Production of Bioactive Cellulose Films Reconstituted from Ionic Liquids

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Production of Bioactive Cellulose Films Reconstituted from Ionic Liquids Megan B. Turner, Scott K. Spear, John D. Holbrey, and Robin D. Rogers* Center for Green Manufacturing and Department of Chemistry, The University of Alabama, Tuscaloosa, Alabama 35487 Received April 27, 2004

A new method for introducing enzymes into cellulosic matrixes which can be formed into membranes, films, or beads has been developed using a cellulose-in-ionic-liquid dissolution and regeneration process. Initial results on the formation of thin cellulose films incorporating dispersed laccase indicate that active enzyme-encapsulated films can be prepared using this methodology and that precoating the enzyme with a second, hydrophobic ionic liquid prior to dispersion in the cellulose/ionic liquid solution can provide an increase in enzyme activity relative to that of untreated films, presumably by providing a stabilizing microenvironment for the enzyme. Introduction Cellulose appears to be an ideal support material for many enzyme systems, being both biosourced and biologically compatible. Many examples of cellulose-supported enzymes are known; in most cases, these supported-enzyme systems are prepared with cellulose derivatives and/or covalent binding, via functional linkers, to attach the enzymes on the surfaces of beads or membranes.1,2 Entrapment or encapsulation of an enzyme or protein using other procedures, especially physical entrapment of the biomolecule, without recourse to chemical attachment is certainly desirable, if it can be achieved. The first organic molten salt system investigated as a cellulose solvent was N-ethylpyridinium chloride (mp 118120 °C), in 1934;3 no commercial use of this solvent system seems to have been developed, possibly due to the high melting point of this salt. Husemann and Seifert4 found that N-ethylpyridinium chloride mixed with 50% dimethylformamide or dimethyl sulfoxide has a lower melting point (around 77 °C) and that this mixture dissolves cellulose well. In the late 1970s and early 1980s, researchers at the Helsinki University of Technology began experimenting with the N-ethylpyridinium chloride mixtures for immobilization of enzymes within cellulose fibers,5 microbial cells within cellulose beads,6 and whole cell yeast β-galactosidase within cellulose,7 largely without success. The investigation into the use of ionic liquids (ILs) as alternative solvents has been steadily increasing over the past 5 years.8,9 Properties of existing ILs, including low melting points, wide liquid ranges, and lack of vapor pressure, have encouraged researchers to explore known chemical reactions and processes using ILs in place of volatile organic solvents.8,9 Our interests in ILs lie in the utilization of their unique solvent properties for process applications, for * To whom correspondence should be addressed. E-mail: rdrogers@ bama.ua.edu.

Figure 1. Chemical structure of 1-butyl-3-methylimidazolium chloride ([C4mim]Cl).

example, in liquid-liquid extraction and separation processes10 based on the consideration and matching of solvent and solute solubility parameters.11,12 For example, we have demonstrated that the IL 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) can be used to readily dissolve cellulose without derivatization (Figure 1).13,14 More recently, Wu and co-workers have shown that the IL 1-allyl-3-methylimidazolium chloride also can be used in a similar manner, as a solvent to dissolve15 or derivatize cellulose.16 To determine whether the ready dissolution of cellulose in molten [C4mim]Cl could be used as a processing route for encapsulation of enzymatic species in biocompatible cellulosic membranes or beads, for reactive and sensing applications, two questions needed to be answered. First, can cellulose materials containing encapsulated enzymes be prepared using the [C4mim]Cl dissolution process, and if so, do the enzymes supported in such a cellulosic matrix remain active? As a model system, we chose to investigate the entrapment of laccase from Rhus Vernificera (E.C. 1.10.3.2) within reconstituted cellulosic films that were made using the [C4mim]Cl-cellulose dissolution and regeneration process.13 Here we demonstrate the proof of concept, focusing on a single cellulose solvent, [C4mim]Cl, and its use to incorporate actiVe biomolecules directly into underivatized cellulose resulting in the formation of biologically active membranes. Experimental Section Preparation of ILs. The ILs, [C4mim]Cl and 1-(2hydroxypropyl)-3-methylimidazolium chloride, were pre-

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Turner et al.

