Enzymatic resolution of (R, S)-Naproxen in water-saturated ionic liquid

A water-saturated ionic liquid has been exploited for resolution of (R, S)-Naproxen by lipase-catalyzed hydrolysis to enhance the conversion and facilitate product recovery. From the enantioselectivity and activity of lipase, water-saturated [bmim]PF₆ (1-butyl-3-methylimidazolium hexafluorophosphate) was selected as the best reaction medium. To prevent the dissolution of lipase in the ionic liquid, a weakly polar, amorphous multiporous silica YWG-C₆H₅ was used as a support for immobilization. The production of (S)-Naproxen was initially performed in a batch reactor containing 20 mL of substrate solution. After 72 h reaction, 98.2% enantiomeric excess of the (S)-Naproxen was obtained with 28.3% hydrolysis conversion. The unconventional solvent properties of ionic liquids have been exploited in reaction medium recycling, product recovery and water recruiting schemes. In a repetitive batch reaction system, the immobilized lipase could be repeatedly used for 5 times with only a slight reduction in reaction conversion.     

Investigation of ionic liquids as reaction media for enzymatic enantioselective acylation of amines

The use of ionic liquids as reaction media for lipase-catalyzed enantioselective acylation of 1-phenylethylamine (1) and 2-phenyl-1-propylamine (2) with 4-pentenoic acid was investigated. The best performing ionic liquid for each of these amines as well as its solvent properties were very different. Preparative scale kinetic resolution of 1 was performed efficiently in 1-butyl-2,3-dimethylimidazolium trifluoromethanesulphonate.     

The bioreduction of a β-tetralone to its corresponding alcohol by the yeast Trichosporon capitatum MY1890 and bacterium Rhodococcus erythropolis MA7213 in a range of ionic liquids

The stereospecific reduction of 6-Br-β-tetralone to its corresponding alcohol (S)-6-Br-β-tetralol was carried out by the yeast Trichosporon capitatum MY1890 and by the bacterium Rhodococcus erythropolis MA7213, using a range of ionic liquids chosen for the diversity of their composition. The decrease in cell viability of both types of cell upon exposure to ionic liquids was found to be between that determined for cells residing purely in fermentation media, and cells residing in a two-phase mixture of media and organic solvent (toluene). For T. capitatum MY1890 bioconversions, the water miscible hydrophilic ionic liquid [Emim][TOS] gave a reaction profile comparable to that observed in the previously studied water-ethanol (10% v/v) system, in terms of overall rate of reaction (0.2 g (prod) L^-1 h^-1) and conversion (100%). Of the hydrophobic ionic liquids evaluated, [Oc₃MeN][BTA] gave the best conversion of 60%, but at a much reduced rate, suggesting solute mass transfer from the ionic liquid phase was rate limiting. For bioconversions carried out with R. erythropolis MA7213 employing 20% v/v [Emim][TOS] as a co-solvent, the conversion yield doubled, and a four-fold increase in initial rate was found compared to the standard ethanol co-solvent. This was attributed to improved cell viability and reduced aggregation of the R. erythropolis MA7213 compared to T. capitatum MY1890. Overall, this study demonstrates the feasibility of using ionic liquids for whole cell biocatalysis, however, no obvious link is apparent between the physico-chemical properties of ionic liquids, their influence on cell viability, and their efficacy as media for bioconversions.     

Biocatalytic transformations in ionic liquids

Room temperature ionic liquids are non-volatile, thermally stable and highly polar; they are also moderately hydrophilic solvents. Here, we discuss their use as reaction media for biocatalysis. Enzymes of widely diverging types are catalytically active in ionic liquids or aqueous biphasic ionic liquid systems. Lipases, in particular, maintain their activity in anhydrous ionic liquid media; the (enantio)selectivity and operational stability are often better than in traditional media. The unconventional solvent properties of ionic liquids have been exploited in biocatalyst recycling and product recovery schemes that are not feasible with traditional solvent systems.     

