— — —

+, Totally miscible; -, immiscible; ±, partially miscible (see Note 2).

+, Totally miscible; -, immiscible; ±, partially miscible (see Note 2).

From an overall point of view, three different operational strategies have been applied for enzymatic processes in ILs: (1) pure ILs in monophasic systems; (2) water-IL mixtures in monophasic systems; and (3) pure ILs in biphasic systems.

1. As pure solvents in monophasic systems, different water-immiscible ILs (e.g., 1-butyl-3-methylimidazolium hexafluorophosphate [bmim+ PF6], 1-ethyl-3-methylimidazolium Ms[trifluoromethane] sulfonylamide [emim+ NTf2-]) have been assayed as reaction media for biocatalytic reactions, by using both free or immobilized enzymes with excellent results (4-25). On the contrary and in all cases, no enzymatic activity was observed when anhydrous water-miscible ILs were used as reaction media. Furthermore, an additional new concept should be discussed. Classically, immobilized enzymes obtained by chemical or physical attachment onto solid supports have been considered advantageous over free enzyme molecules because they facilitate the recovery and reuse of the biocata-lyst, allowing continuous processes to be designed. However, in the case of water-immiscible ILs, free enzyme molecules suspended in these media behaved as anchored or immobilized biocatalysts, because they cannot be separated by liquid-liquid extraction (i.e., with buffer or aqueous solutions) (6,7,9,12-14,18,19). To eliminate protein molecules from ILs, it is necessary filter the enzyme-IL solution through ultrafiltration membranes with a cut-off lower than the molecular weight of the enzyme (22). ILs form a strong ionic matrix and the added enzyme molecules could be considered as included rather than dissolved in the media (see Fig. 2). ILs therefore should be regarded as being liquid enzyme immobilization supports, rather than reaction media (6). In this way, after an enzymatic transformation process in ILs, the products can be recovered by liquid-liquid extraction with solvents (5-8,12,13,17,19-24), or by reduced pressure (14), pervaporation (18), or by extraction with supercritical carbon dioxide (scCO2) (15,25), enabling the enzyme-IL system to be reused in consecutive operation cycles. Several authors have described the excellent stability of free enzymes in water-immiscible ILs towards reuse (up to 2300-fold half-life time with respect to classical organic solvents) (6,9,19,24). In this context, even if enzyme derivatives immobilized onto solid supports (e.g., Novozyme®, a commercial lipase derivative from Novo Nordisk) have been widely assayed in pure IL systems, the classical advantage that they have over free enzymes (e.g., the protective effect of the solid support against denaturative conditions) could be in doubt, because of the exceptional stabilization of free enzymes towards continuous reuse provided by ILs (e.g., free CALB in bmim+ PF6- exhibited a half-life time 2300 times greater than that observed in hexane at 50°C) (6). Also, it has been demonstrated that both free and immobilized Candida antarctica lipase B suspended in bmim+ NTf2- showed similar profiles of activity decay in continuous operation under anhydrous and extremely harsh conditions, such as scCO2 at 150°C and 10 MPa (25). In spite of these facts and because of the relative higher viscosity of ILs compared with water or organic solvents, novel materials, based on high-density metal oxides (e.g., WO3/TiO2, WO3/ZrO2) have been assayed as suitable supports for lipase immobilization by adsorption. Such enzyme derivatives have been used in ILs systems with excellent results as regards activity and enantioselectivity (17).

2. Water-miscible ILs (e.g., #-ethylpyridinium trifluoroacetate, 1,3-dimethyl-imidazolium methyl sulfate) have been described as suitable reaction media for enzymatic transformations when they were assayed in monophasic mixtures with water or aqueous solutions. In these conditions, IL-water mixtures should be considered as ionic solutions of organic salts. Several enzymes, such as subtili-sin, lipase, and P-galactosidase, were successfully assayed in these systems for the resolution of aminoacid esters, regioselective acetylation of glucose and transglycosylation of lactose, respectively (11,21,22). Hydrolytic enzymes exhibited behavior in aqueous solution of water-miscible ILs similar to that observed in classical water miscible organic solvents (e.g., A/,#-dimethylformamide, aceto-nitrile), characterized by maximal synthetic activity at a high water content, gradually decreasing until full enzyme deactivation at a low water content.