Table 1. Components of IL-Coated Laccase Cellulose Films [C4mim]Cl (g)

cellulose (% w/w)a

secondary IL

laccase added (% w/w)b

appearance

15 15 15

4.75 4.75 4.75

IL1, [C3-OHmim]Cl; 3.75 g IL2, [C1im]Cl; 3.75 g IL3, [C4mim][Tf2N]; 3.75 g

2.78 2.78 2.78

transparent, flexible transparent, flexible opaque,c flexible

a w/w: wt cellulose (dry)/wt [C mim]Cl IL. b w/w: wt laccase (dry)/wt cellulose (dry). c The film formed is initially opaque and becomes transparent after 4 storage in DI H2O for 24 h.

pared using the literature procedures.17,18 Purity of the material was confirmed by 1H and 13C NMR spectroscopy. Preparation of IL-Regenerated Cellulose Membranes. Microcrystalline cellulose (4.75% w/w), purchased from Sigma (St. Louis, MO), was dissolved in [C4mim]Cl using microwave pulse heating.13 Caution: ionic liquids are heated with exceptional efficiency by microwaVes,19 and care must be taken to aVoid excess localized heating that can induce cellulose pyrolysis. Upon dissolution of the cellulose, characterized by the formation of a clear, viscous solution, the matrix was allowed to cool to room temperature. The resulting supercooled liquid (the mp of pure [C4mim]Cl is 66 °C)20 was used for the immediate preparation of films. (Prolonged storage of the supercooled solutions results in slow crystallization of the solvent.) R. Vernificera laccase purchased from Aldrich (Milwaukee, WI; 2.78% w/w enzyme/cellulose) was promptly added to the supercooled cellulose/IL matrix and gently stirred to ensure homogeneous dispersion. Films, having a thickness between 100 and 150 µm, were then formed by casting the solution onto a glass plate using coating rods purchased from R&D Specialties (Webster, NY). After casting, the films were immediately washed with deionized (DI) water, leaching the water-soluble [C4mim]Cl and producing colorless, flexible films. Upon completion of this procedure, transparent and flexible but structurally weak films were obtained and stored at 4 °C in a hydrated form until use. If films were allowed to air-dry, they shrank considerably and took on the appearance of cellophane. Preparation of IL-Precoated Laccase, IL-Regenerated Cellulose Membranes. Microcrystalline cellulose (0.71 g) was dissolved in 15 g of [C4mim]Cl using microwave pulse heating as described above.13 Separately, laccase (2.78% w/w enzyme/cellulose), in its lyophilized form, was added to 3.75 g of one of the following secondary ILs: 1-(2-hydroxypropyl)-3-methylimidazolium chloride (IL1), protonated methylimidazolium chloride (IL2), or 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide (IL3) (Figure 2). The resulting solution was immediately added to the supercooled [C4mim]Cl/cellulose solution and gently mixed (Table 1). (Complete homogenization of the two ILs during brief mixing is unlikely to occur due to the high viscosity of the [C4mim]Cl/cellulose solution as well as the minimal processing time.) Upon integration of the IL-coated enzyme, the cellulose solutions were cast into thin (100-150 µm) films using coating rods and reconstituted into DI water, leaching the water-soluble ILs. Following reconstitution, the films again appeared flexible and translucent except for examples where the secondary IL was the hydrophobic IL3. In these cases, the resultant films were initially opaque. After equilibrating in DI H2O for 24 h, the films reverted to a

Figure 2. ILs studied: IL1, 1-(2-hydroxypropyl)-3-methylimidazolium chloride; IL2, protonated methylimidazolium chloride; and IL3, 1-butyl3-methylimidazolium bis(trifluoromethanesulfonyl)imide.

