Protein Stabilization and Enzyme Activation in Ionic Liquids: Specific Ion Effects

Abstract

There are still debates on whether the hydration of ions perturbs the water structure, and what is the degree of such disturbance; therefore, the origin of Hofmeister effect on protein stabilization continues being questioned. For this reason, it is suggested to use the ‘specific ion effect’ instead of other misleading terms such as Hofmeister effect, Hofmeister series, lyotropic effect, and lyotropic series. In this review, we firstly discuss the controversial aspect of inorganic ion effects on water structures, and several possible contributors to the specific ion effect of protein stability. Due to recent overwhelming attraction of ionic liquids (ILs) as benign solvents in many enzymatic reactions, we further evaluate the structural properties and molecular-level interactions in neat ILs and their aqueous solutions. Next, we systematically compare the specific ion effects of ILs on enzyme stability and activity, and conclude that (a) the specificity of many enzymatic systems in diluted aqueous IL solutions is roughly in line with the traditional Hofmeister series albeit some exceptions; (b) however, the specificity follows a different track in concentrated or neat ILs because other factors (such as hydrogen-bond basicity, nucelophilicity, and hydrophobicity, etc) are playing leading roles. In addition, we demonstrate some examples of biocatalytic reactions in IL systems that are guided by the empirical specificity rule.

INTRODUCTION

As a new type of designable solvents, ionic liquids (ILs) have gained tremendous focus in biocatalysis, aiming to replace conventional volatile organic solvents and their solutions. A number of enzymatic systems have been evaluated in neat ILs or IL solutions; these enzymatic systems include various hydrolases (EC 3, e.g. lipases, proteases, thermolysin, α-chymotrypsin, lysozyme, β-glycosidase, cellulase, epoxide hydrolase and penicillin amidase), oxidoreductases (EC 1, e.g. horseradish peroxidase, alcohol dehydrogenase, laccase and lignin peroxidase), lyases (EC 4, e.g. oxynitrilase), and whole cells.^1–4 To improve the stability and activity of enzymes, a variety of methods have been developed by different groups, such as enzyme immobilization (on solid support, sol–gel, or cross-linked enzyme aggregates), physical or covalent attachment to PEG, rinsing with n-propanol methods (PREP and EPRP), water-in-IL microemulsions, IL coating, and the design of enzyme-compatible ILs.^{5, 6}

More importantly, several mechanistic overviews^7–9 have highlighted some important properties of ILs that influence the enzyme’s behaviors in ionic solvents; these properties include IL polarity, hydrogen-bond (H-bond) basicity and nucleophilicity of anions, Hofmeister series, IL hydrophobicity, and IL viscosity, etc. However, there is a mixed understanding of the existence of specific ion effect of ILs on protein stabilization and enzyme activation, and how the specific ion effect is different in diluted solutions of ILs from that in concentrated or neat ILs. The present review aims to survey relevant literatures surrounding the theme of specific ion effect of ILs, and provide mechanistic insights into how different factors contribute to the specific ion effect at different IL concentrations. In particular, we discuss the specific ion effect of inorganic ions in aqueous solutions, followed by the structural properties of ILs and their aqueous solutions, and then the specific ion effects in aqueous ILs and concentrated/neat ILs, and lastly some examples of ion specificity-guided biocatalytic reactions.

SPECIFIC ION EFFECT OF INORGANIC IONS IN AQUEOUS SOLUTIONS

In 1888, Franz Hofmeister proposed the ion specificity based on his observation of ions exhibiting sequential capabilities in precipitating the proteins (globulins from blood serum and hen’s egg).^{10, 11} The order of these ions in salting out proteins is so called the ‘Hofmeister series’ (Fig 1) although later this concept also became associated with other phenomena in physical, colloid, polymer and surface chemistry.^{12, 13} At low concentrations (< 0.1 M), ions affect the protein stability and enzyme activity primarily through electrostatic interactions.^{14, 15} At higher salt concentrations (usually > 0.1–0.3 M,^{14, 15} but not too concentrated such as up to 3.0 M¹⁶), the Hofmeister ion effect becomes important when the ionic dispersion forces exceed the electrostatic forces. The Cremer group¹⁷ observed that at low salt concentrations (< 200–300 mM), the charge pairing between anions and the positively charged lysozyme surface (pH 9.4) is gradually reaching its saturation; at this stage, the liquid–liquid phase transition temperature of lysozyme is directly related to the size and hydration thermodynamics of the anions and thus follows an inverse Hofmeister series (ClO₄⁻ > SCN⁻ > I⁻ > NO₃⁻ > Br⁻ > Cl⁻). Under higher salt conditions, the liquid–liquid phase transition is influenced by the polarizability of the anions and thus exhibits a direct Hofmeister series (Cl⁻ > NO₃⁻ > Br⁻ > ClO₄⁻ > I⁻ > SCN⁻). On the other hand, the Falconer group¹⁸ suggested that anion and cation effects on the structural stability of lysozyme at pH 7 follow the Hofmeister series at high concentrations (> 20 mM), but fail to follow the Hofmeister (or inverse Hofmeister) series at low concentrations (< 5 mM).

Fig. 1.

The Hofmeister series as an order of the ion effect on protein stability^{22, 39} (viscosity B-coefficients in dm³·mol⁻¹ at 25 ºC are taken from the Marcus collection⁴⁰ except those of EtSO₄⁻ and MeSO₄⁻ were from Ref;⁴¹ the positions of EtSO₄⁻ and MeSO₄⁻ are based on the consideration of B-coefficients, NMR B′-coefficients⁴² and enzyme stability studies^43–46).

Several theories have been developed to understand the Hofmeister series, including salt-in and salt-out interactions,^{19, 20} water-structure changes (low/high density water) and protein preferential hydration,^{14, 21–26} hydrophobic interactions,^26–28 excluded volume,^29–31 preferential interactions,^32–34 electrostatic interactions,^{35, 36} ionic dispersion potentials,^{37, 38} etc. However, there is a lack of unified theory that can fully interpret the Hofmeister effect due to the complex nature of ion-water-protein interactions.

Early literatures often related the protein stability with the hydration behavior of ions.^{19, 37} Highly hydrated ions (e.g. Mg²⁺, Ca²⁺, Li⁺, CH₃COO⁻, SO₄²⁻, and HPO₄²⁻) tend to interact strongly with water molecules and increase the ‘structuring of water’, resulting in a lower fluidity (or a higher viscosity) of the solution than that of pure water. For this reason, these ions are referred as ‘structure-makers’ or ‘kosmotropes’ (see Fig 1). In contrast, some other ions are poorly hydrated in aqueous solutions, such as SCN⁻, I⁻, NO₃⁻, BF₄⁻, Cs⁺, (NH₂)₃C⁺ (guanidinium), and (CH₃)₄N⁺ (tetramethylammonium). These ions have weak interactions with water molecules and reduce the ‘structuring of water’, leading to a higher fluidity of the solution. Thus, this effect is called the ‘negative hydration’,^{47, 48} and these ions are often denoted as ‘structure-breakers’ or ‘chaotropes’ (see Fig 1). Based on this theory, the capacity of an ion in strengthening the ‘water structure’, known as kosmotropicity (vs. chaotropicity), is directly associated with the degree of ion hydration. As discussed in our earlier review,⁴⁹ the ion kosmotropicity can be quantified by various thermodynamic parameters including Jones-Dole viscosity B-coefficients, structural entropies, structural volumes, structural heat capacities, NMR B′-coefficients, and ion mobility, etc. These parameters provide valuable information of the interactions involved in the ion hydration from different aspects, and possibly reveal the mechanism behind some phenomena and properties. Jones-Dole viscosity B-coefficients are the most commonly used and widely available parameter for evaluating the ion kosmotropicity. The B-coefficients can be derived from the Jones-Dole empirical equation (eqn 1) of the relative viscosities of electrolyte solutions as functions of their concentrations,⁵⁰

$$\eta /{\eta }_0=1+A{c}^{1/2}+Bc+D{c}^2\dots$$ (1)

where η is the viscosity of the solution and η₀ is the viscosity of the solvent (both of them have the same unit, for example Pa·s), while c is the molar concentration (mol·cm⁻³). The A-coefficient (also known as the Falkenhagen coefficient⁵¹), representing the solute-solute or electrostatic interactions, can be calculated theoretically. However, A-values are usually small and negligible for non-electrolytes;⁴⁰ therefore, they are often neglected in the calculations. The B-coefficient represents the solute-solvent interactions (short-range dispersion forces), while D-coefficient indicates the solute-solute interactions as well as the solute-solvent interactions.⁵² For most salts at low concentrations [(< 0.5 M)⁴⁰ or (< 0.1 M for binary strong electrolytes)⁵³], the D or higher coefficients can be neglected although they are required at higher concentrations.⁴⁰ Positive B-values typically indicate ions as kosmotropes since strongly hydrated ions exhibit a larger change in viscosity with concentration, while negative B-coefficients imply chaotropes for weakly hydrated ions.⁴⁰ However, hydrophobic solutes tend to have unusually large B-coefficients due to so called ‘hydrophobic hydration’.⁴⁹ For example, tetramethylammonium cation (Me₄N⁺) has a positive B-value as high as 0.123,⁴⁰ but this ion is considered as a structure-breaker.^{23, 54–58} Some groups^{40, 59–61} recommend the use of first derivatives of B-values over temperature because the sign of dB/dT could be more indicative in measuring the structure-making or breaking property than the sign or quantity of B-coefficients. The negative sign of dB/dT means structure-making (kosmotropic) while the positive sign suggests structure-breaking (chaotropic). In aqueous solutions of inorganic salts, many studies (see our earlier review¹⁶) have suggested that the ion effect on the enzyme activity follows the Hofmeister series: kosmotropic anions and chaotropic cations stabilize the enzyme, while chaotropic anions and kosmotropic cations destabilize it.

However, some contradictory experimental results argued whether the water structure is indeed influenced by the presence of ions.^{62, 63} Leberman and Soper⁶⁴ found that some salts [e.g. 2 M Na₂SO₄ and 2 M (NH₄)₂SO₄] disturbed more water H-bonding than 4 M NaCl and 4 M NH₄Cl based on the water-water HH correlation functions obtained from the neutron diffraction using isotope substitution. Nucci and Vanderkooi⁶⁵ examined the temperature excursion infrared response of the O–H stretch of aqueous salt solutions by a two-state H-bonding model, and found that ions do change the H-bond network of water and there is a strong correlation between salt effects on the Hofmeister series. They also noted that the specific ion–protein interactions cannot be excluded, and could be a co-factor along with the changes in bulk solvation properties. Thomas and Elcock⁶⁶ conducted molecular dynamics (MD) simulations (1 μs) of the unbiased association of pairs of hydrophobic molecules (methane– methane and neopentane–neopentane) in different salt solutions, and found that the Hofmeister effects can be quantitatively predicted from the H-bond ratio from simulations of pure salt solutions containing no hydrophobic solute. Thus, they indicated that salt-induced changes in water structure is more important than preferential interactions between salt and hydrophobic solutes to the understanding of Hofmeister effects. On the other hand, the Saykally group⁶⁷ measured the oxygen K-edge X-ray absorption spectrum (XAS) of aqueous sodium halide solutions (up to 4 M), and found ions greatly perturb the electronic structure of adjacent water molecules because of the direct perturbation of unoccupied orbitals on water by anions; however, such perturbation is not necessarily due to any significant distortion of the H-bond network beyond the first solvation shell. This group⁶⁸ further confirmed monovalent cations (such as Li⁺, Na⁺, K⁺, NH₄+ and C(NH₂)₃+) cause no considerable perturbation of the unoccupied molecular orbitals of water molecules in the vicinity of cations while the XAS spectral changes are mainly due to water-chloride interactions; however, they also observed that divalent cations (i.e. Mg²⁺ and Ca²⁺) induce a redistribution of charge among water molecules in the solvation shell and result in spectra changes. Krekeler and Delle Site⁶⁹ conducted first-principle Car–Parrinello molecular dynamics of the hydration of monovalent and divalent ions, and suggested the preferential orientation of water molecules is only seen in the first shell and the water–water interaction plays a critical role in the first shell regardless of the size or the charge of ions. Based on the orientational correlation time of H₂O molecules in 1–6 M solutions of three salts [i.e. Mg(ClO₄)₂, NaClO₄, and Na₂SO₄] acquired from the femtosecond pump-probe spectroscopy, Omta et al^{70, 71} suggested that ions have negligible effect on the H-bond structure in liquid water. Their results indicate the anion interactions with water molecules (OH···ClO₄- and OH···SO₄²⁻). Therefore, simple ions have no significant impact on the water structure, at least beyond the first hydration shell; even di- and tri-valent ions cause no appreciable change to the density or orientation of water more than two water molecules (5 Å) away.⁷²

While the debate on the effect of ions on water structure continues, some groups suggest the bulk water structure is not greatly affected by ions and thus the kosmotropicity concept should be abandoned;⁷³ instead, the Hofmeister series should be explained by the ion impact on protein hydration, and direct ion-protein interactions. Before we discuss other explanations of Hofmeister effect, it is necessary to outline how protein/enzyme molecules interact with solutes (denaturants or stabilizers). As shown in Fig 2, solute 1 is a denaturant that has a stronger interaction with protein molecules than with water molecules. Solute 1 excludes those co-factors of enzymes that are essential for the enzyme activity. This category (solute 1) includes chaotropic anions (because they are less hydrated), kosmotropic cations (such as Ca²⁺ strongly salts in the peptide group¹⁹), organic solutes (especially hydrophilic ones including urea³³), and other ions (such as guanidinium GdnH⁺) that have strong interactions with protein surface. The strong interactions may expose the hydrophobic cores of the protein, causing its denaturation. Solute 2 is a stabilizer that has a stronger interaction with water molecules than with protein molecules. This category includes kosmotropic cations and anions (because they are strongly hydrated and salt out nonpolar groups;¹⁹ they ‘drag’ water molecules away from the protein which allows the protein to refold⁷⁴), and organic solutes that have weak binding interactions with the protein.