Toward advanced ionic liquids. Polar,enzyme-friendly solvents for biocatalysis

Ionic liquids, also called molten salts, are mixtures of cations and anions that melt below 100°C. Typical ionic liquids are dialkylimidazolium cations with weakly coordinating anions such as (MeOSO₃) or (PF₆). Advanced ionic liquids such as choline citrate have biodegradable, less expensive, and less toxic anions and cations. Deep eutectic solvents are also included in the advanced ionic liquids. Deep eutectic solvents are mixtures of salts such as choline chloride and uncharged hydrogen bond donors such as urea, oxalic acid, or glycerol. For example, a mixture of choline chloride and urea in 1:2 molar ratio liquefies to form a deep eutectic solvent. Their properties are similar to those of ionic liquids. Water-miscible ionic liquids as cosolvents with water enhance the solubility of substrates or products. Although traditional water-miscible organic solvents also enhance solubility, they often inactivate enzymes, while ionic liquids do not. The enhanced solubility of substrates can increase the rate of reaction and often increases the regioor enantioselectivity. Ionic liquids can also be solvents for non-aqueous reactions. In these cases, they are especially suited to dissolve polar substrates. Polar organic solvent alternatives inactivate enzymes, but ionic liquids do not even when they have similar polarities. Besides their solubility properties, ionic liquids and deep eutectic solvents may be greener than organic solvents because ionic liquids are nonvolatile, and can be made from nontoxic components. This review covers selected examples of enzyme catalyzed reaction in ionic liquids that demonstrate their advantages and unique properties, and point out opportunities for new applications. Most examples involve hydrolases, but oxidoreductases and even whole cell reactions have been reported in ionic liquids.     

Synthesis of benzylidenated hexopyranosides in ionic liquids

The synthesis of 4,6-O-benzylidenated monosaccharides and disaccharides has been studied using ionic liquids as a unique solvent alternative. An examination of several imidazolium ionic liquids indicates that the benzylidenation of hexopyranosides in 3-butyl-1-methylimidazolium tetrafluoroborate, [bmim]BF₄, gives the highest yields for most of the substrates, and that this solvent system could be readily recycled.     

Recent advances of enzymatic reactions in ionic liquids

The tremendous potential of room temperature ionic liquids as an alternative to environmentally harmful ordinary organic solvents is well recognized. Ionic liquids, having no measurable vapor pressure, are an interesting class of tunable and designer solvents, and they have been used extensively in a wide range of applications including enzymatic biotransformation. In fact, ionic liquids can be designed with different cation and anion combinations, which allow the possibility of tailoring reaction solvents with specific desired properties, and these unconventional solvent properties of ionic liquids provide the opportunity to carry out many important biocatalytic reactions that are impossible in traditional solvents. As compared to those observed in conventional organic solvents, the use of enzymes in ionic liquids has presented many advantages such as high conversion rates, high enantioselectivity, better enzyme stability, as well as better recoverability and recyclability. To date, a wide range of pronounced approaches have been taken to further improve the performance of enzymes in ionic liquids. This review presents the recent technological developments in which the advantages of ionic liquids as a medium for enzymes have been gradually realized.     

Thermal stability enhancement of Candida rugosa lipase using ionic liquids

The thermal stability of Candida rugosa (C. rugosa) lipase was investigated and compared in n-hexane, benzene, dibutyl-ether as well as [bmim]PF₆ and [omim]PF₆ ionic liquids and the effect of solvent polarity and water activity were evaluated. Deactivation of the enzyme followed a series-type kinetic model. First order deactivation rate constants and the ratios of specific activities were determined and the kinetics of deactivation were studied. Among the organic solvents, the best stability was observed in n-hexane with a half-life of 6.5 h at water activity of 0.51. In ionic liquids, however, even longer half lives were obtained, and the enzyme was stable in these solvents at 50°C. The highest half-life times were obtained in [bmim]PF₆ (12.3 h) and [omim]PF₆ (10.6 h). A direct correlation was found between solvent polarity and thermal stability since the higher the polarity of the solvent, the lower was the stability decrease at 50°C comparing to that at 30°C.     