transparent appearance and droplets of the hydrophobic IL3 were observed at the bottom of the beaker. Determination of Laccase Activity. Circular disks (d ) 1.60 cm, A ) 2.01 cm2) were cut from each film and immersed in solutions containing 2.8 mL of 20 mM phosphate buffer, pH 7.13, and 0.054 mg of substrate, reduced syringaldazine, and incubated for 24 h at 27 °C. Following the incubation period, each sample was removed from the reaction solution, washed in DI H2O, and mounted vertically on a microscope slide for UV/vis spectroscopic measurement. The films adhere to the slide through cohesive forces between the flexible, wetted film and the glass surface. Samples were scanned from 300 to 700 nm on a Varian Cary 3C UV-visible spectrophotometer. Specific activities were calculated using individual absorbance intensity measurements, the extinction coefficient for oxidized syringaldazine (65 000 M-1 cm-1), and a film thickness of 0.1 cm. Enzyme Leaching Determination. Aliquots of DI H2O solution were collected from the regeneration process as well as from subsequent membrane washings. These solutions, including samples from the preparation of films containing both uncoated and [C4mim][Tf2N]-coated laccase, were measured spectrophotometrically from 200 to 500 nm. Laccase concentrations (mg) were calculated from a calibration curve constructed from spectrophotometric measurements of known concentrations (mg) of laccase. Results and Discussion These materials were studied in an attempt to ease the challenge facing homogeneous and liquid-liquid biphasic catalytic systems, namely, separation and isolation of the catalysts from products during reaction workup. Heterogenization of catalysts on solid supports is an attractive option for controlling catalyst identity as well as separation and recovery of the catalysts from products during reaction workup, one of the primary challenges facing homogeneous

Production of Bioactive Cellulose Films

Biomacromolecules, Vol. 5, No. 4, 2004 1381 Table 2. Activity of Laccase in Aqueous Solution and Entrapped within an IL-Regenerated Film

Figure 3. Cellulose film containing entrapped laccase (2.78% w/w) formed using the IL-dissolution and reconstitution treatment, before (left) and after (right) contacting and incubation in aqueous syringaldazine solution (10.71 mg/L).

Figure 4. UV/vis absorbance spectra of laccase-catalyzed oxidation in IL-reconstituted cellulose films. The peak centered at 371 nm represents the reduced form of syringaldazine, and the peak at 555 nm is indicative of the oxidized form of syringaldazine. (A) ILregenerated cellulose film, without laccase, following contact with syringaldazine solution (10.71 mg/L); (B) IL-regenerated cellulose film containing laccase (2.78% w/w) following contact with syringaldazine solution (10.71 mg/L); (C) IL-regenerated cellulose film with laccase (2.78% w/w) following contact with buffer solution containing no syringaldazine. Scheme 1. Chemical Structures of the Substrate (left) and Product (right) of the Studied Laccase-Catalyzed Oxidation

and liquid-liquid biphasic catalysis. With a growing interest in performing viable enzymatic reactions in IL solutions and an awareness of some of the challenges faced in isolating products from these and other IL systems, we reasoned that an approach to encapsulation of enzymes within cellulose polymer membranes using ionic liquids might prove to be useful for these systems. Laccase is a copper-containing redox enzyme that is produced by various classes of fungi and is responsible for the degradation of polyphenolic compounds, such as lignin.21 Laccase was used in these experiments as a model enzyme system. The activity of the enzymes incorporated into the cellulose films was monitored using the syringaldazine oxidation assay (Scheme 1).21 The reaction can be followed colorimetrically by UV/vis spectroscopy to quantitate the increase of reaction product, oxidized syringaldazine.21 The product absorbs in the visible region (λmax ) 555 nm) and has a characteristic pink color, whereas an aqueous solution of the substrate, reduced syringaldazine, is visibly colorless but produces a characteristic absorption band in the UV region (λmax ) 371 nm) (Figure 3).21 The UV/vis absorbance of the films was measured between 300 and 700 nm in order to monitor both the increase in product formation as well as the loading of the substrate onto the film (Figure 4). Even in the absence of enzyme, syringaldazine was absorbed into

form of enzyme

specific activity (µM/min/mg laccase)

% residual activity

native (aqueous) native (entrapped) [C4mim][Tf2N]-coated (entrapped)