Fig. 2.

Illustration of interactions between solutes and protein.

Several major alternative explanations of Hofmeister series are discussed below. The first theory is the preferential hydration of proteins.^{14, 21–26} Strongly hydrated anions tend to strongly interact with water molecules; as a result, they preferentially hydrated by water molecules instead of interacting directly with the enzyme surface. On the contrary, weakly hydrated anions have a low water affinity and a high polarizability, and therefore bind to the protein-water interface resulting in protein destabilization. Through examining the aqueous potassium salt solutions using femtosecond optical Kerr effect spectroscopy, Hou et al.⁷⁵ found that the hyperpolarizability of six aqueous anions increased in the order: HPO₄²⁻ < HSO₄⁻ < CO₃²⁻ < CH₃COO⁻ < NO₃⁻ < SCN⁻, which correlates with the Hofmeister series (except CO₃²⁻). The role of cations is different. The presence of kosmotropic cations tends to minimize the effect of kosmotropic anions because a strong ion-pairing affinity between kosmotropic cations and anions decreases the amount of free anions in the solution. In aqueous solutions, ion pairs are easily formed between cations and anions with similar water affinity, such as kosmotrope-kosmotrope and chaotrope-chaotrope; the strengthen of these interactions (known as the ‘law of matching water affinity’) is in a decreasing order of kosmotrope-kosmotrope > kosmotrope-water > water-water > chaotrope-water > chaotrope-chaotrope.^{21, 53} Zhang et al.⁷⁶ examined the hydration and interactions of a globular protein (bovine serum albumin, BSA) in concentrated salt solutions (up to 3.0 M) by small-angle neutron scattering (SANS). They suggested a hydration shell with a hydration level of ~0.30 g g⁻¹ protein; they also indicated that the effective protein–protein interactions in concentrated salt solutions can be evaluated by the second virial coefficient, which follows the reverse order of the Hofmeister series: i.e. (NH₄)₂SO₄ < Na₂SO₄ < NaOAc < NaCl < NaNO₃ < NaSCN. To study the specific ion effect on interfacial water structure neighboring to a BSA monolayer adsorbed at the air/water interface, the Cremer group⁷⁷ employed the vibrational sum frequency spectroscopy (VSFS) and suggested that specific anion effects are controlled by the charge state of the interfacial layer rather than its detailed chemical structure: for the positively charged protein layer at pH 2 and 3, more chaotropic anions induced more attenuation of water structure; for the protein layer at its isoelectric point (pH 5), more chaotropic anions lead to greater increase in water structure (although it’s weak); for the negatively charged protein layer (pH 9), no obvious effect could be detected.

A second important account for the Hofmeister series is the direct interactions between ions and protein. In aqueous solutions, protein molecules may interact with water molecules and ions via a variety of hydrophilic, polar, or charged moieties. In particular, the charge groups include dehydrated, chaotropic amide and amino groups, and the hydrated, kosmotropic carboxyl groups. Based on the ‘law of matching water affinity’, chaotropic anions have a greater affinity towards chaotropic amide of the peptide group, whilst the interaction between kosmotropic cations and the kosmotropic carboxyl moiety is weak due to the presence of water molecules in their nearest hydration shells.⁷⁸ This explains the opposite trend of cations and anions in influencing the protein stability, and also the stronger effect of anions than cations. Sedlák et al.⁷⁸ further pointed out that the hydration condition determines the direct interactions between the ions and the protein peptide bonds, which leads to Hofmeister effects of protein stability; the protein stability is more correlated with anion charge density than cation charge density. Gokarn et al.¹⁵ observed that anions selectively and preferentially accumulate at the surface of hen-egg white lysozyme even at low (< 0.1 M) salt concentrations. At a given ion normality of 50 mN, the protein’s effective charge (Q*) decreased in the order F⁻ > Cl⁻ > Br⁻ > NO₃⁻ > I⁻ > SCN⁻ > ClO₄⁻ ≫ SO₄²⁻, which corresponds to the opposite order of anion association to the protein surface, and thus suggests that the SO₄²⁻ anion interacts directly with the protein surface although it is highly hydrated. On the other hand, the cations have no apparent impact on the effective charge of the protein, which is almost unchanged for all the cations studied (Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, GdnH⁺, and Ca²⁺). On the contrary, the Jungwirth group⁷⁹ suggested that the destabilizing effect of weakly hydrated Hofmeister anions (such as Br⁻ or I⁻) is not caused by the direct interactions with the backbone amide groups, but rather due to the affinity of large soft ions toward hydrophobic groups and residues of proteins. A further study by this group and the Cremer group⁸⁰ examined the specific binding sites of Hofmeister ions with an uncharged 600-residue elastin-like polypeptide, and suggested that the interaction between large soft anions (SCN⁻ and I⁻) and the polypeptide backbone through a hybrid binding site comprising the amide nitrogen and the adjacent α-carbon. Cl⁻ anions have a much weaker binding to this site, SO₄²⁻ is excluded from the backbone as well as hydrophobic side chains of the polypeptide. The Gibb group⁸¹ found that chaotropic anions have a strong affinity towards the hydrophobic concavity, which surpasses the affinity between anions and amide groups; therefore, they implied that protein solubilization in solutions of chaotropes is mainly due to the direct binding of chaotropes to concavity in the molten globule state of a protein. Paterová et al.⁸² conducted the NMR and MD studies of ion interactions with capped and uncapped triglycine, and noted (a) a direct Hofmeister series for the capped peptide, which means that strongly hydrated ions (e.g., SO₄²⁻) are repelled from the peptide bond while weakly hydrated ions (e.g., I⁻ and SCN⁻) interact with the peptide bond, and (b) a reversed Hofmeister series for the uncapped peptide due to anion interactions with the positively charged, uncapped N-terminus. It is also suggested that the same specific anion effect could be extrapolated for interactions with the positively charged side chains of lysine, arginine, and (protonated) histidine. Based on a two-scale MD simulation approach, Schwierz et al.⁸³ observed a direct Hofmeister series for anions at the negatively charged hydrophobic surfaces or positive polar surfaces, but a reversal effect for the negative polar or positive nonpolar surfaces. As reviewed by Yang,⁹ the direct interactions between ions and enzyme may lead to several changes to the enzyme including surface pH, net charge, active site and catalytic mechanism.

Thirdly, it has been known that the Hofmeister ions affect the surface tension and surface potential at the air–water interface.²² Based on a surface-bulk partitioning model to assess the Hofmeister effect on the surface tension of water, Pegram and Record^{84, 85} suggested that those anions (such as SCN⁻) that interact favorably with protein surface exposing protein surface to water, tend to accumulate at the air–water interface; other anions (such as F⁻) that are excluded from protein surface and cause dehydration of protein surface, tend to be excluded from the air–water interface. A recent phenomenological theory developed by groups of Dér and Ramsden^{86, 87} indicates that the Hofmeister effect could be explained by the salt-induced changes of hydrophobic/hydrophilic properties of protein–water interfaces, quantitatively by the protein–water interfacial tension. This theory establishes the correlation between interfacial tension and protein structural stability, which is associated with protein conformational fluctuations. Therefore, this theory could interpret the salt effects on protein conformation, dynamics as well as stability, and could even explain the unusual observation of chaotropes stabilizing some proteins.

Due to the ongoing controversial discussion on the origin of Hofmeister series, Friedman⁸⁸ suggested the use of term ‘specific ion effect’ instead of other misleading terms such as Hofmeister effect, Hofmeister series, lyotropic effect, and lyotropic series.

STRUCTURAL PROPERTIES OF ILS AND THEIR AQUEOUS SOLUTIONS

Imidazolium ILs could form H-bonded polymeric supramolecules, so-called organized ‘nano-structures’, with polar and non-polar regions in solid, liquid and solution states, or even in the gas phase.^{90, 91} For imidazolium-based ILs, each cation coordinates with at least three anions while each anion coordinates with three cations, resulting in a H-bonded polymeric network like [(R₁R₂Im)_x(X)_x-n]ⁿ⁺[(R₁R₂IM)_x-n(X)_x]ⁿ⁻ (where R₁R₂IM represents 1,3-dialkylimidazolium cation, and X is the anion). As shown in Fig 3, upon the addition of more solvent molecules (such as acetonitrile, chloroform or water), the supramolecular network turns into various stages of structures such as aggregates and inclusion compounds, charged and neutral clusters, triple ions, contact ion pairs, solvent-shared ion pairs and loose ion pairs.⁸⁹ Watanabe et al.⁹² probed the structures of protic and aprotic ILs ([MMIM][Tf₂N], [MIM][Tf₂N] and [Im][Tf₂N]) by high-energy total scattering (HETS) experiments and MD simulations, and found that the closest cation–anion orientation varies without substantial longer range ordering of r > 12 Å by the N-methyl substitution to proton, resulting in the second layer consisting of ions of the same sign configuration changes. Additionally, they noticed that the O atoms of Tf₂N⁻ anions preferentially form H-bonds with the NH hydrogens of the protic imidazolium and the F atoms locate right above and below the imidazolium ring, and also the NH···O H-bond is short and linear while the C₂H···O bond is long and bent. Very recently, the Ludwig group⁹³ studied the H-bonding in [Cholinium][Tf₂N] [Cholinium = (2-hydroxyethyl)-trimethylammonium] by far infrared spectra, and observed H-bonding between ions of like charge (in addition to H-bonding between cations and anions), i.e. forming cooperative H-bonds as OH···OH···O=S (O=S in Tf₂N⁻ anion) between hydroxyl groups of two choliniums resembling those in alcohol dimers. When comparing with [Me₃NPr][Tf₂N], the enhanced H-bond network in [Cholinium][Tf₂N] leads to a higher melting temperature, a larger viscosity and a lower conductivity.

Fig. 3.

2D illustration of the structure of a neat IL to its infinite dilution in the presence of other solvent molecules. Most of these structures have been confirmed by experiments and/or simulations (red spheres = anions, blue spheres = cations, black spots = solvent molecules and the lines represent the hydrogen bonds and/or other weaker interactions) (Reproduced by permission from Ref,⁸⁹ © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).

Many ILs contain hydrophilic and lipophilic segments, which turn these ILs into amphiphilic compounds. The self-organization of amphiphilic ILs in solutions to form aggregates and micelles have been reported by both experimental and computer simulation methods, which has been reviewed by a number of literatures.^{94, 95} The aggregation property of dialkylimidazolium ILs has been shown to be similar to that of alkyltrimethylammonium salts (cationic surfactants) despite the higher self-organization ability and long-range ordering of ILs. Since the subject has been extensively reviewed, we only discuss a few recent examples herein. The Voth group⁹⁶ conducted MD simulations of ILs, and found that OMIM⁺ cations are more prone to aggregate in water and form micelle-like structures than BMIM⁺ cations, while BMIM⁺ interacts stronger with water than OMIM⁺ leading the slower rotation of water at x_w > 0.61 (x_w is the mole fraction of water). Additionally, they noticed that changing the anion from BF₄⁻ to Cl⁻ also slows the diffusion of cations and water molecules because Cl⁻ anions tend to have a stronger electrostatic interaction with other particles in IL/water mixtures; since at low water mole fractions, the water structure depends on the strength of water–anion attractions, water molecules are more likely to form clusters in [OMIM][BF₄]/water mixtures than in [OMIM]Cl/water mixtures at low concentrations. Greaves et al.⁹⁷ examined the structures of aqueous protic ILs by small- and wide-angle X-ray scattering (SWAXS) and IR spectroscopy, and observed nanostructured aggregates in neat protic ILs; these aggregate structures are maintained upon dilution with minimal change in the size, and the water is present predominately as bulk water. Azadbakht et al.⁹⁸ determined the critical micelle concentrations (cmc) of [C₁₈MIM][BF₄] and [C₁₈MIM][PF₆] as 0.04 mM and 0.02 mM respectively based on the tensiometry method; the smaller cmc value for the latter IL was explained as the PF₆⁻ ion has a larger size and more ability in forming H-bonds with water than BF₄⁻ does, and thus minimizes the surface charge of cations. On the contrary, based on ¹H NMR chemical shift analysis, Inoue and Misono⁹⁹ found that higher solvophilicity of polyoxyethylene (POE)-type nonionic surfactants in [BMIM][PF₆] (vs [BMIM][BF₄]) was due to weaker H-bond interaction between BMIM⁺ and PF₆⁻ than that between BMIM⁺ and BF₄⁻.

It has been known that the hydration of organic cations is quite different from inorganic ions. Due to the hydrophobic nature of their alkyl groups, large organic cations (such as tetraalkylammoniums) in aqueous solutions are surrounded by water molecules forming “cagelike” structures, so called the ‘hydrophobic hydration’.¹⁰⁰ The hydrophobic hydration results in a negative enthalpy change, due to multiple van der Waals interactions, and a negative entropy change due to the increased order in the surrounding water. As discussed by Wen,¹⁰⁰ tetraalkylammonium cations are highly hydrated; for example, the hydration numbers of Me₄N⁺, Et₄N⁺, n-Pr₄N⁺ and n-Bu₄N⁺ are 16, 21, 27 and 32 respectively (or 25, 30, 35, and 40 respectively based on other studies). Despite these organic cations are large in size, single-charged, they are not necessarily chaotropes because of the hydrophobic hydration.^{28, 101, 102} Marcus⁵⁴ indicated that Me₄N⁺ is a chaotrope, Et₄N⁺ is a borderline ion, n-Pr₄N⁺, n-Bu₄N⁺ and n-Pe₄N⁺ are kosmotropes. A similar classification was confirmed by Kay et al⁵⁵ and other groups.^{23, 56–58} In addition, these organic cations have larger B-coefficients than inorganic ions, even chaotropic Me₄N⁺ ions have B-coefficients of 0.123.^{49, 103} As pointed out in a review by von Hippel and Schleich,¹⁰⁴ Me₄N⁺, Et₄N⁺, n-Pr₄N⁺, n-Bu₄N⁺ and n-Pe₄N⁺ ions exhibit an increasing order of destabilizing the ‘native’ form of collagen and ribonuclease; this is consistent with more kosmotropic cations destabilizing the protein. The Rogers group¹⁰⁵ determined phase diagrams of kosmotropic inorganic salts (K₃PO₄, K₂HPO₄, K₂CO₃, KOH, and (NH₄)₂SO₄) in salting out ILs, and further established the chaotropicity of ILs decreasing in the order of [Bu₄P]Cl > [Bu₄N]Cl ≫ [BuPy]Cl ≫ [BDMIM]Cl [BMIM]Cl.