Optimization of lipase-catalyzed glucose fatty acid ester synthesis in a two-phase system containing ionic liquids and t-BuOH

Selective lipase-catalyzed synthesis of glucose fatty acid esters in two-phase systems consisting of an ionic liquid (1-butyl-3-methyl imidazolium tetrafluoroborate [BMIM][BF₄] or 1-butyl-3-methyl imidazolium hexafluorophosphate [BMIM][PF₆]) and t-butanol as organic solvent was investigated. The best enzyme was commercially available lipase B from Candida antarctica (CAL-B), but also lipase from Thermomyces lanuginosa (TLL) gave good conversion. After thorough optimization of several reaction conditions (chain-length and type of acyl donor, temperature, reaction time, percentage of co-solvent) conversions up to 60% could be achieved using fatty acid vinyl ester as acyl donors in [BMIM][PF₆] in the presence of 40% t-BuOH with CAL-B at 60 °C.     

Enzymatic selective acylation of glycosides in ionic liquids: significantly enhanced reactivity and regioselectivity

The enzymatic selective acylations of carbohydrates in ionic liquids were explored in both organic solvents and ionic liquids to see any significant differences in terms of reactivity and regioselectivity between two different classes of reaction media. Monoprotected glycosides (methyl-6-O-trityl-glucosides and galactosides) were chosen as the substrates with Candida rugosa lipase as an acylation enzyme. Two organic solvents, THF and chloroform, and two ionic liquids, [BMIM]⁺PF₆⁻ ([BMIM]⁺ = 1-butyl-3-methylimidazolium) and [MOEMIM]⁺PF₆⁻ ([MOEMIM]⁺ = 1-methoxyethyl-3-methylimidazolium), were employed as reaction media. The enzymatic reactions were performed in the presence of vinyl acetate at room temperature. It was observed that the reactions in ionic liquids took place more rapidly and more selectively than those in conventional organic solvents.     

Lipase-catalyzed syntheses of sugar esters in non-aqueous media

The lipase-catalyzed reaction is useful to obtain sugar esters with chemically defined structures and will contribute to the synthesis of sugar-based compounds by a chemo-enzymatic pathway. The synthesis of sugar esters in nonaqueous media has been attempted for a quarter century. To facilitate the reactions, they have been performed either in an organic solvent with/without a polar adjuvant or in an ionic liquid, or by using a hydrophobic sugar derivative. In this review, the following points are discussed: (1) various synthetic methods of sugar esters; (2) role of the solvents or adjuvants; and (3) improvement in the productivity.     

Lipase-Catalyzed Synthesis and Hydrolysis of Cholesterol Oleate in Aot/Isooctane Microemulsions

We have examined a lipase-catalyzed bidirectional ester synthesis/hydrolysis reaction in a water-in-oil microemulsion system. The reactants were cholesterol (alcohol), oleic acid (acid) and cholesterol oleate (ester), and the solvent system consisted of sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/isooctane/water. The reactions were assayed by using [³H]oleic acid, [³H]cholesterol, or [³H]cholesterol oleate for the synthesis and hydrolysis reactions, respectively (separate incubations). The lipase that we used derived from Candida cylindracea, and was used at a concentration of 0.1mg/ml microemulsion. The reactions were performed at 22°C as the reactions proceeded more slowly at higher temperatures. With the initial reactant concentrations set to 10 mM cholesterol, 1 min oleic acid, and 1 mM cholesterol oleate, it was observed that the optimal [H₂O]/[AOT] ratio was at about 9 both for the esterification reaction and for the hydrolysis reaction (after 24 h). The hydrolysis reaction was slower than the synthesis reaction at all [H₂O]/[AOT] ratios studied (0-20), but the difference in reaction yield for the synthesis and the hydrolysis reactions became smaller as the reaction time increased (up to 11 days). When the reaction yield was followed as a time function, it was observed that about 80% of the oleic acid was esterified within 3 days of reaction ([H₂O]/[AOT] ratio of 6), whereas the corresponding value of 80% hydrolysis of cholesterol oleate was reached within 11 days. The results of the present study indicate that by choosing optimal reactant concentrations and reaction conditions, it is at least in part possible to determine the direction of the lipase-catalyzed synthesis/hydrolysis reaction.     