0.298 0.052 0.086

100 18 29

the cellulose films from reaction solutions, as shown by the growth in the absorbance band at 371 nm (Figure 4). The presence of an absorbance band at 371 nm is indicative of adsorption of the substrate on the films upon contact with aqueous reaction solvents. Adsorbance of the substrate onto the cellulose films in the absence of enzyme demonstrates the ability of the porous membrane to facilitate substrate transport necessary for enzymatic activity and indicates that the immobilized laccase is solely responsible for subsequent substrate oxidation (Figure 4). Initially, films were prepared by dissolving cellulose in [C4mim]Cl as described above followed by dispersion of the enzyme directly in the hot (∼100 °C) solution; the colorimetric assay showed no laccase activity. A number of factors may contribute to the loss of enzymatic activity: (a) thermal denaturation of laccase resulting from the high-temperature processing, (b) increased structural constraints placed on the enzyme from the polymer encapsulation material, (c) deactivation of the laccase due to the presence of the [C4mim]Cl,24 and (d) enzyme leaching. To assess whether thermal shock was responsible, cellulose/IL solutions were initially cooled to room temperature, resulting in the formation of a viscous supercooled liquid, before dispersing the enzyme. Films prepared in this way did exhibit enzyme activity when placed in aqueous syringaldazine, and formation of the product was observed in the UV/vis spectra. This observation is a strong indication that thermal shock is an important factor to consider when processing delicate biological molecules. Thus, a method of preparing enzymatically active films, overcoming total thermal denaturation, was achieved. Laccase-catalyzed oxidation of syringaldazine within the cellulose film was easily discerned and measured. However, the rate of evolution of color within the films was slow in comparison to solution phase reaction with the native enzyme (Table 2). This may be due to a constrained local environment induced by the support matrix. Liu et al. have observed similar results with bovine liver catalase doped silica matrixes but were able to enhance enzymatic activity through a modification in their preparation procedure that allowed the researchers to avoid known protein denaturants and other inhibitory environments.22 While the IL-regenerated laccase/ cellulose films described here demonstrate biological activity, optimization of their preparation should serve to increase the enzymatic activity. While thermal shock appeared to be a contributing factor to the inactivation of laccase within the film, we have demonstrated that this can be overcome, at least in part, by using the cold processing methodology. However, the effect of IL-induced denaturation on the enzyme appears to be a

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major factor resulting in lowered activity compared to that of laccase in aqueous solutions.23 The IL [C4mim]Cl has been shown to have a significant denaturing effect on some enzymes, even in aqueous solution, due in part to the intrinsically high Cl- ion concentration and activity in the reaction media.24 Interactions between Clions and charged amino acid residues on the exterior of some proteins can threaten the integrity of the secondary structure and, thus, the activity of the enzyme. Further, proper hydration, leading to the structural stability, of the enzyme is necessary in most cases to achieve activity comparable to that observed in aqueous systems.25 Not all ILs show the same ability to denature proteins. In fact, Flowers et al. have demonstrated the use of the IL ethylammonium nitrate (EAN) as a protein renaturant.26 In contrast to the imidazolium ILs, EAN27 has a much stronger hydrogen bond donor group (N-H) than the weak C-H donors of the 1,3-dialkylimidazolium cations. One potential solution to the problem of denaturation of laccase in our cellulose films would be to change the dissolution solvent to provide an environment conducive to sustained protein conformation. This is not possible, however, as we know that the ILs proven to dissolve cellulose also have a denaturing effect on some proteins. A possible method for stabilizing enzymes in these systems might be to provide a “protective” precoating for the enzyme. It has previously been suggested that coating an enzyme in a hydrophobic IL can improve its stability in organic reaction media.28 To test whether coating of the enzyme with one IL during processing with a second IL solvent could help protect the enzyme, through sustentation of the conformation and hydration of the enzyme,2 laccase was precoated with an IL prior to its addition into the [C4mim]Cl/cellulose solution. Three ILs, having diverse solvent properties, were tested: 1-(2-hydroxypropyl)-3-methylimidazolium chloride18 (IL1), in which secondary hydroxyl group and strong N-H hydrogen-bond donating functionalities are introduced in the IL, protonated 1-H-3-methylimidazolium chloride (IL2), and the hydrophobic 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide (IL3) (Figure 2). To prepare these films, cellulose was dissolved in [C4mim]Cl as previously described and separately laccase (2.78% w/w) was dispersed in either IL1, IL2, or IL3. The two solutions were combined, gently stirred, and promptly cast into films using coating rods (Table 1). Each system containing the IL-precoated laccases, when tested with syringaldazine solution, showed activity for the oxidation of syringaldazine (Figure 5). IL1-treated laccase cellulose films produced levels of oxidized syringaldazine comparable to those of the uncoated laccase films, indicating that IL1 has neither added beneficial nor detrimental effects on the enzyme (Figure 5). In contrast, both the N-H hydrogen-bonding and hydrophobic IL additives, IL2 and IL3, respectively, lead to a significant increase in relative endpoint concentrations (calculated for a film thickness of 0.1 cm using the extinction coefficient of oxidized syringaldazine (526nm ) 65 000 M-1 cm-1)21) of oxidized syringaldazine (Figure 5). (The extinction coefficient used here corresponds to oxidized syringaldazine in aqueous solution,