The interactions between ILs and water molecules provide valuable insights into the IL hydration behavior. Typically, there is a strong H-bonding interaction between water molecules with basic anions of ILs (such as Cl⁻) as confirmed by negative excess chemical potentials of aqueous ILs.¹⁰⁶ Mele et al.¹⁰⁷ examined the cation–cation, cation–water, and cation–anion interactions in [BMIM][BF₄] (with 0–0.52 mole fraction of water) by NMR spectroscopy through intermolecular nuclear Overhauser enhancements (NOEs), and found that increasing water content in IL progressively increases H-bonds between the cation and water (as H-bond acceptor) instead of C(sp²)–H···F interactions, and also increases the H-bonds between anion and water (as H-bond donor). In addition, they indicated that the presence of tight ion pairs in the neat IL even with a small amounts of water. The Koga group¹⁰⁸ suggested that the influence of BMIM⁺ cation on water structure is similar to that of fructose or increased temperature, where water molecules interaction with the cation leading to the reduction of H-bonds of bulk water region. Xu et al.¹⁰⁹ compared the relative chemical shifts of protons in [EMIM][BF₄] upon dilution with water, and found the strength of H-bonds between water and three aromatic protons decreasing in the order of (C2)H···O > (C4)H···O > (C5)H···O; they also suggested that the ion pairs of this IL are dissociated rapidly when x_water > 0.9. Singh and Kumar¹¹⁰ compared the changes of OH (water) and CH (imidazoliums) vibrational stretching bands in aqueous mixtures of ILs using FT-IR spectroscopy, and observed that the blue shift of OH bands usually increases with the IL concentration and decreases in the order of different ILs: [BMIM][CH₃SO₄] > [BMIM][C₈H₁₇SO₄] > [BMIM][BF₄] > [OMIM]Cl. A higher blue shift of OH bands represents a stronger interruption of H-bonding network of water. In addition, the high hydration numbers of these ILs (14.3, 18.7, 12.7 and 12.8 respectively) along with ¹H NMR spectra of aqueous ILs imply the significant interactions between water and alkyl chains, imidazolium rings and anions (i.e. hydrophobic hydration of cations and hydration of anions). The Mu group¹¹¹ evaluated the H-bonding interactions between [EMIM][OAc] and several deuterated solvents including D₂O in their whole concentrations by attenuated total reflectance infrared spectroscopy (ATR-IR) and ¹H NMR, and reported that with the increase in deuterated solvent concentration, the H-bonding interaction among IL molecules decreases while that between IL and solvent molecules increases. Zhang et al.¹¹² studied the H-bonding interactions between [EMIM][EtSO₄] and water by ATR-IR, ¹H NMR spectroscopy, and quantum chemical calculations, and noted that with the increase in water content, H-bonding of –SO₃ group (in ethyl sulfate) and water is strengthened while H-bonding between C-H (in cations) and water is weakened; water preferentially interacts with ethyl sulfate anions. At high contents (x_water > 0.6), water molecules begin to interact with the hydrogen atoms on the imidazolium ring, yielding a stable new complex. They also suggested a decreasing order of interaction strength as EMIM⁺ – water – EtSO₄⁻ > EMIM⁺ –SO₄⁻ > EtSO₄^{− –} water > EMIM⁺ – water. Bernardes et al.¹¹³ investigated the aqueous solutions of [EMIM][EtSO₄] by MD simulations, and obtained several interesting results: (1) Four distinct structural regimes were identified with four concentration ranges: isolated water molecules (x_water < 0.5); chain-like water aggregates (0.5 < x_water < 0.8); bicontinuous system (0.8 < x_water < 0.95); and isolated ions or small ion clusters (x_water > 0.95), respectively. (2) Two different percolation limits were identified: (a) that of water in the IL network (x_water 0.8), and (b) that of the IL in water (x_water 0.95), upon further dilution, the polar IL network begins to break into smaller aggregates and loses its continuous nature. (3) When x_water = 0.996, 60% of cations and anions become isolated, which implies that at this concentration the solvation energy of EMIM⁺ and EtSO₄⁻ by water obviously compensates the electrostatic interaction energy between the cation and anion, leading to their separation. Danten and coworkers¹¹⁴ examined the interactions of water in BMIM⁺ – based ILs (carrying anions of BF₄⁻, PF₆⁻, OTf⁻, and Tf₂N⁻) using density functional theory (DFT) calculations as well as vibrational spectroscopic tools (IR absorption and Raman scattering), and found that water molecules preferentially interact with two distinct anions by forming associations of type (A···H–O–H···A) at low water concentrations, not in the form of H-bonding of water either with F-atoms (PF₆⁻ and BF₄⁻ anions) or with the O-atoms of the sulfonyl groups (OTf⁻ and Tf₂N⁻ anions). The strength of water–anion interaction in water diluted in ILs decreases in the order of OTf⁻ > Tf₂N⁻ BF₄⁻ > PF₆⁻. Ficke and Brennecke¹¹⁵ determined the excess enthalpies of binary IL and water systems, and found several interesting interactions: (a) appending a hydroxyl group to the ethyl chain of EMIM⁺ cation increases IL/IL interactions; (b) electron-withdrawing fluorine groups on the OTf⁻ anion lead to drastically increased weaker IL/water interactions when comparing with the MeSO₃⁻ anion; (c) increasing the cation’s alkyl chain length from ethyl to butyl reduces the cation/water interactions. The Castner group¹¹⁶ determined the diffusivity of water in [BMPyrr][Tf₂N] and [BMPyrr][OTf] using the pulsed-gradient spin-echo NMR method, and reported that the ratio of water diffusivity to that of cation (D_water/D_cation) is about 10–20, implying that hydrodynamic descriptions are not useful on the molecular scale, and this ratio decreases with increasing temperature for both ILs.

The Dupont group⁹¹ proposed that the inclusion of other molecules and macromolecules into the polymeric IL network results in the formation of polar and nonpolar regions; the aqueous solution of free enzymes could be surrounded by the IL network, which supports the retaining of native structures of proteins by preserving the essential water of proteins and the preferential solvophobic interactions. When the enzyme-in-water droplets are dissolved (or dispersed) into the IL network (in polar regions), the enzyme’s active conformation could be conserved by the network (see Fig 4).¹¹⁷ The inclusion of enzyme molecules in such highly ordered supramolecular structures of ILs prevents the protein from thermal unfolding.¹¹⁸ However, since enzymes are not soluble in most common ILs, enzyme molecules (in particular, immobilized enzymes) are practically suspended in reaction media with low or little water; as a result, the IL network theory is not always suitable for explaining the enzyme activity and stability. The impact of individual anions on enzyme inactivation also cannot be explained by the IL network.

Fig. 4.

Enzymes with a small amount of water are firmly trapped in the network of ILs (Reproduced by permission from Ref,¹¹⁷ © 2007 the Biochemical Society).

SPECIFIC ION EFFECT OF ILS ON PROTEIN STRUCTURES AND ENZYME ACTIVITIES

Aqueous IL solutions

In diluted aqueous solutions, hydrophilic ILs become (partially) dissociated and solvated individual ions, and these individual ions may interact with the enzyme directly. In aqueous solutions of inorganic salts, many studies (see our earlier review¹⁶) have concluded that the ion effect on the enzyme activity followed the ion kosmotropicity (Hofmeister series): kosmotropic anions and chaotropic cations stabilize the enzyme, while chaotropic anions and kosmotropic cations destabilize it. A list of studies are compiled in Table 1 (in terms of protein stability, and enzyme activity/stability) and some representative examples are discussed in details below. A series of studies^{42, 43, 49, 119–124} in our laboratory have demonstrated that the same principle is loosely applicable to the enzyme activity in diluted IL aqueous solutions. In our first study, the activities of Amano protease P6 (from Aspergillus melleus) in 0.7 M IL aqueous solutions were affected by anions in a decreasing order of CH₃COO⁻, CF₃COO⁻ > Cl⁻, Br⁻ > OTs⁻ > BF₄⁻ (which is coherent with the decreasing order of anion’s kosmotropicity), and affected by cations in a decreasing order of EMIM⁺, BuPy⁺ > BMIM⁺ > EtPy⁺.¹¹⁹ In a second study,⁴³ our group carried out the kinetic hydrolysis of enantiomeric phenylalanine methyl ester catalyzed by Bacillus licheniformis protease in aqueous solutions of several hydrophilic ILs (0.5 M). The protease enantioselectivity was in a decreasing order with these anions: PO₄³⁻ > citrate³⁻, CH₃COO⁻, EtSO₄⁻, CF₃COO⁻ > Br⁻ > OTs⁻, BF₄⁻ (decreasing kosmotropicity), and in the presence of these cations: EMIM⁺ > BMIM⁺ > HMIM⁺ (decreasing chaotropicity). The overall IL kosmotropicity can be measured by the δ value (difference in viscosity B-coefficients of anion and cation). In general, a high enzyme enantioselectivity was observed in the solution of IL with a high δ value. After measuring the NMR B′-coefficients of a number of ions (see Fig 5, which is consistent with Fig 1 in general), our group⁴² further found a linear correlation between the enzyme enantioselectivity in aqueous solution and the δ′ parameter (difference in NMR B′-coefficients of anion and cation) of ILs, suggesting that high enzyme enantiomeric ratios (E) could be achieved in solutions of ILs with high δ′ values. Other groups^125–127 also reported low/no activities of β-glycosidase in aqueous solutions of [BMIM][BF₄], which could be explained by the chaotropic nature of BF₄⁻ in solutions¹²⁷ (Note: in neat or concentrated ILs containing anions of BF₄⁻, the chaotropic property of anion may not influence the enzyme activity; therefore, many studies observed certain enzyme activities in BF₄⁻ based ILs). Our group¹²⁰ also conducted the enzymatic hydrolysis of DL-phenylalanine methyl ester in aqueous solutions of ILs (0.5 M) containing anions of chiral- or ω-amino acids, and reported higher enantiomeric excess (ee) and yields in ILs containing anions of D-amino acids rather than in those containing anions of L-isomers. The likely explanation is that amino acid anions are more kosmotropic than zwitterionic amino acids,¹²¹ and D-amino acids are more kosmotropic than L-isomers.¹²² The use of ILs with kosmotropic anions (OAc⁻ and CF₃COO⁻) in activating hydrolases in aqueous solutions was further demonstrated in two of our studies.^{123, 124}

Table 1.

Ion specificity on the enzyme stabilities and activities in ILs.

