted against the (kcat /KM)L, as seen in Fig. 23. One can observe an increase in the (kcat /KM)L/ (kcat /KM)D with increasing the (kcat /KM)L. The catalytic efficiency (kcat /KM)L is attributable to the enzyme structure [11]. Therefore, the higher the native structure of enzymes is kept, the larger the enantioselectivity becomes.

Conclusion
In this chapter, the solvent effect of ionic liquids on the enantioselectivity of α-chymotrypsin has been described. The kcat and KM in ionic liquids were sensitively influenced by the reaction conditions such as water content and reaction temperature. As a result, the enantioselectivity, (kcat /KM)L/ (kcat /KM)D, changed in the wide range. The kcat and KM correspond upon the conformational changes in the enzyme and the overall dissociation constant of all enzyme-bound species, respectively. Consequently, the major change of those parameters indicates that ionic liquids strongly affect the structure of enzyme molecules and the distribution of substrates and products between the reaction medium and the active site of enzymes under a requisite condition. By using the variation of enantioselectivity in ionic liquids, the optical resolution of biological active compounds and the synthesis of nonnative chiral compounds such as D-amino acid derivatives can effectively be carried out. Furthermore, it is expected that the ionic liquid, which exhibits the enantioselectivity suitable for a certain asymmetric synthesis, is prepared by tailoring the constituents of ionic liquids, since chemical and physical properties of ionic liquids can be changed by the appropriate modification of organic cations and anions, which are constituents of ionic liquids.

Conclusion
In this chapter, the solvent effect of ionic liquids on the enantioselectivity of α-chymotrypsin has been described. The k cat and K M in ionic liquids were sensitively influenced by the reaction conditions such as water content and reaction temperature. As a result, the enantioselectivity, (k cat /K M ) L / (k cat /K M ) D , changed in the wide range. The k cat and K M correspond upon the conformational changes in the enzyme and the overall dissociation constant of all enzyme-bound species, respectively. Consequently, the major change of those parameters indicates that ionic liquids strongly affect the structure of enzyme molecules and the distribution of substrates and products between the reaction medium and the active site of enzymes under a requisite condition. By using the variation of enantioselectivity in ionic liquids, the optical resolution of biological active compounds and the synthesis of nonnative chiral compounds such as Damino acid derivatives can effectively be carried out. Furthermore, it is expected that the ionic liquid, which exhibits the enantioselectivity suitable for a certain asymmetric synthesis, is prepared by tailoring the constituents of ionic liquids, since chemical and physical properties of ionic liquids can be changed by the appropriate modification of organic cations and anions, which are constituents of ionic liquids.