Turner et al.

Figure 5. Concentration of laccase-catalyzed oxidized syringaldazine. The solid bar represents uncoated laccase entrapped in cellulose film, the lined bar represents IL1-coated ([C3-OHmim]Cl) laccase entrapped in cellulose film, the cross-hashed bar represents IL2-coated ([C1mim]Cl) laccase entrapped in a cellulose film, and the open bar represents IL3-coated ([C4mim][Tf2N]) laccase in a cellulose film.

Figure 6. Absorbance intensity of the syringaldazine reaction product (oxidized syringaldazine) at various time intervals. (A) 1 h, uncoated laccase; (A′) 1 h, IL3-coated laccase; (B) 3 h, uncoated laccase; (B′) 3 h, IL3-coated laccase; (C) 5 h, uncoated laccase; (C′) 5 h, IL3coated laccase.

but because the reconstituted films contain approximately 95% solution, the use of this value should be sufficient for the calculation of the concentration of product within the film.) Further studies comparing enzymatic activity in uncoated laccase-containing films to that in IL3-coated laccase-containing films were conducted (Figure 6). Films were prepared and introduced to reaction solutions as previously described and then measured at various time intervals. The data collected confirm the expected results: increasing accumulation of product with increasing time and higher product accumulation at each time interval in the IL3coated laccase films as compared to the uncoated laccase films (Figure 6). These results demonstrate that doping the [C4mim]Cl/ cellulose solution with a secondary-IL-coated laccase enhances its activity within a film. Both ILs, IL2 and IL3, should interact with the enzyme’s microenvironment although affecting its activity through different mechanisms. IL2 was chosen as it contains a relatively strong hydrogenbond donor group (N-H) similar to EAN, in which association of the cation with the enzyme through hydrogen-bonding interactions might occur. In contrast, IL3 is a hydrophobic system and the hydrophobic microenvironment should be preserved through the cellulose film processing procedure which does not allow sufficient time for diffusion mixing of the hydrophilic and hydrophobic ionic liquids. Having addressed the issues of thermal denaturation and solution-induced denaturation, experimentation into enzyme leaching was conducted to determine the cause of reduced catalytic activity compared to that of laccase in an aqueous

Production of Bioactive Cellulose Films

Figure 7. Enzyme leaching is not detected during experimental procedures despite the film, containing laccase, being in constant contact with an aqueous solution. Leaching was only observed during the film processing procedure.