Enzyme	Condition	Activity or stability series	Key factor(s)	Ref
Aqueous IL solutions
Protein stability (stability of native fold)
Hen egg white lysozyme	Thermal unfolding transition temperatures Tm in 1.0 M ILs	[EMIM]Cl > [BMIM]Cl > [HMIM]Cl and [HO-EMIM]Cl > [HO-PMIM]Cl > [HO-HMIM]Cl	Hofmeister series and hydrophobic effect	147
Cytochrome c	Stability in ILs containing 20% (wt) water	Anions: H2PO4− > Bu2PO4− > OAc− > Lactate− > MeSO4−, Cations: Cholinium+ > BMPyrr+ > BMIM+	Hofmeister series	45, 128, 129
Ribonuclease A (RNase A)	Thermal stability of in aqueous solution of ILs (typically 0–2 M)	Anions: SO42− > HPO42− > Cl− > EtSO4− > BF4− ~ Br− > MeSO4− > OTf− > SCN− ~ dca− > Tf2N− Cations: K+ > Na+ ~ Me4N+ > Li+ > Et4N+ ~ EMIM+ > BMPyrr+ > BMIM+ ~ Pr4N+ > HMIM+ ~ Bu4N+ and K+ > Na+ ~ Me4N+ > Cholinium+ > EMIM+ ~ Guanidinium+ > BMIM+	Hofmeister series	44, 132
Cyclic dipeptides	Transfer free energies ΔG′tr (positive values) from water to aqueous ILs (30–70% v/v)	[Et3NH][HSO4] > [Et2NH2][HSO4] > [Et3NH][OAc] > [Et2NH2][OAc] > [Et3NH][H2PO4] > [Et2NH2][H2PO4]	Biocompatibility is reverse of ΔG′tr order, and follows the Hofmeister series	138
Stability of horseradish peroxidase (HRP)	50 μM HRP incubated in up to 1.0 M ILs for 60 min	Anions: Cl− > Br− > NO3− > BF4− > OTf− > SCN− > dca− Cations: Me4N+ > Cholinium+ > EMIM+ > BMPip+ > BMPyrr+ > BMIM+ > BuPy+ > HMIM+	Hofmeister series	139
Enzyme activity and stability
Chloroperoxidase from Caldariomyces fumago	Oxidation of 1,2-dihydronaphthalene in 10–30% (v/v) ILs	[MMIM][MeSO4] > [BMIM][MeSO4] ≫ [BMIM][BF4]	Hofmeister series	148
Amano protease P6 (from Aspergillus melleus)	Hydrolytic activity in 0.7 M IL aqueous solutions	Anions: CH3COO−, CF3COO− > Cl−, Br− > OTs− > BF4− Cations: EMIM+, BuPy+ > BMIM+ > EtPy+	Hofmeister series in general	119
Bacillus licheniformis protease	Enantioselectivity in 0.5 M ILs	Anions: PO43− > citrate3−, CH3COO−, EtSO4−, CF3COO− > Br− > OTs−, BF4− Cations: EMIM+ > BMIM+ > HMIM+	Hofmeister series	43
Immobilized Candida antarctica lipase B (Novozym 435)	Enantioselective hydrolysis: initial rate in phosphate buffer containing 10–25% (v/v) ILs	Anions: BF4− > Cl−, Br− > NO3− > HSO4− (same BMIM+ cations) Cations: EMIM+ > PMIM+ > BMIM+ (same BF4− anions)	Anions: possible H-bond basicity and nucleophilicity Cations: Hofmeister series	149
CALB	Hydrolytic activity in up to 0.06 M IL	Br− > Cl− > OTf− > OAc− > CH3SO3− > HSO4− (same [BMIM]+ cation); EMIM+ > BMIM+ > OMIM+	Thermodynamic water activity, H-bond basicity (anions), hydrophobic interaction (cations)	150
Penicillium expansum lipase	Activity, 4.14% (w/v) ILs	(a) cation effect: [MMIM][MeSO4] > [EMIM][MeSO4] > [BMIM][MeSO4], [Me4N][OAc] > [Bu4N][OAc], [Me3NH][MeSO3] > [Bu4N][MeSO3], and [Me3NH][H2PO4] > [Et3NH][H2PO4] > [Bu3NH][H2PO4]; (b) anion effect: [Cholinium][OAc] > [Cholinium][MeSO3] > [Cholinium][NO3], [Bu4N][OAc] > [Bu4N][MeSO3].	(a) Hofmeister series (b) Hofmeister series	137
Papain	Enantioselective hydrolysis: activity and enantioselectivity in phosphate buffer containing 15% (v/v) ILs	Anions: BF4− > OAc− > NO3− > Cl− > HSO4− (same BMIM+ cations) Cation: (activity) C2MIM+ > C3MIM+ > C4MIM+ > C5MIM+ > C6MIM+, (enantioselectivity) C2MIM+ < C3MIM+ < C4MIM+ < C5MIM+ < C6MIM+ (same BF4− anions)	Anions: possible H-bond basicity Cations: Hofmeister series and hydrophobic effect	151
3α-hydrosteroid dehydrogenase from Pseudomonas testosterone	Enzyme activity in 10% (v/v) ILs	[BMIM][Lactate] > [EMIM][OTf] > [BMIM][BF4] > [BMIM][OTf]	Hofmeister series	152
Alcohol dehydrogenase ADH-‘A’ from hodococcus ruber	Reduction conversion of acetophenone in 20% (v/v) ILs	[EMIM][OAc] > [BMIM][OAc] > [EMIM][MeSO3]	Hofmeister series	153
Mesophilic alcohol dehydrogenase from yeast	(a) Activity in up to 600 mM IL; (b) Thermal stability in 150 mM ILs	(a) [BMIM]Cl > [BMIM][BF4] ≫ [MIm][BF4], [MIm]Cl (b) [MIm]Cl > [MIm][BF4] > [BMIM][BF4] > [BMIM]Cl	(a) Competition with substrate a (b) Not strictly following Hofmeister series	154
Thermoanaerobacter brockii alcohol dehydrogenase	(a) Activity, up to ~700 mM IL; (b) Thermal stability in 150 mM ILs	(a) [BMIM]Cl, [BMIM][BF4] ≫ [MIm][BF4], [MIm]Cl (b) [MIm]Cl > [MIm][BF4] > [BMIM]Cl > [BMIM][BF4]	(a) Competition with substrate a (b) Hofmeister series	155
Yeast alcohol dehydrogenase	Enzymatic efficiency kcat/KM in 0.5 M ILs	(a) anion effect: Cl− > Br− > EtSO4− > OTf− > BF4− > dca− > SCN− (same EMIM+ cation) (b) cation effect: Na+ > Me4N+ > Cholinium+ > EMIM+ > Et4N+ > Bu4N+ > Guanidinium+ > BMIM+ (same Cl− anion)	Hofmeister series and hydrophobic interaction	140
β-glucosidases, xylanase E2, arabinofuranosidase F1	Hydrolytic activity; up to 20% (v/v) IL	Me2PO4− > OAc− > Et2PO4− (same [EMIM]+ cation)	Not following Hofmeister series	156
Laccase from Aspergillus	Activity in 10% (v/v) ILs at pH 9.0	[C4MIM]Cl > [C8MIM]Cl > [C10MIM]Cl	Hofmeister series and hydrophobic interaction	157
Mushroom tyrosinase	Oxidation activity in up to 10% (v/v) ILs Stability in up to 2% (v/v) ILs	[BMIM][PF6] > [BMIM][BF4] > [BMIM][MeSO4] [BMIM][BF4] > [BMIM][PF6] > [BMIM][MeSO4]	Not following Hofmeister series	46
Mushroom tyrosinase	Activity, 5.85% (w/v) ILs Stability, 5% (w/v) ILs	(a) cation effect: [MMIM][MeSO4] > [EMIM][MeSO4] > [BMIM][MeSO4], [Me4N][OAc] > [Bu4N][OAc], and [Me3NH][H2PO4] > [Et3NH][H2PO4] (b) anion effect: [Cholinium][OAc] < [Cholinium][MeSO3] < [Cholinium][NO3] and [Bu4N][OAc] < [Bu4N][MeSO3] Stability: [MMIM][MeSO4] > [EMIM][MeSO4] > [BMIM][MeSO4]	Activity: (a) Hofmeister series (b) Reverse Hofmeister series (kosmotropic anions interact with Cu2+ of metalloenzyme Stability: Hofmeister series	137
Lysozyme from chicken egg white	Residual activity after incubation in 0–1.0 M ILs at 25 °C for 30 min	Cl−, BF4− > > OTf− (same EMIM+ cation) (however, above 1.0 M, lysozyme is more stable in BF4− than in Cl−)	Hofmeister series	158
Concentrated or neat ILs
Protein stability (stability of native fold)
Aβ16–22 peptide	Amyloid fibrilization in 90% (v/v) ILs	Rate of amyloid fibrilization (protein destabilization rate): HSO4−, H2PO4− > CF3COO− > lactate− > OTf− > CH3SO3− (same Et3NH+ cation)	Reverse Hofmeister series	159
Enzyme activity and stability
Candida antarctica lipase B (CALB)	Enantioselectivity of resolution of 1-phenylethanol via transesterification	Anions: Tf2N−, CF3SO3− ≫ PF6−, BF4− (same BMIM+ cation) Cations: OMIM+ > HMIM+ > BMIM+ (same BF4− anion)	Anions: unknown Cations:hydrophobicity	160
Free CALB	Transesterification activity in ILs with 2% v/v water	Anions: BF4− > Tf2N− (same EMIM+ cation)  PF6− > Tf2N− (same BMIM+ cation) Cations: EMIM+ > BMIM+ (Tf2N− anion)	unknown	161
Novozym 435 (Immobilized CALB)	Stability in ILs at 30 °C	Anions: OAc− > PF6− > NO3− (same BMIM+ cation)  OAc− > CH3SO3− > NO3− (same MMEP+ cation) Cations: MMEP+ > BMIM+ (OAc− or NO3− anions)	unknown	162
Novozym 435	Transesterification activity	BF4 > PF6 ≫ Lactate > NO3 (same BMIM+ cation)	unknown	163
Novozym 435	Asymmetric ammonolysis: initial rate and enantioselectivity	C3MIM+ < C4MIM+ < C5MIM+ < C6MIM+ > C7MIM+ > C8MIM+ (same BF4− anions)	Hydrophobicity	164
Novozym 435	Initial rate and enantioselectivity of acylation with controlled water activity	[C4MIM][PF6] > [C8MIM][BF4] > [C7MIM][BF4] > [C6MIM][BF4] > [C5MIM][BF4] > [C4MIM][BF4]	Hydrophobicity	165
Novozym 435	Transesterification activity in neat ILs	Tf2N− > PF6− > BF4− > dca− (same BMIM+ cation)	Hydrophobicity and H-bond basicity	166
Novozym 435	Transesterification of triolein and methanol	Anions: Tf2N− > PF6− > BF4− Cations: C18MIM+ > C12MIM+ > C8MIM+ > C4MIM+	Hydrophobicity	167
α-chymotrypsin	Stability in ILs (2% v/v water and 50 °C)	PF6− > BF4− (same BMIM+ cation)	Hydrophobicity	168
PEG complex of lipase PS from Pseudomonas cepacia	Initial rate of alcoholysis in ILs containing 1% (v/v) water	OMIM+ > HMIM+ > BMIM+ (same PF6− anion)	Hydrophobicity	169, 170
Bacillus stearothermophilus esterase immobilized on Celite	Transesterification activity at aw = 0.11	Tf2N− > BF4− > PF6− (same BMIM+ cation)	unknown	171
Bacillus subtilis esterase immobilized on Celite	Transesterification activity at aw = 0.11	Tf2N− > PF6− > BF4− (same BMIM+ cation)	Hydrophobicity	171
Candida rugosa lipase	Esterification of 2-substituted-propanoic acids and 1-butanol	Reaction rate: [BMIM][PF6] > [ONIM][PF6] > [BMIM][BF4] Enantioselectivity: [ONIM][PF6] > [BMIM][PF6] > [BMIM][BF4]	Hydrophobicity and H-bond basicity	172
Penicillin G amidase (PGA)	(a) Stability in ILs with 1–20% water (aw = 0.66–0.80) (b) Activity in ILs (aw = ~0.80)	(a) Stability: BF4− > PF6− > MeSO4; BMIM+ > OMIM+ (b) Activity: [BMIM][PF6] > [BMIM][BF4] > [BMIM][MeSO4] (PGA in ILs requires an optimal hydration aw = ~0.80)	(a) anions: H-bond basicity and hydrophobic interaction (b) H-bond basicity	173
Alcohol dehydrogenase from Rhodococcus erythropolis	Initial rates in 10% (v/v) ILs Half-time stability in 10% (v/v) ILs	(a) reduction of 4′-Br-2,2,2-trifluoroacetophenone [BMIM][PF6] > [EMIM][OTs] > [BMIM][BF4] (b) reduction of 6-Br-β-tetralone [BMIM][PF6] > [BMIM][BF4] > [EMIM][OTs] Stability: [EMIM][EtSO4] > [BMIM][PF6] > [EMIM][OTs] > [BMIM][BF4]	H-bond basicity and hydrophobic interaction unknown	174
Glucose dehydrogenase 103 Lipase from Burkholderia cepacia	Half-time stability in 10% (v/v) ILs Transesterification activity	[EMIM][EtSO4] > [BMIM][BF4] > [BMIM][PF6] > [EMIM][OTs] PF6− > Tf2N− > OTf− > BF4− > CH3SO3− ~ Cl− (same [BMIM]+ cation)	unknown H-bond basicity and Hydrophobicity	174 175
Feruloyl esterase A from Aspergillus niger	Esterification activity (15% v/v aqueous buffer)	PF6− > BF4− (same [BMIM]+ cation)	H-bond basicity	176
Naringinase from Penicilliun decumbens	Hydrolytic activity catalyzed by the enzyme immobilized on IL sol–gel matrices	OMIM+ > BMIM+ > EMIM+ > C2OHMIM+ > BIM+	Hydrophobicity	177
Endo-1,4-β-D-glucanase from Aspergillus niger	Hydrolysis of cellulose azure	[(HOCH2CH2)3MeN][MeSO4] > [BMIM]Cl ≫ [BMIM][MeSO4] (neat ILs); [(HOCH2CH2)3MeN][MeSO4] ≫ [BMIM][MeSO4] > [BMIM]Cl (1.0 M)	H-bond basicity and anion nucleophilicity (neat ILs); Hofmeister series (1.0 M ILs)	178

Note:

^aDue to structural similarity between MIm (1-methylimidazolium) and substrate adenine moiety (NADP⁺).

Fig. 5.

NMR B′-coefficients of some ions.⁴²

Recently, Fujita et al.^{45, 128, 129} evaluated the stability of cytochrome c in ILs containing 20% (wt) water and its relevance to the kosmotropicity of individual ions; the cation’s effect on the protein stability followed a decreasing order of Cholinium⁺ > BMPyrr⁺ > BMIM⁺, which is also a decreasing order of cation chaotropicity; the anion’s effect on the protein stability followed a decreasing order of H₂PO₄⁻ > Bu₂PO₄⁻ > OAc⁻ > lactate⁻ > MeSO₄⁻, which is the decreasing order of anion kosmotropicity (B-coefficients at 25 °C: H₂PO₄⁻ = 0.340,⁴⁰ OAc⁻ = 0.246,⁴⁰ MeSO₄⁻ = 0.188⁴¹; lactate might be considered as a kosmotropic anion¹³⁰). This group¹³¹ further dissolved various metallo proteins (cytochrome c, peroxidase, ascorbate oxidase, azurin, pseudoazurin and D-fructose dehydrogenase) in hydrated [Cholinium][H₂PO₄] (with 30 wt% water), and observed that proteins maintained their active sites and secondary structures in the ionic medium. In addition, they found that some proteins retained their activities in hydrated [Cholinium][H₂PO₄] and D-fructose dehydrogenase showed substantially improved thermal stability in the ionic solution.

