Biocatalysis in Ionic Liquids

In recent years, the industrial use of biocatalysis has been reviewed several times [1-3, 23], Also, since the first reports [17, 24] on biocatalysis in ILs appeared in 2000 this field has often been reviewed, recently for example by the groups of Sheldon [14,21], Kragl [6], and Kazlauskas [7]. Comprehensive tables of the various enzymatic reactions attempted in ILs can be found in these excellent reviews (see, for example. Ref. [6]). Here only general trends will be outlined and illustrated by selected examples. 

Lipases are knovm for their tolerance of hydrophobic media and are now widely used, also on an industrial scale [25], e.g., for enantioselective (de)acylations of alcohols and amines. Hence, it is not surprising that the most explored enzymes in I Ls are lipases. 

Lipase-catalyzed polyester synthesis has received considerable interest [31] due to the harsh conditions used in traditional chemical polymerization ( 200 C), and has also been subjected to reaction in ILs. Both CaLB and PcL lipases have been found to catalyze polyester synthesis in [BMIM][BF4] and [BMIM][PF6], but this approach does not seem to offer any advantages over state-of-the-art lipase-mediated polyester synthesis [32, 33] [Eq. (3)]. Still, the polydispersity index of the polymers formed in ILs were remarkably affected, giving values close to 1, which indicate a very narrow molecular weight range compared to material prepared by conventional polymerization processes [33]. 

In the first report on (isolated) enzymatic catalysis in an IL, Erbeldinger and co-workers described the thermolysin-catalyzed synthesis of Z-aspartame in [BMIM][PF ] [17]. By condensation of Z-Asp-OH and H-Phe-OMe, the sweetener could be prepared in 95% yield, which is similar to the yield that has been obtained in traditional organic solvents [Eq. (4)]. A 5% water content was found critical for the enzymatic activity. Moreover, removal of the water by vacuum resulted in easy isolation of the product by precipitation [17]. Further, in another application of a protease, Adler-creutz and co-workers used a-chymotrypsin to study transesterification of Ac-Phe-OEt with 1-butanol [34] [Eq. (5)]. They found that at low water activity (a ), the 

It has been demonstrated that yeast cells and other microorganisms are active in the presence of ILs [6, 37]. In the very first report on biocatalysis in ILs, Lye and coworkers showed that a toluene-water system has a more detrimental effect on the microorganism Rhodococcus R312 than a system of [BMIM][PF6] and water, which is well tolerated [24]. Consequently, a better yield for the hydration of 1,3-dicyanobenzene [Eq. (7)] to 3-cyanobenzamide was obtained with the microorganism in the two-phase IL/water system than in the toluene system. 

An important number of organic reactions are now catalyzed by whole cells or isolated enzymes. However, there are still problems associated with the solubility, yield and selectivity of these biotransformations. Ever since the solubilization of alkaline phosphatase in a mixture of [Et3NH][N03] and water (4 1), it has been known that enzymes can be stable in ionic liquids. Recent research shows that ionic liquids can be used efficiently as a medium for biocatalytic processes.  

Kim performed similar transesterifications, observing that lipases were up to 25 times more enantioselective in ionic liquids ([bmim][PF6] and [emim]piF4]) than in conventional organic solvents.  

The incubation of enzymes in ionic liquids can also lead to increased reactivity. Sheldon showed that CaLB could be heated as a suspension in [bmim][PF6] before use. Higher transesterification activity was observed than for the untreated enzyme, both when the enzyme was free (SP 525, 120% activity after 20 h incubation) 

Intellectual property is also a concern in the future development of ionic liquids. Given that one of the main targets of these solvents is use in industrial processes, a relatively large number of patents are already protecting this field. Patents cover mainly (1) the preparation and/or new types of ionic liquids and (2) the use of ionic liquids as materials (solvent, catalyst, extraction medium, etc.). 

Most tetrafluoroborate and hexafluorophosphate ionic liquids are patent free however, owing to their cost, their insufficient stability toward hydrolysis and issues concerning their disposal, such ionic liquids are not useful for industrial applications. These problems prompted researchers to develop new types of ionic liquids with different anions, many of them being protected by state-of-the-matter patents, for example, HSOJ (Ref. 184), PF3(C2F5)J (Ref 185) and borate derivatives. To solve the problem of the high cost of imidazolium-based ionic liquids, one patent claims broad coverage of cheap phosphonium ionic liquids.  

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In order to broaden the field of biocatalysis in ionic liquids, other enzyme classes have also been screened. Of special interest are oxidoreductases for the enan-tioselective reduction of prochiral ketones [40]. Formate dehydrogenase from Candida boidinii was found to be stable and active in mixtures of [MMIM][MeS04] with buffer (Entry 12) [41]. So far, however, we have not been able to find an alcohol dehydrogenase that is active in the presence of ionic liquids in order to make use of another advantage of ionic liquids that they increase the solubility of hydrophobic compounds in aqueous systems. On addition of 40 % v/v of [MMIM][MeS04] to water, for example, the solubility of acetophenone is increased from 20 mmol to 200 mmol L ... 