Kinetic resolution of ibuprofen catalyzed by Candida rugosa lipase in ionic liquids

Candida rugosa lipase-catalyzed esterification of ibuprofen with 1-propanol was conducted in seven ionic liquids and the results were compared with those in isooctane. Although the enzyme showed comparable or higher activity in some ionic liquids compared to that in isooctane, only in the case of [BMIM]PF6 was the enantioselectivity (E = 24.1) almost twice that (E = 13.0) of isooctane. In another six ionic liquids the enzyme enantioselectivity was much poorer (E = 1.1-6.4). At the same conversion of 30%, E of [BMIM]PF6 is more than triple that of isooctane. The lipase stability in [BMIM]PF6 was improved by 25% of that in isooctane. It was concluded that [BMIM]PF6 could be applied to substitute the conventional organic solvent (isooctane) in the esterification of ibuprofen.     

Lipase-catalyzed synthesis of phenolic lipids from fish liver oil and dihydrocaffeic acid

The enzymatic synthesis of phenolic lipids by lipase-catalyzed transesterification of dihydrocaffeic acid (DHCA) with fish liver oil was investigated in a selected organic solvent medium. These synthesized phenolic lipids have potential use as nutraceutical products. Using a molar ratio of 1:8 DHCA to fish liver oil in hexane:2-butanone mixtures of 75:25 and 85:15 (v/v), the lipase-catalyzed reaction resulted in maximum conversion of 55.8 and 65.4%, respectively. The maximum conversion of phenolic monoacylglycerols in hexane:2-butanone mixture of 75:25 and 85:15 (v/v) was 40.3 and 37.7%, respectively; using the same solvent mixtures, the conversions of the phenolic diacylglycerol were 15.8 and 36.8%, respectively. Hexane:2-butanone mixture of 75:25 (v/v) was, therefore, the best organic solvent mixture for the production of phenolic monoacylglycerols, while that of 85:15 (v/v) was best for the production of phenolic diacylglycerols. The phenolic lipids produced from the fish liver oil and DHCA demonstrated antioxidant property as indicated by its free radical scavenging capacity.     

Lipase-catalyzed synthesis of phenolic lipids from fish liver oil and dihydrocaffeic acid

The enzymatic synthesis of phenolic lipids by lipase-catalyzed transesterification of dihydrocaffeic acid (DHCA) with fish liver oil was investigated in a selected organic solvent medium. These synthesized phenolic lipids have potential use as nutraceutical products. Using a molar ratio of 1:8 DHCA to fish liver oil in hexane:2-butanone mixtures of 75:25 and 85:15 (v/v), the lipase-catalyzed reaction resulted in maximum conversion of 55.8 and 65.4%, respectively. The maximum conversion of phenolic monoacylglycerols in hexane:2-butanone mixture of 75:25 and 85:15 (v/v) was 40.3 and 37.7%, respectively; using the same solvent mixtures, the conversions of the phenolic diacylglycerol were 15.8 and 36.8%, respectively. Hexane:2-butanone mixture of 75:25 (v/v) was, therefore, the best organic solvent mixture for the production of phenolic monoacylglycerols, while that of 85:15 (v/v) was best for the production of phenolic diacylglycerols. The phenolic lipids produced from the fish liver oil and DHCA demonstrated antioxidant property as indicated by its free radical scavenging capacity.     

Biocatalytic synthesis of ascorbyl esters and their biotechnological applications

Ascorbyl fatty acid esters act both as antioxidants and surfactants. These esters are obtained by acylation of vitamin C using different acyl donors in presence of chemical catalysts or lipases. Lipases have been used for this reaction as they show high regioselectivity and can be used under mild reaction conditions. Insolubility of hydrophilic ascorbic acid in non-polar solvents is the major obstacle during ascorbic acid esters synthesis. Different strategies have been invoked to address this problem viz. use of polar organic solvents, ionic liquids, and solid-phase condensation. Furthermore, to improve the yield of ascorbyl fatty acid esters, reactions were performed by (1) controlling water content in the reaction medium, (2) using vacuum to remove formed volatile side product, and (3) employing activated acyl donors (methyl, ethyl or vinyl esters of fatty acids). This mini-review offers a brief overview on lipase-catalyzed syntheses of vitamin C esters and their biotechnological applications. Also, wherever possible, technical viability, scope, and limitations of different methods are discussed.     