Constantinescu et al.^{44, 132} concluded that the thermal stability of ribonuclease A (RNase A) in aqueous solution of ILs (typically 0–2 M) follows the Hofmeister series. In their study, differential scanning calorimetry (DSC) was employed to measure the effect of ILs on the thermal denaturation of RNase A near 60 °C. In terms of decreasing protein stability, the cation series are

K⁺ > Na⁺ ~ Me₄N⁺ > Li⁺ > Et₄N⁺ ~ EMIM⁺ > BMPyrr⁺ > BMIM⁺~Pr₄N⁺ > HMIM⁺ ~ Bu₄N⁺ and K⁺ > Na⁺ ~ Me₄N⁺ > Cholinium⁺ > EMIM⁺ ~ Guanidinium⁺ > BMIM⁺

and the anion series follows

SO₄²⁻ > HPO₄²⁻ > Cl⁻ > EtSO₄⁻ > BF₄⁻ ~ Br⁻ > MeSO₄⁻ > OTf⁻ > SCN⁻ ~ dca⁻ > Tf₂N⁻

The cation series suggests the higher the cation’s hydrophobicity, the higher the cation’s kosmotropicity, and the lower the protein stability in general. The anion series offers the opposite: the higher the anion’s kosmotropicity, the higher the protein stability in general (with small differences in the position of neighboring ions from our earlier discussion). Constatinescu et al.¹³² also indicated ILs could improve the stability of the native state, accelerate refolding, and suppress irreversible aggregation; in addition, all ILs evaluated could suppress protein aggregation under certain conditions, regardless of their protein stabilizing/destabilizing effect. Yang et al.⁴⁶ found that mushroom tyrosinase is more active in aqueous [BMIM][BF₄] than in aqueous [BMIM][MeSO₄]; however, the enzyme stability follows a decreasing order of KMeSO₄ > NaBF₄ > KPF₆. Yang et al.¹³³ determined the activity and stability of alkaline phosphatase in up to 1.0 M inorganic salt solutions; they found the initial reaction rate or V_max/K_m exhibits a bell-shaped relationship with the (B₋ –B₊) values of the salts, where B₋ and B₊ are Jones–Dole viscosity B-coefficients for anions and cations respectively, and the highest activities are obtained by salts (such as NaCl, KCl, and KNO₃) where the anion and cation have similar kosmotropic/chaotropic properties. This effect is likely due to the influence of cations and anions on the enzyme’s surface pH, active site, and catalytic mechanism. The enzyme’s thermal stability increases with the B₋ or (B₋ – B₊) values, where anions seem to be more essential to the enzyme stabilization. Such a correlation may be explained by the ion effect on the enzyme surface solvation, as well as the ion interaction with surface and internal structure of the enzyme.

The Hinderberger group¹³⁴ probed the impact of ILs on the tertiary structure of human serum albumin (HSA) by using continuous wave electron paramagnetic resonance (EPR) spectroscopy and nanoscale distance measurements with double electron–electron resonance (DEER) spectroscopy. They observed that the protein begins to unfold in 15% (v/v) [BMIM][BF₄] and more hydrophobic alkyl chains promote strong protein-IL interactions; however, the binding capacity and the tertiary structure of HSA is mostly maintained in 25% (v/v) [Cholinium][H₂PO₄]. This can be explained by the Hofmeister series: [BMIM][BF₄] contains a kosmotropic cation and a chaotropic anion while [Cholinium][H₂PO₄] consists of a chaotropic cation and a kosmotropic anion. Urea is a non-ionic chaotrope, and is a known protein denaturant that preferentially interacts with the protein surface and interrupts H-bonds of proteins.¹³⁵ Attri et al.¹³⁶ observed that [Et₃NH][OAc] reduces the denaturing property of urea on α-chymotrypsin in aqueous solutions based on studies using circular dichroism (CD), fluorescence and NMR methods; the likely reason is that kosmotropic acetate ion interacts with urea and water via H-bonds, minimizing the urea-enzyme interactions. The Yang group¹³⁷ found that the activity of Penicillium expansum lipase in 4.14% (w/v) ILs follows the Hofmeister series: for cations [MMIM][MeSO₄] > [EMIM][MeSO₄] > [BMIM][MeSO₄], [Me₄N][OAc] > [Bu₄N][OAc], [Me₃NH][MeSO₃] > [Bu₄N][MeSO₃], and [Me₃NH][H₂PO₄] > [Et₃NH][H₂PO₄] > [Bu₃NH][H₂PO₄]; for anions [Cholinium][OAc] > [Cholinium][MeSO₃] > [Cholinium][NO₃], [Bu₄N][OAc] > [Bu₄N][MeSO₃]. They also observed a similar Hofmeister cation effect on mushroom tyrosinase, for activity in 5.85%, (w/v) ILs: [MMIM][MeSO₄] > [EMIM][MeSO₄] > [BMIM][MeSO₄], [Me₄N][OAc] > [Bu₄N][OAc], and [Me₃NH][H₂PO₄] > [Et₃NH][H₂PO₄]; for stability in 5% (w/v) ILs: [MMIM][MeSO₄] > [EMIM][MeSO₄] > [BMIM][MeSO₄].

Attri and Venkatesu¹³⁸ determined the transfer free energies (ΔG′_tr) of a homologous series of cyclic dipeptides from water to aqueous protic ILs (30%, 50% and 70%, v/v) from solubility measurements at 25 °C under atmospheric pressure. They observed that ΔG′_tr values are positive in all cases studied, and decrease in the order of [Et₃NH][HSO₄] > [Et₂NH₂][HSO₄] > [Et₃NH][OAc] > [Et₂NH₂][OAc] > [Et₃NH][H₂PO₄] > [Et₂NH₂][H₂PO₄]. A higher ΔG′_tr value indicates a stronger unfavorable interaction between an IL and cyclic dipeptide; therefore, the biocompatibility of these ILs is the reverse order of the above ΔG′_tr sequence, which follows the Hofmeister series: kosmotropic anions and chaotropic cations stabilize proteins [viscosity B-coefficients (dm³ mol⁻¹ at 25 °C):⁴⁰ H₂PO₄⁻ (0.340) > OAc⁻ (0.246) > HSO₄⁻ (0.127), Et₃NH⁺ (0.385) > Et₂NH₂⁺ (0.293)]. Lu et al.¹³⁹ found that anodic peak current of horseradish peroxidase (HRP) at bare glassy carbon electrode (GCE) can be correlated with the catalytic activity and the secondary structure of HRP. Therefore, the current signals in the presence of ILs could quantify the impact of ions on the structural stability of HRP. The effect of cations and anions (up to 1.0 M) on the HRP structural stability seems to follow the Hofmeister series:

(Cations) Me₄N⁺ > Cholinium⁺ > EMIM⁺ > BMPip⁺ > BMPyrr⁺ > BMIM⁺ > BuPy⁺ > HMIM⁺,

(Anions) Cl⁻ > Br⁻ > NO₃⁻ > BF₄⁻ > OTf⁻ > SCN⁻ > dca⁻

Weibels et al.¹⁴⁰ evaluated the activities of yeast alcohol dehydrogenase in 0.5 M ILs, and noticed that all ILs studied lower the turnover number (k_cat) when comparing with the reaction in buffer whilst the apparent dissociation constant of the substrate (K_M) varies. Overall, the enzymatic efficiency k_cat/K_M follows the series below:

(Anion) Cl⁻ > Br⁻ > EtSO₄⁻ > OTf⁻ > BF₄⁻ > dca⁻ > SCN⁻ (same EMIM⁺ cation)

(Cation) Na⁺ > Me₄N⁺ > Cholinium⁺ > EMIM⁺ > Et₄N⁺ > Bu₄N⁺ > Guanidinium⁺ > BMIM⁺ (same Cl⁻ anion)

This group argued that the observed Hofmeister series could be explained by the hydrophobic interactions as a controlling factor for ion-specific effects on the enzymatic activity.

Yan et al.¹⁴¹ studied the interaction bovine serum albumin (BSA) and [C_nMIM]Br (n = 4, 6, 8, 10) (up to 8.0 mM) by fluorescence, UV–Vis and FT-IR spectroscopy, as well as the density functional theory (DFT). Their data suggest that these ILs bind with BSA through two types of interactions: (a) H-bonding between cationic headgroups and Asp/Glu amino acid residues at the BSA surface, and hydrophobic interaction between cationic hydrocarbon chains and hydrophobic amino acid residues in the core of BSA. Since the hydrophobic interaction increases with the alkyl chain length, it is the predominated interaction of [C₁₀MIM]Br with BSA; on the other hand, H-bonding and van der Waals force are primary interactions between [C_nMIM]Br (n = 4, 6, 8) with BSA. An excellent review by Yang⁹ systematically discussed the possible mechanisms of Hofmeister effects of ILs on the enzyme activity and stability. The above experimental studies have shown that the kosmotropic effect of ILs on enzymes may be applicable to diluted aqueous solutions of ILs,^{16, 43, 119} as well as some concentrated ILs (such as 20 wt% water⁴⁵). However, it is not quite clear if such an effect exists in neat or concentrated ILs, and how the IL hydrophobicity may influence the kosmotropicity. For example, PF₆⁻ is a chaotropic anion,⁴⁹ and denatures enzymes when dissolved in aqueous solutions as Na⁺ or K⁺ salt (more denaturing than BF₄⁻ and MeSO₄⁻ for mushroom tyrosinase⁴⁶). However, PF₆⁻ based ILs (such as [BMIM][PF₆]) are hydrophobic, and thus the solubility and degree of dissociation of ILs in water become limited. Meanwhile, it is also known PF₆⁻ based ILs containing low water contents are usually enzyme stabilizing.¹ Therefore, the Hofmeister effect may not be suitable for explaining the enzyme’s behaviors in these hydrophobic ILs or their mixtures with water. Without sufficient water to hydrate them, kosmotropic or borderline anions (such as acetate, lactate and chloride) of ILs bearing high H-bond basicities tend to interact strongly with enzymes causing their inactivation (see a later section H-bond basicity and nucleophilicity of anions). Consequently, the enzyme stabilization/activation kosmotropic anions (such as OAc⁻ and Cl⁻) in diluted aqueous solutions become enzyme-inactivating agents in ILs with low water contents (see a simple illustration in Fig 6). For example, several papers^{43, 119, 123, 142} have reported the enzyme activation at low-concentrations of chloride-based ILs in water, but inactivation at high concentrations.

Fig. 6.

Illustration of interactions of enzyme in acetate-containing IL: (a) between acetate anion with water molecules in diluted IL solution, and (b) between acetate anion and enzyme molecule in concentrated IL.

On the other hand, there are a number of studies that indicate enzyme activities in aqueous ILs do not follow Hofmeister series or even follow a reverse order (see Table 1). A few selected examples are discussed below. The Yang group¹³⁷ found that the activity of mushroom tyrosinase in 5.85% (w/v) ILs follows a reverse Hofmeister series: [Cholinium][OAc] < [Cholinium][MeSO₃] < [Cholinium][NO₃] and [Bu₄N][OAc] < [Bu₄N][MeSO₃]; the likely explanation is that kosmotropic anions interact with Cu²⁺ of the metalloenzyme, resulting in lower activities. Curto et al.¹⁴³ observed the activity of lactate oxidase in 0.5 M choline-based ILs follows a decreasing order with anions as Cl⁻ > H₂PO₄⁻ > NO₃⁻ > Levulinate⁻ > HCOO⁻. The viscosity B-coefficients for these anions at 25 °C (in dm³ mol⁻¹) are: Cl⁻ (−0.005), H₂PO₄⁻ (0.340), NO₃⁻ (−0.043) and HCOO⁻ (0.052).⁴⁰ The B-coefficient for Levulinate⁻ is unknown, but is estimated to be greater than that of butanoate (0.419).⁴⁰ Therefore, the kosmotropicity of these anions based on B-coefficients and known Hofmeister series¹⁶ can be listed in a decreasing order of Levulinate⁻ > H₂PO₄⁻ > HCOO⁻ > Cl⁻ > NO₃⁻, which is not in agreement with the order of lactate oxidase activities. This group¹⁴³ also measured the secondary structure of lactate oxidase by CD spectroscopy, and found a considerable decrease of α-helices and increase of β-sheets in hydrated [Cholinium][H₂PO₄] (25%, w/w). However, the changes in its secondary structure lead no appreciable impact on the activity and stability of lactate oxidase. Baker et al.¹⁴⁴ examined the equilibrium unfolding behavior of site-specific tetramethylrhodamine-labelled yeast cytochrome c in aqueous ILs (up to 2.5 M), and found the protein denaturation is highly anion-dependent. However, they noted that Hofmeister theory seems inadequate for providing reason explanations, and more complex factors (such as H-bonding and other specific solvent–solute interactions) should be considered. Kumar and Venkatesu¹⁴⁵ observed the transition temperature (T_m) of myoglobin decreasing in 0.01 – 0.04 M [BMIM]⁺ – based ILs in the order of anions as Br⁻ > Cl⁻> HSO₄⁻ > SCN⁻ > CH₃COO⁻ > I⁻; this sequence is not consistent with the known Hofmeister series. Similarly, this group¹⁴⁶ further determined the T_m values of α-chymotrypsin from fluorescent measurements in 0.01 M salt solutions, which decrease for the sodium salts in the order of SO₄²⁻ > Br⁻ > I⁻ > SCN⁻ > CH₃COO⁻ > Cl⁻, and for [BMIM]⁺ –based ILs in the order of CH₃COO⁻ > Br⁻ > Cl⁻ > HSO₄⁻ > SCN⁻ > I⁻. These sequences do not seem to follow the Hofmeister series. [BMIM]⁺-based ILs carrying anions of CH₃COO⁻, Cl⁻ and Br⁻ enhance the thermal stability of α-chymotrypsin, while HSO₄⁻, SCN⁻ and I⁻ containing ILs act as protein denaturants.