3. The third strategy assayed for enzyme-catalyzed reactions in ILs involves the use of these solvents as liquid or solid enzyme immobilization supports in nonaqueous biphasic systems (15,16,25). In such cases, the ability of ILs to retain free enzyme as a homogeneous phase (catalytic phase), whereas substrates and products reside largely in another phase (extractive phase), have successfully been assayed for lipase-catalyzed kinetic resolution in nonaqueous environments (e.g., organic solvents and scCO2). For the classical concept of immobilized enzymes, ILs with melting points higher than room temperature [e.g., 1-(3'-phenylpropyl)-3-methyl-imidazolonium hexafluorophosphate melts at 53°C] can also be used to easily obtain solid enzyme-IL particles then to be apply in organic reaction media (16).

2. Materials

1. 1-Butyl-3-methylimidzolium hexafluorophosphate (bmim+ PF6-; Solvent Innovation GmbH).

2. 1-Ethyl-3-methylimidzolium bis(trifluoromethane)sulfonylamide (emim+ NTf2-; Sigma Aldrich Chemical Co.).

3. 1-Buthyl-3-methylimidzolium bis(trifluoromethane)sulfonylamide (bmim+ NTf2-; Merck).

4. 1, 3-Dimethylimidazolium methylsulfate (mmim+ MeSO4-; Merck).

5. Themoslysins substrate solution: dissolve 100 mmol of Cbz-L-aspartate and 500 mmol of L-phenylalanine ethyl ester into the wet IL, and shake the mixture mechanically at room temperature until a clear solution is obtained

6. The mobile phase for Z-aspartame synthesis will consist of acetonitrile/water (60/40 [v/v]), containing the aqueous fraction 0.2% (w/v) triethylamine, which is adjusted to pH 2.5 with orthophosphoric acid.

7. CAL-B substrate solution: for the butyl butyrate synthesis. Dissolve 90 ||L (0.71 mmol) of vinyl butyrate and 330 |L (3.63 mmol) of 1-butanol in 900 |L of dry emim+ NTf2- into a screw-capped test tube. A clear solution must be obtained.

8. Reaction mixture for the CALB-catalyzed kinetic resolution of 2-pentanol: dissolve 138 |L (1.25 mmol) of vinyl propionate and 138 |L (1.25 mmol) of rac-2-pentanol in 900 |L of dry bmim+ NTf2- in a screw-capped test tube. A clear solution must be obtained.

9. Reaction mixture for the P-galactosidase-catalyzed #-acetyllactosamine synthesis Add 2.5 mL of mmim+ MeSO4- to a screw-capped test tube containing 4 mL of 1.5 M #-acetylglucosamine in 0.1 Mpotassium phosphate buffer, pH 7.3, and 2.5 mL of 250 mM lactose in 0.1 M potassium phosphate buffer, pH 7.3. Shake the resulting mixture mechanically at room temperature until a clear solution is obtained.

10. Reaction mixture for the polyester synthesis. Dissolve separately 200 |imol of divinyl adipate and 200 |imol of 1,4-butanediol in screw-capped test tubes containing 1 mL of dry bmim+ PF6-, respectively. Shake both mixtures mechanically for 30 min at 50°C to reach full solubilization of the monomers.

2.2. Enzymes

1. Thermolysin (EC; Sigma-Aldrich).

2. Candida antarctica lipase B (CALB; EC; Novo Nordik).

3. Pseudomonas cepacia lipase (PS-C; EC; Sigma).

4. Bacillus licheniforms subtilisin (Alcalase; EC; Novo Nordik).

3. Methods

3.1. Thermolysin-Catalyzed Z-Aspartame Synthesis in Bmim+ PF6- (4)

1. Into a screw-capped test tube containing 5 mL of bmim+ PF6-, add 200 |L of water and shake the mixture mechanically at room temperature until a clear solution is obtained (see Note 4).