solution. It is generally accepted that enzymes prefer aqueous environments to those of ILs, creating a distinct possibility for enzyme leaching during the reconstitution and subsequent washing processes. Spectrophotometric measurements of aliquots of the DI H2O used in the regeneration and washing of the cellulose films accounted for a loss of 16.6 and 13.2% enzyme in the uncoated laccase membrane and IL3-coated laccase membrane, respectively. IL3-coated laccase may demonstrate increased enzyme activity by allowing the enzyme to remain more closely associated with the cellulose polymer, effectively decreasing its leaching. It is of importance to note that once the films are used in reactions, the pink color indicative of the reaction product is distributed throughout the film yet it is neither visually nor spectrophotometrically detected in the reaction media. This observation leads us to believe that once the film has been initially formed and residual [C4mim]Cl has leached from the film, there is no subsequent enzyme leaching (Figure 7). Determining the specific activity for laccase was approached in a methodical manner and takes into account the data collected from enzyme leaching experiments. Laccase was examined in three different environments: in its native state in aqueous solution, uncoated (native) entrapped within a cellulose film, and IL3-coated laccase entrapped within a cellulose film (Table 2). The specific activity of laccase in each environment was calculated using the absorbance intensities at 555 nm that were spectrophotometrically measured, the extinction coefficient of oxidized syringaldazine (65 000 M-1 cm-1), and a film thickness of 0.1 cm. As expected, the highest specific activity was shown in the aqueous environment, followed by the IL3-coated laccase which retained 29% activity and finally the uncoated laccase which retained 18% activity. While these residual activities are a low percentage of the original activity, they are comparable to reported values for cellulose-immobilized enzymes.29 Summary The results described here demonstrate successful incorporation of an active enzyme into cellulose films prepared using the [C4mim]Cl IL processing route. Three fundamental points should be considered:

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Through solution processing, biomolecules can be physically entrapped within a cellulose matrix and demonstrate sustained activity. This process alleviates time-consuming chemistry involved in the functionalization of cellulose as well as subsequent covalent attachment of the desired biomolecule to the cellulose. The enzyme is entrapped while the material is being formed, producing low-leaching bioactive films. Enzyme activity has been demonstrated to be maintained by cold processing, a method made possible by the innate characteristics of the imidazolium ILs, preventing thermal denaturation of the enzyme. The unique features of [C4mim]Cl allow for both the ease of processing at room temperature and the formation of many types of materials (e.g., membranes, beads, hollow fibers, etc.). Opportunities exist utilizing this method for the formation of new, heterogeneously supported enzymatic systems for reactions and sensing systems using biocompatible and renewable cellulose matrixes. Optimization of material processing indicates improvement in bioactivity. Activity can be improved by pretreatment of the enzyme, providing a less denaturing microenvironment, and particularly enhanced with a hydrophobic IL coating. A hydrophobic [C4mim][Tf2N] IL coating serves to protect the hydration sphere of the enzyme during processing. This coating effectively protects the enzyme from the high Cl- ion concentration found in [C4mim]Cl, allowing higher activity to be maintained. Acknowledgment. This research has been supported by the U.S. Environmental Protection Agency’s STAR program through Grant Number RD-83143201-0. (Although the research described in this article has been funded in part by the EPA, it has not been subjected to the Agency’s required peer and policy review and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred.) References and Notes (1) (a) Tiller, J.; Berlin, P.; Klemm, D. Biotechnol. Appl. Biochem. 1999, 30, 155-162. (b) Inoue, Y.; Tsuchiyama, H.; Tran, L. T.; Kosugi, N.; Kimura, A. J. Fermentation Bioeng. 1992, 73, 116-120. (c) Roy, I.; Gupta, M. N. Process Biotechnol. 2003, 39, 325-332. (2) Gupta, M. N. Eur. J. Biochem. 1992, 230, 25-32. (3) Graenacher, C. Cellulose Solution. U.S. Patent 1,943,176, 1934. (4) Husemann, E.; Siefert, E. Makromol. Chem. 1969, 128, 288-291. (5) Linko, Y.-Y.; Pohjola, L.; Viskari, R.; Linko, M. FEBS Lett. 1976, 62, 77-80. (6) Linko, Y.-Y.; Poutanen, K.; Weckstro¨m, L.; Linko, P. Enzyme Microb. Technol. 1979, 1, 26-30. (7) Weckstro¨m, L.; Linko, Y.-Y.; Linko, P. In Food Process Engineering. Vol. 2: Enzyme Engineering in Food Processing; Linko, P., Larinkari, J., Eds.; Applied Science Publishers: London, 1980; pp 148-151. (8) Ionic Liquids: Industrial Applications for Green Chemistry; Rogers, R. D., Seddon, K. R., Eds.; ACS Symposium Series 818; American Chemical Society: Washington, DC, 2002. Ionic Liquids as Green SolVents; Rogers, R. D., Seddon, K. R., Eds.; ACS Symposium Series 856; American Chemical Society: Washington, DC, 2003. (9) Ionic Liquids in Synthesis; Wasserscheid, P., Welton, T., Eds.; WileyVCH: Weinheim, 2002. (10) Huddleston, J. G.; Willauer, H. D.; Swatloski, R. P.; Visser, A. E.; Rogers, R. D. Chem. Commun. 1998, 1765-1766. (11) Abraham, M. H.; Zissimos, A. M.; Huddleston, J. G.; Willauer, H. D.; Rogers, R. D.; Acree, W. E., Jr. Ind. Eng. Chem. Res. 2003, 42, 413-418. (12) Anderson, J. L.; Ding, J.; Welton, T.; Armstrong, D. W. J. Am. Chem. Soc. 2002, 124, 14247-14254.