Enzyme activation by low concentrations of ILs

A number of studies reported that enzymes are activated/stabilized by low concentrations of ILs. The Rogers group¹⁴² observed that the cellulase’s fluorescence intensity associated with tryptophan increased in low concentrations of [BMIM]Cl (up to ~10%) and then drastically decreased at higher salt concentrations. Our group observed that proteases could be activated by a low concentration (e.g. 0.5 M) of [EMIM][EtSO₄]⁴³ or [BMIM][CF₃COO].¹²³ Baker and Heller¹⁷⁹ studied the structures of human serum albumin (HSA) and equine heart cytochrome c in aqueous [BMIM]Cl by CD spectroscopy and small-angle neutron scattering measurements. They found that both proteins maintain most of their higher-order structures in up to 25% (v/v) [BMIM]Cl, and become highly denatured in 50% (v/v) [BMIM]Cl; in addition, HSA dimerizes at high concentrations of [BMIM]Cl, while cytochrome c exclusively retains the monomeric form. Domínguez et al.¹⁸⁰ found that laccase from Trametes versicolor could be activated by 10% (v/v) [BMIM]Cl, but inactivated by the same concentration of [EMIM][EtSO₄] (slight inactivation) or [HMIM]Br (substantial inactivation).

Yang et al.⁴⁶ reported that the activity of mushroom tyrosinase increases with IL concentration at up to 5% (v/v) for [BMIM][BF₄] and 2% (v/v) for [BMIM][MeSO₄], and then declines with a higher IL concentration. The catalytic efficiency (V_max/K_m) increases initially with IL content and then decreases, showing a bell-shaped relationship with the IL concentration. Choline acetate is an IL consisting of a kosmotropic anion and a chaotropic cation. The Huang group¹⁸¹ found that at low concentrations (up to 5 mM), choline acetate could improve the hydrolytic activity of Candida rogusa lipase in AOT/water/isooctane reverse micelles (Fig 7), and cause no lipase conformational changes as evidenced by fluorescence spectra. Infrared spectra suggest stronger H-bonds between choline acetate and water than those between water molecules; as a result, the addition of a low content of choline acetate improves the nucleophilicity of water, accelerating the attack of water molecules on the acyl enzyme intermediate and increasing the lipase’s catalytic efficiency.

Fig. 7.

Illustration of choline acetate influencing the nucleophilicity of water molecules near the lipase in AOT reverse micelles (Adapted from Ref,¹⁸¹ with permission from Elsevier).

Li et al.¹⁸² examined the hydrolytic activity of Candida rugosa lipase in aqueous solutions of a serious ILs [C_nMIM]X (n = 2, 4, 6, 8, 10, or 12; X = Cl⁻, Br⁻, BF₄⁻ or PF₆⁻), and found that the lipase activities increase with the IL contents to optimum concentrations and then decline with higher IL concentrations. In general, the optimum concentrations decrease with the alkyl chain length of cations, and are several-fold lower than their corresponding critical micelle concentration (CMC). Filice et al.¹⁸³ studied the activities of immobilized lipases in a low concentration (0.01 M) of ILs (based on BF₄⁻, PF₆⁻, NO₃⁻, and MeSO₄⁻), and observed that some IL solutions could activate the lipases. For example, an engineered variant (σ-L230C) of Geobacillus thermocatenolatus lipase (GTL) showed seven-fold improvement of activity in [EMIM][PF₆] and five-fold improvement of activity in [EMIM][MeSO₄] during the monodeacetylation of peracetylated glucal; this lipase variant also exhibited a higher regioselectivity in the hydrolysis of peracetylated glucal (from 78% to 96% yield of the C-3 monodeprotected product) in the [BDMIM][PF₆] solution. In addition, the addition of [EMIM][PF₆] improved the regioselectivity of Candida rugosa lipase in the hydrolysis of peracetylated thymidine (from 72% to 81% yield of C-5 monodeprotected product), however, the use of BF₄⁻ –based ILs generally led to lower enzyme activities. CD and fluorescence measurements suggested that a low concentration of ILs could cause conformational changes in the tertiary structure of the lipase.

Concentrated or neat ILs

As discussed earlier, cations and anions of concentrated or neat ILs form complex polymeric network through interactions like electrostatic attractions and H-bonding (Fig 3). Therefore, several key properties, such as H-bond basicity and nucleophilicity of anions and IL hydrophobicity, begin to play critical roles in enzyme stabilization and activation. Some examples are listed in Table 1 and a few representative studies are discussed in-depth below.

H-bond basicity and nucleophilicity of anions

H-bond basicity and nucleophilicity are two different concepts,^† but are often closely related. For molecules containing the same nucleophilic atoms of the same charge, the stronger base is usually the stronger nucleophile in aprotic solvents. Relying on the solvatochromic measurements, several studies have suggested the order of anion’s basicity as the following (in decreasing orders):

• Basicity series #1:¹⁸⁴ OTf⁻ (CF₃SO₃⁻) > Tf₂N⁻ > PF₆⁻

• Basicity series #2:¹⁸⁵ Cl⁻ > Br⁻ > SCN⁻ > OAc⁻ > I⁻ > NO₃⁻ > OTf⁻ > ClO₄⁻ > BF₄

• Basicity series #3:¹⁸⁶ Cl⁻ > Br⁻ > OAc⁻ > OTf⁻ > ClO₄⁻ > BF₄⁻

• Basicity series #4:¹⁸⁷ Cl⁻ > Br⁻ > CH₃OSO₃⁻ > SCN⁻ > BF₄⁻ ~ OTf⁻ > PF₆⁻

• Basicity series #5:¹⁸⁸ OAc⁻, Me₂PO₄⁻, MeHPO₃⁻ > EtSO₄⁻ > MeSO₄⁻ > BF₄⁻ > Tf₂N⁻ > PF₆⁻

Based on the above series and other discussions in literatures,^{189, 190} a summary of the basicity of selected anions is illustrated in Fig 8. These anions are divided into three categories (basic, neutral and acidic), and some of them are ranked in the order of basicity. Basic anions include halides, acetate, dicyanamide (dca⁻), lactate and methyl sulfate; these anions are good H-bond donors and tend to form H-bonds with proteins resulting in enzyme denaturation and/or inactivation at high salt concentrations. Neutral anions include those tending to form hydrophobic ILs (Tf₂N⁻ and PF₆⁻) and others tending to form hydrophilic ILs (BF₄⁻, OTf⁻, SCN⁻, NO₃⁻ and CH₃SO₃⁻). These anions have weak abilities in forming H-bonds; i.e., if enzymes are inactivated in ILs containing neutral anions, the H-bond basicity is unlikely the main reason. Acidic anions (such as amphoteric H₂PO₄⁻ and HSO₄⁻) are not common anions in ILs for biocatalysis. However, the Ohno group^{45, 128} found that choline dihydrogen phosphate (m.p. 119°C) containing 20% (wt) water could dissolve and stabilize cytochrome c.

Fig. 8.

Comparison of H-bond basicity of selected anions in ILs.

Bernson and Lindgren¹⁹¹ dissolved lithium salts LiX in poly(propylene glycol) (MW = 3000) with hydroxy end-groups. Using IR spectroscopy, they observed that the shifts of –– OH stretching band depend on the strength of H-bond formed between the – OH group and the anion, as well as the coordination of cations with the -OH group. The strength of anion coordination is further dependent on the H-bond basicity of the anion, and is summarized from the IR band shifts as (in an increasing order),

PF₆⁻ < BF₄⁻ < ClO₄⁻ < OTf⁻ < I⁻ < Br⁻ < Cl⁻

In general, this basicity series is consistent with the basicity order from solvatochromic measurements (Fig 8). From experimental data of IR and ESI-MS, Dupont⁹¹ suggested the strength of H-bond basicity in a similar increasing order of

BPh₄⁻ < PF₆⁻ < BF₄⁻ < CF₃COO⁻

On the other hand, the ionic association strength of LiX salts was also examined in a variety of aprotic solvents including glymes (see a short review in the Supporting Information of Ref¹⁹²). The approximate ionic association strength in aprotic solvents is listed below in an increasing order:^{192, 193}

beti⁻, Tf₂N⁻ < PF₆⁻ < ClO₄⁻, I⁻ < SCN⁻ < BF₄⁻ < CF₃SO₃⁻ < Br⁻ < NO₃⁻ < CF₃COO⁻ < Cl⁻

This order represents the strength of an anion in interacting with solvated cations through ionic attraction, or could be implied to represent the strength of interactions between the anions and charged regions of macromolecules (such as proteins). This ionic association strength series resembles the anion’s H-bond basicity order in Fig 8.

In the following sections, a number of enzymatic reactions in ILs demonstrate how the nucleophilicity and basicity of anions contribute to the enzyme activity and stability. The first group of examples focused on the effect of anion’s nucleophilicity. Kaar et al¹⁶² observed that free Candida rugosa lipase was only active in hydrophobic [BMIM][PF₆], but inactive in all hydrophilic ILs based on NO₃⁻, OAc⁻ and CF₃COO⁻ during the transesterification of methylmethacrylate with 2-ethyl-1-hexanol. They indicated that the latter three anions are more nucleophilic than PF₆⁻, and thus could interact with the enzyme causing the protein conformation changes. In this example, the solvent hydrophobicity is another important factor in influencing the enzyme activity (see a later section ‘Hydrophobicity’). Hernández-Fernández et al¹⁹⁴ reported that the stability of CALB (lipase B from Candida antarctica) in ILs was in the following order: [HMIM][PF₆] > [HMIM][Tf₂N] > [HMIM][BF₄], and [BMIM][PF₆] > [BMIM][dca], and the stability of Penicillin G acylase was in a similar order of [BMIM][Tf₂N] > [BMIM][PF₆] > [BMIM][BF₄]. They explained the decreasing stability were in general consistent with the increasing order of nucleophilicity in Fig 8 (PF₆⁻ < BF₄⁻ < Tf₂N⁻ < dca⁻), where the more nucleophilic anions tend to interact with the positively charged sites on enzymes and to modify the enzyme’s conformation. On the other hand, they also pointed out that the enzyme stability was in agreement with the hydrophobicity of ILs: both enzymes were more stable in hydrophobic ILs than in hydrophilic ones. However, in another study, a contradictory result was reported. Irimescu and Kato¹⁹⁵ carried out the CALB-catalyzed enantioselective acylation of 1-phenylethylamine with 4-pentenoic acid, and found that the reaction rates relied on the type of IL anions (reaction rates in a decreasing order of OTf⁻ > BF₄⁻ > PF₆⁻, same cations). Thus, this example implies a higher anion nucleophilicity leading to a higher enzymatic activity. In a second acylation reaction of 2-phenyl-1-propylamine with 4-pentenoic acid, however, Irimescu and Kato¹⁹⁵ observed that PF₆⁻ based ILs afforded fastest reaction rates, followed by OTf⁻ and BF₄⁻ based ILs. The rather confusion findings may be due to the fact that the enzymatic reaction is affected by multiple factors of ILs such as nucleophilicity, hydrophobicity, viscosity and impurity. Lee et al¹⁹⁶ measured the initial transesterification rates of three lipases (Novozym^® 435, Rhizomucor miehei lipase, and Candida rugosa lipase) in different ILs under the same water activity (a_w), and observed the anion effect on the initial rates followed a decreasing order of Tf₂N⁻ > PF₆⁻ > OTf⁻ > SbF₆⁻ ~ BF₄⁻. They explained that OTf⁻ and BF₄⁻ are more nucleophilic than PF₆⁻. The second factor could the IL hydrophobicity because lipases seemed more active in hydrophobic ILs than in hydrophilic ones.

The second group of examples focused on the effect of the anion’s H-bond basicity. [BMIM]Cl could effectively dissolve cellulose^{197, 198} because chloride ions (as H-acceptors) interact with the cellulose -OH group and break the H-bonding network of cellulose.¹⁹⁹ Because of the same reason, this IL induced the inactivation of cellulase (from Trichoderma reesei).¹⁴² Similarly, Lee et al²⁰⁰ observed a dramatic decrease of the lipase activity in [OMIM][Tf₂N] in a higher concentration of [OMIM]Cl. Based on the multiple salvation interactions, [BMIM]Cl showed the largest H-bond basicity among ILs considered in a study by Anderson et al,²⁰¹ and thus could dissolve complex polar molecules such as cyclodextrins and antibiotics.²⁰² Lou et al¹⁶⁴ reported that Novozym^® 435 showed no ammonolysis activity towards (R,S)-p-hydroxyphenylglycine methyl ester in [BMIM]Br and [BMIM][NO₃], implying the denaturing nature of these two ILs. Lau et al¹⁶³ suggested that the low CALB activity in [BMIM][Lactate] was caused by the secondary structure changes of the protein, which was further triggered by the H-bonding interaction between lactate anions and peptide chains. Dicyanamide (dca⁻) based ILs such as [BMIM][dca] are capable of dissolving carbohydrates,^{203, 204} however, [BMIM][dca] is an enzyme-denaturing IL^{166, 205, 206} probably due to the high H-bond basicity of the anion. Fujita et al⁴⁵ detected a low stability of cytochrome c in [BMIM][MeSO₄], [BMIM][Lactate] and [BMIM][OAc] all containing 20 wt% water, implying the high H-bond basicity and enzyme-denaturing nature of MeSO₄⁻, lactate and OAc⁻. Our group²⁰⁷ also suggested both free and immobilized CALB in [EMIM][OTf] were about as inactive as in [BMIM][dca]. Bermejo et al²⁰⁸ observed that free CALB lost 35% of its initial activity once being dissolved in [HOPMIm][NO₃], but maintained 80% of the remaining activity after 3 months of incubation in this IL. The CALB activity loss in [HOPMIm][NO₃] was primarily due to the denaturing effect of NO₃⁻ as discussed earlier. On the other hand, the less denaturing property of this IL (vs. [BMIM][NO₃]) may be explained by two reasons: (1) the HOPMIm⁺ cation is larger than BMIM⁺, and as a result, the molar concentration of NO₃⁻ in [HOPMIm][NO₃] is lower than that in [BMIM][NO₃]; (2) [HOPMIm][NO₃] contains a hydroxyl group, which may favorably interact with NO₃⁻ and thus reduce the interaction between NO₃⁻ and the lipase. Zeuner et al.¹⁷⁶ carried out the esterification of glycerol with sinapic acid catalyzed by Feruloyl esterase A from Aspergillus niger (15% v/v aqueous buffer, 18% v/v glycerol, and 67% v/v IL), and found the enzyme is active in PF₆⁻ –based ILs ([BMIM][PF₆] and [(HOCH₂CH₂)MIM][PF₆] but inactive in BF₄⁻ –based ILs. The COSMO-RS simulations suggest that BF₄⁻ is a stronger H-bond acceptor than PF₆⁻, disrupting the H-bond based enzyme structure. The Yang group²⁰⁹ obtained up to 86% conversion of corn oil to biodiesel in [BMIM][PF₆] catalyzed by Penicillium expansum lipase; however, they obtained no enzymatic activity in other ILs containing anions of MeSO₄⁻, OAc⁻, NO₃⁻, and H₂PO₄⁻. Bekhouche et al.²¹⁰ examined the activity and stability of formate dehydrogenase from Candida boidinii (FDH, EC: 1.2.1.2) in three ILs (i.e. [MMIM][Me₂PO₄], [BMIM][OAc], [MMIM][CH₃HPO₂(OCH₃)]) by activity assays and steady-state fluorescence spectroscopy (using iodide as the dynamic quencher or acrylamide as the static quencher). They found the third IL is more denaturing than the first two and each IL induces a different denaturation mechanism. The enzymatic activity was reduced in the presence of 30% (v/v) [MMIM][Me₂PO₄], 10% [BMIM][OAc] or 10% [MMIM][CH₃HPO₂(OCH₃)], and was totally inactivated in 70% (v/v) [MMIM][Me₂PO₄], 30% [BMIM][OAc] or 20% [MMIM][CH₃HPO₂(OCH₃)].