Environmentally friendly, efficient resolution of racemic secondary alcohols by lipase-catalyzed enantioselective transesterification in ionic liquids in the presence of organic bases

The lipase-catalyzed enantioselective transesterification of racemic secondary alcohols was studied using vinyl acetate as acyl donor in two imidazolium-based ionic liquids vs. hexane (Scheme), both in the absence and presence of catalytic amounts of organic bases such as triethylamine (Et(3)N) or pyridine. The organic bases generally enhanced both the rate and enantioselectivity of the reaction. Further, the system 1-butyl-3-methyl-1H-imidazolium hexafluorophosphate/Candida antarctica lipase B ([bmim][PF(6)]/CALB) could be readily recycled four times without significant loss in activity or enantioselectivity.     

Lipase-catalyzed transesterification in several reaction systems: An application of room temperature lonic liquids for bi-phasic production ofn-butyl acetate

Organic solvents are widely used in biotransformation systems. There are many efforts to reduce the consumption of organic solvents because of their toxicity to the environment and human health. In recent years, several groups have started to explore novel organic solvents called room temperature ionic liquids in order to substitute conventional organic solvents. In this work, lipase-catalyzed transesterification in several uni-and bi-phasic systems was studied. Two representative hydrophobic ionic liquids based on 1-butyl-3-methylimidazolum coupled with hexafluorophosphate ([BMIM][PF₆]) and bis[(trifluoromethylsulfonyl) imide] ([BMIM] [Tf₂N]) were employed as reaction media for the transesterification ofn-butanol. The commercial lipase, Novozym 435, was used for the transesterification reaction with vinyl acetate as an acyl donor, The conversion yield was increased around 10% in a water/[BMIM][Tf₂N], bi-phasic system compared with that in a water/hexane system. A higher distribution of substrates into the water phase is believed to enhance the conversion yield in a water/[BMIM][Tf₂N] system. Partion coefficients of the substrates in the water/[BMIM][Tf₂N] bi-phasic system were higher than three times that found in the water/hexane system, while n-butyl acetate showed a similar distribution in both systems. Thus, RTILs appear to be a promising substitute of organic solvents in some biotransformation systems.     

Lipase-catalyzed glycerolysis in ionic liquids directed towards diglyceride synthesis

This work examined the lipase-catalyzed glycerolysis of triglycerides (TG) in a list of commercially available ionic liquids (ILs) with varied cations and anions for the purpose of developing an efficient reaction protocol for diglyceride (DG) production and to understand whether ILs could assist the reaction systems. The reaction performances (reaction rate, TG conversion and DG yield) were found to be greatly dependent on the structure and property of ILs. The reactions in [TOMA·Tf₂N] and Ammoeng 120 produced comparable yield of DG to those most efficient conventional systems but with less by-products. Temperature enhancement generally yields positive effect on the conversion of TG, which was much more significant for the ILs with high viscosity. Unusually, increasing substrate concentration in many types of ILs led to enhanced conversion and yield; whereas the increase of glycerol/TG ratio resulted in a dramatic improvement of the reactions in the ILs with strong acidic anions. This work also sorted out some promising IL candidates, namely the ILs with good DG formation selectivity and the ones being able to achieve high TG conversion, which offered possibility to design binary IL systems. Overall, this study presented the first attempt concerning evaluation and characterization of lipase-catalyzed glycerolysis of TG for DG production in IL-based systems.     

Study on the potential anti-cancer activity of phosphonium and ammonium-based ionic liquids

The anti-cancer activity and cytotoxicity of phosphonium and ammonium-based ionic liquids (ILs) have been determined for the first time via NCI’s in vitro 60 human tumor cell lines. The preliminary SAR showed that the chain length of alkyl substitution on the cations plays crucial role towards anti-tumor activity and cytotoxicity of these ionic liquids. In general, phosphonium-based ILs were found to be more active and less cytotoxic as compared to ammonium ILs. Cell line data and hollow fiber study has demonstrated the potential of ILs to be developed as therapeutic agent.