An ionic liquid can be used as a pure solvent or as a co-solvent. An enzyme-ionic liquid system can be operated in a single phase or in multiple phases. Although most research has focused on enzymatic catalysis in ionic liquids, application to whole cell systems has also been reported (272). Besides searches for an alternative non-volatile and polar media with reduced water and orgamc solvents for biocatalysis, significant attention has been paid to the dispersion of enzymes and microorganisms in ionic liquids so that repeated use of the expensive biocatalysts can be realized. Another incentive for biocatalysis in ionic liquid media is to take advantage of the tunability of the solvent properties of the ionic liquids to achieve improved catalytic performance. Because biocatalysts are applied predominantly at lower temperatures (occasionally exceeding 100°C), thermal stability limitations of ionic liquids are typically not a concern. Instead, the solvent properties are most critical to the performance of biocatalysts. 

Biocatalysis in ionic liquids was first reported in 2000 [7, 8, 9]. The early work involved ionic liquids composed of a 1,3-dialkylimidazolium or N-alkylpyridinium cation and a weakly-coordinating anion (Figure 10.1). More recently, attention is shifting toward new structural types. A number of reviews of this rapidly expanding subject have appeared [10, 11, 12, 13, 14]. 

Various scenarios can be envisaged for biocatalysis in ionic liquids (Figure... 

Lipases, which are noted for their tolerance of organic solvents, were obvious candidates for biocatalysis in ionic liquids. Indeed, stable microbial lipases, such as CaLB [8, 54, 55, 56] and Pseudomonas cepacia lipase (PcL) [28, 55, 57] were cat-alytically active in the ionic liquids of the l-alkyl-3-methylimidazolium and 1-alkylpyridinium families, in combination with anions such as [BF4], [PF6], [TfO] and [ Tf2N]. Early results were not always consistent, which may be caused by impurities that result from the preparation of the ionic liquid. Lipase-mediated transesterification reactions (Figure 10.3) in these ionic liquids proceeded with an efficiency comparable to that in tert-butyl alcohol [8], dioxane [57], or toluene... 

Proteases have received less attention than lipases, but in one of the earliest papers on biocatalysis in ionic liquids it was noted that the activity loss of thermo-lysin during preincubation proceeded much more slowly in [BMIm][PF6] than in ethyl acetate [8]. The storage stability of a-chymotrypsin in the ionic liquid [EMIm][ Tf2N] was compared with that in water, 3 M sorbitol, and 1-propanol. The residual hydrolytic activity (after dilution with aqueous buffer) was measured vs time, and structural changes were monitored by fluorescence and CD spectroscopy as well as DSC [98]. The enzyme s life-time in [EMIm][ Tf2N] at 30°C was more than twice that in 3 M sorbitol, six times as long as that in water, and 96 times as long as that in 1-propanol. 

Most studies of biocatalysis in ionic liquids have been concerned with the use of isolated enzymes. It should not be overlooked, however, that the first report on biocatalysis and ionic liquids involved a whole-cell preparation Rhodococcus R312 in a biphasic [BMIm][PF(s]-water system [7]. It was shown, using a nitrile hydrolysis test reaction, that the microorganism maintained its activity better in ionic liquid than in a biphasic toluene-water system. 

Reetz, M.T., Wiesenhoefer, W., Francio, G., and Leitner, W., Biocatalysis in ionic liquids batchwise and continuous flow processes using supercritical carbon dioxide as the mobile phase, Chem. Commun. 992-993 (2002). 

Chiappe, C., Leandri, E., Lucchesi, S., Pieraccini, D., Hammock, B.D., and Morisseau, C. 2004. Biocatalysis in ionic liquids The stereoconvergent hydrolysis of trans- -methylstyrene oxide catalyzed by soluble epoxide hydrolase. Journal of Molecular Catalysis B Enzymatic, 27 243 8. 

Sheldon RA, Madeira R, Sorgedrager Ml et al (2002) Biocatalysis in ionic liquids. Green Chem 4 147-151... 

Kim K-W, Song B, Choi M-Y et al (2001) Biocatalysis in ionic liquids markedly enhanced enantioselectivity of lipase. Org Lett 3 1507-1509... 

Rantwijk FV, Sheldon RA (2007) Biocatalysis in ionic liquids. Chem Rev 107 2757-2785... 

Park S, Kazlauskas RJ (2003) Biocatalysis in ionic liquids — advantages beyond green. Curr Opin Biotechnol 14 432-A37... 

Tassues APM, Pinho B, Rodriguez O, Macedo EA. Biocatalysis in ionic liquid degradation of phenol by laccase. Procedia Eng 2012 42 226-30. 

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