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(13) Swatloski, R. P.; Spear, S. K.; Holbrey, J. D.; Rogers, R. D. J. Am. Chem. Soc. 2002, 124, 4974-4975. (14) Swatloski, R. P.; Holbrey, J. D.; Spear, S. K.; Rogers, R. D. In Molten Salts XIII; Trulove, P. C., De Long, H. C., Mantz, R. A., Stafford, G. R., Matsunaga, M., Eds.; The Electrochemical Society: Pennington, NJ, 2002; pp 155-164. (15) Ren, Q.; Wu, J.; Zhang, J.; He, J. S.; Guo, M. L. Acta Polym. Sin. 2003, 3, 448-451. (16) Wu, J.; Zhang, J.; Zhang, H.; He, J.; Ren, Q.; Guo, M. Biomacromolecules 2004, 5, 266-268. (17) Huddleston, J. G.; Visser, A. E.; Reichert, W. M.; Willauer, H. D.; Broker, G. A.; Rogers, R. D. Green Chem. 2001, 3, 156-164. (18) Holbrey, J. D.; Turner, M. B.; Reichert, W. M.; Rogers, R. D. Green Chem. 2003, 5, 443-447. (19) Varma, R. S.; Namboordiri, V. V. Chem. Commun. 2001, 643. (20) Holbrey, J. D.; Reichert, W. M.; Nieuwenhuyzen, M.; Johnson, S.; Seddon, K. R.; Rogers, R. D. Chem. Commun. 2003, 1636-1637.

Turner et al. (21) Harkin, J. M.; Obst, J. R. Science 1973, 180, 296-298. (22) Liu, D.-M.; Chen, I.-W. Acta Mater. 1999, 47, 4535-4544. (23) Hinckley, G.; Mozhaev, V. V.; Budde, C.; Khmelnitsky, Y. L. Biotechnol. Lett. 2002, 24, 2083-2087. (24) Turner, M. B.; Spear, S. K.; Huddleston, J. G.; Holbrey, J. D.; Rogers, R. D. Green Chem. 2003, 5, 443-447. (25) Klibanov, A. M. Chemtech 1986, 6, 354-359. (26) Summers, C. A.; Flowers, R. A. Protein Sci. 2000, 9, 2001-2008. (27) Garlitz, J. A.; Summers, C. A.; Flowers, R. A.; Borgstahl, G. E. O. Acta Crystallogr. 1995, D55, 2037-2038. (28) Kim, M.-J.; Lee, J. K. J. Org. Chem. 2002, 67, 6845-6847. (29) Varavinti, S.; Chaokasem, N.; Shobsngob, S. World J. Microbiol. Biotechnol. 2001, 17, 721-725.

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