Hydrophobicity

‘Hydrophobicity’ could be considered as a subset concept of ‘polarity’. However, it is practically important to differentiate ‘hydrophobicity’ from ‘polarity’ because the former one is often related to the miscibility with water.²¹¹ The hydrophobicity of ILs can be quantified by the log P scale, a concept derived from the partition coefficient of ILs between 1-octanol and water. The partition coefficient (K_OW or P) is a ratio of concentrations of un-ionized compound between the two phases. The log P is defined as the partition coefficient at the unlimited dilution concentration of solute,

$$\text{log}P=\underset{c\to 0}{lim}{K}_{OW}=\underset{c\to 0}{lim}\frac{C^o}{C^w}$$ (2)

where C^o is the IL concentration in the octanol phase and C^w is the IL concentration in the aqueous phase. For the simplicity, it is common to use extremely low concentrations of IL in the experiment instead of extrapolating the IL concentration to zero (eqn 2). However, since ILs dissociate into ions in water and current K_OW values were reported as the ratio of concentrations of both undissociated and dissociated ILs in two phases, most log P values of ILs (Table 2) should be strictly called log D, where D is the distribution coefficient, the ratio of the total concentrations of all forms of IL (ionized and un-ionized) between two phases. Alternatively, the intrinsic partition coefficients of ILs should be calculated from the apparent partition coefficients (D).²¹²

Table 2.

log P (or log K_OW at low concentrations ^a) values of ILs at 25 °C

Solvent		log P/log KOW	Reference
1	dichloromethane	1.25	selected value by Ref 213
2	THF	0.46	selected value by Ref 213
3	t-butanol	0.35	selected value by Ref 213
4	acetone	−0.24	selected value by Ref 213
5	acetonitrile	−0.34	selected value by Ref 213
6	[EMIM][Tf2N]	−1.18	214
		log KOW (−1.05 to −0.96) (0.28−2.8 mM)	calculated from Ref 215
7	[BMIM][Tf2N]	0.11	166
		log KOW (−0.96 to −0.21) (0.15−2.2 mM)	calculated from Ref 215
		0.33	216
		−1.74	212
8	[HMIM][Tf2N]	0.64	166
		log KOW (0.15 to 0.22) (0.32−0.38 mM)	calculated from Ref 215
		0.65	216
9	[OMIM][Tf2N]	0.79	214
		log KOW (0.80−1.05) (0.099−0.21 mM)	calculated from Ref 215
10	[EMMIM][Tf2N]	log KOW (−1.15 to −0.92) (0.32−2.9 mM)	calculated from Ref 215
11	[PMMIM][Tf2N]	log KOW (−0.92 to −0.62) (1.4−2.8 mM)	calculated from Ref 215
12	[HMMIM][Tf2N]	log KOW (0.13 to 0.25) (0.36−0.49 mM)	calculated from Ref 215
13	[BMIM][PF6]	−1.66	calculated from Ref 215
		−2.39	162, 216
		−2.38	172, 212
		−2.06	214
		−2.35	165
14	[HMIM][PF6]	−1.86	216
15	[OMIM][PF6]	−0.35	214
		−1.33	216
16	[ONIM][PF6]	−2.19	172
17	[BMIM]Cl	−2.40	calculated from Ref 215
18	[BMIM]Br	−2.48	calculated from Ref 215
19	[EMIM][OAc]	−2.53	166
20	[BMIM][OAc]	−2.77	162
21	[EMIM][CF3COO]	−2.75	166
22	[HMIM][ CF3COO]	−2.30	166
23	[BMIM][NO3]	−2.90	162
		−2.42	calculated from Ref 215
24	[BMIM][dca]	−2.32	166
25	[EMIM][BF4]	−2.57	166
26	[BMIM][BF4]	−2.51	166
		−2.44	165, 172
		−2.52	calculated from Ref 215
27	[OMIM][BF4]	−1.34	166
		−1.14	214
28	[EtPy][ CF3COO]	−2.57	166
29	[EtPy][Tf2N]	−0.90	166
30	[BuPy][Tf2N]	−0.26	166
31	[Cholinium][Tf2N]	log KOW = −0.57 (calculated value)	217b

Note:

^alog K_OW values calculated from Ref²¹⁵ were converted from initial values of K_OW measured at room temperature (24 ± 2 °C), and the concentration range given for each log K_OW was the IL concentration range in water phase;

^bThis reference also provides K_OW values for a number of pyridinium and imidazolium ILs based on Tf₂N⁻ and B(CN)₄⁻.

From a practical point of view, the log P values (or log K_OW at low concentrations) of ILs in Table 2 are valuable for comparing the hydrophobicity of ILs with conventional organic solvents. In general, ILs are very hydrophilic in nature based on the negative log P values (or log K_OW) for most ILs (including water-immiscible Tf₂N⁻ and PF₆⁻ ones); however, by convention, we usually refer those ILs that are poorly miscible with water (e.g. Tf₂N⁻ and PF₆⁻ types) as hydrophobic ILs. The discrepancy between different measurements of the same ILs might be caused by different initial concentrations of ILs (as high concentrations leading to higher K_OW values^{212, 215}), and different experimental techniques.

The Russell group¹⁶² measured the log P values (< −2.0) of several ILs, and suggested that they are very hydrophilic in nature based on the Laane’s scale;^218–220 they also observed that free lipase (Candida rugosa) was only active in hydrophobic [BMIM][PF₆] (log P = −2.39), but inactive in other hydrophilic ILs including [BMIM][CH₃COO] (log P = −2.77), [BMIM][NO₃] (log P = −2.90) and [BMIM][CF₃COO].¹⁶² Similarly, Nara et al²²¹ achieved higher transesterification activities of lipases in [BMIM][PF₆] than in [BMIM][BF₄]. The Goto group also reported higher activities of PEG-modified lipase²²² and subtilisin²²³ in more hydrophobic ILs such as [EMIM][Tf₂N]. Zhang et al²²⁴ reported low penicillin acylase stabilities in [BMIM][BF₄] and [BMIM][dca]. Lou and Zong¹⁶⁵ studied the enantioselective acylation of (R,S)-1-trimethylsilylethanol with vinyl acetate catalyzed by lipases in several ILs, and indicated the activity, enantioselectivity and thermostability of Novozym^® 435 increasing with the IL hydrophobicity ([BMIM][PF₆] > [OMIM][BF₄] > [C₇MIM][BF₄] > [HMIM][BF₄] > [C₅MIM][BF₄] > [BMIM][BF₄]). Paljevac et al²²⁵ reported that the cellulase activity decreased in the order of IL hydrophobicity: [BMIM][PF₆] > [BMIM][BF₄] > [BMIM]Cl. The Víllora group²²⁶ observed a lower stability of penicillin G acylase in [BMIM][BF₄] than in hydrophobic ILs (Tf₂N⁻ and PF₆⁻), particularly in the absence of substrate. A recent study²²⁷ on the alcoholysis of vinyl butyrate and 1-butanol by free CALB suggested that the lipase activities were generally much lower in water-miscible ILs (such as BF₄⁻, dca⁻, NO₃⁻ and OAc⁻, etc.) than in water-immiscible ones (PF₆⁻ and Tf₂N⁻), and the enzyme’s activities increased with the cation’s hydrophobicity (EMIM⁺ < BMIM⁺ < HMIM⁺ < OMIM⁺). Ha et al²²⁸ also found Novozym^® 435 was less active and stable in hydrophilic ILs (BF₄⁻ and OTf⁻) than in other hydrophobic ILs (Tf₂N⁻ and PF₆⁻). Lee et al¹⁹⁶ reported that Novozym^® 435 was more thermally stable in hydrophobic ILs than in hydrophilic ones following the order of [BMIM][Tf₂N] > [BMIM][PF₆] > [BMIM][OTf] > [BMIM][BF₄] > [BMIM][SbF₆]. Shen et al²²⁹ noticed that during the kinetic resolution of racemic cyanohydrins, Amano lipase PS showed a high enantioselectivity (80% ee_p) in hydrophobic [OMIM][PF₆], but poor enantioselectivities (< 5% ee_p) in hydrophilic [HMIM][BF₄] and [HMIM]Cl. Hernández-Fernández et al¹⁹⁴ concluded that both free CALB and penicillin G acylase (PGA) were more stable in hydrophobic ILs than in hydrophilic ones: in the case of CALB, the stability was in a decreasing order of [HMIM][PF₆] > [HMIM][Tf₂N] > [HMIM][BF₄], and [BMIM][PF₆] > [BMIM][dca], as well as [OMIM][PF₆] > [HMIM][PF₆] > [BMIM][PF₆]; in the case of PGA, the stability was in a decreasing order of [BMIM][Tf₂N] > [BMIM][PF₆] > [BMIM][BF₄]. However, the hydrophobic cations showed an adverse effect on the PGA stability: [EMIM][Tf₂N] > [BMIM][Tf₂N], and [BMIM][PF₆] > [OMIM][PF₆]. The effect of nucleophilicity of these anions has been discussed previously. These examples implied that the high hydrophobicity (large log P) of ILs could be beneficial to the enzyme stabilization.

Through a systematic investigation of Novozym^® 435-catalyzed transesterification in over 20 ILs, our group¹⁶⁶ observed that the lipase activity increased with the log P value of ILs to a maximum, and then declined with a further increase in log P (a bell shape). Our previous discussion implied that the enzyme is active in hydrophobic solvents (with a high log P). However, a higher log P of the solvent also means a more thermodynamic ground-state stabilization of substrates,²³⁰ which might reduce the conversion of substrates. This could explain the decreasing reaction rate in very hydrophobic ILs. Similarly, Lou et al¹⁶⁴ found the initial rates of Novozym^® 435-catalyzed ammonolysis of (R,S)-p-hydroxyphenylglycine methyl ester increased with the hydrophobicity of BF₄⁻ based ILs to a maximum (C₃MIM⁺ < C₄MIM⁺ < C₅MIM⁺ < C₆MIM⁺), and then decreased with a further increase in the IL hydrophobicity (C₆MIM⁺ > C₇MIM⁺ > C₈MIM⁺).

As discussed previously, the stabilization of substrates could be one reason. But the possibility of hydrophobic interactions between large IL molecules and the enzyme cannot be fully excluded. For example, the Atkin group²³¹ investigated the stability and activity of hen’s egg white lysozyme in aqueous solutions of four protic ILs (25–75 wt%); the protein denaturing-renaturing CD experiments and the activity measurements of lysozyme indicated that the highest catalytic activity and most complete refolding was achieved in solutions of [(EtOH)NH₃][HCOO], followed by [PrNH₃][HCOO], and then [EtNH₃][HCOO] and [(MeOEt)NH₃][HCOO]. It is believed that the protein-IL interactions include the electrostatic interaction of IL cations with negatively charged residues in the protein, H-bonds between amine protons and the protein, as well as the hydrophobic interactions between alkyl chains in ILs and hydrophobic regions of the protein. Since electrostatic interactions between [(EtOH)NH₃]⁺ and lysozyme is about the same as for [EtNH₃]⁺, the hydroxyl group in [(EtOH)NH₃]⁺ probably reduces the strength of hydrophobic interactions with the protein. Another possibility is that the hydroxyl group interacts with the anion formate via H-bonds, reducing the interaction of formate with the protein. The IL viscosity-induced mass transport was not a limiting factor in the study because [(EtOH)NH₃][HCOO] is several times more viscous that other three ILs. In summary, the hydrophobicity factor of ILs is a combination effect of anion’s H-bond basicity and cation’s hydrophobic effect.