2. Start the reaction by adding 50 |L of 1% (w/v) thermolysin solution in water (see Fig. 3).

3. Mix mechanically the reaction mixture (i.e., by a rotary disk shaker) for up to 50 h at 37°C to obtain full product conversion.

Fig. 2. Operational strategy for enzyme catalysis in monophasic ILs systems, including recycling and IL recovery processes.

4. To follow the enzyme reaction, 100 |L aliquots can be taken from the reaction mixture at appropriate times, and dissolved in 1.5 mL of mobile phase.

5. Analyze samples by high-performance liquid chromatography (HPLC) using a LiChrospher RP-18 column (25-cm length and 3.9-mm internal diameter, 5-|im particle size, and 10-nm pore size; Merck) in isocratic conditions at 1 mL/min flow rate. Elution profiles can be easily monitored at 257 nm by a DAD or UV detector.

6. To extract all product and remaining substrates, the full reaction mixture should be washed with water. Add a 10-fold excess water volume to the reaction mixture and shake vigorously for 15 min to carry out liquid-liquid extraction of the remaining substrates. Decant the aqueous phase. Remove the remaining water into the IL phase by vacuum evaporation, leading to precipitation of the product. Recover product crystals by filtration.

7. The resulting thermolysin-bmim+ PF6- system can be reused by adding an aqueous solution of fresh substrates.

3.2. CALB-Catalyzed Butyl Butyrate Synthesis in Emim+ NTf2- (6,7)

1. Start the reaction by adding 20 |L of 1 % (w/v) CALB solution in water (see Fig. 4).

2. Mechanically mix the reaction mixture (e.g., by a rotary disk shaker) and incubate at 40°C for up to 1 h to obtain full product conversion.

3. To follow the reaction, 20-|L aliquots can be taken from the reaction mixture at regular appropriate intervals of time and suspended in 1 mL of hexane (see Note 5). Shake the biphasic mixture obtained vigorously for 3 min to extract all substrates and products into the hexane phase. Then, mix 400 |L of hexane extract with 600 |L

Aspartam Synthese
Fig. 3. Thermolysin-catalyzed carbobenzoxy-l-aspartyl-l-phenylalanyl methyl ester (Z-aspartame) synthesis from Z-l-aspartate and l-phenylalanine methyl ester.

CH,-CHO 1-Butanol

Vinyl butyrate Candida antarctica Lipase B

Butyl butyrate

Fig. 4. Lipase-catalyzed butyl butyrate synthesis.

of 10 mM propyl acetate (internal standard) solution in hexane, and analyze 1 |L of the resulting solution chromatographically. Recover all the IL aliquots in a test tube for cleaning and reuse (see Subheading 3.6.).

4. Analysis of samples can be carried out by gas chromatography (GC) semicapillary Nukol column (15 m x 0.53 mm x 0.5 |m; Supelco) and a flame ionization detector. Chromatographic conditions are as follows: carrier gas (N2) at 8 kPa (20 mL/min total flow); temperature program: 45°C, 4 min, 8°C/min, 133°C; split ratio, 5:1; detector, 220°C. Retention time of peaks are as follows: propyl acetate, 3.2 min; vinyl butyrate, 4.3 min; 1-butanol, 6.6 min; butyl butyrate, 7.7 min , and butyric acid, 13.5 min.

5. To extract all product and remaining substrates, the full reaction mixture should be washed with hexane. Add a 30-fold excess hexane volume to the reaction mixture and shake vigorously for 15 min to give liquid-liquid extraction of the product and remaining substrates.

6. The resulting CALB-emim+ NTF2- system can be reused by adding fresh substrates, while the enzyme activity remain practically constant.

3.3. CALB-Catalyzed Kinetic Resolution of 2-Pentanol in bmim+ NTf2- (20)

1. Start the reaction by adding 20 |L of 1 % (w/v) CALB solution in water (see Fig. 5).

2. The reaction mixture should be mechanically mixed (e.g., by a rotary disk shaker) and incubated at 60°C for up to 3 h to obtain full product conversion.

un y rac-2-Pentanol

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