Klähn et al.^{232, 233} carried out MD simulations of CALB in imidazolium or guanidinium–based ILs containing anions of NO₃⁻, BF₄⁻ or PF₆⁻. They confirmed that the CALB stability is mainly influenced by anions and follows a decreasing order of PF₆⁻ > BF₄⁻ ≫ NO₃⁻, and long decyl side chains, polar methoxy groups and guanidinium-based cations induce more CALB destabilization than short methyl groups, other non-polar groups and imidazolium-based cations. Two destabilization mechanisms are identified: (a) Destabilization of protein surface by Coulomb interactions with anions carrying a localized charge and strong polarization, or with polar cations. This type of destabilization shows a roughening of the protein surface, loss of compactness, and unraveling of α-helices. Smaller anions and a high anion surface charge lead to stronger Coulomb interactions. (b) Destabilization of protein core by direct hydrophobic interactions of protein core with long alkyl chains or hydrophobic ILs, which leads to a disintegration of β-sheets, diffusion of ions into CAL-B and increasing protein–IL van der Waals interactions. Due to van der Waals interactions with the aliphatic residues at the active site entrance, the butyl group of [BMIM]⁺ cations can easily diffuse into the active site of CALB; this could affect the binding between substrate molecules and active sites.

Other factors

Since hydrophobicity is not the only factor in controlling the hydrolase activity, complications arose in interpreting some biocatalytic reactions. De Diego et al²³⁴ conducted the transesterification of vinyl propionate and 1-butanol catalyzed by free and immobilized lipases from Candida antarctica (CALA and CALB), Thermomyces lanuginosus (TLL) and Rhizomuncor miehei (RML). Most of the enzyme preparations (except free CALA) showed higher activities in more hydrophobic [OMIM][PF₆] than in [BMIM][PF₆], but lower activities in other more hydrophobic based ILs ([OMIM][BF₄] < [HMIM][BF₄] < [BMIM][BF₄], and [BDMIM][PF₆] < [BDMIM][BF₄]). Another study by Irimescu and Kato¹⁹⁵ on the lipase-catalyzed acylation of primary amines indicated lower reaction rates in ILs with longer alkyl chains in cations, and the water miscibility of ILs was not a main factor in influencing the reaction rate. Some studies also obtained relatively high enzyme activities in hydrophilic ILs (such as [BMIM][BF₄], [EMIM][BF₄], [BMIM][OTf] and [MMIM][MeSO₄]).^{163, 235–239} The Bruce group²⁴⁰ evaluated the activities of proteases (chymotrypsin and subtilisin) dissolved in several protic hydroxylalkylammonium-based ILs (containing ~1–2 wt% water), and found that subtilisin was only active in diethanolammonium chloride and chymotrypsin was inactive in these protic ILs. They further indicated that subtilisin retained its secondary and tertiary structures in diethanolammonium chloride as confirmed by the far and near UV CD spectra. Therefore, multiple factors must be considered when explaining the enzymatic systems like these.⁶

Amyloid fibrilization represents a process where the peptide assembles from monomers to oligomers and then into fibrils; this process is associated with the protein destabilization since the development of amyloid fibrils results from the formation of intramolecular H-bonds. The

Byrne group¹⁵⁹ determined the rates of amyloid fibrilization of Aβ16–22 peptide in 90% (v/v) protic triethylammonium-based ILs, and found these rates decrease with IL anions in the order of HSO₄⁻, H₂PO₄⁻ > CF₃COO⁻ > lactate⁻ > OTf⁻ > CH₃SO₃⁻. The reverse of this series is the protein stabilization order, which is roughly a reverse Hofmeister series. The competitive H-bonding between the anion and water contributes to the self-assembly of Aβ16–22 peptide into amyloid fibrils; kosmotropic anions leads to faster amyloid fibrilization (“salt-in”) while chaotropic anions such as mesylate) suppress the formation of amyloid fibrilization (“salt-out”).

ION SPECIFICITY-GUIDED BIOCATALYTIC APPLICATIONS

The empirical ion specificity rules at different concentrations of ILs could provide some general guidance for designing/selecting enzyme-compatible ionic solvents. [EMIM][OAc] contains a chaotropic cation and kosmotropic anion, a unique combination for enzyme stabilization when it is used at low concentrations.^{43, 49, 119} Our group¹²⁴ carried out the enzymatic chiral hydrolysis of amino acid esters catalyzed by Bacillus licheniformis protease in [EMIM][OAc] solutions, and obtained high enantioselectivities in up to 4.0 M [EMIM][OAc] despite lower yields of L-amino acid beyond 2.0 M IL concentrations. In addition, Wang et al.²⁴¹ found that aqueous solutions of [BMIM][OAc] are highly compatible with cellulases. At first, they observed a high stability of a mixture of cellulases and β-glucosidase in [EMIM][OAc] solutions; after incubated in 15% and 20% (w/v) [EMIM][OAc] aqueous solutions at 50°C for 3 h, the enzyme mixture still retained 77% and 65% of its original activity respectively. In addition, the cellulase mixture exhibited a high activity in 15% [EMIM][OAc], leading to 91% conversion of Avicel^® cellulose and up to 54% conversion of yellow poplar biomass into reducing sugars.

Bekhouche et al.²⁴² suggested that [MMIM][Me₂PO₄] consists of a chaotropic cation and a kosmotropic anion, and found that formate dehydrogenase (FDH) from Candida boidinii maintained 76% of its activity in 20% (v/v) [MMIM][Me₂PO₄] (vs 100% activity in carbonate buffer). This group also indicated that FDH grafted with ILs (e.g. [Cholinium]Cl, [HO-EMIM]Cl and [HO-PrMIM]Cl) via covalent coupling exhibited a more tolerance to ionic media (such as in 70% (v/v) [MMIM][Me₂PO₄], the modified enzymes retained ca. 30–45% of their activity in aqueous buffer). They also concluded that more chaotropic grafted cation (such as cholinium) leads to a higher stabilizing effect on the enzyme in aqueous media. Thomas et al.¹⁵⁶ found that xylanase and the arabinofurosidases maintained high or even enhanced hydrolytic activities in up to 20% (v/v) aqueous solutions of [MMIM][Me₂PO₄], [EMIM][Me₂PO₄] and [EMIM][OAc]. Yamamoto et al.²⁴³ evaluated aqueous solutions of N-alkylpyridinium chlorides and N-alkyl-N-methylpyrrolidinium chlorides for the refolding of denatured lysozyme, and found that less hydrophobic and chaotropic ILs (i.e. N-ethyl, N-butyl and N-hexylpyridinium chlorides, and N-butyl-N-methylpyrrolidinium chloride) could suppress protein aggregation and thus are effective for refolding the protein (46–69% yields). On the other hand, although more hydrophobic ILs such as N-octylpyridinium chloride and N-dodecylpyridinium chloride could fully prevent aggregation at lower concentrations, these salts interact directly with the protein via hydrophobic interactions and are not effective in improving refolding yields.

Hydroxyl- or ether-functionalized cations tend to be less hydrophobic and more chaotropic; as a result, such a modification often leads to more enzyme-compatible ILs. As shown in Fig 9, each of the Ammoeng family ILs is an ionic mixture containing multiple alkyloxy groups, which have both hydrophilic and hydrophobic properties like polyethylene glycols (PEGs). The Xu group^244–249 judiciously selected a group of commercial tetraammonium-based ILs as reaction media for the enzymatic glycerolysis. In particular, Ammoeng 100 (also known as [CPMA][MeSO₄]^‡) and 102 are capable of dissolving triglycerides and have shown to be lipase-compatible during the glycerolysis reaction;^{245, 246} trioctylmethylammonium bis(trifluoromethylsulfonyl)imide ([TOMA][Tf₂N]) and its mixture with Ammoeng 102 have also been evaluated as suitable solvents for the enzymatic glycerolysis.^248–250 De Diego et al²³⁴ have further confirmed higher transesterification activities of both free and immobilized CALB in [CPMA][MeSO₄] than in several PF₆⁻ and BF₄⁻ based ILs; however, the other two lipases from Thermomyces lanuginosus (TLL) and Rhizomuncor miehei (RML) seemed less active in [CPMA][MeSO₄] than in PF₆⁻ and BF₄⁻ based ILs. Xu and co-workers^{245, 247} utilized the Conductor-like Screening Model for Real Solvents (COSMS-RS) to derive various parameters (such as misfit, H-bonding and van der Waals interaction energy) to understand the multiple interactions in ILs; the model also provides guidance in designing the structures of cations and anions.²⁵¹ Similarly, the Kroutil group¹⁵³ found that alcohol dehydrogenase is more active in hydroxyl-functionalized ILs than ordinary ILs, even at 50–90% (v/v) IL concentrations; the enzyme activity decreased in the order of [(HO-Et)₃MeN][MeSO₄] > Ammoeng 101 > Ammoeng 100 > Ammoeng 102. The Kragl group²⁵² found an IL in the Ammoeng family - Ammoeng 110 (Fig 9d) –is quite effective in forming aqueous two-phase (ATP) for the purification of active enzymes (two different alcohol dehydrogenases); the IL is capable of stabilizing the enzymes and enhancing the solubility of hydrophobic substrates. It is interesting to mention that oxygen-containing ILs (such as Ammoeng series, and [C₂OHmim]Cl) were used as additives in the enantioselective hydrolysis of diester malonates by pig liver esterase (PLE), and less than 1% of these ILs and 10% isopropanol/water were sufficient to improve the activity of PLE (up to four times) as well as the enantioselectivity.²⁵³

Fig. 9.

Structures of tetraammonium-based ILs (Ammoeng^TM series).

Based on the lyoprotectant effect of tris(hydroxymethyl)aminomethane (Tris) as excipient in horseradish peroxidase lyophilization,²⁵⁴ Das et al²⁵⁵ mimicked the structure of Tris and rationally designed a new IL known as tetrakis(2-hydroxyethyl)ammonium triflouromethanesulfonate (Fig 10); they reported that horseradish peroxidase in this new IL was 10 times more active than in methanol and at least 30–240-fold more active than in conventional ILs. Abe et al²⁵⁶ synthesized an alkyloxy-containing hydrophobic IL named 2-methoxyethyl(tri-n-butyl)phosphonium bis(trifluoromethane)sulfonamide ([MeOCH₂CH₂-Bu₃P][Tf₂N]), and observed a faster reaction rate (lipase PS-catalyzed transesterification of secondary alcohols) in this IL than in diisopropyl ether. Vafiadi et al²⁵⁷ employed two functionalized ILs [C₂OHmim][PF₆] and [C₅O₂mim][PF₆] as solvents for the feruloyl esterase-catalyzed esterification of glycerol with sinapic acid, and achieved high conversion yields (72.5% and 76.7% respectively in two ILs under optimal conditions). These two ILs are considered as amphiphilic (hydrophilic cation and hydrophobic anion), and have relatively low viscosities.

Fig. 10.

Structure of tetrakis(2-hydroxyethyl)ammonium triflouromethanesulfonate.

Li et al.²⁵⁸ covalently attached ether-functionalized ILs (containing carboxylic acid group) to Candida rugosa lipase (CRL) by using the coupling reagent N,N′-carbodiimide (see Fig 11). The modified lipase showed improved catalytic activity, thermostability, organic solvent tolerance, and adaptability to temperature and pH changes in olive oil hydrolysis reaction. In particular, a higher CRL activity is associated with more kosmotropic anions of ILs (H₂PO₄⁻ > Cl⁻ > BF₄⁻), and the use of a small glycol molecule (PEG 350 vs PEG 750) leads to a more active enzyme. The CD spectra suggest that the chemical modification by ILs resulted in an increase in β-sheet and a decrease in α-helix content of secondary structures of CRL.

Fig. 11.

Candida rugosa lipase (CRL) modified by covalent linkage to a glycol-functionalized IL (Reproduced with permission from Ref.²⁵⁸ Copyright (2015) American Chemical Society).

SUMMARY

Molecular level structures of ILs and their solutions are controlled by complex interactions of electrostatic attraction, H-bonds and dispersion forces depending on the concentration of ILs. Clearly, there is a need for more experimental and simulation studies to further visualize the microstructures of ILs and IL solutions. The interactions between proteins and IL solutions depend on some microscopic properties such as ion hydration, ion effect on protein hydration, and direct interactions between ions and proteins, and could be influenced by some macroscopic parameters such as viscosity B-coefficients of ions, H-bond basicity, and hydrophobicity. In diluted aqueous IL solutions, the ion specificity of many enzymatic systems is in line with the traditional Hofmeister series/kosmotropicity despite a number of exceptions, however, the specificity in concentrated or neat ILs is determined by H-bond basicity and nucelophilicity of anions, IL hydrophobicity and other factors. Due to the complex nature of many enzymatic systems, the specific ion effect may provide some empirical guidelines but not universal rules. Hopefully, these simple guidelines could lead to more custom design of enzyme-compatible ILs and biocatalytic systems.

NOTATION

IL Cations

EMIM⁺

1-ethyl-3-methylimidazolium

BMIM⁺

1-butyl-3-methylimidazolium

BIM⁺

1-butylimidazolium

BDMIM⁺

1-butyl-2,3-dimethylimidazolium

PMIM⁺

1-methyl-3-propylimidazolium

HMIM⁺

1-hexyl-3-methylimidazolium

OMIM⁺

1-octyl-3-methylimidazolium

ONIM⁺

1-nonyl-3-octylimidazolium

C₁₈MIM⁺

1-methyl-3-octadecylimidazolium

BMPip⁺

1-butyl-1-methylpiperidinium

BMPyrr⁺

1-butyl-1-methylpyrrolidinium

EtPy⁺

1-ethylpyridinium

BuPy⁺

1-butylpyridinium

Me₃NPr⁺

N,N,N-trimethyl-N-propylammonium

IL Anions

BF₄⁻

tetrafluoroborate

PF₆⁻

hexafluorophosphate

OAc⁻

acetate

Tf₂N⁻

bis(trifluoromethane)sulfonamide, (CF₃SO₂)₂N⁻

beti–

bis(perfluoroethylsulfonyl)imide, (C₂F₅SO₂)₂N⁻

OTf⁻

triflate (i.e. trifluoromethanesulfonate)

dca⁻

dicyanamide

MeSO₄⁻

methyl sulfate

EtSO₄⁻

ethyl sulfate

OTs⁻

tosylate